Department of Ophthalmology
University of Texas Medical Branch at Galveston
Galveston, TX

HARRY E. BOVAY, JR.  RESEARCH PROJECT

Novel therapy for ischemic retinopathy

Current Research Interests

Ischemic retinopathies, such as diabetic retinopathy, retinopathy of prematurity, and
retinal vascular occlusion, affect a large population of patients, often resulting in irreversible vision loss due to the development and growth of abnormal new vessels after a period of retinal ischemia. This process is referred as retinal neovascularization. At present, therapies for ischemic retinopathy mainly target retinal neovascularization and the treatments are limited, not always effective, have considerable side effects, and are expensive. Dr. Zhang’s goal is to develop a novel, effective and inexpensive approach to treat ischemic retinopathy – to selectively eliminate abnormal blood vessels in the retina without affecting normal blood vessels.

Plans for 2024

Given that retinal neuronal injury significantly contributes to vision loss because of losing their normal light sensing and relaying functions as well as causing vasculopathy through their crosstalk to vessels, it would be very beneficial to understand molecular mechanisms of retinal neuronal dysfunction/injury in ischemic retinopathy and develop neuroprotective therapy. In the renewal application, we will further determine and characterize the role of Epac1 in the injury of retinal inner neurons in ischemic retinopathy and elucidate its underlying mechanisms.

Specific Aims:
AIM 1. To determine and characterize the role of Epac1 in the injury of retinal inner
neurons (in particular bipolar cells) during ischemic retinopathy.
AIM 2. To elucidate mechanisms underlying Epac1-induced neuronal injury in ischemic
retinopathy.

Progress in 2023

In 2023, Dr. Zhang studied 5 genes (Synpo, Upp1, Ch25h, Csrp1, Pfkp) that are likely involved in retinal neovascularization. His laboratory determined their roles in angiogenesis using in vitro tube formation assay but did not find any of them played a significant role in this process. Therefore, they did not further investigate their roles in retinal neovascularization in vivo.  He further explored the ER stress/PERK pathway in retinal neovascularization in a mouse model of ischemic retinopathy (OIR model) and found that phosphorylated PERK (p-PERK) was only upregulated during retinal ischemia but not immediately after vessel degeneration and p-PERK was mainly increased in the ganglion cell layer (GCL), inner nuclear layer (INL), and neovessels. Moreover, Dr. Zhang identified a novel role of Epac1 in mediating the injury of retinal inner neurons in ischemic retinopathy. One manuscript based on the data generated from RRF support was published in IOVS. Three abstracts were presented in the ISER 2023 conference and two abstracts were presented in the ARVO annual conference on May 2023.

2023 Publications

Progress in 2022

Throughout 2022, Dr. Zhang investigated a novel role of ER stress/PERK pathway in retinal neovascularization during ischemic retinopathy. This pathway has been involved in many diseases but its role in ischemic retinopathy (IR) is unknown. His lab demonstrated that PERK was activated in the retina during retinal ischemia, and inhibition of PERK with a specific inhibitor GSK2606414 reduced neovascularization while promoting vascular repair. Moreover, PERK inhibition in IR attenuated the loss of retinal ganglion cells. Mechanistically, PERK inhibition preserved astrocyte network, prevented Müller cell activation and downregulated the recruitment / proliferation of macrophage / microglia although it did not affect the upregulation of canonical angiogenic pathways in IR. One manuscript based on data generated from RRF support was published and four abstracts were presented as posters during ARVO’s annual meeting in May, 2022. One abstract poster was awarded an ARVO Knights Templar Eye Foundation Travel Grant.

Specific Aims: Aim 1. To validate protein expression of the selected genes that are upregulated in endothelial cell during ischemic retinopathy and determine their localization in the retina. Aim 2. To determine the role of the selected molecules in angiogenesis.

Progress in 2021

Dr. Zhang used single-cell RNA sequencing technology (scRNAseq) to investigate the heterogeneity of endothelial cells in ischemic retinopathy and identify and characterize the features of putative endothelial cells for neovascularization. His laboratory also identified 77 candidate molecules that could be potentially used as biomarkers for neovascular endothelial cells or as targets for the intervention. Two manuscripts based on data generated from RRF support have been published in a high impact journal (Acta Neuropathol Commun) and one abstract has been presented as posters in the ARVO annual conference on May 2021. This poster was awarded an ARVO Retina Research Foundation/Joseph M. and Eula C. Lawrence Travel Grant.

Progress in 2020

Commensal gut microbiota experts profound influence on our body and regulates the processes of many diseases.  Dr. Zhang has initially assessed the potential role of gut microbiota in retinal neovascularization during ischemic retinopathy using 16SrRNA sequencing to assess bacterial microbiota composition in the colon feces in a mouse model of oxygen-induced retinopathy (OIR). His research team found although total bacterial abundance in the colon was not changed in OIR mice, microbiota composition was changed in OIR neovascularization.  This work warrants further investigation into the role of gut microbiota in ischemic retinopathy.  In 2020’s grant period, one manuscript based on data generated was prepared  and submitted for publication and two abstracts were presented as posters at the May 2020 ARVO annual conference.  Dr. Zhang’s paper has now been published in Acta Neuropathologia Communications.

Progress in 2019

At present, therapies for retinal neovascularization are limited, not always effective, have considerable side effects, and are expensive.  In 2019, the goal of Dr. Zhang’s project was to develop a novel, effective, and inexpensive approach to selectively kill abnormal blood vessels in the retina without affecting normal blood vessels. His prior research found that blocking a protein called Epac1 prevented retinal neovascularization while accelerating vascular repair in ischemic retinopathy. Further investigation of Epac1-mediated retinal neovascularization was performed using in-vivo and in-vitro models.  Dr. Zhang’s studies demonstrated that Epac1 acted as a novel inhibitor of y-secretase/Notch signaling pathway while enhancing VEGF signaling to promote retinal neovascularization.  This work significantly advances knowledge on mechanisms of retinal neovascularization and suggests that Epac1 is a novel drug target for treating ischemic retinopathy.  Together with previous findings, Dr. Zhang’s work was accepted for publication in Science Advances, a Tier 1 journal.  Another manuscript based on data generated from RRF support also was published in IOVS, and two poster abstracts were presented at the ARVO annual conference in May, 2019.

Progress in 2018

Interestingly, clinic evidence suggests that retinal neovascularization is associated with the predominance of potentially pathogenic microorganisms over probiotics in the gut microbiome. Moreover, during Dr. Zhang’s 2018 RRF research period,  his laboratory found that endotoxin from pathogenic bacteria  exaggerated retinal neovascularization while beneficial metabolite (short chain fatty acid) from probiotic bacteria attenuated retinal neovascularization. This information suggests that gut microbiota plays a potential role in retinal neovascularization in ischemic retinopathy. Dr. Zhang’s research institution, UTMB, has a sterile mouse facility, making it possible to use germ-free mice for his research. Germ-free mice specifically are very valuable in addressing the role of gut bacteria in health and diseases.

Ischemic retinopathies, such as diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusion, affect a large population of patients, often resulting in irreversible vision loss due to the development and growth of abnormal new vessels after a period of retinal ischemia. The process is called retinal neovascularization. In 2018, Dr. Zhang’s research goal was to develop a novel approach to selectively remove abnormal blood vessels in the retina without affecting normal blood vessels. Commensal gut microbiota exerts profound influence on our body and regulates the processes of many diseases. The roles of gut microbiota in ischemic retinopathies are completely unknown. However, clinic evidence suggests that retinal neovascularization is associated with the predominance of potentially pathogenic microorganisms over probiotics in the gut microbiome. The research proposed to deplete gut bacteria with antibiotics to test the hypothesis that gut microbiota has a critical role in retinal neovascularization in ischemic retinopathy. During the project,it was determined that it is not easy to achieve an optimal dosage of antibiotic cocktails in neonatal pups, and it is not conclusive whether depleting gut bacteria will affect neovascularization or not. However, pups treated with lipopolysaccharides, which is one of the disease-producing products from pathogenic microorganisms, developed more severe retinal neovascularization while pups treated with butyrate, which is a beneficial metabolite from probiotics, attenuated retinal neovascularization. These data support our hypothesis that gut bacteria may affect retinal neovascularization in ischemic retinopathy through their diseaseproducing products or beneficial metabolites. We have made significant progresses in the project as well as substantially extended this project and tested mechanisms involved in retinal neuronal injury during ischemic retinopathy. One manuscript based on data generated from RRF support has been published in IOVS and three abstracts have been presented as poster in the ARVO annual conference on May 2018.

Progress in 2017

The goal of Dr. Zhang’s project in 2017 was to develop a novel approach to selectively remove abnormal blood vessels in the retina without affecting normal blood vessels.  His research previously identified that Epac1, highly expressed in endothelial cells of abnormal new blood vessels in the retina, plays an important role in the process of retinal neovascularization. Adenosine A2A receptor (A2AR) is a Gs protein-coupled receptor which is activated upon binding to adenosine and stimulates production of cAMP, one of the most common and universal second messengers to regulate many physiological and pathological processes. In a mouse model of ischemic retinopathy, studies showed that A2AR expression was significantly increased in retinas and retinal vessels during ischemic retinopathy, which were associated with increase in cAMP production. A2AR antagonist istradefylline was approved to treat Parkinson’s disease (PD) in Japan.  Further, it was shown that istradefylline treatment attenuated cAMP production, Epac1 activation and retinal neovascularization in ischemic retinopathy. This work supports the possibility of treating ischemic retinopathy by repositioning istradefylline for ophthalmic use. We have successfully fulfilled the project as stated in the application as well as substantially extended this project and tested several mechanisms involved in retinal neuronal injury during ischemic retinopathy. Three manuscripts based on data generated from RRF support were published. One abstract was presented at the ARVO annual conference on May 2017 and selected as a “Hot Topic” by ARVO Program Committee.

Progress in 2016

Dr. Zhang previously had developed and synthesized 12 novel Epac inhibitors based on the structure of ESI-09. In a mouse model of ischemic retinopathy, Dr. Zhang showed that nanoparticle-formulated ESI-09 effectively eliminated abnormal vessels while promoting the physiological vascular repair. Additionally, his studies suggested that the adenosine A_2A receptor may potentially serve as an upstream regulator of the cAMP/pac1 pathway. Since A_2AR antagonist istradefylline has been used to treat Parkinson’s disease in Japan, he plans to further investigate the role of A_2AR in activation of cAMP/Epac pathway and test whether A_2AR antagonists can be used to safely and effectively prevent and eliminate neovascularization without inhibiting the process of vascular repair.

Progress in 2015

Dr. Zhang’s results indicated that targeting Epac alone is sufficient to achieve his research goal to specifically eliminate retinal neovascularization while sparing or even accelerating normal vascular repair. He made some modifications of the original plan to only target Epac1 in retinal neovascularization but substantially extend his study to understand the actions of Epac1 in this process. In addition, he developed in vitro 3D angiogenesis assay that allows him to examine the anti-angiogenic effect of testing agents in a pathophysiological environment. With these modifications, he not only accomplished the goal of the project to develop novel and specific treatment for retinal neovascularization but also provided further insights of mechanisms by which activation of Epac1 induces retinal neovascularization. In addition, since the formulation only contained Epac inhibitor, engaging pharmaceutical companies to develop future anti-Epac therapy might be facilitated because clinical trials testing both Epac inhibitor and Fn14 inhibitors would be expensive.

Dr. Zhang proposed to use the nanotechnology developed during 2014 funding to deliver drugs to inhibit Fn14 and/or Epac to selectively prevent and eliminate neovascularization without inhibiting the vascular repairing process. His aims were: 1) to generate FDA-approved biocompatible nanoparticles loaded with drugs to block Fn14 and Epac individually or simultaneously and demonstrate their efficacies in inhibiting angiogenesis using in vitro assays; 2) to investigate the effects of these nanoparticles in treating retinal neovascularization using his established animal model of ischemic retinopathy.

Progress in 2014

The goal of this project was to develop a novel approach for topical delivery of anti-angiogenic drugs to selectively kill abnormal blood vessels in the retina without affecting normal blood vessels. Dr. Zhang found in 2014 that two proteins known as Fn14 and Epac1, which are highly expressed in endothelial cells of abnormal new blood vessels in retina, are involved in the process of retinal neovascularization. He determined whether blocking Fn14 can specifically eliminate retinal neovascularization and developed nanoparticles targeting Fn14 for the delivery of anti-neovascularization drug. He tested the hypothesis that nanoparticles-mediated delivery of drugs to block Fn14 and Epac1 may allow for safe and effective treatment of retinal neovascularization without impairing normal vascular repair.