Jarrod W. Barnes

Shot-noise Limited Faraday Rotation Spectroscopy for Detection of Nitric Oxide Isotopes in Breath, Urine, and Blood

Measurement of NO and/or its metabolites in the various body compartments has transformed our understanding of biology. The inability of the current NO measurement methods to account for naturally occurring and experimental NO isotopes, however, has prevented the scientific community from fully understating NO metabolism in vivo. Here we present a mid-IR Faraday rotation spectrometer (FRS) for detection of NO isotopes. The instrument utilizes a novel dual modulation/demodulation (DM) FRS method which exhibits noise performance at only 2 times the fundamental quantum shot-noise level and provides the record sensitivity in its class. This is achieved with a system that is fully autonomous, robust, transportable, and does not require cryogenic cooling. The DM-FRS enables continuous monitoring of nitric oxide isotopes with the detection limits of 3.72 ppbv/Hz1/2 to14NO and 0.53 ppbv/Hz1/2 to15NO using only 45 cm active optical path. This DM-FRS measurement method can be used to improve the performance of conventional FRS sensors targeting other radical species. The feasibility of the instrument to perform measurements relevant to studies of NO metabolism in humans is demonstrated.

Breath Analysis in Pulmonary Arterial Hypertension

BACKGROUND Pulmonary arterial hypertension (PAH) is a progressive and devastating condition characterized by vascular cell proliferation and is associated with several metabolic derangements. We hypothesized that metabolic derangements in PAH can be detected by measuring metabolic by-products in exhaled breath. METHODS We collected breath and blood samples from patients with PAH at the time of right-sided heart catheterization (n=31) and from healthy control subjects (n=34). Breath was analyzed by selected ion flow tube-mass spectrometry in predetermined training and validation cohorts. RESULTS Patients with PAH were 51.5±14 years old, and 27 were women (85%). Control subjects were 38±13 years old, and 22 were women (65%). Discriminant analysis in the training set identified three ion peaks (H3O+29+, NO+56+, and O2+98+) and the variable age that correctly classified 88.9% of the individuals. In an independent validation cohort, 82.8% of the individuals were classified correctly. The concentrations of the volatile organic compounds 2-propanol, acetaldehyde, ammonia, ethanol, pentane, 1-decene, 1-octene, and 2-nonene were different in patients with PAH compared with control subjects. Exhaled ammonia was higher in patients with PAH (median [interquartile range]: 94.7 parts per billion (ppb) [70-129 ppb] vs 60.9 ppb [46-77 ppb], P<.001) and was associated with right atrial pressure (ρ=0.57, P<.001), mean pulmonary artery pressure (ρ=0.43, P=.015), cardiac index by thermodilution (ρ=-0.39, P=.03), pulmonary vascular resistance (ρ=0.40, P=.04), mixed venous oxygen (ρ=-0.59, P<.001), and right ventricular dilation (ρ=0.42, P=.03). CONCLUSIONS Breathprint is different between patients with PAH and healthy control subjects. Several specific compounds, including ammonia, were elevated in the breath of patients with PAH. Exhaled ammonia levels correlated with severity of disease.

O-Linked β-N-Acetylglucosamine Transferase Directs Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension

Background— Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Methods and Results— Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway. We measured the levels of O-linked &bgr;-N-acetylglucosamine modification, O-linked &bgr;-N-acetylglucosamine transferase (OGT), and O-linked &bgr;-N-acetylglucosamine hydrolase in control and IPAH cells and tissues. Our data suggest that the activation of the hexosamine biosynthetic pathway directly increased OGT levels and activity, triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-linked &bgr;-N-acetylglucosamine modification levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1. Conclusions— Our data demonstrate that hexosamine biosynthetic pathway flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of host cell factor-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH.

Novel methods in pulmonary hypertension phenotyping in the age of precision medicine (2015 Grover Conference series)

Among pulmonary vascular diseases, pulmonary hypertension (PH) is the best studied and has been the focus of our work. The current classification of PH is based on a relatively simple combination of patient characteristics and hemodynamics. This leads to inherent limitations, including the inability to customize treatment and the lack of clarity from a more granular identification based on individual patient phenotypes. Accurate phenotyping of PH can be used in the clinic to select therapies and determine prognosis and in research to increase the homogeneity of study cohorts. Rapid advances in the mechanistic understanding of the disease, improved imaging methods, and innovative biomarkers now provide an opportunity to define novel PH phenotypes. We have recently shown that altered metabolism may affect nitric oxide levels and protein glycosylation, the peripheral circulation (which may provide insights into the response to therapy), and exhaled-breath analysis (which may be useful in disease evaluation). This review is based on a talk presented during the 2015 Grover Conference and highlights the relevant literature describing novel methods to phenotype pulmonary arterial hypertension patients by using approaches that involve the pulmonary and systemic (peripheral) vasculature. In particular, abnormalities in metabolism, the pulmonary and peripheral circulation, and exhaled breath in PH may help identify phenotypes that can be the basis for a precision-medicine approach to PH management. These approaches may also have a broader scope and may contribute to a better understanding of other diseases, such as asthma, diabetes, and cancer.

Isolation and analysis of sugar nucleotides using solid phase extraction and fluorophore assisted carbohydrate electrophoresis.

Graphical abstract

Abnormal Glucose Metabolism and High-Energy Expenditure in Idiopathic Pulmonary Arterial Hypertension

Rationale: Insulin resistance has emerged as a potential mechanism related to the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH). However, direct measurements of insulin and glucose metabolism have not been performed in patients with IPAH to date. Objectives: To perform comprehensive metabolic phenotyping of humans with IPAH. Methods: We assessed plasma insulin and glucose, using an oral glucose tolerance test and estimated insulin resistance, and &bgr;‐cell function in 14 patients with IPAH and 14 control subjects matched for age, sex, blood pressure, and body mass index. Body composition (dual‐energy X‐ray absorptiometry), inflammation (CXC chemokine ligand 10, endothelin‐1), physical fitness (6‐min walk test), and energy expenditure (indirect calorimetry) were also assessed. Measurements and Main Results: Patients with IPAH had a higher rate of impaired glucose tolerance (57 vs. 14%; P < 0.05) and reduced glucose‐stimulated insulin secretion compared with matched control subjects (IPAH: 1.31 ± 0.76 &mgr;U/ml · mg/dl vs. control subjects: 2.21 ± 1.27 &mgr;U/ml · mg/dl; P < 0.05). Pancreatic &bgr;‐cell function was associated with circulating endothelin‐1 (r = ‐0.71, P < 0.01) and CXC chemokine ligand 10 (r = ‐0.56, P < 0.05). Resting energy expenditure was elevated in IPAH (IPAH: 32 ± 3.4 vs. control subjects: 28.8 ± 2.9 kcal/d/kg fat‐free mass; P < 0.05) and correlated with the plasma glucose response (r = 0.51, P < 0.01). Greater insulin resistance was associated with reduced 6‐minute walk distance (r = 0.55, P < 0.05). Conclusions: Independent of age, sex, blood pressure, and body mass index, patients with IPAH have glucose intolerance, decreased insulin secretion in response to glucose, and elevated resting energy expenditure. These abnormalities are associated with circulating markers of inflammation and vascular dysfunction.

Is Pulmonary Hypertension a Metabolic Disease

Pulmonary hypertension (PH) is a heterogeneous disorder likely to be composed of overlapping syndromes with varying origins and heterogeneous pathobiology and presenting with many phenotypes (1). Knowledge of the underlying pathobiology is necessary for understanding clinical disease manifestations and for devising specific and effective therapies. Enhanced pulmonary vascular cell proliferation, dysregulated cell apoptosis, increased angiogenesis, and vasoconstriction are hallmarks of the disease (Figure 1) (2) and lead to structural and morphological changes within the lung vasculature, including vascular remodeling and arterial wall narrowing. These dysfunctional processes lead to a progressive increase in pulmonary vascular resistance and, ultimately, right ventricular failure and death (3). At the molecular level, genetic factors and derangements in signaling pathways, cytokines, chemokines, and growth factors have been linked to the pathobiology of PH (4). Figure 1. Select identified metabolic derangements in pulmonary hypertension (PH). Dysregulated carbohydrate, lipid, and amino acid metabolism impacts the central phenotypic features of PH. On alteration, these derangements govern the changes in cell proliferation, ... More recently, metabolic dysregulation has emerged as a major area of research in the pathobiology of PH (Figure 1). Just like cancer, PH is characterized by cell proliferation, apoptotic resistance, and increased angiogenesis (5). Also much like patients with cancer, patients with PH exhibit excessive cellular glucose uptake and increased glycolytic metabolism compared with healthy individuals (6, 7). Patients with PH also exhibit alterations in levels of leptin, adiponectin, high-density lipoprotein cholesterol, and insulin resistance (8–11). Cancer cells exposed to these metabolic alterations reprogram their metabolism and protein homeostasis to adapt to nutrient stress conditions, as well as to establish tumor development, progression, and survival. Often, these cells undergo changes in glycosylation (i.e., O-linked N-acetylglucosamine modification of proteins and hyaluronan production) and lipid metabolism (12–14). These observed metabolic changes in cancer are increasingly becoming recognized in the pathogenesis of PH as well (15, 16). One such example is dysregulation in sphingosine 1-phosphate (S1P) metabolism, which is increasingly recognized for its direct involvement in cell proliferation. Two lipid kinases, sphingosine kinase (SphK) 1 and 2, catalyze the conversion of the sphingolipid, sphingosine, to S1P. SphK1 has been linked to several signaling pathways involved in cancer cell proliferation and survival (17). Overexpression of SphK1 has been observed in many tumor tissues, which results in the accumulation of S1P, increased cell proliferation, apoptotic resistance, and disease development and progression (18). Conversely, a reduction in SphK1 activity and subsequent S1P levels is associated with increased cellular ceramide levels, which have been linked to apoptosis and cell cycle arrest (18). Indeed, the homeostatic balance between ceramide and S1P levels (the ceramide/S1P rheostat) is a gauge for cell death or survival. A recent report made a possible link between SphKs and PH (19). In this issue of the Journal, Chen and colleagues (pp. 1032–1043) examined the SphK1/S1P pathway in PH and show that it promotes pulmonary arterial smooth muscle cell (PASMC) proliferation (20). This finding was identified using a combination of rodent models of hypoxia-mediated PH, human explanted lungs, and isolated human PASMCs. By investigating the functional consequences of altering SphKs or S1P in PH, the authors show that overexpression of SphK1 or S1P stimulation promoted PASMC proliferation, whereas loss of SphK1 blocked the PASMC proliferation in PH. In human PH lungs and PASMCs, SphK1 (but not SphK2) was upregulated, which was consistent with their findings of increased S1P levels. In addition, the protective effect of SphK1 deficiency on hypoxia-induced PH was highlighted. These findings of the direct involvement of the SphK signaling pathway in PH vascular proliferation demonstrate the homeostatic balance that is required for sphingosine metabolism in PH. The molecular changes in the SphK1/S1P metabolic pathway, guided by the current knowledge of its role in cancer cell proliferation, may open new avenues to identify its role as a contributor to pulmonary vascular remodeling and a potential therapeutic target in PH. In spite of the novel findings in this report, many questions remain unanswered. Are the metabolic features described here universal in PH? Or do they represent a novel phenotype? Because there were only a few samples from patients with PH analyzed in this study, it will be interesting to determine whether the same observations of increased SphK1 activity and S1P levels holds true in a larger population of patients with PH. What are the main contributors to the increased SphK1 and S1P levels (i.e., hypoxia, altered glucose, lipid, or protein metabolism)? Will targeting the SphK1 activity result in a reduction or reversal of the pulmonary vascular proliferation? Alternatively, could the direct delivery of exogenous ceramide or stimulation of ceramide synthesis slow or reverse the process? Addressing these questions will be necessary to determine the fate of this molecular pathway as a novel therapeutic target in PH. In the meantime, however, the findings by Chen and colleagues invite us to explore deeper the global effects of altered metabolism in the pathogenesis of PH, a journey that will hopefully open doors to new therapeutic targets in this deadly disease (3).

Pulmonary Hypertension and Precision Medicine through the “Omics” Looking Glass

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A Faraday rotation spectrometer for study of NO isotopes in breath

Nitric oxide isotope sensing in breath using a transportable Faraday rotation spectrometer(FRS) is reported. Sensitivity of 0.49 ppbv/Hz^1/2 for ¹⁵NO and 3.59 ppbv/Hz^1/2 for ¹⁴NO were achieved using a quantum cascade laser based dual-modulation FRS.

O-GlcNAc Transferase Directs Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension

Background— Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Methods and Results— Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway. We measured the levels of O-linked &bgr;-N-acetylglucosamine modification, O-linked &bgr;-N-acetylglucosamine transferase (OGT), and O-linked &bgr;-N-acetylglucosamine hydrolase in control and IPAH cells and tissues. Our data suggest that the activation of the hexosamine biosynthetic pathway directly increased OGT levels and activity, triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-linked &bgr;-N-acetylglucosamine modification levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1. Conclusions— Our data demonstrate that hexosamine biosynthetic pathway flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of host cell factor-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH.