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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Using an mGluR2 FRET-based binding assay, binders of the transmembrane region devoid of functional activity were identified. It is reported that slight chemical modifications of these SAMs can dramatically change activity of the resulting analogues without altering their affinities. Starting from compound 1, three mGluR2 NAMs showing also mGluR3 PAM activities were obtained. SAMs therefore represent a useful approach to explore the chemical space for GPCR allosteric modulator identification.

P. chabaudi AS strain in laboratory mice provides an accessible and useful model for investigating antigenic variation in malaria parasites. Evidence that P. chabaudi AS undergoes antigenic variation is summarized. A live indirect fluorescent test (IFAT) detects a variable antigen on the surface of schizont-infected erythrocytes. Five different variable antigen types (VATS) (detected using the live IFAT) are described from a cloned mosquito transmitted parent population. Even during the rising primary parasitaemia VATS switch at high and variable rates (1.2-1.6%). Work towards cloning genes coding for the variable antigen is briefly summarized. Acquired immunity to blood-stage P. chabaudi AS is initially mediated through Th1 CD4+ T cells and after the primary parasitaemia there is a switch to Th2 CD4+ T cells. Acquired immune effector mechanisms are discussed in the context of antigenic variation by the parasite.

Cyr61 is a secreted, cysteine-rich heparin-binding protein that is associated with extracellular matrix and cell surface, and has been demonstrated to be proangiogenic in vitro. In the present study we evaluated the angiogenic effect of human Cyr61 in an adenoviral context in the rabbit ischemic hindlimb model. For this purpose, three randomized groups of New Zealand White rabbits received intramuscular injections of 5 x 10(8) infectious units of an adenovirus carrying either the Cyr61 gene (Ad-Cyr61), the vascular endothelial growth factor gene (Ad-VEGF(165)) used as the angiogenic gene of reference, or no transgene (Ad-Null), 10 days after femoral artery excision in one limb. Perfusion of the ischemic limb was evaluated before adenoviral treatment (day 10) and 30 days postinjection (day 40). Angiographic, hemodynamic, and histologic parameters indicated that animals in the Ad-Cyr61 group had significantly better perfusion than in the Ad-Null group. Interestingly, this improvement exceeded that achieved with Ad-VEGF(165). In conclusion, Cyr61 gene transfer appears potent in stimulating limb revascularization, thereby promoting great improvement in tissue perfusion in the ischemic limb. These findings indicate that Cyr61 could be a promising therapeutic candidate for treating severe peripheral ischemic diseases.

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