April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Global Network Analysis of Diabetic Rat Retina Using 18O Isotopic Labeling and 2D LC-MS/MS Identifies Significantly Dysregulated Novel Targets
Author Affiliations & Notes
  • Hossein Ameri
    Ophthalmology and Visual Sciences,
    University of Texas Medical Branch, Galveston, Texas
  • Jonathan M. Starkey
    Endocrinology,
    University of Texas Medical Branch, Galveston, Texas
  • Srijita Banerjee
    Endocrinology,
    University of Texas Medical Branch, Galveston, Texas
  • Yanhua Zhao
    Endocrinology,
    University of Texas Medical Branch, Galveston, Texas
  • Wanda LeJeune
    Endocrinology,
    University of Texas Medical Branch, Galveston, Texas
  • Chandra Sekhar Rao Kadiyala
    Pharmacology, Case Western Reserve University, Cleveland, Ohio
  • Timothy S. Kern
    Pharmacology, Case Western Reserve University, Cleveland, Ohio
  • Masaru Miyagi
    Pharmacology, Case Western Reserve University, Cleveland, Ohio
  • Ronald G. Tilton
    Endocrinology,
    University of Texas Medical Branch, Galveston, Texas
  • Footnotes
    Commercial Relationships  Hossein Ameri, None; Jonathan M. Starkey, None; Srijita Banerjee, None; Yanhua Zhao, None; Wanda LeJeune, None; Chandra Sekhar Rao Kadiyala, None; Timothy S. Kern, None; Masaru Miyagi, None; Ronald G. Tilton, None
  • Footnotes
    Support  Juvenile Diabetes Research Foundation
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 3553. doi:
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      Hossein Ameri, Jonathan M. Starkey, Srijita Banerjee, Yanhua Zhao, Wanda LeJeune, Chandra Sekhar Rao Kadiyala, Timothy S. Kern, Masaru Miyagi, Ronald G. Tilton; Global Network Analysis of Diabetic Rat Retina Using 18O Isotopic Labeling and 2D LC-MS/MS Identifies Significantly Dysregulated Novel Targets. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3553.

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

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Abstract

Purpose: : To analyze the global retinal proteome in diabetic rats and identify novel dysregulated proteins which may contribute significantly to the pathophysiology of diabetic retinopathy.

Methods: : Diabetes was induced in male, Sprague-Dawley rats using intraperitoneal injection of streptozotocin (STZ). Rats were sacrificed 2 months after the onset of hyperglycemia. Total proteins were extracted from the retinas of 5 diabetic and 5 age- and sex-matched control rats. Control and diabetic samples were digested overnight in trypsin in H216O and H218O, respectively. Total peptides from both experimental groups were pooled and separated by 2D liquid chromatography, followed by peptide sequencing using mass spectrometry. Significant proteins (Student’s t-test, q*=0.05, with a Benjamini-Hochberg correction for multiple testing) were determined following log transformation and mean centering then filtering with at least a 50 % change in normalized diabetes/control isotope ratio. Significantly altered proteins were analyzed with Ingenuity Pathway Analysis (IPA). Real time PCR was used for validation of dysregulated protein of interest using an additional group of 4 control and 3 diabetic rats.

Results: : From 2,973 proteins identified, 118 were dysregulated in diabetic retinas. These proteins are involved in organ development, cell to cell signaling, cancer, cellular development, amino acid metabolism, drug metabolism, cell morphology and cell death. The most significant dysregulation was in glutathione metabolism pathway. IPA network analysis identified significant signaling hubs, including: HNF4A, SFRS2, NF-ΚB, TNF, GRB2 and TGFB1. In addition, SNCA, HSPA1A and YWHAB, were significantly down regulated, whereas FABP5 and LGALS3 were markedly up regulated. Further validation of a protein of interest, HSPA1A, by real time PCR confirmed the down-regulation in retina of diabetic rats at 2 months.

Conclusions: : Global retinal proteome analysis could identify dysregulated proteins not previously recognized by conventional methods. In our study canonical pathway analysis demonstrated significant dysregulation of several pathways, the most significant of which is glutathione metabolism. Down-regulation of HSPA1A in diabetic retina, validated by PCR, is an example of novel findings in our study.

Keywords: diabetic retinopathy • retina • diabetes 
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