Endothelial cell-selective adhesion molecule deficiency exhibits increased pulmonary vascular resistance due to impaired endothelial nitric oxide signaling

Abstract

Endothelial cell-selective adhesion molecule (ESAM) is a member of tight junction molecules, highly abundant in the heart and the lung, and plays a role in regulating endothelial cell permeability. We previously reported that mice with genetic ESAM deficiency (ESAM^-/-) exhibit coronary microvascular dysfunction leading to the development of left ventricular diastolic dysfunction. Here, we hypothesize that ESAM^-/- mice display impairments in the pulmonary vasculature, affecting the overall pulmonary vascular resistance (PVR). We utilized ESAM^-/- mice and employed isolated, ventilated, and perfused whole lung preparation to assess PVR independently of cardiac function. PVR was assessed in response to stepwise increases in flow, and also in response to perfusion of the endothelium-dependent agonist, bradykinin, the thromboxane analog, U46619, and the nitric oxide (NO) donor sodium nitroprusside (SNP). We found that PVR, at every applied flow rate, is significantly elevated in ESAM^-/- mice compared with WT mice. Bradykinin-induced reduction in PVR and U46619-induced increase in PVR were both diminished in ESAM^-/- mice, whereas SNP-induced responses were similar in wild-type (WT) and ESAM^-/- mice. Inhibition of NO synthase with N(ω)-nitro-l-arginine methyl ester increased agonist-induced PVR in WT but not in ESAM^-/- mice. Pulmonary arteries isolated from ESAM^-/- mice exhibited a reduced level of phospho-Ser473-Akt and phospho-Ser1177-eNOS. Furthermore, in human lung microvascular endothelial cells cultured under flow conditions, we found that siRNA-mediated knockdown of ESAM impaired fluid shear stress-induced endothelial cell alignment. Thus, we suggest that ESAM plays an important role in the endothelium-dependent, flow/shear stress- and vasoactive agonist-stimulated, and NO-mediated maintenance of PVR in mice.NEW & NOTEWORTHY Our study reveals a novel role for ESAM in contributing to the maintenance of pulmonary vascular resistance under normal physiological conditions. Employing mice with global genetic deficiency of ESAM and using isolated whole lung preparation, we show significant impairments in nitric oxide-mediated pulmonary artery function. In vitro cell culture studies demonstrate impaired fluid shear stress-induced cell alignment in human lung endothelial cells after siRNA-mediated ESAM knockdown.

Keywords: ESAM; nitric oxide synthase; pulmonary artery; pulmonary vascular resistance.

Figure 1.

Increased pulmonary vascular resistance in ESAM^−/− mice. Representative ultrasound color Doppler images in parasternal long axis mode and traces of pulmonary artery blood flow velocity (A), with summary data of calculated pulmonary acceleration time (PAT) to pulmonary ejection time (PET) ratio (PAT/PET; B) in WT (n = 9) and ESAM^−/− (n = 10). C: summary data of Fulton’s index in WT (n = 6) and ESAM^−/− (n = 4). D: schematic representation of the experimental setup for the isolated, perfused, and ventilated whole lung preparation. E: representative traces for pulmonary vascular perfusion pressure measurement in response to stepwise increases in perfusion flow rates in WT and ESAM^−/− mice. Summary data of pulmonary perfusion pressure (F) and calculated pulmonary vascular resistance (G) in response to stepwise increases in perfusion flow in WT (n = 6) and ESAM^−/− (n = 6) mice. Data presented as means ± SE and analyzed using t test with Welsh’s correction (B and C) or two-way ANOVA with repeated measures (F and G); *P < 0.05. ESAM, endothelial cell-selective adhesion molecule; WT, wild-type.

Figure 2.

Impaired endothelium-dependent, agonist-stimulated reduction of PVR in ESAM^−/− mice. A: schema for the ex vivo whole lung preparation setup used in drug administration experiments. Representative traces (B) and summary data of changes in perfusion pressure in response to injection of cumulative concentration of bradykinin (C) or sodium nitroprusside (SNP) (D) in WT (n = 6) and ESAM^−/− (n = 6) mice. Note that a sudden increase in perfusion pressure occurs at the onset of drug injections, which is considered an artifact. E: summary data for the generated vascular passive force (F_Passive) in response to stepwise stretching of isolated pulmonary arteries (WT, n = 10 vessels isolated from 4 animals; ESAM^−/− n = 5 vessels isolated from 2 animals) in calcium-free PSS. Representative traces (F) and summary data showing the bradykinin-induced response in the absence or presence of NO synthase inhibitor, L-NAME in WT (G) and ESAM^−/− (H) mice. Data are presented as means ± SE and analyzed using two-way ANOVA with repeated measures; *P < 0.05, **P < 0.01. ESAM, endothelial cell-selective adhesion molecule; L-NAME, N(ω)-nitro-l-arginine methyl ester; NO, nitric oxide; PSS, physiological saline solution; PVR, pulmonary vascular resistance; WT, wild-type.

Figure 3.

Impaired NO-mediated signaling in pulmonary arteries. Representative traces (A) and summary graph for basal (B) and U46619-induced changes in perfusion pressure (C), both in the absence and presence of L-NAME, in WT (n = 5) and ESAM^−/− (n = 5) mice. Representative images of Western immunoblots for protein abundance of total eNOS and Akt and also pS1177-eNOS, pT308-Akt, and pS473-Akt in the pulmonary artery (D and E) isolated from WT and ESAM^−/− mice. Summary graphs (E) representing the ratio of phosphorylated and total proteins (eNOS and Akt) and total proteins normalized for housekeeping HSP90 from the pulmonary artery. Data presented as means ± SE and analyzed with an ordinary two-way ANOVA, where the interaction between factors reaches significance (P = 0.0169), followed Tukey’s post hoc multiple comparisons (C) or unpaired t test with Welch’s correction (B and E); *P < 0.05. ESAM, endothelial cell-selective adhesion molecule; NO, nitric oxide; L-NAME, N(ω)-nitro-l-arginine methyl ester; WT.

Figure 4.

VE-cadherin and PECAM coimmunoprecipitate with endothelial cell-selective adhesion molecule (ESAM) while shear stress-induced endothelial cell alignment is impaired after ESAM knockdown. Representative western immunoblotting for ESAM/VE-cadherin and ESAM/PECAM complexes, as well as control for ESAM in cultured endothelial cells (A). Representative Western immunoblot (B) and immunocytochemical staining (C) show the effectiveness of ESAM knockdown in HMLVEC. VE-cadherin was used to co-label endothelial cells. Scale bar: 20 μm. Representative images (D) after 4, 24, 48, and 72 h of culture under flow and summary data (E) from triplicate experiments after 72 h of culture of HLMVEC with control (CTRL) or ESAM siRNA transfection. Scale bar: 100 μm. The ImageJ directionality plugin was used to measure cell orientation angle distribution relative to the flow direction. F: a schema depicts the proposed mechanisms through which ESAM contributes to agonist-induced and shear stress-stimulated and NO-mediated regulation of pulmonary vascular resistance (PVR). HLMVEC, human lung microvascular endothelial cell; IPel, immunoprecipitation eluate; IPfil, immunoprecipitation filtrate; NO, nitric oxide; WCL, whole cell lysates.