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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2003 as doi:10.1096/fj.02-0640fje. |
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Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität, D-60590 Frankfurt am Main, Germany; and
* Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
2Correspondence: Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: fleming{at}em.uni-frankfurt.de
SPECIFIC AIMS
The objective of this study was to identify the signal transduction cascade linking epoxyeicosatrienoic acid (EET) generation by cytochrome P450 (CYP) 2C9 to endothelial cell proliferation and angiogenesis. Endothelial cell tube formation was studied in a fibrin gel and vascular network development was determined in the chick chorioallantoic membrane.
PRINCIPAL FINDINGS
1. Effect of CYP 2C9 overexpression on endothelial cell proliferation
To determine the effect of CYP 2C9 on endothelial cell proliferation, human umbilical vein endothelial cells were infected with either CYP 2C9 sense or CYP 2C9 antisense adenoviruses. The expression of CYP 2C9 protein was observed only in cells infected with the CYP 2C9 sense adenoviruses; expression was maximal 24 h after infection and decreased slowly over the next 14 days. Forty-eight hours after infection, proliferation of CYP 2C9-overexpressing endothelial cells was 50% greater than that of control cells infected with the CYP 2C9 antisense adenoviruses and cultured in the presence of 2% FCS. CYP 2C9-induced proliferation was prevented by the CYP 2C9 inhibitor sulfaphenazole.
2. Involvement of the epidermal growth factor (EGF) receptor in the CYP 2C-induced cell proliferation
The CYP 2C9-induced proliferation of human endothelial cells was abrogated by AG1478 (500 nM), an inhibitor of the EGF receptor tyrosine kinase, without affecting the proliferation of control cells (Fig. 1
A). As cyclin D1 expression is significantly increased in endothelial cells transiently overexpressing CYP 2C9, we determined the effect of AG1478 on cyclin D1 expression in virally infected cells. A threefold increase in the expression of cyclin D1 was observed in CYP 2C9-overexpressing cells (Fig. 1B
) but not in cells infected with the control virus (data not shown). An increase in cyclin D1 expression was not observed in cells incubated with AG1478 (Fig. 1B
).
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3. Effect of 11,12-EET on phosphorylation of the EGF receptor
To determine whether a CYP product could elicit the release of an EGF receptor agonist from the endothelial cell surface, we measured EGF/heparin binding EGF (HB-EGF) levels in the supernatant of human umbilical vein endothelial cells. Application of 11,12-EET (1 µM) to endothelial cells elicited rapid accumulation of EGF/HB-EGF in the cell supernatant (Fig. 1C
). We next determined the effects of 11,12-EET on phosphorylation of the ErbB4 receptor, which is highly expressed in human endothelial cells and can be activated by HB-EGF. 11,12-EET induced the time-dependent tyrosine phosphorylation of the ErbB4 receptor, first evident 
5 min after stimulation (Fig. 1D
) and peaking at up to 10-fold over basal levels after 30 min.
4. Effect of CYP 2C9 overexpression on Akt phosphorylation
In human endothelial cells, CYP 2C9 overexpression was associated with a fourfold increase in the phosphorylation of Akt assessed 12 h after adenoviral infection, an effect prevented by AG1478.
5. Effect of CYP 2C9 on angiogenesis
We next determined endothelial tube formation in a fibrin gel seeded with human umbilical vein endothelial cells infected with the control (CYP 2C9 antisense) or the CYP 2C9 (sense) virus. After 14 days, CYP 2C9 overexpression was associated with a 6.4 ± 2.3-fold increase (P<0.05, n=4) in endothelial cell tube formation. Exogenous application of 11,12-EET to otherwise untreated endothelial cells also enhanced tube formation by 5.73 ± 2.6-fold over 14 days (P<0.001, n=3). To assess vessel development in the chick chorioallantoic membrane (CAM), methylcellulose discs containing either solvent (0.5% ethanol) or 11,12-EET (1 µmol/disk) were applied. After 5 days a significant induction of angiogenesis was observed in the 11,12-EET-treated membranes (Fig. 2
). The response to 11,12-EET was similar to that observed in CAMs with pellets containing EGF or VEGF (Fig. 2)
. To determine the involvement of the EGF receptor in 11,12-EET-induced angiogenesis, CAMs were treated with pellets containing 11,12-EET alone or in combination with AG1478 or an EGF receptor-neutralizing antibody. Both AG 1478 and the EGF receptor-neutralizing antibody prevented the 11,12-EET-induced increase in vessel formation (Fig. 2)
.
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CONCLUSIONS AND SIGNIFICANCE
The results of the present investigation demonstrate that the overexpression of a CYP epoxygenase (CYP 2C9) and the application of 11,12-EET to endothelial cells induce angiogenesis. This effect, in an in vitro assay of endothelial cell tube formation as well as in the developing vasculature of the chick chorioallantoic membrane, is linked to the activation of the EGF receptor.
CYP epoxygenase-derived EETs are important modulators of intracellular signal transduction cascades, but it is unclear how EETs can initiate their effects on cell signaling. At least three modes of cell activation by EETs have been proposed. The first involves the activation of a putative extra- or intracellular EET receptor; the second, the incorporation of EETs into the plasma membrane, where they may associate with effector molecules; and a third mode of cell activation involves the EGF receptor. Although 14,15-EET was initially suggested to act as an intracellular second messenger after activation of the EGF receptor, during the preparation of this manuscript, 14,15-EET was reported to activate metalloproteinases in a kidney epithelial cell line and release heparin binding epidermal growth factor-like growth factor (HB-EGF, an EGF receptor ligand. Since 11,12-EET- and CYP 2C9-induced activation of the EGF receptor as well as the phosphorylation of Akt and increased expression of cyclin D1 observed in the present study was sensitive to an EGF receptor inhibitor, it appears that EETs can also elicit EGF signaling in endothelial cells. Whether activation of the EGF receptor by 11,12-EET in endothelial cells involves the activation of a metalloproteinase or can be attributed to EET-induced changes in G-protein, tyrosine kinase, or tyrosine phosphatase activity remains to be determined.
The CYP 2C9 product 11,12-EET activates multiple signal transduction pathways in native and cultured endothelial cells, and overexpression of a CYP 2C epoxygenase in endothelial cells enhances proliferation via a signal transduction cascade involving a MAP kinase phosphatase-1-mediated reduction in JNK activity and an increase in cyclin D1 expression (Fig. 3
). The angiogenic response and vascular maturation, however, involve much more than an increase in endothelial cell number; numerous additional processes, including matrix degradation, cell migration, and the recruitment of mural cells are also essential. Given the complexity of the angiogenic process, it is essential to demonstrate that any putative angiogenic factor is able to accelerate the formation of fully mature blood vessels in a network. Indeed, in the CAM assay 11,12-EET accelerated vascular network formation, including the convergence of vessels in a classical "spoke wheel" and the bending of large vessels. These effects observed in response to 11,12-EET were directly comparable with those of EGF.
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Although the results of the present study indicate that 11,12-EET is a potent angiogenic factor, CYP 2C9 generates not only EETs; like some other CYP epoxygenases, this enzyme generates reactive oxygen species in amounts sufficient to affect intracellular signaling. Because reactive oxygen species have been reported to trans-activate the EGF receptor and the proliferative response to some growth factors also requires the production of oxygen-derived free radicals, it is tempting to speculate that EETs and CYP-derived free radicals may act synergistically to promote angiogenesis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0640fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0640fje 
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