eNOS-NO-induced small blood vessel relaxation requires EHD2-dependent caveolae stabilization

Abstract

Endothelial nitric oxide synthase (eNOS)-related vessel relaxation is a highly coordinated process that regulates blood flow and pressure and is dependent on caveolae. Here, we investigated the role of caveolar plasma membrane stabilization by the dynamin-related ATPase EHD2 on eNOS-nitric oxide (NO)-dependent vessel relaxation. Loss of EHD2 in small arteries led to increased numbers of caveolae that were detached from the plasma membrane. Concomitantly, impaired relaxation of mesenteric arteries and reduced running wheel activity were observed in EHD2 knockout mice. EHD2 deletion or knockdown led to decreased production of nitric oxide (NO) although eNOS expression levels were not changed. Super-resolution imaging revealed that eNOS was redistributed from the plasma membrane to internalized detached caveolae in EHD2-lacking tissue or cells. Following an ATP stimulus, reduced cytosolic Ca2+ peaks were recorded in human umbilical vein endothelial cells (HUVECs) lacking EHD2. Our data suggest that EHD2-controlled caveolar dynamics orchestrates the activity and regulation of eNOS/NO and Ca2+ channel localization at the plasma membrane.

Energy and Dynamics of Caveolae Trafficking

“…Additionally, an increase in fatty acid uptake was detected in EHD2 knockout adipocytes and mouse embryonic fibroblasts. Detailed electron microscopy and tomography further supported the idea that EHD2 is essential for correct membrane stabilization of caveolae as EHD2-lacking tissues contained large numbers of detached caveolae (Matthaeus et al, 2019 , 2020 ; Fan et al, 2020 ; Webb et al, 2020 ). Additionally, we observed an increased detachment of caveolae due to EHD2 removal resulting in reduced calcium entry and a resultant lack of activated eNOS and NO generation in endothelial cells.…”

“…Furthermore, it was shown that in 3T3 fibroblasts EHD2 loss can be rescued by other EHD proteins (Yeow et al, 2017 ). However, a global EHD2 knockout mouse did not exhibit the same observations in adipocytes, fibroblasts, or endothelial cells (Matthaeus et al, 2019 , 2020 ).…”

“…In fixed cells, however, caveolar vesicles can be distinguished by EM or electron tomography as vesicles containing an enclosed lipid bilayer (Popescu et al, 2006 ; Hubert et al, 2020b ; Matthaeus et al, 2020 ; Webb et al, 2020 ). Novel super resolution imaging techniques such as Stochastic Optical Reconstruction microscopy (STORM) and Stimulated Emission Depletion (STED) microscopy with resolution limits up to 40 nm are also able to identify caveolar vesicles (Platonova et al, 2015 ; Tachikawa et al, 2017 ; Yeow et al, 2017 ; Khater et al, 2018 ; Matthaeus et al, 2019 ). Of special interest with regard to caveolar vesicle trafficking is the application of STED and structured-illumination microscopy (SIM) to live cells allowing single caveolae to be tracked throughout the cell.…”

“…Additionally, we observed an increased detachment of caveolae due to EHD2 removal resulting in reduced calcium entry and a resultant lack of activated eNOS and NO generation in endothelial cells. This lead to reduced blood vessel relaxation in EHD2 knockout mice and reduced running wheel endurance (Matthaeus et al, 2019 ). Taken together, these in vivo data clearly indicate that EHD2 oligomerization at the caveolar neck is an essential cell function with severe physiological consequences when EHD2 is missing or its ATPase function is impaired.…”

EHBP1 and EHD2 regulate Dll4 caveolin-mediated endocytosis during blood vessel development

“…In mice, global EHD2 deletion does not affect viability, in contrast with the embryonic lethality of a Notch1 or Dll4 knockout models [54,55]. However, loss of EHD2 has been shown to increase the number of caveolae that were detached from the membrane and significantly reduced production of endothelial nitric oxide, potentially indicating a preference in endothelial function [56]; loss of Notch function has also been associated with reduced endothelial nitric oxide synthase activity [57]. In line with these observations, we observed that EHD2 knockdown resulted in an elevated number of detached caveolae in ECs.…”