Differential processes of vascular smooth muscle cell differentiation within elastic and muscular arteries of rats and rabbits: An immunofluorescence study of desmin and vimentin distribution

Summary Two main populations of smooth muscle cells exist in the arterial media of adult mammals with respect to expression of two intermediate filament proteins: vimentin-positive/desmin-negative cells (V+/D-) and vimentin-positive/desmin-positive ones (V+/D+). However, it is still not understood how this phenotypic diversity is established. Since the proportion and the distribution patterns of the two muscle cell populations depend both on the type of blood vessel and the species examined, the aim of the present study was to determine and to compare their developmental origin in various artery segments from two different species. Using confocal scanning laser microscopy and sections stained by means of immunofluorescence, the distribution patterns of desmin and vimentin were compared in transverse sections of thoracic and abdominal aortas (elastic arteries) and of the femoral artery (muscular artery) of newborn and adult rats (n=12) and rabbits (n=12). The comparison of sections labelled with specific antibodies showed the existence of a subpopulation of smooth muscle cells in the aortas, but not in the femoral artery which expressed desmin in newborns but not in adults. These data suggest that the phenotype of smooth muscle cells in elastic arteries but not in muscular arteries is modulated during development.     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

Smoothelin in adult and developing human arteries and myocardium

The aim of this investigation was to study, with immunohistochemical methods, the distribution of the novel cytoskeletal protein smoothelin in human cardiovascular tissues, the possible changes during the development of the cardiovascular system and its correlation to the intermediate filament proteins desmin and vimentin. Smoothelin was detected in smooth muscle cells of the fetal coronary arteries. In very young subjects (up to 3 months of age), only a few cells in the media of the elastic arteries contained smoothelin, whereas it was present in most smooth muscle cells in the muscular arteries. In individuals older than 1 year, most smooth muscle cells in the media of all blood vessels contained smoothelin. In vessels with a developed intima, smoothelin was present in a variable proportion of the smooth muscle cells. With few exceptions, smoothelin was more frequently detected than desmin in medial smooth muscle cells. Smoothelin and vimentin were codistributed in the smooth muscle cells of the media in most vessels. In the cardiomyocytes (fetal to adult age), the smoothelin antibody detected epitopes located at the Z-disc level but not in the intercalated discs. In conclusion, smoothelin is more widely distributed in the muscular arteries than in the elastic arteries early in life, and thus exhibits a variable distribution during postnatal development of vascular tissues. In the adult, smoothelin is detected in the media of most vascular smooth muscle cells, both in muscular and elastic arteries, and is not necessarily codistributed with either desmin or vimentin. Evidence that smoothelin is present in human striated cardiomyocytes is also presented. Accepted: 16 July 1999     

Expression of intermediate filament-associated proteins paranemin and synemin in chicken development

The expression of two intermediate filament-associated proteins, paranemin (280,000 mol wt) and synemin (230,000 mol wt), was investigated with respect to the expression of two core intermediate filament proteins, desmin and vimentin, in various embryonic and adult chicken muscle and nonmuscle cells. All developing muscle cells, regardless of their type, simultaneously express desmin, vimentin, paranemin, and synemin. However, a difference is observed in the expression of paranemin in adult muscle. This protein is removed during differentiation of both fast and slow skeletal muscle, visceral smooth muscle, and the smooth muscle of muscular arteries, but remains in mature myocardial cells, cardiac conducting fibers, and the smooth muscle cells of elastic arteries. Some of these cells express vimentin, others desmin, and still others a mixture of the two. On the other hand, synemin is expressed in all the above types of adult muscle cells except myocardial cells. Adult myocardial cells also lack vimentin, and its presence is gradually reduced after hatching. Since in adult striated muscle all expressed intermediate filament proteins are found predominantly in association with the peripheries of myofibrillar Z discs, these results suggest that a change in the composition of skeletal and cardiac muscle Z discs occurs during chicken development and maturation. Erythrocytes that express synemin and vimentin do not express paranemin, while both embryonic and adult Schwann cells co- express paranemin and vimentin, but not synemin. Endothelial cells of muscular vessels express paranemin, while those of elastic vessels do not, and neither contains synemin. Paranemin and synemin are not expressed in neurons, epithelial, and most glial cells, suggesting that these two polypeptides are expressed only in conjunction with desmin or vimentin. These results suggest that the composition of intermediate filaments changes during chicken development, not only with respect to their core subunit proteins but also with respect to two associated polypeptides, particularly in muscle cells.     

Intermediate filament heterogeneity in normal and hypercholesterolemic rabbit vascular smooth muscle cells

We have examined the intermediate filament (IF) protein content of vascular smooth muscle (SM) cells from several arteries and veins in rabbits and quantitated the changes which occur in SM cell expression of these proteins in response to cholesterol feeding. Cells from control rabbit arteries expressed 30% of their IF protein as desmin, while veins expressed 50% as desmin. During development of diet-induced atherosclerosis, morphological changes in arterial SM cells in the intima correlate with changes in IF expression. There is a significant increase in total IF protein content, vimentin increased differentially in thoracic aorta and desmin in pulmonary artery. In abdominal aorta both increase equally. Cholesterol feeding also resulted in changes in the expression of subspecies of desmin, vimentin, and actin in the thoracic arch. Although cholesterol feeding did not produce obvious morphological changes in the veins examined, venous SM IF protein expression was also altered. In the vena cava of cholesterol-fed rabbits there was an increase in vimentin expression without the parallel increase in desmin that occurred in the arterial system. These studies show that cholesterol feeding of rabbits induces measurable changes in the amounts of IF proteins in both arterial atherosclerotic lesions and venous SM cells.     

Localization of vimentin and desmin in BHK21/C13 cells and in baby hamster kidney

Specific antibodies against the intermediate filament protein subunits, desmin and vimentin, were used to characterize the fibroblastic tissue culture cell line BHK21/C13 and the cells comprising baby hamster kidney (BHK). The BHK21/C13 cells have previously been shown to contain desmin and vimentin by biochemical techniques. The results from double immunofluorescence analysis show that both immunologically distinct intermediate filament subunit proteins are expressed simultaneously within the same BHK21/C13 cell, and that the filamentous patterns are very similar, if not superimposable even in cells treated with colchicine. There are some cells that may contain vimentin only. Double immunofluorescence on cryostat sections of BHKs and preparations of dissociated kidney cells demonstrate that the cells, most likely smooth muscle, comprising the blood vessel walls contain vimentin and desmin simultaneously. The simultaneous expression of vimentin and desmin is not a phenomenon which is restricted to tissue culture cells. Thus, the simultaneous presence of these two intermediate filament proteins within the BHK21/C13 cell may not be the result of growth in tissue culture.     

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells.

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle alpha-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3-0.5% of cells were found to be desmin-positive, and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.     

Diagnosis of human childhood rhabdomyosarcoma of antibodies to desmin, the structural protein of muscle specific intermediate filaments

Four embryonal rhabdomyosarcomas, one tumor diagnosed as an undifferentiated sarcoma, probably a rhabdomyosarcoma, and six different non-muscular sarcomas were investigated with antibodies specific for different intermediate filament types. The tumor cells in the rhabdomyosarcomas and the undifferentiated tumor were stained clearly by antibodies to desmin, the intermediate filament type characteristic of muscle. The staining of tumor cell by antibodies to vimentin, the intermediate filament type characteristic of certain cell types of mesenchymal origin including myoblasts, was different in these 5 cases. In one case of embryonal rhabdomyosarcoma nearly all tumor cells were stained, but in the remaining cases few or no tumor cells were positive with the vimentin antibody. In these rhabdomyosarcomas not only the large rhabdomyoblasts, but also the small undifferentiated cells were labeled by antibodies to desmin. In the latter cell type the desmin filaments were arranged typically in coils. In contrast, tumor cells in the non-muscular mesenchymal sarcomas were stained only by antibodies to vimentin but not by antibodies to desmin or prekeratin. The retention of the desmin marker characteristic of normal muscle in cases of rhabdomyosarcoma not only allowed the undifferentiated desmin-positive sarcoma to be classified as rhabdomyosarcoma but also suggests that the use of antibodies to desmin could be very helpful in the future for the diagnosis of undifferentiated rhabdomyosarcomas.     

Correlation of endothelial vimentin content with hemodynamic parameters

 In mammalian species, vimentin is the sole intermediate filament protein of endothelial cells lining the chambers of the heart and the inner surface of large blood vessels. Obvious quantitative differences in the vimentin-like immunoreactivity of endothelial cells observed in different vascular segments led us to undertake a systematic survey on the endothelial content of vimentin throughout the heart chambers, the vena cava, the pulmonary trunk, and the aorta of the pig. Immunostaining and immunoblotting showed that vimentin in endothelial cells of cardiovascular segments exposed to high shear stress and blood pressure (pulmonary trunk, aorta, left ventricle) is approximately 2- to -3-fold higher than in endothelial cells exposed to lower levels of hemodynamic stress (vena cava, left and right atria, right ventricle). Throughout the aorta, an approximately 1.5-fold increase in the vimentin contents was observed in a proximal to distal direction. The total endothelial amount of vimentin was determined to be 1.2% (inferior vena cava) and 2–3.5% (aorta) of total cellular protein. These data support the notion that the endothelial vimentin cytoskeleton can adapt to different hemodynamic loads, indicating that vimentin might help endothelial cells to withstand the mechanical forces exerted by blood flow and blood pressure. Accepted: 2 February 1998     

Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.     

Expression of intermediate filament proteins in fetal and adult human lung tissues

The expression patterns of intermediate filament proteins in fetal and normal or nonpathological adult human lung tissues are described using (chain-specific) monoclonal antibodies. In early stages of development (9-10 weeks and 25 weeks of gestation) only so-called simple cytokeratins such as cytokeratins 7 (minor amounts). 8, 18 and 19 are detected in bronchial epithelial cells. At later stages of development, the cytokeratin expression patterns become more complex. The number of bronchial cells positive for cytokeratin 7 increases, but basal cells in the bronchial epithelium remain negative. These latter cells show, however, expression of cytokeratin 14 in the third trimester of gestation. Developing alveolar epithelial cells express cytokeratins 7, 8, 18 and 19. In adult human bronchial epithelium cytokeratins 4 (varying amounts), 7, 8, 13 (minor amounts), 14, 18 and 19 can be detected, with the main expression of cytokeratins 7, 8, and 18 in columnar cells and the main expression of cytokeratin 14 in basal cells. Vimentin is detected in all mesenchymal tissues. In addition, fetal lung expresses vimentin in bronchial epithelium, however, to a lesser extent with increasing age, resulting in the expression of vimentin in only few scattered bronchial cells at birth. Also in adult bronchial epithelium the expression of vimentin is noticed in part of the basal and columnar epithelial cells. Desmin filaments, present in smooth muscle cells of the lung, appear to alter their protein structure with age. In early stages of development smooth muscle cells surrounding blood vessels are partly reactive with some cytokeratin antibodies and with a polyclonal desmin antibody. At week 9-10 and week 25 of gestation a monoclonal antibody to desmin, however, is not reactive with blood vessel smooth muscle cells but is only reactive with smooth muscle cells surrounding bronchi. With increasing age the reactivity of cytokeratin antibodies with smooth muscle cells in blood vessels decreases, while the reactivity with the monoclonal desmin antibody increases. Our results show that during differentiation profound changes in the intermediate filament expression patterns occur in the different cell types of the developing lung.     

Intermediate filaments in the testis of the teleost mosquito fish Gambusia affinis holbrooki: a light and electron microscope immunocytochemical study and Western blotting analysis

Summary A light and electron microscope immunocytochemical study and Western blotting analysis has been performed on intermediate filaments (vimentin, desmin and cytokeratins) in the testis of the teleost fish Gambusia affinis holbrooki. An immunoreaction to vimentin was observed in the epithelium of the efferent ducts, testicular canal and their surrounding peritubular cells. Positive vimentin immunostaining was also observed in the cells located around seminiferous tubules (boundary cells), Leydig cells, interstitial fibroblasts, chromatophores, and blood vessel endothelial cells. In contrast to mammals, no vimentin immunoreactivity was found in the Sertoli cells. Immunoreactivity to desmin was weak in the epithelial cells of the efferent ducts and testicular canal and intense in the peritubular cells that surrounded these ducts. Desmin immunoreactivity was also observed in the seminiferous tubule boundary cells. The immunoreactivity was weak in the boundary cells that surrounded germ cell cysts containing spermatogonia or spermatocytes and intense in the boundary cells around cysts with elongated or mature spermatids. Immunoreactivity towards cytokeratins was observed only in testicular blood vessels. Cytokeratin immunolabelling was intense in the endothelium and weak in the vascular smooth muscle cells. No cytokeratin immunoreactivity was found in the Sertoli cells, germ cells, interstitial cells or in the efferent duct epithelium. The absence of intermediate filaments in the Sertoli cells, the absence of cytokeratins in the epithelium of the sperm excretory ducts, and the presence of desmin filaments in these epithelial cells are the most important differences with regards to the intermediate filament phenotype in mammalian testes.     

Visualization of intermediate filaments in living cells using fluorescently labeled desmin

Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.     

Intermediate filaments in smooth muscle

The intermediate filament (IF) network is one of the three cytoskeletal systems in smooth muscle. The type III IF proteins vimentin and desmin are major constituents of the network in smooth muscle cells and tissues. Lack of vimentin or desmin impairs contractile ability of various smooth muscle preparations, implying their important role for smooth muscle force development. The IF framework has long been viewed as a fixed cytostructure that solely provides mechanical integrity for the cell. However, recent studies suggest that the IF cytoskeleton is dynamic in mammalian cells in response to various external stimulation. In this review, the structure and biological properties of IF proteins in smooth muscle are summarized. The role of IF proteins in the modulation of smooth muscle force development and redistribution/translocation of signaling partners (such as p130 Crk-associated substrate, CAS) is depicted. This review also summarizes our latest understanding on how the IF network may be regulated in smooth muscle. cytoskeleton; force development; vimentin; desmin     

Comparison of the numbers of plasmalemmal vesicles in arterial and venous endothelia of rats

The numbers of plasmalemmal vesicles in endothelial cells of rat blood vessels were determined on electron microscopic sections. In all vessels examined which included aorta and carotid and femoral arteries, vena cava and femoral vein, and lung and brain capillaries, the numbers were of the same order of magnitude. For arteries the numbers were about double those for the corresponding veins. About one-third of all vesicles could be stained with ruthenium red after its infusion into the vessels. The results make it improbable that differences in numbers of 'transport' vesicles in different types of blood vessel contribute significantly to the selective accumulation of atherogenic plasma proteins in arteries.     

Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo.

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structurees provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Differential Location of Different Types of Intermediate-Sized Filaments in Various Tissues of the Chicken Embryo

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11–20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to – and specific for – epithelial cells; vimentin filaments are seen – at this stage of embryogenesis – only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structures provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Coexpression of vimentin and desmin in gastric leiomyomas. An immunohistochemical study in paraffin sections

The aim of the study was to evaluate the incidence of the reactions for vimentin and desmin in gastric leiomyomas routinely processed in formalin and embedded i paraffin. The material studied included four benign leiomyomas, seven malignant leiomyomas and three malignant epithelioid leiomyomas. A positive reaction to vimentin was found in 13 out 14 leiomyomas under study. The number of neoplastic cells showing vimentin expression was larger in malignant, especially epithelioid leiomyomas than in non-malignant leiomyomas. A positive reaction for desmin in neoplastic cells was found in 9 leiomyomas 64%. One non-malignant leiomyoma showed a moderate reaction. In the remaining eight cases the reaction was weak and occurred in single neoplastic cells. Coexpression of vimentin and desmin in neoplastic cells occurred in 8 out of 9 leiomyomas with a positive reaction for desmin. Coexpression of vimentin and desmin occurred also in the smooth muscle cells of blood vessels in all 14 cases. A weak reaction for desmin or its lack in the tumour cells of leiomyomas with its marked expression in the smooth muscle cells of the blood vessels and gastric wall outside the tumor points rather to a small number of desmin filaments in the neoplastic cells than to their destruction by fixation in formalin. The occurrence of the reaction to desmin only in a limited number of neoplastic cells questions the reliability of its use in the oligopiopsy material.     

Smoothelin and intermediate filament proteins in human aortocoronary saphenous vein by-pass grafts

The aim of this immunohistochemical investigation was to study the distribution of the novel cytoskeletal protein smoothelin and the intermediate filament proteins vimentin and desmin in normal human great saphenous vein and in human aortocoronary by-pass vein grafts. Smoothelin was present in most smooth muscle cells in the media of the native vein. In the neointima of the vein grafts that had been in situ for three months or more, smoothelin was, in general, present only in few smooth muscle cells. Desmin was distributed in the same pattern as smoothelin in the native great saphenous vein. When desmin and smoothelin were present in the neointima, smoothelin was detected in more cells than desmin. Vimentin was present in most cells in all wall layers of both the native saphenous vein and the vein grafts. Vascular smooth muscle cells containing vimentin but not desmin or smoothelin are the principal cells in the neointima of human aortocoronary vein grafts. In some grafts, however, all three cytoskeletal proteins were detected in the neointima. The distribution of smoothelin and desmin in aortocoronary vein grafts support the postulate that these proteins are expressed mainly in the contractile smooth muscle cell phenotype.     

Distribution of myosin, desmin and vimentin in smooth muscle cells of human embryonic vessels

Immunofluorescent study of embryonal vessels of man using antibodies to myosin, desmin and vimentin showed heterogeneity of smooth muscle cells. It is supposed that the use of desmin as a marker of cell differentiation can increase the role of modified phenotypes in the development of the pathological process in the vascular wall.