Human and Murine High Endothelial Venule Cells Phagocytose Apoptotic Leukocytes

Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0–6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosisin vivo.Mice were treated with a natural corticosterone (4-pregnene-11β,21-diol-3,20-dione) at levels detected during stress or malnutrition (93–180 μg serum cortisol/dl). At 4–12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.     

Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway

Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.     

Increased Monocytic Adhesion by Senescence in Human Umbilical Vein Endothelial Cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.     

Cytokines and Cell Adhesion Molecules in Tumor-Endothelial Cell Interaction and Metastasis

Metastasis is a multistep process in which a metastatic tumor cell detaches from the primary tumor, invades the surrounding tissues, passes through supporting structures such as interstitial stroma and extracellular matrix, and enters the lymphatic or blood circulation (Poste and Fidler, 1980). Only a few of the neoplastic cells released into the circulation, that survive hemodynamic pressure and host defense mechanisms, will form metastases. The arrest of tumor cells in the capillary bed of secondary organs through an interaction with vascular or lymphatic endothelium and subendothelial basement membrane is followed by their extravasation into the tissue parenchyma, and then micro-metastasis formation. Therefore cell-cell and cell-substrate adhesions occur at different moments in this process. With the recent identification and characterization of cell surface molecules, it has become of particular interest to clarify their role in tumor progression and metastasis (Albelda, 1993).     

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.     

Cytokines and Cell Adhesion Molecules in Tumor-Endothelial Cell Interaction and Metastasis

Metastasis is a multistep process in which a metastatic tumor cell detaches from the primary tumor, invades the surrounding tissues, passes through supporting structures such as interstitial stroma and extracellular matrix, and enters the lymphatic or blood circulation (Poste and Fidler, 1980). Only a few of the neoplastic cells released into the circulation, that survive hemodynamic pressure and host defense mechanisms, will form metastases. The arrest of tumor cells in the capillary bed of secondary organs through an interaction with vascular or lymphatic endothelium and subendothelial basement membrane is followed by their extravasation into the tissue parenchyma, and then micro-metastasis formation. Therefore cell-cell and cell-substrate adhesions occur at different moments in this process. With the recent identification and characterization of cell surface molecules, it has become of particular interest to clarify their role in tumor progression and metastasis (Albelda, 1993).     

The Neural Stem/Progenitor Cell Marker Nestin Is Expressed in Proliferative Endothelial Cells,but Not in Mature Vasculature

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Transgenic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization. (J Histochem Cytochem 58:721–730, 2010)     

骨髓内皮细胞产物对CFU-F、CFU-GM和WEHI-3细胞生长的作用

为了研究骨髓内皮细胞的造血调控功能,采用串联超波技术脞小鼠骨髓内皮细胞无血清条件培养液（ｍＢＭＥＣ－ＣＭ）中制血出不同相对分子质量（Ｍｇｔ）范围的超滤组分,进行细胞集落形成实难,检测了ｍＢＭＥＣ－ＣＭ及其超滤组分对骨髓成纤维祖细胞（ＣＦＵ－Ｆ）、粒巨噬系造血祖细胞（ＣＦＵ－ＧＭ）和小鼠业单系白现细胞系ＷＥＨＩ－３细胞生长的作用。结果显示：ｍＢＭＥＣ－ＣＭ抑制ＣＦＵ－Ｆ的生长,对ＣＦＵ－ＧＭ和ＷＥＨ     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

Expression of Cell Adhesion Molecules and Cytokines in Murine Antigen-Induced Arthritis

Adhesion molecules and cytokines are important in chronic inflammatory conditions such as rheumatoid arthritis (RA) by virtue of their role in cell activation and emigration. Using immunohistochemical techniques we studied the expression of adhesion molecules and cytokines in cryopreserved sections of murine knee joint in the course of antigen-induced arthritis, an animal model of human RA. Various adhesion molecules and cytokines are expressed in the arthritic joint tissue. LFA-I, Mac-1, CD44, ICAM-I and P-selectin were strongly expressed in the acute phase and to a lesser degree in the chronic phase of arthritis. VLA-4 and VCAM-I appeared to be moderately expressed on day 1, L-selectin between days 1 and 3. LFA-I, Mac-I, CD44, a4-integrin, ICAM-I and the selectins were found expressed on cells of the synovial infiltrate, LFA-1, Mac-1 and ICAM-I on the synovial lining layer, and VCAM-I and P-selectin on endothelial cells. Expression of E-selectin could be demonstrated throughout the experiment at a low level in cells of the acute cell infiltrate. Cytokines, especially IL-2, IL-4, IL-6, TNF, and IFN-7, were heavily expressed during the acute phase of arthritis in cellular infiltrate. Taken together these data demonstrate that cytokines and their activation of adhesion molecules contribute to cell infiltration and activation during the initial phase of arthritis and to the induction and progression of tissue destruction in arthritic joints. These molecules might be potential targets for novel therapeutic strategies in inflammatory and arthritic disorders.     

凝血酶与血管内皮细胞

凝血酶不仅在凝血过程中起主导作用,还可以引发多种凝血酶受体介导的分子和细胞间相互作用,在动脉粥样硬化和再狭窄形成中起着重要的作用。现就凝血酶及其受体的特性,以及对血管内皮细胞的作用,包括通透性改变、内皮生长因子、基质金属蛋白酶、黏附分子表达作一综述。     

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34^LowCD31^PosVEGFR-3^PosPODOPLANIN^Pos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.     

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. ¹). Human tissue samples are available only in a restricted number of laboratories or companies ^2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND ⁴ (from cerebral cortex) and cerebEND ⁵ (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- ^4,6-9 and estrogen-treatment ¹⁰ as well as to pro-infammatory mediators, such as TNFalpha ^5,8. Moreover, we have studied the pathology of multiple sclerosis ¹¹ and hypoxia ^12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.     

细胞因子组合对肝癌细胞HepG2生长的影响

目的:探讨细胞因子组合在体外对肝癌细胞HepG2生长的抑制作用。方法:采用正交试验法,3种细胞因子(IFN-α2a、IL-2和GM-CSF)分别选择3种浓度,共分为9组组合,利用MTT比色法,检测细胞因子组合处理96 h后,对HepG2细胞生长的抑制作用,筛选3种细胞因子最佳组合,并进行形态学观察。结果:处理96 h后,不同浓度细胞因子组合对HepG2细胞的生长均有抑制作用,最佳组合为A1B1C1,其抑制率为64.61%。显微镜下可见,细胞变圆,胞浆中颗粒增多,增殖变慢,细胞间接触不紧密,细胞周围碎片增多。结论:已筛选到3种细胞因子的最佳组合,其在体外能够抑制HepG2细胞的生长。     

脐血造血细胞的体外扩增：2.造血细胞生长因子和培养液更换的作用

研究了造血干细胞生长因子、白介素-3、白介素-6、粒-巨噬细胞集落刺激因子、粒细胞集落刺激因子及红细胞生成素对脐血造血细胞体外培养的影响及其剂量关系,考察了造血细胞因子单独与联合作用对造血细胞体外培养的影响,证实细胞因子组合使用比细胞因子单独使用效果更好,发现SCF+IL-3+IL-6+GM-CSF+G-CSF+EPO组合对总细胞扩增最佳,SCF+IL-3+IL6+GM-CSF组合对CFU-GM扩增最佳。实验发现培养液更换可大大提高脐血造血细胞总数和祖细胞数产出。在每天更换50%培养液下,脐血总细胞数在第三周扩增了27倍,祖细胞数扩增了21倍。     

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5^cre:ZsGreen^l/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.