Timothy N. Bachman

Acute hemodynamic effects of inhaled sodium nitrite in pulmonary hypertension associated with heart failure with preserved ejection fraction

BACKGROUND Pulmonary hypertension (PH) is associated with poor outcomes, yet specific treatments only exist for a small subset of patients. The most common form of PH is that associated with left heart disease (Group 2), for which there is no approved therapy. Nitrite has shown efficacy in preclinical animal models of Group 1 and 2 PH, as well as in patients with left heart failure with preserved ejection fraction (HFpEF). We evaluated the safety and efficacy of a potentially novel inhaled formulation of nitrite in PH-HFpEF patients as compared with Group 1 and 3 PH. METHODS Cardiopulmonary hemodynamics were recorded after acute administration of inhaled nitrite at 2 doses, 45 and 90 mg. Safety endpoints included change in systemic blood pressure and methemoglobin levels. Responses were also compared with those administered inhaled nitric oxide. RESULTS Thirty-six patients were enrolled (10 PH-HFpEF, 20 Group 1 pulmonary arterial hypertension patients on background PH-specific therapy, and 6 Group 3 PH). Drug administration was well tolerated. Nitrite inhalation significantly lowered pulmonary, right atrial, and pulmonary capillary wedge pressures, most pronounced in patients with PH-HFpEF. There was a modest decrease in cardiac output and systemic blood pressure. Pulmonary vascular resistance decreased only in Group 3 PH patients. There was substantial increase in pulmonary artery compliance, most pronounced in patients with PH-HFpEF. CONCLUSIONS Inhaled nitrite is safe in PH patients and may be efficacious in PH-HFpEF and Group 3 PH primarily via improvements in left and right ventricular filling pressures and pulmonary artery compliance. The lack of change in pulmonary vascular resistance likely may limit efficacy for Group 1 patients. TRIAL REGISTRATION ClinicalTrials.gov NCT01431313 FUNDING. This work was supported in part by the NIH grants P01HL103455 (to MAS and MTG), R01HL098032 (to MTG), and R01HL096973 (to MTG), and Mast Therapeutics, Inc.

TSP1–CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction

Aims Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1−/− mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1–CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. Methods and results We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1–CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47−/− mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47−/− pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1–CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. Conclusions In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1–CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Development of a Mouse Model of Metabolic Syndrome, Pulmonary Hypertension, and Heart Failure with Preserved Ejection Fraction

&NA; Pulmonary hypertension (PH) associated with heart failure with preserved ejection fraction (PH‐HFpEF; World Health Organization Group II) secondary to left ventricular (LV) diastolic dysfunction is the most frequent cause of PH. It is an increasingly recognized clinical complication of the metabolic syndrome. To date, no effective treatment has been identified, and no genetically modifiable mouse model is available for advancing our understanding for PH‐HFpEF. To develop a mouse model of PH‐HFpEF, we exposed 36 mouse strains to 20 weeks of high‐fat diet (HFD), followed by systematic evaluation of right ventricular (RV) and LV pressure‐volume analysis. The HFD induces obesity, glucose intolerance, insulin resistance, hyperlipidemia, as well as PH, in susceptible strains. We observed that certain mouse strains, such as AKR/J, NON/shiLtJ, and WSB/EiJ, developed hemodynamic signs of PH‐HFpEF. Of the strains that develop PH‐HFpEF, we selected AKR/J for further model validation, as it is known to be prone to HFD‐induced metabolic syndrome and had low variability in hemodynamics. HFD‐treated AKR/J mice demonstrate reproducibly higher RV systolic pressure compared with mice fed with regular diet, along with increased LV end‐diastolic pressure, both RV and LV hypertrophy, glucose intolerance, and elevated HbA1c levels. Time course assessments showed that HFD significantly increased body weight, RV systolic pressure, LV end‐diastolic pressure, biventricular hypertrophy, and HbA1c throughout the treatment period. Moreover, we also identified and validated 129S1/SvlmJ as a resistant mouse strain to HFD‐induced PH‐HFpEF. These studies validate an HFD/AKR/J mouse model of PH‐HFpEF, which may offer a new avenue for testing potential mechanisms and treatments for this disease.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor

&NA; Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high‐fat diet (HFD) in some inbred mouse strains. We conducted a genome‐wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD‐induced PH. Mice from 36 inbred and wild‐derived strains were fed with regular diet or HFD for 20 weeks beginning at 6‐12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end‐systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest‐path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH‐related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high‐fat feeding. Network‐based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest‐path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high‐fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network‐based approach adds evidence for a role for Egfr in murine PH.

Effects of acute intravenous iloprost on right ventricular hemodynamics in rats with chronic pulmonary hypertension

The inotropic effects of prostacyclins in chronic pulmonary arterial hypertension (PAH) are unclear and may be important in directing patient management in the acute setting. We sought to study the effects of an acute intravenous (IV) infusion of iloprost on right ventricular (RV) contractility in a rat model of chronic PAH. Rats were treated with monocrotaline, 60 mg/kg intraperitoneally, to induce PAH. Six weeks later, baseline hemodynamic assessment was performed with pressure-volume and Doppler flow measurements. In one group of animals, measurements were repeated 10–15 minutes after IV infusion of a fixed dose of iloprost (20 μg/kg). A separate group of rats underwent dose-response assessment. RV contractility and RV–pulmonary artery coupling were assessed by the end-systolic pressure-volume relationship (ESPVR) and end-systolic elastance/effective arterial elastance (Ees/Ea). RV cardiomyocytes were isolated, and intracellular cAMP (cyclic adenosine monophosphate) concentration was measured with a cAMP-specific enzyme immunoassay kit. Animals had evidence of PAH and RV hypertrophy. Right ventricle/(left ventricle + septum) weight was 0.40 ± 0.03. RV systolic pressure (RVSP) was 39.83 ± 1.62 mmHg. Administration of iloprost demonstrated an increase in the slope of the ESPVR from 0.29 ± 0.02 to 0.42 ± 0.05 (P < .05). Ees/Ea increased from 0.63 ± 0.07 to 0.82 ± 0.06 (P < .05). The RV contractility index (max dP/dt normalized for instantaneous pressure) increased from 94.11 to 114.5/s (P < .05), as did the RV ejection fraction, from 48.0% to 52.5% (P < .05). This study suggests a positive inotropic effect of iloprost on a rat model of chronic PAH.

Targeting Pulmonary Endothelial Hemoglobin α Improves Nitric Oxide Signaling and Reverses Pulmonary Artery Endothelial Dysfunction

&NA; Pulmonary hypertension is characterized by pulmonary endothelial dysfunction. Previous work showed that systemic artery endothelial cells (ECs) express hemoglobin (Hb) &agr; to control nitric oxide (NO) diffusion, but the role of this system in pulmonary circulation has not been evaluated. We hypothesized that up‐regulation of Hb &agr; in pulmonary ECs contributes to NO depletion and pulmonary vascular dysfunction in pulmonary hypertension. Primary distal pulmonary arterial vascular smooth muscle cells, lung tissue sections from unused donor (control) and idiopathic pulmonary artery (PA) hypertension lungs, and rat and mouse models of SU5416/hypoxia‐induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Cocultures of human pulmonary microvascular ECs and distal pulmonary arterial vascular smooth muscle cells, lung tissue from control and pulmonary hypertensive lungs, and a mouse model of chronic hypoxia‐induced PH were used. Immunohistochemical, immunoblot analyses, spectrophotometry, and blood vessel myography experiments were performed in this study. We find increased expression of Hb &agr; in pulmonary endothelium from humans and mice with PH compared with controls. In addition, we show up‐regulation of Hb &agr; in human pulmonary ECs cocultured with PA smooth muscle cells in hypoxia. We treated pulmonary ECs with a Hb &agr; mimetic peptide that disrupts the association of Hb &agr; with endothelial NO synthase, and found that cells treated with the peptide exhibited increased NO signaling compared with a scrambled peptide. Myography experiments using pulmonary arteries from hypoxic mice show that the Hb &agr; mimetic peptide enhanced vasodilation in response to acetylcholine. Our findings reveal that endothelial Hb &agr; functions as an endogenous scavenger of NO in the pulmonary endothelium. Targeting this pathway may offer a novel therapeutic target to increase endogenous levels of NO in PH.

Development of an Ex Vivo Ovine Ventricular Assist Device Model for Intraventricular Visualization of the Inflow Cannula

eft ventricular assist devices (LVADs) are increasingly eing employed in the management of heart failure. dequate device function requires unimpeded blood ow from the left ventricle (LV) to the device via the nflow cannula (IC). Malposition may lead to complicaions including hemolysis, thrombus generation and entricular suction events. Current models to study C function are limited to live animal models. Due o difficulties with visualization through blood, develpment of systems for live intracardiac videoscopy ave been limited. We developed an ex vivo model llowing visualization of the IC from within the entricle that is currently being utilized to study IC esign and function. A non-beating, ex vivo, asanguineous ovine cardiac odel was designed, as shown in Figure 1A. Fresh 8to 0-month-old ovine hearts were obtained from a slaugherhouse (Zrile Meat, West Middlesex, PA). The dome of he left atrium was cannulated with a modified t-tube onnector secured with a 4-0 polypropylene pursetring suture. The heart was connected to the flow loop Figure 1B). An LV vent was inserted through the scending aorta and secured with a 0-silk tie. A 30° ideoscope (Karl Storz Industrial–America, Culver City, A) connected to a camera (Model PV420DN; Sony orp., New York, NY) was advanced through the t-tube onnector into the LV. The LV apex was cored using a 11 blade. The beveled IC (Thoratec Corp., Pleasanton, A) was inserted and secured with pledgeted 2-0 Ticron orizontal mattress sutures. This was attached to a Biomeicus centrifugal pump (Model BP80; Medtronic, Inc., inneapolis, MN). The flow loop was primed with distilled water at oom temperature and seeded with neutrally buoyant articles (Amberlite IRA-96; Sigma-Aldrich, St. Louis, O) for flow visualization. Flow through the heart as initiated by gradually increasing the pump speed nd the aortic vent was opened intermittently allowng air bubbles to escape. A resistor on the inflow ubing was used to adjust the flow entering the left trium. The IC position and pump speed were then djusted to create different clinically relevant hemoynamic scenarios. Interactions between the IC, the entricle and the path of fluid flow were captured on ideo from within the ventricle. Two-dimensional rans-epicardial echocardiography (Vingmed, Rosklde, Denmark) was also utilized to provide correponding echocardiographic images. As demontrated in Figure 2A, a ventricular suction event was

Biomechanical and Hemodynamic Measures of Right Ventricular Diastolic Function: Translating Tissue Biomechanics to Clinical Relevance

Background Right ventricular (RV) diastolic function has been associated with outcomes for patients with pulmonary hypertension; however, the relationship between biomechanics and hemodynamics in the right ventricle has not been studied. Methods and Results Rat models of RV pressure overload were obtained via pulmonary artery banding (PAB; control, n=7; PAB, n=5). At 3 weeks after banding, RV hemodynamics were measured using a conductance catheter. Biaxial mechanical properties of the RV free wall myocardium were obtained to extrapolate longitudinal and circumferential elastic modulus in low and high strain regions (E1 and E2, respectively). Hemodynamic analysis revealed significantly increased end‐diastolic elastance (Eed) in PAB (control: 55.1 mm Hg/mL [interquartile range: 44.7–85.4 mm Hg/mL]; PAB: 146.6 mm Hg/mL [interquartile range: 105.8–155.0 mm Hg/mL]; P=0.010). Longitudinal E1 was increased in PAB (control: 7.2 kPa [interquartile range: 6.7–18.1 kPa]; PAB: 34.2 kPa [interquartile range: 18.1–44.6 kPa]; P=0.018), whereas there were no significant changes in longitudinal E2 or circumferential E1 and E2. Last, wall stress was calculated from hemodynamic data by modeling the right ventricle as a sphere: stress=Pressure×radius2×thickness. Conclusions RV pressure overload in PAB rats resulted in an increase in diastolic myocardial stiffness reflected both hemodynamically, by an increase in Eed, and biomechanically, by an increase in longitudinal E1. Modest increases in tissue biomechanical stiffness are associated with large increases in Eed. Hemodynamic measurements of RV diastolic function can be used to predict biomechanical changes in the myocardium.

Chemokine receptor patterns and right heart failure in mechanical circulatory support

BACKGROUND Right ventricular failure (RVF) complicates 9% to 44% of left ventricular assist device (LVAD) implants post-operatively. Current prediction scores perform only modestly in validation studies, and do not include immune markers. Chemokines are inflammatory signaling molecules with a fundamental role in cardiac physiology and stress adaptation. In this study we investigated chemokine receptor regulation in LVAD recipients who develop RVF. METHODS Expression of chemokine receptor (CCR) genes 3 to 8 were examined in the peripheral blood of 111 LVAD patients, collected 24 hours before implant. RNA was isolated using a PAXgene protocol. Gene expression was assessed using a targeted microarray (RT2 Profiler PCR Array; Qiagen). Results were expressed as polymerase chain reaction (PCR) cycles to threshold and normalized to the average of 3 control genes, glyceraldehyde phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and β2-microglobulin (B2M). Secondary outcomes studied were 1-year mortality and long-term RV failure (RVF-LT). RESULTS CCR3, CCR4, CCR6, CCR7 and CCR8 were downregulated in LVAD recipients with RVF. Within this cohort of patients, CCR4, CCR7 and CCR8 were further downregulated in those who required RV mechanical support. In addition, under-expression of CCR3 to CCR8 was independently associated with an increased risk of mortality at 1 year, even after adjusting for RVF. CCR expression did not predict RVF-LT in our patient cohort. CONCLUSIONS Pre-LVAD CCR downregulation is associated with RVF and increased mortality after implant. Inflammatory signatures may play a major role in prognostication in this patient population.

Three-Dimensional virtual anatomic fit study for an implantable pediatric ventricular assist device

An innovative pediatric ventricular assist device (PVAD) is being developed to treat young patients (2.5kg-15kg) with severe heart failure that otherwise have very few options due to their small size. To optimize the design of the PVAD for the target patient population, three-dimensional anatomical compatibility studies must be conducted. The aim of this project was to evaluate the utility of three dimensional reconstructions to obviate fit studies in human subjects. Serial CT scans of the thorax of one child were obtained as part of routine treatment. The images were enhanced by adjusting the contrast of the images and segmented semi-automatically prior to 3-D reconstruction. The results were visualized as surface renderings of the rib cage and heart. This data was then amended with solid models of the implantable hardware, including the PVAD and cannulae. Manipulation of the relative orientation of the components revealed surgical challenges that may be anticipated and motivated design modifications to improve the anatomic compatibility. Unique challenges associated with these data sets include the availability of pediatric CT images and difficulty of segmentation due to the small scale of the anatomic features as compared to the resolution of the images.