Pathway activation profiling reveals new insights into age-related macular degeneration and provides avenues for therapeutic interventions

Abstract

Age-related macular degeneration (AMD) is a major cause of blindness in older people and is caused by loss of the central region of the retinal pigment epithelium (RPE). Conventional methods of gene expression analysis have yielded important insights into AMD pathogenesis, but the precise molecular pathway alterations are still poorly understood. Therefore we developed a new software program, "AMD Medicine", and discovered differential pathway activation profiles in samples of human RPE/choroid from AMD patients and controls. We identified 29 pathways in RPE-choroid AMD phenotypes: 27 pathways were activated in AMD compared to controls, and 2 pathways were activated in controls compared to AMD. In AMD, we identified a graded activation of pathways related to wound response, complement cascade, and cell survival. Also, there was downregulation of two pathways responsible for apoptosis. Furthermore, significant activation of pro-mitotic pathways is consistent with dedifferentiation and cell proliferation events, which occur early in the pathogenesis of AMD. Significantly, we discovered new global pathway activation signatures of AMD involved in the cell-based inflammatory response: IL-2, STAT3, and ERK. The ultimate aim of our research is to achieve a better understanding of signaling pathways involved in AMD pathology, which will eventually lead to better treatments.

Figure 1. Pathway activation strength (PAS) for selected pathways

PAS values have been calculated according to OncoFinder algorithm. PAS presented on this figure passed the following filters PAS<−1.5 and PAS>1.5 in both datasets. Blue bars represent PAS average for each pathway, and error bars represents standard deviation A. PAS derived from GSE50195 dataset. B. PAS derived from GSE50195 that cell-based inflammatory responses within the RPE-choroid are a core feature of AMD. However, cellular sources and targets of pro-inflammatory secreted factors are still need to be determined along with the regulatory mechanism of the chemokine network.

Figure 2. Heatmap of differentially activated pathways shown in Figure 1A

Complement factor H genetic background (rs1061170 SNP) for PAS values derived from GSE50195 dataset shown for high-risk YH/HH and low-risk YY genotype. Blue shading indicates pathway downregulation; red shading indicates pathway upregulation. Samples with names ending in CTRL indicates control samples; samples with names ending in ARM indicates AMD samples.

Figure 3. Comparison of GSE29801 derived PAS distribution and GSE50195 derived PAS distribution

Box plots of GSE29801 (right) derived PAS and GSE50195 (left) derived PAS for each pathway. All PAS values for each pathway from two independent data sets are comparable; moreover box plots for GSE50195 derived PAS lay inside of box plots for GSE50195 derived PAS. Box plot whiskers represent min and max values for each pathways.

Figure 4. An example of how multiple pathways are activated and down-regulated during AMD

This figure also serves as a working hypothesis for the pathogenesis of AMD. Proposed steps and interactions are as follows: A. Environmental Stress in the form of aging, obesity, inflammation, or diet causes B. senescence and loss of proliferation of the retinal pigment epithelial cells leading to C. activation of the MAPK, ERK, p38 and AKT pathways in the cytoplasmic components of the cells. This cellular senescence also has several consequences, primary of which are D. upregulation of the SASP, interleukin, and inflammatory cytokine networks, and E. downregulation of the caspase cascade and mitochondrial apoptosis. These pathways also interact; for example the upregulation of the SASP, interleukin, and inflammatory cytokine pathways causes downregulation of the caspase and mitochondrial apoptosis pathways. Green arrows represent upregulated pathways, red arrows represent downregulated pathways, and blue dotted arrows represent connected pathways.