Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor-beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells.

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Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.

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.

The cellular composition of human atherosclerotic plaques was analyzed by immunologic techniques. Plaques were removed from the internal carotid artery during surgery, and a panel of monoclonal antibodies was used to identify cell types. Macrophages stained by Anti-Leu-M3 were found throughout the plaque, particularly in the lipid core region, where 60% of the cells reacted with this antibody. T cells expressing the T3 antigen were most abundant in the fibrous cap, where they constituted 20% of the cell population. T cells were also isolated from the plaque and detected by a rosetting test; many of these T cells were activated, as indicated by the expression of HLA-DR. Other types of leukocytes were uncommon in the plaque. An antibody to the intermediate filament protein, desmin, was used as a marker for smooth muscle cells since some, but not all, vascular smooth muscle cells contain this protein. The desmin-positive cells were uncommon in the nonatherosclerotic intima but were more numerous in the plaque. In conclusion, atherosclerotic plaques are heterogeneous with respect to cellular composition. The smooth muscle cell dominates in the fibrous cap, which also contains many T cells; the lipid core is dominated by macrophages. We suggest that interactions between smooth muscle cells and blood-borne cells are important in the pathogenesis of atherosclerosis.

Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque.

Thirty years ago, John French wrote a seminal review describing the unique properties of the arterial intima.1 His major point was that the smooth muscle cells of the intima have a unique morphology (Fig 1⇓). French also pointed out that intimal formation appeared during normal development and aging as well as in the response of arteries to almost any imaginable injury, including atherosclerosis.



 Figure 1. 
Top left, Histology of intima vs media. Coronary artery is from an adult patient with idiopathic dilated cardiomyopathy. In the absence of atherosclerosis, there remains diffuse intimal hyperplasia. The intima is several cell layers thick and composed of smooth muscle cells and is separated from the distinct media by the internal elastic lamina (arrows) (hematoxylin and eosin, original magnification ×200). Top right, Advanced atherosclerosis of the left circumflex coronary artery. This lesion was treated by percutaneous balloon angioplasty 23 days before the patient’s death. Note the complex nature of the lesion with a distinct fibrous cap (FC), intimal hyperplasia (IHP), intramural hemorrhage (H), and a necrotic core (NC) composed of inflammatory cells and accumulated lipid (hematoxylin and eosin, original magnification ×100). Bottom left, Myxomatous tissue. Directional coronary atherectomy specimen from a restenotic lesion shows stellate-shaped smooth muscle cells. Smooth muscle cells with this appearance are often regarded as evidence of proliferation. Note the homogeneity of these cells and the absence of inflammatory and endothelial cells in this region (hematoxylin and eosin, original magnification ×200). Bottom right, Directional coronary atherectomy specimen from a primary lesion. Again, note the presence of stellate-shaped smooth muscle cells but a denser connective tissue matrix (hematoxylin and eosin, original magnification ×200).



This article attempts to update French’s review. We will discuss the developmental origins of the intima and suggest that the arterial intima is a distinct tissue with a long and …

The Intima: Soil for Atherosclerosis and Restenosis

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.

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

Cytoskeleton of rat aortic smooth muscle cells. Normal conditions and experimental intimal thickening.

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.

W. S. Bloom

Papers

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

Cytoskeleton of rat aortic smooth muscle cells. Normal conditions and experimental intimal thickening.

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

The cytoskeleton of rat aortic smooth muscle cells: normal conditions and experimental intimal thickening

The cytoskeleton of rat aortic smooth muscle cells (SMC) in vitro: Relationships to culture conditions and replicative activity