Retina-derived extracellular vesicles in diabetic retinopathy: their potential role in pathogenesis and therapy

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Project summary. Extracellular vesicles are cell-derived membranous structures harboring a variety of biomolecules. Their secretion and cargo are determined in part by the physiological or pathological conditions that the parent cell is exposed to. Recently, increased levels of extracellular vesicles in plasma have been associated with the pathogenesis of diabetic retinopathy, but from where those plasma extracellular vesicles originated from, and whether they affect the development of the disease is still under investigation. Early in my postdoctoral training, I established a modified protocol to isolate extracellular vesicles from small tissues, including retina. Using this protocol, I isolated extracellular vesicles of retina explants from nondiabetic and diabetic mice and found that diabetic retina-derived extracellular vesicles induce ICAM-1, an important adhesion molecule, in retinal endothelial cells in vitro. This induction is inhibited by pharmacological blockage of TLR4, a transmembrane protein that has been proposed as an extracellular vesicles cell receptor. I also found that diabetic retina-derived extracellular vesicles activate circulating leukocytes isolated from nondiabetic animals. This work laid the foundation of my ongoing research. I want to determine the mechanism(s) of how retina-derived extracellular vesicles induce ICAM-1 in retinal endothelial cells and activate circulating leukocytes using a mouse model of diabetes. I propose 3 specific aims: Aim 1, To determine if a diabetes-modified visual cycle activity in photoreceptor cells modulates the retina- derived extracellular vesicles phenotype, which lead to the induction of ICAM-1 in retinal endothelial cells and activation of circulating leukocytes. Aim 2, To determine if TLR4 is the common receptor present in retinal endothelial cells and leukocytes and is triggered upon binding of retina-derived extracellular vesicles, leading to the induction of ICAM-1 in retinal endothelial cells and activation of circulating leukocytes in diabetes. Aim 3, To determine if rod-derived extracellular vesicles are key contributors to the retinal vascular pathology characteristic of diabetic retinopathy. These studies will be conducted in vivo using mice genetically deficient in key proteins of the visual cycle and pharmacological means to inhibit extracellular vesicles secretion and TLR4 receptor in diabetes. This proposal is novel because it focuses on (i) the role of retina (and rod)-derived extracellular vesicles in the pathogenesis of the retinopathy, (ii) the role of the visual cycle in the cargo sorting of retina (and rod)-derived extracellular vesicles, and (iii) the role of TLR4 as a receptor of retina (and rod)-derived extracellular vesicles. The insights learned from these studies can lead to the development of novel and effective targeted therapies to inhibit the etiopathogenesis of diabetic retinopathy.