Project Summary. Vascular smooth muscle cells (VSMCs) of a synthetic phenotype meet their energy requirements largely via aerobic glycolysis. Hypoxia-inducible factor-1α (Hif-1α) induces a complex transcriptional program that facilitates glycolysis in the setting of low oxygen tension. In normoxia, Hif-1α undergoes proteasomal degradation via prolyl hydroxylation and ubiquitination. In VSMCs engaged in aerobic glycolysis, however, Hif-1α is stabilized by mechanisms that remain unclear. Preliminary data provide insight into the mechanism of this normoxic stabilization of Hif-1α, showing that Hif-1α-dependent aerobic glycolysis remains the primary source of ATP; that conditioned media obtained from VSMCs contains low-molecular-weight factors that stabilize Hif-1α; and that initial identification of these factors indicates that they comprise the family of branched-chain keto-acids (BCKAs), α-ketoisocaproate (KIC), α- keto-β-methylvalerate (KMV), and α-ketoisovalerate (KIV), derived from their parent branched-chain essential amino acids (BCAAs), leucine, valine, and isoleucine, respectively. Given these preliminary results, the central hypothesis of this proposal is that synthetic VSMCs engage in aerobic glycolysis through the effect of BCKAs on Hif-1α stabilization. To address this hypothesis, we propose the following specific aims: 1) we will examine the determinants of BCKA synthesis in VSMCs and their regulation; 2) we will examine the effect of BCKAs on Hif-1α stabilization and explore potential underlying molecular mechanisms; and 3) we will examine the effect of BCKAs on VSMC phenotype and examine the relationship between phenotype switching and metabolic re-programming. The role of BCKAs on VSMC phenotype and metabolism will also be studied in animal models of pulmonary hypertension. The results of these studies should provide useful insight into molecular mechanisms underlying Hif-1α stabilization and aerobic glycolysis in VSMCs, the role of BCKAs in that process, and the relationship between BCKA- dependent aerobic glycolysis and VSMC phenotype and pathophenotype.