The Experts below are selected from a list of 867 Experts worldwide ranked by ideXlab platform
Sander S Rensen - One of the best experts on this subject based on the ideXlab platform.
-
Smoothelin-B Deficiency Results in Reduced Arterial Contractility, Hypertension, and Cardiac Hypertrophy in Mice
Circulation, 2008Co-Authors: Sander S Rensen, Petra Niessen, Jan M. Van Deursen, Ben J. A. Janssen, Edwin Heijman, Evelien Hermeling, Merlijn J. Meens, Natascha Lie, Marion J.j. Gijbels, Gustav J. StrijkersAbstract:Background— Smoothelins are actin-binding proteins that are abundantly expressed in healthy visceral (Smoothelin-A) and vascular (Smoothelin-B) smooth muscle. Their expression is strongly associated with the contractile phenotype of smooth muscle cells. Analysis of mice lacking both Smoothelins (Smtn-A/B−/− mice) previously revealed a critical role for Smoothelin-A in intestinal smooth muscle contraction. Here, we report on the generation and cardiovascular phenotype of mice lacking only Smoothelin-B (Smtn-B−/−). Methods and Results— Myograph studies revealed that the contractile capacity of the saphenous and femoral arteries was strongly reduced in Smtn-B−/− mice, regardless of the contractile agonist used to trigger contraction. Arteries from Smtn-A/B−/− compound mutant mice exhibited a similar contractile deficit. Smtn-B−/− arteries had a normal architecture and expressed normal levels of other smooth muscle cell–specific genes, including smooth muscle myosin heavy chain, α-smooth muscle actin, and smo...
-
Smoothelin in vascular smooth muscle cells
Trends in cardiovascular medicine, 2007Co-Authors: Guillaume J.j.m. Van Eys, Petra Niessen, Sander S RensenAbstract:Smoothelin-A and -B have only been found in fully differentiated contractile smooth muscle cells. They are increasingly used to monitor the smooth muscle cell differentiation process to a contractile or synthetic phenotype. Vascular-specific Smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis. Recently obtained data show that Smoothelin deficiency results in a considerable loss of contractile potential and hence in impaired smooth muscle function and suggest that Smoothelins are part of the contractile apparatus.
-
Contribution of serum response factor and myocardin to transcriptional regulation of Smoothelins
Cardiovascular Research, 2006Co-Authors: Sander S Rensen, Petra Niessen, Xiaochun Long, Pieter A Doevendans, Joseph M MianoAbstract: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.
-
Smoothelin-a is essential for functional intestinal smooth muscle contractility in mice.
Gastroenterology, 2005Co-Authors: Petra Niessen, Sander S Rensen, Pieter A Doevendans, Jan M. Van Deursen, Joris G. De Man, Ann De Laet, Jean-marie Vanderwinden, Thilo Wedel, Darren J. Baker, Marten H. HofkerAbstract:Background & Aims: In patients with chronic intestinal pseudo-obstruction, intestinal motility is disturbed by either nervous or myogenic aberrations. The cause of the myogenic form is unknown, but it is likely to originate in the contractile apparatus of the smooth muscle cells. Smoothelins are actin-binding proteins that are expressed abundantly in visceral (Smoothelin-A) and vascular (Smoothelin-B) smooth muscle. Experimental data indicate a role for Smoothelins in smooth muscle contraction. A Smoothelin-deficient mouse model may help to establish the role of Smoothelin-A in intestinal contraction and provide a model for myogenic chronic intestinal pseudo-obstruction. Methods: We used gene targeting to investigate the function of Smoothelin-A in intestinal tissues. By deletion of exons 18, 19, and 20 from the Smoothelin gene, the expression of both Smoothelin isoforms was disrupted. The effects of the deficiency were evaluated by pathologic and physiologic analyses. Results: In Smoothelin-A/B knockout mice, the intestine was fragile and less flexible compared with wild-type littermates. The circular and longitudinal muscle layers of the intestine were hypertrophic. Deficiency of Smoothelin-A led to irregular slow wave patterns and impaired contraction of intestinal smooth muscle, leading to hampered transport in vivo. This caused obstructions that provoked intestinal diverticulosis and occasionally intestinal rupture. Conclusions: Smoothelin-A is essential for functional contractility of intestinal smooth muscle. Hampered intestinal transit in Smoothelin-A/B knockout mice causes obstruction, starvation, and, ultimately, premature death. The pathology of mice lacking Smoothelin-A is reminiscent of that seen in patients with chronic intestinal pseudo-obstruction.
-
Biochemical evidence for interaction between Smoothelin and filamentous actin
Experimental cell research, 2004Co-Authors: Petra Niessen, Sander S Rensen, Sophie Clément, Lionel Fontao, Christine Chaponnier, Birgit E.j. Teunissen, Guillaume J.j.m. Van Eys, Giulio GabbianiAbstract:The two major isoforms of Smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs). Based on these findings, we hypothesized that Smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin. The interaction between Smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin [alpha-SKA]) was investigated using several in vitro assays. Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts. In rat embryonic fibroblasts, transfected Smoothelins-A and -B associated with stress fibers. In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled Smoothelin, showed binding of Smoothelin-A to actin filaments, but not to monomeric G-actin. A truncated Smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent. ELISA results showed that the binding of Smoothelin to actin has no significant isoform specificity. Our results indicate an interaction between Smoothelin and actin filaments. Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin.
Guillaume J.j.m. Van Eys - One of the best experts on this subject based on the ideXlab platform.
-
Histopathological Evidence for Irradiation Angiopathy in Head and Neck Cancer
International Journal of Otolaryngology and Head & Neck Surgery, 2015Co-Authors: Nobuhiro Uwa, Hiroyuki Hao, Yoshitane Tsukamoto, Tomonori Terada, Kosuke Sagawa, Takeshi Mohri, Takashi Daimon, Hiroshi Doi, Yohei Sotsuka, Guillaume J.j.m. Van EysAbstract:Objective: To evaluate the incidence of cervical angiopathy caused by radiation therapy for head and neck cancer. Methods: Segments of 57 cervical arteries were obtained during surgery for head and neck malignant tumors and divided into two groups (irradiated group and non-irradiated group) based on the treatment prior to vascular resection. In order to evaluate vascular injury after radiation therapy, we examined the degree of medial atrophy, medial fibrosis, smooth muscle cell (SMC) differentiation in the media and intima, intimal hyperplasia and endothelial cell (EC) injury. Sections of arterial segments were stained with hematoxylin-eosin, Elastica van Gieson and Masson’s trichrome, and immunohistochemistry for α-smooth muscle actin (α-SMA), Smoothelin, S100A4 and CD31 in the resected vessels was conducted. Results: The median interval between the completion of radiation therapy and vascular resection was nine months. No significant differences were observed between the two groups in terms of medial atrophy, medial fibrosis and intimal hyperplasia. The ratio of the Smoothelin-positive area per α-SMA-positive area in the media and the S100A4-positive proportion in the intima, indicating the degree of differentiation of the medial SMC and dedifferentiation of the intimal SMC, respectively, showed no significant differences, despite the tendency toward a lower Smoothelin-positive area per α-SMA-positive area in the media of the irradiated arteries. The EC coverage revealed on CD31 immunohistochemistry was significantly decreased, with mural thrombus adhesion, in the irradiated group. Conclusions: The ECs of small arteries are damaged by irradiation. Although we did not confirm the statistical significance of medial SMC dedifferentiation, a decreased expression of Smoothelin tended to be observed in the media of the irradiated arteries. Our findings provide histopathological evidence of irradiation angiopathy in head and neck cancer and may help to improve the surgical safety of microvascular anastomosis and determine the treatment strategy for head and neck tumors.
-
Smoothelin in vascular smooth muscle cells
Trends in cardiovascular medicine, 2007Co-Authors: Guillaume J.j.m. Van Eys, Petra Niessen, Sander S RensenAbstract:Smoothelin-A and -B have only been found in fully differentiated contractile smooth muscle cells. They are increasingly used to monitor the smooth muscle cell differentiation process to a contractile or synthetic phenotype. Vascular-specific Smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis. Recently obtained data show that Smoothelin deficiency results in a considerable loss of contractile potential and hence in impaired smooth muscle function and suggest that Smoothelins are part of the contractile apparatus.
-
Biochemical evidence for interaction between Smoothelin and filamentous actin
Experimental cell research, 2004Co-Authors: Petra Niessen, Sander S Rensen, Sophie Clément, Lionel Fontao, Christine Chaponnier, Birgit E.j. Teunissen, Guillaume J.j.m. Van Eys, Giulio GabbianiAbstract:The two major isoforms of Smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs). Based on these findings, we hypothesized that Smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin. The interaction between Smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin [alpha-SKA]) was investigated using several in vitro assays. Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts. In rat embryonic fibroblasts, transfected Smoothelins-A and -B associated with stress fibers. In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled Smoothelin, showed binding of Smoothelin-A to actin filaments, but not to monomeric G-actin. A truncated Smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent. ELISA results showed that the binding of Smoothelin to actin has no significant isoform specificity. Our results indicate an interaction between Smoothelin and actin filaments. Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin.
-
Adventitial myofibroblasts play no major role in neointima formation after angioplasty.
Scandinavian cardiovascular journal : SCJ, 2003Co-Authors: Michael Maeng, Guillaume J.j.m. Van Eys, Henrik Mertz, Søren Nielsen, Klaus Rasmussen, G. T. EspersenAbstract:Myofibroblasts migrating from adventitia have been suggested to constitute a majority of neointimal cells after angioplasty. We sought to examine this hypothesis by use of Smoothelin, which is a marker for the quiescent smooth muscle cell (SMC) phenotype while not expressed by myofibroblasts. Balloon angioplasty was performed in left iliac arteries of 25 rabbits that were killed after 3-56 days. Arterial cross-sections were immunostained for alpha-actin (general marker), Smoothelin (quiescent SMC phenotype), and Ki-67 (proliferative phenotype). Adventitial cells became transiently actin-positive (myofibroblasts) but did not express Smoothelin at any time point. In media, angioplasty induced transient proliferation and coinciding transient decrease in Smoothelin expression. Neointimal cells, present 7 days after angioplasty, were initially proliferating and Smoothelin-negative but changed to non-proliferating, Smoothelin-positive cells after 56 days where 82 +/- 10% of cells stained positive for Smoothelin. This phenotypic modulation of medial and intimal cells began in media and moved gradually towards the lumen. At late follow-up, the majority of intimal cells are Smoothelin-positive indicating that adventitial myofibroblasts play no major role for neointima formation.
-
expression of the Smoothelin gene is mediated by alternative promoters
Cardiovascular Research, 2002Co-Authors: Sander S Rensen, Pieter A Doevendans, Victor L. Thijssen, C. J. M. De Vries, S D Deterawadleigh, Guillaume J.j.m. Van EysAbstract:Objective: Two major isoforms of Smoothelin have been reported, a 59-kDa Smoothelin-A in visceral smooth muscle cells and a 110-kDa Smoothelin-B in vascular smooth muscle cells. The present study was undertaken to investigate the expression of these Smoothelin isoforms in different smooth muscle tissues and to determine how they are generated. Methods: Western blotting with a new, well-defined, Smoothelin antibody was used to confirm the existence of two major Smoothelin isoforms. Northern blotting, RT-PCR, primer extension and 5′RACE were applied to analyse the expression of these isoforms in human and mouse. Promoter reporter assays were carried out to establish the existence of a dual promoter system governing the expression pattern of the gene. Results: Antibody C6G confirmed the existence of two Smoothelin proteins. Northern blotting showed that in vascular tissues a larger Smoothelin transcript is generated than in visceral tissue. The cDNA of this larger Smoothelin-B was cloned. Computer analysis of the open reading frame suggests an α-helical structure of 130 amino acids at the amino terminus of Smoothelin-B. The Smoothelin gene was cloned and sequenced. It comprises about 25 kb and contains 21 exons. The translational start of Smoothelin-B is located in exon 2, whereas transcription and translation of the previously described Smoothelin-A starts inside exon 10. Smoothelin-A and -B were demonstrated to be generated by two physically separated promoters. Splice variants within the calponin homology domain at the 3′ end of the gene were found for both isoforms. Conclusions: Two major Smoothelin isoforms are generated from a single gene by a dual promoter system in a tissue specific manner. Further variation in the Smoothelin proteins is achieved by alternative splicing in the calponin homology domain.
Mahul B. Amin - One of the best experts on this subject based on the ideXlab platform.
-
Limited Smoothelin expression within the muscularis mucosae: validation in bladder diverticula
Human pathology, 2011Co-Authors: Donna E. Hansel, Gladell P. Paner, Nalan Nese, Mahul B. AminAbstract:Smoothelin, a marker of differentiated smooth muscle, is diffusely expressed by bladder muscularis propria and is negative to only weakly and focally expressed in muscularis mucosae. We used bladder diverticula, which lack muscularis propria and frequently demonstrate hyperplastic muscularis mucosae, to evaluate the use of Smoothelin immunoreactivity in diagnostic pathology. Diverticula from 40 patients (21 with benign features, 19 with neoplastic features) were studied. Immunohistochemistry was performed using Smoothelin antibody (clone R4A, 1:150 dilution; Abcam, Cambridge, MA); and tissue was scored as 0 (no expression), 1+ (moderate expression b10% of cells), 2+ expression (moderate expression N10% of cells), and 3+ (robust diffuse expression). All diverticula contained muscularis mucosae of varying caliber; staining in diverticular muscularis mucosae was compared with historic results in the muscularis mucosae of cystectomy specimens. Hyperplastic muscularis mucosae occurred in 31 (78%) of 40 cases. Smoothelin immunoreactivity in the diverticular muscularis mucosae included 0 (16/40, or 40%); 1+ (11/40, or 27.5%); 2+ (13/40, or 32.5%); and 3+ (0/40, or 0%), with a slightly higher 2+ expression level in hyperplastic versus nonhyperplastic muscularis mucosae (35% versus 22%). Adjacent normal muscularis propria, present in 12 specimens, demonstrated 3+ muscularis propria immunoreactivity. Comparison between diverticula with benign and neoplastic features showed no significant difference in Smoothelin immunoreactivity. No correlation was evident with Smoothelin immunohistochemistry and muscle caliber. Smoothelin immunoreactivity in bladder diverticula confirms the limited nature of Smoothelin expression in the muscularis mucosae and represents a useful ancillary technique in the proper histopathologic evaluation of diverticular and nondiverticular bladder carcinomas. A strong and robust staining of smooth, rounded muscle with Smoothelin remains a useful diagnostic adjunct in the reliable recognition of muscularis propria.
-
Diagnostic use of antibody to Smoothelin in the recognition of muscularis propria in transurethral resection of urinary bladder tumor (TURBT) specimens.
The American journal of surgical pathology, 2010Co-Authors: Gladell P. Paner, Donna E. Hansel, Nalan Nese, Jeffrey G. Brown, Shawn Lapetino, Ruta Gupta, Steven S. Shen, Mahul B. AminAbstract:Accurate recognition of muscularis propria invasion by urothelial carcinoma is vital as it serves as a crossroad between conservative and aggressive clinical management. Recently, there has been attention to the hyperplastic pattern of muscularis mucosae which may mimic the muscularis propria. We have earlier shown that Smoothelin, a marker of terminally differentiated smooth muscle cells, is relatively specific for muscularis propria (positive staining) and is variably negative to weak in muscularis mucosae. The earlier study was based on cystectomy specimen slides in which the bladder cancer was not present. Pathologic staging in transurethral resection of urinary bladder tumor (TURBT) specimens is complicated by limited, unoriented, or highly cauterized samples. Herein, we test the capability of Smoothelin to recognize muscularis propria in TURBT specimens to further substantiate its diagnostic applicability in routine practice. Representative sections from 70 TURBTs were immunostained with Smoothelin, and muscularis propria was evaluated in H&E slides and the corresponding Smoothelin immunohistochemistry slides using double-blinded analysis. In 31/70 (44%) cases, muscularis propria was involved by invasive carcinoma. Cautery artifact was present in 46/70 (66%) cases, which did not seem to affect Smoothelin immunohistochemistry staining of the muscularis propria. Muscularis propria was present by H&E in 48/70 (69%) cases and 48/70 (69%) cases had muscularis propria by Smoothelin immunohistochemistry-based 2 (+) or 3 (+) positivity in larger muscle bundles with round regular contours. Desmoplastic response to invasive carcinoma stained negatively for Smoothelin. The sensitivity, specificity, positive predictive value, and negative predictive value of Smoothelin based on comparison with morphology in TURBT specimens was 98%, 95%, 98%, and 95%, respectively. This study confirms the relatively high sensitivity and specificity for Smoothelin in MP, including in TURBT specimens. Immunoreactivity is retained despite the presence of thermal tissue injury, desmoplasia, or involvement by carcinoma. Our data confirm the use of Smoothelin in the accurate distinction between muscularis propria and muscularis mucosae or desmoplastic reactions, thereby facilitating appropriate pathologic stage designation in often challenging TURBT specimens.
-
Diagnostic utility of antibody to Smoothelin in the distinction of muscularis propria from muscularis mucosae of the urinary bladder: a potential ancillary tool in the pathologic staging of invasive urothelial carcinoma.
The American journal of surgical pathology, 2009Co-Authors: Gladell P. Paner, Shawn Lapetino, Steven S. Shen, Girish Venkataraman, Güliz A. Barkan, Marcus L. Quek, Mahul B. AminAbstract:Accurate recognition of urinary bladder muscularis propria (MP) invasion by urothelial carcinoma is crucial as it is the critical crossroad between conservative and aggressive management for the patient. It is now widely known that an inconsistent layer of muscularis mucosae (MM) muscle exists in the lamina propria, which can mimic the MP muscle, particularly when hyperplastic, making staging extremely challenging in some limited, unoriented, or highly cauterized specimens. Smoothelin is a novel smooth muscle-specific contractile protein expressed only by fully differentiated smooth muscle cells, and not by proliferative or noncontractile smooth muscle cells and myofibroblasts. We performed immunohistochemical staining in the bladder for Smoothelin to: (a) evaluate its expression in MM and MP muscle in cystectomy specimens and by comparing the staining pattern with smooth muscle actin (SMA), (b) study MP variations in the bladder trigone and at the ureteric insertion in the bladder wall, and (c) assess the staining pattern of MM and MP in a representative group of transurethral resection of bladder tumor specimens. In contrast to SMA, which equitably stained both types of muscle fibers, Smoothelin displayed striking differential immunoreactivity between MM and MP muscle. With Smoothelin, the MM muscle (including hyperplastic forms) typically showed absent (19/42, 45%) or weak and focal (18/42, 43%) staining, whereas the MP muscle typically showed strong and diffuse staining (36/42, 86%). Smoothelin accentuated individual muscle fibers within groups of MP bundles only, a feature which was evident in both MM and MP stained by SMA. When only strong and diffuse immunoreactivity in muscle was set as a threshold for positivity, 100% specificity and positive predictive value of Smoothelin for MP (vs. MM) was achieved in our study. Smoothelin staining confirmed the morphologic variations in MP muscle in the bladder wall of the trigone and at the ureteric insertion. In addition to the well-defined muscle layers of MM and MP, SMA staining revealed a continuous band of ill-defined haphazardly oriented compact spindle cells that were immediately subjacent to the urothelium in all cases. These spindle cells blended with the morphologically recognizable thin slender fascicles of the MM muscle. We designate this hitherto uncharacterized thin layer of SMA-positive [muscle-specific actin positive (6/6), Masson trichrome stain predominantly blue (5/6)] and Smoothelin-negative cells as suburothelial band of myofibroblasts. In all 10 transurethral resection of bladder tumor sections, Smoothelin staining was in agreement with the routine light microscopic presence and absence of MP muscle. In conclusion, the relatively distinct immunohistochemical staining pattern of Smoothelin between MP and MM (including its hyperplastic forms) makes it a robust and attractive marker to be incorporated in the contemporary diagnostic armamentarium for the sometimes difficult area of staging bladder urothelial carcinoma.
Frans C. S. Ramaekers - One of the best experts on this subject based on the ideXlab platform.
-
Mechanisms of Neointima Formation and Remodeling in the Porcine Coronary Artery
Circulation, 2001Co-Authors: Thomas Christen, Guillaume J.j.m. Van Eys, Frans C. S. Ramaekers, Vitali Verin, Marie-luce Bochaton-piallat, Youri Popowski, Philippe Debruyne, Edoardo Camenzind, Giulio GabbianiAbstract:Background—To characterize the cells responsible for neointima formation after porcine coronary artery wall injury, we studied the expression of smooth muscle cell (SMC) differentiation markers in 2 models: (1) self-expanding stent implantation resulting in no or little interruption of internal elastic lamina and (2) percutaneous transluminal coronary angioplasty (PTCA) resulting in complete medial rupture and exposure of adventitia to blood components. Methods and Results—The expression of α-smooth muscle (SM) actin, SM myosin heavy chain isoforms 1 and 2, desmin, and Smoothelin was investigated by means of immunohistochemistry and Western blots in tissues of the arterial wall collected at different time points and in cell populations cultured from these tissues. The expression of Smoothelin, a marker of late SMC differentiation, was used to discriminate between SMCs and myofibroblasts. Both stent- and PTCA-induced neointimal tissues and their cultured cell populations expressed all 4 markers. The advent...
-
Smoothelin in adult and developing human arteries and myocardium.
Histochemistry and cell biology, 1999Co-Authors: Brengt Johansson, Frans C. S. Ramaekers, Anders Eriksson, Lars-eric Van De ThornellAbstract:The aim of this investigation was to study, with immunohistochemical methods, the distribution of the novel cytoskeletal protein Smoothelin in human cardiovascular tissues, the possible changes during the development of the cardiovascular system and its correlation to the intermediate filament proteins desmin and vimentin. Smoothelin was detected in smooth muscle cells of the fetal coronary arteries. In very young subjects (up to 3 months of age), only a few cells in the media of the elastic arteries contained Smoothelin, whereas it was present in most smooth muscle cells in the muscular arteries. In individuals older than 1 year, most smooth muscle cells in the media of all blood vessels contained Smoothelin. In vessels with a developed intima, Smoothelin was present in a variable proportion of the smooth muscle cells. With few exceptions, Smoothelin was more frequently detected than desmin in medial smooth muscle cells. Smoothelin and vimentin were codistributed in the smooth muscle cells of the media in most vessels. In the cardiomyocytes (fetal to adult age), the Smoothelin antibody detected epitopes located at the Z-disc level but not in the intercalated discs. In conclusion, Smoothelin is more widely distributed in the muscular arteries than in the elastic arteries early in life, and thus exhibits a variable distribution during postnatal development of vascular tissues. In the adult, Smoothelin is detected in the media of most vascular smooth muscle cells, both in muscular and elastic arteries, and is not necessarily codistributed with either desmin or vimentin. Evidence that Smoothelin is present in human striated cardiomyocytes is also presented.
-
Smoothelin and intermediate filament proteins in human aortocoronary saphenous vein by-pass grafts.
The Histochemical journal, 1999Co-Authors: Bengt Johansson, Frans C. S. Ramaekers, Anders Eriksson, Lars-eric ThornellAbstract:The aim of this immunohistochemical investigation was to study the distribution of the novel cytoskeletal protein Smoothelin and the intermediate filament proteins vimentin and desmin in normal human great saphenous vein and in human aortocoronary by-pass vein grafts. Smoothelin was present in most smooth muscle cells in the media of the native vein. In the neointima of the vein grafts that had been in situ for three months or more, Smoothelin was, in general, present only in few smooth muscle cells. Desmin was distributed in the same pattern as Smoothelin in the native great saphenous vein. When desmin and Smoothelin were present in the neointima, Smoothelin was detected in more cells than desmin. Vimentin was present in most cells in all wall layers of both the native saphenous vein and the vein grafts. Vascular smooth muscle cells containing vimentin but not desmin or Smoothelin are the principal cells in the neointima of human aortocoronary vein grafts. In some grafts, however, all three cytoskeletal proteins were detected in the neointima. The distribution of Smoothelin and desmin in aortocoronary vein grafts support the postulate that these proteins are expressed mainly in the contractile smooth muscle cell phenotype.
-
Localization of Smoothelin in avian smooth muscle and identification of a vascular-specific isoform
FEBS letters, 1997Co-Authors: X.h.t. Wehrens, G.j.j.m. Van Eys, Frans C. S. Ramaekers, B Mies, Mario Gimona, J.v. SmallAbstract:Smoothelin is a smooth muscle-specific protein of minor abundance first identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibodies to Smoothelin together with antibodies to other smooth muscle proteins showed that Smoothelin was co-distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the finding that Smoothelin, unlike desmin, was readily extracted by Triton X-100 as well as under conditions that solubilized myosin, β-actin and filamin, we conclude that Smoothelin is most likely associated with the actin cytoskeleton. Western blot analysis of gizzard smooth muscle tissue revealed an immunoreactive protein band with an apparent molecular weight of 59 kDa that separated into 3–4 isolated variants, while avian vascular muscle showed a polypeptide band of 95 kDa. These results point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 60 kDa filamin-binding protein, described by Maekawa and Sakai (FEBS Lett. 221, 68–72, 1987). As compared to other smooth muscle markers, such as calponin and SM22, Smoothelin appeared very late during differentiation in the chick gizzard, on about the 18th embryonic day.
-
Smoothelin expression characteristics: Development of a smooth muscle cell in vitro system and identification of a vascular variant
Cell structure and function, 1997Co-Authors: G.j.j.m. Van Eys, M.c.w. Voller, Erika D.j. Timmer, X.h.t. Wehrens, J.v. Small, Jack A. Schalken, Frans C. S. Ramaekers, F. T. L. Van Der LoopAbstract:Recently we described a protein, Smoothelin, that has been exclusively found in smooth muscle cells (SMC). The human cDNA has been cloned from a colon cDNA library and the putative protein sequence was deduced. Smoothelin does not belong to a known protein family but shows a partial homology with members of the spectrin family. Transfection studies revealed that Smoothelin has an affinity for actin and is either capable of forming filamentous structures or colocalizes with such structures. The protein is expressed in visceral as well as vascular tissues of all vertebrate classes. A study on the distribution of Smoothelin in the vascular and placental system showed that Smoothelin expression was largely restricted to the muscular pulsating blood vessels. Therefore, we hypothesized that Smoothelin is expressed in contractile SMC only (36, 37). No expression of Smoothelin was observed in established cell lines of SMC. In tissue explants Smoothelin mRNA concentration decreases to undetectable levels within 12 hours after dissection as was in general the case in primary cell cultures. Here we report on continued Smoothelin expression for several passages observed in a human prostate primary cell culture system. Smoothelin was demonstrated to colocalize with actin stress fibers but not with desmin filaments. This culture system offers opportunities to study the cytological localization of Smoothelin, interactions with other proteins and should provide a system to test the promoter of the Smoothelin gene. On immunoblots the molecular weight of Smoothelin differed between visceral and vascular smooth muscle tissue with apparent molecular weights of respectively 59 kDa and 94 kDa. There is no evidence for the existence of another gene coding for the 94 kDa Smoothelin. Thus, posttranslational modification, alternative splicing and dual promoter control are the alternatives for the expression of two isoforms of Smoothelin.
Petra Niessen - One of the best experts on this subject based on the ideXlab platform.
-
Smoothelin-B Deficiency Results in Reduced Arterial Contractility, Hypertension, and Cardiac Hypertrophy in Mice
Circulation, 2008Co-Authors: Sander S Rensen, Petra Niessen, Jan M. Van Deursen, Ben J. A. Janssen, Edwin Heijman, Evelien Hermeling, Merlijn J. Meens, Natascha Lie, Marion J.j. Gijbels, Gustav J. StrijkersAbstract:Background— Smoothelins are actin-binding proteins that are abundantly expressed in healthy visceral (Smoothelin-A) and vascular (Smoothelin-B) smooth muscle. Their expression is strongly associated with the contractile phenotype of smooth muscle cells. Analysis of mice lacking both Smoothelins (Smtn-A/B−/− mice) previously revealed a critical role for Smoothelin-A in intestinal smooth muscle contraction. Here, we report on the generation and cardiovascular phenotype of mice lacking only Smoothelin-B (Smtn-B−/−). Methods and Results— Myograph studies revealed that the contractile capacity of the saphenous and femoral arteries was strongly reduced in Smtn-B−/− mice, regardless of the contractile agonist used to trigger contraction. Arteries from Smtn-A/B−/− compound mutant mice exhibited a similar contractile deficit. Smtn-B−/− arteries had a normal architecture and expressed normal levels of other smooth muscle cell–specific genes, including smooth muscle myosin heavy chain, α-smooth muscle actin, and smo...
-
Smoothelin in vascular smooth muscle cells
Trends in cardiovascular medicine, 2007Co-Authors: Guillaume J.j.m. Van Eys, Petra Niessen, Sander S RensenAbstract:Smoothelin-A and -B have only been found in fully differentiated contractile smooth muscle cells. They are increasingly used to monitor the smooth muscle cell differentiation process to a contractile or synthetic phenotype. Vascular-specific Smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis. Recently obtained data show that Smoothelin deficiency results in a considerable loss of contractile potential and hence in impaired smooth muscle function and suggest that Smoothelins are part of the contractile apparatus.
-
Contribution of serum response factor and myocardin to transcriptional regulation of Smoothelins
Cardiovascular Research, 2006Co-Authors: Sander S Rensen, Petra Niessen, Xiaochun Long, Pieter A Doevendans, Joseph M MianoAbstract: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.
-
Smoothelin-a is essential for functional intestinal smooth muscle contractility in mice.
Gastroenterology, 2005Co-Authors: Petra Niessen, Sander S Rensen, Pieter A Doevendans, Jan M. Van Deursen, Joris G. De Man, Ann De Laet, Jean-marie Vanderwinden, Thilo Wedel, Darren J. Baker, Marten H. HofkerAbstract:Background & Aims: In patients with chronic intestinal pseudo-obstruction, intestinal motility is disturbed by either nervous or myogenic aberrations. The cause of the myogenic form is unknown, but it is likely to originate in the contractile apparatus of the smooth muscle cells. Smoothelins are actin-binding proteins that are expressed abundantly in visceral (Smoothelin-A) and vascular (Smoothelin-B) smooth muscle. Experimental data indicate a role for Smoothelins in smooth muscle contraction. A Smoothelin-deficient mouse model may help to establish the role of Smoothelin-A in intestinal contraction and provide a model for myogenic chronic intestinal pseudo-obstruction. Methods: We used gene targeting to investigate the function of Smoothelin-A in intestinal tissues. By deletion of exons 18, 19, and 20 from the Smoothelin gene, the expression of both Smoothelin isoforms was disrupted. The effects of the deficiency were evaluated by pathologic and physiologic analyses. Results: In Smoothelin-A/B knockout mice, the intestine was fragile and less flexible compared with wild-type littermates. The circular and longitudinal muscle layers of the intestine were hypertrophic. Deficiency of Smoothelin-A led to irregular slow wave patterns and impaired contraction of intestinal smooth muscle, leading to hampered transport in vivo. This caused obstructions that provoked intestinal diverticulosis and occasionally intestinal rupture. Conclusions: Smoothelin-A is essential for functional contractility of intestinal smooth muscle. Hampered intestinal transit in Smoothelin-A/B knockout mice causes obstruction, starvation, and, ultimately, premature death. The pathology of mice lacking Smoothelin-A is reminiscent of that seen in patients with chronic intestinal pseudo-obstruction.
-
Biochemical evidence for interaction between Smoothelin and filamentous actin
Experimental cell research, 2004Co-Authors: Petra Niessen, Sander S Rensen, Sophie Clément, Lionel Fontao, Christine Chaponnier, Birgit E.j. Teunissen, Guillaume J.j.m. Van Eys, Giulio GabbianiAbstract:The two major isoforms of Smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs). Based on these findings, we hypothesized that Smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin. The interaction between Smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin [alpha-SKA]) was investigated using several in vitro assays. Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts. In rat embryonic fibroblasts, transfected Smoothelins-A and -B associated with stress fibers. In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled Smoothelin, showed binding of Smoothelin-A to actin filaments, but not to monomeric G-actin. A truncated Smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent. ELISA results showed that the binding of Smoothelin to actin has no significant isoform specificity. Our results indicate an interaction between Smoothelin and actin filaments. Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin.