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.

Fig 1. EHD2 is expressed in endothelial cells and stabilizes caveolae at the plasma membrane.

(A) Cryostat section of adult C57BL/6N small arteries in brown adipose tissue stained against EHD2 and actin. (B) EHD2 and eNOS stained in aorta cryostat sections obtained from C57BL/6N mice illustrating co-localization of both proteins within the endothelium. (C) Cryostat sections of lymphatic vessels were stained against EHD2 and actin. (D) In situ hybridization of a C57BL/6N E18.5 embryo showed strong EHD2 expression in blood vessels. Cryostat sections of adult C57BL/6N mesenteric arteries (E) and HUVEC cells (F) stained against Cav1 and EHD2 revealed EHD2 localization at caveolae. (G) Representative EM images of EHD2 del/+ and EHD2 del/del arteries in which caveolae number, size and diameter were determined. (H) EHD2 del/del mice showed an increased number of detached caveolae compared to EHD2 del/+, in which mainly membrane-bound caveolae were found (n(del/+) = 142/3; n(del/del) = 251/3, graphs illustrate each replicate with mean +/- SE, column bar graph illustrates mean, t-test or Mann Whitney U test were used to calculate significance, * P<0.05; ** P<0.001; *** P<0.0001). White arrows point to caveolae.

Fig 2. Loss of EHD2 results in impaired relaxation of mesenteric arteries.

(A) Representative histological staining of EHD2 del/+ and del/del mesenteric arteries revealed no severe morphological differences. (B) Summary of mesenteric artery diameter in EHD2 del/+ and del/del mice (n(del/+) = 33; n(del/del) = 35). (C) Example traces illustrating time course of active force measurements of EHD2 del/+ and del/del mesenteric arteries that were stimulated by phenylephrine (PE) and acetylcholine (ACh). Summary of data on relaxation (D) and contraction (E) of mesenteric arteries isolated from EHD2 del/+ and del/del mice (n(del/+) = 8/5; n(del/del) = 11/5). The data demonstrate reduced relaxation of EHD2 del/del arteries to ACh. Active force traces of EHD2 del/+ (F, H) or EHD2 del/del (G, H) mesenteric arteries treated with L-NAME (gray traces, untreated: n(del/+) = 17/5; n(del/del) = 36/5; L-NAME treated: n(del/+) = 8/5; n(del/del) = 5/5). (I) Application of NO donor SNP caused similar dose-dependent relaxations of EHD2 del/+ and del/del mesenteric arteries (n = 6, c(PE) = 1 μM). (J, K) Tail cuff blood pressure measurements of EHD2 del/+ and del/del mice (50 weeks old, n = 5) revealed no differences between EHD2 del/+ and del/del mice. Graphs illustrate each replicate with mean +/- SE, column bar and line graphs illustrate mean + SE, t-test or Mann Whitney U test were used to calculate significance, * P<0.05; n.s. not significant.

Fig 3. Decreased NO concentrations and impairment of eNOS localization in EHD2 del/del mesenteric arteries.

(A) NO abundance was determined by DAF staining of EHD2 del/+ and del/del mesenteric arteries either untreated or pre-treated with L-NAME. (B) DAF staining intensity is significantly reduced for EHD2 del/del arteries compared to EHD2 del/+ (L-NAME: n(del/+) = 51/3; n(del/del) = 43/3; untreated: n(del/+) = 121/3; n(del/del) = 127/3). (C) STED imaging of eNOS (blue) and Cav1 (magenta) in EHD2 del/+ mesenteric artery cryostat sections illustrates eNOS localization at the plasma membrane of the small arteries in close proximity to caveolae (merge). (D) EHD2 del/del mesenteric arteries showed reduced eNOS staining at the plasma membrane, instead eNOS was detected within the cytosol of endothelial cells. Scale bar 4 μm. See also S4 Fig. (E) Plasma membrane eNOS staining was analyzed and revealed decreased eNOS fluorescence intensity in EHD2 del/del mesenteric arteries compared to EHD2 del/+ (n(del/+) = 83/3; n(del/del) = 81/3). (F) Analysis of total eNOS protein concentration in tissue lysates obtained from EHD2 del/+ and del/del small vessels by Western Blot. Calmodulin was used as loading control. Graphs illustrate each replicate with mean +/- SE, t-test or Mann Whitney U test were used to calculate significance, *** P<0.0001; n.s. not significant.

Fig 4. EHD2 knockdown HUVECs showed eNOS localization in detached caveolae.

(A) HUVECs were stained against Cav1 (gray) and eNOS (red) and STED microscopy was used to analyze single caveolae. Overview of cells was obtained by confocal imaging (left image). STED was then applied on a 20 μm² area of the flat part of the cell (right image). (B, C) Membrane-bound caveolae appear in ring-like structures, detached caveolae from the plasma membrane are illustrated as filled vesicle-like shape (scale bar 200 nm). (D) Characteristic Cav1 fluorescence intensity (FI) plot profile of ring-like membrane bound and vesicle-like detached caveolae corresponds to surface plots and indicates for both caveolae shapes. (E) eNOS fluorescence intensity was measured for single caveolae in EHD2 control and EHD2 siRNA-treated HUVECs and revealed increased eNOS intensity in the absence of EHD2 (n(Negt. CTRL) = 61/2, n(EHD2 siRNA) = 64/2). (F) EM images illustrate characteristic caveolae shapes in HUVEC control and EHD2 siRNA treated cells, whereby EHD2 knockdown HUVECs revealed an increased number of detached caveolae (n(Negt CTRL) = 106/2, n(EHD2 si) = 104/2, scale bar 200 nm, graphs represent all replicates with mean+/- SE, Mann Whitney U test were used to calculate significance, *** P<0.0001).

Fig 5. EHD2 siRNA treated HUVEC showed decreased NO generation and cytosolic Ca²⁺ response.

(A) DAF staining of either non-sense siRNA or EHD2 siRNA (siRNA#1 or siRNA#2) treated HUVECs after acetylcholine stimulation revealed significantly reduced DAF fluorescence intensity in EHD2 siRNA-treated HUVECs. (B) Pre-incubation with L-NAME abolished NO production in all investigated HUVECs (L-NAME: n(Negt. CTRL) = 34/3, n(siRNA#1) = 37/3; n(siRNA#2) = 14/3); untreated: n(Negt. CTRL) = 34/3, n(siRNA#1) = 46/3; n(siRNA#2) = 37/3)). (C) Cytosolic Ca²⁺ recording in HUVEC treated with EHD2 siRNA. Ca²⁺ response was triggered by 30 μM ATP as illustrated in the examples traces. (D, E) Reduced Ca²⁺ peaks (as obtained by integration) and duration were observed in EHD2 knockdown HUVECs. (F) Fura2 fluorescence intensity excited at 380 nm was used as marker for overall cytosolic Ca²⁺ load (n(Negt. CTRL) = 11/3, n(siRNA#1) = 6/3; n(siRNA#2) = 6/3). (G) Reduction of phosphorylation level of eNOS-Ser1177 in HUVEC treated with EHD2 siRNA compared to non-sense siRNA after acetylcholine stimulation (n(Negt. CTRL) = 8, n(siRNA#1) = 8; n(siRNA#2) = 7). (H) Phosphorylation of AKT-Thr308 and AKT-Ser473 in HUVEC revealed no difference between EHD2 siRNA or control siRNA treated cells (n(Negt. CTRL) = 10, n(siRNA#1) = 11; n(siRNA#2) = 10). (I) Schematic overview of acetylcholine-triggered Ca²⁺ response and eNOS activation. After acetylcholine binding to the G protein coupled muscarinic M3 receptor or ATP binding to P2 purinergic receptor (1), PLC is activated and triggers IP₃ production (2). IP₃ binds to the IP₃ receptor within the ER membrane and induces local Ca²⁺ release via IP₃ receptors (3), which correlates to the first peak observed in the Ca²⁺ recordings. Local intracellular Ca²⁺ close to the plasma membrane then activates Ca²⁺ channels localized within caveolae (4, store operated Ca²⁺ entry, SOCE). Increased Ca²⁺ influx increases the cytosolic Ca²⁺ concentration (second peak, longer duration). After Ca²⁺ binds to calmodulin (CaM) (5), CaM binds to eNOS (6) followed by the disruption of eNOS-Cav1 interaction and translocation of eNOS into the cytosol. Ca²⁺/CaM further induces autophosphorylation of CamKII resulting in phosphorylation of Ser1177 of eNOS (7). Activated eNOS catalyzes the conversion of L-arginine to L-citrulline leading to NO production (8). For comparison to the situation in EHD2 knockout cells, see S6 Fig. Graph illustrates each replicate with mean +/- SE, box plots indicate mean with whiskers from min to max, t-test or Mann Whitney U test were used to calculate significance, * P<0.05; ** P<0.001; *** P<0.0001; n.s. not significant.

Fig 6. EHD2 del/del mice showed reduced running wheel activity.

(A) Heart paraffin sections illustrated no apparent morphological changes in EHD2 del/del compared to EHD2 del/+ mice. (B) Echocardiography measurement of 20 weeks old mice showed an increased left ventricle wall size in EHD2 del/del mice (n = 6). (C) Recorded running wheel activity of EHD2 del/+ and del/del mice during the night phase (n(del/+) = 7, n(del/del) = 8). (D) Measured running distance of EHD2 del/+ and del/del mice over two weeks revealed significantly reduced total running distance for EHD2 del/del mice compared to the control group. (E) Heart rate of EHD2 del/+ and del/del mice before and after the running wheel training (n = 6–12) was not impaired. (F) Body weight was not influenced by the running wheel exercise in EHD2 del/+ and del/del mice (n = 6–12). (G) Left ventricle wall size is aligned after two weeks of running wheel training in EHD2 del/+ and del/del mice (n = 6–12). Graphs illustrate each replicate with mean +/- SE, t-test or Mann Whitney U test were used to calculate significance, * P<0.05; ** P<0.001. LVPW–left ventricular posterior wall thickness, IVS–interventricular septum.