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  • Titill er á ensku Transcriptional Effects of mTOR Inhibitors on Vascular Smooth Muscle Cells
  • Bakkalár
  • Útdráttur er á ensku

    Introduction: Cardiac allograft vasculopathy (CAV) is a rapidly progressing arteriosclerotic disease in heart transplant recipients and the major cause of late graft failure. A characteristic feature of CAV is intimal hyperplasia of coronary arteries due to vascular smooth muscle cell (VSMC) proliferation. Currently, the only treatment available to prevent CAV is mechanistic target of rapamycin (mTOR) inhibitors. However, little is known about their mechanism of action in VSMCs beyond signaling alterations. The aim of this study was to determine the transcriptional effects of mTOR inhibitors in VSMCs. Candidate transcripts were identified by a microarray analysis of human coronary arteries transplanted to immunodeficient mice and treated with mTOR inhibitors in vivo. This genome-wide approach identified two major groups of proliferation- and inflammation-related genes that were downregulated by mTOR blockade. We investigated if selected transcripts were mTOR-dependent in model experimental systems of cultured coronary arteries and VSMCs.
    Materials and methods: Human coronary arteries and VSMCs were cultured in vitro and treated with the mTOR inhibitor rapamycin at various concentrations and durations. Cultured cells were also treated with tumor necrosis factor (TNF) to induce pro-inflammatory responses similar to known NFκB activation in arteries immediately after transplantation and during organ culture. Real-time quantitative PCR was used to evaluate the effects on gene expression.
    Results: In cultured VSMCs, the cell proliferation genes CCNA2, HIST1H3G, MKI67, and TOP2A and the inflammatory gene TLR3 were downregulated by rapamycin, with a peak effect after 24 hours of treatment. In contrast, the inflammatory chemokines CXCL9 and CXCL10 were upregulated by rapamycin in TNF-treated VSMCs with a peak effect after 72 hours of treatment, contradictory to the microarray results. In cultured coronary arteries, downregulation was observed for MKI67 and TOP2A and the inflammatory genes IFI30 and SPP1 after 72 hours of rapamycin treatment.
    Conclusions: The regulation of several cell proliferation genes by mTOR in cultured arteries and cells is consistent with the microarray analysis of coronary artery grafts in humanized mice, and suggests that the in vitro models can be used to further study mechanisms of transcriptional control by mTOR inhibitors in VSMCs. The weak and inconsistent effects observed on inflammatory gene expression in vitro may indicate false positive results of the microarray analysis, although an alternative explanation is that de-differentiation of cultured arteries and VSMCs is not suitable for this aspect of mTOR biology and that further in vivo studies are required.

  • 18.5.2015

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