Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

OBJECTIVE: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)-a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)-a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis. CONCLUSIONS: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.

Inflammasome activation in acute lung injury.

Inflammasomes are multiprotein complexes tasked with sensing endogenous or exogenous inflammatory signals and integrating this signal into a downstream response. Inflammasome activation has been implicated in a variety of pulmonary diseases, including pulmonary hypertension, bacterial pneumonia, COPD, and asthma. Of increasing interest is the contribution of inflammasome activation in the context of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Inflammasome activation in both the lung parenchyma and resident immune cells generates intereukin-1ß (IL-1ß) and IL-18, both of which drive the cascade of lung inflammation forward. Blockade of these responses has been shown to be beneficial in animal models and is a focus of translational research in the field. In this review, we will discuss the assembly and regulation of inflammasomes during lung inflammation, highlighting therapeutically viable effector steps. We will examine the importance of IL-1ß and IL-18, two key products of inflammasome activation, in ALI, as well as the contribution of the pulmonary endothelial cell to this process. Finally, we will explore translational research moving toward anti-inflammasome therapies for ALI/ARDS and speculate toward future directions for the field.

Efficacy and safety of decontamination for N95 respirator reuse: a systematic literature search and narrative synthesis.

PURPOSE: Under times of supply chain stress, the availability of some medical equipment and supplies may become limited. The current pandemic involving severe acute respiratory syndrome coronavirus 2 has highlighted limitations to the ordinary provision of personal protective equipment (PPE). For perioperative healthcare workers, N95 masks provide a stark example of PPE in short supply necessitating the creation of scientifically valid protocols for their decontamination and reuse. METHODS: We performed a systematic literature search of MEDLINE, Embase, Cochrane CENTRAL databases, and ClinicalTrials.gov to identify peer-reviewed articles related to N95 mask decontamination and subsequent testing for the integrity of mask filtration and facial seal. To expand this search, we additionally surveyed the official statements from key health agencies, organizations, and societies for relevant citations. RESULTS: Our initial database search resulted in five articles that met inclusion criteria, with 26 articles added from the expanded search. Our search did not reveal any relevant randomized clinical trials or cohort studies. We found that moist mask heating (65-80°C at 50-85% relative humidity for 20-30 min) and vaporous hydrogen peroxide treatment were supported by the literature to provide consistent viral decontamination without compromising mask seal and filtration efficiency. Other investigated decontamination methods lacked comprehensive scientific evidence for all three of these key criteria. CONCLUSIONS: N95 mask reprocessing using either moist heat or vaporous hydrogen peroxide is recommended to ensure healthcare worker safety.

RéSUMé: OBJECTIF: Lorsque les chaînes d'approvisionnement sont mises sous pression, la disponibilité de certains équipements et fournitures médicaux pourrait devenir restreinte. La pandémie actuelle du syndrome respiratoire aigu sévère du coronavirus 2 a mis en lumière les limites de l'approvisionnement usuel des équipements de protection individuelle (EPI). Pour les travailleurs de la santé périopératoires, les masques N95 sont un exemple frappant d'EPI pouvant rapidement venir à manquer et nécessitant l'élaboration de protocoles scientifiquement rigoureux pour leur décontamination et leur réutilisation. MéTHODE: Nous avons réalisé une recherche de littérature systématique dans les bases de données MEDLINE, Embase, Cochrane CENTRAL et sur ClinicalTrials.gov afin d'identifier les articles révisés par les pairs portant sur la décontamination des masques N95 et les tests subséquents pour vérifier l'intégrité de la filtration du masque et son étanchéité sur le visage. Afin d'étendre notre recherche, nous avons également passé en revue les énoncés officiels émanant des agences de santé, ainsi que des organismes et sociétés médicales majeurs pour en extraire les citations pertinentes. RéSULTATS: Notre recherche initiale des bases de données nous a permis d'extraire cinq articles respectant nos critères d'inclusion, et 26 articles ont été ajoutés à la suite de notre recherche étendue. Notre recherche n'a pas découvert d'études cliniques randomisées ou d'études de cohorte pertinentes. Nous avons observé que la décontamination du masque par chaleur humide (65­80°C à une humidité relative de 50­85 % pendant 20-30 min) et le traitement par vapeur de peroxyde d'hydrogène constituaient les deux mesures endossées par la littérature. En effet, ces modalités offrent une décontamination virale constante sans pour autant compromettre l'étanchéité du masque ou son efficacité de filtration. Les autres méthodes de décontamination étudiées ne possédaient pas de données probantes scientifiques exhaustives quant à ces trois critères clés. CONCLUSION: Le retraitement des masques N95 à l'aide de chaleur humide ou de vapeur de peroxyde d'oxygène est recommandé pour assurer la sécurité des travailleurs de la santé.

CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

The mast cell-B cell axis in lung vascular remodeling and pulmonary hypertension.

Over past years, a critical role for the immune system and, in particular, for mast cells in the pathogenesis of pulmonary hypertension (PH) has emerged. However, the way in which mast cells promote PH is still poorly understood. Here, we investigated the mechanisms by which mast cells may contribute to PH, specifically focusing on the interaction between the innate and adaptive immune response and the role of B cells and autoimmunity. Experiments were performed in Sprague-Dawley rats and B cell-deficient JH-KO rats in the monocrotaline, Sugen/hypoxia, and the aortic banding model of PH. Hemodynamics, cell infiltration, IL-6 expression, and vascular remodeling were analyzed. Gene array analyses revealed constituents of immunoglobulins as most prominently regulated mast cell-dependent genes in the lung in experimental PH. IL-6 was shown to link mast cells to B cells, as 1) IL-6 was upregulated and colocalized with mast cells and was reduced by mast-cell stabilizers and 2) IL-6 or mast cell blockade reduced B cells in lungs of monocrotaline-treated rats. A functional role for B cells in PH was demonstrated in that either blocking B cells by an anti-CD20 antibody or B-cell deficiency in JH-KO rats attenuated right ventricular systolic pressure and vascular remodeling in experimental PH. We here identify a mast cell-B cell axis driven by IL-6 as a critical immune pathway in the pathophysiology of PH. Our results provide novel insights into the role of the immune system in PH, which may be therapeutically exploited by targeted immunotherapy.

Is basic science disappearing from medicine? The decline of biomedical research in the medical literature.

Explosive growth in our understanding of genomics and molecular biology have fueled calls for the pursuit of personalized medicine, the notion of harnessing biologic variability to provide patient-specific care. This vision will necessitate a deep understanding of the underlying pathophysiology in each patient. Medical journals play a pivotal role in the education of trainees and clinicians, yet we suspected that the amount of basic science in the top medical journals has been in decline. We conducted an automated search strategy in PubMed to identify basic science articles and calculated the proportion of articles dealing with basic science in the highest impact journals for 8 different medical specialties from 1994 to 2013. We observed a steep decline (40-60%) in such articles over time in almost all of the journals examined. This rapid decline in basic science from medical journals is likely to affect practitioners' understanding of and interest in the basic mechanisms of disease and therapy. In this Life Sciences Forum, we discuss why this decline may be occurring and what it means for the future of science and medicine.

Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.

The pathophysiology of pulmonary hypertension in left heart disease.

Pulmonary hypertension (PH) is characterized by elevated pulmonary arterial pressure leading to right-sided heart failure and can arise from a wide range of etiologies. The most common cause of PH, termed Group 2 PH, is left-sided heart failure and is commonly known as pulmonary hypertension with left heart disease (PH-LHD). Importantly, while sharing many clinical features with pulmonary arterial hypertension (PAH), PH-LHD differs significantly at the cellular and physiological levels. These fundamental pathophysiological differences largely account for the poor response to PAH therapies experienced by PH-LHD patients. The relatively high prevalence of this disease, coupled with its unique features compared with PAH, signal the importance of an in-depth understanding of the mechanistic details of PH-LHD. The present review will focus on the current state of knowledge regarding the pathomechanisms of PH-LHD, highlighting work carried out both in human trials and in preclinical animal models. Adaptive processes at the alveolocapillary barrier and in the pulmonary circulation, including alterations in alveolar fluid transport, endothelial junctional integrity, and vasoactive mediator secretion will be discussed in detail, highlighting the aspects that impact the response to, and development of, novel therapeutics.

TRPV4 Is Required for Hypoxic Pulmonary Vasoconstriction.

BACKGROUND: Hypoxic pulmonary vasoconstriction (HPV) is critically important in regionally heterogeneous lung diseases by directing blood toward better-oxygenated lung units, yet the molecular mechanism of HPV remains unknown. Transient receptor potential (TRP) channels are a large cation channel family that has been implicated in HPV, specifically in the pulmonary artery smooth muscle cell (PASMC) Ca and contractile response to hypoxia. In this study, the authors probed the role of the TRP family member, TRPV4, in HPV. METHODS: HPV was assessed by using isolated perfused mouse lungs or by intravital microscopy to directly visualize pulmonary arterioles in mice. In vitro experiments were performed in primary human PASMC. RESULTS: The hypoxia-induced pulmonary artery pressure increase seen in wild-type mice (5.6 ± 0.6 mmHg; mean ± SEM) was attenuated both by inhibition of TRPV4 (2.8 ± 0.5 mmHg), or in lungs from TRPV4-deficient mice (Trpv4) (3.4 ± 0.5 mmHg; n = 7 each). Functionally, Trpv4 mice displayed an exaggerated hypoxemia after regional airway occlusion (paO2 71% of baseline ± 2 vs. 85 ± 2%; n = 5). Direct visualization of pulmonary arterioles by intravital microscopy revealed a 66% reduction in HPV in Trpv4 mice. In human PASMC, inhibition of TRPV4 blocked the hypoxia-induced Ca influx and myosin light chain phosphorylation. TRPV4 may form a heteromeric channel with TRPC6 as the two channels coimmunoprecipitate from PASMC and as there is no additive effect of TRPC and TRPV4 inhibition on Ca influx in response to the agonist, 11,12-epoxyeicosatrienoic acid. CONCLUSION: TRPV4 plays a critical role in HPV, potentially via cooperation with TRPC6.

From the Journal archives: Understanding the mechanism(s) regulating hypoxic pulmonary vasoconstriction: how an early study has led to novel translational approaches.

Hypoxic pulmonary vasoconstriction (HPV) is a fundamental physiological process whereby ventilation/perfusion matching is optimized through the constriction of the pulmonary circulation supplying poorly ventilated lung units. In their 1981 paper in the Journal, Noble, Kay, and Fisher used a series of animal experiments to show that alveolar carbon dioxide (CO2) plays a critical role in the regulation of hypoxic pulmonary vasoconstriction. At physiological concentrations, CO2 potentiates the HPV response, and the absence of alveolar CO2 blunts HPV. The enhancement of HPV by CO2 resulted in reduced perfusion of specific hypoxic lung regions, thereby improving systemic oxygenation in lung-ventilated dogs. AUTHORS: William H. Noble, J. Colin Kay, Joseph A. Fisher CITATION: Can Anaesth Soc J 1981; 28: 422-30. PURPOSE: To determine the dominant effect of variations in alveolar carbon dioxide tension on hypoxic pulmonary vasoconstriction. PRINCIPAL FINDINGS: The group found that 1) increasing alveolar carbon dioxide concentrations enhanced hypoxic pulmonary vasoconstriction; 2) this enhancement improved oxygenation in ventilated dogs with regional alveolar hypoxia; and 3) this enhanced oxygenation was not due to increased cardiac output. CONCLUSIONS: Increased alveolar carbon dioxide enhances hypoxic pulmonary vasoconstriction. In clinical scenarios where hypoventilated or hypoxic lung regions exist, e.g., one-lung ventilation or lung consolidation, permissive hypercapnea may improve oxygenation.

Crosstalk between GABAA receptors in astrocytes and neurons triggered by general anesthetic drugs.

General anesthetic drugs cause cognitive deficits that persist after the drugs have been eliminated. Astrocytes may contribute to such cognition-impairing effects through the release of one or more paracrine factors that increase a tonic inhibitory conductance generated by extrasynaptic γ-aminobutyric acid type A (GABAA) receptors in hippocampal neurons. The mechanisms underlying this astrocyte-to-neuron crosstalk remain unknown. Interestingly, astrocytes express anesthetic-sensitive GABAA receptors. Here, we tested the hypothesis that anesthetic drugs activate astrocytic GABAA receptors to initiate crosstalk leading to a persistent increase in extrasynaptic GABAA receptor function in neurons. We also investigated the signaling pathways in neurons and aimed to identify the paracrine factors released from astrocytes. Astrocytes and neurons from mice were grown in primary cell cultures and studied using in vitro electrophysiological and biochemical assays. We discovered that the commonly used anesthetics etomidate (injectable) and sevoflurane (inhaled) stimulated astrocytic GABAA receptors, which in turn promoted the release paracrine factors, that increased the tonic current in neurons via a p38 MAPK-dependent signaling pathway. The increase in tonic current was mimicked by exogenous IL-1ß and abolished by blocking IL-1 receptors; however, unexpectedly, IL-1ß and other cytokines were not detected in astrocyte-conditioned media. In summary, we have identified a novel form of crosstalk between GABAA receptors in astrocytes and neurons that engages a p38 MAPK-dependent pathway. Brief commentary BACKGROUND: Many older patients experience cognitive deficits after surgery. Anesthetic drugs may be a contributing factor as they cause a sustained increase in the function of "memory blocking" extrasynaptic GABAA receptors in neurons. Interestingly, astrocytes are required for this increase; however, the mechanisms underlying the astrocyte-to-neuron crosstalk remain unknown. TRANSLATIONAL SIGNIFICANCE: We discovered that commonly used general anesthetic drugs stimulate GABAA receptors in astrocytes, which in turn release paracrine factors that trigger a persistent increase in extrasynaptic GABAA receptor function in neurons via p38 MAPK. This novel form of crosstalk may contribute to persistent cognitive deficits after general anesthesia and surgery.

Bilateral nephrectomy impairs cardiovascular function and cerebral perfusion in a rat model of acute hemodilutional anemia.

Anemia and renal failure are independent risk factors for perioperative stroke, prompting us to assess the combined impact of acute hemodilutional anemia and bilateral nephrectomy (2Nx) on microvascular brain Po2 (PBro2) in a rat model. Changes in PBro2 (phosphorescence quenching) and cardiac output (CO, echocardiography) were measured in different groups of anesthetized Sprague-Dawley rats (1.5% isoflurane, n = 5-8/group) randomized to Sham 2Nx or 2Nx and subsequently exposed to acute hemodilutional anemia (50% estimated blood volume exchange with 6% hydroxyethyl starch) or time-based controls (no hemodilution). Outcomes were assessed by ANOVA with significance assigned at P < 0.05. At baseline, 2Nx rats demonstrated reduced CO (49.9 ± 9.4 vs. 66.3 ± 19.3 mL/min; P = 0.014) and PBro2 (21.1 ± 2.9 vs. 32.4 ± 3.1 mmHg; P < 0.001) relative to Sham 2Nx rats. Following hemodilution, 2Nx rats demonstrated a further decrease in PBro2 (15.0 ± 6.3 mmHg, P = 0.022). Hemodiluted 2Nx rats did not demonstrate a comparable increase in CO after hemodilution compared with Sham 2Nx (74.8 ± 22.4 vs. 108.9 ± 18.8 mL/min, P = 0.003) that likely contributed to the observed reduction in PBro2. This impaired CO response was associated with reduced fractional shortening (33 ± 9 vs. 51 ± 5%) and increased left ventricular end-systolic volume (156 ± 51 vs. 72 ± 15 µL, P < 0.001) suggestive of systolic dysfunction. By contrast, hemodiluted Sham 2Nx animals demonstrated a robust increase in CO and preserved PBro2. These data support the hypothesis that the kidney plays a central role in maintaining cerebral perfusion and initiating the adaptive increase in CO required to optimize PBro2 during acute anemia.NEW & NOTEWORTHY This study has demonstrated that bilateral nephrectomy acutely impaired cardiac output (CO) and microvascular brain Po2 (PBro2), at baseline. Following acute hemodilution, nephrectomy prevented the adaptive increase in CO associated with acute hemodilution leading to a further reduction in PBro2, accentuating the degree of cerebral tissue hypoxia. These data support a role for the kidney in maintaining PBro2 and initiating the increase in CO that optimized brain perfusion during acute anemia.

Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death.

First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death.