f Hypoxia-inducible factors 1 and 2 (HIF-1 and -2) control oxygen supply to tissues by regulating erythropoiesis, angiogenesis and vascular homeostasis. HIFs are regulated in response to oxygen availability by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. In this study, we used a genetic approach to investigate the endothelial PHD2/HIF axis in the regulation of vascular function. We found that inactivation of Phd2 in endothelial cells specifically resulted in severe pulmonary hypertension (ϳ118% increase in right ventricular systolic pressure) but not polycythemia and was associated with abnormal muscularization of peripheral pulmonary arteries and right ventricular hypertrophy. Concurrent inactivation of either Hif1a or Hif2a in endothelial cell-specific Phd2 mutants demonstrated that the development of pulmonary hypertension was dependent on HIF-2␣ but not HIF-1␣. Furthermore, endothelial HIF-2␣ was required for the development of increased pulmonary artery pressures in a model of pulmonary hypertension induced by chronic hypoxia. We propose that these HIF-2-dependent effects are partially due to increased expression of vasoconstrictor molecule endothelin 1 and a concomitant decrease in vasodilatory apelin receptor signaling. Taken together, our data identify endothelial HIF-2 as a key transcription factor in the pathogenesis of pulmonary hypertension.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by innumerous fluid-filled cysts and progressive deterioration of renal function. Previously, we showed that periostin, a matricellular protein involved in tissue repair, is markedly overexpressed by cyst epithelial cells. Periostin promotes cell proliferation, cyst growth, interstitial fibrosis, and the decline in renal function in PKD mice. Here, we investigated the regulation of these processes by the integrin-linked kinase (ILK), a scaffold protein that links the extracellular matrix to the actin cytoskeleton and is stimulated by periostin. Pharmacologic inhibition or shRNA knockdown of ILK prevented periostin-induced Akt/mammalian target of rapamycin (mTOR) signaling and ADPKD cell proliferation Homozygous deletion of ILK in renal collecting ducts (CD) of ; mice caused tubule dilations, apoptosis, fibrosis, and organ failure by 10 weeks of age. By contrast, ; mice had normal renal morphology and function and survived >1 year. Reduced expression of ILK in ; mice, a rapidly progressive model of ADPKD, decreased renal Akt/mTOR activity, cell proliferation, cyst growth, and interstitial fibrosis, and significantly improved renal function and animal survival. Additionally, CD-specific knockdown of ILK strikingly reduced renal cystic disease and fibrosis and extended the life of mice, a slowly progressive PKD model. We conclude that ILK is critical for maintaining the CD epithelium and renal function and is a key intermediate for periostin activation of signaling pathways involved in cyst growth and fibrosis in PKD.

BackgroundPulmonary hypertension (PH), a common clinical problem characterized by increased pulmonary artery (PA) pressure, is frequently triggered by hypoxia. Key mediators of cellular hypoxia responses are hypoxia-inducible factors (HIF)-1 and -2, the activity of which is regulated by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. Although both transcription factors are expressed in the lung, little is known about their cell type-specific roles in the pathogenesis of PH.Methods and ResultsHere we used a genetic approach to investigate the role of endothelial PHD2/HIF axis in the regulation of PA pressure. Endothelial cell specific HIF activation was achieved by crossing Vecadherin (Cdh5)-Cre transgenics to Phd2 floxed mice (ePhd2), while the contribution of each HIF isoform was assessed by generating double mutants lacking Phd2 and Hif-2 (ePhd2Hif2) or Phd2 and Hif-1 (Phd2Hif1). Right ventricular systolic pressure (RVSP) was measured via insertion of a 1.4F Mikro-tip catheter transducer into a surgically exposed right internal jugular vein. ePhd2 mice showed activation of HIF-signaling as shown by immunoblot analysis of lung tissue for HIF-1 and HIF-2. These mice developed spontaneous PH (RVSP, ePhd2: 54.3±6.9 vs Cre-: 24.8±2.2 mm Hg, P=0.005), which was associated with right ventricular hypertrophy (RVH) (Fulton Index, ePhd2: 0.52 vs Cre-: 0.28, P=0.0004) and early mortality. While morphologic analysis of ePhd2 lungs did not demonstrate plexiform or lumen-obliterating lesions, enhanced muscularization of peripheral PAs was detected in mutants compared to controls, as indicated by an increase in the number of arteries with diameters <100 µm that stained positive for αSMA (22.1±1.6 vs. 7.6±1.5 muscularized vessels/10 hpf, P<0.0001). The PH phenotype was maintained in ePhd2Hif1 mutants but was reversed in ePhd2Hif2 mutants. To assess the contribution of endothelial HIF-2 in hypoxia induced PH, endothelial Hif2 single mutants or Cre-littermates were exposed to normobaric hypoxia (10% O2) for 4 weeks. In contrast to controls, eHif2 mutants were protected from development of PH and RVH. Bone marrow transplantation studies showed no contribution from hematopoietic HIF-2 in hypoxia induced PH. Because hypoxia regulates endothelin 1 (EDN1), a potent vasoconstrictor but also apelin (APLN), a vasodilatory peptide acting through binding to the apelin G-protein-coupled receptor (APLNR), we assessed the role of endothelial HIF-2 axis in the regulation of these molecules. Endothelial deletion of Phd2 resulted in 6.4-fold induction of pulmonary Edn1 mRNA (P=0.029), but not Apln mRNA. In contrast, Aplnr was downregulated by 2.5-fold in ePhd2 mutants (P=0.037). A similar pattern of expression was detected in ePhd2Hif1 mice, whereas simultaneous deletion of Hif2a and Phd2 reversed these changes. To investigate the differences between acute and chronic hypoxia, we examined the effects of acute HIF activation on Edn1 and Apln/Aplnr gene expression in viv...