Tumor microvessel density was evaluated using CD31 as an endothelial marker

Survival IGF-TD expression vector is the key ingredient to suppress RGC loss in experimental glaucoma. Based on our observations in the RT-PCR experiments that showed upregulation of inflammatory message in glaucoma groups which was inhibited by IGF-TD, we propose a role for upregulation of inflammatory pathways in the pathogenesis of RGC loss in the setting of glaucoma. In the saline injected glaucoma group, over 5 to 20 fold increase in the messages for TNF-, IL-1 and MCP-1 were detected as compared with the non-glaucomatous saline group. In contrast, in the hNPIGF-TD group the levels of TNF-, IL-1 and MCP-1 were slightly higher but not statistically different from non-glaucomatous saline group. Interestingly, the levels of these cytokines were tapered in the hNP and hNPTD groups suggesting that transplantation of cells themselves exert some intrinsic anti-inflammatory effect as well. RT-PCR data also show tapering of the angiogenic pathways. Several studies have shown that IGF-1 could induce neovascularization and other related pathology. Native IGF-1 is considered a mitogen that may stimulate or support the development of retinal neovascularization, possibly through upregulation of vascular endothelial growth factor. In the saline-injected glaucoma group increased expression of VEGF-A, C and D and cognate receptors, VEGFR-2 and R-3 were detected, as compared with the no glaucoma group. In the hNPIGF-TD group, the levels of these cytokines and receptors were tapered and closer to the no glaucoma. In the glaucoma groups transplanted with hNP and hNPTD, the levels of VEGF-C, VEGFR-2 and R-3 were similar to saline-injected glaucoma group. Interestingly, the levels of VEGF-A and D were tapered suggesting that hNP cells have an intrinsic ability to down-regulate these pathways. Our data are in contrast to prior studies that have implicated IGF-1 as a pro-angiogenic factor.39 Our observations suggest that activation of IGF-1R negatively regulates inflammatory and angiogenic pathways; these may independent of neurotrophic pathways stimulated by IGF-TD. This is in concert with previous studies that have indicated that neovascularization is almost always accompanied by inflammation. Angiogenic and inflammatory pathways may go hand in hand in the glaucomatous eye regardless of CF-101 whether xenografts are introduced. It is likely that the microenvironment of the glaucomatous eye promotes a pro-inflammatory and pro-angiogenic environment, which is negatively regulated by IGF-1 signaling pathways and acts concurrently to activation of NTFs. As an effort to dissect IGF-1-regulated pathways, previous studies have shown PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783938 that one of these pathways may involve phosphoinositide 3-kinase signaling. IGF-1 through activation of its receptor, IGF-R1 promotes survival of neurons via the PI3K pathway. Our in vitro study indicated that the survival rate of primary cultured RGCs significantly decreased when inhibitors of IGF-1R signaling pathways were applied. These findings are in accord with previous studies that demonstrate that blockage of IGF-1R signaling PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 interrupts survival. We observed that microbead-injected glaucomatous eyes developed a pro-inflammatory milieu. We have therefore hypothesized that elevation of IOP may setup inflammatory responses which may lead up to RGC death. Downregulation of inflammatory pathways by IGF-1 may be one mechanism for its neurotropic effects. In corollary to this, we have observed increased inflammatory cells on histologica