Ps-b06-8: exploring the role of necroptosis in hypertension

Objective: Altered immune cell activation plays a key role in promoting hypertension. A major question remains as to the immune system becomes activated during hypertension. A possibility is necroptosis, which a newly described form of cell death alerts the immune system to dying cells, causing inflammation. Using mice genetically unable to undergo necroptosis, we aimed to determine whether necroptosis contributes to inflammation and fibrosis or alters blood pressure during hypertension. To determine whether factors present during hypertension impact necroptosis, we examined whether Angiotensin II sensitizes human and murine cells to necroptosis. Design and Methods: Monocytes were sorted from peripheral blood mononuclear cells from 3 normotensive and 3 untreated hypertensive participants diagnosed with 24-h blood pressure (BP) monitoring. Necroptosis was induced in human monocytes using hTNFα (10ug/ml), SMAC mimetic (500 nM), zVAD (20uM), and in primary neonatal murine cardiomyocytes and fibroblasts from 3 litters of C57BL/6J mice using mTNFα (10ug/ml), SMAC mimetic (100 nM), zVAD (10uM), in the presence or absence of angiotensin II (Ang-II, 5 ug/ml). Viability of human cells was measured using propidium iodide by flow cytometry, and in murine cells using CellTitre-Glo Luminescent Cell Viability Assay. Hypertension was induced in male Mlkl -/- mice, which cannot undergo necroptosis, and wild-type (WT) littermate controls by implanting minipumps containing Ang-II (0.5 mg/kg/day for 28 days; n = 6/genotype). Blood pressure (BP) was measured weekly by tail-cuff. Results: Monocytes from hypertensive participants were not more susceptible to necroptosis than normotensive participants (% viability ± SD; 48% ± 0.11 vs 51% ± 0.04, P = 0.79). This was not altered by Ang-II treatment in normotensive (51% ± 0.04 vs 56% ± 0.11 with Ang-II, P = 0.98) or hypertensive participants (48% ± 0.11 vs 56% ± 0.18 with Ang-II, P = 0.92). Moreover, Ang-II did not alter the viability of primary murine cardiomyocytes in vitro (42% ± 23 vs 40.1 ± 17.8 with Ang-II, P = 0.79). Primary 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: Our findings showed that, compared to normotensive participants, monocytes from hypertensive participants were not more susceptible to necroptosis. Furthermore, Ang-II did not directly act on the necroptosis pathway in both mouse cardiac fibroblasts or human monocytes. Changes in BP were not observed between WT and MLKL knockout mice upon Ang-II treatment, suggesting necroptosis does not impact BP regulation. Further analyses are being performed to determine whether inflammation or tissue fibrosis are altered in the absence of necroptosis.