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Abstract: Objective: Basic fibroblast growth factor (bFGF)-stimulated proliferation of coronary smooth muscle cells (cSMC) contributes to the pathogenesis of arteriosclerosis and restenosis. However, the molecular mechanisms involved are not fully understood. We have shown previously that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) are required for the bFGF-stimulated mitogenic process in bovine cSMC. In this study, we determined the PKC isoform(s) involved and investigated their functional role in the bFGF-stimulated signaling and cell cycle progression in human and bovine cSMC. Methods and Results: Downregulation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibited bFGF-induced DNA synthesis, the activation of MAPK, and the expression of c-myc, demonstrating the involvement of PMA-sensitive PKC isoforms in growth factor-induced proliferation and the MAPK pathway. The PMA-sensitive classical PKC isoforms α, β,  γ and novel PKC isoforms δ and ɛ were found in human cSMC. Whereas blocking of the classical PKC isoforms had no influence, the suppression of PKC δ by genetic and pharmacological approaches inhibited the bFGF-stimulated c-Raf1–MEK–MAPK–c-myc signaling and DNA synthesis in cSMC. In contrast to PKC ɛ, our results showed that bFGF activated PKC δ by phosphorylation in a time-dependent manner. In addition, inhibition of PKC δ induced a hypophosphorylation of the retinoblastoma protein and suppression of the cyclins D1 and A, demonstrating the importance of PKC δ for bFGF-induced cell cycle progression through the G1 phase in cSMC. Conclusions: Our results show that PKC δ is required for the bFGF-stimulated c-Raf1–MEK–MAPK–c-myc signaling pathway involved in the proliferation of cSMC. Therefore, it may be an interesting therapeutic target for preventing proliferative vascular disorders. [Copyright &y& Elsevier]

Provides information on a study that investigated the vascular collagenous matrix in wild-type and granulocyte macrophage colony-stimulating factor-deficient mice. Methodology of the study; Results and discussion on the study.

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P<0.05), but subsequently increased fourfold, attaining levels double that of non-injured controls in the 10-day group (P<0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis. [Copyright &y& Elsevier]