Peritubular myoid cells of human and rat testis are smooth muscle cells that contain desmin-type intermediate filaments

We studied the cytoskeletal composition of human and rat testicular myoid cells by using immunofluorescence microscopy with polyclonal and monoclonal antibodies. In adult human and rat testis, the peritubular myoid cell layer was brightly positive for desmin, the muscle type of intermediate filament protein, and a faint reaction was also seen with antibodies to vimentin, the intermediate filament protein of fibroblasts and diverse other mesenchymal cells. The desmin-positive myoid cell layer could already be identified in newborn rat testis but was more compact in appearance 23 days after birth. Both squash preparations and cultured cells from adult rat seminiferous tubules revealed distinct cell populations positive for desmin. The adult myoid cells of both species also showed a strong reaction with antibodies to myosin and p230, a nonerythroid avian alpha-spectrin analogue. The immunostaining results could be confirmed by the western blotting technique: Experiments with isolated seminiferous tubules showed a specific reaction with a 55,000-dalton and a 58,000-dalton polypeptide when desmin and vimentin antibodies were used, respectively. The present results show that the peritubular myoid cells are genuine smooth muscle cells with desmin-type intermediate filament cytoskeleton and suggest that these cells can be identified by this feature before their ultrastructural maturation.     

Mesenchymal and muscle-specific intermediate filaments (vimentin and desmin) in relation to differentiation in childhood rhabdomyosarcomas

Twenty-one childhood rhabdomyosarcomas were divided into three groups on the basis of cytologic composition. The tumors in group P consisted entirely of primitive mesenchymal cells, whereas those in groups M and W were characterized by the additional presence of numerous round rhabdomyoblasts and strap cells, respectively. The tumors were studied for the universal mesenchymal intermediate filament vimentin, and for the muscle-specific intermediate filament desmin. Vimentin positivity, which tended to be more prominent in primitive tumor cells, was found in all tumors, whereas desmin was found especially in round rhabdomyoblasts and strap cells. Desmin-positive primitive cells were found only in groups M and W, not in group P. It was concluded that the differentiation from primitive mesenchymal cells to morphologically recognizable myogenic tumor cells is accompanied by an increase in desmin positivity and, presumably, a decrease in vimentin positivity. Moreover, the observations suggest the existence of a group of "committed" cells that are morphologically primitive, but desmin-positive. These cells might play an important role in the observed further differentiation of rhabdomyosarcomas under chemotherapy.     

The detection of smooth muscle antibodies reacting with intermediate filaments of desmin type

Some human smooth muscle antibodies (SMA) react with cytoskeletal intermediate filament (IMF) antigens. The smooth muscle tissue contains two types of IMF: vimentin and desmin filaments. In this study, SMA of anti-IMF type in 52 patients' sera have been classified into anti-vimentin filament and anti-desmin filament types according to their immunofluorescence staining patterns on rat testis. This classification is based on the fact that the arterial walls of testis contains both vimentin and desmin whereas the myoid cell layer surrounding the seminiferous tubuli contains only desmin. Four out of the 52 sera gave the anti-desmin staining pattern and 40 sera showed the anti-vimentin type of staining. Thirty-two sera were further classified by using cultured human rhabdomyosarcoma (RD) cells as targets. Nine sera reacted with the intermediate filaments of the RD cells. Among these were 3 out of the 4 sera that gave the anti-desmin filament staining pattern. The anti-desmin specificity of SMA was confirmed in 1 serum by the immunoblotting technique. These results indicate that while human anti-desmin filament antibodies exist, most human SMA of anti-IMF type react with vimentin filaments.     

Disease mutations in the “head” domain of the extra-sarcomeric protein desmin distinctly alter its assembly and network-forming properties

The intermediate filament protein desmin generates an extra-sarcomeric network in myocytes. Mutations in the desmin gene cause myofibrillar myopathy characterized by desmin-positive aggregates and myofibrillar dissolution. Past analysis revealed that the non-α-helical amino-terminal “head” domain of desmin is a vital coordinator of protein assembly. We have now characterized assembly and network-forming properties of five recently discovered myopathy-causing mutations residing in this domain. In vitro analyses with recombinant proteins show that two mutant variants residing in a conserved nonapeptide motif “SSYRRTFGG”—Ser13Phe and Arg16Cys—interfere with assembly by forming filamentous aggregates. Consistent with in vitro data, both mutant proteins are unable to generate a bona fide filament system in cells lacking an intermediate filament cytoskeleton. In cells expressing vimentin or desmin, both mutants firstly fail to integrate into the endogenous filament network and secondly severely affect its cellular localization. The other three mutations—Ser2Iso, Ser46Phe, and Ser46Tyr—influence in vitro filament properties less severely, but in vivo, Ser46Phe and Ser46Tyr impair de novo filament formation. These effects of the “head” mutant proteins on endogenous intermediate filament system and their competition for binding to cellular anchoring structures might explain part of the molecular mechanism that causes disease.     

Distribution of intermediate filament proteins in normal and diseased human glomeruli.

The distribution of intermediate filament proteins (vimentin, desmin, and cytokeratin) was studied by means of immunofluorescence in the normal human and rat glomerulus and in pathologic human glomeruli. Antifibronectin antibodies were used as mesangial markers. In normal human glomeruli, vimentin antibodies stained endothelial cells, podocytes, and mesangial cells; desmin antibodies, surprisingly, stained podocytes. In normal rat glomeruli, the pattern of vimentin staining was the same as in humans, but desmin antibodies stained both mesangial cells and podocytes. In human and rat glomeruli cytokeratin staining was confined to segments of Bowman's capsule. In human pathologic glomeruli, vimentin and desmin antibodies stained the structures that were positive in normal glomeruli, giving a characteristic pattern for each pathologic condition examined. These results are compatible with the mesenchymal origin of podocytes and mesangial cells and suggest that both cells have smooth muscle-like phenotypic features. Mesangial cells may have slightly different differentiation paths in humans and rats, leading to a distinct expression of intermediate filament proteins.     

Proteins of intermediate filaments. An immunohistochemical and biochemical approach to the classification of soft tissue tumors

The intermediate filament cytoskeleton of various types of human soft tissue tumors was analyzed by immunofluorescence microscopy with the use of specific antibodies against cytokeratins, vimentin, and desmin, as well as by one- and two-dimensional gel electrophoresis of high-salt buffer- and detergent-resistant cytoskeletal preparations. All leiomyomas as well as a leiomyosarcoma contained desmin. Leiomyomas of both gastrointestinal and uterine derivation and the retroperitoneal leiomyosarcoma showed strong reaction for desmin in the smooth muscle cells, but the latter two exhibited also vimentin staining. In embryonal rhabdomyosarcomas, desmin prevailed in the large, apparently well-differentiated rhabdomyoblasts; whereas the smaller, less differentiated tumor cells preferentially contained vimentin. Cells of malignant fibrous histiocytomas were characterized by their content of vimentin as the only intermediate filament protein present. In alveolar soft part sarcoma, a rare tumor of hitherto unknown histogenesis, vimentin and desmin co-existed within the same tumor cells, indicating, together with chemical determinations, the myogenic derivation of this neoplasm. The results show that immunologic and biochemical analysis of proteins associated with the intermediate filament cytoskeleton is a useful adjunct in the diagnosis of diverse neoplasms, particularly those with equivocal histologic features, and thus aids in the histogenetic classification of soft tissue tumors.     

Coexpression of intermediate filaments in squamous cell carcinomas of upper aerodigestive tract before and after radiation and chemotherapy

Frozen sections of 48 squamous cell carcinomas and seven undifferentiated carcinomas of the upper aerodigestive tract were investigated immunohistochemically with monoclonal antibodies specific for keratin, vimentin, desmin, neurofilaments, and glial fibrillary acidic proteins. In nine squamous cell carcinomas (19%) and six undifferentiated carcinomas (86%) obtained before treatment coexpression of keratin and vimentin was detected in some tumor cells by double immunofluorescence studies. Nine squamous cell carcinomas expressed neurofilaments in scattered tumor cells. Coexpression of vimentin or neurofilaments was seen especially in the peripheral cell layer of the tumor nests and did not seem to correlate with the degree of differentiation. Three undifferentiated carcinomas additionally expressed desmin, and one tumor contained neurofilaments. Glial fibrillary acidic proteins were not detected. Increased coexpression of keratin with vimentin, desmin, or neurofilaments was seen in some tumors that were studied before and after radiation/chemotherapy, suggesting that the intermediate filament profile of tumor cells can be altered by external influences.     

Effects of extracellular matrix on phenotype modulation and MAPK transduction of rat aortic smooth muscle cells in vitro

The transition of arterial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype may play an essential role in the formation of atherosclerotic and restenotic lesions. This process includes a prominent structural reorganization and allows cells to acquire the ability to migrate, proliferate, and secrete extracellular matrix components. According to Western blotting analysis and immunohistochemical and morphological observations, laminin not only retains SMCs in a contractile state but also possibly stimulates cells to transform a synthetic to a contractile phenotype at an early stage, mediated by P38 MAPK signal transduction. However, fibronectin promotes SMCs to transform from a contractile to a synthetic phenotype, mediated by the ERK MAPK signal pathway. The localization of smooth muscle alpha -actin, myosin heavy chain isoform SM2, and vimentin in explant-isolated rat SMCs was affected by a substrate of fibronectin and laminin and also by ERK MAP kinase inhibitor (PD098059) and P38 MAPK inhibitor (SB203580). Furthermore, vimentin may play a much more important role in differentiation than desmin in phenotype modulation in rat aortic smooth muscle cells.     

Alterations in intermediate filaments expression in disc cells from the rat temporomandibular joint following exposure to continuous compressive force

The articular disc in the temporomandibular joint (TMJ) that serves in load relief and stabilizing in jaw movements is a dense collagenous tissue consisting of extracellular matrices and disc cells. The various morphological configurations of the disc cells have given us diverse names, such as fibroblasts, chondrocyte-like cells and fibrochondrocytes; however, the characteristics of these cells have remained to be elucidated in detail. The disc cells have been reported to exhibit heterogeneous immunoreaction patterns for intermediate filaments including glial fibrillary acidic protein (GFAP), nestin and vimentin in the adult rat TMJ. Because these intermediate filaments accumulate in the disc cells as tooth eruption proceeds during postnatal development, it might be surmised that the expression of these intermediate filaments in the disc cells closely relates to mechanical stress. The present study was therefore undertaken to examine the effect of a continuous compressive force on the immunoexpression of these intermediate filaments and an additional intermediate filament - muscle-specific desmin - in the disc cells of the TMJ disc using a rat experimental model. The rats wore an appliance that exerts a continuous compressive load on the TMJ. The experimental period with the appliance was 5 days as determined by previous studies, after which some experimental animals were allowed to survive another 5 days after removal of the appliance. Histological observations demonstrated that the compressive force provoked a remarkable acellular region and a decrease in the thickness of the condylar cartilage of the mandible, and a sparse collagen fiber distribution in the articular disc. The articular disc showed a significant increase in the number of desmin-positive cells as compared with the controls. In contrast, immunopositive cells for GFAP, nestin and vimentin remained unchanged in number as well as intensity. At 5 days after removal of the appliance, both the disc and cartilage exhibited immunohistological and histological features in a recovery process. These findings indicate that the mature articular cells are capable of producing desmin instead of the other intermediate filaments against mechanical stress. The desmin-positive disc cells lacked α-smooth muscle actin (α-SMA) in this study, even though desmin usually co-exists with α-SMA in the vascular smooth muscle cells or pericytes. Because the precursor of a pericyte has such an immunoexpression pattern during angiogenesis, there is a further possibility that the formation of new vessels commenced in response to the extraordinary compressive force.     

Cytomorphology of atherosclerosis: smooth muscle cells in formation and regression of atherosclerotic lesions

Human fibrous atherosclerotic plaques from the superficial femoral artery, obtained at surgery and autopsy, were examined by transmission electron microscopy and immunofluorescence microscopy for antibodies to intermediate filament proteins (IF) vimentin, and desmin. The results showed that smooth muscle cells (SMC) of human fibrous plaques accounted for the predominant cell type, demonstrating a spectrum of phenotypes, with prevalence of cells with prominent rough endoplasmic reticulum, implying a synthetic phenotype. In regressing atherosclerotic plaques, contractile SMC were predominant and, along with them, few SMC with abundant cellular organelles were observed, displaying ultrastructural manifestation of collagen phagocytosis. SMC of both progressing and regressing atherosclerotic lesions, regardless of the morphological phenotype of the cell, contained vimentin IF and lacked desmin IF, the marker of differentiated SMC. These observations suggest the existence of a distinctive subpopulation of SMC in the human arterial intima that differs substantially from normal medial SMC by ultrastructure, biochemistry, and function.     

Intermediate filament proteins in developing human arteries

 The distribution of intermediate filament proteins in adult human blood vessels and in human fetal elastic arteries is relatively well-known. However, the distribution of these proteins in the course from neonate to adult has not been established. In this investigation, human postnatal arteries were studied with immunohistochemistry, using antibodies targeted on the intermediate filament proteins desmin, vimentin and cytokeratins 8, 18 and 19. Vimentin was present in most smooth muscle cells in all vessels and at all ages. The proportions of desmin-expressing cells increased in the elastic arteries during the first year of life and was higher in the pulmonary trunk than in the aorta. In the muscular arteries, the proportion of desmin-labelled cells increased in the coronary and the deep femoral arteries, but remained constant in the renal and the cerebral arteries. Cytokeratins were detected in the pulmonary trunk earlier than in the aorta. Cytokeratins were present throughout the wall of the ductus arteriosus, but desmin was present only in some cells. Thus, there are postnatal changes in the distribution of intermediate filament proteins in the elastic arteries and in some muscular arteries, whereas the intermediate filament pattern remains unchanged in other muscular arteries. Accepted: 31 July 1998     

Elucidation of GlcNAc‐binding properties of type III intermediate filament proteins,using GlcNAc‐bearing polymers

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin belong to type III intermediate filament family and are expressed in mesenchymal cells, skeletal muscle cells, astrocytes and peripheral neurons, respectively. Vimentin and desmin possess N‐acetyl‐d ‐glucosamine (GlcNAc)‐binding properties on cell surfaces. The rod II domain of these proteins is a GlcNAc‐binding site, which also exists in GFAP and peripherin. However, the GlcNAc‐binding activities and behaviors of these proteins remain unclear. Here, we characterized the interaction and binding behaviors of these proteins, using various well‐defined GlcNAc‐bearing polymers synthesized by radical polymerization with a reversible addition‐fragmentation chain transfer reagent. The small GlcNAc‐bearing polymers strongly interacted with HeLa cells through vimentin expressed on the cell surface and interacted with vimentin‐, desmin‐, GFAP‐ and peripherin‐transfected vimentin‐deficient HeLa cells. These proteins present high affinity to GlcNAc‐bearing polymers, as shown by surface plasmon resonance. These results show that type III intermediate filament proteins possess GlcNAc‐binding activities on cell surfaces. These findings provide important insights into novel cellular functions and physiological significance of type III intermediate filaments.     

Desmin is a specific marker for rhabdomyosarcomas of human and rat origin.

Putative human rhabdomyosarcoma (RMS) has been divided into two groups according to desmin content. Twenty-five tumors with histologic features consistent with but not necessarily sufficient to prove a diagnosis of RMS were desmin-positive. More than 95% of the tumor cells were desmin-positive, suggesting a muscle origin and supporting the diagnosis of RMS. Nine tumors for which the preferred first histologic diagnosis was also RMS were desmin-negative. Reexamination of the original histologic slides together with results from intermediate filament typing resulted in a diagnosis other than RMS for all tumors in this second group, and in several instances other tests were used to prove the correctness of the final diagnosis. The results on human material were extended to a rat model system in which RMS was induced by nickel sulfide. Again, all 24 tumors tested were desmin-positive. Vimentin was coexpressed in a varying percentage of tumor cells in RMS of human and rat origin. The results show that desmin is an excellent marker for rhabdomyosarcoma, yielding few if any false-positive or false-negative results in frozen or alcohol-fixed material.     

Coexpression of intermediate filament polypeptides in human fetal and adult tissues

Tissues from human fetuses (12 to 14 weeks) were studied by using immunohistochemical methods, with special emphasis on coexpression of intermediate filaments. Well-characterized antibodies, monoclonal as well as polyclonal were used. Indirect immunoperoxidase staining disclosed simultaneous expression of cytokeratin and vimentin, or vimentin and desmin in several tissues, whereas some other tissues coexpressed three classes of intermediate filaments, i.e., cytokeratin, vimentin, and desmin. Coexpression of cytokeratin and vimentin was seen in immature tubules of the kidney localized in the blastema and in one case in a small area of the epithelium of the tongue. Coexpression of vimentin and desmin was found in stromal cells of the medulla of the kidney, in stromal cells of the decidua (maternal tissue) and in muscle cells in blood vessels of small intestine, kidney and decidua. Coexpression of cytokeratin, vimentin, and desmin was present in mesothelial cells of serosa, pleura and pericardium, in stroma of umbilical cord and placental villi, in muscle cells of small intestine, tongue, and heart, and in muscle cells of blood vessels of lung, heart, umbilical cord, and placental villi. Mesothelium and reactive submesothelial stroma cells also coexpressed three classes of intermediate filament polypeptides. In some cases, immunoperoxidase results were confirmed by double labeling immunofluorescence microscopy or by immunoblotting experiments. The results of this study indicate that coexpression of different types of intermediate filaments is a more general phenomenon in fetal tissues than previously realized and it also occurs in some reactive proliferative lesions in the adult.     

Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro

Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Here, we report the pathogenic effects of a novel heterozygous R350P desmin missense mutation, which resides in the evolutionary highly conserved coil 2B domain of the alpha-helical coiled-coil desmin rod domain, on the assembly of desmin intermediate filaments (IF) in cultured cells and in vitro. By transfection experiments, we show that R350P desmin is incapable of de novo formation of a desmin IF network in vimentin-free BMGE+H, MCF7 and SW13 cells and that it disrupts the endogenous vimentin cytoskeleton in 3T3 fibroblast cells. Hence, transfected cells displayed abnormal cytoplasmic protein aggregates reminiscent of desmin-positive protein deposits seen in the immunohistochemical and ultrastructural analysis of skeletal muscle derived from the index patient of the affected family. To study the functional effects of the R350P desmin mutation at the protein level, we performed in vitro assembly studies with wild-type (WT) and mutant desmin protein. Our analysis revealed that the in vitro assembly process of R350P desmin is already disturbed at the unit length filament level and that further association reactions generate huge, tightly packed protein aggregates. On assessing the pathogenic effects of R350P desmin in various mixtures with WT desmin, we show that a ratio of 1 : 3 (R350P desmin/WT desmin) is sufficient to effectively block the normal polymerization process of desmin IFs. Our findings indicate that the heterozygous R350P desmin mutation exerts a dominant negative effect on the ordered lateral arrangement of desmin subunits. This disturbance of the lateral packing taking place in the first phase of assembly is ultimately leading to abnormal protein aggregation.     

Desmin-containing stellate cells in rat liver; distribution in normal animals and response to experimental acute liver injury

Using immunohistochemical methods, we have confirmed that the perisinusoidal cells in rat liver express the intermediate filament protein, desmin, and we have used this marker for identification of the cells on light microscopy. The study has been extended to quantify the response of perisinusoidal cells to acute liver injury induced by carbon tetrachloride. A significant increase in desmin-positive cells was observed in areas of necrosis as early as 48 h following the administration of a single bolus of carbon tetrachloride. This reached a peak at 72 h, with a five-fold increase in desmin-positive cells in areas of necrosis. These observations are consistent with the hypothesis that perisinusoidal cells are involved in the response to acute liver injury. Anti-desmin antibodies are of potential value in further characterizing the functional role of perisinusoidal cells in normal and diseased liver.     

Intermediate filaments in smooth muscle tumours

Antisera to the intermediate filaments vimentin and desmin react with fixed paraffin embedded tissue. Benign uterine myomas contain both classes of filaments. Gastrointestinal "smooth muscle tumours" however often lack desmin even when they appear histologically benign. In the sarcomas examined vimentin was the only class of intermediate filament present. The diagnostic and histogenetic implications of these findings are discussed.     

Immunohistochemistry of cytoskeletal filaments in the diagnosis of soft tissue tumors

There is abundant evidence that intermediate filaments can be used as cell type specific markers both for normal tissue and for tumors. The results of intermediate filament typing in soft tissue tumors and its diagnostic relevance is shown. This system can also be used to solve other problems in surgical pathology. One of the most useful applications of intermediate filament typing is to differentiate the round cell tumors of children; thus, rhabdomyo-sarcomas are desmin positive, malignant lymphomas contain only vimentin, and neuroblastomas show positivity for neurofilaments.