© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model
Terrence Donnelly Vascular Biology Laboratory, St. Michael's Hospital, and Departments of Medicine and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
stewartd{at}smh.toronto.on.ca
* Corresponding author. Dexter H.C. Man Chair of Cardiology, University of Toronto and Head, Division of Cardiology, St. Michael's Hospital, 30 Bond Street, Toronto, Ontario M5B 1 W8, Canada. Tel.: +1-416-864-5724; fax: +1-416-864-5419
Objective: Angiogenesis is a complex multistep process that involves endothelial cell (EC) migration, proliferation and differentiation into vascular tubes. NO has been reported to be a downstream mediator in the angiogenic response to a variety of growth factors, but the mechanisms by which NO promotes neovessel formation is not clear. We hypothesized that NO directly contributes to EC migration and capillary tube formation. Methods: Since previous studies have noted important biological differences between NO produced pharmacologically by NO-donor compounds compared to that from NO synthase (NOS), we used a cell-based gene transfer approach to increase NO production in a co-culture model of in vitro angiogenesis. Rat smooth muscle cells (SMCs) were transfected with plasmids containing VEGF121, VEGF165 (SMCVEGF), endothelial NOS (SMCeNOS) or the empty vector (SMCCont). Expression of the eNOS in SMCeNOS was confirmed by Northern analysis, NADPH-diaphorase activity, and nitrite/nitrate levels, whereas VEGF production was confirmed using ELISA. Calf pulmonary artery ECs (CPAECs) were cultured on the fibrin matrix with (co-culture) or without underlying SMCs (monoculture). Results: Co-culture of ECs with SMCCont had no effect on EC differentiation compared with EC in monoculture (differentiation index, DI=2.8±3.4 vs. 2.1±2.8, respectively, NS). In contrast, co-culture with SMCeNOS resulted in the formation of extensive capillary-like structures within 48 h (DI=17.2±5.9, P<0.001 versus SMCCont), which was significantly inhibited using a NOS inhibitor, L-NAME (3 mM) (DI=4.5±3.04, P<0.001 versus SMCeNOS). Similarly, SMCVEGF121 induced an angiogenic response (DI=14.2±3.8), which was also significantly inhibited by L-NAME (DI=5.9±1.8, P<0.05). In using the Boyden chamber model, SMCeNOS, but not SMCCont increased EC migration to a similar extent as SMCVEGF121, and both were significantly inhibited with L-NAME. Conclusions: These data support an important paracrine role for endogenously produced NO in EC migration and differentiation in vitro, and suggest that the cell-based eNOS gene transfer may be a useful approach to increase new blood vessel formation in vivo.
KEYWORDS Angiogenesis; Extracellular matrix; Gene therapy; Growth factors; Nitric oxide; Smooth muscle