LDLR Degradation Research Articles

Abstract 158: The Role Of Ehbp1 In Hypercholesterolemia

Background: Despite aggressive lowering of cholesterol, conventional lipid-lowering therapies only modestly lower cardiovascular risk in the population. Therefore, identifying novel hypercholesterolemia-risk targets is of critical importance. Human genome-wide association studies (GWAS) have revealed that several single-nucleotide polymorphisms (SNPs) in EHBP1 , the gene that encodes EH domain binding protein 1 (EHBP1), an endocytic trafficking regulator, are associated with serum cholesterol. However, the mechanisms underlying this association and the role of EHBP1 in cholesterol metabolism are unknown. We hypothesize that EHBP1 is a novel hypercholesterolemia risk factor. The goal of this study is to explore the role of EHBP1 in hypercholesterolemia and its underlying mechanisms. Methods: Hepatocyte EHBP1 was silenced by intravenous injection (IV) of AAV8-H1-shEhbp1 in wild-type (WT), Apoe-/- and Ldlr-/- mice, followed by Western Diet feeding to induce hypercholesterolemia and atherosclerosis. Primary murine and human hepatocytes were isolated for in vitro mechanistic studies. We also leveraged publicly available human genetic studies and unbiased scRNA datasets to support our study. Results: Individuals with the EHBP1 rs10496099 T>C variant have decreased serum cholesterol and reduced liver EHBP1 expression. Consistent with the human data, our in vivo study with hypercholesterolemic mice shows that EHBP1 silencing in hepatocytes decreases plasma total and LDL/VLDL cholesterol. Our in vitro study with primary hepatocytes reveals that EHBP1 deficiency eliminates sortilin (SORT1)-mediated PCSK9 secretion and induces LDLR-mediated LDL uptake. Mechanistically, we found that EHBP1 silencing abolishes the homeostasis of the retromer complex, a protein complex that helps retrograde and stabilize sortilin. Conclusion: Hepatocyte EHBP1 promotes sortilin-mediated PCSK9 secretion by maintaining retromer homeostasis, leading to LDLR degradation and hypercholesterolemia. Our study elucidates a novel link between EHBP1 and other cholesterol-associated loci (e.g., SORT1 , PCSK9 , and LDLR) and provides new mechanistic insight into the role of EHBP1 in hepatic cholesterol metabolism and hypercholesterolemia.

Legacy perfluoro-alkyl substances impair LDL-cholesterol uptake independently from PCSK9-function

Perfluoro-alkyl substances (PFAS) are pollutants, whose exposure was associated with altered levels of low-density lipoproteins (LDL) in humans. Here we investigated this clinical outcome in two groups of young male adults residing in areas of respectively low and high environmental exposure to perfluoro-octanoic-acid (PFOA). From the Regional Authority data on pollution areas, 38 not-exposed and 59 exposed age-matched participants were evaluated for serum levels of total cholesterol (Total-Chol), LDL-Chol, high-density lipoprotein cholesterol (HDL-Chol), triglycerides (Tgl) and chromatography quantified PFOA. Human hepato-carcinoma cell line HepG2 was exposed to PFOA or perfluoro-octane-sulfonate (PFOS), as legacy PFAAs, and C6O4 as new generation compound. Fluorimetry was used to evaluate the cell-uptake of labelled-LDL. Proprotein Convertase Subtilisin/Kexin 9 (PCSK9)-mediated LDL-receptor (LDL-R) degradation and sub-cellular localization of LDL-R were evaluated by western blot analysis. Serum levels of PFOA, were positively and significantly correlated with Total-Chol (ρ = 0.312, P = 0.002), LDL-Chol (ρ = 0.333, P = 0.001) and Tgl (ρ = 0.375, P < 0.001). Participants with high serum LDL-Chol and Tgl levels, according to the cardiovascular risk, were more prevalent in exposed compared to not-exposed subjects (respectively: 23.7% vs 5.3%, P = 0.023 and 18,6% vs 0%, P = 0.006). Exposure of HepG2 cells to PFOA or C6O4 100 ng/mL was associated with a significantly lower LDL uptake than controls but no major impact of any PFAAs on PCSK9-mediated LDL-R degradation was observed. Compared to controls, exposure to PFAS showed an unbalanced LDL-R partition between membrane and cytoplasm. Endocytosis inducer sphingosine restored LDL-R partition only in samples exposed to C6O4. These data suggest a novel endocytosis-based mechanism of altered lipid trafficking associated with the exposure to legacy PFAS.

MicroRNA 33A controls SREBP-2 and LXR dependent regulation of the LDL receptor pathway

Background and Aims : Coordinated cellular mechanisms that regulate LDLR transcription and degradation remain largely obscure. We sought to assess the effect of miR-33a on the LDLR pathway given the concerted action of SREBP-2 and miR-33a in elevating cellular cholesterol levels.

A novel mouse model of diabetes, atherosclerosis and fatty liver disease using an AAV8-PCSK9-D377Y injection and dietary manipulation in db/db mice

Preclinical mouse models of cardiometabolic diseases are crucial to study the pathological mechanisms of cardiometabolic diseases and to explore potential new therapeutic agents. Using double-knockouts in the background of ApoE−/− or Ldlr−/− mice requires an extensive amount of breeding and is costly. A significant breakthrough in atherosclerosis research is the use of AAV8-PCSK9-D377Y (a gain-of-function mutant of PCSK9 which promotes LDLR degradation) injection which can induce hyperlipidemia, increased endothelial stiffness, vascular calcification, aneurysm, and atherosclerotic plaque development in normal C57BL/6J mice. The purpose of this study was to assess the possibility that the injection of AAV8-PCSK9 vectors in db/db mice (a well-established animal model of type 2 diabetes mellitus) produces a novel mouse model of diabetes, atherosclerosis and fatty liver disease to study the pathomechanisms of cardiometabolic disease and its complications. Db/db mice were injected with AAV8-PCSK9-D377Y (AAV8-PCSK9 for simplicity) or AAV8-control and fed with high-cholesterol diets for 8 weeks. Levels of total cholesterol (TC) and triglyceride (TG) were significantly elevated in AAV8-PCSK9-injected mice compared to the controls. AAV8-PCSK9 injection led to increased serum level of PCSK9, serious liver steatosis, hypercholesterolemia and atherosclerotic plaque as determined by aortic arch/roots histopathological staining, with Oil Red O, Masson-trichrome and hematoxylin-eosin staining. RNA sequencing and bioinformatics were used to assess the global gene expression in liver tissues. We conclude that AAV8-PCSK9 injection in db/db mice is a promising and time-efficient approach to induce diabetic atherosclerosis with fatty liver. This mouse model can be a new one to investigate the etiology and therapeutics of atherosclerosis with diabetes and fatty liver beyond the traditional model established in ApoE−/− mice or LDLR−/− mice receiving streptozotocin (STZ) injection.

Modifying pH-sensitive PCSK9/LDLR interactions as a strategy to enhance hepatic cell uptake of low-density lipoprotein cholesterol (LDL-C).

LDL-receptor (LDLR)-mediated uptake of LDL-C into hepatocytes is impaired by lysosomal degradation of LDLR, which is promoted by proprotein convertase subtilisin/kexin type 9 (PCSK9). Cell surface binding of PCSK9 to LDLR produces a complex that translocates to an endosome, where the acidic pH strengthens the binding affinity of PCSK9 to LDLR, preventing LDLR recycling to the cell membrane. We present a new approach to inhibit PCSK9-mediated LDLR degradation, namely, targeting the PCSK9/LDLR interface with a PCSK9-antagonist, designated Flag-PCSK9PH, which prevents access of WT PCSK9 to LDLR. In HepG2 cells, Flag-PCSK9PH, a truncated version (residues 53-451) of human WT PCSK9, strongly bound LDLR at the neutral pH of the cell surface but dissociated from it in the endosome (acidic pH), allowing LDLR to exit the lysosomes intact and recycle to the cell membrane. Flag-PCSK9PH thus significantly enhanced cell-surface LDLR levels and the ability of LDLR to take up extracellular LDL-C.

305 Usefulness of CHA2DS2-VASc scoring system for predicting risk of embolism in patients with cardiac tumours: a single-centre study

Abstract Aims The link between pro-protein convertase subtilisin/kexin 9 (PCSK9) and inflammatory-related disease has been supported in several experimental and human studies, which show how the protein could acts independent its canonical mechanism, by activation of inflammatory, apoptotic and immune pathways. Among these, TLR4/NFKB signalling pathway has been found to be one of the main pathways mediating the PCSK9-induced increase of pro-inflammatory and pro-thrombotic molecules, such as TNF-alpha, IL-6, IL-1, MCP-1, and TF. To investigate the direct involvement of hTLR4 in recognition of exogenous human recombinant (hr) PCSK9. Methods and results Experiments were carried out in three cell line: (i) HEK293-hTLR4-GFP cell lines obtained by stable co-transfection of HEK293 cells with the pUNO1-hTLR4-GFP, a plasmid expressing the hTLR4 gene fused to a GFP gene; (ii) HEK293-hTLR4+ cells obtained by co-transfection of hTLR4 receptor and its accessory proteins MD-2 and CD14, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene; (iii) HEK293-Null2 cells, a cell line lacking TLR4 expression, used as negative control. NFkB activity were measured by the SEAP reporter gene assay using a fluorescence detection method, while localization of hTLR4 and exogenous hrPCSK9 by confocal microscopy. hrPCSK9 (1 µg/mL) activates NFkB in HEK293-hTLR4+ cells [SEAP activity-OD 620 nm: from a baseline of 0.18 ± 0.06 to 0.68 ± 0.05 (N = 8), P &amp;lt; 0.001] to an extent comparable to lipopolysaccharide, the specific agonist of TLR4. Co-localization of hrPCSK9 and TLR4s was clearly documented by quantitative confocal microscopy in HEK293-hTLR4-GFP cell line, that comprises the analysis of more than 80 fields in ∼2.105 cells/well and showing a percentage of co-localization of ∼4% in membrane spots (P &amp;lt; 0.01). Conclusions Our data support the results reported in previous studies that attribute to PCSK9 a pathophysiological role in development of chronic inflammation and related-diseases such as atherothrombosis. A behavior that extends far beyond LDLR degradation, via a mechanism that might be mediated at least in part by recognition of PCSK9 by the TLRs and later activation of NFkB intracellular pathway. Future studies will be important to better investigate the specific binding site engaged in the PCSK9-TLR interaction and to discriminate the intracellular transduction pathways involved in this response, in order to provide a theoretical basis for the development of innovative therapeutic strategies.

306 Involvement of toll-like receptor 4 in PCSK9-mediated biological effects in cell culture models

Abstract Aims The link between pro-protein convertase subtilisin/kexin 9 (PCSK9) and inflammatory-related disease was supported in several experimental and human studies, which show how the protein could acts independent its canonical mechanism, by activation of inflammatory, apoptotic and immune pathways. Among these, TLR4/NFKB signalling pathway was found to be one of the main pathways mediating the PCSK9-induced increase of pro-inflammatory and pro-thrombotic molecules, such as TNF-alpha, IL-6, IL-1, MCP-1, and TF. To investigate the direct involvement of hTLR4 in recognition of exogenous human recombinant (hr) PCSK9. Methods Experiments were carried out in three cell line: (i) HEK293-hTLR4-GFP cell lines obtained by stable co-transfection of HEK293 cells with the pUNO1-hTLR4-GFP, a plasmid expressing the hTLR4 gene fused to a GFP gene; (ii) HEK293-hTLR4+ cells obtained by co-transfection of hTLR4 receptor and its accessory proteins MD-2 and CD14, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene; (iii) HEK293-Null2 cells, a cell line lacking TLR4 expression, used as negative control. NFkB activity were measured by the SEAP reporter gene assay using a fluorescence detection method, while localization of hTLR4 and exogenous hrPCSK9 by confocal microscopy. Results hrPCSK9 (1 µg/mL) activates NFkB in HEK293-hTLR4+ cells [SEAP activity-OD 620 nm: from a baseline of 0.18 ± 0.06 to 0.68 ± 0.05 (N = 8), P &amp;lt; 0.001] to an extent comparable to Lipopolysaccharide (LPS), the specific agonist of TLR4. Colocalization of hrPCSK9 and TLR4s was clearly documented by quantitative confocal microscopy in HEK293-hTLR4-GFP cell line, that comprises the analysis of more than 80 fields in ∼2.105 cells/well and showing a percentage of co-localization of ∼4% in membrane spots (P &amp;lt; 0.01). Conclusions Our data support the results reported in previous studies that attribute to PCSK9 a pathophysiological role in development of chronic inflammation and related-diseases such as atherothrombosis. A behaviour that extends far beyond LDLR degradation, via a mechanism that might be mediated at least in part by recognition of PCSK9 by the TLRs and later activation of NFKB intracellular pathway. Future studies will be important to better investigate the specific binding site engaged in the PCSK9-TLR interaction and to discriminate the intracellular transduction pathways involved in this response, in order to provide a theoretical basis for the development of innovative therapeutic strategies.

Lipoprotein compartmentalisation as a regulator of PCSK9 activity

Lipoprotein compartmentalisation and PCSK9-mediated LDLR degradation. PCSK9 binds to the extracellular domain of the LDLR to promote its degradation through the lysosomal compartment. PCSK9 exists in a free and lipoprotein-bound form in the circulation. PCSK9 association with LDL is thought to sequester PCSK9 and inhibit PCSK9-mediated LDLR degradation. The functional consequence of the interaction between PCSK9 and HDL is currently unknown. PCSK9 probably shuttles between a lipoprotein-bound pool of PCSK9 and circulating free-PCSK9.

Structural and Molecular Interaction Studies on Familial Hypercholesterolemia Causative PCSK9 Functional Domain Mutations Reveals Binding Affinity Alterations with LDLR

Familial hypercholesterolemia (FH) is a metabolic disease caused by the inherited pathogenic mutations in PCSK9 gene. This study has evaluated the potential of diverse computational methods in predicting the PCSK9 clinical variants and also studied the impacts of mutation on protein phenotype and function. Our findings show the superior prediction ability of FATHMM over CADD, M-CAP, SIFT and Polyphen methods, in screening PCSK9 missense mutations causative to FH. Computational 3D mapping of PCSK9 variants located in prodomain (K83T), catalytic domain (D204N) and c-terminal domains (K494E) revealed mutant residue induced disturbances in the tertiary structure of protein. These variants are also predicted to impair the free energy dynamics, hence stability of PCKS9 as per the consensual predictions made by protein structure-based prediction algorithms like SAAFEC and MAESTRO. Molecular docking assay by PIZMA algorithm showed the increased binding affinity between PCSK9 variants and LDLR molecules. Furthermore, the results from PDBsum method have also suggested changes in interfacing residues, interface area, salt bridge and ionic bond interactions confirming the findings from docking analysis. The PCSK9 functional domain mutations could increase its binding affinity with LDLR, eventually promoting LDLR degradation in lysosomes and elevate circulating cholesterol levels in body. This study supports the application of comprehensive computational assessment of FH causative PCSK9 mutations before undertaking labor intensive functional biology investigations.

INFLAMMATORY MACROPHAGES DERIVED EXOSOMAL MIR-222 PROMOTED THECA CELL STEROIDOGENESIS BY MODULATING MYLIP EXPRESSION

To explore the role of M1 macrophages (M1M) in the linkage between low-grade inflammation and ovarian dysfunction in obesity. In vitro experimental laboratory study. Raw264.7 macrophage was treated with lipopolysaccharide (LPS) to establish a model of M1M. Exosome (Exo) secreted by macrophage was isolated by ultracentrifugation and identified by transmission electron microscopy (TEM), western blot, and flow nanoanalyzer. The primary murine ovary theca cell (TC) was co-incubated with PKH67 fluorescence-labeled Exo to detect whether Exo secreted by macrophage could be uptaken by TC. The expression of CYP11A1 and CYP17A1 were analyzed by qPCR and western blot. Medium testosterone and progesterone level were analyzed. High-throughput sequencing was performed to analyze the expression of exosomal miRNA (miRNA-seq). In order to select key differential exosomal miRNA, the GSE97652 (species: mouse) was downloaded from the GEO DataBases. The data set contains 7 replications in both experimental group and control group. The experimental group is obese mouse adipose tissue macrophage (ATM)-derived Exo, and the control group is control mouse ATM -derived Exo. The overlap between miRNA-seq and GSE97652 differentially expressed miRNA was analyzed according to the Fold change > 2, p < 0.01 standard, which determined miR-222 and MYLIP for subsequent experiments. Next, gain-of-function experiments were performed to examine their role in TC steroidogenesis function with the involvement of the cholesterol metabolism pathway. TEM, Flow NanoAnalyzer and western blot analysis confirmed the successful extraction of Exo. After co-cultured TC with PKH67-labeled Exo, green fluorescence could be detected in TC which demonstrated that TC was able to ingest Exo secreted by the macrophage. M1M increased the expression of CYP11A1 and the production of progesterone in TC which is dependent upon M1-Exo. The result of miRNA-seq showed that there were 30 upregulated miRNA and 112 down-regulated miRNA in M1M derived Exo compared with control Exo according to the standard of Fold change > 2(p < 0.05). A total of two differentially expressed miRNA, namely miR-222 and miR-155, were selected from the venn diagram of miRNA-seq and GSE97652. QPCR verified that miR-222 in both M1M derived Exo and M1M derived Exo treated TC were significantly upregulated. Subsequently, we predicted the target gene of miR-222 through five different databases. MYLIP, with the highest target score was selected, which has been identified as a key factor promoting LDLR degradation. M1M derived Exo was verified to display a high expression level of miR-222, and M1M derived Exo mediated TC steroidogenesis depended miR-222. The overexpression of miR-222 resulted in the down-regulation of MYLIP expression and up-regulation of LDLR expression, followed by increased cholesterol influx, CYP11A1 overexpression and progesterone overproduction. M1M derived Exo can be ingested by TC. M1M promote TC CYP11A1 overexpression and progesterone overproduction through Exo by delivering miR-222 into TC.

Three Musketeers for Lowering Cholesterol: Statins, Ezetimibe and Evolocumab.

Coronary heart disease (CHD) is closely related to hypercholesterolemia, and lowering serum cholesterol is currently the most important strategy in reducing CHD. In humans, the serum cholesterol level is determined mainly by three metabolic pathways, namely, dietary cholesterol intake, cholesterol synthesis, and cholesterol degradation in vivo. An intervention that targets the key molecules in the three pathways is an important strategy in lowering serum lipids. Statins inhibit 3-hydroxyl-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) to reduce low-density lipoprotein (LDL) by about 20% to 45%. However, up to 15% of patients cannot tolerate the potential side effects of high statin dosages, and several patients also still do not reach their optimal LDL goals after being treated with statins. Ezetimibe inhibits cholesterol absorption by targeting the Niemann-Pick C1-like 1 protein (NPC1L1), which is related to cholesterol absorption in the intestines. Ezetimibe lowers LDL by about 18% when used alone and by an additional 25% when combined with statin therapy. The proprotein convertase subtilisin/kexin type 9 (PCSK9) increases hepatic LDLR degradation, thereby reducing the liver's ability to remove LDL, which can lead to hypercholesterolemia. Evolocumab, which is a PCSK9 monoclonal antibody, can reduce LDL from baseline by 53% to 56%. The three drugs exert lipid-lowering effects by regulating the three key pathways in lipid metabolism. Combining any with the two other drugs on the basis of statin treatment has improved the lipid-lowering effect. Whether the combination of the three musketeers will reduce the side effects of monotherapy and achieve the lipid-lowering effect should be studied further in the future.

17β-Estradiol Inhibits PCSK9-Mediated LDLR Degradation Through GPER/PLC Activation in HepG2 Cells.

Plasma levels of PCSK9 are significantly higher in postmenopausal women. Pharmacologically increased estrogen levels have been shown to lower PCSK9 and LDL-C levels in animals and humans. The action of estrogen suggests that it has the ability to prevent PCSK9-mediated LDLR degradation in liver cells. However, little is known about how estrogen alters PCSK9-mediated LDLR degradation. Here, we report that 17β-estradiol (βE2) reduces PCSK9-mediated LDLR degradation by a mechanism that involves activation of the G protein-coupled estrogen receptor (GPER). In cultured HepG2 cells, βE2 prevented the internalization of PCSK9, which subsequently lead to PCSK9-mediated LDLR degradation. The altered LDLR levels also resulted in an increase in LDL uptake that was not observed in the absence of PCSK9. In addition, we showed that clathrin was rapidly increased in the presence of PCSK9, and this increase was blocked by βE2 incubation, suggesting rapid recruitment of clathrin in HepG2 cells. PLCγ activation and intracellular Ca2+ release were both increased due to the rapid effect of estrogen. By using a GPER antagonist G15, we demonstrated that the GPER mediates the action of estrogen. Together, the data from this in vitro study demonstrate that estrogen can regulate LDLR levels mainly through GPER activation, which prevents PCSK9-dependent LDLR degradation in HepG2 cells.

PCSK9-D374Y mediated LDL-R degradation can be functionally inhibited by EGF-A and truncated EGF-A peptides: An in vitro study

Background and aimsProprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR) through the LDLR epidermal growth factor-like repeat A (EGF-A) domain and induces receptor internalization and degradation. PCSK9 has emerged as a novel therapeutic target for hypercholesterolemia. Clinical studies with PCSK9 inhibiting antibodies have demonstrated strong LDL-c lowering effects, but other therapeutic approaches using small molecule inhibitors for targeting PCSK9 functions may offer supplementary therapeutic options. The aim of our study was to evaluate the effect of synthetic EGF-A analogs on mutated (D374Y) PCSK9-D374Y mediated LDLR degradation in vitro. MethodsHuh7 human hepatoma cells were transiently transfected to overexpress the gain-of-function D374Y PCSK9 mutation, which has been associated with severe hypercholesterolemia in humans. ResultsTransient transfection of cells with PCSK9-D374Y expression vector very effectively enhanced degradation of mature LDLR in Huh7. Treatment with both EGF-A and EGF-A truncated peptides inhibited this effect and showed increased LDLR protein in Huh7 cells transfected with PCSK9-D374Y in a clear concentration dependent manner. Huh7 transfected cells treated with increasing concentration of EGF-A analogs also showed an increase internalization of labeled Dil-LDL. ConclusionsThe result of our study shows that EGF-A analogs are able to effectively hamper the enhanced degradation of LDLR in liver cells expressing PCSK9-D374Y.

Pharmacological Inhibition And Hepatic-Deficiency Of Pcsk9 Reduce Post-Prandial Lipemia In Mice

Background and Aims: Postprandial lipemia (PPL) is associated with atherosclerosis development and cardiovascular diseases. PCSK9 reduces plasma cholesterol by promoting LDLR degradation. We previously showed that PCSK9-deficiency reduced PPL by altering the intestinal chylomicron production and by increasing their hepatic catabolism. Our study aims: i) to assess the relative importance of hepatic or intestinal PCSK9 in PPL regulation ; ii) to assess the efficacy of an anti-PCSK9 monoclonal antibody on PPL in C57BL/6J and LDLR-/- mice ; iii) to determine whether PCSK9-deficiency also reduced PPL in insulinopenic streptozotocin(STZ)-induced diabetic mice.

Proprotein convertase subtilisin/kexin type 9 and lipid metabolism.

The purpose of this review is to highlight the recent findings of one of the most promising therapeutic targets in LDL cholesterol (LDL-C) management, proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum cargo receptor, surfeit locus protein 4 interacts with PCSK9 and regulates its exit from endoplasmic reticulum and its secretion. Once secreted, PCSK9 binds to heparin sulfate proteoglycans on the hepatocyte surface and this binding is required for PCSK9-LDL receptor (LDLR) complex formation and LDLR degradation. Posttranscriptionally, recent work has shown that PCSK9 gets degraded in lysosomes by activation of the glucagon receptor signaling, providing more data on the hormonal regulation of PCSK9. Finally, human studies with PCSK9 inhibitors offered more evidence on their benefits and safe use. Recent work on the regulation of PCSK9 has enhanced our understanding of its biology, which may provide important information for future PCSK9-based therapies.

Safety and efficacy of alirocumab: A meta analysis of 12 randomized controlled trials.

Background and Objective:Hypercholesterolemia is one of the major risk factor for atherosclerotic coronary heart disease, especially coronary heart disease. Most effective class of medications for prevention of cardiovascular events and LDL-C reduction are the statins. Approximately only one fourth of these high risk patients had achieved LDL-C levels <70 mg/dL with statins. Monoclonal antibody targeting PCSK9 is a novel class of drug used in the treatment of Hypercholesterolemia. Alirocumab is one such human monoclonal antibody directed against PCSK9. Binding of PCSK9 to the LDL-R on the hepatocytes promotes LDL-R degradation. Inhibition of PCSK9 binding to LDL-R leads to increased number of LDL-Rs to clear LDL, thus decreasing LDL-C levels. The purpose of this systematic study is to assess the safety and efficacy of Alirocumab in adults with hypercholesterolemia and Familial hypercholesterolemia.Materials and Methods:We searched Medline, PubMed Central database, Google scholar, EBSCO, Wiley library, conference proceedings and Clinical trials.gov registry through March 2017. Phase 3 randomized, controlled trials (RCTs) using Alirocumab in adults with hypercholesterolemia and Familial Hypercholesterolemia were selected.Results:In twelve RCTs comprising of 6019 patients included in the meta-analysis, significant favorable changes in LDL-C and HDL-C were found.Limitations:Results were derived from study level data rather than patient level data.Conclusions:Alirocumab substantially reduced the LDL-C level by over 50 %, increased the HDL-C level, and resulted in favorable changes in other lipids.

Key aspects of PCSK9 inhibition beyond LDL lowering.

Our primary objective is to review the most recent findings on the biology of PCSK9 and on two key aspects of PCSK9 inhibition beyond LDL control of great clinical relevance: the regulation of lipoprotein (a) circulating levels by PCSK9 inhibitors and the putative diabetogenic effects of these novel therapies. The reality of two distinct extracellular and intracellular pathways by which PCSK9 decreases the abundance of the LDLR at the surface of many cell types, most importantly hepatocytes, has recently been established. In contrast, the exact mechanisms by which PCSK9 inhibitors lower the circulating levels of lipoprotein (a) remain a point of major dispute. Despite strong indications from genetic studies that PCSK9 inhibition should increase diabetes risk, no such effect has been observed in clinical trials, and in-vitro and in-vivo studies do not clarify this issue. The trafficking pathways by which PCSK9 enhance LDLR degradation via the endolysosomal extracellular route or via the Golgi-lysosomal intracellular route remain to be fully elucidated. The mechanisms by which PCSK9 inhibitors reduce lipoprotein (a) also merit additional research efforts. The role of PCSK9 on glucose metabolism should likewise be studied in depth.

Association between plasma levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) and lipids with rs7903146 polymorphisms of the TCF7L2 gene in diabetic patients

The rs7903146 polymorphism of TCF7L2 gene is known as the strongest genetic risk factor for type 2 diabetes mellitus (T2DM). The polymorphism is in association with clinical profile of T2DM patients. PCSK9 is a serine protease that promotes LDLR degradation and regulates circulating levels of lipids. The association of this polymorphism with PCSK9 and metabolic profile of diabetic and healthy subjects was investigated. This cross-sectional study was performed on 132 T2DM patients and the same number of healthy subjects. All the participants were genotyped for the rs7903146 single nucleotide polymorphism by the PCR-RFLP method. Metabolic profile including plasma levels of PCSK9, triglycerides, total cholesterol, non-HDL cholesterol, LDL cholesterol, HDL cholesterol, fasting plasma glucose, and HBA1C was measured. PCSK9, total cholesterol, and LDL-C levels were lower in the diabetic patients as compared to the healthy subjects. There were also direct and significant associations between PCSK9 and TG, TC, LDL-C, and non HDL-C in the subjects. Values of plasma glucose, HbA1c, PCSK9, TC, and LDL-C were higher in patients with TT genotype, but the differences were not statistically significant for all. A positive Spearman correlation was found between PCSK9 levels and the genotypes in all the participants. The results confirm the association of rs7903146 in the TCF7L2 gene with metabolic parameters and PCSK9. The T allele was associated with higher lipid and PCSK9 levels.

Allium tuberosum Reverses PCSK9-Mediated LDLR Degradation by Inhibition of HNF1α

Allium tuberosum Reverses PCSK9-Mediated LDLR Degradation by Inhibition of HNF1α

Translational and Therapeutic Approaches to the Understanding and Treatment of Dyslipidemia.

Circulating lipoproteins containing cholesterol and triglycerides are major risk factors for many cardiovascular diseases (CVD). Many therapeutic strategies targeting lipid metabolism have been successfully used to treat dyslipidemia and subsequently reduce cardiovascular risk. Most notably, statins, which effectively lower low-density lipoprotein-cholesterol (LDL-C), have been widely used and have been shown to substantially reduce cardiovascular events. More recently, a new class of LDL-lowering drugs, the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, has been developed with highly promising results. Many other therapeutic approaches aiming to modulate lipids are also being considered. These include antisense oligonucleotides (ASOs) designed to inhibit well-characterized gene targets involved in dyslipidemia, modulators of high-density lipoprotein (HDL) function and reverse cholesterol transport (RCT) pathways, and triglyceride-lowering drugs. We herein highlight recent basic, translational, and clinical studies published in ATVB that further elucidates the mechanisms and therapeutic potential of these approaches. LDL-receptor (LDLR) is a cell surface receptor that was found to be mutated in patients with familial hypercholesterolemia (FH).1 FH is a genetic disorder typically characterized by hypercholesterolemia, ectopic cholesterol deposition, and premature CVD. Hepatic LDLR is essential for the uptake of circulating LDL-C, which cannot be effectively cleared in FH patients. Conversely, increasing LDLR levels, particularly in the liver, enhances LDL-C clearance. Recent evidence identified PCSK9, a protein secreted by the liver, as a mediator of LDLR degradation by interacting with its extracellular domain and promoting its trafficking to the lysosome, rather than allowing normal recycling to the cell surface.2,3 PCSK9 gain-of-function mutations in humans were associated with decreased LDLR levels, and thus increased circulating LDL-C, causing autosomal dominant hypercholesterolemia.4,5 In contrast, loss-of-function mutations in PCSK9 were associated with lower LDL-C levels and reduced CVD events.6 Therefore, PCSK9 became a …