Response Of Smooth Muscle Cells Research Articles

Role of ATP-sensitive potassium channels and inflammatory response of basilar artery smooth muscle cells in subarachnoid hemorrhage of rabbit and immune-modulation by shikonin

ObjectiveTo investigate the role of inflammatory response, oxidative damage and changes of ATP-sensitive potassium channels (sKATP) in basilar artery (BA) smooth muscle cells (SMCS) of rabbits in subarachnoid hemorrhage (SAH) model. MethodsTime course studies on inflammatory response by real-time PCR, oxidative process and function of isolated basilar artery after SAH in New Zealand White rabbits were performed. Basilar artery smooth muscle cells (BASMCs) in each group were obtained and whole-cell patch-clamp technique was applied to record cell membrane capacitance and KATP currents. The morphologies of basal arteries were analyzed. Protective effect of shikonin were also determine by same parameters. ResultsInflammatory cytokines levels were highest at 24h compare to 72h after SAH whereas the oxidative damage and cell death marker were at highest peak at 72h. Oxidative damage peak coincided with significant alterations in cell membrane capacitance, KATP currents and morphological changes in basilar arteries. Shikokin pretreatment attenuated early inflammatory response at 24h and associated oxidative damage at 72h. Finally, shikonin attenuated morphological changes in basilar arteries and dysfunction. ConclusionCurrents of ATP-sensitive potassium channels in basilar smooth muscle cells decreased after SAH by putative oxidative modification from immediate inflammatory response and can be protected by shikonin pretreatment.

Rho kinase inhibitors reduce voltage-dependent Ca2+ channel signaling in aortic and renal microvascular smooth muscle cells.

Voltage-dependent L-type Ca2+ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30-90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16-65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 (P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction (P < 0.05). Changes in intracellular Ca2+ concentration ([Ca2+]i) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca2+]i in A7r5 cells was suppressed by 1-10 µM Y-27632 (P < 0.05), but 10 µM Y-27632 was required to suppress Ca2+ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca2+]i. Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca2+]i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.

Alcohol promotes waste clearance in the CNS via brain vascular reactivity

The efficient clearance of the interstitial waste metabolites is essential for the normal maintenance of brain homeostasis. The brain lacks the lymphatic clearance system. Thus, the drainage of waste metabolites in the brain is dependent on a slow flow of cerebrospinal fluid (CSF) system. Glymphatic system claims the direct bulk flow transport of small size water-soluble waste metabolites into to the perivenous space by aquaporin-4 water channels of the astrocyte end-feet, but it did not address the diffusive clearance of large size waste metabolites. Here, we addressed the clearance mechanisms of large size waste metabolites from interstitial fluid to perivascular space as well as from CSF subarachnoid into perivascular space via the paravascular drainage. A low dose ethanol acting as a potent vasodilator promotes the dynamic clearance of waste metabolites through this perivascular-perivenous drainage path. We observed that ethanol-induced increased in vascular endothelial and smooth muscle cell reactivity regulated the enhanced clearance of metabolites. Here, activation of endothelial specific nitric oxide synthase (eNOS) by ethanol and generation of vasodilator nitric oxide mediates the interactive reactivity of endothelial-smooth muscle cells and subsequent diffusion of the CNS waste metabolites towards perivascular space. Detection of tracer dye (waste metabolite) in the perivenous space and in the blood samples further confirmed the improved clearance of waste metabolites through this unraveled interstitial-perivascular-perivenous clearance path. Our results suggest that alcohol intake at low-dose levels may promote clearance of neurological disease associated entangled proteins.

Airway Smooth Muscle-Specific Transcriptomic Signatures of Glucocorticoid Exposure.

Glucocorticoids, commonly used asthma controller medications, decrease symptoms in most patients, but some remain symptomatic despite high-dose treatment. The physiological basis underlying the glucocorticoid response, especially in asthma patients with severe, refractory disease, is not fully understood. We sought to identify differences between the transcriptomic response of airway smooth muscle (ASM) cells derived from donors with fatal asthma and donors without asthma to glucocorticoid exposure and to compare ASM-specific changes with those observed in other cell types. In cells derived from nine donors with fatal asthma and eight donors without asthma, RNA sequencing was used to measure ASM transcriptome changes after exposure to budesonide (100 nM 24 h) or control vehicle (DMSO). Differential expression results were obtained for this dataset, as well as 13 publicly available glucocorticoid-response transcriptomic datasets corresponding to seven cell types. Specific genes were differentially expressed in response to glucocorticoid exposure (7,835 and 6,957 in ASM cells derived from donors with fatal asthma and donors without asthma, respectively; adjusted P value < 0.05). Transcriptomic changes in response to glucocorticoid exposure were similar in ASM derived from donors with fatal asthma and donors without asthma, with enriched ontological pathways that included cytokine- and chemokine-related categories. A comparison of glucocorticoid-induced changes in the nonasthma ASM transcriptome with those observed in six other cell types showed that ASM has a distinct glucocorticoid-response signature that is also present in ASM cells from donors with fatal asthma.

Dihomo-γ-linolenic acid inhibits several key cellular processes associated with atherosclerosis

Atherosclerosis and its complications are responsible for one in three global deaths. Nutraceuticals show promise in the prevention and treatment of atherosclerosis but require an indepth understanding of the mechanisms underlying their actions. A previous study showed that the omega-6 fatty acid, dihomo-γ-linolenic acid (DGLA), attenuated atherosclerosis in the apolipoprotein E deficient mouse model system. However, the mechanisms underlying such protective effects of DGLA are poorly understood and were therefore investigated. We show that DGLA attenuates chemokine-driven monocytic migration together with foam cell formation and the expression of key pro-atherogenic genes induced by three pro-inflammatory cytokines in human macrophages. The effect of DGLA on interferon-γ signaling was mediated via inhibition of signal transducer and activator of transcription-1 phosphorylation on serine 727. In relation to anti-foam cell action, DGLA inhibits modified LDL uptake by both macropinocytosis and receptor-mediated endocytosis, the latter by reduction in expression of two key scavenger receptors (SR-A and CD36), and stimulates cholesterol efflux from foam cells. DGLA also improves macrophage mitochondrial bioenergetic profile by decreasing proton leak. Gamma-linolenic acid and prostaglandin E1, upstream precursor and key metabolite respectively of DGLA, also acted in an anti-atherogenic manner. The actions of DGLA extended to other key atherosclerosis-associated cell types with attenuation of endothelial cell proliferation and migration of smooth muscle cells in response to platelet-derived growth factor. This study provides novel insights into the molecular mechanisms underlying the anti-atherogenic actions of DGLA and supports further assessments on its protective effects on plaque regression in vivo and in human trials.

Proliferative Pathways of Vascular Smooth Muscle Cells in Response to Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia (IH) and is a risk factor for cardiovascular diseases (e.g., atherosclerosis) and chronic inflammatory diseases (CID). The excessive proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the progression of atherosclerosis. Hypoxia-inducible factor-1 and nuclear factor-κB are thought to be the main factors involved in responses to IH and in regulating adaptations or inflammation pathways, however, further evidence is needed to demonstrate the underlying mechanisms of this process in VSMCs. Furthermore, few studies of IH have examined smooth muscle cell responses. Our previous studies demonstrated that increased interleukin (IL)-6, epidermal growth factor family ligands, and erbB2 receptor, some of which amplify inflammation and, consequently, induce CID, were induced by IH and were involved in the proliferation of VSMCs. Since IH increased IL-6 and epiregulin expression in VSMCs, the same phenomenon may also occur in other smooth muscle cells, and, consequently, may be related to the incidence or progression of several diseases. In the present review, we describe how IH can induce the excessive proliferation of VSMCs and we develop the suggestion that other CID may be related to the effects of IH on other smooth muscle cells.

Metformin inhibits LPS-induced inflammatory response in VSMCs by regulating TLR4 and PPAR-γ.

This study aims to explore whether the inhibitory role of metformin could inhibit LPS-induced inflammatory response in vascular smooth muscle cells (VSMCs) and its underlying mechanism. VSMCs were extracted from aorta of Sprague Dawley rats. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to detect VSMCs viability after treatment with different concentrations of metformin. Levels of monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in VSMCs were detected by ELISA (enzyme-linked immunosorbent assay) and qRT-PCR (quantitative Real time-polymerase chain reaction). Protein and mRNA levels of toll like receptor 4 (TLR4) and peroxisome proliferators activated receptor γ (PPAR-γ) in VSMCs were detected by Western blot and qRT-PCR, respectively. Finally, VSMCs were treated with the PPAR-γ antagonist GW9662 and inflammatory indicators in cells were detected. No significant difference in VSMCs viability was found after 0-2 mM metformin treatment or 500 μg/L LPS induction for 24 h. After 500 μg/L LPS induction in VSMCs for 24 h, levels of MCP-1, TNF-α and IL-6 were remarkably elevated. Both mRNA and protein levels of TLR4 in VSMCs were upregulated after 500 μg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin. Knockdown of TLR4 remarkably inhibited LPS-induced inflammatory response in VSMCs, manifesting as decreased levels of MCP1, TNF-α and IL-6, which were further downregulated after combination treatment of TLR4 knockdown and 20 mM metformin. Furthermore, both mRNA and protein levels of PPAR-γ in VSMCs were downregulated after 500 μg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin. GW9662 treatment resulted in elevated expressions of MCP-1, TNF-α and IL-6, which were reversed by metformin treatment. Metformin can effectively inhibit the mRNA and protein expressions of IL-6, MCP-1, and TNF-α in LPS-induced VSMCs. The anti-inflammatory effects of metformin inhibit the inflammatory response through downregulating rely on the downregulation of TLR4 expression and upregulation ofng PPAR-γ activity.

FGF signaling contributes to atherosclerosis by enhancing the inflammatory response in vascular smooth muscle cells.

The contractile to synthetic phenotypic switching of vascular smooth muscle cells (VSMCs) in response to fibroblast growth factor (FGF) has been previously described. However, the role of the inflammatory response induced by FGF signaling in VSMCs and its occurrence in atherosclerosis remains unclear. In the present study, FGF signaling promoted a contractile to secretory phenotypic transition in VSMCs. VSMCs (primary human aortic smooth muscle cells) treated with FGF exhibited a decrease in the protein expression levels of factors involved in contractility and the secretion of various chemokines was increased, as assessed by reverse transcription-quantitative PCR and ELISA. Additionally, inhibition of FGF signaling by silencing FGF receptor substrate 2 (FRS2) decreased the protein expression levels of various chemokines. Furthermore, VSMCs in the medial layers of arteries from apolipoprotein E-deficient mice and human atherosclerotic samples exhibited an increase in FGF signaling that was identified to be associated with an increase in the protein expression levels of pro-inflammatory molecules, including C-C motif chemokine ligand 2, C-X-C motif chemokine ligand (CXCL) 9, CXCL10 and CXCL11, compared with wild-type mice and healthy control samples, respectively. The present results suggested that FGF signaling induced dedifferentiation of contractile VSMCs and the transition to a secretory phenotype, which may be involved in the progression of atherosclerosis. Collectively, the present results suggested that the FGF signaling pathway may represent a novel target for the treatment of atherosclerosis.

Effects of Vasopressin and Norepinephrine on Wound Healing of Vascular Smooth Muscle and Endothelium

ObjectivesHemorrhagic shock as a result of trauma or surgical bleeding is frequently temporized with administration of vasopressors in order to augment systemic vascular resistance while resuscitation measures are employed. This mimics the treatment strategy of other forms of shock where systemic vascular resistance is low and vasopressors are heavily relied upon to resuscitate the patient. Blood vessels respond to direct exposure to vasopressin and norepinephrine via receptors on smooth muscle and endothelial cells and ultimately result in vasoconstriction which contributes to a low flow state and increases shear stress within vessels. The effect of vasopressors on vascular cells with regard to direct cellular injury and repair has not been well‐studied. The objective of this study is to examine the in vitro effect of vasopressin and norepinephrine on the rate of healing of vascular cells after wounding.MethodsWounding and measurement of healing rate of cells were performed using Electric Cell‐substrate Impedance Sensing (ECIS) technology. A specified area of a monolayer of cells was wounded via high electrical charge, and healing of the cell layer was assessed via measurement of resistance (ohms) over time. Pig vascular smooth muscle cells (SMCs) and pig vascular endothelial cells (ECs) were seeded in 96‐well plates. Immediately after wounding, cells were treated with either lysine‐vasopressin (LVP; 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 ηM; n=6/group), or norepinephrine (NE; 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 μM; n=6/group). Data were recorded continuously for another 18 hours. Statistical comparisons were made using two‐way ANOVA and Tukey HSD post hoc analysis(alpha level = 0.05).ResultsWhen SMCs were injured, NE treatment significantly suppressed healing rate over the course of 10 hours (Time × Dose, F(133,665) = 1.955, p&lt;0.0001), whereas LVP had minimal effect on healing of SMCs (F(133,665) = 0.6346, p=0.999). When ECs were injured, both NE (F(133,665) = 3.716, p&lt;0.0001), and LVP (F(133,665) = 3.058, p&lt;0.0001) significantly suppressed rate of healing.ConclusionsNE significantly suppressed the wound healing response of both SMCs and ECs, whereas LVP suppressed wound healing of ECs only. This strongly suggests that the use of different vasopressors during treatment and resuscitation can elicit unique effects on vascular healing in the body. We plan to further explore the physiological effects of vasopressors on post‐injury healing of blood vessels to aid in optimizing resuscitation strategies that reduce vascular injury and end organ damage in the period after recovery from trauma and shock.Support or Funding InformationThis project was funded by the U.S. Army Medical Command. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulating smooth muscle cell response by the release of TGFβ2 from tubular scaffolds for vascular tissue engineering

Tissue engineering has gained considerable attention in the development of small diameter tissue engineered vascular grafts (TEVGs) for treating coronary heart disease. A properly designed acellular and biodegradable TEVG must encourage the infiltration and growth of vascular smooth muscle cells (SMCs). Our group has previously shown that increasing levels of TGFβ2 can differentially modulate SMC migration and proliferation. In this study, tubular electrospun scaffolds loaded with TGFβ2 were fabricated using various ratios of gelatin/polycaprolactone (PCL), resulting in scaffolds with porous nano-woven architecture suitable for tissue ingrowth. Scaffold morphology, degradation rate, TGβ2 release kinetics, and bioactivity were assessed. TGFβ2 was successfully integrated into the electrospun biomaterial that resulted in a differential release profile depending on the gelatin/PCL ratio over the course of 42 days. Higher TGFβ2 elution was obtained in scaffolds with higher gelatin content, which may be related to the biodegradation of gelatin in culture media. The biological activity of the released TGFβ2 was evaluated by its ability to affect SMC proliferation as a function of its concentration. SMCs seeded on TGFβ2-loaded scaffolds also showed higher densities and infiltration after 5 days in culture as compared to scaffolds without TGFβ2. Our results demonstrate that the ratio of synthetic and natural polymers in electrospun blends can be used to tune the release of TGFβ2. This method can be used to intelligently modulate the SMC response in gelatin/PCL scaffolds making the TGFβ2-loaded conduits attractive for cardiovascular tissue engineering applications.

Necrotic cell debris induces a NF-κB-driven inflammasome response in vascular smooth muscle cells derived from abdominal aortic aneurysms (AAA-SMC)

Abdominal aortic aneurysm (AAA) is a multi-factorial progressive vascular disease with life-threatening complications. Increasing evidence suggests that smooth muscle cell (SMC) dysfunction and cell death contribute to dilatation and rupture of the aorta by inducing an inflammatory response. The exact mechanism of this response however, is incompletely understood. We here investigated in vitro the capacity of autologous necrotic cell debris (CD) to induce inflammasome components and inflammatory mediators in aortic SMC (AAA-SMC) isolated from patients with AAA undergoing surgical repair. AAA-SMCs were additionally primed with Interferon- γ (IFN-γ) before treatment with CD in order to mimic the proinflammatory status caused by higher IFN-γ concentrations that have been demonstrated in the wall of AAAs.Real-time RT-PCR revealed that CD significantly increased NLRP3 and IL1B mRNA expression in different SMC cultures within 6 h of exposure. Priming of the AAA-SMC with IFN-γ significantly increased expression of NLRP3, AIM2, IFI16 and CASP1 mRNAs, whereas IL1B mRNA was reduced. Additional exposure of IFN-γ-primed AAA-SMC to CD for 6–24 h, further augmented expression of AIM2, NLRP3, and Caspase-1 protein levels. Analysis of the SMC supernatants by ELISA revealed CD-induced release of the senescence-associated cytokines IL-6 and MCP-1 in native and IFN-γ-primed SMC, whereas no secretion of Interleukin-(IL) 1α and IL-1β secretion were observed. Our results implicate a role of necrotic cell debris derived from dead neighboring cells in SMC dysfunction and in inflammatory response of AAA tissue.

Migration versus proliferation as contributor to in vitro wound healing of vascular endothelial and smooth muscle cells

Wound closure, as a result of collective cell growth, is an essential biological response to injury. In the field of vascular biology, the response of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) to injury and substrate surface is important in therapeutic clinical treatment interventions such as angioplasty and atherectomy. Specifically, the mechanism by which cells close wounds (i.e. proliferation versus migration) in response to injury stimuli is of interest to better modulate recurrent vascular stenosis, prevent thrombus formation, occlusion, and life-threatening cardiovascular events. Here, we examine growth extent and temporal sequence of events following wound or gap introduction to a confluent monolayer of vascular SMCs or ECs. Significant differences in the preferred mechanisms of these cells to close wounds or gaps were observed; after 48 h, 73% of SMC wound closure was observed to be due to proliferation, while 75% of EC wound closure resulted from migration. These mechanisms were further modulated via addition or removal of extracellular matrix substrate and injury, with ECs more responsive to substrate composition and less to injury, in comparison to SMCs. Our results indicate that ECs and SMCs heal wounds differently, and that the time and mode of injury and associated substrate surface all impact this response.

Untargeted Metabolic Profiling Cell-Based Approach of Pulmonary Artery Smooth Muscle Cells in Response to High Glucose and the Effect of the Antioxidant Vitamins D and E.

Pulmonary arterial hypertension (PAH) is a multi-factorial disease characterized by the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Excessive reactive oxygen species (ROS) formation resulted in alterations of the structure and function of pulmonary arterial walls, leading to right ventricular failure and death. Diabetes mellitus has not yet been implicated in pulmonary hypertension. However, recently, variable studies have shown that diabetes is correlated with pulmonary hypertension pathobiology, which could participate in the modification of pulmonary artery muscles. The metabolomic changes in PASMCs were studied in response to 25 mM of D-glucose (high glucose, or HG) in order to establish a diabetic-like condition in an in vitro setting, and compared to five mM of D-glucose (normal glucose, or LG). The effect of co-culturing these cells with an ideal blood serum concentration of cholecalciferol-D3 and tocopherol was also examined. The current study aimed to examine the role of hyperglycemia in pulmonary arterial hypertension by the quantification and detection of the metabolomic alteration of smooth muscle cells in high-glucose conditions. Untargeted metabolomics was carried out using hydrophilic interaction liquid chromatography and high-resolution mass spectrometry. Cell proliferation was assessed by cell viability and the [3H] thymidine incorporation assay, and the redox state within the cells was examined by measuring reactive oxygen species (ROS) generation. The results demonstrated that PASMCs in high glucose (HG) grew, proliferated faster, and generated higher levels of superoxide anion (O2·−) and hydrogen peroxide (H2O2). The metabolomics of cells cultured in HG showed that the carbohydrate pathway, especially that of the upper glycolytic pathway metabolites, was influenced by the activation of the oxidation pathway: the pentose phosphate pathway (PPP). The amount of amino acids such as aspartate and glutathione reduced via HG, while glutathione disulfide, N6-Acetyl-L-lysine, glutamate, and 5-aminopentanoate increased. Lipids either as fatty acids or glycerophospholipids were downregulated in most of the metabolites, with the exception of docosatetraenoic acid and PG (16:0/16:1(9Z)). Purine and pyrimidine were influenced by hyperglycaemia following PPP oxidation. The results in addition showed that cells exposed to 25 mM of glucose were oxidatively stressed comparing to those cultured in five mM of glucose. Cholecalciferol (D3, or vitamin D) and tocopherol (vitamin E) were shown to restore the redox status of many metabolic pathways.

Involvement of inducible nitric oxide synthase and estrogen receptor ESR2 (ERβ) in the vascular dysfunction in female type 1 diabetic rats

AimsInflammation is involved in diabetes-related vascular dysfunction. Estrogen receptor ESR2/ERβ induces the expression of inducible nitric oxide (NO) synthase (iNOS) and inflammation. The present study investigated the effect of alloxan-induced type 1 diabetes on the iNOS and ESR2 expression and the effect of the chronic iNOS inhibition on the vascular smooth muscle dysfunction in diabetic female rats. In addition, we evaluated the involvement of ESR2 in iNOS expression. Main methodsAlloxan-induced diabetic female rats were treated or not with iNOS inhibitor (L-NIL). iNOS and ESR2 immunostaining, S-nitrosylated proteins and IL-1β protein expression in aorta and plasmatic NO levels were analyzed. Contractile response to noradrenaline was analyzed in endothelium-denuded aorta. iNOS mRNA expression was analyzed in isolated aortic smooth muscle cells (ASMCs) of female rats, incubated with 22 mM glucose and an ESR2 antagonist. Key findingsAortic iNOS and ESR2 immunostaining, S-nitrosylated proteins, IL-1β protein expression and plasmatic NO levels were all increased, whereas noradrenaline-induced contraction was reduced in aorta of diabetic female rats. With the exception of iNOS and ESR2 immunostaining, all these parameters were corrected by L-NIL treatment. High glucose increased iNOS mRNA expression in ASMCs, which was reduced by an ESR2 antagonist. SignificanceWe demonstrated that increased iNOS-NO contributed to the impairment of the contractile response of aortic smooth muscle cells in female type 1 diabetic rats and that increased expression of iNOS may involve the participation of ESR2/ERβ.

ERK and miRNA-1 target Cx43 expression and phosphorylation to modulate the vascular protective effect of angiotensin II

Aims and methodsWe previously reported that angiotensin II (AngII) restores the vascular reactivity diminished by hemorrhagic shock. In this study, we investigated whether the beneficial effects of AngII are related to regulation of gap junctions (GJs) and connexin43 (Cx43), and the implication of MAPK signaling and microRNA (miR-1) in this process. Key findingsOur results show that after hemorrhagic shock or hypoxia, the blockade of GJs or knockdown of Cx43 inhibits the AngII-induced increase in vascular reactivity of superior mesenteric arteries and the contractile response of vascular smooth muscle cells (VSMCs). AngII treatment increases Cx43 expression and phosphorylation at Ser262, and restores gap-junctional communication (GJIC) between VSMCs after hypoxia. The AngII-induced up-regulation of Cx43 expression and phosphorylation is blocked in cells transduced with ERK-siRNA, but is not blocked in cells transduced with p38-siRNA. miR-1 levels are elevated after hypoxia; AngII treatment reverses the up-regulation of miR-1, while ERK-siRNA abolishes that effect of AngII. In hypoxic cells, transfection of a miR-1 mimic into VSMCs decreases Cx43 expression and VSMC reactivity, whereas a miR-1 inhibitor increases both. Also in hypoxic cells, miR-1 eliminates the restoration effects of AngII on Cx43 expression and VSMC reactivity. SignificanceAngII provides protection of vascular function through the restoration of the expression and phosphorylation of Cx43 and its mediated GJIC in VSMCs. It is ERK that mediates the AngII-induced phosphorylation of Cx43 at Ser262. Additionally, miR-1 is involved in this process, and AngII may exert its protective effect partially by inhibiting miR-1 elevation via ERK signaling.

Electrochemical fabrication of a biomimetic elastin-containing bi-layered scaffold for vascular tissue engineering

Biomimetic tissue-engineered vascular grafts (TEVGs) have immense potential to replace diseased small-diameter arteries (<4 mm) for the treatment of cardiovascular diseases. However, biomimetic approaches developed thus far only partially recapitulate the physicochemical properties of the native vessel. While it is feasible to fabricate scaffolds that are compositionally similar to native vessels (collagen and insoluble elastic matrix) using freeze-drying, these scaffolds do not mimic the aligned topography of collagen and elastic fibers found in native vessels. Extrusion-based scaffolds exhibit anisotropic collagen orientation but these scaffolds are compositionally dissimilar (cannot incorporate insoluble elastic matrix). In this study, an electrochemical fabrication technique was employed to develop a biomimetic elastin-containing bi-layered collagen scaffold which is compositionally and structurally similar to native vessels and the effect of insoluble elastin incorporation on scaffold mechanics and smooth muscle cell (SMC) response was investigated. Further, the functionality of human umbilical vein endothelial cells (HUVECs) on the scaffold lumen surface was assessed via immunofluorescence. Results showed that incorporation of insoluble elastin maintained the overall collagen alignment within electrochemically aligned collagen (ELAC) fibers and this underlying aligned topography can direct cellular orientation. Ring test results showed that circumferential orientation of ELAC fibers significantly improved scaffold mechanics. Real-time PCR revealed that the expression of α-smooth muscle actin (Acta2) and myosin heavy chain (MyhII) was significantly higher on elastin containing scaffolds suggesting that the presence of insoluble elastin can promote contractility in SMCs. Further, mechanical properties of the scaffolds significantly improved post-culture indicating the presence of a mature cell-synthesized and remodeled matrix. Finally, HUVECs expressed functional markers on collagen lumen scaffolds. In conclusion, electrochemical fabrication is a viable method for the generation of a functional biomimetic TEVG with the potential to be used in bypass surgery.

Magnesium ion leachables induce a conversion of contractile vascular smooth muscle cells to an inflammatory phenotype.

Phenotype switching is a characteristic response of vascular smooth muscle cells (vSMCs) to the dynamic microenvironment and contributes to all stages of atherosclerotic plaque. Here, we immersed pure magnesium and AZ31 alloy in the completed medium under cell culture condition, applied the resultant leaching extracts to the isolated contractile rat aortic vSMCs and investigated how vSMCs phenotypically responded to the degradation of the magnesium-based stent materials. vSMCs became more proliferative and migratory but underwent more apoptosis when exposed to the degradation products of pure magnesium; while the AZ31 extracts caused less cell division but more apoptosis, thus slowing cell moving and growing. Noticeably, both leaching extracts dramatically downregulated the contractile phenotypic genes at mRNA and protein levels while significantly induced the inflammatory adhesive molecules and cytokines. Exogenously added Mg ions excited similar transformations of vSMCs. With the liberation or supplementation of Mg2+ , the expression patterns of the pro-contractile transactivator myocardin and the pro-inflammatory transcriptional factor kruppel-like factor 4 (KLF4) were reversed. Overall, the degradation of the Mg-based materials would evoke a shift of the contractile vSMCs to an inflammatory phenotype via releasing Mg ions to induce a transition from the phenotypic control of vSMCs by the myocardin to that by the KLF4. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 988-1001, 2019.

Direct application of mechanical stimulation to cell adhesion sites using a novel magnetic-driven micropillar substrate.

Cells change the traction forces generated at their adhesion sites, and these forces play essential roles in regulating various cellular functions. Here, we developed a novel magnetic-driven micropillar array PDMS substrate that can be used for the mechanical stimulation to cellular adhesion sites and for the measurement of associated cellular traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9μm, respectively. Sufficient quantities of iron particles were successfully embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field. We established two methods to apply magnetic fields to the micropillars (Suresh 2007). Applying a uniform magnetic field of 0.3T bent all of the pillars by ~4μm (Satcher et al. 1997). Creating a magnetic field gradient in the vicinity of the substrate generated a well-defined local force on the pillars. Deflection of the micropillars allowed transfer of external forces to the actin cytoskeleton through adhesion sites formed on the pillar top. Using the magnetic field gradient method, we measured the traction force changes in cultured vascular smooth muscle cells (SMCs) after local cyclic stretch stimulation at one edge of the cells. We found that the responses of SMCs were quite different from cell to cell, and elongated cells with larger pre-tension exhibited significant retraction following stretch stimulation. Our magnetic-driven micropillar substrate should be useful in investigating cellular mechanotransduction mechanisms.

The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension.

Autophagy Protects From Uremic Vascular Media Calcification.

Chronic kidney disease and diabetes mellitus are associated with extensive media calcification, which leads to increased cardiovascular morbidity and mortality. Here, we investigated the role of autophagy in the pathogenesis of uremic vascular media calcification. DBA/2 mice were fed with high-phosphate diet (HPD) in order to cause vascular calcification. DBA/2 mice on standard chow diet were used as control. In parallel, autophagy and its response to rapamycin, 3-methyladenine (3-MA), and bafilomycin were studied in an in vitro model using mouse vascular smooth muscle cells (MOVAS). DBA/2 mice on HPD developed severe vascular media calcification, which is mirrored in vitro by culturing MOVAS under calcifying conditions. Both, in vitro and in vivo, autophagy significantly increased in MOVAS under calcifying conditions and in aortas of HPD mice, respectively. Histologically, autophagy was located to the aortic Tunica media, but also vascular endothelial cells, and was found to continuously increase during HPD treatment. 3-MA as well as bafilomycin blocked autophagy in MOVAS and increased calcification. Vice versa, rapamycin treatment further increased autophagy and resulted in a significant decrease of vascular calcification in vitro and in vivo. Rapamycin reduced Runx2 transcription levels in aortas and MOVAS to control levels, whereas it increased α-smooth muscle actin and Sm22α transcription in MOVAS to control levels. Furthermore, rapamycin-treated HPD mice survived significantly longer compared to HPD controls. These findings indicate that autophagy is an endogenous response of vascular smooth muscle cells (VSMC) to protect from calcification in uremia. Induction of autophagy by rapamycin protects cells and mice from uremic media calcification possibly by inhibiting osteogenic transdifferentiation of VSMC.