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A. H. Lester, W. Y. Boadi, G. W. McCollum, J. S. Penn; The Effect of Penetrating Ocular Injury on Angiostatic Vitreous Proteins. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2960.
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Penetrating ocular needle injury (POI) induces angiostatic activity in the vitreous humor and alters the retinal and vitreous humor levels of several proteins known to regulate angiogenesis. The purpose of this study was to investigate the presence of known angiostatic proteins in vitreous isolated from rats following POI by Western blot analysis.
To generate POI, a dry 30-gauge needle was inserted twice, just posterior to the temporal ora of the left eyes of six-week-old Sprague-Dawley rats. The right eyes served as non-POI controls. Vitreous from both eyes was harvested 24 hours after needle injury and pooled separately. Vitreous homogenates were prepared and centrifuged (100,000 x g), and the supernatants containing soluble vitreous protein of POI and non-POI eyes were fractionated with a cation-exchange resin. BrdU incorporation was used to assay human retinal microvascular endothelial cell proliferation, and POI fractions containing angiostatic activity were identified by this assay. Extensive genomics and proteomics analysis of the vitreous from POI and non-POI eyes led to the identification of eight candidate angiostatic proteins - angiostatin, endostatin, PAI-1, PEDF, thrombospondin, TIMP-1, TIMP-2, and TIMP-3. Western blot analysis was used to investigate the presence of the candidates in the POI fractions with angiostatic activity.
Fractions from non-POI vitreous protein showed no significant angiostatic activity. However, angiostatic activity was identified in seven of 25 vitreous protein fractions (8, 9, 11, 12, 13, 17, and 18) isolated from rats receiving POI. Four of the eight candidate angiostatic proteins, endostatin, PAI-1, PEDF, and TIMP-2, were identified in one or more of these angiostatic vitreous protein fractions.
Endostatin, PAI-1, PEDF, and TIMP-2 are angiostatic factors that are elevated in vitreous protein isolated from rats receiving POI. The presence of these angiostatic proteins may explain the angiostasis associated with some, but not all, POI vitreous fractions. Additional studies will determine the specific relationship between known angiostatic factors and the POI-induced angiostatic capacity of the vitreous. Complimentary studies will also be designed to identify as-yet unknown angiostatic factors.
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