Abstract
Loss of barrier integrity precedes the development of pathologies such as metastasis, inflammatory disorders, and blood-retinal barrier breakdown present in neovascular age-related macular degeneration. Rap1 GTPase is involved in regulating both endothelial and epithelial cell junctions; the specific role of Rap1A vs. Rap1B isoforms is less clear. Compromise of retinal pigment epithelium barrier function is a contributing factor to the development of AMD. We utilized shRNA of Rap1 isoforms in cultured human retinal pigment epithelial cells, along with knockout mouse models to test the role of Rap1 on promoting RPE barrier properties, with emphasis on the dynamic junctional regulation that is triggered when the adhesion between cells is challenged. In vitro, Rap1A shRNA reduced steady-state barrier integrity, whereas Rap1B shRNA affected dynamic junctional responses. In a laser-induced choroidal neovascularization (CNV) model of macular degeneration, Rap1b−/− mice exhibited larger CNV volumes compared to wild-type or Rap1a−/−. In vivo, intravitreal injection of a cAMP analog (8CPT-2′-O-Me-cAMP) that is a known Rap1 activator significantly reduced laser-induced CNV volume, which correlated with the inhibition of CEC transmigration across 8CPT-2′O-Me-cAMP-treated RPE monolayers in vitro. Rap1 activation by 8CPT-2′-O-Me-cAMP treatment increased recruitment of junctional proteins and F-actin to cell-cell contacts, increasing both the linearity of junctions in vitro and in cells surrounding laser-induced lesions in vivo. We conclude that in vitro, Rap1A may be important for steady state barrier integrity, while Rap1B is involved more in dynamic junctional responses such as resistance to junctional disassembly induced by EGTA and reassembly of cell junctions following disruption. Furthermore, activation of Rap1 in vivo inhibited development of choroidal neovascular lesions in a laser-injury model. Our data suggest that targeting Rap1 isoforms in vivo with 8CPT-2′-O-Me-cAMP may be a viable pharmacological means to strengthen the RPE barrier against the pathological choroidal endothelial cell invasion that occurs in macular degeneration.
Highlights
The barriers created by epithelial and endothelial cell sheets in the body are critical to maintain physiological homeostasis by functioning to limit movement of fluids, solutes, macromolecules, and the passage of other cells or pathogens from one side of a monolayer to the other
There was negligible amount of positive TUNEL staining of cryosections of injected eyes (Fig. S3E). These measurements all indicate that toxicity of 8CPT-cAMP was low. These results indicate that activation of both Rap1 isoforms in retinal pigment epithelium (RPE) with 8CPT-cAMP treatment can inhibit transmigration of choroidal endothelial cells (CECs) across the epithelium in vitro, and this correlated with decreased choroidal neovascularization (CNV) volume in vivo
Whether active Rap1 contributed to enhanced barrier function of the RPE in vivo, and importantly, whether activation of Rap1A or 1B was relevant to mechanisms relating to choroidal neovascularization, remained to be determined
Summary
The barriers created by epithelial and endothelial cell sheets in the body are critical to maintain physiological homeostasis by functioning to limit movement of fluids, solutes, macromolecules, and the passage of other cells or pathogens from one side of a monolayer to the other. Tight junctions and adherens junctions are sites of adhesion between adjacent cells, and the transmembrane protein components of these structures comprise the physical barrier of the paracellular pathway. Transmembrane proteins, such as occludin, members of the claudin family, and cadherins, act as protein scaffolds for cytoplasmic proteins such as ZO-1, b-, a-, and p120catenin, some of which bind to the actin cytoskeleton [1]. This linkage between junctional complexes and the F-actin cytoskeleton is critical for the dynamic opening and resealing of junctions, and is necessary to allow rapid responses to cellular events. Small GTPases of the Rho family are regulators of cell junctions [2,3]; how this occurs relates to the ability of Rho GTPase signaling to affect actin cytoskeleton remodeling [4]
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