Alveolar Protein Levels Research Articles

Von Willebrand factor in experimental malaria‐associated acute respiratory distress syndrome

BackgroundMalaria‐associated acute respiratory distress syndrome (MA‐ARDS) is a lethal complication of severe malaria, characterized by marked pulmonary inflammation. Patient studies have suggested a link between von Willebrand factor (VWF) and malaria severity. ObjectivesTo investigate the role of VWF in the pathogenesis of experimental MA‐ARDS. MethodsPlasmodium bergheiNK65‐E (PbNK65) parasites were injected in Vwf+/+ and Vwf−/− mice. Pathological parameters were assessed following infection. ResultsIn accordance with patients with severe malaria, plasma VWF levels were increased and ADAMTS13 activity levels were reduced in experimental MA‐ARDS. ADAMTS13‐ and plasmin‐independent reductions of high molecular weight VWF multimers were observed at the end stage of disease. Thrombocytopenia was VWF‐independent because it was observed in both Vwf+/+ and Vwf−/− mice. Interestingly, Vwf−/− mice had a shorter survival time compared with Vwf+/+ controls following PbNK65 infection. Lung edema could not explain this shortened survival because alveolar protein levels in Vwf−/− mice were approximately two times lower than in Vwf+/+ controls. Parasite load, on the other hand, was significantly increased in Vwf−/− mice compared with Vwf+/+ mice in both peripheral blood and lung tissue. In addition, anemia was only observed in PbNK65‐infected Vwf−/− mice. Of note, Vwf−/− mice presented with two times more reticulocytes, a preferential target of the parasites. ConclusionsThis study suggests that parasite load together with malarial anemia, rather than alveolar leakage, might contribute to shortened survival in PbNK65‐infected Vwf−/− mice. VWF deficiency is associated with early reticulocytosis following PbNK65 infection, which potentially explains the increase in parasite load.

Absence of lung fibrosis after a single pulmonary delivery of lipid nanocapsules in rats.

Lipid nanocapsules (LNCs) are potential drug carriers for pulmonary delivery since they can be nebulized without any structural or functional changes, and the aerosols produced are highly compatible with pulmonary drug delivery in human beings. The alveolar surface tension, in vitro cytotoxicity, biodistribution and pulmonary toxicity in rats of a single endotracheal spray of LNCs or paclitaxel-loaded LNCs were studied. In vitro cytotoxicity of LNCs after a spray remained unchanged. Biodistribution study showed a homogeneous repartition in the lungs in rats with an improvement in lung retention of the radiolabeled tracer loaded in LNCs compared to the absence of LNCs with a lung half-time of 8.8±0.7 hours. Bronchoalveolar fluid analysis revealed transient 7-day alveolar inflammation, reaching a maximum between days 2 and 4, characterized by a peak of granulocytes at day 1 followed by a peak of lymphocytes at day 3. Alveolar protein levels were increased at days 3 and 7. Acute inflammation was increased with paclitaxel-loaded LNCs in comparison with blank LNCs but dropped out at day 7. No histological pulmonary lesion was observed at day 60. LNCs lowered surface tension to a greater degree than Curosurf® in a physicochemical model of the pulmonary alveolus. A single pulmonary delivery of LNCs induces a short-term alveolar inflammation with no residual lesions in rats at day 60. These data permit to start the study of LNCs in surfactant replacement therapy.

Bubble CPAP Support after Discontinuation of Mechanical Ventilation Protects Rat Lungs with Ventilator-Induced Lung Injury

Background: Bubble continuous positive airway pressure (BCPAP) has been used in neonates with respiratory distress for decades, but its lung-protective effect and underlying mechanism has not been investigated. Objectives: To test the hypothesis that BCPAP use after extubation decreases lung injury and that alterations to lung nitric oxide synthase (NOS) 3 expression may be one of the underlying mechanisms. Methods: We compared gas exchange, lung injury severity, and lung NOS expression among rats with ventilator-induced lung injury (VILI) treated with either BCPAP or spontaneous breathing. After high tidal volume ventilation for 30 min, the rats were randomly divided to three groups: a control group underwent spontaneous breathing (n = 7), and two BCPAP groups were treated with the bubble technique with either a 2.5-mm-diameter (n = 7) or a 5.5-mm-diameter (n = 7) expiratory limb for 2 h. Results: The bubble technique (2.5 and 5.5 mm diameter combined) resulted in a significantly higher Pa<smlcap>O</smlcap>₂, decreased alveolar protein levels (1.01 vs. 1.43 mg/kg, p < 0.05), a lower lung injury score (3.87 vs. 4.86, p < 0.05), and decreased NOS3 expression (1.99 vs. 3.32, p < 0.05) compared to spontaneous breathing in the control group. BCPAP with a 2.5-mm-diameter and with a 5.5-mm-diameter expiratory limb was not different with regard to gas exchange, alveolar protein levels, and lung injury scores, but there was a trend for lower NOS3 expression in the 5.5-mm group (1.41 vs. 2.56, p = 0.052). Conclusions: BCPAP decreases lung injury in rats with VILI after stopping mechanical ventilation. Attenuation of lung NOS3 expression may be one of the underlying mechanisms.

Innovative Hematopoietic Gene-Therapy Concepts for Hereditary Pulmonary Alveolar Proteinosis Utilizing Hematopoietic Stem Cell Derived Macrophages

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colony-stimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce pulmonary transplantation of gene corrected hematopoietic stem cell (HSC)-derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, selective pulmonary engraftment of healthy donor cells upon pulmonary transplantation of MPs was demonstrated by flow- and chip cytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as lung function and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface marker, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following pulmonary transplantation of MPs differentiated from lentivirally corrected Csf2rb -/- HSCs. In vitro these gene-corrected HSCs expanded up to 1045-fold while differentiating into typical alveolar macrophages with F4/80, CD11b, CD11c, CD68, as well as Csf2rb mRNA and protein (CD131) expression and reconstitution of GM-CSF receptor signaling. Transplanted herPAP mice displayed significant improvement of biomarkers in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, administration of human CD34+ cell-derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 human MPs led to long-term pulmonary engraftment and reduced alveolar protein levels by 50-70%. CT scans six months after transplantation revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities and normalisation of inspiratory to expiratory lung volume ratio.Furthermore, to genetically correct human CSF2RA-patient derived CD34+ cells, we have generated SIN-lentiviral vectors expressing a codon-optimized human CSF2RA-cDNA in combination with EGFP (Lv.EFS.CSF2RA.EGFP) from an EFS1a-promoter. BaF3 cells transduced with this vector showed stable and longterm (>3 month) expression of CSF2RA (CD116) by flow cytometry and survived in hGM-CSF dependant assays even at low concentrations of GM-CSF (5 ng/ml) confirming the formation of functional hybrid receptors of the murine GM-CSF receptor ß-chain with the transgene. Characterization of GM-CSF receptor downstream signalling revealed 5-6-fold increased STAT5 phosphorylation by Western blot in response to hGM-CSF (over control cells). Conferring this vector to CD34+ cells of CSF2RA-deficient patients yielded efficient CD116 (CSF2RA) expression, and rescued hGM-CSF dependent colony formation as well as monocytoid differentiation. Of note, clonogenic growth by G-CSF control treatment revealed no differences in colony formation or differentiation capacity when compared to uncorrected patient samples. Furthermore, healthy Lv.EFS.CSF2RA.EGFP transduced CD34+ samples, while showing robust CD116 overexpression, exhibited no aberrations in biological functions such as colony formation or in vitro differentiation towards macrophages.Thus, we here describe an innovative, cause directed and highly effective hematopoietic gene therapy approach to herPAP. Given the longevity of the transplanted MP population in our model, the strategy also may serve as a proof-of-principle to incorporate long-lived differentiated, cell sources into current hematopoietic gene therapy concepts. DisclosuresNo relevant conflicts of interest to declare.

Dexamethasone Attenuates VEGF Expression and Inflammation but Not Barrier Dysfunction in a Murine Model of Ventilator–Induced Lung Injury

BackgroundVentilator–induced lung injury (VILI) is characterized by vascular leakage and inflammatory responses eventually leading to pulmonary dysfunction. Vascular endothelial growth factor (VEGF) has been proposed to be involved in the pathogenesis of VILI. This study examines the inhibitory effect of dexamethasone on VEGF expression, inflammation and alveolar–capillary barrier dysfunction in an established murine model of VILI.MethodsHealthy male C57Bl/6 mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with an inspiratory pressure of 10 cmH2O (“lower” tidal volumes of ∼7.5 ml/kg; LVT) or 18 cmH2O (“higher” tidal volumes of ∼15 ml/kg; HVT). Dexamethasone was intravenously administered at the initiation of HVT–ventilation. Non–ventilated mice served as controls. Study endpoints included VEGF and inflammatory mediator expression in lung tissue, neutrophil and protein levels in bronchoalveolar lavage fluid, PaO2 to FiO2 ratios and lung wet to dry ratios.ResultsParticularly HVT–ventilation led to alveolar–capillary barrier dysfunction as reflected by reduced PaO2 to FiO2 ratios, elevated alveolar protein levels and increased lung wet to dry ratios. Moreover, VILI was associated with enhanced VEGF production, inflammatory mediator expression and neutrophil infiltration. Dexamethasone treatment inhibited VEGF and pro–inflammatory response in lungs of HVT–ventilated mice, without improving alveolar–capillary permeability, gas exchange and pulmonary edema formation.ConclusionsDexamethasone treatment completely abolishes ventilator–induced VEGF expression and inflammation. However, dexamethasone does not protect against alveolar–capillary barrier dysfunction in an established murine model of VILI.

Pharmacological Intervention to the Inflammatory Response from Decompression Sickness in Rats

Venous bubbles resulting from experimental decompression sickness (DCS) may cause an inflammatory-like reaction with activation of granulocytes and release of metabolites from arachidonic acid. The release of cyclooxygenase and lipoxygenase pathway mediated metabolites, namely thromboxane B2 (TXB2) and leukotriene E4 (LTE4) likely contribute to this overall DCS response. In the present study we examined the effect on DCS outcome of several agents affecting both pathways. Indomethacin and acetylsalicylic acid were administered to study the cyclooxygenase pathway mediators, Zafirlukast and Zileuton to study inhibition of the lipoxygenase pathway, and isoproterenol for its beta-agonist effects. The agents were administered to randomly selected Sprague-Dawley rats prior to compression to 683 kPa for 60 min. Following 60 min recovery post-decompression, DCS evaluation included: gross symptoms; pulmonary edema; bronchoalveolar lavage and pleural fluid protein; white blood cell and differential cell counts; and urine, bronchoalveolar lavage, and plasma TXB2 and LTE4 analysis. The results indicate that both Zafirlukast and Zileuton reduced the reported DCS symptoms, pulmonary edema, pleural and bronchoalveolar lavage protein levels, white blood cell counts in the pleural and bronchoalveolar lavage, and leukotriene levels in the bronchoalveolar lavage vs. that of vehicle-treated rats exposed to compression/decompression. The effect of these agents on pleural and bronchial alveolar protein levels demonstrated protective effects on microvascular permeability. Acetylsalicylic acid and indomethacin treatment had less effect on reducing inflammatory-induced changes. The effect of inflammatory-like responses to DCS can be altered with pharmacological intervention given prior to compression.

Response of mouse lung to crocidolite asbestos. 2. Pulmonary fibrosis after long fibres

To determine the cellular and fibrogenic responses of the lung to long asbestos fibres, mice were instilled intratracheally with 0.1 mg of a sample of long crocidolite fibres. Animals were killed at intervals to 20 weeks with 3H thymidine injected one h before death. Following bronchoalveolar lavage, an increase in polymorph neutrophils (PMN) and alveolar macrophages (AM) was found during the first week, accompanied by elevated glucosaminidase and alveolar protein levels. Although the PMN number dropped, some were always recovered by lavage to 20 weeks. Early multifocal necrosis of bronchiolar epithelium was followed by a large increase in labelling of epithelial cells and underlying fibroblasts. Epithelial overgrowth of luminal long fibres and inflammatory exudates was followed by giant cell and granuloma formation in the interstitium. After four weeks collagen levels were significantly increased and fibrosis was seen in these peribronchiolar locations. A few small fibres were observed in AM but no evidence of fibrosis was seen in alveolar walls. These findings suggest that injury to bronchial and bronchiolar epithelium allows long fibres to reach the interstitium where subsequent macrophage-fibroblast interactions result in a severe fibrotic reaction that resembles the bronchiolar component of human asbestosis.

Alveolar epithelial changes in rabbits after a 21-day exposure to 60% O2.

This study characterizes the biochemical and physiological effects of prolonged exposure of rabbits to sublethal (60%) O2 concentrations. After 3 wk in 60% O2, rabbits had arterial PO2 values of 69 +/- 2 vs. 79 +/- 3 Torr for control animals (means +/- SE; P less than 0.05) and a small but significant rise in pulmonary wet weight-to-dry weight ratios (5.6 +/- 0.3 vs. 4.1 +/- 0.3; P less than 0.05). Alveolar permeability to solute, lung compliance, total lung capacity, and alveolar protein levels were unchanged from control, but the amount of lavagable alveolar phospholipid was 90% higher in the O2-exposed rabbits. The lipid biosynthetic ability of isolated alveolar type II pneumocytes, measured by radiolabeled precursor [3H]choline incorporation, indicated that type II cells isolated from hyperoxic animals synthesized phosphatidylcholine at a rate 110% higher than those from control animals. Laser flow cytometric analyses of isolated type II cells showed a significant increase in type II cell diameter, based on time-of-flight measurements, and an average 60% increase in lipid content per cell, based on phosphine-3R fluorescence intensity. These findings indicate that exposure to 60% O2 for 21 days results in a decrease in arterial PO2 and induces several important biochemical and morphological changes in alveolar type II pneumocytes.

Effects of injury and repair of the pulmonary endothelium on lung metastasis after bleomycin

Acute endothelial injury induced by bleomycin has been shown to enhance the localization and metastasis of circulating tumour cells. In the present study we wished to determine whether increased metastases to the lung is related to the degree of endothelial damage as indicated by morphology and protein leakage to alveoli and whether the progression to repair with pulmonary fibrosis also effects metastatic tumour growth. C57b1/6 mice were injected with a single intravenous dose of bleomycin (120 mg/kg). After 5 days, severe enothelial injury was demonstrated by morphology and by increased levels of protein in lung lavage fluid. When [131I]-iododeoxyuridine labeled syngeneic fibrosarcoma cells were injected intravenously at this time, a 9-fold increase in their localization was detected 24 h later in bleomycin-treated lungs compared with saline controls. By electron microscopy tumour cells were observed at sites of denuded vascular basement membrane. There was also a significant increase in the number of gross metastases which developed subsequently and in the percentage of lung occupied by tumour in the bleomycin group. Animals examined 10 days after bleomycin showed less endothelial damage and a smaller increase in tumour cell localization and metastases. At 21 days, when endothelial structure and alveolar protein levels had returned to normal, and at 6 weeks, when there was focal fibrosis, no increase in tumour cell localization or metastases was found. It is concluded that damage to the pulmonary endothelium is a key factor in enhancing the trapping of circulating tumour cells and increasing metastatic tumour growth after bleomycin.

Responses of mouse lung to irradiation: 2. Levels of alveolar protein in lung lavage fluid following neutrons or X-rays

Alveolar protein exudate is shown to exhibit a biphasic increase after irradiation. During the first 6 weeks after doses of between 5 and 28 Gy X-rays or 3.5 and 14 Gy neutrons, mean alveolar protein levels increase to about 4 times control levels. Over this period there was no suggestion of any dose-response nor of an RBE greater than 1. It seems probable that this response is unrelated to cell killing by radiation. After sublethal doses there was recovery from this first phase of leakage. However, although after 5 Gy X-rays this recovery was complete, after higher doses a second and greater increase in alveolar protein took place. After 10 Gy X-rays and 8 Gy neutrons this peaked at about 15 weeks post-irradiation and 20-30 times control levels. This second dose-dependent response corresponded with the times of animal death after doses close to the LD50. A comparison of these changes in alveolar protein exudate with previously published data on surfactant level suggests that the surfactant and protein responses are largely unrelated.