What determines the switch between atrophic and neovascular forms of age related macular degeneration? - the role of BMP4 induced senescence

Abstract

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, targets the retinal pigment epithelium (RPE), a monolayer of cells at the back of the eye. As AMD progresses, it can develop into two distinct forms of late AMD: "dry," atrophic AMD, characterized by RPE senescence and geographic RPE loss, and "wet," neovascular AMD, characterized by RPE activation with abnormal growth of choroidal vessels. The genetic and molecular pathways that lead to these diverse phenotypes are currently under investigation. We have found that bone morphogenetic protein-4 (BMP4) is differentially expressed in atrophic and neovascular AMD. In atrophic AMD, BMP4 is highly expressed in RPE, and mediates oxidative stress induced RPE senescencein vitro via Smad and p38 pathways. In contrast, in neovascular AMD lesions, BMP4 expression in RPE is low, possibly a result of local expression of pro-inflammatory mediators. Thus, BMP4 may be involved in the molecular switch determining which phenotypic pathway is taken in the progression of AMD.

Keywords: BMP4; age related macular degeneration; oxidative stress; retinal pigment epithelial cell; senescence.

Figure 1.

Diagram illustrating the progression of early age related macular degeneration (AMD) into 2 divergent late stages and the potential role of BMP4 as a switch between these pathways. Chronic stressors such as oxidative stress can promote the expression of BMP4 in the retinal pigment epithelium (RPE) and induce RPE senescence as part of the phenotype of early AMD. If BMP4 expression is sustained, it could lead to RPE apoptosis and geographic atrophy. In other individuals, activation of the senescence activated secretory pathway and expression of pro-inflammatory mediators could result in increased expression of interleukin (IL)-8, decreased expression of BMP4 and increased expression of vascular endothelial growth factor (VEGF) resulting in neovascular AMD with choroidal angiogenesis.

Figure 2.

Expression of BMP4 in late stages of age related macular degeneration (AMD). Immunohistochemical stains for BMP4 (red chromogen) in retinal pigment epithelium (RPE)/choroid tissue sections from donor eyes with hematoxylin counterstain. In (A) a control individual without AMD shows no apparent BMP4 staining in RPE or choroid. In (B) an individual with late dry AMD, away from a region of geographic atrophy shows prominent BMP4 immunoreactivity in RPE and in the accumulated drusen material between the RPE and the choroid. In (C) an individual with neovascular form of late AMD shows no apparent BMP4 staining in the RPE or the neovascular lesion between the RPE and retina. In (D) an individual with neovascular form of late AMD that further progressed to scar with loss of neovascular channels shows re-expression of BMP4 staining in cells within and adjacent to the lesion. Note loss of most cells in RPE layer. The institutional review board (IRB) of the University of Southern California approved our use of human donor eyes. All procedures conformed to the Declaration of Helsinki forresearch involving human subjects.

Figure 3.

IL-8 protein concentration in culture medium measured by ELISA. ARPE-19 cells were treated with 150 uM H₂O₂ in culture medium with 10% fetal bovine serum for 2 hours and allowed to recover in stressor-free ARPE medium for 22 hours. The procedure was repeated to generate the next treatment cycle. The twice treated cells were allowed to stay in 1% serum ARPE medium for 72 hours after stress before proceeding to further analytic assays. The culture media from control and senescent RPE cells were collected and used directly for ELISA measurement. IL-8 secretion level was measured in pg/ml using human IL-8 ELISA Kit (BioLegend, Inc., San Diego, CA) according to manufacturer's instructions. The level of IL-8 secretion shown here was averaged from a triplicate of each sample and from 3 independent repeats of H₂O₂ treatments. Student's t test was used for statistical analysis (**; p < 0.0005).