Elucidating the Role of EPAC-Rap1 Signaling Pathway in the Blood-Retinal Barrier

Abstract

Abnormalities in retinal vascular permeability can result in macular edema, the buildup of fluid in the retina, which causes the retina to swell and thicken, and leads to neuronal cell death and vision loss. Increased retinal vascular permeability is found in many eye pathologies, including diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinal vein occlusion (RVO). The pathological changes in vascular permeability are driven by elevated levels of growth factors such as vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines such as tumor necrosis factor (TNF-α). The use of VEGF-neutralizing antibodies as a form of treatment improves vision in about half of patients treated. However, these treatments are invasive, secondary complications are observed, and half of the patients treated do not improve. Therefore, understanding mechanisms that block vascular permeability and restore the blood-retinal barrier (BRB) are important for developing novel therapies promoting barrier restoration. The small guanosine triphosphatase (GTPase) Rap1 and its guanine-nucleotide-exchange factor (GEF), EPAC, have been identified as key regulators of barrier formation, promoting cell-cell adhesion, cell-extracellular matrix (ECM) adhesion, and the assembly of junctional complexes in human umbilical vein endothelial cells (HUVEC) and epithelial cells. However, it is unknown whether the EPAC-Rap1 signaling pathway regulates the tight junctions (TJs) of retinal endothelial cells and the relationship between EPAC-Rap1 and VEGF signaling in retinal permeability is poorly understood. The goals of this thesis are to: 1) determine whether EPAC-Rap1 signaling regulates the barrier properties of the BRB, and 2) gain insight into the mechanisms of endothelial cell permeability to identify novel therapeutic targets. To investigate the role of EPAC-Rap1 signaling in the BRB, EPAC was activated using the membrane permeable cAMP analog, 8-pCPT-2´-O-Me-cAMP-AM (8CPT-AM). 8CPT-AM treatment blocked and, importantly, reversed permeability induced by VEGF and TNF-α both in vivo and in vitro and promoted TJ organization in bovine retinal endothelial cells (BREC). Additionally, inhibition of EPAC2 or knockdown of Rap1B led to an increase in basal permeability. Rap1B knockdown alone recapitulated the TJ disorganization observed with VEGF. Furthermore, pretreatment of cells with 8CPT-AM prior to VEGF, decreased VEGF-induced phosphorylation of VEGFR2 and Erk1/2. Together, these data show that activation of EPAC-Rap1 signaling can promote and restore barrier properties by blocking permeability inducing agents, attenuate VEGF-Erk1/2 signaling, and promote organization of the TJ complex. To further explore and gain insight into the mechanisms of VEGF signaling in retinal endothelial permeability, mass-spectrometry based phosphoproteomics was performed on BREC. 1,724 proteins were identified. From these proteins, 107 phosphoproteins that had a 25% phosphorylation change in the presence of VEGF and were present in more than one mass spectrometry analysis were identified and used to develop protein interaction networks using Cytoscape. One of the protein interaction networks obtained consists of the proteins EPAC, rasip1, and afadin (AF-6), all of which show significant changes in phosphorylation in the presence of VEGF. The results presented here provide new evidence that EPAC-Rap1 signaling contributes to the barrier properties of the BRB via regulation of the TJ complex. Additionally, the phosphoproteome analysis suggests how VEGF may increase permeability through phosphorylation and regulation of the EPAC/Rap/rasip1 pathway. Overall, the results from this thesis expand our knowledge on the role of EPAC2 and Rap1B in regulating retinal endothelial barrier properties and permeability. Further, these data identify specific targets for novel therapies addressing abnormalities in retinal vascular permeability.