Showing papers on "Smoothelin published in 2010"

Shear stress modulation of smooth muscle cell marker genes in 2-D and 3-D depends on mechanotransduction by heparan sulfate proteoglycans and ERK1/2.

Abstract: Background During vascular injury, vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms. Methodology/principal findings Exposure to 8 dyn/cm(2) laminar flow shear stress (2-dimensional, 2-D) for 15 h significantly reduced expression of alpha-smooth muscle actin (alpha-SMA), smooth muscle protein 22 (SM22), SM myosin heavy chain (SM-MHC), smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH(2)O, approximately 0.05 dyn/cm(2), 3-D), and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of alpha-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor) and heparinase III (an enzyme to cleave heparan sulfate) significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of alpha-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs). Conclusions/significance The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation.

Clinical utility of immunohistochemistry in the diagnoses of urinary bladder neoplasia.

Abstract: Urothelial carcinomas demonstrate diverse morphologic and immunologic features that frequently lead to diagnostic challenges. Recent advances have identified a number of immunohistochemical stains that, when used in the context of a panel, can be a valuable tool in properly classifying primary urothelial carcinoma and carcinomas secondarily involving the urinary bladder. In addition, new biomarkers prove helpful in the staging of bladder carcinoma. In this article, we review the clinical utility of immunohistochemistry in a series of diagnostic scenarios, including flat urothelial lesions with atypia, rare variants of urothelial carcinoma, primary adenocarcinoma versus secondary colorectal tumors, distinguishing prostate from urothelial carcinoma, and the utility of smoothelin in staging bladder carcinoma. Emphasis is placed on panels of commonly used biomarkers to establish diagnoses.

Antiproliferative and apoptotic effects of the herbal agent Pygeum africanum on cultured prostate stromal cells from patients with benign prostatic hyperplasia (BPH).

Abstract: BACKGROUND. Previous reports show that the herbal agent Pygeum africanum (PA) used to treat benign prostatic hyperplasia (BPH) inhibits proliferation of prostate stromal cells from BPH tissues. To determine underlying mechanisms, we compared proliferative and apoptotic responses to PA between BPH and non-BPH prostate stromal cells with a focus on the specific reaction displayed by stromal cell subsets. An interaction of PA with growth factors and hormones was also investigated. METHODS. Primary prostate stromal cells from BPH/LUTS patients undergoing open prostatectomy (n ¼ 3) and patients without benign prostatic hyperplasia (BPH) undergoing cystectomy (n ¼ 3) were treated with PA. Cells were characterized by immunofluorescence. Sensitivity to PA was determined using proliferation assays. Apoptosis, transforming growth factor B1 (TGFB1), fibroblast growth factor 2 (FGF2), vimentin, a smooth muscle actin (aSMA), and smoothelin expression were examined after PA treatment. Cell immunophenotype and proliferation were tested after incubating cells with PA plus either FGF2, TGFB1, vascular endothelial growth factor (VEGF), dihydrotestosterone (DHT) or 17b-estradiol (E2). RESULTS. Antiproliferative potency and apoptosis induced by PA on stromal cells were increased in BPH versus non-BPH cells. Apoptosis targeted aSMAþ cells, more abundant in BPH cells. Downregulation of TGFB1 expression was induced by PA. FGF2 increased cells sensitivity to PA. Incubation with other mitogenic factors like VEGF, DHT, and E2 decreased sensitivity to PA. Both TGFB1 and E2 blocked the antiproliferative activity of PA. CONCLUSIONS. Results suggest that PA is antiproliferative and apoptotic on proliferative prostate fibroblasts and myofibroblasts but not on smooth muscle cells. Mechanisms of action include TGFB1 downregulation and inhibition of FGF2 specific signaling. Prostate 70: 1044– 1053, 2010. # 2010 Wiley-Liss, Inc.

Cyclosporine up-regulates Kruppel-like factor-4 (KLF4) in vascular smooth muscle cells and drives phenotypic modulation in vivo.

Abstract: Cyclosporine A (CSA, calcineurin inhibitor) has been shown to block both vascular smooth muscle cell (VSMC) proliferation in cell culture and vessel neointimal formation following injury in vivo. The purpose of this study was to determine molecular and pathological effects of CSA on VSMCs. Using real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunofluorescence microscopy, we show that CSA up-regulated the expression of Kruppel-like factor-4 (KLF4) in VSMCs. KLF4 plays a key role in regulating VSMC phenotypic modulation. KLF4 antagonizes proliferation, facilitates migration, and down-regulates VSMC differentiation marker gene expression. We show that the VSMC differentiation marker genes smooth muscle α-actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD) are all down-regulated by CSA in VSMC monoculture, whereas cyclin-dependent kinase inhibitor-1A (CDKN1A) and matrix metalloproteinase-3 (MMP3) are up-regulated. CSA did not affect the abundance of the VSMC microRNA (MIR) markers MIR143 and MIR145. Administration of CSA to rat carotid artery in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, and smooth muscle myosin heavy chain (MYH11) mRNA levels. The tumor suppressor genes KLF4, p53, and CDKN1A, however, were up-regulated, as well as MMP3, MMP9, and collagen-VIII. CSA-treated arteries showed remarkable remodeling, including breakdown of the internal elastic lamina and reorientation of VSMCs, as well as increased KLF4 immunostaining in VSMCs and endothelial cells. Altogether, these data show that cyclosporin up-regulates KLF4 expression and promotes phenotypic modulation of VSMCs.

Molecular and functional effects of organismal ageing on smooth muscle cells derived from bone marrow mesenchymal stem cells

Abstract: Aims Bone marrow-derived smooth muscle cells (BM-SMCs) have high potential as an autologous cell source of vascular progenitors but normal cell function and turnover frequency may decline with age. In this study we set out to study the effects of organismal ageing on the molecular and functional properties of BM-SMCs. Methods and results To address this issue, we employed a smooth muscle α-actin promoter (αSMA) driving expression of enhanced green fluorescence protein (EGFP) to isolate SMCs from bone marrow of neonatal (nBM-SMCs) or adult (aBM-SMCs) sheep and examined their proliferation potential and contractility. Compared with nBM-SMCs, aBM-SMCs exhibited lower clonogenicity and proliferation potential that could be improved significantly by addition of basic fibroblast growth factor. Vascular constructs from aBM-SMCs showed reduced ability to generate force and contract fibrin hydrogels and this function could be partially restored by addition of transforming growth factor-β1. They also exhibited lower receptor- and non-receptor-mediated vascular contractility and mechanical strength, which was comparable to that of tissue constructs prepared with vascular SMCs from neonatal umbilical veins. In agreement with the contractile properties and mechanical strength of vascular constructs, aBM-SMCs displayed significantly lower expression of αSMA, smoothelin, desmin, type I collagen, and tropoelastin transcripts compared with nBM-SMCs. Conclusion Understanding the effects of organismal ageing on BM-SMCs and the properties of the resulting vascular constructs may lead to innovative ways to facilitate application of these cells in the treatment of cardiovascular disease which is especially prevalent in the elderly.

Pitfalls in the use of smoothelin to identify muscularis propria invasion by urothelial carcinoma.

Abstract: Studies predominantly performed on cystectomy specimens have shown that antibodies against smoothelin can distinguish between muscularis mucosae (MM) (negative or weak stain) and muscularis propria (MP) (strong stain). However, studies on diagnostically difficult (transurethral resection) specimens have not been performed. We studied 34 transurethral resection cases where outside pathologists questioned the presence of MP invasion. Upon expert review of the H&E slides, there was no MP invasion in 18 cases. Smoothelin in MM was negative in 8/18 (44%), weakly positive (1+) in 5/18 (28%), moderately positive (2+) in 4/18 (22%), and moderately/strongly (2-3+) positive in 1/18 (6%). Smoothelin in uninvolved MP present in 8 cases was: 2+ in 2/8 (25%) and 3+in 6/8 (75%). Smoothelin expression in MM was weaker than in MP in 7/8 (88%) cases where both were present. Of 16 tumors with MP invasion, smoothelin in involved MP was: 1+ in 1/16 (6%), 2+ in 3/16 (19%), 2 to 3+ in 9/16 (56%), and 3+ in 3/16 (19%). Smoothelin expression in concurrent uninvolved MP was similar. Our data confirm the relatively distinct staining pattern of smoothelin between MM and MP. However, due to the overlap of intensity between MM and MP, caution should be maintained while using smoothelin immunohistochemistry as a diagnostic tool for MP invasion.

Diagnostic use of antibody to smoothelin in the recognition of muscularis propria in transurethral resection of urinary bladder tumor (TURBT) specimens.

Abstract: 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.

Oxidized low density lipoprotein-induced transdifferentiation of bone marrow-derived smooth muscle-like cells into foam-like cells in vitro.

Abstract: Oxidized-low density lipoprotein (ox-LDL) is believed to contribute to atherogenesis in part by being taken up into smooth muscle cells (SMC) via specific scavenger receptors; however, it is not clear whether ox-LDL receptor(s) are expressed in bone marrow-derived smooth muscle-like cells (SMLCs) and whether they play a role in the process of SMLC development. Therefore, we examined the ox-LDL-induced transdifferentiation of SMLCs that is mediated by lectin-like ox-LDL receptor-1 (LOX-1). Smooth muscle progenitor cells (SMPCs) from bone marrow mesenchymal stem cells (BMSCs) were isolated using a tissue-specific promoter sorting method with a mouse SM22_ promoter (_480 bp)/green fluorescent protein recombinant plasmid. The SMPCs were myocardin+CD105+KDR+CD45(-)CD34(-), but did not express SMC-specific markers alpha-smooth muscle actin (alpha-SMA), SM22, smooth muscle myosin heavy chain (SM-MHC) and smoothelin. After long-term culture with platelet-derived growth factor-BB (PDGF-BB), SMPCs expressed alpha-SMA, SM22 and SM-MHC and differentiated into SMLCs. When SMLCs were incubated with different concentrations of ox-LDL, LOX-1 expression on the surface of SMLCs gradually increased with the increase of the ox-LDL concentration, but myocardin and SMC-specific marker genes decreased, accordingly. Furthermore, receptor-mediated endocytosis was enhanced and lipid droplets accumulated in the cytoplasm of SMLCs. A subpopulation of myocardin+CD105+KDR+CD45(-)CD34(-) SMPCs exist in BMSCs that can differentiate into SMLCs under induction with PDGF-BB. Moreover, LOX-1 contributes to the ox-LDL-induced transdifferentiation of bone marrow-derived SMLCs into foam-like cells.

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

Abstract: Introduction: Vasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts. Methods: The transgenic mouse strain Tβ RIIΔk-fib is characterized by balanced ligand-dependent upregulation of TGF-β signaling. Aortic and cardiac tissues were examined with histologic, biochemical, and isolated organ bath studies. Vascular and perivascular architecture was examined by hematoxylin and eosin (H&E) and special stains including immunostaining for TGF-β1 and phospho-Smad2/3 (pSmad2/3). Confirmatory aortic smooth muscle cell proliferation, phenotype, and functional assays, including signaling responses to exogenous TGF-β and endothelin-1, were performed. Aortic ring contractile responses to direct and receptor-mediated stimulation were assessed. Results: Aortic ring contractility and relaxation were diminished compared with wild-type controls, and this was associated with aortic adventitial fibrosis confirmed histologically and with Sircol assay. TGF-β1 and pSmad 2/3 expression was increased in the adventitia and smooth muscle layer of the aorta. Aortic smooth muscle cells from transgenic animals showed significant upregulation of TGF-β- responsive genes important for cytoskeletal function, such as transgelin and smoothelin, which were then resistant to further stimulation with exogenous TGF-β1. These cells promoted significantly more contraction of free floating type I collagen lattices when compared with the wildtype, but were again resistant to exogenous TGF-β1 stimulation. Aortic ring responses to receptor-mediated contraction were reduced in the transgenic animals. Specifically, bosentan reduced endothelin-mediated contraction in wild-type animals, but had no effect in transgenic animals, and endothelin axis gene expression was altered in transgenic animals. Transgenic mice developed cardiac fibrosis. Conclusions: The histologic, biochemical, and functional phenotype of this transgenic mouse model of scleroderma offers insight into the altered biomechanical properties previously reported for large elastic arteries in human SSc and suggests a role for perturbed TGF-β and endothelin activity in this process.

Smoothelin immunohistochemistry is a useful adjunct for assessing muscularis propria invasion in bladder carcinoma.

Abstract: Bovio I M, Al-Quran S Z, Rosser C J, Algood C B, Drew P A & Allan R W (2010) Histopathology 56, 951–956 Smoothelin immunohistochemistry is a useful adjunct for assessing muscularis propria invasion in bladder carcinoma Aims: To prospectively evaluate the utility of smoothelin immunohistochemical expression for the evaluation of muscularis propria (MP) in diagnostic transurethral resection of bladder tumour (TURBT) specimens and cystectomies. Methods and results: Smoothelin immunohistochemistry was performed on a total of 26 TURBT and cystectomy specimens. All but two cases (24/26) demonstrated strong (3+) or moderate (2+) immunoreactivity of the MP with smoothelin. Muscularis mucosae (MM) never displayed strong (3+) reactivity, and in only one case did the MM have moderate (2+) reactivity; in this case the MP had strong (3+) reactivity. MM intensity mirrored the intensity of reactivity of blood vessels in all cases (26/26). Using moderate or strong immunoreactivity as a cut-off, smoothelin had a sensitivity of 92% for detecting MP and a specificity of 97% for distinguishing between MP and MM. In all unequivocal MP-invasive and lamina proporia-invasive cases by haematoxylin and eosin (H&E), smoothelin immunohistochemistry confirmed the original light microscopic diagnosis. In four cases in which there was equivocal MP involvement by H&E, smoothelin helped establish MP invasion. Conclusions: Smoothelin immunohistochemistry has diagnostic utility in the evaluation of MP invasion in urothelial carcinoma. Smoothelin could be used as an adjunct to traditional H&E-stained light microscopy and may help reduce the number of equivocal diagnoses.

Both nongenomic and genomic effects are involved in estradiol's enhancing the phenotype of smooth muscle cells in cultured prostate stromal cells.

Abstract: BACKGROUND Stromal smooth muscle cells (SMCs) play an important role in the pathogenesis and clinical symptom of benign prostatic hyperplasia. We had reported that estrogen enhances the phenotype of SMC in cultured prostate stromal cells (PRSCs). Here we further investigate the mechanism by which estrogen affects the differentiation of PRSCs. METHODS Primary cultured PRSCs were stimulated with E2 or BSA-E2. The mRNA level of SMC-specific genes, smoothelin, and SM-MHC were measured by qRT-PCR. The SM-MHC protein was measured by Western blot. The mRNA and protein levels of TGF-β1 were measured by qRT-PCR and ELISA. The MAPK inhibitor PD98059, the estrogen receptor antagonist ICI182,780 and neutralizing antibody to TGF-β1 were used to reveal the mechanism of estrogen effect. RESULTS E2 and BSA-E2 significantly up-regulate the expression of SMC-specific genes in PRSCs. Both forms of estrogen could increase the expression of TGF-β1, which can be blocked by pre-treating with PD98059. Moreover, PD98059 and TGF-β1 neutralizing antibody could abrogate the effect of BSA-E2 on cell differentiation. However, they could only inhibit part of E2-induced SMC phenotype enhancement. ICI182,780 could partially suppress the pro-differentiation effect of E2 but had no influence on the effect of BSA-E2. Combined treatment with ICI182,780 and PD98059 can completely abrogate the effect of E2. CONCLUSIONS Estrogen could promote the expression of TGF-β1 in PRSCs through nongenomic activation of MAPK pathway, and in turn enhance the SMC phenotype. Besides for this nongenomic effect, estrogen can also enhance the SMC phenotype through classical genomic action. Prostate 70: 317–332, 2010. © 2009 Wiley-Liss, Inc.

Single and multiple CH (calponin homology) domain containing multidomain proteins in Dictyostelium discoideum: an inventory.

Abstract: We present an inventory of single or multiple calponin homology (CH) domain containing proteins of Dictyostelium discoideum. A multiple alignment and a phylogenetic tree of all 60 CH domains found in 36 proteins showed that most CH domains can be assigned to one of 6 types. We have then distributed the proteins into several classes according to the type and arrangement of the CH domains. Most proteins belong to the class of ABD (actin-binding domain)-forming CH tandems (CH1–CH2) of the α-actinin and fimbrin families or to the class of CH3 domain-bearing proteins. There are a few examples of proteins with a single CH1 or CH2 domain, one with a CH1–CH1 doublet and a single representative of the CHe class of microtubule-binding proteins. A comparison with CH domain proteins in Homo sapiens suggests that while the individual domains are available in both species, the existence of identical multidomain proteins in toto is rare. Fimbrin 1, α-actinin and EB1 appear as perfect orthologs in both species, whereas filamin and interaptin may represent ancestral forms of human filamin and nesprins. In four more cases (NAV/Unc-53-, smoothelin-, transgelin- and Gas2-related proteins) functional data are needed in order to establish a potential relationship with a human counterpart. Although extensive data exist for a few of the D. discoideum CH proteins, most remain to be characterized and our analysis may help predicting some of their properties.

Smoothelin is a specific and robust marker for distinction of muscularis propria and muscularis mucosae in the gastrointestinal tract.

Abstract: AIMS: As tumour specimens and biopsy specimens become smaller, recognition of anatomical structures relevant for staging is increasingly challenging. So far no marker is known that reliably discriminates between muscularis propria (MP) and muscularis mucosae (MM) of the gastrointestinal tract. Recently, smoothelin expression has been shown to differ in MP and MM of the urinary bladder. We aimed to analyse the expression of smoothelin in the gastrointestinal tract in MP and MM in order to define a novel diagnostic tool to identify MM bundles. METHODS AND RESULTS: The expression of smoothelin was analysed immunohistochemically in comparison with alpha-smooth muscle actin (alpha-SMA) in specimens from colon, stomach and oesophagus (n = 107). In contrast to alpha-SMA, which equally stained MM and MP, absent or significantly weaker smoothelin expression was found in MM was found, which was particularly valid in colon and gastric specimens. CONCLUSIONS: The combination of smoothelin and SMA represents a robust marker to discriminate MM from MP in the gastrointestinal tract.

Singlet CH domain containing human multidomain proteins: an inventory

Abstract: The actin cytoskeleton presents the basic force in processes such as cytokinesis, endocytosis, vesicular trafficking and cell migration. Here, we list 30 human singlet CH (calpononin homology/actin binding) containing multidomain molecules, each encoded by one gene. We show the domain distributions as given by the SMART program. These mosaic proteins organize geographically the placement of selected proteins in proximity within the cell. In most instances, their precise location, their actin binding capacity by way of the singlet CH (or by other domains?) and their physiological functions need further elucidation. A dendrogram based solely on the relationship for the human singlet CH domains (in terms of AA sequences) for the various molecules that possess the domain, implies that the singlet descended from a common ancestor which in turn sprouted three main branches of protein products. Each branch bifurcated multiple times thus accounting for a cornucopia of products. Wherever, additional (unassigned), highly homologous regions exist in related proteins (e.g., in LIM and LMO7 or in Tangerin and EH/BP1), these unrecognized domain regions await assignment as specific functional domains. Frequently genes coding multidomain proteins duplicated. The varying modular nature within multidomain proteins should have accelerated evolutionary changes to a degree not feasible to achieve by means of mere post-duplication mutational changes.