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Sample records for circulating smooth muscle

  1. SMOOTH MUSCLE STEM CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vascular smooth muscle cells (SMCs) originate from multiple types of progenitor cells. In the embryo, the most well-studied SMC progenitor is the cardiac neural crest stem cell. Smooth muscle differentiation in the neural crest lineage is controlled by a combination of cell intrinsic factors, includ...

  2. Anti-smooth muscle antibody

    MedlinePlus

    ... medlineplus.gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the ...

  3. Smooth Muscle Strips for Intestinal Tissue Engineering

    PubMed Central

    Walthers, Christopher M.; Lee, Min; Wu, Benjamin M.; Dunn, James C. Y.

    2014-01-01

    Functionally contracting smooth muscle is an essential part of the engineered intestine that has not been replicated in vitro. The purpose of this study is to produce contracting smooth muscle in culture by maintaining the native smooth muscle organization. We employed intact smooth muscle strips and compared them to dissociated smooth muscle cells in culture for 14 days. Cells isolated by enzymatic digestion quickly lost maturity markers for smooth muscle cells and contained few enteric neural and glial cells. Cultured smooth muscle strips exhibited periodic contraction and maintained neural and glial markers. Smooth muscle strips cultured for 14 days also exhibited regular fluctuation of intracellular calcium, whereas cultured smooth muscle cells did not. After implantation in omentum for 14 days on polycaprolactone scaffolds, smooth muscle strip constructs expressed high levels of smooth muscle maturity markers as well as enteric neural and glial cells. Intact smooth muscle strips may be a useful component for engineered intestinal smooth muscle. PMID:25486279

  4. Vascular smooth muscle in hypertension.

    PubMed

    Winquist, R J; Webb, R C; Bohr, D F

    1982-06-01

    The cause of the elevated arterial pressure in most forms of hypertension is an increase in total peripheral resistance. This brief review is directed toward an assessment of recent investigations contributing information about the factors responsible for this increased vascular resistance. Structural abnormalities in the vasculature that characterize the hypertensive process are 1) changes in the vascular media, 2) rarefication of the resistance vessels, and 3) lesions of the intimal vascular surface. These abnormalities are mainly the result of an adaptive process and are secondary to the increase in wall stress and/or to pathological damage to cellular components in the vessel wall. Functional alterations in the vascular smooth muscle are described as changes in agonist-smooth muscle interaction or plasma membrane permeability. These types of changes appear to play a primary, initiating role in the elevation of vascular resistance of hypertension. These alterations are not the result of an increase in wall stress and they often precede the development of high blood pressure. The functional changes are initiated by abnormal function of neurogenic, humoral, and/or myogenic changes that alter vascular smooth muscle activity. PMID:6282652

  5. Calcium Signaling in Smooth Muscle

    PubMed Central

    Hill-Eubanks, David C.; Werner, Matthias E.; Heppner, Thomas J.; Nelson, Mark T.

    2011-01-01

    Changes in intracellular Ca2+ are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca2+ signal is a reflection of the source of Ca2+ (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca2+ entry may be detected optically as graded elevations in intracellular Ca2+, junctional Ca2+ transients, Ca2+ flashes, or Ca2+ sparklets, whereas release of Ca2+ from intracellular stores may manifest as Ca2+ sparks, Ca2+ puffs, or Ca2+ waves. These diverse Ca2+ signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression). PMID:21709182

  6. An invertebrate smooth muscle with striated muscle myosin filaments

    PubMed Central

    Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio; Márquez, Gustavo; Méndez, Franklin; Padrón, Raúl; Craig, Roger

    2015-01-01

    Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components. PMID:26443857

  7. Mechanics of Vascular Smooth Muscle.

    PubMed

    Ratz, Paul H

    2015-01-01

    Vascular smooth muscle (VSM; see Table 1 for a list of abbreviations) is a heterogeneous biomaterial comprised of cells and extracellular matrix. By surrounding tubes of endothelial cells, VSM forms a regulated network, the vasculature, through which oxygenated blood supplies specialized organs, permitting the development of large multicellular organisms. VSM cells, the engine of the vasculature, house a set of regulated nanomotors that permit rapid stress-development, sustained stress-maintenance and vessel constriction. Viscoelastic materials within, surrounding and attached to VSM cells, comprised largely of polymeric proteins with complex mechanical characteristics, assist the engine with countering loads imposed by the heart pump, and with control of relengthening after constriction. The complexity of this smart material can be reduced by classical mechanical studies combined with circuit modeling using spring and dashpot elements. Evaluation of the mechanical characteristics of VSM requires a more complete understanding of the mechanics and regulation of its biochemical parts, and ultimately, an understanding of how these parts work together to form the machinery of the vascular tree. Current molecular studies provide detailed mechanical data about single polymeric molecules, revealing viscoelasticity and plasticity at the protein domain level, the unique biological slip-catch bond, and a regulated two-step actomyosin power stroke. At the tissue level, new insight into acutely dynamic stress-strain behavior reveals smooth muscle to exhibit adaptive plasticity. At its core, physiology aims to describe the complex interactions of molecular systems, clarifying structure-function relationships and regulation of biological machines. The intent of this review is to provide a comprehensive presentation of one biomachine, VSM. PMID:26756629

  8. Leiomodin and tropomodulin in smooth muscle

    NASA Technical Reports Server (NTRS)

    Conley, C. A.

    2001-01-01

    Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker alpha-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca(2+), both SM-Lmod and Tmod are found near alpha-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.

  9. Genetic differences in airway smooth muscle function.

    PubMed

    Martin, James G; Jo, Taisuke

    2008-01-01

    The genetic basis for airway smooth muscle properties is poorly explored. Contraction and relaxation are altered in asthmatic airway smooth muscle, but the basis for the alterations and the role that muscle-specific susceptibility genes may play is largely unexplored. Alterations in the beta-adrenergic receptor, signaling pathways affecting inositol phosphate metabolism, adenylyl and guanylyl cyclase activity, and contractile proteins such as the myosin heavy chain are all suggested by experimental model systems. Significant changes in proliferative and secretory capacities of asthmatic smooth muscle are also demonstrated, but their genetic basis also requires elucidation. Certain asthma-related genes such as ADAM33, although potentially important for smooth muscle function, have been incompletely explored. PMID:18094088

  10. TRPC channels in smooth muscle cells.

    PubMed

    Gonzalez-Cobos, Jose C; Trebak, Mohamed

    2010-01-01

    Transient receptor potential canonical (TRPC) proteins constitute a family of seven (TRPC1-7) nonselective cation channels within the wider TRP superfamily. TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 channels are expressed in vascular smooth muscle cells from human vessels of all calibers and in smooth muscle from organs such as the uterus and the gastrointestinal tract. TRPC channels have recently emerged as important players in the control of smooth muscle function. This review will focus on the retrospective analysis of studies proposing contributions of TRPC channels to native calcium entry pathways in smooth muscle and to physiological and pathophysiological responses with emphasis on the vascular system. PMID:20515740

  11. Caveolae in smooth muscles: nanocontacts

    PubMed Central

    Popescu, LM; Gherghiceanu, Mihaela; Mandache, E; Cretoiu, D

    2006-01-01

    Smooth muscle cell (SMC) caveolae have been investigated by quantitative and qualitative analysis of transmission electron microscopy (TEM) images of rat stomach, bladder and myometrium, guinea pig taenia coli, human ileum, and rat aortic SMCs. Ultrathin (below 30 nm) serial sections were used for examination of caveolar morphology and their connections with SMC organelles. Average caveolar diameter was smaller in vascular SMCs (70 nm, n=50) than in visceral SMCs (77 nm, n=100), but with the same morphology. Most of the caveolae, featured as flask-shaped plasma membrane (PM) invaginations, opened to the extracellular space through a 20 nm stoma (21, 3nm) having a 7 nm thick diaphragm. A small percentage of caveolae (3%), gathered as grape-like clusters, did not open directly to the extracellular space, but to irregular PM pockets having a 20-30 nm opening to the extracellular space. In visceral SMCs, caveolae were disposed in 4 - 6 rows, parallel to myofilaments, whilst aortic SMCs caveolae were arranged as clusters. This caveolar organization in rows or clusters minimizes the occupied volume, providing more space for the contractile machinery. The morphometric analysis of relative volumes (% of cell volume) showed that caveolae were more conspicuous in visceral than in vascular SMCs (myometrium - 2.40%; bladder - 3.66%, stomach - 2.61%, aorta - 1.43%). We also observed a higher number of caveolae per length unit of cell membrane in most visceral SMCs compared to vascular SMCs (myometrium - 1.06/μm, bladder - 0.74/μm, aorta - 0.57/μm, stomach - 0.48/μm). Caveolae increase the cellular perimeter up to 15% and enlarge the surface area of the plasma membrane about 80% in SMCs. Three-dimensional reconstructions (15μ3) showed that most caveolae, in both visceral and vascular SMCs, have nanocontacts with SR (87%), or with mitochondria (10%), and only 3%, apparently, have no contact with these organelles. Usually, 15 nm wide junctional spaces exist between caveolae

  12. Autonomic Modification of Intestinal Smooth Muscle Contractility

    ERIC Educational Resources Information Center

    Montgomery, Laura E. A.; Tansey, Etain A.; Johnson, Chris D.; Roe, Sean M.; Quinn, Joe G.

    2016-01-01

    Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe…

  13. Interstitial Cells: Regulators of Smooth Muscle Function

    PubMed Central

    Sanders, Kenton M.; Ward, Sean M.; Koh, Sang Don

    2014-01-01

    Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα+ cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues. PMID:24987007

  14. Autonomic modification of intestinal smooth muscle contractility.

    PubMed

    Montgomery, Laura E A; Tansey, Etain A; Johnson, Chris D; Roe, Sean M; Quinn, Joe G

    2016-03-01

    Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe this spontaneous activity and its modification by agents associated with parasympathetic and sympathetic nerve activity. A section of the rabbit small intestine is suspended in an organ bath, and the use of a pressure transducer and data-acquisition software allows the measurement of tension generated by the smooth muscle of intestinal walls. The application of the parasympathetic neurotransmitter ACh at varying concentrations allows students to observe an increase in intestinal smooth muscle tone with increasing concentrations of this muscarinic receptor agonist. Construction of a concentration-effect curve allows students to calculate an EC50 value for ACh and consider some basic concepts surrounding receptor occupancy and activation. Application of the hormone epinephrine to the precontracted intestine allows students to observe the inhibitory effects associated with sympathetic nerve activation. Introduction of the drug atropine to the preparation before a maximal concentration of ACh is applied allows students to observe the inhibitory effect of a competitive antagonist on the physiological response to a receptor agonist. The final experiment involves the observation of the depolarizing effect of K(+) on smooth muscle. Students are also invited to consider why the drugs atropine, codeine, loperamide, and botulinum toxin have medicinal uses in the management of gastrointestinal problems. PMID:26873897

  15. Endothelial and smooth muscle histamine receptors

    SciTech Connect

    Blank, R.S.; Hollis, T.M.

    1986-03-01

    Histamine is produced within the vascular wall and mediates a variety of normal and pathologic vascular responses. The interaction of histamine with its vascular cell receptors has been shown to affect factors such as actin cable formation, cyclase activities, prostacyclin synthesis, cell motility, and proliferation. In addition, abundant evidence exists to implicate an arterial nascent histamine pool in the control of vessel wall permeability under conditions of stress and injury. However, endothelial and smooth muscle cell histamine receptors have been only incompletely characterized. The authors report here the time-dependent, saturable, and trypsin sensitive binding of /sup 3/H-histamine to the endothelial cell surface. The K/sub d/ for endothelial and smooth muscle cell histamine receptors are 0.70 and 2.80 ..mu..M respectively. Histamine binding to smooth muscle cells also exhibited saturation with concentrations of /sup 3/H-histamine up to 4 ..mu..M. While the smooth muscle cell H/sub 1/ receptor binding was negligible, the H/sub 2/ receptor appeared to represent a relatively low affinity, high capacity site for histamine binding. The uptake of /sup 3/H-histamine in both cell types displayed kinetics consistent with that of fluid-phase pinocytosis.

  16. On the thermodynamics of smooth muscle contraction

    NASA Astrophysics Data System (ADS)

    Stålhand, Jonas; McMeeking, Robert M.; Holzapfel, Gerhard A.

    2016-09-01

    Cell function is based on many dynamically complex networks of interacting biochemical reactions. Enzymes may increase the rate of only those reactions that are thermodynamically consistent. In this paper we specifically treat the contraction of smooth muscle cells from the continuum thermodynamics point of view by considering them as an open system where matter passes through the cell membrane. We systematically set up a well-known four-state kinetic model for the cross-bridge interaction of actin and myosin in smooth muscle, where the transition between each state is driven by forward and reverse reactions. Chemical, mechanical and energy balance laws are provided in local forms, while energy balance is also formulated in the more convenient temperature form. We derive the local (non-negative) production of entropy from which we deduce the reduced entropy inequality and the constitutive equations for the first Piola-Kirchhoff stress tensor, the heat flux, the ion and molecular flux and the entropy. One example for smooth muscle contraction is analyzed in more detail in order to provide orientation within the established general thermodynamic framework. In particular the stress evolution, heat generation, muscle shorting rate and a condition for muscle cooling are derived.

  17. Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

    PubMed Central

    Perrino, Brian A

    2016-01-01

    An increase in intracellular Ca2+ is the primary trigger of contraction of gastrointestinal (GI) smooth muscles. However, increasing the Ca2+ sensitivity of the myofilaments by elevating myosin light chain phosphorylation also plays an essential role. Inhibiting myosin light chain phosphatase activity with protein kinase C-potentiated phosphatase inhibitor protein-17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation is considered to be the primary mechanism underlying myofilament Ca2+ sensitization. The relative importance of Ca2+ sensitization mechanisms to the diverse patterns of GI motility is likely related to the varied functional roles of GI smooth muscles. Increases in CPI-17 and MYPT1 phosphorylation in response to agonist stimulation regulate myosin light chain phosphatase activity in phasic, tonic, and sphincteric GI smooth muscles. Recent evidence suggests that MYPT1 phosphorylation may also contribute to force generation by reorganization of the actin cytoskeleton. The mechanisms responsible for maintaining constitutive CPI-17 and MYPT1 phosphorylation in GI smooth muscles are still largely unknown. The characteristics of the cell-types comprising the neuroeffector junction lead to fundamental differences between the effects of exogenous agonists and endogenous neurotransmitters on Ca2+ sensitization mechanisms. The contribution of various cell-types within the tunica muscularis to the motor responses of GI organs to neurotransmission must be considered when determining the mechanisms by which Ca2+ sensitization pathways are activated. The signaling pathways regulating Ca2+ sensitization may provide novel therapeutic strategies for controlling GI motility. This article will provide an overview of the current understanding of the biochemical basis for the regulation of Ca2+ sensitization, while also discussing the functional importance to different smooth muscles of the GI tract. PMID:26701920

  18. Tracheobronchial smooth muscle atrophy and separation.

    PubMed

    Mehta, Atul C; Zaki, Khawaja Salman; Banga, Amit; Singh, Jarmanjeet; Gildea, Thomas R; Arrossi, Valeria

    2015-01-01

    We report a case series involving 4 patients with chronic obstructive pulmonary disease who were on an appropriate medical regimen including a high dose of inhaled corticosteroids (ICS). During bronchoscopy, patients were found to have an excessive dynamic collapse of the posterior wall and its separation from the ends of the adjacent cartilaginous rings. This was causing a near-total occlusion of the tracheal and bronchial lumen during exhalation, thereby presenting with an obstructive pattern on the pulmonary functions. We suspect that this was caused by the atrophy of the smooth muscles of the tracheobronchial wall. We reviewed the literature to explore the mechanisms causing atrophy of the bronchial smooth muscle, focusing on the potential role of long-term ICS use. PMID:26138002

  19. Reaction of human smooth muscle antibody with thyroid cells

    PubMed Central

    Biberfeld, Gunnel; Fagraeus, Astrid; Lenkei, Rodica

    1974-01-01

    Sera from cases of active chronic hepatitis or acute hepatitis containing smooth muscle antibodies reacted by immunofluorescence with the membrane region of sectioned thyroid cells from thyrotoxic glands. With non-toxic glands the reaction was negative or weak. The prerequisite for a positive reaction was that the complement of the sera had been heat-inactivated. Absorption with smooth muscle antigen abolished the reaction of smooth muscle antibody positive sera with thyroid cells. Some smooth muscle antibody negative sera from cases with disorders other than liver disease were found to give a similar immunofluorescence staining of the membrane region of sectioned thyroid cells, but these antibodies were not absorbed with smooth muscle antigen. Culture of thyroid cells was found to increase the number of cells reacting with smooth muscle antibody. In contrast, the thyroid cell antigen reacting with smooth muscle antibody negative sera was lost during culture. PMID:4619977

  20. Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders

    PubMed Central

    Brozovich, F.V.; Nicholson, C.J.; Degen, C.V.; Gao, Yuan Z.; Aggarwal, M.

    2016-01-01

    The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function. PMID:27037223

  1. Cortex phellodendri Extract Relaxes Airway Smooth Muscle

    PubMed Central

    Jiang, Qiu-Ju; Chen, Weiwei; Dan, Hong; Tan, Li; Zhu, He; Yang, Guangzhong; Shen, Jinhua; Peng, Yong-Bo; Zhao, Ping; Xue, Lu; Yu, Meng-Fei; Ma, Liqun; Si, Xiao-Tang; Wang, Zhuo; Dai, Jiapei; Qin, Gangjian; Zou, Chunbin; Liu, Qing-Hua

    2016-01-01

    Cortex phellodendri is used to reduce fever and remove dampness and toxin. Berberine is an active ingredient of C. phellodendri. Berberine from Argemone ochroleuca can relax airway smooth muscle (ASM); however, whether the nonberberine component of C. phellodendri has similar relaxant action was unclear. An n-butyl alcohol extract of C. phellodendri (NBAECP, nonberberine component) was prepared, which completely inhibits high K+- and acetylcholine- (ACH-) induced precontraction of airway smooth muscle in tracheal rings and lung slices from control and asthmatic mice, respectively. The contraction induced by high K+ was also blocked by nifedipine, a selective blocker of L-type Ca2+ channels. The ACH-induced contraction was partially inhibited by nifedipine and pyrazole 3, an inhibitor of TRPC3 and STIM/Orai channels. Taken together, our data demonstrate that NBAECP can relax ASM by inhibiting L-type Ca2+ channels and TRPC3 and/or STIM/Orai channels, suggesting that NBAECP could be developed to a new drug for relieving bronchospasm. PMID:27239213

  2. Changes of smooth muscle contractile filaments in small bowel atresia

    PubMed Central

    Gfroerer, Stefan; Fiegel, Henning; Ramachandran, Priya; Rolle, Udo; Metzger, Roman

    2012-01-01

    AIM: To investigate morphological changes of intestinal smooth muscle contractile fibres in small bowel atresia patients. METHODS: Resected small bowel specimens from small bowel atresia patients (n = 12) were divided into three sections (proximal, atretic and distal). Standard histology hematoxylin-eosin staining and enzyme immunohistochemistry was performed to visualize smooth muscle contractile markers α-smooth muscle actin (SMA) and desmin using conventional paraffin sections of the proximal and distal bowel. Small bowel from age-matched patients (n = 2) undergoing Meckel’s diverticulum resection served as controls. RESULTS: The smooth muscle coat in the proximal bowel of small bowel atresia patients was thickened compared with control tissue, but the distal bowel was unchanged. Expression of smooth muscle contractile fibres SMA and desmin within the proximal bowel was slightly reduced compared with the distal bowel and control tissue. There were no major differences in the architecture of the smooth muscle within the proximal bowel and the distal bowel. The proximal and distal bowel in small bowel atresia patients revealed only minimal differences regarding smooth muscle morphology and the presence of smooth muscle contractile filament markers. CONCLUSION: Changes in smooth muscle contractile filaments do not appear to play a major role in postoperative motility disorders in small bowel atresia. PMID:22791945

  3. Collagen formation by transformed smooth muscle cells after arterial injury.

    PubMed

    Chidi, C C; DePalma, R G

    1981-01-01

    Twenty-five normocholesterolemic rabbits were sacrificed at intervals up to 60 days after the thoracic aortas were de-endothelialized. Ultrastructural studies of both the re-endothelialized and nonendothelialized intima were done. The smooth muscle cells in the re-endothelialized intima showed segmental structural changes typically associated with transformation to a secretory cell type; abundant accumulations of collagen were in juxtaposition with these cells. The nonendothelialized intima did not demonstrate similar smooth muscle cell changes and collagen accumulation. These observations suggest that regenerating endothelial cells and intimal smooth muscle cells interact to cause smooth muscle cell transformation and collagen accumulation during arterial repair. PMID:7455897

  4. Caveolar nanospaces in smooth muscle cells

    PubMed Central

    Gherghiceanu, Mihaela; Popescu, L M

    2006-01-01

    Caveolae, specialized membrane nanodomains, have a key role in signaling processes, including calcium handling in smooth muscle cells (SMC). We explored the three-dimensional (3D) architecture of peripheral cytoplasmic space at the nanoscale level and the close spatial relationships between caveolae, sarcoplasmic reticulum (SR), and mitochondria, as ultrastructural basis for an excitation-contraction coupling system and, eventually, for excitation - transcription coupling. About 150 electron micrographs of SMC showed that superficial SR and peripheral mitochondria are rigorously located along the caveolar domains of plasma membrane, alternating with plasmalemmal dense plaques. Electron micrographs made on serial ultrathin sections were digitized, then computer-assisted organellar profiles were traced on images, and automatic 3D reconstruction was obtained using the ‘Reconstruct’ software. The reconstruction was made for 1 μm3 in rat stomach (muscularis mucosae) and 10 μm3 in rat urinary bladder (detrusor smooth muscle). The close appositions (about 15 nm distance) of caveolae, peripheral SR, and mitochondria create coherent cytoplasmic nanoscale subdomains. Apparently, 80% of caveolae establish close contacts with SR and about 10% establish close contacts with mitochondria in both types of SMC. Thus, our results show that caveolae and peripheral SR build Ca2+release units in which mitochondria often could play a part. The caveolae-SR couplings occupy 4.19% of the cellular volume in stomach and 3.10% in rat urinary bladder, while caveolae-mitochondria couplings occupy 3.66% and 3.17%, respectively. We conclude that there are strategic caveolae-SR or caveolae-mitochondria contacts at the nanoscale level in the cortical cytoplasm of SMC, presumably responsible for a vectorial control of free Ca2+ cytoplasmic concentrations in definite nanospaces. This may account for slective activation of specific Ca2+ signaling pathways. PMID:16796817

  5. Epigenetic regulation of smooth muscle cell plasticity

    PubMed Central

    Liu, Renjing; Leslie, Kristen L.; Martin, Kathleen A.

    2014-01-01

    Smooth muscle cells (SMC) are the major cell type in blood vessels. Their principle function in the body is to regulate blood flow and pressure through vessel wall contraction and relaxation. Unlike many other mature cell types in the adult body, SMC do not terminally differentiate but retain a remarkable plasticity. They have the unique ability to toggle between a differentiated and quiescent “contractile” state and a highly proliferative and migratory “synthetic” phenotype in response to environmental stresses. While there have been major advances in our understanding of SMC plasticity through the identification of growth factors and signals that can influence the SMC phenotype, how these regulate SMC plasticity remains unknown. To date, several key transcription factors and regulatory cis elements have been identified that play a role in modulating SMC state. The frontier in understanding the molecular mechanisms underlying SMC plasticity has now advanced to the level of epigenetics. This review will summarize the epigenetic regulation of SMC, highlighting the role of histone modification, DNA methylation, and our most recent identification of a DNA demethylation pathway in SMC that is pivotal in the regulation of the SMC phenotypic state. Many disorders are associated with smooth muscle dysfunction, including atherosclerosis, the major underlying cause of stroke and coronary heart disease, as well as transplant vasculopathy, aneurysm, asthma, hypertension, and cancer. An increased understanding of the major regulators of SMC plasticity will lead to the identification of novel target molecules that may, in turn, lead to novel drug discoveries for the treatment of these diseases. PMID:24937434

  6. Epigenetic regulation of smooth muscle cell plasticity.

    PubMed

    Liu, Renjing; Leslie, Kristen L; Martin, Kathleen A

    2015-04-01

    Smooth muscle cells (SMC) are the major cell type in blood vessels. Their principal function in the body is to regulate blood flow and pressure through vessel wall contraction and relaxation. Unlike many other mature cell types in the adult body, SMC do not terminally differentiate but retain a remarkable plasticity. They have the unique ability to toggle between a differentiated and quiescent "contractile" state and a highly proliferative and migratory "synthetic" phenotype in response to environmental stresses. While there have been major advances in our understanding of SMC plasticity through the identification of growth factors and signals that can influence the SMC phenotype, how these regulate SMC plasticity remains unknown. To date, several key transcription factors and regulatory cis elements have been identified that play a role in modulating SMC state. The frontier in understanding the molecular mechanisms underlying SMC plasticity has now advanced to the level of epigenetics. This review will summarize the epigenetic regulation of SMC, highlighting the role of histone modification, DNA methylation, and our most recent identification of a DNA demethylation pathway in SMC that is pivotal in the regulation of the SMC phenotypic state. Many disorders are associated with smooth muscle dysfunction, including atherosclerosis, the major underlying cause of stroke and coronary heart disease, as well as transplant vasculopathy, aneurysm, asthma, hypertension, and cancer. An increased understanding of the major regulators of SMC plasticity will lead to the identification of novel target molecules that may, in turn, lead to novel drug discoveries for the treatment of these diseases. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity. PMID:24937434

  7. Regeneration and Maintenance of Intestinal Smooth Muscle Phenotypes

    NASA Astrophysics Data System (ADS)

    Walthers, Christopher M.

    Tissue engineering is an emerging field of biomedical engineering that involves growing artificial organs to replace those lost to disease or injury. Within tissue engineering, there is a demand for artificial smooth muscle to repair tissues of the digestive tract, bladder, and vascular systems. Attempts to develop engineered smooth muscle tissues capable of contracting with sufficient strength to be clinically relevant have so far proven unsatisfactory. The goal of this research was to develop and sustain mature, contractile smooth muscle. Survival of implanted SMCs is critical to sustain the benefits of engineered smooth muscle. Survival of implanted smooth muscle cells was studied with layered, electrospun polycaprolactone implants with lasercut holes ranging from 0--25% porosity. It was found that greater angiogenesis was associated with increased survival of implanted cells, with a large increase at a threshold between 20% and 25% porosity. Heparan sulfate coatings improved the speed of blood vessel infiltration after 14 days of implantation. With these considerations, thicker engineered tissues may be possible. An improved smooth muscle tissue culture technique was utilized. Contracting smooth muscle was produced in culture by maintaining the native smooth muscle tissue organization, specifically by sustaining intact smooth muscle strips rather than dissociating tissue in to isolated smooth muscle cells. Isolated cells showed a decrease in maturity and contained fewer enteric neural and glial cells. Muscle strips also exhibited periodic contraction and regular fluctuation of intracellular calclium. The muscle strip maturity persisted after implantation in omentum for 14 days on polycaprolactone scaffolds. A low-cost, disposable bioreactor was developed to further improve maturity of cultured smooth muscle cells in an environment of controlled cyclical stress.The bioreactor consistently applied repeated mechanical strain with controllable inputs for strain

  8. Emergence of airway smooth muscle functions related to structural malleability

    PubMed Central

    Fredberg, Jeffrey J.

    2011-01-01

    The function of a complex system such as a smooth muscle cell is the result of the active interaction among molecules and molecular aggregates. Emergent macroscopic manifestations of these molecular interactions, such as the length-force relationship and its associated length adaptation, are well documented, but the molecular constituents and organization that give rise to these emergent muscle behaviors remain largely unknown. In this minireview, we describe emergent properties of airway smooth muscle that seem to have originated from inherent fragility of the cellular structures, which has been increasingly recognized as a unique and important smooth muscle attribute. We also describe molecular interactions (based on direct and indirect evidence) that may confer malleability on fragile structural elements that in turn may allow the muscle to adapt to large and frequent changes in cell dimensions. Understanding how smooth muscle works may hinge on how well we can relate molecular events to its emergent macroscopic functions. PMID:21127211

  9. Vascular Smooth Muscle Cells in Atherosclerosis.

    PubMed

    Bennett, Martin R; Sinha, Sanjay; Owens, Gary K

    2016-02-19

    The historical view of vascular smooth muscle cells (VSMCs) in atherosclerosis is that aberrant proliferation of VSMCs promotes plaque formation, but that VSMCs in advanced plaques are entirely beneficial, for example preventing rupture of the fibrous cap. However, this view has been based on ideas that there is a homogenous population of VSMCs within the plaque, that can be identified separate from other plaque cells (particularly macrophages) using standard VSMC and macrophage immunohistochemical markers. More recent genetic lineage tracing studies have shown that VSMC phenotypic switching results in less-differentiated forms that lack VSMC markers including macrophage-like cells, and this switching directly promotes atherosclerosis. In addition, VSMC proliferation may be beneficial throughout atherogenesis, and not just in advanced lesions, whereas VSMC apoptosis, cell senescence, and VSMC-derived macrophage-like cells may promote inflammation. We review the effect of embryological origin on VSMC behavior in atherosclerosis, the role, regulation and consequences of phenotypic switching, the evidence for different origins of VSMCs, and the role of individual processes that VSMCs undergo in atherosclerosis in regard to plaque formation and the structure of advanced lesions. We think there is now compelling evidence that a full understanding of VSMC behavior in atherosclerosis is critical to identify therapeutic targets to both prevent and treat atherosclerosis. PMID:26892967

  10. ASIC proteins regulate smooth muscle cell migration.

    PubMed

    Grifoni, Samira C; Jernigan, Nikki L; Hamilton, Gina; Drummond, Heather A

    2008-03-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na(+)Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration. PMID:17936312

  11. Smooth muscle cell calcium activation mechanisms

    PubMed Central

    Berridge, Michael J

    2008-01-01

    Smooth muscle cell (SMC) contraction is controlled by the Ca2+ and Rho kinase signalling pathways. While the SMC Rho kinase system seems to be reasonably constant, there is enormous variation with regard to the mechanisms responsible for generating Ca2+ signals. One way of dealing with this diversity is to consider how this system has been adapted to control different SMC functions. Phasic SMCs (vas deferens, uterus and bladder) rely on membrane depolarization to drive Ca2+ influx across the plasma membrane. This depolarization can be induced by neurotransmitters or through the operation of a membrane oscillator. Many tonic SMCs (vascular, airway and corpus cavernosum) are driven by a cytosolic Ca2+ oscillator that generates periodic pulses of Ca2+. A similar oscillator is present in pacemaker cells such as the interstitial cells of Cajal (ICCs) and atypical SMCs that control other tonic SMCs (gastrointestinal, urethra, ureter). The changes in membrane potential induced by these cytosolic oscillators does not drive contraction directly but it functions to couple together individual oscillators to provide the synchronization that is a characteristic feature of many tonic SMCs. PMID:18787034

  12. Nox regulation of smooth muscle contraction

    PubMed Central

    Ritsick, Darren R.; Edens, William A.; Finnerty, Victoria; Lambeth, J. David

    2007-01-01

    The catalytic subunit, gp91phox (a.k.a., Nox2) of the NADPH-oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology is being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction. PMID:17561091

  13. Neurotrophin and Neurotrophin Receptors in Vascular Smooth Muscle Cells

    PubMed Central

    Donovan, Michael J.; Miranda, Rajesh C.; Kraemer, Rosemary; McCaffrey, Timothy A.; Tessarollo, Lino; Mahadeo, Debbie; Sharif, Setareh; Kaplan, David R.; Tsoulfas, Pantelis; Parada, Luis; Toran-Allerand, C. Dominique; Hajjar, David P.; Hempstead, Barbara L.

    1995-01-01

    The neurotrophins, a family of related polypeptide growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 and NT-4/5 promote the survival and differentiation of distinctive sets of embryonic neurons. Here we define a new functional role for neurotrophins, as autocrine or local paracrine mediators of vascular smooth muscle cell migration. We have identified neurotrophins, and their cognate receptors, the trk tyrosine kinases, in human and rat vascular smooth muscle cells in vivo. In vitro, cultured human smooth muscle cells express BDNF; NT-3; and trk A, B, and C Similarly, rat smooth muscle cells expressed all three trk receptors as well as all four neurotrophins. Moreover, NGF induces cultured human smooth muscle cell migration at subnanomolar concentrations. In the rat aortic balloon deendothelialization model of vascular injury, the expression of NGF, BDNF, and their receptors trk A and trk B increased dramatically in the area of injury within 3 days and persisted during the formation of the neointima. In human coronary atherosclerotic lesions, BDNF, NT-3, and NT-4/5, and the trk B and trk C receptors could be demonstrated in smooth muscle cells. These findings suggest that neurotrophins play an important role in regulating the response of vascular smooth muscle cells to injury. ImagesFigure 1Figure 2Figure 3Figure 5Figure 6Figure 7Figure 8 PMID:7639328

  14. Modeling the dispersion effects of contractile fibers in smooth muscles

    NASA Astrophysics Data System (ADS)

    Murtada, Sae-Il; Kroon, Martin; Holzapfel, Gerhard A.

    2010-12-01

    Micro-structurally based models for smooth muscle contraction are crucial for a better understanding of pathological conditions such as atherosclerosis, incontinence and asthma. It is meaningful that models consider the underlying mechanical structure and the biochemical activation. Hence, a simple mechanochemical model is proposed that includes the dispersion of the orientation of smooth muscle myofilaments and that is capable to capture available experimental data on smooth muscle contraction. This allows a refined study of the effects of myofilament dispersion on the smooth muscle contraction. A classical biochemical model is used to describe the cross-bridge interactions with the thin filament in smooth muscles in which calcium-dependent myosin phosphorylation is the only regulatory mechanism. A novel mechanical model considers the dispersion of the contractile fiber orientations in smooth muscle cells by means of a strain-energy function in terms of one dispersion parameter. All model parameters have a biophysical meaning and may be estimated through comparisons with experimental data. The contraction of the middle layer of a carotid artery is studied numerically. Using a tube the relationships between the internal pressure and the stretches are investigated as functions of the dispersion parameter, which implies a strong influence of the orientation of smooth muscle myofilaments on the contraction response. It is straightforward to implement this model in a finite element code to better analyze more complex boundary-value problems.

  15. Serotonin augments smooth muscle differentiation of bone marrow stromal cells.

    PubMed

    Hirota, Nobuaki; McCuaig, Sarah; O'Sullivan, Michael J; Martin, James G

    2014-05-01

    Bone marrow stromal cells (BMSCs) contain a subset of multipotent stem cells. Here, we demonstrate that serotonin, a biogenic amine released by platelets and mast cells, can induce the smooth muscle differentiation of BMSCs. Brown Norway rat BMSCs stimulated with serotonin had increased expression of the smooth muscle markers smooth muscle myosin heavy chain (MHC) and α actin (α-SMA) by qPCR and Western blot, indicating smooth muscle differentiation. This was accompanied by a concomitant down-regulation of the microRNA miR-25-5p, which was found to negatively regulate smooth muscle differentiation. Serotonin upregulated serum response factor (SRF) and myocardin, transcription factors known to induce contractile protein expression in smooth muscle cells, while it down-regulated Elk1 and Kruppel-like factor 4 (KLF4), known to induce proliferation. Serotonin increased SRF binding to promoter regions of the MHC and α-SMA genes, assessed by chromatin immunoprecipitation assay. Induction of smooth muscle differentiation by serotonin was blocked by the knock-down of SRF and myocardin. Transforming growth factor (TGF)-β1 was constitutively expressed by BMSCs and serotonin triggered its release. Inhibition of miR-25-5p augmented TGF-β1 expression, however the differentiation of BMSCs was not mediated by TGF-β1. These findings demonstrate that serotonin promotes a smooth muscle-like phenotype in BMSCs by altering the balance of SRF, myocardin, Elk1 and KLF4 and miR-25-5p is involved in modulating this balance. Therefore, serotonin potentially contributes to the pathogenesis of diseases characterized by tissue remodeling with increased smooth muscle mass. PMID:24595007

  16. Sphingosylphosphorylcholine inhibits macrophage adhesion to vascular smooth muscle cells.

    PubMed

    Wirrig, Christiane; McKean, Jenny S; Wilson, Heather M; Nixon, Graeme F

    2016-09-01

    Inflammation in de-endothelialised arteries contributes to the development of cardiovascular diseases. The process that initiates this inflammatory response is the adhesion of monocytes/macrophages to exposed vascular smooth muscle cells, typically stimulated by cytokines such as tumour necrosis factor-α (TNF). The aim of this study was to determine the effect of the sphingolipid sphingosylphosphorylcholine (SPC) on the interaction of monocytes/macrophages with vascular smooth muscle cells. Rat aortic smooth muscle cells and rat bone marrow-derived macrophages were co-cultured using an in vitro assay following incubation with sphingolipids to assess inter-cellular adhesion. We reveal that SPC inhibits the TNF-induced adhesion of macrophages to smooth muscle cells. This anti-adhesive effect was the result of SPC-induced changes to the smooth muscle cells (but not the macrophages) and was mediated, at least partly, via the sphingosine 1-phosphate receptor subtype 2. Lipid raft domains were also required. Although SPC did not alter expression or membrane distribution of the adhesion proteins intercellular adhesion molecule-1 and vascular cellular adhesion protein-1 in smooth muscle cells, SPC preincubation inhibited the TNF-induced increase in inducible nitric oxide synthase (NOS2) resulting in a subsequent decrease in nitric oxide production. Inhibiting NOS2 activation in smooth muscle cells led to a decrease in the adhesion of macrophages to smooth muscle cells. This study has therefore delineated a novel pathway which can inhibit the interaction between macrophages and vascular smooth muscle cells via SPC-induced repression of NOS2 expression. This mechanism could represent a potential drug target in vascular disease. PMID:27402344

  17. Tobacco constituents are mitogenic for arterial smooth-muscle cells

    SciTech Connect

    Becker, C.G.; Hajjar, D.P.; Hefton, J.M.

    1985-07-01

    Tobacco glycoprotein (TGP) purified from flue-cured tobacco leaves, tar-derived material (TAR), the water soluble, nondialyzable, delipidized extract of cigarette smoke condensate, rutin-bovine serum albumin conjugates, quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells, but not adventitial fibroblasts. The mitogenicity appears to depend on polyphenol epitopes on carrier molecules. Ellagic acid, another plant polyphenol, inhibited arterial smooth-muscle proliferation. These results suggest that a number of ubiquitous, plant-derived substances may influence smooth-muscle cell proliferation in the arterial wall.

  18. Caffeine relaxes smooth muscle through actin depolymerization.

    PubMed

    Tazzeo, Tracy; Bates, Genevieve; Roman, Horia Nicolae; Lauzon, Anne-Marie; Khasnis, Mukta D; Eto, Masumi; Janssen, Luke J

    2012-08-15

    Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle. PMID:22683573

  19. Smooth muscle signalling pathways in health and disease

    PubMed Central

    Kim, H R; Appel, S; Vetterkind, S; Gangopadhyay, S S; Morgan, K G

    2008-01-01

    Smooth muscle contractile activity is a major regulator of function of the vascular system, respiratory system, gastrointestinal system and the genitourinary systems. Malfunction of contractility in these systems leads to a host of clinical disorders, and yet, we still have major gaps in our understanding of the molecular mechanisms by which contractility of the differentiated smooth muscle cell is regulated. This review will summarize recent advances in the molecular understanding of the regulation of smooth muscle myosin activity via phosphorylation/dephosphorylation of myosin, the regulation of the accessibility of actin to myosin via the actin-binding proteins calponin and caldesmon, and the remodelling of the actin cytoskeleton. Understanding of the molecular ‘players’ should identify target molecules that could point the way to novel drug discovery programs for the treatment of smooth muscle disorders such as cardiovascular disease, asthma, functional bowel disease and pre-term labour. PMID:19120701

  20. Origins of increased airway smooth muscle mass in asthma.

    PubMed

    Berair, Rachid; Saunders, Ruth; Brightling, Christopher E

    2013-01-01

    Asthma is characterized by both chronic inflammation and airway remodeling. Remodeling--the structural changes seen in asthmatic airways--is pivotal in the pathogenesis of the disease. Although significant advances have been made recently in understanding the different aspects of airway remodeling, the exact biology governing these changes remains poorly understood. There is broad agreement that, in asthma, increased airway smooth muscle mass, in part due to smooth muscle hyperplasia, is a very significant component of airway remodeling. However, significant debate persists on the origins of these airway smooth muscle cells. In this review article we will explore the natural history of airway remodeling in asthma and we will discuss the possible contribution of progenitors, stem cells and epithelial cells in mesenchymal cell changes, namely airway smooth muscle hyperplasia seen in the asthmatic airways. PMID:23742314

  1. Stimulation of aortic smooth muscle cell mitogenesis by serotonin

    SciTech Connect

    Nemecek, G.M.; Coughlin, S.R.; Handley, D.A.; Moskowitz, M.A.

    1986-02-01

    Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 ..mu..M serotonin with increased incorporation of (/sup 3/H)thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 ..mu..M. At a concentration of 1 ..mu..M, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was approx. = 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors.

  2. Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure.

    PubMed

    Krebs, Luke T; Norton, Christine R; Gridley, Thomas

    2016-02-01

    The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice. PMID:26742650

  3. Ablation of smooth muscle myosin heavy chain SM2 increases smooth muscle contraction and results in postnatal death in mice.

    PubMed

    Chi, Mei; Zhou, Yingbi; Vedamoorthyrao, Srikanth; Babu, Gopal J; Periasamy, Muthu

    2008-11-25

    The physiological relevance of smooth muscle myosin isoforms SM1 and SM2 has not been understood. In this study we generated a mouse model specifically deficient in SM2 myosin isoform but expressing SM1, using an exon-specific gene targeting strategy. The SM2 homozygous knockout (SM2(-/-)) mice died within 30 days after birth, showing pathologies including segmental distention of alimentary tract, retention of urine in renal pelvis, distension of bladder, and the development of end-stage hydronephrosis. In contrast, the heterozygous (SM2(+/-)) mice appeared normal and reproduced well. In SM2(-/-) bladder smooth muscle the loss of SM2 myosin was accompanied by a concomitant down-regulation of SM1 and a reduced number of thick filaments. However, muscle strips from SM2(-/-) bladder showed increased contraction to K(+) depolarization or in response to M3 receptor agonist Carbachol. An increase of contraction was also observed in SM2(-/-) aorta. However, the SM2(-/-) bladder was associated with unaltered regulatory myosin light chain (MLC20) phosphorylation. Moreover, other contractile proteins, such as alpha-actin and tropomyosin, were not altered in SM2(-/-) bladder. Therefore, the loss of SM2 myosin alone could have induced hypercontractility in smooth muscle, suggesting that distinctly from SM1, SM2 may negatively modulate force development during smooth muscle contraction. Also, because SM2(-/-) mice develop lethal multiorgan dysfunctions, we propose this regulatory property of SM2 is essential for normal contractile activity in postnatal smooth muscle physiology. PMID:19011095

  4. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

    PubMed

    Ma, Yun-Yun; Sun, Lin; Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

  5. Piperine Congeners as Inhibitors of Vascular Smooth Muscle Cell Proliferation.

    PubMed

    Mair, Christina E; Liu, Rongxia; Atanasov, Atanas G; Wimmer, Laurin; Nemetz-Fiedler, Daniel; Sider, Nadine; Heiss, Elke H; Mihovilovic, Marko D; Dirsch, Verena M; Rollinger, Judith M

    2015-08-01

    Successful vascular healing after percutaneous coronary interventions is related to the inhibition of abnormal vascular smooth muscle cell proliferation and efficient re-endothelialization. In the search for vascular smooth muscle cell anti-proliferative agents from natural sources we identified piperine (1), the main pungent constituent of the fruits from Piper nigrum (black pepper). Piperine inhibited vascular smooth muscle cell proliferation with an IC50 of 21.6 µM, as quantified by a resazurin conversion assay. Investigations of ten piperamides isolated from black pepper fruits and 15 synthesized piperine derivatives resulted in the identification of three potent vascular smooth muscle cell proliferation inhibitors: the natural alkaloid pipertipine (4), and the two synthetic derivatives (2E,4E)-N,N-dibutyl-5-(3,5-dimethoxyphenyl)penta-2,4-dienamide (14) and (E)-N,N-dibutyl-3-(naphtho[2,3-d][1,3]dioxol-5-yl)acrylamide (20). They showed IC50 values of 3.38, 6.00, and 7.85 µM, respectively. Furthermore, the synthetic compound (2E,4E)-5-(4-fluorophenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-one (12) was found to be cell type selective, by inhibiting vascular smooth muscle cell proliferation with an IC50 of 11.8 µM without influencing the growth of human endothelial cells. PMID:26132851

  6. Muscarinic receptor size on smooth muscle cells and membranes

    SciTech Connect

    Collins, S.M.; Jung, C.Y.; Grover, A.K.

    1986-08-01

    The loss of (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) binding following high-energy radiation was used to compare the muscarinic receptor size on single smooth muscle cells isolated by collagenase digestion from the canine stomach and on plasma membranes derived from intact gastric smooth muscle without exposure to exogenous proteolysis. Radiation inactivation of galactose oxidase (68 kdaltons), yeast alcohol dehydrogenase (160 kdaltons), and pyruvate kinase (224 kdaltons) activities were used as molecular-weight standards. Radiation inactivation of (/sup 3/H)QNB binding to rat brain membranes, which gave a target size of 86 kdaltons, served as an additional control. In isolated smooth muscle cells, the calculated size of the muscarinic receptor was 80 +/- 8 kdaltons. In contrast, in a smooth muscle enriched plasma membrane preparation, muscarinic receptor size was significantly smaller at 45 +/- 3 kdaltons. Larger molecular sizes were obtained either in the presence of protease inhibitors (62 +/- 4 kdaltons) or by using a crude membrane preparation of gastric smooth muscle 86 +/- 7 kdaltons).

  7. Smooth Muscle Enriched Long Noncoding RNA (SMILR) Regulates Cell Proliferation

    PubMed Central

    Ballantyne, Margaret D.; Pinel, Karine; Dakin, Rachel; Vesey, Alex T.; Diver, Louise; Mackenzie, Ruth; Garcia, Raquel; Welsh, Paul; Sattar, Naveed; Hamilton, Graham; Joshi, Nikhil; Dweck, Marc R.; Miano, Joseph M.; McBride, Martin W.; Newby, David E.; McDonald, Robert A.

    2016-01-01

    Background— Phenotypic switching of vascular smooth muscle cells from a contractile to a synthetic state is implicated in diverse vascular pathologies, including atherogenesis, plaque stabilization, and neointimal hyperplasia. However, very little is known about the role of long noncoding RNA (lncRNA) during this process. Here, we investigated a role for lncRNAs in vascular smooth muscle cell biology and pathology. Methods and Results— Using RNA sequencing, we identified >300 lncRNAs whose expression was altered in human saphenous vein vascular smooth muscle cells following stimulation with interleukin-1α and platelet-derived growth factor. We focused on a novel lncRNA (Ensembl: RP11-94A24.1), which we termed smooth muscle–induced lncRNA enhances replication (SMILR). Following stimulation, SMILR expression was increased in both the nucleus and cytoplasm, and was detected in conditioned media. Furthermore, knockdown of SMILR markedly reduced cell proliferation. Mechanistically, we noted that expression of genes proximal to SMILR was also altered by interleukin-1α/platelet-derived growth factor treatment, and HAS2 expression was reduced by SMILR knockdown. In human samples, we observed increased expression of SMILR in unstable atherosclerotic plaques and detected increased levels in plasma from patients with high plasma C-reactive protein. Conclusions— These results identify SMILR as a driver of vascular smooth muscle cell proliferation and suggest that modulation of SMILR may be a novel therapeutic strategy to reduce vascular pathologies. PMID:27052414

  8. Inhibitory action of relaxin on human cervical smooth muscle.

    PubMed

    Norström, A; Bryman, I; Wiqvist, N; Sahni, S; Lindblom, B

    1984-09-01

    The influence of purified porcine relaxin on contractility of human cervical smooth muscle was investigated in vitro. Strips of cervical tissue were obtained by needle biopsy from pregnant and nonpregnant women and were mounted in a superfused organ chamber for isometric measurement of contractile activity. Relaxin (0.005-25 micrograms/ml) inhibited the spontaneous contractions in cervical strips from 18% of nonpregnant, 68% of early pregnant, and in 100% of term pregnant women. These results indicate that relaxin has an inhibitory action on cervical smooth muscle and that this effect is more constantly detected as pregnancy proceeds. PMID:6746858

  9. Bronchospasm and its biophysical basis in airway smooth muscle

    PubMed Central

    Fredberg, Jeffrey J

    2004-01-01

    Airways hyperresponsiveness is a cardinal feature of asthma but remains unexplained. In asthma, the airway smooth muscle cell is the key end-effector of bronchospasm and acute airway narrowing, but in just the past five years our understanding of the relationship of responsiveness to muscle biophysics has dramatically changed. It has become well established, for example, that muscle length is equilibrated dynamically rather than statically, and that non-classical features of muscle biophysics come to the forefront, including unanticipated interactions between the muscle and its time-varying load, as well as the ability of the muscle cell to adapt rapidly to changes in its dynamic microenvironment. These newly discovered phenomena have been described empirically, but a mechanistic basis to explain them is only beginning to emerge. PMID:15084229

  10. Airway smooth muscle in the pathophysiology and treatment of asthma

    PubMed Central

    Solway, Julian

    2013-01-01

    Airway smooth muscle (ASM) plays an integral part in the pathophysiology of asthma. It is responsible for acute bronchoconstriction, which is potentiated by constrictor hyperresponsiveness, impaired relaxation and length adaptation. ASM also contributes to airway remodeling and inflammation in asthma. In light of this, ASM is an important target in the treatment of asthma. PMID:23305987

  11. A smooth muscle-like origin for beige adipocytes.

    PubMed

    Long, Jonathan Z; Svensson, Katrin J; Tsai, Linus; Zeng, Xing; Roh, Hyun C; Kong, Xingxing; Rao, Rajesh R; Lou, Jesse; Lokurkar, Isha; Baur, Wendy; Castellot, John J; Rosen, Evan D; Spiegelman, Bruce M

    2014-05-01

    Thermogenic UCP1-positive cells, which include brown and beige adipocytes, transform chemical energy into heat and increase whole-body energy expenditure. Using a ribosomal profiling approach, we present a comprehensive molecular description of brown and beige gene expression from multiple fat depots in vivo. This UCP1-TRAP data set demonstrates striking similarities and important differences between these cell types, including a smooth muscle-like signature expressed by beige, but not classical brown, adipocytes. In vivo fate mapping using either a constitutive or an inducible Myh11-driven Cre demonstrates that at least a subset of beige cells arise from a smooth muscle-like origin. Finally, ectopic expression of PRDM16 converts bona fide vascular smooth muscle cells into Ucp1-positive adipocytes in vitro. These results establish a portrait of brown and beige adipocyte gene expression in vivo and identify a smooth muscle-like origin for beige cells. PMID:24709624

  12. Smooth Muscle-Mediated Connective Tissue Remodeling in Pulmonary Hypertension

    NASA Astrophysics Data System (ADS)

    Mecham, Robert P.; Whitehouse, Loren A.; Wrenn, David S.; Parks, William C.; Griffin, Gail L.; Senior, Robert M.; Crouch, Edmond C.; Stenmark, Kurt R.; Voelkel, Norbert F.

    1987-07-01

    Abnormal accumulation of connective tissue in blood vessels contributes to alterations in vascular physiology associated with disease states such as hypertension and atherosclerosis. Elastin synthesis was studied in blood vessels from newborn calves with severe pulmonary hypertension induced by alveolar hypoxia in order to investigate the cellular stimuli that elicit changes in pulmonary arterial connective tissue production. A two- to fourfold increase in elastin production was observed in pulmonary artery tissue and medial smooth muscle cells from hypertensive calves. This stimulation of elastin production was accompanied by a corresponding increase in elastin messenger RNA consistent with regulation at the transcriptional level. Conditioned serum harvested from cultures of pulmonary artery smooth muscle cells isolated from hypertensive animals contained one or more low molecular weight elastogenic factors that stimulated the production of elastin in both fibroblasts and smooth muscle cells and altered the chemotactic responsiveness of fibroblasts to elastin peptides. These results suggest that connective tissue changes in the pulmonary vasculature in response to pulmonary hypertension are orchestrated by the medial smooth muscle cell through the generation of specific differentiation factors that alter both the secretory phenotype and responsive properties of surrounding cells.

  13. New insights in endothelial and smooth muscle cell communication.

    PubMed

    Conejo, Víctor Arana; De Haro, Roberto; Sosa-Melgarejo, Jorge; Méndez, José D

    2007-01-01

    Based on immunohistochemical techniques against connexins and the intercellular flux of staining molecules, it has previously been shown that electrotonic communication occurs among endothelial and vascular smooth muscle cells, this due to the presence of myoendothelial gap junctions. The aim of this study was to evaluate the density of myoendothelial contacts in the left coronary and internal mammary arteries as well as in the left saphenous vein by means of electron microscopy, the distance between both cells participating in an myoendothelial contact with a semi-automatic image analysis system and the presence of homocellular and heterocellular gap junctions between endothelial and smooth muscle cells by using the immunohistochemical technique and confocal microscopy in thoracic aorta were also analyzed. The results are that all blood vessels studied present myoendothelial contacts, while density studies show that they are more abundant in the saphenous vein. The myoendothelial contact distance is constant and in no case the cytoplasmic processes reach the plasma membrane of the partner cell toward which they are advanced. Homocellular gap junctions were found between smooth muscle cells and between endothelial cells. Heterocellular gap junctions were absent, evidencing the possibility that signaling molecules between endothelial and smooth muscle cells may be transferred through plasma membranes as was once thought and not necessarily by electrotonic communication. PMID:17383847

  14. Estimates of activation in arterial smooth muscle.

    PubMed

    Singer, H A; Kamm, K E; Murphy, R A

    1986-09-01

    We have previously described the onset of a "latch" state in the swine carotid media after K+ depolarization. This state was characterized by maintained stress after a decrease in shortening velocities and in the level of cross-bridge phosphorylation. The present experiments were designed to determine whether there were changes in other mechanical properties in swine carotid media associated with the onset of the latch state. Medial strips (less than 500 microM thick), incubated in physiological salt solution (PSS) at 37 degrees C at their optimal length (Lo), were subjected to ramp stretches (5.86 mm/s) of 5% Lo. The active stress (Sa) response to stretch was computed by subtraction of the passive element contribution (as determined from identical stretches after 30 min incubation in Ca2+-free PSS) from the total response in the activated muscle. Transitions in the total and active stress responses to stretch were observed in strips stimulated with 109 mM K+ for 1 min or longer and were interpreted as yielding of the contractile apparatus. Active dynamic stiffness (dS/dLo) calculated from the initial 1% Lo portion of the stretch response, correlated linearly with active stress over a wide range. Maximal stress and dynamic stiffness were reached by 1 min and were maintained for at least 30 min in K+-depolarized preparations. However, yield stress increased significantly between 1 and 10 min, and there was a large increase in the length at which yield was observed (1.09 +/- 0.06 to 1.86 +/- 0.10% Lo; n = 9). These increases were maintained between 10 and 30 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3752237

  15. A modified force-velocity equation for smooth muscle contraction.

    PubMed

    Wang, J; Jiang, H; Stephens, N L

    1994-01-01

    It has been suggested that in skeletal muscle the force-velocity relationship may not be a simple hyperbolic one, as defined by Hill's equation. To determine whether smooth muscle demonstrated the same properties, quick-release force-velocity curves were obtained from canine tracheal smooth muscle. The results showed that the observed data points for tracheal smooth muscle systematically deviated from a hyperbola. Such deviation occurred at values of force (P) approaching maximum isometric force (Po) for curves elicited by quick release at 2 and 10 s in the course of isometric contractions. Shortening velocities under a given afterload were overestimated at the high-force end (P > 75% Po) by Hill's equation; this implied that a relationship more complex than a simple hyperbola was involved at high loads. We next focused on finding an equation to also fit those directly measured data points that did not conform to a hyperbola. Our rationale in developing the equation was that a plot of the linearized transform of Hill's equation should yield a straight line over the entire range of loads at which velocities were measured. The plot demonstrated that, in the low-load high-velocity portion of the curve, a peak value was reached at 70-80% Po, which decreased as load increased in the high-load low-velocity portion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8175513

  16. Increased smooth muscle contractility in mice deficient for neuropilin 2.

    PubMed

    Bielenberg, Diane R; Seth, Abhishek; Shimizu, Akio; Pelton, Kristine; Cristofaro, Vivian; Ramachandran, Aruna; Zwaans, Bernadette M M; Chen, Cheng; Krishnan, Ramaswamy; Seth, Meetu; Huang, Lin; Takashima, Seiji; Klagsbrun, Michael; Sullivan, Maryrose P; Adam, Rosalyn M

    2012-08-01

    Neuropilins (NRPs) are transmembrane receptors that bind class 3 semaphorins and VEGF family members to regulate axon guidance and angiogenesis. Although expression of NRP1 by vascular smooth muscle cells (SMCs) has been reported, NRP function in smooth muscle (SM) in vivo is unexplored. Using Nrp2(+/LacZ) and Nrp2(+/gfp) transgenic mice, we observed robust and sustained expression of Nrp2 in the SM compartments of the bladder and gut, but no expression in vascular SM, skeletal muscle, or cardiac muscle. This expression pattern was recapitulated in vitro using primary human SM cell lines. Alterations in cell morphology after treatment of primary visceral SMCs with the NRP2 ligand semaphorin-3F (SEMA3F) were accompanied by inhibition of RhoA activity and myosin light chain phosphorylation, as well as decreased cytoskeletal stiffness. Ex vivo contractility testing of bladder muscle strips exposed to electrical stimulation or soluble agonists revealed enhanced tension generation of tissues from mice with constitutive or SM-specific knockout of Nrp2, compared with controls. Mice lacking Nrp2 also displayed increased bladder filling pressures, as assessed by cystometry in conscious mice. Together, these findings identify Nrp2 as a mediator of prorelaxant stimuli in SMCs and suggest a novel function for Nrp2 as a regulator of visceral SM contractility. PMID:22688055

  17. Human vascular smooth muscle cells express a urate transporter.

    PubMed

    Price, Karen L; Sautin, Yuri Y; Long, David A; Zhang, Li; Miyazaki, Hiroki; Mu, Wei; Endou, Hitoshi; Johnson, Richard J

    2006-07-01

    An elevated serum uric acid is associated with the development of hypertension and renal disease. Renal regulation of urate excretion is largely controlled by URAT1 (SLC22A12), a member of the organic anion transporter superfamily. This study reports the specific expression of URAT1 on human aortic vascular smooth muscle cells, as assessed by reverse transcription-PCR and Western blot analysis. Expression of URAT1 was localized to the cell membrane. Evidence that the URAT1 transporter was functional was provided by the finding that uptake of 14C-urate was significantly inhibited in the presence of probenecid, an organic anion transporter inhibitor. It is proposed that URAT1 may provide a mechanism by which uric acid enters the human vascular smooth muscle cell, a finding that may be relevant to the role of uric acid in cardiovascular disease. PMID:16775029

  18. Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.

    PubMed Central

    Guilford, W H; Dupuis, D E; Kennedy, G; Wu, J; Patlak, J B; Warshaw, D M

    1997-01-01

    Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin. Images FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 PMID:9138552

  19. Focal Ca2+ transient detection in smooth muscle.

    PubMed

    Young, John S; Amos, Robert J; Brain, Keith L

    2009-01-01

    Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients. PMID:19564842

  20. Smooth muscle FGF/TGFβ cross talk regulates atherosclerosis progression.

    PubMed

    Chen, Pei-Yu; Qin, Lingfeng; Li, Guangxin; Tellides, George; Simons, Michael

    2016-01-01

    The conversion of vascular smooth muscle cells (SMCs) from contractile to proliferative phenotype is thought to play an important role in atherosclerosis. However, the contribution of this process to plaque growth has never been fully defined. In this study, we show that activation of SMC TGFβ signaling, achieved by suppression of SMC fibroblast growth factor (FGF) signaling input, induces their conversion to a contractile phenotype and dramatically reduces atherosclerotic plaque size. The FGF/TGFβ signaling cross talk was observed in vitro and in vivo In vitro, inhibition of FGF signaling increased TGFβ activity, thereby promoting smooth muscle differentiation and decreasing proliferation. In vivo, smooth muscle-specific knockout of an FGF receptor adaptor Frs2α led to a profound inhibition of atherosclerotic plaque growth when these animals were crossed on Apoe(-/-) background and subjected to a high-fat diet. In particular, there was a significant reduction in plaque cellularity, increase in fibrous cap area, and decrease in necrotic core size. In agreement with these findings, examination of human coronary arteries with various degrees of atherosclerosis revealed a strong correlation between the activation of FGF signaling, loss of TGFβ activity, and increased disease severity. These results identify SMC FGF/TGFβ signaling cross talk as an important regulator of SMC phenotype switch and document a major contribution of medial SMC proliferation to atherosclerotic plaque growth. PMID:27189169

  1. Transdifferentiation of human endothelial progenitors into smooth muscle cells.

    PubMed

    Ji, HaYeun; Atchison, Leigh; Chen, Zaozao; Chakraborty, Syandan; Jung, Youngmee; Truskey, George A; Christoforou, Nicolas; Leong, Kam W

    2016-04-01

    Access to smooth muscle cells (SMC) would create opportunities for tissue engineering, drug testing, and disease modeling. Herein we report the direct conversion of human endothelial progenitor cells (EPC) to induced smooth muscle cells (iSMC) by induced expression of MYOCD. The EPC undergo a cytoskeletal rearrangement resembling that of mesenchymal cells within 3 days post initiation of MYOCD expression. By day 7, the reprogrammed cells show upregulation of smooth muscle markers ACTA2, MYH11, and TAGLN by qRT-PCR and ACTA2 and MYH11 expression by immunofluorescence. By two weeks, they resemble umbilical artery SMC in microarray gene expression analysis. The iSMC, in contrast to EPC control, show calcium transients in response to phenylephrine stimulation and a contractility an order of magnitude higher than that of EPC as determined by traction force microscopy. Tissue-engineered blood vessels constructed using iSMC show functionality with respect to flow- and drug-mediated vasodilation and vasoconstriction. PMID:26874281

  2. Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

    SciTech Connect

    Hurtado, Paola A.; Vora, Siddharth; Sume, Siddika Selva; Yang, Dan; Hilaire, Cynthia St.; Guo Ying; Palamakumbura, Amitha H.; Schreiber, Barbara M.; Ravid, Katya; Trackman, Philip C.

    2008-02-01

    Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-{alpha}-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.

  3. Smooth muscle titin forms in vitro amyloid aggregates.

    PubMed

    Bobylev, Alexandr G; Galzitskaya, Oxana V; Fadeev, Roman S; Bobyleva, Liya G; Yurshenas, Darya A; Molochkov, Nikolay V; Dovidchenko, Nikita V; Selivanova, Olga M; Penkov, Nikita V; Podlubnaya, Zoya A; Vikhlyantsev, Ivan M

    2016-07-01

    Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimer's disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700-4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles. PMID:27129292

  4. Ultrastructural Changes of the Smooth Muscle in Esophageal Atresia.

    PubMed

    Al-Shraim, Mubarak M; Eid, Refaat A; Musalam, Adel Osman; Radad, Khaled; Ibrahim, Ashraf H M; Malki, Talal A

    2015-01-01

    Esophageal atresia (EA) with or without tracheo-esophageal fistula (TEF) is a relatively rare congenital anomaly. Despite the advances in the management techniques and neonatal intensive care, esophageal dysmotility remains a very common problem following EA/TEF repair. Our current study aimed to describe the most significant ultrastructural changes of the smooth muscle cells (SMCs) trying to highlight some of the underlying mechanisms of esophageal dysmotility following EA/TEF repair. Twenty-three biopsies were obtained from the tip of the lower esophageal pouch (LEP) of 23 patients during primary repair of EA/TEF. Light microscopic examination was performed with hematoxylin and eosin (HE), and Van Gieson's stains. Ultrastructural examination was done using transmission electron microscopy (TEM). Histopathological examination showed distortion of smooth muscle layer and deposition of an abundant amount of fibrous tissue in-between smooth muscles. Using TEM, SMCs exhibited loss of the cell-to-cell adhesion, mitochondrial vacuolation, formation of myelin figures, and apoptotic fragmentation. There were also plasmalemmal projections and formation of ghost bodies. Interestingly, SMCs were found extending pseudopodia-like projections around adjacent collagen fibers. Engulfed collagen fibers by SMCs underwent degradation within autophagic vacuoles. Degeneration of SMCs and deposition of abundant extracellular collagen fibers are prominent pathological changes in LEP of EA/TEF. These changes might contribute to the pathogenesis of esophageal dysmotility in patients who have survived EA/TEF. PMID:26548437

  5. Contribution of intestinal smooth muscle to Crohn's disease fibrogenesis.

    PubMed

    Severi, C; Sferra, R; Scirocco, A; Vetuschi, A; Pallotta, N; Pronio, A; Caronna, R; Di Rocco, G; Gaudio, E; Corazziari, E; Onori, P

    2014-01-01

    Mesenchymal cells transdifferentiation and extracellular matrix deposition are involved in the fibrotic process of Crohn's disease (CD). Mesenchymal smooth muscle cells (SMCs) de-differentiation, driven by Platelet-derived growth factor (PDGF) that counteracts Transforming growth factor (TGF-β) has been studied in vascular muscle. The role of SMCs in intestinal fibrogenesis is still not clearly elucidated. Aim of the study was to evaluate the possible myogenic contribution to CD fibrotic process through the comparative analysis of histological, morphometric and molecular alterations occurring in human smooth muscle. Full thickness specimens were obtained from CD (non-involved and stenotic tracts) and healthy (control) ileum. Tissues were processed for histological and immunohistochemical (IHC) analyses and SMCs were isolated from the muscularis propria for morphofunctional and molecular (qPCR) analyses. CD stenotic ileum showed a significant increased thickness of all layers compared to CD non-involved and control ileum. IHC revealed an overexpression of α-smooth muscle actin and collagens I-III throughout all intestinal layers only in stenotic tracts. The two growth factors, PDGF and TGF-β, showed a progressive increase in expression in the muscle layer from CD non-involved to stenotic tracts. Freshly isolated SMCs presented alterations in CD non-involved tracts that progressively increased in the stenotic tracts consisting in a statistical increase in mRNA encoding for PDGF-β and collagen III, paralleled to a decrease in TGF-β and Tribbles-like protein-3 mRNA, and altered morphofunctional parameters consisting in progressive decreases in cell length and contraction to acetylcholine. These findings indicate that intrinsic myogenic alterations occur in CD ileum, that they likely precede stricture formation, and might represent suitable new targets for anti-fibrotic interventions. PMID:25578979

  6. A novel transgenic marker for migrating limb muscle precursors and for vascular smooth muscle cells.

    PubMed

    Tidhar, A; Reichenstein, M; Cohen, D; Faerman, A; Copeland, N G; Gilbert, D J; Jenkins, N A; Shani, M

    2001-01-01

    A unique pattern of LacZ expression was found in a transgenic mouse line, likely due to regulatory elements at the site of integration. Two new genes flanking the transgene were identified. At early stages of development, the transgene is transiently expressed in ventro-lateral demomyotomal cells migrating from the somites into the limb buds. At late developmental stages and in the adult, lacZ staining marks vascular smooth muscle cells throughout the vascular bed, with the exception of the major elastic arteries, and in pericytes. No expression was detected in skeletal and smooth muscles. Different patterns of expression in vascular smooth muscles was observed at distinct levels of the vascular tree, in arteries as well as in veins. Vessel injury, resulting in stimulation of smooth muscle cells proliferation and migration, is associated with transgene down-regulation. After the formation of neointima thickening, it is reactivated. This transgenic insertion may therefore be used as a useful marker to identify novel physiological cues or genetic elements involved in the regulation of the vascular smooth muscle phenotype(s). It may also provide an experimental tool for studying vasculature and the involvement of pericytes in regulating microvascular homeostasis. PMID:11146508

  7. Circulating micrornas as potential biomarkers of muscle atrophy

    NASA Astrophysics Data System (ADS)

    Wang, Fei

    2016-07-01

    Noninvasive biomarkers with diagnostic value and prognostic applications have long been desired to replace muscle biopsy for muscle atrophy patients. Growing evidence indicates that circulating microRNAs are biomarkers to assess pathophysiological status. Here, we show that the medium levels of six muscle-specific miRNAs (miR-1/23a/206/133/499/208b, also known as myomiRs) were all elevated in the medium of starved C2C12 cell (P < 0.01). And, the level of miR-1 and miR-23a were all elevated in the serum of hindlimb unloaded mice (P < 0.01). miR-23a levels were negatively correlated with both muscle mass and muscle fiber cross section area in muscle atrophy patients, indicating that they might represent the degree of muscle atrophy. Collectively, our data indicated that circulating myomiRs could serve as promising biomarkers for muscle atrophy.

  8. Hydrogen peroxide induced responses of cat tracheal smooth muscle cells

    PubMed Central

    Bauer, V; Oike, M; Tanaka, H; Inoue, R; Ito, Y

    1997-01-01

    The effects of hydrogen peroxide H2O2 (10−6 and 10−3 M) on membrane potential, membrane currents, intracellular calcium concentration, resting muscle tone and contractions elicited by electrical field stimulation (EFS) and carbachol were examined in cat tracheal strips and isolated smooth muscle cells. H2O2 (10−4 and 10−5 M) enhanced the amplitude of contractions and excitatory junction potentials (e.j.p.) evoked by EFS without changing muscle tone and resting membrane potential of the tracheal smooth muscle, and enhanced the contraction induced by carbachol (10−8 M). At an increased concentration (10−3 M), H2O2 elevated resting muscle tone and marginally hyperpolarized the membrane in the majority of the cells. In 51 out of 56 cells examined, H2O2 (10−6–10−3 M) elicited an outward current at a holding potential of −40 mV and enhanced the frequency of the spontaneous transient outward current (STOC). In 20 cells the outward current was preceded by a small inward current. In the other cells, H2O2 elicited only an inward current or did not affect the background current. In Ca2+ free solution the action of H2O2 on the resting muscle tone, STOCs, background current and on the current induced by ramp depolarization was significantly reduced. H2O2 (10−4 M) increased the intracellular ionized calcium concentration both in the absence and presence of external Ca2+. However, the effect developed faster and was of a higher amplitude in the presence of external Ca2+. These results suggest that H2O2 increases intracellular Ca2+, with a subsequent augmentation of stimulation-evoked contractions, and enhances Ca2+ and voltage-sensitive potassium conductance. PMID:9222542

  9. K(V)7 potassium channels: a new therapeutic target in smooth muscle disorders.

    PubMed

    Stott, Jennifer B; Jepps, Thomas A; Greenwood, Iain A

    2014-04-01

    Potassium channels are key regulators of smooth muscle tone, with increases in activity resulting in hyperpolarisation of the cell membrane, which acts to oppose vasoconstriction. Several potassium channels exist within smooth muscle, but the KV7 family of voltage-gated potassium channels have been identified as being crucial mediators of this process in a variety of smooth muscle. Recently, KV7 channels have been shown to be involved in the pathogenesis of hypertension, as well as being implicated in other smooth muscle disorders, providing a new and inviting target for smooth muscle disorders. PMID:24333708

  10. Adaptive response of pulmonary arterial smooth muscle to length change.

    PubMed

    Syyong, Harley; Cheung, Christine; Solomon, Dennis; Seow, Chun Y; Kuo, Kuo H

    2008-04-01

    Hypervasoconstriction is associated with pulmonary hypertension and dysfunction of the pulmonary arterial smooth muscle (PASM) is implicated. However, relatively little is known about the mechanical properties of PASM. Recent advances in our understanding of plastic adaptation in smooth muscle may shed light on the disease mechanism. In this study, we determined whether PASM is capable of adapting to length changes (especially shortening) and regain its contractile force. We examined the time course of length adaptation in PASM in response to step changes in length and to length oscillations mimicking the periodic stretches due to pulsatile arterial pressure. Rings from sheep pulmonary artery were mounted on myograph and stimulated using electrical field stimulation (12-16 s, 20 V, 60 Hz). The length-force relationship was determined at L(ref) to 0.6 L(ref), where L(ref) was a reference length close to the in situ length of PASM. The response to length oscillations was determined at L(ref), after the muscle was subjected to length oscillation of various amplitudes for 200 s at 1.5 Hz. Release (or stretch) of resting PASM from L(ref) to 0.6 (and vice versa) was followed by a significant force recovery (73 and 63%, respectively), characteristic of length adaptation. All recoveries of force followed a monoexponential time course. Length oscillations with amplitudes ranging from 5 to 20% L(ref) caused no significant change in force generation in subsequent contractions. It is concluded that, like many smooth muscles, PASM possesses substantial capability to adapt to changes in length. Under pathological conditions, this could contribute to hypervasoconstriction in pulmonary hypertension. PMID:18218913

  11. Angiogenesis is induced by airway smooth muscle strain.

    PubMed

    Hasaneen, Nadia A; Zucker, Stanley; Lin, Richard Z; Vaday, Gayle G; Panettieri, Reynold A; Foda, Hussein D

    2007-10-01

    Angiogenesis is an important feature of airway remodeling in both chronic asthma and chronic obstructive pulmonary disease (COPD). Airways in those conditions are exposed to excessive mechanical strain during periods of acute exacerbations. We recently reported that mechanical strain of human airway smooth muscle (HASM) led to an increase in their proliferation and migration. Sustained growth in airway smooth muscle in vivo requires an increase in the nutritional supply to these muscles, hence angiogenesis. In this study, we examined the hypothesis that cyclic mechanical strain of HASM produces factors promoting angiogenic events in the surrounding vascular endothelial cells. Our results show: 1) a significant increase in human lung microvascular endothelial cell (HMVEC-L) proliferation, migration, and tube formation following incubation in conditioned media (CM) from HASM cells exposed to mechanical strain; 2) mechanical strain of HASM cells induced VEGF expression and release; 3) VEGF neutralizing antibodies inhibited the proliferation, migration, and tube formations of HMVEC-L induced by the strained airway smooth muscle CM; 4) mechanical strain of HASM induced a significant increase in hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein, a transcription factor required for VEGF gene transcription; and 5) mechanical strain of HASM induced HIF-1alpha/VEGF through dual phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and ERK pathways. In conclusion, exposing HASM cells to mechanical strain induces signal transduction pathway through PI3K/Akt/mTOR and ERK pathways that lead to an increase in HIF-1alpha, a transcription factor required for VEGF expression. VEGF release by mechanical strain of HASM may contribute to the angiogenesis seen with repeated exacerbation of asthma and COPD. PMID:17693481

  12. Stimulant actions of volatile anaesthetics on smooth muscle

    PubMed Central

    Rang, H. P.

    1964-01-01

    A number of volatile anaesthetics, and some compounds synthesized in the search for new anaesthetics, have been tested on guinea-pig intestinal smooth muscle in vitro. All the compounds produced a contractile response. This effect did not correlate well with convulsant activity in vivo among the compounds tested. Two kinds of stimulant effect were distinguishable: (1) Rapid, transient contractions, abolished by cocaine or lachesine; most of the anaesthetics in clinical use had this action. (2) Slow, sustained contractions, unaffected by cocaine or lachesine; this effect predominated among the fluorinated ring compounds. Hexamethonium and mepyramine did not affect the contractile response to any of the compounds. The first type of effect presumably represents excitation of postganglionic nerve cells, while the second type is a direct action on the muscle cell. The action of perfluorobenzene, which is of the latter kind, was studied further. Adrenaline and lack of calcium diminished the contraction in parallel with the contraction to histamine, which suggests that the cell membrane was the site of action; in contrast to the stimulant action of histamine or acetylcholine, the effect was highly temperature-sensitive, being almost abolished by cooling to 32° C, and enhanced at 40° C. The depressant action of anaesthetics on smooth muscle is affected very little by temperature changes. These findings are discussed in relation to other observations which suggest a stimulant action of volatile anaesthetics on excitable tissues. Protein denaturation is tentatively suggested as a mechanism of action. PMID:14190470

  13. Epithelial modulation of preterm airway smooth muscle contraction.

    PubMed

    Panitch, H B; Wolfson, M R; Shaffer, T H

    1993-03-01

    To determine if epithelium from immature airways can modulate the responsiveness of smooth muscle, we studied paired trachealis muscle strips from preterm sheep. The epithelium was removed from one strip and left undisturbed in the other. Concentration-effect (CE) curves to acetylcholine (ACh), KCl, and isoproterenol were obtained. To evaluate maturational effects, responses to ACh and isoproterenol were studied in trachealis strips from adult airways. Maximal stress (Po) to ACh increased after epithelium removal in preterm (P < 0.05) but not adult strips. Epithelium removal caused a leftward shift of the ACh CE curves in both preterm and adult strips (P < 0.001) and a decrease in the dose required to achieve a one-half maximal response (ED50) in both preterm (P < 0.005) and adult strips (P < 0.05). The magnitude of the change in Po as well as in the ED50 for ACh between preterms and adults was similar. Epithelium removal did not alter either the Po or the CE curves of preterm strips stimulated by KCl. Response to isoproterenol in precontracted strips was enhanced in the presence of an intact epithelium in both groups (P < 0.05). These data demonstrate that preterm airway epithelium is able to modulate the responsiveness of smooth muscle. Additionally, the magnitude of the effect is unchanged with maturation. We speculate that damage of airway epithelium from mechanical ventilation may contribute to the increased incidence of airway hyperreactivity observed in preterm infants. PMID:8482688

  14. Interaction of Vascular Smooth Muscle Cells Under Low Shear Stress

    NASA Technical Reports Server (NTRS)

    Seidel, Charles L.

    1998-01-01

    The blood vessel wall consists of three cellular layers, an outer adventitial, a middle medial and an inner intimal layer. When the blood vessel forms in the embryo it begins as a tube composed of a single cell type called endothelial cells. Over time, other cells are recruited from the surrounding tissue to form additional layers on the outer surface of the endothelial tube. The cells that are recruited are called mesenchymal cells. Mesenchymal cells are responsible for the production of connective tissue that holds the blood vessel together and for developing into vascular smooth muscle cells that are responsible for regulating the diameter of the vessel (1) and therefore, blood flow. In a fully developed blood vessel, the endothelial cells make- up the majority of cells in the intimal layer while the mesenchymal cells make-up the majority of cells in the medial and adventitial layers. Within the medial layer of a mature vessel, cells are organized into multiple circular layers of alternating bands of connective tissue and cells. The cell layer is composed of a mixture of mesenchymal cells that have not developed into smooth muscle cells and fully developed smooth muscle cells (2). The assembly and organization of complex tissues is directed in part by a signaling system composed of proteins on the cell surface called adhesion molecules. Adhesion molecules enable cells to recognize each other as well as the composition of the connective tissue in which they reside (3). It was hypothesized that the different cell types that compose the vascular wall possess different adhesion molecules that enable them to recognize each other and through this recognition system, form the complex layered organization of the vascular wall. In other words, the layered organization is an intrinsic property of the cells. If this hypothesis is correct then the different cells that make up the vessel wall, when mixed together, should organize themselves into a layered structure

  15. Ionic conductances regulating the excitability of colonic smooth muscles

    PubMed Central

    Koh, Sang Don; Ward, Sean M.; Sanders, Kenton M.

    2012-01-01

    The tunica muscularis of the gastrointestinal (GI) tract contains two layers of smooth muscle cells (SMC) oriented perpendicular to each other. SMC express a variety of voltage-dependent and voltage-independent ionic conductance(s) that develop membrane potential and control excitability. Resting membrane potentials (RMP) vary through the GI tract but generally are within the range of −80 to −40mV. RMP sets the ‘gain’ of smooth muscle and regulates openings of voltage-dependent Ca2+ channels. A variety of K+ channels contribute to setting RMP of SMC. In most regions RMP is considerably less negative than the K+ equilibrium potential, due to a finely tuned balance between background K+ channels and non-selective cation channels (NSCC). Variations in expression patterns and openings of K+ channels and NSCC account for differences of the RMP in different regions of the GI tract. Smooth muscle excitability is also regulated by interstitial cells (interstitial cells of Cajal (ICC) and PDGFRα+ cells) that express additional conductances and are electrically coupled to SMC. Thus, ‘myogenic’ activity results from the integrated behavior of the SMC/ICC/PDGFRα+ cell (SIP) syncytium. Inputs from excitatory and inhibitory motor neurons are required to produce the complex motor patterns of the gut. Motor neurons innervate three cell-types in the SIP, and receptors, second messenger pathways and ion channels in these cells mediate post-junctional responses. Studies of isolated SIP cells have begun to unravel the mechanisms responsible for neural responses. This review discusses ion channels that set and regulate RMP of SIP cells and how neurotransmitters regulate membrane potential. PMID:22726670

  16. MicroRNA regulation of airway smooth muscle function.

    PubMed

    Sun, Maoyun; Lu, Quan

    2016-06-01

    Airway smooth muscle (ASM) controls airway narrowing and plays a pivotal role in the pathogenesis of asthma. MicroRNAs are small yet powerful gene tuners that regulate diverse cellular processes. Recent studies have demonstrated the versatile role of microRNAs in regulating multiple ASM phenotypes that are critically involved in asthma pathogenesis. These ASM phenotypes include proliferation, cell size, chemokine secretion, and contractility. Here we review microRNA-mediated regulation of ASM functions and discuss the potential of microRNAs as a novel class of therapeutic targets to improve ASM function for asthma therapy. PMID:26812790

  17. Osteogenic potential of alpha smooth muscle actin expressing muscle resident progenitor cells.

    PubMed

    Matthews, Brya G; Torreggiani, Elena; Roeder, Emilie; Matic, Igor; Grcevic, Danka; Kalajzic, Ivo

    2016-03-01

    Heterotopic ossification (HO) is a pathological process where bone forms in connective tissues such as skeletal muscle. Previous studies have suggested that muscle-resident non-myogenic mesenchymal progenitors are the likely source of osteoblasts and chondrocytes in HO. However, the previously identified markers of muscle-resident osteoprogenitors label up to half the osteoblasts within heterotopic lesions, suggesting other cell populations are involved. We have identified alpha smooth muscle actin (αSMA) as a marker of osteoprogenitor cells in bone and periodontium, and of osteo-chondro progenitors in the periosteum during fracture healing. We therefore utilized a lineage tracing approach to evaluate whether αSMACreERT2 identifies osteoprogenitors in the muscle. We show that in the muscle, αSMACreERT2 labels both perivascular cells, and satellite cells. αSMACre-labeled cells undergo osteogenic differentiation in vitro and form osteoblasts and chondrocytes in BMP2-induced HO in vivo. In contrast, Pax7CreERT2-labeled muscle satellite cells were restricted to myogenic differentiation in vitro, and rarely contributed to HO in vivo. Our data indicate that αSMACreERT2 labels a large proportion of osteoprogenitors in skeletal muscle, and therefore represents another marker of muscle-resident cells with osteogenic potential under HO-inducing stimulus. In contrast, muscle satellite cells make minimal contribution to bone formation in vivo. PMID:26721734

  18. Epstein-Barr Virus-Associated Smooth Muscle Tumor.

    PubMed

    Dekate, Jyoti; Chetty, Runjan

    2016-07-01

    Immunodeficient individuals are prone to develop a number of opportunistic infections and unique neoplasms. Epstein-Barr virus-associated smooth muscle tumor is an uncommon neoplasm associated with immunodeficiency. It has been described in patients infected with human immunodeficiency virus, in the posttransplant setting, and in those with congenital immunodeficiency. Different anatomic sites can be involved by Epstein-Barr virus-associated smooth muscle tumor, and even multiple locations can contain these unique lesions within the same patient. The presence of variable numbers of intratumoral lymphocytes and primitive round cell areas are the unique defining features for this tumor. Histopathologic features may vary considerably in terms of cellular atypia, mitotic activity, and necrosis, with no correlation to the clinical behavior. Demonstration of Epstein-Barr virus infection by in situ hybridization within tumor cell remains critical for the diagnosis. The mechanism for Epstein-Barr virus infection of progenitor cells and neoplastic transformation has been an area of interest and conjecture. Different treatment strategies are proposed according to underlying disease status. This paper reviews the clinicopathologic features of this uncommon neoplasm with detailed discussion of the role of Epstein-Barr virus in the pathogenesis. PMID:27362573

  19. Calcium oscillations in human mesenteric vascular smooth muscle.

    PubMed

    Navarro-Dorado, Jorge; Garcia-Alonso, Mauricio; van Breemen, Cornelis; Tejerina, Teresa; Fameli, Nicola

    2014-02-28

    Phenylephrine (PE)-induced oscillatory fluctuations in intracellular Ca(2+) concentration ([Ca(2+)]i) of vascular smooth muscle have been observed in many blood vessels isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically-stimulated [Ca(2+)]i oscillations in human mesenteric artery smooth muscle. In arterial segments preloaded with Fluo-4 AM and mounted on a myograph on the stage of a confocal microscope, we observed PE-induced oscillations in [Ca(2+)]i, which initiated and maintained vasoconstriction. These oscillations present some variability, possibly due to compromised health of the tissue. This view is corroborated by our ultrastructural analysis of the cells, in which we found only (5 ± 2)% plasma membrane-sarcoplasmic reticulum apposition, markedly less than measured in healthy tissue from laboratory animals. We also partially characterized the oscillations by using the inhibitory drugs 2-aminoethoxydiphenyl borate (2-APB), cyclopiazonic acid (CPA) and nifedipine. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca(2+) and inositol 1,4,5-trisphosphate receptor (IP3R) in the maintenance of the Ca(2+) oscillations observed in human blood vessels. PMID:24508261

  20. Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

    PubMed Central

    Hinz, Boris; Celetta, Giuseppe; Tomasek, James J.; Gabbiani, Giulio; Chaponnier, Christine

    2001-01-01

    To evaluate whether α-smooth muscle actin (α-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of α-SMA, with that of lung fibroblasts (LFs), expressing high levels of α-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of α-SMA–positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for α-SMA. In a second approach, we measured the isotonic contraction of SCF- and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGFβ1 increased α-SMA expression and lattice contraction by SCFs to the levels of LFs; TGFβ-antagonizing agents reduced α-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with α-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with α-cardiac and β- or γ-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased α-SMA expression is sufficient to enhance fibroblast contractile activity. PMID:11553712

  1. Abnormal tracheal smooth muscle function in the CF mouse

    PubMed Central

    Wallace, Helen L; Southern, Kevin W; Connell, Marilyn G; Wray, Susan; Burdyga, Theodor

    2013-01-01

    Increased airway smooth muscle (ASM) contractility is thought to underlie symptoms of airway hyperresponsiveness (AHR). In the cystic fibrosis (CF) airway, ASM anomalies have been reported, but have not been fully characterized and the underlying mechanisms are largely unknown. We examined ASM in an adult CF mouse tracheal ring preparation, and determined whether changes in contractility were associated with altered ASM morphology. We looked for inherent changes in the cellular pathways involved in contractility, and characterized trachea morphology in the adult trachea and in an embryonic lung culture model during development. Results showed that that there was a reduction in tracheal caliber in CF mice as indicated by a reduction in the number of cartilage rings; proximal cross-sectional areas of cftr−/− tracheas and luminal areas were significantly smaller, but there was no difference in the area or distribution of smooth muscle. Morphological differences observed in adult trachea were not evident in the embryonic lung at 11.5 days gestation or after 72 h in culture. Functional data showed a significant reduction in the amplitude and duration of contraction in response to carbachol (CCh) in Ca-free conditions. The reduction in contraction was agonist specific, and occurred throughout the length of the trachea. These data show that there is a loss in the contractile capacity of the CF mouse trachea due to downregulation of the pathway specific to acetylcholine (ACh) activation. This reduction in contraction is not associated with changes in the area or distribution of ASM. PMID:24400140

  2. Relaxant effect of 6-deoxyclitoriacetal on smooth muscle preparations.

    PubMed

    Itthipanichpong, C; Ruangrungsi, N; Saibundasak, K

    2001-06-01

    The pharmacological effect of 6-deoxyclitoriacetal (6-DA), a rotenoid compound isolated from the roots of Clitoria macrophylla Wall. (Papilionaceae), was examined on different smooth muscle preparations. 6-Deoxyclitoriacetal 0.2 mg/ml produced a significant decrease in the spontaneous contraction of isolated rat uterus. It also suppressed the contraction induced by acetylcholine 5x10(-6) M and oxytocin 5x10(-3) IU/ml. The cumulative contractile responses of rat aortic strips caused by serotonin 10(-8)-10(-4) M and norepinephrine 10(-11)-10(-7) M were reduced by 6-DA 0.4 mg/ml. In calcium free Kreb's solution, 6-DA inhibited the aortic contraction produced by a cumulative dose of calcium chloride (0.1-30 mM). In guinea-pig ileum, 6-DA 0.15 mg/ml exerted the spasmolytic activity by inhibition of the contractile response evoked by various contractile agents e.g. acetylcholine 10(-9)-10(-5) M, serotonin 10(-9)-10(-5) M and histamine 10(-9)-10(-5) M. All of the results indicated that 6-DA could induce a smooth muscle relaxant effect by interference with intracellular calcium metabolism. PMID:11529336

  3. Pericytes are progenitors for coronary artery smooth muscle.

    PubMed

    Volz, Katharina S; Jacobs, Andrew H; Chen, Heidi I; Poduri, Aruna; McKay, Andrew S; Riordan, Daniel P; Kofler, Natalie; Kitajewski, Jan; Weissman, Irving; Red-Horse, Kristy

    2015-01-01

    Epicardial cells on the heart's surface give rise to coronary artery smooth muscle cells (caSMCs) located deep in the myocardium. However, the differentiation steps between epicardial cells and caSMCs are unknown as are the final maturation signals at coronary arteries. Here, we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes, mural cells associated with microvessels, and that these cells are present in adults. During development following the onset of blood flow, pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1. Deletion of Notch3 disrupts caSMC differentiation. Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature, but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling. Our data are the first demonstration that pericytes are progenitors for smooth muscle, and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease. PMID:26479710

  4. MURC deficiency in smooth muscle attenuates pulmonary hypertension

    PubMed Central

    Nakanishi, Naohiko; Ogata, Takehiro; Naito, Daisuke; Miyagawa, Kotaro; Taniguchi, Takuya; Hamaoka, Tetsuro; Maruyama, Naoki; Kasahara, Takeru; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

    2016-01-01

    Emerging evidence suggests that caveolin-1 (Cav1) is associated with pulmonary arterial hypertension. MURC (also called Cavin-4) is a member of the cavin family, which regulates caveolar formation and functions together with caveolins. Here, we show that hypoxia increased Murc mRNA expression in the mouse lung, and that Murc-null mice exhibited attenuation of hypoxia-induced pulmonary hypertension (PH) accompanied by reduced ROCK activity in the lung. Conditional knockout mice lacking Murc in smooth muscle also resist hypoxia-induced PH. MURC regulates the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through Rho/ROCK signalling. Cav1 suppresses RhoA activity in PASMCs, which is reversed by MURC. MURC binds to Cav1 and inhibits the association of Cav1 with the active form of Gα13, resulting in the facilitated association of the active form of Gα13 with p115RhoGEF. These results reveal that MURC has a function in the development of PH through modulating Rho/ROCK signalling. PMID:27546070

  5. MicroRNAs Dynamically Remodel Gastrointestinal Smooth Muscle Cells

    PubMed Central

    Park, Chanjae; Yan, Wei; Ward, Sean M.; Hwang, Sung Jin; Wu, Qiuxia; Hatton, William J.; Park, Jong Kun; Sanders, Kenton M.; Ro, Seungil

    2011-01-01

    Smooth muscle cells (SMCs) express a unique set of microRNAs (miRNAs) which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI) SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM) layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF), and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract. PMID:21533178

  6. Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells.

    PubMed Central

    Yu, J. C.; Pickard, J. D.; Davenport, A. P.

    1995-01-01

    1. Endothelin (ET) has been implicated in cerebrovasospasm for example, following subarachnoid haemorrhage, and blocking the interaction of ET with its receptors on cerebral vessels, may be of therapeutic benefit. The aim of our study was to characterize endothelin receptor sub-types on medial smooth muscle cells of human cerebral vessels. Cultures of vascular smooth muscle cells were explanted from human cerebral resistance vessels and characterized as human brain smooth muscle cells (HBSMCs). 2. Over a 48 h incubation period, HBSMC cultures secreted comparable levels of immunoreactive (IR) big endothelin-1 (big ET-1) and IR endothelin (ET): 12.7 +/- 10.3 and 8.3 +/- 5.6 pmol/10(6) cells, respectively (mean +/- s.e. mean from three different individuals), into the culture medium. 3. Total RNA was extracted from cultures of human brain smooth muscle cells. Reverse-transcriptase polymerase chain reaction (RI-PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ETA (299 base pairs) and ETB (428 base pairs) (n = 3 different cultures). 4. Autoradiography demonstrated the presence of specific binding sites for [125I]-ET-1 which labels all ET receptors, and [125I]-PD151242, an ETA subtype-selective antagonist which exclusively labels ETA receptors, but no specific-binding was detected using ETB subtype-selective [125I]-BQ3020 (n = 3 different cultures, in duplicate). 5. In saturation binding assays, [123I]-ET-1 bound with high affinity: KD = 0.8 +/- 0.1 nM and Bmax = 690 +/- 108 fmol mg-1. A one-site fit was preferred and Hill slopes were close to unity over the concentration range (10(-12) to 10(-8) M). [125I]-PD151242 also bound with similar affinity: KD = 0.4 +/- 0.1 nM and Bmax = 388 +/- 68 fmol mg-1 (mean +/- s.e. mean, n = 3 different cultures). Again, a one-site fit was preferred and Hill slopes were close to unity over the concentration range. Unlabelled PD

  7. Role of magnesium in activation of smooth muscle.

    PubMed

    Paul, R J; Rüegg, J C

    1988-10-01

    We studied the effects of Mg2+-free solutions on isometric force (F0) and unloaded shortening velocity (Vus) in contractions elicited by Ca2+ or by ATP after thiophosphorylation by adenosine 5'-O-(3-thiotriphosphate (ATP gamma S) in chemically skinned guinea pig taenia coli smooth muscle. In Mg2+-free solutions, increasing Ca2+ did not increase Fo above resting levels. At the peak of a control contraction elicited by Ca2+, transfer to Mg2+-free (but Ca2+-containing) solutions resulted in a rapid relaxation and concomitant dephosphorylation of myosin. After ATP gamma S, a contracture required neither Mg2+ nor Ca2+ in the solutions for control levels of Fo. Vus in the Mg2+-free solutions after ATP gamma S was approximately 50% of control and could be restored to near control levels by addition of Mg2+ but not Ca2+. After ATP gamma S, pretreatment with 4 mM EDTA and contracture in 0.1 mM EDTA-containing solutions decreased Fo to 70-80% of control and Vus to 50-60% of control. Our results suggest that the relatively high requirement for Mg2+ for contraction in skinned smooth muscle largely reflects the Mg2+ dependence of myosin kinase and not for actin-myosin interaction. The dependence of Fo on Mg2+ (in the presence of excess ATP) in taenia coli is less than that reported for skeletal muscle. Appreciable force can be maintained with no added Mg2+ in the presence of 4 mMEDTA, and thus it appears that ATP4- can be a substrate for contraction after ATP gamma S treatment. In addition, our data imply that any Ca2+-dependent regulatory mechanism that does not involve myosin phosphorylation/dephosphorylation, if present, requires Mg2+ for expression. PMID:3140671

  8. Phosphate and ADP differently inhibit coordinated smooth muscle myosin groups.

    PubMed

    Hilbert, Lennart; Balassy, Zsombor; Zitouni, Nedjma B; Mackey, Michael C; Lauzon, Anne-Marie

    2015-02-01

    Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo--suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state. PMID:25650929

  9. Regulation of vascular tone and arterial blood pressure: role of chloride transport in vascular smooth muscle.

    PubMed

    Hübner, Christian A; Schroeder, Björn C; Ehmke, Heimo

    2015-03-01

    Recent studies suggest that primary changes in vascular resistance can cause sustained changes in arterial blood pressure. In this review, we summarize current knowledge about Cl(-) homeostasis in vascular smooth muscle cells. Within vascular smooth muscle cells, Cl(-) is accumulated above the electrochemical equilibrium, causing Cl(-) efflux, membrane depolarization, and increased contractile force when Cl(-) channels are opened. At least two different transport mechanisms contribute to raise [Cl(-)] i in vascular smooth muscle cells, anion exchange, and cation-chloride cotransport. Recent work suggests that TMEM16A-associated Ca(2+)-activated Cl(-) currents mediate Cl(-) efflux in vascular smooth muscle cells leading to vasoconstriction. Additional proteins associated with Cl(-) flux in vascular smooth muscle are bestrophins, which modulate vasomotion, the volume-activated LRRC8, and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl(-) transporters and Cl(-) channels in vascular smooth muscle cells (VSMCs) significantly contribute to the physiological regulation of vascular tone and arterial blood pressure. PMID:25588975

  10. Rho-kinase mediated cytoskeletal stiffness in skinned smooth muscle

    PubMed Central

    Lan, Bo; Wang, Lu; Zhang, Jenny; Pascoe, Chris D.; Norris, Brandon A.; Liu, Jeffrey C.-Y.; Solomon, Dennis; Paré, Peter D.; Deng, Linhong

    2013-01-01

    The structurally dynamic cytoskeleton is important in many cell functions. Large gaps still exist in our knowledge regarding what regulates cytoskeletal dynamics and what underlies the structural plasticity. Because Rho-kinase is an upstream regulator of signaling events leading to phosphorylation of many cytoskeletal proteins in many cell types, we have chosen this kinase as the focus of the present study. In detergent skinned tracheal smooth muscle preparations, we quantified the proteins eluted from the muscle cells over time and monitored the muscle's ability to respond to acetylcholine (ACh) stimulation to produce force and stiffness. In a partially skinned preparation not able to generate active force but could still stiffen upon ACh stimulation, we found that the ACh-induced stiffness was independent of calcium and myosin light chain phosphorylation. This indicates that the myosin light chain-dependent actively cycling crossbridges are not likely the source of the stiffness. The results also indicate that Rho-kinase is central to the ACh-induced stiffness, because inhibition of the kinase by H1152 (1 μM) abolished the stiffening. Furthermore, the rate of relaxation of calcium-induced stiffness in the skinned preparation was faster than that of ACh-induced stiffness, with or without calcium, suggesting that different signaling pathways lead to different means of maintenance of stiffness in the skinned preparation. PMID:24072407

  11. Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity

    PubMed Central

    Chen, Lihua; DeWispelaere, Allison; Dastvan, Frank; Osborne, William R. A.; Blechner, Christine; Windhorst, Sabine; Daum, Guenter

    2016-01-01

    Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration. PMID:27176050

  12. In vitro proliferation of aortic smooth muscle cells from spontaneously hypertensive and normotensive rats.

    PubMed

    Pang, S C

    1989-06-01

    The characteristics and proliferation of aortic smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied in culture. Smooth muscle cells were isolated from the tunica media of the thoracic aorta by an explant method. Immunofluorescence microscopy showed that 93-95 per cent of cells were positively labelled with antibodies raised against smooth muscle actin, indicating that these were smooth muscle cells. The proliferative activity was compared between aortic smooth muscle cells from hypertensive and normotensive rats in culture by thymidine incorporation and cell number determinations. The results demonstrate that aortic smooth muscle cells from hypertensive rats grew faster than those from normotensive rats in culture. The increased proliferative activity of cultured aortic smooth muscle cells from hypertensive rats was detectable even when they were cultured in a chemically defined serum-free medium. These data have shown that an increased proliferative activity of aortic smooth muscle cells from hypertensive rats can occur in culture conditions without the influence of arterial pressure or other stimuli as in intact animals. The mechanisms underlying the accelerated proliferative activity of aortic smooth muscle cells from genetically hypertensive rats in vitro remain to be determined. PMID:2754547

  13. Natural Bile Acids and Synthetic Analogues Modulate Large Conductance Ca2+-activated K+ (BKCa) Channel Activity in Smooth Muscle Cells

    PubMed Central

    Dopico, Alejandro M.; Walsh, John V.; Singer, Joshua J.

    2002-01-01

    Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle. PMID:11865021

  14. Potassium and insulin affect the contractility of abomasal smooth muscle.

    PubMed

    Türck, G; Leonhard-Marek, S

    2010-08-01

    Abomasal displacement is a frequent and important disease of high yielding dairy cows. Although several factors are related to its occurrence, the pathogenesis of the condition is still inadequately understood, particularly in regard to K(+) and insulin homeostasis. For this reason the aim was to investigate the effects of K(+) and insulin concentrations on in vitro motility of abomasal smooth muscle. The second aim was to determine whether the in vivo change in K(+) and insulin levels might be sufficient to induce reduced abomasal motility. Muscle strips were isolated from the abomasum of slaughtered cows and incubated in buffer solution under isometric conditions. Results show that a decrease in extracellular K(+) (between 5 and 1 mmol/L) or an increase in extracellular insulin concentrations (to 21 mU/L or higher) were able to affect the contraction activity of abomasal muscles. Contraction activity given as median (25th, 75th percentiles) changed from 28.1 mN/min (2.5, 49.9) at 5 mmol/L of K(+) to 9.4 mN/min (0.6, 35.7) at 1 mmol/L of K(+), and from 34.5 mN/min (10.8, 112.4) at 0 mU/L of insulin to 12.0 mN/min (7.6, 49.8) at 120 mU/L of insulin. Because the effect of insulin could be abolished by barium, glybenclamide, or ouabain, the underlying mechanisms of the insulin action could be an increased K(+) conductance or an increased Na/K-ATPase activity or both. Low K(+) or high insulin concentrations both reduced the activity of the circular muscle of the abomasal corpus (i.e., of the part that is responsible for the propulsion of abomasal chymus) and might play an important role in the pathogenesis of abomasal displacement. PMID:20655424

  15. Simulated Hypergravity Alters Vascular Smooth Muscle Cell Proliferation and Motility

    NASA Technical Reports Server (NTRS)

    Hunt, Shameka; Bettis, Barika; Harris-Hooker, Sandra; Sanford, Gary L.

    1997-01-01

    The cellular effects of gravity are poorly understood due to its constancy and nonavailability of altered gravitational models. Such an understanding is crucial for prolonged space flights. In these studies, we assessed the influence of centrifugation at 6G (HGrav) on vascular smooth muscle (SMC) mobility and proliferation. Cells were: (a) plated at low density and subjected to HGrav for 24-72 hr for proliferation studies, or (b) grown to confluency, subjected to HGrav, mechanically denuded and monitored for cell movement into the denuded area. Controls were maintained under normogravity. SMC showed a 50% inhibition of growth under HGrav and 10% serum; HGrav and low serum resulted in greater growth inhibition. The rate of movement of SMC into the denuded area was 2-3-fold higher under HGrav in low serum compared to controls, but similar in 10% serum. These studies show that HGrav has significant effects on SMC growth and mobility, which are dependent on serum levels.

  16. Smooth muscle relaxing flavonoids and terpenoids from Conyza filaginoides.

    PubMed

    Mata, R; Rojas, A; Acevedo, L; Estrada, S; Calzada, F; Rojas, I; Bye, R; Linares, E

    1997-02-01

    Activity-guided fractionation of the smooth muscle relaxing, chloroform-methanol (1:1) extract of Conyza filaginoides (D.C.) Hieron (Asteraceae) led to the isolation of three flavonoids (quercetin 3-glucoside, rutin, and pinostrobin), one sterol (alpha-spinasterol), a sesquiterpenoid (beta-caryophyllene 4,5-alpha-oxide), and two triterpenoids (erythrodiol and 3-beta-tridecanoyloxy-28-hydroxyolean-12-ene). 3-beta-Tridecanoyloxy-28-hydroxy-olean-12-ene is a new naturally occurring terpenoid. All the isolated compounds induced a concentration-dependent inhibition of the spontaneous contractions of rat ileum. The spasmolytic activity exhibited by the extract and active principles tends to support the traditional use of C filaginoides as an antispasmodic agent. PMID:9063094

  17. The Pivotal Role of Airway Smooth Muscle in Asthma Pathophysiology

    PubMed Central

    Ozier, Annaïg; Allard, Benoit; Bara, Imane; Girodet, Pierre-Olivier; Trian, Thomas; Marthan, Roger; Berger, Patrick

    2011-01-01

    Asthma is characterized by the association of airway hyperresponsiveness (AHR), inflammation, and remodelling. The aim of the present article is to review the pivotal role of airway smooth muscle (ASM) in the pathophysiology of asthma. ASM is the main effector of AHR. The mechanisms of AHR in asthma may involve a larger release of contractile mediators and/or a lower release of relaxant mediators, an improved ASM cell excitation/contraction coupling, and/or an alteration in the contraction/load coupling. Beyond its contractile function, ASM is also involved in bronchial inflammation and remodelling. Whereas ASM is a target of the inflammatory process, it can also display proinflammatory and immunomodulatory functions, through its synthetic properties and the expression of a wide range of cell surface molecules. ASM remodelling represents a key feature of asthmatic bronchial remodelling. ASM also plays a role in promoting complementary airway structural alterations, in particular by its synthetic function. PMID:22220184

  18. Airway hyperresponsiveness; smooth muscle as the principal actor

    PubMed Central

    Lauzon, Anne-Marie; Martin, James G.

    2016-01-01

    Airway hyperresponsiveness (AHR) is a defining characteristic of asthma that refers to the capacity of the airways to undergo exaggerated narrowing in response to stimuli that do not result in comparable degrees of airway narrowing in healthy subjects. Airway smooth muscle (ASM) contraction mediates airway narrowing, but it remains uncertain as to whether the smooth muscle is intrinsically altered in asthmatic subjects or is responding abnormally as a result of the milieu in which it sits. ASM in the trachea or major bronchi does not differ in its contractile characteristics in asthmatics, but the more pertinent peripheral airways await complete exploration. The mass of ASM is increased in many but not all asthmatics and therefore cannot be a unifying hypothesis for AHR, although when increased in mass it may contribute to AHR. The inability of a deep breath to reverse or prevent bronchial narrowing in asthma may reflect an intrinsic difference in the mechanisms that lead to softening of contracted ASM when subjected to stretch. Cytokines such as interleukin-13 and tumor necrosis factor-α promote a more contractile ASM phenotype. The composition and increased stiffness of the matrix in which ASM is embedded promotes a more proliferative and pro-inflammatory ASM phenotype, but the expected dedifferentiation and loss of contractility have not been shown. Airway epithelium may drive ASM proliferation and/or molecular remodeling in ways that may lead to AHR. In conclusion, AHR is likely multifactorial in origin, reflecting the plasticity of ASM properties in the inflammatory environment of the asthmatic airway. PMID:26998246

  19. Oxygen mediates vascular smooth muscle relaxation in hypoxia.

    PubMed

    Dada, Jessica; Pinder, Andrew G; Lang, Derek; James, Philip E

    2013-01-01

    The activation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and other ligands has been extensively investigated for many years. In the present study we considered the effect of molecular oxygen (O2) on sGC both as a direct ligand and its affect on other ligands by measuring cyclic guanosine monophosphate (cGMP) production, as an index of activity, as well as investigating smooth muscle relaxation under hypoxic conditions. Our isolated enzyme studies confirm the function of sGC is impaired under hypoxic conditions and produces cGMP in the presence of O2, importantly in the absence of NO. We also show that while O2 could partially affect the magnitude of sGC stimulation by NO when the latter was present in excess, activation by the NO independent, haem-dependent sGC stimulator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was unaffected. Our in vitro investigation of smooth muscle relaxation confirmed that O2 alone in the form of a buffer bolus (equilibrated at 95% O2/5% CO2) had the ability to dilate vessels under hypoxic conditions and that this was dependent upon sGC and independent of eNOS. Our studies confirm that O2 can be a direct and important mediator of vasodilation through an increase in cGMP production. In the wider context, these observations are key to understanding the relative roles of O2 versus NO-induced sGC activation. PMID:23451175

  20. Oxygen Mediates Vascular Smooth Muscle Relaxation in Hypoxia

    PubMed Central

    Lang, Derek; James, Philip E.

    2013-01-01

    The activation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and other ligands has been extensively investigated for many years. In the present study we considered the effect of molecular oxygen (O2) on sGC both as a direct ligand and its affect on other ligands by measuring cyclic guanosine monophosphate (cGMP) production, as an index of activity, as well as investigating smooth muscle relaxation under hypoxic conditions. Our isolated enzyme studies confirm the function of sGC is impaired under hypoxic conditions and produces cGMP in the presence of O2, importantly in the absence of NO. We also show that while O2 could partially affect the magnitude of sGC stimulation by NO when the latter was present in excess, activation by the NO independent, haem-dependent sGC stimulator 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was unaffected. Our in vitro investigation of smooth muscle relaxation confirmed that O2 alone in the form of a buffer bolus (equilibrated at 95% O2/5% CO2) had the ability to dilate vessels under hypoxic conditions and that this was dependent upon sGC and independent of eNOS. Our studies confirm that O2 can be a direct and important mediator of vasodilation through an increase in cGMP production. In the wider context, these observations are key to understanding the relative roles of O2 versus NO-induced sGC activation. PMID:23451175

  1. Airway hyperresponsiveness; smooth muscle as the principal actor.

    PubMed

    Lauzon, Anne-Marie; Martin, James G

    2016-01-01

    Airway hyperresponsiveness (AHR) is a defining characteristic of asthma that refers to the capacity of the airways to undergo exaggerated narrowing in response to stimuli that do not result in comparable degrees of airway narrowing in healthy subjects. Airway smooth muscle (ASM) contraction mediates airway narrowing, but it remains uncertain as to whether the smooth muscle is intrinsically altered in asthmatic subjects or is responding abnormally as a result of the milieu in which it sits. ASM in the trachea or major bronchi does not differ in its contractile characteristics in asthmatics, but the more pertinent peripheral airways await complete exploration. The mass of ASM is increased in many but not all asthmatics and therefore cannot be a unifying hypothesis for AHR, although when increased in mass it may contribute to AHR. The inability of a deep breath to reverse or prevent bronchial narrowing in asthma may reflect an intrinsic difference in the mechanisms that lead to softening of contracted ASM when subjected to stretch. Cytokines such as interleukin-13 and tumor necrosis factor-α promote a more contractile ASM phenotype. The composition and increased stiffness of the matrix in which ASM is embedded promotes a more proliferative and pro-inflammatory ASM phenotype, but the expected dedifferentiation and loss of contractility have not been shown. Airway epithelium may drive ASM proliferation and/or molecular remodeling in ways that may lead to AHR. In conclusion, AHR is likely multifactorial in origin, reflecting the plasticity of ASM properties in the inflammatory environment of the asthmatic airway. PMID:26998246

  2. Engineering smooth muscle tissue with a predefined structure.

    PubMed

    Kim, B S; Mooney, D J

    1998-08-01

    Nonwoven meshes of polyglycolic acid (PGA) fibers are attractive synthetic extracellular matrices (ECMs) for tissue engineering and have been used to engineer many types of tissues. However, these synthetic ECMs lack structural stability and often cannot maintain their original structure during tissue development. This makes it difficult to design an engineered tissue with a predefined configuration and dimensions. In this study, we investigated the ability of PGA fiber-based matrices bonded at their fiber crosspoints with a secondary polymer, poly-L-lactic acid (PLLA), to resist cellular contractile forces and maintain their predefined structure during the process of smooth muscle (SM) tissue development in vitro. Physically bonded PGA matrices exhibited a 10- to 35-fold increase in the compressive modulus over unbonded PGA matrices, depending on the mass of PLLA utilized to bond the PGA matrices. In addition, the bonded PGA matrices degraded much more slowly than the unbonded matrices. The PLLA bonding of PGA matrices had no effect on the ability of cells to adhere to the matrices. After 7 weeks in culture, the bonded matrices maintained 101 +/- 4% of their initial volume and an approximate original shape while the unbonded matrices contracted to 5 +/- 1% of their initial volume with an extreme change in their shape. At this time the bonded PGA matrices had a high cellularity, with smooth muscle cells (SMCs) and ECM proteins produced by these cells (e.g., elastin) filling the pores between PGA fibers. This study demonstrated that physically bonded PGA fiber-based matrices allow the maintenance of the configuration and dimensions of the original matrices and the development of a new tissue in a predefined three-dimensional structure. This approach may be useful for engineering a variety of tissues of various structures and shapes, and our study demonstrates the importance of matching both the initial mechanical properties and the degradation rate of a matrix to

  3. Role of smooth muscle cell mineralocorticoid receptor in vascular tone.

    PubMed

    Tarjus, Antoine; Belozertseva, Ekaterina; Louis, Huguette; El Moghrabi, Soumaya; Labat, Carlos; Lacolley, Patrick; Jaisser, Frédéric; Galmiche, Guillaume

    2015-08-01

    Identification of the mineralocorticoid receptor (MR) in the vasculature (i.e., endothelial and smooth muscle cells) raised the question of its role in vascular function and blood pressure control. Using a mouse model with conditional inactivation of MR in vascular smooth muscle cell (VSMC) (MR(SMKO)), we have recently shown that the VSMC MR is crucial for aldosterone-salt-induced carotid stiffening. In the present study, we have investigated the specific contribution of the VSMC MR in the regulation of vascular tone in large vessels. In MR(SMKO) mice, contractions induced by potassium chloride and calcium (Ca(2+)) are decreased in the aorta, whereas contraction is normal in response to phenylephrine and caffeine. The difference in response to Ca(2+) suggests that the VSMC-specific deficiency of the MR modifies VSM Ca(2+) signaling but without altering the intracellular Ca(2+) store handling. The relaxation induced by acetylcholine is not affected by the absence of MR. However, the relaxation induced by Ach in the presence of indomethacin and the relaxation induced by sodium nitroprussiate are significantly reduced in MR(SMKO) mice compared to controls. Since endothelial nitric oxide synthase (eNOS) activity is increased in mutant mice, their altered relaxation reflects impairment of the nitric oxide (NO) signaling pathway. In addition to altered NO and Ca(2+) signaling, the activity of myosin light chain and its regulators, myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), is reduced. In conclusion, MR expressed in VSMC is required for NO and Ca(2+) signaling pathways and contractile protein activity leading to an altered contraction/relaxation coupling. PMID:25262754

  4. Circular smooth muscle contributes to esophageal shortening during peristalsis

    PubMed Central

    Vegesna, Anil K; Chuang, Keng-Yu; Besetty, Ramashesai; Phillips, Steven J; Braverman, Alan S; Barbe, Mary F; Ruggieri, Michael R; Miller, Larry S

    2012-01-01

    AIM: To study the angle between the circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) fibers in the distal esophagus. METHODS: In order to identify possible mechanisms for greater shortening in the distal compared to proximal esophagus during peristalsis, the angles between the LSM and CSM layers were measured in 9 cadavers. The outer longitudinal layer of the muscularis propria was exposed after stripping the outer serosa. The inner circular layer of the muscularis propria was then revealed after dissection of the esophageal mucosa and the underlying muscularis mucosa. Photographs of each specimen were taken with half of the open esophagus folded back showing both the outer longitudinal and inner circular muscle layers. Angles were measured every one cm for 10 cm proximal to the squamocolumnar junction (SCJ) by two independent investigators. Two human esophagi were obtained from organ transplant donors and the angles between the circular and longitudinal smooth muscle layers were measured using micro-computed tomography (micro CT) and Image J software. RESULTS: All data are presented as mean ± SE. The CSM to LSM angle at the SCJ and 1 cm proximal to SCJ on the autopsy specimens was 69.3 ± 4.62 degrees vs 74.9 ± 3.09 degrees, P = 0.32. The CSM to LSM angle at SCJ were statistically significantly lower than at 2, 3, 4 and 5 cm proximal to the SCJ, 69.3 ± 4.62 degrees vs 82.58 ± 1.34 degrees, 84.04 ± 1.64 degrees, 84.87 ± 1.04 degrees and 83.72 ± 1.42 degrees, P = 0.013, P = 0.008, P = 0.004, P = 0.009 respectively. The CSM to LSM angle at SCJ was also statistically significantly lower than the angles at 6, 7 and 8 cm proximal to the SCJ, 69.3 ± 4.62 degrees vs 80.18 ± 2.09 degrees, 81.81 ± 1.75 degrees and 80.96 ± 2.04 degrees, P = 0.05, P = 0.02, P = 0.03 respectively. The CSM to LSM angle at 1 cm proximal to SCJ was statistically significantly lower than at 3, 4 and 5 cm proximal to the SCJ, 74.94 ± 3.09 degrees vs 84.04 ± 1

  5. Pullulan-based hydrogel for smooth muscle cell culture.

    PubMed

    Autissier, Aude; Letourneur, Didier; Le Visage, Catherine

    2007-08-01

    A hydrogel was prepared from pullulan and evaluated as a novel biomaterial for vascular engineering. Using a crosslinking process with sodium trimetaphosphate in aqueous solution, homogeneous, transparent, and easy-to-handle pullulan gels were obtained with water-content higher than 90%. A circular punch was used to cut 6-mm diameter and 2-mm thickness discs for cell culture. Environmental scanning electron microscopy analysis of hydrated gels revealed a smooth surface, on which rabbit vascular smooth muscle cells were successfully seeded. The absence of cytotoxicity was evidenced by a live/dead assay. Fluorescence-labeled cells were observed adhering and progressively spreading out on the surface of the material. Cellular proliferation was followed for up to 1 week using an MTT assay. In addition, a complete in vitro degradation of the gels was achieved in 3 h upon incubation in a pullulanase solution (44 U/mL). In conclusion, we have shown the feasibility of preparing a biocompatible pullulan-based hydrogel that could support vascular cell culture. Based on these promising results, future studies will focus on the seeding of vascular cells on tubular-shaped hydrogels and the in vivo implantation of these new biomaterials. PMID:17295223

  6. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

  7. HB-EGF-Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse.

    PubMed

    Wang, Qing; Li, Hequan; Yao, Yinan; Lu, Guohua; Wang, Yuehong; Xia, Dajing; Zhou, Jianying

    2016-03-01

    The airway smooth muscle (ASM) cells' proliferation, migration, and their progenitor's migration are currently regarded as causative factors for ASM remodeling in asthma. Heparin-binding epidermal growth factor (HB-EGF), a potent mitogen and chemotactic factor, could promote ASM cell proliferation through MAPK pathways. In this study, we obtained primary ASM cells and their progenitors from C57BL/6 mice and went on to explore the role of HB-EGF in these cells migration and the underlying mechanisms. We found that recombinant HB-EGF (rHB-EGF) intratracheal instillation accelerated ASM layer thickening in an OVA-induced asthmatic mouse. Modified Boyden chamber assay revealed that rHB-EGF facilitate ASM cell migration in a dose-dependent manner and ASM cells from asthmatic mice had a greater migration ability than that from normal counterparts. rHB-EGF could stimulate the phosphorylation of ERK1/2 and p38 in ASM cells but further migration assay showed that only epidermal growth factor receptor inhibitor (AG1478) or p38 inhibitor (SB203580), but not ERK1/2 inhibitor (PD98059), could inhibit rHB-EGF-mediated ASM cells migration. Actin cytoskeleton experiments exhibited that rHB-EGF could cause actin stress fibers disassembly and focal adhesions formation of ASM cells through the activation of p38. Finally, airway instillation of rHB-EGF promoted the recruitment of bone marrow-derived smooth muscle progenitor cells, which were transferred via caudal vein, migrating into the airway from the circulation. These observations demonstrated that ASM remodeling in asthma might have resulted from HB-EGF-mediated ASM cells and their progenitor cells migration, via p38 MAPK-dependent actin cytoskeleton remodeling. PMID:26826248

  8. Unregulated smooth-muscle myosin in human intestinal neoplasia.

    PubMed

    Alhopuro, Pia; Phichith, Denis; Tuupanen, Sari; Sammalkorpi, Heli; Nybondas, Miranda; Saharinen, Juha; Robinson, James P; Yang, Zhaohui; Chen, Li-Qiong; Orntoft, Torben; Mecklin, Jukka-Pekka; Järvinen, Heikki; Eng, Charis; Moeslein, Gabriela; Shibata, Darryl; Houlston, Richard S; Lucassen, Anneke; Tomlinson, Ian P M; Launonen, Virpi; Ristimäki, Ari; Arango, Diego; Karhu, Auli; Sweeney, H Lee; Aaltonen, Lauri A

    2008-04-01

    A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia. PMID:18391202

  9. Differentiation of Murine Bone Marrow-Derived Smooth Muscle Progenitor Cells Is Regulated by PDGF-BB and Collagen

    PubMed Central

    Lin, Clifford; Yuan, Yifan; Courtman, David W.

    2016-01-01

    Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor—BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers α-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-α (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of α-SMA and SM22-α, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure. PMID:27258003

  10. The LIM protein leupaxin is enriched in smooth muscle and functions as an serum response factor cofactor to induce smooth muscle cell gene transcription.

    PubMed

    Sundberg-Smith, Liisa J; DiMichele, Laura A; Sayers, Rebecca L; Mack, Christopher P; Taylor, Joan M

    2008-06-20

    Leupaxin is a LIM domain-containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor-dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix-dependent signals may regulate phenotypic switching of smooth muscle cells. PMID:18497331

  11. Immune/Inflammatory Response and Hypocontractility of Rabbit Colonic Smooth Muscle After TNBS-Induced Colitis

    PubMed Central

    Zhang, Yonggang; Li, Fang; Wang, Hong; Yin, Chaoran; Huang, JieAn; Mahavadi, Sunila; Murthy, Karnam S.

    2016-01-01

    Background The contractility of colonic smooth muscle is dysregulated due to immune/inflammatory responses in inflammatory bowel diseases. Inflammation in vitro induces up-regulation of regulator of G-protein signaling 4 (RGS4) expression in colonic smooth muscle cells. Aims To characterize the immune/inflammatory responses and RGS4 expression pattern in colonic smooth muscle after induction of colitis. Methods Colitis was induced in rabbits by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Innate/adaptive immune response RT-qPCR array was performed using colonic circular muscle strips. At 1–9 weeks after colonic intramuscular microinjection of lentivirus, the distal and proximal colons were collected, and muscle strips and dispersed muscle cells were prepared from circular muscle layer. Expression levels of RGS4 and NFκB signaling components were determined by Western blot analysis. The biological consequences of RGS4 knockdown were assessed by measurement of muscle contraction and phospholipase C (PLC)-β activity in response to acetylcholine (ACh). Results Contraction in response to ACh was significantly inhibited in the inflamed colonic circular smooth muscle cells. RGS4, IL-1, IL-6, IL-8, CCL3, CD1D, and ITGB2 were significantly up-regulated, while IL-18, CXCR4, CD86, and C3 were significantly down-regulated in the inflamed muscle strips. RGS4 protein expression in the inflamed smooth muscles was dramatically increased. RGS4 stable knockdown in vivo augmented ACh-stimulated PLC-β activity and contraction in colonic smooth muscle cells. Conclusion Inflamed smooth muscle exhibits up-regulation of IL-1-related signaling components, Th1 cytokines and RGS4, and inhibition of contraction. Stable knockdown of endogenous RGS4 in colonic smooth muscle increases PLC-β activity and contractile responses. PMID:26879904

  12. BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

    PubMed Central

    de Jesus Perez, Vinicio A.; Ali, Ziad; Alastalo, Tero-Pekka; Ikeno, Fumiaki; Sawada, Hirofumi; Lai, Ying-Ju; Kleisli, Thomas; Spiekerkoetter, Edda; Qu, Xiumei; Rubinos, Laura H.; Ashley, Euan; Amieva, Manuel; Dedhar, Shoukat

    2011-01-01

    We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)–β-catenin (βC) and Wnt–planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt–dependent manner, induces βC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt–PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1–mediated motility. Transfection of a Dvl mutant that binds βC without activating RhoA-Rac1 not only prevents BMP-2–mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent βC activity. Interfering with the Dvl-dependent Wnt–PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2–mediated tandem activation of Wnt–βC and Wnt–PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations. PMID:21220513

  13. miR-145 and miR-143 Regulate Smooth Muscle Cell Fate Decisions

    PubMed Central

    Cordes, Kimberly R.; Sheehy, Neil T.; White, Mark; Berry, Emily; Morton, Sarah U.; Muth, Alecia N.; Lee, Ting-Hein; Miano, Joseph M.; Ivey, Kathryn N.; Srivastava, Deepak

    2009-01-01

    SUMMARY microRNAs are regulators of myriad cellular events, but evidence for a single microRNA that can efficiently differentiate multipotent cells into a specific lineage or regulate direct reprogramming of cells into an alternate cell fate has been elusive. Here, we show that miR-145 and miR-143 are co-transcribed in multipotent cardiac progenitors before becoming localized to smooth muscle cells, including neural crest stem cell–derived vascular smooth muscle cells. miR-145 and miR-143 were direct transcriptional targets of serum response factor, myocardin and Nkx2.5, and were downregulated in injured or atherosclerotic vessels containing proliferating, less differentiated smooth muscle cells. miR-145 was necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. Furthermore, miR-145 and miR-143 cooperatively targeted a network of transcription factors, including Klf4, myocardin, and Elk-1 to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells. PMID:19578358

  14. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

    PubMed

    Shankaran, Mahalakshmi; King, Chelsea L; Angel, Thomas E; Holmes, William E; Li, Kelvin W; Colangelo, Marc; Price, John C; Turner, Scott M; Bell, Christopher; Hamilton, Karyn L; Miller, Benjamin F; Hellerstein, Marc K

    2016-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  15. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  16. Calcium ion-regulated thin filaments from vascular smooth muscle.

    PubMed Central

    Marston, S B; Trevett, R M; Walters, M

    1980-01-01

    Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 x 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g. PMID:6446898

  17. The comparative effects of aminoglycoside antibiotics and muscle relaxants on electrical field stimulation response in rat bladder smooth muscle.

    PubMed

    Min, Chang Ho; Min, Young Sil; Lee, Sang Joon; Sohn, Uy Dong

    2016-06-01

    It has been reported that several aminoglycoside antibiotics have a potential of prolonging the action of non-depolarizing muscle relaxants by drug interactions acting pre-synaptically to inhibit acetylcholine release, but antibiotics itself also have a strong effect on relaxing the smooth muscle. In this study, four antibiotics of aminoglycosides such as gentamicin, streptomycin, kanamycin and neomycin were compared with skeletal muscle relaxants baclofen, tubocurarine, pancuronium and succinylcholine, and a smooth muscle relaxant, papaverine. The muscle strips isolated from the rat bladder were stimulated with pulse trains of 40 V in amplitude and 10 s in duration, with pulse duration of 1 ms at the frequency of 1-8 Hz, at 1, 2, 4, 6, 8 Hz respectively. To test the effect of four antibiotics on bladder smooth muscle relaxation, each of them was treated cumulatively from 1 μM to 0.1 mM with an interval of 5 min. Among the four antibiotics, gentamicin and neomycin inhibited the EFS response. The skeletal muscle relaxants (baclofen, tubocurarine, pancuronium and succinylcholine) and inhibitory neurotransmitters (GABA and glycine) did not show any significant effect. However, papaverine, had a significant effect in the relaxation of the smooth muscle. It was suggested that the aminoglycoside antibiotics have inhibitory effect on the bladder smooth muscle. PMID:27260628

  18. Functional effects of KCNQ K+ channels in airway smooth muscle

    PubMed Central

    Evseev, Alexey I.; Semenov, Iurii; Archer, Crystal R.; Medina, Jorge L.; Dube, Peter H.; Shapiro, Mark S.; Brenner, Robert

    2013-01-01

    KCNQ (Kv7) channels underlie a voltage-gated K+ current best known for control of neuronal excitability, and its inhibition by Gq/11-coupled, muscarinic signaling. Studies have indicated expression of KCNQ channels in airway smooth muscle (ASM), a tissue that is predominantly regulated by muscarinic receptor signaling. Therefore, we investigated the function of KCNQ channels in rodent ASM and their interplay with Gq/11-coupled M3 muscarinic receptors. Perforated-patch clamp of dissociated ASM cells detected a K+ current inhibited by the KCNQ antagonist, XE991, and augmented by the specific agonist, flupirtine. KCNQ channels begin to activate at voltages near resting potentials for ASM cells, and indeed XE991 depolarized resting membrane potentials. Muscarinic receptor activation inhibited KCNQ current weakly (~20%) at concentrations half-maximal for contractions. Thus, we were surprised to see that KCNQ had no affect on membrane voltage or muscle contractility following muscarinic activation. Further, M3 receptor-specific antagonist J104129 fumarate alone did not reveal KCNQ effects on muscarinic evoked depolarization or contractility. However, a role for KCNQ channels was revealed when BK-K+ channel activities are reduced. While KCNQ channels do control resting potentials, they appear to play a redundant role with BK calcium-activated K+ channels during ASM muscarinic signaling. In contrast to effect of antagonist, we observe that KCNQ agonist flupirtine caused a significant hyperpolarization and reduced contraction in vitro irrespective of muscarinic activation. Using non-invasive whole animal plethysmography, the clinically approved KCNQ agonist retigabine caused a transient reduction in indexes of airway resistance in both wild type and BK β1 knockout (KO) mice treated with the muscarinic agonist. These findings indicate that KCNQ channels can be recruited via agonists to oppose muscarinic evoked contractions and may be of therapeutic value as bronchodilators

  19. Functional effects of KCNQ K(+) channels in airway smooth muscle.

    PubMed

    Evseev, Alexey I; Semenov, Iurii; Archer, Crystal R; Medina, Jorge L; Dube, Peter H; Shapiro, Mark S; Brenner, Robert

    2013-01-01

    KCNQ (Kv7) channels underlie a voltage-gated K(+) current best known for control of neuronal excitability, and its inhibition by Gq/11-coupled, muscarinic signaling. Studies have indicated expression of KCNQ channels in airway smooth muscle (ASM), a tissue that is predominantly regulated by muscarinic receptor signaling. Therefore, we investigated the function of KCNQ channels in rodent ASM and their interplay with Gq/11-coupled M3 muscarinic receptors. Perforated-patch clamp of dissociated ASM cells detected a K(+) current inhibited by the KCNQ antagonist, XE991, and augmented by the specific agonist, flupirtine. KCNQ channels begin to activate at voltages near resting potentials for ASM cells, and indeed XE991 depolarized resting membrane potentials. Muscarinic receptor activation inhibited KCNQ current weakly (~20%) at concentrations half-maximal for contractions. Thus, we were surprised to see that KCNQ had no affect on membrane voltage or muscle contractility following muscarinic activation. Further, M3 receptor-specific antagonist J104129 fumarate alone did not reveal KCNQ effects on muscarinic evoked depolarization or contractility. However, a role for KCNQ channels was revealed when BK-K(+) channel activities are reduced. While KCNQ channels do control resting potentials, they appear to play a redundant role with BK calcium-activated K(+) channels during ASM muscarinic signaling. In contrast to effect of antagonist, we observe that KCNQ agonist flupirtine caused a significant hyperpolarization and reduced contraction in vitro irrespective of muscarinic activation. Using non-invasive whole animal plethysmography, the clinically approved KCNQ agonist retigabine caused a transient reduction in indexes of airway resistance in both wild type and BK β1 knockout (KO) mice treated with the muscarinic agonist. These findings indicate that KCNQ channels can be recruited via agonists to oppose muscarinic evoked contractions and may be of therapeutic value as

  20. Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

    PubMed Central

    Park, Song-Young; Gifford, Jayson R.; Andtbacka, Robert H. I.; Trinity, Joel D.; Hyngstrom, John R.; Garten, Ryan S.; Diakos, Nikolaos A.; Ives, Stephen J.; Dela, Flemming; Larsen, Steen; Drakos, Stavros

    2014-01-01

    Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s−1·mg−1, P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g−1·min−1, P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s−1·mg−1, P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production. PMID:24906913

  1. PDMS Elastic Micropost Arrays for Studying Vascular Smooth Muscle Cells

    PubMed Central

    Cheng, Qi; Sun, Zhe; Meininger, Gerald; Almasri, Mahmoud

    2015-01-01

    This paper describes the design, modeling, fabrication and characterization of a micromachined array of high-density 3-dimensional microposts (100×100) made of flexible material (silicone elastomers) for use to measure quantitatively the cellular traction force and contractile events in isolated vascular smooth muscle cells (VSMCs). The micropost array was fabricated with diameters ranged from 3 to 10 μm, with edge to edge spacing of 5, 7 and 10 μm, and with a height to diameter aspect ratio up to 10. VSMCs exerted larger basal traction forces when they were grown on stiffer micropost arrays. These basal traction forces were 80% larger in control VSMCs than in VSMCs in which integrin linked kinase (ILK) was knocked down using shRNA. The addition of Angiotensin II (ANGII) led to VSMC contraction as evidenced by an increased traction force exerted on the microposts under the cell. This ANGII induced contractile response and change in traction force on the microposts was not observed in VSMCs lacking ILK. Following treatment of VSMCs with Cytochalasin D to depolymerize the actin cytoskeleton, the VSMCs exhibited relaxation that was apparent as a significant reduction in the measured traction force exerted on microposts under the cell. Overall, this study demonstrates the usefulness of micropost arrays for study of the contractile responsiveness of VSMC and the results indicate that ILK plays a critical role in the signaling pathways leading to the generation of substrate traction force in VSMC. PMID:26451074

  2. Tensile Properties of Contractile and Synthetic Vascular Smooth Muscle Cells

    NASA Astrophysics Data System (ADS)

    Miyazaki, Hiroshi; Hasegawa, Yoshitaka; Hayashi, Kozaburo

    Tensile properties of vascular smooth muscle cells (VSMCs) of synthetic and contractile phenotypes were determined using a newly developed tensile test system. Synthetic and contractile VSMCs were isolated from the rabbit thoracic aorta with an explant and an enzymatic digestion method, respectively. Each cell floated in Hanks' balanced salt solution of 37°C was attached to the fine tips of a pair of micropipettes with a cell adhesive and, then, stretched at the rate of 6µm/sec by moving one of the micropipettes with a linear actuator. Load applied to the cell was measured with a cantilever-type load cell; its elongation was determined from the distance between the micropipette tips using a video dimension analyzer. The synthetic and contractile VSMCs were not broken even at the tensile force of 2.4µN and 3.4µN, respectively. Their stiffness was significantly higher in contractile phenotype (0.17N/m) than in synthetic one (0.09N/m). The different tensile properties between synthetic and contractile cells are attributable to the differences in cytoskeletal structures and contractile apparatus.

  3. Origin and differentiation of vascular smooth muscle cells

    PubMed Central

    Wang, Gang; Jacquet, Laureen; Karamariti, Eirini; Xu, Qingbo

    2015-01-01

    Vascular smooth muscle cells (SMCs), a major structural component of the vessel wall, not only play a key role in maintaining vascular structure but also perform various functions. During embryogenesis, SMC recruitment from their progenitors is an important step in the formation of the embryonic vascular system. SMCs in the arterial wall are mostly quiescent but can display a contractile phenotype in adults. Under pathophysiological conditions, i.e. vascular remodelling after endothelial dysfunction or damage, contractile SMCs found in the media switch to a secretory type, which will facilitate their ability to migrate to the intima and proliferate to contribute to neointimal lesions. However, recent evidence suggests that the mobilization and recruitment of abundant stem/progenitor cells present in the vessel wall are largely responsible for SMC accumulation in the intima during vascular remodelling such as neointimal hyperplasia and arteriosclerosis. Therefore, understanding the regulatory mechanisms that control SMC differentiation from vascular progenitors is essential for exploring therapeutic targets for potential clinical applications. In this article, we review the origin and differentiation of SMCs from stem/progenitor cells during cardiovascular development and in the adult, highlighting the environmental cues and signalling pathways that control phenotypic modulation within the vasculature. PMID:25952975

  4. Airway smooth muscle and bronchospasm: fluctuating, fluidizing, freezing

    PubMed Central

    Krishnan, Ramaswamy; Trepat, Xavier; Nguyen, Trang T. B.; Lenormand, Guillaume; Oliver, Madavi; Fredberg, Jeffrey J.

    2008-01-01

    We review here four recent findings that have altered in a fundamental way our understanding of airways smooth muscle (ASM), its dynamic responses to physiological loading, and their dominant mechanical role in bronchospasm. These findings highlight ASM remodeling processes that are innately out-of-equilibrium and dynamic, and bring to the forefront a striking intersection between topics in condensed matter physics and ASM cytoskeletal biology. By doing so, they place in a new light the role of enhanced ASM mass in airway hyper-responsiveness as well as in the failure of a deep inspiration to relax the asthmatic airway. These findings have established that (i) ASM length is equilibrated dynamically, not statically; (ii) ASM dynamics closely resemble physical features exhibited by so-called soft glassy materials; (iii) static force-length relationships fail to describe dynamically contracted ASM states; (iv) stretch fluidizes the ASM cytoskeleton. Taken together, these observations suggest that at the origin of the bronchodilatory effect of a deep inspiration, and its failure in asthma, may lie glassy dynamics of the ASM cell. PMID:18514592

  5. Mechanisms of BDNF regulation in asthmatic airway smooth muscle.

    PubMed

    Aravamudan, Bharathi; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S

    2016-08-01

    Brain-derived neurotrophic factor (BDNF), a neurotrophin produced by airway smooth muscle (ASM), enhances inflammation effects on airway contractility, supporting the idea that locally produced growth factors influence airway diseases such as asthma. We endeavored to dissect intrinsic mechanisms regulating endogenous, as well as inflammation (TNF-α)-induced BDNF secretion in ASM of nonasthmatic vs. asthmatic humans. We focused on specific Ca(2+) regulation- and inflammation-related signaling cascades and quantified BDNF secretion. We find that TNF-α enhances BDNF release by ASM cells, via several mechanisms relevant to asthma, including transient receptor potential channels TRPC3 and TRPC6 (but not TRPC1), ERK 1/2, PI3K, PLC, and PKC cascades, Rho kinase, and transcription factors cAMP response element binding protein and nuclear factor of activated T cells. Basal BDNF expression and secretion are elevated in asthmatic ASM and increase further with TNF-α exposure, involving many of these regulatory mechanisms. We conclude that airway BDNF secretion is regulated at multiple levels, providing a basis for autocrine effects of BDNF under conditions of inflammation and disease, with potential downstream influences on contractility and remodeling. PMID:27317689

  6. Molecular mechanisms of increased vascular smooth muscle contraction in SHR

    SciTech Connect

    Sharma, R.V.; Aqel, M.B.; Butters, C.; McEldoon, J.; Bhalla, R.C.

    1986-03-01

    The isometric tension development and /sup 45/Ca influx in response to NE and methoxamine stimulation were significantly (P < .05) increased in SHR caudal arteries as compared to WKY. Estimation of /sup 3/H-prazosin binding to the membranes isolated from caudal artery of WKY and SHR showed a single class of high affinity binding sites with Kd values: SHR, 128 +/- 14 pM; WKY, 141 +/- 19 pM and the Bmax values; SHR, 108 +/- 14 fmoles/mg protein; WKY, 113 +/- 21 fmoles/mg protein. Nifedipine inhibition of caudal artery contractions in response to NE stimulation was significantly greater (P < .05) in SHR as compared to WKY. On the other hand, there were no differences between WKY and SHR caudal artery rings either in the isometric tension development, /sup 45/Ca influx or nifedipine inhibition in response to K/sup +/-depolarization. Their results indicate that the increased vascular smooth muscle contraction in SHR in response to NE-stimulation may be due to increased Ca/sup 2 +/ influx through the receptor operated Ca/sup 2 +/ channels.

  7. Extracellular calcium sensing in rat aortic vascular smooth muscle cells

    SciTech Connect

    Smajilovic, Sanela; Hansen, Jakob Lerche; Christoffersen, Tue E.H.

    2006-10-06

    Extracellular calcium (Ca2+o) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca2+o stimulates proliferation of the cells. The effects of Ca2+o were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca2+o-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca2+o-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.

  8. Upregulation of decorin by FXR in vascular smooth muscle cells

    SciTech Connect

    He Fengtian; Zhang Qiuhong; Kuruba, Ramalinga; Gao Xiang; Li Jiang; Li Yong; Gong Wei; Jiang, Yu; Xie Wen; Li Song

    2008-08-08

    Decorin is a member of the family of small leucine-rich proteoglycans that are present in blood vessels and synthesized by vascular smooth muscle cells (VSMCs). Decorin plays complex roles in both normal vascular physiology and the pathogenesis of various types of vascular disorders. However, the mechanisms of regulation of decorin expression in vasculature are not clearly understood. Particularly little information is available about a role of nuclear receptors in the regulation of decorin expression. In the present study, we report that activation of vascular FXR by a specific ligand resulted in upregulation of decorin at the levels of both mRNA and protein. FXR appears to induce decorin expression at a transcriptional level because (1) upregulation of decorin mRNA expression was abolished by the treatment of a transcription inhibitor, actinomycin D; and (2) decorin promoter activity was significantly increased by activation of FXR. Functional analysis of human decorin promoter identified an imperfect inverted repeat DNA motif, IR8 (-2313TGGTCAtagtgtcaTGACCT-2294), as a likely FXR-responsive element that is involved in decorin regulation.

  9. Traction in smooth muscle cells varies with cell spreading

    NASA Technical Reports Server (NTRS)

    Tolic-Norrelykke, Iva Marija; Wang, Ning

    2005-01-01

    Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

  10. Contraction of gut smooth muscle cells assessed by fluorescence imaging.

    PubMed

    Tokita, Yohei; Akiho, Hirotada; Nakamura, Kazuhiko; Ihara, Eikichi; Yamamoto, Masahiro

    2015-03-01

    Here we discuss the development of a novel cell imaging system for the evaluation of smooth muscle cell (SMC) contraction. SMCs were isolated from the circular and longitudinal muscular layers of mouse small intestine by enzymatic digestion. SMCs were stimulated by test agents, thereafter fixed in acrolein. Actin in fixed SMCs was stained with phalloidin and cell length was determined by measuring diameter at the large end of phalloidin-stained strings within the cells. The contractile response was taken as the decrease in the average length of a population of stimulated-SMCs. Various mediators and chemically identified compounds of daikenchuto (DKT), pharmaceutical-grade traditional Japanese prokinetics, were examined. Verification of the integrity of SMC morphology by phalloidin and DAPI staining and semi-automatic measurement of cell length using an imaging analyzer was a reliable method by which to quantify the contractile response. Serotonin, substance P, prostaglandin E2 and histamine induced SMC contraction in concentration-dependent manner. Two components of DKT, hydroxy-α-sanshool and hydroxy-β-sanshool, induced contraction of SMCs. We established a novel cell imaging technique to evaluate SMC contractility. This method may facilitate investigation into SMC activity and its role in gastrointestinal motility, and may assist in the discovery of new prokinetic agents. PMID:25837933

  11. Human colonic smooth muscle: electrical and contractile activity in vitro.

    PubMed Central

    Gill, R C; Cote, K R; Bowes, K L; Kingma, Y J

    1986-01-01

    Extracellular electrical and contractile activities were recorded in vitro from strips of human colonic smooth muscle obtained at the time of surgery. Serosal electrical activity of longitudinally oriented strips from the taenia and intertaenial region was characterised by continuous oscillation at a frequency of 28 +/- 1/min. Contractions were marked electrically by a series of oscillations upon which spikes were superimposed. The electrical activity recorded from the submucosal surface of circularly oriented strips exhibited oscillations at 24 +/- 4/min, a frequency significantly lower (p less than 0.001) than that recorded from the serosal surface of similar preparations. The contractile force and frequency was dependent upon the part of the colon from which the strip originated; the most powerful contractions were recorded from strips of sigmoid colon. The contractile frequency of circularly oriented strips from the right colon was 6.3 +/- 0.6/min, significantly higher (p less than 0.001) than that of strips from the left colon (3.4 +/- 0.3/min). Stretching these strips caused an increase in contractile frequency to that of the electrical oscillation. PMID:3699550

  12. LAT1 regulates growth of uterine leiomyoma smooth muscle cells.

    PubMed

    Xia Luo; Coon, John S; Su, Emily; Pearson, Elizabeth Kerry; Ping Yin; Ishikawa, Hiroshi; Bulun, Serdar E

    2010-09-01

    L-type amino acid transporter 1 (LAT1) and LAT2 were shown to encode system L, which mediates the Na(+)-independent transport of branched-chain and aromatic amino acids. We demonstrated previously that LAT2 is a progesterone receptor target gene involved in leiomyoma growth. The role of LAT1 in the regulation of human uterine leiomyoma growth, however, remains unelucidated. We herein investigated the function of LAT1 and its progesterone-mediated regulation within human uterine leiomyoma smooth muscle (LSM) cells (n = 8) and tissues (n = 29). In vivo, LAT1 expression was higher in leiomyoma than in myometrial tissue. LAT1 knockdown augmented cell proliferation and viability. Treatment of LSM cells with RU486 markedly increased LAT1 messenger RNA (mRNA) levels but decreased proliferation in a dose-dependent manner. L-type amino acid transporter 1 as a downstream target, however, did not entirely account for this antiproliferative effect of RU486 on LSM cells. Taken together, LAT1 may have a critical and complex role in regulating human leiomyoma cell growth. PMID:20601542

  13. Hydraulic Conductivity of Smooth Muscle Cell-Initiated Arterial Cocultures.

    PubMed

    Mathura, Rishi A; Russell-Puleri, Sparkle; Cancel, Limary M; Tarbell, John M

    2016-05-01

    The purpose of the study was to examine the effects of arterial coculture conditions on the transport properties of several in vitro endothelial cell (EC)-smooth muscle cell (SMC)-porous filter constructs in which SMC were grown to confluence first and then EC were inoculated. This order of culturing simulates the environment of a blood vessel wall after endothelial layer damage due to stenting, vascular grafting or other vascular wall insult. For all coculture configurations examined, we observed that hydraulic conductivity (L p) values were significantly higher than predicted by a resistances-in-series (RIS) model accounting for the L p of EC and SMC measured separately. The greatest increases were observed when EC were plated directly on top of a confluent SMC layer without an intervening filter, presumably mediated by direct EC-SMC contacts that were observed under confocal microscopy. The results are the opposite of a previous study that showed L p was significantly reduced compared to an RIS model when EC were grown to confluency first. The physiological, pathophysiological and tissue engineering implications of these results are discussed. PMID:26265460

  14. Airway smooth muscle growth from the perspective of animal models.

    PubMed

    Martin, James G; Ramos-Barbón, David

    2003-09-16

    Airway smooth muscle maintains airway tone and may assist in adjusting ventilation distribution within the normal lung. Alterations in the properties or the quantity of ASM are likely responsible for some instances of airways hyperresponsiveness to bronchoconstrictive stimuli that is a characteristic of diseases such as asthma. Morphometric studies have shown an increase in the mass of ASM in human asthmatic airways. Animal models have been developed that confirm that ASM can be induced to grow by allergic sensitization and challenge. Growth is in large part by hyperplasia as measured by incorporation of bromodeoxyuridine as a marker of the S-phase of the cell cycle. T cells, in particular CD4+ cells, may participate in the stimulation of growth of ASM by allergen challenge. The growth factors responsible for the increase in ASM are as yet unidentified but two mediators associated with allergic airway responses, cysteinyl leukotrienes and endothelin, have been implicated using specific receptor antagonists. The links between T cells and the biochemical mediators of growth have not been established. PMID:14516730

  15. Arterial Myogenic Activation through Smooth Muscle Filamin A.

    PubMed

    Retailleau, Kevin; Arhatte, Malika; Demolombe, Sophie; Peyronnet, Rémi; Baudrie, Véronique; Jodar, Martine; Bourreau, Jennifer; Henrion, Daniel; Offermanns, Stefan; Nakamura, Fumihiko; Feng, Yuanyi; Patel, Amanda; Duprat, Fabrice; Honoré, Eric

    2016-03-01

    Mutations in the filamin A (FlnA) gene are frequently associated with severe arterial abnormalities, although the physiological role for this cytoskeletal element remains poorly understood in vascular cells. We used a conditional mouse model to selectively delete FlnA in smooth muscle (sm) cells at the adult stage, thus avoiding the developmental effects of the knockout. Basal blood pressure was significantly reduced in conscious smFlnA knockout mice. Remarkably, pressure-dependent tone of the resistance caudal artery was lost, whereas reactivity to vasoconstrictors was preserved. Impairment of the myogenic behavior was correlated with a lack of calcium influx in arterial myocytes upon an increase in intraluminal pressure. Notably, the stretch activation of CaV1.2 was blunted in the absence of smFlnA. In conclusion, FlnA is a critical upstream element of the signaling cascade underlying the myogenic tone. These findings allow a better understanding of the molecular basis of arterial autoregulation and associated disease states. PMID:26923587

  16. Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.

    PubMed

    Thiel, William H; Esposito, Carla L; Dickey, David D; Dassie, Justin P; Long, Matthew E; Adam, Joshua; Streeter, Jennifer; Schickling, Brandon; Takapoo, Maysam; Flenker, Katie S; Klesney-Tait, Julia; Franciscis, Vittorio de; Miller, Francis J; Giangrande, Paloma H

    2016-04-01

    Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-β phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease. PMID:26732878

  17. Identification of possible adenosine receptors in vascular smooth muscle

    SciTech Connect

    Doctrow, S.R.

    1985-01-01

    Adenosine is a vasodilator and has been implicated in increased blood flow in tissues that undergo energy deficiency. During conditions such as hypoxia and ischemia, adenosine is produced and is said to increase blood flow by relaxing the vascular smooth muscle (VSM) lining the resistance vessels. The goal of this research was to identify receptors that might be responsible for adenosine-mediated VSM relaxation. When an insoluble fraction from calf aortic VSM was incubated with /sup 32/P-ATP, two components were phosphorylated. One was identified as myosin light chain by MW, pl, and immunoprecipitation. The other product was identified as phosphatidylinositol-4-phosphate (DPI) by tic. Both phosphorylations were inhibited by adenosine and by 5'-chloro-5'-deoxyadenosine (Cl-Ado). DPI production was much more sensitive to the nucleosides than was myosin phosphorylation. Neither inhibition involved change in cAMP production. Phosphatidylinositol (Pl) kinase in the VSM membranes required magnesium, was activated and solubilized by Triton X-100, and phosphorylated both endogenous and exogenous Pl. Cl-Ado inhibited Pl kinase in a manner competitive with respect to ATP and noncompetitive with respect to Pl. Adenosine and adenosine analogs modified in the ribose ring were inhibitors with potencies comparable to that of Cl-Ado. Adenine nucleotides and purine-modified adenosine analogs were weaker inhibitors than Cl-Ado.

  18. PDT-induced apoptosis in arterial smooth muscles cells

    NASA Astrophysics Data System (ADS)

    Nyamekye, Isaac; Renick, R.; Gilbert, C.; McEwan, Jean R.; Evan, G.; Bishop, Christopher C. R.; Bown, Stephen G.

    1995-03-01

    PDT kills smooth muscle cells (SMC) in vivo and thus prevents intimal hyperplasia after angioplasty. It causes little inflammation and structural integrity of the artery is not compromised. We have studied the process of the SMC death in vitro. Cultured rat SMC (cell line sv40 ATCC) were sensitized with aluminum disulphonated phthalocyanine (AlS2Pc), and then irradiated with 675 nm laser light (2.5 J/cm2). Controls were studied using only sensitizer or laser for treatment. The cells were incubated and the dying process observed with a time lapse video and microscope system. PDT caused a characteristic pattern of death. Cells lost contact with neighbors, shrank, and showed hyperactivity and membrane ruffling. The cells imploded into active and condensed membrane bound vesicles which were terminally reduced to residual bodies. These are the morphological changes of apoptosis. The control cells which were given AlS2Pc alone or laser alone showed no death. PDT induced cultured arterial SMC death by apoptosis rather than necrosis. An apoptotic mechanism of cell death in vivo would explain the relative lack of inflammation and local tissue destruction in the face of massive death.

  19. Arteriolar vascular smooth muscle cells: mechanotransducers in a complex environment.

    PubMed

    Hill, Michael A; Meininger, Gerald A

    2012-09-01

    Contraction of small artery (diameters typically less than 250 μm) vascular smooth muscle cells (VSMCs) plays a critical role in local control of blood flow and arterial pressure through its affect on vascular caliber. Specifically, contraction of small arteries in response to increased intraluminal pressure is referred to as the myogenic response and represents an important role for mechanotransduction. Critical questions remain as to how changes in pressure are sensed by VSMCs and transduced across the cell membrane to tune the contractile state of the cell. Recent studies suggest a pivotal role for interactions between VSMCs and extracellular matrix (ECM) proteins. Thus, pressure-induced deformation of ECM proteins and their cell surface receptors (for example, integrins) may initiate contraction and cytoskeletal remodeling through modulation of ion channels, membrane depolarization, increased intracellular Ca(2+) and actomyosin crossbridge cycling. Importantly, it is argued that the contractile properties of small artery VSMCs reflect an intimate and integrated interaction with their extracellular environment and the three-dimensional structure of the vessel wall. PMID:22677491

  20. SREBP inhibits VEGF expression in human smooth muscle cells

    SciTech Connect

    Motoyama, Koka; Fukumoto, Shinya . E-mail: sfukumoto@med.osaka-cu.ac.jp; Koyama, Hidenori; Emoto, Masanori; Shimano, Hitoshi; Maemura, Koji; Nishizawa, Yoshiki

    2006-03-31

    Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate expression of genes encoding enzymes for lipid biosynthesis. SREBPs are activated by HMG-CoA reductase inhibitors (statins). Statins have been also reported to suppress vascular endothelial growth factor (VEGF) expression in vascular smooth muscle cells (VSMCs). Therefore, we hypothesized that SREBPs are involved in statin-mediated regulation of VEGF production in VSMCs. SREBP1 was robustly expressed, and was activated by atorvastatin in VSMCs, as demonstrated by increased levels of the mature nuclear form of SREBP1, and increased promoter activities of a reporter containing sterol regulatory elements by atorvastatin. Moreover, overexpression of SREBP1a dose-dependently suppressed VEGF promoter activity. Site-specific mutation or deletion of the proximal Sp1 sites reduced the inhibitory effects of SREBP1a on VEGF promoter activity. These data demonstrated that SREBP1, activated by atorvastatin, suppressed VEGF expression through the indirect interaction with the proximal tandem Sp1 sites in VSMCs.

  1. Dense-body aggregates as plastic structures supporting tension in smooth muscle cells.

    PubMed

    Zhang, Jie; Herrera, Ana M; Paré, Peter D; Seow, Chun Y

    2010-11-01

    The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells. PMID:20709732

  2. Large conducting potassium channel reconstituted from airway smooth muscle.

    PubMed

    Savaria, D; Lanoue, C; Cadieux, A; Rousseau, E

    1992-03-01

    Microsomal fractions were prepared from canine and bovine airway smooth muscle (ASM) by differential and gradient centrifugations. Surface membrane vesicles were characterized by binding assays and incorporated into planar lipid bilayers. Single-channel activities were recorded in symmetric or asymmetric K+ buffer systems and studied under voltage and Ca2+ clamp conditions. A large-conductance K(+)-selective channel (greater than 220 pS in 150 mM K+) displaying a high Ca2+, low Ba2+, and charybdotoxin (CTX) sensitivity was identified. Time analysis of single-channel recordings revealed a complex kinetic behavior compatible with the previous schemes proposed for Ca(2+)-activated K+ channels in a variety of biological surface membranes. We now report that the open probability of the channel at low Ca2+ concentration is enhanced on in vitro phosphorylation, which is mediated via an adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition to this characterization at the molecular level, a second series of pharmacological experiments were designed to assess the putative role of this channel in ASM strips. Our results show that 50 nM CTX, a specific inhibitor of the large conducting Ca(2+)-dependent K+ channel, prevents norepinephrine transient relaxation on carbamylcholine-precontracted ASM strips. It was also shown that CTX reversed the steady-state relaxation induced by vasoactive intestinal peptide and partially antagonized further relaxation induced by cumulative doses of this potent bronchodilatator. Thus it is proposed that the Ca(2+)-activated K+ channels have a physiological role because they are indirectly activated on stimulation of various membrane receptors via intracellular mechanisms. PMID:1372487

  3. Pharmacological modulation of sarcoplasmic reticulum function in smooth muscle.

    PubMed

    Laporte, Régent; Hui, Adrian; Laher, Ismail

    2004-12-01

    The sarco/endoplasmic reticulum (SR/ER) is the primary storage and release site of intracellular calcium (Ca2+) in many excitable cells. The SR is a tubular network, which in smooth muscle (SM) cells distributes close to cellular periphery (superficial SR) and in deeper aspects of the cell (deep SR). Recent attention has focused on the regulation of cell function by the superficial SR, which can act as a buffer and also as a regulator of membrane channels and transporters. Ca2+ is released from the SR via two types of ionic channels [ryanodine- and inositol 1,4,5-trisphosphate-gated], whereas accumulation from thecytoplasm occurs exclusively by an energy-dependent sarco-endoplasmic reticulum Ca2+-ATPase pump (SERCA). Within the SR, Ca2+ is bound to various storage proteins. Emerging evidence also suggests that the perinuclear portion of the SR may play an important role in nuclear transcription. In this review, we detail the pharmacology of agents that alter the functions of Ca2+ release channels and of SERCA. We describe their use and selectivity and indicate the concentrations used in investigating various SM preparations. Important aspects of cell regulation and excitation-contractile activity coupling in SM have been uncovered through the use of such activators and inhibitors of processes that determine SR function. Likewise, they were instrumental in the recent finding of an interaction of the SR with other cellular organelles such as mitochondria. Thus, an appreciation of the pharmacology and selectivity of agents that interfere with SR function in SM has greatly assisted in unveiling the multifaceted nature of the SR. PMID:15602008

  4. Caveolin-3 Promotes a Vascular Smooth Muscle Contractile Phenotype

    PubMed Central

    Gutierrez-Pajares, Jorge L.; Iturrieta, Jeannette; Dulam, Vipin; Wang, Yu; Pavlides, Stephanos; Malacari, Gabriella; Lisanti, Michael P.; Frank, Philippe G.

    2015-01-01

    Epidemiological studies have demonstrated the importance of cardiovascular diseases in Western countries. Among the cell types associated with a dysfunctional vasculature, smooth muscle (SM) cells are believed to play an essential role in the development of these illnesses. Vascular SM cells are key regulators of the vascular tone and also have an important function in the development of atherosclerosis and restenosis. While in the normal vasculature, contractile SM cells are predominant, in atherosclerotic vascular lesions, synthetic cells migrate toward the neointima, proliferate, and synthetize extracellular matrix proteins. In the present study, we have examined the role of caveolin-3 in the regulation of SM cell phenotype. Caveolin-3 is expressed in vivo in normal arterial SM cells, but its expression appears to be lost in cultured SM cells. Our data show that caveolin-3 expression in the A7r5 SM cell line is associated with increased expression of contractility markers such as SM α-actin, SM myosin heavy chain but decreased expression of the synthetic phenotype markers such as p-Elk and Klf4. Moreover, we also show that caveolin-3 expression can reduce proliferation upon treatment with LDL or PDGF. Finally, we show that caveolin-3-expressing SM cells are less sensitive to apoptosis than control cells upon treatment with oxidized LDL. Taken together, our data suggest that caveolin-3 can regulate the phenotypic switch between contractile and synthetic SM cells. A better understanding of the factors regulating caveolin-3 expression and function in this cell type will permit the development of a better comprehension of the factors regulating SM function in atherosclerosis and restenosis. PMID:26664898

  5. Agonist-induced Ca2+ Sensitization in Smooth Muscle

    PubMed Central

    Artamonov, Mykhaylo V.; Momotani, Ko; Stevenson, Andra; Trentham, David R.; Derewenda, Urszula; Derewenda, Zygmunt S.; Read, Paul W.; Gutkind, J. Silvio; Somlyo, Avril V.

    2013-01-01

    Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca2+]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca2+ sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca2+-sensitized force are not well understood. Using permeabilized blood vessels from PRG(−/−) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca2+-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5′-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca2+ transient and phasic force components and the onset of Ca2+-sensitized force. PMID:24106280

  6. Regulation of smooth muscle cell phenotype by glycosaminoglycan identity.

    PubMed

    Qu, Xin; Jimenez-Vergara, Andrea Carolina; Munoz-Pinto, Dany J; Ortiz, Diana; McMahon, Rebecca E; Cristancho, Deissy; Becerra-Bayona, Silvia; Guiza-Arguello, Viviana; Grande-Allen, K Jane; Hahn, Mariah S

    2011-03-01

    The retention of lipoproteins in the arterial intima is an initial event in early atherosclerosis and occurs, in part, through interactions between negatively charged glycosaminoglycans (GAGs) and the positively charged residues of apolipoproteins. Smooth muscle cells (SMCs) which infiltrate into the lipoprotein-enriched intima have been observed to transform into lipid-laden foam cells. This phenotypic switch is associated with SMC acquisition of a macrophage-like capacity to phagocytose lipoproteins and/or of an adipocyte-like capacity to synthesize fatty acids de novo. The aim of the present work was to explore the impact of GAG identity on SMC foam cell formation using a scaffold environment intended to be mimetic of early atherosclerosis. In these studies, we focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HAIMW, ∼400 kDa), the levels and/or distribution of each of which are significantly altered in atherosclerosis. DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HAIMW gels. Similarly, only SMCs in DS constructs maintained increased expression of the adipocyte marker A-FABP relative to HAIMW gels over 35 days of culture. The increased SMC foam cell phenotype in DS hydrogels was reflected in a corresponding decrease in SMC myosin heavy chain expression in these constructs relative to HAIMW gels at day 35. In addition, this DS-associated increase in foam cell formation was mirrored in an increased SMC synthetic phenotype, as evidenced by greater levels of collagen type I and glucose 6-phosphate dehydrogenase in DS gels than in HAIMW gels. Combined, these results support the increasing body of literature that suggests a critical role for DS-bearing proteoglycans in early atherosclerosis. PMID:21094702

  7. Distribution of a lanthanide (147 Pm) in vascular smooth muscle.

    PubMed

    Weiss, G B; Goodman, F R

    1976-08-01

    In order to ascertain whether trivalent rare earth ions such as lanthanum (La+++) penetrate the cell membrane under physiological conditions, the extracellular and cellular distribution of promethium (147 Pm), a carrier-free rare earth radioisotope, was examined in rabbit aortic smooth muscle. As the duration of incubation was lengthened, uptake of 147Pm continued to increase; it was inhibited by La+++ and other rare earth ions (Nd+++, Lu+++) only when the 147 Pm/rare earth concentration ratio exceeded 1:10(6). However, equally high concentrations of Ca++ had no effect on 147Pm uptake. Efflux of 147Pm was only transiently increased by 1.5 mM La+++, and exposure to 0.05 mM EDTA elicited an increased 147Pm efflux with both transient and maintained components. The magnitude of the EDTA-induced increase in 147 Pm efflux was similar over a 30-fold range of EDTA concentration (0.05-1.5 mM); the limiting factor for 147Pm efflux is the rate of 147Pm desorption from the tissue rather than the extracellular concentration of EDTA. Loss of 147Pm in the presence of 0.05 mM EDTA could be described in terms of two specific washout components (the more rapid of which included 147Pm within the extracellular space and the slower of which had half-times of washout of approximately 7-10 minutes). Uptake of 147Pm was inhibited by lowering the incubation solution temperature to 0 degrees C or by procaine. However, concentrations of metabolic inhibitors (iodoacetate and dinitrophenol) which diminish loss of Ca++ from the cell did not decrease either the uptake or efflux of 147Pm. Thus, significant quantities of 147Pm do not appear to be accumulated within the cell or transported out of the cell; distribution of 147Pm can be most simply described in terms of a binding at and desorption from surface acessible fiber sites. PMID:820850

  8. Interaction of chicken gizzard smooth muscle calponin with brain microtubules.

    PubMed

    Fujii, T; Hiromori, T; Hamamoto, M; Suzuki, T

    1997-08-01

    Calponin, a major actin-, tropomyosin-, and calmodulin-binding protein in smooth muscle, interacted with tubulin, a main constituent of microtubules, in a concentration-dependent fashion in vitro. The apparent K(d) value of calponin to tubulin was calculated to be 5.2 microM with 2 mol of calponin maximally bound per 1 mol of tubulin. At low ionic strength, tubulin bound to calponin immobilized on Sepharose 4B, and the bound protein was released at about 270 mM NaCl. Chemical cross-linking experiments showed that a 1:1 molar covalent complex of calponin and tubulin was produced. The amount of calponin bound to microtubules decreased with increasing ionic strength or Ca2+ concentration. The addition of calmodulin or S100 to the mixture of calponin and microtubule proteins caused the removal of calponin from microtubules in the presence of Ca2+, but not in the presence of EGTA. Calponin-related proteins including tropomyosin, SM22, and caldesmon had little effect on the calponin binding to microtubules, whereas MAP2 inhibited the binding. Interestingly, there was little, if any, effect of mycalolide B-treated actin on the binding of calponin to microtubules. Furthermore, only about 20% of calponin-F-actin interaction was inhibited in the presence of an excess amount of tubulin (4 mol per mol of calponin), indicating that tubulin binds to calponin at a different site from that of actin. Compared with MAP2, calponin had little effect on microtubule polymerization. PMID:9378712

  9. Length adaptation of smooth muscle contractile filaments in response to sustained activation.

    PubMed

    Stålhand, Jonas; Holzapfel, Gerhard A

    2016-05-21

    Airway and bladder smooth muscles are known to undergo length adaptation under sustained contraction. This adaptation process entails a remodelling of the intracellular actin and myosin filaments which shifts the peak of the active force-length curve towards the current length. Smooth muscles are therefore able to generate the maximum force over a wide range of lengths. In contrast, length adaptation of vascular smooth muscle has attracted very little attention and only a handful of studies have been reported. Although their results are conflicting on the existence of a length adaptation process in vascular smooth muscle, it seems that, at least, peripheral arteries and arterioles undergo such adaptation. This is of interest since peripheral vessels are responsible for pressure regulation, and a length adaptation will affect the function of the cardiovascular system. It has, e.g., been suggested that the inward remodelling of resistance vessels associated with hypertension disorders may be related to smooth muscle adaptation. In this study we develop a continuum mechanical model for vascular smooth muscle length adaptation by assuming that the muscle cells remodel the actomyosin network such that the peak of the active stress-stretch curve is shifted towards the operating point. The model is specialised to hamster cheek pouch arterioles and the simulated response to stepwise length changes under contraction. The results show that the model is able to recover the salient features of length adaptation reported in the literature. PMID:26925813

  10. In vivo recording of electrical activity of canine tracheal smooth muscle.

    PubMed

    Kondo, T; Tamura, K; Onoe, K; Takahira, H; Ohta, Y; Yamabayashi, H

    1992-01-01

    Electrical activity of the tracheal smooth muscle was studied using extracellular bipolar electrodes in 37 decerebrate, paralyzed, and mechanically ventilated dogs. A spontaneous oscillatory potential that consisted of a slow sinusoidal wave of 0.57 +/- 0.13 (SD) Hz mean frequency but lacked a fast spike component was recorded from 15 dogs. Lung collapse accomplished by bilateral pneumothoraxes evoked or augmented the slow potentials that were associated with an increase in tracheal muscle contraction in 26 dogs. This suggests that the inputs from the airway mechanoreceptors reflexly activate the tracheal smooth muscle cells. Bilateral vagal transection abolished both the spontaneous and the reflexly evoked slow waves and provided relaxation of the tracheal smooth muscle. Electrical stimulation of the distal nerve with a train pulse (0.5 ms, 1-30 Hz) evoked slow-wave oscillatory potentials accompanied by a contraction of the tracheal smooth muscle in all the experimental animals. Our observations in this in vivo study confirm that the electrical activity of tracheal smooth muscle consists of slow oscillatory potentials and that tracheal contraction is at least partly coupled to the slow-wave activity of the smooth muscle. PMID:1537706

  11. Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network

    PubMed Central

    2014-01-01

    Background Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors. Results Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other’s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation. Conclusions Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors. PMID:25015411

  12. Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function.

    PubMed

    Roffel, A F; Elzinga, C R; Van Amsterdam, R G; De Zeeuw, R A; Zaagsma, J

    1988-08-01

    Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle. PMID:3215279

  13. Mebendazole Reduces Vascular Smooth Muscle Cell Proliferation and Neointimal Formation Following Vascular Injury in Mice

    PubMed Central

    Wang, Jintao; Wang, Hui; Guo, Chiao; Luo, Wei; Lawler, Alyssa; Reddy, Aswin; Wang, Julia; Sun, Eddy B.; Eitzman, Daniel T.

    2014-01-01

    Mebendazole is an antihelminthic drug that exerts its effects via interference with microtubule function in parasites. To determine the utility of mebendazole as a potential treatment for vascular diseases involving proliferation of vascular smooth muscle cells, the effects of mebendazole on vascular smooth muscle cell proliferation were tested in vitro and in a mouse model of arterial injury. In vitro, mebendazole inhibited proliferation and migration of murine vascular smooth muscle cells and this was associated with altered intracellular microtubule organization. To determine in vivo effects of mebendazole following vascular injury, femoral arterial wire injury was induced in wild-type mice treated with either mebendazole or placebo control. Compared with placebo-treated mice, mebendazole-treated mice formed less neointima at the site of injury. Mebendazole is effective at inhibiting vascular smooth muscle cell proliferation and migration, and neointimal formation following arterial injury in mice. PMID:24587248

  14. ROLE OF ACETYLCHOLINE IN RHYTHMIC SPONTANEOUS CONTRACTIONS OF RAT'S DUODENAL SMOOTH MUSCLE

    EPA Science Inventory

    The purpose of the study is to reexamine the role of endogenous acetylcholine in spontaneous contractions of smooth muscle whose contractions are associated with cell metabolism. The study does not attempt to define the role of endogenous acetylcholine.

  15. Cyclic GMP alters Ca exchange in vascular smooth muscle

    SciTech Connect

    Magliola, L.; Bailey, B.; Jones, A.W.

    1986-03-05

    Contraction and /sup 42/K efflux from vascular smooth muscle stimulated either by norepinephrine (NE) or by K-depolarization is dependent on an increase in cytosolic Ca concentration. The purpose of this study was to determine if cyclic GMP (cGMP) inhibited these processes and if inhibition was secondary to the action of cGMP on Ca movements. Basal cGMP content of rat aorta was 1.2 fmol/mg wet wt. Sodium nitroprusside (NP) increased cGMP approx.2-fold at 1 nM and approx.750-fold at 1 ..mu..M with no effect on cAMP levels. A 5 min pretreatment with NP (1 ..mu..M) completely prevented tension development induced by 3 ..mu..M NE. The same concentration of NP also inhibited NE-stimulated /sup 42/K and /sup 45/Ca efflux > 90 and > 80%, respectively. Removal of NP in the continued presence of NE (3 ..mu..M) caused recovery of the /sup 42/K efflux response to approx.75% of control with a half-time of approx.2.5 min. NP (1 ..mu..M) also caused a rapid relaxation of aorta contracted with 3 ..mu..M NE and a loss of the /sup 42/K efflux response with half-times of 2-3 min. In contrast, 100 ..mu..M NP produced only a 50% inhibition of contraction induced by high K (55 mM). Also, NP (1 ..mu..M) inhibited K-stimulated /sup 42/K efflux only approx.25%. These results demonstrate both a concentration- and a time-dependent relationship between increases in cGMP induced by NP and decreases in NE-stimulated contraction, /sup 42/K and /sup 45/Ca effluxes. They also indicate that the sensitivity of NE-induced contraction and /sup 42/K efflux to NP is greater than that induced by high K. These studies suggest that cGMP modulates the control sites for Ca exchange in the plasma membrane and sarcoplasmic reticulum.

  16. 3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells

    PubMed Central

    Luo, Tong; Chen, Huan; Kassab, Ghassan S.

    2016-01-01

    Aims The 3D geometry of individual vascular smooth muscle cells (VSMCs), which are essential for understanding the mechanical function of blood vessels, are currently not available. This paper introduces a new 3D segmentation algorithm to determine VSMC morphology and orientation. Methods and Results A total of 112 VSMCs from six porcine coronary arteries were used in the analysis. A 3D semi-automatic segmentation method was developed to reconstruct individual VSMCs from cell clumps as well as to extract the 3D geometry of VSMCs. A new edge blocking model was introduced to recognize cell boundary while an edge growing was developed for optimal interpolation and edge verification. The proposed methods were designed based on Region of Interest (ROI) selected by user and interactive responses of limited key edges. Enhanced cell boundary features were used to construct the cell’s initial boundary for further edge growing. A unified framework of morphological parameters (dimensions and orientations) was proposed for the 3D volume data. Virtual phantom was designed to validate the tilt angle measurements, while other parameters extracted from 3D segmentations were compared with manual measurements to assess the accuracy of the algorithm. The length, width and thickness of VSMCs were 62.9±14.9μm, 4.6±0.6μm and 6.2±1.8μm (mean±SD). In longitudinal-circumferential plane of blood vessel, VSMCs align off the circumferential direction with two mean angles of -19.4±9.3° and 10.9±4.7°, while an out-of-plane angle (i.e., radial tilt angle) was found to be 8±7.6° with median as 5.7°. Conclusions A 3D segmentation algorithm was developed to reconstruct individual VSMCs of blood vessel walls based on optical image stacks. The results were validated by a virtual phantom and manual measurement. The obtained 3D geometries can be utilized in mathematical models and leads a better understanding of vascular mechanical properties and function. PMID:26882342

  17. Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

    PubMed

    Hsiao, Amy Y; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

    2015-01-01

    The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments. PMID:25734774

  18. Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein.

    PubMed

    Xu, Long; Ting-Lou; Lv, Nonghua; Zhu, Xuan; Chen, Youxiang; Yang, Jing

    2009-08-01

    Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway. PMID:19409890

  19. Adult human arterial smooth muscle cells in primary culture. Modulation from contractile to synthetic phenotype.

    PubMed

    Thyberg, J; Nilsson, J; Palmberg, L; Sjölund, M

    1985-01-01

    Smooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [3H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1-2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3-4 days. It was accompanied by initiation of DNA replication and mitosis. The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis. PMID:3967287

  20. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration.

    PubMed

    Hedges, J C; Dechert, M A; Yamboliev, I A; Martin, J L; Hickey, E; Weber, L A; Gerthoffer, W T

    1999-08-20

    Smooth muscle cells are exposed to growth factors and cytokines that contribute to pathological states including airway hyperresponsiveness, atherosclerosis, angiogenesis, smooth muscle hypertrophy, and hyperplasia. A common feature of several of these conditions is migration of smooth muscle beyond the initial boundary of the organ. Signal transduction pathways activated by extracellular signals that instigate migration are mostly undefined in smooth muscles. We measured migration of cultured tracheal myocytes in response to platelet-derived growth factor, interleukin-1beta, and transforming growth factor-beta. Cellular migration was blocked by SB203580, an inhibitor of p38(MAPK). Time course experiments demonstrated increased phosphorylation of p38(MAPK). Activation of p38(MAPK) resulted in the phosphorylation of HSP27 (heat shock protein 27), which may modulate F-actin polymerization. Inhibition of p38(MAPK) activity inhibited phosphorylation of HSP27. Adenovirus-mediated expression of activated mutant MAPK kinase 6b(E), an upstream activator for p38(MAPK), increased cell migration, whereas overexpression of p38alpha MAPK dominant negative mutant and an HSP27 phosphorylation mutant blocked cell migration completely. The results indicate that activation of the p38(MAPK) pathway by growth factors and proinflammatory cytokines regulates smooth muscle cell migration and may contribute to pathological states involving smooth muscle dysfunction. PMID:10446196

  1. Recruitment of β-catenin to N-cadherin is necessary for smooth muscle contraction.

    PubMed

    Wang, Tao; Wang, Ruping; Cleary, Rachel A; Gannon, Olivia J; Tang, Dale D

    2015-04-01

    β-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the β-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of β-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by β-catenin knockdown. In addition, the expression of the β-catenin armadillo domain disrupted the recruitment of β-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the β-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of β-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of β-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of β-catenin with N-cadherin is regulated by actin polymerization during contractile activation. PMID:25713069

  2. Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

    PubMed Central

    Hsiao, Amy Y.; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

    2015-01-01

    The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments. PMID:25734774

  3. Increased Vascular Smooth Muscle Contractility in TRPC6−/− Mice

    PubMed Central

    Dietrich, Alexander; Mederos y Schnitzler, Michael; Gollasch, Maik; Gross, Volkmar; Storch, Ursula; Dubrovska, Galyna; Obst, Michael; Yildirim, Eda; Salanova, Birgit; Kalwa, Hermann; Essin, Kirill; Pinkenburg, Olaf; Luft, Friedrich C.; Gudermann, Thomas; Birnbaumer, Lutz

    2005-01-01

    Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6−/− smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6−/− smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone. PMID:16055711

  4. Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens.

    PubMed Central

    Nakazawa, K.; Higo, K.; Abe, K.; Tanaka, Y.; Saito, H.; Matsuki, N.

    1993-01-01

    1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]-nimodipine binding. Trifluoperazine and W-7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation. 4. Ba2+ current flowing through voltage-gated Ca2+ channels was measured under whole-cell voltage-clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration-range where inhibition of [3H]-nimodipine binding was observed. Neither voltage-dependence nor the inactivation time course of Ba2+ current were affected by these compounds. 5. The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W-7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds. PMID:8495236

  5. OUABAIN- AND MARINOBUFAGENIN-INDUCED PROLIFERATION OF HUMAN UMBILICAL VEIN SMOOTH MUSCLE CELLS AND A RAT VASCULAR SMOOTH MUSCLE CELL LINE, A7R5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the growth-promoting effects of 2 sodium pump-selective cardiotonic steroids, ouabain and marinobufagenin, on cultured cells from vascular smooth muscle (VSMCs) from human umbilical vein and a rat VSMC line, A7r5. Both ouabain and marinobufagenin activated proliferation of these cells in...

  6. From depolarization-dependent contractions in gastrointestinal smooth muscle to aortic pulse-synchronized contractions

    PubMed Central

    Marion, Sarah B; Mangel, Allen W

    2014-01-01

    For decades, it was believed that the diameter of gastrointestinal smooth muscle cells is sufficiently narrow, and that the diffusion of calcium across the plasma membrane is sufficient, to support contractile activity. Thus, depolarization-triggered release of intracellular calcium was not believed to be operative in gastrointestinal smooth muscle. However, after the incubation of muscle segments in solutions devoid of calcium and containing the calcium chelator ethylene glycol tetraacetic acid, an alternative electrical event occurred that was distinct from normal slow waves and spikes. Subsequently, it was demonstrated in gastrointestinal smooth muscle segments that membrane depolarization associated with this alternative electrical event triggered rhythmic contractions by release of intracellular calcium. Although this concept of depolarization-triggered calcium release was iconoclastic, it has now been demonstrated in multiple gastrointestinal smooth muscle preparations. On the basis of these observations, we investigated whether a rhythmic electrical and mechanical event would occur in aortic smooth muscle under the same calcium-free conditions. The incubation of aortic segments in a solution with no added calcium plus ethylene glycol tetraacetic acid induced a fast electrical event without corresponding tension changes. On the basis of the frequency of these fast electrical events, we pursued, contrary to what has been established dogma for more than three centuries, the question of whether the smooth muscle wall of the aorta undergoes rhythmic activation during the cardiac cycle. As with depolarization-triggered contractile activity in gastrointestinal smooth muscle, it was “well known” that rhythmic activation of the aorta does not occur in synchrony with the heartbeat. In a series of experiments, however, it was demonstrated that rhythmic contractions occur in the aortic wall in synchrony with the heartbeat and share a common pacemaker with the heart

  7. Smooth muscle NOS, colocalized with caveolin-1, modulates contraction in mouse small intestine

    PubMed Central

    El-Yazbi, Ahmed F; Cho, Woo Jung; Cena, Jonathan; Schulz, Richard; Daniel, Edwin E

    2008-01-01

    Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca2+] through L-type Ca2+ channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 μM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-β cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-ω-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 μM) blocked the responses to KCl and LNNA confirming the role of L-type Ca2+ channels. ODQ (1 μM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism. PMID:18400048

  8. Developmental changes in expression of contractile and cytoskeletal proteins in human aortic smooth muscle.

    PubMed

    Glukhova, M A; Frid, M G; Koteliansky, V E

    1990-08-01

    To describe phenotypic changes of human aortic smooth muscle cells (SMCs), proportion of smooth muscle and nonmuscle variants of actin, myosin heavy chains (MHCs), vinculin, and caldesmon, during prenatal and several months of postnatal development was determined. In aortic SMCs from 9-10-week-old fetus, both nonmuscle and smooth muscle-specific variants of all four proteins were present, however, the nonmuscle forms were more abundant. During development, a shift towards the expression of muscle-specific variants was observed, although the time course of changes in protein variant content was not similar for all the proteins studied. By the 24th week of gestation, fractional content of alpha-smooth muscle actin and smooth muscle MHCs was rather close to that in the mature SMCs, and comprised approximately 80 and 90%, respectively, of the levels characteristic of SMCs from adult aortic media. On the contrary, fractional ratio of meta-vinculin and 150-kDa caldesmon was still rather low in the aorta from the 24-week-old fetus, did not increase in a 2-month-old child aorta, and did not reach the level characteristic of mature SMCs even in the 6-month-old child aorta. Thus changes in alpha-smooth muscle actin and smooth muscle MHC fractional content occur mainly during the prenatal period of development, before the 24th week of gestation; while meta-vinculin and the 150-kDa caldesmon proportion increases mainly in the postnatal period, during several months after birth. In the "Discussion," phenotypes of SMCs from developing aorta were compared to those from different layers of the adult aortic wall. PMID:2376586

  9. Smooth muscle and purinergic contraction of the human, rabbit, rat, and mouse testicular capsule.

    PubMed

    Banks, Frederick C L; Knight, Gillian E; Calvert, Robert C; Turmaine, Mark; Thompson, Cecil S; Mikhailidis, Dimitri P; Morgan, Robert J; Burnstock, Geoffrey

    2006-03-01

    The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors. PMID:16280417

  10. A Simple, Inexpensive Model to Demonstrate How Contraction of GI Longitudinal Smooth Muscle Promotes Propulsion

    ERIC Educational Resources Information Center

    Lujan, Heidi L.; DiCarlo, Stephen E.

    2015-01-01

    Peristalis is a propulsive activity that involves both circular and longitudinal muscle layers of the esophagus, distal stomach, and small and large intestines. During peristalsis, the circular smooth muscle contracts behind (on the orad side) the bolus and relaxes in front (on the aborad side) of the bolus. At the same time, the longitudinal…

  11. Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate.

    PubMed

    Zhao, Lei; Wang, Zhe; Ruan, Ye-Chun; Zhou, Wen-Liang

    2012-07-01

    In the earlier study, sodium orthovanadate (SOV) has been reported to be a powerful inhibitor of (Na(+), K(+)) adenosine triphosphatase, exhibit widespread actions on the renal and cardiovascular systems, induces smooth muscle contraction by inhibiting the phosphorylation of the protein tyrosine phosphatases. In the current study, we aimed to investigate the cellular mechanisms by which SOV facilitated contractile response of vas deferens smooth muscle and its potential therapeutic advantage. Exogenous application of ATP and NA-caused contraction was strengthened by pretreatment with SOV. This facilitation was inhibited not by bath with the inhibitor of P2 receptor, PPADS, or the inhibitor of α1 receptor, Prazosin, but by bath with the protein tyrosine kinase inhibitor, Genistein. SOV induced a sustained increase in intracellular Ca(2+) of smooth muscle cells, which was abolished by 100 μM Genistein or Ca(2+)-free solution. The facilitation of SOV could also be inhibited by the selective inhibitors of TRP channel, 2-APB and non-selective cation channel, Gd(3+), Ni(+). The in vivo study showed that peritoneal injection of SOV in dystrophic mice (mdx mice) enhanced the contraction of vas deferens smooth muscle stimulated by electrical field stimulation, ATP, noradrenaline, or KCl. The above results suggest that SOV facilitates the concentration of vas deferens smooth muscle through the tyrosine phosphorylation activated the non-selective cation channels, which has potential use in the therapy for muscle dysfunction. PMID:22476902

  12. Pleomorphic rhabdomyosarcoma showing smooth-muscle and fibrohistiocytic differentiation: a single case report.

    PubMed

    Eyden, Brian

    2010-02-01

    Rhabdomyosarcoma has traditionally been subclassified into alveolar, embryonal, and pleomorphic variants. Less commonly, spindle-cell, neuroendocrine, sclerosing, and lipid-rich or clear-cell subtypes are seen. The author recently encountered a myogenic sarcoma, with all the common markers of rhabdomyosarcoma, but expressing the unusual features of alpha-smooth-muscle actin and abundant rough endoplasmic reticulum (rER). This myogenic sarcoma, therefore, exhibited four lines of differentiation, and is documented here. The patient was a 65-year-old man with an inguinal soft tissue mass. Following surgical excision, the patient was given radiotherapy and was well without disease after 6 years. The tumor was positive for vimentin, desmin, alpha-smooth-muscle actin, alpha-sarcomeric actin, myogenin, MyoD1, and CD68. Cytoplasm was dominated by abundant rER intermingled with lipid droplets and lysosomes. Cell surfaces exhibited microvillous processes and focal adhesions, but no lamina. Subplasmalemmal smooth-muscle-type myofilaments with focal densities and rare sarcomeric filaments were seen. The low level of expression of some markers was interpreted as consistent with a poorly differentiated tumor. Given the four lines of differentiation--striated muscle, smooth muscle, fibroblastic, and histiocytic--a name reflecting its phenotype would be pleomorphic rhabdomyosarcoma showing smooth-muscle and fibrohistiocytic differentiation. PMID:20070153

  13. Cell shape and the presentation of adhesion ligands guide smooth muscle myogenesis.

    PubMed

    Zhang, Douglas; Sun, Michael B; Lee, Junmin; Abdeen, Amr A; Kilian, Kristopher A

    2016-05-01

    The reliable generation of smooth muscle cells is important for a number of tissue engineering applications. Human mesenchymal stem cells (MSCs) are a promising progenitor of smooth muscle, with high expression of smooth muscle markers observed in a fraction of isolated cells, which can be increased by introduction of soluble supplements that direct differentiation. Here we demonstrate a new micropatterning technique, where peptides of different ligand affinity can be microcontact printed onto an inert background, to explore MSC differentiation to smooth muscle through controlled biochemical and biophysical cues alone. Using copper-catalyzed alkyne-azide cycloaddition (CuAAC), we patterned our surfaces with RGD peptide ligands-both a linear peptide with low integrin affinity and a cyclic version with high integrin affinity-for the culture of MSCs in shapes with various aspect ratios. At low aspect ratio, ligand affinity is a prime determinant for smooth muscle differentiation, while at high aspect ratio, ligand affinity has less of an effect. Pathway analysis reveals a role for focal adhesion turnover, Rac1, RhoA/ROCK, and calpain during smooth muscle differentiation of MSCs in response to cell shape and the affinity of the cell adhesion interface. Controlling integrin-ligand affinity at the biomaterials interface is important for mediating adhesion but may also prove useful for directing smooth muscle myogenesis. Peptide patterning enables the systematic investigation of single to multiple peptides derived from any protein, at different densities across a biomaterials surface, which has the potential to direct multiple MSC differentiation outcomes without the need for soluble supplements. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1212-1220, 2016. PMID:26799164

  14. Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

    PubMed Central

    Kullmann, F. Aura; Daugherty, Stephanie L.; de Groat, William C.; Birder, Lori A.

    2015-01-01

    We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions. The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release. The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to

  15. Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development.

    PubMed

    Belkin, V M; Belkin, A M; Koteliansky, V E

    1990-11-01

    A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA-1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA-1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1

  16. Has cervical smooth muscle any physiological role in the human?

    PubMed

    Bryman, I; Norström, A; Lindblom, B

    1985-01-01

    Strips of human cervical tissue were obtained by needle biopsy and contractile activity was registered isometrically in a tissue chamber perfused by Krebs-Ringer bicarbonate buffer. The most frequently encountered pattern of contractile activity was high frequency-short duration. Prostaglandin (PG)E2, PGI2 and 6-keto-PGF1 alpha had an inhibitory effect on the muscular activity. Cervical muscle from pregnant women was more sensitive to PGE2 than specimens from non-pregnant women. PGF2 alpha had no apparent effect on cervical contractility in non-pregnant and early pregnant patients. In late pregnancy, however, PGF2 alpha inhibited muscle contractions. The present results point to a physiological role of the cervical muscles for the control of cervical competence during pregnancy. The inhibitory effect of PGs on the muscle activity may promote cervical dilatation and retraction. PMID:3893038

  17. Membrane currents that govern smooth muscle contraction in a ctenophore.

    PubMed

    Bilbaut, A; Hernandez-Nicaise, M L; Leech, C A; Meech, R W

    1988-02-11

    Ctenophores are transparent marine organisms that swim by means of beating cilia; they are the simplest animals with individual muscle fibres. Predatory species, such as Beroe ovata, have particularly well-developed muscles and are capable of an elaborate feeding response. When Beroe contacts its prey, the mouth opens, the body shortens, the pharynx expands, the prey is engulfed and the lips then close tightly. How this sequence, which lasts 1 s, is accomplished is unclear. The muscles concerned are structurally uniform and are innervated at each end by a neuronal nerve net with no centre for coordination. Isolated muscle cells studied under voltage-clamp provide a solution to this puzzle. We find that different groups of muscle cells have different time-dependent membrane currents. Because muscle contraction depends upon calcium entry during each action potential, these different currents produce different patterns of contraction. We conclude that in a simple animal such as a ctenophore, a sophisticated set of membrane conductances can compensate for the absence of an elaborate system of effectors. PMID:2448648

  18. Basic fibroblast growth factor: its role in the control of smooth muscle cell migration.

    PubMed Central

    Jackson, C. L.; Reidy, M. A.

    1993-01-01

    The formation of an intimal lesion in an injured artery is the consequence of the replication and migration of smooth muscle cells. Recent studies have implicated basic fibroblast growth factor (bFGF) as an important mediator of replication in the arterial media, and platelet-derived growth factor as an important mediator of migration. However, the degree of arterial trauma produced during injury has a significant influence on the time of onset of intimal thickening, suggesting that factors released from damaged smooth muscle cells may affect migration. We have investigated the role of one of these factors, bFGF, in smooth muscle cell migration in vivo. We found that 1) deendothelialization of the rat carotid artery results in significantly more migration when it is accompanied by traumatic injury to the underlying smooth muscle; 2) the rate of migration in arteries that have been gently deendothelialized is significantly stimulated by systemic injection of bFGF; and 3) inhibition of bFGF with a blocking antibody significantly reduces the amount of migration after traumatic deendothelializing injury with a balloon catheter. These findings suggest that bFGF plays an important role in the mediation of smooth muscle cell migration after arterial injury. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8213998

  19. Phenotypic and Functional Changes of Endothelial and Smooth Muscle Cells in Thoracic Aortic Aneurysms

    PubMed Central

    Malashicheva, Anna; Kostina, Daria; Kostina, Aleksandra; Irtyuga, Olga; Voronkina, Irina; Smagina, Larisa; Ignatieva, Elena; Gavriliuk, Natalia; Uspensky, Vladimir; Moiseeva, Olga; Vaage, Jarle; Kostareva, Anna

    2016-01-01

    Thoracic aortic aneurysm develops as a result of complex series of events that alter the cellular structure and the composition of the extracellular matrix of the aortic wall. The purpose of the present work was to study the cellular functions of endothelial and smooth muscle cells from the patients with aneurysms of the thoracic aorta. We studied endothelial and smooth muscle cells from aneurysms in patients with bicuspid aortic valve and with tricuspid aortic valve. The expression of key markers of endothelial (CD31, vWF, and VE-cadherin) and smooth muscle (SMA, SM22α, calponin, and vimentin) cells as well extracellular matrix and MMP activity was studied as well as and apoptosis and cell proliferation. Expression of functional markers of endothelial and smooth muscle cells was reduced in patient cells. Cellular proliferation, migration, and synthesis of extracellular matrix proteins are attenuated in the cells of the patients. We show for the first time that aortic endothelial cell phenotype is changed in the thoracic aortic aneurysms compared to normal aortic wall. In conclusion both endothelial and smooth muscle cells from aneurysms of the ascending aorta have downregulated specific cellular markers and altered functional properties, such as growth rate, apoptosis induction, and extracellular matrix synthesis. PMID:26904289

  20. Estradiol attenuates directed migration of vascular smooth muscle cells in vitro.

    PubMed Central

    Kolodgie, F. D.; Jacob, A.; Wilson, P. S.; Carlson, G. C.; Farb, A.; Verma, A.; Virmani, R.

    1996-01-01

    Although the cardiovascular benefits of the hormone estrogen are at least, in part, mediated by its antiproliferative effect on vascular smooth muscle, its action on the migration of these cells is unknown. To explore this relationship, female rat aortic smooth muscle cells were grown in hormone-free medium, and the effect of various concentrations of beta-estradiol on directed cellular migration was measured in vitro using a microwell Boyden chamber apparatus. Migration of smooth muscle cells to the known chemoattractants platelet-derived growth factor, insulin-like growth factor-1, and fibronectin (all at peak doses for migratory activity) was attenuated by beta-estradiol (0.5 to 10 ng/ml) in a concentration-dependent manner relative to control cells treated with vehicle (0.01% ethanol). This response was insensitive to pretreatment with indomethacin and was stereospecific (17 alpha-estradiol lacked response). Like beta-estradiol, the synthetic estrogen diethylstilbestrol attenuated directed smooth muscle cell migration whereas the male hormone testosterone was ineffective. Additional studies showed that beta-estradiol-mediated suppression of migration was inhibited by the anti-estrogen ICI 164,384 and the gene transcription inhibitor actinomycin D. These are the first results demonstrating a reduction in directed smooth muscle cell migration by beta-estradiol. The mechanism of this estrogen-mediated response appears to involve conventional estrogen receptors. PMID:8774151

  1. Captopril augments acetylcholine-induced bronchial smooth muscle contractions in vitro via kinin-dependent mechanisms.

    PubMed

    Agrawal, Naman; Akella, Aparna; Deshpande, Shripad B

    2016-06-01

    Angiotensin converting enzyme (ACE) inhibitors therapy is aassociated with bothersome dry cough as an adverse effect. The mechanisms underlying this adverse effect are not clear. Therefore, influence of captopril (an ACE inhibitor) on acetylcholine (ACh)-induced bronchial smooth muscle contractions was investigated. Further, the mechanisms underlying the captopril-induced changes were also explored. In vitro contractions of rat bronchial smooth muscle to cumulative concentrations of ACh were recorded before and after exposure to captopril. Further, the involvement of kinin and inositol triphosphate (IP₃) pathways for captopril-induced alterations were explored. ACh produced concentration-dependent (5-500 µM) increase in bronchial smooth muscle contractions. Pre-treatment with captopril augmented the ACh-induced contractions at each concentration significantly. Pre-treatment with aprotinin (kinin synthesis inhibitor) or heparin (inositol triphosphate, IP₃-inhibitor), blocked the captopril-induced augmentation of bronchial smooth muscle contractions evoked by ACh. Further, captopril-induced augmentation was absent in calcium-free medium. These results suggest that captopril sensitizes bronchial smooth muscles to ACh-induced contractions. This sensitization may be responsible for dry cough associated with captopril therapy. PMID:27468462

  2. Inhibition of the Ca sup 2+ -ATPase of vascular smooth muscle sarcoplasmic reticulum by superoxide radicals

    SciTech Connect

    Suzuki, Yuichiro; Ford, G.D. )

    1991-03-15

    The effect of oxygen free radicals generated by hypoxanthine plus xanthine oxidase on the Ca{sup 2+}-ATPase of sarcoplasmic reticulum from bovine aortic smooth muscle were studied. Exogenous hypoxanthine plus xanthine oxidase produced an hypoxanthine concentration dependent inhibition of the Ca{sup 2+}-ATPase. The inhibition could be completely blocked by superoxide dismutase but not by either mannitol or deferoxamine. Direct addition of reagent hydrogen peroxide in the {mu}M range did not cause significant inhibition. These results suggest that superoxide is the primary damaging species. Additionally, 1.16 {plus minus} 0.17 mU/g wet wt of xanthine oxidase activity were detected in the post-nuclear supernatant of bovine aortic smooth muscle, suggesting the existence of a possible intracellular source of superoxide. This value was calculated to be approximately 5 mU/ml by using a usual value of vascular smooth muscle cellular volume. Thus the level of endogenous xanthine oxidase resident in vascular smooth muscle is comparable with the level of exogenous xanthine oxidase used in the present study. These findings suggest a potential role of xanthine oxidase-generated superoxide in free radical injury to vascular smooth muscle.

  3. Influences on vascular wall smooth muscle cells with novel short-duration thermal angioplasty

    NASA Astrophysics Data System (ADS)

    Kunio, M.; Shimazaki, N.; Arai, T.; Sakurada, M.

    2012-02-01

    We investigated the influences on smooth muscle cells after our novel short-duration thermal angioplasty, Photo-thermo Dynamic Balloon Angioplasty (PTDBA), to reveal the mechanism that can suppress neo-intimal hyperplasia after PTDBA. We obtained the sufficient arterial dilatations by short-duration heating (<=15 s, <70°C) and low dilatation pressure (<0.4 MPa) without arterial injuries in our previous in vivo studies. Smooth muscle cells, which play most important role in chronic treatment effects, were heated during PTDBA and stretch-fixed after PTDBA. The dead cell rate by heating, estimated by Arrhenius equation with A=2.5x1016 s-1 and Ea=1.17×105 J mol-1, was 15.7+/-2.2% after PTDBA. The measured deformation rate of smooth muscle cells' nuclei was 1.6+/-0.1 after PTDBA in vivo. We found that the expression of smooth muscle cells' growth factor after PTDBA was inhibited 0.52 fold compared to that after the conventional balloon angioplasty in vivo. The measured neo-intimal hyperplasia occupancy rate was less than 20% after PTDBA in vivo. We prospect that the inhibition of the growth factor's expression by stretch-fixing may result to suppress the neo-intimal hyperplasia. In addition, the decrease of smooth muscle cells' density in the vessel media by heating might be another reason for the neo-intimal hyperplasia suppression.

  4. Urinary Bladder Smooth Muscle Engineered from Adipose Stem Cells and a Three Dimensional Synthetic Composite

    PubMed Central

    Jack, Gregory S.; Zhang, Rong; Lee, Min; Xu, Yuhan; Wu, Ben; Rodríguez, Larissa V.

    2009-01-01

    Human adipose stem cells were cultured in smooth muscle inductive media and seeded into synthetic bladder composites to tissue engineer bladder smooth muscle. 85:15 poly-lactic-glycolic acid bladder dome composites were cast using an electropulled microfiber luminal surface combined with an outer porous sponge. Cell seeded bladders expressed smooth muscle actin, myosin heavy chain, calponinin, and caldesmon via RT-PCR and immunoflourescence. Nude rats (n=45) underwent removal of half their bladder and repair using: (i) augmentation with the adipose stem cell seeded composites, (ii) augmentation with a matched acellular composite, or (iii) suture closure. Animals were followed for 12 weeks post-implantation and bladders were explanted serially. Results showed that bladder capacity and compliance were maintained in the cell seeded group throughout the 12 weeks, but deteriorated in the acellular scaffold group sequentially with time. Control animals repaired with sutures regained their baseline bladder capacities by week 12, demonstrating a long term limitation of this model. Histological analysis of explanted materials demonstrated viable adipose stem cells and increasing smooth muscle mass in the cell seeded scaffolds with time. Tissue bath stimulation demonstrated smooth muscle contraction of the seeded implants but not the acellular implants after 12 weeks in vivo. Our study demonstrates the feasibility and short term physical properties of bladder tissue engineered from adipose stem cells. PMID:19345408

  5. Hypertrophy and hyperplasia of smooth muscle cells of small intramyocardial arteries in spontaneously hypertensive rats.

    PubMed

    Amann, K; Gharehbaghi, H; Stephen, S; Mall, G

    1995-01-01

    Hearts of stroke-prone spontaneously hypertensive rats (SHR) were investigated by means of stereology and were compared with those of normotensive. Wistar-Kyoto controls. At the age of 9 months, hypertensive rats showed cardiac hypertrophy, marked myocardial fibrosis, activation of nonvascular interstitium, focal myocytial degeneration, reduction of capillarization, and microarteriopathy of small intramyocardial arteries. Stereologically, a significant increase in the total left ventricular arterial wall volume (+180% versus controls) was found in SHR hearts. By using new stereological techniques, the orientator and the nucleator, we investigated whether this significant increase in total left ventricular arterial wall volume was due to hyperplasia of smooth muscle cells in addition to the process of vascular smooth muscle cell hypertrophy that is common in SHR. Additionally, the nuclear size and ratio of cell volume to nuclear volume were determined using another new stereological technique, the selector. The stereological data indicate a significant increase in mean cell and nuclear volumes as well as in the total number of left ventricular arterial smooth muscle cells of SHR. Additionally, the total length of intramyocardial arteries was also significantly increased in hypertensive rats. The volume and number of arterial smooth muscle cells per arterial length were significantly (P < .001 and P < .05, respectively) higher in SHR than in normotensive controls. Thus, we conclude that hypertrophy and hyperplasia of smooth muscle cells are involved in intramyocardial arterial growth processes in hypertensive heart remodeling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7843743

  6. Phenotypic and Functional Changes of Endothelial and Smooth Muscle Cells in Thoracic Aortic Aneurysms.

    PubMed

    Malashicheva, Anna; Kostina, Daria; Kostina, Aleksandra; Irtyuga, Olga; Voronkina, Irina; Smagina, Larisa; Ignatieva, Elena; Gavriliuk, Natalia; Uspensky, Vladimir; Moiseeva, Olga; Vaage, Jarle; Kostareva, Anna

    2016-01-01

    Thoracic aortic aneurysm develops as a result of complex series of events that alter the cellular structure and the composition of the extracellular matrix of the aortic wall. The purpose of the present work was to study the cellular functions of endothelial and smooth muscle cells from the patients with aneurysms of the thoracic aorta. We studied endothelial and smooth muscle cells from aneurysms in patients with bicuspid aortic valve and with tricuspid aortic valve. The expression of key markers of endothelial (CD31, vWF, and VE-cadherin) and smooth muscle (SMA, SM22α, calponin, and vimentin) cells as well extracellular matrix and MMP activity was studied as well as and apoptosis and cell proliferation. Expression of functional markers of endothelial and smooth muscle cells was reduced in patient cells. Cellular proliferation, migration, and synthesis of extracellular matrix proteins are attenuated in the cells of the patients. We show for the first time that aortic endothelial cell phenotype is changed in the thoracic aortic aneurysms compared to normal aortic wall. In conclusion both endothelial and smooth muscle cells from aneurysms of the ascending aorta have downregulated specific cellular markers and altered functional properties, such as growth rate, apoptosis induction, and extracellular matrix synthesis. PMID:26904289

  7. Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.

    PubMed

    Nair, Dileep G; Han, T Y; Lourenssen, S; Blennerhassett, Michael G

    2011-05-01

    Intestinal inflammation causes an increased intestinal wall thickness, in part, due to the proliferation of smooth muscle cells, which impairs the contractile phenotype elsewhere. To study this, cells from the circular muscle layer of the rat colon (CSMC) were isolated and studied, both in primary culture and after extended passage, using quantitative PCR, Western blot analysis, and immunocytochemistry. By 4 days in vitro, both mRNA and protein for the smooth muscle marker proteins α-smooth muscle actin, desmin, and SM22-α were reduced by >50%, and mRNA for cyclin D1 was increased threefold, evidence for modulation to a proliferative phenotype. Continued growth caused significant further decrease in expression, evidence that phenotypic loss in CSMC was proportional to the extent of proliferation. In CSMC isolated at day 2 of trinitrobenzene sulfonic acid-induced colitis, flow cytometry and Western blotting showed that these differentiated markers were reduced in mitotic CSMC, while similar to control in nonmitotic CSMC. By day 35 post-trinitrobenzene sulfonic acid, when inflammation has resolved, CSMC were hypertrophic, but, nonetheless, showed markedly decreased expression of smooth muscle protein markers per cell. In vitro, day 35 CSMC displayed an accelerated loss of phenotype and increased thymidine uptake in response to serum or PDGF-BB. Furthermore, carbachol-induced expression of phospho-AKT (a marker of cholinergic response) was lost from day 35 CSMC in vitro, while retained in control cells. Therefore, proliferation reduces the expression of smooth-muscle-specific markers in CSMC, possibly leading to altered contractility. However, inflammation-induced proliferation in vivo also causes lasting changes that include unexpected priming for an exaggerated response to proliferative stimuli. Identification of the molecular mechanisms of intestinal smooth muscle cell phenotypic modulation will be helpful in reducing the detrimental effects of inflammation. PMID

  8. Cigarette Smoke Modulates Vascular Smooth Muscle Phenotype: Implications for Carotid and Cerebrovascular Disease

    PubMed Central

    Jabbour, Pascal M.; Tjoumakaris, Stavropoula I.; Gonzalez, Fernando; Hasan, David M.; Rosenwasser, Robert H.; Owens, Gary K.; Koch, Walter J.; Dumont, Aaron S.

    2013-01-01

    Background The role of smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation and pathogenesis of stroke has not been determined. Cigarette smoke is a major risk factor for atherosclerosis, but potential mechanisms are unclear, and its role in SMC phenotypic modulation has not been established. Methods and Results In cultured cerebral vascular SMCs, exposure to cigarette smoke extract (CSE) resulted in decreased promoter activity and mRNA expression of key SMC contractile genes (SM-α-actin, SM-22α, SM-MHC) and the transcription factor myocardin in a dose-dependent manner. CSE also induced pro-inflammatory/matrix remodeling genes (MCP-1, MMPs, TNF-α, IL-1β, NF-κB). CSE increased expression of KLF4, a known regulator of SMC differentiation, and siKLF4 inhibited CSE induced suppression of SMC contractile genes and myocardin and activation of inflammatory genes. These mechanisms were confirmed in vivo following exposure of rat carotid arteries to CSE. Chromatin immune-precipitation assays in vivo and in vitro demonstrated that CSE promotes epigenetic changes with binding of KLF4 to the promoter regions of myocardin and SMC marker genes and alterations in promoter acetylation and methylation. Conclusion CSE exposure results in phenotypic modulation of cerebral SMC through myocardin and KLF4 dependent mechanisms. These results provides a mechanism by which cigarette smoke induces a pro-inflammatory/matrix remodeling phenotype in SMC and an important pathway for cigarette smoke to contribute to atherosclerosis and stroke. PMID:23967268

  9. Diffuse and uncontrolled vascular smooth muscle cell proliferation in rapidly progressing pediatric moyamoya disease.

    PubMed

    Reid, Amy J; Bhattacharjee, Meenakshi B; Regalado, Ellen S; Milewicz, Allen L; El-Hakam, Lisa M; Dauser, Robert C; Milewicz, Dianna M

    2010-09-01

    Moyamoya disease is a rare stroke syndrome of unknown etiology resulting from stenosis or occlusion of the supraclinoid internal carotid artery (ICA) in association with an abnormal vascular network in the basal ganglia. Although the highest incidence of moyamoya disease is in pediatric patients, pathology reports have been primarily limited to adult samples and describe occlusive fibrocellular lesions in the intimae of affected arteries. We describe the case of a young girl with primary moyamoya disease who presented at 18 months of age with right hemiparesis following an ischemic stroke. Angiography showed stenosis of the distal left ICA, left middle cerebral artery, and right ICA. An emergent left-sided dural inversion was performed. Recurrent strokes and alternating hemiplegia necessitated a right dural inversion 6 months later. Nonetheless, her aggressive disease proved uniquely refractory to surgical revascularization, and she succumbed to recurrent strokes and neurological deterioration at 2.5 years of age. Pathological specimens revealed a striking bilateral occlusion of the anterior carotid circulation resulting from intimal proliferation of smooth muscle cells (SMCs). Most strikingly, the ascending aorta and the superior mesenteric artery demonstrated similar intimal proliferation, along with SMC proliferation in the media. The systemic pathology involving multiple arteries in this extremely young child, the first case of its kind available for autopsy, suggests that globally uncontrolled SMC proliferation, in the absence of environmental risk factors and likely resulting from an underlying genetic alteration, may be a primary etiologic event leading to moyamoya disease. PMID:20809708

  10. Isolation and characterization of the inositol trisphosphate receptor from smooth muscle

    SciTech Connect

    Chadwick, C.C.; Saito, A.; Fleischer, S. )

    1990-03-01

    The release of Ca{sup 2+} from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca{sup 2+} release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP{sub 3}) is believed to mobilize Ca{sup 2+} from internal stores and thereby modulate contraction. The authors describe the isolation of an IP{sub 3} receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, and the IP{sub 3} receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The receptor is an oligomer of a single polypeptide with a M{sub r} of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of {approx}250 {times} 250 {angstrom} with fourfold symmetry. The IP{sub 3} receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding.

  11. Mechanical properties of the rabbit iris smooth muscles.

    PubMed

    Yamaji, Kazutsuna; Yoshitomi, Takeshi; Usui, Shiro; Ohnishi, Yoshitaka

    2003-02-01

    The study focuses on obtaining the visco-elastic properties of the iris sphincter and dilator muscles. Two kinds of experiments were performed: the isometric contraction experiment and the isotonic quick release experiment. The length-tension relationship was obtained from the former experiment. This relationship clarified the contribution of each muscle in determining the statics of the pupil. The viscous and serial elastic properties were obtained from the latter experiment. The viscosity could be expressed by the expanded Hill's equation as a function of velocity and contractile tension. We argue that serial elasticity is independent of contractile tension. These properties provide insights into the pupillary mechanism. PMID:12536003

  12. Ranolazine inhibits voltage-gated mechanosensitive sodium channels in human colon circular smooth muscle cells.

    PubMed

    Neshatian, Leila; Strege, Peter R; Rhee, Poong-Lyul; Kraichely, Robert E; Mazzone, Amelia; Bernard, Cheryl E; Cima, Robert R; Larson, David W; Dozois, Eric J; Kline, Crystal F; Mohler, Peter J; Beyder, Arthur; Farrugia, Gianrico

    2015-09-15

    Human jejunum smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs) express the SCN5A-encoded voltage-gated, mechanosensitive sodium channel NaV1.5. NaV1.5 contributes to small bowel excitability, and NaV1.5 inhibitor ranolazine produces constipation by an unknown mechanism. We aimed to determine the presence and molecular identity of Na(+) current in the human colon smooth muscle and to examine the effects of ranolazine on Na(+) current, mechanosensitivity, and smooth muscle contractility. Inward currents were recorded by whole cell voltage clamp from freshly dissociated human colon SMCs at rest and with shear stress. SCN5A mRNA and NaV1.5 protein were examined by RT-PCR and Western blots, respectively. Ascending human colon strip contractility was examined in a muscle bath preparation. SCN5A mRNA and NaV1.5 protein were identified in human colon circular muscle. Freshly dissociated human colon SMCs had Na(+) currents (-1.36 ± 0.36 pA/pF), shear stress increased Na(+) peaks by 17.8 ± 1.8% and accelerated the time to peak activation by 0.7 ± 0.3 ms. Ranolazine (50 μM) blocked peak Na(+) current by 43.2 ± 9.3% and inhibited shear sensitivity by 25.2 ± 3.2%. In human ascending colon strips, ranolazine decreased resting tension (31%), reduced the frequency of spontaneous events (68%), and decreased the response to smooth muscle electrical field stimulation (61%). In conclusion, SCN5A-encoded NaV1.5 is found in human colonic circular smooth muscle. Ranolazine blocks both peak amplitude and mechanosensitivity of Na(+) current in human colon SMCs and decreases contractility of human colon muscle strips. Our data provide a likely mechanistic explanation for constipation induced by ranolazine. PMID:26185330

  13. Assays for in vitro monitoring of proliferation of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cells.

    PubMed

    Goncharova, Elena A; Lim, Poay; Goncharov, Dmitry A; Eszterhas, Andrew; Panettieri, Reynold A; Krymskaya, Vera P

    2006-01-01

    Vascular and airway remodeling, which are characterized by airway smooth muscle (ASM) and pulmonary arterial vascular smooth muscle (VSM) proliferation, contribute to the pathology of asthma, pulmonary hypertension, restenosis and atherosclerosis. To evaluate the proliferation of VSM and ASM cells in response to mitogens, we perform a [3H]thymidine incorporation assay. The proliferation protocol takes approximately 48 h and includes stimulating cells synchronized in G0/G1 phase of the cell cycle with agonists, labeling cells with [3H]thymidine and examining levels of [3H]thymidine incorporation by scintillation counting. Although using radiolabeled [3H]thymidine incorporation is a limitation, the greatest benefit of the assay is providing reliable and statistically significant data. PMID:17406550

  14. Stretch activates myosin light chain kinase in arterial smooth muscle

    SciTech Connect

    Barany, K.; Rokolya, A.; Barany, M. )

    1990-11-30

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol (32P)phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the (32P)phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in (32P)phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

  15. Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension

    PubMed Central

    An, Changlong; Bhetwal, Bhupal P.; Sanders, Kenton M.; Somlyo, Avril V.; Perrino, Brian A.

    2015-01-01

    Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to

  16. Cannabinoid CB{sub 1} receptor inhibition decreases vascular smooth muscle migration and proliferation

    SciTech Connect

    Rajesh, Mohanraj; Mukhopadhyay, Partha; Hasko, Gyoergy; Pacher, Pal

    2008-12-26

    Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli and chemoattractants such as platelet-derived growth factor (PDGF) are key events in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation and migration in various cell types through cannabinoid receptors. Here we investigated the effects of CB{sub 1} receptor antagonist rimonabant (SR141716A), which has recently been shown to have anti-atherosclerotic effects both in mice and humans, on PDGF-induced proliferation, migration, and signal transduction of human coronary artery smooth muscle cells (HCASMCs). PDGF induced Ras and ERK 1/2 activation, while increasing proliferation and migration of HCASMCs, which were dose dependently attenuated by CB{sub 1} antagonist, rimonabant. These findings suggest that in addition to improving plasma lipid alterations and decreasing inflammatory cell migration and inflammatory response, CB{sub 1} antagonists may exert beneficial effects in atherosclerosis and restenosis by decreasing vascular smooth muscle proliferation and migration.

  17. Smooth muscle relaxant activity of Crocus sativus (saffron) and its constituents: possible mechanisms.

    PubMed

    Mokhtari-Zaer, Amin; Khazdair, Mohammad Reza; Boskabady, Mohammad Hossein

    2015-01-01

    Saffron, Crocus sativus L. (C. sativus) is rich in carotenoids and used in traditional medicine for treatment of various conditions such as coughs, stomach disorders, amenorrhea, asthma and cardiovascular disorders. These therapeutic effects of the plant are suggested to be due to its relaxant effect on smooth muscles. The effect of C. sativus and its constituents on different smooth muscles and the underlying mechanisms have been studied. Several studies have shown the relaxant effects of C. sativus and its constituents including safranal, crocin, crocetin and kaempferol on blood vessels. In addition, it was reported that saffron stigma lowers systolic blood pressure. The present review highlights the relaxant effects of C. sativus and its constituents on various smooth muscles. The possible mechanisms of this relaxing effect including activation of ß2-adrenoceptors, inhibition of histamine H1 and muscarinic receptors and calcium channels and modulation of nitric oxide (NO) are also reviewed. PMID:26468456

  18. Smooth muscle relaxant activity of Crocus sativus (saffron) and its constituents: possible mechanisms

    PubMed Central

    Mokhtari-Zaer, Amin; Khazdair, Mohammad Reza; Boskabady, Mohammad Hossein

    2015-01-01

    Saffron, Crocus sativus L. (C. sativus) is rich in carotenoids and used in traditional medicine for treatment of various conditions such as coughs, stomach disorders, amenorrhea, asthma and cardiovascular disorders. These therapeutic effects of the plant are suggested to be due to its relaxant effect on smooth muscles. The effect of C. sativus and its constituents on different smooth muscles and the underlying mechanisms have been studied. Several studies have shown the relaxant effects of C. sativus and its constituents including safranal, crocin, crocetin and kaempferol on blood vessels. In addition, it was reported that saffron stigma lowers systolic blood pressure. The present review highlights the relaxant effects of C. sativus and its constituents on various smooth muscles. The possible mechanisms of this relaxing effect including activation of ß2-adrenoceptors, inhibition of histamine H1 and muscarinic receptors and calcium channels and modulation of nitric oxide (NO) are also reviewed. PMID:26468456

  19. The role of mechanotransduction on vascular smooth muscle myocytes cytoskeleton and contractile function

    PubMed Central

    Ye, George J.C.; Nesmith, Alexander P.; Parker, Kevin Kit

    2016-01-01

    Smooth muscle exhibits a highly organized structural hierarchy that extends over multiple spatial scales to perform a wide range of functions at the cellular, tissue, and organ levels. Early efforts primarily focused on understanding vascular smooth muscle function through biochemical signaling. However, accumulating evidence suggests that mechanotransduction, the process through which cells convert mechanical stimuli into biochemical cues, is requisite for regulating contractility. Cytoskeletal proteins that comprise the extracellular, intercellular, and intracellular domains are mechanosensitive and can remodel their structure and function in response to external mechanical cues. Pathological stimuli such as malignant hypertension can act through the same mechanotransductive pathways to induce maladaptive remodeling, leading to changes in cellular shape and loss of contractile function. In both health and disease, the cytoskeletal architecture integrates the mechanical stimuli and mediates structural and functional remodeling in the vascular smooth muscle. PMID:25125187

  20. Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions.

    PubMed

    Wolfbauer, G; Glick, J M; Minor, L K; Rothblat, G H

    1986-10-01

    A possible mechanism for the formation of smooth muscle foam cells in the atherosclerotic lesion was explored. Cultured macrophages (J774 cell line) were induced to form cytoplasmic cholesteryl ester inclusions by exposure to acetylated low density lipoprotein in the presence of cholesterol-rich phospholipid dispersions. The macrophages were disrupted by brief sonication, and the inclusions were isolated by flotation. When these inclusions were placed in direct contact with cultured smooth muscle cells, cellular uptake of the inclusions in a time- and dose-dependent manner was observed. Light and electron microscopy indicated the presence of lipid inclusions throughout the cytoplasm of the cells. Uptake of inclusion lipid by the smooth muscle cells was inhibited by several metabolic inhibitors, indicating that the process is dependent on metabolic activity. A modest but significant hydrolysis of the cholesteryl ester was observed, showing that the stored cholesteryl esters are metabolically available. PMID:3020555

  1. Low density lipoprotein uptake by an endothelial-smooth muscle cell bilayer

    SciTech Connect

    Alexander, J.J.; Miguel, R.; Graham, D. )

    1991-03-01

    To study the interaction of endothelial and smooth muscle cells, and the means by which such interaction may affect lipid permeability of the arterial wall, cell bilayers were established by use of a transwell culture system. After confluent growth of both cell types had been achieved, iodine 125 bound to low-density lipoprotein (10 ng protein/ml) was added to the media of the upper well. After a 3-hour incubation period, the iodine 125-bound low-density lipoprotein content of the upper and lower media demonstrated an impedance to lipoprotein movement across the endothelial cell monolayer as compared to the bare porous polycarbonate filter of the transwell (p less than 10(-6)). The presence of smooth muscle cells in the bottom well significantly enhanced the permeability of the endothelial cell layer (p less than 10(-60)). This effect remained unchanged over a 9-day time course. Membrane binding and cellular uptake of low-density lipoprotein by endothelial cells was not altered by smooth muscle cells, indicating that this change in permeability could not be easily attributed to changes in receptor-mediated transport or transcytosis. Membrane binding (p less than 0.02) and cellular uptake (p less than 10(-6)) of low-density lipoprotein by smooth muscle cells in the bilayer, when adjusted for counts available in the smooth muscle cell media, were both reduced in the early incubation period as compared to isolated smooth muscle cells. The disproportionate reduction in uptake as compared to binding would suggest that this was not entirely a receptor-dependent process.

  2. Augmented Vascular Smooth Muscle Cell Stiffness and Adhesion when Hypertension is Superimposed on Aging

    PubMed Central

    Sehgel, Nancy L.; Sun, Zhe; Hong, Zhongkui; Hunter, William C.; Hill, Michael A.; Vatner, Dorothy E.; Vatner, Stephen F.; Meininger, Gerald A.

    2014-01-01

    Hypertension and aging are both recognized to increase aortic stiffness, but their interactions are not completely understood. Most prior studies have attributed increased aortic stiffness to changes in extracellular matrix proteins that alter mechanical properties of the vascular wall. Alternatively, we hypothesized that a significant component of increased vascular stiffness in hypertension is due to changes in the mechanical and adhesive properties of vascular smooth muscle cells, and that aging would augment the contribution from vascular smooth muscle cells compared to the extracellular matrix. Accordingly, we studied aortic stiffness in young (16 wks) and old (64 wks) spontaneously hypertensive rats and Wistar-Kyoto wild-type controls. Systolic and pulse pressures were significantly increased in young spontaneously hypertensive rats, compared to young Wistar-Kyoto rats, and these continued to rise in old spontaneously hypertensive rats, compared to age-matched controls. Excised aortic ring segments exhibited significantly greater elastic moduli in both young and old spontaneously hypertensive rats vs. Wistar-Kyoto rats. Vascular smooth muscle cells were isolated from the thoracic aorta, and stiffness and adhesion to fibronectin were measured by atomic force microscopy. Hypertension increased both vascular smooth muscle cell stiffness and vascular smooth muscle cell adhesion, and these increases were both augmented with aging. By contrast, hypertension did not affect histological measures of aortic collagen and elastin, which were predominantly changed by aging. This supports the concept that stiffness and adhesive properties of vascular smooth muscle cells are novel mechanisms contributing to the increased aortic stiffness occurring with hypertension superimposed on aging. PMID:25452471

  3. In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells.

    PubMed

    Zaniboni, Andrea; Bernardini, Chiara; Bertocchi, Martina; Zannoni, Augusta; Bianchi, Francesca; Avallone, Giancarlo; Mangano, Chiara; Sarli, Giuseppe; Calzà, Laura; Bacci, Maria Laura; Forni, Monica

    2015-09-01

    Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes. PMID:26135800

  4. A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

    PubMed

    Turner, Elizabeth C; Huang, Chien-Ling; Sawhney, Neha; Govindarajan, Kalaimathi; Clover, Anthony J P; Martin, Kenneth; Browne, Tara C; Whelan, Derek; Kumar, Arun H S; Mackrill, John J; Wang, Shaohua; Schmeckpeper, Jeffrey; Stocca, Alessia; Pierce, William G; Leblond, Anne-Laure; Cai, Liquan; O'Sullivan, Donnchadh M; Buneker, Chirlei K; Choi, Janet; MacSharry, John; Ikeda, Yasuhiro; Russell, Stephen J; Caplice, Noel M

    2016-05-01

    Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC.  In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement.  PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368. PMID:26840832

  5. Myosin light chain phosphorylation in contraction of gastric antral smooth muscle from neonate and adult rabbits.

    PubMed

    Ierardi, J A; Paul, D A; Ryan, J P

    1996-01-01

    The decreased contractility of gastric antral smooth muscle in the neonate has been attributed to reduced levels of activator calcium. It is generally accepted that calcium-dependent myosin light chain phosphorylation (MLCP) is the key step in the initiation of force development in smooth muscle. In this study, we investigated the relationship between MLCP and force development in gastric antral smooth muscle from neonatal (4-6 d old) and adult rabbits. We tested the hypothesis that the reduced force development of circular smooth muscle from the neonate would be accompanied by decreased levels of MLCP, as compared with data from adult animals. Full thickness muscle strips oriented parallel to the circular muscle layer were examined for their contractile response to acetylcholine (ACh) (10(-8) M to 10(-3) M) or 10(-4) M ACh only. In the latter study, tissues were rapidly frozen in a dry ice-acetone slurry for subsequent MLCP determination. MLCP was determined at times corresponding to 5, 10, 15, 30, and 60 s of stimulation. For each age group, maximal active force developed at an ACh concentration of 10(-4) M and was significantly greater in tissues from adults (1.86 +/- 0.24 N/m2, adult; 0.95 +/- 0.05 N/m2, neonate; p < 0.05). In contrast, no significant differences were observed with respect to basal or agonist-stimulated levels of MLCP. The data suggest that factors other than levels of MLCP contribute to the reduced force-generating capacity of antral smooth muscle from the neonate. PMID:8825402

  6. Induced Pluripotent Stem Cell-derived Vascular Smooth Muscle Cells: Methods and Application

    PubMed Central

    Dash, Biraja C.; Jiang, Zhengxin; Suh, Carol; Qyang, Yibing

    2015-01-01

    Vascular smooth muscle cells (VSMCs) play a major role in the pathophysiology of cardiovascular diseases. The advent of induced pluripotent stem cell (iPSC) technology and their capability to differentiation into virtually every cell type in the human body make this field a ray of hope for vascular regenerative therapy and for understanding disease mechanism. In this review, we first discuss the recent iPSC technology and vascular smooth muscle development from embryo and then examine different methodology to derive VSMCs from iPSCs and their applications in regenerative therapy and disease modeling. PMID:25559088

  7. [Influence of nanosize particles of cobalt ferrite on contractile responses of smooth muscle segment of airways].

    PubMed

    Kapilevich, L V; Zaĭtseva, T N; Nosarev, A V; D'iakova, E Iu; Petlina, Z R; Ogorodova, L M; Ageev, B G; Magaeva, A A; Itin, V I; Terekhova, O G; Medvedev, M A

    2012-02-01

    Contractile responses of airways segments of porpoises inhaling nanopowder CoFe2O4 were stidued by means of a mechanographic method. Inhalation of the nanosize particles of CoFe2O4 in vivo and in vitro testing the nanomaterial on isolated smooth muscles led to potentiation histaminergic, cholinergic contractile activity in airways of porpoises and to strengthening of adrenergic relaxing answers. Nanosize particles vary amplitude of hyperpotassium reductions in smooth muscle segments of airways similarly to the effect of depolymerizing drug colchicine. PMID:22650066

  8. Thrombospondin-1 limits ischemic tissue survival by inhibiting nitric oxide–mediated vascular smooth muscle relaxation

    PubMed Central

    Isenberg, Jeff S.; Hyodo, Fuminori; Matsumoto, Ken-Ichiro; Romeo, Martin J.; Abu-Asab, Mones; Tsokos, Maria; Kuppusamy, Periannan; Wink, David A.; Krishna, Murali C.

    2007-01-01

    The nitric oxide (NO)/cGMP pathway, by relaxing vascular smooth muscle cells, is a major physiologic regulator of tissue perfusion. We now identify thrombospondin-1 as a potent antagonist of NO for regulating F-actin assembly and myosin light chain phosphorylation in vascular smooth muscle cells. Thrombospondin-1 prevents NO-mediated relaxation of precontracted vascular smooth muscle cells in a collagen matrix. Functional magnetic resonance imaging demonstrated that an NO-mediated increase in skeletal muscle perfusion was enhanced in thrombospondin-1–null relative to wild-type mice, implicating endogenous thrombospondin-1 as a physiologic antagonist of NO-mediated vasodilation. Using a random myocutaneous flap model for ischemic injury, tissue survival was significantly enhanced in thrombospondin-1–null mice. Improved flap survival correlated with increased recovery of oxygen levels in the ischemic tissue of thrombospondin-1–null mice as measured by electron paramagnetic resonance oximetry. These findings demonstrate an important antag-onistic relation between NO/cGMP signaling and thrombospondin-1 in vascular smooth muscle cells to regulate vascular tone and tissue perfusion. PMID:17082319

  9. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  10. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells

    PubMed Central

    Brun, Juliane; Lutz, Katrin A.; Neumayer, Katharina M. H.; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K.; Hart, Melanie L.

    2015-01-01

    The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1–2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

  11. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

    PubMed

    Brun, Juliane; Lutz, Katrin A; Neumayer, Katharina M H; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

    2015-01-01

    The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

  12. Embracing change: striated-for-smooth muscle replacement in esophagus development.

    PubMed

    Krauss, Robert S; Chihara, Daisuke; Romer, Anthony I

    2016-01-01

    The esophagus functions to transport food from the oropharyngeal region to the stomach via waves of peristalsis and transient relaxation of the lower esophageal sphincter. The gastrointestinal tract, including the esophagus, is ensheathed by the muscularis externa (ME). However, while the ME of the gastrointestinal tract distal to the esophagus is exclusively smooth muscle, the esophageal ME of many vertebrate species comprises a variable amount of striated muscle. The esophageal ME is initially composed only of smooth muscle, but its developmental maturation involves proximal-to-distal replacement of smooth muscle with striated muscle. This fascinating phenomenon raises two important questions: what is the developmental origin of the striated muscle precursor cells, and what are the cellular and morphogenetic mechanisms underlying the process? Studies addressing these questions have provided controversial answers. In this review, we discuss the development of ideas in this area and recent work that has shed light on these issues. A working model has emerged that should permit deeper understanding of the role of ME development and maturation in esophageal disorders and in the functional and evolutionary underpinnings of the variable degree of esophageal striated myogenesis in vertebrate species. PMID:27504178

  13. Conditional deletion of the relaxin receptor gene in cells of smooth muscle lineage affects lower reproductive tract in pregnant mice.

    PubMed

    Kaftanovskaya, Elena M; Huang, Zaohua; Lopez, Carolina; Conrad, Kirk; Agoulnik, Alexander I

    2015-04-01

    Relaxin hormone secreted into the circulation during pregnancy was discovered through its effects on pubic symphysis relaxation and parturition. Genetic inactivation of the relaxin gene or its cognate relaxin family peptide receptor 1 (RXFP1) in mice caused failure of parturition and mammary nipple enlargement, as well as increased collagen fiber density in the cervix and vagina. However, the relaxin effect on discrete cells and tissues has yet to be determined. Using transgenic mice with a knockin LacZ reporter in the Rxfp1 allele, we showed strong expression of this gene in vaginal and cervical stromal cells, as well as pubic ligament cells. We produced a floxed Rxfp1 allele that was used in combination with the Tagln-cre transgene to generate mice with a smooth muscle-specific gene knockout. In pregnant females, the ROSA26 reporter activated by Tagln-cre was detected in smooth muscle cells of the cervix, vagina, uterine artery, and in cells of the pubic symphysis. In late pregnant females with conditional gene ablation, the length of pubic symphysis was significantly reduced compared with wild-type or heterozygous Rxfp1(+/-) females. Denser collagen content was revealed by Masson trichrome staining in reproductive tract organs, uterine artery, and pubic symphysis. The cervical and vaginal epithelium was less developed than in heterozygous or wild-type females, although nipple size was normal and the dams were able to nurse their pups. In summary, our data indicate that relaxin/RXFP1 signaling in smooth muscle cells is important for normal collagen turnover and relaxation of the pubic symphysis during pregnancy. PMID:25715795

  14. Nitric Oxide-mediated Relaxation by High K in Human Gastric Longitudinal Smooth Muscle.

    PubMed

    Kim, Young Chul; Choi, Woong; Yun, Hyo-Young; Sung, Rohyun; Yoo, Ra Young; Park, Seon-Mee; Yun, Sei Jin; Kim, Mi-Jung; Song, Young-Jin; Xu, Wen-Xie; Lee, Sang Jin

    2011-12-01

    This study was designed to elucidate high-K(+)induced response of circular and longitudinal smooth muscle from human gastric corpus using isometric contraction. Contraction from circular and longitudinal muscle stripes of gastric corpus greater curvature and lesser curvature were compared. Circular smooth muscle from corpus greater curvature showed high K(+) (50 mM)-induced tonic contraction. On the contrary, however, longitudinal smooth muscle strips showed high K(+) (50 mM)-induced sustained relaxation. To find out the reason for the discrepancy we tested several relaxation mechanisms. Protein kinase blockers like KT5720, PKA inhibitor, and KT5823, PKG inhibitor, did not affect high K(+)-induced relaxation. K(+) channel blockers like tetraethylammonium (TEA), apamin (APA), glibenclamide (Glib) and barium (Ba(2+)) also had no effect. However, N(G)-nitro-L-arginine (L-NNA) and 1H-(1,2,4) oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC) and 4-AP (4-aminopyridine), voltage-dependent K(+) channel (K(V)) blocker, inhibited high K(+)-induced relaxation, hence reversing to tonic contraction. High K(+)-induced relaxation was observed in gastric corpus of human stomach, but only in the longitudinal muscles from greater curvature not lesser curvature. L-NNA, ODQ and K(V) channel blocker sensitive high K(+)-induced relaxation in longitudinal muscle of higher portion of corpus was also observed. These results suggest that longitudinal smooth muscle from greater curvature of gastric corpus produced high K(+)-induced relaxation which was activated by NO/sGC pathway and by K(V) channel dependent mechanism. PMID:22359479

  15. Orai channel-mediated Ca2+ signals in vascular and airway smooth muscle.

    PubMed

    Spinelli, Amy M; Trebak, Mohamed

    2016-03-15

    Orai (Orai1, Orai2, and Orai3) proteins form a family of highly Ca(2+)-selective plasma membrane channels that are regulated by stromal-interacting molecules (STIM1 and STIM2); STIM proteins are Ca(2+) sensors located in the membrane of the endoplasmic reticulum. STIM and Orai proteins are expressed in vascular and airway smooth muscle and constitute the molecular components of the ubiquitous store-operated Ca(2+) entry pathway that mediate the Ca(2+) release-activated Ca(2+) current. STIM/Orai proteins also encode store-independent Ca(2+) entry pathways in smooth muscle. Altered expression and function of STIM/Orai proteins have been linked to vascular and airway pathologies, including restenosis, hypertension, and atopic asthma. In this review we discuss our current understanding of Orai proteins and the store-dependent and -independent signaling pathways mediated by these proteins in vascular and airway smooth muscle. We also discuss the current studies linking altered expression and function of Orai proteins with smooth muscle-related pathologies. PMID:26718630

  16. Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

    SciTech Connect

    Costa-Silva, Bruno; Coelho da Costa, Meline; Melo, Fernanda Rosene; Neves, Cynara Mendes; Alvarez-Silva, Marcio; Calloni, Giordano Wosgrau; Trentin, Andrea Goncalves

    2009-04-01

    The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.

  17. Cytotoxicity of some oxysterols on human vascular smooth muscle cells was mediated by apoptosis.

    PubMed

    Miyashita, Y; Shirai, K; Ito, Y; Watanabe, J; Urano, Y; Murano, T; Tomioka, H

    1997-01-01

    A decrease in smooth muscle cells is observed in advanced atherosclerotic lesion. To understand this mechanism, we selected oxysterols as candidates for toxic lipid, and examined their cytotoxicity on human cultured vascular smooth muscle cells, together with the manner of cell death. In the presence of 7-ketocholesterol or 7 beta-hydroxycholesterol (50 mumol/L), the percentage of detached cells increased significantly with dose dependency, and an increase in detached cell number and DNA nick detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling study (TUNEL) preceded an increase in lactate dehydrogenase released into the medium. DNA extracted from smooth muscle cells incubated with 7-ketocholesterol or 7 beta-hydroxycholesterol showed a laddering pattern on agarose electrophoresis. In the presence of 7-ketocholesterol or 7 beta-hydroxycholesterol, fragmented DNA quantified by the quantitative sandwich enzyme immunoassay was significantly increased. From these results, it is proposed that 7-ketocholesterol and 7 beta-hydroxycholesterol are toxic to smooth muscle cells, and that this cytotoxicity is mediated by apoptosis. PMID:9638517

  18. ACTIVATION OF GATA-4 BY SEROTONIN IN PULMONARY ARTERY SMOOTH MUSCLE CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serotonin (5-HT) is a mitogen of pulmonary artery smooth muscle cells (PASMC) and plays an important role in the development of pulmonary hypertension. Signal transduction initiated by 5-HT involves serotonin transporter (SERT)-dependent generation of reactive oxygen species (ROS) and activation of...

  19. Intercellular ultrafast Ca2+ wave in vascular smooth muscle cells: numerical and experimental study

    PubMed Central

    Quijano, J. C.; Raynaud, F.; Nguyen, D.; Piacentini, N.; Meister, J. J.

    2016-01-01

    Vascular smooth muscle cells exhibit intercellular Ca2+ waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca2+ wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca2+ wave and it was suggested to be the result of the interplay between membrane potential and Ca2+ dynamics which depended on influx of extracellular Ca2+, cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca2+ wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca2+ wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca2+ wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca2+ waves in smooth muscle cells. PMID:27507785

  20. Effects of sumatriptan nasal spray (Imigran) on isolated rat's tracheal smooth muscle.

    PubMed

    Cheng, Li-Hsiang; Wu, Pei-Chuan; Liu, Shao-Cheng; Chiu, Feng-Shiang; Chu, Yueng-Hsiang; Chang, Ying-Nan; Wang, Hsing-Won

    2015-10-01

    Sumatriptan (Imigran) is a potent and highly selective 5-HT1 receptor agonist often used in treating acute migraine. Intranasal sumatriptan is well absorbed and is generally effective in relieving headache. However, the effects of Imigran given intratracheally have rarely been well explored. We aimed to verify the effect of Imigran, which acts on the tracheal smooth muscle directly in vitro. We examined the effectiveness of Imigran on isolated rat tracheal smooth muscle by testing: (1) effect on tracheal smooth muscle resting tension; (2) effect on contraction caused by 10(-6) M methacholine as a parasympathetic mimetic; (3) effect of the drugs on electrically induced tracheal smooth muscle contractions. The results indicated that the addition of methacholine to the incubation medium caused the trachea to contract in a dose-dependent manner. The addition of Imigran at doses of 10(-5) M or above elicited a significant relaxation response to 10(-6) M methacholine-induced contraction. Imigran could inhibit electrical field stimulation-induced spike contraction. It also had a minimal effect on the basal tension of trachea as the concentration increased. The study indicated high concentrations of Imigran could cause bronchodilation to reduce asthma attacks not only by blocking parasympathetic tone, but also by directly antagonizing the effect of cholinergic receptors. PMID:25394582

  1. Anti-cholinergic effect of singulair on isolated rat's tracheal smooth muscle.

    PubMed

    Cheng, Li-Hsiang; Kao, Chuan-Hsiang; Wang, Chih-Hung; Chu, Yueng-Hsiang; Wang, Jia-Yi; Wang, Hsing-Won

    2012-08-01

    Singulair (Montelukast) is a potent and selective leukotriene D(4) receptor antagonist, often used in treating inflammatory conditions of the respiratory system such as allergic rhinitis and asthma. However, the effects of singulair given intratracheally have rarely been well explored. To verify the effect of singulair, which acts on the tracheal smooth muscle directly in vitro. We used our preparation to test the effects of singulair on isolated rat's tracheal smooth muscle. The following assessments of singulair were performed: (1) effect on the tracheal smooth muscle resting tension, (2) effect on contraction caused by 10(-6) M methacholine as a parasympathetic mimetic, and (3) effect of the drugs on electrically induced tracheal smooth muscle contractions. The results indicated that the addition of methacholine to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of singulair at doses of 10(-5) M or above elicited a significant relaxation response to 10(-6) M methacholine-induced contraction. Singulair could not inhibit electrical field stimulation-induced spike contraction. It also had a minimal effect on the basal tension of trachea as the concentration increased. This study showed that the high concentrations of singulair also had an anti-cholinergic effect for relieving symptoms of asthma. PMID:22203119

  2. Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

    PubMed

    Choi, Jong Seob; Piao, Yunxian; Seo, Tae Seok

    2014-01-01

    The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function. PMID:24120039

  3. A specific gastrin receptor on plasma membranes of antral smooth muscle.

    PubMed

    Baur, S; Bacon, V C

    1976-12-20

    Plasma membranes with a 17 fold enrichment in 5'-nucleotidase over homogenate were prepared from antral smooth muscle. A specific gastrin receptor on the plasma membranes has been demonstrated. By Scatchard analysis receptor has a Kaff of 2x10(9)M(-1) and a binding capacity of 5x10(-14) moles/mg of membrane protein. PMID:15625862

  4. Epstein-Barr virus-associated smooth muscle tumor of the tonsil.

    PubMed

    Suwansirikul, Songkiet; Sukpan, Kornkanok; Sittitrai, Pichit; Suwiwat, Supaporn; Khunamornpong, Surapan

    2012-06-01

    Smooth muscle tumors of the tonsil are rare. Recently, the occurrence of Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) has been increasingly recognized in immunocompromised patients, mainly post-transplantation and AIDS patients. The clinicopathologic features of EBV-SMT are different from conventional smooth muscle tumors. To the best of our knowledge, EBV-SMT involving the tonsil in an AIDS patient has not been reported. A 27-year-old man presented with a 2.2cm right tonsillar mass six months after AIDS diagnosis. The tumor was composed of a cellular proliferation of oval to spindle-shaped cells with mitotic count up to 10 in 10 high-power fields. The diagnosis of EBV-SMT was confirmed by in situ hybridization for EBV-encoded RNA (EBER) transcripts. Synchronous lesions were also detected in the liver and peritoneum by an abdominal computed tomographic scan. EBV-SMT should be included in the differential diagnoses of a mesenchymal tumor in immunocompromised patients, and in the differential diagnoses of a smooth muscle tumor occurring in uncommon sites including the tonsil. PMID:21885224

  5. Intercellular ultrafast Ca(2+) wave in vascular smooth muscle cells: numerical and experimental study.

    PubMed

    Quijano, J C; Raynaud, F; Nguyen, D; Piacentini, N; Meister, J J

    2016-01-01

    Vascular smooth muscle cells exhibit intercellular Ca(2+) waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca(2+) wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca(2+) wave and it was suggested to be the result of the interplay between membrane potential and Ca(2+) dynamics which depended on influx of extracellular Ca(2+), cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca(2+) wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca(2+) wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca(2+) wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca(2+) waves in smooth muscle cells. PMID:27507785

  6. Endothelial Cells Direct Mesenchymal Stem Cells Toward a Smooth Muscle Cell Fate

    PubMed Central

    Lin, Cho-Hao

    2014-01-01

    Under defined conditions, mesenchymal stem cells can differentiate into unique cell types, making them attractive candidates for cell-based disease therapies. Ischemic diseases would greatly benefit from treatments that include the formation of new blood vessels from mesenchymal stem cells. However, blood vessels are complex structures composed of endothelial cells and smooth muscle cells, and their assembly and function in a diseased environment is reliant upon joining with the pre-existing vasculature. Although endothelial cell/smooth muscle cell interactions are well known, how endothelial cells may influence mesenchymal stem cells and facilitate their differentiation has not been defined. Therefore, we sought to explore how endothelial cells might drive mesenchymal stem cells toward a smooth muscle fate. Our data show that cocultured endothelial cells induce smooth muscle cell differentiation in mesenchymal stem cells. Endothelial cells can promote a contractile phenotype, reduce proliferation, and enhance collagen synthesis and secretion. Our data show that Notch signaling is essential for endothelial cell-dependent differentiation, and this differentiation pathway is largely independent of growth factor signaling mechanisms. PMID:24914692

  7. A mechanochemical 3D continuum model for smooth muscle contraction under finite strains.

    PubMed

    Stålhand, J; Klarbring, A; Holzapfel, G A

    2011-01-01

    This paper presents a modelling framework in which the mechanochemical properties of smooth muscle cells may be studied. The activation of smooth muscles is considered in a three-dimensional continuum model which is key to realistically capture the function of hollow organs such as blood vessels. On the basis of a general thermodynamical framework the mechanical and chemical phases are specialized in order to quantify the coupled mechanochemical process. A free-energy function is proposed as the sum of a mechanical energy stored in the passive tissue, a coupling between the mechanical and chemical kinetics and an energy related purely to the chemical kinetics and the calcium ion concentration. For the chemical phase it is shown that the cross-bridge model of Hai and Murphy [1988. Am. J. Physiol. Cell Physiol. 254, C99-C106] is included in the developed evolution law as a special case. In order to show the specific features and the potential of the proposed continuum model a uniaxial extension test of a tissue strip is analysed in detail and the related kinematics and stress-stretch relations are derived. Parameter studies point to coupling phenomena; in particular the tissue response is analysed in terms of the calcium ion level. The model for smooth muscle contraction may significantly contribute to current modelling efforts of smooth muscle tissue responses. PMID:20946904

  8. Pulmonary surfactant in the airway physiology: a direct relaxing effect on the smooth muscle.

    PubMed

    Calkovska, A; Uhliarova, B; Joskova, M; Franova, S; Kolomaznik, M; Calkovsky, V; Smolarova, S

    2015-04-01

    Beside alveoli, surface active material plays an important role in the airway physiology. In the upper airways it primarily serves in local defense. Lower airway surfactant stabilizes peripheral airways, provides the transport and defense, has barrier and anti-edematous functions, and possesses direct relaxant effect on the smooth muscle. We tested in vitro the effect of two surfactant preparations Curosurf® and Alveofact® on the precontracted smooth muscle of intra- and extra-pulmonary airways. Relaxation was more pronounced for lung tissue strip containing bronchial smooth muscle as the primary site of surfactant effect. The study does not confirm the participation of ATP-dependent potassium channels and cAMP-regulated epithelial chloride channels known as CFTR chloride channels, or nitric oxide involvement in contractile response of smooth muscle to surfactant.By controlling wall thickness and airway diameter, pulmonary surfactant is an important component of airway physiology. Thus, surfactant dysfunction may be included in pathophysiology of asthma, COPD, or other diseases with bronchial obstruction. PMID:25583659

  9. Distinct function of estrogen receptor α in smooth muscle and fibroblast cells in prostate development.

    PubMed

    Vitkus, Spencer; Yeh, Chiuan-Ren; Lin, Hsiu-Hsia; Hsu, Iawen; Yu, Jiangzhou; Chen, Ming; Yeh, Shuyuan

    2013-01-01

    Estrogen signaling, through estrogen receptor (ER)α, has been shown to cause hypertrophy in the prostate. Our recent report has shown that epithelial ERα knockout (KO) will not affect the normal prostate development or homeostasis. However, it remains unclear whether ERα in different types of stromal cells has distinct roles in prostate development. This study proposed to elucidate how KO of ERα in the stromal smooth muscle or fibroblast cells may interrupt cross talk between prostate stromal and epithelial cells. Smooth muscle ERαKO (smERαKO) mice showed decreased glandular infolding with the proximal area exhibiting a significant decrease. Fibroblast ERαKO mouse prostates did not exhibit this phenotype but showed a decrease in the number of ductal tips. Additionally, the amount of collagen observed in the basement membrane was reduced in smERαKO prostates. Interestingly, these phenotypes were found to be mutually exclusive among smERαKO or fibroblast ERαKO mice. Compound KO of ERα in both fibroblast and smooth muscle showed combined phenotypes from each of the single KO. Further mechanistic studies showed that IGF-I and epidermal growth factor were down-regulated in prostate smooth muscle PS-1 cells lacking ERα. Together, our results indicate the distinct functions of fibroblast vs. smERα in prostate development. PMID:23204329

  10. Modeling smooth muscle cell proliferation of coronary artery expanded with a drug eluting stent

    NASA Astrophysics Data System (ADS)

    Lyu, Suping

    2010-03-01

    The drug eluting coronary stent is for the treatment of narrowed coronary artery. A high strength balloon is used to open the narrowed vessel and leave behind a tiny metal mesh, or stent, to mechanically prevent the vessel from re-narrowing and biologically slow down proliferation of the smooth muscle cells. However, the drug eluting stents that had better performance also more seriously prevented the healing processes of the vessels, which could cause serious thrombotic reactions. In this study, we assume the healing process is controlled by proper proliferation of smooth cells. We also assume that the inflammation reactions and mechanical traction drive the smooth muscle cells to proliferate while the drug loaded in the stents drives the processes at the opposite direction. Numerical calculation was applied to the system. The drug distribution and elution durations, inflammation reactions and mechanical traction were discussed.

  11. The induction of YAP expression following arterial injury is crucial for smooth muscle phenotypic modulation and neointima formation

    PubMed Central

    Wang, Xiaobo; Hu, Guoqing; Gao, Xiangwei; Wang, Yong; Zhang, Wei; Harmon, Erin Yund; Zhi, Xu; Xu, Zhengping; Lennartz, Michelle R.; Barroso, Margarida; Trebak, Mohamed; Chen, Ceshi; Zhou, Jiliang

    2012-01-01

    Objective Abnormal proliferation and migration of vascular smooth muscle cells (SMCs) are the key events in the progression of neointima formation in response to vascular injury. The goal of this study is to investigate the functional role of a potent oncogene YAP in smooth muscle phenotypic modulation in vitro and in vivo. Methods and Results In vitro in cell culture and in vivo in both mouse and rat arterial injury models YAP expression is significantly induced and correlated with the vascular SMC synthetic phenotype. Over-expression of YAP promotes SMC migration and proliferation while attenuating smooth muscle contractile gene expression. Conversely, knocking-down endogenous YAP in SMCs up-regulates smooth muscle gene expression but attenuates SMC proliferation and migration. Consistent with this, knocking-down YAP expression in a rat carotid balloon injury model and genetic deletion of YAP specifically in vascular SMCs in mouse after carotid artery ligation injury attenuates injury-induced smooth muscle phenotypic switch and neointima formation. Conclusions YAP plays a novel integrative role in smooth muscle phenotypic modulation by inhibiting smooth muscle-specific gene expression while promoting smooth muscle proliferation and migration in vitro and in vivo. Blocking the induction of YAP would be a potential therapeutic approach for ameliorating vascular occlusive diseases. PMID:22922963

  12. Airway smooth muscle NOX4 is upregulated and modulates ROS generation in COPD.

    PubMed

    Hollins, Fay; Sutcliffe, Amanda; Gomez, Edith; Berair, Rachid; Russell, Richard; Szyndralewiez, Cédric; Saunders, Ruth; Brightling, Christopher

    2016-01-01

    The burden of oxidative stress is increased in chronic obstructive pulmonary disease (COPD). However, whether the intra-cellular mechanisms controlling the oxidant/anti-oxidant balance in structural airway cells such as airway smooth muscle in COPD is altered is unclear. We sought to determine whether the expression of the NADPH oxidase (NOX)-4 is increased in airway smooth muscle in COPD both in vivo and primary cells in vitro and its role in hydrogen peroxide-induced reactive oxygen species generation. We found that in vivo NOX4 expression was up-regulated in the airway smooth muscle bundle in COPD (n = 9) and healthy controls with >20 pack year history (n = 4) compared to control subjects without a significant smoking history (n = 6). In vitro NOX4 expression was increased in airway smooth muscle cells from subjects with COPD (n = 5) compared to asthma (n = 7) and upregulated following TNF-α stimulation. Hydrogen peroxide-induced reactive oxygen species generation by airway smooth muscle cells in COPD (n = 5) was comparable to healthy controls (n = 9) but lower than asthma (n = 5); and was markedly attenuated by NOX4 inhibition. Our findings demonstrate that NOX4 expression is increased in vivo and in vitro in COPD and although we did not observe an intrinsic increase in oxidant-induced reactive oxygen species generation in COPD, it was reduced markedly by NOX4 inhibition supporting a potential therapeutic role for NOX4 in COPD. PMID:27435477

  13. AMPK Dilates Resistance Arteries via Activation of SERCA and BKCa Channels in Smooth Muscle.

    PubMed

    Schneider, Holger; Schubert, Kai Michael; Blodow, Stephanie; Kreutz, Claus-Peter; Erdogmus, Serap; Wiedenmann, Margarethe; Qiu, Jiehua; Fey, Theres; Ruth, Peter; Lubomirov, Lubomir T; Pfitzer, Gabriele; Mederos Y Schnitzler, Michael; Hardie, D Grahame; Gudermann, Thomas; Pohl, Ulrich

    2015-07-01

    The protective effects of 5'-AMP-activated protein kinase (AMPK) on the metabolic syndrome may include direct effects on resistance artery vasomotor function. However, the precise actions of AMPK on microvessels and their potential interaction are largely unknown. Thus, we set to determine the effects of AMPK activation on vascular smooth muscle tone and the underlying mechanisms. Resistance arteries isolated from hamster and mouse exhibited a pronounced endothelium-independent dilation on direct pharmacological AMPK activation by 2 structurally unrelated compounds (PT1 and A769662). The dilation was associated with a decrease of intracellular-free calcium [Ca(2+)]i in vascular smooth muscle cell. AMPK stimulation induced activation of BKCa channels as assessed by patch clamp studies in freshly isolated hamster vascular smooth muscle cell and confirmed by direct proof of membrane hyperpolarization in intact arteries. The BKCa channel blocker iberiotoxin abolished the hyperpolarization but only partially reduced the dilation and did not affect the decrease of [Ca(2+)]i. By contrast, the sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor thapsigargin largely reduced these effects, whereas combined inhibition of SERCA and BKCa channels virtually abolished them. AMPK stimulation significantly increased the phosphorylation of the SERCA modulator phospholamban at the regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular smooth muscle cell by a decrease of [Ca(2+)]i. This is achieved by calcium sequestration via SERCA activation, as well as activation of BKCa channels. There is in part a mutual compensation of both calcium-lowering mechanisms. However, SERCA activation which involves an AMPK-dependent phosphorylation of phospholamban is the predominant mechanism in resistance vessels. PMID:26034200

  14. Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells.

    PubMed

    Stringa, E; Knäuper, V; Murphy, G; Gavrilovic, J

    2000-06-01

    Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35 degrees C to mimic physiological conditions. PDGF-BB pre-treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by alpha2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on alphavbeta3 integrin. alphavbeta3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-beta tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix. PMID:10806116

  15. Electrical properties of purinergic transmission in smooth muscle of the guinea-pig prostate.

    PubMed

    Lam, Michelle; Mitsui, Retsu; Hashitani, Hikaru

    2016-01-01

    Prostatic smooth muscle develops spontaneous myogenic tone which is modulated by autonomic neuromuscular transmission. This study aimed to investigate the role of purinergic transmission in regulating electrical activity of prostate smooth muscle and whether its contribution may be altered with age. Intracellular recordings were simultaneously made with isometric tension recordings in smooth muscle preparations of the guinea-pig prostate. Immunostaining for P2X1 receptors on whole mount preparations was also performed. In prostate preparations which generated spontaneous slow waves, electrical field stimulation (EFS)-evoked excitatory junction potentials (EJPs) which were abolished by guanethidine (10 μM), α-β-methylene ATP (10 μM) or pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS, 10 μM) but not phentolamine (1 μM). Consistently, immunostaining revealed the expression of P2X1 receptors on prostatic smooth muscle. EJPs themselves did not cause contractions, but EJPs could sum to trigger a slow wave and associated contraction. Yohimbine (1 μM) and 3,7-dimethyl-1-propargylxanthine (DMPX, 10 μM) but not propranolol (1 μM) potentiated EJPs. Although properties of EJPs were not different between young and aging guinea-pig prostates, ectoATPase inhibitor ARL 67156 (100 μM) augmented EJP amplitudes by 64.2 ± 29.6% in aging animals, compared to 22.1 ± 19.9% in young animals. These results suggest that ATP released from sympathetic nerves acts on P2X1 purinoceptors located on prostate smooth muscle to evoke EJPs, while pre-junctional α2-adrenergic and adenosine A2 receptors may play a role in preventing excessive transmitter release. Age-related up-regulation of enzymatic ATP breakdown may be a compensatory mechanism for the enhanced purinergic transmission which would cause hypercontractility arising from increased ATP release in older animals. PMID:26657181

  16. A new enzymic method for the isolation and culture of human bladder body smooth muscle cells.

    PubMed

    Ma, F -H; Higashira, H; Ukai, Y; Hanai, T; Kiwamoto, H; Park, Y C; Kurita, T

    2002-01-01

    Cultured cells of the human urinary bladder smooth muscle are useful for investigating bladder function, but methods for culturing them are not well developed. We have now established a novel enzymic technique. The smooth muscle layer was separated out and incubated with 0.2% trypsin for 30 min at 37 degrees C. The samples were then minced and incubated with 0.1% collagenase for 30 min and centrifuged at 900 g. The pellets were resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and centrifuged at 250 g. The smooth muscle cells from the supernatant were cultured in RPMI-1640 containing 10% FCS. The cells grew to confluence after 7-10 days, forming the "hills and valleys" growth pattern characteristic of smooth muscle cells. Immunostaining with anti-alpha-actin, anti-myosin, and anti-caldesmon antibodies demonstrated that 99% of the cells were smooth muscle cells. To investigate the pharmacological properties of the cultured cells, we determined the inhibitory effect of muscarinic receptor antagonists on the binding of [3H]N-methylscopolamine to membranes from cultured cells. The pKi values obtained for six antagonists agreed with the corresponding values for transfected cells expressing the human muscarinic M2 subtype. Furthermore, carbachol produced an increase in the concentration of cytoplasmic free Ca2+ an action that was blocked by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 selective antagonist. This result suggests that these cells express functional M3 muscarinic receptors, in addition to M2 receptors. The subcultured cells therefore appear to be unaffected by our new isolation method. PMID:11835427

  17. A novel bronchial ring bioassay for the evaluation of small airway smooth muscle function in mice.

    PubMed

    Liu, John Q; Yang, Dennis; Folz, Rodney J

    2006-08-01

    Advances in our understanding of murine airway physiology have been hindered by the lack of suitable, ex vivo, small airway bioassay systems. In this study, we introduce a novel small murine airway bioassay system that permits the physiological and pharmacological study of intrapulmonary bronchial smooth muscle via a bronchial ring (BR) preparation utilizing BR segments as small as 200 microm in diameter. Using this ex vivo BR bioassay, we characterized small airway smooth muscle contraction and relaxation in the presence and absence of bronchial epithelium. In control BRs, the application of mechanical stretch is followed by spontaneous bronchial smooth muscle relaxation. BRs pretreated with methacholine (MCh) partially attenuate this stretch-induced relaxation by as much as 42% compared with control. MCh elicited a dose-dependent bronchial constriction with a maximal tension (E(max)) of 8.7 +/- 0.2 mN at an EC(50) of 0.33 +/- 0.02 microM. In the presence of nifedipine, ryanodine, 2-aminoethoxydiphenyl borate, and SKF-96365, E(max) to MCh was significantly reduced. In epithelium-denuded BRs, MCh-induced contraction was significantly enhanced to 11.4 +/- 1.0 mN with an EC(50) of 0.16 +/- 0.04 microM (P < 0.01). Substance P relaxed MCh-precontracted BR by 62.1%; however, this bronchial relaxation effect was completely lost in epithelium-denuded BRs. Papaverine virtually abolished MCh-induced constriction in both epithelium-intact and epithelium-denuded bronchial smooth muscle. In conclusion, this study introduces a novel murine small airway BR bioassay that allows for the physiological study of smooth muscle airway contractile responses that may aid in our understanding of the pathophysiology of asthma. PMID:16648239

  18. Rapid effects of phytoestrogens on human colonic smooth muscle are mediated by oestrogen receptor beta.

    PubMed

    Hogan, A M; Collins, D; Sheehan, K; Zierau, O; Baird, A W; Winter, D C

    2010-05-14

    Epidemiological studies have correlated consumption of dietary phytoestrogens with beneficial effects on colon, breast and prostate cancers. Genomic and non-genomic mechanisms are responsible for anti-carcinogenic effects but, until now, the effect on human colon was assumed to be passive and remote. No direct effect on human colonic smooth muscle has previously been described. Institutional research board approval was granted. Histologically normal colon was obtained from the proximal resection margin of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended under 1g of tension in organ baths containing oxygenated Krebs solution at 37 degrees C. After an equilibration period, tissues were exposed to diarylpropionitrile (DPN) (ER beta agonist) and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (ER alpha agonist) or to the synthetic phytoestrogen compounds genistein (n=8), daidzein (n=8), fisetin (n=8) and quercetin (n=8) in the presence or absence of fulvestrant (oestrogen receptor antagonist). Mechanism of action was investigated by inhibition of downstream pathways. The cholinergic agonist carbachol was used to induce contractile activity. Tension was recorded isometrically. Phytoestrogens inhibit carbachol-induced colonic contractility. In keeping with a non-genomic, rapid onset direct action, the effect was within minutes, reversible and similar to previously described actions of 17 beta oestradiol. No effect was seen in the presence of fulvestrant indicating receptor modulation. While the DPN exerted inhibitory effects, PPT did not. The effect appears to be reliant on a p38/mitogen activated protein kinase mediated induction of nitric oxide production in colonic smooth muscle. The present data set provides the first description of a direct effect of genistein, daidzein, fisetin and quercetin on human colonic smooth muscle. The presence of ER in colonic smooth muscle has been functionally proven and the beta

  19. Molecular and functional significance of Ca2+-activated Cl− channels in pulmonary arterial smooth muscle

    PubMed Central

    Forrest, Abigail S.; Ayon, Ramon J.; Wiwchar, Michael; Angermann, Jeff E.; Pritchard, Harry A. T.; Singer, Cherie A.; Valencik, Maria L.; Britton, Fiona; Greenwood, Iain A.

    2015-01-01

    Abstract Increased peripheral resistance of small distal pulmonary arteries is a hallmark signature of pulmonary hypertension (PH) and is believed to be the consequence of enhanced vasoconstriction to agonists, thickening of the arterial wall due to remodeling, and increased thrombosis. The elevation in arterial tone in PH is attributable, at least in part, to smooth muscle cells of PH patients being more depolarized and displaying higher intracellular Ca2+ levels than cells from normal subjects. It is now clear that downregulation of voltage-dependent K+ channels (e.g., Kv1.5) and increased expression and activity of voltage-dependent (Cav1.2) and voltage-independent (e.g., canonical and vanilloid transient receptor potential [TRPC and TRPV]) Ca2+ channels play an important role in the functional remodeling of pulmonary arteries in PH. This review focuses on an anion-permeable channel that is now considered a novel excitatory mechanism in the systemic and pulmonary circulations. It is permeable to Cl− and is activated by a rise in intracellular Ca2+ concentration (Ca2+-activated Cl− channel, or CaCC). The first section outlines the biophysical and pharmacological properties of the channel and ends with a description of the molecular candidate genes postulated to encode for CaCCs, with particular emphasis on the bestrophin and the newly discovered TMEM16 and anoctamin families of genes. The second section provides a review of the various sources of Ca2+ activating CaCCs, which include stimulation by mobilization from intracellular Ca2+ stores and Ca2+ entry through voltage-dependent and voltage-independent Ca2+ channels. The third and final section summarizes recent findings that suggest a potentially important role for CaCCs and the gene TMEM16A in PH. PMID:26064450

  20. Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling.

    PubMed

    Tang, Dale D

    2015-01-01

    Asthma is characterized by airway hyperresponsiveness and airway remodeling, which are largely attributed to increased airway smooth muscle contractility and cell proliferation. It is known that both chemical and mechanical stimulation regulates smooth muscle contraction. Recent studies suggest that contractile activation and mechanical stretch induce actin cytoskeletal remodeling in smooth muscle. However, the mechanisms that control actin cytoskeletal reorganization are not completely elucidated. This review summarizes our current understanding regarding how actin-associated proteins may regulate remodeling of the actin cytoskeleton in airway smooth muscle. In particular, there is accumulating evidence to suggest that Abelson tyrosine kinase (Abl) plays a critical role in regulating airway smooth muscle contraction and cell proliferation in vitro, and airway hyperresponsiveness and remodeling in vivo. These studies indicate that Abl may be a novel target for the development of new therapy to treat asthma. PMID:26517982

  1. A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion

    PubMed Central

    Kuckwa, Jessica; Fritzen, Katharina; Buttgereit, Detlev; Rothenbusch-Fender, Silke; Renkawitz-Pohl, Renate

    2016-01-01

    The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. PMID:26657767

  2. Two-pore-domain potassium channels in smooth muscles: new components of myogenic regulation.

    PubMed

    Sanders, Kenton M; Koh, Sang Don

    2006-01-01

    Gastrointestinal (GI) smooth muscles are influenced by many levels of regulation, including those provided by enteric motor neurones, hormones and paracrine substances. The integrated contractile responses to these regulatory mechanisms depend heavily on the state of excitability of smooth muscle cells. Resting ionic conductances and myogenic responses to agonists and physical parameters, such as stretch, are important in establishing basal excitability. This review discusses the role of 2-pore-domain K+ channels in contributing to background conductances and in mediating responses of GI muscles to enteric inhibitory nerve stimulation and stretch. Murine GI muscles express TREK-1 channels and display a stretch-dependent K+ (SDK) conductance that is also activated by nitric oxide via a cGMP-dependent mechanism. Cloning and expression of mTREK-1 produced an SDK conductance that was activated by cGMP-dependent phosphorylation at serine-351. GI muscle cells also express TASK-1 and TASK-2 channels that are inhibited by lidocaine and external acidification. These conductances appear to provide significant background K+ permeability that contributes to the negative resting potentials of GI muscles. PMID:16239268

  3. A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion.

    PubMed

    Kuckwa, Jessica; Fritzen, Katharina; Buttgereit, Detlev; Rothenbusch-Fender, Silke; Renkawitz-Pohl, Renate

    2016-01-15

    The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. PMID:26657767

  4. Protein kinases in vascular smooth muscle tone--role in the pulmonary vasculature and hypoxic pulmonary vasoconstriction.

    PubMed

    Ward, Jeremy P T; Knock, Greg A; Snetkov, Vladimir A; Aaronson, Philip I

    2004-12-01

    Hypoxic pulmonary vasoconstriction (HPV) is an adaptive mechanism that in the normal animal diverts blood away from poorly ventilated areas of the lung, thereby maintaining optimal ventilation-perfusion matching. In global hypoxia however, such as in respiratory disease or at altitude, it causes detrimental increases in pulmonary vascular resistance and pulmonary artery (PA) pressure. The precise intracellular pathways and mechanisms underlying HPV remain unclear, although it is now recognised that both an elevation in smooth muscle intracellular [Ca2+] and a concomitant increase in Ca2+ sensitivity are involved. Several key intracellular protein kinases have been proposed as components of the signal transduction pathways leading to development of HPV, specifically Rho kinase, non-receptor tyrosine kinases (NRTK), p38 mitogen activated protein (MAP) kinase, and protein kinase C (PKC). All of these have been implicated to a greater or lesser extent in pathways leading to Ca2+ sensitisation, and in some cases regulation of intracellular [Ca2+] as well. In this article, we review the role of these key protein kinases in the regulation of vascular smooth muscle (VSM) constriction, applying what is known in the systemic circulation to the pulmonary circulation and HPV. We conclude that the strongest evidence for direct involvement of protein kinases in the mechanisms of HPV concerns a central role for Rho kinase in Ca2+ sensitisation, and a potential role for Src-family kinases in both modulation of Ca2+ entry via capacitative Ca2+ entry (CCE) and activation of Rho kinase, though others are likely to have indirect or modulatory influences. In addition, we speculate that Src family kinases may provide a central interface between the proposed hypoxia-induced generation of reactive oxygen species by mitochondria and both the elevation in intracellular [Ca2+] and Rho kinase mediated Ca2+ sensitisation. PMID:15556675

  5. Airway smooth muscle changes in the nitrofen-induced congenital diaphragmatic hernia rat model.

    PubMed

    Belik, Jaques; Davidge, Sandra T; Zhang, Wei; Pan, Jingyi; Greer, John J

    2003-05-01

    In the fetal rat, nitrofen induces congenital diaphragmatic hernia (CDH) and pulmonary vascular remodeling similar to what is observed in the human condition. Airway hyperactivity is common in infants with CDH and attributed to the ventilator-induced airway damage. The purpose of this study was to test the hypothesis that airway smooth muscle mechanical properties are altered in the nitrofen-induced CDH rat model. Lungs from nitrofen-exposed fetuses with hernias (CDH) or intact diaphragm (nitrofen) and untreated fetuses (control) were studied on gestation d 21. The left intrapulmonary artery and bronchi were removed and mounted on a wire myograph, and lung expression, content, and immunolocalization of cyclooxygenases COX-1 and COX-2 were evaluated. Pulmonary artery muscle in the CDH group had significantly (p < 0.01) lower force generation compared with control and nitrofen groups. In contrast, the same generation bronchial smooth muscle of the CDH and nitrofen groups developed higher force compared with control. Whereas no differences were found in endothelium-dependent pulmonary vascular muscle tone, the epithelium-dependent airway muscle relaxation was significantly decreased (p < 0.01) in the CDH and nitrofen groups. The lung mRNA levels of COX-1 and COX-2 were increased in the CDH and nitrofen groups. COX-1 vascular and airway immunostaining, as well as COX-1 and COX-2 lung protein content, were increased in the CDH group. This is the first report of airway smooth muscle abnormalities in the nitrofen-induced fetal rat model of CDH. We speculate that congenital airway muscle changes may be present in the human form of this disease. PMID:12612200

  6. beta. -adrenergic relaxation of smooth muscle: differences between cells and tissues

    SciTech Connect

    Scheid, C.R.

    1987-09-01

    The present studies were carried out in an attempt to resolve the controversy about the Na/sup +/ dependence of ..beta..-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of ..beta..-adrenergic agents, including a stimulatory effect on /sup 45/Ca efflux, were dependent on the presence of a normal transmembrane Na/sup +/ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of ..beta..-adrenergic agents was Na/sup +/ independent. Uncertainty remained as to whether these discrepancies reflected differences between cells and tissues or differences between species. Thus, in the present studies, the authors utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na/sup +/ dependence of ..beta..-adrenergic relaxation. They found that elimination of a normal Na/sup +/ gradient abolished ..beta..-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Thus the controversy concerning the mechanisms of ..beta..-adrenergic relaxation may reflect inherent differences between tissues and cells.

  7. Nitric oxide mediates stretch-induced Ca2+ oscillation in smooth muscle.

    PubMed

    Zheng, Ji; Zhai, Kui; Chen, Yingxiao; Zhang, Xu; Miao, Lin; Wei, Bin; Ji, Guangju

    2016-06-15

    The stretching of smooth muscle tissue modulates contraction through augmentation of Ca(2+) transients, but the mechanism underlying stretch-induced Ca(2+) transients is still unknown. We found that mechanical stretching and maintenance of mouse urinary bladder smooth muscle strips and single myocytes at 30% and 18% beyond the initial length, respectively, resulted in Ca(2+) oscillations. Experiments indicated that mechanical stretching remarkably increased the production of nitric oxide (NO) as well as the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) oscillations and contractility increases were completely abolished by the NO inhibitor L-NAME or eNOS (also known as NOS3) gene inactivation. Moreover, exposure of eNOS-knockout myocytes to exogenous NO donor induced Ca(2+) oscillations. The stretch-induced Ca(2+) oscillations were greatly inhibited by the selective inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor xestospongin C and partially inhibited by ryanodine. Moreover, the stretch-induced Ca(2+) oscillations were also suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, but not by the soluble guanylyl cyclase (sGC) inhibitor ODQ. These results suggest that stretching myocyte and maintenance at a certain length results in Ca(2+) oscillations that are NO dependent , and sGC and cGMP independent, and results from the activation of PI3K in smooth muscle. PMID:27189081

  8. Combined electric field and gap junctions on propagation of action potentials in cardiac muscle and smooth muscle in PSpice simulation.

    PubMed

    Sperelakis, Nicholas

    2003-10-01

    Propagation of action potentials in cardiac muscle and smooth muscle were simulated using the PSpice program. Excitation was transmitted from cell to cell along a strand of 6 cells (cardiac muscle) or 10 cells (smooth muscle) either not connected (control) or connected by low-resistance tunnels (gap-junction connexons). A significant negative cleft potential (V(jv) ) develops in the narrow junctional cleft when the pre-JM fires. V(jc) depolarizes the postjunctional membrane (post-JM) to threshold by a patch-clamp action. With few connecting tunnels, cell-to-cell transmission by the EF mechanism was facilitated. With many tunnels, propagation was dominated by the low-resistance mechanism, and propagation velocity (theta) became very fast and nonphysiological. In conclusion, when the 2 mechanisms for cell-to-cell transfer of excitation were combined, the two mechanisms facilitated each other in a synergistic manner. When there were many connecting tunnels, the tunnel mechanism was dominant. PMID:14661164

  9. Steroids and antihistamines synergize to inhibit rat's airway smooth muscle contractility.

    PubMed

    Liu, Shao-Cheng; Chu, Yueng-Hsiang; Kao, Chuan-Hsiang; Wu, Chi-Chung; Wang, Hsing-Won

    2015-06-01

    Both glucocorticoids and H1-antihistamines were widely used on patients with allergic rhinitis (AR) and obstructive airway diseases. However, their direct effects on airway smooth muscle were not fully explored. In this study, we tested the effectiveness of prednisolone (Kidsolone) and levocetirizine (Xyzal) on isolated rat trachea submersed in Kreb's solution in a muscle bath. Changes in tracheal contractility in response to the application of parasympathetic mimetic agents were measured. The following assessments of the drug were performed: (1) effect on tracheal smooth muscle resting tension; (2) effect on contraction caused by 10(-6) M methacholine; (3) effect of the drug on electrical field stimulation (EFS) induced tracheal smooth muscle contractions. The result revealed sole use of Kidsolone or Xyzal elicited no significant effect or only a little relaxation response on tracheal tension after methacholine treatment. The tension was 90.5 ± 7.5 and 99.5 ± 0.8 % at 10(-4) M for Xyzal and 10(-5) M for Kidsolone, respectively. However, a dramatically spasmolytic effect was observed after co-administration of Kidsolone and Xyzal and the tension dropped to 67.5 ± 13.6 %, with statistical significance (p < 0.05). As for EFS-induced contractions, Kidsolone had no direct effect but Xyzal could inhibit it, with increasing basal tension. In conclusion, using glucocorticoids alone had no spasmolytic effect but they can be synergized with antihistamines to dramatically relax the trachea smooth muscle within minutes. Therefore, for AR patients with acute asthma attack, combined use of those two drugs is recommended. PMID:25115316

  10. Depolarization-induced contractile activity of smooth muscle in calcium-free solution.

    PubMed

    Mangel, A W; Nelson, D O; Rabovsky, J L; Prosser, C L; Connor, J A

    1982-01-01

    In calcium-free solution, strips of cat intestinal muscle developed slow, rhythmic electrical potential changes that triggered contractions. Some strips failed to develop spontaneous electrical activity in calcium-free solution but responded with contractions to depolarization by direct electrical stimulation or by treatment with barium chloride, potassium chloride, or acetylcholine. Similar results were obtained with segments of cat stomach, colon, esophagus, bladder, uterus, and vena cava, as well as with rabbit vena cava. In calcium-free saline, rat small intestinal muscle showed fast electrical activity with accompanying development of a tetanuslike contraction. After 60 min in calcium-free solution, cat small intestinal muscle retained 17.7% of its original concentration of calcium. It is concluded that in some smooth muscles, depolarization-triggered release of intracellular calcium does not require an associated influx of calcium. PMID:7058877

  11. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  12. Airway smooth muscle responsiveness from dogs with airway hyperresponsiveness after O/sub 3/ inhalation

    SciTech Connect

    Jones, G.L.; O'Byrne, P.M.; Pashley, M.; Serio, R.; Jury, J.; Lane, C.G.; Daniel, E.E.

    1988-07-01

    Airway hyperresponsiveness occurs after inhalation of O3 in dogs. The purpose of this study was to examine the responsiveness of trachealis smooth muscle in vitro to electrical field stimulation, exogenous acetylcholine, and potassium chloride from dogs with airway hyperresponsiveness after inhaled O3 in vivo and to compare this with the responsiveness of trachealis muscle from control dogs. In addition, excitatory junction potentials were measured with the use of single and double sucrose gap techniques in both groups of dogs to determine whether inhaled O3 affects the release of acetylcholine from parasympathetic nerves in trachealis muscle. Airway hyperresponsiveness developed in all dogs after inhaled O3 (3 ppm for 30 min). The acetylcholine provocative concentration decreased from 4.11 mg/ml before O3 inhalation to 0.66 mg/ml after O3 (P less than 0.0001). The acetylcholine provocative concentration increased slightly after control inhalation of dry room air. Airway smooth muscle showed increased responses to both electrical field stimulation and exogenous acetylcholine but not to potassium chloride in preparations from dogs with airway hyperresponsiveness in vivo. The increased response to electrical field stimulation was not associated with a change in excitatory junctional potentials. These results suggest that a postjunctional alteration in trachealis muscle function occurs after inhaled O3 in dogs, which may account for airway hyperresponsiveness after O3 in vivo.

  13. Smooth Muscle-Targeted Overexpression of Peroxisome Proliferator Activated Receptor-γ Disrupts Vascular Wall Structure and Function

    PubMed Central

    Kleinhenz, Jennifer M.; Murphy, Tamara C.; Pokutta-Paskaleva, Anastassia P.; Gleason, Rudolph L.; Lyle, Alicia N.; Taylor, W. Robert; Blount, Mitsi A.; Cheng, Juan; Yang, Qinglin; Sutliff, Roy L.; Hart, C. Michael

    2015-01-01

    Activation of the nuclear hormone receptor, PPARγ, with pharmacological agonists promotes a contractile vascular smooth muscle cell phenotype and reduces oxidative stress and cell proliferation, particularly under pathological conditions including vascular injury, restenosis, and atherosclerosis. However, pharmacological agonists activate both PPARγ-dependent and -independent mechanisms in multiple cell types confounding efforts to clarify the precise role of PPARγ in smooth muscle cell structure and function in vivo. We, therefore, designed and characterized a mouse model with smooth muscle cell-targeted PPARγ overexpression (smPPARγOE). Our results demonstrate that smPPARγOE attenuated contractile responses in aortic rings, increased aortic compliance, caused aortic dilatation, and reduced mean arterial pressure. Molecular characterization revealed that compared to littermate control mice, aortas from smPPARγOE mice expressed lower levels of contractile proteins and increased levels of adipocyte-specific transcripts. Morphological analysis demonstrated increased lipid deposition in the vascular media and in smooth muscle of extravascular tissues. In vitro adenoviral-mediated PPARγ overexpression in human aortic smooth muscle cells similarly increased adipocyte markers and lipid uptake. The findings demonstrate that smooth muscle PPARγ overexpression disrupts vascular wall structure and function, emphasizing that balanced PPARγ activity is essential for vascular smooth muscle homeostasis. PMID:26451838

  14. Se Enhances MLCK Activation by Regulating Selenoprotein T (SelT) in the Gastric Smooth Muscle of Rats.

    PubMed

    Li, Jia-Ping; Zhou, Jing-Xuan; Wang, Qi; Gu, Gao-Qin; Yang, Shi-Jin; Li, Cheng-Ye; Qiu, Chang-Wei; Deng, Gan-Zhen; Guo, Meng-Yao

    2016-09-01

    Selenium (Se), a nutritionally essential trace element, is associated with health and disease. Selenoprotein T (SelT) was identified as a redoxin protein with a selenocystein, localizing in the endoplasmic reticulum. The myosin light chain kinase (MLCK) and myosin light chain (MLC) play key roles in the contraction process of smooth muscle. The present study was to detect the effect and mechanism of SelT on the contraction process of gastric smooth muscle. The WT rats were fed with different Se concentration diets, and Se and Ca(2+) concentrations were detected in the gastric smooth muscle. Western blot and qPCR were performed to determine SelT, CaM, MLCK, and MLC expressions. MLCK activity was measured by identifying the rates of [γ-32P]ATP incorporated into the MLC. The results showed Se and Ca(2+) concentrations were enhanced with Se intake in gastric smooth muscle tissues. With increasing Se, SelT, CaM, MLCK and MLC expressions increased, and MLCK and MLC activation improved in gastric smooth muscle tissue. The SelT RNA interference experiments showed that Ca(2+) release, MLCK activation, and MLC phosphorylation were regulated by SelT. Se affected the gastric smooth muscle constriction by regulating Ca(2+) release, MLCK activation, and MLC phosphorylation through SelT. Se plays a major role in regulating the contraction processes of gastric smooth muscle with the SelT. PMID:26779623

  15. Migration of smooth muscle cells from the arterial anastomosis of arteriovenous fistulas requires Notch activation to form neointima.

    PubMed

    Liang, Ming; Wang, Yun; Liang, Anlin; Mitch, William E; Roy-Chaudhury, Prabir; Han, Guofeng; Cheng, Jizhong

    2015-09-01

    A major factor contributing to failure of arteriovenous fistulas (AVFs) is migration of smooth muscle cells into the forming neointima. To identify the source of smooth muscle cells in neointima, we created end-to-end AVFs by anastomosing the common carotid artery to the jugular vein and studied neural crest-derived smooth muscle cells from the carotid artery, which are Wnt1-positive during development. In Wnt1-cre-GFP mice, smooth muscle cells in the carotid artery but not the jugular vein are labeled with GFP. About half of the cells were GFP-positive in the neointima, indicating their migration from the carotid artery to the jugular vein in AVFs created in these mice. As fibroblast-specific protein-1 (FSP-1) regulates smooth muscle cell migration, we examined FSP-1 in failed AVFs and polytetrafluoroethylene grafts from patients with end-stage kidney disease or from AVFs in mice with chronic kidney disease. In smooth muscle cells of AVFs or polytetrafluoroethylene grafts, FSP-1 and activation of Notch1 are present. In smooth muscle cells, Notch1 increased RBP-Jκ transcription factor activity and RBP-Jκ stimulated FSP-1 expression. Conditional knockout of RBP-Jκ in smooth muscle cells or general knockout of FSP-1 suppressed neointima formation in AVFs in mice. Thus, the artery of AVFs is the major source of smooth muscle cells during neointima formation. Knockout of RBP-Jκ or FSP-1 ameliorates neointima formation and might improve AVF patency during long-term follow-up. PMID:25786100

  16. HEF-19-induced relaxation of colonic smooth muscles and the underlying mechanisms

    PubMed Central

    Wei, Yuan-Yuan; Sun, Lu-Lu; Fu, Shou-Ting

    2013-01-01

    AIM: To investigate the relaxant effect of chromane HEF-19 on colonic smooth muscles isolated from rabbits, and the underlying mechanisms. METHODS: The relaxant effect and action mechanisms of HEF-19 were investigated using descending colon smooth muscle of the rabbits. Preparations 1 cm long were mounted in 15-mL tissue baths containing Tyrode’s solution, maintained at 37 ± 0.5 °C and aerated with a mixture of 5% CO2 in oxygen (Carbogen). The tension and amplitude of the smooth muscle strips were recorded after adding HEF-19 (10-6, 10-5 and 10-4 mol/L). After cumulative administration of four antispasmodic agents, including acetylcholine chloride (Ach) (10-4 mol/L), histamine (10-4 mol/L), high-K+ (60 mmol/L) and BaCl2 (8.2 mmol/L), HEF-19 (3 × 10-7-3 × 10-4 mol/L) was added to investigate the relaxant effect of HEF-19. CaCl2 (10-4-2.5 × 10-3 mol/L) was added cumulatively to the smooth muscle preparations pretreated with and without HEF-19 (1 × 10-6 or 3 × 10-6 mol/L) and verapamil (1 × 10-7 mol/L) to study the mechanisms involved. Finally, phasic contraction was induced with ACh (15 × 10-6 mol/L), and CaCl2 (4 × 10-3 mol/L) was added to the smooth muscle preparations pretreated with and without HEF-19 (3 × 10-6 mol/L or 1 × 10-5 mol/L) and verapamil (1 × 10-7 mol/L) in calcium-free medium to further study the underlying mechanisms. RESULTS: HEF-19 (1 × 10-6, 1 × 10-5 and 1 × 10-4 mol/L) suppressed spontaneous contraction of rabbit colonic smooth muscles. HEF-19 (3 × 10-7-3 × 10-4 mol/L) relaxed in a concentration-dependent manner colonic smooth muscle preparations pre-contracted with BaCl2, high-K+ solution, Ach or histamine with respective EC50 values of 5.15 ± 0.05, 5.12 ± 0.08, 5.58 ± 0.16 and 5.25 ± 0.24, thus showing a spasmolytic activity. HEF-19 (1 × 10-6 mol/L and 3 × 10-6 mol/L) shifted the concentration-response curves of CaCl2 to the right and depressed the maximum response to CaCl2. The two components contracted by Ach were

  17. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells

    PubMed Central

    Patsch, Christoph; Challet-Meylan, Ludivine; Thoma, Eva C.; Urich, Eduard; Heckel, Tobias; O’Sullivan, John F; Grainger, Stephanie J; Kapp, Friedrich G.; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H. C.; He, Wei; Pan, Wei; Prummer, Michael; Warren, Curtis R.; Jakob-Roetne, Roland; Certa, Ulrich; Jagasia, Ravi; Freskgård, Per-Ola; Adatto, Isaac; Kling, Dorothee; Huang, Paul; Zon, Leonard I; Chaikof, Elliot L.; Gerszten, Robert E.; Graf, Martin; Iacone, Roberto; Cowan, Chad A.

    2015-01-01

    The use of human pluripotent stem cells for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies over 80% within six days. Upon purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. PMID:26214132

  18. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

    PubMed

    Patsch, Christoph; Challet-Meylan, Ludivine; Thoma, Eva C; Urich, Eduard; Heckel, Tobias; O'Sullivan, John F; Grainger, Stephanie J; Kapp, Friedrich G; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H C; He, Wei; Pan, Wei; Prummer, Michael; Warren, Curtis R; Jakob-Roetne, Roland; Certa, Ulrich; Jagasia, Ravi; Freskgård, Per-Ola; Adatto, Isaac; Kling, Dorothee; Huang, Paul; Zon, Leonard I; Chaikof, Elliot L; Gerszten, Robert E; Graf, Martin; Iacone, Roberto; Cowan, Chad A

    2015-08-01

    The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. PMID:26214132

  19. Vanilloid and Melastatin Transient Receptor Potential Channels in Vascular Smooth Muscle

    PubMed Central

    Earley, Scott

    2010-01-01

    The mammalian transient receptor potential (TRP) superfamily consists of six subfamilies that are defined by structural homology: TRPC (conventional or canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), and TRPML (mucoliptin). This review focuses on channels belonging to the vanilloid (V) and melastatin (M) TRP subfamilies. The TRPV subfamily consists of six members (TRPV1–6) and the TRPM subfamily has eight (TRPM1–8). The basic biophysical properties of these channels are briefly described. All of these channels except TRPV5, TRPV6, and TRPM1 are reportedly present in arterial smooth muscle from various segments of the vasculature. Studies demonstrating involvement of TRPV1, TRPV2, TRPV4, TRPM4, TRPM7 and TRPM8 in regulation of arterial smooth muscle function are reviewed. The functions of TRPV3, TRPM2, TRPM3, and TRPM6 channels in arterial myocytes have not been reported. PMID:20536737

  20. Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells

    PubMed Central

    Chen, Qi; Zhang, Hui; Liu, Yang; Adams, Susanne; Eilken, Hanna; Stehling, Martin; Corada, Monica; Dejana, Elisabetta; Zhou, Bin; Adams, Ralf H.

    2016-01-01

    Mural cells of the vessel wall, namely pericytes and vascular smooth muscle cells, are essential for vascular integrity. The developmental sources of these cells and molecular mechanisms controlling their progenitors in the heart are only partially understood. Here we show that endocardial endothelial cells are progenitors of pericytes and vascular smooth muscle cells in the murine embryonic heart. Endocardial cells undergo endothelial–mesenchymal transition and convert into primitive mesenchymal progenitors expressing the platelet-derived growth factor receptors, PDGFRα and PDGFRβ. These progenitors migrate into the myocardium, differentiate and assemble the wall of coronary vessels, which requires canonical Wnt signalling involving Frizzled4, β-catenin and endothelial cell-derived Wnt ligands. Our findings identify a novel and unexpected population of progenitors for coronary mural cells with potential relevance for heart function and disease conditions. PMID:27516371

  1. Visualization of Head–Head Interactions in the Inhibited State of Smooth Muscle Myosin

    PubMed Central

    Wendt, Thomas; Taylor, Dianne; Messier, Terri; Trybus, Kathleen M.; Taylor, Kenneth A.

    1999-01-01

    The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited. PMID:10613897

  2. Cyclic RNA has-circ-000595 regulates apoptosis of aortic smooth muscle cells

    PubMed Central

    ZHENG, CHENGFEI; NIU, HUI; LI, MING; ZHANG, HONGKUN; YANG, ZHENGGANG; TIAN, LU; WU, ZIHENG; LI, DONGLIN; CHEN, XUDONG

    2015-01-01

    Aortic aneurysm is a cardiovascular condition with a serious risk of mortality and the dismal prognosis of any type of major cardiovascular disease. The present study found that tissues from aortic aneurysm patients and hypoxic aortic smooth muscle cells showed aberrant high expression of the cyclic RNA hsa-circ-000595, as demonstrated by polymerase chain reaction array screening. Knockdown of hsa-circ-000595 resulted in a decreased apoptotic rate of human aortic smooth muscle cells. Furthermore, it was determined that miR-19a is a target of hsa-circ-000595. The results of the present study laid an epigenetic foundation for exploring the underlying mechanisms of the development of aortic aneurysm. PMID:26324352

  3. Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells.

    PubMed

    Chen, Qi; Zhang, Hui; Liu, Yang; Adams, Susanne; Eilken, Hanna; Stehling, Martin; Corada, Monica; Dejana, Elisabetta; Zhou, Bin; Adams, Ralf H

    2016-01-01

    Mural cells of the vessel wall, namely pericytes and vascular smooth muscle cells, are essential for vascular integrity. The developmental sources of these cells and molecular mechanisms controlling their progenitors in the heart are only partially understood. Here we show that endocardial endothelial cells are progenitors of pericytes and vascular smooth muscle cells in the murine embryonic heart. Endocardial cells undergo endothelial-mesenchymal transition and convert into primitive mesenchymal progenitors expressing the platelet-derived growth factor receptors, PDGFRα and PDGFRβ. These progenitors migrate into the myocardium, differentiate and assemble the wall of coronary vessels, which requires canonical Wnt signalling involving Frizzled4, β-catenin and endothelial cell-derived Wnt ligands. Our findings identify a novel and unexpected population of progenitors for coronary mural cells with potential relevance for heart function and disease conditions. PMID:27516371

  4. The relaxant effect of Nigella sativa on smooth muscles, its possible mechanisms and clinical applications

    PubMed Central

    Keyhanmanesh, Rana; Gholamnezhad, Zahra; Boskabady, Mohammad Hossien

    2014-01-01

    Nigella sativa (N. sativa) is a spice plant which has been traditionally used for culinary and medicinal purposes. Different therapeutic properties including the beneficial effects on asthma and dyspnea, digestive and gynecology disorders have been described for the seeds of N. sativa. There is evidence of the relaxant effects of this plant and some of its constituents on different types of smooth muscle including rabbit aorta, rabbit jejunum and trachea. The relaxant effect of N. sativa could be of therapeutic importance such as bronchodilation in asthma, vasodilation in hypertension and therapeutic effect on digestive or urogenital disorders. Therefore in the present article, the relaxant effects of N. sativa and its constituents on smooth muscles and its possible mechanisms as well as clinical application of this effect were reviewed. PMID:25859297

  5. Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

    NASA Astrophysics Data System (ADS)

    Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

    1986-08-01

    Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

  6. Vasopressin induced production of inositol trisphosphate and calcium efflux in a smooth muscle cell line

    SciTech Connect

    Doyle, V.M.; Rueegg, U.T.

    1985-08-30

    Phosphatidylinositol metabolism and /sup 45/Ca/sup 2 +/ efflux were examined in a vascular smooth muscle cell line (A7r5). (Arg 8)Vasopressin stimulated the rapid formation (measurable at 1 sec) of inositol phosphates in a concentration-dependent manner. The time course for formation of inositol phosphates was similar to that for /sup 45/Ca/sup 2 +/ efflux from preloaded cells. The efflux of /sup 45/Ca/sup 2 +/ in response to (Arg8)vasopressin could be inhibited by a vasopressin antagonist. This supports the hypothesis that inositol 1,4,5-trisphosphate plays a role in vasopressin stimulated calcium mobilization from an intracellular source in cultured vascular smooth muscle cells.

  7. The Inhibitory Mechanism of Gentamicin on Electrical Field Stimulation Response in Rat Bladder Smooth Muscle

    PubMed Central

    Min, Chang Ho; Wang, YiYi; Bae, Jinhyung; Han, Jung Hoon

    2015-01-01

    To see the inhibitory mechanism of gentamicin in response to electrical field stimulation (EFS) using the rat bladder smooth muscle, atropine or guanethidine was treated but had no effect. Methylsergide, a non-selective 5-HT1, 5-HT2 receptor antagonist was also treated but had on effect. Kinase inhibitors, such as chelerythrine (PKC inhibitor), ML-9 (MLCK inhibitor), or Y27632 (rho kinase inhibitor) were pretreated before gentamicin treatment, but did not have effect. For U73122, a phospholipase C (PLC) inhibitor however, the inhibitory effect to gentamicin was significantly attenuated in all frequencies given by the EFS. Therefore gentamicin induced inhibitory effect on EFS response in rat bladder smooth muscle was not mediated by the activation of adrenergic, cholinergic, or serotonergic receptor. The inhibition of gentamicin might be mediated through the PLC dependent pathway, but not through the PKC, MLCK or rho kinase dependent pathway. PMID:26330761

  8. Purification of phenylethanoids from Brandisia hancei and the antiproliferative effects on aortic smooth muscle.

    PubMed

    He, Z D; Huang, Y; Yao, X; Lau, C W; Law, W I; Chen, Z Y

    2001-08-01

    The present study describes the isolation and purification of acteoside, 2'-acetylacteoside, poliumoside and brandioside, four phenylethanoid glycosides from Brandisia hancei. We examined their effects on the proliferation of cultured A7r5 rat aortic smooth muscle cells. The proliferative response was measured from the [(3)H]-thymidine incorporation into DNA. All four glycosides suppressed the proliferative response in the presence of 2 % or 5 % fetal bovine serum in a concentration-dependent manner. The rank order of effectiveness for inhibition of cell proliferation was: brandioside > or = poliumoside > 2'-acetylacteoside > or = acteoside. The acetyl group at position 2' of glucose does not seem necessary for the anti-proliferative effects of acteoside and 2'-acetylacteoside, while the hydroxy groups of the aromatic rings appear to play a role. Inhibition of smooth muscle cell proliferation by phenylethanoids indicates that these compounds may have preventative effects on arteriosclerosis. PMID:11509971

  9. Smooth muscle-specific drug targets for next generation Drug-eluting stent

    PubMed Central

    Tang, Rui; Chen, Shiyou

    2015-01-01

    The occurrence of stent thrombosis is one of the major obstacles limiting the long-term clinical efficacy of percutaneous coronary intervention. The anti-smooth muscle proliferation drugs coated on drug-eluting stents (DES) often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, due to their non-specific effect on endothelial cells (EC). Therefore, identification of therapeutic targets that differentially regulate vascular smooth muscle cell (VSMC) and EC proliferation may lead to the development of ideal drugs for next generation DES. Our recent studies have shown that CTP synthase 1 (CTPS1) differentially regulates the proliferation of VSMC and EC following vascular injury. Therefore, CTPS1 inhibitors are promising agents for DES. In addition to CTPS1, other factors have also shown cell-specific effects on VSMC and/or EC proliferation and thus may become potential molecular targets for developing drugs to coat stents. PMID:24325297

  10. Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle-alpha-actin gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Smooth muscle a actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells(vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a ...

  11. BMP-2 gene expression and effects on human vascular smooth muscle cells.

    PubMed

    Willette, R N; Gu, J L; Lysko, P G; Anderson, K M; Minehart, H; Yue, T

    1999-01-01

    Bone morphogenetic proteins (BMPs) and their serine/threonine kinase receptors have been identified in atherosclerotic arteries and vascular smooth muscle cells, respectively. Thus, BMPs (the largest subfamily of the TGF-beta superfamily) have been implicated in the pathogenesis of atherosclerosis. However, the origins of BMP biosynthesis and the functional roles of BMP in blood vessels are unclear. The present study explored BMP-2 gene expression in various human blood vessels and vascular cell types. Functional in vitro studies were also performed to determine the effects of recombinant human BMP-2 on migration (transwell assay) and proliferation ([3H]-thymidine incorporation) of human aortic vascular smooth muscle cells (HASMC). RT-PCR experiments revealed BMP-2 gene expression in normal and atherosclerotic human arteries as well as cultured human aortic and coronary vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs) and human macrophages. In cellular migration studies, incubation with BMP-2 produced efficacious (smooth muscle cell response to vascular injury. PMID:10213907

  12. Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells.

    PubMed Central

    Edwards, I. J.; Wagner, W. D.; Owens, R. T.

    1990-01-01

    Arterial dermatan sulfate proteoglycan has been shown to increase with atherosclerosis progression, but factors responsible for this increase are unknown. To test the hypothesis that smooth muscle cell proteoglycan synthesis may be modified by macrophage products, pigeon arterial smooth muscle cells were exposed to the media of either cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D1. Proteoglycans radiolabeled with [35S]sulfate and [3H]serine were isolated from culture media and smooth muscle cells and purified following precipitation with 1-hexadecylpyridinium chloride and chromatography. Increasing concentrations of macrophage-conditioned media were associated with a dose-response increase in [35S]sulfate incorporation into secreted proteoglycans, but there was no change in cell-associated proteoglycans. Incorporation of [3H]serine into total proteoglycan core proteins was not significantly different (5.2 X 10(5) dpm and 5.5 X 10(5) disintegrations per minute (dpm) in control and conditioned media-treated cultures, respectively), but selective effects were observed on individual proteoglycan types. Twofold increases in dermatan sulfate proteoglycan and limited degradation of chondroitin sulfate proteoglycan were apparent based on core proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoinhibition studies indicated that interleukin-1 was involved in the modulation of proteoglycan synthesis by macrophage-conditioned media. These data provide support for the role of macrophages in alteration of the matrix proteoglycans synthesized by smooth muscle cells and provide a mechanism to account for the reported increased dermatan sulfate/chondroitin sulfate ratios in the developing atherosclerotic lesion. Images Figure 6 PMID:2316626

  13. Membrane properties of the smooth-muscle fibres of the guinea-pig portal vein.

    PubMed

    Ito, Y; Kuriyama, H

    1971-05-01

    The membrane activities and the various characteristic constants of the smooth-muscle membrane of the guinea-pig portal vein were investigated with the micro-electrode technique.1. The mean membrane potential was -37 mV. Spontaneous discharges appeared as regular bursts of short trains of spikes alternating with silent periods, as a mixture of single spikes and bursts of spikes appearing continuously, or as regular spikes with low frequency.2. Spontaneous spikes with overshoot were frequently observed. The maximum rate of rise of the spike was 3.7 V/sec. The shapes of the spikes were classified into three different types, i.e. pace-maker type of spike, monophasic spike and spike with a hump during the falling phase.3. Tetrodotoxin (10(-5) g/ml.) did not influence the patterns of the spontaneous train discharges nor the shape of the spike.4. Extracellularly applied outward current elicited spikes which were either monophasic or had a hump on the falling phase. Inward current elicited break excitation of the spike.5. Current-voltage relations, produced by application of inward current pulses to the tissue and measured at various distances from the stimulating partition, were linear.6. The smooth-muscle membrane of portal vein showed cable-like properties. The mean space constant of the membrane was 0.52 mm; the mean time constant of the membrane calculated from the electrotonic potential was 330 msec.7. Conduction velocity of the spike measured by insertion of two micro-electrodes was 0.58 cm/sec.8. The time constant of the foot of the propagated spike was 27 msec. The time constant of the membrane calculated from the time constant of the foot of the spike and the conduction velocity was 310 msec.9. The membrane properties of longitudinal smooth muscle of the portal vein were discussed in comparison with other veins and various visceral smooth muscles. PMID:5580862

  14. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

    SciTech Connect

    Joo, Hyung Joon; Seo, Ha-Rim; Jeong, Hyo Eun; Choi, Seung-Cheol; Park, Jae Hyung; Yu, Cheol Woong; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2014-07-11

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.

  15. Transforming growth factor-beta as a differentiating factor for cultured smooth muscle cells.

    PubMed

    Gawaziuk, J P; X; Sheikh, F; Cheng, Z-Q; Cattini, P A; Stephens, N L

    2007-10-01

    The aim of the present study was to determine whether the development of supercontractile smooth muscle cells, contributing to the nonspecific hyperreactivity of airways in asthmatic patients, is due to transforming growth factor (TGF)-beta. In cultured smooth muscle cells starved by removal of 10% foetal bovine serum for 7 days, growth arrest was seen; 30% became elongated and demonstrated super contractility. Study of conditioned medium suggested that the differentiating factor was TGF-beta. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on conditioned medium from the arrested cells. Two protein bands were identified as matrix metalloproteinase (MMP)-2 and TGF-beta1. To determine second messenger signalling by SMAD2, Western blotting and confocal microscopy were employed. Conditioned medium from arrested cultures showed the presence of MMP-2 and TGF-beta1, as revealed by SDS-PAGE; 68- and 25-kDa bands were seen. Differentiation was confirmed by upregulation of marker proteins, smooth muscle type myosin heavy chain and myosin light chain kinase. Confirmation was obtained by downregulating these proteins with decorin treatment, which reduces the levels of active TGF-beta and an adenoviral dominant-negative vector coding for a mutated type II TGF-beta-receptor. Activation of second messenger signalling was demonstrated immunocytochemically by the presence of phosphorylated SMAD2 and SMAD4. Transforming growth factor-beta is likely to be the differentiating factor responsible for the development of these supercontractile smooth muscle cells. The development of such cells in vivo after cessation of an asthmatic attack could contribute to the nonspecific hyperreactivity of airways seen in patients. PMID:17596270

  16. Neuropilin 1 Is Essential for Gastrointestinal Smooth Muscle Contractility and Motility in Aged Mice

    PubMed Central

    Yamaji, Maiko; Mahmoud, Marwa; Evans, Ian M.; Zachary, Ian C.

    2015-01-01

    Background and Aims Neuropilin 1 (NRP1) is a non-tyrosine kinase receptor for vascular endothelial growth factor (VEGF) and class 3 semaphorins, playing a role in angiogenesis and neuronal axon guidance, respectively. NRP1 is expressed in smooth muscle cells (SMC) but the functional role of NRP1 in SMC has not been elucidated. We therefore investigated the biological relevance of NRP1 in SMC in vivo by generating mice with SMC-specific Nrp1 deficiency. Methods Conditional gene targeting generated SMC-specific Nrp1 knockout mice (Nrp1SMKO) in which Cre recombinase is driven by the smooth muscle-specific myosin heavy chain (smMHC) promoter. Results SMC-specific Nrp1 deficiency resulted in a significant reduction in intestinal length by 6 months, and, by 18 months, in severe constipation, and enlargement of the intestine consistent with chronic intestinal pseudo-obstruction. These effects were associated with significant thinning of the intestinal smooth muscle, and decreased intestinal contractility. Expression of contractile proteins was reduced in Nrp1SMKO mice, including the smMHC isoform, SMB, whereas we observed a significant increase in the expression of the small-conductance calcium-activated potassium channel 3 (SK3/KCa2.3), implicated in negative regulation of smooth muscle contraction. Conclusions Nrp1 deficiency in visceral SMC results in adult-onset defects in gastrointestinal contractility and motility and causes a shift to a less contractile SMC phenotype. These findings indicate a new role for Nrp1 in the maintenance of the visceral SMC contractile phenotype required for normal GI motility in aged mice. PMID:25659123

  17. Calcifying nanoparticles promote mineralization in vascular smooth muscle cells: implications for atherosclerosis

    PubMed Central

    Hunter, Larry W; Charlesworth, Jon E; Yu, Sam; Lieske, John C; Miller, Virginia M

    2014-01-01

    Background Nano-sized complexes of calcium phosphate mineral and proteins (calcifying nanoparticles [CNPs]) serve as mineral chaperones. Thus, CNPs may be both a result and cause of soft tissue calcification processes. This study determined if CNPs could augment calcification of arterial vascular smooth muscle cells in vitro. Methods CNPs 210 nm in diameter were propagated in vitro from human serum. Porcine aortic smooth muscle cells were cultured for up to 28 days in medium in the absence (control) or presence of 2 mM phosphate ([P] positive calcification control) or after a single 3-day exposure to CNPs. Transmission electron-microscopy was used to characterize CNPs and to examine their cellular uptake. Calcium deposits were visualized by light microscopy and von Kossa staining and were quantified by colorimetry. Cell viability was quantified by confocal microscopy of live-/dead-stained cells and apoptosis was examined concurrently by fluorescent labeling of exposed phosphatidylserine. Results CNPs, as well as smaller calcium crystals, were observed by transmission electron-microscopy on day 3 in CNP-treated but not P-treated cells. By day 28, calcium deposits were visible in similar amounts within multicellular nodules of both CNP- and P-treated cells. Apoptosis increased with cell density under all treatments. CNP treatment augmented the density of apoptotic bodies and cellular debris in association with mineralized multicellular nodules. Conclusion Exogenous CNPs are taken up by aortic smooth muscle cells in vitro and potentiate accumulation of smooth-muscle-derived apoptotic bodies at sites of mineralization. Thus, CNPs may accelerate vascular calcification. PMID:24920905

  18. Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

    PubMed Central

    Waisberg, Daniel Reis; Parra, Edwin Roger; Barbas-Filho, João Valente; Fernezlian, Sandra; Capelozzi, Vera Luiza

    2012-01-01

    OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of interleukin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling. These events seem to be

  19. Immunolocalization of BMP-6, a novel TGF-beta-related cytokine, in normal and atherosclerotic smooth muscle cells.

    PubMed

    Schluesener, H J; Meyermann, R

    1995-03-01

    We have analyzed expression of a novel transforming growth factor type beta (TGF-beta)-related cytokine, bone morphogenetic protein-6 (BMP-6) in normal and atherosclerotic brain arteries. BMP-6 immunoreactivity was detected in smooth muscle cells of normal cerebral blood vessels. It is also expressed by smooth muscle cells of intimal plaques in atherosclerotically changed blood vessels. The BMPs regulate tissue modeling and remodeling and aberrant expression of BMPs might contribute to smooth muscle cell migration, proliferation, tissue reorganization and macrophage attraction, which are known mechanisms of atherosclerotic plaque formation. PMID:7605353

  20. Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media.

    PubMed Central

    Gabbiani, G; Kocher, O; Bloom, W S; Vandekerckhove, J; Weber, K

    1984-01-01

    Actin of smooth muscle cells of rat and human aortic media shows a predominance of the alpha-isoform. In experimental rat aortic intimal thickening, in human atheromatous plaque, and in cultured aortic smooth muscle cells, there is a typical switch in actin expression with a predominance of the beta-form and a noticeable amount of gamma-form. This pattern of actin expression represents a new reliable protein-chemical marker of experimental and human atheromatous smooth muscle cells. Images PMID:6690475

  1. Long-Term Expression of Human Adenosine Deaminase in Vascular Smooth Muscle Cells of Rats: A Model for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Lynch, Carmel M.; Clowes, Monika M.; Osborne, William R. A.; Clowes, Alexander W.; Dusty Miller, A.

    1992-02-01

    Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli β-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

  2. Mechanism of action of barium ion on rat aortic smooth muscle.

    PubMed

    Hansen, T R; Dineen, D X; Petrak, R

    1984-03-01

    The mechanism of action of barium ion on the aortic smooth muscle of the normal rat was investigated using in vitro calcium-depleted aortic strips. Aortic strips were depleted of calcium by repeated exposure to norepinephrine in a calcium-free bathing solution. Although calcium depletion abrogated the response of strips to catecholamines and depolarizing agents, the response to barium chloride remained quantitatively intact. The calcium influx blocker D 600 prevented the contractile response to barium but not to catecholamines, whereas phentolamine prevented the response to catecholamines but not barium. The strip response to barium was depressed by a twofold increase in extracellular magnesium concentration whether the strip was intact or calcium depleted. Although increased concentrations of calcium in the extracellular medium inhibited the contractile response to potassium ion, increases in barium merely potentiated the potassium contracture. These findings indicate that barium produces its contractile effect on vascular smooth muscle by a direct intracellular interaction with the contractile or regulatory proteins. Barium enters these cells via calcium influx channels and is probably not sequestered in a physiologically releasable pool. Unlike calcium, barium does not stabilize the smooth muscle sarcolemma when present in high concentration. PMID:6703038

  3. Statin-exposed vascular smooth muscle cells secrete proteoglycans with decreased binding affinity for LDL.

    PubMed

    Meyers, C Daniel; Tannock, Lisa R; Wight, Thomas N; Chait, Alan

    2003-11-01

    Retention of LDL in the artery intima is mediated by extracellular matrix proteoglycans and plays an important role in the initiation of atherosclerosis. Compared with quiescent cells, proliferating smooth muscle cells secrete proteoglycans with elongated glycosaminoglycan side chains, which have an increased binding affinity to LDL. Because 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) decrease smooth muscle cell proliferation, we hypothesized that statin exposure would decrease both the size and LDL binding affinity of vascular proteoglycans. Monkey aortic smooth muscle cells grown in culture were exposed to simvastatin (10 and 100 microM) and cerivastatin (0.1 and 1 microM), and newly secreted proteoglycans were quantified and characterized. Both simvastatin and cerivastatin caused a concentration-dependent reduction in cell growth and reduced 35SO4 incorporation into secreted proteoglycans, on both an absolute and a per cell basis. Interestingly, statin exposure increased the apparent molecular weight and hydrodynamic size of secreted proteoglycans. However, proteoglycans secreted from statin-exposed cells demonstrated a reduction in binding affinity to LDL. Thus, statins may induce atheroprotective changes in vascular proteoglycans and lower LDL retention in the vessel wall. These findings suggest a mechanism whereby statins may benefit atherosclerosis in a manner unrelated to serum LDL lowering. PMID:12923222

  4. Allosteric interactions of three muscarine antagonists at bovine tracheal smooth muscle and cardiac M2 receptors.

    PubMed

    Roffel, A F; Elzinga, C R; Meurs, H; Zaagsma, J

    1989-03-01

    The kinetics of [3H]dexetimide dissociation from muscarine receptors in bovine cardiac left ventricular and tracheal smooth muscle membranes were studied in the absence and presence of three muscarine antagonists. It was found that [3H]dexetimide dissociation from cardiac muscarine receptors was monophasic and very fast (half life less than 1 min) and was slowed by the cardioselective muscarine antagonists, gallamine, methoctramine and AF-DX 116, concentration dependently. [3H]Dexetimide dissociation from tracheal muscarine receptors was biphasic, with a fast phase (half-life less than 1 min) followed after 4-5 min by a slow phase (half-life = 38.5 min). The fast component, but not the slow component, was slowed by the muscarine antagonists with concentration dependencies very similar to those found in the heart. We conclude from these data that the major population of tracheal smooth muscle muscarine receptors resembles the cardiac M2 type not only with respect to equilibrium binding affinities but also with respect to the secondary, allosteric binding site on the muscarine receptor. The results also imply that the cardiac receptor subtype is much more sensitive to allosteric modulation than the glandular/smooth muscle receptor subtype. PMID:2714370

  5. IGF-1 Has Plaque-Stabilizing Effects in Atherosclerosis by Altering Vascular Smooth Muscle Cell Phenotype

    PubMed Central

    von der Thüsen, Jan H.; Borensztajn, Keren S.; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; van Berkel, Theo J.C.; Biessen, Erik A.L.

    2011-01-01

    Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype. PMID:21281823

  6. IGF-1 has plaque-stabilizing effects in atherosclerosis by altering vascular smooth muscle cell phenotype.

    PubMed

    von der Thüsen, Jan H; Borensztajn, Keren S; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; van Berkel, Theo J C; Biessen, Erik A L

    2011-02-01

    Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype. PMID:21281823

  7. Bladder Smooth Muscle Cells Differentiation from Dental Pulp Stem Cells: Future Potential for Bladder Tissue Engineering.

    PubMed

    Song, Bing; Jiang, Wenkai; Alraies, Amr; Liu, Qian; Gudla, Vijay; Oni, Julia; Wei, Xiaoqing; Sloan, Alastair; Ni, Longxing; Agarwal, Meena

    2016-01-01

    Dental pulp stem cells (DPSCs) are multipotent cells capable of differentiating into multiple cell lines, thus providing an alternative source of cell for tissue engineering. Smooth muscle cell (SMC) regeneration is a crucial step in tissue engineering of the urinary bladder. It is known that DPSCs have the potential to differentiate into a smooth muscle phenotype in vitro with differentiation agents. However, most of these studies are focused on the vascular SMCs. The optimal approaches to induce human DPSCs to differentiate into bladder SMCs are still under investigation. We demonstrate in this study the ability of human DPSCs to differentiate into bladder SMCs in a growth environment containing bladder SMCs-conditioned medium with the addition of the transforming growth factor beta 1 (TGF-β1). After 14 days of exposure to this medium, the gene and protein expression of SMC-specific marker (α-SMA, desmin, and calponin) increased over time. In particular, myosin was present in differentiated cells after 11 days of induction, which indicated that the cells differentiated into the mature SMCs. These data suggested that human DPSCs could be used as an alternative and less invasive source of stem cells for smooth muscle regeneration, a technology that has applications for bladder tissue engineering. PMID:26880982

  8. Globular adiponectin reduces vascular calcification via inhibition of ER-stress-mediated smooth muscle cell apoptosis

    PubMed Central

    Lu, Yan; Bian, Yunfei; Wang, Yueru; Bai, Rui; Wang, Jiapu; Xiao, Chuanshi

    2015-01-01

    Objective: This study aims to explore the mechanism of globular adiponectin inhibiting vascular calcification. Methods: We established drug-induced rat vascular calcification model, globular adiponectin was given to observe the effect of globular Adiponectin on the degree of calcification. The markers of vascular calcification and apoptosis were also investigated. Meanwhile, the in vitro effect of globular Adiponectin on vascular calcification was also evaluated using primary cultured rat vascular smooth muscle cells. Results: We found that globular adiponectin could inhibit drug-induced rat vascular calcification significantly in vivo. The apoptosis of vascular smooth muscle cells was also reduced. The possible mechanism could be the down-regulation of endoplasmic reticulum stress by globular adiponectin. Experiments in primary cultured vascular smooth muscle cells also confirmed that globular adiponectin could reduce cell apoptosis to suppress vascular calcification via inhibition of endoplasmic reticulum stress. Conclusions: This study confirmed that globular adiponectin could suppress vascular calcification; one of the mechanisms could be inhibition of endoplasmic reticulum stress to reduce cell apoptosis. It could provide an effective method in the therapy of vascular calcification-associated diseases. PMID:26045760

  9. Cardiac myofibroblasts express alpha smooth muscle actin during right ventricular pressure overload in the rabbit.

    PubMed Central

    Leslie, K. O.; Taatjes, D. J.; Schwarz, J.; vonTurkovich, M.; Low, R. B.

    1991-01-01

    A number of changes occur in contractile proteins and mechanical performance of the heart within 2 weeks of right ventricular pressure overload in 8- to 12-week-old rabbits. These changes are accompanied by increases in collagen concentration and the ratio of type I to type III collagen. The purpose of the present study was to evaluate the evolution of these connective tissue changes morphologically and to characterize the interstitial cells that might be responsible. The myocardium is infiltrated by mononuclear inflammatory cells 2 days after banding, accompanied by focal myocyte necrosis. By 7 days, the inflammatory infiltrates subside and the damaged myocytes seen at 2 days are replaced by new collagen and a population of spindle-shaped cells, with ultrastructural features of myofibroblasts. A significant proportion of these cells contain alpha smooth muscle actin by immunohistochemical analysis. At 14 days, there is a large increase in stainable collagen with complex remodeling and reduplication of the collagen fiber network of the interstitium. Alpha smooth muscle actin-containing myofibroblasts persist, but their immunoreactivity appears reduced compared with day 7. The authors hypothesize that the interstitial fibroblasts that acquire smooth-muscle-like features in this model play a critical role in the heart's response to severe and sudden mechanical stress and are at least partly responsible for the changes in connective tissue that occur as a result of pressure overload in this model. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1853934

  10. Electrospun elastin-like polypeptide enriched polyurethanes and their interactions with vascular smooth muscle cells.

    PubMed

    Blit, Patrick H; Battiston, Kyle G; Yang, Meilin; Paul Santerre, J; Woodhouse, Kimberly A

    2012-07-01

    In vascular tissue, elastin is an essential extracellular matrix protein that plays an important biomechanical and biological signalling role. Native elastin is insoluble and is difficult to extract from tissues, which results in its relatively rare use for the fabrication of vascular tissue engineering scaffolds. Recombinant elastin-like polypeptide-4 (ELP4), which mimics the structure and function of native tropoelastin, represents a practical alternative to the native elastic fibre for vascular applications. In this study, electrospinning was utilized to fabricate fibrous scaffolds which were subsequently surface modified with ELP4 and used as substrates for smooth muscle cell culture. ELP4 surface modified materials demonstrated enhanced smooth muscle cell (SMC) adhesion and maintenance of cell numbers over a 1-week period relative to controls. SMCs seeded on the ELP4 surface modified materials were also shown to exhibit the cell morphology and biological markers of a contractile phenotype including a spindle-like morphology, actin filament organization and smooth muscle myosin heavy chain expression. Competitive inhibition experiments demonstrated that the elastin-laminin cell surface receptor and its affinity for the VGVAPG peptide sequence on ELP4 molecules are likely involved in the initial SMC contact with the ELP4 modified materials. Elastin-like polypeptides show promise as surface modifiers for candidate scaffolds for engineering contractile vascular tissues. PMID:22459513

  11. Endothelin B receptors on human endothelial and smooth-muscle cells show equivalent binding pharmacology.

    PubMed

    Flynn, M A; Haleen, S J; Welch, K M; Cheng, X M; Reynolds, E E

    1998-07-01

    We have described the pharmacologic profiles of endothelin B receptors in human endothelial cells and vascular and nonvascular smooth-muscle cells. First, by amplifying endothelin B receptor numbers through the use of phosphoramidon and intact cell-binding techniques, we demonstrated the presence of these receptors in human umbilical vein endothelial cells (100% endothelin B receptors), human aortic smooth-muscle cells (22% endothelin B, 78% endothelin A receptors), and human bronchial smooth-muscle cells (55% endothelin B, 45% endothelin A receptors) by using [125I]-endothelin-1 radioligand binding. The typical binding profiles of the endothelin B receptors were established through competition binding curve analysis with endothelin-1, endothelin-3, sarafotoxin 6c, and the endothelin A receptor-selective antagonist BQ-123. In the presence of BQ-123, a diverse group of antagonists, including PD 142893, BQ-788, SB 209670, and Ro 47-0203, were used to probe for binding differences indicative of multiple endothelin B-receptor subtypes. The results indicate a rank order of potency for the antagonists of BQ-788 > SB 209670 > PD 142893 > Ro 47-0203 for each cell line, and that between any of these human cell lines, measurements of [125I]-endothelin-1-binding antagonism for each of the four test compounds differed by less than twofold. Although this study cannot discount the possibility of more than one endothelin B-receptor subtype in humans, it does indicate that these tissues express receptors that show equivalent binding pharmacology. PMID:9676729

  12. Cell, matrix changes and alpha-smooth muscle actin expression in repair of the canine meniscus.

    PubMed

    Kambic, H E; Futani, H; McDevitt, C A

    2000-01-01

    Processes in the repair of a crevice in the knee joint meniscus were investigated in 10 dogs. Two 2-mm cylindrical plugs from each medial meniscus were removed, rendered acellular by freezing and thawing, and then reinserted into the meniscus. Dogs were euthanized at intervals of 3-52 weeks after surgery. The crevice between the plug and meniscus at 3 weeks after surgery was filled with a tissue containing alpha-smooth muscle actin-positive cells. One year after surgery, the plug had remodeled and was populated with spindle-shaped and fibrochondrocyte-like cells. The plug had an appearance intermediate between that of hyaline and fibrocartilage at this time, with a seamless integration in sites between the remodeled plug and the surrounding meniscus. alpha-smooth muscle actin-positive cells were concentrated at the interface of the remodeled plug and adjacent meniscus and at the surface of the plug. Therefore, remodeling of both the plug and meniscal tissue and the participation of alpha-smooth muscle actin-positive cells appear essential for integration of the plug into the adjacent meniscal tissue. Cells in the superficial zone of the meniscus seem to be active in the repair process. A change in both the phenotype of the cells and the quality of the matrix toward a more hyaline state appears to be an integral part of the remodeling process in the meniscus. PMID:11208183

  13. Advanced glycation endproducts regulate smooth muscle cells calcification in cultured HSMCs

    PubMed Central

    He, Hu-Qiang; Liu, Yong; Zeng, Hong; Sun, Xiao-Lei; Zhang, Lei; Zhang, Xue-Lin; Liao, Wen-Jun; Zhou, Xiang-Yu; He, Yan-Zheng

    2015-01-01

    Objective: To investigate the mechanism of Advanced glycation end products (AGEs) promoting the calcification of smooth muscle cells. Methods: The successfully cultured smooth muscle cells were divided into three groups: normal culture group (group A), calcified culture group (group B), calcification + AGEs group (group C); the concentration of intracellular calcium ion was detected in each group; the promotion of AGEs on the calcification of HSMCs was confirmed by VON KOSSA staining; and the expressions of β-catenin, RAGE, β-catenin, OPG and E-cadherin protein were detected by immunofluorescence and western blot. Results: The morphology of the cells in each group showed that the amount of calcified plaques in calcification + AGES group were significantly higher than the calcification group. VON KOSSA staining showed that with increasing concentrations of AGE-BSA, the amount of its calcification gradually increased. Calcium concentration in Calcification + 20 mg/L AGEs group was significantly higher, followed by 40 mg/L AGEs group. The expression of β-catenin increased with the increasing concentrations of AGEs. Conclusion: AGEs can promote the calcification of human femoral artery smooth muscle cells, with a concentration gradient effect. With increasing concentrations of AGEs, the expression of RAGE increased, indicating that AGEs-induced HSMCs proliferation was correlated with RAGE expression. PMID:26722411

  14. Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice

    SciTech Connect

    Orlandi, Augusto . E-mail: orlandi@uniroma2.it; Ferlosio, Amedeo; Gabbiani, Giulio; Spagnoli, Luigi Giusto; Ehrlich, Paul H.

    2005-12-10

    Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less {alpha}2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of {alpha}-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express {alpha}-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Anti-{alpha}2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC-PLCs; anti-{alpha}1 and anti-{alpha}2 integrin antibodies induced a similar slight inhibition in TI-SMC-PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs.

  15. Steroids augment relengthening of contracted airway smooth muscle: potential additional mechanism of benefit in asthma.

    PubMed

    Lakser, O J; Dowell, M L; Hoyte, F L; Chen, B; Lavoie, T L; Ferreira, C; Pinto, L H; Dulin, N O; Kogut, P; Churchill, J; Mitchell, R W; Solway, J

    2008-11-01

    Breathing (especially deep breathing) antagonises development and persistence of airflow obstruction during bronchoconstrictor stimulation. Force fluctuations imposed on contracted airway smooth muscle (ASM) in vitro result in its relengthening, a phenomenon called force fluctuation-induced relengthening (FFIR). Because breathing imposes similar force fluctuations on contracted ASM within intact lungs, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. While this bronchoprotective effect appears to be impaired in asthma, corticosteroid treatment can restore the ability of deep breaths to reverse artificially induced bronchoconstriction in asthmatic subjects. It has previously been demonstrated that FFIR is physiologically regulated through the p38 mitogen-activated protein kinase (MAPK) signalling pathway. While the beneficial effects of corticosteroids have been attributed to suppression of airway inflammation, the current authors hypothesised that alternatively they might exert their action directly on ASM by augmenting FFIR as a result of inhibiting p38 MAPK signalling. This possibility was tested in the present study by measuring relengthening in contracted canine tracheal smooth muscle (TSM) strips. The results indicate that dexamethasone treatment significantly augmented FFIR of contracted canine TSM. Canine tracheal ASM cells treated with dexamethasone demonstrated increased MAPK phosphatase-1 expression and decreased p38 MAPK activity, as reflected in reduced phosphorylation of the p38 MAPK downstream target, heat shock protein 27. These results suggest that corticosteroids may exert part of their therapeutic effect through direct action on airway smooth muscle, by decreasing p38 mitogen-activated protein kinase activity and thus increasing force fluctuation-induced relengthening. PMID:18768574

  16. Smooth muscle cell progenitors are primed to muscularize in pulmonary hypertension

    PubMed Central

    Sheikh, Abdul Q.; Misra, Ashish; Rosas, Ivan O.; Adams, Ralf H.; Greif, Daniel M.

    2015-01-01

    Excess and ectopic smooth muscle cells (SMCs) are central to cardiovascular disease pathogenesis, but underlying mechanisms are poorly defined. For instance, pulmonary hypertension (PH) or elevated pulmonary artery blood pressure is a devastating disease with distal extension of smooth muscle to normally unmuscularized pulmonary arterioles. We identify novel SMC progenitors that are located at the pulmonary arteriole muscular-unmuscular border and express both SMC markers and the undifferentiated mesenchyme marker platelet-derived growth factor receptor-β (PDGFR-β). We term these cells “primed” because in hypoxia-induced PH, they express the pluripotency factor Kruppel-like factor 4 (KLF4), and in each arteriole, one of them migrates distally, dedifferentiates, and clonally expands, giving rise to the distal SMCs. Furthermore, hypoxia-induced expression of the ligand PDGF-B regulates primed cell KLF4 expression, and enhanced PDGF-B and KLF4 levels are required for distal arteriole muscularization and PH. Finally, in PH patients, KLF4 is markedly up-regulated in pulmonary arteriole smooth muscle, especially in proliferating SMCs. In sum, we have identified a pool of SMC progenitors that are critical for the pathogenesis of PH, and perhaps other vascular disorders, and therapeutic strategies targeting this cell type promise to have profound implications. PMID:26446956

  17. Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro.

    PubMed

    Morioka, Hideo; Morii, Takeshi; Vogel, Tikva; Hornicek, Francis J; Weissbach, Lawrence

    2003-07-01

    Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by alpha(v)-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes. PMID:12799192

  18. Cavernosum smooth muscle relaxation induced by Schisandrol A via the NO-cGMP signaling pathway.

    PubMed

    Liu, W; Choi, B R; Bak, Y O; Zhang, L T; Zhou, L X; Huang, Y R; Zhao, C; Park, J K

    2016-01-01

    To evaluate the effect of Schisandrol A on rabbit corpus cavernosum smooth muscle and elucidate the potential mechanism. Penises were obtained from healthy male New Zealand White rabbits (2.5-3.0 kg). The pre-contracted penis with phenylephrine (Phe, 10 µM) was treated with accumulative concentrations of Schisandrol A (10-7, 10-6, 10-5 and 10-4 M). The change in intracavernosum pressure (ICP) and tension was recorded, cyclic nucleotides in the cavernosum tissue were measured by radioimmunoassay, mRNA level and expression of endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS) were measured by real time PCR and western blot respectively. The corpus cavernosum smooth muscle relaxation induced by Schisandrol A was in a dose-dependent manner. Pre-treatment with NOS inhibitor (Nω nitro-L-arginine-methyl ester, L-NAME) or guanylyl cyclase inhibitor (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, ODQ) significantly diminished the relaxation. The cyclic guanosine monophosphate (cGMP) level was significantly increased in the cavernosum tissue. Real time PCR and western blot showed the mRNA level and expression of eNOS and nNOS was also upregulated. Schisandrol A relaxes the cavernosum smooth muscle by activating NO-cGMP signaling pathway. It may be a new promising treatment for erectile dysfunction and cardiovascular disease. PMID:27064883

  19. Effect of controlled adventitial heparin delivery on smooth muscle cell proliferation following endothelial injury.

    PubMed

    Edelman, E R; Adams, D H; Karnovsky, M J

    1990-05-01

    Continuous intravenous infusion of heparin suppresses smooth muscle cell proliferation in rats after endothelial injury but may lead to hemorrhage and other complications. The anticoagulant property has been removed from chemically modified heparin without loss of antiproliferative effect but use of such compounds is still limited. In this study ethylene-vinyl acetate copolymer matrices containing standard and modified heparin were placed adjacent to rat carotid arteries at the time of balloon dendothelialization. After 14 days arterial occlusion by smooth muscle cell proliferation was defined. Matrix delivery of both heparin compounds effectively diminished this proliferation in comparison to controls without producing systemic anticoagulation or side effects. In addition, this mode of therapy appeared more effective than the administration of the same agents by either intravenous pumps or heparin/polymer matrices placed in a subcutaneous site distant from the injured carotid artery. Thus, heparin's inhibition of smooth muscle cell proliferation after vascular injury might be most effective within the microenvironment of the injured vessel wall, and the accelerated atherosclerosis or restenosis that often follows angioplasty and other vascular interventions might best be treated with site-specific therapy. PMID:2339120

  20. The role of protein breakdown in growth, quiescence, and starvation of vascular smooth muscle cells.

    PubMed

    Libby, P; O'Brien, K V

    1984-03-01

    Protein accumulation in growing cells may be due in part to a reduction in the rate of protein breakdown. Previous studies of the relation of cell proliferation to protein degradation often produced growth arrest by conditions that may involve nutritional deprivation. However, nutrient lack can itself accelerate proteolysis and produce negative protein balance. We therefore reexamined the relation between growth and protein breakdown using a more selective method for limiting cell growth. We produced quiescent cell cultures using a chemically defined, serum-free medium supplemented with hormones and nutrients. Such media can maintain viability and near neutral protein balance in cultured vascular smooth muscle cells, in part because of reduced breakdown of cellular protein. We then compared rates of protein degradation in these quiescent but not starving cells, to those of cultures stimulated to grow by addition of mitogenic substances. Platelet-derived growth factor, fibroblast growth factor, or fetuin added to insulin-containing medium stimulated growth of smooth muscle cells, but further reduced protein breakdown only slightly. Contrary to the implications of certain previous studies, our results show that proliferating cells can accumulate protein without an appreciable reduction in the rates of protein breakdown. Thus, while accelerated proteolysis appears to be an important adaptation to adverse nutritional conditions, growth of smooth muscle cells does not require changes in overall protein breakdown, but occurs primarily through an increase in protein synthesis. PMID:6365933

  1. Steroid receptors in canine and human female genital tract tumours with smooth muscle differentiation.

    PubMed

    Millán, Y; Gordon, A; de los Monteros, A Espinosa; Reymundo, C; de las Mulas, J Martín

    2007-01-01

    The expression of oestrogen receptor-alpha (ERalpha) and progesterone receptor (PR) was examined in 32 canine genital tract tumours diagnosed as smooth muscle tumours (benign or malignant, pure or mixed). The immunohistochemical expression of calponin was used to assess the smooth muscle differentiation of the tumours. Nineteen human uterine leiomyomas were also examined. Calponin expression was detected in 89.3% of canine and 100% of human genital tract tumours diagnosed as leiomyomas, as well as in the majority of other tumours examined (canine or human, genital or extragenital, benign or malignant) with the exception of canine negative control tumours (cutaneous fibroma and hepatoid gland adenoma). ERalpha was found in 56.3% of canine and 52.6% of human leiomyomas, while PR was found in 84.4% of canine and 94.7% of human tumours. These results indicate that calponin is a good marker for differentiating neoplasia of the canine genital system of uncertain origin, as in human patients. They also show that canine tumours with smooth muscle differentiation of the genital tract of the bitch express steroid hormone receptors, a finding that opens up the possibility of hormone therapy. PMID:17362977

  2. Exposure of immature rats to hyperoxia increases tracheal smooth muscle stress generation in vitro.

    PubMed

    Hershenson, M B; Wylam, M E; Punjabi, N; Umans, J G; Schumacker, P T; Mitchell, R W; Solway, J

    1994-02-01

    Recently, we demonstrated that chronic exposure to hyperoxia causes in vivo airway muscarinic receptor hyperresponsiveness in the developing rat [Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L263-L269, 1992]. To test whether airway cholinergic hyperresponsiveness might result from intrinsic alterations in smooth muscle contractility, we measured the effect of in vivo hyperoxia on the contractile force elicited by acetylcholine (ACh) of isometrically mounted tracheal rings in vitro. Tracheal rings were obtained from 3-wk-old rats exposed to air or to > 95% O2 for 8 days. Muscarinic responses were determined by measuring the force elicited by exposure to increasing concentrations of ACh. Responses were normalized to the morphometrically determined tracheal smooth muscle cross-sectional area in a plane perpendicular to the axis of force generation. In vivo O2 exposure significantly increased maximal ACh-induced stress generation (response to 10(-3) M ACh: air, 15.92 +/- 1.37 g/mm2; O2, 21.78 +/- 1.52 g/mm2; P = 0.010). The ACh-induced stress generation of cylinders from hyperoxic rats was substantially reduced by both epithelial removal and treatment with the cyclooxygenase inhibitor indomethacin. We conclude that in vivo hyperoxic exposure increases tracheal smooth muscle contractile function in vitro and that epithelium-derived prostaglandin(s) contributes to the observed increase in maximal contractile responsiveness. PMID:8175585

  3. Alteration by phosphatidyl serine of tension responses and 45Ca distribution in aortic smooth muscle.

    PubMed

    Goodman, F R; Weiss, G B; Goth, A

    1976-07-01

    The effects of phosphatidyl serine (PS) on 45Ca distribution, 45Ca movements and contractions were examined in rabbit aortic smooth muscle. Contractile responses to submaximal concentrations of norepinephrine and histamine were potentiated by prior exposure to PS, but equivalent responses to potassium were unaffected. Addition of PS to the incubation solution decreased 45Ca uptake; exposure of aortic strips to PS during washout of either 45Ca or promethium (147Pm) resulted in maintained increases in efflux. These PS-induced alterations in net loss of 45Ca or 147Pm can be attributed to a decreased membrane reuptake and/or rebinding. However, the presence of PS during the washout significantly reduced the increases in 45Ca efflux rate elicited with either 0.05 mM concentrations of Ca++ or ethylenediamine tetraacetic acid. Thus, in rabbit aortic smooth muscle, exogenous PS can alter the availability and/or exchangeability of a membrane-bound Ca++ fraction. By specifically increasing the affinity for Ca++ at relevant membrane sites or stores. PS may enhance the ability of vascular smooth muscle to respond to stimulatory agents that mobilize Ca++ from these sites and, in this manner, potentiate contractile responses. PMID:933004

  4. Heparin fragments inhibit human vascular smooth muscle cell proliferation in vitro

    SciTech Connect

    Selden, S.C.; Johnson, W.V.; Maciag, T.

    1986-03-01

    The authors have examined the effect of heparin on human abdominal aortic smooth muscle cell growth. Cell proliferation was inhibited by more than 90% at a concentration of 20 ..mu..g/ml in a 12 day growth assay using heparin from Sigma, Upjohn or Calbiochem. Additionally, 200 ..mu..g/ml Upjohn heparin inhibits /sup 3/H-thymidine incorporation by 50% in short term assays using serum or purified platelet-derived growth factor (25-100ng/ml) to initiate the cell cycle. Homogeneous size classes of heparin fragments were prepared by nitrous acid cleavage and BioGel P-10 filtration chromatography. Deca-, octa-, hexa-, tetra-, and di-saccharides inhibited proliferation by 90% at concentrations of 280, 320, 260, 180 and 100 ..mu..g/ml, respectively, in a 12 day growth assay. These data confirm the work of Castellot et.al. and extend the range of inhibitory fragments down to the tetra- and di-saccharide size. These data suggest, therefore, that di-saccharide subunit of heparin is sufficient to inhibit vascular smooth muscle cell proliferation. The authors are now examining the role of the anhydromannose moiety on the reducing end of the nitrous acid generated fragments as a possible mediator of heparin-induced inhibition of vascular smooth muscle cell proliferation.

  5. Bladder Smooth Muscle Cells Differentiation from Dental Pulp Stem Cells: Future Potential for Bladder Tissue Engineering

    PubMed Central

    Song, Bing; Jiang, Wenkai; Alraies, Amr; Liu, Qian; Gudla, Vijay; Oni, Julia; Wei, Xiaoqing; Sloan, Alastair; Ni, Longxing; Agarwal, Meena

    2016-01-01

    Dental pulp stem cells (DPSCs) are multipotent cells capable of differentiating into multiple cell lines, thus providing an alternative source of cell for tissue engineering. Smooth muscle cell (SMC) regeneration is a crucial step in tissue engineering of the urinary bladder. It is known that DPSCs have the potential to differentiate into a smooth muscle phenotype in vitro with differentiation agents. However, most of these studies are focused on the vascular SMCs. The optimal approaches to induce human DPSCs to differentiate into bladder SMCs are still under investigation. We demonstrate in this study the ability of human DPSCs to differentiate into bladder SMCs in a growth environment containing bladder SMCs-conditioned medium with the addition of the transforming growth factor beta 1 (TGF-β1). After 14 days of exposure to this medium, the gene and protein expression of SMC-specific marker (α-SMA, desmin, and calponin) increased over time. In particular, myosin was present in differentiated cells after 11 days of induction, which indicated that the cells differentiated into the mature SMCs. These data suggested that human DPSCs could be used as an alternative and less invasive source of stem cells for smooth muscle regeneration, a technology that has applications for bladder tissue engineering. PMID:26880982

  6. Myosin Phosphatase Isoforms as Determinants of Smooth Muscle Contractile Function and Calcium Sensitivity of Force Production

    PubMed Central

    DIPPOLD, RACHAEL P.; FISHER, STEVEN A.

    2014-01-01

    The dephosphorylation of myosin by the MP causes smooth muscle relaxation. MP is also a key target of signals that regulate vascular tone and thus blood flow and pressure. Here, we review studies from the past two decades that support the hypothesis that the regulated expression of MP subunits is a critical determinant of smooth muscle responses to constrictor and dilator signals. In particular, the highly regulated splicing of the regulatory subunit Mypt1 Exon 24 is proposed to tune sensitivity to NO/cGMP-mediated relaxation. The regulated transcription of the MP inhibitory subunit CPI-17 is proposed to determine sensitivity to agonist-mediated constriction. The expression of these subunits is specific in the microcirculation and varies in developmental and disease contexts. To date, the relationship between MP subunit expression and vascular function in these different contexts is correlative; confirmation of the hypothesis will require the generation of genetically engineered mice to test the role of MP subunits and their isoforms in the specificity of vascular smooth muscle responses to constrictor and dilator signals. PMID:24112301

  7. CCN1 suppresses pulmonary vascular smooth muscle contraction in response to hypoxia

    PubMed Central

    Lee, Seon-jin; Zhang, Meng; Hu, Kebin; Lin, Ling; Zhang, Duo

    2015-01-01

    Abstract Pulmonary vasoconstriction and increased vascular resistance are common features in pulmonary hypertension (PH). One of the contributing factors in the development of pulmonary vasoconstriction is increased pulmonary artery smooth muscle cell (PASMC) contraction. Here we report that CCN1, an extracellular matrix molecule, suppressed PASMC contraction in response to hypoxia. CCN1 (Cyr61), discovered in past decade, belongs to the Cyr61-CTGF-Nov (CCN) family. It carries a variety of cellular functions, including angiogenesis and cell adhesion, death, and proliferation. Hypoxia robustly upregulated the expression of CCN1 in the pulmonary vessels and lung parenchyma. Given that CCN1 is a secreted protein and functions in a paracine manner, we examined the potential effects of CCN1 on the adjacent smooth muscle cells. Interestingly, bioactive recombinant CCN1 significantly suppressed hypoxia-induced contraction in human PASMCs in vitro. Consistently, in the in vivo functional studies, administration of bioactive CCN1 protein significantly decreased right ventricular pressure in three different PH animal models. Mechanistically, protein kinase A–pathway inhibitors abolished the effects of CCN1 in suppressing PASMC contraction. Furthermore, CCN1-inhibited smooth muscle contraction was independent of the known vasodilators, such as nitric oxide. Taken together, our studies indicated a novel cellular function of CCN1, potentially regulating the pathogenesis of PH. PMID:26697179

  8. Hypertonic upregulation of amino acid transport system A in vascular smooth muscle cells.

    PubMed

    Chen, J G; Klus, L R; Steenbergen, D K; Kempson, S A

    1994-08-01

    The A10 line of vascular smooth muscle cells has Na+ dependent transport systems for alanine, proline, and Pi, whereas uptake of leucine, myo-inositol and D-glucose is Na+ independent. When A10 cells were incubated for 4 h in medium made hypertonic by addition of sucrose, there was a marked increase in Na(+)-dependent transport of alanine and proline but no change in Na(+)-dependent Pi uptake or Na(+)-independent uptake of leucine and inositol. Intracellular alanine content was increased 61% by the hypertonic treatment. Other nonpenetrating solutes, such as cellobiose and mannitol, reproduced the effect of sucrose, but urea, a penetrating solute, did not. Studies with 2-(methylamino)-isobutyric acid revealed that the upregulation by hypertonicity involved only system A. Increases in alanine and proline uptake also occurred after incubating the cells in isotonic medium containing 0.1 mM ouabain, suggesting that an increase in intracellular Na+ may be part of the intracellular signal for upregulation of system A. Hypertonic upregulation of Na(+)-dependent alanine transport occurred also in primary cultures of vascular smooth muscle cells. The response was blocked by actinomycin D and cycloheximide, indicating that gene transcription and protein synthesis play important roles in the mechanism leading to increased alanine uptake. We conclude that vascular smooth muscle cells, during prolonged hypertonic stress, activate system A and accumulate specific neutral amino acids which may act as organic osmolytes to help maintain normal cell volume. PMID:8074188

  9. Smooth muscle BK channel activity influences blood pressure independent of vascular tone in mice

    PubMed Central

    Sachse, Gregor; Faulhaber, Jörg; Seniuk, Anika; Ehmke, Heimo; Pongs, Olaf

    2014-01-01

    The large conductance voltage- and Ca2+-activated K+ (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKβ1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKβ1 knockout (BKβ1−/−) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKβ1−/− mice independent of genetic background, BKβ1−/− strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKβ1+/+ controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKβ1−/− strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKβ1+/+ strain A level. In contrast, loss of BKβ1 did not affect plasma aldosterone in strain C mice. Smooth muscle-restricted restoration of BKβ1 expression increased blood pressure in BKβ1−/− strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways. PMID:24687584

  10. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence.

    PubMed

    Wang, Zhe; Wen, Yan; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-03-15

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 10(6) cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  11. The role of TRPP2 in agonist-induced gallbladder smooth muscle contraction.

    PubMed

    Zhong, Xingguo; Fu, Jie; Song, Kai; Xue, Nairui; Gong, Renhua; Sun, Dengqun; Luo, Huilai; He, Wenzhu; Pan, Xiang; Shen, Bing; Du, Juan

    2016-04-01

    TRPP2 channel protein belongs to the superfamily of transient receptor potential (TRP) channels and is widely expressed in various tissues, including smooth muscle in digestive gut. Accumulating evidence has demonstrated that TRPP2 can mediate Ca(2+) release from Ca(2+) stores. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remains unclear. In this study, we used Ca(2+) imaging and tension measurements to test agonist-induced intracellular Ca(2+) concentration increase and smooth muscle contraction of guinea pig gallbladder, respectively. When TRPP2 protein was knocked down in gallbladder muscle strips from guinea pig, carbachol (CCh)-evoked Ca(2+) release and extracellular Ca(2+) influx were reduced significantly, and gallbladder contractions induced by endothelin 1 and cholecystokinin were suppressed markedly as well. CCh-induced gallbladder contraction was markedly suppressed by pretreatment with U73122, which inhibits phospholipase C to terminate inositol 1,4,5-trisphosphate receptor (IP3) production, and 2-aminoethoxydiphenyl borate (2APB), which inhibits IP3 recepor (IP3R) to abolish IP3R-mediated Ca(2+) release. To confirm the role of Ca(2+) release in CCh-induced gallbladder contraction, we used thapsigargin (TG)-to deplete Ca(2+) stores via inhibiting sarco/endoplasmic reticulum Ca(2+)-ATPase and eliminate the role of store-operated Ca(2+) entry on the CCh-induced gallbladder contraction. Preincubation with 2 μmol L(-1) TG significantly decreased the CCh-induced gallbladder contraction. In addition, pretreatments with U73122, 2APB or TG abolished the difference of the CCh-induced gallbladder contraction between TRPP2 knockdown and control groups. We conclude that TRPP2 mediates Ca(2+) release from intracellular Ca(2+) stores, and has an essential role in agonist-induced gallbladder muscle contraction. PMID:26660312

  12. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence

    PubMed Central

    Wang, Zhe; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-01-01

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 106 cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  13. Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

    SciTech Connect

    Gerthoffer, W.T.; Murphey, K.A.; Khoyi, M.A.

    1988-08-01

    Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased /sup 45/Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on /sup 45/Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.

  14. Perturbed equilibrium of myosin binding in airway smooth muscle and its implications in bronchospasm.

    PubMed

    Fredberg, J J; Inouye, D S; Mijailovich, S M; Butler, J P

    1999-03-01

    In asthma, the mechanisms relating airway obstruction, hyperresponsiveness, and inflammation remain rather mysterious. We show here that regulation of airway smooth muscle length corresponds to a dynamically equilibrated steady state, not the static mechanical equilibrium that had been previously assumed. This dynamic steady state requires as an essential feature a continuous supply of external mechanical energy (derived from tidal lung inflations) that acts to perturb the interactions of myosin with actin, drive the molecular state of the system far away from thermodynamic equilibrium, and bias the muscle toward lengthening. This mechanism leads naturally to the suggestion that excessive airway narrowing in asthma may be associated with the destabilization of that dynamic process and its resulting collapse back to static equilibrium. With this collapse the muscle undergoes a phase transition and virtually freezes at its static equilibrium length. This mechanism may help to elucidate several unexplained phenomena including the multifactorial origins of airway hyperresponsiveness, how allergen sensitization leads to airway hyperresponsiveness, how hyperresponsiveness can persist long after airway inflammation is resolved, and the inability in asthma of deep inspirations to relax airway smooth muscle. PMID:10051279

  15. Interference with PPARγ Function in Smooth Muscle Causes Vascular Dysfunction and Hypertension

    PubMed Central

    Halabi, Carmen M.; Beyer, Andreas M.; de Lange, Willem J.; Keen, Henry L.; Baumbach, Gary L.; Faraci, Frank M.; Sigmund, Curt D.

    2008-01-01

    Summary Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand activated transcription factor playing a critical role in metabolism. Thiazolidinediones, high affinity PPARγ ligands used clinically to treat type-II diabetes, have been reported to lower blood pressure and provide other cardiovascular benefits. Some mutations in PPARγ cause type-II diabetes and severe hypertension. We tested the hypothesis that PPARγ in vascular muscle plays a role in the regulation of vascular tone and blood pressure. Transgenic mice expressing dominant negative mutations in PPARγ under the control of a smooth muscle-specific promoter exhibit a loss of responsiveness to nitric oxide and striking alterations in contractility in the aorta, hypertrophy and inward remodeling in the cerebral microcirculation, and systolic hypertension. These results identify PPARγ as pivotal in vascular muscle as a regulator of vascular structure, vascular function and blood pressure, potentially explaining some of the cardioprotective effects of thiazolidinediones. PMID:18316027

  16. Inward rectifier potassium conductance regulates membrane potential of canine colonic smooth muscle.

    PubMed

    Flynn, E R; McManus, C A; Bradley, K K; Koh, S D; Hegarty, T M; Horowitz, B; Sanders, K M

    1999-07-01

    1. The membrane potential of gastrointestinal smooth muscles determines the open probability of ion channels involved in rhythmic electrical activity. The role of Ba2+-sensitive K+ conductances in the maintenance of membrane potential was examined in canine proximal colon circular muscle. 2. Application of Ba2+ (1-100 microM) to strips of tunica muscularis produced depolarization of cells along the submucosal surface of the circular muscle layer. Significantly higher concentrations of Ba2+ were needed to depolarize preparations from which the submucosal and myenteric pacemaker regions were removed. 3. Elevation of extracellular [K+]o (from 5.9 to 12 mM) brought membrane potentials closer to EK (the Nernst potential for K+ ions), suggesting activation of a K+ conductance. This occurred at potentials much more negative than the activation range for delayed rectifier channels (Kv). 4. Forskolin (1 microM) caused hyperpolarization and a leftward shift in the dose-response relationship for Ba2+, suggesting that forskolin may activate a Ba2+-sensitive conductance. 5. Patch-clamp recordings from interstitial cells of Cajal (ICC) revealed the presence of a Ba2+-sensitive inward rectifier potassium conductance. Far less of this conductance was present in smooth muscle cells. 6. Kir2.1 was expressed in the circular muscle layer of the canine proximal colon, duodenum, jejunum and ileum. Kir2.1 mRNA was expressed in greater abundance along the submucosal surface of the circular muscle layer in the colon. 7. These results demonstrate that ICC express a Ba2+-sensitive conductance (possibly encoded by Kir2.1). This conductance contributes to the generation and maintenance of negative membrane potentials between slow waves. PMID:10373706

  17. Activation of the retinoid X receptor modulates angiotensin II-induced smooth muscle gene expression and inflammation in vascular smooth muscle cells.

    PubMed

    Lehman, Allison M B; Montford, John R; Horita, Henrick; Ostriker, Allison C; Weiser-Evans, Mary C M; Nemenoff, Raphael A; Furgeson, Seth B

    2014-11-01

    The retinoid X receptor (RXR) partners with numerous nuclear receptors, such as the peroxisome proliferator activated receptor (PPAR) family, liver X receptors (LXRs), and farnesoid X receptor (FXR). Although each heterodimer can be activated by specific ligands, a subset of these receptors, defined as permissive nuclear receptors, can also be activated by RXR agonists known as rexinoids. Many individual RXR heterodimers have beneficial effects in vascular smooth muscle cells (SMCs). Because rexinoids can potently activate multiple RXR pathways, we hypothesized that treating SMCs with rexinoids would more effectively reverse the pathophysiologic effects of angiotensin II than an individual heterodimer agonist. Cultured rat aortic SMCs were pretreated with either an RXR agonist (bexarotene or 9-cis retinoic acid) or vehicle (dimethylsulfoxide) for 24 hours before stimulation with angiotensin II. Compared with dimethylsulfoxide, bexarotene blocked angiotensin II-induced SM contractile gene induction (calponin and smooth muscle-α-actin) and protein synthesis ([(3)H]leucine incorporation). Bexarotene also decreased angiotensin II-mediated inflammation, as measured by decreased expression of monocyte chemoattractant protein-1 (MCP-1). Activation of p38 mitogen-activated protein (MAP) kinase but not extracellular signal-related kinase (ERK) or protein kinase B (Akt) was also blunted by bexarotene. We compared bexarotene to five agonists of nuclear receptors (PPARα, PPARγ, PPARδ, LXR, and FXR). Bexarotene had a greater effect on calponin reduction, MCP-1 inhibition, and p38 MAP kinase inhibition than any individual agonist. PPARγ knockout cells demonstrated blunted responses to bexarotene, indicating that PPARγ is necessary for the effects of bexarotene. These data demonstrate that RXR is a potent modulator of angiotensin II-mediated responses in the vasculature, partially through inhibition of p38. PMID:25169989

  18. Pathway of Programmed Cell Death and Oxidative Stress Induced by β-Hydroxybutyrate in Dairy Cow Abomasum Smooth Muscle Cells and in Mouse Gastric Smooth Muscle

    PubMed Central

    Wang, Zhe; Zhang, Naisheng; Xie, Guanghong

    2014-01-01

    The administration of exogenous β-hydroxybutyrate (β-HB), as well as fasting and caloric restriction, is a condition associated with β-HB abundance and decreased appetite in animals. Increased β-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of β-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous β-HB on the stomach, mice were injected intraperitoneally with β-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of β-HB. We found that β-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. β-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. β-HB also promoted AIF, EndoG release and p53 expression. β-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. β-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with β-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that β-HB causes ROS generation through a Ca2+-dependent mechanism and that intracellular Ca2+ levels play a critical role in β-HB -induced apoptotic cell death. The impact of β-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of β-HB on smooth muscle. We propose that β-HB is a possible cause of some stomach diseases, including bovine LDA. PMID:24801711

  19. Reactivity of tracheal smooth muscles in albino rats with experimental diabetes mellitus treated with a new complex compound of oxovanadium (IV) and isonicotinic acid hydrazide.

    PubMed

    Khafiz'yanova, R Kh; Minnebaev, M M; Gallyamov, R M; Latypov, R S; Gosmanov, A R; Aleeva, G N

    2003-06-01

    We studied functional properties of tracheal smooth muscle cells in rats with diabetes mellitus. Reactivity of tracheal smooth muscles increased in rats with experimental alloxan-induced diabetes mellitus. A new complex compound of oxovanadium (IV) and isonicotinic acid hydrazide affected reactivity of tracheal smooth muscles in albino rats with experimental type I diabetes mellitus. This new organic vanadium-containing compound reduced contractility of tracheal smooth muscles in rats and potentiated relaxation of smooth muscle cells in the trachea in response to exogenous nitric oxide. PMID:12937677

  20. Protein kinase A regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle.

    PubMed

    Harraz, Osama F; Welsh, Donald G

    2013-07-01

    Recent investigations have identified that T-type Ca(2+) channels (CaV3.x) are expressed in rat cerebral arterial smooth muscle. In the study reported here, we isolated the T-type conductance, differentiated the current into the CaV3.1/CaV3.2 subtypes and determined whether they are subject to protein kinase regulation. Using patch clamp electrophysiology, whole-cell Ba(2+) current was monitored and initially subdivided into nifedipine-sensitive and -insensitive components. The latter conductance was abolished by T-type Ca(2+) channel blockers and was faster with leftward shifted activation/inactivation properties, reminiscent of a T-type channel. Approximately 60% of this T-type conductance was blocked by 50 µM Ni(2+), a concentration that selectively interferes with CaV3.2 channels. Subsequent work revealed that the whole-cell T-type conductance was subject to protein kinase A (PKA) modulation. Specifically, positive PKA modulators (db-cAMP, forskolin, isoproterenol) suppressed T-type currents and evoked a hyperpolarized shift in steady-state inactivation. Blocking PKA (with KT5720) masked this suppression without altering the basal T-type conductance. A similar effect was observed with stHt31, a peptide inhibitor of A-kinase anchoring proteins. A final set of experiments revealed that PKA-induced suppression targeted the CaV3.2 subtype. In summary, this study revealed that a T-type Ca(2+) channel conductance can be isolated in arterial smooth muscle, and differentiated into CaV3.1 and CaV3.2 components. It also showed that vasodilatory signaling cascades inhibit this conductance by targeting CaV3.2. Such targeting would impact Ca(2+) dynamics and consequent tone regulation in the cerebral circulation. PMID:23613468

  1. TNF-α induces phenotypic modulation in cerebral vascular smooth muscle cells: implications for cerebral aneurysm pathology.

    PubMed

    Ali, Muhammad S; Starke, Robert M; Jabbour, Pascal M; Tjoumakaris, Stavropoula I; Gonzalez, L Fernando; Rosenwasser, Robert H; Owens, Gary K; Koch, Walter J; Greig, Nigel H; Dumont, Aaron S

    2013-10-01

    Little is known about vascular smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. Tumor necrosis factor-alpha (TNF-α) has been associated with aneurysms, but potential mechanisms are unclear. Cultured rat cerebral SMCs overexpressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, smooth muscle myosin heavy chain), while dominant-negative cells suppressed expression. Tumor necrosis factor-alpha treatment inhibited this contractile phenotype and induced pro-inflammatory/matrix-remodeling genes (monocyte chemoattractant protein-1, matrix metalloproteinase-3, matrix metalloproteinase-9, vascular cell adhesion molecule-1, interleukin-1 beta). Tumor necrosis factor-alpha increased expression of KLF4, a known regulator of SMC differentiation. Kruppel-like transcription factor 4 (KLF4) small interfering RNA abrogated TNF-α activation of inflammatory genes and suppression of contractile genes. These mechanisms were confirmed in vivo after exposure of rat carotid arteries to TNF-α and early on in a model of cerebral aneurysm formation. Treatment with the synthesized TNF-α inhibitor 3,6-dithiothalidomide reversed pathologic vessel wall alterations after induced hypertension and hemodynamic stress. Chromatin immunoprecipitation assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4-dependent alterations in promoter regions of myocardin, SMCs, and inflammatory genes. In conclusion, TNF-α induces phenotypic modulation of cerebral SMCs through myocardin and KLF4-regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory/matrix-remodeling phenotype, which has important implications for the mechanisms behind intracranial aneurysm formation. PMID:23860374

  2. Nitric oxide suppresses a Ca(2+)-stimulated Cl- current in smooth muscle cells of opossum esophagus.

    PubMed

    Zhang, Y; Vogalis, F; Goyal, R K

    1998-05-01

    Nitric oxide (NO) hyperpolarizes visceral smooth muscles. Using the patch-clamp technique, we investigated the possibility that NO-mediated hyperpolarization in the circular muscle of opossum esophagus results from the suppression of a Ca(2+)-stimulated Cl- current. Smooth muscle cells were dissociated from the circular layer and bathed in high-K+ Ca(2+)-EGTA-buffered solution. Macroscopic ramp currents were recorded from cell-attached patches. Contaminating K(+)-channel currents were blocked with tetrapentylammonium chloride (200 microM) added to all solutions. Raising bath Ca2+ concentration above 150 nM in the presence of A-23187 (10 microM) activated a leak current (IL-Ca) with an EC50 of 1.2 microM at -100 mV. The reversal potential (Erev) of IL-Ca (-8.5 +/- 1.8 mV, n = 8) was significantly different (P < 0.05) from Erev of the background current (+4.2 +/- 1.2 mV, n = 8). Equimolar substitution of 135 mM Cl- in the pipette solution with gluconate significantly shifted Erev of IL-Ca to +16.6 +/- 3.4 mV (n = 4) (P < 0.05 compared with background), whereas replacement of total Na+ with Tris+ suppressed IL-Ca but did not affect Erev (-15 +/- 3 mV, n = 3; P > 0.05). IL-Ca was inhibited by DIDS (500 microM). Diethylenetriamine-NO adduct (200 microM), a NO donor, and 8-bromo-cGMP (200 microM) suppressed IL-Ca by 59 +/- 15% (n = 5) and 62 +/- 21% (n = 4) at -100 mV, respectively. We conclude that in opossum esophageal smooth muscle NO-mediated hyperpolarization may be produced by suppression of a Ca(2+)-stimulated Cl(-)-permeable conductance via formation of cGMP. PMID:9612270

  3. The role of K+ conductances in regulating membrane excitability in human gastric corpus smooth muscle

    PubMed Central

    Lee, Ji Yeon; Ko, Eun-ju; Ahn, Ki Duck; Kim, Sung

    2015-01-01

    Changes in resting membrane potential (RMP) regulate membrane excitability. K+ conductance(s) are one of the main factors in regulating RMP. The functional role of K+ conductances has not been studied the in human gastric corpus smooth muscles (HGCS). To examine the role of K+ channels in regulation of RMP in HGCS we employed microelectrode recordings, patch-clamp, and molecular approaches. Tetraethylammonium and charybdotoxin did not affect the RMP, suggesting that BK channels are not involved in regulating RMP. Apamin, a selective small conductance Ca2+-activated K+ channel (SK) blocker, did not show a significant effect on the membrane excitability. 4-Aminopyridine, a Kv channel blocker, caused depolarization and increased the duration of slow wave potentials. 4-Aminopyridine also inhibited a delayed rectifying K+ current in isolated smooth muscle cells. End-product RT-PCR gel detected Kv1.2 and Kv1.5 in human gastric corpus muscles. Glibenclamide, an ATP-sensitive K+ channel (KATP) blocker, did not induce depolarization, but nicorandil, a KATP opener, hyperpolarized HGCS, suggesting that KATP are expressed but not basally activated. Kir6.2 transcript, a pore-forming subunit of KATP was expressed in HGCS. A low concentration of Ba2+, a Kir blocker, induced strong depolarization. Interestingly, Ba2+-sensitive currents were minimally expressed in isolated smooth muscle cells under whole-cell patch configuration. KCNJ2 (Kir2.1) transcript was expressed in HGCS. Unique K+ conductances regulate the RMP in HGCS. Delayed and inwardly rectifying K+ channels are the main candidates in regulating membrane excitability in HGCS. With the development of cell dispersion techniques of interstitial cells, the cell-specific functional significance will require further analysis. PMID:25591864

  4. Effect of pinaverium bromide on stress-induced colonic smooth muscle contractility disorder in rats

    PubMed Central

    Dai, Yun; Liu, Jian-Xiang; Li, Jun-Xia; Xu, Yun-Feng

    2003-01-01

    AIM: To investigate the effect of pinaverium bromide, a L-type calcium channel blocker with selectivity for the gastrointestinal tract on contractile activity of colonic circular smooth muscle in normal or cold-restraint stressed rats and its possible mechanism. METHODS: Cold-restraint stress was conducted on rats to increase fecal pellets output. Each isolated colonic circular muscle strip was suspended in a tissue chamber containing warm oxygenated Tyrode-Ringer solution. The contractile response to ACh or KCl was measured isometrically on ink-writing recorder. Incubated muscle in different concentrations of pinaverium and the effects of pinaverium were investigated on ACh or KCl-induced contraction. Colon smooth muscle cells were cultured from rats and [Ca2+]i was measured in cell suspension using the Ca2+ fluorescent dye fura-2/AM. RESULTS: During stress, rats fecal pellet output increased 61% (P < 0.01). Stimulated with ACh or KCl, the muscle contractility was higher in stress than that in control. Pinaverium inhibited the increment of [Ca2+]i and the muscle contraction in response to ACh or KCl in a dose dependent manner. A significant inhibition of pinaverium to ACh or KCl induced [Ca2+]i increment was observed at 10-6 mol/L. The IC50 values for inhibition of ACh induced contraction for the stress and control group were 1.66 × 10-6 mol/L and 0.91 × 10-6 mol/L, respectively. The IC50 values for inhibition of KCl induced contraction for the stress and control group were 8.13 × 10-7 mol/L and 3.80 × 10-7 mol/L, respectively. CONCLUSION: Increase in [Ca2+]i of smooth muscle cells is directly related to the generation of contraction force in colon. L-type Ca2+ channels represent the main route of Ca2+ entry. Pinaverium inhibits the calcium influx through L-type channels; decreases the contractile response to many kinds of agonists and regulates the stress-induced colon hypermotility. PMID:12632518

  5. Stromal cells in the human gut show ultrastructural features of fibroblasts and smooth muscle cells but not myofibroblasts.

    PubMed

    Eyden, Brian; Curry, Alan; Wang, Guofeng

    2011-07-01

    The free spindled cells of the lamina propria of the gut have been reported as showing fibroblastic, smooth-muscle and myofibroblastic differentiation. A precise understanding of the differentiation of these cells is essential for appreciating their functions, and this paper addresses this question using ultrastructural analysis. Histologically normal samples from different areas of the gastrointestinal tract were studied. Both subepithelial stromal cells, lying immediately beneath the basal lamina, and the deeper interstitial stromal cells, were studied. Subepithelial and interstitial cells had comparable features, reinforcing the idea that these formed a single reticulum of cells. Two major cell types were identified. Some were smooth-muscle cells, on the basis of abundant myofilaments with focal densities, glycogen, an irregular cell surface, focal lamina and multiple attachment plaques alternating with plasmalemmal caveolae. Some cells had a lesser expression of these markers, especially of myofilaments, and were regarded as poorly differentiated smooth-muscle cells and descriptively referred to as 'myoid'. Other cells were fibroblastic to judge by prominent rough endoplasmic reticulum, an absence of myofilaments and lamina, but presence of focal adhesions. The fibronexus junctions of true myofibroblasts were not seen. The study emphasises that the smooth-muscle actin immunoreactivity in this anatomical site resides in smooth-muscle cells and not in myofibroblasts, a view consistent with earlier ultrastructural and immunostaining results. The recognition that these cells are showing smooth-muscle or fibroblastic but not true myofibroblastic differentiation should inform our understanding of the function of these cells. PMID:20662992

  6. A network of 2-4 nm filaments found in sea urchin smooth muscle. Protein constituents and in situ localization.

    PubMed

    Pureur, R P; Coffe, G; Soyer-Gobillard, M O; de Billy, F; Pudles, J

    1986-01-01

    In this report the coisolation of two proteins from sea urchin smooth muscle of apparent molecular weights (Mr) 54 and 56 kD respectively, as determined on SDS-PAGE, is described. Like the intermediate filament proteins, these two proteins are insoluble in high ionic strength buffer solution. On two-dimensional gel electrophoresis and by immunological methods it is shown that these proteins are not related (by these criteria) to rat smooth muscle desmin (54 kD) or vimentin (56 kD). Furthermore, in conditions where both desmin and vimentin assemble in vitro into 10 nm filaments, the sea urchin smooth muscle proteins do not assemble into filaments. Ultrastructural studies on the sea urchin smooth muscle cell show that the thin and thick filaments organization resembles that described in the vertebrate smooth muscle. However, instead of 10 nm filaments, a network of filaments, 2-4 nm in diameter, is revealed, upon removal of the thin and thick filaments by 0.6 M KCl treatment. By indirect immunofluorescence microscopy, and in particular by immunocytochemical electron microscopy studies on the sea urchin smooth muscle cell, it is shown that the antibodies raised against both 54 and 56 kD proteins appear to specifically label these 2-4 nm filaments. These findings indicate that both the 54 and 56 kD proteins might be constituents of this category of filaments. The possible significance of this new cytoskeletal element, that we have named echinonematin filaments, is discussed. PMID:3509996

  7. Effects of Gingko biloba extract (EGb 761) on vascular smooth muscle cell calcification induced by β-glycerophosphate.

    PubMed

    Li, En-Gang; Tian, Jun; Xu, Zhong-Hua

    2016-05-01

    Objective To investigate the effects of Gingko biloba extract (EGb 761) on calcification induced by β-glycerophosphate in rat aortic vascular smooth muscle cells. Methods Rat aortic vascular smooth muscle cells were cultured with various concentrations of EGb 761 and β-glycerophosphate for 7 days. Calcium content in the cells, alkaline phosphatase activity, cell protein content, NF-κB activation, and reactive oxygen species production were assayed, respectively. Results The calcium depositions of vascular smooth muscle cells of the β-glycerophosphate group were significantly higher than those of the control group (p < 0.01), and were inhibited by EGb 761 in a concentration-dependent manner (p < 0.05). Data showed β-glycerophosphate induced the enhanced expression of alkaline phosphatase, up-regulated the NF-κB activity and increased reactive oxygen species production of vascular smooth muscle cells while these decreased when administrated with EGb 761(p < 0.05). Conclusions EGb 761 significantly reduced deposition of calcium induced by β-glycerophosphate in rat aortic vascular smooth muscle cells. It not only reduced the deposition of calcium, but also inhibited osteogenic transdifferentiation, which may be associated with decreasing expression of alkaline phosphatase, down-regulating the NF-κB activity, and reducing reactive oxygen species production of vascular smooth muscle cells, and may have the potential to serve as a role for vascular calcification in clinical situations. PMID:26908182

  8. Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

    PubMed Central

    Ames, Juliana; Chokshi, Mithil; Aiad, Norman; Sanyal, Sonali; Kawabata, Kimihito C.; Levental, Ilya; Sundararaghavan, Harini G.; Burdick, Jason A.; Janmey, Paul; Miyazono, Kohei; Wells, Rebecca G.; Jones, Peter L.

    2015-01-01

    Abstract Although there are many studies focusing on the molecular pathways underlying lung vascular morphogenesis, the extracellular matrix (ECM)–dependent regulation of mesenchymal cell differentiation in vascular smooth muscle development needs better understanding. In this study, we demonstrate that the paired related homeobox gene transcription factor Prx1 maintains the elastic ECM properties, which are essential for vascular smooth muscle precursor cell differentiation. We have found that Prx1null mouse lungs exhibit defective vascular smooth muscle development, downregulated elastic ECM expression, and compromised transforming growth factor (TGF)–β localization and signaling. Further characterization of ECM properties using decellularized lung ECM scaffolds derived from Prx1 mice demonstrated that Prx1 is required to maintain lung ECM stiffness. The results of cell culture using stiffness-controlled 2-D and 3-D synthetic substrates confirmed that Prx1-dependent ECM stiffness is essential for promotion of smooth muscle precursor differentiation for effective TGF-β stimulation. Supporting these results, both decellularized Prx1null lung ECM and Prx1WT (wild type) ECM scaffolds with blocked TGF-β failed to support mesenchymal cell to 3-D smooth muscle cell differentiation. These results suggest a novel ECM-dependent regulatory pathway of lung vascular development wherein Prx1 regulates lung vascular smooth muscle precursor development by coordinating the ECM biophysical and biochemical properties. PMID:26064466

  9. Actions of 4-aminopyridine on vascular smooth muscle tissues of the guinea-pig

    PubMed Central

    Hara, Yusuke; Kitamura, Kenji; Kuriyama, Hirosi

    1980-01-01

    1 Effects of 4-aminopyridine (4-AP) and procaine on the membrane and contractile properties of smooth muscle cells of the guinea-pig pulmonary artery and portal vein were observed. 2 The membrane potential and length constant of smooth muscle cells of the guinea-pig pulmonary artery were -53.2 mV and 1.2 mm, respectively, and those of the portal vein were -52.6 mV and 0.71 mm, respectively. The membrane was electrically quiescent in the pulmonary artery and it was electrically active in the portal vein. 3 Both 4-AP and procaine depolarized the membrane, increased the membrane resistance and suppressed the rectifying properties in both tissues. Both agents evoked a graded response from the muscle membranes of the pulmonary artery by outward current pulse. Procaine had a greater effect than 4-AP on the above membrane properties. 4 4-AP (10-5 M) produced contraction without depolarization of the membrane. The contraction evoked by 10-5 M 4-AP was completely suppressed but that evoked by 5 × 10-4 M 4-AP was only partly suppressed by phentolamine (10-7 M). However, the contraction evoked by procaine was not suppressed by phentolamine. 5 4-AP enhanced but procaine suppressed the amplitude of 118 mM [K]0-induced contraction. 6 The results suggest that 4-AP and procaine suppress K-conductance of the muscle membrane, and 4-AP but not procaine increases noradrenaline release from the nerve terminal. Presumably intracellular free Ca concentrations are also modified by these agents. The effects of 4-AP and procaine on the vascular muscle were compared with those on other excitable tissues. PMID:7357204

  10. Evidence for 5-HT7 receptors mediating relaxation of human colonic circular smooth muscle

    PubMed Central

    Prins, Nicolaas H; Briejer, Michel R; Van Bergen, Patrick J E; Akkermans, Louis M A; Schuurkes, Jan A J

    1999-01-01

    5-HT4 receptors mediate relaxation of human colon circular muscle. However, after 5-HT4 receptor blockade (SB 204070 10 nM), 5-HT still induced a relaxation (pEC50 6.3). 5-HT4 receptors were sufficiently blocked, as the curves to 5-HT obtained in the presence of 10 and 100 nM SB 204070 were indistinguishable. This 5-HT-induced relaxation was tetrodotoxin-insensitive, indicative of a smooth muscle relaxant 5-HT receptor. This, and the rank order of potency (5-CT=5-MeOT=5-HT) suggested involvement of 5-HT1 or 5-HT7 receptors. Mesulergine, a 5-HT7 receptor antagonist at nanomolar concentrations, and a 5-HT1 receptor antagonist at micromolar concentrations, competitively antagonized the 5-HT-induced relaxation (pKB 8.3) and antagonized the relaxation to 5-CT. Methysergide antagonized the 5-HT-induced relaxation (pA2 7.6). It is concluded that the profile of the smooth muscle inhibitory 5-HT receptor resembles that of the 5-HT7 receptor. These data provide the first evidence for functional human 5-HT7 receptors. PMID:10556917

  11. A role of stretch-activated potassium currents in the regulation of uterine smooth muscle contraction.

    PubMed

    Buxton, Iain L O; Heyman, Nathanael; Wu, Yi-ying; Barnett, Scott; Ulrich, Craig

    2011-06-01

    Rates of premature birth are alarming and threaten societies and healthcare systems worldwide. Premature labor results in premature birth in over 50% of cases. Preterm birth accounts for three-quarters of infant morbidity and mortality. Children that survive birth before 34 weeks gestation often face life-long disability. Current treatments for preterm labor are wanting. No treatment has been found to be generally effective and none are systematically evaluated beyond 48 h. New approaches to the treatment of preterm labor are desperately needed. Recent studies from our laboratory suggest that the uterine muscle is a unique compartment with regulation of uterine relaxation unlike that of other smooth muscles. Here we discuss recent evidence that the mechanically activated 2-pore potassium channel, TREK-1, may contribute to contraction-relaxation signaling in uterine smooth muscle and that TREK-1 gene variants associated with human labor and preterm labor may lead to a better understanding of preterm labor and its possible prevention. PMID:21642947

  12. Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

    PubMed

    Curtis, Tim M; Tumelty, James; Stewart, Michael T; Arora, A Rakha; Lai, F Anthony; McGahon, Mary K; Scholfield, C Norman; McGeown, J Graham

    2008-02-01

    Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle. PMID:17574671

  13. Increase in passive stiffness at reduced airway smooth muscle length: potential impact on airway responsiveness.

    PubMed

    Bossé, Ynuk; Solomon, Dennis; Chin, Leslie Y M; Lian, Kevin; Paré, Peter D; Seow, Chun Y

    2010-03-01

    The amplitude of strain in airway smooth muscle (ASM) produced by oscillatory perturbations such as tidal breathing or deep inspiration (DI) influences the force loss in the muscle and is therefore a key determinant of the bronchoprotective and bronchodilatory effects of these breathing maneuvers. The stiffness of unstimulated ASM (passive stiffness) directly influences the amplitude of strain. The nature of the passive stiffness is, however, not clear. In this study, we measured the passive stiffness of ovine ASM at different muscle lengths (relative to in situ length, which was used as a reference length, L(ref)) and states of adaptation to gain insights into the origin of this muscle property. The results showed that the passive stiffness was relatively independent of muscle length, possessing a constant plateau value over a length range from 0.62 to 1.25 L(ref). Following a halving of ASM length, passive stiffness decreased substantially (by 71%) but redeveloped over time ( approximately 30 min) at the shorter length to reach 65% of the stiffness value at L(ref), provided that the muscle was stimulated to contract at least once over a approximately 30-min period. The redevelopment and maintenance of passive stiffness were dependent on the presence of Ca(2+) but unaffected by latrunculin B, an inhibitor of actin filament polymerization. The maintenance of passive stiffness was also not affected by blocking myosin cross-bridge cycling using a myosin light chain kinase inhibitor or by blocking the Rho-Rho kinase (RhoK) pathway using a RhoK inhibitor. Our results suggest that the passive stiffness of ASM is labile and capable of redevelopment following length reduction. Redevelopment and maintenance of passive stiffness following muscle shortening could contribute to airway hyperresponsiveness by attenuating the airway wall strain induced by tidal breathing and DI. PMID:20008114

  14. Loss of Notch3 Signaling in Vascular Smooth Muscle Cells Promotes Severe Heart Failure Upon Hypertension.

    PubMed

    Ragot, Hélène; Monfort, Astrid; Baudet, Mathilde; Azibani, Fériel; Fazal, Loubina; Merval, Régine; Polidano, Evelyne; Cohen-Solal, Alain; Delcayre, Claude; Vodovar, Nicolas; Chatziantoniou, Christos; Samuel, Jane-Lise

    2016-08-01

    Hypertension, which is a risk factor of heart failure, provokes adaptive changes at the vasculature and cardiac levels. Notch3 signaling plays an important role in resistance arteries by controlling the maturation of vascular smooth muscle cells. Notch3 deletion is protective in pulmonary hypertension while deleterious in arterial hypertension. Although this latter phenotype was attributed to renal and cardiac alterations, the underlying mechanisms remained unknown. To investigate the role of Notch3 signaling in the cardiac adaptation to hypertension, we used mice with either constitutive Notch3 or smooth muscle cell-specific conditional RBPJκ knockout. At baseline, both genotypes exhibited a cardiac arteriolar rarefaction associated with oxidative stress. In response to angiotensin II-induced hypertension, the heart of Notch3 knockout and SM-RBPJκ knockout mice did not adapt to pressure overload and developed heart failure, which could lead to an early and fatal acute decompensation of heart failure. This cardiac maladaptation was characterized by an absence of media hypertrophy of the media arteries, the transition of smooth muscle cells toward a synthetic phenotype, and an alteration of angiogenic pathways. A subset of mice exhibited an early fatal acute decompensated heart failure, in which the same alterations were observed, although in a more rapid timeframe. Altogether, these observations indicate that Notch3 plays a major role in coronary adaptation to pressure overload. These data also show that the hypertrophy of coronary arterial media on pressure overload is mandatory to initially maintain a normal cardiac function and is regulated by the Notch3/RBPJκ pathway. PMID:27296994

  15. MODULAR STRUCTURE OF SMOOTH MUSCLE MYOSIN LIGHT CHAIN KINASE: HYDRODYNAMIC MODELING AND FUNCTIONAL IMPLICATIONS†

    PubMed Central

    Mabuchi, Yasuko; Mabuchi, Katsuhide; Stafford, Walter F.; Grabarek, Zenon

    2010-01-01

    Smooth muscle myosin light chain kinase (smMLCK) is a calcium/calmodulin dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot: P29294) contains the catalytic/regulatory domain, three immunoglobulin related motifs (Ig), one fibronectin related motif (Fn3), a repetitive, proline rich segment (PEVK) and, at the N-terminus, a unique F-actin binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147 and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distance (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with the program HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3 and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of 40 nm or more. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells. PMID:20196616

  16. Expression of Potassium Channels in Uterine Smooth Muscle Cells from Patients with Adenomyosis

    PubMed Central

    Shi, Jing-Hua; Jin, Li; Leng, Jin-Hua; Lang, Jing-He

    2016-01-01

    Background: Adenomyosis (AM) has impaired contraction. This study aimed to explore the expression of potassium channels related to contraction in myometrial smooth muscle cells (MSMCs) of AM. Methods: Uterine tissue samples from 22 patients (cases) with histologically confirmed AM and 12 (controls) with cervical intraepithelial neoplasia were collected for both immunohistochemistry and real-time polymerase chain reaction to detect the expression of large conductance calcium- and voltage-sensitive K+ channel (BKCa)-α/β subunits, voltage-gated potassium channel (Kv) 4.2, and Kv4.3. Student's t-test was used to compare the expression. Results: The BKCa-α/β subunits, Kv4.2, and Kv4.3 were located in smooth muscle cells, glandular epithelium, and stromal cells. However, BKCa-β subunit expression in endometrial glands of the controls was weak, and Kv4.3 was almost undetectable in the controls. The expression of BKCa-α messenger RNA (mRNA) (0.62 ± 0.19-fold decrease, P < 0.05) and Kv4.3 mRNA (0.67 ± 0.20-fold decrease, P < 0.05) decreased significantly in the MSMCs of the control group compared with the AM group. However, there were no significant differences in BKCa-β subunit mRNA or Kv4.2 mRNA. Conclusions: The BKCa-α mRNA and the Kv4.3 mRNA are expressed significantly higher in AM than those in the control group, that might cause the abnormal uterus smooth muscle contractility, change the microcirculation of uterus to accumulate the inflammatory factors, impair the endometrium further, and aggravate the pain. PMID:26830992

  17. Leptin augments coronary vasoconstriction and smooth muscle proliferation via a Rho-kinase-dependent pathway.

    PubMed

    Noblet, Jillian N; Goodwill, Adam G; Sassoon, Daniel J; Kiel, Alexander M; Tune, Johnathan D

    2016-05-01

    Leptin has been implicated as a key upstream mediator of pathways associated with coronary vascular dysfunction and disease. The purpose of this investigation was to test the hypothesis that leptin modifies the coronary artery proteome and promotes increases in coronary smooth muscle contraction and proliferation via influences on Rho kinase signaling. Global proteomic assessment of coronary arteries from lean swine cultured with obese concentrations of leptin (30 ng/mL) for 3 days revealed significant alterations in the coronary artery proteome (68 proteins) and identified an association between leptin treatment and calcium signaling/contraction (four proteins) and cellular growth and proliferation (35 proteins). Isometric tension studies demonstrated that both acute (30 min) and chronic (3 days, serum-free media) exposure to obese concentrations of leptin potentiated depolarization-induced contraction of coronary arteries. Inhibition of Rho kinase significantly reduced leptin-mediated increases in coronary artery contractions. The effects of leptin on the functional expression of Rho kinase were time-dependent, as acute treatment increased Rho kinase activity while chronic (3 day) exposure was associated with increases in Rho kinase protein abundance. Proliferation assays following chronic leptin administration (8 day, serum-containing media) demonstrated that leptin augmented coronary vascular smooth muscle proliferation and increased Rho kinase activity. Inhibition of Rho kinase significantly reduced these effects of leptin. Taken together, these findings demonstrate that leptin promotes increases in coronary vasoconstriction and smooth muscle proliferation and indicate that these phenotypic effects are associated with alterations in the coronary artery proteome and dynamic effects on the Rho kinase pathway. PMID:26975316

  18. Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers.

    PubMed

    Jia, Lin; Prabhakaran, Molamma P; Qin, Xiaohong; Ramakrishna, Seeram

    2014-09-01

    Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration. PMID:24682037

  19. Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle.

    PubMed

    Wang, Jian; Laurier, Lisanne G; Sims, Stephen M; Preiksaitis, Harold G

    2003-06-01

    Transient receptor potential channel (TRPC) genes encode Ca(2+)-permeable channels mediating capacitative Ca(2+) entry (CCE), which maintains intracellular Ca(2+) stores. We compared TRPC gene expression and CCE in human esophageal body (EB) and lower esophageal sphincter (LES), because these smooth muscles have distinct contractile functions that are likely associated with different Ca(2+) regulatory mechanisms. Circular layer smooth muscle cells were grown in primary culture. Transcriptional expression of TRPC genes was compared by semiquantitative RT-PCR. CCE was measured by fura 2 Ca(2+) fluorescence after blockade of sarcoplasmic reticulum Ca(2+)-ATPase with thapsigargin. mRNA for TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 was identified in EB and LES. TRPC3 and TRPC4 were more abundant in LES than EB. Basal concentration of free intracellular Ca(2+) ([Ca(2+)](i)) was similar in cells from LES (138 +/- 8 nmol/l) and EB (110 +/- 6 nmol/l) and increased with ACh (10 micromol/l; 650 +/- 28 and 590 +/- 21 nmol/l, respectively). With zero Ca(2+) in bath, thapsigargin (2 micromol/l) increased [Ca(2+)](i) more in LES (550 +/- 22 nmol/l) than EB (250 +/- 15 nmol/l, P < 0.001). Subsequent external application of 1 mmol/l Ca(2+) increased [Ca(2+)](i) more in LES (585 +/- 35 nmol/l) than EB (295 +/- 21 nmol/l, P < 0.001), indicating enhanced CCE in LES. This demonstrates CCE and TRPC transcriptional expression in human esophageal smooth muscle. In LES cells, enhanced CCE and expression of TRPC3 and TRPC4 may contribute to the physiological characteristics that distinguish LES from EB. PMID:12736151

  20. Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate.

    PubMed

    Kunit, Thomas; Gratzke, Christian; Schreiber, Andrea; Strittmatter, Frank; Waidelich, Raphaela; Rutz, Beata; Loidl, Wolfgang; Andersson, Karl-Erik; Stief, Christian G; Hennenberg, Martin

    2014-10-01

    Smooth muscle contraction may be critical for lower urinary tract symptoms (LUTS) in patients with benign prostate hyperplasia and requires stable anchorage of the cytoskeleton to the cell membrane. These connections are regulated by focal adhesion kinase (FAK). Here, we addressed the involvement of FAK in the regulation of smooth muscle contraction in hyperplastic human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Expression of FAK and focal adhesion proteins was assessed by Western blot analysis and immunohistochemical stainings. Effects of the FAK inhibitors PF-573228 and Y-11 on contraction of prostate strips were examined in the organ bath. Expression of FAK and focal adhesion proteins (integrin-5α, paxilin, and c-Src) was detected by Western blot analysis in prostate samples. By double immunofluorescence staining with calponin and pan-cytokeratin, expression of FAK was observed in stromal and epithelial cells. Immunoreactivity for FAK colocalized with integrin-5α, paxilin, talin, and c-Src. Stimulation of prostate tissues with the α1-adrenergic agonist phenylephrine increased the phosphorylation state of FAK at Tyr³⁹⁷ and Tyr⁹²⁵ with different kinetics, which was blocked by the α1-adrenoceptor antagonist tamsulosin. Norepinephrine and phenylephrine induced concentration-dependent contractions of prostate strips. Both FAK inhibitors PF-573228 and Y-11 significantly inhibited norepinephrine- and phenylephrine-induced contractions. Finally, PF-573228 and Y-11 inhibited contractions induced by electric field stimulation, which was significant at the highest frequency. In conclusion, α1-adrenergic smooth muscle contraction or its regulation involves FAK in the human prostate. Consequently, FAK may be involved in the pathophysiology of LUTS and in current or future LUTS therapies. PMID:25056351

  1. ROLE OF CELLULAR BIOENERGETICS IN SMOOTH MUSCLE CELL PROLIFERATION INDUCED BY PLATELET-DERIVED GROWTH FACTOR

    PubMed Central

    Perez, Jessica; Hill, Bradford G.; Benavides, Gloria A.; Dranka, Brian P.; Darley-Usmar, Victor M.

    2013-01-01

    SYNOPSIS Abnormal smooth muscle cell proliferation is a hallmark of vascular disease. Although growth factors are known to contribute to cell hyperplasia, the changes in metabolism associated with this response, particularly mitochondrial respiration, remain unclear. Given the increased energy requirements for proliferation, we hypothesized that platelet-derived growth factor (PDGF) would stimulate glycolysis and mitochondrial respiration and that this elevated bioenergetic capacity is required for smooth muscle cell hyperplasia. To test this hypothesis, cell proliferation, glycolytic flux, and mitochondrial oxygen consumption were measured after treatment of primary rat aortic smooth muscle cells with PDGF. PDGF increased basal and maximal rates of glycolytic flux and mitochondrial oxygen consumption; enhancement of these bioenergetic pathways led to a substantial increase in the mitochondrial reserve capacity. Interventions with the PI3K inhibitor LY-294002 or the glycolysis inhibitor 2-deoxy-D-glucose abrogated PDGF-stimulated proliferation and prevented augmentation of glycolysis and mitochondrial reserve capacity. Similarly, when L-glucose was substituted for D-glucose, PDGF-dependent proliferation was abolished, as were changes in glycolysis and mitochondrial respiration. Interestingly, lactate dehydrogenase protein levels and activity were significantly increased after PDGF treatment. Moreover, L-lactate substitution for D-glucose was sufficient for increasing the mitochondrial reserve capacity and cell proliferation after treatment with PDGF; these effects were inhibited by the lactate dehydrogenase inhibitor, oxamate. These data suggest that glycolysis, by providing substrates that enhance the mitochondrial reserve capacity, plays an essential role in PDGF-induced cell proliferation, underscoring the integrated metabolic response required for proliferation of VSMC in the diseased vasculature. PMID:20331438

  2. Activity of sap from Croton lechleri on rat vascular and gastric smooth muscles.

    PubMed

    Froldi, G; Zagotto, G; Filippini, R; Montopoli, M; Dorigo, P; Caparrotta, L

    2009-08-01

    The effects of red sap from Croton lechleri (SdD), Euphorbiaceae, on vascular and gastric smooth muscles were investigated. SdD, from 10 to 1000 microg/ml, induced concentration-dependent vasoconstriction in rat caudal arteries, which was endothelium-independent. In arterial preparations pre-constricted by phenylephrine (0.1 microM) or KCl (30 mM), SdD also produced concentration-dependent vasoconstriction. To study the mechanisms implicated in this effect we used selective inhibitors such as prazosin (0.1 microM), an antagonist of alpha(1)-adrenoceptors, atropine (0.1 microM), an antagonist of muscarinic receptors, and ritanserin (50 nM), a 5-HT(2A) antagonist; none of these influenced vasoconstriction caused by SdD. Likewise, nifedipine (50 nM), an inhibitor of L-type calcium channels, did not modify the action of SdD. Capsaicin (100 nM), an agonist of vanilloid receptors, also did not affect vasoconstriction by SdD. We also investigated the action of SdD (10-1000 microg/ml) on rat gastric fundus; per se the sap slightly increased contractile tension. When the gastric fundus was pre-treated with SdD (100 microg/ml) the contraction induced by carbachol (1 microM) was increased, whereas that by KCl (60mM) or capsaicin (100 nM) were unchanged. The data shows that SdD increased contractile tension in a concentration-dependent way, both on vascular and gastric smooth muscles. The vasoconstriction is unrelated to alpha(1), M, 5-HT(2A) and vanilloid receptors as well as L-type calcium channels. SdD increased also contraction by carbachol on rat gastric fundus. Thus for the first time, experimental data provides evidence that sap from C. lechleri owns constricting activity on smooth muscles. PMID:19406630

  3. Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain.

    PubMed

    Espinoza-Fonseca, L Michel; Colson, Brett A; Thomas, David D

    2014-10-01

    We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylated RLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle. PMID:25091814

  4. Mechanism of action of calcium antagonists on myocardial and smooth muscle membranes.

    PubMed

    Zakhari, S

    1986-01-01

    Although calcium channel blockers vary considerably in their chemical structure and pharmacologic profile, the widely accepted mechanism of their action is an inhibition of Ca2+ influx - via voltage-activated slow channels - into smooth and cardiac muscle. Other ways of Ca2+ entry, such as passive diffusion and Na+/Ca2+ and K+/Ca2+ exchange, are not affected by these compounds. However, various blockers exert a slightly different inhibitory action on the slow channels, which indicates that various binding sites in the cell membranes, or even in intracellular sites, may be involved. Potential sites of action of calcium channel blockers in the myocardium include: the slow calcium channels; Na+/Ca2+ channels; mitochondria; sarcoplasmic reticulum; myofilaments; and calcium efflux. However, experimental evidence has been given for only the first site, and the third and fourth sites are still controversial. In the vascular smooth muscles, calcium channel blockers could possibly block the potential-dependent or receptor-operated channels, or bind to calmodulin. Again, only the first site of action has been experimentally proven. An important feature of calcium channel blockers is their different affinities for various tissues. For instance, cinnarizine and its difluorinated derivative flunarizine are 1000 times more effective in blocking slow channels in vascular smooth muscles than those in the myocardium. Even within the same system, such as the cardiovascular system, differences in tissue specificity are encountered. Thus, while nimodipine acts preferentially on cerebral vessels, diltiazem has more affinity for the coronary vasculature. Tissue specificity is exhibited even for different myocardial structures; thus, while verapamil affects the nodal and conductive tissues in the myocardium (hence its use as an antiarrhythmic agent), nifedipine is almost devoid of such activity. Organ selectivity of calcium channel blockers is one of the attractive features of this group

  5. Platelet-activating factor biosynthesis in rat vascular smooth muscle cells.

    PubMed

    Tomlinson, P R; Croft, K; Harris, T; Stewart, A G

    1994-01-01

    The ability of platelet-activating factor (PAF) receptor antagonists to protect rats from the cardiovascular collapse induced by large doses of endothelin 1 led us to examine the capacity of rat cultured vascular smooth muscle cells to produce PAF and also to evaluate its potential functional roles in this cell type. Adenosine triphosphate and the vasoactive peptides, endothelin 1, angiotensin II, and arginine vasopressin, each elicited an increase in the PAF level in extracts of rat cultured vascular smooth muscle cells as determined by bioassay. PAF was not detectable (above 20 fmol/mg protein) in the supernatants of these cells. The identity of the bioactivity as PAF was confirmed by GC/MS which indicated that more than 80% of the PAF was 1-O-hexadecyl-2-acetyl-3-sn-glyceryl-phosphorylcholine. Exogenous PAF (100 nM) elicited increases in intracellular calcium that were inhibited by WEB 2086 (10 microM). Endothelin 1, at a concentration which stimulated PAF synthesis, (1 nM), elicited increases in intracellular calcium levels that were not inhibited by WEB 2086 (10 microM). Thus, endogenous PAF is unlikely to be involved in the endothelin-1-induced calcium increases. Although WEB 2086 (3-100 microM) inhibited concentration dependently fetal calf serum (10% v/v) induced [3H]-thymidine incorporation, reaching a maximum effect at 30 microM of 40-50% reduction, in parallel experiments WEB 2086 had no effect on serum-induced increases in cell numbers. We conclude that PAF is produced and retained by cultured rat vascular smooth muscle and that it is unlikely to contribute to the signaling of increases in intracellular calcium or proliferation. PMID:8148465

  6. Immunostaining of macrophages, endothelial cells and smooth muscle cells in the atherosclerotic mouse aorta

    PubMed Central

    Menon, Prashanthi; Fisher, Edward A.

    2016-01-01

    The atherosclerotic mouse aorta consists of a heterogeneous population of cells, including macrophages, endothelial cells (EC) and smooth muscle cells (SMC), that play critical roles in cardiovascular disease. Identification of these vascular cells in the vessel wall is important to understanding their function in pathological conditions. Immunohistochemistry is an invaluable technique used to detect the presence of cells in different tissues. Here, we describe immunohistochemical techniques commonly used for the detection of the vascular cells in the atherosclerotic mouse aorta using cell specific markers. PMID:26445786

  7. Effect of some smooth muscle relaxant drugs on calcium-related phenomena.

    PubMed

    Ronca-Testoni, S; Hrelia, S; Hakim, G; Ronca, G; Rossi, C A

    1984-04-30

    Some smooth muscle relaxant drugs devoid of anticholinergic action have been tested for their interaction with calmodulin, calmodulin-stimulated cyclic nucleotide phosphodiesterase activity, and uterine membrane binding sites for nitrendipine and adenosine. The myolytic activity of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems. Trimebutine maleate does not bind either to calmodulin or to nitrendipine and adenosine receptors. Tiropramide has no effect on calmodulin-dependent systems and on Ca2+ channels but it shows a competition for the A2-type adenosine receptors. PMID:6329247

  8. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo

    NASA Astrophysics Data System (ADS)

    Simons, Michael; Edelman, Elazer R.; Dekeyser, Jean-Luc; Langer, Robert; Rosenberg, Robert D.

    1992-09-01

    SYNTHETIC antisense oligonucleotides have been used to dissect gene function in vitro. Technical difficulties prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense c-myb oligonu-cleotide to suppress intimal accumulation of rat carotid arterial smooth muscle cells. Our results suggest that antisense oligonucleotides can be used to define the in vivo biological role of specific macromolecules in the blood vessel wall and could potentially serve as a new class of therapeutic agents for cardiovascular disorders.

  9. Vitamin E mediated response of smooth muscle cell to oxidant stress.

    PubMed

    Azzi, A; Boscoboinik, D; Clément, S; Ozer, N; Ricciarelli, R; Stocker, A

    1999-09-01

    Oxidant stress is associated with diminution of antioxidant molecules, such as alpha-tocopherol. Alpha-tocopherol specifically decreases, in a concentration dependent way, the proliferation of vascular smooth muscle cells. At the same concentrations (10-50 microM) it induces inhibition of protein kinase C (PKC) activity. The latter event is not due to a decrease in PKC level or to alpha-tocopherol binding to PKC, but it results from increase of protein phosphatase 2A1 activity. In vitro data, as well as at a cellular level, demonstrates that protein phosphatase 2A1 is activated, in its trimeric structure--but not as a dimer by alpha-tocopherol. This activation is followed by PKC-alpha dephosphorylation. The activation of protein phosphatase 2A1 and deactivation of PKC-alpha affect the AP1 transcription factor, resulting in a change in the composition and the binding of this factor to DNA. By transfecting smooth muscle cell with a construct containing three TRE (TPA responsive elements), the promoter thymidine kinase and the reporter gene chloramphenicol-acetyl-transferase a modulation of gene expression by alpha-tocopherol is observed. Beta-tocopherol does not cause any of the responses observed with alpha-tocopherol and R,R,R-alpha-tocopherol is twice as potent as all-rac-alpha-tocopherol. When added together, beta-tocopherol prevents the effects of alpha-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal. By differential display analysis it has been found that several genes of smooth muscle cells are differentially transcribed in the presence of alpha-tocopherol but not beta-tocopherol. In particular, the gene of alpha-tropomyosin shows a transient enhancement of transcription as a function of the cell cycle time. Alpha-tropomyosin translation is also increased by alpha-tocopherol and not by beta-tocopherol. Because no changes of mRNA stability can be observed

  10. Interaction of smooth muscle relaxant drugs with calmodulin and cyclic nucleotide phosphodiesterase.

    PubMed

    Ronca-Testoni, S; Hrelia, S; Hakim, G; Rossi, C A

    1985-01-15

    Some smooth muscle relaxant drugs with an unknown mechanism of action have been tested for their interaction with calmodulin and with calmodulin-induced cyclic nucleotide phosphodiesterase (PDE) activity. The affinity of these drugs for calmodulin does not parallel their inhibitory effect on the calmodulin activation of PDE. The lack of parallelism could be due to a binding of the drugs to different sites on calmodulin; furthermore a binding of papaverine, octylonium bromide and felodipine to PDE molecule might also be considered to explain their inhibitory effect on PDE basal activity. The myolytic effect of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems. PMID:2981701

  11. De Novo ACTA2 Mutation Causes a Novel Syndrome of Multisystemic Smooth Muscle Dysfunction

    PubMed Central

    Milewicz, Dianna M.; Østergaard, John R.; Ala-Kokko, Leena M.; Khan, Nadia; Grange, Dorothy K.; Mendoza-Londono, Roberto; Bradley, Timothy J.; Olney, Ann Haskins; Adès, Lesley; Maher, Joseph F.; Guo, Dongchuan; Buja, L. Maximilian; Kim, Dong; Hyland, James C.; Regalado, Ellen S.

    2011-01-01

    Smooth muscle cells (SMCs) contract to perform many physiological functions, including regulation of blood flow and pressure in arteries, contraction of the pupils, peristalsis of the gut and voiding of the bladder. SMC lineage in these organs is characterized by cellular expression of the SMC isoform of α-actin, encoded by the ACTA2 gene. We report here on a unique and de novo mutation in ACTA2, R179H, that causes a syndrome characterized by dysfunction of SMCs throughout the body, leading to aortic and cerebrovascular disease, fixed dilated pupils, hypotonic bladder, malrotation and hypoperistalsis of the gut and pulmonary hypertension. PMID:20734336

  12. Incidental seminal vesicle smooth muscle neoplasm of unknown malignancy following robotic-assisted laparoscopic prostatectomy.

    PubMed

    Samadi, David B; Chughtai, Bilal; Akhavan, Ardavan; Guru, Khurshid; Rehman, Jamil

    2008-06-01

    Primary soft tissue sarcomas of the genitourinary tract are rarely seen, especially in the seminal vesicle. While sarcomas have been reported in the seminal vesicle, this is the first report of a smooth muscle neoplasm, of uncertain malignant potential, involving the seminal vesicle. The finding was incidental, following robotic-assisted radical retropubic prostatectomy for prostate cancer. To our knowledge, this is also the first report of a primary seminal vesicle tumor found following radical prostatectomy. A clinical case review and a brief review of the literature are presented. PMID:18570719

  13. Loss of serum response factor induces microRNA-mediated apoptosis in intestinal smooth muscle cells

    PubMed Central

    Park, C; Lee, M Y; Slivano, O J; Park, P J; Ha, S; Berent, R M; Fuchs, R; Collins, N C; Yu, T J; Syn, H; Park, J K; Horiguchi, K; Miano, J M; Sanders, K M; Ro, S

    2015-01-01

    Serum response factor (SRF) is a transcription factor known to mediate phenotypic plasticity in smooth muscle cells (SMCs). Despite the critical role of this protein in mediating intestinal injury response, little is known about the mechanism through which SRF alters SMC behavior. Here, we provide compelling evidence for the involvement of SRF-dependent microRNAs (miRNAs) in the regulation of SMC apoptosis. We generated SMC-restricted Srf inducible knockout (KO) mice and observed both severe degeneration of SMCs and a significant decrease in the expression of apoptosis-associated miRNAs. The absence of these miRNAs was associated with overexpression of apoptotic proteins, and we observed a high level of SMC death and myopathy in the intestinal muscle layers. These data provide a compelling new model that implicates SMC degeneration via anti-apoptotic miRNA deficiency caused by lack of SRF in gastrointestinal motility disorders. PMID:26633717

  14. [Regulation of the differentiation and proliferation of smooth muscle cells by the sex hormones].

    PubMed

    Guiochon-Mantel, A

    2000-06-01

    Steroids effects are mediated by their receptors. These proteins define the large family of steroid hormone receptors, characterized by the presence of 3 functional domains: a transactivation domain, a DNA-binding domain and a ligand-binding domain. Receptor activation induces the modulation of transcription of specific genes, and as a consequence, the modulation of production of specific proteins. Sex steroid receptors are located in the nucleus. This nuclear localization is in fact a dynamic situation, resulting from a continuous shuttling of the receptor between the cytoplasm and the nucleus. The recent discovery that an additional estrogen receptor is present in various tissues has advanced our understanding of the mechanism underlying estrogen signalling. Non genomic effects of steroids have also been described. Sex steroids inhibit proliferation of smooth muscle cells. On the contrary, they stimulate proliferation of tumoral muscle cells. The mechanisms of sex steroid effects on cellular proliferation are complex, and may involve transcriptional or non transcriptional phenomena. PMID:10939122

  15. Peripheral Airway Smooth Muscle, but Not the Trachealis, Is Hypercontractile in an Equine Model of Asthma.

    PubMed

    Matusovsky, Oleg S; Kachmar, Linda; Ijpma, Gijs; Bates, Genevieve; Zitouni, Nedjma; Benedetti, Andrea; Lavoie, Jean-Pierre; Lauzon, Anne-Marie

    2016-05-01

    Heaves is a naturally occurring equine disease that shares many similarities with human asthma, including reversible antigen-induced bronchoconstriction, airway inflammation, and remodeling. The purpose of this study was to determine whether the trachealis muscle is mechanically representative of the peripheral airway smooth muscle (ASM) in an equine model of asthma. Tracheal and peripheral ASM of heaves-affected horses under exacerbation, or under clinical remission of the disease, and control horses were dissected and freed of epithelium to measure unloaded shortening velocity (Vmax), stress (force/cross-sectional area), methacholine effective concentration at which 50% of the maximum response is obtained, and stiffness. Myofibrillar Mg(2+)-ATPase activity, actomyosin in vitro motility, and contractile protein expression were also measured. Horses with heaves had significantly greater Vmax and Mg(2+)-ATPase activity in peripheral airway but not in tracheal smooth muscle. In addition, a significant correlation was found between Vmax and the time elapsed since the end of the corticosteroid treatment for the peripheral airways in horses with heaves. Maximal stress and stiffness were greater in the peripheral airways of the horses under remission compared with controls and the horses under exacerbation, potentially due to remodeling. Actomyosin in vitro motility was not different between controls and horses with heaves. These data demonstrate that peripheral ASM is mechanically and biochemically altered in heaves, whereas the trachealis behaves as in control horses. It is therefore conceivable that the trachealis muscle may not be representative of the peripheral ASM in human asthma either, but this will require further investigation. PMID:26473389

  16. Effect of airway inflammation on smooth muscle shortening and contractility in guinea pig trachealis.

    PubMed

    Mitchell, R W; Ndukwu, I M; Arbetter, K; Solway, J; Leff, A R

    1993-12-01

    We studied the effect of either 1) immunogenic inflammation caused by aerosolized ovalbumin or 2) neurogenic inflammation caused by aerosolized capsaicin in vivo on guinea pig tracheal smooth muscle (TSM) contractility in vitro. Force-velocity relationships were determined for nine epithelium-intact TSM strips from ovalbumin-sensitized (OAS) vs. seven sham-sensitized controls and TSM strips for seven animals treated with capsaicin aerosol (Cap-Aer) vs. eight sham controls. Muscle strips were tethered to an electromagnetic lever system, which allowed isotonic shortening when load clamps [from 0 to maximal isometric force (Po)] were applied at specific times after onset of contraction. Contractions were elicited by supramaximal electrical field stimulation (60 Hz, 10-s duration, 18 V). Optimal length for each muscle was determined during equilibration. Maximal shortening velocity (Vmax) was increased in TSM from OAS (1.72 +/- 0.46 mm/s) compared with sham-sensitized animals (0.90 +/- 0.15 mm/s, P < 0.05); Vmax for TSM from Cap-Aer (0.88 +/- 0.11 mm/s) was not different from control TSM (1.13 +/- 0.08 mm/s, P = NS). Similarly, maximal shortening (delta max) was augmented in TSM from OAS (1.01 +/- 0.15 mm) compared with sham-sensitized animals (0.72 +/- 0.14 mm, P < 0.05); delta max for TSM from Cap-Aer animals (0.65 +/- 0.11 mm) was not different from saline aerosol controls (0.71 +/- 0.15 mm, P = NS). We demonstrate Vmax and delta max are augmented in TSM after ovalbumin sensitization; in contrast, neurogenic inflammation caused by capsaicin has no effect on isolated TSM contractility in vitro. These data suggest that airway hyperresponsiveness in vivo that occurs in association with immunogenic or neurogenic inflammation may result from different effects of these types of inflammation on airway smooth muscle. PMID:8279571

  17. Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration

    PubMed Central

    Upadhyayula, Pavan; Chen, Robert Y.; Chooljian, Marc S.; Li, Ju; Kung, Sunny; Jiang, Kevin P.; Conboy, Irina M.

    2014-01-01

    The regenerative capacity of skeletal muscle declines with age. Previous studies suggest that this process can be reversed by exposure to young circulation, but systemic age-specific factors responsible for this phenomenon are largely unknown. Here we report that oxytocin- a hormone best known for its role in lactation, parturition, and social behaviors - is required for proper muscle tissue regeneration and homeostasis, and that plasma levels of oxytocin decline with age. Inhibition of oxytocin signaling in young animals reduces muscle regeneration, whereas systemic administration of oxytocin rapidly improves muscle regeneration by enhancing aged muscle stem cell activation/proliferation throughactivation of the MAPK/ERK signalling pathway. We further show that the genetic lack of oxytocin does not cause a developmental defect in muscle, but instead leads to premature sarcopenia. Considering that oxytocin is an FDA approved drug, this work reveals a potential novel and safe way to combat or prevent skeletal muscle aging. PMID:24915299

  18. Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration.

    PubMed

    Elabd, Christian; Cousin, Wendy; Upadhyayula, Pavan; Chen, Robert Y; Chooljian, Marc S; Li, Ju; Kung, Sunny; Jiang, Kevin P; Conboy, Irina M

    2014-01-01

    The regenerative capacity of skeletal muscle declines with age. Previous studies suggest that this process can be reversed by exposure to young circulation; however, systemic age-specific factors responsible for this phenomenon are largely unknown. Here we report that oxytocin--a hormone best known for its role in lactation, parturition and social behaviours--is required for proper muscle tissue regeneration and homeostasis, and that plasma levels of oxytocin decline with age. Inhibition of oxytocin signalling in young animals reduces muscle regeneration, whereas systemic administration of oxytocin rapidly improves muscle regeneration by enhancing aged muscle stem cell activation/proliferation through activation of the MAPK/ERK signalling pathway. We further show that the genetic lack of oxytocin does not cause a developmental defect in muscle but instead leads to premature sarcopenia. Considering that oxytocin is an FDA-approved drug, this work reveals a potential novel and safe way to combat or prevent skeletal muscle ageing. PMID:24915299

  19. Stretch-dependent potassium channels in murine colonic smooth muscle cells.

    PubMed

    Koh, S D; Sanders, K M

    2001-05-15

    Gastrointestinal muscles are able to maintain negative resting membrane potentials in spite of stretch. We investigated whether stretch-dependent K+ channels might contribute to myogenic regulation of smooth muscle cells from the mouse colon. Negative pressure applied to on-cell membrane patches activated K+ channels that were voltage independent and had a slope conductance of 95 pS in symmetrical K+ gradients. The effects of negative pressure on open probability were graded as a function of pressure and reversible when atmospheric pressure was restored. Cell elongation activated K+ channels with the same properties as those activated by negative pressure, suggesting that the channels were stretch-dependent K+ (SDK) channels. Channels with the same properties were maximally activated by patch excision, suggesting that either an intracellular messenger or interactions with the cytoskeleton regulate open probability. Internal 4-aminopyridine, Ca2+ (10(-8) to 10(-6) M), and tetraethylammonium (internal or external) were without effect on SDK channels. Nitric oxide donors (and cell-permeant cGMP analogues) activated SDK channels, suggesting that these channels may mediate a portion of the enteric inhibitory neural response in colonic muscles. In summary, SDK channels are an important conductance expressed by colonic muscle cells. SDK channels may stabilize membrane potential during dynamic changes in cell length and mediate responses to enteric neurotransmitters. PMID:11351024

  20. Phorbol 12,13-dibutyrate-induced, protein kinase C-mediated contraction of rabbit bladder smooth muscle.

    PubMed

    Wang, Tanchun; Kendig, Derek M; Trappanese, Danielle M; Smolock, Elaine M; Moreland, Robert S

    2012-01-01

    Contraction of bladder smooth muscle is predominantly initiated by M(3) muscarinic receptor-mediated activation of the G(q/11)-phospholipase C β-protein kinase C (PKC) and the G(12/13)-RhoGEF-Rho kinase (ROCK) pathways. However, these pathways and their downstream effectors are not well understood in bladder smooth muscle. We used phorbol 12,13-dibutyrate (PDBu), and 1,2-dioctanoyl-sn-glycerol (DOG), activators of PKC, in this investigation. We were interested in dissecting the role(s) of PKC and to clarify the signaling pathways in bladder smooth muscle contraction, especially the potential cross-talk with ROCK and their downstream effectors in regulating myosin light chain phosphatase activity and force. To achieve this goal, the study was performed in the presence or absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850) myosin phosphatase target subunit (MYPT1) were measured during PDBu or DOG stimulation using site specific antibodies. PDBu-induced contraction in bladder smooth muscle involved both activation of PKC and PKC-dependent activation of ROCK. CPI-17 as a major downstream effector, is phosphorylated by PKC and ROCK during PDBu and DOG stimulation. Our results suggest that Thr(696) and Thr(850)-MYPT1 phosphorylation are not involved in the regulation of a PDBu-induced contraction. The results also demonstrate that bladder smooth muscle contains a constitutively active isoform of ROCK that may play an important role in the regulation of bladder smooth muscle basal tone. Together with the results from our previous study, we developed a working model to describe the complex signaling pathways that regulate contraction of bladder smooth muscle. PMID:22232602

  1. Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle.

    PubMed

    Léguillette, Renaud; Zitouni, Nedjma B; Govindaraju, Karuthapillai; Fong, Laura M; Lauzon, Anne-Marie

    2008-09-01

    Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (nu(max)) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of nu(max) versus [MgADP] was significantly greater for tonic (-0.51+/-0.04) than phasic muscle myosin (-0.15+/-0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092+/-0.022 pN for phasic muscle and 0.084+/-0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state. PMID:18614813

  2. Your Muscles

    MedlinePlus

    ... Homework? Here's Help White House Lunch Recipes Your Muscles KidsHealth > For Kids > Your Muscles Print A A ... and skeletal (say: SKEL-uh-tul) muscle. Smooth Muscles Smooth muscles — sometimes also called involuntary muscles — are ...

  3. Hypercholesterolemic diet induces vascular smooth muscle cell apoptosis in sympathectomized rats via intrinsic pathway.

    PubMed

    Hachani, Rafik; Dab, Houcine; Feriani, Anouar; Saber, Sami; Sakly, Mohsen; Vicaut, Eric; Callebert, Jacques; Sercombe, Richard; Kacem, Kamel

    2014-07-01

    In this study, we intend to investigate the role of hypercholesterolemic diet, a high risk factor for atherosclerosis, on vascular cell apoptosis in rats that have been previously sympathectomized. Thus, newborn male Wistar rats received injections of guanethidine for sympathectomy. Sham received injections of vehicle. The two groups were fed 1% cholesterol diet for 3months. Sympathectomy alone group was also exploited. Apoptosis in abdominal aortic tissue was identified by TUNEL method and conventional agarose gel electrophoresis to detect specific DNA fragmentation. Caspases 3 and 9, Bcl-2, Bax and cytochrome c were examined by immunoblotting. Oil Red O staining was used to reveal lipid in the arterial wall. Vascular smooth muscle cells (VSMCs) and macrophages were identified by immunostaining for α-smooth muscle actin and rat macrophage marker (ED1), respectively. The efficacy of sympathectomy was evaluated by analysis of perivascular sympathetic fibers. Our study showed that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, 1) increased aortic TUNEL-positive cells compared to sham and sympathectomy alone groups, 2) illustrated a typical apoptotic DNA ladder on agarose gel electrophoresis, 3) induced Bax translocation from cytosol to mitochondria, 4) enhanced cytochrome c release from mitochondria to cytosol, 5) increased expression of active caspases 3 and 9, and 6) decreased Bcl-2 expression. VSMCs are identified as the major cell type exhibiting apoptosis in this model. Taken together, it can be concluded that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, induces vascular cell apoptosis in an intrinsic pathway. PMID:24708922

  4. Regulatory effects of eicosanoids on thymidine uptake by vascular smooth muscle cells of rats

    SciTech Connect

    Uehara, Y.; Ishimitsu, T.; Kimura, K.; Ishii, M.; Ikeda, T.; Sugimoto, T.

    1988-12-01

    To define the roles of eicosanoids in vascular smooth muscle cells (VSMC) growth, we examined the effects of exogenous eicosanoids on (/sup 3/H)thymidine uptake by cultured VSMC of Wistar rats. Stable prostacyclin (PGI2) analog, OP-41483, significantly decreased the incorporation of (/sup 3/H)thymidine into deoxyribonucleic acid (DNA) of VSMC in a dose dependent manner from 10(-8) to 10(-4) M. Prostaglandin E2 (PGE2) and PGD2 ranging from 10(-8) to 10(-4) M also dose-dependently decreased the (/sup 3/H)thymidine uptake by VSMC. In contrast, stable thromboxane A2 analog, STA2, significantly increased the incorporation of (/sup 3/H)thymidine into DNA in a dose dependent manner from 10(-8) to 10(-4) M. The dose response curve of STA2 was shifted toward a lowered response when 10(-5) M PGI2 analog, PGE2 or PGD2 was added in the culture medium. Thus, it is indicated that vasodepressor eicosanoids decrease the proliferation of VSMC, whereas vasoconstrictor TXA2 enhances the VSMC growth. Vascular smooth muscle cells possibly autoregulate the cell proliferation through the eicosanoids generation.

  5. Digestive smooth muscle mitochondrial myopathy in patients with mitochondrial-neuro-gastro-intestinal encephalomyopathy (MNGIE).

    PubMed

    Blondon, Hugues; Polivka, Marc; Joly, Francisca; Flourie, Bernard; Mikol, Jacqueline; Messing, Bernard

    2005-01-01

    We report 3 new cases of Mitochondrial-Neuro-Gastro-Intestinal Encephalomyopathy (MNGIE) (or Pseudo-Obstruction-Leukoencephalopathy-Intestinal-Pseudoobstruction Syndrome [POLIP]), a rare disease that associates chronic intestinal pseudo-obstruction (CIPO) and neurological symptoms. A review of the 72 reported cases together with these 3 cases revealed that this condition was associated with (a) a specific cluster of neurological symptoms including leukoencephalopathy (96%), polyneuropathy (96%), ophthalmoplegia (91%) and hearing loss (55%); (b) a CIPO syndrome with the presence of small bowel diverticulae (53%); and (c) mitochondrial cytopathy in 36 of the 37 tested patients (2 of our 3 cases), and thymidine phosphorylase gene mutations in all the 37 tested patients (2 of our cases). The etiology of POLIP/MNGIE syndrome appears therefore to be due to a mitochondrial cytopathy secondary to thymidine phosphorylase gene mutation(s). In 3 cases, including 2 of our 3 patients, mitochondrial abnormalities were evidenced at the ultrastructural level in digestive smooth muscle demonstrating that the pathogenesis of gastrointestinal involvement was directly related to mitochondrial alterations in digestive smooth muscle cells. PMID:16294144

  6. Physiological functions of transient receptor potential channels in pulmonary arterial smooth muscle cells.

    PubMed

    Yang, Xiao-Ru; Lin, Mo-Jun; Sham, James S K

    2010-01-01

    The transient receptor potential (TRP) gene superfamily, which consists of 7 subfamilies with at least 28 mammalian homologues, is known to encode a wide variety of cation channels with diverse biophysical properties, activation mechanisms, and physiological functions. Recent studies have identified multiple TRP channel subtypes, belonging to the canonical (TRPC), melastatin-related (TRPM), and vanilloid-related (TRPV) subfamilies, in pulmonary arterial smooth muscle cells (PASMCs). They operate as specific Ca(2+) pathways responsive to stimuli, including Ca(2+) store depletion, receptor activation, reactive oxygen species, growth factors, and mechanical stress. Increasing evidence suggests that these channels play crucial roles in agonist-induced pulmonary vasoconstriction, hypoxic pulmonary vasoconstriction, smooth muscle cell proliferation, vascular remodeling, and pulmonary arterial hypertension. This chapter highlighted and discussed these putative physiological functions of TRP channels in pulmonary vasculatures. Since Ca(2+) ions regulate many cellular processes via specific Ca(2+) signals, future investigations of these novel channels will likely uncover more important regulatory mechanisms of pulmonary vascular functions in health and in disease states. PMID:20204726

  7. Proteomic network analysis of human uterine smooth muscle in pregnancy, labor, and preterm labor

    PubMed Central

    Ulrich, Craig; Quilici, David R.; Schlauch, Karen A.; Buxton, Iain L. O.

    2015-01-01

    The molecular mechanisms involved in human uterine quiescence during gestation and the induction of labor at term or preterm are not completely known. Preterm delivery is associated with major morbidity and mortality and current efforts to prevent delivery until term are largely ineffective. Identification and semi-quantification of proteomic changes in uterine smooth muscle during pregnancy will allow for targeted research into how quiescence is maintained and what changes are associated with induction of labor. Examining preterm labor in this context will provide potential therapeutic targets for the management of preterm labor. We have recently performed two dimensional liquid chromatography coupled with tandem mass spectrometry on myometrial proteins isolated from pregnant patients in labor, pregnant patients not in labor, and pregnant patients in labor preterm. Using a conservative false discovery rate of 1% we have identified 2132 protein groups using this method and semi-quantitative spectral counting shows 201 proteins that have disparate levels of expression in preterm laboring samples. To our knowledge this is the first large scale proteomic study examining human uterine smooth muscle and this initial work has provided a target list for future experiments that can address how changing protein levels are involved in the induction of labor at term and preterm. PMID:26413312

  8. Cyclooxygenase-2 in Endothelial and Vascular Smooth Muscle Cells Restrains Atherogenesis in Hyperlipidemic Mice

    PubMed Central

    Tang, Soon Yew; Monslow, James; Todd, Leslie; Lawson, John; Puré, Ellen; FitzGerald, Garret A.

    2014-01-01

    Background Placebo controlled trials of nonsteroidal antinflammatory drugs (NSAIDs) selective for inhibition of COX-2 reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages. Methods and Results Here, selective depletion of COX-2 in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) depressed biosynthesis of prostaglandin (PG)I2 and PGE2, elevated blood pressure and accelerated atherogenesis in Ldlr knockout (KO) mice. Deletion of COX-2 in VSMCs and ECs coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, α-smooth muscle actin and matrix-rich fibrosis was also apparent in lesions of the mutants. Conclusions Although atherogenesis is accelerated in global COX-2 KOs, consistent with evidence of risk transformation during chronic NSAID administration, this masks the contrasting effects of enzyme depletion in macrophages versus VSMCs and ECs. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk. PMID:24519928

  9. Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells

    PubMed Central

    Jeong, Ji Young; Son, Younghae; Kim, Bo-Young; Eo, Seong-Kug; Rhim, Byung-Yong

    2015-01-01

    We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype. PMID:26557022

  10. Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells.

    PubMed

    Jeong, Ji Young; Son, Younghae; Kim, Bo-Young; Eo, Seong-Kug; Rhim, Byung-Yong; Kim, Koanhoi

    2015-11-01

    We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype. PMID:26557022

  11. Thin-film dielectric elastomer sensors to measure the contraction force of smooth muscle cells

    NASA Astrophysics Data System (ADS)

    Araromi, O.; Poulin, A.; Rosset, S.; Favre, M.; Giazzon, M.; Martin-Olmos, C.; Liley, M.; Shea, H.

    2015-04-01

    The development of thin-film dielectric elastomer strain sensors for the characterization of smooth muscle cell (SMC) contraction is presented here. Smooth muscle disorders are an integral part of diseases such as asthma and emphysema. Analytical tools enabling the characterization of SMC function i.e. contractile force and strain, in a low-cost and highly parallelized manner are necessary for toxicology screening and for the development of new and more effective drugs. The main challenge with the design of such tools is the accurate measurement of the extremely low contractile cell forces expected as a result of SMC monolayer contraction (as low as ~ 100 μN). Our approach utilizes ultrathin (~5 μm) and soft elastomer membranes patterned with elastomer-carbon composite electrodes, onto which the SMCs are cultured. The cell contraction induces an in-plane strain in the elastomer membrane, predicted to be in the order 1 %, which can be measured via the change in the membrane capacitance. The cell force can subsequently be deduced knowing the mechanical properties of the elastomer membrane. We discuss the materials and fabrication methods selected for our system and present preliminary results indicating their biocompatibility. We fabricate functional capacitive senor prototypes with good signal stability over the several hours (~ 0.5% variation). We succeed in measuring in-plane strains of 1 % with our fabricated devices with good repeatability and signal to noise ratio.

  12. Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries.

    PubMed

    Yip, Ham; Chan, Wing-Yee; Leung, Pan-Cheung; Kwan, Hiu-Yee; Liu, Cuiling; Huang, Yu; Michel, Villaz; Yew, David Tai-Wai; Yao, Xiaoqiang

    2004-12-01

    TRPC channels are a group of Ca(2+)-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca(2+) influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3-6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure. PMID:15538613

  13. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.

    PubMed Central

    Wickham, T J; Segal, D M; Roelvink, P W; Carrion, M E; Lizonova, A; Lee, G M; Kovesdi, I

    1996-01-01

    A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer. PMID:8794324

  14. Smooth Muscle α Actin (Acta2) and Myofibroblast Function during Hepatic Wound Healing

    PubMed Central

    Rockey, Don C.; Weymouth, Nate; Shi, Zengdun

    2013-01-01

    Smooth muscle α actin (Acta2) expression is largely restricted to smooth muscle cells, pericytes and specialized fibroblasts, known as myofibroblasts. Liver injury, associated with cirrhosis, induces transformation of resident hepatic stellate cells into liver specific myofibroblasts, also known as activated cells. Here, we have used in vitro and in vivo wound healing models to explore the functional role of Acta2 in this transformation. Acta2 was abundant in activated cells isolated from injured livers but was undetectable in quiescent cells isolated from normal livers. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. Inhibition of Acta2 using several different techniques had no effect on cytoplasmic actin isoform expression, but led to reduced cellular motility and contraction. Additionally, Acta2 knockdown was associated with a significant reduction in Erk1/2 phosphorylation compared to control cells. The data indicate that Acta2 is important specifically in myofibroblast cell motility and contraction and raise the possibility that the Acta2 cytoskeleton, beyond its structural importance in the cell, could be important in regulating signaling processes during wound healing in vivo. PMID:24204762

  15. Cellular and Molecular Mechanisms of Phenotypic Switch in Gastrointestinal Smooth Muscle.

    PubMed

    Scirocco, Annunziata; Matarrese, Paola; Carabotti, Marilia; Ascione, Barbara; Malorni, Walter; Severi, Carola

    2016-02-01

    As a general rule, smooth muscle cells (SMC) are able to switch from a contractile phenotype to a less mature synthetic phenotype. This switch is accompanied by a loss of differentiation with decreased expression of contractile markers, increased proliferation as well as the synthesis and the release of several signaling molecules such as pro-inflammatory cytokines, chemotaxis-associated molecules, and growth factors. This SMC phenotypic plasticity has extensively been investigated in vascular diseases, but interest is also emerging in the field of gastroenterology. It has in fact been postulated that altered microenvironmental conditions, including the composition of microbiota, could trigger the remodeling of the enteric SMC, with phenotype changes and consequent alterations of contraction and impairment of gut motility. Several molecular actors participate in this phenotype remodeling. These include extracellular molecules such as cytokines and extracellular matrix proteins, as well as intracellular proteins, for example, transcription factors. Epigenetic control mechanisms and miRNA have also been suggested to participate. In this review key roles and actors of smooth muscle phenotypic switch, mainly in GI tissue, are described and discussed in the light of literature data available so far. J. Cell. Physiol. 231: 295-302, 2016. © 2015 Wiley Periodicals, Inc. PMID:26206426

  16. Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.

    PubMed

    Pugh, Raymond J; Slee, Joshua B; Farwell, Sara Lynn N; Li, Yaqiu; Barthol, Trista; Patton, Walter A; Lowe-Krentz, Linda J

    2016-03-01

    Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells. PMID:26769966

  17. Complex I dysfunction underlies the glycolytic switch in pulmonary hypertensive smooth muscle cells

    PubMed Central

    Rafikov, Ruslan; Sun, Xutong; Rafikova, Olga; Louise Meadows, Mary; Desai, Ankit A.; Khalpey, Zain; Yuan, Jason X.-J.; Fineman, Jeffrey R.; Black, Stephen M.

    2015-01-01

    ATP is essential for cellular function and is usually produced through oxidative phosphorylation. However, mitochondrial dysfunction is now being recognized as an important contributing factor in the development cardiovascular diseases, such as pulmonary hypertension (PH). In PH there is a metabolic change from oxidative phosphorylation to mainly glycolysis for energy production. However, the mechanisms underlying this glycolytic switch are only poorly understood. In particular the role of the respiratory Complexes in the mitochondrial dysfunction associated with PH is unresolved and was the focus of our investigations. We report that smooth muscle cells isolated from the pulmonary vessels of rats with PH (PH-PASMC), induced by a single injection of monocrotaline, have attenuated mitochondrial function and enhanced glycolysis. Further, utilizing a novel live cell assay, we were able to demonstrate that the mitochondrial dysfunction in PH-PASMC correlates with deficiencies in the activities of Complexes I–III. Further, we observed that there was an increase in mitochondrial reactive oxygen species generation and mitochondrial membrane potential in the PASMC isolated from rats with PH. We further found that the defect in Complex I activity was due to a loss of Complex I assembly, although the assembly of Complexes II and III were both maintained. Thus, we conclude that loss of Complex I assembly may be involved in the switch of energy metabolism in smooth muscle cells to glycolysis and that maintaining Complex I activity may be a potential therapeutic target for the treatment of PH. PMID:26298201

  18. Effects of trimebutine on cytosolic Ca2+ and force transitions in intestinal smooth muscle.

    PubMed

    Nagasaki, M; Kobayashi, T; Tamaki, H

    1991-04-01

    The effects of trimebutine maleate on cytosolic free Ca2+ and force transitions in the guinea-pig taenia cecum were studied by fura-2 fluorometry and tension recording. The addition of 80 mM K+ induced a transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) and tension, followed by a sustained increase. Trimebutine (10 microM) suppressed both [Ca2+]i elevation and tension development. The tonic responses were more potently inhibited than the phasic responses. Phasic components gradually increased as the added K+ increased (10-40 mM). The relationship between the peak increases in [Ca2+]i and tension was not affected by trimebutine (10 microM). This means that trimebutine does not affect the Ca2+ sensitivity of contractile elements. In a high K+ and Ca(2+)-free medium, carbachol (10 microM) or caffeine (30 mM) caused transient [Ca2+]i elevation and tension development in the smooth muscle. Trimebutine (10 microM) decreased the amplitude of both responses. Trimebutine (10 microM) inhibited the spontaneous fluctuations in [Ca2+]i and motility of taenia cecum in the presence of tetrodotoxin (TTX; 0.3 microM). These results suggest that trimebutine has two types of inhibitory actions on intestinal smooth muscle; one, the inhibition of Ca2+ influx through voltage-dependent calcium channels, and the other, the inhibition of Ca2+ release from intracellular storage sites. PMID:1868878

  19. Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction

    PubMed Central

    Koopmans, Tim; Kumawat, Kuldeep; Halayko, Andrew J; Gosens, Reinoud

    2016-01-01

    A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma. PMID:27468699

  20. Receptor-based differences in human aortic smooth muscle cell membrane stiffness

    NASA Technical Reports Server (NTRS)

    Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.

    2001-01-01

    Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (P<0.001). Mechanical characteristics of vascular smooth muscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

  1. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    SciTech Connect

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier; Noppe, Gauthier; Horman, Sandrine; Morel, Nicole

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  2. Effect of gamma-aminobutyric acid on neurally mediated contraction of guinea pig trachealis smooth muscle.

    PubMed

    Tamaoki, J; Graf, P D; Nadel, J A

    1987-10-01

    To determine whether gamma-aminobutyric acid (GABA) affects the contractile properties of airway smooth muscle and, if so, what the mechanism of action is, the authors studied guinea pig tracheal rings under isometric conditions in vitro. GABA and related substances, baclofen and muscimol, had no effect on the resting tension but reversibly depressed contractions induced by electrical field stimulation in a dose-dependent fashion, IC50 values (mean +/- S.E.) being 5.6 +/- 1.4 X 10(-6) M, 6.8 +/- 0.9 X 10(-6) M and 8.5 +/- 1.5 X 10(-5) M, respectively. In contrast, GABA did not alter the response to exogenous acetylcholine or the nonadrenergic noncholinergic inhibitory component. Pretreatment of tissues with bicuculline antagonized the inhibitory effect of GABA as well as that of baclofen. This inhibitory effect was not modified by propranolol, phentolamine, hemicholinium-3 or naloxone, but it was blocked by the Cl channel blocker furosemide and by the substitution of external Cl. These results suggest that GABA decreases the contractile response of airway smooth muscle to cholinergic nerve stimulation by inhibiting the evoked release of acetylcholine and that this effect is exerted by activating Cl-dependent, bicuculline-sensitive GABA receptors. PMID:3668869

  3. Effects of dexamethasone on the synthesis and secretion of galaptin by vascular smooth muscle cells

    SciTech Connect

    Sanford, G.L.; Harris-Hooker, S.A.

    1986-05-01

    The effects of dexamethasone (Dex) on the synthesis and secretion of beta-galactoside specific lectin (galaptin) was examined in cultured primate aortic smooth muscle cells (SMC). SMC cells were treated with 0.15 ..mu..M Dex during their proliferative phase to confluency, and after reaching confluency. Both cultures were labeled with (/sup 3/H)-phenylalanine (phe) for 24 h following exposure to Dex. Incorporation of phe into galaptin increased twofold in the medium from Dex treated confluent cultures, when serum was present. No change was found in incorporation when serum was removed prior to Dex treatment. Phe incorporation into total protein was also increased twofold by Dex treatment of SMC in the presence of serum, but there was a 1.4-fold increase when serum was absent. Dex did not affect the incorporation of phe into either total protein or galaptin in the cell layer of confluent cultures in the presence of serum, but caused a twofold increase in its absence. There was no effect of Dex on the incorporation of phe into galaptin or total protein in either the medium or cell layer of cultures given Dex during their proliferative phase. Dex retarded the growth of SMC, and lowered the total protein content of the cell layer. The results show that vascular smooth muscle cells synthesize and secrete galaptin and that Dex acts directly on confluent SMC to increase galaptin synthesis and secretion. Serum seems to modulate the effect of Dex.

  4. Smooth muscle actin isoforms: a tug of war between contraction and compliance.

    PubMed

    Arnoldi, Richard; Hiltbrunner, Anita; Dugina, Vera; Tille, Jean-Christophe; Chaponnier, Christine

    2013-01-01

    In higher vertebrates, smooth muscle (SM) contains two tissue-specific actin isoforms: α-SMA and γ-SMA, which predominate in vascular and visceral SM, respectively. Whether α-SMA has been extensively studied and recognized for its contractile activity in SM and SM-like cells such as myofibroblasts, myoepithelial and myoid cells, the distribution and role of γ-SMA remained largely unknown. We developed a new specific monoclonal antibody against γ-SMA and confirmed that γ-SMA predominates in the visceral system and is minor in the vascular system, although more expressed in highly compliant veins than in stiff arteries. Contrary to α-SMA, γ-SMA is absent from myofibroblasts in vitro, and in fibrotic diseases in vivo. We raised the hypothesis that, whereas α-SMA is responsible for the "contractile" activity, γ-SMA would be involved in the "compliance" of SM and SM-like cells. Several models support this hypothesis, namely veins vs. arteries and the physiological modifications occurring in the uterus and mammary glands during pregnancy and lactation. Our results suggest that, in addition to enteric smooth muscles, γ-SMA is expressed in all the tissues submitted to an important dilation including veins, gravid uterus, and lactating mammary glands. The hypothesis of two complementary mechanical roles for the two SMA isoforms is sustained by their different intracellular distributions and by functional assays. PMID:23915964

  5. Comparison of doxycycline and minocycline in the inhibition of VEGF-induced smooth muscle cell migration

    PubMed Central

    Yao, Jianhua S.; Shen, Fanxia; Young, William L.; Yang, Guo-Yuan

    2007-01-01

    Smooth muscle migration plays an important role during angiogenesis and vascular remodeling. In this study, we examined the effects of doxycycline and minocycline on vascular endothelial growth factor (VEGF)-induced human aortic smooth muscle cell (HASMCs) migration, and explored the mechanisms in which doxycycline or minocycline inhibit HASMC migration. We demonstrated that both doxycycline and minocycline attain consistent anti-angiogenic effects in the inhibition of HASMC migration via a different signal pathway (p<0.05). This effect is through attenuating VEGF-induced matrix metalloproteinase-9 (MMP-9) activity (p<0.05). Doxycycline could increase tissue inhibitors of metalloproteinases-1 (TIMP-1) expression while minocycline down-regulated PI3K/Akt phosphorylation in HASMC. Our study suggests that doxycycline has a stronger ability to inhibit MMP secretion in HASMC by up-regulating endogenous MMPs inhibitor TIMP-1, while minocycline implements anti-angiogenic effect through inhibiting HASMC migration by down-regulating PI3K/Akt pathway. PMID:17145119

  6. Alpha smooth muscle actin in the cycling ovary - an immunohistochemical study.

    PubMed

    Hirschberg, Ruth M; Plendl, Johanna; Kaessmeyer, Sabine

    2012-01-01

    In the ovary with its cyclically developing and regressing functional bodies and the associated intense neovascularisation and remodelling, alpha-smooth muscle actin (SMA) immunolocalisation has been frequently used as a marker to establish vessel hierarchy, in angiogenesis studies, or in studies characterising ovarian neoplasms in various species. The present study aims at detection of alpha-SMA-immunolocalisation within all structural components of the cycling bovine ovary in order to complement the hitherto available data. 27 ovaries, mainly of dairy cows ranging from 23 to 118 months of age and displaying all major stages of follicle and corpora lutea development, were collected at the abattoir and subjected to routine HE and trichrome staining as well as alpha-SMA immunohistochemistry. For this purpose, the specimens were pooled to form groups of the respective stage of corpus luteum development. The ovarian stroma displayed a notable alpha-SMA-reactivity, particularly surrounding the functional bodies. The study revealed specialised vascular modifications such as multi-directionally arranged vascular smooth muscle layers, vascular sphincters and distinct epitheloid modifications of the media in ovarian arteries. Alpha-SMA-reactivity of the microcirculation within corpora lutea of various stages allowed inferences on respective angiogenic properties. The findings were discussed focussing on functional interpretations. PMID:22538540

  7. Peptides PHI and VIP: comparison between vascular and nonvascular smooth muscle effect in rabbit uterus

    SciTech Connect

    Bardrum, B.; Ottesen, B.; Fahrenkrug, J.

    1986-07-01

    The distribution and effects of the two neuropeptides, vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine amide (PHI), on vascular and nonvascular smooth muscle in the urogenital tract of nonpregnant rabbit female, were investigated. Immunoreactive VIP and PHI were present in all regions except the ovary with the highest concentration in the uterin cervix. By using in vitro tension recordings of myometrial specimens, it was demonstrated that both peptides displayed a dose-dependent inhibition of the mechanical activity. The dose-response curves of VIP and PHI were superimposable with and ID50 of 3 x 10 Y mol/l, and their combined effect was additive. In addition, the influence of the two peptides on myometrial blood flow (MBF) was investigated by the xenon-133 washout technique. Both peptides were found to increase MBF with the same potency and efficacy. Their combined effect was additive. In conclusion VIP and PHI are present in the rabbit urogenital tract, and the two peptides are equipotent inhibitors of mechanical nonvascular and vascular smooth muscle activity in the uterus.

  8. Magnesium relaxes arterial smooth muscle by decreasing intracellular Ca2+ without changing intracellular Mg2+.

    PubMed Central

    D'Angelo, E K; Singer, H A; Rembold, C M

    1992-01-01

    Elevations in extracellular [Mg2+] ([Mg2+]o) relax vascular smooth muscle. We tested the hypothesis that elevated [Mg2+]o induces relaxation through reductions in myoplasmic [Ca2+] and myosin light chain phosphorylation without changing intracellular [Mg2+] ([Mg2+]i). Histamine stimulation of endothelium-free swine carotid medial tissues was associated with increases in both Fura 2- and aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and force. Elevated [Mg2+]o decreased myoplasmic [Ca2+] and force to near resting values. However, elevated [Mg2+]o only transiently decreased myosin phosphorylation values: sustained [Mg2+]o-induced decreases in myoplasmic [Ca2+] and force were associated with inappropriately high myosin phosphorylation values. The elevated myosin phosphorylation during [Mg2+]o-induced relaxation was entirely on serine 19, the Ca2+/calmodulin-dependent myosin light chain kinase substrate. Myoplasmic [Mg2+] (estimated with Mag-Fura 2) did not significantly increase with elevated [Mg2+]o. These results are consistent with the hypothesis that increased [Mg2+]o induces relaxation by decreasing myoplasmic [Ca2+] without changing [Mg2+]i. These data also demonstrate dissociation of myosin phosphorylation from myoplasmic [Ca2+] and force during Mg(2+)-induced relaxation. This finding suggests the presence of a phosphorylation-independent (yet potentially Ca(2+)-dependent) mechanism for regulation of force in vascular smooth muscle. Images PMID:1602005

  9. Potentiation of carbachol-induced detrusor smooth muscle contractions by β-adrenoceptor activation

    PubMed Central

    Klausner, Adam P; Rourke, Keith F; Miner, Amy S; Ratz, Paul H

    2011-01-01

    In strips of rabbit bladder free of urothelium, the β-adrenoceptor agonist, isoproterenol, significantly reduced basal detrusor smooth muscle tone and inhibited contractions produced by low concentrations of the muscarinic receptor agonist, carbachol. During a carbachol concentration-response curve, instead of inhibiting, isoproterenol strengthened contractions produced by high carbachol concentrations. Thus, the carbachol concentration-response curve was shifted by isoproterenol from a shallow, graded relationship, to a steep, switch-like relationship. The tyrosine kinase inhibitor, genistein, inhibited carbachol-induced contractions only in the presence of isoproterenol. Contraction produced by a single high carbachol concentration (1 µM) displayed 1 fast and 1 slow peak. In the presence of isoproterenol, the slow peak was not strengthened, but was delayed, and U-0126 (mitogen-activated protein kinase kinase inhibitor) selectively inhibited this delay concomitantly with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. Isoproterenol reduced ERK phosphorylation only in the absence of carbachol. These data support the concept that, by inhibiting weak contractions, potentiating strong contractions, and producing a more switch-like concentration-response curve, β-adrenoceptor stimulation enhanced the effectiveness of muscarinic receptor-induced detrusor smooth muscle contraction. Moreover, β-adrenoceptor stimulation changed the cellular mechanism by which carbachol produced contraction. The potential significance of multi-receptor and multi-cell crosstalk is discussed. PMID:19374847

  10. Use of an alpha-smooth muscle actin (SMAA) GFP reporter to identify an osteoprogenitor population

    PubMed Central

    Kalajzic, Zana; Li, Haitao; Wang, Li-Ping; Jiang, Xi; Lamothe, Katie; Adams, Douglas J.; Aguila, Hector L.; Rowe, David W.; Kalajzic, Ivo

    2008-01-01

    Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of α-smooth muscle actin (α-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of SMAA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of α-SMA+ cells towards osteoprogenitor lineage. Utilizing α-SMAGFP transgene, α-SMAGFP+ positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the α-SMAGFP+ population derived from bone marrow or adipose tissue. In adipose tissue, α-SMA+ cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, α-SMA+/CD45−/Sca1+ progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts. PMID:18571490

  11. Metabolomic Profiling of Cellular Responses to Carvedilol Enantiomers in Vascular Smooth Muscle Cells

    PubMed Central

    Wang, Mingxuan; Bai, Jing; Chen, Wei Ning; Ching, Chi Bun

    2010-01-01

    Carvedilol is a non-selective β-blocker indicated in the treatment of hypertension and heart failure. Although the differential pharmacological effects of individual Carvedilol enantiomer is supported by preceding studies, the cellular response to each enantiomer is not well understood. Here we report the use of GC-MS metabolomic profiling to study the effects of Carvedilol enantiomers on vascular smooth muscle cells (A7r5) and to shed new light on molecular events underlying Carvedilol treatment. The metabolic analysis revealed alternations in the levels of 8 intracellular metabolites and 5 secreted metabolites in A7r5 cells incubated separately with S- and R-Carvedilol. Principal component analysis of the metabolite data demonstrated the characteristic metabolic signatures in S- and R-Carvedilol-treated cells. A panel of metabolites, including L-serine, L-threonine, 5-oxoproline, myristic acid, palmitic acid and inositol are closely correlated to the vascular smooth muscle contraction. Our findings reveal the differentiating metabolites for A7r5 cells incubated with individual enantiomer of Carvedilol, which opens new perspectives to employ metabolic profiling platform to study chiral drug-cell interactions and aid their incorporation into future improvement of β-blocker therapy. PMID:21124793

  12. Airway hyperresponsiveness in asthma: a problem of limited smooth muscle relaxation with inspiration.

    PubMed Central

    Skloot, G; Permutt, S; Togias, A

    1995-01-01

    We hypothesized that hyperresponsiveness in asthma is caused by an impairment in the ability of inspiration to stretch airway smooth muscle. If the hypothesis was correct, we reasoned that the sensitivity to inhaled methacholine in normal and asthmatic subjects should be the same if the challenge was carried out under conditions where deep inspirations were prohibited. 10 asthmatic and 10 normal subjects received increasing concentrations of inhaled methacholine under conditions where forced expirations from a normal end-tidal inspiration were performed. When no deep inspirations were allowed, the response to methacholine was similar in the normal and asthmatic subjects, compatible with the hypothesis we propose. Completely contrary to our expectations, however, was the marked responsivity to methacholine that remained in the normal subjects after deep breaths were initiated. 6 of the 10 normal subjects had > 20% reduction in forced expiratory volume in one second (FEV 1) at doses of methacholine < 8 mg/ml, whereas there was < 15% reduction with 75 mg/ml during routine challenge. The ability of normal subjects to develop asthmatic responses when the modulating effects of increases in lung volume was voluntarily suppressed suggests that an intrinsic impairment of the ability of inspiration to stretch airway smooth muscle is a major feature of asthma. PMID:7593627

  13. Possible Mechanisms for Functional Antagonistic Effect of Ferula assafoetida on Muscarinic Receptors in Tracheal Smooth Muscle

    PubMed Central

    Kiyanmehr, Majid; Boskabady, Mohammad Hossein; Khazdair, Mohammad Reza; Hashemzehi, Milad

    2016-01-01

    Background The contribution of histamine (H1) receptors inhibitory and/or β-adrenoceptors stimulatory mechanisms in the relaxant property of Ferula assa-foetida. (F. asafoetida) was examined in the present study. Methods We evaluated the effect of three concentrations of F. asafoetida extract (2.5, 5, and 10 mg/mL), a muscarinic receptors antagonist, and saline on methacholine concentration-response curve in tracheal smooth muscles incubated with β-adrenergic and histamine (H1) (group 1), and only β-adrenergic (group 2) receptors antagonists. Results EC50 values in the presence of atropine, extract (5 and 10 mg/mL) and maximum responses to methacholine due to the 10 mg/mL extract in both groups and 5 mg/mL extract in group 1 were higher than saline (P < 0.0001, P = 0.0477, and P = 0.0008 in group 1 and P < 0.0001, P = 0.0438, and P = 0.0107 in group 2 for atropine, 5 and 10 mg/mL extract, respectively). Values of concentration ratio minus one (CR-1), in the presence of extracts were lower than atropine in both groups (P = 0.0339 for high extract concentration in group 1 and P < 0.0001 for other extract concentrations in both groups). Conclusion Histamine (H1) receptor blockade affects muscarinic receptors inhibitory property of F. asafoetida in tracheal smooth muscle PMID:27540324

  14. Integration of signal pathways for stretch-dependent growth and differentiation in vascular smooth muscle.

    PubMed

    Albinsson, Sebastian; Hellstrand, Per

    2007-08-01

    The vascular smooth muscle phenotype is regulated by environmental factors, such as mechanical forces, that exert effects on signaling to differentiation and growth. We used the mouse portal vein in organ culture to investigate stretch-dependent activation of Akt, ERK, and focal adhesion kinase (FAK), which have been suggested to be involved in the regulation of stretch-dependent protein synthesis. The role of actin polymerization in these signaling events was examined using the actin-stabilizing agent jasplakinolide. Stretch caused a biphasic activation of FAK at 5-15 min and 24-72 h, which may reflect first a direct phosphorylation of preexisting focal adhesions followed by a rearrangement of focal adhesions to accommodate for the increased mechanical load. Phosphorylation of ERK was increased by acute stretch but then decreased, and Akt did not have a distinct peak in stretch-induced phosphorylation. Inhibition of ERK, phosphatidylinositol 3-kinase, or mammalian target of rapamycin reduced global but not contractile protein synthesis with maintained stretch sensitivity. Stabilization of actin filaments with jasplakinolide, in unstretched portal veins, resulted in increased ERK phosphorylation and global protein synthesis as well as the synthesis of contractile proteins. In contrast, stretch during culture with jasplakinolide did not affect FAK phosphorylation or contractility. Therefore, remodeling of smooth muscle cells to adapt to stretch requires a dynamic cytoskeleton. PMID:17507430

  15. beta. -Adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

    SciTech Connect

    van Koppen, C.J.; Hermanussen, M.W.; Verrijp, K.N.; Rodrigues de Miranda, J.F.; Beld, A.J.; Lammers, J.W.J.; van Ginneken, C.A.M.

    1987-06-29

    Specific binding of (/sup 125/I)-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K/sub d/ = 5.3 +/- 0.9 pmol/l and R/sub T/ = 78 +/- 7 fmol/g tissue). The ..beta../sub 1/-selective antagonists atenolol and LK 203-030 inhibited specific (/sup 125/I)-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous BETA/sub 2/-adrenoceptor population. In one subject using LK 203-030 a small ..beta../sub 1/-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK/sub H/- and pK/sub L/-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD/sub 2/-value of 6.63 +/- 0.19. 32 references, 4 figures, 2 tables.

  16. Purification and partial characterization of glycosaminoglycans and proteoglycans from cultured rabbit smooth muscle cells

    SciTech Connect

    Sabatino, R.D.

    1985-01-01

    Glycosaminoglycans synthesized by cultured rabbit smooth muscle cells were isolated after incorporation of (/sup 3/H)-glucosamine into glycosaminoglycans in the presence or absence of 10% fetal bovine serum. Glycosaminoglycans were quantitated by two-dimensional electrophoresis after proteolytic digestion of the cell layers and media. The results show that the presence of serum has no effect on the chondroitin sulfate, heparan sulfate and dermatan sulfate content of the cell layers. The incorporation of (/sup 3/H)-glucosamine into hyaluronic acid of the cell layers was three times higher in the presence of serum. In the medium , the quantity of hyaluronic was two times higher in the presence of serum while the other glycosaminoglycans remained unchanged. The incorporation of (/sup 3/H)-glucosamine into hyaluronic acid was unaffected by the presence of serum. Specific proteoglycans were isolated from medium after with (/sup 35/S)-sulfate and (/sup 3/H)-serine by isopycnic ultracentrifugation and chromatography on Sepharose CL-4B and DEAE-cellulose. Preparations contained a chondroitin sulfate proteoglycan, a condroitin sulfate-dermatan sulfate proteoglycan and a heparan sulfate proteoglycan. Glycosaminoglycans and proteoglycans synthesized by rabbit aorta smooth muscle cells are similar to those from human aorta.

  17. Dual mechanism of action of nicorandil on rabbit corpus cavernosal smooth muscle tone.

    PubMed

    Hsieh, G C; Kolasa, T; Sullivan, J P; Brioni, J D

    2001-08-01

    The potential of ATP-sensitive potassium channel openers (KCOs) for the treatment of male erectile dysfunction has recently been suggested based on positive clinical outcomes following intra-cavernosal administration of pinacidil. Agents that increase the levels of cGMP via elevation of nitric oxide (NO) nitroglycerin, for example, are also effective in improving erectile function preclinically and clinically. The aim of the present study was to determine the effects and mechanism of the action of nicorandil on rabbit corpus cavernosum. The in vitro regulation of smooth muscle tone was assessed in isolated cavernosal tissues pre-contracted with phenylephrine. Nicorandil, but not its major metabolite, relaxed phenylephrine-precontracted cavernosum smooth muscle with an EC(50) of 15 microM. The effects of nicorandil were only partially reversed by the K(ATP) channel blocker glyburide (10 microM) or by a soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (ODQ, 3 microM). However, a combination of ODQ and glyburide completely blocked the relaxant effects of nicorandil. The results of the present study indicate that nicorandil can relax rabbit cavernosal tissue in vitro via a mechanism that involves activation of K(ATP) channels and stimulation of soluble guanylate cyclase. PMID:11494082

  18. Calcium entry blocking drugs, 'calcium antagonists' and vascular smooth muscle function.

    PubMed

    Bou, J; Llenas, J; Massingham, R

    1983-09-01

    The group of drugs known as "calcium antagonists' is under extensive investigation in experimental animals and man and a re-evaluation of their pharmacological properties is overdue. Recent proposals to adopt the more specific nomenclature of calcium entry blockers for some of these compounds (Vanhoutte & Bohr, 1981) should be supported since there is much confusion in the literature with this class of compound. In this review, which concentrates on vascular smooth muscle, only nifedipine, verapamil, their close chemical analogues and diltiazem are recognised as being relatively selective calcium entry blocking drugs. Whilst definitive evidence for calcium entry blockade must include the demonstration of a selective inhibition of Ca2+-influx into a tissue over a range of concentrations also inhibiting contraction, it is nevertheless possible to define several simple pharmacological criteria which may aid in the identification of such activity. These criteria include the selective antagonism of K+ and Ca2+-induced contractions, relative to those of noradrenaline in suitable vascular smooth muscle preparations and a selective inhibition of alpha 2- as opposed to alpha 1-adrenoreceptor mediated pressor responses in, for example, pithed rat preparations. Recent pharmacological and biochemical studies have identified 3 major subgroups of "calcium antagonist' drugs but the compounds within each subgroup varies with the technique adopted. It is therefore suggested that a combination of both pharmacological and ligand-binding studies be used for purposes of classification. Which mechanism, if any, of inhibiting calcium entry is therapeutically most desirable remains an important question for future research. PMID:6315739

  19. Vasopressin induces release of arachidonic acid from vascular smooth muscle cells

    SciTech Connect

    Grillone, L.R.; Clark, M.A.; Heckman, G.; Schmidt, D.; Stassen, F.L.; Crooke, S.T.

    1986-05-01

    Cultured smooth muscle cells (A-10), derived from rat thoracic aorta, have vascular (V/sub 1/) vasopressin receptors. They have previously shown that these receptors mediate phosphatidylinositol turnover, Ca/sup 2 +/ efflux, and inhibition of isoproterenol-induced increases in cAMP. Here they studied the effect of vasopressin on arachidonic acid metabolism of A-10 cells. Cells were incubated for 18-20 hr with (/sup 3/H)-arachidonic acid (80 Ci/mmol). Vasopressin stimulated release of arachidonic acid in a time- and dose-dependent manner. Significant release of arachidonic acid was observed after 4 min with 10/sup -9/ M vasopressin. Maximum release was reached 4 min after addition of 10/sup -7/ M vasopressin (1100 dpm/10/sup 6/ cells). About 800 dmp were released after 1 and 4 min with 10/sup -7/ M and 10/sup -8/ M vasopressin, respectively. The vasopressin-stimulated release of arachidonic acid was blocked by the specific V/sub 1//V/sub 2/ vasopressin antagonist d(CH2)5D-Tyr(Et)VAVP. These data indicate that vascular smooth muscle cells increase arachidonic acid release in response to vasopressin. This response is likely mediated by V/sub 1/ receptors.

  20. Heparin induces the expression of specific matrix proteins by human intestinal smooth muscle cells

    SciTech Connect

    Cochran, D.L.; Perr, H.; Graham, M.F.; Diegelmann, R.F.

    1986-03-01

    Human intestinal smooth muscle (HISM) cells have recently been identified as the major cell type responsible for stricture formation in Crohn's disease. Heparin, a sulfated glycosaminoglycan, has been shown to be a key modulator of vascular smooth muscle cell (VSMC) growth both in vivo and in vitro and to affect the phenotypic expression of proteins made by VSMC. Heparin has also been shown to effect the growth of HISM cells and in this report the authors demonstrate that heparin also has very specific effects on proteins released by HISM cells in vitro. Examination of the proteins in the culture medium of heparin-treated HISM cells observed at 3 time points following sparse plating and proliferation revealed an increase in /sup 35/S-methionine-labeled 200, 37, and 35 kd proteins. A transient effect on a 48 kd protein was observed in substrate-attached material left on the culture dish after the cells were removed with EGTA. No effects on intracellular labeled proteins could be demonstrated. The protein phenotype of HISM cells exposed to heparin appears very similar to that observed in VSMC. The release of specific proteins following exposure to heparin does not appear to be species specific. This response to heparin may reflect a significant influence of this glycosaminoglycan on the phenotypic expression of these cells.

  1. A new scoring system using multiple immunohistochemical markers for diagnosis of uterine smooth muscle tumors

    PubMed Central

    Rath-Wolfson, Lea; Rosenblat, Yevgenia; Halpern, Marisa; Herbert, M; Hammel, I; Gal, Rivka; Leabu, M; Koren, Rumelia

    2006-01-01

    The diagnosis of uterine smooth muscle neoplasms by light microscopy is difficult. Multiple classification schemes have been proposed based on mitotic rate, nuclear atypia, and the presence or absence of necrosis. None of these classification systems has been entirely successful. This study was undertaken to evaluate the use of selected immunohistochemical and histochemical markers in differentiating these tumors, in addition to accepted morphologic criteria. Ten cases of each of the following: leiomyosarcomas (LMS), atypical leiomyomas (AL), cellular leiomyomas (CL) and usual leiomyomas (UL), were classically evaluated for histological diagnosis and were stained for Ki-67 (MIB-1), bcl-2 and p53 using monoclonal antibodies and the avidin-biotin peroxidase method, and argyrophilic nucleolar organizer region (AgNORs). The number of stained cells was counted in the most positively stained region in a 4 mm2 square cover glass mounted on each slide. The mean value was calculated for each group of tumors. The data for Ki-67 (MIB-1), bcl-2, p53 and AgNOR staining respectively, were significantly higher in LMS by comparison to UL, CL or AL. Because many singular cases had superimposed data being difficult to diagnose, a new scoring system for pathological evaluation was created. The results obtained by this scoring system suggest that immunohistochemical markers Ki-67 (MIB-1), bcl-2, p53 together with the AgNOR staining could be useful, by the scoring system, as an adjunct to the current accepted morphologic criteria in differentiating smooth muscle tumors of the uterus. PMID:16563231

  2. Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells.

    PubMed

    Iyer, Dharini; Gambardella, Laure; Bernard, William G; Serrano, Felipe; Mascetti, Victoria L; Pedersen, Roger A; Talasila, Amarnath; Sinha, Sanjay

    2015-04-15

    The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFβ1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening. PMID:25813541

  3. Oestrogen signalling in white adipose progenitor cells inhibits differentiation into brown adipose and smooth muscle cells.

    PubMed

    Lapid, Kfir; Lim, Ajin; Clegg, Deborah J; Zeve, Daniel; Graff, Jonathan M

    2014-01-01

    Oestrogen, often via oestrogen receptor alpha (ERα) signalling, regulates metabolic physiology, highlighted by post-menopausal temperature dysregulation (hot flashes), glucose intolerance, increased appetite and reduced metabolic rate. Here we show that ERα signalling has a role in adipose lineage specification in mice. ERα regulates adipose progenitor identity and potency, promoting white adipogenic lineage commitment. White adipose progenitors lacking ERα reprogramme and enter into smooth muscle and brown adipogenic fates. Mechanistic studies highlight a TGFβ programme involved in progenitor reprogramming downstream of ERα signalling. The observed reprogramming has profound metabolic outcomes; both female and male adipose-lineage ERα-mutant mice are lean, have improved glucose sensitivity and are resistant to weight gain on a high-fat diet. Further, they are hypermetabolic, hyperphagic and hyperthermic, all consistent with a brown phenotype. Together, these findings indicate that ERα cell autonomously regulates adipose lineage commitment, brown fat and smooth muscle cell formation, and systemic metabolism, in a manner relevant to prevalent metabolic diseases. PMID:25330806

  4. Epithelium-generated neuropeptide Y induces smooth muscle contraction to promote airway hyperresponsiveness.

    PubMed

    Li, Shanru; Koziol-White, Cynthia; Jude, Joseph; Jiang, Meiqi; Zhao, Hengjiang; Cao, Gaoyuan; Yoo, Edwin; Jester, William; Morley, Michael P; Zhou, Su; Wang, Yi; Lu, Min Min; Panettieri, Reynold A; Morrisey, Edward E

    2016-05-01

    Asthma is one of the most common chronic diseases globally and can be divided into presenting with or without an immune response. Current therapies have little effect on nonimmune disease, and the mechanisms that drive this type of asthma are poorly understood. Here, we have shown that loss of the transcription factors forkhead box P1 (Foxp1) and Foxp4, which are critical for lung epithelial development, in the adult airway epithelium evokes a non-Th2 asthma phenotype that is characterized by airway hyperresponsiveness (AHR) without eosinophilic inflammation. Transcriptome analysis revealed that loss of Foxp1 and Foxp4 expression induces ectopic expression of neuropeptide Y (Npy), which has been reported to be present in the airways of asthma patients, but whose importance in disease pathogenesis remains unclear. Treatment of human lung airway explants with recombinant NPY increased airway contractility. Conversely, loss of Npy in Foxp1- and Foxp4-mutant airway epithelium rescued the AHR phenotype. We determined that NPY promotes AHR through the induction of Rho kinase activity and phosphorylation of myosin light chain, which induces airway smooth muscle contraction. Together, these studies highlight the importance of paracrine signals from the airway epithelium to the underlying smooth muscle to induce AHR and suggest that therapies targeting epithelial induction of this phenotype may prove useful in treatment of noneosinophilic asthma. PMID:27088802

  5. Oestrogen signalling in white adipose progenitor cells inhibits differentiation into brown adipose and smooth muscle cells

    PubMed Central

    Clegg, Deborah J.; Zeve, Daniel; Graff, Jonathan M.

    2016-01-01

    Oestrogen, often via oestrogen receptor alpha (ERα) signalling, regulates metabolic physiology, highlighted by post-menopausal temperature dysregulation (hot flashes), glucose intolerance, increased appetite and reduced metabolic rate. Here we show that ERα signalling has a role in adipose lineage specification in mice. ERα regulates adipose progenitor identity and potency, promoting white adipogenic lineage commitment. White adipose progenitors lacking ERα reprogramme and enter into smooth muscle and brown adipogenic fates. Mechanistic studies highlight a TGFβ programme involved in progenitor reprogramming downstream of ERα signalling. The observed reprogramming has profound metabolic outcomes; both female and male adipose-lineage ERα-mutant mice are lean, have improved glucose sensitivity and are resistant to weight gain on a high-fat diet. Further, they are hypermetabolic, hyperphagic and hyperthermic, all consistent with a brown phenotype. Together, these findings indicate that ERα cell autonomously regulates adipose lineage commitment, brown fat and smooth muscle cell formation, and systemic metabolism, in a manner relevant to prevalent metabolic diseases. PMID:25330806

  6. Complex I dysfunction underlies the glycolytic switch in pulmonary hypertensive smooth muscle cells.

    PubMed

    Rafikov, Ruslan; Sun, Xutong; Rafikova, Olga; Meadows, Mary Louise; Desai, Ankit A; Khalpey, Zain; Yuan, Jason X-J; Fineman, Jeffrey R; Black, Stephen M

    2015-12-01

    ATP is essential for cellular function and is usually produced through oxidative phosphorylation. However, mitochondrial dysfunction is now being recognized as an important contributing factor in the development cardiovascular diseases, such as pulmonary hypertension (PH). In PH there is a metabolic change from oxidative phosphorylation to mainly glycolysis for energy production. However, the mechanisms underlying this glycolytic switch are only poorly understood. In particular the role of the respiratory Complexes in the mitochondrial dysfunction associated with PH is unresolved and was the focus of our investigations. We report that smooth muscle cells isolated from the pulmonary vessels of rats with PH (PH-PASMC), induced by a single injection of monocrotaline, have attenuated mitochondrial function and enhanced glycolysis. Further, utilizing a novel live cell assay, we were able to demonstrate that the mitochondrial dysfunction in PH-PASMC correlates with deficiencies in the activities of Complexes I-III. Further, we observed that there was an increase in mitochondrial reactive oxygen species generation and mitochondrial membrane potential in the PASMC isolated from rats with PH. We further found that the defect in Complex I activity was due to a loss of Complex I assembly, although the assembly of Complexes II and III were both maintained. Thus, we conclude that loss of Complex I assembly may be involved in the switch of energy metabolism in smooth muscle cells to glycolysis and that maintaining Complex I activity may be a potential therapeutic target for the treatment of PH. PMID:26298201

  7. Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols

    PubMed Central

    Yang, Libang; Geng, Zhaohui; Nickel, Thomas; Johnson, Caitlin; Gao, Lin; Dutton, James; Hou, Cody; Zhang, Jianyi

    2016-01-01

    Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs. PMID:26771193

  8. May Sonic Hedgehog proteins be markers for malignancy in uterine smooth muscle tumors?

    PubMed

    Garcia, Natalia; Bozzini, Nilo; Baiocchi, Glauco; da Cunha, Isabela Werneck; Maciel, Gustavo Arantes; Soares Junior, José Maria; Soares, Fernando Augusto; Baracat, Edmund Chada; Carvalho, Katia Candido

    2016-04-01

    Several studies have demonstrated that the Sonic Hedgehog signaling pathway (SHH) plays an important role in tumorigenesis and cellular differentiation. We analyzed the protein expression of SHH pathway components and evaluated whether their profile could be useful for the diagnosis, prognosis, or prediction of the risk of malignancy for uterine smooth muscle tumors (USMTs). A total of 176 samples (20 myometrium, 119 variants of leiomyoma, and 37 leiomyosarcoma) were evaluated for the protein expression of the SHH signaling components, HHIP1 (SHH inhibitor), and BMP4 (SHH target) by immunohistochemistry. Western blot analysis was performed to verify the specificity of the antibodies. We grouped leiomyoma samples into conventional leiomyomas and unusual leiomyomas that comprise atypical, cellular, mitotically active leiomyomas and uterine smooth muscle tumors of uncertain malignant potential. Immunohistochemical analysis showed that SMO, SUFU, GLI1, GLI3, and BMP4 expression gradually increased depending on to the histologic tissue type. The protein expression of SMO, SUFU, and GLI1 was increased in unusual leiomyoma and leiomyosarcoma samples compared to normal myometrium. The inhibitor HHIP1 showed higher expression in myometrium, whereas only negative or basal expression of SMO, SUFU, GLI1, and GLI3 was detected in these samples. Strong expression of SHH was associated with poorer overall survival. Our data suggest that the expression of SHH proteins can be useful for evaluating the potential risk of malignancy for USMTs. Moreover, GLI1 and SMO may serve as future therapeutic targets for women with USMTs. PMID:26997437

  9. ( sup 3 H)QNB binding and contraction of rabbit colonic smooth muscle cells

    SciTech Connect

    Ringer, M.J.; Hyman, P.E.; Kao, H.W.; Hsu, C.T.; Tomomasa, T.; Snape, W.J. Jr. )

    1987-11-01

    The authors used radioligand binding and studies of cell contraction to characterize muscarinic receptors on dispersed smooth muscle cells from rabbit proximal and distal colon. Cells obtained after serial incubations in collagenase were used to measure binding of tritiated quinuclidinyl benzilate (({sup 3}H)QNB). At 37{degree}C, specific ({sup 3}H)QNB binding was saturable and linearly related to cell number. Nonlinear regression analysis was used to determine the affinity of ({sup 3}H)QNB for its receptor. The IC{sub 50} for the muscarinic agonists bethanechol and oxotremorine were 80 and 0.57 {mu}M, respectively. Hill coefficients were 0.67 for both, suggesting more complex interaction involving receptors of different affinities. In studies of cell contraction, bethanechol stimulated a dose-dependent decrease in cell length with half the maximal contraction occurring at 100 pM. These results suggest that (1) contraction is mediated by binding of bethanechol to M{sub 2}-muscarinic receptors and that (2) there are a large number of spare receptors in colonic smooth muscle.

  10. Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

    PubMed Central

    An, S.S.; Bai, T.R.; Bates, J.H.T.; Black, J.L.; Brown, R.H.; Brusasco, V.; Chitano, P.; Deng, L.; Dowell, M.; Eidelman, D.H.; Fabry, B.; Fairbank, N.J.; Ford, L.E.; Fredberg, J.J.; Gerthoffer, W.T.; Gilbert, S.H.; Gosens, R.; Gunst, S.J.; Halayko, A.J.; Ingram, R.H.; Irvin, C.G.; James, A.L.; Janssen, L.J.; King, G.G.; Knight, D.A.; Lauzon, A.M.; Lakser, O.J.; Ludwig, M.S.; Lutchen, K.R.; Maksym, G.N.; Martin, J.G.; Mauad, T.; McParland, B.E.; Mijailovich, S.M.; Mitchell, H.W.; Mitchell, R.W.; Mitzner, W.; Murphy, T.M.; Paré, P.D.; Pellegrino, R.; Sanderson, M.J.; Schellenberg, R.R.; Seow, C.Y.; Silveira, P.S.P.; Smith, P.G.; Solway, J.; Stephens, N.L.; Sterk, P.J.; Stewart, A.G.; Tang, D.D.; Tepper, R.S.; Tran, T.; Wang, L.

    2008-01-01

    Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored. PMID:17470619

  11. A Synthetic Chloride Channel Relaxes Airway Smooth Muscle of the Rat

    PubMed Central

    Yau, Kwok-hei; Mak, Judith Choi-wo; Leung, Susan Wai-sum; Yang, Dan; Vanhoutte, Paul M.

    2012-01-01

    Synthetic ion channels may have potential therapeutic applications, provided they possess appropriate biological activities. The present study was designed to examine the ability of small molecule-based synthetic Cl– channels to modulate airway smooth muscle responsiveness. Changes in isometric tension were measured in rat tracheal rings. Relaxations to the synthetic chloride channel SCC-1 were obtained during sustained contractions to KCl. The anion dependency of the effect of SCC-1 was evaluated by ion substitution experiments. The sensitivity to conventional Cl– transport inhibitors was also tested. SCC-1 caused concentration-dependent relaxations during sustained contractions to potassium chloride. This relaxing effect was dependent on the presence of extracellular Cl– and HCO3−. It was insensitive to conventional Cl– channels/transport inhibitors that blocked the cystic fibrosis transmembrane conductance regulator and calcium-activated Cl– channels. SCC-1 did not inhibit contractions induced by carbachol, endothelin-1, 5-hydroxytryptamine or the calcium ionophore A23187. SCC-1 relaxes airway smooth muscle during contractions evoked by depolarizing solutions. The Cl– conductance conferred by this synthetic compound is distinct from the endogenous transport systems for chloride anions. PMID:23049786

  12. Smooth Muscle Cell Stiffness Syndrome”—Revisiting the Structural Basis of Arterial Stiffness

    PubMed Central

    Sehgel, Nancy L.; Vatner, Stephen F.; Meininger, Gerald A.

    2015-01-01

    In recent decades, the pervasiveness of increased arterial stiffness in patients with cardiovascular disease has become increasingly apparent. Though, this phenomenon has been well documented in humans and animal models of disease for well over a century, there has been surprisingly limited development in a deeper mechanistic understanding of arterial stiffness. Much of the historical literature has focused on changes in extracellular matrix proteins—collagen and elastin. However, extracellular matrix changes alone appear insufficient to consistently account for observed changes in vascular stiffness, which we observed in our studies of aortic stiffness in aging monkeys. This led us to examine novel mechanisms operating at the level of the vascular smooth muscle cell (VSMC)—that include increased cell stiffness and adhesion to extracellular matrix—which that may be interrelated with other mechanisms contributing to arterial stiffness. We introduce these observations as a new concept—the Smooth Muscle Cell Stiffness Syndrome (SMCSS)—within the field of arterial stiffness and posit that stiffening of vascular cells impairs vascular function and may contribute stiffening to the vasculature with aging and cardiovascular disease. Importantly, this review article revisits the structural basis of arterial stiffness in light of these novel findings. Such classification of SMCSS and its contextualization into our current understanding of vascular mechanics may be useful in the development of strategic therapeutics to directly target arterial stiffness. PMID:26635621

  13. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration

    SciTech Connect

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping Wang, Hong

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation and migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that Ipt

  14. Calphostin-C induction of vascular smooth muscle cell apoptosis proceeds through phospholipase D and microtubule inhibition.

    PubMed

    Zheng, Xi-Long; Gui, Yu; Du, Guangwei; Frohman, Michael A; Peng, Dao-Quan

    2004-02-20

    Calphostin-C, a protein kinase C inhibitor, induces apoptosis of cultured vascular smooth muscle cells. However, the mechanisms are not completely defined. Because apoptosis of vascular smooth muscle cells is critical in several proliferating vascular diseases such as atherosclerosis and restenosis after angioplasty, we decided to investigate the mechanisms underlying the calphostin-C-induced apoptotic pathway. We show here that apoptosis is inhibited by the addition of exogenous phosphatidic acid, a metabolite of phospholipase D (PLD), and that calphostin-C inhibits completely the activities of both isoforms of PLD, PLD1 and PLD2. Overexpression of either PLD1 or PLD2 prevented the vascular smooth muscle cell apoptosis induced by serum withdrawal but not the calphostin-C-elicited apoptosis. These data suggest that PLDs have anti-apoptotic effects and that complete inhibition of PLD activity by calphostin-C induces smooth muscle cell apoptosis. We also report that calphostin-C induced microtubule disruption and that the addition of exogenous phosphatidic acid inhibits calphostin-C effects on microtubules, suggesting a role for PLD in stabilizing the microtubule network. Overexpressing PLD2 in Chinese hamster ovary cells phenocopies this result, providing strong support for the hypothesis. Finally, taxol, a microtubule stabilizer, not only inhibited the calphostin-C-induced microtubule disruption but also inhibited apoptosis. We therefore conclude that calphostin-C induces apoptosis of cultured vascular smooth muscle cells through inhibiting PLD activity and subsequent microtubule polymerization. PMID:14660552

  15. Graded effects of unregulated smooth muscle myosin on intestinal architecture, intestinal motility and vascular function in zebrafish.

    PubMed

    Abrams, Joshua; Einhorn, Zev; Seiler, Christoph; Zong, Alan B; Sweeney, H Lee; Pack, Michael

    2016-05-01

    Smooth muscle contraction is controlled by the regulated activity of the myosin heavy chain ATPase (Myh11). Myh11 mutations have diverse effects in the cardiovascular, digestive and genitourinary systems in humans and animal models. We previously reported a recessive missense mutation, meltdown (mlt), which converts a highly conserved tryptophan to arginine (W512R) in the rigid relay loop of zebrafish Myh11. The mlt mutation disrupts myosin regulation and non-autonomously induces invasive expansion of the intestinal epithelium. Here, we report two newly identified missense mutations in the switch-1 (S237Y) and coil-coiled (L1287M) domains of Myh11 that fail to complement mlt Cell invasion was not detected in either homozygous mutant but could be induced by oxidative stress and activation of oncogenic signaling pathways. The smooth muscle defect imparted by the mlt and S237Y mutations also delayed intestinal transit, and altered vascular function, as measured by blood flow in the dorsal aorta. The cell-invasion phenotype induced by the three myh11 mutants correlated with the degree of myosin deregulation. These findings suggest that the vertebrate intestinal epithelium is tuned to the physical state of the surrounding stroma, which, in turn, governs its response to physiologic and pathologic stimuli. Genetic variants that alter the regulation of smooth muscle myosin might be risk factors for diseases affecting the intestine, vasculature, and other tissues that contain smooth muscle or contractile cells that express smooth muscle proteins, particularly in the setting of redox stress. PMID:26893369

  16. Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells

    SciTech Connect

    Mong, S.; Miller, J.; Wu, H.L.; Crooke, S.T.

    1988-02-01

    A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. (/sup 3/H)LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the (/sup 3/H)myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of (/sup 3/H)inositol-trisphosphate was rapid and the induction of biosynthesis of (/sup 3/H)inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

  17. Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds

    PubMed Central

    Morris, G. E.; Bridge, J. C.; Eltboli, O. M. I.; Lewis, M. P.; Knox, A. J.; Aylott, J. W.; Brightling, C. E.; Ghaemmaghami, A. M.

    2014-01-01

    Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment. PMID:24793171

  18. The effects of cannabidiol on the antigen-induced contraction of airways smooth muscle in the guinea-pig.

    PubMed

    Dudášová, A; Keir, S D; Parsons, M E; Molleman, A; Page, C P

    2013-06-01

    (-)-Δ(9)-Tetrahydrocannabinol has been demonstrated to have beneficial effects in the airways, but its psychoactive effects preclude its therapeutic use for the treatment of airways diseases. In the present study we have investigated the effects of (-)-cannabidiol, a non-psychoactive component of cannabis for its actions on bronchial smooth muscle in vitro and in vivo. Guinea-pig bronchial smooth muscle contractions induced by exogenously applied spasmogens were measured isometrically. In addition, contractile responses of bronchial smooth muscle from ovalbumin-sensitized guinea-pigs were investigated in the absence or presence of (-)-cannabidiol. Furthermore, the effect of (-)-cannabidiol against ovalbumin-induced airway obstruction was investigated in vivo in ovalbumin-sensitized guinea-pigs. (-)-Cannabidiol did not influence the bronchial smooth muscle contraction induced by carbachol, histamine or neurokinin A. In contrast, (-)-cannabidiol inhibited anandamide- and virodhamine-induced responses of isolated bronchi. A fatty acid amide hydrolase inhibitor, phenylmethanesulfonyl fluoride reversed the inhibitory effect of (-)-cannabidiol on anandamide-induced contractions. In addition, (-)-cannabidiol inhibited the contractile response of bronchi obtained from allergic guinea-pigs induced by ovalbumin. In vivo, (-)-cannabidiol reduced ovalbumin-induced airway obstruction. In conclusion, our results suggest that cannabidiol can influence antigen-induced airway smooth muscle tone suggesting that this molecule may have beneficial effects in the treatment of obstructive airway disorders. PMID:23428645

  19. Calponin isoforms CNN1, CNN2 and CNN3: Regulators for actin cytoskeleton functions in smooth muscle and non-muscle cells.

    PubMed

    Liu, Rong; Jin, J-P

    2016-07-01

    Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and many types of non-muscle cells. Three homologous genes, CNN1, CNN2 and CNN3, encoding calponin isoforms 1, 2, and 3, respectively, are present in vertebrate species. All three calponin isoforms are actin-binding proteins with functions in inhibiting actin-activated myosin ATPase and stabilizing the actin cytoskeleton, while each isoform executes different physiological roles based on their cell type-specific expressions. Calponin 1 is specifically expressed in smooth muscle cells and plays a role in fine-tuning smooth muscle contractility. Calponin 2 is expressed in both smooth muscle and non-muscle cells and regulates multiple actin cytoskeleton-based functions. Calponin 3 participates in actin cytoskeleton-based activities in embryonic development and myogenesis. Phosphorylation has been extensively studied for the regulation of calponin functions. Cytoskeleton tension regulates the transcription of CNN2 gene and the degradation of calponin 2 protein. This review summarizes our knowledge learned from studies over the past three decades, focusing on the evolutionary lineage of calponin isoform genes, their tissue- and cell type-specific expressions, structure-function relationships, and mechanoregulation. PMID:26970176

  20. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases. PMID:23084979

  1. Cultured rat vascular smooth muscle cells are resistant to methylamine toxicity: no correlation to semicarbazide-sensitive amine oxidase

    NASA Technical Reports Server (NTRS)

    Langford, S. D.; Trent, M. B.; Boor, P. J.

    2001-01-01

    Methylamine (MA), a component of serum and a metabolite of nicotine and certain insecticides and herbicides, is metabolized by semicarbazide-sensitive amine oxidase (SSAO). MA is toxic to cultured human umbilical vein and calf pulmonary artery endothelial cells. Endothelial cells, which do not exhibit endogenous SSAO activity, are exposed to SSAO circulating in serum. In contrast, vascular smooth muscle cells (VSMC) do exhibit innate SSAO activity both in vivo and in vitro. This property, together with the critical localization of VSMC within the arterial wall, led us to investigate the potential toxicity of MA to VSMC. Cultured rat VSMC were treated with MA (10-5 to 1 M). In some cultures, SSAO was selectively inhibited with semicarbazide or MDL-72145 [(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine]. Cytotoxicity was measured via MTT, vital dye exclusion, and clonogenic assays. MA proved to be toxic to VSMC only at relatively high concentrations (LC(50) of 0.1 M). The inhibition of SSAO with semicarbazide or MDL-72145 did not increase MA toxicity, suggesting that the production of formaldehyde via tissue-bound, SSAO-mediated MA metabolism does not play a role in the minimal toxicity observed in isolated rat VSMC. The omission of fetal calf serum (FCS), which contains high SSAO activity, from media similarly showed little effect on cytotoxicity. We conclude that VSMC--in contrast to previous results in endothelial cells--are relatively resistant to MA toxicity, and SSAO does not play a role in VSMC injury by MA.

  2. Oxygen-Sensitive Calcium Channels in Vascular Smooth Muscle and Their Possible Role in Hypoxic Arterial Relaxation

    NASA Astrophysics Data System (ADS)

    Franco-Obregon, A.; Urena, J.; Lopez-Barneo, J.

    1995-05-01

    We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering O_2 tension (PO_2) directly influences Ca2+ homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca2+ whose frequency depends on extracellular Ca2+ influx. Switching from a PO_2 of 150 to 20 mmHg leads to a reversible attenuation of the Ca2+ oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+ through voltage-dependent channels, which can account for the inhibition of the Ca2+ oscillations. Low PO_2 selectively inhibits L-type Ca2+ channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO_2 on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of O_2-sensitive, voltage-dependent, Ca2+ channels in vascular smooth muscle that may critically contribute to the local regulation of circulation.

  3. Androgens are powerful non-genomic inducers of calcium sensitization in visceral smooth muscle.

    PubMed

    González-Montelongo, Maria C; Marín, Raquel; Gómez, Tomás; Díaz, Mario

    2010-01-01

    Androgens are recognized as genotropic inducers of a number of physiological functions mainly associated with the development of sexual characteristics. However, as in the case of estrogens, the number of studies evidencing androgen actions in non-reproductive tissues has steadily grown over the past years. Here, we show that androgens acutely ( approximately 30min) alter the frequency spectrum of peristaltic activity of intestinal smooth muscle and augment the amplitude agonist-induced contractile activity. Maximal stimulation occurred at physiological concentrations of androgens with EC(50) values in the picomolar range. Androgen-induced potentiation was prevented by preincubation with androgen receptor (AR) antagonists but unaffected by cycloheximide plus actinomycin D, indicating that potentiation was mediated by ARs via a non-genomic mechanism. The effects of androgens were mimicked by polyamines and were completely blocked by inhibitors of polyamine synthesis. Using ionomycin-permeabilized intestinal smooth muscle preparations, we demonstrate that androgens exert their effects by inducing a mechanism of sensitization to calcium and not by altering intracellular calcium homeostasis. Correspondingly, the potentiation of mechanical activity induced by androgens was accompanied by an increase in the phosphorylation of the regulatory myosin light chain (LC(20)) within the same time-course than calcium sensitization and mechanical potentiation. The pursuit of potential signalling pathways linking androgen receptor activation with calcium sensitization revealed that mechanical potentiation of intestinal muscle by androgens involve activation of the Rho pathway, whose downstream effector, Rho-associated kinase (ROCK), is eventually responsible for displacement of the phosphorylation/dephosphorylation state of LC(20) towards its phosphorylated form. PMID:19800357

  4. Effects of hypoxia and glucose-removal condition on muscle contraction of the smooth muscles of porcine urinary bladder

    PubMed Central

    NAGAI, Yuta; KANEDA, Takeharu; MIYAMOTO, Yasuyuki; NURUKI, Takaomi; KANDA, Hidenori; URAKAWA, Norimoto; SHIMIZU, Kazumasa

    2015-01-01

    To elucidate the dependence of aerobic energy metabolism and utilization of glucose in contraction of urinary bladder smooth muscle, we investigated the changes in the reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity, and determined the phosphocreatine (PCr) and ATP contents of the porcine urinary bladder during contractions induced by high K+ or carbachol (CCh) and with and without hypoxia (achieved by bubbling N2 instead of O2) or in a glucose-free condition. Hyperosmotic addition of 65 mM KCl (H-65K+) and 1 µM CCh induced a phasic contraction followed by a tonic contraction. A glucose-free physiological salt solution (PSS) did not change the subsequent contractile responses to H-65K+ and CCh. However, hypoxia significantly attenuated H-65K+- and CCh-induced contraction. H-65K+ and CCh induced a sustained increase in PNred fluorescence, representing glycolysis activity. Hypoxia enhanced H-65K+- and CCh-induced increases in PNred fluorescence, whereas glucose-free PSS decreased these increases, significantly. In the presence of H-65K+, hypoxia decreased the PCr and ATP contents; however, the glucose-free PSS did not change the PCr contents. In conclusion, we demonstrated that high K+- and CCh-induced contractions depend on aerobic metabolism and that an endogenous substrate may be utilized to maintain muscle contraction in a glucose-free PSS in the porcine urinary bladder. PMID:26369431

  5. Mechanisms of the nitroglycerine-induced vasodilation in vascular smooth muscles of the rabbit and pig.

    PubMed Central

    Itoh, T; Kuriyama, H; Ueno, H

    1983-01-01

    The effects of nitroglycerine (NG) on the mechanical responses of small pieces of intact and skinned smooth muscles of the mesenteric artery, were investigated, as were the Ca fluxes of isolated smooth muscle cells of the coronary artery. NG (10(-6)-10(-5) M) inhibited both phasic and tonic components of the K-induced contraction; however, the tonic component was more sensitive to NG. The minimum concentration of NG required to decrease significantly the tonic response evoked by 39 mM-external K was 10(-8) M. NG (10(-5) M) reduced the number of oscillatory contractions evoked by 10(-5) M-noradrenaline (NAd). After complete removal of stored Ca, the addition of Ca did not produce contraction in polarized muscle (5.9 mM-external K), yet depolarized muscles (128 mM-external K) did contract. Addition of NG (10(-5) M) with Ca produced no change in the resting tone in polarized muscles but inhibited the contraction in depolarized muscles. After application of NG (10(-5) M), caffeine or NAd consistently produced smaller contraction in both polarized and depolarized muscles in Ca-free solution. NG (10(-5) M) inhibited the Na-free contraction evoked by prolonged treatment with Na-free solution. Contractions evoked by repetitive applications of 10(-5) M-NAd or 10 mM-caffeine persisted longer in Na-free, Ca-free (EGTA) solution than those observed in Ca-free Krebs solution, but when NG was added to the Na-free, Ca-free (EGTA) solution, the contractions ceased more rapidly than in the absence of NG. In chemically skinned muscles, 10(-5) M-NG had no effect on the pCa-tension relationship. The absolute amplitude of Ca-induced contraction was also not affected by 10(-5) M-NG. In these muscles, when the amount of stored Ca was estimated from the amplitude of the caffeine-induced contraction, Ca accumulation into and release from store sites were unaffected by 10(-5) M-NG. The effects of 10(-5) M-NG on the accumulation and efflux of 45Ca in isolated cell suspensions prepared from

  6. Epstein–Barr virus-associated iris smooth muscle tumor with epithelioid morphology in AIDS patients: a case report

    PubMed Central

    Suwan, Yanin; Rojanaporn, Duangnate; Teekhasaenee, Chaiwat; Keelawat, Somboon

    2016-01-01

    Importance Report of an acquired immunodeficiency syndrome (AIDS) patient with Epstein–Barr virus (EBV)-associated iris smooth muscle tumor. Observations A 14-year-old African American female diagnosed with AIDS developed a painless iris mass in the right eye for 10 months. Iridocyclectomy was performed, and the pathology indicated EBV-associated iris smooth muscle tumor with epithelioid morphology. Immunohistochemical stains and in situ hybridization for EBV-encoded ribonucleic acid are very useful diagnostic tools for definite diagnosis. At 14-month follow-up, the patient did not have any tumor recurrence. Conclusion This is the case report of EBV-associated iris smooth muscle tumor in a person diagnosed with AIDS with a unique epithelioid morphologic feature. PMID:27099533

  7. Individual sympathetic postganglionic neurons coinnervate myenteric ganglia and smooth muscle layers in the gastrointestinal tract of the rat.

    PubMed

    Walter, Gary C; Phillips, Robert J; McAdams, Jennifer L; Powley, Terry L

    2016-09-01

    A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days postinjection, animals were euthanized and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n = 154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/tertiary plexus. Very few CSMG neurons projected exclusively (i.e., ∼100% of an arbor's varicose branches) to myenteric plexus (∼2%) or smooth muscle (∼14%). With less stringent inclusion criteria (i.e., ≥85% of an axon's varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (∼13%) or smooth muscle (∼27%). The majority (i.e., ∼60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that coinnervated extensively (>15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ∼87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively. J. Comp. Neurol. 524:2577-2603, 2016. © 2016 Wiley Periodicals, Inc. PMID:26850701

  8. Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells.

    PubMed Central

    Papapetropoulos, A.; Go, C. Y.; Murad, F.; Catravas, J. D.

    1996-01-01

    1. While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sGC) activation, the mechanisms responsible for this phenomenon in intact cells remain unclear. In the present study, possible mechanisms of tolerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 microM SNP over a 15 min period in the presence of a phosphodiesterase (PDE) inhibitor. 2. Pretreatment of rat aortic smooth muscle cells with 10-500 microM SNP led to a dose-dependent downregulation of cyclic GMP accumulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 microM SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 microM oxyhaemoglobin. 3. Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast, resulted in downregulation of the SNP-induced cyclic GMP accumulation in a time- and concentration-dependent manner, that was first evident after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H89. 4. beta 1 sGC steady state mRNA levels of S-nitroso N-acetylpenicillamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as indicated by Northern blot analysis. However, Western blot analysis revealed that alpha 1 protein levels were decreased in zaprinast, but not in SNP, SNAP or 8Br-cyclic GMP pretreated cells. 5. While thiol depletion did not prevent the development of tolerance, pretreatment of cells with SNP in the presence of reducing agents partially or completely restored the ability of cells to respond to SNP. 6. We conclude that tolerance to SNP results from two distinct mechanisms: an early onset, NO-mediated event that is reversed by reducing agents and a more delayed, PKA-sensitive process

  9. Glucose and glucosamine regulate growth factor gene expression in vascular smooth muscle cells.

    PubMed Central

    McClain, D A; Paterson, A J; Roos, M D; Wei, X; Kudlow, J E

    1992-01-01

    We have investigated the regulation of the expression of two growth factors found in vascular smooth muscle, transforming growth factor alpha (TGF alpha) and basic fibroblast growth factor (bFGF). Cells cultured in medium containing 30 mM glucose exhibited a 2-fold increase in TGF alpha mRNA and a 3-fold increase in bFGF mRNA compared with cells grown in normal (5.5 mM) glucose. Glucosamine was more potent than glucose, leading to a 6-fold increase in TGF alpha mRNA. TGF alpha protein levels were also increased by glucosamine treatment, and the predominant species present was the membrane-bound precursor form of TGF alpha. To examine further the regulation of growth factors by sugars, cultured rat aortic smooth muscle cells were transfected with a plasmid construct consisting of a 1.2-kilobase-pair fragment of the TGF alpha promoter linked to a luciferase reporter gene. Increasing the concentration of glucose in the culture medium from 5.5 mM to 30 mM led to a rapid, 1.7-fold increase in the activity of the TGF alpha promoter. Glucosamine was much more potent than glucose in this stimulation, with 2 mM glucosamine causing a 12-fold increase in TGF alpha promoter activity. Insulin had no effect on luciferase activity in either the presence or the absence of added sugars. The glucose response element of the TGF alpha gene maps to a 130-base-pair segment that includes three potential binding sites for the transcription factor Sp1. We conclude that high glucose concentrations such as are reached in diabetes mellitus can stimulate the transcription of the genes for growth factors in vascular smooth muscle cells. This signaling pathway apparently involves the metabolism of glucose to glucosamine. This effect could be representative of nutritional regulation of a family of genes and could contribute to the toxicity of hyperglycemia and the vascular complications of diabetes. Images PMID:1518840

  10. Expression of fibronectin variants in vascular and visceral smooth muscle cells in development.

    PubMed

    Glukhova, M A; Frid, M G; Shekhonin, B V; Balabanov, Y V; Koteliansky, V E

    1990-09-01

    Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo. PMID:2202605

  11. Exploring smooth muscle phenotype and function in a bioreactor model of abdominal aortic aneurysm

    PubMed Central

    2013-01-01

    Background Vascular smooth muscle cells (SMC) are central to arterial structure and function yet their involvement in the progression of abdominal aortic aneurysm (AAA) disease is not well studied. The progressive and silent nature of AAA in man essentially restricts research to the use of “end-stage” tissue recovered during surgical repair. This study aimed to generate an ex vivo model of AAA using protease-treated porcine carotid arteries maintained in a novel bioreactor, and to compare the structural and functional changes in SMC cultured from the recovered vessels with those from human tissue acquired at elective surgical repair. Methods Freshly isolated porcine arteries were pretreated with collagenase and/or elastase before culturing under flow in a bioreactor for 12 days. Human end-stage aneurysmal tissue and saphenous veins from age-matched controls were collected from patients undergoing surgery. SMC were cultured and characterised (immunocytochemistry, measurement of spread cell area) and assessed functionally at the level of proliferation (cell-counting) and matrix-metalloproteinase (MMP) secretion (gelatin zymography). Cellular senescence was investigated using β-galactosidase staining and apoptosis was quantified using a fluorescence-based caspase 3 assay. Results Co-expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain confirmed all cell populations as SMC. Porcine SMC harvested and cultivated after collagenase/elastase pretreatment displayed a prominent “rhomboid” morphology, increased spread area (32%, P < 0.01), impaired proliferation (47% reduction, P < 0.05), increased senescence (52%, P < 0.001), susceptibility to apoptosis and reduced MMP-2 secretion (60% decrease, P < 0.01) compared with SMC from vehicle, collagenase or elastase pre-treated vessels. Notably, these changes were comparable to those observed in human AAA SMC which were 2.4-fold larger than non-aneurysmal SMC (P < 0.001) and

  12. Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase.

    PubMed

    Pato, M D; Sutherland, C; Winder, S J; Walsh, M P

    1993-07-01

    Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase

  13. Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods.

    PubMed

    Mazza, G; Roßmanith, E; Lang-Olip, I; Pfeiffer, D

    2016-08-01

    Even though umbilical cord arteries are a common source of vascular smooth muscle cells, the lack of reliable marker profiles have not facilitated the isolation of human umbilical artery smooth muscle cells (HUASMC). For accurate characterization of HUASMC and cells in their environment, the expression of smooth muscle and mesenchymal markers was analyzed in umbilical cord tissue sections. The resulting marker profile was then used to evaluate the quality of HUASMC isolation and culture methods. HUASMC and perivascular-Wharton's jelly stromal cells (pv-WJSC) showed positive staining for α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as α-SMA+ , SM-MHC+ , CD10- cells, which are additionally negative for endothelial markers (CD31 and CD34). Enzymatic isolation provided primary HUASMC batches with 90-99 % purity, yet, under standard culture conditions, contaminant CD10+ cells rapidly constituted more than 80 % of the total cell population. Contamination was mainly due to the poor adhesion of HUASMC to cell culture plates, regardless of the different protein coatings (fibronectin, collagen I or gelatin). HUASMC showed strong attachment and long-term viability only in 3D matrices. The explant isolation method achieved cultures with only 13-40 % purity with considerable contamination by CD10+ cells. CD10+ cells showed spindle-like morphology and up-regulated expression of α-SMA and SM-MHC upon culture in smooth muscle differentiation medium. Considering the high contamination risk of HUASMC cultures by CD10+ neighboring cells and their phenotypic similarities, precise characterization is mandatory to avoid misleading results. PMID:25535117

  14. Bronchodilatory and B-adrenergic effects of methanolic and aqueous extracts of Althaea root on isolated tracheobronchial smooth rat muscle

    PubMed Central

    Alani, Behrang; Zare, Mohammad; Noureddini, Mahdi

    2015-01-01

    Background: The smooth muscle contractions of the tracheobronchial airways are mediated through the balance of adrenergic, cholinergic and peptidergic nervous mechanisms. This research was designed to determine the bronchodilatory and B-adrenergic effects of methanolic and aqueous extracts of root Althaea on the isolated tracheobronchial smooth muscle of the rat. Materials and Methods: In this experimental study, 116 tracheobronchial sections (5 mm) from 58 healthy male Sprague-Dawley rats were dissected and divided into 23 groups. The effect of methanolic and aqueous extracts of the root Althaea was assayed at different concentrations (0.2, 0.6, 2.6, 6.6, 14.6 μg/ml) and epinephrine (5 μm) in the presence and absence of propranolol (1 μM) under one g tension based on the isometric method. This assay was recorded in an organ bath containing Krebs-Henseleit solution for tracheobronchial smooth muscle contractions using potassium chloride (KCl) (60 mM) induction. Results: Epinephrine (5 μm) alone and root methanolic and aqueous extract concentrations (0.6-14.6 μg/ml) reduced tracheobronchial smooth muscle contractions induced using KCl (60 mM) in a dose dependent manner. Propranolol inhibited the antispasmodic effect of epinephrine on tracheobronchial smooth muscle contractions, but could not reduce the antispasmodic effect of the root extract concentrations. Conclusion: The methanolic and aqueous extracts of Althaea root inhibited the tracheobronchial smooth muscle contractions of rats in a dose dependent manner, but B-adrenergic receptors do not appear to engage in this process. Understanding the mechanism of this process can be useful in the treatment of pulmonary obstructive diseases like asthma. PMID:25879003

  15. Prostanoid receptors EP2, EP4, and FP are regulated by estradiol in bovine oviductal smooth muscle.

    PubMed

    Huang, Na; Liu, Bo; Dong, Zhiheng; Mao, Wei; Zhang, Nan; Li, Changyou; Cao, Jinshan

    2015-09-01

    Gamete and embryo transport is an important function of the oviduct. This transport involves both smooth muscle contraction and epithelial cell secretions, the former of which is mediated by prostaglandins (PGs) and their receptors. Our aim was to study the regulation of prostaglandin E2 and prostaglandin F2α receptors (EP2, EP4, and FP receptor) by estradiol in bovine oviduct smooth muscle. EP2, EP4, and FP receptor mRNA and protein expression was investigated using real-time RT-PCR and Western blot analyses, respectively. To evaluate the contraction or relaxation of cultured bovine oviductal smooth muscle tissue, peristalsis was used to assess contractile activity. EP2, EP4, and FP receptor mRNA and protein expression was increased in oviductal smooth muscle tissue after treatment with different concentrations of estradiol for various durations. The expression of all receptors peaked at an estradiol concentration of 10(-11)mol/L after 8h of treatment, whereas no increase in expression was observed after fulvestrant (a selective antagonist of E2 receptor) treatment, indicating that E2 interacts with specific E2 nuclear receptors to regulate EP2, EP4, and FP receptor expression. Although PGF2α and PGE2 induced both contraction and relaxation, no significant differences were found in contractility between the estradiol-treated and control groups, with both groups of cultured smooth muscle strips showing similar vitality. In conclusion, estradiol increases EP2, EP4, and FP receptor mRNA and protein expression in bovine oviductal smooth muscle when added for different periods of time and at different concentrations. Additionally, E2 is transported intracellularly and interacts with specific E2 nuclear receptors to regulate their expression. PMID:26319698

  16. Time-dependent changes in Ca2+ sensitivity during phasic contraction of canine antral smooth muscle.

    PubMed Central

    Ozaki, H; Gerthoffer, W T; Publicover, N G; Fusetani, N; Sanders, K M

    1991-01-01

    1. Relationships between cytosolic Ca2+ concentration ([Ca2+]cyt), myosin light chain (MLC) phosphorylation and muscle tension were examined in circular smooth muscle of canine gastric antrum. 2. Electrical slow waves induced a transient increase in [Ca2+]cyt and muscle tension. [Ca2+]cyt increased before the initiation of contraction and reached a maximum before the peak of the phasic contractions. Following the first Ca2+ transient, a second rise in [Ca2+]cyt was often observed. The second Ca2+ transient was of similar magnitude to the first, but only in some cases was this increase in [Ca2+]cyt associated with a second phase of contraction. Relaxation occurred more rapidly than the restoration of resting levels of [Ca2+]cyt. 3. Acetylcholine (ACh; 3 x 10(-7) M) increased the amplitude of Ca2+ transients, caused MLC phosphorylation and increased the force of contraction. The decay of contraction and MLC dephosphorylation preceded that of [Ca2+]cyt. 4. Increasing external K+ (to 25-40 mM) caused a sustained increase in [Ca2+]cyt, but little change in resting tension. This suggests that the Ca2+ sensitivity decreased as [Ca2+]cyt increased. Increasing K+ to 59.5 mM further increased the level of [Ca2+]cyt, induced MLC phosphorylation and caused a transient contraction. When normal levels of K+ were restored, the rates of MLC dephosphorylation and relaxation exceeded the rate of decay in [Ca2+]cyt. 5. Removal of external Ca2+ in depolarized muscles decreased [Ca2+]cyt below the resting level without affecting resting tension. Readmission of Ca2+ to depolarized muscles caused force to develop at [Ca2+]cyt levels below the original resting level, suggesting that Ca2+ sensitivity was increased when the resting level of [Ca2+]cyt was decreased. 6. The phosphatase inhibitor, calyculin-A (10(-6) M), induced tonic contraction and MLC phosphorylation without an increase in [Ca2+]cyt. During these contractures, electrical activity caused transient increases in [Ca2+]cyt and

  17. The effects of aluminum, iron, chromium, and yttrium on rat intestinal smooth muscle in vitro.

    PubMed

    Cunat, L; Membre, H; Marchal, L; Chaussidon, M; Burnel, D

    1998-01-01

    The modification of peristaltic activity in the presence of several metal ions has been investigated in the rat intestine by the isolated organ technique. The metals tested modify the intestinal movements: aluminum, chromium, and yttrium cause a decrease of amplitude, while iron showed no effect. By use of microscopic techniques, the presence of yttrium hydroxide was observed in the intestinal tissues. Iron also appears as a precipitate outside of the intestinal serosal, which may explain why iron did not modify the peristaltism. Chromium and aluminum were not apparent to microscope, despite being detected and quantified in the tissues by means of atomic emission spectrometer. We conclude that the trivalent ions of these elements may operate differently on the mechanisms of intestinal contractions: yttrium precipitates in intercellular spaces, iron precipitates outside the intestines, and chromium and aluminum remain in solution and are distributed homogeneously in the smooth intestinal muscle. PMID:9845462

  18. Local electromechanical properties of different phenotype models of vascular smooth muscle cells using force microscopy

    NASA Astrophysics Data System (ADS)

    Thompson, Gary; Reukov, Vladimir; Nikiforov, Maxim; Guo, Senli; Ovchinnikov, Oleg; Jesse, Stephen; Kalinin, Sergei; Vertegel, Alexey

    2010-03-01

    Vascular smooth muscle cells (VSMCs) exist as a spectrum of diverse phenotypes raning between contractile and synthetic, the latter being associated with disease states. Different VSMC phenotypes, modeled using serum-starvation, exhibit characteristic electromechanical responses that can be distinguished using band excitation piezoresponse force microscopy (BEPFM), which maps information at the same rate as the atomic force microscope (AFM) scan performed simultaneously. BEPFM image formation mechanism in the culture medium is determined using excitation steps from 1 mV to 100 V. High voltage improves contrast between cells and collagen-coated substrates. Viscoelasticity from AFM stress relaxation experiments and local elasticity from force maps correlate to BEPFM data providing a map of local mechanical properties on different VSMCs.

  19. MicroRNA-141 inhibits vascular smooth muscle cell proliferation through targeting PAPP-A

    PubMed Central

    Zhang, Yudong; Chen, Bainan; Ming, Liu; Qin, Hongsong; Zheng, Liu; Yue, Zhang; Cheng, Zhixin; Wang, Yannan; Zhang, Dawei; Liu, Chunmei; Bin, Wang; Hao, Qingzhi; Song, Fuchen; Ji, Bo

    2015-01-01

    It is well known that ox-LDL plays key roles in the development of atherosclerosis, partly by inducing vascular smooth muscle cells (VSMCs) proliferation. Recent findings have revealed that microRNAs, a class of small noncoding RNAs, could regulate cell proliferation in many physiological and pathological conditions. However, the role and function of miRNAs on ox-LDL induced VSMC proliferation are not fully elucidated. In this study, we showed that ox-LDL could suppress miR-141 expression and inhibition of miR-141 could promote VSMCs proliferation. Moreover, we found that PAPPA was the direct target gene of miR-141. Overexpression of PAPPA impaired the miR-141-induced inhibition of proliferation in the VSMCs. Taken together; miR-141 may play important roles in ox-LDL-induced abnormal proliferation of the VSMC. PMID:26823756

  20. Tissue-Engineered Vascular Rings from Human iPSC-Derived Smooth Muscle Cells.

    PubMed

    Dash, Biraja C; Levi, Karen; Schwan, Jonas; Luo, Jiesi; Bartulos, Oscar; Wu, Hongwei; Qiu, Caihong; Yi, Ting; Ren, Yongming; Campbell, Stuart; Rolle, Marsha W; Qyang, Yibing

    2016-07-12

    There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust, patient-specific tissue model systems for studying the pathogenesis of vascular disease, and for developing novel therapeutic interventions. Here, we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore, we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system, extendable to study other vascular proliferative diseases for drug screening. Thus, this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications. PMID:27411102

  1. Rare Presentations of Epstein-Barr Virus--Associated Smooth Muscle Tumor in Children.

    PubMed

    Arva, Nicoleta C; Schafernak, Kristian T

    2016-01-01

    Epstein-Barr virus (EBV) has oncogenic potential and has been implicated in the etiology of a wide range of malignancies. Certain EBV-driven neoplasms, such as smooth muscle tumors (SMTs), manifest typically in immunocompromised patients. In children, these neoplasms have been encountered in the setting of primary immune disorders, specifically severe combined and common variable immunodeficiency syndromes. Human immunodeficiency virus infection and posttransplant immunosuppression, in particular liver and kidney transplantation, likewise increase the risk in the pediatric population. The location of these neoplasms appears related to the type of immunodeficiency: in acquired immunodeficiency syndrome they are frequently located intracranially or intraspinally, whereas after transplant they usually involve the liver or lung. We report 2 distinct cases of EBV-related SMT, unique through their coassociated immunosuppressive state or location: the 1st occurred in a patient with immunodeficiency secondary to NEMO gene mutation following hematopoietic stem cell transplantation; the 2nd developed in the orbit after heart transplant. PMID:26230054

  2. Dual effects of trimebutine on electrical responses of gastric smooth muscles in the rat.

    PubMed

    Xue, L; Fukuta, H; Yamamoto, Y; Suzuki, H

    1995-12-27

    The effects of trimebutine on the electrical properties of smooth muscle membranes were studied in the isolated rat stomach, the objective being to elucidate the dual actions of this drug on gastric motility. Transmural nerve stimulation elicited a cholinergic excitatory junction potential (e.j.p.) and a nonadrenergic noncholinergic inhibitory junction potential (i.j.p.), and trimebutine inhibited the e.j.p. more than the i.j.p., with no significant change in the acetylcholine-induced depolarization. Trimebutine reduced the interval and, at high concentrations, the amplitude of slow waves. In enzymatically dispersed single cells, the Ca2+ current elicited by depolarization of the membrane was also inhibited by trimebutine. Thus, trimebutine increases slow wave frequency and inhibits cholinergic transmission and Ca2+ influx. The former would enhance while the latter two would depress gastric motility. PMID:8788418

  3. Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding

    PubMed Central

    Qiu, Juhui; Zheng, Yiming; Hu, Jianjun; Liao, Donghua; Gregersen, Hans; Deng, Xiaoyan; Fan, Yubo; Wang, Guixue

    2014-01-01

    Vascular smooth muscle cells (VSMCs) have critical functions in vascular diseases. Haemodynamic factors are important regulators of VSMC functions in vascular pathophysiology. VSMCs are physiologically active in the three-dimensional matrix and interact with the shear stress sensor of endothelial cells (ECs). The purpose of this review is to illustrate how haemodynamic factors regulate VSMC functions under two-dimensional conditions in vitro or three-dimensional co-culture conditions in vivo. Recent advances show that high shear stress induces VSMC apoptosis through endothelial-released nitric oxide and low shear stress upregulates VSMC proliferation and migration through platelet-derived growth factor released by ECs. This differential regulation emphasizes the need to construct more actual environments for future research on vascular diseases (such as atherosclerosis and hypertension) and cardiovascular tissue engineering. PMID:24152813

  4. Orthogonally oriented scaffolds with aligned fibers for engineering intestinal smooth muscle

    PubMed Central

    Kobayashi, Masae; Lei, Nan Ye; Wang, Qianqian; Wu, Benjamin M.; Dunn, James C.Y.

    2015-01-01

    Controlling cellular alignment is critical in engineering intestines with desired structure and function. Although previous studies have examined the directional alignment of cells on the surface (x-y plane) of parallel fibers, quantitative analysis of the cellular alignment inside implanted scaffolds with oriented fibers has not been reported. This study examined the cellular alignment in the x-z and y-z planes of scaffolds made with two layers of orthogonally oriented fibers. The cellular orientation inside implanted scaffolds was evaluated with immunofluorescence. Quantitative analysis of coherency between cell orientation and fiber direction confirmed that cells aligned along the fibers not only on the surface (x-y plane) but also inside the scaffolds (x-z & y-z planes). Our study demonstrated that two layers of orthogonally aligned scaffolds can generate the histological organization of cells similar to that of intestinal circular and longitudinal smooth muscle. PMID:26001072

  5. Vascular smooth muscle cell differentiation from human stem/progenitor cells.

    PubMed

    Steinbach, Sarah K; Husain, Mansoor

    2016-05-15

    Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promote angiogenesis and wound healing. However, VSMCs are derived from diverse embryonic sources which may influence their role in the development of vascular disease and in its therapeutic modulation. Despite progress in understanding the mechanisms of VSMC differentiation, there remains a shortage of robust methods for generating lineage-specific VSMCs from pluripotent and adult stem/progenitor cells in serum-free conditions. Here we describe a method for differentiating pluripotent stem cells, such as embryonic and induced pluripotent stem cells, as well as skin-derived precursors, into lateral plate-derived VSMCs including 'coronary-like' VSMCs and neural crest-derived VSMC, respectively. We believe this approach will have broad applications in modeling origin-specific disease vulnerability and in developing personalized cell-based vascular grafts for regenerative medicine. PMID:26678794

  6. Measurement of PLGA-NP interaction with single smooth muscle cells using optical tweezers

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Mondal, Argha; Homayoni, Homa; Nguyen, Kytai; Mohanty, Samarendra

    2012-10-01

    For intervention of cardiovascular diseases, biodegradable and biocompatible, poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) are emerging as agents of choice for controlled and targeted drug delivery. Therefore development of PLGA-NP with optimal physico-chemical properties will allow efficient binding and thus delivery of drug to targeted cells under various patho-physiological conditions. The force kinetics and its dependence on size of the NPs will be crucial for designing the NPs. Since optical tweezers allow non-contact, highly sensitive force measurement with high spatial and temporal resolution, we utilized it for studying interaction forces between magnetic PLGA nanoparticles with smooth muscle cells (SMC). In order to investigate effect of size, interaction force for 200 to 1100nm PLGA NP was measured. For similar interaction duration, the force was found to be higher with increase in size. The rupture force was found to depend on time of interaction of SMC with NPs.

  7. Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells

    SciTech Connect

    Takahashi, Yoichiro; Watanabe, Hiroyuki; Murakami, Manabu; Ono, Kyoichi; Munehisa, Yoshiko; Koyama, Takashi; Nobori, Kiyoshi; Iijima, Toshihiko; Ito, Hiroshi

    2007-10-05

    We investigated the functional role of STIM1, a Ca{sup 2+} sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca{sup 2+} entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1{sup E87A}, produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.

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

    SciTech Connect

    Helkin, Alex; Maier, Kristopher G.; Gahtan, Vivian

    2015-09-04

    Introduction: The 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. Methods: Human 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. Results: TSP-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 relevance: The 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

  9. Use of liposome-mediated DNA transfection to determine promoter activity in smooth muscle cells.

    PubMed

    Fabunmi, R P

    1999-01-01

    The transfer and expression of DNA plasmids containing promoter fragments of heterologous genes linked to reporter cDNAs in mammalian cells has become an invaluable technique for studying the regulation of gene expression. Several reporter genes such as luciferase, β-galactosidase, chloramphenicol acetyl transferase, and green flourescent protein are ideal to study promoter activities as their gene products are not endogenous to smooth muscle cells (SMC) and their expression can be readily detected using convenient assays (1). Among these genes, a popular choice is the firefly luciferase, as its expression can be easily detected in cells using a highly sensitive chemiluminescent assay (2). The firefly luciferase catalyses a rapid, ATP-dependent oxidation of the substrate, luciferin, which then emits light. Reactions catalyzed by firefly luciferase are: [Formula: see text]. PMID:21341022

  10. The impact of vitamin D on asthmatic human airway smooth muscle.

    PubMed

    Hall, Sannette C; Fischer, Kimberly D; Agrawal, Devendra K

    2016-02-01

    Asthma is a chronic heterogeneous disorder, which involves airway inflammation, airway hyperresponsiveness (AHR) and airway remodeling. The airway smooth muscle (ASM) bundle regulates the broncho-motor tone and plays a critical role in AHR as well as orchestrating inflammation. Vitamin D deficiency has been linked to increased severity and exacerbations of symptoms in asthmatic patients. It has been shown to modulate both immune and structural cells, including ASM cells, in inflammatory diseases. Given that current asthma therapies have not been successful in reversing airway remodeling, vitamin D supplementation as a potential therapeutic option has gained a great deal of attention. Here, we highlight the potential immunomodulatory properties of vitamin D in regulating ASM function and airway inflammation in bronchial asthma. PMID:26634624

  11. The role of mechanotransduction on vascular smooth muscle myocytes' [corrected] cytoskeleton and contractile function.

    PubMed

    Ye, George J C; Nesmith, Alexander P; Parker, Kevin Kit

    2014-09-01

    Smooth muscle (SM) exhibits a highly organized structural hierarchy that extends over multiple spatial scales to perform a wide range of functions at the cellular, tissue, and organ levels. Early efforts primarily focused on understanding vascular SM (VSM) function through biochemical signaling. However, accumulating evidence suggests that mechanotransduction, the process through which cells convert mechanical stimuli into biochemical cues, is requisite for regulating contractility. Cytoskeletal proteins that comprise the extracellular, intercellular, and intracellular domains are mechanosensitive and can remodel their structure and function in response to external mechanical cues. Pathological stimuli such as malignant hypertension can act through the same mechanotransductive pathways to induce maladaptive remodeling, leading to changes in cellular shape and loss of contractile function. In both health and disease, the cytoskeletal architecture integrates the mechanical stimuli and mediates structural and functional remodeling in the VSM. PMID:25125187

  12. Ablation of SM22alpha decreases contractility and actin contents of mouse vascular smooth muscle.

    PubMed

    Zeidan, Asad; Swärd, Karl; Nordström, Ina; Ekblad, Eva; Zhang, Janet C L; Parmacek, Michael S; Hellstrand, Per

    2004-03-26

    The actin-binding protein SM22alpha marks contractile differentiation in smooth muscle, but its function is unknown. We tested its role in arterial contractility and stretch-sensitive vascular protein synthesis. Active stress in depolarised mesenteric resistance arteries and portal veins was reduced by 40% in SM22alpha(-/-) mice. Passive and active arterial circumference-force relationships were shifted leftwards, whereas alpha(1)-adrenergic responses were increased. Actin contents were 10-25% lower in vessels from SM22alpha(-/-) mice, but protein composition was otherwise similar. Synthesis of SM22alpha, calponin and alpha-actin, but not beta-actin, was sensitive to stretch. Ablation of SM22alpha did not affect stretch sensitivity of any of these proteins. Thus, SM22alpha plays a role in contractility, possibly by affecting actin filament organisation. PMID:15044015

  13. Relaxant Action of Plumula Nelumbinis Extract on Mouse Airway Smooth Muscle

    PubMed Central

    Tan, Li; Chen, Weiwei; Wei, Ming-Yu; Shen, Jinhua; Yu, Meng-Fei; Yang, Guangzhong; Guo, Donglin; Qin, Gangjian; Ji, Guangju; Liu, Qing-Hua

    2015-01-01

    The traditional herb Plumula Nelumbinis is widely used in the world because it has many biological activities, such as anti-inflammation, antioxidant, antihypertension, and butyrylcholinesterase inhibition. However, the action of Plumula Nelumbinis on airway smooth muscle (ASM) relaxation has not been investigated. A chloroform extract of Plumula Nelumbinis (CEPN) was prepared, which completely inhibited precontraction induced by high K+ in a concentration-dependent manner in mouse tracheal rings, but it had no effect on resting tension. CEPN also blocked voltage-dependent L-type Ca2+ channel- (VDCC-) mediated currents. In addition, ACh-induced precontraction was also completely blocked by CEPN and partially inhibited by nifedipine or pyrazole 3. Besides, CEP