Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors

Abstract

Age‐related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen‐associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen‐associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely‐tuned mechanism regulating directional apical:basal sorting and secretion of drusen‐associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE‐derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.

Extracellular Vesicles fromPRPF31+/−iPSC-Derived RPE Have a Distinct RNA Profile Compared to Those From Healthy RPE

“…Additionally, there were 9,166 poly-A RNAs enriched in apical EVs and 9,027 enriched in basal EVs (Figure 4D). These data are consistent with the concept that RPE apical and basal EVs exhibit distinct transcriptomes as well as protein contents (31, 32).…”

“…Previous studies have reported that proteins found within EVs reflect the polarity of the RPE (31, 32). In order to determine if RNAs within RPE EVs reflect polarity, we compared all apically and basally released miRNAs and Poly-A RNAs among PRPF31 +/+ and PRPF31 +/− EVs.…”

“…Several groups have evaluated the use of RNAs in EVs from the vitreous, aqueous and serum as biomarkers of uveal melanoma, age-related macular degeneration, and glaucoma (26)(27)(28)(29)(30). The protein contents of EVs produced by the RPE have also been studied (31,32).…”

A Surgical Kit for Stem Cell-Derived Retinal Pigment Epithelium Transplants: Collection, Transportation, and Subretinal Delivery

“…hiPSC culture and retinal organoid differentiation were conducted as previously described (Zhong et al, 2014). hiPSCderived RPE was generated according to Flores-Bellver et al (2021). Briefly, retinal organoids at 60 days of differentiation were used to isolate retinal pigment epithelium (RPE) tissue.…”

“…We previously demonstrated that human-induced pluripotent stem cells (hiPSC) can form three-dimensional retinal organoids containing neural retina and retinal pigment epithelium (RPE) cells in vitro (Zhong et al, 2014;Flores-Bellver et al, 2021). To generate hiPSC-derived RPE monolayers to use as substrate for our RPE transplants, we harvested the RPE spheroids from retinal organoids (Figure 1A), and cultured the dissociated RPE cells on 10 μm thick transparent polyester membranes.…”