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Biji Mathew, Sriram Ravindran, Lieanne A. Torres, Mohansrinivas Chinnakesavalu, Sophie Tran, Raj Patel, Steven Roth; Mechanisms of Action of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Retinal Ischemia. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2895.
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© ARVO (1962-2015); The Authors (2016-present)
We demonstrated the neuroprotective effects of mesenchymal stem cells (MSCs) and their media. However, most effects of MSCs are paracrine. We hypothesized that the neuroprotective effect of MSC media was due to extracellular vesicles (EVs). The mechanisms of action of EVs in retina remain incompletely determined. Heparin sulfate proteoglycans(HSPGs) receptors on the plasma membrane are actively involved in viral endocytosis believed to resemble EVs entrance. We hypothesized that HSPGs are essential for EVs endocytosis in retinal neurons. We also hypothesized there is cell-specific differential uptake of EVs and distribution in retina.
We used an in vitro simulated ischemia model using inner retinal neurons and an in vivo rat ischemia model to test our hypothesis. Human MSC EVs (hMSC EVs) were isolated by precipitation and centrifugation and the size and concentration determined using Nanoparticle Tracking Analysis. Additional experiments determined the mechanism of endocytosis of EVs into retinal neurons. We examined in vitro temperature dependence, saturation, blockade of HSPGs, and involvement of caveolins or clathrins in the receptor-mediated endocytosis. A separate set of experiments evaluated the uptake and distribution of EVs in rat retina by injecting fluorescently labeled EVs into vitreous and imaging in vivo over 4 weeks.
EV endocytosis was saturable, dose and temperature-dependent, and occurred via cell surface HSPGs mediated by the caveolar endocytic pathway. EVs significantly attenuuated death of retinal cells subjected to oxygen glucose deprivation in vitro. EVs were taken up by retinal neurons, retinal ganglion cells, and microglia in vivo after injection into vitreous. They were present in the vitreous for four weeks after intravitreal administration, with saturable binding to vitreous components. Injection of EVs 24 h after ischemia in vivo significantly restored retinal function and prevented apoptotic cell death and neuro-inflammation.
MSC-EVs are endocytosed by retinal cells in a receptor-mediated, dose-dependent and saturable manner, and they show strong promise for treatment of retinal ischemic conditions. Our findings on the involvement of HSPGs on the target cell surface in EV endocytosis and the binding of EVs to the vitreous serve as a basis for the development of engineered EVs for targeting or enhanced delivery and/or functionality.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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