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Kathleen Chirco, S Scott Whitmore, Shemin Zeng, Grefachew Workalemahu, Lawrence A Potempa, Sara E Miller, Edwin M Stone, Budd A Tucker, Robert F Mullins; Monomeric C-reactive Protein and Altered Human Endothelial Cell Function in Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6554.
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© ARVO (1962-2015); The Authors (2016-present)
C-reactive protein (CRP) is an acute phase molecule that primarily exists in two functionally independent forms: net anti-inflammatory pentameric CRP (pCRP) and pro-inflammatory monomeric CRP (mCRP). Previous studies have suggested a link between elevated total CRP in the choroid, CFH genotype, and age-related macular degeneration (AMD) status; however, the exact form and functional consequences of CRP on choroidal cells remains unknown. In the current study, we employed immunohistochemical, biochemical, and functional assays to evaluate the role of mCRP on choroidal endothelial cells in the context of AMD.
Genotyped human donor macula sections (n=42) were labeled using antibodies specific to mCRP (3H12) and pCRP (1D6), and the labeling patterns were compared, masked to donor genotype, to those of sections incubated with secondary antibody only. To quantitate these differences in CRP levels based on AMD risk, ELISA (n=10) and semi-native Western blotting (n=14) was performed using RPE-choroid tissue from genotyped donors. RNA-Seq (n=3 per group) and Western blotting (n=18) of human RPE-choroid organ cultures treated with mCRP (20µg/mL) or media alone were utilized to assess gene expression and protein level changes, respectively.
Donors homozygous for the high-risk CFH (Y402H) allele had elevated mCRP immunolabeling within the choriocapillaris and Bruch’s membrane compared to those with the low-risk genotype, which is in agreement with semi-native Western blotting analysis. Additionally, the ELISA data show that, on average, total CRP levels in HH donor choroids are 189% higher than the levels in YY donor choroids. Organ cultures treated with mCRP (20µg/mL) exhibit a dramatic increase in expression of inflammatory genes including ICAM-1 (fold change=2.1; p<0.05), with a corresponding increase in ICAM-1 protein levels (p<0.05).
Our data indicate that mCRP is the more abundant form of the protein in human choroid, and that mCRP levels are elevated in individuals with the high-risk CFH genotype. Moreover, pro-inflammatory mCRP significantly affects endothelial cell phenotypes in vitro and ex vivo, suggesting a potential role for mCRP in choroidal vascular dysfunction in the pathogenesis of AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Human donors homozygous for the high-risk CFH allele have increased mCRP immunoreactivity (B) in the choroid compared to low-risk eyes (E).
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