Abstract.A monoclonal antibody (anti-ctsm-1) recognizing exclusively a-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of a-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-ctsm-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 a-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 a-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-ctsm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, a-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, a-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma, a-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. FinaUy, ~t-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-asm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions. T HOUGH actin is one of the most conserved eukaryotic proteins, it is expressed in mammals and birds as six isoforms characterized by two-dimensional (2D)-PAGE and amino acid sequence analysis (14,49,56,57,59). Four of them represent differentiation markers of muscle tissues and two are found practically in all cells (56,57). Actin isoforms show >90% overall sequence homology, but only 50-60% homology in their 18 NH2-terminal residues (56, 57). The NH2-terminal region of actin appears to be a major antigenic region (2, 5, 34) and may be involved in the interaction of actin with other proteins such as myosin (31).Little is presently known about the function of actin isoforms. It has been shown that the relative proportions of actin isoforms are different in smooth muscles of different organs (12, 47; Skalli, O., J. Vandekerckhove, and G. Gabbiani, manuscript submitted for publication) and change within the same population of smooth muscle cells (SMCs) t during development (23, 24), pathological situations (11, 21), and different culture conditions (35,46,50). In noumuscle cells, a difference in the proportions of 13-and y-cytoplasmic actins has been reported between normal and tumoral T-lympho-1. Abbreviation used in this paper: SMC, smooth muscle cell. cytes (28). All these studies have been performed on cell populations by means ...

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.

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Objective-Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species.We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis. Methods and Results-By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a "hill-and-valley" growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-␤2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection. Conclusions-Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media.The study of these subpopulations will be useful for understanding the mechanisms of restenosis. Key Words: intimal thickening Ⅲ restenosis Ⅲ endothelial cells Ⅲ myosin Ⅲ smoothelin S mooth muscle cell (SMC) replication and migration from the media into the intima are essential processes during the development and evolution of atheromatous plaque and restenosis. 1 There is now substantial experimental evidence to support the assumption that SMCs from the arterial wall of several species are phenotypically heterogeneous and that certain subsets of medial SMCs are particularly prone to accumulate within the intima under appropriate stimuli. [1][2][3] Two distinct SMC populations have been identified in the rat arterial media: spindle-shaped SMCs (S-SMCs) and epithelioid SMCs, with both exhibiting distinct biological features. 4 Epithelioid SMCs are capable of replicating in the absence of serum 4,5 and exhibit high migratory activity 5 that is correlated with increased tissue plasminogen activator (tPA) expression. 6 Epithelioid SMCs have been shown to be the predominant component of intimal thickening (IT). 7 Spindle-shaped and epithelioid clones can be recovered from adult rat normal media (NM) and IT, albeit in different proportions according to the origin. 5,8 These clones, irrespective of their origin, exhibit phenotypic features similar to those of the corresponding whole-cell populations, thus providing evidence that the NM contains cells capable of displaying each of the 2 phenotypes in vitro.To extend the notion of SMC heterogeneity to other species and to a well-accepted model for human athe...