S100A Proteins In The Pathogenesis Of Experimental Corneal Neovascularization

Purpose:The S100A protein family is involved in various inflammatory processes. Several members like S100A4, S100A6 and A13 are proved to be pro-angiogenic in tumor development. Current study examines whether S100A proteins are involved in the pathogenesis of inflammation-associated corneal neovascularization (CorNV). Further research evaluates the effects and mechanism of S100A8/9 proteins in neovascularization by experiments in vitro and in vivo.Methods:We used 10-0 nylon suture-(S) or chemical burn (CB)-induced CorNV models in Balb/c or C57B1/6 mice for a microarray analysis of the genome-wide expression pattern. At different time points after suturing, we conducted histopathological examinations to detect the infiltration of inflammatory cells into the corneal stroma. Representative members of the S100A family (S100A4, S100A6, S100A8, S100A9, and S100A13), pro-inflammatory cytokines (IL-1β, IL-6, transforming growth factorβ1, and MIP-2), and pro-angiogenic factors (fibroblast growth factor and vascular endothelial growth factor) were detected with reverse-transcription quantitative PCR (RT-QPCR). We used immunofluorescence to monitor neutrophils or macrophages infiltration and S100A8 or S100A9 protein deposition in neovascularized corneas. Antibody-mediated neutrophil depletion or S100A8 depletion in mice was performed to evaluate the role of neutrophils and S100A proteins in suture-induced corneal neovascularization (S-CorNV). Cell proliferation, tube formation and transfer experimental were used to examine the angiogenic effect of S100A8/9 proteins in HUVEC cells. In vivo, Matrigel were injected subcutaneously in mice to compare the S100A8/9 proteins effect on the growth of new blood vessels by quantitative hemoglobin and histological sections.Results:Microarray profiling revealed that S100A4, S100A6, S100A8, S100A9, and S100A13 were upregulated in both CorNV models, with S100A8 and S100A9 manifesting the most significant changes compared to the normal animals. An RT-QPCR assay of these S100A genes and cytokine genes in the S-CorNV corneas showed that the changes were time-dependent, reaching the apex at day 5. Immunofluorescence analysis demonstrated that neutrophils and macrophages produce S100A8 and S100A9. The depletion of neutrophils beginning one day before S-CorNV induction decreased disease severity and S100A8/9 deposition in the neovascularized corneas. The extent of upregulation of other detected S100A genes and pro-inflammatory or pro-angiogenic genes was also decreased by neutrophil depletion. Subconjunctival administration of S100A8 antibodies also significantly inhibited the growth of vessels and inflammation in the S-CorNV model. In vitro, S100A8/9 proteins (lOug/ml) could promote the proliferation, migration and tube formation of HUVEC cells. In in vivo Matrigel vasculogenesis model, Matrigel plug containing S100A8/9 proteins generated more neovascularization than control group. Moreover, higher hemoglobin content and more local infiltration of inflammatory cells were observed in S100A8/9 supplemented Matrigel plug.Conclusions:We determined that S100A proteins are involved in the inflammatory CorNV model and S100A8 or S100A9 in particular might be employed as targets in managing diseases involving this pathological process. Action of S100A8/9 proteins in neovascularization might be via direct effect on vascular endothelial cells.