Hyperreflective foci, OCT progression indicators in age-related macular degeneration, include transdifferentiated retinal pigment epithelium

Purpose By optical coherence tomography (OCT) imaging, hyperreflective foci (HRF) indicate progression risk for advanced age-related macular degeneration (AMD) and are in part attributable to ectopic retinal pigment epithelium (RPE). We hypothesized that ectopic RPE are molecularly distinct from in-layer cells and that their cross-retinal course follows Müller glia. Method In clinical OCT (61 eyes, 44 AMD patients, 79.4 ± 7.7 years; 29 female; follow-up = 4.7 ± 0.9 years) one HRF type, RPE plume, was reviewed. Twenty eyes of 20 donors characterized by ex vivo OCT were analyzed by histology (normal, 4; early/intermediate AMD, 7; geographic atrophy (GA), 6; 3 neovascular AMD (nvAMD). Cryosections were stained with antibodies to retinoid (RPE65, CRALPB) and immune (CD68, CD163) markers. In published RPE cellular phenotypes, red immunoreactivity was assessed semi-quantitatively by one observer (none, some cells, all cells). Results Trajectories of RPE plume and cellular debris paralleled Müller glia, whether ordered or subsident, near atrophy borders. RPE corresponding to HRF lost immunoreactivity for retinoid markers and gained immunoreactivity for immune markers. Aberrant immunoreactivity appeared in individual in-layer RPE cells and extended to all abnormal phenotypes. Müller glia remained CRALBP-positive. Plume cells approached and contacted retinal capillaries. Conclusion Gain- and loss-of-function starts with individual in-layer RPE cells and extends to all abnormal phenotypes. Down-regulated RPE retinoid handling may impair rod vision while Müller glia sustain cone vision. Evidence for RPE transdifferentiation, possibly due to ischemia, supports a proposed process of epithelial-mesenchyme transition. Data can propel new biomarkers and therapeutic strategies for AMD. Précis Hyperreflective foci (HRF) are OCT progression risk indicators in age-related macular degeneration. Abnormal RPE cells including some that correspond to HRF lose immunoreactivity for retinoid markers and gain immunoreactivity for immune markers, indicating molecular transdifferentiation. ### Competing Interest Statement T.A. receives research funding from Novartis and is a stockholder of MacRegen Inc. K.B.F. is a consultant to Genentech, Optovue, Zeiss, Heidelberg Engineering, Allergan, and Novartis. C.A.C. receives research funds from Genentech/ Hoffman LaRoche and Heidelberg Engineering and is a stockholder of MacRegen Inc. ### Clinical Trial n.a. ### Funding Statement This work was supported by National Institutes of Health (Bethesda, MD, USA) grant R01EY015520 (CAC); The Macula Foundation, Inc., New York, NY; an anonymous donor to AMD research at UAB; unrestricted funds to the Department of Ophthalmology from Research to Prevent Blindness, Inc.; and EyeSight Foundation of Alabama. Acquisition of human tissues for immunohistochemistry was funded by NIH grant R01EY015520 (CAC) and R01EY027948 (CAC, TA) and IZKF Würzburg (N-304, TA). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Study of a clinical cohort and use of human tissue samples was approved by institutional review board at Vitreous Macula Retina and University of Alabama at Birmingham, respectively, and complied with the Health Insurance Portability and Accountability Act of 1996 and the Declaration of Helsinki. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Available on request. * AMD : Age-related macular degeneration OCT : optical coherence tomography HRF : hyperreflective foci BLamD : basal laminar deposit BrM : Bruch’s membrane Ch : choroid ChC : choriocapillaris cRORA : complete RPE and outer retinal atrophy d : drusen ELM : external limiting membrane EZ : ellipsoid zone fv scar : fibrovascular scar GCL : Ganglion cell layer HFL : Henle fiber layer ILM : internal limiting membrane IPL : inner plexiform layer INL : inner nuclear layer IS : inner segments of photoreceptors iRORA : complete RPE and outer retinal atrophy IZ : interdigitation zone nvAMD : neovascular AMD ONL : outer nuclear layer OPL : outer plexiform layer OS : outer segments of photoreceptors R : retina RPE : retinal pigment epithelium Sc : sclera SRS : subretinal space x : detachment