Is cytochrome P450 3A4 regulated by menstrual cycle hormones in control endometrium and endometriosis?

The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10 μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10 μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10 μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.     

Losartan inhibits endothelial-to-mesenchymal transformation in mitral valve endothelial cells by blocking transforming growth factor-β-induced phosphorylation of ERK

Adult cardiac valve endothelial cells (VEC) undergo endothelial to mesenchymal transformation (EndMT) in response to transforming growth factor-β (TGFβ). EndMT has been proposed as a mechanism to replenish interstitial cells that reside within the leaflets and further, as an adaptive response that increases the size of mitral valve leaflets after myocardial infarction. To better understand valvular EndMT, we investigated TGFβ-induced signaling in mitral VEC, and carotid artery endothelial cells (CAEC) as a control. Expression of EndMT target genes α-smooth muscle actin (α-SMA), Snai1, Slug, and MMP-2 were used to monitor EndMT. We show that TGFβ-induced EndMT increases phosphorylation of ERK (p-ERK), and this is blocked by Losartan, an FDA-approved antagonist of the angiotensin II type 1 receptor (AT1), that is known to indirectly inhibit phosphorylation of ERK (p-ERK). Blocking TGF-β-induced p-ERK directly with the MEK1/2 inhibitor RDEA119 was sufficient to prevent EndMT. In mitral VECs, TGFβ had only modest effects on phosphorylation of the canonical TGF-β signaling mediator mothers against decapentaplegic homolog 3 (SMAD3). These results indicate a predominance of the non-canonical p-ERK pathway in TGFβ-mediated EndMT in mitral VECs. AT1 and angiotensin II type 2 (AT2) were detected in mitral VEC, and high concentrations of angiotensin II (AngII) stimulated EndMT, which was blocked by Losartan. The ability of Losartan or MEK1/2 inhibitors to block EndMT suggests these drugs may be useful in manipulating EndMT to prevent excessive growth and fibrosis that occurs in the leaflets after myocardial infarction.     

Knockout RAGE alleviates cardiac fibrosis through repressing endothelial-to-mesenchymal transition (EndMT) mediated by autophagy

Endothelial-to-mesenchymal transition (EndMT) has been shown to contribute to cardiac fibrosis and heart failure (HF). Recent studies have demonstrated that EndMT is regulated by autophagy, and we previously showed suppression of excessive autophagy and alleviation of cardiac fibrosis in HF mice with inactivated receptor for advanced glycation end products (RAGE). Thus, we investigated whether reduced cardiac fibrosis due to RAGE knockout occurred by inhibiting EndMT mediated by excessive autophagy. We found a decrease in endothelial cells (CD31⁺/VE-Cadherin⁺) and an increase in cells co-expressing CD31 and α-smooth muscle actin (α-SMA, myofibroblast marker) at 8 weeks in heart tissue of mice subjected to transverse aortic constriction (TAC), which implied EndMT. Knockout RAGE decreased EndMT accompanied by decreased expression of autophagy-related proteins (LC3BII/I and Beclin 1), and alleviated cardiac fibrosis and improved cardiac function in TAC mice. Moreover, 3-methyladenine (3-MA) and chloroquine (CQ), inhibitors of autophagy, attenuated EndMT, and cardiac fibrosis in TAC mice. Importantly, EndMT induced by AGEs could be blocked by autophagy inhibitor in vivo and in vitro. These results suggested that AGEs/RAGE-autophagy-EndMT axis involved in the development of cardiac fibrosis and knockout RAGE ameliorated cardiac fibrosis through decreasing EndMT regulated by autophagy, which could be a promising therapeutic strategy for HF.Subject terms: Heart failure, Experimental models of disease     

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.     

The inhibitory effect of ginsan on TGF-β mediated fibrotic process

Transforming growth factor-beta (TGF-β) plays a central role in the development of fibrosis by stimulating extracellular matrix accumulation, and signals either directly or indirectly through types I, II, and III (TβRI, II, and III) TGF-β receptor complexes. Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. Here, we examine whether ginsan regulates the fibrogenic process by interfering with TGF-β signaling pathways. TGF-β treatment of murine or human normal lung fibroblasts enhanced the levels of several fibrotic markers, including smooth muscle alpha actin (α-SMA), collagen-1, and fibronectin. Interestingly, ginsan treatment either before or after TGF-β administration led to significant reductions in all of α-SMA, collagen-1, and fibronectin expression levels. Ginsan not only inhibited phosphorylation of Smad2 and Smad3, but also attenuated pERK and pAKT signaling induced by TGF-β. Moreover, ginsan restored TβRIII protein expression, which was significantly downregulated by TGF-β, but reduced TβRI and TβRII protein levels. In a murine model of bleomycin (BLM)-induced pulmonary fibrosis, ginsan significantly suppressed accumulation of collagen, α-SMA, and TGF-β. These data collectively suggest that ginsan acts as an effective anti-fibrotic agent in the treatment of pulmonary fibrosis by blocking multiple TGF-β signaling pathways.     

A novel mechanism of Smads/miR-675/TGFβR1 axis modulating the proliferation and remodeling of mouse cardiac fibroblasts

Cardiac fibroblasts (CFs) can over-proliferate during the progression of cardiac fibrosis, accompanied by a net accumulation of extracellular matrix proteins. Based on the profibrotic actions of transforming growth factor beta 1 (TGFβ1), investigating the mechanisms of TGFβ1 function in CFs may provide new directions to treatment for cardiac fibrosis. microRNAs (miRNAs) could control CFs proliferation or remodeling via binding to 3′-untranslated region of messenger RNA (mRNA) to negatively regulate gene expression. In the present study, we downloaded several microarray analyses results from GEO attempting to identify miRNAs and possible downstream targets that may be involved in TGF-β1 function in CFs and to detect the cellular and molecular functions of the identified miRNA–mRNA axis. Here, we identified miR-675 as a downregulated miRNA by TGFβ1 by bioinformatics analyses and experimental verification. Upon TGFβ1 stimulation, the protein levels of Α-SMAΑ-SMA, collagen I, and POSTN, and the secreted collagen in the cell culture supernatant significantly increased, whereas the miR-675 expression decreased. Smads mediate TGFβ1-induced suppression on miR-675 via binding miR-675 promoter region. miR-675 overexpression could inhibit, whereas miR-675 inhibition could enhance TGFβ1-induced mouse CFs (MCF) remodeling and proliferation. TGFβ receptor 1 (TGFβR1), a critical receptor in TGFβ1/Smad signaling, is a direct downstream target of miR-675. TGFβR1 overexpression significantly reverses the effect of miR-675 overexpression on MCF remodeling and proliferation. In summary, miR-675 targets TGFβR1 to attenuate TGFβ1-induced MCF remodeling and proliferation. We demonstrate a novel mechanism of the Smads/miR-675/TGFβR1 axis modulating TGFβ1-induced MCF remodeling and proliferation.     

TGF-β-Operated Growth Inhibition and Translineage Commitment into Smooth Muscle Cells of Periodontal Ligament-Derived Endothelial Progenitor Cells through Smad- and p38 MAPK-Dependent Signals

The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-β (TGF-β) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-β signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-β1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-β1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-β-induced Smad-dependent signaling, suppressed the TGF-β1-induced growth inhibition and SMC markers expression, but did not the TGF-β1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-β1-induced downregulation of EC marker expression. In addition, the TGF-β1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-β1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-β1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.     

Dapagliflozin alleviates cardiac fibrosis through suppressing EndMT and fibroblast activation via AMPKα/TGF-β/Smad signalling in type 2 diabetic rats

Diabetic cardiomyopathy (DCM) is one of the leading causes of heart failure in patients with diabetes mellitus, with limited effective treatments. The cardioprotective effects of sodium-glucose cotransporter 2(SGLT2) inhibitors have been supported by amounts of clinical trials, which largely fills the gap. However, the underlying mechanism still needs to be further explored, especially in terms of its protection against cardiac fibrosis, a crucial pathophysiological process during the development of DCM. Besides, endothelial-to-mesenchymal transition (EndMT) has been reported to play a pivotal role in fibroblast multiplication and cardiac fibrosis. This study aimed to evaluate the effect of SGLT2 inhibitor dapagliflozin (DAPA) on DCM especially for cardiac fibrosis and explore the underlying mechanism. In vivo, the model of type 2 diabetic rats was built with high-fat feeding and streptozotocin injection. Untreated diabetic rats showed cardiac dysfunction, increased myocardial fibrosis and EndMT, which was attenuated after treatment with DAPA and metformin. In vitro, HUVECs and primary cardiac fibroblasts were treated with DAPA and exposed to high glucose (HG). HG-induced EndMT in HUVECs and collagen secretion of fibroblasts were markedly inhibited by DAPA. Up-regulation of TGF-β/Smad signalling and activity inhibition of AMPKα were also reversed by DAPA treatment. Then, AMPKα siRNA and compound C abrogated the anti-EndMT effects of DAPA in HUVECs. From above all, our study implied that DAPA can protect against DCM and myocardial fibrosis through suppressing fibroblast activation and EndMT via AMPKα-mediated inhibition of TGF-β/Smad signalling.     

EndMT: A promising and controversial field

The endothelial to mesenchymal transition (EndMT) is the process by which endothelial cells lose a portion of their cellular features and obtain certain characteristics of mesenchymal cells, including loss of tight junctions, increased motility, and increased secretion of extracellular matrix proteins. EndMT is involved in cardiac development and a variety of diseases processes, such as vascular or tissue fibrosis and tumor. However, its role in specific diseases remains under debate. This review summarizes EndMT-related diseases, existing controversies, different types of EndMT, and molecules and signaling pathways associated with the process.     

Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl₄-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.     

Response gene to complement 32 is essential for fibroblast activation in renal fibrosis

Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-β (TGF-β). TGF-β is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-β-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.