Plasma Transforming Growth Factor-β1 (TGF-β1) Levels Increase with Age and Aortic Stenosis Progression and Low-Dose Galunisertib Attenuates Disease Progression in Mice

Objective: Aortic stenosis (AS) is an age-related degenerative valvular disease that ultimately leads to heart failure and sudden death. Platelet-derived TGF-β1 is a major source of plasma TGF-β1 and contributes to AS progression in LDLR ^-/-ApoB ^100/100(LA100), a mouse model that spontaneously develops AS that simulates human pathology, including fibrosis and high shear gradient. Although risk factors, such as hypercholesteremia and hyperglycemia, are like those for coronary artery disease, lowering cholesterol and glucose has not been successful in stopping progression, and there is no biomarker for early detection of AS. In this study, we measured plasma TGF-β1 levels as a function of age in LA100 mice and human AS patients and tested a new approach of partial inhibition of TGF-β signaling to inhibit AS progression in LA100 mice, while limiting cardiotoxicity.

Methods: We measured plasma TGF-β1 levels in controls, severe AS patients, and LA100 mice before they developed AS (2-months of age), when they developed moderate AS (1-year), and when they developed severe AS (2-years). LA100 mice at different stages of AS were treated with low-dose galunisertib, an inhibitor of the TGF-β receptor, ALK5.

Results: Plasma levels of total TGF-β1 were significantly higher in AS patients (n=34) compared to healthy controls (n=22) and patient controls (n=47) (p<0.001 for AS patients vs. healthy controls and p=0.049 for AS patients vs. patient controls). Age was positively correlated with total TGF-β1 levels in AS patients (r=0.475; p=0.005), but not patient or healthy controls (r=0.12, p=0.43). LA100mice developed AS with aging and plasma TGF-β1 levels in LA100mice (n=21-52; ages 2 months-2 years) were significantly higher than those in age-matched WT mice (n=23); (p<0.0001). Plasma TGF-β1 levels correlated with age in LA100 mice (R=0.6, p=<0.0001). Low-dose oral galunisertib was administered at a concentration of 1 µM in drinking water, giving an average daily dose of ~0.06 mg/kg, compared to a human cancer treatment dose of ~4.2 mg/kg daily. Galunisertib treatment: a) halted AS progression at all stages in LA100 mice compared to vehicle-treated control mice (p<0.05, n=9-13), b) did not change plasma TGF-β1 levels, c) inhibited TGF-β1 signaling as shown by decreased Smad2 phosphorylation in valve cells compared to vehicle-treated animals. An age-dependent increase in neutrophil to lymphocyte ratio (NY/LY), a marker of inflammation, was observed in LA100 mice compared to control mice (n=12-31; p<0.01), and the NY/LY ratio correlated with total TGF-β1 plasma levels (R=0.16; p=0.020). Galunisertib treatment reduced NY/LY ratios in LA100 mice (p=0.03).

Conclusions: 1. Increases in plasma TGF-β1 levels may be a valuable surrogate marker of AS progression. 2. Low-dose galunisertib attenuates AS progression in the LA100 mouse model without producing the cardiotoxicity associated with higher doses and thus may warrant further study in human AS patients.