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.

Smooth muscle proliferation has been recognized as central to the pathology of both major forms of vascular disease: atherosclerosis and hypertension. Recent advances in our knowledge of mechanisms of control of proliferation suggest that events occurring in adult animals may recapitulate portions of the developmental biology of the smooth muscle cell. This review attempts to consider the current state of knowledge of the mechanisms controlling smooth muscle proliferation in these two diseases, to put that knowledge into the context of what is known about smooth muscle biology, and to offer two hypotheses on the possible roles of smooth muscle developmental biology in manifestations of atherosclerosis and hypertension in adult humans.

Replication of smooth muscle cells in vascular disease.

Cellular accumulation in the intima of injured artery has generally been attributed to smooth muscle cell proliferation. Since smooth muscle cells in normal artery are found mainly in the media, migration of smooth muscle cells into the intima has been considered a necessary prerequisite for subsequent myointimal thickening. The nondividing medial cells would appear to have no role in the reparative process. We have investigated in the rat ballooned carotid the possibility that nondividing cells might also contribute to injury-induced intimal thickening. All proliferating smooth muscle cells were labeled by 3H-thymidine given by continuous intraperitoneal infusion. The amounts of 3H-thymidine used were not toxic and did not inhibit smooth muscle cell proliferation. Autoradiograms performed on histological cross-sections showed a progressive decrease in the fraction of unlabeled cells at 3, 7, and 14 days after carotid injury. However, the actual number of nondividing cells remained constant. The calculated growth fraction for the 14-day period was 40%. A substantial number of unlabeled cells was observed in the intima. These data have led us to conclude that only a small fraction of smooth muscle cells in an artery proliferate in response to the injury stimulus, and do so shortly after injury, or not at all. Furthermore, nondividing, as well as proliferating smooth muscle cells, can migrate and contribute, in a substantial way, to the increase in intimal smooth muscle cell number.

Significance of quiescent smooth muscle migration in the injured rat carotid artery.

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.

Mesangial cell proliferation is common in glomerulonephritis but it is unclear if proliferation is associated with any in vivo alteration in phenotype. We investigated whether mesangial of mesangial proliferative nephritis induced with antibody to the Thy-1 antigen present on mesangial cells. At day 3 glomeruli displayed de novo immunostaining for alpha-smooth muscle actin in a mesangial pattern, correlating with the onset of proliferation, and persisting until day 14. An increase in desmin and vimentin in mesangial regions was also noted. Immunoelectron microscopy confirmed that the actin-positive cells were mesangial cells, and double immunolabeling demonstrated that the smooth muscle actin-positive cells were actively proliferating. Northern analysis of isolated glomerular RNA confirmed an increase in alpha and beta/gamma actin mRNA at days 3 and 5. Complement depletion or platelet depletion prevented or reduced proliferation, respectively; these maneuvers also prevented smooth muscle actin and actin gene expression. Studies of five other experimental models of nephritis confirmed that smooth muscle actin expression is a marker for mesangial cell injury. Thus, mesangial cell proliferation in glomerulonephritis in the rat is associated with a distinct phenotypic change in which mesangial cell assume smooth muscle cell characteristics.

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Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation.

Actin of smooth muscle cells of rat and human aortic media shows a predominance of the alpha-isoform. In experimental rat aortic intimal thickening, in human atheromatous plaque, and in cultured aortic smooth muscle cells, there is a typical switch in actin expression with a predominance of the beta-form and a noticeable amount of gamma-form. This pattern of actin expression represents a new reliable protein-chemical marker of experimental and human atheromatous smooth muscle cells.

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J Vandekerckhove

Papers

Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media.