Exosomes from vascular smooth muscle cells of diabetic origin promote a pro‐inflammatory macrophage phenotype

Abstract

Diabetes Mellitus accounts for over 350,000 deaths annually, within the United States alone. One of the most common co-morbidities of diabetes is heart disease. Vascular inflammation among diabetic patients leads to a two-to-four fold increase in risk of heart attack and stroke. Two specific micro-RNAs (miRNA), miR-221 and miR-222, promote vascular smooth muscle cell (VSMC) proliferation and are elevated in VSMCs of diabetic subjects. Diabetic VSMC derived exosomes (DVEs) also exhibit elevated levels of miR-221 and miR-222 compared to exosomes isolated from normal VSMCs (NVEs), suggesting a novel paracrine signaling method mediating the cardiovascular complications of diabetes. The objective of this study is to determine whether this mechanism alters macrophage phenotype. Macrophages exist in multiple phenotypes, including a pro-inflammatory state (M1) and a resolving state (M2). We hypothesize that exposure of macrophages to DVEs will promote polarization toward the M1 phenotype. CD14+ human monocytes were differentiated to macrophages by a 6-day serum incubation. Macrophages were then co-incubated with DVEs or NVEs overnight. M1 versus M2 polarization was assessed using a score based on expression of seven RNA markers of macrophage phenotype. Additional studies were performed using DVEs and NVEs isolated from VSMCs following knockdown of miR-221/222. Exposure of macrophages to DVEs promoted a significant increase in M1 polarization. This polarization was diminished in the DVEs isolated from VSMCs following miR-221/222 knockdown. DVE exposure was also associated with diminished expression of an RNA-binding protein, quaking (QKI), a known target of miR-221/222 whose deficiency is associated with M1 polarization. We conclude that exosomal transfer of miR-221/222 from diabetic VSMCs to macrophages promotes M1 polarization through down-regulation of QKI. These findings suggest that this paracrine signaling mechanism represents a novel mechanism underlying the cardiovascular complications of diabetes.