American Journal of Physiology - Lung Cellular and Molecular Physiology

Ventilator-induced lung injury (VILI) is an inflammatory process that can be attenuated by lung protective ventilation strategies. Our objectives to further investigate the pathogenesis of ALI and VILI and the mechanism of lung protection in these syndromes were: 1) to determine if plasma measurements of soluble TNF receptor I (sTNFRI) and II (sTNFRII) would predict the development of ALI and mortality in a small single center trial; 2) to test the predictive value of these markers and of TNF-α in a larger, broader group of patients with ALI; 3) to test the hypothesis that low tidal volume ventilation (LTVV) would be associated with a decrease in plasma levels of TNF-α, sTNFRI, and sTNFRII. In the single center study, sTNFRI and II levels were higher in patients at risk for and with ALI, but they did not predict the development of the syndrome. In the multicenter trial sTNFRI and II were strongly associated with mortality (OR 5.76/1 log10 increment in receptor level; 95% CI 2.63–12.6 and OR 2.58; 95% CI 1.05–6.31, respectively) and morbidity measured as fewer nonpulmonary organ failure-free and ventilator-free days. The LTVV strategy was associated with an attenuation of plasma sTNFRI levels. In vitro, stimulated A549 cells release sTNFRI but not sTNRFII. In conclusion, plasma levels of sTNFRI and II can serve as biomarkers for morbidity and mortality in patients with ALI. Furthermore, LTVV is associated with a specific decrease in sTNFRI levels. This suggests that one beneficial effect of LTVV may be to attenuate alveolar epithelial injury.

An in vivo rat model was used to evaluate the effects of Escherichia coli pneumonia on lung function and surfactant in bronchoalveolar lavage (BAL). Total extracellular surfactant was increased in infected rats compared with controls. BAL phospholipid content in infected rats correlated with the severity of alveolar-capillary leak as reflected in lavage protein levels ( R²= 0.908, P < 0.0001). Western blotting showed that levels of surfactant protein (SP)-A and SP-D in BAL were significantly increased in both large and small aggregate fractions at 2 and 6 h postinstillation of E. coli. SP-B was also increased at these times in the large aggregate fraction of BAL, whereas SP-C levels were increased at 2 h and decreased at 6 h relative to controls. The small-to-large (S/L) aggregate ratio (a marker inversely proportional to surfactant function) was increased in infected rats with >50 mg total BAL protein. There was a significant correlation ( R²= 0.885, P < 0.0001) between increasing S/L ratio in BAL and pulmonary damage assessed by total protein. Pulmonary volumes, compliance, and oxygen exchange were significantly decreased in infected rats with >50 mg of total BAL protein, consistent with surfactant dysfunction. In vitro surface cycling studies with calf lung surfactant extract suggested that bacterially derived factors may have contributed in part to the surfactant alterations seen in vivo.

Heme oxygenase (HO)-1 catalyzes the oxidative cleavage of heme to yield equimolar amounts of biliverdin, iron, and carbon monoxide. HO-1 is a stress response protein, the induction of which is associated with protection against oxidative stress. The mechanism(s) of protection is not completely elucidated, although it is suggested that one or more of the catalytic by-products provide antioxidant functions either directly or indirectly. The involvement of reactive oxygen species in apoptosis raised the question of a possible role for HO-1 in programmed cell death. Using the tetracycline-regulated expression system, we show here that conditional overexpression of HO-1 prevents tumor necrosis factor-α-induced apoptosis in murine L929 fibroblasts. Inhibition of apoptosis was not observed in the presence of tin protoporphyrin, a specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. Interestingly, exogenous administration of a low concentration of carbon monoxide also prevented tumor necrosis factor-α-induced apoptosis in L929 fibroblasts. Inhibition of tumor necrosis factor-α-induced apoptosis by HO-1 overexpression was reversed by 1 H-(1,2,4)oxadiazolo(4,3- a)quinoxalin-1-one, an inhibitor of guanylate cyclase, which is a target enzyme for carbon monoxide. Taken together, our data suggest that the antiapoptotic effect of HO-1 may be mediated via carbon monoxide.

The discovery of the gaseous molecule nitric oxide in 1987 unraveled investigations on its functional role in the pathogenesis of a wide spectrum of biological and pathological processes. At that time, the novel concept that an endogenous production of a gaseous substance such as nitric oxide can impart such diverse and potent cellular effects proved to be very fruitful in enhancing our understanding of many disease processes including lung disorders. Interestingly, we have known for a longer period of time that there exists another gaseous molecule that is also generated endogenously; the heme oxygenase (HO) enzyme system generates the majority if not all of the endogenously produced carbon monoxide. This enzyme system also liberates two other by-products, bilirubin and ferritin, each possessing important biological functions and helping to define the uniqueness of the HO enzyme system. In recent years, interest in HO has emerged in numerous disciplines including the central nervous system, cardiovascular physiology, renal and hepatic systems, and transplantation. We review the functional role of HO in lung biology and its real potential application to lung diseases.

Mặc dù nhiều bệnh lý phổi liên quan đến tình trạng thiếu oxy, sự suy giảm tế bào biểu mô loại II phế nang (ATII), và rối loạn chức năng hoạt chất bề mặt phổi, tác động của sự hạn chế O₂ đến các con đường chuyển hóa cần thiết để duy trì năng lượng của tế bào trong các tế bào ATII vẫn chưa được nghiên cứu kỹ lưỡng. Bài báo này trình bày kết quả của các thử nghiệm có mục tiêu nhằm xác định các quá trình chuyển hóa cụ thể góp phần vào cân bằng năng lượng sử dụng các tế bào ATII nguyên phát và một dòng tế bào ATII mẫu, tế bào biểu mô phổi chuột 15 (MLE-15), được nuôi cấy trong điều kiện bình thường (normoxic) và thiếu oxy (hypoxic). Các tế bào MLE nuôi cấy trong điều kiện bình thường thể hiện tỷ lệ tiêu thụ O₂ mạnh mẽ (OCR) kèm theo sự sản xuất ATP và sản xuất lactate ngoại bào hạn chế, cho thấy sự phụ thuộc vào phosphoryl hóa oxy hóa để sản xuất ATP. Việc không gắn kết dược lý của quá trình hô hấp đã làm tăng OCR trong các nền văn hóa bình thường lên 175% so với mức cơ bản, cho thấy khả năng dự trữ hô hấp đáng kể. Tuy nhiên, khi tiếp xúc với điều kiện thiếu oxy trong 20 giờ, mức tiêu thụ O₂ cơ bản giảm còn 60% so với tỷ lệ bình thường, và các tế bào chỉ duy trì khoảng 50% khả năng dự trữ hô hấp bình thường, cho thấy sự ức chế chức năng ty thể, mặc dù mức ATP nội bào vẫn giữ gần mức bình thường. Hơn nữa, trong khi sự tiếp xúc với điều kiện thiếu oxy kích thích tổng hợp và lưu trữ glycogen trong MLE-15, tốc độ glycolysis (như được đo bằng sản xuất lactate) không được tăng đáng kể trong các tế bào, mặc dù có sự tăng cường biểu hiện của một số enzyme liên quan đến glycolysis. Những kết quả này chủ yếu được tái hiện trong các tế bào ATII nguyên phát của chuột, chứng minh rằng MLE-15 phù hợp để mô hình hóa chuyển hóa ATII. Khả năng của các tế bào ATII duy trì mức ATP trong điều kiện thiếu oxy mà không tăng cường glycolysis cho thấy rằng các tế bào này cực kỳ hiệu quả trong việc bảo tồn ATP để giữ cân bằng năng lượng sinh học trong điều kiện hạn chế O₂.

Bạch cầu đa nhân trung tính (PMN) đóng vai trò trung gian gây tổn thương cấp tính ở phổi do thiếu máu/tái tưới máu (I/R) vẫn tiếp tục là nguyên nhân chính gây tử vong trong y học chăm sóc đặc biệt. Trong nghiên cứu này, chúng tôi báo cáo rằng việc hít oxit carbon (CO) liều thấp và tiêm tĩnh mạch resolvin D1 (RvD1) ở chuột có thể làm giảm tổn thương phổi cấp tính do PMN gây ra từ I/R. Việc hít CO (125–250 ppm) và RvD1 (250–500 ng) đều làm giảm sự thâm nhập của PMN vào phổi và cải thiện bảo vệ phổi. Trong mẫu máu toàn phần của chuột, CO và RvD1 giảm mạnh tổng hợp PMN-tiểu cầu, từ đó giảm leukotrien (LTs) và thromboxan B₂(TxB₂) trong phổi chịu tác động I/R. Với mẫu máu toàn phần của người, CO (125–250 ppm) làm giảm tổng hợp PMN-tiểu cầu, sự biểu hiện của phân tử bám dính, cytokine LTs cũng như TxB₂. RvD1 (1–100 nM) cũng giảm phụ thuộc liều tổng hợp PMN-tiểu cầu kích thích bởi yếu tố hoạt hóa tiểu cầu trong mẫu máu người. Trong nghiên cứu trên động vật không phải người (khỉ đầu chó) bị nhiễm Streptococcus pneumoniae ở phổi, CO hít vào làm giảm đáng kể LTs cysteinyl trong nước tiểu. Những kết quả này chứng minh việc bảo vệ phổi bằng CO hít liều thấp cũng như RvD1 giảm tổn thương mô cấp tính bởi PMN, tương tác PMN-tiểu cầu và sản xuất LTs cysteinyl và TxB₂. Cùng nhau, chúng gợi ý một vai trò điều trị tiềm năng của CO hít liều thấp trong bảo vệ cơ quan, như đã chứng minh qua các mô hình tổn thương phổi khởi tạo I/R trên chuột, nhiễm trùng khỉ đầu chó và mẫu máu toàn phần của người.

The systemic vasculature in and surrounding the lung is proangiogenic, whereas the pulmonary vasculature rarely participates in neovascularization. We studied the effects of the proangiogenic ELR+ CXC chemokine MIP-2 (macrophage inflammatory protein-2) on endothelial cell proliferation and chemotaxis. Mouse aortic, pulmonary arterial, and lung microvascular endothelial cells were isolated and subcultured. Proliferation ([³H]thymidine uptake) and migration (Transwell chemotaxis) were evaluated in each cell type at baseline and upon exposure to MIP-2 (1–100 ng/ml) without and with exposure to hypoxia (24 h)-reoxygenation. Baseline proliferation did not vary among cell types, and all cells showed increased proliferation after MIP-2. Aortic cell chemotaxis increased markedly upon exposure to MIP-2; however, neither pulmonary artery nor lung microvascular endothelial cells responded to this chemokine. Assessment of CXCR₂, the G protein-coupled receptor through which MIP-2 signals, displayed no baseline difference in mRNA, protein, or cell surface expression among cell types. Exposure to hypoxia increased expression of CXCR₂of aortic endothelial cells only. Additionally, aortic cells, compared with pulmonary cells, showed significantly greater protein and activity of cathepsin S, a proteolytic enzyme important for cell motility. Thus the combined effects of increased cathepsin S activity, providing increased motility and enhanced CXCR₂expression after hypoxia, both contribute to the proangiogenic phenotype of systemic arterial endothelial cells.

We tested the hypothesis that tumor necrosis factor (TNF)-α induces a peroxynitrite (ONOO⁻)-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated β-actin (NO₂-β-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. β-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted β-actin was quantified in terms of its nitrotyrosine/β-actin ratio with antinitrotyrosine and anti-β-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO₂-β-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with β-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/β-actin ratio at 0.5 h with minimal association of the NO₂-β-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/β-actin ratio and permeability were prevented by the anti-ONOO⁻ agent Urate. The data indicate that TNF induces an ONOO⁻-dependent barrier dysfunction, which is associated with the generation of NO₂-β-actin.

Lung barrier dysfunction is a cardinal feature of the acute respiratory distress syndrome (ARDS). Alcohol abuse, which increases the risk of ARDS two- to fourfold, induces transforming growth factor (TGF)-β1, which increases epithelial permeability and impairs granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent barrier integrity in experimental models. We hypothesized that the relative balance of GM-CSF and TGF-β1 signaling regulates lung epithelial barrier function. GM-CSF and TGF-β1 were tested separately and simultaneously for their effects on lung epithelial cell barrier function in vitro. TGF-β1 alone caused an ∼25% decrease in transepithelial resistance (TER), increased paracellular flux, and was associated with projections perpendicular to tight junctions (“spikes”) containing claudin-18 that colocalized with F-actin. In contrast, GM-CSF treatment induced an ∼20% increase in TER, decreased paracellular flux, and showed decreased colocalization of spike-associated claudin-18 with F-actin. When simultaneously administered to lung epithelial cells, GM-CSF antagonized the effects of TGF-β1 on epithelial barrier function in cultured cells. Given this, GM-CSF and TGF-β1 levels were measured in bronchoalveolar lavage (BAL) fluid from patients with ventilator-associated pneumonia and correlated with markers for pulmonary edema and patient outcome. In patient BAL fluid, protein markers of lung barrier dysfunction, serum α2-macroglobulin, and IgM levels were increased at lower ratios of GM-CSF/TGF-β1. Critically, patients who survived had significantly higher GM-CSF/TGF-β1 ratios than nonsurviving patients. This study provides experimental and clinical evidence that the relative balance between GM-CSF and TGF-β1 signaling is a key regulator of lung epithelial barrier function. The GM-CSF/TGF-β1 ratio in BAL fluid may provide a concentration-independent biomarker that can predict patient outcomes in ARDS.

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.