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.

Hypomethylation of the LncRNA H19 promoter accelerates osteogenic differentiation of vascular smooth muscle cells by activating the Erk1/2 pathways.

OBJECTIVE: Vascular calcification is a common chronic kidney disease complication. This study aimed to investigate the function of long non-coding RNA (LncRNA) H19 in vascular calcification to explore new therapeutic strategies. METHODS: We induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) using ß-glycerophosphate. Then, we detected the LncRNA H19 promoter methylation status and Erk1/2 pathways using methylation-specific polymerase chain reaction and western blotting, respectively. RESULTS: Compared with the control group, high phosphorus levels induced VSMC calcification, accompanied by increases in LncRNA H19 and the osteogenic marker Runx2 and reduction of the contractile phenotype marker SM22a. LncRNA H19 knockdown inhibited osteogenic differentiation and calcification of VSMCs. However, the suppressed role of VSMC calcification caused by shRNA H19 was partially reversed by simultaneous activation of the Erk1/2 pathways. Mechanically, we found that the methylation rate of CpG islands in the LncRNA H19 promoter region was significantly lower in the high-phosphorus group, and the hypomethylation state elevated LncRNA H19 levels, which in turn regulated phosphorylated Erk1/2 expression. CONCLUSIONS: LncRNA H19 promoted osteogenic differentiation and calcification of VSMCs by regulating the Erk1/2 pathways. Additionally, hypomethylation of LncRNA H19 promoter CpG islands upregulated LncRNA H19 levels and subsequently activated Erk1/2 phosphorylation.

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.

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.

Anji white tea relaxes precontracted arteries, represses voltage-gated Ca2+ channels and voltage-gated K+ channels in the arterial smooth muscle cells: Comparison with green tea main component (-)-epigallocatechin gallate.

ETHNOPHARMACOLOGICAL RELEVANCE: Tea (Camellia sinensis) is a favorite drink worldwide. Tea extracts and green tea main component (-)-epigallocatechin gallate (EGCG) are recommended for various vascular diseases. Anji white tea is a very popular green tea. Its vascular effect profile, the mechanisms, and the contribution of EGCG to its integrated effect need elucidation. AIM: To characterize the vasomotion effects of Anji white tea and EGCG, and to explore possible involvement of voltage-gated Ca2+ channels (VGCCs) and voltage-gated K+ (Kv) channels in their vasomotion effects. MATERIALS AND METHODS: Anji white tea water soaking solution (AJWT) was prepared as daily tea-making process and concentrated to a concentration amounting to 200 mg/ml of dry tea leaves. The tension of rat arteries including aorta, coronary artery (RCA), cerebral basilar artery (CBA), intrarenal artery (IRA), intrapulmonary artery (IPA) and mesenteric artery (MA) was recorded with myographs. In arterial smooth muscle cells (ASMCs) freshly isolated from RCA, the levels of intracellular Ca2+ were measured with Ca2+-sensitive fluorescent probe fluo 4-AM, and Kv currents were recorded with patch clamp. The expressions of VGCCs and Kv channels were assayed with RT-qPCR and immunofluorescence staining. RESULTS: At 0.4-12.8 mg/ml of dry tea leaves, AJWT profoundly relaxed all tested arteries precontracted with various vasoconstrictors about half with a small transient potentiation on the precontractions before the relaxation. KCl-induced precontraction was less sensitive than precontractions induced by phenylephrine (PE), U46619 and serotonin (5-HT). IPA was less sensitive to the relaxation compared with other arteries. AJWT pretreatment for 1 h, 24 h and 72 h time-dependently inhibited the contractile responses of RCAs. In sharp contrast, at equivalent concentrations according to its content in AJWT, EGCG intensified the precontractions in most small arteries, except that it induced relaxation in PE-precontracted aorta and MA, U46619-precontracted aorta and CBA. EGCG pretreatment for 1 h and 24 h did not significantly affect RCA contractile responses. In RCA ASMCs, AJWT reduced, while EGCG enhanced, intracellular Ca2+ elevation induced by depolarization which activates VGCCs. Patch clamp study showed that both AJWT and EGCG reduced Kv currents. RT-qPCR and immunofluorescence staining demonstrated that both AJWT and EGCG reduced the expressions of VGCCs and Kv channels. CONCLUSION: AJWT, but not EGCG, consistently induces vasorelaxation. The vasomotion effects of either AJWT or EGCG vary with arterial beds and vasoconstrictors. Modulation of VGCCs, but not Kv channels, contributes to AJWT-induced vasorelaxation. It is suggested that Anji white tea water extract instead of EGCG may be a promising food supplement for vasospastic diseases.

Atherosclerotic three-layer nanomatrix vascular sheets for high-throughput therapeutic evaluation.

In vitro atherosclerosis models are essential to evaluate therapeutics before in vivo and clinical studies, but significant limitations remain, such as the lack of three-layer vascular architecture and limited atherosclerotic features. Moreover, no scalable 3D atherosclerosis model is available for making high-throughput assays for therapeutic evaluation. Herein, we report an in vitro 3D three-layer nanomatrix vascular sheet with critical atherosclerosis multi-features (VSA), including endothelial dysfunction, monocyte recruitment, macrophages, extracellular matrix remodeling, smooth muscle cell phenotype transition, inflammatory cytokine secretion, foam cells, and calcification initiation. Notably, we present the creation of high-throughput functional assays with VSAs and the use of these assays for evaluating therapeutics for atherosclerosis treatment. The therapeutics include conventional drugs (statin and sirolimus), candidates for treating atherosclerosis (curcumin and colchicine), and potential gene therapy (miR-146a-loaded liposomes). The high efficiency and flexibility of the scalable VSA functional assays should facilitate drug discovery and development for atherosclerosis.

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.

Shh-Gli2-Runx2 inhibits vascular calcification.

BACKGROUND: In patients with chronic kidney disease (CKD), vascular calcification (VC) is common and is associated with a higher risk of all-cause mortality. Shh, one ligand for Hedgehog (Hh) signaling, participates in osteogenesis and several cardiovascular diseases. However, it remains unclear whether Shh is implicated in the development of VC. METHODS: Inorganic phosphorus 2.6 mM was used to induce vascular smooth muscle cells (VSMCs) calcification. Mice were fed with adenine diet supplement with 1.2% phosphorus to induce VC. RESULTS: Shh was decreased in VSMCs exposed to inorganic phosphorus, calcified arteries in mice fed with an adenine diet, as well as radial arteries from patients with CKD presenting VC. Overexpression of Shh inhibited VSMCs ostosteoblastic differentiation and calcification, whereas its silencing accelerated these processes. Likewise, mice treated with smoothened agonist (SAG; Hh signaling agonist) showed alleviated VC, and mice treated with cyclopamine (CPN; Hh signaling antagonist) exhibited severe VC. Additionally, overexpression of Gli2 significantly reversed the pro-calcification effect of Shh silencing on VSMCs, suggesting that Shh inhibited VC via Gli2. Mechanistically, Gli2 interacted with Runx2 and promoted its ubiquitin proteasomal degradation, therefore protecting against VC. Of interest, the pro-degradation effect of Gli2 on Runx2 was independent of Smurf1 and Cullin4B. CONCLUSIONS: Our study provided deeper insight to the pathogenesis of VC, and Shh might be a novel potential target for VC treatment.

Ganglioside GM3 Protects Against Abdominal Aortic Aneurysm by Suppressing Ferroptosis.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular condition, but approved medical therapies to prevent AAA progression and rupture are currently lacking. Sphingolipid metabolism disorders are associated with the occurrence and development of AAA. It has been discovered that ganglioside GM3, a sialic acid-containing type of glycosphingolipid, plays a protective role in atherosclerosis, which is an important risk factor for AAA; however, the potential contribution of GM3 to AAA development has not been investigated. METHODS: We performed a metabolomics study to evaluated GM3 level in plasma of human patients with AAA. We profiled GM3 synthase (ST3GAL5) expression in the mouse model of aneurysm and human AAA tissues through Western blotting and immunofluorescence staining. RNA sequencing, affinity purification and mass spectrometry, proteomic analysis, surface plasmon resonance analysis, and functional studies were used to dissect the molecular mechanism of GM3-regulating ferroptosis. We conditionally deleted and overexpressed St3gal5 in smooth muscle cells (SMCs) in vivo to investigate its role in AAA. RESULTS: We found significantly reduced plasma levels of GM3 in human patients with AAA. GM3 content and ST3GAL5 expression were decreased in abdominal aortic vascular SMCs in patients with AAA and an AAA mouse model. RNA sequencing analysis showed that ST3GAL5 silencing in human aortic SMCs induced ferroptosis. We showed that GM3 interacted directly with the extracellular domain of TFR1 (transferrin receptor 1), a cell membrane protein critical for cellular iron uptake, and disrupted its interaction with holo-transferrin. SMC-specific St3gal5 knockout exacerbated iron accumulation at lesion sites and significantly promoted AAA development in mice, whereas GM3 supplementation suppressed lipid peroxidation, reduced iron deposition in aortic vascular SMCs, and markedly decreased AAA incidence. CONCLUSIONS: Together, these results suggest that GM3 dysregulation promotes ferroptosis of vascular SMCs in AAA. Furthermore, GM3 may constitute a new therapeutic target for AAA.

Selenium Deficiency Promotes Dilatation of the Aorta by Increasing Expression and Activity of Vascular Smooth Muscle Cell Derived Matrix Metalloproteinase-2.

OBJECTIVE: Selenium (Se) is a key part of the body's oxidation defence system. However, it is unclear whether Se affects the development of aortic aneurysm (AA). An animal experiment was conducted to clarify the role of Se in AA development. METHODS: C57BL/6N male mice were fed with a Se deficient (Se-D, < 0.05 mg/kg), Se adequate (Se-A, 0.2 mg/kg), or Se supplemented (Se-S, 1 mg/kg) diet for 8 weeks. Subsequently, an AA murine model (Se-D, n = 11; Se-A, n = 12; Se-S, n = 15) was established using angiotensin II (Ang II, 1 mg/kg/min) for four weeks plus ß-aminopropionitrile (BAPN, 1 mg/mL) for the first two weeks. Saline replaced Ang II, and BAPN was removed during the modelling process for sham mice (Se-A, n = 9). To determine whether Se deficiency promoted aortic dilation via matrix metalloproteinase-2 (MMP-2), the non-specific MMP inhibitor doxycycline (Dox, 100 mg/kg/day) was given to Se-D AA mice (n = 7) for two weeks. RESULTS: The maximum aortic diameter in Se-D AA model mice was significantly increased compared with Se-A AA model mice. MMP-2 expression and activity in the aortic media of Se-D AA model mice was significantly increased compared with Se-A AA model mice. A large number of vascular smooth muscle cells (VSMCs) were found aggregating in the media of the non-dilated aorta of Se-D AA model mice, which was completely inhibited by Dox. The percentage of VSMCs in aortic media of Se-D AA model mice was significantly higher than in Se-A AA model mice. The maximum aortic diameter and occurrence rate of AA in Se-D AA model mice with Dox were significantly reduced compared with Se-D AA model mice. CONCLUSION: Se deficiency promoted dilatation of the aorta in AA model mice by increasing expression and activity of VSMC derived MMP-2, causing abnormal aggregation and proliferation of VSMCs in aortic media.

Trimethylamine N-oxide promotes abdominal aortic aneurysm by inducing vascular inflammation and vascular smooth muscle cell phenotypic switching.

OBJECTIVE: Inflammation and vascular smooth muscle cell (VSMC) phenotypic switching are implicated in the pathogenesis of abdominal aortic aneurysm (AAA). Trimethylamine N-oxide (TMAO) has emerged as a crucial risk factor in cardiovascular diseases, inducing vascular inflammation and calcification. We aimed to evaluate the effect of TMAO on the formation of AAA. APPROACH AND RESULTS: Here, we showed that TMAO was elevated in plasma from AAA patients compared with nonaneurysmal subjects by liquid chromatography‒mass spectrometry (LC‒MS) detection. Functional studies revealed that increased TMAO induced by feeding a choline-supplemented diet promoted Ang II-induced AAA formation. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot analyses revealed that TMAO induced macrophage infiltration and inflammatory factor release. Conversely, inhibition of TMAO by supplementation with DMB suppressed AAA formation and the inflammatory response. Molecular studies revealed that TMAO regulated VSMC phenotypic switching. Flow cytometry analyses showed that TMAO induces macrophage M1-type polarization. Furthermore, pharmacological intervention experiments suggested that the nuclear factor-κB (NF-κB) signaling pathway was critical for TMAO to trigger AAA formation. CONCLUSIONS: TMAO promotes AAA formation by inducing vascular inflammation and VSMC phenotypic switching through activation of the NF-κB signaling pathway. Thus, TMAO is a prospective therapeutic AAA target.

Efficacy and mechanism of Shenqi Compound in inhibiting diabetic vascular calcification.

BACKGROUND: Shenqi Compound (SQC) has been used in clinic for several decades in the prevention and treatment of diabetes and its complications. But this is merely a heritage of experience. The primary aim of this study is to scientifically validate the therapeutic effects of SQC on diabetic vascular calcification (DVC) in an animal model and, simultaneously, uncover its potential underlying mechanisms. METHOD: Spontaneous diabetic rat- Goto Kakizaki (GK) rats were selected for rat modeling. We meticulously designed three distinct groups: a control group, a model group, and an SQC treatment group to rigorously evaluate the influence of SQC. Utilizing a comprehensive approach that encompassed methods such as pathological staining, western blot analysis, qRT-PCR, and RNA sequencing, we thoroughly investigated the therapeutic advantages and the underlying mechanistic pathways associated with SQC in the treatment of DVC. RESULT: The findings from this investigation have unveiled the extraordinary efficacy of SQC treatment in significantly mitigating DVC. The underlying mechanisms driving this effect encompass multifaceted facets, including the restoration of aberrant glucose and lipid metabolism, the prevention of phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteogenic-like states, the subsequent inhibition of cell apoptosis, the modulation of inflammation responses, the remodeling of the extracellular matrix (ECM), and the activation of the Hippo-YAP signaling pathway. Collectively, these mechanisms lead to the dissolution of deposited calcium salts, ultimately achieving the desired inhibition of DVC. CONCLUSION: Our study has provided compelling and robust evidence of the remarkable efficacy of SQC treatment in significantly reducing DVC. This reduction is attributed to a multifaceted interplay of mechanisms, each playing a crucial role in the observed therapeutic effects. Notably, our findings illuminate prospective directions for further research and potential clinical applications in the field of cardiovascular health.

Chrysanthemum coronarium L. Extract Attenuates Homocysteine-Induced Vascular Inflammation in Vascular Smooth Muscle Cells.

Hyperhomocysteinemia is a main risk factor for phenotypic modulation of vascular smooth muscle cells (VSMCs) and atherosclerosis. Phenotypic switching and proliferation of VSMCs are related to the progression of vascular inflammation. Chrysanthemum coronarium L. is a leafy vegetable with various biological functions, such as antioxidative, anti-inflammatory, and antiproliferative effects. In this study, we aimed to identify the mechanisms underlying the therapeutic and preventive effects of C. coronarium L. extract (CC) in regulating homocysteine (Hcy)-induced vascular inflammation in human aortic VSMCs. CC did not exhibit cytotoxicity and inhibited Hcy-stimulated VSMC proliferation and migration. In addition, CC promoted Hcy-induced expression of VSMC contractile phenotype proteins, including alpha-smooth muscle actin, calponin, and smooth muscle 22α. CC also decreased Hcy-induced accumulation of reactive oxygen species and expression of inflammatory markers nicotinamide adenine dinucleotide phosphate oxidase-4 and soluble epoxide hydrolase. These results showed that CC attenuates Hcy-induced inflammatory responses, highlighting its potential as a therapeutic or preventive target for Hcy-induced vascular inflammation.

Discovery of Salidroside as a Novel Non-Coding RNA Modulator to Delay Cellular Senescence and Promote BK-Dependent Apoptosis in Cerebrovascular Smooth Muscle Cells of Simulated Microgravity Rats.

Cardiovascular aging has been reported to accelerate in spaceflights, which is a great potential risk to astronauts' health and performance. However, current exercise routines are not sufficient to reverse the adverse effects of microgravity exposure. Recently, salidroside (SAL), a valuable medicinal herb, has been demonstrated to display an important role for prevention and treatment in cardiovascular and other diseases. In the present work, Sprague-Dawley rats with four-week tail-suspension hindlimb-unloading were used to simulate microgravity effects on the cardiovascular system. We found that intragastrical administration of SAL not only significantly decreased the expressions of senescence biomarkers, such as P65 and P16, but also obviously increased the expressions of BK-dependent apoptotic genes, including the large-conductance calcium-activated K+ channel (BK), Bax, Bcl-2, and cleaved caspase-3, in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In addition, relative non-coding RNAs were screened, and a luciferase assay identified that SAL increased apoptosis by activating LncRNA-FLORPAR, inhibiting miR-193, and then triggering the activity of the BK-α subunit. Our work indicated that SAL is a novel non-coding RNA modulator for regulating the LncRNA-FLORPAR sponging miR-193 pathway, which significantly promoted BK-dependent apoptosis and delayed cerebrovascular aging-like remodeling during simulated microgravity exposure. Our findings may provide a new approach to prevent cardiovascular aging in future spaceflights.

Astragaloside IV restrains pyroptosis and fibrotic development of pulmonary artery smooth muscle cells to ameliorate pulmonary artery hypertension through the PHD2/HIF1α signaling pathway.

BACKGROUND: Astragaloside (AS)-IV, extracted from traditional Chinese medicine Astragalus mongholicus, has been widely used in the anti-inflammatory treatment for cardiovascular disease. However, the mechanism by which AS-IV affects pulmonary artery hypertension (PAH) development remains largely unknown. METHODS: Monocrotaline (MCT)-induced PAH model rats were administered with AS-IV, and hematoxylin-eosin staining and Masson staining were performed to evaluate the histological change in pulmonary tissues of rats. Pulmonary artery smooth muscle cells (PASMCs) were treated by hypoxia and AS-IV. Pyroptosis and fibrosis were assessed by immunofluorescence, western blot and enzyme-linked immunosorbent assay. RESULTS: AS-IV treatment alleviated pulmonary artery structural remodeling and pulmonary hypertension progression induced by MCT in rats. AS-IV suppressed the expression of pyroptosis-related markers, the release of pro-inflammatory cytokine interleukin (IL)-1ß and IL-18 and fibrosis development in pulmonary tissues of PAH rats and in hypoxic PAMSCs. Interestingly, the expression of prolyl-4-hydroxylase 2 (PHD2) was restored by AS-IV administration in PAH model in vivo and in vitro, while hypoxia inducible factor 1α (HIF1α) was restrained by AS-IV. Mechanistically, silencing PHD2 reversed the inhibitory effect of AS-IV on pyroptosis, fibrosis trend and pyroptotic necrosis in hypoxia-cultured PASMCs, while the HIF1α inhibitor could prevent these PAH-like phenomena. CONCLUSION: Collectively, AS-IV elevates PHD2 expression to alleviate pyroptosis and fibrosis development during PAH through downregulating HIF1α. These findings may provide a better understanding of AS-IV preventing PAH, and the PHD2/HIF1α axis may be a potential anti-pyroptosis target during PAH.

Sodium-glucose cotransporter 2 inhibitor canagliflozin alleviates vascular calcification through suppression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome.

AIMS: Vascular calcification (VC) is prevalent in pathological processes such as diabetes, chronic kidney disease (CKD), and atherosclerosis, but effective therapies are still lacking by far. Canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor, has been approved for the treatment of type 2 diabetes mellitus and exhibits beneficial effects against cardiovascular disease. However, the effect of CANA on VC remains unknown. In this study, we hypothesize that CANA protects against VC. METHODS AND RESULTS: Micro-computed tomography analysis and alizarin red staining revealed that CANA treatment prevented aortic calcification in CKD rats and in VitD3-overloaded mice. Moreover, CANA alleviated the calcification of rat and human arterial rings. Alizarin red staining revealed that calcification of rat and human vascular smooth muscle cells (VSMCs) was attenuated by CANA treatment and this phenomenon was confirmed by calcium content assay. In addition, CANA downregulated the expression of osteogenic differentiation markers Runx2 and BMP2. Of interest, qPCR and western blot analysis revealed that CANA downregulated the expression of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), and the downstream signalling molecules Caspase-1 and IL-1ß in VSMCs as well. Both NLRP3 inhibitor MCC950 and knockdown of NLRP3 by siRNA independently resulted in decreased calcification of VSMCs. By contrast, activation of NLRP3 exacerbated VSMC calcification, and this effect was prevented by the addition of CANA. CONCLUSIONS: Our study for the first time demonstrates that CANA exerts a protective effect on VC at least partially via suppressing the NLRP3 signalling pathway. Therefore, supplementation of CANA as well as inhibition of NLRP3 inflammasome presents a potential therapy for VC.

Phosphorus May Induce Phenotypic Transdifferentiation of Vascular Smooth Muscle Cells through the Reduction of microRNA-145.

Phosphorus is a vital element for life found in most foods as a natural component, but it is also one of the most used preservatives added during food processing. High serum phosphorus contributes to develop vascular calcification in chronic kidney disease; however, it is not clear its effect in a population without kidney damage. The objective of this in vivo and in vitro study was to investigate the effect of high phosphorus exposure on the aortic and serum levels of miR-145 and its effect on vascular smooth muscle cell (VSMCs) changes towards less contractile phenotypes. The study was performed in aortas and serum from rats fed standard and high-phosphorus diets, and in VSMCs exposed to different concentrations of phosphorus. In addition, miR-145 silencing and overexpression experiments were carried out. In vivo results showed that in rats with normal renal function fed a high P diet, a significant increase in serum phosphorus was observed which was associated to a significant decrease in the aortic α-actin expression which paralleled the decrease in aortic and serum miR-145 levels, with no changes in the osteogenic markers. In vitro results using VSMCs corroborated the in vivo findings. High phosphorus first reduced miR-145, and afterwards α-actin expression. The miR-145 overexpression significantly increased α-actin expression and partially prevented the increase in calcium content. These results suggest that miR-145 could be an early biomarker of vascular calcification, which could give information about the initiation of the transdifferentiation process in VSMCs.

HMGB1-induced activation of ER stress contributes to pulmonary artery hypertension in vitro and in vivo.

BACKGROUND: HMGB1 and ER stress have been considered to participate in the progression of pulmonary artery hypertension (PAH). However, the molecular mechanism underlying HMGB1 and ER stress in PAH remains unclear. This study aims to explore whether HMGB1 induces pulmonary artery smooth muscle cells (PASMCs) functions and pulmonary artery remodeling through ER stress activation. METHODS: Primary cultured PASMCs and monocrotaline (MCT)-induced PAH rats were applied in this study. Cell proliferation and migration were determined by CCK-8, EdU and transwell assay. Western blotting was conducted to detect the protein levels of protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor-4 (ATF4), seven in absentia homolog 2 (SIAH2) and homeodomain interacting protein kinase 2 (HIPK2). Hemodynamic measurements, immunohistochemistry staining, hematoxylin and eosin staining were used to evaluate the development of PAH. The ultrastructure of ER was observed by transmission electron microscopy. RESULTS: In primary cultured PASMCs, HMGB1 reduced HIPK2 expression through upregulation of ER stress-related proteins (PERK and ATF4) and subsequently increased SIAH2 expression, which ultimately led to PASMC proliferation and migration. In MCT-induced PAH rats, interfering with HMGB1 by glycyrrhizin, suppression of ER stress by 4-phenylbutyric acid or targeting SIAH2 by vitamin K3 attenuated the development of PAH. Additionally, tetramethylpyrazine (TMP), as a component of traditional Chinese herbal medicine, reversed hemodynamic deterioration and vascular remodeling by targeting PERK/ATF4/SIAH2/HIPK2 axis. CONCLUSIONS: The present study provides a novel insight to understand the pathogenesis of PAH and suggests that targeting HMGB1/PERK/ATF4/SIAH2/HIPK2 cascade might have potential therapeutic value for the prevention and treatment of PAH.

Vascular protection afforded by zinc supplementation in human coronary artery smooth muscle cells mediated by NRF2 signaling under hypoxia/reoxygenation.

Zinc (Zn) has antioxidant, anti-inflammatory and anti-proliferative actions, with Zn dysregulation associated with coronary ischemia/reperfusion injury and smooth muscle cell dysfunction. As the majority of studies concerning Zn have been conducted under non-physiological hyperoxic conditions, we compare the effects of Zn chelation or supplementation on total intracellular Zn content, antioxidant NRF2 targeted gene transcription and hypoxia/reoxygenation-induced reactive oxygen species generation in human coronary artery smooth muscle cells (HCASMC) pre-adapted to hyperoxia (18 kPa O2) or normoxia (5 kPa O2). Expression of the smooth muscle marker SM22-α was unaffected by lowering pericellular O2, whereas calponin-1 was significantly upregulated in cells under 5 kPa O2, indicating a more physiological contractile phenotype under 5 kPa O2. Inductively coupled plasma mass spectrometry established that Zn supplementation (10 µM ZnCl2 + 0.5 µM pyrithione) significantly increased total Zn content in HCASMC under 18 but not 5 kPa O2. Zn supplementation increased metallothionein mRNA expression and NRF2 nuclear accumulation in cells under 18 or 5 kPa O2. Notably, NRF2 regulated HO-1 and NQO1 mRNA expression in response to Zn supplementation was only upregulated in cells under 18 but not 5 kPa. Furthermore, whilst hypoxia increased intracellular glutathione (GSH) in cells pre-adapted to 18 but not 5 kPa O2, reoxygenation had negligible effects on GSH or total Zn content. Reoxygenation-induced superoxide generation in cells under 18 kPa O2 was abrogated by PEG-superoxide dismutase but not by PEG-catalase, and Zn supplementation, but not Zn chelation, attenuated reoxygenation-induced superoxide generation in cells under 18 but not 5kPaO2, consistent with a lower redox stress under physiological normoxia. Our findings highlight that culture of HCASMC under physiological normoxia recapitulates an in vivo contractile phenotype and that effects of Zn on NRF2 signaling are altered by oxygen tension.

Betulinaldehyde inhibits vascular remodeling by regulating the microenvironment through the PLCγ1/Ca2+/MMP9 pathway.

BACKGROUND: Vascular remodeling plays a crucial role in the pathogenesis of several cardiovascular diseases (CVDs). Unfortunately, current drug therapies offer limited relief for vascular remodeling. Therefore, the development of innovative therapeutic strategies or drugs that target vascular remodeling is imperative. Betulinaldehyde (BA) is a triterpenoid with diverse biological activities, but its effects on vascular remodeling remain unclear. OBJECTIVE: This study aimed to investigate the role of BA in vascular remodeling and its mechanism of action, providing valuable information for future applications of BA in the treatment of CVDs. METHODS: Network pharmacology was used to predict the key targets of BA in vascular remodeling. The effect of BA on vascular remodeling was assessed in a rat model of balloon injury using hematoxylin and eosin staining, Masson staining, immunohistochemistry staining, and Western blotting. A phenotypic transformation model of vascular smooth muscle cells (VSMCs) was induced by platelet-derived growth factor-BB, and the functional impacts of BA on VSMCs were assessed via CCK-8, EdU, Wound healing, Transwell, and Western blotting. Finally, after manipulation of phospholipase C gamma1 (PLCγ1) expression, Western blotting and Ca2+ levels determination were performed to investigate the potential mechanism of action of BA. RESULTS: The most key target of BA in vascular remodeling, matrix metalloproteinase 9 (MMP9), was identified through network pharmacology screening. Vascular remodeling was alleviated by BA in vivo and its effects were associated with decreased MMP9 expression. In vitro studies indicated that BA inhibited VSMC proliferation, migration, phenotypic transformation, and downregulated MMP9 expression. Additionally, BA decreased PLCγ1 expression and Ca2+ levels in VSMCs. However, after pretreatment with a phospholipase C agonist, BA's effects on down-regulating the expression of PLCγ1 and Ca2+ levels were inhibited, while the expression of MMP9 increased compared to that in the BA treatment group. CONCLUSION: This study demonstrated the critical role of BA in vascular remodeling. These findings revealed a novel mechanism whereby BA mediates its protective effects through MMP9 regulation by inhibiting the PLCγ1/Ca2+/MMP9 signaling pathway. Overall, BA may potentially be developed into a novel medication for CVDs and may serve as a promising therapeutic strategy for improving recovery from CVDs by targeting MMP9.