In the current study, we introduced an hfRPE cell culture model with a proteome that resembles the proteome of the AH from patients with drusen and RPD. Eight of eleven proteins with differential abundance between the controls and dry AMD patients in SWATH-MS analysis (Table
2) were previously considered to be major components or regulators in drusen [
27,
28]. Among these proteins, APOA1, CTSD, and CLUS were found to be elevated via IF and WB analysis in the RPE cell cultures treated with 4-HNE compared to the levels in the control cultures. In all the patient AH samples, various immunoglobulins were identified, which accounted for one-third of the total protein abundance (based on spectral counts). This high abundance of endogenous immunoglobulins detected in AH has also been observed in other body fluids (e.g., blood), suggesting that immunologic dysregulation is one of the major pathogenic mechanisms of dry AMD. Complement factor H, one of the most important soluble (secretory) complement regulatory proteins and a major inhibitor of alternative pathways providing protection against harmful complement activation [
29], was decreased in both the AH from patients with drusen and RPD and the conditioned media from both apical and basal baths of cells exposed to oxidative stress in the present study. In a previous histologic study using AMD donor eyeballs, CFH immunoreactivity was identified in drusen, the sub-RPE space, along Bruch’s membrane, and in the walls of choriocapillaris [
30]. While some authors have reported decreased CFH expression in Bruch’s membrane/choroid complexes in cases of both early AMD and geographic atrophy [
31], others have found no difference in the amount of CFH proteins in Bruch’s membrane/choroid tissue samples from AMD patients and controls [
32]. Reduced expression of CFH has been observed in senescent or oxidative stressed RPE cell lines (ARPE-19 cells) [
33,
34] suggesting that degenerative RPE cells might not be capable of synthesizing complement regulators for self-preservation under conditions of a dysregulated complement pathway; however, the changes in the intracellular expression level of CFH have not been described in RPE from donor eyeballs or polarized hfRPE cultures. Thus, we speculate that secreted CFH, regardless of its intracellular expression level in RPE, decreases during the progression of AMD, leading to the accumulation sub-RPE deposits [
35]. Various oxidation products, including advanced glycation end products such as carboxymethyllysine and reactive aldehydes such as carboxyethylpyrrole, malondialdehyde, and 4-HNE, have been shown to accumulate in the retina and cause damage to the retina during the progression of degeneration, as observed during AMD [
36‐
38]; thus, all the phenotypes and pathologies of the RPE observed in vivo cannot be recapitulated by the application of any single agent to a culture system in vitro. However, we were able to demonstrate the compensatory role of our in vitro culture system, which reflects proteomics data from dry AMD patients, in the present study.
The RPE cell is a highly polarized cell types that produces and secretes proteins onto at basolateral and apical surface, meeting differential demands on either side of the photoreceptors and choriocapillaris and holding a large majority of secreted proteins on the basolateral side [
14,
15]. In the RPE cultures exposed to oxidative stress in the present study, however, the ratios of secreted proteins between chambers corresponding to the basal and apical surfaces were reversed; the total protein concentrations in the conditioned media from the apical and basal chambers in the in vitro culture model exposed to oxidative stress were different (apical>basal) from those in the control cultures (basal>apical). Likewise, each AH sample (control, drusen, and RPD) exhibited a different total protein concentration (RPD > drusen>control). Epithelial-mesenchymal transition (EMT) is a complicated phenomenon through which polarized epithelial cells lose their polarity and cell-cell adhesions and acquire the mesenchymal characteristics of motility and invasiveness [
39,
40]. Thus, it is plausible that a dysfunctional or stressed RPE cell may lose its polarity and increasingly misdirectionally secrete proteins onto the apical surface rather than onto the basolateral surface, resulting in accumulation in the subretinal space (forming RPD) rather than in the sub-RPE space. We speculate that different locations or subtypes of material deposition are associated with different statuses of the RPE, especially the maintenance of proper polarity. In terms of their composition, drusen and RPD have been shown to share several common components, including membranous vesicles, vitronectin, CFH, and apolipoproteins [
6,
41‐
43]; however, their lipid composition is distinct, with a higher concentration of unesterified cholesterol being found in RPD [
44‐
46].