Mouse Aortic Smooth Muscle Cells Research Articles

Polystyrene nanoplastics accelerate atherosclerosis: Unraveling the impact on smooth muscle cells through KIF15-mediated migration

Microplastics and nanoplastics (MNPs) originating from plastic pollution pose potential threats to cardiovascular health, with prior studies linking MNPs to atherosclerosis. Our earlier research elucidated how nanoplastics enhance macrophages' phagocytic activity, leading to the formation of foam cells and an elevated risk of atherosclerosis. However, the specific influence of MNPs on smooth muscle cells (SMCs) in the context of MNP-induced atherosclerosis remains poorly understood. In this study, ApoE knockout (ApoE-/-) male mice with a high-fat diet were orally exposed to environmentally realistic concentrations of 2.5–250 mg/kg polystyrene nanoplastics (PS-NPs, 50 nm) for consecutive 19 weeks. Cardiovascular toxicity was comprehensively assessed through histopathological, transcriptomic, and proteomic analyses, while mechanisms underlying this toxicity were explored through in vitro studies. Herein, hematoxylin and eosin staining revealed accelerated atherosclerotic plaque development in ApoE-/- mice exposed to PS-NPs. Multi-omics analysis identified kinesin family member 15 (KIF15) as a pivotal target molecule. Both in vitro and in vivo experiments affirmed the specific upregulation of KIF15 in mouse aortic SMCs exposed to PS-NPs. Furthermore, in vitro experiments demonstrated that PS-NPs can promote the migration ability of MOVAS cells. Knockdown of Kif15 revealed its role in reducing MOVAS cell migration, with subsequent exposure to PS-NPs reversing the increased migration ability. This suggests that PS-NPs promote SMC migration by upregulating KIF15, and the migration of SMCs is closely associated with atherosclerosis outcomes. This study significantly advances our understanding of MNP-induced cardiovascular toxicity, providing valuable insights for risk assessment of human MNP exposure.

Quercetin Attenuates KLF4-Mediated Phenotypic Switch of VSMCs to Macrophage-like Cells in Atherosclerosis: A Critical Role for the JAK2/STAT3 Pathway.

The objective of this study was to elucidate the protective role of quercetin in atherosclerosis by examining its effect on the phenotypic switch of vascular smooth muscle cells (VSMCs) to macrophage-like cells and the underlying regulatory pathways. Aorta tissues from apolipoprotein E-deficient (ApoE KO) mice fed a high-fat diet (HFD), treated with or without 100 mg/kg/day quercetin, were analyzed for histopathological changes and molecular mechanisms. Quercetin was found to decrease the size of atherosclerotic lesions and mitigate lipid accumulation induced by HFD. Fluorescence co-localization analysis revealed a higher presence of macrophage-like vascular smooth muscle cells (VSMCs) co-localizing with phospho-Janus kinase 2 (p-JAK2), phospho-signal transducer and activator of transcription 3 (p-STAT3), and Krüppel-like factor 4 (KLF4) in regions of foam cell aggregation within aortic plaques. However, this co-localization was reduced following treatment with quercetin. Quercetin treatment effectively inhibited the KLF4-mediated phenotypic switch in oxidized low-density lipoprotein (ox-LDL)-loaded mouse aortic vascular smooth muscle cells (MOVAS), as indicated by decreased expressions of KLF4, LGALS3, CD68, and F4/80, increased expression of alpha smooth muscle actin (α-SMA), reduced intracellular fluorescence Dil-ox-LDL uptake, and decreased lipid accumulation. In contrast, APTO-253, a KLF4 activator, was found to reverse the effects of quercetin. Furthermore, AG490, a JAK2 inhibitor, effectively counteracted the ox-LDL-induced JAK2/STAT3 pathway-dependent switch to a macrophage-like phenotype and lipid accumulation in MOVAS cells. These effects were significantly mitigated by quercetin but exacerbated by coumermycin A1, a JAK2 activator. Our research illustrates that quercetin inhibits the KLF4-mediated phenotypic switch of VSMCs to macrophage-like cells and reduces atherosclerosis by suppressing the JAK2/STAT3 pathway.

Ligustrazine alleviates the progression of coronary artery calcification by inhibiting caspase-3/GSDME mediated pyroptosis.

Coronary artery calcification (CAC) is an early marker for atherosclerosis and is mainly induced by the osteoblast-like phenotype conversion of vascular smooth muscle cells (VSMCs). Recent reports indicate that NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis plays a significant role in the calcification of vascular smooth muscle cells (VSMCs), making it a promising target for treating calcific aortic valve disease (CAC). Ligustrazine, or tetramethylpyrazine (TMP), has been found effective in various cardiovascular and cerebrovascular diseases and is suggested to inhibit NLRP3-mediated pyroptosis. However, the function of TMP in CAC is unknown. Herein, influences of TMP on β-glycerophosphate (β-GP)-stimulated VSMCs and OPG-/- mice were explored. Mouse Aortic Vascular Smooth Muscle (MOVAS-1) cells were stimulated by β-GP with si- caspase-3, si- Gasdermin E (GSDME) or TMP. Increased calcification, reactive oxygen species (ROS) level, Interleukin-1beta (IL-1β) and Interleukin-18 (IL-18) levels, lactate dehydrogenase (LDH) release, enhanced apoptosis, and activated cysteine-aspartic acid protease-3 (caspase-3)/GSDME signaling were observed in β-GP-stimulated MOVAS-1 cells, which was sharply alleviated by si-caspase-3, si-GSDME or TMP. Furthermore, the impact of TMP on the β-GP-induced calcification and injury in MOVAS-1 cells was abolished by raptinal, an activator of caspase-3. Subsequently, OPG-/- mice were dosed with TMP or TMP combined with raptinal. Calcium deposition, increased nodules, elevated IL-1β and IL-18 levels, upregulated CASP3 and actin alpha 2, smooth muscle (ACTA2), and activated caspase-3/GSDME signaling in OPG-/- mice were markedly alleviated by TMP, which were notably reversed by the co-administration of raptinal. Collectively, TMP mitigated CAC by inhibiting caspase-3/GSDME mediated pyroptosis.

High estrogen induces trans-differentiation of vascular smooth muscle cells to a macrophage-like phenotype resulting in aortic inflammation via inhibiting VHL/HIF1a/KLF4 axis.

Estrogen is thought to have a role in slowing down aging and protecting cardiovascular and cognitive function. However, high doses of estrogen are still positively associated with autoimmune diseases and tumors with systemic inflammation. First, we administered exogenous estrogen to female mice for three consecutive months and found that the aorta of mice on estrogen develops inflammatory manifestations similar to Takayasu arteritis (TAK). Then, in vitro estrogen intervention was performed on mouse aortic vascular smooth muscle cells (MOVAS cells). Stimulated by high concentrations of estradiol, MOVAS cells showed decreased expression of contractile phenotypic markers and increased expression of macrophage-like phenotypic markers. This shift was blocked by tamoxifen and Krüppel-like factor 4 (KLF4) inhibitors and enhanced by Von Hippel-Lindau (VHL)/hypoxia-inducible factor-1α (HIF-1α) interaction inhibitors. It suggests that estrogen-targeted regulation of the VHL/HIF-1α/KLF4 axis induces phenotypic transformation of vascular smooth muscle cells (VSMC). In addition, estrogen-regulated phenotypic conversion of VSMC to macrophages is a key mechanism of estrogen-induced vascular inflammation, which justifies the risk of clinical use of estrogen replacement therapy.

Abstract 2141: An IL12/IL23 Positive Feedback Loop Modulates Smooth Muscle Differentiation Via Setd4 During Macrophage-mediated Inflammation

Diabetic patients have increased risk of vascular disease (e.g., atherosclerosis), which is driven by macrophage-mediated inflammation. In diseases such as atherosclerosis, smooth muscle cells (SMC) undergo phenotypic modulation from a mature, contractile to a synthetic phenotype, thus leading to increased neointima formation. We hypothesize that pro-inflammatory macrophages as in the setting of diabetes epigenetically pattern SMC during vascular disease. In this study, we use single cell-RNA sequencing (scRNA-seq) of human arteries, loss of function experiments, and mouse models of disease to identify epigenetic mechanisms that regulate SMC phenotype. We identified the H3K4 and H3K9 histone lysine methyltransferase Setd4, which was increased in aortic SMC from diabetic mice and in our scRNA-seq dataset from human atherosclerotic arteries. siRNA mediated knockdown of Setd4 in mouse aortic SMC increased expression of the early SMC differentiation markers, Acta2 and Tagln (p<0.05). To model the pro-inflammatory environment in vitro, supernatant from LPS-stimulated macrophages increased Setd4 expression and inhibited smooth muscle gene expression ( Acta2, Tagln, Cnn1, Myh11 ), which was reversed by siRNA-mediated knockdown of Setd4 in SMC (p<0.05). IL23 increased Setd4 expression in SMC and enrichment at smooth muscle gene promoters, while deceasing SMC-specific gene expression (p<0.05). Treatment of SMC with an IL23 receptor neutralizing antibody prevented downregulation of SMC gene expression by macrophage supernatant (p<0.05). Intriguingly, ELISA of mouse aortic SMC supernatant revealed that IL23 increased SMC secretion of IL12, which also inhibited SMC gene expression via Setd4 (p<0.05). In conclusion, we demonstrate that pro-inflammatory macrophage IL23 represses smooth muscle gene expression via SETD4, thereby shifting the SMC to a synthetic, diseased phenotype. IL23 also stimulates vascular SMC secretion of IL12, which further increases production of inflammatory cytokines from macrophages and leads to additional repression of smooth muscle gene expression. Thus, targeting the IL23-SETD4 axis in SMC may be a potential therapy for the treatment of atherosclerosis, especially in patients who also have diabetes.

Functional profiling of resident cells during vascular aging

Introduction: Depending on surrounding cell types, substrate mechanics, and chemical agonists, vascular cell types – including endothelial (ECs) and smooth muscle cells (VSMC) — modulate their behavior to maintain homeostasis. Aging perturbs this homeostasis resulting in EC and SMC dysfunction, marked by impaired barrier integrity and pathological ECM remodeling. Endothelial-to-mesenchymal transition (EndMT) in ECs and “dedifferentiation” of VSMCs towards a synthetic phenotype are phenomenon under investigation as cellular drivers of the widespread dysregulation seen with aging. The overall goal of this project is to understand the effect of cross-talk between ECM and growth factor signaling on vascular cell plasticity during aging. Materials and Methods: Human aortic endothelial cells (HAECs) were seeded onto transwell inserts after seven days treatment with or without TGF-β. After growing to confluence on the insert, cells were subjected to a FITC-dextran transwell assay. A parallel group of cells was seeded onto fibronectin coated glass-bottom dishes and stained for the expression of focal adhesion proteins and cell-type markers. Mouse aortic smooth muscle cells (maSMCs) were isolated from C57Bl6 mice at 3 (young), 6 (early middle-aged; EMA), 9 (middle-age; MA), or greater than 18 months of age (old). Cells were seeded onto dye-quenched (DQ) collagen type I (Col-I) substrate before gain of fluorescence was measured in proximity to the cell as well as in the surrounding media. A parallel group of cells were allowed to grow in media containing FITC Col-I, and gain of fluorescence was measured in proximity to the cells. Each cell type (3M, 6M, 9M, and 18M) in both experiments (DQ and FITC) were further treated with MMP inhibitor, TG2 inhibitor and TGF-β1 to measure their impacts on Col-I turnover. Results: Barrier function was not impaired in ECs cultured with TGF-β1, relative to their untreated counterparts. Mouse aortic SMCs isolated from older mice had decreased rates of Col-I cleavage and were more susceptible to MMP inhibition than their younger counterparts. Conclusions: maSMCs from older mice have lower Col-I turnover than their younger counterparts. This is consistent with bulk tissue behavior, as it is well known that relative collagen levels increase in the elastic vasculature as we age. TGF-β1 treatment did not have an effect on permeability of 4kd FITC-dextran, it is feasible that there will be an impact on the larger 70kd fragments. Travis Brady is supported by the NASEM Ford Foundation Pre-doctoral Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1054: Toll-like Receptor 4 Is A New Regulator Of Smooth Muscle Cell Phenotypic Transitions

Objectives: There is growing evidence that smooth muscle cell ( SMC ) phenotypic transitions have a critical role in cardiovascular pathologies, including atherosclerosis. However, molecular mechanisms regulating different aspects of these transitions are still not fully elucidated. We previously found that the pluripotency factor OCT4 plays a protective role in SMC in atherogenesis and neointioma formation after vascular injury. Using a bioinformatics approach, we identified Toll-like receptor 4 ( TLR4 ), an integral component of the innate immune system, pivotal in eliciting inflammatory responses, as an indirect target of OCT4 in SMC. However, it is not yet elucidated whether OCT4/TLR4 axis has a role in SMC transitions. Methods and Results: First, we used blocking siRNA against Oct4 and different downstream targets of the TLR4 signaling ( Trif , Irf3 ), as well as a TLR4 inhibitor TAK242 (Resatorvid) in cultured mouse aortic SMC treated with the oxidized phospholipid, POVPC. These experiments demonstrated that blocking TLR4 signaling in wild-type SMC resulted in suppression of SMC contractile genes and up-regulation of LGALS3 (SMC transition marker). In contrast, TAK242 treatment decreased LGALS3 levels in OCT4 deficient SMC, indicating that TLR4, at least in part, is responsible for the SMC phenotype in OCT4 deficient SMC. Next, using the bioinformatics approach and Chromatin Immunoprecipitation assays, we identified the EST Proto-oncogene 1 ( ETS1 ) transcriptional factor as a target of OCT4 that directly regulates TLR4. Interestingly, on the other hand, we found that overexpression of TLR4 led to OCT4 activation in SMC, and, oppositely, the inhibition of TLR4 signaling by TAK242 resulted in significant decreases in the POVPC-induced OCT4 activation, indicating that TLR4 signaling is critical for OCT4 activation in SMC. Using an SMC-lineage tracing mouse model combined with a TLR4-IRES-YFP reporter, we demonstrated that TLR4 is highly expressed in the healthy arteries of young mice but not in the phenotypically modulated SMC within the atherosclerotic plaques. Conclusions: These results suggest a negative feedback loop between TLR4 and OCT4 regulating SMC phenotypic transitions.

Integrative analysis of the lncRNA-miRNA-mRNA interactions in smooth muscle cell phenotypic transitions.

Objectives: We previously found that the pluripotency factor OCT4 is reactivated in smooth muscle cells (SMC) in human and mouse atherosclerotic plaques and plays an atheroprotective role. Loss of OCT4 in SMC in vitro was associated with decreases in SMC migration. However, molecular mechanisms responsible for atheroprotective SMC-OCT4-dependent effects remain unknown. Methods: Since studies in embryonic stem cells demonstrated that OCT4 regulates long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), making them candidates for OCT4 effect mediators, we applied an in vitro approach to investigate the interactions between OCT4-regulated lncRNAs, mRNAs, and miRNAs in SMC. We used OCT4 deficient mouse aortic SMC (MASMC) treated with the pro-atherogenic oxidized phospholipid POVPC, which, as we previously demonstrated, suppresses SMC contractile markers and induces SMC migration. Differential expression of lncRNAs, mRNAs, and miRNAs was obtained by lncRNA/mRNA expression array and small-RNA microarray. Long non-coding RNA to mRNA associations were predicted based on their genomic proximity and association with vascular diseases. Given a recently discovered crosstalk between miRNA and lncRNA, we also investigated the association of miRNAs with upregulated/downregulated lncRNA-mRNA pairs. Results: POVPC treatment in SMC resulted in upregulating genes related to the axon guidance and focal adhesion pathways. Knockdown of Oct4 resulted in differential regulation of pathways associated with phagocytosis. Importantly, these results were consistent with our data showing that OCT4 deficiency attenuated POVPC-induced SMC migration and led to increased phagocytosis. Next, we identified several up- or downregulated lncRNA associated with upregulation of the specific mRNA unique for the OCT4 deficient SMC, including upregulation of ENSMUST00000140952-Hoxb5/6 and ENSMUST00000155531-Zfp652 along with downregulation of ENSMUST00000173605-Parp9 and, ENSMUST00000137236-Zmym1. Finally, we found that many of the downregulated miRNAs were associated with cell migration, including miR-196a-1 and miR-10a, targets of upregulated ENSMUST00000140952, and miR-155 and miR-122, targets of upregulated ENSMUST00000155531. Oppositely, the upregulated miRNAs were anti-migratory and pro-phagocytic, such as miR-10a/b and miR-15a/b, targets of downregulated ENSMUST00000173605, and miR-146a/b and miR-15b targets of ENSMUST00000137236. Conclusion: Our integrative analyses of the lncRNA-miRNA-mRNA interactions in SMC indicated novel potential OCT4-dependent mechanisms that may play a role in SMC phenotypic transitions.

ALAS2 overexpression alleviates oxidative stress-induced ferroptosis in aortic aneurysms via GATA1 activation.

Aortic aneurysm, characterized by abnormal dilation of the aorta, poses significant health risks. This study aims to investigate the interaction between 5-aminolevulinate synthase 2 (ALAS2) and GATA-binding protein 1 (GATA1) in ferroptosis and oxidative stress responses in aortic aneurysm. A weighted gene co-expression network analysis (WGCNA) was performed on the differentially expressed genes (DEGs) within the GSE9106 dataset to identify the key module. Subsequently, protein-protein interaction (PPI) network analysis was performed on the key module. Mouse aortic vascular smooth muscle cells (MOVAS) were treated with hydrogen peroxide (H2O2) to induce oxidative stress, and ferroptosis inducers and inhibitors were added to evaluate their effects on iron content and oxidative stress markers. Through a series of in vitro cellular experiments, we assessed cell viability, expression levels of GATA1 and iron mutation-associated proteins, as well as cellular phenotypes such as inflammatory responses and apoptosis rates. Three candidate genes (ALAS2, GYPA, and GYPB) were upregulated in the thoracic aortic aneurysm (TAA) samples of the GSE9106 dataset. The H2O2 treatment increased the MOVAS cells' iron content and oxidative stress, upregulated ALAS2 protein levels, and decreased the ferroptosis-related protein levels. ALAS2 overexpression reversed H2O2-induced apoptosis and increased the inflammatory cytokine levels. Additionally, the knockdown of GATA1 partially reversed the protective mechanism of overexpressed ALAS2 on H2O2-induced ferroptosis. ALAS2 overexpression reduced H2O2-induced oxidative damage and iron-induced apoptosis in MOVAS cells, and GATA1 knockdown partially reversed this protective effect. These findings suggested that the ALAS2 and GATA1 regulatory pathways may be potential therapeutic targets in aortic aneurysms.

Huanglian Jiedu decoction inhibits vascular smooth muscle cell-derived foam cell formation by activating autophagy via suppressing P2RY12

Ethnopharmacological relevanceHuanglian Jiedu Decoction (HLJDD) is a Chinese medicine with a long history of therapeutic application. It is widely used in treating atherosclerosis (AS) in Chinese medicine theory and clinical practice. However, the mechanism of HLJDD in treating AS remains unclear. Aim of the studyTo investigate the efficacy and mechanism of HLJDD in treating AS. Materials and methodsAS was induced on high-fat diet-fed ApoE−/− mice, with the aorta pathological changes evaluated with lipid content and plaque progression. In vitro, foam cells were induced by subjecting primary mouse aortic vascular smooth muscle cells (VSMCs) to oxLDL incubation. After HLJDD intervention, VSMCs were assessed with lipid stack, apoptosis, oxidative stress, and the expression of foam cell markers. The effects of P2RY12 were tested by adopting clopidogrel hydrogen sulfate (CDL) in vivo and transfecting P2RY12 over-expressive plasmid in vitro. Autophagy was inhibited by Chloroquine or transfecting siRNA targeting ATG7 (siATG7). The mechanism of HLJDD treating atherosclerosis was explored using network pharmacology and validated with molecular docking and co-immunoprecipitation. ResultsHLJDD exhibited a dose-dependent reduction in lipid deposition, collagen loss, and necrosis within plaques. It also reversed lipid accumulation and down-regulated the expression of foam cell markers. P2RY12 inhibition alleviated AS, while P2RY12 overexpression enhanced foam cell formation and blocked the therapeutic effects of HLJDD. Network pharmacological analysis suggested that HLJDD might mediate PI3K/AKT signaling pathway-induced autophagy. P2RY12 overexpression also impaired autophagy. Similarly, inhibiting autophagy counteracted the effect of CDL, exacerbated AS in vivo, and promoted foam cell formation in vitro. However, HLJDD treatment mitigated these detrimental effects by suppressing the PI3K/AKT signaling pathway. Immunofluorescence and molecular docking revealed a high affinity between P2RY12 and PIK3CB, while co-immunoprecipitation assays illustrated their interaction. ConclusionsHLJDD inhibited AS in vivo and foam cell formation in vitro by restoring P2RY12/PI3K/AKT signaling pathway-suppressed autophagy. This study is the first to reveal an interaction between P2RY12 and PI3K3CB.

Primary Mouse Aortic Smooth Muscle Cells Exhibit Region- and Sex-Dependent Biological Responses In Vitro.

Despite advancements in elucidating biological mechanisms of cardiovascular remodeling, cardiovascular disease (CVD) remains the leading cause of death worldwide. When stratified by sex, clear differences in CVD prevalence and mortality between males and females emerge. Regional differences in phenotype and biological response of cardiovascular cells are important for localizing the initiation and progression of CVD. Thus, to better understand region and sex differences in CVD presentation, we have focused on characterizing in vitro behaviors of primary vascular smooth muscle cells (VSMCs) from the thoracic and abdominal aorta of male and female mice. VSMC contractility was assessed by traction force microscopy (TFM; single cell) and collagen gel contraction (collective) with and without stimulation by transforming growth factor-beta 1 (TGF-β1) and cell proliferation was assessed by a colorimetric metabolic assay (MTT). Gene expression and TFM analysis revealed region- and sex-dependent behaviors, whereas collagen gel contraction was consistent across sex and aortic region under baseline conditions. Thoracic VSMCs showed a sex-dependent sensitivity to TGF-β1-induced collagen gel contraction (female > male; p = 0.025) and a sex-dependent proliferative response (female > male; p < 0.001) that was not apparent in abdominal VSMCs. Although primary VSMCs exhibit intrinsic region and sex differences in biological responses that may be relevant for CVD presentation, several factors-such as inflammation and sex hormones-were not included in this study. Such factors should be included in future studies of in vitro mechanobiological responses relevant to CVD differences in males and females.

Carbonylation of Runx2 at K176 by 4-Hydroxynonenal Accelerates Vascular Calcification.

Vascular calcification, which is characterized by calcium deposition in arterial walls and the osteochondrogenic differentiation of vascular smooth muscle cells, is an actively regulated process that involves complex mechanisms. Vascular calcification is associated with increased cardiovascular adverse events. The role of 4-hydroxynonenal (4-HNE), which is the most abundant stable product of lipid peroxidation, in vascular calcification has been poorly investigated. Serum was collected from patients with chronic kidney disease and controls, and the levels of 4-HNE and 8-iso-prostaglandin F2α were measured. Sections of coronary atherosclerotic plaques from donors were immunostained to analyze calcium deposition and 4-HNE. A total of 658 patients with coronary artery disease who received coronary computed tomography angiography were recruited to analyze the relationship between coronary calcification and the rs671 mutation in aldehyde dehydrogenase 2 (ALDH2). ALDH2 knockout (ALDH2-/-) mice, smooth muscle cell-specific ALDH2 knockout mice, ALDH2 transgenic mice, and their controls were used to establish vascular calcification models. Primary mouse aortic smooth muscle cells and human aortic smooth muscle cells were exposed to medium containing β-glycerophosphate and CaCl2 to investigate cell calcification and the underlying molecular mechanisms. Elevated 4-HNE levels were observed in the serum of patients with chronic kidney disease and model mice and were detected in calcified artery sections by immunostaining. ALDH2 knockout or smooth muscle cell-specific ALDH2 knockout accelerated the development of vascular calcification in model mice, whereas overexpression or activation prevented mouse vascular calcification and the osteochondrogenic differentiation of vascular smooth muscle cells. In patients with coronary artery disease, patients with ALDH2 rs671 gene mutation developed more severe coronary calcification. 4-HNE promoted calcification of both mouse aortic smooth muscle cells and human aortic smooth muscle cells and their osteochondrogenic differentiation in vitro. 4-HNE increased the level of Runx2 (runt-related transcription factor-2), and the effect of 4-HNE on promoting vascular smooth muscle cell calcification was ablated when Runx2 was knocked down. Mutation of Runx2 at lysine 176 reduced its carbonylation and eliminated the 4-HNE-induced upregulation of Runx2. Our results suggest that 4-HNE increases Runx2 stabilization by directly carbonylating its K176 site and promotes vascular calcification. ALDH2 might be a potential target for the treatment of vascular calcification.

Empagliflozin ameliorates vascular calcification in diabetic mice through inhibiting Bhlhe40-dependent NLRP3 inflammasome activation.

Type 2 diabetes mellitus (T2DM) patients exhibit greater susceptibility to vascular calcification (VC), which has a higher risk of death and disability. However, there is no specific drug for VC therapy. NLRP3 inflammasome activation as a hallmark event of medial calcification leads to arterial stiffness, causing vasoconstrictive dysfunction in T2DM. Empagliflozin (EMPA), a sodium-glucose co-transporter 2 inhibitor (SGLT2i), restrains hyperglycemia with definite cardiovascular benefits. Given the anti-inflammatory activity of EMPA, herein we investigated whether EMPA protected against VC in the aorta of T2DM mice by inhibiting NLRP3 inflammasome activation. Since db/db mice receiving a normal diet developed VC at the age of about 20 weeks, we administered EMPA (5, 10, 20 mg·kg-1·d-1, i.g) to 8 week-old db/db mice for 12 weeks. We showed that EMPA intervention dose-dependently ameliorated the calcium deposition, accompanied by reduced expression of RUNX2 and BMP2 proteins in the aortas. We found that EMPA (10 mg·kg-1·d-1 for 6 weeks) also protected against VC in vitamin D3-overloaded mice, suggesting the protective effects independent of metabolism. We showed that EMPA (10 mg·kg-1·d-1) inhibited the abnormal activation of NLRP3 inflammasome in aortic smooth muscle layer of db/db mice. Knockout (KO) of NLRP3 significantly alleviated VC in STZ-induced diabetic mice. The protective effects of EMPA were verified in high glucose (HG)-treated mouse aortic smooth muscle cells (MOVASs). In HG-treated NLRP3 KO MOVASs, EMPA (1 μM) did not cause further improvement. Bioinformatics and Western blot analysis revealed that EMPA significantly increased the expression levels of basic helix-loop-helix family transcription factor e40 (Bhlhe40) in HG-treated MOVASs, which served as a negative transcription factor directly binding to the promotor of Nlrp3. We conclude that EMPA ameliorates VC by inhibiting Bhlhe40-dpendent NLRP3 inflammasome activation. These results might provide potential significance for EMPA in VC therapy of T2DM patients.

TRIM65 promotes vascular smooth muscle cell phenotypic transformation by activating PI3K/Akt/mTOR signaling during atherogenesis

Background and aimsTripartite motif (TRIM65) is an important member of the TRIM protein family, which is a newly discovered E3 ligase that interacts with and ubiquitinates various substrates and is involved in diverse pathological processes. However, the function of TRIM65 in atherosclerosis remains unarticulated. In this study, we investigated the role of TRIM65 in the pathogenesis of atherosclerosis, specifically in vascular smooth muscle cells (VSMCs) phenotype transformation, which plays a crucial role in formation of atherosclerotic lesions. Methods and ResultsBoth non-atherosclerotic and atherosclerotic lesions during autopsy were collected singly or pairwise from each individual (n = 16) to investigate the relationship between TRIM65 and the development of atherosclerosis. In vivo, Western diet-fed ApoE−/− mice overexpressing or lacking TRIM65 were used to assess the physiological function of TRIM65 on VSMCs phenotype, proliferation and atherosclerotic lesion formation. In vitro, VSMCs phenotypic transformation was induced by platelet-derived growth factor-BB (PDGF-BB). TRIM65-overexpressing or TRIM65-abrogated primary mouse aortic smooth muscle cells (MOASMCs) and human aortic smooth muscle cells (HASMCs) were used to investigate the mechanisms underlying the progression of VSMCs phenotypic transformation, proliferation and migration. Increased TRIM65 expression was detected in α-SMA-positive cells in the medial and atherosclerotic lesions of autopsy specimens. TRIM65 overexpression increased, whereas genetic knockdown of TRIM65 remarkably inhibited, atherosclerotic plaque development. Mechanistically, TRIM65 overexpression activated PI3K/Akt/mTOR signaling, resulting in the loss of the VSMCs contractile phenotype, including calponin, α-SMA, and SM22α, as well as cell proliferation and migration. However, opposite phenomena were observed when TRIM65 was deficient in vivo or in vitro. Moreover, in cultured PDGF-BB-induced TRIM65-overexpressing VSMCs, inhibition of PI3K by treatment with the inhibitor LY-294002 for 24 h markedly attenuated PI3K/Akt/mTOR activation, regained the VSMCs contractile phenotype, and blocked the progression of cell proliferation and migration. ConclusionsTRIM65 overexpression enhances atherosclerosis development by promoting phenotypic transformation of VSMCs from contractile to synthetic state through activation of the PI3K/Akt/mTOR signal pathway.

Macrophage-mediated downregulation of lncRNA Carmn in mouse abdominal aortic aneurysm

The long noncoding RNA (lncRNA) CARMN (cardiac mesoderm enhancer associated noncoding RNA) is a highly conserved lncRNA that expresses primarily by smooth muscle cells (SMCs). Recent literature demonstrates that CARMN plays a critical role in the differentiation and maintaining of the contractile state of vascular SMCs. Because aortic SMCs show diminished contractile proteins in abdominal aortic aneurysms (AAAs), we hypothesize that the expression of CARMN is downregulated in the aortic wall affected by aneurysm. In this study, we analyzed publicly available single-cell or bulk RNA sequencing data comparing healthy and aneurysmal mouse aortic tissues. In both healthy and diseased aortas, Carmn expression was enriched in SMCs characterized by the high expression of SMC-specific contractile proteins including Myh11 and Acta2. Carmn expression levels varied among the sub-clusters of SMCs and consequently along the aortic tree. Comparing to the corresponding sham aorta, aortas from 3 distinct AAA models contained less Carmn. To validate the Carmn downregulation, we induced AAA using the Angiotensin II and CaCl2 models. In situ hybridization showed that Carmn mRNA located in the nuclei of SMCs and became downregulated within a few days following the aneurysm induction. Mechanistically, we tested whether Carmn expression is regulated by infiltrating macrophages --- the predominant inflammatory cells found in aneurysmal tissues --- by treating healthy mouse aortic SMCs with media conditioned by macrophages primed with pro-inflammatory or anti-inflammatory cytokines. PCR analysis showed that inflammatory macrophages reduced the expression of Carmn and contractile genes including Myh11 and Acta2. Taken together, our results from bioinformatic and experimental analyses demonstrate that Carmn is downregulated in different AAA models, likely by inflammatory macrophages. The negative regulation of Carmn in AAA tissues may explain at least in part the loss of SMC contractile state during the pathogenesis of this progressive degenerative disease.

L-type calcium ion channel-mediated activation of autophagy in vascular smooth muscle cells via thonningianin A (TA) alleviates vascular calcification in type 2 diabetes mellitus

Vascular calcification (VC) is associated with increased morbidity and mortality, especially among people with type 2 diabetes mellitus (T2DM). The pathogenesis of vascular calcification is incompletely understood, and until now, there have been no effective therapeutics for vascular calcification. The L-type calcium ion channel in the cell membrane is vital for Ca2+ influx. The effect of L-type calcium ion channels on autophagy remains to be elucidated. Here, the natural compound thonningianin A (TA) was found to ameliorate vascular calcification in T2DM via the activation of L-type calcium ion channels. The results showed that TA had a concentration-dependent ability to decrease the transcriptional and translational expression of the calcification-related proteins runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteopontin (OPN) (P < 0.01) via ATG7-dependent autophagy in β-glycerophosphate (β-GP)- and high glucose (HG)-stimulated primary mouse aortic smooth muscle cells (MASMCs) and alleviate aortic vascular calcification in VitD3-stimulated T2DM mice. However, nifedipine, an inhibitor of L-type calcium ion channels, reversed TA-induced autophagy and Ca2+ influx in MASMCs. Molecular docking analysis revealed that TA was located in the hydrophobic pocket of Cav1.2 α1C and was mainly composed of the residues Ile, Phe, Ala and Met, which confirmed the efficacy of TA in targeting the L-type calcium channel of Cav1.2 on the cell membrane. Moreover, in an in vivo model of vascular calcification in T2DM mice, nifedipine reversed the protective effects of TA on aortic calcification and the expression of the calcification-related proteins RUNX2, BMP2 and OPN (P < 0.01). Collectively, the present results reveal that the activation of cell membrane L-type calcium ion channels can induce autophagy and ameliorate vascular calcification in T2DM. Thonningianin A (TA) can target and act as a potent activator of L-type calcium ion channels. Thus, this research revealed a novel mechanism for autophagy induction via L-type calcium ion channels and provided a potential therapeutic for vascular calcification in T2DM.

Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization.

Cell migration is essential for many biological and pathological processes. Establishing cell polarity with a trailing edge and forming a single lamellipodium at the leading edge of the cell is crucial for efficient directional cell migration and is a hallmark of mesenchymal cell motility. Lamellipodia formation is regulated by spatial-temporal activation of the small GTPases Rac and Cdc42at the front edge, and RhoA at the rear end. At a molecular level, partitioning-defective (Par) protein complex comprising Par3, Par6, and atypical Protein Kinase (aPKC isoforms ζ and λ/ι) regulates front-rear axis polarization. At the front edge, integrin clustering activates Cdc42, prompting the formation of Par3/Par6/aPKC complexes to modulate MTOC positioning and microtubule stabilization. Consequently, the Par3/Par6/aPKC complex recruits Rac1-GEF Tiam to activate Rac1, leading to lamellipodium formation. At the rear end, RhoA-ROCK phosphorylates Par3 disrupting its interaction with Tiam and inactivating Rac1. RhoA activity at the rear end allows the formation of focal adhesions and stress fibers necessary to generate the traction forces that allow cell movement. Nox1-based NADPH oxidase is necessary for PDGF-induced migration in vitro and in vivo for many cell types, including fibroblasts and smooth muscle cells. Here, we report that Nox1-deficient cells failed to acquire a normal front-to-rear polarity, polarize MTOC, and form a single lamellipodium. Instead, these cells form multiple protrusions that accumulate Par3 and active Tiam. The exogenous addition of H2O2 rescues this phenotype and is associated with the hyperactivation of Par3, Tiam, and Rac1. Mechanistically, Nox1 deficiency induces the inactivation of PP2A phosphatase, leading to increased activation of aPKC. These results were validated in Nox1y/- primary mouse aortic smooth muscle cells (MASMCs), which also showed PP2A inactivation after PDGF-BB stimulation consistent with exacerbated activation of aPKC. Moreover, we evaluated the physiological relevance of this signaling pathway using a femoral artery wire injury model to generate neointimal hyperplasia. Nox1y/- mice showed increased staining for the inactive form of PP2A and increased signal for active aPKC, suggesting that PP2A and aPKC activities might contribute to reducing neointima formation observed in the arteries of Nox1y/- mice.

Buxu Tongyu Granule Alleviates Myocardial Ischemia by Activating Vascular Smooth Muscle Cell Soluble Guanylate Cyclase to Inhibit Abnormal Vasomotion

Myocardial ischemia is a serious threat to human health, and vascular dysfunction is its main cause. Buxu Tongyu (BXTY) Granule is an effective traditional Chinese medicine (TCM) for treating myocardial ischemia. However, the underlying mechanism of BXTY is still unclear. In this study, we demonstrate that BXTY ameliorates myocardial ischemia by activating the soluble guanylate cyclase (sGC)–3′,5′-cyclic guanosine monophosphate (cGMP)–protein kinase G (PKG) signaling pathway in vascular smooth muscle cells (VSMCs) to dilate the arteries. BXTY was given by gavage for ten consecutive days before establishing an animal model of acute myocardial ischemia in mice via the intraperitoneal injection of pituitrin. The results showed that BXTY alleviated the symptoms of myocardial ischemia induced by pituitrin in mice, including electrocardiogram abnormalities and changes in plasma enzymes. In addition, BXTY dilated pre-constricted blood vessels and inhibited the vasoconstriction of the superior mesenteric artery in a dose-dependent but endothelial-independent manner. These effects were eliminated by pre-incubating vascular rings with the sGC inhibitors NS 2028 or ODQ, or with the PKG inhibitor KT 5823. Moreover, BXTY increased the protein expression of sGC-β1 and the intracellular second messenger cGMP level in mouse aortic vascular smooth muscle cells (MOVAs). NS 2028 or ODQ reversed these effects of BXTY. The expression level of the cGMP downstream effector protein PKG-1 increased after treating MOVAs with BXTY. NS 2028, ODQ, or KT 5823 also reversed this effect of BXTY. In conclusion, BXTY can improve the symptoms of acute myocardial ischemia in mice, and activating the sGC–cGMP–PKG pathway in VSMCs to induce vasodilation is its key pharmacodynamic mechanism.

Silicate ions as soluble form of bioactive ceramics alleviate aortic aneurysm and dissection.

Aortic aneurysm and dissection (AAD) are leading causes of death in the elderly. Recent studies have demonstrated that silicate ions can manipulate multiple cells, especially vascular-related cells. We demonstrated in this study that silicate ions as soluble form of bioactive ceramics effectively alleviated aortic aneurysm and dissection in both Ang II and β-BAPN induced AAD models. Different from the single targeting therapeutic drug approaches, the bioactive ceramic derived approach attributes to the effect of bioactive silicate ions on the inhibition of the AAD progression through regulating the local vascular microenvironment of aorta systematically in a multi-functional way. The in vitro experiments revealed that silicate ions did not only alleviate senescence and inflammation of the mouse aortic endothelial cells, enhance M2 polarization of mouse bone marrow-derived macrophages, and reduce apoptosis of mouse aortic smooth muscle cells, but also regulate their interactions. The in vivo studies further confirm that silicate ions could effectively alleviate senescence, inflammation, and cell apoptosis of aortas, accomplished with reduced aortic dilation, collagen deposition, and elastin laminae degradation. This bioactive ceramic derived therapy provides a potential new treatment strategy in attenuating AAD progression.

Chlorin e6 (Ce6)-loaded plaque-specific liposome with enhanced photodynamic therapy effect for atherosclerosis treatment

Recently, photodynamic therapy (PDT) has been considered as a new strategy for atherosclerosis treatment. Targeted delivery of photosensitizer could significantly reduce its toxicity and enhance its phototherapeutic efficiency. CD68 is an antibody that can be conjugated to nano-drug delivery systems to actively target plaque sites, owing to its specific binding to CD68 receptors that are highly expressed on the surfaces of macrophage-derived foam cells. Liposomes are very popular nanocarriers due to their ability to encapsulate a wide range of therapeutic compounds including drugs, microRNAs and photosensitizers, and their ability to be surface-modified with targeting moieties leading to the development of nanocarriers with an improved targeted ability. Hence, we designed a Ce6-loaded liposomes using the film dispersion method, followed by the conjugation of CD68 antibody on the liposomal surface through a covalent crosslinking reaction, forming CD68-modified Ce6-loaded liposomes (CD68-Ce6-mediated liposomes). Flow cytometry results indicated that Ce6-containing liposomes were more effective in promoting intracellular uptake after laser irradiation. Furthermore, CD68-modified liposomes significantly strengthened the cellular recognization and thus internalization. Different cell lines have been incubated with the liposomes, and the results showed that CD68-Ce6-mediated liposomes had no significant cytotoxicity to coronary artery endothelial cells (HCAEC) under selected conditions. Interestingly, they promoted autophagy in foam cells through the increase in LC3-Ⅰ, LC3-Ⅱ expression and the decrease in p62 expression, and restrained the migration of mouse aortic vascular smooth muscle cells (MOVAS) in vitro. Moreover, the enhancement of atherosclerotic plaque stability and the reduction in the cholesterol content by CD68-Ce6-mediated liposomes were dependent on transient reactive oxygen species (ROS) generated under laser irradiation. In summary, we demonstrated that CD68-Ce6-mediated liposomes, as a photosensitizer nano-drug delivery system, have an inhibitory effect on MOVAS migration and a promotion of cholesterol efflux in foam cells, and thereby, represent promising nanocarriers for atherosclerosis photodynamic therapy.