Robert N. Weinreb - US grants

Eicosanoids are ubiquitous biological substances which have many potent actions throughout the body. In the eye, two classes of eicosanoids, prostaglandins and leukotrienes, are thought to be mediators of inflammation and may contribute to the regulation of intraocular pressure. When administered topically, they disrupt the blood-aqueous barrier and also influence intraocular pressure. In the proposed studies we will evaluate eicosanoid formation, metabolism and action in tissues of the inflow and outflow pathways of the eye and its modulation by pharmacologic agents used in the treatment of glaucoma. We will also examine the effects of laser irradiation of the iris and trabecular meshwork, other treatment modalities in glaucoma, on the release of eicosanoids into aqueous humor and its pharmacologic modification. Trabecular tissue, cells derived from this tissue and ciliary body tissue will be incubated in the presence and absence of radiolabeled arachidonic acid; the products formed will be measured either by radioimmunoassay or by conversion of radiolabeled substrate after separation of products by high performance liquid chromatography. The results of these studies will enhance our understanding of glaucoma (particularly primary open angle, inflammatory and steroid glaucoma) and its treatment with pharmacologic agents and the laser. They also will be relevant to our understanding of uveitis and its treatment with anti-inflammatory drugs, and cataracts and their prevention.

Eicosanoids are ubiquitous biological substances which have many potent actions throughout the body. In the eye, two classes of eicosanoids, prostaglandins and leukotrienes, are thought to be mediators of inflammation and may contribute to the regulation of intraocular pressure. Topically administered prostaglandins reduce intraocular pressure in man, most likely by increasing uveoscleral outflow. The role of these agents in the pharmacologic treatment of glaucoma is being evaluated. Further, the intraocular pressure- lowering effects of other drugs, such as epinephrine, can be accounted for, in part, by their effects on uveoscleral outflow; this effect may be mediated endogenously-produced prostaglandins. In the proposed studies, we will evaluate eicosanoid formation, metabolism and action in tissues of the inflow and outflow pathways of the eye and its modulation by pharmacologic agents used in the treatment of glaucoma, other substances, flow and laser irradiation. Trabecular meshwork and ciliary muscle, and cells derived from these tissues, will be incubated in the presence and absence of radiolabeled arachidonic acid; the products formed will be measured either by radioimmunoassay or by conversion of radiolabeled substrate after separation of products by high performance liquid chromatography. These studies also will evaluate the modulation of eicosanoid biosynthesis by phospholipase A2 and C and the role of cyclic nucleotides and calcium in these cellular processes. Since drugs that increase uveoscleral outflow may do so by direct effects on the ciliary muscle, we will evaluate the morphology of the ciliary muscle, the proposed site of action of prostaglandins, using light and electron microscopic methods. The results of these studies will enhance our understanding of glaucoma (particularly primary open-angle, inflammatory and steroid glaucoma) and its treatment with pharmacologic agents and the laser. These studies also are relevant to our understanding of uveitis and its treatment with anti-inflammatory drugs.

Laser scanning tomography will be employed to quantitatively assess the thickness of the nerve fiber layer. A pinhole is located at a plane conjugate to the focal plane of the scanning laser beam. This confocal arrangement assures that only light originating from the illuminated focal plane on the retina passes through the pinhole and is detected by the photomultiplier. Consequently, images with high spatial resolution in all directions are obtained. By capturing and processing 32 images spaced between 10 and 30 microns in depth, a pseudo three dimensional image of the area being observed can be generated. In the polarization dependent imaging mode, the contrast of nerve fiber layer images is enhanced. In order to evaluate the thickness of the nerve fiber layer from polarization dependent measurements, the nerve fibers are approximated by an array of parallel thin cylindrical rods immersed in a medium of different refractive index (Muller-cell cytoplasm). The obtained data will yield point by point thickness measurements of the nerve fiber layer. To assess the accuracy of this technique, unfixed and fixed post-mortem human eyes will be studied. Fixed tissue will be examined with light microscopy to determine the accuracy of the methods and calibrate the instrument for clinical measurements in patient eyes. Next, nerve fiber layer thickness will be measured, and the reproducibility determined for the eyes of normal subjects and glaucoma patients. These data will be compared with standard photographic techniques for assessing the presence of the nerve fiber layer.

DESCRIPTION (Investigator's Abstract) "This is an application to become a participating Clinical Center in the Ocular Hypertension Treatment Study (OHTS). The complete details of the OHTS rationale, design, and methods are contained in the OHTS Manual of Procedures. This proposal provides complete documentation of the ability of the University of California, San Diego, to screen large numbers of ocular hypertensive patients and to enroll at least 50 eligible patients over a 24-month period. Rigby Slight, M.D., backup for the Principal Investigator, is an Associate Clinical Professor of Ophthalmology and a full time glaucoma specialist in private practice in the area who will refer or follow patients screened for enrollment and randomization to treatment in the OHTS clinical trial. Documentation is provided of the capabilities of the proposed investigators and their staff for the performance of the study in accord with the details of the OHTS Manual of Procedures, the nature and extent of their commitment to the University of California, San Diego."

This application describes an ancillary study to the Ocular Hypertension Treatment Study (OHTS), a multicenter clinical trial sponsored by the National Eye Institute, to determine the effectiveness of confocal scanning laser ophthalmoscopy to objectively evaluate the optic disc in ocular hypertensive patients. Specific Aims: 1. To evaluate the use of confocal scanning laser imaging techniques to detect glaucomatous optic nerve damage and change in ocular hypertensive patients. 2. To investigate the quantitative temporal relationship between optic nerve topography and measurable visual field loss. 3. To evaluate the effect of treatment on optic nerve topography. 4. To compare topographic optic disc parameters in African-American and white ocular hypertensive patients. Methods: Six of the 21 OHTS study centers will participate in this ancillary study; at least 400 ocular hypertensive patients will be included. Confocal laser scanning ophthalmoscopy will be used to measure optic disc and peripapillary topography at the randomization visit, and annually thereafter for the duration of the study. A Confocal Scanning Laser Ophthalmoscopy Reading Center will be established at University of California, San Diego to process and analyze the images. To ensure that this ancillary study will not jeopardize recruitment, retention, implementation of the primary trial or assessment of the primary findings, the reading center will be masked to identity and treatment status of all patients. These data will be evaluated quantitatively for evidence of glaucomatous damage and change, and compared with other structural and functional measures of glaucomatous optic neuropathy obtained in the OHTS. This study will evaluate whether these advances in fundus imaging technology can improve the precision with which we can detect and monitor glaucomatous optic neuropathy.

Topical administration of certain prostaglandins (PGs) has been known for more than twenty years to reduce intraocular pressure in several non-human species. However, their clinical use has been limited by early experimental observations of blood-aqueous barrier disruption with concomitant anterior chamber cells and flare. During the past several years, PG analogs have been developed which retain the ocular hypotensive effect but have minimal, if any, influence on the blood-aqueous barrier in human patients. These agents are now known to reduce intraocular pressure by increasing uveoscleral outflow. Although the biologic basis for the increased facility is unknown, altered turnover of ciliary muscle extracellular matrix linked with G-protein activation may be involved. The proposed studies will continue our investigations of eicosanoid actions on the outflow pathways through an analysis of PG modulation of extracellular matrix metabolism in human ciliary smooth muscle cells. PG-mediated effects on ciliary muscle cell production of extracellular matrix molecules including collagen types I, III, IV, fibronectin, and laminin will be determined. Changes in mRNA coding for these molecules or their subunits also will be determined. Ciliary muscle from monkey eyes treated in vivo with topical PGs will be analyzed for the alterations in the specific content of extracellular matrix molecules, matrix-degrading enzymes, regulatory molecules (TIMPs) and messages for these molecules or their subunits. In parallel experiments, PG-treated eyes will be sectioned and processed to determine the distribution of these molecules and their corresponding messages by immunocytochemistry and in situ hybridization. The role of G-proteins in mediating the responses of ciliary muscle cells to PGs will be investigated using agonist induction of GTP-gamma-S hydrolysis in ciliary muscle membranes The identity of specific G-protein alpha subunits activated by PGs in ciliary smooth muscle will be determined using a covalently-binding GTP analogue. These studies will provide new information about extracellular matrix biology in the ciliary muscle and will enhance our understanding of PG ocular hypotensive action.

animal tissue; model design /development; eye; biological products; Primates;

DESCRIPTION (provided by applicant): We have recently identified key structural similarities between aqueous humor outflow in mouse eyes, including a well-defined uveoscleral outflow pathway, and human eyes. We also have developed novel methods for measuring aqueous humor dynamics in the mouse, and discovered that they also have similar physiological characteristics to humans. Moreover, we have shown that mouse intraocular pressure, like human intraocular pressure, is reduced by topical administration of a widely-used prostaglandin analogue (latanoprost), and that latanoprost increases uveoscleral outflow in the mouse eye. These similarities indicate that the mouse eye is highly suitable for studying biologic mechanisms influencing intraocular pressure and how it changes under different conditions. Our technical advances now enable us to use transgenic mice to ascertain how activating or altering specific genes influences intraocular pressure. Specific Aim 1. To determine age-related changes in intraocular pressure, conventional outflow, uveoscleral outflow, and prostaglandin-mediated pressure lowering in normal and transgenic mice with abnormal collagen metabolism. Specific Aim 2. To determine prostaglandin-mediated alterations of scleral matrix metalloproteinase gene transcription, protein biosynthesis, and transscleral-permeability in vitro and in vivo. Specific Aim 3. To determine the role of specific signal transduction events in the intraocular pressure lowering and aqueous dynamics changes induced by topical prostaglandin treatment of mouse eyes. The results of these studies have important implications for the discovery of new treatments for glaucoma and other vision-threatening eye diseases.

DESCRIPTION (provided by applicant): This proposal addresses the idea that activation of sirtuin-1 (Sirt1), a deacetylase that can regulate gene expression, will increase retinal ganglion cell (RGC) survival and visual system function in glaucoma. Activation of Sirt1 is essential to caloric restriction-induced reduction of age-related morbidity and increase of lifespan. This enzyme exerts its effects on metabolism, mitochondrial function, and other cell functions largely by deacetylation of histones and other proteins. Caloric restriction-induced protection against the normal age-related loss of retinal ganglion cells by Sirt1 activation suggests that the sirtuins are highly relevant to glaucoma. Growing evidence indicates that many, though not all, of the salutary benefits of caloric restriction can be induced by feeding with resveratrol, a sirtuin activator and natural compound, that is enriched in certain foods including red wine and peanuts. Our overall hypothesis is that increased Sirt1 activation will protect against glaucomatous damage in the retina, optic nerve, and brain of mouse models of glaucoma. Our general approach will be to investigate the effect of increasing Sirt1 activation by caloric restriction or by dietary resveratrol supplementation and to identify the role of Sirt1 by the use of transgenic mice lacking SIRT1 expression in their RGCs. Specific Aims will be: 1) to determine whether Sirt1 activation will reduce RGC loss of Thy-1 gene expression and RGC death following optic nerve injury;2) to determine the biological basis of Sirt1-mediated RGC protection by evaluating retinal lipid peroxidation, mitochondrial integrity and function, and acetylation of proteins (including histones and transcription factors) critically involved in gene regulation;and 3) to determine whether Sirt1 activation preserves the structural integrity and function of the central visual system pathway. PUBLIC HEALTH RELEVANCE: The present proposal evaluates the hypothesis that increased activation of sirtuin-1 protects against glaucomatous damage in the retina, optic nerve, and brain. The data generated will significantly enhance our basic understanding of sirtuin function in glaucoma, and assess whether targeting sirtuin-1 can lead to a novel glaucoma treatment.

PROJECT SUMMARY Glaucoma results in vision loss due to damage of the optic nerve that is irreversible if undetected or untreated. The most common form of glaucoma is primary open angle glaucoma (POAG). While glaucoma affects all races, persons of African descent are disproportionately affected; studies show African Americans (AAs) are about four to five times more likely than Caucasian Americans to develop the disease. Glaucoma is the leading cause of irreversible blindness in Americans of African descent, and the second leading cause in all Americans. The lack of understanding about the etiology of POAG impedes our ability to identify and treat it early in its development. Evidence of genetic contribution in the pathogenesis of POAG is well established. Since POAG tends to run in families, it is critical to identify the genetic basis of the disease in order to develop effective therapies for early intervention. While genome wide association studies (GWAS) for glaucoma have been completed for Caucasian populations, evidence from other studies suggests that a GWAS of glaucoma specific genes to the African- American population will yield unique and important findings for both this population and for glaucoma in general. A better understanding of the relationship among the stage of disease, the rate of change, ancestry, and other important risk factors being tracked in the ongoing African Descent and Glaucoma Study (ADAGES) will allow us to evaluate the relationship between genetics, visual loss and structural damage in this high-risk cohort. The scientific plan for this new study focuses on glaucoma in ~2000 African-Americans by detailed phenotyping of new subjects, acquisition of samples from both new and established previously phenotyped study subjects for a repository, establishment of a data coordinating center, and genome wide association studies. The recruitment, enrollment, and phenotyping of both established and new subjects occurs at four clinical centers, University of California (UCSD), New York Eye and Ear Infirmary New York University of Medicine, a private practice in the Atlanta area, and the University of Alabama at Birmingham. UCSD is also the location for the Data Coordinating Center and the Repository with Robert N. Weinreb as Principal Investigator. CSMC will do the genotyping with a GWAS panel of ~2.5 million single nucleotide polymorphisms (SNPs) plus an exome set of ~300,000 SNPs using the Illumina Omni2.5 plus exome platform under the direction of Jerome Rotter and Kent Taylor. CSMC will also provide GWAS and exome data for 3435 controls. Comparison and confirmation of the GWAS and exome SNPs and data associated with glaucoma will be compared and confirmed with the University of Pennsylvania's similar glaucoma study.

? DESCRIPTION (provided by applicant): The overall objective of the NEI Mentored Clinical Scientist Development Program Award at UCSD is to develop outstanding clinician scientists to successfully compete at the national level for NIH grants and emerge as leaders within academic Ophthalmology. Through this institutional career development award, we will mentor clinician scientists toward independent academic appointments in ophthalmology. Since 1991, the University of California San Diego Shiley Eye Center has excelled in mentoring the career development of academic ophthalmologists in all fields, particularly in the field of glaucoma. Many of these individuals now lead successful research and clinical careers around the world; they include Department Chairs, Division Chiefs and impactful academicians. We will amplify these strengths through a structured program of mentoring, research and education to identify and mentor the next generation of academic clinician scientists, expand our emphasis to career development across all subspecialty areas of ophthalmology. With an emphasis on recruiting strong applications from women and minority applicants, Scholars will be selected each year after their completion of a post-residency clinical fellowship in ophthalmology. The Scholar will undertake these activities in the area of basic and/or clinical sciences appropriate to their scientific focus, and by partnering with active clinician scientist mentors to facilitate career development. Each Scholar will be trained in clinically relevant research, statistics, scientific writing, grant preparation, ethics, leadership and the responsible conduct of research. Additional aspects of the curriculum of each Scholar will be individualized by the Program Director (PI), Research Director, Education Director, Executive Committee, Mentoring Group, and lead mentor, based upon the Scholar's selected primary research track and his/her skills, background and career goals. Each Scholar will create an original research program and through this Award develop an independent academic career. We propose six primary research tracks: (1) Visual Neuroscience, (2) Genomics and Proteomics, (3) Bioengineering, (4) Stem cell biology, (5) Computational Ophthalmology (and Telemedicine), and (6) Clinical (human subjects) Research.

Project Summary Optical coherence tomography angiography (OCTA) has been introduced recently as a non- invasive, 3-dimensional imaging method to visualize and quantify microvasculature throughout the retina. The proposed study evaluates the clinical utility of OCTA measurements compared to standard structural measurements of the optic nerve head (ONH), retinal nerve fiber layer (RNFL) and macula measured using the current clinical imaging standard, spectral domain optical coherence tomography (SDOCT). Our research, and that of others, has shown that superficial retinal vessel density (proportion of measured area composed of blood vessels) in the ONH region and macula is less dense in primary open angle glaucoma (POAG) eyes than in healthy eyes. Moreover, diagnostic accuracy is improved with increasing disease severity. Research from our laboratory suggests that the diagnostic accuracy of vessel density is similar to that of SDOCT-measured RNFL thickness, and that vessel density is reduced in retinal regions associated with localized visual field (VF) defects. These cross-sectional results strongly suggest that OCTA measurements reflect damage to tissues relevant to the pathophysiology of POAG. In a longitudinal study, moreover, the mean rate of change in macula vessel density is significantly faster in POAG eyes than in glaucoma suspect or healthy eyes. The current study provides a unique opportunity to extend for up to 8 years longitudinal OCTA data that has already been collected from 250 eyes over 2 years, and to investigate vessel density change over time in glaucoma suspect and POAG eyes, as well as to compare it to other imaging modalities and glaucoma-related visual field change. The aims of the study are 1) to assess the longitudinal temporal and topographic relationship in glaucoma between loss of superficial retinal vessel density, loss of RNFL thickness, loss of neuroretinal rim width, and loss of ganglion cell complex (GCC) thickness in glaucoma suspect and glaucoma eyes of varying disease severity, and 2) to assess the longitudinal temporal and topographic relationship between superficial loss of macula and ONH retinal vessel density, loss of RNFL thickness , loss of neuroretinal rim width, and loss GCC thickness with the development and progression of central and peripheral VF defects in glaucoma. Comparison of the time course of changes in vessel density relative to changes in RNFL thickness, neuroretinal rim width, ganglion cell layer thickness and visual function will provide information about the pathophysiology of glaucoma that can improve early detection and accurate monitoring of advanced disease compared to currently available and widely accepted methods. Enhanced diagnosis and detection of progression should lead to earlier and more effective treatment, thus reducing the rate of disease worsening and preventing loss of vision-related quality of life (including blindness).

PROJECT SUMMARY/ABSTRACT Glaucoma affects more than 70 million people worldwide and is the world's leading cause of irreversible blindness. The only current method to delay its development and progression is by lowering intraocular pressure (IOP), achieved with topical administration of eyedrops. Adherence rates for glaucoma eyedrop administration are poor, in many cases below 50%, resulting in disease progression, eventual blindness, and a more than 2-fold increase in healthcare costs. African Americans and Latinos carry a significantly higher glaucoma burden compared with Caucasians. Minorities have additionally been found to have disproportionately lower rates of medication adherence. Previously studied interventions aimed at improving glaucoma adherence had key limitations that particularly affect minorities, including unreliable self-reported measures of adherence, lack of consideration of individual circumstances influencing glaucoma medication management, and developing/testing interventions in predominantly Caucasian populations. Health information technology has experienced rapid advancement in the last decade with the electronic health record (EHR), the proliferation of accessory mobile health technologies, and the advancement of artificial intelligence. Although their integration holds great promise to enable screening tools for diagnosis and risk prediction, successful integration to aid minority populations in real-world settings depends on: understanding how the collected information relates to the patient's other (e.g. clinical) data and the patient's socio-cultural context; seamless information exchange and interoperability with the EHR, the central portal of healthcare delivery; and integration of algorithmic findings into workflows involving both providers and patients to deliver information and/or recommendations in a simple, actionable manner. Glaucoma is a complex chronic disease, spanning decades of patients' lives and requiring ongoing monitoring and evaluation, thus making it an ideal application for the use of health IT to reduce racial disparities. In this proposal, we aim to accomplish this by: demonstrating the effectiveness of a flexible electronic eyedrop sensor to generate granular digital signatures of an individual's adherence and contextualizing this data in a socio- cultural context with patient interviews (Aim 1), combining adherence data with EHR variables to construct machine learning models to predict IOP control and enhance clinical risk stratification (Aim 2), and prototyping a dynamic dashboard for intervention coordination (Aim 3). Altogether, success of this innovative, comprehensive, culturally-tailored, and scalable health IT framework will improve medication adherence and slow disease progression among minorities, therefore narrowing this important racial health disparity.