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.

A major functional component of the blood-brain barrier is P-glycoprotein. In principle, inhibition of this efflux transporter would permit greater distribution of its substrates into the brain and increased central effects. Tariquidar and elacridar, potent and selective P-glycoprotein inhibitors, were investigated in this regard using the opioid loperamide as an in vivo probe in mice. Pretreatment with both inhibitors converted intravenous loperamide from a drug without central effects to one producing antinociception. Radiolabeled loperamide tissue distribution studies indicated that inhibition was associated with increased uptake into brain and testes in the absence of changes in plasma levels, along with enhanced efflux of rhodamine 123 from CD3e ϩ T-lymphocytes. However, with tariquidar, the loperamide dose-response curves for testes/plasma and brain/ plasma concentration ratios were shifted 6-(p ϭ 0.07) and 25-fold (p Ͻ 0.01) to the right, respectively (ED 50 ϭ 1.48 and 5.65 mg/kg), compared with the rhodamine 123 efflux curve (ED 50 ϭ 0.25 mg/kg). Less pronounced shifts were noted with elacridar where the brain/plasma ratio was shifted only 2-fold relative to the rhodamine 123 efflux data (ED 50 ϭ 2.36 versus 1.34 mg/kg, respectively; p Ͻ 0.01). These results indicate that the P-glycoprotein localized in the blood-brain barrier and, to a lesser extent, the testes-blood barrier is more resistant to inhibition than at other tissue sites such as the lymphocyte; moreover, the extent of this effect depends on the inhibitor. Such resistance can be overcome by a sufficiently high dose of an inhibitor; however, whether this is safely attainable in the clinical situation remains to be determined.

Background Pulmonary arterial hypertension (PAH) is a proliferative disease of the pulmonary vasculature which preferentially affects females. Estrogens, such as the metabolite 16α-hydroxyestrone (16αOHE), may contribute to PAH pathogenesis; and, alterations in cellular energy metabolism associate with PAH. We hypothesized that 16αOHE promotes heritable PAH (HPAH) via miR-29 family upregulation, and that antagonism of miR-29 would attenuate pulmonary hypertension in transgenic mouse models of Bmpr2 mutation. Methods and Results MicroRNA (miR) array profiling of human lung tissue found elevation of miRs associated with energy metabolism, including the miR-29 family, among HPAH patients. miR-29 expression was 2-fold higher in Bmpr2 mutant mice lungs at baseline compared to controls, and 4 to 8-fold higher in Bmpr2 mice exposed to 16αOHE 1.25 μg/hr for 4 weeks. Blot analyses of Bmpr2 mouse lung protein showed significant reductions in PPARγ and CD36 in those mice exposed to 16αOHE, as well as from protein derived from HPAH lungs compared to controls. Bmpr2 mice treated with anti-miR-29 (α-miR29) (20mg/kg injections for 6 weeks) had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism compared to controls. PASMCs derived from Bmpr2 murine lungs demonstrated mitochondrial abnormalities, which improved with α-miR29 transfection in vitro; endothelial-like cells derived from HPAH patient iPS cell lines were similar, and improved with α-miR29 treatment. Conclusions 16αOHE promotes the development of HPAH via upregulation of miR-29, which alters molecular and functional indices of energy metabolism. Antagonism of miR-29 improves in vivo and in vitro features of HPAH, and reveals a possible novel therapeutic target.

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.

Serotonergic anorexigens are the primary pharmacologic risk factor associated with pulmonary arterial hypertension (PAH), and the resulting PAH is clinically indistinguishable from the heritable form of disease, associated with BMPR2 mutations. Both BMPR2 mutation and agonists to the serotonin receptor HTR2B have been shown to cause activation of SRC tyrosine kinase; conversely, antagonists to HTR2B inhibit SRC trafficking and downstream function. To test the hypothesis that a HTR2B antagonist can prevent BMRP2 mutation induced PAH by restricting aberrant SRC trafficking and downstream activity, we exposed BMPR2 mutant mice, which spontaneously develop PAH, to a HTR2B antagonist, SB204741, to block the SRC activation caused by BMPR2 mutation. SB204741 prevented the development of PAH in BMPR2 mutant mice, reduced recruitment of inflammatory cells to their lungs, and reduced muscularization of their blood vessels. By atomic force microscopy, we determined that BMPR2 mutant mice normally had a doubling of vessel stiffness, which was substantially normalized by HTR2B inhibition. SB204741 reduced SRC phosphorylation and downstream activity in BMPR2 mutant mice. Gene expression arrays indicate that the primary changes were in cytoskeletal and muscle contractility genes. These results were confirmed by gel contraction assays showing that HTR2B inhibition nearly normalizes the 400% increase in gel contraction normally seen in BMPR2 mutant smooth muscle cells. Heritable PAH results from increased SRC activation, cellular contraction, and vascular resistance, but antagonism of HTR2B prevents SRC phosphorylation, downstream activity, and PAH in BMPR2 mutant mice.