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Transcriptomic profiles of CD4+ T lymphocytes from RhoA wild type (WT) and RhoA G17V mice were generated by deep sequencing in duplicate, using Illumina poly-A RNA seq.

ABSTRACT: The goal of this study is to identify transcriptomic effects of RhoA G17V expression in mouse CD4+ T lymphocytes. To this end, mice conditionally expressing RhoA G17V were generated by replacing exon 3 of RhoA with a minigene containing RhoA exons 3 to 6 flanked by loxP sites followed by a neomycin selection cassette and a mutant Rhoa exon 3 encoding the G17V substitution. These mice were then crossed with the CD4CreERT2 line (Tg(Cd4-cre/ERT2)11Gnri, Jackson Laboratories), which expresses a 4-hydroxi-tamoxifen-inducible form of the Cre recombinase under the control of the mouse Cd4 promoter to generate conditional inducible CD4 specific RhoA G17V mice (Rhoaco-G17V/+ CD4CreERT2). Mice were treated with vehicle only or Tamoxifen to induce expression of the RhoA G17V in CD4+ T cells. CD4CreERT2 mice were included as a negative control. CD4+ T cells were isolated from single cell suspensions of spleen by negative selection using the mouse naive CD4+ T Cell Isolation Kit (Milteny#130-104-453) according to the manufacturer’s protocol. Total RNA was extracted and standard Illumina poly-A RNA seq was performed. Naive mouse CD4+ T cells expressing RhoA G17V displayed an enrichment in a Tfh gene expression program.

ORGANISM(S): Mus musculus

PROVIDER: GSE101340 | GEO | 2017/09/01

SECONDARY ACCESSION(S): PRJNA394012

REPOSITORIES: GEO

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Project description:The small GTPase RhoA regulates a variety of cellular processes, including cell motility, proliferation, survival and permeability. In addition, there are reports suggesting that the RhoA-ROCK axis plays a role in VEGF-mediated angiogenesis, whereas other work has shown opposite effects. To elucidate this conflicting data, we examined HUVEC and HCAEC after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a variety of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. We observed that expression of active as well as wild-type RhoA but not expression of dominant-negative RhoA significantly increased endothelial cell death as well as inhibited endothelial cell proliferation, migration, tube formation and angiogenic sprouting of endothelial cells in vitro and reduced HUVEC-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized inhibitory RhoA effects and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 had no significant effect on RhoA-related effects, but again increased angiogenic sprouting and migration of control-treated cells. In line with these data, VEGF did not activate RhoA in HUVEC as measured by a FRET-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology (GO) enrichment analyses revealed that constitutively active RhoA induces a differentially expressed gene pattern that is enriched for GO biological process terms such as mitotic nuclear division, cell proliferation, cell motility and cell adhesion and includes a significant decrease in VEGFR-2 and NOS3 expression. Thus, our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and anti-angiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.

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Transcriptomic profiles of CD4+ T lymphocytes from RhoA wild type (WT) and RhoA G17V mice were generated by deep sequencing in duplicate, using Illumina poly-A RNA seq.

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