TGF-β1 expression is associated with invasion and metastasis of intrahepatic cholangiocarcinoma

Background

Transforming growth factor (TGF)-β is involved in many physiologic processes, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we analyzed the correlation between transforming growth factor beta 1 (TGF-β1) expression and prognosis in intrahepatic cholangiocarcinoma.

Results

We examined the expression of TGF-β1 in 78 intrahepatic cholangiocarcinomas by immunohistochemistry and correlated the expression with clinicopathological parameters. TGF-β1 was expressed in 37 of 78 (47.4 %) intrahepatic cholangiocarcinomas. The expression of TGF-β1 was significantly correlated with lymph node metastasis, distant metastasis, and tumour recurrence. Patients with TGF-β1-positive tumours had significantly shorter survival time. In a multivariant analysis, the expression of TGF-β1 and the tumour stage were independent prognostic factors.

Conclusions

Our data suggest that expression of TGF-β1 is a novel prognostic marker for intrahepatic cholangiocarcinoma.     

Biomarkers of TGF-β Signaling Pathway and Prognosis of Pancreatic Cancer

Background

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

Method

We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

Results

The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

Conclusion

Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.     

Myofibroblast Differentiation and Enhanced Tgf-B Signaling in Cystic Fibrosis Lung Disease

Rationale

TGF-β, a mediator of pulmonary fibrosis, is a genetic modifier of CF respiratory deterioration. The mechanistic relationship between TGF-β signaling and CF lung disease has not been determined.

Objective

To investigate myofibroblast differentiation in CF lung tissue as a novel pathway by which TGF-β signaling may contribute to pulmonary decline, airway remodeling and tissue fibrosis.

Methods

Lung samples from CF and non-CF subjects were analyzed morphometrically for total TGF-β₁, TGF-β signaling (Smad2 phosphorylation), myofibroblast differentiation (α-smooth muscle actin), and collagen deposition (Masson trichrome stain).

Results

TGF-β signaling and fibrosis are markedly increased in CF (p<0.01), and the presence of myofibroblasts is four-fold higher in CF vs. normal lung tissue (p<0.005). In lung tissue with prominent TGF-β signaling, both myofibroblast differentiation and tissue fibrosis are significantly augmented (p<0.005).

Conclusions

These studies establish for the first time that a pathogenic mechanism described previously in pulmonary fibrosis is also prominent in cystic fibrosis lung disease. The presence of TGF-β dependent signaling in areas of prominent myofibroblast proliferation and fibrosis in CF suggests that strategies under development for other pro-fibrotic lung conditions may also be evaluated for use in CF.     

Expression of TGF-β3 in isolated fibroblasts from foreskin

Background:

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s).

Methods:

We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

Conclusions:

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.Key Words: Fibroblasts, Gene expression, TGF-β3     

Marked alveolar apoptosis/proliferation imbalance in end-stage emphysema

Background

Apoptosis has recently been proposed to contribute to the pathogenesis of emphysema.

Methods

In order to establish if cell fate plays a role even in end-stage disease we studied 16 lungs (9 smoking-associated and 7 α1antitrypsin (AAT)-deficiency emphysema) from patients who had undergone lung transplantations. Six unused donor lungs served as controls. Apoptosis was evaluated by TUNEL analysis, single-stranded DNA laddering, electron microscopy and cell proliferation by an immunohistochemical method (MIB1). The role of the transforming growth factor (TGF)-β1 pathway was also investigated and correlated with epithelial cell turnover and with the severity of inflammatory cell infiltrate.

Results

The apoptotic index (AI) was significantly higher in emphysematous lungs compared to the control group (p ≤ 0.01), particularly if only lungs with AAT-deficiency emphysema were considered (p ≤ 0.01 vs p = 0.09). The proliferation index was similar in patients and controls (1.9 ± 2.2 vs 1.7 ± 1.1). An increased number of T lymphocytes was observed in AAT-deficiency lungs than smoking-related cases (p ≤ 0.05). TGF-β1 expression in the alveolar wall was higher in patients with smoking-associated emphysema than in cases with AAT-deficiency emphysema (p ≤ 0.05). A positive correlation between TGF-βRII and AI was observed only in the control group (p ≤ 0.005, r² = 0.8). A negative correlation was found between the TGF-β pathway (particularly TGF-βRII) and T lymphocytes infiltrate in smoking-related cases (p ≤ 0.05, r² = 0.99)

Conclusion

Our findings suggest that apoptosis of alveolar epithelial cells plays an important role even in end-stage emphysema particularly in AAT-deficiency disease. The TGFβ-1 pathway does not seem to directly influence epithelial turnover in end-stage disease. Inflammatory cytokine different from TGF-β1 may differently orchestrate cell fate in AAT and smoking-related emphysema types.     

Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

Background

The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells.

Methodology

In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected.

Conclusions

Statins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs.     

Liver Cancer-Derived Hepatitis C Virus Core Proteins Shift TGF-Beta Responses from Tumor Suppression to Epithelial-Mesenchymal Transition

Background

Chronic hepatitis C virus (HCV) infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC) development. TGF-β is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-β signaling.

Principal Findings

We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-β responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-β was still able to induce an epithelial to mesenchymal transition (EMT), a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-β responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-β growth inhibitory effects to tumor promoting responses.

Conclusion/Significance

Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-β, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-β responses from cytostatic effects to EMT development.     

Polymorphisms and Plasma Level of Transforming Growth Factor-Beta 1 and Risk for Preeclampsia: A Systematic Review

Background

Transforming growth factor-beta 1 (TGF-β1) is thought to be involved in the pathogenesis of preeclampsia (PE), but the results are inconsistent among studies. This article aims to compile an overview of the studies about the associations of TGF-β 1 polymorphism and plasma level with PE risk and to provide recommendations for future research.

Methods and Results

The databases PubMed, Embase and Web of Science were searched up to December 2013. Five studies investigating the associations of four polymorphisms with the risks of PE were involved. A meta-analysis was conducted for the 869T>C polymorphism and PE risk. The results show that genotype TT of 869T>C polymorphism is a protective factor of PE (pooled odds ratio = 0.73, 95% CI: 0.56, 0.95). Eight case-control studies reported the plasma level of TGF-β 1. The substantial heterogeneity among studies may be attributed to the differences in the blood sample processing and the TGF-β 1 analysis kits. The results suggest that plasma TGF-β 1 level in the second trimester was significantly lower in the PE group than in the normal pregnancy group, but was significantly higher in the PE group during the third trimester.

Conclusions

The current results support that the TGF-β 1 869 T>C polymorphism was associated with the risk of PE. However, the number of eligible studies is small and more studies are needed to clarify whether this association can be detected on larger sample sizes and different populations. Owing to the heterogeneity between studies, no conclusion on the association between plasma TGF-β 1 level and PE risk can be drawn from this review. Further studies about the TGF-β 1 levels at different stages of pregnancy and the development of TGF-β 1 assay methodology are required to reveal the role of TGF-β 1 in the pathological development of PE.     

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

Background/Objective

Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.

Methodology/Principal Findings

In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.

Conclusion/Significance

Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.     

TGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells

Background

Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E₂) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.

Methodology/Principal Findings

Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E₂ as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E₂ secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E₂ and TGF-β1, and E₂ treatment in turn restored the inhibition of TGF-β1 on GJIC.

Conclusions

Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E₂ secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.     

Inhibition of the K+ Channel KCa3.1 Reduces TGF-β1-Induced Premature Senescence,Myofibroblast Phenotype Transition and Proliferation of Mesangial Cells

Objective

K_Ca3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K_Ca3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

Methods & Materials

Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml) and TGF-β1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K_Ca3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K_Ca3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05.

Results

K_ca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G₀-G₁ phase and the expression of K_ca3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01).

Conclusion

Targeted disruption of K_Ca3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.     

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Transforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.

Methods

Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.

Results

TGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.

Conclusions

RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.     

Activated Effects of Parathyroid Hormone-Related Protein on Human Hepatic Stellate Cells

Background & Aims

After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP) has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-β1), which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC) and LX-2 cell lines.

Methods

TGF-β1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR) after HSCs or LX-2 cells were treated with PTHrP(1–36) or TGF-β1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGF-β1 secretion was measured with enzyme-linked immunosorbent assay (ELISA) of HSC cell culture media.

Results

In cultured human HSCs, mRNA and protein levels of α-SMA, collagen I, MMP-2, and TGF-β1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGF-β1 secretion in cultured media from HSCs.

Conclusions

PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-β1, highlighting the potential effects of PTHrP in the liver.     

Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β

Background

Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease.

Methods

C57BL/6 wild-type (WT) and SP-D−/− mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D.

Results

Dp-challenged SP-D−/− mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D−/− mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D−/− mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D−/− mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D−/− mice.

Conclusion

These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0143-9) contains supplementary material, which is available to authorized users.     

Vitamin D Deficiency Aggravates Chronic Kidney Disease Progression after Ischemic Acute Kidney Injury

Background

Despite a significant improvement in the management of chronic kidney disease (CKD), its incidence and prevalence has been increasing over the years. Progressive renal fibrosis is present in CKD and involves the participation of several cytokines, including Transforming growth factor-β1 (TGF-β1). Besides cardiovascular diseases and infections, several studies show that Vitamin D status has been considered as a non-traditional risk factor for the progression of CKD. Given the importance of vitamin D in the maintenance of essential physiological functions, we studied the events involved in the chronic kidney disease progression in rats submitted to ischemia/reperfusion injury under vitamin D deficiency (VDD).

Methods

Rats were randomized into four groups: Control; VDD; ischemia/reperfusion injury (IRI); and VDD+IRI. At the 62 day after sham or IRI surgery, we measured inulin clearance, biochemical variables and hemodynamic parameters. In kidney tissue, we performed immunoblotting to quantify expression of Klotho, TGF-β, and vitamin D receptor (VDR); gene expression to evaluate renin, angiotensinogen, and angiotensin-converting enzyme; and immunohistochemical staining for ED1 (macrophages), type IV collagen, fibronectin, vimentin, and α-smooth mucle actin. Histomorphometric studies were performed to evaluate fractional interstitial area.

Results

IRI animals presented renal hypertrophy, increased levels of mean blood pressure and plasma PTH. Furthermore, expansion of the interstitial area, increased infiltration of ED1 cells, increased expression of collagen IV, fibronectin, vimentin and α-actin, and reduced expression of Klotho protein were observed. VDD deficiency contributed to increased levels of plasma PTH as well as for important chronic tubulointerstitial changes (fibrosis, inflammatory infiltration, tubular dilation and atrophy), increased expression of TGF-β1 and decreased expression of VDR and Klotho protein observed in VDD+IRI animals.

Conclusion

Through inflammatory pathways and involvement of TGF-β1 growth factor, VDD could be considered as an aggravating factor for tubulointerstitial damage and fibrosis progression following acute kidney injury induced by ischemia/reperfusion.     

Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

Background

We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-β1).

Methodology/Principal Findings

VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-β1 was able to reverse this effect. ASA (2 mM) induced TGF-β1 secretion; however it was unable to induce Smad activation. ASA increased p38^MAPK phosphorylation in a TGF-β1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-β1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged.

Conclusions/Significance

In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-β1 and p38^MAPK. Pre-surgical serum levels of TGF- β1 from bypass patients who took aspirin at antiplatelet doses did not change.