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he role of lipoprotein-X in the development of glomerulosclerosis in familial LCAT deficienty Lynn, Edward G. 1997

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T H E R O L E O F LIPOPROTEIN-X IN T H E D E V E L O P M E N T OF G L O M E R U L O S C L E R O S I S IN FAMILIAL L C A T D E F I C I E N C Y by EDWARD G LYNN B . G S , Simon Fraser University, 1995 A T H E S I S SUBMITTED IN PARTIAL F U L F I L L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard  T H E UNIVERISITY O F BRITISH C O L U M B I A June 1997 ®Edward G Lynn, 1997  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his  or her representatives.  It is  understood  that  copying or  publication of this "thesis for financial gain shall not be allowed without my written permission.  C^2 Department of The University of British Columbia! Vancouver, Canada Date  DE-6 (2/88)  ABSTRACT Progressive glomerulosclerosis is a major complication in patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency.  The absence of  active LCAT in the plasma of these patients leads to the formation of an abnormal plasma lipoprotein, lipoprotein-X (Lp-X). Renal biopsies of these patients have revealed lipid deposition, macrophage infiltration, the presence of foam cells, and mesangial cell proliferation in affected glomeruli. The objective of this project was to examine the role of Lp-X in the development of glomerulosclerosis. Our results have demonstrated that Lp-X is taken up by cultured rat mesangial cells and that the lipid component of Lp-X is metabolized intracellularly.  W e have also  investigated the role of apolipoproteins in the uptake of Lp-X. Both apo C-l and 0-1II inhibited Lp-X uptake while C-ll (1.5 fold) and E (4 fold), as well as all four apolipoproteins combined (1.5 fold), stimulated this process. W e have also observed that cell surface proteoglycans are involved in modulating the uptake of this abnormal lipoprotein.  Lp-X, either alone or combined with rat peritoneal  macrophages, has no effect of the proliferation of mesangial cells.  ii  TABLE OF  CONTENTS  Page ABSTRACT TABLE OF CONTENTS LIST O F T A B L E S LIST O F F I G U R E S LIST O F A B B R E V I A T I O N S ACKNOWLEDGEMENTS DEDICATION  ii iii vii viii ix xi xii  1. INTRODUCTION  1  1.1 Lipoproteins  1  1.1.1 The major lipoprotein classes  1  1.1.2 Structure of lipoproteins  3  1.1.3 Apolipoproteins  4  1.2 Receptor-mediated uptake of lipoproteins  4  1.2.1 LDL receptor  4  1.2.2 LDL-receptor related protein (LRP)  6  1.2.3 Scavenger receptor  6  1.3 Lecithin:cholesterol acyltransferase  1.4 Familial LCAT deficiency  7  10  1.4.1 Genetic basis of LCAT deficiency  10  1.4.2 Lipoprotein-X  10  1.4.3 Clinical symptoms in L C A T deficiency  12  1.4.3.1 Corneal opacities  12  iii  1.4.3.2 Hemolytic anemia  12  1.4.3.3 Proteinuria  13  1.4.3.4 Coronary artery disease and L C A T deficiency  14  1.5 Glomerulosclerosis  16  1.5.1 Glomerular mesangial cells  17  1.5.2 Macrophages  17  1.5.3 Lipid accumulation  18  1.5.4 Extracellular matrix expansion  19  1.6 Rationale of the study  19  1.7 Hypotheses and objective  20  1.8 Specific aims  20  2. M A T E R I A L S A N D M E T H O D S  21  2.1 Tissue Culture  21  2.1.1 Isolation and culture of rat mesangial cells  21  2.1.2 Isolation and culture of rat peritoneal macrophages  22  2.2 Preparation of plasma lipoproteins  23  2.2.1 Isolation of lipoprotein-X  23  2.2.2 Preparation of radiolabeled lipoprotein-X  25  2.3 Preparation of reconstituted lipoprotein-X (rLp-X)  25  iv  2.3.1 Preparation of rLp-X  25  2.3.2 Preparation of radiolabeled rLp-X  26  2.4 Uptake and metabolism of lipoprotein-X (Lp-X) by mesangial cells  26  2.4.1 Uptake of Lp-X and rLp-X by mesangial cells  26  2.4.2 Metabolism of lipids by mesangial cells  26  2.4.3 Effect of cytochalasin, suramin, and polyinosinic acid on the uptake of rLp-X  27  2.4.4 Effect of Lp-X and rLp-X on mesangial cell proliferation  28  2.5 Effect of reconstituted lipoprotein-X on macrophages  28  2.5.1 Lipid analyses  28  2.5.2 Oil Red O (ORO) staining  28  2.6 Effect of reconstituted lipoprotein-X and macrophages on Mesangial cell proliferation  29  3. R E S U L T S  30  3.1 Uptake and metabolism of lipoprotein-X by mesangial cells  30  3.1.1 Uptake of Lp-X  30  3.1.2 Metabolism of Lp-X lipids  30  3.1.3 Uptake and metabolism of rLp-X  33  3.1.3.1 Uptake of rLp-X  33  3.1.3.2 Metabolism of rLp-X lipids  36  3.2 The effect of apolipoproteins on the uptake of lipoprotein-X  v  '  36  3.3 The effect of suramin, cytochalasin, and polyinosinic acid on the uptake of reconstituted lipoprotein-X  39  3.4 Effect of lipoprotein-X on macrophages  42  3.4.1 Lipid accumulation in macrophages  42  3.4.2 Foam cell formation  42  3.5 Mesangial cell proliferation  45  3.5.1 Effect of Lp-X and rLp-X  45  3.5.2 Combined effect of rLp-X and macrophages  45  4. DISCUSSION  48  4.1 Uptake and metabolism of lipoprotein-X in mesangial cells  48  4.1.1 Role of apolipoproteins and LDL receptor in Lp-X uptake  49  4.1.2 Role of scavenger receptor, phagocytosis, and proteoglycans in the uptake of Lp-X  49  4.1.3 Possible mechanisms for the uptake of Lp-X  50  4.2 Effect of reconstituted lipoprotein-X on macrophages  51  4.3 Mesangial cell proliferation  53  4.3.1 The effect of Lp-X  53  4.3.2 The combined effect of macrophages and Lp-X  54  4.4 Conclusion  54  5. L I T E R A T U R E CITED  56  vi  LIST OF TABLES Table  Page  1.  The four major classes of human plasma lipoproteins  2  2.  Chemical composition of normal human plasma lipoproteins  2  3.  Distribution of plasma apolipoproteins in normal humans  5  4.  Lipid concentrations in the kidneys of L C A T deficient patients  15  5.  Effect of apolipoproteins on the size of rLp-X  40  6.  Effects of suramin, cytochalasin and polyinosinic acid on the uptake of reconstituted Lp-X  41  vii  LIST O F  FIGURES  Figure  Page  1. The L C A T reaction  8  2. Lipoprotein electrophoresis  24  3. The uptake of Lp-X by rat mesangial cells  31  4. Metabolism of Lp-X by rat mesangial cells  32  5. The uptake of [ H]cholesteryl hexadecyl ether (CHE) rLp-X by rat mesangial cells  34  6. The uptake of [ H]FC-or [ H]PC-rLp-X by rat mesangial cells  35  7. Metabolism of rLp-X by rat mesangial cells  37  3  3  3  8. The effect of apolipoproteins on the uptake of Lp-X by rat mesangial cells  38  9. Effect of rLp-X on cellular lipids in rat peritoneal macrophages  43  10. Induction of foam cell formation by Lp-X  44  11. The effect of Lp-X and rLp-X on the incorporation of [ H]thymidine into rat mesangial cells  46  12. Combined effect of macrophage-conditioned medium and rLp-X on [ H]thymidine incorporation by rat mesangial cells  47  3  3  viii  LIST OF ABBREVIATIONS  apo  Apolipoprotein  BSA  Bovine serum albumin  CAD  Coronary artery disease  CE  Cholesteryl ester  CETP  Cholesteryl ester transfer protein  CHE  Cholesteryl hexadecyl ether  CM  Chylomicron  FBS  Fetal bovine serum  FC  Free (unesterified) cholesterol  FED  Fish Eye Disease  HBSS  Hank's buffered saline solution  HDL  High density lipoprotein  LCAT  Lecithin:cholesterol acyltransferase  LDL  Low density lipoprotein  Lp-X  Lipoprotein-X  LPC  Lysophosphatidylcholine  LRP  LDL-receptor related protein  NaBr  Sodium bromide  NaOH  Sodium hydroxide  ORO  Oil Red 0  PBS  Phosphate buffered saline  PC  phosphatidylcholine  PDGF  platelet-derived growth factor  PL  phospholipid  rLp-X  reconstituted lipoprotein-X  TG  triglyceride  TGF-p  transforming growth factor p  UC  unesterified cholesterol  VLDL  very low density lipoprotein  \  X  ACKNOWLEDGEMENTS  Numerous individuals aided me during this project.  First and foremost I  wish to thank my supervisor, Dr. Karmin O, for giving me the opportunity  to  further my education, for her invaluable encouragement and guidance, and for without whom I would undoubtedly be in a much different place than I am today. I also thank Drs. Jiri Frohlich and Haydn Pritchard for their constructive criticism and for creating an enjoyable research environment.  Drs. Richard Hegele,  Alexander Magil, and Urs Steinbrecher's valuable advice and suggestions greatly benefited this project.  I thank Dr. Patrick Choy for his help, for allowing me to  work in his laboratory for two months and for letting me experience the occasional blizzard.  I also thank Chung Yee Har and Ding Z. Fang for their technical  assistance. Ayyobi,  I benefited from the suggestions and advice of Lida Adler, Amir  Kit Chow, Michael Koon,  Scott  Lear, Ming  Li, and  Mohammed  Moghadesian, all of whom also created a friendly working environment.  xi  To my family  xii  1. INTRODUCTION 1.1 LIPOPROTEINS Plasma lipoproteins are heterogeneous particles composed of lipids and proteins.  They vary widely in size but almost all appear to be microemulsions  (Edelstein et al., 1979). They play a crucial role in the transport and metabolism of plasma lipids.  1.1.1 The major lipoprotein classes Lipoproteins have been traditionally divided into four main classes based on their density by sequential ultracentrifugation (Havel and Kane, 1995). Table 1 lists the major lipoprotein classes along with their corresponding densities and sizes. Table 2 summarizes the composition of the major lipoprotein classes. The two classes (Table 1) with the largest lipoproteins, whose cores consist mainly of triglycerides (Table 2), are the chylomicrons and the very low density lipoproteins (VLDL). Chylomicrons are derived from dietary fat and its B apolipoprotein (apo) is primarily apo B-48. V L D L is synthesized and secreted by hepatocytes and contains apo B-100. The two classes with smaller lipoproteins, which mainly contain cholesteryl esters in their cores, are low density lipoproteins (LDL) and high density lipoproteins (HDL).  Elevated levels of LDL and/or low  concentrations of HDL are well-known risk factors of coronary artery disease (CAD) (Lavie and Milani, 1997). The mature forms of LDL and HDL are not directly  secreted by  hepatocytes. Mature LDL and HDL are mainly produced by metabolic processes  1  Table 1. The four major classes of human plasma lipoproteins.  Class  chylomicrons VLDL LDL HDL  Density (g/mL)  Diameter (nm)  Molecular Weight (Da)  0.93 0.93 - 1.006 1.006 - 1.063 1.063- 1.210  7 5 - 1200 30 - 80 18 - 2 5 5 -• 12  5 0 - 100 x 1 0 1 0 - 8 0 x10 2,300,000 175-360,000 6  6  VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein. Adapted from Havel and Kane (1995).  Table 2. Chemical composition of normal human plasma lipoproteins.  Surface components Cholesterol PL Apo  CM VLDL LDL HDL  2 7 8 4  7 18 22 35  2 8 22 55  Core lipids TG CE  86 55 6 3  3 12 42 13  Surface components and core lipids are given as percentage of dry mass (% grams). Apo, apolipoprotein; C E , cholesteryl ester; C M , chylomicron; PL, phospholipid; T G , triglyceride. Adapted from Havel and Kane (1995).  2  occurring in the plasma. LDL are derived from the metabolic products of V L D L while the components of HDL are secreted with chylomicrons and VLDL. Some components of HDL are also secreted independently  as HDL precursors  (Hamilton era/., 1991).  1.1.2 Structure of lipoproteins Lipoproteins are composed of two major components, lipids and proteins. A typical lipoprotein particle is spherical and consists of a polar lipid monolayer surrounding a core of nonpolar lipids. The surface monolayer consists primarily of phospholipids, unesterified cholesterol, and proteins. albumin and apolipoproteins.  These proteins include  Apolipoproteins solubilize the lipoprotein and  enable its transport via the circulatory system.  The central core of lipoprotein  particles consists primarily of triglycerides and cholesteryl esters (Gotto et  al.,  1986). Phospholipids are amphipathic, possessing polar and nonpolar regions. The polar or hydrophilic head of the phospholipid is exposed to the aqueous environment (plasma) and the nonpolar, hydrophobic tail faces the central core of the lipoprotein.  Like the phospholipids, the protein components of the surface  monolayer have amphipathic characteristics. These amphipathic properties result from regions containing both polar and nonpolar amino acid residues (Gotto et al., 1986). The fatty acyl chains of phospholipids and the nonpolar amino acid side chains of proteins are excluded from the aqueous environment  by their  hydrophobicity. These hydrophobic forces generate the association of polar lipids  3  and proteins with lipoproteins.  1.1.3 Apolipoproteins Most of the apolipoproteins, except apolipoprotein B, are relatively soluble in an aqueous environment.  These water soluble apolipoproteins are able to  exchange readily between lipoprotein particles as well as with other lipid surfaces. The exchange of phospholipids and nonpolar lipids among lipoproteins requires the presence of specific transfer proteins (Fielding, 1990).  Table 3 lists the  apolipoproteins and their distribution in lipoproteins. From Table 3, it is clear that the major apolipoprotein associated with LDL is apo B-100 while apo E is associated primarily with HDL and to a lesser extent V L D L and LDL.  1.2 Receptor-mediated uptake of lipoproteins 1.2.1 LDL receptor The catabolism of apolipoprotein B-containing lipoproteins occurs by receptor-mediated endocytosis (Havel and Hamilton, 1988). The LDL receptor is a transmembrane protein that recognizes the apo B-100 on lipoprotein surfaces, which includes partially catabolized V L D L and LDL. LDL receptor also binds to lipoproteins containing apo E. Once bound to the LDL receptor the lipoproteins are endocytosed via coated-pits on the plasma membrane.  The lipoproteins  dissociate from the receptor in the acidic environment of endosomes, allowing the LDL receptor to return to the cell surface for reuse.  4  Table 3. Distribution of plasma apolipoproteins in normal humans.  Distribution in Lipoproteins (%)  Apo  HDL  A-l A-ll B-48 B-100 C-l C-l I C-lll D E-ll, E-lll, & E-IV  >99 >99  -  97 60 60 100 50  LDL  VLDL  CM  MW (kDa)  <1  10  >99 6 2 30 20  -  -  -  29.0 17.4 241.0 512.7 6.6 8.9 8.8 19.0  10  20  10  34.1  -  88  -  -  <1 <1  1 10 10  apo, apolipoprotein; MW, molecular weight. Adapted from Havel and Kane (1995) and Gotto etal., 1986.  5  The amount of LDL receptor is regulated  by the concentration  of  intracellular cholesterol. High levels of cholesterol lead to the down-regulation of LDL receptor while low levels of cholesterol increase LDL receptor synthesis (Brown and Goldstein, 1975). The liver is the principal site of LDL removal from the blood (Goldstein and Brown, 1987). The apo E binding domain of nascent chylomicrons is unavailable for binding to LDL receptor (Havel and Kane, 1995). However, as chylomicrons are metabolized to chylomicron remnants, the apo E binding domain becomes available for interaction with LDL receptor. The apo B and E binding domains are also unavailable in nascent VLDL. The apo B and E binding domains gradually become exposed during lipolysis. The LDL receptor has a much lower affinity for apo B-100 than for apo E (Havel and Hamiliton, 1988).  1.2.2 LDL-receptor related protein (LRP) Some studies have suggested that the LDL-receptor related protein (LRP) is a chylomicron remnant receptor (Kowal et al., 1989). apo E but not apo B-48.  L R P is thought to bind  V L D L receptor is a member of the L R P family.  The  V L D L receptor specifically recognizes apo E as its ligand. It has recently been shown that V L D L receptor is able to mediate chylomicron remnant  uptake  (Niemeir et al., 1996).  1.2.3 Scavenger receptor Scavenger receptors take up lipoproteins whose protein components have  6  been modified, such as oxidized LDL. Scavenger receptor mediates endocytosis and lysosomal catabolism of modified lipoproteins (Kowal et al.,  1989).  The  scavenger receptor is found primarily on macrophages (Beisiegel and St. Clair, 1996).  1.3 LECITHIN:CHOLESTEROL ACYLTRANSFERASE Lecithin:cholesterol acyltransferase (LCAT) (EC 2.3.1.43) catalyzes the transfer of a fatty acid from the sn  2  position of lecithin (phosphatidylcholine) to the  3-hydroxyl group of cholesterol, forming lysophosphatidylcholine and cholesteryl ester in plasma (Figure 1) (Glomset and Wright, 1964). Apo A-l is an important cofactor in this LCAT reaction (Jonas, 1991).  The L C A T gene consists of 6  exons and 5 introns and has been localized to chromosome 16 (Kuivenhoven et al., 1997). L C A T is a glycoprotein of approximately 63 kDa that is synthesized and secreted by the liver. 25% of the molecular mass of LCAT is due to carbohydrate (Marcel, 1982).  LCAT circulates in the blood primarily bound to HDL and is  responsible for esterifying cholesterol in plasma.  Normal L C A T exhibits two  activities in plasma. a - L C A T activity is specific for HDL while p-LCAT activity is specific for LDL and to a smaller extent V L D L (Carlson and Holmquist, 1985; Funke  et  al.,  1991).  The  preferred  7  substrate  of  LCAT  is  HDL.  PHOSPHATIDYLCHOLINE  LYSOPHOSPHATIDYLCHOLINE  L C A T apo A-l  CHOLESTEROL  CHOLESTERYL ESTER  Figure 1. The L C A T reaction. Lecithin:cholesterol acyltransferase (LCAT) catalyzes the transfer of a fatty acid from the sn position of phosphatidylcholine to the 3-hydroxyl group of cholesterol. Apo A-l is an important cofactor in this reaction. This reaction produces lysophosphatidylcholine and cholesteryl ester. 2  8  Less than 10% of LCAT activity is associated with LDL (Kuivenhoven et  al.,  1997). Apo A-l is the most effective cofactor in promoting L C A T activity. Apo A-ll, A-IV, C-ll and C-lll can also, to a lesser extent, stimulate this reaction (Jonas, 1991; O a n d Frohlich, 1995). Determination of L C A T activity can be approached using proteoliposomes (Dobiasova and Schutzova, 1986; O and Frohlich, 1995). L C A T activity can be measured  with  proteoliposomes  containing  unesterified  cholesterol,  phosphatidylcholine, and apo A - l . Assaying for either the decrease in unesterified cholesterol or the increase in free fatty acids will give a measure of the esterase and phospholipase activity of LCAT, respectively (O and Frohlich, 1995; O et al., 1993). Reverse cholesterol transport is the system by which excess extrahepatic cholesterol is transported to the liver (Fielding, 1990).  The HDL cholesteryl  esters produced by the L C A T reaction are transferred to LDL and VLDL.  LDL  and V L D L are in turn taken up by the liver, completing the transport of excess extrahepatic cholesterol to the liver.  This transfer of cholesterol esterified by  L C A T from HDL to the lower density lipoproteins requires the presence of cholesteryl ester transfer protein (CETP). LCAT, HDL and C E T P are believed to be vital in reverse cholesterol transport (Havel and Kane, 1995).  9  1.4 FAMILIAL LCAT DEFICIENCY 1.4.1 Genetic basis of LCAT deficiency Familial L C A T deficiency is an autosomal recessive disorder resulting from various point mutations in the LCAT gene. Thirty-five mutations dispersed among the six exons of the L C A T gene have been identified (Kuivenhoven et al., 1997). Recently, a mutation in intron 4 of the LCAT gene has also been reported (Kuivenhoven et al., 1996).  These mutations lead either to the expression of  inactive L C A T or to the absence of L C A T protein. Mutations in the LCAT gene that only lead to a deficiency in a - L C A T activity (associated with HDL) gives rise to Fish Eye Disease (FED), or partial L C A T deficiency. Familial L C A T deficiency results from the absence of both a L C A T and p-LCAT activities (Kuivenhoven et al., 1997).  1.4.2 Lipoprotein-X One unique feature of LCAT deficiency is the presence of an abnormal plasma  lipoprotein,  lipoprotein-X  (Lp-X),  in  patients'  plasma.  The  total  concentration of Lp-X in the plasma of patients is 0.3 - 1.5 mmol/L (Glomset et al., 1995).  Lp-X is also present in the plasma of patients with cholestatic liver  disease (Hamilton et al., 1971) and is associated with the LDL fraction (1.190 mg/mL < d < 1.063 g/mL) upon ultracentrifugation.  In both of these diseases Lp-  X appears to have an identical structure and chemical composition (Seidel et al., 1974). In LCAT deficiency, Lp-X arises from the surface of chylomicron remnants 10  that are not further catabolized due to the absence of active L C A T (Sabesin, 1982).  In cholestatic liver disease, Lp-X is thought to originate from the large  amounts of bile cholesterol and phosphatidylcholine present in the plasma due to the obstruction of the bile ducts (Miller, 1990; Sabesin, 1982). Unlike normal plasma lipoproteins, Lp-X is a bilayer vesicle and has a diameter of 30 to 70 nm (Miller, 1990; Sabesin, 1982; Seidel et al.,  1974).  Another unique feature of Lp-X is its high concentration of unesterified cholesterol (30% w/w) and phospholipid (60% w/w) (Miller, 1990; Sabesin, 1982; Seidel et al.,  1974).  Phosphatidylcholine is the  major  sphingomyelin comprises the minor fraction.  phospholipid in Lp-X while Cholesteryl esters (2% w/w),  triglycerides (2% w/w), and proteins (6% w/w) are the remaining constituents of this particle (Miller, 1990; Sabesin, 1982; Seidel et al.,  1974).  The major  constituents of the protein fraction are albumin (40%) and the C apolipoproteins (60%). Lp-X also contains small amounts of apo E (O and Frohlich, 1995; Miller, 1990; Sabesin, 1982; Seidel et al., 1974). The cholesteryl esters, triglycerides, and albumin form the core of Lp-X while the apolipoproteins, phospholipids, and cholesterol are on its surface. third  unique  feature  of  Lp-X is  electrophoresis (Sabesin, 1982).  its  cathodic  migration  on  A  agarose gel  Normal plasma lipoproteins have an anodic  migration. Unlike the other lipoproteins that are taken up by hepatocytes, Lp-X is mainly taken up by the reticuloendothelial system (Walli and Seidel, 1984).  11  1.4.3 Clinical symptoms in LCAT deficiency Clinical abnormalities in familial LCAT deficiency include corneal opacities, anemia, and proteinuria (Glomset et al., 1995).  1.4.3.1 Corneal opacities Corneal opacities are present in all patients with LCAT deficiency since early childhood. Corneal opacities consist of grayish dots in the corneal stroma, giving the cornea a cloudy or misty appearance. While the material making up the dots has yet to be determined, ultrastructural examination of sections obtained by superficial keratectomy revealed the presence of numerous vacuoles (Bron et al.,  1975; Bethell et al.,  membranous deposits.  1975).  Many of these vacuoles contained  Although the presence of excess lipids has not been  verified chemically or morphologically, crystals that may be cholesterol have been visualized in the cornea.  Examination of the cornea obtained from a patient  revealed excess unesterified cholesterol and phospholipid (Glomset era/., 1995).  1.4.3.2 Hemolytic anemia Hematological data and  bone  marrow  studies  have  indicated  that  moderate hemolysis in addition to low compensatory erythropoiesis leads to the anemia seen in LCAT deficient patients (Funke et al.,  1991).  Studies have  shown that the half-life of erythrocytes in patients is greatly reduced ( 1 6 - 1 7 days versus 23 - 35 days in normal subjects) (Glomset et al., 1995). Erythrocytes in LCAT deficiency have an abnormal appearance and lipid composition (Gjone et 12  al., 1968). Analyses of whole erythrocytes in some patients showed nearly twice the normal amount of unesterified cholesterol and phosphatidylcholine.  Such  abnormalities may contribute to hemolytic anemia. Patients had normal amounts of total phospholipid (Murayama et al.,  1984).  1.4.3.3 Proteinuria LCAT deficient patients frequently present with proteinuria.  Proteinuria is  usually detected early in life (20 - 30 years of age) and remains moderate (0.5 to 1.5 mg protein per milliliter urine) for many years (Gjone, 1974). proteinuria increases in severity as renal function deteriorates. may contain protein, hyaline casts and erythrocytes.  However,  Patients' urine  The level of albumin in  patients' serum decreases considerably with the onset of renal failure (Glomset et al., 1995). The  majority of  patients  develop  potentially life threatening complication.  progressive glomerulosclerosis, a  Light-microscopy of renal biopsies has  revealed the presence of foam cells in glomerular tufts of affected glomeruli (Stokke et al., 1974). Arterioles have thickened intimas and exhibit a narrowing of the lumens (Hovig and Gjone, 1973).  Deposits of lipid material have been  found in the renal arteries and arterioles.  Lipid accumulated in glomeruli  consisted mainly of unesterified cholesterol and phosphatidylcholine (Table 4). In Table 4 it can be seen that the cortex and glomeruli in the kidneys of some L C A T deficient patients have a higher ratio (0.7 and 0.9, respectively) of unesterified cholesterol to phosphatidylcholine relative to control (0.5 and 0.6, respectively).  13  Electron microscopy of renal biopsies revealed capillary lumens that were partly filled with a meshwork of membranes (Magil et al., 1982).  Capillary walls  appeared abnormal and endothelial cells were often missing. The basal lamina had an  irregular  thickness, endothelial foot  processes were  fused, and  membrane-surrounded particles were present in both the subendothelial and subepithelial regions.  Immunofluorescence has identified complement (C3)  deposits in the mesangium (Imbasciata et al., 1986).  1.4.3.4 Coronary artery disease and LCAT deficiency In the past an increased risk of coronary artery disease (CAD) was believed to be nonexistent in familial L C A T deficiency (Kuivenhoven et al., 1996; Glomset et al.,  1995).  While there were only a few cases of C A D in these  patients, calcification in the aorta had been demonstrated and postmortem examinations had revealed atherosclerosis of the aorta and large arteries (Gjone, 1974; Stokke era/., 1974). Renal arteries and arterioles changes.  had also shown early atherosclerotic  Histological examinations revealed fibrosis and hyalinization of renal  arterial walls accompanied by marked narrowing of the vessel lumens (Hovig and Gjone, 1973). Studies using electron microscopy of sections from renal and iliac arteries and from the aorta showed the presence of lipid-like deposits. These deposits were present in the different layers of the vessel walls. Foam cells were found in the lipid-like deposits and there was smooth muscle cell proliferation in  14  Table 4. Lipid Concentrations in the kidneys of LCAT deficient patients.  Concentration (nmol lipid/mg protein)  Kidneys  UC  PL  TG  UC/PL  Cortex, control Cortex, patient Glomeruli, control Glomeruli, patient  50 170 57 416  109 257 103 455  4 9 14 9  0.5 0.7 0.6 0.9  UC, unesterified cholesterol; PL, total phospholipid; T G , triglyceride. Adapted from Glomset et al., (1995).  15  the intimal layer (Glomset et al., 1995).  Lipid analysis of an atheroma from a  renal artery showed that only 35% of the total cholesterol was esterified, in contrast to 75% found in patients who died from other causes (Smith, 1965). Recently, a case has been reported where a 53 year-old man with partial L C A T deficiency (FED) presented with premature coronary artery disease (CAD) in the absence of other risk factors (Kuivenhoven et al., 1996). F E D patients lack ot-LCAT activity but still retain p-LCAT activity (Carlson and Holmquist, 1985). This finding  puts in doubt the belief that there  is no increased risk  atherosclerosis in LCAT deficiency and F E D (Kuivenhoven  et  al.,  of  1996).  Kuivenhoven et al. speculate that inactive or partially active LCAT increases the risk for atherosclerosis.  This would support the protective role of active L C A T  against C A D via its action in reverse cholesterol transport.  However, no  conclusive statement can be made regarding F E D or L C A T deficiency and the risk of C A D until more cases are identified.  1.5 GLOMERULOSCLEROSIS A major complication of L C A T deficiency is glomerulosclerosis, which can lead to renal failure.  The mechanisms by which L C A T deficiency leads to  glomerulosclerosis are still unclear.  Some researchers have suggested that  glomerulosclerosis and atherosclerosis may share a similar pathology (Diamond and Karnovsky, 1988). Arterial intima becomes thickened in atherosclerosis and contains vascular smooth muscle cells (SMC), macrophage foam cells, collagen fibers,  and  glycosaminoglycans,  eventually  16  leading  to  necrosis.  In  glomerulosclerosis  there  is  mesangial  matrix  expansion,  mesangial  cell  proliferation, macrophage infiltration, and the presence of foam cells (Diamond and Karnovsky, 1988).  1.5.1 Glomerular mesangial cells Glomerular  mesangial cells have several functions.  They  provide  structural support for capillary loops, modulate glomerular filtration by its smooth muscle cell activity,  and generate  vasoactive agents  (Schlondorff,  1987).  Mesangial cells are also able to synthesize and break down structural elements such as collagen, produce various growth factors and cytokines, endocytose macromolecules (including immune complexes and lipoproteins), and modulate glomerular injury through cell proliferation and production of extracellular matrix (Schlondorff, 1987). The analogy between glomerulosclerosis and atherosclerosis is largely based on the origin and functional properties of the glomerular mesangial cell. There are two distinct functional types of mesangial cells. 5 - 10% of mesangial cells are bone-marrow derived and are responsible for the phagocytosis of macromolecules entering the mesangium. The second type of mesangial cell is smooth muscle cell-like and shares many similarities with vascular smooth muscle cells (Schlondorff, 1987).  1.5.2 Macrophages The mechanisms by which macrophage infiltration and the presence of  17  foam cells may cause renal damage are as yet not fully understood. Glomerular macrophages are a locally activated mononuclear cell population which produces reactive oxygen species that may contribute to macrophage-mediated glomerular injury (Van Goor et al., 1994). Macrophages are a rich source of cytokines that modulate mesangial cell proliferation and extracellular matrix expansion (Van Goor et al., 1994). Renal biopsies of LCAT deficient patients revealed macrophage infiltration and the presence of foam cells in affected glomeruli.  Foam cells have an  important pathogenic role in atherosclerosis. If glomerulosclerosis is analogous to atherosclerosis, then foam cells may also have an important role in the development of glomerulosclerosis in these patients.  1.5.3 Lipid accumulation Observations in experimental animals and in patients with genetically determined and acquired hyperlipidemias suggest that lipids can damage the kidney and hence lead to glomerulosclerosis (Walli et al., 1993).  There are  strong indications that lipoproteins play a vital role in mesangial cell damage and in the development of progressive renal disease. It has been reported that lipid accumulation in perfused rat kidney was caused by the direct deposition of lipoprotein-X (O et al., 1997). Mesangial cells are able to take up LDL and apo Econtaining lipoproteins (VLDL) through receptor-mediated pathways. It has been suggested that rat mesangial cells possess both LDL receptor and scavenger receptor (Coritsidis et al., 1991).  18  1.5.4 Extracellular matrix expansion In the normal kidney, collagens IV, V, and laminin have been localized to the basement membrane of the glomerular tuft, Bowman's capsule, and in the mesangium (Striker et al., 1984). Fibronectin has been localized to the basement membrane region and the mesangium.  Striker et al. (1984) studied the  composition of the extracellular matrix in glomerulosclerosis.  In this study they  found that the glomerular basement membranes and mesangial areas within glomeruli principally consisted of collagen IV. scleroses, multi-lamination  They also found  significant  of Bowman's capsule basement membranes, and  periglomerular fibrosis. The sclerotic mass consisted of interstitial collagen (type III), as opposed to type IV. Glomerulosclerosis in LCAT deficiency is histologically characterized by the  deposition  of  lipids,  especially  unesterified  cholesterol  and  phosphatidylcholine, in the glomerular membrane. It is also characterized by the expansion of extracellular matrix, infiltration of macrophages, the presence of foam cells, and mesangial cell proliferation (Diamond and Karnovsky, 1988).  1.6 RATIONALE OF THE STUDY Patients with familial  LCAT deficiency experience corneal opacities,  anemia and proteinuria (Glomset et al., 1995; Frohlich and Pritchard, 1992). Proteinuria progresses dramatically as renal function deteriorates. patients develop glomerulosclerosis.  Most of these  The pathogenesis of glomerulosclerosis  19  may involve multiple factors. proliferation  The deposition of lipids, foam cell formation,  of mesangial cells,  and extracellular  matrix  expansion  could  contribute to the development of glomerulosclerosis.  1.7 HYPOTHESES AND OBJECTIVE Lipoprotein-X phosphatidylcholine.  has  high  levels  of  unesterified  cholesterol  and  These two lipids are also elevated in affected glomeruli.  Hence, Lp-X may play a role in the accumulation of lipids in the kidneys of these patients. The objective of this project is to examine the uptake and metabolism of lipoprotein-X in mesangial cells and to study its effect on mesangial cell proliferation.  1.8 SPECIFIC AIMS • To examine the uptake of Lp-X by mesangial cells. • To examine the metabolic fate of Lp-X lipids. • To determine whether Lp-X is able to induce foam cell formation. • To examine the  effect  of  Lp-X and  proliferation.  20  macrophages on  mesangial cell  2. MATERIALS and METHODS 2.1 TISSUE CULTURE 2.1.1 Isolation and culture of rat mesangial cells Mesangial cells were isolated from rat kidney glomeruli by a differential sieving technique and collagenase digestion (Harper et al.,  1984).  Dawley rats (350 +/- 25 g) were anesthetized with halothane.  The kidneys were  then removed and glomeruli isolated as follows.  Sprague-  First, any adhering tissue was  removed from the kidney, followed by the removal of the cortices from the medulla.  The cortical portion was minced into millimeter square pieces and  passed through a series of stainless steel sieves with decreasing pore sizes (200 u-pore, 150 u-pore and 75 u-pore).  The resulting glomeruli remained on top of  the 75 u-pore sieve and were stripped of their capsules as well as being 100% free of tubular tissue (Kreisberg et al.,  1978).  Isolated glomeruli were then  resuspended with 10 mL Hank's Buffered Saline Solution (HBSS) containing 1% antibiotic-antimycotic and washed twice. Glomeruli were then incubated in 10 mL 0.2% trypsin-1% antibiotic-antimycotic in H B S S at 37°C. After twenty minutes the glomeruli were centrifuged at 1,000 rpm for one minute.  The trypsin was then  removed and the glomeruli resuspended in 0.1% collagenase-1% antibioticantimycotic in H B S S and incubated at 37°C for 40 minutes. The glomeruli were then washed and cultured in 10 mL RPMI-1640 (containing 20% heat-inactivated fetal bovine serum (FBS), 1% antibiotic-antimycotic, 5 pg/mL bovine insulin, 5 pg/mL human transferrin, and 5 ng/mL sodium selenite) at 37°C.  Media was  changed every two to three days. For experiments only cells between the fifth 21  and eleventh passages were used. The homogeneity of the cell culture was confirmed by immunofluorescence staining (Harper et al., 1984), which was kindly performed by Dr. Alexander Magil, and by the addition of the epithelial cytotoxin puromycin to the culture medium. Immunofluorescent staining was negative for cytokeratin (rat leukocyte antigen) and positive for vimentin and smooth muscle cell specific actin. Unlike epithelial cells, mesangial cells are resistant to puromycin (100 u.g/mL). comprised > 99% of the cell culture.  Mesangial cells  Cell viability was greater than 95% when  tested with trypan blue.  2.1.2 Isolation and culture of rat peritoneal macrophages RPMI-1640 medium, containing 10% F B S and 1% antibiotic-antimycotic, was injected (15 mL) into the peritoneal cavity of Sprague-Dawley rats (350 ± 25 g) after cervical dislocation. The abdomen was massaged for five minutes and the medium removed using a hypodermic needle.  The medium was then  centrifuged at 1,000 rpm for 10 minutes to pellet the cells. After centrifugation, the cells were resuspended and the cell density was determined using light microscopy and a hemocytometer. Macrophages were plated at a density of 1 - 6 x10 cells/35mm dish and incubated at 37°C for 2 h. 6  Within the first 2 h of  incubation macrophages are able to attach to the culture dish.  Washing and  changing the medium after 2 h removed any other cell types that may have been present  (Adams,  1979).  Fresh  culture  medium  macrophages were used immediately for experiments. 22  was  then  added  and  Macrophage-conditioned  medium  was  prepared  by  incubating  rat  peritoneal macrophages in the presence or absence of reconstituted Lp-X (rLp-X) for 24 h at 37°C. The macrophage-conditioned medium was collected and used immediately for experiments.  2.2 Preparation of plasma lipoproteins 2.2.1 Isolation of Lipoprotein-X All procedures and methods were approved by the University Ethical Review Committee. There is no difference  between the composition and properties  of  lipoprotein-X isolated from familial LCAT deficiency and cholestatic liver disease (Seidel et al., 1974). Since cholestatic liver disease is more common than familial LCAT deficiency, sera were collected from patients with cholestatic liver disease (O and Frohlich, 1995).  Lp-X positive sera were identified by their cathodic  migration on 1% agarose gel (Figure 2). Lp-X was prepared by ultracentrifugation and column chromatography.  In  brief, a fraction with a density of 1.019 - 1.063 g/mL was prepared by sequential ultracentrifugation (Sabesin, 1982; Miller, 1990). The Lp-X present in the 1.019 < d < 1.063 g/mL fraction was separated from LDL by gel filtration chromatography, using a matrix of Superose 6B beads (Pharmacia).  Lp-X was eluted off the  column as a distinct peak using a buffer of 0.15 M NaCl / 10 mM Tris-HCl, pH 7.4. The peak was identified by measuring absorbency at 280 nm. The purity of Lp-X  was  determined  by  the  mobility  23  of  the  isolated  Lp-X  1  2  3  HDL-+VLDL->*  LDL->*  Figure 2. Lipoprotein electrophoresis. Samples were loaded on a 1% agarose gel and electrophoresed for 35 minutes. The lipoproteins were stained with Sudan Black. The arrows indicate the position of individual lipoproteins. Lane 1, sera from controls; Lane 2, pooled sera from obstructive jaundice patients; Lane 3, LDL isolated from normal serum (lane 3). From O and Frohlich, 1995.  24  on 1% agarose gel, as well as by the ratio ( >95%) of total cholesterol to unesterified cholesterol. Free and total cholesterol were measured enzymatically using commercially available kits (Boehringer-Mannheim).  2.2.2 Preparation of radiolabeled Lp-X For experiments  requiring  radiolabeling,  3  Lp-X was equilibrated  with  •>  [ H]cholesterol- (0.05 pCi/nmol) or dipalmitoyl phosphatidyl-[Me- Hjcholine (0.05 uCi/nmol)-coated filter disks at 4°C overnight.  This method has been used  successfully to label Lp-X and other lipoproteins by our laboratory as well as other investigators (O and Frohlich, 1995; O et al., 1993; Dobiasova and Schutzova, 1986). Various concentrations of labeled Lp-X were added directly to the culture medium.  2.3 PREPARATION OF RECONSTITUTED LIPOPROTEIN-X (rLp-X) 2.3.1 Preparation of rLp-X rLp-X consisted of a 1:1 molar ratio of phosphatidylcholine (PC) and cholesterol, which is similar to the ratio found in Lp-X. To prepare rLp-X the lipids were dissolved and mixed in chloroform and then dried under nitrogen gas. Dried lipids were resuspended with 250 pL absolute ethanol and rapidly injected into 30 mL H B S S while vortexing.  The preparation was concentrated to 4 mL with an  Amicon stirred ultrafiltration cell using a YM-100 membrane. The free cholesterol content of the rLp-X preparation was determined by commercially available enzymatic kits. 25  2.3.2 Preparation of radiolabeled rLp-X 3  rLp-X was  labeled with [ H]cholesterol  (0.05  u/Ji/nmol),  dipalmitoyl  phosphatidyl-[Me- H]choline (0.05 ^Ci/nmol), or [ H]cholesteryl hexadecyl ether 3  (0.3  u/Ji/nmol).  3  Radiolabeled rLp-X are  ([ H]cholesterol-labeled 3  3  rLp-X), [ H]PC 3  referred  to  as  rLp-X (dipalmitoyl  [ H]FC  rLp-X  3  phosphatidyl-[Me-  H]choline-labeled rLp-X) and [ H]CHE rLpX ([ H]cholesteryl hexadecyl ether3  3  labeled rLp-X), respectively.  2.4 UPTAKE AND METABOLISM OF LIPOPROTEIN-X (LP-X) BY MESANGIAL CELLS 2.4.1 Uptake of Lp-X and rLp-X by mesangial cells Mesangial cells were rendered quiescent by overnight preincubation in serum-free RPMI-1640. Cells were pulse-labeled with either [ H]FC- or [ H]PC3  3  labeled Lp-X or rLp-X. After incubation the cells were washed with phosphate buffered saline (PBS) containing 1% albumin, followed by two washes with P B S . The cells were lysed in 1 mL 0.2 N NaOH and then neutralized with glacial acetic acid.  The uptake of Lp-X was determined by measuring the  radioactivity  associated with cells.  2.4.2 Metabolism of lipids by mesangial cells The metabolism of the cholesterol and phosphatidylcholine in Lp-X in mesangial cells was also measured. Quiescent mesangial cells were incubated with [ H]cholesterol-labeled Lp-X (0.05 nCi/nmol) or dipalmitoyl phosphatidyl-[Me3  26  3  H]choline-labeled Lp-X (0.05 pCi/nmol). After incubation, aliquots of cell lysates  were used for lipid extraction.  Cellular lipids were extracted by a solvent  containing chloroform:methanol:water (4:2:3, v/v/v) ( 0 et al., 1993; Wheeler et al.,  1990), followed by centrifugation for ten minutes at 175 x g.  After  centrifugation the organic phase (lower layer) was carefully removed and then dried under nitrogen gas. The dried lipids were resuspended in chloroform.  Free cholesterol and  cholesteryl ester, or phosphatidylcholine and lysophosphatidylcholine were separated by thin layer chromatography (O et al., 1997). Radioactivity associated with individual lipids was determined using liquid scintillation counting.  2.4.3 Effects of cytochalasin, suramin, and polyinosinic acid on the uptake of rLp-X Quiescent cells were pretreated with cytochalasin D and polyinosinic acid overnight and with suramin for two hours.  After pretreatment,  media was  removed and fresh media with the corresponding agents (cytochalasin D, polyinosinic acid, suramin) were added in addition to radiolabeled Lp-X.  After  incubation, the medium was removed and the cells washed with 2 mL P B S containing 1% B S A followed by two more washes with 2 mL P B S . The cells were lysed in 1 mL 0.2 N NaOH. The lysate was then neutralized with 10.5 pL glacial acetic acid.  Radioactivity associated with cells was measured by liquid  scintillation counting.  27  2.4.4 Effect of Lp-X and rLp-X on mesangial cell proliferation 3  The incorporation of  [ HJthymidine into cellular DNA was used to  determine the effect of lipoprotein-X on mesangial cell proliferation (Gupta et al., 1992).  Quiescent mesangial cells were pulse-labeled with [ H]thymidine (1 3  juCi/mL) at 37°C in the presence or absence of Lp-X or rLp-X for different time periods. After incubation, the medium was removed and the cells washed three times with 2 mL P B S . The cells were lysed in 1 mL 0.2 N NaOH. The lysate was then neutralized with glacial acetic acid. The radioactivity associated with cellular DNA was measured by liquid scintillation counting.  2.5 EFFECT OF RECONSTITUTED LIPOPROTEIN-X ON MACROPHAGES 2.5.1 Lipid analyses Rat peritoneal macrophages were incubated in the presence or absence of rLp-X for 24 h at 37°C. After incubation, the medium was removed and the cells were washed three times with 2 mL P B S . The cells were then lysed in 1 mL 0.2 N NaOH.  After neutralization with acetic acid, cellular lipids were extracted as  described above.  Total cholesterol and free cholesterol were assayed using  enzymatic kits.  2.5.2 Oil Red O (ORO) staining Macrophages cultured on cover-slips were stained with Oil Red O (ORO) after 24 h incubation in the presence or absence of rLp-X (Luna, 1968). Coverslips were first fixed in 10% neutral formalin for 15 minutes. 28  They were then  rinsed in distilled water and left in 100% propylene glycol for 2 minutes followed by staining in O R O for 10 minutes.  Macrophages were then treated in 60%  propylene glycol for 1 minute and rinsed in distilled water.  The macrophages  were examined using oil immersion and light microscopy. Macrophages with 10 or more lipid droplets were identified as foam cells.  2.6 EFFECT OF RECONSTITUTED LIPOPROTEIN-X AND MACROPHAGES ON MESANGIAL CELL PROLIFERATION The combined effect of rLp-X and macrophages on mesangial cell proliferation was also determined by measuring [ H]thymidine incorporation (1 3  uGi/mL).  Quiescent mesangial cells were cultured in macrophage-conditioned  medium in the presence or absence of rLp-X (100 - 400 nmol FC/mL).  After  incubation the medium was removed and the cells washed three times with 2 mL P B S . The cells were then lysed in 1 mL 0.2 N NaOH. After neutralization with acetic acid, the radioactivity associated with the cellular DNA was determined by liquid scintillation counting.  29  3. RESULTS 3.1 UPTAKE AND METABOLISM OF LIPOPROTEIN-X BY MESANGIAL CELLS 3.1.1 Uptake of Lp-X The uptake of Lp-X by rat mesangial cells was studied by incubating cells with either [ H]FC- or [ H]PC- labeled Lp-X for 10 hours. Cells were washed once 3  3  with P B S containing 1% B S A followed by two additional washes with P B S before measuring the radioactivity associated with cells. A s seen in Figure 3, uptake of labeled Lp-X was in a concentration dependent manner.  The uptake of  radiolabeled Lp-X appeared to reach a plateau at 75 - 125 nmol cholesterol/mL Lp-X.  3.1.2 Metabolism of Lp-X lipids The metabolic fate of the lipids in Lp-X was examined in Figure 4. Mesangial cells were concentrations  of  65  incubated with [ H]FC3  nmol  Lp-X  or  cholesterol/mL  [ H]PC-labeled Lp-X at 3  and  110  nmol  Lp-X  cholesterol/mL. It is evident from these experiments that the cholesterol in Lp-X is esterified by mesangial cells.  In addition, phosphatidylcholine in Lp-X was  metabolized to lysophosphatidylcholine.  When treated with high levels (110  nmol/mL) of Lp-X, the cells showed a significant increase in their uptake of unesterified  cholesterol  and  phosphatidylcholine,  concentration (65 nmol/mL) of Lp-X.  30  relative  to  the  This increased uptake most  lower likely  Figure 3. The uptake of L p - X by rat mesangial cells. Mesangial cells were pulse-labeled with various amounts of [ H]cholesterol or dipalmitoyl phosphatidyl[Me- H]choline labeled Lp-X. After 10 h incubation the radioactivity associated with cells was determined as described in Materials and Methods. Preliminary experiments have shown that 10 h incubation is within the linear range of Lp-X uptake (data not shown). Results are expressed as the mean ± standard deviation from five separate experiments. 3  3  31  20 C  1  r  8  Lp-X (nmol cholesterol/mL) Figure 4. The metabolism of L p - X by rat mesangial cells. Mesangial cells were incubated with [ H]cholesterol labeled Lp-X (65 nmol/mL or 110 nmol cholesterol/mL). After 10 h cellular lipids were extracted and the radioactivity associated with the unesterified (free) cholesterol (FC) and cholesteryl ester (CE) was determined. In separate experiments, cells were incubated with phosphatidyl-[Me- H]choline labeled Lp-X (65 nmol cholesterol/mL or 110 nmol cholesterol/mL). After 10 h, the radioactivity associated with phosphatidylcholine and lysophosphatidylcholine was determined. Results obtained from five separate experiments incubated with 65 nmol/mL Lp-X are expressed as 100 percentile and depicted as mean ± standard deviation. 3  3  32  contributed  to  the  significant  increase  in  cholesteryl  ester  and  lysophosphatidylcholine.  3.1.3 Uptake and metabolism of rLp-X 3.1.3.1 Uptake of rLp-X In order to demonstrate that Lp-X lipids were indeed entering the cell (and not just simply exchanging with plasma membrane lipids) rLp-X labeled with [ H]cholesteryl hexadecyl ether (CHE) was prepared. [ H]CHE has been shown 3  3  to remain inside liposomes and does not readily exchange with membrane lipids (Bally et al., 1990). Additionally, C H E is not subject to esterase activity once it is internalized by the cell (Bally et al., 1990).  Figure 5 (Panel A) shows that the  uptake of [ H]CHE rLp-X was time dependent. 3  Uptake of [ H]CHE rLp-X (100 3  nmol cholesterol/mL) began to level off after 20 h incubation. A s shown in Figure 5 (Panel B), the uptake of [ H]CHE rLp-X by mesangial cells was concentration 3  dependent.  These results suggest that the uptake of Lp-X lipids was not  facilitated by a simple exchange mechanism. The ability of mesangial cells to take up and metabolize lipids in rLp-X was compared to the uptake and metabolism of Lp-X lipids.  In Figure 6 cells were  treated with rLp-X, labeled with either [ H]FC or [ H]PC, and their uptake by 3  3  mesangial cells was examined. Like Lp-X (Figure 3), the uptake of labeled rLp-X was concentration dependent. However, at the concentrations tested the uptake of rLp-X does not appear to be saturable (Figure 6).  33  5000  0  1  0  1  1  0  10  1  L  20 Time (h)  1  -  30  1  1  50 100 rLp-X (nmol cholesterol/mL)  150  Figure 5. The uptake of r^Hjcholesteryl hexadecyl ether (CHE) rLp-X by rat mesangial cells. Cells were incubated with [ H]CHE rLp-X for various time periods (100 nmol cholesterol/mL) (Panel A) or various concentrations (Panel B). After incubation, the radioactivity associated with the cells was determined. Results are expressed as D P M per mg of cellular proteins and are depicted as the as mean ± standard deviation from five separate experiments. 3  34  FC-labeled rLp-X PC-labeled rLp-X  0  25  50  75  100  125  rLp-X (nmol cholesterol/mL)  Figure 6. The uptake of [ H]FC or r'rfJPC rLp-X by rat mesangial cells. Mesangial cells were incubated with various amounts of [ H]cholesterol or dipalmitoyl phosphatidyl-[Me- H]choline rLp-X. After 10 h, the radioactivity associated with the cells was determined. The uptake of rLp-X is expressed as D P M per mg of cellular proteins. Each point represents the mean ± standard deviation from five separate experiments. 3  3  3  35  3.1.3.2 Metabolism of rLp-X lipids Similar  to  results  phosphatidylcholine  in  found  in  rLp-X were  Figure  metabolized  3, to  the  cholesterol  cholesteryl  ester  and and  lysophosphatidylcholine, respectively (Figure 7). Cells treated with 120 nmol/mL rLp-X showed a significant increase in the uptake of unesterified cholesterol and phosphatidylcholine relative to cells treated with 70 nmol/mL rLp-X. In turn, the higher levels of cholesterol and phosphatidylcholine most likely led to the significant increases in cholesteryl ester (10 nmol cholesterol/mg protein) and lysophosphatidylcholine (11 nmol cholesterol/mg protein) (Figure 7).  3.2 THE EFFECT OF APOLIPOPROTEINS ON THE UPTAKE OF LIPOPROTEIN-X The role of apolipoproteins in Lp-X uptake was examined as shown in Figure 8. [ H]CHE rLp-X was incubated with mesangial cells in the presence or 3  absence of individual apolipoproteins or with all four apolipoproteins combined. From Figure 8 it can be seen that both apolipoproteins C-l and C-l 11 have a significant inhibitory effect.  In contrast, apo C-ll (1.5 fold) and E (4 fold)  significantly stimulated the uptake of [ H]CHE rLp-X. In the presence of all four 3  apolipoproteins the uptake of [ H]CHE rLp-X by mesangial cells was enhanced 3  1.5 fold. Since  the  size  of  rLp-X  may  be  affected  by  the  presence  of  apolipoproteins, and in turn affect the rate of uptake, the diameter of each rLp-X preparation  was  measured  by  quasi-elastic  36  light  scattering  analysis  10  —•— FC CE - - * -- P C — A — LPC  0  25 50 75 100 rLp-X (nmol cholesterol/mL)  125  Figure 7. Metabolism of rLp-X by rat mesangial cells. Mesangial cells were incubated with [ H]FC-rLp-X (70 nmol cholesterol/mL or 120 nmol cholesterol/mL). After 10 h the cellular lipids were extracted and the radioactivity associated with unesterified (free) cholesterol (FC) and cholesteryl ester (CE) was determined. In separate experiments, cells were incubated with phosphatidyl-[Me- H]choline rLp-X (70 nmol cholesterol/mL or 120 nmol cholesterol/mL) for 10 h. The radioactivity associated with phosphatidylcholine and lysophosphatidylcholine was determined. Results obtained from five separate experiments incubated with 70 nmol/mL liposomes are expressed as 100 percentile and depicted as mean ± standard deviation. 3  3  37  500 450 400 2 c o o o  350 300 250 200 150 100 50 -  o L Control  ApoCI  ApoCII  ApoCI II  ApoE  Mix  Figure 8. The effect of human apolipoproteins on the uptake of Lp-X by rat mesangial cells. A n aliquot of [ H]CHE rLp-X (100 nmol/mL) was mixed with individual or all apolipoproteins. The mixture (Mix) contained 100 nmol cholesterol/mL and 4-10 pg apolipoprotein/mL. This was then incubated with the mesangial cells. After 10 h the radioactivity associated with the cells was measured. Cells incubated without apolipoproteins were used as control. The results are expressed as a percentage of control and each point represents the mean ± standard deviation from five separate experiments. By student's t test * p < 0.01, where n = 5.  38  (Mayer et al.,  1986).  A s seen in Table 5, the presence or absence of  apolipoprotein(s) had no significant effect on particle size.  3.3 THE EFFECT OF SURAMIN, CYTOCHALASIN, AND POLYINOSINIC ACID ON THE UPTAKE OF RECONSTITUTED LIPOPROTEIN-X Cell surface heparan sulfate proteoglycans play a role in the uptake of normal plasma lipoproteins (Hurt-Camejo et al., 1990; Vijayagopal et al., 1992; Ismail et al., 1994). Hence, the involvement of heparan sulfate proteoglycans in the uptake of Lp-X by mesangial cells was examined. Suramin is an agent that blocks the interaction between proteoglycans and lipoprotein ligands (Ismail et al., 1994). Cells treated with suramin (0.5 and 1.0 mg/mL) showed a significant 15 to 20% reduction in the uptake of rLp-X, as shown in Table 6. This result suggests that a portion of Lp-X uptake involves interactions with cell surface proteoglycans. Cytochalasin D is an agent that blocks cytoskeleton dependent uptake (Ismail et al.,  1994; Ting et al.,  1995).  In order to investigate the role of  phagocytosis in Lp-X uptake, mesangial cells were treated with cytochalasin D. A s seen in Table 6, this compound had no significant effect on the uptake of rLpX. By treating cells with polyinosinic acid, an inhibitor of lipoprotein uptake in smooth muscle cells (Ismail et al., 1994), the role of scavenger receptor in the uptake of rLp-X was investigated. Polyinosinic acid had no effect on the uptake  39  Table 5. Effect of apolipoproteins on the size of rLp-X.  Mean diameter (nm)  rLp-X rLp-X rLp-X rLp-X rLp-X rLp-X  + + + + +  apo apo apo apo apo  103 104 105 102 104 105  C-l C-l I C-lll E C-l, C-ll, C-lll, E  The size distribution of [ H]cholesterol (FC), [ H]phosphatidylcholine (PC) or [ H]cholesteryl hexadecyl ether (CHE) rLp-X was determined using quasi-elastic light scattering in the absence or presence of apolipoproteins (10-15 ug/mL). The amounts of apolipoproteins were chosen to match the concentrations normally associated with Lp-X (Seidel et al., 1974). The mean diameter of particles was obtained using Gaussian analysis from two separate experiments. 3  3  3  40  Table 6. Effects of suramin, cytochalasin and polyinosinic acid on the uptake of reconstituted Lp-X.  Total Uptake (% of control)  Control + suramin 0.5 mg/mL + suramin 1 mg/mL + cytochalasin D 15 pg/mL + cytochalasin D 30 pg/mL + polyinosinic acid 25 pg/mL + polyinosinic acid 50 pg/mL  100 85 ± 3 * 80 ± 10* 122 ± 2 7 104 ± 8 103 ± 2 0 102 ± 5  Mesangial cells were preincubated with suramin (2 h), cytochalasin D (18 h), or polyinosinic acid (18 h) in RPMI-1640 medium. [ H]CHE labeled rLp-X (100 nmol cholesterol/mL) was added to the culture medium and cells were incubated at 37 °C for 10 hours. The amounts of suramin, cytochalasin D, and polyinosinic acid used above were previously shown to be effective by other investigators (Ismail et al., 1994; Ting et al., 1995). The radioactivity associated with the cells was determined by liquid scintillation counting. The results are expressed as mean ± standard deviation from three separate experiments. The * P < 0.05 when compared with control. 3  41  of rLp-X by mesangial cells (Table 6), indicating that scavenger receptor is not involved in the uptake of Lp-X.  3.4 EFFECT OF LIPOPROTEIN-X ON MACROPHAGES 3.4.1 Lipid accumulation in macrophages Macrophages treated with rLp-X (177 - 387 nmol FC/mL) showed a significant increase in both cellular total cholesterol (162%) and cholesteryl ester (223% to 245%) relative to control.  There was a slight, but not significant,  increase in free cholesterol (117%) relative to control, as shown in Figure 9.  3.4.2 Foam cell formation Macrophages cultured on cover-slips, treated with 120 nmol FC/mL rLp-X, stained with O R O revealed the presence of foam cells, which comprised 10% of the macrophage population.  Foam cells could not be detected in macrophages  not treated with rLp-X. Figure 10 shows the induction of foam cell formation by rLp-X.  Foam cells were identified as being any macrophage containing 10 or  more lipid droplets. Figure 10 was magnified 100 X under light microscopy using oil immersion.  42  2 200  H Control  Q_ O)  E 150 2 03 •*-» 100 CO  I1177 nmol FC/mL  0)  o o o  P  • 387 nmol FC/mL  50  „ TC  FC  CE  Figure 9. Effect of rLp-X on cellular lipids in rat peritoneal macrophages. Macrophages were plated at a density of 2 x 10 cells/35 mm dish and incubated for 24 h at 37°C in the presence or absence of rLp-X (177 - 387 nmol FC/mL). The results are from five separate experiments and are expressed as percentage of control and depicted as mean ± standard deviation. By student's t test, * p < 0.05 and ** p < 0.005, where n = 5. 6  43  Figure 10. Induction of foam cell formation by Lp-X. (200 X magnification) Rat peritoneal macrophages were incubated with 120 nmol cholesterol/mL rLp-X for 24 h and then stained with Oil Red 0 and examined under light microscopy using oil immersion. The long, thick arrow indicates a foam cell while the short,, thin arrow indicates a macrophage cell identical to those seen in control treatments.  44  3.5 MESANGIAL CELL PROLIFERATION 3.5.1 Effect of Lp-X and rLp-X The effect of Lp-X on mesangial cell proliferation was examined by measuring the incorporation of [ H]thymidine into cellular DNA. 3  Lp-X at low  concentrations had no effect on the incorporation of radioactivity, while at higher concentrations Lp-X had a slight inhibitory, but insignificant, effect. These results are shown in Figure 11. In order to examine the effect of the lipids in Lp-X on cell proliferation, cells were treated with rLp-X.  In this study, rLp-X had no significant effect on the  incorporation of [ H]thymidine into cellular DNA (Figure 11). 3  These results  indicate that Lp-X and rLp-X, at the concentrations tested (5 - 200 nmol/mL), do not affect mesangial cell proliferation.  3.5.2 Combined effect of rLp-X and macrophages Mesangial cells treated with macrophage-conditioned medium in the presence or absence of rLp-X showed no change in [ H]thymidine incorporation 3  relative to control (Figure 12).  45  250  0  5  10 25 50 nmol cholesterol/mL  100  200  Figure 11. The effect of Lp-X and rLp-X on the incorporation of [ H]thymidine into rat mesangial cells. Mesangial cells were incubated with [ HJthymidine in the presence or absence of Lp-X or liposomes. After 10 h the radioactivity associated with the cells was determined. No serum was present in the culture. Results are expressed as a percentage of control and each point represents the mean ± standard deviation from five separate experiments. 3  46  250 .5 o  200  k  150 100  o o o  50 0 L Control  100  200 nmol/mL  nmol/mL  400 nmol/mL  Figure 12. C o m b i n e d effect of macrophage-conditioned medium and rLp-X o n [ H]thymidine incorporation by rat mesangial cells. Mesangial cells were incubated in macrophage-conditioned medium in the presence of rLp-X (100 400 nmol FC/mL) and pulsed with [ H]thymidine for 24 h. After incubation the radioactivity associated with the cells was measured. No serum was present in the culture. The results are expressed as percentage of control and depicted as mean ± standard deviation from four separate experiments. 3  3  47  4. DISCUSSION One of the major clinical features of familial L C A T deficiency is progressive glomerulosclerosis (Magil et al.,  1982; O and Frohlich, 1995). A typical feature of  this disorder is the presence of an abnormal plasma lipoprotein, Lp-X. Although the  pathogenesis of glomerulosclerosis may  involve multiple  factors,  the  increased amounts of phosphatidylcholine and unesterified cholesterol seen in affected glomeruli may be due to the accumulation of Lp-X in the kidney. Lp-X has unusually high levels of these two lipids. Renal biopsies of L C A T deficient patients have shown mesangial cell proliferation and the presence of foam cells in addition to the accumulation of lipids in the mesangial region.  It has recently  been reported that lipid accumulation in perfused rat kidney was caused by a direct deposition of Lp-X (O et al., 1997).  The objective of this project was to  examine the uptake and metabolism of Lp-X in mesangial cells as well as its effect on mesangial cell function.  4.1 UPTAKE AND METABOLISM OF LIPOPROTEIN-X IN MESANGIAL CELLS One of the key events in the development of glomerulosclerosis is the accumulation of lipids in the kidney (Striker and Striker, 1985; Diamond and Karnovsky,  1988).  The  results  obtained  from  this  study  have  clearly  demonstrated that Lp-X is taken up in a concentration dependent manner by rat mesangial cells (Figure 3). (Figure 4).  Furthermore, the lipids in Lp-X are metabolized  The uptake of Lp-X by mesangial cells may contribute to the lipid  48  accumulation  in the  kidneys of  LCAT  deficient  patients,  leading to  the  development of glomerulosclerosis.  4.1.1 Role of apolipoproteins in Lp-X uptake The apolipoproteins C-l, C-ll, C-lll, and E are associated with Lp-X. Reconstituted  Lp-X was  prepared  in  order  to  examine  the  role  these  apolipoproteins play in the uptake of Lp-X by mesangial cells. rLp-X contains a similar lipid content to Lp-X. Apolipoproteins  E and  C-ll stimulated  the  uptake  of  apolipoproteins C-l and C-lll inhibited this process (Figure 8).  rLp-X  while  These results  suggest that the apolipoproteins (C-l, C-ll, C-lll, and E) associated with Lp-X may play a significant role in modulating its uptake.  The significant effect of  apolipoproteins on the uptake of Lp-X also suggests that the mechanism of Lp-X uptake may involve one of the known lipoprotein receptors.  4.1.2 The role of scavenger receptor, phagocytosis, and proteoglycans in the uptake of Lp-X Additional experiments were performed to determine whether scavenger receptor or cytoskeleton-dependent phagocytosis are involved in the uptake of Lp-X by mesangial cells. The results indicated that scavenger receptor and nonreceptor mediated phagocytosis do not play a role in Lp-X uptake (Table 6). Neither polyinosinic acid, which inhibits scavenger receptor (Coritsidis et  al.,  1991),  for  nor  cytochalasin  D,  which  disrupts  49  the  machinery  required  phagocytosis, affected the uptake of Lp-X. Furthermore, the role of cell surface heparan sulfate proteoglycans in the uptake of Lp-X by mesangial cells was examined. Heparan sulfate proteoglycans have been shown to be involved in the uptake of normal plasma lipoproteins (Hurt-Camejo et al., 1990; Vijayagopal et al., 1992; Ismail et al., 1994). treated with suramin reduced Lp-X uptake by 15 to 20% (Table 6).  Cells  Suramin  competes with positively charged particles for sites on the negatively charged heparan sulfate  proteoglycans.  These results  indicate  that cell surface  proteoglycans may play a role in the uptake of Lp-X by mesangial cells. Abnormalities in the function of scavenger receptor, phagocytosis, or cell surface proteoglycans could lead to intracellular lipid accumulation. This would in turn lead to the accumulation of lipids in the kidney and possibly contribute to the development of glomerulosclerosis. Whether the uptake of Lp-X by mesangial cells  is  enhanced  or  attenuated  by  other  putative  participants  (monocyte/macrophages, extracellular matrix expansion, and various cytokines) in the development of glomerulosclerosis still remains to be clarified.  4.1.3 Possible mechanisms for the uptake of Lp-X Rat mesangial cells have LDL receptor on its surface, which is responsible for the uptake of LDL and V L D L (Coritsidis et al., 1991; Schlondorff, 1993). The LDL receptor recognizes its lipoprotein ligands by virtue of their associated apolipoprotein B or E (Havel and Kane, 1995; Coritsidis et al., 1991; Schlondorff, 1993).  Apolipoprotein  E is associated with Lp-X (Sabesin,  50  1982).  The  stimulatory effect of apolipoprotein E on the uptake of Lp-X (Figure 8) suggests that LDL receptor may be involved in this process.  If we are able to obtain  sufficient Lp-X samples then competition studies will be done in order to clarify the role of LDL-R in Lp-X uptake. Alternatively, other receptors that recognize apolipoprotein E could also be possible routes for Lp-X uptake.  An example of a receptor other than LDL  receptor that recognizes apo E as a ligand is LRP. L R P is thought to bind apo E (Kowal et al., 1989). Besides its role in lipoprotein metabolism, lipoprotein lipase (LPL) may also help in the association of lipoproteins with cell surfaces (Rutledge et al., 1997). LPL, possibly in conjunction with cell surface proteoglycans, could be involved in the uptake of Lp-X by mesangial cells. Complement components are sometimes present in glomerular injury (Adler et al., 1984). Complement may play a role in Lp-X uptake by an as yet unknown mechanism.  However, Lp-X  may just simply fuse with the cell plasma membrane. Clearly, further studies are needed to examine and identify the mechanism(s) by which Lp-X is taken up into mesangial cells.  4.2 EFFECT OF RECONSTITUTED LIPOPROTEIN-X ON MACROPHAGES Infiltration of macrophages and the formation of foam cells have been found in the affected glomeruli of patients with L C A T deficiency.  In vivo the  uptake of Lp-X by infiltrated macrophages could lead to foam cell formation. Foam cells are a rich source of cytokines and growth factors. One of the growth factors secreted by macrophages is transforming growth factor p (TGF-p). T G F -  51  (3 can act as a stimulator of mesangial cell proliferation as well as induce matrix production in vitro (MacKay et al., 1989). extracellular  matrix  expansion  are  key  Both mesangial cell proliferation and events  in  the  development  of  glomerulosclerosis. The process of matrix production may in turn lead to or involve the upregulation of P D G F (platelet-derived growth factor) p-receptor in mesangial cells (Van Goor et al., 1994). Macrophages are able to secrete P D G F , a cytokine that could play an important role in the progression of glomerulosclerosis. It has been shown  that  PDGF  selectively  induces  mesangial  cell  proliferation  and  extracellular matrix accumulation (Floege et al., 1993). In addition to contributing to lipid accumulation in mesangial cells, Lp-X may also contribute to the accumulation of lipids in macrophages. The effect of rLp-X on cellular lipid levels in macrophages was examined.  Macrophages  cultured in the presence of rLp-X showed an increase in cellular total cholesterol and cholesteryl esters relative to control (Figure 9).  rLp-X does not contain  cholesteryl ester. These results suggested that the increase in cholesteryl ester was do to the uptake of unesterified cholesterol from rLp-X by macrophages. Excess cellular cholesterol is esterified and stored as lipid droplets. The accumulation of cellular lipids in macrophages could lead to the formation of foam cells.  O R O staining of macrophages treated with rLp-X  revealed the presence of foam cells (Figure 10). Foam cells were not detected in untreated macrophages. Two of the major events in glomerulosclerosis are the accumulation of 52  lipids and the presence of foam cells in affected glomeruli. The results obtained from these experiments indicate that rLp-X is able to cause lipid accumulation in macrophages. This could contribute to the accumulation of lipids in the kidneys of L C A T deficient patients.  Results obtained from this study have shown that  macrophage lipid accumulation via the uptake of Lp-X may lead to the formation of  foam  cells.  Foam  cells  may  contribute  to  the  development  of  glomerulosclerosis by the secretion of growth factors and cytokines.  4.3 MESANGIAL CELL PROLIFERATION Increased mesangial cell proliferation is one of the key events in the development of glomerulosclerosis.  This proliferation  may be due to the  accumulation of Lp-X lipids by mesangial cells or to the combined effect of Lp-X and macrophages. In this study these two possible mechanisms were tested.  4.3.1 The effect of Lp-X Considerable amounts of intracellular lipid are acquired by the mesangial cell when treated with Lp-X. This accumulation of lipid could possibly lead to enhanced cell proliferation. However, the results obtained in this study indicated that Lp-X does not induce cell proliferation, as measured by the incorporation of [ H]thymidine (Figure 11). Increasing the time of incubation (up to 48 h) did not 3  alter mesangial cell proliferation  (data not shown).  Since mesangial cell  proliferation is one of the characteristics of glomerulosclerosis, it may be possible that Lp-X affects proliferation indirectly.  53  4.3.2 The combined effect of macrophages and Lp-X Treatment of macrophages with rLp-X led to the formation of foam cells. Lp-X  may  affect  mesangial  cell  proliferation  macrophages/foam cells to secrete growth factors. mesangial  cells  were  incubated  in  indirectly  by  inducing  To examine this possibility,  macrophage-conditioned  medium.  Macrophage-conditioned medium in the presence or absence of rLp-X had no effect on the incorporation of [ H]thymidine by mesangial cells (Figure 12). 3  These  results suggest that other factors must be involved in mesangial cell proliferation. In addition to macrophages, further agents present in plasma in vivo may be required to stimulate mesangial cell proliferation.  Whether macrophages are  involved in this process is still uncertain.  4.4 CONCLUSION Lipid accumulation, macrophage infiltration, mesangial  cell proliferation  are four  key  foam cell formation,  events  in the  and  development  of  glomerulosclerosis. This study has examined whether Lp-X is involved in these events and thus able to contribute to the development of glomerulosclerosis in L C A T deficient patients. The results obtained from this study have clearly shown that Lp-X is taken up  by  rat  mesangial  cells  and  that  the  lipid  components  of  Lp-X,  phosphatidylcholine and cholesterol, are metabolized to lysophosphatidylcholine and cholesteryl ester, respectively. The uptake of Lp-X may involve cell surface  54  heparan sulfate proteoglycans.  The accumulation of lipids in mesangial cells  could contribute to the lipid accumulation seen in glomerulosclerosis. Lp-X is able to induce foam cell formation  in macrophages.  The uptake of Lp-X by  macrophages may also contribute to the accumulation of lipids in the kidneys of these patients.  However, the results obtained in this study do not suggest that  Lp-X, alone or combined with macrophages, is able to induce mesangial cell proliferation.  These results indicate that other factors are involved in this  process. Further studies are needed to examine the mechanisms involved in mesangial cell proliferation and to investigate the role of extracellular matrix expansion in the pathogenesis of glomerulosclerosis in familial L C A T deficiency.  55  5. LITERATURE CITED Adams DO. Macrophages. Methods in Enzymology. 58: 494-506, 1979. Adler S, Baker P J , Pritzl P, Couser W G . Detection of terminal complement components in experimental immune glomerular injury. Kidney Int 26(6): 830837, 1984. Bally MB, Nayar R, Masin D, Hope M J , Cullis P R , Mayer LD. Liposomes with entrapped doxorubicin exhibit extended blood residence times. Biochim Biophys Acta 1023: 133-139, 1990. Bethell W , McCulloch C , Ghose M. Lecithin:cholesterol acyltransferase deficiency. Light and electron microscopic finding from two corneas. Can J Ophthalmol 10: 494-501, 1975. Beisiegel U, St Clair R W . An emerging understanding of the interaction of plasma lipoproteins with the arterial wall that leads to the development of atherosclerosis. Current Opinion on Lipidology 7: 265-268, 1996. Bron A F , Lloyd J K , Fosbrooke A S , Winder A F , Tripathi R C . Primary lecithin:cholesterol acyltransferase deficiency disease. Lancet 1: 928-929, 1975. Brown M S , Goldstein JL. Regulation of the activity of the low density lipoprotein receptor in human fibroblasts. Cell 6(3): 307-316, 1975. Carlson LA, Holmquist L. Evidence for deficiency of high density lipoprotein lecithin:cholesterol acyltransferase activity (a-LCAT) in fish-eye disease. Acta M e d Scand 218: 189-196, 1985. Coritsidis G , Rifici V , Gupta S, Rie J , Shan Z, Neugarten J , Schlondorff D. Preferential binding of oxidized LDL to rat glomeruli in vivo and cultured mesangial cells in vitro. Kidney International 39: 858-866, 1991. Diamond JR, Karnovsky M J . Focal and segmental glomerulosclerosis: Analogies to atherosclerosis. Kidney Int 33: 917-924, 1988. Dobiasova M. Lecithin:cholesterol acyltransferase and the regulation of endogenous cholesterol transport. Adv Lipid Res 20: 107-194, 1983. Dobiasova M, Schutzova M. Cold labeled substrate and estimation of cholesterol esterification rate in lecithin:cholesterol acyltransferase radioassay. Phyaiol. Bohemoslov 35: 319-327, 1986.  56  Edelstein C, Dezdy F, Scanu A M , Shen BW. Apolipoproteins and the structural organization of plasma lipoproteins: Human plasma high density lipoprotein-3. J Lipid Res 20: 143-153, 1979. Fielding C J . Lecithin:Cholesterol acyltransferase. In: Advances in Cholesterol Research. Esfahanie M, Swaney J (eds). Caldwell, N J , Telford, 1990, pp. 270. Floege J , Eng E, Young BA, Alpers C E , Barrett TB, Bowen-Pope DF, et al. Infusion of platelet-derived growth factor or basic fibroblast growth facto induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. J Clin Invest 92: 2952-62, 1993. Frohlich J , Pritchard P H . Analysis of familial syndromes. Mol Cell Biochem, 113: 141-149, 1992.  hypoalphalipoproteinemia  Funke HA, von Eckardstein A, Pritchard P H , Karas M, Albers J J , Assmann G. A frameshift mutation in the human apolipoprotein A - l gene causes high density lipoprotein deficiency, partial lecithin:cholesterol acyltransferase deficiency and corneal opacities. J Clin Invest 87: 371-376, 1991. Funke HA, von Eckardstein A, Pritchard P H , Albers J J , Kastelein J J P , Droste C , Assmann G . A molecular defect causing fish eye disease: an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of a-LCAT activity. Proc Natl Acad Sci USA 88: 4855-4859, 1991. Gjone E. Familial lecithin:cholesterol acyltransferase deficiency: A clinical survey. ScandJ Clin Lab Invest 33: suppl 137, 73, 1974. Gjone E, Torsvik H, Norum KR. Familial plasma cholesterol ester deficiency: A study of the erythrocytes. Scand J Clin Lab Invest 21: 327-332, 1968. Glomset J A , Assmann G , Gjone E, Norum K R . Lecithin:cholesterol Acyltransferase Deficiency and Fish Eye Disease. In: The Metabolic and Molecular Bases of Inherited Disease. Scriver C R , Beaudet A L , Sly W S , Valle D (eds). McGraw-Hill, Inc., New York, 1995, pp. 1933-1951. Glomset JA, Wright JL. Some properties of a cholesterol esterifying enzyme in human plasma. Biochim Biophys Acta 89: 266-276, 1964. Goldstein J L , Brown M S . Regulation of low density lipoprotein-receptors: implications for pathogenesis and therapy of hypercholesterolemia and atherosclerosis. Circulation 76(3): 504-507, 1987. Gotto A M , Pownall HR, Havel R J . Methods Enzymol 128: 3-41, 1986.  Introduction to the plasma lipoproteins.  57  Gupta S, Rifici V, Crowley S, Brownlee M, Shan Z, Schlondorff D. Interactions of LDL and modified LDL with mesangial cells and matrix. Kidney International 41: 1161-1169, 1992. Hamilton RL, Havel R J , Kane J P , Blaurock A E , Sata T. Cholestasis: Lamellar structure of the abnormal human serum lipoprotein. Science 172: 475-478, 1971. Hamilton RL, Moorehouse A , Havel R J . Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions J Lipid Res 33: 529, 1991. Harper PA, Robinson J M , Richard L, Hoover T C , Wright T C , Karnovsky M J . Improved methods for culturing rat glomerular cells. Kidney International 26: 875880, 1984. Havel R J , Hamilton R. Hepatocytic lipoprotein lipoprotein catabolism. Hepatology 8: 1689, 1988.  receptors  and intracellular  Havel R J , Kane J P . Structure and Metabolism of Plasma Lipoproteins. In: The Metabolic and Molecular Bases of Inherited Disease. Scriver C R , Beaudet AL, Sly W S , Valle D (eds). McGraw-Hill, Inc., New York, 1995, pp 1841-1851. Hovig T, Gjone E. Familial lecithin:cholesterol acyltransferase deficiency: Ultrastructural aspects of a new syndrome with particular reference to lesions in the kidneys and the spleen. Acta Pathol Microbiol Scan 81:681, 1973. Hurt-Camejo E, Bondjers G , Camejo G . Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages. Journal of Lipid Research 31: 443-454, 1990. Imbasciata E, Paties C, Scarpioni L, Mihatsch M J . Renal lesions in familial lecithin-cholesterol acyltransferase deficiency. Am J Nephrol 6: 66-70, 1986. Ismail NAE, Alavi MZ, Moore S. Lipoprotein-proteoglycan complexes from injured rabbit aortas accelerate lipoprotein uptake by arterial smooth muscle cells. Atherosclerosis 105: 79-87, 1994. Jonas A . Lecithin:cholesterol acyltransferase in the metabolism of high-density lipoproteins. Biochim Biophys Acta 1084: 205-220, 1991. Kowal R C , Herz J , Goldstein JL, Esser V, Brown M S . Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoproteins. Proc Natl Acad Sci USA 86: 5810-5814, 1989.  58  Kreisberg J l , Hoover RL, Karnovsky M J . Isolation and characterization of rat glomerular epithelial cells in vitro. Kidney International 14:21-30, 1978. Kuivenhoven JA, Pritchard H, Hill J , Frohlich J , Assmann G , Kastelein J . The molecular pathology of lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes. J Lipid Res 38: 191-205, 1997. Kuivenhoven JA, Stalenhoef A F H , Hill J S , Demacker P N M , Errami A, Kastelein J J P , Pritchard P H . Two Novel Molecular Defects in the LCAT Gene Are Associated With Fish Eye Disease. Arterioscler Thromb Vase Biol 16: 294-303, 1996. Kuivenhoven JA, Wiebusch H, Pritchard P H , Funke H, Benne R, Assmann G , Kastelein J P . A mutation in a lariat branchpoint sequence causes intron retention: a novel molecular basis for hereditary disease. J Clin Invest 98: 358-364, 1996. Lavie C J , Milani RV. Effects of cardiac rehabilitation, exercise training, and weight reduction on exercise capacity, coronary risk factors, behavioral characteristics, and quality of life in obese coronary patients. Am J Cardiol 79(4): 397-401, 1997. Lowry O H , Rosebrough N J , Farr A L , Randall R J . Protein measurement with Folin phenol reagent. J Biol Chem 193: 265-275, 1951. Luna L G . Manual of Histological Staining Methods of the Armed Forces Institute of Pathology. McGraw-Hill, New York. 1968. MacKay K, Striker L J , Stauffer J W , Doi T, Agodoa L J , Striker G E . Transforming growth factor-p. Murine glomerular receptors and responses of isolated glomerular cells. J Clin Invest 83: 1160-7, 1989. Magil A , Chase W , Frohlich J . Unusual renal biopsy findings in a patient with familial lecithin:cholesterol acyltransferase deficiency. Hum Pathol 13: 283-285, 1982. Marcel YL. Lecithin:cholesterol acyltransferase and intravascular transport. Adv Lipid Res 19: 85-136, 1982. Mayer LD, Hope M J , Cullis PR. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858: 161-168, 1986. Miller J P . Dyslipoproteinaemia of liver disease. Bailliere's Clin Endocrinol Metab 4: 804-833, 1990.  59  Murayama N, Asano Y , Hosoda S, Maesawa M, Saito M, Takaku F, Sugihara T, Miyashima K, Yawata Y . Decreased sodium influx and abnormal red cell membrane lipids in a patient with familial plasma lecithin:cholesterol acyltransferase deficiency. Am J Hematol 16: 129-137, 1984. Niemeir A, Gafvels M, Heeren J , Meyer N, Angelin B, Beisiegel V. V L D L receptor mediates the uptake of human chylomicron remnants in vitro. J Lipid Res 37: 1733-1742, 1996. O K, Frohlich J . Role of lecithin:cholesterol acyltransferase and apolipoprotein A-l in cholesterol esterification in lipoprotein-X in vitro. Journal of Lipid Research 36: 2344-2354, 1995. O K, Hill J S , Wang X , Pritchard P H . Recombinant lecithin:cholesterol acyltransferase containing Thr123 - He mutation esterifies cholesterol in low density lipoprotein but not in high density lipoprotein. Journal of Lipid Research 34: 81-88, 1993. O K, Ly M, Fang DZ, Frohlich J , Choy P C . Effect of lipoprotein-X on lipid metabolism in rat kidney. Molecular and Cellular Biochemistry (in press), 1997. Rutledge J C , Woo M M , Rezai A A , Curtiss LK, Goldberg IJ. Lipoprotein lipase increases lipoprotein binding to the artery wall and increases endothelial layer permeability by formation of Iipolysis products. Circ Res 80(6): 819-828, 1997. Sabesin S M . Cholestatic lipoproteins-their Gastroenterology 83: 704-709, 1982.  pathogenesis  and significance.  Schlondorff D. The glomerular mesangial cell: an expanding role for a specialized pericyte. FASEBJ 1: 272-281, 1987. Schlondorff D. Cellular mechanisms of lipid injury in the glomerulus. Am J Kidney Dis 22: 72-82, 1993. Seidel D, Gjone E, Blomhoff J P , Geisen HP. Plasma lipoproteins in patients with familial plasma lecithin:cholesterol acyltransferase (LCAT) deficiency- studies on the apolipoprotein composition of isolated fractions with identification of Lp-X. Horm Metab Res Suppl, 4: 6-11, 1974. Smith E B . The influence of age and atherosclerosis on the chemistry of aortic intima. J Atheroscler Res 5: 224, 1965.  60  Stokke KT, Bjerve K S , Blomhoff J P , Oystese B, Flatmark A, Norum KR, Gjone E. Familial lecithin: cholesterol acyltransferase deficiency: Studies on lipid composition and morphology of tissues. Scand J Clin Lab Invest 33: 93-100, 1974. Striker LM, Kellen PD, Chi E, Striker G E . The Composition of Glomerulosclerosis. I. Studies in Focal Sclerosis, Crescentic Glomerulonephritis, and Membranoproliferative Glomerulonephritis. Lab Invest 51: 181-192, 1984. Striker G E , Striker LJ. Biology of Disease: Glomerular cell culture. Lab Invest 53: 122-131, 1985. Ting B H P , Lee A S , Hochmuth R M . Effect of cytochalasin D on the mechanical properties and morphology of human neutrophils. Ann Biomed Engl 23: 666-671, 1995. Van Goor H, Ding G , Kees-Folts D, Grand J , Schreiner G F , Diamond JR. Biology of Disease: Macrophages and Renal Disease. Lab Invest 71: 456-464, 1994. Vijayagopal P, Srinivasan S R , Radhakrishnamurthy B, Berenson G S . Lipoprotein-proteoglycan complexes from atherosclerotic lesions promote cholesteryl ester accumulation in human monocytes/macrophages. Arteriosclerosis Thrombosis 12: 237-249, 1992. Walli A K , Grone E, Miller B, Grone H-J, Thiery J , Seidel D. Role of Lipoproteins in Progressive Renal Disease. Am J Hypertens 6: 358S-366S, 1993. Walli A K , Seidel D. Role of Lipoprotein-X in the Pathogenesis of Cholestatic Hypercholesterolemia. J Clin Invest 74: 867-879, 1984. Wheeler DC, Persaud J W , Fernando R, Sweny P, Varghese Z, Moorhead J F . Effects of low-density lipoproteins on mesangial cell growth and viability. Nephr Dial Tranplant 5: 185-191, 1990.  61  

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