© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Nitric oxide and angiogenic activity of endothelial cells: direct or VEGF-dependent effect?
aDepartment of Cell Biochemistry, Institute of Molecular Biology and Biotechnology, Jagiellonian University, Kraków, Poland
bLaboratory of Molecular Genetics and Genetic Engineering, Institute of Molecular Biology and Biotechnology, Jagiellonian University, Kraków, Poland
cDepartment of Vascular Surgery, University of Vienna, Vienna, Austria
jdulak{at}mol.uj.edu.pl
* Corresponding author. Tel.: +48-12-252-6375; fax: +42-12-252-6902
Received 25 June 2002; accepted 25 June 2002
In a recent interesting paper Babaei and Stewart [1] demonstrated that coculture of endothelial cells (ECs) with smooth muscle cells (SMCs) may result in the formation of extensive capillary-like structure. However, the effect was observed only when SMCs were previously transfected with vector containing the endothelial nitric oxide synthase (eNOS) cDNA. Such an in vitro angiogenic-like events were abrogated by L-NAME, a NOS inhibitor. A similar angiogenic response has been observed when SMCs were transfected with plasmids containing VEGF121 cDNA, the effect again being inhibited by L-NAME. Similarly, in a Boyden chamber model the EC migration was potently enhanced in the presence of SMC transfected with eNOS or VEGF121, and was significantly attenuated by L-NAME.
Much evidence indicates that NO plays an integral role in VEGF signaling (see Ref. [1]). It has been convincingly demonstrated that NO may be involved in EC proliferation, migration, protease release and increased vascular permeability, the effects important for initiation of angiogenesis. Thus, the recent report by Babaei and Stewart [1] is in keeping with previous observations. The authors state that this is the demonstration that NO exerts a direct proangiogenic activity. They claim that NO generated by eNOS or by NO donors facilitate EC migration, even in the absence of growth factors [1].
We want, however, to propose another explanation for those phenomena. A number of recent studies demonstrated that NO induces VEGF synthesis in numerous cell types, among them SMCs ([2–6] and Refs. therein). We have shown that induction of inducible NOS (iNOS) [2–4] or treatment of SMCs with different NO donors [5] induces VEGF expression in rat or human VSMCs, acting at the level of transcriptional activation. Additionally, we have recently demonstrated that VSMCs engineered to overexpress plasmids containing either the iNOS [5] or eNOS gene [2,5] generated significantly more VEGF than cells transfected with control vectors. Such an enhanced VEGF synthesis was attenuated when eNOS or iNOS-transfected cells were treated with the NOS inhibitor L-NAME, but not the inactive enantiomer, D-NAME. More importantly, we have finally demonstrated that the conditioned media from SMC transfected with eNOS or iNOS enhanced the proliferation of human umbilical vein endothelial cells (HUVECs), the effect being abrogated by pretreatment with anti-VEGF neutralizing antibodies [5].
In their study, Babaei and Stewart used only inhibitors of NOS activity and did not investigate the possibility that overexpression of eNOS enhanced VEGF production [1]. Thus, we postulate that the direct angiogenic effect of NO has not been yet proven.
The increased morphogenic events observed by Babaei and Stewart in the co-culture of ECs and SMCs transfected with VEGF expression vectors were also abolished by L-NAME. This observations add to the previous studies demonstrating that VEGF induces angiogenesis by enhancing NO generation in endothelial cells [1].
Endothelial cells are the target, not the main source of VEGF. Microvascular endothelial cells express VEGF and can release some amounts of this growth factor. Macrovascular endothelial cells, like HUVECs, do not release detectable quantities of VEGF into the culture media. Interestingly, however, it has been also recently demonstrated that exogenous VEGF can induce VEGF expression in microvascular endothelial cells [7]. The detailed mechanisms behind this event have not been yet clarified. Therefore, it would be worthwhile to elucidate whether the NO-dependent effects in ECs rely on VEGF expression in those cells (Fig. 1). Accordingly, it still remains to be proven whether NO really exerts a direct angiogenic effect in ECs or whether this action of NO is mediated by autocrine activity of VEGF.
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Studies in the authors’ laboratories are supported by grants from the Polish State Committee for Scientific Research.
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- Babaei S., Stewart D.J. Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model. Cardiovasc. Res. (2002) 55:190–200.
[Abstract/Free Full Text] - Dulak J., Jozkowicz A., Dembinska-Kiec A., Guevara I., Zdzienicka A., Zmudzinska-Grochot D., Florek I., Wojtowicz A., Szuba A., Cooke J.P. Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. (2000) 20:659–666.
[Abstract/Free Full Text] - Dulak J., Jozkowicz A., Dichtl W., Alber H., Schwarzacher S.P., Pachinger O., Weidinger F. Vascular endothelial growth factor synthesis in vascular smooth muscle cells is enhanced by 7-ketocholesterol and lysophosphatidylcholine independently of their effect on nitric oxide generation. Atherosclerosis (2001) 159:325–332.[CrossRef][Web of Science][Medline]
- Dulak J., Jozkowicz A., Frick M., Alber H.F., Dichtl W., Schwarzacher S.P., Pachinger O., Weidinger F. Vascular endothelial growth factor: angiogenesis, atherogenesis or both? J. Am. Coll. Cardiol. (2001) 38:2137–2138.
[Free Full Text] - Jozkowicz A., Cooke J.P., Guevara I., Huk I., Funovics P., Pachinger O., Weidinger F., Dulak J. Genetic augmentation of nitric oxide synthase increases the vascular generation of VEGF. Cardiovasc. Res. (2001) 51:773–783.
[Abstract/Free Full Text] - Dulak J., Jozkowicz A., Foresti R., Kasza A., Frick M., Huk I., Green C.J., Pachinger O., Weidinger F., Motterlini R. Heme oxygenase activity modulates vascular endothelial growth factor synthesis in vascular smooth muscle cells. Antioxid. Redox. Signal (2002) 4:229–240.[CrossRef][Web of Science][Medline]
- Vega-Diaz B., Herron G.S., Michel S. An autocrine loop mediates expression of vascular endothelial growth factor in human dermal microvascular endothelial cells. J. Invest. Dermatol. (2001) 116:525–530.[CrossRef][Web of Science][Medline]
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