类固醇受体辅活化子-3缺失对核因子-κB表达及活性的影响

目的 观察类固醇受体辅活化子(SRC)-3蛋白缺失对脂多糖(LPS)诱导的炎症反应中核因子-κB(NF-κB)表达及活性的影响.方法 健康清洁野生型(SRC-3+/+)小鼠、SRC-3基因敲除(SRC-3-/-)小鼠各20只,雌性,体质量约20 g,分为SRC-3+/+组和SRC-3-/-组,每组设正常(N)、1 h、4 h、12 h四个时相点,每个时相点各5只小鼠.采用腹腔注射5 mg/kg体质量LPS构建炎症反应动物模型,Western blot测定肝组织IκB-α、NF-κB p65/p50和干扰素调控因子-1(IRF-1)的蛋白表达.结果 LPS刺激后早期两组小鼠肝组织IκB-α蛋白水平显著降低,其中SRC-3+/+组小鼠下降更明显;两组小鼠肝组织NF-κB p65/p50蛋白表达和核转位程度均显著增加,其蛋白表达水平SRC-3-/-组小鼠高于SRC-3+/+组,而核转位程度却显著低于SRC-3+/+组;无论是SRC-3+/+组还是SRC-3-/-组小鼠,LPS刺激引起IRF-1蛋白表达水平显著增加,在相应时相点SRC-3-/-组小鼠均显著低于SRC-3+/+组.结论 SRC-3可通过干预NF-κB的活性来调控炎症反应,其蛋白缺失减轻炎症反应程度可能是其导致IκB-α降解障碍和NF-κB活性下降所致.     

氯胺酮调节炎症反应中类固醇受体辅活化子的表达

目的观察脂多糖(LPS)腹腔注射后类固醇受体辅活化子(SRC)家族蛋白表达的早期变化及氯胺酮的影响,探讨氯胺酮调节炎症反应的分子机制。方法将15只健康C57/129雄性小鼠随机分为正常对照组、LPS组、LPS 氯胺酮组,每组5只。LPS 5 mg/kg,腹腔注射；氯胺酮10 mg/kg,肌肉注射,LPS注射后15 min给予。LPS注射后4 h脱臼处死,取肝、脾、肺,采用Western blot测定SRC家族(SRC-1、GRIP-1、NCoA-3)在蛋白水平的变化。结果LPS组与正常对照组相比,肝组织SRC-1蛋向表达水平显著降低,GRIP-1、NCoA-3显著升高；肺组织SRC-1、NCoA -3显著增加,GRIP-1显著降低；脾组织GRIP-1显著降低,NCoA-3显著增加。LPS 氯胺酮组肝组织SRC-1显著高于LPS组,低于正常对照组,肺组织显著低于LPS组,与正常对照组相比明显升高；肝组织GRIP-1显著低于LPS组,高于正常对照组,而脾、肺组织显著增加,与正常对照组相比无显著差别；NCoA-3蛋白表达水平显著低于LPS组,脾、肺组织明显高于正常对照组,而肝组织相差不显著。结论LPS刺激下肝、脾、肺组织SRC家族成员的变化不同,氯胺酮可抑制LPS诱导的SRC表达变化,其对炎症反应的抑制可能是一种间接作用。     

类固醇受体辅活化子-3缺失对脂多糖诱导炎症反应的影响

目的 观察类固醇受体辅活化子(SRC)-3蛋白缺失对脂多糖(LPS)诱导炎症反应的影响.方法 健康清洁野生型(SRC-3~(+/+))小鼠、SRC-3基因敲除(SRC-3~(-/-))小鼠各20只,雌性,体质量约20 g,分为SRC-3~(+/+)组和SRC-3~(-/-)组,每组设正常(N)、1h、4h、12h四个时相点,每个时相点各5只小鼠.采用腹腔注射5 mg/kg体质量LPS构建炎症反应动物模型,观察各时相点重要脏器的病理变化并测定血清TNF-α、IL-1β、IL-6和IL-10浓度.结果 LPS腹腔注射后,SRC-3~(-/-)组小鼠全身情况好于SRC-3~(+/+)组小鼠,但两组的肝、脾、肾、心肌和胸腺病理均未见明显差别.LPS腹腔注射后两组血清TNF-α、IL-1β、IL-6和IL-10的水平均显著升高,但SRC-3~(-/-)组小鼠血清TNF-α、IL-1β、IL-6水平显著低于SRC-3~(+/+)组小鼠,IL-10水平却显著高于SRC-3~(+/+)组.结论 SRC-3蛋白与致炎细胞因子的分泌和释放有关,SRC-3蛋白缺失可减轻炎症反应程度,抑制LPS诱导的TNF-α、IL-1β和IL-6分泌和释放.     

类固醇受体辅活化子-3基因敲除对炎症反应中活化蛋白-1的影响

目的 观察类固醇受体辅活化子(SRC)-3基因敲除对脂多糖(LPS)诱导的炎症反应中活化蛋白-1(AP-1)表达水平及活性的影响.方法 健康清洁野生型(SRC-3+/+)小鼠、SRC-3基因敲除(SRC-3-/-)小鼠各20只,雌性,体质量约20 g,分为SRC-3+/+组和SRC-3-/-组,每组设正常(N)、1 h...     

新生小鼠全身炎症反应综合征时肺组织糖皮质激素受体的表达及意义

目的 检测小鼠全身炎症反应综合征(SIRS)时肺组织糖皮质激素受体(GR)的表达,探讨GR在内毒素引起SIRS过程中的作用.方法 40只新生小鼠随机分为4组,每组10只:实验组A、B、C及对照组D.实验组以尾静脉注射脂多糖(LPS)法建立SIRS模型,其中实验组A予麻醉后尾静脉注射LPS(5 mg/kg);实验组B、C均先予LPS预处理:实验组B予腹腔注射LPS(0.25 mg/kg),24 h后处理同实验组A;实验组C予腹腔注射LPS(0.25 mg/kg),24 h后再次腹腔注射LPS(0.5 mg/kg),第2次注射24 h后处理同实验组A;对照组D仅予以麻醉.各组末次注射24 h后采集肺脏标本,行HE染色肺组织病理评分,免疫组化检测GR蛋白、逆转录聚合酶链反应法(RT-PCR)检测GR mRNA的表达.结果 (1)小鼠肺组织病理评分实验组A(6.44±1.16)、B(4.12±1.07)和C组(4.10±1.31)均高于对照组D(2.52±0.93,P均＜0.05);LPS预处理组B、C的肺组织病理评分明显低于实验组A(P均＜0.05).(2)对照组D小鼠肺组织GR蛋白及GR mRNA的表达水平高于实验组A,低于预处理组B和C,差异有统计学意义(P均＜0.01).结论 新生小鼠SIRS时肺组织GR表达减少,其与SIRS的发展、转归相关;LPS重复刺激可使新生小鼠产生耐受性,耐受性的产生与GR的表达调高相关.     

Alpha-黑素细胞刺激素对内毒素血症小鼠肝肺组织表达高迁移率族蛋白1的抑制作用

目的 研究Alpha-黑素细胞刺激素( α-MSH)对内毒素血症小鼠肝、肺组织中表达高迁移率族蛋白1(HMGB1)的调控作用.方法 腹腔内注射(ip)LPS(25 g/kg)和D-Gal(100 mg/kg)建立内毒素血症小鼠模型,分别在LPS刺激小鼠1、2、3 h后腹腔注射α-MSH(2.5 mg/kg),24 h后处死小鼠,取小鼠肺、脑、脾、肝、肾组织,应用RT-PCR和Western blot的方法,从基因、蛋白两个水平检测α-MSH对HMGB1表达的调节作用.结果 HMGB1 mRNA及蛋白在内毒素血症小鼠肺、脑、脾、肝、肾中均有表达,但在肝、肺中的表达量高于其他组织;于LPS刺激小鼠3 h之内腹腔注射α-MSH,能显著下调HMGB1 mRNA及蛋白的表达,并且1 h之内给药效果最理想.结论 α-MSH能有效抑制内毒素血症小鼠肝、肺组织中HMGB1的表达.     

脂多糖刺激前后小鼠肺肝脾组织中Toll样等受体基因表达情况

目的了解脂多糖(LPS)对肺、肝、脾组织中与LPS信号转导有关的受体的基因表达情况的影响。方法LPS刺激前后取小鼠肺、肝、脾组织总RNA，采用逆转录-聚合酶链反应(RT-PCR)半定量测定脂多糖结合蛋白(LBP)、CD14、Toll样受体2(TLR2)、TLR4和肿瘤坏死因子-α(TNF-α)等基因的表达情况。结果正常组织中，肺有TLR2的表达，肝有TLR2、CD14、LBP的表达，脾有TLR2的表达；注射LPS后的5h内，肺组织TLR2、TLR4、CD14和TNF-α，肝组织TLR2、CD14、LPB和TNF-α，脾组织TLR2、TLR4、CD14和TNF-α的基因表达均呈逐渐增加趋势。结论正常情况下TLR2等基因在不同组织中的表达有差异，LPS刺激5h内各基因的表达提高，从而提高机体对LPS等病原体模式识别分子的反应性。     

内毒素性休克大鼠肺组织糖皮质激素受体mRNA的表达

目的 探讨内毒素(LPS)性休克大鼠肺组织糖皮质激素受体(GR)mRNA的表达.方法 56只SD大鼠,随机(随机数字法)分IPS休克组(16只)、LPS+血管活性肠肽(vasosctive intestinal peptide,VIP)组(16只)、LPS+VIP+糖皮质激素(GC)组(16只)和对照组(8只).尾静脉注射LPS制作休克模型,尾静脉注射LPS 10 mg/kg后,分别于15 min内注射VIP 5 nmol/kg或VIP 5 nmol/ks+甲基强的松龙3 mg/kg,对照组注射等容量生理盐水,分别于沣射后6 h和24 h处死,留取肺标本,观察肺组织病理变化,RT-PCR榆测肺组织GR mRNA的表达.结果 (1)肺组织病理改变:LPS组见肺泡腔结构破坏、肺泡间隔增宽、炎性细胞浸润、出血、间质水肿,细胞器破坏,LPS+VIP组和IPS+VIP+GC组病变较轻.(2)GR mRNA的表达:注射LPS后6 h,LPS组和LPs+VIP组GR mRNA表达下降,与对照组相比,差异具有统计学意义(P＜0.05).LPS组下降较LPS+VIP组稍明显,但差异无统计学意义(P＞0.05).24 h GR mRNA表达恢复.LPS+VIP+GC组GR mRNA表达6 h增加,24 h GR mRNA表达更高(P＜0.05).结论 LPS致肺损伤时,肺组织GR mRNA的表达减少.VIP和GC可抵抗炎症反应、减轻肺损伤,其机制可能与增强GR mRNA的表达有关.     

脂多糖诱导大鼠主动脉内皮细胞蛋白C受体和蛋白酶活化受体1的表达及血必净注射液的干预作用

目的 观察脂多糖(LPS)诱导的大鼠主动脉内皮细胞蛋白C受体(EPCR)和蛋白酶活化受体1(PAR1)的表达,并探讨血必净注射液对其的干预作用.方法 采用组织贴块法培养Wistar大鼠主动脉内皮细胞,培养1周后用流式细胞仪鉴定内皮细胞纯度.按1∶3传代至3～4代时,随机将细胞分为空白对照组、LPS刺激组(1 mg/L)、血必净干预组(LPS 1 mg/L,血必净注射液10 g/L),活化蛋白C(APC)干预组(LPS 1 mg/L,APC 0.1 mg/L).分别在12、24、48、72 h采用逆转录-聚合酶链反应(RT-PCR)测定内皮细胞EPCR和PAR1的mRNA表达;用流式细胞仪检测EPCR和PAR1的蛋白表达.结果 12 h时各组间EPCR和PAR1蛋白表达水平比较差异均无统计学意义(P均>0.05);LPS刺激组EPCR和PAR1 mRNA表达则均降低,APC和血必净干预后两值均升高(P<0.05或P<0.01).24～72 h LPS刺激组EPCR的mRNA和蛋白表达水平均明显低于空白对照组(P<0.05或P<0.01),EPCR的表达随LPS作用时间延长而减少,呈明显的负相关;而血必净干预组EPCR表达明显高于LPS刺激组(P均<0.01).LPS刺激组PAR1的mRNA和蛋白表达水平较空白对照组有所降低(P<0.05或P<0.01),用血必净干预后,PAR1的mRNA和蛋白表达水平有所增加(P均<0.01).与APC干预组比较,血必净干预组EPCR和PAR1的蛋白表达增加更为明显.结论 血必净注射液能从基因和蛋白水平增加由LPS诱导大鼠主动脉EPCR和PAR1的表达作用,可能与其保护内皮细胞的功能抑制其凋亡有关.     

AngⅡ及其受体拮抗剂调节大鼠肺微血管内皮细胞ACE2蛋白表达的体外实验

目的 观察血管紧张肽(angiotensin,Ang )Ⅱ及其受体拮抗剂对体外培养的大鼠肺微血管内皮细胞(pulmonary microvascular endothelial cells, PMVEC)内血管紧张肽转化酶(angiotensin converting enzyme,ACE)2表达的影响.方法 体外培养大鼠PMVEC,随机分为4组.正常对照组;AngⅡ组:以浓度为10-7 mol/L的AngⅡ与之分别孵育3 h、6 h、12 h和24 h;脂多糖(lipopolysaccharide,LPS)组:给予10-2 mg/mL的LPS刺激细胞24 h;AngⅡ1型受体(angiotensin Ⅱ type 1 receptor,AT1R)拮抗剂[Sar1,Ile8]-Ang Ⅱ干预组:使用[Sar1,Ile8]-Ang Ⅱ预处理细胞30 min后再给予10-7 mol/L Ang Ⅱ或10-2 mg/mL LPS刺激24 h;采用RT-PCR、Western blot方法检测ACE2表达的变化情况.结果 与正常对照组相比,AngⅡ刺激组于不同时相点ACE2表达均显著降低(P<0.05),ACE2表达与AngⅡ刺激的时间呈负相关;LPS组ACE2蛋白表达亦显著降低(P<0.05).[Sar1,Ile8]-Ang Ⅱ干预组ACE2蛋白表达较LPS/Ang Ⅱ组显著增加(P<0.05).结论 AngⅡ可以降低体外培养PMVEC中ACE2的表达,并与Ang Ⅱ刺激时间呈负相关;LPS亦可以显著降低ACE2蛋白表达;AT1R拮抗剂可以降低AngⅡ及脂多糖对ACE2蛋白表达的抑制作用,AngⅡ和ACE2相互作用可能与急性肺损伤(acute lung injury,ALI)时PMVEC损伤的发生、发展有关.     

敲除腺苷A2A受体基因对慢性低O2高CO2模型小鼠前额叶皮质细胞凋亡及磷酸化p38MAPK蛋白的影响

目的 观察敲除腺苷A2A受体基因对慢性低O2高CO2模型小鼠前额叶皮质细胞凋亡以及磷酸化p38丝裂原活性蛋白激酶（p-p38MAPK）蛋白表达的影响。 方法 采用随机数字表法将16只腺苷A2A受体野生型（+/+）小鼠和16只腺苷A2A受体基因敲除型（-/-）小鼠各分为2个亚组,分别是对照-野生基因组、4周低O2高CO2-野生基因组（简称模型-野生基因组）、对照-基因敲除组、4周低O2高CO2-基因敲除组（简称模型-基因敲除组）,每组各8只小鼠。将模型-野生基因组、模型-基因敲除组小鼠置于常压低O2高CO2动物舱内,舱内O2浓度维持在9%~11%水平,CO2浓度维持在5.5%~6.5%水平,每天干预8 h,每周干预6 d,持续干预4周。于4周制模结束后采用原位末端标记法(TUNEL)检测各组小鼠前额叶皮质细胞凋亡情况,采用蛋白免疫印迹法检测各组小鼠前额叶皮质磷酸化p38MAPK蛋白表达水平。 结果 两模型亚组前额叶皮质凋亡细胞数量均较相应的对照亚组明显增加(P<0.05),并且模型-野生基因组皮质凋亡情况较模型-基因敲除组更显著(P<0.05);两模型亚组前额叶皮质p-p38MAPK蛋白表达均较相应的对照亚组明显上调(P<0.05),并且模型-野生基因组p-p38MAPK蛋白表达上调幅度较模型-基因敲除组更显著(P<0.05)。 结论 敲除腺苷A2A受体基因能抑制慢性低O2高CO2模型小鼠前额叶皮质p38MAPK信号转导通路活化,减少前额叶皮质神经细胞凋亡,为改善慢性阻塞性肺疾病（COPD）患者认知功能提供更多实验依据。     

Impaired hemostasis and protection against thrombosis in protease-activated receptor 4-deficient mice is due to lack of thrombin signaling in platelets.

Platelets from protease-activated receptor 4 (PAR4)-deficient mice are unresponsive to thrombin, and Par4-/- mice have prolonged bleeding times and are protected against thrombosis. However, in addition to its role in platelets, PAR4 contributes to thrombin signaling in cells in the blood vessel wall that might participate in hemostasis and thrombosis, such as endothelial cells. To determine whether the hemostatic and thrombotic phenotypes of Par4-/- mice were due to loss of PAR4 function in hematopoietic vs. other cell types, tail bleed times and thromboplastin-induced pulmonary embolism were examined in lethally irradiated mice reconstituted with Par4+/+ or Par4-/- bone marrow. In Par4+/+ and Par4-/- mice reconstituted with Par4+/+ marrow, the median tail bleed times were 2.0 and 1.7 min, respectively, vs. > 10 min for both Par4+/+ and Par4-/- mice reconstituted with Par4-/- marrow. In the pulmonary embolism model, Par4+/+ and Par4-/- mice reconstituted with Par4+/+ marrow survived a median of 3.7 and 2.8 min, respectively, after administration of thromboplastin, vs. > 20 min for both Par4+/+ and Par4-/- mice reconstituted with Par4-/- marrow. Further, the phenotype of mice reconstituted with Par4-/- marrow was almost as dramatic as that seen in Nf-e2-/- mice, which lack platelets. These data strongly suggest that increased tail bleed times and protection against thrombosis in Par4-/- mice are accounted for by lack of PAR4 function in platelets, emphasize the importance of thrombin signaling in platelets among the multiple pathways and cell types that govern hemostasis and thrombosis.     

Differential effects of delta9-tetrahydrocannabinol and methanandamide in CB1 knockout and wild-type mice

Mice devoid of CB1 cannabinoid receptors (CB1-/- mice) provide a unique opportunity to further investigate the role of CB1 receptors in exocannabinoid and endocannabinoid effects. CB1-/- mice (N = 18) and their wild-type littermates (CB1+/+ mice; N = 12) were placed in standard mouse operant chambers and trained to lever press under a fixed ratio 10 schedule of reinforcement. When stable lever press responding under the fixed ratio 10 schedule had been established, cannabinoids and noncannabinoids were administered to both groups. CB1+/+ mice acquired the lever press response more readily than CB1-/- mice. Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) decreased lever press responding in CB1+/+ mice only, whereas methanandamide, a metabolically stable endocannabinoid analog, produced similar response rate decreases in both genotypic groups. Similar to Delta(9)-THC, another endocannabinoid analog, (R)-(20-cyano-16,16-dimethyl docosa-cis-5,8,11,14-tetraeno)-1'-hydroxy-2'-propylamine (O-1812), decreased responding in CB1+/+ mice, but not in CB1-/- mice. The CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A) blocked the effects of Delta(9)-THC, but not those of methanandamide. Because methanandamide binds poorly to CB2 receptors, these results suggest possible non-CB1, non-CB2 mechanisms of action for methanandamide-induced behavioral disruption of lever press responding. Ethanol and morphine elicited greater response decreases in CB1-/- mice than in CB1+/+ mice, suggesting a possible role of CB1 receptors in the rate disruptive effects of these drugs. In contrast, diazepam did not produce between group differences, suggesting that CB1 receptors are not involved in diazepam-induced disruption of lever press responding.     

Urokinase is required for T lymphocyte proliferation and activation in vitro

We have previously demonstrated that urokinase-deficient (uPA-/-) mice do not increase lung T lymphocyte number and fail to mount protective immune responses during pulmonary Cryptococcus neoformans infection. These observations suggest a previously unconsidered role for urokinase-type plasminogen activator (uPA) in T lymphocyte-mediated immune responses. Accordingly, we sought to determine whether uPA is required for T cell receptor-mediated (TCR-mediated) lymphocyte proliferation and activation. Splenocytes from uPA-/- and uPA+/+ mice were stimulated with concanavalin A (Con A). The uPA-/- mice had diminished T cell proliferation as compared with uPA+/+ mice. Coculturing uPA-/- T cells with uPA+/+ accessory cells led to the restoration of proliferation. Similarly, T cell proliferation induced by CD3 cross-linking was diminished in uPA-/- mice as compared with uPA+/+ mice. T lymphocyte activation, defined as the induced expression of antigens and the elaboration of cytokines, was determined. The expression of CD69 and that of CD49d were diminished in response to Con A stimulation in uPA-/- mice as compared with uPA+/+ mice. The elaboration of cytokines in response to Con A was also altered in the uPA-/- mice. The production of the Th1 cytokines interferon-gamma and interleukin-12 was diminished in uPA-/- mice as compared with uPA+/+ mice. The uPA-/- mice produced increased amounts of interleukin-10, a Th2 cytokine. We conclude that the lack of uPA results in impaired T cell activation and proliferation in response to TCR-mediated signaling and the expression of a less Th1-polarized profile of cytokines. These findings suggest that the inability of uPA-/- mice to combat Cryptococcus neoformans infection may be caused by the impairment of T lymphocyte immune responses in the absence of uPA.     

乌司他丁对脂多糖致大鼠急性肺损伤肺组织糖皮质激素受体的影响

目的探讨乌司他丁(UTI)对脂多糖(LPS)致大鼠急性肺损伤(ALI)组织糖皮质激素受体(GR)的影响,并了解其可能机制。方法清洁级成年雄性SD大鼠54只,随机分为3组:空白对照组(Con组,n=18),LPS组(n=18),脂多糖+乌司他丁组(LPS+UTI组,n=18)。各组又按照不同的时间点被分为4 h、8 h、12 h三个亚组。在各时间点处死大鼠,利用Western blot检测大鼠肺组织中GR的表达,实时荧光定量PCR检测GR m RNA的表达,同时酶联免疫吸附法(ELISA)检测肺组织匀浆液中肿瘤坏死因子(TNF)-α和白细胞介素(IL)-10的浓度以及肺组织病理学的变化。结果与对照组相比,LPS组GR蛋白表达在各时间点降低(P<0.05),LPS+UTI组GR蛋白在各时间点的表达水平高于LPS组,低于Con组(P<0.05);与对照组相比,LPS组GR m RNA表达降低(P<0.05),LPS+UTI组GR m RNA在各时间点的表达水平略高于LPS组(P>0.05),低于Con组(P<0.05);与对照组相比,LPS组TNF-α的表达在各时间点升高(P<0.05),LPS+UTI组TNF-α的表达水平在各时间点低于LPS组,高于Con组(P<0.05);与对照组相比,LPS组IL-10的表达在各时间点升高(P<0.05),LPS+UTI组IL-10的表达水平在各时间点高于LPS组(P<0.05)。组织病理学检查显示:LPS组肺组织见肺泡结构破坏,炎性细胞浸润,肺泡间隔增宽,出血,水肿,LPS+UTI组肺组织病理损害较LPS组明显减轻。结论 UTI能提高GR的表达;TNF-α和IL-10出现高峰的时间不同。     

The angiotensin II AT1 receptor-associated protein Arap1 is involved in sepsis-induced hypotension

Introduction

Hypotension in septic patients results from hypovolemia, vasodilatation and hyporeactivity to vasoconstrictors, such as angiotensin II. The AT1 receptor-associated protein 1 (Arap1) is expressed in vascular smooth muscle cells and increases the surface expression of the AT1-receptor in vitro. We hypothesized that dysregulation of Arap1 may contribute to vascular hyporeactivity to angiotensin II during endotoxemia.

Methods

Arap1-deficient mice were used to assess the role of Arap1 in sepsis-induced hypotension. The isolated perfused kidney was used as an in vitro model to determine the relevance of Arap1 for vascular resistance and sensitivity to angiotensin II.

Results

During endotoxemia, mean arterial blood pressure (MAP) decreased in both genotypes, with the time course of sepsis-induced hypotension being markedly accelerated in Arap1-/- compared to +/+ mice. However, baseline MAP was similar in Arap1-/- and wildtype mice (102 ± 2 vs.103 ± 2 mmHg; telemetry measurements; n = 10; P = 0.66). Following lipopolysaccharide (LPS) injections (3 mg/kg), Arap1 expression was successively down-regulated in the wildtype mice, reaching levels below 10% of baseline expression. The endotoxemia-related decline in Arap1 expression could be recapitulated in cultured mesangial cells by incubation with pro-inflammatory cytokines, such as tumor necrosis factor α and interferon γ. Plasma renin concentration was increased in Arap1-/- mice compared to wildtype mice (66 ± 6 vs. 41 ± 4 ng AngI/ml/h; n = 23; P = 0.001), presumably contributing to preserved MAP under baseline conditions. The sensitivity of the vasculature to angiotensin II was reduced in Arap1-/- compared to +/+ mice, as determined in the isolated perfused kidney.

Conclusions

Our data suggest that down-regulation of Arap1 expression during sepsis contributes to the development of hypotension by causing reduced vascular sensitivity to angiotensin II.     

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.