We explore the interaction between estrogen and PCSK9 and their collective impact on lipid metabolism, especially concerning the regulation of low-density lipoprotein receptor levels. Utilizing both animal and cellular models, including ovariectomized mice and HepG2 cell lines, we demonstrate that estrogen deficiency leads to a disruption in lipid metabolism, characterized by elevated levels of total cholesterol and LDL-C. The study commences with mice undergoing ovariectomy, followed by a diet regimen comprising either high-fat diet or normal feed for a four-week duration. Key assessments include analyzing lipid metabolism, measuring PCSK9 levels in the bloodstream, and evaluating hepatic low-density lipoprotein receptor expression. We will also conduct correlation analyses to understand the relationship between PCSK9 and various lipid profiles. Further, a subset of ovariectomized mice on high-fat diet will undergo treatment with either estrogen or PCSK9 inhibitor for two weeks, with a subsequent re-evaluation of the earlier mentioned parameters. Our findings reveal that estrogen inhibits PCSK9-mediated degradation of low-density lipoprotein receptor, a process crucial for maintaining lipid homeostasis. Through a series of experiments, including immunohistochemistry and western blot analysis, we establish that PCSK9 is involved in lipid metabolism disorders caused by estrogen deficiency and that estrogen regulates PCSK9 and low-density lipoprotein receptor at post-transcriptional level. The study provides a mechanism for the involvement of PCSK9 in elucidating the disorders of lipid metabolism caused by estrogen deficiency due to perimenopause and ovarian decline.

BACKGROUND: Familial hypercholesterolemia (FH) is a common inherited metabolic disease that causes premature atherosclerosis, cardiovascular disease, and even death at a young age. Approximately 95% of FH-causing genetic variants that have been identified are in the LDLR gene. However, only 10% of the FH population worldwide has been diagnosed and adequately treated, due to the existence of numerous unidentified variants, uncertainties in the pathogenicity scoring of many variants, and a substantial number of individuals lacking access to genetic testing.
OBJECTIVE: The aim of this study was to identify a novel variant in the LDLR gene that causes FH in a Chinese family, thereby expanding the spectrum of FH-causing variants.
METHODS: Patients were recruited from Beijing Anzhen Hospital, Capital Medical University. FH diagnosis was made according to the Dutch Lipid Clinical Network (DLCN) criteria. Whole-exome sequencing (WES) was conducted to identify the FH-causing variant in the proband, and amplicon sequencing was used to verify the variant in his family members.
RESULTS: A three-generation Chinese family was recruited, and two FH patients were clinically diagnosed, both without known FH-causing variants. These two FH patients and another possible patient carried a novel variant, NC_000019.9(NM_000527.5):c.89_92dup (NP_000518.1:p.Phe32Argfs*21), in the ligand-binding domain of the low-density lipoprotein (LDL) receptor that led to a frameshift. The FH adults in the family showed severe clinical symptoms and statin therapy resistance.
CONCLUSIONS: This study identified a novel pathogenic LDLR variant, c.89_92dup, associated with severe FH clinical manifestations and statin therapy resistance.

Familial hypercholesterolemia (FH) is a genetic disorder affecting the metabolism of lipoprotein, characterized by elevated levels of plasma concentrations of low-density lipoprotein cholesterol (LDLC). The most common FH cause is mutations within the gene that encodes for the LDL receptor (LDLR) protein. Two induced pluripotent stem cell (iPSC) lines were generated from patients with FH, each carrying a single heterozygous mutation in the LDLR gene, one is a missense mutation, c.631C > T, and the other is a splice-site mutation, c.313 + 1G > A. Both iPSC lines exhibited strong expression of pluripotency markers, demonstrated the ability to differentiate into derivatives of the three germ layers, and maintained normal karyotypes. These derived iPSC lines represent powerful tools for in vitro modeling FH and offer a promising platform for therapeutic development.

This study investigated the effects of far-infrared (FIR) irradiation on low-density lipoprotein cholesterol (LDL-C) uptake by human hepatocellular carcinoma G2 (HepG2) cells via the regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). FIR irradiation for 30 min significantly decreased PCSK9 expression (p < 0.01) in HepG2 cells. FIR irradiation substantially increased the low-density lipoprotein receptor (p < 0.0001) and LDL-C uptake (p < 0.01). Activation of transient receptor potential vanilloid (TRPV) channels mimicked the effects of FIR irradiation, significantly decreasing the protein expression of PCSK9 (p < 0.05). Conversely, inhibition of TRP channels using ruthenium red reversed the reduction in PCSK9 protein expression following FIR irradiation (p < 0.01). The specific activation of TRPV4 using 4α-PDD mimicked the effect of FIR irradiation (p < 0.01), whereas PCSK9 reduction by FIR irradiation was significantly reversed by the inhibition of TRPV4 using RN1734 (p < 0.05). These findings implied that FIR irradiation emitted from a ceramic lamp specifically increased TRPV4 activity. These findings provide insights into a novel therapeutic approach using FIR irradiation for LDL-C regulation and its implications for cardiovascular health.

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).
CONCLUSIONS: 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.

Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

Improving target versus off-target ratio in nanomedicine remains a major challenge for increasing drug bioavailability and reducing toxicity. Active targeting using ligands on nanoparticle surfaces is a key approach but has limited clinical success. A potential issue is the integration of targeting ligands also changes the physicochemical properties of nanoparticles (passive targeting). Direct studies to understand the mechanisms of active targeting and off-targeting in vivo are limited by the lack of suitable tools. Here, the biodistribution of a representative active targeting liposome is analyzed, modified with an apolipoprotein E (ApoE) peptide that binds to the low-density lipoprotein receptor (LDLR), using zebrafish embryos. The ApoE liposomes demonstrated the expected liver targeting effect but also accumulated in the kidney glomerulus. The ldlra-/- zebrafish is developed to explore the LDLR-specificity of ApoE liposomes. Interestingly, liver targeting depends on the LDLR-specific interaction, while glomerular accumulation is independent of LDLR and peptide sequence. It is found that cationic charges of peptides and the size of liposomes govern glomerular targeting. Increasing the size of ApoE liposomes can avoid this off-targeting. Taken together, the study shows the potential of the zebrafish embryo model for understanding active and passive targeting mechanisms, that can be used to optimize the design of nanoparticles.

This study aimed to develop an innovative dosage form for 10-hydroxycamptothecin (HCPT), a chemotherapeutic agent with limited aqueous solubility and stability, to enhance its parenteral delivery and targeting to hepatic cancer. We formulated HCPT into a nanoemulsion using tributyrin, a dietary component with histone deacetylase inhibitor activity. The resulting HCPT-loaded tributyrin nanoemulsion (Tri-HCPT-E) underwent extensive evaluations. Tri-HCPT-E significantly improved the aqueous solubility, stability, and anti-cancer activities in HepG2 cells. Pharmacokinetic studies confirmed the increased stability and hepatic targeting, with Tri-HCPT-E leading to a 120-fold increase in plasma exposure of intact HCPT and a 10-fold increase in hepatic exposure compared to the commercial free solution. Co-administration of 17α-ethynylestradiol, an up-regulator of low-density lipoprotein (LDL) receptor, further enhanced the distribution and metabolism of HCPT, demonstrating an association between the LDL receptor pathway and hepatic targeting. Most importantly, Tri-HCPT-E exhibited superior in vivo anti-cancer efficacy in a mouse xenograft model compared to the commercial formulation, without causing escalated hepatic or renal toxicity. In conclusion, formulating HCPT into a nanoemulsion with tributyrin has proven to be an innovative and effective strategy for targeted hepatic cancer chemotherapy while tributyrin, a pharmacologically active dietary component, has emerged as a promising functional excipient for drug delivery.

UNASSIGNED: Low-density lipoprotein receptor (LDL-R) gene polymorphisms have been indicated to be correlated with ischemic cerebrovascular disease including ischemic stroke susceptibility. However, the results from each published study are inconsistent.
UNASSIGNED: All eligible case-control studies that met the search terms were retrieved in PubMed, Embase, Wanfang Med Online and Chinese National Knowledge Infrastructure (CNKI) databases. We identified seven independent case-control studies with a total of 10,355 subjects from Chinese population up to May 2023. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the susceptibility of ischemic stroke.
UNASSIGNED: Meta-analysis results indicated that rs1122608 polymorphism of LDL-R gene significantly decreased ischemic stroke risk under dominant model (OR = 0.69, 95% CI = 0.54-0.87), heterozygote comparison (OR = 0.69, 95% CI = 0.53-0.92) and allele comparison (OR = 0.74, 95% CI = 0.65-0.84) in overall analysis. Furthermore, pooled analysis showed that significant associations were observed between rs688 polymorphism and ischemic stroke risk in heterozygote carriers (OR = 1.71, 95% CI = 1.07-2.71) and dominant model (OR = 1.67, 95% CI = 1.04-2.68) in Chinese population.
UNASSIGNED: Our comprehensive meta-analysis on the role of LDL-R gene rs1122608 and rs688 polymorphisms in the risk of ischemic stroke revealed that the rs1122608 polymorphism was associated with a decreased risk, while the rs688 polymorphism was associated with an increased risk of ischemic stroke in Chinese population. Further multicenter studies were needed to confirm the effect on the susceptibility of ischemic stroke.

Nucleic acid-based therapeutics encapsulated into lipid nanoparticles (LNPs) can potentially target the root cause of genetic skin diseases. Although nanoparticles are considered impermeable to skin, research and clinical studies have shown that nanoparticles can penetrate into skin with reduced skin barrier function when administered topically. Studies have shown that epidermal keratinocytes express the low-density lipoprotein receptor (LDLR) that mediates endocytosis of apolipoprotein E (apoE)-associated nanoparticles and that dermal fibroblasts express mannose receptors. Here we prepared LNPs designed to exploit these different endocytic pathways for intracellular mRNA delivery to the two most abundant skin cell types, containing: (i) labile PEG-lipids (DMG-PEG2000) prone to dissociate and facilitate apoE-binding to LNPs, enabling apoE-LDLR mediated uptake in keratinocytes, (ii) non-labile PEG-lipids (DSPE-PEG2000) to impose stealth-like properties to LNPs to enable targeting of distant cells, and (iii) mannose-conjugated PEG-lipids (DSPE-PEG2000-Mannose) to target fibroblasts or potentially immune cells containing mannose receptors. All types of LNPs were prepared by vortex mixing and formed monodisperse (PDI ∼ 0.1) LNP samples with sizes of 130 nm (±25%) and high mRNA encapsulation efficiencies (≥90%). The LNP-mediated transfection potency in keratinocytes and fibroblasts was highest for LNPs containing labile PEG-lipids, with the addition of apoE greatly enhancing transfection via LDLR. Coating LNPs with mannose did not improve transfection, and stealth-like LNPs show limited to no transfection. Taken together, our studies suggest using labile PEG-lipids and co-administration of apoE when exploring LNPs for skin delivery.