A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.

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A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.

D uring recent years, it has become increasingly evident that arterial smooth muscle cells occur in at least two distinct states, usually referred to as a synthetic and a contractile phenotype. Synthetic-state cells have a fibroblast-like appearance, and their main functions are to proliferate and to produce extracellular matrix components. They are found in the embryo and the young growing organism, where they take part in the formation of the vessel wall. Contractile-state cells have a musclelike appearance and contract in response to chemical and mechanical stimuli. They predominate in the vessels of adults and are primarily involved in the control of blood pressure and flow. However, these cells are able to return to a synthetic phenotype, and this appears to be an important early event in atherogenesis. This review briefly summarizes the present knowledge concerning the regulation of differentiated properties and proliferation of arterial smooth muscle cells. Most of the discussion will deal with studies on cultured cells, which so far are the most abundant. Particular attention will be paid to the role of extracellular matrix components like fibronectin and laminin and of polypeptide mitogens like platelet-derived growth factor (PDGF). It is believed that future studies in this area will help to widen our understanding of vasculogenesis and the initial stages in the pathogenesis of atherosclerosis.

Regulation of differentiated properties and proliferation of arterial smooth muscle cells.

Background The adventitia undergoes remodeling changes after a deep medial coronary injury. Because this process is associated with the formation of adventitial myofibroblasts, which resemble medial smooth muscle (SM) cells, we have examined myofibroblast involvement in the development of neointima. Methods and Results In a porcine model, severe endoluminal coronary injury resulted in fibroblast proliferation and adventitial remodeling. Significant adventitial responses were associated with increased neointimal formation (P<.01). To examine the contribution of adventitial cells to the development of neointima, proliferating cells were labeled with bromodeoxyuridine (BrdU) at 12 and 24 hours after injury, and their subsequent localization was determined by immunohistochemistry (n=24). At 2 to 3 days after severe injury, the adventitia contained numerous BrdU-labeled cells (37±4%), whereas the media demonstrated infrequent labeled cells (4±1%). Adventitial cells lacked α-SM actin and desmin, which distingui...

Adventitial Myofibroblasts Contribute to Neointimal Formation in Injured Porcine Coronary Arteries

Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-β1 (TGF-β1) induction of the putative fibroblast contractile marker, α-smooth muscle actin (α-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-β1 (10 ng/ml; 6 days) increased α-SMA protein content by ninefold as detected by Western blots but did not affect β-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-β1-induced increase of α-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of α-SMA protein. Northern analyses for α-SMA and β-actin indicated that TGF-β1 selectively increased mRNA for α-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular α-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-β1 treatment. TGF-β1 increased α2 and β1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the α2 and β1 integrin subunits, TGF-β1 failed to increase α-SMA protein content in all three types of collagen matrices. These data indicate that TGF-β1-induced increases of α-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.

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The compliance of collagen gels regulates transforming growth factor-β induction of α-smooth muscle actin in fibroblasts

During atheromatous plaque formation or restenosis after angioplasty, smooth muscle cells (SMCs) migrate from the media toward the intima, where they proliferate and undergo phenotypic changes. The mechanisms that regulate these phenomena and, in particular, the phenotypic modulation of intimal SMCs have been the subject of numerous studies and much debate during recent years. One view is that any SMCs present in the media could undergo phenotypic modulation. Alternatively, the seminal observation of Benditt and Benditt that human atheromatous plaques have the features of a monoclonal or an oligoclonal lesion has led to the hypothesis that a predisposed, medial SMC subpopulation could play a crucial role in the production of intimal thickening. The presence of a distinct SMC population in the arterial wall implies that under normal conditions, SMCs are phenotypically heterogeneous. The concept of SMC heterogeneity is gaining wider acceptance, as shown by the increasing number of publications on this subject. In this review, we discuss the in vitro studies that demonstrate the presence of distinct SMC subpopulations in arteries of various species, including humans. Their specific features and their regulation will be highlighted. Finally, the relevance of an atheroma-prone phenotype to intimal thickening formation will be discussed.

Arterial Smooth Muscle Cell Heterogeneity Implications for Atherosclerosis and Restenosis Development

Cytoskeletal features of arterial smooth muscle cells (SMC) vary characteristically during development and during atheromatous plaque formation (Gabbiani et al., 1984; Kocher et al., 1985). We have analyzed the cytoskeletal features of rat aortic SMC placed in culture in the presence of 10% foetal calf serum (thus containing growth factors probably playing a role in SMC development and atheroma formation), as compared to SMC freshly isolated from the rat aortic media. Under these conditions, SMC show a typical cytoskeletal remodeling characterized by: 1) increased content of vimentin per cell, increased number of cells containing only vimentin, and decreased number of vimentin plus desmin containing cells; 2) decreased contents of actin, tropomyosin and myosin; 3) a switch in the pattern of actin isoforms with the appearance of a beta-type predominance. Some of these changes (e.g. increase of vimentin and decrease of alpha-type actin) are seen already in cells entering for the first time in S-phase after plating. Pulse-chase experiments with 3H-thymidine (3H-TdR) indicate that vimentin containing SMC possess a higher replicative activity than vimentin plus desmin containing SMC, thus explaining the selection of vimentin containing cells during culture. Our results indicate that during culture SMC develop features similar to those observed in normal foetal SMC or in SMC present in atheromatous plaques; this model may be useful for the understanding of mechanisms leading to SMC differentiation and to atheroma formation.

P Ropraz

Papers

Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationships to culture conditions and analogies to in vivo situations.