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Expression data of murine capillary endothelial cells after MICAL2 gene knock-down by means of shRNA-expressing lentiviral vectors.

ABSTRACT: Molecules interacting with CasL 2 (MICAL2) belongs to a three-member family of multi-domain flavoprotein monooxygenase enzymes that catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. MICAL2 is over-expressed, being a negative prognostic factor, in aggressive gastric, kidney, prostate, bladder, breast and endometrial human cancer. MICAL2 is expressed in cancer-associated neo-angiogenic capillaries. The study of MICAL2 knock-down in endothelial cells shed light on the transcriptional impact of the gene on endothelial cell biology. To explore the transcriptional impact of the lack of Mical2 in endothelial cells, we performed genome-wide expression profiling of Mical2 knock-down SVEC4-10 (ATTC CRL-2181) cells (M2KD). Reference cells were: Mical3 knock-down (M3KD), obtained with same strategy; untreated SVEC4-10 cells ((WT), and cells transduced with insert-free viral vector (empty).

ORGANISM(S): Mus musculus

PROVIDER: GSE120494 | GEO | 2019/04/29

REPOSITORIES: GEO

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Project description:Sphingosine 1-phosphate (S1P) influences T cell migration into and out of secondary lymphoid organs; however, its mechanism of action remains uncertain. Our previous research shows that agonism of the S1P receptor S1P1 inhibits the egress of T lymphocytes from the peripheral tissues into afferent lymphatics. To better define the mechanism of inhibition, we developed an in vitro model to characterize T cell transendothelial migration across lymphatics. Two commercially available endothelial cell lines (MS-1 and SVEC4-10) were characterized by flow cytometry, real time RT-PCR, and Affymetrix Gene Array. These cell lines were grown to confluent monolayers in transwell systems, on either the upper or lower surface of the transwell insert. T cells were isolated from the spleens of (C57BL/6 x C3H/HeJ)F1, S1P1 KO, or S1P1 KO littermate controls, and either treated with the S1P receptor modulator FTY720 or left untreated. Cells were migrated to chemokines (CCL19 or CCL21) for 4 hours, and migration quantified. Flow cytometry, RT-PCR, and array results identified MS-1 as a blood vascular endothelial cell line, expressing high levels of CD31, CD34, and ICAM-1 as well as other endothelial cell markers; while SVEC4-10 closely resemble a lymphatic phenotype, expressing LYVE-1, VEGFR-3, and podoplanin. T cells efficiently migrate across MS-1, whether grown on the upper or lower surface; whereas migration across SVEC4-10 only occurs when cells are grown on the lower surface of the transwell (iSVEC), recapitulating basal (abluminal) to apical (luminal) migration that occurs in vivo. FTY720 inhibits T cell migration across iSVEC, but not across MS-1. Inhibition is due to drug effects only on T cells but not endothelial cells. S1P1 KO T cells treated with FTY720 are not inhibited in their migration across the iSVEC line, showing that S1P1 stimulation is required for migration inhibition. The in vitro model developed here is the first to use endothelial cell lines to analyze the regulation of T cell migration across lymphatic endothelium. The results show there is directional control of T cell migration across lymphatic cells, such that T cells only migrate from a basal to apical direction. Agonism of S1P1 specifically inhibits migration, while absence of the receptor does not. These findings have important implications for the use of S1P1 agonists in transplantation, as inhibition of cell entry into afferent lymphatics and lymph nodes could impede the development of graft rejection. We used microarrays to detail the global gene expression of these two cell lines in order to beter determine their phenotype as blood vascular or lymphatic endothelial cell line for use in our newly-developed in vitro model. Experiment Overall Design: MS-1 or SVEC4-10 endothelial cell lines were grown in culture flasks for 3-5 days in standard culture medium (see protocol below) until reaching confluence. Cells were then gently dissociated from flask and cells were placed in Trizol reagent for RNA isolation.

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Expression data of murine capillary endothelial cells after MICAL2 gene knock-down by means of shRNA-expressing lentiviral vectors.

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