Astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice

Numerous cytokines participate in the occurrence and development of inflammation and renal interstitial fibrosis. Previous studies confirmed that TGF-β1 overexpressed in diabetic nephropathy. As a downstream signal protein of TGF-β1 family, SMAD has an important role in the process of α-SMA mediated renal interstitial fibrosis. This study aimed to study astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissue of diabetic KKAy mice, to reveal its potential impact on renal interstitial fibrosis. 20 type II diabetic KKAy mice were randomly equally divided into model group and astragaloside group, while 10 male C57BL/6J mice were selected as the control. Astragaloside at 40 mg/(kg•d) was given when the KKAy mice fed with high-fat diet to 14 weeks old. The mice were killed at 24 weeks old and the kidney tissue samples were collected. Pathology morphological changes were observed. TGF-β1, SMAD2/3, and α-SMA expression levels were determined by immunohistochemistry. Compared with control, mice kidney in model group appeared obvious fibrosis and up-regulated blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression (P < 0.05). Mice in astragaloside group exhibited alleviated renal interstitial fibrosis compared with the model. Its blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression levels were significantly lower than the model group (P < 0.05). Astragaloside can delay the renal fibrosis process in diabetic mice by influencing the TGF-β/SMADS signaling pathway and down-regulating TGF-β1, SMAD2/3, and α-SMA expression.     

miR-144 regulates transforming growth factor-β1 iduced hepatic stellate cell activation in human fibrotic liver

Objectives: Activation of hepatic stellate cells (HSCs) into collagen producing myofibroblasts is critical for pathogenesis of liver fibrosis. Transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators for HSC transdifferentiation. Recent studies have shown effect of microRNAs (miRNAs) on regulating TGF-β1-induced HSC activation during liver fibrosis. Here, we aimed to explore the roles of miR-144 and miR-200c in human liver fibrosis. Methods: Expression of TGF-β1 was detected in 42 fibrotic and 18 normal human liver tissues by quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry, and its correlation with α-smooth muscle actin (α-SMA) was calculated. miR-144 and miR-200c expression level in fibrotic liver tissues were also detected by qRT-PCR. The correlation of TGF-β1 expression with miR-200c and miR-144 in the fibrotic liver was analyzed. Results: The results showed that TGF-β1 expression was much higher in fibrotic liver than that in normal liver tissues (P<0.05). TGF-β1 protein high expressing liver fibrosis showed α-SMA positive cells in the liver parenchyma indicating activated HSCs. Expression of TGF-β1 in fibrotic liver was significantly correlated with α-SMA expression (R=0.633, P<0.001). Furthermore, miR-144 was less expressed in liver fibrosis (P<0.05) and was significantly correlated with expression of TGF-β1 in fibrotic liver tissues (R=-0.442, P<0.01). However, miR-200c did not show significant difference between normal and fibrotic liver (P=0.48) and correlation with TGF-β1 expression (R=0.106, P=0.51). Conclusion: All the results indicate that miR-144 can be a novel regulator of TGF-β1-induced HSC activation during liver fibrosis.     

Protective effects of calcitriol on diabetic nephropathy are mediated by down regulation of TGF-β1 and CIP4 in diabetic nephropathy rat

Objective: To explore the protective effects of calcitriol on diabetic nephropathy by modulating the expressions of transforming growth factor-beta 1 (TGF-β1) and Cdc42 interacting protein-4 (CIP4). Methods: Streptozotocin-induced diabetic nephropathy rats (n=36) were randomly divided into control group (control-H, control-M, control-L) and calcitriol group (calcitriol-H, calcitriol-M, calcitriol-L). The expression of TGF-β1 gradually decreased in control-H, control-M and control-L subgroups by injection of different virus vectors. Peanut oil and calcitriol were given to control and calcitriol group, respectively. The expressions of TGF-β1 and CIP4 in kidney, the pathology, and the renal function and lipid profiles were compared between control and calcitriol treatment groups. Results: In the control group, the higher level of TGF-β1 was associated with more severe glomerular pathology (P<0.05). There is a positive correlation between the expression of CIP4 and TGF-β1. Control-H subgroup had significant more severe kidney disease, higher levels of cholesterol, triglyceride, blood glucose, blood urea nitrogen (BUN) and creatinine (Cr) than control-M and control-L subgroups. After calcitriol treatment, the expression of TGF-β1 and CIP4 were significantly decreased compared to the corresponding control subgroups (all P<0.05). Renal fibrosis and pathological changes were markedly improved. The levels of cholesterol, triglyceride, blood glucose, BUN and Cr were significantly reduced (P<0.05). Conclusion: Calcitriol may protect diabetic nephropathy from fibrosis via inhibition of TGF-β1 and CIP4.     

Development of a KLD-12 polypeptide/TGF-β1-tissue scaffold promoting the differentiation of mesenchymal stem cell into nucleus pulposus-like cells for treatment of intervertebral disc degeneration

Objective: To develop tissue engineering scaffolds consisting of self-assembling KLD-12 polypeptide/TGF-β1 nanofiber gel, for the induction of mesenchymal stem cell (MSCs) differentiation into nucleus pulposus (NP)-like cells. Methods: The release of TGF-β1 from KLD-12 polypeptide gels containing varying TGF-β1 concentrations was detected by ELISA. MSCs were isolated with a density gradient method and their differentiation into NP-like cells was analyzed in KLD-12 polypeptide/TGF-β1- or KLD-12 polypeptide control nanofiber-gel 3D-cultures. The Alcianblue method, Real-time quantitative PCR (RT-qPCR), and immunocytochemistry were used to measure the expression of extracellular matrix (ECM) molecules, such as aggrecan, glycosaminoglycans (GAGs), and type II collagen. Results: ELISA results documented favorable time-dependent release characteristics of TGF-β1 in the KLD-12 polypeptide/TGF-β1 gel scaffolds. The results of CCK-8 cell proliferation assay showed the TGF-β1 containing scaffolds induced higher growth rate in MSCs compared to the control group. Calcein-AM/PI fluorescent staining showed: the cells in the gel grew well, maintaining the circular shape of cells, and the spindle and fusiform shape of cells on the gel edges. The cell viability displayed a survival rate of 89.14% ± 2.468 for the TGF-β1 group with no significant difference between the two groups at 14 d of culture. The production of ECM was monitored showing higher expression of GAGs in the TGF-β1 group (P < 0.01) with highest amounts at 10 d and 14 d compared to 4 d and 7 d (P < 0.05). Real-time PCR results revealed that the expression levels of collagen II and aggrecan mRNA were higher in the TGF-β1 group (P < 0.05). Finally, immunocytochemical staining of collagen II confirmed the higher expression levels. Conclusion: A scaffold containing a KLD-12 polypeptide/TGF-β1-nanofiber gel and MSCs differentiated into NP-like cells is able to produce ECM and has the potential to serve as a three-dimensional (3-D) support scaffold for the filling of early postoperative residual cavities and the treatment of intervertebral disc degeneration.     

Relationship of TLR2, TLR4 and tissue remodeling in chronic rhinosinusitis

In order to explore the role of innate immunity in the remodeling of CRS (chronic rhinosinusitis), we investigated the correlation between TLR2, TLR4 and remodeling involved cytokines and histopathological features. Immunohistochemical staining was applied to detect the expression of TLR2, TLR4 and TGF-β1. Masson staining was used for observing the collagen deposition. The other histopathologic features of remodeling were observed by hemotoxylin and eosin (HE) staining. Nasal epithelial cell culture was used to elucidate the effect of TLR2, TLR4 agonists and inhibitors on the expression of TGF-β1 and MMP-9. The association study showed that the significantly higher expression of TLR2, TLR4, TGF-β1 and collagen appeared in CRSsNP (chronic rhinosinusitis without nasal polyps) patients compared with CRSwNP (chronic rhinosinusitis with nasal polyps) patients. In CRSsNP, patients with a severe epithelial damage (score 3) had a significantly higher expression of TLR2 than patients with mild epithelial damage (score ≤ 2) (P < 0.05). Moreover the expression of TLR2 correlated negatively with squamous hyperplasia in CRSsNP, and positively with gland hyperplasia in CRSwNP. The expression of TLR2 and TLR4 was closely related to neutrophil infiltration in CRSsNP (P < 0.01). TGF-β1 was downregulated by TLR2 agonist in CRSwNP and upregulated by TLR4 agonist in CRSsNP (P < 0.05). MMP-9 was upregulated by TLR4 agonist in CRSwNP (P < 0.05). TLR2 and TLR4 had close relationship with TGF-β1 and the histologic features of remodeling, especially collagen deposition and neutrophil infiltration in CRSsNP. The innate immunity could influence the histologic characteristics and involved cytokines through TLR2 and TLR4 in the remodeling of CRS.     

Serum amyloid A, but not C-reactive protein, stimulates vascular proteoglycan synthesis in a pro-atherogenic manner

Inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) are predictive of cardiac disease and are proposed to play causal roles in the development of atherosclerosis, in which the retention of lipoproteins by vascular wall proteoglycans is critical. The purpose of this study was to determine whether SAA and/or CRP alters vascular proteoglycan synthesis and lipoprotein retention in a pro-atherogenic manner. Vascular smooth muscle cells were stimulated with either SAA or CRP (1 to 100 mg/L) and proteoglycans were then isolated and characterized. SAA, but not CRP, increased proteoglycan sulfate incorporation by 50 to 100% in a dose-dependent manner (P < 0.0001), increased glycosaminoglycan chain length, and increased low-density lipoprotein (LDL) binding affinity (K_d, 29 μg/ml LDL versus 90 μg/ml LDL for SAA versus control proteoglycans; P < 0.005). Furthermore, SAA up-regulated biglycan via the induction of endogenous transforming growth factor (TGF)-β. To determine whether SAA stimulated proteoglycan synthesis in vivo, ApoE^−/− mice were injected with an adenovirus expressing human SAA-1, a null virus, or saline. Mice that received adenovirus expressing SAA had increased TGF-β concentrations in plasma and increased aortic biglycan content compared with mice that received either null virus or saline. Thus, SAA alters vascular proteoglycans in a pro-atherogenic manner via the stimulation of TGF-β and may play a causal role in the development of atherosclerosis.     

Knockdown of elF3a inhibits collagen synthesis in renal fibroblasts via Inhibition of transforming growth factor-β1/Smad signaling pathway

Renal fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM). The eukaryotic translation initiation factor (eIF) 3a is the largest subunit of the eIF3 complex and has been involved in pulmonary fibrosis. However, the role of eIF3a in rental fibrosis is still unclear. Therefore, in this study, we investigated the role of eIF3a in rental fibrosis and explored the underlying mechanism. Our study found that eIF3a was up-regulated in renal fibrotic tissues and transforming growth factor (TGF)-β1-treated HK-2 cells. In addition, knockdown of eIF3a significantly inhibited TGF-β1-induced expression levels of α-smooth muscle actin (α-SMA) and collagen I. Furthermore, knockdown of eIF3a attenuated TGF-β1-induced Smad3 activation in HK-2 cells. Taken together, these results suggest that knockdown of eIF3a inhibits collagen synthesis in renal fibroblasts via inhibition of TGF-β1/Smad signaling pathway, and eIF3a may be a potential molecular target for the treatment of renal fibrosis.     

Aldosterone and TGF-β1 synergistically increase PAI-1 expression in hepatic stellate cells of rats

Objective: Aldosterone is related to the fibrosis of several organs, but the specific mechanism underlying the aldosterone induced hepatic fibrosis is still unclear. Methods: Separation, culture and identification of primary hepatic stellate cells (HSCs): The fluids and digestives used in the present study were able to completely remove blood cells, digest hepatocytes and matrix, and effectively separate HSCs. The in situ perfusion was performed at 2 steps: in situ perfusion with pre-perfusion fluid and ex vivo perfusion with enzyme-containing perfusion fluid. Influence of Ald on PAI-1 and Smad expressions in HSCs: cells were divided into control group, Ald group (10^-6 M), spironolactone (SPI) group and Ald+SPI group, and the mRNA and protein expressions of PAI-1 and Smad were detected. Ald induced type I collagen expression in HSCs: Immunohistochemistry was performed to detect type I collagen expression in the supernatant of control group, Ald group (10^-6 M), TGF-β₁ group, and Ald+TGF-β₁ group. Influence of Ald and TGF-β₁ on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10^-6 M), TGF-β₁ group, and Ald+TGF-β₁ group, and the mRNA and protein expressions of PAI-1 were determined by RT-PCR and Western blot assay, respectively. Synergistic effect of Ald and TGF-β₁ on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10^-6), TGF-β₁ group, Ald (10^-6 M)+TGF-β₁ group, Ald (10^-7 M)+TGF-β₁ group and Ald (10^-8 M)+TGF-β₁ group, and the mRNA and protein expressions of PAI-1 were detected by RT-PCR and Western blot assay, respectively. Results: The survival rate, purity, markers and activation of HSCs were determined after separation. Influence of Ald on PAI-1 expression in HSCs: PAI-1 expression increased in HSCs of Ald group, SPI group and Ald+API group, and the PAI-1 expression in Ald group and Ald+SPI group was significantly higher than in control group (P<0.01). Influence of Ald on Smad expression in HSCs: Smad expression in Ald group, TGF-β₁ group and ALD+TGF-β₁ group was markedly higher than in control group (P<0.05). Smad expression in ALD+TGF-β₁ group increased significantly when compared with Ald group (P<0.01). Ald induced type I collagen expression in HSCs: type I collagen expression in Ald group, TGF-β₁ group and ALd+TGF-β₁ group was dramatically higher than in control group (P<0.05), and it in ALd+TGF-β₁ group was also significantly different from that in Ald group and TGF-β₁ group (P<0.01). Synergistic effects of Ald and TGF-β₁ on PAI expression in HSCs: PAI-1 expression in treated cells was markedly higher than in control group (P<0.01). PAI-1 expression in 10^-6 M Ald+5 ng/ml TGF-β₁ group increased dramatically as compared to Ald group and TGF-β₁ group (P<0.01), but the increased PAI-1 expression reduced after SPI treatment. Ald at different concentrations exerts synergistic effect with TGF-β₁ to increase PAI-1 expression in HSCs: PAI-1 expression in HSCs after different treatments increased markedly as compared to control group (P<0.01). Significant difference in PAI-1 expression was observed in 10^-6 M Ald+50 pg/ml TGF-β₁ group and 10^-6 M Ald group (P<0.01), PAI-1 expression in 10^-7 M Ald+50 pg/ml TGF-β₁ group was significantly higher than in 50 pg/ml TGF-β₁ group (P<0.01), but the PAI-1 expression in 10^-7 M Ald+50 pg/ml TGF-β₁ group was similar to that in 10^-6 M Ald group (P>0.05). Conclusion: Aldosterone is able to activate HSCs and increase PAI-1 expression during hepatic fibrosis, which may be inhibited by spironolactone. Aldosterone and TGF-β₁ may synergistically act on HSCs to increase PAI-1 expression as compared to treatment with aldosterone or TGF-β₁ alone. Aldosterone or TGF-β₁ alone may slightly increase PAI-1 expression in HSCs, which can be inhibited by spironolactone.     

Evaluation of TGF-β1, CCL5/RANTES and sFas/Apo-1 urine concentration in children with ureteropelvic junction obstruction

Introduction

The aim of this study was to evaluate changes in expression of soluble biomarkers tumor factor growth-β1 (TGF-β1), CCL5/RANTES, and sFas/Apo-1 in the urine of patients undergoing ureteropyeloplasty for ureteropelvic junction (UPJ) obstruction. These factors are connected with different processes ongoing in the obstructive uropathy. If their urine concentrations correlate with AP diameter of the renal pelvis and differential function of the affected kidney, they can be helpful in making a decision on corrective surgery.

Material and methods

Creatinine, TGF-β1, CCL5/RANTES, and sFas/Apo-1 levels were measured in the urine from the bladder and renal pelvis of 45 patients undergoing ureteropyeloplasty and from bladders of 25 patients undergoing inguinal herniorrhaphy.

Results

Levels of examined biomarkers were higher in the renal pelvis and bladder of children with UPJ obstruction as compared to controls: TGF-β1 in older children and adolescents (p < 0.05), CCL5/RANTES in the youngest and older children (p < 0.05), and sFas/Apo-1 in all patients (p < 0.05). Twelve months after surgery their levels in the bladder decreased: TGF-β1 in younger and older children (p < 0.05), CCL5/RANTES in the youngest patients and adolescents (p < 0.05), and sFas/Apo-1 in the youngest and older children (p < 0.05). A significant decrease in the AP diameter of the renal pelvis post-operatively (32.09 mm vs. 18.72 mm) (p < 0.01) and significant improvement in renal function (36.94% vs. 42.76%) (p < 0.05) were observed in the examined group.

Conclusions

Mean TGF-β1, CCL5/RANTES, and sFas/Apo-1 urine levels are significantly increased in patients with UPJ and decreased 1 year after ureteropyeloplasty. Bladder concentrations of examined factors may be clinically useful markers of obstruction.     

Activation of AMP-Activated Protein Kinase Prevents TGF-β1–Induced Epithelial-Mesenchymal Transition and Myofibroblast Activation

Transforming growth factor (TGF)-β contributes to tubulointerstitial fibrosis. We investigated the mechanism by which TGF-β exerts its profibrotic effects and specifically the role of AMP-activated protein kinase (AMPK) in kidney tubular epithelial cells and interstitial fibroblasts. In proximal tubular epithelial cells, TGF-β1 treatment causes a decrease in AMPK phosphorylation and activation together with increased fibronectin and α-smooth muscle actin expression and decreased in E-cadherin. TGF-β1 causes similar changes in interstitial fibroblasts. Activation of AMPK with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside, metformin, or overexpression of constitutively active AMPK markedly attenuated TGF-β1 functions. Conversely, inhibition of AMPK with adenine 9-β-d-arabinofuranoside or siRNA-mediated knockdown of AMPK (official name PRKAA1) mimicked the effect of TGF-β1 and enhanced basal and TGF-β1–induced phenotypic changes. Importantly, we found that tuberin contributed to the protective effects of AMPK and that TGF-β1 promoted cell injury by blocking AMPK-mediated tuberin phosphorylation and activation. In the kidney cortex of TGF-β transgenic mice, the significant decrease in AMPK phosphorylation and tuberin phosphorylation on its AMPK-dependent activating site was associated with an increase in mesenchymal markers and a decrease in E-cadherin. Collectively, the data indicate that TGF-β exerts its profibrotic action in vitro and in vivo via inactivation of AMPK. AMPK and tuberin activation prevent tubulointerstitial injury induced by TGF-β. Activators of AMPK provide potential therapeutic strategy to prevent kidney fibrosis and progressive kidney disease.Tubulointerstitial fibrosis is a prominent pathologic feature of progressive renal disease that culminates in loss of renal function. Inflammatory and metabolic insults result in kidney fibrosis in which transforming growth factor (TGF)-β plays a prominent role. Tubular epithelial cells and interstitial fibroblast contribute to this process by secreting and remodeling the extracellular matrix. In progressive fibrotic renal disease, TGF-β causes proximal tubular epithelial cells (PTECs) to acquire mesenchymal cell characteristics sometimes referred to as epithelial-mesenchymal transition (EMT).¹ In the presence of TGF-β, interstitial fibroblasts are also activated, differentiate to myofibroblasts, and contribute to the accumulation of extracellular matrix proteins. Accumulation of matrix proteins progressively destroys the normal kidney tissue architecture and disrupts blood flow and nephron function.² The increase in TGF-β1 levels is causally linked to the activation of profibrotic signaling pathways initiated by angiotensin, glucose, and oxidative stress.³ There is substantial evidence supporting a role for AMP-activated protein kinase (AMPK) in multiple diseases, including diabetes mellitus,^4,5 metabolic syndrome,^6–9 and cancer.^10,11 However, the role of AMPK in renal disease is underexplored. AMPK is a phylogenetically conserved serine/threonine kinase that regulates diverse cellular functions.¹² AMPK is heterotrimeric complex comprising a catalytic α (α1, α2) subunit and two regulatory subunits β (β1, β2) and γ (γ1, γ2, γ3). The activity and subunit composition of AMPK are expressed in a cell- and tissue-specific manner, with the α₁ and α₂ subunits expressed in the kidney, including tubular epithelial cells and glomerular cells.^13,14 AMPK activation leads to its phosphorylation at Thr172 in the catalytic domain of the α-subunit. AMPK can also be activated independent of changes in the AMP/ATP ratio.^15–17 On activation, AMPK turns on ATP-generating catabolic pathways and turns off ATP-consuming anabolic pathways.We investigated the role of AMPK in mediating the effect of TGF-β1 in human and murine PTECs, rat kidney interstitial fibroblast cells, and TGF-β1 transgenic mouse model. We provide strong evidence that TGF-β induces EMT phenotype through inactivation of AMPK and that activation of AMPK prevents the effects of TGF-β.     

Transforming Growth Factor-β–Independent Role of Connective Tissue Growth Factor in the Development of Liver Fibrosis

We previously identified transforming growth factor (TGF)-β signaling as a fibronectin-independent mechanism of type I collagen fibrillogenesis following adult liver injury. To address the contribution of TGF-β signaling during the development of liver fibrosis, we generated adult mice lacking TGF-β type II receptor (TGF-βIIR) from the liver. TGF-βIIR knockout livers indeed showed a dominant effect in reducing fibrosis, but fibrosis still remained approximately 45% compared with control and fibronectin knockout livers. Unexpectedly, this was accompanied by significant up-regulation of connective tissue growth factor mRNA levels. Organized type I collagen networks in TGF-βIIR knockout livers colocalized well with fibronectin. We provide evidence that elimination of TGF-βIIR is not sufficient to completely prevent liver fibrosis. Our results indicate a TGF-β–independent mechanism of type I collagen production and suggest connective tissue growth factor as its potent mediator. We advocate combined elimination of TGF-β signaling and connective tissue growth factor as a potential therapeutic target by which to attenuate liver fibrosis.Liver fibrosis is defined as an abnormal response to persistent liver injury, characterized by the excessive accumulation of collagenous extracellular matrices (ECMs).^{1, 2} Liver fibrosis affects tens of millions of people worldwide and is of great clinical significance because normal liver architecture is disrupted and liver function is ultimately impaired. Because there is no effective treatment of liver fibrosis, many patients develop progressive liver cirrhosis, eventually requiring a liver transplant.^{3, 4}There is a long-standing concept that cells in culture cannot form a collagen fibril network without the ECM glycoprotein fibronectin.⁵ We recently established an adult mouse model lacking liver fibronectin and demonstrated that fibronectin-null livers, in fact, formed collagen fibril networks similarly to wild-type mice in response to carbon tetrachloride–induced chronic liver injury. The networks were found to be nucleated by type V collagen, induced by elevated local transforming growth factor (TGF)-β bioavailability.⁶ Therefore, we identified two mechanisms of collagen fibrillogenesis in response to liver injury: both fibronectin and TGF-β–signaling mediated.Early in the fibrogenic process, inflammatory cytokines are important in initiating repair following injury. TGF-β acts as a fibrogenic master cytokine and plays a pivotal role in the progression of a variety of chronic fibrotic diseases by promoting myofibroblastic differentiation, stimulating synthesis of ECMs, and down-regulating ECM degradation.⁷ In TGF-β–mediated signaling, ligand TGF-βs bind to TGF-β type I and type II receptors that form heterotetrameric complexes. On ligand binding, downstream Smad signaling pathways are initiated. Activated (phosphorylated) Smads translocate to the nucleus where they are involved in the regulation of gene expression.^{8, 9} Currently, several monoclonal antibodies and small molecules targeting TGF-β are in the process of clinical application for chronic fibrotic diseases, including liver fibrosis.¹⁰ However, these studies were initiated without knowledge of how TGF-β signaling exerts its action in the development of liver fibrosis.Elevated TGF-β activity in chronic fibrotic diseases is often accompanied by elevated expression of a matricellular protein, connective tissue growth factor (CTGF/CCN2).¹¹ The manifestation of CTGF/CCN2 functions in vivo involves cooperative interactions with costimulatory factors in the microenvironment, such as TGF-β and fibronectin. One hypothesis arising from the in vivo models is that fibrosing liver injuries are exacerbated by the action of TGF-β–mediated CTGF/CCN2.¹² Here, we addressed the extent to which fibrosis is dependent on the TGF-β/CTGF/CCN2 axis in chronic liver injury using an adult mouse model lacking liver TGF-β type II receptors (TGF-βIIR).     

Cross-talk between TGF-β/Smad pathway and Wnt/β-catenin pathway in pathological scar formation

TGF-β1 is a key factor in the process of wound healing, which is regulated by TGF-β/Smad pathway. We previously demonstrated that TGF-β1 contributed to pathological scar formation. And previous studies also suggested Wnt/β-catenin pathway might be involved in wound healing. However, their role and relation in pathological scar formation remains not very clear. For evaluating TGF-β1 and β-catenin, key factors of the two signal pathways, immunohistochemistry, western blot analysis and RT-PCR were used. Simultaneously, immunohistochemistry were used to evaluate Smad2, Smad3 and Wnt-1, which were also the important factors. We found that they all significantly accumulated in pathological scars compared with normal skins (P<0.05), that implied the two signal pathways both contributed to pathological scar formation. Meanwhile, β-catenin expression showed a tendency to increase first and then decrease under the influence of different concentrations of TGF-β1 (P<0.01). It is possible that there is a complicated interaction between the two signal pathways in pathological scar formation (both synergy and antagonism).     

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (−) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-β activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-β1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-β activation, we examined the role of latent TGF-β-binding proteins (LTBPs), key regulators of TGF-β bioavailability and activation, in TGF-β1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-β1 complexes in Thy-1 (−) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-β1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-β1. Expression of LTBP-4 restored TGF-β1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-β-binding site or only the TGF-β-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-β activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-β1 activation in bleomycin-induced lung fibrosis.     

FGFR4 and TGF-β1 Expression in Hepatocellular Carcinoma: Correlation with Clinicopathological Features and Prognosis

Objective: To investigate the expression and correlation of transforming growth factor-β1 (TGF-β1) and fibroblast growth factor receptor 4 (FGFR4) in human hepatocellular carcinoma (HCC) and the relationship with clinicopathological features and prognosis.Materials and methods: The expression of TGF-β1 and FGFR4 in 126 HCC samples was detected immunohistochemically. Combined with clinical postoperative follow-up data, the expression of TGF-β1 and FGFR4 in HCC and the relationship with the prognosis of patients were analyzed by statistically.Results: The positive expression rate of TGF-β1 was 84.1% (106/126) in tumors, and that in peritumoral liver tissues was 64.3% (81/126); the positive expression rate of FGFR4 in tumors was 74.6% (94/126) and that in peritumoral liver tissues was 57.1% (72/126). The expression of TGF-β1 and FGFR4 in the carcinoma tissues was significantly higher than that in peritumoral liver tissues (p < 0.05). Intratumoral TGF-β1 and FGFR4 expression was associated with TNM stage (p < 0.05). TGF-β1 and FGFR4 expression levels didn''t significantly correlate with other clinicopathological parameters, including age, sex, tumor size, serum AFP level, tumor differentiation, lymph node metastasis, etc. (p > 0.05). TGF-β1 expression was positively correlated with FGFR4 expression (r = 0.595, p < 0.05). Patients with positive FGFR4 or TGF-β1 expression had shorter overall survival compared with negative expression (p < 0.05).Conclusions: The expression of TGF-β1 and FGFR4 could make synergy on the occurrence and progression of HCC, and may be used as prognosis indicators for HCC patients.     

Endothelial-Myofibroblast Transition Contributes to the Early Development of Diabetic Renal Interstitial Fibrosis in Streptozotocin-Induced Diabetic Mice

Diabetic nephropathy is the leading cause of chronic renal failure. Myofibroblasts play a major role in the synthesis and secretion of extracellular matrix in diabetic renal fibrosis. Increasing evidence suggests that endothelial cells may undergo endothelial-myofibroblast transition under physiological and pathophysiological circumstances. Therefore, this study investigates whether endothelial-myofibroblast transition occurs and contributes to the development of diabetic renal interstitial fibrosis. Diabetes was induced by administration of streptozotocin to Tie2-Cre;LoxP-EGFP mice, an endothelial lineage-traceable mouse line generated by crossbreeding B6.Cg-Tg(Tek-cre)12F1v/J mice with B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J mice. The endothelial-myofibroblast transition was also studied in MMECs (a mouse pancreatic microvascular endothelial cell line) and primary cultures of CD31⁺/EYFP⁻ (enhanced yellow fluorescent protein) endothelial cells isolated from adult normal α-smooth muscle actin promoter-driven-EYFP (α-SMA/EYFP) mouse kidneys. Confocal microscopy demonstrated that 10.4 ± 4.2 and 23.5 ± 7.4% of renal interstitial myofibroblasts (α-SMA⁺) in 1- and 6-month streptozotocin-induced diabetic kidneys were of endothelial origin (EGFP⁺/α-SMA⁺ cells), compared with just 0.2 ± 0.1% of myofibroblasts in vehicle-treated Tie2-Cre;LoxP-EGFP mice (P < 0.01). Confocal microscopy and real-time PCR showed that transforming growth factor (TGF)-β1 induced de novo expression of α-SMA and loss of expression of VE-cadherin and CD31 in MMECs and primary cultures of renal endothelial cells in a time- and dose-dependent fashion. These findings demonstrate that the endothelial-myofibroblast transition occurs and contributes to the early development and progression of diabetic renal interstitial fibrosis and suggest that the endothelial-myofibroblast transition may be a therapeutic target.Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the Western world. Approximately 25 to 35% of patients with type 1 diabetes¹ and 5 to 10% of patients with type II diabetes² develop DN. Glomerulosclerosis and interstitial fibrosis are the key morphological features of DN, and both correlate well with the development and progression of renal disease.³ Myofibroblasts play a major role in the synthesis and secretion of extracellular matrix in the development and progression of renal fibrosis. In DN, cells expressing α-smooth muscle actin (α-SMA), the putative marker of myofibroblasts, are located primarily in the renal interstitium and to a lesser extent in glomeruli in association with mesangial proliferation.⁴ The number of myofibroblasts is inversely correlated with renal function in DN.⁵Importantly however, the origin of myofibroblasts in DN remains unclear. It is generally believed that myofibroblasts may be derived from resident fibroblasts, epithelial cells through the epithelial-myofibroblast transition, mesangial cells, or bone marrow-derived cells. Interestingly, increasing evidence suggests that endothelial cells may undergo endothelial-myofibroblast transition (EndoMT) under physiological and pathophysiological circumstances^6,7 and thereby give rise to myofibroblasts. Arciniegas et al⁸ demonstrated that transforming growth factor (TGF)-β1 can induce aortic endothelial cells to differentiate into α-SMA⁺ cells in vitro, suggesting a novel role for TGF-β1 in atherogenesis. Moreover, embryonic endothelial cells have been shown to transdifferentiate into mesenchymal cells expressing α-SMA in vitro and in vivo,⁹ and vascular endothelium-derived cells contain α-SMA in restenosis,¹⁰ inflammation, and hypertension,¹¹ suggesting that myofibroblasts may be of endothelial origin.The involvement of TGF-β1 in renal fibrosis, including DN, has been the subject of extensive investigation.¹² TGF-β1 exerts its biological effects by signaling through TGF-β type II and type I receptors,¹³ and their downstream effectors, R-Smads (Smad2 and Smad3). TGF-β/Smad2/3 signaling pathways are activated in human DN¹⁴ and diabetic mouse kidneys.^15,16 Smad3-null mice are resistant to streptozocin (STZ)-induced DN.¹⁷ It remains largely unknown whether TGF-β1 can induce EndoMT in microvascular endothelial cells in DN, one of the major microvascular complications of diabetes and whether Smad3 plays a pivotal role in the process of TGF-β1-induced EndoMT.In this study we investigated whether EndoMT occurs and contributes to the development of renal interstitial fibrosis in STZ-induced DN in an endothelial lineage-traceable mouse line, the Tie2-Cre;LoxP-EGFP mouse. We also assess whether a specific inhibitor for Smad3 (SIS3)¹⁸ can inhibit TGF-β-induced EndoMT in a mouse microvascular endothelial cell line (MMECs).     

Inflammatory Bone Loss in Experimental Periodontitis Induced by Aggregatibacter actinomycetemcomitans in Interleukin-1 Receptor Antagonist Knockout Mice

The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.     

Dermal Transforming Growth Factor-β Responsiveness Mediates Wound Contraction and Epithelial Closure

Stromal-epithelial interactions are important during wound healing. Transforming growth factor-β (TGF-β) signaling at the wound site has been implicated in re-epithelization, inflammatory infiltration, wound contraction, and extracellular matrix deposition and remodeling. Ultimately, TGF-β is central to dermal scarring. Because scarless embryonic wounds are associated with the lack of dermal TGF-β signaling, we studied the role of TGF-β signaling specifically in dermal fibroblasts through the development of a novel, inducible, conditional, and fibroblastic TGF-β type II receptor knockout (Tgfbr2^dermalKO) mouse model. Full thickness excisional wounds were studied in control and Tgfbr2^dermalKO back skin. The Tgfbr2^dermalKO wounds had accelerated re-epithelization and closure compared with controls, resurfacing within 4 days of healing. The loss of TGF-β signaling in the dermis resulted in reduced collagen deposition and remodeling associated with a reduced extent of wound contraction and elevated macrophage infiltration. Tgfbr2^dermalKO and control skin had similar numbers of myofibroblastic cells, suggesting that myofibroblastic differentiation was not responsible for reduced wound contraction. However, several mediators of cell-matrix interaction were reduced in the Tgfbr2^dermalKO fibroblasts, including α1, α2, and β1 integrins, and collagen gel contraction was diminished. There were associated deficiencies in actin cytoskeletal organization of vasodilator-stimulated phosphoprotein-containing lamellipodia. This study indicated that paracrine and autocrine TGF-β dermal signaling mechanisms mediate macrophage recruitment, re-epithelization, and wound contraction.Skin wound healing in adult humans after full-thickness injury results in scar tissue, which is prone to contracture, loss in elasticity, and altered tensile strength. The characteristics of a healed wound, unlike the original dermal tissue, include an extracellular matrix without the normal basket weave organization of collagen fibers that can be further exaggerated as a hypertrophic scar. In scars, there is a loss of normal tissue architecture and function due, at least in part, to altered cell-matrix interaction and the assembly of the actin cytoskeleton that occurs during wound repair.^1,2 The actin cytoskeleton is important in processes that are fundamental to wound healing: migration; contraction; adhesion; and proliferation.³ The vasodilator-stimulated phosphoprotein (VASP) is associated with filamentous actin formation and likely plays a role in cell adhesion and motility.⁴ VASP may also be involved in the intracellular signaling pathways that regulate integrin-extracellular matrix interactions.The importance of stromal-epithelial interactions in development and wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the transforming growth factor-β (TGF-β) family. TGF-β1, β2, and β3 isoforms signal by binding to the TGF-β receptors type I and type I at the cell surface. The ligand-receptor complex formed stimulates multiple parallel signal pathways in the cytoplasm. TGF-β is a growth inhibitor for many cell types and stimulates extracellular matrix expression in mesenchymal cells. In the injured skin, macrophages, endothelium, fibroblasts, and epithelia are all sources of elevated TGF-β expression. TGF-β signaling at the wound site is thought to be important for extracellular matrix deposition and remodeling. The application of TGF-β1 to wounds has been shown to accelerate wound healing.⁵ In apparent contradiction, a genetic knockout of the TGF-β effector, Smad3, in mice exhibited accelerated skin and lens wound healing.^6–8 However, the epithelial and stromal compartments can respond differentially to TGF-β signaling, and the specific mechanism of TGF-β action on any individual cell type within tissues is still not understood. Wounding of embryonic skin, in contrast to that in adults, is not associated with remodeling deficiencies associated with scarring. Interestingly, embryonic dermal fibroblasts lack expression of TGF-β receptor type II.^9,10In the present study, we specifically address the role of TGF-β signaling in dermal fibroblasts by the development of a novel dermal fibroblast-inducible, conditional, TGF-β type II receptor knockout (Tgfbr2^dermalKO) mouse model. The generation of Tgfbr2^dermalKO mice provided an opportunity to test the role of TGF-β in stromal cells in wound healing. The full-thickness skin excision wounds in Tgfbr2^dermalKO mice had accelerated re-epithelization compared with control, impaired collagen organization associated with a lack of wound contraction, and reduced macrophage recruitment. The studies indicate that paracrine and autocrine TGF-β dermal responsiveness mechanisms impact skin wound healing.