HDL Binding Research Articles

Abstract 18666: SR-BI and Tyrosine Kinase Inhibitors: A Novel Pathway for Atherosclerotic Cardiovascular Disease Therapeutic Development

Introduction: Cardiovascular disease (CVD) is the leading cause of death in the U.S., where coronary artery disease (CAD) accounts for 42.1% of all CVD deaths. Current therapeutics focus on lowering LDL-C, as previous attempts to raise HDL-C were not successful in altering CVD outcomes. However, the therapeutic potential of HDL has not been fully explored. Variants in SCARB1, the gene that encodes HDL receptor Scavenger Receptor B1 (SR-BI), are associated with dyslipidemia and atherosclerotic CVD (ASCVD), and we first to identified Mendelian inheritance of SCARB1 variants that cause severe early-onset CAD and dyslipidemia. Limited clinical evidence suggests imatinib reduces total cholesterol in chronic myeloid leukemia (CML). Additionally, TKI treatment in atherosclerotic mice reduced total cholesterol (imatinib) and atherosclerotic lesions (dasatinib). Yet, no data is available on the effects of TKIs on HDL and RCT. Hypothesis: Our findings suggest that HDL function (vs. HDL-C concentration) in reverse cholesterol transport (RCT) may be a promising target for cholesterol-based therapy and studying TKIs in RCT may provide a novel mechanism for therapeutic development. Methods: We performed a high throughput drug screen with 788 FDA-approved compounds, using HepG2 cells to measure endogenous HDL binding. We identified four compounds that significantly increased HDL binding, of which, imatinib was the only one to increase SR-BI. Wildtype C57Bl/6 mice on a high fat, high cholesterol diet, underwent imatinib treatment for 4 weeks and measured biweekly serum lipids. Results: We have found that imatinib and dasatinib significantly enhance HDL binding, where imatinib increased SR-BI, ABCA1, and ABCG1 in vitro. Furthermore, in vivo imatinib treatment decreased plasma total cholesterol, HDL-C and triglyceride levels, and elevated hepatic SR-BI. Knockdown of SR-BI in HepG2 cells ameliorated the imatinib-induced HDL binding increase suggesting an SR-BI-specific mechanism. Mass spectrometry revealed a novel SR-BI methionine oxidation site with imatinib treatment. Conclusions: Our data supports the exploration of TKI-mediated SR-B1 regulation, HDL metabolism, and RCT mechanism to identify new therapeutic targets for dyslipidemia and ASCVD.

Acquired Chylomicronemia Due to Autoantibodies Against GPIHBP1

Lead Author's Financial Disclosures Nothing to disclose. Study Funding None. Background/Synopsis We report a case of recurrent triglyceride-induced pancreatitis that was found to be due to autoantibodies to glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) in the setting of biopsy-proven membranous lupus nephritis. Objective/Purpose This case report highlights a very rare cause of acquired chylomicronemia and discusses the treatment of GPIHBP1 autoantibody-induced hypertriglyceridemia. Methods A 12-year-old female with no medical history was admitted to the hospital for triglyceride induced pancreatitis. Her initial lipids consisted of: triglycerides 4070, total cholesterol 290, HDLc 9, and non-HDL-c 281 mg/dL. After inpatient treatment, she was started on fenofibrate and referred to lipid clinic. Despite fenofibrate 160 mg daily, icosapent ethyl 2 g twice a day, and a strict low-fat diet, the patient continued to experience triglyceride-induced pancreatitis. Her triglycerides fluctuated between 1200 - 2300 mg/dL. Genotyping was negative for familial chylomicronemia (deficiency/mutation in LPL, APOC2, APOA5, LFM-1, and GPIHBP1). Therefore, the patient was presumed to have genotype-negative familial chylomicronemia syndrome. Over the next 2 years, the patient had 15 hospital admissions for triglyceride-induced pancreatitis. At age 14 she was started on alirocumab 75 mg injections every 14 days, which decreased, but did not eliminate, the frequency of pancreatitis episodes. Her triglyceride levels subsequently ranged 650 - 991 mg/dL. During a hospital admission at age 15, the patient was found to have nephrotic syndrome with an ANA titer >1:2560. Renal biopsy confirmed membranous lupus nephritis. She was treated with pulse-dose steroids, mycophenolate mofetil, and hydroxychloroquine. Her lupus diagnosis prompted testing for GPIHBP1 autoantibodies,which were strongly positive. Results Two months after our patient's diagnosis of GPIHBP1 autoantibody induced hypertriglyceridemia, she continues to receive treatment with prednisone 40 mg daily, mycophenolate mofetil 1000 mg twice a day, and hydroxychloroquine 200 mg daily and has received two doses of rituximab-pvvr 1000 mg. Her triglycerides remain elevated 1400 - 2200 mg/dL. Conclusions This case highlights a recently described entity in the literature, “GPIHBP1 autoantibody syndrome,” wherein patients with unexplained chylomicronemia are found to have autoantibodies to GPIHBP1, causing hypertriglyceridemia resistant to traditional medical therapy. Of the 33 known patients worldwide with GPIHBP1 autoantibody syndrome, immunosuppression with rituximab and mycophenolate are reported to be effective therapies.Our patient is currently undergoing immunosuppression, but triglyceride levels have yet to decrease. Additional time and/or rituximab doses may be needed in order to maximally suppress autoantibody production and, in turn, reduce triglyceride levels. Nothing to disclose.

Abstract 423: Imatinib Promotes Reverse Cholesterol Transport And Elevates Sr-bi

Dyslipidemia is a cardiovascular risk factor for coronary artery disease and atherosclerosis that is characterized by elevated serum cholesterol and lipid levels. Although high-density lipoprotein-associated cholesterol (HDL-C) is associated with reduced risk of cardiovascular events, targeted therapy to increase HDL-C levels have been unsuccessful in altering outcomes of associated atherosclerotic disease. Single nucleotide polymorphisms in SCARB1 , the gene that encodes HDL receptor Scavenger Receptor B1 (SR-BI), are associated with dyslipidemia and atherosclerotic cardiovascular disease. We were the first to identify inherited mutations in SCARB1 that segregate with disease in a family with severe coronary artery disease and dyslipidemia, including elevated HDL. Our findings suggest that HDL function (vs. HDL-C concentration) may be a promising target for cholesterol-based therapy. Here, we performed an unbiased high throughput drug screen with 788 FDA-approved compounds, using HepG2 cells to measure endogenous HDL binding. We identified five compounds that significantly increased endogenous HDL binding: imatinib, trimethoprim, eszopiclone, clemastine, and mepenzolate, of which, imatinib was the only compound to increase SR-BI expression. Imatinib is a tyrosine kinase inhibitor that is a chemotherapeutic agent designed to treat individuals with chronic myeloid leukemia. Limited clinical evidence suggests a reduction in total cholesterol with 400mg/day imatinib. Additionally, imatinib treatment (150mg/kg) in mouse models of atherosclerosis reduces total cholesterol. Yet, no data is available on the effects of imatinib on HDL and reverse cholesterol transport. We have found that imatinib promotes HDL binding and SR-BI expression in vitro . Furthermore, in wildtype C57Bl/6 mice on a high fat, high cholesterol diet, imatinib treatment (50mg/kg) was sufficient to decrease plasma total cholesterol, HDL-C and triglyceride levels and elevate hepatic SR-BI expression. In summary, our data supports the exploration of imatinib-mediated SR-BI regulation, HDL metabolism, and RCT pathway to identify new therapeutic targets for dyslipidemia.

Abstract 14320: High Density Lipoprotein Affinity and Transport of Small RNAs in Atherosclerosis

HDL have many alternative functions outside of cholesterol efflux likely due to its diverse cargo of proteins, lipids, and small non-coding RNAs (sRNA). We have reported that HDL transports microRNAs (miRNA) and other sRNA classes, including tRNA-derived sRNAs (tDR), which are produced during cell stress. We hypothesize that HDL-tDR transport is conferred by apolipoprotein A-I (apoA-I) RNA binding capacity. HDL-miRNAs have previously been shown to be altered in many diseases; however, changes to HDL-tDRs in diseases such as cardiovascular disease, have not been reported. Based on sRNA sequencing, tDR-GlyGCC was identified as one of the most abundant sRNAs on HDL and HDL-tDR-GlyGCC levels were found to be significantly increased in humans with advanced atherosclerosis, as determined by coronary artery calcium scores. Moreover, we found that macrophages readily export tDR-GlyGCC to HDL. To determine the binding properties of HDL-tDR-GlyGCC, microscale thermophoresis (MST) and electromobility shift assays were completed. HDL were determined to have the strongest binding affinity to tDR-GlyGCC (K d =48nM) compared to other sRNAs, including miR-223-3p (K d =372nM), suggesting that HDL has preferences for distinct sRNAs. As sRNAs can be post-transcriptionally modified, we investigated if RNA modifications inhibit HDL binding. Both 2- O -methylation and locked-nucleic acid modifications diminished HDL binding while other chemical forms of tDR-GlyGCC, namely dsRNA and ssDNA, were found to completely block binding. To determine if apoA-I confers RNA binding capacity of HDL, MST studies were completed on lipid-free apoA-I and single unilamellar lipid vesicles (SUV), and while apoA-I was found to bind moderately, SUVs failed to bind tDR-GlyGCC. Interestingly, reconstitution of SUVs with apoA-I rescued tDR-GlyGCC binding. Moreover, chemical cross-linking of 4-thiouridine-containing tDR-GlyGCC to apoA-I caused both protein size shift and dimerization as determined by western blotting. Crosslinked proteins were also used to uncover potential apoA-I RNA binding domains, as determined by LC-MS/MS. These results support that apoA-I RNA binding capacity confers HDL-tDR-GlyGCC acceptance from macrophages and transport in atherosclerosis.

Inherited Variants in SCARB1 Cause Severe Early-Onset Coronary Artery Disease.

[Figure: see text].

Cell Signaling in Diabetic Dyslipidemia

Diabetes accompanies several metabolic complications and poor production and clearance of lipoprotein is one of them. Type 2 diabetes mellitus is linked with atherosclerosis, though exact mechanism is still unclear. There are different complications of type 2 diabetes mellitus. Dyslipidemia is one of them. It has been documented that deranged lipid profile is caused by insulin resistance. Dyslipidemia arises due to low level of high density lipoproteins and high level of triglycerides. Diabetic dyslipidimia occurs due to imbalance in reverse cholesterol transport mechanism which is a major risk for cardiovascular diseases. Reverse cholesterol transport is regulated by cell surface receptors scavenger receptor B1 and ATP binding cassette protein-1. These are HDL binding receptors and help to maintain balance of HDL. Genetic and epigenetic factors influence the regulation of cell surface receptors. It plays a significant role in lipid homeostasis. HDL receptors are encoded by SCARB1 and ABCA1 genes. Peroxisome proliferator activated receptor alpha (PPARα) is a down-stream transcriptional activator of SCARB1, ABCA1 and ApoA1 genes.

Human variant of scavenger receptor BI (R174C) exhibits impaired cholesterol transport functions

AbstractHDL and its primary receptor, scavenger receptor class B type I (SR-BI), work together to promote the clearance of excess plasma cholesterol, thereby protecting against atherosclerosis. Human variants of SR-BI have been identified in patients with high HDL-cholesterol levels, and at least one variant has been linked to cardiovascular disease. Therefore, while often regarded as beneficial, very high levels of HDL-cholesterol may result from impaired cholesterol clearance through SR-BI and contribute to cardiovascular risk. In this study, we characterized the function of a rare human variant of SR-BI, resulting in the substitution of arginine-174 with cysteine (R174C), which was previously identified in a heterozygous individual with high levels of HDL-cholesterol. We hypothesized that the R174C-SR-BI variant has impaired cholesterol transport functions, which were assessed in COS-7 cells after transient transfection with full-length WT or R174C-SR-BI. Although R174C-SR-BI was expressed at levels comparable to the WT receptor, HDL binding, cholesteryl hexadecyl ether uptake, free cholesterol efflux, and modulation of membrane cholesterol were disrupted in the presence of R174C-SR-BI. We further examined the role of salt bridges as a potential mechanism for R174C-SR-BI dysfunction. If translatable, this human variant could lead to increased plasma HDL-cholesterol levels, impaired cholesterol clearance, and increased cardiovascular disease risk.

On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity

Angiopoietin-like (ANGPTL) 8 is a secreted inhibitor of LPL, a key enzyme in plasma triglyceride metabolism. It was previously reported that ANGPTL8 requires another member of the ANGPTL family, ANGPTL3, to act on LPL. ANGPTL3, much like ANGPTL4, is a physiologically relevant regulator of LPL activity, which causes irreversible inactivation of the enzyme. Here, we show that ANGPTL8 can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states. In contrast to the augmented inhibitory effect of the ANGPTL3/ANGPTL8 complex on LPL activity, the ANGPTL4/ANGPTL8 complex is less active compared with ANGPTL4 alone. In our experiments, all three members of the ANGPTL family use the same mechanism to inactivate LPL, which involves dissociation of active dimeric LPL to monomers. This inactivation can be counteracted by the presence of glycosylphosphatidylinositol-anchored HDL binding protein 1, the endothelial LPL transport protein previously known to protect LPL from spontaneous and ANGPTL4-catalyzed inactivation. Our data demonstrate that ANGPTL8 may function as an important metabolic switch, by forming complexes with ANGPTL3, or with ANGPTL4, in order to direct the flow of energy from triglycerides in blood according to the needs of the body.

Abstract 045: SR-BI and PCPE2 Modulate Lipid Trafficking in Adipocytes

Adipose tissue undergoes distinct structural remodeling in response to increases in fat mass, as in obesity. During this process, the extracellular matrix (ECM) plays a pivotal role with respect to both adipocyte receptor signaling and structural remodeling. One ECM protein, procollagen endopeptidase enhancer 2 (PCPE2), has been shown by our lab to work in partnership with the scavenger receptor class B type I (SR-BI) to regulate adipocyte cholesterol homeostasis. To investigate the functional impact of the partnership between SR-BI and PCPE2, we studied mature adipocytes differentiated from murine embryonic fibroblasts (MEF) and compared to our adipose specific PCPE2 knockout mice. Our studies showed the presence of an SR-BI-PCPE2 protein complex based on the co-immunoprecipitation of PCPE2 with SR-BI in both differentiated MEFs and mouse adipose tissue. Furthermore, stimulated emission depletion (STED) microscopy showed SR-BI and PCPE2 localized on the lipid droplet surface separated by distances ranging from 24 – 90 nm, suggesting that the SR-BI-PCPE2 complex may contain other proteins. Most significantly, a loss of SR-BI function was shown in PCPE2-deficient adipocytes by the SR-BI mediated HDL binding and cholesteryl ester (CE) uptake assays. Compared to differentiated MEFs from Ldlr -/- mice, binding of HDL to differentiated MEFs from Ldlr -/- Pcpe2 -/- mice was reduced by 75%, and HDL cholesteryl oleyl ether uptake was reduced by 50%. Disruption of adipocyte SR-BI function in the absence of PCPE2 also reduced free cholesterol efflux indicating substantial disruption to intracellular cholesterol homeostasis. Moreover, analysis of RNAseq from several human clinical studies show that PCPE2 mRNA expression is positively correlated with fat mass in specific adipose depots, while negatively correlated with plasma insulin, glucose and triglyceride levels, with the latter two more robust in type 2 diabetic populations (FDR < 0.004). Overall, these results suggest that the partnership between SR-BI and PCPE2 in adipocytes plays an important role in cholesterol trafficking and homeostasis which influences fat mass expansion, glucose tolerance and insulin resistance.

Abstract 548: Separation of Lipoprotein Particles Utilizing Asymmetric Flow Field-flow Fractionation and Quantitation of Apolipoprotein L1 in Human Plasma with IMER-UPLC-MS/MS

Apolipoprotein L1 (ApoL1) is an intriguing protein and was shown to be involved in numerous diseases states such as chronic kidney disease (CKD), cardiovascular diseases(CVD), Human Immunodeficiency Virus associated Nephropathy (HIVAN), systemic lupus erythematosus (SLE) (associated collapsing glomerulopathy), and type 2 diabetes(especially for people of African descent). People of African ancestry were shown to have either one or two alleles which contribute to their susceptibility of diseases related to CKD. ApoL1 is characterized as a HDL binding protein. In this work we studied ApoL1 binding to HDL by using a gentle size fractionation technique, asymmetric flow field-flow fractionation (AF4), and liquid chromatography (LC) with quantitative tandem mass spectrometry (MS/MS) detection. We injected 50 uL of whole plasma onto the AF4 system, where smaller HDL particles eluted first followed by the larger HDL, and then by LDL, IDL and VLDL particles. The lipoproteins were separated into 40 fractions and quantitatively analyzed using on-line trypsin digestion coupled with LC-MS/MS analysis. We analyzed plasma samples both with and without purification by UC. When HDL particles were separated by UC, apoL1 was found in the higher density HDL fractions, as was also found by previous studies. When HDL separated using AF4 with or without UC, apoL1 eluted in the larger HDL size region (12-13 nm). With UC vs. without UC, the apoL1 peptide signal intensities were reduced by more than 50%, indicating significant loss of apoL1 from the surface of HDL particles during UC. Our data on apoL1 provides further evidence for the fact that size and density are not directly interconvertible physical characteristics of lipoproteins. Furthermore, our data shows that apoL1 is also an example of exchangeable apolipoproteins whose binding can be significantly diminished due to the effect of intense shear forces during UC separation. This work also demonstrates the advantages of utilizing AF4-IMER-UPLC-MS/MS methodology to separate lipoprotein particles and study of their apolipoprotein composition.

Acute coronary syndrome remodels the antiplatelet aggregation properties of HDL particle subclasses

Background Although HDLs have antithrombotic effects by reducing platelet activation, the relationship between HDL levels and the risk of acute coronary syndrome (ACS) is unclear, as HDL particles are heterogeneous in composition and biological properties. Objective To characterize the effects of HDL2 and HDL3 subclasses from ACS patients and non-coronary artery disease (CAD) subjects on platelet activation. Methods We measured platelet aggregation and ex vivo thrombus formation, analyzed signaling pathways by flow cytometry, and performed a targeted lipidomics analysis on HDL subclasses. Results Analysis of human platelet aggregation in suspension, adhesion on von Willebrand factor and thrombus formation on collagen under arterial shear demonstrated that HDL2 from ACS patients had higher antiplatelet potency than HDL3 from ACS patients and HDL from non-CAD subjects. HDL binding to scavenger receptor class B type I was essential for this effect. A lipidomics analysis revealed that HDL2 from ACS patients had more oxidized polyunsaturated fatty acids (PUFAs). An inverse correlation between the concentrations of 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), the eicosapentaenoic acid metabolite 18-hydroxyeicosapentaenoic acid (18-HEPE) and hydroxyeicosatetraenoic acid isomers in HDL2 and platelet aggregation was observed. This relationship was further demonstrated by the direct inhibitory effects of 18-HEPE, 9-HODE, 13-HODE, 17-hydroxydocosahexaenoic acid and 14-hydroxydocosahexaenoic acid on collagen-related peptide-induced platelet aggregation, indicating that oxidized PUFAs contribute to the antithrombotic effect of ACS HDL2. Conclusions Our data shed new light on the antiplatelet effects of HDL subclasses, and suggest physiological adaptation through the modulation of HDL properties in ACS patients that may limit their platelet-dependent thrombotic risk.

MicroRNA-24 aggravates atherosclerosis by inhibiting selective lipid uptake from HDL cholesterol via the post-transcriptional repression of scavenger receptor class B type I

Background and aimsLiver scavenger receptor class B type I (SR-BI) exerts atheroprotective effects through selective lipid uptake (SLU) from high-density lipoprotein cholesterol (HDL-C). Low hepatic SR-BI expression leads to high HDL-C levels in the circulation and an increased risk of atherosclerosis. Furthermore, macrophage SR-BI mediates bidirectional cholesterol flux and may protect against atherogenesis. Previous studies have revealed that miR-24 is closely related to cardiovascular disease (CVD) progression. We aimed to investigate the molecular mechanisms by which miR-24 participates in SR-BI-mediated selective HDL cholesteryl ester (HDL-CE) uptake and further atherogenesis in apoE−/− mice. MethodsBioinformatic predictions and luciferase reporter assays were utilized to detect the association between miR-24 and the SR-BI 3′ untranslated region (3′ UTR), and RT-PCR and western blotting were used to evaluate SR-BI mRNA and protein expression, respectively. The effects of miR-24 on Dil-HDL uptake were determined by flow cytometry assay. Double-radiolabeled HDL (125I-TC-/[3H] CEt-HDL) was utilized to measure the effects of miR-24 on HDL and CE binding and SLU in HepG2 and PMA-treated THP-1 cells. In addition, total cholesterol (TC) levels in HepG2 cells were analyzed using enzymatic methods, and macrophage lipid content was evaluated by high-performance liquid chromatography (HPLC) assay. Small interfering RNA (siRNA) and pcDNA3.1(−)-hSR-BI plasmid transfection procedures were utilized to confirm the role of SR-BI in the effects of miR-24 on Dil-HDL uptake, SLU and cholesterol levels in both cell types. Hepatic SR-BI level in apoE−/− mice was measured by western blotting. Liver TC, FC and CE levels and plasma triglycerides (TG), TC and HDL-C levels were evaluated enzymatically using commercial test kits. Atherosclerotic lesion sizes were measured using Oil Red O and hematoxylin-eosin staining. ResultsmiR-24 directly repressed SR-BI expression by targeting its 3′UTR. In addition, miR-24 decreased Dil-HDL uptake and SLU in HepG2 and THP-1 macrophages. In the presence of HDL, miR-24 decreased TC levels in HepG2 cells and TC, free cholesterol (FC) and CE levels in macrophages. Overexpression and down-regulation assays showed that SR-BI mediated the effects of miR-24 on Dil-HDL uptake, SLU and cholesterol levels. Lastly, miR-24 administration decreased hepatic SR-BI expression and promoted atheromatous plaque formation in apoE−/− mice, findings in line with those of our in vitro studies. ConclusionsThese findings indicate that miR-24 accelerates atherogenesis by repressing SR-BI-mediated SLU from HDL-C.

High-Density Lipoprotein Function in Cardiovascular Disease and Diabetes Mellitus.

Cardiovascular disease (CVD) is the most common cause of death worldwide.1 Major CVD risk factors are hypertension, smoking, physical inactivity, abnormal glucose levels/diabetes mellitus, and dyslipidemia. Among these, dyslipidemia characterized by a low level of HDL-C (high-density lipoprotein cholesterol) is strongly and inversely correlated with CVD risk,2–4 and low HDL-C levels is part of the atherogenic dyslipidemia complex associated with diabetes mellitus.5 These observations triggered intense interest in increasing HDL-C levels for therapeutic intervention of CVD.6,7 However, several recent lines of evidence now suggest that the association between HDL-C levels and CVD status may be indirect or more complicated than previously recognized.7 Thus, human genetic studies demonstrate that genetically altered HDL-C levels do not necessarily translate to an altered risk of CVD.8–12 Phase III clinical trials of drugs that elevate HDL-C, such as niacin and CETP (cholesteryl ester transfer protein) inhibitors, also have largely failed to reduce CVD events in statin-treated subjects with established CVD.13–15 For several CETP inhibitors, improvement in CVD prevention was unsuccessful because of off-target effects (torcetrapib) or lack of efficacy (dalcetrapib and evacetrapib).14,16–18 The exception is the recent REVEAL trial (Randomized Evaluation of the Effects of Anacetrapib Through Lipid-Modification), which included more subjects and was performed for longer than previous CETP inhibitor trials. The REVEAL demonstrated that the CETP inhibitor anacetrapib exhibited beneficial effects on cardiovascular outcomes on top of those of statin therapy, although the risk reduction was moderate19,20 and might have been due, at least in part, to a reduction in non-HDL-C rather than elevated HDL-C.21 Considering these cumulative observations, it remains uncertain whether increased HDL-C directly impacts atherosclerosis and the …

The role of plasma lipoprotein lipase, hepatic lipase and GPIHBP1 in the metabolism of remnant lipoproteins and small dense LDL in patients with coronary artery disease

BackgroundThe relationship between plasma lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), glycosylphosphatidylinositol anchored HDL binding protein1 (GPIHBP1) concentration and the metabolism of remnant lipoproteins (RLP) and small dense LDL (sdLDL) in patients with coronary artery disease (CAD) is not fully elucidated. MethodsOne hundred patients who underwent coronary angiography were enrolled. The plasma LPL, HTGL and GPIHBP1 concentrations were determined by ELISA. The time dependent changes in those lipases, lipids and lipoproteins were studied at a time-point just before, and 15min, 4h and 24h after heparin administration. ResultsThe LPL concentration exhibited a significant positive correlation with HDL-C, and inversely correlated with TG and RLP-C. The HTGL concentration was positively correlated with RLP-C and sdLDL-C. The HTGL ratio of the pre-heparin/post-heparin plasma concentration and sdLDL-C/LDL-C ratio were significantly greater in CAD patients than in non-CAD patients. GPIHBP1 was positively correlated with LPL and inversely correlated with RLP-C and sdLDL-C. ConclusionThe HTGL concentration was positively correlated with RLP-C and sdLDL-C, while LPL and GPIHBP1 were inversely correlated with RLP-C and sdLDL-C. These results suggest that elevated HTGL is associated with increased CAD risk, while elevated LPL is associated with a reduction of CAD risk.

Mutating a conserved cysteine in GPIHBP1 reduces amounts of GPIHBP1 in capillaries and abolishes LPL binding

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease-chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.

VEGF-A Regulates Cellular Localization of SR-BI as Well as Transendothelial Transport of HDL but Not LDL.

Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of 125I-HDL but not 125I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells. The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall.

Triglyceride-Rich Lipoproteins and Remnants: Targets for Therapy?

It is now evident that elevated circulating levels of triglycerides in the non-fasting state, a marker for triglyceride (TG)-rich remnant particles, are associated with increased risk of premature cardiovascular disease (CVD). Recent findings from basic and clinical studies have begun to elucidate the mechanisms that contribute to the atherogenicity of these apoB-containing particles. Here, we review current knowledge of the formation, intravascular remodelling and catabolism of TG-rich lipoproteins and highlight (i) the pivotal players involved in this process, including lipoprotein lipase, glycosylphosphatidylinositol HDL binding protein 1 (GPIHBP1), apolipoprotein (apo) C-II, apoC-III, angiopoietin-like protein (ANGPTL) 3, 4 and 8, apoA-V and cholesteryl ester transfer protein; (ii) key determinants of triglyceride (TG) levels and notably rates of production of very-low-density lipoprotein 1 (VLDL1) particles; and (iii) the mechanisms which underlie the atherogenicity of remnant particles. Finally, we emphasise the polygenic nature of moderate hypertriglyceridemia and briefly discuss modalities for its clinical management. Several new therapeutic strategies to attenuate hypertriglyceridemia have appeared recently, among which those targeted to apoC-III appear to hold considerable promise.

Myristic acid is associated to low plasma HDL cholesterol levels in a Mediterranean population and increases HDL catabolism by enhancing HDL particles trapping to cell surface proteoglycans in a liver hepatoma cell model

BackgroundHDL-C plasma levels are modulated by dietary fatty acid (FA), but studies investigating dietary supplementation in FA gave contrasting results. Saturated FA increased HDL-C levels only in some studies. Mono-unsaturated FA exerted a slight effect while poly-unsaturated FA mostly increased plasma HDL-C. AimsThis study presents two aims: i) to investigate the relationship between HDL-C levels and plasma FA composition in a Sicilian population following a “Mediterranean diet”, ii) to investigate if FA that resulted correlated with plasma HDL-C levels in the population study and/or very abundant in the plasma were able to affect HDL catabolism in an “in vitro” model of cultured hepatoma cells (HepG2). Resultsplasma HDL-C levels in the population correlated negatively with myristic acid (C14:0, β = −0.24, p < 0.01), oleic acid (C18:1n9, β = −0.22, p < 0.01) and cis-11-Eicosenoic (C20:1n9, β = −0.19, p = 0.01) and positively with palmitoleic acid (C16:1, β = +0.19, p = 0.03). HepG2 cells were conditioned with FA before evaluating HDL binding kinetics, and only C14:0 increased HDL binding by a non-saturable pathway. After removal of heparan sulphate proteoglycans (HSPG) by heparinases HDL binding dropped by 29% only in C14:0 conditioned cells (p < 0.05). C14:0 showed also the highest internalization of HDL-derived cholesteryl esters (CE, +32% p = 0.01 vs. non-conditioned cells). ConclusionsC14:0 was correlated with decreased plasma HDL-C levels in a Mediterranean population. C14:0 might reduce HDL-C levels by increasing HDL trapping to cell surface HSPG and CE stripping from bound HDL. Other mechanisms are to be investigated to explain the effects of other FA on HDL metabolism.

Serum amyloid A impairs the antiinflammatory properties of HDL.

HDL from healthy humans and lean mice inhibits palmitate-induced adipocyte inflammation; however, the effect of the inflammatory state on the functional properties of HDL on adipocytes is unknown. Here, we found that HDL from mice injected with AgNO3 fails to inhibit palmitate-induced inflammation and reduces cholesterol efflux from 3T3-L1 adipocytes. Moreover, HDL isolated from obese mice with moderate inflammation and humans with systemic lupus erythematosus had similar effects. Since serum amyloid A (SAA) concentrations in HDL increase with inflammation, we investigated whether elevated SAA is a causal factor in HDL dysfunction. HDL from AgNO3-injected mice lacking Saa1.1 and Saa2.1 exhibited a partial restoration of antiinflammatory and cholesterol efflux properties in adipocytes. Conversely, incorporation of SAA into HDL preparations reduced antiinflammatory properties but not to the same extent as HDL from AgNO3-injected mice. SAA-enriched HDL colocalized with cell surface-associated extracellular matrix (ECM) of adipocytes, suggesting impaired access to the plasma membrane. Enzymatic digestion of proteoglycans in the ECM restored the ability of SAA-containing HDL to inhibit palmitate-induced inflammation and cholesterol efflux. Collectively, these findings indicate that inflammation results in a loss of the antiinflammatory properties of HDL on adipocytes, which appears to partially result from the SAA component of HDL binding to cell-surface proteoglycans, thereby preventing access of HDL to the plasma membrane.

Identification and analysis of anti-HDL scFv-antibodies obtained from phage display based synthetic antibody library

ObjectiveIn epidemiological studies plasma high density lipoprotein cholesterol (HDL-C) levels are found to correlate inversely with atherosclerotic cardiovascular events. HDL consists of different subpopulations and they vary in their anti-atherogenic properties. The aim of this study is to isolate coronary artery disease (CAD) specific anti-HDL scFv-antibodies. Design and methodsTo obtain CAD specific HDL binders, we used phage displayed synthetic antibody libraries to enrich specific antibodies against HDL isolated from CAD patients. The antibodies were affinity purified. Their capability to recognize apolipoproteins A-I and A-II, various HDL forms differing in lipid/protein ratios and plasma HDL, was studied using time-resolved fluorescence based immunoassay. ResultsUsing different selection strategies and immunoassay based screening we obtained altogether 1200 clones displaying HDL binding activity. By sequencing 337, we identified 264 unique antibodies against HDL. A set of 61 antibodies were selected for further analysis. We found a variety of antibodies with different binding profiles, including apoA-I binding antibodies either in lipid-dependent or lipid-independent manner and binders against apoA-II. Several antibodies were able to discriminate between HDL derived from CAD patients and healthy controls. A majority of the antibodies were immunoreactive with HDL in plasma. ConclusionThe novel HDL recognizing antibodies isolated from synthetic antibody phage library have displayed interesting HDL-binding characteristics suggesting that, in addition to use as research tools, a part of them might be useful for the development of diagnostic methods for CAD risk assessment.