Multi-omics approaches for revealing the complexity of cardiovascular disease.

The development and progression of cardiovascular disease (CVD) can mainly be attributed to the narrowing of blood vessels caused by atherosclerosis and thrombosis, which induces organ damage that will result in end-organ dysfunction characterized by events such as myocardial infarction or stroke. It is also essential to consider other contributory factors to CVD, including cardiac remodelling caused by cardiomyopathies and co-morbidities with other diseases such as chronic kidney disease. Besides, there is a growing amount of evidence linking the gut microbiota to CVD through several metabolic pathways. Hence, it is of utmost importance to decipher the underlying molecular mechanisms associated with these disease states to elucidate the development and progression of CVD. A wide array of systems biology approaches incorporating multi-omics data have emerged as an invaluable tool in establishing alterations in specific cell types and identifying modifications in signalling events that promote disease development. Here, we review recent studies that apply multi-omics approaches to further understand the underlying causes of CVD and provide possible treatment strategies by identifying novel drug targets and biomarkers. We also discuss very recent advances in gut microbiota research with an emphasis on how diet and microbial composition can impact the development of CVD. Finally, we present various biological network analyses and other independent studies that have been employed for providing mechanistic explanation and developing treatment strategies for end-stage CVD, namely myocardial infarction and stroke.

Addressing the heterogeneity in liver diseases using biological networks.

The abnormalities in human metabolism have been implicated in the progression of several complex human diseases, including certain cancers. Hence, deciphering the underlying molecular mechanisms associated with metabolic reprogramming in a disease state can greatly assist in elucidating the disease aetiology. An invaluable tool for establishing connections between global metabolic reprogramming and disease development is the genome-scale metabolic model (GEM). Here, we review recent work on the reconstruction of cell/tissue-type and cancer-specific GEMs and their use in identifying metabolic changes occurring in response to liver disease development, stratification of the heterogeneous disease population and discovery of novel drug targets and biomarkers. We also discuss how GEMs can be integrated with other biological networks for generating more comprehensive cell/tissue models. In addition, we review the various biological network analyses that have been employed for the development of efficient treatment strategies. Finally, we present three case studies in which independent studies converged on conclusions underlying liver disease.

Glucosylceramide synthase deficiency in the heart compromises ß1-adrenergic receptor trafficking.

AIMS: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function. METHODS AND RESULTS: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to ß-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of ß1-adrenergic receptors. CONCLUSIONS: Our findings suggest that cardiac glycosphingolipids are required to maintain ß-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.

Sacubitril/valsartan (Entresto) utilisation and prescribing patterns in the context of a reimbursement application system.

AIMS: Entresto (sacubitril/valsartan) is used to treat symptomatic chronic heart failure with reduced ejection fraction. Given its high potential budget impact, the Health Services Executive introduced a reimbursement application system (RAS) to ensure its appropriate use. The aim of this study was to evaluate the utilisation of Entresto in Ireland and compare patient characteristics to those of the pivotal PARADIGM-HF trial. METHODS: We used dispensed claims data from the Primary Care Reimbursement Services, clinical data obtained from the RAS, and data from published studies of Entresto utilisation. Differences in the baseline characteristics in the study populations vs the Entresto arm of the PARADIGM-HF trial were analysed. We also investigated cardiovascular medication use in the 6 months pre- and post-Entresto initiation. RESULTS: In 2018, there were 1043 individuals receiving Entresto, corresponding to an expenditure of €1.2 million. Patients prescribed Entresto in Ireland were older, had lower left ventricular ejection fraction and were more symptomatic than those in the PARADIGM-HF trial. Irish patient characteristics were reflective of Entresto-treated populations in other real-world studies. More than 63% of patients were commenced on the lowest Entresto dose. Entresto initiation was associated with a reduction in the use of other medications for heart failure. CONCLUSION: The utilisation of Entresto has been steadily increasing in Ireland since its reimbursement approval. The expenditure in the first year was substantially lower than predicted, and the RAS is an example of how health technology management can facilitate appropriate and cost-effective use of medicines.

Trends in strong opioid prescribing in Ireland: A repeated cross-sectional analysis of a national pharmacy claims database between 2010 and 2019.

PURPOSE: Significant increases in opioid utilisation have been reported in many countries in recent decades. This study investigated strong opioid prescribing in Irish General Medical Services (GMS) patients over a 10-year period. METHODS: A retrospective repeated cross-sectional analysis of a national pharmacy claims database between January 2010 and December 2019 was conducted. Strong opioid prescribing in GMS patients was evaluated, including by route of administration, age (16-64 years and ≥65 years) and gender. Measures of consumption included prescribing prevalence and defined daily dose (DDD)/1000 population/day. Prevalence ratios (PRs) with 95% confidence intervals (CIs), and percentage and absolute changes were determined. RESULTS: Strong opioid prescribing prevalence increased from 14.43% in 2010 to 16.28% in 2019, with the greatest increase in the ≥65 years age group. Tramadol was the most frequently prescribed product, constituting 63.9% of total strong opioid prescribing. The prescribing prevalence of oxycodone increased from 0.95% in 2010 to 2.68% in 2019 (PR 2.81, 95% CI 2.76, 2.87), with steep increases in oxycodone-naloxone since it became available (PR 5.23, 95% CI 4.98, 5.50). The prescribing prevalence of tapentadol increased from 0.18% to 1.58% between 2012 (first complete year available for reimbursement) and 2019 (PR 8.79, 95% CI 8.43, 9.16). Strong opioid prescribing was highest in females aged ≥65 years. CONCLUSIONS: This study found an overall increase in strong opioid prescribing in Ireland between 2010 and 2019, particularly in older adults. Tramadol was the most frequently prescribed product, with oxycodone and tapentadol prescribing increasing markedly over the study period.

Upregulation of airway smooth muscle calcium-sensing receptor by low-molecular-weight hyaluronan.

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20-25 g) exposed to either bromine (Br2) or Cl2 (600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2 or Cl2 exposure, produced higher AHR compared with Br2 or Cl2 alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2 resulted in membrane potential depolarization (Vm Air: 62 ± 3 mV; 3 h post Br2:-45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2 and nifedipine-induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2 reversed the effects of Br2 and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vm depolarization, [Ca2+]i increase, and Ca-SR expression to a similar extent as exposure to Br2 and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure.

Bromine (Br2) is an organohalide found in nature and is integral to many manufacturing processes. Br2 is toxic to living organisms, and high concentrations can prove fatal. To meet industrial demand, large amounts of purified Br2 are produced, transported, and stored worldwide, providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br2 exposure, we have surveyed the lung proteomes of C57BL/6 male mice and human lung-derived microvascular endothelial cells (HMECs) at 24 h following exposure to Br2 in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 h after Br2 exposure. These experiments revealed significant increases in the filtration coefficient (Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br2 and Br-lipid-treated HMECs exhibited differential levels of zona occludens-1 that were found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadherin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br2 toxicity.

Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats.

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.

Organ Donation and the Ars Moriendi.

Organ donation is rightly understood as a gift that is a genuine act of love. Organ donation as an act of love requires it to be an act of freedom that honors the integrity of the human person who is in the process of dying. However, the process of organ donation, by necessity, inserts a third party of interest whose primary aim is to assist someone other than the dying person. Caregivers can become "organ focused" instead of "patient focused." The procurement of organs potentially results in the commodification of the potential organ donor. Furthermore, death is not a momentary event but rather an ontological change in the person where the union of body and soul becomes divided. This Catholic understanding of death is important to assess the impact of organ donation on the process of dying. Family members of organ donors often have traumatic memories associated with the organ donation process, potentially overshadowing the ars moriendi-the art of dying. SUMMARY: While organ donation is an act of love, the donation process can be distraction from the care of the dying patient, who may be treated differently than other dying patients who are not organ donors. A Catholic understanding of death is helpful in assessing the impact of the organ donation process.

Organ Donation and Declaration of Death: Combined Neurologic and Cardiopulmonary Standards.

Prolonged survival after the declaration of death by neurologic criteria creates ambiguity regarding the validity of this methodology. This ambiguity has perpetuated the debate among secular and nondissenting Catholic authors who question whether the neurologic standards are sufficient for the declaration of death of organ donors. Cardiopulmonary criteria are being increasingly used for organ donors who do not meet brain death standards. However, cardiopulmonary criteria are plagued by conflict of interest issues, arbitrary standards for candidacy, and the lack of standardized protocols for organ procurement. Combining the neurological and cardiopulmonary standards into a single protocol would mitigate the weaknesses of both and provide greater biologic and moral certainty that a donor of unpaired vital organs is indeed dead. SUMMARY: Before a person's organs can be used for transplantation, he or she must be declared "brain-dead." However, sometimes when someone is declared brain-dead, that person can be maintained on life-support for days or even weeks. This creates some confusion about whether the person has truly died. For patients who have a severe neurologic injury but are not brain-dead, organ donation can also occur after his or her heart stops beating. However, this protocol is more ambiguous and lacks standardized protocols. We propose that before a person can donate organs, he or she must first be declared brain-dead, and then his or her heart must irreversibly stop beating before organs are taken.

Formation of chlorinated lipids post-chlorine gas exposure.

Exposure to chlorine (Cl2) gas can occur during accidents and intentional release scenarios. However, biomarkers that specifically indicate Cl2 exposure and Cl2-derived products that mediate postexposure toxicity remain unclear. We hypothesized that chlorinated lipids (Cl-lipids) formed by direct reactions between Cl2 gas and plasmalogens serve as both biomarkers and mediators of post-Cl2 gas exposure toxicities. The 2-chloropalmitaldehyde (2-Cl-Pald), 2-chlorostearaldehyde (2-Cl-Sald), and their oxidized products, free- and esterified 2-chloropalmitic acid (2-Cl-PA) and 2-chlorostearic acid were detected in the lungs and plasma of mouse and rat models of Cl2 gas exposure. Levels of Cl-lipids were highest immediately post-Cl2 gas exposure, and then declined over 72 h with levels remaining 20- to 30-fold higher at 24 h compared with baseline. Glutathione adducts of 2-Cl-Pald and 2-Cl-Sald also increased with levels peaking at 4 h in plasma. Notably, 3-chlorotyrosine also increased after Cl2 gas exposure, but returned to baseline within 24 h. Intranasal administration of 2-Cl-PA or 2-Cl-Pald at doses similar to those formed in the lung after Cl2 gas exposure led to increased distal lung permeability and inflammation and systemic endothelial dysfunction characterized by loss of eNOS-dependent vasodilation. These data suggest that Cl-lipids could serve as biomarkers and mediators for Cl2 gas exposure and toxicity.

Respiratory syncytial virus infection increases chlorine-induced airway hyperresponsiveness.

Exposure to chlorine (Cl2) damages airway and alveolar epithelia resulting in acute lung injury and reactive airway hyperresponsiveness (AHR) to methacholine. However, little is known about the effect of preexisting respiratory disease on Cl2-induced lung injury. By using a murine respiratory syncytial virus (RSV) infection model, we found that preexisting RSV infection increases Cl2 (187 ppm for 30 min)-induced lung inflammation and airway AHR at 24 h after exposure (5 days after infection). RSV infection and Cl2 exposure synergistically induced oxygen desaturation and neutrophil infiltration and increased MCP-1, MIP-1ß, IL-10, IFN-γ, and RANTES concentrations in the bronchoalveolar lavage fluid (BALF). In contrast, levels of type 2 cytokines (i.e., IL-4, IL-5, IL-9, and IL-13) were not significantly affected by either RSV infection or Cl2 exposure. Cl2 exposure, but not RSV infection, induced AHR to methacholine challenge as measured by flexiVent. Moreover, preexisting RSV infection amplified BALF levels of hyaluronan (HA) and AHR. The Cl2-induced AHR was mitigated by treatment with inter-α-trypsin inhibitor antibody, which inhibits HA signaling, suggesting a mechanism of HA-mediated AHR from exacerbated oxidative injury. Our results show for the first time that preexisting RSV infection predisposes the lung to Cl2-induced injury. These data emphasize the necessity for further research on the effects of Cl2 in vulnerable populations and the development of appropriate treatments.

Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury.

Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca(2+), and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca(2+), blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca(2+) channels of airway smooth muscle cells, increasing their contractility and thus causing AHR.

Nitrite therapy improves survival postexposure to chlorine gas.

Exposure to relatively high levels of chlorine (Cl2) gas can occur in mass-casualty scenarios associated with accidental or intentional release. Recent studies have shown a significant postexposure injury phase to the airways, pulmonary, and systemic vasculatures mediated in part by oxidative stress, inflammation, and dysfunction in endogenous nitric oxide homeostasis pathways. However, there is a need for therapeutics that are amenable to rapid and easy administration in the field and that display efficacy toward toxicity after chlorine exposure. In this study, we tested whether nitric oxide repletion using nitrite, by intramuscular injection after Cl2 exposure, could prevent Cl2 gas toxicity. C57bl/6 male mice were exposed to 600 parts per million Cl2 gas for 45 min, and 24-h survival was determined with or without postexposure intramuscular nitrite injection. A single injection of nitrite (10 mg/kg) administered either 30 or 60 min postexposure significantly improved 24-h survival (from ∼20% to 50%). Survival was associated with decreased neutrophil accumulation in the airways. Rendering mice neutropenic before Cl2 exposure improved survival and resulted in loss of nitrite-dependent survival protection. Interestingly, female mice were more sensitive to Cl2-induced toxicity compared with males and were also less responsive to postexposure nitrite therapy. These data provide evidence for efficacy and define therapeutic parameters for a single intramuscular injection of nitrite as a therapeutic after Cl2 gas exposure that is amenable to administration in mass-casualty scenarios.

Postexposure aerosolized heparin reduces lung injury in chlorine-exposed mice.

Chlorine (Cl2) is a highly reactive oxidant gas that, when inhaled, may cause acute lung injury culminating in death from respiratory failure. In this study, we tested the hypothesis that exposure of mice to Cl2 causes intra-alveolar and systemic activation of the coagulation cascade that plays an important role in development of lung injury. C57Bl/6 mice were exposed to Cl2 (400 for 30 min or 600 ppm for 45 min) in environmental chambers and then returned to room air for 1 or 6 h. Native coagulation (NATEM) parameters such as blood clotting time and clot formation time were measured in whole blood by the viscoelastic technique. D-dimers and thrombin-anti-thrombin complexes were measured in both plasma and bronchoalveolar lavage fluid (BALF) by ELISA. Our results indicate that mice exposed to Cl2 gas had significantly increased clotting time, clot formation time, and D-dimers compared with controls. The thrombin-anti-thrombin complexes were also increased in the BALF of Cl2 exposed animals. To test whether increased coagulation contributed to the development of acute lung injury, mice exposed to Cl2 and returned to room air were treated with aerosolized heparin or vehicle for 20 min. Aerosolized heparin significantly reduced protein levels and the number of inflammatory cells in the BALF at 6 h postexposure. These findings highlight the importance of coagulation abnormities in the development of Cl2-induced lung injury.

TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury.

The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.

Chlorine induces the unfolded protein response in murine lungs and skin.

Chlorine (Cl2) is an important industrial chemical. Accidental full body exposure to Cl2 poses an environmental, occupational, and public health hazard characterized mainly by injury to the lung, skin, and ocular epithelia. The cellular mechanisms underlying its acute toxicity are incompletely understood. This study examined whether whole body exposure of BALB/c mice to Cl2 in environmental chambers leads to the up-regulation of the unfolded protein response (UPR) in their lungs and skin. Shaved BALB/c mice were exposed to a sublethal concentration of Cl2 (400 ppm for 30 min) and returned to room air for 1 or 6 hours and killed. IL-6 and TNF-α were increased significantly at 1 and 6 hours after Cl2 exposure in the lungs and at 6 hours in the skin. These changes were accompanied by increased UPR signaling (i.e., activation of protein kinase RNA-like endoplasmic reticulum kinase, inositol-requiring enzyme 1 α, and activating transcription factor 6α) at these time points. The expression of hepcidin, which regulates tissue accumulation and mobilization of iron, was increased in the skin and lungs of Cl2-exposed mice. The data shown herein indicate for the first time the up-regulation of UPR signaling and hepcidin in the skin and lungs of Cl2-exposed mice, which persisted when the mice were returned to room air for 6 hours.

Chlorine gas exposure increases susceptibility to invasive lung fungal infection.

Chlorine (Cl2) is a highly irritating and reactive gas with potential occupational and environmental hazards. Acute exposure to Cl2 induces severe epithelial damage, airway hyperreactivity, impaired alveolar fluid clearance, and pulmonary edema in the presence of heightened inflammation and significant neutrophil accumulation in the lungs. Herein, we investigated whether Cl2 exposure affected the lung antimicrobial immune response leading to increased susceptibility to opportunistic infections. Mice exposed to Cl2 and challenged intratracheally 24 h thereafter with the opportunistic mold Aspergillus fumigatus demonstrated an >500-fold increase in A. fumigatus lung burden 72 h postchallenge compared with A. fumigatus mice exposed to room air. Cl2-exposed A. fumigatus challenged mice also demonstrated significantly higher lung resistance following methacholine challenge and increased levels of plasma proteins (albumin and IgG) in the bronchoalveolar lavage fluid. Despite enhanced recruitment of inflammatory cells to the lungs of Cl2-exposed A. fumigatus challenged mice, these cells (>60% of which were neutrophils) demonstrated a profound impairment in generating superoxide. Significantly higher A. fumigatus burden in the lungs of Cl2 exposed mice correlated with enhanced production of IL-6, TNF-α, CXCL1, CCL2, and CCL3. Surprisingly, however, Cl2-exposed A. fumigatus challenged mice had a specific impairment in the production of IL-17A and IL-22 in the lungs compared with mice exposed to room air and challenged with A. fumigatus. In summary, our results indicate that Cl2 exposure markedly impairs the antimicrobial activity and inflammatory reactivity of myeloid cells in the lung leading to increased susceptibility to opportunistic pathogens.

Cardiomyocyte-specific PCSK9 deficiency compromises mitochondrial bioenergetics and heart function.

AIMS: Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9-/- mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. METHODS AND RESULTS: Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9-/- mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9-/- hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9-/- mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9-/- mice. Circulating lipid levels were unchanged in CM-Pcsk9-/- mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9-/- mice had an increased number of mitochondria-endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. CONCLUSION: PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Post-exposure antioxidant treatment in rats decreases airway hyperplasia and hyperreactivity due to chlorine inhalation.

We assessed the safety and efficacy of combined intravenous and aerosolized antioxidant administration to attenuate chlorine gas-induced airway alterations when administered after exposure. Adult male Sprague-Dawley rats were exposed to air or 400 parts per million (ppm) chlorine (a concentration likely to be encountered in the vicinity of industrial accidents) in environmental chambers for 30 minutes, and returned to room air, and they then received a single intravenous injection of ascorbic acid and deferoxamine or saline. At 1 hour and 15 hours after chlorine exposure, the rats were treated with aerosolized ascorbate and deferoxamine or vehicle. Lung antioxidant profiles, plasma ascorbate concentrations, airway morphology, and airway reactivity were evaluated at 24 hours and 7 days after chlorine exposure. At 24 hours after exposure, chlorine-exposed rats had significantly lower pulmonary ascorbate and reduced glutathione concentrations. Treatment with antioxidants restored depleted ascorbate in lungs and plasma. At 7 days after exposure, in chlorine-exposed, vehicle-treated rats, the thickness of the proximal airways was 60% greater than in control rats, with twice the amount of mucosubstances. Airway resistance in response to methacholine challenge was also significantly elevated. Combined treatment with intravenous and aerosolized antioxidants restored airway morphology, the amount of airway mucosubstances, and airway reactivity to control levels by 7 days after chlorine exposure. Our results demonstrate for the first time, to the best of our knowledge, that severe injury to major airways in rats exposed to chlorine, as characterized by epithelial hyperplasia, mucus accumulation, and airway hyperreactivity, can be reversed in a safe and efficacious manner by the post-exposure administration of ascorbate and deferoxamine.