A new 165-SNP low-density lipoprotein cholesterol polygenic risk score based on next generation sequencing outperforms previously published scores in routine diagnostics of familial hypercholesterolemia.

Genetic diagnosis of familial hypercholesterolemia (FH) remains unexplained in 30 to 70% of patients after exclusion of monogenic disease. There is now a growing evidence that a polygenic burden significantly modulates LDL-cholesterol (LDL-c) concentrations. Several LDL-c polygenic risk scores (PRS) have been set up. However, the balance between their diagnosis performance and their practical use in routine practice is not clearly established. Consequently, we set up new PRS based on our routine panel for sequencing and compared their diagnostic performance with previously-published PRS. After a meta-analysis, four new PRS including 165 to 1633 SNP were setup using different softwares. They were established using two French control cohorts (MONA LISA n=1082 and FranceGenRef n=856). Then the explained LDL-c variance and the ability of each PRS to discriminate monogenic negative FH patients (M-) versus healthy controls were compared with 4 previously-described PRS in 785 unrelated FH patients. Between all PRS, the 165-SNP PRS developed with PLINK showed the best LDL-c explained variance (adjusted R²=0.19) and the best diagnosis abilities (AUROC=0.77, 95%CI=0.74-0.79): it significantly outperformed all the previously-published PRS (p<1 × 10-4). By using a cut-off at the 75th percentile, 61% of M- patients exhibited a polygenic hypercholesterolemia with the 165-SNP PRS versus 48% with the previously published 12-SNP PRS (p =3.3 × 10-6). These results were replicated using the UK biobank. This new 165-SNP PRS, usable in routine diagnosis, exhibits better diagnosis abilities for a polygenic hypercholesterolemia diagnosis. It would be a valuable tool to optimize referral for whole genome sequencing.

Pathogenic gain-of-function mutations in the prodomain and C-terminal domain of PCSK9 inhibit LDL binding.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds and mediates endo-lysosomal degradation of low-density lipoprotein receptor (LDLR), limiting plasma clearance of cholesterol-rich LDL particles in liver. Gain-of-function (GOF) point mutations in PCSK9 are associated with familial hypercholesterolemia (FH). Approximately 30%-40% of PCSK9 in normolipidemic human plasma is bound to LDL particles. We previously reported that an R496W GOF mutation in a region of PCSK9 known as cysteine-histidine-rich domain module 1 (CM1) prevents LDL binding in vitro [Sarkar et al., J. Biol. Chem. 295 (8), 2285-2298 (2020)]. Herein, we identify additional GOF mutations that inhibit LDL association, localized either within CM1 or a surface-exposed region in the PCSK9 prodomain. Notably, LDL binding was nearly abolished by a prodomain S127R GOF mutation, one of the first PCSK9 mutations identified in FH patients. PCSK9 containing alanine or proline substitutions at amino acid position 127 were also defective for LDL binding. LDL inhibited cell surface LDLR binding and degradation induced by exogenous PCSK9-D374Y but had no effect on an S127R-D374Y double mutant form of PCSK9. These studies reveal that multiple FH-associated GOF mutations in two distinct regions of PCSK9 inhibit LDL binding, and that the Ser-127 residue in PCSK9 plays a critical role.

The effect of drug loading and multiple administration on the protein corona formation and brain delivery property of PEG-PLA nanoparticles.

The presence of protein corona on the surface of nanoparticles modulates their physiological interactions such as cellular association and targeting property. It has been shown that α-mangostin (αM)-loaded poly(ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles (NP-αM) specifically increased low density lipoprotein receptor (LDLR) expression in microglia and improved clearance of amyloid beta (Aß) after multiple administration. However, how do the nanoparticles cross the blood‒brain barrier and access microglia remain unknown. Here, we studied the brain delivery property of PEG-PLA nanoparticles under different conditions, finding that the nanoparticles exhibited higher brain transport efficiency and microglia uptake efficiency after αM loading and multiple administration. To reveal the mechanism, we performed proteomic analysis to characterize the composition of protein corona formed under various conditions, finding that both drug loading and multiple dosing affect the composition of protein corona and subsequently influence the cellular uptake of nanoparticles in b.End3 and BV-2 cells. Complement proteins, immunoglobulins, RAB5A and CD36 were found to be enriched in the corona and associated with the process of nanoparticles uptake. Collectively, we bring a mechanistic understanding about the modulator role of protein corona on targeted drug delivery, and provide theoretical basis for engineering brain or microglia-specific targeted delivery system.

Interplay of Low-Density Lipoprotein Receptors, LRPs, and Lipoproteins in Pulmonary Hypertension.

The low-density lipoprotein receptor (LDLR) gene family includes LDLR, very LDLR, and LDL receptor-related proteins (LRPs) such as LRP1, LRP1b (aka LRP-DIT), LRP2 (aka megalin), LRP4, and LRP5/6, and LRP8 (aka ApoER2). LDLR family members constitute a class of closely related multifunctional, transmembrane receptors, with diverse functions, from embryonic development to cancer, lipid metabolism, and cardiovascular homeostasis. While LDLR family members have been studied extensively in the systemic circulation in the context of atherosclerosis, their roles in pulmonary arterial hypertension (PAH) are understudied and largely unknown. Endothelial dysfunction, tissue infiltration of monocytes, and proliferation of pulmonary artery smooth muscle cells are hallmarks of PAH, leading to vascular remodeling, obliteration, increased pulmonary vascular resistance, heart failure, and death. LDLR family members are entangled with the aforementioned detrimental processes by controlling many pathways that are dysregulated in PAH; these include lipid metabolism and oxidation, but also platelet-derived growth factor, transforming growth factor ß1, Wnt, apolipoprotein E, bone morpohogenetic proteins, and peroxisome proliferator-activated receptor gamma. In this paper, we discuss the current knowledge on LDLR family members in PAH. We also review mechanisms and drugs discovered in biological contexts and diseases other than PAH that are likely very relevant in the hypertensive pulmonary vasculature and the future care of patients with PAH or other chronic, progressive, debilitating cardiovascular diseases.

Bile acid coordinates microbiota homeostasis and systemic immunometabolism in cardiometabolic diseases.

Cardiometabolic disease (CMD), characterized with metabolic disorder triggered cardiovascular events, is a leading cause of death and disability. Metabolic disorders trigger chronic low-grade inflammation, and actually, a new concept of metaflammation has been proposed to define the state of metabolism connected with immunological adaptations. Amongst the continuously increased list of systemic metabolites in regulation of immune system, bile acids (BAs) represent a distinct class of metabolites implicated in the whole process of CMD development because of its multifaceted roles in shaping systemic immunometabolism. BAs can directly modulate the immune system by either boosting or inhibiting inflammatory responses via diverse mechanisms. Moreover, BAs are key determinants in maintaining the dynamic communication between the host and microbiota. Importantly, BAs via targeting Farnesoid X receptor (FXR) and diverse other nuclear receptors play key roles in regulating metabolic homeostasis of lipids, glucose, and amino acids. Moreover, BAs axis per se is susceptible to inflammatory and metabolic intervention, and thereby BAs axis may constitute a reciprocal regulatory loop in metaflammation. We thus propose that BAs axis represents a core coordinator in integrating systemic immunometabolism implicated in the process of CMD. We provide an updated summary and an intensive discussion about how BAs shape both the innate and adaptive immune system, and how BAs axis function as a core coordinator in integrating metabolic disorder to chronic inflammation in conditions of CMD.

Improvement of Definite Diagnosis of Familial Hypercholesterolemia Using an Expanding Genetic Analysis.

Background: The deeper understanding of the complex hereditary basis of familial hypercholesterolemia (FH) has raised the rationale of genetic testing, which has been underutilized in clinical practice. Objectives: The present study aimed to explore the variant spectrum of FH in an expanding manner and compare its diagnostic performance. Methods: A total of 169 Chinese individuals (124 index cases and 45 relatives) with clinical definite/probable FH were consecutively enrolled. Next-generation sequencing was performed for genetic analysis of 9 genes associated with hypercholesterolemia (major genes: LDLR, APOB, and PCSK9; minor genes: LDLRAP1, LIPA, STAP1, APOE, ABCG5, and ABCG8) including the evaluations of small-scale variants and large-scale copy number variants (CNVs). Results: Among the 169 clinical FH patients included, 98 (58.0%) were men. A total of 85 (68.5%) index cases carried FH-associated variants. The proportion of FH caused by small-scale variants in LDLR, APOB, and PCSK9 genes was 62.1% and then increased by 6.5% when other genes and CNVs were further included. Furthermore, the variants in LDLR, APOB, and PCSK9 genes occupied 75% of all FH-associated variants. Of note, there were 8 non-LDLR CNVs detected in the present study. Conclusions: LDLR, APOB, and PCSK9 genes should be tested in the initial genetic screening, although variants in minor genes also could explain phenotypic FH, suggesting that an expanding genetic testing may be considered to further explain phenotypic FH.

Coronary Artery Disease Genetics Enlightened by Genome-Wide Association Studies.

Many cardiovascular diseases are facilitated by strong inheritance. For example, large-scale genetic studies identified hundreds of genomic loci that affect the risk of coronary artery disease. At each of these loci, common variants are associated with disease risk with robust statistical evidence but individually small effect sizes. Only a minority of candidate genes found at these loci are involved in the pathophysiology of traditional risk factors, but experimental research is making progress in identifying novel, and, in part, unexpected mechanisms. Targets identified by genome-wide association studies have already led to the development of novel treatments, specifically in lipid metabolism. This review summarizes recent genetic and experimental findings in this field. In addition, the development and possible clinical usefulness of polygenic risk scores in risk prediction and individualization of treatment, particularly in lipid metabolism, are discussed.

MLb-LDLr: A Machine Learning Model for Predicting the Pathogenicity of LDLr Missense Variants.

Untreated familial hypercholesterolemia (FH) leads to atherosclerosis and early cardiovascular disease. Mutations in the low-density lipoprotein receptor (LDLr) gene constitute the major cause of FH, and the high number of mutations already described in the LDLr makes necessary cascade screening or in vitro functional characterization to provide a definitive diagnosis. Implementation of high-predicting capacity software constitutes a valuable approach for assessing pathogenicity of LDLr variants to help in the early diagnosis and management of FH disease. This work provides a reliable machine learning model to accurately predict the pathogenicity of LDLr missense variants with specificity of 92.5% and sensitivity of 91.6%.

High-Fat Diet Enhances Neutrophil Adhesion in LDLR-Null Mice Via Hypercitrullination of Histone H3.

Neutrophil adhesion on the atheroprone femoral artery of high-fat diet-fed low-density lipoprotein receptor-null mice was enhanced more than in wild-type mice. The inhibition of histone H3 citrullination of neutrophils reversed the enhancement of neutrophil adhesion, suggesting that hypercitrullination contributes to enhanced neutrophil adhesion. Furthermore, pemafibrate reduced the citrullination of histone H3 in these mice. Therefore, the hypercitrullination of histone H3 in neutrophils contributes to atherosclerotic vascular inflammation.

B Cells in Atherosclerosis: Mechanisms and Potential Clinical Applications.

Because atherosclerotic cardiovascular disease is a leading cause of death worldwide, understanding inflammatory processes underpinning its pathology is critical. B cells have been implicated as a key immune cell type in regulating atherosclerosis. B-cell effects, mediated by antibodies and cytokines, are subset specific. In this review, we focus on elaborating mechanisms underlying subtype-specific roles of B cells in atherosclerosis and discuss available human data implicating B cells in atherosclerosis. We further discuss potential B cell-linked therapeutic approaches, including immunization and B cell-targeted biologics. Given recent evidence strongly supporting a role for B cells in human atherosclerosis and the expansion of immunomodulatory agents that affect B-cell biology in clinical use and clinical trials for other disorders, it is important that the cardiovascular field be cognizant of potential beneficial or untoward effects of modulating B-cell activity on atherosclerosis.

S-Allylmercaptocysteine improves alcoholic liver disease partly through a direct modulation of insulin receptor signaling.

Alcoholic liver disease (ALD) causes insulin resistance, lipid metabolism dysfunction, and inflammation. We investigated the protective effects and direct regulating target of S-allylmercaptocysteine (SAMC) from aged garlic on liver cell injury. A chronic ethanol-fed ALD in vivo model (the NIAAA model) was used to test the protective functions of SAMC. It was observed that SAMC (300 mg/kg, by gavage method) effectively ameliorated ALD-induced body weight reduction, steatosis, insulin resistance, and inflammation without affecting the health status of the control mice, as demonstrated by histological, biochemical, and molecular biology assays. By using biophysical assays and molecular docking, we demonstrated that SAMC directly targeted insulin receptor (INSR) protein on the cell membrane and then restored downstream IRS-1/AKT/GSK3ß signaling. Liver-specific knock-down in mice and siRNA-mediated knock-down in AML-12 cells of Insr significantly impaired SAMC (250 µmol/L in cells)-mediated protection. Restoration of the IRS-1/AKT signaling partly recovered hepatic injury and further contributed to SAMC's beneficial effects. Continuous administration of AKT agonist and recombinant IGF-1 in combination with SAMC showed hepato-protection in the mice model. Long-term (90-day) administration of SAMC had no obvious adverse effect on healthy mice. We conclude that SAMC is an effective and safe hepato-protective complimentary agent against ALD partly through the direct binding of INSR and partial regulation of the IRS-1/AKT/GSK3ß pathway.

Novel vectors and approaches for gene therapy in liver diseases.

Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body's total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

Therapeutic Antibody Against Phosphorylcholine Preserves Coronary Function and Attenuates Vascular 18F-FDG Uptake in Atherosclerotic Mice.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of 18F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

Lipoprotein(a) Cellular Uptake Ex Vivo and Hepatic Capture In Vivo Is Insensitive to PCSK9 Inhibition With Alirocumab.

Lipoprotein(a) (Lp[a]) is the most common genetically inherited risk factor for cardiovascular disease. Many aspects of Lp(a) metabolism remain unknown. We assessed the uptake of fluorescent Lp(a) in primary human lymphocytes as well as Lp(a) hepatic capture in a mouse model in which endogenous hepatocytes have been ablated and replaced with human ones. Modulation of LDLR expression with the PCSK9 inhibitor alirocumab did not alter the cellular or the hepatic uptake of Lp(a), demonstrating that the LDL receptor is not a major route for Lp(a) plasma clearance. These results have clinical implications because they underpin why statins are not efficient at reducing Lp(a).

Design, synthesis, and biological evaluation of novel tetrahydroprotoberberine derivatives (THPBs) as proprotein convertase subtilisin/kexin type 9 (PCSK9) modulators for the treatment of hyperlipidemia.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulators may attenuate PCSK9-induced low-density lipoprotein receptor (LDLR) degradation in lysosome and promote the clearance of circulating low-density lipoprotein cholesterol (LDL-C). A novel series of tetrahydroprotoberberine derivatives (THPBs) were designed, synthesized, and evaluated as PCSK9 modulators for the treatment of hyperlipidemia. Among them, eight compounds exhibited excellent activities in downregulating hepatic PCSK9 expression better than berberine in HepG2 cells. In addition, five compounds 15, 18, 22, (R)-22, and (S)-22 showed better performance in the low-density lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-LDL) uptake assay, compared with berberine at the same concentration. Compound 22, selected for in vivo evaluation, demonstrated significant reductions of total cholesterol (TC) and LDL-C in hyperlipidemic hamsters with a good pharmacokinetic profile. Further exploring of the lipid-lowering mechanism showed that compound 22 promoted hepatic LDLR expression in a dose-dependent manner in HepG2 cells. Additional results of human ether-à-go-go related gene (hERG) inhibition assay indicated the potential druggability for compound 22, which is a promising lead compound for the development of PCSK9 modulator for the treatment of hyperlipidemia.

Aggravated Atherosclerosis and Vascular Inflammation With Reduced Kidney Function Depend on Interleukin-17 Receptor A and Are Normalized by Inhibition of Interleukin-17A.

Effective therapy of atherosclerotic complications in patients with chronic kidney disease (CKD) is an unmet clinical need. Cardiovascular events are the most common cause of death. At a glomerular filtration rate ≤60 ml/min, these events are increased also after correction for common risk factors. Previous studies have reported enhanced vascular inflammation in mice and recently also in humans. Our current data show, in a mouse model of atherosclerosis in moderate renal impairment, that interleukin-17 receptor A is instrumental in this condition, and blockade of this pathway can normalize arterial inflammation even in advanced atherosclerosis.

LDLR rs688 TT Genotype and T Allele Are Associated with Increased Susceptibility to Coronary Artery Disease-A Case-Control Study.

PURPOSE: The low-density lipoprotein receptor is responsible for the binding and uptake of plasma LDL particles and plays a critical role in maintaining cellular cholesterol homeostasis. LDLR gene SNP rs688 has been reported to be associated with increased plasma total and LDL cholesterol in several populations and can lead to elevated plasma LDL levels, resulting in an increased risk for atherosclerosis and coronary artery disease. This study aimed to explore genetic LDLR variant rs688 for its potential roles in coronary artery disease. METHODOLOGY: This study recruited 200 coronary artery disease patients and 200 healthy individuals. Genotyping of LDLR-rs688C > T gene variations was performed using the allele specific PCR method. Correlation of LDLR-rs688C > T gene variants with different clinicopathological features of coronary artery disease patients was performed. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were applied to evaluate the correlation of this microRNA polymorphism with coronary artery disease risk. RESULTS: A significant difference was observed in genotype distribution among the coronary artery disease and matched healthy controls (p = 0.003). The frequencies of all three genotypes CC, CT, TT reported in the patient samples were 14%, 65% and 21% and in the healthy controls samples were 18%, 73% and 9%, respectively. The increased risk of developing CAD in Indian patients was found to be associated with LDLR rs688 TT genotype (OR = 3.0, 95% CI, 1.43 × 6.2; p = 0.003) RR 1.87 (1.20⁻2.91) p = 0.0037) and also the increased risk of developing CAD was reported to be associated with LDLR rs688 T allele (OR = 0.74, 95% CI, 1.57⁻0.97; p = 0.03) RR 0.85 (0.73⁻0.99) p = 0.03) compared to the C allele. Therefore, it was observed that more than a 3.0- and 0.74-fold increase risk of developing CAD was associated with TT genotype and T allele in Indian coronary artery disease patients. CONCLUSION: The findings indicated that LDLR rs688 TT genotype and T allele are associated with an increased susceptibility to coronary artery disease patients. LDLR-rs688C > T gene variation can be used as a predisposing genetic marker for coronary artery disease. Further studies with larger sample sizes are necessary to confirm our findings.

Uncovering a Predictive Molecular Signature for the Onset of NASH-Related Fibrosis in a Translational NASH Mouse Model.

BACKGROUND & AIMS: The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis. METHODS: A time-course study in low-density lipoprotein-receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets. RESULTS: High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis. CONCLUSIONS: An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames.

Biomacromolecules as carriers in drug delivery and tissue engineering.

Natural biomacromolecules have attracted increased attention as carriers in biomedicine in recent years because of their inherent biochemical and biophysical properties including renewability, nontoxicity, biocompatibility, biodegradability, long blood circulation time and targeting ability. Recent advances in our understanding of the biological functions of natural-origin biomacromolecules and the progress in the study of biological drug carriers indicate that such carriers may have advantages over synthetic material-based carriers in terms of half-life, stability, safety and ease of manufacture. In this review, we give a brief introduction to the biochemical properties of the widely used biomacromolecule-based carriers such as albumin, lipoproteins and polysaccharides. Then examples from the clinic and in recent laboratory development are summarized. Finally the current challenges and future prospects of present biological carriers are discussed.

Apolipoprotein E Deficiency Increases Remnant Lipoproteins and Accelerates Progressive Atherosclerosis, But Not Xanthoma Formation, in Gene-Modified Minipigs.

Deficiency of apolipoprotein E (APOE) causes familial dysbetalipoproteinemia in humans resulting in a higher risk of atherosclerotic disease. In mice, APOE deficiency results in a severe atherosclerosis phenotype, but it is unknown to what extent this is unique to mice. In this study, APOE was targeted in Yucatan minipigs. APOE-/- minipigs displayed increased plasma cholesterol and accumulation of apolipoprotein B-48-containing chylomicron remnants on low-fat diet, which was significantly accentuated upon feeding a high-fat, high-cholesterol diet. APOE-/- minipigs displayed accelerated progressive atherosclerosis but not xanthoma formation. This indicates that remnant lipoproteinemia does not induce early lesions but is atherogenic in pre-existing atherosclerosis.