NADPH oxidase 4 (NOX4) is a member of the NADPH oxidase gene family that regulates cellular differentiation, innate immunity and tissue fibrosis. Transforming growth factor-β (TGF-β1) is known to induce expression of NOX4 mRNA in mesenchymal cells. However, the mechanisms of transcriptional regulation of NOX4 are not well understood. In this study, we examined the transcriptional regulation of NOX4 in human lung fibroblasts by TGF-β1. Five promoter-reporter constructs containing DNA fragments of 0.74kb, 1.35kb, 1.84kb, 3.97kb and 4.76kb upstream from the transcriptional start site (TSS) of the human NOX4 gene were generated and their relative responsiveness to TGF-β1 analyzed. TGF-β1-induced NOX4 gene promoter activation requires a region between -3.97kb and -4.76kb. Bioinformatics analysis revealed a 15bp AP-1/Smad binding element in this region. Mutation or deletion of either the AP-1 or the Smad element attenuated TGF-β1 responsiveness of the -4.76kb NOX4 promoter. Furthermore, insertion of this AP-1/Smad box conferred TGF-β1 inducibility to the non-responsive -3.97kb NOX4 promoter construct. Chromatin immunoprecipitation analysis indicated that phospho-Smad3 and cJun associate with this element in a TGF-β1-inducible manner. These results demonstrate that the AP-1/Smad box located between 3.97kb and 4.76kb upstream of the TSS site of the NOX4 promoter is essential for NOX4 gene transcription induced by TGF-β1 in human lung fibroblasts. Our study provides insights into the molecular mechanisms of NOX4 gene expression, informing novel therapeutic approaches to interfere with upregulation of NOX4 in diseases characterized by activation of the TGF-β1/NOX4 pathway.

Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine that plays important roles in cervical tumor formation, invasion, progression, and metastasis. TGF-β1 functions as a tumor inhibitor in precancerous lesions and early stage cancers of cervix whereas as a tumor promoter in later stage. This switch from a tumor inhibitor to a tumor promoter might be due to various alterations in TGF-β signaling pathway, such as mutations or loss of expression of TGF-β receptors and SMAD proteins. Additionally, the oncoproteins of human papillomaviruses have been shown to stimulate TGF-β1 expression, which in turn suppresses host immune surveillance. Thus, in addition to driving tumor cell migration and metastasis, TGF-β1 is believed to play a key role in promoting human papillomavirus infection by weakening host immune defense. In this article, we will discuss the role of TGF-β1 in the expression, carcinogenesis, progression, and therapy in cervical cancers. A better understanding of this cytokine in cervical carcinogenesis is essential for critical evaluation of this cytokine as a potential prognostic marker and therapeutic target.

Myofibroblasts are a key cell type in wound repair, cardiovascular disease, and fibrosis and in the tumor-promoting microenvironment. The high accumulation of myofibroblasts in reactive stroma is predictive of the rate of cancer progression in many different tumors, yet the cell types of origin and the mechanisms that regulate proliferation and differentiation are unknown. We report here, for the first time to our knowledge, the characterization of normal human prostate-derived mesenchymal stem cells (MSCs) and the TGF-β1-regulated pathways that modulate MSC proliferation and myofibroblast differentiation. Human prostate MSCs combined with prostate cancer cells expressing TGF-β1 resulted in commitment to myofibroblasts. TGF-β1-regulated runt-related transcription factor 1 (RUNX1) was required for cell cycle progression and proliferation of progenitors. RUNX1 also inhibited, yet did not block, differentiation. Knockdown of RUNX1 in prostate or bone marrow-derived MSCs resulted in cell cycle arrest, attenuated proliferation, and constitutive differentiation to myofibroblasts. These data show that RUNX1 is a key transcription factor for MSC proliferation and cell fate commitment in myofibroblast differentiation. This work also shows that the normal human prostate gland contains tissue-derived MSCs that exhibit multilineage differentiation similar to bone marrow-derived MSCs. Targeting RUNX1 pathways may represent a therapeutic approach to affect myofibroblast proliferation and biology in multiple disease states.

The pathophysiological events that lead to renal interstitial fibrogenesis are incompletely understood. Epoxyeicosatrienoic acid (EET), an arachidonic acid metabolite, has anti-inflammatory and profibrinolytic functions. Soluble epoxide hydrolase (sEH) converts EET to less active dihydroxyeicosatrienoic acid. Here, we tested the hypothesis that sEH deficiency would prevent tubulointerstitial fibrosis and inflammation induced by unilateral ureteral obstruction (UUO) in mouse kidneys. The loss of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis as demonstrated by reduced collagen deposition and myofibroblast formation at 3 and 10 days after UUO. The inflammatory response was prevented as demonstrated by decreased influx of neutrophil and macrophage, expression of inflammatory cytokines, and chemotactic factors in sEH-deficient UUO kidneys. Pharmacological inhibition of sEH also prevented inflammation and fibrosis after UUO. Next, we delved into the molecular mechanisms piloting the beneficial effects of sEH deficiency in renal fibrosis. UUO upregulated profibrotic factors associated with transforming growth factor (TGF)-β1/Smad3 signaling, oxidative stress, and NF-κB activation, and downregulated antifibrotic factors including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPARγ, but the loss of sEH prevented these adverse effects in UUO kidneys. Furthermore, administration of PPAR antagonists enhanced myofibroblast formation and activation of Smad3 and NF-κB p65, effects that were prevented by sEH deficiency in UUO kidneys. These data demonstrate that loss of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys via activation of PPAR isoforms and downregulation of NF-κB, TGF-β1/Smad3, and inflammatory signaling pathways. Our data suggest the potential use of sEH inhibitors in treating fibrotic diseases.

Macrophage infiltration is a prominent feature of the innate immune response to kidney injury. The persistence of macrophages is associated with tubulointerstitial fibrosis and progression of chronic kidney disease. Macrophages are known to be major producers of transforming growth factor-β1 (TGF-β1), especially in the setting of phagocytosis of apoptotic cells. TGF-β1 has long been implicated as a central mediator of tissue scarring and fibrosis in many organ disease models, including kidney disease. In this study, we show that homozygous deletion of Tgfb1 in myeloid lineage cells in mice heterozygous for Tgfb1 significantly reduces kidney Tgfb1 mRNA expression and Smad activation at late time points after renal ischemia-reperfusion injury. However, this reduction in kidney Tgfb1 expression and signaling results in only a modest reduction of isolated fibrosis markers and does not lead to decreased interstitial fibrosis in either ischemic or obstructive injury models. Thus, targeting macrophage-derived TGF-β1 does not appear to be an effective therapy for attenuating progressive renal fibrosis after kidney injury.

BACKGROUND

Prevention of chronic kidney allograft injury (CAI) is a major goal in improving kidney allograft survival; however, the mechanisms of CAI are not clearly understood. The current study investigated whether alternatively activated M2-type macrophages are involved in the development of CAI.

METHODS

A retrospective study examined kidney allograft protocol biopsies (at 1 h and at years 1, 5, and 10--a total of 41 biopsies) obtained from 13 children undergoing transplantation between 1991 and 2008 who were diagnosed with CAI: interstitial fibrosis and tubular atrophy (IF/TA) not otherwise specified (IF/TA-NOS).

RESULTS

Immunostaining identified a significant increase in interstitial fibrosis with accumulation of CD68 + CD163+ M2-type macrophages. CD163+ cells were frequently localized to areas of interstitial fibrosis exhibiting collagen I deposition and accumulation of α-smooth muscle actin (SMA) + myofibroblasts. There was a significant correlation between interstitial CD163+ cells and the parameters of interstitial fibrosis (p < 0.0001), and kidney function (r =-0.82, p < 0.0001). The number of interstitial CD163+ cells at years 1 and 5 also correlated with parameters of interstitial fibrosis at years 5 and 10 respectively. Notably, urine CD163 levels correlated with interstitial CD163+ cells (r = 0.79, p < 0.01) and parameters of interstitial fibrosis (p < 0.0001). However, CD3+ T lymphocytic infiltration did not correlate with macrophage accumulation or fibrosis. In vitro, dexamethasone up-regulated expression of CD163 and cytokines (TGF-β1, FGF-2, CTGF) in human monocyte-derived macrophages, indicating a pro-fibrotic phenotype.

CONCLUSIONS

Our findings identify a major population of M2-type macrophages in patients with CAI, and suggest that these M2-type macrophages might promote the development of interstitial fibrosis in IF/TA-NOS.

Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H(+)-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients' dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-β signalling and increased TGF-β1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL.

Epithelial-mesenchymal transition (EMT) is an important factor in cancer invasiveness and metastatic progression. During EMT, cancer cells acquire stem cell properties. The role of EMT and stemness in colon cancer has not been fully understood. We aimed to demonstrate the clinical significance of EMT and the stem cell phenotype in colorectal cancer. Two hundred and thirty-one surgically resected colon cancer cases were included in the present study. mRNAs of E-cadherin, TWIST1 and SNAI1 were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) (n=109). Immunohistochemical staining was performed for six markers (ALDH1, TGF-β1, E-cadherin, β-catenin, TWSIT1 and SNAI1) (n=231). We assessed clinicopathological characteristics according to the expression of the stem cell phenotype and EMT markers. Based on the results of qRT-PCR, TWIST1 and SNAI1 significantly influenced node metastasis (P=0.04 and P=0.02, respectively). High TWIST1 and SNAI1 mRNA expression was associated with poor overall survival according to the univariate analysis (P<0.01 and P=0.01, respectively) and the multivariate analysis (P=0.04 and P=0.04, respectively). ALDH1 expression as detected by immunohistochemical staining was associated with high nodal stage, advanced clinical stage, lymphatic invasion and poor survival (P=0.01, P=0.04, P<0.05 and P<0.01, respectively) and with the expression of TGF-β1 and β-catenin. In conclusion, in human colorectal cancer, the EMT markers TWIST1 and SNAI1 are suggested as important markers of poor prognosis. Their expression is associated with the expression of putative stem cell marker ALDH1, and ALDH1 is associated with the expression of TGF-β1.

SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 10(9) CFU/ml) or high (1 × 10(11) CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4(+) ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3(+) CD4(+) CD8(-) T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4(+) ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3(+) CD4(+) CD8(-) cells and Peyer's patch CD3(+) CD4(-) CD8(-) and CD3(-) CD4(-) CD8(+) cells. The percentage of ileal intraepithelial CD3(+) CD4(-) CD8(+) cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4(+) ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor β1 (TGF-β1), and ileal IL-10 was upregulated in the low-dose piglets after F4(+) ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3(+) CD4(+) CD8(-) T cells, the expansion of Peyer's patch CD3(+) CD4(-) CD8(-) and CD3(-) CD4(-) CD8(+) cells, and the attenuation of F4(+) ETEC-induced increase in CD3(+) CD4(+) CD8(+) T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4(+) ETEC challenge, thus eliciting a strong proinflammatory response.

OBJECTIVE

Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids. However, the mechanisms of the action of those triterpenoids are poorly understood. In this study, we aimed to determine the antifibrotic potential of other triterpenes, betulin and betulinic acid, and to characterize their influence on the signal transduction pathways involved in ethanol-activated hepatic stellate cells (HSCs).

METHODS

Investigated was the influence of preincubation of rat HSCs with betulin and betulinic acid, at non-toxic concentrations, on ethanol-induced toxicity, migration, and several markers of HSC activation such as smooth muscle α-actin (α-SMA) and procollagen I expression, release of reactive oxygen species (ROS) and cytokines: tumor necrosis factor-α (TNF-α) and tumor growth factor-β1 (TGF-β1), and production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). To assess the mechanism of the action of those triterpenes, intracellular signals such as nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol were examined.

RESULTS

In vitro, betulin, but not betulinic acid, protected HSCs against ethanol toxicity. However, both betulin and betulinic acid inhibited the production of ROS by HSCs treated with ethanol and inhibited their migration as well as ethanol-induced TNF-α, and TGF-β1, production. Betulin and betulinic acid down-regulated ethanol-induced production of TIMP-1 and TIMP-2. Betulin and betulinic acid, also decreased ethanol-induced activity of MMP-2. In ethanol-induced HSCs, betulin inhibited the activation of the p38 MAPK and the JNK transduction pathways, while betulinic acid inhibited the JNK transduction pathway only. They also significantly inhibited phosphorylation of IκB and Smad 3 and attenuated the activation of TGF-β1 and NFκB/IκB transduction signaling.

CONCLUSIONS

The results indicated that betulin and betulinic acid inhibited ethanol-induced activation of HSCs on different levels, acting as antioxidants, inhibitors of cytokine production, and inhibitors of TGF-β, and NFκB/IκB transduction signaling. Betulin was also inhibitor of both JNK and p38 MAPK signal transduction, while betulinic acid inhibited only JNK. The remarkable inhibition of several markers of HCS activation makes triterpenes, especially betulin, promising agents for anti-fibrotic combination therapies.

BACKGROUND

Dysregulation of CD4 (+) T cell subsets participates in the pathogenesis of IgA nephropathy (IgAN). FoxP3 (+) regulatory T cells (Treg) and Th17 cells are two novel subsets of CD4 (+) T cells. This study aims to investigate Treg/Th17 balance in IgAN patients.

METHODS

Peripheral frequencies of Th17 and Treg functional subsets - CD45RA (+) FoxP3(low) resting Treg (rTreg) and CD45RA(-)FoxP3(high) activated Treg (aTreg) were assessed in 63 adult IgAN patients. Expression of transcription factors (FoxP3 and RORγt) and related cytokines of Treg and Th17 were analysed. Renal expression of FoxP3 and IL-17A were detected by immunohistochemistry.

RESULTS

Compared with normal controls, IgAN patients had decreased frequency of CD45RA(-)FoxP3(high) aTreg subset (p < 0.05), increased frequency of Th17 (p < 0.05) and decreased ratio of Treg/Th17 (p < 0.05). Frequency of aTreg subset correlated with SBP(r = - 0.57, p < 0.05), DBP (r = - 0.50, p < 0.05), eGFR (r = 0.68, p < 0.05) and 24 h proteinuria (r = - 0.58, p < 0.05). RORγtmRNA/FoxP3mRNA ratio increased in IgAN (p < 0.05). Serum IL-17A, IL-21, IL-23, IL-1β and IL-6 elevated while IL-10 decreased in IgAN (p < 0.05), and serum IL-17A correlated with 24 h proteinuria (r = 0.35, p < 0.05). Serum TGF-β1 wasn't different between the two groups. Renal interstitial infiltration of FoxP3 (+) mononuclear cells were observed in IgAN patients, particularly prominent in those with>> 25% tubular atrophy/interstitial fibrosis. Tubular IL-17A expression was found in 34 out of 63 IgAN patients. Compared with 29 patients without IL-17A expression, these patients had lower renal function, greater proteinuria, and more severe tubulointerstitial damage.

CONCLUSIONS

Imbalance of Treg/Th17 found in IgAN may play a role in disease pathogenesis and progression.

Myocardial fibrosis is characterized by significant extracellular matrix (ECM) deposition. The specific cellular mediators that contribute to the development of fibrosis are not well understood. Using a model of fibrosis with Angiotensin II (AngII) infusion, our aim was to characterize the cellular elements involved in the development of myocardial fibrosis. Male C57Bl/6 and Tie2-GFP mice were given AngII (2.0 mg/kg/min) or saline (control) via mini osmotic pumps for up to 7 days. Hearts were harvested, weighed and processed for analysis. Cellular infiltration and collagen deposition were quantified. Immunostaining was performed for specific markers of leukocytes (CD45, CD11b), myofibroblasts (SMA), endothelial cells (vWF) and hematopoietic progenitor cells (CD133). Bone marrow (BM) origin of infiltrating cells was assessed using GFP(+) chimeric animals. Relative qRT-PCR was performed for pro-fibrotic cytokines (transforming growth factor (TGF)-β1, CTGF) as well as the chemokine stromal-derived factor (SDF)-1α. Myocardial-infiltrating cells were grown in vitro. AngII exposure resulted in multifocal myocardial cellular infiltration, which preceded extensive ECM deposition. A limited number of myocardial-infiltrating cells were positive for leukocyte markers but were significantly positive for myofibroblast (SMA) and endothelial cell (vWF) markers. However, using Tie2-GFP mice, where endothelial cells are GFP(+), myocardial-infiltrating cells were not GFP(+). Transcript levels for SDF-1α were significantly elevated at 1 day of AngII exposure suggesting that hematopoietic progenitor cells may be recruited. This was confirmed by positive CD133 staining of infiltrating cells and evident GFP(+) cellular infiltration when exposing GFP(+) BM chimeras to AngII. Furthermore, a significant number of CD133(+)/SMA(+) cells were grown in vitro from the myocardium of AngII-exposed animals (P<0.01). Myocardial ECM deposition is preceded by the infiltration of the myocardium with hematopoietic cells that express mesenchymal markers. These data suggest that mesenchymal progenitor cells are recruited, and may have a primary role, in the initiation of myocardial fibrosis.

OBJECTIVE

Animal studies show that transforming growth factor-β1 (TGF-β1) is an important mediator of atrial fibrosis and atrial fibrillation (AF). This study investigated the role of TGF-β1 in human AF and the mechanism of atrial-selective fibrosis.

RESULTS

Atrial specimens from 17 open heart surgery patients and left atrial and ventricular specimens from 17 explanted hearts were collected to assess the relationship between TGF-β1, AF, and differential atrial vs. ventricular TGF-β1 levels. A transgenic mouse model overexpressing active TGF-β1 was used to study the mechanisms underlying the resultant atrial-selective fibrosis. Higher right atrial total TGF-β1 levels (2.58 ± 0.16-fold, P < 0.0001) and active TGF-β1 (3.7 ± 0.7-fold, P = 0.013) were observed in those that developed post-operative AF. Although no ventricular differences were observed, 11 explanted heart failure hearts exhibited higher atrial TGF-β1 levels than 6 non-failing hearts (2.30 ± 0.87 fold higher, P < 0.001). In the transgenic mouse, TGF-β1 receptor-1 kinase blockade resulted in decreased atrial expression of fibrosis-related genes. By RNA microarray analyses in that model, 80 genes in the atria and only 2 genes in the ventricle were differentially expressed. Although these mice atria, but not the ventricles, exhibited increased expression of fibrosis-related genes and phosphorylation of Smad2, there were no differences in TGF-β1 receptor levels or Smads in the atria compared with the ventricles.

CONCLUSIONS

TGF-β1 mediates selective atrial fibrosis in AF that occurs via TGF-β Receptor 1/2 and the classical Smad pathway. The differential atrial vs. ventricular fibrotic response occurs at the level of TGF-β1 receptor binding or phosphorylation.

The cardiac response to myocardial injury includes fibrotic and hypertrophic processes and a key mediator in this response is transforming growth factor-β1 (TGF-β1). Caveolin-1 (cav1), the main structural protein of caveolae, is an inhibitor of the TGF-β1 signaling pathway. To examine the role of cav1 in cardiac repair, cav1 deficient (Cav1(-/-)) and wild type (WT) mice were subjected to cryoinjury of the left ventricle (LV). At baseline the two groups exhibited no inflammation, similar collagen content, and similar cardiac function. After injury, Cav1(-/-) animals displayed enhanced TGF-β1 signaling, as reflected by a 3-fold increase in the activation of the Smad2-dependent pathway and more widespread collagen deposition in the heart. Qualitative and quantitative analyses indicated that collagen deposition peaked in the WT LV 14days after injury, accompanied by increased mRNA abundance for procol1a2 (2-fold) and procol3a1 (3-fold). Collagen deposition was further enhanced in Cav1(-/-) mice, which was accompanied by reduced expression of matrix metalloproteinases MMP-8 (3-fold) and -13 mRNA (2-fold). The levels of expression of inflammatory markers of acute phase were similar between the strains However, macrophage clearance in the damaged region was delayed in Cav1(-/-) mice. We observed a 4-fold decrease in collagen deposition in Cav1(-/-) mice injected with a cav1 scaffolding domain peptide (CSD) and a 2-fold decrease in WT mice treated with the CSD. We conclude that cav1 has a direct role in reducing TGF-β1 signaling and as such might be an appropriate target for therapies to influence cardiac remodeling.

The role of a small transforming growth factor beta (TGF-β)-induced TIAF1 (TGF-β1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-β1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-β receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid β (Aβ) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aβ. To test this hypothesis, in vitro analysis showed that TGF-β-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aβ and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.

This study tested the hypothesis that CD4(+)CD25(+)CD127(low) regulatory T (Treg) cells might induce immunosuppressive properties in apoptotic neutrophils. Treg cells are recognized as a major subset of immune cells possessing potent suppressive properties directed at T effector cells. However, Treg cells have recently been found to inhibit neutrophil function and promote their apoptosis. One of the mechanisms of action of Treg cells is the induction of other suppressor cell populations according to an infectious tolerance model. We showed that LPS-activated Treg cells promote generation of IL-10 and TGF-β1, inhibit IL-6 production by PMNs and induce the expression of heme oxygenase-1 (HO-1) and the suppressor of cytokine signaling 3 molecule (SOCS3). However, CD3/CD28-activated Treg cells were seen to promote TGF-β1 production, as well as IDO and HO-1 expression by PMNs. These findings suggest that Treg cells might play an important role in the direct control of innate immune responses through the induction of neutrophils with immunosuppressive properties that generate IL-10, TGF-β1, IDO and HO-1.

The mechanisms underlying the pathogenesis of idiopathic pulmonary fibrosis (IPF) involve multiple pathways, such as inflammation, epithelial mesenchymal transition, coagulation, oxidative stress, and developmental processes. The small GTPase, RhoA, and its target protein, Rho-kinase (ROCK), may interact with other signaling pathways known to contribute to pulmonary fibrosis. This study aimed to determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice. Our results showed that the Aschcroft score and hydroxyproline content of the bleomycin-treated mouse lung decreased in response to fasudil treatment. The number of infiltrated inflammatory cells in the bronchoalveolar lavage fluid (BALF) was attenuated by fasudil. In addition, fasudil reduced the production of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), and plasminogen activator inhibitor-1 (PAI-1) mRNA and protein expression in bleomycin-induced pulmonary fibrosis. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

BACKGROUND

Cirrhosis is a long-term consequence of chronic hepatic injury with fibrosis. No effective therapy is currently available for decompensated cirrhosis except liver transplantation. Hence, we investigated the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) on hepatic fibrosis in a thioacetamide (TAA)-induced cirrhotic rat model.

METHODS

The BM-MSCs were injected directly into the right liver lobe twice, at 6 and 8 weeks during the 12-week TAA administration, in thioacetamide (TAA)-induced cirrhotic rats model, and hepatic fibrosis was evaluated. At 12 weeks, the effect of BM-MSCs on hepatic fibrosis was analyzed histomorphologically using the Laennec fibrosis scoring system, and the collagen proportionate area was quantified. Cirrhosis-related factors, such as transforming growth factor β1 (TGF-β1), type 1 collagen (collagen-1), α-smooth muscle actin (α-SMA), and P-Smad3/Smad3 expression levels, were evaluated using real-time polymerase chain reaction and western blot assays.

RESULTS

According to the Laennec fibrosis scoring system, histological improvement was observed in hepatic fibrosis after BM-MSC treatment (P <0.01). The percentage of the collagen proportionate area decreased from 16.72 ± 5.51 to 5.06 ± 1.27 after BM-MSC treatment (P <0.01). The content of hepatic hydroxyproline was significantly lower in the BM-MSC treated group (46.25 ± 13.19) compared to the untreated cirrhotic group (85.81 ± 17.62; P <0.01). BM-MSC administration significantly decreased TGF-β1, collagen-1, and α-SMA expression in TAA-induced cirrhotic rats (P <0.01). We also confirmed P-Smad3/Smad3, downstream effectors of the TGF-β1 signaling pathway, and found that MSC transplantation inhibited Smad3 phosphorylation.

CONCLUSIONS

BM-MSC treatment attenuated hepatic fibrosis in rats with TAA-induced cirrhosis, raising the possibility of the clinical use of BM-MSCs in the treatment of cirrhosis.

Oxidative stress is implicated in the pathogenesis of diabetic kidney injury. SS-31 is a mitochondria-targeted tetrapeptide that can scavenge reactive oxygen species (ROS). Here, we investigated the effect and molecular mechanism of mitochondria-targeted antioxidant peptide SS-31 on injuries in diabetic kidneys and mouse mesangial cells (MMCs) exposed to high-glucose (HG) ambience. CD-1 mice underwent uninephrectomy and streptozotocin treatment prior to receiving daily intraperitoneal injection of SS-31 for 8 wk. The diabetic mice treated with SS-31 had alleviated proteinuria, urinary 8-hydroxy-2-deoxyguanosine level, glomerular hypertrophy, and accumulation of renal fibronectin and collagen IV. SS-31 attenuated renal cell apoptosis and expression of Bax and reversed the expression of Bcl-2 in diabetic mice kidneys. Furthermore, SS-31 inhibited expression of transforming-growth factor (TGF)-β1, Nox4, and thioredoxin-interacting protein (TXNIP), as well as activation of p38 MAPK and CREB and NADPH oxidase activity in diabetic kidneys. In vitro experiments using MMCs revealed that SS-31 inhibited HG-mediated ROS generation, apoptosis, expression of cleaved caspase-3, Bax/Bcl-2 ratio, and cytochrome c (cyt c) release from mitochondria. SS-31 normalized mitochondrial potential (ΔΨm) and ATP alterations, and inhibited the expression of TGF-β1, Nox4, and TXNIP, as well as activation of p38 MAPK and CREB and NADPH oxidase activity in MMCs under HG conditions. SS-31 treatment also could reverse the reduction of thioredoxin (TRX) biologic activity and upregulate expression of thioredoxin 2 (TRX2) in MMCs under HG conditions. In conclusion, this study demonstrates a protective effect of SS-31 against HG-induced renal injury via an antioxidant mechanism in diabetic nephropathy.

OBJECTIVE

Low-grade systemic inflammation and adipose tissue inflammatory macrophages are frequently detected in patients with obesity and type 2 diabetes. Whether inflammatory macrophages also increase in skeletal muscle of individuals with metabolic disorders remains controversial. Here, we assess whether macrophage polarisation markers in skeletal muscle of humans correlate with insulin sensitivity in obesity and type 2 diabetes.

METHODS

Skeletal muscle biopsies were obtained from individuals of normal weight and with normal glucose tolerance (NGT), and overweight/obese individuals with or without type 2 diabetes. Insulin sensitivity was determined by euglycaemic-hyperinsulinaemic clamps. Expression of macrophage genes was analysed by quantitative RT-PCR.

RESULTS

Gene expression of the inflammatory macrophage phenotype marker cluster of differentiation (CD)11c was higher in muscle of type 2 diabetes patients (p = 0.0069), and correlated with HbA1c (p = 0.0139, ρ = 0.48) and fasting plasma glucose (p = 0.0284, ρ = 0.43), but not after correction for age. Expression of TGFB1, encoding the anti-inflammatory marker TGF-β1, correlated inversely with HbA1c (p = 0.0095, ρ = -0.50; p = 0.0484, ρ = -0.50) and fasting plasma glucose (p = 0.0471, ρ = -0.39; p = 0.0374, ρ = -0.52) in two cohorts, as did HbA1c with gene expression of macrophage galactose-binding lectin (MGL) (p = 0.0425, ρ = -0.51). TGFB1 expression was higher in NGT individuals than in individuals with type 2 diabetes (p = 0.0303), and correlated with low fasting plasma insulin (p = 0.0310, ρ = -0.42). In exercised overweight/obese individuals, expression of genes for three anti-inflammatory macrophage markers, MGL (p = 0.0031, ρ = 0.71), CD163 (p = 0.0268, ρ = 0.57) and mannose receptor (p = 0.0125, ρ = 0.63), correlated with high glucose-disposal rate.

CONCLUSIONS

Muscle expression of macrophage genes reveals a link between inflammatory macrophage markers, age and high glycaemia, whereas anti-inflammatory markers correlate with low glycaemia and high glucose-disposal rate.

Transforming growth factor β (TGF-β) is known to play a key role in intestinal fibrosis; however, the underlying mechanisms are not well understood. TGF-β signal transduction is through TGF-β receptors, including the TGF-β type 1 receptor. Most cell types contain a TGF-β type 1 receptor form known as activin receptor-like kinase 5 (ALK5), which propagates the signal to the nucleus through the phosphorylation of Smad2 and Smad3 proteins. Therefore, we assessed the effect of the disruption of TGF-β/ALK5/Smad signalling by an ALK5 inhibitor (SD-208) in two experimental animal models of intestinal fibrosis: anaerobic bacteria- and trinitrobenzensulphonic acid-induced colitis. In addition, isolated myofibroblasts were pretreated with SD-208 and exposed to recombinant TGF-β1. Finally, myofibroblasts were transfected with ALK5, Smad2, and Smad3-specific siRNA. Up-regulation of ALK5 and TIMP-1, phosphorylation of Smad2 and Smad3 proteins, and increased intestinal wall collagen deposition were found in both experimental animal models. These effects were decreased by SD-208. TGF-β1 treatment also induced phosphorylation of Smad2 and Smad3 and up-regulation of ALK5 protein, TIMP-1, and α2 type 1 collagen gene expression in isolated myofibroblasts. Again these effects were inhibited by SD-208. Also, ALK5, Smad2, and Smad3 siRNA abolished the induction of TIMP-1 and α2 type 1 collagen. Our findings provide evidence that the TGF-β/ALK5/Smad pathway participates in the pathogenesis of experimental intestinal fibrosis. These data show promise for the development of an effective therapeutic intervention in this condition.

Posttranslational modification of proteins could regulate their multiple biological functions. Transforming growth factor-β receptor I and II (ALK5 and TGF-βRII), which are glycoproteins, play important roles in the renal tubular epithelial-mesenchymal transition (EMT). In the present study, we examined the role of core fucosylation of TGF-βRII and ALK5, which is regulated by α-1,6 fucosyltransferase (Fut8), in the process of EMT of cultured human renal proximal tubular epithelial (HK-2) cells. The typical cell model of EMT induced by TGF-β1 was constructed to address the role of core fucosylation in EMT. Core fucosylation was found to be essential for both TGF-βRII and ALK5 to fulfill their functions, and blocking it with Fut8 small interfering RNA greatly reduced the phosphorylation of Smad2/3 protein, caused the inactivation of TGF-β/Smad2/3 signaling, and resulted in remission of EMT. More importantly, even with high levels of expressions of TGF-β1, TGF-βRII, and ALK5, blocking core fucosylation also could attenuate the EMT of HK-2 cells. Thus blocking core fucosylation of TGF-βRII and ALK5 may attenuate EMT independently of the expression of these proteins. This study may provide new insight into the role of glycosylation in renal interstitial fibrosis. Furthermore, core fucosylation may be a novel potential therapeutic target for treatment of renal tubular EMT.

Recently identified as a key component of the murine periodontal ligament (PDL), periostin has been implicated in the regulation of collagen fibrillogenesis and fibroblast differentiation. We investigated whether periostin protein is expressed in the human PDL in situ and the mechanisms regulating periostin expression in PDL fibroblasts in vitro. With immunohistochemistry, periostin protein was identified in the PDL, with expression lower in teeth with reduced occlusal loading. In vitro application of uniaxial cyclic strain to PDL fibroblasts elevated periostin mRNA levels, depending on the age of the patient. Treatment with transforming growth factor-beta1 (TGF-β1) also significantly increased periostin mRNA levels, an effect attenuated by focal adhesion kinase (FAK) inhibition. FAK-null fibroblasts contained no detectable periostin mRNA, even after stimulation with cyclic strain. In conclusion, periostin protein is strongly expressed in the human PDL. In vitro, periostin mRNA levels are modulated by cyclic strain as well as TGF-β1 via FAK-dependent pathways.