Vascular Smooth Muscle Cell Activation Research Articles

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.

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.

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.

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.

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

Selenium (Se) is a key part of the body's oxidation defence system. However, it is unclear whether Se affects the development of aortic aneurysm (AA). An animal experiment was conducted to clarify the role of Se in AA development. C57BL/6N male mice were fed with a Se deficient (Se-D, < 0.05 mg/kg), Se adequate (Se-A, 0.2 mg/kg), or Se supplemented (Se-S, 1 mg/kg) diet for 8 weeks. Subsequently, an AA murine model (Se-D, n = 11; Se-A, n = 12; Se-S, n = 15) was established using angiotensin II (Ang II, 1 mg/kg/min) for 4 weeks plus β-aminopropionitrile (BAPN, 1 mg/mL) for the first 2 weeks. Saline replaced Ang II, and BAPN was removed during the modelling process for sham mice (Se-A, n = 9). To determine whether Se deficiency promotes aortic dilation via matrix metalloproteinase-2 (MMP-2), the non-specific MMP inhibitor doxycycline (Dox, 100 mg/kg/day) was given to Se-D AA mice (n = 7) for 2 weeks. Maximum aortic diameter in Se-D AA model mice was significantly increased compared with Se-A AA model mice. MMP-2 expression and activity in aortic media of Se-D AA model mice was significantly increased compared with Se-A AA model mice. A large number of vascular smooth muscle cells (VSMCs) were found aggregating in the media of non-dilated aorta of Se-D AA model mice, which was completely inhibited by Dox. The percentage of VSMCs in aortic media of Se-D AA model mice was significantly higher than in Se-A AA model mice. Maximum aortic diameter and occurrence rate of AA in Se-D AA model mice with Dox were significantly reduced compared with Se-D AA model mice. Se deficiency promoted dilatation of the aorta in AA model mice by increasing expression and activity of VSMC derived MMP-2, causing abnormal aggregation and proliferation of VSMCs in aortic media.

Redox Dysregulation of Vascular Smooth Muscle Sirtuin-1 in Thoracic Aortic Aneurysm in Marfan Syndrome.

Thoracic aortic aneurysms (TAAs) are abnormal aortic dilatations and a major cardiovascular complication of Marfan syndrome. We previously demonstrated a critical role for vascular smooth muscle (VSM) SirT1 (sirtuin-1), a lysine deacetylase, against maladaptive aortic remodeling associated with chronic oxidative stress and aberrant activation of MMPs (matrix metalloproteinases). In this study, we investigated whether redox dysregulation of SirT1 contributed to the pathogenesis of TAA using fibrillin-1 hypomorphic mice (Fbn1mgR/mgR), an established model of Marfan syndrome prone to aortic dissection/rupture. Oxidative stress markers 3-nitrotyrosine and 4-hydroxynonenal were significantly elevated in aortas of patients with Marfan syndrome. Moreover, reversible oxidative post-translational modifications (rOPTM) of protein cysteines, particularly S-glutathionylation, were dramatically increased in aortas of Fbn1mgR/mgR mice, before induction of severe oxidative stress markers. Fbn1mgR/mgR aortas and VSM cells exhibited an increase in rOPTM of SirT1, coinciding with the upregulation of acetylated proteins, an index of decreased SirT1 activity, and increased MMP2/9 activity. Mechanistically, we demonstrated that TGFβ (transforming growth factor beta), which was increased in Fbn1mgR/mgR aortas, stimulated rOPTM of SirT1, decreasing its deacetylase activity in VSM cells. VSM cell-specific deletion of SirT1 in Fbn1mgR/mgR mice (SMKO-Fbn1mgR/mgR) caused a dramatic increase in aortic MMP2 expression and worsened TAA progression, leading to aortic rupture in 50% of SMKO-Fbn1mgR/mgR mice, compared with 25% of Fbn1mgR/mgR mice. rOPTM of SirT1, rOPTM-mediated inhibition of SirT1 activity, and increased MMP2/9 activity were all exacerbated by the deletion of Glrx (glutaredoxin-1), a specific deglutathionylation enzyme, while being corrected by overexpression of Glrx or of an oxidation-resistant SirT1 mutant in VSM cells. Our novel findings strongly suggest a causal role of S-glutathionylation of SirT1 in the pathogenesis of TAA. Prevention or reversal of SirT1 rOPTM may be a novel therapeutic strategy to prevent TAA and TAA dissection/ruptures in individuals with Marfan syndrome, for which, thus far, no targeted therapy has been developed.

BRD4770 inhibits vascular smooth muscle cell proliferation via SUV39H2, but not EHMT2 to protect against neointima formation.

The behavior of vascular smooth muscle cells (VSMCs) contributes to the formation of neointima. We previously found that EHMT2 suppressed autophagy activation in VSMCs. BRD4770, an inhibitor of EHMT2/G9a, plays a critical role in several kinds of cancers. However, whetherand how BRD4770 regulates the behavior of VSMCs remain unknown. In this study, we evaluate the cellular effect of BRD4770 on VSMCs by series of experiments in vivo and ex vivo. We demonstrated that BRD4770 inhibited VSMCs' growth by blockage in G2/M phase in VSMCs. Moreover, our results demonstrated that the inhibition of proliferation was independent on autophagy or EHMT2 suppression which we previous reported. Mechanistically, BRD4770 exhibited an off-target effect from EHMT2 and our further study reveal that the proliferation inhibitory effect by BRD4770 was associated with suppressing on SUV39H2/KTM1B. In vivo, BRD4770 was also verified to rescue VIH. Thus, BRD4770 function as a crucial negative regulator of VSMC proliferation via SUV39H2 and G2/M cell cycle arrest and BRD4770 could be a molecule for the therapy of vascular restenosis.

Alterations of the Ca2+ clearing mechanisms by type 2 diabetes in aortic smooth muscle cells of Zucker diabetic fatty rat.

Type 2 Diabetes Mellitus (T2DM) is a rapidly rising disease with cardiovascular complications constituting the most common cause of death among diabetic patients. Chronic hyperglycemia can induce vascular dysfunction through damage of the components of the vascular wall, such as vascular smooth muscle cells (VSMCs), which regulate vascular tone and contribute to vascular repair and remodeling. These functions are dependent on intracellular Ca2+ changes. The mechanisms by which T2DM affects Ca2+ handling in VSMCs still remain poorly understood. Therefore, the objective of this study was to determine whether and how T2DM affects Ca2+ homeostasis in VSMCs. We evaluated intracellular Ca2+ signaling in VSMCs from Zucker Diabetic Fatty rats using Ca2+ imaging with Fura-2/AM. Our results indicate that T2DM decreases Ca2+ release from the sarcoplasmic reticulum (SR) and increases the activity of store-operated channels (SOCs). Moreover, we were able to identify an enhancement of the activity of the main Ca2+ extrusion mechanisms (SERCA, PMCA and NCX) during the early stage of the decay of the ATP-induced Ca2+ transient. In addition, we found an increase in Ca2+ entry through the reverse mode of NCX and a decrease in SERCA and PMCA activity during the late stage of the signal decay. These effects were appreciated as a shortening of ATP-induced Ca2+ transient during the early stage of the decay, as well as an increase in the amplitude of the following plateau. Enhanced cytosolic Ca2+ activity in VSMCs could contribute to vascular dysfunction associated with T2DM.

Abstract 607: Activation Of Yes-associated Protein(yap)/pdz-binding Motif (taz) Signaling By Angiotensin II Contributes To Hypertensive Cardiacand Vascular Remodeling

Vascular remodeling is pathogenic basis of hypertension and end organ injury, 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 Hippo pathway and crucial for control of 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 were 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)b/SMAD fibrotic signaling. Ang II induced Rho A, extracellular signal-regulated kinase 1/2 and YAP/TAZ activation in VSMCs, either Rho kinase inhibitor fasudi, or ERK inhibitor PD98059 suppressed Ang II activation of YAP/TAZ . Verteporfin or fasudi also inhibited Ang II-induce VSMC proliferation and fibrotic TGFβ1/SMAD1/2 pathway. These results demonstrate that Ang II activates YAP/TAZ via Rho kinase/ERK1/2 pathway in VSMCs, which may contribute to Ang II-induced cardiac and vascular remodeling in hypertension. Our results suggest that YAP/TAZ plays a critical role in hypertension and hypertensive end organ injury, targeting YAP/TAZ pathway may be a new strategy for prevention and treatment of hypertension and cardiovascular diseases.

Orally administered sodium nitrite prevents the increased α-1 adrenergic vasoconstriction induced by hypertension and promotes the S-nitrosylation of calcium/calmodulin-dependent protein kinase II

The unsatisfactory rates of adequate blood pressure control among patients receiving antihypertensive treatment calls for new therapeutic strategies to treat hypertension. Several studies have shown that oral sodium nitrite exerts significant antihypertensive effects, but the mechanisms underlying these effects remain unclear. While these mechanisms may involve nitrite-derived S-nitrosothiols, their implication in important alterations associated with hypertension, such as aberrant α1-adrenergic vasoconstriction, has not yet been investigated. Here, we examined the effects of oral nitrite treatment on vascular responses to the α1-adrenergic agonist phenylephrine in two-kidney, one clip (2K1C) hypertensive rats and investigated the potential underlying mechanisms. Our results show that treatment with oral sodium nitrite decreases blood pressure and prevents the increased α1-adrenergic vasoconstriction in 2K1C hypertensive rats. Interestingly, we found that these effects require vascular protein S-nitrosylation, and to investigate the specific S-nitrosylated proteins we performed an unbiased nitrosoproteomic analysis of vascular smooth muscle cells (VSMCs) treated with the nitrosylating compound S-nitrosoglutathione (GSNO). This analysis revealed that GSNO markedly increases the nitrosylation of calcium/calmodulin-dependent protein kinase II γ (CaMKIIγ), a multifunctional protein that mediates the α1-adrenergic receptor signaling. This result was associated with reduced α1-adrenergic receptor-mediated CaMKIIγ activity in VSMCs. We further tested the relevance of these findings in vivo and found that treatment with oral nitrite increases CaMKIIγ S-nitrosylation and blunts the increased CaMKIIγ activity induced by phenylephrine in rat aortas. Collectively, these results are consistent with the idea that oral sodium nitrite treatment increases vascular protein S-nitrosylation, including CaMKIIγ as a target, which may ultimately prevent the increased α1-adrenergic vasoconstriction induced by hypertension. These mechanisms may help to explain the antihypertensive effects of oral nitrite and hold potential implications in the therapy of hypertension and other cardiovascular diseases associated with abnormal α1-adrenergic vasoconstriction.

Vascular-Parenchymal Cross-Talk Promotes Lung Fibrosis through BMPR2 Signaling.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive lung scarring. IPF-related pulmonary vascular remodeling and pulmonary hypertension (PH) result in a particularly poor prognosis. Objectives: To study the pathogenesis of vascular remodeling in fibrotic lungs and its contribution to progression of fibrosis. Methods: We used an experimental model of lung fibrosis associated with PH by transient overexpression of active TGF-β1 (transforming growth factor-β1). Samples from patients with fibrotic lung diseases were analyzed in depth using immunostaining, gene expression, and gene mutations. Measurements and Main Results: We found a reduction in endothelial cells (ECs) and activation of vascular smooth muscle cells (VSMCs) in fibrotic lungs. Coculturing fibroblasts with VSMCs or ECs from fibrotic lungs induced fibrotic phenotypes in fibroblasts. IPF fibroblasts induced EC death and activation of VSMCs in coculture systems. Decreased concentrations of BMPR2 (bone morphogenic protein receptor 2) and its signaling were observed in ECs and VSMCs from fibrotic lungs in both rats and humans. On fibroblasts treated with media from VSMCs, BMPR2 suppression in VSMCs led to fibrogenic effects. Tacrolimus activated BMPR2 signaling and attenuated fibrosis and PH in rodent lungs. Whole-exome sequencing revealed rare mutations in PH-related genes, including BMPR2, in patients with IPF undergoing transplantation. A unique missense BMPR2 mutation (p.Q721R) was discovered to have dysfunctional effects on BMPR2 signaling. Conclusions: Endothelial dysfunction and vascular remodeling in PH secondary to pulmonary fibrosis enhance fibrogenesis through impaired BMPR2 signaling. Tacrolimus may have value as a treatment of advanced IPF and concomitant PH. Genetic abnormalities may determine the development of PH in advanced IPF.

BACH1 deficiency prevents neointima formation and maintains the differentiated phenotype of vascular smooth muscle cells by regulating chromatin accessibility.

The transcription factor BTB and CNC homology 1(BACH1) has been linked to coronary artery disease risk by human genome-wide association studies, but little is known about the role of BACH1 in vascular smooth muscle cell (VSMC) phenotype switching and neointima formation following vascular injury. Therefore, this study aims to explore the role of BACH1 in vascular remodeling and its underlying mechanisms. BACH1 was highly expressed in human atherosclerotic plaques and has high transcriptional factor activity in VSMCs of human atherosclerotic arteries. VSMC-specific loss of Bach1 in mice inhibited the transformation of VSMC from contractile to synthetic phenotype and VSMC proliferation and attenuated the neointimal hyperplasia induced by wire injury. Mechanistically, BACH1 suppressed chromatin accessibility at the promoters of VSMC marker genes via recruiting histone methyltransferase G9a and cofactor YAP and maintaining the H3K9me2 state, thereby repressing VSMC marker genes expression in human aortic smooth muscle cells (HASMCs). BACH1-induced repression of VSMC marker genes was abolished by the silencing of G9a or YAP. Thus, these findings demonstrate a crucial regulatory role of BACH1 in VSMC phenotypic transition and vascular homeostasis and shed light on potential future protective vascular disease intervention via manipulation of BACH1.

Inhibition of mitochondrial phosphate carrier prevents high phosphate-induced superoxide generation and vascular calcification

Vascular calcification is a serious complication of hyperphosphatemia that causes cardiovascular morbidity and mortality. Previous studies have reported that plasmalemmal phosphate (Pi) transporters, such as PiT-1/2, mediate depolarization, Ca2+ influx, oxidative stress, and calcific changes in vascular smooth muscle cells (VSMCs). However, the pathogenic mechanism of mitochondrial Pi uptake in vascular calcification associated with hyperphosphatemia has not been elucidated. We demonstrated that the phosphate carrier (PiC) is the dominant mitochondrial Pi transporter responsible for high Pi-induced superoxide generation, osteogenic gene upregulation, and calcific changes in primary VSMCs isolated from rat aortas. Notably, acute incubation with high Pi markedly increased the protein abundance of PiC via ERK1/2- and mTOR-dependent translational upregulation. Genetic suppression of PiC prevented Pi-induced ERK1/2 activation, superoxide production, osteogenic differentiation, and vascular calcification of VSMCs in vitro and aortic rings ex vivo. Pharmacological inhibition of mitochondrial Pi transport using butyl malonate (BMA) or mersalyl abolished all pathologic changes involved in high Pi-induced vascular calcification. BMA or mersalyl also effectively prevented osteogenic gene upregulation and calcification of aortas from 5/6 subtotal nephrectomized mice fed a high-Pi diet. Our results suggest that mitochondrial Pi uptake via PiC is a critical molecular mechanism mediating mitochondrial superoxide generation and pathogenic calcific changes, which could be a novel therapeutic target for treating vascular calcification associated with hyperphosphatemia.