Abstract 617: Wnt-Containing Microvesicles Are Upregulated in Vascular Smooth Muscle Cell Cultures and Plasma From SM22-Cre;LRP6(fl/fl);LDLR-/- Mice

Abstract

When fed a high fat diet (HFD) typical of westernized societies, male LDL receptor -deficient (LDLR-/-) mice develop obesity and insulin-resistant diabetes, with concomitant arteriosclerotic calcification and fibrosis. We recently demonstrated that noncanonical Wnt signals and ligands (including Wnt4, Wnt7b, and Wnt10a) were upregulated with LRP6 deficiency in aortic vascular smooth muscle (VSM) of LDLR-/- mice, with concomitant worsening of vascular disease. Because Wnt ligands are hydrophobic due to fatty acid acylation, we examined whether lipidaceous microvesicles (MVs) containing Wnt ligands were elaborated by VSM, and whether LRP6 impacted plasma MV numbers and contents. Microvesicles were isolated using both polymer based- and differential ultracentrifugation - based purification methods, and Malvern NS300 NanoSight nanoparticle tracking analysis implemented to quantify vesicle concentrations, focusing upon the 50-200 nm size range of exosomes and mineralizing vesicles. As compared to Cre-negative control cultures, SM22-Cre;LRP6(fl/fl);LDLR-/- VSM elaborated 2.9-fold fold higher numbers of MVs over a 2 day period (143 +/-27 million/ml +/- vs. 49+/- 5 million/ml; p = 0.01). ML141 -- a cdc42/rac1 antagonist that reduces VSM calcification and noncanonical Wnt signals activated with LRP6 deficiency — reduces MV numbers in VSM cultures (42+/- 10 million / ml with 10 uM ML141 vs.120 +/- 19 million/ ml vehicle; p< 0.001). Consistent with primary VSM results, a 2-fold increase in the circulating plasma MV concentration was observed in aging SM22-Cre;LRP6(fl/fl);LDLR-/- mice vs. LRP6(fl/fl);LDLR-/- sibling controls (4.7+/- 1.1 billion/ml vs. 2.3+/-0.3 billion/ml, p = 0.07, n = 7-8 per genotype). Western blot analysis of extracts prepared from plasma MVs confirmed the presence of Wnt5a/b, Wnt10a, Wnt4 in this circulating nanoparticle population, and that Wnt4 was significantly increased in the MV fraction with LRP6 deficiency (1.4-fold, p = 0.01). Thus, Wnt-containing microvesicles are elaborated by VSM in vitro and in vivo. Quantitative characterization of plasma MV protein and lipid composition may yield novel biomarkers of pro-sclerotic Wnt signaling in diabetic vascular disease.