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Showing papers on "Smoothelin published in 2006"


Journal ArticleDOI
TL;DR: Adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.
Abstract: Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific α actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.

325 citations


Journal ArticleDOI
TL;DR: Both classical and novel smooth muscle markers and transmission electron microscopy are used to investigate muscular alterations in severe colorectal motility disorders, suggesting specific defects of smooth muscle cells involved in the pathogenesis of gastrointestinal motility Disorders.
Abstract: Histopathological studies of gastrointestinal motility disorders have mainly focused on enteric nerves and interstitial cells of Cajal, but rarely considered the enteric musculature. Here we used both classical and novel smooth muscle markers and transmission electron microscopy (TEM) to investigate muscular alterations in severe colorectal motility disorders. Full-thickness specimens from Hirschsprung's disease, idiopathic megacolon, slow-transit constipation and controls were stained with haematoxylin/eosin (HE) and Masson's trichrome (MT), incubated with antibodies against smooth muscle alpha-actin (alpha-SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin (SM) and histone deacetylase 8 (HDAC8) and processed for TEM. Control specimens exhibited homogeneous immunoreactivity for all antibodies. Diseased specimens showed normal smooth muscle morphology by HE and MT. While anti-alpha-SMA staining was generally normal, immunoreactivity for SMMHC, HDAC8 and/or SM was either absent or focally lacking in Hirschsprung's disease (80%), idiopathic megacolon (75%) and slow-transit constipation (70%). Ultrastructurally, clusters of myocytes with noticeably decreased myofilaments were observed in all diseases. SMMHC and the novel smooth muscle markers SM and HDAC8 often display striking abnormalities linked to the smooth muscle contractile apparatus unnoticed by both routine stainings and alpha-SMA, suggesting specific defects of smooth muscle cells involved in the pathogenesis of gastrointestinal motility disorders.

81 citations


Journal ArticleDOI
TL;DR: Gene expression studies in the human MCA leading to the ischemic region is similar to that seen after MCA occlusion in rats, and new genes are found that support the dynamic changes that occur in the MCA distributing to theIschemic area.
Abstract: We have investigated the gene expression in human middle cerebral artery (MCA) after ischemia. Ischemic stroke affects the perfusion in the affected area and experimental cerebral ischemia results in upregulation of vasopressor receptors in the MCA leading to the ischemic area. We obtained human MCA samples distributing to the ischemic area, 7-10 days post-stroke. The gene expression was examined with real-time polymerase chain reaction (PCR) and microarray, proteins were studied with immunohistochemistry. We investigated genes previously shown to be upregulated in animal models of cerebral ischemia (e.g. ET(A), ET(B), AT1, AT2, and 5-HT(2A/1B/1D)). Their mRNA expression was increased compared with controls, consistent with findings in experimental stroke. Immunohistochemistry showed upregulation of the receptors localized on the smooth muscle cells. The gene expression was profiled with microarray and seven genes chosen for further investigation with real-time PCR; ELK3, LY64, Metallothionin IG, POU3F4, Actin alpha2, RhoA and smoothelin. Six of these were regulated the same way when confirming array expression with real-time PCR. Gene expression studies in the human MCA leading to the ischemic region is similar to that seen after MCA occlusion in rats. We found new genes that support the dynamic changes that occur in the MCA distributing to the ischemic region.

69 citations


Journal ArticleDOI
TL;DR: Intimal SMCs of all situations exhibit a phenotypic profile, suggesting that they have modulated into myofibroblasts (MFs), and the high accumulation of alpha-SMA-positive MFs in erosions compared with stable plaques correlates with the higher appearance of thrombotic complications in this situation.

65 citations


Journal ArticleDOI
TL;DR: To the authors' knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system and the A(1)AR was characterized by Western blot analysis using an A( 1)AR-specific antibody.
Abstract: Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.

43 citations


Journal ArticleDOI
TL;DR: LPP appears to be a myocardin-, RhoA/ROK-dependent SMC differentiation marker that plays a role in regulating SMC migration.
Abstract: Lipoma preferred partner (LPP) has been identified as a protein highly expressed in smooth muscle (SM) tissues. The aim of the present study was to determine mechanisms that regulate LPP expression in an in vitro model of SM cell (SMC) differentiation and in stent-induced pig coronary vessel injury. All trans-retinoic acid treatment of A404 cells induced a strong increase in LPP, as well as SM α-actin, SM myosin heavy chain, and smoothelin mRNA levels, in a Rho kinase (ROK)-dependent manner. Adenovirus mediated overexpression of myocardin in A404 cells significantly increased LPP mRNA expression. Interestingly, inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors strongly inhibited myocardin mRNA expression in retinoic acid–treated A404 cells or human iliac vein SMCs. LPP silencing with short interfering RNA significantly decreased SMC migration. LPP expression was also markedly decreased in focal adhesion kinase (FAK)-null cells known to have impaired migration but rescued with inducib...

42 citations


Journal ArticleDOI
TL;DR: Smoothelin-A expression is controlled by an intragenic promoter whose activity is, in part, dependent on two CArG boxes that bind SRF, which shows a role for SRF/myocardin in regulating smootheli-A whereas the higher smoothelins-B expression appears to beSRF/ myocardin-independent.
Abstract: Objective: Smoothelin-A and -B isoforms are highly restricted to contractile smooth muscle cells (SMCs). Serum response factor (SRF) and myocardin are essential for contractile SMC differentiation. We evaluated the contribution of SRF/myocardin to transcriptional regulation of smoothelins. Methods: Rat vascular SMCs were transfected with smoothelin-A and smoothelin-B promoter reporter constructs and promoter activity was analyzed. The effects of mutations in the smoothelin-A promoter CArG-boxes and co-transfections with a myocardin expression plasmid were assessed. Electrophoretic mobility shift assays and chromatin immunoprecipitations were performed to investigate SRF-binding to the smoothelin-A CArG-boxes. Results: Smoothelin promoter activity was detected in vascular SMCs. Comparative sequence analysis revealed two conserved CArG elements in the smoothelin-A promoter that bind SRF as shown by chromatin immunoprecipitation. The proximal CArG-near bound SRF stronger than CArG-far in gel shift assays. Mutagenesis studies also indicated that CArG-near is more important than CArG-far in regulating smoothelin-A promoter activity. Myocardin augmented smoothelin-A promoter activity 2.5-fold in a CArG-near-dependent manner. In contrast, myocardin had little effect on the smoothelin-B promoter. Conclusion: Smoothelin-A expression is controlled by an intragenic promoter whose activity is, in part, dependent on two CArG boxes that bind SRF. Our data show a role for SRF/myocardin in regulating smoothelin-A whereas the higher smoothelin-B expression appears to be SRF/myocardin-independent.

37 citations


Journal ArticleDOI
TL;DR: Increased expression of smoothelin in patients with detrusor instability and sensory urge implies that the etiology of these dysfunctions includes changes in myogenic parameters, and supports the new classification of the International Continence Society for overactive bladder.

24 citations


Dissertation
01 Jan 2006
TL;DR: It is suggested that Notch receptors modulate vascular SMC phenotype in vitro and cyclic strain enhances SMC differentiation in part through inhibition of Notch receptor expression.
Abstract: Notch receptor-ligand interactions are a highly conserved mechanism, originally described in developmental studies using Drosophilae, that regulate inter-cell communication and dictate, in part, vascular smooth muscle cell (VSMC) fate in response to mechanical stimuli. VSMC differentiation is a crucial developmental process regulating angiogenesis and vasculogenesis, with phenotypic modulation of SMC a key factor in vascular pathology. It has been previously shown that cyclic strain decreases the proliferation of VSMC'S in vitr-u. Tnus, citaraci-erizauon of &e mechani~rils~ "~i ir~l i iirligi ~ b i fk~e~l i iai i~i -~ state of VSMC is of critical importance in determining the cell fate response of these cells to various mechanical stimuli. We investigated the role of Notch 1 and 3 receptor signaling in controlling vascular SMC differentiation in vitro, and established a role for cyclic strain induced changes in Notch in mediating this response. The expression of smouth muscle cell specific a-atin, calponin, myosin and smoothdin was examined by immunocytochemistry, Western blot analysis and quantitative real time PCR in human vascular SMC cultured under static conditions following over-expression of constitutively active Notch 1 and 3 receptors. The effect of equibiaxial cyclic strain (10% 24 h) on the expression of Notch receptors and SMC differentiation was subsequently determined using a Flexercell Tension Plus Unit. Over-expression of constitutively active Notch intracellular receptors (Notch 1 IC and 3 IC) resulted in a significant downregulation of a-actin, calponin, myosin and smoothelin expression, an effect that was significantly attenuated following inhibition of Notch mediated CBF- 1/RBP-Jk dependent signaling by co+xpressia of RPMS-T-I. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 and 3 IC expression, concomitant with a significant increase in smooth muscle cell a-actin, calponin, myosin and smoothelin expression. Moreover, this cyclic strain-induced increase in SMC differentiation marker expression was further enhanced following inhibition of CBF-1/RBP-Jk dependent signaling with RPMS-1. These findings suggest that Notch receptors modulate vascular SMC phenotype in vitro and cyclic strain enhances SMC differentiation in part through inhibition of Notch receptor expression. The Notch signaling pathway may therefore represent a novel mechanism for targeting vascular disorders in which SMC phenotypic diversity occurs in vivo.