The distribution of hypoxic inducible factor in choroidal neovascular membranes from patients with age–related macular degeneration

Abstract

Abstract: : Purpose:Up regulation of pro–angiogenic cytokine expression occurring secondary to chronic hypoxia in physiologic and pathophysiologic conditions is mediated by the transcription regulators known as hypoxic inducible factors (HIF). The present study was undertaken to investigate the expression of HIF occurring in choroidal neovascularization (CNV) secondary to age–related macular degeneration (AMD). Methods: Nine CNV membranes, which were surgically removed from patients with AMD, were fixed in formalin, embedded in wax and serially sectioned for histochemical and immunohistochemical evaluation. Immunohistochemical analysis used monoclonal antibodies against markers for HIF–1 and HIF–2, cell markers CD34 (endothelial cells), wide screening cytokeratins (RPE) and CD68 (macrophages). Secondary antibody amplification was performed using Dako Envision and positive immunoreactivity was visualised with nova red substrate. Results:Cellular immunoreactivity for members of the hypoxic inducible factor (HIF) was found in 8 of the 9 specimens studied. Histological evaluation revealed the immunoreactivity to be co–localised with pigmented cells and not vascular cells. Immunohistochemical analysis revealed areas within the CNV membranes, which were predominately immuno–positive for CD68 and cytokeratin positive cells indicating the presence of RPE and/or macrophages showed highest immunoreactivity for HIFs. Faint, diffuse immunoreactivity for HIF was also found in areas of the intact, differentiated RPE monolayer. No immunochemical co–localisation was observed with HIF and the endothelial cell marker CD34 in any of the membranes studied. Conclusions:The localization of HIF expression within CNV suggests that hypoxia is a major stimulus for the development of angiogenesis in AMD and that in this context CNV is just one of the components of a sub–macular wound healing response.

Keywords: age–related macular degeneration • choroid: neovascularization • wound healing