Cultured Porcine Coronary Artery Smooth Muscle Cells

Abstract

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...

Regulation and characteristics of vascular smooth muscle cell phenotypic diversity

“…For example, within the bovine pulmonary artery, four SMC phenotypes with distinct marker protein expression profiles and different morphologies have been described. 18 The existence of distinct SMC populations within the same artery has also been demonstrated in the rat, 14 the pig, 6,15 and in humans. 19 In all these studies, in vivo differences were maintained in vitro.…”

“…13 More detailed information on the nature of SMC diversity has been obtained by analyses of primary cultures. 6,14,15 These studies show that immediately after enzymatic digestion, SMCs contain not only different amounts of contractile proteins, but also that some SMCs do not express particular contractile markers at all.…”

“…The expression levels of these contractile marker proteins gradually decrease when SMCs are cultured, although the extent to which their expression is downregulated differs among the markers. 6 Markers that are upregulated in the synthetic phenotype are rare. Instead, disappearance of proteins associated with the contractile phenotype is generally taken as characteristic of the synthetic phenotype (figure 2).…”

“…Its expression is more uniformly and more rapidly downregulated in cultured vascular SMCs modulating towards a synthetic phenotype. 6 Both SM-MHC and smoothelin have been shown to be absent in myofibroblasts in models of arterial injury. 8 SMemb/non-muscle MHC isoform-B and, in rodents, CRBP-1 represent suitable synthetic SMC markers, since these proteins are quickly and markedly upregulated in proliferating SMCs.…”

“…These, almost invariably, reveal highly heterogeneous staining patterns and intensities between adjacent SMCs. 6,12 These differences become more distinct after vascular injury. Although the overall expression level of contractile SMC phenotype markers after vascular injury initially goes down, the response is remarkably heterogeneous between the different SMCs in the vessel wall.…”