Monomeric C-reactive protein (mCRP) in human donor choroids: relationship with CFH genotype and effects on endothelial cell function

Abstract

 

Purpose

 

Previous reports showed an increase in overall levels of C-reactive protein (CRP) in the choroids of human donor eyes with AMD (Bhutto et al., 2011) and eyes homozygous for the CFH (Y402H) gene polymorphism (Johnson et al., 2006). In this study, we employed immunohistochemical analysis and functional assays to study the localization of pro-inflammatory mCRP versus anti-inflammatory pCRP in the human macula and to determine the effect these two conformations of CRP have on endothelial cells and retinal pigment epithelium (RPE) cells in vitro.

 

Methods

 

Cryostat sections of genotyped human donor maculas (n=12) were labeled using antibodies specific for mCRP (3H12) or pCRP (1D6), and their pattern of labeling was compared to sections incubated with secondary antibody only. Scratch closure assays were performed to quantify migration of the chorioretinal endothelial cell line RF/6A and ARPE-19 cells during incubation with: media only, pCRP (20µg/mL), boiled pCRP, mCRP (20µg/mL), or boiled mCRP. VEGF (5µg/mL) was used as a positive control for RF/6A cell migration. To study the effect of CRP on cell permeability, transepithelial resistance (TER) assays were performed on both cell types using the same conditions.

 

Results

 

mCRP was found to localize mainly to the choriocapillaris and Bruch’s membrane, with qualitatively increased staining in tissue from donors with a high-risk CFH (Y402H) genotype compared to those with a low-risk genotype. pCRP was not detected in any of the evaluated samples regardless of genotype. In functional assays, mCRP markedly promotes RF/6A cell migration after 24 hours (p<0.05), while it increases the permeability of confluent endothelial cell layers within 15 minutes (p<0.05). Migration and TER of ARPE-19 cells were not significantly altered by mCRP or pCRP treatment.

 

Conclusions

 

Our data suggests that pro-inflammatory mCRP is the major conformation of CRP present in the aging macula. The elevated CRP in the choroids of high-risk CFH donor eyes appears to be predominantly due to mCRP. In addition, mCRP, but not pCRP, has a significant effect on endothelial cell phenotypes in vitro, suggesting a potential role for mCRP in choroidal vascular dysfunction in AMD pathogenesis.  

 

mCRP increases RF/6A cell migration 24 hours after treatment