Inactivation of platelet-derived TGF-β1 attenuates aortic stenosis progression in a robust murine model

Abstract

Aortic stenosis (AS) is a degenerative heart condition characterized by fibrosis and narrowing of aortic valves (AV), resulting in high wall shear stress (WSS) across valves. AS is associated with high plasma levels of transforming growth factor-β1 (TGF-β1), which can be activated by WSS to induce organ fibrosis, but the cellular source of TGF-β1 is not clear. Here, we show that platelet-derived TGF-β1 plays an important role in AS progression. We first established an aggressive and robust murine model of AS, using the existing Ldlr−/−Apob100/100 (LDLR) breed of mice, and accelerated AS progression by feeding them a high-fat diet (HFD). We then captured very high resolution images of AV movement and thickness and of blood flow velocity across the AV, using a modified ultrasound imaging technique, which revealed early evidence of AS and distinguished different stages of AS progression. More than 90% of LDLR animals developed AS within 6 months of HFD. Scanning electron microscopy and whole-mount immunostaining imaging of AV identified activated platelets physically attached to valvular endothelial cells (VEC) expressing high phosphorylated Smad2 (p-Smad2). To test the contribution of platelet-derived TGF-β1 in AS, we derived LDLR mice lacking platelet TGF-β1 (TGF-β1platelet-KO-LDLR) and showed reduced AS progression and lower p-Smad2 and myofibroblasts in their AV compared with littermate controls fed the HFD for 6 months. Our data suggest that platelet-derived TGF-β1 triggers AS progression by inducing signaling in VEC, and their subsequent transformation into collagen-producing-myofibroblasts. Thus, inhibiting platelet-derived TGF-β1 might attenuate or prevent fibrotic diseases characterized by platelet activation and high WSS, such as AS.

COX-2 Is Downregulated in Human Stenotic Aortic Valves and Its Inhibition Promotes Dystrophic Calcification

“…In agreement with the data in AVs, we found that in human AVICs isolated from non-calcific AVs, COX-2 inhibition with celecoxib promotes AVICs trans-differentiation towards a myofibroblast phenotype and the formation of calcific nodules, even in absence of TGF-β1, a multifunctional cytokine involved in collagen deposition and calcification of the AV [24]. Noteworthy, celecoxib also increased the levels of calcification measured by Alizarin Red when AVICs were grown in osteogenic condition.…”

Oscillatory shear potentiates latent TGF-β1 activation more than steady shear as demonstrated by a novel force generator

“…These responses were almost completely blocked by anti-TGF-β1 antibody, which showed the specificity of active TGF-β1-generated by OSS. We recently showed HUVEC cultured with the activated form of platelet-derived TGF-β1 induced EndoMT markers, α-SMA and vimentin 19 . In addition, incubation of HUVEC with OSS induced higher periostin expression than cells cultured with SS (data not shown).…”

“…We recently showed that shear-activated platelet TGF-β1 directly contributes to aortic stenosis (AS) progression in a robust mouse model 19 . EndoMT is a phenomenon induced by TGF-β1 signaling 17 , and we found platelets are physically attached with valvular endothelial cells inducing mesenchymal transition 19 . These results suggest further studies should determine whether OSS-induced TGF-β1 activation around valve leaflets contributes to AS progression.…”

Platelets

“…In AS, the WSS and accompanying turbulent blood flow through the AV can activate the endothelium, which activates platelets and stimulates their adhesion to the leaflets ( 10 ). This review explores the following ways that platelets are involved in accelerating the pathogenesis of AS: 1) activated platelets’ release of transforming growth factor (TGF)- that promotes the conversion of VECs and VICs to collagen-producing-myofibroblasts, which increases fibrosis and mineralization ( 11 ); 2) the role of Dickkopf-1 (DKK-1) glycoprotein and lymphangiogenesis in AS; 3) release of ADP from activated platelets, which promotes the osteogenic transition of VICs and leads to AV mineralization ( 10 ); 4) retention and oxidation of lipids that play an active role in VIC osteogenic transition by activating the nuclear factor-kappa B (NF- B) inflammation pathway ( 8 ).…”