Desmuslin, an intermediate filament protein that interacts with alpha -dystrobrevin and desmin.
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Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, alpha1-syntrophin, and the sarcoglycan complex. The precise role of alpha-dystrobrevin in skeletal muscle has not yet been determined. To study alpha-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and alpha-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity. (+info)
Long-term effect of low energy laser irradiation on infarction and reperfusion injury in the rat heart.
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Low-energy laser irradiation (LELI) has been found to modulate biological processes. The present study investigated the effect of LELI on infarct size after chronic myocardial infarction (MI) and ischemia-reperfusion injury in rats. The left anterior descending (LAD) coronary artery was ligated in 83 rats to create MI or ischemia-reperfusion injury. The hearts of the laser-irradiated (LI) rats received irradiation after LAD coronary artery occlusion and 3 days post-MI. At 14, 21, and 45 days post-LAD coronary artery permanent occlusion, infarct sizes (percentage of left ventricular volume) in the non-laser-irradiated (NLI) rats were 52 +/- 12 (SD), 47 +/- 11, and 34 +/- 7%, respectively, whereas in the LI rats they were significantly lower, being 20 +/- 8, 15 +/- 6, and 10 +/- 4%, respectively. Left ventricular dilatation (LVD) in the chronic infarcted rats was significantly reduced (50-60%) in LI compared with NLI rats. LVD in the ischemia-reperfusion-injured LI rats was significantly reduced to a value that did not differ from intact normal noninfarcted rats. Laser irradiation caused a significant 2.2-fold elevation in the content of inducible heat shock proteins (specifically HSP70i) and 3.1-fold elevation in newly formed blood vessels in the heart compared with NLI rats. It is concluded that LELI caused a profound reduction in infarct size and LVD in the rat heart after chronic MI and caused complete reduction of LVD in ischemic-reperfused heart. This phenomenon may be partially explained by the cardioprotective effect of the HSP70i and enhanced angiogenesis in the myocardium after laser irradiation. (+info)
Venous neointimal hyperplasia in polytetrafluoroethylene dialysis grafts.
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BACKGROUND: Vascular access dysfunction is the most important cause of morbidity and hospitalization in the hemodialysis population in the United States at a cost of $1 billion per annum. Venous neointimal hyperplasia (VNH) characterized by stenosis and subsequent thrombosis accounts for the overwhelming majority of pathology resulting in polytetrafluoroethylene (PTFE) dialysis graft failure. Despite the magnitude of the problem and the enormity of the cost ($1 billion), there are currently no effective therapies for the prevention or treatment of venous neointimal hyperplasia in PTFE dialysis grafts. METHODS: Tissue samples were collected from the graft-vein anastomosis of stenotic PTFE grafts during surgical revision. Specimens were graded using standard light microscopy and immunohistochemistry for the magnitude of neointimal hyperplasia and for the expression of specific cell types, cytokines, and matrix proteins. RESULTS: VNH was characterized by the (1) presence of smooth muscle cells/myofibroblasts, (2) accumulation of extracellular matrix components, (3) angiogenesis within the neointima and adventitia, and (4) presence of an active macrophage cell layer lining the PTFE graft material. Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were expressed by smooth muscle cells/myofibroblasts within the venous neointima, by macrophages lining both sides of the PTFE graft, and by vessels within the neointima and adventitia. CONCLUSIONS: Our results suggest that macrophages, specific cytokines (bFGF, PDGF, and VEGF), and angiogenesis within the neointima and adventitia are likely to contribute to the pathogenesis of VNH in PTFE dialysis grafts. Interventions aimed at these specific mediators and processes may be successful in reducing the very significant human and economic costs of vascular access dysfunction. (+info)
Mechanical properties and structure of carotid arteries in mice lacking desmin.
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OBJECTIVE: Our aim was to determine in desmin homozygous mutant mice the viscoelastic properties, the mechanical strength and the structure of the carotid artery. METHODS: To assess the viscoelastic properties of large arteries, we have performed an in vivo analysis of the diameter-, and distensibility-pressure curves of the common carotid artery (CCA) in homozygous (Des -/-), heterozygous (Des +/-) and wild-type (Des +/+) mice. To evaluate the mechanical strength, we have measured the in vitro intraluminal pressure producing the rupture of the carotid artery wall. The structure analysis of the arterial wall was based on histology and electronic microscopy. RESULTS: A lower distensibility and an increase of arterial wall viscosity were observed in Des -/- compared with Des +/+. Arterial thickness of Des -/- was similar to those of Des +/+, without changes in elastin and collagen contents. Electron microscopy revealed that the perimeter of cellular fingerlike-projections was smaller in Des -/-, indicating that the cells have lost part of their connections to the extracellular matrix. The rupture pressure was significantly lower in Des -/- (1500+/-200 mmHg) compared with Des +/+ (2100+/-80 mmHg) indicating a lower mechanical strength of the vascular wall. No significant difference was found between Des +/- and Des +/+. CONCLUSION: The desmin is essential to maintain proper viscoelastic properties, structure and mechanical strength of the vascular wall. (+info)
Pleomorphic rhabdomyosarcoma in adults: a clinicopathologic study of 38 cases with emphasis on morphologic variants and recent skeletal muscle-specific markers.
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Pleomorphic rhabdomyosarcoma (PRMS) is a rare and controversial tumor of skeletal muscle phenotype. Diagnostic criteria for PRMS by combined histology and currently available immunohistochemistry have not been clearly defined. We report 38 pleomorphic rhabdomyosarcomas in adults, explore morphologic variants, and discuss our experience with both specific and nonspecific skeletal muscle markers in these tumors. Clinical data, morphology, and immunohistochemistry were reviewed. Electron microscopy was performed. Of 38 cases, there were 28 males and 10 females. Patient ages ranged from 21 to 81 years (median = 54 y; mean = 51 y). Tumors were located in the lower extremity (n = 18), abdomen/retroperitoneum (n = 6), chest/abdominal wall (n = 5), spermatic cord/testes (n = 4), upper extremity (n = 3), and one each in the mouth and orbit. Tumor sizes ranged from 1.5 to 15.0 cm (mean = 7.3 cm; median = 6.8 cm). The cases were divided into three variants, each with large, atypical, pleomorphic polygonal rhabdomyoblasts (PRMB) with abundant eosinophilic cytoplasm in varying numbers and different morphologic backgrounds of round or spindled rhabdomyoblasts (RMB). 1. Classic PRMS: Predominantly atypical PRMB in sheets (n = 8). 2. Round cell PRMS: Clusters of PRMB throughout the tumor with a background of slightly atypical, medium-sized, round, blue RMB (n = 13). 3. Spindle cell PRMS: Scattered PRMB in a predominance of atypical spindled RMB arranged in a storiform growth pattern (n = 17). Immunohistochemistry revealed the following: myoglobin (37/38), MyoD1 (19/36), skeletal muscle myogenin (myf4; 19/34), fast skeletal muscle myosin (4/5), desmin (36/38), muscle-specific actin (MSA; 25/35), smooth muscle actin (SMA; 15/33), and muscle specific myogenin (myf3; 25/35). Immunohistochemistry was supportive of skeletal muscle differentiation with at least one positive skeletal muscle-specific marker (myoglobin, MyoD1, fast skeletal muscle myosin, or myf4). In addition, all cases had some positivity for nonspecific muscle markers (desmin, MSA, SMA, myf3). Electron microscopy (EM), performed on eight selected cases from all three morphologic groups, demonstrated definitive skeletal muscle differentiation in all cases. Follow-up, available on 30 (79%) cases, revealed that 70% of patients died of disease (mean 20 months, range 1 month-108 months), 3% were alive with disease at 12 months (n = 1); and 27% had no evidence of disease (mean, 83 mo; range, 18 to 108 mo). PRMS, a tumor of predominantly middle-aged adult males in the lower extremity, can be diagnosed by the morphologic presence of scattered PRMB with immunohistochemical evidence of at least one skeletal muscle-specific marker. There are three morphologic variants of PRMS. The appropriate diagnosis of PRMS is significant as it is a high-grade sarcoma, with an aggressive clinical course. (+info)
The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells.
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Bone marrow stem cells (BMC) develop into hematopoietic and mesenchymal lineages but have not been known to differentiate into glomerular cells. To investigate whether such differentiation is possible, a search was made for donor glomerular cells in lethally irradiated C57BL/6j (B6) mice given transplants of BMC from syngeneic mice transgenic for green fluorescence protein (GFP) ([GFP-->B6] mice). After the recipients of donor BMC manifested GFP-positive cells in their glomeruli, the numbers of such cells increased markedly, in a time-dependent manner, from 2 wk to 24 wk after bone marrow transplantation. Immunohistochemical analyses revealed that most GFP-positive cells in the glomeruli were neither macrophages nor T cells. With the use of a laser-scanning confocal microscope, GFP-positive cells were observed within the mesangium of [GFP-->B6] mice. Furthermore, indirect immunofluorescence assays demonstrated that desmin-positive cells in the glomeruli of these chimeric mice were also positive for GFP. Among glomerular cells isolated from [GFP-->B6] mice 24 wk after bone marrow transplantation and then cultured, the majority of cells (approximately 84%) stained for desmin and approximately 60% of the desmin-positive cells expressed GFP. In addition, these GFP-positive cells in the cultures contracted in response to angiotensin II stimulation. These results suggest that bone marrow-derived cells may have the potential to differentiate into glomerular mesangial cells. (+info)
Expression of R120G-alphaB-crystallin causes aberrant desmin and alphaB-crystallin aggregation and cardiomyopathy in mice.
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Upregulation of alphaB-crystallin (CryAB), a small heat shock protein, is associated with a variety of diseases, including the desmin-related myopathies. CryAB, which binds to both desmin and cytoplasmic actin, may participate as a chaperone in intermediate filament formation and maintenance, but the physiological consequences of CryAB upregulation are unknown. A mutation in CryAB, R120G, has been linked to a familial desminopathy. However, it is unclear whether the mutation is directly causative. We created multiple transgenic mouse lines that overexpressed either murine wild-type CryAB or the R120G mutation in cardiomyocytes. Overexpression of wild-type CryAB was relatively benign, with no increases in mortality and no induction of desmin-related cardiomyopathy even in a line in which CryAB mRNA expression was increased approximately 104-fold and the protein level increased by 11-fold. In contrast, lines expressing the R120G mutation were compromised, with a high-expressing line exhibiting 100% mortality by early adulthood. Modest expression levels resulted in a phenotype that was strikingly similar to that observed for the desmin-related cardiomyopathies. The desmin filaments in the cardiomyocytes were overtly affected, myofibril alignment was significantly impaired, and a hypertrophic response occurred at both the molecular and cellular levels. The data show that the R120G mutation causes a desminopathy, is dominant negative, and results in cardiac hypertrophy. (+info)
Injury induces dedifferentiation of smooth muscle cells and increased matrix-degrading metalloproteinase activity in human saphenous vein.
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Long-term patency of human saphenous vein bypass grafts is low because of intimal thickening and superimposed atherosclerosis. Matrix-degrading metalloproteinases (MMPs) and changes in vascular smooth muscle cell (VSMC) phenotype are thought to be essential for the VSMC migration that contributes to intimal thickening. We examined VSMC phenotype and MMP activity in saphenous veins obtained before and after surgical manipulation. Surgical preparation of the veins significantly increased pro-MMP-1 expression by 2-fold and significantly reduced tissue inhibitor of MMPs (TIMP)-2 expression, whereas MMP-3 and TIMP-1 were unaffected. Furthermore, caseinolytic and gelatinolytic activities measured by in situ zymography were dramatically elevated by injury. The expression of desmin and smoothelin was significantly decreased by injury, whereas vimentin expression was significantly increased. In addition, these changes in phenotype and MMP activity were localized to a subpopulation of VSMCs, the circumferential medial VSMCs. Our data show that surgical preparative injury induces phenotypic modulation of a subpopulation of medial VSMCs to a synthetic phenotype and increases MMP activity. This may favor matrix degradation, VSMC migration, and the subsequent intimal thickening that leads to graft failure. (+info)