Regulation and function of the Na+/H+ exchanger NHE1 in endothelial cells
Na+/H+ exchanger NHE1 is a plasma membrane protein that maintains intracellular pH and cell volume by exchanging extracellular Na+ for intracellular H+ playing role in proliferation, survival and migration of different cell types, but its role in endothelial function is not completely understood. The current work was aimed to examine NHE1’s involvement in endothelial cell functions, particularly angiogenesis. Measurements of the intracellular pH of endothelial cells from the lungs of total Nhe1-/- mice, confirmed that NHE1 is the most important acid-extruder responsible for the pH recovery after cytoplasmic acidification of endothelial cells. In vitro downregulation of NHE1 in primary human umbilical vein endothelial cells reduced endothelial cell proliferation by 35%, without affecting survival. Silencing of NHE1 inhibited chemotaxis induced by sphingosine-1-phosphate by 32%. Knockdown of In agreement with a proliferative and migratory role of NHE1, its involvement in VEGF-induced angiogenesis was demonstrated in vitro and in vivo. Downregulation of NHE1 impaired VEGF-induced spheroid sprouting by 56%. Moreover, a Matrigel plug assay in constitutive Nhe1-/- mice revealed an inhibition of VEGF-induced angiogenesis by 22% compared to wild type littermates. Endothelial cell-specific Nhe1-/- mice were generated using the Cre/loxP-system by mating Nhe1-floxed mice with the VE-Cadherin-Cre-recombinase transgenic mice. However, angiogenesis in these mice using Matrigel plug assay shall be further investigated. Together, these findings portray NHE1 as a player in angiogenesis. Results of this thesis also show that NHE1 is upregulated during hypoxia (1% O2) through HIF2α-dependent mechanisms, which could potentiate NHE1’s role in ischemia-induced angiogenesis. Therefore, in vivo models like hind limb ischemia could yet highlight the role of NHE1 in angiogenesis. The generated endothelial cell-specific Nhe1-/- mice are a valuable tool for future in vivo studies.