MFGE8 is down-regulated in cardiac fibrosis and attenuates endothelial-mesenchymal transition through Smad2/3-Snail signalling pathway.

Endothelial-mesenchymal transition (EndMT) is a major source of transformed cardiac fibroblasts, which is reported to play a key role in cardiac fibrosis (CF), a pathogenesis of cardiovascular diseases such as heart failure, myocardial infarction and atrial fibrillation. Nonetheless, the specific mechanism underlying the progression of EndMT to CF is still largely unknown. In this study, we aimed to investigate the role of milk fat globule-EGF factor 8 (MFGE8), a kind of soluble glycoprotein, in TGF-β1-induced EndMT. In animal experiments, the expression of MFGE8 was found down-regulated in the left ventricle and aorta of rats after transverse aortic constriction (TAC) compared with the sham group, especially in endothelial cells (ECs). In in vitro cultured ECs, silencing MFGE8 with small interfering RNA (siRNA) was found to promote the process of TGF-β1-induced EndMT, whereas administration of recombinant human MFGE8 (rh-MFGE8) attenuated the process. Moreover, activated Smad2/3 signalling pathway after TGF-β1 treatment and EndMT-related transcription factors, such as Snail, Twist and Slug, was potentiated by MFGE8 knock-down but inhibited by rh-MFGE8. In conclusion, our experiments indicate that MFGE8 might play a protective role in TGF-β1-induced EndMT and might be a potential therapeutic target for cardiac fibrosis.

Wang B ，Ge Z ，Wu Y ，Zha Y ，Zhang X ，Yan Y ，Xie Y ... -  《-》

Inhibition of BRD4 attenuates transverse aortic constriction- and TGF-β-induced endothelial-mesenchymal transition and cardiac fibrosis.

Cardiac fibrosis (CF), a process characterized by potentiated proliferation of cardiac fibroblasts and excessive secretion and deposition of extracellular matrix (ECM) from the cells, contributes strongly to the pathogenesis of a series of cardiovascular (CV) diseases, including AMI, heart failure and atrial fibrillation. Endothelial-mesenchymal transition (EndMT), one of the sources of transformed cardiac fibroblasts, has been reported as a key factor involved in CF. However, the molecular basis of EndMT has not been thoroughly elucidated to date. At the posttranscriptional level, of the three epigenetic regulators, writer and eraser are reported to be involved in EndMT, but the role of reader in the process is still unknown. In this study, we aimed to explore the role of Bromodomain-containing protein 4 (BRD4), an acetyl-lysine reader protein, in EndMT-induced CF and related mechanisms. We found that BRD4 was upregulated in endothelial cells (ECs) in the pressure-overload mouse heart and that its functional inhibitor JQ1 potently attenuated the TAC-induced CF and preserved cardiac function. In umbilical vein endothelial cells (HUVECs) and mouse aortic endothelial cells (MAECs), bothJQ1 and shRNA-mediated silencing of BRD4 blocked TGF-β-induced EC migration, EndMT and ECM synthesis and preserved the EC sprouting behavior, possibly through the downregulation of a group of transcription factors specific for EndMT (Snail, Twist and Slug), the Smads pathway and TGF-β receptor I. In the absence of TGF-β stimulation, ectopic expression of BRD4 alone could facilitate EndMT, accelerate migration and increase the synthesis of ECM. In vivo, JQ1 also attenuated TAC-induced EndMT and CF, which was consistent with JQ1's intracellular mechanisms of action. Our results showed that BRD4 plays a critical role in EndMT-induced CF and that targeting BRD4 might be a novel therapeutic option for CF.

Song S ，Liu L ，Yu Y ，Zhang R ，Li Y ，Cao W ，Xiao Y ，Fang G ，Li Z ，Wang X ，Wang Q ，Zhao X ，Chen L ，Wang Y ，Wang Q ... -  《-》

MFGE8 attenuates Ang-II-induced atrial fibrosis and vulnerability to atrial fibrillation through inhibition of TGF-β1/Smad2/3 pathway.

Atrial fibrillation (AF) is characterized by potentiated growth of atrial fibroblasts and excessive deposition of the extracellular matrix. Atrial fibrosis has emerged as a hallmark of atrial structural remodeling linked to AF. Nonetheless, the specific mechanism underlying the progression of atrial fibrosis to AF is still largely unknown. MFGE8 (milk fat globule-EGF factor 8) is a soluble glycoprotein associated with many human diseases. Recently, a number of studies revealed that MFGE8 plays a crucial role in heart disease. Yet, MFGE8 regulation and function in the process of atrial fibrosis and vulnerability to AF remain unexplored. In this study, we found that the expression of MFGE8 was downregulated in the atriums of patients with AF compared with individuals without AF. In addition, the expression of MFGE8 was lower in atriums of angiotensin II (Ang-II)-stimulated rats as compared with the sham group. In vitro, silencing of MFGE8 by small interfering RNA significantly increased Ang-II-induced atrial fibrosis, whereas administration of recombinant human MFGE8 (rhMFGE8) attenuated the atrial fibrosis. Moreover, we found that the activated TGF-β1/Smad2/3 pathway after Ang-II treatment was significantly potentiated by the MFGE8 knockdown but inhibited by rhMFGE8 in vitro. Inhibition of integrin β3 which is the receptor for MFGE8, suppressed the TGF-β1/Smad2/3 activating effects of the MFGE8 knockdown in Ang-II-treated rat atrial fibroblasts. Finally, we administered rhMFGE8 to rats; it attenuated atrial fibrosis and remodeling and further reduced AF vulnerability induced by Ang-II, indicating that MFGE8 might have the potential both as a novel biomarker and as a therapeutic target in atrial fibrosis and AF.

Ge Z ，Chen Y ，Wang B ，Zhang X ，Yan Y ，Zhou L ，Zhang Y ，Xie Y ... -  《-》

Foxm1 is a critical driver of TGF-β-induced EndMT in endothelial cells through Smad2/3 and binds to the Snail promoter.

Endothelial-to-mesenchymal transition (EndMT) was first reported in heart development. Recent studies have shown that EndMT also occurs in the progression of cardiac fibrosis. Herein, we demonstrated a critical role of the Forkhead Box M1 (Foxm1) transcription factor in transforming growth factor beta (TGF-β)-induced EndMT in endothelial cells (ECs) and a possible underlying molecular mechanism. Foxm1 was induced in ECs following TGF-β stimulation. Using both pharmacological and molecular approaches to inhibit Foxm1 function can attenuate the TGF-β-induced EndMT and cell migration. In contrast, lentivirus-mediated overexpression of Foxm1 allowed EndMT to proceed despite the absence of TGF-β in ECs. Moreover, we found that the activation of the Smad2/3 signaling pathway and EndMT-related transcription factors played important roles in the pathogenesis of Foxm1-mediated EndMT. Further analysis revealed that Foxm1 bound to and increased the promoter activity of the Snail gene encoding a critical transcriptional regulator of EndMT. In conclusion, our results identify FOXM1 as a driver of TGF-β-induced EndMT and underscore the therapeutic potential of targeting FOXM1 for cardiac fibrosis.

Song S ，Zhang R ，Cao W ，Fang G ，Yu Y ，Wan Y ，Wang C ，Li Y ，Wang Q ... -  《-》

TGF-β1-induced SMAD2/3/4 activation promotes RELM-β transcription to modulate the endothelium-mesenchymal transition in human endothelial cells.

Endothelial-to-mesenchymal transition (EndMT), which is characterized by increased proliferation, migration and invasion of endothelial cells, increased expression of mesenchymal markers and reduced expression of endothelial markers, has been reported to be closely related to the pathogenesis of several diseases, including pulmonary fibrosis. Resistin-like molecule-β (RELM-β), also known as "found in inflammatory zone 2″ (FIIZ2), plays an essential role in airway remodeling and pulmonary fibrosis; however, its role and mechanism in EndMT remain unclear. Herein, we used TGF-β1-induced EndMT cell model in human umbilical vein endothelial cells (HUVECs) and human primary pulmonary artery endothelial cells (HPAECs) to investigate the function and mechanism of RELM-β in TGF-β1-induced EndMT in endothelial cell lines. We found that TGF-β1 stimulation significantly upregulated RELM-β expression; RELM-β knockdown could attenuate TGF-β1-induced cell proliferation and migration of endothelial cell lines and changes in protein levels of EndMT markers. SB432542, an inhibitor of SMADs, could partially reverse TGF-β1-induced RELM-β expression, endothelial cell migration and changes in EndMT marker protein levels. SMADs complex exerted its effects through SMAD2/3/4 complex mediating RELM-β transcription. In conclusion, TGF-β1 induces RELM-β transcription to promote EndMT in HUVECs and HPAECs through activation of SMAD2/3/4; blocking SMADs-mediated RELM-β transcription might ameliorate TGF-β1-induced EndMT in endothelial cells.

Jiang Y ，Zhou X ，Hu R ，Dai A ... -  《-》