Retinal neovascularization is a complex process that is not completely understood. In retinal neovascularization, there are abnormally formed blood vessels and fibrous tissue that grow from the retina or the optic disk along the posterior surface of the vitreous. These retinal capillaries do not normally invade the vitreous or extend beyond the inner plexiform layer of the retina. However, in retinal diseases, e.g., diabetic retinopathy, the disruption of the balance between angiogenic and anti-angiogenic factors favors neovascularization, which enters the sub-retinal space, where leakage and bleeding lead to retinal detachment and photoreceptor death . Clearly, the process of retinal neovascularization is mediated by the regulation of several angiogenic inducers, inhibitors, their respective receptors, and transcriptional factors. VEGF is a potent angiogenic and permeability enhancing factor causally linked to neovascularization in cancer, wound healing, diabetic retinopathy, and age-related macular degeneration [14, 34]. Several reports have shown that VEGF is produced by several cell types within the eye (retinal pigment epithelial cells, glial cells, retinal capillary pericytes, endothelial cells, Müller cells and ganglion cells) [ 35 ] and play a role in the development of retinal neovascularization [13, 36, 7]. Aiello et al.  showed that VEGF played a significant role in mediating retinal neovascularization in diabetic patients. Using animal models of ischemic retinopathy and iris neovascularization, several researchers found that VEGF expression is upregulated in the retina during neovascularization [13, 7, 8, 38]. Furthermore, inhibiting VEGF using VEGF receptor chimeric proteins, neutralizing antibodies, and antisense oligonucleotides in these animal models could reduce both retinal and iris neovascularization. Finding increased VEGF levels during retinal neovascularization has resulted in a controversy as to whether VEGF itself is sufficient to stimulate retinal neovascularization.
Increase VEGF expression could be the result of the retina microenvironment (hypoxia or ischemia) as well as changes in other factors such as pigment epithelium-derived factor (PEDF). The balance between the expression of VEGF and pigment epithelium-derived factor (PEDF) in modulating retinal neovascularization was investigated by Zhang and co-workers . These researchers found a reciprocal interaction between VEGF and PEDF in retina; significant increases in VEGF expression was observed following silencing of the PEDF gene. Hypoxia has been shown to upregulate VEGF mRNA in retinal endothelial cells, pericytes, RPE cells, Muller cells and ganglion cells [14, 40]. Our studies show that both HUVEC and HRPC express basal levels of VEGF and VEGFR-2 mRNA and protein, which was significantly increased by hypoxia treatment. Additionally, we found that co-cultures of these cell lines resulted in a synergistic increase in the expression of both VEGF and VEGFR-2, similar to that induced in single cultures by hypoxia. In fact, co-cultures treated with hypoxia showed a pronounced increase in the expression of both VEGF and VEGFR-2. However, contacting co-culture did not show a difference in response to hypoxia treatment from that of non-contacting co-cultures. Recently, Dardix and coworkers  also investigated the effects of co-culture (with retina pigmented epithelial cells - RPE) and hypoxia on endothelial cell expression of VEGF. These researchers reported that contact co-cultures upregulated both VEGF mRNA and protein expression to the same degree as hypoxia alone. In contrast to our findings, these researchers found that non-contacting co-cultures did not show an effect. However, these researchers used RPE cells for co-cultures as well as utilized CoCl2, a hypoxia mimetic to study the effect of hypoxia. In our studies we used human retinal progenitor cells (HRPC) cells for co-cultures; these cells express and secrete VEGF, among other soluble factors those have direct effects on endothelial cells . In addition, we also used hypoxic culture conditions rather than the hypoxia mimetic, which may activate additional mechanisms not seen with the hypoxia mimetic.
Hence, our studies are consistent with the idea that hypoxia-inducing VEGF expression will lead to an enhanced neovascular response. These data not only highlight the important angiogenic properties of VEGF, but they also demonstrate the autocrine ability of VEGF to regulate the synthesis of multiple angiogenic proteins . The autocrine effect of VEGF on retinal elements previously has been reported, for both retinal glial cells [43, 44] and RPE cells , but not for the HRPC. To our knowledge, however, the regulation of VEGF receptor expression has not yet been investigated in retinal neovascularization induced by hypoxia co-culture system. Several lines of evidence indicate that enhanced VEGF expression in response to hypoxia is due to transcriptional activation [45, 46]. Furthermore, this is the first time HRPC cells have been shown to have VEGF receptors (VEGFR-2) and induced the expression after hypoxia treatment. If similar autocrine functions of VEGF were present in HRPC cells, this would explain our observation of increased expression of VEGF and VEGFR-2 receptor after hypoxia and co-cultured conditioned.
It is well-established that hypoxia will stimulate neovascularization through inducing HIF1α, which translocates into the nucleus, binds to the hypoxia-response element, and up-regulates the expression of VEGF and other angiogenic factors . Several studies have shown that hypoxia induces VEGF expression in matured retinal cells, which may play a major role in development of many retinal diseases. Our data from nuclear staining pattern and the immunoblotting of nuclear extracts indicate that HIF-1α, present in the cytoplasm of HRPC treated with normoxia HUVEC-CM, did translocate to the nucleus when cells were treated with hypoxia HUVEC-CM. HIF-1 may also have a function in retina that is unrelated to hypoxia because the constitutive level of HIF-1 expression in adult normoxic retina is much higher than that found for heart and lung, where it was undetectable by immunoblot or immunohistochemistry. VEGF is also constitutively expressed in the retina and it is possible that this is driven by the constitutive expression of HIF-1.
Another mechanism that may be activated by hypoxia treatment is NF-κB, which has also been suggested to play an important role in the induction of retinal neovascularization [21, 23]. In the present study, we found that hypoxia has increases expression of NFκB mRNA and protein in HRPC/HUVEC cultured and co-cultured conditions. The hypoxic sensitivity of the NF-κB pathway and a wide variety of in vitro studies have demonstrated that exposure of various cell types to hypoxia activates NF κB signaling pathways. There are several reports that NFκB was activated in retinal glial cells, vascular endothelial cells, pericytes, and macrophage/microglia, all of which participate in neovascular disorders such as diabetic retinopathy. In the retina, NF-κB is localized in sub-retinal membranes and in microvessels and is activated very early in the course of development of retinopathy in diabetes. The activation of NFKB in retinal pericytes is responsible for the hyperglycemia-induced accelerated loss of pericytes observed in diabetic retinopathy. An inhibitor of NF-κB, PDTC inhibited NF-KB activation and reduced retinal neovascularization without discernible toxicity in a mouse model [45, 46]. It also revealed that NF-KB might provide a basis for a specific and effective therapeutic regimen for various diseases associated with undesired angiogenesis. Normally, NFκB is present in the cytoplasm as a complex with members of the IkB inhibitor family. The hypoxia-induced activation of NFκB could result in the degradation of IkB. This in turn exposes the nuclear localization signal, allowing NFκB to be transported into the nucleus. In this study, we investigated the effects of normoxia/hypoxia exposed HUVEC-CM on NFκB nuclear translocation. Our studies showed that the HRPC cells did not significantly affect the nuclear translocation of NFκB when the cells were exposed with normoxia HUVEC-CM. However, nuclear translocation of NFκB was induced by hypoxia HUVEC-CM. Since nuclear localization of NF-κB is thought to be equivalent to NF-κB activation, we have shown that NF-κB was activated in HRPC cells by hypoxia HUVEC-CM, which may have triggered the transcription of NF-κB dependent genes and regulated the expression retinal neovascularization related genes.