4PBA Restores Signaling of a Cysteine-substituted Mutant BMPR2 Receptor Found in Patients with Pulmonary Arterial Hypertension.

Mutations in the gene encoding BMPR2 (bone morphogenetic protein type 2 receptor) are the major cause of heritable pulmonary arterial hypertension (PAH). Point mutations in the BMPR2 ligand-binding domain involving cysteine residues (such as C118W) are causative of PAH and predicted to cause protein misfolding. Using heterologous overexpression systems, we showed previously that these mutations lead to retention of BMPR2 in the endoplasmic reticulum but are partially rescued by chemical chaperones. Here, we sought to determine whether the chemical chaperone 4-phenylbutyrate (4PBA) restores BMPR2 signaling in primary cells and in a knockin mouse harboring a C118W mutation. First, we confirmed dysfunctional BMP signaling in dermal fibroblasts isolated from a family with PAH segregating the BMPR2 C118W mutation. After BMP4 treatment, the induction of downstream signaling targets (Smad1/5, ID1 [inhibitor of DNA binding 1], and ID2) was significantly reduced in C118W mutant cells. Treatment with 4PBA significantly rescued Smad1/5, ID1, and ID2 expression. Pulmonary artery smooth muscle cells isolated from the lungs of heterozygous mice harboring the Bmpr2 C118W mutation exhibited significantly increased proliferation. In the presence of 4PBA, hyperproliferation was dramatically reduced. Furthermore, in vivo, 4PBA treatment of Bmpr2 C118W mice partially rescued Bmpr2 expression, restored downstream signaling, and improved vascular remodeling. These findings demonstrate in primary cells and in a knockin mouse that the repurposed small-molecule chemical chaperone 4PBA might be a promising precision medicine approach to treat PAH in patients with specific subtypes of BMPR2 mutation involving cysteine substitutions in the ligand-binding domain.

Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice.

Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.

Hematopoietic stem cell transplantation alters susceptibility to pulmonary hypertension in Bmpr2-deficient mice.

Increasing evidence suggests that patients with pulmonary arterial hypertension (PAH) demonstrate abnormalities in the bone marrow (BM) and hematopoietic progenitor cells. In addition, PAH is associated with myeloproliferative diseases. We have previously demonstrated that low-dose lipopolysaccharide (LPS) is a potent stimulus for the development of PAH in the context of a genetic PAH mouse model of BMPR2 dysfunction. We hypothesized that the hematopoietic progenitor cells might be driving disease in this model. To test this hypothesis, we performed adoptive transfer of BM between wild-type (Ctrl) and heterozygous Bmpr2 null (Mut) mice. Sixteen weeks after BM reconstitution, mice were exposed to low-dose chronic LPS (0.5 mg/kg three times a week for six weeks). Mice underwent right heart catheterization and tissues were removed for histology. After chronic LPS dosing, Ctrl mice in receipt of Mut BM developed PAH, whereas Mut mice receiving Ctrl BM were protected from PAH. BM histology demonstrated an increase in megakaryocytes and there was an increase in circulating platelets in Ctrl mice receiving Mut BM. These findings demonstrate that the hematopoietic stem cell compartment is involved in the susceptibility to PAH in the Mut mouse. The results raise the possibility that hematopoietic stem cell transplantation might be a potential treatment strategy in genetic forms of PAH.

Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension.

BACKGROUND: Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein-coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model. METHODS: In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr1]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model. RESULTS: ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein- and ß-arrestin-dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and pulmonary vascular remodeling in monocrotaline-exposed rats. CONCLUSIONS: These results show that ELA is an endogenous agonist of the human apelin receptor, exhibits a cardiovascular profile comparable to apelin, and is downregulated in human disease and rodent PAH models, and exogenous peptide can reduce the severity of cardiopulmonary remodeling and function in PAH in rats. This study provides additional proof of principle that an apelin receptor agonist may be of therapeutic use in PAH in humans.

TNFα drives pulmonary arterial hypertension by suppressing the BMP type-II receptor and altering NOTCH signalling.

Heterozygous germ-line mutations in the bone morphogenetic protein type-II receptor (BMPR-II) gene underlie heritable pulmonary arterial hypertension (HPAH). Although inflammation promotes PAH, the mechanisms by which inflammation and BMPR-II dysfunction conspire to cause disease remain unknown. Here we identify that tumour necrosis factor-α (TNFα) selectively reduces BMPR-II transcription and mediates post-translational BMPR-II cleavage via the sheddases, ADAM10 and ADAM17 in pulmonary artery smooth muscle cells (PASMCs). TNFα-mediated suppression of BMPR-II subverts BMP signalling, leading to BMP6-mediated PASMC proliferation via preferential activation of an ALK2/ACTR-IIA signalling axis. Furthermore, TNFα, via SRC family kinases, increases pro-proliferative NOTCH2 signalling in HPAH PASMCs with reduced BMPR-II expression. We confirm this signalling switch in rodent models of PAH and demonstrate that anti-TNFα immunotherapy reverses disease progression, restoring normal BMP/NOTCH signalling. Collectively, these findings identify mechanisms by which BMP and TNFα signalling contribute to disease, and suggest a tractable approach for therapeutic intervention in PAH.

Praziquantel reverses pulmonary hypertension and vascular remodeling in murine schistosomiasis.

RATIONALE: Schistosomiasis is the most common worldwide cause of pulmonary arterial hypertension. The anti-schistosome drug praziquantel has been shown to reverse the liver fibrosis associated with Schistosoma mansoni in mice. OBJECTIVES: We sought to determine whether praziquantel reverses established pulmonary vascular remodeling and pulmonary hypertension in a mouse model of S. mansoni. METHODS: Mice were infected percutaneously with S. mansoni. At 17 weeks after infection mice were either killed or received two doses of praziquantel or vehicle by oral gavage. Treated mice were studied at 25 weeks after infection. MEASUREMENTS AND MAIN RESULTS: Vehicle-treated mice demonstrated significant increases in right ventricular systolic pressures (RVSP) and right ventricular hypertrophy (RVH) at 25 weeks, accompanied by pulmonary vascular remodeling. The degree of vascular remodeling correlated with proximity to granulomas. The elevation of RVSP and RVH at 25 weeks was dependent on the presence of eggs in the lung. Praziquantel eliminated the production of eggs in feces and led to clearance of eggs from the lung and to a lesser extent from liver. Praziquantel prevented the rise in RVSP and RVH seen in vehicle-treated mice and reversed established pulmonary vascular remodeling. Praziquantel significantly reduced lung mRNA expression of IL-13, IL-8, and IL-4, but did not reduce serum cytokine levels. CONCLUSIONS: The development of pulmonary hypertension associated with S. mansoni infection can be prevented by praziquantel, and established vascular remodeling can be reversed. The mechanism involves clearance of lung eggs and reduced local expression of lung cytokines.

Pulmonary vascular disease associated with schistosomiasis.

In this article we focus on the pathogenesis and clinical characteristics of schistosomiasis infection on the lung vasculature. Overall, the basic biology and understanding of Schistosoma immune responses and their effect on the cardiopulmonary system is limited in both animal and human models, which hinders clinical care and drug development. The inflammatory response to the eggs in the lung appears to contribute to the remodeling of the pulmonary vessels. Portal hypertension caused by parasitemia also appears to contribute to the development of pathophysiologic alterations of the pulmonary vascular bed. Antischistosomal therapy, praziquantel, used for pulmonary hypertension secondary to schistosomiasis usually has no effect, but it is given to prevent further progression of disease. Currently, there are no clinical trials for the treatment of pulmonary vascular disease secondary to schistosomiasis. Specialty drugs such as phosphodiesterase type 5 or tyrosine kinase inhibitors exhibit some interesting activity, yet are prohibitively expensive, lack safety and efficacy studies in schistosomiasis endemic populations, and tend to be limited by safety, efficacy, route of administration and compliance problems.

Regulation of growth differentiation factor 15 expression by intracellular iron.

Growth differentiation factor 15 (GDF15) is a divergent member of the transforming growth factor-beta superfamily and has been identified in different contexts as a hypoxia-inducible gene product and as a molecule involved in hepcidin regulation. The biology of iron and oxygen is closely related, and known regulatory pathways involving hypoxia-inducible factor (HIF) and iron-regulatory proteins (IRPs) are responsive to both these stimuli. We therefore sought to characterize the regulation of GDF15 by iron and oxygen and to define the involvement or otherwise of HIF and IRP pathways. Here we show that GDF15 is strongly up-regulated by stimuli that deplete cells of iron and that this response is specifically antagonized by the reprovision of iron. GDF15 exhibits greater sensitivity to iron depletion than hypoxia, and responses to hypoxia and iron depletion are independent of HIF and IRP activation, suggesting a novel mechanism of regulation. We also report significant induction of serum GDF15 in iron-deficient subjects and after administration of an iron chelator to normal subjects. These findings indicate that GDF15 can be induced by pathophysiologic changes in iron availability, raising important questions about the mechanism of regulation and its role in iron homeostasis.

Variability in end-tidal PCO2 and blood gas values in humans.

The aims of this study were: (1) to determine the within-subject, between-day variability in end-tidal P(CO2) (P(ET,CO2)); (2) to determine whether the within-subject, between-day variability in P(ET,CO2) was caused by variations in metabolic acid-base status; and (3) to determine whether between-subject variations in blood gas variables arose predominantly through variations in respiratory or renal pH control mechanisms. Fourteen healthy males were studied, of whom 12 provided adequate data for further analysis. Each subject was studied on at least six different occasions, with each visit separated by at least 1 week. On each occasion, P(ET,CO2) was determined over a 4-10 min period using a fine nasal catheter taped just inside the nose, and an arterialised capillary blood sample was obtained from each ear and analysed for blood gases. The following results were obtained. (1) P(ET,CO2) showed a standard deviation (S.D.) for the within-subject, between-day variation of 1.06 mmHg. (2) Less than 5% (P = NS) of the variability in P(ET,CO2) could be explained by underlying variations in metabolic acid-base status. (3) The variation in blood gas values between individuals did not fit a pattern associated with either a predominantly respiratory or a predominantly renal origin. Furthermore, the pattern of variation in the blood gas data suggested that variations in the renal controller for pH and the respiratory controller for pH were not independent. The precise origins of variability in blood gas regulation remain obscure.