1-磷酸鞘氨醇受体2在LPS介导的内皮细胞高通透性中的作用

目的观察1-磷酸鞘氨醇受体2（S1PR2）在脂多糖（LPS）刺激后表达的变化,探讨其在LPS介导的内皮细胞高通透性中的作用。方法用不同浓度和时间的LPS刺激培养的ECV304细胞,采用RT-PCR方法检测S1PR2mRNA的变化,Western blot方法检测S1PR2蛋白的变化,TRITC荧光标记白蛋白漏出法测定单层内皮细胞的通透系数Pa值。结果与对照组相比,S1PR2mRNA和S1PR2蛋白在LPS（500ng/ml）刺激24h后以及在LPS（1000ng/ml）刺激12h显著增加（P〈0.01）;S1PR2拮抗剂JTE-013能显著降低LPS引起的内皮细胞通透性增高（P〈0.05）。结论 S1PR2在LPS引起的内皮细胞通透性增高中起着重要的作用。     

1-磷酸鞘氨醇对血小板活化因子所致大鼠肠系膜微血管通透性增高的影响

目的:探讨1-磷酸鞘氨醇(S1P)对血小板活化因子(PAF)引起的大鼠肠系膜微血管通透性增高的影响。方法:本研究拟采用大鼠在体肠系膜微血管灌注的方法,通过测定微静脉的静水传导性(Lp),观察S1P对外源性PAF引起的微血管通透性增高的影响;并利用激光共聚焦显微镜技术,观察S1P对PAF引起的微血管荧光强度变化以及血管内皮细胞钙粘蛋白(VE-cadherin)变化的影响。结果:给予10nmol/LPAF作用后,大鼠肠系膜微血管Lp值明显增高,而经1μmol/L S1P预处理后,再给予PAF并未引起Lp的明显变化;PAF作用微血管后可见微血管内皮细胞间隙打开,微血管荧光强度明显增加,大量红色荧光微球(FMs)分布于内皮细胞间隙之中,S1P预处理后并未见内皮细胞间隙打开及FMs的明显积聚,微血管荧光强度与正常对照值比较无显著差异。结论:PAF可增加微血管的通透性,改变内皮细胞VE-cadherin正常结构,导致粘附连接断裂,细胞间隙形成,血管通透性增加可能与此结构变化有关。S1P能改善PAF引起的血管通透性增高,其作用与加强内皮细胞间粘附连接,抑制细胞间隙打开有关。     

IL-2／IFN-γ融合蛋白基因质粒联合HBVDNA疫苗治疗HBV转基因小鼠效果评价

目的评价融合蛋白基因质粒（pFP）联合在体电脉冲（EP）介导的HBVDNA疫苗（pS2．S）免疫HBV转基因（Tg）小鼠的治疗效果。方法HBVTg小鼠随机分成3组，每组5只，分别为pS2．S＋pFP、pS2．S＋pcDNA3．1免疫治疗和pcDNA3．1对照组。联合免疫质粒总剂量40μg／只，按1：1比例混合接种。初次免疫后第4、8周分别进行第一、二次增强免疫，免疫前后分别检测血清学、组织学及HBV特异性免疫应答。结果HBVDNA血清定量检测结果，pS2．S＋pFP组免疫第8周（13317±2539）拷贝／ml、第12周（6462±3359）拷贝／ml时分别较免疫前（36159±7769）拷贝／ml明显减低，差异具有统计学意义（P〈0．01）；pS2．S＋pcDNA3．1组第4周（20618±9523）拷贝／ml及第8周（23818±5319）拷贝／ml时均较其免疫前水平（36090±4421）拷贝／ml明显降低（P〈0．01），但不能持续到12周（27691＋13071）拷贝／ml。并明显高于此时pS2．S＋pFP组水平，差异有统计学意义（P〈0．01）；观察终点（12周）pS2．S＋pFP组强小鼠个体血清HBVDNA及肝组织HBsAg表达水平降低的同时，伴随其血清ALT水平及HBsAg特异性IFN-γ分泌细胞数目的升高。结论，Th1类细胞因子IL-2／IFN-γ融合蛋白基因表达质粒能够增强HBVDNA疫苗抑制强鼠HBVDNA复制和表达的治疗作用。     

p38丝裂原活化蛋白激酶抑制剂SB203580对严重烧伤大鼠离体库普弗细胞促炎性细胞因子分泌的影响

目的探讨p38丝裂原活化蛋白激酶抑制剂SB203580对严重烧伤大鼠离体库普弗细胞促炎性细胞因子肿瘤坏死因子（tumor necrosis factor，TNF）-α和白细胞介素（interleukin，IL）-1β分泌的影响。方法健康成年的SD大鼠10只分为假烫组和烧伤组，假烫或烧伤24h后处死分离出库普弗细胞。37℃5％CO2条件下培养60min后加入SB203580，18h后用25ng/孔的脂多糖（lipopolysaccharide，LPS）刺激。酶联免疫吸附法（enzyme-linked immunosorbent assay，ELISA）测定库普弗细胞上清液TNF-α和IL-1β的含量。结果烧伤大鼠的离体库普弗细胞在LPS刺激后分泌的TNF-α和IL-1β量较假烫组增高显著[（2．847±0．398）ng／ml vs（1．232±0．101）ng／ml，P〈0．01；（742．1914±103．7009）pg／ml vs （320.5462±26.3022）pg／ml，P〈0.01）]。体外使用SB203580既可以抑制烧伤大鼠的离体库普弗细胞分泌TNF-α和IL-1β[（0.1021±0.018）ng／ml vs（2．847±0.398）ng／ml，P〈0．01；（26 .7167±4.9213）pg／ml vs （742.1914±103.7009）pg／ml，P〈0.01）]，也可以抑制正常大鼠的离体库普弗细胞分泌TNF-α和IL-1β[（0.113±0．032）ng／ml vs （1．232±0.101）ng／ml，P〈0．01；（30.2427±8.9803）pg／ml vs（320.5462±26．3022）pg／ml，P〈0.01）]。结论p38MAPK信号转导通路介导了严重烧伤后库普弗细胞TNF-α和IL-1β的产生，在烧伤后全身炎症反应的发生中发挥着重要的调控作用。     

休克期大面积切痂对严重烧伤大鼠细胞免疫功能影响的研究

目的 观察休克期大面积切痂对严重烧伤大鼠细胞免疫功能的影响，探索改善烧伤后机体免疫功能紊乱的有效方法。方法 将大鼠分成休克期切痂组（A组）、常规切痂组（B组）和正常对照组（C组）。A、B组造成30％TBSAⅢ度烫伤，C组不烫伤。A组伤后第6h、B组伤后第4d切痂，并于伤后第1、5、9d各活杀10只，取材送检，观察其免疫指标的变化。结果 （1）A、B组与C组比较：A、B组烫伤大鼠各时相点CD3^＋T细胞变化不大（P〉0．05），但CD4^＋T细胞、CD4^＋／CD8^＋比值明显下降、CD8^＋T细胞增高（P〈0．05或P〈0．01）。NK细胞活性明显下降（P〈0．05或P〈0.01），外周血CD25^＋T淋巴细胞表达及经活化后脾脏CD25^＋T淋巴细胞表达明显下降（P〈0．05或P〈0．01）。（2）A组与B组比较：A组CD4^＋T细胞、CD4^＋／CD8^＋比值明显升高、CD8^＋T细胞降低（P〈0．05或P〈0．01），NK细胞活性明显升高（P〈0．05或P〈0．01），外周血CD25^＋T淋巴细胞表达及经活化后脾脏CD25^＋T淋巴细胞表达均明显升高（P〈0．05或P〈0．01）。结论 （1）大鼠烫伤后细胞免疫状况发生了明显变化。（2）休克期切痂可以改善烫伤大鼠T淋巴细胞亚群分布，提高NK细胞活性，增加外周血CD25^＋T淋巴细胞的表达。提高经活化后脾脏CD25^＋T淋巴细胞数。从而改善烫伤大鼠伤后机体的细胞免疫功能。     

p38丝裂原活化蛋白激酶对严重烧伤大鼠肝脏激活蛋白-1和核因子-κB的影响

目的观察严重烧伤后肝脏p38丝裂原活化蛋白激酶（mitogen—activated protein kinase，MAPK）对激活蛋白（activating protein，AP）-1和核因子（nuclear factor，NF）-κB的影响。方法采用30％TBSAⅢ度烫伤动物模型，健康成年的雄性SD大鼠24只，随机分为假烫组、烧伤对照组和烧伤＋SB203580组。观察烧伤24h后肝脏IκBα含量的变化，并采用凝胶电泳迁移率变化分析（electrophoretic mobility shift assay，EMSA）法检测肝脏AP-1和NF-κB DNA结合活性的变化。结果大鼠烧伤24h后肝脏IκBα含量明显下降（2086±684 vs 4138±1160，P〈0．05），肝脏AP-1和NF-κB DNA结合活性均显著增强，使用p38MAPK抑制剂SB203580能显著抑制肝脏AP-1 DNA结合活性的上升（469±84 vs 1306±486，P〈0．01），但对肝脏IκBα含量和NF-κB DNA结合活性的变化无明显影响。结论严重烧伤后，肝脏中活化的p38MAPK通过激活转录因子AP-1进而引起细胞因子的产生，在烧伤后肝损伤的发生中起重要作用，p38MAPK这一作用与IκBα降解引起的NF-κB DNA结合活性增强无关。     

c—Jun氨基末端激酶选择性抑制剂对哮喘小鼠肺组织IL-2表达的影响

目的探讨c-Jun氨基末端激酶选择性抑制剂（SP600125）对哮喘小鼠肺组织白介素-2（IL-2）表达的影响。方法30只BALB／c小鼠随机分为对照组、哮喘组和SP600125组，制作哮喘模型及干预处理后，处死小鼠，取肺组织，采用免疫组织化学方法和Western blot方法检测各组小鼠肺组织内IL-2的表达。结果免疫组化结果显示，哮喘组小鼠肺组织IL-2表达的平均光密度值为0．76＋0．11，显著高于对照组0．25±0．04，P〈0．01；而SP600125组小鼠肺组织IL-2表达的平均光密度值为0．45±0．08，与哮喘组相比明显降低，P〈0．01。Westernblot结果显示，哮喘组小鼠肺组织IL-2表达的平均光密度值为1．26±0．21，显著高于对照组0．36±0．05，P〈0．01；而SP600125组小鼠肺组织IL-2表达的平均光密度值为0．88±0．11，与哮喘组相比明显降低，P〈0．01。结论c．Jun氨基末端激酶选择性抑制剂能降低哮喘小鼠肺组织IL-2的表达。     

T细胞诱导的结肠炎中多形螺旋线虫感染对CD4^＋T细胞增殖的影响

目的 研究肠道多形螺旋线虫对T细胞诱导的小鼠结肠炎CD4^±T细胞增殖情况的影响。方法 用羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）染色的卵清蛋白（OVA）特异性CD4＾±辅助性T细胞转入重度联合免疫缺陷（SCID）小鼠中，制作小鼠实验性肠炎模型。将实验模型小鼠分为多形螺旋线虫感染组和无感染组（每组n=5），观察多形螺旋线虫感染7d后小鼠结肠炎性反应的组织学变化；以流式细胞仪检测感染3、5、7d小鼠肠系膜淋巴结中CD4＾＋T细胞CFSE的阴性率，判定肠系膜淋巴结中CD4＾＋T细胞的增殖情况。结果 与无感染组比较，感染组小鼠第7天时有螺旋线虫感染结肠炎性反应明显加重，黏膜固有层细胞浸润增多，结肠上皮破损增加，病理评分明显升高（5．20±0．84比2．00±0．71，P〈0．05）。感染3、5、7d后，感染组小鼠肠系膜淋巴结中CD4±T细胞增殖均比无感染组明显增强，CFSE的阴性率升高[3d：（7．03±1．61）％比（2．32±0．62）％，5d：（55．05±13．41）％比（29．10±2．23）％，7d：（76．97±1．89）％比（43．87±5．56）％，均P〈0．05]。结论 多形螺旋线虫感染在CD4＾＋T细胞诱导的小鼠实验性结肠炎的早期阶段促进了炎性反应的加重，可能与促进CD4＾＋T细胞的增殖有关。     

1-磷酸鞘氨醇通过提高心脏微血管密度缓解压力负荷诱导的小鼠心力衰竭

目的：探究1-磷酸鞘氨醇（sphingosine-1-phosphate, S1P）通过调节心脏微血管密度对压力负荷诱导的小鼠心力衰竭的作用及相关机制。方法：将8周龄的雄性C57BL/6小鼠随机分为4组：假手术（sham）组、sham+2-乙酰基-5-四羟基丁基咪唑（2-acetyl-5-tetrahydroxybutyl imidazole, THI; S1P裂解酶抑制剂）组、主动脉弓缩窄术（transverse aortic constriction, TAC）组和TAC+THI组。TAC手术后1周给予THI灌胃处理，实验终点检测各组小鼠血浆和心脏匀浆组织中S1P水平；心脏超声和Millar导管检测心功能；HE染色检测各组小鼠心脏肥大程度，Masson染色观察各组小鼠心脏间质和管周纤维化程度，CD31和麦胚凝集素免疫荧光染色观察各组小鼠心肌细胞横截面积和心脏微血管密度；RT-qPCR检测心房钠尿肽（atrial natriuretic peptide, ANP）、脑钠肽（brain natriuretic peptide, BNP）、I型胶原蛋白（collagen type I）、...     

miR-126在1713-雌二醇抑制血管内皮细胞VCAM-1蛋白表达中的作用研究

目的探讨17β-雌二醇（17β-estradiol，E2）对于血管内皮细胞中血管细胞黏附分子1（VCAM-1）的表达影响，探索miR-126在其中的作用。方法培养人脐静脉血管内皮细胞，以脂多糖LPS刺激诱导炎症反应，给予不同浓度梯度的E2处理不同时间，应用免疫印迹法观察VCAM-1的蛋白表达。转染miR-126的抑制物沉默miR-126表达后，观察E2对VCAM．1蛋白表达的影响变化。结果与对照组比较，LPS可诱导VCAM-1蛋白表达增加（854．0±21．3）％，n：3，P〈0．01）。分别给予不同浓度的E2（10^-8～10^-6mol／L）预处理后，均能抑制VCAM-1蛋白表达。抑制率分别为（44．3±4．0）％（n=3，P〈0．05）、（45．14±7．1）％（n=3，P〈0．05）和（65．9±7．2）％（n=3，P〈0．01）；E2（10^-8mol／L）分别处理内皮细胞24、48、72h后，可呈时间依赖性抑制LPS所诱导的VCAM．1的蛋白表达，抑制率分别为（52．1±4．4）％、（59．54-7．7）％和（64．5＋6．5）％（n=3，P〈0．05）。转染miR-126的抑制物可明显抑制miR-126表达[抑制率为（75．8±6．7）％，n=3，P〈0．01]，该抑制物可明显抑制17β-雌二醇对VCAM-1蛋白的抑制作用。结论E2通过miR-126下调LPS诱导的血管内皮细胞VCAM-1蛋白表达。     

Focal adhesion kinase mediates porcine venular hyperpermeability elicited by vascular endothelial growth factor

Focal adhesion kinase (FAK) is known to mediate endothelial cell adhesion and migration in response to vascular endothelial growth factor (VEGF). The aim of this study was to explore a potential role for FAK in VEGF regulation of microvascular endothelial barrier function. The apparent permeability coefficient of albumin ( P _a) was measured in intact isolated porcine coronary venules. Treating the vessels with VEGF induced a time- and concentration-dependent increase in P _a. Inhibition of FAK through direct delivery of FAK-related non-kinase (FRNK) into venular endothelium did not alter basal barrier function but significantly attenuated VEGF-elicited hyperpermeability. Furthermore, cultured human umbilical vein endothelial monolayers displayed a similar hyperpermeability response to VEGF which was greatly attenuated by FRNK. Western blot analysis showed that VEGF promoted FAK phosphorylation in a time course correlating with that of venular hyperpermeability. The phosphorylation response was blocked by FRNK treatment. In addition, VEGF stimulation caused a significant morphological change of FAK from a punctate pattern to an elongated, dash-like staining that aligned with the longitudinal axis of the cells. Taken together, the results suggest that FAK contributes to VEGF-elicited vascular hyperpermeability. Phosphorylation of FAK may play an important role in the signal transduction of vascular barrier response to VEGF.     

Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery

Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.     

The protein kinase MEK1/2 mediate vascular endothelial growth factor- and histamine-induced hyperpermeability in porcine coronary venules

Mitogen-activated protein kinases (MAPKs) have been implicated in the signal transduction of the endothelial response to growth factors and inflammatory stimuli. The objective of this study was to test the hypothesis that the p42/44 MAPK pathway plays a common role in mediating the microvascular hyperpermeability response to vascular endothelial growth factor (VEGF) and histamine. The apparent permeability coefficient of albumin was measured in isolated and perfused coronary venules. Application of VEGF induced a rapid increase in venular permeability, and the effect was blocked by PD98059 and UO126, selective inhibitors of the mitogen-activated protein kinase kinase MEK1/2, in a dose-dependent pattern. The same MEK1/2 inhibitors dose-dependently attenuated the increase in venular permeability caused by histamine. In addition, the increases in venular permeability caused by agents that are known to activate the nitric oxide pathway, including the calcium ionophore ionomycin, the nitric oxide donor S -nitroso- N -acetylpenicillamine, and the protein kinase G activator 8-bromo-cGMP, were significantly attenuated in venules pretreated with the MEK1/2 inhibitors. Furthermore, transfection of venules with active MEK1 increased baseline permeability. In contrast, transfection of active ERK1, a downstream target of MEK1/2, did not significantly alter the basal permeability of venules. Moreover, inhibition of ERK1/2 with a specific inhibiting peptide did not prevent the hyperpermeability response to VEGF or histamine. The results suggest that activation of MEK1/2 may play a central role in the signal transduction of microvascular hyperpermeability in response to growth factors and inflammatory mediators.     

Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice

Previous experiments using cultured endothelial monolayers indicate that Rho-family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin-based contractility and junctional distribution of the Ca²⁺-dependent endothelial cell adhesion molecule, VE-cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng ml⁻¹) inactivated 50–90 % of all endothelial Rho proteins within 60–90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F-actin in cultured endothelial monolayers and in mouse aortic endothelium in situ . Also, VE-cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y-27632 (30 μ m ) for 90–120 min induced F-actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE-cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability ( L _p) but did not attenuate the transient increase in L _p of microvessels exposed to bradykinin. Perfusion of microvessels with Y-27632 (30 μ m ) for up to 100 min reduced basal L _p but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B-mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA-mediated contractile mechanisms are not involved in bradykinin- or PAF-induced hyperpermeability of intact microvessels.     

Neutrophil extravasation in rat mesenteric venules induced by the chemotactic peptide N-formyl-methionyl-luecylphenylalanine (fMLP), with special attention to a barrier function of the vascular basal lamina for neutrophil migration

The present study was performed to investigate morphologically the process of neutrophil extravasation induced by the synthetic bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in venules of the rat mesentery by the combined use of intravital microscopy and transmission electron microscopy (TEM). Special attention was given to the interaction of the neutrophils with the endothelial cells and endothelial basal lamina. By intravital microscopy, the rolling and adhesion of leukocytes were observed within 3 min in preparations activated by fMLP. Neutrophils remained in the vascular wall for more than 30 min even after penetration of the endothelium. In this period, neutrophils migrating between endothelial cells and their basal lamina were often observed by TEM. After 40 min application of fMLP, some of the migrating neutrophils parted from the vessel wall into the surrounding connective tissues. There were neutrophils which passed through the small pore of the basal lamina with a cytoplasmic constriction. These findings indicate that the endothelial basal lamina acts as a physical barrier against neutrophil extravasation, thus resulting in the transient retainment of neutrophils beneath the endothelial cells of the venular wall.     

FTY720 improves functional recovery after spinal cord injury by primarily nonimmunomodulatory mechanisms

Spinal cord injury (SCI) is an incapacitating injury that can result in limited functional recovery. We have previously shown increases in the lysophospholipid mediator, sphingosine-1-phosphate (S1P), in the spinal cord after contusion injury. To apply S1P receptor modulation to the treatment of SCI, we examined the therapeutic effects of FTY720, an S1P receptor agonist, on locomotor recovery after SCI in mice. Oral administration of FTY720 shortly after contusion SCI significantly improved motor function recovery, as assessed by both Basso Mouse Scale scores and Rotarod Performance test results. FTY720 induced lymphopenia and reduced T-cell infiltration in the spinal cord after SCI but did not affect the early infiltration of neutrophils and the activation of microglia. In addition, plasma levels and mRNA expression of inflammatory cytokines in the spinal cord after SCI were not attenuated by FTY720. Vascular permeability and astrocyte accumulation were both decreased by FTY720 in the injured spinal cord. The therapeutic effects of FTY720 were not solely dependent on immune modulation, as confirmed by the demonstration that FTY720 also ameliorated motor function after SCI in mice with severe combined immunodeficiency. Finally, the S1P(1) receptor agonist, SEW2871, partly mimicked the therapeutic effect of FTY720. Our data highlight the importance of immune-independent functions of FTY720 in decreasing vascular permeability and astrogliosis in the injured spinal cord and promoting locomotor function recovery after SCI.     

Attenuation of vascular permeability by methylnaltrexone: role of mOP-R and S1P3 transactivation

Endothelial cell (EC) barrier dysfunction (i.e., increased vascular permeability) is observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that preserve vascular integrity have important clinical therapeutic implications. We examined the effects of methylnaltrexone (MNTX), a mu opioid receptor (mOP-R) antagonist, on human pulmonary EC barrier disruption produced by edemagenic agents including morphine, the endogenous mOP-R agonist DAMGO, thrombin, and LPS. Pretreatment of EC with MNTX (0.1 muM, 1 h) or the uncharged mOP-R antagonist naloxone attenuated morphine- and DAMGO-induced barrier disruption in vitro. However, MNTX, but not naloxone, pretreatment of EC inhibited thrombin- and LPS-induced barrier disruption, indicating potential mOP-R-independent effects of MNTX. In addition, intravenously delivered MNTX attenuated LPS-induced vascular hyperpermeability in the murine lung. We next examined the mechanistic basis for this MNTX barrier protection and observed that silencing of mOP-R attenuated the morphine- and DAMGO-induced EC barrier disruption, but not the permeability response to either thrombin or LPS. Because activation of the sphingosine 1-phosphate receptor, S1P(3), is key to a number of barrier-disruptive responses, we examined the role of this receptor in the permeability response to mOP-R ligation. Morphine, DAMGO, thrombin, and LPS induced RhoA/ROCK-mediated threonine phosphorylation of S1P(3), which was blocked by MNTX, suggesting S1P(3) transactivation. In addition, silencing of S1P(3) receptor expression (siRNA) abolished the permeability response to each edemagenic agonist. These results indicate that MNTX provides barrier protection against edemagenic agonists via inhibition of S1P(3) receptor activation and represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.     

Free oxygen radicals decrease electrical resistance of microvascular endothelium in brain

The effect of free oxygen radicals on the electrical resistance of brain venular endothelium was studied in anesthetized frogs. The technique allowed continuous recording of the electrical resistance of the vascular wall reflecting its ionic permeability. The oxygen radicals were generated by an enzymatic reaction between xanthine oxidase and hypoxanthine supplied to the surface of the exposed brain. Electrical resistance of the venular endothelium decreased within 1-2 s after the reaction was initiated. Hypoxanthine (1 mM) and xanthine oxidase at a concentration of 10, 25, 50, 100, and 250 mU ml-1 lowered resistance to 1.0, 0.9, 0.8, 0.5 and 0.2 X control value, respectively, within a 3 min period of administration. The effect induced by 25 and 50 mU ml-1 of xanthine oxidase was readily reversible, whereas that induced by the two highest concentrations was irreversible within the observation time. The response was totally blocked by allopurinol as well as by superoxide dismutase plus catalase. Pretreatment with methylprednisolone or BW755C (an inhibitor of cyclo- and lipoxygenase) did not inhibit the response, nor did removal of calcium or magnesium from the extracellular medium. Free oxygen radicals are powerful agents that rapidly induce dynamic changes in the electrical resistance of brain vessels, supporting the notion that they may be important mediators of vascular endothelial damage in the brain.     

Bone Morphogenetic Protein-Modulator BMPER Regulates Endothelial Barrier Function

The endothelium serves as a selective barrier and controls the exchange of nutrients, hormones, and leukocytes between blood and tissues. Molecular mechanisms contributing to the pathogenesis of endothelial barrier dysfunction remain incompletely understood. Accumulating evidence implicates bone morphogenetic protein (BMP)-modulator BMPER as a key regulator in endothelial biology. Herein, we analyze the impact of BMPER in the control of endothelial barrier function. To assess the role of BMPER in vascular barrier function in mice, we measured the leakage of Evans blue dye from blood into interstitial lung tissue. BMPER^+/? mice exhibited a significantly higher degree of vascular leak compared with wild-type siblings. In accordance with our in vivo observation, siRNA-based BMPER knockdown in human umbilical endothelial cells increased endothelial permeability measured by FITC-dextran passage in transwell assays. Mechanistically, BMPER knockdown reduced the expression of VE-cadherin, a pivotal component of endothelial adherens junctions. Conversely, recombinant human BMPER protein upregulated VE-cadherin protein levels and improved endothelial barrier function in transwell assays. The effects of BMPER knockdown on VE-cadherin expression and endothelial permeability were induced by enhanced BMP activity. Supporting this notion, activation of BMP4-Smad-Id1 signaling reduced VE-cadherin levels and impaired endothelial barrier function in vitro. In vivo, Evans blue dye accumulation was higher in the lungs of BMP4-treated C57BL/6 mice compared to controls indicating that BMP4 increased vascular permeability. High levels of BMPER antagonized BMP4-Smad5-Id1 signaling and prevented BMP4-induced downregulation of VE-cadherin and endothelial leakage, suggesting that BMPER exerts anti-BMP effects and restores endothelial barrier function. Taken together, this data demonstrates that BMPER-modulated BMP pathway activity regulates VE-cadherin expression and vascular barrier function.