Pulmonary arterial hypertension is characterized by vascular remodeling associated with obliteration of pulmonary arterioles and formation of plexiform lesions comprised of hyperproliferative endothelial and vascular smooth muscle cells. Here, we describe a novel, microRNA-dependent association between APLN and FGF2 pathways in the pulmonary artery endothelial cells (PAECs), where disruption of APLN signaling results in a robust increase in FGF2 expression. We show that this link is mediated by two microRNAs, miR-424 and miR-503, that are regulated by APLN and significantly downregulated in PAH. MiR-424 and miR-503 exert anti-proliferative effects by targeting FGF2 and FGFR1. Overexpression of miR-424 and miR-503 in PAECs promoted cellular quiescence and inhibited the capacity of PAEC conditioned media to induce proliferation of pulmonary artery smooth muscle cells. We show that reconstitution of miR-424 and miR-503 can ameliorate pulmonary hypertension in experimental models. These studies demonstrate the importance of APLN-miR-424/503-FGF axis in maintaining pulmonary vascular homeostasis.

Rationale The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. Objective This study aimed to characterize the embryonic lethal phenotype of the Apj−/− mice and define the involved downstream signaling targets. Methods and Results We report the first characterization of the embryonic lethality of the Apj−/− mice. Greater than half of the expected Apj−/− embryos died in utero due to cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj−/− embryos surviving to later stages demonstrated incomplete vascular maturation due to a deficiency of vascular smooth muscle cells, and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, non-canonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of MEF2 (myocyte enhancer factor 2). Apj−/− mice have greater endocardial Hdac4 and Hdac5 nuclear localization, and reduced expression of the MEF2 transcriptional target Klf2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj−/− embryos and endothelial cells. Conclusions Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.