PARP-1 Inhibition Prevents Oxidative and Nitrosative Stress–Induced Endothelial Cell Death via Transactivation of the VEGF Receptor 2

Abstract

Objective-PARP-1, a DNA base repair enzyme, is activated by DNA breaks induced by oxidative (ROS) and nitrosative (RNS) stress. By consuming NAD ϩ , PARP-1 activation can lead to ATP depletion and cell death. Studies suggest that inhibiting PARP-1 activity can attenuate pathologies associated with vascular smooth muscle and endothelial dysfunction. PARP-1 inhibition can also activate the prosurvival serine/threonine kinase, Akt. Vascular endothelial growth factor (VEGF) regulates endothelial cell survival via Akt activation downstream of VEGF receptor 2 (VEGFR2) activation. Here we investigated the hypothesis that PARP-1 inhibition protects human umbilical vein endothelial cells (HUVECs) from ROS-and RNS-induced cell death by limiting NAD ϩ depletion and by activating a prosurvival signaling pathway via VEGFR2 phosphorylation. Methods and Results-We activated PARP-1 in HUVECs by treatment with hydrogen peroxide (H 2 O 2 ) and peroxynitrite (ONOO Ϫ ). Both depleted HUVECs of NAD ϩ and ATP, processes that were limited by the PARP-1 inhibitor, PJ34. ONOO Ϫ and H 2 O 2 -induced cell death and apoptosis were attenuated in cells treated with PJ34 or PARP-1 siRNA. PARP-1 inhibition increased Akt, BAD, and VEGFR2 phosphorylation in HUVECs and in PJ34-treated rabbit aortas. The VEGFR2-specific tyrosine kinase inhibitor SU1498 decreased PARP-1 inhibition-mediated phosphorylation of VEGFR2 and Akt, and also reversed survival effects of PJ34. Finally, PARP-1 inhibition protected cells from death induced by serum starvation, evidence for a role in cell survival independent of energy protection. Conclusions-PARP-1 inhibition prevents ROS-and RNS-induced HUVEC death by maintaining cellular energy in the form of NAD ϩ and ATP, and also by activating a survival pathway via VEGFR2, Akt, and BAD phosphorylation. Key Words: PARP inhibition Ⅲ endothelial cell survival Ⅲ VEGF receptor 2 Ⅲ oxidative stress S everal studies have shown that inhibiting poly-ADP ribose polymerase-1 (PARP-1) attenuates organ dysfunction in settings such as postmyocardial infarction remodeling, 1 ischemia-reperfusion injury, 2 diabetic retinopathy, 3 septic shock, 4 diabetes, 3,5 and atherosclerosis. 6 A major feature of atherosclerosis includes increased levels of reactive nitrosative species (RNS) and reactive oxygen species (ROS) associated with damage to cell membranes and DNA. 7,8 It is not surprising therefore that PARP-1 inhibition was also shown to limit endothelial dysfunction and atherosclerosis in the ApoE Ϫ/Ϫ mouse. 6,9 High levels of ROS and RNS, such as occur in ischemia-reperfusion injury, inflammation, and diabetes mellitus, induce DNA single-strand breaks and activate poly-ADP ribose polymerase-1 (PARP-1). 10 PARP-1 is a zinc finger protein that belongs to a family of 18 identified genes that transcribe poly(ADP-ribose) polymerases, enzymes that catalyze the covalent transfer of poly-ADP units from NAD ϩ to acceptor proteins. PARP-1 has 3 functional domains: a DNA-binding domain (containing a nuclear localization signal), an automodificatio...

Reciprocal regulation of actin cytoskeleton remodelling and cell migration by Ca2+ and Zn2+: role of TRPM2 channels

“…S4E-H). This can be attributed to TRPM2-independent effects of PJ34, as reported previously (Mathews and Berk, 2008). This finding might be of clinical relevance because poly(ADP-ribose) polymerase (PARP) inhibitors are currently being evaluated for cancer therapy (Michels et al, 2014).…”

AMPKα2 exerts its anti-inflammatory effects through PARP-1 and Bcl-6

“…Aberrant activity and/or expression of PARP-1 are present in ECs under abnormally elevated levels of radical oxygen species (ROS), TNF-α, or angiotensin II to cause vascular inflammation (26)(27)(28). Such proinflammatory conditions are linked to decreases in AMPK activity and Bcl-6 expression.…”

Molecular Biology of Atherosclerosis

“…ADP-ribosylation of histones alters their conformation and allows greater access to damaged DNA for repair enzymes. Hydrogen peroxide, although less reactive than other ROS, can also activate PARP-1 (perhaps through formation of highly reactive hydroxyl radical), but to a lesser extent than superoxide and peroxynitrite (1153). PARP-1 is an intriguing enzyme that is rapidly activated 500-fold by binding to single-strand DNA breaks.…”