Cleveland Clinic Cole Eye Institute is at the forefront of innovative basic, clinical and translational research on the physiology of vision and the pathological conditions that lead to vision loss. The department of ophthalmic research is currently chaired by Bela Anand-Apte, MBBS, PhD.
The faculty in the department are involved in multi-disciplinary and highly collaborative approaches using both basic science and clinical investigation, which serves as the basis for exploring and evaluating treatment strategies to slow and prevent vision loss. Disorders currently being investigated include retinitis pigmentosa, macular degeneration (both inherited and age-related forms), diabetic retinopathy, retinopathy of prematurity, ciliopathies, glaucoma, uveal melanoma, amblyopia and corneal disorders such as infections, wound healing and repair. There is a concentrated focus on innovative strategies to improve imaging modalities for the eye as diagnostic as well as intra-operative tools. Stem cell approaches for therapeutics are also being investigated.
One of the many strengths of the department of ophthalmology is the close interaction between basic science researchers and clinicians who are committed to achieving a common goal of preventing vision loss. This cohesive community of investigators fosters innovation through collaboration and allows the movement of ideas from both bench to bedside and bedside to bench.
- Understanding the basic molecular mechanisms of ocular neovascularization with a special focus on Tissue Inhibitors of Metalloproteinases-3 (TIMP-3) and Sorsby Fundus Dystrophy
- Examination of the physiological and pathological pathways that regulate breakdown of the blood-retinal barrier in diabetic retinopathy
Research and Innovations
With the discovery of TIMP-3 mutations being causative in an inherited retinal degeneration, Sorsby Fundus Dystrophy, in which patients developed florid choroidal neovascularization, we focus our studies on understanding this disease and identifying potential therapeutic approaches to prevent or slow vision loss.
We have made significant progress in being able to dissect out the mechanisms by which mutations in TIMP-3 cause the Sorsby fundus dystrophy phenotype as well as identifying the regions of TIMP-3 that are responsible for angiogenesis inhibition. We have been awarded a patent for the use of TIMP-3 peptides for the inhibition of angiogenesis in a number of diseases in which neovascularization plays a major role. For the most part we have focused our efforts on the regulation of neovascularization in the eye with some activities in tumor angiogenesis.
Using both human, animal in vivo and in vitro studies we have identified insulin and betacellulin to play a role in the development of macular edema in patients with diabetes. We have established a novel transgenic zebrafish model that can be used for high throughput screening of drugs that effect retinal and brain vascular leakage. Our ultimate goal is the understanding, prevention and/or reversal of angiogenesis and retinal vascular permeability, in an effort to control the devastating blinding consequences of ocular diseases.
Lab Staff Members
- Bela Anand-Apte, Principal Investigator
- Jian-Hua Qi, Project Staff
- Mariya Ali, Lead Research Technologist
- Lana Pollock, Post-doctoral Fellow
- Alyson Wolk, Graduate Student
- Allison Grenell, Graduate Student
- Shiming Luo, Medical Student
- Srinidhi Singuri, Medical Student
- Allison Mancuso, Undergraduate Student
Corneal Disease Diagnosis & Treatment Lab
- Simulation-based diagnosis and treatment of corneal disease
- Computational modeling
- Elasticity imaging and corneal biomechanics
Corneal Wound Healing, Diseases & Ocular Surface Lab
- Identify and characterize the growth factor-receptor systems through which the functions of corneal, immune, and other cells of the anterior segment of the eye are controlled during development, homeostasis, and wound healing.
- Understand at the molecular and cellular level, the factors that lead to corneal opacity, and its resolution, after injury, surgery or infection
- Explore the mechanism of epithelial basement membrane regeneration after injury and the importance of the corneal epithelial basement membrane in modulating epithelial-stromal interactions in the cornea, including development of myofibroblasts associated with corneal stromal opacity.
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Lab Staff Members
- Steven E. Wilson, MD, Principal Investigator
- Paramananda Saikia, PhD, Research Associate
- Rodrigo Carlos de Oliveira, MD, Postdoctoral Fellow
- Sofia Murillo, Researcher
- The function of TULP1 in photoreceptor cells of the retina
- Pharmacogenetics of neovascular age-related macular degeneration
- Genetic analysis of inherited retinal diseases
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Innate Immunity and Inflammation Lab
My lab focuses on understanding the innate defense and immunoregulatory functions of corneal epithelial cells. Epithelial tissues form a protective lining of the body, including the outer surfaces that are exposed to the environment, as well as the inner body cavities, glands, and ducts. Through constitutive and inducible expression of danger- and pathogen-associated molecular pattern recognition receptors, innate defense molecules, cytokines and chemokines, epithelial cells serve as an essential component of the innate immune system to stop pathogens right at the points of entry before they can cause diseases, regulate regional immune and inflammatory responses, and maintain tissue homeostasis. Recently, we discovered that the C-terminal fragments of a cytokeratin protein, K6, in corneal epithelial cells are antimicrobial, and that endogenous K6 regulates signaling pathways to control production of inflammatory mediators. These are unexpected functions for keratin proteins since they have long been viewed as the building blocks of cytoskeleton that maintain structural integrity and resilience of epithelial cells. Given the wide distribution of K6 among different epithelial tissues, as well as the structural and functional similarities shared among different keratins, our studies aim to advance understanding of epithelial innate immune functions of the cornea and other sites of the body and to contribute to biocompatible anti-infective and immunoregulatory drug development in the post-antibiotic era.
Lab Staff Members
- K.P. Connie Tam, PhD, Principal Investigator
- Jonathan Chan, PhD, Research Associate
- Yan Sun-Beck, MD, Research Associate
Neurovascular Development Lab
The Rao lab is interested in understanding how neurons and vasculature pattern themselves and interact with each other during development as well as in disease. Specifically, we are studying the role of circadian clocks and their contribution to retinal neurovascular development and function. We use the mouse eye as our model system mainly because of the presence of multiple vascular networks and their close association with the neurons. This interface between the neuronal and vascular systems is important for normal function and disruption can lead to pathologies. Circadian disruption is associated with wide range of metabolic syndromes and more recently has been implicated in the progression of certain diseases. Not much is known about the contribution of the circadian clock on retinal function. Our goal is to identify the molecular targets of the circadian clock within this retinal neurovascular unit and study the effects of the loss of these modulators on retinal development and maintenance. Though our studies are focused in the eye, our findings will have implications for the design of novel biological therapies for any tissue within the body.
- Circadian regulation of neuronal and vascular development in the eye. We have recently demonstrated that environmental light plays an important role in the regulation of neuronal and vascular development in the eye. Based on our findings we proposed that light exposure during development is required to establish or entrain a circadian clock within the retina. Circadian clocks are biochemical oscillators that oscillate in phase with the solar day and night cycle. Thus a circadian oscillator could regulate and coordinate processes like cell cycle entry and exit or secretion of growth factors and hormones. Retinal development is a coordinated process of timed cell division, differentiation and growth. We hypothesize that this kind of synchronized activity within groups of cells must be due to the early timing cues provided by light. We use a variety of genetic, molecular and biochemical techniques to dissect the function of circadian clocks within different cell types of the retina.
- Circadian regulation of endocrine function. An exciting and new area of research that has emerged from our analysis, is the observation that the circadian clock directly regulates enzymes that are responsible for controlling local thyroid hormone availability. The control of endocrine systems by the circadian clock is not surprising, since it allows for a systemic coordination of various physiological target systems according to the time of the day. Our data specifically shows that an enzyme called Dio2 (Deiodinase 2) is targeted by the circadian clock. Dio2 is responsible for locally converting the prohormone T4 to its active state. This results in a spatial and temporal control of T3 mediated response, which is not always the case with the systemic release of the thyroid hormone. This interaction between the circadian clock and Dio2 prompted us to investigate if Dio2 has any role in ocular vasculature. We have identified a novel signaling axis that is controlled by thyroid hormone in regulation of arterial and venous blood vessels. Current research is focused on identifying the downstream targets of thyroid hormone that play a significant role in vascular development and homeostasis. Our data suggest that the notch signaling is affected by thyroid hormone signaling. Disruption in Notch pathway in humans results in arterial venous malformation. We will investigate if these malformations in an animal model can be rescued by providing thyroid hormone during the right time in development. The information gained from these experiments will help us to determine if manipulation of thyroid hormone can be used to treat certain vascular diseases. Moreover, thyroid hormone signaling is extremely important in maintaining energy balance and controlling metabolism. The molecular information that we gain from this analysis can also be used in non-vascular tissues where energy metabolism is critical for the proper function of the tissue.
- Role of circadian clock in inflammation. The ocular vasculature is closely associated with resident macrophages and microglial cells. We are interested in understanding if circadian clocks within the microglia can regulate secretion of inflammatory molecules within the retina. Age related changes within the retina can lead to many retinal pathologies. Some pathologies have been associated with increased inflammation in the retina. We will investigate if clock disruption within the microglial cells affect or contribute to retinal pathologies. The ultimate goal of this project is to define a functional role for microglial clocksin maintenance and repair of neurons and vasculature.
Research & Innovation
Our research was the first analysis that demonstrated a link between environmental light and proper development of the eye. Since then we have demonstrated that light exposure in first trimester is a risk factor for the development of severe retinopathy. We can use the information that we gather from these projects to consider early interventions in treatment of retinopathy of prematurity where it could have an enormous benefit. Furthermore, our current research investigating the role of clock genes which are important in the generation and maintenance of circadian rhythms will uncover novel roles for these genes and will provide us new targets for treatments of proliferative retinopathy.
In other words ...
Every living organism, have an internal timing mechanism that allows for certain physiological, behavioral and biochemical processes to occur in a rhythmic manner to coincide with the external environment. There is a growing body of evidence that shows a correlation between disruption of the internal timing and its negative impact on behavior and pathophysiology. We are interested in understanding how these internal timing cues affect tissue development and function and why do these disruptions lead to certain diseases. Our primary goal is to use the gained information to identify the targets of these clocks and design novel therapeutic that can be used to directly manipulate the targets. Though we study the eye, clocks are present in all the tissues within the body and this knowledge can be applied to any tissue within the body.
Lab Staff Members
- Sujata Rao, PhD, Principal Investigator
- Onkar Sawant, PhD, Research Associate
- Vijay Jidigam, PhD, Postdoctoral Fellow
- Rebecca Fuller, BS, Technician
- Mitchell Boshkos, Research Student
Goals and Projects
- The long-term goal of our uveal melanoma project is the development of a multi-antibody immunological assay for UM metastasis that will complement current cytogenetic and genetic prognostic methods and establish for the first time methods to detect and quantify circulating uveal melanoma tumor cells.
- The long-term goals of our glaucoma projects are to better understand the molecular mechanisms of glaucomatous vision loss, identify therapeutic targets and develop a panel of blood-borne glaucoma biomarkers.
- The long-term goal of our age-related macular degeneration (AMD) project is the development of molecular technology for assessing AMD risk and monitoring AMD therapeutics.
Research and Innovations
Uveal Melanoma (UM) is the most common primary malignancy of the eye and has a high mortality rate (40%). Survival rates have not improved in part because the pathobiology is poorly understood and primary UM tumors (pUM) can metastasize before diagnosis and UM micrometastases can lie dormant for years. Earlier detection of metastatic pUM is critical for earlier interventions. About 95% of primary uveal melanoma (pUM) originate in the capillary-rich uveal tract (i.e., the iris, ciliary body, and choroid), which facilitates hematogenous dissemination. The most common site of metastases is the liver (93%), but the majority of patients exhibit multiple sites. Our laboratory has completed the most extensive proteomic characterization of primary uveal melanoma tumors (pUM) on record as of 2019. Using LC MS/MS iTRAQ technology, we have analyzed 100 pUM specimens (45 that metastasized and 55 that were non-metastasizing). The pUM were collected from enucleated eyes in collaboration with ophthalmic pathologist Sarah Coupland, MBBS, PhD (Director, North West Cancer Research Centre, University of Liverpool, UK) and Cleveland Clinic ophthalmic oncologist Arun Singh, MD. The metastatic status of pUM was determined by cytogenetic and gene expression analyses, and clinical survival data. Thirteen choroid specimens excised from UM eyes distant from the pUM were utilized as a pooled control. Tryptic digests were labeled with unique iTRAQ tags, fractionated by RPHPLC at pH10 and subjected to LC MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer. The study resulted in the quantification of 3952 tumor proteins and the identification of a large number of differentially expressed (DE) proteins with Met/NoMet ratios exhibiting p values less than or equal to 0.05 (adjusted test), including 119 DE proteins that are predicted to be plasma membrane/cell surface proteins. Prediction modeling of the 100 proteomic datasets using DE protein predictors has provided discriminatory accuracies (C-statistics) and metastatic status prediction success rates greater than 90% with as little as 12-16 cell surface DE proteins. We are now developing an immunoassay for UM metastasis using multiple antibodies to select DE proteins as a prognostic tool for characterizing UM tumor biopsies and for the detection and quantification of pUM circulating in the blood.
Glaucoma is a multifactorial optic neuropathy and a leading cause of blindness worldwide. Glaucomatous damage to the visual system can occur at normal and elevated levels of intraocular pressure (IOP). Age and IOP are risk factors for the neuropathy, but the identification of molecular risk factors for IOP elevation and glaucomatous vision loss are a high priority. Previous proteomic analyses in our laboratory have demonstrated that cochlin, a protein associated with deafness, is abnormally expressed in human trabecular meshwork (TM) from primary open angle glaucoma donors, suggesting that this protein may contribute to obstruction of the aqueous humor (AH) outflow pathway through the TM and elevated IOP. Other proteomic studies in the laboratory have demonstrated peptidyl arginine deiminase 2 (PAD2) in human POAG optic nerve and in monkey experimental glaucoma optic nerve head (ONH) and retina. Still other proteomic studies in the laboratory have shown that treating cultured human TM cells with either transforming growth factor beta 2 (TGFb2) or dexamethasone significantly altered the abundance of TM proteins and identified many proteins not previously associated with TGFb2-signaling or glucocorticoid-signaling in the eye. We have completed a preliminary quantitative proteomic study of experimental glaucoma (EG) versus control eye differences within the ONH and retina from 3 monkeys with unilateral, laser-induced high IOP (IOP Max >28 mm Hg). Glaucoma-altered proteins in the ONH strongly support the connective tissue deformation and remodeling evident in the EG monkey and strongly implicated myelin-associated neurodegeneration. Glaucoma-altered proteins in the retina implicated dysfunction in the mitochondria, oxidative stress response, cytoskeletal/connective tissue organization, and transport and regulatory processes. Up to 1819 proteins were quantified in the same tissues from one mild IOP EG monkey (IOP Max < 20 mm Hg). Both the direction and character of glaucoma-induced proteomic alterations were vastly different in mild IOP versus high IOP monkey EG eyes. Site-specific deimination differences in the ONH and retina were also detected between mild and high IOP as well as quantitative differences in PAD2. Deimination has been linked to cancer, immune and neurodegenerative disorders and to the regulation of multiple cellular processes. We hypothesize that the proteomic changes we observed at mild IOP are representative of real differences between those at high IOP and that they contribute to molecular mechanisms of early EG pathology. Efforts are now focused on using additional animals to rigorously compare glaucoma-induced proteomic alterations in monkey ocular tissues, including deimination, at mild IOP versus those at high IOP.
Age-Related Macular Degeneration (AMD) is a complex disease and a major cause of vision loss in the elderly. Of those with early AMD, clinicians cannot predict who will progress to advanced disease and severe visual loss. Only a fraction of early/mid-stage AMD patients progress to advanced AMD, with neovascular or “wet” AMD being more prevalent than advanced dry AMD (also known as geographic atrophy). Effective molecular biomarkers would facilitate early clinical assessment of AMD progression, the monitoring of AMD therapeutics and help prevent or slow severe visual loss. Growing evidence supports AMD as an inflammatory disease involving oxidative stress. A host of oxidative protein modifications have been associated with AMD, including adducts derived from docosahexaenoate-lipids such as carboxyethylpyrrole (CEP). Our laboratory participated in the initial immunodetection of elevated CEP in AMD ocular tissues and AMD plasma. CEP-protein stimulates neovascularization in vivo, mice immunized with CEP-protein develop a dry AMD-like phenotype, and anti-CEP antibodies have utility in monitoring the efficacy of select pharmacological interventions. However, no significant mass spectrometric evidence supports the presence of CEP-protein adducts in vivo despite the strong CEP immunoreactivity demonstrated in AMD plasma and ocular tissues. We hypothesize that CEP adducts are metabolically altered in vivo to structures that remain recognized by anti-CEP antibodies. This raises questions like “What is the molecular identity of the protein modification(s) responsible for anti-CEP immunoreactivity and do they have bioactivities?” Toward answers to such questions, we are structurally and functionally characterizing a peptide modification we have identified on 43 plasma peptides captured by anti-CEP immunoaffinity chromatography. This lysine modification exhibits a mass addition of 120.0206, rather than the 122.0362 expected for CEP and has been detected in 16 proteins and at 6 lysine residues in human serum albumin that correspond to CEP modification sites in our authentic CEP-bovine serum albumin standard. Preliminary analyses suggest it is more abundant in AMD than control plasma.
Lab Staff Members
- John W. Crabb PhD, Principal Investigator
- Geeng-Fu Jang PhD, Project Scientist
- Jack Crabb BS, Research Computer Analyst
Regeneration/Cell Therapy Lab
Goals and Projects
The primary goal of Dr. Alex Yuan’s laboratory is to characterize the wound healing and regenerative response of the retina and to develop novel methods for retinal repair. The retina is comprised of a multi-layered, complex network of neurons that receive light stimuli and transmit that information to the brain. In response to mechanical, chemical, or photic damage, the retina forms scar tissue, which interrupts the normal connections between neurons. This disruption is permanent and once vision has been compromised, it cannot be restored. However, there are some organisms such as teleost fish that are capable of retinal repair following injury. Our lab is studying the retina repair process following laser induced retinal injury in fish. We hypothesize that there are molecular pathways that are modified or lost in mammals which, if restored, may allow the mammalian retina to regenerate or may facilitate the reintegration of transplanted cells into the normal retina.
- Characterize the role of the leukocyte adhesion molecule, Alcama, during zebrafish retinal regeneration. Our lab studies retinal regeneration in zebrafish using a targeted laser injury model. Using this model, we are able to precisely place lesions in the retina. Laser injury has a benefit of selectively damaging the outer retina and is also desirable due to its rapid and facile application compared to other methods. Following outer retina (photoreceptor) injury, Muller glia rapidly proliferate and some of the resulting daughter cells migrate from the inner nuclear layer to the outer nuclear layer to replenish the damaged photoreceptors. The remaining Muller glia start to express the cell surface protein, Alcama in response to injury. During development, Alcama plays a role in axonal guidance and cellular migration. We hypothesize that Alcama plays a similarly important role during retinal regeneration in zebrafish. Interestingly, using a similar mouse model, we were unable to detect Alcama expression following injury. We are currently characterizing the role of Alcama in zebrafish and look to determine if Alcama can aid in facilitating retinal regeneration in mice and other mammals.
- Optic nerve regeneration model. In addition to our work on retinal regeneration, we are also collaborating with Dr. Frank Papay’s laboratory on developing an ocular transplantation model. The first step in this process is to successfully establish optic nerve regeneration following the complete transection of the optic nerve. We have developed a method to maintain the retina blood flow while completely transecting the optic nerve and are currently evaluating different small molecules that may facilitate the regrowth of axons through the injury site.
- Evaluation of a novel self-assembling hydrogel for sustained intravitreal drug delivery. In collaboration with Dr. Vivek Kumar’s group (New Jersey Institute of Technology), we are evaluating the anti-angiogenic properties of a self-assembling hydrogel and its ability to promote sustained delivery of bevacizumab (Avastin) and steroids using a rat model of retinal neovascularization.
Lab Staff Members
- Alex Yuan, MD, PhD, Principal Investigator
- Rose DiCicco, Laboratory Manager and Lead Technician
- Kristin Allan, Graduate Student in Molecular Medicine
- Michael Ramos, Technician
Retinal Cell Biology Lab
Goals and Projects
Research in the laboratory of Brian D. Perkins, PhD, uses zebrafish and induced pluripotent stem cell (iPSC) models to study photoreceptor degeneration and regeneration.
Inherited retinal diseases lead to irreversible loss of rod and cone photoreceptors and frequently result in blindness. Leber’s Congenital Amaurosis (LCA) is the most common blinding disorder in children. Unfortunately, the body has no way to replace those cells once they are gone. Mutations in the gene for Centrosomal Protein of 290 kDa (CEP290) are one of the leading causes of LCA, as well as other ciliopathies such as Joubert Syndrome (JBTS) and Bardet-Biedl Syndrome (BBS). Importantly, photoreceptor degeneration is one of the most common manifestations in ciliopathies. The long-term goals of this laboratory are to understand the mechanisms regulating the formation of photoreceptor outer segments and to understand how to regenerate photoreceptors in genetic models of retinal degeneration. Our laboratory utilizes a complementary approach of zebrafish mutants in cep290 and several other cilia genes, as well as 3D retinal cups generated from patient-derived human iPSCs. Zebrafish cep290 mutants undergo retinal degeneration and we are exploring the mechanisms that lead to photoreceptor loss and attempting to identify signals that can trigger regeneration in these mutant zebrafish. We are also using iPSCs created from patients with CEP290 mutations to make retinal organoids (also known as “retinas-in-a-dish”) in order to establish the mechanisms that determine disease severity. Our goal is to combine knowledge from stem cell derived retina and zebrafish to develop better therapies for LCA patients in order to restore vision.
Lab Staff Members
- Brian D. Perkins, PhD, Director
- Ping Song, PhD, Project Staff
- Joe Fogerty, PhD, Project Staff
- Rachel Stupay, PhD, Postdoctoral Fellow
- Lauren Cianciolo, Post baccalaureate
Retinal Degenerations Lab
- Studying of mechanisms underlying the structural and physiological changes due to ageing in the retina and retinal pigment epithelium (RPE) as model systems
- Understanding how age-related macular degeneration (AMD) pathology affects RPE cellular function, with focus on etiological drivers of RPE degeneration
Research and Innovation
Age-related macular degeneration (AMD) etiology is complex, and we know that it includes both a genetic component and environmental risk, the strongest being advanced age. As the retina and retinal pigment epithelium (RPE) ages, many structural and physiological changes occur. Although these changes are well known, the underlying mechanisms involved in them are frequently poorly understood. It is also important to dissect the changes due to aging from the ones due to pathology. My long-term goal is to understand how age-related macular degeneration (AMD) pathology affects RPE cellular function. To this end, my research is focusing on aging and oxidative stress function, an etiological driver of RPE degeneration in AMD pathology. Specifically, we are investigating the antioxidant mechanisms regulated by DJ-1. DJ-1 is a multifunctional protein that plays an essential role in the oxidative stress response in neurodegenerative disorders such as familial and sporadic PD, amyotrophic lateral sclerosis, Alzheimer, and Huntington’s disease. My work has demonstrated that the aging DJ-1 KO mice display degeneration of retina and RPE cells, changes in mitochondria structure and function, and increased inflammation, all hallmarks of AMD. We hypothesize that limiting the formation of reactive oxygen species within the RPE may effectively prevent or reduce RPE dysfunction observed in AMD patients. In this context, my lab will analyze RPE antioxidant and mitochondrial functions regulated by DJ-1. I also intend to test the therapeutic potential of DJ-1 in animal models that recapitulate features of AMD. My lab looks to model AMD by superimposing additional etiological drivers (e.g. inflammation, cigarette smoking, light exposure) to our animal model. Finally, my lab aims to perform ex-vivo imaging, histology, Immunohistology and of eyes from donor with AMD. The aim of this research is to correlate morphological and molecular signatures of RPE/photoreceptors degeneration with ex-vivo imaging findings. The crosstalk study between RPE degeneration, mitochondrial function, oxidative stress, autophagy and DJ-1 function is novel and has translational potential.
Lab Staff Members
- Vera L. Bonilha, PhD, Director
- Caroline Milliner, Research Laboratory Assistant
- Sanghamitra Bhattacharyya, PhD, Research Associate
- Mala Upadhyay, PhD, Postdoctoral Fellow
Retinopathy of Prematurity Lab
Laboratory Goals and Projects
- Eradicate retinopathy of prematurity by using a proangiogenic strategy during phase one to direct the normal sequential growth of blood vessels.
- Define the metabolic basis of liver induced retinovascular plasticity.
- Understand the molecular mechanism of oxygen-induced retinopathy.
The long term goal of this laboratory is to eradicate retinopathy of prematurity (ROP) - the most common form of infant blindness worldwide, accounting for 150,000 blind children annually. Survival after premature birth requires oxygen supplementation that is paradoxically associated with toxicity to premature developing tissues, such as the lung alveoli, nephrons of the kidney, cerebral cortex, and retinal capillaries. The direct relationship of oxygen saturation to disease severity in clinical trials as well as in preclinical investigations has placed the oxygen sensitive transcription factor hypoxia inducible factor (HIF) as a central mediator of retinovascular growth and development. We have definitively demonstrated the safety and efficacy of HIF stabilization in the prevention of oxygen-induced retinopathy (OIR) via HIF prolyl hydroxylase inhibition (PHi) in preclinical models in two different species, achieving a protected phenotype for both retina and lung simultaneously by systemic PHi.
- Synergy between the liver and the retina. We have extensively tested dozens of small molecules with varying efficacy in stabilizing HIF. Among these, we have determined that carbonyl glycines, hydrazones, and benzolamides are three classes of drugs capable of initiating protection against hyperoxia, but each has varied specificities for liver versus eye versus other organs. For example, dimethyloxaloylglycine is able to target only the liver yet still protects retinovascular tissue whereas other compounds such as Roxadustat work synergistically with both the liver and the retina to create protection against oxygen toxicity. A western blot using both dose and time response did not see changes in retinal HIF concentrations after intraperitoneal injection of DMOG. Instead we saw clear upregulation of HIF-1 in the liver. Using a luciferase-ODD in vivo reporter gene, we clearly found that DMOG targeted only the liver. Surprised by this result, we next conditionally ablated hepatic HIF1A to demonstrate that hepatic HIF-1α, with DMOG as a drug, was necessary and sufficient to transduce protection. We further proved this result by comparing two carbonyl amides- DMOG and Roxadustat using RNA seq. Transcriptional profile was nearly identical between the two drugs in the liver, but they were vastly different in the retina where DMOG had little transcriptional effect that had no overlap with Roxadustat. Finally, using LC-MS 1 and MS 2 we definitively demonstrated that DMOG was so labile as to not survive the first pass in the liver but rather never entered the blood. Roxadustat on the other hand not only entered blood but also entered retina. Therefore Roxadustat could override the hepatic HIF-1α KO and worked synergistically to provide near total protection to the retina. In summary, western blot, luc-ODD reporter gene analysis, RNA-seq, conditional KO experiments, and LC-MS confirmed that indeed a visceral organ could protect a distal capillary bed and provided the notion that low and intermittent dosage of HIF stabilizers might be safe and effective in fragile premature infants in hyperoxia.
- Metabolic basis of liver induced retinovascular plasticity. Transcriptional and knockout studies of PHi animals described above have determined a unique liver-eye axis that directs “remote” protection against oxygen-induced retinopathy and cardiac ischemia, respectively. We are using targeted and untargeted metabolite profiling to determine how the liver might contribute to protection of a distal capillary bed such as in the retina. After uncovering no protein based/hepatokines induced protection from the liver we used untargeted metabolite profiling to link 1) retinal serine/glycine levels and 2) activation of both the hepatic urea cycle and retinal serine/1-carbon metabolism to hepatic HIF-1 dependent, providing a metabolic phenotype of mice protected by pharmaceutical HIF stabilization against oxygen toxicity.
- Metabolic basis of oxygen induced retinopathy. Using both cells in culture and animal models, we have determined that hyperoxia induces a pronounced shift in metabolism of retinal Müller glia and retina in general. Despite the well known fact that HIF stabilization induces anaerobic glycolysis, we were stunned to find that hyperoxia also restricts flow of glycolytic carbon into the TCA cycle. Instead, Müller cells use glutamine-fueled anaplerosis to generate energy. For every mole of glutamine deamidated to glutamate and further deaminated to α-ketoglutarate, 2 moles of ammonia are released providing the hypothesis that oxygen toxicity to the retina may involve ammonia toxicity and therefore links nitrogen balance through transamination of 2-oxoacids and upregulation of the urea cycle as two metabolic, liver dependent pathways that might explain the synergy of liver/retina by certain classes of HIF stabilizers.
The concept of pro-angiogenic strategy preventing pathologic angiogenesis is novel. This concept is translational and applies to all forms of ischemic disease, offering the potential to prevent vascular loss before it happens even in the setting of stimuli that creates ischemia, such as in ROP or diabetes.
Lab Staff Members
- Jonathan E. Sears, MD, Principal Investigator
- Charandeep Singh, PhD, Postdoctoral Fellow
- George Hoppe, PhD, Project Staff
- Vincent Tran, BS Research Technician
- Youstina Bolok, MS, Research Technician
- Leah McCollum, BS, Research Technician
Visual Electrophysiology Lab
- X-linked retinoschisis. X-linked retinoschisis (XLRS) is a prevalent early onset retinal disorder affecting primarily males. XLRS is caused by mutations in the gene Retinoschisin (RS1). We have been working with three mouse models that carry disease-causing mutations in Rs1. These mice develop an early onset and severe phenotype that varies across the different genetic models. Current studies focus on the very early stages of the disease, using an array of electrophysiological and imaging modalities. This work involves collaborations with investigators at the Burke Medical Research Institute and the University of Illinois at Chicago Eye and Ear Infirmary and is supported by the NIH.
- Genetics of Age-Related Macular Degeneration. Age-related macular degeneration (AMD) impacts central vision with a prevalence that increases with age. Prior genetic studies have focused on European populations. Recognizing that the prevalence and presentation of AMD varies across different ethnic populations we are working with the VA Million Veteran Program to determine whether the genetic underpinnings of AMD may differ across different ethnic groups. In veterans of European descent, we have replicated the major genetic risk factors noted in earlier work, and have also identified additional AMD risk alleles. In Veterans of African descent, however, we do not see major contributions from the most prominent AMD risk alleles previously identified in European populations. Current studies are designed to understand these differences and also to expand our analysis to additional eye diseases. This work involves collaboration with investigators at Case Western Reserve University, Buffalo VA Medical Center, and Providence VA Medical Center, and supported by the intramural research program of the United States Department of Veterans Affairs.
Lab Staff Members
- Neal S. Peachey, Ph.D., Director
- Minzhong Yu, Project Scientist
- Craig Beight, Research Technician