Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado School of Medicine, Aurora, Colorado.
Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado School of Medicine, Aurora, Colorado.
Ann Thorac Surg. 2021 Aug;112(2):519-525. doi: 10.1016/j.athoracsur.2020.08.068. Epub 2020 Nov 13.
Proinflammatory activation of toll-like receptor-4 (TLR4) drives phenotypic changes in aortic valve interstitial cells (AVICs) and produces a fibrogenic phenotype that mediates valvular fibrosis and contributes to aortic stenosis. Prior work identified upregulated Wnt signaling in AVICs taken from valves affected by aortic stenosis. Our purpose was to determine the contribution of Wnt signaling to TLR4-dependent fibrogenic activity in isolated human AVICs.
Human AVICs were isolated from hearts explanted for cardiac transplantation (N = 4). To test whether Wnt signaling contributed to TLR4-dependent fibrogenic activity, AVICs were treated with Wnt inhibitor (Dkk1) prior to TLR4 activation (LPS) and fibrogenic markers assessed. To determine the mediator of TLR4-to-Wnt signaling, expression of the key Wnt ligand, Wnt3a, was assessed after TLR4 activation and neutralizing antibodies confirmed the identity of the mediator. Fibrogenic activity was assessed after AVICs were treated with recombinant Wnt3a. Statistics were by analysis of variance (P < .05).
TLR4 activation upregulated in vitro collagen deposition, type IV collagen and MMP2 expression, and Dkk1 inhibited these responses (P < .05). Expression of Wnt3a was upregulated after TLR4 activation (P < .05). Anti-Wnt3a neutralizing antibodies abrogated TLR4-dependent type IV collagen and MMP2 expression (P < .05). Wnt3a upregulated type IV collagen and MMP2 expression independent of TLR4 activation (P < .05).
This study found that TLR4-dependent fibrogenic activity was mediated through Wnt signaling. The mediator of profibrogenic TLR4-to-Wnt signaling was a key Wnt ligand, Wnt3a. The abrogation of TLR4-induced fibrogenic activity in human AVICs by Wnt blockade illustrates a potential therapeutic role for Wnt inhibition in treatment and/or prevention of aortic stenosis.
Toll 样受体-4（TLR4）的促炎激活驱动主动脉瓣间质细胞（AVICs）的表型变化，并产生纤维生成表型，介导瓣膜纤维化并导致主动脉瓣狭窄。先前的工作发现，在主动脉瓣狭窄患者的瓣膜中，AVICs 的 Wnt 信号上调。我们的目的是确定 Wnt 信号在分离的人 AVICs 中 TLR4 依赖性纤维生成活性中的作用。
从心脏移植（N=4）中取出心脏，分离人 AVICs。为了测试 Wnt 信号是否有助于 TLR4 依赖性纤维生成活性，在 TLR4 激活（LPS）之前，AVICs 用 Wnt 抑制剂（Dkk1）处理，并评估纤维生成标记物。为了确定 TLR4 到 Wnt 信号的介质，在 TLR4 激活后评估关键 Wnt 配体 Wnt3a 的表达，并使用中和抗体确认介质的身份。在 AVICs 用重组 Wnt3a 处理后评估纤维生成活性。统计学分析采用方差分析（P<.05）。
TLR4 激活上调了体外胶原蛋白沉积、IV 型胶原蛋白和 MMP2 的表达，而 Dkk1 抑制了这些反应（P<.05）。TLR4 激活后 Wnt3a 的表达上调（P<.05）。抗 Wnt3a 中和抗体消除了 TLR4 依赖性 IV 型胶原蛋白和 MMP2 的表达（P<.05）。Wnt3a 独立于 TLR4 激活而上调 IV 型胶原蛋白和 MMP2 的表达（P<.05）。
本研究发现 TLR4 依赖性纤维生成活性是通过 Wnt 信号介导的。促纤维生成 TLR4 到 Wnt 信号的介质是关键的 Wnt 配体 Wnt3a。Wnt 阻断剂阻断 TLR4 诱导的人 AVICs 纤维生成活性，说明了 Wnt 抑制在治疗和/或预防主动脉瓣狭窄中的潜在治疗作用。
