Disruption of the apelin-APJ system worsens hypoxia-induced pulmonary hypertension

Abstract

Objective: The G-protein-coupled receptor APJ and its ligand apelin are highly expressed in the pulmonary vasculature, but their function in this vascular bed is unclear. We hypothesized that disruption of apelin signaling would lead to worsening of the vascular remodeling associated with pulmonary hypertension (PH).

Methods and results: We found that apelin-null mice developed more severe PH compared with wild-type mice when exposed to chronic hypoxia. Micro-computed tomography of the pulmonary arteries demonstrated significant pruning of the microvasculature in the apelin-null mice. Apelin-null mice had a significant reduction of serum nitrate levels. This was secondary to downregulation of endothelial nitric oxide synthase (eNOS), which was associated with reduced expression of Kruppel-like factor 2 (KLF2), a known regulator of eNOS expression. In vitro knockdown studies targeting apelin in human pulmonary artery endothelial cells demonstrated decreased eNOS and KLF2 expression, as well as impaired phosphorylation of AMP-activated kinase and eNOS. Moreover, serum apelin levels of patients with PH were significantly lower than those of controls.

Conclusions: These data demonstrate that disruption of apelin signaling can exacerbate PH mediated by decreased activation of AMP-activated kinase and eNOS, and they identify this pathway as a potentially important therapeutic target for treatment of this refractory human disease.

Figure 1

Apelin-null mice are more susceptible to hypoxia-induced PH. A, Pulmonary apelin and APJ mRNA expression in chronic hypoxia–exposed mice. Graphs depict mouse apelin and APJ expression in response to hypoxia at 1 and 3 weeks (WK). n=5 mice in each group. B, Measurement of RVSPs in wild-type (+/+) and apelin-null (−/−) mice demonstrated a greater increase in RVSP in the apelin-null mice in response to hypoxia. n=7 to 10 mice per group. C, Staining for smooth muscle α-actin (red) demonstrated increased muscularization in hypoxia-treated apelin-null mice. Counterstaining is for von Willebrand Factor (brown). Graph represents the percentage of muscularization of the alveolar wall arteries.

Figure 2

Apelin-null mice develop significant loss of pulmonary microvasculature in response to hypoxia. A, Microcomputed tomography was performed in hypoxia-treated wild-type (+/+) and apelin-null (−/−) mice (n=6 per group). Whole lung images and magnified images to visualize the pulmonary microvasculature are shown. Larger pulmonary arteries (>75 μm) are depicted in purple, whereas the smaller pulmonary arteries (<75 μm) are depicted in gray. B, Quantification of pulmonary arteries by size of the vessels. Density of vessels that were <75 μm (left graph) or between 75 and 100 μm (right graph) before and after chronic hypoxia exposure was determined. n.s. indicates not significant; WT, wild-type; KO, knockout. C, Morphometric analyses of lung sections were performed to quantify the number of vessels <75 μm.

Figure 3

Decreased NO synthesis in apelin-null mice. A, Serum nitrate levels of wild-type (+/+) and apelin-null (−/−) mice. B, Expression of eNOS in the lungs was determined by quantitative polymerase chain reaction methods. n=7 mice in each group. Shown is a Western blot of whole lung lysates with eNOS antibody from wild-type (+/+) and apelin-null (−/−) mice (top panel). GAPDH loading control is also shown (bottom panel). C, Apelin mRNA levels in PAECs transfected with control or apelin siRNA. D, eNOS mRNA (graph) and protein expression in PAECs after siRNA knockdown of apelin. A representative Western blot shows eNOS (top panel) and GAPDH control (bottom panel). E, Expression of KLF2 in the lungs was determined by quantitative polymerase chain reaction methods. n=7 mice in each group. Shown is a representative Western blot of whole lung lysates with KLF2 antibody from wild-type (+/+) and apelin-null (−/−) mice (top panel). GAPDH loading control is also shown (bottom panel). F, siRNA knockdown of apelin in PAECs led to decreased KLF2 mRNA expression.

Figure 4

Apelin regulates phosphorylation of AMPK and its downstream targets in PAECs. A, PAECs transfected with either control or apelin siRNA were subjected to hypoxia and lysed for protein detection of phosphorylated AMPKα, acetyl-CoA carboxylase (ACC), and eNOS. The ratio of phosphorylated (p−) to total protein is depicted in the graphs from 3 independent experiments. *P<0.05. B, Increase in KLF2 mRNA expression in response to hypoxia in PAECs was abrogated with apelin knockdown. *P<0.05, **P<0.01.

Figure 5

Serum apelin level is decreased in patients with PH. Serum apelin level was measured from patients with documented PH versus healthy controls.