Wound healing occurs through the processes of cell proliferation (mitosis) and/or cell migration. The corneal endothelium in humans is known to be mostly non-proliferative. As a result, wounds in the corneal endothelium heal by migration. A 21-amino acid peptide called Endothelin-1 (ET-1) is studied in the Crawford research lab and is a known mitogen, i.e. increases cell proliferation. Data suggests that Endothelin-1 can stimulate cell proliferation in the corneal endothelium. This fact led to the first hypothesis that ET-1 stimulated wound closure in this cell layer. I explored this hypothesis using a bovine model of corneal endothelial cells isolated and grown in 10% calf-serum DMEM media. Half the cells were treated with ET-1 and the other half were left in media alone (control). The cells were wounded mechanically and allowed to heal for 72-hours with photo micrographs taken every 24-hour to record wound closure. The data suggested that ET-1 stimulated wound closure because the cells treated with ET-1 had a higher percent wound closure when compared to the control. This experiment was modified to test my second hypothesis, that inhibition of cell proliferation would inhibit wound closure. In this experiment, half the cells were treated with 5-Flurouracil, an inhibitor of cell proliferation. The data showed that the prescence of 5-FU decreased the percentage of wound closure, but did not eliminate it. ET-1 was still able to increase wound closure in the presence of 5-FU by 10.8% compared to the cells treated with 5-FU alone. My third hypothesis then became that ET-1 stimulates cell migration. I explored this hypothesis using a chemotaxis assay. The endothelial cells were loaded with Calcein-AM, a fluorescent dye, and migration was examined with a Chemotx chemotaxis system. The cells were treated with serum free media (control), 0.1 nM ET-1, 1 nM ET-1, 10 nM ET-1, or serum (positive control). They were allowed to incubate for 48-hours and fluorescence of migrated cells was recorded. The data suggests that ET-1 stimulates wound healing, promotes wound closure in the absence of proliferation, and that ET-1 stimulates cell migration.
Advisor(s) or Committee Chair
Dr. Kenneth Crawford
Frazier, Leah B., "The Effects of Endothelin-1 on Cell Migration of Corneal Endothelial Cells" (2012). Honors College Capstone Experience/Thesis Projects. Paper 377.