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M. C. Markowski, E. S. Wittchen, K. W. T. Burridge, S. J. Budd, M. E. Hartnett; Upregulation of Rap1 in Human RPE Causes Increased Barrier Integrity. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1865.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the role of Rap1, a GTPase, on RPE barrier property. Since, RPE barrier dysfunction occurs in several human diseases, we were interested in studying Rap1 activation because it has been shown to be involved in the cytoskeleton and barrier properties of other epithelial cells.
ARPE-19 and human fetal RPE cells were cultured and plated at a density of 1.8 x 105 cells/cm2 (determined as total confluence) on 12-well filters (12 mm diameter, 0.4 µm pore) in growth media. After 1 week, the media was changed to contain 1% FBS to avoid interference caused by high serum concentration. Transepithelial electrical resistance (TER) was measured using an Endohm-12 chamber and EVOM voltmeter. Net TER measurements were calculated by subtracting the value of a blank Transwell insert from the value of each filter with plated cells. When net TER values stabilized, rap1 activity was modulated in ARPE and human fetal RPE by transfecting with adeno viral constructs. Cells were transfected with either a constitutively active construct to induce Rap1 activity (G12V) or a dominant negative form (17N) to reduce Rap1 activity. As controls, cells were transfected with wildtype Rap1, GFP alone, or untreated. TER was measured 24-48 hours after transfection of each virus. Results were analyzed by paired two-sample t-tests.
Cells transfected with constitutively active virus (G12V) showed a significant increase in TER within 24 hours when compared to untreated and GFP transfected cells (p = .003). In addition, cells transfected with dominant negative virus (17N) showed a significant decrease in TER within 24 hours when compared to controls (p = .011). After 48 hours, cells transfected with G12V exhibited TER measurements similar to before treatment. However, cells transfected with 17N virus, had continued decreases in TER measurements compared to at 24 hours (p=.02). There were no significant differences between untreated, GFP, and wildtype treated controls.
Overexpressing constitutively active Rap1 increases barrier property of RPE cells as measured by TER within a 24 period after treatment whereas inhibiting Rap1 activity decreases TER measurements for a period of 48-72 hours after treatment.
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