<p>Epac1 inhibits PKR to reduce NLRP3 inflammasome proteins in retinal endothelial cells</p>

Abstract

Purpose: Inflammation has been strongly associated with retinal damage in diseases such as diabetic retinopathy. Several studies have reported that high glucose exposure induces damage to the retinal vasculature. We and others have shown that high glucose can activate the NOD-like receptor family, pyrin domain containing family member 3 (NLRP3) pathway, leading to increased levels of cleaved caspase 1 and IL-1β to activate a number of inflammatory pathways in the retina. Methods: We used retinal endothelial cells grown in normal (5 mM) or high (25 mM) glucose or retinal lysates from endothelial cell-specific knockout mice for exchange protein activated by cAMP 1 (Epac1). Human recombinant protein kinase R (PKR) or C16, a PKR inhibitor, was used on the cells to dissect PKR and NLRP3 signaling. Results: Using retinal endothelial cells (REC) in high glucose and whole retinal lysates from endothelial cell-specific knockout of Epac1, we demonstrate that Epac1 regulates PKR phosphorylation. Using an Epac1 agonist or PKR inhibition with C16, we demonstrated that loss of PKR resulted in reduced NLRP3, cleaved caspase 1, and IL-1β levels. Furthermore, despite the addition of recombinant human PKR, Epac1 was still able to significantly reduce NLRP3 signaling. Conclusion: Overall, these studies demonstrated that PKR regulates the NLRP3 inflammasome in REC, and that Epac1 inhibition of PKR can reduce activation of the NLRP3 inflammasome.

The Protein Kinase R Inhibitor C16 Alleviates Sepsis-Induced Acute Kidney Injury Through Modulation of the NF-κB and NLR Family Pyrin Domain-Containing 3 (NLPR3) Pyroptosis Signal Pathways

“…PKR inhibition has been shown to decrease inflammation and attenuate lung injury in LPS-challenged mice [ 12 , 13 ]. The present in vitro study showed that inhibition of PKR exerted a protective effect through regulation of inflammatory response in macrophages and in retinal endothelial cells [ 8 , 25 ]. The imidazolo-oxindole derivative C16 is the first selective and potent PKR inhibitor to be described.…”

PKR Promotes Oxidative Stress and Apoptosis of Human Articular Chondrocytes by Causing Mitochondrial Dysfunction through p38 MAPK Activation—PKR Activation Causes Apoptosis in Human Chondrocytes

“…The concentration of SC 79 was selected by our preliminary tests (data not shown). The concentrations of ZLN005, C16, Pifithrin-μ and mito TEMPO were referred from published studies [23,24,25,26]. Anti-p38, anti-p-p38, anti-p53, anti-p-p53, anti-AKT, anti-p-AKT, anti-PGC-1α, and anti-β-actin were all bough from Cell Signaling Technology (Danvers, MA, USA).…”

Targeting Pyroptotic Cell Death Pathways in Retinal Disease

“…Endothelial cells line the retinal microvasculature and comprise the highly selective inner BRB ( 65 ). NLRP3/caspase-1 activation and IL-1β release have been recorded in retinal endothelial cells (RECs) in various in vivo and in vitro models of DR ( 42 , 43 , 67 ). Pyroptotic cell death and caspase-1 activity were markedly increased in human retinal microvascular endothelial cells (HRMECs) incubated in 30 mM high glucose compared to controls ( 44 ).…”