血管收縮素轉化□II–血管收縮素1-7途徑於心臟細胞中之調控機制探討

Abstract

血管收縮素II（angiotensin II, Ang II）是腎素-血管收縮素系統（renin-angiotensin system, RAS）中一個重要的作用因子。Ang II調控心血管功能的恆定被認為與MMPs與TIMPs的平衡協調相關，而後者影響到組織中ECM的代謝。血管收縮素1-7（angiotensin 1-7, Ang 1-7）也是一個RAS中具有生物活性的胜□，它被認為扮演拮抗Ang II的角色。然而，Ang 1-7在心臟細胞中對MMPs和TIMPs的調控仍然未知，而Ang 1-7在拮抗Ang II媒介的MMPs和TIMPs表現的能力也需要被確認。據此，我們檢視人類心臟纖維母細胞（human cardiofibroblasts, HCF）和心臟心肌細胞（human cardiomyocytes, HCM）在經由Ang II或Ang 1-7刺激後MMPs和TIMPs的基因表現樣態。而結果顯示Ang 1-7和Ang II在初代培養的人類心臟細胞中對MMPs和TIMPs的基因調控作用具有相反和拮抗性作用。這些效應導致了MMPs/TIMPs的比例在Ang II刺激後上升和Ang 1-7處理後的降低，而這個效應可能具有心臟細胞的種類依存性關係。這些結果指出了Ang 1-7可能在減弱Ang II誘發ECM重塑所造成心臟損傷的角色。 Ang II與Ang 1-7兩者是RAS系統內重要的調控性胜□。RAS系統中，ACE2（angiotensin converting enzyme II）酵素以Ang II作為受質降解產生Ang 1-7。Ang II被指出在心臟結構性重塑過程中扮演重要的角色，而Ang 1-7則扮演拮抗Ang II的功能。許多的研究顯示Ang II可能具有調控心血管系統中ACE2的表現。然而，這些報導的結果並不一致。在本研究中，將使用HCF細胞分析Ang II與Ang 1-7對ACE2在轉錄與轉譯層面的調控效應。結果顯示Ang II可以增加ACE2的表現，而此作用可能是藉由Ang II第一型受器（Ang II type I receptor; AT1R）所媒介。Ang II所媒介的ACE2表現增加可以被AT1R下游標的－NADPH oxidase與ERK-MAPK（extracellular signal-regulated protein kinases-mitogen-activated protein kinase）訊息途徑的拮抗劑所阻斷。此外，Ang 1-7也可以在HCF細胞中增加ACE2的表現量，而此作用可以被Mas受器之阻斷劑所抑制。我們結果顯示HCF細胞受Ang 1-7刺激所造成的ACE2表現增加可能不具有Ang II-AT1R訊息途徑依存性關係。這些結果顯示Ang II誘導的ACE2表現可能會增加Ang II轉化生成Ang 1-7，接著ACE2的表現又可進一步的藉由Ang 1-7所促進。根據這個結果，我們認為此心臟細胞中ACE2的正向類回饋式的迴路調控可能有助於維持心臟中RAS系統的穩定狀態。 Ang II依存性訊息傳遞路徑被認為與AF病程中的間質性纖維化有關，而Ang II可被血管收縮素轉化□II（angiotensin converting enzyme II, ACE2）進一步的降解。因此，我們於AF模式豬隻的心房中檢視ACE2表現量，並探討其於AF期間纖維化病變過程之關聯。在組織學的分析方面，經由Masson's trichrome染色證實在AF心房組織間質空間中有較多的ECM累積，且其第一型膠原蛋白有顯著性增加。在AF心房組織中的ACE2基因與蛋白質表現量有顯著增加，然而細胞外信號調節激□2（extracellular signal-regulated kinase 2, ERK2）和活化型ERK2則在AF組織中有顯著提升。我們認為在AF組織中ACE2的減少可能導因於Ang II依賴性訊息傳遞途徑的回饋調控，此外，結果指出AF的心房纖維化病變可能經由ACE與ACE2間的拮抗性調控所誘發。

Angiotensin II (Ang II) is a critical effector in renin-angiotensin system (RAS). Ang II modulates the cardiovascular homeostasis, and matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) related metabolism of extracellular matrix (ECM). Angiotensin 1-7 (Ang 1-7) is also a bioactive peptide in RAS, which is considered to play a role of opposing effects to Ang II. However, the modulation of MMPs and TIMPs by Ang 1-7 is largely unclear in cardiocytes, and the counteractive effects of Ang 1-7 on Ang II-mediated MMPs and TIMPs expression need to be identified. In the present study, we examined the transcript expression of MMPs and TIMPs in human cardiac fibroblasts (HCF) and cardiac myocytes (HCM) after Ang II or Ang 1-7 stimulation, and analyzed the antagonistic effects of Ang 1-7 to Ang II. Our results suggest that Ang 1-7 and Ang II have opposite and antagonistic effects on regulation of MMPs and TIMPs transcription in primary cultures of human cardiocytes. These effects lead to increased MMPs/TIMPs ratios after Ang II stimulation and decreased MMPs/TIMPs ratios after Ang 1-7 stimulation, which effects partial may depend of the type of cardiac cells. These results might raise a potential role of Ang 1-7 to attenuate the heart damage in Ang II-induced ECM remodeling. Ang II and Ang 1-7, both are critical regulatory peptides in RAS. In RAS, Ang II is a substrate for angiotensin converting enzyme II (ACE2) to generate Ang 1-7. Ang II has been documented to play an important role in the progression of cardiac structural remodeling, and Ang 1-7 plays an antagonized effect of Ang II. Numerous studies implied that Ang II may modulate the expression of ACE2 in cardiovascular system. However, lots of reports showed controversial results. In the present study, HCF cells were used to test the regulatory effects of Ang II and Ang 1-7 on the ACE2 expression at transcriptional and translational level. The results show that Ang II could up-regulate ACE2 expression and this action may modulate through the activation of Ang II type I receptor (AT1R). Ang II-mediated ACE2 upregulation could be blocked by the antagonists of downstream targets of AT1R, NADPH oxidase and extracellular signal-regulated protein kinases-mitogen-activated protein kinase (ERK-MAPK) pathway cascades. Additionally, Ang 1-7 also could up-regulate ACE2 expression in the HCF cells and this upregulation could be inhibited by Mas receptor blocker. Our result shows that the ACE2 upregulation in HCF cells stimulated by Ang 1-7 might be independent to Ang II-AT1R pathway. In conclusion, we showed that Ang II-induced ACE2 may increase Ang 1-7 formation from Ang II and then the ACE2 expression is further enhanced by the Ang 1-7. According to the results, we proposed that the positive feedback-like loop on the ACE2 regulation of cardiac cells may help heart to maintain a steady state of RAS. Ang II dependent signaling pathways have been implicated in interstitial fibrosis during the development of AF. However, Ang II could be further degraded by ACE2. Hence, we examined expression of ACE2 in the fibrillating atria of pigs and its involvement in fibrotic pathogenesis during AF. In the histological examinations, extensive accumulation of extracellular matrix in the interstitial space of the atria, as evidenced by Masson's trichrome stain, were found in fibrillating atria. The relative amount of collagen type I in the atria with AF was significantly increased as compared with that in the sinus rhythm (SR). ACE2 gene and protein expression in the AF subjects were significantly decreased compared with those in the SR subjects, whereas expression of extracellular signal-regulated protein kinases 2 (ERK2) and activated ERK2 were significantly greater in the AF subjects. We proposed that decreasing ACE2 expression during AF may cause feedback regulation of the Ang II–dependent signaling pathway. In addition, our results suggest that atrial fibrosis in AF may be induced by antagonistic regulation between ACE and ACE2.