Goals and Projects:
Age-Related Macular Degeneration
Age-related macular degeneration (AMD) is a complex disease and the leading cause of blindness in the elderly. Only a fraction of early/mid-stage AMD (also known as “dry” AMD) patients progress to advanced AMD, with neovascular or “wet” AMD being more prevalent than advanced dry AMD (also known as geographic atrophy). Currently clinicians cannot predict which patients will progress to advanced AMD.
Objective molecular markers for clinical prognostics could help prevent or slow with severe visual loss. Genomic markers alone are insufficient as many individuals carrying AMD risk genotypes never develop impaired vision. A long-term goal of the laboratory is the development of molecular technology for assessing AMD risk and monitoring AMD therapeutics.
Glaucoma is a multifactorial optic neuropathy and a leading cause of blindness worldwide. Glaucomatous damage to the visual system can occur at normal and elevated levels of intraocular pressure (IOP). While age and IOP are risk factors for the development and progression of the neuropathy, the identification of additional risk factors for IOP elevation and/or glaucomatous vision loss and their mechanism are a
high priority. A long-term goal of the laboratory is to establish a panel of blood-borne biomarkers for glaucomatous damage to the trabecular meshwork (TM) and visual system that will lead to improved clinical assessment of risk for visual loss from primary open angle glaucoma (POAG) and to improved glaucoma patient management.
Research and Innovations
Age-Related Macular Degeneration
Growing evidence supports AMD as an inflammatory disease involving oxidative stress. A host of oxidative protein modifications have been associated with AMD, including modifications derived from lipids such as carboxyethylpyrrole (CEP). Our laboratory participated in the discovery of elevated CEP in AMD ocular tissues and the initial demonstration that CEP stimulates neovascularization, independent of VEGF.
Others have shown that oxidative modifications derived from sugars such as carboxymethyllysine (CML) and pentosidine (and known as advanced glycation end products) are elevated in AMD ocular tissues; CML also stimulates neovascularization but through VEGF. Our biomarker analyses have confirmed the
AMD biomarker potential of plasma CEP adducts and CEP autoantibodies and demonstrated that CEP and genomic AMD biomarkers used together are more effective in assessing AMD risk than when used alone. Recently we have shown that light-induced CEP adducts in rat retina and plasma is significantly decreased by pretreatment with a serotonin 5-HT1A receptor agonist. These results support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics. We have also demonstrated the AMD biomarker potential of plasma protein CML and pentosidine. CML plus CEP or CEP plus pentosidine provided significantly improved discrimination accuracy between AMD and controls. Ongoing studies indicate that these markers in combination with genomic AMD markers and CEP significantly improve AMD risk prediction. Using LC MS/MS iTRAQ technology, we have identified proteomic changes with AMD progression in macular Bruch’s membrane/choroid and established a test set of 99 protein biomarker candidates. We are in the process of verifying AMD biomarker candidates in plasma using targeted quantitative proteomics.
Previous qualitative proteomic analyses in the laboratory demonstrated that cochlin, a protein associated with deafness, was abnormally expressed in human trabeculectomy tissues from POAG donors, suggesting it possibly could obstruct the aqueous humor (AH) outflow pathway and contribute
to elevated IOP. Our previous qualitative proteomic analyses found peptidyl arginine deiminase 2 (PAD2) in human POAG optic nerve. Subsequent analyses demonstrated elevated PAD2 in POAG optic nerve and revealed myelin basic protein as a major deiminated protein, suggesting deamination may contribute to demyelination and visual loss in POAG. We demonstrated the feasibility of global quantitative proteomic analysis of in vivo retinal ganglion cells purified from rats with laser induced unilateral experimental glaucoma. Transforming growth factor beta 2 (TGF2) is often elevated in AH and TM of POAG patients. Accordingly, we quantified TGF2-induced proteomic changes in vitro in TM cells using LC MS/MS iTRAQ technology and found that TGF2-treatment significantly altered the abundance of TM proteins, including 40 not previously associated with TGF2-signaling in the eye. Glucocorticoids (GCs) are common anti-inflammatory agents that can cause ocular hypertension and secondary glaucoma. Accordingly, we quantified Dexamethasone (Dex)-induced proteomic changes in vitro in TM cells and found Dex-treatment also significantly altered the abundance of TM proteins, including 38 not previously associated with GC-signaling in the eye. These results expand the repertoire of proteins known to participate in TGF2-signaling and GC-signaling, demonstrate similarities in proteomic changes induced by steroids and TGF2 and identify glaucoma biomarker candidates. Current global quantitative proteomic studies of AH are directed toward identify biomarker candidates for glaucomatous damage to human TM in patients with ocular hypertension and POAG. In addition we are pursuing global quantitative proteomic analysis of optic nerve head, orbital optic nerve, retina and peripapillary sclera from Rhesus Macaques with laser-induced, unilateral, mild and high IOP early experimental glaucoma. The results from these ongoing global analyses have already identified biomarker candidates for targeted quantitative proteomic analyses of human and monkey glaucomatous and normal plasma. In the long term, this research will verify a subset of biomarker candidates as blood biomarkers for glaucomatous damage to the TM and/or to the visual system.
John S Crabb, BA
Research Computing Analyst
Geeng-Fu Jang, PhD
Crabb, JW, M Miyagi, X Gu, K Shadrach, KA West, H Sakaguchi, M Kamei, A Hasan, L Yan, ME Rayborn, RG Salomon, JG Hollyfield (2002) Drusen Proteome Analysis: an Approach to the Etiology of Age-Related Macular Degeneration Proc Natl Acad Sci USA 99:14682-14687.
Miyagi M, H Sakaguchi, RM Darrow, L Yan, KA West, KS Aulak, DJ Stuehr, JG Hollyfield, DT Organisciak and JW Crabb (2002) Evidence that Light Modulates Protein Nitration in Rat Retina. Molecular and Cellular Proteomics 1: 293-303.
Golovleva I, S Bhattacharya, Z.Wu, N Shaw, Y Yang, K Andrabi, KA West , MS Burstedt, K Forsman, G Holmgren, O Sandgren, N Noy , J Qin and JW.Crabb (2003) Disease Causing Mutations in the Cellular Retinaldehyde-binding Protein Tighten as well as Abolish Retinoid Interactions. J Biol Chem 278: 12397-12402.
Bhattacharya SK, EJ Rockwood, SD Smith, VL Bonilha, JS Crabb, RW Kuchtey, NG Robertson, NS Peachey, CC Morton and JW Crabb(2005) Proteomics Reveals Cochlin Deposits Associated With Glaucomatous Trabecular Meshwork. J Biol Chem 280: 6080 – 6084.
EbrahemQ, K Renganathan, J Sears, A Vasanji, X Gu, L Lu, RG Salomon, JW Crabb, B Anand-Apte (2006) Carboxyethylpyrrole Oxidative Protein Modifications Stimulate Neovascularization: Implications for Age-Related Macular Degeneration Proc Nal Acad Sci USA 103: 13480-13484
Gu J, GJT Pauer, X Yue, U Narendra, GM Sturgill, J Bena, X Gu, NS Peachey, RG Salomon, SA Hagstrom, W Crabb and the Clinical Genomic and Proteomic AMD Study Group (2009) Assessing Susceptibility To Age-Related Macular Degeneration With Proteomic And Genomic Biomarkers. Mol & Cell Proteomics 8:1338-49.
Ni J, X Yuan, J Gu, X Yue, X Gu, RH Nagaraj, JW Crabb and The Clinical Genomic and Proteomic AMD Study Group (2009) Plasma Protein Pentosidine And Carboxymethyllysine, Biomarkers For Age-Related Macular Degeneration. Mol & Cell Proteomics 8: 1921-33.
Yuan X, X Gu, JS Crabb, X Yue, K Shadrach, JG Hollyfield and JW Crabb (2010) Quantitative Proteomics: Comparison of the Macular Bruch’s Membrane/Choroid Complex from Age-related Macular Degeneration and Normal Eyes. Mol & Cell Proteomics 9: 1031-1046.
Bollinger KE, Crabb JS, Yuan X, Putliwala T, Clark AF, Crabb JW (2011) Quantitative Proteomics: TGFβ2-Signaling in Trabecular Meshwork Cells. Invest Ophthal Visual Sci 52, 8287-8291.
Bollinger KE, Crabb JS, Yuan X, Putliwala T, Clark AF, Crabb JW (2012) Proteomic Similarities in Steroid Responsiveness In Normal and Glaucomatous Trabecular Meshwork Cells. Mol Vision 18: 2001-2011.
Renganathan K, J Gu, ME Rayborn, JS Crabb, RG Salomon, RJ Collier, MA Kapin, C Romano, JG Hollyfield, and JW Crabb (2013) CEP Biomarkers As Potential Tools for Monitoring Therapeutics, PLoS One 8:e76325.
Gu X, Hu Z, Ebrahem Q, Crabb JS, Mahfouz R, Radivoyevitch T, Crabb JW, Saunthararajah Y. (2014) Runx1 Regulation of Pu.1 Corepressor/Coactivator Exchange Identifies Specific Molecular Targets for Leukemia Differentiation Therapy. J Biol Chem 289: 14881-95.
Kim YW, Yakubenko VP, West XZ, Gugiu GB, Kutralanathan R, Biswas S, Gao D, Crabb JW, Salomon RG, Podrez E, Byzova T (2015) Receptor-Mediated Mechanism Controlling Tissue Levels of Bioactive Lipid Oxidation Products. Circ Res 117: 321-332.
Crabb JW, Hu B, Crabb JS, Triozzi P, Saunthararajah Y, Tubbs R, Singh AD (2015) iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors. PLoS ONE 10: e0135543.
Tayou J, Wang Q, Jang GF, Pronin AN, Orlandi C, Martemyanov KA, Crabb JW, Slepak VZ (2016) Regulator of G-protein Signaling 7 (RGS7) can exist in a homo-oligomeric form that is regulated by Gαo and R7-binding protein. J Biol Chem 291, 9133-47
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