Pentamethylquercetin attenuates angiotensin II-induced abdominal aortic aneurysm formation by blocking nuclear translocation of C/EBPß at Lys253.

BACKGROUND: Pentamethylquercetin (PMQ) is a natural polymethyl flavonoid that possesses anti-apoptotic and other biological properties. Abdominal aortic aneurysm (AAA), a fatal vascular disease with a high risk of rupture, is associated with phenotypic switching and apoptosis of medial vascular smooth muscle cells (VSMCs). This study aimed to investigate the protective effects of PMQ on the development of AAA and the underlying mechanism. METHODS: ApoE-/- mice were continuously infused with angiotensin II (Ang II) for 4 weeks to develop the AAA model. Intragastric administration of PMQ was initiated 5 days before Ang II infusion and continued for 4 weeks. In vitro, VSMCs were cultured and pretreated with PMQ, stimulated with Ang II. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the roles and mechanisms of PMQ on the phenotypic switching and apoptosis of VSMCs. RESULTS: PMQ dose-dependently reduced the incidence of Ang II-induced AAA, aneurysm diameter enlargement, elastin degradation, VSMCs phenotypic switching and apoptosis. Furthermore, PMQ also inhibited phenotypic switching and apoptosis in Ang II-stimulated VSMCs. PMQ exerted protective effects by regulating the C/EBPß/PTEN/AKT/GSK-3ß axis. AAV-mediated overexpression of PTEN reduced the therapeutic effects of PMQ in the AAA model mice, suggesting that the effects of PMQ on Ang II-mediated AAA formation were related to the PTEN/AKT/GSK-3ß axis. PMQ inhibited VSMCs phenotypic switching and apoptosis by bounding to C/EBPß at Lys253 with hydrogen bond to regulate C/EBPß nuclear translocation and PTEN/AKT/GSK-3ß axis, thereby inhibiting Ang II-induced AAA formation. CONCLUSIONS: Pentamethylquercetin inhibits angiotensin II-induced abdominal aortic aneurysm formation by bounding to C/EBPß at Lys253. Therefore, PMQ prevents the formation of AAA and reduces the incidence of AAA.

Glucagon-like peptide-1 receptor agonist exendin 4 ameliorates diabetes-associated vascular calcification by regulating mitophagy through the AMPK signaling pathway.

BACKGROUND: Vascular calcification (VC) is a complication in diabetes mellitus (DM) patients. Osteogenic phenotype switching of vascular smooth muscle cells (VSMCs) plays a critical role in diabetes-related VC. Mitophagy can inhibit phenotype switching in VSMCs. This study aimed to investigate the role of the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin 4 (EX4) in mitophagy-induced phenotype switching. MATERIALS AND METHODS: The status of VC in T2DM mice was monitored using Von Kossa and Alizarin Red S (ARS) staining in mouse aortic tissue. Human aortic smooth muscle cells were cultured in high glucose (HG) and ß-glycerophosphate (ß-GP) conditioned medium. Accumulation of LC3B and p62 was detected in the mitochondrial fraction. The effect of EX4 in vitro and in vivo was investigated by knocking down AMPKα1. RESULTS: In diabetic VC mice, EX4 decreased the percentage of von Kossa/ARS positive area. EX4 inhibited osteogenic differentiation of HG/ß-GP-induced VSMCs. In HG/ß-GP-induced VSMCs, the number of mitophagosomes was increased, whereas the addition of EX4 restored mitochondrial function, increased the number of mitophagosome-lysosome fusions, and reduced p62 in mitochondrial frictions. EX4 increased the phosphorylation of AMPKα (Thr172) and ULK1 (Ser555) in HG/ß-GP-induced VSMCs. After knockdown of AMPKα1, ULK1 could not be activated by EX4. The accumulation of LC3B and p62 could not be reduced after AMPKα1 knockdown. Knockdown of AMPKα1 negated the therapeutic effects of EX4 on VC of diabetic mice. CONCLUSION: EX4 could promote mitophagy by activating the AMPK signaling pathway, attenuate insufficient mitophagy, and thus inhibit the osteogenic phenotype switching of VSMCs.

Chloroquine and cinchonine affect rat vascular smooth muscle tonus through calcium channels - in silico and in vitro approaches.

BACKGROUND: In the present study, two structurally similar alkaloids from trees of Cinchona genus, chloroquine and cinchonine, were examined for their vasorelaxant effects in a model of phenylephrine-induced smooth muscle contractions. METHODS: Potential mechanisms of action associated with endothelial vasorelaxant compounds, voltage-gated Ca2+ channels (LTCCs), and inositol triphosphate receptors were examined in isolated rat aortic rings. Also, an in silico approach was used to predict the activity of the two test compounds. RESULTS: Experimental results revealed that both chloroquine and cinchonine significantly decrease phenylephrine-induced smooth muscle contractions, although to a different extent. Evaluated mechanisms of action indicate that endothelium is not involved in the vasorelaxant action of the two tested alkaloids. On the other hand, voltage-gated Ca2+ channels were found to be the dominant way of action associated with the vasorelaxant action of chloroquine and cinchonine. Finally, IP3R is found to have only a small impact on the observed activity of the tested compounds. CONCLUSION: Molecular docking studies predicted that chloroquine possesses a significant activity toward a suitable model of LTCCs, while cinchonine does not. The results of the present study point to the fact that great caution should be paid while administering chloroquine to vulnerable patients, especially those with cardiovascular disorders (Tab. 3, Fig. 3, Ref. 28).

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

USP18 Curbs the Progression of Metabolic Hypertension by Suppressing JAK/STAT Pathway.

Hypertension is a pathological state of the metabolic syndrome that increases the risk of cardiovascular disease. Managing hypertension is challenging, and we aimed to identify the pathogenic factors and discern therapeutic targets for metabolic hypertension (MHR). An MHR rat model was established with the combined treatment of a high-sugar, high-fat diet and ethanol. Histopathological observations were performed using hematoxylin-eosin and Sirius Red staining. Transcriptome sequencing was performed to screen differentially expressed genes. The role of ubiquitin-specific protease 18 (USP18) in the proliferation, apoptosis, and oxidative stress of HUVECs was explored using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. Moreover, USP18 downstream signaling pathways in MHR were screened, and the effects of USP18 on these signaling pathways were investigated by western blotting. In the MHR model, total cholesterol and low-density lipoprotein levels increased, while high-density lipoprotein levels decreased. Moreover, high vessel thickness and percentage of collagen were noted along with increased malondialdehyde, decreased superoxide dismutase and catalase levels. The staining results showed that the MHR model exhibited an irregular aortic intima and disordered smooth muscle cells. There were 78 differentially expressed genes in the MHR model, and seven hub genes, including USP18, were identified. USP18 overexpression facilitated proliferation and reduced apoptosis and oxidative stress in HUVECs treated with Ang in vitro. In addition, the JAK/STAT pathway was identified as a USP18 downstream signaling pathway, and USP18 overexpression inhibited the expression of JAK/STAT pathway-related proteins. Conclusively, USP18 restrained MHR progression by promoting cell proliferation, reversing apoptosis and oxidative stress, and suppressing the JAK/STAT pathway.

IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

SP2509 functions as a novel ferroptosis inhibitor by reducing intracellular iron level in vascular smooth muscle cells.

Previous studies have shown that ferroptosis of vascular smooth muscle cells (VSMCs) is involved in the development of aortic dissection (AD) and that histone methylation regulates this process. SP2509 acts as a specific inhibitor of lysine-specific demethylase 1 (LSD1), which governs a variety of biological processes. However, the effect of SP2509 on VSMC ferroptosis and AD remains to be elucidated. This aim of this study was to investigate the role and underlying mechanism of SP2509-mediated histone methylation on VSMC ferroptosis. Here, a mouse model of AD was established, and significantly reduced levels of H3K4me1 and H3K4me2 (target of SP2509) were found in the aortas of AD mice. In VSMCs, SP2509 treatment led to a dose-dependent increase in H3K4me2 levels. Furthermore, we found that SP2509 provided equivalent protection to ferrostatin-1 against VSMC ferroptosis, as evidenced by increased cell viability, decreased cell death and lipid peroxidation. RNA-sequencing analysis and subsequent experiments revealed that SP2509 counteracted cystine deficiency-induced response to inflammation and oxidative stress. More importantly, we demonstrated that SP2509 inhibited the expression of TFR and ferritin to reduce intracellular iron levels, thereby effectively blocking the process of ferroptosis. Therefore, our findings indicate that SP2509 protects VSMCs from multiple stimulus-induced ferroptosis by reducing intracellular iron levels, thereby preventing lipid peroxidation and cell death. These findings suggest that SP2509 may be a promising drug to alleviate AD by reducing iron deposition and VSMC ferroptosis.

Shizukaol C alleviates trimethylamine oxide-induced inflammation through activating Keap1-Nrf2-GSTpi pathway in vascular smooth muscle cell.

BACKGROUND: Cardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation. PURPOSE: The purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation. METHODS: The effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay. RESULTS: Our data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation. CONCLUSION: Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.

12,13-diHOME attenuates high glucose-induced calcification of vascular smooth muscle cells through repressing CPT1A-mediated HMGB1 succinylation.

Diabetes is closely associated with vascular calcification (VC). Exorbitant glucose concentration activates pro-calcific effects in vascular smooth muscle cells (VSMCs). This study enrolled 159 elderly patients with type 2 diabetes and divided them into three groups, T1, T2 and T3, according to brachial-ankle pulse wave velocity(BaPWV). There were statistically significant differences in the waist circumference, waist hip ratio, systolic blood pressure, 12,13-diHOME (a lipokin) concentration among T1, T2 and T3. 12,13-diHOME levels were positively correlated to high density lipoprotein cholesterol and total cholesterol, but negatively correlated to with waist circumference, waist hip ratio, systolic blood pressure and baPWV. Studies in vitro showed that 12,13-diHOME effectively inhibits calcification in VSMCs under high glucose conditions. Notably, 12,13-diHOME suppressed the up-regulation of carnitine O-palmitoyltransferase 1 (CPT1A) and CPT1A-induced succinylation of HMGB1. The succinylation of HMGB1 at the K90 promoted the protein stability and induced the enrichment of HMGB1 in cytoplasm, which induced the calcification in VSMCs. Together, 12,13-diHOME attenuates high glucose-induced calcification in VSMCs through repressing CPT1A-mediated HMGB1 succinylation.

Carrageenan maintains the contractile phenotype of vascular smooth muscle cells by increasing macromolecular crowding in vitro.

BACKGROUND: The contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype. PURPOSE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR). METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR. RESULTS: Notably, 225 µg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis. CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.

Effects of BNT162b2 mRNA Covid-19 vaccine on vascular function.

The effects of Covid-19 vaccines on vascular function are still controversial. We evaluated the effects of BNT162b2 vaccine (BioNTech and Pfizer) on endothelial function assessed by flow-mediated vasodilation (FMD) and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation (NID). This study was a prospective observational study. A total of 23 medical staff at Hiroshima University Hospital were enrolled in this study. FMD and NID were measured before vaccination and two weeks and six months after the 2nd dose of vaccination. FMD was significantly smaller two weeks after the 2nd dose of vaccination than before vaccination (6.5±2.4% and 8.2±2.6%, p = 0.03). FMD was significantly larger at six months than at two weeks after the 2nd dose of vaccination (8.2±3.0% and 6.5±2.4%, p = 0.03). There was no significant difference between FMD before vaccination and that at six months after the 2nd dose of vaccination (8.2±2.6% to 8.2±3.0%, p = 0.96). NID values were similar before vaccination and at two weeks, and six months after vaccination (p = 0.89). The BNT162b2 Covid-19 vaccine temporally impaired endothelial function but not vascular smooth muscle function, and the impaired endothelial function returned to the baseline level within six months after vaccination.

Nardilysin in vascular smooth muscle cells controls blood pressure via the regulation of calcium dynamics.

Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cell (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca2+ dynamics.

Curcumol reduces lower limb arteriosclerosis in rats by inhibiting human arterial smooth muscle cell activity.

Cardiovascular diseases, particularly those involving arterial stenosis and smooth muscle cell proliferation, pose significant health risks. This study aimed to investigate the therapeutic potential of curcumol in inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced human aortic smooth muscle cell (HASMC) proliferation, migration and autophagy. Using cell viability assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and Western Blot analyses, we observed that curcumol effectively attenuated PDGF-BB-induced HASMC proliferation and migration in a concentration-dependent manner. Furthermore, curcumol mitigated PDGF-BB-induced autophagy, as evidenced by the downregulation of LC3-II/LC3-I ratio and upregulation of P62. In vivo experiments using an arteriosclerosis obliterans model demonstrated that curcumol treatment significantly ameliorated arterial morphology and reduced stenosis. Additionally, curcumol inhibited the activity of the KLF5/COX2 axis, a key pathway in vascular diseases. These findings suggest that curcumol has the potential to serve as a multi-target therapeutic agent for vascular diseases.

NGF increases Connexin-43 expression and function in pulmonary arterial smooth muscle cells to induce pulmonary artery hyperreactivity.

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.

Exploring the anti-atherosclerosis mechanism of ginsenoside Rb1 by integrating network pharmacology and experimental verification.

Ginsenoside Rb1 is the major active constituent of ginseng, which is widely used in traditional Chinese medicine for the atherosclerosis treatment by anti-inflammatory, anti-oxidant and reducing lipid accumulation. We explored cellular target and molecular mechanisms of ginsenoside Rb1 based on network pharmacology and in vitro experimental validation. In this study, we predicted 17 potential therapeutic targets for ginsenoside Rb1 with atherosclerosis from public databases. We then used protein-protein interaction network to screen the hub targets. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment showed that the effects of ginsenoside Rb1 were meditated through multiple targets and pathways. Next, molecular docking results revealed that in the 10 core targets, CCND1 has the highest binding energy with ginsenoside Rb1. Vascular cell proliferation plays a critical role in atherosclerosis development. However, the effect and direct target of ginsenoside Rb1 in regulating vascular cell proliferation in atherosclerosis remains unclear. Edu straining results indicated that ginsenoside Rb1 inhibited the cell proliferation of endothelial cells, macrophages, and vascular smooth muscle cells. The protein immunoprecipitation (IP) analysis showed that ginsenoside Rb1 inhibited the vascular cell proliferation by suppressing the interaction of CCDN1 and CDK4. These findings systematically reveal that the anti-atherosclerosis mechanism of ginsenoside Rb1 by integrating network pharmacology and experimental validation, which provide evidence to treat atherosclerosis by using ginsenoside Rb1 and targeting CCND1.

Gualou-Xiebai herb pair and its active ingredients act against atherosclerosis by suppressing VSMC-derived foam cell formation via regulating P2RY12-mediated lipophagy.

BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid accumulation in the aortic wall and the formation of foam cells overloaded with large lipids inclusions. Currently, Western medicine is primarily used to improve lipid metabolism disorders and reduce inflammatory reactions to delay AS progression, but these medicines come with serious side effects and drug resistance. Gualou-Xiebai (GLXB) is a renowned herb pair that has been proven effective against AS. However, the potential molecular mechanism through which GLXB exerts the anti-atherosclerotic effects of increasing lipophagy in vascular smooth muscle cells (VSMCs) remains unknown. PURPOSE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS. METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays. RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPß abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12. CONCLUSION: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.

In vitro Evaluation of the Calcification Inhibitory Properties of Policosanol, Genistein, and Vitamin D (Reduplaxin®) either Alone or in Combination.

INTRODUCTION: The process of vascular calcification has severe clinical consequences in a number of diseases, including diabetes, atherosclerosis, and end-stage renal disease. In the present study, we investigated the effect of policosanol (Poli), genistein (Gen), and vitamin D (VitD) separately and in association to evaluate the possible synergistic action on inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs). METHODS: Primary human VSMCs were cultured with either growth medium or growth medium supplemented with calcium and phosphorus (calcification medium) in combination with Poli, Gen, and VitD. Alizarin Red staining, mineralization, and the protein expression of RUNX2 and superoxide dismutase-2 (SOD2) were investigated. RESULTS: All three substances tested were effective at reducing osteogenic differentiation of VSMCs in a dose-dependent manner. Poli+Gen, Poli+VitD, Gen+VitD treatment induced a greater inhibition of calcification and RUNX2 expression compared to single compounds treatments. Moreover, the association of Poli+Gen+VitD (Reduplaxin®) was more effective at inhibiting VSMCs mineralization and preventing the increase in RUNX2 expression induced by calcification medium but not modified SOD2 expression. CONCLUSIONS: The association of Pol, Gen, and VitD (Reduplaxin®) has an additive inhibitory effect on the calcification process of VSMCs induced in vitro by a pro-calcifying medium.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2.

BACKGROUND: Panax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs). METHODS: The present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured. RESULTS: Treatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS. CONCLUSION: Taken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

Moxonidine Increases Uptake of Oxidised Low-Density Lipoprotein in Cultured Vascular Smooth Muscle Cells and Inhibits Atherosclerosis in Apolipoprotein E-Deficient Mice.

This study aimed to investigate the effect of the sympatholytic drug moxonidine on atherosclerosis. The effects of moxonidine on oxidised low-density lipoprotein (LDL) uptake, inflammatory gene expression and cellular migration were investigated in vitro in cultured vascular smooth muscle cells (VSMCs). The effect of moxonidine on atherosclerosis was measured by examining aortic arch Sudan IV staining and quantifying the intima-to-media ratio of the left common carotid artery in apolipoprotein E-deficient (ApoE-/-) mice infused with angiotensin II. The levels of circulating lipid hydroperoxides in mouse plasma were measured by ferrous oxidation-xylenol orange assay. Moxonidine administration increased oxidised LDL uptake by VSMCs via activation of α2 adrenoceptors. Moxonidine increased the expression of LDL receptors and the lipid efflux transporter ABCG1. Moxonidine inhibited mRNA expression of inflammatory genes and increased VSMC migration. Moxonidine administration to ApoE-/- mice (18 mg/kg/day) decreased atherosclerosis formation in the aortic arch and left common carotid artery, associated with increased plasma lipid hydroperoxide levels. In conclusion, moxonidine inhibited atherosclerosis in ApoE-/- mice, which was accompanied by an increase in oxidised LDL uptake by VSMCs, VSMC migration, ABCG1 expression in VSMCs and lipid hydroperoxide levels in the plasma.

GALNT3 protects against phosphate-induced calcification in vascular smooth muscle cells by enhancing active FGF23 and inhibiting the wnt/ß-catenin signaling pathway.

Vascular calcification (VC) acts as a notable risk factor in the cardiovascular system. Disorder of phosphorus (Pi) metabolism promotes VC. Recent findings show that polypeptide N-acetylgalactosaminyltransferase 3(GALNT3) is Pi responsive and with potent effects on Pi homeostasis. However, whether GALNT3 is involved in high Pi-induced VC remains unclear. The present study investigated the potential role of GALNT3 as a novel regulator of VC. In vitro, human aortic smooth muscle cells (HASMCs) calcification was induced by inorganic Pi, while in vivo, C57BL/6 J mice were used to determine the effects of GALNT3 on Vitamin D3-induced medial arterial calcification. Alizarin red staining, Von Kossa staining, calcium and alkaline phosphatase (ALP) activity were performed to test VC. We showed that expression of GALNT3 was increased in the calcified HASMCs and aortas of the calcified mice.In vitro, overexpression of GALNT3 increased the levels of active full-length FGF23, accompanied by suppression of the osteoblast-related factors (Runx2 and BMP2), and further inhibited the formation of calcified nodules. Moreover, the protein levels of Wnt3a and active ß-catenin were determined and it was found that GALNT3 significantly inhibited their expression. LiCl, a Wnt/ß-catenin signaling activator, was observed to reverse the protective effect of GALNT3 overexpression. The opposite results were observed in the GALNT3 knockdown cells. In vivo, overexpression of GALNT3 by adeno-associated virus decreased the serum Pi and slowed the formation of aortic calcification in the calcified mice. In conclusion, our results indicate that GALNT3 counteracts high Pi-induced osteoblastic differentiation of VSMCs and protects against the initiation and progression of VC by inhibiting the Wnt/ß-catenin signaling pathway.