Loredana Ciuclan

A novel murine model of severe pulmonary arterial hypertension

RATIONALE The complex pathologies associated with severe pulmonary arterial hypertension (PAH) in humans have been a challenge to reproduce in mice due to the subtle phenotype displayed to PAH stimuli. OBJECTIVES Here we aim to develop a novel murine model of PAH that recapitulates more of the pathologic processes, such as complex vascular remodeling and cardiac indices, that are not characteristic of alternative mouse models. METHODS Inhibition of vascular endothelial growth factor receptor (VEGFR) with SU5416 combined with 3 weeks of chronic hypoxia was investigated. Hemodynamics, cardiac function, histological assessment of pulmonary vasculature, and molecular pathway analysis gauged the extent of PAH pathology development. MEASUREMENTS AND MAIN RESULTS The combination of VEGFR inhibition with chronic hypoxia profoundly exacerbated all measures of PAH-like pathology when compared with hypoxia alone (> 45 mm Hg right ventricular pressure, > 0.35 right ventricular hypertrophy). The changes in pulmonary vascular remodeling in response to hypoxia were further enhanced on SU5416 treatment. Furthermore, hypoxia/SU5416 treatment steadily decreased cardiac output, indicating incipient heart failure. Molecular analysis showed a dysregulated transforming growth factor-β/bone morphogenetic protein/Smad axis in SU5416- and/or hypoxia-treated mice as well as augmented induction of IL-6 and Hif-1α levels. These changes were observed in accordance with up-regulation of Tph1 and Pdgfr gene transcripts as well as a rise in platelet-rich serotonin. Biomarker analysis in response to VEGFR inhibition and/or hypoxia revealed distinct signatures that correlate with cytokine profiles of patients with idiopathic PAH. CONCLUSIONS These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.

Bone morphogenetic protein receptor II regulates pulmonary artery endothelial cell barrier function

Mutations in bone morphogenetic protein receptor II (BMPR-II) underlie most heritable cases of pulmonary arterial hypertension (PAH). However, less than half the individuals who harbor mutations develop the disease. Interestingly, heterozygous null BMPR-II mice fail to develop PAH unless an additional inflammatory insult is applied, suggesting that BMPR-II plays a fundamental role in dampening inflammatory signals in the pulmonary vasculature. Using static- and flow-based in vitro systems, we demonstrate that BMPR-II maintains the barrier function of the pulmonary artery endothelial monolayer suppressing leukocyte transmigration. Similar findings were also observed in vivo using a murine model with loss of endothelial BMPR-II expression. In vitro, the enhanced transmigration of leukocytes after tumor necrosis factor α or transforming growth factor β1 stimulation was CXCR2 dependent. Our data define how loss of BMPR-II in the endothelial layer of the pulmonary vasculature could lead to a heightened susceptibility to inflammation by promoting the extravasation of leukocytes into the pulmonary artery wall. We speculate that this may be a key mechanism involved in the initiation of the disease in heritable PAH that results from defects in BMPR-II expression.

Activity of the Estrogen-Metabolizing Enzyme Cytochrome P450 1B1 Influences the Development of Pulmonary Arterial Hypertension

Background— Pulmonary arterial hypertension (PAH) is a hyperproliferative vascular disorder observed predominantly in women. Estrogen is a potent mitogen in human pulmonary artery smooth muscle cells and contributes to PAH in vivo; however, the mechanisms attributed to this causation remain obscure. Curiously, heightened expression of the estrogen-metabolizing enzyme cytochrome P450 1B1 (CYP1B1) is reported in idiopathic PAH and murine models of PAH. Methods and Results— Here, we investigated the putative pathogenic role of CYP1B1 in PAH. Quantitative reverse transcription–polymerase chain reaction, immunoblotting, and in situ analysis revealed that pulmonary CYP1B1 is increased in hypoxic PAH, hypoxic+SU5416 PAH, and human PAH and is highly expressed within the pulmonary vascular wall. PAH was assessed in mice via measurement of right ventricular hypertrophy, pulmonary vascular remodeling, and right ventricular systolic pressure. Hypoxic PAH was attenuated in CYP1B1−/− mice, and the potent CYP1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene (TMS; 3 mg · kg−1 · d−1 IP) significantly attenuated hypoxic PAH and hypoxic+SU5416 PAH in vivo. TMS also abolished estrogen-induced proliferation in human pulmonary artery smooth muscle cells and PAH–pulmonary artery smooth muscle cells. The estrogen metabolite 16&agr;-hydroxyestrone provoked human pulmonary artery smooth muscle cell proliferation, and this mitogenic effect was greatly pronounced in PAH–pulmonary artery smooth muscle cells. ELISA analysis revealed that 16&agr;-hydroxyestrone concentration was elevated in PAH, consistent with CYP1B1 overexpression and activity. Finally, administration of the CYP1B1 metabolite 16&agr;-hydroxyestrone (1.5 mg · kg−1 · d−1 IP) caused the development of PAH in mice. Conclusions— Increased CYP1B1-mediated estrogen metabolism promotes the development of PAH, likely via the formation of mitogens, including 16&agr;-hydroxyestrone. Collectively, this study reveals a possible novel therapeutic target in clinical PAH.

Imatinib Attenuates Hypoxia-induced Pulmonary Arterial Hypertension Pathology via Reduction in 5-Hydroxytryptamine through Inhibition of Tryptophan Hydroxylase 1 Expression

RATIONALE Whether idiopathic, familial, or secondary to another disease, pulmonary arterial hypertension (PAH) is characterized by increased vascular tone, neointimal hyperplasia, medial hypertrophy, and adventitial fibrosis. Imatinib, a potent receptor tyrosine kinase inhibitor, reverses pulmonary remodeling in animal models of PAH and improves hemodynamics and exercise capacity in selected patients with PAH. OBJECTIVES Here we use both imatinib and knockout animals to determine the relationship between platelet-derived growth factor receptor (PDGFR) and serotonin signaling and investigate the PAH pathologies each mediates. METHODS We investigated the effects of imatinib (100 mg/kg) on hemodynamics, vascular remodeling, and downstream molecular signatures in the chronic hypoxia/SU5416 murine model of PAH. MEASUREMENTS AND MAIN RESULTS Treatment with imatinib reduced all measures of PAH pathology observed in hypoxia/SU5416 mice. In addition, 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) expression were reduced compared with the normoxia/SU5416 control group. Imatinib attenuated hypoxia-induced increases in Tph1 expression in pulmonary endothelial cells in vitro via inhibition of the PDGFR-β pathway. To better understand the consequences of this novel mode of action for imatinib, we examined the development of PAH after hypoxic/SU5416 exposure in Tph1-deficient mice (Tph1(-/-)). The extensive changes in pulmonary vascular remodeling and hemodynamics in response to hypoxia/SU5416 were attenuated in Tph1(-/-) mice and further decreased after imatinib treatment. However, imatinib did not significantly further impact collagen deposition and collagen 3a1 expression in hypoxic Tph1(-/-) mice. Post hoc subgroup analysis suggests that patients with PAH with greater hemodynamic impairment showed significantly reduced 5-HT plasma levels after imatinib treatment compared with placebo. CONCLUSIONS We report a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-β signaling. Our data reveal interplay between PDGF and 5-HT pathways within PAH, demonstrating TPH1-dependent imatinib efficacy in collagen-mediated mechanisms of fibrosis.

MicroRNA-140-5p and SMURF1 regulate pulmonary arterial hypertension

Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been demonstrated to promote pulmonary arterial endothelial cell dysfunction and induce pulmonary arterial smooth muscle cell (PASMC) proliferation, leading to the development of pulmonary arterial hypertension (PAH). MicroRNAs (miRs) mediate higher order regulation of cellular function through coordinated modulation of mRNA targets; however, miR expression is altered by disease development and drug therapy. Here, we examined treatment-naive patients and experimental models of PAH and identified a reduction in the levels of miR-140-5p. Inhibition of miR-140-5p promoted PASMC proliferation and migration in vitro. In rat models of PAH, nebulized delivery of miR-140-5p mimic prevented the development of PAH and attenuated the progression of established PAH. Network and pathway analysis identified SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) as a key miR-140-5p target and regulator of BMP signaling. Evaluation of human tissue revealed that SMURF1 is increased in patients with PAH. miR-140-5p mimic or SMURF1 knockdown in PASMCs altered BMP signaling, further supporting these factors as regulators of BMP signaling. Finally, Smurf1 deletion protected mice from PAH, demonstrating a critical role in disease development. Together, these studies identify both miR-140-5p and SMURF1 as key regulators of disease pathology and as potential therapeutic targets for the treatment of PAH.

Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II

Previous studies from our group have demonstrated that bone morphogenetic protein receptor-II (BMPR-II), expressed on pulmonary artery endothelial cells, imparts profound anti-inflammatory effects by regulating the release of proinflammatory cytokines and promoting barrier function by suppressing the transmigration of leukocytes into the pulmonary vessel wall. Here we demonstrate that, in mice with endothelial-specific loss of BMPR-II expression (L1Cre(+);Bmpr2(f/f)), reduction in barrier function and the resultant pulmonary hypertension observed in vivo are the result of increased leukocyte recruitment through increased CXCR1/2 signaling. Loss of endothelial expressed BMPR-II leads to elevated plasma levels of a wide range of soluble mediators important in regulating leukocyte migration and extravasation, including the CXCR1/2 ligand, KC. Treatment of L1Cre(+);Bmpr2(f/f) mice with the CXCR1/2 antagonist SCH527123 inhibits leukocyte transmigration into lung and subsequently reverses the pulmonary hypertension. Our data have uncovered a previously unrecognized regulatory function of BMPR-II, which acts to regulate the expression of CXCR2 on endothelial cells, suggesting that increased CXCR2 signaling may also be a feature of the human pathology and that CXCR1/2 pathway antagonists may represent a novel therapeutic approach for treating pulmonary hypertension because of defects in BMPR-II expression.

Treatment with Anti–Gremlin 1 Antibody Ameliorates Chronic Hypoxia/SU5416–Induced Pulmonary Arterial Hypertension in Mice

The expression of the bone morphogenetic protein antagonist, Gremlin 1, was recently shown to be increased in the lungs of pulmonary arterial hypertension patients, and in response to hypoxia. Gremlin 1 released from the vascular endothelium may inhibit endogenous bone morphogenetic protein signaling and contribute to the development of pulmonary arterial hypertension. Here, we investigate the impact of Gremlin 1 inhibition in disease after exposure to chronic hypoxia/SU5416 in mice. We investigated the effects of an anti-Gremlin 1 monoclonal antibody in the chronic hypoxia/SU5416 murine model of pulmonary arterial hypertension. Chronic hypoxic/SU5416 exposure of mice induced upregulation of Gremlin 1 mRNA in lung and right ventricle tissue compared with normoxic controls. Prophylactic treatment with an anti-Gremlin 1 neutralizing mAb reduced the hypoxic/SU5416-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy. Importantly, therapeutic treatment with an anti-Gremlin 1 antibody also reduced pulmonary vascular remodeling and right ventricular hypertrophy indicating a role for Gremlin 1 in the progression of the disease. We conclude that Gremlin 1 plays a role in the development and progression of pulmonary arterial hypertension in the murine hypoxia/SU5416 model, and that Gremlin 1 is a potential therapeutic target for pulmonary arterial hypertension.

Genetic ablation of PI3Kγ results in defective IL‐17RA signalling in T lymphocytes and increased IL‐17 levels

The signalling molecule PI3Kγ has been reported to play a key role in the immune system and the inflammatory response. In particular, it facilitates the migration of haemato‐poietic cells to the site of inflammation. In this study, we reveal a novel role for PI3Kγ in the regulation of the pro‐inflammatory cytokine IL‐17. Loss of PI3Kγ or expression of a catalytically inactive mutant of PI3Kγ in mice led to increased IL‐17 production both in vitro and in vivo in response to various stimuli. The kinetic profile was unaltered from WT cells, with no effect on proliferation or other cytokines. Elevated levels of IL‐17 were not due to an aberrant expansion of IL‐17‐producing cells. Furthermore, we also identified an increase in IL‐17RA expression on PI3Kγ−/− CD4+ T cells, yet these cells exhibited impaired PI3K‐dependent signalling in response to IL‐17A, and subsequent NF‐κB phosphorylation. In vivo, instillation of recombinant IL‐17 into the airways of mice lacking PI3Kγ signalling also resulted in reduced phosphorylation of Akt. Cell influx in response to IL‐17 was also reduced in PI3Kγ−/− lungs. These data demonstrate PI3Kγ‐dependent signalling downstream of IL‐17RA, which plays a pivotal role in regulating IL‐17 production in T cells.

T5 MicroRNA-140–5p Regulates Disease Phenotype in Experimental Pulmonary Arterial Hypertension via SMURF1

Introduction and objectives Clinical therapies for the treatment of pulmonary arterial hypertension (PAH) target vasoconstriction. However, the proliferative pulmonary vascular remodelling that drives disease persists contributing to significant patient morbidity and mortality. MicroRNA (miR) are short non-coding RNA that mediate post-transcriptional regulation of mRNA targets. We hypothesise that dysregulation of miR leads to de-repression of cellular targets central to disease pathogenesis. We sought to identify dysregulated circulating miR in patients with PAH, determine their phenotypic effect using in vitro and in vivo models and identify key mechanistic regulators that may represent novel therapeutic targets. Methods Two patient cohorts were used to identify and validate differential expression of miR in whole blood by microarray and single assay qPCR. Binding site and network analysis was used to identify key miR targets. Effect of miR on identified targets and disease phenotype was determined in pulmonary artery smooth muscle cells (PASMC) and in the monocrotaline (MCT) and Sugen5416 plus Hypoxia (SuHx) models of PAH. Results Expression of miR-140–5p was reduced in whole blood samples from patients with PAH and experimental models of PAH. Network and pathway analysis identified key regulators of TGFß and PDGF signalling as miR-140–5p targets. Transfection with miR-140–5p inhibitor resulted in increased proliferation and migration of PASMC and de-repression of key targets. Nebulised delivery of miR-140–5p mimic prevented the development of PAH in the MCT rat model and attenuated progression of established PAH in MCT and SuHx rat models. In experimental models levels of SMURF1 protein correlated inversely with miR-140–5p. Direct regulation of SMURF1 by miR-140–5p was demonstrated in vitro by 3’UTR luciferase activity. Both miR-140–5p mimic and SMURF1 siRNA increased BMP response element activity identifying SMURF1 as a key negative regulator of BMP signalling in PASMC. Genetic ablation of SMURF1 in C57BL6 mice conferred allele dependent protection from SuHx induced PAH. Finally, whole blood mRNA and pulmonary vascular immunoreactivity of SMURF1 was increased in patients with PAH. Conclusions These studies suggest that miR-140–5p and SMURF1 are key regulators of BMP signalling and disease pathology in PAH and highlight SMURF1 as a potential novel therapeutic target.

E microRNA-140-5p and SMURF1 Regulate Pulmonary Arterial Hypertension

Background Clinical therapies for the treatment of pulmonary arterial hypertension (PAH) target vasoconstriction. However, the proliferative pulmonary vascular remodelling that drives disease persists contributing to significant patient morbidity and mortality. MicroRNA (miR) are short non-coding RNA that mediate post-transcriptional regulation of mRNA targets. We hypothesise that dysregulation of miR leads to de-repression of cellular targets central to disease pathogenesis. Objective To identify dysregulated circulating miR in patients with PAH, determine their phenotypic effect using in vitro and in vivo models and identify key mechanistic regulators that may represent novel therapeutic targets. Methods and results Expression of miR-140-5p was reduced in whole blood samples from patients with PAH. Reduced expression of miR-140-5p at the time of diagnosis identified patients with adverse clinical characteristics. Transfection with miR-140-5p inhibitor resulted in increased proliferation PASMC and de-repression of key targets. Nebulised delivery of miR-140-5p mimic attenuated progression of established PAH in the SuHx rat model. Network and pathway analysis identified SMAD Specific E3 Ubiquitin Protein Ligase 1 (SMURF1) as a key miR-140-5p target. Whole blood mRNA and pulmonary vascular immunoreactivity of SMURF1 was increased in patients with PAH implicating SMURF1 in the pathology of human disease. Direct regulation of SMURF1 by miR-140-5p was demonstrated in-vitro by 3’UTR luciferase activity. Both miR-140-5p mimic and SMURF1 siRNA increased BMP response element activity identifying SMURF1 as a key negative regulator of BMP signalling in PASMC. Genetic ablation of SMURF1 in C57BL6 mice conferred allele dependent protection from SuHx induced PAH. Conclusions These studies suggest that miR-140 and SMURF1 are key regulators of BMP signalling and disease pathology in PAH and highlight SMURF1 as a potential novel therapeutic target.