Amani A. Fawzi - US grants

DESCRIPTION (provided by applicant): A team with world-class expertise in ophthalmic disease, imaging, retinal pathology, and biomedical engineering will utilize hyperspectral imaging to provide, for the first time, in vivo molecular probing, validated with histopathologic correlation, of age-related macular degeneration (AMD). Their system, a specially modified fundus camera harnessed to cutting-edge biomedical image analysis techniques, will revolutionize the field by identifying the distribution and spectral signature of the various chromophores and fluorophores associated with AMD lesions. Drusen, the hallmark lesions of AMD, are biochemically heterogeneous and are key to understanding this disease. However, their composition cannot be determined in vivo with even the current highest resolution techniques. Hyperspectral imaging data, now uniquely clinically attainable with a new snapshot hyperspectral device, offers a vision of spectral biopsy of the retina. This data is encoded in a four-dimensional "hypercube," with two spatial (x and y coordinates) and two spectral (amplitude and wavelength) dimensions. This complex data cube can be explored with advanced unsupervised mathematical tools that search out the dominant spectral signatures in the data matrix. By dissecting the spectral reflectance and autofluorescence (AF) signatures from drusen and other AMD lesions, this research will achieve in vivo spectral classifications of these lesions. To accomplish this, the research team plans to acquire hyperspectral, photographic, AF and infrared scanning laser ophthalmoscope (SLO), and spectral domain optical coherence tomography (SD-OCT) images of all phenotypes of AMD, with analysis of the hyperspectral images by an optimized matrix factorization protocol to recover dominant spectra and their spatial distributions. Hyperspectral AF images will also be acquired simultaneously with the same device by adding appropriate excitation and barrier filters. Further, hyperspectral imaging of genotyped donor eyes will provide histopathologic confirmation of the components of drusen and other AMD lesions that correlate with the drusen spectral signatures, as well as robust genotype/phenotype correlations. Long-term goals: We envision an integrated imaging system for AMD and ophthalmologic care that incorporates this paradigm shift in technology. Having such a system of in vivo molecular probes will be instrumental in research. The insights so obtained will be of high value in clinical diagnosis and treatment. In addition, such a system will accelerate translational research with sensitive and early outcome testing of prospective therapeutic agents, saving sight and thereby providing enormous benefit to society. PUBLIC HEALTH RELEVANCE: Hyperspectral imaging with a specially modified retinal camera will provide, for the first time, molecular probing of living eye tissue, validated by examination of donor eye tissue, in the study of age-related macular degeneration (AMD). Identification of the distribution and biochemical nature of AMD lesions will be uniquely instrumental in understanding AMD, the leading cause of blindness in our country. Insights so obtained will be highly valuable in the clinical care of AMD patients and will result in saving sight, with enormous benefit to our society.

DESCRIPTION (provided by applicant): Diabetic retinopathy (DR) is a leading cause of irreversible blindness among working-age adults, which is a typical type of ischemia driven retinal disease characterized by microvascular damage to the retina in patients with diabetes. DR progresses through a sequence of recognizable stages, which begin with structural and functional derangement of the retinal microcirculation even before the early clinical signs occur. The earliest clinical signs of DR are microaneurysms and dot intraretinal hemorrhages resulting from damage to the capillary pericytes and endothelial cells. This capillary damage leads to an increase in retinal vascular permeability, localized loss of capillaries with resulting ischemia, and, in the final stage of DR, the growth of abnormal retinal blood vessels (pathological retinal neovascularization) known as proliferative diabetic retinopathy (PDR). Our long term goal is to provide clinical comprehensive functional and anatomical assessment of retinal vessels in humans. In the proposed project, we first address the need for technology by developing multimodal technologies based on functional photoacoustic ophthalmoscopy (PAOM), optical coherence tomography (OCT)/optical Doppler tomography (ODT). The multimodal imaging technology will be validated and optimized through imaging animal models. Then we will test the hypothesis that the multimodal imaging technology based on PAOM and OCT/ODT can provide comprehensive functional information for the early diagnosis of DR before clinical signs occur in the oxygen induced retinopathy (OIR) rat model. To further test the hypothesis we will apply intervention at the hemodynamic threshold (the earliest hemodynamic changes signifying DR) found by the proposed imaging system on the OIR rat model to show the early intervention benefits - after the time point of hemodynamic threshold interventions cannot prevent PDR. Aim 1. Develop a PAOM to measure sO2 in retinal vessels. PAOM provides accurate quantification of sO2 in retinal vessels by directly sensing the different optical absorption of oxy- and deoxy-hemoglobins. A powerless contact lens integrated with an ultrasonic transducer will be developed for imaging the eye. Aim 2. Develop a dual beam spectral domain OCT to image retinal hemodynamics. The OCT system features two probing beams separated by a controlled distance on retina. Thus, effects of the Doppler angle in blood flow measurement are eliminated and the absolute blood flow velocity can be measured in real-time. Aim 3. Integrate POAM and OCT to provide multimodal functional imaging of both sO2 and blood flow of retinal blood vessels. Validate and optimize the integrated system by imaging phantoms and the eyes of normal rats and rabbits. Aim 4. Test the hypothesis using the developed technology by studying the variation of retinal vascular functions during ischemic retinopathy development in the OIR rat model. PUBLIC HEALTH RELEVANCE: The proposed research will provide a powerful multimodal functional retinal imaging tool that enables the early diagnosis of diabetic retinopathy before clinical signs occur. It also provides a unique tool for the research on the pathological pathways of diabetic retinopathy and the development of new therapies.

DESCRIPTION (provided by applicant): Retinopathy of prematurity (ROP) and bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH) are two conditions that co-exist in many neonates and together contribute to a high rate of morbidity. Current therapeutic approaches address each condition separately, without considering the potentially shared vascular mechanism. In this proposal, we seek to dissect the role of cyclic GMP pathway in the pathogenesis of ROP. We will test the hypothesis that there is a window of opportunity to treat ROP using phosphodiesterase-5 (PDE5) inhibitors that is concurrent with the suggested treatment window for BPD-PH. By studying the effects of manipulating cGMP on the developing neurovascular elements of the retina in neonatal mice, we will examine the potential for reversible adverse effects on this delicate process. We will then use the oxygen-induced retinopathy (OIR) mouse and rat models as test beds for human ROP, in order to dissect the role of cGMP on the different phases of OIR. We hypothesize that systemic use of PDE5 inhibitors will have a beneficial role stabilizing HIF1¿ and promoting normal retinal vascular development in the initial vaso- obliterative phase of OIR, while simultaneously promoting normal lung vascular development and preventing BPD-PH. We further hypothesize that initiating PDE5 inhibition during the angiogenic second phase of OIR may be associated with detrimental enhancement of retinal angiogenesis through HIF1¿ while being ineffective in BPD-PH. The study proposed herein will allow us to identify a safe and effective therapeutic window for the use of PDE5 inhibitors in neonatal mouse and rat OIR model, and pave the way towards an enhanced understanding of the role played by cGMP in retinal photoreceptor and vascular development. The mechanistic experiments proposed herein will capitalize on the interdisciplinary expertise of the two clinician-scientist co-PI's. Dr. Fawzi is a clinician-scientst retinal specialist with special expertise in retinal degenerations, electrophysiology and retinal vascular diseases, and she is NIH funded to study novel functional retinal imaging modalities in ischemic retinopathies. Dr. Farrow is a clinician-scientist neonatologist with an established NIH-funded pulmonary vascular laboratory with a special expertise in cGMP signaling and animal models of pulmonary hypertension. This study capitalizes on a successful collaborative effort using techniques already established in the respective laboratories, as evidenced by the preliminary data presented here. Success of the current proposal, will allow further proof of principle studies in larger animal models, as a pre-requisite for human clinical trials. PDE5 inhibitors are readily available and are already being used in term infants with pulmonary hypertension, where the pharmacokinetics have been validated and the drug shown to be safe. The extension of the use of PDE inhibitors to preterm neonates to prevent BPD-PH and ROP will be the long-term goal of these investigators, who are closely collaborating at the Lurie Children's Hospital and Northwestern University.

? DESCRIPTION: Diabetic retinopathy is a leading cause of blindness, while diabetic nephropathy is a leading cause of end-stage renal disease. Both of these conditions are manifestations of diabetic microvascular disease and, hence, would greatly benefit from a clinical tool that is able to predict patients at risk for disease progression and can monitor oxygen supply and demand in real time. We will test the hypothesis that imaging of the retinal metabolic rate of oxygen, using an innovative OCT approach, can provide a much-needed, non- invasive biomarker of diabetic microvascular disease. In this proposal, we will establish a normative database of retinal metabolic rate in healthy subjects, before proceeding to examining diabetic patients with various complications of diabetic retinopathy before and after pharmaco-therapy. We hypothesize that functional imaging will be able to explain the response to pharmaco-therapy, as well as correlate with diabetic microvascular disease stages. In addition, we will explore the potential renal effects of antiVEGF on this population of patients using rigorous monitoring of renal function. The experiments proposed herein will capitalize on the interdisciplinary expertise of the PI's at Northwestern University. Dr. Fawzi is an NIH-funded clinician-scientist retinal specialist with special expertise in retinal imaging and retinal vasculr diseases. Dr. Zhang is an NIH-funded biomedical engineer, member of the ANSI laser safety committee whose expertise is in functional optical imaging and novel retinal imaging tools. Dr. Zhang has an established laboratory at Northwestern, with strong collaborative ties to Dr. Fawzi, as they share an NIH-funded research program. The current proposal will allow these researchers to validate their proof-of-principle animal data. The research collaboration includes co-I Dr. Jampol, a world-class ophthalmologist clinician-scientist. Nephrologist, Co-I (Dr. Quaggin) is a world-class clinician-scientist and expert on the glomerular function of VEGF and its signaling partners. In addition to fulfilling an important and clinically relevant need, succes of this proposal will provide quantitative tools to study retinal blood flow, oxygen delivery and extraction, in addition to vessel area, allowing researchers to study functional relationships between retinal vessel geometry and systemic micro- and macro-vascular diseases. We believe that our proposal will provide a paradigm shift in patient management, as well as greatly improve our understanding of the pathophysiology of diabetic retinopathy.

Diabetic retinopathy (DR) and other forms of retinal ischemia are a leading cause of blindness in working age adults. There is a need to identify the mechanisms that control the transition from angiogenesis to fibrosis in these conditions. This would be a first step towards new therapies to address progressive vascular endothelial damage that ultimately leads to pre-retinal fibrosis and traction detachment with poor visual and anatomic outcomes. To address this gap in our knowledge, we have designed a series of experiments that build logically on our preliminary data, which shows evidence for endothelin-1 (ET-1) involvement in fibrovascular human surgical membranes. We hypothesize that dysregulated endothelial ET-1 is particularly important in the pathogenesis of ischemia-induced retinopathy and its complications. To examine this hypothesis, we will study animal models that span the entire spectrum of retinal ischemia, including streptozotocin (STZ)-induced diabetes and models of developmental ischemia- the oxygen induced retinopathy (OIR, to replicate angiogenesis in ischemia) and the limited hyperoxia-induced proliferative retinopathy (l-HIPR), which replicates the angiofibrotic end-stages of severe ischemia. We will use these models to test the hypothesis that dysregulation of vascular endothelial cell-derived ET-1 is critically involved in the promotion of vascular pathology, using an inducible, targeted vascular endothelial ET-1 knockout transgenic mouse (ET-1Eko). In aim 1, we subject this ET-1Eko mouse to the STZ-induced diabetes and the developmental models of ischemia to study the role of endothelial ET-1 dysregulation in angiogenesis and fibrosis. In aim 2, we will cross the ET-1Eko with a transgenic reporter mouse model to focus on the endothelial- to-mesenchymal transition in the retinal vessels and the surrounding pericytes, glia and neurons. Finally, in aim 3, we will focus on developing pharmacologic interventions geared towards ET-1 receptors to improve retinal pathology in models of DR, ischemia- induced angiogenesis and fibrosis (OIR and l-HIPR), as a first step towards translating our findings to the bedside. The mechanistic experiments proposed herein will capitalize on the interdisciplinary expertise of the clinician- scientist PI and her collaborators. Dr. Fawzi is a clinician-scientist retina surgeon with special expertise in non- invasive retinal imaging, animal models of ischemia and retinal vascular diseases. Her co-investigator, Dr. Schnaper is a clinician-scientist, pediatric nephrologist, who is a world expert in fibrogenic signaling. Finally, the group also capitalizes on novel imaging technologies in Dr. Zhang?s lab at Northwestern University, another collaborator.

This proposal aims to study retinal blood flow, detected by non-invasive imaging, during medical treatments for diabetic retinopathy. Success of this proposal will lead to the development of non-invasive measurements that can guide clinicians as they manage patients with sight threatening complications of diabetic retinopathy.