NITRIC-OXIDE SYNTHASE INHIBITORS ATTENUATE TRANSFORMING-GROWTH-FACTOR-BETA(1)-STIMULATED CAPILLARY ORGANIZATION IN-VITRO

Angiogenesis is a complex process involving endothelial cell (EC) prol iferation, migration, differentiation, and organization into patent ca pillary networks. Nitric oxide (NO), and EC mediator, has been reporte d to be antigenic as well as proangiogenic in different models of in v ivo angiogenesis. Our aim was to investigate the role of NO in capilla ry organization using rat microvascular ECs (RFCs) grown in three-dime nsional (3D) collagen gels. RFCs placed in 3D cultures exhibited exten sive tube formation in the presence of transforming growth factor-beta (1). Addition of the NO synthase (NOS) inhibitors L-nitro-arginine met hylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L ) inhibited tube formation and the accumulation of nitrite in the medi a by approximately 50%. Incubation of the 3D cultures with excess L-ar ginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synt hesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor -beta(1) to suppress EC proliferation in 2D cultures. Reverse transcri ptase-polymerase chain reaction revealed the constitutive expression o f all three NOS isoforms, neuronal, inducible, and endothelial NOSs, i n 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D culture s of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bro mo-cGMP, restored capillary tube formation. Thus, the autocrine produc tion of NO and the activation of soluble guanylate cyclase are necessa ry events in the process of differentiation and in vitro capillary tub e organization of RFCs.