Elizabeth K.-Y. Walker

ABCA1 mRNA and Protein Distribution Patterns Predict Multiple Different Roles and Levels of Regulation

Mutations in ABCA1 cause the allelic disorders familial hypolipoproteinemia and Tangier Disease. To identify where ABCA1 was likely to have a functional role, we determined the cellular and tissue-specific patterns of murine ABCA1 expression. RT-PCR and Western blot analysis on dissected murine tissues demonstrated broad expression of ABCA1 mRNA and protein in many tissues with prominent protein expression in liver, testis, and adrenal tissue. In situ hybridization and immunohistochemistry experiments demonstrated specific patterns of ABCA1 expression at the cellular level, with hepatocytes, the epithelial lining of the digestive system and bladder, the proximal convoluted tubule of the kidney, and Purkinje and cortical pyramidal neurons containing abundant ABCA1 protein. Significant discordance between relative mRNA and protein expression patterns suggests the possibility of post-transcriptional regulation of ABCA1 expression in selected cells or tissues. We also show that ABCA1 protein levels are up-regulated specifically in the liver after exposure to an atherogenic diet for 7 days, supporting a major role for the liver in dietary modulation of HDL-C levels. Our observations show that ABCA1 is expressed in a pattern consistent with its role in HDL-C metabolism. Additionally, ABCA1 may have important functional roles in other cell types independent of HDL-C regulation.

Ablation of Matrix Metalloproteinase-9 Increases Severity of Viral Myocarditis in Mice

Background— Coxsackievirus B3 (CVB3) causes human myocarditis, which can result in cardiac damage, maladaptive remodeling, and heart failure. Matrix metalloproteinases (MMP)-8 and -9 have been identified in virus-infected myocardium, but their particular roles and underlying mechanisms of effect are unknown. For the first time, we examine the severity of CVB3-induced myocarditis in MMP-8–and MMP-9–deficient mice. Methods and Results— CVB3-infected MMP-8 and MMP-9 knockout (KO) mice and corresponding wild-type (WT) mice were euthanized and harvested at 9 days after infection. Expression of MMP-2, -8, -12, and -13 and tissue inhibitors of MMPs was assessed by zymography or immunoblotting on harvested hearts, and in situ hybridization was performed to detect active infection. Infected MMP-9 KO mice had greater myocardial injury and foci of infection than WT mice despite similar pancreatic infection. Increased fibrosis (10.6±2.7% versus 7.1±2.6%, P=0.04), viral titer, as well as decreased cardiac output, were evident in MMP-9 KO compared with WT mice as assessed by picrosirius red staining, plaque assay, and echocardiography, respectively. Immune infiltration was also greatly increased in MMP-9 KO compared with WT mice (15.2±12.6% versus 2.0±3.0%, P<0.002). Myocardial interferon-&bgr;1, interferon-&ggr;, interleukin-6, interleukin-10, and macrophage inflammatory protein-1&agr; expression was elevated in MMP-9 KO mice as measured by quantitative real-time polymerase chain reaction and ELISA. In contrast, MMP-8 KO mice had the same degree of cardiac injury, fibrosis, and viral infection as their WT counterparts. Conclusions— During acute CVB3 infection, MMP-9 appears necessary to halt virus propagation in the heart, promote proper immune infiltration and remodeling, and preserve cardiac output.

CXCL10 Inhibits Viral Replication Through Recruitment of Natural Killer Cells in Coxsackievirus B3-Induced Myocarditis

Coxsackievirus (CV)B3 is the primary cause of viral myocarditis. We previously observed CXC chemokine ligand 10 (CXCL10) upregulation in the myocardium early in infection. However, the impact of CXCL10 in CVB3-induced myocarditis is unknown. Using isolated primary mouse cardiomyocytes we demonstrated for the first time that cardiomyocytes can express CXCL10 on interferon-&ggr; stimulation. To explore the role of CXCL10 in CVB3-induced myocarditis, both CXCL10 transgenic and knockout mice were used. Following CVB3 challenges, the viral titer in the hearts inversely correlated with the levels of CXCL10 at early phase of infection before visible immune infiltration. Furthermore, as compared with the control mice, the decreased virus titers in the CXCL10 transgenic mouse hearts led to less cardiac damage and better cardiac function and vice verse in the knockout mice. This antiviral ability of CXCL10 might be through recruitment of natural killer (NK) cells to the heart and increased interferon-&ggr; expression early in infection. At day 7 postinfection, with massive influx of mononuclear cells the expression of CXCL10 enhanced the infiltration of CXCR3+ cells, CD4+, and CD8+ T cells, as well as the expression of associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines failed to alter the viral clearance and mice survival. These results suggest the protective role of CXCL10 during the early course of CVB3 infection, which is attributed to the recruitment of NK cells. Nonetheless, CXCL10-directed chemoattractant effect is not sufficient for host to clear the virus in the heart.

A comparison of biologically variable ventilation to recruitment manoeuvres in a porcine model of acute lung injury

BackgroundBiologically variable ventilation (return of physiological variability in rate and tidal volume using a computer-controller) was compared to control mode ventilation with and without a recruitment manoeuvre – 40 cm H2O for 40 sec performed hourly; in a porcine oleic acid acute lung injury model.MethodsWe compared gas exchange, respiratory mechanics, and measured bronchoalveolar fluid for inflammatory cytokines, cell counts and surfactant function. Lung injury was scored by light microscopy. Pigs received mechanical ventilation (FIO2 = 0.3; PEEP 5 cm H2O) in control mode until PaO2 decreased to 60 mm Hg with oleic acid infusion (PaO2/FIO2 <200 mm Hg). Additional PEEP to 10 cm H2O was added after injury. Animals were randomized to one of the 3 modes of ventilation and followed for 5 hr after injury.ResultsPaO2 and respiratory system compliance was significantly greater with biologically variable ventilation compared to the other 2 groups. Mean and mean peak airway pressures were also lower. There were no differences in cell counts in bronchoalveolar fluid by flow cytometry, or interleukin-8 and -10 levels between groups. Lung injury scoring revealed no difference between groups in the regions examined. No differences in surfactant function were seen between groups by capillary surfactometry.ConclusionsIn this porcine model of acute lung injury, various indices to measure injury or inflammation did not differ between the 3 approaches to ventilation. However, when using a low tidal volume strategy with moderate levels of PEEP, sustained improvements in arterial oxygen tension and respiratory system compliance were only seen with BVV when compared to CMV or CMV with a recruitment manoeuvre.

Bosentan Enhances Viral Load via Endothelin-1 Receptor Type-A–Mediated p38 Mitogen-Activated Protein Kinase Activation While Improving Cardiac Function During Coxsackievirus-Induced Myocarditis

Reduced cardiac output is one of the consequences of myocarditis. Bosentan, an endothelin-1 receptor (ET1R) antagonist, could be useful to reduce cardiac afterload, preserving cardiac output. In this study, we investigated the potential therapeutic use of bosentan in an animal model of viral myocarditis. Using a mouse model of coxsackievirus B3 (CVB3)-induced myocarditis, we demonstrated preserved ejection fraction (EF) and fractional shortening (FS) by treatment with bosentan (68±5.8% EF and 40±3.7% FS for treated versus 48±2.2% EF and 25±2.6% FS for controls; P=0.028). However, bosentan enhanced cardiac viral load (10.4±6.7% in the bosentan group versus 5.0±5.5% in control group; P=0.02), likely through enhancement of p38 mitogen-activated protein kinase (MAPK) phosphorylation (0.77±0.40% ATF2 activation in the bosentan group versus 0.03±0.02% in controls; P=0.0002), mediated by endothelin receptor type-A. We further demonstrate that a water soluble inhibitor of p38 MAPK, SB203580 HCl, is a potent inhibitor of virus replication in the heart (0.28% antisense viral genome stained area for 3 mg/kg dose versus 2.9% stained area for controls; P=0.01), attenuates CVB3-induced myocardial damage (blinded cardiac histopathologic scores of 1.8±1.6 and 2.05±1.2 for the 3 mg/kg and 10 mg/kg doses, respectively, versus 3.25±1.2 for the controls), and preserves cardiac function (69±3.5% EF for 3 mg/kg dose and 71±6.7% EF for 10 mg/kg dose versus 60±1.5% EF control; P=0.038 and P=0.045, as compared to control, respectively). Bosentan, a prescribed vasodilator, improves cardiac function but enhances viral load and myocarditis severity through ETRA mediated p38 MAPK activation; p38 MAPK is a desirable antiviral target. Caution must be exercised during treatment of suspected infectious myocarditis with supportive vasoactive remedies.

Biologically variable ventilation improves gas exchange and respiratory mechanics in a model of severe bronchospasm.

Objective:Mechanical ventilation can be lifesaving for status asthmaticus, but how best to accomplish mechanical ventilation is unclear. Biologically variable ventilation (mechanical ventilation that emulates healthy variation) and conventional control mode ventilation (monotonously regular) were compared in an animal model of bronchospasm to determine which approach yields better gas exchange and respiratory mechanics. Design:A randomized prospective trial of biologically variable ventilation vs. control mode ventilation in swine. Setting:University research laboratory. Subjects:Eighteen farm-raised pigs. Interventions:Methacholine was administered as a nebulized aerosol to initiate bronchospasm, defined as doubling of peak inspiratory pressure and respiratory system resistance, and then randomized (n = 9 each group) to either continue control mode ventilation or switch to biologically variable ventilation at the same minute ventilation. Over the next 4 hrs, hemodynamics, blood gases, respiratory mechanics, and carbon dioxide expirograms were recorded hourly. At end-experiment, tracheobronchial lavage was undertaken to determine interleukin-6 and -10 concentrations. Measurements and Main Results:Measurements of physiologic variables and inflammatory cytokines showed that biologically variable ventilation significantly improved gas exchange, with greater arterial oxygen tensions (p = .006; group × time interaction), lower arterial carbon dioxide tensions (p = .0003; group effect), lower peak inspiratory pressures (p = .0001; group × time), greater static compliance (p = .0001; group × time), greater dynamic compliance (p = .0001; group × time), and lower total respiratory system resistance (p = .028; group × time), compared with conventional ventilation. The appearance of inflammatory cytokines in bronchoalveolar lavage fluid (interleukin-6 and -10) was not affected by mode of ventilation. Conclusions:In this experimental model, biologically variable ventilation was superior to control mode ventilation in terms of gas exchange and respiratory mechanics during severe bronchospasm.

Neutralizing anti-4-1BBL treatment improves cardiac function in viral myocarditis

Coxsackievirus B3 (CVB3) is the most common causative agent of infectious myocarditis. Chronic inflammation, loss of contractile tissue, and maladaptive remodeling all contribute to dilated cardiomyopathy and heart failure. The 4-1BB receptor is a costimulatory molecule expressed by T cells and cardiomyocytes. We infected mice with CVB3 to examine if virus infection triggers 4-1BB activation and whether inhibition of this pathway will reduce inflammation and improve heart function. Echocardiography was performed on days 3, 9, 30 and at 10 weeks post-infection (pi) and ejection fraction (EF), left ventricular (LV) wall thickness, contractility, and internal cardiac dimensions were measured. At day 9, reduced rate of wall thickening (30±17 vs 70±19%), increased LV wall thickness (0.15±0.04 vs 0.09±0.01 cm in diastole and 0.19±0.04 vs 0.15±0.02 cm in systole), and reduced cardiac volume (0.013±0.004 vs 0.023±0.003 ml in diastole and 0.004±0.002 ml vs 0.007±0.001 ml in systole) were observed in infected hearts as compared with shams. At 14 days pi, CVB3-infected mice were randomly assigned to receive either anti-4-1BBL neutralizing (M522) or control antibodies (Ab) for 8 weeks. Cardiac damage, fibrosis, and inflammation were assessed by histological stains and immunohistochemistry. Polymerase chain reaction (PCR) was utilized to detect matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12 expressions. At 10 weeks pi, M522 treatment improved LV wall thickening rate (−10±13 vs −49±16%, expressed as percentage change from baseline) and reduced diastolic LV posterior wall thickness (17±10 vs 57±47%, expressed as percentage change from baseline), cardiac damage as assessed by histological scores (0 vs 1.3±1.5), fibrosis by collagen volume fraction (3.2±0.6 vs 4.9±2.2%), overall inflammation (5.9±1.3 vs 8.5±4.1%), and T-cell infiltration (1.3±0.9 vs 4.3±3.8%) as compared to control. MMP-12 was highly increased during acute and chronic myocarditis, but was significantly decreased by M522 treatment. Thus, long-term inhibition of the 4-1BB pathway reduces cardiac damage, remodeling, and inflammation during viral myocarditis.

Physiological noise versus white noise to drive a variable ventilator in a porcine model of lung injury.

Purpose: Variable ventilation is superior to control mode ventilation in a number of circumstances. The nature of the breathing file used to deliver the variable rate and tidal volume has not been formally examined.Methods: We compared two different noise files in a randomized prospective trial of variable ventilation. Pigs were anesthetized, intubated, and mechanically ventilated. Oleic acid was infused to introduce lung injury. The animals were ventilated at a tidal volume of 7 mL·kg−1, in variable mode, with either physiologically-derived noise (variability file − 1,587 breath intervals-obtained from a spontaneously breathing volunteer;n=10) or a variability file of identical length derived from computergenerated white noise (n=10).Results: The physiologically-derived noise had a power law α-exponent of −0.27 and a Hölder exponent of −0.38, indicative of auto-correlated noise. The computer-generated noise had an α-exponent of −0.52 and a Hölder exponent of −0.49, indicative of white noise. Both files showed multifractal characteristics. There were no differences between groups, at any time period, for PaO2, PaCO2, and static or dynamic respiratory system compliance. No differences were observed between groups for wet:dry lung weight ratios or for interleukin-8 in bronchoalveolar lavage fluid.Conclusion: This study demonstrates that the nature of the variability files, chosen to drive the variable ventilator, had no effect on indices of gas exchange or respiratory mechanics in this model. A considerable overlap of the multifractal files existed. The potential to drive a variable ventilator using algorithmderived files with multifractal characteristics, thereby eliminating the requirement to use physiologically-derived signals, is discussed.RésuméObjectif: La ventilation en mode variable est supérieure à la ventilation en mode contrôlée dans plusieurs situations. La nature du fichier de respiration utilisé pour engendrer la fréquence et le volume courant variable n’a pas été évaluée de façon formelle.Méthode: Nous avons comparé deux fichiers de bruit différents dans une étude prospective randomisée de la ventilation en mode variable. Les cochons ont été anesthésiés, intubés et ventilés mécaniquement. Ils ont reçu une perfusion d’acide oléique afin de provoquer une lésion pulmonaire. Les animaux ont été ventilés à un volume courant de 7 mL·kg−1, en mode variable, avec soit du bruit de provenance physiologique (fichier de variabilité — 1587 intervalles de respiration — obtenus d’un volontaire respirant spontanément ; n=10) ou un fichier de variabilité de longueur identique dérivé d’un bruit blanc généré par ordinateur (n=10).Résultats: Le bruit d’origine physiologique avait un exposant α de la loi de puissance de −0,27 et un exposant de Hölder de −0,38, ce qui indique un bruit auto-corrélé. Le bruit généré par ordinateur avait un exposant α de −0,52 et un exposant de Hölder de −0,49, ce qui indique un bruit blanc. Les deux fichiers ont montré des caractéristiques multifractales. Il n’y a pas eu de différence entre les groupes, à n’importe quelle période de temps, pour la PaO2, la PaCO2, et la conformité statique et dynamique du système respiratoire. Aucune différence n’a été observée entre les groupes en ce qui touche aux rapports de poids oedème pulmonaire/poumon sec ou pour l’interleukine 8 dans le liquide de lavage bronchoalvéolaire.Conclusion: Cette étude démontre que la nature des fichiers de variabilité sélectionnés pour entraîner le respirateur en mode variable n’a pas eu d’effet sur les indices d’échange gazeux ou de mécanique respiratoire dans ce modèle. Un chevauchement considérable est apparu dans les fichiers multifractals. La possibilité d’entraîner un respirateur en mode variable avec des fichiers dérivés d’algorithmes avec des caractéristiques multifractales, éliminant ainsi le besoin de recourir à des signaux d’origine physiologique, est discutée ici.

Variable ventilation compared with recruitment manoeuvres in a porcine model of ARDS

INTRODUCTION A negative consequence of mechanical ventilation using lower tidal volumes (VT) in patients with the acute respiratory distress syndrome (ARDS) is alveolar collapse. Recruitment manoeuvres (RMsthe application of high levels of positive airway pressure for short periods of time) have been proposed to inflate these atelectatic areas of lung. In the present study, we compared biologically variable ventilation (BVV) with conventional control mode ventilation (CMV), and to CMV with RMs – 40 cm H2O positive pressure for 40 sec (CMVRM) hourly for 5 hrs. BVV is a unique version of CMV that uses a computer-controller to return physiological variability or “noise” to mechanical ventilators. By varying the frequency and VT of delivered breaths (via changes in peak airway pressure), BVV may provide an alternative means to recruit atelectatic areas seen with ARDS.