Low Density Lipoprotein Metabolism Research Articles

Protective Effect of Patchouli Alcohol Against High-Fat Diet Induced Hepatic Steatosis by Alleviating Endoplasmic Reticulum Stress and Regulating VLDL Metabolism in Rats.

Nonalcoholic fatty liver disease (NAFLD) is currently the most common chronic hepatic disorder worldwide. The earliest stage of NAFLD is simple steatosis, which is characterized by the accumulation of triglycerides in hepatocytes. Inhibition of steatosis is a potential treatment for NAFLD. Patchouli alcohol (PA) is an active component of Pogostemon cablin (Blanco) Benth. (Labiatae), which is a medicinal food in Asia countries and proved to possess hepatoprotective effect. This research aimed to investigate the effectiveness of PA against high fat diet (HFD)-induced hepatic steatosis in rats. In this study, male Sprague Dawley rats were fed a HFD for 4 weeks to induce NAFLD. Oral administration with PA significantly reduced pathological severity of steatosis in HFD-fed rats. It was associated with suppressing endoplasmic reticulum (ER) stress and regulating very low-density lipoprotein (VLDL) metabolism. Our data showed that PA treatment effectively attenuated ER stress by inhibiting the activation of protein kinase-like ER kinase (PERK), inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1), and activating transcription factor 6 (ATF6). Moreover, PA decreased hepatic VLDL uptake by suppressing very low-density lipoprotein receptor (VLDLR) expression. It also restored VLDL synthesis and export by increasing apolipoprotein B100 (apoB 100) secretion and microsomal triglyceride-transfer protein (MTP) activity. Taken together, PA exerted a protective effect on the treatment of NAFLD in HFD-fed rats and may be potential therapeutic agent acting on hepatic steatosis.

Structure and Function of Proprotein Convertase Subtilisin/kexin Type 9 (PCSK9) in Hyperlipidemia and Atherosclerosis.

One of the important factors in Low-Density Lipoprotein (LDL) metabolism is the LDL receptor (LDLR) by its capacity to bind and subsequently clear cholesterol derived from LDL (LDL-C) in the circulation. Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is a newly discovered serine protease that destroys LDLR in the liver and thereby controls the levels of LDL in plasma. Inhibition of PCSK9-mediated degradation of LDLR has, therefore, become a novel target for lipid-lowering therapy. We review the current understanding of the structure and function of PCSK9 as well as its implications for the treatment of hyperlipidemia and atherosclerosis. New treatments such as monoclonal antibodies against PCSK9 may be useful agents to lower plasma levels of LDL and hence prevent atherosclerosis. PCSK9's mechanism of action is not yet fully clarified. However, treatments that target PCSK9 have shown striking early efficacy and promise to improve the lives of countless patients with hyperlipidemia and atherosclerosis.

CircRNAs: Potential Targets for the Prevention and Treatment of Cerebrovascular Diseases

circRNAs, as a kind of non-coding RNA, are characterized by rich content, high stability, and tissue specificity, and have become a research focus in recent years. A large number of studies have found that circRNA and miRNA have the strongest binding capacity among competing endogenous RNAs, playing a role as a miRNA molecular sponge in cellular gene expression and playing an important role in many diseases. At present, studies have shown that circRNAs are closely related to cerebrovascular diseases and play a crucial role in vascular endothelial cells, metabolism of low-density lipoprotein, smooth muscle cells, inflammatory oxidative stress, ischemia-reperfusion, and other regulatory mechanisms. In order to systematically understand circRNA and its function, in order to understand the research progress of circRNA in regulating the molecular mechanism of cerebrovascular diseases, and in order to find potential markers and drug molecules in the field of cerebrovascular disease occurrence, development, diagnosis, and treatment targets, we reviewed the research progress of circRNA in the development of cerebrovascular disease.

N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression.

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

The New Face of Hyperlipidemia and the Role of PCSK9 Inhibitors.

To review the clinical rationale for use of proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors in clinical practice. Despite widespread use of statins for lipid lowering, many patients at high cardiovascular risk continue to demonstrate unsatisfactory cholesterol levels and experience clinical events. This highlights the ongoing need to develop additional strategies to achieve more effective risk reduction in a greater number of patients. Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in the regulation of low-density lipoprotein metabolism. Inhibitory approaches that reduce PCSK9 activity have been demonstrated to produce substantive reductions in LDL cholesterol levels, when administered as either monotherapy or in addition to statin therapy. More recently, PCSK9 monoclonal antibodies have been reported to reduce cardiovascular event rates in large scale clinical trials. Increasing evidence suggests that PCSK9 inhibitors can produce effective lipid lowering in high risk patients. Ongoing work will identify those patients most likely to derive cost effective risk reduction with their use.

Nanoparticles with High-Surface Negative-Charge Density Disturb the Metabolism of Low-Density Lipoprotein in Cells.

Endocytosis is an important pathway to regulate the metabolism of low-density lipoprotein (LDL) in cells. At the same time, engineering nanoparticles (ENPs) enter the cell through endocytosis in biomedical applications. Therefore, a crucial question is whether the nanoparticles involved in endocytosis could impact the natural metabolism of LDL in cells. In this study, we fabricated a series of gold nanoparticles (AuNPs) (13.00 ± 0.69 nm) with varied surface charge densities. The internalized AuNPs with high-surface negative-charge densities (HSNCD) significantly reduced LDL uptake in HepG-2, HeLa, and SMMC-7721 cells compared with those cells in control group. Notably, the significant reduction of LDL uptake in cells correlates with the reduction of LDL receptors (LDL-R) on the cell surface, but there is no change in protein and mRNA of LDL-Rs. The cyclic utilization of LDL-R in cells is a crucial pathway to maintain the homoeostasis of LDL uptake. The release of LDL-Rs from LDL/LDL-R complexes in endosomes depended on reduction of the pH in the lumen. AuNPs with HSNCD hampered vacuolar-type H+-ATPase V1 (ATPaseV1) and ATPaseV0 binding on the endosome membrane, blocking protons to enter the endosome by the pump. Hence, fewer freed LDL-Rs were transported into recycling endosomes (REs) to be returned to cell surface for reuse, reducing the LDL uptake of cells by receptor-mediated endocytosis. The restrained LDL-Rs in the LDL/LDL-R complex were degraded in lysosomes.

Analysis of mycobacterial infection-induced changes to host lipid metabolism in a zebrafish infection model reveals a conserved role for LDLR in infection susceptibility

Changes to lipid metabolism are well-characterised consequences of human tuberculosis infection but their functional relevance are not clearly elucidated in these or other host-mycobacterial systems. The zebrafish-Mycobacterium marinum infection model is used extensively to model many aspects of human-M. tuberculosis pathogenesis but has not been widely used to study the role of infection-induced lipid metabolism. We find mammalian mycobacterial infection-induced alterations in host Low Density Lipoprotein metabolism are conserved in the zebrafish model of mycobacterial pathogenesis. Depletion of LDLR, a key lipid metabolism node, decreased M. marinum burden, and corrected infection-induced altered lipid metabolism resulting in decreased LDL and reduced the rate of macrophage transformation into foam cells. Our results demonstrate a conserved role for infection-induced alterations to host lipid metabolism, and specifically the LDL-LDLR axis, across host-mycobacterial species pairings.

Pathophysiology of Diabetic Dyslipidemia

Accumulating clinical evidence has suggested serum triglyceride (TG) is a leading predictor of atherosclerotic cardiovascular disease, comparable to low-density lipoprotein (LDL)-cholesterol (C) in populations with type 2 diabetes, which exceeds the predictive power of hemoglobinA1c. Atherogenic dyslipidemia in diabetes consists of elevated serum concentrations of TG-rich lipoproteins (TRLs), a high prevalence of small dense low-density lipoprotein (LDL), and low concentrations of cholesterol-rich high-density lipoprotein (HDL)2-C. A central lipoprotein abnormality is an increase in large TG-rich very-low-density lipoprotein (VLDL)1, and other lipoprotein abnormalities are metabolically linked to increased TRLs. Insulin critically regulates serum VLDL concentrations by suppressing hepatic VLDL production and stimulating VLDL removal by activation of lipoprotein lipase. It is still debated whether hyperinsulinemia compensatory for insulin resistance is causally associated with the overproduction of VLDL. This review introduces experimental and clinical observations revealing that insulin resistance, but not hyperinsulinemia stimulates hepatic VLDL production. LDL and HDL consist of heterogeneous particles with different size and density. Cholesterol-depleted small dense LDL and cholesterol-rich HDL2 subspecies are particularly affected by insulin resistance and can be named “Metabolic LDL and HDL,” respectively. We established the direct assays for quantifying small dense LDL-C and small dense HDL(HDL3)-C, respectively. Subtracting HDL3-C from HDL-C gives HDL2-C. I will explain clinical relevance of measurements of LDL and HDL subspecies determined by our assays. Diabetic kidney disease (DKD) substantially worsens plasma lipid profile thereby potentiated atherogenic risk. Finally, I briefly overview pathophysiology of dyslipidemia associated with DKD, which has not been so much taken up by other review articles.

Associations Between Soluble LDLR and Lipoproteins in a White Cohort and the Effect of PCSK9 Loss-of-Function.

Elevated circulating cholesterol-rich low-density lipoprotein (LDL) particles increase coronary artery disease risk. Cell-surface hepatic LDL receptors (LDLRs) clear 70% of these particles from circulation. The ectodomain of LDLR is shed into circulation, preventing it from removing LDL particles. The role that LDLR ectodomain shedding plays as a regulatory mechanism is unknown. We describe LDLR shedding via the relationships between circulating soluble LDLRs (sLDLRs) and serum lipoproteins, serum proprotein convertase subtilin/kexin type 9 (PCSK9; a negative regulator of LDLR), and clinical parameters in a white Canadian population. Population-based, cross-sectional study. Clinical Research Center, The Ottawa Hospital, and Faculty of Medicine, University of Ottawa. Two hundred seventy-three white Canadians. None. sLDLR measured by ELISA; serum lipids and PCSK9, PCSK9 genotypes, and clinical parameters from previous analyses. sLDLRs correlated strongly with triglycerides (TG; r = 0.624, P < 0.0001) and moderately with LDL cholesterol (r = 0.384, P < 0.0001), and high-density lipoprotein cholesterol (r = -0.307, P = 0.0003). Only TG correlations were unaffected by PCSK9 variations. sLDLR levels were significantly elevated in those with TG >50th or LDL cholesterol >75th percentiles. Serum sLDLR levels correlate with several lipoprotein parameters, especially TG, and the presence of PCSK9 loss-of-function variants alters sLDLR levels and correlations, except for TG. Ectodomain LDLR shedding has a role in LDL metabolism, distinct from PCSK9, with interplay between these two pathways that regulate cell-surface LDLRs. Findings suggest alteration of LDLR shedding could emerge as a target to treat dyslipidemia.

Lipid Target in Very High-Risk Cardiovascular Patients: Lesson from PCSK9 Monoclonal Antibodies.

The role of low-density lipoproteins (LDLs) as a major risk factor for cardiovascular disease has been demonstrated by several epidemiological studies. The molecular basis for LDLs in atherosclerotic plaque formation and progression is not completely unraveled yet. Pharmacological modulation of plasma LDL-C concentrations and randomized clinical trials addressing the impact of lipid-lowering interventions on cardiovascular outcome have clearly shown that reducing plasma LDL-C concentrations results in a significant decrease in major cardiovascular events. For many years, statins have represented the most powerful pharmacological agents available to lower plasma LDL-C concentrations. In clinical trials, it has been shown that the greater the reduction in plasma LDL-C concentrations, the lower the rate of major cardiovascular events, especially in high-risk patients, because of multiple risk factors and recurrent events. However, in a substantial number of patients, the recommended LDL target is difficult to achieve because of different factors: genetic background (familial hypercholesterolemia), side effects (statin intolerance), or high baseline plasma LDL-C concentrations. In the last decade, our understanding of the molecular mechanisms involved in LDL metabolism has progressed significantly and the key role of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged. This protein is an enzyme able to bind the LDL receptors (LDL-R) on hepatocytes, favoring their degradation. Blocking PCSK9 represents an intriguing new therapeutic approach to decrease plasma LDL-C concentrations, which in recent studies has been demonstrated to also result in a significant reduction in major cardiovascular events.

(Pro)renin Receptor Inhibition Reprograms Hepatic Lipid Metabolism and Protects Mice From Diet-Induced Obesity and Hepatosteatosis

An elevated level of plasma LDL (low-density lipoprotein) is an established risk factor for cardiovascular disease. Recently, we reported that the (pro)renin receptor ([P]RR) regulates LDL metabolism in vitro via the LDLR (LDL receptor) and SORT1 (sortilin-1), independently of the renin-angiotensin system. To investigate the physiological role of (P)RR in lipid metabolism in vivo. We used N-acetylgalactosamine modified antisense oligonucleotides to specifically inhibit hepatic (P)RR expression in C57BL/6 mice and studied the consequences this has on lipid metabolism. In line with our earlier report, hepatic (P)RR silencing increased plasma LDL-C (LDL cholesterol). Unexpectedly, this also resulted in markedly reduced plasma triglycerides in a SORT1-independent manner in C57BL/6 mice fed a normal- or high-fat diet. In LDLR-deficient mice, hepatic (P)RR inhibition reduced both plasma cholesterol and triglycerides, in a diet-independent manner. Mechanistically, we found that (P)RR inhibition decreased protein abundance of ACC (acetyl-CoA carboxylase) and PDH (pyruvate dehydrogenase). This alteration reprograms hepatic metabolism, leading to reduced lipid synthesis and increased fatty acid oxidation. As a result, hepatic (P)RR inhibition attenuated diet-induced obesity and hepatosteatosis. Collectively, our study suggests that (P)RR plays a key role in energy homeostasis and regulation of plasma lipids by integrating hepatic glucose and lipid metabolism.

Genetic epidemiology of autosomal recessive hypercholesterolemia in Sicily: Identification by next-generation sequencing of a new kindred

Autosomal recessive hypercholesterolemia (ARH) is a rare inherited lipid disorder. In Sardinia, differently from other world regions, the mutated allele frequency is high. It is caused by mutations in the low-density lipoprotein receptor adaptor protein 1 gene. Fourteen different mutations have been reported so far; in Sardinia, 2 alleles (ARH1 and ARH2) explain most of the cases. Four ARH patients, all carriers of the ARH1 mutation, have been identified in mainland Italy and 2 in Sicily. The objectives of the study were to improve the molecular diagnosis of familial hypercholesterolemia (FH) and to estimate the frequency of the ARH1 allele in 2 free-living Sicilian populations. We sequenced by targeted next-generation sequencing 20 genes related to low-density lipoprotein metabolism in 50 hypercholesterolemic subjects. Subjects from 2 free-living populations from Northern (Ventimiglia Heart Study, 848 individuals) and Southern Sicily (Zabut Zabùt Aging Project, 1717 individuals) were genotyped for ARH1 allele. We identified 1 homozygous carrier of the ARH1 mutation among the 50 hypercholesterolemic outpatients. Population-based genotyping of ARH1 in 2565 subjects allowed the identification of 1 heterozygous carrier. The overall estimated allele frequency of ARH1 in Sicily was 0.0002 (0.02%). The identification of a new case of ARH in Sicily among 50 clinically diagnosed FH highlights the importance of next-generation sequencing analysis as tool to improve the FH diagnosis. Our results also indicate that ARH1 carrier status is present in ∼1:2500 of Sicilian inhabitants, confirming that ARH is extremely rare outside Sardinia.

Targeting pro-protein convertase subtilisin kexin-9 as a novel therapy of hypercholesterolemia

Reducing low density lipoprotein (LDL) cholesterol level is an established primary and secondary prevention strategy for coronary heart disease. However, not all patients are able to achieve their LDL targets as recommended by the guidelines. Over the last 10 years, high plasma LDL level is known to be associated with a higher level of pro-protein convertase subtilisin kexin-9 (PCSK-9). Loss-of-function mutations in the PCSK-9 gene is associated with lower plasma LDL level and cardiovascular risk. Since its discovery in 2003, PCSK-9 has triggered many researchers to design a PCSK-9 inhibitor to reduce LDL cholesterol through competitive inhibition of this molecule. Some phase III clinical trials have showed promising results of PCSK-9 inhibitor efficacy in lowering LDL level and improving clinical outcome. This article aims to discuss the role of PCSK-9 in LDL metabolism and the efficacy of PCSK-9 inhibitor in reducing plasma LDL level.

A genome-wide sirna screen as a tool to unveil new players in hepatic high density lipoprotein and low density lipoprotein metabolism

A genome-wide sirna screen as a tool to unveil new players in hepatic high density lipoprotein and low density lipoprotein metabolism

THE INFLUENCE OF LOW-DENSITY LIPOPROTEINS ON NEOCORTICAL AMYLOID LOAD IN THE AUSTRALIAN IMAGING BIOMARKER AND LIFESTYLE STUDY

In the last decade, attention has turned to the possible link between lipid metabolism and Alzheimer's disease (AD). Previous work using animals highlighted the need for further investigation into the mechanisms for AD pathogenesis. Recently, metabolic syndrome has been considered a risk factor for AD. This cluster of disorders, each with their own associated risk for AD, includes abnormal lipid and cholesterol metabolism as one of its core components. Cholesterol is considered to be important in sporadic forms of AD. A large proportion of cholesterol traffic is carried in the plasma as low density lipoprotein (LDL), which is usually rapidly cleared by the liver. It is also thought to have roles in the synthesis and clearance of amyloid-beta (Aβ). However, the actual mechanism in AD pathogenesis is poorly understood. In this cross-sectional comparison, we examined the plasma LDL lipoprotein profiles of clinically classified healthy control and Alzheimer's affected individuals from the Australian Imaging, Biomarker and Lifestyle (AIBL) study. These individuals had also undergone several other characterisations including PiB-PET imaging for determination of brain amyloid load. The data indicate that the APOE ε4 participants tended to have higher median levels of larger plasma LDL species while also being associated with higher amyloid load. These individuals also showed high variations in large LDL levels, indicating altered regulation of LDL metabolism. We also identified a possible association with LDL levels in non-ε4 participants and neocortical amyloid load, suggesting that plasma LDL is involved with AD pathogenesis. APOE ε4 influences the levels of plasma LDL species and is associated with higher amyloid load in the brain. The larger LDL species show positive association with higher brain amyloid load, supporting a metabolic link between brain and periphery.

GLP‐1 Mediates the Intrinsic Gut‐Liver Metabolic Signaling in Anti‐VLDL Overproduction and Insulin Resistance in Vagotomized Mice

Perturbations in hepatic lipid and very low density lipoprotein (VLDL) metabolism are involved in the pathogenesis of obesity and hepatic insulin resistance. In this study, our objective was to investigate the intrinsic peripheral gut‐liver vagal nerve circuit in the regulation of hepatic VLDL secretion and insulin sensitivity in sub‐diaphragmatic vagotomized mice. Specifically, by disrupting both hepatic and gastric divisions of the vagal nerve and using fast protein liquid chromatography (FPLC) to subclassify the lipoprotein particles, we discovered that vagotomy significantly decreased triglyceride (TG) contents in VLDL particles compared to that in the control mice with sham‐surgery. Vagotomy further prevented overproduction of VLDL‐TG and VLDL‐apoB induced by an acute fat load. Espousing both sham control and vagotomized mice to a long term high fat diet (HFD) for 22 weeks, we found that vagotomy protected mice from HFD induced hyperlipidemia, obesity, hepatic steatosis and insulin resistance. In an attempt to delineate the molecular mechanism for the anti‐hyperlipidemia and anti‐insulin resistant effect of vagotomy, we investigated the gut hormones and discovered that plasma glucagon‐like peptide‐1(GLP‐1) was significantly higher in the vagotomised mice compared to sham controls, which was associated with significant reduction on mRNA and protein expression of hepatic stearoyl CoA desaturase‐1 (SCD‐1), an enzyme that catalyzes the synthesis of oleyl CoA from stearolyl CoA. In vitro, pre‐treating a rat hepatoma cell line, McA‐7777 cells, with GLP‐1 inhibited SCD‐1 and Fasn mRNA expression which resulted in significant reduction on hepatic de novo lipid synthesis and VLDL secretion. Vagotomy further sensitized insulin sensitivity by upregulating expression of insulin receptors and enhanced activation of downstream signaling molecule, AKT. In conclusion, we demonstrated that increased secretion of GLP‐1 in vagotomized mice mediates the intrinsic gut‐liver metabolic signaling in preventing VLDL overproduction and insulin resistance induced by HFD. GLP‐1 inhibits SCD‐1 activity which in turn inhibits hepatic de novo lipogenesis and VLDL biogenesis. These novel findings, for the first time, provide mechanistic rationale for the application of vagotomy in clinical treatment in patients with obesity and diabetes.Support or Funding InformationHatch Funds from USDA/NIFA to Q. Su

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation: A NOVEL MECHANISM CAUSING FAMILIAL HYPERCHOLESTEROLEMIA

Familial hypercholesterolemia (FH) is characterized by severely elevated low density lipoprotein (LDL) cholesterol. Herein, we identified an FH patient presenting novel compound heterozygote mutations R410S and G592E of the LDL receptor (LDLR). The patient responded modestly to maximum rosuvastatin plus ezetimibe therapy, even in combination with a PCSK9 monoclonal antibody injection. Using cell biology and molecular dynamics simulations, we aimed to define the underlying mechanism(s) by which these LDLR mutations affect LDL metabolism and lead to hypercholesterolemia. Our data showed that the LDLR-G592E is a class 2b mutant, because it mostly failed to exit the endoplasmic reticulum and was degraded. Even though LDLR-R410S and LDLR-WT were similar in levels of cell surface and total receptor and bound equally well to LDL or extracellular PCSK9, the LDLR-R410S was resistant to exogenous PCSK9-mediated degradation in endosomes/lysosomes and showed reduced LDL internalization and degradation relative to LDLR-WT. Evidence is provided for a tighter association of LDL with LDLR-R410S at acidic pH, a reduced LDL delivery to late endosomes/lysosomes, and an increased release in the medium of the bound/internalized LDL, as compared with LDLR-WT. These data suggested that LDLR-R410S recycles loaded with its LDL-cargo. Our findings demonstrate that LDLR-R410S represents an LDLR loss-of-function through a novel class 8 FH-causing mechanism, thereby rationalizing the observed phenotype.

Mature proprotein convertase subtilisin/kexin type 9, coronary atheroma burden, and vessel remodeling in heterozygous familial hypercholesterolemia

Proprotein convertase subtilisin/kexin type 9 (PCSK9), an important contributor to low-density lipoprotein metabolism in heterozygous familial hypercholesterolemia (HeFH), exhibits direct proatherogenic effects. PCSK9 circulates as mature and furin-cleaved forms, which differ in its biological activity. However, it remains to be elucidated whether each PCSK9 subtype has different atherogenic properties. To investigate the association of each PCSK9 subtype with coronary atherosclerosis in HeFH. About 204 nonculprit segments in 138 HeFH subjects with coronary artery disease were evaluated by using intravascular ultrasound. Mature, furin-cleaved PCSK9 and total concentration of PCSK9 subtypes were measured by using enzyme-linked immunosorbent assay (BML Inc., Tokyo, Japan). The relationship of these PCSK9 values with intravascular ultrasound measures was investigated. Mature PCSK9 level was positively associated with percent atheroma volume (PAV: r=0.78, P=.003). Despite extensive atheroma under a higher mature PCSK9 level, vessel volume did not change across any mature PCSK9 levels (r=0.05, P=.78). These responses resulted in smaller lumen volume, which was negatively correlated to mature PCSK9 level (r=0.65, P=.009). By contrast, there were no significant relationships of PAV with furin-cleaved (r=0.12, P=.45) and total PCSK9 (r=0.37, P=.25) levels. On multivariate analysis, mature PCSK9 level independently contributed to PAV (odds ratio: 1.45, 95% confidence interval: 1.11-1.67, P=.01). Even in subjects with low-density lipoprotein cholesterol level <2.6mmol/L, greater PAV was still observed in association with an elevated mature PCSK9 level (P=.003). Mature PCSK9 associated with atheroma volume and impaired vessel remodeling in HeFH patients with coronary artery disease. These findings suggest the potential role of mature PCSK9 in propagation of coronary atherosclerosis in HeFH.

Plasma PCSK9 measurement by liquid chromatography-Tandem mass spectrometry and comparison with conventional ELISA.

The combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91±7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5ng/ml. This LC-MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r=0.936, p<0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC-MS/MS or ELISA methods (514.2±217.2 vs. 504.2±231.0ng/ml, respectively- p=NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.

Plasma PCSK9 concentrations during the course of nondiabetic chronic kidney disease: Relationship with glomerular filtration rate and lipid metabolism

The association between proprotein convertase subtilisin/kexin type 9 (PCSK9), a critical regulator of low-density lipoprotein (LDL) metabolism, and kidney function is a matter of debate. We aimed to assess the association of circulating PCSK9 concentrations with both glomerular filtration rate (eGFR) and serum lipid parameters in nondiabetic patients with chronic kidney disease (CKD). Fasting plasma PCSK9 concentrations were measured by ELISA in 94 nondiabetic nondialysis CKD (ND-CKD) patients not receiving statins, at different stages of CKD. Plasma PCSK9 levels were associated neither to eGFR (P=.770) nor to proteinuria (P=.888) at several stages of CKD. In addition, plasma PCSK9 levels did not vary significantly between the different CKD stages. Plasma PCSK9 concentrations were positively correlated with apolipoprotein B (r=0.221; P=.03) and triglycerides (r=0.211; P=.04) but not with total cholesterol, calculated LDL-cholesterol, HDL cholesterol, lipoprotein(a), or CRP. In a homogeneous population of nondiabetic subjects without lipid-lowering therapy, plasma PCSK9 concentrations are not associated to eGFR at several stages of CKD. These data suggest that kidney function per se does not impact significantly PCSK9 metabolism.