S-45-3: PARP INHIBITION AMELIORATES VEGF INHIBITOR-ASSOCIATED VASCULAR DYSFUNCTION VIA TRPM2

Abstract

Hypertension is a common unwanted effect of VEGF inhibitors (VEGFi) commonly used as anti-angiogenic drugs in cancer patients. The combination of VEGFi with olaparib, a PARP inhibitor (PARPi), has been shown to reduce VEGFi-induced hypertension. However underlying molecular mechanisms are unknown. PARP plays major role in the activation of TRPM2, a redox-sensitive calcium channel, which is associated with hypertension-induced vascular dysfunction. Objective: to elucidate whether PARP/TRPM2 axis is involved in the protective mechanisms of the combination VEGFi/PARPi in vascular cells. Design and Methods: Human vascular smooth muscle cells (VSMC), aortic endothelial cells (HAEC), and mice mesenteric arteries were used. Cells/arteries were exposed to axitinib (VEGFi) alone and in combination with olaparib (PARPi). Wire myography assessed vascular function. Reactive oxygen species (ROS) production, Ca2+ influx, protein/gene analysis, PARP activity, and TRPM2 siRNA were assessed in VSMC, and nitric oxide (NO) levels in HAEC. Results: Axitinib increased ROS production in hVSMC (RUL: 0.8 ± 0.2 [Ct] vs. 1.1 ± 0.09 [Axi]), which was followed by an increase in PARP activity (a.u.: 0.09 ± 0.03 [Ct] vs. 0.14 ± 0.004 [Axi]). Axitinib reduced ACh-induced vasodilation (% relaxation: 70.5 [Ct] vs. 34.8 [Axi]), an effect blocked by olaparib. U46619- and ET-1-induced vasoconstriction (% KCl-U4: 101.2 [Ct] vs. 141.4 [Axi]; ET-1: 122.6 [Ct] vs. 152.5 [Axi]) were increased by axitinib, which not observed with the combination axitinib plus olaparib. TRPM2 channel blocker (8-Br-cADPR) attenuated the hypercontractile effects and endothelial dysfunction induced by axitinib in mesenteric arteries. PARP and TRPM2 blockage also overturned the increase in VSMC Ca2+ influx induced by axitinib (AUC: 17541 ± 4708 [Ct] vs. 22249 ± 1438 [Axi]). This was also confirmed by TRPM2 siRNA. Phosphorylation of MLC20 in VSMC (a.u.: 0.028 ± 0.02 [Ct] vs. 0.04 ± 0.01 [Axi]), and eNOS (Thr495) in HAEC (a.u.: 0.99 ± 0.35 [Ct] vs. 1.35 ± 0.10 [Axi]) were enhanced by axitinib and prevented by olaparib and TRPM2 siRNA. HAEC exposed to the combination olaparib and axitinib showed NO levels similar to VEGF-stimulated cells. Pro-inflammatory markers (IL-6, MCP-1 and IL-1B) were also upregulated in axitinib-stimulated VSMC (p < 0.01), which was reduced by ROS and PARP-TRPM2 inhibition. Conclusions: We identify that PARP inhibition, by reducing TRPM2 activation, attenuates the vascular deleterious effects of axitinib. Our study defines novel mechanisms whereby the combination VEGFi/PARPi may reduce vascular dysfunction in VEGFi-treated cancer patients.