Activation of Rap1 inhibits NADPH oxidase‐dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization

Wang, Haibo; Jiang, Yanchao; Shi, Dallas; Quilliam, Lawrence A.; Chrzanowska‐Wodnicka, Magdalena; Wittchen, Erika S.; Li, Dean Y.; Hartnett, M. Elizabeth

doi:10.1096/fj.13-240028

Cited by 25 publications

(39 citation statements)

References 58 publications

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“…21 Herein, we provide evidence that activation of Rap1 through 8CPT can modulate the effects of TNF-a or VEGF on CEC migration and CNV formation through several steps preventing ROS-triggered NF-kB/Rac1 activation.…”

Section: Rap1 Inhibits Tnf-aeinduced Ros Signalsmentioning

confidence: 84%

“…Forty-eight hours after viral transduction, intracellular ROS generation measured with the DCF-diacetate assay was significantly reduced in CECs transduced with Ad-63E and treated with PBS ( Figure 5A), consistent with our published data. 21 Also, in CECs overexpressing active Rap1a and treated with TNF-a, ROS was significantly inhibited compared with CECs treated with TNF-a and control adenovirus, Ad-GFP. In a parallel experiment, TNF-aeinduced p-p65 and GTP-Rac1 were also inhibited in CECs transduced with Ad-63E ( Figure 5B).…”

Section: Activation Of Nf-kb Signaling Regulates Tnf-aemediated Rac1 mentioning

confidence: 93%

“…21 Herein, we determined if activated Rap1 could inhibit TNF-aeinduced ROS generation in CECs and reduce CEC migration. CECs were incubated with an adenovirus expressing either GFP (Ad-GFP) or active Rap1a (Ad-63E).…”

Section: Activation Of Nf-kb Signaling Regulates Tnf-aemediated Rac1 mentioning

confidence: 99%

“…21 CECs were loaded with 5-mm 2 0 ,7 0 -dichlorofluorescein (DCF; Invitrogen) in serum-free medium for 30 minutes at 37 C. After two washes with PBS, cells were pretreated with 100 mmol/L apocynin (Sigma-Aldrich, St. Louis, MO) or PBS for 30 minutes, then incubated with 20 ng/mL TNF-a or PBS for another 30 minutes. ROS generation was measured in a fluorescent plate reader at an excitation of 488 nm and an emission of 520 nm.…”

Section: Ros Generation Assay In Cultured Cecsmentioning

confidence: 99%

“…19 We then found that activation of another GTPase protein, Rap1, increased RPE barrier integrity and reduced CNV volume in a murine laser injury model by inhibiting NADPH oxidaseedependent ROS generation in RPE. 20,21 Herein, we study another pathway that involves ROS generation through the inflammatory cytokine TNF-a, specifically testing the hypothesis that TNF-a induces CEC migration via NADPH oxidaseegenerated ROS, in which activation of Rac1 is downstream of ROS, and that activation of Rap1 prevents TNF-aeinduced CEC migration by reducing ROS generation. On the basis of previous studies in other tissues, we postulate that interactions between inflammatory and oxidative signaling mechanisms overwhelm homeostatic mechanisms in the outer retina, which would normally prevent the formation of CNV.…”

mentioning

confidence: 99%

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Rap1 GTPase Inhibits Tumor Necrosis Factor-α–Induced Choroidal Endothelial Migration via NADPH Oxidase– and NF-κB–Dependent Activation of Rac1

Wang

Fotheringham

Wittchen

et al. 2015

The American Journal of Pathology

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Macrophage-derived tumor necrosis factor (TNF)-a has been found in choroidal neovascularization (CNV) surgically removed from patients with age-related macular degeneration. However, the role of TNF-a in CNV development remains unclear. In a murine laser-induced CNV model, compared with un-lasered controls, TNF-a mRNA was increased in retinal pigment epithelial and choroidal tissue, and TNF-a colocalized with lectin-stained migrating choroidal endothelial cells (CECs). Inhibition of TNF-a with a neutralizing antibody reduced CNV volume and reactive oxygen species (ROS) level around CNV. In CECs, pretreatment with the antioxidant apocynin or knockdown of p22phox, a subunit of NADPH oxidase, inhibited TNF-aeinduced ROS generation. Apocynin reduced TNF-aeinduced NF-kB and Rac1 activation, and inhibited TNF-aeinduced CEC migration. TNF-aeinduced Rac1 activation and CEC migration were inhibited by NF-kB inhibitor Bay11-7082. Overexpression of Rap1a prevented TNF-aeinduced ROS generation and reduced NF-kB and Rac1 activation. Activation of Rap1 by 8-(4-chlorophenylthio) adenosine-2 0 -O-Me-cAMP prevented TNF-aeinduced CEC migration and reduced laser-induced CNV volume, ROS generation, and activation of NF-kB and Rac1. These findings provide evidence that active Rap1a inhibits TNF-aeinduced CEC migration by inhibiting NADPH oxidase-dependent NF-kB and Rac1 activation and suggests that Rap1a de-escalates CNV development by interfering with ROSdependent signaling in several steps of the pathogenic process. One reason may be that treatment is provided too late when CNV is already present. To address this possibility, we focus on understanding early steps in the pathophysiology in neovascular AMD to prevent vision loss. Activation and migration of choroidal endothelial cells (CECs) are early steps in the development of CNV. Activated CECs migrate through the Bruch membrane toward the retinal pigment epithelium (RPE) and also transmigrate the RPE into the sensory retina. At either location, activated CECs can proliferate to form CNV. Severe vision loss occurs when CNV develops in the sensory retina. 3,4 Both inflammation and oxidative stress have been implicated in the development of CNV.

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Section: Rap1 Inhibits Tnf-aeinduced Ros Signalsmentioning

confidence: 84%

Section: Activation Of Nf-kb Signaling Regulates Tnf-aemediated Rac1 mentioning

confidence: 93%

Section: Activation Of Nf-kb Signaling Regulates Tnf-aemediated Rac1 mentioning

confidence: 99%

Section: Ros Generation Assay In Cultured Cecsmentioning

confidence: 99%

mentioning

confidence: 99%

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Rap1 GTPase Inhibits Tumor Necrosis Factor-α–Induced Choroidal Endothelial Migration via NADPH Oxidase– and NF-κB–Dependent Activation of Rac1

Wang

Fotheringham

Wittchen

et al. 2015

The American Journal of Pathology

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Spatial Mechano‐Signaling Regulation of GTPases through Non‐Degradative Ubiquitination

Sewduth,

Carai,

Ivanisevic

et al. 2023

Advanced Science

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Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear‐stressed endothelial cells demonstrated that the non‐degradative ubiquitination of several GTPases is regulated by mechano‐signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non‐degradative ubiquitination fine‐tunes the function of GTPases by modifying their interacting network. Specifically, WWP2‐mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress‐induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial‐specific Wwp2‐knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non‐degradative ubiquitination of GTPases in endothelial mechano‐activation.

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Breakdown of the Retinal Pigmented Epithelium Blood–Retinal Barrier☆

Hartnett¹

2017

Reference Module in Neuroscience and Biobehavioral Psychology

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Activation of Rap1 inhibits NADPH oxidase‐dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization

Cited by 25 publications

References 58 publications

Rap1 GTPase Inhibits Tumor Necrosis Factor-α–Induced Choroidal Endothelial Migration via NADPH Oxidase– and NF-κB–Dependent Activation of Rac1

Rap1 GTPase Inhibits Tumor Necrosis Factor-α–Induced Choroidal Endothelial Migration via NADPH Oxidase– and NF-κB–Dependent Activation of Rac1

Spatial Mechano‐Signaling Regulation of GTPases through Non‐Degradative Ubiquitination

Breakdown of the Retinal Pigmented Epithelium Blood–Retinal Barrier☆

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