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Cardiovascular Research 2002 56(3):489-491; doi:10.1016/S0008-6363(02)00671-5
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Angiogenesis? the answer is NO·

Saeid Babaei, Golnaz Karoubi and Duncan J. Stewart*

Terrence Donnelly Vascular Biology Laboratory, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada

* Corresponding author. Tel.: +1-416-864-5724; fax: +1-416-864-5419 stewartd{at}smh.toronto.on.ca

Received 16 September 2002; accepted 18 September 2002

We thank Drs Dulak and Jozkowicz for their interest in our paper [1]. They raise the question of whether induction of VEGF expression by NO in cultured smooth muscle cells (SMCs) may have contributed to the angiogenic effects of eNOS in our co-culture model. Although, we agree that this is a potential mechanism whereby NO might induce angiogenesis, we would like to point out that NO does not always increase angiogenic factor expression, and may even attenuate hypoxia-induced upregulation of VEGF by inhibition of HIF-1{alpha} in aortic SMCs and pulmonary artery EC or AP-1 in aortic SMCs [2,3].

In order to address this issue in our model, we have since measured VEGF production in response to eNOS overexpression in co-culture after 48 h of incubation. No detectable VEGF levels were observed in the co-culture of SMCeNOS with CPAEC, suggesting that eNOS overexpression did not increase VEGF expression in this model. However, SMCVEGF121 produced high levels of VEGF peptide detected by an ELISA kit (R&D Systems), (265±31 pg/ml per 105 cells, n = 4, each measurement in duplicate) which was not detected in the SMCCont co-cultures. These results confirm our earlier findings that NO generated by NO-donors facilitated EC migration and tube formation, without changing bFGF mRNA expression [4].

Clearly, there are limitations to any in vitro model. We believe our results are important, since they show that, in addition to increasing angiogenic growth factor expression [5], NO can also have direct effects stimulating EC migration and capillary-like tube formation. Formation of new blood vessels is a complex process and requires coordinate regulation of multiple mechanisms, including modifications of the cell–cell interactions and their extracellular matrix, ECs migration/invasion and proliferation and finally maturation into the neovessels. The appended figure is an attempt to illustrate how different angiogenic pathways converge on NO as a downstream mediator (Fig. 1).


Figure 1
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  		Fig. 1 NO involvement in various angiogenic cascades. NO has been suggested to initiate an ‘angiogenic program’, modulating the angiogenic effects of various growth factors including bFGF and VEGF [4–6] through the changes in the ECM proteins in particular integrin [7,8] and EC adhesion molecules [9,10] as well as EC cell migration and proliferation by changes in PKC{delta} activity [10–13]; however, the mechanism(s) by which NO modulates angiogenesis are not fully elucidated. Most intracellular functions of NO are mediated via activation of intracellular receptor guanylyl cyclase, increase in cGMP and further activation of the MAPK pathway. However, NO may also directly upregulate factors involved in the angiogenic cascade. For instance, NO has been implicated to increase ICAM-1 expression leading to cytoskeleton changes contributing to enhancement of EC migration via the PI3K/AKT/NO pathway [14]. NO may also regulate the integrin-dependent signal transduction pathway via its action to stimulate ADP-ribosylation of actin [15,16], and also being involved in the de novo formation of focal adhesions, tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin, and assembly of stress fibers, which are important components in cell locomotion and migration [17].

 


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