ACE2 as therapy for pulmonary arterial hypertension: the good outweighs the bad

Rationale: Endothelial dysfunction plays an integral role in pulmonary hypertension (PH). AMPK (AMP-activated protein kinase) and ACE2 (angiotensin-converting enzyme 2) are crucial in endothelial homeostasis. The mechanism by which AMPK regulates ACE2 in the pulmonary endothelium and its protective role in PH remain elusive. Objectives: We investigated the role of AMPK phosphorylation of ACE2 Ser680 in ACE2 stability and deciphered the functional consequences of this post-translational modification of ACE2 in endothelial homeostasis and PH. Methods: Bioinformatics prediction, kinase assay, and antibody against phospho-ACE2 Ser680 (p-ACE2 S680) were used to investigate AMPK phosphorylation of ACE2 Ser680 in endothelial cells. Using CRISPR-Cas9 genomic editing, we created gain-of-function ACE2 S680D knock-in and loss-of-function ACE2 knockout ( ACE2 −/− ) mouse lines to address the involvement of p-ACE2 S680 and ACE2 in PH. The AMPK–p-ACE2 S680 axis was also validated in lung tissue from humans with idiopathic pulmonary arterial hypertension. Measurements and Main Results: Phosphorylation of ACE2 by AMPK enhanced the stability of ACE2, which increased Ang (angiotensin) 1–7 and endothelial nitric oxide synthase–derived NO bioavailability. ACE2 S680D knock-in mice were resistant to PH as compared with wild-type littermates. In contrast, ACE2 -knockout mice exacerbated PH, a similar phenotype found in mice with endothelial cell–specific deletion of AMPKα2 . Consistently, the concentrations of phosphorylated AMPK, p-ACE2 S680, and ACE2 were decreased in human lungs with idiopathic pulmonary arterial hypertension. Conclusions: Impaired phosphorylation of ACE2 Ser680 by AMPK in pulmonary endothelium leads to a labile ACE2 and hence is associated with the pathogenesis of PH. Thus, AMPK regulation of the vasoprotective ACE2 is a potential target for PH treatment.

This study tested the hypothesis that angiotensin promotes oxidative stress and inflammation in humans via aldosterone and the mineralocorticoid receptor. We measured the effect of intravenous aldosterone (0.7 mug/kg per hour for 10 hours followed by 0.9 mug/kg per hour for 4 hours) and vehicle in a randomized, double-blind crossover study in 11 sodium-restricted normotensive subjects. Aldosterone increased interleukin (IL)-6 (from 4.7+/-4.9 to 9.4+/-7.1 pg/mL; F=4.94; P=0.04) but did not affect blood pressure, serum potassium, or high-sensitivity C-reactive protein. We next conducted a randomized, double-blind, placebo-controlled, crossover study to measure the effect of 3-hour infusion of angiotensin II (2 ng/kg per minute) and norepinephrine (30 ng/kg per minute) on separate days after 2 weeks of placebo or spironolactone (50 mg per day) in 14 salt-replete normotensive subjects. Angiotensin II increased blood pressure (increase in systolic pressure: 13.7+/-7.5 and 15.2+/-9.4 mm Hg during placebo and spironolactone, respectively; P<0.001 for angiotensin II) and decreased renal plasma flow (-202+/-73 and -167+/-112 mL/min/1.73 kg/m(2); P<0.001 for angiotensin II effect) similarly during placebo and spironolactone. Spironolactone enhanced the aldosterone response to angiotensin II (increase of 17.0+/-10.6 versus 9.0+/-5.7 ng/dL; P=0.002). Angiotensin II transiently increased free plasma F(2)-isoprostanes similarly during placebo and spironolactone. Angiotensin II increased serum IL-6 concentrations during placebo (from 1.8+/-1.1 to 2.4+/-1.4 pg/mL; F=4.5; P=0.04) but spironolactone prevented this effect (F=6.4; P=0.03 for spironolactone effect). Norepinephrine increased blood pressure and F(2)-isoprostanes but not aldosterone or IL-6. Aldosterone increases IL-6 in humans. These data suggest that angiotensin II induces IL-6 through a mineralocorticoid receptor-dependent mechanism in humans. In contrast, angiotensin II-induced oxidative stress, as measured by F(2)-isoprostanes, is mineralocorticoid receptor independent and may be pressor dependent.

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.