Bleomycin induces IL-8 and ICAM-1 expression in microvascular pulmonary endothelial cells

To investigate the pathomechanisms of bleomycin-induced early inflammation of lung parenchyma which is known to result in pulmonary fibrosis, we examined the in vitro effect of bleomycin (BLM) on primary human pulmonary microvascular endothelial cells (HMVEC-L).

After incubation of microvascular endothelial cells with BLM we detected an induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) by immunoblotting. Further, after BLM-exposure an increased concentration of interleukin-8 (IL-8) in culture supernatant and an increased expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on the cell surface have been observed. Real-time PCR revealed up-regulated mRNA expression levels of both, IL-8 and ICAM-1 after treatment with BLM. Finally, pre-treatment with a selective p38 MAPK-inhibitor, SB 203580, potently reduced the BLM-induced up-regulation of IL-8 expression but did not show any effect on expression of ICAM-1.

These results demonstrate that BLM induces the expression of pro-inflammatory molecules in the pulmonary microvascular endothelium, which thereby may actively contribute to the development of early inflammation and later fibrosis of the lung. Furthermore, investigating the effect of an inhibitor of p38 MAPK the data indicate the involvement of p38 MAPK-dependent as well as p38 MAPK-independent mechanisms in the effects of BLM on the pulmonary microvasculature.     

AGEs诱发体外大鼠心脏微血管内皮细胞iNOS表达

目的观察体外原代培养心脏微血管内皮细胞内一氧化氮(NO)生成与诱导型一氧化氮合酶(iNOS)表达的变化及相关性。方法采用不同浓度牛血清白蛋白糖基化终末产物(BSA-AGEs)(50~200 mg/L)分别作用于心脏微血管内皮细胞(0~24 h),检测NO生成,免疫组化及Western blot检测iNOS表达。结果随着AGEs浓度增加及作用时间的延长,心脏微血管内皮细胞中NO生成增多,iNOS表达也增多(P<0.05)。结论BSA-AGEs可以诱发心脏微血管内皮细胞产生毒性NO,从而引发以微血管内皮功能障碍为特征的糖尿病性心肌病。     

Distinct effects of high-glucose conditions on endothelial cells of macrovascular and microvascular origins.

Recent studies implicate hyperglycemia as an important cause of macrovascular and ocular complications in diabetes mellitus. In this study, the authors examined the effect of high glucose on macrovascular and microvascular endothelial cell viability and apoptosis in culture. Human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs) were exposed to normal-glucose conditions (NG) and high-glucose conditions (NG supplemented with 25 mM D-glucose) for 72 h in vitro. D-Mannitol was used as an osmotic control. Cell viability was assessed by methlythiazolydiphenyltetrazolium bromide (MTT) assay, and induction of apoptosis was assessed by Hoechst staining. Statistics were analyzed by paired t tests. In HAECs, cell viability was decreased by 12.9% in high-glucose conditions, and apoptotic cells were significantly increased by 77%. However, in HRECs, cell viability was increased by 14.9% in high-glucose conditions, and apoptotic cells were significantly decreased by 33.3%. Mannitol did not show any effect on cell survival or apoptosis ruling out an osmotic effect. High-glucose conditions reduce cell viability and induce apoptosis in HAECs, which may contribute to macrovascular complications associated with diabetes. In contrast, high-glucose increases viability in HRECs and inhibits apoptosis, which may contribute to the development of diabetic retinopathy.     

Comparative study of adhesion molecule expression in cultured human macro- and microvascular endothelial cells

Culture systems as models for disease are only valid as long as they are comparable to in vivo conditions. The phenotype of cultured endothelial cells (ECs) has only been sporadically compared to the corresponding phenotype in vivo. Thus, we compared by immunolocalization the endothelial expression of ICAM-1, VCAM, and E-selectin in vivo in stimulated/unstimulated human umbilical vein endothelial cells (HUVEC) as a model for macrovascular ECs and stimulated/unstimulated HPMEC (human pulmonary microvessel endothelial cells) as a model for pulmonary microvascular ECs with that in human lungs in vivo (normal and ARDS). Proinflammatory stimuli in vitro were used to stimulate conditions relevant for ARDS. ICAM-1 expression in stimulated HUVEC/HPMEC correlated well with in vivo expression (macro- and microvessels). For E-selectin, the staining pattern in macro/microvessels correlated moderately with unstimulated and well with stimulated HUVEC/HPMEC. For VCAM a good correlation was found for stimulated/unstimulated HUVEC and unstimulated HPMEC. The expression patterns in stimulated HUVEC corresponded well for all three molecules with those in vivo. Thus, the expression patterns in vitro are only partially transferable to in vivo conditions. The study suggests that E-selectin- and VCAM-coated beads could potentially serve in the isolation process of arteriolar and venular ECs.     

炎症刺激因子TNF-α诱导转录因子ESE-1在内皮细胞中的表达

目的: 探讨炎症刺激因子TNF-α介导炎症时上调血管内皮细胞Tie 2基因的机制。 方法: 采用RT-PCR、定量PCR以及Western blotting方法检测TNF-α作用于原代人主动脉内皮细胞株（HAECs）、原代人肺动脉内皮细胞株（HPAECs）时Tie 2基因及ETS转录因子mRNA及蛋白的表达。结果: TNF-α可增加血管内皮细胞Tie 2基因的表达。TNF-α不能增加内皮细胞HAECs和HPAECs的NERF-2、ELF表达；TNF-α刺激后约 24 h 第1次检测到ESE-1 mRNA表达。延长刺激作用时间后，发现在18-36h之间可测到ESE-1 mRNA表达，24 h 为高峰，而蛋白质表达在18 h极弱，36 h达峰值。检测中所观察到ESE-1表达在Tie 2表达之前且表达模式类似。结论: Tie 2基因在TNF-α刺激时的上调与NERF-2或ELF介导无关，ESE-1可能在炎症因子刺激后Tie 2的转录调节中起作用。     

Sera from patients with falciparum malaria induce substance P gene expression in cultured human brain microvascular endothelial cells.

Substance P is a pluripotent neuropeptide capable of inducing neurogenic inflammation, immunoregulation, and vasodilatation. In an effort to contribute to the understanding of the pathophysiology of cerebral malaria, we have evaluated the effects of sera obtained from patients suffering from severe or mild malaria and from a healthy donor with no previous history of exposure to malaria on the expression of the substance P gene by cultured human brain microvascular endothelial cells (HBMEC) and human umbilical-vein endothelial cells. PCR, Southern blotting, hybridization with an internal probe, and densitometry demonstrated that treatment of HBMEC with sera from patients with severe malaria caused remarkably increased expression of the substance P gene. In HBMEC, substance P was not significantly influenced by serum from a healthy donor. Substance P was expressed at almost undetectable levels in untreated HBMEC. Treatment of cultured human umbilical-vein endothelial cells with the same sera produced no signal. The influence of different sera on the expression of substance P by HBMEC suggests that substance P expression may be involved in events leading to the development of severe malaria.     

Internalization of Staphylococcus aureus by endothelial cells induces cytokine gene expression.

The ability of the vascular endothelium to elaborate cytokines in response to gram-positive sepsis has received limited attention. This study examined cytokine expression by human umbilical vein endothelial cells (EC) following infection with a gram-positive bacterial pathogen, Staphylococcus aureus. S. aureus infection of EC resulted in the production of interleukin-6 (IL-6) and IL-1 beta. For IL-6, message was detected at 3 h after infection, protein was present at 24 h, and both message and protein persisted for 72 h. IL-1 beta message was detected at 12 h, IL-1 beta protein was detected at 24 h, and both persisted for 72 h. Message for colony-stimulating factor 1 remained unaltered. UV-killed S. aureus also elicited IL-1 beta and IL-6 message and protein expression at 24 and 48 h. Twenty-one clinical isolates of S. aureus were tested, and all induced IL-6 release by 48 h. However, the laboratory strain 8325-4 did not induce cytokine expression at any time point and was internalized by EC 1,000-fold less than other strains were. Internalization of latex beads by EC did not induce IL-6 gene expression. Furthermore, cytochalasin D treatment of the EC prevented IL-1 and IL-6 induction by S. aureus but not by tumor necrosis factor alpha or lipopolysaccharide. These results indicate that S. aureus is a potent inducer of IL-1 and IL-6 in EC and that internalization of S. aureus by EC is necessary for their cytokine expression. Thus, our data suggest that the vascular endothelium may play an important role in the pathogenesis of septicemia caused by gram-positive organisms.     

Acanthamoeba interactions with human brain microvascular endothelial cells

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis, however, the pathogenic mechanisms associated with this disease remain unclear. One of the primary factors in Acanthamoeba encephalitis is the haematogenous spread, followed by invasion of the blood-brain barrier resulting in the transmigration of Acanthamoeba into the central nervous system. In this study, we have used human brain microvascular endothelial cells, which constitute the blood-brain barrier and studied their interactions with Acanthamoeba. Using in vitro cultures, we showed that Acanthamoeba isolates belonging to genotypes T3, T4 and T11, exhibited increased cytotoxicity on human brain microvascular endothelial cells as well as exhibited higher binding and were considered potential pathogens. In contrast, Acanthamoeba isolates belonging to genotypes T2 and T7 exhibited minimal cytotoxicity and significantly less binding to human brain microvascular endothelial cells (P< 0.01). Furthermore, exogenous alpha-mannose inhibited binding but increased cytotoxicity of human brain microvascular endothelial cells. This is the first demonstration of Acanthamoeba interactions with primary human brain microvascular endothelial cells.     

低氧增强小鼠脑微血管内皮细胞CD73表达

目的：探讨低氧对小鼠脑微血管内皮细胞系bEnd.3细胞CD73的表达及活性的影响。 方法： ①构建bEnd.3细胞低氧模型。②用乳酸脱氢酶检测试剂盒测定细胞上清液中乳酸脱氢酶的含量。③高压液相层析法检测细胞 CD73的活性。④半定量RT-PCR法检测细胞CD73的mRNA表达。⑤生物素化bEnd.3细胞表面蛋白，继而用免疫沉淀及Western blot方法检测CD73蛋白表达。 结果： ①低氧24 h之后bEnd.3细胞乳酸脱氢酶漏出显著高于正常对照组(P＜0.01)。②低氧能刺激bEnd.3细胞表面CD73的酶活性增高，并呈时间依赖性(P＜0.05)。③低氧4 h、8 h，bEnd.3细胞CD73 mRNA表达增加（P＜0.05）。④低氧12 h、24 h，bEnd.3细胞表面CD73蛋白表达增加（P＜0.05）。 结论： 低氧刺激小鼠脑微血管内皮细胞bEnd.3 CD73 mRNA、蛋白水平及酶活性的提高。     

Pseudomonas aeruginosa induces apoptosis in human endothelial cells

Pseudomonas aeruginosa has been shown to enter into human endothelial cells in vitro. To ascertain the effects of bacterial intracellular (IC) infection, endothelial cells were exposed to PAK and PAO-1 strains for 1 h and treated with gentamicin in culture medium for different periods. P. aeruginosa induced a significant production of superoxide and hydrogen peroxide by endothelial cells. Concentrations of IC bacteria were reduced progressively with time and no viable PAO-1 was detected at 24 h after infection. However, IC infection led to killing of 32.2%+/-2.9 and 51.8%+/-3.5 of the cells infected with PAK and PAO-1, respectively, as determined by the MTT assay. By three criteria (transmission electron microscopy, DNA electrophoresis and reactivity with annexin V) infected cells exhibited features of apoptosis. Treatment of infected cells with anti-oxidants (catalase, tocopherol and N -acetyl-L-cysteine) significantly decreased the percentage of cell death. In contrast, treatment with aminoguanidine, an inhibitor of inducible NO synthase, increased significantly the killing of PAO-1 infected cells. Based on these results we speculate that in response to P. aeruginosa infection, endothelial cells increase the production of reactive oxygen intermediates to eliminate IC pathogens, but cells do not resist the oxidative stress and die by apoptosis.     

Lovastatin stimulates p75 TNF receptor (TNFR2) expression in primary human endothelial cells

HMG-CoA reductase inhibitors (statins) exert pleiotropic physiological effects. Among others they attenuate cellular responses to genotoxic and inflammatory stress. We investigated the effect of lovastatin on the expression level of TNF receptors (TNFR) in primary human endothelial cells (HUVEC). ELISA, FACS and immunocytochemical analyses show that lovastatin selectively increases the cell surface expression of TNFR2 without affecting the expression level of TNFR1. This effect of lovastatin is independent from inhibition of cell-cycle progression since cells both in G1- and G2-phase showed elevated levels of TNFR2 after lovastatin treatment. To analyze the physiological relevance of lovastatin-mediated upregulation of TNFR2, we investigated the expression of the cell adhesion molecule E-selectin, which is inducible by TNFalpha. While lovastatin on its own did not change the number of HUVEC expressing E-selectin protein, it promoted the TNFalpha-stimulated increase in the percentage of E-selectin expressing endothelial cells in a dose-dependent manner. This indicates that lovastatin sensitizes HUVEC towards TNFalpha-induced signaling by upregulation of TNFR2 expression. Based on the data, we suggest that statins have impact on endothelial responses to inflammatory stress by modulation of the expression of cytokine receptors.     

Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells

Most cases of neonatal bacterial meningitis develop as a result of a hematogenous spread, but it is not clear how circulating bacteria cross the blood-brain barrier. Attempts to answer these questions have been hampered by the lack of a reliable model of the human blood-brain barrier. Human brain microvascular endothelial cells (HBMEC) were isolated and transfected with a pBR322 based plasmid containing simian virus 40 large T antigen (SV40-LT). The transfected HBMEC exhibited similar brain endothelial cell characteristics as the primary HBMEC, i.e. gamma glutamyl transpeptidase and a high transendothelial electrical resistance. Escherischia coli and Citrobacter spp, two important Gram-negative bacilli causing neonatal meningitis, were found to transcytose across primary and transfected HBMEC, without affecting the integrity of the monolayer. In addition, E. coli and C. freundii invaded transfected HBMEC as shown previously with primary HBMEC. We conclude that E. coli and C. freundii are able to invade and transcytose HBMEC and these bacterial-HBMEC interactions are similar between primary and transfected HBMEC. Therefore, our transfected HBMEC should be useful for studying pathogenesis of CNS infections.     

Minimally oxidized low-density lipoprotein induces tissue factor expression in cultured human endothelial cells.

Oxidatively modified low-density lipoprotein is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis through its biologic effects on vascular cells. This study examined the effects of minimally oxidized preparations of LDL (MM-LDL) on tissue factor (TF) expression by cultured human endothelial cells. Low-density lipoprotein purified from normal donors was modified by exposure to iron or by prolonged storage, resulting in levels of thiobarbituric acid-reacting substances of approximately 2.5 to 4 nmoles/mg cholesterol. Preparations had less than 2.5 pg of endotoxin per microgram LDL and had no intrinsic procoagulant activity. This form of modified but not native LDL induced TF expression in endothelial cells in a time- and dose-dependent manner. Peak TF coagulant activity in cells exposed to 40 micrograms/ml MM-LDL were observed at 4 to 6 hours, and ranged from 50 to 500 pg/10(5) cells, compared with less than 10 pg/10(5) cells exposed to native LDL. Northern blot analysis showed TF mRNA levels to increase approximately 30-fold with exposure to MM-LDL for 2 hours. Induction of TF activity was dependent on the concentration of MM-LDL from 1 microgram/ml to 80 micrograms/ml, a range in which cell viability and morphology were unaffected. The findings suggest that minimally oxidized LDL may be a local mediator promoting thrombosis in atherosclerotic lesions.     

Characterization of the adherence of human monocytes to cytokine-stimulated human macrovascular endothelial cells.

At sites of inflammation, interactions between monocytes and vascular endothelium play an important role in the margination and extravasation of monocytes. The aim of this study was to investigate the relative contributions of the CD11/CD18 family of leucocyte adhesion molecules on monocytes and ICAM-1 and ELAM-1 molecules on endothelial cells (EC) to the binding of monocytes to EC stimulated with recombinant interleukin-1 alpha (rIL-1 alpha), rIL-6, recombinant tumour necrosis factor-alpha (rTFN-alpha) or recombinant interferon-gamma (rIFN-gamma). The adhesiveness of EC for monocytes increased 1.8-2.3-fold after incubation of monolayers of venous or arterial EC with rIL-1 alpha or rTNF-alpha for 4 hr, and 1.6-2.0-fold after stimulation of both types of EC with rIL-1 alpha, rTNF-alpha or rIFN-gamma for 24 hr. Incubation with rIL-6 was without effect. The monoclonal antibodies (mAb) against CD11a, b, c and CD18 on monocytes did not inhibit the increase in the number of monocytes bound to rIL-1 alpha-, rTNF-alpha-, or rIFN-gamma-stimulated EC. However, mAb against ELAM-1 expressed on the surface of 4 hr rIL-1 alpha-stimulated EC slightly inhibited (15-21%) the enhanced monocyte binding. ICAM-1, which exhibited marked expression on 24 hr rIL-1 alpha-, rTNF-alpha- or rIFN-gamma-stimulated EC, did not contribute to the enhanced monocyte binding. The percentage of EC-bound monocytes which had stretched out over the surface of cytokine-stimulated venous or arterial EC was significantly increased compared to the percentage found for non-stimulated EC. It was observed that mild fixation of EC as well as treatment of EC with cytochalasin B or mAb against ICAM-1 did not affect the number of monocytes that were bound to EC, but considerably reduced the percentage of EC-bound monocytes with a stretched morphology. It is concluded that the binding of monocytes to cytokine-stimulated EC is dependent on the type of cytokine and the duration of cytokine stimulation. The increase in the binding of monocytes to cytokine-stimulated EC occurred as a result of CD11/CD18- and ICAM-1-independent factors. The subsequent morphological changes, i.e. stretching of monocytes over the surface of EC, required viable EC and ICAM-1.