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J.G. Hollyfield, K.G. Shadrach, H. Sakaguchi, J.A. Pichler, M.E. Rayborn, X. Gu, J.W. Crabb; TIMP–3 Distribution and Activity in the High Molecular Weight Aggregates From Bruch’s Membrane/Choroid Complex of Caucasian and African American Donor Eyes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2316.
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© ARVO (1962-2015); The Authors (2016-present)
As we age, some proteins are damaged and cannot be efficiently degraded, resulting in the accumulation of high molecular weight protein aggregates in ocular tissues. To define the proteins vulnerable to these changes and any regional or ethnic differences that may occur, we have performed proteome analysis on high molecular weight protein aggregates isolated from Bruch’s membrane/choroid complex below the macula, and from peripheral areas in both Caucasian and African American donor eyes. Donor ages were between the 5th to the 9th decade of life.
Selected areas of Bruch’s membrane and choroid were isolated from age–matched Caucasian and African American donor eyes. The tissue was prepared for SDS/PAGE analysis and top bands of the gel were isolated, digested with trypsin and analyzed by mass spectrometry. Other samples were prepared for immunocytochemistry, Western blotting and reverse zymography
TIMP–3, complement factors, vitronectin and crystallins immunoreactivity is present in the high molecular weight aggregates at younger ages in the Caucasian samples than was observed in the African American samples. Further, in Caucasian samples, TIMP–3 is found outside Bruch’s membrane, but in African American samples is restricted to Bruch’s membrane. Western blots reveal the presence of TIMP–3 in the high molecular weight complexes, but reverse zymography of these regions failed to show TIMP–3 functionality.
Aggregation and loss of function can be observed in specific proteins present in Bruch’s membrane in Caucasian tissues earlier than these changes are observed in African American tissues. The early accumulation of these damaged proteins within Bruch’s membrane/choroid complex may alter the hydrolic conductivity and movement of metabolites between the choriocapillaris and RPE.
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