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Serum amyloid P ameliorates radiation-induced oral mucositis and fibrosis Murray, Lynne A; Kramer, Michael S; Hesson, David P; Watkins, Brynmor A; Fey, Edward G; Argentieri, Rochelle L; Shaheen, Furquan; Knight, Darryl A; Sonis, Stephen T Jul 5, 2010

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Murray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Open AccessR E S E A R C HResearchSerum amyloid P ameliorates radiation-induced oral mucositis and fibrosisLynne A Murray*1, Michael S Kramer1, David P Hesson1, Brynmor A Watkins2, Edward G Fey2, Rochelle L Argentieri1, Furquan Shaheen3, Darryl A Knight3 and Stephen T Sonis2,4AbstractPurpose: To evaluate the effect of the anti-fibrotic protein serum amyloid P (SAP) on radiation-induced oral mucositis (OM) and fibrosis in a hamster cheek-pouch model.Experimental Design: Hamsters received a single dose of radiation (40 Gy) to the left everted cheek pouch to induce significant OM. The protective therapeutic potential of SAP was evaluated using varying dosing regimens. The extent of OM was measured using a validated six-point scoring scheme ranging from 0 (normal tissue, no mucositis) to 5 (complete ulceration). Fibrotic remodeling was also visualized histologically and quantified at later time points using collagen gene expression.Results: SAP treatment attenuated the profile of radiation-induced oral mucositis by delaying the time of onset, reducing the peak value, and enhancing the resolution of injury. The peak mucositis score was reduced by approximately 0.5 grade in SAP-treated animals. The number of animal days with a score of ≥ 3 was reduced by 48% in the SAP-treated group, compared with the saline control group (P < 0.01). SAP also inhibited the extent of tissue remodeling and decreased radiation-induced increases in myofibroblast number. Attenuated collagen deposition and gene expression was also observed in the cheek pouches of hamsters treated with SAP at both 16 and 28 days post-radiation.Conclusions: SAP treatment significantly attenuated radiation-induced injury. In particular, SAP attenuated the severity of OM and inhibited pathogenic remodeling. This suggests that SAP may be a useful therapy for the palliation of side effects observed during treatment for head and neck cancer.BackgroundOral mucositis is a common side effect of chemotherapyand radiotherapy, with mucositis occurring to somedegree in more than one-third of patients receiving anti-neoplastic drug therapy [1]. Moderate to severe mucositisoccurs in virtually all patients who receive radiation ther-apy for tumors of the head and neck. It typically begins atcumulative exposures of 15 Gy and then worsens whentotal doses exceed 60 Gy [1-4]. The ensuing injury signifi-cantly impairs quality of life and can hamper the sched-uled course of therapy, thus reducing efficacy oftreatment. The complex pathoetiology is associated withthe induction of a series of biologic pathways within thesubmucosa. Pronounced epithelial cell apoptosis and thegeneration of pro-inflammatory cytokines such as tumornecrosis factor (TNF)-α and interleukin (IL)-1β [5], andpro-fibrotic mediators such as transforming growth fac-tor (TGF)-β [5], result in the breakdown of the oralmucosa, causing formation of ulcerative lesions. Inpatients with granulocytopenia, the ulcerations thataccompany mucositis are common portals of entry forindigenous oral bacteria, often leading to sepsis or bacte-remia [3]. Fibrotic remodeling of the damaged tissue thenserves to seal off the region; however, if the extent ofremodeling is overexuberant, the surrounding tissuebecomes involved, resulting in a loss of elasticity that pro-duces detrimental functional consequences. This, alongwith pronounced mucositis after directed radiotherapy orcertain chemotherapies, can cause the patients to inter-rupt their scheduled treatments, thereby affecting long-* Correspondence: murrayl@medimmune.com© 2010 Murray et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.term survival [3].1 Promedior, Inc, 371 Phoenixville Pike, Malvern, PA, 19355, USAFull list of author information is available at the end of the articleMurray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 2 of 10Serum amyloid P (SAP) is a member of the pentraxinfamily of serum proteins and has been previously shownto reduce fibrosis in experimental models [6-9]. This pro-tein is highly conserved between species. In vitro, SAPhas been described as inhibiting monocyte to fibrocytedifferentiation and macrophage activation [9,10]. Fibro-cytes are bone marrow-derived CD45+ collagen I+ cellsthat are thought to contribute to excess extracellularmatrix (ECM) deposition at sites of fibrosis. In vivo, SAPalso decreases fibrocyte and macrophage numbers inmodels of cardiac and pulmonary fibrosis [6,7]. Morerecently, an association of fibrocytes with both the prog-nosis and severity of interstitial lung disease has beendescribed [11]. SAP also decreases the expression of mac-rophage activation markers in models of pulmonary andrenal fibrosis [8,9].The standard therapy for mucositis is predominantlypalliative, including application of topical analgesics suchas lidocaine and/or systemic administration of narcoticsand antibiotics. In this study, we used an establishedhamster cheek-pouch model of radiation-inducedmucositis to determine if SAP treatment affects either theclinical or pathological hallmarks of the disease. We fur-ther assessed the potential effect of SAP on the down-stream fibrosis mediated by radiation.MethodsAnimalsMale Syrian golden hamsters (CRL) weighing approxi-mately 80-90 g (5-6 weeks old, n = 12 per group) wereused in this study. All experiments were conducted underBiomodels Institutional Animal Care and Use Committeeregulations and protocols.Acute model of radiation-induced mucositisAnimals were given an acute radiation dose of 40 Gydirected to their left buccal cheek pouch, with the rightcheek pouch serving as the non-irradiated control, aspreviously described [12]. Briefly, radiation was gener-ated with a 160 KV potential (18.75 mA) source at a focaldistance of 210 mm, hardened with a 3.0 mm Al filtrationsystem (Kimtron Polaris II, Kimtron Inc, Woodbury, CT,USA). Irradiation targeted the left buccal pouch mucosaat a rate of 1.32 Gy/minute. Before irradiation, animalswere anesthetized with intraperitoneal (i.p.) ket-amine:xylazine (160 mg/kg:8 mg/kg). The left buccalpouch was everted and fixed, and isolated using a leadshield.SAP administrationSAP in 10 mM Tris, 140 mM NaCl buffer (EMD Biosci-(day 0). Animals were dosed daily on days 0 to 7, every 2days on days 0 to 12, or every 2 days on days -1 to 26.Additional groups of animals were dosed every 2 dayswith phosphate-buffered saline (PBS) on days 0 to 12 ordays -1 to 26, and served as vehicle control groups. Thedose level of 2 mg/kg SAP was chosen as this approxi-mately doubles predicted endogenous SAP levels in thehamster.Evaluation of oral mucositisOral mucositis was evaluated from photographs by twoindependent, trained observers who were blinded to thestudy groups. The extent of mucositis was scored visuallyby comparison with a validated photographic scale (clini-cal scoring) (0 = completely healthy pouch with no vaso-dilation or erythema; 1 = light to severe erythema andvasodilation with no visible erosion of mucosa; 2 = severeerythema, vasodilation and erosion of the superficialmucosa; 3 = severe erythema and vasodilation, formationof off-white ulcers in ≥ 1 places with the cumulative sizeof the ulcers equaling approximately one-quarter of thepouch; 4 = severe erythema and vasodilation, cumulativesize of ulcers encompassing approximately half thepouch; 5 = virtually all of the pouch is ulcerated, loss ofpliability with the pouch only partially extractable fromthe mouth).Gene expression analysisFor gene analysis, cheek pouch tissue was homogenizedin lysis buffer (Panomics, Freemont, CA, USA), in accor-dance with the manufacturer's instructions. Owing to thehigh levels of collagen present in the hamster cheekpouch, homogenized samples were passed through a 0.45μm cellulose nitrate plate (Whatman, Piscataway, NJ,USA) before mRNA analysis. mRNA levels were quanti-fied using a branched-DNA technology-based method(QuantiGene Reagent System; Panomics), according tothe manufacturer's protocols. Transcript levels of fibrosisand macrophage-related genes were normalized to β-actin mRNA.HistologyCheek pouches fixed in 10% normal buffered formalin(NBF) were cut into horizontal sections perpendicular tothe long axis of the cheek pouch. Samples were changedto 70% alcohol, embedded in paraffin and cut into sec-tions approximately 5 μm thick. Serial sections werestained with hematoxylin and eosin (H&E) for gross mor-phology, or Masson's trichrome stain for collagen deposi-tion. Sections were examined using am invertedmicroscope and camera (Micromaster; Fisher ScientificPittsburgh, PA, USA).ences, San Diego, CA, USA) was administered by i.p.injection at a dose of 2 mg/kg with dosing initiated 1 daybefore radiation (day -1) or immediately after radiationHistological fibrosis scoringH&E-stained slides were assessed for fibrosis accordingto a six-point scale (0 = no fibrosis noted; 1 = minimalMurray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 3 of 10fibrosis; 2 = mild fibrosis; 3 = moderate fibrosis; 4 =marked fibrosis; 5 = severe fibrosis). Analysis wasdirected by a board-certified veterinary pathologistblinded to the study group designation.ImmunohistochemistryFormalin-fixed, paraffin wax-embedded cheek pouchsections were analyzed for immunohistochemical local-ization of α-smooth muscle actin (α-SMA) expressionusing an indirect immunoperoxidase procedure. Sectionswere dewaxed with xylene, rehydrated in graded concen-trations of ethanol, and blocked with normal horseserum. Mouse anti-α-SMA monoclonal antibody (A5228,clone 1A4; Sigma-Aldrich, Missouri, MO, USA) and con-trol normal rabbit IgG were diluted in PBS to a final con-centration of 5 μg/ml. Anti-α-SMA or IgG were added tohistological sections for 60 min, after which each tissuesection was washed thoroughly with PBS. A secondarybiotinylated donkey-anti-mouse IgG (Jackson Immu-noResearch, West Grove, PA, USA) was added to eachsection for 1 hour. Slides were then thoroughly washedand α-SMA visualized with a commercial staining kit(HRP-Dab Staining Kit; Vector Laboratories, Burlingame,CA, USA).In vitro fibroblast assayNormal human dermal fibroblasts (NHDF) were platedinto 24-well plates (Costar, Corning, NY, USA) at 100,000cells/well, and allowed to adhere for 8 hours. The cellswere then washed with PBS and cultured overnight inserum-free media (Dulbecco's modified Eagle's medium(DMEM) with L-glutamine, penicillin and streptomycin).Cells were then stimulated for 24 hrs in the presence orabsence of platelet-derived growth factor (PDGF) anti-body (200 ng/mL) and/or SAP (1 or 4 μg/mL). Prolifera-tion was assessed a cell proliferation ELISA with 5-bromo-2-deoxyuridine incorporation (Roche AppliedScience, Roche Diagnostic Corporation, Indianapolis, IN,USA).Epithelial to mesenchymal transition assayExperiments were performed in A549 cells grown to 60%confluence in six-well plates (BD Biosciences, Missis-sauga, ON, USA), which were grown in DMEM contain-ing 10% fetal bovine serum (FBS) at 37°C in 5% CO2 in air.Before each experiment, cells were incubated in DMEMwith 0.5% FBS for 24 hours. A549 cells were then incu-bated with or without TGF-β1 (10 ng/mL) or SAP (1, 5 or10 μg/mL) or both for 48 hours. After this, cells werelysed in protein extraction buffer supplemented withphenylmethanesulfonyl fluoride, phosphatase inhibitorthen underwent western blotting analysis with antibodiesdirected against extra domain-A (EDA)-fibronectin(EDA-FN) (MAB1940; Chemicon International, Temec-ula, CA, USA) and E-cadherin (SC-8426, Santa Cruz Bio-technology, Santa Cruz, CA, USA). Hsp90 (BD610418,BD Biosciences, Mississauga, ON, USA) was used as aprotein loading control.StatisticsFor each evaluation day, the scores of the vehicle controlgroup were compared with those of the treated groupsusing non-parametric rank sum analysis. Statistical dif-ferences between treatment groups were determinedusing the Student t-test, Mann-Whitney U test and χ2analysis, with a critical value of 0.05 as appropriate. P val-ues of <0.05, <0.01 or P < 0.005 *referred to as *, ** or ***,respectively) were considered significant.ResultsRadiation-induced fibrosisThe clinical course of mucositis is well characterized inthis hamster cheek-pouch model of radiation-inducedmucositis [12], with peak mucosal breakdown occurringbetween days 14 and 18. The development of ulcerativemucositis in this study was consistent with historical datafrom our laboratory. To assess the extent of downstreamtissue remodeling as it pertains to collagen deposition,hamsters received a single dose of radiation (40 Gy) spe-cifically to the left everted cheek pouch on day 0 and werekilled 16 or 28 days later. Cheek pouches were sectionedand stained with Masson's trichrome to visualize areas ofexcess collagen accumulation (Figure 1). In non-irradi-ated cheek pouches, densitometric analysis showedapproximately 43% trichrome positivity in each of thecheek pouches analyzed (Figure 1A, B). At day 16 in irra-diated cheek pouches, there were large areas of ulcerationdensely infiltrated with neutrophils, with surroundingnecrosis in the muscle layer (Figure 1D). Irregular regionsof fibrosis were observed beneath the muscular layer ofthe buccal pouch (Figure 1C). At day 28, there were fewerregions of epithelial ulceration, and the extent of inflam-mation was also reduced, indicating a degree of mucositisresolution at the histopathological level (Figure 1F). How-ever, the cheek pouches exhibited more pronouncedfibrosis at day 28 (60% trichrome positivity; Figure 1E),with the aberrant ECM being better organized comparedwith the day 16 time point (58% trichrome positivity; Fig-ure 1C). This increase in ECM deposition indicates thatthe hamster cheek-pouch model is suitable to assess theanti-fibrotic potential of SAP as it pertains to radiation-induced fibrosis.cocktail 2, and protease inhibitor cocktail (Sigma-Aldrich). Lysates at a concentration of 50 ng/ml werethen separated by electrophoresis in SDS-PAGE gels andelectrotransferred to nitrocellulose membranes, whichSAP inhibits radiation-induced mucositisThe effect of SAP on radiation-induced mucositis wasinitially assessed by administering SAP (2 mg/kg, i.p.) toMurray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 4 of 10the hamsters every 2 days from days 0-12 after radiationexposure. In control (PBS-treated) animals, the peak inmucositis occurred at day 18, with a mean ± SEMmucositis score of 3.25 ± 0.11 (Figure 2). After the peak atday 18, the extent of mucositis subsided, with a meanmucositis score of 0.81 ± 0.10 on day 6. In the SAP-treated animals, the peak in radiation-induced mucositisalso occurred at day 18. However, the magnitude ofmucositis was lower (2.88 ± 0.09), with significantlyreduced mucositis scores being observed in the SAP-treated (day 0-12) animals at days 14 and 16 comparedwith vehicle controls (P < 0.05). In fact, the SAP-treatedanimals never reached a group mean of 3 throughout thestudy, whereas vehicle-treated animals reached a groupmean of >3 by day 16. Another significant effect of SAPwas observed at day 26, with SAP-treated animals havinga lower mean mucositis score (0.25 ± 0.11) than vehiclecontrol hamsters (P < 0.01). Totaling the number of daysthat animals had a mucositis score of ≥ 3 (severe mucosi-tis and of clinical significance), revealed that animalstreated with SAP had a significantly attenuated durationof severe mucositis (48% relative reduction, P < 0.01).Taken together, these results suggest that SAP treatmentdelays the onset, reduces the peak dampens the totalextent, and promotes resolution of mucositis.SAP inhibits radiation-induced fibrosis in the hamster cheek pouchHistological analysis and quantification of H&E-stainedsections of the cheek pouches of hamsters exposed toradiation, assessing both fibrotic changes and overtinflammation, indicated that there was a time-dependentincrease in overall pathogenic changes, with scores peak-ing at day 16 (2.75 ± 0.25) and showing only a moderatereduction by day 28 (2.25 ± 0.25) (Figure 3). As with vehi-cle controls, hamsters treated with SAP (day 0-12) hadincreased fibrous material at day 16 (2.50 ± 0.29) com-pared with non-irradiated control cheek pouches andcompared with day 8 hamster cheek pouches. However,there was significantly reduced fibrous material in thecheek pouches of animals treated with SAP at day 28(1.50 ± 0.29) compared with vehicle control-treated ani-mals (P < 0.05; Figure 4).Acute dosing with SAP at the time of radiation gives greatest protectionTo determine the optimum dosing strategy of SAP toattenuate radiation-induced mucositis and fibrosis, weassessed the efficacy of various dosing schedules. Ham-sters were treated with SAP immediately after radiation(day 0) and given SAP either every 2 days from day 0 to 12or daily from days 0 to 7. In another group of animals,SAP treatment was initiated 1 day before radiation andwas given every 2 days until the end of the study (days -1to 26). Mucositis scoring indicated that all dosing regi-mens reduced the peak of mucositis and enhanced its res-olution (Figure 3A), thus attenuating the overall time thatanimals actually had severe mucositis (Figure 3B).Figure 2 Effect of serum amyloid P (SAP) on radiation-induced mucositis. Hamsters were exposed to radiation and treated with SAP (2 mg/kg, i.p.) every 2 days from days 0 to 12. Mean mucositis scores were determined in phosphate buffered saline (vehicle)-treated (open Figure 1 Representative histopathology of radiation-induced fi-brosis. Hamsters were exposed to radiation (40 Gy) on day 0. At subse-quent time points, cheek pouches were removed, sectioned and stained with Masson's trichrome to visualize collagen deposition. Rep-resentative cheek pouch sections are shown for (A,B) non-irradiated controls, (C,D) day 16 post-radiation, and (E,F) day 28 post-radiation.ACEBDFAnalysis of procollagen III gene expression in the ham-ster cheek pouches, as a surrogate for the extent of fibro-sis, determined that hamsters treated with SAP dailysymbols; n = 12) and SAP-treated (filled symbols; n = 12) animals from days 6 to 26. *P < 0.05, **P < 0.01 compared with PBS vehicle-treated control irradiated hamster cheek pouches.Murray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 5 of 10from days 0-7 had significantly less procollagen III genetranscript compared with the PBS vehicle-treated or thehamsters treated with the other two SAP dosing strate-gies when measured at day 16 (Figure 5A) or day 28 (Fig-ure 5B). Quantification of myofibroblast number, asvisualized by immunohistochemical staining of α-SMA(Figure 6A-H) confirmed the anti-fibrotic activity of SAPat day 16 in animals dosed on days 0-7 (Figure 7). How-ever there were also significantly fewer α-SMA positivecells at both day 16 and day 28 in animals dosed every 2days from days 0 - 12, and also at day 28 in the animalsdosed with SAP throughout the study (Figure 7).SAP does not promote fibroblast proliferation or epithelial to mesenchymal transitionTo determine the potential mechanistic anti-fibroticactivity of SAP on mesenchymal cells, we initiallyassessed the effect(s) of SAP on fibroblast proliferation,alone or in combination with known proliferative media-tors. As expected, PDGF promoted significant prolifera-tion (Figure 8). By contrast, SAP had no directproliferative effect, nor did it modulate PDGF-inducedproliferation when added concomitantly (Figure 8A). Wealso assessed the effects of SAP on epithelial to mesen-chymal transition (EMT) using the human lung epithelialcell line A549. Incubation with TGF-β1 at 10 ng/mlinduced phenotypic changes consistent with EMT (Fig-ure 8B). Morphologically, the cells lost their rounded cob-blestone appearance and became stellate. Exposure toTGF-β1 also induced expression of the mesenchymalmarker EDA-FN, together with downregulation of theepithelial marker E-cadherin (Figure 8B). Addition ofSAP (1-10 μg/ml) alone had no obvious effect on the phe-notype of A549 cells or on the expression of E-cadherinor EDA-FN (Figure 8B). Similarly, when added at thesame time as TGFβ1, SAP had no effect on TGFβ1-induced changes in cell morphology or on expression ofEDA-FN or E-cadherin (Figure 8B).DiscussionWe have shown that SAP treatment results in a signifi-cant reduction in the two pathological sequelae of clinicalradiotherapy and chemotherapy, mucositis and fibrosis.In the oral mucosa, directed radiation therapy inducesapoptosis of the epithelial barrier which initiates aninflammatory cascade, characterized by nuclear factor(NF)κB activation [13]. The ensuing fibrotic responseserves to provide adequate local wound healing; however,this can be overexuberant and limit tissue elasticity andfunction. SAP treatment dampened the extent of mucosi-tis, as demonstrated by a reduction in the time to onsetFigure 3 Attenuation of radiation-induced mucositis with an al-tered serum amyloid P (SAP) dosing schedule. Hamsters were ex-posed to radiation and treated with SAP (2 mg/kg, i.p. every 2 days from day 0 to 12, daily from days 0 to 7, or every 2 days from days -1 to 26 (n = 12 per group) or vehicle control (phosphate-buffered saline (PBS), i.p. every 2 days from days 0 to 12; n = 12). (A) Mean mucositis scores were determined in PBS (vehicle)-treated (open symbols) and SAP-treated (filled symbols) animals from days 6 to 28. (B) The total number of days that hamsters had a mucositis score of ≥ 3 was calcu-lated. *P < 0.05, **P < 0.01 compared with PBS vehicle-treated control irradiated hamster cheek pouches.A.  4 8 12 16 20 24 2801234 VehicleSAP (2mg/kg) q2d -1 to 26SAP (2mg/kg) qd 0-7SAP (2mg/kg) q2d 0-12Days Post-RadiationMucositis ScoreB.  050100150VehicleSAP q2d D-1 to 26SAP qd D0-7SAP q2d D0-12** ** *Treatment GroupsDays with Score3Figure 4 Reduction in radiation-induced fibrosis mediated by se-rum amyloid P (SAP). Hamsters were exposed to radiation (40 Gy) on day 0. At subsequent time points, cheek pouches were removed, sec-tioned and stained with haematoxylin and eosin, and extent of fibrosis 01234VehicleSAPDay 8 Day 28Day 16*non-irradiatedDays Post-RadiationFibrosis Score (0-5)and attenuation in both the peak and duration of pathol-ogy. SAP also attenuated the fibrotic wound healingresponse.was assessed. Original magnification: left, × 11; right × 200 (region in the inset box). Bars represent the mean ± SEM of four animals per group.Murray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 6 of 10In the present study, oral mucositis was scored usingthe established WHO criteria [14]. Using this scale, ascore of ≥ 3 [represents severe mucositis that affects apatient's quality of life, with 70% of patients requiringfeeding tubes to maintain adequate nutrition [14]. Fur-ther, in approximately 35% of patients with grade 3-4mucositis, subsequent rounds of radiotherapy may bedelayed or prevented, leading to less successful clinicaltherapy. Lower grades of mucositis may also significantlyaffect patient quality of life. Grade 1 is associated withpain that is sufficiently severe to require opioid analgesia.At grade 2, patients have difficulty or an inability to swal-low due to ulcerations in the mouth and throat, which, ifsevere, may necessitate total parenteral nutrition andcoactivator of a large number of genes involved inmucosal injury [16]. The activation of NF-κB due to radi-ation results in upregulation of a variety of genes leadingto increased production of pro-inflammatory cytokinesincluding TNF-α, IL-1β and IL-6 [17]. Targeted radiationhas been shown to increase cyclooxygenase-2 expressionin submucosal tissues, and this parallels the developmentof ulcerative mucositis [18]. In this study, we show thatSAP treatment reduced the number of days that animalshad a mucositis score of ≥ 3, indicating that SAP may pro-vide significant palliative care, allowing the continuationof radiotherapy. Histopathologic assessment of cheekpouches indicated a significant reduction in the extent ofleukocyte infiltration after SAP treatment. SAP hasFigure 5 Attenuated fibrosis-associated gene expression after serum amyloid P (SAP) treatment. Hamsters were exposed to radiation and treated with SAP (2 mg/kg, i.p. every 2 days from days 0 to 12, daily from days 0 to 7, or every 2 days from days -1 to 26) or vehicle control (phosphate-buffered saline (PBS), i.p. every 2 days from days 0-12). Procollagen III gene expression was determined in the irradiated cheek pouches using branched DNA technology at (A) day 16 and (B) day 28. Bars represent the mean ± SEM. of four animals per group.A.  0246810VehicleSAP (2mg/kg) q2d -1 to 26SAP (2mg/kg) qd 0-7SAP (2mg/kg) q2d 0-12Day 16 Fold Change(ACTB Housekeeper)B.  051015VehicleSAP (2mg/kg) q2d -1 to 26SAP (2mg/kg) qd 0-7SAP (2mg/kg) q2d 0-12Day 28 Fold Change(ACTB Housekeeper)rehydration. Early expression of mitogen-activated pro-tein kinase (MAPK) is observed within 8 hours post-radi-ation [15]. MAPK may function with NF-κB as arecently been shown to reduce inflammation in severalexperimental models of fibrosis through an Fc receptor-dependent mechanism [7,9,19]. Therefore the anti-Murray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 7 of 10inflammatory effect of SAP treatment on mucositis mayoccur via a similar mechanism. Owing to the model limi-tations, with limited hamster reagents available, we wereconfined by the number of endpoints we could assess.The pathogenesis of mucositis is multifactorial, involv-ing the interaction of oral mucosal epithelial cells,endothelial cells, connective tissue and the submucosalinfiltrate [5]. There are four interdependent phases: (1) aninflammatory/vascular phase, (2) an epithelial phase, (3)an ulcerative/bacteriological phase, and finally (4) a heal-during apoptosis promotes inflammation via numerouscascades including Toll-like receptor signaling and NFκBactivation. Non-phlogistic clearance of these pro-inflam-matory signals by macrophages acts to switch off theimmune response, thus limiting damage to host tissue[21]. Inadequate clearance by macrophages has beenshown to contribute directly to a number of acute andchronic diseases [22]. SAP clears apoptotic and necroticdebris [9,23], which then helps to promote a quiescentclearance of the damage-associated molecular patterns(DAMPs), thus minimizing the extent of inflammation.Further, inhibiting apoptosis has been shown to reduceoral mucositis [24]. Subsequent studies assessing theeffect(s) of SAP on radiation-induced apoptosis andnecrosis would help to determine if this is part of the pro-tective therapeutic mechanism.The ensuing fibrotic response mediated by radiation isassociated with an increase in ECM deposition. Fibro-blasts, when activated, produce various ECM compo-nents and can also differentiate into myofibroblasts,which are more contractile and more readily synthesizeECM, resulting in a greater loss of tissue elasticity. SAPdecreased the amount of ECM and the number of myofi-broblasts in the cheek pouch after radiation. SAP did notdirectly modulate fibroblast proliferation; therefore thisanti-fibrotic mechanism in vivo is upstream of directfibroblast activity.Another key stromal cell type demonstrated to promotefibrosis is the epithelial cell. Epithelial cells stimulatedwith the prototypic growth factor TGF-β result in a tran-sition of cell phenotype away from the resident epithelialcell and towards a motile, ECM-producing mesenchymalcell. This process is referred to as epithelial to mesenchy-mal transition (EM)T and is understood to contribute tolung, liver and kidney fibrosis [25-27]. TGF-β is the pro-Figure 6 Reduction in radiation-induced α-smooth muscle actin (α-SMA) expression with serum amyloid P (SAP) treatment. Ham-sters were exposed to radiation and treated with SAP (2 mg/kg, i.p. ev-ery 2 days from days 0 to 12, daily from days 0 to 7, or every 2 days from days -1 to 26) or vehicle control (phosphate-buffered saline (PBS), i.p. every 2 days from days 0 to 12). At either 16 or 28 days post-radiation, animals were killed and cheek pouches removed, sectioned and stained with anti-α-SMA to visualize myofibroblast accumulation and vascular smooth muscle cells. (A-H) Representative histopathology from irradiated cheek pouches from the vehicle groups (A) at day 16 and (B) day 28; from the first SAP group (2 mg/kg every 2 days, days -1 to 26) at (C) day 16 and (D) day 28; from the second SAP group (2 mg/kg daily, days 0 to 7) at (E) day 16 and (F) day 28; and from the third SAP group (2 mg/kg every 2 days, days 0 to 12) at day 16 (G) and day 28 (H).ACBDEGFHFigure 7 The total number of α-smooth muscle actin (α-SMA)-positive cells was quantified morphometrically. Bars represent the mean ± SEM of four animals per group. *P < 0.05 compared with PBS (vehicle) control irradiated hamster cheek pouches. Original mag-nification × 400.0200400600800VehicleSAP (2mg/kg) q2d -1 to 26SAP (2mg/kg) qd 0-7SAP (2mg/kg) q2d 0-12non-irradiatedDay 16 Day 28****MeanSMA+ cells per 10hpf viewing/resolution phase [20]. Radiation directly damages cel-lular DNA, which results in cellular apoptosis and alsoinhibits homeostatic cell renewal. The debris generatedtotypic inducer of EMT. However, we determined thatSAP did not modulate TGFβ-induced EMT, nor did SAPhave any direct EMT effects.Murray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 8 of 10Recent studies have indicated that during fibrosis, SAPdirects monocyte to profibrotic fibrocyte or M2 mac-rophage differentiation [8-10]. This suggests that thereduction in myofibroblast number in the cheek pouchafter SAP treatment is due to a mechanism upstream ofdirect modulation of fibroblast activity and may be due tomodulation of monocyte differentiation. Future studiesassessing the monocyte/macrophage phenotype in thecheek pouch should determine if there is also a profi-brotic phenotype associated with the pathology observedin this model, as has been reported clinically [28]. Fur-ther, determining whether SAP can direct the mac-The observed utility of SAP as a therapeutic formucositis in reducing both the acute inflammation andthe downstream fibrosis is exciting. Various potentialtherapeutics have been assessed clinically for oralmucositis including recombinant IL-11 [29], granulocyte-macrophage colony-stimulating factor (GM-CSF) [30,31],G-CSF [32] and TGF-β3 [33]. Currently, the onlyapproved treatment for mucositis is palifermin (Kepiv-ance®; Biovitrum, Stockholm, Sweden), which is approvedfor the treatment of oral mucositis in patients undergoingbone marrow ablation for transplant. Palifermin is arecombinant keratinocyte growth factor and, as with allFigure 8 Serum amyloid P (SAP) has no effect on fibroblast proliferation or transforming growth factor (TGF)-β-mediated epithelial to mes-enchymal transition (EMT). (A) Normal human dermal fibroblasts (NHDF) were stimulated with platelet-derived growth factor (PDGF; 200 ng/mL) for 24 hours in the presence or absence of SAP. Proliferation was quantified using 5-bromo-2-deoxyuridine incorporation. (B) A549 cells were stimu-lated with TGF-β1 in the presence or absence of SAP for 48 hours. The resultant lysates were assessed for the epithelial marker E-cadherin or the mes-enchymal marker extra domain-A (EDA)-fibronectin using western blotting analysis. *P < 0.05, **P < 0.01, ***P < 0.005.A.0.00.20.40.60.81.0control1g/ml SAP4g/ml SAPmedia alone 200 ng/ml PDGF* *** **OD450B.FibronectinEDAECadherinTGFB(ng/mL)SAP(g/mL) 1011051010101510rophage phenotype away from a fibrotic M2 phenotypeand towards a classic M1 macrophage phenotype will beinsightful.the other agents tested clinically, promotes epithelial cellproliferation, thus providing a denser barrier to protectthe underlying mucosal tissue. By contrast, we have dem-Murray et al. Fibrogenesis & Tissue Repair 2010, 3:11http://www.fibrogenesis.com/content/3/1/11Page 9 of 10onstrated that SAP has no direct pro-proliferativeresponse on resident cells, nor does it directly inhibit epi-thelial cell transition during fibrosis. Importantly, SAPhas more profound effects on the downstream responsesmediated by radiotherapy, namely reducing the ensuinginflammation and thus limiting stromal cell activation, asdemonstrated by reduced ECM deposition and α-SMAexpression in vivo.Competing interestsPromedior sponsored this study. Promedior are developing SAP for the treat-ment of fibrotic diseases. LAM, MSK, DPH and RLA were employed by Prome-dior during this study.Authors' contributionsLAM conceived the study, participated in design and coordination, and draftedthe manuscript. MSK, DPH, STS conceived the study, and participated in designand coordination. BAW, EGF, RLA and FS participated in design and coordina-tion, and conducted the studies. DAK participated in design and coordination.All authors read and approved the final manuscript.Author Details1Promedior, Inc, 371 Phoenixville Pike, Malvern, PA, 19355, USA, 2Biomodels and Affiliates, 313 Pleasant Street, Watertown, MA, 02472, USA, 3Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Burrard Street, Vancouver, Canada and 4Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine Diviosn of Oral Medicine, Dana Farber Cancer Institute, Brigham And Women's Hospital, 75 Francis Street, Boston, MA, 02115, USAReferences1. Plevova P: Prevention and treatment of chemotherapy- and radiotherapy-induced oral mucositis: a review.  Oral Oncol 1999, 35:453-470.2. Knox JJ, Puodziunas AL, Feld R: Chemotherapy-induced oral mucositis. Prevention and management.  Drugs Aging 2000, 17:257-267.3. Peterson DE: Research advances in oral mucositis.  Curr Opin Oncol 1999, 11:261-266.4. 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J Pathol 2001, 193:242-247.29. Antin JH, Lee SJ, Neuberg D, Alyea E, Soiffer RJ, Sonis S, Ferrara JL: A phase I/II double-blind, placebo-controlled study of recombinant human Received: 16 April 2010 Accepted: 5 July 2010 Published: 5 July 2010This article is available from: http://www.fibrogenesis.com/content/3/1/11© 2010 Murray et l; icensee BioMed Central Ltd. is an Open Access articl  distributed under th  terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Fibroge esis & Tissu  Repa r 2010, 3:11gamma R-dependent monocyte-macrophage regulation in vivo.  Sci Transl Med 2009, 1:5ra13.10. Pilling D, Buckley CD, Salmon M, Gomer RH: Inhibition of fibrocyte differentiation by serum amyloid P.  J Immunol 2003, 171:5537-5546.interleukin-11 for mucositis and acute GVHD prevention in allogeneic stem cell transplantation.  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Wymenga AN, van der Graaf WT, Hofstra LS, Spijkervet FK, Timens W, Timmer-Bosscha H, Sluiter WJ, van Buuren AH, Mulder NH, de Vries EG: Phase I study of transforming growth factor-beta3 mouthwashes for prevention of chemotherapy-induced mucositis.  Clin Cancer Res 1999, 5:1363-1368.doi: 10.1186/1755-1536-3-11Cite this article as: Murray et al., Serum amyloid P ameliorates radiation-induced oral mucositis and fibrosis Fibrogenesis & Tissue Repair 2010, 3:11

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