三维基质硬化和软化对心肌肌成纤维细胞表型转化的作用及机制研究

Myocardial fibrosis, as the pivotal cause and effect of heart failure, has been one of the major therapeutic targets for treating cardiovascular diseases. The differentiation of cardiac fibroblasts to myofibroblasts, i.e., the cardiac myofibroblast differentiation, plays a critical role in myocardial remodeling and is becoming the research highlight of myocardial fibrosis. The cardiac myofibroblast differentiation has been found to be closely regulated by extracellular matrix stiffness. Despite the remarkable advances in mechanical regulation of cardiac myofibroblast differentiation in two-dimensional microenvironment, few related three-dimensional (3D) studies exist and therefore the effects and mechanisms of matrix stiffness, especially matrix stiffening and softening, on cardiac myofibroblast differentiation in 3D remain elusive. We have previously done many works on hydrogel-based 3D tissue construct fabrication and cell mechanical microenvironment engineering. In this proposal, we plan to develop and apply collagen-alginate hybrid hydrogels for engineering the mechanical microenvironment of cardiac myofibroblasts and investigate the cellular responses to matrix stiffness changes. Collagen-alginate hybrid hydrogels with optimized mechanical stiffening and softening properties are fabricated. The effects of matrix stiffening on cardiac myofibroblast differentiation in 3D are studied by increasing matrix stiffness with calcium chloride in the presence of cells. Myocardial fibrosis models with different degrees of cardiac myofibroblast differentiation are then constructed. After reducing the stiffness of these models with sodium citrate, the effects of matrix softening on dedifferentiation of cardiac myofibroblasts are investigated. Finally, the roles of angiotensin II type 1 receptor, Yes-associated protein and transcriptional co-activator with PDZ binding motif are studied to uncover the matrix stiffness mechanotransduction pathways of cardiac myofibroblasts in 3D. The results would be helpful for understanding the pathological mechanisms of myocardial fibrosis, preventing and reversing myocardial fibrosis during heart failure.

心肌纤维化的预防和逆转是众多心血管病防治的主要目标之一，而心肌肌成纤维细胞（MyoFB）表型调控是预防和逆转心肌纤维化的研究热点。基质硬度是影响心肌MyoFB表型转化的关键因素，但是，目前三维微环境下基质硬化和软化对心肌MyoFB表型转化的作用及及机制不清楚。本项目结合申请人和课题组在体外组织模型构建及三维细胞力学微环境调控方面的研究基础，制备可变化基质硬度的胶原–海藻酸盐复合水凝胶；通过氯化钙作用（提供钙离子使海藻酸盐交联）使包埋心肌MyoFB的复合水凝胶的硬度升高，基于基质硬化构建不同纤维化程度的三维心肌组织模型；通过柠檬酸钠作用（螯合钙离子使海藻酸盐解交联）使上述组织模型的硬度降低，研究三维微环境下基质软化对心肌MyoFB去分化的作用；阐明三维微环境下基质硬度影响心肌MyoFB表型转化的关键信号通路，揭示其作用机制。研究结果将为心肌纤维化的预防和逆转以及心血管病的防治提供重要参考。

纤维化是许多疾病致残致死的主要原因之一，而基质刚度变化是纤维化的一个典型病理特征，其对效应细胞（如心肌中心肌成纤维细胞）的表型转化具有重要影响。但是，目前三维微环境下基质刚度变化对纤维化效应细胞表型转化的影响规律和作用机制不清楚。本项目在系统调研和综述基于仿生生物材料的细胞三维微环境工程的基础上，制备了基质刚度原位可调的胶原-海藻酸盐复合水凝胶，实现了细胞三维微环境中基质刚度硬化和软化的原位调控，以及基质刚度和细胞铺展的独立调控；基于上述复合水凝胶，构建了载有心肌成纤维细胞或星形胶质细胞的体外组织模型；表明了基质刚度变化促使细胞表型发生转化，且三维微环境中细胞表型转化对基质刚度和化学刺激因子的响应与细胞铺展状态密切相关；发现了硬基质刚度可以通过血管紧张素II 1型受体促进心肌成纤维细胞表型转化，而Yes相关蛋白在基质刚度调控的星形胶质细胞力学信号转导过程中可能发挥了关键作用。研究结果将为细胞微环境工程、纤维化力学生物学以及纤维化相关疾病的防治提供有用参考。