DDAH2/ADMA系统经TGF-β1信号通路调控房颤心房纤维化的作用机制

Transforming growth factor-β1 (TGF-β1) mediated atrial fibrosis plays an important role in the occurrence and maintenance of atrial fibrillation (AF). The precise mechanisms that underlie atrial fibrosis are incompletely understood. ADMA is an endogenous nitric oxide synthase inhibitor, which is mainly metabolized by the enzyme dimethyarginine dimethylaminohydrolase (DDAH). Plasma ADMA concentration was increased in patients with AF, and was positively correlated with left atrial diameter in our previous study. In dogs with chronic AF induced by rapid atrial pacing, DDAH activity was reduced, and ADMA was correlated with the extent of interstitial fibrosis. Additionally, exogenous administration of ADMA dose-dependently increased mRNA and protein expression of TGF-β1 in cultured atrial fibroblasts. Therefore, we hypothesized that ADMA participated in the process of atrial fibrosis through TGF-β1 signal pathway; DDAH2 overexpression inhibited upregulation of TGF-β1 signaling, inhibited fibroblast proliferation and collagen synthesis, and finally alleviated the development of atrial fibrosis. In the present study, we planed to: (1) measure atrial ADMA levels, DDAH2 expression and activity in AF dogs, and their correlations with TGF-β1 signaling, MMPs/TIMPs system, and type I/III collagen in AF dogs; (2) determine the effect and molecular mechanisms of ADMA in atrial fibrobalsts proliferation, collagen synthesis and metabolism; (3) produce adenovirus vector encoding human DDAH2 to study the effect of DDAH2 overexpression on TGF-β1 signaling, fibroblast proliferation, collagen synthesis and metabolism. We aimed to clarify the effect and mechanism of DDAH2/ADMA pathway in the regulation of atrial fibrosis during AF, and to identify new potential therapeutic target for the prevention and treatment of AF.

TGF-β1介导的心房纤维化是房颤发生和维持的重要机制。ADMA是内源性的NOS抑制剂，主要由DDAH分解代谢。前期研究发现，房颤患者血浆ADMA升高；房颤犬心房组织DDAH活性下降，ADMA与心房纤维化程度相关；ADMA能够上调犬心房成纤维细胞的TGF-β1表达。因此我们假设：ADMA通过促进TGF-β1信号通路，刺激心房成纤维细胞增殖及胶原合成，参与心房纤维化；而上调DDAH2表达能够抑制ADMA诱导的TGF-β1信号通路，延缓心房纤维化。本研究将：①观察房颤犬心房组织DDAH2/ADMA系统变化，及其与心房纤维化关系；②细胞水平明确ADMA促进TGF-β1信号通路，刺激心房成纤维细胞增殖及胶原合成的作用及分子机制；③观察上调DDAH2表达对ADMA诱导的TGF-β1信号通路及心房纤维化的抑制作用。阐明DDAH2/ADMA系统调控心房纤维化的作用及机制，为房颤防治提供新的干预靶点。

TGF-β1介导的心房纤维化是房颤发生和维持的重要机制。该研究证实：1.房颤犬心房组织ADMA水平与心房胶原容积呈正相关；房颤犬心房组织TGF-β1信号通路中TGF-β1、P-smad2、Smad4的mRNA及蛋白表达水平明显升高，MMP9及I型胶原的蛋白表达较对照组也明显增加。ADMA水平与TGF-β1、P-smad2、MMP9及I型胶原的蛋白表达呈正相关。以上数据初步证实，DDAH2/ADMA系统参与房颤心房胶原的合成，与TGF-β1/P-smad2信号通路有关。2. 进一步的细胞水平研究发现，ADMA能够浓度依赖性的促进犬成纤维细胞的增殖。成纤维细胞中TGF-β1、P-smad2、TAK1 mRNA及蛋白水平随着ADMA浓度增加而逐渐升高，而加用TGF-β1 siRNA或TGF-β受体阻滞剂，能够明显抑制成纤维细胞的增殖程度。上述数据表明，ADMA是通过上调TGF-β1通路相关因子的表达而促进成纤维细胞增殖及胶原合成。3. 利用多种工具酶进行分组干预发现，ADMA能够明显提高成纤维细胞中的Ang II水平。而氯沙坦能够明显降低ADMA引起的TGF-β1、MMP9、I型胶原的mRNA及蛋白表达水平，证实ADMA通过提高Ang II水平进而促进TGF-β1、MMP9、I型胶原的过度表达，而与NOS/NO通路无关。由此证实，Ang II是ADMA调控TGF-β1信号通路中关键环节，阻断Ang II通路能够抑制ADMA的致纤维化作用。4. 构建表达人DDAH2的腺病毒，内源性上调DDAH2表达或给予DDAH2抑制剂能够调控ADMA诱导的TGF-β1信号通路、成纤维细胞增殖及胶原合成。5. 临床试验中发现，在非瓣膜病房颤患者中ADMA水平与左心耳血栓风险的相关。血栓组的血浆ADMA水平明显高于非血栓组。多元回归分析证实，ADMA 水平是预测非瓣膜病房颤患者左心耳血栓形成的独立危险因素。综上，该研究多角度、多层次阐明了DDAH2/ADMA系统调控心房纤维化的作用及机制，为房颤防治提供新的干预靶点。