Inhibition of shiga toxin cytotoxicity in human renal cortical epithelial cells by nitrobenzylthioinosine

Nitrobenzylthioinosine (NBTI), a nucleoside-transport inhibitor, has been found to possess the ability to prevent the cytotoxic action of Shiga toxin (Stx) 1 in human renal cortical epithelial cells (HRCECs), thereby protecting HRCECs from cell death. Further examination revealed that NBTI does not affect either the binding or the endocytosis of Stx1 but alters the intracellular transport of Stx1. Generally, endocytosed Stx1 is thought to be transported from endosomes to the endoplasmic reticulum. In NBTI-treated cells, however, the endocytosed Stx1 is delivered to an early endosome, but no further transportation occurs. Moreover, Stx1 is rapidly excreted from NBTI-treated HRCECs, preventing the accumulation of Stx1. Investigation of the NBTI-mediated protection mechanism against Stx cytotoxicity may provide insights into the analysis of Stx-mediated cell damage and lead to improvements in therapeutic approaches for diseases caused by Stx.     

FVIII production by human lung microvascular endothelial cells

While extrahepatic factor VIII (FVIII) synthesis suffices for hemostasis, the extrahepatic production sites are not well defined. We therefore investigated the ability of the human lungs to produce FVIII. Lungs from heart-beating donors who were declined for transplantation were perfused and ventilated in an isolated reperfusion model for 2 hours. A progressive accumulation of FVIII and von Willebrand factor (VWF) was recorded in the perfusion medium in 3 of 4 experiments. By contrast, factor V, fibrinogen, and immunoglobulin G (IgG) levels remained constant during the perfusion period, indicating that the accumulation of FVIII and VWF was not due to diffusion from the intercellular medium into the vascular system. Purified human lung microvascular endothelial cells produced FVIII during at least 2 passages in vitro. Altogether, these data identify the lung endothelial cells as a FVIII production site in humans.     

PECAM-1 isoform-specific functions in PECAM-1-deficient brain microvascular endothelial cells

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is alternatively spliced generating eight isoforms that only differ in the length of their cytoplasmic domain. Multiple isoforms of PECAM-1 are present in the endothelium and their expression levels are regulated during vascular development and angiogenesis. However, the functional significance of PECAM-1 isoforms during these processes remains largely unknown. We recently showed that mouse brain endothelial (bEND) cells prepared from PECAM-1-deficient (PECAM-1-/-) mice differ in their cell adhesive and migratory properties compared to PECAM-1+/+ bEND cells. Here we demonstrate that the restoration of PECAM-1 expression in these cells affects their adhesive and migratory properties in an isoform-specific manner. Expression of Delta14&15 PECAM-1, the predominant isoform present in the mouse endothelium, in PECAM-1-/- bEND cells activated MAPK/ERKs, disrupted adherens junctions, and enhanced cell migration and capillary morphogenesis in Matrigel. In contrast, expression of Delta15 PECAM-1 in PECAM-1-/- bEND cells had minimal effects on their activation of MAPK/ERKs, migration, and capillary morphogenesis. The effects of PECAM-1 on cell adhesive and migratory properties were mediated in an isoform-specific manner, at least in part, through its interactions with intracellular signaling proteins, including SHP-2 and Src. These results suggest that the impact of PECAM-1 on EC adhesion, migration, and capillary morphogenesis is modulated by alternative splicing of its cytoplasmic domain.     

Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells

Recent evidence from this laboratory indicated that reduced expression of caveolin-1 accompanied the diminished expression of tight junction (TJ)-associated proteins occludin and zonula occludens-1 (ZO-1) following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2 (formerly called MCP-1). Because attenuated caveolin-1 levels have also been correlated with heightened permeability of other endothelia, the objective of this study was to test the hypothesis that reduced caveolin-1 expression is causally linked to the action of CCL2 on BMEC junctional protein expression and barrier integrity. This was achieved using adenovirus to nondestructively deliver caveolin-1 siRNA (Ad-siCav-1) to BMEC monolayers, which model the blood-brain barrier (BBB). Treatment with siRNA reduced the caveolin-1 protein level as well as occludin and ZO-1. Additionally, occludin exhibited dissociation from the cytoskeletal framework. These changes were attended by comparable alterations in adherens junction (AJ)-associated proteins, VE-cadherin and beta-catenin, increased BMEC paracellular permeability, and facilitated the ability of CCL2 to stimulate monocytic transendothelial migration. Furthermore, treating BMECs with cavtratin, a synthetic cell-permeable peptide encoding the caveolin-1 scaffolding domain, antagonized effects of both Ad-siCav-1 and CCL2. These results collectively highlight caveolin-1 loss as a critical step in CCL2-induced modulation of BMEC junctional protein expression and integrity, and possibly serve a crucial role in regulating inflammation at the BBB.     

Induction of cyclooxygenase ii in human synovial microvessel endothelial cells by interleukin-1

Conclusion. IL-1 treatment of HSE cells induces COX II, as demonstrated by both Northern blotting and immunoprecipitation. The induction of COX II expression provides, at least in part, a mechanism for the pronounced increase in prostanoid synthesis observed in HSE cells following incubation with IL-1. The selective up-regulation of HSE COX II by inflammatory cytokines such as IL-1 suggests that development of specific pharmaceutical inhibitors for this novel isozyme may provide significant new therapeutic advantages in the treatment of RA. Objective. To characterize the effects of inter-leukin-1α (IL-1) on prostanoid biosynthesis by human rheumatoid synovium microvessel endothelium (HSE). Methods. HSE cells were treated with cytokines, metabolic inhibitors, and steroids under various conditions, and prostaglandin biosynthesis was determined by radioimmunoassay. Newly synthesized cyclooxygenase (COX) was quantitated by immunoprecipitation of metabolically labeled HSE cell lysates. The effects of IL-1 on levels of messenger RNA (mRNA) for COX II were also determined. Results. IL-1 induced an increase in COX activity (as assessed by prostaglandin E₂ release) that was dose-and time-dependent and was blocked by cycloheximide, actinomycin D, and dexamethasone. IL-1 induced a selective increase in COX II mRNA and biosynthesis of COX II protein that was blocked by dexamethasone. Conclusion. Mortality rates are increased at least 2-fold in RA, and are linked to clinical severity.     

Hypoxia induces angiogenic factors in brain microvascular endothelial cells

Hypoxia is increasingly recognized as an important contributing factor to the development of brain diseases such as Alzheimer's disease (AD). In the periphery, hypoxia is a powerful regulator of angiogenesis. However, vascular endothelial cells are remarkably heterogeneous and little is known about how brain endothelial cells respond to hypoxic challenge. The objective of this study is to characterize the effect of hypoxic challenge on the angiogenic response of cultured brain-derived microvascular endothelial cells. Brain endothelial cell cultures were initiated from isolated rat brain microvessels and subjected to hypoxia (1% O(2)) for various time periods. The results showed that hypoxia induced rapid (≤ 0.5h) expression of hypoxia-inducible factor 1α (HIF-1α) and that cell viability, assessed by MTT assay, was unaffected within the first 8h. Examination of brain endothelial cell cultures for pro- and anti-angiogenic proteins by western blot, RT-PCR and ELISA revealed that within 0.5 to 2h of hypoxia levels of vascular endothelial growth factor and endothelin-1 mRNA and protein were elevated. The expression of heme oxygenase-1 also increased but only after 8h of hypoxia. In contrast, similar hypoxia exposure evoked a decrease in endothelial nitric oxide synthase and thrombospondin-2 levels. Exposure of brain endothelial cell cultures to hypoxia resulted in a significant (p<0.001) decrease (94%) in tube length, an in vitro index of angiogenesis, compared to control cultures. The data indicate that, despite a shift toward a pro-angiogenic phenotype, hypoxia inhibited vessel formation in brain endothelial cells. These results suggest that in brain endothelial cells expression of angiogenic factors is not sufficient for the development of new vessels. Further work is needed to determine what factors/conditions prevent hypoxia-induced angiogenic changes from culminating in the formation of new brain blood vessels and what role this may play in the pathologic changes observed in AD and other diseases characterized by cerebral hypoxia.     

The effects of scleroderma serum on human microvascular endothelial cells. Induction of antibody-dependent cellular cytotoxicity

To investigate the vascular immunopathology of systemic sclerosis, we developed a model consisting of human microvascular endothelial cells, leukocytes, and serum. Sera from 19% of the patients studied mediated antibody-dependent cellular cytotoxicity against endothelial cells. Some sera also mediated cytotoxicity against aortic endothelium and fibroblasts. K lymphocytes, the cells that mediate antibody-dependent cellular cytotoxicity, were identified in the skin of some patients. The sera alone were not cytotoxic or growth inhibitory, and did not affect endothelial prostacyclin production.     

Collagen type 1 retards tube formation by human microvascular endothelial cells in a fibrin matrix

Angiogenesis, or the formation of new microvessels, is often encountered in pathological situations. A fibrinous exudate can often act as a temporary matrix for the ingrowth of these new microvessels. This matrix consists mainly of fibrin, but is mingled with other plasma components and interstitial collagen fibres. In vitro, capillary-like tube formation can be mimicked by exposing human microvascular endothelial cells (hMVECs), seeded on top of a three-dimensional fibrin matrix, to an angiogenic growth factor (e.g. fibroblast growth factor (FGF)-2) and the cytokine tumour necrosis factor (TNF)-. Plasmin activity is required in this process. We investigated whether the angiogenic potential of hMVECs was altered by the presence of collagen. The addition of type I collagen to fibrin matrices dose-dependently inhibited tube-formation. Tube-formation in these fibrin/collagen matrices by hMVECs required matrix metalloprotease (MMP) activity, as well as plasmin activity. On a pure collagen type I matrix, hMVECs were not able to form tube-like structures in the matrix but formed sprouts. This sprouting required MMP activity and was, in contrast to the tube-like structures in a fibrin matrix, not influenced by hypoxia. These data indicate that the interaction between endothelial cells and different matrix components is of importance for the angiogenic potential of these cells.     

Inhibition of p38 mitogen-activated protein kinase ameliorates cytokine up-regulated shigatoxin-1 toxicity in human brain microvascular endothelial cells

Brain injury in hemolytic-uremic syndrome (HUS) may be enhanced by inflammatory cytokine up-regulation of endothelial cell sensitivity to shigatoxin (Stx). The present study investigated whether inflammatory cytokine up-regulation of Stx toxicity could be ameliorated by inhibiting candidate signal transduction pathways. Exposure of human brain endothelial cells (HBECs) to tumor necrosis factor (TNF) greatly increased Stx-1 and Stx-2 cytotoxicity; this was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK), but not c-Jun kinase. SB203580, a specific inhibitor of p38 MAPK, reduced TNF-stimulated Stx cytotoxicity in HBECs, TNF-stimulated (125)Stx-1 binding to intact HBECs, the cellular content of Gb3 (galactose alpha 1,4, galactose ss 1,4, glucose-ceramide) (the Stx receptor), and TNF-stimulated Gb3 synthase and glucosylceramide synthase activities but did not affect lactosylceramide synthase activities or mRNA content. Thus, inhibition of p38 MAPK substantially reduces inflammatory cytokine up-regulation of Stx-receptor synthesis and cell-surface expression, thereby decreasing Stx cytotoxicity. Inhibition of p38 MAPK may be of therapeutic benefit in HUS.     

Des-acyl ghrelin protects microvascular endothelial cells from oxidative stress-induced apoptosis through sirtuin 1 signaling pathway

ObjectiveGhrelin is a stomach-derived hormone. Acylation of ghrelin has been essential for its biological activities such as stimulating appetite. On the other hand, the function of des-acyl ghrelin (Des-G) has not been fully elucidated. The aim of the present study is to examine the anti-apoptotic effect of Des-G on endothelial cells.Materials/MethodsAfter human retinal microvascular endothelial cells (RMECs) were pretreated with or without 100 nmol/L Des-G, apoptosis was induced with 0.1 mmol/L hydrogen peroxide (H₂O₂). For pharmacological inhibition of surtuin 1 (SIRT1) catalytic activity, the cells were treated with 10 μmol/L Ex-527. Inhibition of SIRT1 with siRNA was also performed. The quantitative estimation of DNA fragmentation was used as a marker of apoptosis. Furthermore, total SIRT activity in nuclear extracts, mRNA and protein levels of SIRT1, manganese superoxide dismutase (MnSOD) and catalase were determined.ResultsDes-G pretreatment protected RMECs from oxidative stress-induced apoptosis and increased SIRTs deacetylase activity in nuclear extracts. On the other hand, both pharmacological and siRNA mediated inhibition of SIRT1 attenuated the anti-apoptotic effect of Des-G. Moreover, Des-G increased mRNA and protein levels of SIRT1 and antioxidant enzymes such as MnSOD and CAT, which are downstream targets of SIRT1. Although the treatment of Ex-527 did not alter mRNA expression levels of SIRT1, it decreased mRNA expression levels of antioxidant enzymes in the cells with Des-G pretreatment.ConclusionsOur results suggest that SIRT1 signaling pathway contributes to protective effect of Des-G against oxidative stress-induced apoptosis.     

Monocyte chemoattractant protein-1 alters expression of tight junction-associated proteins in brain microvascular endothelial cells

The chemokine monocyte chemoattractant protein (MCP-1) is recognized to mediate extravasation of mononuclear leukocytes into the brain during a variety of neuroinflammatory conditions. In large part produced by parenchymal neural cells during these disease states, it is unclear how this chemokine can stimulate the migration of circulating leukocytes that lie behind the highly impermeant blood-brain barrier (BBB). Based on the premise that disruption of tight junctions (TJs) could foster leukocyte extravasation, experiments were conducted to test the hypothesis that MCP-1 alters the expression and/or distribution of the TJ-associated proteins zonulae occludens-1 (ZO-1) and occludin in brain microvascular endothelial cells (BMEC) comprising the BBB. Exposure to MCP-1 caused a loss in immunostaining of ZO-1 at inter-endothelial junctional regions in both cultured BMEC and isolated brain microvessels, as well as a similar effect on occludin in cultured BMEC, but did not alter occludin staining in microvessels. In cellular fractionation experiments, ZO-1 associated predominantly with the detergent-resistant cytoskeletal framework (CSK) in both cultured BMEC and brain microvessels, while a slimmer majority of occludin partitioned with the CSK. Following MCP-1 exposure, ZO-1 was reduced in the CSK fraction of cultured BMEC and microvessels, with a shift of ZO-1 to the detergent-soluble fraction in both cases. Occludin exhibited a similar pattern of MCP-1-induced loss and shift from the CSK in cultured BMEC, but remained nearly constant in microvessels. Lastly, expression of caveolin-1, a major structural component of membrane microdomains thought to be functionally complexed with TJs, was additionally altered by MCP-1 treatment of both cultured BMEC and microvessels. These results indicate that, in addition to its chemotactic activity, MCP-1 might alter BBB integrity during CNS inflammation.     

大鼠脑微血管内皮细胞的纯化培养与鉴定

目的探讨获取纯度较高的原代大鼠脑微血管内皮细胞（BMECs）的分离和培养方法。方法选取10只3周龄Wistar大鼠,取大脑,去除白质,剪碎成大约1mm^3大小,通过两次酶消化、20％BSA离心和33％连续密度Percoll梯度离心,获得纯度较高的大脑微血管片段,接种于涂布明胶的35himdish培养皿中,加入含4μg／ml嘌呤霉素的内皮细胞培养基,2d后更换新鲜培养基;采用倒置盟微镜观察内皮细胞生长状况及其形态;免疫组化法检测BMECs标志物Ⅷ因子相关抗原（vWF）的表达,以及神经胶质原纤维酸性蛋白（GFAP）的表达情况。结果培养24h后可见内皮细胞从贴壁的脑微血管片段周围长出,细胞呈梭形;5～6d可形成融合;融合后的BMECs呈典型的“鹅卵石”样外观;免疫组化法检测显示BMECs的vWF表达阳性．GFAP表达阴性,内皮细胞纯度（vWF阳性细胞表达率）达96％以上。结论该方法能够成功获得纯度较高的原代大鼠BMECs。     

Interaction of Shiga toxins with human brain microvascular endothelial cells: cytokines as sensitizing agents.

Neurologic abnormalities are among the most serious extraintestinal complications of infection with Shiga toxin (Stx)-producing bacteria. Histopathologic examination of tissues from patients with extraintestinal sequelae suggested that Stxs damage endothelial cells. It is shown here that human brain microvascular endothelial cells (HBMECs) are relatively resistant to purified Stxs (50% cytotoxic doses [CD50s] >/=10 microgram/mL). Pretreatment of HBMECs with tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, n-butyric acid, or a cAMP analogue resulted in a 103- to 104-fold decrease in CD50 values and a 2- to 4-fold increase in fluoresceinated Stx binding to HBMECs. Treatment of HBMECs with lipopolysaccharides did not significantly alter cytotoxicity or toxin binding. TNF-alpha and IL-1beta treatment was associated with the increased HBMEC expression of the toxin-binding glycolipid globotriaosylceramide. HBMECs did not produce IL-1beta and produced only trace amounts of TNF-alpha when stimulated with purified Stx1 in vitro.     

白藜芦醇抑制缺氧复氧诱导心肌微血管内皮细胞凋亡的研究

目的探讨白藜芦醇对缺氧复氧诱导心肌微血管内皮细胞(CMEC)凋亡的抑制作用及其可能的分子机制。方法体外培养大鼠CMEC,建立缺氧复氧损伤模型,随机分为对照1组、缺氧复氧1组、缺氧复氧+白藜芦醇组(白藜芦醇1组),白藜芦醇分别选择5、10、20、30及40μmol/L浓度,MTT法检测CMEC增殖能力。20μmol/L白藜芦醇为最适浓度,使用磷脂酰肌醇3激酶(PI3K)特异性抑制剂LY294002,后续实验又分为对照2组、缺氧复氧2组、缺氧复氧+白藜芦醇2组(白藜芦醇2组)、缺氧复氧+白藜芦醇+LY294002组(LY294002组)。PI-AnnexinⅤ检测CMEC的凋亡;Western blot法检测细胞蛋白激酶B(Akt)、磷酸化Akt、缺氧诱导因子1α(HIF-1α)蛋白表达或磷酸化水平。结果与缺氧复氧1组比较,不同浓度白藜芦醇1组均可增加CMEC增殖能力,呈剂量依赖性,以白藜芦醇1组20μmol/L保护作用最显著(P<0.05)。与白藜芦醇2组比较,LY294002组CMEC凋亡率明显升高,磷酸化Akt和Akt蛋白、HIF-1α蛋白表达明显下降(P<0.05)。结论白藜芦醇可显著抑制缺氧复氧诱导的CMEC凋亡,其机制可能与PI3K/Akt介导的HIF-1α上调相关。     

Production of hepatocyte growth factor from human lung microvascular endothelial cells induced by interleukin-1beta.

To investigate the possible role of hepatocyte growth factor (HGF) in the reconstruction process following inflammatory damage in lung tissue, we compared HGF production of human lung microvascular endothelial cells (HLWECs) and human umbilical vein endothelial cells (HUVECs) after stimulation by interleukin(IL)-1beta. In an HLMEC-conditioned medium, large amounts of total (single and 2-chain) HGF were detected, and were 26- to 28-fold higher than those in HUVECs or human lung fibroblasts. The production of total HGF increased in a dose-dependent manner (4.7 to 9.2 times) with IL-1beta. In contrast, the amount of HGF in an HUVEC-conditioned medium was unaffected by IL-1beta treatment. The amount of cell-associated HGF also showed a dose-related increase (140% to 160%) in HLMECs, but not in HUVECs with IL1beta. In addition, HGF and c-met (HGF receptor) mRNAs in HLMECs and HUVECs were examined by the RT-PCR method. HGF and c-met mRNAs were clearly detected in HLMECs before and after treatment with IL-1beta, but not in HUVECs. These results suggest that increases in HGF production from HLMECs may play a role in the reconstruction process following inflammatory damage in lung tissue.     

Regulation of adhesion molecule expression by human synovial microvascular endothelial cells in vitro

Objective. To examine the in vitro expression of E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), and platelet–endothelial cell adhesion molecule 1 (PECAM-1) by synovial microvascular endothelial cells (SMEC) in comparison with microvascular neonatal foreskin endothelial cells (FSE) and macrovascular human umbilical vein endothelial cells (HUVE). Methods. Cultured endothelial cells were treated for 4 hours with medium alone or tumor necrosis factor α (TNF α). The expression of endothelial adhesion molecules was evaluated by flow cytometry, cell enzyme-linked immunosorbent assay, and Northern blot analysis. Results. SMEC continuously expressed E-selectin under basal culture conditions, whereas FSE and HUVE did not. TNF α treatment of rheumatoid arthritis (RA) SMEC resulted in sustained peak expression of E-selectin for up to 24 hours, which subsequently declined but remained elevated even at 72 hours. In contrast, peak E-selectin expression in FSE and HUVE occurred between 4 hours and 16 hours after TNF α treatment and then declined to near basal levels by 24–48 hours. SMEC expressed significantly higher levels of ICAM-1 compared with HUVE under basal culture conditions. There was no difference between SMEC, FSE, and HUVE in the expression of P-selectin, VCAM-1, ICAM-2, or PECAM-1. Northern blot analysis demonstrated that the levels of E-selectin expression by TNF α-stimulated endothelial cells correlated with their respective messenger RNA levels. Conclusion. Regulation of E-selectin and ICAM-1 expression in RA synovial endothelium is different from that in neonatal foreskin and human umbilical vein endothelium. The augmented expression of adhesion molecules in RA synovial endothelium may facilitate the recruitment of leukocytes to this site.