Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K+ channel, have been identified in pulmonary arterial hypertension patients. OBJECTIVE: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. METHODS AND RESULTS: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3-mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3-mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3-mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3-mutated rats were characterized by overactivation of ERK1/2 (extracellular signal-regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3-mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3-mutated rats developed age-dependent PH associated with low serum-albumin concentration. CONCLUSIONS: We established the first Kcnk3-mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.

NMDA-Type Glutamate Receptor Activation Promotes Vascular Remodeling and Pulmonary Arterial Hypertension.

BACKGROUND: Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N-methyl-d-aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown. METHODS: We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers. RESULTS: We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. Kv channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries. CONCLUSIONS: These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.

Functional interaction between PDGFß and GluN2B-containing NMDA receptors in smooth muscle cell proliferation and migration in pulmonary arterial hypertension.

In this study, we explored the complex interactions between platelet-derived growth factor (PDGF) and N-methyl-d-aspartate receptor (NMDAR) and their effect on the excessive proliferation and migration of smooth muscle cells leading to obstructed arteries in pulmonary arterial hypertension (PAH). We report lower expression of glutamate receptor NMDA-type subunit 2B (GluN2B), a subunit composing NMDARs expected to affect cell survival/proliferation of pulmonary artery smooth muscle cells (PASMCs), in PAH patient lungs. PASMC exposure to PDGF-BB stimulated immediate increased levels of phosphorylated Src family kinases (SFKs) together with increased phosphorylated GluN2B (its active form) and cell surface relocalization, suggesting a cross talk between PDGFR-recruited SFKs and NMDAR. Selective inhibition of PDGFR-ß or SFKs with imatinib or A-419259, respectively, on one hand, or with specific small-interfering RNAs (siRNAs) on the other hand, aborted PDGF-induced phosphorylation of GluN2B, thus validating the pathway. Selective inhibition of GluN2B using Rö25-6981 and silencing with specific siRNA, in the presence of PDGF-BB, significantly increased both migration and proliferation of PASMCs, thus strengthening the functional importance of the pathway. Together, these results indicate that GluN2B-type NMDAR activation may confer to PASMCs antiproliferative and antimigratory properties. The decreased levels of GluN2B observed in PAH pulmonary arteries could mediate the excessive proliferation of PASMCs, thus contributing to medial hyperplasia and PAH development.

Synergy between nanomaterials and volatile organic compounds for non-invasive medical evaluation.

This article is an overview of the present and ongoing developments in the field of nanomaterial-based sensors for enabling fast, relatively inexpensive and minimally (or non-) invasive diagnostics of health conditions with follow-up by detecting volatile organic compounds (VOCs) excreted from one or combination of human body fluids and tissues (e.g., blood, urine, breath, skin). Part of the review provides a didactic examination of the concepts and approaches related to emerging sensing materials and transduction techniques linked with the VOC-based non-invasive medical evaluations. We also present and discuss diverse characteristics of these innovative sensors, such as their mode of operation, sensitivity, selectivity and response time, as well as the major approaches proposed for enhancing their ability as hybrid sensors to afford multidimensional sensing and information-based sensing. The other parts of the review give an updated compilation of the past and currently available VOC-based sensors for disease diagnostics. This compilation summarizes all VOCs identified in relation to sickness and sampling origin that links these data with advanced nanomaterial-based sensing technologies. Both strength and pitfalls are discussed and criticized, particularly from the perspective of the information and communication era. Further ideas regarding improvement of sensors, sensor arrays, sensing devices and the proposed workflow are also included.

Volatolomics of breath as an emerging frontier in pulmonary arterial hypertension.

There is accumulating evidence in support of the significant improvement in survival rates and clinical outcomes when pulmonary arterial hypertension (PAH) is diagnosed at early stages. Nevertheless, it remains a major clinical challenge and the outcomes are dependent on invasive right heart catheterisation.Resulting from pathophysiological processes and detectable in exhaled breath, volatile organic compounds (VOCs) have been proposed as noninvasive biomarkers for PAH. Studies have confirmed significant alterations of the exhaled VOCs among PAH patients when compared to controls and/or patients with other respiratory diseases. This suggests exhaled breath analysis as a potential noninvasive medical application in the field of PAH.In this article, we review and discuss the progress made so far in the field of exhaled volatolomics (the omics of VOCs) as a potential noninvasive diagnostics of PAH. In addition, we propose a model including possible biochemical pathways on the level of the remodelled artery, in which specific VOCs could be detectable in exhaled breath during the early phases of PAH. We debate the different analytical approaches used and recommend a diagram including a "bottom-top" strategy, from basic to translational studies, required for promoting the field.

Sensor arrays based on nanoparticles for early detection of kidney injury by breath samples.

The outcomes of acute kidney injury (AKI) could be severe and even lethal, if not diagnosed in its early stages and treated appropriately. Blood and urine biomarkers, currently in use as indicators for kidney function, are either inaccurate in various cases or not timely. We report on dramatic changes in exhaled breath composition, associated with kidney dysfunction after ischemic insult in rat models. Gas chromatography linked mass spectrometry examination of breath samples indicated significant elevations in the concentration of three exhaled volatile organic compounds, two to six hours after AKI was surgically induced. Relying on these findings, we introduce an array of sensors, based on organic-layer capped gold nanoparticles, sensitive to odor changes. The ability of the array to detect AKI via breath testing was examined and scored a sensitivity of 96%, only one hour after disease induction. FROM THE CLINICAL EDITOR: In this study, organic-layer capped gold nanoparticle-based biosensors are used to analyse breath samples in an acute kidney injury model, capitalizing on the observation that specific volatile organic compounds are present in breath samples in that condition. The authors report excellent sensitivity in as little as one hour after acute kidney injury. This method, if commercialized, may replace the current blood and urine sample analysis-based tests with a more convenient, rapid and accurate nanotechnology-based method.

Reproducibility and reporting, the routes to progress in breath research-highlights from the Breath Biopsy Conference 2021.

With the continued presence of COVID-19 worldwide, it has been a challenge for the breath research community to progress with clinical studies and travel restrictions have also limited the opportunities to meet up, share ideas and celebrate the latest advances. The Breath Biopsy Conference 2021 offered the chance to catch up with the latest breath research and to share progress that researchers in the community have been able to make in these difficult times. Limited opportunities for clinical research have led many in the field to look more closely at different methods for breath collection and have contributed to the growing calls for consistent standards in how results are reported, shared and even how breath studies themselves are carried out. As such, standardization was a key theme for this year's event and featured prominently in the keynotes, discussions and throughout many of the presentations. With over 900 registrants, almost 400 live attendees and 16 speakers, the Breath Biopsy Conference continues to bring together breath research leaders from around the world. This article provides an overview of the highlights from this event.

Iron Deficiency in Pulmonary Arterial Hypertension: A Deep Dive into the Mechanisms.

Pulmonary arterial hypertension (PAH) is a severe cardiovascular disease that is caused by the progressive occlusion of the distal pulmonary arteries, eventually leading to right heart failure and death. Almost 40% of patients with PAH are iron deficient. Although widely studied, the mechanisms linking between PAH and iron deficiency remain unclear. Here we review the mechanisms regulating iron homeostasis and the preclinical and clinical data available on iron deficiency in PAH. Then we discuss the potential implications of iron deficiency on the development and management of PAH.

Trichloroethylene increases pulmonary endothelial permeability: implication for pulmonary veno-occlusive disease.

Trichloroethylene exposure is a major risk factor for pulmonary veno-occlusive disease. We demonstrated that trichloroethylene alters the endothelial barrier integrity, at least in part, through vascular endothelial (VE)-Cadherin internalisation, and suggested that this mechanism may play a role in the development of pulmonary veno-occlusive disease.

Breath analysis of cancer in the present and the future.

Most of the currently used diagnostics for cancerous diseases have yet to meet the standards of screening, as they are insufficiently accurate and/or invasive and risky. In this review, we describe the rationale, the progress made to date, and the potential of analysing the exhaled volatile organic compounds as a pathway for enabling early diagnosis of cancer and, therefore, for achieving better clinical prognosis and survival rates. The review highlights the major advancements made in this field, from fundamentals, up to translational phases and clinical trials, with a special emphasis on sensing platforms based on nanomaterials. The prospects for breath analysis in early cancerous disease are presented and discussed.

Targeted breath analysis: exogenous volatile organic compounds (EVOC) as metabolic pathway-specific probes.

Breath research has almost invariably focussed on the identification of endogenous volatile organic compounds (VOCs) as disease biomarkers. After five decades, a very limited number of breath tests measuring endogenous VOCs is applied to the clinic. In this perspective article, we explore some of the factors that may have contributed to the current lack of clinical applications of breath endogenous VOCs. We discuss potential pitfalls of experimental design, analytical challenges, as well as considerations regarding the biochemical pathways that may impinge on the application of endogenous VOCs as specific disease biomarkers. We point towards several lines of evidence showing that breath analysis based on administration of exogenous compounds has been a more successful strategy, with several tests currently applied to the clinic, compared to measurement of endogenous VOCs. Finally, we propose a novel approach, based on the use of exogenous VOC (EVOC) probes as potential strategy to measure the activity of metabolic enzymes in vivo, as well as the function of organs, through breath analysis. We present longitudinal data showing the potential of EVOC probe strategies in breath analysis. We also gathered important data showing that administration of EVOC probes induces significant changes compared to previous exposures to the same compounds. EVOC strategies could herald a new wave of substrate-based breath tests, potentially bridging the gap between research tools and clinical applications.

Sensor Array for Detection of Early Stage Parkinson's Disease before Medication.

Early diagnosis of Parkinson's disease (PD) is important because it affects the choice of therapy and is subject to a relatively high degree of error. In addition, early detection of PD can potentially enable the start of neuroprotective therapy before extensive loss of dopaminergic neurons of the substantia nigra occurs. However, until now, studies for early detection of PD using volatile biomarkers sampled only treated and medicated patients. Therefore, there is a great need to evaluate untreated patients for establishing a real world screening and diagnostic technology. Here we describe for the first time a clinical trial to distinguish between de novo PD and control subjects using an electronic system for detection of volatile molecules in exhaled breath (sensor array). We further determine for the first time the association to other common tests for PD diagnostics as smell, ultrasound, and nonmotor symptoms. The test group consisted of 29 PD patients after initial diagnosis by an experienced neurologist, compared with 19 control subjects of similar age. The sensitivity, specificity, and accuracy values of the sensor array to detect PD from controls were 79%, 84%, and 81% respectively, in comparison with midbrain ultrasonography (93%, 90%, 92%) and smell detection (62%, 89%, 73%). The results confirm previous data showing the potential of sensor arrays to detect PD.

Altered Volatile Organic Compound Profile in Transgenic Rats Bearing A53T Mutation of Human α-Synuclein: Comparison with Dopaminergic and Serotonergic Denervation.

Early diagnosis of Parkinson's disease (PD) is of great importance due its progressive phenotype. Neuroprotective drugs could potentially slow down disease progression if used at early stages. Previously, we have reported an altered content of volatile organic compounds (VOCs) in the breath of rats following a 50% reduction in striatal dopamine (DA) content induced by 6-hydroxydopamine. We now report on the difference in the breath-print and content of VOCs between rats with mild and severe lesions of DA neurons, serotonergic neuronal lesions, and transgenic (Tg) rats carrying the PD-producing A53T mutation of the SNCA (α-synuclein) gene. The Tg rats had an increased content of 3-octen-1-ol and 4-chloro-3-methyl phenol in blood, while in brain tissue, hexanal, hexanol, and 2,3-octanedione were present in controls but absent in Tg rats. Levels of 1-heptyl-2-methyl cyclopropane were increased in brain tissue of Tg rats. The data confirm the potential of breath analysis for detection of human idiosyncratic as well as autosomal dominant PD.

Diagnosis and Classification of 17 Diseases from 1404 Subjects via Pattern Analysis of Exhaled Molecules.

We report on an artificially intelligent nanoarray based on molecularly modified gold nanoparticles and a random network of single-walled carbon nanotubes for noninvasive diagnosis and classification of a number of diseases from exhaled breath. The performance of this artificially intelligent nanoarray was clinically assessed on breath samples collected from 1404 subjects having one of 17 different disease conditions included in the study or having no evidence of any disease (healthy controls). Blind experiments showed that 86% accuracy could be achieved with the artificially intelligent nanoarray, allowing both detection and discrimination between the different disease conditions examined. Analysis of the artificially intelligent nanoarray also showed that each disease has its own unique breathprint, and that the presence of one disease would not screen out others. Cluster analysis showed a reasonable classification power of diseases from the same categories. The effect of confounding clinical and environmental factors on the performance of the nanoarray did not significantly alter the obtained results. The diagnosis and classification power of the nanoarray was also validated by an independent analytical technique, i.e., gas chromatography linked with mass spectrometry. This analysis found that 13 exhaled chemical species, called volatile organic compounds, are associated with certain diseases, and the composition of this assembly of volatile organic compounds differs from one disease to another. Overall, these findings could contribute to one of the most important criteria for successful health intervention in the modern era, viz. easy-to-use, inexpensive (affordable), and miniaturized tools that could also be used for personalized screening, diagnosis, and follow-up of a number of diseases, which can clearly be extended by further development.

Programmed Nanoparticles for Tailoring the Detection of Inflammatory Bowel Diseases and Irritable Bowel Syndrome Disease via Breathprint.

Chemical sensors based on programmable molecularly modified gold nanoparticles are tailored for the detection and discrimination between the breathprint of irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD). The sensors are examined in both lab- and real-world clinical conditions. The results reveal a discriminative power accuracy of 81% between IBD and IBS and 75% between Crohn's and Colitis states.

Monolayer-capped gold nanoparticles for disease detection from breath.

The recognition of volatile organic compounds in breath samples is a promising approach for noninvasive safe diagnosis of disease. Spectrometry and spectroscopy methods used for breath analysis suffer from suboptimal accuracy, are expensive and are unsuitable for diagnostics. This article presents a concise review on arrays of monolayer-capped gold nanoparticle (GNP) sensors in conjugation with pattern recognition methods for cost-effective, fast and high-throughput point-of-care diagnostic results from exhaled breath samples. The article starts with a general introduction to the rationale and advantages of breath analysis as well as with a presentation of the utility of monolayer-capped GNP sensors in this field. The article continues with a presentation of the main fabrication and operation principles of these GNP sensors and concludes with selected examples regarding their utility in different fields of medicine, particularly in neurology, infectiology, respiratory medicine and oncology.