Dataset Information

Active Rap1-mediated inhibition of choroidal neovascularization requires interactions with IQGAP1 in choroidal endothelial cells.

ABSTRACT: Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness. The pathophysiology involves activation of choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelial (RPE) monolayer and form choroidal neovascularization (CNV) in the neural retina. The multidomain GTPase binding protein, IQGAP1, binds active Rac1 and sustains activation of CECs, thereby enabling migration associated with vision-threatening CNV. IQGAP1 also binds the GTPase, Rap1, which when activated reduces Rac1 activation in CECs and CNV. In this study, we tested the hypothesis that active Rap1 binding to IQGAP1 is necessary and sufficient to reduce Rac1 activation in CECs, and CNV. We found that pharmacologic activation of Rap1 or adenoviral transduction of constitutively active Rap1a reduced VEGF-mediated Rac1 activation, migration, and tube formation in CECs. Following pharmacologic activation of Rap1, VEGF-mediated Rac1 activation was reduced in CECs transfected with an IQGAP1 construct that increased active Rap1-IQGAP1 binding but not in CECs transfected with an IQGAP1 construct lacking the Rap1 binding domain. Specific knockout of IQGAP1 in endothelial cells reduced laser-induced CNV and Rac1 activation in CNV lesions, but pharmacologic activation of Rap1 did not further reduce CNV compared to littermate controls. Taken together, our findings provide evidence that active Rap1 binding to the IQ domain of IQGAP1 is sufficient to interfere with active Rac1-mediated CEC activation and CNV formation.

SUBMITTER: Ramshekar A

PROVIDER: S-EPMC8238370 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Active Rap1-mediated inhibition of choroidal neovascularization requires interactions with IQGAP1 in choroidal endothelial cells.

Ramshekar Aniket A Wang Haibo H Kunz Eric E Pappas Christian C Hageman Gregory S GS Chaqour Brahim B Sacks David B DB Hartnett M Elizabeth ME

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20210701 7

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness. The pathophysiology involves activation of choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelial (RPE) monolayer and form choroidal neovascularization (CNV) in the neural retina. The multidomain GTPase binding protein, IQGAP1, binds active Rac1 and sustains activation of CECs, thereby enabling migration associated with vision-threatening CNV. IQGAP1 also binds the GTPase, Rap1, which ...[more]

PMID: 34166557

Similar Datasets

Project description:PurposeAge-related macular degeneration (AMD) is the leading cause of central visual loss among patients over the age of 55 years worldwide. Neovascular-type AMD (nAMD) accounts for approximately 10% of patients with AMD and is characterized by choroidal neovascularization (CNV). The proliferation of choroidal endothelial cells (CECs) is one important step in the formation of new vessels. Transcriptional coactivator Yes-associated protein (YAP) can promote the proliferation of multiple cancer cells, corneal endothelial cells, and vascular smooth muscle cells, which participate in angiogenesis. This study intends to reveal the expression and functions of YAP during the CNV process.MethodsIn the study, a mouse CNV model was generated by laser photocoagulation. YAP expression was detected with western blotting and immunohistochemistry. YAP siRNA and ranibizumab, a VEGF monoclonal antibody, were injected intravitreally in CNV mice. The YAP and VEGF expression levels after injection were detected with western blotting. The incidence and leakage area of CNV were measured with fundus fluorescein angiography, choroidal flat mounting, and hematoxylin and eosin (HE) staining. Immunofluorescent double staining was used to detect YAP cellular localization with CD31 (an endothelial cell marker) antibody. Proliferating cell nuclear antigen (PCNA) expression in CNV mice without or with YAP siRNA intravitreal injection and the colocalization of PCNA and CD31 were measured with western blotting and immunofluorescent double staining, respectively.ResultsYAP expression increased following laser exposure, in accordance with vascular endothelial growth factor (VEGF) expression. YAP siRNA and ranibizumab decreased VEGF expression and the incidence and leakage area of CNV. YAP was localized in the vascular endothelium within the CNV site. Additionally, after laser exposure, YAP siRNA inhibited the increased expression of PCNA, which was colocalized with endothelial cells.ConclusionsThis study showed that YAP upregulation promoted CNV formation by upregulating the proliferation of endothelial cells, providing evidence for the molecular mechanisms of CNV and suggesting a novel molecular target for nAMD treatment.

Project description: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.

Dataset Information

Active Rap1-mediated inhibition of choroidal neovascularization requires interactions with IQGAP1 in choroidal endothelial cells.

Publications

Active Rap1-mediated inhibition of choroidal neovascularization requires interactions with IQGAP1 in choroidal endothelial cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets