title: Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model.
abstract: 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 VEGF(121), VEGF(165) (SMC(VEGF)), endothelial NOS (SMC(eNOS)) or the empty vector (SMC(Cont)). Expression of the eNOS in SMC(eNOS) 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 SMC(Cont) 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 SMC(eNOS) resulted in the formation of extensive capillary-like structures within 48 h (DI=17.2+/-5.9, P<0.001 versus SMC(Cont)), which was significantly inhibited using a NOS inhibitor, L-NAME (3 mM) (DI=4.5+/-3.04, P<0.001 versus SMC(eNOS)). Similarly, SMC(VEGF121) 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, SMC(eNOS), but not SMC(Cont) increased EC migration to a similar extent as SMC(VEGF121), 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.
authors.affiliation: Terrence Donnelly Vascular Biology Laboratory, St. Michael's Hospital, Toronto, Ontario, Canada.
authors.forename: Saeid
authors.initials: S
authors.lastname: Babaei
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Stewart
mesh_terms: D000818:Animals; D002417:Cattle; D002454:Cell Differentiation; D002465:Cell Movement; D018920:Coculture Techniques; D016228:Endothelial Growth Factors / Q000235:genetics; D004730:Endothelium, Vascular / Q000201:enzymology*; D015870:Gene Expression; D008222:Lymphokines / Q000235:genetics; D023421:Models, Animal; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D011651:Pulmonary Artery; D051381:Rats; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D016228:Endothelial Growth Factors; D008222:Lymphokines; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat