Activation of Mitochondrial KATP Channels by Diazoxide Stimulates Vascular Repair‐Relevant Functions of Human CD34+ Cells by Activating Endothelial Nitric Oxide Synthase

Abstract

Diazoxide is a selective activator of mitochondrial ATP‐sensitive potassium channel (mitoKATP), which causes mitochondrial depolarization, and is known to be protective in ischemic conditions. Bone marrow derived CD34+ hematopoietic stem/progenitor cells have vascular‐reparative functions and are now being tested for their potential benefit for the treatment of ischemic vascular diseases in clinical trials. We hypothesized that diazoxide stimulates vascular reparative functions of CD34+ cells by mitochondrial mechanisms. The hypothesis was tested by determining the effects of diazoxide on the vascular repair‐relevant functions of CD34+ cells in vitro, and evaluated the mitochondrial mechanisms involved in the beneficial response. Peripheral blood was obtained from healthy individuals (n=58) and mononuclear cells (MNCs) were separated by Ficoll centrifugation. MNCs were enriched for lineage negative cells, which were further enriched for CD34+ cells by immunomagnetic selection. Proliferation and migration functions of CD34+ cells were evaluated by using BrdU‐ELISA, and by Chemotaxis assay, respectively. Mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS), and intracellular nitric oxide (NO) levels were evaluated by flow cytometry using JC‐1, mitoSOX and DAF‐FM, respectively. Diazoxide stimulated proliferation and migration of CD34+ cells in basal conditions, and potentiated these responses induced by hypoxia‐regulated factors, stromal‐derived factor‐1a (SDF) or vascular endothelial growth factor (VEGF), and in 5‐hydroxydecanoate (100 μM) (5HD)‐sensitive manner (P<0.05, n=6). Diazoxide (10 μM) induced mitochondrial depolarization of cells and decreased JC‐1 red fluorescence (P<0.05, n=8, vs untreated) that was reversed by 5HD. Diazoxide elevated intracellular NO levels that accompanied increased cGMP levels. NO release was blocked by a nonspecific NOS inhibitor, L‐NAME (300 μM) or by eNOS‐selective inhibitor, L‐N5‐(1‐Iminoethyl) ornithine (NIO, 10 μM) (P<0.01, n=6), but not by nNOS‐selective inhibitor, N‐ω‐Propyl‐L‐arginine (NPA, 50 nM). NO generation by diazoxide was sensitive to treatment with Ca2+ chelator BAPTA (10 μM), Akt inhibitor, triciribine (5 μM), or PI3K inhibitor, LY294002 (30 μM) (P<0.05, n=6). This was further confirmed by increased eNOS phosphorylation at Ser1177 or dephosphorylation at Thr495 (P<0.05, n=6). Diazoxide increased mitochondrial superoxide levels that was abolished by mitoTEMPOL (100 μM) (P<0.01, n=6), and interestingly, it was partially inhibited by L‐NAME or NPA (P<0.01, n=5) but not by NIO. Effects of diazoxide on proliferation and migration of CD34+ cells (P<0.05 vs untreated, n=6) were inhibited by L‐NAME (P<0.01, n=6) or by mitoTEMPOL (P<0.05, n=4). In conclusion, diazoxide stimulated vascular‐repair‐relevant functions of CD34+ cells in NO and ROS‐dependent mechanisms. Mitochondrial depolarization by diazoxide generates ROS, which in turn stimulates NO release by kinase‐ and Ca2+‐dependent activation of eNOS. Intriguingly, nNOS‐dependent generation of mitochondrial ROS contributes to the activation of eNOS.