Vascular Smooth Muscle Cell Activation Research Articles

TRIM25 activates Wnt/β-catenin signalling by destabilising MAT2A mRNA to drive thoracic aortic aneurysm development.

This study explored the roles of methionine adenosyltransferase 2A (MAT2A) and tripartite motif containing 25 (TRIM25) in the progression of thoracic aortic aneurysm (TAA). The TAA model was established based on the β-aminopropionitrile method. The effects of MAT2A on thoracic aortic lesions and molecular levels were analyzed by several pathological staining assays (hematoxylin-eosin, Verhoeff-Van Gieson, TUNEL) and molecular biology experiments (qRT-PCR, Western blot). Angiotensin II (Ang-II) was used to induce injury in vascular smooth muscle cells (VSMCs) in vitro. The effects of MAT2A, shMAT2A, shTRIM25 and/or Wnt inhibitor (IWR-1) on the viability, apoptosis and protein expressions of VSMCs were examined by CCK-8, Annexin V-FITC/PI and Western blot assays. In TAA mice, overexpression of MAT2A alleviated thoracic aortic injury, inhibited the aberrant expressions of aortic contractile proteins and dedifferentiation markers, and blocked the activation of Wnt/β-catenin pathway. In Ang-II-induced VSMCs, up-regulation of MAT2A increased cellular activity and repressed the expression of β-catenin protein. TRIM25 knockdown promoted activity of VSMCs, inhibited apoptosis, and blocked the Wnt/β-catenin pathway activation by binding to MAT2A. IWR-1 partially counteracted the regulatory effects of shMAT2A. Collectively, TRIM25 destabilises the mRNA of MAT2A to activate Wnt/β-catenin signaling and ultimately exacerbate TAA injury.

KIF11 promotes vascular smooth muscle cell proliferation by regulating cell cycle progression and accelerates neointimal formation after arterial injury in mice.

Background and aims: One of the primary causes of lumen narrowing is vascular injury induced during medical procedures. Vascular injury disrupts the integrity of the endothelium, triggering platelet deposition, leukocyte recruitment, and the release of inflammatory factors. This, in turn, induces the proliferation of vascular smooth muscle cells (VSMCs), leading to neointima formation. However, the molecular mechanism underlying VSMC proliferation following injury remains unknown. KIF11 is critical in regulating the cell cycle by forming bipolar spindles during mitotic metaphase. This process may contribute to VSMCs proliferation and neointima formation following vascular injury. Yet, the function of KIF11 in VSMCs has not been elucidated. This study aims to investigate the role and mechanisms of KIF11 in regulating VSMCs cycle progression and proliferation. Methods: After conducting biological analysis of the transcriptome sequencing data from the mouse carotid artery injury model and the cell transcriptome data of PDGF-BB-induced VSMCs, we identified a potential target gene, KIF11, which may play a crucial role in vascular injury. Then we established a vascular injury model to investigate how changes in KIF11 expression and activity influence in vivo VSMCs proliferation and neointimal formation. In addition, we employed siRNA and specific inhibitors to suppress KIF11 expression and activity in VSMCs cultured in vitro to study the mechanisms underlying VSMCs cycle progression and proliferation. Results: The results of immunohistochemistry and immunofluorescence indicate a significant upregulation of KIF11 expression in the injured vascular. The intraperitoneal injection of the KIF11 specific inhibitor, K858, partially inhibits intimal hyperplasia in the vascular injury model. In vitro experiments further demonstrate that PDGF-BB upregulates KIF11 expression through the PI3K/AKT pathway, and enhances KIF11 activity. Inhibition of both KIF11 expression and activity partially reverses the pro-cycle progression and pro-proliferation effects of PDGF-BB on VSMCs. Additionally, KIF11 overexpression partially counteracts the proliferation arrest and cell cycle arrest induced by inhibiting the PI3K/AKT pathway in VSMCs. Conclusion: Our study highlights the crucial role of KIF11 in regulating the cycle progression and proliferation of VSMCs after vascular injury. A comprehensive understanding of these mechanisms could pave the way for potential therapeutic interventions in treating vascular stenosis.

MiR-140-3p Normalizes Vascular Smooth Muscle Cell Behavior by Inhibiting the RhoA/ROCK Signaling Pathway in Lower-extremity Arteriosclerosis Obliterans.

Excessive vascular smooth muscle cell (VSMC) proliferation and migration are the main contributors to the symptoms of lower-extremity arteriosclerosis obliterans (ASO). Previous studies suggested that microRNAs (miRNAs) regulate VSMC activity. Nevertheless, the molecular mechanisms by which they do so are unclear. The present study aimed to identify the biological processes accounting for the effects of miR-140-3p on VSMCs in ASO. The expression levels of miR-140-3p in clinical samples were analyzed by real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR-140-3p on VSMCs. The transwell® assays and MTT assays were used to assess migration and proliferation. The interaction between RhoA and miR-140-3p was verified using the Dualluciferase reporter assay. Western blot technique was used to identify RhoA, RhoA-associated protein kinase 1 (ROCK1), and ROCK2. We discovered that miR-140-3p inhibited the proliferation, migration, and invasion but promoted the apoptosis of VSMCs, and RhoA was its downstream target gene. RhoA, ROCK1, and ROCK2 were upregulated in vascular tissues damaged by ASO compared to normal, healthy arteries. MiR-140-3p also decreased RhoA, ROCK1, and ROCK2 mRNA and protein expression. Overall, the present work partially elucidated the mechanism by which miR-140-3p regulates VSMC function and offered novel insights into potential therapeutic approaches for patients with lower-extremity arteriosclerosis obliterans.

Inhibition of CIRBP represses the proliferation and migration of vascular smooth muscle cells via inhibiting Rheb/mTORC1 axis

The excessive migration and proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in vascular intimal hyperplasia. CIRBP is involved in the proliferation of various cancer cells. This study was aimed to explore the role of CIRBP in the proliferation and migration of VSMCs. Adenovirus was used to interfere with cold-inducible RNA-binding protein (CIRBP) expression, while lentivirus was used to overexpress Ras homolog enriched in brain (Rheb). Western blotting and qRT-PCR were used to evaluate the expression of CIRBP, Rheb, and mechanistic target of rapamycin complex 1 (mTORC1) activity. The cell proliferation was determined by Ki67 immunofluorescence staining and CCK-8 assay. The wound healing assay was performed to assess cell migration. Additionally, immunohistochemistry was conducted to explore the role of CIRBP in intimal hyperplasia after vascular injury. We found that silencing CIRBP inhibited the proliferation and migration of VSMCs, decreased the expression of Rheb and mTORC1 activity. Restoration of mTORC1 activity via insulin or overexpression of Rheb via lentiviral transfection both attenuated the inhibitory effects of silencing CIRBP on the proliferation and migration of VSMCs. Moreover, Rheb overexpression abolished the inhibitory effect of silencing CIRBP on mTORC1 activity in VSMCs. CIRBP was upregulated in the injured carotid artery. Silencing CIRBP ameliorated intimal hyperplasia after vascular injury. In the summary, silencing CIRBP attenuates mTORC1 activity via reducing Rheb expression, thereby supressing the proliferation and migration of VSMCs and intimal hyperplasia after vascular injury.

Asprosin promotes vascular inflammation via TLR4-NFκB-mediated NLRP3 inflammasome activation in hypertension

Objectiveand design Mild vascular inflammation promotes the pathogenesis of hypertension. Asprosin, a newly discovered adipokine, is closely associated with metabolic diseases. We hypothesized that asprosin might led to vascular inflammation in hypertension via NLRP3 inflammasome formation. This study shows the importance of asprosin in the vascular inflammation of hypertension. MethodsPrimary vascular smooth muscle cells (VSMCs) were obtained from the aorta of animals, including spontaneously hypertensive rats (SHR), Wistar–Kyoto rats (WKY), NLRP3−/− and wild-type mice. Studies were performed in VSMCs in vitro, as well as WKY and SHR in vivo. ResultsAsprosin expressions were up-regulated in VSMCs and media of arteries in SHR. Asprosin overexpression promoted NLRP3 inflammasome activation via Toll-like receptor 4 (TLR4), accompanied with activation of NFκB signaling pathway in VSMCs. Exogenous asprosin protein showed similar roles in promoting NLRP3 inflammasome activation. Knockdown of asprosin restrained NLRP3 inflammasome and p65-NFκB activation in VSMCs of SHR. NLRP3 inhibitor MCC950 or NFκB inhibitor BAY11-7082 attenuated asprosin-caused VSMC proliferation and migration. Asprosin-induced interleukin-1β production, proliferation and migration were attenuated in NLRP3−/− VSMCs. Local asprosin knockdown in common carotid artery of SHR attenuated inflammation and vascular remodeling. ConclusionsAsprosin promoted NLRP3 inflammasome activation in VSMCs by TLR4-NFκB pathway, and thereby stimulates VSMCs proliferation, migration, and vascular remodeling of SHR.

Vascular Smooth Muscle Cell Mechanical Stretch Modulates Tissue Transglutaminase Activity and Cytoskeletal Dynamics

Vascular smooth muscle cells (VSMCs) are exposed to a wide range of mechanical stimuli in both physiological and pathological conditions. These mechanical stimuli are converted into biochemical signals that regulate VSMC contractility and cytoskeletal structures in a process known as mechanotransduction. Thus, the ability of VSMCs to sense and respond to mechanical signals is essential for proper regulation of their structural characteristics and the modulation of vascular tone. We had previously shown that increased tissue transglutaminase 2 (TG2) activity modulates actin dynamics via alterations of the phosphorylation state of cofilin leading to VSMC stiffening and remodeling of the vascular wall. However, whether mechanical forces act through TG2 to modulate VSMC stiffness remains unknown. Herein, we tested the hypothesis that mechanical stretch increases VSMC stiffness by increasing TG2 activity, reducing cofilin activity, and increasing actin cytoskeletal stress fiber formation. To this end, immortalized human aortic vascular smooth muscle cells (HAoSMCs) were cultured and seeded on Uniflex culture plates. HAoSMCs were left unstretched or exposed to cyclic uniaxial stretch using a Flexcell tension system with 11% elongation at 1 Hz for 6 hrs. HAoSMCs were treated without or with the nitric oxide (NO) donor s-nitrosoglutathione (400μmol/L) or cystamine (200μmol/L) under stretched and unstretched conditions, as NO and cystamine are known inhibitors of TG2. All differences reported herein are significant at p<0.05. Our results show that cyclic uniaxial stretch for 6 hrs increases VSMC TG2 content and activity, actin stress fibers (polymerization), and phospho-cofilin/cofilin ratios (cofilin inactivation), relative to the unstretched condition. Notably, the increase in TG2 activity and F-actin content occurring in response to stretch was inhibited by the NO donor or cystamine. As it has been previously shown that cellular stiffness is associated with stress fiber formation, this work suggests that stretch-induced cellular stiffening is potentially mediated by TG2 activity in VSMC. National Institutes of Health grant: R01HL153264 to LM-L and JP. 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 131: Local Pcsk6 Inhibition Attenuates VSMC Response In Vascular Injury And Restenosis

Intro: Vascular smooth muscle cell (VSMC) proliferation following endovascular interventions leads to restenosis, treated with drug-eluting stents. However, current drugs completely abolish vascular healing, cause thrombosis and clinical events. PCSK6 is a key protease in vascular remodeling and VSMCs activation upon vessel injury, via regulation of MMP2/14 activity. Here, we investigate whether local PCSK6 inhibition reduces VSMC proliferation and intimal hyperplasia. Methods: PCSK6 peptide inhibitor was tested in vitro on VSMCs migration, proliferation, apoptosis in comparison to rapamycin and paclitaxel. Internalization of a FITC labelled inhibitor was studied by flow cytometry and immunofluorescence. In vivo , PCSK6 inhibitor was administered at 3mg/kg IP using cationic microbubbles, followed by ultrasound-mediated local delivery in mice developing intimal hyperplasia after carotid ligation (n=8 vs 11). Carotid ligation in Tagln Cre+ /Pcsk6 fl/fl mice investigated the effect of PCSK6 specific ablation in VSMCs on intimal hyperplasia (n=7 vs 9). Carotids were collected from these and constitutive Pcsk6 -/- mice vs. controls for transcriptomic and histological analyses. Results: In vitro , PCSK6 inhibitor was internalized and inhibited both VSMC migration and proliferation (p<0.0001), reducing the expression of PCSK6, MMP2 and GDF15 . In comparison, paclitaxel showed severe VSMC toxicity, whereas rapamycin inhibited more strongly VSMC migration and proliferation than the PCSK6 inhibitor. In vivo, the inhibitor reduced intimal hyperplasia (p=0.026), again, by repressing PCSK6 and MMP2 expression in treated vs. control mice, with a favorable safety profile. Likewise, conditional VSMC Pcsk6 knockouts showed less intimal hyperplasia (p=0.053). In carotids from Pcsk6 -/- mice, cytoskeletal (Synm) and peptidase (Pgpep1l, Klk1) genes were strongly downregulated, showing its importance for vascular matrix composition. Conclusion: Our proof-of-concept translational study shows that local application of a PCSK6 inhibitor is a viable method for specifically modulating VSMC activation in intimal hyperplasia following vascular injury

Long-Term Intrauterine Hypoxia Disrupts Calcium Sparks in Middle Cerebral Arterial Myocytes of Fetal Sheep

Local activation of ryanodine receptors (RyRs) on the sarcoplasmic reticulum in vascular smooth muscle cells (VSMCs) generates rapid subcellular changes in cellular Ca2+ that are commonly known as Ca2+ sparks. These Ca2+ sparks are well regarded for their role in the autoregulation of vascular contractility and cerebral blood flow through activation of potassium channels and feedback modulation of the membrane potential. Gestational hypoxia disrupts cerebrovascular development, increasing the risk of intracranial hemorrhage and stroke in the newborn. Due to the role of RyR’s in regulating neonatal cerebrovascular blood flow, understanding intracellular Ca2+ release patterns in VSMCs can offer insight into hypoxia-related perinatal cerebrovascular disease. The central aim of this study is to determine the extent to which gestational hypoxia disrupts RyR activity. This was achieved through examination of RyR activity in VSMCs of fetal ovine middle cerebral arteries (MCAs). MCAs were isolated from term fetal sheep (~140 days of gestation) delivered by cesarean section from ewes held at low- (700 m) and high-altitude conditions (3801 m) for 100+ days of gestation. Arteries were bathed in a physiological buffer with 5 mm K+ (control) or were treated with either 30 mM K+ (30K), to depolarize myocytes and increase Ca2+ spark events, in the presence or absence of 10 μM ryanodine, to block RyR mediated Ca2+ release. Ca2+ spark activity was measured in situ using line scan confocal microscopy fluorescence imaging techniques. Automated analysis of Ca2+ sparks in the records was conducted using SparkLab 5.8. Membrane depolarization with 30K increased Ca2+ spark activity. Long term hypoxia, however, reduced spark activity in the 30K group. Ryanodine attenuated Ca2+ spark activity in both low- and high-altitude groups. Hypoxia’s influence on the spatial and temporal aspects of Ca2+ sparks in fetal myocytes varied. These findings corroborate previous evidence demonstrating that long-term hypoxia attenuates Ca2+ spark activity. The disruption in Ca2+ spark activity could impact brain blood flow in the neonate, potentially impacting brain development and the likelihood of a traumatic event. R01HL155295, R01HL149608, Advanced Imaging and Microscopy Core Loma Linda University School of Medicine. 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 2065: Activation Of Cgas-sting Axis By DNA Double-strand Breaks In Atherosclerosis: A Possible Link From DNA Damage To Inflammation And Cell Senescence

Aim: Premature atherosclerosis in some progeroid syndromes suggests the involvement of DNA damage in the progression of atherosclerosis. However, causative links between DNA double-strand breaks (DSBs) and atherosclerosis have yet to be determined. The aim of this study was to elucidate the role of DSBs in atherosclerosis using mice and vascular smooth muscle cells (VSMCs) deficient in Ku80, a DSB repair protein. Methods and Results: The plaque size and DSBs were increased in the aorta of Ku80-deficient apolipoprotein E knockout mice (Ku80 +/- ApoE -/- ) compared to those of ApoE -/- control after 4 weeks of a high-fat diet. At the early stages of atherosclerosis (two-week high-fat diet), although the plaque size of Ku80 +/- ApoE -/- was similar to that of ApoE -/- control, the number of DSBs and mRNA levels of inflammatory cytokines such as IL-6 and MCP-1 were significantly increased in the Ku80 +/- ApoE -/- aortas. We further investigated molecular links between DSBs and inflammatory responses using VSMCs isolated from Ku80 +/- mice. In VSMCs from Ku80 +/- mice, IL-6 mRNA level was elevated along with increased cellular senescence markers, such as p16 mRNA and senescence-associated (SA) β-gal activity. Additionally, Ku80 +/- VSMCs showed accumulated cytosolic free DNA, an increased level of cGAMP, and activation of TBK1, interferon regulatory factor 3 (IRF3) and NF-κB, suggesting the activation of cGAS-STING axis, a cytosolic DNA sensing machinery. Activation of cGAS-STING axis was also shown in the aortae from Ku80 +/- ApoE -/- in comparison with those from ApoE -/- control. Silencing IRF3, but not NF-κB attenuated the IL-6 mRNA level in Ku80 +/- VSMCs, so did cGAS-silencing. Of interest, cGAS-silencing also attenuated DNA damage-induced SA β-gal activity in wild-type VSMCs. Furthermore, depletion of either p16 or p53 suppressed IRF3 activation by DNA damage. Conclusions: These results suggest that accumulation of cytosolic DNA induced by DNA damage accelerates atherosclerosis via the activation of cGAS-STING-IRF3 axis. cGAS-STING-IRF3 axis may play a pivotal role as a common signaling pathway that serve as a link from DNA damage to inflammatory and cell senescence response in arterial wall.

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

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

Effects of oleogels (alpha-linolenic acid plus beeswax) extracted supplementation for approaching the therapeutic food ingredient: in vitro model

The dietary fatty acid intake for vascular disease has been highlighted for a decade. This study aimed to determine the effect of palmitic acid and fatty acid-incorporated beeswax (oleogels: OG) supplementation on anti-atherosclerotic effects through vascular smooth muscle cell (VSMC) activity. The A7r5 cells were cultured, cells were treated with three treatments, including 1) control, 2) palmitic acid (PA), and 3) alpha-linolenic acid (ALA) + beeswax, ratio 1:1 (OG) at different concentrations of 0, 25, 50 and 100 μM for 48 hrs. Cell proliferation, cell apoptosis, wound repair, and relative quantity of mRNA level of inflammatory cytokines and angiogenetic transcription factors were determined. The results showed there was a significantly reduced VSMCs proliferation with concentrationdependent (p < 0.05). In the presence of OG at 100 μM, the wound area had healed after 24 hrs (59.2%) compared to PA (28.0%) group treatments. OG and PA supplementation significantly up-regulated eNOS and VEGF but down-regulated the TNF-α, CRP, CD36, iNOS, and NF-κB mRNA expression levels, while PA was a contrasting pattern (p < 0.05). Therefore, supplementation of OG reduced the pro-inflammatory effects of inflammatory cytokines in macrophages and induced VEGF expression in VSMCs, contributing to anti-atherosclerotic effect.

Exploring the effects of Nano-liposomal TGF-β1 on induced pluripotent stem Cell-Derived vascular smooth muscle cells in Tissue-Engineered vascular graft; an in vivo study

This study explores the impact of liposomal Transforming Growth Factor β1 (TGF-β1) on phenotypic switching in Vascular Smooth Muscle Cells (VSMCs) derived from induced pluripotent stem cells (iPSCs) for arterial tissue engineering. We present an innovative approach to reducing graft waiting periods effectively. Using a biomimetic perfusion system, biological scaffolds were developed from sheep's carotid artery to create Tissue-Engineered Vessels (TEVs). VSMCs derived from rabbit iPSCs were reseeded onto these TEVs to promote proliferation. TGF-β1 was encapsulated in liposomes using the thin-film hydration method to enhance VSMC growth. The controlled release of TGF-β1 from these liposomes was quantified through enzyme-linked immunosorbent assay (ELISA). Nano-liposomal TGF-β1 was loaded into the scaffolds at a concentration of 50 ng/mL, with the release monitored through ELISA. The proliferative activity of VSMCs was evaluated using the CCK8 kit. Techniques such as real-time PCR, Western blot, Immunohistochemistry, and Flowcytometry were employed to assess VSMC phenotypic switching. Liposomal TGF-β1 treatment led to a 122.30 % increase in cell proliferation rate in TEVs compared to the control group, indicating no cytotoxicity. ELISA results confirmed a controlled and sustained release of liposomal TGF-β1. Furthermore, we demonstrated the efficacy of liposomal TGF-β1 for sustained delivery over an extended period by implanting the grafts in rabbit aortas and monitoring their performance for four weeks. Our findings highlight the role of Nano-liposomal TGF-β1 in inducing phenotypic transformation within TEVs. This research underscores the promising therapeutic potential of Nano-liposomal TGF-β1 in advancing blood vessel tissue engineering, offering novel pathways for clinical application and addressing the critical need for improved vascular graft outcomes. In conclusion, our study showcases the synergy between a biomimetic environment and Nano-liposomal TGF-β1 on rabbit induced pluripotent stem cell-derived VSMCs, emphasizing the significant increase in SMC proliferation, controlled release of TGF-β1, successful graft implantation, and the pioneering approach in arterial tissue engineering through the development of TEVs from sheep carotid arteries.

No-touch saphenous vein: current understanding of the conduit 'less handled'.

The no-touch technique is an established method to harvest the saphenous vein (NT-SV), which is the most commonly used conduit in coronary artery bypass grafting. Herein, we summarize the foundational evidence, as well as highlight recent innovations and ongoing clinical trials involving NT-SV. Through preservation of perivascular tissue for atraumatic handling and omission of manual distension, the NT-SV maintains endothelial nitrous oxide synthase levels and experiences less vascular smooth muscle cell activation, which translates to slower progression of atherosclerosis and less size mismatch of the graft and target vessel. These biomolecular advantages allow NT-SV to provide superior graft patency compared to conventional skeletonized saphenous vein and approximating that of the radial artery. Nonetheless, the clinical benefits of NT-SV for mortality and reduction in major adverse cardiac and cerebrovascular events are insufficiently studied in the long-term. The drawback of NT-SV is the short-term harvest site complications, which may potentially be addressed by the advent of endoscopic no-touch technique. NT-SV is a promising conduit, and its role will be further clarified in upcoming clinical trials and as follow-up lengthens. However, conduit selection and harvest technique should ultimately be personalized to the individual patient.

Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

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.

Creation of an Atherosclerosis Model Using Palmitic Acid and Oleic Acid in the Vascular Smooth Muscle Cells of Rats.

Atherosclerosis (AS) is a chronic vascular inflammatory disease resulting from vascular endothelial injury and lipid deposition, closely linked to abnormal lipid metabolism within the body. The critical processes involved in atherosclerosis encompass lipid deposition, oxidation, metabolic disruptions, and inflammatory stimulation within the inner vessel wall. Lipid deposition emerges as a pivotal factor triggering these pathological changes, with vascular smooth muscle cells (VSMCs) playing a significant role in the development of AS. Therefore, the goal was to employ lipids, specifically palmitic acid (PA) and oleic acid (OA) solutions, to stimulate VSMCs and create an in vitro atherosclerosis model. This approach allows for the establishment of a rapid and efficient cell model for simulating atherosclerosis in vitro. Primary vascular smooth muscle cells (VSMCs) were isolated and cultured from the thoracic aorta of healthy rats using the tissue-block method. VSMCs were identified through cell climbing slices and immunofluorescence. The growth of VSMCs was observed using light microscopy. The logarithmic growth phase of VSMCs was induced and stimulated by various concentrations of palmitic acid (PA) and oleic acid (OA) ranging from 0 to 650 μmol/L, with a gradient dilution of 50 μmol/L. VSMC activity was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Intracellular lipid deposition was visualized through Oil Red O staining. The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) within VSMCs were quantified using commercially available kits. The optimal conditions for VSMC proliferation were determined to be an OA concentration of 500 μmol/L, a PA concentration of 300 μmol/L, and a culture duration of 48 hours. In comparison to the control group, the presence of lipid droplets within VSMCs became significantly evident following treatment with OA or PA. Furthermore, the levels of TC, TG, and LDL-C increased, while the HDL-C content decreased after treatment with OA or PA. A research model for atherosclerosis (AS) and the early stages of cardiovascular events, specifically lipid deposition, was successfully established through the use of OA and PA solutions. This model has the potential to open up new research avenues for gaining a deeper understanding of the pathogenesis and progression of AS.

Zic family member 3 attenuates oxidative stress-induced vascular smooth muscle cell apoptosis in patients with chronic kidney disease

Neointimal hyperplasia is reportedly essential for arteriovenous fistulas (AVF) in patients undergoing hemodialysis. Oxidative stress is vital in the progression of uremic venous intimal hyperplasia. Studies have suggested that zinc ions obstruct vascular calcification in patients with chronic kidney disease (CKD). Recent studies have shown that the zinc finger protein, Zic family member 3 (ZIC3), is crucial for the earliest cardiovascular progenitors. ZIC3 mutations are associated with congenital heart disease. However, the mechanism of action of ZIC3 in vascular intimal hyperplasia in CKD remains unelucidated. Venous specimens were collected during primary AVF surgery and traumatic amputation, and serum samples were collected from patients with CKD and healthy controls. Mouse vascular smooth muscle cells (VSMCs) were treated with hydrogen peroxide (H2O2) to clarify the role of ZIC3 in CKD. ZIC3 expression was reduced in the veins of patients with uremia and the serum of those with CKD. Zic3 and Bcl2 levels were significantly decreased in mouse VSMCs treated with H2O2·H2O2 inhibited mouse VSMC activity, upregulated Bax, and cleaved caspase 3 expression. Following Zic3 overexpression, Bcl2 expression level and cell viability were elevated, whereas Bax and cleaved caspase 3 expression levels were downregulated. In contrast, Zic3 knockdown yielded the opposite results. Therefore, ZIC3 could be a new therapeutic target in venous neointimal hyperplasia of CKD.

The role of angiotensin II activation of yes-associated protein/PDZ-binding motif signaling in hypertensive cardiac and vascular remodeling

Vascular remodeling is the pathogenic basis of hypertension and end organ injury, and the proliferation of vascular smooth muscle cells (VSMCs) is central to vascular remodeling. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway and crucial for controlling cell proliferation, apoptosis and differentiation. The present study investigated the role of YAP/TAZ in cardiac and vascular remodeling of angiotensin II-induced hypertension. Ang II induced YAP/TAZ activation in the heart and aorta, which was prevented by YAP/TAZ inhibitor verteporfin. Treatment with verteporfin significantly reduced Ang II-induced cardiac and vascular hypertrophy with a mild reduction in systolic blood pressure (SBP), verteporfin attenuated Ang II-induced cardiac and aortic fibrosis with the inhibition of transform growth factor (TGF)β/Smad2/3 fibrotic signaling and extracellular matrix collagen I deposition. Ang II induced Rho A, extracellular signal-regulated kinase 1/2 (ERK1/2) and YAP/TAZ activation in VSMCs, either Rho kinase inhibitor fasudil or ERK inhibitor PD98059 suppressed Ang II-induced YAP/TAZ activation, cell proliferation and fibrosis of VSMCs. Verteporfin also inhibited Ang II-induced VSMC proliferation and fibrotic TGFβ1/Smad2/3 pathway. These results demonstrate that Ang II activates YAP/TAZ via Rho kinase/ERK1/2 pathway in VSMCs, which may contribute to cardiac and vascular remodeling in hypertension. Our results suggest that YAP/TAZ plays a critical role in the pathogenesis of hypertension and end organ damage, and targeting the YAP/TAZ pathway may be a new strategy for the prevention and treatment of hypertension and cardiovascular diseases.

The lysine methyltransferase SMYD2 facilitates neointimal hyperplasia by regulating the HDAC3–SRF axis

Coronary restenosis is an important cause of poor long-term prognosis in patients with coronary heart disease. Here, we show that lysine methyltransferase SMYD2 expression in the nucleus is significantly elevated in serum- and PDGF-BB-induced vascular smooth muscle cells (VSMCs), and in tissues of carotid artery injury-induced neointimal hyperplasia. Smyd2 overexpression in VSMCs (Smyd2-vTg) facilitates, but treatment with its specific inhibitor LLY-507 or SMYD2 knockdown significantly inhibits VSMC phenotypic switching and carotid artery injury-induced neointima formation in mice. Transcriptome sequencing revealed that SMYD2 knockdown represses the expression of serum response factor (SRF) target genes and that SRF overexpression largely reverses the inhibitory effect of SMYD2 knockdown on VSMC proliferation. HDAC3 directly interacts with and deacetylates SRF, which enhances SRF transcriptional activity in VSMCs. Moreover, SMYD2 promotes HDAC3 expression via tri-methylation of H3K36 at its promoter. RGFP966, a specific inhibitor of HDAC3, not only counteracts the pro-proliferation effect of SMYD2 overexpression on VSMCs, but also inhibits carotid artery injury-induced neointima formation in mice. HDAC3 partially abolishes the inhibitory effect of SMYD2 knockdown on VSMC proliferation in a deacetylase activity-dependent manner. Our results reveal that the SMYD2-HDAC3-SRF axis constitutes a novel and critical epigenetic mechanism that regulates VSMC phenotypic switching and neointimal hyperplasia.

Abstract 17777: Sorafenib Aggravates Atherosclerotic Progression Involving With Vascular Smooth Muscle Cell (VSMCs) Phenotypic Switching and Proliferation by PKM2-Mediated Glycolysis

Introduction: As a VEGF-targeting agent, sorafenib has been used to treat a number of solid tumors but can easily lead to adverse vascular effects, including hypertension, myocardiopathy and coronary artery spasm. Our previous study reveals that sorafenib impairs endothelium-dependent vasodilatation, whereas the effect of sorafenib on atherosclerotic progression has not been determined. Hypothesis: Hence, we hypothesize that sorafenib may promote atherosclerotic progression via such adverse vascular effects. Methods: ApoE -/- mice were orally administered by sorafenib for 12 weeks, indicating that sorafenib aggravates atherosclerotic progression via promoting the formation of foam cells and the accumulation of VSMCs in intima. To study the underlying mechanism, we used sorafenib to incubate VSMCs (0, 3 and 6 mg/L) for 48 h. Results: We found that sorafenib promoted the proliferation, migration and synthetic phenotype-related gene expression in VSMCs. More importantly, sorafenib increased lactate production, the extracellular acidification rate, PKM2 expression and PK activity in VSMCs. Interestingly, after deletion of PKM2 in VSMCs by AAV-CRISPR/Cas9 system, decreased lactate production and the extracellular acidification rate were found. Meanwhile, sorafenib-induced the proliferation, migration and phenotypic switching of VSMCs were suppressed. Conclusions: Sorafenib can aggravate atherosclerotic progression and smooth muscle cell phenotypic switching, which may be partly ascribed to PKM2-depended glycolysis.

Angiotensin II reduces glyoxalase 1 activity and expression in vascular smooth muscle cells: Implications for diabetic vascular complications.

Angiotensin II (Ang II), a key mediator of vascular diseases, is linked to methylglyoxal (MGO) formation, a by-product of glucose metabolism implicated in vascular complications. The glyoxalase system, consisting of glyoxalase 1 (Glo1) and reduced glutathione (GSH), is responsible for detoxifying MGO. This study investigated the effect of Ang II on Glo1 activity and expression in vascular smooth muscle cells (VSMCs). Primary VSMCs were isolated from rat aortas and exposed to Ang II under standard or high glucose conditions. We examined Glo1 activity, expression, intracellular GSH, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) levels. We also analyzed the expressions of nuclear factor-κB (NF-κB) p65 and nuclear factor erythroid 2-related factor 2 (Nrf2) as potential regulators of Glo1 expression. The results demonstrated that Ang II reduced Glo1 activity, expression, and GSH levels while increasing MG-H1 levels in VSMCs. Telmisartan and irbesartan, AT1R blockers, restored Glo1 activity, expression, and GSH levels and alleviated MG-H1 levels. Treatment with AT1R blockers or inhibitors targeting signaling pathways involved in Ang II-induced responses mitigated these effects. High glucose exacerbated the reduction in Glo1 activity and expression. In conclusion, this study provides evidence that Ang II reduces Glo1 activity and expression in VSMCs, which may contribute to developing vascular complications in diabetes. AT1R blockers and inhibitors targeting specific signaling pathways show potential in restoring Glo1 function and mitigating MGO-associated damage. These findings highlight the complex interactions between RAS, MGO, and vascular diseases, highlighting potential therapeutic targets for diabetic vascular complications.