An impairment of the vascular endothelium plays a role, either as a primary cause or as a result of organ damage, in most of human diseases. Despite intensive research, molecular mechanisms underlying endothelial dysfunction remain unclear. Potential factors affecting normal endothelial functions could be nicotinamide N-methyltransferase (NNMT) and 1-Methylnicotinamide (MNA). The aim of this work was to explore the role of NNMT in endothelial cells subjected to different types (models) of stressors. Experiments were performed with the use of endothelial cells EA.hy926 in which the NNMT expression was silenced. For EA.hy926 stable transfection, it was used a set of pLKO.1 vectors containing stem-loop cassettes encoding short hairpin RNA (shRNA) targeted to human NNMT. NNMT silenced cells were incubated with increasing concentrations of menadione, ONOO- and H2O2 for 24h and data obtained showed a significant decrease of cell viability in NNMT silenced cell lines compared to controls. Subsequently, metabolic function was evaluated through the Seahorse technology. Surprisingly, NNMT silenced lines showed reduced levels of mitochondrial respiration parameters compared to control line EAhy.926. In the second part of the work, results obtained were confirmed by using Human Aortic Endothelial Cells (HAEC) primary line, in which the MNA production was repressed by novel NNMT inhibitors 5-Amino-1-methylquinoline and 6-Methoxynicotinamide N-methylated. Consistently with data obtained on NNMT silenced EAhy.926 cells, menadione-treated HAECs incubated with NNMT inhibitors showed a significant decrease of cell viability compared to controls. Experiments of Seahorse technology revealed reduced levels of mitochondrial respiration parameters in HAECs treated with NNMT inhibitors compared to control. Taken together, data obtained suggest a possible involvement of NNMT in the bioenergetic regulation of endothelial cells and confirm the protective role of the enzyme in the endothelium.
Nicotinamide N-Methyltransferase Inhibition in Endothelium: Effect on Cell Viability and Respiration / Campagna, Roberto. - (2019 Mar 27).
Nicotinamide N-Methyltransferase Inhibition in Endothelium: Effect on Cell Viability and Respiration
CAMPAGNA, ROBERTO
2019-03-27
Abstract
An impairment of the vascular endothelium plays a role, either as a primary cause or as a result of organ damage, in most of human diseases. Despite intensive research, molecular mechanisms underlying endothelial dysfunction remain unclear. Potential factors affecting normal endothelial functions could be nicotinamide N-methyltransferase (NNMT) and 1-Methylnicotinamide (MNA). The aim of this work was to explore the role of NNMT in endothelial cells subjected to different types (models) of stressors. Experiments were performed with the use of endothelial cells EA.hy926 in which the NNMT expression was silenced. For EA.hy926 stable transfection, it was used a set of pLKO.1 vectors containing stem-loop cassettes encoding short hairpin RNA (shRNA) targeted to human NNMT. NNMT silenced cells were incubated with increasing concentrations of menadione, ONOO- and H2O2 for 24h and data obtained showed a significant decrease of cell viability in NNMT silenced cell lines compared to controls. Subsequently, metabolic function was evaluated through the Seahorse technology. Surprisingly, NNMT silenced lines showed reduced levels of mitochondrial respiration parameters compared to control line EAhy.926. In the second part of the work, results obtained were confirmed by using Human Aortic Endothelial Cells (HAEC) primary line, in which the MNA production was repressed by novel NNMT inhibitors 5-Amino-1-methylquinoline and 6-Methoxynicotinamide N-methylated. Consistently with data obtained on NNMT silenced EAhy.926 cells, menadione-treated HAECs incubated with NNMT inhibitors showed a significant decrease of cell viability compared to controls. Experiments of Seahorse technology revealed reduced levels of mitochondrial respiration parameters in HAECs treated with NNMT inhibitors compared to control. Taken together, data obtained suggest a possible involvement of NNMT in the bioenergetic regulation of endothelial cells and confirm the protective role of the enzyme in the endothelium.File | Dimensione | Formato | |
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