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

2011 ◽  
Vol 300 (5) ◽  
pp. G903-G913 ◽  
Author(s):  
Dileep G. Nair ◽  
T. Y. Han ◽  
S. Lourenssen ◽  
Michael G. Blennerhassett
Keyword(s):  
Smooth Muscle ◽  
Sulfonic Acid ◽  
Molecular Mechanisms ◽  
Muscle Protein ◽  
Rat Colon ◽  
Intestinal Smooth Muscle ◽  
Trinitrobenzene Sulfonic Acid ◽  
Thymidine Uptake

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.

Neuronal nitric oxide inhibits intestinal smooth muscle growth

2010 ◽  
Vol 298 (6) ◽  
pp. G896-G907 ◽  
Author(s):  
Anne-Marie Pelletier ◽  
Shriram Venkataramana ◽  
Kurtis G. Miller ◽  
Brian M. Bennett ◽  
Dileep G. Nair ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Enteric Neurons ◽  
Rat Colon ◽  
Intestinal Smooth Muscle ◽  
Trinitrobenzene Sulfonic Acid ◽  
Thymidine Uptake ◽  
Circular Smooth Muscle

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [3H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [3H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo.

Mitogenic factors promoting intestinal smooth muscle cell proliferation

2010 ◽  
Vol 299 (4) ◽  
pp. C805-C817 ◽  
Author(s):  
Roger D. P. Stanzel ◽  
Sandra Lourenssen ◽  
Dileep G. Nair ◽  
Michael G. Blennerhassett
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Smooth Muscle Cells ◽  
Primary Cultures ◽  
Muscle Cells ◽  
Rat Colon ◽  
Intestinal Smooth Muscle ◽  
Trinitrobenzene Sulfonic Acid

Intestinal smooth muscle cells are normally quiescent, but in the widely studied model of trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat, the onset of inflammation causes proliferation that leads to increased cell number and an altered phenotype. The factors that drive this are unclear and were studied in primary cultures of circular smooth muscle cells (CSMC) from the rat colon. While platelet-derived growth factor (PDGF)-AA, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were ineffective, PDGF-BB and insulin-like growth factor-1 (IGF-1) caused significant increase in [3H]thymidine incorporation, bromodeoxyuridine uptake, and increased CSMC number, with PDGF-BB (≥0.2 nM) substantially more effective than IGF-1. Surprisingly, CSMC lacked expression of PDGF receptor-β (PDGF-Rβ) upon isolation but by 4 days in vitro, CSMC gained expression of PDGF-Rβ as shown by quantitative PCR, Western blot analysis, and immunocytochemistry; these CSMC responded to PDGF-BB but not IGF-1. PDGF-BB caused PDGF-Rβ phosphorylation and mobilization from the surface membrane, leading to activation of both Akt and ERK signaling pathways, which were essential for subsequent proliferation. In contrast, PDGF-AA, FGF, EGF, and IGF-1 were ineffective. In vivo, control CSMC lacked expression of PDGF-Rβ. However, this changed rapidly with TNBS-colitis, and by day 2 when CSMC proliferation in vivo is maximal, freshly isolated CSMC showed on-going PDGF-Rβ phosphorylation that was further increased by exogenous PDGF-BB. This suggests that the onset of PDGF-Rβ expression is a key factor in CSMC growth in vitro and in vivo, where inflammation may damage intrinsic inhibitory mechanisms and thus lead to hyperplasia.

Neural regulation of intestinal smooth muscle growth in vitro

2000 ◽  
Vol 279 (3) ◽  
pp. G511-G519 ◽  
Author(s):  
M. G. Blennerhassett ◽  
S. Lourenssen
Keyword(s):  
Smooth Muscle ◽  
Sympathetic Neurons ◽  
Muscle Growth ◽  
Scorpion Venom ◽  
Enteric Neurons ◽  
Intestinal Smooth Muscle ◽  
Early Event ◽  
Thymidine Uptake

The loss of intrinsic neurons is an early event in inflammation of the rat intestine that precedes the growth of intestinal smooth muscle cells (ISMC). To study this relationship, we cocultured ISMC and myenteric plexus neurons from the rat small intestine and examined the effect of scorpion venom, a selective neurotoxin, on ISMC growth. By 5 days after neuronal ablation, ISMC number increased to 141 ± 13% ( n = 6) and the uptake of [3H]thymidine in response to mitogenic stimulation was nearly doubled. Atropine caused a dose-dependent increase in [3H]thymidine uptake in cocultures, suggesting the involvement of neural stimulation of cholinergic receptors in regulation of ISMC growth. In contrast, coculture of ISMC with sympathetic neurons increased [3H]thymidine uptake by 45–80%, which was sensitive to propranolol (30 μM) and was lost when the neurons were separated from ISMC by a permeable filter. Western blotting showed that coculture with myenteric neurons increased α-smooth muscle-specific actin nearly threefold to a level close to ISMC in vivo. Therefore, factors derived from enteric neurons maintain the phenotype of ISMC through suppression of the growth response, whereas catecholamines released by neurons extrinsic to the intestine may stimulate their growth. Thus inflammation-induced damage to intestinal innervation may initiate or modulate ISMC hyperplasia.

Role of GADD153 (growth arrest- and DNA damage-inducible gene 153) in vascular smooth muscle cell apoptosis

2001 ◽  
Vol 100 (3) ◽  
pp. 275-281 ◽  
Author(s):  
Michiya IGASE ◽  
Takafumi OKURA ◽  
Michitsugu NAKAMURA ◽  
Yasunori TAKATA ◽  
Yutaka KITAMI ◽  
...  
Keyword(s):  
Dna Damage ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Molecular Mechanisms ◽  
Growth Arrest ◽  
Cell Contact ◽  
Inducible Gene

GADD153 (growth arrest- and DNA damage-inducible gene 153) is expressed at very low levels in growing cells, but is markedly induced in response to a variety of cellular stresses, including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations. Forced expression of GADD153 induces cell cycle arrest in many types of cells. It is also reported that GADD153 is directly associated with apoptosis. Recently we have reported that platelet-derived growth factor (PDGF)-BB induces apoptosis in cultured vascular smooth muscle cells (VSMC), but only when 100% confluency is reached. These results suggested that cell–cell contact inhibition (cell growth arrest) may be a critical factor for induction of VSMC apoptosis by PDGF-BB. In the present study, we explored the role of GADD153, one of a number of growth-arrest-related gene products, in the molecular mechanisms of VSMC apoptosis in vitro and in vivo. GADD153 was markedly induced at both the mRNA and protein levels, in parallel with the induction of VSMC apoptosis, after treatment with PDGF-BB. Moreover, overexpression of GADD153 in VSMC significantly reduced cell viability and induced apoptosis. In the carotid artery balloon injury model in rats, GADD153 protein was expressed in apoptotic VSMC which were positively stained by in situ DNA labelling. These results demonstrate an important role for GADD153 in the molecular mechanisms of VSMC apoptosis.

Tu1211 AN ANTISENSE OLIGONUCLEOTIDE FOR MICRORNA-24-3P EXHIBITS SAFE AND EFFICIENT KNOCKDOWN ABILITY IN VITRO AND IN VIVO AND INHIBITS TRINITROBENZENE SULFONIC ACID (TNBS) COLITIS

2020 ◽  
Vol 158 (6) ◽  
pp. S-1019-S-1020
Author(s):  
Artin Soroosh ◽  
Carl R. Rankin ◽  
Kai Fang ◽  
Zulfiqar A. Lokhandwala ◽  
Ivy Ka Man Law ◽  
...  
Keyword(s):  
Antisense Oligonucleotide ◽  
Sulfonic Acid ◽  
Trinitrobenzene Sulfonic Acid ◽  
Tnbs Colitis

scholarly journals Molecular Mechanisms for the Mechanical Modulation of Airway Responsiveness

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Wenwu Zhang ◽  
Susan J. Gunst
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Airway Responsiveness ◽  
Small Gtpase ◽  
Cortical Actin

The smooth muscle of the airways is exposed to continuously changing mechanical forces during normal breathing. The mechanical oscillations that occur during breathing have profound effects on airway tone and airway responsiveness both in experimental animals and humans in vivo and in isolated airway tissues in vitro. Experimental evidence suggests that alterations in the contractile and mechanical properties of airway smooth muscle tissues caused by mechanical perturbations result from adaptive changes in the organization of the cytoskeletal architecture of the smooth muscle cell. The cytoskeleton is a dynamic structure that undergoes rapid reorganization in response to external mechanical and pharmacologic stimuli. Contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins into large macromolecular signaling complexes (adhesomes) that undergo activation to mediate the polymerization and reorganization of a submembranous network of actin filaments at the cortex of the cell. Cortical actin polymerization is catalyzed by Neuronal-Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, which are activated by pathways regulated by paxillin and the small GTPase, cdc42. These processes create a strong and rigid cytoskeletal framework that may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. This model for the regulation of airway smooth muscle function can provide novel perspectives to explain the normal physiologic behavior of the airways and pathophysiologic properties of the airways in asthma.

scholarly journals CHIP Represses Myocardin-Induced Smooth Muscle Cell Differentiation via Ubiquitin-Mediated Proteasomal Degradation

2009 ◽  
Vol 29 (9) ◽  
pp. 2398-2408 ◽  
Author(s):  
Ping Xie ◽  
Yongna Fan ◽  
Hua Zhang ◽  
Yuan Zhang ◽  
Mingpeng She ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Serum Response Factor ◽  
Ex Vivo ◽  
Critical Role ◽  
Physiological Control ◽  
Interacting Protein

ABSTRACT Myocardin, a coactivator of serum response factor (SRF), plays a critical role in the differentiation of vascular smooth muscle cells (SMCs). However, the molecular mechanisms regulating myocardin stability and activity are not well defined. Here we show that the E3 ligase C terminus of Hsc70-interacting protein (CHIP) represses myocardin-dependent SMC gene expression and transcriptional activity. CHIP interacts with and promotes myocardin ubiquitin-mediated degradation by the proteasome in vivo and in vitro. Furthermore, myocardin ubiquitination by CHIP requires its phosphorylation. Importantly, CHIP overexpression reduces the level of myocardin-dependent SMC contractile gene expression and diminishes arterial contractility ex vivo. These findings for the first time, to our knowledge, demonstrate that CHIP-promoted proteolysis of myocardin plays a key role in the physiological control of SMC phenotype and vessel tone, which may have an important implication for pathophysiological conditions such as atherosclerosis, hypertension, and Alzheimer's disease.

scholarly journals Collagen and heparan sulfate coatings differentially alter cell proliferation and attachment in vitro and in vivo

TECHNOLOGY ◽  
2016 ◽  
Vol 04 (03) ◽  
pp. 159-169
Author(s):  
Christopher M. Walthers ◽  
Chase J. Lyall ◽  
Alireza K. Nazemi ◽  
Puneet V. Rana ◽  
James C.Y. Dunn
Keyword(s):  
Tissue Engineering ◽  
Cell Proliferation ◽  
Smooth Muscle ◽  
Heparan Sulfate ◽  
Intestinal Smooth Muscle ◽  
Smooth Muscle Tissue ◽  
Cell Numbers ◽  
Survival And Growth

Tissue engineering is an innovative field of research applied to treat intestinal diseases. Engineered smooth muscle requires dense smooth muscle tissue and robust vascularization to support contraction. The purpose of this study was to use heparan sulfate (HS) and collagen coatings to increase the attachment of smooth muscle cells (SMCs) to scaffolds and improve their survival after implantation. SMCs grown on biologically coated scaffolds were evaluated for maturity and cell numbers after 2, 4 and 6 weeks in vitro and both 2 and 6 weeks in vivo. Implants were also assessed for vascularization. Collagen-coated scaffolds increased attachment, growth and maturity of SMCs in culture. HS-coated implants increased angiogenesis after 2 weeks, contributing to an increase in SMC survival and growth compared to HS-coated scaffolds grown in vitro. The angiogenic effects of HS may be useful for engineering intestinal smooth muscle.

Sign in / Sign up

Export Citation Format

Share Document