Mechanisms Of SC5b-9-induced Pulmonary Microvascular Permeability Dysfunction

Continuous exposure of animals to oxygen at pressures ranging from more than 60²00 kPa causes progressive lung injury, that is pulmonary oxygen toxicity. The pathologic effects of pulmonary oxygen toxicity include destruction of both capillary endothelium and alveolar epithelium, alveolar cell hyperplasia, edema, hemorrhage, arteriolar thickening and hyalinization, fibrin formation, atelectasis, and consolidation with severe impairment of gas exchange, hypoxemia and death. Alternating hyperoxic and normoxic exposure periods to extension of pulmonary oxygen tolerance in man is applicated in hyerbaric oxygen therapy.Oxygen toxicity has been studied extensively for the past several decades. It is generally accepted that oxygen toxicity is caused by oxidative stress, direct oxidative effect of oxygen and free radical damage to membranes. Peroxidation of membrane unsaturated fatty acids, oxidation of structural proteins and inactivation of membrane-bound enzymes can increase membrane permeability and eliminate transmembrane ion gradients with the loss of secretory and other important membrane functions. While pulmonary oxygen toxicity also has typical inflammation changes, which suggests that inflammation plays vital role in the mechanisms of pulmonary oxygen toxicity.The hypothesis that inflammation contribute to the development of pulmonary oxygen toxicity is supported primarily by observations made in animals during multiday exposures to O2 at 101 kPa (1.0 ATA). In rats exposed for more than 2 days, neutrophils accumulate within the pulmonary vasculature, frequently adhering to capillary endothelial cells, and are also more numerous within the lung interstitium. The accumulation of neutrophils in the lungs was associated with rapid exacerbation of lung damage and, conversely, the pathologic effects of oxygen exposure were decreased by prior systemic depletion of neutrophils.Activated neutrophils can release into the surrounding medium a variety of reactive species including super-oxide, hydrogen peroxide, hydroxyl radical, hypochlorous acid, and peroxynitrite.In addition, as a key mediator of inflammation, complement also significantly contributes to tissue damage in various clinical disorders, including systemic inflammatory response syndrome (SIRS) and acute respiratory distress syndrome (ARDS), septic shock, trauma, burns, acid aspiration to the lungs. In principle, when inflammation is involved in the pathogenesis, complement has to be considered as a possible mediator in the disease process. Since blood levels of vitronectin-containing complement complexes SC5b-9 increase in ARDS, the ligation of the endothelial luminal vβ₃ integrin by these complexes increases lung endothelial liquid conductance, which is competitively bloked by RGD containing peptide GRGDSP or anti- vβ₃ monoclonal antibody LM609. Recently, it was reported that both C3a and C5a could increase vascular permeability by activating their specific receptors. These results suggest that multielements of complement system contribute to vascular permeability dysfunction by activating specific reptors.To investigate the mechanisms of pulmonary oxygen toxicity, gene expression studies were conducted to identify the gene exression patterns underlying hyperoxic lung injury. The gene expression ratio ofβ₃ integrin went up significantly with hyperoxic injury, which suggested thatβ₃ integrin may contribute to development of pulmonary oxygen toxicity.We postulated that hyperbaric oxygen exposure may activate complement system, leading to the formation of vitronectin-containing complement complex, SC5b-9. The ligation of the endothelial luminal vβ₃ integrin by these complexes induces cytoskeletal reorganization and further evokes an increae in vascular permeability. To prove our postulation, we performed the following experiments. 1. Microarray analysis of pulmonary oxygen toxicityTo evaluate the molecular and cellular bases of pulmonary oxygen toxicity, changes in gene expression in the c57BL/6 mouse lung after polonged hyperbaric oxygen exposure were investigated. GeneSpring software was used to analysis the data aquired.2. Detection of complement level and pulmonary vascular permeability in oxygen toxicity rat(1) CH50 was used to measure the complement level in rat;(2) Evans Blue Assay was used to detect pulmonary vascular permeability.3. Establishment of SC5b-9-induced endothelial monolayer permeability dysfunction model(1) Purification and identification of SC5b-9;(2) Rat pulmonary microvascular endothelial cell culture and identification;(3) Detection of SC5b-9-induced endothelial monolayer permeability by Evans Blue albumin assay.4. Effects of varying concentrations of SC5b-9 on cytoskeletal changes in pulmonary microvascular endothelial cells.5. Effects of Rho-kinase inhibitor Y-27632 on SC5b-9-induced cytoskeletal changes in pulmonary microvascular endothelial cells.The main resuls are as fllow:1. Microarray analysis of 200 kPa hyperbaric oxygen exposure mice lung: We choose a relatively high threshold (a ratio of 3.0) to identify hyperoxia responsive genes . Using this criteria, 69 genes up-regulated, and 229 genes down-regulated. The highest up-regulated gene isβ₃ integrin, which suggest that it may play an important role in the mechanisms of pulmonary oxygen toxicity.2. CH50 tests showed that the complement levels in 5-hour hyperoxia exposed rats were significantly elevated (P<0.05). Hyperpermeability in hyperoxia exposed rat lungs were identified by Evans Blue Assay (P<0.01).3. We established a in vitro model of SC5b-9-induced endothelial monolayer permeability dyfunction. Endothelial permeability was significantly increased by SC5b-9 in a time- and concentration-dependent manner and was accompanied by changes in the F-actin cytoskeleton. Endothelial monolayer stimulated by 50μg/mL SC5b-9 for just 1 hour resulted in barrier changes. Endothelial monolayer stimulated by 200μg/mL SC5b-9 showed both permeability dysfunction and prominent structure damage. While, Y-27632 showed permeability promoting effect instead of brrier protecting effect.4. SC5b-9 could induce significant cytoskeletal changes. Y-27632 did not show inhibiting effect on permeability dysfunction.Our results showed that hyperbaric oxygen exposure could activate complement system, leading to the formation of vitronectin-containing complement complex, SC5b-9. The ligation of the endothelial luminal vβ₃ integrin by these complexes induces cytoskeletal reorganization and further evokes an increae in vascular permeability. The involvement of vβ₃ integrin and complement activation provides a new area for finding therapeutic measure for pulmonary oxygen toxicity.