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Z Si, AK Palkama, D Velasquez, B Gebhardt, RW Beuerman; Release of Thrombospondin-4 and Other Proteins from Outflow Pathways in the Human Eye . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3280.
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Purpose: To analyze the release of protein from outflow pathways and to correlate protein release with facility of outflow in human cadaver eyes. Method: For each eye, the posterior 1/3 of the globe, the aqueous humor, the lens, and the retina were removed. The resulting anterior segment specimen was attached to a perfusion chamber and infused with DMEM+PSF for 48 hours at 37 C and a perfusion pressure of 11 mmHg. The perfusate was collected under silicone oil at 4, 24, and 48 hr outside the eye (after the perfusate passed through the outflow pathways) and at 48 hr inside the eye. Protein composition and concentration in the perfusates were analyzed using spectrophotometry, SDS-PAGE, and Western blot. Differences in concentration were analyzed by 2-tailed Student's t-test. Thrombospondin-4 (TSP-4) fragments in the cornea, sclera, iris, ciliary body, and pigment layer were analyzed using Western blot. Facility of outflow was measured continuously with an electronic balance. Changes in facility of outflow over time were evaluated by analysis of regression. Results:Total protein concentration in the perfusate collected from outside the eye was similar at 4, 24, and 48 hr. The total protein concentration in the perfusate collected outside the eye (2304.67 363.46 µg/ml) was 6 times the concentration in the perfusate collected inside the eye (365.52 74.19 µg/ml) (p=0.002; n=4) at 48 hr. Two subunits of TSP-4 (110 and 140 kDa) were found in the perfusate collected outside the eye at 4, 24, and 48 hr, but not in the perfusate collected inside the eye at 48 hr. The same two TSP-4 subunits were found in fresh (unperfused) cornea and sclera, but not in perfused cornea and sclera at 48 hr. Mean facility of outflow was 0.19 0.11 µl/min/mmHg ( SEM; n=5), with a slight but significant decrease over time (0.002%/min/mmHg; p=0.0002). Conclusion: As the concentration of TSP-4 decreased in the sclera containing the outflow pathways, the facility of outflow also decreased. Thus, the two subunits of TSP-4 which were released from the outflow pathways might be macromolecules correlated to the facility of outflow in the human eye.
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