FGF2 is one of 23 fibroblast growth factor family members and signals via one of four receptors, FGFR1, 2, 3 and 4, of which FGFR1 is most commonly expressed on endothelial cells . A principal role of secreted FGF2 is to stimulate blood vessel growth although we have shown previously that it can also act as a potent autocrine growth factor to enhance epithelial cell proliferation . In vitro and animal xenograft studies have shown that secretion of epithelial FGF2 in endometrial adenocarcinoma xenografts can enhance tumour growth by enhancing blood vessel size and width . Furthermore antisense targeting of FGF2 in such model systems is known to reduce tissue microvascular density as well as xenograft size .
Endothelial cell differentiation and proliferation are two of the processes required for angiogenesis [11, 21–23]. In the present study we have shown that conditioned medium from endometrial adenocarcinoma cells, which stably express the FP receptor to the levels observed in endometrial adenocarcinomas (FPS cells) and produce FGF2, promotes endothelial network formation (differentiation) and proliferation. Using a specific FGFR1 tyrosine kinase inhibitor and FGF2-immunoneutralised conditioned medium, we showed that the effects of conditioned medium on endothelial cell network formation and proliferation were via FGF2-FGFR1 signalling. We found that although the FGF2-immunoneutralised treatment inhibited network formation and proliferation, it was less effective than the FGFR1 inhibitor SU4984. We believe this difference lies in the residual FGF2 remaining in the FGF2-immunoneutralised CM after neutralisation  which could, albeit to a lesser extent, activate the FGF receptor on HUVECs.
To explore the signalling pathways activated in HUVECs by FGF2, following its release from epithelial cells in response to PGF2α-FP receptor activation, we used small molecule chemical inhibitors of intracellular signalling pathways. We found that conditioned medium from PGF2α-treated Ishikawa FPS cells enhanced endothelial cell network formation via FGFR1 and ERK1/2 independently of PI3K and mTOR. This is in agreement with the observations of Kanda et al. , who demonstrated in murine brain endothelial cells that FGF2 induced endothelial network formation is not dependent on activation of the mTOR pathway  and Sulpice et al  who showed that, in adrenal cortex capillary endothelial cells, ERK1/2 phosphorylation induced by recombinant FGF2 is not mediated via the PI3K pathway . Similarly, Peng et al. showed that FGF2 treatment can induce mTOR phosphorylation in HUVECs .
In contrast, we found that conditioned medium-induced endothelial cell proliferation was dependent on ERK1/2 signalling to mTOR as endothelial cell proliferation could be inhibited with the ERK1/2 kinase inhibitor PD98059 and rapamycin, but not the PI3K inhibitors wortmannin or LY294002. This is in agreement with previous studies showing that the ERK1/2 inhibitor PD98059 can inhibit FGF2- induced angiogenesis  and HUVEC proliferation . Our data indicate that endothelial network formation and proliferation are regulated by distinct signal transduction pathways which are integrated by ERK1/2 signalling.
ERK1/2 is known to be a potent regulator of cell growth, differentiation and development . Once phosphorylated, ERK1/2 can translocate to the nucleus and promote gene transcription . The phosphorylation and activation of ERK1/2 can be modulated via a multitude of intracellular signal transduction pathways. Hence, we investigated conditioned medium signalling to ERK1/2 in HUVECs and found within our experimental paradigm that ERK1/2 was phosphorylated in a time dependent manner. The fact that ERK1/2 is activated by P CM and that the ERK1/2 inhibitor abolished ERK1/2 phosphorylation, as well as endothelial network formation and proliferation, suggests that ERK1/2 could act as a major transcriptional regulator in this model system. This phosphorylation and activation of ERK1/2 was found to be regulated via FGFR1 signalling to c-Src, since co-treatment of HUVECs with P CM and the FGFR1 inhibitor SU4984 or cSrc inhibitor PP2 significantly inhibited ERK phosphorylation. Furthermore ERK1/2 phosphorylation was found to be independent of PI3K and mTOR as neither the PI3K inhibitor LY294002 nor the mTOR inhibitor rapamycin (data not shown) inhibited P CM-induced ERK1/2 phosphorylation. c-Src is a protein tyrosine kinase which co-ordinates a diverse spectrum of receptor-induced signalling to ERK1/2 via the phosphorylation of signalling intermediates such as Ras and Raf . c-Src has been shown to be involved in FGF-2 induced angiogenesis  and a recent study has shown that c-Src, Raf and ERK1/2 are essential for HUVEC lumen formation in vitro . These data suggest that the FGF2-FGFR1-c-Src pathway plays a role in the activation of ERK1/2 by P CM treatment.
Following ERK1/2 activation, mTOR has been shown to be regulated via the tuberous sclerosis complex 1 and 2 (TSC1/2 also called Hamartin and Tuberin) [33, 34]. Phosphorylation of TSC2 by ERK1/2 results in its dissociation from TSC1 and its subsequent degradation via the ubiquitin pathway. This inactivates the inhibitory effect of TSC1/2 on the mTOR pathway and allows cellular proliferation to proceed .
Over the past decade several reports have highlighted the importance of COX enzymes and prostaglandins in regulating vascular function indirectly. This may occur via the activation of ERK1/2 signalling resulting in epithelial or stromal cell production of pro-angiogenic factors which act in a paracrine manner on endothelial cells [4, 36, 37]. This is in agreement with our observations here whereby FGF2, released by Ishikawa FPS cells in response to PGF2α, enhanced the expression of COX-2 in endothelial cells via the FGF2-FGFR1-ERK1/2 pathway. Similarly, FGF2 has been shown to upregulate endothelial COX-2 in murine cerebral microvascular cells leading to an increase in prostaglandin E2 production .
Prostaglandins have been shown to be secreted by endothelial cells and to influence directly endothelial cell function via their receptors on endothelial cells [39–41]. These studies showed that PGE2 present in the endothelial environment can enhance endothelial cell functions [38–41], however in our study we found no significant elevation in PGE2 biosynthesis in response to P CM. Instead, we found that endothelial cells secrete elevated levels of PGF2α following activation by CM from PGF2α-treated Ishikawa FPS cells and that this PGF2α secretion was regulated via the FGF2-FGFR1-ERK1/2-mediated induction of COX-2 since the specific COX-2 inhibitor significantly reduced PGF2α secretion. In order to determine whether COX-1 contributed towards the generation of PGF2α, a general COX inhibitor indomethacin was used. Co-treatment of cells with indomethacin significantly reduced PGF2α secretion to a level below that observed for the specific COX-2 inhibitor suggesting that basal levels of COX-1 may, to a lesser extent, contribute towards the secretion of PGF2α. These data indicate that although PGE2 is secreted in higher quantities than PGF2α by unstimulated HUVECs , under P CM stimulated conditions, prostaglandin F2α is the predominant COX-2 product. Furthermore, this suggests that the endothelial signalling pathways induced by FGF2 are context dependent, i.e. dependent on the nature of the external stimulus, such as cancer conditioned medium, from which the FGF2 originates. We found exogenous prostaglandin F2α was able to stimulate endothelial cell network formation but not proliferation. Interestingly, the effect of exogenous PGF2α on network formation was less than that observed for P CM. We believe that the higher levels of FP receptor in endothelial cells induced by P CM accounts for this difference. It is likely that the upregulated FP receptor in P CM treated HUVECs would enable a greater signalling capacity and ability to form networks compared with HUVECs treated with exogenous PGF2α alone in the absence of growth factors, where FP receptor expression is not induced. Using a specific FP receptor short hairpin RNA (FP shRNA) in an adenoviral delivery system for targeted ablation of endothelial FP receptor, we found that P CM-induced endothelial network formation was regulated by the endothelial FP receptor. In addition, the use of a chemical inhibitor against COX-2 and a specific FP receptor antagonist further confirmed a role for endothelial PGF2α signalling through the endothelial FP receptor in the regulation of P CM-induced endothelial cell network formation.