Abstract
Purpose: :
To determine whether the small GTPase Rap1 regulates the formation and maintenance of retinal pigment epithelial (RPE) cell junctional barrier.
Methods: :
We utilized ARPE-19 cells as an in vitro cell culture model to study retinal pigment epithelial barrier properties. To dissect the role of Rap1 in this process, we used two techniques to inhibit Rap1 function: overexpression of RapGAP protein, which acts as a negative regulator of endogenous Rap1 activity, as well as treatment with engineered, adenovirally-transduced microRNAs designed to knockdown Rap1 protein expression. Transepithelial electrical resistance (TER) and real-time cellular analysis (RTCA) of electrical impedance were used as readouts for monolayer barrier properties. Immunofluorescence microscopy was used to visualize localization of cadherins, both under steadystate conditions, and during junctional reassembly following calcium switch. Choroidal endothelial cell (CEC) transmigration across RPE monolayers was quantified to assess choroidal neovascularization under conditions of Rap1 inhibition in RPE.
Results: :
Both knockdown of Rap1 or inhibition of its activity in RPE reduces steadystate TER and electrical impedance of ARPE-19 cell monolayers. The loss of barrier function is also reflected by the mislocalization of cadherin and formation of gaps within the monolayer. TER measurement and immunofluorescent staining of cadherins after a calcium switch indicate that junctional reassembly kinetics are also impaired after the loss of Rap1 protein or its activity. Furthermore, CEC transmigration is significantly higher in Rap1-knockdown ARPE-19 monolayers compared to control.
Conclusions: :
Rap1 GTPase is an important cellular regulator of RPE cell-cell junctions, and is required for maintenance and formation of barrier function. Our observation that RPE monolayers lacking Rap1 allow greater transmigration of CECs suggests a possible role for potentiating choroidal neovascularization during the pathology of neovascular age-related macular degeneration.
Keywords: age-related macular degeneration • cell adhesions/cell junctions • retinal pigment epithelium