In this study, we found that the increase in PS-, PE-, and mCRP-positive MPs was accompanied by the increase in apoptosis and loss of mCRPs (CD46, CD55, CD59) from 500 μM H
2O
2-treated RPE cells by flow cytometry, suggesting that the decreased mCRPs on the RPE surface result from increased shedding of mCRP-positive MPs. Our Western blot analysis confirms the flow cytometry findings for CD46 and CD59. Although we constantly found that CD55 was present on RPE cell surface and on MPs by flow cytometry (
Figs. 6,
7D,
8D), we were unable to detect CD55 expression in lysates prepared from intact cells (whole-cell lysates) or lysates prepared from MPs by Western blot analysis, using two commercially available antibodies against CD55 (data not shown). One CD55 antibody we used was mouse anti-human CD55 monoclonal antibody from EMD Millipore (Cat#: CBL511; Lot#: 26017014), and another was rabbit anti-human CD55 monoclonal antibody (EPR66890, Cat#: ab133684; Lot#: GR93375-13; Abcam, Cambridge, MA, USA). Consistent with our results, Yang et al.
32 did not detect CD55 expression in both native and cultured human RPE cells by Western blot analysis using a different commercially available antibody, although they did detect CD55 expression on RPE surface by flow cytometry. Thurman et al.
31 reported that 1 mM H
2O
2 reduced the surface expression of CD55 (also known as decay accelerating factor [DAF]) and CD59 in ARPE-19 cells using flow cytometry, but no Western blot analysis of CD46, CD55, and CD59 was reported in their study. The reasons for not detecting CD55 protein expression by Western blot analysis are not clear. One possible explanation could be that CD55 antibodies commercially developed for Western blot analysis are not suitable for CD55 antigen in the RPE cells due to its unique posttranslational modifications. Additional investigations will be required to test this hypothesis in the future studies. Interestingly, Ebrahimi et al.
33 found that cellular CD46 and CD59 proteins were decreased in ARPE-19 cells treated with oxidized low-density lipoproteins and that CD59 and CD46 along with an exosomal marker CD63 were detected in culture supernatant. They suggested that the decreased levels of CD46 and CD59 were in part due to the release of exosomes and apoptotic particles.
33 Two proteomic studies were performed on extracellular vesicles derived from ARPE-19 cells. One type of extracellular vesicles (ARPE-19 membrane blebs) was obtained by centrifuging conditioned medium for 15 minutes at 100
g,
65 whereas another type of extracellular vesicles (exosomes) was obtained by centrifuging conditioned medium for 1 hour at 100,000
g.
60 Both proteomic studies did not demonstrate the detection of CD46, CD55, and CD59 on these vesicles. Although there are no standard procedures available to isolate different types of extracellular vesicles (mainly MPs, exosomes, and apoptotic bodies), the most common protocol for isolation of MPs consists of differential centrifugation with final centrifugal force of 20,000
g.
17,42 In this study, we followed this most common protocol to isolate and analyze MPs from cultured RPE cells derived from donor eyes as well as ARPE-19 cells. We detected PS- and PE-positive MPs, and found the presence of CD46, CD55, and CD59 on the surface of isolated MPs, suggesting the CD46 found in drusen during early AMD
33,36 or CD59 detected in subretinal space
33 could originate from RPE cells through the release of MPs.