Age-related macular degeneration (AMD) is the most common cause of visual impairment of the elderly in the United States. We have previously identified a genetic variant in the chromosome region of HTRA1/ARMS2 was associated with major susceptibility to AMD. This disease genotype results in increased expression of high temperature requirement factor A1 (HTRA1). In our previous funding period, we systematically scanned all single nucleotide polymorphisms (SNPs) related to AMD and investigated their regulation of expression of HTRA1 and ARMS2. Our functional study reveals loss of HTRA1 leads to decreased retinal vascular development and significant down-regulation of vascular endothelial growth factor (VEGF) gene expression in HTRA1 knockout (htra1-/-) mice. Conversely, we show Increased expression of HTRA1 in RPE leads to Bruch's membrane pathology and elevated VEGF expression. We further revealed that the up-regulation of VEGF by HTRA1 is inversely correlated to the down-regulation of a member of the TGF-b family, the growth differentiation factor 6 (GDF6). The long term objectives of this proposal are to characterize the normal function of HTRA1, elucidate molecular mechanism by which HTRA1 contributes increased risk of AMD, and development potential therapies. Our central hypothesis is that HTRA1 contributes to AMD risk by regulation of vascular development, angiogenesis, TGF-b signaling, and extracellular matrix modeling. Guided by this hypothesis, we propose to conduct the following specific aims.
Specific Aim 1 : Generation of monoclonal antibodies to HTRA1 and investigating their therapeutic potential by tissue specific delivery into the RPE cells. Increased HTRA1 expression induced PCV and retinal pigment epithelium atrophy and photoreceptor degeneration. We will generate monoclonal antibodies specifically bind to HTRA1. The selected antibody strains with the high affinity to HTRA1 will be converted into a single chain format (scFv), which will be further """"""""evolved"""""""" to maximize binding specificity and affinity using a state-of-the-art novel protein in vitro evolution technology. We will use a gene therapy strategy to deliver such optimized HTRA1 antibodies into RPE and test its effect on inhibition in HTRA1 transgenic mice and a laser-CNV model.
Specific Aim 2 : To determine the role of TGF-b signal pathway in retinal development and pathology. We show that GDF6 significantly associated with AMD and demonstrated that the GDF6 AMD risk allele is associated with decreased expression of the GDF6 and increased expression of HTRA1. We also showed that TGF-b receptor 1 (TGF-b R1) is associated with AMD. We will generate conditional knockout mice which delete GDF6 and TGF-b R1 in either retina or RPE to examine the role of TGF-b signal pathway in retinal development and pathogenic changes, including ophthalmoscopy, histology, ERG, and expression profile, immunohistochemistry and choroidal neovascularization (CNV) formation.
AMD represents a major public health burden with economical and social impacts. Identification and functional studies of genes that have substantial impact on the risk of AMD may define key molecular pathways involved in its pathogenesis. The experiments we propose will yield novel mechanistic insight relevant to the pathogenesis of both early and late AMD, which may lead to therapies directed at the underlying cause and pre-symptomatic diagnostics to allow for earlier intervention with those therapies.
|Bhisitkul, Robert B; Desai, Shilpa J; Boyer, David S et al. (2016) Fellow Eye Comparisons for 7-Year Outcomes in Ranibizumab-Treated AMD Subjects from ANCHOR, MARINA, and HORIZON (SEVEN-UP Study). Ophthalmology 123:1269-77|
|Dedania, Vaidehi S; Grob, Seanna; Zhang, Kang et al. (2015) Pharmacogenomics of response to anti-VEGF therapy in exudative age-related macular degeneration. Retina 35:381-91|
|Huu, Viet Anh Nguyen; Luo, Jing; Zhu, Jie et al. (2015) Light-responsive nanoparticle depot to control release of a small molecule angiogenesis inhibitor in the posterior segment of the eye. J Control Release 200:71-7|
|Skowronska-Krawczyk, Dorota; Zhao, Ling; Zhu, Jie et al. (2015) P16INK4a Upregulation Mediated by SIX6 Defines Retinal Ganglion Cell Pathogenesis in Glaucoma. Mol Cell 59:931-40|
|Bhisitkul, Robert B; Mendes, Thais S; Rofagha, Soraya et al. (2015) Macular atrophy progression and 7-year vision outcomes in subjects from the ANCHOR, MARINA, and HORIZON studies: the SEVEN-UP study. Am J Ophthalmol 159:915-24.e2|
|Huang, Lv-Zhen; Li, Ying-Jie; Xie, Xue-Feng et al. (2015) Whole-exome sequencing implicates UBE3D in age-related macular degeneration in East Asian populations. Nat Commun 6:6687|
|Zhang, Li; Lim, Siok Lam; Du, Hongjun et al. (2012) High temperature requirement factor A1 (HTRA1) gene regulates angiogenesis through transforming growth factor-? family member growth differentiation factor 6. J Biol Chem 287:1520-6|
|Jones, Alex; Kumar, Sandeep; Zhang, Ning et al. (2011) Increased expression of multifunctional serine protease, HTRA1, in retinal pigment epithelium induces polypoidal choroidal vasculopathy in mice. Proc Natl Acad Sci U S A 108:14578-83|
|Yang, Zhenglin; Tong, Zongzhong; Chen, Yuhong et al. (2010) Genetic and functional dissection of HTRA1 and LOC387715 in age-related macular degeneration. PLoS Genet 6:e1000836|