Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.     

Primary cultures of enzyme-isolated cells from normal and atherosclerotic human aorta

A technique has been developed for isolating cells from the intimal and medial layers of the human aorta by enzymatic dispersion. After mechanical separation of intima, media and adventitia the intima and the media were dispersed by collagenase and elastase. Enzyme-isolated cells seeded in the culture with at a frequency of 30 to 50%. In the primary culture differentiated aortic cells were morphologically heterogenous. It was possible to define four main types of cells according to their shape: polygonal, elongated, asymmetrical and stellate. Polygonal and stellate cells are found only in cultures of grossly normal intima, whereas elongated and asymmetric cells are found in practically all cultures. The ratio of elongated to asymmetric cells in cultures obtained from healthy aorta and atherosclerotic plaque is more or less the same at approximately 3:1. In cultures of fatty streaks the proportion of asymmetric cells exceeds 50%. Using immunofluorescence, all four types of cell were identified as smooth muscle cells. The possible reasons for the cellular polymorphism in primary culture and the prospects of utilizing this culture for the study of cellular aspects of atherosclerosis' pathogenesis are discussed.     

The use of freeze fracturing in combination with light, scanning and transmission electron microscopy for studying the structure of the normal and atherosclerotically altered human aortic intima

Three-dimensional network of smooth muscle cells (SMC) with processes was found in the subendothelial intima of human aorta. The cells were connected with each other through gap junctions. In the direction from the media to the endothelium the number of plasma membrane caveolae increased, their distribution becoming more random. In the fatty streak, the integrity of cellular network was seen destroyed. In the extracellular matrix multilamellar ball-like structures containing large intramembranous particles appeared. In the fibrous plaque, SMCs are completely isolated by connective tissue fibres.     

THE SMOOTH MUSCLE CELL : II. Growth of Smooth Muscle in Culture and Formation of Elastic Fibers

Smooth muscle derived from the inner media and intima of immature guinea pig aorta were grown for up to 8 wk in cell culture. The cells maintained the morphology of smooth muscle at all phases of their growth in culture. After growing to confluency, they grew in multiple overlapping layers. By 4 wk in culture, microfibrils (110 A) appeared within the spaces between the layers of cells. Basement membrane-like material also appeared adjacent to the cells. Analysis of the microfibrils showed that they have an amino acid composition similar to that of the microfibrillar protein of the intact elastic fiber. These investigations coupled with the radioautographic observations of the ability of aortic smooth muscle to synthesize and secrete extracellular proteins demonstrate that this cell is a connective tissue synthetic cell.     

Three-dimensional cytoarchitecture of the media of arteriovenous anastomoses in the rabbit ear

Arteriovenous anastomoses in the rabbit ear were examined with scanning electron microscopy to elucidate the structural differentiation of the media of the shunt. Arterial, intermediate, and venous segments in the shunt and two layers of the media in the intermediate segment were differentiated based on cell shape and cell organization. In the arterial segment, smooth muscle cells were spindle-shaped, either elongated or short, with a few branches, and were arranged circularly or diagonally with respect to the vessel's long axis. There were also stellate muscle cells with radiating processes. In the intermediate segment, the smooth muscle cells of the outer layer of the media were also arranged circularly and resembled the elongated cells in the arterial segments, but they were more irregular in shape and had more processes than those of the arterial segment. The epithelioid cells of the inner layer of the media were oval or polygonal and oriented irregularly with respect to the vessel's long axis, clustering to form longitudinal plicae. The smooth muscle cells of the venous segment were flat with many lateral processes and formed a thin, discontinuous layer. The smooth muscle cells in the arterial segment and those of the outer layer of the intermediate segment exhibited a highly rugged surface texture, indicating their strong contractility; the epithelioid cells and the smooth muscle cells in the venous segment exhibited a generally smooth surface, indicating less contractility. The intermediate segments were supplied with a dense nerve plexus. The intermediate segments, therefore, may be actively involved in the regulation of blood flow under neuronal influence.     

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta.

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.     

Quantitative estimation of lipid-laden cells in atherosclerotic lesions of the human aorta.

The intima of the adult human aorta consists of three sublayers: a muscular layer lying next to the media, a median hyperplastic layer and an innermost connective tissue layer, adjoining the lumen. The cells inhabiting these sublayers were isolated by the method of alcoholic-alkaline dissociation from grossly normal areas, fatty streaks and atherosclerotic plaques. The populations obtained contained cells with different numbers of cytoplasmic inclusions and a number without any. In unaffected intima and in fatty streaks, the cells with lipid inclusions were found predominantly in the outermost intimal layer including the connective tissue and in part of the median hyperplastic layer. In the superficial layer of unaffected intima and the fatty streak, these cells accounted for 15 and 25% of the total cell population, respectively. In the plaque, most cells with lipid inclusions were localized in the median hyperplastic layer of the intima (10%). The muscular layer was characterized by the lowest content of cells with lipid inclusions both in the unaffected intima and atherosclerotic lesions (from 0.75% in unaffected intima to 5% plaques). Among the intimal smooth muscle cells of various shapes, the cells with lipid inclusions were most often found in the stellate cell subpopulation (5-35%). A possible role of stellate cells in atherogenesis is discussed.     

Immunohistochemical and ultrastructural study of aortic lesions in fat-fed quails

A total of 13 forty-day-old male Japanese quails had free access to a atherogenic diet containing 15% corn oil and 2% cholesterol or commercial basal diet for 3 months. Birds fed basal diet showed no significant intimal lesions. These birds had two types of cells, i.e. smooth muscle cell and fibroblast-like cell, in the tunica media of the ascending aorta. While fat-fed birds showed marked lipid-rich intimal lesions in the ascending aorta but not in the abdominal aorta. Some macrophage-derived foam cells, which were stained for lysozyme and OKM1, were demonstrated in the superficial portion of the thickened intima. The majority of the cells in the lipid-rich thickened intima showed ultrastructural character of fibroblast-like cells with or without lipid droplets. Alpha-1-antichymotrypsin was positive for fibroblast-like cells in the thickened intima but not for those in the tunica media of the ascending aorta. These results suggest that metaplasia of the medial fibroblast-like cells is responsible for the development of atherosclerosis in the quail.     

Neutral glycolipids of atherosclerotic plaques and unaffected human aorta tissue

The composition, structure and localization of neutral glycosphingolipids of human aorta taken from subjects who had died after myocardial infarction were studied. Individual glycosphingolipids were purified by high-performance liquid chromatography and were characterized on the basis of their chromatographic mobility, carbohydrate composition, methylation analysis and by 1H-NMR spectroscopy. The main aortic glycosphingolipids were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. Significant differences in the neutral glycosphingolipid composition of intima and media were detected. The neutral glycosphingolipid profile of medial plaques resembled that of unaffected media; however, significant differences were detected between intimal plaques and unaffected intima. Whereas the latter contained trihexosylceramide and globoside as the only neutral glycolipids, the intimal plaque glycolipids consisted mainly of glucosylceramide and also contained appreciable amounts of lactosylceramide which were completely absent in the unaffected intima. In comparison to intimal plaques, unaffected intima is characterized by a much higher content of cerebrosides terminating by beta-galactosyl residues which are known to interact with growth factors and other external stimuli. It thus seems possible that the proliferative activity of smooth muscle cells in atherosclerotic diseases is to some extent associated with their neutral glycolipid profile.     

Immunohistochemical and histoenzymological characteristics of the arterial intimal cells in nonspecific aorto-arteritis

Enzymatic activity of cells, antigenic cellular markers and extracellular matrix of the hyperplastic intima of the aorta and carotid arteries was investigated in non-specific aorto-arteritis by immunomorphological and histochemical techniques. The cells of subendothelial layer of thickened arterial intima contained smooth muscle cell myosin, gave positive reactions to myosin ATP-ase and revealed high activity of thiamine pyrophosphatase. Fibronectin and type IV and V collagen were located in close proximity to these cells. The data obtained make it possible to consider these cells as modified smooth muscle cells. Type III collagen was the prevalent type of extracellular matrix of the thickened intima. A great number of blood vessels of the capillary and precapillary types have been found to penetrate into the intima from the adventitia. A possible role of pericytes surrounding newly formed capillaries as the precursors of subendothelial cell population in the hyperplastic intima is discussed.     

Polymorphism of smooth muscle cells in atheromatous plaques of the human aorta

The expression of cell cytoskeleton proteins in atheromatous plaques of human aorta was investigated using double immunofluorescence technique and a set of antibodies. It was found that in 4 out of 12 plaques some smooth muscle cells (SMC) were stained by monoclonal antibodies to desmin. No such cells were detected in apparently unaffected aortic intima. In addition to typical SMC and these cells, the cells unstained by antisera to smooth muscle myosin but reacting with monoclonal antibodies to vimentin and SMC surface were revealed in all plaques adjacent to the central fatty mass.     

Ultrastructural and immunohistochemical study of experimental atherosclerosis in Japanese quails

A marked plaque was produced at the tunica intima of the ascending aorta in all of the Japanese quails of 9 weeks old which fed on atherogenic diet containing 2% cholesterol for 8 weeks, while no structural changes of aortic wall were observed in Japanese quails which fed on normal basic food for the same period. The media of aorta in normal quails consist of smooth muscle cells, myofibroblast-like cells (MF) cells), and many successive elastic membranes. At the atherosclerotic lesion, many MF cells migrated from media into intima, and a part of smooth muscle cells were also differentiated to MF cells. Moreover, the most migrating MF cells differentiated to foam cells at the intimal thickness regions, and a few other MF cells also differentiated into endothelial cells of newly forming capillaries. By the immunohistochemical stainings, medial smooth muscle cells were negatively stained with anti-vimentin antibody, and the majority of cells in the intima (MF cells, foam cells, and endothelial cells) contained vimentin filaments. These results indicate that MF cells play a very important role in the development of atherosclerosis in Japanese quail. The morphologicals study offers some new insights into the evaluation of Japanese quails as an animal model of atherosclerosis.     

Ultrastructural study of secondary cultures of rat aorta. Cellular aspects]

Cells obtained from the media and intima of ten days rat aorta, after enzymatic dissociation, were grown in subculture for up to three months. Electron microscopic observations demonstrate that these cells maintained the morphology of smooth muscle cells at all phases of their growth in subculture and kept their ability to synthesize and secrete intracellular proteins with better enzymatic features than the cells obtained by proliferation at the periphery of an explant.     

Developmental changes in expression of adhesion-mediating proteins in human aortic smooth muscle.

Phenotypic variability of vascular smooth muscle cells (SMCs) can serve as a good model for studying the mechanisms regulating the expression of adhesion-mediating proteins. To describe phenotypic changes of human aortic SMCs, we have studied the expression of cytodifferentiation-related adhesion-mediating proteins in samples of media from fetal, child and adult human aorta, and in subendothelial intima of normal and atherosclerotic aorta. We have shown that during prenatal and post-natal development vascular SMCs co-ordinately change several times the expression of certain differentiation-related proteins. Our data show the existence of certain groups of proteins whose expression during smooth muscle development might be controlled by two basic mechanisms: selection of genes to be expressed at particular developmental stages and generation of several different protein variants from a single gene via alternative RNA splicing.     

Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity

Summary Recent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epithelioid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.     

Regional functional heterogeneity of aortic smooth muscle cells in rats

The aorta is a magistral artery, which has been traditionally looked upon as a vessel whose properties are invariable throughout its length. However, in the most recent decade, there have been accumulated data that provide evidence that different aorta sections arise from different embryonic origins and that the population of smooth muscle cells making up the vessel’s wall is, consequently, heterogenic. Tracing the fate of smooth muscle cells, the basic components of the vessel, with the aid of genetic marking methods revealed that the cells’ response to various factors is largely determined by the embryonic origin of a certain cell population. However, functional differences between the smooth muscle cells making up different aorta sections remain poorly understood. The aim of the current work was to compare the functional characteristics of the populations of aortic wall smooth muscle cells obtained from the aorta sections differing by their embryonic origin. Towards this end, we obtained smooth muscle cell cultures from the three aorta sections of linear rats, namely, the neural crest derived ascending thoracic aorta, the somites derived descending thoracic aorta, and splanchnic mesoderm derived abdominal aorta. Using immunocytochemistry and Western blotting, the cells from the different regions of aorta were compared on the basis of smooth muscle actin, vimentin, and SM22 content in them. Cell proliferation rate was estimated using the growth curves method. We have demonstrated that the three smooth muscle cell populations arising from different embryonic origins differ in their morphological characteristics as well as by smooth muscle actin and SM22 content. We have shown that smooth muscle cells from the ascending aorta proliferate more actively than the corresponding cells from the descending thoracic aorta. Thus, the functional properties of the populations of rat aortic smooth muscle cells are different and depend on the embryonic origin of the aorta section from which they were obtained.     

Morphogenesis of the intimal thickening observed in nonspecific aortoarteritis

Thickening of blood vessel segment intima (aorta, carotid, femoral and renal arteries) excised from 9 patients during surgery for nonspecific aortoarteritis was studied, using electron microscopic autoradiography. A large number of vessels of capillary and precapillary type were found among cells and in the intracellular substance of thickened intima. Vascular endotheliocytes and pericytes were easily labelled with 3H-uridine. It is suggested that cells appear in the thickened intima due to growth of small vessels of the capillary type, covered with pericytes which turn into fibroblast-like cells producing intracellular substance, and not due to smooth muscle cell migration from the media. In addition, it was found that the lumens of some vessels were filled with fibrillar material and that the cells underlying the vessel stayed apart, not forming a continuous circle. It is suggested that the damage of normal vascular structure can also result in the appearance of free cells.     

Regeneration of the rat aortic intima in the region of a microsurgical suture

Intimal regeneration in the region of microvascular suture of the rat aorta was investigated by LM, SEM and TEM. Endothelial integrity was restored by endothelial cells from the defect edges. No thrombotic masses took part in the formation of the intimal thickening. It is supposed that the core of the intimal thickening formation is a transition of regenerating smooth muscle cells along elastic "railes" from media into intima.     

Angiotensin II-induced growth effects in vascular smooth muscle in cell culture and in the aortic tunica media in organ culture

Several different studies have investigated the growth effects of angiotensin II on vascular smooth muscle cells in culture. However, smooth muscle cells change their phenotype when placed in culture. The objective of the present study was to investigate the effects of angiotensin II on (3)H-thymidine and (3)H-proline incorporation in vascular smooth muscle cells in culture and in the tunica media of blood vessels perfused at normal physiological pressures in organ culture, thus avoiding the phenotypic changes observed in cell culture. The perfusion system consisted of a peristaltic pump and a closed circuit of plastic tubing connected to a culture media bottle where thoracic rat aortae were placed. Angiotensin II induced an increase in (3)H-thymidine and (3)H-proline incorporation in both culture systems. The results suggest that angiotensin II may play a role in mediating cell growth in vascular smooth muscle cells in their 'contractile' as well as in their 'synthetic' phenotype.