Abstract 4371105: Vascular Smooth Muscle Cells lacking the Insulin-like Growth Factor 1 Receptor Produce Pro-Inflammatory Extracellular Vesicles Upon Insulin Stimulation

Introduction: Diabetes Mellitus is associated with an increase in atherosclerotic plaque development. We have previously shown that extracellular vesicles derived from vascular smooth muscle cells (SMCs) of diabetic patients and mouse models contain elevated levels of two microRNAs, miR-221 and -222 (miR-221/222) and promote endothelial cell activation and polarization of macrophages towards the M1 inflammatory phenotype in a miR-221/222 dependent manner. Furthermore, we have found that loss of the insulin-like growth factor 1 receptor (IGFR) coupled with physiological insulin stimulation leads to increased miR-221/222 in SMCs. Hypothesis: Knockdown of the IGFR in VSMCs coupled with physiological insulin stimulation leads to production of extracellular vesicles that promote an inflammatory phenotype in endothelial cells and macrophages. Methods: Human coronary artery SMCs were electroporated with plasmids encoding CRISPR/Cas9 and either IGFR-targeting or non-targeting gRNA to produce SMCs lacking the IGFR (IKD) and controls (NT), respectively. Extracellular vesicles were obtained from these SMCs following incubation with 1 nmol/L insulin (NT+I, IKD+I) or vehicle (NT+V, IKD+V) for 24 hours. Human coronary artery endothelial cells were incubated with extracellular vesicles from these SMCs for 24 hours. Increased adhesion of THP-1 monocytes to human coronary artery endothelial cells was measured in a static assay as an indicator of endothelial activation. Human CD14+ monocytes were differentiated into macrophages and incubated with extracellular vesicles. The inflammatory state of the macrophages was determined by measuring interleukin-6 secretion and calculating a M1(inflammatory):M2 (resolving) macrophage phenotype score based on mRNA makers. Results: Endothelial cells incubated with IKD+I, but not NT+V, NT+I, or IKD+V exhibited significantly increased adhesion of THP-1 monocytes compared to the other those treated with the other extracellular vesicles (Figure A). The macrophages incubated with the IKD+I extracellular vesicles were the only group with detectable secretion of interleukin-6 and exhibited a significant increase M1:M2 phenotype score (Figure B). Conclusions: These data suggest that loss of the IGFR in vascular smooth muscle cells coupled with physiological insulin stimulation promotes secretion of extracellular vesicles that promote vascular inflammation. This represents a novel mechanism for the cardiovascular complications of diabetes.