Abstract-Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage after enzymatic digestion of the media. The effects of heparin, transforming growth factor (TGF)-␤ 1 or TGF-␤ 2 , and all-trans-retinoic acid (tRA) on proliferation, migration, and differentiation of these cells also were examined. Porcine arterial SMCs in culture not only express high levels of ␣-smooth muscle (SM) actin but, contrary to rodent SMCs, also maintain an appreciable expression of SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin, a recently described late differentiation marker of vascular SMCs. We demonstrate for the first time that smoothelin is colocalized with ␣-SM actin in these cells. Finally, we show that in the porcine model, heparin is more potent than TGF-␤ 1 or TGF-␤ 2 and tRA in terms of inhibition of proliferation and migration and of increasing the expression of differentiation markers. This model should be a useful complement to in vivo studies of SMC differentiation and of pathological situations such as restenosis and atheromatosis. (Circ Res. 1999;85:99-107.)Key Words: atheromatosis Ⅲ restenosis Ⅲ actin Ⅲ smoothelin Ⅲ myosin T he arterial intimal thickenings (IT) induced in the rat or rabbit after endothelial lesion are presently the moststudied models for atheroma formation and/or restenosis and have been useful for defining several biological features of smooth muscle cells (SMCs). However, these models have many important limitations (for review, see Reference 1), which could explain the clinical failure of substances that proved to be efficient inhibitors of IT formation in these animal models.The biological features of SMCs in culture also have been systematically studied using cells derived from rat or rabbit arteries, 2-7 but these, too, have shown limitations similar to those observed in in vivo experiments. Among the models in large animals, the pig coronary artery IT has been used more and more. 8,9 Pigs may develop spontaneously coronary atheromatosis with age, and the induction of typical plaques is easily achieved by a cholesterol-rich diet. 10,11 Furthermore, angioplasty and other interventions can be performed in porcine coronary arteries with the same instruments as in humans. Although pig aortic and coronary SMCs have already been studied in vitro, no systematic description of their differentiation features during culture has been published.The present study describes the characterization of differentiation features and several biological properties of cultured porcine left anterior descending (LAD) coronary artery SMCs. We show that, contrary to rat or rabbit arterial SMCs, 2-7 porcine SMCs maintain in culture a high level of differentiati...

Abstract-We have recently shown that all-trans retinoic acid (tRA) modulates arterial smooth muscle cell (SMC) morphologic features and biochemical composition in vitro. It has been proposed that different SMC phenotypes coexist in arteries, which may be retrieved in culture: hence, a differential action of tRA on distinct SMC subsets is conceivable.We have examined the effect of tRA on SMC proliferation, migration, plasminogen activator activity, and ␣-smooth muscle actin expression in 2 phenotypically different rat SMC populations, cultured respectively from the normal aortic media and from the intimal thickening (IT) after endothelial injury. tRA inhibited proliferation and increased migration and tissue-type plasminogen activator activity in both SMC populations, but decreased ␣-smooth muscle actin only in SMC cultured from the IT. The action of tRA is mediated by 2 families of nuclear receptors, RAR and RXR, each containing 3 isoforms, ␣, ␤, and ␥. RAR and RAR-␣ agonists, but not RXR agonists, inhibited SMC proliferation in both cell populations and ␣-smooth muscle actin expression only in IT SMC. When administered intraperitoneally to balloon-injured rats, tRA and RAR-␣ agonists reduced the intimal hyperplasia in the carotid artery. Our results show that tRA and synthetic retinoids can affect the proliferation, migration, and differentiation of SMC in vitro. Key Words: smooth muscle cell heterogeneity Ⅲ smooth muscle cell motility Ⅲ smooth muscle cell differentiation Ⅲ intimal thickening S mooth muscle cell (SMC) differentiation, migration, and proliferation are key factors in the development of atherosclerosis and restenosis after angioplasty. 1,2 These phenomena involve an SMC phenotypic modulation characterized by modification of gene expression. 3 It has been suggested that SMC subsets present in the normal media are particularly prone to undergoing phenotypic modulation, and the concept of SMC heterogeneity is gaining wider acceptance (for review see Reference 4). For instance, SMC populations cultured from the rat aortic media and from the intima 15 days after endothelial injury are characterized by a spindle-shaped and an epithelioid morphology, respectively (for discussion of this point see Reference 5). These 2 phenotypes are also obtained when SMC are cloned from the aortic media or the intima, albeit in different proportions. 6 The regulation of SMC phenotype is thought to be exerted by cytokines, growth factors, and agents regulating differentiation. 7 Within the last category, some agents are known to govern SMC differentiation during embryogenesis, and there is a growing body of evidence suggesting that the genetic programs used during embryogenesis may act also during arterial disease processes. 8,9 Vitamin A plays a crucial role in the regulation of cell growth and differentiation, and its active form, retinoic acid, is involved in signal transduction pathways regulating embryonic development. 10 These effects are mediated by 2 families of nuclear receptors that are ligand-dependent trans...

Abstract. The expression of the constituent o~1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the ~1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of otl-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the etl chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the 131/~/1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the otl chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin odchain gene may be a regulatory element in determining cell differentiation.ASEMENT membranes (BM) l are specialized, sheetlike extracellular matrices dividing tissues into compartments; they form the supporting structure on which epithelial cells lie. BM consists of ubiquitous components: type IV collagen, laminin, nidogen (also known as entactin), and heparan sulfate proteoglycan (perlecan) that are secreted locally by epithelial or parenchymal as well as fibroblastic cells. The BM functions as a dynamic structure in tissular morphogenesis, differentiation, and maintenance of the mature structural and functional steady states; its constituent molecules are able to regulate different types of cell behavior such as adhesion, proliferation, and maintenance of cell polarity either directly or via the delivery of growth/migration signals.Laminin-1, a major constituent of the basement membranes, is the earliest molecule produced in embryogenesis; its potential importance has been largely stressed in the last decade (Timpl and Brown, 1994). Indeed, laminin-1, as well as type IV collagen, has been shown to self-assemble,

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