P95	Uncoupling of the endothelial nitric oxide synthase in bone marrow of diabetic rats – impact on endothelial progenitor cells.
T.Thum, D.Fraccarollo, P.Galuppo, M.Schultheiss, G.Ertl, J.Bauersachs
Julius-Maximilians-Universität, Würzburg, DE.

Background: Diabetes is associated with reduced vascular repair and is a major risk factor for atherosclerosis. Endothelial progenitor cells (EPC) are bone marrow derived cells with the potential to repair endothelial lesions. EPC are translocated to the circulation upon vascular endothelial growth factor (VEGF)/nitric oxide (NO) mediated pathways. Reduction or dysfunction of EPC is mechanistically associated with progression of atherosclerosis. Patients with diabetes mellitus have reduced amounts of circulating EPC, but the underlying events leading to impaired mobilisation of these cells from bone marrow are not known. Methods: We studied EPC levels and bone marrow alterations in rats with streptozotocin-induced diabetes and control animals. EPC were defined as acetylated LDL+/UEA-1+/VEGF receptor-2+/eNOS+ progenitor cells with the capability for migration and incorporation into vascular networks. Results: Diabetic animals had significantly higher plasma glucose concentrations, whereas the number of circulating EPC was reduced to 63±17%. Expression of the endothelial NO synthase (eNOS) was upregulated in bone marrow extracts of diabetic rats versus control animals (1140±74 versus 898±56 densitometric units/µg protein; p<0.05), whereas eNOS phosphorylation was reduced (245±35 versus 369±40 densitometric units/µg protein; p<0.05). The production of reactive oxygen species (ROS) was 5-fold increased in bone marrow of diabetic rats. In addition, NOS inhibition in bone marrow extracts of diabetic animals by N-omega-nitro-L-arginine methyl ester (L-NAME) significantly reduced ROS production as determined by lucigenin-enhanced chemiluminescence, whereas in control animals the opposite effect was observed. Finally, we found a reduced eNOS dimer/monomer ratio in bone marrow of diabetic animals. Conclusions: We describe for the first time the association between reduced circulating EPC and uncoupling of the eNOS in the bone marrow with increased eNOS-dependent ROS formation in diabetes. This may contribute to the pathogenesis of vascular complications in diabetes mellitus.

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