Evidence from various studies suggests that RPE cells are prone to oxidative stress . The oxLDL is a trigger for early events in the pathogenesis of AMD, since the metabolism of oxLDL and ROS generation can promote RPE dysfunction and augmented apoptosis . Serum LDL and oxLDL readily enters the retina through the choriocapillaries via RPE . ApoB, a major protein in the LDL and oxLDL molecules are reportedly present in the cholesterol-containing drusen and the basal deposits in human eyes with AMD . Hcy mediated accumulation of cholesterol in THP-1 macrophages has been shown [36, 37]. However, the accumulation of oxLDL mediated by Hcy warrants more studies. Macrophages and RPE interactions are reported to be associated with AMD pathology [38, 39]. Though, plasma Hcy association with AMD pathology is reported [40–42] the cellular pathology triggered by Hcy at the level of RPE is incompletely understood.
This study therefore focussed on the localized effects of these systemic prooxidants namely Hcy and oxLDL associated with AMD pathology that were evaluated in both ARPE-19 as well as in THP-1 macrophage cells in vitro. An augmented oxLDL uptake by ARPE-19 cells and THP-1 macrophages were observed in cells exposed to Hcy and its thiolactone form along with increased expression of CD36 and PPARγ in both the cells. The CD36 receptor is the principal receptor, responsible for oxLDL uptake and plays an important role in the clearance of oxLDL in ARPE-19 cells . The CD36 transcription and oxLDL uptake could be induced by the activation of PPARγ . Thus, this study reveals that Hcy induces oxLDL uptake that promotes cellular stress. The cellular response in ARPE-19 cells due to Hcy and oxLDL promoting inflammation and chemotaxis of macrophage cells was earlier reported by us . Malfunction of choroidal macrophages and RPE leads to incomplete removal of debris from sub-RPE and transported to the choroid that results in the development of drusen and basal laminar deposits . Further, there is oxidative stress response in RPE with the increased phagocytic and metabolic activity .
PON, an antioxidant enzyme (PON1 and PON3) prevents oxidation of LDL at systemic level . Elevated protein levels and PON activity is reported by us in AMD  and at cellular level (PON2) . As the oxidative stress potentially increases in response to Hcy and HCTL mediated accumulation of oxLDL, the antioxidant PON2 protein expression was evaluated in ARPE-19 and THP-1 macrophages that showed an increased expression, mediated by increased SP1 transcription factor. This is suggestive of a defense mechanism that seems to set in to metabolise the oxLDL and counteract the intracellular oxidative stress. We earlier reported on such a defense in chlorpyrifos induced oxidative stress in ARPE-19 cells in vitro, wherein such an increase in PON enzyme activities and PON2 expression was observed mediated by SP1 transcription factor . There are other similar reports as well [9, 46].
As there is increased oxLDL uptake, accumulation of oxidized cholesteryl esters in lysosomes can occur [47, 48]. Hence, the lysosomal activity in terms of cathepsin D expression in ARPE-19 and THP-1 macrophages was studied. In ARPE-19 cells, the cathepsin D protein expression was significantly increased under oxidative stress. The oxLDL promoted the cathepsin D expression in ARPE-19 cells. The oxidized lipids generated by the prooxidants are known to induce RPE and macrophage degeneration under chronic conditions due to lysosomal destabilization , apart from inflammation and apoptosis were reported . In THP-1 macrophage cells, there was initial drop in cathepsin D expression at short exposure (3 h) but the expression increased with time in vitro (24 and 72 h). Previous reports suggest that uptake of oxLDL in macrophage results in partial lysosomal enzyme inactivation and relocation to the cytosol contributing to poor degradation activity of lysosomes [50, 51].
We further evaluated the key observations in the cultured premature senescent ARPE-19 cells (sARPE-19) to relate closely to AMD pathology. The oxLDL uptake in sARPE-19 was found to be increased under oxidative stress conditions similar to the observation in ARPE-19 cells. The antioxidant response was significant in terms of PON2 protein expression in sARPE-19 under oxidative stress conditions similar to the observations in ARPE-19 cells. However, unlike ARPE-19, NAC treatment in sARPE-19 did not alter the oxLDL uptake that was increased by H2O2 treatment. Higher concentration of NAC needs to be evaluated. A significant increase in CD36 protein expression under the oxidative stress conditions was similar to that of ARPE-19. However, NAC treatment seems to further augment the CD36 expression in sARPE-19 unlike ARPE-19 that showed no such increase. A significant decrease in the cathepsin D protein expression in sARPE-19 was seen similar to ARPE-19 cells (72 h) revealing that the lysosomal activity is drastically reduced under prolonged oxidative stress which improves with the NAC treatment. NAC thus improved the CD36 and cathepsin D expression in the senescence model of ARPE-19 cells exposed to prooxidant metabolites associated with AMD pathology.
Further, PON2 and CD36 expression were found to be increased in RPE of AMD eye tissues compared to control tissues based on immunolocalization, which supports the inference from the in vitro data. There are no reports up to our knowledge that have studied about the PON2 and CD36 expression in RPE of AMD eye tissues. Increased PON2 expression localized to RPE is observed in this study in the AMD eyes for the first time and therefore, PON2 might be considered as disease marker of oxidative stress in AMD, apart from being a systemic marker as reported by us earlier . Prolonged increase in CD36 expression also observed in the AMD eyes results in oxLDL accumulation causes oxidative stress in the RPE cells apart from direct ROS generation by Hcy as reported in the in vitro studies in ARPE-19 . Antioxidant response in terms of PON expression is observed, while treatment with antioxidants can be beneficial in improving the cathepsin D activity and therefore in the rescue of lysosomal activity. Autophagy and proteasomal clearance are thus promoted by the lysosomal activity of hydrolases including cathepsin D in AMD [52, 53]. Though increased cathepsin D immunoreactivity around drusen was reported the enzyme activity is crucial . Loss of cathepsin D activity initiated by oxidative stress contributes to accumulation of undegraded substrates, inflammasome formation leading to RPE dysfunction apoptosis and AMD pathogenesis .