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Endothelial dysfunction is an underlying mechanism for the development of pulmonary arterial hypertension (PAH). Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF) may help repair the dysfunctional endothelium and provide treatment for PAH. To examine this possibility, nanoparticles carrying human recombinant VEGF and SDF (VEGFNP and SDFNP) were aerosolized into the lungs of nude rats at Day 14 after monocrotaline (MCT) injection and analyses were performed at Day 28. The data show that the VEGFNP/SDFNP delivery led to a lower pulmonary arterial pressure and prevented right ventricular hypertrophy in the MCT rats: the right ventricular systolic pressure of the control, MCT, and MCT + VEGFNP/SDFNP treatment groups were 29±2, 70±9, and 44±5 (mean±SD) mmHg, respectively; the pulmonary vascular resistance indices of the groups were 0.6±0.3, 3.2±0.7, and 1.7±0.5, respectively; and the Fulton indices [-RV/(LV + Septum)] were 0.22±0.01, 0.44±0.07, and 0.23±0.02, respectively. The VEGFNP/SDFNP delivery delayed the thickening of distal pulmonary vessels: the number of nearly occluded vessels in a whole lung section from the MCT and MCT + VEGFNP/SDFNP groups were 46±12 and 2±3, respectively. Gene expression analysis of the endothelial cell markers, VE-cadherin, KDR, BMPR2, and eNOS, and smooth cell markers, SM-MHC and α-SMA, indicated significant loss of distal pulmonary vessels in the MCT- treated rats. VEGFNP/SDFNP delivery did not recover the loss, but significantly increased eNOS and decreased α-SMA expression in the MCT-treated lungs. Thus, the therapeutic effect of VEGFNP/SDFNP may be mediated by improving/repairing endothelial function in the PAH lungs., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Pulmonary Circulation published by John Wiley & Sons Ltd on behalf of Pulmonary Vascular Research Institute.)

Hypoxia-inducible factor 1α (HIF1α) is a master regulator of numerous biological processes under low oxygen tensions. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in primary cells remain elusive. Here, we show that HIF1α signaling is activated in several human primary vascular cells under ambient oxygen tensions, and in vascular smooth muscle cells (VSMCs) of normal human lung tissue, which contributed to a relative resistance to further enhancement of glycolytic activity in hypoxia. Mechanistically, aerobic HIFα activation is mediated by paracrine secretion of three branched chain α-ketoacids (BCKAs), which suppress prolyl hydroxylase domain-containing protein 2 (PHD2) activity via direct inhibition and via lactate dehydrogenase A (LDHA)-mediated generation of L-2-hydroxyglutarate (L2HG). Metabolic dysfunction induced by BCKAs was observed in the lungs of rats with pulmonary arterial hypertension (PAH) and in pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH patients. BCKA supplementation stimulated glycolytic activity and promoted a phenotypic switch to the synthetic phenotype in PASMCs of normal and PAH subjects. In summary, we identify BCKAs as novel signaling metabolites that activate HIF1α signaling in normoxia and that the BCKA-HIF1α pathway modulates VSMC function and may be relevant to pulmonary vascular pathobiology., Competing Interests: Competing interests B.M.W discloses a consulting relationship with Change Healthcare (outside the scope of the current topic), an expert witness relationship with United Therapeutics Corporation (outside the scope of the current topic), and co-inventorship on US patent 63/541,939 (outside the scope of the current topic). The other authors declare no competing interests.

Aims: To leverage deep learning on the resting 12-lead electrocardiogram (ECG) to estimate peak oxygen consumption (V˙O2peak) without cardiopulmonary exercise testing (CPET)., Methods and Results: V ˙ O 2 peak estimation models were developed in 1891 individuals undergoing CPET at Massachusetts General Hospital (age 45 ± 19 years, 38% female) and validated in a separate test set (MGH Test, n = 448) and external sample (BWH Test, n = 1076). Three penalized linear models were compared: (i) age, sex, and body mass index ('Basic'), (ii) Basic plus standard ECG measurements ('Basic + ECG Parameters'), and (iii) basic plus 320 deep learning-derived ECG variables instead of ECG measurements ('Deep ECG-V˙O2'). Associations between estimated V˙O2peak and incident disease were assessed using proportional hazards models within 84 718 primary care patients without CPET. Inference ECGs preceded CPET by 7 days (median, interquartile range 27-0 days). Among models, Deep ECG-V˙O2 was most accurate in MGH Test [r = 0.845, 95% confidence interval (CI) 0.817-0.870; mean absolute error (MAE) 5.84, 95% CI 5.39-6.29] and BWH Test (r = 0.552, 95% CI 0.509-0.592, MAE 6.49, 95% CI 6.21-6.67). Deep ECG-V˙O2 also outperformed the Wasserman, Jones, and FRIEND reference equations (P < 0.01 for comparisons of correlation). Performance was higher in BWH Test when individuals with heart failure (HF) were excluded (r = 0.628, 95% CI 0.567-0.682; MAE 5.97, 95% CI 5.57-6.37). Deep ECG-V˙O2 estimated V˙O2peak <14 mL/kg/min was associated with increased risks of incident atrial fibrillation [hazard ratio 1.36 (95% CI 1.21-1.54)], myocardial infarction [1.21 (1.02-1.45)], HF [1.67 (1.49-1.88)], and death [1.84 (1.68-2.03)]., Conclusion: Deep learning-enabled analysis of the resting 12-lead ECG can estimate exercise capacity (V˙O2peak) at scale to enable efficient cardiovascular risk stratification., Competing Interests: Conflict of interest: B.M.W. has consulted for Change Healthcare. P.D.A. and P.B. are supported by grants from Bayer AG and IBM applying machine learning in cardiovascular disease. P.B. serves as a consultant for Novartis and Prometheus Biosciences. P.T.E. receives sponsored research support from Bayer AG and IBM Research; he has also served on advisory boards or consulted for Bayer AG, MyoKardia, and Novartis. S.A.L. receives sponsored research support from Bristol Myers Squibb/Pfizer, Bayer AG, Boehringer Ingelheim, Fitbit, and IBM, and has consulted for Bristol Myers Squibb/Pfizer, Bayer AG, and Blackstone Life Sciences. S.A.L. is now an employee of Novartis Institute for Biomedical Research. The remaining authors have no disclosures., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Elevated mean pulmonary artery pressure (mPAP) is common in patients with hypertrophic cardiomyopathy (HCM) and heart failure symptoms. However, dynamic left ventricular (LV) outflow tract obstruction may confound interpretation of pulmonary hypertension (PH) pathophysiologic features in HCM when relying on resting invasive hemodynamic data alone., Research Question: Do structural changes to the lung vasculature clarify PH pathophysiologic features in patients with HCM with progressive heart failure?, Study Design and Methods: Clinical data and ultrarare lung autopsy specimens were acquired retrospectively from the National Institutes of Health (1975-1992). Patients were included based on the availability of lung tissue and recorded mPAP. Discarded tissue from rejected lung donors served as control specimens. Histomorphology was performed on pulmonary arterioles and veins. Comparisons were calculated using the Student t test and Mann-Whitney U test; Pearson correlation was used to assess association between morphometric measurements and HCM cardiac and hemodynamic measurements., Results: The HCM cohort (n = 7; mean ± SD age, 43 ± 18 years; 71% men) showed maximum mean ± SD LV wall thickness of 25 ± 2.8 mm, mean ± SD outflow tract gradient of 90 ± 30 mm Hg, median mPAP of 25 mm Hg (interquartile range [IQR], 6 mm Hg), median pulmonary artery wedge pressure (PAWP) of 16 mm Hg (IQR, 4 mm Hg), and median pulmonary vascular resistance of 1.8 Wood units (WU; IQR, 2.4 WU). Compared with control samples (n = 5), patients with HCM showed greater indexed pulmonary arterial hypertrophy (20.7 ± 7.2% vs 49.7 ± 12%; P < .001) and arterial wall fibrosis (11.5 ± 3.4 mm vs 21.0 ± 4.7 mm; P < .0001), which correlated with mPAP (r = 0.84; P = .018), PAWP (r = 0.74; P = .05), and LV outflow tract gradient (r = 0.78; P = .035). Compared with control samples, pulmonary vein thickness was increased by 2.9-fold (P = .008) in the HCM group, which correlated with mPAP (r = 0.81; P = .03) and LV outflow tract gradient (r = 0.83; P = .02)., Interpretation: To the best of our knowledge, these data demonstrate for the first time that in patients with obstructive HCM, heart failure is associated with pathogenic pulmonary vascular remodeling even when mPAP is elevated only mildly. These observations clarify PH pathophysiologic features in HCM, with future implications for clinical strategies that mitigate outflow tract obstruction., (Published by Elsevier Inc.)

In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-Seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all aa and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest that elevated glucose and proline avidity underlie increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.

Introduction: Inducible laryngeal obstruction is a common and challenging cause of exertional dyspnea. We report a case of an unanticipated cardiac condition that presented with symptoms suggestive of inducible laryngeal obstruction., Discussion: A 55-year-old man was evaluated for progressive exertional dyspnea and throat tightness, unexplained after multiple medical evaluations. Resting laryngeal examination was suspicious for laryngopharyngeal reflux and mild vocal fold adduction during quiet expiration. Given progressive and refractory symptoms, maximal cardiopulmonary exercise testing with intermittent laryngeal examination was performed. This study excluded laryngeal causes of exercise limitation and led to an unexpected diagnosis of persistent atrial flutter and hypertrophic cardiomyopathy., Conclusion: Cardiopulmonary exercise testing with laryngeal examination can identify unexpected and life-threatening mimics of inducible laryngeal obstruction that may be missed by unmonitored exercise challenges. Suspicion for inducible laryngeal obstruction at rest may not predict the true nature of exercise limitation on cardiopulmonary exercise testing., (Copyright © 2020 The Voice Foundation. All rights reserved.)

The pathobiology of in situ pulmonary thrombosis in acute respiratory distress syndrome (ARDS) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is incompletely characterized. In human pulmonary artery endothelial cells (HPAECs), hypoxia increases neural precursor cell expressed, developmentally downregulated 9 (NEDD9) and induces expression of a prothrombotic NEDD9 peptide (N9 P ) on the extracellular plasma membrane surface. We hypothesized that the SARS-CoV-2-ARDS pathophenotype involves increased pulmonary endothelial N9 P . Paraffin-embedded autopsy lung specimens were acquired from patients with SARS-CoV-2-​​​​​​ARDS ( n  = 13), ARDS from other causes ( n  = 10), and organ donor controls ( n  = 5). Immunofluorescence characterized the expression of N9 P , fibrin, and transcription factor 12 (TCF12), a putative binding target of SARS-CoV-2 and known transcriptional regulator of NEDD9 . We performed RNA-sequencing on normal HPAECs treated with normoxia or hypoxia (0.2% O 2 ) for 24 h. Immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) profiled protein-protein interactions involving N9 P relevant to thrombus stabilization. Hypoxia increased TCF12 messenger RNA significantly compared to normoxia in HPAECs in vitro (+1.19-fold, p  = 0.001; false discovery rate = 0.005), and pulmonary endothelial TCF12 expression was increased threefold in SARS-CoV-2-ARDS versus donor control lungs ( p  < 0.001). Compared to donor controls, pulmonary endothelial N9 P -fibrin colocalization was increased in situ in non-SARS-CoV-2-ARDS and SARS-CoV-2-ARDS decedents (3.7 ± 1.2 vs. 10.3 ± 3.2 and 21.8 ± 4.0 arb. units, p  < 0.001). However, total pulmonary endothelial N9 P was increased significantly only in SARS-CoV-2-ARDS versus donor controls (15 ± 4.2 vs. 6.3 ± 0.9 arb. units, p  < 0.001). In HPAEC plasma membrane isolates, IP-LC-MS identified a novel protein-protein interaction between NEDD9 and the β 3 -subunit of the α v β 3 -integrin, which regulates fibrin anchoring to endothelial cells. In conclusion, lethal SARS-CoV-2-ARDS is associated with increased pulmonary endothelial N9 P expression and N9 P -fibrin colocalization in situ. Further investigation is needed to determine the pathogenetic and potential therapeutic relevance of N9 P to the thrombotic pathophenotype of SARS-CoV-2-ARDS., Competing Interests: George A. Alba: Stock, Verve Therapeutics; Pending US Patents PCT/US2019/059890 and PCT/US2020/066886. Lida P. Hariri: Plaint Therapeutics and Boehringer Ingelheim, consulting feeds; Boehringer Ingelheim, medical advisory board. Bradley A. Maron: Actelion; steering committee (outside the scope of this work); Deerfield Company, investigator‐sponsored research; Regeneron, consultant (outside the scope of this work); US Patent #9,605,047; PCT/US2015/029672. The remaining authors report no conflicts of interest., (© 2022 The Authors. Pulmonary Circulation published by John Wiley & Sons Ltd on behalf of Pulmonary Vascular Research Institute.)

Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in vivo . Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico . Compared with normoxia, hypoxia (0.2% O 2 ) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in vitro . Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in vitro and from patients with CTEPH ex vivo . Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9 -/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in vivo . Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.

Background: In pulmonary hypertension subgroups, elevated pulmonary vascular resistance (PVR) of 3·0 Wood units or more is associated with poor prognosis. However, the spectrum of PVR risk in pulmonary hypertension is not known. To address this area of uncertainty, we aimed to analyse the relationship between PVR and adverse clinical outcomes in pulmonary hypertension., Methods: We did a retrospective cohort study of all patients undergoing right heart catheterisation (RHC) in the US Veterans Affairs health-care system (Oct 1, 2007-Sep 30, 2016). Patients were included in the analyses if data from a complete RHC and at least 1 year of follow-up were available. Both inpatients and outpatients were included, but individuals with missing mean pulmonary artery pressure (mPAP), pulmonary artery wedge pressure, or cardiac output were excluded. The primary outcome measure was time to all-cause mortality assessed by the Veteran Affairs vital status file. Cox proportional hazards models were used to assess the association between PVR and outcomes, and the mortality hazard ratio was validated in a RHC cohort from Vanderbilt University Medical Center (Sept 24, 1998-June 1, 2016)., Findings: The primary cohort (N=40 082; 38 751 [96·7%] male; median age 66·5 years [IQR 61·1-73·5]; median follow-up 1153 days [IQR 570-1971]), included patients with a history of heart failure (23 201 [57·9%]) and chronic obstructive pulmonary disease (13 348 [33·3%]). We focused on patients at risk for pulmonary hypertension based on a mPAP of at least 19 mm Hg (32 725 [81·6%] of 40 082). When modelled as a continuous variable, the all-cause mortality hazard for PVR was increased at around 2·2 Wood units compared with PVR of 1·0 Wood unit. Among patients with a mPAP of at least 19 mm Hg and pulmonary artery wedge pressure of 15 mm Hg or less, the adjusted hazard ratio (HR) for mortality was 1·71 (95% CI 1·59-1·84; p<0·0001) and for heart failure hospitalisation was 1·27 (1·13-1·43; p=0·0001), when comparing PVR of 2·2 Wood units or more to less than 2·2 Wood units. The validation cohort (N=3699, 1860 [50·3%] male, median age 60·4 years [49·5-69·2]; median follow-up 1752 days [IQR 1281-2999]) included 2870 patients [77·6%] with mPAP of at least 19 mm Hg (1418 [49·4%] male). The adjusted mortality HR for patients in the mPAP of 19 mm Hg or more group and with PVR of 2·2 Wood units or more and pulmonary artery wedge pressure of 15 mm or less Hg (1221 [42·5%] of 2870) was 1·81 (95% CI 1·33-2·47; p=0·0002)., Interpretation: These data widen the continuum of clinical risk for mortality and heart failure in patients referred for RHC with elevated pulmonary artery pressure to include PVR of around 2.2 Wood units and higher. Testing the generalisability of these findings in at-risk populations with fewer cardiopulmonary comorbidities is warranted., Funding: None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Pulmonary arterial hypertension (PAH) is a highly morbid disease characterized by elevated pulmonary vascular resistance (PVR) and pathogenic right ventricular remodeling. Endothelial expression of the prometastatic protein NEDD9 is increased in fibrotic PAH arterioles, and NEDD9 inhibition decreases PVR in experimental PAH. We hypothesized that circulating NEDD9 is increased in PAH and informs the clinical profile of patients., Methods: Clinical data and plasma samples were analyzed retrospectively for 242 patients from 5 referral centers (2010-2017): PAH (n = 139; female 82%, 58 [48-67] years), non-PAH pulmonary hypertension (PH) (n = 54; female 56%, 63.4 ± 12.2 years), and dyspnea non-PH controls (n = 36; female 75%, 54.2 ± 14.0 years)., Results: Compared with controls, NEDD9 was increased in PAH by 1.82-fold (p < 0.0001). Elevated NEDD9 correlated with PVR in idiopathic PAH (ρ = 0.42, p < 0.0001, n = 54), connective tissue disease (CTD)-PAH (ρ = 0.53, p < 0.0001, n = 53), and congenital heart disease-PAH (ρ = 0.68, p < 0.0001, n = 10). In CTD-PAH, NEDD9 correlated with 6-minute walk distance (ρ = -0.35, p = 0.028, n = 39). In contrast to the PAH biomarker N-terminal pro-brain natriuretic peptide (n = 38), NEDD9 correlated inversely with exercise pulmonary artery wedge pressure and more strongly with right ventricular ejection fraction (ρ = -0.41, p = 0.006, n = 45) in a mixed population. The adjusted hazard ratio for lung transplant-free survival was 1.12 (95% confidence interval [CI], 1.02-1.22, p = 0.01) and 1.75 (95% CI, 1.12-2.73, p = 0.01) per 1 ng/ml and 5 ng/ml increase in plasma NEDD9, respectively, by Cox proportional hazard model., Conclusions: In PAH, plasma NEDD9 is increased and associates with key prognostic variables. Prospective studies that include hard end points are warranted to validate NEDD9 as a novel PAH biomarker., (Published by Elsevier Inc.)

Background: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis., Methods: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing., Results: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds., Conclusions: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Transcriptomic analysis of pulmonary microvascular endothelial cells from experimental models offers insight into pulmonary arterial hypertension (PAH) pathobiology. However, culturing may alter the molecular profile of endothelial cells prior to analysis, limiting the translational relevance of results. Here we present a novel and validated method for isolating RNA from pulmonary microvascular endothelial cells (PMVECs) ex vivo that does not require cell culturing. Initially, presumed rat PMVECs were isolated from rat peripheral lung tissue using tissue dissociation and enzymatic digestion, and cells were cultured until confluence to assess endothelial marker expression. Anti-CD31, anti-von Willebrand Factor, and anti-α-smooth muscle actin immunocytochemistry/immunofluorescence signal was detected in presumed rat PMVECs, but also in non-endothelial cell type controls. By contrast, flow cytometry using an anti-CD31 antibody and isolectin 1-B4 (from Griffonia simplicifolia) was highly specific for rat PMVECs. We next developed a strategy in which the addition of an immunomagnetic selection step for CD31+ cells permitted culture-free isolation of rat PMVECs ex vivo for RNA isolation and transcriptomic analysis using fluorescence-activated cell sorting. Heterogeneity in the validity and reproducibility of results using commercial antibodies against endothelial surface markers corresponded to a substantial burden on laboratory time, labor, and scientific budget. We demonstrate a novel protocol for the culture-free isolation and transcriptomic analysis of rat PMVECs with translational relevance to PAH. In doing so, we highlight wide variability in the quality of commonly used biological reagents, which emphasizes the importance of investigator-initiated validation of commercial biomaterials., Competing Interests: The authors have declared that no competing interests exist.

Germline mutations involving small mothers against decapentaplegic-transforming growth factor-β (SMAD-TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys 18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1 Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap and, thus, was not contingent on TGF-β signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Rationale: Current methods assessing clinical risk because of exercise intolerance in patients with cardiopulmonary disease rely on a small subset of traditional variables. Alternative strategies incorporating the spectrum of factors underlying prognosis in at-risk patients may be useful clinically, but are lacking., Objective: Use unbiased analyses to identify variables that correspond to clinical risk in patients with exercise intolerance., Methods and Results: Data from 738 consecutive patients referred for invasive cardiopulmonary exercise testing at a single center (2011-2015) were analyzed retrospectively (derivation cohort). A correlation network of invasive cardiopulmonary exercise testing parameters was assembled using |r|>0.5. From an exercise network of 39 variables (ie, nodes) and 98 correlations (ie, edges) corresponding to P <9.5e -46 for each correlation, we focused on a subnetwork containing peak volume of oxygen consumption (pVo 2 ) and 9 linked nodes. K-mean clustering based on these 10 variables identified 4 novel patient clusters characterized by significant differences in 44 of 45 exercise measurements ( P <0.01). Compared with a probabilistic model, including 23 independent predictors of pVo 2 and pVo 2 itself, the network model was less redundant and identified clusters that were more distinct. Cluster assignment from the network model was predictive of subsequent clinical events. For example, a 4.3-fold ( P <0.0001; 95% CI, 2.2-8.1) and 2.8-fold ( P =0.0018; 95% CI, 1.5-5.2) increase in hazard for age- and pVo 2 -adjusted all-cause 3-year hospitalization, respectively, were observed between the highest versus lowest risk clusters. Using these data, we developed the first risk-stratification calculator for patients with exercise intolerance. When applying the risk calculator to patients in 2 independent invasive cardiopulmonary exercise testing cohorts (Boston and Graz, Austria), we observed a clinical risk profile that paralleled the derivation cohort., Conclusions: Network analyses were used to identify novel exercise groups and develop a point-of-care risk calculator. These data expand the range of useful clinical variables beyond pVo 2 that predict hospitalization in patients with exercise intolerance., (© 2018 American Heart Association, Inc.)

Importance: Thermodilution (Td) and estimated oxygen uptake Fick (eFick) methods are widely used to measure cardiac output (CO). They are often used interchangeably to make critical clinical decisions, yet few studies have compared these approaches as applied in medical practice., Objectives: To assess agreement between Td and eFick CO and to compare how well these methods predict mortality., Design, Setting, and Participants: This investigation was a retrospective cohort study with up to 1 year of follow-up. The study used data from the Veterans Affairs Clinical Assessment, Reporting, and Tracking (VA CART) program. The findings were corroborated in a cohort of patients cared for at Vanderbilt University, an academic referral center. Participants were more than 15 000 adults who underwent right heart catheterization, including 12 232 in the Veterans Affairs cohort between October 1, 2007, and September 30, 2013, and 3391 in the Vanderbilt cohort between January 1, 1998, and December 31, 2014., Exposures: A single cardiac catheterization was performed on each patient with CO estimated by both Td and eFick methods. Cardiac output was indexed to body surface area (cardiac index [CI]) for all analyses., Main Outcomes and Measures: All-cause mortality over 90 days and 1 year after catheterization., Results: Among 12 232 VA patients (mean [SD] age, 66.4 [9.9] years; 3.3% female) who underwent right heart catheterization in this cohort study, Td and eFick CI estimates correlated modestly (r = 0.65). There was minimal mean difference (eFick minus Td = -0.02 L/min/m2, or -0.4%) but wide 95% limits of agreement between methods (-1.3 to 1.3 L/min/m2, or -50.1% to 49.4%). Estimates differed by greater than 20% for 38.1% of patients. Low Td CI (<2.2 L/min/m2 compared with normal CI of 2.2-4.0 L/min/m2) more strongly predicted mortality than low eFick CI at 90 days (Td hazard ratio [HR], 1.71; 95% CI, 1.47-1.99; χ2 = 49.5 vs eFick HR, 1.42; 95% CI, 1.22-1.64; χ2 = 20.7) and 1 year (Td HR, 1.53; 95% CI, 1.39-1.69; χ2 = 71.5 vs eFick HR, 1.35; 1.22-1.49; χ2 = 35.2). Patients with a normal CI by both methods had 12.3% 1-year mortality. There was no significant additional risk for patients with a normal Td CI but a low eFick CI (12.9%, P = .51), whereas a low Td CI but normal eFick CI was associated with higher mortality (15.4%, P = .001). The results from the Vanderbilt cohort were similar in the context of a more balanced sex distribution (46.6% female)., Conclusions and Relevance: There is only modest agreement between Td and eFick CI estimates. Thermodilution CI better predicts mortality and should be favored over eFick in clinical practice.

Objectives: We sought to address concerns regarding recurring inpatient laboratory test order practices (daily laboratory tests) through a multifaceted approach to changing ordering patterns., Methods: We engaged in an interdepartmental collaboration to foster mindful test ordering through clinical policy creation, electronic clinical decision support, and continuous auditing and feedback., Results: Annualized daily order volumes decreased from approximately 25,000 to 10,000 during a 33-month postintervention review. This represented a significant change from preintervention order volumes (95% confidence interval, 0.61-0.64; P < 10-16). Total inpatient test volumes were not affected., Conclusions: Durable changes to inpatient order practices can be achieved through a collaborative approach to utilization management that includes shared responsibility for establishing clinical guidelines and electronic decision support. Our experience suggests auditing and continued feedback are additional crucial components to changing ordering behavior. Curtailing daily orders alone may not be a sufficient strategy to reduce in-laboratory costs., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Background: Overuse of clinical laboratory testing in the inpatient setting is a common problem. The objective of this project was to develop an inexpensive and easily implemented intervention to promote rational laboratory use without compromising resident education or patient care., Methods: The study comprised of a cluster-randomized, controlled trial to assess the impact of a multifaceted intervention of education, guideline development, elimination of recurring laboratory orders, unbundling of laboratory panels, and redesign of the daily progress note on laboratory test ordering. The population included all patients hospitalized "general medicine" was duplicated during 2 consecutive months on a general medicine teaching service within a 999-bed tertiary care hospital in Boston, Massachusetts. The primary outcome was the total number of commonly used laboratory tests per patient day during 2 months in 2008. Secondary outcomes included a subgroup analysis of each individual test per patient day, adverse events, and resident and nursing satisfaction., Results: A total of 5392 patient days were captured. The intervention produced a 9% decrease in aggregate laboratory use (rate ratio, 0.91; P = .021; 95% confidence interval, 0.84-0.98). Six instances of delayed diagnosis of acute kidney injury and 11 near misses were reported in the intervention arm., Conclusions: A bundled educational and administrative intervention promoting rational ordering of laboratory tests on a single academic general medicine service led to a modest but significant decrease in laboratory use. To our knowledge, this was the first study to examine the daily progress note as a tool to limit excessive test ordering. Unadjudicated near misses and possible harm were reported with this intervention. This finding warrants further study., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: To test whether the addition of Flavobacterium johnsoniae could increase the strength of saturated Ottawa 30 sand., Methods and Results: A box model was built that simulates groundwater-like flow through a main sand compartment. Strength tests were performed at seven locations and at two depths, 10.8 and 20.3 cm below the top of the tank, using a vane shear device before and after the addition of bacteria. After the addition of Fl. johnsoniae, sand samples were obtained from multiple sampling ports on the vertical sides of the box model. The presence of a bacterial biofilm was confirmed by staining these sand samples with SYTO-9 and Alexa Fluor 633 and viewing with a confocal microscope. The average shear strength increases after the addition of Fl. johnsoniae were 15.2-87.5%, depending on the experimental conditions., Conclusions: Flavobacterium johnsoniae caused a statistically significant increase in the strength of saturated Ottawa 30 sand., Significance and Impact of the Study: Biofilm-forming bacteria can increase the shear strength of saturated sand. The addition of biofilm-forming bacteria to a building site may be an alternate method to mitigate the effects of liquefaction.