Showing papers on "Smoothelin published in 2015"

Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

Abstract: Objective—Marfan’s syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan’s syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Approach and Results—Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of m...

Guidelines for the Isolation and Characterization of Murine Vascular Smooth Muscle Cells. A Report from the International Society of Cardiovascular Translational Research

Abstract: Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.

Differentiated adipose-derived stem cells for bladder bioengineering.

Abstract: Objective. The aim of this study was to characterize and differentiate adipose-derived stem cells (ADSCs) to functional smooth muscle cells (SMCs) as an alternative cell source for bladder engineering. Materials and methods. Rat ADSCs were differentiated into SMCs for 1-6 weeks using induction medium. The changes in contractile genes and protein expression were investigated by real-time polymerase chain reaction, fluorescence-activated cell sorting and Western blot at different time-points. Spontaneous and carbachol-induced contractions of engineered SMC tissue at different stages were investigated to define the optimal duration of induction. Results. ADSCs differentiated into SMCs lost their capacity for expansion and their contractile phenotype, changing to a synthetic phenotype over time. Highest levels of calponin, smoothelin and MyH11 expression were observed in ADSCs induced for 3 weeks. Cells acquired typical SMC morphology when contractile proteins were expressed. However, SMC morphology w...

Inhibitory effect of Puerariae radix flavones on platelet-derived growth factor-BB-induced proliferation of vascular smooth muscle cells via PI3K and ERK pathways

Abstract: Abnormal proliferation of vascular smooth muscle cells (VSMCs) results in intimal thickening of the aorta, which may lead to arteriosclerosis. Therefore, VSMC antiproliferative agents may be efficient in the prevention and treatment of arteriosclerosis. Puerariae radix (PR) is the dried root of Pueraria lobata Ohwi or Pueraria thomsonii Benth. Flavones are the main components of PR and have been shown to have a protective effect on vascular disorders in traditional Chinese medicine treatments. However, the underlying molecular mechanism remains unclear. The aim of the present study was to explore the effect of PR flavone (PRF) on platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation. PDGF-BB (25 ng/ml) and different doses of PRF (10, 50, 100 and 200 ng/ml) were used to treat VSMCs. The results revealed that PRF notably inhibited the PDGF-BB-induced VSMC proliferation and induced a cell cycle arrest at growth 1 phase of the cell cycle. In addition, cell cycle-associated proteins, including cyclin D1, proliferating cell nuclear antigen and cyclin-dependent kinase 4, were found to be downregulated. Furthermore, PRF inhibited the PDGF-BB-stimulated downregulation of VSMC markers, including α-smooth muscle actin, desmin and smoothelin. PDGF-BB upregulated the phosphorylation levels of phosphatidylinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK), which are associated with cell proliferation; however, these were decreased following PRF treatment. These observations indicated that PRF had a suppressive effect on PDGF-BB-induced VSMC proliferation by inhibiting PI3K and ERK pathways.

In situ Analysis of Smoothelin‐like 1 and Calmodulin Interactions in Smooth Muscle Cells by Proximity Ligation

Abstract: The smoothelin-like 1 (SMTNL1) protein is the newest member of the smoothelin family of muscle proteins. Two calmodulin (CaM)-binding domains (CBD1 for Ca-CaM; CBD2 for apo-CaM) have been described for the SMTNL1 protein using in vitro assays. We now demonstrate in situ associations of SMTNL1 and CaM in A7r5 smooth muscle cells using the proximity ligation assay (PLA). We quantified CaM-SMTNL1 proximity events accurately after taking into account variations in protein expression levels. The refined method allows quantification of in situ proximity after transient transfection with an associated error of <10%. The proximity of SMTNL1 and CaM in A7r5 cells could be reduced by scrambling the amino acid sequence and mutation of large hydrophobic amino acids of CBD1. The truncation of CBD2 did not influence SMTNL1 proximity to CaM. Ultimately, we conclude that SMTNL1 forms complex interactions with CaM in smooth muscle cells, with a role for CBD1 and possibly the intrinsically disordered region.

Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy

Abstract: The igf1 gene is alternatively spliced as IGF-IEa and IGF-IEc variants in humans. In fibrostenotic Crohn's disease, the fibrogenic cytokine TGF-β1 induces IGF-IEa expression and IGF-I production in intestinal smooth muscle and results in muscle hyperplasia and collagen I production that contribute to stricture formation. Mechano-growth factor (MGF) derived from IGF-IEc induces skeletal and cardiac muscle hypertrophy following stress. We hypothesized that increased IGF-IEc expression and MGF production mediated smooth muscle hypertrophy also characteristic of fibrostenotic Crohn's disease. IGF-IEc transcripts and MGF protein were increased in muscle cells isolated from fibrostenotic intestine under regulation by endogenous TGF-β1. Erk5 and MEF2C were phosphorylated in vivo in fibrostenotic muscle; both were phosphorylated and colocalized to nucleus in response to synthetic MGF in vitro. Smooth muscle-specific protein expression of α-smooth muscle actin, γ-smooth muscle actin, and smoothelin was increased in affected intestine. Erk5 inhibition or MEF2C siRNA blocked smooth muscle-specific gene expression and hypertrophy induced by synthetic MGF. Conditioned media of cultured fibrostenotic muscle induced muscle hypertrophy that was inhibited by immunoneutralization of endogenous MGF or pro-IGF-IEc. The results indicate that TGF-β1-dependent IGF-IEc expression and MGF production in patients with fibrostenotic Crohn's disease regulates smooth muscle cell hypertrophy a critical factor that contributes to intestinal stricture formation.

Antigenic profile of muscularis mucosae and muscularis propria of the urinary bladder.

Abstract: Background: Differentiation between the muscularis mucosae (MM) and muscularispropria (MP) of the bladder remains challenging. Objective: To identify MM- and MP-specific antigens that could be of potential value for staging of urothelialcarcinomain a pilot study. Method: The expression of 12 protein antigens in 11 human bladder specimens was examined. There were 5 post radical cystectomy specimens and 6 normal bladder autopsy specimens. Antibodies against actin, caldesmon, type IV collagen, cytokeratin, desmin, elastin, fibronectin, filamin, laminin, miotilin, smoothelin, and vimentin were used. Slides were stained with immunohistochemical reagents and assessed using light microscopy. The intensity of the immune reaction within MM and MP was evaluated in a four-level scale as negative, weakly, moderately, or strongly positive. Results: The presence of MM was noticed in 63.6% of the specimens.The expression of desmin, filamin, and smoothelin was stronger within MP compared to MM in all cases. Stronger reaction with anti-type IV collagen antibodies was noticed within MP in 80% of the cases. In the whole study group, the expression of vimentin was stronger within MM than MP. Conclusions: MM and MP cells are of different antigenic characteristics. This can be used in the microscopic diagnostics of selected cases. The results need to be validated in a series of specimens from transurethral resection.

External vibration enhances macromolecular crowding for construction of aligned three-dimensional collagen fibril scaffolds

Abstract: There are many techniques for preparing two-dimensional aligned fibril matrices. However, the critical problem associated with these techniques is the destruction of the native structure (e.g., the α-helix) of the proteins. Moreover, most of these techniques cannot create a three-dimensional (3D), aligned reconstituted collagen fibril matrix in one step. In this study, we used a simple device composed of a pneumatic membrane that generates a tunable vibration frequency to apply physical stimulation to fabricate a 3D, aligned collagen fibril matrix with the characteristic D-period structure of collagen in one step. Using second harmonic images, we demonstrated that the aligned, reconstituted collagen fibrils preserve the native collagen D-period structure. The average angular deviation of fibril alignment was reduced to 25.01 ± 4.2° compared with the 39.7 ± 2.19° of alignment observed for the randomly distributed fibril matrix. In addition, the ultimate tensile strength of the aligned matrix when force was applied in the direction parallel to the fiber orientation was higher than that of the randomly oriented matrix. The aligned reconstituted collagen fibril matrix also enhanced the expression of smoothelin (a specific marker of contractile phenotype) of thoracic aortic smooth muscle cell (A7r5) relative to the randomly distributed collagen fibril matrix.

Histopathological Evidence for Irradiation Angiopathy in Head and Neck Cancer

Abstract: 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.