N-Acetylcysteine Amide Protects Against Oxidative Stress-Induced Microparticle Release From Human Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly affecting tens of millions of people worldwide, and is the most common cause of vision loss in the elderly in the United States. The dry form of AMD accounts for 80% to 90% of all AMD cases.1–4 The hallmarks of dry AMD are the appearance of drusen, and apoptosis of the retinal pigment epithelium (RPE).5–9 Drusen are extracellular lipid- and protein-containing deposits that accumulate mainly between the RPE and Bruch's membrane. Drusen are a hallmark of aging and early AMD, as well as a risk factor for developing late AMD.10–14 Although understanding of the mechanisms of AMD has increased, there is neither a cure nor means of prevention for AMD. Therefore, there is a critical need to identify new mechanisms for AMD to develop preventive and therapeutic strategies for this age-related blinding disease. The deposition of drusen between Bruch's membrane and the RPE layer impairs the RPE cell layer through obstruction of nutrient flow to and from the choroid. It is believed that drusen are formed from cellular materials originating from RPE cells. The suggested mechanisms for drusen formation include cellular budding during apoptosis5,15 and exosomal trafficking during autophagy.16 An additional possible mechanism that has yet to be explored is through the generation of microparticles (MPs) by RPE cells. Cell-derived MPs are small membrane-bound vesicles that range in diameter from 100 to 1000 nm. They are released from activated, injured, and apoptotic parental cells into the extracellular space.17–21 Microparticles shed by other organ systems under pathologic conditions have been noted to contain parental cell components and are capable of potentiating the disease state.17,22–25 The retina is highly susceptible to oxidative stress, which is recognized as a major risk factor for AMD.13,26,27 Retinal pigment epithelium cells spend their life recycling the lipid-rich photoreceptor outer segments, which requires an abundance of antioxidants and repair systems to maintain homeostasis.4,13,26 Disruption of this balance may lead to RPE dysfunction, cell death, and inflammation.3,13,26–28 Complement activation is currently thought to play an important role in AMD development.1,4,29 Membrane complement regulatory proteins (mCRPs: CD46, CD55, and CD59) provide normal cells with a first line of defense against complement attack, and have been identified on human RPE cells in culture30–33 and in situ.30,33–36 Oxidative stress leads to the loss of mCRPs on RPE cells.31 In addition, CD46 is lost from RPE cells at the earliest stage of AMD and has been shown to be present in drusen.33,34,36 However, mechanisms leading to the loss of mCRPs on RPE cells under oxidative stress or during early AMD remain unknown. Therefore, we hypothesized that oxidative stress–induced loss of mCRPs on RPE cells is through the release of MPs that carry mCRPs. N-acetylcysteine amide (NACA; also termed AD4), the amide form of N-acetylcysteine (NAC), is a thiol antioxidant with enhanced properties of lipophilicity, membrane permeability, and antioxidant capacity when compared with NAC.37 Emerging evidence provides strong support for NACA as a protective agent against oxidative stress under numerous pathologic conditions in vitro and in vivo, including a mouse model of retinal degeneration.38 In this study, we investigated if oxidative stress drives MP release from RPE cells, assessed whether these released MPs carry mCRPs, and evaluated the effects of NACA, a thiol antioxidant, on oxidative stress–induced MP release.