The expression of nestin delineates skeletal muscle differentiation in the developing rat esophagus

The muscularis externa of the developing rodent esophagus is initially composed of smooth muscle, and later replaced by skeletal muscle in a craniocaudal progression. There is growing evidence of distinct developmental origins for esophageal smooth and skeletal muscles. However, the identification of skeletal muscle progenitor cells is controversial, and the detailed cell lineage of their descendants remains elusive. In the current study, we carried out multiple labeling immunofluorescence microscopy of nestin and muscle type-specific markers to characterize the dynamic process of rat esophageal myogenesis. The results showed that nestin was transiently expressed in immature esophageal smooth muscle cells in early developing stages. After nestin was downregulated in smooth muscle cells, a distinct population of nestin-positive cells emerged as skeletal muscle precursors. They were mitotically active, and subsequently co-expressed MyoD, followed by the embryonic and later the fast type of skeletal muscle myosin heavy chain. Thus, the cell lineage of esophageal skeletal muscle differentiation was established by an immunotyping approach, which revealed that skeletal myocytes arise from a distinct lineage rather than through transdifferentiation of smooth muscle cells during rat esophageal myogenesis.     

Differentially expressed gene networks in cultured smooth muscle cells from normal and neuropathic bladder.

Neuropathic bladder dysfunction results from abnormal development of the spine, spinal cord injuries, or diseases such as diabetics. Patients with neuropathic bladders often require surgical intervention such as bladder reconstruction to improve incontinence and prevent renal damage. Tissue engineering with ex-vivo cultured bladder cells has been suggested as one means for improving bladder function. However, we previously demonstrated that cultured bladder smooth muscle cells (SMCs) derived from neuropathic bladder exhibit and maintain altered pathologic phenotypes in culture. To identify genes that are responsible for the abnormal neuropathic phenotypes specifically elevated cell proliferation, the expression levels of 1,185 genes were compared between cultured SMCs derived from normal and neuropathic bladders using a cDNA array consisting of well-annotated genes. The expression data were analyzed using several methods to identify differentially expressed genes. The resulting sets of differentially expressed genes were examined by pathway analysis to identify the networks that remain abnormal in the culture-stable phenotype of neuropathic SMCs. A total of 18 genes that are differentially expressed between cultured normal and neuropathic bladder SMCs were identified. Of these 17 were up-regulated greater than 2-fold in neuropathic bladder SMCs, six of them along with one gene that was not up-regulated greater than 2-fold in cultured neuropathic bladder SMCs were confirmed and identified by more stringent analysis methods including significance analysis of microarrays, class comparison, and class prediction analyses. The major dysregulated pathways include fibroblast growth factor signaling, PTEN signaling, and integrin signaling. Our results further suggest that altered neuropathic bladder SMC phenotypes is stable in the culture environments and that SMCs derived from diseased bladders may not be appropriate for tissue engineering purpose without modification of pathologically altered genes expression.     

Embryonic essential myosin light chain regulates fetal lung development in rats

Congenital diaphragmatic hernia (CDH) is currently the most life-threatening congenital anomaly the major finding of which is lung hypoplasia. Lung hypoplasia pathophysiology involves early developmental molecular insult in branching morphogenesis and a late mechanical insult by abdominal herniation in maturation and differentiation processes. Since early determinants of lung hypoplasia might appear as promising targets for prenatal therapy, proteomics analysis of normal and nitrofen-induced hypoplastic lungs was performed at 17.5 days after conception. The major differentially expressed protein was identified by mass spectrometry as myosin light chain 1a (MLC1a). Embryonic essential MLC1a and regulatory myosin light chain 2 (MLC2) were characterized throughout normal and abnormal lung development by immunohistochemistry and Western blot. Disruption of MLC1a expression was assessed in normal lung explant cultures by antisense oligodeoxynucleotides. Since early stages of normal lung development, MLC1a was expressed in vascular smooth muscle (VSM) cells of pulmonary artery, and MLC2 was present in parabronchial smooth muscle and VSM cells of pulmonary vessels. In addition, early smooth muscle differentiation delay was observed by immunohistochemistry of alpha-smooth muscle actin and transforming growth factor-beta1. Disruption of MLC1a expression during normal pulmonary development led to significant growth and branching impairment, suggesting a role in branching morphogenesis. Both MLC1a and MLC2 were absent from hypoplastic fetal lungs during pseudoglandular stage of lung development, whereas their expression partially recovered by prenatal treatment with vitamin A. Thus, a deficiency in contractile proteins MLC1a and MLC2 might have a role among the early molecular determinants of lung hypoplasia in the rat model of nitrofen-induced CDH.     

Bone morphogenetic protein-mediated modulation of lineage diversification during neural differentiation of embryonic stem cells

Embryonic stem cells (ES cells) can give rise to a broad spectrum of neural cell types. The biomedical application of ES cells will require detailed knowledge on the role of individual factors modulating fate specification during in vitro differentiation. Bone morphogenetic proteins (BMPs) are known to exert a multitude of diverse differentiation effects during embryonic development. Here, we show that exposure to BMP2 at distinct stages of neural ES cell differentiation can be used to promote specific cell lineages. During early ES cell differentiation, BMP2-mediated inhibition of neuroectodermal differentiation is associated with an increase in mesoderm and smooth muscle differentiation. In fibroblast growth factor 2-expanded ES cell-derived neural precursors, BMP2 supports the generation of neural crest phenotypes, and, within the neuronal lineage, promotes distinct subtypes of peripheral neurons, including cholinergic and autonomic phenotypes. BMP2 also exerts a density-dependent promotion of astrocyte differentiation at the expense of oligodendrocyte formation. Experiments involving inhibition of the serine threonine kinase FRAP support the notion that these effects are mediated via the JAK/STAT pathway. The preservation of diverse developmental BMP2 effects in differentiating ES cell cultures provides interesting prospects for the enrichment of distinct neural phenotypes in vitro.     

Anatomy of Smooth Muscle Cells in Nonmalignant and Malignant Human Prostate Tissue

Differently graded areas of human prostate adenocarcinoma were examined after Masson's trichrome staining or immunohistochemistry for smooth muscle alpha‐actin, type IV collagen and laminin. In addition, the ultrastructure of the prostatic smooth muscle cells (SMC) during glandular proliferation and epithelial invasion in selected tumors was studied. The SMC formed a thick layer below the epithelial structures in unaffected areas and were closely associated with each other in homotypic interactions. As the tumor grade increased, the SMC gradually lost interactions with each other and became atrophic. With the growth of the epithelial compartment, the SMC initially segregated to the tumor periphery and the intercellular spaces increased. In high grade tumors, the epithelial cancer cells invaded the spaces between the SMC. Immunohistochemical analysis of the basal membrane revealed increased disruption of the usually thick basal membrane, which became thinner and faintly stained with each of the antibodies used. We conclude that most SMC become atrophic following epithelial invasion in human tumors and that degradation of the basal membrane is an important factor in this process. At the ultrastructural level, different SMC phenotypes occur in prostatic tissues during epithelial invasion. Interconversion between these phenotypes is suggested and a probable relationship among them is proposed. Anat Rec, 291:1115–1123, 2008. © 2008 Wiley‐Liss, Inc.     

Molecular regulation of vascular smooth muscle cell differentiation in development and disease

The focus of this review is to provide an overview of the current state of knowledge of molecular mechanisms/processes that control differentiation of vascular smooth muscle cells (SMC) during normal development and maturation of the vasculature, as well as how these mechanisms/processes are altered in vascular injury or disease. A major challenge in understanding differentiation of the vascular SMC is that this cell can exhibit a wide range of different phenotypes at different stages of development, and even in adult organisms the cell is not terminally differentiated. Indeed, the SMC is capable of major changes in its phenotype in response to changes in local environmental cues including growth factors/inhibitors, mechanical influences, cell-cell and cell-matrix interactions, and various inflammatory mediators. There has been much progress in recent years to identify mechanisms that control expression of the repertoire of genes that are specific or selective for the vascular SMC and required for its differentiated function. One of the most exciting recent discoveries was the identification of the serum response factor (SRF) coactivator gene myocardin that appears to be required for expression of many SMC differentiation marker genes, and for initial differentiation of SMC during development. However, it is critical to recognize that overall control of SMC differentiation/maturation, and regulation of its responses to changing environmental cues, is extremely complex and involves the cooperative interaction of many factors and signaling pathways that are just beginning to be understood. There is also relatively recent evidence that circulating stem cell populations can give rise to smooth muscle-like cells in association with vascular injury and atherosclerotic lesion development, although the exact role and properties of these cells remain to be clearly elucidated. The goal of this review is to summarize the current state of our knowledge in this area and to attempt to identify some of the key unresolved challenges and questions that require further study.     

Tuning smooth muscle contraction by molecular motors

As in striated muscle, smooth muscle cells (SMC) contract by Ca²⁺ activated cyclic interaction between actin and type II myosin. However, smooth muscle maintains tone at basal activating Ca²⁺ and low energetic cost during sustained activation. This review analyzes the regulation of phasic and tonic contraction of SMC on the molecular level. Type II myosin is the molecular motor also of smooth muscle contraction. Six myosin heavy chain (MHC) isoenzymes (four smooth muscle, two nonmuscle) and five myosin light chain (MLC) isoforms (two 17 kDa, two 20 kDa, one 23 kDa) are expressed in SMC. These myosin subunits could be generated by alternative splicing or by differential gene expression. Thus different myosin isoenzymes are generated which may be modified posttranslationally by phosphorylation, affecting the contractile state of the SMC. Furthermore, they may be part of distinct contractile systems which are targeted by different second messenger cascades and are recruited differentially during activation, electromechanical, and pharmacomechanical coupling. Low energy consumption, shortening velocity, and MLC20 phosphorylation at low Ca²⁺ activation levels during tone maintenance ("latch") could be explained by a switch from smooth muscle myosin to nonmuscle myosin activation upon prolonged activation.Abbreviations MHC Myosin heavy chains - MLC Myosin light chains - MLCK Myosin light chain kinase - MLCP MLC20 phosphatase - NM Nonmuscle - nt Nucleotide - SM Smooth muscle - SMC Smooth muscle cells     

A novel isoform of the smooth muscle cell differentiation marker smoothelin

Studies on smooth muscle cell differentiation and those on vascular development in mouse and humans have long been hampered by the lack of suitable markers. Here we describe a novel, large isoform of smoothelin, a structural protein of differentiated, contractile smooth muscle cells. The protein, which is highly conserved in mouse and humans, shows homology with other cytoskeleton-associated smooth muscle cell proteins and contains an actinin-type actin-binding domain. Northern blot analysis from various mouse organs identified short and long smoothelin mRNA forms, which exhibit distinct tissue expression patterns. The short form is highly expressed in visceral muscle tissues such as intestine and stomach and is not detectable in brain, while the long mRNA form is expressed in all vascularized organs. These results may provide new tools and approaches to study both smooth muscle cell differentiation and proliferative vascular disease. Received: 25 August 1998 / Accepted: 19 October 1998     

Survey of the Distribution of a Newly Characterized Receptor for Advanced Glycation End Products in Tissues

Advanced glycation end products (AGEs), the final products of nonenzymatic glycation and oxidation of proteins, are found in the plasma and accumulate in the tissues during aging and at an accelerated rate in diabetes. A novel integral membrane protein, termed receptor for AGE (RAGE), forms a central part of the cell surface binding site for AGEs. Using monospecific, polyclonal antibody raised to human recombinant and bovine RAGE, immunostaining of bovine tissues showed RAGE in the vasculature, endothelium, and smooth muscle cells and in mononuclear cells in the tissues. Consistent with these data, RAGE antigen and mRNA were identified in cultured bovine endothelium, vascular smooth muscle, and monocyte-derived macrophages. RAGE antigen was also visualized in bovine cardiac myocytes as well as in cultures of neonatal rat cardiac myocytes and in neural tissue where motor neurons, peripheral nerves, and a population of cortical neurons were positive. In situ hybridization confirmed the presence of RAGE mRNA in the tissues, and studies with rat PC12 pheochromocytes indicated that they provide a neuronal-related cell culture model for examining RAGE expression. Pathological studies of human atherosclerotic plaques showed infiltration of RAGE-expressing cells in the expanded intima. These results indicate that RAGE is present in multiple tissues and suggest the potential relevance of AGE-RAGE interactions for modulating properties of the vasculature as well as neural and cardiac function, prominent areas of involvement in diabetes and in the normal aging process.     

Ultrastructural analysis of the smooth-to-striated transition zone in the developing mouse esophagus: emphasis on apoptosis of smooth and origin and differentiation of striated muscle cells.

The exact mechanism of smooth-to-striated muscle conversion in the mouse esophagus is controversial. Smooth-to-striated muscle cell transdifferentiation vs. distinct differentiation pathways for both muscle types were proposed. Main arguments for transdifferentiation were the failure to detect apoptotic smooth and the unknown origin of striated muscle cells during esophageal myogenesis. To reinvestigate this issue, we analyzed esophagi of 4-day-old mice by electron microscopy and a fine-grained sampling strategy considering that, in perinatal esophagus, the replacement of smooth by striated muscle progresses craniocaudally, while striated myogenesis advances caudocranially. We found numerous (1) apoptotic smooth muscle cells located mainly in a transition zone, where smooth intermingled with developing striated muscle cells, and (2) mesenchymal cells in the smooth muscle portion below the transition zone, which appeared to give rise to striated muscle fibers. Taken together, these results provide further evidence for distinct differentiation pathways of both muscle types during esophagus development.     

Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-beta and PDGF-BB). Expression of smooth muscle protein 22alpha and alpha-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.     

Molecular control of vascular smooth muscle cell differentiation

Changes in the differentiated state of the vascular smooth muscle cell (SMC) including enhanced growth responsiveness, altered lipid metabolism, and increased matrix production are known to play a key role in development of atherosclerotic disease. As such, there has been extensive interest in understanding the molecular mechanisms and factors that regulate differentiation of vascular SMC, and how this regulation might be disrupted in vascular disease. Key questions include determination of mechanisms that control the coordinate expression of genes required for the differentiated function of the smooth muscle cell, and determination as to how these regulatory processes are influenced by local environmental cues known to be important in control of smooth muscle differentiation. Of particular interest, a number of common cis regulatory elements including highly conserved CArG [CC(A/T)₆GG] motifs or CArG-like motifs and a TGF_β control element have been identified in the promoters of virtually all smooth muscle differentiation marker genes characterized to date including smooth muscle α-actin, smooth muscle myosin heavy chain, telokin, and SM22α and shown to be required for expression of these genes both in vivo and in vitro. In addition, studies have identified a number of trans factors that interact with these cis elements, and shown how the expression or activity of these factors is modified by local environmental cues such as contractile agonists that are known to influence differentiation of smooth muscle.     

Identification of proteins from colorectal cancer tissue by two-dimensional gel electrophoresis and SELDI mass spectrometry

We investigated protein profiles obtained from colorectal tumor tissue and adjacent normal mucosa to identify tumor specific changes. Protein extracts of biopsis were separated by two-dimensional gel electrophoresis and >40 low-molecular mass proteins were identified by peptide fingerprinting using surface-enhanced laser desoption/ionization mass spectrometry (SELDI-MS). Among these, PACAP protein, hnrnp A1, flavin reductase, calgizzarin, NDK B (NM23-H2), cyclophilin A and smooth muscle protein 22-alpha showed significantly differential abundancy in the analyzed specimens. In addition, immunohistochemical analysis of tissue distribution and subcellular localization of some of the differentially expressed proteins demonstrated alterations in subcellular protein distribution. Further investigations are in progress to assess whether these differentially expressed proteins are associated with tumor development and tumor progression.     

Heparin inhibits proliferation of myometrial and leiomyomal smooth muscle cells through the induction of alpha-smooth muscle actin, calponin h1 and p27

Mast cells are widely distributed in human tissues, including the human uterus. However, the function of mast cells in uterine smooth muscle has not been clearly established. Mast cells possess secretory granules containing such substances as heparin, serotonin, histamine and many cytokines. To help establish the role of mast cells in the human myometrium, the action of heparin was investigated using smooth muscle cells (SMC) from normal myometrium and from leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by heparin treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under heparin treatment. Western blotting analysis showed that markers of SMC differentiation such as alpha-smooth muscle actin (alpha-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by heparin, whereas cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not changed. Taken together, these results indicate that heparin inhibits the proliferation of myometrial and leiomyomal SMC through the induction of alpha-SMA, calponin h1 and p27. We suggest that heparin from mast cells may induce differentiation in uterine SMC and may influence tissue remodelling and reconstruction during physiological and pathophysiological events.     

Caveolin-3 is a sensitive and specific marker for rhabdomyosarcoma.

Caveolin-3 (Cav-3) is a principal structural protein of caveolae membrane domains. Animal studies have revealed that Cav-3 is expressed in skeletal and cardiac myocytes but absent in other types of cells. Recent studies have shown that abnormalities in the Cav-3 gene are associated with some forms of muscular dystrophy, while skeletal muscle abnormalities have been observed in Cav-3 transgenic and knockout mice. In this study the authors evaluated the distribution of Cav-3 in normal human tissues and compared the expression of Cav-3 with that of myogenin and myoD1 in rhabdomyosarcoma (RMS), malignant mixed mullerian tumor (MMMT), and an array of neoplasms that mimic RMS to assess the utility of Cav-3 as a diagnostic marker for tumors with skeletal muscle differentiation. In nonneoplastic human tissues, crisp membrane staining for Cav-3 was present in cardiac and skeletal myocytes and occasionally in arterial smooth muscle cells and prostatic stromal cells, while other cell types were negative for Cav-3. Eighty-eight percent (21/24) of RMS studied were positive for Cav-3. Positive staining was generally observed in the more maturely differentiated tumor cells but not the primitive tumor cells. Eight of nine cases of MMMT stained strongly with Cav-3 in their rhabdomyosarcomatous component but not in other components. Fifty-four other neoplasms (13 leiomyosarcomas, 8 neuroblastomas, 5 lymphomas, 6 Wilms tumors without skeletal muscle differentiation, 5 Ewing sarcomas, 4 malignant fibrous histiocytomas, 4 angiosarcomas, 6 malignant melanomas, and 3 synovial sarcomas) were negative for Cav-3 expression. Nearly all (96% [23/24]) cases of RMS were positive for myogenin, while 88% (21/24) were positive for myoD1. Primitive tumor cells showed significantly increased expression of myoD1 and myogenin; conversely, more differentiated tumor cells were negative or weakly stained. The rhabdomyosarcomatous component of MMMT stained focally with myogenin and myoD1, in contrast to the strong Cav-3 labeling in these cells. These results demonstrate that Cav-3 is specifically expressed in human cardiac and skeletal myocytes. Furthermore, its high specificity and relatively high sensitivity (88%) for tumors with skeletal muscle differentiation suggest that Cav-3 is a valuable marker for these tumors and may be used to assess the degree of differentiation of RMS and to identify residual tumor cells in post-chemotherapy specimens.     

Spatiotemporal expression of Wnt5a during the development of the striated muscle complex in rats with anorectal malformations

Fecal incontinence and constipation after procedures for anorectal malformations (ARMs) are closely related to the maldevelopment of the striated muscle complex (SMC). Previous studies have demonstrated that myogenic regulatory factors (MRFs) play a significant role in muscle development. Wnt signal pathway is extremely important for MRFs regulation. This study was designed to investigate the spatiotemporal expression pattern of Wnt5a in SMC in ARMs rat embryos. Materials and Methods: Anorectal malformation embryos were induced by ethylene thiourea on embryonic day 10 (E10). Expression levels of protein and mRNA of Wnt5a were confirmed by immunohistochemistry staining, western blot and quantitative real-time PCR (qRT-PCR) between normal rat embryos and embryos with ARMs. Results: Immunostaining revealed that, on embryonic day 17 (E17), the Wnt5a protein was initially expressed in the SMC in normal embryos. With the growth of pregnancy, the positive staining cells gradually increased. The same time-dependent changes of Wnt5a protein were detected in ARMs embryos. Besides, immunostaining showed that Wnt5a had a significant increase in normal embryos compared with ARMs embryos. Similarly, in Western blot and qRT-PCR, the higher expression of Wnt5a protein and mRNA were remarkable in normal embryos during the SMC development, relatively. Conclusion: Our study demonstrated that the downregulation of Wnt5a at the time of SMC development might partly be related to the dysplasia of SMC in ARMs.