Echocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels.
Reversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p < 0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p < 0.05) and correlated with plasma total TGF-β1 levels (r = 0.38, p = 0.05 for PLAs and r = 0.37, p = 0.06 for single leukocytes) and reductions in ejection fraction (r = − 0.42, p = 0.03 for PLAs and r = − 0.37, p = 0.06 for single leukocytes).
p-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-β1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-β1.