There is substantial evidence indicating that the study of cytoskeletal and cytocontractile protein composition in vascular smooth muscle cells (SMCs) can be valuable in tracing structural changes during vascular remodeling. Recent nucleic acid and protein investigations suggest that myosin can be used as a new specific marker for the identification of SMC phenotypes in some pathological conditions affecting the vascular wall. In view of this new information, it would seem timely to review the structural bases of myosin isoform expression in the vascular smooth muscle system as well as the factors involved in its regulation. A puzzling feature has arisen in recent studies on this topic: the presence of non-muscle myosin variants in SMCs during physiological and pathological vascular remodeling. In the response to injury caused by mechanical, chemical and hormonal factors in animals, characterized by proliferation and migration of vascular SMCs from the media to the intima, there is a partial or complete recapitulation of a myosin isoform pattern pertinent to developing vascular smooth muscle tissue. Analysis of myosin isoform content in the vascular wall also demonstrates that: (1) changes in SMC composition may occur independent of medial SMC migration into intima, and (2) the presence of fetal-type SMCs in the neointima is not necessarily related to specific positional changes of medial SMCs.

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Smooth muscle cells (SMCs) of rabbit aorta undergo marked changes in myosin isoform content during development. Analysis of nonmuscle myosin composition at the protein level has permitted the identification of three phases in the SMC differentiation process: fetal, postnatal, and adult. Using monoclonal antibodies specific for smooth muscle and nonmuscle myosins and extra domain A of fibronectin as well as cDNA probes for platelet-derived growth factors (PDGF) and various procollagens, we have evaluated the differentiation pattern of aortic SMCs in two-kidney, one-clip hypertensive rabbits. Morphometric and bromo-deoxyuridine studies indicate that hypertrophy of aortic media along with intimal thickening occurring in hypertensive animals is due to SMC hyperplasia. Western blotting experiments performed on aortic specimens from hypertensive animals with antimyosin antibodies revealed the appearance of a myosin isoform pattern of the "immature" type. Immunofluorescence tests showed that these cells are localized in the thickened intima or distributed in the underlying media (sparsely or in groups). Similarly, the fibronectin variant showing the extra domain A, peculiar to "phenotypically modulated" SMCs, appeared in intimal thickening, and its expression followed the time course of nonmuscle myosin expression. Counting of postnatal-type SMCs in the aortic media revealed that this cell population increases markedly with hypertension (2- up to 15-fold at 4 months) and then declines to near control level in 8-month hypertensive rabbits. Diminution of postnatal-type SMCs at later stages of hypertension was temporally correlated with the slowing down of aortic wall hypertrophy. Average levels of mRNAs, as determined by densitometric analysis in aortas from 1- and 2.5-month hypertensive rabbits, showed an increased expression for PDGF beta receptor (up to twofold), procollagen type I (alpha 1, threefold), procollagen type III (alpha 1, twofold), and fibronectin (up to threefold) compared with controls. Conversely, the steady-state levels of mRNAs for PDGF (A and B chain), PDGF alpha receptor, TGF-beta 1, and procollagen type IV (alpha 1) did not increase significantly. These results provide evidence that in adult renovascular hypertensive rabbits, the hyperplastic growth of aortic SMCs is accompanied by the expansion of an "immature" cell phenotype characteristic of the early stages of development.

Morphological techniques (histology and electron microscopy), as well as immunofluorescence assays, were applied to the study of the localization and smooth muscle cell (SMC) composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits during a 4.5-month period. Vascular segments from different arteries (carotid, coronary, and iliac arteries) or from the same vessel at different levels (aorta) of animals at days 7,15, 30, 40, 60, 90, and 135 showed that the atherosclerotic lesion first became visible at the level of the aortic arch in 60-day-old WHHL animals. Histological examination of serial cryosections from this vascular region indicated that the vascular lesion arose from a cavity in the media layer, located anatomically at the level of the juncture of the ligamentum arteriosum with the aortic arch. This aortic arch cavity is formed during the postnatal closure of the ductus arteriosus and is characterized by the presence of a thickened intima, which was absent in the other vascular regions examined. Immunofluorescence comparison of normal and atherosclerotic tissues from the aortic arch cavity wall with the use of monoclonal antibodies specific for smooth muscle and nonmuscle myosin isoforms revealed the existence of distinct SMC populations. SMCs in the thickened intima showed a myosin isoform pattern peculiar to cells with a degree of maturation intermediate between the fully differentiated and the developing (fetal) aortic SMCs. By contrast, SMCs present in atherosclerotic lesions displayed a predominant fetal-type pattern of myosin isoform expression. The achievement of this myosin isoform content seems to be correlated with the accumulation of lipids in the intima. In the media subjacent to the intimal thickening or atherosclerotic lesion, SMCs primarily displayed an intermediate degree of maturation. In older WHHL animals and at this aortic level, the SMC composition of the atherosclerotic lesion did not change, whereas in the subjacent media, the cells of intermediate type almost disappeared. In the vascular regions in which the atherosclerotic lesion appeared at later stages, such as near the aortic bifurcation, the distribution of fetal and intermediate cell types in the atherosclerotic wall was similar to that taken at the aortic arch level. These results indicate that there is 1) a preferential anatomic site from which atherogenesis initiates in WHHL rabbits; 2) a time correlation between the accumulation of lipids in the wall and the phenotypic change of SMCs toward a poorly differentiated cell type; and 3) the tendency for SMCs to follow the same differentiation pattern in early atherosclerotic lesions, irrespective of the site and time at which they develop. (