Abstract 10357: Pyruvate Dehydrogenase Kinase 4 is a Novel Regulator of Endothelial Cell Mitochondrial Respiration in Diabetes-Impaired Angiogenesis: Implications for Preventing Diabetic Vascular Complications

Abstract

Introduction: Diabetic patients have a greater risk of experiencing chronic wounds and amputation than non-diabetic patients. Ischaemia-driven angiogenesis is essential for tissue repair but is impaired in diabetes due to aberrant endothelial cell (EC) responses to ischaemia. Pyruvate dehydrogenase kinase 4 (PDK4) normally suppresses mitochondrial respiration in ischaemia by phosphorylating the pyruvate dehydrogenase complex (pPDC) to preserve EC function. However, the role of the PDK4/PDC axis in angiogenesis and the effects of diabetes are unexplored. Methods and Results: In human coronary artery endothelial cells (HCAECs), siRNA-mediated PDK4 knockdown reduced tubulogenesis (49%, P <0.01) and migration (51%, P <0.01), revealing an essential role for PDK4 in angiogenesis. HCAECs were exposed to 5 or 25mM glucose for 72h and hypoxia for 6h. High glucose attenuated the induction of PDK4 and pPDC in response to hypoxia. This was associated with an aberrant increase in mitochondrial respiration (20%, P <0.01), measured by the Seahorse Bioanalyser system, and impaired tubulogenesis (52%, P <0.001) and migration (79%, P <0.0001). Lentiviral overexpression of PDK4 rescued this impairment in high glucose and hypoxia. Similarly, treatment with reconstituted high-density lipoprotein (rHDL), a known proangiogenic agent, restored the induction of PDK4 and pPDC in hypoxia and high glucose, and rescued tubulogenesis and migration. Mechanistically, rHDL increased binding of FOXO1 to the PDK4 promoter (78%, P <0.05), assessed by chromatin immunoprecipitation, compared to PBS controls. In a murine model of diabetic wound healing, diabetes impaired the early induction of PDK4 and pPDC in response to wound ischaemia, and reduced wound neovessels. Topical rHDL enhanced wound PDK4 and pPDC levels over 72h post-wounding (68-165%, P <0.05), and increased wound neovascularisation (62%, P <0.05) and wound closure (45%, P <0.001), compared to PBS controls. Conclusion: PDK4 is essential for hypoxia-driven angiogenesis. This response is impaired in diabetes and can be corrected by PDK4 overexpression and rHDL. We have identified a novel regulatory pathway for diabetes-impaired angiogenesis that can be targeted to prevent diabetic vascular complications.