Project Summary. Vascular smooth muscle cells (VSMCs) of a synthetic phenotype meet their energy
requirements largely via aerobic glycolysis. Hypoxia-inducible factor-1a (Hif-1a) induces a complex
transcriptional program that facilitates glycolysis in the setting of low oxygen tension. In normoxia, Hif-1a
undergoes proteasomal degradation via prolyl hydroxylation and ubiquitination. In VSMCs engaged in
aerobic glycolysis, however, Hif-1a is stabilized by mechanisms that remain unclear. Preliminary data
provide insight into the mechanism of this normoxic stabilization of Hif-1a, showing that Hif-1a-dependent
aerobic glycolysis remains the primary source of ATP; that conditioned media obtained from VSMCs
contains low-molecular-weight factors that stabilize Hif-1a; and that initial identification of these factors
indicates that they comprise the family of branched-chain keto-acids (BCKAs), a-ketoisocaproate (KIC), a-
keto-ß-methylvalerate (KMV), and a-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-1a 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-1a 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-1a 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.