Abstract
Purpose :
Next to age-related changes of the refracting optic media and neuroretina, the retinal pigment epithelium (RPE) underlies the physiological aging process of the eye. Previous analyses of the transcriptome and proteome of the aging RPE of the common marmoset Callithrix jacchus (C. jacchus) showed a significant increase in the expression of Cathepsin B (CTSB). CSTB is involved in the regulation of angiogenesis and is increasingly secreted by oxidative stress. To date, the underlying functions of CTSB in physiological as well as pathological states within the RPE remain unknown. The aim of the study was to validate the expression characteristics and functional role of CTSB in the aging RPE in-vitro.
Methods :
The expression of CTSB in the RPE was examined by immunohistochemistry (IHC), Western blot (WB) and qRT-PCR in neonatal (0-3 days), adult (10-22 months) and senile (96-108 months) RPE of C. jacchus. To study effects of different CTSB concentrations, ARPE-19 cells were exposed to recombinant CTSB (rCTSB) in different concentrations (0, 1, 10, 100 ng/ml). The effects on viability (MTT-assay), tight junction proteins (ZO-1, Occludin; IHC), expression of senescence-related proteins like proteins of the p53/MDM2 signaling cascade (qRT-PCR, WB) and proangiogenic factors, e.g. VEGF (IHC, ELISA) were evaluated.
Results :
Increased expression of CTSB in the aging RPE was confirmed on both gene- and protein-level. ARPE-19 cells exposed to rCTSB show decreased cell viability and disrupted staining patterns of ZO-1 and Occludin, indicating a decreased integrity of intercellular tight junctions of RPE cells. Furthermore, a dose-depended alteration of the p53/MDM2 signaling cascade has been demonstrated. An elevated VEGF expression indicates pro-angiogenic properties of rCTSB-exposed RPE cells.
Conclusions :
The study indicates an increased age-related expression of CTSB in the RPE of C. jacchus. ARPE-19 cells exposed to rCTSB revealed alterations of relevant cellular conditions, like viability, intercellular tight junctions, and pro-angiogenic factors. Further studies are mandatory to proof the role of CTSB within the physiological aging process and the suspected conversion into pathoysiological conditions. Supported by the German Research Foundation (DFG; BO-4661/-1)
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.