SC5b-9介导肺微血管内皮通透性障碍及其机制
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摘要
机体长时间吸入绝对压相对较低(60～200 kPa)的高压氧可引起肺损害,即肺型氧中毒。主要病理表现为:肺泡上皮细胞受损,肺微血管内皮通透性增加,蛋白质渗出,肺水肿、肺不张和肺出血,最终导致气体交换障碍而危及生命。高压氧的毒性使它在潜水作业领域和高压氧临床治疗当中的应用受到很大限制,如大深度饱和潜水过程中,为避免发生肺型氧中毒,必须采取措施严格控制氧分压,大大增加了潜水作业的难度;高压氧治疗时必须采用间歇吸氧法,使吸氧效率显著降低。关于肺型氧中毒的发病机制,目前普遍认为是氧化应激、高压氧的直接毒性作用和生物膜受损等,使肺毛细血管通透性增加,肺泡上皮细胞破坏,肺泡表面活性物质减少,肺表面张力增高,造成组织水肿和肺不张。但是,肺型氧中毒具有肺部炎症反应的典型特征,炎症反应在其发病过程中的作用不容忽视。研究表明,肺型氧中毒的发生不仅有氧化应激反应,内皮细胞和上皮细胞的破坏、细胞凋亡等变化,还有炎症细胞的聚集、凝血和纤溶机制的紊乱等诸多因素的参与,各种因素的消长决定了病理过程的发生、发展和转归。
由于补体系统在炎症反应中发挥重要作用,补体可能参与肺型氧中毒的发生。有研究发现,补体复合物沉积于肺泡腔引起的补体活化会触发一系列反应导致非常严重的肺实质损害。血管内注射补体活化血清或C5活化产物均可直接引起急性肺损伤。急性呼吸窘迫综合征时补体活化,可溶性补体复合物SC5b-9与内皮细胞管腔面的αvβ3整合素结合,可能是导致肺微血管内皮屏障功能下降和肺水肿的机制之一。近来的研究表明,补体成分C3a和C5a可以分别通过结合C3aR和C5aR引起人脐静脉内皮通透性增加。离体肺血管研究也发现,SC5b-9特异性结合αvβ3可能激活酪氨酸磷酸化途径,进而导致内皮细胞骨架重组和血管通透性增高。含有RGD (Arg-Gly-Asp)序列的六肽GRGDSP可以竞争性抑制SC5b-9与αvβ3的结合,减轻微血管内皮屏障的破坏;αvβ3的单克隆抗体LM609也可以显著抑制SC5b-9引起的内皮通透性增加。由此可见,多种补体成分可以通过特异性受体介导的途径引起肺血管内皮损伤。
为探索肺型氧中毒的发生机制,本课题首先采用高通量寡核苷酸芯片技术筛选肺型氧中毒小鼠肺组织基因表达谱,发现β3整合素基因显著上调,提示β3整合素在肺型氧中毒发生机制中可能发挥重要作用。
我们推测,高压氧暴露可能激活补体系统,由可溶性补体复合物SC5b-9特异性结合αvβ3整合素,引起内皮细胞骨架重组,进而导致肺微血管内皮通透性增加。为证明上述推论,本课题进行了以下研究:
1.肺型氧中毒的基因表达谱研究
制备c57BL/6小鼠肺型氧中毒模型,采用寡核苷酸芯片技术获取200 kPa纯氧暴露5 h的c57BL/6小鼠肺组织基因表达谱,以GeneSpring软件分析表达谱数据。
2.肺型氧中毒大鼠体内补体活化水平及肺血管通透性检测
(1)CH50法检测肺型氧中毒大鼠体内总补体活性;
(2)伊文思蓝染色法检测肺型氧中毒大鼠肺血管通透性。
3.建立补体复合物SC5b-9造成大鼠肺微血管内皮屏障功能破坏的体外模型
(1)大鼠血清补体复合物SC5b-9的提取、纯化及鉴定;
(2)大鼠肺微血管内皮细胞的体外培养及鉴定;
(3)伊文思蓝标记白蛋白法检测不同浓度(50μg/ml, 100μg/ml, 150μg/ml, 200μg/ml)SC5b-9对肺微血管内皮细胞单层通透性影响的变化特点。
4.补体复合物SC5b-9对肺微血管内皮细胞应力纤维形成和分布的影响以不同浓度SC5b-9刺激肺微血管内皮细胞,观察应力纤维变化特点。
5. Rho激酶抑制剂Y-27632对SC5b-9诱导肺微血管内皮细胞骨架重组的影响以Y-27632阻断Rho激酶,观察SC5b-9刺激肺微血管内皮细胞后应力纤维变化特点。
主要研究结果如下:
1.获得200 kPa纯氧暴露5 h的c57BL/6小鼠肺组织基因表达谱。表达谱数据经GeneSpring软件分析,共计获得表达上调3倍以上的基因69个,表达下调3倍以上的基因229个,在表达上调基因中,以β3整合素基因最为显著(上调7.49倍),这一结果提示β3整合素在肺型氧中毒发生机制中可能发挥重要作用。
2. CH50检测表明高压氧暴露5 h大鼠体内补体活性显著增高,伊文思蓝染色法结果提示高压氧暴露大鼠肺血管通透性增加。
3.建立了补体复合物SC5b-9造成大鼠肺微血管内皮屏障功能破坏的体外模型。SC5b-9对微血管内皮细胞单层通透性的影响具有显著的时间、浓度依赖性,50μg/ml SC5b-9刺激1 h即可引起通透性的改变,200μg/ml SC5b-9不仅可以引起内皮通透性的明显增加,而且可以观察到内皮细胞单层的明显破坏;令人感兴趣的是, Rho激酶抑制剂Y-27632不仅不能阻断SC5b-9对大鼠肺微血管内皮屏障功能的破坏,反而可以促进内皮通透性的增加。
4. SC5b-9可以引起肺微血管内皮细胞骨架重组,Rho激酶阻断剂Y-27632不能阻止SC5b-9诱导的内皮通透性增加。
本课题提示,高压氧暴露可以激活补体系统,产生的可溶性补体复合物SC5b-9通过结合αvβ3整合素引起内皮细胞骨架重构,形成应力纤维,应力纤维将导致内皮细胞收缩,使内皮通透性增加,即补体活化产物SC5b-9通过特异性受体途径介导的肺血管内皮通透性增加是高压氧暴露引起肺微血管内皮通透性障碍的原因之一。αvβ3整合素和补体系统的参与为寻找防治肺型氧中毒的有效手段提供了新的思路。
Continuous exposure of animals to oxygen at pressures ranging from more than 60~200 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 vβ_3 integrin by these complexes increases lung endothelial liquid conductance, which is competitively bloked by RGD containing peptide GRGDSP or anti- vβ_3 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 ofβ_3 integrin went up significantly with hyperoxic injury, which suggested thatβ_3 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 vβ_3 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 toxicity
To 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 isβ_3 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 vβ_3 integrin by these complexes induces cytoskeletal reorganization and further evokes an increae in vascular permeability. The involvement of vβ_3 integrin and complement activation provides a new area for finding therapeutic measure for pulmonary oxygen toxicity.
由于补体系统在炎症反应中发挥重要作用,补体可能参与肺型氧中毒的发生。有研究发现,补体复合物沉积于肺泡腔引起的补体活化会触发一系列反应导致非常严重的肺实质损害。血管内注射补体活化血清或C5活化产物均可直接引起急性肺损伤。急性呼吸窘迫综合征时补体活化,可溶性补体复合物SC5b-9与内皮细胞管腔面的αvβ3整合素结合,可能是导致肺微血管内皮屏障功能下降和肺水肿的机制之一。近来的研究表明,补体成分C3a和C5a可以分别通过结合C3aR和C5aR引起人脐静脉内皮通透性增加。离体肺血管研究也发现,SC5b-9特异性结合αvβ3可能激活酪氨酸磷酸化途径,进而导致内皮细胞骨架重组和血管通透性增高。含有RGD (Arg-Gly-Asp)序列的六肽GRGDSP可以竞争性抑制SC5b-9与αvβ3的结合,减轻微血管内皮屏障的破坏;αvβ3的单克隆抗体LM609也可以显著抑制SC5b-9引起的内皮通透性增加。由此可见,多种补体成分可以通过特异性受体介导的途径引起肺血管内皮损伤。
为探索肺型氧中毒的发生机制,本课题首先采用高通量寡核苷酸芯片技术筛选肺型氧中毒小鼠肺组织基因表达谱,发现β3整合素基因显著上调,提示β3整合素在肺型氧中毒发生机制中可能发挥重要作用。
我们推测,高压氧暴露可能激活补体系统,由可溶性补体复合物SC5b-9特异性结合αvβ3整合素,引起内皮细胞骨架重组,进而导致肺微血管内皮通透性增加。为证明上述推论,本课题进行了以下研究:
1.肺型氧中毒的基因表达谱研究
制备c57BL/6小鼠肺型氧中毒模型,采用寡核苷酸芯片技术获取200 kPa纯氧暴露5 h的c57BL/6小鼠肺组织基因表达谱,以GeneSpring软件分析表达谱数据。
2.肺型氧中毒大鼠体内补体活化水平及肺血管通透性检测
(1)CH50法检测肺型氧中毒大鼠体内总补体活性;
(2)伊文思蓝染色法检测肺型氧中毒大鼠肺血管通透性。
3.建立补体复合物SC5b-9造成大鼠肺微血管内皮屏障功能破坏的体外模型
(1)大鼠血清补体复合物SC5b-9的提取、纯化及鉴定;
(2)大鼠肺微血管内皮细胞的体外培养及鉴定;
(3)伊文思蓝标记白蛋白法检测不同浓度(50μg/ml, 100μg/ml, 150μg/ml, 200μg/ml)SC5b-9对肺微血管内皮细胞单层通透性影响的变化特点。
4.补体复合物SC5b-9对肺微血管内皮细胞应力纤维形成和分布的影响以不同浓度SC5b-9刺激肺微血管内皮细胞,观察应力纤维变化特点。
5. Rho激酶抑制剂Y-27632对SC5b-9诱导肺微血管内皮细胞骨架重组的影响以Y-27632阻断Rho激酶,观察SC5b-9刺激肺微血管内皮细胞后应力纤维变化特点。
主要研究结果如下:
1.获得200 kPa纯氧暴露5 h的c57BL/6小鼠肺组织基因表达谱。表达谱数据经GeneSpring软件分析,共计获得表达上调3倍以上的基因69个,表达下调3倍以上的基因229个,在表达上调基因中,以β3整合素基因最为显著(上调7.49倍),这一结果提示β3整合素在肺型氧中毒发生机制中可能发挥重要作用。
2. CH50检测表明高压氧暴露5 h大鼠体内补体活性显著增高,伊文思蓝染色法结果提示高压氧暴露大鼠肺血管通透性增加。
3.建立了补体复合物SC5b-9造成大鼠肺微血管内皮屏障功能破坏的体外模型。SC5b-9对微血管内皮细胞单层通透性的影响具有显著的时间、浓度依赖性,50μg/ml SC5b-9刺激1 h即可引起通透性的改变,200μg/ml SC5b-9不仅可以引起内皮通透性的明显增加,而且可以观察到内皮细胞单层的明显破坏;令人感兴趣的是, Rho激酶抑制剂Y-27632不仅不能阻断SC5b-9对大鼠肺微血管内皮屏障功能的破坏,反而可以促进内皮通透性的增加。
4. SC5b-9可以引起肺微血管内皮细胞骨架重组,Rho激酶阻断剂Y-27632不能阻止SC5b-9诱导的内皮通透性增加。
本课题提示,高压氧暴露可以激活补体系统,产生的可溶性补体复合物SC5b-9通过结合αvβ3整合素引起内皮细胞骨架重构,形成应力纤维,应力纤维将导致内皮细胞收缩,使内皮通透性增加,即补体活化产物SC5b-9通过特异性受体途径介导的肺血管内皮通透性增加是高压氧暴露引起肺微血管内皮通透性障碍的原因之一。αvβ3整合素和补体系统的参与为寻找防治肺型氧中毒的有效手段提供了新的思路。
Continuous exposure of animals to oxygen at pressures ranging from more than 60~200 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 vβ_3 integrin by these complexes increases lung endothelial liquid conductance, which is competitively bloked by RGD containing peptide GRGDSP or anti- vβ_3 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 ofβ_3 integrin went up significantly with hyperoxic injury, which suggested thatβ_3 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 vβ_3 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 toxicity
To 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 isβ_3 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 vβ_3 integrin by these complexes induces cytoskeletal reorganization and further evokes an increae in vascular permeability. The involvement of vβ_3 integrin and complement activation provides a new area for finding therapeutic measure for pulmonary oxygen toxicity.
引文
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