Smooth Muscle-specific Alpha-actin Research Articles

Su1862 Smooth Muscle Specific Alpha-Actin Plays a Critical Role in the Regulation of Mechanical Stress-Induced Gene Expression in the Colon

Su1862 Smooth Muscle Specific Alpha-Actin Plays a Critical Role in the Regulation of Mechanical Stress-Induced Gene Expression in the Colon

Fetal pulmonary vascular remodeling in a rat model induced by hypoxia and indomethacin

Objective. This study sought to determine the effect of combined treatment of hypoxia plus indomethacin on pulmonary vascular remodeling in fetal rats.Methods. Hypoxia and indomethacin were used to treat pregnant rats during 19–21 days of gestation. The adventitia, media, and intima of pulmonary arteries from fetal rats were assessed. Western blots were used for determining the abundance of smooth muscle specific alpha-actin protein (α-SMA), elastin, and endothelial nitric oxide synthase (eNOS) in lung tissues. Plasma brain-type natriuretic peptide (BNP) levels, reflecting the increased right ventricular load or pulmonary arterial pressure, were detected.Results. The ratio of left ventricular free wall plus septum to right ventricular weight significantly increased in hypoxia plus indomethacin-treated group. The medial thickness percentage of pulmonary arteries of < 100 μm and ≥100 μm in diameter from hypoxia plus indomethacin-treated group was higher than that from control or single treatment group. Vascular elastin area percentage and immunostaining density of eNOS from the combined-treated group were higher than other groups. The relative abundance of α-SMA, elastin, and eNOS and plasma BNP levels in hypoxia plus indomethacin-treated group also significantly increased compared with other groups.Conclusions. Hypoxia and indomethacin had synergistic effect on fetal pulmonary vascular remodeling. This rat model induced by combined treatments can mimic human persistent pulmonary hypertension of the newborn.

Monotypic epithelioid angiomyolipoma of the adrenal gland: an unusual site for a rare extrarenal tumor

Epithelioid angiomyolipoma (AML) is an uncommon renal mesenchymal tumor with malignant potential and is frequently associated with tuberous sclerosis. Extrarenal AMLs are rare, and to the best of our knowledge, this is the first reported case of a primary monotypic epithelioid AML of adrenal gland in a patient without evidence of tuberous sclerosis. The patient is a 42-year old man who presented with retroperitoneal hemorrhage resulting from spontaneous rupture of adrenal mass. Histologically, the tumor showed a prominent component of epithelioid smooth muscle cells with slightly pleomorphic nuclei, sometimes with prominent nucleoli and eosinophilic cytoplasm resembling oncocytic tumors. Epithelioid cells were positive for melanoma (HMB45 and positive MelanA) and smooth muscle markers (alpha-smooth muscle-specific actin), but not for epithelial markers (cytokeratin, EMA). Differential diagnosis from renal cell carcinoma, adrenal gland carcinoma, and metastatic carcinoma is often challenging because of its epithelioid morphology. Because primary and secondary malignant tumors are much more common and aggressive neoplasms, establishing the correct diagnosis has important therapeutic and prognostic implications.

Requirement of an Intermediate Gene Expression for Biphasic ERK1/2 Activation in Thrombin-stimulated Vascular Smooth Muscle Cells

The expression of contractile proteins in vascular smooth muscle cells is controlled by still poorly defined mechanisms. A thrombin-inducible expression of smooth muscle-specific alpha-actin and myosin heavy chain requires transactivation of the epidermal growth factor (EGF) receptor and a biphasic activation of ERK1/2. Here we demonstrate that the sustained second phase of ERK1/2 phosphorylation requires de novo RNA and protein synthesis. Depolymerization of the actin cytoskeleton by cytochalasin D or disruption of transit between the endoplasmic reticulum and the Golgi apparatus by brefeldin A prevented the second phase of ERK1/2 phosphorylation. We thus conclude that synthesis and trafficking of a plasma membrane-resident protein may be critical intermediates. Analysis of the expression of protease-activated receptor 1, heparin-binding EGF (HB-EGF), and the EGF receptor revealed that pro-HB-EGF is significantly up-regulated upon thrombin stimulation. The kinetic of HB-EGF expression closely matched that of the second phase of ERK1/2 phosphorylation. Because inhibition of matrix metalloproteases or of the EGF receptor strongly attenuated the late phase of ERK1/2 phosphorylation, the second phase of ERK1/2 activation is primarily relayed by shedding of EGF receptor ligands. The small interfering RNA-mediated knockdown of HB-EGF expression confirmed an important role of HB-EGF expression in triggering the second phase of ERK1/2 activation. Confocal imaging of a yellow fluorescent protein-tagged HB-EGF construct demonstrates the rapid plasma membrane integration of the newly synthesized protein. These data imply that the hormonal control of contractile protein expression relies on an intermediate HB-EGF expression to sustain the signaling strength within the Ras/Raf/MEK/ERK cascade.

Upregulation of Protein Kinase Cδ in Vascular Smooth Muscle Cells Promotes Inflammation in Abdominal Aortic Aneurysm

The development of abdominal aortic aneurysms (AAAs) involves a complex interplay of extracellular matrix degradation, inflammation, and apoptosis. We have previously shown that protein kinase Cdelta (PKCdelta) plays a critical role in vascular smooth muscle cell (vSMC) apoptosis in the setting of oxidative stresses. Here, we show that PKCdelta is also involved in the signaling that draws inflammatory cells to aneurismal tissue.Immunostaining for monocyte chemotactic factor (MCP)-1 and PKCdelta was performed on paraffin-fixed arterial sections. Enzyme-linked immunosorbent assay to detect MCP-1 produced by vSMCs was performed on media from cultured rat A10 cells after cytokine induction with or without the PKCdelta-specific inhibitor rottlerin. Migration of isolated lymphocytes was evaluated in response to media from activated A10 cells.Human AAAs show widespread and elevated expression of PKCdelta that is not seen in normal aortic tissues. Cytokine stimulation of cultured vSMCs induced vigorous production of the key chemotactant MCP-1, the expression of which was PKCdelta dependent. Stimulated vSMCs were capable of inducing the migration of leukocytes, and this effect was also dependent on PKCdelta activity. Staining of human AAA tissue for MCP-1 showed an expression pattern that was identical to that of PKCdelta and smooth muscle specific alpha-actin.PKCdelta is widely expressed in human AAA vessel walls and mediates MCP-1 expression by vSMCs, which could contribute to the inflammatory process. These findings, coupled with earlier studies of PKCdelta, suggest that PKCdelta plays a central role in the pathogenesis of AAAs and may be a potential target for future therapies.

Enhanced smooth muscle cell adhesion and proliferation on protein‐modified polycaprolactone‐based copolymers

Smooth muscle cells (SMC) were cultured for up to 6 days on copolymer films fabricated from a PCL-PEG-PCL block copolymer or P(epsilon-CL-co-D,L-LA)-PEG-P(epsilon-CL-co-D,L-LA), named P(100/0) and P(70/30), respectively. The films were modified by aminolysis using 1,6-hexanediamine, and fibronectin, fibrinogen, or fibrin layers were subsequently immobilized by physisorption or by covalent coupling using imidoester chemistry. Immobilization of all the tested proteins resulted in significantly enhanced cell adhesion on these polymers. Moreover, we found that covalently immobilized proteins supported significantly greater cell proliferation than physisorbed proteins over 6 days. SMC cultured on P(100/0) films modified by covalently attached fibronectin or fibrin layers proliferated at a rate comparable to that observed on control tissue culture polystyrene. The proposed surface modification schemes were much less efficient in improving cell attachment and proliferation on P(70/30) films. However, prewetting P(70/30) with a phosphate buffer prior to aminolysis significantly improved cell numbers following immobilization of fibronectin. Immunostaining of smooth muscle-specific alpha-actin of SMC grown on protein-modified P(100/0) 8 h and 48 h after cell seeding, confirmed preserved SMC phenotype on all modified surfaces.

76: Atrial natriuretic peptide (ANP) production and receptors in the human uterus and effect on myometrial relaxation

Natriuretic peptides induce uterine relaxation. We identify human myometrial ANP production, ANP receptor expression, and physiology in human tissue and primary cultured cells. In an IRB-approved study, gravid human myometrium was obtained from cesarean section patients (21 to 41 weeks gestation). Isometric tension studies were performed on 2 ×10 mm strips of uterine tissue. ANP dose-curve (beginning at 3 pM) was determined. Western blot analysis was performed on uterine tissue samples for ANP receptors (A, B, and C subtypes). Radioimmunoassay (RIA)measured ANP, cAMP, and cGMP in myometrial tissue, primary myometrial cultured cells, and cell culture media in control cells and oxytocin (30 nM) or nifedipine (10μM) treated cells. In primary cultured myometrial cells, micromolar ANP had little effect on intracellular calcium in the presence or absence of oxytocin. In dramatic contrast, very low dosages (picomolar levels) of ANP have statistically significant effect on human myometrial tissue contraction rate and amplitude. In fresh human myometrial tissue, we demonstrated the presence of three ANP receptor subtypes (A, B, and C). Normalized to total protein loaded and smooth muscle specific alpha-actin, the relative ANP receptor subtype expression was measured in term, preterm, hypertensive, preeclamptic, and normotensive patients in parallel with RIA determination of ANP, cAMP, and cGMP levels. A complex pattern of ANP receptor and ANP concentration was observed. Primary cultured myometrial tissue lack a response to nanomolar ANP. In contrast, fresh myometrial tissue contraction is modulated by picomolar ANP. All ANP receptor subtypes are present in fresh myometrial tissue but may decrease in culture (future experiments). ANP and all three ANP receptor subtypes are present in human myometrial tissue and may serve an important role in treating preterm labor.

Lack of MK2 Inhibits Myofibroblast Formation and Exacerbates Pulmonary Fibrosis

Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle-specific alpha-actin (alpha-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture. In this article, we demonstrate that MK2 also regulates myofibroblasts in vivo. Disruption of MK2 in mice prevented myofibroblast formation in a model of pulmonary fibrosis. However, MK2 disruption and consequent lack of myofibroblast formation exacerbated fibrosis rather than ameliorated it as previously postulated. When mice lacking MK2 (MK2-/-) were exposed to bleomycin, more collagen accumulated and more fibroblasts populated fibrotic regions in their lungs than in similarly treated wild-type mice. While there were many vimentin-positive cells in the bleomycin-treated MK2-/- mouse lungs, few alpha-SMA-positive cells were observed in these lungs compared with wild-type mouse lungs. siRNA against MK2 reduced alpha-SMA expression in wild-type mouse embryonic fibroblasts (MEF), consistent with its suppression in MK2-/- MEF. On the other hand expressing constitutively active MK2 in MK2-/- MEF significantly increased alpha-SMA expression. MK2-/-MEF proliferated at a faster rate and produced more collagen; however, they migrated at a slower rate than wild-type MEF. Overexpressing phosphomimicking HSP27, a target of MK2, did not reverse the effect of MK2 disruption on fibroblast migration. MK2 disruption did not affect Smad2 activation by transforming growth factor-beta. Thus, MK2 appears to mediate myofibroblast differentiation, and inhibiting that differentiation might contribute to fibrosis rather than protect against it.

Novel method to characterize primary cultures of leiomyoma and myometrium with the use of confirmatory biomarker gene arrays

To develop a rapid biomarker method for confirming that leiomyoma and myometrium primary cultures maintain the molecular phenotype of the progenitor tissues. Confirmation of primary cultures from leiomyoma and myometrium tissues. University hospital. Women undergoing hysterectomy for symptomatic leiomyomas. Primary cell cultures, reverse-transcriptase polymerase chain reaction (RT-PCR), microarray, real time RT-PCR, and immunofluorescence. Relative messenger RNA and protein expression in leiomyoma and myometrial cell cultures. We developed primary cell cultures from human leiomyoma and patient-matched myometrium obtained from hysterectomy specimens. In the primary cultures, we confirmed the presence of smooth muscle-specific alpha-actin as well as filamentous actin. Based on microarray analysis, we expected and confirmed, in the progenitor tissue and derived primary cultures, an overexpression of versican (8.31 fold +/- 2.2 SEM and 4.3 fold +/- 1.01 SEM, respectively), transforming growth factor beta-3 (5.66 fold +/- 0.82 SEM and 4.92 fold +/- 0.58 SEM, respectively), and cytochrome P450-26A1 (6.76 fold +/- 0.80 SEM and 6.17 fold +/- 2.02 SEM, respectively), and an underexpression of dermatopontin (-5.6 fold +/- 1.82 SEM and -3.41 +/- 1.20 SEM, respectively). Primary cell cultures offer a reliable in vitro model system for leiomyoma disease, if confirmed. Analysis of a gene array that distinguishes between myometrium and leiomyoma molecular phenotypes offers a rapid and reliable confirmation method, and provides confidence that in vitro findings resemble in vivo disease.

Preparation of primary cultured mesenteric artery smooth muscle cells for fluorescent imaging and physiological studies

In this protocol, we describe a method for isolation and culture of smooth muscle cells derived from the adult rat (or mouse) superior mesenteric artery. Arterial myocytes are obtained by enzymatic dissociation and established in primary culture. The cultured cells retain expression of smooth muscle-specific alpha-actin and physiological responses to agonists. Cultured arterial myocytes (prepared from wild-type or transgenic animals) provide a useful model for studying the regulation of a wide range of vascular smooth muscle responses at the cellular and subcellular levels. Plasmids, RNA interference and antisense oligodeoxynucleotides can be readily introduced into the cells to alter protein expression. Fluorescent dyes can also be introduced to visualize a variety of activities, some of which may be specific to vascular smooth muscle cells. This protocol requires about 3 h on each of 2 consecutive days to complete.

Cyclic strain induces vascular smooth muscle cell differentiation from murine embryonic mesenchymal progenitor cells

Hemodynamic forces play a crucial role in regulating vascular cell phenotype. However, the underlying molecular mechanisms are largely unknown. The objective of this study was to test our hypothesis that cyclic strain could affect smooth muscle cell (SMC) differentiation. A murine embryonic mesenchymal progenitor cell line (C3H/10T1/2) was cultured with or without cyclic strain for 6 days. Changes in cell morphology were studied with fluorescence dye Calcein-AM staining. Expression of specific SMC markers, smooth muscle specific alpha-actin (alpha-SMA), and smooth muscle myosin heavy chain (SMMHC), was determined by real-time polymerase chain reaction (PCR) and Western blot. Transforming growth factor- beta (TGF-beta) was used as a positive control. With cyclic strain, CH3/10T1/2 cells demonstrated spindle-shaped morphology and parallel alignment. Cells exposed to cyclic strain illustrated significantly increased mRNA levels of alpha-SMA and SMMHC by 3- and 2-fold, respectively, compared with static cells (P<.05). In addition, cells cultured under cyclic strain with TGF-beta (2 ng/ml) supplementation demonstrated increased mRNA levels of alpha-SMA and SMMHC by 10- and 2-fold, respectively, compared with static cells (P<.05). Furthermore, protein levels of alpha-SMA and SMMHC were also significantly increased by more than 3-fold in cyclic strain-treated cells compared with static cultures (P<.05). TGF-beta synergistically enhanced the effect of cyclic strain on alpha-SMA mRNA expression in CH3/10T1/2 cells. This is the first study to demonstrate that cyclic strain significantly induces expression of two of the most important SMC markers in a murine embryonic mesenchymal progenitor cell line. Cyclic strain and TGF-beta have a synergistic effect on alpha-SMA mRNA expression.

Spindle epithelial tumor with thymus‐like differentiation (SETTLE): Youngest reported patient

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a very rare thyroid tumor. It is one of a family of tumors arising either from ectopic thymus or remnants of branchial pouches that retain the potential to differentiate along the thymic line. Herein is reported a case of SETTLE in a 2-year-old girl. The patient underwent right thyroid lobectomy for a tumor of the right thyroid lobe. The resected specimen of this tumor revealed a whitish and solid mass. On microscopy, the tumor exhibited an area of spindle cells, glandular epithelium, and mucinous cystic lesions. The following findings were obtained on immunohistochemistry: the spindle cell area was diffusely positive for cytokeratin AE1/3 and vimentin, and partially positive for alpha-smooth muscle-specific actin. The glandular structures consisted of columnar cells and the cystic area was also positive for cytokeratin AE1/3. All three components of the tumor were negative for thyroglobulin, thyroid transcription factor-1, S-100 protein, carcinoembryonic antigen, somatostatin, synaptophysin, and chromogranin A. On the basis of the aforementioned findings, SETTLE was diagnosed. The patient remains disease free to date, 2 years after surgery with no additional treatment. To the best of the authors' knowledge the present SETTLE patient is the youngest yet reported.

Heaves, an asthma-like equine disease, involves airway smooth muscle remodeling

Increased airway smooth muscle mass is a prominent feature of asthmatic airway remodeling. Airway smooth muscle hyperplasia occurs in rodent models of experimental asthma, but the relevance of such finding to spontaneously occurring disease in large mammals is unknown. We examined horses with heaves, a naturally occurring equine asthma related to sensitization and exposure to moldy hay. We hypothesized that airway remodeling occurs in heaves and shares disease mechanisms with asthma. We quantified the airway smooth muscle mass and the numbers of proliferating and apoptotic airway smooth muscle cells in 5 horses with heaves and 5 control horses using morphometric techniques. Cell proliferation was detected in tissue sections by immunostaining for proliferating cell nuclear antigen, and apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of fragmented DNA. Both signals were colocalized with smooth muscle specific alpha-actin. Horses with heaves had a significant increase in the amount of smooth muscle in the airways (nearly triple that of the controls) associated with increased myocyte proliferation (7-fold proliferating cell nuclear antigen-positive airway myocytes) and apoptosis (6-fold). Heaves involves airway smooth muscle growth associated with myocyte hyperplasia, which may contribute to the growth, and increased myocyte apoptosis that may reflect a compensatory mechanism serving to limit the abnormal smooth muscle growth. Airway smooth muscle remodeling in heaves may be involved in the mechanism of airway hyperresponsiveness and chronic lung function impairment in a way comparable to human asthma.

Small intestinal submucosa as a tunica albuginea graft material.

We evaluated the morphological, immunological and functional response to small intestinal submucosa grafting of the tunica albuginea to determine its potential as a grafting material for penile surgery. Male New Zealand White rabbits underwent a sham procedure (6) or tunical excision and grafting with small intestinal submucosa (6). The erectile response to the intracavernous vasoactive agents sodium nitroprusside plus a papaverine, phentolamine and prostaglandin E1 combination (Sigma Chemical Co., St. Louis, Missouri) was evaluated 45-day postoperatively. The area under the graft was evaluated for stromal collagen and smooth muscle content by Masson's trichrome stain. Protein expression of smooth muscle specific alpha-actin and the inflammatory markers inducible nitric oxide synthase (NOS) and transforming growth factor-beta1 (TGF-beta1) was evaluated by immunohistochemical methods. Total RNA was extracted from the corpora cavernosum underlying the small intestinal submucosa graft and reverse transcriptase-polymerase chain reaction (RT-PCR) was done using an Access system (Promega, Madison, Wisconsin) with gene specific primers for inducible NOS, TGF-beta1 and vascular endothelial growth factor (VEGF). Grafting of the tunica albuginea with small intestinal submucosa had no significant effect on the magnitude or duration of the erectile response to intracavernous vasoactive agents. Histological examination demonstrated no inflammatory changes in the tunica albuginea or corporeal tissue underlying the area of the small intestinal submucosa graft and there was no appreciable alteration in smooth muscle or collagen content. The 2 groups showed intense positive immunostaining to alpha-actin. Weak expression of TGF-beta1 predominantly associated with smooth muscle fibers was identified in the 2 groups of rabbits by immunostaining and RT-PCR. No significant inducible NOS was detected by immunostaining or RT-PCR in either group. Strong VEGF expression was observed in grafted rabbits. The most noticeable (3-fold) increase in expression was detected in splice variant 165. Small intestinal submucosa grafting of the tunica albuginea preserves the duration and magnitude of the erectile response to vasoactive agents. This type of tunical grafting does not stimulate a significant inflammatory response, or cause corporeal fibrosis or loss of cavernous smooth muscle content. Stimulating VEGF may facilitate wound healing and the maintenance of normal erectile function.

Urethral replacement using cell seeded tubularized collagen matrices.

Acellular collagen matrices derived from bladder submucosa have been used successfully as an off-the-shelf biomaterial for urethral replacement, experimentally and clinically in an onlay fashion. We investigated whether collagen matrices, either alone or with autologous cells, could be used for tubularized urethral replacement. Acellular collagen matrices were processed and tubularized. Ten rabbits underwent an open bladder biopsy with subsequent cell expansion. Autologous bladder cells were grown and seeded onto the pre-configured tubular matrices. A 1 cm. long urethral segment was excised in 24 male rabbits. Urethroplasty was performed with the tubularized collagen matrices seeded with cells in 12 animals and without cells in 12. Serial urethrography was performed preoperatively and at 1, 2, 3 and 6 months postoperatively. Retrieved urethras were analyzed grossly, histologically, immunocytochemically and with Western blots. Contractility and the presence of neurotransmitter receptors were confirmed with organ bath studies. Serial urethrography confirmed the maintenance of a wide urethral caliber without any signs of strictures in animals implanted with the cell seeded matrices. The urethral segments replaced with the collagen scaffolds without cells demonstrated strictures and graft collapse at all time points. The implanted cell seeded matrices had a normal urethral architecture by 1 month, consisting of a transitional cell layer surrounded by muscle cell fiber bundles with increasing cellular organization with time. Epithelial and smooth muscle phenotypes were confirmed immunocytochemically and with Western blot analyses using pancytokeratins AE1/AE3 and smooth muscle specific alpha-actin antibodies. Formation of a transitional cell layer was confirmed in the matrices implanted without cells but only scant unorganized muscle fiber bundles were present, mostly at the anastomotic sites. Organ bath studies demonstrated the capacity for contractility along with cholinergic and adrenergic specific receptors in the tissue engineered scaffolds compared to controls. These results show that collagen matrices seeded with cells form normal urethral tissue can be used for tubularized replacement, whereas tubularized collagen matrices alone without cells lead to poor tissue formation and strictures. The collagen matrices seeded with cells may offer a useful alternative in the future for patients requiring a tubularized urethral segment replacement.

Small Intestinal Submucosa As A Tunica Albuginea Graft Material

We evaluated the morphological, immunological and functional response to small intestinal submucosa grafting of the tunica albuginea to determine its potential as a grafting material for penile surgery. Male New Zealand White rabbits underwent a sham procedure (6) or tunical excision and grafting with small intestinal submucosa (6). The erectile response to the intracavernous vasoactive agents sodium nitroprusside plus a papaverine, phentolamine and prostaglandin E1 combination (Sigma Chemical Co., St. Louis, Missouri) was evaluated 45-day postoperatively. The area under the graft was evaluated for stromal collagen and smooth muscle content by Masson's trichrome stain. Protein expression of smooth muscle specific alpha-actin and the inflammatory markers inducible nitric oxide synthase (NOS) and transforming growth factor-beta1 (TGF-beta1) was evaluated by immunohistochemical methods. Total RNA was extracted from the corpora cavernosum underlying the small intestinal submucosa graft and reverse transcriptase-polymerase chain reaction (RT-PCR) was done using an Access system (Promega, Madison, Wisconsin) with gene specific primers for inducible NOS, TGF-beta1 and vascular endothelial growth factor (VEGF). Grafting of the tunica albuginea with small intestinal submucosa had no significant effect on the magnitude or duration of the erectile response to intracavernous vasoactive agents. Histological examination demonstrated no inflammatory changes in the tunica albuginea or corporeal tissue underlying the area of the small intestinal submucosa graft and there was no appreciable alteration in smooth muscle or collagen content. The 2 groups showed intense positive immunostaining to alpha-actin. Weak expression of TGF-beta1 predominantly associated with smooth muscle fibers was identified in the 2 groups of rabbits by immunostaining and RT-PCR. No significant inducible NOS was detected by immunostaining or RT-PCR in either group. Strong VEGF expression was observed in grafted rabbits. The most noticeable (3-fold) increase in expression was detected in splice variant 165. Small intestinal submucosa grafting of the tunica albuginea preserves the duration and magnitude of the erectile response to vasoactive agents. This type of tunical grafting does not stimulate a significant inflammatory response, or cause corporeal fibrosis or loss of cavernous smooth muscle content. Stimulating VEGF may facilitate wound healing and the maintenance of normal erectile function.

Gβγ Mediate Differentiation of Vascular Smooth Muscle Cells

Proliferation and subsequent dedifferentiation of vascular smooth muscle (VSM) cells contribute to the pathogenesis of atherosclerosis and postangioplastic restenosis. The dedifferentiation of VSM cells in vivo or in cell culture is characterized by a loss of contractile proteins such as smooth muscle-specific alpha-actin and myosin heavy chain (SM-MHC). Serum increased the expression of contractile proteins in neonatal rat VSM cells, indicating a redifferentiation process. RNase protection assays defined thrombin as a serum component that increases the abundance of SM-MHC transcripts. Additionally, serum and thrombin transiently elevated cytosolic Ca(2+) concentrations, led to a biphasic extracellular signal-regulated kinase (ERK) phosphorylation, up-regulated a transfected SM-MHC promoter construct, and induced expression of the contractile proteins SM-MHC and alpha-actin. Pertussis toxin, N17-Ras/Raf, and PD98059 prevented both the serum- and thrombin-induced second phase ERK phosphorylation and SM-MHC promoter activation. Constitutively active Galpha(q), Galpha(i), Galpha(12), and Galpha(13) failed to up-regulate SM-MHC transcription, whereas Gbetagamma concentration-dependently increased the SM-MHC promoter activity. Furthermore, the Gbetagamma scavenger beta-adrenergic receptor kinase 1 C-terminal peptide abolished the serum-mediated differentiation. We conclude that receptor-mediated differentiation of VSM cells requires Gbetagamma and an intact Ras/Raf/MEK/ERK signaling.

TIME DEPENDENT SMOOTH MUSCLE REGENERATION AND MATURATION IN A BLADDER ACELLULAR MATRIX GRAFT: HISTOLOGICAL STUDIES AND IN VIVO FUNCTIONAL EVALUATION

We evaluated the time dependence of smooth muscle regeneration and restoration of in vivo functional properties in bladder augmented with a bladder acellular matrix graft. A total of 45 Sprague-Dawley rats underwent augmentation cystoplasty with a bladder acellular matrix graft. Two rats each were sacrificed at various intervals within the first 21 days and 6 each were sacrificed at 4, 8 and 12 weeks. This second group underwent preoperative and postoperative assessment of bladder function, including cystometry, electrostimulation and stimulation with ice water, potassium and carbachol, as well as labeling of the bladder wall by the injection of fluorescent microspheres. After sacrifice slides of the bladders prepared for hematoxylin and eosin, trichrome, KI67, vimentin, desmin, smooth muscle specific alpha-actin and fluorescent microspheres were evaluated. Within 2 weeks the number of cells in the matrix as well as the proliferation index increased rapidly and then decreased gradually. Erythrocytes and inflammatory cells were found in the matrix within 2 to 4 days, followed by fibroblasts. A bladder host-to-matrix shift was evident by the appearance of microspheres in the matrix. Cell marker expression indicated the early appearance of vimentin and alpha-actin within the first 10 days. Distinct desmin expression was observed later, when the first smooth muscle cells were recognized. Functional evaluation revealed restored bladder function at 12 weeks. The time dependent increase of muscle cell markers during smooth muscle cell regeneration in a bladder acellular matrix graft is concordant with the progressive restoration of bladder function. These results may support the bladder acellular matrix graft concept for clinical application.

Plexiform fibrohistiocytic tumor of the foot

The first case of plexiform fibrohistiocytic tumor in the foot is presented in this article. The tumor developed on the dorsum of the left foot in a 14-year-old female. This tumor was originally described in 1988 by Enzinger and Zhang. Their study indicated that this tumor has a female predominance, median age of 14.5 years, 63% located in the upper extremities, 37.5% recurrence rate, and 3% metastasis rate. These tumors are very unique with a nodular pattern and a cellular component of histiocytes, fibroblasts, and multinucleated giant cells. Typically they are located within the deep dermis and subcutaneous tissue. Immunohistochemical preparations show that the tumor does not stain for S-100 protein, desmin, cytokeratin, factor VIII-related protein, or lysozyme. However, it does stain for alpha-1-antitrypsin, alpha-1-antichymotrypsin, alpha-smooth muscle-specific actin, vimentin, and CD68 antibody.

Expression of smooth muscle alpha-actin in mesenchymal cells during formation of avian endocardial cushion tissue: a role for transforming growth factor beta3.

During early cardiac morphogenesis, outflow tract (OT) and atrio-ventricular (AV) endothelial cells differentiate into mesenchymal cells, which have characteristics of smooth muscle-like myofibroblasts, and which form endocardial cushion tissue, the primordia of valves, and septa in the adult heart. During this embryonic event, transforming growth factor beta3 (TGF beta3) is an essential element in the progression of endothelial-transformation into mesenchyme. TGF beta(s) are known to be a potent inducer for mesodermal differentiation and a promoter for differentiation of endothelial cells into smooth muscle-like cells. Using a monoclonal antibody against smooth muscle-specific alpha-actin (SMA), we examined the immunohistochemical staining of this form of actin in avian endocardial cushion tissue formation. To determine whether TGF beta3 initiates the expression of SMA, the pre-migratory AV endothelial monolayer was cultured with or without chicken recombinant TGF beta3 and the expression of SMA was examined immunochemically. Migrating mesenchymal cells expressed SMA beneath the cell surface membrane. These cells showed a reduction of endothelial specific marker antigen, QH1. Stationary endothelial cells did not express SMA. The deposition of SMA in the mesenchymal tissue persisted until the end of the fetal period. Pre-migratory endothelial cells cultured in complete medium (CM199) that contained TGF beta3 expressed SMA, whereas cells cultured in CM199 alone did not. At the onset of the endothelial-mesenchymal transformation, migrating mesenchymal cells express SMA and the expression of this form of actin is upregulated by TGF beta3. The induction of the expression of SMA by TGF beta3 is one of the initial events in the cytoskeletal reorganization in endothelial cells which separate from one another during the initial phenotypic change associated with the endothelial-mesenchymal transformation.