May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
A New in vitro Model of Bruch's Membrane Aging: A Basis for the Study of Age–Related RPE Defects
Author Affiliations & Notes
  • J.V. Glenn
    Ophthalmology Research Centre, Queen's University of Belfast, Belfast, United Kingdom
  • H. Mahaffy
    Ophthalmology Research Centre, Queen's University of Belfast, Belfast, United Kingdom
  • N. Frizzell
    Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC
  • S. Thorpe
    Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC
  • A.A. Hussain
    Department of Ophthalmology, St Thomas' Hospital Medical School, London, United Kingdom
  • M. Boulton
    School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
  • K. Wu
    School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
  • W.J. Curry
    Ophthalmology Research Centre, Queen's University of Belfast, Belfast, United Kingdom
  • A.W. Stitt
    Ophthalmology Research Centre, Queen's University of Belfast, Belfast, United Kingdom
  • Footnotes
    Commercial Relationships  J.V. Glenn, None; H. Mahaffy, None; N. Frizzell, None; S. Thorpe, None; A.A. Hussain, None; M. Boulton, None; K. Wu, None; W.J. Curry, None; A.W. Stitt, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 3006. doi:
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      J.V. Glenn, H. Mahaffy, N. Frizzell, S. Thorpe, A.A. Hussain, M. Boulton, K. Wu, W.J. Curry, A.W. Stitt; A New in vitro Model of Bruch's Membrane Aging: A Basis for the Study of Age–Related RPE Defects . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3006.

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

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Abstract

Abstract: : Purpose:Bruch’s membrane is known to become altered with ageing. Advanced glycation end products (AGEs) have been shown to accumulate with age in the retinal pigment epithelium (RPE)–Bruch’s membrane–choroid axis. The current study has sought to develop and evaluate a new model of physiologically relevant ageing of RPE substrate with direct relevance to AGE formation on human Bruch’s membrane. Methods:Bruch’s membrane–immobilised AGEs were quantified in human post–mortem specimens using a combination of gas chromatography–mass spectrometry (GC–MS)(n=12), reverse phase HPLC (n= 40) and immunohistochemistry. Matrigel basement membrane extract was AGE–modified (AGE–BM) and degrees of adduct formation assessed. How AGE–BM influenced hydraulic conductivity on porous membranes was also determined and compared to clinical specimens. In a complementary cell–based study, RPE interactions with native or AGE–modified BM were investigated using the ARPE–19 cell–line. Confluent RPE were also grown up to 1 month on control or AGE–BM and proteomic analysis performed on cell extracts. Changes in mRNA/protein expression were confirmed using real–time RT–PCR and immunohistochemistry. Results: GC–MS, HPLC and immunohistochemistry analysis of Bruch’s membrane demonstrated an increase in AGE (CML, pentosidine) levels with ageing (p<0.005) and these modifications were mirrored in the in vitro aged Bruch’s membrane model. Hydraulic conductivity was reduced in AGE–BM when compared to controls, a finding also reflected in previous studies on human Bruch’s membrane. RPE spreading was significantly impaired on AGE–BM compared to controls (p<0.001). Proteomic analysis revealed marked alterations in levels of several proteins, including matrix metalloproteinase MMP–2, vimentin and several BM component proteins. These changes were confirmed by RT–PCR (p<0.001) and immunohistochemistry. Conclusions:The current study shows that a range of defined AGE adducts are increased with age in human Bruch’s membrane. Proteomic profiles identified a range of post–translationally modified and up/down regulated proteins in RPE exposed to AGE–BM. AGE–modification of Bruch’s membrane may have an important pathophysiological role in RPE age–related dysfunction.

Keywords: retinal pigment epithelium • extracellular matrix • Bruch's membrane 
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