Mice Lacking Platelet TGF-β1 Are Protected from Aortic Stenosis Progression

Introduction and Hypothesis: Aortic stenosis (AS) is an age-related disease primarily affecting the elderly and is characterized by accelerated fibrosis in the aortic valve resulting in stiffening and narrowing of the valve opening and causing high wall shear stress (WSS) across the valves. We have previously shown shear stress activates TGF-β1 released from platelets and found higher levels of TGF-β1in both patients and mice with AS, indicating that platelet TGF-β1 may be involved in AS progression. To test this hypothesis, we used genetic approaches and new technology to characterize AS progression noninvasively and assessed the role of platelet-derived TGF-β1 and its activation by high shear across the valve and its consequent contribution to aortic stenosis progression.

Methods: We used a hyperlipidemia LDLR^-/-ApoB^100/100 mouse model that spontaneously develops AS. To test the contribution of platelet-derived TGF-β1 in AS progression, theLDLR^-/-ApoB^100/100 mice were crossed with PF4CreTgfb1^flox/flox mice to delete platelet TGF-β1, and the resulting PF4CreLDLR^-/-ApobB^100/100Tgfb1^f/f mice were designated as final genotype. LDLR^-/-ApobB^100/100Tgfb1^f/f mice, in which TGF-β1 is normally expressed in the whole animal, including platelets, served as littermate controls. Both groups of mice received a very high fat (HF) diet containing 1.25% cholesterol (6-times higher cholesterol concentration than the 0.2% present in Western diet) commencing at ~6 weeks of age for up to 6 months. AS progression was monitored using a new modified view of echocardiographic imaging of the aortic valves to precisely measure fractional valve opening (Cusp Separation/Left Ventricular Outflow Tract). Velocity of blood flow across the aortic valve (AV Peak Velocity) was measured by a combination of color and Pulse-wave Doppler, and WSS was calculated using the Bernoulli's equation (WSS=4μ*v/r), where r=fractional valve opening/2, μ=blood viscosity, and v=AV Peak Velocity.

Results: The final genotype mice had ~40% lower total plasma TGF-β1 than littermate controls (2.2 ± 0.06 ng/ml, n=45 final genotype, 3.8 ± 0.2 ng/ml, n=14, littermate controls; p<0.0001), and ~80% lower total platelet TGF-β1 (6.0 ± 0.9 ng/5x10⁸platelets vs. 27.2 ± 1.4 ng/5x10⁸platelets, n=5; p<0.0001). These mice also showed significantly lower AS progression than littermate controls after 6 months on HF diet as measured by improvements in all three AS progression parameters [AV Peak Velocity (1460 ± 62 mm/s, in final genotype; n=15 vs. 1767 ± 120 mm/s, in littermate controls, n=7; p=0.02), fractional valve opening (0.56 ± 0.02, in final genotype; n=15 vs. 0.47 ± 0.03, n=7, littermate controls; p=0.03) and WSS (690 ± 40 dyn/cm², in final genotype; n=15 vs. 924 ± 90 dyn/cm², n=7, littermate controls; p=0.01)]. Final genotype mice also had significantly lower TGF-β1 signaling events, as measured by phophorylated-Smad2 staining in valvular cells (2226 ± 457 pixel², in final genotype; n=4 vs. 7041 ± 964 pixel², n=3, littermate controls; p<0.001). They also had less valve thickening (28545 ± 4555 pixel², in final genotype; n=4 vs. 52405 ± 18703 pixel², n=3, littermate controls; p=0.02) at 6 months as measured by histology. Whole mount confocal imaging of the valves showed expression and translocation of phosphorylated-Smad2 to the nucleus of valvular cells, and scanning electron microscopy showed activated platelets in direct contact with valvular cells, indicating direct crosstalk between platelets and valvular cells via TGF-β1 signaling. We also found a subset of valvular cells co-expressing vimentin, a fibroblast-specific marker with IsolectinB4, an endothelial cell-specific marker. These double-positive cells appear to be migrating inwards towards the spongiosa layer of the valve and also co-express α-SMA, suggesting myofibroblast transformation to produce excess collagen. These processes were less frequent in the final genotype mice than the WT littermate controls.

Conclusion: The final genotype mice have reduced AS progression compared to littermate controls. Thus, platelet TGF-β1 contributes to AS progression, and blocking TGF-β1 release and activation may have beneficial effects on preventing AS progression.