Abstract

In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood. We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action. We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion. Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling.

Highlights

  • RATIONALE: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling

  • SMILR and this downstream axis were found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy

  • SMILR depletion and overexpression were previously shown to decrease and increase, respectively, proliferation induced by stimulation of human saphenous vein SMCs (HSVSMCs) with IL1-PDGF.[17]

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Summary

Methods

The authors declare that all supporting data and materials are available within the article (and its in the Online Data Supplement) and available from the corresponding author on reasonable request. Expanded information about materials and methods are available in the Online Data Supplement. All studies comply with the Declaration of Helsinki, that the locally appointed ethics committee has approved the research protocol and that informed consent has been obtained from the subjects. Primary human saphenous vein SMCs (HSVSMCs) were isolated via explant technique from consented patients and cultured as previously described.[17] All procedures had local ethical approval (15/ES/0094). All qRT-PCR data were analyzed via the 2-ΔΔCt method.[18] This method uses a house keeping gene and UBC (ubiquitin C) was selected as a housekeeping gene due to its stability across all groups and conditions studied. Data are graphed as relative quantification normalized to the UBC housekeeping gene.[18]

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