Sara Romero-Vázquez, Alfredo Adan, Blanca Molins.
PURPOSE. The retinal pigment epithelium (RPE) of the outer blood retinal barrier (oBRB) is the prime target in age-related macular degeneration (AMD). C-reactive protein (CRP) is an acute phase reactant and a serum biomarker of chronic inflammation. It presents two isoforms with distinct biological functionalities: the pentameric (pCRP) and the monomeric (mCRP) form, being the latter the tissue-associated form. Some studies have suggested the implication of CRP in the pathophysiology of AMD, however its role remains unknown. Previous studies in our group have shown that addition of mCRP to the apical surface of RPE disrupts its integrity in vitro. As RPE cells are highly polarized, we aimed to determine whether the effect of CRP depends on its topological localization.
METHODS. ARPE-19, a spontaneously raised human RPE cell line, cells were seeded on transwell inserts. Cell monolayer integrity was assessed by measuring transepithelial electrical resistance (TEER) three times per week. After a three week culture, cells were treated apically or basolaterally with mCRP or pCRP for 48 hours. In addition of TEER, the paracellular permeability was determined by measuring the diffusion rate of FITC-dextran and ZO-1 distribution was determined by immunofluorescence. These experiments were replicated in primary porcine RPE cells.
RESULTS. In ARPE-19 cells, TEER was significantly reduced by apical and basolateral treatment with mCRP, but only apical exposure increased paracellular permeability and altered ZO-1 expression. By contrast, treatment with pCRP had no effect on barrier integrity. In primary RPE cells apical mCRP treatment reduced TEER and basal mCRP increased paracellular permeability, while ZO-1 expression was not affected. By contrast, pCRP has no significant effect on primary porcine RPE cells, regardless of its topological localization.
CONCLUSION. The effect of mCRP on barrier disruption may depend on the RPE domain stimulated, whereas pCRP may not have any effect on these cells.