Abstract 16458: Paracrine Crosstalk Between Paltelets And Vascular Endothelial Cell is Mediated via MicroRNAs

Abstract

Majority of extracellular miRNAs constituting plasma miRNA pool are released by platelets. This study investigated the miRNA signature of leukocyte depleted platelets (LDP) isolated from T2DM or healthy subjects and explored the mechanism of miRNA mediated vascular crosstalk. LDPs, obtained by leukocyte filtration of platelet rich plasma (PRP), were isolated from blood withdrawn from 6 T2DM patients (male, age: 54.8±7.6 yrs; HbA1c: 8.9±0.6%; BMI: 30.9±2.8kg/m2) and 10 healthy volunteers (male, age: 49.2±6.8yrs; HbA1c: 5.3±0.2%; BMI: 26.6±3.8kg/m2). Total RNA isolated from diabetic or healthy LDPs was subjected to PCR based miRNA profiling. Matrigel capillary formation and genomic BrdU incorporation were measured as readouts of angiogenesis and cell proliferation respectively. Cellular metabolic activities were measured by WST-1 assay. Student t-test was applied to compare groups and p<0.05 considered significant. Total 735 (97%) miRNAs were detected (threshold< 29 Ct) in all LDPs. Levels of 19.5% miRNAs were decreased in T2DM platelets, whereas 7.5% miRNAs showed increase at 2-50 fold cutoff value. Compared to healthy LDPs (n=10), decreased levels of 14 highly platelet enriched miRNAs candidates including miR-21 and miR-22 were confirmed by RTPCR validation of individual T2DM LDPs irrespective of aspirin intake or presence of coronary heart disease (n=11). Thrombin stimulation of washed healthy platelets increased miRNA release into cell supernatant and confocal microscopy confirmed the cellular uptake of platelet released micro-particles into human coronary artery endothelial cells within 2-24h. In addition, endothelial overexpression of miR-21 or miR-22 decreased cell proliferation and metabolic activity, whereas miR-22 also attenuated capillary formation capacity. MiR-21 and miR-22 modulation inversely regulated expression of cell-cycle regulator gene CDK-6 at mRNA and protein levels. Diabetic platelets show distinct miRNA signature compared to healthy platelets and lower levels of miRNAs in diabetic platelets may represent their active secretion during diabetes. Released miRNAs may mediate crosstalk with vascular cells by influencing cellular gene expression and function upon their uptake.