FUNDING PERIOD: APRIL 1, 2008 - MARCH 31, 2010

Justine R. Smith, M.D., Ph.D.
Oregon Health and Science University
Portland, OR
Novel Molecular Regulators of Choroidal Neovascularization in Age-Related Macular Degeneration
$100,000

Age-related macular degeneration or AMD - the leading cause of blindness in adults aged over 60 years - is characterized by damage to the portion of the retina responsible for reading vision. One key player in the development of AMD is the endothelial cell. Endothelial cells line the blood vessels of the body. In the most severe form of AMD, endothelial cells grow from the tissue adjacent to the retina, which is known as the choroid, into the retina. The new blood vessels formed by these choroidal endothelial cells leak blood cells and proteins into the retina. Recently it has been realized that endothelial cells in different parts of the body differ in terms of the molecules they contain. Work from my laboratory has showed that choroidal endothelial cells have a unique molecular composition. This observation implies that it should be possible to develop drugs against molecules that are uniquely expressed in choroidal endothelial cells for treating severe AMD. In this project, we will study three molecules that are produced at high levels by choroidal endothelial cells. We will test the ability of these molecules to control the formation of blood vessels by choroidal endothelial cells in a culture dish. My research group has developed a technique for isolation of endothelial cells of the choroid from human eyes that are supplied to us by the Lions Eye Bank of Oregon. We will also examine the expression of these molecules in eyes donated by patients with AMD to the Casey Eye Institute at death. Our studies should establish whether manipulating the levels of these molecules could be used therapeutically in patients with severe AMD.

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Dequan Chen, Ph.D.
Mayo Clinic
Rochester, MN
The gene structure of ARMS2/LOC387715 and its expression in human eyes
$100,000

Chromosomal ARMS2 (LOC387715)/HTRA1 locus in 10q26.13 region has been consistently associated with age-related macular degeneration (AMD) in different populations across the world. Previously some researchers proposed that the AMD causative gene of 10q26.13 was HTRA1 rather than ARMS2. However, recent experimental data strongly supported that it's the upstream ARMS2 rather than the downstream HTRA1 that is the causative one for AMD. In NCBI database, ARMS2 is a gene with a structure of exon1-intron-exon2 and the predicted mRNA is composed of the 2 exons and encodes a predicted protein of 107 amino acids (aa) or 11.4 kDa. Our experiments using anti-ARMS2 recombinant protein antibodies, RT-PCR and second generation 5' and 3' RACE analysis (rapid amplification of cDNA ends), failed to support the above gene structure and expression paradigm: (a) multiple native cellular and eye tissue protein bands with molecular weights bigger than 12 kDa were immunoreactive with purified rabbit antibodies against our bacterial expressed recombinant ARMS2 protein which has the same aa sequence as that of the predicted ARMS2, and (b) multiple cDNA bands were amplified by both nested 5' RLM-RACE and 3' RLM-RACE, and the band sizes are much bigger than the ones expected from the predicted ARMS2 mRNA. We believe that the gene structure and the transcription profiles of ARMS2 in nature are different from what current NCBI database has predicted, and that expression changes of ARMS2 coupled with its genetic variation (rs10490924) may be a mechanism for human AMD development. In this proposal, we will determine all the possible but unknown native exons of ARMS2; determine all the full-length transcribed mRNA(s) and the gene structure of ARMS2; and determine whether there are differences of ARMS2 expression in human eye tissues between control and AMD donors and between different rs10490924 genotypes. The results obtained advance our knowledge regarding ARMS2 native gene structure, transcription profiles and expression changes between control and AMD or different genotypes. The results will also form a good basis for our future determination of the normal function(s) of ARMS2 and the pathobiological mechanisms of chromosomal ARMS2/HTRA1 locus in AMD development.

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Derek Van Der Kooy, Ph.D.
University of Toronto
Toronto, Ontario
Canada
Retinal Stem Cells for Transplantation in Macular Degeneration
$100,000

Normal vision relies on the function of cells, called cone photoreceptors and the retinal pigment epithelial cells.  In patients suffering from macular degeneration these cells die.  Current treatments are effective in slowing down macular degeneration, but unfortunately, they do not reverse the degeneration that has already occurred.  To restore normal vision, these lost cells will need to be replaced. 
Stem cell therapy offers the promise of an inexhaustible supply of retinal cells that can be used for transplant into eyes affected by macular degeneration.  Our lab was the first to describe the isolation of retinal stem cells from adult mouse and adult human eyes.   We were also able to show that these cells can generate cone photoreceptors and retinal pigment epithelial cells.  When these retinal stem cells are transplanted into mouse eyes, they are able to integrate into the host retina and develop correctly.

We propose to isolate mouse adult retinal stem cells and enrich for cone photoreceptors by manipulating specific cellular pathways as well as with a cell sorting technique.  Finally, we will test the ability of transplanted adult retinal stem cells to restore vision in mice that lack functioning cone photoreceptors. 

In the future it should be possible to isolate a few retinal stem cells from a patient's eye, expand them in culture and then use them to replace cells that are lost due to macular degeneration.  One advantage of our approach is that the cells to be transplanted into a patient's eye could be derived from that same patient.  Thus we would be getting around the problem of tissue rejection which occurs when transplanted cells are derived from a different individual.  Furthermore, these retinal stem cells are obtained from adult eyes, so we completely avoid the use of human embryos or embryonic stem cell lines.  It is our hope that this approach will lead to a cure for macular degeneration.

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Sally Temple, Ph.D.
Regenerative Research Foundation
Rensselaer, NY
The role of Sox2 in AMD
$100,000

I have been involved in stem cell biology for nervous system diseases for nearly two decades.  Five years ago, my mother was diagnosed with Age-related Macular Degeneration (AMD), a degenerative disease of the retina. This personal experience has committed me to apply my skills in stem cell and molecular biology to work on AMD, which is so prevalent amongst our growing elderly population.
 
We have discovered that a gene critical for producing the normal eye is actually turned on abnormally in the retina of AMD patients. This gene, named Sox2, has the property of controlling other genes, including one that is already associated with AMD -- a gene called alphaB-crystallin. We suggest that the abnormal expression of Sox2 leads to abnormal expression of other genes such as alphaB-crystallin, and that this sequence contributes to AMD. Interestingly, we have found that the form of the Sox2 gene expressed in AMD is an unusual variant. This might be an excellent target to single out for AMD treatment.

The exploration of progenitor genes in AMD is a unique approach. Our findings of Sox2 expression and the new Sox2 variant are novel and likely to be important. If we can identify the components of the molecular pathway operating in AMD, then we will have a deeper understanding of the disease mechanism, and new avenues to pursue for therapeutics.

We propose to conduct the following specific aims: we will determine whether Sox2 is expressed in the retina of patients with an AMD diagnosis, and whether that is the Sox2 variant we have described in our early, preliminary studies. We will also turn on and off Sox2 gene expression in human retinal pigment epithelial cells growing in tissue culture, and test whether this increases and decreases alphaB-crystallin protein, respectively. If Sox2 does indeed emerge through our studies as an AMD associated gene, then we will be able in the future to trace this molecular pathway to determine earlier steps in the disease pathway. This will contribute to our understanding of the progression of AMD, and help us identify ways to slow or even halt this devastating degenerative disease.

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Jeffrey H. Stern, M.D., Ph.D.
Regenerative Research Foundation
Rensselaer, NY
alphaB-crystallin and the Amyloid-beta mediated RPE stress response in Age related Macular Degeneration
$100,000

As a young scientist involved in vision research, I was inspired to become an MD in order to treat retinal disease. Now as a researcher and clinician, I am actively studying age-related macular degeneration (AMD), which is a highly prevalent disease and the major cause of blindness in the elderly in the US. My objective is to uncover the process underlying AMD so that we can combat this disease more successfully.
Other researchers have discovered that a protein present in the Alzheimer’s disease brain is also present in the back of the eye (the retina) of patients with AMD. Our research has shown a second protein, named alphaB-crystallin, is also present. These two proteins can bind together to produce a compound that can be toxic to nervous system cells and promote the growth of blood vessels; both these processes could stimulate AMD progression. Therefore it is very important that we examine whether these proteins do indeed interact in AMD and whether this interaction has the detrimental effect we anticipate.
 
This project is unique because to our knowledge no one else is studying the interaction of these two proteins in the context of AMD. In addition, we will be using cutting edge tools to examine whether targeting these proteins will help increase retinal survival and improve function. This proposed study could reveal new ways to combat the progression of AMD.

Our aims in this study are to find out if these two proteins are made by retinal cells in AMD patients and if they bind together to make a new compound. We will find out whether altering the levels of each of these proteins will improve the health of retinal cells in a culture dish. Finally, we will test a new potential drug for AMD that lowers the Alzheimer’s disease protein to see if it also improves the health of human retinal cells in a culture dish.  If these culture studies are successful, they could pave the way towards clinical trials in the future.

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Neal S. Peachey, Ph.D.
The Cleveland Clinic Foundation
Cleveland, OH
The Role of Complement Regulation in Maintaining Outer Retinal Integrity
$100,000

Recent studies have indicated that patients with certain genetic features are more likely to develop AMD. These important advances suggest that we may be able to arrest the disease process at an early stage, instead of focusing on symptoms encountered in late disease stages.  Before this goal can be realized, however, it will be important to place these genetic findings into context.  Several of the genes implicated in AMD are known to be involved in the complement pathways of the immune system.  We will use mouse models that lack key members of these complement pathways, which are predicted to predispose these mice to developing more severe retinal abnormalities.  In Aim 1, we will compare the severity of outer retinal defects encountered in a mouse model of AMD due to the absence of the MCP1 (monocyte chemoattractant protein-1) gene with those found in mice also lacking one of two proteins involved in complement regulation.  Because the double mutant mice are expected to have higher rates of complement activation, we will be able to test the hypothesis that complement regulation is important for maintaining outer retinal integrity.  In Aim 2, we will study the same mouse models after using high light exposure to increase the activation the complement pathways.  In this aim, we will be able to test the hypothesis that biochemicals normally generated in the outer retina by the interaction of photoreceptor proteins, light and oxygen provide an important trigger to develop outer retinal abnormalities.  If these hypotheses are supported, we will expand our studies to include additional mouse models that involve key steps of complement activation.  Negative results, where similar results are obtained in these different mouse models, would indicate that some other aspect(s) of the complement pathway plays more important role for initial development of outer retinal abnormalities.

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Jonathan L Haines, Ph.D.
Vanderbilt University Medical Center
Nashville, TN
Genetic examination of AMD in the Midwestern Amish
$100,000

To better treat or even prevent age-related macular degeneration (AMD), we must first understand all the components of what cause this disease. Our research focuses on finding all the genetic (an individual's DNA makeup) components of this disease.  Recently a lot of progress has been made in this area of research, but it is far from complete. Therefore we are proposing a novel approach within AMD research by examining a population (the Amish) who because of their large inter-related family and community structures are more likely to share environmental exposures and underlying genetic structure than other average Americans. The nature of this population is a powerful tool to help us identify genes for involvement in macular degeneration. Our approach complements other work within this field, but uniquely gives us the opportunity to reduce the amount of normal differences that studies within the general population are accustomed to having. This should make it easier to find the remaining genes involved in AMD. Our first aim is to examine our sample population for the already known genetic and environmental influences on AMD already identified by our group and other previous research. Accounting for what is known is important to understanding new components of the disease. Once this is accomplished, we can begin in earnest the search for novel disease factors.  It is our goal to identify these novel factors to better our understanding and to provide other researchers within the scientific community the information needed to develop better treatments and preventative measures.

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Jinbo Liu, M.D.
Case Western Reserve University
Cleveland, OH
Identification of autoreactive T cells in AMD Patients
$100,000

Because the back of the eye where light is detected is a site where light induced modifications of occular components can occur as individuals age, such modifications accumulate.  Becase our immune system is designed to deistinguish any thing that differs from our normal body components, these modifications can cause the immune system to attack the back of the eye and compromise vision.  The experiments in this proposal will adress the mechanisms underlying this immune attack and the information could lead to treatment approaches which are not currently available.

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Dwight Stambolian, M.D., Ph.D.
University of Pennsylvania
Philadelphia, PA
Potential Therapy of Age-Related Macular Degeneration with Small Molecules
$100,000

Age-related macular degeneration (AMD) has no current treatment to return the vision deficit back to normal.  In the case of wet AMD, treatment is directed at the terminal stages making it impossible to return vision to 20/20.  For the dry type, the only treatment is a special vitamin preparation that is only effective in slowing the progression of AMD in some people.  We are looking for a small molecule which can penetrate the eye and diminish the eye inflammation that occurs in AMD.  If successful, this small molecule can be administered at the earlier stages of AMD to halt progression to the later stages and eventual blindness.  In order to identify such a molecule, we will screen a small molecule library and confirm its efficacy in a test tube before moving onto animal models.

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Luba Robman, M.D., Ph.D.
Centre for Eye Research Australia
Melbourne, Australia
Genetic risks for AMD in Australians of  Northern and Southern European origin
$100,000

This study will investigate whether our underlying risk of age-related macular degeneration (AMD), which is pre-determined by our genes, could be modified by changes in diet. This AHAF specific proposed project is to genotype AMD cases and controls from the examined largest Australian cohort of participants of Anglo-Celtic and Mediterranean origin (n=22,386) in order to define their genetic risk for AMD. We will identify the AMD genes in elderly from this cohort (about 8,000 people), and correlate genetic status of the participants with their dietary history and condition of the retina.  There are many strong characteristics of the proposed study: it has a uniquely large sample of elderly in their 70's and 80's, this is the age the most affected by AMD; it has participants with the contrast phenotypes and dietary patterns that are typical for Mediterranean area (Greek and Italian) and United Kingdom, and it has AMD status already defined and DNA prepared for genetic analysis. The differential dietary recommendations would be an important public health initiative. It would be a first step towards personalized medical care, based upon knowing one's susceptibility risk to a disease using genetic and other risk factor information.

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Roxana A. Radu, M.D.
University of California, Los Angeles
Los Angeles, CA
Analysis of A2E-degradation and Complement Activation in Two New Animals Models for Age-related Macular Degeneration
$100,000

Age-related Macular Degeneration (AMD) is the leading cause of severe vision loss in people over 60 years of age. In AMD, the central part of the retina (macula) deteriorates for unknown causes. The macula contains the highest density of light sensitive cells, such as rods and cones, in the retina.  Every time we 'look at' something, we are using our maculas.  Individuals with macular degeneration lose the ability to perform basic tasks such reading, driving or recognizing faces. Among the risk factors for AMD are advancing age, cigarette smoking and genetic predisposition. Other factors, including inflammation, oxidative stress and phototoxicity, are also important to the pathogenesis of AMD.  A pathologic feature of AMD is accumulation of toxic lipofuscin pigments, derived from vitamin A, in cells of the retinal pigment epithelium (RPE). This layer of cells adjacent to the photoreceptors is critical for the normal function and survival of rods and cones. Lipofuscin is a natural byproduct of photoreceptor turnover, and slowly accumulates in everyone's RPE.  However, patients with AMD may have biochemical defects leading to accelerated accumulation or delayed clearance of lipofuscin pigments.  When the burden of these pigments gets high, the RPE becomes sick, and loses the ability to support the photoreceptors. In time, this leads to photoreceptor death and subsequent blindness.

Our understanding of how the identified predisposing factors cause AMD is hampered by the scarcity of suitable animal models to study. Recent observations by several research groups showed that the gene for complement factor H (CFH), a component of the innate immune system, is a strong susceptibility locus for AMD. However, the mechanism by which dysfunction of CFH causes AMD is not known.  The current proposal will test the hypothesis that abnormal metabolism of vitamin A and its derivatives could lead to overt activation of the complement system. We will test this hypothesis using the abca4 null mutant mouse, a well established animal model for lipofuscin-based maculopathies developed by our group. Further, we will investigate the biochemical and molecular mechanisms used by the RPE to deal with abnormal build-up of vitamin A-based toxic compounds such as A2E. Toward this goal, we will generate two new mouse models for AMD. These mouse genetic models are expected to accelerate the onset and increase the severity of retinal pathology compared to existing mouse models.  Studying these mice will help us to understand the relationship between lipofuscin accumulation, complement activation, and retinal degeneration in AMD. Finally, these mouse models may be valuable tools for us and other investigators to develop new treatments for AMD.

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Zhenglin Yang, M.D.
John Moran Eye Centera
Salt Lake City, UT
Functional Study of HTRA1 and Age-related Macular Degeneration
$100,000

AMD represents a major public health burden with economical and social impacts.  Identification and functional studies of HTRA1 that have substantial impact on the genetic risk of the AMD may define key molecular pathways involved in its pathogenesis. The experiments we propose will yield novel mechanistic insights  relevant to the pathogenesis of both early and late AMD.  This may lead to therapies directed at the underlying cause and pre-symptomatic diagnostics to allow for earlier intervention with those therapies.

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FUNDING PERIOD: APRIL 1, 2007 – MARCH 31, 2009

Jayakrishna Ambati, M.D.
University of Kentucky
Lexington, KY
Project: The Role of SPARC in Macular Propensity for CNV
$100,000

Age-related macular degeneration is as common as cancer in the United States. The principal cause of vision loss AMD is choroidal neovascularization (CNV), the growth of abnormal blood vessels (angiogenesis) beneath the retina. CNV is devastating because it occurs almost always in the macula, the central retina that provides fine vision. The reason for this macular propensity is unknown. We have identified a protein called SPARC that is present almost exclusively in the macula and increased in AMD. We have also shown that the presence of SPARC is necessary for VEGF-A, a potent molecule that promotes angiogenesis, to perform this function. We have also shown that the presence or absence of SPARC determines whether VEGF-A acts via VEGF receptor-1, which suppresses angiogenesis, or VEGF receptor-2, which promotes angiogenesis. We will determine the levels of SPARC, VEGF-A and the activity of VEGF receptors in the macula and peripheral retina in patients with or without AMD, and whether blocking SPARC reduces CNV in animal models. We will determine whether the differential distribution of SPARC underlies the macular propensity of CNV and whether modulating SPARC will reduce the attractiveness of the macula for CNV development, thereby permitting improved vision in patients with AMD.

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Chi-Chao Chan, M.D.
Institution: National Eye Institute
Bethesda, MD
Project: Chaperone and Age-Related Macular Degeneration Model
$100,000

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the USA and developed countries. Currently there is no treatment for the disease. AMD results from damage to the retina, a neuronal tissue in the eye that is responsible for vision. We have established a mouse strain, which shows many AMD features. We hypothesize that these features may result from accumulation of certain toxic proteins in retinal cells. We propose to study this mouse model and identify the specific toxic proteins. We plan to find therapies to eliminate these toxic proteins and treat AMD.

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Albert O. Edwards, M.D., Ph.D.
Mayo Clinic
Dallas, TX
Project: Genome-wide scan of nsSNPs
$100,000

The discovery of genetic variation increasing the risk of macular degeneration has revolutionized our understanding of how patients lose vision from this condition. These discoveries have relied upon the study of changes in our DNA sequence in large groups of persons with and without macular degeneration. The genetic risks discovered to date change the sequence of proteins in our bodies. We believe that additional variation in protein sequence will be found to alter the risk of developing macular degeneration or its complications that lead to blindness. We propose to systematically screen all known common variation changing the sequence of proteins to determine if they alter the risk of developing AMD. These experiments are expected to discover new pathways for developing AMD and improve our understanding of how the disease develops.

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Ali Hafezi-Moghadam, M.D., Ph.D.
Harvard Medical School
Boston, MA
Project: Role of a Novel VEGF Effector on CNV in a Model of AMD Recommended:
$100,000

Age-related macular degeneration (AMD) is the leading cause of vision loss in people over age 55 in the US.  This disease causes a loss of central vision, making people with AMD unable to read, drive, or recognize faces.  In the wet form of AMD, abnormal, leaky blood vessels develop under the retina, causing damage to the surrounding tissue.  Important new therapeutic strategies target VEGF, a molecule that plays a role in producing the leakiness of the blood vessels in AMD.  Since it is not completely understood how VEGF achieves these effects, the objective of this proposal is to investigate the molecular links between VEGF and retinal vascular leakage.  We identified a molecule released by immune cells that is a likely culprit in the damage to retinal blood vessels, causing their leakiness.  We hypothesize that VEGF is largely producing its destructive effects in AMD directly through this molecule.  Our preliminary data support this hypothesis, as we find that blocking this molecule greatly reduces the size of vascular lesions formed in a laser-induced model of AMD.  We propose to further investigate these molecular links in tissues from human patients.  Our approach may provide a more effective and specific target for treatment, which would mean a substantial benefit to AMD patients.

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Josephine Hoh, Ph.D.
Yale University
New Haven, CT
Project: AMD beyond Complement and Serine Protease Pathways
$100,000

Age-related macular degeneration (AMD) has a complex etiology and is broadly classified as either dry or wet. The dry form is more common, accounting for approximately 90% of patients, and does not typically result in blindness. About 10% of patients have the wet form, in which central vision will be destroyed. We have identified two major genetic factors predisposing some individuals to the more aggressive wet form of AMD and others to the slowly progressing dry type. Together with the new risk factors we will discover, it will be possible to detect who is at increased risk and allow those affected to take preventive measures.

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Xuri Li, Ph.D.
NIH/NEI
Bethesda, MD
Project: PDGF - C/D in Choroidal Neovascularization and AMD Therapy
$100,000

Reagents that can suppress the growth of undesired blood vessels have shown beneficial effects for some AMD patients. However, none of them can halt or reverse the course of the disease. New anti-angiogenic reagents are therefore still needed. The platelet-derived growth factor C (PDGF-C) and PDGF-D are two promising candidate molecules to be targeted. Our previous and current work has shown that both PDGF-C and PDGF-D are potent angiogenic factors with potential important roles in choroidal neovascularization (CNV). This study is thus designed to  test the role of PDGF-C and PDGF-D in the development and growth of undesired new blood vessels in the choroids and retina. Multiple approaches including different CNV mouse models, PDGF-C deficient and transgenic mice, neutralizing antibodies and siRNA, etc, will be used to achieve our scientific goals. Results derived from this study investigating the angiogenic nature of PDGF-C and PDGF-D in CNV formation and progression, as well as the anti-angiogenic effects of PDGF-C/D antagonists alone and in combination with other angiogenesis inhibitors will provide not only new insights into the basic molecular and cellular mechanisms of CNV formation in neovascular AMD, but also possibilities of novel therapy for the treatment of AMD patients. 

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Andrew Lotery, M.D.
University of Southhampton
Southampton, UK
Project: Study of Complement and HLA Related AMD
$100,000

Age related macular degeneration (AMD) is the commonest cause of blindness in the western world. As current treatments are extremely limited, it is vital to improve understanding of this disease in order to develop better treatments. Happily, progress  is being made. Recent work on complement factor H (CFH) and by us on Human Leucocyte Antigens (HLA) have highlighted the importance of inflammation in the development of AMD. Our proposed research will use modern molecular genetic techniques to test whether 1) there are other, related complement genes which also cause AMD, 2) there is a microbial trigger activating the complement cascade in AMD and 3) investigate how HLA associations lead to AMD. If these experiments are successful, our understanding of the biology of complement and “HLA related age related macular degeneration” could be radically altered. This offers the best hope for tailoring current AMD management and developing novel treatments for this prevalent cause of blindness.

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Xiaoxi Qiao, M.D., Ph.D.
Indiana University
Indianapolis, IN
Project: TFPI-VLDLR Pathway in Retina Angiogenesis
$100,000

Age-related macular degeneration (AMD) is the most common cause of severe and irreversible vision loss in older adults in the US. The key lesion in the eye is abnormal vasal growth in the retina. Although several risk factors have been named, what molecule triggers the abnormal vasal growth is still not clear. A recent study reported that similar abnormal vasal growth was found in the eyes of a mutant mouse which lacks very low density lipid receptor (vldlr). The current proposal is to examine the role of vldlr as well as its known up and down stream molecules on the abnormal vasal growth in the eye. We will use this mutant mouse model and look for potential differences between the normal control and the mutant mice. We will also use retinal vascular cell culture to determine if vldlr and related molecules are the key pathway to stimulate abnormal vasal growth. Identify key molecules responsible for abnormal vasal growth will provide us new targets to develop better treatment strategy for AMD.  

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Dennis W. Schultz, Ph.D.
Oregon Health & Science University
Portland, OR
Project: Characterization of 10q26 AMD Protein
Recommended: $100,000

Age-related macular degeneration (AMD) is the most common disease leading to blindness in the United States. It is responsible for about half of all registered cases of blindness. Because our elderly population is growing, this will only become a bigger problem in the future. Furthermore, there are not as yet, any really good treatments available and early diagnosis is not yet possible. A region on chromosome 10 has been shown to be the most significant portion of our genome associated with AMD. However, the exact protein associated with AMD has not yet been identified. The preponderance of evidence, however, points to one of two proteins, LOC387715 and HtrA1. We hypothesize that the AMD gene on chromosome 10 is one of these two proteins. However, it is not possible to use genetic or statistical techniques to determine which protein is more likely to be associated with AMD. We will use human donor eyes to determine where these proteins are located and to determine which tissues in the eye express these proteins. We will also extract these proteins from human retinal tissue and cells to study their size and quantity. Through looking at where these proteins are expressed and located inside the back of the eye, where AMD occurs, we hope to obtain evidence that will determine which protein is associated with AMD. This will help us in the future to develop assays to determine which individuals are most likely to get AMD and to develop treatments that will better alleviate this devastating disorder.

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Robin L. Seitzman, Ph.D.
University of California, Los Angeles
Los Angeles, CA
Project: Single Nucleotide Polymorphisms and AMD in Older Women
$100,000

Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in persons 65 years of age and older in the developed world. The cause of AMD is not known; however, it is believed that a combination of genetic and environmental factors is responsible for the majority of cases. Therefore, studies involving genes relevant to eye tissues and function, and determining if their effects are modified by the presence of other AMD risk factors such as smoking. This may reveal important information regarding the cause of this important disease. Some of our preliminary research suggests that bone disease and AMD may have common risk factors. Therefore, genes relevant to bone disease and metabolism may be associated with AMD risk. Using data from the Study of Osteoporotic Fractures, the goals of our study are to determine whether variations in 15 genes related to bone metabolism and expressed in eye tissues are associated with incident AMD in women 75 years of age and older, and whether genetic effects may be modified by environmental risk factors for AMD such as smoking, nutritional factors, or reproductive hormone exposures.

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Allen Taylor, Ph.D.
Tufts University
Boston, MA
Project: Carbohydrate, Dietary Patterns and AMD rick in 2 Cohorts
$100,000

Age related macular degeneration (AMD) is due to damage to the most sensitive portion of the retina, the macula.  It is in this zone that light information is received and converted to signals that are then interpreted by the brain. Estimates vary for prevalence of AMD, but it appears that over 15% of the elderly are subject to some visual loss due to AMD. With the aging of our population, along with increasing diabetes and obesity, this proportion is sure to grow significantly.   It is the major cause of non remediable blindness. The personal, societal, and public health ramifications of such an epidemic are horrific. We obtained preliminary evidence that limiting dietary simple carbohydrates is associated with prolonged retina function and less indication of macular damage. Since at present there are no intervention trials which specifically test the hypothesis that limiting simple carbohydrates is associated with prolonged retina function, we seek to use data from existing large well executed ophthalmic/nutritional/epidemiologic studies to test the hypothesis.  had we set out to do this work from the beginning it would cost many millions of dollars. By using existing data sets, we can accomplish this mission with a fraction of the cost.  This grant will help us accomplish this objective.   

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Heping Xu, Ph.D.
University of Aberdeen
Foresterhill, Aberdeen, UK
Project: Retinal Pigment Epithelial Cells and Complement
$100,000

Age-related macular degeneration (AMD) is the largest cause of untreatable blindness in developed countries. How the disease is started is not known. Recent evidence suggests that patients who carry variants of certain gene (namely complement regulatory factor H (CFH) gene) are at higher risk of developing AMD, whereas another gene (namely complement factor B (CFB) gene) variation can reduce the risk of AMD. The products of these two genes are CFH and CFB proteins, which are both important in complement activation (a kind of inflammation). CFH/CFB proteins are normally produced in the liver and distributed in the blood stream. Patients with AMD usually have no other diseases related to CFH or CFB dysfunction apart from the eye. We speculate that these genes also are active in the eye with local protein productions, this may serve as a regulatory system to prevent unwanted inflammation. When these genes are not functioning properly as in some variants of the genes then AMD might develop due to damage from local inflammation. The project is to investigate how CFH/CFB protein production is controlled in the eye in normal and disease conditions. It will help us to identify factors that are responsible for AMD development, and develop new treatments.

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Caroline J. Zeiss, Ph.D.
Yale School of Medicine
New Haven, CT
Project: Interaction of diet and genotype in the pathogenesis of AMD
$100,000

Age-related macular degeneration is the predominant cause of vision loss in adults over 65. There is no single cause for this disease, and it results from complex interactions of genetic predisposition and environmental influences. Consequently, it is difficult to predict, and to treat. Lack of effective treatment stems from both the complexity of the disease, and lack of good animal models in which to study it. We have been able to obtain a mouse model that carries a mutation in an important macular degeneration gene  - Factor H. Using this mouse as the basis of our experiments, we will superimpose additional mutations (ApoE and CCr2) and environmental conditions (high fat diet). We expect that these interventions hasten onset of AMD in mice. We will also determine whether mice carrying a mutation in Tlr4, a gene that has recently been associated with AMD, develop AMD-like lesions after normal or high fat feeding. The intent of this is to develop a robust early onset mouse model of AMD for therapeutic testing. These experiments also provide insight into how diet interacts with predisposing genetic burden to create AMD pathology. Understanding these relationships will enhance the capacity of the clinician to council at-risk patients.

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FUNDING PERIOD: APRIL 1, 2006 – MARCH 31, 2008
GRANTS IN EXTENSION

Steve Abcouwer, Ph.D.
Pennsylvania State University College of Medicine
Hershey, PA
Project: Effect of Mutant Fibulin Expression on RPE Cell Function
$100,000

A single genetic mutation in the fibulin-3 gene causes inherited early-onset macular degenerative disease known as Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD). Similar mutations in other fibulins (5 and 6) are linked to age-related macular degeneration (AMD). These mutations cause the expression of abnormal forms of fibulin proteins. How these abnormal proteins contribute to macular degeneration is unknown. He provides a testable hypothesis of how expression of a mutant protein might cause retinal pigment epithelial (RPE) cell dysfunction leading to macular degeneration. Expression of fibulin-3 mutant protein lead to accumulation of this protein in an intracellular organelle called the endoplasmic reticulum (ER), causing stress to this organelle. The hypothesis that the expression of mutant fibulin proteins compromises the ability of RPE cells to perform one of their primary functions, swallowing and processing of outer segments that are shed from photoreceptor cells. Evidence suggests that the rate of outer segments shedding and the ability of the RPE to remove and process the outer segments are key factors in the progression of AMD. The proposed studies would expand the understanding of these vital RPE functions, and could point the way to AMD treatments that act by alleviating protein aggregation.

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Qiuyun Chen, Ph.D.
Cleveland Clinic Foundation 
Cleveland, OH
Project: Identification of Human Retinal Lutein Binding Proteins
$200,000

The long-term goal of this project is to study the molecular basis for therapeutic manipulation of lutein levels in the retina in order to treat age-related macular degeneration (AMD), the leading cause of incurable blindness in the western world.  Lutein is one of the few carotenoids that are selectively accumulated in the human macula.  Low concentrations of lutein in the retina have been found to correlate with a high risk of developing AMD.  However, the exact molecular mechanisms underlying the protective function of carotenoids are unresolved.  One reason this problem persists is the lack of knowledge of how and why lutein is selectively accumulated in the macula.  Dr. Chen’s hypothesis is that specific binding proteins facilitate the transport and retention of lutein in the retina.  To test this hypothesis, she will identify lutein binding proteins from human retina by searching for genes that when expressing will produce proteins that specifically bind lutein.  Once the gene(s) are identified, she will determine what type of proteins they are and whether their distribution in the retina correlates with the lutein distribution pattern. 

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Michael Gorin, M.D., Ph.D,
University of Pittsburgh
Pittsburgh, PA
Project: Linkage and Association Studies for Macular Degeneration
$150,000

Age-related macular degeneration (ARM) is a major cause of vision loss in the elderly. It is thought that smoking and diet may contribute to the risk of developing the condition but it is clear that heredity plays a major role. Variations in two genes, CFH and PLEKHA1/LOC387715, have been found to strongly contribute to the risk of developing ARM, but there are additional genes that probably influence a person’s chances of having this condition and how they will progress to vision loss. Dr. Gorin is investigating the genetic variations that contribute to ARM so that he can eventually understand the causes of this complex condition. He studies the genetic variations that are shared among ARM-affected individuals within families as well as compare the frequencies of genetic variations in ARM-affected individuals with those in unaffected persons who are matched in age, gender, and exposures. His long-term goals are to develop new preventive therapies that can slow or halt the development of this disease and to be able to provide these treatments to those who are at greatest risk before they experience vision-threatening changes.

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Jeffrey Gross, Ph.D.
University of Texas at Austin
Austin, TX
Project: A Genetic Model of RPE Dysfunction in ARMD
$150,000

In patients with the dry form of age-related macular degeneration (ARMD), a substance called drusen builds up in cells of the retinal pigment epithelium (RPE) – the pigmented layer at the back of the eye.  Drusen poisons RPE cells by inhibiting their ability to degrade protein and lipid components of photoreceptors – the light sensitive cells of the retina.  Normally, RPE cells help to maintain the survival of photoreceptors by continually removing their tips, which have accumulated cellular damage over time.  Dr. Gross will study the mechanisms that RPE cells utilize to facilitate photoreceptor degradation, focusing on a protein complex called the vacuolar ATPase that is necessary for degradative processes in other cell types.   Dr. Gross predicts that mutations in the vacuolar ATPase complex lead to ARMD in humans.  To test this prediction, Dr. Gross will analyze the effects of vacuolar ATPase mutations in zebrafish, an animal model system in which human diseases can be studied.  Zebrafish vacuolar ATPase mutants show severe ARMD-like pathologies in their eyes and are therefore an excellent animal model system in which ARMD progression can be further understood. 

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John O’Brien, Ph.D.
The University of Texas Health Science Center at Houston
Houston, TX
Project: Physiological Uncoupling of Cone Gap Junctions
$200,000

Photoreceptors are joined to each other by gap junctions, channels that permit exchange of small molecules between adjacent cells.  During cell death, such as occurs in macular degeneration, death-promoting factors may pass through gap junctions to adjacent cells and trigger death in otherwise healthy adjacent photoreceptors.  This process can speed the progression of disease and hasten vision loss.  Treatments that close these gap junctions may delay the progression of disease.  This project will examine the physiological mechanisms that close cone-cone gap junctions.  By studying biochemical properties of the cone photoreceptor gap junction protein, connexin35, Dr. O’Brien can estimate the functional state of the gap junctions.  He will now use these studies to identify the signaling pathways that change the functional state of cone gap junctions.  This information will be used to develop pharmacological interventions to close cone gap junctions, reducing the exchange of small molecules between photoreceptors.  It is hoped that these studies will identify drugs that can be used to close cone gap junctions selectively.  Such drugs could provide a treatment strategy that is complementary to existing treatments for macular degeneration by reducing the rate at which the degeneration spreads.

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