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Background: The Ca 2+ -independent contraction of vascular smooth muscle is a leading cause of cardiovascular and cerebrovascular spasms. In the previous study, we demonstrated the involvement of Src family protein tyrosine kinase Fyn and Rho-kinase in the sphingosylphosphorylcholine (SPC)-induced abnormal and Ca 2+ -independent contraction of vascular smooth muscle, but the specific mechanism has not been completely clarified., Methods: Paxillin knockdown human coronary artery smooth muscle cells (CASMCs) and smooth muscle-specific paxillin knockout mice were generated by using paxillin shRNA and the tamoxifen-inducible Cre-LoxP system, respectively. CASMCs contraction was observed by time-lapse recording. The vessel contractility was measured by using a myography assay. Fyn, Rho-kinase, and myosin light chain activation were assessed by immunoprecipitation and western blotting. The paxillin expression and actin stress fibers were visualized by histological analysis and immunofluorescent staining., Results: The SPC-induced abnormal contraction was inhibited in paxillin knockdown CASMCs and arteries of paxillin knockout mice, indicating that paxillin is involved in this abnormal contraction. Further study showed that paxillin knockdown inhibited the SPC-induced Rho-kinase activation without affecting Fyn activation. In addition, paxillin knockdown significantly inhibited the SPC-induced actin stress fiber formation and myosin light chain phosphorylation. These results suggest that paxillin, as an upstream molecule of Rho-kinase, is involved in the SPC-induced abnormal contraction of vascular smooth muscle., Conclusions: The present study demonstrated that paxillin participates in the SPC-induced abnormal vascular smooth muscle contraction by regulating Rho-kinase activation. Video Abstract., (© 2024. The Author(s).)

Vascular calcification (VC) has emerged as a key predictor of cardiovascular events in patients with chronic kidney disease (CKD). In recent years, an expanding body of research has put forth the concept of accelerated vascular aging among CKD patients, highlighting the significance of vascular cells senescence in the process of VC. Within the milieu of uremia, senescent vascular endothelial cells (VECs) release extracellular microvesicles (MV) that promote vascular smooth muscle cells (VSMCs) senescence, thereby triggering the subsequent osteogenic phenotypic switch and ultimately contributing to the VC process. In addition, senescent vascular progenitor or stem cells with diminished ability to differentiate into VECs and VSMCS, compromise the repair of vascular integrity, on the other hand, release a cascade of molecules associated with senescence, collectively known as the senescence-associated secretory phenotype (SASP), perpetuating the senescence phenomenon. Furthermore, SASP triggers the recruitment of monocytes and macrophages, as well as adjacent VECs and VSMCs into a pro-adhesive and pro-inflammatory senescent state. This pro-inflammatory microenvironment niche not only impacts the functionality of immune cells but also influences the differentiation of myeloid immune cells, thereby amplifying the reduced ability to effectively clear senescent cells of senescent macrophages, promoted calcification of VSMCs. The objective of this paper is to provide a comprehensive review of the contribution of vascular cell senescence to the emergence and advancement of VC. Gaining a comprehensive understanding of the involvement of cellular senescence within the vessel wall is pivotal, especially when it comes to its intersection with VC. This knowledge is essential for advancing groundbreaking anti-aging therapies, aiming to effectively mitigate cardiovascular diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fang, Zhao, Huang, Yang, Liu and Zhang.)

Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3-7 mM) but not with β-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, β-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.

Background: Tar is the main toxic of cigarettes, and its effect on atherosclerosis progression and the underlying mechanisms remain largely unknown. Vascular smooth muscle cells (VSMCs) play a key role in atherogenesis and plaque vulnerability. The present study sought to investigate the mechanism of atherosclerosis progression through tar-induced VSMC necroptosis, a recently described form of necrosis., Methods: The effect of tar on atherosclerosis progression and VSMC necroptosis was examined in ApoE -/- mice and cultured VSMCs. The role of necroptosis in tar-induced plaque development was evaluated in RIPK3-deletion mice (ApoE -/- RIPK3 -/- ). The key proteins of necroptosis in carotid plaques of smokers and non-smokers were also examined. Quantitative proteomics of mice aortas was conducted to further investigate the underlying mechanism. Pharmacological approaches were then applied to modulate the expression of targets to verify the regulatory process of tar-induced necroptosis., Results: Tar administration led to increased atherosclerotic plaque area and reduced collagen and VSMCs in ApoE -/- mice. The expression of RIPK1、RIPK3、and MLKL in VSMCs of plaques were all increased in tar-exposed mice and smokers. RIPK3 deletion protected against VSMC loss and plaque progression stimulated by tar. In mechanistic studies, quantitative proteomics analysis of ApoE -/- mice aortas suggested that tar triggered endoplasmic reticulum (ER) stress. PERK-eIF2α-CHOP axis was activated in tar-treated VSMCs and atherosclerotic plaque. Inhibition of ER stress using 4PBA significantly reduced plaque progression and VSMC necroptosis. Further study revealed that ER stress resulted in calcium (Ca 2+ ) release into mitochondria and cytoplasm. Elevated Ca 2+ levels lead to mitochondrial dysfunction and excessive reactive oxygen species (ROS) production, which consequently promote RIPK3-dependent necroptosis. In addition, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) activated by cytosolic Ca 2+ overload binds to RIPK3, accounting for necroptosis., Conclusion: The findings revealed that cigarette tar promoted atherosclerosis progression by inducing RIPK3-dependent VSMC necroptosis and identified novel avenues of ER stress and Ca 2+ overload., (© 2024. The Author(s).)

Mature vascular smooth muscle cells (VSMC) exhibit a remarkable degree of plasticity, a characteristic that has intrigued cardiovascular researchers for decades. Recently, it has become increasingly evident that the chromatin remodeler SWItch/Sucrose Non-Fermentable (SWI/SNF) complex plays a pivotal role in orchestrating chromatin conformation, which is critical for gene regulation. In this review, we provide a summary of research related to the involvement of the SWI/SNF complexes in VSMC and cardiovascular diseases (CVD), integrating these discoveries into the current landscape of epigenetic and transcriptional regulation in VSMC. These novel discoveries shed light on our understanding of VSMC biology and pave the way for developing innovative therapeutic strategies in CVD treatment.

Background: During sepsis, serve vascular dysfunctions lead to life-threatening multiple organ failure, due to vascular smooth muscle cells (VSMC) impairments, resulting in vasoplegia, hypotension and hypoperfusion. In addition, septic patients have an altered cell metabolism that leads to lactic acidosis. Septic patients suffering from lactic acidosis have a high risk of mortality. In addition, septic survivors are at risk of secondary vascular disease. The underlying mechanisms of whether and how lactic acidosis leads to the changes in VSMCs is not well understood. The aim of this study was to comprehensively investigate the effect of lactic acidosis on VSMCs and additionally compare the effects with those induced by pure acidosis and sodium lactate., Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated for 48 h with lactic acidosis (LA_pH 6.8), hydrochloric acid (HCl_pH 6.8), sodium lactate (Na + -lactate_pH 7.4) and the respective controls (ctrl._pH 7.4; hyperosmolarity control: mannitol_pH 7.4) and comparatively analyzed for changes in (i) transcriptome, (ii) energy metabolism, and (iii) phenotype., Results: Both types of acidosis led to comparable and sustained intracellular acidification without affecting cell viability. RNA sequencing and detailed transcriptome analysis revealed more significant changes for lactic acidosis than for hydrochloric acidosis, with lactate being almost ineffective, suggesting qualitative and quantitative synergism of acidosis and lactate. Bioinformatic predictions in energy metabolism and phenotype were confirmed experimentally. Lactic acidosis resulted in strong inhibition of glycolysis, glutaminolysis, and altered mitochondrial respiration which reduced cellular ATP content, likely due to increased TXNIP expression and altered NAD + /NADH ratio. Hydrochloric acidosis induced significantly smaller effects without changing the NAD + /NADH ratio, with the ATP content remaining constant. These metabolic changes led to osteo-/chondrogenic/senescent transdifferentiation of VSMCs, with the effect being more pronounced in lactic acidosis than in pure acidosis., Conclusions: Overall, lactic acidosis exerted a much stronger effect on energy metabolism than pure acidosis, whereas lactate had almost no effect, reflecting the qualitative and quantitative synergism of acidosis and lactate. As a consequence, lactic acidosis may lead to acute functional impairments of VSMC, sustained perturbations of the transcriptome and cellular dedifferentiation. Moreover, these effects may contribute to the acute and prolonged vascular pathomechanisms in septic patients., (© 2024. The Author(s).)

Atherosclerosis is a chronic inflammatory disease leading to various vascular diseases. Vascular smooth muscle cell (VSMC) senescence promotes atherosclerotic inflammation and the formation of plaque necrosis core, in part through telomere damage mediated by a high-fat diet. Our previous research found that paeonol, a potential anti-inflammatory agent extracted from Cortex Moutan, could significantly improve VSMCs dysfunction. However, the impact of paeonol on the senescence of VSMCs remains unexplored. This study presents the protective effects of paeonol on VSMCs senescence, and its potential activity in inhibiting the progression of atherosclerosis in vivo and in vitro. Sirtuin 1 (SIRT1) is a nuclear deacetylase involved in cell proliferation, senescence, telomere damage, and inflammation. Here, SIRT1 was identified as a potential target of paeonol having anti-senescence and anti-atherosclerosis activity. Mechanistic studies revealed that paeonol binds directly to SIRT1 and then activates the SIRT1/P53/TRF2 pathway to inhibit VSMCs senescence. Our results suggested that SIRT1-mediated VSMCs senescence is a promising druggable target for atherosclerosis, and that pharmacological modulation of the SIRT1/P53/TRF2 signaling pathway by paeonol is of potential benefit for patients with atherosclerosis.

Pathological vascular remodeling is a hallmark of various vascular diseases. Previous research has established the significance of andrographolide in maintaining gastric vascular homeostasis and its pivotal role in modulating endothelial barrier dysfunction, which leads to pathological vascular remodeling. Potassium dehydroandrographolide succinate (PDA), a derivative of andrographolide, has been clinically utilized in the treatment of inflammatory diseases precipitated by viral infections. This study investigates the potential of PDA in regulating pathological vascular remodeling. The effect of PDA on vascular remodeling was assessed through the complete ligation of the carotid artery in C57BL/6 mice. Experimental approaches, including rat aortic primary smooth muscle cell culture, flow cytometry, bromodeoxyuridine (BrdU) incorporation assay, Boyden chamber cell migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay, were employed to evaluate the influence of PDA on the proliferation and motility of smooth muscle cells (SMCs). Molecular docking simulations and co-immunoprecipitation assays were conducted to examine protein interactions. The results revealed that PDA exacerbates vascular injury-induced pathological remodeling, as evidenced by enhanced neointima formation. PDA treatment significantly increased the proliferation and migration of SMCs. Further mechanistic studies disclosed that PDA upregulated myeloid differentiation factor 88 (MyD88) expression in SMCs and interacted with T-cadherin (CDH13). This interaction augmented proliferation, migration, and extracellular matrix deposition, culminating in pathological vascular remodeling. Our findings underscore the critical role of PDA in the regulation of pathological vascular remodeling, mediated through the MyD88/CDH13 signaling pathway., (Copyright © 2024 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Introduction: Hyperuricemia may be involved in the phenotypic transformation of vascular smooth muscle cells, thus promoting the occurrence of atherosclerosis, and autophagy may be one of the important links, but little is known about the specific molecular mechanism., Methods: We established a mouse model of hyperuricemia and studied the relationship between changes in autophagy levels and the phenotypic transformation of muscle cells., Results: Our study found that high uric acid levels promote the phenotypic transformation of muscle cells by inhibiting autophagy, thus enhancing their proliferation and migration abilities. If autophagy is restored, phenotypic transformation can be reversed by reducing the levels of the transcription factor Kruppel-like factor 4., Conclusion: Uric acid may induce the phenotypic transformation of muscle cells and promote the occurrence of atherosclerosis by disrupting normal autophagy., (© 2023 S. Karger AG, Basel.)

Backgruound: Glucagon-like peptide-1 receptor agonist (GLP-1RA), which is a therapeutic agent for the treatment of type 2 diabetes mellitus, has a beneficial effect on the cardiovascular system., Methods: To examine the protective effects of GLP-1RAs on proliferation and migration of vascular smooth muscle cells (VSMCs), A-10 cells exposed to angiotensin II (Ang II) were treated with either exendin-4, liraglutide, or dulaglutide. To examine the effects of GLP-1RAs on vascular calcification, cells exposed to high concentration of inorganic phosphate (Pi) were treated with exendin-4, liraglutide, or dulaglutide., Results: Ang II increased proliferation and migration of VSMCs, gene expression levels of Ang II receptors AT1 and AT2, proliferation marker of proliferation Ki-67 (Mki-67), proliferating cell nuclear antigen (Pcna), and cyclin D1 (Ccnd1), and the protein expression levels of phospho-extracellular signal-regulated kinase (p-Erk), phospho-c-JUN N-terminal kinase (p-JNK), and phospho-phosphatidylinositol 3-kinase (p-Pi3k). Exendin-4, liraglutide, and dulaglutide significantly decreased the proliferation and migration of VSMCs, the gene expression levels of Pcna, and the protein expression levels of p-Erk and p-JNK in the Ang II-treated VSMCs. Erk inhibitor PD98059 and JNK inhibitor SP600125 decreased the protein expression levels of Pcna and Ccnd1 and proliferation of VSMCs. Inhibition of GLP-1R by siRNA reversed the reduction of the protein expression levels of p-Erk and p-JNK by exendin-4, liraglutide, and dulaglutide in the Ang II-treated VSMCs. Moreover, GLP-1 (9-36) amide also decreased the proliferation and migration of the Ang II-treated VSMCs. In addition, these GLP-1RAs decreased calcium deposition by inhibiting activating transcription factor 4 (Atf4) in Pi-treated VSMCs., Conclusion: These data show that GLP-1RAs ameliorate aberrant proliferation and migration in VSMCs through both GLP-1Rdependent and independent pathways and inhibit Pi-induced vascular calcification.

Restenosis after angioplasty is caused usually by neointima formation characterized by aberrant vascular smooth muscle cell (VSMC) dedifferentiation. Myeloid-derived growth factor (MYDGF), secreted from bone marrow-derived monocytes and macrophages, has been found to have cardioprotective effects. In this study we investigated the effect of MYDGF to postinjury neointimal formation and the underlying mechanisms. Rat carotid arteries balloon-injured model was established. We found that plasma MYDGF content and the level of MYDGF in injured arteries were significantly decreased after balloon injury. Local application of exogenous MYDGF (50 μg/mL) around the injured vessel during balloon injury markedly ameliorated the development of neointimal formation evidenced by relieving the narrow endovascular diameter, improving hemodynamics, and reducing collagen deposition. In addition, local application of MYDGF inhibited VSMC dedifferentiation, which was proved by reversing the elevated levels of osteopontin (OPN) protein and decreased levels of α-smooth muscle actin (α-SMA) in the left carotid arteries. We showed that PDGF-BB (30 ng/mL) stimulated VSMC proliferation, migration and dedifferentiation in vitro; pretreatment with MYDGF (50-200 ng/mL) concentration-dependently eliminated PDGF-BB-induced cell proliferation, migration and dedifferentiation. Molecular docking revealed that MYDGF had the potential to bind with sphingosine-1-phosphate receptor 2 (S1PR2), which was confirmed by SPR assay and Co-IP analysis. Pretreatment with CCG-1423 (Rho signaling inhibitor), JTE-013 (S1PR2 antagonist) or Ripasudil (ROCK inhibitor) circumvented the inhibitory effects of MYDGF on VSMC phenotypic switching through inhibiting S1PR2 or its downstream RhoA-actin monomers (G-actin) /actin filaments (F-actin)-MRTF-A signaling. In summary, this study proves that MYDGF relieves neointimal formation of carotid arteries in response to balloon injury in rats, and suppresses VSMC dedifferentiation induced by PDGF-BB via S1PR2-RhoA-G/F-actin-MRTF-A signaling pathway. In addition, our results provide evidence for cross talk between bone marrow and vasculature., (© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

While arterial tone is generally determined by the phosphorylation of Ser 19 in myosin light chain (p-MLC2), Thr 18 /Ser 19 diphosphorylation of MLC2 (pp-MLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-β and MYPT1 levels were higher in MA than in PA. Interestingly, the level of pp-MLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 μM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 μM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.

Collateral circulation is essential for blood resupply to the ischemic heart, which is dictated by the contractile phenotypic restoration of vascular smooth muscle cells (VSMC). Here we investigate whether S-nitrosylation of AMP-activated protein kinase (AMPK), a key regulator of the VSMC phenotype, impairs collateral circulation. In rats with collateral growth and development, nitroglycerin decreases coronary collateral blood flow (CCBF), inhibits vascular contractile phenotypic restoration, and increases myocardial infarct size, accompanied by reduced AMPK activity in the collateral zone. Nitric oxide (NO) S-nitrosylates human recombinant AMPKγ1 at cysteine 131 and decreases AMP sensitivity of AMPK. In VSMCs, exogenous expression of S-nitrosylation-resistant AMPKγ1 or deficient NO synthase (iNOS) prevents the disruption of VSMC reprogramming. Finally, hyperhomocysteinemia or hyperglycemia increases AMPKγ1 S-nitrosylation, prevents vascular contractile phenotypic restoration, reduces CCBF, and increases the infarct size of the heart in Apoe -/- mice, all of which is rescued in Apoe -/- /iNOS sm-/- mice or Apoe -/- mice with enforced expression of the AMPKγ1-C130A mutant following RI/MI. We conclude that nitrosative stress disrupts coronary collateral circulation during hyperhomocysteinemia or hyperglycemia through AMPK S-nitrosylation., (© 2023. The Author(s).)

Type A aortic dissection (AD) is a catastrophic cardiovascular disease. Hypoxia-inducible factor-1α (HIF-1α) and autophagy are reported to be upregulated in the AD specimens. However, the interaction between HIF-1α and autophagy in the pathogenesis of AD remains to be explored. HIF-1α and LC3 levels are evaluated in 10 AD and 10 normal aortic specimens. MDC staining, autophagic vacuoles, and autophagic flux are detected in human aortic smooth muscle cells (HASMCs) under hypoxia treatment. CCK-8, transwell, and wound healing assay are used to identify proliferation and migration under hypoxia treatment. Furthermore, 3-MA is used to inhibit autophagy in hypoxia-treated HASMCs. This study reveals that AD tissues highly express HIF-1α and the LC3. Autophagy is induced under hypoxia in a time-dependent manner, and autophagy is positively related to HIF-1α in HASMCs. Moreover, the proliferation and migration of HASMCs are enhanced by hypoxia, whereas the knockdown of HIF-1α attenuates this effect. Additionally, inhibiting autophagy with 3-MA ameliorates hypoxia-induced proliferation and migration of HASMCs. In summary, the above results indicate that HIF-1α facilitates HASMC proliferation and migration by upregulating autophagy in a hypoxic microenvironment. Thus, inhibition of autophagy may be a novel therapeutic target for the prevention and treatment of AD., (© 2023 Wiley-VCH GmbH.)

Arterial calcification is a hallmark of vascular pathology in the elderly and in individuals with chronic kidney disease (CKD). Vascular smooth muscle cells (VSMCs), after attaining a senescent phenotype, are implicated in the calcifying process. However, the underlying mechanism remains to be elucidated. Here, we reveal an aberrant upregulation of transcriptional factor GATA6 in the calcified aortas of humans, mice with CKD and mice subjected to vitamin D3 injection. Knockdown of GATA6, via recombinant adeno-associated virus carrying GATA6 shRNA, inhibited the development of arterial calcification in mice with CKD. Further gain- and loss-of function experiments in vitro verified the contribution of GATA6 in osteogenic differentiation of VSMCs. Samples of human aorta exhibited a positive relationship between age and GATA6 expression and GATA6 was also elevated in the aortas of old as compared to young mice. Calcified aortas displayed senescent features with VSMCs undergoing premature senescence, blunted by GATA6 downregulation. Notably, abnormal induction of GATA6 in senescent and calcified aortas was rescued in Sirtuin 6 (SIRT6)-transgenic mice, a well-established longevity mouse model. Suppression of GATA6 accounted for the favorable effect of SIRT6 on VSMCs senescence prevention. Mechanistically, SIRT6 inhibited the transcription of GATA6 by deacetylation and increased degradation of transcription factor Nkx2.5. Moreover, GATA6 was induced by DNA damage stress during arterial calcification and subsequently impeded the Ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair process, leading to accelerated VSMCs senescence and osteogenic differentiation. Thus, GATA6 is a novel regulator in VSMCs senescence. Our findings provide novel insight in arterial calcification and a potential new target for intervention., (Copyright © 2023. Published by Elsevier Inc.)

Circular RNAs (circRNAs) regulate the function of vascular smooth muscle cells (VSMCs) in atherosclerosis (AS) progression. We aimed to explore the role of circUSP9X in oxidized low-density lipoprotein (ox-LDL)-induced VSMCs. Cell proliferation was assessed using cell counting kit-8 and EDU assays. Cell migration was evaluated using Transwell and wound healing assays. The interaction between circUSP9X or STIM1 and miR-599 was analyzed using dual-luciferase reporter and RNA pull-down assays. Their levels were examined using quantitative real-time PCR. CircUSP9X and STIM1 expression was increased, whereas miR-599 expression was reduced in the serum of patients with AS and ox-LDL-stimulated VSMCs. Overexpression of circUSP9X facilitated the proliferation and migration of VSMCs induced by ox-LDL. CircUSP9X sponged miR-599, which targeted STIM1. MiR-599 reversed the effects induced by circUSP9X, and STIM1 reversed the effects induced by miR-599. Taken together, CircUSP9X promoted proliferation and migration in ox-LDL-treated VSMCs via the miR-599/STIM1 axis, providing a theoretical basis for the role of circUSP9X/miR-599/STIM1 axis in AS.

Background: In-stent restenosis hardly limits the therapeutic effect of the percutaneous vascular intervention. Although the restenosis is significantly ameliorated after the application of new drug-eluting stents, the incidence of restenosis remains at a high level., Objective: Vascular adventitial fibroblasts (AFs) play an important role in intimal hyperplasia and subsequent restenosis. The current study was aimed to investigate the role of nuclear receptor subfamily 1, group D, member 1 (NR1D1) in the vascular intimal hyperplasia., Methods and Results: We observed increased expression of NR1D1 after the transduction of adenovirus carrying Nr1d1 gene (Ad-Nr1d1) in AFs. Ad-Nr1d1 transduction significantly reduced the numbers of total AFs, Ki-67-positive AFs, and the migration rate of AFs. NR1D1 overexpression decreased the expression level of β-catenin and attenuated the phosphorylation of the effectors of mammalian target of rapamycin complex 1 (mTORC1), including mammalian target of rapamycin (mTOR) and 4E binding protein 1 (4EBP1). Restoration of β-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Surprisingly, the restoration of mTORC1 activity by insulin could also reverse the decreased expression of β-catenin, attenuated proliferation, and migration in AFs induced by NR1D1 overexpression. In vivo , we found that SR9009 (an agonist of NR1D1) ameliorated the intimal hyperplasia at days 28 after injury of carotid artery. We further observed that SR9009 attenuated the increased Ki-67-positive AFs, an essential part of vascular restenosis at days 7 after injury to the carotid artery., Conclusion: These data suggest that NR1D1 inhibits intimal hyperplasia by suppressing the proliferation and migration of AFs in a mTORC1/β-catenin-dependent manner.

Vascular calcification (VC) is an age-related complication characterised by calcium-phosphate deposition in the arterial wall driven by the osteogenic transformation of vascular smooth muscle cells (VSMCs). The JAK-STAT pathway is an emerging target in inflammation. Considering the relationship between VC and inflammation, we investigated the role of JAK-STAT signalling during VSMC calcification. Human aortic smooth muscle cells (HASMCs) were cultured in high-inorganic phosphate (Pi) medium for up to 7 days; calcium deposition was determined via Alizarin staining and colorimetric assay. Inflammatory factor secretion was evaluated via ELISA and JAK-STAT members' activation using Western blot or immunohistochemistry on HASMCs or calcified aortas of Vitamin D-treated C57BL6/J mice, respectively. The JAK-STAT pathway was blocked by JAK Inhibitor I and Von Kossa staining was used for calcium deposits in murine aortic rings. During Pi-induced calcification, HASMCs released IL-6, IL-8, and MCP-1 and activated JAK1-JAK3 proteins and STAT1. Phospho-STAT1 was detected in murine calcified aortas. Blocking of the JAK-STAT cascade reduced HASMC proliferation and pro-inflammatory factor expression and release while increasing calcium deposition and osteogenic transcription factor RUNX2 expression. Consistently, JAK-STAT pathway inhibition exacerbates mouse aortic ring calcification ex vivo. Intriguingly, our results suggest an alternative link between VSMC inflammation and VC.

The pathological feature of hypoxic pulmonary hypertension (PH) is pulmonary vascular remodeling (PVR), primarily attributed to the hyperproliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Existing PH-targeted drugs have difficulties in reversing PVR. Therefore, it is vital to discover a new regulatory mechanism for PVR and develop new targeted drugs. G protein-coupled receptor 146 (GPR146) is believed to participate in this process. This study aimed to investigate the role of GPR146 in PASMCs during PH. We investigated the role of GPR146 in PVR and its underlying mechanism using hypoxic PASMCs and mouse model (Sugen 5416 (20 mg/kg)/hypoxia). In our recent study, we have observed a significant increase in the expression of GPR146 protein in animal models of PH as well as in patients diagnosed with pulmonary arterial hypertension (PAH). Through immunohistochemistry, we found that GPR146 was mainly localized in the smooth muscle and endothelial layers of the pulmonary vasculature. GPR146 deficiency induction exhibited protective effects against hypoxia-induced elevation of right ventricular systolic blood pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling in mice. In particular, the deletion of GPR146 attenuated the hypoxia-triggered proliferation of PASMCs. Furthermore, 5-lipoxygenase (5-LO) was related to PH development. Hypoxia and overexpression of GPR146 increased 5-LO expression, which was reversed through GPR146 knockdown or siRNA intervention. Our study discovered that GPR146 exhibited high expression in the pulmonary vessels of pulmonary hypertension. Subsequent research revealed that GPR146 played a crucial role in the development of hypoxic PH by promoting lipid peroxidation and 5-LO expression. In conclusion, GPR146 may regulate pulmonary vascular remodeling by promoting PASMCs proliferation through 5-LO, which presents a feasible target for PH prevention and treatment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

This study aimed to investigate the effects of ulinastatin on the apoptosis and (Sirt1/FoxO3a) pathway of vascular smooth muscle cells (VSMC) in aortic dissection (AD) rats. For this purpose a rat model of aortic dissection (AD) was constructed by giving drinking water containing 0.08% β-aminopropionitrile (BAPN) to rats, HE staining was used to observe the pathological changes of the aorta in AD rats; the diseased blood vessels of AD rats were taken for primary culture and passage of VSMCs, the morphology of VSMCs was observed, and VSMCs were identify with immunofluorescence staining; VSMCs were treated with culture media containing 0, 1000, 2000, 3000, 4000, 5000, 6000, 7000 U/mL ulinastatin, and MTT kit was used to determine the effect of ulinastatin on VSMC proliferation in AD rats; the VSMC of AD rats were divided into blank group (normal culture), ulinastatin group (medium containing 5000 U/mL ulinastatin), Sirt1 inhibitor group (medium containing 1 μmol/L EX527), ulinastatin + Sirt1 inhibitor group (medium containing 5000 U/mL ulinastatin, 1 μmol/L EX527), flow cytometry was used to detect the VSMC apoptosis in each group, WB was used to detect the expression of VSMC apoptosis-related proteins and Sirt1/FoxO3a pathway-related proteins in each group. Findings suggested that the aortic wall of AD rats was thickened, and the dissection false cavity appeared; VSMC mostly presented different shapes such as triangles and stars, the immunofluorescence staining results showed that α-SMA was arranged in the cytoplasm in the form of myofilaments, showing green fluorescence, and the nucleus showed blue fluorescence, and the rate of positive cells was more than 95%; various doses of ulinastatin had a certain inhibitory effect on the proliferation of VSMC, and 5000 U/mL ulinastatin had a higher proliferation inhibition rate; compared with the blank group, the VSMC apoptosis rate, Caspase-3, Bax protein, Sirt1/FoxO3a pathway related protein expression in the ulinastatin group were significantly increased, and the Bcl-2 protein expression was significantly decreased (P<0.05), the VSMC apoptosis rate, Caspase-3, Bax protein, Sirt1/FoxO3a pathway related protein expression in the Sirt1 inhibitor group were significantly decreased, and the Bcl-2 protein expression was significantly increased (P<0.05); compared with the ulinastatin group, the VSMC apoptosis rate, Caspase-3, Bax protein, Sirt1/FoxO3a pathway related protein expression in the ulinastatin + Sirt1 inhibitor group were significantly decreased, and the Bcl-2 protein expression was significantly increased (P<0.05). It was concluded that ulinastatin can inhibit the proliferation of VSMCs in AD rats and promote their apoptosis, which may be achieved by activating the Sirt1/FoxO3a pathway.

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 22 α . 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.

Vascular smooth muscle cells (VSMCs) phenotypic switching is identified as enhanced dedifferentiation, proliferation, and migration ability of VSMCs, in which microRNAs have been identified as important regulators of the process. The present study is aimed to explore the pathophysiological effect of miR-122 on VSMC phenotypic modulation. Here, the result showed that the decreased miR-122 expression was found in VSMCs subjected to platelet-derived growth factor-BB (PDGF-BB) treatment. Next, we investigated the response of miR-122 knockdown in VSMCs with PDGF-BB stimulation. MiR-122 silencing showed increased proliferation and migration capability, whereas attenuated the differentiation markers expression. The above results were reversed by miR-122 overexpression. Finally, we further demonstrated that FOXO3 was an important target for miR-122. Collectively, we demonstrated that miR-122 silencing promoted VSMC phenotypic modulation partially through upregulated FOXO3 expression that indicated miR-122 may be a novel therapeutic target for neointimal formation., Competing Interests: None.

Aim: Increased level of serum fibroblast growth factor 23 (FGF23) is a hallmark of abnormal phosphate metabolism in patients with chronic kidney disease (CKD) and is recently shown to be associated with the risk of cardiovascular disease even in those without CKD. This study investigated the association between serum FGF23 levels and vascular function in patients with type 2 diabetes., Methods: This was a cross-sectional study involving 283 Japanese patients with type 2 diabetes. Flow-mediated dilatation (FMD) and nitroglycerin-mediated dilatation (NMD) of the brachial artery were measured via ultrasonography to evaluate vascular endothelial and smooth muscle functions, respectively. Serum intact FGF23 levels were determined via a sandwich enzyme-linked immunosorbent assay., Results: The median values of FMD, NMD, and serum FGF23 were 6.0%, 14.0%, and 27.3 pg/mL, respectively. The serum FGF23 levels were inversely associated with NMD but not with FMD, and the association was independent of atherosclerotic risk factors, estimated glomerular filtration rate (eGFR), and serum phosphate levels. Furthermore, the relationship between serum FGF23 levels and NMD was modified by kidney function, which was pronounced in subjects with normal kidney function (eGFR ≥ 60 mL/min/1.73 m 2 )., Conclusion: Serum FGF23 levels are independently and inversely associated with NMD in patients with type 2 diabetes, particularly in those with normal kidney function. Our results indicate that FGF23 is involved in vascular smooth muscle dysfunction and that increased serum levels of FGF23 may serve as a novel biomarker for vascular smooth muscle dysfunction in patients with type 2 diabetes.

Context: Gastrodin has been used as antihypertension therapy in China; however, the mechanisms underlying the effects of gastrodin have yet to be fully elucidated., Objective: To explore the therapeutic efficiency of gastrodin as an antihypertensive and determine the mechanisms underlying this effect., Materials and Methods: C57BL/6 mice were continuously administered angiotensin II (Ang II) (500 ng/kg/min) to induce hypertension. Mice were randomly divided into control, Ang II and Ang II + gastrodin groups. Mice received intragastric administration of gastrodin (5 mg/kg) or double distilled water once a day for 4 weeks. Blood pressure, pulse wave velocity (PWV), thickness of the abdominal aorta, pathological morphology and differential expression transcripts (DETs) were assessed. Abdominal aorta rings and primary isolated vascular smooth muscle cells were subjected to Ang II stimulation to induce hypertension as ex vivo and in vitro models, respectively. Vascular ring tension, release of Ca 2+ and levels of proteins involved in the myosin light chain kinase (MLCK)/phospho-myosin light chain 2 (p-MLC 2 ) pathway were determined., Results: Gastrodin treatment attenuated increases in blood pressure, PWV and thickness of the abdominal aorta. Treatment with gastrodin resulted in 2785 DETs and the enrichment of vascular contraction and calcium signalling pathways. Gastrodin treatment attenuated Ang II-induced vasoconstriction, produced a norepinephrine-precontracted vasodilation effect (attenuated by verapamil), and reduced intracellular Ca 2+ release. Furthermore, gastrodin suppressed activation of the MLCK/p-MLC 2 pathway in vivo and in vitro ., Conclusions: Gastrodin treatment lowers blood pressure, suppresses Ang II-induced vascular contraction and MLCK/p-MLC 2 pathway activation, thereby demonstrating the mechanisms underlying the therapeutic efficacy of gastrodin as an antihypertensive.

A human aorta from a female donor affected by Klippel-Trenaunay syndrome was retrieved during a surgery for organ donation for transplant. The aorta was preserved in refrigerated Belzer UW organ preservation solution and tested within a few hours for mechanical characterization with and without vascular smooth muscle activation. KCl and Noradrenaline were used as vasoactive agents in bubbled Krebs-Henseleit buffer solution at 37 °C. A quasi-static and a dynamic mechanical characterization of the full wall and the three individual layers were carried out for strips taken in longitudinal and circumferential directions. The full wall in the descending portion of the aorta underwent mechanical tests with and without smooth muscle activation. Results were compared to data obtained from healthy aortas and show a reduced stiffness of the full wall in circumferential direction. Also, a significant reduction of the response to vasoactive agents in circumferential direction was observed, while the longitudinal response was similar to healthy cases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)