Factor Xa induces mitogenesis of vascular smooth muscle cells via autocrine production of epiregulin

Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell.     

Transforming growth factor β1 involvement in the conversion of fibroblasts to smooth muscle cells in the rabbit bladder serosa

In an attempt to identify the growth factors or cytokines involved in the serosal thickening that occurs in rabbit bladder subjected to partial outflow obstruction, the following growth factors – transforming growth factor β1, platelet-derived growth factor, epidermal growth factor, granulocyte colony-stimulating factor and granulocyte–monocyte colony-stimulating factor – were delivered separately onto the serosal surface of the intact bladder via osmotic minipumps. The proliferative/differentiative cellular response of the rabbit bladder wall was evaluated by bromodeoxyuridine incorporation and immunofluorescence staining with a panel of monoclonal antibodies to cytoskeletal proteins (desmin, vimentin, keratins 8 and 18 and non-muscle myosin) and to smooth muscle (α-actin, myosin and SM22) proteins. Administration of the transforming growth factor, but not of the other growth factors/cytokines, was effective in inducing serosal thickening. Accumulating cells in this tissue were identified as myofibroblasts, i.e. cells showing a mixed fibroblast–smooth muscle cell differentiation profile. The phenotypic pattern of myofibroblasts changed in a time-dependent manner: 21 days after the growth factor delivery, small bundles of smooth muscle cells were found admixed with myofibroblasts, as occurs in the obstructed bladder. These ‘ectopic’ muscle structures displayed a variable proliferating activity and expressed an immature smooth muscle cell phenotype. The complete cellular conversion to smooth muscle cells was not achieved if transforming growth factor β1 was delivered to fibroblasts of subcutaneous tissue. These findings suggest a tissue-specific role for this growth factor in the cellular conversion from myofibroblast to smooth muscle cells. © 1998 Chapman & Hall     

Deubiquitinating enzyme A20 negatively regulates NF-κB signaling in skeletal muscle in mdx mice

Duchenne muscular dystrophy (DMD) is caused by the lack of a functional dystrophin protein that results in muscle fiber membrane disruption and, ultimately, degeneration. Regeneration of muscle fibers fails progressively, and muscle tissue is replaced with connective tissue. As a result, DMD causes progressive limb muscle weakness and cardiac and respiratory failure. The absence of dystrophin from muscle fibers triggers the chronic activation of the nuclear factor of kappa B (NF-κB). Chronic activation of NF-κB in muscle leads to infiltration of macrophages, up-regulation of the ubiquitin-proteosome system, and down-regulation of the helix-loop-helix muscle regulatory factor, MyoD. These processes, triggered by NF-κB activation, promote muscle degeneration and failure of muscle regeneration. A20 (TNFAIP3) is a critical negative regulator of NF-κB. In this study, we characterize the role of A20 in regulating NF-κB activation in skeletal muscle, identifying a novel role in muscle regeneration. A20 is highly expressed in regenerating muscle fibers, and knockdown of A20 impairs muscle differentiation in vitro, which suggests that A20 expression is critically important for regeneration of dystrophic muscle tissue. Furthermore, down-regulation of the classic pathway of NF-κB activation is associated with up-regulation of the alternate pathway in regenerating muscle fibers, suggesting a mechanism by which A20 promotes muscle regeneration. These results demonstrate the important role of A20 in muscle fiber repair and suggest the potential of A20 as a therapeutic target to ameliorate the pathology and clinical symptoms of DMD.     

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.     

An angiogenic extract from skeletal muscle stimulates monocyte and endothelial cell chemotaxis in vitro.

The purpose of this study was to determine whether the extraction of skeletal muscle with a combination of ethanol and hydrochloric acid yields a product capable of stimulating angiogenesis. The resulting extract stimulated inflammation in the rabbit corneal assay, which was followed by capillary formation. In order to determine whether the observed angiogenesis was stimulated by a factor(s) acting directly on the endothelial cells versus a factor(s) recruiting macrophages that in turn release factors acting on endothelial cells, the muscle extract was tested for endothelial cell and monocyte chemotaxis activity in vitro. The muscle extract stimulated significant endothelial cell chemotaxis at concentrations between 94 and 750 micrograms of protein/ml and significant monocyte chemotaxis at concentrations between 8 and 75 micrograms of protein/ml. Polyacrylamide gel electrophoresis suggests that basic fibroblast growth factor and transforming growth factor-beta may be present in this acid/ethanol extract of skeletal muscle.     

THE ELONGATION OF MYOFIBRILS FROM THE INDIRECT FLIGHT MUSCLE OF DROSOPHILA

Myofibrils which lengthen by several per cent in the presence of ATP and magnesium ions were prepared by teasing indirect flight muscle of Drosophila in solutions containing ethylenediaminetetraacetate. A study was made of the hydrogen ion, magnesium ion, ATP, and potassium chloride concentrations with which this effect could be observed. The lack of elongation with pyrophosphate and several nucleoside triphosphates suggests that the lengthening is ATP specific. A relaxing factor system comparable to that described for rabbit muscle was not demonstrable, as elongated fibrils did not shorten with calcium ions, carnosine, or digitonin.     

Is muscle co-activation a predisposing factor for low back pain development during standing? A multifactorial approach for early identification of at-risk individuals

Purpose and scopeLow back pain development has been associated with static standing postures in occupational settings. Previous work has demonstrated gluteus muscle co-activation as a predominant pattern in previously asymptomatic individuals who develop low back pain when exposed to 2-h of standing. The purpose of this work was to investigate muscle co-activation as a predisposing factor in low back pain development while including a multifactorial approach of clinical assessment tools and psychosocial assessments to identify individuals who are at risk for pain development during standing.ResultsForty percent of participants developed low back pain during the 2-h of standing. Pain developers demonstrated bilateral gluteus medius and trunk flexor–extensor muscle co-activation prior to reports of pain development. Pain developers and non-pain developers demonstrated markedly different patterns of muscle activation during the 2-h of standing. A novel screening test of active hip abduction was the only clinical assessment tool that predicted pain development.ConclusionsGluteus medius and trunk muscle co-activation appears to be a predisposing rather than adaptive factor in low back pain development during standing. A combination of a positive active hip abduction test and presence of muscle co-activation during standing may be useful for early identification of at-risk individuals.     

Biological study of muscle degenerating hemorrhagic factor from the venom of Vipera aspis aspis (aspic viper)

1. A hemorrhagic factor was isolated from Vipera a. aspis venom by gel filtration, ion-exchange chromatography and affinity chromatography. 2. Molecular weight of the purified factor was determined to be 67,000 Da, which was composed of 552 amino acid residues. 3. The minimum hemorrhagic dose (MHD) was measured to be 0.11 micrograms per mouse with subcutaneous injection. The hemorrhagic activity was inhibited by chelating agents and reductant. 4. The hemorrhagic factor possesses proteolytic activity against dimethylcasein. 5. Serum creatine phosphokinase level was rapidly increased within 30 min following injections of this preparation into the mice thighs. 6. Actin, one of the main components of muscle fiber, is apparently digested by the hemorrhagic factor. The pathological findings are further reported in this paper.     

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.     

Human umbilical vein endothelium-derived cells retain potential to differentiate into smooth muscle-like cells

Mouse embryonic stem-derived cells were recently shown to differentiate into endothelial and smooth muscle cells. In the present study, we investigated whether human umbilical vein endothelium-derived cells retain the potential to differentiate into smooth muscle cells. Examination of biochemical markers, including basic calponin, SM22alpha, prostaglandin E synthase, von Willebrand factor, and PECAM-1, as well as cell contractility, showed that whereas endothelium-derived cells cultured with fibroblast growth factor can be characterized as endothelial cells, when deprived of fibroblast growth factor, a significant fraction differentiates into smooth muscle-like cells. Reapplication of fibroblast growth factor reversed this differentiation. Activin A was up-regulated in fibroblast growth factor-deprived, endothelium-derived cells; moreover, the inhibitory effects of exogenous follistatin and overexpressed Smad7 on smooth muscle-like differentiation confirmed that the differentiation was driven by activin A signaling. These findings indicate that when deprived of fibroblast growth factor, human umbilical vein endothelium-derived cells are capable of differentiating into smooth muscle-like cells through activin A-induced, Smad-dependent signaling, and that maintenance of the endothelial cell phenotype and differentiation into smooth muscle-like cells are reciprocally controlled by fibroblast growth factor-1 and activin A.