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Background and aims: Familial hypercholesterolemia (FH) and other disorders with similar features are common genetic disorders that remain underdiagnosed and undertreated, due in part to the cost of screening. The aim of this study was to design and implement a whole gene targeted NGS panel for the molecular diagnosis of FH and statin intolerance with an emphasis on high quality variant calling, including copy number analysis. Methods: A whole gene panel for hybridisation-based short read NGS was designed for the dominant FH-genes low density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proproteinconvertas subtilisin/kexin type 9 (PCSK9), apolipoprotein E (APOE) and the recessive FH-genes low density lipoprotein receptor adaptor protein 1 (LDLRAP1), ATP binding cassette subfamily member 5/8 (ABCG5/8) and lipase A, lysosomal acid type (LIPA), as well as solute carrier organic anion transporter family member 1B1 (SLCO1B1), not an FH gene but linked to statin intolerance. Polygenetic risk score markers were also included. The panel was used for screening of a Swedish FH-study population (n = 133). Results: The panel sequencing resulted in high coverage and confident variant calling of included genes. Known causal variants were found in common dominant FH-genes in 43 % of the cohort. Copy number variants were found in LDLR in 10 individuals and a whole gene deletion of SLCO1B1 in one individual. In addition, coding variants in recessive genes and rare non-coding intronic and untranslated region variants were found in a large proportion of the study individuals highlighting the need for extended gene panels. Conclusions: This new tool can be used for a comprehensive high-quality molecular genetic analysis according to guidelines for the diagnosis and treatment of FH.

Neurodegenerative disorders of the aging population are characterized by progressive accumulation of neuronal proteins such as α-synuclein (α-syn) in Parkinson's Disease (PD) and Amyloid ß (Aß) and Tau in Alzheimer's disease (AD) for which no treatments are currently available. The ability to regulate the expression at the gene transcription level would be beneficial for reducing the accumulation of these proteins or regulating expression levels of other genes in the CNS. Short interfering RNA molecules can bind specifically to target RNAs and deliver them for degradation. This approach has shown promise therapeutically in vitro and in vivo in mouse models of PD and AD and other neurological disorders; however, delivery of the siRNA to the CNS in vivo has been achieved primarily through intra-cerebral or intra-thecal injections that may be less amenable for clinical translation; therefore, alternative approaches for delivery of siRNAs to the brain is needed. Recently, we described a small peptide from the envelope protein of the rabies virus (C2-9r) that was utilized to deliver an siRNA targeting α-syn across the blood brain barrier (BBB) following intravenous injection. This approach showed reduced expression of α-syn and neuroprotection in a toxic mouse model of PD. However, since receptor-mediated delivery is potentially saturable, each allowing the delivery of a limited number of molecules, we identified an alternative peptide for the transport of nucleotides across the BBB based on the apolipoprotein B (apoB) protein targeted to the family of low-density lipoprotein receptors (LDL-R). We used an 11-amino acid sequence from the apoB protein (ApoB11) that, when coupled with a 9-amino acid arginine linker, can transport siRNAs across the BBB to neuronal and glial cells. To examine the value of this peptide mediated oligonucleotide delivery system for PD, we delivered an siRNA targeting the α-syn (siα-syn) in a transgenic mouse model of PD. We found that ApoB11 was effective (comparable to C2-9r) at mediating the delivery of siα-syn into the CNS, co-localized to neurons and glial cells and reduced levels of α-syn protein translation and accumulation. Delivery of ApoB11/siα-syn was accompanied by protection from degeneration of selected neuronal populations in the neocortex, limbic system and striato-nigral system and reduced neuro-inflammation. Taken together, these results suggest that systemic delivery of oligonucleotides targeting α-syn using ApoB11 might be an interesting alternative strategy worth considering for the experimental treatment of synucleinopathies.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its effect on the LDLR, and LDL itself inhibits this uptake, though how it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, cell-surface heparin-like molecules (HLMs) can partly explain this difference, consistent with heparan sulfate proteoglycans (HSPGs) acting as coreceptors for PCSK9. We also show that HLMs can interact with either PCSK9 or LDL to modulate the inhibitory activity of LDL on PCSK9 uptake, with such inhibition rescued by competition with the entire PCSK9 prodomain, but not its truncated variants. Additionally, we show that the gain-of-function PCSK9 variant, S127R, located in the prodomain near the HSPG binding site, exhibits increased affinity for HLMs, potentially explaining its phenotype. Overall, our findings suggest a model where LDL acts as a negative regulator of PCSK9 function by decreasing its uptake via direct interactions with either the LDLR or HLMs.

Elevated plasma LDL cholesterol is the dominant risk factor for the development of atherosclerosis and cardiovascular disease. Deficiency in the LDL receptor (LDLR) is a major cause of familial hypercholesterolemia in humans, and the LDLR knockout mouse is a major animal model of atherosclerosis. Here we report the generation and characterization of an ldlr mutant zebrafish as a new animal model to study hypercholesterolemia and vascular lipid accumulation, an early event in the development of human atherosclerosis. The ldlr mutant zebrafish were characterized by activated SREBP-2 pathway and developed moderate hypercholesterolemia when fed a normal diet. However, a short-term, 5-day feeding of ldlr mutant larvae with a high-cholesterol diet (HCD) resulted in exacerbated hypercholesterolemia and accumulation of vascular lipid deposits. Lomitapide, an inhibitor of apoB lipoprotein secretion, but not the antioxidant probucol, significantly reduced accumulation of vascular lipid deposits in HCD-fed ldlr mutant larvae. Furthermore, ldlr mutants were defective in hepatic clearance of lipopolysaccharides, resulting in reduced survival. Taken together, our data suggest that the ldlr knockout zebra-fish is a versatile model for studying the function of the LDL receptor, hypercholesterolemia, and related vascular pathology in the context of early atherosclerosis.

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. [ABSTRACT FROM AUTHOR]

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.

Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLRW483X in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-κB and p38/MAPK signaling pathways.

Angiopoietin-like protein (ANGPTL)3 regulates plasma lipids by inhibiting LPL and endothelial lipase (EL). ANGPTL3 inactivation lowers LDL-C independently of the classical LDLR-mediated pathway and represents a promising therapeutic approach for individuals with homozygous familial hypercholesterolemia due to LDLR mutations. Yet, how ANGPTL3 regulates LDL-C levels is unknown. Here, we demonstrate in hyperlipidemic humans and mice that ANGPTL3 controls VLDL catabolism upstream of LDL. Using kinetic, lipidomic, and biophysical studies, we show that ANGPTL3 inhibition reduces VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation. This suggests that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production. Mechanistically, we discovered that EL is a key mediator of ANGPTL3's novel pathway. Our experiments revealed that, although dispensable in the presence of LDLR, EL-mediated processing of VLDL becomes critical for LDLR-independent particle clearance. In the absence of EL and LDLR, ANGPTL3 inhibition perturbed VLDL catabolism, promoted accumulation of atypical remnants, and failed to reduce LDL-C. Taken together, we uncover ANGPTL3 at the helm of a novel EL-dependent pathway that lowers LDL-C in the absence of LDLR.

Abstract Background Green tea drinking has been proven to lower lipid and exert cardiovascular protection, while the potential mechanism has not been fully determined. This study was to investigate whether the beneficial impact of epigallocatechingallate (EGCG), a type of catechin in green tea on lipids is associated with proprotein convertase subtilisin/kexin type 9 (PCSK9) pathways. Methods We studied the effects and underlying molecular mechanism of EGCG or green tea on regulating cholesterol from human, animal and in vitro. Results In the age- and gender-matched case control observation, we found that individuals with frequent tea consumption (n = 224) had the lower plasma PCSK9 and low density lipoprotein cholesterol (LDL-C) levels compared with ones without tea consumption (n = 224, p

Cardiovascular disease responsible for number 1 of death worldwide. Many genetic and environmental factors influence the development of CVDs. PCSK9 protein responsible for degradations of LDLR which lead to increase blood level of bad cholesterol. Gain of function mutation in PCSK9gene result in hypercholesterolemia and loss of function lead to hypocholesterolemia. Aim of this study: to investigate the polymorphism of rs775278198 of PCSK9 gene and serum level of PCSK9 with their effect on LDL-C level in CVDs patients. Materials and methods: a common SNP PCSK9 gene was studied using Real- Time PCR and technique to show association between PCSK9 SNP with risk of developing CVDs. fifty of cardiovascular diseases patients who were clinically diagnosed by physicians and fifty apparently healthy individuals were conducted within this study. Blood samples were collected from all subjects after overnight fasting. Genomic DNA was extracted from blood samples and analyzed for rs775278198 SNP in PCSK9 gene with specific primers and probes using Real-Time PCR technique. Also, serum levels of PCSK9 were evaluated using Elisa kit supplied by Bioassay Technology Laboratory. In addition LDL-C levels were measured by lipid profile test kit (CHOL2) supplied by Roch. Identification of PCSK9 SNP polymorphism and PCSK9 serum levels with LDL-C levels in this study success percentage of the methods. Results: in Real Time PCR, CVDs patients appeared the TT (20 vs 4, OR=6, p=0.028) genotypes were significantly higher in patients when compared to controls group (20 vs 4%, OR=6, P=0.028) .In comparison of the serum levels of PCSK9 in CVDs patient were higher than PCSK9 serum levels in controls group (p value =f0.000005) at the same time there is a positive relationship between PCSK9 serum levels and LDL-C levels as measured by Pearson Correlation. [ABSTRACT FROM AUTHOR]

Abstract Background Familial hypercholesterolemia (FH), an autosomal dominant genetic disorder, is underdiagnosed and undertreated. The majority of FH cases are caused by low density lipoprotein receptor (LDL-R) gene mutations. The C308Y mutation in LDL-R results in approximately 70% loss of LDL-R activity, leading to the elevation of low density lipoprotein-cholesterol (LDL-C) and an increased risk of premature coronary heart disease (CHD). The aim of this study was to identify FH cases by cascade screening in family members and relatives of a 37-year old male with premature CHD and hypercholesterolemia. Methods Clinical exam, blood lipid profiling and genomic DNA sequencing of all exons of LDL-R were performed for the proband and his 14 family members and relatives. FH diagnosis was carried out using the Dutch Lipid Clinic Network (DLCN) criteria. Results Lipid profiling showed that 9 individuals, including the proband, had hypercholesterolemia. All these 9 subjects had a G > A substitution at nucleotide 986 in exon 7 resulting in the C308Y mutation as determined by DNA sequencing, and all those carrying the mutation were diagnosed as having definite FH under the DLCN criteria. However, most (7/9) did not have suggestive clinical manifestations of CHD. Conclusions The C308Y mutation was discovered in multiple family members and relatives for the first time in mainland China. Cascade screening is key for the confirmatory diagnosis of FH. Our hypothesis that the C308Y is a common variant in the population of Southern China origin warrants further validation by screening for the C308Y mutation in a large population.

Chylomicron metabolism is critical for determining plasma levels of triacylglycerols (TAGs) and cholesterol, both of which are risk factors for CVD. The rates of chylomicron secretion and remnant clearance are controlled by intracellular and extracellular factors, including apoC-III. We have previously shown that human apoC-III overexpression in mice (apoC-IIITg mice) decreases the rate of chylomicron secretion into lymph, as well as the TAG composition in chylomicrons. We now find that this decrease in chylomicron secretion is not due to the intracellular effects of apoC-III, but instead that primary murine enteroids are capable of taking up TAG from TAG-rich lipoproteins (TRLs) on their basolateral surface; and via Seahorse analyses, we find that mitochondrial respiration is induced by basolateral TRLs. Furthermore, TAG uptake into the enterocyte is inhibited when excess apoC-III is present on TRLs. In vivo, we find that dietary TAG is diverted from the cytosolic lipid droplets and driven toward mitochondrial FA oxidation when plasma apoC-III is high (or when basolateral substrates are absent). We propose that this pathway of basolateral lipid substrate transport (BLST) plays a physiologically relevant role in the maintenance of dietary lipid absorption and chylomicron secretion. Further, when apoC-III is in excess, it inhibits BLST and chylomicron secretion.

Neurodegenerative disorders of the aging population are characterized by progressive accumulation of neuronal proteins such as α-synuclein (α-syn) in Parkinson's Disease (PD) and Amyloid ß (Aß) and Tau in Alzheimer's disease (AD) for which no treatments are currently available. The ability to regulate the expression at the gene transcription level would be beneficial for reducing the accumulation of these proteins or regulating expression levels of other genes in the CNS. Short interfering RNA molecules can bind specifically to target RNAs and deliver them for degradation. This approach has shown promise therapeutically in vitro and in vivo in mouse models of PD and AD and other neurological disorders; however, delivery of the siRNA to the CNS in vivo has been achieved primarily through intra-cerebral or intra-thecal injections that may be less amenable for clinical translation; therefore, alternative approaches for delivery of siRNAs to the brain is needed. Recently, we described a small peptide from the envelope protein of the rabies virus (C2-9r) that was utilized to deliver an siRNA targeting α-syn across the blood brain barrier (BBB) following intravenous injection. This approach showed reduced expression of α-syn and neuroprotection in a toxic mouse model of PD. However, since receptor-mediated delivery is potentially saturable, each allowing the delivery of a limited number of molecules, we identified an alternative peptide for the transport of nucleotides across the BBB based on the apolipoprotein B (apoB) protein targeted to the family of low-density lipoprotein receptors (LDL-R). We used an 11-amino acid sequence from the apoB protein (ApoB11) that, when coupled with a 9-amino acid arginine linker, can transport siRNAs across the BBB to neuronal and glial cells. To examine the value of this peptide mediated oligonucleotide delivery system for PD, we delivered an siRNA targeting the α-syn (siα-syn) in a transgenic mouse model of PD. We found that ApoB11 was effective (comparable to C2-9r) at mediating the delivery of siα-syn into the CNS, co-localized to neurons and glial cells and reduced levels of α-syn protein translation and accumulation. Delivery of ApoB11/siα-syn was accompanied by protection from degeneration of selected neuronal populations in the neocortex, limbic system and striato-nigral system and reduced neuro-inflammation. Taken together, these results suggest that systemic delivery of oligonucleotides targeting α-syn using ApoB11 might be an interesting alternative strategy worth considering for the experimental treatment of synucleinopathies.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its effect on the LDLR, and LDL itself inhibits this uptake, though how it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, cell-surface heparin-like molecules (HLMs) can partly explain this difference, consistent with heparan sulfate proteoglycans (HSPGs) acting as coreceptors for PCSK9. We also show that HLMs can interact with either PCSK9 or LDL to modulate the inhibitory activity of LDL on PCSK9 uptake, with such inhibition rescued by competition with the entire PCSK9 prodomain, but not its truncated variants. Additionally, we show that the gain-of-function PCSK9 variant, S127R, located in the prodomain near the HSPG binding site, exhibits increased affinity for HLMs, potentially explaining its phenotype. Overall, our findings suggest a model where LDL acts as a negative regulator of PCSK9 function by decreasing its uptake via direct interactions with either the LDLR or HLMs.

Context: Cassia semen (Cs), a seed of Cassia obtusifolia L. (Leguminosae), is a popular functional beverage. Previous studies reported that Cs displayed antioxidant, antifungal and strong liver protective effects. Objective: This study evaluates the hepatoprotective effects of Cs on non-alcoholic fatty liver disease (NAFLD). Materials and methods: Seventy-two male Wistar rats raised with high-fat diet (HFD) were randomly allotted into model, metformin (0.2 g/kg) and Cs (0.5, 1, and 2 g/kg)-treated groups. Another 12 rats were raised with normal feed as control group; all the rats were orally administrated with drugs and vehicle for 6 weeks. Alanine transferase (ALT), aspartate transaminase (AST), triglycerides (TG), total cholesterol (TC), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8 and low density lipoprotein receptor (LDL-R) mRNA levels were measured at the end of the experiment. Results: Twelve weeks of HFD administration significantly increased the levels of AST, ALT, TG, TC, TNF-α, IL-6, IL-8 and MDA, decreased SOD (199.42 vs. 137.70 U/mg protein) and GSH (9.76 vs. 4.55 mg/g protein) contents, compared to control group. Cs administration group significantly decreased the elevated biomarkers with the ED50 = 1.2 g/kg for NAFLD rats. Cs treatment also prevents the decreased expression of LDL-R mRNA, and improved the histopathological changes compared to model group. Conclusions: The hepatoprotective effect of Cs on NAFLD may possibly be due to its antioxidant effect. Cs may become a potent hepatoprotective agent in clinical therapy in the future.

In this study, we present the status regarding molecular genetic testing for mutations in the genes encoding the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) as causes of autosomal dominant hypercholesterolemia (ADH) in Norway. We have extracted data from the laboratory information management system at Unit for Cardiac and Cardiovascular Genetics, Oslo University Hospital for the period 1993–2020. This laboratory is the sole laboratory performing molecular genetic testing for ADH in Norway. A total of 29,449 unrelated hypercholesterolemic patients have been screened for mutations in the LDLR gene, in the APOB gene and in the PCSK9 gene. Of these, 2818 (9.6%) were heterozygotes and 11 were homozygotes or compound heterozygotes. Most of the 264 different mutations identified were found in the LDLR gene. Only two and three mutations were found in the APOB gene or in the PCSK9 gene, respectively. Several founder mutations were identified. After testing of 14,230 family members, a total of 8811 heterozygous patients have been identified. Of these, 94.0% had a mutation in the LDLR gene, 5.4% had a mutation in the APOB gene and 0.6% had a mutation in the PCSK9 gene. A large proportion of Norwegian ADH patients have been provided with a molecular genetic diagnosis. Norway is probably only second to the Netherlands in this respect. A molecular genetic diagnosis may form the basis for starting proper preventive measures and for identifying affected family members by cascade genetic screening. [Display omitted] • 29,449 patients have been screened for mutations in the LDLR gene, APOB gene and PCSK9 gene. • 2818 (9.6%) were heterozygotes and 11 were homozygotes or compound heterozygotes. • Mutations were found in the LDLR gene (94.0%), APOB gene (5.4%) and PCSK9 gene (0.6%). • 14,230 family members were tested. • A total of 8811 heterozygous patients have been diagnosed. [ABSTRACT FROM AUTHOR]

Background. Trigonella foenum graecum L. (Fenugreek, FG) is used in many countries as a medicinal plant. Evidence has suggested the hypolipidemic effect of Fenugreek; however, its actual mechanism has not been determined yet. Objectives. The purpose of our research was to investigate the effect of Fenugreek on lipid profile, liver histology and LDL receptor gene expression in male hamsters fed with high cholesterol diet. Methods. These animals were given normal diet (ND), high cholesterol diet (HCD: 2% cholesterol and 0.5% cholic acid added to ND), HCD supplemented with 2g and 8g fenugreek per 100g ND (HCD+FG2 and HCD+FG8) respectively for four weeks. At the end of treatment, serum lipids, lipoproteins and liver enzymes were measured. Finally, LDL receptor (LDLR) gene expression was determined in the liver of the studied animals using Real Time-PCR method and liver histological changes were evaluated by H&E staining method. Results. A significant reduction was observed in serum triglyceride (p<0.01), total cholesterol, low and very low density cholesterol, aspartate and alanine transaminases in HCD+FG8 group (p<0.05) compared with HCD group, while serum level of HDL-c (p<0.01) increased. In addition, LDLR gene expression in HCD+FG8 group increased 7.8 folds. The results confirm the protection effect of liver tissue in HCD+FG8 group against pathological changes. There was no significant change in LDLR gene expression in HCD+FG2 group. In conclusion, fenugreek ameliorated dyslipidemia by up-regulation of LDLR gene expression and can be used as a protective agent against hypercholesterolemia. [ABSTRACT FROM AUTHOR]

Background: Pathogenic variants in the low density lipoprotein receptor gene are associated with familial hypercholesterolemia. Some of these variants can result in incorrect folding of the LDLR protein, which is then accumulated inside the cell and cannot fulfill its function to internalize LDL particles. We analyzed the functional impact of 10 LDLR variants localized in the beta-propeller of epidermal growth factor precursor homology domain. The experimental part of the work was complemented by a structural analysis on the basis of 3D LDLR protein structure.Methods: T-Rex Chinese hamster ovary cells transfected with the human LDLR gene were used for live cell imaging microscopy, flow cytometry, and qRT-PCR analysis.Results: Our results showed that the analyzed LDLR protein variants can be divided into three groups. (1) The variants buried inside the 3D protein structure expressing proteins accumulated in the endoplasmic reticulum (ER) with no or reduced plasma membrane localization and LDL particle internalization, and associated with an increased gene expression of ER-resident chaperones. (2) The variants localized on the surface of 3D protein structure with slightly reduced LDLR plasma membrane localization and LDL particle internalization, and associated with no increased mRNA level of ER-resident chaperones. (3) The variants localized on the surface of the 3D protein structure but expressing proteins with cell responses similar to the group 1.Conclusion: All analyzed LDLR variants have been evaluated as pathogenic but with different effects on protein localization and function, and expression of genes associated with ER stress.

Elevated plasma LDL cholesterol is the dominant risk factor for the development of atherosclerosis and cardiovascular disease. Deficiency in the LDL receptor (LDLR) is a major cause of familial hypercholesterolemia in humans, and the LDLR knockout mouse is a major animal model of atherosclerosis. Here we report the generation and characterization of an ldlr mutant zebrafish as a new animal model to study hypercholesterolemia and vascular lipid accumulation, an early event in the development of human atherosclerosis. The ldlr mutant zebrafish were characterized by activated SREBP-2 pathway and developed moderate hypercholesterolemia when fed a normal diet. However, a short-term, 5-day feeding of ldlr mutant larvae with a high-cholesterol diet (HCD) resulted in exacerbated hypercholesterolemia and accumulation of vascular lipid deposits. Lomitapide, an inhibitor of apoB lipoprotein secretion, but not the antioxidant probucol, significantly reduced accumulation of vascular lipid deposits in HCD-fed ldlr mutant larvae. Furthermore, ldlr mutants were defective in hepatic clearance of lipopolysaccharides, resulting in reduced survival. Taken together, our data suggest that the ldlr knockout zebra­fish is a versatile model for studying the function of the LDL receptor, hypercholesterolemia, and related vascular pathology in the context of early atherosclerosis.—

Cholesterol-rich LDL (LDL-C) is a major atherogenic lipoprotein. Elevated content of LDL-C in serum is associated with the higher risk of atherosclerosis. The plasma LDL-C level is regulated by LDL receptor. The T allele of rs2228671 in LDLR may be associated with decreased LDL-C levels. We investigated the association of rs2228671 of LDLR with myocardial infarction (MI) in people from Fars province of Iran. In this study 248 cases with MI and 256 healthy blood donors were tested for their rs2228671 LDLR polymorphism by PCR-RFLP method. The CC genotype of the rs2228671 single nucleotide polymorphism tended to be more common in patients while TT showed a higher frequency in the control group. Patients with anteroseptal hypokinesia had a significantly higher frequency of the CC genotype compared with other patients (p=0.04, OR 8.217 and 95% CI 0.4755 to 142). Also frequency of C allele was increased as compared with that of the T allele in patients with anetroseptal hypokinesia (p=0.05, OR 7.637 and 95% CI 0.4367 to 124.6). There was also a significant increase of CT genotype in patients with abnormal heart rate (p=0.014). A significantly higher frequency of the CC genotype in patients with anetroseptal hypokinesia and its decrease in patients with abnormal heart rate suggest a complex relationship between LDLR variants and complications of MI. [ABSTRACT FROM AUTHOR]

Background: Pathogenic variants in the low density lipoprotein receptor gene are associated with familial hypercholesterolemia. Some of these variants can result in incorrect folding of the LDLR protein, which is then accumulated inside the cell and cannot fulfill its function to internalize LDL particles. We analyzed the functional impact of 10 LDLR variants localized in the beta-propeller of epidermal growth factor precursor homology domain. The experimental part of the work was complemented by a structural analysis on the basis of 3D LDLR protein structure. Methods: T-Rex Chinese hamster ovary cells transfected with the human LDLR gene were used for live cell imaging microscopy, flow cytometry, and qRT-PCR analysis. Results: Our results showed that the analyzed LDLR protein variants can be divided into three groups. (1) The variants buried inside the 3D protein structure expressing proteins accumulated in the endoplasmic reticulum (ER) with no or reduced plasma membrane localization and LDL particle internalization, and associated with an increased gene expression of ER-resident chaperones. (2) The variants localized on the surface of 3D protein structure with slightly reduced LDLR plasma membrane localization and LDL particle internalization, and associated with no increased mRNA level of ER-resident chaperones. (3) The variants localized on the surface of the 3D protein structure but expressing proteins with cell responses similar to the group 1. Conclusion: All analyzed LDLR variants have been evaluated as pathogenic but with different effects on protein localization and function, and expression of genes associated with ER stress. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are one of the more promising technologies for efficiently delivering nucleic acids in vivo. Hepatocytes are the primary target cells of LNPs that are delivered via the apolipoprotein E (ApoE)-low density lipoprotein receptor (LDLR) pathway, an endogenous targeting pathway. This robust targeting mechanism results in the specific and efficient delivery of nucleic acids to hepatocytes. Trivalent N- acetyl-D-galactosamine (GalNAc) is known to be a high-affinity exogenous ligand against the asialoglycoprotein receptor (ASGPR), which is highly expressed on hepatocytes. In this study, we report that the kinetics of the hepatic uptake process between the two types of targeting pathways are different. Rapid blood clearance, accumulation to the space of Disse and a subsequent slow cellular uptake was observed in the case of the endogenous ApoE-LDLR pathway. On the other hand, both blood clearance and cellular uptake were more gradual in the case of the exogenous GalNAc-ASGPR pathway. Interactions between ApoE-bound LNPs and hepatic heparan sulfate proteoglycans (HSPGs) were involved in the rapid blood clearance and accumulation to the space of Disse in the case of the endogenous pathway. The findings presented here contribute to a more precise understanding of the mechanism of hepatic uptake and to the rational design of hepatocyte-targeting nanoparticles. Unlabelled Image [ABSTRACT FROM AUTHOR]

Background: Green tea drinking has been proven to lower lipid and exert cardiovascular protection, while the potential mechanism has not been fully determined. This study was to investigate whether the beneficial impact of epigallocatechingallate (EGCG), a type of catechin in green tea on lipids is associated with proprotein convertase subtilisin/kexin type 9 (PCSK9) pathways.Methods: We studied the effects and underlying molecular mechanism of EGCG or green tea on regulating cholesterol from human, animal and in vitro.Results: In the age- and gender-matched case control observation, we found that individuals with frequent tea consumption (n = 224) had the lower plasma PCSK9 and low density lipoprotein cholesterol (LDL-C) levels compared with ones without tea consumption (n = 224, p < 0.05). In the high fat diet (HFD) fed rats, EGCG administration significantly lowered circulating PCSK9 concentration and liver PCSK9 expression, along with up-regulated LDL receptor (LDLR) expression but decreased level of LDL-C. In hepatic cell study, similar results were obtained regarding the impact of EGCG on LDLR and PCSK9 expression. The assay transposase-accessible chromatic with high-throughput sequencing (ATAC-seq) and subsequent results suggested that two transcription factors, hepatocyte nuclear factor-1α (HNF-1α) and forkhead box class O (FoxO) 3a involved in inhibitory action of EGCG on PCSK9 expression.Conclusions: The present study demonstrates that EGCG suppresses PCSK9 production by promoting nuclear FoxO3a, and reducing nuclear HNF1α, resulting in up-regulated LDLR expression and LDL uptake in hepatocytes. Thereby inhibiting liver and circulating PCSK9 levels, and ultimately lowering LDL-C levels. [ABSTRACT FROM AUTHOR]

Objective To investigate the relationship between polymorphism and mutation of LRP5 rs41494349 and rs2306862 locus gene and bone mineral density(BMD) in postmenopausal women with type 2 diabetes(T2 DM). Methods The data of postmenopausal Han women in Xinjiang were collected. According to the result of blood glucose and BMD,women were divided into 4 groups: normal glucose tolerance with normal bone mass group(group A), T2 DM with normal bone mass group(group B),normal glucose tolerance with abnormal bone mass group(group C), and T2 DM with abnormal bone mass group(group D). The clinical indexes and LRP5 rs41494349 and rs2306862 locus gene polymorphism were measured. Results(1) Compared to those in group A, the levels of FPG and HbA1 c% in group B and D increased. The levels of TG,BMD(L1-4), and BMD(femoral neck) in group C and D reduced. (2) Mutant(CT/TT) compared with wild-type(CC) in rs2306862 locus: P increased in group B, and BMD(L1-4) and P decreased in group D(P<0.05). Mutant(AG/GG) compared with wild-type(AA) in rs41494349 locus: BMD(L1-4) in group D decreased. (3) Multivariate linear regression analysis showed that menopause years and BMI were the influential factors of BMD(L1-4) and BMD(femoral neck). TG was the influential factor of BMD(femoral neck).Rs41494339 and rs2306862 locus in LRP5 gene polymorphism were the influential factors of BMD(femoral neck). Conclusion LRP5 rs41494339 and rs2306862 locus gene polymorphisms and mutations may be involved in the occurrence and development of OP by affecting BMD and bone metabolism markers in postmenopausal women with T2 DM. [ABSTRACT FROM AUTHOR]

Pro-protein convertase subtilisin/kexin type 9 (PCSK9) is secreted mostly by hepatocytes and to a lesser extent by the intestine, pancreas, kidney, adipose tissue, and vascular cells. PCSK9 has been known to interact with the low-density lipoprotein receptor (LDLR) and chaperones the receptor to its degradation. In this manner, targeting PCSK9 is a novel attractive approach to reduce hyperlipidaemia and the risk for cardiovascular diseases. Recently, it has been recognised that the effects of PCSK9 in relation to cardiovascular complications are not only LDLR related, but that various LDLR-independent pathways and processes are also influenced. In this review, the various LDLR dependent and especially independent effects of PCSK9 on the cardiovascular system are discussed, followed by an overview of related PCSK9-polymorphisms and currently available and future therapeutic approaches to manipulate PCSK9 expression.

BACKGROUND: Small heterodimer partner (SHP) is an important transcriptional factor involved in the regulation of glucose, lipid, and bile acid metabolism in the liver. SHP has been reported to be down-regulated in ovariectomized (Ovx) mice and up-regulated by estrogens suggesting a link between estrogens and SHP. The aim of the present study was to determine the effects of exercise training on SHP and key molecular markers of cholesterol and bile acid homeostasis in Ovx rats under cholesterol feeding. METHODS: Our main experimental group was composed of Ovx rats fed a high-cholesterol diet (Ovx-Chol) that was compared to a group of Ovx rats fed a standard diet (Ovx-SD) and a group of sham operated rats fed the cholesterol diet (Sham-Chol). These three groups of Ovx and sham rats were subdivided into either voluntary wheel running (Tr) or sedentary (Sed) groups for 5 weeks. The mRNA expression of all genes was measured by quantitative real-time polymerase chain reaction. RESULTS: Liver total cholesterol levels were not affected by exercise training in any of the experimental conditions. Cholesterol feeding in both sham and Ovx rats resulted in significantly higher hepatic cholesterol accumulation than in Ovx-SD (P < 0.001). Hepatic low-density lipoprotein-receptor (LDL-R) involved in cholesterol uptake from circulation was not influenced by training. A main effect of training was, however, found for transcripts of SHP and cholesterol 7 alpha-hydroxylase (CYP7A1, P < 0.05). CYP7A1 is the main gene involved in bile acid biosynthesis from cholesterol. CONCLUSION: These results suggest that voluntary wheel running modulates cholesterol metabolism in Ovx animals through up-regulation of SHP and bile acid formation.

Hypercholesterolemia is a well-known risk factor for a wide range of diseases in developed countries. Here, we report that mice lacking functional LDLR (low density lipoprotein receptor), an animal model of human familial hypercholesterolemia, show circadian abnormalities. In free running behavioral experiments in constant darkness, these mice showed a prolonged active phase and distinctly bimodal rhythms. Even when the circadian rhythms were entrained by light and dark cycles, these mice showed a significant attenuation of behavioral onset intensity at the start of the dark period. Further, we hypothesized that the combination of hypercholesterolemia and circadian abnormalities may affect cardiovascular disease progression. To examine this possibility, we generated LDLR-deficient mice with impaired circadian rhythms by simultaneously introducing a mutation into Period2, a core clock gene, and found that these mice showed a significant enlargement of artery plaque area with an increase in inflammatory cytokine IL-6 levels. These results suggest that circadian dysfunction may be associated with the development or progression of cardiovascular diseases.

Abstract Background The present study is aimed to evaluate difference of lipid metabolism related gene single nucleotide polymorphisms (SNPs) with ischemic stroke (IS) in Han and Uighur population of Xinjiang, China. Methods Four hundred eight patients with ischemic stroke and 347 unrelated healthy individuals of age and sex matched were genotyped for Apolipoprotein A5 (ApoA5), lipoprotein lipase (LPL), Cholesteryl ester transfer protein (CETP) and low-density lipoprotein receptor (LDL-R) genes. Their mutation difference was analyzed by SNaP shot techniques. GeneMapper4.1 SPSS20.0 software was used for data management and analysis. Using a single locus analysis, the distribution difference of genotype loci in ischemic stroke cases and controls were detected to assess the genetic risk factors of ischemic stroke. Results Significance differences of genotype distribution in ischemic stroke cases and controls were observed in LDLR rs688 in Han and Uighur population in recessive model from analysis of single gene locus. It also was found that dramatic difference of triglyceride (TG) of LPL rs328 and systolic blood pressure in CETP rs708277 of total population. In binary logistic regression analysis of total studied population, ischemic stroke was observed significantly associated with LDLR rs688 both addictive model (TT/CC, adjusted OR = 1.47, 95% CI = 1.04–2.07) and recessive model (TT/CT + CC, adjusted Odds ratio (OR) = 2.66, 95% Confidence Interval (CI) = 1.37–5.14). In Han population, ischemic stroke was observed significantly associated with rs688 both in addictive model (TT/CC, adjusted OR = 3.27, 95% CI = 1.06–10.05). In Uighur population, no significant association was found between gene polymorphisms and the risk of ischemic stroke. Combined analysis of multiple gene and loci, interaction effects of LDLR rs688 C/T, ApoA5 rs662799 A/G and CETP rs708272 C/T denoted a significant influence on IS susceptibility. Conclusion Single nucleotide polymorphisms of lipid metabolism relative gene were significantly associated with the morbidity of ischemic stroke in Han population. The interaction effects of rs688 C/T with ApoA5 rs662799 A/G and CETP rs708272 C/T promoted the occurrence of IS.

The farnesoid X receptor (FXR) plays critical roles in plasma cholesterol metabolism, in particular HDL-cholesterol (HDL-C) homeostasis. Obeticholic acid (OCA) is a FXR agonist being developed for treating various chronic liver diseases. Previous studies reported inconsistent effects of OCA on regulating plasma cholesterol levels in different animal models and in different patient populations. The mechanisms underlying its divergent effects have not yet been thoroughly investigated. The scavenger receptor class B type I (SR-BI) is a FXR-modulated gene and the major receptor for HDL-C. We investigated the effects of OCA on hepatic SR-BI expression and correlated such effects with plasma HDL-C levels and hepatic cholesterol efflux in hyperlipidemic hamsters. We demonstrated that OCA induced a time-dependent reduction in serum HDL-C levels after 14 days of treatment, which was accompanied by a significant reduction of liver cholesterol content and increases in fecal cholesterol in OCA-treated hamsters. Importantly, hepatic SR-BI mRNA and protein levels in hamsters were increased to 1.9- and 1.8-fold of control by OCA treatment. Further investigations in normolipidemic hamsters did not reveal OCA-induced changes in serum HDL-C levels or hepatic SR-BI expression. We conclude that OCA reduces plasma HDL-C levels and promotes transhepatic cholesterol efflux in hyperlipidemic hamsters via a mechanism involving upregulation of hepatic SR-BI.

Context:Cassia semen (Cs), a seed of Cassia obtusifolia L. (Leguminosae), is a popular functional beverage. Previous studies reported that Cs displayed antioxidant, antifungal and strong liver protective effects. Objective: This study evaluates the hepatoprotective effects of Cs on non-alcoholic fatty liver disease (NAFLD). Materials and methods: Seventy-two male Wistar rats raised with high-fat diet (HFD) were randomly allotted into model, metformin (0.2 g/kg) and Cs (0.5, 1, and 2 g/kg)-treated groups. Another 12 rats were raised with normal feed as control group; all the rats were orally administrated with drugs and vehicle for 6 weeks. Alanine transferase (ALT), aspartate transaminase (AST), triglycerides (TG), total cholesterol (TC), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8 and low density lipoprotein receptor (LDL-R) mRNA levels were measured at the end of the experiment. Results: Twelve weeks of HFD administration significantly increased the levels of AST, ALT, TG, TC, TNF-α, IL-6, IL-8 and MDA, decreased SOD (199.42 vs. 137.70 U/mg protein) and GSH (9.76 vs. 4.55 mg/g protein) contents, compared to control group. Cs administration group significantly decreased the elevated biomarkers with the ED50 = 1.2 g/kg for NAFLD rats. Cs treatment also prevents the decreased expression of LDL-R mRNA, and improved the histopathological changes compared to model group. Conclusions: The hepatoprotective effect of Cs on NAFLD may possibly be due to its antioxidant effect. Cs may become a potent hepatoprotective agent in clinical therapy in the future. [ABSTRACT FROM AUTHOR]

Background: Familial hypercholesterolemia (FH), an autosomal dominant genetic disorder, is underdiagnosed and undertreated. The majority of FH cases are caused by low density lipoprotein receptor (LDL-R) gene mutations. The C308Y mutation in LDL-R results in approximately 70% loss of LDL-R activity, leading to the elevation of low density lipoprotein-cholesterol (LDL-C) and an increased risk of premature coronary heart disease (CHD). The aim of this study was to identify FH cases by cascade screening in family members and relatives of a 37-year old male with premature CHD and hypercholesterolemia. Methods: Clinical exam, blood lipid profiling and genomic DNA sequencing of all exons of LDL-R were performed for the proband and his 14 family members and relatives. FH diagnosis was carried out using the Dutch Lipid Clinic Network (DLCN) criteria. Results: Lipid profiling showed that 9 individuals, including the proband, had hypercholesterolemia. All these 9 subjects had a G > A substitution at nucleotide 986 in exon 7 resulting in the C308Y mutation as determined by DNA sequencing, and all those carrying the mutation were diagnosed as having definite FH under the DLCN criteria. However, most (7/9) did not have suggestive clinical manifestations of CHD. Conclusions: The C308Y mutation was discovered in multiple family members and relatives for the first time in mainland China. Cascade screening is key for the confirmatory diagnosis of FH. Our hypothesis that the C308Y is a common variant in the population of Southern China origin warrants further validation by screening for the C308Y mutation in a large population. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin kexin 9 (PCSK9) regulates lipid metabolism by degrading low-density lipoprotein receptor on the surface of hepatocytes. PCSK9-mediated lipid degradation is associated with lipophagy. Lipophagy is a process by which autophagosomes selectively sequester lipid-droplet-stored lipids and are delivered to lysosomes for degradation. Lipophagy was first discovered in hepatocytes, and its occurrence provides important fundamental insights into how lipid metabolism regulates cellular physiology and pathophysiology. Furthermore, PCSK9 may regulate lipid levels by affecting lipophagy. This review will discuss recent advances by which PCSK9 mediates lipid degradation via the lipophagy pathway and present lipophagy as a potential therapeutic target for atherosclerosis. • Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation; • Lipophagy plays an important role in the prevention of atherosclerosis by removing excessive lipids; • PCSK9 participates in lipid metabolism through the lipophagy pathway, and thus affects atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Chemotherapy remains a primary treatment method for advanced pancreatic cancer. However, chemotherapy resistance can influence the therapeutic effect of pancreatic cancer. The resistance mechanism of chemotherapeutic agents such as gemcitabine, which is an agent typically used to treat pancreatic cancer, is complicated and can be influenced by genes and the environment. Oridonin is a tetracyclic diterpenoid compound extracted from the traditional Chinese herb Rabdosia labtea. Oridonin may overcome drug resistance in pancreatic cancer, but researching pancreatic cancer drug resistance of chemotherapy by oridonin is not completely understood. Purpose: The present study aimed to assess the impact of oridonin on multidrug resistance proteins, apoptosis-associated proteins and energy metabolism in gemcitabine-resistant PANC-1 (PANC-1/Gem) pancreatic cancer cells. Methods: Gemcitabine resistance in PANC-1/Gem cells was induced using a concentration gradient of gemcitabine. Cell Counting Kit-8 assays were used to detect the impact of gemcitabine and oridonin on the proliferation of PANC-1 and PANC-1/Gem cells. Western blot analysis and immunofluorescence were used to detect the expression of multidrug resistance proteins, apoptosis-associated proteins and low-density lipoprotein receptor protein 1 (LRP1) proteins in PANC-1/Gem cells. The effects of gemcitabine and oridonin on PANC-1/Gem cells apoptosis were detected using flow cytometry. Animal xenograft tumor assays were used to detect the effect of gemcitabine and oridonin on pancreatic cancer in vivo. Furthermore, the ATP Assay kit was used to determine the effects of gemcitabine and oridonin on ATP levels in PANC-1/Gem cells. Immunofluorescence assays were used to detect the effects of gemcitabine and oridonin on the expression of low-density lipoprotein receptor protein 1 (LRP1) in PANC-1/Gem cells. In addition, LRP1 expression was knocked down in PANC-1/Gem cells via lentiviral vector-mediated RNA silencing. Clone formation assays and Western blot analysis were used to detect the effect of LRP1 knockdown on the proliferation of PANC-1/Gem cells. Results: The present results demonstrate that oridonin overcomes PANC-1/Gem cells gemcitabine reistance by regulating GST pi and LRP1/ERK/JNK signaling. Conclusion: In conclusion, the present study indicated that oridonin could overcome gemcitabine resistance in PANC-1/Gem cells by regulating GST pi and LRP1/ ERK/JNK signaling, inducing cell apoptosis. Therefore, oridonin with gemcitabine may be a promising preoperative treatment for patients who suffer from pancreatic cancer. [ABSTRACT FROM AUTHOR]

Hypercholesterolemia represents a leading cause in the development of atherosclerotic plaques, increasing the risk for ACVS. It actually counts as a major cause of cardiovascular disease etiopathogenesis. The causes of hypercholesterolemia are multifactorial, spanning from genetic constitution, age, sex, to sedentary lifestyle and diets rich in sugars and lipids. Although dietary restriction in saturated fats, increased exercise, and other modification in lifestyle represent a first-line approach to treat very initial stages in hypercholesterolemia, most patients will require the addition of pharmacological agents. Pharmacological approaches include inhibition of cholesterol synthesis, decreased fat absorption from the GI tract, and increased degradation of FA. These strategies present a series of side effects, low therapeutic efficiency in some patients, and reduced tolerability. One of the major goals in treatment for hypercholesterolemia is to decrease the levels of low density lipoproteins (LDL), while maintaining those of high density lipoproteins (HDL). LDL particles contain about 80% of lipids, most of it cholesterol and cholesteryl esters, and 20% of the ApoB-100 protein. LDL carries cholesterol to the tissues, to be incorporated to biological membranes, or to be transformed to steroids. Excess of LDL translates into increased levels of circulating cholesterol particles and accumulation in certain tissues, especially vascular tissue, initiating a fatty streak, which may evolve to an atheroma, causing a series of cardiovascular problems, including impaired circulation, high blood pressure, increased cardiac workload, and coronary artery disease. It is essential to prevent LDL accumulation into the bloodstream to avoid the formation of these fatty streaks and the initiation of a cascade that will lead to the development of atherosclerosis. In healthy individuals. Under physiological conditions, LDL is effectively removed from circulation through receptor-mediated endocytosis. LDL clearance involves binding to its receptor, LDLR, which enables the internalization of the LDL particle and drives its degradation in lysosomes. Once the LDL particle is degraded, the free receptor recycles to the plasma membrane, and captures new LDL particles. Adequate levels of LDLR are essential to remove the excess of cholesterol-laden LDL. Proprotein convertase, subtilysin kexin type 9 (PCSK-9), expressed in liver and intestine, binds to LDLR, and internalized. Once inside the cell, PCSK-9 catalyzes the proteolysis of LDLR, preventing its recycling to the cell surface, and effectively decreasing the number of LDLR, notoriously decreasing the ability to clear LDL from circulation. Levels of PCSK-9 varies with age, gender, and levels of insulin, glucose, and triglycerides. Loss-of-function mutations in PCSK-9 gene invariably translates into lower levels of LDL, and decreased risk of developing coronary artery disease. Conversely, increased activity or expression of this enzyme leads to hypercholesterolemia. Inhibition of PCSK9 has proven to be successful in decreasing LDL levels and risk of the development of hypercholesterolemia with its associated higher risk for ASCVD. Patient with gain-of-function mutations in the PCSK9 undoubtedly benefit from therapies based on PCSK-9 inhibitors. However, millions of patients show statin intolerance, or cannot be efficiently controlled by statins alone- the most prevalent therapy for hypeprcholesterolemia. This commentary will evaluate the possibilities, caveats and future directions in the treatment of hypercholesterolemia, and therapies with combination of drugs. [ABSTRACT FROM AUTHOR]

Purpose: Quercetin, the most widely distributed flavonoid, has been shown to have multiple properties and beneficial effects on various metabolic diseases. Thus, our aim was to investigate the underlying mechanism whereby quercetin regulates renal lipid accumulation and ameliorates early diabetic renal injuries in Leprdb,/Leprdb, (db/db) mice, a model of type 2 diabetes. Methods: db/db mice were administered either 50 mg/kg or 100 mg/kg quercetin by oral gavage once a day to evaluate its effects on early stage diabetic nephropathy; mice were sacrificed at the end of the 10th week after intervention; a similar number of db/db and db/m mice were used as controls. During the experimental study, the general status of the animals was observed daily; body weight and blood glucose concentrations were measured at bi-weekly intervals. Biochemical parameters of lipid metabolism were measured by automatic biochemical analyzer. Renal function parameters were performed using commercial kits. Early renal histological changes and lipid accumulation were demonstrated by H&E staining and Oil-Red-O staining, respectively. Moreover, the expression of key proteins in the low-density lipoprotein receptors (LDLr)-SREBP-2-SREBP cSCAP signaling pathway in the kidneys of diabetic mice was detected by Western blot assay. Results: Compared with diabetic controls, quercetin not only ameliorated albuminuria and urinary albumin-to-creatinine ratio, but also decreased blood urea nitrogen and glucose, serum cholesterol, triglycerides, and low-density lipoprotein cholesterol, whereas it had no remarkable effect on the high-density lipoprotein cholesterol in diabetic db/db mice. Additionally, the evidently down regulated expression of LDLr, HMGCR, SREBP-2, and SCAP subsequently attenuated the renal lipid profile change and lipid droplet accumulation, resulting in the alleviation of renal injury of db/db mice. Conclusion: Quercetin safely and efficiently alleviates early diabetic renal injuries, possibly through improving the lipid metabolism via SCAP-SREBP2-LDLr signaling pathway. [ABSTRACT FROM AUTHOR]

Phytosterol metabolism is unknown in the hypercholesterolemia of genetic origin. We investigated the metabolism of phytosterols in a cholesterol-free, phytosterol-containing standard diet in hypercholesterolemic mice knockouts for low density lipoprotein receptor (LDLR) and apolipoprotein E (apoE) mice compared to wild-type mice (controls). Phytosterols were measured in mice tissues by GCMS. ApoE-KO mice absorbed less phytosterols than LDLR-KO and the latter absorbed less phytosterols than control mice, because the intestinal campesterol content was low in both KO mice, and sitosterol was low in the intestine in apoE-KO mice as compared to LDLR-KO mice. Although the diet contained nine times more sitosterol than campesterol, the concentration of sitosterol was lower than that of campesterol in plasma in LDLR-KO, and in the liver in controls and in LDLR-KO, but only in apoE-KO. On the other hand, in the intestine sitosterol was higher than campesterol in controls, and in LDLR-KO but with a tendency only in apoE-KO. Because of the high dietary supply of sitosterol, sitosterol was better taken up by the intestine than campesterol, but the amount of sitosterol was lower than that of campesterol in the liver, while in the whole body the amounts of these phytosterols do not differ from each other. Therefore, via intestinal lymph less sitosterol than campesterol was transferred to the body. However, as compared to controls, in apoE-KO mice, but not in LDLR-KO mice, the increase in campesterol and sitosterol in plasma and in the whole body indicating that apoE-KO mice have a marked defect in the elimination of both phytosterols from the body. [ABSTRACT FROM AUTHOR]

Abstract Statins are effective drugs used to prevent and treat cardiovascular diseases but their effects in the absence of low density lipoprotein receptor (LDLR) and on the risk of diabetes are not yet well characterized. The aim of this study was to clarify systemic and pleiotropic effects of rosuvastatin on cardiovascular and diabetic phenotypes. IGF-II/LDLR−/−ApoB100/100 hypercholesterolemic prediabetic mice were used to test the effects of rosuvastatin on plasma glucose, insulin, lipids, atherosclerosis and liver steatosis. To get a more comprehensive view about changes in gene expression RNA-sequencing was done from the liver. Rosuvastatin significantly reduced plasma cholesterol in hypercholesterolemic mice in the absence of LDLR but had no effects on atherosclerosis at aortic sinus level or in coronary arteries. Rosuvastatin also significantly reduced liver steatosis without any harmful effects on glucose or insulin metabolism. RNA-sequencing showed relatively specific effects of rosuvastatin on genes involved in cholesterol metabolism together with a significant anti-inflammatory gene expression profile in the liver. In addition, significant changes were found in the expression of Perilipin 4 and 5 which are involved in lipid droplet formation in the liver. For the first time it could be shown that Tribbles proteins are affected by rosuvastatin treatment in the hyperlipidemic mice. Rosuvastatin had several positive effects on hypercholesterolemic mice showing early signs of diabetes, many of which are unrelated to cholesterol and lipoprotein metabolism. These results increase our understanding about the systemic and pleiotropic effects of rosuvastatin in the absence of LDLR expression. Highlights • Rosuvastatin treatment reduced plasma cholesterol in mice lacking LDLR. • Rosuvastatin treatment was able to reduce steatosis in mice lacking LDLR. • Rosuvastatin treatment did not have any harmful effects on glucose metabolism. • RNA-seq showed the specificity of rosuvastatin at the transcriptional level. • Tribbles proteins in the liver are affected by rosuvastatin treatment. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • LDLR can be degraded by PCSK9 and can lead to abnormal lipid homeostasis. • PCSK9 inhibitors provided protective effects against hypercholesterolemia. • PCSK9 inhibitors are associated with a reduction in cardiovascular events. • PCSK9 inhibitors improved lipid metabolism greater than standard treatment. Abstract Low density lipoprotein cholesterol (LDL-C) is a well-established risk factor for cardiovascular disease. Although there are several developed lipid lowering drugs such as statins and fenofibrates, many patients do not achieve an adequate response. Recently, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have been developed as a new therapeutic strategy for cholesterol regulation. PCSK9 binds to low density lipoprotein receptors (LDLR) and initiates LDLR degradation, elevating LDL-C. Therefore, PCSK9 inhibition could exert beneficial effects on cardiovascular disease outcomes. This review comprehensively summarizes and discusses the effects of PCSK9 inhibitors on lipid metabolism and cardiovascular function comparatively with current lipid lowering drugs. This review also details essential information regarding the cardiovascular benefits of PCSK9 inhibition which could encourage further clinical studies. [ABSTRACT FROM AUTHOR]

Lipoprotein (a) [Lp(a)] is independently associated with CVD risk. Evolocumab, a monoclonal antibody (mAb) to proprotein convertase subtilisin/kexin type 9 (PCSK9), decreases Lp(a). The potential mechanisms were assessed. A pooled analysis of Lp(a) and LDL cholesterol (LDL-C) in 3,278 patients from 10 clinical trials (eight phase 2/3; two extensions) was conducted. Within each parent study, biweekly and monthly doses of evolocumab statistically significantly reduced Lp(a) at week 12 versus control (P < 0.001 within each study); pooled median (quartile 1, quartile 3) percent reductions were 24.7% (40.0, 3.6) and 21.7% (39.9, 4.2), respectively. Reductions were maintained through week 52 of the open-label extension, and correlated with LDL-C reductions [with and without correction for Lp(a)-cholesterol] at both time points (P < 0.0001). The effect of LDL and LDL receptor (LDLR) availability on Lp(a) cell-association was measured in HepG2 cells: cell-associated LDL fluorescence was reversed by unlabeled LDL and Lp(a). Lp(a) cell-association was reduced by coincubation with LDL and PCSK9 and reversed by adding PCSK9 mAb. These studies support that reductions in Lp(a) with PCSK9 inhibition are partly due to increased LDLR-mediated uptake. In most situations, Lp(a) appears to compete poorly with LDL for LDLR binding and internalization, but when LDLR expression is increased with evolocumab, particularly in the setting of low circulating LDL, Lp(a) is reduced.

The purpose of the study was to explore the effect and the underlying mechanism of YangXue QingNao Wan (YXQNW) and Silibinin Capsules (SC), the two Chinese medicines, on cognitive impairment in older people with familial hyperlipidaemia. Fourteen month-old female LDLR (+/-) golden Syrian hamsters were used with their wild type as control. YXQNW (0.5 g/kg/day), SC (0.1 g/kg/day), or YXQNW (0.5 g/kg/day) + SC (0.1 g/kg/day) were administrated orally for 30 days. To assess the effects of the two drugs on plasma lipid content and cognitive ability, plasma TC, TG, LDL-C, and HDL-C were measured, and Y maze task was carried out both before and after administration. After administering of the drugs for 30 days, to evaluate the effect of the two drugs on disturbed blood flow caused by hyperlipidemia, the cerebral blood flow (CBF) was measured. To assess blood–brain barrier integrity, albumin leakage in middle cerebral artery (MCA) area was determined. To evaluate the effect of the drugs on impaired microvessels, the number and morphology of microvessels were assessed in hippocampus area. To further evaluate the ultrastructure of microvessels in hippocampus, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were carried out. To assess the profiles of claudin-5 and occludin in hippocampus, we performed immunofluorescence. Finally, to assess the expression of claudin-5, JAM-1, occludin and ZO-1 in hippocampus, western blot was carried out. The results showed that YXQNW, SC, and YXQNW + SC improved cognitive impairment of aged LDLR (+/-) golden Syrian hamsters without lowering plasma TC and LDL-C. YXQNW, SC, and YXQNW + SC attenuated albumin leakage in MCA area and neuronal damage in hippocampus, concomitant with an increase in CBF, a decrease of perivascular edema and an up-regulated expression of claudin-5, occludin and ZO-1. In conclusion, YXQNW, SC, and YXQNW + SC are able to improve cognitive ability in aged LDLR (+/-) golden Syrian hamsters via mechanisms involving maintaining blood–brain barrier integrity. These findings provide evidence suggesting YXQNW or SC as a potential regime to counteract the cognitive impairment caused by familial hypercholesterolemia.

The transcription factors hepatic nuclear factor (HNF)1α and HNF1β can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1β) to examine the effects of knockdown of HNF1α or HNF1β on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1β, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (∼50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (∼1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1β did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1β infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1β, is the primary positive regulator of PCSK9 transcription in mouse liver.

Perlecan is a major heparan sulfate (HS) proteoglycan in the arterial wall. Previous studies have linked it to atherosclerosis. Perlecan contains a core protein and three HS side chains. Its core protein has five domains (DI–DV) with disparate structures and DII is highly homologous to the ligand-binding portion of LDL receptor (LDLR). The functional significance of this domain has been unknown. Here, we show that perlecan DII interacts with LDL. Importantly, the interaction largely relies on O-linked glycans that are only present in the secreted DII. Among the five repeat units of DII, most of the glycosylation sites are from the second unit, which is highly divergent and rich in serine and threonine, but has no cysteine residues. Interestingly, most of the glycans are capped by the negatively charged sialic acids, which are critical for LDL binding. We further demonstrate an additive effect of HS and DII on LDL binding. Unlike LDLR, which directs LDL uptake through endocytosis, this study uncovers a novel feature of the perlecan LDLR-like DII in receptor-mediated lipoprotein retention, which depends on its glycosylation. Thus, perlecan glycosylation may play a role in the early LDL retention during the development of atherosclerosis.

There is a drastic increase in the number of people suffering from coronary artery disease (CAD) worldwide with Indians being no exception. Being a developing country and experiencing a dramatic shift in lifestyle and eating habits, urbanization and industrialization, all these factors have collectively predisposed the Indian population towards CAD and the prevalence data are quite alarming. Genetic studies have disclosed the role of genes in CAD susceptibility and severity. One such gene is proprotein convertase subtilisin/kexin type 9 (PCSK9) which is sought to modulate the cholesterol levels and hence, has implications in CAD. We aim to explore the association of PCSK9 A/G (rs505151) polymorphism and hence, the susceptibility towards CAD in the north Indian population. Five-hundred angiographically confirmed CAD patients and 500 healthy individuals as control were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Statistical analysis revealed a significant association with the G allele with odds ratio (OR)=1.50, 95% confidence interval (CI)=1.22-1.85 and P=0.000. Also, a strong association was observed for CAD risk with OR=1.590, 95% CI=1.106-2.284 and P=0.012. However, the homozygous GG mutant genotype was found to be completely absent from our population. Analysis of the dominant model also revealed an association with CAD risk. Our work demonstrated for the first time the association of PCSK9 A/G (rs505151) polymorphism with CAD risk in the north Indian population. [ABSTRACT FROM AUTHOR]

Abstract Familial hypercholesterolemia (FH) is a genetic disorder resulting from mutations in genes encoding proteins involved in the metabolism of low density lipoproteins (LDL) and characterized by premature cardiovascular disease due to the exposure to high levels of LDL-cholesterol (LDL-C) from birth. Thus, the early identification of FH subjects, followed by appropriate treatment is essential to prevent or at least delay the onset of cardiovascular events. However, FH is largely underdiagnosed; in addition, FH patients are frequently not adequately treated, despite the availability of several pharmacological therapies to significantly reduce LDL-C levels. Current guidelines recommend LDL-C targets for FH (either heterozygotes [HeFH] or homozygotes [HoFH]) <100 mg/dL (<2.6 mmol/L) for adults or <70 mg/dL (<1.8 mmol/L) for adults with CHD or diabetes, and <135 mg/dL (<3.5 mmol/L) for children. With the pharmacological options now available, which include statins as a first approach, ezetimibe, and the recently approved monoclonal antibodies targeting PCSK9, the guideline recommended LDL-C target levels can be achieved in the majority of heterozygous FH subjects, while for the most severe forms of homozygous FH, the addition of therapies such as lomitapide either with or without apheresis may be required. Highlights • Familial hypercholesterolemia (FH) is underdiagnosed and undertreated. • Current guidelines clearly indicate LDL-C goal for FH subjects. • Combination of available lipid-lowering therapies improves the management of FH. • Novel emerging therapies for FH are currently under development. [ABSTRACT FROM AUTHOR]

Copyright of Karbala Journal of Pharmaceutical Sciences is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background Low density lipoprotein receptor ( LDLR ) is a membrane bound receptor maintaining cholesterol homeostasis along with Apolipoprotein B ( APOB ), Proprotein Convertase Subtilisin/Kexin type 9 ( PCSK9 ) and other genes of lipid metabolism. Any pathogenic variation in these genes alters the function of the receptor and leads to Familial Hypercholesterolemia (FH) and other cardiovascular diseases. Objective This study was aimed at screening the LDLR , APOB and PCSK9 genes in Hypercholesterolemic patients to define the genetic spectrum of FH in Indian population. Methods Familial Hypercholesterolemia patients (n = 78) of South Indian Tamil population with LDL cholesterol and Total cholesterol levels above 4.9 mmol/l and 7.5 mmol/l with family history of Myocardial infarction were involved. DNA was isolated by organic extraction method from blood samples and LDLR , APOB and PCSK9 gene exons were amplified using primers that cover exon-intron boundaries. The amplicons were screened using High Resolution Melt (HRM) Analysis and the screened samples were sequenced after purification. Results This study reports 20 variations in South Indian population for the first time. In this set of variations 9 are novel variations which are reported for the first time, 11 were reported in other studies also. The in silico analysis for all the variations detected in this study were done to predict the probabilistic effect in pathogenicity of FH. Conclusion This study adds 9 novel variations and 11 recurrent variations to the spectrum of LDLR gene mutations in Indian population. All these variations are reported for the first time in Indian population. This spectrum of variations was different from the variations of previous Indian reports. [ABSTRACT FROM AUTHOR]

Mutations in the low density lipoprotein receptor gene (LDLR) frequently impair folding and intracellular traffic of the receptor protein, resulting in the development of a monogenic disorder, familial hypercholesterolemia (FH). Identification of novel LDLR mutations requires confirmation of their functional importance in distinguishing pathogenic mutations from neutral changes in the aminoacid sequence. To elaborate a system for evaluation of the effect of mutation on the folding and intracellular transport of the LDLR, as well as its ability to bind low density lipoprotein (LDL), we constructed a plasmid containing LDLR cDNA and the gene of enhanced green fluorescent protein (EGFP). Confocal microscopy has shown that, upon transient transfection of HEK293 cells with the plasmid, the recombinant fusion protein LDLR-EGFP is transported onto the cellular membrane and binds labeled LDL. This construct will be further modified by site-directed mutagenesis to reproduce the LDLR missense mutations most common in the population of northwest Russia so as to study the subcellular localization and function of the modified chimeric protein. [ABSTRACT FROM AUTHOR]