Proceedings of The Physiological Society

Mitochondria: Form and function (London, UK) (2017) Proc Physiol Soc 38, PC01

Poster Communications

Exosomal Transfer of MicroRNA in Vascular Smooth Muscle Cell Proliferation is Mitochondrial-Dependent

P. Coats1, Z. Al-Sulti1

1. SIPBS, University of Strathclyde, Glasgow, United Kingdom.

Exosomes are widely recognised as important autocrine/ paracrine mediators of cell function. Vascular smooth muscle (VSM) cells undergo phenotypic transformation from a contractile to a synthetic hyperproliferative phenotype in atherosclerosis and re-stenosis associated with percutaneous coronary interventions. We have previously shown mitochondrial bioenergetics are upregulated in synthetic hyperproliferative VSM cells. In this work we aimed to study potential correlation between the synthetic hyperproliferative VSM cell and exosomal-dependent signalling. All experiments were undertaken in VSM cells isolated from aorta harvested from 12 week old male Sprague Dawley rats. Exosomes were isolated from VSM cells using a sucrose gradient-ultracentrifuge technique and confirmed by zestier and western blotting for CD markers. miRNA and RNA measured using standard qRT-PCR. Cell proliferation measured using 3H thymidine incorporation. Total exosomal release was increased in synthetic VSM cells vs. wild type (WT) cells. Likewise, total protein and RNA within exosomes in synthetic VSM was significantly greater when compared with WT VSM cells (Table 1.). Addition of exosomes isolated from WT or synthetic hyperproliferative cells to VSM cell cultures resulted in 4.7 ± 2.3% and 23.4 ± 4.6% respectively, p<0.05. qRT-PCR of exosomal contents (synthetic VSM cells vs. WT VSM cells) highlighted a significant reduction in pro-apoptotic genes (Bnip3, SOD1, SOD2), a reduction in significant tumour suppressor genes (Pmaip1, p53) and significant reduction in cell cycle regulator Cdkn2a. Likewise, RNA for PI3K, 4EBP1 and mTOR were all significantly greater in exosomes isolated from synthetic VSM cell vs. WT VSM cells. miRNA sequencing highlighted significant differences in exosome contents. Notably a 10-fold increase in pro-mitogenic miR21 and 3.5-fold loss of anti-mitogenic miR145, synthetic VSM cell vs. WT VSM cells. The use of selective anti-miRs/ miR mimetics in cell proliferation assays confirmed that both miR145/ miR21 are regulators of VSM cell phenotype and proliferation. Inhibition of mitochondrial bioenergetics or mitochondrial dynamics (fission-fusion) with the DRP-1 inhibitor MDivi-1 normalised exosomal yield, exosomal contents, RNA and miRNA similar to that measured in WT cells. Our results implicate exosomes and their RNA/ miRNA contents as crucial mediators of cell proliferation in VSM cells with a synthetic hyperproliferative phenotype. VSM cell proliferation is in part regulated by mitochondrial bioenergetics/ dynamics. Significantly, we have for the first time demonstrated a relationship between mitochondrial function, exosomal trafficking and VSM cell proliferation.

Where applicable, experiments conform with Society ethical requirements