Vascular smooth muscle cell (VSMC) hypertrophy is a characteristic feature of hypertensive vessels that contributes to abnormal vessel tone and structure. In intact aorta, the contractile agonist angiotensin II (ang II) regulates VSMC mass by stimulating an increase in protein synthesis. An in vitromodel of hypertrophy using cultured rat aortic VSMC is well defined, in which ang II induces a ~hypertrophic growth response~ consisting of an increase in protein synthesis and cell size without changes in DNA synthesis or cell number. There has been considerable interest in identifying the mechanisms and cellular signaling pathways whereby ang II stimulates VSMC hypertrophy. Previous work has shown that ang II activates a variety of second messengers, including Ca2+, protein kinase C (PKC), tyrosine kinases, and mitogen activated protein (MAP) kinases. MAP kinases are key mediators of cell growth and have been shown to regulate ribosomal kinases and initiation factors that are crucial for protein synthesis and hypertrophic and hyperplastic growth responses. Our preliminary data indicate that ang II causes a rapid Ca2+ dependent activation of MAP kinase in VSMC. Thus, the major hypothesis of this proposal is that Ca2+ -dependent Map kinase activation is a key mediator of ang II-induced VSMC hypertrophy. A series of experiments with a synthetic MEK inhibitor will determine whether inhibition of MAP kinases blocks rate limiting steps involved in VSMC protein synthesis and translation (as assessed by the regulation of the translation initiation factor eIF-4F) that are crucial to the ang II-induced hypertrophic growth response. A series of pharmacological, biochemical and molecular studies are designed to identify Ca2+ dependent components of the MAP kinase pathway (Shc, Ras, Raf) that are activated by ang II in VSMC. Finally, cellular mechanisms, by which Ca2+ regulates the MAP kinase pathway will be determined. These studies will concentrate on Ca2+ -dependent forms of PKC and calmodulin by use of pharmacological and antisense oligonucleotide inhibitors of PKC and calmodulin affinity chromatography. The proposed investigations are of fundamental importance and will critically test the major hypothesis. Future development of therapeutic strategies specifically targeted to signaling pathways involved in VSMC hypertrophy may have important clinical implications in the treatment of hypertension and atherosclerosis.
