Analysis of Glomerulosclerosis and Atherosclerosis in Lecithin Cholesterol Acyltransferase-deficient Mice*

Gilles LambertDagger §, Naohiko SakaiDagger §, Boris L. VaismanDagger , Edward B. NeufeldDagger , Benoit MarteynDagger , Chi-Chao Chan||, Beverly Paigen**, Enrico LupiaDagger Dagger , Alton ThomasDagger Dagger , Liliane J. StrikerDagger Dagger , Joan Blanchette-Mackie§§, Gyorgy Csako¶¶, John N. Brady||||, Rene Costello¶¶, Gary E. StrikerDagger Dagger , Alan T. RemaleyDagger , H. Bryan Brewer Jr.Dagger , and Silvia Santamarina-FojoDagger

From the Dagger  Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, the || Laboratory of Immunology, NEI, National Institutes of Health, Bethesda, Maryland 20892, the ** Jackson Laboratory, Bar Harbor, Maine 04609, the Dagger Dagger  Renal Cell Biology Laboratory, University of Miami School of Medicine, Miami, Florida 33101, the §§ Lipid Cell Biology Laboratory, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the ¶¶ Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, and the |||| Laboratory of Receptor Biology and Gene Expression, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, September 15, 2000, and in revised form, December 22, 2000

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (-70 to -94%) and non-HDL cholesterol (-48 to -85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (-35% to -99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

As the key enzyme responsible for the esterification of free cholesterol present in circulating lipoproteins, LCAT1 plays a major role in HDL metabolism (1). Several lines of evidence that include epidemiological data, transgenic animal studies, and, more recently, prospective human clinical trials (2-6) indicate that increased plasma HDL levels protect against the development of atherosclerosis. One of several proposed functions of HDL as an anti-atherogenic lipoprotein is to facilitate reverse cholesterol transport, a process by which cholesterol is transported from peripheral cells to the liver for removal from the body (7, 8). LCAT may play a major role in this process by maintaining a free cholesterol gradient between peripheral cells and the HDL particle surface, thus promoting free cholesterol efflux (1). The newly generated cholesteryl esters (CE) accumulating in the HDL core may be transferred directly to the liver via whole particle and/or selective uptake (9, 10). Alternatively HDL-CE may be transferred to apoB-containing lipoproteins as a result of the activity of cholesterol ester transfer protein (CETP) (8).

The important role that LCAT plays in HDL metabolism has been established by the identification and characterization of patients with LCAT deficiency. Mutations in the human LCAT gene result either in familial LCAT deficiency (FLD) or in fish eye disease (FED). FED patients have a partial deficiency of LCAT function leading to hypoalphalipoproteinemia and the development of corneal opacities (11, 12). FLD patients have a total deficiency of LCAT function (12, 13). In addition to hypoalphalipoproteinemia and corneal opacities, many FLD patients also develop a mild normochromic, normocytic anemia, proteinuria, and/or renal dysfunction (14-16). The mild hemolytic anemia in FLD appears to be secondary to changes in red blood cell membrane lipids resulting from altered cholesterol metabolism (17, 18). Glomerulosclerosis, the major cause of morbidity and mortality in FLD, may ultimately lead to renal failure in the fourth or fifth decade of life (19, 20), but its pathogenesis is poorly understood. Likewise, the role of LCAT in modulating the development of atherosclerosis in these patients remains unclear. Despite reduced plasma HDL, many patients with FLD or FED do not appear to have an increased risk for developing cardiovascular disease (21). Nevertheless, premature coronary heart disease has been documented angiographically and clinically in a subset of these patients (22-27). LCAT overexpression has been shown to enhance aortic atherosclerosis in LCAT transgenic mice (28, 29) but decrease atherogenesis in LCAT transgenic rabbits (30). The mechanisms by which LCAT modulates the development of glomerulosclerosis and atherosclerosis in patients with FLD remain unknown.

We (31) and others (32) have previously described the generation of LCAT-KO mice. On a regular chow diet these animals have markedly reduced total cholesterol, HDL cholesterol, and apoA-I levels (31, 32). In the present study we characterize the lipid, ophthalmologic, hematological, renal, and cardiovascular organ systems of LCAT-KO mice on both regular and high fat/high cholesterol (HF/HC) diets. LCAT-KO mice develop a clinical phenotype similar to human patients with FLD. In addition to severe hypoalphalipoproteinemia, LCAT-KO mice present with normochromic normocytic anemia and glomerulosclerosis. Our findings indicate that the induction of LpX by the HF/HC diet is associated with the development of glomerulosclerosis in these mice. We also report that, in addition to decreased HDL, LCAT deficiency reduces the plasma levels of apoB-containing lipoproteins by up-regulating LDL receptor gene expression. Despite the low plasma HDL levels, these changes in the plasma lipid profile lead to markedly reduced aortic atherosclerosis in mice with LCAT deficiency. These combined findings provide important new insights into the role that LCAT plays in the development of glomerulosclerosis and atherosclerosis in LCAT-deficient states.

    MATERIALS AND METHODS
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Animals and Diet-- Homozygous LCAT knockout mice (31) were backcrossed to strain C57BL/6 for eight generations. LCAT-KO mice were cross-bred with apoE-KO mice (33), LDL receptor KO mice (34), and CETP-Tg mice (35) with C57BL/6 background. Control C57BL/6 mice were obtained from Charles River (Wilmington, MA). The animals were 25-30 g at the beginning of the study. Mice first maintained on a regular NIH-07 chow diet containing 5% fat (Ziegler Brothers Inc., Gardners, PA) were put on a HF/HC diet containing 15% fat (ratio of polyunsaturated to saturated fatty acids = 0.69), 1.25% cholesterol, and 0.5% cholic acid (36).

Measurement of Plasma Lipids, Lipoproteins, Apolipoproteins, LCAT Activity, and Cholesterol Esterification Rate-- Total cholesterol and triglycerides (Sigma), as well as free cholesterol and phospholipids (Wako, Osaka, Japan) concentrations were measured in a 12-µl aliquot of plasma using commercial kits and the Hitachi 911 automated chemistry analyzer (Roche Molecular Biochemicals). HDL cholesterol was determined after dextran sulfate-Mg2+ (Ciba-Corning, Oberlin, OH) or heparin (Sigma) CaCl2 precipitation of apoB containing lipoproteins (37, 38). Mouse apoA-I, A-II, and apoB were quantitated by a sandwich enzyme-linked immunosorbent assay (31, 39). Plasma lipoproteins were analyzed by gel filtration on two Superose 6 columns in series (fast protein liquid chromatography, Amersham Pharmacia Biotech) at 0.3 ml/min in phosphate-buffered saline containing 0.1 mM EDTA and 0.02% sodium-azide. The LCAT activity was determined as previously described (40), using 40 µl of mouse plasma. The cholesterol esterification rate was assessed by determining the rate of esterification of [14C]cholesterol using 125 µl of mouse plasma (41).

Characterization of Lipoprotein Fractions-- Lipoproteins were obtained from 2 ml of pooled plasma by sequential ultracentrifugation (42), and their morphology was assessed by negative staining electron microscopy (43). Plasma lipoprotein electrophoresis as well as immunofixation electrophoresis were carried out in agarose gels (REP Vis Cholesterol kit, Helena Laboratories, Beaumont, TX). Rabbit anti-mouse apoC-I, C-II, and C-III goat anti-human (Biodesign International, Kennebunk, ME), and sheep anti-mouse albumin-IgG fraction (The Binding Site Inc., San Diego, CA) were used for immunofixation electrophoresis. Patterns were developed by staining lipids (Fat Red 7B, Sigma) and/or proteins (Amido Black 10B; Paragon Blue, Beckman Coulter Inc., Brea, CA). LpX in mouse plasma was specifically analyzed by the polyanion precipitation method (44). Briefly, after electrophoresis of plasma samples in a 0.5% agar gel (Agar Noble, Difco Labs, Detroit, MI), lipoproteins were precipitated in a solution containing heparin and MgCl2. Gels were evaluated by visually observing the development of white precipitates, by staining for lipids with Fat Red 7B, and by immunofixation using the above antibodies.

Eye Examination-- Mice were killed, and the eyes were enucleated immediately. The right eye was fixed in 4% glutaraldehyde for 30 min and then transferred into 10% buffered formalin for at least 24 h before processing. The tissue was embedded in methacrylate and sectioned through the central-vertical plane. The sections of the corneas were stained with hematoxylin/eosin, periodic acid-Schiff, or oil red-O (Sigma) before examination.

Urine, Hematology, and Blood Chemistry Tests-- Urine samples were obtained from the mice immediately prior to sacrifice and kept frozen at -80 °C prior to analysis. Electrophoresis of the urine samples was done in 1% agarose gel (Paragon SPE kit, Beckman Coulter Inc., CA), followed by Amido Black 10B staining. Protein fractions were quantified by scanning the gels with an EDC densitometer (Helena Labs). Complete blood count was determined on an Abbot Cell Dyn 3500 automated analyzer (Santa Clara, CA). Erythrocyte osmotic fragility was determined using the Unopette kit (Becton Dickinson, Franklin Lakes, NJ). Erythrocytes were stained with methylene blue (Sigma), and the cells containing stained RNA precipitates (i.e. reticulocytes) were counted manually under microscope. Target cells were counted manually under microscope. Serum total bilirubin, albumin, blood urea nitrogen, and creatinine levels were measured using commercial kits and the Hitachi 917 automated analyzer (Roche Molecular Biochemicals).

Light and Electron Microscopy Analysis of Renal Lesions-- The kidneys were perfusion fixed as previously described (45). Briefly, the aorta was cannulated below the renal arteries, and the kidneys were perfused with buffered 2% paraformaldehyde. For light microscopy coronal sections were embedded in methacrylate, and sections were stained with periodic acid Schiff. Alternatively the sections were embedded in OCT compound, frozen, and stained with oil red-O. A subset of kidneys were fixed in 3% paraformaldehyde, 0.2% glutaraldehyde, 0.2 M sodium cacodylate, pH 7.4, and sections were stained in 0.05% filipin (Sigma). Slides were analyzed with a Zeiss microscope, using excitation filter BP 360 for filipin, without the observer being aware of the animal grouping. Lesions were scored on a 1-4+ grading scale (45). For electron microscopy, cortical regions containing glomeruli were sectioned, and photographs were obtained of at least three glomeruli in each mouse.

Histological Analysis of Aortic Lesions-- Hearts and upper aortas were dissected and placed in 0.9% saline, followed by fixation in 4% formaldehyde for 24 h and then 10% formaldehyde for 5 days. The mean aortic lesion areas were quantitated from five cross-sections/mouse as previously described (46).

Preparation of Labeled Targets for Hybridization-- Mice livers (~0.5 g) were harvested, submerged in RNA later solution (Ambion, Austin, TX), and frozen at -70 °C. Total RNA was isolated with Trizol (Life Technologies, Inc.). Poly (A)+ RNA was purified using oligo(dT) conjugated latex beads (Qiagen, Valencia, CA), and 5 µg was denaturated with 100 pmol of GGCCAGTGAATTGTAATACGACGACTCACTAT- AGGGAGGCGG-d(T)24 primer at 70 °C for 10 min. Reverse transcription and second strand cDNA synthesis were performed using the Superscript Choice System (Life Technologies, Inc.). The resulting cDNA was extracted with phenol:chloroform:isoamylalcohol (25:24:1) (Ambion). In vitro transcription was performed using the BioArray-HighYield-RNA-transcript-labeling kit (Enzo, Farmingdale, NY) with 1 µg of cDNA as a template. The resulting biotinilated RNA (cRNA) was purified with Rneasy columns (Qiagen), and 50 µg cRNA was fragmented in 40 mM Tris-HCl, pH 8.0, 100 mM KOAc, 30 mM MgOAc (Sigma) for 35 min at 95 °C in 75 µl. The quality of total RNA, poly(A)+ RNA, cDNA, cRNA, and fragmented cRNA was judged by running aliquots on a Tris borate/EDTA/urea 6% polyacrylamide gel (Novex, San Diego, CA), followed by SYBRGold (Molecular Probes, Eugene, OR) staining. The efficiency of the in vitro transcription was ascertained on a Test1 oligonucleotide array (Affymetrix, Santa Clara, CA).

Array Hybridization-- A set of four oligonucleotide arrays (Mu6500 GeneChip, Affymetrix) were used for hybridization. The oligo array cartridges were prehybridized with 200 µl of 1× hybridization buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, and 0.01% Tween 20 (all reagents, Sigma)) for 10 min at 45 °C/60 rpm on a rotisserie. Fragmented cRNA was brought to 1 ml in 1× hybridization buffer with 0.1 mg/ml herring sperm DNA (Promega, Madison, WI), 0.5 mg/ml acetylated bovine serum albumin (Life Technologies, Inc.), as well as 50 pM B2, 1.5 pM BioB, 5 pM BioC, 25 pM BioD, and 100 pM Cre control oligonucleotides (Affymetrix). The mixture was incubated for 5 min at 99 °C and centrifuged to pellet debris. Prehybridization solution was removed from the cartridges, and 200 µl of the fragmented RNA solution was added and hybridized for 16 h at 45 °C/60 rpm. After hybridization, the samples were removed, and the cartridges were washed, stained with Streptavidin Phycoerythrin, and rinsed on a fluidix station using the EukGE-WS1 protocol. The arrays were scanned on a Hewlett Packard confocal laser scanner and visualized using the GeneChip 3.3 software (Affymetrix) to determine the expression of each gene and their differential expression between mice. Comparison analysis between three untreated LCAT-KO mice and three controls (i.e. a total of nine comparisons) was performed. The genes differentially expressed (i.e. more than 2-fold change) in at least seven comparisons were listed.

Western Blot Analysis-- Mouse livers (~0.5 g) were cut into small pieces and transferred in 5 ml of phosphate-buffered saline 1× containing a protease inhibitor mixture P2714 (Sigma). The suspensions were homogenized and centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatants were centrifuged in a TI-60 rotor at 40,000 rpm using a L8-70M ultracentrifuge (Beckman, Palo Alto, CA) for 1 h at 4 °C. Pellets were resuspended in lauryl dodecyl sulfate sample buffer (1× final concentration). Liver proteins (10 µg) were resolved on Nu-PAGE 4-12% Bis-Tris gels in MES-SDS buffer (Novex, San Diego, CA) under nonreducing conditions. Proteins were transferred onto an Immobilon membrane (Millipore, Bedford, MA), probed with polyclonal anti-human-LDLr-IgG (RDI, Flanders, NJ) using Vectastain PK6101 (Vector, Burlingame, CA). ApoA-I, apoA-II, and apoE levels of LCAT-KO and control mice plasma samples (0.1 µl) were similarly also analyzed by Western blot using anti mouse apoA-I, apoA-II, and apoE IgG (Biodesign, Saco, ME).

Statistical Analysis-- Data are expressed as the means ± S.E. The statistical significance of the differences of the mean between two groups was evaluated using the Student's t test. Differences in mean aortic lesion areas were evaluated using the Mann-Whitney test (Instat, GraphPad Software Inc., San Diego, CA).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma LCAT Activity, Lipids, Apolipoproteins, and Lipoproteins-- The changes in the lipid profile and apolipoproteins in the two study groups of mice before and 16 weeks after a HF/HC diet are summarized in Table I. Compared with controls, the plasma LCAT activity was reduced by 99% in LCAT-KO mice. On a regular chow diet the plasma levels of total cholesterol, phospholipids, CE, HDL cholesterol, apoA-I, and apoA-II of LCAT-KO mice were decreased to 25, 54, 7, 14, 10, and 23% of controls, respectively, whereas the plasma triglycerides levels increased by 50% (p < 0.01, all). The non HDL cholesterol and apoB levels were similar in both study groups. On the HF/HC diet, the plasma levels of a total cholesterol, phospholipids, CE, HDL cholesterol, non-HDL cholesterol, apoA-I, apoA-II, and apoB of LCAT-KO mice were decreased to 45, 48, 44, 6, 52, 17, 50, and 45% of controls, respectively (p < 0.01; all), whereas the plasma triglycerides levels remained unchanged. The plasma CE levels dramatically increased as a result of the HF/HC diet in both controls and LCAT-KO mice. Of particular interest was the increase in the CE/total cholesterol ratio from 21 to 76%, indicating an alternate pathway for the esterification of cholesterol in LCAT-KO mice. Fig. 1 illustrates the fast protein liquid chromatography elution profile of the plasma lipoproteins in control and LCAT-KO mice maintained 16 weeks on a HF/HC diet. Most of the cholesterol eluted in the very low/intermediate density lipoproteins (VLDL and IDL) and LDL fractions in both study groups. However, the VLDL cholesterol, IDL cholesterol as well as HDL cholesterol were significantly reduced in LCAT-KO mice. Electron microscopy evaluation of negatively stained lipoprotein fractions revealed discoidal particles forming rouleaux structures (i.e. pre-beta HDL) only in the HDL fraction of LCAT-KO mice (Fig. 1, inset), as previously described (32). On the HF/HC, the morphology of HDL from control mice did not change, whereas there was an increase in the mean diameter of the small spherical particles in the HDL size range of the LCAT-KO mice (10.9 ± 0.4 nm on chow versus 14.5 ± 0.4 nm on HF/HC diet). In addition the proportion of discoidal particles in the HDL fraction from LCAT-KO mice decreased from 14 to 4% after the HF/HC diet (p < 0.05). LDL of LCAT-KO mice were polydisperse consisting of smaller 19.6 ± 1.0 nm as well as normal 33.1 ± 1.4 nm spherical lipoproteins and some larger structures 66.7 ± 1.8 nm in size, whereas those of control mice consisted in particles of 28.0 ± 1.8 nm in diameter. The sizes of the particles present in the LDL fraction of both control, and LCAT-KO mice did not significantly change after the HF/HC challenge. Lipoprotein electrophoresis (Fig. 2A, middle panel) showed a virtual absence of alpha -lipoproteins in the plasma of LCAT-KO mice and increased staining at the application site (indicated by arrows) after a HF/HC diet in both control and LCAT-KO mice (Fig. 2A). On agarose gel, an LpX-like lipoprotein (Fig. 2A, middle panel) migrated in the alpha -globulin fraction (Fig. 2A, left panel). Like human LpX (47), the mouse LpX was shown to contain apoC-I, C-II, and C-III (Fig. 2A, right panel) and albumin (data not shown) by immunofixation. On agar gel, mouse LpX, which migrated anodally (Fig. 2B), was detected only in a subset of LCAT-KO mice (8 of 12) after 16 weeks on the HF/HC diet. LpX was not present in the plasma of LCAT-KO mice on a regular chow diet (6 mice) or control mice on a regular chow (6 mice) or a HF/HC diet (11 mice).

                              
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Table I
Plasma lipids, lipoproteins, apolipoproteins, and LCAT activities in control and LCAT-knockout mice on a regular chow diet as well as a high fat/high cholesterol diet
TC, total cholesterol; PL, phospholipids; TG, triglycerides; CE, cholesteryl esters; HDL-C, HDL-cholesterol; CER, cholesterol esterification rate.


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Fig. 1.   Elution profile of control (open circle ) and LCAT knockout (black-diamond ) mice plasma on fast protein liquid chromatography after 16 weeks HF/HC diet. 50 µl of pooled mouse plasma was applied on two Superose 6 columns in series. The concentration of phospholipids, triglycerides, and cholesterol in each fraction are indicated in the y axis. Inset, left panel, low magnification of HDL fraction (1.063 < d < 1.21) from LCAT KO mouse showing numerous discs present in the fraction. Bar, 10 nm). Right panel, high magnification of HDL fraction from LCAT-KO mouse showing a rouleau of discs associated with HDL particle. Bar, 5nm).


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Fig. 2.   Electrophoresis analysis of LCAT-KO and control mice plasma lipoproteins. A, plasma lipoproteins were separated in agarose gels. Protein staining (left panel), lipoprotein staining (middle panel), and immunofixation with anti-apoCI, CII and CIII antibodies (right panel) are shown. B, plasma lipoproteins were resolved in agar gel followed by precipitation with heparin/MgCl2. ApoC-containing LpX is present only in the plasma of LCAT-KO mice after 16 weeks HF/HC diet. Arrows indicate the point of application.

Eyes-- Sections of the corneas from LCAT-KO and control mice fed a HF/HC diet for 16 weeks were stained with hematoxylin and eosin, periodic acid Schiff, or oil red-O. No ocular abnormalities were found in the LCAT knockout mice compared with controls using these three histological techniques (data not shown).

Blood-- Blood samples from LCAT-KO and control mice were analyzed for cell counts, hemoglobin, hematocrit, and total bilirubin levels. LCAT knockout mice had anemia with decreased red blood cell count (9.6 ± 0.1 versus 10.4 ± 0.1 106/µl), hematocrit (42.4 ± 0.4 versus 46.0 ± 0.5%), hemoglobin levels (13.5 ± 0.1 versus 14.9 ± 0.1 g/dl) as well as white blood cell count (3.63 ± 0.35 versus 5.84 ± 0.61 103/µl) compared with controls (p < 0.005, all). The platelet count, the red blood cell indices (mean corpuscular volume and mean corpuscular hemoglobin), and the plasma bilirubin levels were normal in both study groups (data not shown). Dietary status did not alter blood counts, hemoglobin, hematocrit, and bilirubin levels in either mouse model (data not shown). Despite the absence of hyperbilirubinemia, the proportion of target cells (11.2 ± 2.6 versus 2.3 ± 0.5%, p < 0.01) as well as reticulocytes (13.5 ± 4.1 versus 3.2 ± 1.4%, p < 0.04) was increased in LCAT-KO mice compared with controls, indicating increased red blood cell hemolysis. As described in FLD patients (17, 18, 48), the osmotic fragility tests showed that red blood cells from LCAT-KO mice were more resistant to hemolysis than those of controls (5.3 ± 1.5 versus 43.7 ± 3.1% hemoglobin leakage in 0.5% NaCl and 86.3 ± 3.5 versus 97.7 ± 2.1% in 0.4% NaCl; p < 0.01, all).

Renal Function-- Plasma samples from LCAT-KO and control mice were collected before and after the HF/HC challenge and analyzed for albumin, blood urea nitrogen, and creatinine. On either diet no significant difference between the two groups was detected (data not shown). However, after a HF/HC diet, proteinuria was detected by electrophoresis and immunofixation electrophoresis in 10 of 11 LCAT-KO mice urine samples, whereas the urine samples from all 11 control mice were negative (data not shown).

Kidneys-- Histologic lesions were only present in a subset of LCAT-KO mice on the HF/HC diet (i.e. those presenting with LpX). The kidneys of the other mice were normal. The lesions consisted of an increase in mesangial matrix (Fig. 3, A and B), which involved the glomeruli irregularly, within and between individual glomeruli. Occasional vacuoles were observed in the cellular cytoplasm of cells, most prominent near the vascular pole. There were no tubular or interstitial lesions. Occasional preglomerular arterioles contained periodic acid Schiff positive droplets. There were no lesions of larger blood vessels. Fig. 3 (C and D) shows representative kidney sections from LCAT-KO and control mice after the 16 weeks HF/HC challenge analyzed by electron microscopy. The glomeruli from control animals were unremarkable. In LCAT-KO mice, the endothelial cytoplasm and the vascular spaces were smaller than in controls. Depositions of electron dense material in the capillary lumen and mesangial regions were evident. The mesangial regions contained an increased amount of extracellular matrix, and there were irregular but prominent zones containing lucent lacunae. Occasional cells resembling macrophages were present within the mesangium. No changes were seen in either the peripheral basement membrane or the podocytes. In particular, the pedicels were intact. Unlike controls, a subset of LCAT-KO mice on a HF/HC diet accumulated lipids within their glomeruli, which were detected by oil red-O staining (Fig. 3, E and F). The glomeruli of these LCAT-KO mice had increased free cholesterol content compared with those of controls, as shown on filipin fluorescence-stained sections (Fig. 3, G and H). Renal lesions were only present in LCAT-KO mice in which LpX was detected in plasma. No renal lesions were detected in heterozygous LCAT-KO mice.


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Fig. 3.   Analysis of renal sections from control (A, C, E, and G) and LCAT-KO (B, D, F, and H) mice maintained on a HF/HC diet for 16 weeks. A and B, light microscopy of periodic acid Schiff stained sections embedded in methacrylate. C and D, electron microscopy of frozen sections. E and F, light microscopy of oil red-O stained sections embedded in OCT compound. G and H, fluorescent light microscopy of filipin stained sections embedded in OCT compound.

Effect of LCAT Deficiency on the Plasma Cholesterol Levels and Atherosclerosis in apoE-KO, LDLr-KO, and CETP-Tg Mice-- LCAT deficiency reduced the plasma levels of TC, CE, and non-HDL cholesterol (Fig. 4) in apoE-KO, LDLr-KO, and CETP-Tg mice (p < 0.05; all). The mean aortic lesion area of apoE-KO and LCAT-KO × apoE-KO mice maintained on a regular chow diet and that of LCAT-KO, LDLr-KO, LCAT-KO × LDLr-KO, CETP-Tg, LCAT-KO × CETP-Tg, and control mice maintained 16 weeks on a HF/HC diet was quantitated from five cross- sections of the proximal aortas (Fig. 5). LCAT deficiency significantly reduced the mean aortic lesion area in C57BL/6 (-85%), apoE-KO (-51%), LDLr-KO (-35%), and CETP-Tg (-99%) mice.


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Fig. 4.   Plasma cholesterol levels in LCAT +/+ and LCAT -/- mice on the C57BL, CETP transgenic, apoE knockout, and LDL receptor knockout backgrounds. The feeding status of the animals is indicated in the legend of Fig. 5. *, p < 0.05; **, p < 0.001 versus LCAT +/+ mice of the same genetic background.


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Fig. 5.   Extent of atherosclerosis in the proximal aorta of LCAT +/+ (black-square) and LCAT -/- (open circle ) mice on the C57BL (A), CETP transgenic (B), apoE knockout (C), and LDL receptor knockout (D) backgrounds. The animals (A, B, and D) were maintained for 16 weeks on a HF/HC diet, whereas apoE-knockout mice (C) were fed a regular chow diet. All mice were females and 3 months old at the beginning of the study.

Analysis of Differentially Expressed Hepatic Genes in LCAT-KO Mice-- Table II summarizes the changes in hepatic gene expression induced by LCAT deficiency in mice. Of 6500 possible transcripts, the expressions of 51 transcripts were altered by greater than 2-fold in LCAT-KO compared with control mice. Besides LCAT mRNA, the transcripts of Cyp 7alpha hydroxylase, the rate-limiting enzyme for bile synthesis as well as squalene epoxydase, involved in the cholesterol biosynthetic pathway, were down-regulated in LCAT-KO mice livers. Of particular interest is the up-regulation of the LDL receptor and apoE, whose increased expressions were also confirmed by Western blot hybridization analysis (Fig. 6). Despite no differences in their mRNA levels, apoA-I and apoA-II plasma levels were reduced in LCAT-KO mice.

                              
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Table II
Differentially expressed genes and ESTs in LCAT knockout mice


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Fig. 6.   Immunoblot of hepatic LDLr and plasma apolipoproteins in control (lanes 1 and 2) and LCAT-KO (lanes 3 and 4) mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we have investigated potential mechanisms leading to renal disease and altered atherogenic risk in LCAT-KO and LCAT-KO mice crossed with atherosclerosis-susceptible mouse models. LCAT-KO mice present with a clinical phenotype similar to patients with FLD, including normochromic normocytic anemia, glomerulosclerosis, hypoalphalipoproteinemia, and hypertriglyceridemia. In addition, LCAT deficiency alters the plasma levels of the pro-atherogenic apoB containing lipoproteins by modulating the hepatic expression of the LDLr and apoE, which, despite decreased HDL levels, leads to reduced atherogenic risk.

LCAT deficiency in mice markedly reduces total cholesterol, HDL cholesterol, and apoA-I levels as well as increases pre-beta HDL and triglyceride levels (31, 32). Surprisingly, on the HF/HC diet LCAT-KO mice had significantly lower plasma levels of the proatherogenic apoB-containing lipoproteins. Decreased plasma LDL levels have been reported in some (12, 13, 49-53) but not all (23, 27) patients with LCAT deficiency reflecting genetic variability absent in LCAT-KO mice. Despite the absence of LCAT activity, LCAT-KO mice had a dramatic increase in their plasma CE levels on the atherogenic diet, suggesting enhanced contribution by the enzyme acylCoA:cholesterol acyltransferase responsible for the intracellular esterification of cholesterol. Further analysis of the plasma lipoproteins in LCAT-KO mice revealed that the VLDL density fractions were triglyceride-enriched. LDL were polydisperse consisting of normal spherical lipoproteins as well as large structures of 66.7 nm in diameter previously designated as large molecular weight LDL (54). The two major HDL particles described in FLD (i.e. discoidal particles forming rouleaux and spherical particles) (12) were also present in LCAT knock-out mice with a decreased proportion of discoidal particles and an increase in the mean diameter of the spherical particles after the HF/HC diet. LpX was detected by electrophoresis and immunofixation electrophoresis on agarose and agar gels in a subset of LCAT-KO mice fed a HF/HC diet for 16 weeks. LpX, first described in patients with primary biliary obstruction (55), appear to be bilayer vesicles with diameters of 30-70 nm (47, 56) that contain primarily free cholesterol, phosphatidylcholine, albumin, and apoCs. These particles also accumulate in FLD patients as a result of the high plasma concentrations of free cholesterol and phosphatidylcholine observed in LCAT deficiency (47, 57).

Lipoprotein disorders are frequently associated with alterations in the morphology and lipid composition of the cornea. Corneal opacities are found in several syndromes associated with HDL deficiency including Tangier disease (58), apoA-I deficiency (59, 60) as well as FED and FLD (12, 61). However, analysis of the cornea of LCAT-KO mice with hematoxylin and eosin, periodic acid-Schiff, or oil red-O failed to reveal similar changes.

LCAT-KO mice developed anemia characterized by normochromic normocytic indices, increased target cells, and reticulocyte count findings consulted with hemolysis. The mild hemolytic anemia appears to be secondary to altered red blood cell membrane lipids resulting from changes in plasma cholesterol metabolism. The red blood cell membrane fragility was decreased in LCAT-KO compared with controls, consistent with altered red blood cell membrane lipids as well as an increased proportion of target cells that are more resistant to hemolysis (62).

We further examined the consequences of LCAT deficiency on the morphology and function of the kidneys from mice maintained either on regular or HF/HC diets. Glomerulosclerosis was not detected in LCAT-KO mice on a regular chow diet. In contrast, on the HF/HC diet, a subset of LCAT-KO mice developed renal lesions similar to those previously described in patients with FLD (19, 20, 63-65). Histologically, these lesions were characterized by significant reductions in the vascular space, expanded mesangial region with increased number of mesangial cells and mesangial sclerosis, and increased extracellular matrix with accumulation of lipid droplets and macrophages. Filipin and oil red-O staining demonstrated the accumulation of free cholesterol and polar lipids in the glomeruli. Ultrastructurally, the endothelial cytoplasm and the vascular spaces were smaller in LCAT-KO than in controls, and the mesangial regions contained an increased amount of extracellular matrix. Although significant, the glomerular lesions in LCAT-KO mice fall within the milder spectrum of lesions described in FLD patients. However, most of the tissue analysis has been performed in patients with end stage renal disease. The absence of lesions on a regular chow diet, and the mildness of the lesions after the HF/HC diet, in the LCAT-KO mice, are both consistent with previous observations that the C57Bl/6 mouse strain is resistant to the induction of glomerulosclerosis (66). The plasma albumin, total protein, blood urea nitrogen, and creatinine levels were normal in all study animals, even in LCAT-KO mice found to have renal lesions. In patients with FLD these plasma indicators of renal function remain normal until the development of severe renal disease. Proteinuria, one of the earliest findings in patients with FLD (12, 13, 62) was also detected in 10 of 11 LCAT-KO mice on the HF/HC diet. Thus, the renal lesions in LCAT-KO mice lead to abnormal renal function.

The pathogenesis of renal injury in FLD is poorly understood. Some investigators have suggested that the renal damages observed in FLD may be immune complex- and complement-mediated (20, 65) and may be further exacerbated by the accumulation of oxidized phospholipids in the glomeruli (67). It has also been suggested that large LDL particles as well as LpX trapped in capillary loops induce endothelial damage and vascular injury in the kidneys of FLD patients (51, 54, 63). However, these abnormal lipoproteins are not always detected in FLD (20). It is of interest that LCAT-KO mice developed glomerulosclerosis only while on the HF/HC diet, when the plasma levels of apoB-containing lipoproteins were increased. Both large LDL and LpX were present in the plasma of LCAT-KO mice only on the HF/HC diet. Furthermore, renal lesions were only detected in the group of mice that accumulated LpX. These findings provide strong support for the importance of large LDL and LpX in the development of renal disease in FLD (68).

In patients with LCAT deficiency the atherogenic risk is variable. The mechanisms leading to altered atherosclerosis are poorly understood. Despite low HDL, many FLD and FED patients do not appear to be at increased risk of developing premature coronary artery disease (21). However, some patients presenting with severe coronary artery disease have been reported (22-27, 69). To investigate potential mechanisms leading to this variable atherogenic risk, we examined the consequences of LCAT deficiency on the development of atherosclerosis in LCAT-KO and LCAT-KO mice crossed with atherosclerosis-susceptible mouse models. Despite decreased HDL, LCAT-KO, LCAT-KO × apoE-KO, LCAT-KO × LDLr-KO, and LCAT-KO × CETP-Tg mice had a significant reduction in the mean aortic lesion area compared with their respective controls. Analysis of LCAT-induced changes in other plasma lipoproteins provided some insights into these unexpected findings. In addition to lowering HDL, LCAT deficiency reduced the plasma levels of the pro-atherogenic apoB containing lipoproteins by enhancing the hepatic expression of LDLr and apoE. The marked reduction in the plasma levels of the anti-atherogenic HDL may be offset by a reduction in the plasma levels of the proatherogenic apoB-containing lipoproteins, thus resulting in reduced atherosclerosis despite decreased HDL.

Decreased plasma LDL levels have been reported in most (12, 13, 49-53) but not all (23, 27) patients with LCAT deficiency, reflecting different genetic backgrounds. This same variability in apoB containing lipoproteins levels can be observed in apoE-KO, LDLr-KO, and CETP-Tg mice crossed with LCAT-KO mice, where the apoB levels vary according to genetic background. Interestingly, in contrast to LCAT-deficient patients with normal or reduced apoB levels, patients with increased triglycerides and apoB levels (23, 27) have been shown to have increased cardiac risk. These patients have delayed catabolism of LDL (70). These combined data suggest that, in LCAT-deficient states, the plasma levels of the proatherogenic apoB-containing lipoproteins, rather than anti-atherogenic HDL, modulate atherogenic risk. Additionally, in contrast to patients with Tangier disease, who cannot generate pre-beta HDL (71), patients with LCAT deficiency have increased plasma levels of this nascent lipoprotein particle, the most effective plasma cholesterol acceptor. Thus, despite reduced HDL, the process of reverse cholesterol transport may still function in LCAT deficiency.

In summary, we have characterized the lipid, ophthalmologic, hematological, renal, and cardiovascular organ systems of LCAT-KO mice, a new animal model for FLD. Like human patients with FLD, LCAT deficiency in mice leads to marked hypoalphalipoproteinemia, anemia, and glomerulosclerosis. Our studies provide strong support for the role of LpX in the development of glomerulosclerosis in LCAT deficiency. We also report for the first time that despite decreased HDL, LCAT deficiency protects against the development of diet-induced aortic atherosclerosis in mice. The increased plasma levels of pre-beta HDL as well as the decreased proatherogenic apoB-containing lipoproteins play a pivotal role in determining the atherogenic risk in patients with hypoalphalipoproteinemia due to LCAT deficiency. These combined findings provide new insights into the role that LCAT plays in the development of glomerulosclerosis and atherosclerosis in LCAT-deficient states. The identification of LCAT-KO mice as a mouse model in FLD will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in patients with LCAT deficiency.

    ACKNOWLEDGEMENTS

We thank Natalie Murray for excellent technical assistance as well as Donna James for helping in the preparation of this manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

To whom correspondence should be addressed: Molecular Disease Branch, NHLBI, NIH, Bldg. 10, Rm. 7N115, 10 Center Dr., Bethesda, MD 20892-1666. Tel.: 301-496-6750; Fax: 301-402-0190; E-mail: gl68i@nih.gov.

Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M008466200

    ABBREVIATIONS

The abbreviations used are: LCAT, lecithin cholesterol acyl transferase; HDL, high density lipoprotein; LDL, low density lipoprotein; LDLr, low density lipoprotein receptor; CE, cholesteryl ester(s); CETP, cholesteryl ester transfer protein; FLD, familial LCAT deficiency; FED, fish eye disease; apo, apolipoprotein; HF/HC, high fat/high cholesterol; KO, knockout; LpX, lipoprotein X; MES, 4-morpholineethanesulfonic acid; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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