Bioinspired carbon monoxide delivery using artificial blood attenuates the progression of obliterative bronchiolitis via suppression of macrophage activation by IL-17A.

Carbon monoxide (CO) is expected to attenuate the progression of obliterative bronchiolitis (OB), which is a serious complication after lung transplantation. However, issues in terms of feasible exogenous CO supply, such as continuousness and safety, remain unsolved. Here, we applied nano red blood cells, namely hemoglobin vesicles (Hb-V), as a CO cargo based on the biomimetic concept and investigated the therapeutic potential of CO-loaded Hb-V on OB in orthotopic tracheal transplant model mice. The CO-loaded Hb-V was comprised of negatively charged liposomes encapsulating carbonylhemoglobin with a size of ca. 220 nm. The results of histological evaluation showed that allograft luminal occlusion and fibrosis were significantly ameliorated by treatment with CO-loaded Hb-V compared to treatment with saline, cyclosporine, and Hb-V. The therapeutic effects of CO-loaded Hb-V on OB were due to the suppression of M1 macrophage activation in tracheal allografts, resulting from decreased IL-17A production. Furthermore, the expression of TNF-α and TGF-ß in tracheal allografts was decreased by CO-loaded Hb-V treatment but not saline and Hb-V treatment, indicating that CO liberated from CO-loaded Hb-V inhibits epithelial-mesenchymal transition. These findings suggest that CO-loaded Hb-V exerts strong therapeutic efficacy against OB via the regulation of macrophage activation by IL-17A and TGF-ß-driven epithelial-mesenchymal transition.

Carbon Monoxide as a Therapeutic for Airway Diseases: Contrast and Comparison of Various CO Delivery Modalities.

The quest to find novel strategies to tackle respiratory illnesses has led to the exploration of the potential therapeutic effects of carbon monoxide (CO) as an endogenous signaling molecule and a cytoprotective agent. Further, several studies have demonstrated the pharmacological efficacy of CO in animal models of respiratory disorders, such as acute lung injury and pulmonary hypertension. Because of the gaseous nature of CO and its affinity for multiple targets, its controlled delivery has been a challenge. Past studies have employed different delivery modalities, including CO gas, HO-1 inducers, and CO donors, sometimes leading to substantive variations in the resulting pharmacological effects for various reasons. Herein, this review summarizes and analyzes the differences among the profiles of various CO-delivery modalities in terms of their efficacy, dosing regimen, and pharmacokinetics in airways models. We believe that analysis of these issues will help in understanding the fundamental roles of CO in airways, and eventually, contribute to its development as a medicine for respiratory diseases.

Metal-organic frameworks for therapeutic gas delivery.

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are gaseous signaling molecules (gasotransmitters) that regulate both physiological and pathological processes and offer therapeutic potential for the treatment of many diseases, such as cancer, cardiovascular disease, renal disease, bacterial and viral infections. However, the inherent labile nature of therapeutic gases results in difficulties in direct gases administration and their controlled delivery at clinically relevant ranges. Metal-organic frameworks (MOFs) with highly porous, stable, and easy-to-tailor properties have shown promising therapeutic gas delivery potential. Herein, we highlight the recent advances of MOF-based platforms for therapeutic gas delivery, either by endogenous (i.e., direct transfer of gases to targets) or exogenous (i.e., stimulating triggered release of gases) means. Reports that involve in vitro and/or in vivo studies are highlighted due to their high potential for clinical translation. Current challenges for clinical requirements and possible future innovative designs to meet variable healthcare needs are discussed.

Rectal administration of carbon monoxide inhibits the development of intestinal inflammation and promotes intestinal wound healing via the activation of the Rho-kinase pathway in rats.

The inhalation of carbon monoxide (CO) gas and the administration of CO-releasing molecules were shown to inhibit the development of intestinal inflammation in a murine colitis model. However, it remains unclear whether CO promotes intestinal wound healing. Herein, we aimed to evaluate the therapeutic effects of the topical application of CO-saturated saline enemas on intestinal inflammation and elucidate the underlying mechanism. Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. A CO-saturated solution was prepared via bubbling 50% CO gas into saline and was rectally administrated twice a day after colitis induction; rats were sacrificed 3 or 7 days after induction for the study of the acute or healing phases, respectively. The distal colon was isolated, and ulcerated lesions were measured. In vitro wound healing assays were also employed to determine the mechanism underlying rat intestinal epithelial cell restitution after CO treatment. CO solution rectal administration ameliorated acute TNBS-induced colonic ulceration and accelerated ulcer healing without elevating serum CO levels. The increase in thiobarbituric acid-reactive substances and myeloperoxidase activity after induction of acute TNBS colitis was also significantly inhibited after CO treatment. Moreover, the wound healing assays revealed that the CO-saturated medium enhanced rat intestinal epithelial cell migration via the activation of Rho-kinase. In addition, the activation of Rho-kinase in response to CO treatment was confirmed in the inflamed colonic tissue. Therefore, the rectal administration of a CO-saturated solution protects the intestinal mucosa from inflammation and accelerates colonic ulcer healing through enhanced epithelial cell restitution. CO may thus represent a novel therapeutic agent for the treatment of inflammatory bowel disease.

Lipid-encapsulated upconversion nanoparticle for near-infrared light-mediated carbon monoxide release for cancer gas therapy.

Cancer gas therapy is just in an early stage of research and development. Several important gasotransmitters have proven their therapeutic potentials, but handling, delivery and controlled release of these gases remain very challenging for therapeutic purposes. This research develops a versatile nanosystem that is capable of delivering carbon monoxide (CO) gasotransmitter in the form of photo-responsive carbon monoxide-releasing molecule (CORM) for targeted cancer therapy. The core-shell upconversion nanoparticles (UCNPs) were designed to transfer bio-friendly low energy near infrared (NIR) light to ultraviolet (UV) light and trigger CO release from the loaded CORM. The synthesized delivery system demonstrated its ability to mediate the sustained release of CO upon 808 or 980 nm NIR light excitation. The optimized nanoformulation was efficiently taken up by HCT116 cancer cells and showed dose-dependent cytotoxicity to HCT116 and other cancer cells. Intracellular CO release and subsequent therapeutic action involving ROS production were found to significantly contribute to cell apoptosis. Therefore, the current research demonstrates the potency and efficiency of an NIR-mediated UCNP-based CORM prodrug delivery system for targeted cancer gas therapy.

Carbon monoxide attenuates LPS-induced myocardial dysfunction in rats by regulating the mitochondrial dynamic equilibrium.

Lipopolysaccharide (LPS) induces myocardial dysfunction by damaging the mitochondrial structure in cardiomyocytes. Since low levels of carbon monoxide can confer cytoprotective effects against end-organ damage from endotoxic shock, we tested whether treatment with carbon monoxide-releasing molecule-2 (CORM-2) could ameliorate LPS-induced myocardial dysfunction in rats by maintaining the dynamic equilibrium between the mitochondrial fusion and fission processes. Cardiac function, myocardial histopathology, myocardial enzymes, and changes in myocardial mitochondrial function and mitochondrial fusion-fission protein expression were assessed in rats. The mitochondrial structure and morphology were studied by electron microscopy, and the expression levels of key proteins involved in the mitochondrial dynamics were assessed by Western blot assay. Cardiac dysfunction and increased myocardial enzyme activity together with myocardial pathological damage, mitochondrial dysfunction, and impaired mitochondrial dynamics homeostasis were observed in the LPS-challenged septic rats. However, these observations were reversed by CORM-2, which effectively inhibited cardiac and mitochondrial damage in the LPS-challenged rats and improved the survival rate of the animals. In conclusion, CORM-2 regulates the LPS-induced imbalance of the dynamic mitochondrial fusion and fission processes, thereby effectively ameliorating the LPS-induced myocardial dysfunction and improving the survival of the rats.

The impact of carbon monoxide inhalation on developing noise-induced hearing loss in guinea pigs.

Carbon monoxide (CO) poisoning is one of the most common types of fatal poisonings worldwide. Acute exposure to high levels of CO as well as chronic exposure to low levels of CO and excessive noise can lead to high frequency hearing loss. In this study, twelve guinea pigs were randomly divided into two groups: (1) exposed to noise and (2) exposed to noise plus CO. Auditory brainstem responses (ABRs) were measured prior to the experiment and immediately, 5, 10 and 15 days post exposures. There was a significant difference between the ABR thresholds before and immediately after exposure to noise at frequencies of 4, 8, and 16 kHz and the most threshold shift was observed at 8 kHz. There was also a significant difference between the ABR thresholds before and immediately after exposure to noise and CO at frequencies of 2, 4, 8, and 16 kHz which demonstrated a temporary hearing loss after exposure to noise and CO and the major impact of CO on developing noise induced hearing loss occurred at 8 kHz. No significant difference was observed between the ABR thresholds recorded before conducting the experiments and the ones obtained 5, 10 and 15 days after simultaneous exposure to noise and CO at none of frequencies. Simultaneous exposure to noise and CO contributes to transient hearing loss in guinea pigs with the most evident temporary shift at 8 kHz. The methods were accepted in the Ethics Committee of Iran University of Medical Science (registration No. CTRI/2016/01/017170) on January 18, 2016.

Breathing Micelles for Combinatorial Treatment of Rheumatoid Arthritis.

Breathing process involves inhalation and exhalation of different gases in animals. The gas exchange of the breathing process plays a critical role in maintaining the physiological functions of living organisms. Although artificial breathing materials exhibiting volume expansion and contraction upon alternate exposure to different gases have been well explored, those being able to realize the gas exchange remain elusive. Herein, we report breathing micelles (BM) capable of inhaling nitric oxide (NO) and exhaling carbon monoxide (CO), both of which are endogenous gaseous signaling molecules. We demonstrate that BM can simultaneously scavenge overproduced NO and attenuate proinflammatory cytokines in lipopolysaccharide (LPS)-challenged macrophage cells. In vivo studies revealed that BM outperformed conventional nonsteroidal anti-inflammatory drugs such as dexamethasone (Dexa) in treatment of rheumatoid arthritis (RA) in adjuvant-induced arthritis (AIA) rats, likely due to the combinatorial effect of NO depletion, CO-mediated deactivation of inducible NO synthase (iNOS) and activation of heme oxygenase-1 (HO-1). This work provides new insights into artificial BM for potential biomedical applications.

Chronic Exposure to Low-Dose Carbon Monoxide Alters Hemoglobin Mass and V˙O2max.

By blocking the oxygen binding sites on the hemoglobin molecule, chronic low-dose carbon monoxide (CO) administration may produce similar effects to those of exposure to altitude. PURPOSE: This study aimed to determine the effect of chronic low-dose CO application on hemoglobin mass (Hbmass) and V˙O2max. METHODS: For 3 wk, 11 healthy and moderately trained male subjects inhaled a CO bolus five times per day to increase their HbCO concentration by ~5%. Another 11 subjects received a placebo. Hbmass, serum erythropoietin concentration, ferritin, and basic hematological parameters were determined before and weekly during and until 3 wk after the CO inhalation period. V˙O2max tests on a cycle ergometer were performed before and after the CO administration period. RESULTS: In the CO group, Hbmass increased from 919 ± 69 to 962 ± 78 g in week 3 (P < 0.001) and was maintained for the following 3 wk. Reticulocytes (%) and immature reticulocyte fraction significantly increased after 1 wk. Serum erythropoietin concentration tended to increase after 1 wk (P = 0.07) and was suppressed in the postperiod (P < 0.01). Ferritin decreased during the inhalation period (from 106 ± 37 to 72 ± 37 ng·mL, P < 0.001). V˙O2max tended to increase from 4230 ± 280 to 4350 ± 350 mL·min (P < 0.1) immediately after the inhalation period and showed a significant relationship to the change in Hbmass (y = 4.1x - 73.4, r = 0.70, P < 0.001). CONCLUSIONS: Chronic continuous exposure to low-dose CO enhances erythropoietic processes resulting in a 4.8% increase in Hbmass. The individual changes in Hbmass were correlated to the corresponding changes in V˙O2max. Examination of ethical and safety concerns is warranted before the implementation of low-dose CO inhalation in the clinical/athletic setting as a tool for modifying Hbmass.

Application of the optimized carbon monoxide rebreathing method for the measurement of total haemoglobin mass in chronic liver disease.

BACKGROUND: Anemia is common in liver cirrhosis. This generally infers a fall in total hemoglobin mass (tHb-mass). However, hemoglobin concentration ([Hb]) may fall due to an expansion in plasma volume (PV). The "optimized carbon monoxide rebreathing method" (oCOR) measures tHb-mass directly and PV (indirectly using hematocrit). It relies upon carboxyhemoglobin (COHb) distribution throughout the entire circulation. In healthy subjects, such distribution is complete within 6-8 min. Given the altered circulatory dynamics in cirrhosis, we sought in this pilot study, to assess whether this was true in cirrhosis. The primary aim was to ascertain if the standard timings for the oCOR were applicable to patients with chronic liver disease and cirrhosis. The secondary aim was to explore the applicability of standard CO dosing methodologies to this patient population. METHODS: Sixteen patients with chronic liver parenchymal disease were studied. However, tHb-mass was determined using the standard oCOR technique before elective paracentesis. Three subjects had an inadequate COHb% rise. In the remaining 13 (11 male), mean ± standard deviation (SD) age was 52 ± 13.8 years, body mass 79.1 ± 11.4 kg, height 175 ± 6.8 cm. To these, mean ± SD dose of carbon monoxide (CO) gas administered was 0.73 ± 0.13 ml/kg COHb values at baseline, 6 and 8 min (and "7-min value") were compared to those at 10, 12, 15 and 20 min after CO rebreathing. RESULTS: The "7-min value" for median COHb% (IQR) of 6.30% (6.21%-7.47%) did not differ significantly from those at subsequent time points (8 min: 6.30% (6.21%-7.47%), 10 min: 6.33% (6.00%-7.50%), 12 min: 6.33% (5.90%-7.40%), 15 min: 6.37% (5.80%-7.33%), 20 min: 6.27% (5.70%-7.20%)). Mean difference in calculated tHb-mass between minute 7 and minute 20 was only 4.1 g, or 0.6%, p = .68. No subjects reported any adverse effects. CONCLUSIONS: The oCOR method can be safely used to measure tHb-mass in patients with chronic liver disease and ascites, without adjustment of blood sample timings. Further work might refine and validate appropriate dosing regimens.

Carbon Monoxide Rescues the Developmental Lethality of Experimental Rat Models of Rhabdomyolysis-Induced Acute Kidney Injury.

Many victims, after being extricated from a collapsed building as the result of a disaster, suffer from disaster nephrology, a term that is referred to as the crush syndrome (CS). Recommended treatments, which include dialysis or the continuous administration of massive amounts of fluid are not usually easy in cases of such mass natural disasters. In the present study, we examined the therapeutic performance of a biomimetic carbon monoxide (CO) delivery system, CO-enriched red blood cells (CO-RBCs), on experimental animal models of an acute kidney injury (AKI) induced by traumatic and nontraumatic rhabdomyolysis, including CS and rhabdomyolysis with massive hemorrhage shock. A single CO-RBC treatment was found to effectively suppress the pathogenesis of AKI with the mortality in these model rats being improved. In addition, in further studies using glycerol-induced rhabdomyolysis model rats, the pathogenesis of which is similar to that for the CS, AKI and mortality were also reduced as the result of a CO-RBC treatment. Furthermore, CO-RBCs were found to have renoprotective effects via the suppression of subsequent heme protein-associated renal oxidative injury; the oxidation of myoglobin in the kidneys, the generation of reactive oxygen species by free heme produced from degraded-cytochrome P450 and hemoglobin-associated renal injury. Because CO-RBCs can be prepared and used at both hospitals and at a disaster site, these findings suggest that CO-RBCs have the potential for use as a novel cell therapy against both nontraumatic and traumatic rhabdomyolysis including CS-induced AKI. SIGNIFICANCE STATEMENT: After mass natural and man-made disasters, people who are trapped in collapsed buildings are in danger of acute kidney injury (AKI), including crush syndrome (CS)-related AKI. This paper reports that carbon monoxide-enriched red blood cells (CO-RBCs), which can be prepared at both hospitals and disaster sites, dramatically suppressed the pathogenesis of CS-related AKI, thus improving mortality via suppressing heme protein-associated renal injuries. CO-RBCs have the potential for serving as a practical therapeutic agent against disaster nephrology associated with the CS.

Intermittent low dose carbon monoxide inhalation does not influence glucose regulation in overweight adults: a randomized controlled crossover trial.

NEW FINDINGS: What is the central question of this study? Low dose carbon monoxide (CO) inhalation plays a role in regulating proteins involved in glucose metabolism; does low dose CO improve glucose and insulin responses to an oral glucose tolerance test in overweight adults? What is the main finding and its importance? Five days of intermittent CO inhalation does not alter the glucose or insulin responses to ingestion of a glucose bolus in overweight adults. Low dose CO is utilized in various physiological assessment procedures; these findings allow researchers and clinicians to utilize these procedures without concern of altering glucose metabolism. ABSTRACT: Low dose carbon monoxide (CO) inhalation upregulates several proteins important for glucose metabolism. Such changes could be clinically significant and may be relevant to those who use CO as a research tool. We hypothesized that low dose CO inhalation would improve glucose and insulin responses to an oral glucose bolus in overweight humans. Eleven young adults (5 men, 6 women; body mass index: 25-35 kg m-2 ) were included in this randomized, placebo-controlled, single-blinded crossover study. Following screening, participants completed two 7-day protocols with a 4-week washout. Twenty-four hours prior to and following five consecutive days of either once daily CO (men: 1.2 ml (kg body mass)-1 ; women: 1.0 ml (kg body mass)-1 ) or placebo (room air) inhalation, participants underwent oral glucose tolerance tests (OGTT). For key outcome variables, there were no significant main effects or interactions across condition or time point (mean ± SD), including fasting glucose (mg dl-1 : pre-placebo: 85.2 ± 10.1; post-placebo: 82.9 ± 10.6; pre-CO: 83.6 ± 7.7; post-CO: 84.0 ± 9.0), 2 h post glucose (mg dl-1 : pre-placebo: 100.9 ± 20.0; post-placebo: 98.7 ± 13.1; pre-CO: 94.2 ± 23.2; post-CO: 94.4 ± 14.9), or the Matsuda index (pre-placebo: 16.1 ± 11.5; post-placebo: 20.3 ± 24.7; pre-CO: 15.6 ± 15.3; post-CO: 17.5 ± 16.8). In conclusion, 5 days of low dose CO administration did not influence glucose and insulin responses to an OGTT in overweight adults. Low dose CO inhalation is utilized in a variety of physiological assessment procedures; these findings allow researchers to utilize these procedures without concern of altering glucose metabolism.

Systemic vasoprotection by inhaled carbon monoxide is mediated through prolonged alterations in monocyte/macrophage function.

Carbon monoxide (CO) is anti-inflammatory and protective in models of disease. Its actions in vitro are short-lived but are sustained in vivo. We hypothesize that systemic CO can mediate prolonged phenotype changes in vivo, with a focus on macrophages (Mφs). Mφs isolated from CO treated rats responded to lipopolysaccharide (LPS) with increased IL6, IL10 and iNOS expression but decreased TNF. Conditioned media (CM) collected from peritoneal Mφs isolated from CO treated rats stimulated endothelial cell (EC) proliferation versus CM from Mφs from air treated rats. This effect was mediated by Mφ released VEGF and HMGB1. Inhaled CO reduced LPS induced Mφ M1 inflammatory phenotype for up to 5 days. Mitochondrial oxygen consumption in LPS treated Mφs from CO treated mice was preserved compared to LPS treated Mφs from control mice. Finally, transient reduction of inflammatory cells at the time of inhaled CO treatment eliminated the vasoprotective effect of CO in a rodent carotid injury model. Thus, inhaled CO induces a prolonged mixed phenotype change in Mφs, and potentially other inflammatory cells, that contribute to vasoprotection. These findings demonstrate the ability of inhaled CO to modify Mφs in a sustained manner to mediate its therapeutic actions, supporting the translational potential of inhaled CO.

Carbon monoxide: An emerging therapy for acute kidney injury.

Treating acute kidney injury (AKI) represents an important unmet medical need both in terms of the seriousness of this medical problem and the number of patients. There is also a large untapped market opportunity in treating AKI. Over the years, there has been much effort in search of therapeutics with minimal success. However, over the same time period, new understanding of the underlying pathobiology and molecular mechanisms of kidney injury have undoubtedly helped the search for new therapeutics. Along this line, carbon monoxide (CO) has emerged as a promising therapeutic agent because of its demonstrated cytoprotective, and immunomodulatory effects. CO has also been shown to sensitize cancer, but not normal cells, to chemotherapy. This is particularly important in treating cisplatin-induced AKI, a common clinical problem that develops in patients receiving cisplatin therapies for a number of different solid organ malignancies. This review will examine and make the case that CO be developed into a therapeutic agent against AKI.

Emerging Delivery Strategies of Carbon Monoxide for Therapeutic Applications: from CO Gas to CO Releasing Nanomaterials.

Carbon monoxide (CO) therapy has emerged as a hot topic under exploration in the field of gas therapy as it shows the promise of treating various diseases. Due to the gaseous property and the high affinity for human hemoglobin, the main challenges of administrating medicinal CO are the lack of target selectivity as well as the toxic profile at relatively high concentrations. Although abundant CO releasing molecules (CORMs) with the capacity to deliver CO in biological systems have been developed, several disadvantages related to CORMs, including random diffusion, poor solubility, potential toxicity, and lack of on-demand CO release in deep tissue, still confine their practical use. Recently, the advent of versatile nanomedicine has provided a promising chance for improving the properties of naked CORMs and simultaneously realizing the therapeutic applications of CO. This review presents a brief summarization of the emerging delivery strategies of CO based on nanomaterials for therapeutic application. First, an introduction covering the therapeutic roles of CO and several frequently used CORMs is provided. Then, recent advancements in the synthesis and application of versatile CO releasing nanomaterials are elaborated. Finally, the current challenges and future directions of these important delivery strategies are proposed.

Low Dose Carbon Monoxide Exposure in Idiopathic Pulmonary Fibrosis Produces a CO Signature Comprised of Oxidative Phosphorylation Genes.

Compelling preclinical studies indicate that low-dose carbon monoxide (CO) abrogates experimental lung fibrosis. We recently reported the results of a multicenter, double-blinded, clinical trial of inhaled CO in patients with idiopathic pulmonary fibrosis (IPF). Identifying no significantly changes in metalloproteinase-7 (MMP7) serum concentration, or secondary endpoints of physiologic measurements, hospitalization, death, or patient-reported outcomes. In the present study, we evaluated the effect of low dose CO exposure (100-200 ppm) for 12 weeks on genome-wide gene expression in peripheral blood mononuclear cells (PBMC) derived from these IPF study subjects. We conducted transcriptome profiling on 38 IPF subjects with time points available at 0, 12, and 24 weeks. Total RNA isolated from PBMCs was hybridized onto the Affymetrix Human Gene 2.0 ST Array. We identified 621 genes significantly upregulated in the 24-week CO exposed group compared with the 12-week. Pathway analysis demonstrated association with Oxidative Phosphorylation (adjusted P < 0.05). We identified a clear CO signature dominated with 23 oxidative phosphorylation-related genes (FDR <10%). We confirmed the expression of nine selected gene products using Nanostring's nCounter analysis system. These findings suggest this signature may serve as a potential genomic biomarker for CO exposure and for potential titration of dosage to allow precision testing of therapies in future low dose CO therapeutic studies in IPF.

Modification of the CO-rebreathing method to determine haemoglobin mass and blood volume in patients suffering from chronic mountain sickness.

NEW FINDINGS: What is the central question of this study? Is it necessary to modify the CO-rebreathing method to acquire reliable measurements of haemoglobin mass in patients with chronic mountain sickness? What is the main finding and its importance? The CO-rebreathing method must be modified because of the prolonged CO-mixing time in patients with chronic mountain sickness. After adaptation of the blood sampling method, reliable and valid results were attained. With this modification, it is possible to quantify the extent of polycythaemia and to distinguish between a haemoconcentration and an exclusive enhancement of erythrocyte volume. ABSTRACT: Patients suffering from chronic mountain sickness (CMS) exhibit extremely high haemoglobin concentrations. Their haemoglobin mass (Hbmass), however, has rarely been investigated. The CO-rebreathing protocol for Hbmass determination in those patients might need to be modified because of restricted peripheral perfusion. The aim of this study was to evaluate the CO uptake and carboxyhaemoglobin-mixing time in the blood of CMS patients and to adapt the CO-rebreathing method for this group. Twenty-five male CMS patients living at elevations between 3600 and 4100 m above sea level were compared with ethnically matched healthy control subjects from identical elevations (n = 11) and near sea level (n = 9) and with a Caucasian group from sea level (n = 6). CO rebreathing was performed for 2 min, and blood samples were taken for the subsequent 30 min. After the method was modified, its reliability was evaluated in test-retest experiments (n = 28), and validity was investigated by measuring the Hbmass before and after the phlebotomy of 500 ml (n = 4). CO uptake was not affected by CMS. The carboxyhaemoglobin mixing was completed after 8 min in the Caucasian group but after 14 min in the groups living at altitude. When blood was sampled 14-20 min after inhalation, the typical error of the method was 1.6% (confidence limits 1.2-2.5%). After phlebotomy, Hbmass decreased from 1779 ± 123 to 1650 ± 129 g, and no difference was found between the measured and calculated Hbmass (1666 ± 122 g). When the time of blood sampling was adapted to accommodate a prolonged carboxyhaemoglobin-mixing time, the CO-rebreathing method became a reliable and valid tool to determine Hbmass in CMS patients.

Extracorporeal resuscitation with carbon monoxide improves renal function by targeting inflammatory pathways in cardiac arrest in pigs.

Deleterious consequences like acute kidney injury frequently occur upon successful resuscitation from cardiac arrest. Extracorporeal life support is increasingly used to overcome high cardiac arrest mortality. Carbon monoxide (CO) is an endogenous gasotransmitter, capable of reducing renal injury. In our study, we hypothesized that addition of CO to extracorporeal resuscitation hampers severity of renal injury in a porcine model of cardiac arrest. Hypoxic cardiac arrest was induced in pigs. Animals were resuscitated using a conventional [cardiopulmonary resuscitation (CPR)], an extracorporeal (E-CPR), or a CO-assisted extracorporeal (CO-E-CPR) protocol. CO was applied using a membrane-controlled releasing system. Markers of renal injury were measured, and histopathological analyses were carried out. We investigated renal pathways involving inflammation as well as apoptotic cell death. No differences in serum neutrophil gelatinase-associated lipocalin (NGAL) were detected after CO treatment compared with Sham animals (Sham 71 ± 7 and CO-E-CPR 95 ± 6 ng/mL), while NGAL was increased in CPR and E-CPR groups (CPR 135 ± 11 and E-CPR 124 ± 5 ng/mL; P < 0.05). Evidence for histopathological damage was abrogated after CO application. CO increased renal heat shock protein 70 expression and reduced inducible cyclooxygenase 2 (CPR: 60 ± 8; E-CPR 56 ± 8; CO-E-CPR 31 ± 3 µg/mL; P < 0.05). Caspase 3 activity was decreased (CPR 1,469 ± 276; E-CPR 1,670 ± 225; CO-E-CPR 755 ± 83 pg/mL; P < 0.05). Furthermore, we found a reduction in renal inflammatory signaling upon CO treatment. Our data demonstrate improved renal function by extracorporeal CO treatment in a porcine model of cardiac arrest. CO reduced proinflammatory and proapoptotic signaling, characterizing beneficial aspects of a novel treatment option to overcome high mortality.

Carbon Monoxide Attenuates Lipopolysaccharides (LPS)-Induced Acute Lung Injury in Neonatal Rats via Downregulation of Cx43 to Reduce Necroptosis.

BACKGROUND Acute lung injury (ALI) is one of major causes of death in newborns, making it urgent to improve therapy. Administration of low dose carbon monoxide (CO) plays a protective role in ALI but the mechanisms are not fully understood. This study was designed to test the therapeutic effect of monoxide-releasing molecule 3 (MORM3) in lipopolysaccharide (LPS) induced neonatal ALI and the possibly associated molecular mechanisms. MATERIAL AND METHODS For this study, 3- to 8-day old Newborn Sprague-Dawley rats were subjected to intraperitoneal injection of 3 mg/kg LPS to induce ALI. Then animals received intraperitoneal injection of carbon monoxide-releasing molecules 3 (CORM3) (8 mg/kg) or inactive CORM3 (iCORM3) for 7 consecutive days. Lung tissues were collected for histological examination and total cell counts and protein content in bronchoalveolar lavage fluid (BALF) were measured. Expression of Cx43 and necroptosis-related markers were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. RESULTS LPS exposure induced significant lung injury indicated by histological damage, increased lung wet/dry weight ratio (W/D) and increased total cell counts and protein concentration in BALF. These changes were significantly ameliorated by administration of CORM3 but not iCORM3. LPS also increased necroptosis-related markers RIP1, RIP3, and MLKL and their elevation was blocked by CORM3. CORM3 administration ameliorated LPS induced elevation of Cx43 expression and adenoviral overexpression of Cx43 abolished lung protective effect of CORM3. CORM3 administration attenuated LPS induced activation of extracellular-signal-regulated kinase (ERK) and its protection against necroptosis was abolished by ERK inhibitor U0126. CONCLUSIONS CORM3 attenuates LPS-Induced ALI in neonatal rats and its lung protective effect might be through downregulation of Cx43 to attenuate ERK signaling and ameliorate necroptosis, suggesting CORM3 as a potential therapeutic drug for ALI in neonates.