血管微环境中内皮细胞AMPK抑制心肌纤维化的功能与机制研究

Cardiac fibrosis is the pathological basis of various heart diseases. The progress of cardiac fibrosis is regulated by endothelial cells (ECs) in the vascular niche. ECs are involved in cardiac fibrosis through regulating angiogenesis, transforming to mesenchymal cells (endothelial-to-mesenchymal transition, EndMT) and secreting angiocrine factors. However, the mechanisms by which ECs are regulated during cardiac fibrosis remain largely unknown. AMP-activated protein kinase (AMPK) is an metabolic sensor that is critically involved in cardiac and endothelial homeostasis. Our previous work showed that AMPK activator metformin or AMPK upstream activator SIRT2 could repress aging-related or angiotensin II (Ang II)-induced cardiac perivascular fibrosis, whereas AMPK upstream inhibitor SIRT4 could promote the development of Ang II-induced cardiac perivascular fibrosis. Our preliminary data also revealed that metformin could repress Ang II-induced expression of endothelin-1 (angiocrine factor for cardiac fibrosis) in ECs. Based on these findings, we hypothesize that endothelial AMPK may regulate the angiocrine functions of ECs in the vascular niche to repress cardiac fibrosis. In this project, we will establish Ang II-induced cardiac fibrosis in mice with endothelial-specific AMPK knockout. Then, pathological, cellular and molecular approaches will be applied to answer the following questions: (1) The functions of endothelial AMPK in cardiac fibrosis, (2) The effects of AMPK on angiocrine functions of ECs during cardiac fibrosis and the underlying mechanism, (3) The potential involvement of endothelial AMPK in metformin-mediated inhibition of cardiac fibrosis. Through answering these questions, we could elucidate the functions of endothelial AMPK in cardiac fibrosis, which will improve our understanding of cardiac fibrosis and hopefully provide new strategies for the clinical treatment of cardiac fibrosis.

心肌纤维化是多种心脏疾病共有的病理特征，其发生过程受到血管微环境中内皮细胞的调控。内皮细胞可通过血管新生、内皮间充质转化和分泌血管分泌因子参与心肌纤维化，但内皮细胞功能的调控机制并不清楚。申请人发现代谢调节酶AMPK参与衰老或血管紧张素II诱导的心肌纤维化，初步结果提示这一过程可能涉及内皮细胞的血管分泌功能。因此，血管微环境中内皮细胞AMPK可能通过调节其血管分泌功能来抑制心肌纤维化。本项目拟以内皮细胞特异性AMPK敲除小鼠为研究对象，利用实验病理学、细胞生物学、分子生物学等研究手段，回答以下科学问题：（1）血管微环境中内皮细胞AMPK是否参与心肌纤维化；（2）血管微环境中内皮细胞AMPK是否调控内皮细胞的血管分泌功能来抑制心肌纤维化及其机制；（3）内皮细胞AMPK是否参与二甲双胍对心肌纤维化的抑制作用。探讨这些问题有助于明确内皮细胞AMPK在心肌纤维化中的功能，为防治心肌纤维化提供依据。

心肌纤维化是多种心脏疾病共有的病理特征，其发生过程受到血管微环境中内皮细胞的调控。内皮细胞可通过血管新生、内皮间充质转化和分泌血管分泌因子参与心肌纤维化，但内皮细胞功能的调控机制并不清楚。本项目立项前发现代谢调节酶AMPK参与衰老或血管紧张素II诱导的心肌纤维化，这一过程涉及内皮细胞的血管分泌功能。因此，申请人猜想内皮细胞中AMPK可能通过调节内皮细胞的血管分泌功能来抑制心肌纤维化。本项目以内皮细胞特异性AMPK敲除小鼠为研究对象，利用实验病理学、细胞生物学、分子生物学等研究手段，取得了以下学术发现：（1）构建了内皮细胞特异性AMPKa1敲除小鼠，发现内皮细胞AMPKa1敲除可以促进血管紧张素II诱导的心肌肥厚和心肌纤维化，提示内皮中代谢调节因子AMPK具有心脏保护功能。并且，我们发现内皮细胞AMPK贡献了二甲双胍对心肌肥厚和心肌纤维化的抑制作用。（2）我们在内皮细胞系和小鼠心脏内皮细胞中研究了AMPK对内皮细胞功能的影响，证明了AMPK可以抑制内皮细胞表达EDN1。（3）我们构建了内皮细胞特异性AMPKa1/EDN1双敲小鼠，发现内皮细胞EDN1是AMPK的关键下游分子并参与AMPK在心肌纤维化中的功能。（4）在本项目的基础上，我们进行了适度的拓展，研究了AMPK重要下游分子ECHS1在心肌肥厚中的功能。我们发现AMPK下游ECHS1在心衰病人中显著降低，并且ECHS1调控了巴豆酰化修饰；在小鼠中敲除ECHS1可以促进心肌肥厚和心肌纤维化的发生发展；相关研究结果发表在Circulation杂志。上述学术发现阐明了内皮细胞AMPK在心肌纤维化中的功能和分子机理，为防治心肌纤维化提供一定的理论依据。