Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
DSpace
UBCV
Sauge, Elodie
Doctor of Philosophy - PhD
University of British Columbia
Losartan and telmisartan are blood pressure (BP)-lowering angiotensin II (ANGII) type 1 receptor (AT1) blockers (ARBs) with unexpected therapeutic properties in settings not linked to high BP. ARB pleiotropism is complex, however previous studies have revealed that ARBs can activate endothelial function in vivo by promoting the release of protective vasodilatory nitric oxide (NO) in fashions that may be independent of their ANGII/AT1-blocking effects. As a partial prodrug, losartan is first metabolized into EXP3179, which has traditionally been considered a non-AT1-blocking molecule, followed by conversion into EXP3174, the predominant AT1 blocker. We hypothesized that EXP3179 is the elusive metabolite behind losartan's unexpected NO-activating properties. To test this hypothesis, wild-type and transgenic mice and tissues were treated with losartan, EXP3179 and EXP3174. We showed that contrary to previous beliefs, EXP3179 has similar AT1-blocking and BP-lowering effects as EXP3174, although only the former has the unique ability to enhance endothelial function. We observed that the endothelial function-enhancing effects of EXP3179 were endothelium removal-sensitive, involved the endothelial NO synthase (eNOS) and endothelium-derived hyperpolarizing factor (EDHF) pathways, and are dependent on an intact caveolae/caveolin-1 system, a critical component of the luminal endothelial membrane’s ability to sense flow. As AT1 antagonists did not diminish EXP3179’s effects on vessel reactivity and NO release, ANGII type 2 receptor (AT2) knock-out mice were used to evaluate whether the NO-potentiating effects of losartan and EXP3179 resulted from an ANGII switch towards protective AT2 signalling or direct AT2 activation. We demonstrated that both losartan and EXP3179 act on the endothelium via an AT2-independent mechanism. Finally, we explored the pleiotropic effects of losartan and the most effective ARB at activating the NO pathway, telmisartan, in mouse models of chronic obstructive pulmonary disease (COPD) and allergic asthma. Contrary to previous literature, both drugs failed to improve lung function and remodelling in COPD while showing limited efficacy in improving allergic asthma. In summary, the current thesis provides a better understanding of the complex and diverse pleiotropic properties of ARBs and proposes that direct but selective activation of endothelial function by ARB-like molecules may be behind their protective properties in non-hypertensive settings.
PLEIOTROPISM OF LOSARTAN AND ITS METABOLITES AGAINST CARDIOPULMONARY DISEASES: SMALL CHEMICAL DIFFERENCES, MAJOR BIOLOGICAL IMPACTS by Elodie Sauge M.Sc., University of Lausanne, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmacology and Therapeutics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2024 © Elodie Sauge, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Pleiotropism of losartan and its metabolites against cardiopulmonary diseases: small chemical differences, major biological impacts submitted by Elodie Sauge in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Pharmacology and Therapeutics Examining Committee: Dr. Pascal Bernatchez, Associate Professor, Department of Pharmacology and Therapeutics, UBC Supervisor Dr. Angela Devlin, Professor, Department of Pediatrics, UBC Supervisory Committee Member Dr. Brian Rodrigues, Professor, Faculty of Pharmaceutical Sciences, UBC University Examiner Dr. Kevin Bennewith, Professor, Department of Pathology and Laboratory Medicine, UBC University Examiner Additional Supervisory Committee Members: Dr. Don D. Sin, Professor, Department of Medicine, UBC Supervisory Committee Member Dr. Kelly M. McNagny, Department of Medical Genetics, UBC Supervisory Committee Member iii Abstract Losartan and telmisartan are blood pressure (BP)-lowering angiotensin II (ANGII) type 1 receptor (AT1) blockers (ARBs) with unexpected therapeutic properties in settings not linked to high BP. ARB pleiotropism is complex, however previous studies have revealed that ARBs can activate endothelial function in vivo by promoting the release of protective vasodilatory nitric oxide (NO) in fashions that may be independent of their ANGII/AT1-blocking effects. As a partial prodrug, losartan is first metabolized into EXP3179, which has traditionally been considered a non-AT1-blocking molecule, followed by conversion into EXP3174, the predominant AT1 blocker. We hypothesized that EXP3179 is the elusive metabolite behind losartan's unexpected NO-activating properties. To test this hypothesis, wild-type and transgenic mice and tissues were treated with losartan, EXP3179 and EXP3174. We showed that contrary to previous beliefs, EXP3179 has similar AT1-blocking and BP-lowering effects as EXP3174, although only the former has the unique ability to enhance endothelial function. We observed that the endothelial function-enhancing effects of EXP3179 were endothelium removal-sensitive, involved the endothelial NO synthase (eNOS) and endothelium-derived hyperpolarizing factor (EDHF) pathways, and are dependent on an intact caveolae/caveolin-1 system, a critical component of the luminal endothelial membrane’s ability to sense flow. As AT1 antagonists did not diminish EXP3179’s effects on vessel reactivity and NO release, ANGII type 2 receptor (AT2) knock-out mice were used to evaluate whether the NO-potentiating effects of losartan and EXP3179 resulted from an ANGII switch iv towards protective AT2 signalling or direct AT2 activation. We demonstrated that both losartan and EXP3179 act on the endothelium via an AT2-independent mechanism. Finally, we explored the pleiotropic effects of losartan and the most effective ARB at activating the NO pathway, telmisartan, in mouse models of chronic obstructive pulmonary disease (COPD) and allergic asthma. Contrary to previous literature, both drugs failed to improve lung function and remodelling in COPD while showing limited efficacy in improving allergic asthma. In summary, the current thesis provides a better understanding of the complex and diverse pleiotropic properties of ARBs and proposes that direct but selective activation of endothelial function by ARB-like molecules may be behind their protective properties in non-hypertensive settings. v Lay Summary Losartan has been used since 1995 as a medication to treat high blood pressure. Studies have shown it has additional beneficial properties beyond its blood pressure-lowering effects. One of these properties is its ability to enhance the release of nitric oxide, a protective molecule in blood vessels. Our studies focused on one of losartan's main byproducts, EXP3179, to understand its mechanism of action and how it contributes to the beneficial effects unrelated to blood pressure control. We also investigated the impacts of losartan and another drug from the losartan family on chronic lung diseases. Our findings suggest that EXP3179 has unique effects on blood vessels and may be responsible for some of losartan's beneficial properties seen in preclinical studies. More importantly, we found further evidence to suggest that the protective effects of losartan are not linked to blood pressure, which gives valuable insight into its mechanism of action. vi Preface All experiments using animal models were approved by the UBC animal care committee under protocols A16-0113, A18-0126, A20-0112, A20-0249, A21-0099. Chapter 1: Introduction I wrote Chapter 1 myself. Dr. Pascal Bernatchez proofread and edited it. Chapter 2: Losartan metabolite EXP3179 is a unique blood pressure-lowering AT1R antagonist with direct, rapid endothelium-dependent vasoactive properties I wrote Chapter 2 as the lead 1st author, jointly with Dr. Dmitri Pechkovsky as a co-1st author. Dr. Pascal Bernatchez was the senior author with Dr. Marco A. Ciufolini. I carried out 70% of the experiments, wrote the original draft of the paper, performed data analysis and interpretation, and designed the figures. Dr. Dmitri Pechkovsky performed the in vitro and western blotting experiments and edited the manuscript. Dr. Marco A. Ciufolini and Dr. N.D. Prasad Atmuri provided losartan metabolites, the experimental protocol for their synthesis and NMR figures found in the supplementary data. Dr. Arash Y. Tehrani, Dr. Zoe White, Graham Donen, and Christopher Yuen assisted with myography experiments. Ying Dong and Michael J.A. Walker performed the surgical procedure for blood pressure measurements in rats. Jessica Cait, Dr. Michael Hughes, Dr. Kelly M. McNagny, Dr. Anthony Tam, and Dr. Don Sin assisted with animal training. The manuscript was proofread and edited by Dr. Pascal Bernatchez. This chapter has been published in the journal Vascular Pharmacology. vii Cite: Sauge E, Pechkovsky D, Atmuri NDP, Tehrani AY, White Z, Dong Y, Cait J, Hughes M, Tam A, Donen G, Yuen C, Walker MJA, McNagny KM, Sin DD, Ciufolini MA, Bernatchez P. Losartan metabolite EXP3179 is a unique blood pressure-lowering AT1R antagonist with direct, rapid endothelium-dependent vasoactive properties. Vascul Pharmacol. 2022 Sep 27;147:107112. doi: 10.1016/j.vph.2022.107112. Epub ahead of print. PMID: 36179789. Chapter 3: Losartan and metabolite EXP3179 activate endothelial function without lowering blood pressure in AT2 receptor KO mice I carried out 85% of the experiments in Chapter 3. I designed the experiments, performed all data analysis and interpretation, designed the figures, and wrote the original manuscript. Dr. Zoe White performed the echocardiography experiments, assisted with animal experiments and breeding, and proofread the manuscript. Christopher Yuen assisted with animal experiments. Farah Lizotte and Dr. Pedro Geraldes provided the AT2 KO mice used in this study and provided advice with animal experiments. Dr. Marco A. Ciufolini and Dr. N.D. Prasad Atmuri provided losartan metabolites. The study was conceived by myself and Dr. Pascal Bernatchez, who proofread, edited, and revised the manuscript. This chapter has been published in the European Journal of Pharmacology. Cite : Sauge E, White Z, Lizotte F, Yuen C, Atmuri NDP, Ciufolini MA, Geraldes P, Bernatchez P. Losartan and metabolite EXP3179 activate endothelial function without lowering blood pressure in AT2 receptor KO mice. Eur J Pharmacol. 2024 Aug 15;977:176663. doi: 10.1016/j.ejphar.2024.176663. Epub 2024 May 28. PMID: 38815786. viii Chapter 4: Exploring the Pleiotropic Effects of Telmisartan and Losartan in Mouse Models of COPD and Allergic Asthma I performed 80% of the experiments in Chapter 4. I performed all data analyses and interpretation, designed all figures, and wrote the original manuscript. Dr. Zoe White assisted with FlexiVent experiments and mouse breeding, Dr. Arash Y Tehrani assisted with myography experiments, and Dr. Marine Theret assisted with flow cytometry. Jessica Cait, Dr. Michael Hughes, Dr. Anthony Tam and Dr. Dmitri Pechkovsky assisted with animal experiments and training. Dr. Pascal Bernatchez proofread and edited the manuscript. Chapter 5: Overall conclusions, study impact, and future perspectives I wrote Chapter 5 and Dr. Pascal Bernatchez edited and proofread it. ix Table of Contents Abstract ......................................................................................................................... iii Lay Summary ................................................................................................................. v Preface .......................................................................................................................... vi Table of Contents ......................................................................................................... ix List of Tables ............................................................................................................... xv List of Figures ............................................................................................................ xvi List of Abbreviations .................................................................................................. xix Acknowledgements .................................................................................................. xxiii Chapter 1: Introduction ................................................................................................. 1 1.1 The renin-angiotensin-aldosterone system ....................................................... 1 1.2 Angiotensin II type 1 receptor ............................................................................ 5 1.3 Angiotensin II type 2 receptor ........................................................................... 6 1.4 Angiotensin receptor blockers ........................................................................... 7 1.4.1 Losartan and its metabolism ...................................................................... 8 1.4.2 Losartan metabolite EXP3179 ................................................................. 11 1.4.3 Telmisartan - beyond blood pressure control ........................................... 12 1.5 The endothelium ............................................................................................. 13 1.5.1 The vascular endothelium ........................................................................ 13 1.5.2 Nitric oxide ............................................................................................... 14 1.5.3 Prostacyclin ............................................................................................. 17 1.5.4 Endothelium-derived hyperpolarizing factors ........................................... 18 x 1.5.5 Caveolin-1 ................................................................................................ 19 1.5.6 Endothelial dysfunction ............................................................................ 21 1.6 Chronic lung diseases ..................................................................................... 22 1.6.1 Introduction to chronic obstructive pulmonary disease ............................ 22 1.6.2 Mouse models of COPD .......................................................................... 23 1.6.3 Introduction to asthma ............................................................................. 25 1.6.4 Mouse models of asthma ......................................................................... 26 1.6.5 Role of the endothelium in COPD and asthma ........................................ 27 1.6.6 ARBs in COPD and asthma ..................................................................... 28 1.7 Scope and aims of this thesis ......................................................................... 31 Chapter 2: Losartan Metabolite EXP3179 is a Unique Blood Pressure-Lowering AT1 Antagonist with Direct, Rapid Endothelium-Dependent Vasoactive Properties .... 33 2.1 Chapter summary ........................................................................................... 34 2.2 Introduction ..................................................................................................... 35 2.3 Material and methods ..................................................................................... 37 2.3.1 Chemicals ................................................................................................ 37 2.3.2 Cell Cultures, Angiotensin II Type 1 Receptor (AT1) DNA Construct and Generation of Stably AT1-expressing HEK 293 cells ............................................. 38 2.3.3 Western blotting (WB) .............................................................................. 38 2.3.4 Blood pressure and isometric force measurements ................................. 39 2.3.5 Statistical analyses .................................................................................. 41 2.4 Results ............................................................................................................ 41 xi 2.4.1 Both EXP3179 and EXP3174 block ANGII-AT1 signalling in reconstituted and primary cells .................................................................................................... 41 2.4.2 EXP3179 and EXP3174 share similar BP-lowering properties in rats and mice ................................................................................................................. 44 2.4.3 EXP3179 but not EXP3174 reduces mouse aortic contractility ex vivo in the absence of ANGII ................................................................................................... 46 2.4.4 Role of endothelium and direct effect on SMC ......................................... 51 2.5 Discussion....................................................................................................... 52 Chapter 3: Losartan and Metabolite EXP3179 Activate Endothelial Function Without Lowering Blood Pressure in AT2 Receptor KO Mice ............................................... 56 3.1 Chapter summary ........................................................................................... 57 3.2 Introduction ..................................................................................................... 58 3.3 Material and methods ..................................................................................... 60 3.3.1 Chemicals ................................................................................................ 60 3.3.2 Western blotting (WB) .............................................................................. 61 3.3.3 Animals .................................................................................................... 61 3.3.4 Isometric force and blood pressure (BP) measurements ......................... 61 3.3.5 Echocardiography .................................................................................... 62 3.3.6 Statistical analyses .................................................................................. 64 3.4 Results ............................................................................................................ 64 3.4.1 Pre-treatment with two AT1 agonists does not block the endothelial function-dependent anti-constrictive effect of EXP3179 ......................................... 64 3.4.2 AT2 KO mice release more NO than WT mice ........................................ 67 xii 3.4.3 AT2 KO mice show normal endothelial function response ....................... 70 3.4.4 Losartan did not reduce BP in AT2 KO mice ........................................... 74 3.5 Discussion....................................................................................................... 75 Chapter 4: Exploring the Pleiotropic Effects of Telmisartan and Losartan in Mouse Models of COPD and Allergic Asthma ....................................................................... 78 4.1 Chapter summary ........................................................................................... 79 4.2 Introduction ..................................................................................................... 80 4.3 Material and methods ..................................................................................... 83 4.3.1 Chemicals ................................................................................................ 83 4.3.2 Animals .................................................................................................... 83 4.3.3 CS exposure procedure ........................................................................... 84 4.3.4 Ovalbumin (OVA)-induced allergic lung inflammation .............................. 84 4.3.5 Measurements of lung function ................................................................ 85 4.3.6 Non-invasive blood pressure (BP) measurements ................................... 86 4.3.7 Flow cytometry measurements ................................................................ 86 4.3.8 Statistical analyses .................................................................................. 87 4.4 Results ............................................................................................................ 88 4.4.1 Lung mechanics of mice after chronic smoke exposure .......................... 88 4.4.2 Lung histology, body weights and BP of mice after chronic smoke exposure ................................................................................................................. 88 4.4.3 Lung mechanics of mice after OVA-induced allergic lung inflammation .. 92 4.4.4 Comparison of the effects of losartan, EXP3179 and EXP3174 on eosinophils ............................................................................................................. 95 xiii 4.4.5 Comparison of the effects of losartan, EXP3179 and EXP3174 on B cells .. ................................................................................................................. 95 4.4.6 Comparison of the effects of losartan, EXP3179 and EXP3174 on T cells .. ................................................................................................................. 95 4.4.7 Comparison of the effects of losartan, EXP3179 and EXP3174 on macrophages (MQs) .............................................................................................. 96 4.4.8 Comparison of the effects of losartan, EXP3179 and EXP3174 on leucocytes (CD45+) ............................................................................................... 96 4.4.9 Comparison of the effects of losartan, EXP3179 and EXP3174 on neutrophils ............................................................................................................. 96 4.5 Discussion....................................................................................................... 99 4.5.1 COPD ...................................................................................................... 99 4.5.2 Asthma ................................................................................................... 101 Chapter 5: Overall conclusions, study impact, and future perspectives ............. 103 5.1 Overall conclusion ......................................................................................... 103 5.2 Small chemical changes have big impacts ................................................... 104 5.3 Study limitations ............................................................................................ 105 5.4 Future perspectives of EXP3179 and ARB research .................................... 107 5.5 Conclusion .................................................................................................... 108 Bibliography .............................................................................................................. 110 Appendices ................................................................................................................ 176 xiv Appendix A : Supplementary data for Chapter 2 - Losartan Metabolite EXP3179 is a Unique Blood Pressure-Lowering AT1 Antagonist with Direct, Rapid Endothelium-Dependent Vasoactive Properties ........................................................................... 176 Appendix B : Supplementary data for Chapter 3 - Losartan and metabolite EXP3179 activate endothelial function without lowering blood pressure in AT2 receptor KO mice................................................................................................................................. 188 Appendix C : Supplementary data for Chapter 4 - Exploring the Pleiotropic Effects of Telmisartan and Losartan in Mouse Models of COPD and Allergic Asthma ............ 191 xv List of Tables Table 1: Comparison of pharmacological properties between losartan, EXP3179, EXP3174 and telmisartan .............................................................................................. 10 Table 2: Antibodies used in flow cytometry ................................................................... 87 Table A.1: Baseline systolic BP, diastolic BP, mean arterial BP, and heart rates for hypertensive rats ......................................................................................................... 182 Table A.2: Baseline systolic BP, diastolic BP, mean arterial BP, and heart rates for normotensive rats ........................................................................................................ 182 xvi List of Figures Figure 1.1: The renin-angiotensin-aldosterone system is a series of reactions designed to help regulate blood pressure ....................................................................................... 4 Figure 1.2: Chemical structure of (A) losartan and its metabolites (B) EXP3179 and (C) EXP3174 and (D) telmisartan ........................................................................................ 10 Figure 1.3: Important endothelial mechanisms involved in the pathogenesis of COPD and asthma. ......................................................................................................................... 28 Figure 2.1: Losartan and both derivatives (EXP3174 and EXP3179) inhibit ANGII- ..... 44 Figure 2.2: EXP3179 decreases blood pressure (BP) in normotensive and spontaneously hypertensive rats (SHR).. .............................................................................................. 46 Figure 2.3: EXP3179 reduces PE-induced contraction in aortas from WT mice. .......... 48 Figure 2.4: EXP3179 reduces PE-induced contraction in aortas from eNOS KO mice. 50 Figure 2.5: The vasoactive effect of EXP3179 is lost after endothelium removal and in caveolin-1 KO animals .................................................................................................. 52 Figure 3.1: Schematic representation of losartan first-passage metabolism to aldehyde EXP3179 and carboxylic acid EXP3174, by cytochrome P450. .................................... 60 Figure 3.2: EXP3179 reduces phenylephrine (PE)-induced contraction in aortas from WT mice pre-treated with EXP3174 and angiotensin II (ANGII) .......................................... 67 Figure 3.3: EXP3179 significantly reduces phenylephrine (PE)-induced contraction in aortas from both female and male WT and angiotensin II receptor type 2 (AT2) KO mice. ...................................................................................................................................... 70 xvii Figure 3.4: Losartan reduces phenylephrine (PE)-induced contraction in aortas from both female and male WT and angiotensin II receptor type 2 (AT2) KO mice....................... 73 Figure 3.5: Losartan does not affect blood pressure (BP) in angiotensin II receptor type 2 (AT2) KO mice. ............................................................................................................. 74 Figure 4.1: The effects of chronic smoke exposure on lung function in female mice. ... 90 Figure 4.2: Telmisartan did not prevent emphysema in cigarette smoke (CS)-exposed mice ............................................................................................................................... 91 Figure 4.3: The effects of losartan and its metabolites on lung function in BALB/c female mice exposed to ovalbumin (OVA)-induced allergic inflammation ................................. 93 Figure 4.4: Losartan and its metabolites did not prevent lung inflammation in ovalbumin (OVA)-sensitized mice ................................................................................................... 94 Figure 4.5: Distribution of lung inflammatory cell subtypes among ovalbumin (OVA)-sensitized mouse model treated with losartan, EXP3179 or EXP3174 ......................... 98 Figure 5.1: Current knowledge on EXP3179 endothelium-related mechanism of actions. .................................................................................................................................... 105 Figure A.1: Experimental procedure for in-house synthesized EXP3179 .................... 176 Figure A.2: Experimental procedure for in-house synthesized EXP3174. A................ 177 Figure A.3: NMR Data of synthesized EXP3174 and EXP3179 Analysis .................... 178 Figure A.4: Purity Assessment of synthesized EXP3179 by HPLC. ............................ 179 Figure A.5: Purity Assessment of synthesized EXP3174 by HPLC ............................. 180 Figure A.6: Heart rates (HR) were unchanged during the experiments. ...................... 181 Figure A.7: EXP3179 and EXP3174 applied intraperitoneally (i.p.) reduced blood pressure in wild-type (WT) mice. ................................................................................. 183 xviii Figure A.8: Assessment of vessel and endothelium integrity and smooth muscle contribution for each vessel used for myography experiments with wild-type (WT) mice. .................................................................................................................................... 184 Figure A.9: Representative tracings of PE-induced contractions under different treatment conditions. ................................................................................................................... 185 Figure A.10: EXP3179 reduces PE-induced contraction similarly in the presence and absence of ibuprofen ................................................................................................... 186 Figure A.11: Exposure of aortic rings from WT mice to PI3K inhibitor Wortmannin reversed EXP3179 effects on PE-induced contraction ................................................ 187 Figure B.1: WT and AT2 KO aortic rings respond similarly to acetylcholine (ACh) concentration-response curve ..................................................................................... 188 Figure B.2: EXP3179 reduces phenylephrine (PE)-induced contraction in WT and angiotensin II receptor type 2 (AT2) KO mouse aortas ............................................... 188 Figure B.3: WT and AT2 KO aortic rings respond similarly to acetylcholine (ACh) concentration-response curve. .................................................................................... 189 Figure B.4: Losartan reduces phenylephrine (PE)-induced contraction in aortas from WT and angiotensin II receptor type 2 (AT2) KO mice ...................................................... 189 Figure B.5: WT and angiotensin II receptor type 2 (AT2) KO mice have similar aorta morphology and pulse wave velocity (PWV) at baseline and after 4 weeks of losartan treatment. .................................................................................................................... 190 Figure C.1: Representative gating strategies in naive mice (non-asthmatic). .............. 191 Figure C.2: Representative gating strategies in OVA-SEN mice (asthmatic). ............. 192 xix List of Abbreviations AA - arachidonic acid Abs - antibodies ACE1 - angiotensin-converting enzyme 1 ACE2 - angiotensin-converting enzyme 2 ACEi - angiotensin-converting enzyme inhibitors ACh - acetylcholine AHR - airway hyperresponsiveness Akt - protein kinase B Alum - aluminum ANGII - angiotensin II ARB - angiotensin II type 1 receptor blocker AT1 - angiotensin II type 1 receptor AT2 - angiotensin II type 2 receptor BAL - bronchoalveolar lavage fluid BP - blood pressure cAMP - cyclic adenosine monophosphate CAV-1 - caveolin-1 cGMP - cyclic guanosine monophosphate COPD - chronic obstructive pulmonary disease COX- cyclooxygenase COX-2 - cyclooxygenase-2 CS - cigarette smoke xx DBP - diastolic blood pressure DMSO - dimethyl sulfoxide ECG - electrocardiogram ED - endothelial dysfunction EDHF - endothelium-derived hyperpolarizing factor eNOS - endothelial nitric oxide synthase FMD - flow-mediated dilation GPCR - G-protein-coupled receptor H2O2 - hydrogen peroxide H2S - hydrogen sulphide HDM - house dust mite HEK - human embryonic kidney HR - heart rate IBP - invasive blood pressure Ig - immunoglobulin iNOS - inducible nitric oxide synthase i.p. - intraperitoneal injection i.v. - intravenous injection K+ - potassium KO - knock-out L-NAME - L-NG-Nitroarginine methyl ester LOS - losartan LPS - Lipopolysaccharide xxi MABP - mean arterial blood pressure MLI - mean linear intercept MQ - macrophages NADPH - nicotinamide adenine dinucleotide phosphate nNOS - neuronal nitric oxide synthase NO - nitric oxide NOS - nitric oxide synthase O2•− - superoxide ONOO− - peroxynitrite OVA - ovalbumin PE - phenylephrine PGI2 - prostacyclin PGIS - prostacyclin synthase PPAR-γ - peroxisome proliferator-activated receptor gamma PPAR-δ - peroxisome proliferator-activated receptor delta PWV - pulse wave velocity RAAS - renin-angiotensin-aldosterone system rASMC - rat aortic smooth muscle cells ROS - reactive oxygen species SBP - systolic blood pressure SD - Sprague Dawley SHR - spontaneously hypertensive rats SMC - smooth muscle cells xxii SNP - sodium nitroprusside STJ - sinotubular junction TEM - transendothelial migration TGF - transforming growth factor VEGFR2 - vascular endothelial growth factor receptor 2 VSMC - vascular smooth muscle cells WB - western blotting WT - wild-type xxiii Acknowledgements I would like to sincerely thank the following people for their help and support in completing this PhD thesis. First, I would like to thank my supervisor, Dr. Pascal Bernatchez, for allowing me to work in his laboratory. I am grateful for his continuous support, advice and guidance, as well as his insightful feedback and dedicated time invested in evaluating and refining this work. I also want to thank my supervisory committee members, Dr. Don Sin, Dr. Angela Devlin, and Dr. Kelly McNagny, for their time, encouragement, and valuable feedback during committee meetings. I would like to express my deepest gratitude to Dr. Zoe White, Dr. Dmitri Pechkovsky, and Dr. Anthony Tam for their expertise, collaboration, support and friendship. A special thanks to Dr. Arash Tehrani, Claire Smits, Tatjana Ponomarev, Lubos Bohunek and Lynne Carter for their technical assistance in my research. A big thank you to Chris Yuen and Zeren Sun for creating a positive and fun work environment and our fruitful discussions. My sincere appreciation extends to the Department of Anesthesiology, Pharmacology & Therapeutics, the Providence Airway Centre and the Centre for Heart Lung Innovation of St. Paul’s Hospital for providing the necessary resources and academic environment which facilitated my research. I am grateful to Mitacs-Accelerate, the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada and the Marfan Foundation for their financial support, which enabled me to pursue this research and present my findings at conferences. Finally, I would like to thank my family and friends for their encouragement and support throughout these years. 1 Chapter 1: Introduction 1.1 The renin-angiotensin-aldosterone system The renin-angiotensin-aldosterone system (RAAS) is a complex regulatory system that plays a crucial role in the regulation of blood volume, electrolyte balance, and systemic vascular resistance [1]. It is ubiquitous, involving multiple organ systems such as the kidneys, lungs, liver, systemic vasculature, adrenal cortex, and brain [1]. While the baroreceptor reflex responds to short-term decreases in arterial pressure, RAAS is responsible for both acute and chronic variations [2], [3]. It is a crucial mediator of cardiac, vascular, and renal physiology, regulating vascular tone and salt and water homeostasis [1]. In addition to its primary physiological functions, RAAS is implicated in various physiological conditions, including hypertension, heart failure, other cardiovascular diseases, and chronic kidney disease [4], [5], [6]. Medications that block the overactivation of RAAS were shown to improve outcomes in these conditions [1], [7], [8], [9]. The classical understanding of RAAS involves three significant molecules: renin, angiotensin II (ANGII), and aldosterone. Renin is released in response to decreased renal perfusion and reduced tubular sodium content, and it is the rate-limiting enzyme in RAAS [1], [10]. It acts on angiotensinogen to form angiotensin I [1]. Angiotensin I conversion to ANGII occurs through the action of angiotensin-converting enzyme 1 (ACE1) [1], [6]. ANGII is the primary mediator of RAAS effects, including aldosterone secretion [11]. Aldosterone, produced in the adrenal cortex, acts on mineralocorticoid receptors in the renal cortical collecting duct. It promotes sodium reabsorption through the epithelial sodium channel and influences salt and water homeostasis by regulating thirst and salt 2 intake [12], [13], [14], [15], [16], [17]. All these components work together to elevate arterial pressure in response to decreased renal blood pressure (BP), salt intake to the distal convoluted tubule, and stimulation of adrenergic receptors [1]. Physiological effects of ANGII include vasoconstriction, aldosterone secretion, increased sodium reabsorption, enhanced sympathetic outflow, and the release of vasopressin from the posterior pituitary [18], [19], [20], [21], [22], [23]. ANGII is also implicated in oxidative stress, endothelial dysfunction (ED), fibrosis, and various pathological conditions such as atherosclerosis [24], [25], [26], [27]. It acts mainly through two types of receptors, ANGII type I receptors (AT1) and ANGII type II receptors (AT2), with different and opposing physiological responses [28], [29]. The majority of ANGII actions are exerted through AT1, which is strongly expressed in vascular smooth muscle cells (VSMC) and mediates vasoconstriction [30]. The AT2 receptor is recognized for its role in promoting vasodilation by releasing nitric oxide (NO) and exerting anti-proliferative effects [7], [31].[30], [32] Apart from the AT1 and AT2 receptors, the renin-angiotensin system (RAS) also includes pathways for angiotensin II (ANGII) deactivation. One important enzyme, angiotensin-converting enzyme 2 (ACE2), converts ANGII into angiotensin-(1-7), a peptide that counteracts many of ANGII's effects [33]. Angiotensin-(1-7) interacts with the Mas receptor to induce vasodilation and demonstrates anti-fibrotic and anti-inflammatory properties, as shown in animal studies [34]. ANGII can also be metabolized by aminopeptidases into angiotensin III, which has a higher affinity for AT2 receptors compared to AT1 receptors—by approximately 30-fold. This preferential binding to AT2 receptors results in vasodilation and enhanced natriuresis and bradykinin levels [35], [36]. Additionally, ACE2 converts angiotensin 1 into angiotensin-(1-9), a 3 peptide whose actions are not fully understood but which is further processed into angiotensin-(1-7) by ACE1, contributing to the pool of vasodilatory and protective angiotensin-(1-7) [33]. Overactivation of RAAS is implicated in the pathogenesis of cardiovascular and renal diseases, including primary hypertension [37], [38], [39], [40], [41]. Medications that block different steps of RAAS, such as angiotensin receptor blockers (ARBs), ACE inhibitors (ACEi), and aldosterone antagonists, have proven successful in managing these conditions [1], [42]. A schematic representation of the RAAS signalling pathways is shown in Figure 1.1. In summary, RAAS is a complex regulatory system with critical cardiovascular and renal health implications. Understanding its components and functions is essential for developing effective therapeutic strategies for conditions associated with RAAS dysregulation. 4 Figure 1.1: The renin-angiotensin-aldosterone system is a series of reactions designed to help regulate blood pressure. When blood pressure falls, the kidneys release the enzyme renin into the bloodstream. Renin splits angiotensinogen, a large protein that circulates in the bloodstream, into angiotensin I, which is further split by angiotensin-converting enzyme (ACE) into angiotensin II (ANGII). ANGII has many effects such as binding to angiotensin receptor type 1 (AT1) and angiotensin receptor type 2 (AT2), with a lower affinity for the latter. Binding to AT1 causes vasoconstriction, increasing blood pressure. ANGII also triggers the release of the hormone aldosterone from the adrenal glands, causing the kidneys to retain sodium, thus increasing water retention. In addition, ANGII induces the release of vasopressin, an antidiuretic hormone, from the pituitary gland, which causes water to be retained. This increase in water retention increases blood volume and, therefore, increases blood pressure. Angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB) are antihypertensive medications blocking RAAS signalling. Adapted from www.msdmanuals.com. 5 1.2 Angiotensin II type 1 receptor AT1 is a G-protein coupled receptor (GPCR) widely expressed across various cell types [43]. While the human genome encodes for a single isoform of AT1, rodents express two isoforms, AT1a and AT1b [44]. The activation of AT1 by ANGII results in vasoconstriction, water intake, and sodium retention [45]. Found in the heart, vasculature, kidney, adrenal glands, and central nervous system [46], [47], [48], [49], AT1 is responsible for various physiological actions and, in pathogenic states, triggers a cascade of events leading to inflammation, fibrosis, oxidative stress, tissue remodelling, and elevated blood pressure [44]. The ACE/ANGII/AT1 signalling pathway is associated with oxidative stress, hypertrophy, fibrosis, and inflammation, playing a central role in cardiac and renal diseases [45]. As a member of the GPCR superfamily, AT1 activates phospholipase C through the heterotrimeric Gq protein [44], [50]. It increases intracellular calcium and phosphorylation of proteins, resulting in vascular contraction and secretion of aldosterone from the adrenal cortex cells, as well as cell growth [44], [51]. Beyond its classical actions, AT1, through ANGII, controls cellular adhesion, migration, and intercellular matrix deposition [50]. These influence chronic adaptive changes in vascular and cardiac growth, remodelling, repair, and atherosclerosis [50]. ANGII also enhances nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, leading to the stimulation of reactive oxygen species (ROS) and nitrogen production within the vessel wall [50], [52]. These ROS products, including superoxide (O2•−) and hydrogen peroxide (H2O2), can activate a range of cellular components which subsequently trigger processes like cell growth and the expression of proinflammatory genes [50], [53], [54], [55], [56]. Therefore, 6 AT1 stands as a crucial molecular protagonist, orchestrating a variety of physiological and pathophysiological responses. Understanding its role opens avenues for targeted interventions in cardiovascular, pulmonary, and renal diseases and provides hope for better therapeutic strategies. 1.3 Angiotensin II type 2 receptor AT2 emerges as a distinctive player in RAAS. It presents unique characteristics and is often described as part of the natural protective system of the RAAS [57]. As a GPCR, AT2 is initially prevalent in fetal tissues, with its expression diminishing in adulthood [29], [43]. However, AT2 retains significance in adults, particularly in the aorta and coronary arteries [42]. Pathologically, an increase in AT2 expression occurs in conditions like vascular injury and myocardial infarction, indicating its involvement in cardiovascular responses [58], [59], [60]. Moreover, unlike the vasoconstriction and anti-natriuresis induced by AT1, the stimulation of AT2 by ANGII results in vasodilation via the increase of NO production and release and natriuresis, as well as inhibition of inflammation and fibrosis [29], [61], [62]. AT2 is a crucial component of the RAAS's protective arm. Understanding its dynamic expression, physiological roles, and involvement in pathological conditions highlights AT2 as a potential target for therapeutic interventions in cardiovascular and renal diseases. 7 1.4 Angiotensin receptor blockers Angiotensin II receptor antagonists, commonly referred to as ARBs, are a family of drugs that bind to and inhibit AT1, thereby suppressing RAAS signalling. ARBs interact with AT1 at different sites, but the most critical for receptor blockade and prevention of ANGII actions are AT1’s Arg167, Tyr35, and Trp84 residues, which can be found in all current ARBs. In addition, telmisartan also has Tyr92 as a fourth binding site (Figure 1.1) [63]. This inhibition disrupts the cascade of effects responsible for vascular contraction and sodium retention. Studies also suggest that ARBs can reduce ANGII-induced oxidative stress [64], [65]. In contrast to ACEi, which prevent the cleavage of angiotensin I to ANGII, ARBs target the peripheral actions of ANGII [66]. There are currently eight approved ARBs: losartan (Cozaar: 1995), valsartan (Diovan: 1996), irbesartan (Avapro: 1997), eprosartan (Teveten: 1997), candesartan (Atacand: 1998), telmisartan (Micardis: 1998), olmesartan (Benicar: 2002), and azilsartan (Edarbi: 2011) [66]. Additionally, fimasartan, the latest ARB developed, has been approved in Korea, China, Singapore, and Russia [67]. Even if they share similar chemical structures and activities, their pharmacokinetics vary. Losartan, candesartan, olmesartan, and azilsartan are prodrugs and undergo first-pass metabolism in the liver or gastrointestinal tract to generate a fully-active AT1 antagonist. In contrast, telmisartan, irbesartan, eprosartan, and valsartan are not prodrugs and do not require metabolic activation [68]. Introduced in 1995, ARBs have demonstrated efficacy in lowering BP in both animal models and humans, as well as reducing complications of cardiovascular [69], [70], [71] and kidney diseases [66], [72], [73]. Despite their similar chemical structure, publications from our group and that of others indicate that some ARBs may possess 8 heterogenous signalling activities resulting in endothelial function in an ANGII-independent fashion [74], [75], [76]. Although the cardiovascular benefits of BP are well established for each ARB [66], studies showed their beneficial effects in various diseases not linked to hypertension, such as emphysema and chronic lung diseases [77], cancer [78], [79], Alzheimer’s disease [80], or Marfan syndrome [75], although how ARBs mediate their pleiotropic effects remain poorly understood. It has been reported that the biological actions of all ARBs are not identical [81]. For example, activation of peroxisome proliferator-activated receptor gamma (PPAR-γ) is one of the mechanisms behind the beneficial effects of telmisartan and irbesartan [82], [83]. In addition, the ability of losartan and irbesartan to inhibit cyclooxygenase-2 (COX-2) or the thromboxane A2/prostaglandin endoperoxide H2 receptor is not shared by candesartan [81], [84], [85]. The differences observed between ARBs are likely caused by intrinsic differences in their chemical structures, as high lipophilicity is required to bind to intracellular PPAR-γ [42], [82]. Therefore, some of their beneficial effects might be specific to their molecular structure rather than an ARB class effect [86]. 1.4.1 Losartan and its metabolism The synthesis of imidazole derivatives by Furukawa and colleagues, targeting ANGII-induced vasoconstriction, led to the development of DuP 753 [87]. This compound proved to be a potent ANGII antagonist when administered orally to animals, highlighting its efficacy in blocking RAAS [87], [88], [89]. Later, DuP 753 was renamed losartan and was the first orally active non-peptide ARB approved for use in the United States [90]. Losartan has been a widely prescribed therapy for hypertension since its introduction in 9 1995, and its current indications include the treatment of hypertension, prevention of progression of diabetic nephropathy, and reduction of the risk of stroke in hypertensive patients with left ventricular hypertrophy [90]. Functionally, losartan selectively and competitively blocks the effects of ANGII at the AT1 site, resulting in a compensatory elevation of renin and ANGII levels [91], [92], [93]. It exhibits a high affinity for AT1, being over 10,000 times more selective for AT1 than AT2 [94]. Losartan's inhibitory effects include vasopressin release, adrenal catecholamine release, thirst, cellular hypertrophy, hyperplasia, noradrenergic neurotransmission, and sympathetic tone increase. Furthermore, losartan inhibits ANGII-induced vasoconstriction and the action of aldosterone, leading to reduced blood pressure [94]. Following oral administration, losartan peak plasma concentrations are reached within 1 to 2 hours (Table 1) [94], [95]. Approximately 14% of the administered dose undergoes hepatic conversion to produce the carboxylic acid metabolite, designated as EXP3174, via the hepatic P450 enzyme CYP2C9 [96], [97]. This metabolite is 10- to 40-fold more potent than losartan and has a half-life of 6 to 9 hours (Table 1) [97]. In addition, EXP3179, an intermediate aldehyde metabolite, has also been identified as an active metabolite of losartan. However, EXP3174 is believed to mediate the majority of losartan's AT1-blocking effects [98], [99]. The chemical structure of losartan and its metabolites are shown in Figure 1.2A-1.2C. 10 Table 1: Comparison of pharmacological properties between losartan, EXP3179, EXP3174 and telmisartan. Adapted from Michel M, et al. 2013 and Rothlin R, et al. 2023 [100], [101]. Figure 1.2: Chemical structure of (A) losartan and its metabolites (B) EXP3179 and (C) EXP3174 and (D) telmisartan. All current ARBs have three mandatory cores in the molecule for AT1R blocking and preventing ANGII actions (Arg167 or Lys199, Tyr35, Trp84). Telmisartan has a fourth binding core, Tyr92. 11 1.4.2 Losartan metabolite EXP3179 EXP3179 exhibits diverse pharmacological actions that extend beyond its traditional AT1-blocking effects. One notable action involves the promotion of endothelial nitric oxide synthase (eNOS) phosphorylation through the activation of a vascular endothelial growth factor receptor 2 (VEGFR2)/PI3K/protein kinase B (Akt) pathway, as shown by Watanabe et al. [102]. Moreover, they found that EXP3179 can suppress TNFα-induced caspase-3 activation and endothelial cell apoptosis. Despite its lower affinity for AT1 compared to EXP3174, EXP3179 surprisingly induces Akt and eNOS phosphorylation to levels equivalent to losartan and significantly greater than EXP3174, particularly at clinically relevant doses. This discrepancy in AT1 affinity suggests that the activation of Akt and eNOS by EXP3179 is independent of AT1 blockade [102]. Beyond its role in endothelial function, Krämer et al. showed that EXP3179 has anti-inflammatory and antiaggregatory properties. They found that it inhibits COX-2 mRNA upregulation, downstream prostaglandin generation, and platelet aggregation, which suggests a potential role in modulating inflammatory responses [98]. In addition, Schupp et al. found that the EXP3179 structure is similar to indomethacin, which indicates a potential binding affinity to PPAR-γ, further supporting its anti-inflammatory characteristics [103]. In 2007, EXP3179 was identified as a platelet-inhibitory and potentially vaso-protective molecule that could selectively reduce collagen-dependent human platelet activation, adhesion, and aggregation independent of AT1 antagonism [104]. These findings suggested that EXP3179 could be a potential candidate to prevent cardiovascular events such as atherothrombosis [104]. Moreover, Fortuño et al. demonstrated that EXP3179 blocks NADPH oxidase on a protein kinase C-dependent 12 mechanism and prevents the release of matrix metalloproteinase-9, which could lead to inhibition of matrix degeneration [105]. Hence, EXP3179 may have the ability to stabilize atherosclerotic plaques and positively impact the clinical outcomes of individuals with coronary artery disease [106]. The distinct actions of EXP3179 also extend to myocardial fibrosis in hypertensive models. While EXP3174 demonstrates a stronger antihypertensive effect, EXP3179 emerges as more effective in preventing myocardial fibrosis by downregulating lysyl oxidase expression and inhibiting the transforming growth factor (TGF)-β1-CTGF pathway, as shown by Miguel-Carrasco et al. in 2017 [107]. In summary, many studies have relied on EXP3179 as a biologically active, AT1-independent losartan metabolite responsible for losartan’s pleiotropic activities, as EXP3179 exhibits a multifaceted pharmacological profile, including endothelial protection [102], anti-inflammatory actions, antiaggregatory effects [98], and potential benefits in myocardial fibrosis [107]. However, none of those studies provide evidence of EXP3179‘s AT1 independence to validate these claims. EXP3179’s diverse mechanisms of action suggest therapeutic potential and open avenues for further exploration in cardiovascular and anti-inflammatory interventions. Moreover, additional studies are required to understand the exact mechanisms underlying these beneficial effects. 1.4.3 Telmisartan - beyond blood pressure control Telmisartan is a prominent ARB that has exhibited remarkable effects beyond its classic role as a BP-lowering agent [108]. It has a greater affinity for AT1 than both losartan and its active metabolite EXP3174, and a circulation half-life of 24 hours (Table 1) [100]. Telmisartan also shows greater lipophilicity compared to other ARBs, enabling 13 easier oral absorption and enhancing its ability to penetrate tissues and cells [109]. Experimental and clinical evidence demonstrate telmisartan’s ability to induce regression of cardiac hypertrophy, enhance endothelium-dependent relaxation, and improve arterial stiffness [110], [111], [112], [113]. Notably, these effects are independent of BP regulation and AT1 antagonism [113]. Telmisartan actions have been linked to increased NO availability, anti-inflammatory properties, and antioxidative effects on the endothelium, suggesting its potential as a multifaceted therapeutic agent in vascular diseases [114], [115], [116]. In addition, recent research indicates that telmisartan might possess agonistic properties towards PPAR-γ, potentially conferring it beneficial metabolic effects [117], [118]. Telmisartan’s chemical structure is shown in Figure 1.2D. 1.5 The endothelium 1.5.1 The vascular endothelium The vascular endothelium forms a continuous lining throughout the circulatory system, extending from the heart to the smallest capillaries. The endothelium is not only a physical barrier, but also functions as an endocrine organ, actively executing distinct functions crucial for physiological balance [119]. Under normal conditions, it regulates fluid filtration, blood vessel growth, hemostasis, neutrophil recruitment, and hormone trafficking [120], [121], as well as controlling thrombosis and platelet and leukocyte interactions with the vessel wall [122]. In the lung, the endothelium is located within the lining of the entire pulmonary vasculature, including the arteries, veins, and capillaries that facilitate gas exchange between the bloodstream and the air in the lung alveoli. It forms a barrier that prevents leakage of excess water and macromolecules into the lung 14 interstitium. It also plays a role in the transport of respiratory gases, water and solutes [123]. Moreover, the endothelium plays a critical role in modulating vascular tone by generating vasodilator and growth-stabilizing mediators, including NO. NO is predominant in large conduit arteries, whereas endothelium-derived hyperpolarizing factors (EDHF) are particularly significant in small resistance vessels [124], [125]. Additionally, endothelial cells release prostacyclin (PGI2) that plays a role across various vessel types, but its contribution to vasodilation and vascular homeostasis is especially notable in both large conduit arteries and small resistance vessels. [126]. The balance between these factors is essential for proper vascular function. Their dysregulation is implicated in conditions like hypertension, diabetes mellitus, and atherosclerosis [120], [127], as well as chronic lung diseases, stroke, heart disease, insulin resistance, chronic kidney failure, tumour growth, metastasis, venous thrombosis, and severe viral infectious diseases [120], [128], [129], [130]. Understanding the intricate roles of the vascular endothelium is crucial for the understanding of the pathophysiology of various cardiovascular diseases and developing targeted therapeutic strategies. Ongoing research aims to explore novel pharmacological approaches specifically targeting endothelial cells for more effective treatments and preventive measures. 1.5.2 Nitric oxide NO is a highly reactive and easily diffusible gaseous free radical continuously synthesized from the amino acid L-arginine in endothelial cells by the enzyme nitric oxide 15 synthase (NOS) [131]. It is generated by three subtypes of NOS, each displaying unique expression patterns and functional characteristics: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3) [132]. They undergo distinct regulation, influenced by both post-translational modifications and interactions with regulatory and scaffolding proteins [133]. A variety of agonists, including acetylcholine, histamine, thrombin, serotonin, ADP, bradykinin, norepinephrine, substance P, and isoproterenol can increase the release of NO from the endothelium. In addition, shear stress from blood flow is a primary physiological stimulus of NO synthesis, highlighting its relevance in vascular dynamics [134]. The biological effects that depend on NO are diverse. Notably, it induces vasodilation by modulating vascular dilator tone through the stimulation of soluble guanylyl cyclase, leading to heightened cyclic guanosine monophosphate (cGMP) concentrations [134]. Beyond its vasodilatory role, NO exerts antiplatelet effects, impeding platelet aggregation and adhesion, thereby averting the formation of pathological clots [135]. Furthermore, NO exhibits anti-inflammatory properties by inhibiting the synthesis and expression of cytokines and cell adhesion molecules. This anti-inflammatory effect may be mediated through the inhibition of the nuclear transcription factor κB, which governs the expression of proinflammatory proteins [134]. In addition, NO regulates growth factors, including TGF-β [136], limiting their activity within the vessel wall and platelets on the endothelial surface [134]. eNOS is predominantly expressed in vascular endothelial cells, with additional expression detected in platelets, some neurons in the brain, placental cells, and kidney tubular epithelium [137]. This enzyme catalyzes the synthesis of NO and L-citrulline from 16 L-arginine and O2. eNOS activation is contingent on calcium-calmodulin, with phosphorylation of the Ser1177 residue in the eNOS reductase domain enhancing its activity [132], [138], [139] The multifaceted roles of eNOS include vasodilation [140], [141], [142], antiplatelet effects [143], anti-inflammatory effects [144], and participation in vascular remodelling [145], highlighting its critical role in maintaining vascular homeostasis. Pathologically, ED arises in various disorders, such as essential hypertension, atherosclerosis, chronic obstructive pulmonary disease (COPD), and pre-eclampsia, where diminished NO release disrupts endothelial equilibrium [146]. Conditions including hypertension, hypercholesterolemia, smoking, diabetes mellitus, and heart failure are implicated in reduced NO release. Consequently, therapeutic strategies aiming at enhancing NO release have been explored to counteract ED [131]. The role of NO also extends to specific organ systems. For example, in the cardiovascular system, NO demonstrates direct inotropic effects and may contribute to circulatory dysfunction, especially post-myocardial infarction [147]. In addition, pulmonary function involves the investigation of inhaled NO for potential therapeutic benefits in lung pathologies [148], [149]. In summary, NO is a versatile molecule with critical functions in maintaining vascular homeostasis, and its dysregulation is associated with a wide range of pathological conditions. The endothelium plays a crucial role as a primary source of NO, constitutively expressing eNOS and adapting NO production patterns in response to various endogenous and exogenous stressors. ED occurs when this NO signalling 17 pathway is disrupted, limiting the ability of the vasculature either to produce NO itself or to respond appropriately to its presence [135]. 1.5.3 Prostacyclin PGI2, one of the most significant prostanoids regulating cardiovascular homeostasis, is a circulating hormone continually released by the lungs into the arterial circulation [150]. It is formed by the action of cyclooxygenase (COX) and prostacyclin synthase (PGIS) on arachidonic acid (AA) present in cell membranes throughout the body [151], [152], [153]. PGI2 plays diverse roles in physiological activities, such as platelet aggregation, vasorelaxation, vasoconstriction, local inflammatory responses, and leukocyte-endothelial cell adhesion, thereby modulating the pathogenesis of vascular diseases like thrombosis and atherosclerosis [151], [152], [154], [155]. These effects are mediated through specific cell surface receptors known as IP, a GPCR-generating cyclic adenosine monophosphate (cAMP) to facilitate physiological responses [156]. Additionally, PGI2's effects on vasculature involve the PPAR-delta (PPAR-δ) pathway, revealing a nuanced signalling mechanism beyond the classical IP-cAMP pathway [157]. IP receptors are expressed by endothelial cells, and PGI2 analogs have demonstrated protective functions against endothelial damage, restoring ED in conditions such as diabetes [158], [159]. Furthermore, the cAMP pathway is implicated in the interaction between eNOS and PGI2, suggesting a cross-talk mechanism within endothelial cells [160], [161]. PGI2's vasorelaxant effects are traditionally considered endothelium-independent, acting through IP receptor-mediated cAMP accumulation in VSMC, yet 18 complex interactions between NO and cAMP in endothelial cells may contribute to endothelium-dependent vasorelaxation [158], [160], [161]. Beyond its cardiovascular roles, PGI2 serves as an end product in the metabolism of AA, exhibiting immunomodulatory functions. It regulates both innate and adaptive immunity, displaying primarily anti-inflammatory or immunosuppressive effects [162], [163], [164], [165], [166], [167]. Clinically, PGI2 has a history of use in treating pulmonary arterial hypertension, demonstrating potent vasodilatory effects [168], [169]. Furthermore, in human emphysema lung tissue and after exposure to cigarette smoke (CS) extract, there is a reduced expression of PGIS compared to normal lungs. This indicates that the decrease in PGIS levels may result in harmful effects on lung vasculature in the context of COPD [170]. 1.5.4 Endothelium-derived hyperpolarizing factors The endothelium not only regulates vascular tone through the release of NO and PGI2 but also induces the hyperpolarization of underlying smooth muscle cells (SMC), a process attributed to EDHF. It makes it an essential component in the regulation of vascular homeostasis, contributing significantly to endothelium-dependent relaxation [171], [172]. While NO has long been acknowledged as a primary factor in conduit arteries, the contribution of EDHF is greater at the level of small arteries. Therefore, it plays an important role in the regulation of organ blood flow, peripheral vascular resistance and BP, particularly when the production of NO is compromised. Moreover, altered EDHF responses may contribute to or compensate for endothelial abnormalities associated with various cardiovascular diseases, influencing their pathogenesis [171]. 19 The term EDHF encompasses various molecules, including AA metabolites, H2O2, carbon monoxide and hydrogen sulphide (H2S) [173], [174], [175]. These factors activate different families of potassium (K+) channels, causing hyperpolarization of VSMC and contributing to their relaxation. The involvement of two key populations of endothelial K+ channels, the small- and intermediate-conductance calcium-activated potassium channels (SK(Ca) and IK(Ca), respectively), is crucial in mediating EDHF-induced responses [172]. ED involves not only a decrease in NO synthesis but also alterations in the EDHF pathway. The EDHF-mediated responses are sex-dependent and can be affected by aging, hypertension, atherosclerosis, and diabetes, highlighting its broader implications in vascular health [176]. EDHF's role in endothelium-dependent relaxation is now recognized as a crucial feature of a healthy endothelium [171]. Understanding the vessel-specific nature of EDHF and its modulation by activators or inhibitors is crucial for advancing therapeutic strategies. Restoration of EDHF responses through interventions, such as ACEi, ARBs, estrogen and potassium/calcium intake, has shown promise as potential therapies against cardiovascular diseases [176]. 1.5.5 Caveolin-1 Caveolin-1 (CAV-1) stands as a pivotal member of the caveolin family, alongside caveolin-2 and caveolin-3 [177], [178]. CAV-1 is a multifunctional membrane protein that plays a central role in the formation of caveolae, described as small invaginations on the plasma membrane known for their involvement in various cellular processes [179]. These membrane invaginations, considered a subset of lipid rafts, are highly ordered microdomains enriched in specific lipids [180], [181], [182]. The association of CAV-1 and 20 caveolae provides a platform for compartmentalizing and concentrating signalling molecules [183]. While CAV-1 is ubiquitously expressed, its levels vary across tissues, with the highest expression observed in adipocytes, endothelial cells, fibroblasts, SMC, and various epithelial cells [184]. CAV-1 also exhibits a diverse subcellular distribution, being found in various cellular organelles such as the Golgi apparatus, endoplasmic reticulum, mitochondria, nucleus, peroxisomes and lipid droplets [185]. This broad distribution emphasizes the CAV-1's involvement in numerous cellular functions beyond its role in caveolae. In addition, CAV-1 acts as a key regulator of energy homeostasis, influencing energy metabolism and mediating the signal transduction of glucose and lipid metabolism [186]. Implications extend to metabolic-related diseases, including diabetes [187], [188], obesity [189], cardiovascular disease [190], and cancer [191]. CAV-1 plays a crucial role in the regulation of eNOS. It can act as a negative regulator of eNOS by directly binding to it and inhibiting its enzymatic activity. This interaction is particularly important under basal or resting conditions [192], [193], [194], [195]. This inhibition maintains a tight control over NO levels in the absence of stimulatory signals [193], [194], [195]. Moreover, the CAV-1-eNOS interaction is dynamic and can be modulated in response to various extracellular stimuli. For example, various agonists, such as bradykinin or vascular endothelial growth factor, can trigger the dissociation of CAV-1 from eNOS, relieving the inhibition and allowing eNOS to become active. This activation leads to an increase in NO production, contributing to vasodilation and other downstream effects [196], [197]. 21 Dysregulation of the CAV-1-eNOS interaction has been implicated in cardiovascular diseases, including hypertension and atherosclerosis [198], [199]. Changes in the expression levels or phosphorylation status of CAV-1 can affect eNOS activity and NO bioavailability, contributing to ED and vascular pathologies [200], [201]. Therefore, the dynamic interaction between CAV-1 and eNOS is pivotal, regulating NO production under basal conditions and facilitating the integration of extracellular stimuli into intracellular NO signals [197]. 1.5.6 Endothelial dysfunction ED is a complex state characterized by a shift in endothelial phenotype, manifesting as reduced vasodilation, a proinflammatory state, and prothrombotic properties. This condition is intricately linked to various vascular diseases, including but not limited to hypertension, atherosclerosis, and thrombosis [202]. ED is biochemically defined as a reduction in the amount of bioavailable NO in the vasculature. ROS, particularly O2•−, contribute significantly to the deficiency in NO signalling by reacting readily with NO to form peroxynitrite (ONOO−), a reactive nitrogen species with deleterious effects. Various pathological states can potentiate the overproduction of ROS and subsequent ONOO− formation [203], [204]. The disruption of the delicate balance of NO by free radicals can damage the endothelium, rendering it excessively permeable and allowing toxins to pass into body tissues [202]. While the human body typically possesses an adequate supply of antioxidants from various nutrients to neutralize these free radicals, depletion of antioxidants or the presence of multiple coexistent factors may lead to endothelial injury and an imbalance in NO levels [120]. 22 Additionally, modifications to eNOS itself can alter NO production at the source, leading to a situation referred to as eNOS uncoupling. Disruptions in the interaction between eNOS and its cofactors result in eNOS functioning as a weak NADPH oxidase, producing O2•− instead of NO [203], [204]. Multiple mechanisms contribute to eNOS uncoupling, including the disruptive effects of ONOO− on the eNOS structure and the bioavailability of L-arginine or its inhibitor, asymmetric dimethyl-L-arginine [135]. Moreover, post-translational modifications of eNOS further contribute to pathologic uncoupling and reduction of NO production under oxidative stress conditions [205], [206]. Addressing ED or regulating the functions of eNOS holds the potential to prevent associated vascular disorders that contribute significantly to global mortality [207]. 1.6 Chronic lung diseases 1.6.1 Introduction to chronic obstructive pulmonary disease COPD is a complex and progressive lung condition characterized by chronic pulmonary inflammation, mucus hypersecretion, airway remodelling, and emphysema [208]. Beyond lung inflammation, COPD manifests as a multisystemic disorder, leading to cardiovascular disease, diabetes mellitus, malnutrition, muscle dysfunction, lung cancer, osteoporosis, infections, anemia, and depression [209], [210]. Environmental factors, particularly CS exposure, play a pivotal role in COPD development, activating ROS, inflammatory-oxidative stress, and apoptosis [211].[212] COPD is a significant public health concern, ranked as the third leading cause of death in the United States [211], and significantly impacts patients' daily lives, causing significant morbidity and mortality [213]. While tobacco smoking is widely recognized as a major risk factor for 23 COPD, only around 20% of smokers develop the condition. Recent evidence indicates that several other factors contribute significantly to COPD development. These include air pollution, respiratory infections, malnutrition, chronic asthma, underdeveloped lungs, lower socio-economic status, and genetic susceptibility [214], [215], [216]. In high-income countries, smoking is the primary cause of COPD, accounting for over 70% of cases. However, in low- and middle-income countries, tobacco smoking is responsible for 30–40% of COPD cases, while household air pollution emerges as a substantial risk factor [217]. With its prevalence on the rise, especially in aging populations, COPD poses a considerable economic burden on individuals, families, and societies worldwide [218]. While it remains a major public health challenge, there is currently no treatment to attenuate the disease progression [219], with smoking cessation remaining the primary preventive measure [220], [221]. 1.6.2 Mouse models of COPD CS-induced COPD: In the context of COPD, CS exposure is a major risk factor, commonly used in in vivo studies as the predominant inducer of COPD in mice [222], [223], [224]. Among the various animal models available for the induction of COPD, mice have been the most commonly used species [225], [226]. Nevertheless, studies have shown varying sensitivity levels to CS challenge among mouse strains [226]. In addition, challenges arise from variations in exposure protocols (whole body or nose-only exposure), as well as frequency and duration (once or twice/day, several times/week for various days/weeks/months) and the type of cigarettes used (commercial vs. research 24 cigarettes, with or without a filter) [227], [228], which therefore limits the comparability of studies because of the lack of standardized exposure methods [228]. Lipopolysaccharide (LPS)-induced COPD: LPS is a component found in Gram-negative bacteria cell walls and is present as a contaminant in air pollution, organic dust and CS [229], [230]. It induces a short-term COPD model with human-like features [231], [232]. LPS exposure, either alone or in conjunction with CS, triggers acute exacerbations and contributes to COPD development [233]. In mice, exposure to LPS induces emphysema-like changes as well as airway hyperresponsiveness (AHR) and airway inflammation [234], [235], [236]. Elastase-induced COPD: Elastase, a proteolytic enzyme, induces emphysema by breaking down alveolar tissue [237]. This model is used to induce inflammatory responses to initiate and perpetuate the inflammatory response seen in COPD. The elastase model offers significant benefits, including the immediate loss of alveolar wall structure through a single instillation of the enzyme into the lung and the capability to regulate the disease severity by adjusting the enzyme amount [233], [238]. A diverse range of animals has been employed in the elastase emphysema model to replicate certain features observed in human COPD induced by CS [233], [237], [239]. While the role of elastase in COPD has been established through these models, the direct instillation of elastases into an animal model provides limited insights into the underlying pathogenesis of the disease [232]. 25 1.6.3 Introduction to asthma Asthma is a complex and intricate chronic disease that significantly affects the respiratory system [129]. It is a global health concern, affecting around 300 million individuals across all age groups. Its prevalence is increasing worldwide, leading to approximately 180,000 asthma-related deaths annually [240], [241]. The impact of asthma extends beyond individual suffering, contributing to the loss of productivity in adults and learning impairment in children [242]. Asthma symptoms can range from mild to severe, with varying responses to standard therapies. While mild-to-moderate asthma often responds well to inhaled corticosteroids and long-acting beta-adrenergic agents, some individuals experience severe treatment-refractory asthma. These patients require high-dose corticosteroids to prevent uncontrolled symptoms, posing a significant disease burden and increased risk of exacerbations [243], [244]. Asthma is characterized by chronic inflammation of the airways, AHR, airflow obstruction, and airway wall remodelling [245]. In allergic asthma, eosinophilic inflammation has been considered the central characteristic of asthmatic airways [246]. Asthma's complexity arises from the involvement of various cellular players, including eosinophils, T-lymphocytes, macrophages, mast cells, neutrophils, and epithelial cells. These cells release cellular mediators, such as cytokines, chemokines, and growth factors, contributing to the disease progression [247]. It results in airway remodelling, characterized by mucus hypersecretion, fibrosis, goblet cell hyperplasia/metaplasia, and smooth muscle thickening [248]. In addition, asthma also induces vascular changes, including angiogenesis, vascular dilation, hyperpermeability, sustained inflammation and airway thickening [249]. 26 1.6.4 Mouse models of asthma Ovalbumin (OVA)-induced asthma: Traditionally, OVA has served as a key allergen for inducing both acute and chronic experimental allergic asthma in mouse models [250]. However, concerns have been raised about the clinical relevance of OVA, as it is not a naturally occurring allergen, and the in vivo sensitization process involving aluminum adjuvants differs significantly from human sensitization [251]. Mice are intraperitoneally immunized twice with OVA adsorbed to an aluminum hydroxide adjuvant (Alum) before the challenging phase. During this sensitization phase, the mice produce anti-OVA immunoglobulin (Ig)E antibodies. After this sensitization, the mice are intranasally challenged with OVA, which will make them develop clinical features of asthma [250], [252]. The OVA-induced asthma model replicates many features of asthmatic lungs, including specific IgE levels, Th2-associated eosinophilic inflammation, early and late asthmatic responses, AHR, and tissue remodelling [253]. Nevertheless, researchers have questioned its clinical significance, prompting the exploration of more relevant models. House dust mite (HDM)-induced asthma: In response to the limitations of OVA, researchers have increasingly turned to HDM as a clinically relevant allergen to induce allergic asthma models [254]. The induction of allergic asthma through topically delivered HDM into the airways has become more common, specifically via the intranasal route [253], [255]. In addition, HDM, as a natural allergen, eliminates the need for adjuvants [256]. HDM-induced asthma models exhibit robust characteristics such as airway eosinophilic inflammation, Th2 cytokine production, HDM-specific IgE presence, airway remodelling, and bronchial hyperreactivity, aligning closely with human allergic asthma 27 [251], [254], [257]. Researchers increasingly recognize HDM as a valuable and representative tool for investigating asthma pathogenesis and potential therapeutic interventions [258]. 1.6.5 Role of the endothelium in COPD and asthma Both asthma and COPD are inflammatory disorders, with research focusing mainly on inflammatory cells like neutrophils and eosinophils [259], [260]. However, the role of the endothelial tissue, essential for the transendothelial migration (TEM) of inflammatory cells into the lung parenchyma, has been understudied [129]. Abnormal endothelial function could contribute to increased inflammation and tissue damage in asthma and COPD [129], and ED is observed in both diseases [129], [261]. Investigating the endothelium's involvement in these diseases is crucial for understanding their pathogenesis and exploring potential treatments [129]. Asthma patients exhibit reduced flow-mediated dilation (FMD), indicating a dysfunctional endothelium, which affects airway responses [261]. In COPD, ED is linked to disease severity, reduced lung function, and poor clinical outcomes. It also correlates with exacerbations, potentially contributing to systemic atherosclerosis and increased cardiac events in COPD patients [262], [263], [264], [265]. The endothelium's abnormal behaviour in asthma and COPD involves various pathways. For instance, increased angiogenesis occurs in asthma [249], while emphysema exhibits decreased angiogenesis [129]. In some COPD patients, endothelial apoptosis leads to alveolar destruction, impacting gas transfer. Additionally, dysfunctional endothelial progenitor cells contribute to impaired repair processes [129]. Inflammation likely underlies these endothelial alterations, suggesting that existing treatments targeting 28 inflammation might improve endothelial function. However, more research is necessary to develop effective treatments for ED in asthma and COPD [129]. The different endothelial mechanisms for COPD and asthma are summarized in Figure 1.3. 1.6.6 ARBs in COPD and asthma In the context of COPD, studies revealed that ARBs could potentially alter the course of the disease, decreasing mortality rates and reducing hospitalizations [266], [267]. A retrospective study demonstrated lower mortality in ARB-treated COPD patients [268]. In addition, a population-based cohort study reported that COPD patients already receiving ARBs for cardiovascular disease had lower in-hospital mortality [269]. Figure 1.3: Important endothelial mechanisms involved in the pathogenesis of COPD and asthma. Adapted from Green et al., 2017. 29 Studies suggest that RAAS is implicated in COPD pathogenesis by stimulating proinflammatory mediators [270], [271], [272]. In addition, ROS generation via AT1 contributes to mitochondrial dysfunction, causing oxidative stress and ED observed in COPD patients [273], [274]. Moreover, Bullock et al. found that an increased AT1/AT2 ratio in fibrotic regions surrounding bronchioles correlates with reduced forced expiratory volume in 1 second, suggesting a role for ANGII in bronchoconstriction via AT1 [275]. eNOS plays a pivotal role in vascular tone control in COPD [276]. Notably, serum concentrations of competitive inhibitors of NOS function are increased in COPD, contributing to long-term all-cause mortality risk and heightened airway resistance [277], [278], [279], [280]. Thus, the complex interaction between RAAS and NO in COPD might open new therapeutic avenues for the treatment of COPD. RAAS blockade with ARBs, as well as restoring NO balance in patients with COPD, are promising strategies for decreasing exacerbations, reducing mortality, and addressing the systemic consequences of COPD. In a mouse model of asthma, overexpression of the eNOS gene prevented AHR, inflammation, and cytokine production [281]. Despite the traditional focus on iNOS [282], [283], [284], studies highlight the importance of eNOS in controlling asthmatic disease [285], [286], [287], [288]. Polymorphisms in the eNOS gene can change the enzyme's activity in patients, reducing its ability to produce NO. This reduced NO production can affect inflammation and bronchodilation, which are protective mechanisms, and may contribute to chronic inflammation and airway hyperresponsiveness in asthma [289]. Studies have found specific eNOS gene polymorphisms that are more common in asthma patients compared to controls. These polymorphisms can alter NO production, indicating 30 a genetic predisposition that could impact the development and progression of the disease [290]. Understanding these polymorphisms is crucial for developing targeted therapies to compensate for decreased NO levels. This highlights the potential of eNOS as a promising therapeutic target in managing asthma [281], [291], [292]. Interestingly, studies have shown the activation of RAAS in patients with acute severe asthma [293], [294]. ANGII induces bronchoconstriction and inflammation, contributing to asthma symptoms [295]. Receptors, including AT1 and AT2, play crucial roles in inflammation and airway reactivity, making them potential targets for asthma treatment [296]. Several studies have explored the effects of AT1 and AT2 modulation in allergic asthma, shedding light on their therapeutic potential [241], [296]. The ARBs candesartan and irbesartan were investigated for their effects on allergic asthma. The findings suggest their potential as anti-allergic agents [293]. Moreover, the ARB losartan and novokinin, an AT2 agonist, demonstrated promising results in mitigating airway inflammation and hyperreactivity in asthma models [241]. However, caution is warranted when translating these results into clinical settings considering the varying responses observed in different patient populations [293]. 31 1.7 Scope and aims of this thesis Losartan was the first orally active ARB approved for clinical use and is now a well-established medication in its class. It has been shown to have unexpected therapeutic properties in a range of diseases not linked to hypertension [77], [78], [297], [298], [299], and may regulate other signalling pathways than that of ANGII. Our laboratory has recently shown that losartan can directly increase vascular relaxation via the activation of NO-dependent endothelial function independently of AT1 or BP lowering. Since NO and endothelial function are synonymous with aerobic exercise, tissue homeostasis and health, these may be behind the pleiotropic effects of losartan and other ARBs in many indications. However, results from clinical trials assessing losartan's effects on various diseases are underwhelming compared to promising preclinical results, highlighting the need to better characterize losartan’s mechanism of action. In this thesis, we hypothesize that “the endothelial enhancing functions of losartan metabolites EXP3179 or EXP3174 are behind some of the pleiotropic effects seen with losartan”. First, we attempt to understand how losartan activates NO, which will help design more specific activators of endothelial function. Furthermore, we try to re-purpose losartan, its metabolites or another ARB for chronic lung diseases, such as emphysema and asthma. As losartan is the key ARB used in this thesis, we first characterize the effects of losartan metabolites, EXP3179 and EXP3174, on endothelial function and blood pressure (chapter 2). We show that EXP3179 has unique endothelial function-enhancing properties not shared with losartan or EXP3174. Then, we provide evidence that the chronic endothelium-enhancing effects of losartan and the acute effects of EXP3179 are mostly ANGII/AT2 pathway- and BP-lowering independent (chapter 3). 32 Next, we aim to test whether losartan metabolites are behind some of its pleiotropic properties reported in preclinical studies. We also investigate the effects of telmisartan in chronic lung disease. Here we show that losartan and its metabolites do not have beneficial effects in a model of allergic asthma. In addition, we find that telmisartan does not improve lung function in a mouse model of emphysema (chapter 4). Chapter 5 provides a general summary of the findings and their implications in the current field and the overall significance and contribution of this thesis research. Limitations and future perspectives are also provided based on our findings. 33 Chapter 2: Losartan Metabolite EXP3179 is a Unique Blood Pressure-Lowering AT1 Antagonist with Direct, Rapid Endothelium-Dependent Vasoactive Properties Elodie Sauge MSc1,2*, Dmitri Pechkovsky PhD1,2*, ND Prasad Atmuri PhD3, Arash Y Tehrani PhD1,2, Zoe White PhD1,2, Ying Dong MSc1, Jessica Cait MSc4, Michael Hughes4 PhD, Anthony Tam2 PhD, Graham Donen1,2 MSc, Christopher Yuen, Michael JA Walker PhD1, Kelly M. McNagny PhD4, Don D. Sin PhD2, Marco Ciufolini PhD3#, Pascal Bernatchez PhD1,2# 1Department of Anesthesiology, Pharmacology & Therapeutics, 2Centre for Heart Lung Innovation, 3Department of Chemistry, 4Biomedical Research Centre, University of British Columbia (UBC), Vancouver, Canada. 34 2.1 Chapter summary Background and Purpose: Losartan is an anti-hypertensive angiotensin II (ANGII) type 1 receptor (AT1) blocker (ARB) with many unexpected therapeutic properties, even in non-blood pressure (BP)-related diseases. Administered as a prodrug, losartan undergoes serial metabolism into EXP3179, a metabolite alleged to lack AT1 blocking properties, and EXP3174, the dominant AT1 antagonist. Having observed that losartan can decrease vascular tone in mice with low AT1 expression and inhibit Marfan aortic widening at very high doses, we investigated whether EXP3179 may have unique, AT1-independent effects on vascular tone and endothelial function. Experimental Approach: We compared the AT1 blocking capabilities of EXP3179 and EXP3174 using AT1-expressing cell lines. Their BP lowering and vasoactive properties were studied in normal, hypertensive and transgenic rodents and ex vivo wire myography. Key Results: We observed that both EXP3179 and EXP3174 can fully block (100%) AT1 signalling in vitro and significantly decrease BP in normotensive and spontaneously hypertensive rats. Only EXP3179 prevented PE-induced contraction by up to 65.1 ± 26.4 % (p < 0.01) in L-NAME and endothelium removal-sensitive fashion. The use of transgenic mice revealed that these effects involve the eNOS/caveolin-1 axis and the endothelium-derived hyperpolarizing factor (EDHF). Conclusion and implications: We provide direct structure-activity evidence that EXP3179 is a BP-lowering AT1 blocker with unique endothelial function-enhancing properties not shared with losartan or EXP3174. Therefore, the major pharmacological effects of losartan in patients are likely more complex than simple blockade of AT1 by 35 EXP3174, which helps rationalize its therapeutic and prophylactic properties, especially at very high doses. Reports relying on EXP3179 as an AT1-independent losartan analogue may require careful re-evaluation. 2.2 Introduction Losartan is an angiotensin II (ANGII) type 1 receptor (AT1) blocker (ARB) mainly used for the management of hypertension, heart failure and kidney disease [300]. However, many therapeutic effects have been attributed to losartan in disease settings not typically associated with high blood pressure (BP) or hyperactivation of the ANGII system, such as emphysema and chronic lung diseases [301], Alzheimer’s disease [302], diabetes [303], cancer [304], muscular dystrophy [305] or Marfan syndrome [7], [8]. Some of losartan’s therapeutic properties are controversial [308], go ‘beyond its BP-lowering effects’ [309] particularly at very high doses that offer no additional BP-lowering benefits during chronic renal disease management [310] and have even been suggested to be AT1-independent [311], although how this occurs is poorly understood. Losartan is a prodrug with only mild AT1-blocking properties. After oral administration, metabolism by cytochrome P450 enzymes converts losartan’s 5-hydroxymethyl moiety to carboxaldehyde, a metabolite called EXP3179 and alleged to lack AT1-blocking properties [312]. EXP3179-carboxaldehyde is further converted to a carboxylic acid-containing metabolite called EXP3174 (Figure 2.1A), a 10- to 40-fold more potent AT1 blocker than losartan although only 14% of losartan is typically converted into EXP3174 when administered orally [313]. Early studies have revealed that patients who metabolize losartan into EXP3179 but not EXP3174 show no BP response, 36 which suggested EXP3174 as the dominant losartan metabolite, and EXP3179 as a non-BP lowering tool to study the AT1-independent effects of losartan [314]. Although AT1 blockade by EXP3179 has never been directly characterized, it is nonetheless biologically active [314] with alleged anti-inflammatory, anti-platelet, anti-diabetic properties along with protein kinase B (Akt) and peroxisome proliferator-activated receptor gamma (PPAR-γ) activating abilities, likely due to being chemically similar to PPAR-γ activators and cyclooxygenase blockers [314]. Our group has recently shown that in vivo treatment of mice with losartan drastically enhances nitric oxide (NO)-dependent endothelial function for months, which is typically associated with vascular homeostasis and lower BP [307], [315], [316]. While this may occur via lower ANGII-driven oxidative stress or a switch towards protective ANGII type 2 receptor (AT2) signalling, losartan remained fully active in vessels with low AT1 expression [307], suggesting that potentiation of vascular NO release may be mediated in an AT1-independent manner. Of note, the early development of losartan/DuP753 characterized the unexpected release of vasodilatory substances in cultured cells in fashions not mediated by ANGII receptor blockade [311], which supports the concept that losartan increases NO from vessels [317]. Hence, we studied the hemodynamic and vasoactive effects of AT1-independent EXP3179 and compared them to the AT1 antagonist EXP3174. We perform the first direct comparison of losartan and its first two metabolites on BP regulation and endothelial function activation and provide evidence that both EXP3179 and EXP3174 can block AT1-dependent ANGII signaling in reconstituted cell systems and smooth muscle cells and quickly lower BP in normal and hypertensive mice and rats. In contrast, only EXP3179 but not EXP3174 cause near-37 complete inhibition of aortic ring contractility in fashions that involve age-dependent modulation of Akt, NO, the endothelial NO synthase (eNOS), endothelium-derived hyperpolarizing factor (EDHF) release and an intact caveolae/caveolin-1 system, which are typically associated with endothelial mechanosensory responses to flow [318]. We conclude that the BP lowering and therapeutic effects of losartan are likely more complex than simple EXP3174-driven inhibition of AT1 and may involve direct endothelial stimulation of a non-ANGII/AT1 pathway by EXP3179. Reports that rely on EXP3179 as a non-AT1-blocking compound may require careful re-evaluation. 2.3 Material and methods 2.3.1 Chemicals Losartan (LOS) potassium salt was purchased from BOC Sciences (Shirley, NY, USA). Pure losartan carboxylic acid (EXP3174) and carboxaldehyde (EXP3179) were obtained from Toronto Research Chemicals (Toronto, ON, Canada); Bulk quantities of both EXP3174 and EXP3179, and water-soluble EXP3179 potassium salt were synthesized and prepared for experiments by the UBC Chemistry Department (Figures A.1-A.5). Dimethyl sulfoxide (DMSO), phenylephrine (PE), acetylcholine (ACh), and thiobutabarbital (Inactin) were obtained from Sigma-Aldrich (Oakville, ON, Canada). PD123319 and Wortmannin were purchased from Cayman Chemical (Ann Arbor, MI, USA). Human ANGII was purchased from Abcam (Cambridge, MA, USA). Cell culture reagents were obtained from ThermoFisher Scientific (Waltham, MA, USA). 38 2.3.2 Cell Cultures, Angiotensin II Type 1 Receptor (AT1) DNA Construct and Generation of Stably AT1-expressing HEK 293 cells Normal rat aortic smooth muscle cells (rASMC) were purchased from Applied Biological Materials (ABM, Richmond, BC, Canada) and cultured in DMEM supplemented with 10% FBS and 1% antibiotics in a humidified 37°C incubator with 5% CO2 and 95% room air. Cells were passaged three to seven times for all experiments. rASMCs were grown to 90% confluence in 6-well tissue culture plates and after serum-free medium replenishing incubated in the absence or presence of various concentrations of LOS, EXP3174, EXP3179, and human ANGII for the indicated time periods. Human embryonic kidney (HEK) 293 cells were obtained from Sigma-Aldrich and showed no response to ANGII stimulation. Human AT1-expressing vectors (pCXN2-AT1-YFP; Addgene #101659) were transiently transfected with pCXN2-AT1-YFP or pcDNA3-GFP control plasmids using Lipofectamine-2000 transfection reagent (Invitrogen, ThermoFisher Scientific). The efficiency of the transfection protocol was evaluated by counting YFP- or GFP-positive cells, and it was >80%. After passage 1, cells were cultured with Geneticin (Gibco, ThermoFisher Scientific) according to the manufacturer’s recommendations to establish stable lines. 2.3.3 Western blotting (WB) Cell and mouse tissue protein extract preparation and WB was performed as previously described [319]. Briefly, Akt and ERK1/2 phosphorylation was determined using rabbit polyclonal antibodies (Abs) against pAkt-Ser473 (#4060S) and pERK1/2-Thr202/Tyr204 (#9101S), and mouse mAbs against Akt (#2920S) and p44/42 MAPK 39 (ERK1/2) (#9107S). Rabbit anti-GAPDH (#2118S) Abs were used for a protein loading control (All from Cell Signaling Technology, Danvers, MA). Notoriously non-specific anti-AT1 Abs were not used in this study [320]. Mouse anti-eNOS (#610297) mAbs and rabbit anti-Caveolin1 Abs (#SC-894) were purchased from BD Transduction Laboratories (Lexington, KY) and Santa Cruz Biotechnology (Dallas, TX). Goat DyLight680 anti-rabbit and DyLight800 anti-mouse Abs (#611-144-122 and #611-145-121, Rockland Immunochemicals, Limerick, PA), and the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE) were used for detection, whereas quantification was done with the Odyssey software 1.1 (LI-COR Biotechnology). 2.3.4 Blood pressure and isometric force measurements The study protocol was approved by the UBC Animal Care Committee, and conducted according to the guidelines of the Canadian Council on Animal Care. Animal randomization and treatments were performed in blinded fashions, and no animals were excluded from the study. Previous reports showed a gender difference in the vascular function and release of NO, and females produced bigger variability compared to males [321], [322], [323]. Therefore, only males were used in the present study to limit the number of animals utilized in each experiment. 15 normotensive Sprague Dawley (SD) male rats (10-mo-old) were purchased from Jackson Laboratory (Bar Harbor, ME). 6 spontaneously hypertensive 10-mo-old male rats (SHR) were purchased from Charles River Laboratories (Wilmington, MA). Wild type (WT) C57BL/6 (3-mo to 10-mo old, Stock number: 00664), 10 eNOS knock-out (KO) (3-mo-old, Stock number: 002684) and 5 caveolin-1 (CAV-1)-KO (3-mo-old, Stock number: 007083) were purchased from Jackson 40 Laboratory. 8 WT male mice C57BL/6 (40-weeks-old, Stock number: 701) were purchased from Charles River Laboratories. For invasive BP (IBP) measurements, rats were anesthetized by intraperitoneal (i.p). injection of thiobutabarbital (100 mg/kg) and pentobarbital (15 mg/kg) at the lower right quadrant of the abdomen. The carotid artery was cannulated using a PE50 tube catheter pre-filled with degassed heparinized normal saline (25 IU/mL) attached to a pressure transducer for 3 hours. Water soluble EXP3179 was dissolved in normal saline and injected i.v. at 10 mg/kg dosage. 10 min before drug injection the same volume of normal saline was injected, and systolic (SBP), diastolic (DBP), and mean arterial BP (MABP) were recorded and used as control (baseline) values. EXP3179 effects were calculated as BP changes from baseline in mmHg. The IBP method was not available in mice due to technical limitations, therefore non-invasive BP measuring was used. For non-invasive BP measurements, lightly anesthetized (1.5% v/v isoflurane, 1.5L O2) C57Bl6 male mice were placed on a warming tray with the tail inserted into an inflatable cuff (Kent Scientific CODA2) as previously shown [324] using 100 mM DMSO stock of EXP3174 and EXP3179 diluted in sterile corn oil for i.p. injection (10 mg/kg). Isometric force measurements were performed as previously published [325] using KCl, PE and ACh (10-8 M to 10-4 M) along with DMSO control (vehicle), LOS, EXP3174 or EXP3179 at 15 µM for 30 min. Endothelium denudation was achieved by intraluminal rubbing followed by 30 min for recovery. Percentage constriction for each data point was first calculated relative to the vehicle control average value (set as 100%). Percentage inhibition was obtained by calculating the opposite of the constriction value. For instance, if the shift for the vehicle condition is 3.66 mN, then the value is set at 100 %. If the shift for EXP3179 condition is 1.47 mN, then the percentage constriction for 41 EXP3179 would be (1.47*100)/3.66 = 40.2 %. The inhibition would therefore be calculated as: 100 - 40.2 = 59.8 %. 2 to 4 rings were obtained from each aorta and used as technical replicates, tested simultaneously. The data plotted represent the average of these rings, generating a single representative value for each animal. These values served as the biological replicates in the analysis. 2.3.5 Statistical analyses Comparisons between 2 groups were performed by two-tailed Student’s t-test whereas one-way ANOVA was used to compare multiple groups with Tukey’s post-hoc method used to correct for multiple comparisons with GraphPad Prism Software 8.4.0 (San Diego, CA). Data are presented as the means ± SEM, with a p-value < 0.05 considered significant. 2.4 Results 2.4.1 Both EXP3179 and EXP3174 block ANGII-AT1 signalling in reconstituted and primary cells Stably AT1-expressing HEK 293 cells (HEK-AT1) were incubated with either vehicle DMSO, EXP3174 (positive control), EXP3179 or AT2 antagonist PD123319 at a 25 µM concentration followed by ANGII stimulation for 10 min (0.2 µM). We observed complete (100%) inhibition of ANGII-induced ERK1/2 activation with EXP3174 and EXP3179, whereas DMSO and PD123319 did not block the action of ANGII (Figure 2.1B); the inhibitory effects of EXP3174 and EXP3179 were concentration-dependent, observed in the M range (Figure 2.1C-2.1D), whereas PI3K/Akt activation was not 42 detected (Figure 2.1B). Control-transfected HEK 293 cells did not respond to ANGII (data not shown). In primary rat ASMC cultures, EXP3174, EXP3179 and LOS at 25 µM concentration completely blocked ANGII-induced ERK1/2 phosphorylation after 10 min, and this effect was observed within 1 hr period (Figure 2.1E-2.1F). Both LOS derivatives inhibited ANGII-induced ERK1/2 signalling activation in a concentration-dependent manner in primary rASMC (Figure 2.1G). Hence, EXP3179 and EXP3174 share similar concentration-dependent ANGII/AT1 blocking capabilities. 43 44 2.4.2 EXP3179 and EXP3174 share similar BP-lowering properties in rats and mice Normotensive rats that received an i.p. injection of either EXP3179 or EXP3174 (10 mg/kg) followed by a 3h catheter-based BP measurement period showed significantly decreased SBP and DBP by 35 and 26 mmHg, respectively (p < 0.01 compared to vehicle, Figure 2.2A). To exclude metabolic conversion of EXP3179 to EXP3174 after i.p. injections, water-soluble EXP3179 at a dose of 10 mg/kg was injected intravenously, and SBP, DBP were monitored within 3 hrs period. As shown in Figure 2.2B, EXP3179 effectively decreases both SBP and DBP in normotensive rats within 30 min after injection (33 and 28 mmHg, respectively, p < 0.05). In SHR (Figure 2.2C), i.v. administration of EXP3179 at a dose of 10 mg/kg caused a profound decrease in SBP and DBP (103 and 64 mmHg respectively) within 30 min after injection. Heart rates (HR) were measured during the experiments, and no changes were observed despite the different treatments (Figure A.6). The absolute baseline BP and HR data for normotensive rats and hypertensive rats are shown in Table A.1 and Table A.2, respectively. In addition, both Figure 2.1: Losartan and both derivatives (EXP3174 and EXP3179) inhibit ANGII-induced ERK1/2 signalling pathway activation in AT1-HEK 293 and rASMC. Schematic representation of losartan cascade metabolism to aldehyde EXP3179 and carboxylic acid EXP3174, by cytochrome P450 CYP2C9 (A); Stably AT1-plasmid expressing HEK 293 (AT1-HEK) cells were exposed to 25 uM EXP3174, EXP3179 or PD123319 (AT2 blocker) for 30 min followed by 10 min stimulation with human ANGII (0.2 uM). ERK1/2 and PI3K/Akt signalling activation was determined by WB (B); Representative WB images and bar graph demonstrate that both EXP3174 and EXP3179 efficiently and concentration-dependently inhibit ANGII-induced ERK1/2 signalling activation in AT1-HEK. Data are expressed as means ± SEM of 3 independent experiments. *p<0.009 compared with DMSO Ctrl, and all other EXP3174 or EXP3179 concentrations tested (C and D). Primary rat ASMC were treated with 25 uM EXP3174, EXP3179, and losartan (LOS) for 30 min following stimulation by 0.2uM human ANGII for 10 and 60 minutes (E); Bar graph shows 100% blocking of ANGII-induced and AT1-mediated ERK1/2 signalling activation by both metabolites and LOS after 10 and 60 min of exposure. Data are expressed as means ± SEM of 3 independent experiments (F); Representative WB images and ERK1/2 signalling quantification demonstrate inhibition of ANGII-induced ERK1/2 activation in rASMC by EXP3174 and EXP3179 in a concentration-dependent manner (G). 45 LOS derivatives EXP3174 or EXP3179 injected i.p. at a dose of 10 mg/kg, but not a vehicle control, significantly decreased BP after 3 hrs compared to baseline by 24 and 17 mmHg respectively in WT (C57Bl/6J) mice, measured by a non-invasive tail-cuff method (Figure A.7). Altogether these results strongly support data obtained by in vitro experiments that EXP3179 similar to EXP3174, has AT1-blocking capabilities, and produces BP-lowering effects in rodents. 46 2.4.3 EXP3179 but not EXP3174 reduces mouse aortic contractility ex vivo in the absence of ANGII Despite the absence of ANGII in the organ chambers, pre-exposure of aortic rings isolated from 3- 4-mo-old (young) WT mice to EXP3179 (15 µM) for 30 min in the presence of ibuprofen (a prostacyclin synthesis inhibitor) reduced PE-induced contractility by 59.8 ± 34.2 % on average compared to vehicle controls (p < 0.05) (DMSO treated), which was in stark contrast to losartan or EXP3174 that failed to prevent constriction (Figures 2.3A, 2.3C, and A.8A). Representative tracings of PE-induced contraction under each treatment condition are shown in Figure A.9. The relaxing effect of EXP3179 was significantly attenuated in the presence of NO inhibitor L-NG-Nitroarginine methyl ester (L-NAME) (200µM) on aortas from 3- 4-mo-old (young) animals (Figures 2.3B-2.3C). However, aortic rings obtained from 9- 10-mo-old (aged) C57Bl/6J males, while treatment with EXP3179 also reduced PE-induced constriction by 65.1 ± 26.4 % (p < 0.05) (Figures 2.3D and A.8E), L-NAME did not abrogate this relaxing effect of EXP3179 (Figures 2.3D, 2.3E, and A.8E). These results suggest an age-associated switch of EXP3179-induced aorta-relaxing mechanisms to a NO-independent pathway. Ibuprofen did not significantly affect PE-induced contractility in the presence of EXP3179 (Figures A.10A and A.10B), thereby excluding prostacyclin-dependent mechanisms as the main pathway of EXP3179 action. The EC50 values for PE-induced contraction did not differ Figure 2.2: EXP3179 decreases blood pressure (BP) in normotensive and spontaneously hypertensive rats (SHR). Pharmacodynamics of EXP3174 and EXP3179 were determined by an invasive BP measuring in normotensive Sprague Dawley (SD) rats after single intraperitoneal (i.p.) injection (A) of EXP3174 and EXP3179 at a dose of 10 mg/kg, and DMSO as a vehicle control; and after intravenous injection (i.v.) of water-soluble EXP3179 at a dose of 10 mg/kg in normotensive SD (B) and hypertensive SHR (C) rats. Data are shown as means ± SEM of systolic and diastolic BP changes from baseline after drug injection at the indicated time periods. 47 between treatments for both young and old mice, indicating that the potency of PE-induced contraction was unaffected by the treatments (Figure A.10C). KCl and ACh-induced relaxation curves were performed for each experiment to assess the integrity of the vessels (Figures A.8B- A.8C and A.8F-A.8G). At the end of each experiment, a sodium nitroprusside (SNP) concentration-response curve was performed to assess the responsiveness of aortic SMC to NO stimulation. None of the drugs showed significant differences compared to controls in both groups of animals (Figures A.8D and A.8H). 48 Figure 2.3: EXP3179 reduces PE-induced contraction in aortas from WT mice. PE concentration-response curves after vehicle and drug incubation (15uM) of thoracic aorta sections from 4-mo-old (young) WT mice before (A) and after L-NAME incubation (200uM) (B). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments performed (C). PE-induced contraction curves after vehicle and EXP3179 incubation (15uM) of thoracic aorta sections from 10-mo-old (aged) WT mice before and after L-NAME incubation (200uM) (D). Bar graphs represent the difference in contractility between the baseline and the maximum PE-induced constriction (E). Data are expressed as means ± SEM, One-way ANOVA with Tukey’s Post Hoc tests, n = 5 – 10, *p < 0.05. 49 To mimic accelerated aging of the vasculature, 3-mo-old (young) male eNOS-null mice were used. Aortic rings isolated from these mice responded to EXP3179 treatment in the same manner compared to aged WT control animals. EXP3179 prevented PE-induced constriction by 58.3 ± 31.5 % (p < 0.001) alone, and by 69.7 ± 29.7 % (p < 0.0001) in the presence of L-NAME (Figures 2.4A-2.4C). As NO-generation capabilities played a role in the EXP3179 effects, and it was reported age-associated eNOS deficiencies in vasculature [326], we therefore examined eNOS protein expression in the tissues of young and aged WT mice by WB. Indeed, Figure 2.4D demonstrates that eNOS protein was significantly decreased in highly vascularized lung tissues from aged (12-mo-old) WT males compared with young animals (p < 0.05). In contrast, CAV-1 protein was not significantly different between the 2 groups of mice (Figure 2.4D). 50 As we found in previously published studies, CAV-1 plays a critical role in eNOS activation and NO generation by vasculature [17], [25]. We explored EXP3179’s effects on PE-induced contractility in aortas from CAV-1 KO mice. When aortic rings from CAV1-KO animals were stimulated with either DMSO (vehicle) control or EXP3179, as mentioned above, no significant difference in PE-induced contraction was observed Figure 2.4: EXP3179 reduces PE-induced contraction in aortas from eNOS KO mice. PE concentration-response curves after vehicle and drug incubation (15uM) of thoracic aorta sections from eNOS KO mice before (A) and after L-NAME incubation (200uM) (B). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments performed (C). Data are expressed as means ± SEM, One-way ANOVA with Tukey’s Post Hoc tests, n = 5, ***p < 0.001, ****p < 0.0001. (D) eNOS and caveolin-1 (CAV-1) proteins expression in lung tissues isolated from 4 young and 4 aged mice by WB. GAPDH protein was used as a loading control. Bar graph represents means ± SEM of eNOS expression index. *p < 0.01 compared with young mice by Wilcoxon test, n = 8. 51 compared to vehicle controls, and L-NAME did not exacerbate EXP3179 vs vehicle differences (Figures 2.5A). 2.4.4 Role of endothelium and direct effect on SMC The role of the direct effect of EXP3179 on SMC was examined after endothelial denudation, and EXP3179 had little vasodilatory effect in WT vessels from young mice (Figure 2.5B). In vessels from young WT mice able to release NO, a 10 min pre-incubation with wortmannin, an inhibitor of established eNOS activator PI3K [329] (n = 5-6, 0.1uM), increased PE-induced contraction by 160.9 ± 80.3 %, but also reduced the 69.5 ± 39.7 % EXP3179-dependent inhibition of contractility to only 25.6 ± 55.5 % (p < 0.05) (Figure A.11). Mechanisms of EXP3179 action, therefore, involve PI3K-Akt signaling and caveolin-1/caveolae regulation of NO release, which changes with age. Losartan has been shown to impact EDHF release [330], [331]. In our study, we observed a change in mechanism with age, as the NO-dependent constriction is decreased when the animals get older. As prostacyclin-dependent mechanisms were excluded, this suggests a switch towards EDHF release in an age-dependent manner, which was confirmed using young eNOS-KO mice to mimic old WT animal vessels. 52 2.5 Discussion Through structure-function-based evidence, we propose that EXP3179 is not only able to block AT1 and lower BP similarly to EXP3174 but also has the unique ability to promote direct endothelial NO-dependent inhibition of vessel constriction in age-dependent fashions, even in the absence of exogenous ANGII, a prerequisite for an antagonist to mediate its normal activity. These unexpected observations lend credence to observations showing that losartan may have AT1-independent BP-lowering abilities [332]. Although the main target of losartan is AT1, a G-protein-coupled receptor (GPCR), Figure 2.5: The vasoactive effect of EXP3179 is lost after endothelium removal and in caveolin-1 KO animals. PE concentration-response curves after vehicle and drug incubation (15uM) of thoracic aorta sections from caveolin-1 (CAV-1) KO mice, before and after nitric oxide generation blocking by L-NAME (200uM) (A). PE concentration-response curves after vehicle and drug incubation (15uM) of thoracic aorta sections show that EXP3179 is not capable of inhibiting PE-induced contraction after endothelium removal (B). Graph plots are mean ± SEM compared with vehicle, multiple t-tests, n = 6. 53 recent studies have revealed that this receptor may have G-protein-independent signalling activities [333], suggesting that GPCRs may exist in different active conformations depending on the structures of the ligands binding their receptor. Other ARBs have been shown to behave as inverse and biased agonists [333], [334]. Whether protective AT2 plays a key role in these observations is unknown, however, losartan is far more effective at blocking AT1 than AT2, and a protective switch towards AT2 signalling requires the presence of ANGII, a key condition that was not met in our myography system. The observations that a losartan metabolite can activate the endothelial NO and EDHF systems in Akt- and caveolin-1/caveolae-dependent fashions bear some resemblance to the signalling events elicited during flow-induced vasodilation, a hallmark of aerobic exercise. Laminar sheering of the endothelium is known to elicit caveolae-dependent endothelial mechano-sensory signalling events at the luminal membrane that result in Akt and eNOS activation followed by vasodilation [335], a process also called endothelial mechanotransduction or mechanosensing of extracellular forces. Losartan pleiotropism that results in direct endothelial function activation may also imply superior BP lowering effects than what can be achieved via inhibition of the ANGII/AT1 axis alone. We have previously shown that aged vessels respond well to eNOS/caveolin-1 antagonism, resulting in a profound inhibition of constriction [336], although EXP3179 appears superior at inducing EDHF release in these settings. Herein, the observation that aging affects the NO/EDHF balance of a vessel is intriguing, particularly following agonist stimulation, and suggests that losartan metabolism activates a secondary non-NO-dependent system to reduce vascular tone in older vessels, which could benefit aging 54 patients with endothelial dysfunction. Endothelial function has robust anti-remodeling properties, whereas endothelial dysfunction is linked to a range of cardiovascular and pulmonary pathologies. We previously showed that nitric oxide synthase (NOS) inhibition rendered losartan inactivate in settings of Marfan syndrome-associated aortic root widening [5], [9]. The contribution of the direct release of endothelial cell-derived vasoactive substances to losartan BP-lowering and other therapeutic properties warrants further investigation. Many studies have relied on EXP3179 as a biologically active, AT1-independent losartan metabolite responsible for its anti-inflammatory, antiaggregatory [338] and anti-oxidative stress effects [339] while also acting as a partial PPAR- agonist [340]. However, none of those studies provide evidence of EXP3179‘s AT1 independence to substantiate these claims. Early regulatory documents on losartan bioavailability polymorphism revealed how individuals who metabolize losartan into EXP3179 but not EXP3174 do not show the expected BP reduction normally observed in those with EXP3174, which is, to our knowledge, the only reasonable source behind the misconception that EXP3179 does not block AT1. However, a careful literature review revealed that a nonpeptide 5’ carboxyaldehyde derivative of losartan – the same chemical structure as EXP3179 – can block AT1 and lower BP after IV injections [341]. Moreover, another 5’ carboxaldehyde derivative of losartan called EXP132, which shares the same IUPAC name as EXP3179, also shows robust AT1 antagonistic and BP-lowering properties [342]. With these data in mind, we propose that EXP132 and EXP3179 are the same carboxaldehyde-containing compounds capable of blocking AT1 and lowering BP. We also suggest that studies relying on EXP3179 as a non-BP-lowering, non-AT1-55 blocking molecule may therefore need careful re-evaluation. Combined with early studies looking at the BP-lowering effects of losartan that mention the release of vasodilatory substances with losartan in cultured cells [343], we propose that BP lowering caused by orally-administered losartan is likely more complex than simple AT1 inhibition by EXP3174, and suggest that the established beneficial effects of very high dose losartan in kidney disease – beyond obvious hemodynamic effects [344] – may be attributable to a sufficient blood concentration of EXP3179. Our data have important implications for the true nature of losartan metabolism, its mechanism of action in young and aged vasculature, and the structure-activity relationship that may result in the activation of a new vascular signalling pathway, as well as its therapeutic properties in BP- and non-BP-related disease settings, at both normal and very high doses. 56 Chapter 3: Losartan and Metabolite EXP3179 Activate Endothelial Function Without Lowering Blood Pressure in AT2 Receptor KO Mice Elodie Saugea, b, Zoe Whitea, b, Farah Lizottec, Christopher Yuena, b, Prasad Atmurid, Marco A. Ciufolinid, Pedro Geraldesc, Pascal Bernatcheza, b a Department of Anesthesiology, Pharmacology & Therapeutics, d Department of Chemistry, University of British Columbia (UBC), Vancouver, Canada b Centre for Heart Lung Innovation, University of British Columbia (UBC), Vancouver, Canada. c Research Centre of the Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Québec, Canada 57 3.1 Chapter summary Background and Purpose: We have documented protective endothelial function activation via acute and chronic use of angiotensin II (ANGII) receptor type 1 (AT1) blockers (ARB) losartan, making it relevant to cardiovascular diseases. Losartan is a prodrug and we showed that losartan metabolite EXP3179 enhances nitric oxide (NO) levels in endothelial cells. However, the underlying mechanisms remain unknown. Since blood pressure (BP) lowering by ARBs is alleged to require an ANGII-dependent switch towards ANGII receptor type 2 (AT2) signalling and is associated with increased endothelial NO release, we investigated the contribution of AT2 to the biological properties of losartan and EXP3179. Experimental Approach: Two AT1 ligands were used to block the AT1-dependent endothelium-enhancing effects of EXP3179. AT2-null mice were used to evaluate the acute and chronic effects of EXP3179 (20uM) and losartan (0.6 g/l) respectively, on endothelial function activation. Key Results: Blocking AT1 receptors did not attenuate EXP3179’s effects on NO and EDHF-dependent endothelial function activation. Ex vivo experiments with EXP3179 demonstrated significant reductions in PE-induced contractility in both WT and AT2 KO mouse aortic rings. In vivo, chronic treatment with losartan decreased PE-induced contraction by 74.9 ± 18.4 % in WT (p < 0.0001) and 47.3 ± 52.1 % in AT2 KO (p < 0.05), compared to controls. Conclusion and implications: Our data suggest that the endothelial function-enhancing effects of losartan and EXP3179 are pleiotropic and mostly independent of the 58 ANGII/AT2 pathway. These findings have implications for understanding the diverse actions of AT1 blockers in cardiovascular health. 3.2 Introduction The vascular endothelium is a specialized monolayer of cells that regulates many critical aspects of the circulatory system. It acts as a protective barrier between the blood and the vascular wall by regulating vessel permeability and tone, as well as ensuring homeostasis through its anti-coagulant and anti-adhesion properties [345]. The regulation of vascular homeostasis by the endothelium is accomplished via the production of three vasodilatory substances, nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factors (EDHF) [126]. NO is the primary endothelium-derived vasodilator in large conduit vessels, such as the aorta, diffusing within the vessel wall to induce smooth muscle cell (SMC) relaxation in response to agonist-induced stimulation or shear stress [345]. Impaired endothelium-dependent vasodilation, caused by reduced bioavailability of vasodilators, is a characteristic of endothelial dysfunction, which is linked to the development of cardiovascular diseases like heart failure and hypertension [128], [346], [347]. Angiotensin II (ANGII) type 1 receptor (AT1) blockers (ARB) are a class of medications mainly used for the management of hypertension, but they have proven to be beneficial in the treatment of a range of cardiovascular and renal conditions [1], [348] beyond blood pressure (BP) regulation [297], [349], [350] and simple blockade of AT1 [1], [348], [351]. The first-in-class ARB losartan is a complex prodrug and is first metabolized by cytochrome P450 enzymes to losartan-carboxaldehyde, a metabolite called EXP3179. 59 EXP3179 is further converted to a carboxylic acid-containing metabolite called EXP3174 [95] (Figure 3.1). Our group recently showed that both EXP3179 and EXP3174 metabolites can block AT1 and lower BP in rodents, but only EXP3179 improved endothelial function ex vivo despite its structural similarities with losartan and EXP3174 [352]. Some of the pleiotropic effects of losartan are thought to be the results of EXP3179, although the mechanisms behind these effects remain unclear [314]. AT2 is one of the main components of the renin-angiotensin-aldosterone system (RAAS) and is believed to have protective effects in many pathologies [353]. AT2 has the same endogenous ligand, ANGII, as AT1 however its effect is frequently described as opposite to those of AT1 [57]. In adults, research indicates that AT2 expression level rises in response to vascular injury and proinflammatory cytokines [353], [354], [355]. It has beneficial effects on the heart, promoting NO production [356], resulting in protective responses such as anti-proliferative, natriuretic, and antifibrotic effects, as well as vasodilation [357]. Since ARBs have been shown to promote compensatory AT2 activation switching [358], [359], resulting in unique downstream signalling, ARBs like losartan may also promote endothelial function activation through a similar AT2 switch or a direct AT2 activation. Here we first investigated the AT1-independent effects of losartan metabolite EXP3179 on the endothelium. We also examined the direct effect of EXP3179 on the endothelium, distinct from its interactions with AT1. Moreover, we treated mouse aortic rings with EXP3179 in the absence of AT2 and ANGII. Finally, we administered losartan to AT2 knock-out (KO) mice to evaluate its impact on endothelial function in the absence 60 of AT2. Our data suggest a mostly AT2-independent mechanism underlying EXP3179 and losartan beneficial impacts on the endothelium. 3.3 Material and methods 3.3.1 Chemicals Losartan potassium salt was purchased from BOC Sciences (Shirley, NY, USA). Bulk quantities of both EXP3174 and EXP3179 were synthesized and prepared for experiments by the UBC Chemistry Department, as previously published [352]. Dimethyl sulfoxide (DMSO), phenylephrine (PE), and acetylcholine (ACh) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Human ANGII was purchased from Abcam (Cambridge, MA, USA). For chronic treatment, patient formulation of losartan was purchased at St. Paul’s Hospital pharmacy. Losartan (0.6 g/l) was administered in drinking water ad libitum and replaced twice a week. Figure 3.1: Schematic representation of losartan first-passage metabolism to aldehyde EXP3179 and carboxylic acid EXP3174, by cytochrome P450. 61 3.3.2 Western blotting (WB) Normal rat aortic SMC (rASMC) were purchased from Applied Biological Materials (ABM, Richmond, BC, Canada). Cell culture, protein extract preparation and WB were performed as previously described [352]. Briefly, ERK1/2 phosphorylation was determined using rabbit polyclonal antibodies (Abs) against pERK1/2-Thr202/Tyr204 (#9101S) and mouse Abs against p44/42 MAPK (ERK1/2) (#9107S). The Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE) was used for detection. 3.3.3 Animals The study protocol was approved by the UBC Animal Care Committee, and conducted according to the guidelines of the Canadian Council on Animal Care. Wild type (WT) C57BL/6 were purchased from the Jackson Laboratory and bred in-house similarly to AT2 KO mice. 6- to 8-week-old WT mice were used for the EXP3174- and ANGII-blocking experiments. For chronic experiments, 8-week-old mice were randomly assigned to receive losartan (0.6 g/l, 2 fresh preparations per week) in drinking water or vehicle (regular water) for 4 weeks. Both males and females were used for each experiment. 3.3.4 Isometric force and blood pressure (BP) measurements Isometric force measurements were performed as previously published [357], [360] using KCl, PE and ACh (10−8 M to 10−4 M). EXP3179 (20 μM) was added for 30 minutes along with the vessels during direct stimulation experiments. During the EXP3174- and ANGII-blocking experiments, 6 aortic rings were cut per animal, and 2 62 rings were used per condition. DMSO was used as a control. EXP3174 [20 μM] or ANGII [1 μM] was added for 15 min before adding EXP3179 [20 μM] for 30 min. Percentage constriction for each data point was first calculated relative to the vehicle control average value (set as 100 %). Percentage inhibition was obtained by calculating the opposite of the constriction value. The endothelial integrity of each aortic ring was evaluated with ACh before treatment. Vessels from WT mice with an ACh relaxation of < 80% were excluded from the study. To calculate NO contribution, we set the reduction of PE contractility without L-NG-Nitroarginine methyl ester (L-NAME) as 100 %. The difference in PE contractility with and without L-NAME was determined for each condition, and a percentage was calculated using these values. EDHF contribution represents the complement value of NO contribution (EDHF = 100 – NO contribution for a condition). For non-invasive BP measurements, lightly anesthetized (1.5% v/v isoflurane, 1.5 L O2) mice were placed on a warming tray with the tail inserted into an inflatable cuff (Kent Scientific CODA2) as previously shown [361]. 2 to 4 rings were obtained from each aorta and used as technical replicates, tested simultaneously. The data plotted represent the average of these rings, generating a single representative value for each animal. These values served as the biological replicates in the analysis. 3.3.5 Echocardiography A Vevo 2100 ultrasound system (VisualSonics, Toronto, ON, Canada) equipped with an MS550 transducer was used for mouse echocardiography. The transducer had a central frequency of 40 MHz, a focal length of 7.0 mm and a frame rate of 233 frames per second. The maximal field of view of two-dimensional (2D) imaging was 11 x 12.8 mm. 63 Anesthesia was induced by putting the mouse in an induction chamber using 1.5% isoflurane and 1.5 L/min 100% oxygen for 2-4 minutes. Once the animal lost its righting reflex, it was laid supine on a heated platform under 2-3% isoflurane. All four limbs were taped to electrocardiogram (ECG) electrodes on a heated platform and heart rate, ECG and respiratory rate were monitored. Aortic diameters for the sinotubular junction (STJ) were measured in B-mode, and all diameter measurements were recorded at the onset of the QRS complex across 6-10 cardiac cycles. Pulse wave velocity (PWV) was determined using the protocol established by Lee et al. 2016 [362]. Ascending and descending aortic peak velocities were measured across the aortic arch using PW Doppler mode. PWV was calculated as aortic arch distance/transit time (cm·s-1). PW Doppler flow volumes were obtained in ascending aorta (at the level of the sinus of Valsalva) and the time (T1) from the onset of the QRS complex to the onset of the ascending aortic Doppler waveform was measured. In the same 2D image, PW Doppler flow volumes were measured in the descending aorta (at the level of the left subclavian artery) and the time (T2) from the onset of the QRS complex to the onset of the Doppler waveform was measured. On the same image plane, the PW Doppler volumes were measured in the descending aorta (at the level of the left subclavian artery) and the time (T2) from the onset of the QRS complex to the onset of the Doppler waveform was measured. Obtained values for T1 and T2 were averaged over 12-15 cardiac cycles. The aortic arch distance was measured between the two PWV volume readings along the central axis of the aortic arch using B-mode, and the transit time was calculated by T2 –T1 (ms); (i.e. PWV = [aortic arch distance] / [T2-T1]). 64 3.3.6 Statistical analyses Comparisons between 2 groups were performed by two-tailed Student’s t-test whereas one-way ANOVA or two-way ANOVA was used to compare multiple groups with one or two independent variables respectively. Tukey’s post-hoc method was used to correct for multiple comparisons with GraphPad Prism Software 9.5.1 (San Diego, CA). Significance in BP changes was calculated with a paired sample t-test. Data are presented as the means ± SEM, with a p-value < 0.05 considered significant. 3.4 Results 3.4.1 Pre-treatment with two AT1 agonists does not block the endothelial function-dependent anti-constrictive effect of EXP3179 As AT1 antagonist EXP3174 and ANGII are two high-affinity AT1 ligands, we investigated their ability to block the endothelial function-activating effects of EXP3179 by pre-incubating aortic rings with them before the EXP3179 challenge. Despite pre-treatment of WT aortic rings with EXP3174 [20 µM] or vehicle (DMSO), we observed similar EXP3179-dependent inhibition of PE-induced contractility (72.5 ± 16.3 % inhibition with EXP3179 alone vs 62.8 ± 10.8 % inhibition after pre-treatment with EXP3174, p < 0.0001 and p < 0.001 respectively) (Figures 3.2A and 3.2C). After adding the NO synthase (NOS) inhibitor L-NAME (200 µM), PE-induced contraction decreased by 32.9 ± 25.7 % after pre-treatment with EXP3174, in comparison to a 40.0 ± 24.2 % reduction observed with EXP3179 alone (p < 0.05) (Figures 3.2B-3.2C). As prostacyclin synthesis was blocked throughout these experiments by ibuprofen, we concluded that the NO-dependent effects are 52.6 ± 38.7 % after pre-treatment with EXP3174 and 48.2 ± 28.2 % 65 with EXP3179 alone. In addition, the EDHF-dependent anti-constrictive effects of EXP3179 on PE contraction in this experiment were 47.4 ± 38.7 % and 51.8 ± 28.2 %, respectively. Similarly, despite pre-treatment with ANGII [1 μM] or vehicle (DMSO), we noticed a comparable reduction in PE-induced contractility due to EXP3179, with inhibitions of 71.5 ± 12.9 % and 74.3 ± 15.6 % (both p < 0.01) (Figures 3.2D and 3.2F). After L-NAME (200µM), EXP3179 inhibited PE-induced contraction by 62.6 ± 17.3 % when combined with ANGII compared to 63.7 ± 12.7 % when used alone (both p < 0.05) (Figures 3.2E-3.2F), which led us to determine that the NO-dependent effects were 7.6 ± 15.5 % after pre-treatment with ANGII and 9.1 ± 17.7 % with EXP3179 alone and that the EDHF-mediated anti-constrictive effects of EXP3179 on PE-induced contraction reached 92.4 ± 15.5 % and 90.9 ± 17.7 % respectively. Of note, ANGII pre-treatment for 15 min did not cause vessel contraction in mice nor influence PE contraction compared to DMSO (Figure 3.2G). As AT1 ligands EXP3174 and ANGII did not block the effects of EXP3179, this suggests that the latter’s effects may be AT1-independent. In addition, rASMC were used to show that both EXP3179 and EXP3174 block ANGII signalling as previously published [352] (Figure 3.2H), which further implies that the anti-constrictive effects of EXP3179 may be AT1- and ANGII-independent. 66 67 3.4.2 AT2 KO mice release more NO than WT mice We used aortic rings from 3- to 4-month-old AT2 KO mice in absence of ANGII ex vivo to prevent an AT2 switch or direct AT2 activation. In female mice, we observed a significant baseline difference in PE-induced contraction between WT and AT2 KO control groups (p < 0.0001). Upon pre-treatment with EXP3179 (20 μM) for 30 minutes, there was a marked reduction in PE-induced contractility by 61.9 ± 8.1 % in WT mice and 69.3 ± 9.2 % in AT2 KO mice, both highly significant with p-values < 0.0001 compared to vehicle controls (Figures 3.3A and 3.3C). This suggests that EXP3179 effectively reduces vascular contractility regardless of AT2 receptor presence. In the presence of L-NAME (200 µM), EXP3179 decreased PE-induced contraction by 43.3 ± 10.0 % in WT mice and 52.3 ± 14.0 % in AT2 KO mice (both p < 0.01) (Figures 3.3B - 3.3C). This indicates that the NO contribution to the reduction in PE-induced contractility by EXP3179 was 29.2 ± 12.3 % in WT mice and 25.6 ± 11.1 % in AT2 KO mice. The EDHF contribution, calculated as the complement of NO contribution, was 70.8 ± 12.3 % in WT mice and 74.4 ± 11.1 % in AT2 KO mice. Interestingly, the baseline significant difference in PE-induced contraction between WT and AT2 KO mice (p < 0.0001) was no longer significant Figure 3.2: EXP3179 reduces phenylephrine (PE)-induced contraction in aortas from WT mice pre-treated with EXP3174 and angiotensin II (ANGII). PE concentration-response curves after vehicle and EXP3174 incubation (20 uM) of thoracic aorta sections from 2-mo-old WT mice before (A) and after L-NAME incubation (200 uM) (B). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments performed (C). PE concentration-response curves after vehicle and ANGII incubation (1 uM) of thoracic aorta sections from 6-week-old WT mice before (D) and after L-NAME incubation (200 uM) (E). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments performed (F). Representative ANGII incubation and PE-induced contraction experiment with DMSO and ANGII alone (G). Representative western blot showing that losartan and both derivatives (EXP3174 and EXP3179) inhibit ANGII-induced ERK1/2 signalling pathway activation in rat aortic smooth muscle cells (rASMC) (H). Data are expressed as means ± SEM, One-way ANOVA with Tukey’s post hoc tests, *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5-6. 68 after L-NAME treatment. This suggests that AT2 KO mice have slightly higher baseline NO levels compared to WT mice (Figure 3.3C). In contrast to the females, male mice did not exhibit any baseline differences in PE-induced contraction between WT and AT2 KO control groups. Following pre-treatment with EXP3179 (20 μM) for 30 minutes, PE-induced contractility was significantly reduced by 59.3 ± 13.2 % in WT mice and 56.3 ± 12.4 % in AT2 KO mice (both p < 0.01) compared to vehicle controls (Figures 3.3D and 3.3F). This reduction demonstrates the effectiveness of EXP3179 in decreasing vascular contractility in both WT and AT2 KO male mice, with no significant difference between the two groups. When treated with L-NAME, EXP3179 reduced PE-induced contraction by 53.0 ± 17.6 % in WT mice and 28.5 ± 15.9 % in AT2 KO mice (p < 0.01) (Figures 3.3E - 3.3F). This indicates a NO contribution of 12.3 ± 13.5 % in WT mice and 36.9 ± 38.9 % in AT2 KO mice after EXP3179 treatment. The EDHF contribution was calculated to be 87.7 ± 13.5 % in WT mice and 63.1 ± 38.9 % in AT2 KO mice. Notably, the NO contribution to the reduction in PE-induced contractility was almost three times higher in AT2 KO mice than in WT mice, suggesting a compensatory increase in NO-mediated effects in the absence of AT2 receptors. This also aligns with the observation that there is more reversibility in AT2 KO mice after L-NAME treatment compared to WT, indicating a greater reliance on NO pathways in the absence of AT2 receptors. In both female and male mice, ACh relaxation curves showed no difference between the WT and AT2 KO groups, suggesting no endothelium impairment in AT2 KO mice (Figure B.1). Results for both females and males pooled together are shown in Figure B.2. 69 Our findings suggest sex-specific differences in the vascular responses to EXP3179 and the contributions of NO and EDHF to endothelial function. In females, EXP3179's effects on PE-induced contraction are robust and largely independent of AT2 receptors, with significant contributions from both NO and EDHF pathways. In males, while EXP3179 also effectively reduces PE-induced contraction, there is a pronounced reliance on NO pathways in AT2 KO mice, highlighting a potential compensatory mechanism. 70 3.4.3 AT2 KO mice show normal endothelial function response As AT1 antagonism by losartan may cause a switch towards protective AT2 signalling in the presence of ANGII, we treated WT and AT2 KO mice for 4 weeks with either losartan or vehicle. In female mice, we observed a minor, non-significant baseline difference in PE-induced contractility between WT and AT2 KO control groups. Losartan reduced PE-induced significantly by 67.1 ± 20.3 % in WT mice (p < 0.01) and by 71.7 ± 15.0 % in AT2 KO mice (p < 0.05) compared to their respective vehicle controls (Figures 3.4A and 3.4C). This reduction indicates that losartan effectively lowers PE-induced contractility in both WT and AT2 KO female mice, with no significant difference between the two groups. In the presence of L-NAME (200µM), the endothelial function-dependent anti-constrictive effect of losartan on PE contractility was 34.5 ± 15.1 % in WT mice (p < 0.01) and 51.8 ± 10.2 % in AT2 KO mice (p < 0.0001) (Figures 3.4B - 3.4C). This indicates that NO contributed to 52.2 ± 18.8 % of the losartan-induced reduction in PE contractility in WT mice and 23.0 ± 17.3 % in AT2 KO mice. The EDHF contribution was therefore 47.8 ± 18.8 % in WT mice and 77.0 ± 17.3 % in AT2 KO mice. These results suggest a higher reliance on EDHF pathways in AT2 KO female mice compared to WT mice when treated with losartan. Interestingly, the small baseline difference in PE-induced contraction between WT and AT2 KO female mice disappeared after L-NAME treatment, Figure 3.3: EXP3179 significantly reduces phenylephrine (PE)-induced contraction in aortas from both female and male WT and angiotensin II receptor type 2 (AT2) KO mice. PE concentration-response curves after vehicle and drug incubation (20 uM) of thoracic aorta sections from 3- to 4-month-old WT and AT2 KO female mice before (A) and after L-NAME incubation (200 uM) (B). Bar graphs represent the maximum PE-induced contraction in mN from all experiments performed (C). PE concentration-response curves after vehicle and drug incubation (20 uM) of thoracic aorta sections from 3- to 4-month-old WT and AT2 KO male mice before (D) and after L-NAME incubation (200 uM) (E). Bar graphs represent the maximum PE-induced contraction in mN from all experiments performed (F). Data are expressed as means ± SEM, Two-way ANOVA, Tukey multiple comparisons with *p < 0.05, **p < 0.01, ****p < 0.0001, n = 5-6. 71 which suggests that the baseline NO levels might have been slightly higher in AT2 KO mice. No significant differences were observed in the ACh relaxation curves between WT and AT2 KO female mice, and treatment with losartan did not affect these curves (Figure B.3A). This indicates that endothelium-dependent relaxation remains intact and is unaffected by both the absence of AT2 receptors and losartan treatment in female mice. In male mice, a non-significant baseline difference in PE-induced contractility between WT and AT2 KO control groups was observed. Chronic losartan treatment for 4 weeks showed a substantial reduction in PE-induced contraction by 79.8 ± 10.5 % in WT mice (p < 0.001), but only by 45.3 ± 44.0 % in AT2 KO mice (Figures 3.4D and 3.4F). These results indicate that losartan significantly reduces PE-induced contractility in WT male mice but has a less pronounced effect in AT2 KO mice, suggesting that AT2 receptors play a significant role in losartan’s efficacy in male mice. After L-NAME (200 µM) incubation, losartan reduced PE-induced contraction by 35.3 ± 16.8 % in WT mice (p < 0.001) and 29.7 ± 12.2 % in AT2 KO mice (p < 0.05) (Figures 3.4E - 3.4F). This indicates that NO contributed to 59.3 ± 16.9 % of losartan’s reduction in PE contractility in WT mice and 52.1 ± 6.9 % in AT2 KO mice. Therefore, the EDHF contribution was 40.1 ± 16.9 % in WT mice and 47.9 ± 6.9 % in AT2 KO mice. These findings highlight that while both NO and EDHF contribute to the vascular effects of losartan in male mice, the NO contribution is slightly higher in WT compared to AT2 KO mice. Interestingly, the baseline difference in PE-induced contractility between WT and AT2 KO male mice was reduced after L-NAME incubation, indicating a greater reversal in the WT treated group compared to the AT2 KO group. This suggests that NO contributes more significantly to the vascular effects in WT male mice, which is diminished in the absence of AT2 72 receptors. Although all curves relaxed to 100% with ACh, losartan shifted these curves to the left, indicating that losartan-treated male aortic rings responded faster to ACh (Figure B.3B). This enhanced responsiveness suggests that chronic losartan treatment improves the endothelium-dependent relaxation in male mice, regardless of AT2 receptor presence. Results for both females and males pooled together are shown in Figure B.4. Our results indicate sex-specific differences in how losartan affects vascular contractility and endothelial function in WT and AT2 KO mice. In females, losartan reduces PE-induced contraction similarly in both WT and AT2 KO mice, with a notable contribution from both NO and EDHF. In males, losartan's efficacy in reducing PE-induced contraction is significantly greater in WT mice compared to AT2 KO mice, suggesting that AT2 receptors significantly modulate losartan’s effects. Echocardiography was performed, and measurements of PWV and STJ diameter were obtained to evaluate morphological distinctions between WT and AT2 KO mice. No statistically significant differences were observed either at baseline (untreated, NT) or following a 4-week losartan treatment (LOS) (Figure B.5). These findings indicate that the effects of losartan on PE-induced contractility in AT2 KO mice are unrelated to any morphological variations when compared to WT mice. 73 Figure 3.4: Losartan reduces phenylephrine (PE)-induced contraction in aortas from both female and male WT and angiotensin II receptor type 2 (AT2) KO mice. PE concentration-response curves of thoracic aorta sections from 3-month-old WT and AT2 KO female mice treated with losartan (0.6 g/l) for 4 weeks in drinking water before (A) and after L-NAME incubation (200 uM) (B). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments performed (C). PE concentration-response curves of thoracic aorta sections from 3-month-old WT and AT2 KO male mice treated with losartan (0.6 g/l) for 4 weeks in drinking water before (D) and after L-NAME incubation (200uM) (E). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments performed (F). Data are expressed as means ± SEM, Two-way ANOVA, Tukey multiple comparisons with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5–9. 74 3.4.4 Losartan did not reduce BP in AT2 KO mice In WT mice, a 4-week treatment with losartan significantly lowered BP (p < 0.05). Interestingly, AT2 KO BP was not affected by losartan treatment (Figure 3.5A) despite the presence of AT1, the main BP-mediating receptor. These results alone provide evidence that the endothelium-enhancing effects of losartan are BP-independent. Body and kidney weights were measured on the day of euthanasia and their ratio was calculated. Data showed that both males and females AT2 KO mice have larger kidneys compared to WT mice (p < 0.05) (Figure 3.5B), which could affect losartan clearance and the BP results observed in this study. Figure 3.5: Losartan does not affect blood pressure (BP) in angiotensin II receptor type 2 (AT2) KO mice. BP was measured at baseline, 2 weeks and 4 weeks after losartan was added to drinking water (0.6 g/l) (A). Data are shown as mean ± SEM of mean arterial BP changes from baseline, paired sample t-test, *p < 0.05, n = 4-5. Kidneys were weighed during aorta collections. Ratios between kidney and body weights were calculated for both WT and AT2 KO mice (B). Data are shown as mean ± SEM, Student t-test, *p < 0.05, n = 4-5. 75 3.5 Discussion ARBs, such as losartan, are commonly prescribed medications for treating conditions such as hypertension and heart failure, but they are known to mediate a range of biological activities that cannot be rationalized by simple AT1 blockade. Our ex vivo results suggest that the direct effects of EXP3179 on PE-induced contraction are independent of the classical ANGII/AT2 pathway. In contrast to the observed absence of a BP-lowering effect in AT2 KO mice, our study demonstrates that losartan promotes endothelial function in AT2 KO mice, which is primarily independent of a switch towards or direct activation of protective AT2, suggesting a minor role for AT2 in losartan’s endothelial function enhancing activity. Our data further support the concept that ARBs are capable of robust pleiotropic effects independent of BP lowering. Early data revealed that losartan may not lower BP solely through AT1 antagonism but rather in combination with the release of ‘vasodilatory substances’ [332], which we suggest to be through the acute and chronic release of NO and EDHF [363], [364], [365], [366]. The exact mechanisms behind the activation of endothelial function by ARB remains to be elucidated. Even though EXP3179 can block ANGII signalling by binding to AT1, a G-protein-coupled receptor (GPCR) [352], its unique ability to promote direct endothelial NO-dependent inhibition of vessel constriction is likely not mediated by the classic ANGII/AT1/BP pathway. However, studies have suggested that GPCR may exist in different conformations depending on the interaction between the ligand and the receptor [333] and EXP3179 could exhibit inverse and biased agonistic activities towards AT1, as seen with other ARBs [333], [367]. It has been documented that when AT1 is blocked with an ARB, plasma concentrations of ANGII rise through blunted AT1-mediated negative feedback on renin release [368], 76 resulting in endogenous ANGII that might lead to AT2 stimulations and lowering of BP [369], [370]. Other studies also reported that AT2 might play a role in the BP-lowering effects of ARBs via the release of bradykinin [371], [372] a potent BP-lowering cytokine behind some of the anti-hypertensive effects of angiotensin-converting enzyme inhibitors (ACEi) [373]. This lends credence to our findings that long-term treatment with losartan does require the presence of AT2 to lower BP. It is widely accepted that AT2 is protective and upregulated in pathologies such as vascular injury [374], hypertension [375], [376] or myocardial infarction [377], [378]. The contribution of AT2 on losartan’s protective effects in non-pathological conditions was, to our knowledge, unassessed. Here we showed that in AT2 KO mice, losartan was still able to increase endothelial function in vivo, even though the effects of losartan were slightly attenuated. This again suggests that the BP-reducing activity of ARBs and their ability to improve endothelial function are mediated via different mechanisms. The structure of ARBs is loosely derived from the C-terminus of ANGII, particularly Ang5-8 [379], a tetrapeptide initially alleged to lack biological activities but that was later shown to promote AT1/AT2-independent signalling properties that may include NO release in non-BP settings [380], [381]. Whether a new receptor or a more complex inverse or biased agonistic activity is behind these non-antagonistic properties we and others report remains to be determined. It must be noted that the rodent RAAS is not identical to that of humans and therefore more studies are needed to confirm the applicability of our rodent data to human disease settings [382]. Even though we reproduce the lack of BP lowering of ARBs in AT2-null animals [383], monitoring of diurnal and nocturnal variations in BP must be assessed. In conclusion, our data have important implications for the understanding of losartan metabolism and its 77 mechanism of action. We showed that losartan metabolite EXP3179 can activate endothelial function in a non-standard AT1/AT2/ANGII signalling pathway. Moreover, losartan could improve vascular function independently of its BP-lowering activity. These results are crucial to understanding the therapeutic properties of losartan in BP- and non-BP-related disease settings. 78 Chapter 4: Exploring the Pleiotropic Effects of Telmisartan and Losartan in Mouse Models of COPD and Allergic Asthma Elodie Sauge MSc1,2, Zoe White PhD1,2, Arash Y Tehrani PhD1,2, Jessica Cait MSc3, Michael Hughes3 PhD, Marine Theret3 PhD, Dmitri Pechkovsky PhD1,2, Anthony Tam2 PhD, Don D. Sin PhD2, Pascal Bernatchez PhD1,2 1Department of Anesthesiology, Pharmacology & Therapeutics, 2Centre for Heart Lung Innovation, 3Biomedical Research Centre, University of British Columbia (UBC), Vancouver, Canada. 79 4.1 Chapter summary Background and Purpose: Chronic obstructive pulmonary disease (COPD) and asthma are distinct chronic lung conditions. Despite being common in the population, there are currently no effective treatments or cures for individuals dealing with these conditions. COPD is a progressive respiratory disorder that restricts airflow, causing breathing difficulties and ranking as the third leading cause of death worldwide. On the other hand, allergic asthma is an inflammatory lung disease that manifests symptoms such as shortness of breath, chest tightness, and restricted airflow. Recent studies revealed that angiotensin II receptor type 1 (AT1) blockers (ARBs) enhance endothelial function, which plays a crucial role in diverse chronic lung diseases. ARBs, particularly telmisartan and losartan, have been shown to prevent emphysema, lung infiltration and inflammation in models of COPD and asthma. This study aims to investigate the pleiotropic effects of telmisartan and losartan in mouse models of COPD and allergic asthma. Experimental Approach: In the COPD study, female C57Bl/6 mice were exposed to daily cigarette smoke for 6 months starting at 2 months of age. Telmisartan treatment was initiated at 2 months via drinking water (10 mg/kg/d) and was used as a preventative strategy. Lung function was measured using the FlexiVent. Left lungs were fixed in 10% formalin, and emphysema was analyzed by mean linear intercept. Non-treated mice exposed to smoke were used as a control group. In the asthma study, BALB/c female mice were treated with losartan (10 mg/kg), EXP3179 (10 mg/kg) or EXP3174 (10 mg/kg). Allergic airway inflammation was induced by intraperitoneal sensitization and intranasal challenge with ovalbumin (OVA). Lung function was measured using the FlexiVent. Lung 80 inflammation was assessed using flow cytometry and lung histology. Non-treated mice, sensitized or not with OVA, were used as positive and negative control groups, respectively. Key Results: We found that a 6-month treatment with telmisartan did not improve lung functions or emphysema in a mouse model of COPD. In addition, losartan and its main metabolites, EXP3179 and EXP3174, did not improve lung function or lung inflammation in a mouse model of allergic asthma. Conclusion and implications: In our study, telmisartan and losartan failed to improve lung function and remodelling in both chronic lung disease models. These results highlight the importance of dosage and route of administration for each drug. It also shows that more studies are required to improve our understanding of ARBs and their mechanism of action. 4.2 Introduction Chronic obstructive pulmonary disease (COPD), also called emphysema or chronic bronchitis, is a progressive respiratory disorder causing airflow restriction and breathing complications [217]. It is characterized by conditions ranging from chronic airway inflammation to tissue proteolysis [228]. Ethical concerns surrounding studies on COPD patients have led to the use of animal models, particularly mice, guinea pigs, and rats. Animal models provide valuable insights into the basic mechanisms of COPD physiology, pathophysiology, and treatment. Although no animal model perfectly mimics human disease, the variety of available models has provided valuable insights into the complex and diverse pathophysiology of COPD [228]. Additionally, these models have 81 helped improve our understanding of the mechanisms underlying the progression and exacerbations of this disease. Asthma is the most prevalent inflammatory lung disease. It affects 5% to 10% of the population in developed countries, presenting a heterogeneous range of airway diseases [384]. Chronic exposure to environmental triggers induces asthma, marked by inflammation, airway hyperresponsiveness, elevated immunoglobulin (Ig)E levels, and airway remodelling [293], [385]. Clinical symptoms include wheezing, shortness of breath, chest tightness, cough, and restricted airflow [250], [386], [387]. As a long-term inflammatory disease, asthma exhibits typical features like eosinophilic inflammation, mucus overproduction, hyperresponsiveness, and eventual airway remodelling [385]. Allergic asthma, the most common type affecting around 300 million individuals globally, is characterized by obstruction and chronic airway inflammation [384]. In research, murine models have played a pivotal role in elucidating various aspects of asthma pathogenesis and treatment. While the ovalbumin (OVA)-induced asthma model has been widely used, house dust mite (HDM) has emerged as the allergen of choice in recent years. HDM is a crucial environmental aeroallergen and has been identified as a risk factor for persistent asthma in human subjects [388]. These models have greatly contributed to advancing our understanding of allergic asthma and exploring potential therapeutic interventions. Recent studies have shown ARBs’ remarkable pleiotropic effects, suggesting benefits that extend beyond their primary role in hypertension management [77], [297], [351], [360]. Among these ARBs, losartan and telmisartan have emerged as significant therapeutic agents, showing diverse physiological impacts beyond simple AT1 blocking 82 and BP regulation [114], [115], [116]. Their actions have been attributed to increased nitric oxide (NO) availability, anti-inflammatory properties, and antioxidative effects on the endothelium, indicating their potential as heterogeneous therapeutic agents in vascular diseases [114], [115], [116]. Research also demonstrated their ability to increase peroxisome proliferator-activated receptor gamma (PPAR-γ) levels potentially conferring beneficial metabolic effects [117], [118], [389]. Losartan's induction of PPAR-γ has shown preventive and reversal effects on cigarette smoke (CS)-induced emphysema in mouse models, highlighting its potential in addressing lung-related inflammatory conditions [390]. In summary. the pleiotropic effects of ARBs, such as losartan and telmisartan, extend beyond their traditional role in managing hypertension [77], [297], [351], [360]. While their main function is to regulate BP through vascular effects, recent evidence suggests that these medications also impact endothelial function, inflammation, and oxidative stress. These broad-ranging effects make ARBs potential candidates for therapeutic applications beyond cardiovascular diseases, potentially including pulmonary conditions. This study investigated the effects of telmisartan in a COPD disease model and studied the effects of losartan and its main metabolites, EXP3179 and EXP3174, in a model of allergic asthma. The hypothesis was that ARBs could mitigate inflammation and oxidative stress in the pulmonary system, possibly through mechanisms related to vascular protection and endothelial function. However, our findings revealed that telmisartan did not reduce CS-induced emphysema in a mouse model of COPD. Similarly, losartan failed to improve lung function or reduce inflammation in a mouse model of allergic asthma. These results indicate that the efficacy of telmisartan and losartan in 83 treating pulmonary airway diseases like COPD and asthma may be limited, potentially due to differences in disease pathology or local tissue responses. 4.3 Material and methods 4.3.1 Chemicals Pure losartan carboxylic acid (EXP3174) and carboxaldehyde (EXP3179) were obtained from Toronto Research Chemicals (Toronto, ON, Canada); Bulk quantities of both EXP3174 and EXP3179 were synthesized and prepared for experiments by the UBC Chemistry Department. Dimethyl sulfoxide (DMSO), phenylephrine (PE), and acetylcholine (ACh) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Telmisartan was patient formulation-based and purchased at St. Paul’s Hospital pharmacy. Telmisartan (10 mg/kg/day) was delivered in drinking water and replaced twice a week. Dosage was continuously adjusted for body weight and averaged drinking water per day per cage. Losartan, EXP3179 and EXP3174 were diluted in DMSO and mixed with corn oil to avoid precipitates. Stock solutions were kept at 4°C for 1 week. Mice were given a daily intraperitoneal (i.p.) injection of either losartan, EXP3174, or EXP3179 at a dose of 10 mg/kg. 4.3.2 Animals All animals were housed in a 12 h/12 h light/dark cycle, temperature‐regulated facility. All animal procedures were carried out following the guidelines and regulations set by the University of British Columbia (UBC), the Animal Care Committee, and the UBC Animal Ethics Committee, Vancouver, BC. 84 4.3.3 CS exposure procedure 8 female C57BL/6 WT mice were purchased from Jackson Laboratory (stock number: 000664) and randomly divided into 2 different groups; 4 regular water and 4 telmisartan treated. In our study, we used the protocol published by Zhou S, et al. 2013 [391]. Briefly, starting at 2 months of age, telmisartan treatment (10 mg/kg/d) was started via drinking water, and all the mice were exposed to CS generated from 4 research-grade cigarettes (University of Kentucky, Lexington, KY) 5 days per week for 6 months. All smoke exposures were conducted using a standard nose-only smoke exposure system. Animals were not anesthetized during the CS-exposure procedure. Animals were individually weighed twice a week. Lung function was measured using the FlexiVent as described below. Histology of the left lung was performed, via fixed pressure inflation of 30 cm H2O, fixed in 10% formalin, and sections were stained with Masson's trichrome and hematoxylin and eosin. Airspace enlargement was analyzed by mean linear intercept (MLI) [392]. Two animals from the group of 4 regular water mice were excluded from the analyses. One mouse died during the CS-exposed procedure, and another mouse died during the FlexiVent procedure. The mouse that died during the FlexiVent procedure could still be used for lung histology analysis, body weight, and blood pressure measurements. 4.3.4 Ovalbumin (OVA)-induced allergic lung inflammation 20 BALB/c female mice were purchased from Jackson Laboratory (stock number: 000651), and randomly assigned to 5 different groups; 2 Naive (negative control), 3 OVA-SEN (positive control, asthmatic mice), 5 losartan (10 mg/kg), 5 EXP3179 (10 mg/kg) and 85 5 EXP3174 (10 mg/kg). Experiments were started at 12 weeks of age. On days 0 and 7, mice were injected intraperitoneally (i.p.) with 0.5 mg/kg of chicken OVA (grade III; Sigma-Aldrich) complexed with aluminum hydroxide (Alum) (Sigma) at a 2:1 ratio. On day 14, daily i.p. treatments were started for 14 days. On days 21, 22, 23, 25, and 27, mice were intranasally challenged with 2.5 mg/kg of OVA. On day 28, lung function was measured using the FlexiVent as described below. Bronchoalveolar lavage fluid (BAL) was collected by 3 washes with 1 ml of sterile saline. Cells were enumerated and differentiated by flow cytometry. The yield of the BAL collected was recorded and used to normalize results obtained by flow cytometry. The left lung was collected in 10% formalin after fixed pressure inflation of 30 cm H2O. Sections were stained with hematoxylin and eosin. Lung inflammation was assessed on digital images to enable colour contrast measurements using ImageJ software. 4.3.5 Measurements of lung function Lung function was assessed using the FlexiVent (SCIREQ, Montréal, QC, Canada). As previously described [361]. Briefly, mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine before being tracheotomized with an 18-gauge blunted needle. They were mechanically ventilated at a respiratory rate of 150 breaths/minute and a tidal volume of 10 ml/kg, with a pressure limit of 30 cm H2O. Muscle paralysis was achieved using pancuronium bromide (2 mg/kg; Sandoz, Boucherville, QC, Canada) to prevent respiratory efforts during the measurement. The following sequence of measures was repeated three times and averaged for analysis using FlexiWare Software version 7.6 (SCIREQ): deep inflation, Snapshot-150, Quick Prime-3, and pressure/volume-loop 86 to obtain measures for compliance, resistance, tissue damping, elastance, hysteresis and lung inspiratory capacity. 4.3.6 Non-invasive blood pressure (BP) measurements Non-invasive BP measurements were performed in lightly anesthetized (1.5% v/v isoflurane, 1.5L O2) using a tail cuff system (Kent Scientific CODA2). Briefly, mice were placed on a warming tray with the tail inserted into an inflatable cuff. After a 10-minute acclimation period, BP was measured for 5 cycles and mean, systolic and diastolic BP were calculated as the average of the 5 cycles. BP from CS-exposed mice was measured at baseline, 3 months and 6 months after the beginning of the exposure. BP from OVA-induced allergic mice was measured before and 3h after i.p. injections of the drugs. 4.3.7 Flow cytometry measurements BAL samples were centrifuged for 5 min at 14000 rpm and the supernatant was removed. Pellets were resuspended in red cell lysis buffer (ACK lysing buffer), and incubated for 5 min at RT. FACS buffer [1× PBS, 2% fetal bovine serum (FBS), 2 mM EDTA] was added to the cell suspension. Samples were centrifuged for 5 min at 14000 rpm and pellets were resuspended in FACS buffer with 1:1000 Fc Receptor-blocking antibody (AbLab), cells were stained with antibodies listed in Table 2. Eosinophils are gated as CD45+, B220-, CD3-, Ly6G-, SiglecF+, CD11b+, CD11c-; B cells as CD45+, B220+; T cells as CD45+, B220-, CD3+; alveolar macrophages as CD45+, FVD+, B220-, CD3-, Ly6G-, SiglecF+, CD11b+, CD11c+; neutrophils as CD45+, B220-, CD3-, Ly6G+, and CD45 is total CD45+ cells. All flow cytometry data were analyzed using FlowJo 87 10.5.3v. Representative flow plots showing the gating strategies are shown in Figure C.1 and Figure C.2. 4.3.8 Statistical analyses Comparisons between 2 groups were performed by two-tailed Student’s t-test. One-way ANOVA was used to compare multiple groups. Groups of 2 mice were not used for statistical analyses. Tukey’s post-hoc method was used to correct for multiple comparisons with GraphPad Prism Software 9.5.1 (San Diego, CA). Data are presented as the means ± SEM, with a p-value < 0.05 considered significant. Table 2: Antibodies used in flow cytometry Table 2: Antibodies used in flow cytometry 88 4.4 Results 4.4.1 Lung mechanics of mice after chronic smoke exposure Following 6 months of CS exposure, telmisartan-treated mice showed statistically significant differences in pressure-curve loops (p < 0.05), inspiratory capacity (p < 0.05), elastance (p < 0.05) and compliance measurements (p < 0.01) compared to WT untreated controls (Figures 4.1A, 4.1D, 4.1E and 4.1F). In the context of lung function, the plotted resistance indicates the resistance of the central or conducting airways, tissue damping is associated with tissue resistance and reflects the energy dissipation in the alveoli and hysteresis is the difference between the transpulmonary pressure of inhalation (increasing volume) and the pressure of exhalation (decreasing volume). Our results show that CS exposure did not significantly affect resistance, tissue damping and hysteresis when mice were treated with telmisartan (Figures 4.1B, 4.1C and 4.1G).. The effects of telmisartan on lung functions impaired by CS exposure are found to be underwhelming for all parameters. It should be noted that statistical analyses could not include the smoke untreated group due to the low sample size. 4.4.2 Lung histology, body weights and BP of mice after chronic smoke exposure Lung histology did not show significant improvement with telmisartan, showing an MLI of 39.8 in smoked mice and 36.7 when mice were treated with telmisartan for 6 months (p = 0.4476). These results indicate that telmisartan did not reduce CS-induced emphysema significantly (Figures 4.2A-4.2B). BP was measured after 4 months and 6 months of CS exposure. BP results showed that telmisartan was biologically active, significantly reducing mean arterial BP in the telmisartan-treated group at 4 and 6 months 89 (p < 0.001 and p < 0.05, respectively) (Figure 4.2C). Body weights were measured before euthanasia and were similar between the two groups (p = 0.0529) (Figure 4.2D). 90 Figure 4.1: The effects of chronic smoke exposure on lung function in female mice. Pressure-curve loops (A), resistance (B), tissue damping (C), inspiratory capacity (D), elastance (E), compliance (F) and hysteresis (G) were assessed in WT untreated mice compared to smoked exposed mice with or without treatment. Telmisartan treatment did not improve mouse lung function after smoke exposure. Values are expressed as mean ± SEM, Student t-test: WT Untreated vs Smoked Telmisartan, *p < 0.05, **p < 0.01, n = 2 – 7. 91 Figure 4.2: Telmisartan did not prevent emphysema in cigarette smoke (CS)-exposed mice. Airspace enlargement was assessed using H&E staining in control (CS-exposed, untreated mice) and telmisartan-treated mice after 6 months of CS exposure of original magnification (higher panel, bar = 1 mm) and original magnification x4 (lower panel, bar = 500 µm) (A). Quantification of the mean linear intercept (MLI) for control and telmisartan groups (B). Mean arterial blood pressure (BP) was measured at 4 months and 6 months to assess telmisartan biological activity (C). Body weights were measured before euthanasia (D). Data are shown as mean ± SEM, Student t-test, *p < 0.05, ***p < 0.001, n = 3-4. 92 4.4.3 Lung mechanics of mice after OVA-induced allergic lung inflammation In our allergic asthma model, lung functions were measured 24h after the last OVA challenge in all groups: healthy control (Naive, non-asthmatic), OVA-sensitized control (OVA-SEN, asthmatic mice with no treatments), OVA-sensitized treated groups (Losartan, EXP3179, EXP3174, asthmatic mice). Our results showed that OVA-induced inflammation shifted the inspiratory expiratory pressure loop in all groups compared to controls (Figure 4.3A), suggesting a decreased inspiratory capacity in mice with inflammatory asthma. Losartan, EXP3179 and EXP3174 showed no changes in resistance, tissue damping, inspiratory capacity, elastance or compliance compared to untreated OVA-SEN mice (Figures 4.3B - 4.3F). Hysteresis was significantly higher in the losartan-treated group compared to the OVA-SEN control group (p < 0.05) (Figure 4.3G). Together, these results show that none of the drugs tested in this experiment improve lung function in OVA-sensitized mice. In addition, lung histology results suggested that OVA-sensitized mice have increased lung inflammation. Again, none of the drugs tested were able to improve it significantly (Figure 4.4). It should be noted that statistical analyses could not include the naive group due to its low sample size. 93 Figure 4.3: The effects of losartan and its metabolites on lung function in BALB/c female mice exposed to ovalbumin (OVA)-induced allergic inflammation. Pressure-curve loops (A), resistance (B), tissue damping (C), inspiratory capacity (D), elastance (E), compliance (F) and hysteresis (G) were assessed in female BALB/c control mice compared to OVA-sensitized mice with or without treatment. Losartan, EXP3179 and EXP3174 treatment did not improve mouse lung function in our OVA-sensitized mouse model. Values are expressed as mean ± SEM, One-way ANOVA, Tukey multiple comparisons, *p < 0.05, n = 2 – 5. 94 Figure 4.4: Losartan and its metabolites did not prevent lung inflammation in ovalbumin (OVA)-sensitized mice. Airway inflammation was assessed using H&E staining in healthy control (naive), OVA-sensitized mice (OVA-SEN), and treated mice with losartan, EXP3179 or EXP3174 of original magnification (left panel, bar = 2 mm) and original magnification x5 (right panel, bar = 500 µm) (A). Quantification of the inflammation area for each group (B). Data are shown as mean ± SEM, One-way ANOVA, Tukey multiple comparisons, n = 2 - 5. 95 4.4.4 Comparison of the effects of losartan, EXP3179 and EXP3174 on eosinophils OVA-SEN mouse bronchoalveolar lavage fluid (BAL) had a higher number of eosinophils compared to the control group (1076.9 ± 911.7 in naive vs. 3239.9 ± 297.1 in OVA-SEN). Similar results were found in the losartan-, EXP3179- and EXP3174-treated groups (2281.7 ± 717.4, 2319.0 ± 699.9 and 2310.7 ± 542.7, respectively). These results show that none of the treatments affected the eosinophils number in our OVA-sensitized model, however, the animal number in the control group was low which could have affected the results (Figure 4.5A). 4.4.5 Comparison of the effects of losartan, EXP3179 and EXP3174 on B cells B cells were not affected in the OVA-sensitized group compared to the control (74.8 ± 50.5 and 70.0 ± 50.5, respectively). Results were similar in all treated groups with 46.3 ± 9.7, 57.2 ± 11.4 and 37.6 ± 4.1 for losartan, EXP3179 and EXP3174, respectively. The low animal number in our control group could have affected these results (Figure 4.5B). 4.4.6 Comparison of the effects of losartan, EXP3179 and EXP3174 on T cells T cells seemed to be decreased in the OVA-sensitized group compared to control (369.7 ± 157.9 in Naive vs 119.7 ± 34.2 in OVA-SEN). In all treated groups, results were similar to OVA-induced allergic mice, suggesting no improvement from any of the drugs (71.4 ± 14.1 in LOS, 85.8 ± 9.4 in EXP3179 and 98.3 ± 30.7 in EXP3174) (Figure 4.5C). 96 4.4.7 Comparison of the effects of losartan, EXP3179 and EXP3174 on macrophages (MQs) In the control group, MQs were the primary cell type, therefore their abundance was high (9454.7 ± 4553.9). MQs were reduced in the OVA-SEN group, suggesting the presence of other inflammatory cells contributing to the cellular infiltrate. Similar results were found in all treated groups, showing no improvement compared to the OVA-SEN group (45.6 ± 12.5 in LOS, 40.4 ± 11.6 in EXP3179 and 49.4 ± 16.2 in EXP3174) (Figure 4.5D). 4.4.8 Comparison of the effects of losartan, EXP3179 and EXP3174 on leucocytes (CD45+) Taking all leucocytes together, we found that the control group expressed a high number of CD45+ cells (26065.5 ± 14056.7), probably due to its higher number of MQs. In OVA-SEN group, CD45+ cells were reduced (4319.2 ± 335.9), suggesting a lower abundance of leucocytes in BAL of OVA-induced allergic mice. In all treated groups, we found similar results to those of OVA-SEN, with 3132.9 ± 918.7 in LOS, 3399.6 ± 955.9 in EXP3179 and 3097.5 ± 760.9 in EXP3174 (Figure 4.5E). 4.4.9 Comparison of the effects of losartan, EXP3179 and EXP3174 on neutrophils Neutrophils were decreased in OVA-SEN mice compared to Naive as well as in all treated groups. The abundance of neutrophils was 8699.0 ± 4016.9 in Naive group, 69.2 ± 9.8 in OVA-SEN, 133.7 ± 46.5 in LOS, 104.8 ± 47.6 in EXP3179 and 100.4 ± 49.6 in EXP3174 (Figure 4.6F). 97 A discrepancy between the flow cytometry data showing decreased CD45+ leukocytes in the BAL of OVA-treated mice and the histological sections can be observed. This requires careful interpretation of the inflammation data from BAL alone. The analysis of BAL fluid is useful for understanding the cells and substances present in the airways, but it may not give a complete picture of inflammation in the entire lung. The CD45+ leukocytes in BAL mainly show the immune cells that have moved into the alveolar spaces, whereas examining lung tissue sections allows for a broader view, including cells in the interstitial and perivascular spaces. This could explain why histological sections sometimes reveal a more extensive inflammatory response than what is found in BAL. Furthermore, depending exclusively on BAL fluid analysis may result in underestimating the overall inflammatory condition within the lung tissue. For a more thorough understanding, combining BAL analysis with data from lung homogenates or tissue sections could help to assess the distribution and extent of inflammation across different lung compartments. Statistical analyses could not include the naive group in all BAL fluid experiments due to its small sample size. 98 Figure 4.5: Distribution of lung inflammatory cell subtypes among ovalbumin (OVA)-sensitized mouse model treated with losartan, EXP3179 or EXP3174. Cells from bronchoalveolar lavage (BAL) fluid were analyzed for indicated cell populations: eosinophils (A), B cells (B), T cells (C), macrophages (D), all leucocytes (CD45+) (E) and neutrophils (F). Values are expressed as mean ± SEM, One-way ANOVA, Tukey multiple comparisons, n = 2 – 5. 99 4.5 Discussion 4.5.1 COPD Research demonstrated that RAAS plays a role in COPD pathogenesis [393]. Blocking RAAS with ARBs rather than using angiotensin-converting enzyme inhibitors (ACEi) has been shown to decrease COPD complications and mortality [394]. Therefore, we studied whether telmisartan could significantly improve lung function and emphysema in mice chronically exposed to CS. In our study, we could induce emphysema in mice with a 6-month CS exposure protocol. However, we found that telmisartan did not improve lung function or prevent emphysema when given in drinking water for the duration of the experiment, despite evidence that it was biologically active at the dose given. In the literature, conflicting data can be found about the efficacy of ARBs to improve lung function and emphysema in COPD. Podowski et al. evaluated the effects of losartan in the prevention of emphysema progression in mice. They found that losartan reduced or reversed emphysema mediated by transforming growth factor (TGF) β. In addition, losartan also reduced oxidative stress, cell death, and inflammation, as well as improved lung mechanics [77]. In this study, they used fibrillin-deficient mice, which exhibit emphysema because of excessive TGF signalling. In addition to using a different ARB, we also used a different mouse model of COPD which could explain the differences between their study and ours. More recently, Wise et al. showed that in a randomized, placebo-controlled trial, the efficacy of losartan in reducing emphysema progression was underwhelming but might reduce the risk of hospitalizations [395]. 100 Clini et al. found that exhaled NO was reduced in patients with severe COPD compared to patients with mild COPD [396]. In addition, another study showed plasma NO levels and other markers of the endothelium were also reduced in COPD [397]. Our lab recently showed that telmisartan was able to increase endothelial function in a NO-dependent fashion [360], thus making telmisartan a good candidate for the management of COPD. Nevertheless, other studies reported a negative correlation between exhaled NO and lung functions [398]. Similarly, Agusti et al. found that exhaled NO appears to increase during exacerbations of COPD instead of the decreased NO levels described by Clini et al. [399]. These data highlight the difficulties of using NO as a marker of COPD severity but suggest that endothelial function might play an important role in the disease. In 2006, Andreas et al. published a study where they evaluated the effects of irbesartan in 60 patients with COPD. Even though irbesartan affected the hematocrit and the total lung capacity of patients, it did not improve the maximum inspiratory pressure, meaning that the respiratory muscle strength of COPD patients was not significantly improved [400]. However, Raupach et al. showed that irbesartan had positive effects on emphysema severity, lung biomechanics and exercise capacity in a mouse model of emphysema [401]. While our study provides valuable insights into the effects of telmisartan in mice with CS-induced emphysema, it is essential to acknowledge its limitations. Firstly, the pilot study uses a constrained number of mice due to technical challenges associated with the COPD mouse model, potentially influencing our findings. Additionally, comparing results directly with other research groups is challenging, given the diverse animal models of emphysema employed. Variations in drugs, chemicals, cigarettes, CS doses, 101 instruments, exposure protocols, and drug administration methods contribute to the complexity. Furthermore, numerous animal models of emphysema exist, each unable to replicate all human COPD characteristics. Therefore, this study alone may not fully capture the effects of telmisartan. 4.5.2 Asthma Studies reported that ANGII is elevated in the plasma of asthmatic patients [402]. In addition to being a weak bronchoconstrictor itself, it can potentiate the effects of other bronchoconstrictors such as methacholine [295], [402]. Therefore, the use of ARBs was investigated in various studies to assess their anti-remodelling and anti-inflammatory effects. In our study, we used the OVA model to induce allergic asthma, resulting in increased eosinophil infiltration and decreased lung function. We also used losartan and its metabolites, EXP3179 and EXP3174, to evaluate whether these compounds had distinct effects in a mouse model of allergic asthma. We found that none of the drugs were able to improve lung function or lung inflammation despite the existing literature showing the opposite [241]. In their study, Patel et al. investigated the role of AT1 and angiotensin II receptor type 2 (AT2) in an allergic mouse model of asthma and found that losartan reduced inflammation, airway hyperreactivity and methacholine-induced contraction [241]. The discrepancies between our study and theirs could be explained by the protocol used, as well as the drug concentration and route of administration. In 1998, Dicpinigaitis et al. could not demonstrate significant effects on bronchoconstriction of losartan in patients with stable asthma [403]. Despite being in humans, this study correlates with our results suggesting that the dosage of losartan might be crucial, and 102 great variability in results might be observed due to the heterogenous characteristics of the disease. Moreover, other studies showed that telmisartan, irbesartan and candesartan may have potential applications as anti-allergic agents, probably due to their antioxidant and anti-inflammatory effects [293], [296], [404]. Again, it is difficult to directly compare results obtained from different studies as they all differ in terms of animal model used and drug concentration. However, our negative results might have been largely influenced by the route of administration of the drugs. Similarly to our CODP study, there are currently no animal models replicating all the characteristics of human asthma [405], [406]. Therefore, results seen in animals and humans might not always correlate. In addition to the different drugs and routes of administration, the discrepancies between the results of different studies are explained by the use of different protocols and allergens, as well as the animal model itself. This suggests that studying allergic asthma is complex and requires researchers to think wisely while defining their study protocol. Moreover, the ARB chemical structure might play a crucial role in the results seen by the researchers. In future studies, transgenic mouse models could offer promising options to enhance our understanding of the roles played by individual molecules and cytokines in various asthma phenotypes [248]. 103 Chapter 5: Overall conclusions, study impact, and future perspectives 5.1 Overall conclusion The information presented in this document seeks to address the overall aims outlined in this thesis: • Compare the effects of EXP3179 and EXP3174 to those of losartan on vascular tone and endothelial function. • Determine the contribution of AT2 to the biological properties of losartan and EXP3179. • Investigate the pleiotropic effects of telmisartan and losartan in mouse models of COPD and allergic asthma. Completion of the current thesis allows us to have a better understanding of the chemical structure required to enhance endothelial function and address our hypothesis that “the endothelial enhancing functions of losartan metabolites EXP3179 or EXP3174 are behind some of the pleiotropic effects seen with losartan”. Taken together, the results presented in this thesis provide a better understanding of losartan and its metabolites, in particular EXP3179. These data could explain the underwhelming effects of losartan seen in clinical trials due to low concentrations of EXP3179, thus providing important insights as to the chemical structure required to activate endothelial function in mice. It is an important step for future studies and for the development of new drugs with better protective effects than losartan. 104 5.2 Small chemical changes have big impacts Previously, our laboratory showed that the beneficial effects of losartan and other ARBs observed in a mouse model of Marfan syndrome were likely AT1-independent [351]. Moreover, we found that these effects rely on the enhancement of endothelial function through eNOS [360], and ARBs exhibit significant variability in their capacity to induce these NO-enhancing effects in the vasculature [75]. Although other studies explore the pleiotropic effects of ARBs on endothelial function, our research contributes to the limited amount of existing data in this field. In this work, we aim to better understand the underwhelming effects of losartan in clinical trials. We show that despite studies relying on EXP3179 as a biologically active, AT1-independent losartan metabolite, both EXP3174 and EXP3179 can block AT1 and lower BP in rodents [352]. We also investigate EXP3179 and EXP3174 ex vivo on isolated aortic vessels from WT mice. This allows us to remove the impact of circulating ANGII in the system, avoiding potential biological effects of ANGII on its receptors that may be relevant in vivo. Despite the same BP-lowering effect between losartan, EXP3179 and EXP3174, we show that only EXP3179 can improve endothelial function ex vivo after a treatment of 30 minutes. Moreover, we assess losartan and EXP3179 in the absence of AT2 and find that their endothelial-enhancing effects are not decreased. This shows that losartan likely promotes endothelial function activation through a different pathway than the AT2 switch or a direct AT2 activation shown in previous studies as compensatory mechanisms [358], [359]. The current understanding of EXP3179 mechanism of action on the endothelium is summarized in Figure 5.1. 105 5.3 Study limitations The studies presented in this thesis come with limitations. Firstly, while our studies investigate the effects of losartan metabolites ex vivo, the literature is notably sparse regarding the use of EXP3179 in vivo. To our knowledge, there are no existing studies that address the pharmacokinetics or metabolism of EXP3179 in mice. Consequently, the protocols and dosages employed in our research require further validation and optimization. Without comprehensive pharmacokinetic data, it is challenging to ascertain the systemic exposure and biodistribution of EXP3179, which are critical for understanding its potential therapeutic effects and side effects.. Despite using mice Figure 5.1: Current knowledge on EXP3179 endothelium-related mechanism of actions. 106 models and cell cultures, exploring alternative animal models, such as guinea pigs or rats, may be considered. These species, closer to human conditions, particularly in studies involving blood pressure and lung diseases like COPD, could provide valuable insights. However, it is essential to recognize that animal models inherently simplify the complex pathophysiology of diseases, potentially limiting the findings' applicability to human conditions. Moreover, the clinical relevance of certain models and interventions, such as using OVA to induce asthma, is limited. The choice of allergen or sensitization method could impact the generalizability of the findings to human populations. Therefore, it is imperative to replicate these findings using other allergens, such as HDM. In addition, there is a possibility of publication bias, where studies with positive results are more likely to be published than those with neutral or negative findings. This bias could influence the interpretation of our negative results with telmisartan and losartan, as presented in Chapter 4. Acknowledging this, our conclusions about the efficacy of these drugs in reducing pulmonary inflammation and improving lung function should be considered with caution, and further studies are needed to confirm these findings. The relevance of vasoactive responses observed in the aorta to systemic blood pressure regulation remains an open question. While our studies provide insights into how losartan and its metabolites influence aortic contractility, it is unclear how these effects translate to overall blood pressure control, particularly in disease settings characterized by endothelial dysfunction. Future studies should consider testing these drugs in models of atherosclerosis, hypertension, or diabetes, where endothelial dysfunction is more pronounced. Such investigations would help elucidate whether the 107 pharmacological actions of losartan, EXP3179, and EXP3174 on aortic contractility differ in pathological versus healthy conditions. Lastly, due to technical and ethical constraints, some results in this thesis may be limited by sample size, impacting statistical power and result generalizability. Small sample sizes can introduce variability and limit the ability to detect subtle effects. However, as the preliminary results were not encouraging, the deliberately low animal number was maintained. 5.4 Future perspectives of EXP3179 and ARB research Our studies point toward several avenues that allow for a deeper understanding of EXP3179 and its implications. The future of EXP3179 research holds promising possibilities that could significantly enhance our understanding of its therapeutic potential and broaden the horizons of ARB-related investigations. First, the negative outcomes observed in Chapter 4, particularly with the intraperitoneal route of administration, prompt us to explore alternative drug delivery methods. Investigating the use of devices like an Alzet pump for chronic EXP3179 administration could offer a solution, allowing for sustained concentrations over time and potentially altering our results. Given the significant improvement in endothelial function after a brief stimulation of aortic vessels, EXP3179 could be tested in vascular disease models, such as atherosclerosis. Losartan has been shown to have beneficial effects in atherosclerosis [407], [408], therefore unravelling the exact mechanisms behind losartan’s effects by testing EXP3179 could lead to better disease management and drug development. In 108 addition, designing novel drugs with a chemical structure similar to EXP3179 opens avenues toward understanding the structure-function mechanisms of EXP3179 and ARBs in general. To enhance our understanding of the mechanism of action behind the inhibition of vessel contraction after a treatment with EXP3179, phospho-proteomic studies could be performed. Identifying specific targets of EXP3179 would provide valuable insights into the complex cellular pathways involved in its vascular effects. Our study on allergic asthma using OVA revealed unexpected results, including decreased respiratory capacity and lung resistance, as well as a low number of MQs and neutrophils. These results could have been influenced by the animal model chosen. Repeating the experiments using HDM as the allergen to promote allergic asthma offers a promising avenue. Quantifying histology slides and assessing inflammatory markers such as IgE, IL-4, and IL-5 could provide a more comprehensive understanding. Additionally, experimenting with alternative routes of administration, like inhalation, may offer better perspectives. 5.5 Conclusion In conclusion, our study highlights the impact of losartan and telmisartan beyond their conventional roles in managing hypertension. These medications were previously found to have profound effects on endothelial function, inflammation, and oxidative stress, positioning them as promising candidates for diverse therapeutic applications. While our investigation into telmisartan in a COPD model and losartan and its metabolites in allergic asthma did not yield anticipated positive outcomes, it emphasizes the complexities of 109 studying chronic lung diseases. Recognizing the importance of different animal models, and considering chemical differences, dosage, and drug administration protocols are crucial for understanding these diseases comprehensively. 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Hayek, “The Angiotensin-II Receptor Antagonist, Losartan, Inhibits LDL Lipid Peroxidation and 175 Atherosclerosis in Apolipoprotein E-Deficient Mice,” Biochem Biophys Res Commun, vol. 236, no. 3, pp. 622–625, Jul. 1997, doi: 10.1006/BBRC.1997.6844. 176 Appendices Appendix A : Supplementary data for Chapter 2 - Losartan Metabolite EXP3179 is a Unique Blood Pressure-Lowering AT1 Antagonist with Direct, Rapid Endothelium-Dependent Vasoactive Properties Figure A.1: Experimental procedure for in-house synthesized EXP3179. A literature procedure for the preparation of these compounds was modified as follows. Commercial pyridinium dichromate (0.110 mol, 1.7 equiv) was added portionwise to a cold (0 °C) solution of commercial losartan potassium (0.065 mol, 1.0 equiv) in DMF (120 mL) and the mixture was warmed to rt and stirred for overnight (13 h) after which time TLC showed that the reaction was complete. The mixture was cooled to 0°C, quenched with cool water (500 mL) and extracted with EtOAc (3x250 mL). The combined extracts were washed with brine (300 mL), dried (Na2SO4) and evaporated. The residue was dried under a high vacuum and purified by flash column chromatography (0-3% Me0H in CH2Cl2). Evaporation of the collected fractions gave EXP 3179 (22.5 g, 0.053 mol, 82%) as a white solid: R f = 0.4 (10% MeOH/CH2Cl2, visualized by UV); mp 154-156 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.69 (s, 1H), 7.64-7.70 (m, 2H), 7.51-7.60 (m, 2H), 7.02-7.09 (m, 4H), 5.59 (s, 2H), 2.61-2.65 (t, 2H, J = 7.74 Hz), 1.46-1.56 (m, 2H), 1.15-1.31 (m, 2H), 0.78-0.83 (t, 3H, J = 7.5 Hz); 13C NMR (75 MHz, CDCI3) δ 172.0, 164.8, 155.7, 144.1, 143.9, 141.4, 141.4, 127.8, 127.2, 125.3, 125.2, 120.1, 120.1, 67.1, 55.3, 52.7, 51.3, 47.3, 36.7, 33.2, 30.3, 30.1, 14.1; HRMS (Q-TOF) m/z [M ]+ calcd for C22 H21CIN6O 420.1464, found 420.1465; Purity by HPLC >95%. 177 Figure A.2: Experimental procedure for in-house synthesized EXP3174. A literature procedure for the preparation of these compounds was modified as follows. A solution of EXP3179 (15 g, 0.036 mol, 1.0 equiv) in t-BuOH (250 mL) was added dropwise at rt to a solution of sodium chlorite (80%) (29 g, 0.32 mmol, 9 equiv), sodium dihydrogen phosphate monohydride (29.4 g, 021 mol, 6 equiv). The resulting mixture was continued to stir overnight at rt, whereupon TLC showed that the reaction was complete. The mixture was quenched with 1N HCl extracted twice with EtOAc (2x500 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated. The crude solid was dissolved in saturated NaHCO3 solution (250 mL) washed with EtOAC (2x500 mL). The aqueous phase was acidified with 1 M HCI to pH 2-4 at 0°C to get solid, filtered and dried to afford EXP 3174 (9.5 g, 0.022 mol, 61% ) as a white solid: mp 135-143 °C; 1H NMR (300 MHz, DMS0-d6) δ 7.65-7.70 (m, 2H), 7.51-7.59 (m, 2H), 7.07-7.09 (d, 2H, J = 8.13 Hz), 6.95-6.98 (d, 2H, J = 8.13 Hz) 5.59 (s, 2H), 2.50-2.60 (t, 2H, J = 7.65 Hz), 1.45-1.52 (m, 2H), 121-128 (m, 2H), 0.78-0.83 (t, 3H, J = 7.35 Hz); 13C NMR (75 MHz, DMS0-d6) δ 160.7, 152.3, 141.4, 138.7, 136.7, 135.3, 131.5, 131.0, 129.5, 128.2, 1264, 117.7, 47.9, 29.1, 26.2, 22.0,14.0; HRMS (Q-TOF) m/z [M]+ calcd for C22H21Cl N6O2 436.1416, found 436.1415; Purity by HPLC >95%. 178 1H NMR 300 13C NMR NHN NNNNClHOEXP 31791H NMR 300 MHz Solvent: DMSO-d6 NHN NNNNClHOOEXP 317413C NMR 75MHz Solvent: DMSO-d6 Figure A.3: NMR Data of synthesized EXP3174 and EXP3179 Analysis 179 Analysis Info 7/30/2020 1:18:11 PMAcquisition DateAnalysis Name D:\Data\Mass Spec\Jane.LC\577-LCMS-Los-Aldehyde-5.dpra_lcms_30-90_17min_14473. m janezhuMethod Operator577-LCMS-Los-Aldehyde-5 HCTultra PTM DiscoverySystemInstrumentSample NameCommentAcquisition ParameterIon Source Type Ion Polarity Alternating Ion Polarity off ESI Positive Scan End100 m/zStd/Enhanced Scan BeginMass Range Mode 1500 m/zTrap Drive 60.9 40.0 Volt136.0 Volt SkimmerCapillary ExitAccumulation Time Auto MS/MS off 5 Spectra39790 µs AveragesDisplay Reportprinted:Bruker Compass DataAnalysis 4.0 1 of 1Page 7/31/2020 1:28:34 PMNHN NNNNClHOEXP 3179Figure A.4: Purity Assessment of synthesized EXP3179 by HPLC. Analytical HPLC, 10 to 90% MeCN in water over 14 min, flow rate of 0.7 mL/min on a Xterra MS C18 Analytical Column (3.5 um, 4.6 mm x 50 mm). EXP3179 – purity > 95% (MeCN). 180 Figure A.5: Purity Assessment of synthesized EXP3174 by HPLC. Analytical HPLC, 10 to 90% MeCN in water over 14 min, flow rate of 0.7 mL/min on a Xterra MS C18 Analytical Column (3.5 um, 4.6 mm x 50 mm). EXP3174 – purity > 96% (MeCN) Analysis Info 7/30/2020 1:37:24 PMAcquisition DateAnalysis Name D:\Data\Mass Spec\Jane.LC\579-LCMS- Los-Acid-5.dpra_lcms_30-90_17min_14474. m janezhuMethod Operator579-LCMS- Los-Acid-5 HCTultra PTM DiscoverySystemInstrumentSample NameCommentAcquisition ParameterIon Source Type Ion Polarity Alternating Ion Polarity off ESI Positive Scan End100 m/zStd/Enhanced Scan BeginMass Range Mode 1500 m/zTrap Drive 60.9 40.0 Volt136.0 Volt SkimmerCapillary ExitAccumulation Time Auto MS/MS off 5 Spectra7633 µs AveragesDisplay Reportprinted:Bruker Compass DataAnalysis 4.0 1 of 1Page 7/31/2020 1:22:53 PM181 Figure A.6: Heart rates (HR) were unchanged during the experiments. HR were measured in normotensive rats and showed no differences despite the different treatments at a dose of 10 mg/kg (A). Hypertensive rats showed constant HR i.v. administration of EXP3179 at a dose of 10 mg/kg (B). 182 Table 0-1: Baseline systolic BP, diastolic BP, mean arterial BP, and heart rates for normotensive rats Table 0-2: Baseline systolic BP, diastolic BP, mean arterial BP, and heart rates for hypertensive rats A. li li i li i l iTable A.2: Baseline systolic BP, diastolic BP, mean arterial BP, and heart rates for hypertensive rats 183 Figure A.7: EXP3179 and EXP3174 applied intraperitoneally (i.p.) reduced blood pressure in wild-type (WT) mice. Blood pressure was measured at baseline and 3h after i.p. injection of each drug at a concentration of 10mg/kg in WT mice. DMSO was used as the vehicle control. Data are shown as means ± SEM of systolic and diastolic BP changes from baseline 3h after drug injection, n = 7-9. 184 Figure A.8: Assessment of vessel and endothelium integrity and smooth muscle contribution for each vessel used for myography experiments with wild-type (WT) mice. Percentage inhibition of PE-induced constriction by each drug (A). High doses of KCl were used at the beginning of each experiment to assess the vessel integrity. Acetylcholine (ACh) concentration-response curve was used to assess the integrity of the endothelium before drug incubation and sodium nitroprusside (SNP) was used at the end of each experiment to assess the contractility of smooth muscle cells. 3-4-mo-old WT mice (young) (B-D). Similar experiments were performed with aortas of 10-mo-old WT mice (aged) (E-H). Relaxation is expressed as a percentage of PE-induced contraction after each dose of ACh or SNP. Data are expressed as means ± SEM, One-way ANOVA with Tukey’s post hoc tests or unpaired student t-test, n = 5 – 10, *p < 0.05, **p < 0.01. 185 236 240 244 248 252 256 260 264 268 272 276 2804.04.55.05.56.06.57.07.58.08.59.09.5Time (min)mNEXP3174EXP3179LosartanVehicleDrugsPE concentration-response10-8 M to 10-4 Mwash30-minute incubationFigure A.9: Representative tracings of PE-induced contractions under different treatment conditions. The vehicle control is shown in red, losartan in blue, EXP3179 in green, and EXP3174 in orange. Aortic rings were treated with one of the following treatments (15 µM) for 30 minutes before adding PE. The black arrows indicate the addition of drugs or PE to the myograph chambers. The grey arrow indicates the end of the PE concentration-response curve. 186 Figure A.10: EXP3179 reduces PE-induced contraction similarly in the presence and absence of ibuprofen. PE concentration-response curves after vehicle and drug incubation (15uM) of thoracic aorta sections WT mice with and without ibuprofen (a prostacyclin synthesis inhibitor) (A). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments (B). The half-maximal concentrations for PE-induced contraction (EC50) values were calculated for each treatment, as well as for both young and old mice (C). Data are expressed as means ± SEM, two-tailed Student’s t-test (two groups) or One-way ANOVA with Tukey’s Post Hoc tests (4 groups), n = 4-10, *p < 0.05, ***p < 0.001. 187 Figure A.11: Exposure of aortic rings from WT mice to PI3K inhibitor Wortmannin reversed EXP3179 effects on PE-induced contraction. PE concentration-response curves after vehicle and EXP3179 (15uM) and Wortmannin (0.1uM) incubation of thoracic aorta sections (A). Bar graphs represent the difference in contractility between the baseline and the maximum PE-induced contraction in mN (B). Data are expressed as means ± SEM, One-way ANOVA with Tukey’s post hoc tests, n = 5-6, *p < 0.05 compared to DMSO, #p < 0.05, ###p < 0.001 compared to Wortmannin. 188 Appendix B : Supplementary data for Chapter 3 - Losartan and metabolite EXP3179 activate endothelial function without lowering blood pressure in AT2 receptor KO mice Figure B.1: WT and AT2 KO aortic rings respond similarly to acetylcholine (ACh) concentration-response curve. ACh was used to assess the integrity of the endothelium in female mice (A) and male mice (B) before treatment. Relaxation is expressed as a percentage of PE-induced contraction after each dose of ACh. Figure B.2: EXP3179 reduces phenylephrine (PE)-induced contraction in WT and angiotensin II receptor type 2 (AT2) KO mouse aortas. PE concentration-response curves after vehicle and drug incubation (20 uM) of thoracic aorta sections from 3- to 4-month-old WT and AT2 KO mice before (A) and after L-NAME incubation (200 uM) (B). Bar graphs represent the maximum PE-induced contraction in mN from all experiments performed (C). Data are expressed as means ± SEM, Two-way ANOVA, Tukey multiple comparisons with *p < 0.05, **p < 0.01, ****p < 0.0001, n = 10–11. 189 Figure B.3: WT and AT2 KO aortic rings respond similarly to acetylcholine (ACh) concentration-response curve. ACh was used to assess the integrity of the endothelium in female mice (A) and male mice (B) after chronic treatment with losartan. Relaxation is expressed as a percentage of PE-induced contraction after each dose of ACh. Figure B.4: Losartan reduces phenylephrine (PE)-induced contraction in aortas from WT and angiotensin II receptor type 2 (AT2) KO mice. PE concentration-response curves of thoracic aorta sections from 3-mo-old WT and AT2 KO mice treated with losartan (0.6 g/l) for 4 weeks in drinking water before (A) and after L-NAME incubation (200 uM) (B). Bar graphs represent means ± SEM of the maximum PE-induced contraction in mN from all experiments performed (C). Data are expressed as means ± SEM, Two-way ANOVA, Tukey multiple comparisons *p < 0.05, **p < 0.01, ****p < 0.0001, n = 10–14. 190 Figure B.5: WT and angiotensin II receptor type 2 (AT2) KO mice have similar aorta morphology and pulse wave velocity (PWV) at baseline and after 4 weeks of losartan treatment. Echocardiography images of male and female, WT and AT2 KO mice at untreated (NT) baseline (8 weeks of age) and following 4 weeks of losartan treatment (12 weeks of age). Images are taken in Parasternal Long-Axis (PSLA) across the aortic arch (A). Baseline/Untreated (NT) PWV (B) and Sinotubular Junction (STJ) (C) for combined male and female data. WT (n = 4 male; n = 4 female); AT2 KO (n = 5 male; n = 4 female); Data are mean ± SEM, Two-way ANOVA, Tukey multiple comparisons, Scale 1 mm. 191 Appendix C : Supplementary data for Chapter 4 - Exploring the Pleiotropic Effects of Telmisartan and Losartan in Mouse Models of COPD and Allergic Asthma Figure C.1 : Representative gating strategies in naive mice (non-asthmatic). 192 Figure C.2: Representative gating strategies in OVA-SEN mice (asthmatic).
Thesis/Dissertation
eng
Pharmacology and Therapeutics
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Bernatchez, Pascal
Pleiotropism of losartan and its metabolites against cardiopulmonary diseases : small chemical differences, major biological impacts
Text