Role of nitric oxide in capillary organization and differentiation

Angiogenesis is a complex process involving endothelial cell division and proliferation, cytostasis, differentiation and organization into a patent capillary networks. Nitric oxide (NO), an endothelial cell mediator has been reported to be anti- as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using microvascular rat epididymal fat pad capillary endothelial cells (RFCs) grown in either two (2D) or three dimensional (3D) tissue culture. Inhibition of NOS with nitro-1-arginine methyl ester (L-NAME, 1 mM), did not influence proliferation of RFCs, or antagonize the ability of the ability of transforming growth factor-β1 (TGF-β) to suppress RFC proliferation in 2D culture. In 3D culture, TGF-β stimulated extensive capillary tube formation. In contrast to results obtained in 2D, L-NAME and L-monomethyl-nitro-1-arginine (L-NMMA,! mM), markedly inhbited TGF-β induced tube formation (by 75-80%) in 3D culture. The inhibitory effects of L-NAME were partially reversed with L, but not D-arginine. Interestingly, in face of NOS blockade, co-treatment with the NO donor, sodium nitroprusside (SNP, 100 μM) or 8-bromocGMP (1 mM) resored capillary tube formation. Moreover, méthylène blue (100 μM), an inhibitor of soluble guanylyl cyclase (sGC), attenuated TGF-β induced tube formation. Thus, the autocrine production of NO and activation of sGC are necessary events in the process of capillary organization and differentiation in RFCs.