In the present study, we investigated the effects of RPE-EC interaction in both contact and noncontact coculture models, representing pathologic and normal conditions, respectively, on the angiogenic behavior of EC per se. After coculture in direct contact, ECs were separated from RPE cells using either anti-CD31– or anti-VEGFR2–coated magnetic beads. This procedure enabled us to examine the angiogenic properties of ECs after direct contact with RPE cells. ECs cocultured in contact with RPE, followed by separation from the mixed culture, exhibited enhanced angiogenic properties, including increased expression of
VEGF and
MMP2 at both mRNA and protein levels. Furthermore, ECs grown in contact with RPE cells demonstrated upregulated mRNA levels of several other proangiogenic genes, which were also upregulated after exposure of solo ECs to hypoxia (see
Table 1). Of note, the levels of endothelial PEDF, a potent inhibitor of angiogenesis counteracting VEGF,
29 were not affected by growing ECs in contact coculture with RPE. Interestingly, neither mRNA nor protein levels of EC VEGFR2 were affected by contact coculture (data not shown). However, enhanced VEGFR2–mediated signaling is possible in view of the functional studies of Matrigel tube formation, indicating accelerated tube formation rate induced by exposure of ECs to contact coculture with RPE. It must be emphasized that the proangiogenic effects described above were observed only in ECs grown in direct contact with RPE cells; none of these effects could be observed in ECs separated from RPE cells after noncontact coculture. This profound importance of RPE-EC contact was evident in both mRNA and protein assays of VEGF and MMP2, as well as in Matrigel tube formation assay. Our findings indicating the significance of RPE-EC contact are in agreement with those of Peterson et al.,
11 who demonstrated that direct contact of ECs with RPE and/or RPE extracellular matrix enhances EC transmigration through the RPE barrier. This enhanced migration was found to be associated with increased activity of the GTPase Rac1 in the ECs, mediated by activation of PI3K and Akt-1.
11 It must be noted that Hartnett et al.,
30 who investigated the effect of EC-RPE interaction on the RPE barrier function, discovered that coculture with EC induced RPE barrier dysfunction in both contact and noncontact coculture. The observation that ECs residing in close proximity with RPE exerted a deleterious effect on RPE barrier function independent of direct contact with the RPE cells indicated that this effect was mediated by a soluble factor, and VEGF was proposed to be a likely candidate.
30 However, in view of the fact that inhibition of VEGF was only partially effective in the restoration of RPE barrier function,
30 involvement of other molecular mechanisms in this process cannot be excluded. For example, involvement of FGF2 in CNV is suggested by a recent study showing that combined inhibition of VEGF and FGF2 is more effective in inhibiting RPE-induced angiogenesis in an in vitro model, compared with exclusive inhibition of VEGF.
31 The latter observation is an agreement with the data of Geisen et al.
9 showing that RPE-induced enhancement of EC proliferation, observed to be independent of direct EC-RPE contact, was not mediated by VEGF.