Molecular And Immunopathology Of Experimental And Clinical Ocular Diseases

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We identify and topographically localize inflammatory, degenerative, and malignant cells, as well as their products in patient specimens and animal tissues. We analyze these cells and their products mainly by routine pathology, immunohistochemistry, and molecular pathology. The application of cutting-edge technology, such as microdissection combined with molecular techniques including PCR, RT-PCR, RQ-PCR and genotyping, allows us to provide more accurate pathological diagnosis (assessment) and pathogenesis of the disease. It also guides us in selecting the most appropriate treatment for patients. We learn a great deal about the mechanisms of different ocular diseases from various animal models. Using animal models, we can also assess the efficacy of different therapeutic agents for various ocular diseases. In FY2012, we accomplished the following in our research: 1. Molecular Pathology of Age-Related Macular Degeneration (AMD): AMD is the leading cause of vision loss among the elderly in the world. It is projected that AMD will afflict 3 million Americans over the age of 50 by the year 2020. The pathology of AMD is characterized by the accumulation of soft drusen, RPE and photoreceptor degeneration, geographic atrophy, and/or exudation with choroidal neovascularization. While several risk factors, including age, race, smoking, and diet have been linked to AMD, the etiology and pathogenesis of the disease remain largely unclear. Treatment options for geographic atrophy AMD are extremely limited and for neovascular AMD include intravitreal injections of anti-vascular endothelial growth factor (VEGF). Current knowledge demonstrates that AMD development is strongly influenced by genetic factors. We and other investigators have reported significant associations between AMD and single nucleotide polymorphisms in CFH, CX3CR1, and ARMS2/HtrA1 over the past years. Growing evidence also suggests that inflammatory and immunological elements (e.g., macrophages and microglia), apoptosis, cholesterol trafficking, angiogenesis (e.g., VEGF expression), and oxidative stress (e.g., the role of mitochondria) play an important role in AMD development. In FY2012, using microdissection and quantitative RT-PCR for the signature chemokine transcripts of M1 and M2 macrophages on macular tissues in autopsied AMD and normal eyes, we found advanced AMD macula had a higher M1 to M2 ratio compared to the normal controls. Macrophages in the two AMD-associated choroidal neovascular membranes of patients were polarized toward either M1 or M2 phenotypes. A pathological shift of macrophage polarization may have a potential role in AMD pathogenesis. With Dr. Zhang of UC San Diego, we reviewed the association between AMD and immunological or inflammatory genes, with particular focus on the genes related to innate immunity. Gene-gene and gene-environment interactions are found as significant covariates in AMD pathology. Studies of copy number variation, epigenetics and microRNA may provide further evidence for understanding molecular mechanisms in AMD. In collaboration with Drs. Thomas Ferguson of Washington University and Rachel Caspi of NEI/LI, we discovered and reported the rd8 mutation of Crb1 gene contamination in several vendor stocks of C57BL/6N mice and embryonic stem cells. This contamination could confound ocular induced mutant phenotypes, including our Ccl2/Cx3cr1 double deficient (DKO rd8) mice. However, our DKO rd8 mice showed focal RPE and photoreceptor degeneration as well as elevated ocular A2E levels, in addition to the prominent retinal dystrophic lesions seen in rd8 mice. We demonstrated that AAV-5 mediated sFLT01 gene therapy arrested retinal lesions of these mice. We also published anti-inflammatory recombinant TSG-6 stabilized the progression of focal retinal degeneration in DKO rd8 mice. We believe that DKO rd8 mice can still be used for appropriate experimental interventional studies by treating one eye and using the contralateral eye as the control. 2. Ocular Lymphoma: We published primary vitreoretinal lymphoma (PVRL) in The Oncologist, as a report from an international primary CNS lymphoma (PCNSL) collaborative group. PVRL commonly masquerades as posterior uveitis and has a unique tropism for the retina and CNS. It is often a fatal disease because of a close association with PCNSL. An accurate diagnosis is required to treat the patient. Due to the disease rarity, it requires international and multicenter, collaborative efforts. Priorities for future studies include: the application of transcriptional and proteomic profiling studies to ocular specimens to elucidate mechanisms, the development of new treatment approaches using animal model, clinical trials to evaluate and compare existing therapeutic options, and investigation of new ophthalmic imaging modalities, such as OCT and intraocular IL-10 levels to follow treatment responses. Treatment recommendations outside the context of clinical trials include chemotherapy and radiation therapies (local, systemic or combined), and are generally based on PVRL with or without CNS involvement. We examined molecular markers for the diagnosis of PVRL. We retrospectively evaluated 208 cases with a clinical diagnosis of masquerade syndrome at NEI from 1998 to 2010. The efficiencies for molecular analysis (microdissection and PCR), cytology, and cytokine measurement (the ratio of ocular IL-10 and IL-6 levels) are 99%, 89%, and 87%, respectively. The study indicates that IgH and TCR genes are reliable biomarkers for PVRL. In collaboration with Drs. Smith of Oregon Health & Science University and Coupland of Liverpool in UK, we reported an association between IL-10-1082 SNP and PVRL / PCNSL. More PVRL expressed one copy of the IL-10-1082 G→A SNP with the GA genotype compared to controls. The frequencies of the three genotypes (AA, AG, GG) significantly differed in PVRL versus controls and in PCNSL versus controls. In PVRLs, the vitreal IL-10/IL-6 ratio was higher in IL-10-1082 AG and IL-10-1082 AA patients, compared to IL-10-1082 GG patients. IL-10 mRNA expression was higher in IL-10-1082 AG and IL-10-1082 AA PCNSLs, compared to IL-10-1082 GG PCNSLs. The findings suggest that the IL-10-1082 A allele is a risk factor for higher IL-10 levels in PVRLs and PCNSLs. Higher IL-10 levels have been correlated with more aggressive disease in both PVRLs and PCNSLs, making this finding an important and potentially clinically significant observation. 3. New Pathology and Pathogenesis of Ocular Diseases: In collaboration with Dr. Singh of Cleveland Clinic, we reported the novel ophthalmic manifestations and pathology of paraneoplastic vitelliform retinopathy, an atypical melanoma-associated retinopathy. For the first time, the new findings illustrate direct focal damage of the inner nuclear and outer plexiform layers in the retina. In collaboration with Drs. Jager of Leiden University Medical Center in Netherlands and Honavar of L.V. Prasad Eye Institute in India, we reviewed the genetics of three most common intraocular tumors uveal melanoma, retinoblastoma and PVRL. Genetic studies on specific molecules and pathways could help to reveal more detailed features of intraocular tumors, which provide hints for identifying therapeutic targets. 4. Experimental Models for Various Ocular Diseases: In collaboration with Drs. Caspi, Egwuagu, Hejtmancik, and Nussenblatt of the NEI, Drs. Warren Leonard of NHLBI, Dr. Nicholas Restifo of NCI, Drs. Steven Holland and Philip Murphy of NIAID, and Dr. Charles Venditti of NHGRI, different models and mechanisms of ocular inflammation and genetic diseases have been studied and/or published. The data is further described in the annual reports of these senior investigators.