Endothelial Nitric Oxide Synthase Uncoupling Impairs Endothelial Progenitor Cell Mobilization and Function in Diabetes

Endothelial Nitric Oxide Synthase Uncoupling Impairs Endothelial Progenitor Cell Mobilization and Function in Diabetes Thomas Thum 1 , Daniela Fraccarollo 1 , Maximilian Schultheiss 1 , Sabrina Froese 1 , Paolo Galuppo 1 , Julian D. Widder 1 2 , Dimitrios Tsikas 3 , Georg Ertl 1 and Johann Bauersachs 1 1 Universität Würzburg, Universitatsklinikum, Medizinische Klinik I, Würzburg, Germany 2 Division of Cardiology, Emory School of Medicine, Atlanta, Georgia 3 Institute for Clinical Pharmacology, Medical School Hannover, Hannover, Germany Address correspondence and reprint requests to Dr. med. Thomas Thum or PD Dr. med. Johann Bauersachs, Universitatsklinikum, Medizinische Klinik I, Josef-Schneider Str. 2, 97080 Würzburg, Germany. E-mail: thum_t{at}klinik.uni-wuerzburg.de or bauersachs_j{at}medizin.uni-wuerzburg.de Abstract Uncoupling of the endothelial nitric oxide synthase (eNOS) resulting in superoxide anion (O 2 − ) formation instead of nitric oxide (NO) causes diabetic endothelial dysfunction. eNOS regulates mobilization and function of endothelial progenitor cells (EPCs), key regulators of vascular repair. We postulate a role of eNOS uncoupling for reduced number and function of EPC in diabetes. EPC levels in diabetic patients were significantly reduced compared with those of control subjects. EPCs from diabetic patients produced excessive O 2 − and showed impaired migratory capacity compared with nondiabetic control subjects. NOS inhibition with N G -nitro- l -arginine attenuated O 2 − production and normalized functional capacity of EPCs from diabetic patients. Glucose-mediated EPC dysfunction was protein kinase C dependent, associated with reduced intracellular BH 4 (tetrahydrobiopterin) concentrations, and reversible after exogenous BH 4 treatment. Activation of NADPH oxidases played an additional but minor role in glucose-mediated EPC dysfunction. In rats with streptozotocin-induced diabetes, circulating EPCs were reduced to 39 ± 5% of controls and associated with uncoupled eNOS in bone marrow. Our results identify uncoupling of eNOS in diabetic bone marrow, glucose-treated EPCs, and EPCs from diabetic patients resulting in eNOS-mediated O 2 − production. Subsequent reduction of EPC levels and impairment of EPC function likely contributes to the pathogenesis of vascular disease in diabetes. BH4, tetrahydrobiopterin CFU, colony forming unit EBM, endothelial basal medium eNOS, endothelial nitric oxide synthase EPC, endothelial progenitor cell HPLC, h