Knockdown of LINC01128 Downregulates FUT8 to Inhibit OxLDL-Induced Vascular Smooth Muscle Cell Apoptosis in Atherosclerosis.

BACKGROUND: The apoptosis of vascular smooth muscle cells (VSMCs) contributes to the progression of atherosclerosis (AS). Long intergenic non-protein coding RNA 1128 (LINC01128) has been implicated in AS, and this study aims to uncover the role and mechanism of LINC01128 in regulating oxidized low-density lipoprotein (oxLDL)-induced VSMCs. METHODS: The position of LINC01128 in cells and its target genes were predicted using bioinformatics. The localization of LINC01128 in human VSMCs was determined through fluorescence in situ hybridization. VSMCs were transfected, and the interaction between LINC01128 and fucosyltransferase 8 (FUT8) was validated by chromatin immunoprecipitation assay. The apoptotic VSMC model was established using oxLDL. LINC01128 expression in VSMCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and FUT8 expression was detected by qRT-PCR and western blot. VSMC viability, migration, invasion abilities, and apoptosis were assessed using cell counting kit-8, transwell assay, and flow cytometry, respectively. RESULTS: OxLDL (200 µg/mL) upregulated the expression of LINC01128 and FUT8 mRNA, as well as FUT8 protein, in VSMCs. LINC01128 was expressed in the nucleus of VSMCs and bound to FUT8. Knockdown of LINC01128 alleviated the inhibitory effects of oxLDL (200 µg/mL) on viability, migration, and invasion, and mitigated the promotion of apoptosis and FUT8 expression in VSMCs. On the other hand, FUT8 overexpression enhanced the suppressive effects of oxLDL (200 µg/mL) on viability, migration, and invasion activities, and amplified the facilitating effect of oxLDL on apoptosis in VSMCs. Moreover, FUT8 overexpression reversed the impact of LINC01128 silencing on viability, migration, invasion, and apoptosis in oxLDL-stimulated VSMCs. CONCLUSION: The knockdown of LINC01128 downregulates FUT8, inhibiting the progression of VSMCs in AS.

LDLR c.415G > A causes familial hypercholesterolemia by weakening LDLR binding to LDL.

BACKGROUND: Familial hypercholesterolemia (FH) is a prevalent hereditary disease that can cause aberrant cholesterol metabolism. In this study, we confirmed that c.415G > A in low-density lipoprotein receptor (LDLR), an FH-related gene, is a pathogenic variant in FH by in silico analysis and functional experiments. METHODS: The proband and his family were evaluated using the diagnostic criteria of the Dutch Lipid Clinic Network. Whole-exome and Sanger sequencing were used to explore and validate FH-related variants. In silico analyses were used to evaluate the pathogenicity of the candidate variant and its impact on protein stability. Molecular and biochemical methods were performed to examine the effects of the LDLR c.415G > A variant in vitro. RESULTS: Four of six participants had a diagnosis of FH. It was estimated that the LDLR c.415G > A variant in this family was likely pathogenic. Western blotting and qPCR suggested that LDLR c.415G > A does not affect protein expression. Functional studies showed that this variant may lead to dyslipidemia by impairing the binding and absorption of LDLR to low-density lipoprotein ( LDL). CONCLUSION: LDLR c.415G > A is a pathogenic variant in FH; it causes a significant reduction in LDLR's capacity to bind LDL, resulting in impaired LDL uptake. These findings expand the spectrum of variants associated with FH.

TT Genotype of TLR4 rs1928295 Is a Risk Factor of Overweight/Obesity in Han Chinese Children Aged 7-12 Years and Can Interact with Dietary Patterns to Affect the Incidence of Central Obesity and Lipid Profile, Systolic Blood Pressure Levels.

Previous studies have found that TLR4 rs1928295 polymorphism is associated with Body Mass Index in European and American Indian adults. This study evaluates the relationship between this locus polymorphism, obesity-related parameters and dietary patterns in Chinese Han Children. A total of 798 children aged 7-12 years were included in this cross-sectional study. An improved Multiple Ligase Detection Reaction was used for genotyping. Dietary patterns were identified by principal component factor analysis. The overweight/obesity rate of the TT genotype was greater than those of the CC/CT genotype (p = 0.032 and 0.048 in boys and girls, respectively). Boys of the TT genotype could interact with protein and cholesterol intake to increase low density lipoprotein (LDL) levels (p = 0.02, 0.015, respectively), while girls of the TT genotype could interact with total energy intake to increase triglyceride (TG) (p = 0.018) levels. Boys predisposed to a healthy balance dietary pattern (HBDP) and girls predisposed to an egg/fruit/fish dietary pattern (EFDP) were significantly associated with lower rates of central obesity (p = 0.045, 0.028). Boys carrying the TT genotype and predisposed to animal food dietary pattern (AFDP) had a higher level of low-density lipoprotein (p = 0.017) and systolic pressure (p = 0.044). Our results indicated that the TT genotype of TLR4 rs1928295 is a potential risk factor for obesity in Chinese Han children and is associated with dietary patterns.

Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells.

We previously described the role of low-density lipoprotein (LDL) in aggressiveness in papillary thyroid cancer (PTC). Moreover, the MAPK signaling pathway in the presence of BRAF V600E mutation is associated with more aggressive PTC. Although the link between MAPK cascade and LDL receptor (LDLR) expression has been previously described, it is unknown whether LDL can potentiate the adverse effects of PTC through it. We aimed to investigate whether the presence of LDL might accelerate the oncogenic processes through MAPK pathway in presence or absence of BRAF V600E in two thyroid cell lines: TPC1 and BCPAP (wild-type and BRAF V600E, respectively). LDLR, PI3K-AKT and RAS/RAF/MAPK (MEK)/ERK were analyzed via Western blot; cell proliferation was measured via MTT assay, cell migration was studied through wound-healing assay and LDL uptake was analyzed by fluorometric and confocal analysis. TPC1 demonstrated a time-specific downregulation of the LDLR, while BCPAP resulted in a receptor deregulation after LDL exposition. LDL uptake was increased in BCPAP over-time, as well as cell proliferation (20% higher) in comparison to TPC1. Both cell lines differed in migration pattern with a wound closure of 83.5 ± 9.7% after LDL coculture in TPC1, while a loss in the adhesion capacity was detected in BCPAP. The siRNA knockdown of LDLR in LDL-treated BCPAP cells resulted in a p-ERK expression downregulation and cell proliferation modulation, demonstrating a link between LDLR and MAPK pathway. The modulation of BRAF-V600E using vemurafenib-impaired LDLR expression decreased cellular proliferation. Our results suggest that LDLR regulation is cell line-specific, regulating the RAS/RAF/MAPK (MEK)/ERK pathway in the LDL-signaling cascade and where BRAF V600E can play a critical role. In conclusion, targeting LDLR and this downstream signaling cascade, could be a new therapeutic strategy for PTC with more aggressive behavior, especially in those harboring BRAF V600E.

The impact of sociodemographic status on the association of classical cardiovascular risk factors with coronary artery disease: a stratified Mendelian randomization study.

OBJECTIVES: Low socioeconomic status (SES) is associated with cardiovascular risk factors and increased coronary artery disease (CAD) risk. We tested whether SES is an effect modifier of the association between classical cardiovascular risk factors and CAD using SES-stratified Mendelian Randomization in European-ancestry participants from UK Biobank. STUDY DESIGN AND SETTING: We calculated weighted genetic risk scores (GRS) for the risk factors body mass index (BMI), systolic blood pressure, low-density lipoprotein cholesterol, and triglycerides. Participants were stratified by Townsend deprivation index score. Logistic regression models were used to investigate associations between GRSs and CAD occurrence. Additionally, stratification based on GRS-adjusted Townsend deprivation index residuals was conducted to correct for possible collider-stratification bias. RESULTS: In a total sample size of N = 446,485, with 52,946 cases, the risk for CAD per standard deviation increase in genetically influenced BMI was highest in the group with the lowest 25% SES (odds ratio: 1.126, 95% confidence interval: 1.106-1.145; odds ratio: 1.081, 95% confidence interval: 1.059-1.103 in high SES), remaining similar after controlling for possible collider-stratification bias. The effects of genetically influenced systolic blood pressure, low-density lipoprotein cholesterol, and triglyceride on CAD were similar between SES groups. CONCLUSION: CAD risk attributable to increased BMI is not homogenous and could be modified by SES. This emphasizes the need of tailor-made approaches for BMI-associated CAD risk reduction.

[Primary disorders of lipid metabolism: their place in current dyslipidemia guidelines and treatment innovations]. / Primäre Fettstoffwechselstörungen ­ Stellenwert in aktuellen Dyslipidämieleitlinien und therapeutische Innovationen.

According to current guidelines, the selection and intensity of lipid-effective therapies are based on the risk to be treated. The sole clinical categories of primary and secondary prevention of cardiovascular diseases result in over- and under-treatment, which may be a contributory cause of incomplete implementation of current guidelines in everyday practice. For the extent of benefit in cardiovascular outcome studies with lipid-lowering drugs, the importance of dyslipdemia for the pathogenesis of atherosclerosis-related diseases is crucial. Primary lipid metabolism disorders are characterized by life-long increased exposure to atherogenic lipoproteins. This article describes the relevance of new data for low density lipoprotein-effective therapy: inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), adenosine triphosphate (ATP) citrate lyase with bempedoic acid, and ANGPTL3 with special consideration of primary lipid metabolism disorders, which are insufficiently taken into account, or not taken into account at all, in current guidelines. This is due to their apparently low prevalence rate and thus the lack of large outcome studies. The authors also discuss the consequences of increased lipoprotein (a), which cannot be sufficiently reduced until the ongoing intervention studies examining antisense oligonucleotides and small interfering RNA (siRNA) against apolipoprotein (a) are completed. Another challenge in practice is the treatment of rare, massive hypertriglyceridemia, especially with the aim of preventing pancreatitis. For this purpose, the apolipoprotein C3 (ApoC3) antisense oligonucleotide volenasorsen is available, which binds to the mRNA for ApoC3 and lowers triglycerides by around three quarters.

A novel nonsynonymous SNP in the OLR1 gene associated with litter size in Guizhou white goats.

Oxidized low-density lipoprotein receptor-1 (OLR1) encodes a low-density lipoprotein receptor belonging to the C-type lectin superfamily, which is closely related to reproduction. OLR1 is associated with fecundity in Awassi sheep. However, its effect on litter size has not been investigated in goats. In this study, OLR1 sequences and their mRNA expression levels in the gonadal axis of Guizhou white goats were evaluated to investigate the relationship between gene polymorphisms and litter size. In addition, the potential effects of a nonsynonymous substitution were evaluated using a bioinformatics approach. The expression levels of OLR1 were highest in the uterus of mothers with multiple kids and highest in the ovaries of mothers with single kids. OLR1 mRNA expression levels in the ovaries of mothers with single kids were two times higher than in the ovaries of mothers with multiple kids. The sequencing results revealed five SNPs in OLR1; however, only g.294 T > A, g.2260 T > C, and g.2268 C > T were significantly associated with litter size (P < 0.05). Linkage disequilibrium was detected between g.2260 T > C and g.2268 C > T (r2 = 0.322, D' = 0.6). Additionally, goats with the Hap 1/1 diplotype had a greater litter size than others (P < 0.05). g.2260 T > C was a nonsynonymous mutation that resulted in the replacement of valine with alanine at the amino acid residue 54 of the OLR1 protein. Bioinformatic analyses revealed that the p.V54A locus was relatively conserved in cloven-hoofed species. Mutations at this locus could change the local conformation and reduce the stability of OLR1, affecting its half-life and the litter size of the nanny goat. These findings confirm that OLR1 affects goat kidding traits and provide a novel insight into the regulatory mechanism underlying the effect of OLR1 on litter size.

PCSK9-D374Y Suppresses Hepatocyte Migration through Downregulating Free Cholesterol Efflux Rate and Activity of Extracellular Signal-Regulated Kinase.

Proprotein convertase subtilisin/kexin type 9 can mediate the intracellular lysosomal degradation of the low-density lipoprotein receptor protein in hepatocytes and decrease the liver's ability to scavenge low-density lipoprotein cholesterol from circulation, resulting in high levels of cholesterol in the circulatory system. Current studies have primarily focused on the relationship between PCSK9 and blood lipid metabolism; however, the biological function of PCSK9 in hepatocytes is rarely addressed. In this study, we evaluate its effects in the human hepatoma carcinoma cell line HepG2, including proliferation, migration, and free cholesterol transport. PCSK9-D374Y is a gain-of-function mutation that does not affect proliferation but significantly suppresses the migration and cholesterol efflux capacity of these cells. The suppression of the transmembrane outflow of intracellular-free cholesterol regulates small G proteins and the suppression of extracellular signal-regulated kinase. In summary, PCSK9-D374Y affects hepatocyte features, including their migration and free cholesterol transport capabilities.

Abdominal Obesity, Excessive Adiposity, and the Taq1B CETP Variant Are Positively Associated with Serum Lipid Levels in Mexican Women.

INTRODUCTION: Obesity is a prevalent multifactorial disease whose main complication is dyslipidemia. Serum lipid levels also depend on genetic factors including the Taq1B variant of the CETP gene, which is suggested to be influenced by environmental factors and adiposity. Therefore, this study aimed to determine the effect of the Taq1B CETP variant on serum lipid levels associated with anthropometrical variables. METHODS: 165 women from western Mexico were enrolled in this cross-sectional study. Weight and body fat were measured by bioimpedance and waist circumference with a measuring tape. Serum lipid levels were determined by dry chemistry. The Taq1B CETP variant was analyzed by allelic discrimination. RESULTS: Women with abdominal obesity and the B1B2/B2B2 genotype had significantly higher total cholesterol levels (195.17 [185.95-204.39] vs. 183 mg/dL [169.83-196.16], p = 0.007) and low density lipoprotein (118.84 [110.65-127.03] vs. 113.84 mg/dL [102.37-125.31], p = 0.037) than carriers of the B1B1 genotype. Likewise, subjects with excessive adiposity and the B1B2/B2B2 genotype showed significantly higher total cholesterol levels (195.05 [186.04-204.06] vs. 182.40 mg/dL [169.03-195.76], p = 0.003) than those with the B1B1 genotype. CONCLUSION: Women with abdominal obesity or excessive adiposity, who are also carriers of the B1B2/B2B2 genotype, have higher serum lipid levels than women with the B1B1 genotype.

Normal range CAG repeat size variations in the HTT gene are associated with an adverse lipoprotein profile partially mediated by body mass index.

Tandem cytosine-adenine-guanine (CAG) repeat sizes of 36 or more in the huntingtin gene (HTT) cause Huntington's disease (HD). Apart from neuropsychiatric complications, the disease is also accompanied by metabolic dysregulation and weight loss, which contribute to a progressive functional decline. Recent studies also reported an association between repeats below the pathogenic threshold (<36) for HD and body mass index (BMI), suggesting that HTT repeat sizes in the non-pathogenic range are associated with metabolic dysregulation. In this study, we hypothesized that HTT repeat sizes < 36 are associated with metabolite levels, possibly mediated through reduced BMI. We pooled data from three European cohorts (n = 10 228) with genotyped HTT CAG repeat size and metabolomic measurements. All 145 metabolites were measured on the same targeted platform in all studies. Multilevel mixed-effects analysis using the CAG repeat size in HTT identified 67 repeat size metabolite associations. Overall, the metabolomic profile associated with larger CAG repeat sizes in HTT were unfavorable-similar to those of higher risk of coronary artery disease and type 2 diabetes-and included elevated levels of amino acids, fatty acids, low-density lipoprotein (LDL)-, very low-density lipoprotein- and intermediate density lipoprotein (IDL)-related metabolites while with decreased levels of very large high-density lipoprotein (HDL)-related metabolites. Furthermore, the associations of 50 metabolites, in particular, specific very large HDL-related metabolites, were mediated by lower BMI. However, no mediation effect was found for 17 metabolites related to LDL and IDL. In conclusion, our findings indicate that large non-pathogenic CAG repeat sizes in HTT are associated with an unfavorable metabolomic profile despite their association with a lower BMI.

The Extent to Which Lipid Nanoparticles Require Apolipoprotein E and Low-Density Lipoprotein Receptor for Delivery Changes with Ionizable Lipid Structure.

Lipid nanoparticles (LNPs) have delivered therapeutic RNA to hepatocytes in humans. Adsorption of apolipoprotein E (ApoE) onto these clinical LNP-mRNA drugs has been shown to facilitate hepatocyte entry via the low-density lipoprotein receptor (LDLR). Since ApoE-LDLR trafficking is conserved in mice, non-human primates, and humans, characterizing this mechanism eased clinical transition. Recently, LNPs have delivered mRNA to non-hepatocytes in mice and non-human primates, suggesting they can target new cell types via ApoE- and LDLR-independent pathways. To test this hypothesis, we quantified how 60 LNPs delivered mRNA with cell type resolution in wild-type mice and three knockout mouse strains related to lipid trafficking: ApoE-/-, LDLR-/-, and PCSK9-/-. These data suggest that the hydrophobic tail length of diketopiperazine-based lipids can be changed to drive ApoE- and LDLR-independent delivery in vivo. More broadly, the results support the hypothesis that endogenous LNP trafficking can be tuned by modifying lipid chemistry.

Association of Leptin and Leptin receptor Gene polymorphisms with Insulin resistance in pregnant women: A cross-sectional study.

Introduction: Leptin, along with its receptor, are linked with mechanisms affecting a diverse array of pregnancy-specific pathologies that include gestational diabetes and intrauterine growth restriction. The goal of the study was to examine if there was a link between the leptin (LEP)/leptin receptor (LEPR) gene polymorphism and insulin resistance in pregnant women, and to determine the extent to which the leptin gene polymorphism could cause insulin resistance.. Methods: 208 pregnant women participated in this cross-sectional study of which 74 were insulin resistant cases and 134 were insulin sensitive controls. The study was carried out from December 2018 to December 2020 at a charitable hospital in Mangalore, Karnataka, India. Genotyping of leptin and its receptor gene were carried out using the Polymerase Chain Reaction- Restriction fragment Length Polymorphism (PCR-RFLP) method. Serum levels of leptin, insulin, and C peptide were assayed using Enzyme Linked Immuno Sorbent Assay (ELISA). Statistical analysis was carried out using SPSS 23. Results: Insignificant association was observed between leptin receptor gene polymorphisms and insulin resistance, and leptin gene and insulin resistant women. There was no significant difference in the serum leptin levels among the cases and control (61.62±29.23 and 59.88±22.25). However, fasting blood sugar, insulin, C peptide, Triglycerides (TG), and very low-density Lipoprotein (VLDL) levels were significantly higher in cases as compared to controls (p=0.0068, p<0.0001, p<0.0001 and 0.01 respectively). Homeostatic Model Assessment for Insulin Resistance (HOMA IR) was greater in subjects with homozygous dominant, 'GG' of LEPR (p=0.0409) and hyperinsulinemia (p=0.023) as compared to other genotypes. However, hyperglycaemia was observed in subjects with homozygous dominant, 'AA' of leptin gene (p=0.0173). Conclusion: No significant association was found between leptin and leptin receptor gene polymorphisms with insulin resistance in pregnancy. However, genotyping of these genes may be useful in predicting insulin resistance and gestational diabetes in pregnancy.

Universal screening for familial hypercholesterolemia in 2 populations.

PURPOSE: In Europe, >2 million individuals with familial hypercholesterolemia (FH) are currently undiagnosed. Effective screening strategies for FH diagnosis in childhood are urgently needed. We assessed the overall performances of 2 different FH screening programs in children: universal screening program with opt-out and opt-in type participation. METHODS: We analyzed the data from 2 independent populations based on >166,000 individuals screened for hypercholesterolemia. Genetic analyses of FH-related genes were finalized in 945 children and 99 parents. RESULTS: A total of 305 (32.3%) children were genotyped as positive or with a variant of uncertain significance in FH-related genes. For low-density lipoprotein cholesterol levels of 3.5 mmol L (135.3 mg/dL), the overall sensitivity and specificity for confirming FH were 90.5% and 55.3%, respectively. As part of child-parent screening, in >90% of the families, the parent with reported higher cholesterol levels was positive for the familial genetic variant. The cohort-based prevalence of FH from the opt-out universal screening program was estimated to be 1 in 431 individuals (95% CI = 1/391-1/472). CONCLUSION: Universal 3-step FH screening approach in children enabled detection of most children and their parents in every generation screened at reasonable costs. Opt-out screening strategy might be preferable over opt-in screening strategy.

miR-125a-3p aggravates ox-LDL-induced HUVEC injury through BAMBI.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by the formation of atherosclerotic plaque in the intima of arteries. Among the known regulators of atherosclerosis, microRNAs (miRNAs) have been reported to play critical roles in lipoprotein homeostasis and plaque formation. But the roles of microRNA-125a-3p (miR-125a-3p) in the pathogenesis of AS remain unknown. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to construct the vascular injury model of AS pathogenesis in vitro. miR-125a-3p and BMP and activin membrane-bound inhibitor (BAMBI) expression levels in HUVECs were then measured by quantitative real-time polymerase chain reaction and western blot. The viability and apoptosis of HUVECs were analyzed by Cell Counting Kit-8 assay, TUNEL assay, and flow cytometry, respectively. The relationship between BAMBI 3'-untranslated region and miR-125a-3p was validated by dual luciferase reporter gene assay. miR-125a-3p expression was raised in HUVECs induced with ox-LDL. In HUVECs, miR-125a-3p enhanced the effects of ox-LDL treatment on repressing the viability and promoting the apoptosis of cells. Additionally, BAMBI was confirmed as a direct target of miR-125a-3p and BAMBI overexpression reversed the effects of miR-125a-3p on HUVECs. miR-125a-3p aggravates the dysfunction of HUVECs induced by ox-LDL via BAMBI, which implies that miR-125a-3p is involved in the pathogenesis of AS.

Abnormal inflammatory traits and downregulated caveolin-1 expression in monocytes of psoriasis patients may be associated with psoriatic inflammation and atherosclerosis.

BACKGROUND: Monocytes and macrophages are implicated in inflammation and atherosclerosis, whereas monocytes are involved in psoriasis lesion formation. We previously reported a psoriatic inflammation-associated significant decrease in the membrane protein caveolin-1 (CAV-1) in psoriasis patient monocytes. However, the phenotype of circulating monocytes and their macrophage differentiation in psoriasis patients remain unclear. OBJECTIVE: We sought to clarify circulating monocyte and monocyte-derived macrophage (MDM) phenotypes in psoriasis patients with and without comorbidities. METHODS: Thirty-one patients with psoriasis vulgaris and 28 control subjects were included. Surface macrophage markers and inflammatory status were examined in circulating monocytes and MDMs from both groups. Expression of CD36, which mediates macrophage uptake of oxidized low-density lipoprotein (oxLDL), was evaluated in these cells. CAV-1-silenced monocytes were differentiated into macrophages to investigate the effects of CAV-1 downregulation on psoriatic inflammation and atherosclerosis. RESULTS: Macrophage surface markers were detectable in circulating monocytes. A significant M1 shift was detected in monocytes and MDMs in psoriasis patients, including those without cardiovascular disease risk factors, as compared to controls. MDMs of psoriasis patients had more CD36-expressing cells, which are associated with atherosclerosis risk. Additionally, CAV-1-silencing in monocytes increased the likelihood of M1-biased macrophage differentiation and increased pro-inflammatory cytokine production. CONCLUSIONS: Monocytes from psoriasis patients were more likely to differentiate into M1-dominant macrophages, correlating with inflammatory status and CAV-1 expression. These aberrant inflammatory monocytes not only contribute to psoriatic inflammation by producing psoriatic cytokines, but also have a phenotype that could increase atherosclerosis risk by uptake of oxLDL and formation of foam cells.

Downregulation of low-density lipoprotein receptor mRNA in lymphatic endothelial cells impairs lymphatic function through changes in intracellular lipids.

Rationale: Impairment in lymphatic transport is associated with the onset and progression of atherosclerosis in animal models. The downregulation of low-density-lipoprotein receptor (LDLR) expression, rather than increased circulating cholesterol level per se, is involved in early atherosclerosis-related lymphatic dysfunction. Enhancing lymphatic function in Ldlr-/- mice with a mutant form of VEGF-C (VEGF-C 152s), a selective VEGFR-3 agonist, successfully delayed atherosclerotic plaque onset when mice were subsequently fed a high-fat diet. However, the specific mechanisms by which LDLR protects against lymphatic function impairment is unknown. Methods and results: We have thus injected wild-type and Pcsk9-/- mice with an adeno-associated virus type 1 expressing a shRNA for silencing Ldlr in vivo. We herein report that lymphatic contractility is reduced upon Ldlr dowregulation in wild-type mice only. Our in vitro experiments reveal that a decrease in LDLR expression at the mRNA level reduces the chromosome duplication phase and the protein expression of VEGFR-3, a membrane-bound key lymphatic marker. Furthermore, it also significantly reduced the levels of 18 lipid subclasses, including key constituents of lipid rafts as well as the transcription of several genes involved in cholesterol biosynthesis and cellular and metabolic processes. Exogenous PCSK9 only reduces lymphatic endothelial-LDLR at the protein level and does not affect lymphatic endothelial cell integrity. This puts forward that PCSK9 may act upon lymphatic muscle cells to mediate its effect on lymphatic contraction capacity in vivo. Conclusion: Our results suggest that treatments that specifically palliate the down regulation of LDLR mRNA in lymphatic endothelial cells preserve the integrity of the lymphatic endothelium and sustain lymphatic function, a prerequisite player in atherosclerosis.

High fat diet-induced obesity prolongs critical stages of the spermatogenic cycle in a Ldlr-/-.Leiden mouse model.

Obesity can disturb spermatogenesis and subsequently affect male fertility and reproduction. In our study, we aim to elucidate at which cellular level of adult spermatogenesis the detrimental effects of obesity manifest. We induced high fat diet (HFD) obesity in low-density lipoprotein receptor knock-out Leiden (Ldlr-/-.Leiden) mice, and studied the morphological structure of the testes and histologically examined the proportion of Sertoli cells, spermatocytes and spermatids in the seminiferous tubules. We examined sperm DNA damage and chromatin condensation and measured plasma levels of leptin, testosterone, cholesterol and triglycerides. HFD-induced obesity caused high plasma leptin and abnormal testosterone levels and induced an aberrant intra-tubular organisation (ITO) which is associated with an altered spermatids/spermatocytes ratio (2:1 instead of 3:1). Mice fed a HFD had a higher level of tubules in stages VII + VIII in the spermatogenic cycle. The stages VII + VII indicate crucial processes in spermatogenic development like initiation of meiosis, initiation of spermatid elongation, and release of fully matured spermatids. In conclusion, HFD-induced obese Ldlr-/-.Leiden mice develop an aberrant ITO and alterations in the spermatogenic cycle in crucial stages (stages VII and VII). Thereby, our findings stress the importance of lifestyle guidelines in infertility treatments.

LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis.

Treatments for pulmonary fibrosis (PF) are ineffective because its molecular pathogenesis and therapeutic targets are unclear. Here, we show that the expression of low-density lipoprotein receptor (LDLR) was significantly decreased in alveolar type II (ATII) and fibroblast cells, whereas it was increased in endothelial cells from systemic sclerosis-related PF (SSc-PF) patients and idiopathic PF (IPF) patients compared with healthy controls. However, the plasma levels of low-density lipoprotein (LDL) increased in SSc-PF and IPF patients. The disrupted LDL-LDLR metabolism was also observed in four mouse PF models. Upon bleomycin (BLM) treatment, Ldlr-deficient (Ldlr-/-) mice exhibited remarkably higher LDL levels, abundant apoptosis, increased fibroblast-like endothelial and ATII cells and significantly earlier and more severe fibrotic response compared to wild-type mice. In vitro experiments revealed that apoptosis and TGF-ß1 production were induced by LDL, while fibroblast-like cell accumulation and ET-1 expression were induced by LDLR knockdown. Treatment of fibroblasts with LDL or culture medium derived from LDL-pretreated endothelial or epithelial cells led to obvious fibrotic responses in vitro. Similar results were observed after LDLR knockdown operation. These results suggest that disturbed LDL-LDLR metabolism contributes in various ways to the malfunction of endothelial and epithelial cells, and fibroblasts during pulmonary fibrogenesis. In addition, pharmacological restoration of LDLR levels by using a combination of atorvastatin and alirocumab inhibited BLM-induced LDL elevation, apoptosis, fibroblast-like cell accumulation and mitigated PF in mice. Therefore, LDL-LDLR may serve as an important mediator in PF, and LDLR enhancing strategies may have beneficial effects on PF.

Circular RNA_0033596 aggravates endothelial cell injury induced by oxidized low-density lipoprotein via microRNA-217-5p /chloride intracellular channel 4 axis.

In recent years, the modulatory functions of some circular RNAs (circRNAs) in the pathogenesis of atherosclerosis (AS) have been reported. Nonetheless, the role of circular RNA_0033596 (circ_0033596) in AS and its mechanism remains unclarified. In this study, oxidized low-density lipoprotein (ox-LDL) was applied to treat human umbilical vein endothelial cells (HUVECs) to establish a cell model of endothelial cell injury. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to detect the expression of circ_0033596, microRNA-217-5p (miR-217-5p), and chloride intracellular channel 4 (CLIC4) in HUVECs. The binding sites between circ_0033596 and miR-217-5p, as well as between miR-217-5p and CLIC4 mRNA 3'UTR were determined through a dual-luciferase reporter gene assay. It was found that circ_0033596 expression was increased in ox-LDL-induced HUVECs. After ox-LDL stimulation, HUVEC viability and cell cycle progression were inhibited, and the apoptosis was promoted, while circ_0033596 overexpression aggravated these effects. MiR-217-5p was identified as a downstream target of circ_0033596, and circ_0033596 negatively regulated miR-217-5p expression. CLIC4 was identified as miR-217-5p's downstream target gene and could be positively modulated by circ_0033596. All in all circ_0033596 aggravates ox-LDL-induced HUVEC apoptosis by regulating the miR-217-5p/CLIC4 axis, by which circ_0033596 participates in the pathogenesis of AS.