Sex-dependent influence of endogenous estrogen in pulmonary hypertension.

RATIONALE: The incidence of pulmonary arterial hypertension is greater in women, suggesting estrogens may play a role in the disease pathogenesis. Experimentally, in males, exogenously administered estrogen can protect against pulmonary hypertension (PH). However, in models that display female susceptibility, estrogens may play a causative role. OBJECTIVES: To clarify the influence of endogenous estrogen and sex in PH and assess the therapeutic potential of a clinically available aromatase inhibitor. METHODS: We interrogated the effect of reduced endogenous estrogen in males and females using the aromatase inhibitor, anastrozole, in two models of PH: the hypoxic mouse and Sugen 5416/hypoxic rat. We also determined the effects of sex on pulmonary expression of aromatase in these models and in lungs from patients with pulmonary arterial hypertension. MEASUREMENTS AND MAIN RESULTS: Anastrozole attenuated PH in both models studied, but only in females. To verify this effect was caused by reduced estrogenic activity we confirmed that in hypoxic mice inhibition of estrogen receptor α also has a therapeutic effect specifically in females. Female rodent lung displays increased aromatase and decreased bone morphogenetic protein receptor 2 and Id1 expression compared with male. Anastrozole treatment reversed the impaired bone morphogenetic protein receptor 2 pathway in females. Increased aromatase expression was also detected in female human pulmonary artery smooth muscle cells compared with male. CONCLUSIONS: The unique phenotype of female pulmonary arteries facilitates the therapeutic effects of anastrozole in experimental PH confirming a role for endogenous estrogen in the disease pathogenesis in females and suggests aromatase inhibitors may have therapeutic potential.

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.

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-ß 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-ß signaling. Our data reveal interplay between PDGF and 5-HT pathways within PAH, demonstrating TPH1-dependent imatinib efficacy in collagen-mediated mechanisms of fibrosis.

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-ß/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.

Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity.

Cardiac tissues express constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in redox signaling. Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A2AR in the regulation of cardiac ROS production remains unknown. We found that knockout of A2AR significantly decreased (39+/-8%) NADPH-dependent O2- production in mouse hearts compared to age (10 weeks)-matched wild-type controls. This was accompanied by a significant decrease in Nox2 (a catalytic subunit of NADPH oxidase) protein expression, and down-regulation of ERK1/2, p38MAPK, and JNK phosphorylation (all P<0.05). In wild-type mice, intraperitoneal injection of the selective A2AR antagonist SCH58261 (3-10 mg/kg body weight for 90 min) inhibited phosphorylation of p47phox (a regulatory subunit of Nox2), which was accompanied by a down-regulated cardiac ROS production (48+/-8%), and decreased JNK and ERK1/2 activation by 54+/-28% (all P<0.05). In conclusion, A2AR through MAPK signaling regulates p47phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A2AR activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.

Critical overview of the management of neonatal jaundice in the UK.

AIM: To determine the current management of early neonatal jaundice in the UK and to evaluate whether the current practices are evidence based. METHODS: A questionnaire survey was carried out among identified lead paediatricians of neonatal intensive care units. RESULTS: The survey found markedly differing practices for the recognition, investigation and treatment of neonatal jaundice. This applies particularly to confirmation of the clinical suspicion of jaundice; use of invasive and non-invasive technologies for diagnosis; preferred wavelength and intensity of light used for treatment; and whether birth weight, gestational age and postnatal age should influence treatment. CONCLUSION: The study found a lack of consistency in the management of jaundiced infants in the UK. The evidence-based practice currently available does not appear to have been incorporated into treatment protocols.

Ritonavir and Topical Ocular Corticosteroid Induced Cushing's Syndrome in an Adolescent With HIV-1 Infection.

Cushing's syndrome after topical ocular corticosteroid use is extremely rare. We describe a case of symptomatic Cushing's syndrome in an adolescent male with sight-threatening vernal keratoconjunctivitis on antiretroviral therapy for HIV-1 infection that included ritonavir, a potent cytochrome p450 CYP3A4 inhibitor. CYP3A4 inhibition reduces the metabolism of exogenous corticosteroids leading to suppression of endogenous steroid production and Cushing's syndrome.

Identification of a Novel Allosteric Inhibitory Site on Tryptophan Hydroxylase 1 Enabling Unprecedented Selectivity Over all Related Hydroxylases.

Pulmonary arterial hypertension (PAH) has demonstrated multi-serotonin receptor dependent pathologies, characterized by increased tone (5-HT1B receptor) and complex lesions (SERT, 5-HT1B, 5-HT2B receptors) of the pulmonary vasculature together with right ventricular hypertrophy, ischemia and fibrosis (5-HT2B receptor). Selective inhibitors of individual signaling elements - SERT, 5-HT2A, 5HT2B, and combined 5-HT2A/B receptors, have all been tested clinically and failed. Thus, inhibition of tryptophan hydroxylase 1 (TPH1), the rate limiting step in 5-HT synthesis, has been suggested as a more broad, and thereby more effective, mode of 5-HT inhibition. However, selectivity over non-pathogenic enzyme family members, TPH2, phenylalanine hydroxylase, and tyrosine hydroxylase has hampered therapeutic development. Here we describe the site/sequence, biochemical, and biophysical characterization of a novel allosteric site on TPH1 through which selectivity over TPH2 and related aromatic amino acid hydroxylases is achieved. We demonstrate the mechanism of action by which novel compounds selectively inhibit TPH1 using surface plasma resonance and enzyme competition assays with both tryptophan ligand and BH4 co-factor. We demonstrate 15-fold greater potency within a human carcinoid cell line versus the most potent known TPH1/2 non-specific inhibitor. Lastly, we detail a novel canine in vivo system utilized to determine effective biologic inhibition of newly synthesized 5-HT. These findings are the first to demonstrate TPH1-selective inhibition and may pave the way to a truly effective means to reduce pathologic 5-HT and thereby treat complex remodeling diseases such as PAH.

Optimization of Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors for Duration of Action, as an Inhaled Therapy for Lung Remodeling in Pulmonary Arterial Hypertension.

A series of potent PDGFR inhibitors has been identified. The series was optimized for duration of action in the lung. A novel kinase occupancy assay was used to directly measure target occupancy after i.t. dosing. Compound 25 shows 24 h occupancy of the PDGFR kinase domain, after a single i.t. dose and has efficacy at 0.03 mg/kg, in the rat moncrotaline model of pulmonary arterial hypertension. Examination of PK/PD data from the optimization effort has revealed in vitro:in vivo correlations which link duration of action in vivo with low permeability and high basicity and demonstrate that nonspecific binding to lung tissue increases with lipophilicity.

Targeting the serotonin pathway for the treatment of pulmonary arterial hypertension.

As we uncover the complex pathophysiology underlying idiopathic and familial pulmonary arterial hypertension, multiple disease associated pathways, cell types and processes reveal links to elements of the serotonin system. Beyond the original 'serotonin hypothesis' observed with anorexigens, and the latterly demonstrated association with vascular tone and pulmonary artery smooth muscle cell proliferation, recent studies suggest links to BMPR2, PDGF and RhoK pathways, as well as an impact upon more complex lesion formation and pathologic bone marrow progenitor mobilization. Clinical experience with antagonists targeting the various elements of the serotonin pathway has been unsatisfactory, yet perhaps this is less than surprising given our expanding knowledge around serotonin production and signaling biology, which indicate opportunities for novel therapeutic options.

Reduced response to the formalin test and lowered spinal NMDA glutamate receptor binding in adenosine A2A receptor knockout mice.

Adenosine is a neuromodulator with complex effects on pain pathways. Mice lacking the adenosine A2A receptor are hypoalgesic, and have altered analgesic responses to receptor-selective opioid agonists. These and other findings suggest a role for the adenosine A2A receptor in sensitizing afferent fibres projecting to the spinal cord. To test this hypothesis formalin (20 microl, 5%) was injected into the paw and nociceptive responses were measured in wildtype and adenosine A2A receptor knockout mice. There was a significant reduction in nociception associated with sensory nerve activation in the knockout mice as measured by time spent biting/licking the formalin-injected paw and number of flinches seen during the first phase, but only the number of flinches was reduced during the second inflammatory phase. In addition, the selective adenosine A2A antagonist SCH58261 (3 and 10 mg/kg) also antagonised both phases of the formalin test. We also labelled NMDA glutamate and NK1 receptors in spinal cord sections as an indirect measure of nociceptive transmission from peripheral sites to the spinal cord. [3H]-Substance P binding to NK1 receptors was unaltered but there was a substantial reduction in binding of [3H]-MK801 to NMDA glutamate receptors in all regions of the spinal cord from knockout mice. The decrease in NMDA glutamate receptor binding may reflect reduced peripheral sensory input to the spinal cord during development and could relate to the hypoalgesia in this genotype. These results support a key role for the adenosine A2A receptor in peripheral nociceptive pathways.