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.     

Molecules mediating adhesion of T and B cells, monocytes and granulocytes to vascular endothelial cells.

Leucocytes interact with vascular endothelial cells (EC), and adhesion between these two cell types in vitro is modulated by phorbol ester. Monocytes were found to display the highest basal adhesion to EC, followed by Epstein-Barr virus-immortalized normal B cells (EBV-B), T cells and granulocytes. Phorbol ester treatment increased the adhesion of all types of leucocytes, except monocytes. In the presence of this compound, monoclonal antibody 60.3 to GP90 (CD18, a leucocyte-adhesion protein which is non-covalently associated to either GP160, GP155, or GP130) was found to inhibit the adhesion of the four types of leucocytes to a considerable extent, while anti-lymphocyte function-associated antigen-1 (LFA-1) antibody to GP160 (CD11a) inhibited the adhesion of T and B cells only. Antibody 60.1 to GP155 (CD11b) had a major inhibitory activity exclusively on granulocytes, while antibody LB-2, which recognizes a distinct adhesion molecule (GP84) and, in contrast to the previous antibodies, reacts with EC, mainly inhibited adhesion of EBV-B and did not increase the inhibition obtained with antibody 60.3 alone. Fab fragments of antibody 60.3 inhibited leucocyte adhesion more efficiently, in either the absence or presence of phorbol ester, than the intact antibody molecule. It is concluded the GP90, either alone or associated to the larger glycoproteins, mediates the adhesion in all types of leucocytes, while GP84 mediates the adhesion of the activated B cells.     

Effects of magnetic cell separation on monocyte adhesion to endothelial cells under flow

Studies on monocyte adhesion are frequently limited by spontaneous changes of CD11b and CD62L during cell purification. Most isolation protocols for flow cytometric analysis that overcome this problem cannot be used when large numbers of living cells are needed for functional adhesion assays. This study investigated whether magnetic cell separation of monocytes with a paramagnetic bead against CD33 is a feasible method combining high yield with a low degree of spontaneous activation. As determined by flow cytometry, isolation of magnetically tagged monocytes at 4 degrees C did not alter the expression of CD11b and CD62L when compared to whole blood controls. Warming the cells slowly to room temperature immediately before starting the adhesion assay in a parallel plate flow chamber at 37 degrees C prevented further upregulation of adhesion molecules due to rewarming. When adhesion of magnetically tagged monocytes was compared with untouched monocytes that had been isolated via depletion of contaminating leukocytes, videomicroscopy showed that labelling CD33 neither affected rolling nor firm adhesion to human umbilical venous endothelial cells under flow. Finally, the subsequent upregulation of tissue factor expression on adherent monocytes indicates that magnetically separated monocytes responded properly to activating stimuli during cell adhesion. We conclude that magnetic cell separation via CD33 represents a feasible method for cell separation whenever large numbers of non-activated monocytes are needed for adhesion assays under flow.     

Methylene blue modulates adhesion molecule expression on microvascular endothelial cells

Objective and design

As methylene blue (MB) has been recently proposed to preserve blood pressure in case of vasoplegic syndrome and shock, an entity directly related to systemic inflammation, we aimed to elucidate the effect of MB on the expression of adhesion-molecules in endothelial-cells.

Materials and treatment

Human microvascular endothelial-cells (HuMEC-1) were treated with 10, 30 or 60 µM MB for 30 min and 2 h each. Additionally, the treated HuMEC-1 were co-cultured with either human peripheral blood mononuclear cells (PBMCs) or Jurkat cells (human T-lymphocytes) for 2 h.

Methods

HuMEC-1 were analyzed after MB treatment and after co-culture experiments for expression of different adhesion-molecules (ICAM-1, VCAM-1, L-selectin, E-selectin) via FACS measurement and western blot analysis. The supernatants of the experiments were analyzed with regard to the soluble forms of the adhesion molecules.

Results

We found that MB is able to modulate the expression of adhesion-molecules on EC. Administration of MB increases the expression of E-selectin and VCAM-1 depending on the dosage and time of exposure. ICAM-1 measurements provide evidence that different circulating blood cells can differently alter the adhesion-molecule expression on EC after MB exposure.

Conclusion

Our results provide evidence regarding the immunomodulatory effect of MB upon endothelial-cells after inflammation.     

Differential contribution of neutrophilic granulocytes and macrophages to nitrosative stress in a host-parasite animal model

Tyrosine nitration is a hallmark for nitrosative stress caused by the release of reactive oxygen and nitrogen species by activated macrophages and neutrophilic granulocytes at sites of inflammation and infection. In the first part of the study, we used an informative host-parasite animal model to describe the differential contribution of macrophages and neutrophilic granulocytes to in vivo tissue nitration. To this purpose common carp (Cyprinus carpio) were infected with the extracellular blood parasite Trypanoplasma borreli (Kinetoplastida). After infection, serum nitrite levels significantly increased concurrently to the upregulation of inducible nitric oxide synthase (iNOS) gene expression. Tyrosine nitration, as measured by immunohistochemistry using an anti-nitrotyrosine antibody, dramatically increased in tissues from parasite-infected fish, demonstrating that elevated NO production during T. borreli infection coincides with nitrosative stress in immunologically active tissues. The combined use of an anti-nitrotyrosine antibody with a panel of monoclonal antibodies specific for several carp leukocytes, revealed that fish neutrophilic granulocytes strongly contribute to in vivo tissue nitration most likely through both, a peroxynitrite- and an MPO-mediated mechanism. Conversely, fish macrophages, by restricting the presence of radicals and enzymes to their intraphagosomal compartment, contribute to a much lesser extent to in vivo tissue nitration. In the second part of the study, we examined the effects of nitrosative stress on the parasite itself. Peroxynitrite, but not NO donor substances, exerted strong cytotoxicity on the parasite in vitro. In vivo, however, nitration of T. borreli was limited if not absent despite the presence of parasites in highly nitrated tissue areas. Further, we investigated parasite susceptibility to the human anti-trypanosome drug Melarsoprol (Arsobal), which directly interferes with the parasite-specific trypanothione anti-oxidant system. Arsobal treatment strongly decreased T. borreli viability both, in vitro and in vivo. All together, our data suggest an evolutionary conservation in modern bony fish of the function of neutrophilic granulocytes and macrophages in the nitration process and support the common carp as a suitable animal model for investigations on nitrosative stress in host-parasite interactions. The potential of T. borreli to serve as an alternative tool for pharmacological studies on human anti-trypanosome drugs is discussed.     

柯萨基B3病毒感染增强单个核细胞与心脏微血管内皮细胞的粘附

目的：研究柯萨基Ｂ３病毒（Ｃｏｘ Ｂ３）感染对内皮细胞表面ＩＣＡＭ－１表达的诱导作用,及对内皮细胞与单个核细胞粘附的影响,探讨病毒性心肌炎心肌细胞免疫损伤的机理。方法：采用流式细胞仪分析内皮细胞ＩＣＡＭ－１表达;粘附试验和单克隆抗体抑制试验观察单个核细胞与内皮细胞的粘附。结果：１００和４００ ＴＣＩＤ５０／ｍｌ Ｃｏｘ Ｂ３感染内皮细胞２４和４８ｈ,均可显著增加单个核细胞与内皮细胞的粘附（Ｐ〈０．０     

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.     

Dexamethasone affects cytokine-mediated adhesion of HL-60 human promyelocytic leukemia cells to cultured dermal microvascular endothelial cells

Leukocyte endothelial adhesion (LEA) is the prelude to a complex cascade of reactions following an immunological challenge. Recently, LEA has been implicated in the molecular basis of several dermatological disorders. While the role of proinflammatory cytokines, such as interleukin-1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), in LEA has been investigated using nondermal models, limited data exist regarding their effects on LEA in dermal models. This study shows that cotreatment of cultured human dermal endothelial cells (CADMEC) with IL-1beta and TNF-alpha resulted in a marked increase in the adherence of human promyelocytic leukemia (HL-60) cells to CADMEC and an increase in expression of intercellular adhesion molecule-1 and E-selectin. Pretreatment of CADMEC with dexamethasone, a long-lasting glucocorticoid, resulted in a decrease in both HL-60 cell adhesion to CADMEC and adhesion molecule expression. Taken together, these data demonstrate that LEA may play a role in inflammatory skin conditions and in the mechanisms underlying the potential use of glucocorticoids as a treatment option.     

Effect of ultraviolet light on the expression of adhesion molecules and T lymphocyte adhesion to human dermal microvascular endothelial cells

In order to determine the effect of ultraviolet radiation (UVR) on the cell adhesion molecules expressed in human dermal microvascular endothelial cells (HDMEC), the cells were exposed to varying UVR doses and the cell surface was examined for expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM- 1), and E-selectin. The effect of UVB irradiation on the binding of T lymphocytes to HDMEC was also examined. UVA irradiation did not affect the surface expression of ICAM-1, VCAM-1, or E-selectin on the HDMEC. However, following UVB exposure, ELISA demonstrated a significant increase in the baseline ICAM-1 cell surface expression on the HDMEC. However, no induction of either E-selectin or VCAM-1 was noted. UVB also significantly augmented ICAM-1 induction by IL-1alpha and TNF-alpha. VCAM-1 was induced by stimulating HDMEC with IL-1alpha following a UVB irradiation dose of 100 mJ/cm2. Flow cytometric analysis of the HDMEC stimulated with IL-1alpha for 24h demonstrated that 12% of the cells expressed VCAM-1 but either IL-1alpha or UVB irradiation alone failed to induce VCAM-1 expression. Enhancement of T cell-HDMEC binding by IL-1alpha or TNF-alpha treatment was not significantly affected after UVB irradiation. This study demonstrated that UVB irradiation can alter ICAM-1 and VCAM-1 expression on the HDMEC surface and that augmentation of ICAM-1 expression and the IL-1alpha-dependent induction of VCAM-1 following UVB exposure might be important steps in the pathogenesis of sunburn.     

Tumor cell adhesion under hydrodynamic conditions of fluid flow

Current evidence indicates that tumor cell adhesion to the microvasculature in host organs during formation of distant metastases is a complex process involving various types of cell adhesion molecules. Recent results have shown that stabilization of tumor cell adhesion to the microvascular vessel wall is a very important step for successful tumor cell migration and colonization of host organs. We are beginning to understand the influences of fluid flow and local shear forces on these adhesive interactions and cellular responses within the circulation. Mechanosensory molecules or molecular complexes can transform shear forces acting on circulating tumor cells into intracellular signals and modulate cell signaling pathways, gene expression and other cellular functions. Flowing tumor cells can interact with microvascular endothelial cells mediated mainly by selectins, but the strength of these bonds is relatively low and not sufficient for stable cell adhesions. Integrin-mediated tumor cell adhesion and changes in the binding affinity of these adhesion molecules appear to be required for stabilized tumor cell adhesion and subsequent cell migration into the host organ. Failure of the conformational affinity switch in integrins results in breaking of these bonds and recirculation or mechanical damage of the tumor cells. Various cell signaling molecules, such as focal adhesion kinase, pp60src or paxillin, and cytoskeletal components, such as actin or microtubules, appear to be required for tumor cell adhesion and its stabilization under hydrodynamic conditions of fluid flow.     

Differential reactivity of brain microvascular endothelial cells to TNF reflects the genetic susceptibility to cerebral malaria

Upon infection with Plasmodium berghei ANKA (PbA), various inbred strains of mice exhibit different susceptibility to the development of cerebral malaria (CM). Tumor necrosis factor-α (TNF) and interferon-γ (IFN-γ) have been shown to be crucial mediators in the pathogenesis of this neurovascular complication. Brain microvascular endothelial cells (MVEC) represent an important target of both cytokines. In the present study, we show that brain MVEC purified from CM-susceptible (CM-S) CBA / J mice and CM-resistant (CM-R) BALB / c mice exhibit a different sensitivity to TNF. CBA / J brain MVEC displayed a higher capacity to produce IL-6 and to up-regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in response to TNF than BALB / c brain MVEC. In contrast, no difference was found in the induction of E-selectin after TNF challenge. CM-S brain MVEC were also significantly more sensitive to TNF-induced lysis. This differential reactivity to TNF was further substantiated by comparing TNF receptor expression on CM-S and CM-R brain MVEC. Although the constitutive expression of TNF receptors was comparable on cells from the two origins, TNF induced an up-regulation of both p55 and p75 TNF receptors in CM-S, but not in CM-R brain MVEC. A similar regulation was found at the level of TNF receptor mRNA, but not for receptor shedding. Although a protein kinase C inhibitor blocked the response to TNF in both the brain MVEC, an inhibitor of protein kinase A selectively abolished the response to TNF in CM-R, but not CM-S brain MVEC, suggesting a differential protein kinase involvement in TNF-induced activation of CM-S and CM-R brain MVEC. These results indicate that brain MVEC purified from CM-S and CM-R mice exhibit distinctive sensitivity to TNF. This difference may be partly due to a differential regulation of TNF receptors and via distinct protein kinase pathways.     

Fibrinogen and von Willebrand factor mediated platelet adhesion to polystyrene under flow conditions

The roles of adsorbed fibrinogen (Fg) and von Willebrand factor (VWF) in mediating platelet adhesion to synthetic surfaces under flow were investigated using polystyrene (PS) as a model hydrophobic surface. We measured platelet adhesion to PS pre-adsorbed with Fg, VWF, normal plasma, afibrinogenemic plasma, VWF-deficient plasma and deficient plasmas with various concentrations of added Fg or VWF. Platelets in a red blood cell suspension were passed through a flow chamber at either low (50 or 100 s(-1)) or high (500 or 1000 s(-1)) shear. Adhesion to PS pre-adsorbed with afibrinogenemic plasma was very low under both low and high shear conditions, but was restored in a dose-dependent manner with addition of Fg. Less than 20 ng/cm(2)of adsorbed Fg was sufficient to support full-scale platelet adhesion under flow. At high shear rate, platelet adhesion on PS pre-adsorbed with VWF-deficient plasma was much less than on PS pre-adsorbed with normal plasma, but adhesion to PS pre-adsorbed with VWF-deficient plasma with added VWF was very similar to adhesion to PS pre-adsorbed with normal plasma. At low shear, adhesion to PS pre-adsorbed with VWF-deficient plasma was the same as on PS pre-adsorbed with normal plasma. As little as 1 ng/cm(2) of VWF adsorbed from plasma made platelet adhesion higher under high shear than under low shear. The effects of adsorbed Fg and VWF on the morphologies of platelets that adhered from suspensions flowing at high shear rates were also investigated. The lack of either Fg or VWF resulted in marked decreases in the extent of platelet spreading. Real-time observation of platelet adhesion under an epifluorescent microscope showed that platelets adhered to the surface in a linear pattern aligned in the direction of flow under high shear conditions.     

Clinico-morphological tests to assess the function of neutrophilic granulocytes

Clinico-morphological tests for the estimation of neutrophil granulocyte function were compared by their reproducibility, reliability and availability for standard clinical laboratories, and the results were analysed. Lysosomal-cationic test was offered for clinical practice, as it gives qualitative and quantitative assessment of the phagocytosis process, allows prediction of complications before their clinical onset, performance of rapid evaluation of the therapy used, reveals shifts in the level of non-specific resistance of the body. Methods of application of lysosomal-cationic test in the pathoanatomic practice have been developed for the study of intravital biopsy samples and surgical material.     

缺氧条件下间充质干细胞以旁分泌方式影响脑微血管内皮细胞的功能

目的：探讨缺氧条件下间充质干细胞（MSCs）以旁分泌方式对脑微血管内皮细胞（BMECs）的增殖、迁移和细胞单层通透性的影响。 方法:分离、培养并鉴定人MSCs和大鼠BMECs，ELISA检测细胞条件培养基中VEGF和MMP-9的含量，跨内皮电阻（TEER）检测反映脑BMECs单层通透性的变化，观察不同条件培养基对缺氧BMECs增殖、迁移和通透性的影响。结果:VEGF和MMP-9在MSCs条件培养基中的含量显著高于BMECs条件培养基，缺氧处理的MSCs条件培养基中VEGF和MMP-9含量比正常MSCs条件培养基显著增高；MSCs条件培养基能明显促进BMECs在缺氧条件下的增殖和迁移，但也使BMECs的TEER显著降低（50.5％±2.6％，P<0.05），这些作用在不同程度上能被VEGF抗体和MMP-9抑制剂所抑制。结论:缺氧条件下MSCs可通过旁分泌方式促进BMECs增殖和迁移，但同时也增加了BMECs单层的通透性。     

Steady unidirectional laminar flow inhibits monolayer formation by human and rat microvascular endothelial cells.

Endothelialization of artificial vascular grafts is rapid and complete in numerous animal models, including dogs and rats, but not in human patients. One possible explanation for this well-known, yet puzzling observation might be that monolayer formation of human endothelial cells (ECs), and of canine or rodent ECs, is affected differently by flow-induced shear stress. To begin testing this hypothesis, the authors wounded confluent monolayers of cultured rat and human ECs and exposed these cultures for 20 h to unidirectional steady laminar shear stress of 10 dyn/cm(2) induced by fluid flow perpendicular to the wound boundaries. In comparison to experimental control cultures simultaneously maintained under static (no-flow) conditions, flow-induced shear stress attenuated the monolayer formation (sheet migration) in both human and rat ECs. In brief, compared to control, the average human EC monolayer formation under shear was reduced by 33% whereas the average rat EC monolayer formation was reduced by 34%. Furthermore, the cell responses showed a dependence on fluid flow direction that differed per species. When exposed to shear stress, human EC monolayer formation was reduced by 16% in the upstream direction (opposing the direction of flow) and reduced by 50% in the downstream direction (with the direction of flow), whereas rat EC monolayer formation was reduced by 64% upstream and showed no change downstream. These findings suggest that although overall monolayer formation is inhibited by fluid-induced shear stress to the same extent in both species, there are cell type- and/or species-dependent migration responses to fluid-induced shear stress, and that different flow conditions possibly contribute to species-specific patterns of endothelialization.