Internalization Of Low Density Lipoprotein Research Articles

Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis

The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr-/- mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Decreased levels and function of circulating endothelial progenitor cells in unruptured intracranial saccular aneurysm patients

Circulating endothelial progenitor cells (EPCs) play a critical role in maintaining endothelial integrity and keeping vascular homeostasis. Previously, we reported that EPCs were involved in repair and remodeling of aneurismal wall. In the present study, we verified this hypothesis by investigating the proliferative ability and count of EPCs in peripheral blood of patients with unruptured intracranial aneurysms (UIAs). Twenty-four patients with UIAs (UIA group) and 24 negative controls (control group) were included in this study. Peripheral blood monocytes (PBMCs) were harvested and selectively cultured. The colony-forming ability of cultured cells was analyzed and the biological functions were examined by testing the adsorption of ulex europaeus agglutinin-1 labeled by fluorescein isothiocyanate and acetylated low-density lipoprotein internalization. The migratory and adhesive ability of cultured EPCs were assessed. In vitro cultured PBMCs were identified as EPCs by examining surface markers CD34, CD133 and vascular endothelial growth factor receptor 2 using flow cytometry. EPCs from UIA group possessed significantly decreased proliferative, migratory and adhesive capacities compared with EPCs from control group. Furthermore, EPCs count in UIA group was significantly decreased. Collectively, these results indicated that the circulating EPCs of UIA patients may be involved in intracranial aneurysm repair and remodeling.

A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia

Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.

A. actinomycetemcomitans LPS Enhances Foam Cell Formation Induced by LDL

The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages.

Receptor-mediated endocytosis and trafficking between endosomal–lysosomal vacuoles in Giardia lamblia

The early branching Giardia lamblia has highly polarized vacuoles, located underneath the plasma membrane, which have at least some of the characteristics of endosomes and of lysosomes. These peripheral vacuoles (PVs) are necessary for nutrient uptake and the maintenance of plasma membrane composition, but whether they carry out sorting and segregation of receptors and ligands is a matter of debate. Here, we showed that the internalization of low-density lipoprotein (LDL) to the PVs is highly dynamic in trophozoites with a rate similar to the internalization of the low-density lipoprotein receptor-related protein 1. Moreover, by analyzing receptor-mediated and fluid-phase endocytosis in living cells, we showed that after endocytosis LDL but not dextran moved laterally between the PVs. We speculate on PV functional heterogeneity and maturation in this parasite.

Mutations in the SORT1 gene are unlikely to cause autosomal dominant hypercholesterolemia

ObjectiveTo study whether mutations in the SORT1 gene could be a cause of autosomal dominant hypercholesterolemia and to study the effect of sortilin on the binding and internalization of low density lipoprotein (LDL). Methods842 unrelated hypercholesterolemic subjects without mutations in genes known to cause autosomal dominant hypercholesterolemia, were screened for mutations in the SORT1 gene by DNA sequencing. Transfections of wild-type or mutant SORT1 plasmids in HeLa T-REx cells and the use of siRNA were used to study the effect of sortilin on the number of cell-surface LDL receptors and on the binding and internalization of LDL. ResultsA total of 45 mutations in the SORT1 gene were identified of which 15 were missense mutations. Eight of these were selected for in vitro studies, of which none had a major impact on the amount of LDL bound to the cell surface. There was a positive correlation between the amount of sortilin on the cell surface and the amount of LDL bound. The observation that a mutant sortilin which is predominantly found on the cell surface rather than in post-Golgi compartments, bound very high amounts of LDL, indicates that sortilin does not increase the binding of LDL through an intracellular mechanism. Rather, our data indicate that sortilin binds LDL on the cell surface. ConclusionEven though sortilin binds and internalizes LDL by receptor-mediated endocytosis, mutations in the SORT1 gene are unlikely to cause autosomal dominant hypercholesterolemia and may only have a marginal effect on plasma LDL cholesterol levels.

Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs

Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.

AAK1 Regulates Numb Function at an Early Step in Clathrin‐Mediated Endocytosis

Numb is an endocytic protein that is proposed to influence clathrin-coated pit assembly, although its mode of action and the mechanisms that regulate its activity are unknown. In this study, we show that Numb binds to and is phosphorylated by adaptor-associated kinase 1 (AAK1), a key endocytic kinase. We find that AAK1 redistributes Numb to perinuclear endosomes when overexpressed, while kinase depletion causes Numb to accumulate at the plasma membrane. Overexpression of a Numb point mutant (T102A) that lacks the AAK1 phosphorylation site potently disrupts transferrin and low-density lipoprotein internalization but does not impact EGF uptake. Consistent with Numb redistribution results, we find that T102A Numb no longer localizes to perinuclear endosomes. Instead, it is enriched at the plasma membrane where it shows elevated levels of colocalization with coated pit markers. Collectively, these observations demonstrate that Numb endocytic activity is regulated by AAK1 and that phosphorylation may be a critical step in promoting coated pit maturation.

Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins

The uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (∼ ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake.

Effect of mutations in the PCSK9 gene on the cell surface LDL receptors

The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene is involved in the post-transcriptional regulation of the low-density lipoprotein (LDL) receptors (LDLR). Mutations in the PCSK9 gene have been associated with both hypocholesterolemia and hypercholesterolemia through 'loss-of-function' and 'gain-of-function' mechanisms, respectively. We have studied the effect of the four loss-of-function mutations R46L, G106R, N157K and R237W and the two gain-of-function mutations S127R and D374Y on the autocatalytic activity of PCSK9, as well as on the amount of the cell surface LDLR and internalization of LDL in transiently transfected HepG2 cells. The two groups of mutations did not differ with respect to autocatalytic activity of PCSK9, but they did differ with respect to the amount of cell surface LDLR and internalization of LDL. The four loss-of-function mutations had a 16% increased level of cell surface LDLR and a 35% increased level of internalization of LDL as compared with WT-PCSK9. The two gain-of-function mutations had a 23% decreased level of cell surface LDLR and a 38% decreased level of internalization of LDL as compared with WT-PCSK9. Our studies have also shown that transfer of media from transiently transfected HepG2 cells to untransfected HepG2 cells, reduces the amount of cell surface LDLR and internalization of LDL in the untransfected cells within 20 min of media transfer. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the transfected cells and degrades LDLR both in transfected and untransfected cells.

Retention of Mutant Low Density Lipoprotein Receptor in Endoplasmic Reticulum (ER) Leads to ER Stress

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and retention of mutant LDLR are poorly understood. In the present study we show that the G544V mutant LDLR associates with the chaperones Grp78, Grp94, ERp72, and calnexin in the ER of transfected Chinese hamster ovary cells. Retention of the mutant LDLR was shown to cause ER stress and activation of the unfolded protein response. We observed a marked increase in the activity of two ER stress sensors, IRE1 and PERK. These results show that retention of mutant LDLR in ER induces cellular responses, which might be important for the clinical outcome of familial hypercholesterolemia.

Hyaluronan forms complexes with low density lipoprotein while also inducing foam cell infiltration in the dermis

Xanthoma is a foam cell infiltrating lesion similar to atherosclerosis. Glycosaminoglycans and proteoglycans have long been considered to play a role in atherogenesis. The purpose of this study is to investigate the role of hyaluronan, the main dermal glycosaminoglycan, in xanthoma formation. The complex formation of low density lipoprotein (LDL) with hyaluronan was investigated by assaying the cholesterol level of precipitates that were formed by incubating LDL, hyaluronan and cetylpyridinium chloride in the presence of Ca2+. The uptake of LDL by mouse peritoneal macrophages was studied by assaying the cellular cholesterol esterification activity. The responsible receptor for the LDL internalization was examined by saturating hyaluronan receptor and blocking class A macrophage scavenger receptor (CD204). Hyaluronan was injected into the dorsal skin of diet-induced hypercholesterolemic rabbits to reveal the xanthoma inducing activity of hyaluronan. Cetylpyridinium chloride precipitated hyaluronan, which had formed complexes with LDL. The macrophages incorporated hyaluronan-LDL complexes and oxidized LDL via CD204. Foam cell infiltration and cholesterol accumulation were induced by intradermal injections of hyaluronan in diet-induced hypercholesterolemic rabbits. Hyaluronan, like other sulfated glycosaminoglycans, retains LDL by forming a complex. Via macrophage scavenger receptors, macrophages incorporate not only LDL-hyaluronan complexes, but also oxidized LDL, which has been oxidized during the retention time.

Adaptor Protein ARH Is Recruited to the Plasma Membrane by Low Density Lipoprotein (LDL) Binding and Modulates Endocytosis of the LDL/LDL Receptor Complex in Hepatocytes

ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 degrees C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.

Oxidation of Low-Density Lipoproteins Induces Amyloid-like Structures That Are Recognized by Macrophages

The macrophage scavenger receptor CD36 plays a key role in the initiation of atherosclerosis through its ability to bind to and internalize oxidized low-density lipoproteins (oxLDL). Prompted by recent findings that the CD36 receptor also recognizes amyloid fibrils formed by beta-amyloid and apolipoprotein C-II, we investigated whether the oxidation of low-density lipoproteins (LDL) generates characteristic amyloid-like structures and whether these structures serve as CD36 ligands. Our studies demonstrate that LDL oxidized by copper ions, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), or ozone react with the diagnostic amyloid dyes thioflavin T and Congo Red and bind to serum amyloid P component (SAP), a universal constituent of physiological amyloid deposits. X-ray powder diffraction patterns for native LDL show a diffuse powder diffraction ring with maximum intensity corresponding to an atomic spacing of approximately 4.7 A, consistent with the spacing between beta-strands in a beta-sheet. Ozone treatment of LDL generates an additional diffuse powder diffraction ring with maximum intensity indicating a spacing of approximately 9.8 A. This distance is consistent with the presence of cross-beta-structure, a defining characteristic of amyloid. Evidence that these cross-beta-amyloid structures in oxLDL are recognized by macrophages is provided by the observation that SAP strongly inhibits the association and internalization of (125)I-labeled copper-oxidized LDL by peritoneal macrophages. The ability of SAP to bind to amyloid-like structures in oxLDL and prevent lipid uptake by macrophages highlights the potential importance of these structures and suggests an important preventative role for SAP in foam cell formation and early-stage atherosclerosis.

The Modular Adaptor Protein ARH Is Required for Low Density Lipoprotein (LDL) Binding and Internalization but Not for LDL Receptor Clustering in Coated Pits

ARH is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues. Individuals lacking ARH (ARH-/-) have severe hypercholesterolemia due to impaired hepatic clearance of LDL. Immortalized lymphocytes, but not fibroblasts, from ARH-deficient subjects fail to internalize LDL. To further define the role of ARH in LDLR function, we compared the subcellular distribution of the LDLR in lymphocytes from normal and ARH-/- subjects. In normal lymphocytes LDLRs were predominantly located in intracellular compartments, whereas in ARH-/- cells the receptors were almost exclusively on the plasma membrane. Biochemical assays and quantification of LDLR by electron microscopy indicated that ARH-/- lymphocytes had >20-fold more LDLR on the cell surface and a approximately 27-fold excess of LDLR outside of coated pits. The accumulation of LDLR on the cell surface was not due to failure of receptors to localize in coated pits since the number of LDLRs in coated pits was similar in ARH-/- and normal cells. Despite the dramatic increase in cell surface receptors, LDL binding was only 2-fold higher in the ARH-/- lymphocytes. These findings indicate that ARH is required not only for internalization of the LDL.LDLR complex but also for efficient binding of LDL to the receptor and suggest that ARH stabilizes the associations of the receptor with LDL and with the invaginating portion of the budding pit, thereby increasing the efficiency of LDL internalization.

Autosomal Recessive Hypercholesterolemia

Autosomal recessive hypercholesterolemia (ARH) presents with a clinical phenotype similar to that of classical homozygous familial hypercholesterolemia (FH) caused by defects in the low-density lipoprotein (LDL) receptor gene but is more variable, generally less severe, and more responsive to lipid-lowering therapy than homozygous FH; furthermore, FH is inherited with a dominant pattern. The approximately 50 known affected ARH individuals are mostly of Sardinian or Middle Eastern origin, but rare cases of ARH have occurred worldwide. The physiological defect in ARH is a failure of some, but not all, cell types to mediate LDL receptor-dependent internalization of LDL and is caused by mutations in the gene for a putative adaptor protein called ARH. In affected cells, the LDL receptor gene is normal but LDL receptor protein accumulates at the cell surface; this also occurs in livers of recombinant mice lacking ARH, providing an explanation for the failure of clearance of LDL from plasma in ARH patients. The structural features of the ARH protein and its capacity to interact with the internalization sequence of the LDL receptor, plasma membrane phospholipids, and the clathrin endocytic machinery suggest that it plays a key role in the LDL receptor pathway.

A Splice Mutation in a Syrian Autosomal Recessive Hypercholesterolemia Family Causes a Two-Nucleotide Deletion of mRNA

To the Editor: Autosomal recessive hypercholesterolemia (ARH) is a rare Mendelian dyslipidemia characterized by markedly elevated plasma low-density lipoprotein (LDL) cholesterol levels. ARH is caused by mutations in a protein that contains a phosphotyrosine binding domain (PTB) required for internalization of LDL in the liver.1,2 LDL receptor function is defective in liver and lymphocytes but not in fibroblasts from ARH patients. We reported a Syrian ARH family earlier in Circulation Research and identified a mutation in the splice-acceptor site of intron 1 that converts the guanine of AG into cytosine AC.3 Splice mutations may cause cryptic site activation, intron retention, creation of new splice sites, or exon skipping.4 However, we had no access to ARH gene-expressing tissue. We therefore constructed ARH gene fragments from an affected and a nonaffected individual and transfected the fragments into hepatocellular carcinoma cells (HEPG2). DNA fragments containing the 3′ part of intron 1, exon 2, the entire intron 2, exon 3, and the 5′ part of intron 3 were PCR-amplified from nonaffected and affected probands. Both fragments were subcloned into TOPO-XL vector (Invitrogen). Fragments containing the 5′ noncoding part of exon 1, exon 1, and the 5′ part of intron 1 were also amplified and were subcloned upstream to the previously cloned fragment. The final partial ARH gene, with and without mutation, was cloned into phCMV3/Xi expression vector (Gene Therapy Systems, San Diego, Calif). We did not include the entire intron 1 because we were concerned that the entire intron …

Molecular mechanisms of autosomal recessive hypercholesterolemia

Autosomal recessive hypercholesterolemia (ARH) is a rare Mendelian dyslipidemia characterized by markedly elevated plasma LDL levels, xanthomatosis, and premature coronary artery disease. LDL receptor function is normal, or only moderately impaired in fibroblasts from ARH patients, but their cultured lymphocytes show increased cell-surface LDL binding, and impaired LDL degradation, consistent with a defect in LDL receptor internalization. Recently, the disorder was shown to be caused by mutations in a phosphotyrosine binding domain protein, ARH, which is required for internalization of low density lipoproteins in the liver. This review summarizes the findings of new investigations into the pathophysiology and molecular genetics of ARH. All mutations that have been characterized to date preclude the synthesis of a full-length protein. GST-pulldown experiments indicate that the phosphotyrosine binding domain of ARH interacts with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR, and that conserved motifs in the C-terminal portion of the protein bind to clathrin and to the beta2-adaptin subunit of AP-2. The available data suggest that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery.

Effect of Cyclosporine A on the Binding Affinity and Internalization of Low-Density Lipoproteins in Human Skin Fibroblasts

The aim of this study was to elucidate the possible causes of elevated low-density lipoprotein (LDL)-cholesterol levels in patients with transplants who were treated with the immunosuppressant drug cyclosporine A (CSA). The binding and internalization of (125)I-LDL in the presence or absence of CSA at varying concentrations (5-15 microg/mL) within human skin fibroblasts were determined. In addition, the effect of LDL-associated CSA on the binding of LDL to its receptor was determined. CSA decreases LDL internalization without altering the extent and affinity of its binding to the LDL receptor. CSA did not alter the number of available LDL binding sites. Furthermore, the association of CSA with LDL did not affect the binding affinity of LDL to its receptor, suggesting that this binding may not be a mechanism by which CSA affects the subsequent clearance of LDL from the bloodstream. These findings suggest that CSA may cause an increase in plasma LDL-cholesterol in patients with transplants, thereby inhibiting LDL particle internalization without altering LDL receptor binding.