Vascular Smooth Muscle Cells In Culture Research Articles

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Antiapoptotic Effect of β1 Blockers in Ascending Thoracic Aortic Smooth Muscle Cells: The Role of HSP70 Expression.

Heat shock proteins (HSPs) play an important role in the cellular adaptation to stress, a requisite for cell survival. The aortic wall appears to be a target for increased expression of HSPs during surgical stress. We aimed to define the expression and function of aortic HSP70 in 31 patients with normal ascending thoracic aortic diameter who underwent aortic valve replacement due to aortic valve stenosis and in 35 patients with dilated ascending thoracic aorta who underwent replacement of an ascending thoracic aortic aneurysm. To elucidate responsible signaling mechanisms we used an in vitro model of rat hypoxic aortic vascular smooth muscle cell (AVSMC) cultures. We demonstrated an increase in AVSMC HSP70 and an attenuation of the apoptotic markers (TUNEL-positive nuclei, caspase-3 activity, Bax/Bcl2 ratio) in aortic wall tissue specimens from both aortic valve stenosis and ascending thoracic aortic aneurysm patients on β1 blockade with metoprolol. In vitro, metoprolol treatment of hypoxic rat AVSMCs increased nitric oxide (NO) production, induced heat shock factor 1 transport to the nucleus, upregulated HSP70, decreased p53 phosphorylation and attenuated apoptosis. Blockade of NO production, resulted in decreased HSP70 and prevented the metoprolol-induced anti-apoptotic response of hypoxic AVSMCs. We demonstrate an anti-apoptotic effect of metoprolol dependent on NO-induced HSP70 expression, and thus augmentation of HSP70 expression should be considered as a therapeutic approach to limit apoptosis in the human ascending thoracic aorta of patients undergoing cardiac surgery.

Effect of Ang III on Nuclear Factor Kappa Beta in Wistar Rat VSMCs

ObjectiveWe investigated whether angiotensin (Ang) III induces nuclear factor kappa beta (NF‐kb) phosphorylation in isolated rat vascular smooth muscle cells (VSMCs).BackgroundThe molecular mechanisms by which Ang III induces various biological effects have not been fully investigated. Most studies have shown that NF‐kb mediates Ang II inflammatory responses effects in VSMCs. Inflammation of vascular smooth muscle cells (VSMCs) is a critical action associated with cardiovascular diseases including hypertension. The role of Ang III to induce NF‐kb in VSMCs is unknown and is a focus of these studies.MethodsPrimary cultures of VSMCs were isolated from the thoracic aorta of adult Wistar rats by the explant technique. VSMCs were treated with Ang III ranging in concentration from 0.1 nM to 1000 nM for 10 minutes or with 100 nM Ang III for 1 minute to 30 minutes. The western blotting technique was used to determine the effects of the peptide on NF‐kb protein phosphorylation.ResultsConcentration studies showed that Ang III caused a dose‐dependent increase in NF‐kb protein phosphorylation. The effects of the peptide on NF‐kb phosphorylation were maximal between 10 nM and 100 nM. The peptide's effects were rapid and significant; occurring within minutes of treatment and the maximal effects on NF‐kb phosphorylation was observed by 10 minutes.ConclusionThese findings provide insight into the molecular nature of the actions of Ang III and offer possible mechanism by which Ang III physiological actions occur in VSMCs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phenotypic Modulation of Cultured Primary Human Aortic Vascular Smooth Muscle Cells by Uremic Serum.

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular disease (CVD). The high concentration of circulating uremic toxins and alterations in mineral metabolism and hormone levels produce vascular wall remodeling and significant vascular damage. Medial calcification is an early vascular event in CKD patients and is associated to apoptosis or necrosis and trans-differentiation of vascular smooth muscle cells (VSMC) to an osteogenic phenotype. VSMC obtained from bovine or rat aorta and cultured in the presence of increased inorganic phosphate (Pi) have been extensively used to study these processes. In this study we used human aortic VSMC primary cultures to compare the effects of increased Pi to treatment with serum obtained from uremic patients. Uremic serum induced calcification, trans-differentiation and phenotypic remodeling even with normal Pi levels. In spite of similar calcification kinetics, there were fundamental differences in osteochondrogenic marker expression and alkaline phosphatase induction between Pi and uremic serum-treated cells. Moreover, high Pi induced a dramatic decrease in cell viability, while uremic serum preserved it. In summary, our data suggests that primary cultures of human VSMC treated with serum from uremic patients provides a more informative model for the study of vascular calcification secondary to CKD.

Cyclic Mechanical Stretch Induced Smooth Muscle Cell Changes in Cerebral Aneurysm Progress by Reducing Collagen Type IV and Collagen Type VI Levels

Background/Aims: Cerebral aneurysm growth is characterized by continuous structural weakness of local smooth muscle cells, though the mechanism is unclear. In this study, we examine protein changes in cerebral aneurysm and human brain vascular smooth muscle cells after cyclic mechanical stretch. We further explore the relationship between the smooth muscle cell changes and reductions in the levels of collagen types IV and VI. Methods: Saccular cerebral aneurysms (n=10) were collected, and temporal artery samples were used as controls. Quantitative proteomics were analyzed and histopathological changes were examined. Smooth muscle cells were cultured in a flexible silicone chamber and subjected to 15% cyclic mechanical stretch. The effect of stretch on the cell viability, function, gene and protein expression were further studied for the understanding the molecular mechanism of aneurysm development. Results: Proteomics analysis revealed 92 proteins with increased expression and 88 proteins with decreased expression compared to the controls (p<0.05). KEGG pathway analysis showed that the change in focal adhesion and extracellular matrix-receptor interaction, suggesting the involvement of collagen type IV and VI. The aneurysm tissue exhibited fewer smooth muscle cells and lower levels of collagen type IV and VI. Human brain vascular smooth muscle cell culture showed spindle-like cells and obvious smooth muscle cell layer. Cell proteomics analysis showed that decreased expression of 118 proteins and increased expression of 32 proteins in smooth muscle cells after cyclic mechanical stretch. KEGG pathway analysis indicated that focal adhesion and ECM-receptor interaction were involved. After cyclic mechanical stretch, collagen type IV and IV expression were decreased. Moreover, the stretch induced MMP-1 and MMP-3 expression elevation. Conclusion: We demonstrated that collagen type IV and VI were decreased in cerebral aneurysms and continuous cyclic mechanical stretch induced smooth muscle cell changes. Smooth muscle cell protection provides an additional therapeutic option to prevent the growth of cerebral aneurysms.

Inhibition of arterial medial calcification and bone mineralization by extracellular nucleotides: The same functional effect mediated by different cellular mechanisms.

Arterial medial calcification (AMC) is thought to share some outward similarities to skeletal mineralization and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. ATP and UTP have previously been shown to inhibit bone mineralization. This investigation compared the effects of extracellular nucleotides on calcification in VSMCs with those seen in osteoblasts. ATP, UTP and the ubiquitous mineralization inhibitor, pyrophosphate (PPi ), dose dependently inhibited VSMC calcification by ≤85%. Culture of VSMCs in calcifying conditions was associated with an increase in apoptosis; treatment with ATP, UTP, and PPi reduced apoptosis to levels seen in non-calcifying cells. Extracellular nucleotides had no effect on osteoblast viability. Basal alkaline phosphatase (TNAP) activity was over 100-fold higher in osteoblasts than VSMCs. ATP and UTP reduced osteoblast TNAP activity (≤50%) but stimulated VSMC TNAP activity (≤88%). The effects of extracellular nucleotides on VSMC calcification, cell viability and TNAP activity were unchanged by deletion or inhibition of the P2Y2 receptor. Conversely, the actions of ATP/UTP on bone mineralization and TNAP activity were attenuated in osteoblasts lacking the P2Y2 receptor. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) hydrolyses ATP and UTP to produce PPi . In both VSMCs and osteoblasts, deletion of NPP1 blunted the inhibitory effects of extracellular nucleotides suggesting involvement of P2 receptor independent pathways. Our results show that although the overall functional effect of extracellular nucleotides on AMC and bone mineralization is similar there are clear differences in the cellular mechanisms mediating these actions.

Mitochondrial regulation of exosomal transported miRNA play a central role in vascular smooth muscle cell proliferation and migration associated with atherogenesis and re-stenosis

Vascular smooth muscle (VSM) cell proliferation and migration are the hallmark of atherosclerosis and re-stenosis. In this study, we have investigated the role of mitochondrial bioactivity in determining the content of exosomes released by vascular smooth muscle cells and the biological effect these exosomes have on vascular smooth muscle (VSM) cell proliferation and migration. VSM cells were isolated from 12 week old male Sprague-Dawley rats. Experiments were undertaken using day zero isolated (contractile phenotype), 21 day cultured (synthetic phenotype) and 21 day mitochondrial incompetent (synthetic phenotype- Rho cell). The effect of balloon angioplasty on rat aorta structural remodelling was also studied. Inhibition of mitochondrial network formation with the DRP1 inhibitor MDivi (10 uM) inhibited angioplasty-dependent remodelling. This observation was confirmed in VSM cell cultures where MDivi significantly reduced proliferation and migration. Total exosomal release was significantly greater in the 21 day cultured cells when compared with quiesced non-proliferating cells and 21 day cultured Rho cells (480±20 ng, 520±10 ng and 420±10 ng per ml respectively) and total exosomal RNA yield was 70.2±10.2 ng/ul, 118.7±2.4 ng/ul and 70.8±4.7 ng/ul respectively. Mitochondrial function significantly influenced miRNA and mRNA measured in exosomes. When compared with the hyperproliferative synthetic VSM cell miR-21 expression was reduced by 88±12.1% and miR-145 expression increased by 73±19.8%. We also measured a 7-fold decrease and 6.6-fold decrease mTOR, PI3K and 4EBP1 respectively. A significant increase expression of P53, cdkn2a and ROS scavenging proteins including SOD1 and SOD2 were measured in 21 day cultured Rho cells vs. 21 day hyperproliferative VSM cells. In this study we have further correlated VSM cell hyperproliferative phenotype with mitochondrial function. Moreover, further demonstrated mitochondrial function/VSM cell phenotype with exosomal release and cargo that potentially drives the hyperproliferative/migratory phenotype central to atherosclerosis and re-stenosis.

Docosahexaenoic Acid Inhibits Vascular Smooth Muscle Cell Proliferation Induced by Glucose Variability.

Background:Vascular Smooth Muscle cells (VSMC) enact crucial roles in early vasculogenesis and sustenance of vascular integrity. However, aberrant proliferation of VSMC followed by migration into the blood vessel wall leads to the formation of vascular lesions accounting for the degeneration and remodelling of vascular basement membrane. In diabetes, hyperglycaemia accelerates VSMC proliferation and contributes to the initiation and progression of atherosclerotic lesions. Recently, acute glucose fluctuations have been implicated in the abnormal VSMC proliferation and complications of diabetic atherosclerosis. Docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid (PUFA) has been shown to inhibit proliferation of several cell types implicating several different mechanisms. In the present study, we have investigated the effects of DHA on VSMC proliferation induced by stable and intermittent high glucose levels.Method:Confluent cultures of rat aortic VSMCs were treated with DHA for 24 hrs and then exposed to stable high glucose (25 mmol/L, SHG) or intermittent high glucose (5 mmol/L and 25 mmol/L alternating every 12 hrs, IHG) for 72 hrs. Cell proliferation was examined by the MTT viability assay, while apoptosis process was evaluated by the Hoechst staining, flow cytometry and caspase-3 activity assays.Results:Our data demonstrated that the hyper proliferation induced by stable and intermittent high glucose levels was significantly inhibited by the DHA pre-treatment. DHA significantly increased caspase-3 activity, resulting in enhanced DNA fragmentation and apoptosis.Conclusion:Our results suggest that DHA reduced the high glucose-induced proliferation of VSMC and induced cell apoptosis.

Collagen XIV and a related recombinant fragment protect human vascular smooth muscle cells from calcium-/phosphate-induced osteochondrocytic transdifferentiation

Transdifferentiation of vascular smooth muscle cells (VSMC) promotes the development of vascular calcifications such as arteriosclerosis. The aim was to investigate effects of specific extracellular matrix (ECM) components on transdifferentiation of VSMC to identify novel ECM-based therapeutic tools.Human collagens I & IV (CI, CIV) along with collagen XIV (CXIV) and a CXIV-derived fragment (CXIV-F), both of which induce differentiation, were applied in an in-vitro model of calcium-/phosphate (Ca/P)-induced osteochondrocytic transdifferentiation of human and murine VSMC. Transdifferentiation was determined by RT-PCR and calcium contents of VSMC cultures. Signaling pathways involved were determined by western-blot and luciferase reporter plasmid assays.Under normal culture conditions, CI induced VSMC proliferation and a more epithelioid/synthetic phenotype while CIV and predominantly CXIV provoked opposite effects. CIV and CXIV further blocked Ca/P-induced osteochondrocytic transdifferentiation of VSMC displayed e.g. by reduced gene expressions of Runx2, Sox9, osterix and increased expressions of αSMA and SM22α. This involved impaired activation of ERK1/2, NF-ĸB and Wnt-signaling. Similar preventive effects were achieved by applying CXIV-F. Impaired preventive effects of CXIV by co-treatment with a cluster of differentiation (CD)44 agonist propose CD44 as a CXIV-target structure on VSMC.In conclusion, CXIV and CXIV-F interfere with osteochondrocytic transdifferentiation of VSMC and should be further explored as potential therapeutic tools in vascular calcification.

Abstract 617: Inhibitors of Beta-Catenin Signaling Limit Growth of Vascular Smooth Muscle Cells

Smooth muscle cell (SMC) growth is essential for artery formation during development and significantly contributes to neointima formation after vascular injury in adulthood. We recently showed, using a tissue-specific deletion and knock-in of mutant alleles, that beta-catenin (b-ctn) signaling function is essential for SMC growth and artery formation; moreover, protein interactions mediated by its C-terminus domain are required for assembly of the arterial wall, while b-ctn N-terminus interactions are dispensable. Inhibitors of b-ctn have been developed, but their effects on vascular SMC growth have not been fully tested. We hypothesize that inhibitors that disrupt protein interactions mediated by the b-ctn C-terminus domain will impair b-ctn signaling and limit growth of vascular SMCs. We evaluated growth of mouse aortic SMCs and human coronary artery SMCs in culture using AlamarBlue after exposure to increasing concentrations (0.01 to 10 micromolar) of several validated b-ctn inhibitors or vehicle control: PKF118-310 (disrupts the b-ctn/TCF interaction), ICG001 (disrupts the b-ctn C-terminus/CBP interaction), XAV939 (promotes the b-ctn destruction complex), and carnosic acid (disrupts the b-ctn N-terminus/BCL9 interaction). We also evaluated the effect of these inhibitors on b-ctn transcriptional activity in SMCs using a TOPflash reporter system. We found that PKF118-310 (p&lt;0.05 vs. vehicle), ICG001 (p&lt;0.05 vs. vehicle), and XAV939 (p&lt;0.05 vs. vehicle), but not carnosic acid, limit mouse (n=16 independent cultures) and human (n=8 independent cultures) SMC growth in a dose-response manner. PKF118-310 exhibited the most potent inhibitory effect. We also found that PKF118-310, ICG001, and XAV939, but not carnosic acid, inhibit b-ctn transcriptional activity in arterial SMCs in culture. In conclusion, pharmacological inhibition of b-ctn signaling, particularly blocking the b-ctn C-terminus output, inhibits growth of vascular SMCs in culture, providing a rationale to test these inhibitors in models of vascular injury as they hold promise as novel therapies for cardiovascular disease.

Effects of p53-knockout in vascular smooth muscle cells on atherosclerosis in mice.

In vitro and in vivo evidence has indicated that the tumor suppressor, p53, may play a significant role in the regulation of atherosclerotic plaque formation. In vivo studies using global knockout mice models, however, have generated inconclusive results that do not address the roles of p53 in various cell types involved in atherosclerosis. In this study, we have specifically ablated p53 in vascular smooth muscle cells (VSMC) in the ApoE-/- mouse model to investigate the roles of p53 in VSMC in atherosclerotic plaque formation and stability. We found that p53 deficiency in VSMC alone did not affect the overall size of atherosclerotic lesions. However, there was a significant increase in the number of p53-/- VSMC in the fibrous caps of atherosclerotic plaques in the early stages of plaque development. Loss of p53 results in migration of VSMC at a faster rate using wound healing assays and augments PDGF-induced formation of circular dorsal ruffles (CDR), known to be involved in cell migration and internalization of surface receptors. Furthermore, aortic VSMC from ApoE-/- /p53-/- mice produce significantly more podosomes and are more invasive. We conclude that p53-/- VSMC are enriched in the fibrous caps of lesions at early stages of plaque formation, which is caused in part by an increase in VSMC migration and invasion as shown by p53-/- VSMC in culture having significantly higher rates of migration and producing more CDRs and invasive podosomes.

TRPM8 downregulation by angiotensin II in vascular smooth muscle cells is involved in hypertension.

Angiotensin II (AngII)-induced injury of vascular smooth muscle cells (VSMCs) serves an important role in hypertension and other cardiovascular disorders. Transient receptor potential melastatin8 (TRPM8) is a thermally‑regulated Ca2+‑permeable channel that is activated by reduced body temperature. Although several recent studies have revealed the regulatory effect of TRPM8 in vascular tone and hypertension, the precise role of TRPM8 in dysfunction of vascular smooth muscle cells (VSMCs) induced by AngII remains elusive. In the present study, the possible function of TRPM8 in AngII‑induced VSMCs malfunction invivo and invitro was investigated. In the aortae from rats that had undergone a two‑kidney one‑clip operation, which is a widely‑used renovascular hypertension model, the mRNA and protein levels of TRPM8 were reduced. In addition, exogenous AngII treatment decreased TRPM8 mRNA and protein expression levels in primary cultures of rat VSMCs. TRPM8 activation by menthol, a pharmacological agonist, in VSMCs, significantly attenuated the AngII‑induced increase in reactive oxygen species and H2O2 production. In addition, TRPM8 activation reduced the AngII‑induced upregulation of NADPH oxidase (NOX)1 and NOX4 in VSMCs. Furthermore, TRPM8 activation relieved the AngII‑induced activation of ras homolog gene family, member A‑rho associated protein kinase2 and janus kinase2 signaling pathways in VSMCs. In conclusion, the results presented in the current study indicated that TRPM8 downregulation by AngII in VSMCs may be involved in hypertension.

NADPH oxidase (NOX) 1 mediates cigarette smoke-induced superoxide generation in rat vascular smooth muscle cells

Smoking is a well-established risk factor for cardiovascular diseases. Oxidative stress is one of the common etiological factors, and NADPH oxidase (NOX) has been suggested as a potential mediator of oxidative stress. In this study, cigarette smoke (CS)-induced superoxide production was characterized in vascular smooth muscle cells (VSMC). CS was prepared in forms of cigarette smoke extract (CSE) and total particulate matter (TPM). Several molecular probes for reactive oxygen species were trialed, and dihydroethidium (DHE) and WST-1 were chosen for superoxide detection considering the autofluorescence, light absorbance, and peroxidase inhibitory activity of CS. Both CSE and TPM generated superoxide in a VSMC culture system by stimulating cells to produce superoxide and by directly producing superoxide in the aqueous solution. NOX, specifically NOX1 was found to be an important cellular source of superoxide through experiments with the NOX inhibitors diphenyleneiodonium (DPI) and VAS2870 as well as isoform-specific NOX knockdown. NOX inhibitors and the superoxide dismutase mimetic TEMPOL reduced the cytotoxicity of CSE, thus suggesting the contribution of NOX1-derived superoxide to cytotoxicity. Since NOX1 is known to mediate diverse pathological processes in the vascular system, NOX1 may be a critical effector of cardiovascular toxicity caused by smoking.

Biomaterials patterned with discontinuous microwalls for vascular smooth muscle cell culture: biodegradable small diameter vascular grafts and stable cell culture substrates

The medial layer of small diameter blood vessels contains circumferentially aligned vascular smooth muscle cells (vSMC) that possess contractile phenotype. In tissue-engineered constructs, these cellular characteristics are usually achieved by seeding planar scaffolds with vSMC, rolling the cell-laden scaffold into a tubular structure, and maturing the construct in a pulsatile bioreactor, a lengthy process that can take up to two months. During the maturation phase, the cells circumferentially orient, their contractile protein expression increases, and they obtain a contractile phenotype. Generating cell culture platforms that enable the rapid production of directionally oriented vSMC with increased contractile protein expression would be a major step forward for blood vessel tissue engineering and would greatly facilitate the in vitro study of vSMC biology. Previously, we developed a micropatterned cell culture surface that promotes orientation and contractile protein expression of vSMC. Herein, we explore two potential applications of this technology. First, we fabricate tubular and biodegradable scaffolds that possess the micropatterning on their exterior surface. When vSMC are seeded on these scaffolds, they initially proliferate in order to fill the microchannels and as confluence is reached the cells align in the direction of the micropatterning resulting in a biodegradable scaffold that is inhabited by circumferentially aligned vSMC within a week. Second, we illustrate that we can generate biostable cell culture surfaces that allow the in vitro study of the cells in a more contractile state. Specifically, we explore contractile protein expression of cells cultured on the micropatterned surfaces with the addition of soluble transforming growth factor beta one (TGFβ1).

Porphyromonas gingivalis Lipopolysaccharide Stimulation of Vascular Smooth Muscle Cells Activates Proliferation and Calcification.

Porphyromonas gingivalis (Pg) is a major etiologic agent of periodontitis, whose virulence has been attributed to different factors, including lipopolysaccharide (LPS). Vascular ectopic calcification as a well-known major risk factor for adverse cardiovascular diseases is a highly prevalent vascular pathophenotype, and vascular smooth muscle cells (VSMCs) play an important role in mediating vascular calcification. It was hypothesized that Pg-LPS may stimulate vascular calcification through a direct effect on VSMC function. To test this hypothesis, the effect of Pg-LPS on VSMC calcification was determined. Primary cultures of VSMCs were obtained and identified by immunochemistry in vitro. The proliferation and alkaline phosphatase (ALP) activity of VSMCs were measured using a cell counting kit and an ALP activity test. Mineral deposition was examined using alizarin red staining. Gene (e.g. ALP, core binding factor α1 [Cbfα1], bone sialoprotein [BSP], and osteopontin [OPN]) expression levels altered by Pg-LPS were determined by reverse transcription-polymerase chain reaction array. Pg-LPS could increase the proliferation of VSMCs at different times and enhance ALP activity of VSMCs after 1 day. Alizarin red staining and quantification showed that, with Pg-LPS treatment, VSMCs displayed more obvious calcification nodules. When stimulated with Pg-LPS, the expression of specific osteogenic genes (e.g., ALP, Cbfα1, BSP, and OPN) was significantly promoted in the presence or absence of mineralization-inducing medium, whereas the expression of the OPN gene was inhibited in the mineralization induction group at day 7. Pg-LPS can stimulate VSMC calcification, which results in vascular calcification, further proving the precise relationship between periodontitis and vascular calcification.

ECM-mimetic heparin glycosamioglycan-functionalized surface favors constructing functional vascular smooth muscle tissue in vitro

Contractile vascular smooth muscle accounts for the normal physiological function of artery. Heparin, as a native glycosaminoglycan, has been well known for its important function in promoting or maintaining the contractile phenotype of vascular smooth muscle cells (VSMCs). In this study, heparin-functionalized non-woven poly(ε-caprolactone) (PCL) mat was fabricated by a facile and efficient surface modification protocol, which enables the control of surface heparin density within a broad range. Surface heparization remarkably increased the hydrophilicity of PCL, and reduced platelet adhesion. MTT assay showed that VSMC proliferation was evidently inhibited on the heparin-functionalized PCL surface in a dose-dependent manner. Gene analysis confirmed that surface heparization also promoted the transition of VSMCs from synthetic phenotype to contractile one. Furthermore, with a proper surface density of heparin, it allowed VSMCs to grow in a certain rate, while exhibiting contractile phenotype. Culture of VSMCs on a modified PCL mat with moderate heparin density (PCL-Hep-20) for 2 days resulted in a confluent layer of contractile smooth muscle cells. These data suggest that the heparin-modified PCL scaffolds may be a promising candidate to generate functional vascular tissues in vitro.

Isolation of Murine Coronary Vascular Smooth Muscle Cells.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22α, α-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

Gas6 protein: its role in cardiovascular calcification.

BackgroundCardiovascular calcifications can be prevented by vitamin K and are accelerated by vitamin K antagonists. These effects are believed to be mainly mediated by the vitamin K-dependent matrix Gla protein. Another vitamin K-dependent protein, Gas6, is also expressed in vascular smooth muscle cells (VSMC). In vitro Gas6 expression was shown to be regulated in VSMC calcification and apoptotic processes.MethodsWe investigated the role of Gas6 in vitro using VSMC cultures and in vivo in young and old Gas6-deficient (Gas6-/-) and wildtype (WT) mice. In addition, Gas6-/- and WT mice were challenged by (a) warfarin administration, (b) uninephrectomy (UniNX) plus high phosphate diet, or (c) UniNX plus high phosphate plus electrocautery of the residual kidney.ResultsIn vitro VSMC from WT and Gas6-/- mice exposed to warfarin showed increased apoptosis and calcified similarly. In vivo, aortic, cardiac and renal calcium content in all groups was similar, except for a lower cardiac calcium content in Gas6-/- mice (group a). Von Kossa staining revealed small vascular calcifications in both WT and Gas6-/- mice (groups a-c). In aging, non-manipulated mice, no significant differences in vascular calcification were identified between Gas6-/- and WT mice. Gas6-/- mice exhibited no upregulation of matrix Gla protein in any group. Cardiac output was similar in all treatment groups.ConclusionsTaken together, in our study Gas6 fails to aggravate calcification against the previous assumption.

Zoledronate upregulates MMP-9 and -13 in rat vascular smooth muscle cells by inducing oxidative stress.

BackgroundBisphosphonates, including zoledronate, target osteoclasts and are widely used in the treatment of osteoporosis and other bone resorption diseases, despite side effects that include damaging the stomach epithelium. Beneficial and adverse effects on other organ systems, including the cardiovascular system, have also been described and could impact on the use of bisphosphonates as therapeutic agents. Vascular smooth muscle cells (VSMCs) are major constituents of the normal vascular wall and have a key role in intimal thickening and atherosclerosis, in part by secreting MMPs that remodel the extracellular matrix and cleave cell surface proteins or secreted mediators. In this study, we investigated the effects of zoledronate on MMP expression.MethodsRat VSMCs were stimulated by PDGF (50 ng/mL) plus TNF-α (10 ng/mL) or left unstimulated for a further 24 hours in serum-free medium. In other series of experiments, cells were pre-treated either with SC-514 (50 μM) or with apocynin (20 nM) for 2 hours, then zoledronate (100 μM) was added into 2% fetal calf serum containing medium for 24 hours.Results and discussionUsing isolated rat VSMCs in culture, zoledronate (100 μM) increased MMP-9 and -13 mRNA expressions but inhibited MMP-2 expression. MMP-9 and MMP-13 up-regulation was shown to depend on the NF-κB pathway; and this was activated by zoledronate. Furthermore, zoledronate elevated the levels of reactive oxygen species detected by either dichlorofluorescein in isolated VSMCs or lucigenin enhanced chemiluminescence in rat aortic rings in vitro. Apocynin, an inhibitor of NADPH oxidase, reversed NF-κB activation and MMP-9 and MMP-13 up-regulation by zoledronate.ConclusionWe conclude that zoledronate increases MMP-9 and MMP-13 expressions in rat VSMCs dependent upon stimulation of the NF-κB pathway by reactive oxygen species. Effects on MMP expression may contribute to the pharmacologic profile of bisphosphonates.

Phenotypic switching of vascular smooth muscle cells in the 'normal region' of aorta from atherosclerosis patients is regulated by miR-145.

Switching of vascular smooth muscle cells (VSMCs) from a contractile phenotype to an adverse proliferative phenotype is a hallmark of atherosclerosis or vascular restenosis. However, the genetic modulators responsible for this switch have not been fully elucidated in humans nor have they been correlated with clinical abnormalities. This study investigated genetic mechanisms involved in phenotypic switching of VSMCs at non‐defect areas of the aorta in patients with atherosclerosis. Aortic wall samples were obtained from patients with (N = 53) and without (N = 27) atherosclerosis undergoing cardiovascular surgery. Vascular smooth muscle cell cultures were generated, and expression of microRNA‐145 (miR‐145), its target gene Kruppel‐Like Factor 5 (KLF5) and Myocardin (MYOCD, a smooth muscle‐specific transcriptional coactivator) were analysed using RT‐qPCR, along with expression of relevant proteins. Vascular smooth muscle cells were transduced with miR‐145 inhibitor and mimic to determine the effect of miR‐145 expression on VSMC proliferation. miR‐145 expression decreased while KLF5 expression increased in atherosclerotic aortas. Atherosclerotic samples and VSMCs had decreased expression of contractile markers calponin and alpha smooth muscle actin (α‐SMA) and MYOCD. miR‐145 inhibitor‐transduced VSMCs from non‐atherosclerotic patients showed decreased expression of calponin and α‐SMA and increased proliferation compared with non‐transduced controls, and these levels were close to those of atherosclerotic patients. miR‐145 mimic‐transduced VSMCs from atherosclerotic patients showed increased expression of calponin and α‐SMA and decreased proliferation compared with non‐transduced controls, and these levels were close to those found in non‐atherosclerotic patients. These data demonstrate that miR‐145 modulates the phenotypic switch of VSMCs from a contractile to a proliferative state via KLF5 and MYOCD in atherosclerosis.