Smooth Muscle Cell Physiology Research Articles

MIRO1 controls energy production and proliferation of smooth muscle cells.

The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.

Regional transcriptomic profiling reveals immune system enrichment in nonfailing atria and all chambers of the failing human heart.

The different chambers of the human heart demonstrate regional physiological traits and may be differentially affected during pathological remodeling, resulting in heart failure. Few previous studies, however, have characterized the different chambers at a transcriptomic level. We, therefore, conducted whole tissue RNA sequencing and gene set enrichment analysis of biopsies collected from the four chambers of adult failing (n = 8) and nonfailing (n = 11) human hearts. Atria and ventricles demonstrated distinct transcriptional patterns. When compared with nonfailing ventricles, the transcriptional pattern of nonfailing atria was enriched for many gene sets associated with cardiogenesis, the immune system and bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β), MAPK/JNK, and Wnt signaling. Differences between failing and nonfailing hearts were also determined. The transcriptional pattern of failing atria was distinct compared with that of nonfailing atria and enriched for gene sets associated with the innate and adaptive immune system, TGF-β/SMAD signaling, and changes in endothelial, smooth muscle cell, and cardiomyocyte physiology. Failing ventricles were also enriched for gene sets associated with the immune system. Based on the transcriptomic patterns, upstream regulators associated with heart failure were identified. These included many immune response factors predicted to be similarly activated for all chambers of failing hearts. In summary, the heart chambers demonstrate distinct transcriptional patterns that differ between failing and nonfailing hearts. Immune system signaling may be a hallmark of all four heart chambers in failing hearts and could constitute a novel therapeutic target.NEW & NOTEWORTHY The transcriptomic patterns of the four heart chambers were characterized in failing and nonfailing human hearts. Both nonfailing atria had distinct transcriptomic patterns characterized by cardiogenesis, the immune system and BMP/TGF-β, MAPK/JNK, and Wnt signaling. Failing atria and ventricles were enriched for gene sets associated with the innate and adaptive immune system. Key upstream regulators associated with heart failure were identified, including activated immune response elements, which may constitute novel therapeutic targets.

Chitinase 3 like 1 is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability.

Atherosclerotic cerebrovascular disease underlies the majority of ischaemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals. Using two independent human endarterectomy biobanks, we found that the understudied glycoprotein, chitinase 3 like 1 (CHI3L1), is up-regulated in patients with carotid disease compared to healthy controls. Further, CHI3L1 levels were found to stratify individuals based on symptomatology and histopathological evidence of an unstable fibrous cap. Gain- and loss-of-function studies in cultured human carotid artery smooth muscle cells (SMCs) showed that CHI3L1 prevents a number of maladaptive changes in that cell type, including phenotype switching towards a synthetic and hyperproliferative state. Using two murine models of carotid remodelling and lesion vulnerability, we found that knockdown of Chil1 resulted in larger neointimal lesions comprised by de-differentiated SMCs that failed to invest within and stabilize the fibrous cap. Exploratory mechanistic studies identified alterations in potential downstream regulatory genes, including large tumour suppressor kinase 2 (LATS2), which mediates macrophage marker and inflammatory cytokine expression on SMCs, and may explain how CHI3L1 modulates cellular plasticity. CHI3L1 is up-regulated in humans with carotid artery disease and appears to be a strong mediator of plaque vulnerability. Mechanistic studies suggest this change may be a context-dependent adaptive response meant to maintain vascular SMCs in a differentiated state and to prevent rupture of the fibrous cap. Part of this effect may be mediated through downstream suppression of LATS2. Future studies should determine how these changes occur at the molecular level, and whether this gene can be targeted as a novel translational therapy for subjects at risk of stroke.

The H2S-generating enzyme 3-mercaptopyruvate sulfurtransferase regulates pulmonary vascular smooth muscle cell migration and proliferation but does not impact normal or aberrant lung development

Along with nitric oxide (NO), the gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H2S) are emerging as potentially important players in newborn physiology, as mediators of newborn disease, and as new therapeutic modalities. Several recent studies have addressed H2S in particular in animal models of bronchopulmonary dysplasia (BPD), a common complication of preterm birth where oxygen toxicity stunts lung development. In those studies, exogenous H2S attenuated the impact of oxygen toxicity on lung development, and two H2S-generating enzymes were documented to affect pulmonary vascular development. H2S is directly generated endogenously by three enzymes, one of which, 3-mercaptopyruvate sulfurtransferase (MPST), has not been studied in the lung. In a hyperoxia-based animal model of BPD, oxygen exposure deregulated MPST expression during post-natal lung development, where MPST was localized to the smooth muscle layer of the pulmonary vessels in developing lungs. siRNA-mediated abrogation of MPST expression in human pulmonary artery smooth muscle cells in vitro limited baseline cell migration and cell proliferation, without affecting apoptosis or cell viability. In vivo, MPST was dispensable for normal lung development in Mpst−/−mice, and MPST did not contribute to stunted lung development driven by hyperoxia exposure, assessed by design-based stereology. These data demonstrate novel roles for MPST in pulmonary vascular smooth muscle cell physiology. The potential caveats of using Mpst−/- mice to study normal and aberrant lung development are also discussed, highlighting the possible confounding, compensatory effects of other H2S-generating enzymes that are present alongside MPST in the smooth muscle compartment of developing pulmonary vessels.

Imaging and optogenetic modulation of vascular mural cells in the live brain.

Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood-brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer's disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.

Non-coding RNAs: key regulators of smooth muscle cell fate in vascular disease.

The vascular smooth muscle cell (SMC) is one of the most plastic cells in the body. Understanding how non-coding RNAs (ncRNAs) regulate SMC cell-fate decision making in the vasculature has significantly enhanced our understanding of disease development, and opened up exciting new avenues for potential therapeutic applications. Recent studies on SMC physiology have in addition challenged our traditional view on their role and contribution to vascular disease, mainly in the setting of atherosclerosis as well as aneurysm disease, and restenosis after angioplasties. The impact of SMC behaviour on vascular disease is now recognized to be context dependent; SMC proliferation and migration can be harmful or beneficial, whereas their apoptosis, senescence, and switching into a more macrophage-like phenotype can promote inflammation and disease progression. This is in particular true for atherosclerosis-related diseases, where proliferation of SMCs was believed to promote lesion formation, but may also prevent plaque rupture by stabilizing the fibrous cap. Based on newer findings of genetic lineage tracing studies, it was revealed that SMC phenotypic switching can result in less-differentiated forms that lack classical SMC markers while exhibiting functions more related to macrophage-like cells. This switching can directly promote atherogenesis. The aim of this current review is to summarize and discuss how ncRNAs (mainly microRNAs and long ncRNAs) are involved in SMC plasticity, and how they directly affect vascular disease development and progression. Finally, we want to critically assess where potential future therapies could be useful to influence the burden of vascular diseases.

160 - Poldip2 Activates the Rho Guanine Nucleotide Exchange Factor Ect2 To Regulate Vascular Smooth Muscle Cell Proliferation

Introduction NADPH oxidase (Nox)-mediated generation of reactive oxygen species (ROS) and Rho GTPase signaling play a vital role in vascular smooth muscle cell (VSMC) physiology and their aberrant signaling contributes to vascular disease. Polymerase δ-interacting protein 2 (Poldip2) modulates both Nox and Rho activity, yet the mechanisms are unknown. Here we explore how Poldip2 enhances Rho GTPase activation. Methods A nucleotide-free-RhoA pulldown followed by mass spectrometry was used to identify Rho activators, guanine nucleotide exchange factors (GEFs), that are stimulated upon Poldip2 overexpression. These pulldowns followed by western blot were subsequently used to examine the activity of the identified GEF. Co-immunoprecipitation experiments were performed to look for direct interaction and immuno-fluorescence microscopy was used to examine subcellular localization and cytokinesis defects (via multinucleated cells). Finally, proliferation was examined by counting cells post-treatment with siRNA. Results The RhoGEF, Epithelial cell transforming sequence 2 (Ect2), is activated by overexpression of Poldip2 (>2-fold, p=2e-7) in VSMCs. However, no interaction was observed between Poldip2 and Ect2, nor did Poldip2 regulate Ect2 subcellular localization. Nox4 is not required for Poldip2-driven Ect2 activation, as Ect2 activation in Nox4 knockout VSMCs is even more dramatic (>5 fold, p=0.001) than in wild type cells. Instead, depletion of Nox1 inhibits the activation of Ect2 by Poldip2. Consistent with ROS derived from Nox1 activating Ect2, the ROS scavenger N-acetyl-cysteine blocks Poldip2-driven Ect2 activation. Depletion of Ect2 or Poldip2 inhibits VSMC proliferation (p Conclusions Poldip2 activates Ect2, via ROS generated by Nox1, to regulate VSMC cytokinesis and promote proliferation.

Thrombospondin-1 differentially regulates microRNAs in vascular smooth muscle cells

Thrombospondin-1 (TSP-1) is an important regulator of vascular smooth muscle cell (VSMC) physiology and gene expression. MicroRNAs (microRNA), small molecules that regulate protein translation, have emerged as potent regulators of cell function. MicroRNAs have been shown to be involved in intimal hyperplasia, atherosclerosis, and upregulated in the vasculature in diabetes. The purpose of this study was to identify microRNAs regulated by TSP-1 in vascular smooth muscle cells (VSMCs). Human VSMCs were treated for 6 h with basal media or TSP-1 both supplemented with 0.2% FBS. Cells were then snap frozen and RNA extracted. An Affymetrix GeneChip microRNA array analysis was performed in triplicate on three separate collections. Confirmatory qrtPCR was performed. Data were analyzed by ANOVA or t test, with significance set at p < 0.05. MicroRNAs identified were subjected to KEGG pathway analysis using the DIANA tools miRPath online tool. TSP-1 upregulated 22 microRNAs and downregulated 18 microRNAs in VSMCs (p < 0.05). The most upregulated microRNA was miR-512-3p (45.12 fold). The microRNA most downregulated by TSP-1 was miR-25-5p, which was decreased by 9.61. Of note, five members of the mir-17-92 cluster were downregulated. KEGG analysis revealed that thirty-three cellular signaling pathways were impacted by these microRNAs and that nine pathways were relevant to vascular disease. MicroRNAs regulate protein expression at the level of translation and may represent a significant mechanism by which TSP-1 regulates VSMC function. Several of the microRNAs identified have a role in vascular function. The miR-17-92 cluster family, which was found to exhibit reduced expression in this study, is known to be involved in angiogenesis and vascular function. TSP-1 regulates multiple microRNAs in VSMCs adding a new layer of complexity to TSP-1 regulation of VSMC function.

Thrombospondin-1, -2 and -5 have differential effects on vascular smooth muscle cell physiology

IntroductionThe thrombospondins (TSPs) are matricellular proteins that exert multifunctional effects by binding cytokines, cell-surface receptors and other proteins. TSPs play important roles in vascular pathobiology and are all expressed in arterial lesions. The differential effects of TSP-1, -2, and -5 represent a gap in knowledge in vascular smooth muscle cell (VSMC) physiology. Our objective is to determine if structural differences of the TSPs imparted different effects on VSMC functions critical to the formation of neointimal hyperplasia. We hypothesize that TSP-1 and -2 induce similar patterns of migration, proliferation and gene expression, while the effects of TSP-5 are different. MethodsHuman aortic VSMC chemotaxis was tested for TSP-2 and TSP-5 (1–40 μg/mL), and compared to TSP-1 and serum-free media (SFM) using a modified Boyden chamber. Next, VSMCs were exposed to TSP-1, TSP-2 or TSP-5 (0.2–40 μg/mL). Proliferation was assessed by MTS assay. Finally, VSMCs were exposed to TSP-1, TSP-2, TSP-5 or SFM for 3, 6 or 24 h. Quantitative real-time PCR was performed on 96 genes using a microfluidic card. Statistical analysis was performed by ANOVA or t-test, with p < 0.05 being significant. ResultsTSP-1, TSP-2 and TSP-5 at 20 μg/mL all induce chemotaxis 3.1 fold compared to serum-free media. TSP-1 and TSP-2 induced proliferation 53% and 54% respectively, whereas TSP-5 did not. In the gene analysis, overall, cardiovascular system development and function is the canonical pathway most influenced by TSP treatment, and includes multiple growth factors, cytokines and proteases implicated in cellular migration, proliferation, vasculogenesis, apoptosis and inflammation pathways. Conclusions and relevanceThe results of this study indicate TSP-1, -2, and -5 play active roles in VSMC physiology and gene expression. Similarly to TSP-1, VSMC chemotaxis to TSP-2 and -5 is dose-dependent. TSP-1 and -2 induces VSMC proliferation, but TSP-5 does not, likely due conservation of N-terminal domains in TSP-1 and -2. In addition, TSP-1, -2 and -5 significantly affect VSMC gene expression; however, little overlap exists in the specific genes altered. This study further delineates TSP-1, -2 and -5's contributions to processes related to VSMC physiology.

P84 H2S inhibits NO generation and this inhibition contributes to H2S-induced muscle contraction in mouse colon smooth muscle

H 2 S is generated by CSE in the muscle layers of the mouse colon. The aim of this study was to study the effect of H 2 S on mouse colon circular smooth muscle cells and investigate a possible link between changes in the electrical activity and the muscle tone. Result: Superfusion of NaHS (0.2 mM) had no significant effect on resting membrane potential but inhibited ongoing myoelectric complexes of muscle cells and induced a biphasic change in muscle tone: initial muscle contraction followed by relaxation. Washing out NaHS restored the ongoing myoelectric complexes but did not restore resting muscle tone suggesting that relaxation was not associated with electric activity of smooth muscle cells. NaHS reduced the frequency and amplitude of spontaneous inhibitory junction potentials (IJPs) with a time course similar to muscle contraction. Also, NaHS reduced NO-mediated slow-IJPs evoked by field stimulation suggesting H 2 S inhibits NO production. We further tested NO generation in wild type and CSE-KO mouse preparations. Both tissue levels of NO and the amplitude of evoked slow-IJPs were significantly higher in muscle layers of CSE-KO mice. Our results suggest that the action of H 2 S on colonic smooth muscle cell physiology involving brief contraction and irreversible relaxation, inhibition of myoelectrical activity, and inhibition of NO likely occurs via three independent mechanisms. The inhibition of NO contributes to the H 2 S-induced muscle contraction phase (Support: NIH DK17238).

Thrombospondin-1 Differentially Regulates MicroRNAs in Vascular Smooth Muscle Cells

Thrombospondin-1 (TSP-1) is an important regulator of vascular smooth muscle cell (VSMC) physiology and gene expression. MicroRNAs (microRNA), small molecules that regulate protein translation, have emerged as potent regulators of cell function. MicroRNAs have been shown to be involved in intimal hyperplasia, atherosclerosis, and upregulated in the vasculature in diabetes. The purpose of this study was to identify microRNAs regulated by TSP-1 in vascular smooth muscle cells (VSMCs). Human VSMCs were treated for 6 h with basal media or TSP-1 both supplemented with 0.2% FBS. Cells were then snap frozen and RNA extracted. An Affymetrix GeneChip microRNA array analysis was performed in triplicate on three separate collections. Confirmatory qrtPCR was performed. Data were analyzed by ANOVA or t test, with significance set at p < 0.05. MicroRNAs identified were subjected to KEGG pathway analysis using the DIANA tools miRPath online tool. TSP-1 upregulated 22 microRNAs and downregulated 18 microRNAs in VSMCs (p < 0.05). The most upregulated microRNA was miR-512-3p (45.12 fold). The microRNA most downregulated by TSP-1 was miR-25-5p, which was decreased by 9.61. Of note, five members of the mir-17-92 cluster were downregulated. KEGG analysis revealed that thirty-three cellular signaling pathways were impacted by these microRNAs and that nine pathways were relevant to vascular disease. MicroRNAs regulate protein expression at the level of translation and may represent a significant mechanism by which TSP-1 regulates VSMC function. Several of the microRNAs identified have a role in vascular function. The miR-17-92 cluster family, which was found to exhibit reduced expression in this study, is known to be involved in angiogenesis and vascular function. TSP-1 regulates multiple microRNAs in VSMCs adding a new layer of complexity to TSP-1 regulation of VSMC function.

Regulation of IGF-1 but not TGF-β1 by NGF in the smooth muscle of the inflamed urinary bladder

Intraperitoneal injection of cyclophosphamide (CYP) causes hemorrhagic cystitis with excess growth of muscular layer leading to bladder hypertrophy; this could be attributable to changes in the expression profiles of growth factors in the inflamed urinary bladder. The growth factors characterized in the current study include nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β1. We found that following CYP injection for 8h and 48h, the mRNA levels of all three factors were increased in the inflamed bladder when compared to control. The level of NGF mRNA was mainly increased in the urothelium layer while the levels of IGF-1 mRNA and TGF-β1 mRNA were increased in the smooth muscle layer. The level of NGF high affinity receptor TrkA mRNA was also increased in both the urothelium and the smooth muscle layers during bladder inflammation. When we blocked NGF action with NGF neutralizing antibody in vivo, we found that the up-regulation of IGF-1 in the inflamed bladder was reversed while the up-regulation of TGF-β1 was not affected by NGF neutralization. The effect of NGF on regulating IGF-1 expression was further confirmed in bladder smooth muscle culture showing that exogenous NGF increased the mRNA level of IGF-1 after 30min to 1h stimulation. These results suggested that bladder inflammation induced region-specific changes in the expression profiles of NGF, IGF-1 and TGF-β1. The up-regulation of NGF in the urothelium may have a role in affecting bladder smooth muscle cell physiology by regulating IGF-1 expression.

FHL2 Protein Is a Novel Co-repressor of Nuclear Receptor Nur77

The three members of the NR4A orphan nuclear receptor subfamily Nur77, Nurr1, and NOR-1, regulate a variety of biological functions including vascular disease and metabolism. In this study, we identified Four and a half LIM domains protein-2 (FHL2) as a novel interacting protein of NR4A nuclear receptors by yeast two-hybrid screen and co-immunoprecipitation studies. Each of the four LIM domains of FHL2 can bind Nur77, and both the amino-terminal domain and the DNA binding domain of Nur77 are involved in the interaction between FHL2 and Nur77. FHL2 represses Nur77 transcriptional activity in a dose-dependent manner, and short hairpin RNA-mediated knockdown of FHL2 results in increased Nur77 transcriptional activity. ChIP experiments on the enolase3 promoter revealed that FHL2 inhibits the association of Nur77 with DNA. FHL2 is highly expressed in human endothelial and smooth muscle cells, but not in monocytes or macrophages. To substantiate functional involvement of FHL2 in smooth muscle cell physiology, we demonstrated that FHL2 overexpression increases the growth of these cells, whereas FHL2 knockdown results in reduced DNA synthesis. Collectively, these studies suggest that association of FHL2 with Nur77 plays a pivotal role in vascular disease.

11 Generation of developmental origin-specific vascular smooth muscle cells from human embryonic stem cells

Heterogeneity is a hallmark of vascular smooth muscle cell (SMC) development, physiology and pathology. This could be attributed to the diverse embryological origins of vascular SMCs. In order to uncover...

BFGF induces S1P1 receptor expression and functionality in human pulmonary artery smooth muscle cells

Sphingosine-1-phosphate (S1P), acting through five closely related G-protein coupled receptors termed S1P1-5, has recently emerged as a possible regulator of smooth muscle cell (SMC) physiology with the potential to induce contraction, proliferation and stress fiber formation. In the present study, real-time quantitative PCR was used to determine the expression patterns of S1P receptor subtypes in human primary pulmonary artery smooth muscle cells (PASMC). We report here that subconfluent PASMC express predominantly S1P2 and S1P3 receptors and we show that S1P1 receptor mRNA levels are significantly up-regulated following basic fibroblast growth factor (bFGF) treatment. As a consequence, increased responsiveness, as measured by impedance and ERK1/2 phosphorylation, was observed upon stimulation with a specific S1P1 receptor agonist SEW2871. We therefore demonstrate, for the first time, that a growth factor that was previously shown to be involved in physiological and pathological changes of SMC function induced S1P1 receptor expression and we propose that S1P1 receptor up-regulation could contribute to vascular remodeling.

Saphenous Vein Grafts: to Use or Not to Use?

The choice of the graft conduit is crucial to the success of coronary artery bypass grafting (CABG) because the patency of a coronary conduit is closely associated with an uneventful postoperative course and better long-term patient survival. From the beginning of coronary bypass surgery venous conduits particularly the great saphenous vein (GSV) has been the most frequently used coronary conduit. However, over the last decade or so, coronary bypass graft surgery with arterial revascularization of all diseased coronaries has shown to be efficient because arterial grafts have better long-term patency, especially left internal mammary artery (LIMA), compared with venous grafts. Early vein graft failure coupled with occlusion is the most important limitation of saphenous vein grafts. Nevertheless, vein grafting is still an integral part of cardiac surgical practice. This review provides a summary of the patency rates, technical features and certain characteristics of the venous conduits. It also examines the current understanding and knowledge of venous histology, vein graft pathology and the associated endothelial and smooth muscle cell physiology and pharmacology. In addition, the existing and the emerging strategies to combat and control vein graft intimal hyperplasia and accelerated atherosclerosis are reviewed in detail.

Activation of mitogenic and antimitogenic pathways in cyclically stretched arterial smooth muscle.

Biophysical forces regulate vascular smooth muscle cell (VSMC) physiology and evoke vascular remodeling. Two VSMC autocrine molecules, insulin-like growth factor I (IGF-I) and nitric oxide (NO), are implicated in remodeling attributable to VSMC hyperplasia. We investigated the role of in vitro cyclic stretch on rat VSMC IGF-I, NO, and cellular growth. Cyclic stretch (1 Hz at 120% resting length for 48 h) stimulated VSMC proliferation 2.5-fold vs. unstretched cells and was accompanied by a 1.8-fold increase in VSMC IGF-I secretion. Despite activation of this proliferative pathway, cyclic stretch induced inducible (i) nitric oxide synthase (NOS) expression and a twofold increase in NO secretion, a molecule with documented antiproliferative effects. Cytokine treatment enhanced iNOS expression and NO secretion while inhibiting vascular growth by approximately 50% in static cells. Cytokine treatment of stretched VSMC enhanced NO secretion 2.5-fold while inhibiting growth by approximately 80%. Exogenous IGF-I increased NOS activity 1.5-fold and NO secretion 8.5-fold in static cells. In turn, iNOS inhibition increased IGF-I secretion 1.6-fold and enhanced VSMC growth 1.6-fold in stretched cells. An NO donor (sodium nitroprusside) similarly inhibited VSMC proliferation in static (24%) and stretched (50%) VSMC while also inhibiting IGF-I secretion from stretched cells by approximately 35%. Thus cyclic stretch stimulates mitogenic (IGF-I) and antimitogenic (NO) pathways in VSMC. These two molecules regulate each other's secretory rates, providing tight regulation of VSMC proliferation. These data may have profound implications in understanding vascular growth alterations in vascular injury and hypertension.

HIV envelope gp120 activates human arterial smooth muscle cells.

There have been increasing reports of acute coronary thrombotic events in patients with HIV. Although these clinical events have been attributed primarily to dyslipidemia associated with protease inhibitor therapy, autopsy studies in children with HIV suggest the presence of an underlying arteriopathy. This study demonstrates that the HIV envelope protein, gp120, activates human arterial smooth muscle cells to express tissue factor, the initiator of the coagulation cascade. The induction of tissue factor by gp120 is mediated by two biologically relevant coreceptors for HIV infection, CXCR4 and CCR5, and is also dependent on the presence of functional CD4. Induction of tissue factor by gp120 requires activation of mitogen-activating protein kinases, activation of protein kinase C, and generation of reactive oxygen species, signaling pathways that have protean effects on smooth muscle cell physiology. The activation of smooth muscle cells by gp120 may play an important role in the vascular, thrombotic, and inflammatory responses to HIV infection.

An in vitro model for videoimaging of human bladder smooth muscle cell contractions.

Knowledge regarding human bladder smooth muscle cell (SMC) physiology is very limited. Only a few specific medical therapies for bladder disorders have therefore been established. The objective of this study was to develop a model for videomicroscopy of bladder SMC contractions. Cells were isolated from human cystoprostatectomy specimens and cultured in a modified EMEM medium. These cells were identified as SMCs by means of immunohistochemistry. For videomicroscopy, the culture flasks were coated with a viscous agent to allow cell contraction. Contractions were visualized by means of a cell culture microscope with a time-lapse videosystem. For cholinergic stimulation of the cells, acetylcholine, in concentrations ranging from 100 microM to 10 mM, was applied. The percentage of contracting cells within the observation field was evaluated for quantitative analysis. In control experiments without contractile stimulant 6% of the cells were observed to contract. Stimulation with acetylcholine induced a significant dose-dependent increase to 47% in contracting cells. These results demonstrated that videomicroscopy is an appropriate tool to investigate the contraction mechanisms of bladder SMCs. This model offers the possibility of studying drug effects on the human detrusor in vitro.

Chlamydia pneumoniae activates nuclear factor kappaB and activator protein 1 in human vascular smooth muscle and induces cellular proliferation.

Observational data strongly suggest an association between Chlamydia pneumoniae and atherosclerotic cardiovascular disease. However, few studies have mechanistically linked C. pneumoniae to vascular remodeling. The purpose of the present study was to examine the mechanistic relationship between C. pneumoniae and human vascular smooth muscle cell (VSMC) physiology. We sought to determine the influence of human VSMC infection by C. pneumoniae on (1) VSMC proliferation and (2) activation of the proinflammatory and proliferative transcription factors nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). C. pneumoniae was grown and isolated from Hep 2 cells. Human aortic VSMCs were inoculated with C. pneumoniae in the presence and absence of the azalide antibiotic azithromycin. Cell proliferation was assayed by direct cell counting 48 h following infection. Two hours following infection, nuclear extracts were isolated, and activation of both NF-kappaB and AP-1 was assessed by electrophoretic mobility shift assay. Compared with control, C. pneumoniae infection stimulated VSMC proliferation (P < 0.05) and induced both NF-kappaB and AP-1 DNA binding activity. These effects were eliminated by concurrent treatment with azithromycin. VSMC infection with C. pneumoniae activates proliferative intracellular signals and stimulates cell growth. These data implicate C. pneumoniae as a pathogenic mediator and a potential therapeutic target in the prevention of atherosclerotic disease.