Gallic Acid Can Promote Low-Density Lipoprotein Uptake in HepG2 Cells via Increasing Low-Density Lipoprotein Receptor Accumulation.

Gallic acid (GA) is a type of polyphenolic compound that can be found in a range of fruits, vegetables, and tea. Although it has been confirmed it improves non-alcoholic fatty liver disease (NAFLD), it is still unknown whether GA can improve the occurrence of NAFLD by increasing the low-density lipoprotein receptor (LDLR) accumulation and alleviating cholesterol metabolism disorders. Therefore, the present study explored the effect of GA on LDLR and its mechanism of action. The findings indicated that the increase in LDLR accumulation in HepG2 cells induced by GA was associated with the stimulation of the epidermal growth factor receptor-extracellular regulated protein kinase (EGFR-ERK1/2) signaling pathway. When the pathway was inhibited by EGFR mab cetuximab, it was observed that the activation of the EGFR-ERK1/2 signaling pathway induced by GA was also blocked. At the same time, the accumulation of LDLR protein and the uptake of LDL were also suppressed. Additionally, GA can also promote the accumulation of forkhead box O3 (FOXO3) and suppress the accumulation of hepatocyte nuclear factor-1α (HNF1α), leading to the inhibition of proprotein convertase subtilisin/kexin 9 (PCSK9) mRNA expression and protein accumulation. This ultimately results in increased LDLR protein accumulation and enhanced uptake of LDL in cells. In summary, the present study revealed the potential mechanism of GA's role in ameliorating NAFLD, with a view of providing a theoretical basis for the dietary supplementation of GA.

Targeting PCSK9 to upregulate MHC-II on the surface of tumor cells in tumor immunotherapy.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9), the last member of the proprotein convertase family, functions as a classic regulator of low-density lipoprotein (LDL) by interacting with low-density lipoprotein receptor (LDLR). Recent studies have shown that PCSK9 can affect the occurrence and development of tumors and can be used as a novel therapeutic target. However, a comprehensive pan-cancer analysis of PCSK9 has yet to be conducted. METHODS: The potential oncogenic effects of PCSK9 in 33 types of tumors were explored based on the datasets of The Cancer Genome Atlas (TCGA) dataset. In addition, the immune regulatory role of PCSK9 inhibition was evaluated via in vitro cell coculture and the tumor-bearing mouse model. Finally, the antitumor efficacy of targeted PCSK9 combined with OVA-II vaccines was verified. RESULTS: Our results indicated that PCSK9 was highly expressed in most tumor types and was significantly correlated with late disease stage and poor prognosis. Additionally, PCSK9 may regulate the tumor immune matrix score, immune cell infiltration, immune checkpoint expression, and major histocompatibility complex expression. Notably, we first found that dendritic cell (DC) infiltration and major histocompatibility complex-II (MHC-II) expression could be upregulated by PCSK9 inhibition and improve CD8+ T cell activation in the tumor immune microenvironment, thereby achieving potent tumor control. Combining PCSK9 inhibitors could enhance the efficacies of OVA-II tumor vaccine monotherapy. CONCLUSIONS: Conclusively, our pan-cancer analysis provided a more comprehensive understanding of the oncogenic and immunoregulatory roles of PCSK9 and demonstrated that targeting PCSK9 could increase the efficacy of long peptide vaccines by upregulating DC infiltration and MHC-II expression on the surface of tumor cells. This study reveals the critical oncogenic and immunoregulatory roles of PCSK9 in various tumors and shows the promise of PCSK9 as a potent immunotherapy target.

Multifaceted roles of Meg3 in cellular senescence and atherosclerosis.

BACKGROUND AND AIMS: Long noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long noncoding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis. METHODS AND RESULTS: By analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr-/- mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified antisense oligonucleotides attenuated the formation of atherosclerotic lesions by 34.9% and 20.1% in male and female mice, respectively, revealed by en-face Oil Red O staining, which did not correlate with changes in plasma lipid profiles. Real-time quantitative PCR analysis of cellular senescence markers p21 and p16 revealed that Meg3 deficiency aggravates hepatic cellular senescence but not cellular senescence at aortic roots. Human Meg3 transgenic mice were generated to examine the role of Meg3 gain-of-function in the development of atherosclerosis induced by PCSK9 overexpression. Meg3 overexpression promotes atherosclerotic lesion formation by 29.2% in Meg3 knock-in mice independent of its effects on lipid profiles. Meg3 overexpression inhibits hepatic cellular senescence, while it promotes aortic cellular senescence likely by impairing mitochondrial function and delaying cell cycle progression. CONCLUSIONS: Our data demonstrate that Meg3 promotes the formation of atherosclerotic lesions independent of its effects on plasma lipid profiles. In addition, Meg3 regulates cellular senescence in a tissue-specific manner during atherosclerosis. Thus, we demonstrated that Meg3 has multifaceted roles in cellular senescence and atherosclerosis.

Multiple Immunomodulatory Strategies Based on Targeted Regulation of Proprotein Convertase Subtilisin/Kexin Type 9 and Immune Homeostasis against Hepatocellular Carcinoma.

Immunotherapy is the most promising systemic therapy for hepatocellular carcinoma. However, the outcome remains poor. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a role in altering cell-surface protein levels, potentially undermining the efficacy of immunotherapy against tumors. This highlights its potential as a target for antitumor therapy. Herein, CaCO3-based nanoparticles coencapsulated with DOX, an immunogenic cell death (ICD) inducer, and evolocumab was developed to enhanced the efficacy of immunotherapy. The obtained DOX/evolocumab-loaded CaCO3 nanoparticle (named DECP) exhibits a good capacity of acid neutralization and causes ICD of cancer cells. In addition, DECP is able to evaluate the cell-surface level of MHC-I, a biomarker that correlates positively with patients' overall survival. Upon intravenous injection, DECP accumulates within the tumor site, leading to growth inhibition of hepa1-6 bearing subcutaneous tumors. Specifically, DECP treatment causes augmented ratios of matured dendritic cells, tumor-infiltrating CD8+ T cells and natural killing cells, while concurrently depleting Foxp3+ regulatory T cells. Peritumoral delivery of DECP enhances the immune response of distant tumors and exhibits antitumor effects when combined with intravenous αPD-L1 therapy in a bilateral tumor model. This study presents CaCO3-based nanoparticles with multiple immunomodulatory strategies against hepatocellular carcinoma by targeting PCSK9 inhibition and modulating immune homeostasis in the unfavorable TME.

PCSK9 plasma concentration is associated with epicardial adipose tissue volume and metabolic control in patients with type 1 diabetes.

Patients with type 1 diabetes (T1D) have a greater risk of cardiovascular disease. Proconvertase subtilisin-kexin 9 (PCSK9) is involved in the atherosclerosis process. This study aimed to determine the relationship between PCSK9 levels and epicardial adipose tissue (EAT) volume and cardiometabolic variables in patients with T1D. This was an observational cross-sectional study including 73 patients with T1D. Clinical, biochemical and imaging data were collected. We divided the patients into two groups according to their glycemic control and the EAT index (iEAT) percentile. We performed a correlation analysis between the collected variables and PCSK9 levels; subsequently, we performed a multiple regression analysis with the significant parameters. The mean age was 47.6 ± 8.5 years, 58.9% were men, and the BMI was 26.9 ± 4.6 kg/m2. A total of 31.5%, 49.3% and 34.2% of patients had hypertension, dyslipidemia and smoking habit, respectively. The PCSK9 concentration was 0.37 ± 0.12 mg/L, which was greater in patients with worse glycemic control (HbA1c > 7.5%), dyslipidemia and high EAT volume (iEAT > 75th percentile). The PCSK9 concentration was positively correlated with age (r = 0.259; p = 0.027), HbA1c (r = 0.300; p = 0.011), insulin dose (r = 0.275; p = 0.020), VLDL-C level (r = 0.331; p = 0.004), TG level (r = 0.328; p = 0.005), and iEAT (r = 0.438; p < 0.001). Multiple regression analysis revealed that 25% of the PCSK9 variability was explained by iEAT and HbA1c (p < 0.05). The PCSK9 concentration is associated with metabolic syndrome parameters, poor glycemic control and increased EAT volume in patients with T1D.

Sesamin alleviates lipid accumulation induced by oleic acid via PINK1/Parkin-mediated mitophagy in HepG2 cells.

Sesamin, a special compound present in sesame and sesame oil, has been reported a role in regulating lipid metabolism, while the underlying mechanisms remain unclear. Autophagy has been reported associated with lipid metabolism and regarded as a key modulator in liver steatosis. The present work aimed to investigate whether sesamin could exert its protective effects against lipid accumulation via modulating autophagy in HepG2 cells stimulated with oleic acid (OA). Cell viability was evaluated using the CCK-8 method, and triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein, cholesterol (LDL-C), alanine aminotransferase (ALT), along with aspartate aminotransferase (AST) were assessed by oil red O staining, transmission electron microscopy (TEM), and biochemical kits to investigate the lipid-lowering effects of sesamin. Differentially expressed genes were screened by RNA sequencing and validated using real-time quantitative PCR and Western blot. Autophagy and mitophagy related molecules were analyzed employing TEM, Western blot, and immunofluorescence. The data shows that in HepG2 cells stimulated by OA, sesamin reduces levels of TG, TC, LDL-C, ALT, and AST while elevating HDL-C, alleviates the lipid accumulation and improves fatty acid metabolism through modulating the levels of fat metabolism related genes including PCSK9, FABP1, CD36, and SOX4. Sesamin restores the suppressed autophagy in HepG2 cells caused by OA, which could be blocked by autophagy inhibitors. This indicates that sesamin improves fatty acid metabolism by enhancing autophagy levels, thereby mitigating the intracellular lipid accumulation. Furthermore, sesamin significantly enhances the mitophagy and improves mitochondrial homeostasis via activating the PINK/Parkin pathway. These data suggest that sesamin alleviates the excessive lipid accumulation in HepG2 caused by OA by restoring the impaired mitophagy via the PINK1/Parkin pathway, probably playing a preventive or therapeutic role in hepatic steatosis.

Arenobufagin modulation of PCSK9-mediated cholesterol metabolism induces tumor-associated macrophages polarisation to inhibit hepatocellular carcinoma progression.

BACKGROUND: The tumor microenvironment (TME) of hepatocellular carcinoma is heterogeneous enough to be prone to drug resistance and multidrug resistance during treatment, and reprogramming of cholesterol metabolism in TME mediates tumor-associated macrophages (TAMs) polarization, which has an impact on the regulation of malignant tumor progression. Arenobufagin (ARBU) was extracted and isolated from toad venom (purity ≥98 %), which is the main active ingredient of the traditional Chinese medicine Chan'su with good anti-tumor effects. PURPOSE: To investigate the regulatory effect of ARBU on lipid metabolism in tumor microenvironment, interfere with macrophage polarization, and determine its mechanism of action on liver cancer progression. METHODS: In this study, the inhibitory effect of ARBU on the proliferation of Hepa1-6 in C57 mice and the safety of administration were evaluated by establishing a transplanted tumor model of Hepa1-6 hepatocellular carcinoma mice and using 5-FU as a positive control drug. In addition, we constructed a co-culture system of Hepa1-6 cells and primary mouse macrophages to study the effects of ARBU on the polarization phenotypic transformation of macrophages and the proliferation and migration of hepatoma cells. The influence of ARBU on the metabolism of lipids in the hepatocellular carcinoma mouse model was investigated by combining it with lipidomics technology. The influence of ARBU on the PCSK9/LDL-R signaling pathway and macrophage polarization, which regulate cholesterol metabolism, was tested by using qRT-PCR, gene editing, IF, and WB. CONCLUSION: ARBU significantly inhibited the proliferation of Hepa1-6 in vivo and in vitro, regulated cholesterol metabolism, and promoted the M1-type polarization of macrophages in the tumor microenvironment. ARBU inhibits cholesterol synthesis in the TME through the PCSK9/LDL-R signaling pathway, thereby blocking macrophage M2 polarization, promoting apoptosis of the tumor cells, and inhibiting their proliferation and migration.

Olive (Olea europaea L.) Seed as New Source of Cholesterol-Lowering Bioactive Peptides: Elucidation of Their Mechanism of Action in HepG2 Cells and Their Trans-Epithelial Transport in Differentiated Caco-2 Cells.

The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates enormous quantities of waste and by-products, which can be exploited as new raw materials to obtain innovative ingredients and therefore make the olive production more sustainable. In a previous study, we decided to foster olive seeds by generating two protein hydrolysates using food-grade enzymes, alcalase (AH) and papain (PH). These hydrolysates have shown, both in vitro and at the cellular level, antioxidant and antidiabetic activities, being able to inhibit the activity of the DPP-IV enzyme and modulate the secretion of GLP-1. Given the multifunctional behavior of peptides, both hydrolysates displayed dual hypocholesterolemic activity, inhibiting the activity of HMGCoAR and impairing the PPI of PCSK9/LDLR, with an IC50 equal to 0.61 mg/mL and 0.31 mg/mL for AH and PH, respectively. Furthermore, both samples restored LDLR protein levels on the membrane of human hepatic HepG2 cells, increasing the uptake of LDL from the extracellular environment. Since intestinal bioavailability is a key component of bioactive peptides, the second objective of this work is to evaluate the capacity of AH and PH peptides to be transported by differentiated human intestinal Caco-2 cells. The peptides transported by intestinal cells have been analyzed using mass spectrometry analysis, identifying a mixture of stable peptides that may represent new ingredients with multifunctional qualities for the development of nutraceuticals and functional foods to delay the onset of metabolic syndrome, promoting the principles of environmental sustainability.

Discovery of (2-(4-Substituted phenyl)quinolin-4-yl)(4-isopropylpiperazin-1-yl)methanone Derivatives as Potent Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors.

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an increasingly important role in the treatment of hyperlipidemia. In pursuit of potent small molecules that block the PCSK9/low-density lipoprotein receptor (LDLR) protein-protein interaction (PPI), a series of 2-phenylquinoline-4-carboxylic acid derivatives were designed and synthesized based on previously derived molecules. In the in vitro PPI inhibition test, compounds M1, M12, M14, M18 and M27 exhibited potent activities with IC50 values of 6.25 µM, 0.91 µM, 2.81 µM, 4.26 µM and 0.76 µM, respectively, compared with SBC-115337 (IC50 value of 9.24 µM). Molecular docking and molecular dynamics simulations revealed the importance of hydrophobic interactions in the binding of inhibitors to the PPI interface of PCSK9. In LDLR expression and LDL uptake assays, the tested compounds M1, M12 and M14 were found to restore LDLR expression levels and to increase the extracellular LDL uptake capacity of HepG2 cells in the presence of exogenous PCSK9. Collectively, novel small-molecule PCSK9/LDLR PPI inhibitors (especially M12) with in vitro lipid lowering ability, were discovered as lead compounds for further development of hypolipidemic drugs.

Proprotein convertase subtilisin/kexin 9 inhibitor downregulates microRNA-130a-3p expression in hepatocytes to alleviates atherosclerosis progression.

Proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors have been shown to regulate lipid metabolism and reduce the risk of cardiovascular events. This study explores the effect and potential mechanism of PCSK9 inhibitors on lipid metabolism and coronary atherosclerosis. HepG2 cells were incubated with PCSK9 inhibitor. ApoE-/- mice were fed with a high fat to construct an atherosclerosis model, and then treated with PCSK9 inhibitor (8 mg/kg for 8 w). PCSK9 inhibitor downregulated microRNA (miRNA)-130a-3p expression in a dose-dependent manner. And, miR-130a-3p could bind directly to the 3' untranslated region (3'-UTR) region of LDLR to down-regulate LDLR expression in HepG2 cells, as confirmed by the luciferase reporter gene assay. In addition, miR-130a-3p overexpression significantly attenuated the promoting effect of PCSK9 inhibitor on LDLR and DiI-LDL uptake in HepG2 cells. More importantly, in vivo experiments confirmed that PCSK9 inhibitor could significantly inhibit miR-130a-3p levels and promote LDLR expression in liver tissues, thus regulating serum lipid profile and alleviating the progression of coronary atherosclerosis. PCSK9 inhibitor could moderately improve coronary atherosclerosis by regulating miR-130a-3p/LDLR axis, providing an exploitable strategy for the treatment of coronary atherosclerosis.

Schisandrin A in Schisandra chinensis Upregulates the LDL Receptor by Inhibiting PCSK9 Protein Stabilization in Steatotic Model.

Schisandra chinensis extract (SCE) protects against hypocholesterolemia by inhibiting proprotein convertase subtilisin/kexin 9 (PCSK9) protein stabilization. We hypothesized that the hypocholesterolemic activity of SCE can be attributable to upregulation of the PCSK9 inhibition-associated low-density lipoprotein receptor (LDLR). Male mice were fed a low-fat diet or a Western diet (WD) containing SCE at 1% for 12 weeks. WD increased final body weight and blood LDL cholesterol levels as well as alanine transaminase and aspartate aminotransferase expression. However, SCE supplementation significantly attenuated the increase in blood markers caused by WD. SCE also attenuated WD-mediated increases in hepatic LDLR protein expression in the obese mice. In addition, SCE increased LDLR protein expression and attenuated cellular PCSK9 levels in HepG2 cells supplemented with delipidated serum (DLPS). Non-toxic concentrations of schisandrin A (SA), one of the active components of SCE, significantly increased LDLR expression and tended to decrease PCSK9 protein levels in DLPS-treated HepG2 cells. High levels of SA-mediated PCSK9 attenuation was not attributable to reduced PCSK9 gene expression, but was associated with free PCSK9 protein degradation in this cell model. Our findings show that PCSK9 secretion can be significantly reduced by SA treatment, contributing to reductions in free cholesterol levels.

Macrophage KLF15 prevents foam cell formation and atherosclerosis via transcriptional suppression of OLR-1.

BACKGROUND: Macrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis. METHODS: We used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and ApoE-/-- mice on a HFD. We subsequently injected mice with AAV-KLF15 and AAV-LacZ to assess the role of KLF15 in the development of atherosclerosis in vivo. Oil Red O, H&E, and Masson's trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively. RESULTS: We determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis. CONCLUSION: Macrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.

HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

Identification of 4-amino-2-Pyridones as new potent PCSK9 inhibitors: From phenotypic hit discovery to in vivo tolerability.

Among the strategies to overcome the underperformance of statins in cardiovascular diseases (CVDs), the development of drugs targeting the Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is considered one of the most promising. However, only anti-PCSK9 biological drugs have been approved to date, and orally available small-molecules for the treatment of hypercholesterolemic conditions are still missing on the market. In the present work, we describe the application of a phenotypic approach to the identification and optimization of 4-amino-2-pyridone derivatives as a new chemotype with anti-PCSK9 activity. Starting from an in-house collection of compounds, functional assays on HepG2 cells followed by a chemistry-driven hit optimization campaign, led to the potent anti-PCSK9 candidate 5c. This compound, at 5 µM, totally blocked PCSK9 secretion from HepG2 cells, significantly increased LDL receptor (LDLR) expression, and acted cooperatively with simvastatin by reducing its induction of PCSK9 expression. Finally, compound 5c also proved to be well tolerated in C57BL/6J mice at the tested concentration (40 mg/kg) with no sign of toxicity or behavior modifications.

Lindenane sesquiterpenoid dimers from Chloranthus japonicus improve LDL uptake by regulating PCSK9 and LDLR.

UPLC-TOF-MS/MDF directed phytochemical research of Chloranthus japonicus led to the isolation of 46 lindenane sesquiterpenoid dimers, which included 13 new analogs. Their structures with absolute configurations were elucidated by analysis of spectroscopic data. Fourteen compounds with ester chains significantly decreased PCSK9 protein level in medium of HepG2 cells, especially for compounds 14 and 29 (5 µM) with inhibition rates of 69.0% and 72.8%, respectively. Compound 14 in HepG2 cells was evaluated via DiI-LDL uptake assays and found to increase LDL uptake by upregulating LDLR mRNA and protein level. Meanwhile, 14 decreased the secretion of PCSK9 protein in medium and downregulated intracellular PCSK9 protein and mRNA level. The discovery of these natural small molecule compounds provides a novel structure basis for design PCSK9 regulators, making them a promising lead for development of new lipid-lowering agents.

Familial hypercholesterolemia and its manifestations: Practical considerations for general practitioners.

Familial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism, affecting almost 1 in 250 individuals worldwide. It is usually inherited via the autosomal dominant way and is characterized by aberrantly high total and low-density lipoprotein cholesterol (LDL-C) concentrations from early childhood, predisposing to increased risk of premature atherosclerotic cardiovascular disease (ASCVD), mostly coronary heart disease (CHD). Despite its high prevalence in the general population and the high ASCVD risk, FH is often underdiagnosed and undertreated. Genetic diagnosis is not always necessary since specific criteria, taking into account the patient's individual and family history, clinical signs, and untreated LDL-C concentrations, may be used for prompt diagnosis. Except for CHD, which may be already evident at diagnosis, leading to increased mortality, other non-CHD morbidities, such as stroke, peripheral artery disease, carotid artery stenosis, and aortic valve calcification may be also present, substantiating the need for prompt intervention. Statins constitute the mainstay of treatment both in adults and children >8 years old. In cases of statin intolerance or not achieving the LDL-C target despite maximally tolerated statin dose, ezetimibe and/or proprotein convertase subtilisin-kexin type 9 inhibitors may be used. The advent of recently approved medications, such as inclisiran and bempedoic acid, either as monotherapy or as add-on therapy to statins, has further enhanced the therapeutic armamentarium that can be used in FH patients. The purpose of this narrative review is to provide practical considerations regarding the diagnostic and therapeutic approach to FH patients.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

The modulatory effects on enterohepatic cholesterol metabolism of novel cholesterol-lowering peptides from gastrointestinal digestion of Xuanwei ham.

The aim of this study was to investigate the effects and mechanism of in vitro protein digestive products of Xuanwei ham with different ripening periods on cholesterol metabolism and hypercholesterolemia. The results showed that compared with other gastrointestinal digestion (GID) groups, the GID group of Xuanwei ham with 3-year ripening period (XWH3-GID) inhibited the expression of Niemann-Pick C1-like 1 (NPC1L1) and acetyl-CoA acetyltransferase 2 (ACAT2) through hepatocyte nuclear factor 1-alpha (HNF-1α), which in turn effectively inhibited cholesterol absorption in Caco-2 cell monolayers. Following absorption by Caco-2 cell monolayers, the XWH3-GID group suppressed the expression and secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) via HNF-1α, which enhanced the protein expression and fluorescence intensity of low density lipoprotein receptor (LDLR) on the HepG2 cell membrane, and thus promoted the uptake of low density lipoprotein (LDL). Importantly, three novel peptides (LFP, PKF and VPFP) derived from titin were identified after intestinal epithelial transport in the XWH3-GID group, which could exert cholesterol-lowering effects through inhibiting intestinal cholesterol absorption and promoting peripheral hepatic LDL uptake, and effectively ameliorate western diet-induced hypercholesterolemia in ApoE-/- mice. These results suggest that Xuanwei ham with 3-year ripening period can be used as a source of cholesterol-lowering peptides and has potential to intervene in hypercholesterolemia.

The Lin28b/Wnt5a axis drives pancreas cancer through crosstalk between cancer associated fibroblasts and tumor epithelium.

Bidirectional signal transduction between tumor epithelial cells and tumor microenvironment (TME) is important for tumor development. Here we show that Lin28b/let-7 pathway is indispensable for modulating the expression of Wnt5a in tumor epithelium, which could be secreted and then up-regulates Lin28b in cancer-associated fibroblasts (CAFs). Moreover, we demonstrate that Lin28b in CAFs promoted growth of PDAC by inducing cytokine PCSK9's production. Using an orthotopic mouse model of PDAC, we find that depletion of Lin28b in CAFs reduced tumor weight, highlighting the importance of Lin28b in PDAC stroma. Thus, our study shows that the Lin28b-Wnt5a axis plays a critical role in bidirectional crosstalk between pancreatic tumor epithelium and TME and results in a pro-|tumorigenic contexture.

Confounding Effects of Tamoxifen: Cautionary and Practical Considerations for the Use of Tamoxifen-Inducible Mouse Models in Atherosclerosis Research-Brief Report.

BACKGROUND: In recent years, fate-mapping lineage studies in mouse models have led to major advances in vascular biology by allowing investigators to track specific cell populations in vivo. One of the most frequently used lineage tracing approaches involves tamoxifen-inducible CreERT-LoxP systems. However, tamoxifen treatment can also promote effects independent of Cre recombinase activation, many of which have not been fully explored. METHODS: To elucidate off-target effects of tamoxifen, male and female mice were either unmanipulated or injected with tamoxifen or corn oil. All mice received PCSK9 (proprotein convertase subtilisin/kexin type 9)-AAV (adeno-associated virus) injections and a modified Western diet to induce hypercholesterolemia. After 2 weeks, serum cholesterol and liver morphology were assessed. To determine the duration of any tamoxifen effects in long-term atherosclerosis experiments, mice received either 12 days of tamoxifen at baseline or 12 days plus 2 sets of 5-day tamoxifen boosters; all mice received PCSK9-AAV injections and a modified Western diet to induce hypercholesterolemia. After 24 weeks, serum cholesterol and aortic sinus plaque burden were measured. RESULTS: After 2 weeks of atherogenic treatment, mice injected with tamoxifen demonstrated significantly reduced serum cholesterol levels compared with uninjected- or corn oil-treated mice. However, there were no differences in PCSK9-mediated knockdown of LDL (low-density lipoprotein) receptors between the groups. Additionally, tamoxifen-treated mice exhibited significantly increased hepatic lipid accumulation compared with the other groups. Finally, the effects of tamoxifen remained for at least 8 weeks after completion of injections, with mice demonstrating persistent decreased serum cholesterol and impaired atherosclerotic plaque formation. CONCLUSIONS: In this study, we establish that tamoxifen administration results in decreased serum cholesterol, decreased plaque formation, and increased hepatic lipid accumulation. These alterations represent significant confounding variables in atherosclerosis research, and we urge future investigators to take these findings into consideration when planning and executing their own atherosclerosis experiments.