Abstract

BACKGROUNDIn hypoxic and pseudohypoxic rodent models of pulmonary arterial hypertension (PAH), hypoxia-inducible factor (HIF) inhibition reduces disease severity. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PAH in humans, indicating the involvement of other mechanisms. METHODSPseudohypoxia was investigated in primary human lung endothelial cells by silencing PHD2, and in Tie2-Cre/Phd2 knockout mice, a rodent model of PAH. Lung vascular endothelial cells from PAH patients, and lung tissue from both SU5416/hypoxia PAH rats and PAH patients, were examined for validation. RESULTSPHD2 silencing or inhibition, while activating HIF2, induces apoptosis-resistance, hypo-proliferation, and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activates AKT and ERK, inhibits JNK, and reduces AIP1 (ASK1-interacting protein 1), all independent of HIF2. Like PHD2, AIP1 silencing affects these same kinase pathways and produces a similar dysfunctional endothelial cell phenotype, which can be partially reversed by AKT inhibition. These findings are corroborated in lung tissues of rodent PAH models and pulmonary vascular endothelial cells and tissues from PAH patients. CONCLUSIONSPHD2 deficiency in lung vascular endothelial cells induces an apoptosis-resistant, inflammatory, and hypo-proliferative phenotype. AKT activation and AIP1 loss, but not HIF signaling, drive these aberrant phenotypic changes. Our study suggests that HIF blockade alone may not suffice for PAH therapy; targeting PHD2, AKT, and AIP1 has the potential for developing more effective treatment. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/578286v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1fd0a0borg.highwire.dtl.DTLVardef@19e6ba9org.highwire.dtl.DTLVardef@1ae2827org.highwire.dtl.DTLVardef@ce31ec_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPHD2 silencing in human lung vascular endothelial cells suppresses apoptosis, inhibits proliferation, and activates STAT signaling, effects that persist despite HIF2 inhibition or knockdown. C_LIO_LIPHD2 silencing activates AKT and ERK, inhibits JNK, and decreases AIP1, all independently of HIF2 C_LIO_LILike PHD2, AIP1 silencing led to similar alterations in kinase signaling and endothelial cell phenotypes, which are partially reversed by ATK inhibition. C_LIO_LIThese in vitro findings align with observations in lung vascular endothelial cells and tissues from rodent models of PAH as well as PAH patients. C_LI