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Endothelial cells |
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A NEW EXPERIMENTAL MODEL FOR STUDYING OF HUMAN ENDOTHELIAL CELLS Recent studies have identified the link between inflammation and angiogenesis. Angiogenesis is the formation of new capillaries from preexisting vasculature by migration and proliferation of endothelial cells. Angiogenesis is a fundamental process required for a number of physiological and pathological events. Angiogenesis occurs during embryonic development, wound healing, and the menstruation cycle, unregulated angiogenesis is seen under pathological conditions such as tumor growth, diabetic retinopathy and psoriasis. The progression of angiogenesis is controlled by a delicate balance between the positive and negative regulators of this process. To trigger angiogenesis, either the production of proangiogenic factors must increase or the level of inhibitors must decrease. Now, it has been realized that the inflammatory cells as well as a variety of mediators, including cytokines, chemokines and enzymes, may facilitate angiogenesis and promote the growth, invasion, and metastasis of tumor cells. So, studying of the properties of endothelial cells and of the influence of biologically active mediators upon endothelial cells is a very actual problem. Endothelial cells (EC) are currently used as in
vitro model systems for various physiological and pathological
processes, especially in angiogenesis research.
The purpose of this study was to assess the
suitability of using EA.hy 926 line for investigating endothelium in an
in vitro situation closely modeling the in vivo status. Recent results
from our laboratory have shown that EA.hy 926 cells is a good in vitro
model for the analysis of growth factor activity, drug screening and the
determination of the potential of anti-cancer compounds and/or
angiogenic inhibitors.
Fig.3 Reduced proliferation of EA.hy 926 cells in response to anti-cancer compound- epirubicin (light curve). Flowcytometric analysis clearly differentiates normal cells (dark curve). Single parameter histogram is shown. Endothelial cells EA.hy 926 express
a wide spectrum of surface molecules involved in multiple vascular
functions. Quantitative modulations of molecule expression in function
of experimental conditions are a component of EC characterization and
could provide information about its implication in cellular
pathophysiologic processes. The same human macro-vascular EC line is EA.hy926 was
used for construction of experimental model for studying
transendothelial migration of mononuclear cells from blood vessels. As an example of researches using such an experimental model we show the following description of the results of studying transendothelial migration induced alterations of surface molecules expression on human monocytic cells THP-1. INTRODUCTION Leukocyte migration to sites of inflammation is crucial to the cellular functions of leukocytes in the innate and adaptive immunity Leukocyte migration not only facilitates the exit of leukocytes from the vascular lumen but numerous other responses can also occur as a consequence within leukocytes and endothelial cells. Such changes induced in leukocytes might facilitate their onward migration through venular walls, as well as their responsiveness, behavior, differentiation state and survival at sites of inflammation. Circulating monocytes following recruitment to tissues, can differentiate into macrophages or myeloid dendritic cells. and contribute significantly to immune defense or to the pathogenesis of the inflammatory diseases. Passage of monocytes across the endothelial lining into sites of inflammation has been shown to be regulated by many endogenous and exogenous factors, including cytokines. We therefore constructed an in vitro model system to examine phenotypic characteristics of human monocytic THP-1 cells that migrated through an endothelial cells (EA.hy 926) monolyaers in the presence or absence of TNFα, IFNγ and IL-4. We have chosen TNFα, IFNγ and IL-4 because of their importance as mediators during acute and chronic inflammation. MATERIALS AND METHODS EA.hy 926 human endothelial cells was a gift from Dr. Cora-Jean C. Edgell (University of North Carolina, USA).Cells were grown as monolyers in tissue culture flasks incubated in 100% humidity and 5% CO2 at 37oC in DMEM/F12 (“Sigma”, USA) medium supplemented with 10% FBS («ICN», USA), , penicillin (100 U/ml) and streptomycin (100 μg/ml) («Samson», RF), 2 мМ L-glutamine («Flow Laboratories», England) and HAT («ICN», США). Cell monolyers were harvested with EDTA (0.1%) in PBS («Biolot», RF) centrifuged at low speed (250 g, 5 min) and resuspended in fresh medium prior to culture on permeable filters in twenty-four well Transwell® tissue culture plates with 8.0 μm pore size (Becton Dickinson, USA). Cells (0.3 x 106 /ml) in 150 μl were grown to confluence on the upper surfaces of these filters in Transwell plates three days using the same conditions outlined for the flasks (fig.1). Monolauer integrity was assessed by microscopy. THP-1 monocytic cells were maintained in RPMI-1640 medium («Biolot», RF) supplemented with 10% FBS («ICN», USA), , penicillin (100 U/ml) and streptomycin (100 μg/ml) («Samson», RF), 2 мМ L-glutamine («Flow Laboratories», England). Monocytes (0.3 x 106 cells) in 150 μl of medium were placed in the upper chamber, above the naked filter or above the endothelial cells monolayers cultured on filters. Culture medium consisted of 1.5% FCS in DMEM/F12 medium. In some experiments, either 50 IU/ml of recombinant TNFα, 500 IU/ml of recombinant IFNγ (“Sanitas”, Lithuania) or 50 IU/ml of recombinant IL-4 (“Becton Dickinson”, USA) in 350 μl DMEM/F12 with 1.5% FCS, was places in the lower chambers. Medium alone was placed in lower chamber as a negative control in each experiment. After 72 h incubation, transmigrated cells from lower chamber and nonmigrated cells from upper chamber were collected, washed and prepared for flow cytometry staining. In all experiments cell viability was greater than 98% as assessed by trepan blue exclusion. Cell surface multi-color staining of monocytes was performed as recommended by the manufacturer with PE-conjugated antibodies against CD11b, PerCP-conjugated anti-CD14 Ab and mouse anti-HLA-DR Ab, followed by detection with FITC-conjugated anti-mouse IgG. Cells were analyzed on a FACSCalibur cytometer (“Becton Dickinson”, USA). All data are presented as mean fluorescence intensity ± SD for at least three independent experiments. Student’s t-tests were used for comparisons.
RESULTS We compared the phenotypic characteristics of the surface of human monocytes after migration through EC barriers with that through naked filters. The expression of several surface molecules (CD11b and HLA-DR) was significantly increased on the migrated through endothelium monocytes in comparison with monocytes migrated through naked filters. In contrast, no statistically significant change in monocyte CD14 expression was observed (Fig. 4). The changes in the expression of surface markers might be a result of adhesion of monocytes to endothelium. This did not occur; all surface markers studied were expressed to similar extents on the adherent cells (nonmigrated monocytes from upper chamber) and initial populations. During monocytes migration through the EC barrier, both monocytes and endothelium actively participate in the regulating of this process because their interaction is accompanied by bidirectional signaling in both cell types. Previous findings indicated that TNFα, IFNγ or IL-4 could regulate EC responsiveness and function. We next therefore studied phenotypic characteristics of monocytes undergoing transendothelial migration in the presence of cytokines. Only presence of TNFα and IFNγ in lower chamber led to a significant increase in CD11b, HLA-DR and CD14 expression on migrated through EC monolyers monocytes (Fig. 5, 6). With IL-4, only small differences were noted in CD11b and HLA-DR expression (Fig. 7). In the presence any of the cytokines, phenotypic changes on the monocytes migrated through the naked filters were minimal and significantly less than that on the monocytes migrated through the cellular barriers (Table 1).
Table 1. Changes of surface markers expression after
migration of monocytic cells THP-1 in presence and in absence of
cytokines into low chamber. Monocytes activation and/or differentiation are a key event in host defense. Our findings indicate that monocyte migration is accompanied by changes expression of function-associated surface antigens (Fig.8). This phenotype seems to be induced by interactions between monocytes and ECs and this change is enhanced by cytokines (Fig.9). Taken together, the results suggest that during monocytes migration through the EC barrier, endothelium actively participate in the regulation of the monocytes responsiveness, behavior, differentiation state and survival at sites of inflammation.
Fig.4.Changes in surface markers expression after
migration of monocytic cells THP-1 in presence (transmigration) and in
absence (migration) of endothelial cells monolayer (without cytokines).
Fig.5.Changes in surface markers expression after
migration of monocytic cells THP-1 in presence (transmigration) and in
absence (migration) of endothelial cells monolayer (with TNFα in the low
chamber).
Fig.6.Changes in surface markers expression after
migration of monocytic cells THP-1 in presence (transmigration) and in
absence (migration) of endothelial cells monolayer (with IFNγ in the low
chamber).
Fig.7.Changes in surface markers expression after
migration of monocytic cells THP-1 in presence (transmigration) and in
absence (migration) of endothelial cells monolayer (with IL-4 in the low
chamber).
Fig. 8. Histograms show the surface markers mean fluorescence intensity of monocytic cells THP-1 migrated in presence (transmigration) and in absence (migration) of endothelial cells monolayer (without cytokines). a) HLA-DR, b) CD11b, c) CD14.
Fig. 9. Histograms show the surface markers mean fluorescence intensity of monocytic cells THP-1 migrated in presence (transmigration) and in absence (migration) of endothelial cells monolayer (with TNFα in the low chamber). a) HLA-DR, b) CD11b, c) CD14. ACKNOWLEDGEMENTS
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