For the second part of our study, we investigated the effects of Sema3A on RPE when cocultured in the presence of the most potent angiogenesis factor, VEGF
165. According to our results (ELISA results), Sema3A did not affect VEGF
165 or PEDF secretion. In the presence of exogenous and endogenous VEGF
165, Sema3A inhibited VEGF
165-induced RPE behavior, including proliferation, migration, cell cycle arrest, JNK and p38MAPK phosphorylation, and cocultured HUVEC tube formation. In the present study, phosphorylation of JNK and p38MAPK was not completely inhibited by Sema3A. This could be explained by that JNK and p38MAPK signal pathways can be induced by a lot of growth factors, inflammatory factors and stress, such as epithelium growth factor, TGF-beta, interlukin-1, and so on. And several studies have demonstrated that VEGF induces the upregulation of several kinds of angiogenesis and inflammation factors.
39,40 In our study, Sema3A only inhibited the VEGF
165 induced JNK and p38MAPK phosphorylation partially, but not all of the upregulated signal pathways induced by all of the factors, thus, the phosphorylation of JNK and p38MAPK are only partially inhibited. Besides the behavior study of the RPE cells, we also detected the expression pattern of the two crucial receptors, VEGFR2 and Nrp1, which are important in the function of VEGF
165. In the present study, the results showed that both VEGF
165 and Sema3A can induce the upregulation of these two receptors. While under the treatment of both stimulation factors, the two receptors remained higher comparing with the untreated group, but had no significant difference compared with the VEGF
165- and Sema3A-treated group. As the ligands for VEGFR2 and Nrp1, exogenous application of VEGF
165 and Sema3A can induce the increase of the receptors is as expected. While under the costimulation, although the protein level of the two receptors is slightly higher than separately treatment groups, there was no significant difference between the control groups. The comparable elevation of the two receptors can be explained by maintaining the balance of the two receptors, and restrict the utilization of VEGF
165. Based on the previously published articles and our results, the mechanism of the Sema3A inhibit the function of VEGF
165 most likely includes competition of Sema3A with VEGF
165 for their b1 binding site on Nrp1.
41,42 As we stated before, VEGF family members mediate their downstream effects by binding to neuropilins and forming complexes with VEGF receptors, which is analogous to the Sema3-neuropilin-plexin complex.
43 While Nrp1 is not required for VEGF
165 function, it can enhance the signaling of VEGF
165 through VEGFR2, one of its receptor tyrosine kinases,
43 which was supported by our Western blot analysis, showing that VEGFR2 downstream signaling through JNK and p38MAPK phosphorylation was downregulated. Also, in spite of the upregulation of VEGFR2 under the treatment of VEGF
165 and Sema3A, there is no increase of phosphorylation of JNK and p38MAPK, which could be the results of simultaneously increase of Nrp1 receptor, which blocks the utilization of VEGF
165. Another study by Guttmann-Raviv et al.
19 also suggested that Sema3A can induce inhibitory effects on signaling mediated directly by Nrp1. And recent findings indicate that the range of growth factors that bind to Nrp1 is not limited to semaphorin and VEGF
165; it also includes others, such as VEGF
121,
44 placental growth factor,
45 hepatocyte growth factor,
46 and fibroblast growth factor 2.
47 All of these growth factors play important roles in proliferative and neovascular eye diseases. Thus, Sema3A can compete with these factors for binding to Nrp1 and inhibit their signal pathways, just as it inhibited VEGF
165 function. Besides the in vitro study, we also demonstrated that Sema3A can inhibit PVR formation in a dose-dependent manner, which provide new information for clinical PVR treatment. In our study, we also used the coculture method to explore the effect of Sema3A on tube formation. As expected, Sema3A inhibited HUVECs tube formation in the presence of both endogenous and exogenous VEGF
165 and we further confirmed that Sema3A can inhibit the function of VEGF
165 not only in RPE cells, but also in HUVECs. Although the previous studies and our results showed the mechanisms of Sema3A on the function of VEGF
165-induced RPE cells activities in a certain extent, more experiments need to be done to demonstrate that the affinities of sema3A and VEGF to Nrp1 and VEGFR2, to make clear spectrum on the binding studies.