Dimitrios Toumpanakis

Angiopoietin-1 Protects against Airway Inflammation and Hyperreactivity in Asthma

RATIONALE The angiopoietins (Ang) comprise a family of growth factors mainly known for their role in blood vessel formation and remodeling. The best-studied member, Ang-1, exhibits antiapoptotic and antiinflammatory effects. Although the involvement of Ang-1 in angiogenesis is well recognized, little information exists about its role in respiratory physiology and disease. On the basis of its ability to inhibit vascular permeability, adhesion molecule expression, and cytokine production, we hypothesized that Ang-1 administration might exert a protective role in asthma. OBJECTIVES To determine changes in the expression of Ang and to assess the ability of Ang-1 to prevent the histologic, biochemical, and functional changes observed in an animal model of asthma. METHODS To test our hypothesis, a model of allergic airway disease that develops after ovalbumin (OVA) sensitization and challenge was used. MEASUREMENTS AND MAIN RESULTS Ang-1 expression was reduced at the mRNA and protein levels in lung tissue of mice sensitized and challenged with OVA, leading to reduced Tie2 phosphorylation. Intranasal Ang-1 treatment prevented the OVA-induced eosinophilic lung infiltration, attenuated the increase in IL-5 and IL-13, and reduced eotaxin and vascular cell adhesion molecule 1 expression. These antiinflammatory actions of Ang-1 coincided with higher levels of IkappaB and decreased nuclear factor-kappaB binding activity. More importantly, Ang-1 reversed the OVA-induced increase in tissue resistance and elastance, improving lung function. CONCLUSIONS We conclude that Ang-1 levels are decreased in asthma and that administration of Ang-1 might be of therapeutic value because it prevents the increased responsiveness of the airways to constrictors and ameliorates inflammation.

DNA repair systems in malignant mesothelioma

Malignant mesothelioma (MM) is an aggressive tumor of serosal surfaces with increasing incidence and poor prognosis. Asbestos exposure is the main cause of MM and asbestos-induced DNA damage is critical for MM pathogenesis. The present review summarizes the implications of DNA repair systems in MM development, focusing on gene expression alterations and single nucleotide polymorphisms of genes encoding for DNA repair enzymes. The involvement of DNA repair systems in MM improves understanding of MM pathogenesis and provides novel therapeutical targets.

Inspiratory resistive breathing induces MMP-9 and MMP-12 expression in the lung

Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1β, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1β and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.

Tiotropium bromide exerts anti-inflammatory effects during resistive breathing, an experimental model of severe airway obstruction

Introduction Resistive breathing (RB), a hallmark of obstructive airway diseases, is characterized by strenuous contractions of the inspiratory muscles that impose increased mechanical stress on the lung. RB is shown to induce pulmonary inflammation in previous healthy animals. Tiotropium bromide, an anticholinergic bronchodilator, is also shown to exert anti-inflammatory effects. The effect of tiotropium on RB-induced pulmonary inflammation is unknown. Methods Adult rats were anesthetized, tracheostomized and breathed spontaneously through a two-way non-rebreathing valve. Resistances were connected to the inspiratory and/or expiratory port, to produce inspiratory resistive breathing (IRB) of 40% or 50% Pi/Pi,max (40% and 50% IRB), expiratory resistive breathing (ERB) of 60% Pe/Pe,max (60% ERB) or combined resistive breathing (CRB) of both 40% Pi/Pi,max and 60% Pe/Pe,max (40%/60% CRB). Tiotropium aerosol was inhaled prior to RB. After 6 h of RB, mechanical parameters of the respiratory system were measured and bronchoalveolar lavage (BAL) was performed. IL-1β and IL-6 protein levels were measured in lung tissue. Lung injury was estimated histologically. Results In all, 40% and 50% IRB increased macrophage and neutrophil counts in BAL and raised IL-1β and IL-6 lung levels, tissue elasticity, BAL total protein levels and lung injury score. Tiotropium attenuated BAL neutrophil number, IL-1β, IL-6 levels and lung injury score increase at both 40% and 50% IRB. The increase in macrophage count and protein in BAL was only reversed at 40% IRB, while tissue elasticity was not affected. In all, 60% ERB raised BAL neutrophil count and total protein and reduced macrophage count. IL-1β and IL-6 levels and lung injury score were increased. Tiotropium attenuated these alterations, except for the decrease in macrophage count and the increase in total protein level. In all, 40%/60% CRB increased macrophage and neutrophil count in BAL, IL-1β and IL-6 levels, tissue elasticity, total protein in BAL and histological injury score. Tiotropium attenuated the aforementioned alterations. Conclusion Tiotropium inhalation attenuates RB-induced pulmonary inflammation.

The differential effects of inspiratory, expiratory, and combined resistive breathing on healthy lung

Combined resistive breathing (CRB) is the hallmark of obstructive airway disease pathophysiology. We have previously shown that severe inspiratory resistive breathing (IRB) induces acute lung injury in healthy rats. The role of expiratory resistance is unknown. The possibility of a load-dependent type of resistive breathing-induced lung injury also remains elusive. Our aim was to investigate the differential effects of IRB, expiratory resistive breathing (ERB), and CRB on healthy rat lung and establish the lowest loads required to induce injury. Anesthetized tracheostomized rats breathed through a two-way valve. Varying resistances were connected to the inspiratory, expiratory, or both ports, so that the peak inspiratory pressure (IRB) was 20%–40% or peak expiratory (ERB) was 40%–70% of maximum. CRB was assessed in inspiratory/expiratory pressures of 30%/50%, 40%/50%, and 40%/60% of maximum. Quietly breathing animals served as controls. At 6 hours, respiratory system mechanics were measured, and bronchoalveolar lavage was performed for measurement of cell and protein concentration. Lung tissue interleukin-6 and interleukin-1β levels were estimated, and a lung injury histological score was determined. ERB produced significant, load-independent neutrophilia, without mechanical or permeability derangements. IRB 30% was the lowest inspiratory load that provoked lung injury. CRB increased tissue elasticity, bronchoalveolar lavage total cell, macrophage and neutrophil counts, protein and cytokine levels, and lung injury score in a dose-dependent manner. In conclusion, CRB load dependently deranges mechanics, increases permeability, and induces inflammation in healthy rats. ERB is a putative inflammatory stimulus for the lung.

Dose- and time-dependent effects of lipopolysaccharide on technetium-99-m-labeled diethylene-triamine pentaacetatic acid clearance, respiratory system mechanics and pulmonary inflammation.

Intratracheal administration of lipopolysaccharide (LPS) in animals is a commonly used model of acute lung injury, characterized by increased alveolar-capillary membrane permeability causing protein-rich edema, inflammation, deterioration of lung mechanical function and impaired gas exchange. Technetium-99-m-labeled diethylene-triamine pentaacetatic acid (99mTc-DTPA) scintigraphy is a non-invasive technique to assess lung epithelial permeability. We hypothesize that the longer the exposure and the higher the dose of LPS the greater the derangement of the various indices of lung injury. After 3, 6 and 24 h of 5 or 40 μg LPS intratracheally administration, 99mTc-DTPA was instilled in the lung. Images were acquired for 90 min with a γ-camera and the radiotracer clearance was estimated. In another subgroup, the mechanical properties of the respiratory system were estimated with the forced oscillation technique and static pressure-volume curves, 4.5, 7.5 and 25.5 h post-LPS (iso-times with the end of 99mTc-DTPA scintigraphy). Bronchoalveolar lavage (BAL) was performed and a lung injury score was estimated by histology. Lung myeloperoxidase (MPO) activity was measured. 99mTc-DTPA clearance increased in all LPS challenged groups compared with control. DTPA clearance presented a U-shape time course at the lower dose, while LPS had a declining effect over time at the larger dose. At 7.5 and 25.5 h post-LPS, tissue elasticity was increased and static compliance decreased at both doses. Total protein in the BAL fluid increased at both doses only at 4.5 h Total lung injury score and MPO activity were elevated in all LPS-treated groups. There is differential time- and dose-dependency of the various indices of lung injury after intratracheally LPS instillation in rats.

Can resistive breathing injure the lung? Implications for COPD exacerbations

In obstructive lung diseases, airway inflammation leads to bronchospasm and thus resistive breathing, especially during exacerbations. This commentary discusses experimental evidence that resistive breathing per se (the mechanical stimulus) in the absence of underlying airway inflammation leads to lung injury and inflammation (mechanotransduction). The potential implications of resistive breathing-induced mechanotrasduction in COPD exacerbations are presented along with the available clinical evidence.

Antioxidant Supplementation Alters Cytokine Production From Monocytes

We studied in 10 healthy subjects the effect of chronic enteral supplementation of antioxidants (vitamins E, C, A, allopurinol, and N-acetylcysteine) on cytokine production by monocytes at rest, end exercise (60-min cycling at 60% of maximum oxygen consumption), and 60 min post-exercise (recovery). The percentage and the mean fluorescent intensity (MFI) of both unstimulated and lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6-producing monocytes were detected using flow cytometry. Antioxidants decreased the percentage of unstimulated IL-6-producing monocytes following exercise, while their MFI increased at rest. The percentage of LPS-stimulated monocytes increased after exercise and they produced more IL-6 both at rest and following exercise. The percentage of unstimulated and LPS-stimulated IL-1beta-producing monocytes was not affected by antioxidants. The MFI of IL-1beta-produced unstimulated monocytes was increased after antioxidants both at rest and following exercise. After antioxidants, LPS-stimulated monocytes produced more IL-1beta following exercise. Antioxidants decreased the percentage of TNF-alpha spontaneously-produced monocytes following exercise, which produced more TNF-alpha at recovery. Antioxidants did not affect the percentage of LPS-stimulated monocytes producing TNF-alpha, while LPS-stimulated production of TNF-alpha increased both at rest and following exercise. Antioxidants differentially affect TNF-alpha, IL-1beta, and IL-6 production by monocytes, with a general tendency of augmenting cytokine production.

Immune cell response to strenuous resistive breathing: comparison with whole body exercise and the effects of antioxidants

Background/hypothesis Whole body exercise (WBE) changes lymphocyte subset percentages in peripheral blood. Resistive breathing, a hallmark of diseases of airway obstruction, is a form of exercise for the inspiratory muscles. Strenuous muscle contractions induce oxidative stress that may mediate immune alterations following exercise. We hypothesized that inspiratory resistive breathing (IRB) alters peripheral blood lymphocyte subsets and that oxidative stress mediates lymphocyte subpopulation alterations following both WBE and IRB. Patients and methods Six healthy nonathletes performed two WBE and two IRB sessions for 45 minutes at 70% of VO2 maximum and 70% of maximum inspiratory pressure (Pimax), respectively, before and after the administration of antioxidants (vitamins E, A, and C for 75 days, allopurinol for 30 days, and N-acetylcysteine for 3 days). Blood was drawn at baseline, at the end of each session, and 2 hours into recovery. Lymphocyte subsets were determined by flow cytometry. Results Before antioxidant supplementation at both WBE end and IRB end, the natural killer cell percentage increased, the T helper cell (CD3+ CD4+) percentage was reduced, and the CD4/CD8 ratio was depressed, a response which was abolished by antioxidants only after IRB. Furthermore, at IRB end, antioxidants promoted CD8+ CD38+ and blunted cytotoxic T-cell percentage increase. CD8+ CD45RA+ cell percentage changes were blunted after antioxidant supplementation in both WBE and IRB. Conclusion We conclude that IRB produces (as WBE) changes in peripheral blood lymphocyte subsets and that oxidative stress is a major stimulus predominantly for IRB-induced lymphocyte subset alterations.