Vascular Smooth Muscle Growth Research Articles

PH-sensing GPR68 inhibits vascular smooth muscle cell proliferation through Rap1A.

Phenotypic transformation of vascular smooth muscle (VSM) from a contractile state to a synthetic, proliferative state is a hallmark of cardiovascular disease (CVD). In CVD, diseased tissue often becomes acidic from altered cellular metabolism secondary to compromised blood flow, yet the contribution of local acid/base imbalance to the disease process has been historically overlooked. In this study, we examined the regulatory impact of the pH-sensing G protein-coupled receptor GPR68 on vascular smooth muscle (VSM) proliferation in vivo and in vitro in wild-type (WT) and GPR68 knockout (KO) male and female mice. Arterial injury reduced GPR68 expression in WT vessels and exaggerated medial wall remodeling in GPR68 KO vessels. In vitro, KO VSM cells showed increased cell cycle progression and proliferation compared to WT VSM cells, and GPR68-inducing acidic exposure reduced proliferation in WT cells. mRNA and protein expression analyses revealed increased Rap1A in KO cells compared to WT cells, and RNA silencing of Rap1A reduced KO VSM cell proliferation. In sum, these findings support a growth-inhibitory capacity of pH-sensing GPR68 and suggest a mechanistic role for the small GTPase Rap1A in GPR68-mediated VSM growth control. These results shed light on GPR68 and its effector Rap1A as potential targets to combat pathologic phenotypic switching and proliferation in VSM.

Protease‐Activated Receptor‐2 Differentially Regulates Vascular Smooth Muscle Cell Proliferation in Cyclic AMP‐Dependent Protein Kinase/Phosphoinositide 3‐Kinase‐Dependent Manner

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide, despite decades of extensive research. One key facet in CVD pathogenesis is the phenotypic transition of affected vascular smooth muscle (VSM) from a quiescent, contractile state to a synthetic, proliferative state. Protease‐activated receptors (PARs) are of emerging interest in CVD; however, their potential impact on VSM phenotypic switching inherent in CVD is unclear. The overall objective of the current study was to determine the role of PAR2 and PAR4 in phenotypic switching and VSM growth. We hypothesized that PAR2 and PAR4 increase following arterial injury in vivo and after growth stimulation in vitro, that they serve to protect against VSM cell growth, and that they signal primarily through specific protein kinases in phenotype‐specific fashion. In rat carotid arteries, PAR2 expression and activity increased following mechanical distension injury compared to uninjured controls. In rat primary VSM cells, PAR2 expression also increased in response to growth stimulation. Differential expression of cyclic GMP‐dependent protein kinase PKG‐1 and the PKG target VASP distinguished low passage (P3‐P6) from high P (P10‐P15) VSM cells, with low P cells showing elevated PKG‐1, phosphorylated (Ser239) VASP and total VASP compared to high P cells. Pharmacologic PAR2 agonism promoted DNA synthesis and proliferation in low P cells but decreased DNA synthesis and proliferation in high P cells. Western blotting analyses showed increased expression of MEK1/2 MAPK, PKA, and PI3K in low P cells versus high P cells. Specific protein kinase blockade revealed that PAR2 differentially controls proliferation of phenotypically distinct low versus high P VSM cells predominantly through PKA and PI3K. Our findings highlight potential (patho)physiological relevance of PAR2 and PAR2‐driven PKA/PI3K in the control of VSM cell growth and provide evidence for continued study of VSM PAR2 in CVD.

MEOX2-mediated regulation of Cathepsin S promotes cell proliferation and motility in glioma

Nuclear transcription factor Mesenchyme Homeobox 2 (MEOX2) is a homeobox gene that is originally discovered to suppress the growth of vascular smooth muscle and endothelial cells. However, whether or not it is connected to cancer is yet unknown. Here, we report that MEOX2 functions as a tumor-initiating element in glioma. Bioinformatic analyses of public databases and investigation of MEOX2 expression in patients with glioma demonstrated that MEOX2 was abundant at both mRNA and protein levels in glioma. MEOX2 expression was shown to be inversely linked with the prognosis of glioma patients. MEOX2 inhibition changed the morphology of glioma cells, inhibited cell proliferation and motility, whereas had no effect on cell apoptosis. Besides, silencing MEOX2 also hampered the epithelial-mesenchymal transition (EMT), focal adhesion formation, and F-actin assembly. Overexpression of MEOX2 exhibited opposite effects. Importantly, RNA-sequencing, ChIP-qPCR assay, and luciferase reporter assay revealed Cathepsin S (CTSS) as a novel transcriptional target of MEOX2 in glioma cells. Consistently, MEOX2 causes glioma tumor development in mice and greatly lowers the survival period of tumor-bearing mice. Our findings indicate that MEOX2 promotes tumorigenesis and progression of glioma partially through the regulation of CTSS. Targeting MEOX2-CTSS axis might be a promising alternative for the treatment of glioma.

Conjugated Metabolites of Hydroxytyrosol and Tyrosol Contribute to the Maintenance of Nitric Oxide Balance in Human Aortic Endothelial Cells at Physiologically Relevant Concentrations.

Nitric oxide (NO) is an important signaling molecule involved in many pathophysiological processes. NO mediates vasodilation and blood flow in the arteries, and its action contributes to maintaining vascular homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Dietary antioxidants and their metabolites have been found to be directly and/or indirectly involved in the modulation of the intracellular signals that lead to the production of NO. The purpose of this study was to investigate the contribution of conjugated metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to the release of NO at the vascular level, and the related mechanism of action, in comparison to their parental forms. Experiments were performed in human aortic endothelial cells (HAEC) to evaluate the superoxide production, the release of NO and production of cyclic guanosine monophosphate (cGMP), the activation of serine/threonine-protein kinase 1 (Akt1), and the activation state of endothelial nitric oxide synthase (eNOS). It was observed that the tested phenolic compounds enhanced NO and cGMP concentration, inhibiting its depletion caused by superoxide overproduction. Moreover, some of them enhanced the activation of Akt (TYR, HT metabolites) and eNOS (HT, HVA, TYR-S, HT-3S). Overall, the obtained data showed that these compounds promote NO production and availability, suggesting that HT and TYR conjugated metabolites may contribute to the effects of parental extra virgin olive oil (EVOO) phenolics in the prevention of cardiovascular diseases.

Metalloproteinases in atherosclerosis

Atherosclerosis underlies most cardiovascular diseases, and is accepted as a primary cause of mortality worldwide. Proteases have been implicated in the development and progression of atherosclerosis, due to their ability to provoke focal destruction of the vascular extracellular matrix. Members of the metalloproteinase family, especially matrix metalloproteinases (MMPs), and their endogenous tissue inhibitors (TIMPs) have been suggested to perform complex dual roles during late-stage progression and rupture of atherosclerotic plaques. Proposed favourable actions of metalloproteinases include the promotion of vascular smooth muscle growth and survival which stabilises plaques, while conversely extracellular matrix destruction alongside interminable monocyte/macrophage accumulation can encourage plaque rupture. This review provides a summary of the cogent evidence connecting the contribution of individual metalloproteinases to atherosclerotic plaque development, progression, and instability. Topics discussed include structural, functional and cell-specific diversity of MMP members; evidence from animal models of atherosclerosis and comparisons with findings in humans; the dual role of MMPs and the requirement to selectively target individual MMPs; and the need for efficient surrogate markers of MMP inhibition. Accordingly, as our knowledge of the complex roles individual MMPs play especially during the progression and rupture of atherosclerotic plaques expands, new impetus is required for clinical trials evaluating the therapeutic potential of selective MMP inhibition, which could limit cardiovascular morbidity and mortality worldwide.

The effects of a growth hormone-releasing hormone antagonist and a gastrin-releasing peptide antagonist on intimal hyperplasia of the carotid artery after balloon injury in a diabetic rat model

IntroductionArterial restenosis after angioplasty/stenting has hindered coronary artery disease treatment, especially in diabetics. We theorized that gastrin-releasing peptide (GRP) antagonists and growth hormone-releasing hormone (GHRH) antagonists might decrease neointimal hyperplasia and restenosis in diabetic rats after common carotid arterial balloon injury. MethodsTwo separate experiments were conducted to test the effects of a GRP antagonist (RC-3095) and a GHRH antagonist (MZ-4-71) on vascular smooth muscle (VSM) growth. In a preliminary in vitro experiment non-injured human aortic vascular smooth muscle (VSM) proliferation was compared between growth media and control. In a second in vivo experiment, intimal and medial area, intima/media ratio (IM) and percent stenosis were compared between injured carotid arteries in twelve Zucker type II obese rats treated with subcutaneously injected RC-3095, MZ-4-71, or control media. ResultsIn the in vitro experiment, decreased VSM cell growth was observed in GRP antagonist (p < 0.05) and GHRH antagonist groups (p < 0.05) compared to the control group. In the in vivo experiment, the GRP antagonist group had a decreased IM ratio (1.63 ± 0.41, p < 0.05) and an increased area of stenosis (98.78% ± 1.48 p = NS) compared to control (2.38 ± 1.09) while the GHRH antagonist group had decreased IM ratio (1.33 ± 0.58 SD, p < 0.05) and percent area of stenosis (78.84% ± 24.97, p < 0.05) compared to control (2.38 ± 1.09). ConclusionsThe significant decrease in both IM ratio and percent area of stenosis in the GHRH antagonist group supports the hypothesis that this peptide may reduce neointimal hyperplasia and restenosis.

Ca2+/Calmodulin-Dependent Protein Kinase II in Vascular Smooth Muscle.

Ca2+-dependent signaling pathways are central regulators of differentiated vascular smooth muscle (VSM) contractile function. In addition, Ca2+ signals regulate VSM gene transcription, proliferation, and migration of dedifferentiated or "synthetic" phenotype VSM cells. Synthetic phenotype VSM growth and hyperplasia are hallmarks of pervasive vascular diseases including hypertension, atherosclerosis, postangioplasty/in-stent restenosis, and vein graft failure. The serine/threonine protein kinase Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous mediator of intracellular Ca2+ signals. Its multifunctional nature, structural complexity, diversity of isoforms, and splice variants all characterize this protein kinase and make study of its activity and function challenging. The kinase has unique autoregulatory mechanisms, and emerging studies suggest that it can function to integrate Ca2+ and reactive oxygen/nitrogen species signaling. Differentiated VSM expresses primarily CaMKIIγ and -δ isoforms. CaMKIIγ isoform expression correlates closely with the differentiated phenotype, and some studies link its function to regulation of contractile activity and Ca2+ homeostasis. Conversely, synthetic phenotype VSM cells primarily express CaMKIIδ and substantial evidence links it to regulation of gene transcription, proliferation, and migration of VSM in vitro, and vascular hypertrophic and hyperplastic remodeling in vivo. CaMKIIδ and -γ isoforms have opposing functions at the level of cell cycle regulation, proliferation, and VSM hyperplasia in vivo. Isoform switching following vascular injury is a key step in promoting vascular remodeling. Recent availability of genetically engineered mice with smooth muscle deletion of specific isoforms and transgenics expressing an endogenous inhibitor protein (CAMK2N) has enabled a better understanding of CaMKII function in VSM and should facilitate future studies.

Potassium Channels in Regulation of Vascular Smooth Muscle Contraction and Growth.

Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca2+ channels (VGCC), Ca2+ influx through VGCC, intracellular Ca2+, and VSM contraction. Membrane potential also affects release of Ca2+ from internal stores and the Ca2+ sensitivity of the contractile machinery such that K+ channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. VSM cells express multiple isoforms of at least five classes of K+ channels that contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression, and function of large conductance, Ca2+-activated K+ (BKCa) channels, intermediate-conductance Ca2+-activated K+ (KCa3.1) channels, multiple isoforms of voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and inward-rectifier K+ (KIR) channels in both contractile and proliferating VSM cells.

Functionalized polyethylene naphthalate for cytocompatibility improvement

This work is focused on the characterization of functionalized polyethylene naphthalate (PEN) with Ar plasma treatment. The changes of the surface contact angle and the surface free energy, the surface morphology and roughness were studied using electrokinetic analysis, goniometry, AFM and XPS method. Ablation of PEN surface layers was determined by gravimetry. Plasma treated samples were consequently metallized; the surface morphology and cross-section of polymer/metal were studied with SEM and FIB-SEM analysis. The plasma treatment induced globular surface morphology connected with significant mass loss and wettability (contact angle) changes. Electrokinetic analysis confirmed dramatic changes in surface chemistry after plasma treatment depending on plasma power and exposure time. Also the ablated polymer mass increased with the increasing power and time. The plasma treatment increases oxygen concentration on the PEN surface. Consequently deposited gold layer and its FIB-SEM analysis confirmed homogeneous electrically continuous layer formation for a wide interval of plasma power and time used for the pretreatment. Plasma treatment was applied for significant enhancement of vascular smooth muscle growth cell adhesion and proliferation on PEN.

Insulin promotes vascular smooth muscle cell proliferation and apoptosis via differential regulation of tumor necrosis factor-related apoptosis-inducing ligand.

Insulin regulates glucose homeostasis but can also promote vascular smooth muscle (VSMC) proliferation, important in atherogenesis. Recently, we showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) stimulates intimal thickening via accelerated growth of VSMCs. The aim of the present study was to determine whether insulin-induced effects on VSMCs occur via TRAIL. Expression of TRAIL and TRAIL receptor in response to insulin and glucose was determined by polymerase chain reaction. Transcriptional activity was assessed using wild-type and site-specific mutations of the TRAIL promoter. Chromatin immunoprecipitation studies were performed. VSMC proliferation and apoptosis was measured. Insulin and glucose exposure to VSMC for 24 h stimulated TRAIL mRNA expression. This was also evident at the transcriptional level. Both insulin- and glucose-inducible TRAIL transcriptional activity was blocked by dominant-negative specificity protein-1 (Sp1) overexpression. There are five functional Sp1-binding elements (Sp1-1, Sp1-2, Sp-5/6 and Sp1-7) on the TRAIL promoter. Insulin required the Sp1-1 and Sp1-2 sites, but glucose needed all Sp1-binding sites to induce transcription. Furthermore, insulin (but not glucose) was able to promote VSMC proliferation over time, associated with increased decoy receptor-2 (DcR2) expression. In contrast, chronic 5-day exposure of VSMC to 1 µg/mL insulin repressed TRAIL and DcR2 expression, and reduced Sp1 enrichment on the TRAIL promoter. This was associated with increased cell death. The findings of the present study provide a new mechanistic insight into how TRAIL is regulated by insulin. This may have significant implications at different stages of diabetes-associated cardiovascular disease. Thus, TRAIL may offer a novel therapeutic solution to combat insulin-induced vascular pathologies.

The angiotensin receptor blocker losartan reduces coronary arteriole remodeling in type 2 diabetic mice

Cardiovascular complications are a leading cause of morbidity and mortality in type 2 diabetes mellitus (T2DM) and are associated with alterations of blood vessel structure and function. Although endothelial dysfunction and aortic stiffness have been documented, little is known about the effects of T2DM on coronary microvascular structural remodeling. The renin–angiotensin–aldosterone system plays an important role in large artery stiffness and mesenteric vessel remodeling in hypertension and T2DM. The goal of this study was to determine whether the blockade of AT1R signaling dictates vascular smooth muscle growth that partially underlies coronary arteriole remodeling in T2DM. Control and db/db mice were given AT1R blocker losartan via drinking water for 4weeks. Using pressure myography, we found that coronary arterioles from 16-week db/db mice undergo inward hypertrophic remodeling due to increased wall thickness and wall-to-lumen ratio with a decreased lumen diameter. This remodeling was accompanied by decreased elastic modulus (decreased stiffness). Losartan treatment decreased wall thickness, wall-to-lumen ratio, and coronary arteriole cell number in db/db mice. Losartan treatment did not affect incremental elastic modulus. However, losartan improved coronary flow reserve. Our data suggest that Ang II–AT1R signaling mediates, at least in part, coronary arteriole inward hypertrophic remodeling in T2DM without affecting vascular mechanics, further suggesting that targeting the coronary microvasculature in T2DM may help reduce cardiac ischemic events.

Differential Effects of IFN-β on the Survival and Growth of Human Vascular Smooth Muscle and Endothelial Cells.

It has been documented that interferon (IFN)-β is effective against the genesis of atherosclerosis or hyperplastic arterial disease in animal model. The main mechanism of the efficacy was antiproliferative action on the growth of vascular smooth muscle cells (SMC). To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC). IFN-β enhanced SMC apoptosis in serum starved medium. Conversely, EC apoptosis induced by serum and growth factor deprivation was inhibited by IFN-β. The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities. Anti-apoptotic bcl-2 mRNA was also up-regulated in EC. IFN-β inhibited SMC growth in a dose dependent manner. However, the growth of EC was rather enhanced by a low dose of IFNs. The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells. The growth stimulation on EC was considered to link with increase of S and G2/M phase cells. SMC produced IFN-β in response to various stimulants. However, IFN-β was not induced in EC. These suggested that endogenous IFN-β from SMC may act on EC and affect to EC functions. In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth. These effects were considered to contribute to a cure against hyperplastic arterial diseases as the mechanisms in the efficacy of IFN-β.

Regulation of Renal Fibrosis by Smad3 Thr388 Phosphorylation

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

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Estrogen, vascular estrogen receptor and hormone therapy in postmenopausal vascular disease

Cardiovascular disease (CVD) is less common in premenopausal women than men of the same age or postmenopausal women, suggesting vascular benefits of estrogen. Estrogen activates estrogen receptors ERα, ERβ and GPR30 in endothelium and vascular smooth muscle (VSM), which trigger downstream signaling pathways and lead to genomic and non-genomic vascular effects such as vasodilation, decreased VSM contraction and growth and reduced vascular remodeling. However, randomized clinical trials (RCTs), such as the Women's Health Initiative (WHI) and Heart and Estrogen/progestin Replacement Study (HERS), have shown little vascular benefits and even adverse events with menopausal hormone therapy (MHT), likely due to factors related to the MHT used, ER profile, and RCT design. Some MHT forms, dose, combinations or route of administration may have inadequate vascular effects. Age-related changes in ER amount, distribution, integrity and post-ER signaling could alter the vascular response to MHT. The subject's age, preexisting CVD, and hormone environment could also reduce the effects of MHT. Further evaluation of natural and synthetic estrogens, phytoestrogens, and selective estrogen-receptor modulators (SERMs), and the design of appropriate MHT combinations, dose, route and ‘timing’ could improve the effectiveness of conventional MHT and provide alternative therapies in the peri-menopausal period. Targeting ER using specific ER agonists, localized MHT delivery, and activation of specific post-ER signaling pathways could counter age-related changes in ER. Examination of the hormone environment and conditions associated with hormone imbalance such as polycystic ovary syndrome may reveal the causes of abnormal hormone-receptor interactions. Consideration of these factors in new RCTs such as the Kronos Early Estrogen Prevention Study (KEEPS) could enhance the vascular benefits of estrogen in postmenopausal CVD.

Abstract 297: Cyclic GMP Reduces Vascular Smooth Muscle Migration through Inhibition of TGF-ß1/Smad Signaling

Abnormal vascular smooth muscle (VSM) growth remains an elemental foundation of vasculoproliferative disorders including atherosclerosis and restenosis. Many second messenger, cytokine, and growth factor signals mediate control of VSM growth, and among these is transforming growth factor (TGF)-β1, a pluripotent cytokine with wide-ranging yet often opposite effects in VSM. Cyclic nucleotide signaling also exerts powerful growth control of VSM, and our previous work has helped establish a biological link between cyclic GMP and TGF-β1 in injured carotid arteries. The current study characterized the influence of cyclic GMP on TGF-β1 and its receptor-activated Smad3 in rat primary VSM cells. The heme-dependent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY41) significantly increased cyclic GMP and site-specific phosphorylation of vasodilator-activated serum phosphoprotein (VASP) in manner indicative of active protein kinase G (PKG) and PKA signaling. Recombinant TGF-β1 (10 ng/ml) significantly stimulated phospho-Smad3 (Ser 423/425 ) and decreased inhibitory Smad7 in VSM cell homogenates, and using flow cytometry significantly increased cells in G 2 /M and expression of cyclins D and E and Cdk2 and Cdk4 while decreasing expression of inhibitory p21 and p27 after 24 hours compared to vehicle controls. TGF-β1 also significantly increased cell numbers compared to controls after 48 hours, thus confirming growth promoting capacities of TGF-β1 in VSM. In cell lysates double-sandwich ELISA revealed that BAY41 significantly reduces total and active TGF-β1, and Western analyses showed it significantly decreases total and phospho-Smad3 Ser423/425 expression and reduces MMP-2 and MMP-9 expression and activity (via column zymography) in both cell lysates and conditioned media after 1 and 48 hours. BAY41 also significantly reduced serum- and PDGF-stimulated cell migration between 6 and 18 hours using an in vitro scrape injury and a transwell assay. In comparison, inclusive effects of BAY41 were replicated by its prototype YC-1 and by the heme-independent sGC activator BAY 60-2770. These data clearly support growth protective capacities of cGMP in VSM and propose it operates through attenuation of TGF-β1/Smad3 signaling.

Stretch‐sensitive microRNA expression and signaling events in vascular smooth muscle

Vascular smooth muscle cells are constantly exposed to mechanical force by the blood pressure, which is thought to regulate smooth muscle growth, differentiation and contractile function. We have previously shown that expression of small non‐coding RNAs, microRNAs (miRNAs), is essential for regulation of smooth muscle phenotype including stretch‐dependent contractile differentiation.In this study we have investigated the effect of mechanical stretch on miRNA expression and the role of stretch‐sensitive miRNAs for intracellular signaling in smooth muscle.Mechanical stretch was applied to mouse portal veins in organ culture ex vivo by attaching a gold weight at one end of the vessel for 24h. MiScript whole genome miRNA PCR arrays analysis, comparing miRNA levels in stretched versus non‐stretched portal veins, revealed a dramatic decrease in the miR‐144/451 cluster level with no change in other active miRNA strands. Furthermore, we could confirm that these miRNAs target the AMPK signaling pathway, which correlates with an increased AMPK activation in stretched portal veins.In conclusion, our results suggest that reduced expression of miR‐144/451 in stretched portal veins could participate in the regulation of AMPK signaling. This effect may represent a negative feedback mechanism, limiting stretch‐induced growth in vascular smooth muscle.

Inhibition of Vascular Smooth Muscle Growth by the Soluble Guanylyl Cyclase Activator BAY 60–2770

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are key components of pathologic vessel growth and efforts aimed at their reduction are clinically essential. Cyclic nucleotide signaling has long been studied for its growth‐mitigating properties, and in the current study we hypothesized that the novel soluble guanylyl cyclase activator BAY 60–2770 (BAY) inhibits proliferation and migration of rat primary VSMCs through PKA and PKG. BAY significantly reduced cell numbers in concentration‐dependent fashion after 72 h (0.1–10 μM) and S‐phase cell numbers (~40%) after 16 h with no effects on cell viability. Interestingly, BAY caused nominal reduction (~20%) in cell migration after 16 h. Mechanistically, BAY significantly induced cGMP in concentration‐and time‐dependent fashion without apparent effects on cAMP. In‐Cell Western analysis showed that BAY significantly increased phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at Ser157 and Ser239, respective indicators of PKA and PKG signaling. Lastly, BAY doubled PKG activity without observable effects on PKA activity. These data suggest that BAY has capacity to inhibit VSMC proliferation through cGMP/PKG/VASP signaling. This work was supported by NHLBI grants HL59976 and HL81720 and by an ECU Undergraduate Research and Creative Activity Award.

Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

Blood pressure (BP) is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM) and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN). In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i), which forms a complex with calmodulin, activates myosin light chain (MLC) kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC). PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK), a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs) in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in concert with antioxidants, MMP inhibitors and cytokine antagonists to reduce VSM hyperactivity in certain forms of HTN that do not respond to Ca2+ channel blockers.

Involvement of IL-1 and Oncostatin M in Acanthosis Associated With Hypertensive Leg Ulcer

Hypertensive leg ulcer (HLU) is an inflammatory disease characterized by intense pain, alteration of vascularization, and skin necrosis. The optimal treatment relies on surgical removal of necrotic tissues covered by a split-skin graft. We studied the histomorphology of the lesions and investigated the involvement of inflammatory cells and cytokines to further define the physiopathology of HLU. We report epidermis acanthosis and a preferential occlusion of the precapillary arterioles with infiltration of neutrophils, macrophages, and T lymphocytes in the dermis. OSM, IL-1β, and IL-6 were overexpressed in the ulcer, whereas the Th17-derived cytokines were not. In vitro, the addition of IL-1β and OSM promoted acanthosis and destructuring of reconstructed epidermis. Exogenous IL-1β and OSM synergistically induced epidermal acanthosis in mice. These data show that OSM and IL-1β are not only a biological characteristic signature of HLU, but these cytokines reflect a specific inflammatory state, directly involved in the pathogenesis. We suggest that anti-cytokine biotherapies could be an alternative strategy to surgery to treat HLU.