To isolate the genes involved in the cell cycle G1 phase progression of arterial smooth muscle cells (SMCs), a cDNA clone (M11) was previously selected by differential hybridization screening of a mid-G1 serum-stimulated SMC cDNA library. The delay of induction after mitogenic stimulation, time of expression, and need for new protein synthesis for full expression made it possible to classify this gene in the "delayed early" gene group. Determination of the partial M11 cDNA sequence showed full homology with the osteopontin gene (secreted phosphoprotein 1, 2ar), an Arg-Gly-Asp-containing extracellular matrix protein. Osteopontin mRNA was also detected in the aorta at levels as high as in the kidney but lower than in bone, two tissues in which it has been previously detected. In vitro analysis of osteopontin expression in serum-stimulated quiescent SMCs and asynchronously cycling SMCs demonstrated that osteopontin overexpression was associated with SMC proliferation. In view of our results, the high osteopontin expression observed by others in the injured carotid artery could be explained by the involvement of SMCs in the proliferative process. Taken together, these results suggest that osteopontin may play an important role in pathological processes that are associated with arterial SMC proliferation, such as atherosclerosis or restenosis.

The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin I1 or 111. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP 753) but not by CGP 421 12A. In parallel, a transient and dose-dependent induction of czfos was demonstrated not only with angiotensins I1 and I11 but also with angiotensin I. Both angiotensins I1 and 111 exerted their maximal effect at 1 pM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-jos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin 11, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins I1 and 111 act directly on smooth muscle cells.The effect of hypertension on the arterial wall is characterized by an increase in smooth muscle mass due to cellular hypertrophy and/or hyperplasia (for review, see [l]). In chronic hypertension models such as spontaneously hypertensive rats (SHR) or two-kidney one-clip Goldblatt hypertensive rats, the increased mass of smooth muscle cells (SMC) in aortas is due principally to SMC hypertrophy [2-41. In contrast, an increase in medial SMC number, resulting from their proliferation, is accounted for by the increase in smooth muscle mass in models of acute severe hypertension such as aortic coarctation [5, 61. Factors inducing in vivo arterial SMC hypertrophy are not yet well defined. However, some reports suggest that the vasoconstrictor peptide angiotensin (Ang) I1 may play a role in this induction. Indeed, administration of angiotensin-converting enzyme (ACE) inhibitors decreases SMC hypertrophy in the aorta of SHR during the development of hypertension. As this effect is not entirely mediated by reduction in blood pressure, it has been postulated that local angiotensin may participate in the development of SMC hypertrophy [7, 81. In vitro data corroborate the role of AngII in SMC hypertrophy, since AngII induces an increase in cell volume and protein content in quiescent aortic SMC in culture [9 -111. In addition