Induction of tissue factor expression in human monocyte/endothelium cocultures

Summary. Induction of tissue factor (TF) expression on monocyctes and endothelial cells is central to the development of septic coagulopathy. Serum concentrations of endotoxin in septic patients who develop disseminated intravascular coagulation (DIC) do not, however, reach the levels that would directly stimulate TF expression on either monocytes or endothelium. We show, using an in vitro coculture system, that the interaction of monocytes with endothelium induces the expression of significant levels of TF. Unstimulated cocultures of monocytes (2 × 10⁴/well) and endothelial cells (2 × 10⁴/well) produced 35.3± 8.5 mU of PCA/well, representing a 5-fold increase over the combined PCA of each cell type cultured alone (7.1 ± 1.5 mU, n = 6, P < 0.001). Significant enhancement was also found in the presence of low concentrations of LPS. Induction of TF protein was confirmed by Western blotting. Fixation of monocytes with paraformaldehyde completely abolished TF induction in cocultures, whereas fixation of endothelium had no effect, suggesting that TF induction occurred in monocytes rather than endothelial cells. Induction of TF in cocultures could be further augmented by preincubating the endothelial cells with IFN-γ. When endothelium was prestimulated with 500U/ml IFN-γ there was 142 ±11% increase over unstimulated cocultures (n = 5, P< 0.01). TF induction was inhibited by 32 ± 6% in the presence of anti-ICAM-1 mAb (n = 5, P < 001). Our results suggest that monocyte interactions with vascular endothelium, regulated by inflammatory cytokines, and mediated by adhesive ligand binding, leads to the induction of functional monocyte TF protein, which may be responsible for the initiation of DIC in sepsis.     

Role of SPan-1 antigen in adhesion of human colon cancer cells to vascular endothelium

Recently E-selectin (ELAM-1, endothelial leukocyte adhesion molecule-1) was shown to recognize not only sialyl Lewis X but also sialyl Lewis A, and these carbohydrate antigens may be involved in the process of the adhesion between cancer cells and endothelial cells in cancer metastasis. To investigate the contribution of sialylated carbohydrate antigen, SPan-1, and sialic acid to the adhesion of human colon cancer cells to endothelial cells, adhesion assay using HUVECs (human umbilical vein endothelial cells) was performed. The adhesion was significantly inhibited by pretreatment with anti-E-selectin antibody, indicating that this adhesion was thought to be mediated by E-selectin. When these cancer cells were pretreated with SPan-1 antibody, the adhesion was significantly inhibited in a concentration-dependent manner. The adhesion was also inhibited by pretreatment with neuraminidase. These findings suggest that the SPan-1 antigen plays a significant role in the adhesion of human colon cancer cells to endothelial cells, and sialylation of the terminal structure of carbohydrate antigens is important in this adhesion.Part of this study was presented at the Second Osaka International Symposium on Gastroenterology held in Osaka, Japan on November 22–23, 1993.     

Prolonged E-selectin induction by monocytes potentiates the adhesion of flowing neutrophils to cultured endothelial cells

We investigated the hypothesis that the infiltration of monocytes into inflamed tissue or damaged vessels would induce a secondary accumulation of neutrophils. Confluent human umbilical vein endothelial cells (HUVEC) and blood monocytes (0.5 or 0.05 monocytes/endothelial cell) were co-incubated for 4 or 24 h. The adhesion of neutrophils flowing over HUVEC was then analysed by video microscopy. Co-incubation caused up to a 40-fold increase in neutrophil adhesion, dependent upon monocyte/HUVEC ratio and duration of incubation. At the lower monocyte/HUVEC ratio, rolling adhesion alone was induced after 4 h co-incubation; however, the full repertoire of rolling, immobilization and migration of neutrophils was observed at all other combinations of co-culture ratio and exposure time. After maximal stimulation by monocytes, antibody blockade of the neutrophil integrin CD18 inhibited neutrophil arrest and migration and revealed underlying rolling adhesion. Rolling was supported by endothelial E-selectin as demonstrated by the almost total abolition of adhesion by a blocking antibody. In a direct comparison, monocytes, tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were assessed for their ability to induce endothelial expression of E-selectin. E selectin was significantly increased by all agents at 4 h, but monocytes alone were able to maintain high levels of E-selectin expression for 24 h. We conclude that monocytes can induce prolonged neutrophil adhesion and migration by activating endothelial cells and causing expression of E-selectin.     

同型半胱氨酸对血管内皮细胞E-选择素、IL-6分泌的影响

目的探讨同型半胱氨酸（homocysteine,Hcy）对人脐静脉内皮细胞E-选择素、白细胞介素6（IL6）分泌的影响。方法在人脐静脉内皮细胞ECV304培养基中加入不同浓度Hcy,孵育不同时间,用ELISA法分别测定培养上清液中E-选择素及IL6的含量。结果终浓度为250μmol/L的Hcy孵育内皮细胞,E选择素的分泌在4h显著上调,6h达峰值;IL6的分泌在6h开始升高,24h达峰值,之后随着时间的延长逐渐下降。Hcy呈浓度依赖性（50μmol/L～500μmol/L）促进内皮细胞E选择素及IL6的分泌。结论Hcy能刺激血管内皮细胞分泌E选择素及IL6。     

蜂胶水提物预处理对血管内皮细胞的保护作用

目的观察蜂胶水提物对损伤血管内皮细胞的保护作用,探讨蜂胶抗动脉粥样硬化的作用及其机制。方法用50 μg/L TNF-α诱导体外培养脐静脉内皮细胞损伤,用50、100、200 mg/L蜂胶水提物分别干预6、12、24 h,分为对照组、模型组、蜂胶低浓度组、蜂胶中浓度组、蜂胶高浓度组、氟伐他汀钠组、联合组,采用流式细胞仪检测细胞间黏附分子1(ICAM-1)和血管细胞黏附分子1(VCAM-1)的表达。结果与对照组比较,模型组ICAM-1和VCAM-1表达明显升高;与模型组比较,蜂胶低浓度组、蜂胶中浓度组和蜂胶高浓度组ICAM-1和VCAM-1明显降低(P0.01)。12 h时与氟伐他汀钠组比较,联合组ICAM-1和VCAM-1表达明显降低(P0.01)。结论蜂胶水提物能降低ICAM-1和VCAM-1的表达。与氟伐他汀钠联合应用,对血管内皮细胞损伤有协同保护作用。     

糖耐量受损患者血清可溶性E选择素和血管细胞黏附分子1的水平

用ELISA法测定 62例糖耐量受损(IGT)患者、24名正常糖耐量 (NGT)者和 23例 2型糖尿病患者可溶性E选择素和可溶性血管细胞黏附分子 1(sVCAM 1)的血清水平,发现IGT患者sVCAM 1浓度明显高于NGT者,提示IGT患者已发生不同程度的血管内皮细胞功能障碍。     

消化系统不同器官原发癌白细胞介素1及肿瘤坏死因子α的比较

[目的]对消化系统不同器官之胃癌、肝癌、结肠癌患者进行白细胞介素1(IL-1)、肿瘤坏死因子α(TNF-α)检测,比较3组间及治疗前、后是否存在差异.[方法]采用放射免疫分析法,对胃癌11例、肝癌15例、结肠癌12例患者化疗前、后进行IL-1、TNFα检测.[结果]胃癌、肝癌患者的IL-1、TNF-α检测值比正常值增加4倍,而结肠癌仅增加2倍,与前2组比较差异有统计学意义(P＜0.05).化疗后与化疗前比较,胃癌、肝癌患者的IL-1、TNF-α值均显著下降(P＜0.05),结肠癌患者亦有下降但无统计学意义:3组间比较差异无统计学意义(P＞0.05).[结论]IL1、TNF-α主要与组织器官的炎症程度相关,炎症的启动因子不同则宿主产生的细胞因子和炎症因子参与炎症反应存在较大差异,而近端结肠癌与患者的背景基因和遗传因素相关.     

西罗莫司对缺氧/复氧诱导内皮细胞-中性粒细胞黏附的影响及机制

目的　探讨西罗莫司对缺氧 复氧后血管内皮细胞表面黏附分子的表达和中性粒细胞 内皮细胞黏附的影响及机制。方法　采用 β N 乙酰氨基己糖苷酶比色法检测黏附率 ,流式细胞术检测内皮细胞表面黏附分子E 选择素、细胞间黏附分子 1(ICAM 1)的表达 ,Fenton反应测定活性氧 (reactiveoxygenspecies,ROS)的含量 ,Western杂交法检测内皮细胞c JunN端激酶 (JNK)及核因子 κB[nuclearfactor κB ,NF κB(P6 5 ) ]蛋白的表达。结果　血管内皮细胞经缺氧 复氧处理后ROS释放增多 ,JNK及NF κB(P6 5 )蛋白表达增加 ,E 选择素、ICAM 1的表达上调 ,其表面中性粒细胞的黏附增加 ,西罗莫司显著抑制缺氧 复氧的上述作用。结论　西罗莫司抑制缺氧 复氧后血管内皮细胞与中性粒细胞的黏附 ,并可能通过抑制ROS、JNK、NF κB的信号转导途径实现     

消化系癌血清微量元素与T细胞亚群相关性研究的价值

目的探讨消化系癌血清微量元素与Ｔ细胞亚群关系的临床意义．方法用原子吸收分光光度法测定食管癌３１２例和健康人１００例血清Ｚｎ,Ｃｕ,Ｆｅ,Ｍｎ,Ｃａ．４８例癌患者和健康人外周血Ｔ细胞亚群用间接免疫荧光法测定．结果消化系癌患者血清中Ｃｕ含量,Ｃｕ／Ｚｎ比,ＣＤ８＋显著高于健康人（Ｐ＜００１）;Ｚｎ,Ｃａ含量,ＣＤ３＋,ＣＤ４＋,ＣＤ４＋／ＣＤ８＋比值明显低于健康人（Ｐ＜００５～Ｐ＜００１）;Ⅲ～Ⅳ期及转移癌患者Ｃｕ含量,Ｃｕ／Ｚｎ比,ＣＤ８＋明显高于Ⅰ～Ⅱ期癌患者（Ｐ＜００５～Ｐ＜００１）．直线相关分析表明：血清Ｃｕ,Ｃｕ／Ｚｎ比值与ＣＤ４＋,ＣＤ４＋／ＣＤ８＋比成负相关,Ｚｎ与ＣＤ４＋／ＣＤ８＋比成正相关（Ｐ＜００５）．多因素Ｌｏｇｉｓｔｉｃ回归分析表明,Ｃｕ,Ｃｕ／Ｚｎ比升高,发生消化系癌的相对危险度升高;Ｚｎ,Ｃａ含量升高,发生消化系癌相对危险度降低．结论对消化系癌患者适量补Ｚｎ,调节和改善宿主抗肿瘤免疫力;Ｃｕ／Ｚｎ比对消化系癌阳性诊断率为７０％,特异性为７３％．因此,Ｃｕ／Ｚｎ值可做为消化系癌的一项诊断指标,并对从高危人群中筛检消化系癌患者有一定的意义     

Relationship between the clinical manifestations of sickle cell disease and the expression of adhesion molecules on white blood cells

BACKGROUND: The severity of sickle cell disease (SCD) increases with leukocyte count. The biological basis could be that leukocyte adherence to vascular endothelium mediated by adhesion molecules (AMs) facilitates vaso-occlusion, the basic pathological process in SCD. OBJECTIVE: To find out if there is a relationship between expression of AMs by leukocytes and the clinical manifestations of SCD. METHODS: Flow cytometry was used to study the relationship between leukocyte AM expression and disease manifestations in 100 patients with homozygous (HbSS) sickle cell disease and 34 genotype HbAA controls. The effect of hydroxyurea therapy on AM expression was also examined. We excluded HbSS patients with any other disease, pregnancy in the previous 3 months, or Haemogloben F (HbF) > or = 10%. RESULTS: Patients with complications of SCD showed high expression of alphaMbeta integrin by the neutrophils; and l-selectin by lymphocytes and neutrophils (P < 0.03). CD18 was highly expressed by neutrophils in patients with sickle nephropathy (P = 0.018), and l-selectin by lymphocytes in those with stroke (P = 0.03). Monocyte l-selectin increased in sickle cell crisis relative to steady state (P = 0.04). Expression of alphaLbeta2 integrin by neutrophils, monocytes, and lymphocytes decreased within a month of hydroxyurea therapy (P < 0.05), with symptomatic improvement in the patients and no more than 3.3% rise in HbF level. CONCLUSIONS: The findings suggest that in SCD (1): High steady-state expression of alphaMbeta2 integrin and l-selectin by leukocytes predisposes to severe manifestations. (2) Increased leukocyte AM expression above steady-state levels could be important in the genesis of crisis. (3) The early symptomatic improvement that follows hydroxyurea therapy is mediated via mechanisms independent of increased HbF, and may involve reduced AM expression in leukocytes. (4) Other treatment modalities that reduce leukocyte AM expression might also confer clinical benefit.     

多效生长因子在消化系肿瘤中的表达及意义

多效生长因子(pleiotrophin,PTN)是分泌性肝素结合细胞因子,PTN基因作为一个原癌基因,与肿瘤的发生和发展关系密切。PTN可分泌到细胞外液(包括血液)中,与肝素具有高亲和性。PTN在细胞分裂、血管生成、神经系统发育和神经系统损伤性疾病以及肿瘤等方面均有作用。近年来研究表明PTN在肿瘤的发生和发展中起着重要的作用,PTN高表达于大多数肿瘤中,尤其是消化系统肿瘤,其表达水平可以作为肿瘤诊断、治疗、预后评价的指标。本文结合国内外文献对PTN在消化系统肿瘤的表达水平及其意义作一概述。     

福辛普利对高脂诱发动脉粥样硬化血管内皮细胞黏附分子-1 mRNA表达的影响

目的利用高脂诱发的动脉粥样硬化模型观察了血管紧张素转换酶抑制剂福辛普利(fosinopril)的抗动脉粥样硬化作用.方法将建立动脉粥样硬化模型日本大耳白兔32只,随机分为三组对照组8只;高胆固醇组11只;福辛普利组13只.检测如下指标⑴计算主动脉粥样斑块面积.⑵测定血浆脂蛋白水平及低密度脂蛋白(LDL)氧化易感性.⑶RT-PCR检测组织中血管内皮细胞黏附分子(VCAM-1) mRNA表达的水平.结果福辛普利组和高脂组与对照组相比总胆固醇和甘油三酯水平明显升高,但二组间差异无显著性(P>0.05).福辛普利组动脉粥样斑块面积明显低于高胆固醇组[(26±5.4)% 比 (41±9.6)%,P<0.05]; 与高脂组比较福辛普利明显延长CuSO4诱导的LDL脂蛋白氧化反应的潜伏期(150 min 与240 min,P<0.001).福辛普利组主动脉VCAM-1/GAPDH mRNA比值低于高胆固醇组(0.90±0.35和1.40±0.51,P<0.05).福辛普利组明显降低了主动脉组织中VCAM-1的mRNA的表达.结论血管紧张素转换酶抑制剂福辛普利通过抑制低密度脂蛋白的氧化及黏附分子VCAM-1的表达延缓早期的动脉粥样硬化病变.     

Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium

In pathological conditions such as sickle cell disease, falciparum malaria and diabetes, an abnormal adherence of erythrocytes to endothelium is concomitant with loss of phospholipid asymmetry resulting in phosphatidylserine (PS) exposure. We have investigated the involvement of PS in this interaction by studying adhesion of human erythrocytes, treated with Ca2+-ionophore A23187 in combination with N-ethylmaleimide, to human umbilical vein endothelial cells in a flow-based assay. Results showed that erythrocytes which exposed PS, massively adhered to HUVEC in a Ca2+-dependent manner. This adhesion was inhibited by PS liposomes and by annexin V, giving clear evidence of the PS dependence of these interactions.     

Insulin enhances vascular cell adhesion molecule-1 expression in human cultured endothelial cells through a pro-atherogenic pathway mediated by p38 mitogen-activated protein-kinase

Aims/hypothesis Although hyperinsulinaemia in Type 2 diabetes in states of insulin resistance is a risk factor for atherosclerotic vascular disease, underlying mechanisms are poorly understood. We tested the hypothesis that insulin increases monocyte-endothelial interactions, which are implicated in atherosclerosis.Methods We treated human umbilical vein endothelial cells with insulin (10^–10 to 10^–7 mol/l) for 0 to 24 h. To dissect potentially implicated signal transduction pathways, we treated endothelial cells with known pharmacological inhibitors of two distinct insulin signalling pathways: the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor wortmannin (3×10^–8 to 10^–6 mol/l), involved in insulin-induced endothelial nitric oxide synthase stimulation, and the p38 mitogen-activated protein (p38MAP) kinase inhibitor SB-203580 (10^–7 to 2×10^–6 mol/l). We measured adhesion molecule expression by cell surface enzyme immunoassays and U937 monocytoid cell adhesion in rotational adhesion assays.Results At pathophysiological concentrations (10^–9 to 10^–7 mol/l), insulin concentration-dependently induced vascular cell adhesion molecule (VCAM)-1 (average increase: 1.8-fold) peaking at 16 h. By contrast, the expression of intercellular adhesion molecule-1 and E-selectin were unchanged. The effect on VCAM-1 was paralleled by increased U937 cell adhesion. In the absence of cytotoxicity, wortmannin significantly potentiated the effect of insulin alone on VCAM-1 surface expression and monocytoid cell adhesion, whereas SB-203580 (10^–6 mol/l) completely abolished such effects.Conclusions/interpretation These observations indicate that insulin promotes VCAM-1 expression in endothelial cells through a p38MAP-kinase pathway, amplified by the PI3-kinase blockage. This could contribute to explaining the increased atherosclerosis occurring in subjects with hyperinsulinaemia, or in states of insulin resistance, which feature a defective PI3-kinase pathway.Abbreviations VCAM-1 vascular cell adhesion molecule-1 - ICAM-1 intercellular adhesion molecule-1 - PI3-kinase phosphatidylinositol 3-kinase - MAP mitogen-activated protein - NO nitric oxide - EIA enzyme immunoassays     

Elevated levels of soluble E-selectin in patients with IDDM and NIDDM: relation to metabolic control

Summary The adhesion of leucocytes to the endothelium, an early step in atherogenesis, is mediated by cell adhesion molecules. In this study we evaluated the concentration of soluble adhesion molecules in patients with insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) and studied its relation to glycaemic control. Soluble adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) were measured in 31 diabetic patients (18 with IDDM and 13 with NIDDM), 20 hyperlipoproteinaemic patients (10 with type IIa and 10 with type IIb) and 20 healthy subjects. Increased E-selectin concentrations were found in the patients with IDDM and NIDDM and in the hyperlipoproteinaemic patients when compared to the control subjects (p<0.01 for all the groups). ICAM-1 was found to be elevated only in the patients with NIDDM (p<0.01). No significant differences in VCAM-1 concentration were found in the different groups of subjects. The concentration of plasma E-selectin was positively correlated with the glycated haemoglobin (r=0.54, p<0.01) in patients with IDDM and NIDDM. In the same patients E-selectin was not related to the concentrations of plasma lipids in spite of the fact that it was found to be elevated in hyperlipoproteinaemic subjects. The results though preliminary suggest that in diabetic patients the concentration of soluble adhesion molecules and especially of E-selectin may be related to metabolic control.Abbreviations IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus - ICAM-1 intercellular adhesion molecule-1 - VCAM-1 vascular adhesion molecule-1 - AGE advanced glycation end products     

Adhesion molecule expression on CD34+ progenitor cells from normal and aplastic anaemia bone marrow

Summary. Aplastic anaemia (AA) is a disease of bone marrow failure. Evidence has been produced for both a stem cell and a stromal cell defect in this disease. The contribution of deficient or defective cell adhesion molecules (CAMs) has not been determined. CAMs have been shown to be important in stem cell-stromal cell interactions and maintenance of haemopoiesis.
In this study the expression of CAMs (LFA-1, LFA-3, ICAM-1, VLA-4, CD44, sLe^x and L-selectin) on CD34+ progenitor cells from 10 normal donors and eight patients with AA was investigated using double immunofluorescence. There was no significant difference in the percentage of CD34⁺ cells that were CAM⁺ between normal and AA bone marrow, suggesting that abnormal CAM expression on AA progenitor cells is not responsible for nor contributes to the pathogenesis of the disease. However, these findings do not exclude abnormal CAM function on progenitor cells, or abnormal expression or function of CAM ligands or counter-receptors on AA stromal cells.     

霉酚酸酯抑制内皮细胞表面粘附分子的表达

目的 :观察霉酚酸酯 (MMF)对炎症因子刺激下内皮细胞表面粘附分子ICAM 1表达的影响。　 方法 :以TNFα(2 0 μg/L)刺激内皮细胞 ,细胞表面的ICAM 1蛋白表达以流式细胞术检测 ,内皮细胞ICAM 1mRNA表达采用逆转录 半定量PCR法进行测定。　 结果 :TNFα(2 0 μg/L)刺激内皮细胞 2 4h ,内皮细胞表面的粘附分子ICAM 1的蛋白和mRNA表达明显上升。MMF可以抑制内皮细胞的ICAM 1蛋白和mRNA的表达 ,这一抑制作用随着MMF剂量的增加而增强。　 结论 :MMF可以抑制内皮细胞粘附分子ICAM 1的表达     

Malignant degeneration of Barrett''s esophagus: the role of the Ki-67 proliferation fraction, expression of E-cadherin and p53

Barrett's columnar epithelium with dysplasia is the most important risk factor for adenocarcinoma of the distal esophagus. The molecular mechanisms responsible for progression of columnar metaplasia to dysplasia and invasive carcinoma are mostly unknown. We investigated expression of the tumor suppressor gene p53, E-cadherin expression and cell proliferation in the metaplasia-dysplasia-carcinoma sequence of esophageal adenocarcinoma. In 24 patients with R0-resected adenocarcinomas of the distal esophagus we evaluated the expression of E-cadherin (antibody HECD-1), mutated p53 (antibody DO1) and cell proliferation (antibody MiB1) by immunohistochemistry in sections of adenocarcinoma, columnar metaplasia, with and without dysplasia, and in squamous epithelium of the esophagus. No p53 immunoreactivity was seen in sections of normal squamous epithelium or columnar metaplasia. Fifty per cent of invasive adenocarcinomas stained positive for mutated p53. The p53 expression correlated with the T-category (P = 0.048) and the N-category (P = 0.024). There was a significant decrease in the expression of E-cadherin from columnar metaplasia to dysplasia and to esophageal adenocarcinoma (P < 0.0001). Expression of E-cadherin in columnar metaplasia without dysplasia was similar to that seen in normal squamous epithelium of the esophagus. The Ki-67 proliferation fraction increased significantly from normal squamous epithelium to columnar metaplasia to dysplasia and to invasive carcinoma (P < 0.001), with a marked expansion of the proliferative component. There was no correlation between cell proliferation, E-cadherin expression and the tumor stage. In contrast to the alterations in the p53 expression, a decreased E-cadherin expression and the expansion of the proliferative component represent an early phenomenon in the malignant degeneration of Barrett's esophagus. This might aid in the early detection of esophageal adenocarcinoma.