Jennifer A. Johnson

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

A potential role for insulin resistance in experimental pulmonary hypertension

Patients with pulmonary arterial hypertension have increased prevalence of insulin resistance. We aimed to determine whether metabolic defects are associated with bone morphogenic protein receptor type 2 (Bmpr2) mutations in mice, and whether these may contribute to pulmonary vascular disease development. Metabolic phenotyping was performed on transgenic mice with inducible expression of Bmpr2 mutation, R899X. Phenotypic penetrance in Bmpr2R899X was assessed in a high-fat diet model of insulin resistance. Alterations in glucocorticoid responses were assessed in murine pulmonary microvascular endothelial cells and Bmpr2R899X mice treated with dexamethasone. Compared to controls, Bmpr2R899X mice showed increased weight gain and demonstrated insulin resistance as assessed by the homeostatic model assessment insulin resistance (1.0±0.4 versus 2.2±1.8) and by fat accumulation in skeletal muscle and decreased oxygen consumption. Bmpr2R899X mice fed a high-fat diet had strong increases in pulmonary hypertension penetrance (seven out of 11 versus three out of 11). In cell culture and in vivo experiments, Bmpr2 mutation resulted in a combination of constitutive glucocorticoid receptor activation and insensitivity. Insulin resistance is present as an early feature of Bmpr2 mutation in mice. Exacerbated insulin resistance through high-fat diet worsened pulmonary phenotype, implying a possible causal role in disease. Impaired glucocorticoid responses may contribute to metabolic defects.

ACE2 Improves Right Ventricular Function in a Pressure Overload Model

Background Right ventricular (RV) dysfunction is a complication of pulmonary hypertension and portends a poor prognosis. Pharmacological therapies targeting RV function in pulmonary hypertension may reduce symptoms, improve hemodynamics, and potentially increase survival. We hypothesize that recombinant human angiotensin-converting enzyme 2 (rhACE2) will improve RV function in a pressure overload model. Results rhACE2 administered at 1.8 mg/kg/day improved RV systolic and diastolic function in pulmonary artery banded mice as measured by in vivo hemodynamics. Specifically, rhACE2 increased RV ejection fraction and decreased RV end diastolic pressure and diastolic time constant (p<0.05). In addition, rhACE2 decreased RV hypertrophy as measured by RV/LV+S ratio (p<0.05). There were no significant negative effects of rhACE2 administration on LV function. rhACE2 had no significant effect on fibrosis as measured by trichrome staining and collagen1α1 expression. In pulmonary artery banded mice, rhACE2 increased Mas receptor expression and normalized connexin 37 expression. Conclusion In a mouse RV load-stress model of early heart failure, rhACE2 diminished RV hypertrophy and improved RV systolic and diastolic function in association with a marker of intercellular communication. rhACE2 may be a novel treatment for RV failure.

Physiologic and molecular consequences of endothelial Bmpr2 mutation

BackgroundPulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.MethodsIn vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2delx4+ or Bmpr2R899X mutation.ResultsTransgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2delx4+ and Bmpr2R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells.ConclusionsBmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.

Oxidative injury is a common consequence of BMPR2 mutations

Hereditary pulmonary arterial hypertension (PAH) is usually caused by mutations in BMPR2. Mutations are found throughout the gene, and common molecular consequences of different types of mutation are not known. Knowledge of common molecular consequences would provide insight into the molecular etiology of the disease. The objective of this study was to determine the common molecular consequences across classes of BMPR2 mutation. Increased superoxide and peroxide production and alterations in genes associated with oxidative stress were a common consequence of stable transfection of the vascular smooth muscle cells, with three distinct classes of BMPR2 mutation, in the ligand binding domain, the kinase domain and the cytoplasmic tail domain. Measurement of oxidized lipids in whole lung from transgenic mice expressing a mutation in the BMPR2 cytoplasmic tail showed a 50% increase in isoprostanes and a two-fold increase in isofurans, suggesting increased reactive oxygen species (ROS) of mitochondrial origin. Immunohistochemistry on BMPR2 transgenic mouse lung showed that oxidative stress was vascular-specific. Electron microscopy showed decreased mitochondrial size and variability in the pulmonary vessels from BMPR2-mutant mice. Measurement of oxidized lipids in urine from humans with BMPR2 mutations demonstrated increased ROS, regardless of disease status. Immunohistochemistry on hereditary PAH patient lung confirmed oxidative stress specific to the vasculature. Increased oxidative stress, likely of mitochondrial origin, is a common consequence of BMPR2 mutation across mutation types in cell culture, mice and humans.

Interaction between bone morphogenetic protein receptor type 2 and estrogenic compounds in pulmonary arterial hypertension.

The majority of heritable pulmonary arterial hypertension (HPAH) cases are associated with mutations in bone morphogenetic protein receptor type 2 (BMPR2). BMPR2 mutation carries about a 20% lifetime risk of PAH development, but penetrance is approximately three times higher in females. Previous studies have shown a correlation between estrogen metabolism and penetrance, with increased levels of the estrogen metabolite 16α-hydroxyestrone (16αOHE) and reduced levels of the metabolite 2-methoxyestrogen (2ME) associated with increased risk of disease. The goal of this study was to determine whether 16αOHE increased and 2ME decreased penetrance of disease in Bmpr2 mutant mice and, if so, by what mechanism. We found that 16αOHE:2ME ratio was high in male human HPAH patients. Bmpr2 mutant male mice receiving chronic 16αOHE had doubled disease penetrance, associated with reduced cardiac output. 2ME did not have a significant protective effect, either alone or in combination with 16αOHE. In control mice but not in Bmpr2 mutant mice, 16αOHE suppressed bone morphogenetic protein signaling, probably directly through suppression of Bmpr2 protein. Bmpr2 mutant pulmonary microvascular endothelial cells were insensitive to estrogen signaling through canonical pathways, associated with aberrant intracellular localization of estrogen receptor α. In both control and Bmpr2 mutant mice, 16αOHE was associated with suppression of cytokine expression but with increased alternate markers of injury, including alterations in genes related to thrombotic function, angiogenesis, planar polarity, and metabolism. These data support a causal relationship between increased 16αOHE and increased PAH penetrance, with the likely molecular mechanisms including suppression of BMPR2, alterations in estrogen receptor translocation, and induction of vascular injury and insulin resistance–related pathways.

Copy-number variation in BMPR2 is not associated with the pathogenesis of pulmonary arterial hypertension

BackgroundCopy-number variations (CNVs) are structural variations in the genome involving 1 kb to 3 mb of DNA. CNV has been reported within intron 1 of the BMPR2 gene. We propose that CNV could affect phenotype in familial and/or sporadic pulmonary arterial hypertension (PAH) by altering gene expression.Methods97 human DNA samples were obtained which included 24 patients with familial PAH, 18 obligate carriers (BMPR2 mutation positive), 20 sporadic PAH patients, and 35 controls. Two sets of primers were designed within the CNV, and two sets of control primers were designed outside the CNV. Quantitative PCR was performed to quantify genomic copies of CNV and control sequences.ResultsA CNV in BMPR2 was present in one African American negative control subject.ConclusionWe conclude that the CNV in intron 1 in BMPR2 is unlikely to play a role in the pathogenesis of either familial or sporadic PAH.Trial RegistrationNIH NCT00091546.

A Case Series and Review of Histoplasmosis Infection in the Neck

An endemic fungus of the Ohio and Mississippi River valleys, Histoplasma capsulatum is found in soil contaminated with bird or bat droppings. Inhalation of H capsulatum spores leads to infection, which can either be asymptomatic, present as an acute influenzalike illness, or progress to disseminated life-threatening disease. Although most cases of pulmonary histoplasmosis are associated with hilar or mediastinal lymphadentis confined within the thorax, clinical presentation in the neck has been reported. Complications from histoplasmosis are caused by inflammation of pulmonary or regional lymph nodes. One specific complication of inflammation from histoplasmosis is the development of a mediastinal granuloma, characterized by a mediastinal mass (3-10 cm) comprised mostly of caseous mediastinal lymph nodes that have matted together and broken down into a single semiliquid encapsulated lesion. 1 Mediastinal granulomas are often located in the right paratracheal region and less frequently in the left paratracheal, subcarinal, or hilar regions. They are usually asymptomatic and are often discovered incidentally on chest radiography. Rarely, mediastinal granulomas produce symptoms related to superior vena cava or esophageal compression or from fistula formation to esophagus, bronchus, or skin. 1 We describe 4 patients with cervical manifestations of histoplasmosis, review existing literature, and provide suggestions for diagnosis and treatment of this unusual disease presentation. This study was approved by the institutional review board of Vanderbilt University, Nashville, Tennessee.

A 48-Year-Old Man With Paralysis and Hypotension

A 48-year-old man presented to the emergency depaItment with acute paralysis from the umbilicus down. The patient was watching television and drinking beer with friends when he developed the sudden onset of bilateral lower extremity paralysis associated with loss of bowel control. The patient denied any trauma, chest pain, abdominal pain, or syncope. His medical history was significant for hypertension, tobacco abuse, posttraumatic stress disorder, and depreSSion with suicide attempts. He had no history ofsurgery. His medications included felodipine, hydrochlorothiazide, lisinopril, and quetiapine.

A potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a deadly disease with no cure. Alternate conversion of angiotensin II (AngII) to angiotensin-(1–7) (Ang-(1–7)) by angiotensin-converting enzyme 2 (ACE2) resulting in Mas receptor (Mas1) activation improves rodent models of PAH. Effects of recombinant human (rh) ACE2 in human PAH are unknown. Our objective was to determine the effects of rhACE2 in PAH. We defined the molecular effects of Mas1 activation using porcine pulmonary arteries, measured AngII/Ang-(1–7) levels in human PAH and conducted a phase IIa, open-label pilot study of a single infusion of rhACE2 (GSK2586881, 0.2 or 0.4 mg·kg−1 intravenously). Superoxide dismutase 2 (SOD2) and inflammatory gene expression were identified as markers of Mas1 activation. After confirming reduced plasma ACE2 activity in human PAH, five patients were enrolled in the trial. GSK2586881 was well tolerated with significant improvement in cardiac output and pulmonary vascular resistance. GSK2586881 infusion was associated with reduced plasma markers of inflammation within 2–4 h and increased SOD2 plasma protein at 2 weeks. PAH is characterised by reduced ACE2 activity. Augmentation of ACE2 in a pilot study was well tolerated, associated with improved pulmonary haemodynamics and reduced markers of oxidant and inflammatory mediators. Targeting this pathway may be beneficial in human PAH. The ACE2–Ang-(1–7)–Mas1 axis is probably involved in the pathophysiology of human pulmonary arterial hypertension http://ow.ly/pgS530jOxnd