A83-01藉由調控Notch及Wnt訊息傳遞增加心臟前趨細胞促進心臟自我修復

Abstract

摘要 藉由增加內生性心臟前驅細胞的數量，而促進心臟自我再生可能是一個有潛力的方法去促使受傷後的心臟恢復其功能。我們先前研究已發現A83-01（為一個乙型轉化生長因子第一型受體抑制劑）能於離體擴增一群心臟前驅細胞。本研究目標是探討A83-01促進出生後小鼠心臟前驅細胞增殖的機制，並且在活體評估其對於受傷後心臟的作用。 實驗利用Nkx2.5 enhancer-eGFP 基因轉殖報告老鼠，藉由酵素分離心臟細胞，經由流式細胞分選儀分選出表達GFP之Nkx2.5+心臟前驅細胞進行培養。由溴化去氧尿苷嵌入DNA試驗，搭配流式細胞方法，檢測增生細胞的比例。梅生三色染色/膠質纖維染色則是用於定量出心肌與纖維化區域的面積。 藉由小鼠全基因cDNA 微陣列分析比較細胞分別處理DMSO及A83-01後其基因表現差異，發現A83-01處理會增加Birc5, FIGF和 DLK1的轉錄表現量，並且減少Fzd6 及WISP1的表現。利用siRNA降解沉默Birc5及FIGF均能顯著抑制A83-01之促進增生作用。然而只有Birc5可以直接增殖Nkx2.5+心臟前驅細胞。若同時給予Birc5及A83-01，並無產生協同作用。A83-01促進心臟前驅細胞增殖作用可被MEK/ERK 抑制劑 (PD0325901)抑制﹔但不受PI3K/Akt 抑制劑 (LY294002)影響。另外，在活體動物實驗中，於isoproterenol誘導心衰竭的小鼠，連續每日腹腔注射一劑A83-01 (10 mg/kg)三日後，藉由心臟超音波及梅生三色染色/膠質纖維染色發現A83-01可以保護心肌並改善小鼠的心臟功能。總結來說，本篇研究闡述A83-01能經由抑制smad2/3 訊息傳遞途徑，增加Birc5 (survivin)表達，來產生MEK/ERK依存性地促進心臟前驅細胞增殖作用。於心臟受傷後的活體小鼠，A83-01能藉由活化Birc5，增加心臟前驅細胞來補償損失的心肌細胞，並促進心肌細胞的存活，產生保護心臟功能之優點。

Abstract Approaches to enhance cardiac regeneration via repopulating resident cardiac progenitor cells (CPC) may be a potential strategy to restore cardiac dysfunction in post-injured heart. Our previous study found A83-01, a TGFβRI inhibitor, could expand a population of CPC in vitro. The goals of the present study were to investigate the underline mechanism of proliferative effect of A83-01, a TGFβRI inhibitor, on postnatal CPC and to assess the in vivo effect on post-injured hearts. We isolated Nkx2.5+ CPC from Nkx2.5 enhancer-eGFP transgenic reporter mice by enzymatic method and sorted by FACS-ariaII for culture. BrdU-pulse-labeling assay was performed to count the percentage of the proliferative cells by flow cytometry. Masson’s Trichrome staining (MTS) was used to quantify the area of myocardium and fibrosis. Using mouse genome wide cDNA microarray to analyze the alteration in the transcriptional profile between DMSO- and A83-01- treated groups, it was found A83-01 could up-regulate Birc5, FIGF and DLK1 as well as down-regulated Fzd6 and WIPS1. Using siRNAs to knockdown Bicr5 and FIGF could inhibit A83-01-mediated proliferative effect. However, only Birc5 could directly proliferate Nkx2.5+ CPC without producing synergistic effect as co-administrated with A83-01. Furthermore, the proliferative effect of A83-01 could be markedly inhibited by MEK/ERK inhibitor (PD0325901), but not by PI3K/Akt inhibitor (LY294002). Moreover, administration of A83-01 (10 mg/kg, i.p., QD, 3 days) in vivo in heart failure mice induced by isoproterenol could improve cardiac function monitored by echocardiography and preserved myocardium observed by MTS. In conclusion, the present study demonstrated that A83-01 could proliferate Nkx2.5+ CPC in a MEK/ERK-dependent manner mainly through blocking smad2/3 signaling to up-regulate Birc5 (survivin). The activation of Birc5 may mainly contribute to the cardiac benefit of A83-01 in post-injured heart via the expansion of endogenous CPC to compensate the lost myocytes as well as the enhancement of survival signaling in myocytes.