急性肺损伤肺微血管内皮细胞间隙连接蛋白40与血管通透性的关系研究
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摘要
为探索急性肺损伤(ALI)时肺血管内皮细胞间隙连接蛋白40(Cx40)的表达,连接通道(GJC)功能的状态改变,细胞损伤以及细胞内钙浓度的变化与内皮细胞通透性之间的关系,本研究进行了下列三部分实验。
实验一.急性肺损伤兔肺组织通透性改变与Cx40的表达研究
目的:观察ALI时家兔肺组织通透性与微血管内皮细胞Cx40表达的关系
方法:应用胸部战伤方法建立家兔急性肺损伤模型,将伤后经过简单救治存活下来的家兔随机分为6组,每组6例,每例动物在伤前、伤后6、12、24、48,72小时取动脉血检测IL-8和TNF-α含量;于各时段分别处死动物,取肺组织做HE病理和免疫组织化学染色(Cx40);伊文思蓝漏出率法测肺微血管通透性;干湿比法测肺的含水量。
结果:HE染色见伤肺肺泡腔内大量渗出液体和红细胞的渗出聚集,肺泡隔增厚、断裂、融合,大量白细胞渗出、聚集,毛细血管扩张充血。对侧肺组织肺泡腔内液体渗出、红细胞聚集,肺泡隔内白血病浸润,毛细血管扩张充血。伤后12小时、24小时、48小时和72小时血清中TNF-α和IL-8含量均高于致伤前(p<0.05)。伤后24小时以后各时间点,肺组织含水量均有明显升高(p<0.05),持续至72小时。肺组织伊文思蓝漏出率随着时间的发展升高(p<0.05)。血管内皮细胞膜Cx40染色较伤前减弱,OD值显著减小(p<0.05),并与伊文思蓝漏出率呈负相关(γ=-0.934, P<0.05,γ2=0.8723)。
实验二.急性肺损伤兔肺微血管内皮细胞通透性改变与Cx40的关系研究
目的:观察离体培养的兔肺微血管内皮细胞通透性改变与Cx40表达、GJC功能之间的关系。
方法:组织块贴壁法分离、培养兔肺微血管内皮细胞。细胞分为三组,对照组、创伤血清处理组和阻滞剂组。在阻滞剂组,细胞间隙连接通道阻滞剂1-庚醇(0.5 mM)加入到培养液中。1小时后,阻滞剂组和创伤血清组的20%胎牛血清的培养液均被更换为含有20%创伤血清的培养液。对照组则只更换培养液。三组细胞继续孵育6小时后,进入实验观察。分别做划痕实验、Cx40蛋白免疫印迹和单层细胞通透性检测。
结果:培养的内皮细胞融合成单层,呈典型的铺路石样镶嵌状排列。Cx40蛋白印迹见创伤血清组与阻滞剂组蛋白表达水平较对照组有明显降低,且两组之间亦不同,阻滞剂组蛋白水平较创伤血清组低(对照组为1±0.07,创伤血清组为0.55±0.15,阻滞剂组为0.43±0.13,n=4,p<0.05)。三组细胞划痕边缘的细胞均被染料LY染色,LY在对照组细胞扩散得最远,显色的周边细胞的数量最多,创伤血清组次之,阻滞剂组显色的细胞数量最少。相邻细胞与边缘细胞的比值的差异具有统计学意义(23±5,11±3 ver 7±2,n=4,p<0.05)。三组细胞伊文思蓝清除率均随着时间的延长而增加,且三组细胞间亦有不同,阻滞剂组清除率高于创伤血清组和对照组。组间差别和时间点差别均有显著性差异。
实验三急性肺损伤兔肺微血管内皮细胞Cx40对细胞内Ca~(++)浓度的影响研究
目的:观察急性肺损伤时离体培养的兔肺微血管内皮细胞Cx40与内皮细胞损伤、细胞内Ca~(++)浓度变化之间的关系。
方法:兔肺微血管内皮细胞的培养和分组同实验二。三组细胞继续孵育6小时后,提取培养液测定LDH含量,加入fluo-3AM负载检测细胞内Ca~(++)钙浓度。
结果:通过共聚焦显微镜的观察,三组细胞荧光强度不相同,阻滞剂组荧光最强,对照组最弱。创伤血清组和阻滞剂组细胞内Ca~(++)浓度均显著高于对照组,(nmol/L, 494.8±41.94, 569.8±6.70 versus 158.8±13.09, p<0.05)。创伤血清组和阻滞剂组细胞培养液中LDH含量均显著高于对照组(IU/L, 165.88±16.17, 186.4±24.91 versus 51.31±13.27, p<0.05),且血清组与阻滞剂组之间的差别亦有统计学意义。此外,三组细胞内Ca~(++)含量与培养液中LDH的浓度还存在正相关关系(γ=0.997, p< 0.05,γ2=0.994)。
结论:
1.在动物实验中,肺组织通透性升高与肺组织微血管内皮细胞Cx40表达呈负相关关系。在离体培养的肺微血管内皮细胞,富含炎症介质的创伤血清能够抑制Cx40的水平和GJC的功能,与此同时肺血管内皮细胞通透性升高,二者存在负相关关系,Cx40水平和GJC功能的下降能够明显增加肺微血管内皮细胞的通透性。
2.创伤因素所致的Cx40水平和GJC功能的抑制,与内皮细胞胞浆内Ca~(++)浓度和和细胞损伤是负相关关系。
3.创伤发生后炎症介质可能是通过抑制Cx40水平和GJC功能,减少细胞间Ca~(++)的流动和调节,升高细胞内Ca~(++)浓度,使血管内皮细胞通透性增加。这为预防和减轻急性肺损伤提供一个新的思路。
The permeability of pulmonary microvascular endothelial cells increases markedly after acute lung injury via paracellular gap. Connexin40 is a primary component of pulmonary microvascular endothelial cells gap junction channel and mediates intercellular communication. However, the relationship between Connexin40 and the permeability of pulmonary microvascular endothelial cells is still unknown. Therefore, we determined whether Connexin40 affected rabbits’pulmonary microvascular endothelial cells permeability after acute lung injury induced by gunshot trauma.
Part I: rabbit lung tissue permeability and the expression of Cx40 in ALI
Objective Establish acute lung injury animal model following gunshot chest trauma and investigate the relationship of Connexin40 inmunohistochemistry and lung tissue permeability.
Methods: We used an acute lung injury model in New Zealand rabbits following gunshot chest trauma and correlated Connexin40 inmunohistochemistry in gunshot lung tissue with Evans blue leak rate. Lung tissue water content and serum concentration of TNF alfa and IL 8 were measured.
Results: Lung tissue water content and serum concentration of TNF alfa and IL 8 increased significantly in post time compared with pre gunshot (p<0.05). Connexin40 expression time dependently decreased, whereas Evans blue leak rate increased. Connexin40 expression and Evans blue leak rate exhibited a strong inverse correlation (γ=-0.934, p <0.05).
PartⅡ: The relationship of Cx40 and rabbit pulmonary microvascular endothelial cells permeability in acute lung injury
Objective: Investigate the relationship of gap junction channel Connexin40 and rabbit pulmonary microvascular endothelial cells permeability in acute lung injury
Methods: Cultured pulmonary microvessel endothelial cells were divided into three groups, control (Gcontrol), injured serum (Gserum), and blocker agent (Gblocker). Gap junction channel function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer, and expression of Cx40 was measured by western blot.
Results: Injured serum decreased gap junction channel function and the level of Cx40 and the gap junction channel blocker aggravated this effect. Similarly, pulmonary microvascular endothelial cells permeability increased significantly in Gserum and Gblocker compared with Gcontrol (p <0.05).
PartⅢ: The relationship of PMVECs Cx40 and change of intracullar free calcium concentration in acute lung injury
Objective: Investigate the relationship of PMVECs Cx40 and intracullar free calcium concentration in cells
Methods: Cultured pulmonary microvessel endothelial cells were cultured and divided into three groups as described in partⅡ. Intracullar free calcium concentration was measured by fluo-3am, and the degree of cell injury was assessed by LDH concentration in medium.
Results: When intervented by injured serum and gap junction blocker in vitro, intracellular free calcium concentration increased (p<0.05), medium LDH concentration in medium increased and the degree of cell injury deteriorated (p<0.05).
Conclusions:
1. Down-regulation of pulmonary microvascular endothelial cells Connexin40 expression contributes to the increase of lung tissue microvascular permeability after acute lung injury.
2. Down-regulation of PMVECs gap junction Cx40 expression and gap junction channel opening contribute to the increase of PMVECs permeability after ALI.
3. Down-regulation of PMVECs Cx40 expression and gap junction channel opening contribute to the increase of intracellular calcium concentration, and overload calcium increased PMVECs permeability and made cells injured.
实验一.急性肺损伤兔肺组织通透性改变与Cx40的表达研究
目的:观察ALI时家兔肺组织通透性与微血管内皮细胞Cx40表达的关系
方法:应用胸部战伤方法建立家兔急性肺损伤模型,将伤后经过简单救治存活下来的家兔随机分为6组,每组6例,每例动物在伤前、伤后6、12、24、48,72小时取动脉血检测IL-8和TNF-α含量;于各时段分别处死动物,取肺组织做HE病理和免疫组织化学染色(Cx40);伊文思蓝漏出率法测肺微血管通透性;干湿比法测肺的含水量。
结果:HE染色见伤肺肺泡腔内大量渗出液体和红细胞的渗出聚集,肺泡隔增厚、断裂、融合,大量白细胞渗出、聚集,毛细血管扩张充血。对侧肺组织肺泡腔内液体渗出、红细胞聚集,肺泡隔内白血病浸润,毛细血管扩张充血。伤后12小时、24小时、48小时和72小时血清中TNF-α和IL-8含量均高于致伤前(p<0.05)。伤后24小时以后各时间点,肺组织含水量均有明显升高(p<0.05),持续至72小时。肺组织伊文思蓝漏出率随着时间的发展升高(p<0.05)。血管内皮细胞膜Cx40染色较伤前减弱,OD值显著减小(p<0.05),并与伊文思蓝漏出率呈负相关(γ=-0.934, P<0.05,γ2=0.8723)。
实验二.急性肺损伤兔肺微血管内皮细胞通透性改变与Cx40的关系研究
目的:观察离体培养的兔肺微血管内皮细胞通透性改变与Cx40表达、GJC功能之间的关系。
方法:组织块贴壁法分离、培养兔肺微血管内皮细胞。细胞分为三组,对照组、创伤血清处理组和阻滞剂组。在阻滞剂组,细胞间隙连接通道阻滞剂1-庚醇(0.5 mM)加入到培养液中。1小时后,阻滞剂组和创伤血清组的20%胎牛血清的培养液均被更换为含有20%创伤血清的培养液。对照组则只更换培养液。三组细胞继续孵育6小时后,进入实验观察。分别做划痕实验、Cx40蛋白免疫印迹和单层细胞通透性检测。
结果:培养的内皮细胞融合成单层,呈典型的铺路石样镶嵌状排列。Cx40蛋白印迹见创伤血清组与阻滞剂组蛋白表达水平较对照组有明显降低,且两组之间亦不同,阻滞剂组蛋白水平较创伤血清组低(对照组为1±0.07,创伤血清组为0.55±0.15,阻滞剂组为0.43±0.13,n=4,p<0.05)。三组细胞划痕边缘的细胞均被染料LY染色,LY在对照组细胞扩散得最远,显色的周边细胞的数量最多,创伤血清组次之,阻滞剂组显色的细胞数量最少。相邻细胞与边缘细胞的比值的差异具有统计学意义(23±5,11±3 ver 7±2,n=4,p<0.05)。三组细胞伊文思蓝清除率均随着时间的延长而增加,且三组细胞间亦有不同,阻滞剂组清除率高于创伤血清组和对照组。组间差别和时间点差别均有显著性差异。
实验三急性肺损伤兔肺微血管内皮细胞Cx40对细胞内Ca~(++)浓度的影响研究
目的:观察急性肺损伤时离体培养的兔肺微血管内皮细胞Cx40与内皮细胞损伤、细胞内Ca~(++)浓度变化之间的关系。
方法:兔肺微血管内皮细胞的培养和分组同实验二。三组细胞继续孵育6小时后,提取培养液测定LDH含量,加入fluo-3AM负载检测细胞内Ca~(++)钙浓度。
结果:通过共聚焦显微镜的观察,三组细胞荧光强度不相同,阻滞剂组荧光最强,对照组最弱。创伤血清组和阻滞剂组细胞内Ca~(++)浓度均显著高于对照组,(nmol/L, 494.8±41.94, 569.8±6.70 versus 158.8±13.09, p<0.05)。创伤血清组和阻滞剂组细胞培养液中LDH含量均显著高于对照组(IU/L, 165.88±16.17, 186.4±24.91 versus 51.31±13.27, p<0.05),且血清组与阻滞剂组之间的差别亦有统计学意义。此外,三组细胞内Ca~(++)含量与培养液中LDH的浓度还存在正相关关系(γ=0.997, p< 0.05,γ2=0.994)。
结论:
1.在动物实验中,肺组织通透性升高与肺组织微血管内皮细胞Cx40表达呈负相关关系。在离体培养的肺微血管内皮细胞,富含炎症介质的创伤血清能够抑制Cx40的水平和GJC的功能,与此同时肺血管内皮细胞通透性升高,二者存在负相关关系,Cx40水平和GJC功能的下降能够明显增加肺微血管内皮细胞的通透性。
2.创伤因素所致的Cx40水平和GJC功能的抑制,与内皮细胞胞浆内Ca~(++)浓度和和细胞损伤是负相关关系。
3.创伤发生后炎症介质可能是通过抑制Cx40水平和GJC功能,减少细胞间Ca~(++)的流动和调节,升高细胞内Ca~(++)浓度,使血管内皮细胞通透性增加。这为预防和减轻急性肺损伤提供一个新的思路。
The permeability of pulmonary microvascular endothelial cells increases markedly after acute lung injury via paracellular gap. Connexin40 is a primary component of pulmonary microvascular endothelial cells gap junction channel and mediates intercellular communication. However, the relationship between Connexin40 and the permeability of pulmonary microvascular endothelial cells is still unknown. Therefore, we determined whether Connexin40 affected rabbits’pulmonary microvascular endothelial cells permeability after acute lung injury induced by gunshot trauma.
Part I: rabbit lung tissue permeability and the expression of Cx40 in ALI
Objective Establish acute lung injury animal model following gunshot chest trauma and investigate the relationship of Connexin40 inmunohistochemistry and lung tissue permeability.
Methods: We used an acute lung injury model in New Zealand rabbits following gunshot chest trauma and correlated Connexin40 inmunohistochemistry in gunshot lung tissue with Evans blue leak rate. Lung tissue water content and serum concentration of TNF alfa and IL 8 were measured.
Results: Lung tissue water content and serum concentration of TNF alfa and IL 8 increased significantly in post time compared with pre gunshot (p<0.05). Connexin40 expression time dependently decreased, whereas Evans blue leak rate increased. Connexin40 expression and Evans blue leak rate exhibited a strong inverse correlation (γ=-0.934, p <0.05).
PartⅡ: The relationship of Cx40 and rabbit pulmonary microvascular endothelial cells permeability in acute lung injury
Objective: Investigate the relationship of gap junction channel Connexin40 and rabbit pulmonary microvascular endothelial cells permeability in acute lung injury
Methods: Cultured pulmonary microvessel endothelial cells were divided into three groups, control (Gcontrol), injured serum (Gserum), and blocker agent (Gblocker). Gap junction channel function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer, and expression of Cx40 was measured by western blot.
Results: Injured serum decreased gap junction channel function and the level of Cx40 and the gap junction channel blocker aggravated this effect. Similarly, pulmonary microvascular endothelial cells permeability increased significantly in Gserum and Gblocker compared with Gcontrol (p <0.05).
PartⅢ: The relationship of PMVECs Cx40 and change of intracullar free calcium concentration in acute lung injury
Objective: Investigate the relationship of PMVECs Cx40 and intracullar free calcium concentration in cells
Methods: Cultured pulmonary microvessel endothelial cells were cultured and divided into three groups as described in partⅡ. Intracullar free calcium concentration was measured by fluo-3am, and the degree of cell injury was assessed by LDH concentration in medium.
Results: When intervented by injured serum and gap junction blocker in vitro, intracellular free calcium concentration increased (p<0.05), medium LDH concentration in medium increased and the degree of cell injury deteriorated (p<0.05).
Conclusions:
1. Down-regulation of pulmonary microvascular endothelial cells Connexin40 expression contributes to the increase of lung tissue microvascular permeability after acute lung injury.
2. Down-regulation of PMVECs gap junction Cx40 expression and gap junction channel opening contribute to the increase of PMVECs permeability after ALI.
3. Down-regulation of PMVECs Cx40 expression and gap junction channel opening contribute to the increase of intracellular calcium concentration, and overload calcium increased PMVECs permeability and made cells injured.
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[28] Le Cras TD, Spitzmiller RE, Albertine KH, Greenberg JM, Whitsett JA, Akeson AL. VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice. Am J Physiol Lung Cell Mol Physiol 2004 Jul 287;L134-L142.
[29] Cullen VC, Mackarel AJ, Hislip SJ, O'Connor CM, Keenan AK. Investigation of vascular endothelial growth factor effects on pulmonary endothelial monolayer permeability and neutrophil transmigration. Gen Pharmacol 2000 Sep 1935;149-57.
[30] Feng D, Nagy JA, Hipp J, Dvorak HF, Dvorak AM. Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin. J Exp Med 1996 May 1 183;1981-6.
[31] Suttorp N, Nolte A, Wilke A, Drenckhahn D. Human neutrophil elastase increases permeability of cultured pulmonary endothelial cell monolayers. Int J Microcirc Clin Exp 1993 Dec 1913;187-203.
[32] Peterson MW, Walter ME, Nygaard SD. Effect of neutrophil mediators on epithelial permeability. Am J Respir Cell Mol Biol 1995 Dec 1913;719-27.
[33] Raats CJ, Van Den BJ, Berden JH. Glomerular heparan sulfate alterations: mechanisms and relevance for proteinuria. Kidney Int 2000 Feb 1957;385-400.
[34] Inoue Y, Tanaka H, Ogura H, et al. A neutrophil elastase inhibitor, sivelestat, improvesleukocyte deformability in patients with acute lung injury. J Trauma 2006 May 1960;discussion 943.
[35] Piantadosi CA, Schwartz DA. The acute respiratory distress syndrome. Ann Intern Med 2004 Sep 21 141;460-70.
[36] Kevil CG, Oshima T, Alexander B, Coe LL, Alexander JS. H(2)O(2)-mediated permeability: role of MAPK and occludin. Am J Physiol Cell Physiol 2000 Jul 279;C21-C30.
[37] Zhao X, Alexander JS, Zhang S, et al. Redox regulation of endothelial barrier integrity. Am J Physiol Lung Cell Mol Physiol 2001 Oct 281;L879-L886.
[38] Tong Q, Zheng L, Kang Q, et al. Upregulation of hypoxia-induced mitogenic factor in bacterial lipopolysaccharide-induced acute lung injury. FEBS Lett 2006 Apr 17 580;2207-15.
[39] Connelly L, Palacios-Callender M, Ameixa C, Moncada S, Hobbs AJ. Biphasic regulation of NF-kappa B activity underlies the pro- and anti-inflammatory actions of nitric oxide. J Immunol 2001 Mar 15 166;3873-81.
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[42] House C, Kemp BE. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 1987 Dec 18 238;1726-8.
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[44] Wu MH, Yuan SY, Granger HJ. The protein kinase MEK1/2 mediate vascular endothelial growth factor- and histamine-induced hyperpermeability in porcine coronary venules. J Physiol 2005 Feb 15 563;95-104.
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[48] Park JH, Okayama N, Gute D, Krsmanovic A, Battarbee H, Alexander JS. Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton. Am J Physiol 1999 Dec 277;C1066-C1074.
[49] Yuan Y, Meng FY, Huang Q, Hawker J, Wu HM. Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function. Am J Physiol 1998 Jul 275;H84-H93.
[50] Konstantoulaki M, Kouklis P, Malik AB. Protein kinase C modifications of VE-cadherin, p120, and beta-catenin contribute to endothelial barrier dysregulation induced by thrombin. Am J Physiol Lung Cell Mol Physiol 2003 Aug 285;L434-L442.
[51] Chandrasekharan UM, Yang L, Walters A, Howe P, DiCorleto PE. Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation. J Biol Chem 2004 Nov 5 279;46678-85.
[52] Mehta D, Rahman A, Malik AB. Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J Biol Chem 2001 Jun 22 276;22614-20.
[53] Paria BC, Vogel SM, Ahmmed GU, et al. Tumor necrosis factor-alpha-induced TRPC1 expression amplifies store-operated Ca2+ influx and endothelial permeability. Am J Physiol Lung Cell Mol Physiol 2004 Dec 287;L1303-L1313.
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[55] Muzaffar S, Jeremy JY, Angelini GD, Stuart-Smith K, Shukla N. Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries. Thorax 2003 Jul 1958;598-604.
[56] Fackler ML. Wound ballistics. A review of common misconceptions. JAMA 1988 May 13;259(18):2730-6.
[57] Bruzzone R, Haefliger JA, Gimlich RL, Paul DL. Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell 1993 Jan;4(1):7-20.
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[26] Wysolmerski RB, Lagunoff D. Regulation of permeabilized endothelial cell retraction by myosin phosphorylation. Am J Physiol 1991 Jul 261;C32-C40.
[27] Kaner RJ, Ladetto JV, Singh R, Fukuda N, Matthay MA, Crystal RG. Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema. Am J Respir Cell Mol Biol 2000 Jun 1922;657-64.
[28] Le Cras TD, Spitzmiller RE, Albertine KH, Greenberg JM, Whitsett JA, Akeson AL. VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice. Am J Physiol Lung Cell Mol Physiol 2004 Jul 287;L134-L142.
[29] Cullen VC, Mackarel AJ, Hislip SJ, O'Connor CM, Keenan AK. Investigation of vascular endothelial growth factor effects on pulmonary endothelial monolayer permeability and neutrophil transmigration. Gen Pharmacol 2000 Sep 1935;149-57.
[30] Feng D, Nagy JA, Hipp J, Dvorak HF, Dvorak AM. Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin. J Exp Med 1996 May 1 183;1981-6.
[31] Suttorp N, Nolte A, Wilke A, Drenckhahn D. Human neutrophil elastase increases permeability of cultured pulmonary endothelial cell monolayers. Int J Microcirc Clin Exp 1993 Dec 1913;187-203.
[32] Peterson MW, Walter ME, Nygaard SD. Effect of neutrophil mediators on epithelial permeability. Am J Respir Cell Mol Biol 1995 Dec 1913;719-27.
[33] Raats CJ, Van Den BJ, Berden JH. Glomerular heparan sulfate alterations: mechanisms and relevance for proteinuria. Kidney Int 2000 Feb 1957;385-400.
[34] Inoue Y, Tanaka H, Ogura H, et al. A neutrophil elastase inhibitor, sivelestat, improvesleukocyte deformability in patients with acute lung injury. J Trauma 2006 May 1960;discussion 943.
[35] Piantadosi CA, Schwartz DA. The acute respiratory distress syndrome. Ann Intern Med 2004 Sep 21 141;460-70.
[36] Kevil CG, Oshima T, Alexander B, Coe LL, Alexander JS. H(2)O(2)-mediated permeability: role of MAPK and occludin. Am J Physiol Cell Physiol 2000 Jul 279;C21-C30.
[37] Zhao X, Alexander JS, Zhang S, et al. Redox regulation of endothelial barrier integrity. Am J Physiol Lung Cell Mol Physiol 2001 Oct 281;L879-L886.
[38] Tong Q, Zheng L, Kang Q, et al. Upregulation of hypoxia-induced mitogenic factor in bacterial lipopolysaccharide-induced acute lung injury. FEBS Lett 2006 Apr 17 580;2207-15.
[39] Connelly L, Palacios-Callender M, Ameixa C, Moncada S, Hobbs AJ. Biphasic regulation of NF-kappa B activity underlies the pro- and anti-inflammatory actions of nitric oxide. J Immunol 2001 Mar 15 166;3873-81.
[40] Ohsugi S, Iwasaki Y, Takemura Y, et al. An inhaled inducible nitric oxide synthase inhibitor reduces damage of Candida-induced acute lung injury. Biomed Res 2007 Apr 1928;91-9.
[41] Adhikari NK, Burns KE, Friedrich JO, Granton JT, Cook DJ, Meade MO. Effect of nitric oxide on oxygenation and mortality in acute lung injury: systematic review and meta-analysis. BMJ 2007 Apr 14 334;779.
[42] House C, Kemp BE. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 1987 Dec 18 238;1726-8.
[43] Parker PJ, Murray-Rust J. PKC at a glance. J Cell Sci 2004 Jan 15 117;131-2.
[44] Wu MH, Yuan SY, Granger HJ. The protein kinase MEK1/2 mediate vascular endothelial growth factor- and histamine-induced hyperpermeability in porcine coronary venules. J Physiol 2005 Feb 15 563;95-104.
[45] Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiol Rev 2006 Jan 1986;279-367.
[46] Kolosova IA, Ma SF, Adyshev DM, et al. Role of CPI-17 in the regulation ofendothelial cytoskeleton. Am J Physiol Lung Cell Mol Physiol 2004 Nov 287;L970-L980.
[47] Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev 2004 Jul 1984;869-901.
[48] Park JH, Okayama N, Gute D, Krsmanovic A, Battarbee H, Alexander JS. Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton. Am J Physiol 1999 Dec 277;C1066-C1074.
[49] Yuan Y, Meng FY, Huang Q, Hawker J, Wu HM. Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function. Am J Physiol 1998 Jul 275;H84-H93.
[50] Konstantoulaki M, Kouklis P, Malik AB. Protein kinase C modifications of VE-cadherin, p120, and beta-catenin contribute to endothelial barrier dysregulation induced by thrombin. Am J Physiol Lung Cell Mol Physiol 2003 Aug 285;L434-L442.
[51] Chandrasekharan UM, Yang L, Walters A, Howe P, DiCorleto PE. Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation. J Biol Chem 2004 Nov 5 279;46678-85.
[52] Mehta D, Rahman A, Malik AB. Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J Biol Chem 2001 Jun 22 276;22614-20.
[53] Paria BC, Vogel SM, Ahmmed GU, et al. Tumor necrosis factor-alpha-induced TRPC1 expression amplifies store-operated Ca2+ influx and endothelial permeability. Am J Physiol Lung Cell Mol Physiol 2004 Dec 287;L1303-L1313.
[54] Serfilippi G, Ferro TJ, Johnson A. Activation of protein kinase C mediates altered pulmonary vasoreactivity induced by tumor necrosis factor-alpha. Am J Physiol 1994 Sep 267;L282-L290.
[55] Muzaffar S, Jeremy JY, Angelini GD, Stuart-Smith K, Shukla N. Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries. Thorax 2003 Jul 1958;598-604.
[56] Fackler ML. Wound ballistics. A review of common misconceptions. JAMA 1988 May 13;259(18):2730-6.
[57] Bruzzone R, Haefliger JA, Gimlich RL, Paul DL. Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell 1993 Jan;4(1):7-20.
[58] Harada A, Sekido N, Akahoshi T, Wada T, Mukaida N, Matsushima K. Essential involvement of interleukin-8 (IL-8) in acute inflammation 1. J Leukoc Biol 1994 Nov;56(5):559-64.
[59] Christ GJ, Moreno AP, Melman A, Spray DC. Gap junction-mediated intercellular diffusion of Ca2+ in cultured human corporal smooth muscle cells. Am J Physiol 1992 Aug 263;C373-C383.
[60] Tzima E, Irani-Tehrani M, Kiosses WB, et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 2005 Sep 15;437(7057):426-31.
[61] Chen SF, Fei X, Li SH. A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries 1. Microvasc Res 1995 Jul;50(1):119-28.
[62] Simon AM, McWhorter AR, Chen H, Jackson CL, Ouellette Y. Decreased intercellular communication and connexin expression in mouse aortic endothelium during lipopolysaccharide-induced inflammation. J Vasc Res 2004 Jul;41(4):323-33.
[63] Figueroa XF, Alvina K, Martinez AD, et al. Histamine reduces gap junctional communication of human tonsil high endothelial cells in culture. Microvasc Res 2004 Nov;68(3):247-57.
[64] Lidington D, Tyml K, Ouellette Y. Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43. J Cell Physiol 2002 Dec;193(3):373-9.
[65] Tonon R, D'Andrea P. The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: evidence for a Ca2+-dependent mechanism. Biorheology 2002;39(1-2):153-60.
[66] Garcia-Dorado D, Inserte J, Ruiz-Meana M, et al. Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion 1. Circulation 1997 Nov 18;96(10):3579-86.
[67] Danpure CJ. Lactate dehydrogenase and cell injury 1. Cell Biochem Funct 1984 Jul;2(3):144-8.
[68] Garrigues C, Goupil-Feuillerat N, Cocaign-Bousquet M, Renault P, Lindley ND, Loubiere P. Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity 1. Metab Eng 2001 Jul;3(3):211-7.
[69] Chopra J, Joist JH, Webster RO. Loss of 51chromium, lactate dehydrogenase, and 111indium as indicators of endothelial cell injury 1. Lab Invest 1987 Nov;57(5):578-84.
[70] Ng LC, Gurney AM. Store-operated channels mediate Ca(2+) influx and contraction in rat pulmonary artery. Circ Res 2001 Nov 9;89(10):923-9.