Showing papers on "Smoothelin published in 2011"

Influence of Low Intensity Laser Irradiation on Isolated Human Adipose Derived Stem Cells Over 72 Hours and Their Differentiation Potential into Smooth Muscle Cells Using Retinoic Acid

Abstract: Human adipose derived stem cells (hADSCs), with their impressive differentiation potential, may be used in autologous cell therapy or grafting to replace damaged tissues. Low intensity laser irradiation (LILI) has been shown to influence the behaviour of various cells, including stem cells. This study aimed to investigate the effect of LILI on hADSCs 24, 48 or 72 h post-irradiation and their differentiation potential into smooth muscle cells (SMCs). hADSCs were exposed to a 636 nm diode laser at a fluence of 5 J/cm2. hADSCs were differentiated into SMCs using retinoic acid (RA). Morphology was assessed by inverted light and differential interference contrast (DIC) microscopy. Proliferation and viability of hADSCs was assessed by optical density (OD), Trypan blue staining and adenosine triphosphate (ATP) luminescence. Expression of stem cell markers, β1-integrin and Thy-1, and SMC markers, smooth muscle alpha actin (SM-αa), desmin, smooth muscle myosin heavy chain (SM-MHC) and smoothelin, was assessed by immunofluorescent staining and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Morphologically, hADSCs did not show any differences and there was an increase in viability and proliferation post-irradiation. Immunofluorescent staining showed expression of β1-integrin and Thy-1 72 h post-irradiation. RT-PCR results showed a down regulation of Thy-1 48 h post-irradiation. Differentiated SMCs were confirmed by morphology and expression of SMC markers. LILI at a wavelength of 636 nm and a fluence of 5 J/cm2 does not induce differentiation of isolated hADSCs over a 72 h period, and increases cellular viability and proliferation. hADSCs can be differentiated into SMCs within 14 days using RA.

Detection of smoothelin expression in the urinary bladder is strongly dependent on pretreatment conditions: a critical analysis with possible consequences for cancer staging.

Abstract: Distinguishing urinary bladder muscularis propria (MP) from muscularis mucosae (MM) is crucial in bladder cancer staging. Immunohistochemical staining for the smooth muscle-specific protein smoothelin has been reported to be a robust marker for MP. The aim of this study was to investigate how smoothelin immunostaining in the bladder varies with pretreatment techniques and if it can be used to discriminate between MM and MP. Immunohistochemistry (IHC) for smoothelin was performed on nontumoral sections from 18 cystectomy specimens using three different pretreatment protocols. The immunoreactivity of MM, MP and blood vessels was scored semiquantitatively. Staining intensity depended strongly on the different pretreatment protocols used. Heat-induced epitope retrieval (HIER) in alkaline buffer resulted in the strongest staining with a moderate or strong immunostaining of the MP in 18/18 (100%) of cases, but in 11/18 (61%), the MM was moderately or strongly stained. HIER in acidic buffer resulted in a suboptimal staining of the MP. Enzymatic pretreatment resulted in absent or weak staining. In conclusion, smoothelin IHC is strongly dependent on epitope retrieval, and smoothelin staining did not discriminate reliably between MP and MM with any of the tested pretreatment protocols.

Characterization of smooth muscle differentiation of purified human skeletal muscle-derived cells.

Abstract: The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle-derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low-serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT-PCR, Western blot and immunofluorescence studies. Western blot and immunofluorescence studies demonstrated the purified hMDCs cultured in SMIM for 4 weeks and expressed significant amount of smooth muscle myosin heavy chain (MHC) and α-smooth muscle actin (ASMA). The cells cultured in low-serum medium for 4 weeks also expressed ASMA, while the control group did not. RT-PCR analysis showed increased gene expression of smooth muscle markers, such as ASMA, Calponin, SM22, Caldesmon, Smoothelin and MHC when purified hMDCs were exposed to SMIM for 2 and 4 weeks when compared to the controls. In conclusion, we confirmed the smooth muscle differentiation capability of purified hMDCs. The gene expression of smooth muscle differentiation of purified hMDCs was characterized. These cells may be potential biomaterials for human tissue regeneration.

Expression of smoothelin and smooth muscle actin in the skin

Abstract: Introduction: Smoothelin is a cytoskeletal protein of differentiated smooth muscle cells with contractile capacity, distinguishing it from other smooth muscle proteins, such as smooth muscle actin (SMA). Objective: To evaluate the expression of smoothelin and SMA in the skin in order to establish specific localizations of smoothelin in smooth muscle cells with high contractile capacity and in the epithelial component of cutaneous adnexal structures. Methods: Immunohistochemical analysis (smoothelin and SMA) was performed in 18 patients with normal skin. Results: SMA was expressed by the vascular structures of superficial, deep, intermediate and adventitial plexuses, whereas smoothelin was specifically expressed in the cytoplasm of smooth muscle cells of the deepest vascular plexus and in no other plexus of the dermis. The hair erector muscle showed intense expression of smoothelin and SMA. Cells with nuclear expression of smoothelin and cytoplasmic expression of SMA were observed in the outer root sheath of the inferior portion of the hair follicles and intense cytoplasmic expression in cells of the dermal sheath to SMA. Conclusions: We report the first study of smoothelin expression in normal skin, which differentiates the superficial vascular plexus from the deep. The deep plexus comprises vessels with high contractile capacity, which is important for understanding dermal hemodynamics in normal skin and pathological processes. We suggest that the function of smoothelin in the outer root sheath may be to enhance the function of SMA, which has been related to mechanical stress. Smoothelin has not been studied in cutaneous pathology; however we believe it may be a marker specific for the diagnosis of leiomyomas and leiomyosarcomas of the skin. Also, smoothelin could differentiate arteriovenous malformations of cavernous hemangioma of the skin.

ADAMTS13—marker of contractile phenotype of arterial smooth muscle cells lost in benign nephrosclerosis

Abstract: Background Hypertensive nephrosclerosis alone and in combination with other renal diseases is a leading cause of terminal renal insufficiency Histologic lesions manifest as benign nephrosclerosis (bN) with arteriolar hyalinosis and later fibrosis Procoagulant micromilieus have been implicated in fibrosis Hyalinosis is considered to consist of plasma insudation possibly containing procoagulant factors like von Willebrand factor (VWF) Therefore, it is hypothesized that VWF cleaving protease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif, 13) is normally expressed by arteriolar vascular smooth muscle cells (VSMCs) and diminished in bN and that this reduction contributes to fibrosis in bN Methods ADAMTS13 expression was examined by immunohistochemistry and quantitative real-time polymerase chain reaction in VSMCs of various human organs Fifty-four specimens with and seven without bN were immunostained for ADAMTS13, VWF, CD61 and VSMC differentiation markers in arteriolar walls Results Expression of ADAMTS13 is confirmed in VSMCs In bN, ADAMTS13 immunostaining of arterial VSMCs correlated inversely with fibrotic but not hyalinotic lesions Smooth muscle myosin heavy chain showed an inverse correlation with hyalinotic, as opposed to fibrotic lesions of bN Smoothelin showed an inverse correlation with both hyalinotic and fibrotic lesions of bN VWF was absent in normal controls and hyalinotic lesions, but present exclusively in fibrotic lesions in 7/54 (13%) bN cases CD61 was absent in all arteriolar walls Conclusions The present results establish ADAMTS13 as a novel marker of contractile VSMCs that is retained in early hyalinotic bN but partially lost later in fibrotic bN Loss of ADAMTS13 and accumulation of VWF in fibrotic but not hyalinotic arteriolar walls could further propagate fibrosis in bN

Immunohistochemical staining for smoothelin in the duplicated versus the true muscularis mucosae of Barrett esophagus.

Abstract: Background: The muscularis mucosa underlying the metaplastic mucosa of Barrett esophagus is frequently duplicated, with an intervening layer of lamina propria between the superficial or neomuscularis mucosa (NMM) and the deep/true muscularis mucosa (TMM). This duplication causes difficulties with accurate staging of superficially invasive carcinoma in biopsy specimens and endoscopic mucosal resections (EMRs), as invasion underneath the superficial muscle layers may be mistaken for submucosal invasion. Mucosal resections or other ablative nonsurgical therapies can be curative in patients with esophageal intramucosal carcinoma, whereas patients with submucosal invasion are recommended for esophagectomy. Therefore, the accurate staging of such specimens is crucial. Smoothelin is a novel smooth muscle protein expressed only by fully differentiated smooth muscle cells and not by proliferative or noncontractile smooth muscle cells and fibroblasts. It has been suggested that in the bladder, immunohistochemistry for smoothelin may help separate hyperplastic muscularis mucosa from the true muscularis propria. We hypothesized that in the esophagus, immunohistochemistry for smoothelin would differentiate the NMM from the TMM. Design: Thirty cases of EMRs for Barrett esophagus-related neoplasia were retrieved from the archives of the pathology department, St Michael's Hospital. Immunohistochemical staining for smoothelin was performed to evaluate differential staining in the TMM versus NMM. Fifteen cases were stained for smooth muscle actin and smooth muscle myosin. The staining score was evaluated on a scale from 0 to 3 according to the percentage and intensity of staining. Results: Immunohistochemical staining results for smoothelin were as follows: the NMM showed weak focal staining (+ 1) in 23 of 30 cases (82%), and moderate staining (+ 2) in 7 of 30 cases (12%), and the TMM showed very strong and diffuse staining (+ 3) in 30 of 30 cases. No cases showed negative (0) staining in the NMM. With smooth muscle actin and myosin, strong and diffuse staining was observed with similar intensity in both the TMM and NMM in 15 of 15 cases. Conclusions: In our study, smoothelin staining in the NMM is significantly weaker than that seen in the true/deep muscularis mucosa. This pattern is similar to that reported for the muscularis mucosae of the urinary bladder. Although smoothelin can readily distinguish the 2 layers, its value might be limited by the need to simultaneously compare the 2 layers. Although this might be of use in EMR specimens in which both layers are visible, use in biopsies may be limited as the residual staining in the NMM may inhibit definitive evaluation. This issue may be resolved by the use of appropriate standard controls, individual optimization of the antibody, and the use of an automated digital assessment.

In response--a modified staining protocol for Smoothelin immunostaining.

Abstract: Dear Editor, In response to the article by Lindh et al. [1] in which they have discussed the utility of Smoothelin and have concluded that Smoothelin should be used cautiously, we would like to share our experience on the usefulness of Smoothelin staining. We performed a study assessing the utility of Smoothelin immunostaining in distinguishing muscularis mucosa (MM) from muscularis propria (MP) in 103 urinary bladder specimens, including 90 TURBTS and 13 cystectomies (results yet to be published). Immunohistochemical staining was performed using the Dako Autostainer Plus platform (Dako, Glostrup, Denmark) in order to validate the anti-Smoothelin primary antibody (clone R4A; Menarini Diagnostics, Wokingham, UK) for purposes of our study. Heat-induced epitope retrieval (HIER) was performed using the Dako PT link (Dako). Sections were incubated in the appropriate antigen retrieval buffer for 20 min at 97°C and were then allowed to cool to 65°C before transferring to distilled water. Two antigen retrieval buffers were used to establish the optimal HIER protocol. These HIER buffers were as follows:

Smoothelin in bladder and gastrointestinal tract again.

Abstract: Sir: We read with great interest the papers by Montani et al. and Bovio et al. on smoothelin expression in gastrointestinal tract and bladder, demonstrating its usefulness for distinguishing muscularis mucosae from the muscular propia layer. However, neither paper mentioned that, although smoothelin is a cytoplasmatic protein, its nuclear expression has been reported in smooth muscle cells of the gastrointestinal tract and in muscle tumours. The R4A clone used in both studies identifies two isoforms: smoothelin A for differentiated contractile smooth muscle cells of organs with muscle layers, and smoothelin B for those of blood vessels. Figures in the papers show vascular structures with slight-to-moderate smoothelin positivity, i.e. structures with incomplete differentiation and contractile capacity. These findings, which were not reported by Montani et al. and were noted only briefly by Bovio et al., warrant wider interpretation. In this regard, the use of smoothelin to evaluate vascular plexuses in the skin allowed our group to differentiate between superficial (smoothelin-negative) and deep (smoothelin-positive) vascular plexuses. We believe that the determination of smoothelin expression would allow vascular plexuses in the bladder and gastrointestinal tract to be characterized.