Statins inhibit angiotensin II/Smad pathway and related vascular fibrosis, by a TGF-β-independent process

We have recently described that in an experimental model of atherosclerosis and in vascular smooth muscle cells (VSMCs) statins increased the activation of the Smad pathway by transforming growth factor-β (TGF-β), leading to an increase in TGF-β-dependent matrix accumulation and plaque stabilization. Angiotensin II (AngII) activates the Smad pathway and contributes to vascular fibrosis, although the in vivo contribution of TGF-β has not been completely elucidated. Our aim was to further investigate the mechanisms involved in AngII-induced Smad activation in the vasculature, and to clarify the beneficial effects of statins on AngII-induced vascular fibrosis. Infusion of AngII into rats for 3 days activates the Smad pathway and increases fibrotic-related factors, independently of TGF-β, in rat aorta. Treatment with atorvastatin or simvastatin inhibited AngII-induced Smad activation and related-fibrosis. In cultured rat VSMCs, direct AngII/Smad pathway activation was mediated by p38 MAPK and ROCK activation. Preincubation of VSMCs with statins inhibited AngII-induced Smad activation at all time points studied (from 20 minutes to 24 hours). All these data show that statins inhibited several AngII-activated intracellular signaling systems, including p38-MAPK and ROCK, which regulates the AngII/Smad pathway and related profibrotic factors and matrix proteins, independently of TGF-β responses. The inhibitory effect of statins on the AngII/Smad pathway could explain, at least in part, their beneficial effects on hypertension-induced vascular damage.     

Blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathways by statins reduces the expression of bFGF, HGF, and TGF-β as angiogenic factors in mouse osteosarcoma

The tumor microenvironment plays a critical role in modulating malignant behavior and can dramatically influence cancer treatment strategies. We investigated whether statins inhibit the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and transforming growth factor-β (TGF-β) mRNA in the mouse osteosarcoma cell line LM8. We found that statins significantly inhibited mRNA expressions of bFGF, HGF, and TGF-β, and bFGF, HGF, and TGF-β secretions at concentrations that did not have antiproliferative effects on LM8 cells, but had no effect on the mRNA expression and secretion of VEGF. The inhibition of bFGF, HGF, and TGF-β mRNA expression, and bFGF, HGF, TGF-β secretions was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination with statins. Furthermore, statins reduced the membrane localization of K-Ras, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our research indicates that statins inhibit GGPP biosynthesis in the mevalonate pathway, and then inhibit signal transduction in the Ras/ERK and Ras/Akt pathways, thereby inhibiting bFGF, HGF, TGF-β expression in LM8 cells. These results suggest that statins are potentially useful as anti-angiogenic agents for the treatment of osteosarcoma.     

Oxidative stress promotes autophagic cell death in human neuroblastoma cells with ectopic transfer of mitochondrial PPP2R2B (Bβ2)

Background

Bone morphogenetic proteins (BMPs) are pleiotropic members of the TGF-beta superfamily which regulate many biological processes during development and adult tissue homeostasis and are implicated in the pathogenesis of a number of human diseases. Their involvement in both normal and aberrant physiology creates a need for rapid, sensitive and methodologically simple assays to evaluate their activity from a variety of biological samples. Previously alkaline phosphatase based assays, ELISA and luciferase based bioassays have been developed to evaluate either individual or total BMP activity. In this paper, we describe a highly sensitive, rapid and specific cell based assay for the simultaneous quantification of total and isoform specific BMP activity from biological samples.

Results

A C2C12 cell line stably transfected with a reporter plasmid consisting of the BMP response element (BRE) from the Id1 promoter fused to a luciferase reporter gene was generated. Exposure of this cell line to human recombinant BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10 induced the expression of luciferase which was quantified using a luminometer. This assay was specific for BMP activity as the other TGF-β superfamily members TGF-β 1, Nodal and Mullerian Inhibiting Substance (MIS) did not induce the reporter. Pretreatment of samples with isoform specific BMP blocking antibodies coupled with isoform specific titration analysis allowed the simultaneous identification and quantification of BMP4, BMP6 and BMP9 in serum samples.

Conclusion

The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.     

The differential proliferative response of fetal and adult human skin fibroblasts to TGF-β is retained when cultured in the presence of fibronectin or collagen

Background

Transforming growth factor-β is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-β inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-β on fetal and adult cells.

Methods

Human fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-β was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors.

Results

Fetal skin fibroblast-proliferation was inhibited by TGF-β, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-β on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK–ERK pathway.

Conclusions

Fetal and adult human skin fibroblasts retain their differential proliferative response to TGF-β when cultured in the presence of fibronectin and unpolymerized or polymerized collagen.

General significance

The interplay between TGF-β and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

Background

TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT).

Methods

The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis.

Results

After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed.

Conclusion

Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling.     

Elastic band resistance training influences transforming growth factor-ß receptor I mRNA expression in peripheral mononuclear cells of institutionalised older adults: the Vienna Active Ageing Study (VAAS)

Background

Ageing, inactivity and obesity are associated with chronic low-grade inflammation contributing to a variety of lifestyle-related diseases. Transforming growth factor-β (TGF-β) is a multimodal protein with various cellular functions ranging from tissue remodelling to the regulation of inflammation and immune functions. While it is generally accepted that aerobic exercise exerts beneficial effects on several aspects of immune functions, even in older adults, the effect of resistance training remains unclear. The aim of this study was to investigate whether progressive resistance training (6 months) with or without nutritional supplementation (protein and vitamins) would influence circulating C-reactive protein and TGF-β levels as well as TGF-β signalling in peripheral mononuclear cells (PBMCs) of institutionalised adults with a median age of 84.5 (65.0–97.4) years.

Results

Elastic band resistance training significantly improved performance as shown by the arm-lifting test (p?=?0.007), chair stand test (p?=?0.001) and 6-min walking test (p?=?0.026). These results were paralleled by a reduction in TGF-β receptor I (TGF-βRI) mRNA expression in PBMCs (p?=?0.006), while circulating inflammatory markers were unaffected. Protein and vitamin supplementation did not provoke any additional effects. Interestingly, muscular endurance of upper and lower body and aerobic performance at baseline were negatively associated with changes in circulating TGF-β at the early phase of the study. Furthermore, drop-outs of the study were characterised not only by lower physical performance but also higher TGF-β and TGF-βRI mRNA expression, and lower miRNA-21 expression.

Conclusions

Progressive resistance training with elastic bands did not influence chronic low-grade inflammation but potentially affected TGF-β signalling in PBMCs through altered TGF-βRI mRNA expression. There appears to be an association between physical performance and TGF-β expression in PBMCs of older adults, in which the exact mechanisms need to be clarified.

     

Cholest-4-en-3-one attenuates TGF-β responsiveness by inducing TGF-β receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells

Purpose: The transforming growth factor-beta (TGF-β) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-β signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro.

Methods: The inhibitory effects of cholest-4-en-3-one on TGF-β-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-β receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation.

Results: Cholest-4-en-3-one attenuated TGF-β signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-β-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling.

Conclusions: Our results suggest that cholest-4-en-3-one inhibits TGF-β signaling may be due, in part to the translocation of TGF-β receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-β deficiency.     

Transforming growth factor-β modulates pancreatic cancer associated fibroblasts cell shape,stiffness and invasion

Background

Tumor microenvironment consists of the extracellular matrix (ECM), stromal cells, such as fibroblasts (FBs) and cancer associated fibroblasts (CAFs), and a myriad of soluble factors. In many tumor types, including pancreatic tumors, the interplay between stromal cells and the other tumor microenvironment components leads to desmoplasia, a cancer-specific type of fibrosis that hinders treatment. Transforming growth factor beta (TGF-β) and CAFs are thought to play a crucial role in this tumor desmoplastic reaction, although the involved mechanisms are unknown.

Beclin 1 regulates neuronal transforming growth factor-β signaling by mediating recycling of the type I receptor ALK5

Background

Beclin 1 is a key regulator of multiple trafficking pathways, including autophagy and receptor recycling in yeast and microglia. Decreased beclin 1 levels in the CNS result in neurodegeneration, an effect attributed to impaired autophagy. However, neurons also rely heavily on trophic factors, and signaling through these pathways requires the proper trafficking of trophic factor receptors.

Results

We discovered that beclin 1 regulates signaling through the neuroprotective TGF-β pathway. Beclin 1 is required for recycling of the type I TGF-β receptor ALK5. We show that beclin 1 recruits the retromer to ALK5 and facilitates its localization to Rab11⁺ endosomes. Decreased levels of beclin 1, or its binding partners VPS34 and UVRAG, impair TGF-β signaling.

Conclusions

These findings identify beclin 1 as a positive regulator of a trophic signaling pathway via receptor recycling, and suggest that neuronal death induced by decreased beclin 1 levels may also be due to impaired trophic factor signaling.

     

Involvement of polyamines in epidermal growth factor (EGF), transforming growth factor (TGF)-α and -β1 action on culture of L6 and fetal bovine myoblasts

• 1.1. α-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase significantly abolished stimulation of protein synthesis evoked by EGF, TGF-α or -β 1 in L6 and fetal bovine myoblasts.
• 2.2. The participation of polyamines in early events evoked by growth factors was shown by a significant stimulation of ornithine decarboxylase and Sdenosylmethionine decarboxylase activity as well as increased concentration of spermidine and spermine in L6 cells exposed to TGF-α and EGF.
• 3.3. TGF-β 1 at a high concentration (1 ng/ml) increased protein synthesis in L6 myoblasts but inhibited it in fetal bovine myoblasts. Metabolic effects of TGF-β 1 in L6 cells was associated with an enhancement of decarboxylase activities, however there were no significant changes in cellular polyamine concentrations. Presented data suggest that polyamines are involved in the signal transduction pathway of EGF, TGF-α, and -β 1 in L6 and fetal bovine myoblasts.

     

The clinical and biological roles of transforming growth factor beta in colon cancer stem cells: A systematic review

Background

Transforming growth factor beta (TGF-β) is a multipurpose cytokine, which plays a role in many cellular functions such as proliferation, differentiation, migration, apoptosis, cell adhesion and regulation of epithelial to mesenchymal transition. Despite many studies having observed the effect that TGF-β plays in colorectal cancer, its role in the colorectal stem cell population has not been widely observed.

TLR3 promotes MMP-9 production in primary human airway epithelial cells through Wnt/β-catenin signaling

Background

Airway epithelial cells (AEC) act as the first line of defence in case of lung infections. They constitute a physical barrier against pathogens and they participate in the initiation of the immune response. Yet, the modalities of pathogen recognition by AEC and the consequences on the epithelial barrier remain poorly documented.

Method

We investigated the response of primary human AEC to viral (polyinosinic-polycytidylic acid, poly(I:C)) and bacterial (lipopolysaccharide, LPS) stimulations in combination with the lung remodeling factor Transforming Growth Factor-β (TGF-β).

Results

We showed a strong production of pro-inflammatory cytokines (Interleukin (IL)-6, Tumor Necrosis Factor α, TNFα) or chemokines (CCL2, CCL3, CCL4, CXCL10, CXCL11) by AEC stimulated with poly(I:C). Cytokine and chemokine production, except CXCL10, was Toll Like Receptor (TLR)-3 dependent and although they express TLR4, we found no cytokine production after LPS stimulation. Poly(I:C), but not LPS, synergised with TGF-β for the production of matrix metalloproteinase-9 (MMP-9) and fibronectin. Mechanistic analyses suggest the secretion of Wnt ligands by AEC along with a degradation of the cellular junctions after poly(I:C) exposure, leading to the release of β-catenin from the cell membrane and stimulation of the Wnt/β-catenin pathway.

Conclusion

Our results highlight the cross talk between TGF-β and TLR signaling in bronchial epithelium and its impact on the remodeling process.

     

Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

Background

Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) contribute to this effect.

Methods

Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing), were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β₁ concentrations were measured in culture supernatants by ELISA.

Results

Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM) of initial area vs. 48.0% ± 0.4 at 48 hours; P < 0.001 and 41.5% ± 0.6 vs. 60.6% ± 0.3 at 48 hours; P < 0.001, respectively). Fixed platelets had no effect in the system. Both TGF-β₁ and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β₁ release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction.

Conclusion

We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.     

Nf1 RasGAP Inhibition of LIMK2 Mediates a New Cross-Talk between Ras and Rho Pathways

Background

Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways.

Methodology/Principal Findings

In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity: the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition: in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator.

Conclusions/Significance

Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases.     

Growth hormone exacerbates diabetic renal damage in male but not female rats

Background

Human and animal studies support the idea that there are sex differences in the development of diabetic renal disease. Our lab and others have determined that in addition to Ang II (through the AT₁R), growth hormone (GH) contributes to renal damage in models of renal failure; however, the impact of sex and GH on the mechanisms initiating diabetic renal disease is not known. This study examined the effect of sex and GH on parameters of renal damage in early, uncontrolled streptozotocin (STZ)-induced diabetes.

Methods

Adult male and female Sprague–Dawley rats were injected with vehicle (control), STZ, or STZ?+?GH and euthanized after 8 weeks.

Results

Mild but significant glomerulosclerosis (GS) and tubulointerstitial fibrosis (TIF) was observed in both kidneys from male and female diabetic rats, with GH significantly increasing GS and TIF by 30% and 25% in male rats, but not in female rats. STZ increased TGF-β expression in both kidneys from male and female rats; however, while GH had no further effect on TGF-β protein in diabetic females, GH increased TGF-β protein in the male rat’s kidneys by an additional 30%. This sex-specific increase in renal injury following GH treatment was marked by increased MCP-1 and CD-68+ cell density. STZ also reduced renal MMP-2 and MMP-9 protein expression in both kidneys from male and female rats, but additional decreases were only observed in GH-treated diabetic male rats. The sex differences were independent of AT₁R activity.

Conclusions

These studies indicate that GH affects renal injury in diabetes in a sex-specific manner and is associated with an increase in pro-inflammatory mediators.

     

Neovessel formation promotes liver fibrosis via providing latent transforming growth factor-β

Aim

Hepatic fibrosis and angiogenesis occur in parallel during the progression of liver disease. Fibrosis promotes angiogenesis via inducing vascular endothelial growth factor (VEGF) from the activated hepatic stellate cells (HSCs). In turn, increased neovessel formation causes fibrosis, although the underlying molecular mechanism remains undetermined. In the current study, we aimed to address a role of endothelial cells (ECs) as a source of latent transforming growth factor (TGF)-β, the precursor of the most fibrogenic cytokine TGF-β.

Methods

After recombinant VEGF was administered to mice via the tail vein, hepatic angiogenesis and fibrogenesis were evaluated using immunohistochemical and biochemical analyses in addition to investigation of TGF-β activation using primary cultured HSCs and liver sinusoidal ECs (LSECs).

Results

In addition to increased hepatic levels of CD31 expression, VEGF-treated mice showed increased α-smooth muscle actin (α-SMA) expression, hepatic contents of hydroxyproline, and latency associated protein degradation products, which reflects cell surface activation of TGF-β via plasma kallikrein (PLK). Liberating the PLK-urokinase plasminogen activator receptor complex from the HSC surface by cleaving a tethering phosphatidylinositol linker with its specific phospholipase C inhibited the activating latent TGF-β present in LSEC conditioned medium and subsequent HSC activation.

Conclusion

Neovessel formation (angiogenesis) accelerates liver fibrosis at least in part via provision of latent TGF-β that activated on the surface of HSCs by PLK, thereby resultant active TGF-β stimulates the activation of HSCs.