Erika S Wittchen1, M Elizabeth Hartnett. 1. Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Abstract
PURPOSE: To determine whether the small GTPase Rap1 regulates the formation and maintenance of the retinal pigment epithelial (RPE) cell junctional barrier. METHODS: An in vitro model was used to study RPE barrier properties. To dissect the role of Rap1, two techniques were used to inhibit Rap1 function: overexpression of RapGAP, which acts as a negative regulator of endogenous Rap1 activity, and treatment with engineered, adenovirally-transduced microRNAs to knockdown Rap1 protein expression. Transepithelial electrical resistance (TER) and real-time cellular analysis (RTCA) of impedance were used as readouts for barrier properties. Immunofluorescence microscopy was used to visualize localization of cadherins under steady state conditions and also during junctional reassembly after calcium switch. Finally, choroidal endothelial cell (CEC) migration across RPE monolayers was quantified under conditions of Rap1 inhibition in RPE. RESULTS: Knockdown of Rap1 or inhibition of its activity in RPE reduces TER and electrical impedance of the RPE monolayers. The loss of barrier function is also reflected by the mislocalization of cadherins 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. Furthermore, CEC transmigration is significantly higher in Rap1-knockdown RPE monolayers compared with control. CONCLUSIONS: Rap1 GTPase is an important regulator of RPE cell junctions, and is required for maintenance of barrier function. This 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.
PURPOSE: To determine whether the small GTPase Rap1 regulates the formation and maintenance of the retinal pigment epithelial (RPE) cell junctional barrier. METHODS: An in vitro model was used to study RPE barrier properties. To dissect the role of Rap1, two techniques were used to inhibit Rap1 function: overexpression of RapGAP, which acts as a negative regulator of endogenous Rap1 activity, and treatment with engineered, adenovirally-transduced microRNAs to knockdown Rap1 protein expression. Transepithelial electrical resistance (TER) and real-time cellular analysis (RTCA) of impedance were used as readouts for barrier properties. Immunofluorescence microscopy was used to visualize localization of cadherins under steady state conditions and also during junctional reassembly after calcium switch. Finally, choroidal endothelial cell (CEC) migration across RPE monolayers was quantified under conditions of Rap1 inhibition in RPE. RESULTS: Knockdown of Rap1 or inhibition of its activity in RPE reduces TER and electrical impedance of the RPE monolayers. The loss of barrier function is also reflected by the mislocalization of cadherins 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. Furthermore, CEC transmigration is significantly higher in Rap1-knockdown RPE monolayers compared with control. CONCLUSIONS:Rap1 GTPase is an important regulator of RPE cell junctions, and is required for maintenance of barrier function. This 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.
Authors: Haibo Wang; Yanchao Jiang; Dallas Shi; Lawrence A Quilliam; Magdalena Chrzanowska-Wodnicka; Erika S Wittchen; Dean Y Li; M Elizabeth Hartnett Journal: FASEB J Date: 2013-09-16 Impact factor: 5.191
Authors: Haibo Wang; Xiaokun Han; Colin A Bretz; Silke Becker; Deeksha Gambhir; George W Smith; R Jude Samulski; Erika S Wittchen; Lawrence A Quilliam; Magdalena Chrzanowska-Wodnicka; M Elizabeth Hartnett Journal: Mol Ther Methods Clin Dev Date: 2016-08-24 Impact factor: 6.698
Authors: Erika S Wittchen; Eiichi Nishimura; Manabu McCloskey; Haibo Wang; Lawrence A Quilliam; Magdalena Chrzanowska-Wodnicka; M Elizabeth Hartnett Journal: PLoS One Date: 2013-09-10 Impact factor: 3.240