April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Quantitative Proteomic Analysis of TGF-β2-Treated Human Trabecular Meshwork Cells
Author Affiliations & Notes
  • K. E. Bollinger
    Ophthalmology, Medical College of Georgia, Augusta, Georgia
  • J. S. Crabb
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • X. Yuan
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • X. Yue
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • A. F. Clark
    Cell Biology & Genetics, University of North Texas HSC, Fort Worth, Texas
  • J. W. Crabb
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 3207. doi:
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      K. E. Bollinger, J. S. Crabb, X. Yuan, X. Yue, A. F. Clark, J. W. Crabb; Quantitative Proteomic Analysis of TGF-β2-Treated Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3207.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Transforming growth factor (TGF)-β2 is elevated in the aqueous humor of patients with primary open angle glaucoma (POAG), elevates intraocular pressure in perfusion cultured eyes, and may be involved in the pathogenesis of POAG. Toward a better understanding of POAG pathology, we evaluated quantitative proteomic changes in cultured trabecular meshwork (TM) cells following TGF-β2 treatment.

Methods: : Primary cultures of human TM cells from 2 POAG and 2 non-glaucomatous donors were treated with or without TGF-β2 (5 ng/ml) for 48 h. Protein was extracted in SDS buffer, digested with trypsin, peptides labeled with iTRAQ tags, and TGF-β2-treated TM samples were combined with their corresponding untreated samples. Following strong cation exchange chromatography, peptides were analyzed by LC MS/MS and proteins were identified using the Mascot search engine and the Swiss-Protein database. Relative protein quantification from iTRAQ labeling utilized code written in R. Immunoblotting was used to corroborate differential expression of select proteins.

Results: : A total of 653 proteins were quantified with 2 or more peptides from TGF-β2-treated TM cells. No definitive proteomic differences were detected between TGF-β2-treated POAG and control TM cells. The expression of over 30 proteins changed significantly following TGF-β2-treatment, including increased expression of SPARC, α-crystallin B, tropomysin α1 and collagen α1(III) and decreased expression of CD9 antigen, guanine nucleotide-binding protein G(i) α-2, and superoxide dismutase [Mn].

Conclusions: : Almost 3-fold more proteins were significantly increased than decreased following TGF-β2 treatment. Proteins increased in abundance may contribute to ECM and cytoskeletal abnormalities that facilitate outflow resistance in POAG TM. Proteins reduced in abundance may compromise cellular adhesion, signaling and defense mechanisms in the TM. The results provide new insights to possible molecular mechanisms of POAG.CR:

Keywords: trabecular meshwork • proteomics • aqueous 
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