Abstract 049: Endothelial-Derived Microparticles as Biomarkers and Mediators of Endothelial Cell Injury in VEGF Inhibitor-Treated Cancer Patients: Implications in Hypertension

Abstract

Vascular endothelial growth factor receptor inhibitors (VEGFRi), used as anti-angiogenic drugs to treat cancer, induce severe hypertension although the underlying molecular mechanisms are still unclear. Endothelial microparticles (MPs) are biomarkers of endothelial injury and are also functionally active since they influence downstream target cell signalling and function. We questioned whether MP status is altered in cancer patients treated with VEGFRi and whether they influence endothelial cell function associated with vascular dysfunction in hypertension. Plasma MPs were isolated from cancer patients before and after treatment with VEGFRi. Human aortic endothelial cells (HAEC) were stimulated with human plasma-isolated MPs (10 6 MPs/mL). MP characterization was assessed by flow cytometry; protein and gene expression by immunoblotting and qPCR; ROS and NO production by lucigenin and immunofluorescence. Patients treated with VEGFRi had significantly increased in endothelial cell-derived MPs (EMP) (0.19±0.02 Pre vs. 0.31±0.04 Post-treatment). HAEC exposed to post-treatment MPs increased pre-pro-ET-1 mRNA (4.10±0.97 vs. 10.57±3.54), corroborating the raise in ET-1 levels (μg/mL: 631.9±46.1 vs. 780.2±37.2) observed in HAEC stimulated with vatalanib (VEGFRi). Post-treatment MPs increased ROS generation in HAEC (100.0 vs. 5 min- 123.4±7.7, 30 min- 162.4±22.5, 60 min- 183.9±44.7), effects that were attenuated by ET A and ET B receptor blockers. VEGFRi post-treatment MPs increased phosphorylation of the inhibitory site of eNOS (Thr 495 ) (100.0 vs. 181.3±18.7) and decreased NO levels in HAEC (100.0 vs. 72.7±9.9) which was inhibited by ET B receptor blockade (eNOS: 109.8±14.9; NO: 142.1±25.9). Gene expression of proinflammatory mediators was increased in HAEC exposed to post-treatment MPs (1.0 vs. TNF-α: 5.4±1.8, MCP-1: 2.1±0.3, iNOS: 3.1±0.8, COX2: 2.4±0.4, ICAM1: 2.7±0.6), effects inhibited by BQ123 and BQ788. In conclusion, our data identify EMPs as biomarkers of VEGFi-induced endothelial injury and important mediators of ET-1-sensitive redox-regulated endothelial cell signalling. These molecular processes may play a role in vascular dysfunction associated with hypertension in VEGFRi-treated cancer patients.