Abstract P084: Exploring The Role Of Necroptosis In Hypertension

Abstract

Objective: Altered immune cell activation plays a key role in promoting hypertension. How the immune system becomes activated during hypertension is unknown. A possibility is necroptosis, a newly described form of cell death that alerts the immune system to dying cells, causing inflammation. Using Mlkl -/- mice that are genetically unable to undergo necroptosis, we explored the role of necroptosis in inflammation or blood pressure regulation during hypertension. We also examined whether Ang-II sensitizes human and murine cells to necroptosis. Methods: Monocytes were sorted from peripheral blood mononuclear cells of 3 normotensive and 3 untreated hypertensive participants diagnosed with 24h BP monitoring. Primary murine cardiomyocytes and fibroblasts were obtained from 3 litters of neonatal C57BL/6J mice. Necroptosis was induced using TNF-alpha, SMAC mimetic and zVAD in the presence or absence of Ang-II. Viability of human cells was measured using propidium iodide and in murine cells with CellTitre-Glo Viability Assay. Hypertension was induced in male Mlkl -/- mice and WT littermate controls by implanting minipumps containing Ang-II (0.5mg/kg/day, 28 days; n=6/group). BP was measured weekly by tail-cuff. Results: Monocytes from hypertensive participants were not more susceptible to necroptosis than normotensive participants (P=0.79). This was not altered by Ang-II treatment in normotensive (P=0.98) or hypertensive participants (P=0.92). Moreover, Ang-II did not alter the viability of primary murine fibroblasts in vitro (P=0.79). Mouse cardiac fibroblasts were unable to undergo necroptosis. In vivo, there was no difference in BP of Ang-II-treated Mlkl -/- and WT control mice at 4 weeks (P=0.87). Conclusions: We show that monocytes from hypertensive participants were not more susceptible to necroptosis, compared to normotensive participants. Furthermore, Ang-II did sensitize mouse cardiac fibroblasts or human monocytes to necroptosis. No changes in BP were observed between Ang-II treated WT and MLKL knockout mice, suggesting necroptosis does not impact BP regulation. Further analyses are being performed to determine if inflammation or fibrosis are altered when necroptosis is inhibited.