Abstract. 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 a-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-ill (TGFfll) to rats results in the formation of a granulation tissue in which a-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-a, despite their profibrotic activity, do not induce a-SM actin in myofibroblasts. In situ hybridization with an a-SM actin probe shows a high level of ot-SM actin mRNA expression in myofibmblasts of TGFBl-induced granulation tissue. Moreover, TGFfll induces cz-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 TGFfll results in a decrease of a-SM actin expression by fibmblasts in replicative and nonreplicative conditions. These results suggest that TGFfll plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of ot-SM actin in these cells.

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.

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...

Abstract-We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S-and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with ␣-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S-and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein.In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes. Key Words: 2D-PAGE Ⅲ stent Ⅲ endothelial cells Ⅲ mts1 Ⅲ ␣-smooth muscle actin Ⅲ smoothelin T he concept of smooth muscle cell (SMC) phenotypic heterogeneity has been validated in several species including man (for review see 1 ). We have recently extended this notion to the porcine coronary artery (CA) and isolated from the normal media 2 distinct SMC populations: spindleshaped (S) with the classical "hills-and-valleys" growth pattern and rhomboid (R), which grows as a monolayer. 2 R-SMCs display enhanced proliferative, migratory and proteolytic activities as well as poor level of differentiation compared with S-SMCs. R-SMCs are recovered in higher proportion when SMCs are cultured from the intimal thickening (IT) induced after experimental stent implantation compared with the normal media, indicating that they are crucial for arterial repair, and could represent an atheromaprone phenotype.Our aim was to further characterize the phenotypic features of S-and R-SMCs, to identify them in vivo and possibly to verify their presence in atheromatous plaque and restenotic lesions. We have analyzed protein extracts by means of 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by identification of differentially expressed proteins using tandem mass spectrometry (M...