Interactions of high-density lipoprotein 3 with brain capillary endothelial cells

High-density lipoprotein 3 (HDL3) binds to capillary endothelial cells when their lumen surfaces are exposed to 125I-HDL3 by post-mortem perfusion of whole brain. Kinetic studies of binding of HDL3 to isolated membranes show that HDL3 binds only to endothelial membranes with high affinity (Kd = 7 micrograms/ml). Trypsin treatment of membranes abolishes HDL3 binding. High-affinity binding sites for HDL3 were recovered when endothelial cells from bovine brain capillaries were maintained in culture (Kd = 13 micrograms/ml HDL3 protein). The characteristics of the binding were preserved up to the 6th passage. Competition experiments using isolated luminal membranes or cultured endothelial cells indicate that only HDL3 and not LDL or methylated LDL, are able to compete binding of 125I-HDL3. Furthermore, the inhibition of 125I-HDL3 binding by lipoprotein A-I and lipoprotein A-I:A-II strongly suggests that apolipoprotein A-I is implicated in the formation of HDL3-receptor complexes. The binding is increased by loading cells with free cholesterol or LDL cholesterol. In addition, surface-bound 125I-HDL3 remains sensitive to mild trypsin treatment after subsequent incubation of BBCE at 37 degrees C. HDL3 bound to the cell surface is not endocytosed, but rather rapidly released into the medium after binding (t1/2 = 5 min).     

Interaction of high density lipoprotein with its receptor on cultured fibroblasts and macrophages. Evidence for reversible binding at the cell surface without internalization

Cultured extrahepatic cells possess a specific high affinity receptor for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. Current results suggest that HDL receptors on cultured human fibroblasts and mouse peritoneal macrophages promote reversible binding of HDL to the cell surface without internalization of lipoprotein particles. When 125I-HDL3 was bound to cultured cells at 0 degrees C and then warmed to 37 degrees C after removal of unbound lipoprotein, most of the cell surface-bound HDL was released rapidly (t1/2 = 3 min) into the medium without entering a cellular pool that was inaccessible to digestion by trypsin at 0 degrees C. This lack of internalization of HDL was evident under conditions where internalization of 125I-low density lipoprotein and 125I-transferrin were readily detected. When cells were exposed to 125I-HDL3 at 37 degrees C, only a trace amount of iodinated apoprotein remained associated with cells after treatment of cells with trypsin. Fibroblasts treated with medium containing increasing concentrations of cholesterol exhibited a dose-dependent increase in reversible, trypsin-sensitive binding of 125I-HDL3 at 37 degrees C without an attendant increase in trypsin-resistant binding. These results suggest that reversible binding of HDL to its cell-surface receptor without subsequent endocytosis of receptor-HDL complexes is the mechanism by which HDL receptors facilitate cholesterol transport from cells.     

Identification and characterization of a high density lipoprotein-binding protein in cell membranes by ligand blotting

Cholesterol efflux from cultured cells can be mediated through binding of high density lipoprotein (HDL) to a cell-surface site which shows many characteristics of a biological receptor. To determine whether a specific protein forms a component of this site, cell membrane proteins were analyzed by ligand blotting using 125I-HDL3. Results demonstrated that membranes from a number of cell types possess a protein with an apparent molecular mass of 110 kDa that binds HDL and apoA-I and apoA-II proteoliposomes, but not low density lipoprotein, acetylated low density lipoprotein, or apoE proteoliposomes. The binding activity of this protein was increased by loading cells with cholesterol and was abolished by trypsin treatment of intact cell monolayers. These results suggest that HDL binds with specificity to a cell-surface protein which is regulated by intracellular cholesterol levels. Since HDL binding to intact cell monolayers shows the same characteristics, the 110-kDa binding protein may represent the proposed HDL receptor that functions to facilitate transport of cholesterol from cells to HDL particles.     

Regulation of low-density lipoprotein receptors in cultured bovine adrenocortical cells

Bovine adrenocortical cells in monolayer culture produce cortisol and respond to corticotropin (ACTH) by an increase in cortisol secretion. Several lines of evidence are indicative that much of the cholesterol that serves as precursor for steroid hormone biosynthesis by these cells is derived from low-density lipoprotein (LDL) cholesterol that is taken up endocytotically by means of specific receptors localized in bovine adrenocortical plasma membranes. ACTH stimulated this process concomitant with an increase in steroid production. In the absence of LDL, ACTH had no effect on steroid biosynthesis. ACTH action in bovine adrenocortical cells resulted in an increase in the number of LDL receptor sites in the membrane fractions, whereas the dissociation constant for LDL binding was not changed. Chloroquine and NH₄Cl, considered to be inhibitors of lysosomal degradative activity, caused an increase in the number of [¹²⁵I]iodoLDL binding sites in the plasma membrane but the effect of ACTH was still apparent in the presence of these agents. These results are suggestive that the lifetime of the LDL receptor is increased when lysosomal activity is inhibited. When aminoglutethimide was added to block cholesterol side-chain cleavage activity and inhibit steroid production, the number of [¹²⁵I]iodoLDL binding sites in the membrane fractions prepared from bovine adrenocortical cells cultured in the presence of ACTH was reduced to 50% of that in cells maintained in aminoglutethimide-free medium. However, under these conditions the number of binding sites was still significantly greater than in cells maintained in the absence of ACTH. The effects of aminoglutethimide on uptake and degradation of [¹²⁵I]iodoLDL were similar to the effects on the number of [¹²⁵I]iodoLDL binding sites. Based on these results, we conclude that the action of ACTH to stimulate LDL metabolism in bovine adrenocortical cells results from an increase in the number of LDL binding sites in the plasma membranes. This action of ACTH appears to be, at least in part, independent of cholesterol utilization for cortisol biosynthesis. However, the effect of aminoglutethimide is indicative that changes in the intracellular cholesterol concentration might modulate the action of ACTH to increase the number of LDL binding sites and therefore to stimulate LDL degradation.     

Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts.

An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals.     

Demonstration and structural comparison of receptors for insulin-like growth factor-I and -II (IGF-I and -II) in brain and blood-brain barrier

Specific receptors for insulin-like growth factors (IGF) I and II on microvessel-free rat brain cell membranes (RBCM) and in the microvessels that constitute the blood-brain barrier (BBB) were identified and characterized by means of affinity cross-linking techniques and specific anti-receptor antibodies. Two different models of BBB were examined: isolated rat brain capillaries and cultured bovine brain microvessel endothelial cells. Cross-linking with 125-I-IGF-I, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), revealed an alpha subunit of apparent Mr = 138,000 in both BBB preparations, compared to 120,000 in RBCM. Cross-linking was inhibited by unlabeled IGF and insulin, but not by antibody directed against the IGF-II receptor. When 125-I-IGF-II was cross-linked, followed by SDS-PAGE under reducing conditions, a major band of apparent Mr = 250,000 was identified in RBCM and both BBB preparations. This band, which migrated with an approximately equivalent Mr in both brain and BBB membranes, was inhibited by unlabeled IGF and by antibody specific for the IGF-II receptor. Thus, both rat and bovine brain microvessels possess classical Type I and II IGF receptors. While the alpha subunit of the Type I receptor of brain is smaller than that of the BBB, the Type II receptor of brain and BBB appear to be structurally and immunologically identical.     

Distinction in the mode of receptor-mediated endocytosis between high density lipoprotein and acetylated high density lipoprotein: evidence for high density lipoprotein receptor-mediated cholesterol transfer

The interactions of high density lipoprotein (HDL) and acetylated high density lipoprotein (acetyl-HDL) with isolated rat sinusoidal liver cells have been investigated. Cellular binding of 125I-acetyl-HDL at 0 degrees C demonstrated the presence of a specific, saturable membrane-associated receptor. This receptor was affected neither by formaldehyde-treated albumin nor by low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by the cells, indicating that the receptor for acetyl-HDL constitutes a distinct class among the scavenger receptors for chemically modified proteins. Parallel binding experiments using 125I-HDL also revealed the presence on these cells of a receptor for unmodified HDL. The ligand specificities of these two receptors were similar to each other except that the acetyl-HDL receptor was sensitive to polyanions such as dextran sulfate and fucoidin. Interaction of HDL with the cells at 37 degrees C was totally different from that of acetyl-HDL. Cellular binding of HDL was not accompanied by subsequent intracellular degradation of its apoprotein moiety, whereas its cholesterol moiety was significantly transferred to the cells. In contrast, acetyl-HDL was endocytosed and underwent lysosomal degradation as a holoparticle. This shift in receptor-recognition from the HDL receptor to the acetyl-HDL receptor was accomplished by acetylation of approximately 8% of the total lysine residues of HDL apoprotein. This unique difference in endocytic behavior between HDL and acetyl-HDL suggests a potential link of the HDL receptor to HDL-mediated cholesterol transfer in sinusoidal liver cells.     

Uptake of irradiated human low density lipoprotein by cultured Chinese hamster V79 cells

Specific binding and degradation of native and gamma-rays irradiated (100-2000 rad; 100 rad/min; 137Cs) human low density lipoprotein by Chinese hamster V79 cells and mouse peritoneal macrophage line, J774G were studied. Low density lipoproteins were labeled with 125I for studying the specific binding and subsequent degradation. The specific binding and degradation of irradiated 125I-low density lipoproteins (mixed with irradiated native lipoprotein) by Chinese hamster V79 cells are considerably reduced. The uptake depends on the concentration of thiobarbutaric acid-reactive products generated in the irradiated lipoproteins which in turn depends on the concentration of carotenoids. In contrast the rate of uptake of oxidized low density lipoproteins is enhanced by Chinese hamster macrophages. The alteration in the surface amino groups of apo-B of low density lipoprotein either due to direct damage of peptide bonds by gamma-rays or via interaction with lipid peroxides (generated in the core upon irradiation) are invoked as possible mechanisms for the reduction in specific binding and subsequent degradation by V79 cells.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

LDL receptors in coated vesicles isolated from bovine adrenal cortex: binding sites unmasked by detergent treatment

Coated vesicles isolated from bovine adrenal cortex contain specific binding sites that recognize 125I-labeled human low density lipoprotein (LDL). These sites share the properties of the functional LDL receptors previously demonstrated on the surface of adrenal cells and in unfractionated adrenal membranes. Approximately 90% of the LDL receptors of the isolated coated vesicles were initially masked. Binding of 125I-LDL increased 10 fold after the vesicles were disrupted with the detergent octylglucoside. The LDL receptors of intact coated vesicles were also shielded from destruction by pronase; proteolytic destruction occurred only after the vesicles had been disrupted with octylglucoside. The adrenal coated vesicles measured 60 nm in diameter, suggesting that they were derived from the Golgi apparatus. Like the previously studied coated vesicles from brain and other tissues, the coated vesicles from adrenal cortex contained clathrin as the major protein component. In contrast to the coated vesicles of adrenal cortex, however, the brain coated vesicles failed to reveal masked LDL receptor activity when treated with octylglucoside. The current data indicate that isolated coated vesicles from the adrenal cortex contain LDL receptors and that these receptors exist in a masked form, apparently because their binding sites face the interior of the vesicle.     

A comparative study of surface binding of human low density and high density lipoproteins to human fibroblasts: regulation by sterols and susceptibility to proteolytic digestion.

Binding of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein (HDL) was determined in cultured human fibroblasts from a normal subject and two subjects with homozygous familial hypercholesterolemia (HFH). Binding was assayed at 0 degree C to minimize the internalization of labeled lipoproteins. The binding of LDL and of HDL were compared following interventions reported to affect LDL binding in normal fibroblast. LDL binding to normal cells increased two to three fold 24 hours after transfer from medium containing whole fetal calf serum to medium containing lipoprotein-deficient fetal calf serum. This increase was completely blocked in the presence of cycloheximide (200 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). This increased capacity of normal fibroblasts to bind LDL could be reduced 70-80% by a subsequent 18-hour incubation with cholesterol (50 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). In contrast, no significant change in HDL binding to normal fibroblasts was observed after any of these interventions. HFH cells to show any significant change in either LDL binding or HDL binding following these interventions. These results suggest that HDL binding sites on normal fibroblasts are for the most part distinct from LDL binding sites. They also support the conclusion that LDL binding sites on HFH cells are for the most part qualitatively different from those on normal cells.     

Binding of apolipoprotein A-I and A-II after recombination with phospholipid vesicles to the high density lipoprotein receptor of luteinized rat ovary

To determine the apolipoprotein specificity of high density lipoprotein (HDL) receptor, apolipoprotein A-I (apo-AI) and apolipoprotein A-II (apo-AII) purified from high density lipoprotein3 (HDL3) were reconstituted into dimyristoyl phosphatidylcholine vesicles (DMPC) and their ability to bind to luteinized rat ovarian membranes was examined. Both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC were shown to bind to ovarian membranes with Kd = 2.87 and 5.70 micrograms of protein/ml, respectively. The binding of both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC was inhibited by unlabeled HDL3, apo-A-I.DMPC, apo-A-II.DMPC, apo-C-I.DMPC, apo-C-II.DMPC, apo-C-III1.DMPC, and apo-C-III2.DMPC, but not by DMPC vesicles, bovine serum albumin.DMPC or low density lipoprotein. Since the binding labeled apo-A-I.DMPC and apo-A-II.DMPC was inhibited by the DMPC complexes of apo-C groups, the direct binding of 125I-apo-C-III1.DMPC was also demonstrated with Kd = 9.6 micrograms of protein/ml. In addition, unlabeled apo-A-I.DMPC, and apo-A-II.DMPC, as well as apo-C.DMPC, inhibited 125I-HDL3 binding. 125I-apo-A-I, 125I-apo-A-II, and 125I-apo-C-III1 in the absence of DMPC also bind to the membranes. These results suggest that HDL receptor recognizes apolipoprotein AI, AII, and the C group and that the binding specificity of the reconstituted lipoproteins is conferred by their apolipoprotein moiety rather than the lipid environment. In vivo pretreatment of rats with human chorionic gonadotropin resulted in an increase of 125I-apo-A-I.DMPC, 125I-apo-A-II.DMPC, and 125I-apo-C-III1.DMPC binding activities. However, no induction of binding activity was observed when the apolipoprotein was not included in DMPC vesicles. An examination of the equilibrium dissociation constant and binding capacity for 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC after human chorionic gonadotropin treatment revealed that the increase in binding activity was due to an increase in the number of binding sites rather than a change in the binding affinity. These results further support our contention that apo-A-I, apo-A-II, and the apo-C group bind to HDL receptor. In conclusion, the HDL receptor of luteinized rat ovary recognizes apolipoproteins A-I, A-II, and the C group but not low density lipoprotein, and the binding is induced by human chorionic gonadotropin in vivo.     

Detection and quantitation of low density lipoprotein (LDL) receptors in human liver by ligand blotting, immunoblotting, and radioimmunoassay. LDL receptor protein content is correlated with plasma LDL cholesterol concentration

Low density lipoprotein (LDL) receptor activity has been detected and identified in human liver samples by ligand blotting with biotinylated lipoproteins and by immunoblotting with a monoclonal antibody raised against the bovine adrenal LDL receptor. The molecular weight of the human liver LDL receptor, approximately 132,000 on nonreduced polyacrylamide gels, is identical to that of LDL receptors detected in normal human skin fibroblasts by the same methods. LDL receptor-dependent binding activity in human liver samples has been semi-quantitated by integrating the areas under the peaks after scanning photographs of ligand blots, and receptor protein determined by radioimmunoassay with purified bovine adrenal LDL receptor protein as the standard. There was a highly significant correlation between the values obtained by each method for seven different liver samples (r = 0.948). The LDL receptor protein content of liver membranes from 10 subjects as determined by radioimmunoassay was inversely related to the plasma LDL cholesterol concentration (r = 0.663, p = 0.05) but not to other plasma lipid values, including total plasma cholesterol, high density lipoprotein cholesterol, or plasma triglyceride concentrations.     

Cellular localization and characterization of proteins that bind high density lipoprotein.

High density lipoprotein (HDL) stimulates excretion of excess intracellular cholesterol from cells, presumably by interacting with a cell-surface receptor. A 110 kDa membrane protein that is a candidate for the HDL receptor has been identified by ligand blot analysis. In this study we determined the cellular localization of this and other HDL-binding proteins and characterized their properties. The plasma membranes (PM) of cultured bovine aortic endothelial cells were labeled with trace amounts of [3H]cholesterol, and cell homogenates were fractionated on sucrose and Percoll gradients. Ligand blot analysis of homogenates of cultured bovine aortic endothelial cells demonstrated that cells contain multiple proteins that bind HDL3, including a major membrane protein with an apparent M(r) of 110 kDa and two minor ones with M(r) of 105 and 130 kDa. The gradient distribution of the 105, 110, and 130 kDa HDL-binding proteins mirrored that of labeled cholesterol and 5'-nucleotidase, both PM markers. Treatment of intact cells with the water-soluble cross-linker bis(sulfosuccinimidyl)suberate abolished the HDL binding activity of the 110 and 130 kDa proteins but not that of the 105 kDa protein. These findings suggest that the 105, 110, and 130 kDa HDL-binding proteins are localized to the PM and that at least two of these proteins are exposed to the extracellular fluid. Solubilized 110 and 130 kDa proteins were retained on wheat-germ agglutinin and abrin lectin columns, showing that they are glycoproteins. The cellular localization and physical properties of the 110 and 130 kDa proteins suggest that they may play a role in binding of HDL to the cell surface.     

Epinephrine decreases low density lipoprotein processing and lipid synthesis in cultured human fibroblasts

The effect of epinephrine on 125I-low density lipoprotein (LDL) uptake and cholesterol metabolism was investigated after a 24 hours pretreatment of cultured human fibroblasts. Epinephrine decreased LDL uptake (binding + internalization) and degradation in a dose-dependent manner. Cholesterol synthesis from 14C sodium acetate and cholesterol esterification measured by 14C oleic acid incorporation into cholesteryl esters were also decreased. These results are in agreement with the general view that epinephrine increases cyclic AMP intracellular level, as it was previously demonstrated that dibutyryl cyclic AMP or isoproterenol treatment of cultured fibroblasts had similar effect on these pathways. The decrease in LDL processing induced by epinephrine could be involved in the worsening effect of epinephrine on atherosclerosis.     

Changes in cholesterol metabolism in cultured fibroblasts from patients with Niemann-Pick disease

Cultured skin fibroblasts from 6 patients with Niemann-Pick disease type A were investigated for cholesterol metabolism. An increase in cholesterol synthesis from ¹⁴C-sodium acetate was observed in all cases. A decrease in ¹⁴C-oleic acid incorporation into cholesteryl esters was found in 5 of 6 cases. ¹²⁵I-low density lipoprotein binding was significantly reduced in 3 of 4 investigated cases.     

Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes

An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).     

Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of circulating lipoprotein triglyceride molecules, is synthesized in myocytes and adipocytes but functions while bound to heparan sulfate proteoglycans (HSPGs) on the luminal surface of vascular endothelial cells. This requires transfer of LPL from the abluminal side to the luminal side of endothelial cells. Studies were performed to investigate the mechanisms of LPL transcytosis using cultured monolayers of bovine aortic endothelial cells. We tested whether HSPGs and members of the low density lipoprotein (LDL) receptor superfamily were involved in transfer of LPL from the basolateral to the apical side of cultured endothelial cells. Heparinase/heparinitase treatment of the basolateral cell surface or addition of heparin to the basolateral medium decreased the movement of LPL. This suggested a requirement for HSPGs. To assess the role of receptors, we used either receptor-associated protein, the 39-kDa inhibitor of ligand binding to the LDL receptor-related protein and the very low density lipoprotein (VLDL) receptor, or specific receptor antibodies. Receptor-associated protein reduced (125)I-LPL and LPL activity transfer across the monolayers. When the basolateral surface of the cells was treated with antibodies, only anti-VLDL receptor antibodies inhibited transcytosis. Moreover, overexpression of the VLDL receptor using adenoviral-mediated gene transfer increased LPL transcytosis. Thus, movement of active LPL across endothelial cells involves both HSPGs and VLDL receptor.     

Characteristics of chylomicron binding and lipid uptake by endothelial cells in culture.

Bovine vascular endothelial cells bind chylomicrons via a high affinity membrane receptor site. Subsequent to binding, the chylomicron apoprotein was neither internalized nor degraded by either sparse or confluent (contact-inhibited) cells. However, the adsorption of chylomicrons was associated with interiorization of chylomicron cholesteryl ester and triglyceride and the hydrolysis of these lipids to free cholesterol and unesterified fatty acids by a lysosome-dependent pathway. This pathway was active in both subconfluent and contact-inhibited cells. The chylomicron free cholesterol so produced inhibited endogeneous cholesterol synthesis measured in terms of the incorporation of [1-14C]-acetate into sterol. An excess of high density lipoprotein was 2- to 3-fold more effective in reducing both binding of chylomicrons and interiorization of chylomicron lipid than was low density lipoprotein. Chylomicron binding was not "down-regulated" by preincubation of the cells with low density lipoprotein or chylomicrons. The results are discussed in the context of cholesterol sources for contact-inhibited endothelial cells which do not interiorize low density lipoprotein cholesterol.