It has long been thought that hypoxia is a stimulus for human vascular smooth muscle cell (HVSMC) proliferation occurring in pulmonary hypertension, however, the mechanisms involved are unknown. Based on our novel finding that hypoxia directly stimulates proliferation in cultured HVSMC, the main objective of this proposal is to determine the role of hypoxia-inducible transcription factors (HIFs) in the O2-dependent regulation of HVSMC proliferation. Numerous studies have demonstrated a crucial role of HIF-1, a dimer consisting of alpha and beta subunits, in hypoxia-inducible gene transcription. Our preliminary data indicate that hypoxia induces increased nuclear levels of HIF-1alpha and HIF-2alpha protein (by Western blot analysis), increased nuclear HIF complexes which bind to a hypoxia response element (by EMSA), and enhanced HIF-dependent reporter gene expression, in HVSMC. Furthermore, transient overexpression of HIF-1alpha or HIF-2alpha stimulates HVSMC proliferation. Based on these findings, the proposed studies will test the hypothesis that hypoxia induces human VSMC proliferation via upregulation of HIFs, and will further explore the role of downstream signaling pathways in their mitogenic effect. Transient and stable transfection techniques, adenoviral vectors, antisense oligonucleotides, electrophoretic mobility shift assays, Western blot and promoter activity analysis will be used to address the following specific aims: to determine whether HIF transcription factors mediate the proliferative responses of human VSMC to hypoxia; to characterize HIF-dependent gene promoters that are involved in the subsequent mitogenic effect. Elucidating the roles of hypoxia-inducible transcription factors in human VSMC proliferation will enhance our understanding of the pathogenesis of vascular diseases such as pulmonary hypertension, and may lead to identification of novel therapeutic targets for pharmacologic intervention in this disease.