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Vascular endothelial growth factor induces cyclooxygenase-dependent proliferation of endothelial cells via the VEGF-2 receptor¹

Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin 2, Ireland

²Correspondence: Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland. E-mail: jmurphy{at}rcsi.ie

SPECIFIC AIMS

We investigated the relationship between vascular endothelial growth factor (VEGF) and the enzyme cyclooxygenase (COX) to determine whether VEGF could modulate COX activity in human vascular endothelial cells and whether COX was a downstream effector of the angiogenic response to VEGF.

PRINCIPAL FINDINGS

1. VEGF induces COX-1 and -2
Endothelial cells (EC) were incubated with VEGF₁₆₅ (50 ng/ml) for varying periods, then assayed for 6-keto-PGF₁ generation and COX protein expression. After 3 h incubation, COX-2 protein expression was increased, and this was accompanied by an increase in 6-keto-PGF₁ generation that was completely inhibited by the specific COX-2 inhibitor NS398. Prolonged exposure (8–10 h) to VEGF also resulted in an increase in COX-1 protein expression that was accompanied by 6-keto-PGF₁ generation.

2. VEGF-induced COX induction is mediated via the VEGFR-2
Peptides derived from VEGFR-2 that block VEGF binding to the receptor inhibited VEGF-mediated COX-2 expression and PGI₂ formation (Fig. 1 ). The scrambled form of the peptide had no effect up to a concentration of 1 mM. The peptide also inhibited COX-1 protein expression and PGI₂ formation after prolonged exposure to VEGF. The VEGFR-2 peptide had no effect on induction of COX activity by PMA.

View larger version (18K): [in this window] [in a new window]   Figure 1. VEGFR-2 peptide blocks VEGF-induced COX-2 expression. EC were grown to confluence on 6- or 24-well plates, serum starved in M199/2.5% medium overnight, and the wells were treated with aspirin 200 µM for 45 min to inactivate all endogenous COX activity. VEGF (50 ng/ml) was added in the presence or absence of VEGFR-2 peptide for 3 h in serum-free M199 medium. The supernatant was analyzed for 6-keto-PGF1 and the cells were lysed for Western blot of COX-2. Data represent the mean ± SE of duplicate determinations from four experiments.

3. COX- and VEGF-dependent cell proliferation
HUVEC were grown to 50–60% confluence in 96-well tissue culture plates, serum starved (2.5% FBS) overnight, and VEGF was added for 8–10 h in the presence or absence of the COX inhibitors NS398 (COX-2) and SC560 (COX-1). When applied alone, SC560 was partially effective whereas NS398 had no effect. When combined, however, SC560 and NS398 inhibited cell proliferation induced by VEGF even at concentrations of 10 nM. A similar effect was seen with aspirin. No significant inhibition of proliferation was seen in non-VEGF-treated cells. The PGI₂ analog iloprost, but not dinoprostone or U44619, reversed the inhibitory effect of the combined NS398/SC560 combination and aspirin in a dose-dependent manner.

4. COX and VEGF induced angiogenesis
To further address the functional relationship between VEGF and COX isoforms, we examined the effect of the COX inhibitors in an angiogenesis model (Fig. 2 ). Blood vessel growth occurred over 14 days in control wells and was increased by exogenous VEGF 20 ng/ml. In this model, angiogenesis was unaffected by NS398 and almost completely inhibited by the COX-1 inhibitor SC560. This inhibitory effect was partially reversed when SC560 was removed on day 7 of the 14 day assay. Moreover, the addition of the PGI₂ analog iloprost from the outset partially reversed the inhibitory effect of SC560. Similar results were observed in VEGF-treated cells, NS398 did not affect tubule formation whereas SC560 significantly inhibited microtubule formation induced by VEGF. Figure 3 outlines a diagrammatic representation of our findings.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effect of VEGF and COX inhibitors in an in vitro model of angiogenesis. Angiogenesis was measured as microtubule formation over 14 days. The preparation was fixed and stained for either EC protein CD31 or von Willebrand factor. The wells were visualized by phase contrast microscopy and the tubules over five fields were counted. VEGF (20 ng/ml) and the COX inhibitors NS398 (NS) and SC560 (SC, each at 1 µM) were added to the media. The media were changed and fresh VEGF or inhibitors were added every 3 days. SC560 inhibited blood vessel formation whereas NS398 had no effect. The inhibitory effect of SC560 was reversed (rev) when the drug was removed on day 7 or by the addition of iloprost (+ilo) (ANOVA, F=161.2). *P < 0.01; ***P < 0.001; +++P < 0.001 inhibitors vs. control.

View larger version (13K): [in this window] [in a new window]   Figure 3. No caption available.

CONCLUSIONS

COX-2 has been implicated in the pathogenesis of several cancers, particularly colon cancer. First, COX-2 is expressed in human colonic tumors where its expression is linked to survival. Second, inhibition of COX-2 in the Mn-/- murine model of colon cancer suppresses tumor formation. Inhibition of COX-2 has been reported to suppress growth in colon cancer cell lines and to induce apoptosis in some tumors. The findings in these and other cell types are consistent with a role for cyclooxygenase products in regulating cell survival. Indeed, 15-deoxy, delta 12,14- PGJ₂ promotes cell death whereas PGI₂ promotes cell survival. These effects are mediated by a series of nuclear orphan receptors, including peroxisomal proliferator activator receptors (PPAR) (for GI₂) and (for 15-deoxy, delta 12,14-PGJ₂). However, whether these are responsible for their effects on cell growth and survival is unclear.

COX-2 is also expressed in the vasculature surrounding colon cancers (24), and there is evidence that inhibition of COX-1 and -2 suppresses angiogenesis. Arguably, this is the explanation for the beneficial effects of COX inhibitors. As VEGF is generated at the site of tumors and is a potent angiogenic factor, we explored the relationship between COX and VEGF. Several reports have shown an increase in cyclooxygenase activity in human endothelial cells treated with VEGF. Similarly, we showed that VEGF increased PGI₂ formation by EC over 3 h and that this was COX-2 dependent.

VEGF acts on two receptors, VEGFR-1 and VEGFR-2, in EC. VEGFR-2 transduces much of the signaling in EC, leading to changes in cell morphology, actin reorganization, membrane ruffling, and proliferation. Inhibition of VEGFR-2 activation by several different strategies including the VEGFR-2 dominant-negative mutant, neutralizing antibodies directed against VEGFR-2 or VEGF, ribozymes against VEGFR-2 or antisense strategies against VEGF, and recently described pharmacological strategies all lead to decreased tumor angiogenesis and tumor growth. However, VEGFR-1 may also contribute to EC migration and new vessel formation around tumors.

Our experiments show that VEGF-dependent COX-2 induction mediated through the VEGFR-2 as a peptide corresponding to the receptor binding site blocked VEGF-induced COX-2 expression. VEGF activates p42/44 (ERK1/2) MAP kinase, a pathway that has been linked to induction of COX-2. Indeed, the promoter of the COX-2 gene contains several elements that are sensitive to ERK1/2 activation, including Ets-1, AP-1, and CREB. We cannot exclude that some of the signaling occurred via the VEGF-1 receptor. However, much of the signal was attenuated by blocking the VEGFR-2.

COX-1 played an important role in the proliferative response to VEGF in EC in that the response was blocked by the combination of selective COX isoform inhibitors. Similarly, in the stomach, inhibition of both isoforms is necessary for mucosal injury to occur, which suggests that the two isoforms can compensate for one another. In an angiogenesis assay where VEGF promotes formation of microtubules, only the COX-1 inhibitor prevented microtubule formation. The two assays differ in one important respect in that the angiogenesis assay is performed over 14 days and the proliferation assay over 8–10 h. It is possible that an acute-phase gene such as COX-2 is less relevant over a prolonged period.

Our results are consistent with recent reports that inhibition of COX suppresses angiogenesis. Although angiogenesis has been attributed to prostaglandin generation by COX-2, both COX-1 and COX-2 have been implicated and the response has both prostaglandin-dependent and -independent components. In our experiments, the inhibition of VEGF-induced EC proliferation was overcome by the addition of an analog of prostacyclin, the most abundant product of these cells. Thus, the effect of the COX inhibitors could be explained at least in part by suppression of product formation. There are two types of receptors for prostacyclin: surface G-protein-coupled receptors and PPARs. As the prostacyclin GPCR is not expressed in EC, prostacyclin may interact with PPAR to mediate its protective effects.

In conclusion, COX induction and PG formation are downstream effectors of VEGF-dependent EC activation and angiogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0757fje ; to cite this article, use FASEB J. (May 29, 2001) 10.1096/fj.00-0757fje

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