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R.A. Sack, S. Sathe, M. Laniado-Schwartzman, A.R. Beaton, A. Mesentsev, C. Morris, B.I. Bogart; Closed Eye Tear Fluid Contains a Potent High Molecular Weight Angiogenic Factor . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1385.
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Purpose: Overnight eye closure is known to induce a sub-clinical inflammation and result in the accumulation in closed eye tear fluid (CTF) of a wide range of bioactive proteins. This study was designed to characterize the principal angiogenic factors in CTF. Methods: Capillary tube collected CTF samples were recovered from 10 donors over a several week period and centrifuged. The resultant supernatants were pooled and on occasion individually separated into 24 molecular weight fractions using a preparative molecular sieve TSK 3000 HPLC. These fractions ranged in size from > 500 kDa to <10 kDa. Samples were tested for angiogenic activity based on the capacity to induce capillary-like tubes of endothelial cells grown on MatrigelTM. Fractions that exhibited bioactivity were subjected to 2-D electrophoretic characterization and western blot probing. Active fractions were further purified using isoelectric focusing and/or affinity chromatography. Results: Neat open eye and CTF samples on bioassay exhibit negligible to very low levels of net angiogenic activity. After HPLC separation, high levels of previously masked angiogenic activity could be detected in the CTF eluent in two distinct fractions. Minor activity eluted in a post-lysozyme fraction (<14,000 kDa). 2-D electrophoresis and western blot probing reveals this fraction to be complex and to contain IL-8. This angiogenic factor is known to accumulate in CTF. The majority of the angiogenic activity, however, elutes in a protein-sparse >200 kDa fraction. 2D gel analysis reveals a highly complex mixture of proteins. Comparative analysis of this and adjacent eluting non-active HPLC fractions allows the exclusion of the vast majority of proteins and reveals trace levels of several protein species that are endemic only to the bioactive fraction. Results suggest a bioactivity activity in the ng to sub ng range. Conclusions: CTF contains a yet to be identified highly potent high molecular weight (> 200 kDa) protein or complex that appears to be the principal angiogenic factor in CTF.
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