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The low-density lipoprotein (LDL) receptor family consists of several related scavenger receptors that not only function as important cargo transporters, but also inform the cell of changes in its environment by mediating signaling responses. The LDL receptor was the first family member to be characterized and its function seems to be restricted to lipoprotein metabolism. By contrast, lipoprotein metabolism does not appear to be the exclusive function of the other characterized LDL receptor family members. It is now apparent that cargo transport by members of the LDL receptor family is closely associated with regulation of cellular physiology and cellular signaling events. Here, we focus on the diverse biological activities of certain members of this family.

Although very low density lipoprotein (VLDL) receptor (VLDLr) knockout mice have been reported to have no lipoprotein abnormalities, they develop less adipose tissue than control mice when fed a high calorie diet. Mice that are deficient in adipose tissue expression of lipoprotein lipase (LpL) also have less fat, but only when crossed with ob/ob mice. We hypothesized that the VLDLr, a protein that will bind and transport LpL, is required for optimal LpL actions in vivo and that hypertriglyceridemia due to VLDLr deficiency is exacerbated by either LpL deficiency or VLDL overproduction. Fasted VLDLr knockout (VLDLr0) mice were more hypertriglyceridemic than controls (2-fold greater triglyceride levels). The hypertriglyceridemia due to VLDLr0 was even more evident when VLDLr0 mice were crossed with heterozygous LpL-deficient (LpL1) and human apolipoprotein B (apoB) transgenic mice. This was due to an increase in apoB48-containing VLDL. [3H]VLDL turnover studies showed that VLDL-triglyceride clearance in VLDLr0/LpL1 mice was impaired by 50% compared with LpL1 mice. VLDLr0/LpL1 mice had less LpL activity in postheparin plasma, heart, and skeletal muscle. Infection of mice with an adenovirus-expressing receptor-associated protein, an inhibitor of the VLDLr, reduced LpL activity in wild type but not VLDLr0 mice. Therefore, the VLDLr is required for normal LpL regulation in vivo, and the disruption of VLDLr results in hypertriglyceridemia associated with decreased LpL activity.

We examined whether very low-density lipoprotein receptors (VLDLR) on the oocyte membrane contributed to changes in VLDL uptake into the yolk by the ovarian follicle during the ovulation cycle of the laying hen. White and yellow (F5-F1) follicles were collected from laying hens at 14-16 h (when the yellow follicles actively uptake yolk) and 0-2 h (when the yolk uptake is less active) before ovulation and subjected to binding experiments with 125I-VLDL and assays for aromatase activity. The preparations from white follicles did not bind to 125I-VLDL, while yellow follicle preparations bound to the radioligand. The 125I-VLDL bindings of F3-F1 yellow follicles were higher at 14-16 h than 0-2 h before ovulation. Values of the dissociation constant (Kd) and the maximum binding capacity (Bmax) of VLDLR calculated from the 125I-VLDL bindings to the oocyte membranes showed profiles related to the changes in 125I-VLDL bindings; in F3 and F2 follicles Bmax values were higher at 14-16 h than 0-2 h before ovulation and Kd values were higher at 0-2 h than 14-16 h before ovulation. High aromatase activities of the F3-F1 yellow follicles of 14-16 h before ovulation decreased 0-2 h before ovulation, which was a change consistent with the change in 125I-VLDL bindings of the same follicles. These results suggest that the yolk VLDL uptake during the ovulation cycle is regulated by changes in VLDL binding underlined by changes in Bmax and Kd of VLDLR. Estrogen produced by the aromatase of the theca layer of the yellow follicles might facilitate VLDL binding by affecting VLDLR on the oocyte membrane of the same follicles in a paracrine manner.

Background: Salvianolic acid A (SalA) has been shown to display robust protection against endothelial injury. VLDL receptor (VLDLr) is expressed at high levels in the endothelial cells. However its endothelial biological function has not been completely elucidated. Here, we investigated molecular effects of SalA on endothelial VLDLr expression, ER stress, and apoptosis under hypoxia condition. Methods: Human umbilical vein endothelial cells (HUVECs) pretreated with SalA were subjected to hypoxia stimulation. Endothelial ER stress and apoptosis were examined. The mRNA levels were tested by real-time RT-PCR, and the protein levels were determined by immunoblot analysis. Results: Pretreatment of HUVECs with SalA markedly attenuated hypoxia-induced endothelial ER stress and apoptosis. Hypoxia resulted in enhancement of VLDLr expression, which was effectively inhibited by SalA pretreatment. Furthermore, luciferase reporter gene assays indicated that SalA inhibited vldlr gene promoter activity, and ChIP assays showed that hypoxia increase the recruitment of HIF-1α to the vldlr gene promoter, and this process was hampered markedly by pretreatment of SalA. Finally, overexpression of VLDLr abolished SalA-mediated protection of endothelial cells from ER stress and apoptosis. Knockdown of VLDLr mimicked SalA protective effect. Conclusion: These results for the first time demonstrate that SalA protects against hypoxia-induced endothelial ER stress and apoptosis through inhibiting recruitment of HIF-1α to vldlr gene promoter and thus suppressing VLDLr expression.

Tissue factor pathway inhibitor (TFPI) contains three Kunitz-type proteinase inhibitor domains and is a potent inhibitor of tissue factor-mediated coagulation. Here, we report that TFPI inhibits the proliferation of basic fibroblast growth factor-stimulated endothelial cells. A truncated form of TFPI, containing only the first two Kunitz-type proteinase inhibitor domains, has very little antiproliferative activity, suggesting that the carboxyl-terminal region of TFPI is responsible for this activity. Binding studies revealed that full-length TFPI, but not the truncated TFPI molecule, is recognized by the very low density lipoprotein receptor (VLDL receptor) indicating that this receptor is a novel high affinity endothelial cell receptor for TFPI. The antiproliferative activity of TFPI on endothelial cells is inhibited by the receptor-associated protein, a known antagonist of ligand binding by the VLDL receptor, and by anti-VLDL receptor antibodies. These results confirm that the antiproliferative activity of TFPI is mediated by the VLDL receptor and suggest that this receptor-ligand system may be a useful target for the development of new anti-angiogenic and antitumor agents.

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

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.

Plasminogen activator inhibitor 1 (PAI-1) is a major inhibitor of urokinase-type plasminogen activator (uPA). In this study, we explored the role of PAI-1 in cell signaling. In MCF-7 cells, PAI-1 did not directly activate the mitogen-activated protein (MAP) kinases, extracellular signal–regulated kinase (ERK) 1 and ERK2, but instead altered the response to uPA so that ERK phosphorylation was sustained. This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr). When MCF-7 cells were treated with uPA–PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA–PAI-1R76E complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (By sustaining ERK phosphorylation, PAI-1 converted uPA into an MCF-7 cell mitogen. This activity was blocked by receptor-associated protein and not observed with uPA–PAI-1R76E complex, demonstrating the importance of the VLDLr. uPA promoted the growth of other cells in which ERK phosphorylation was sustained, including β3 integrin overexpressing MCF-7 cells and HT 1080 cells. The MEK inhibitor, PD098059, blocked the growth-promoting activity of uPA and uPA–PAI-1 complex in these cells. Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment. The kinetics of ERK phosphorylation in response to uPAR ligation determine the function of uPA and uPA–PAI-1 complex as growth promoters.

The low-density lipoprotein receptor (LDLR) family is composed of a class of single transmembrane glycoproteins, generally recognized as cell surface endocytic receptors, which bind and internalize extracellular ligands for degradation by lysosomes. Structurally, members of the LDLR family share homology within their extracellular domains, which are highlighted by the presence of clusters of ligand-binding repeats. Recently, information regarding the structural and functional elements within their cytoplasmic tails has begun to emerge, which suggests that members of the LDLR family function not only in receptor-mediated endocytosis, but also in transducing signals that are important during embryonic development and the pathogenesis of Alzheimer’s disease. This review focuses on recent knowledge of the structural and functional aspects of LDLR family members in endocytosis and signal transduction. The relationship of these functions to the development of the neuronal system and in the pathogenesis of Alzheimer’s disease is specifically discussed.

The formation of complexes between the minor receptor group human rhinovirus HRV2 and two recombinant soluble receptor fragments derived from the human very low density lipoprotein receptor (VLDLR) and containing ligand-binding repeats 1–3 (MBP·VLDLR1–3) or 1–8 (MBP·VLDLR1–8) fused to the carboxyl terminus of the maltose-binding protein was analyzed by affinity capillary electrophoresis. At low molar ratios of receptor/virus, the peaks corresponding to substoichiometric complexes were broad indicating heterogeneity. When the receptors were present in molar excess with respect to the virus, the peaks were sharp, suggesting saturation of all binding sites. For the determination of the stoichiometry, constant amounts of receptor were incubated with increasing amounts of virus, and the peak areas corresponding to free receptor were measured and plotted versus total virus concentration. Extrapolation of the linear part of the resulting curve to zero concentration of free receptor enabled quantitation of the molar ratios of the components present in the complex. Using this method, we determined that about 60 molecules of MBP·VLDLR1–3 but only about 30 molecules of MBP·VLDLR1–8 were bound per virion.

In order to determine the contribution of the low density lipoprotein receptor (LDL-R) to the removal of apoB-containing native lipoproteins by macrophages, we compared the uptake of beta-VLDL in peritoneal macrophages (MPM) from wild type mice and mice lacking the LDL-R. The d

Apolipoprotein E (apoE) polymorphism is associated with onset of Alzheimer's disease (AD). We found seven polymorphisms in apoE receptor 2 (ApoER2), an apoE-binding receptor, in Japanese sporadic AD patients, but no association of ApoER2 polymorphisms with AD. We consider that the functions of ApoER2 in the brain may be compensated for by those of other apoE-binding receptors such as VLDL receptor.

The discovery of an ever growing number of low density lipoprotein (LDL) receptor gene family members has triggered research into many different directions. Here we first summarize the results of classical studies on the role of the LDL receptor in cholesterol transport, the structure/function relationships delineated with the help of LDL receptor mutations in familial hypercholesterolemia, and the elegant way in which cells regulate cholesterol at the transcriptional level. The second part deals with a multifunctional, structurally very close relative, the very low density lipoprotein (VLDL) receptor. While it is involved in lipoprotein transport in certain tissues and species, detailed studies on its function have generated new knowledge about the growing spectrum of ligands and about exciting and unexpected aspects of receptor biology. In particular, these investigations have elucidated the roles of LDL receptor gene family members in ligand-mediated signal transduction. In the third part of this review article, we provide first insight into the roles of the VLDL receptor and of another small relative, the so-called apolipoprotein E receptor-2, in such signaling processes. These findings suggest that to date, only the tip of an iceberg has been uncovered.

Correct positioning of neurons during embryonic development of the brain depends, among other processes, on the proper transmission of the reelin signal into the migrating cells via the interplay of its receptors with cytoplasmic signal transducers. Cellular components of this signaling pathway characterized to date are cell surface receptors for reelin like apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR), and cadherin-related neuronal receptors, and intracellular components like Disabled-1 and the nonreceptor tyrosine kinase Fyn, which bind to the intracellular domains of the ApoER2 and VLDL receptor or of cadherin-related neuronal receptors, respectively. Here we show that ApoER2, but not VLDLR, also binds the family of JNK-interacting proteins (JIPs), which act as molecular scaffolds for the JNK-signaling pathway. The ApoER2 binding domain on JIP-2 does not overlap with the binding sites for MLK3, MKK7, and JNK. These results suggest that ApoER2 is able to assemble a multiprotein complex containing Disabled-1 and JIPs, together with their binding partners, to the cell surface of neurons. This complex might participate in ApoER2-specific reelin signaling and thus would explain the different phenotype of mice lacking the ApoER2 from that of VLDLR-deficient mice.

Although a large amount of cholesterol is known to be needed for brain maturation and differentiation, cholesterol metabolism during these periods remains unclear. To elucidate the developmental regulation of cholesterol metabolism in the brain, we investigated the expression of 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), low-density-lipoprotein (LDL) receptor and very-low-density-lipoprotein (VLDL)/apolipoprotein E (apo E) receptor (VLDL receptor) using RNase protection assay (RPA) to quantitate mRNA levels in mouse brain, liver and kidney during development. Messenger RNA levels of HMG-CoA reductase in the brain decreased with age, and those levels at -5 (5 days before birth) and 5 days after birth were significantly higher than the control level of adult mice. The period from -5 to 5 days might correspond to stages of active biogenesis of the membranes of brain cells. The mRNA level of HMG-CoA reductase in the liver was also high at -5 days; a finding that correlated with cell proliferation. On the other hand, mRNA levels of the LDL and VLDL receptors in the brain did not change markedly during development. These results suggest that de novo cholesterol biosynthesis in brain cells plays a major role in the supply of cholesterol to the developing brain, rather than the uptake of cholesterol from serum lipoproteins through lipoprotein receptors.

Objectives: The metabolism of triglyceride-rich lipoproteins (TRL) is, in part, mediated by lipoprotein receptors (such as low density lipoprotein receptor-related protein [LRP] and very low density lipoprotein [VLDL] receptors), which recognize TRL remnants after specific binding with apolipoprotein E. The purpose of this study was to explore the association of the genetic polymorphisms of remnant receptors with lipid, lipoprotein, and apolipoprotein levels including remnant-like particle-cholesterol (RLP-C). Design and Methods: Using polymerase chain reaction-amplified DNA, VLDL receptor tetranucleotide repeat polymorphism, LRP trinucleotide repeat polymorphism, and LRP exon 3 polymorphism were analyzed in normal adults (control group: n = 161) and in patients with coronary artery disease (CAD group: n = 102). Results: The allelic distributions of VLDL receptor triple repeat polymorphism, LRP tetranucleotide repeat polymorphism, and LRP exon 3 polymorphism in Koreans were similar to those of Japanese but were significantly different from those of other ethnic groups. There were no significant differences in the allele frequencies of the polymorphisms between the control and CAD groups. VLDL receptor polymorphism in the control group (p = 0.0403) and LRP exon 3 polymorphism in the CAD group ( p = 0.0459) showed significant associations with lipoprotein (a) [Lp(a)] levels. Conclusions: The results of the present study demonstrated significant interracial distribution of remnant receptor polymorphisms. There was no association between the remnant receptor polymorphisms and the RLP-C levels. However, the polymorphisms showed a significant association with Lp(a), which may suggest that the Lp(a) metabolism is in part mediated by the uptake through the remnant receptors.

A major gene effect on the fasting insulin level and insulin resistance has been suggested in previous studies. Several candidate genes for insulin resistance in rare syndromes have been proposed. However, there has been limited success in finding genes for common forms of insulin resistance. There is accumulating evidence of a relationship between insulin resistance and a disturbance of free fatty acid (FFA) metabolism. The very-low-density lipoprotein (VLDL) receptor, which is associated with FFA metabolism, could serve as a possible candidate gene for insulin resistance. We performed linkage analyses between the VLDL receptor gene and fasting insulin and the homeostasis model assessment (HOMA) insulin resistance index (fasting insulin x fasting glucose/22.5) in 1,050 sibpairs participating in the phase II physical examination of the National Heart, Lung, and Blood Institute Family Heart Study (FHS). Data analyses were completed using the SIBPAL component of the SAGE software package (SAGE, Statistical Analysis for Genetic Epidemiology, Version 3.1; Computer program package available from the Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH, 1997). We did not find evidence for linkage of the fasting insulin or the HOMA insulin resistance index with a polymorphic marker at the VLDL locus (P = .316 and .402, respectively). Adjustment of fasting insulin and the HOMA insulin resistance index for the body mass index (BMI) did not change the results (P = .319 and .472, respectively). In conclusion, no evidence was found for a linkage between a locus controlling the fasting insulin level or HOMA insulin resistance index and a VLDL polymorphism in the present study. Additional adjustment of fasting insulin or the HOMA insulin resistance index for the BMI did not change the linkage results significantly.

The low density lipoprotein receptor-related protein (LRP) has been reported to regulate cellular migration. In this study, an antisense RNA expression strategy was used to reduce LRP to undetectable levels in HT 1080 fibrosarcoma cells. The LRP-deficient cells demonstrated increased levels of cell-surface uPAR, higher levels of uPA in conditioned medium, increased migration on vitronectin-coated surfaces, and increased invasion of Matrigel. LRP-deficient cells also demonstrated increased levels of phosphorylated extracellular signal-regulated kinase (ERK) in the absence of exogenous stimulants. Antibodies which block binding of endogenously produced uPA to uPAR reduced ERK phosphorylation and migration of LRP-deficient cells to the levels observed with control cells. Inhibitors of ERK activation, including PD098059 and dominant-negative MEK1, also decreased the migration of LRP-deficient but not control cells. By contrast, constitutively active MEK1 stimulated the migration of control but not LRP-deficient cells. Although Matrigel invasion by LRP-deficient cells was inhibited by the proteinase inhibitor, aprotinin, PD098059 in combination with aprotinin was necessary for an optimal effect. Expression of the VLDL receptor in LRP-deficient cells reversed the changes in cellular migration and invasion. These studies demonstrate that binding of endogenously produced uPA to uPAR may serve as a major determinant of basal levels of activated ERK and, by this mechanism, regulate cellular migration and invasion. By regulating the uPA/uPAR system, LRP may also regulate ERK activation, cellular migration, and invasion.

Rat HDL are known to increase testosterone production by cultured Leydig cells either following gonadotropin stimulation or cholesteryl ester depletion. However, rat HDL contain apolipoprotein E and have a high affinity for the members of the low density receptor family such as LDL receptor, LDL receptor related protein and VLDL receptor. In contrast with the adrenal cells, the contribution of apo A-I and apo E pathways in HDL cholesterol uptake has not been yet evidenced in rat Leydig cells. Recent data provided evidence that hCG stimulates scavenger receptor BI expression in testes. In order to investigate if testosterone production can be stimulated by apo E depleted HDL, we compared the level of testosterone stimulation by HDL with or without apo E first, in presence of saturating dose of hCG (1 IU/ml) and second, after depletion of cholesterol synthesis by pravastatin, an inhibitor of HMG-CoA reductase. In presence of hCG, HDL with or without apo E increased testosterone production respectively by 37 and 25%. Pravastatin at 100 μg/ml inhibited the cholesterol synthesis and the testosterone production by 25% and decreased the cholesteryl content by 25%. The addition of HDL with or without apo E (50 μg protein HDL/ml) completely overcame the depletion of cellular cholesteryl esters and the inhibition of testosterone production induced by pravastatin. In the presence of heparin, apo E depleted HDL overcame the testosterone production induced by pravastatin, indicating that uptake of HDL without apo E via a secretion of apo E by the cells themselves was not involved. Therefore, in absence of apo E, it is suggested that rat Leydig cells used HDL to regulate steroidogenesis via an apolipoprotein A-I pathway.

The potential role of the very low density lipo- protein (VLDL) receptor in mediating VLDL-induced plas- minogen activator inhibitor-1 (PAI-1) expression was stud- ied in vitro. Cultured endothelial cells incubated with VLDL showed an increased secretion of PAI-1. This response to VLDL could be completely prevented by the receptor-asso- ciated protein (RAP) and partially blocked by rabbit poly- clonal anti-VLDL receptor IgG. Furthermore, Chinese ham- ster ovary (CHO) control cells and cells overexpressing the VLDL receptor were transiently transfected with a PAI-1 promoter-reporter construct and incubated with VLDL. The PAI-1 promoter activity in response to VLDL was signif- icantly higher in the VLDL receptor overexpressing cells compared to the control cells. Addition of RAP completely blocked the VLDL-activated PAI-1 transcription. Electromo- bility shift assay was performed to investigate whether the enhanced PAI-1 promoter activity seen in the VLDL recep- tor overexpressing cells in response to VLDL involved in- duction of the previously described VLDL-inducible fac- tor(s) binding to the 2 675 to 2 653 region of the PAI-1 promoter. We found that the binding of the VLDL-inducible factor in VLDL receptor overexpressing cells was markedly enhanced by addition of VLDL as compared to control cells where no increased binding could be seen in response to VLDL. In summary, these results indicate that the VLDL receptor is a strong candidate for mediating VLDL effects on PAI-1 synthesis and secretion in cells expressing this re- ceptor.— Nilsson, L., M. Gafvels, L. Musakka, K. Ensler, D. K. Strickland, B. Angelin, A. Hamsten, and P. Eriksson. VLDL activation of plasminogen activator inhibitor-1 (PAI-1) ex- pression: involvement of the VLDL receptor. J. Lipid Res. 1999. 40: 913-919.

We have shown previously that human apolipoprotein (apo)C1 transgenic mice exhibit hyperlipidaemia, due primarily to an impaired clearance of very-low-density lipoprotein (VLDL) particles from the circulation. In the absence of at least the low-density-lipoprotein receptor (LDLR), it was shown that APOC1 overexpression in transgenic mice inhibited the hepatic uptake of VLDL via the LDLR-related protein. In the present study, we have now examined the effect of apoC1 on the binding of lipoproteins to both the VLDL receptor (VLDLR) and the LDLR. The binding specificity of the VLDLR and LDLR for apoC1-enriched lipoprotein particles was examined in vivo through adenovirus-mediated gene transfer of the VLDLR and the LDLR [giving rise to adenovirus-containing (Ad)-VLDLR and Ad-LDLR respectively] in APOC1 transgenic mice, LDLR-deficient (LDLR-/-) mice and wild-type mice. Remarkably, Ad-VLDLR treatment did not reduce hyperlipidaemia in transgenic mice overexpressing human APOC1, irrespective of both the level of transgenic expression and the presence of the LDLR, whereas Ad-VLDLR treatment did reverse hyperlipidaemia in LDLR-/- and wild-type mice. On the other hand, Ad-LDLR treatment strongly decreased plasma lipid levels in these APOC1 transgenic mice. These results suggest that apoC1 inhibits the clearance of lipoprotein particles via the VLDLR, but not via the LDLR. This hypothesis is corroborated by in vitro binding studies. Chinese hamster ovary (CHO) cells expressing the VLDLR (CHO-VLDLR) or LDLR (CHO-LDLR) bound less APOC1 transgenic VLDL than wild-type VLDL. Intriguingly, however, enrichment with apoE enhanced dose-dependently the binding of wild-type VLDL to CHO-VLDLR cells (up to 5-fold), whereas apoE did not enhance the binding of APOC1 transgenic VLDL to these cells. In contrast, for binding to CHO-LDLR cells, both wild-type and APOC1 transgenic VLDL were stimulated upon enrichment with apoE. From these studies, we conclude that apoC1 specifically inhibits the apoE-mediated binding of triacylglycerol-rich lipoprotein particles to the VLDLR, whereas apoC1-enriched lipoproteins can still bind to the LDLR. The variability in specificity of these lipoprotein receptors for apoC1-containing lipoprotein particles provides further evidence for a regulatory role of apoC1 in the delivery of lipoprotein constituents to different tissues on which these receptors are located.

The epsilon4 allele of the apolipoprotein E gene (APOE) constitutes a major genetic susceptibility factor for Alzheimer disease (AD). Recently, a polymorphic triplet (CGG) in the very low density lipoprotein receptor (VLDL-R) gene, coding for a receptor binding only apoE-containing lipoproteins, was associated with AD in a Japanese population but not in Caucasian American populations. We explored this association and the potential interaction with the APOE polymorphism in a Caucasian sample of sporadic AD and control subjects of similar ages of European origin. The allelic distribution of the VLDL-R polymorphism differed significantly between Japanese and Caucasian populations (p < 0.0001). However, in our population, the presence of at least one VLDL-R 5 repeat allele increased significantly (p < 0.0003) the probability to develop AD after 65 years of age in APOE epsilon4 allele bearers.

The discovery in 1992 of a member of the low density lipoprotein receptor (LDLR) family with eight ligand binding repeats (LR8) has raised more questions than have been answered to date. Here, we summarize the current status of knowledge about this intriguing molecule, generally termed VLDL receptor, at the molecular biological, cell biological, and physiological levels. On one hand, the wealth of reports concerning the role(s) of this receptor in lipoprotein metabolism in mammalian systems has revealed partially conflicting details, particularly in regards to its natural ligand(s) and site of action. On the other hand, molecular genetic and biochemical studies in the chicken have clearly demonstrated the multiple roles of LR8 in the physiology and reproduction of egg-laying species, and have generated insights into the evolutionary aspects of the LDLR gene family.

The very low density lipoprotein (VLDL) recep- tor is a member of the low density lipoprotein supergene family of receptors in which differential splicing of mRNA has been reported. We present several lines of evidence showing that bovine aortic endothelial cells exclusively ex- press a VLDL receptor isoform that lacks the O-linked sugar domain i ) Western and receptor-associated protein (RAP) ligand blotting gave a single band of about 99 kDa in membrane extracts of bovine aortic endothelial cells (BAEC). ii ) Screening of the BAEC cDNA library with the previously characterized human VLDL receptor cDNA as a probe gave several C-terminal-positive clones; all lacked the 84 nucleotides corresponding to exon 16. Polymerase chain reaction (PCR) confirmed that VLDL receptor cDNA en- coding exon 16 was absent from the library. iii ) Reverse transcription (RT)-PCR analysis of the BAEC mRNA using a pair of oligonucleotide primers that flank the deletion gave only one band of 136 nt. iv ) Semiquantitative RT-PCR anal- ysis showed that only the non-O-glycosylated variant was ex- pressed in BAEC. Cell-binding studies with antibodies against the N-terminal domain showed that the BAEC VLDL receptor is present at the plasma membrane, suggesting that the non-glycosylated variant could be functional. In addi- tion, RT-PCR performed in bovine tissues showed that the variant containing the O-linked sugar domain is preferen- tially expressed in heart, brain, and skeletal muscle, whereas the non-O-glycosylated spliced variant is found in all tissues analyzed. Taken together these results suggest that the differential splicing of the VLDL receptor is cell- and tissue- specific and that the functions of the receptor could de- pend on the cell type.— Magrane, J., M. Reina, R. Pagan, A. Luna, R. P. Casaroli-Marano, B. Angelin, M. Gafvels, and S. Vilaro. Bovine aortic endothelial cells express a variant of the very low density lipoprotein receptor that lacks the O- linked sugar domain. J. Lipid Res. 1998. 39: 2172-2181.

The very low density lipoprotein receptor (VLDLR) gene contains an exon encoding a region of clustered serine and threonine residues immediately outside the membrane-spanning sequence, and this region has been proposed to be the site of clustered O-linked carbohydrate chains. Two forms of VLDLR transcripts, with and without the O-linked sugar region, are generated through alternative splicing. Reverse transcription polymerase chain reaction with RNAs from various rabbit tissues revealed that the VLDLR transcript with the O-linked sugar region (type-1 VLDLR) is the major transcript in heart and muscle, while the VLDLR transcript without the O-linked sugar region (type-2 VLDLR) predominates in non-muscle tissues, including cerebrum, cerebellum, kidney, spleen, adrenal gland, testis, ovary, and uterus. Hamster fibroblasts expressing type-2 VLDLR bound with relatively low affinity to beta-migrating very low density lipoprotein compared with type-1 VLDLR-transfected cells. In contrast, the internalization, dissociation, and degradation of the ligand were not significantly impaired in either type of VLDLR-transfected cell. The receptor proteins in type-2 VLDLR-transfected cells underwent rapid degradation and accumulated in the culture medium, while those in type-1 VLDLR-transfected cells were stable and resistant to proteolytic cleavage. Analysis of the O-linked sugars of both types of transfected cells suggested that the O-linked sugar region is the major site for O-glycosylation.

Research into lipoprotein metabolism has developed because understanding lipoprotein metabolism has important clinical indications. Lipoproteins are risk factors for cardiovascular disease. Recent advances include the identification of factors in the synthesis and secretion of triglyceride rich lipoproteins, chylomicrons (CM) and very low density lipoproteins (VLDL). These included the identification of microsomal transfer protein, the cotranslational targeting of apoproteinB (apoB) for degradation regulated by the availability of lipids, and the characterization of transport vesicles transporting primordial apoB containing particles to the Golgi. The lipase maturation factor 1, glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 and an angiopoietin-like protein play a role in lipoprotein lipase (LPL)-mediated hydrolysis of secreted CMs and VLDL so that the right amount of fatty acid is delivered to the right tissue at the right time. Expression of the low density lipoprotein (LDL) receptor is regulated at both transcriptional and post-transcriptional level. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has a pivotal role in the degradation of LDL receptor. Plasma remnant lipoproteins bind to specific receptors in the liver, the LDL receptor, VLDL receptor and LDL receptor-like proteins prior to removal from the plasma. Reverse cholesterol transport occurs when lipid free apoAI recruits cholesterol and phospholipid to assemble high density lipoprotein (HDL) particles. The discovery of ABC transporters (ABCA1 and ABCG1) and scavenger receptor class B type I (SR-BI) provided further information on the biogenesis of HDL. In humans HDL-cholesterol can be returned to the liver either by direct uptake by SR-BI or through cholesteryl ester transfer protein exchange of cholesteryl ester for triglycerides in apoB lipoproteins, followed by hepatic uptake of apoB containing particles. Cholesterol content in cells is regulated by several transcription factors, including the liver X receptor and sterol regulatory element binding protein. This review summarizes recent advances in knowledge of the molecular mechanisms regulating lipoprotein metabolism.

Lp(a) is a major inherited risk factor associated with premature heart disease and stroke. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to influence plasminogen activation as well as its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that fibroblasts expressing the human VLDL receptor can mediate endocytosis of Lp(a), leading to its degradation within lysosomes. In contrast, fibroblasts deficient in this receptor are not effective in catabolizing Lp(a). Lp(a) degradation was prevented by antibodies against the VLDL receptor, and by RAP, an antagonist of ligand binding to the VLDL receptor. Catabolism of Lp(a) was inhibited by apolipoprotein(a), but not by LDL or by monoclonal antibodies against apoB100 that block LDL binding to the LDL receptor, indicating that apolipoprotein(a) mediates Lp(a) binding to this receptor. Removal of Lp(a) antigen from the mouse circulation was delayed in mice deficient in the VLDL receptor when compared with control mice, indicating that the VLDL receptor may play an important role in Lp(a) catabolism in vivo. We also demonstrate the expression of the VLDL receptor in macrophages present in human atherosclerotic lesions. The ability of the VLDL receptor to mediate endocytosis of Lp(a) could lead to cellular accumulation of lipid within macrophages, and may represent a molecular basis for the atherogenic effects of Lp(a).

Lipoprotein lipase (LPL) promotes the binding and internalization of beta-VLDL (very low density lipoprotein) by many cell types. We examined the function of receptors in the LDL receptor family (LRF) and heparan sulfate proteoglycans (HSPG) in the metabolism of LPL-associated beta-VLDLa by rat vascular smooth muscle cells (VSMCs) in culture. These cells express LDL receptor-related protein and the VLDL receptor, but not the LDL receptor. LPL greatly increased the binding of 125I-labeled beta-VLDL to VSMCs at 4 degrees C. Binding was almost entirely inhibited by heparin, but essentially unaffected by the potent LRF-antagonist, receptor-associated protein (RAP), indicating that LRFs do not contribute significantly to the VSMC binding capacity for LPL-associated beta-VLDL. At 37 degrees C, RAP inhibited the rapid internalization of LPL-associated 125I-labeled beta-VLDL and the digestion of the beta-VLDL into trichloroacetic acid soluble radioactivity; these processes still occurred, but at a decreased rate. RAP did not inhibit the ability of beta-VLDL-LPL complex to stimulate VSMC ACAT activity. Furthermore, in Oil red-O histochemistry experiments, which model foam cell transformation in vitro, RAP paradoxically increased cholesteryl ester storage in VSMCs treated with beta-VLDL and LPL under specific cell culture conditions. These results support a model in which the internalization of LPL-associated beta-VLDL by VSMCs is mediated by two pathways, one involving LRFs and a second that is independent of LRFs, probably involving direct uptake by HSPG. The LRF-dependent pathway leads to less cellular storage of cholesteryl ester and thus may be antiatherogenic under certain conditions.

The properties of the very-low-density lipoprotein (VLDL) receptor have been studied in Chinese hamster ovary (CHO) cells stably transfected with human VLDL-receptor cDNA and compared with those of the low-density lipoprotein (LDL) receptor expressed under the same conditions. Immunoblotting showed that the cells produced a mature VLDL receptor protein, of apparent Mr 123000 on non-reduced and 158000 on reduced gels, that was less extensively glycosylated than the LDL receptor. The VLDL receptor was more slowly processed than the LDL receptor, with only approx. 70% of the precursor being converted into the mature protein. Nevertheless, the majority of the receptor in the cells was in the mature form, and most of this was present on the cell surface. The human VLDL receptor bound rabbit very-low-density lipoprotein with beta electrophoretic mobility (betaVLDL), but not human LDL, and uptake through the receptor led to stimulation of oleate incorporation into cholesteryl esters. At 37 degrees C, the characteristics of VLDL-receptor-mediated uptake and degradation of betaVLDL were essentially the same as those mediated by the LDL receptor. However, the VLDL receptor apparently did not show the increase in affinity and decrease in binding of betaVLDL on cooling to 4 degrees C that was exhibited by the LDL receptor. Thus the overexpressed VLDL receptor in CHO cells appears to behave as a lipoprotein receptor with similar, but not identical, properties to the LDL receptor.

Acquired VLDL receptor deficiency in experimental nephrosis. Nephrotic syndrome (NS) is commonly associated with elevation of plasma very low density lipoprotein (VLDL) and triglyceride concentrations. VLDL receptor (VLDL-R) is a novel protein that specifically binds and internalizes VLDL particles and is primarily distributed in heart, skeletal muscle, brain and adipose tissue. Based on these properties, VLDL-R is thought to play a role in VLDL and triglyceride metabolism. The present study was undertaken to test the hypothesis that elevation of plasma VLDL in NS may be, in part, related to VLDL-R deficiency. To this end, heart and skeletal muscle VLDL-R protein (Western blot) and mRNA (Northern blot) were measured at various points in the course of puromycin-induced NS in rats. The results were compared with those obtained in the placebo-treated normal control animals. The NS group showed a significant decline in VLDL-R protein (relative to total plasma membrane protein mass) in the heart and skeletal muscle paralleling the rise in plasma VLDL and triglyceride concentrations. The fall in VLDL-R protein was accompanied by a parallel decline in VLDL-R mRNA in the heart but not skeletal muscle. VLDL-R protein was directly related to proteinuria and inversely related to plasma VLDL and triglyceride concentrations. In conclusion, puromycin-induced NS in rats is associated with profound reduction in heart and skeletal muscle VLDL receptor protein. Acquired VLDL-R deficiency, shown for the first time here, may contribute to elevation of plasma concentration of triglyceriderich VLDL in the nephrotic rat. Recognition of this abnormality reveals another dimension of the complex dysregulation of lipid metabolism in NS. The precise mechanism responsible for NS-induced VLDL-R deficiency in this model is not clear and awaits further investigation.

Down-regulation of VLDL receptor expression in chronic experimental renal failure. VLDL receptor (VLDL-R) is a novel member of the LDL receptor gene family with distinct tissue distribution and function. It binds and internalizes VLDL particles and is primarily expressed in skeletal muscle, heart, brain and adipose tissue, which use fatty acids for energy production or storage. CRF is associated with elevated serum triglyceride and VLDL concentrations and depressed VLDL and chylomicron clearance. We have recently shown marked down-regulation of lipoprotein lipase expression in CRF. This study was conducted to test the hypothesis that VLDL-R expression may be similarly depressed in CRF. To this end, VLDL-R mRNA (Northern blot) and protein mass (Western blot) of skeletal muscle (soleus) and heart were measured in male Sprague-Dawley rats six weeks after 5/6 nephrectomy (CRF group) or sham operation (NL group). A group of erythropoietin (EPO)-treated (150 U/kg twice weekly) CRF animals was included to determine the possible effect of EPO-deficiency anemia (EPO-CRF group). Subgroups of animals were studied at weeks 1, 3 and 6. The CRF group showed a fivefold increase in plasma triglyceride concentration. This was associated with an impressive fourfold reduction in heart and skeletal muscle VLDL-R mRNA and protein mass. VLDL-R mRNA levels in the heart and skeletal muscle were directly related to creatinine clearance and inversely related to serum triglyceride and VLDL concentrations. EPO therapy led to a mild improvement in CRF hypertriglyceridemia but failed to improve VLDL-R expression. Thus, the rise in plasma triglyceride and VLDL concentrations in CRF animals was associated with marked down-regulation of VLDL-R expression. Down-regulation of VLDL-R expression, shown here for the first time, reveals another facet of disturbed lipid metabolism in CRF.

SummaryThe plasminogen activation system is thought to be important in cell migration processes. A role for this system during smooth muscle cell migration after vascular injury has been suggested from several animal studies. However, not much is known about its involvement in human vascular remodelling. We studied the involvement of the plasminogen activation system in human smooth muscle cell migration in more detail using an in vitro wound assay and a matrix invasion assay. Inhibition of plasmin activity or inhibition of urokinase-type plasminogen activator (u-PA) activity resulted in approximately 40% reduction of migration after 24 h in the wound assay and an even stronger reduction (70-80%) in the matrix invasion assay. Migration of smooth muscle cells in the presence of inhibitory antibodies against tissue-type plasminogen activator (t-PA) was not significantly reduced after 24 h, but after 48 h a 30% reduction of migration was observed, whereas in the matrix invasion assay a 50% reduction in invasion was observed already after 24 h. Prevention of the interaction of u-PA with cell surface receptors by addition of soluble u-PA receptor or α2-macroglobulin receptor associated protein (RAP) to the culture medium, resulted in a similar inhibition of migration and invasion. From these results it can be concluded that both u-PA and t-PA mediated plasminogen activation can contribute to in vitro human smooth muscle cell migration and invasion. Furthermore, the interaction between u-PA and its cell surface receptor appears also to be involved in this migration and invasion process. The inhibitory effects on migration and invasion by the addition of RAP suggests an involvement of a RAP sensitive receptor of the LDL receptor family, possibly the LDL-receptor related protein (LRP) and/or the VLDL receptor.

Atherosclerotic lesions are characterized by the presence of lipid-loaded cells that are derived from monocyte/macrophages and smooth muscle cells. At least four major lipoprotein receptors have been characterized that could be involved in arterial lipid uptake. These receptors include: scavenger receptor(s), the LDL receptor-related protein/alpha 2-macroglobulin receptor, the LDL receptor and the VLDL receptor. With the exception of the LDL receptor, all of these receptors are expressed in atherosclerotic lesions. Whereas scavenger receptors are mostly expressed in macrophages, the LDL receptor-related protein may play an important role in mediating lipid uptake in smooth muscle cells. It appears that no single receptor pathway is solely responsible for the increased lipid uptake in lesion cells, but several redundant mechanisms may contribute to the uptake and degradation of lipoproteins in atherosclerotic lesions.

To elucidate the regulation of very low density lipoprotein (VLDL) receptor gene expression, we administered to rabbits for 14 days gemfibrozil, a fabric acid derivative and a lipid lowering drug that is also included among peroxisome proliferators. VLDL receptor mRNA levels were examined by Northern blot analysis. The VLDL receptor mRNA levels in retroperitoneal adipose tissue and in gastrocnemius muscle were increased 6.9-fold and 3.7-fold, respectively, with gemfibrozil treatment, but no marked changes were observed in the heart, the organ in which VLDL receptor is most highly expressed. In the liver, VLDL receptor mRNA was not detected either before or after gemfibrozil administration. Lipoprotein lipase (LPL) and long-chain acyl coenzyme A synthetase (ACS) mRNA levels were also increased in parallel in adipose tissue. The enhanced expression of VLDL receptor mRNA may contribute to the increase of triglyceride-rich lipoprotein catabolism in peripheral tissues such as adipose tissue and muscles.

The plasminogen activation system is known to be involved in several cell migration processes. A role for plasminogen activators in smooth muscle cell migration after vascular injury has been suggested from several animal studies. However, not much is known about their involvement in human vascular remodelling. In this study, the role of the plasminogen activation system in human smooth muscle cells was investigated in more detail using an in vitro wound assay. Confluent cultures were wounded by stripping the cells away from their extracellular matrix with the use of Millipore strips. After wounding, cells start to migrate from the wounded edge into the denuded area. Migration was followed during 24 hours and quantified by counting the number of cells that migrated into the denuded area. Migration was inhibited to 61.9 + 7.1 % of control by the addition of the plasmin inhibitor aprotinin (100 KIU/mL) to the culture medium. Blocking the activity of u-PA or t-PA using specific polyclonal antibodies reduced migration to 57.8 ±6.1% with anti-uPA and had no significant effect on migration with anti-tPA. Interference in the uPA/uPAR interaction by addition of soluble uPAR had a similar effect as for anti-uPA. A possible involvement of LRP or the VLDL receptor was shown by adding the receptor antagonist RAP to the culture medium, which inhibited migration to 61.0 ± 7.9 % of control. These results suggest an involvement of the plasminogen activation system and particularly surface bound u-PA in human smooth muscle cell migration.

Liver directed gene transfer with adenoviral vectors is being considered for the treatment of several metabolic diseases, including familial hypercholesterolaemia (FH). Gene replacement therapy of human low density lipoprotein (LDL) receptor gene into the murine model of FH transiently corrected the dyslipidaemia; however, humoral and cellular immune responses to LDL receptor developed--possibly contributing to the associated hepatitis and extinguishing of transgene expression. We evaluated an alternative strategy of ectopic expression in the liver of the very low density lipoprotein (VLDL) receptor, which is homologous to the LDL receptor but has a different pattern of expression. Infusion of recombinant adenoviruses containing the VLDL receptor gene corrected the dsylipidaemia in the FH mouse and circumvented immune responses to the transgene leading to a more prolonged metabolic correction.

Isolation and characterization of a human cDNA demonstrated a novel lipoprotein receptor designated apolipoprotein E receptor 2 (apoER2). The new receptor consists of five functional domains resembling the low density lipoprotein (LDL) and very low density lipoprotein (VLDL) receptors. LDL receptor deficient Chinese hamster ovary cells expressing human apoER2 bound apoE rich beta-migrating VLDL with high affinity and internalized. LDL was bound with much lower affinity to these cells. The 4.5- and 8.5-kb mRNAs for the receptor were most highly expressed in human brain and placenta. In rabbit tissues, multiple species of the mRNA with 4, 4.5, 5.5, 8.5, and 11 kb were detected most intensely in brain and testis and, to a much lesser extent, in ovary, but were undetectable in other tissues. In rat adrenal pheochromocytoma PC12 cells, the receptor mRNA was induced by treatment of the cells with nerve growth factor. The receptor transcripts were detectable most intensely in the cerebellar cortex, choroid plexus, ependyma, hippocampus, olfactory bulb and, to a much lesser extent, in the cerebral cortex as revealed by in situ hybridization histochemistry. In the cerebellar cortex, the receptor transcripts were densely deposited in Purkinje cell somata.

Abstract In this article we describe the cellular distribution of the very low density lipoprotein receptor (VLDLR), a transmembrane protein that is expressed at high concentrations in skeletal muscle, heart, adipose tissue, and brain but in only trace amounts in the liver. Indirect immunofluorescence localization studies were performed in murine and bovine tissues using a rabbit polyclonal anti-human VLDLR antibody. Immunoreactive VLDLR protein was detected in the endothelium of capillaries and small arterioles but not in veins or venules of bovine skeletal muscle, heart, ovary, and brain. In the liver, there was intense staining of the capillaries and arterioles that supply the capsule and hepatic vessels but no staining of the sinusoidal surfaces. We failed to detect any signal from nonendothelial cells in the liver or peripheral organs. The VLDLR was also expressed at high levels on the endothelial surface of bovine coronary arteries; in contrast, little or no staining was seen in aortic endothelium. Antibody staining of cultured bovine coronary artery endothelial cells demonstrated punctate cell-surface staining, as well as staining of large and small cytoplasmic vesicles. This tissue and cell pattern of expression suggests that the VLDLR plays a role in the transport of VLDL or another plasma constituent from the vascular compartment to adjacent tissues.

The VLDL receptor is made up of five functional domains that resemble the LDL receptor. In mammals, the receptor is highly expressed in muscle and fat cells, while in chicken, it is abundant in oocytes. The extremely high degree of amino acid conservation of the VLDL receptor during the evolution suggests that the receptor plays an essential role in vertebrates. Recent studies on the chicken VLDL receptor revealed that the receptor plays a key role in the uptake of yolk precursors in oocytes and mediates the growth of oocytes. The VLDL receptor is an essential receptor in avian species and the receptor-deficient mutant hens are sterile and exhibit severe hyperlipidemia with aortic atherosclerosis.

The VLDL receptor has been described as a new member of the LDL receptor supergene family that specifically binds VLDL in vitro via apolipoprotein E and lipoprotein lipase. Both apolipoprotein E and lipoprotein lipase are constituents of chylomicron remnants, another triglyceride-rich lipoprotein which has been proposed as a physiological ligand for the VLDL receptor. We used human chylomicron remnants to study their uptake into LDL, receptor-deficient Chinese hamster ovary cells overexpressing the human VLDL receptor. The uptake into these cells was compared to that into cells transfected with an empty transfection vector. Human chylomicron remnants were produced in vitro by hydrolysis with lipoprotein lipase, and were labeled with 125I. The uptake of these remnants into the cells overexpressing the VLDL receptor was found to be about 3-fold higher than the uptake into the control cells. The addition of a surplus of either apolipoprotein E or inactivated lipoprotein lipase to the remnants led to an increase in particle uptake. The chylomicron remnant uptake was inhibited by addition of the 39 kDa receptor associated protein These in vitro experiments strongly support the idea that the VLDL receptor is a physiological receptor for chylomicron remnants. The increase of receptor-mediated uptake induced by the addition of apoE or lipoprotein lipase underlines the role of these two proteins in this process.

We investigated the mechanism by which granulocyte-macrophage colony-stimulating factor (GM-CSF) lowers plasma cholesterol levels. Recombinant human GM-CSF (rhGM-CSF) was administered to normal and Watanabe heritable hyperlipidemic (WHHL) rabbits. Treatment with rhGM-CSF reduced the levels of cholesterol and triglyceride in these animals. In vitro colony assay for hematopoietic progenitors indicated that rhGM-CSF was capable of supporting granulocyte-macrophage colony formation in rabbits, suggesting that rhGM-CSF stimulates macrophage function even in rabbits. Northern blot analysis of rabbit very-low density lipoprotein (VLDL) receptor showed that rhGM-CSF elevated the levels of VLDL receptor mRNA 2.6- and 1.8-fold in muscles of normal WHHL rabbits, respectively, 1.5 hours after a single injection. Increases of 1.5- and 1.4-fold were observed in muscles of these rabbits after 5 days of administration. No changes were found in the LDL receptor mRNA levels in liver, spleen or bone marrow. These findings show that the lowering of lipids by GM-CSF may be mediated through the up-regulation of the VLDL receptor mRNA and the enhancement of macrophage function.

Una comunicación referente a esta línea de trabajo, titulada «La activación de PPARβ/δ previene la hipertrigliceridemia causada por una dieta rica en grasas. Implicación de la AMPK y de la vía PGC-1α-lipina 1-PPARα», fue presentada en el XXIV Congreso Nacional de la SEA (Sevilla 2011) y fue galardonada con una mención especial., [ES]: [Introducción]: El consumo excesivo de alimentos hipercalóricos y de alto contenido en grasas saturadas produce una dislipidemia aterogénica. En este estudio hemos analizado los efectos del activador de PPARβ/δ GW501516 sobre la hipertrigliceridemia inducida por una dieta rica en grasas. [Metodología]: Ratones macho fueron distribuidos aleatoriamente en 3 grupos: control (dieta estándar), dieta grasa (high fat diet [HFD], 35% grasa en peso, 58% kcal procedentes de grasa) y dieta grasa más GW501516 (3 mg/kg/día). La duración del tratamiento fue de 3 semanas. [Resultados]: La HFD causó hipertrigliceridemia acompañada de una reducción de los niveles hepáticos de la proteína AMPK fosforilada y de los niveles de ARNm Pgc-1α y lipina 1. Estos efectos fueron revertidos por el tratamiento con GW501516. El mantenimiento de la AMPK fosforilada tras el tratamiento con GW501516 podría deberse al aumento de la relación AMP/ATP. GW501516 incrementó los niveles de proteína lipina 1 nuclear acompañado por una amplificación de la vía PGC-1α-PPARα y un aumento de la actividad de unión al ADN de PPARα, así como el incremento en la expresión de los genes diana de PPARα implicados en la β-oxidación de ácidos grasos. GW501516 también aumentó los niveles plasmáticos de β-hidroxibutirato, producto final de la β-oxidación hepática. Finalmente, GW501516 incrementó los niveles del ligando endógeno de PPARα, 16:0/18:1-fosfatidilcolina, y aumentó la expresión del receptor de las VLDL en hígado. [Conclusión]: El efecto hipotrigliceridemiante de GW501516 en ratones sometidos a HFD se acompaña de un aumento de los niveles de la AMPK fosforilada y de un aumento de la vía PGC-1α-lipina 1-PPARα., [EN]: [Introduction]: Excessive consume of hypercaloric and high in saturated fat food causes an atherogenic dyslipidemia. In this study we analyzed the effects of PPARβ/δ activator GW501516 on the hypertriglyceridemia induced by a high-fat diet. [Methods]: Male mice were randomized in three groups: control (standard chow), high fat diet (HFD, 35% fat by weight, 58% Kcal from fat) and high fat diet plus GW501516 (3 mg/Kg/day). Treatment duration was three weeks. [Results]: HFD-induced hypertriglyceridemia was accompanied by a reduction in hepatic levels of phospho-AMPK and in PGC-1α and Lipin 1 mRNA levels. All these effects were reversed by GW501516 treatment. The lack of changes in phospho-AMPK levels after GW501516 treatment in HFD-fed animals could be the result of an increase in the AMP/ATP ratio. GW501516 treatment also increased Lipin 1 protein levels in the nucleus, led to the amplification of the PGC-1α-PPARα pathway and increased PPARα DNA-binding activity, as well as the expression of PPARα-target genes involved in fatty acid oxidation. GW501516 also increased β-hydroxibutirate plasmatic levels, a hepatic β-oxidation end product. Finally, GW501516 increased the hepatic levels of the PPARα endogenous ligand 16:0/18:1-PC and the expression of the VLDL receptor. [Conclusion]: These data indicate that the hypotriglyceridemic effect of GW501516 in mice subjected to HFD-fed mice is accompanied by an increase in phospho-AMPK levels and the amplification of the PGC-1α-Lipin 1-PPARα pathway.

The non-nephrotic chronic kidney disease (CKD) or ESRD results in marked reductions of lipoprotein lipase and VLDL receptor in skeletal muscle and adipose tissue, downregulation of hepatic lipase, LDL receptor-related protein, Apo-A1 and LCAT in the liver, up-regulation of ACAT in the liver, renal and arterial tissues, reduction of plasma HDL, oxidative modification of LDL and HDL, and impaired antioxidant, anti-inflammatory and reverse cholesterol transport capacities of HDL. Together these abnormalities lead to impaired clearance of chylomicrons and VLDL, accumulation of proinflammatory and atherogenic chylomicron and VLDL remnants, formation of small dense LDL, hypertriglyceridemia, reduced availability of lipid fuel for energy production and storage. These events contribute to systemic inflammation, accelerated atherosclerosis, cachexia, and impaired exercise capacity. Plasma cholesterol is usually within or below the normal limits and only occasionally elevated in patients with ESRD or non-nephrotic CKD. This accounts for the reported failure of the randomized clinical trials of cholesterol-lowering agents to reduce the risk of adverse cardiovascular outcomes and overall mortality in hemodialysis patients.

The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well as the 39-kDa receptor-associated protein (RAP). Ligand blotting experiments using RAP and immunoblotting experiments using an anti-VLDL receptor IgG detected the VLDL receptor in detergent extracts of human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells. To gain insight into the role of the VLDL receptor in the vascular endothelium, its ligand binding properties were further characterized. In vitro binding experiments documented that lipoprotein lipase (LpL), a key enzyme in lipoprotein catabolism, binds with high affinity to purified VLDL receptor. In addition, urokinase complexed with plasminogen activator-inhibitor type I (uPA.PAI-1) also bound to the purified VLDL receptor with high affinity. To assess the capacity of the VLDL receptor to mediate the cellular internalization of ligands, an adenoviral vector was used to introduce the VLDL receptor gene into a murine embryonic fibroblast cell line deficient in the VLDL receptor and the LDL receptor-related protein, another endocytic receptor known to bind LpL and uPA.PAI-1 complexes. Infected fibroblasts that express the VLDL receptor mediate the cellular internalization of 125I-labeled LpL and uPA.PAI-1 complexes, leading to their degradation. Non-infected fibroblasts or fibroblasts infected with the lacZ gene did not internalize these ligands. These studies confirm that the VLDL receptor binds to and mediates the catabolism of LpL and uPA.PAI-1 complexes. Thus, the VLDL receptor may play a unique role on the vascular endothelium in lipoprotein catabolism by regulating levels of LpL and in the regulation of fibrinolysis by facilitating the removal of urokinase complexed with its inhibitor.

The low-density lipoprotein (LDL) receptor plays a crucial role in cholesterol metabolism. A related protein, designated the very low density lipoprotein (VLDL) receptor, that specifically binds apolipoprotein (apo) E has recently been characterized and shown to be expressed in heart, muscle and adipose tissue and the human monocyte-macrophage cell line THP-1. The VLDL receptor binds and internalizes VLDL and intermediate density lipoprotein from Watanabe heritable hyperlipidemic (WHHL) rabbits as well as beta-migrating VLDL from cholesterol-fed rabbits but not LDL from WHHL rabbits. Chinese hamster ovary (CHO) cells transfected with the rabbit VLDL receptor cDNA have now been shown to bind or internalize VLDL (d1.006 g/ml) isolated from fasted normolipidemic human subjects with lower affinity than WHHL-VLDL or rabbit beta-VLDL. However, binding and internalization were markedly enhanced when fasted human VLDL was preincubated with either recombinant human apoE (3/3) or lipoprotein lipase (LPL) in CHO cells overexpressing the rabbit or human VLDL receptor. CHO cells transfected with both the rabbit VLDL receptor cDNA and the human LPL cDNA effectively bound, internalized, and degraded fasted human VLDL without pretreatment. Treatment of heparinase reduced the effect of LPL-mediated binding at 4 degrees C, but the inhibitory effect was lower at 37 degrees C. Pseudomonas LPL also enhanced the binding of human fasted VLDL to the VLDL receptor at 37 degrees C in CHO cells overexpressing the human VLDL receptor. Taken together, LPL causes the enhancement of triglyceride-rich lipoproteins binding to the VLDL receptor via both the formation of bridge between lipoproteins and heparan sulfate proteoglycans and its lipolytic effect. Ligand blot analysis showed that the apparent molecular mass of the VLDL receptor is 118 kDa, which is smaller than that of the LDL receptor. These results indicate that the VLDL receptor recognizes both triglyceride-rich lipoproteins that are also relatively rich in apoE, as well as the remnants of triglyceride-rich lipoproteins after catabolism and the interaction with heparan sulfate proteoglycans by LPL. The VLDL receptor may thus function as a receptor for remnants of triglyceride-rich lipoproteins in extrahepatic tissues.