Vascular Smooth Muscle Cell Line Research Articles

Role of miRNAs regulation of Orai1 and store operated Ca2+ entry in vascular restenosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación Background The main cause of restenosis is attributed to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Orai1 is one of the main molecular components of Store-Operated Calcium Entry (SOCE), playing a key role in VSMCs proliferation during neointima formation and vascular remodeling. Recent studies have demonstrated the implication of microRNAs (miRNAs) in VSMCs proliferation and migration, however, the role of these miRNAs as regulators of SOCE during vascular remodeling has not been described. Purpose This study aims to determine whether miRNAs regulate the intracellular Ca2+ handling, focusing on SOCE, during neointima formation. Methods Experiments were conducted in an animal rat model following the "Principles of laboratory animal care" and according to the Spanish law. The neointima development was assessed after balloon injury of carotid arteries and confirmed by haematoxylin and eosin staining, and αSMA staining of tissue sections up to 3 weeks after surgery. miRNAs array and RT-qPCR was used to examine the expression of miRNAs. Dual-Luciferase reporter assay was performed in A7R5 VSMCs line. Results Injured arteries showed significant increase in the expression of Orai1, as compared to non-injured arteries, indicating their possible participation in the neointima layer formation. Microarray of miRNAs analysis of the injured carotids compared to control carotids showed significant increase in the expression of miRNAs related to signalling pathways involving cell proliferation and migration, cell differentiation, or VSMCs contraction. Of these, we confirmed the significant increase of miR-18a-5p, miR-20a-5p, miR-20b-5p and miR-17-5p, which belong to widely studied miR-17-92 cluster involved in different cell proliferation. Moreover, we found that mimics of those miRNAs were able to regulate differentially the activity of the Orai1 promotor in A7R5 cell line, indicating that Orai1 can be targeted by miRNAs during in VSMCs. Conclusions In conclusion, our data confirms that Orai1 is upregulated during neointima formation associated with miRNAs dysregulation. Moreover, we found that Orai1-dependent SOCE can be finely regulated by miRNAs during restenosis.

Enhanced isradipine sensitivity in vascular smooth muscle cells due to hypoxia-induced Cav1.2 splicing and RbFox1/Fox2 downregulation.

Calcium influx via the L-type voltage-gated Cav1.2 calcium channel in smooth muscle cells regulates vascular contraction. Calcium channel blockers (CCBs) are widely used to treat hypertension by inhibiting Cav1.2 channels. Using the vascular smooth muscle cell line, A7r5 and primary culture of cerebral vascular smooth muscle cells, we found that the expression and function of Cav1.2 channels are downregulated during hypoxia. Furthermore, hypoxia induces structural changes in Cav1.2 channels via alternative splicing. The expression of exon 9* is upregulated, whereas exon 33 is downregulated. Such structural alterations of Cav1.2 channels are caused by the decreased expression of RNA-binding proteins RNA-binding protein fox-1 homolog 1 and 2 (RbFox1 and RbFox2). Overexpression of RbFox1 and RbFox2 prevents hypoxia-induced exon 9* inclusion and exon 33 exclusion. Importantly, such structural alterations of the Cav1.2 channel partly contribute to the enhanced sensitivity of Cav1.2 to isradipine (a CCB) under hypoxia. Overexpression of RbFox1 and RbFox2 successfully reduces isradipine sensitivity in hypoxic smooth muscle cells. Our results suggest a new strategy to manage ischemic diseases such as stroke and myocardial infarction.

Two methods of isolation of rat aortic smooth muscle cells with high yield

Abstract Vascular smooth muscle cells (VSMCs) are an integral part of blood vessels and are the focus of intensive research in vascular biology, translational research, and cardiovascular diseases. Though immortalized vascular smooth muscle cell lines are available, their use is limited, underscoring the need for primary VSMCs. There are several methods for isolating primary cells from mice. However, the isolation method from rat blood vessels requires optimization, given the differences in the aorta of mice and rats. Here we compare two methods for VSMCs isolation from rats: enzymatic digestion and the “block” method. We observed a significantly higher yield of VSMCs using the enzymatic digestion method. We further confirmed that VSMCs expressed well-established VSMC-specific markers (calponin) with both methods and observed the persistence of this marker up to 9 passages, suggesting a continuation of the secretory phenotype of VSMCs. Overall, this work compares two methods and demonstrates a practical and effective method for isolating VSMCs from rat aorta, providing vascular biologists with a valuable and reliable experimental tool.

Tunable electrospun scaffolds of polyacrylonitrile loaded with carbon nanotubes: from synthesis to biological applications.

Growing cells in a biomimetic environment is critical for tissue engineering as well as for studying the cell biology underlying disease mechanisms. To this aim a range of 3D matrices have been developed, from hydrogels to decellularized matrices. They need to mimic the extracellular matrix to ensure the optimal growth and function of cells. Electrospinning has gained in popularity due to its capacity to individually tune chemistry and mechanical properties and as such influence cell attachment, differentiation or maturation. Polyacrylonitrile (PAN) derived electrospun fibres scaffolds have shown exciting potential due to reports of mechanical tunability and biocompatibility. Building on previous work we fabricate here a range of PAN fibre scaffolds with different concentrations of carbon nanotubes. We characterize them in-depth in respect to their structure, surface chemistry and mechanical properties, using scanning electron microscopy, image processing, ultramicrotomic transmission electron microscopy, x-ray nanotomography, infrared spectroscopy, atomic force microscopy and nanoindentation. Together the data demonstrate this approach to enable finetuning the mechanical properties, while keeping the structure and chemistry unaltered and hence offering ideal properties for comparative studies of the cellular mechanobiology. Finally, we confirm the biocompatibility of the scaffolds using primary rat cardiomyocytes, vascular smooth muscle (A7r5) and myoblast (C2C12) cell lines.

Inhibition of miR-33a-5p in Macrophage-like Cells In Vitro Promotes apoAI-Mediated Cholesterol Efflux.

Atherosclerosis is caused by cholesterol accumulation within arteries. The intima is where atherosclerotic plaque accumulates and where lipid-laden foam cells reside. Intimal foam cells comprise of both monocyte-derived macrophages and macrophage-like cells (MLC) of vascular smooth muscle cell (VSMC) origin. Foam cells can remove cholesterol via apoAI-mediated cholesterol efflux and this process is regulated by the transporter ABCA1. The microRNA miR-33a-5p is thought to be atherogenic via silencing ABCA1 which promotes cholesterol retention and data has shown inhibiting miR-33a-5p in macrophages may be atheroprotective via enhancing apoAI-mediated cholesterol efflux. However, it is not entirely elucidated whether precisely inhibiting miR-33a-5p in MLC also increases ABCA1-dependent cholesterol efflux. Therefore, the purpose of this work is to test the hypothesis that inhibition of miR-33a-5p in cultured MLC enhances apoAI-mediated cholesterol efflux. In our study, we utilized the VSMC line MOVAS cells in our experiments, and cholesterol-loaded MOVAS cells to convert this cell line into MLC. Inhibition of miR-33a-5p was accomplished by transducing cells with a lentivirus that expresses an antagomiR directed at miR-33a-5p. Expression of miR-33a-5p was analyzed by qRT-PCR, ABCA1 protein expression was assessed via immunoblotting, and apoAI-mediated cholesterol efflux was measured using cholesterol efflux assays. In our results, we demonstrated that lentiviral vector-mediated knockdown of miR-33a-5p resulted in decreasing expression of this microRNA in cultured MLC. Moreover, reduction of miR-33a-5p in cultured MLC resulted in de-repression of ABCA1 expression, which caused ABCA1 protein upregulation in cultured MLC. Additionally, this increase in ABCA1 protein expression resulted in enhancing ABCA1-dependent cholesterol efflux through increasing apoAI-mediated cholesterol efflux in cultured MLC. From these findings, we conclude that inhibiting miR-33a-5p in MLC may protect against atherosclerosis by promoting ABCA1-dependent cholesterol efflux.

Hesperetin Relaxes Depolarizing Contraction in Human Umbilical Vein by Inhibiting L-Type Ca2+ Channel.

To study hesperetin-induced vasorelaxation after depolarizing contraction in human umbilical veins (HUVs) to elucidate the role of L-type Ca2+ channel (LTCC) and related signaling pathway. Isometric tension recording was performed in HUV rings pre-contracted with K+. Hesperetin relaxing mechanism was investigated using a LTCC opener (BayK8644) and blockers of cyclic nucleotides and phosphodiesterases (PDEs). Whole-cell patch-clamping in A7r5 cells, a rat vascular smooth muscle cell line, was performed to study the effect of hesperetin on LTCC current. After depolarizing precontraction, hesperetin induced HUV relaxation concentration-dependently and endothelium-independently; 1 mmol/L hesperetin reduced denuded HUV ring tension by 68.7% ± 4.3% compared to matching vehicle, osmolality, and time controls (P<0.0001). Importantly, hesperetin competitively inhibited BayK8644-induced contraction, shifting the half maximal effective concentration of BayK8644 response from 1.08 nmol/L [95% confidence interval (CI) 0.49-2.40] in vehicle control to 11.30 nmol/L (95% CI 5.45-23.41) in hesperetin (P=0.0001). Moreover, hesperetin elicited further vasorelaxation in denuded HUV rings pretreated with inhibitors of soluble guanylyl cyclase, adenylyl cyclase, PDE3, PDE4, and PDE5 (P<0.01), while rings pretreated with PDE1 inhibitors could not be relaxed by hesperetin (P>0.05). However, simultaneously applying inhibitors of soluble guanylyl cyclase and adenylyl cyclase could not inhibit hesperetin's effect (P>0.05). In whole-cell patch-clamping, hesperetin rapidly decreased LTCC current in A7r5 cells to 66.7% ± 5.8% (P=0.0104). Hesperetin diminishes depolarizing contraction of human vascular smooth muscle through inhibition of LTCC, and not cyclic nucleotides nor PDEs. Our evidence supports direct LTCC interaction and provides additional basis for the use of hesperetin and its precursor hesperidin as vasodilators and may lead to future vasodilator drug development as a treatment alternative for cardiovascular diseases.

Dissecting the Impact of Vascular Smooth Muscle Cell ABCA1 versus ABCG1 Expression on Cholesterol Efflux and Macrophage-like Cell Transdifferentiation: The Role of SR-BI.

Cholesterol-laden macrophages are recognized as a major contributor to atherosclerosis. However, recent evidence indicates that vascular smooth muscle cells (VSMC) that accumulate cholesterol and transdifferentiate into a macrophage-like cell (MLC) phenotype also play a role in atherosclerosis. Therefore, removing cholesterol from MLC may be a potential atheroprotective strategy. The two transporters which remove cholesterol from cells are ABCA1 and ABCG1, as they efflux cholesterol to apoAI and HDL, respectively. In this study, the well-characterized immortalized VSMC line MOVAS cells were edited to generate ABCA1- and ABCG1-knockout (KO) MOVAS cell lines. We cholesterol-loaded ABCA1-KO MOVAS cells, ABCG1-KO MOVAS cells, and wild-type MOVAS cells to convert cells into a MLC phenotype. When we measured apoAI- and HDL-mediated cholesterol efflux in these cells, we observed a drastic decrease in apoAI-mediated cholesterol efflux within ABCA1-KO MOVAS MLC, but HDL-mediated cholesterol efflux was only partially reduced in ABCG1-KO MOVAS cells. Since SR-BI also participates in HDL-mediated cholesterol efflux, we assessed SR-BI protein expression in ABCG1-KO MOVAS MLC and observed SR-BI upregulation, which offered a possible mechanism explaining why HDL-mediated cholesterol efflux remains maintained in ABCG1-KO MOVAS MLC. When we used lentivirus for shRNA-mediated knockdown of SR-BI in ABCG1-KO MOVAS MLC, this decreased HDL-mediated cholesterol efflux when compared to ABCG1-KO MOVAS MLC with unmanipulated SR-BI expression. Taken together, these major findings suggest that SR-BI expression in MLC of a VSMC origin plays a compensatory role in HDL-mediated cholesterol efflux when ABCG1 expression becomes impaired and provides insight on SR-BI demonstrating anti-atherogenic properties within VSMC/MLC.

MiR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells.

Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.

Role of Uric Acid in Vascular Remodeling: Cytoskeleton Changes and Migration in VSMCs.

The mechanisms by which hyperuricemia induces vascular dysfunction and contributes to cardiovascular disease are still debated. Phenotypic transition is a property of vascular smooth muscle cells (VSMCs) involved in organ damage. The aim of this study was to investigate the effects of uric acid (UA) on changes in the VSMC cytoskeleton, cell migration and the signals involved in these processes. MOVAS, a mouse VSMC line, was incubated with 6, 9 and 12 mg/dL of UA, angiotensin receptor blockers (ARBs), proteasome and MEK-inhibitors. Migration property was assessed in a micro-chemotaxis chamber and by phalloidin staining. Changes in cytoskeleton proteins (Smoothelin B (SMTB), alpha-Smooth Muscle Actin (αSMA), Smooth Muscle 22 Alpha (SM22α)), Atrogin-1 and MAPK activation were determined by Western blot, immunostaining and quantitative reverse transcription PCR. UA exposition modified SMT, αSMA and SM22α levels (p < 0.05) and significantly upregulated Atrogin-1 and MAPK activation. UA-treated VSMCs showed an increased migratory rate as compared to control cells (p < 0.001) and a re-arrangement of F-actin. Probenecid, proteasome inhibition and ARBs prevented the development of dysfunctional VSMC. This study shows, for the first time, that UA-induced cytoskeleton changes determine an increase in VSMC migratory rate, suggesting UA as a key player in vascular remodeling.

Effect of miRNA-218-5p on Proliferation, Migration, Apoptosis and Inflammation of Vascular Smooth Muscle Cells in Abdominal Aortic Aneurysm and Extracellular Matrix Protein.

To explore the effects of miRNA-218-5p on inflammation and extracellular matrix proteins of vascular smooth muscle cell line in abdominal aortic aneurysm (AAA). miR-218-5p expression was detected with RT-qPCR. The proliferative activity of vascular smooth muscle cells (VSMCs) was detected with CCK-8, the migration was detected by Transwell, and the apoptosis was detected with flow cytometry. The expression levels of inflammatory factors (IL-1β and IL-18) were detected by ELISA. The expression levels of proteins (MMP-9 and Netrin-1) and ADAMTS5 were detected by Western blot. The targeting relationship between miR-218-5p and ADAMTS5 was verified with dual-luciferase reporter assay. Up-regulating miR-218-5p could significantly inhibit the proliferation and migration of VSMCs and induced the apoptosis (P<0.05). Down-regulating miR-218-5p could significantly promote the proliferation and migration of VSMCs and inhibit the apoptosis (P<0.05). Up-regulating miR-218-5p could inhibit the expression levels of THP-1 cytoinflammatory factors (IL-8 and IL-1β), MMP-9 and netrin-1. ADAMTS5 was the target gene of miR-218-5p. When there were both overexpression of ADAMTS5 and upregulation of miR-218-5p, the upregulation of miR-218-5p could alleviate the effects of overexpression of ADAMTS5 on the proliferation, migration and apoptosis of VSMCs. miR-218-5p/ADAMTS-5 molecular axis regulates the proliferation, migration, and apoptosis of VSMCs, as well as the expression of THP-1 cell inflammatory molecules and extracellular matrix molecules.

Abstract 9535: The Effect of Vascular Smooth Muscle Cell miR-33a Expression on apoAI-Mediated Cholesterol Efflux and Macrophage-Like Cell Transdifferentiation

Background: ABCA1 removes cellular cholesterol by participating in apoAI-mediated cholesterol efflux. MiR-33a is an SREBP-2 intronic miRNA that silences ABCA1, which impairs ABCA1-dependent cholesterol efflux. Thus, miR-33a expression may be atherogenic and previous data suggests inhibiting miR-33a in certain cells is atheroprotective. However, current data is scant on whether miR-33a expression in vascular smooth muscle cells (VSMC) is atherogenic. Moreover, cholesterol accumulation in VSMC results in these cells to transdifferentiate into an atherogenic macrophage-like cell (MLC). Therefore, ablating miR-33a expression in VSMC may be atheroprotective via hindering cholesterol accumulation and MLC transdifferentiation in these cells by enhancing apoAI-mediated cholesterol efflux. Methods: An immortalized VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells via using CRISPR/Cas9. We used these cells and parental wild-type (WT) MOVAS cells in our studies to assess whether apoAI-mediated cholesterol efflux is increased in MLC of VSMC origin when miR-33a expression is ablated. We also analyzed whether miR-33a ablation rapidly restores VSMC phenotype in MLC of VSMC origin when cholesterol-loaded cells were exposed to apoAI. Results: We used PCR to confirm that miR-33a was deleted in miR-33a KO MOVAS cells. We also showed that miR-33a-5p and miR-33a-3p expression was absent in miR-33a KO MOVAS cells via RT-PCR. SREBP-2 functionality remained intact in miR-33a KO MOVAS cells, as determined by upregulation of SREBP-2 target genes in serum-starved conditions. When we cholesterol-loaded miR-33a KO MOVAS cells to induce MLC transdifferentiation, we observed increased apoAI-mediated cholesterol efflux when compared to WT MOVAS cells exposed to the same conditions. Based on RT-qPCR analysis, we also observed a rapid restoration in VSMC phenotype when cholesterol-loaded miR-33a KO MOVAS cells were incubated with apoAI when compared to WT MOVAS cells treated with the same conditions. Conclusions: MiR-33a expression in VSMC appears to promote MLC transdifferentiation by impairing apoAI-mediated cholesterol efflux. Future studies are underway to determine if these effects are mediated by miR-33a-5p and/or miR-33a-3p.

2,3-Diphospho-D-glyceric acid inhibits calciprotein particle growth and calcification in MOVAS cells but not in MC3T3-E1 cells

Anionic polyphosphate compounds are well-known to inhibit the formation of hydroxyapatite crystals. Vascular calcification (VC) is associated with an increase in crystalline calciprotein particles (CPPs) in the bloodstream. 2,3-Diphospho-D-glyceric acid (2,3-DPG) is a highly anionic Poly-P compound found in the concave center of erythrocytes where it binds to hemoglobin, thereby reducing oxygen affinity. Since 2.3-DPG exists in plasma, we hypothesized that 2,3-DPG may act as an endogenous inhibitor of VC. In comparison to widely recognized calcification inhibitors such as myo-inositol hexakisphosphate, 2,3-DPG significantly delayed the formation of crystalline CPPs. Additionally, 2,3-DPG inhibited calcification in a mouse vascular smooth muscle cell line (MOVAS) without cytotoxic effects. Taken together with the result that 2,3-DPG did not impact bone-like nodule formation in mouse osteoblast-like MC3T3-E1 cells, these results suggest that 2,3-DPG may inhibit VC selectively.

Yohimbine, an α2-Adrenoceptor Antagonist, Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway.

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.

Glycolaldehyde induces synergistic effects on vascular inflammation in TNF-α-stimulated vascular smooth muscle cells.

Atherosclerosis is a chronic inflammatory disease that contributes to disease progression is associated with the expression of adhesion molecules in vascular smooth muscle cells (VSMCs). Glycolaldehyde (GA) has been shown to impair cellular function in various disorders, including diabetes, and renal diseases. This study investigated the effect of GA on the expression of adhesion molecules in the mouse VSMC line, MOVAS-1. Co-incubation of VSMCs with GA (25-50 μM) dose-dependently increased the protein and mRNA level of Vcam-1 and ICAM-1. Additionally, GA upregulated intracellular ROS production and phosphorylation of MAPK and NK-κB. GA also elevated TNF-α-induced PI3K-AKT activation. Furthermore, GA enhanced TNF-α-activated IκBα kinase activation, subsequent IκBα degradation, and nuclear translocation of NF-κB. These findings suggest that GA stumulated VSMC adhesive capacity and the induction of VCAM-1 and ICAM-1 in VSMCs through inhibition of MAPK and NF-κB signaling pathways, providing insights into the effect of GA to induce inflammation within atherosclerotic lesions.

Ginsenoside Rg-1 prevents elevated cytosolic Ca2+ via store-operated Ca2+ entry in high-glucose–stimulated vascular endothelial and smooth muscle cells

BackgroundGinsenoside Rg-1 (Rg-1), a triterpenoid saponin abundantly present in Panax ginseng, is a type of naturally occurring steroid with known anti-diabetic and anti-inflammatory effects. In this study, we sought to confirm the effects and mechanisms of action of Rg-1 on store-operated Ca2+ entry (SOCE) in human vascular endothelial cell line (EA) and murine aortic vascular smooth muscle cell line (MOVAS) cells exposed to high glucose.MethodsCytosolic Ca2+ concentrations in EA and MOVAS cells were measured by monitoring fluorescence of the ratiometric Ca2+-indicator, Fura-2 AM.ResultsHigh glucose significantly increased Ca2+ influx by abnormally activating SOCE in EA and MOVAS cells. Notably, this high glucose-induced increase in SOCE was restored to normal levels in EA and MOVAS cells by Rg-1. Moreover, Rg-1 induced reductions in SOCE in cells exposed to high glucose were significantly inhibited by the plasma membrane Ca2+ ATPase (PMCA) blocker lanthanum, the Na+/K+-ATPase blocker ouabain, or the Na+/Ca2+ exchanger (NCX) blockers Ni2+ and KB-R7943. These observations suggest that the mechanism of action of Rg-1 inhibition of SOCE involves PMCA and Na+/K+-ATPase, and an increase in Ca2+ efflux via NCXs in both EA and MOVAS cells exposed to high glucose.ConclusionsThese findings indicate that Rg-1 may protect vascular endothelial and smooth muscle cells from Ca2+ increases following exposure to hyperglycemic conditions.

Vitamin D Treatment Prevents Uremia-Induced Reductions in Aortic microRNA-145 Attenuating Osteogenic Differentiation despite Hyperphosphatemia.

In chronic kidney disease, systemic inflammation and high serum phosphate (P) promote the de-differentiation of vascular smooth muscle cells (VSMC) to osteoblast-like cells, increasing the propensity for medial calcification and cardiovascular mortality. Vascular microRNA-145 (miR-145) content is essential to maintain VSMC contractile phenotype. Because vitamin D induces aortic miR-145, uremia and high serum P reduce it and miR-145 directly targets osteogenic osterix in osteoblasts, this study evaluated a potential causal link between vascular miR-145 reductions and osterix-driven osteogenic differentiation and its counter-regulation by vitamin D. Studies in aortic rings from normal rats and in the rat aortic VSMC line A7r5 exposed to calcifying conditions corroborated that miR-145 reductions were associated with decreases in contractile markers and increases in osteogenic differentiation and calcium (Ca) deposition. Furthermore, miR-145 silencing enhanced Ca deposition in A7r5 cells exposed to calcifying conditions, while miR-145 overexpression attenuated it, partly through increasing α-actin levels and reducing osterix-driven osteogenic differentiation. In mice, 14 weeks after the induction of renal mass reduction, both aortic miR-145 and α-actin mRNA decreased by 80% without significant elevations in osterix or Ca deposition. Vitamin D treatment from week 8 to 14 fully prevented the reductions in aortic miR-145 and attenuated by 50% the decreases in α-actin, despite uremia-induced hyperphosphatemia. In conclusion, vitamin D was able to prevent the reductions in aortic miR-145 and α-actin content induced by uremia, reducing the alterations in vascular contractility and osteogenic differentiation despite hyperphosphatemia.

Basic fibroblast growth factor uniquely stimulates quiescent vascular smooth muscle cells and induces proliferation and dedifferentiation.

Blood vessels normally remain stable over the long term. However, in atherosclerosis, vascular cells leave the quiescent state and enter an activated state. Here, we investigated the factors that trigger the breakage of the quiescent state by screening growth factors and cytokines using a vascular smooth muscle cell (SMC) line and an endothelial cell (EC) line. Despite known functions of the tested factors, only basic fibroblast growth factor (bFGF) was identified as a potent trigger of quiescence breakage in SMCs, but not ECs. bFGF disrupted tight SMC-monolayers and caused morphological changes, proliferation, and dedifferentiation. Human primary SMCs, but not ECs, also showed similar results. Aberrant SMC proliferation is a critical histological event in atherosclerosis. We, thus, provide further insights into the role of bFGF in vascular pathobiology.

The clinical effects of resveratrol on atherosclerosis treatment and its effect on the expression of NADPH oxidase complex genes in vascular smooth muscle cell line.

Atherosclerosis is a disease that covers the arteries of the middle to large arteries and can affect the aorta to the coronary arteries. Atherosclerosis is a progressive and slow process that begins in childhood and leads to clinical manifestations in adulthood. Increased activity of the NADPH oxidase complex plays an important role in the development of this complication. In this study, the clinical effects of resveratrol were considered on atherosclerosis treatment and its effect on the expression of NADPH oxidase complex genes was performed in the vascular smooth muscle cell line. For this purpose, 120 Chinese Patients prone to atherosclerosis participated for 12 months. These patients were divided into two groups of 60 patients. The first group was treated to the placebo and the second group was treated to 100 mg/day resveratrol for 12 months. Also, the expression of gp91Phox and P22Phox subunits of NADPH oxidase complex was evaluated in vascular smooth muscle cells by real-time PCR technique. The results showed that resveratrol could decrease systolic blood pressure, diastolic blood pressure, cholesterol, triglyceride, and low-density lipoprotein (LDL). It was also able to reduce the expression of gp91Phox and P22Phox subunits of NADPH oxidase complex in the vascular smooth muscle cell line. Therefore, it is possible to introduce resveratrol as an able medicine to treat atherosclerosis. However, more studies are needed to complete the information about the role of resveratrol.

TET2 modulates proliferation and migration of vascular smooth muscle cells via LEMD2/NOX1/NOX4 signaling pathway

Abstract Background Ten-eleven translocation 2 (TET2), a widely reported DNA hydroxymethylation enzyme, is involved in active DNA demethylation. TET2 may play an critical role in numerous cellular processes by regulating the level of DNA hydroxymethylation and altering gene expression. TET2 expression was proved to be down-regulated in aorta of spontaneously hypertensive rats (SHR) compared to Wistar-Kyoto rats (WKY) as well as in VSMCs subjected to15% cyclic stretch compared to 5% cyclic stretch. However, whether TET2 regulates vascular smooth muscle cells (VSMCs) proliferation and migration and its underlying mechanisms remains unclear. Purpose The present study aims to investigate whether TET2 affects VSMC proliferation and migration and its possible underlying mechanisms. Methods TET2 knockdown rat VSMC was established using crispr/Cas9 method. Different expression genes were identified by next generation sequencing (NGS) between TET2 knockdown VSMC and control VSMCs while differentially hydroxymethylated promoter region were identified by hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) sequencing. Expression of mRNA and proteins was detected by qRT-PCR and western blot respectively. The proliferation and migration of VSMCs were assessed by EdU assay, flowcytometry assay, and wound healing assay. LEMD2 overexpression and knockdown stable VSMC lines were established through Lentiviral infection. Results The EdU assay and wound healing assay show that knockdown of TET2 enhanced proliferation and migration of VSMCs. NGS identified 123 differentially expressed genes (91 up-regulated and 32 down-regulated) between TET2 knockdown VSMCs and control VSMCs while hMeDIP sequencing identified 166 genes with differentially hydroxymethylated promoter region (68 up-regulated and 98 down-regulated) between TET2 knockdown VSMCs and control VSMCs. Through comparing these sequencing results, we identified a gene named as LEMD2 simultaneously present in both sequencing results. Expression of LEMD2 at mRNA and protein level was significantly increased in TET2 knockdown VSMCs compared to control VSMCs. Then we successfully established LEMD2 overexpression and knockdown stable VSMC lines and found that overexpression of LEMD2 enhanced proliferation and migration of VSMCs while knockdown of LEMD2 inhibited proliferation and migration of VSMCs. Furthermore, overexpression of LEMD2 up-regulated the expression of NOX1 and down-regulated the expression of NOX4 while knockdown of LEMD2 show the opposite effect. Conclusion The present study confirmed that TET2 modulates VSMC proliferation and migration via LEMD2/NOX1/NOX4. The ROS level may be involved in VSMC proliferation and migration. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National natural science foundation of China

Metformin Suppresses the Progress of Diabetes-Accelerated Atherosclerosis by Inhibition of Vascular Smooth Muscle Cell Migration Through AMPK-Pdlim5 Pathway.

Backgrounds: Our previous work revealed that AMP-activated protein kinase (AMPK) activation inhibits vascular smooth muscle cell migration in vitro by phosphorylating PDZ and LIM domain 5 (Pdlim5). As metformin is an AMPK activator, we used a mouse vascular smooth muscle cell (VSMC) line and a Myh11-cre-EGFP mice to investigate whether metformin could inhibit the migration of VSMCs in vitro and in a wire-injury model in vivo. It is recognized that VSMCs contribute to the major composition of atherosclerotic plaques. In order to investigate whether the AMPK–Pdlim5 pathway is involved in the protective function of metformin against atherosclerosis, we utilized ApoE−/− male mice to investigate whether metformin could suppress diabetes-accelerated atherosclerosis by inhibition of VSMC migration via the AMPK–Pdlim5 pathway.Methods: The mouse VSMC cell line was exogenously transfected wild type, phosphomimetic, or unphosphorylatable Pdlim5 mutant before metformin exposure. Myh11-cre-EGFP mice were treated with saline solution or metformin after these were subjected to wire injury in the carotid artery to study whether metformin could inhibit the migration of medial VSMCs into the neo-intima. In order to investigate whether the AMPK–Pdlim5 pathway is involved in the protective function of metformin against atherosclerosis, ApoE−/− male mice were divided randomly into control, streptozocin (STZ), and high-fat diet (HFD)-induced diabetes mellitus; STZ+HFD together with metformin or Pdlim5 mutant carried the adenovirus treatment groups.Results: It was found that metformin could induce the phosphorylation of Pdlim5 and inhibit cell migration as a result. The exogenous expression of phosphomimetic S177D-Pdlim5 inhibits lamellipodia formation and migration in VSMCs. It was also demonstrated that VSMCs contribute to the major composition of injury-induced neointimal lesions, while metformin could alleviate the occlusion of the carotid artery. The data of ApoE−/− mice showed that increased plasma lipids and aggravated vascular smooth muscle cell infiltration into the atherosclerotic lesion in diabetic mice were observed Metformin alleviated diabetes-induced metabolic disorders and atherosclerosis and also reduced VSMC infiltration in atherosclerotic plaques, while the Pdlim5 phospho-abolished mutant that carried adenovirus S177A-Pdlim5 undermines the protective function of metformin.Conclusions: The activation of the AMPK–Pdlim5 pathway by metformin could interrupt the migratory machine of VSMCs and inhibit cell migration in vitro and in vivo. The maintenance of AMPK activity by metformin is beneficial for suppressing diabetes-accelerated atherosclerosis.