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

Abstract

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.

The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration

“…C). Because Rac1‐biosensors rely on the intrinsic interaction of Rac1‐binding domains of Rac effectors with the active Rac, and this would interfere with the interaction of the active Rac1 moiety in the biosensor with JFC1, and therefore block the putative JFC1‐dependent localization of the Rac1 biosensor, we analyzed active Rac1 localization using a validated antibody that detects the endogenous active form of Rac1 (Rac1‐GTP) but not Rac1‐GDP . First, we confirmed the specificity of the antibody for endogenous Rac1‐GTP showing: (a) that the anti‐Rac1‐GTP antibody efficiently pulls down GTP‐loaded Rac1 (Supplementary Fig.…”

Activation of the Small GTPase Rap1 Inhibits Choroidal Neovascularization by Regulating Cell Junctions and ROS Generation in Rats

“…17,21,22 In CNV, besides the RPE barrier, Rap1 may also regulate the barrier function of CECs to affect angiogenesis. 15,23 Therefore, an alternative interpretation to increased ZO-1 and occludin expression by 8CPT is that CECs have more stable tight junctions, and thus they are less likely to remodel and migrate ( Figure 5). This could yield an inhibition to CNV lesion.…”

“…This result suggests that Rap1a de-escalates CNV development by interfering with ROSdependent signaling. 23 Similarly, in RPE inhibition of Rap1 by Rap1GAP increases ROS generation, whereas activation of Rap1 by 8CPT reduces ROS by interfering with NADPH oxidase assembly. 26 Our results in a rat laser-induced CNV model showed that reducing ROS generation by antioxidant apocynin treatment inhibited CNV formation in rats.…”

Retracted: MicroRNA‑224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1

“…Rac1 is a member of the Ras superfamily of Rho proteins, and it plays an essential role in many cellular processes, including mitogenesis, kinase cascade activation, transcriptional activation, DNA synthesis and cytoskeleton reorganization [37][38][39][40][41][42]. Rac1 overexpression has been found in various types of cancer, such as lung cancer, gastric cancer, pancreatic cancer, bladder cancer and breast cancer [43][44][45][46][47].…”