Application of Metabolomics across the Spectrum of Pulmonary and Critical Care Medicine.

In recent years, metabolomics, the systematic study of small-molecule metabolites in biological samples, has yielded fresh insights into the molecular determinants of pulmonary diseases and critical illness. The purpose of this article is to orient the reader to this emerging field by discussing the fundamental tenets underlying metabolomics research, the tools and techniques that serve as foundational methodologies, and the various statistical approaches to analysis of metabolomics datasets. We present several examples of metabolomics applied to pulmonary and critical care medicine to illustrate the potential of this avenue of research to deepen our understanding of pathophysiology. We conclude by reviewing recent advances in the field and future research directions that stand to further the goal of personalizing medicine to improve patient care.

Collagen 18A1/Endostatin Expression in the Progression of Right Ventricular Remodeling and Dysfunction in Pulmonary Arterial Hypertension.

Numerous studies have demonstrated that endostatin (ES), a potent angiostatic peptide derived from collagen type XVIII α 1 chain and encoded by COL18A1, is elevated in pulmonary arterial hypertension (PAH). It is important to note that elevated ES has consistently been associated with altered hemodynamics, poor functional status, and adverse outcomes in adult and pediatric PAH. This study used serum samples from patients with Group I PAH and plasma and tissue samples derived from the Sugen/hypoxia rat pulmonary hypertension model to define associations between COL18A1/ES and disease development, including hemodynamics, right ventricle (RV) remodeling, and RV dysfunction. Using cardiac magnetic resonance imaging and advanced hemodynamic assessments with pressure-volume loops in patients with PAH to assess RV-pulmonary arterial coupling, we observed a strong relationship between circulating ES levels and metrics of RV structure and function. Specifically, RV mass and the ventricular mass index were positively associated with ES, whereas RV ejection fraction and RV-pulmonary arterial coupling were inversely associated with ES levels. Our animal data demonstrate that the development of pulmonary hypertension is associated with increased COL18A1/ES in the heart as well as the lungs. Disease-associated increases in COL18A1 mRNA and protein were most pronounced in the RV compared with the left ventricle and lung. COL18A1 expression in the RV was strongly associated with disease-associated changes in RV mass, fibrosis, and myocardial capillary density. These findings indicate that COL18A1/ES increases early in disease development in the RV and implicates COL18A1/ES in pathologic RV dysfunction in PAH.

Aberrant long-chain fatty acid metabolism associated with evolving systemic sclerosis-associated pulmonary arterial hypertension.

We sought to investigate differential metabolism in patients with systemic sclerosis (SSc) who develop pulmonary arterial hypertension (PAH) versus those who do not, as a method of identifying potential disease biomarkers. In a nested case-control design, serum metabolites were assayed in SSc subjects who developed right heart catheterization-confirmed PAH (n = 22) while under surveillance in a longitudinal cohort from Johns Hopkins, then compared with metabolites assayed in matched SSc patients who did not develop PAH (n = 22). Serum samples were collected at "proximate" (within 12 months) and "distant" (within 1-5 yr) time points relative to PAH diagnosis. Metabolites were identified using liquid chromatography-mass spectroscopy (LC-MS). An LC-MS dataset from SSc subjects with either mildly elevated pulmonary pressures or overt PAH from the University of Michigan was compared. Differentially abundant metabolites were tested as predictors of PAH in two additional validation SSc cohorts. Long-chain fatty acid metabolism (LCFA) consistently differed in SSc-PAH versus SSc without PH. LCFA metabolites discriminated SSc-PAH patients with mildly elevated pressures in the Michigan cohort and predicted SSc-PAH up to 2 yr before clinical diagnosis in the Hopkins cohort. Acylcholines containing LCFA residues and linoleic acid metabolites were most important for discriminating SSc-PAH. Combinations of acylcholines and linoleic acid metabolites provided good discrimination of SSc-PAH across cohorts. Aberrant lipid metabolism is observed throughout the evolution of PAH in SSc. Lipidomic signatures of abnormal LCFA metabolism distinguish SSc-PAH patients from those without PH, including before clinical diagnosis and in mild disease.NEW & NOTEWORTHY Abnormal lipid metabolism is evident across time in the development of SSc-PAH, and dysregulated long-chain fatty acid metabolism predicts overt PAH.

Fatty acid metabolism promotes TRPV4 activity in lung microvascular endothelial cells in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.

Poor cardiac output reserve in pulmonary arterial hypertension is associated with right ventricular stiffness and impaired interventricular dependence.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterised by poor exercise tolerance. The contribution of right ventricular (RV) diastolic function to the augmentation of cardiac output during exercise is not known. This study leverages pressure-volume (P-V) loop analysis to characterise the impact of RV diastology on poor flow augmentation during exercise in PAH. METHODS: RV P-V loops were measured in 41 PAH patients at rest and during supine bike exercise. Patients were stratified by median change in cardiac index (CI) during exercise into two groups: high and low CI reserve. Indices of diastolic function (end-diastolic elastance (E ed)) and ventricular interdependence (left ventricular transmural pressure (LVTMP)) were compared at matched exercise stages. RESULTS: Compared to patients with high CI reserve, those with low reserve exhibited lower exercise stroke volume (36 versus 49 mL·m-2; p=0.0001), with higher associated exercise afterload (effective arterial elastance (E a) 1.76 versus 0.90 mmHg·mL-1; p<0.0001), RV stiffness (E ed 0.68 versus 0.26 mmHg·mL-1; p=0.003) and right-sided pressures (right atrial pressure 14 versus 8 mmHg; p=0.002). Higher right-sided pressures led to significantly lower LV filling among the low CI reserve subjects (LVTMP -4.6 versus 3.2 mmHg; p=0.0001). Interestingly, low exercise flow reserve correlated significantly with high afterload and RV stiffness, but not with RV contractility nor RV-PA coupling. CONCLUSIONS: Patients with poor exercise CI reserve exhibit poor exercise RV afterload, stiffness and right-sided filling pressures that depress LV filling and stroke work. High afterload and RV stiffness were the best correlates to low flow reserve in PAH. Exercise unmasked significant pathophysiological PAH differences unapparent at rest.

Chinese hamster ovary cell line engineering strategies for modular production of custom extracellular vesicles.

Continuously secreted by all cell types, extracellular vesicles (EVs) are small membrane-bound structures which shuttle bioactive cargo between cells across their external environment. Their central role as natural molecular messengers and ability to cross biological barriers has garnered significant attention in the use of EVs as therapeutic delivery vehicles. Still, harnessing the potential of EVs is faced with many obstacles. A cell line engineering approach can be used to exploit EVs to encapsulate a bespoke cargo of interest. However, full details regarding native EV-loading mechanisms remain under debate, making this a challenge. While Chinese hamster ovary (CHO) cells are well known to be the preferred host for recombinant therapeutic protein production, their application as an EV producer cell host has been largely overlooked. In this study, we engineered CHO DG44 cells to produce custom EVs with bespoke cargo. To this end, genetic constructs employing split green fluorescent protein technology were designed for tagging both CD81 and protein cargoes to enable EV loading via self-assembling activity. To demonstrate this, NanoLuc and mCherry were used as model reporter cargoes to validate engineered loading into EVs. Experimental findings indicated that our custom EV approach produced vesicles with up to 15-fold greater cargo compared with commonly used passive loading strategies. When applied to recipient cells, we observed a dose-dependent increase in cargo activity, suggesting successful delivery of engineered cargo via our custom CHO EVs.

Financial Incentives for Physical Activity and Sports Participation in Young People.

Physical inactivity is a global health problem. Childhood is an opportune time to establish healthy physical activity behaviors, including the participation in organized physical activity, such as sports. We hypothesize that financial incentives can improve young people's participation in physical activity and sports. The design of the incentive and the context in which it operates are crucial to its success.

Improved Survival for Patients with Systemic Sclerosis-associated Pulmonary Arterial Hypertension: The Johns Hopkins Registry.

Rationale: To date, it remains unclear whether recent changes in the management of patients with systemic sclerosis-associated pulmonary hypertension (SSc-PH) have improved survival. Objectives: To describe a cohort of patients with SSc-PH and compare their characteristics and survival between the last two decades. Methods: Patients with SSc-PH prospectively enrolled in the Johns Hopkins Pulmonary Hypertension Center Registry were grouped into two cohorts based on the date of diagnostic right heart catheterization: cohort A included patients whose disease was diagnosed between 1999 and 2010, and cohort B included those whose disease was diagnosed between 2010 and 2021. Patients' characteristics were compared between the two cohorts. Measurements and Main Results: Of 504 patients with SSc-PH distributed almost equally between the two cohorts, 308 (61%) had World Symposium on Pulmonary Hypertension group 1, 43 (9%) had group 2, and 151 (30%) had group 3 disease. Patients with group 1 disease in cohort B had significantly better clinical and hemodynamic characteristics at diagnosis, were more likely to receive upfront combination pulmonary arterial hypertension therapy, and had a nearly 4-year increase in median transplant-free survival in univariable analysis than those in cohort A (P < 0.01). Improved transplant-free survival was still observed after adjusting for patients' baseline characteristics. In contrast, for group 2 or 3 patients with SSc-PH, there were no differences in baseline clinical, hemodynamic, or survival characteristics between the two cohorts. Conclusions: This is the largest single-center study that compares clinical characteristics of patients with SSc-PH between the last two decades. Transplant-free survival has improved significantly for those with group 1 disease over the last decade, possibly secondary to earlier detection and better therapeutic management. Conversely, those with group 2 or 3 disease continue to have dismal prognosis.

Metabolic profiling of in vivo right ventricular function and exercise performance in pulmonary arterial hypertension.

Right ventricular (RV) adaptation is the principal determinant of outcomes in pulmonary arterial hypertension (PAH), however, RV function is challenging to assess. RV responses to hemodynamic stressors are particularly difficult to interrogate without invasive testing. This study sought to identify metabolomic markers of in vivo right ventricular function and exercise performance in PAH. Consecutive subjects with PAH (n = 23) underwent rest and exercise right heart catheterization with multibeat pressure volume loop analysis. Pulmonary arterial blood was collected at rest and during exercise. Mass spectrometry-based targeted metabolomics were performed, and metabolic associations with hemodynamics and comprehensive measures of RV function were determined using sparse partial least squares regression. Metabolite profiles were compared with N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) measurements for accuracy in modeling ventriculo-arterial parameters. Thirteen metabolites changed in abundance with exercise, including metabolites reflecting increased arginine bioavailability, precursors of catecholamine and nucleotide synthesis, and branched-chain amino acids. Higher resting arginine bioavailability predicted more favorable exercise hemodynamics and pressure-flow relationships. Subjects with more severe PAH augmented arginine bioavailability with exercise to a greater extent than subjects with less severe PAH. We identified relationships between kynurenine pathway metabolism and impaired ventriculo-arterial coupling, worse RV diastolic function, lower RV contractility, diminished RV contractility with exercise, and RV dilation with exercise. Metabolite profiles outperformed NT-proBNP in modeling RV contractility, diastolic function, and exercise performance. Specific metabolite profiles correspond to RV functional measurements only obtainable via invasive pressure-volume loop analysis and predict RV responses to exercise. Metabolic profiling may inform discovery of RV functional biomarkers.NEW & NOTEWORTHY In this cohort of patients with pulmonary arterial hypertension (PAH), we investigate metabolomic associations with comprehensive right ventricular (RV) functional measurements derived from multibeat RV pressure-volume loop analysis. Our results show that tryptophan metabolism, particularly the kynurenine pathway, is linked to intrinsic RV function and PAH pathobiology. Findings also highlight the importance of arginine bioavailability in the cardiopulmonary system's response to exercise stress. Metabolite profiles selected via unbiased analysis outperformed N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) in predicting load-independent measures of RV function at rest and cardiopulmonary system performance under stress. Overall, this work suggests the potential for select metabolites to function as disease-specific biomarkers, offers insights into PAH pathobiology, and informs discovery of potentially targetable RV-centric pathways.

Kynurenine pathway metabolism evolves with development of preclinical and scleroderma-associated pulmonary arterial hypertension.

Understanding metabolic evolution underlying pulmonary arterial hypertension (PAH) development may clarify pathobiology and reveal disease-specific biomarkers. Patients with systemic sclerosis (SSc) are regularly surveilled for PAH, presenting an opportunity to examine metabolic change as disease develops in an at-risk cohort. We performed mass spectrometry-based metabolomics on longitudinal serum samples collected before and near SSc-PAH diagnosis, compared with time-matched SSc subjects without PAH, in a SSc surveillance cohort. We validated metabolic differences in a second cohort and determined metabolite-phenotype relationships. In parallel, we performed serial metabolomic and hemodynamic assessments as the disease developed in a preclinical model. For differentially expressed metabolites, we investigated corresponding gene expression in human and rodent PAH lungs. Kynurenine and its ratio to tryptophan (kyn/trp) increased over the surveillance period in patients with SSc who developed PAH. Higher kyn/trp measured two years before diagnostic right heart catheterization increased the odds of SSc-PAH diagnosis (OR 1.57, 95% CI 1.05-2.36, P = 0.028). The slope of kyn/trp rise during SSc surveillance predicted PAH development and mortality. In both clinical and experimental PAH, higher kynurenine pathway metabolites correlated with adverse pulmonary vascular and RV measurements. In human and rodent PAH lungs, expression of TDO2, which encodes tryptophan 2,3 dioxygenase (TDO), a protein that catalyzes tryptophan conversion to kynurenine, was significantly upregulated and tightly correlated with pulmonary hypertensive features. Upregulated kynurenine pathway metabolism occurs early in PAH, localizes to the lung, and may be modulated by TDO2. Kynurenine pathway metabolites may be candidate PAH biomarkers and TDO warrants exploration as a potential novel therapeutic target.NEW & NOTEWORTHY Our study shows an early increase in kynurenine pathway metabolism in at-risk subjects with systemic sclerosis who develop pulmonary arterial hypertension (PAH). We show that kynurenine pathway upregulation precedes clinical diagnosis and that this metabolic shift is associated with increased disease severity and shorter survival times. We also show that gene expression of TDO2, an enzyme that generates kynurenine from tryptophan, rises with PAH development.

Leaf structural traits mediating pre-existing physical innate resistance to sorghum aphid in sorghum under uninfested conditions.

KEY MESSAGE: We found four indicative traits of innate immunity. Sorghum-resistant varieties had a greater trichome, stomatal and chloroplast density, and smaller mesophyll intercellular width than susceptible varieties. The sorghum aphid (SA), Melanaphis sorghi (Theobald), can severely reduce sorghum yield. The contribution of structural traits to SA resistance has not been extensively studied. Moreover, the current screening method for resistance is inherently subjective for resistance and requires infestation in plants. Quantifying the microanatomical basis of innate SA resistance is crucial for developing reliable screening tools requiring no infestation. The goal of this study was to identify structural traits linked to physical innate SA resistance in sorghum. We conducted controlled environment and field experiments under no SA infestation conditions, with two resistant (R. LBK1 and R. Tx2783) and two susceptible (R. Tx7000 and R. Tx430) varieties. Leaf tissues collected at the fifth leaf stage in the controlled environment experiment were analyzed for the epidermal and mesophyll traits using light and transmission electron microscopy. Leaf tissues collected at physiological maturity in the field experiment were analyzed for surface traits using scanning electron microscopy. Our results showed that stomatal density, trichome density, trichome length, and chloroplast density are key leaf structural traits indicative of physical innate SA resistance. We found that resistant varieties had a greater density of trichomes (39%), stomata (31%), and chloroplast (42%), and smaller mesophyll intercellular width (- 52%) than susceptible varieties. However, the chloroplast, mitochondria, and epidermal cell ultrastructural traits were ineffective indicators of SA resistance. Our findings provide the foundation for developing an objective high-throughput method for SA resistance screening. We suggest a follow-up validation experiment to confirm our outcomes under SA infestation conditions.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.

Effectiveness of physical activity interventions for improving depression, anxiety and distress: an overview of systematic reviews.

OBJECTIVE: To synthesise the evidence on the effects of physical activity on symptoms of depression, anxiety and psychological distress in adult populations. DESIGN: Umbrella review. DATA SOURCES: Twelve electronic databases were searched for eligible studies published from inception to 1 January 2022. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Systematic reviews with meta-analyses of randomised controlled trials designed to increase physical activity in an adult population and that assessed depression, anxiety or psychological distress were eligible. Study selection was undertaken in duplicate by two independent reviewers. RESULTS: Ninety-seven reviews (1039 trials and 128 119 participants) were included. Populations included healthy adults, people with mental health disorders and people with various chronic diseases. Most reviews (n=77) had a critically low A MeaSurement Tool to Assess systematic Reviews score. Physical activity had medium effects on depression (median effect size=-0.43, IQR=-0.66 to -0.27), anxiety (median effect size=-0.42, IQR=-0.66 to -0.26) and psychological distress (effect size=-0.60, 95% CI -0.78 to -0.42), compared with usual care across all populations. The largest benefits were seen in people with depression, HIV and kidney disease, in pregnant and postpartum women, and in healthy individuals. Higher intensity physical activity was associated with greater improvements in symptoms. Effectiveness of physical activity interventions diminished with longer duration interventions. CONCLUSION AND RELEVANCE: Physical activity is highly beneficial for improving symptoms of depression, anxiety and distress across a wide range of adult populations, including the general population, people with diagnosed mental health disorders and people with chronic disease. Physical activity should be a mainstay approach in the management of depression, anxiety and psychological distress. PROSPERO REGISTRATION NUMBER: CRD42021292710.

Early mobilisation in critically ill children: Does routine patient screening reduce time to commencing mobilisation?

OBJECTIVE: The objective of this study was to investigate the impact of daily screening for medical readiness to participate in early mobilisation in the paediatric intensive care unit (PICU), on reducing time to mobilisation and to explore the safety-, feasibility-, and patient-level barriers to the practice. METHODS: An interventional study with a historical control group was conducted in a PICU in a tertiary teaching hospital in Australia. The Early Mobilisation Screening Checklist was applied at 24-48 h of PICU stay with the aim to reduce time to commencing mobilisation. All patients aged term to 18 years admitted to the PICU for >48 h were included in this study. Data on time to mobilisation and patient characteristics were collected by an unblinded case note audit of children admitted to the PICU over 5 months in 2018 for the baseline group and over a corresponding period in 2019 for the intervention group. MEASUREMENTS AND MAIN RESULTS: A total of 71 children were enrolled. Survival analysis was used to compare time to mobilisation between groups, and a cox regression model found that children in the intervention group were 1.26 times more likely to participate in mobility, but this was not statistically significant (P = 0.391, log rank test for equality of survival functions). Early mobilisation was safe, with no adverse events reported in 177 participant mobilisation days. Feasibility was demonstrated by 62% of participants mobilising within 72 h of admission. Mechanical ventilation during stay (P = 0.043) and days receiving sedation infusion (% of days) (P = 0.042) were associated with a decreased likelihood of participating in mobility. CONCLUSIONS: Implementation of routine screening alone does not significantly reduce time to commencing mobility in the PICU. Early mobilisation in the PICU is safe and feasible and resulted in no adverse events during mobilisation. Patient characteristics influencing participation in mobility warrant further exploration.

Optimization and Scale Up of Spray Dried CPZEN-45 Aerosol Powders for Inhaled Tuberculosis Treatment.

PURPOSE: Tuberculosis (TB) remains one of the most serious diseases caused by a single organism. Multiple (MDR) and extensively (XDR) drug resistant disease poses a threat to global health and requires new drugs and/or innovative approaches to treatment. A number of drugs have been proposed as inhaled therapy for TB, frequently prepared by spray drying. CPZEN-45 is a novel anti-tubercular drug that has poor oral bioavailability but has shown promise when administered via inhalation. METHODS: Excipient-free CPZEN-45 HCl has been spray dried into a powder with physicochemical characteristics, aerodynamic particle size distribution, and delivered dose suitable for consideration as an inhaled product. RESULTS: The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the powder delivered using a RS01 inhaler were 2.62 ± 0.04 µm and 1.76 ± 0.09, respectively. Additionally, the powder was physically and chemically stable after storage at ambient conditions for >1.5 years with particle size similar to freshly manufactured product. Overages in spray dried powder were recycled the powder and resprayed into drug product likewise resulting in negligible change in quality thus allowing for further preclinical characterization as necessary. CPZEN-45 was scaled up using pilot-scale manufacturing equipment where the density of the powder was increased to facilitate larger delivered doses without affecting the aerodynamic performance properties. CONCLUSION: The spray dried powders were suitable for pharmacokinetics, efficacy and preclinical toxicology studies. The final method of manufacture may be used directly for CGMP particle manufacture to support IND and Phase I clinical trials and beyond.

Insulin-like growth factor binding protein-2: a new circulating indicator of pulmonary arterial hypertension severity and survival.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease that results from cardio-pulmonary dysfunction with the pathology largely unknown. Insulin-like growth factor binding protein 2 (IGFBP2) is an important member of the insulin-like growth factor family, with evidence suggesting elevation in PAH patients. We investigated the diagnostic and prognostic value of serum IGFBP2 in PAH to determine if it could discriminate PAH from healthy controls and if it was associated with disease severity and survival. METHODS: Serum IGFBP2 levels, as well as IGF1/2 levels, were measured in two independent PAH cohorts, the Johns Hopkins Pulmonary Hypertension program (JHPH, N = 127), NHLBI PAHBiobank (PAHB, N = 203), and a healthy control cohort (N = 128). The protein levels in lung tissues were determined by western blot. The IGFBP2 mRNA expression levels in pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) were assessed by RNA-seq, secreted protein levels by ELISA. Association of biomarkers with clinical variables was evaluated using adjusted linear or logistic regression and Kaplan-Meier analysis. RESULTS: In both PAH cohorts, serum IGFBP2 levels were significantly elevated (p < 0.0001) compared to controls and discriminated PAH from controls with an AUC of 0.76 (p < 0.0001). A higher IGFBP2 level was associated with a shorter 6-min walk distance (6MWD) in both cohorts after adjustment for age and sex (coefficient - 50.235 and - 57.336 respectively). Cox multivariable analysis demonstrated that higher serum IGFBP2 was a significant independent predictor of mortality in PAHB cohort only (HR, 3.92; 95% CI, 1.37-11.21). IGF1 levels were significantly increased only in the PAHB cohort; however, neither IGF1 nor IGF2 had equivalent levels of associations with clinical variables compared with IGFBP2. Western blotting shown that IGFBP2 protein was significantly increased in the PAH vs control lung tissues. Finally, IGFBP2 mRNA expression and secreted protein levels were significantly higher in PASMC than in PAEC. CONCLUSIONS: IGFBP2 protein expression was increased in the PAH lung, and secreted by PASMC. Elevated circulating IGFBP2 was associated with PAH severity and mortality and is a potentially valuable prognostic marker in PAH.

Cellular sources of interleukin-6 and associations with clinical phenotypes and outcomes in pulmonary arterial hypertension.

The pro-inflammatory cytokine interleukin (IL)-6 has been associated with outcomes in small pulmonary arterial hypertension (PAH) cohorts composed largely of patients with severe idiopathic PAH (IPAH). It is unclear whether IL-6 is a marker of critical illness or a mechanistic biomarker of pulmonary vascular remodelling. We hypothesised that IL-6 is produced by pulmonary vascular cells and sought to explore IL-6 associations with phenotypes and outcomes across diverse subtypes in a large PAH cohort.IL-6 protein and gene expression levels were measured in cultured pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) from PAH patients and healthy controls. Serum IL-6 was measured in 2017 well-characterised PAH subjects representing each PAH subgroup. Relationships between IL-6 levels, clinical variables, and mortality were analysed using regression models.Significantly higher IL-6 protein and gene expression levels were produced by PASMCs than by PAECs in PAH (p<0.001), while there was no difference in IL-6 between cell types in controls. Serum IL-6 was highest in PAH related to portal hypertension and connective tissue diseases (CTD-PAH). In multivariable modelling, serum IL-6 was associated with survival in the overall cohort (hazard ratio 1.22, 95% CI 1.08-1.38; p<0.01) and in IPAH, but not in CTD-PAH. IL-6 remained associated with survival in low-risk subgroups of subjects with mild disease.IL-6 is released from PASMCs, and circulating IL-6 is associated with specific clinical phenotypes and outcomes in various PAH subgroups, including subjects with less severe disease. IL-6 is a mechanistic biomarker, and thus a potential therapeutic target, in certain PAH subgroups.

Elevated Interleukin-6 Levels Predict Clinical Worsening in Pediatric Pulmonary Arterial Hypertension.

OBJECTIVE: To assess whether circulating interleukin-6 (IL-6) is associated with measures of disease severity and clinical worsening in pediatric pulmonary arterial hypertension (PAH). STUDY DESIGN: IL-6 was measured by enzyme-linked immunosorbent assay in serum samples from a cross-sectional cohort from the National Heart, Lung, and Blood Institute Pulmonary Arterial Hypertension Biobank (n = 175) and a longitudinal cohort from Children's Hospital Colorado (CHC) (n = 61). Associations between IL-6, disease severity, and outcomes were studied with regression and Kaplan-Meier analysis. RESULTS: In analyses adjusted for age and sex, each log-unit greater IL-6 was significantly associated in the Pulmonary Arterial Hypertension Biobank cohort with greater pulmonary vascular resistance indices, lower odds of having idiopathic PAH or treatment with prostacyclin, and greater odds of having PAH associated with a repaired congenital shunt. In the CHC cohort, each log-unit greater IL-6 was significantly associated with greater mean pulmonary arterial pressure over time. Kaplan-Meier analysis in the CHC cohort revealed that IL-6 was significantly associated with clinical worsening (a composite score of mortality, transplant, or palliative surgery) (P = .037). CONCLUSIONS: IL-6 was significantly associated with worse hemodynamics at baseline and over time and may be associated with clinical worsening. IL-6 may provide a less-invasive method for disease monitoring and prognosis in pediatric PAH as well as a potential therapeutic target.

Performance of Critical Care Outcome Prediction Models in an Intermediate Care Unit.

BACKGROUND: Intermediate care units (IMCUs) are heterogeneous in design and operation, which makes comparative effectiveness studies challenging. A generalizable outcome prediction model could improve such comparisons. However, little is known about the performance of critical care outcome prediction models in the intermediate care setting. The purpose of this study is to evaluate the performance of the Acute Physiology and Chronic Health Evaluation version II (APACHE II), Simplified Acute Physiology Score version II (SAPS II) and version 3 (SAPS 3), and Mortality Probability Model version III (MPM0III) in patients admitted to a well-characterized IMCU. MATERIALS AND METHODS: In the IMCU of an academic medical center (July to December 2012), the discrimination and calibration of each outcome prediction model were evaluated using the area under the receiver-operating characteristic and Hosmer-Lemeshow goodness-of-fit test, respectively. Standardized mortality ratios (SMRs) were also calculated. RESULTS: The cohort included data from 628 unique IMCU admissions with an inpatient mortality rate of 8.3%. All models exhibited good discrimination, but only the SAPS II and MPM0III were well calibrated. While the APACHE II and SAPS 3 both markedly overestimated mortality, the SMR for the SAPS II and MPM0III were 0.91 and 0.91, respectively. CONCLUSIONS: The SAPS II and MPM0III exhibited good discrimination and calibration, with slight overestimation of mortality. Each model should be further evaluated in multicenter studies of patients in the intermediate care setting.