Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli

Aims/hypothesis We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-β1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells. Methods THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/−THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA. Results In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF. Conclusions/interpretation Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target. N. A. Wahab and L. Schaefer made an equal contribution to the work reported in this paper     

Kidney-targeting Smad7 gene transfer inhibits renal TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways, and improves diabetic nephropathy in mice

Aims/hypothesis  

The TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes.     

NF-κB Activation Precedes Increases in mRNA Encoding Neurokinin-1 Receptor,Proinflammatory Cytokines,and Adhesion Molecules in Dextran Sulfate Sodium–Induced Colitis in Rats

Nuclear factor kappa B (NF-κ B) plays a key role in initiating inflammation associated with colitis. A systematic study was conducted in the rat DSS colitis model to determine the temporal relationship between NF-κ B activation and expression of substance P (SP), neurokinin-1 receptor (NK-1R), proinflammatory cytokines, and adhesion molecules. Rats were given 5% DSS in their water and sacrificed daily for 6 days. Colon tissue was collected for assessment of histological changes, NF-κ B activation, myeloperoxidase (MPO) activity, and expression of NK-1R, SP, TNFα, IL-1β, VCAM-1, ICAM-1, E-selectin, CINC-1, MIP-1α, and iNOS. NF-κ B activation increased, biphasically, on Day 1 and again on Days 4–6. The mRNA levels for ICAM-1, CINC-1, IL-1β, TNFα, VCAM-1, and NK-1R rose significantly (P< 0.05) by 2–4 days. Increased iNOS mRNA levels, MPO activity, and mucosal damage occurred on Day 6. These data demonstrate that NF-κ B activation substantially precedes the onset of physical disease signs and active inflammation.     

NF-κB-dependent synergistic regulation of CXCL10 gene expression by IL-1β and IFN-γ in human intestinal epithelial cell lines

Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in intestinal epithelial cell lines. Materials and methods CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1β, TNF-α, IFN-γ alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-κB2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-κB were also performed. Results Real-time PCRs and ELISA experiments revealed that IL-1β was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-γ had a delayed kinetics. There was a strong synergistic effect of either TNF-α or IL-1β with IFN-γ both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-κB inhibitor and transfection experiments with a NF-κB-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1β and its synergism with IFN-γ is NF-κB dependent. Conclusion These data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-κB pathway, IL-1β alone and/or in synergism with IFN-γ may play a major role in the induction of CXCL10.     

Celecoxib inhibits production of MMP and NO via down-regulation of NF-κB and JNK in a PGE2 independent manner in human articular chondrocytes

The purpose of this study was to examine the effects of celecoxib on matrix metalloproteinases (MMP-1 and MMP-3), nitric oxide (NO), and the phosphorylation of nuclear factor-κB (NF-κB) and three mitogen-activated protein kinases (MAPKs), (p38, JNK and ERK) in human articular chondrocytes from normal, osteoarthritis, and rheumatoid arthritis cartilages. Celecoxib at 100 nM reduced the IL-1β-induced productions of MMP-1, MMP-3, iNOS, and NO, whereas indomethacin at 100 nM showed no effect. The additional stimulation of prostaglandin E2 (PGE2) failed to restore those productions, while the production of PGE2 were reduced by 1 and 10 μM but not 100 nM of celecoxib. The inhibitors of NF-κB, JNK and p38, but not ERK, decreased IL-1β-enhanced MMP-1, MMP-3 and NO production, respectively, and 100 nM celecoxib down-regulated the phosphorylation of NF-κB and JNK but has no effect on either p38 or ERK. Celecoxib has inhibitory effects on MMP-1, MMP-3 and NO productions, suggesting the protective roles directly on articular chondrocytes. Despite the COX-2 selectivity, celecoxib affects those productions via not PGE2 but NF-κB and JNK MAPK.     

Tumor necrosis factor receptor 2 signaling limits β-adrenergic receptor-mediated cardiac hypertrophy in vivo

The in vivo role of TNF signaling in the genesis of β-adrenergic receptor (β-AR)-mediated cardiac hypertrophy is unknown. Wild-type (WT), TNF receptor 1 (TNFR1)-/- and TNFR2-/- mice were given isoproterenol (ISO, 12.5 μg/kg/h) or saline (SAL) for 1 or 7 days. In WT mice, 7 days of ISO yielded chamber/myocyte hypertrophy and hyperdynamic function without hypertension or fibrosis. WT ISO hearts exhibited an early (1 day) pro-inflammatory response with significant (p < 0.05) activation of nuclear factor (NF)-κB and activator protein 1 (AP-1) and upregulation of TNF, interleukin (IL)-1β and IL-6, inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1), together with increased anti-inflammatory IL-10. This response diminished markedly by 7 days. As compared with WT ISO mice, TNFR1-/- ISO mice exhibited significantly (p < 0.05) less NF-κB and AP-1 activation, less IL-1β, TNF, iNOS and MCP-1 upregulation, but greater IL-10 at 1 day. However, there were no differences in hypertrophy or contractility at 7 days. In contrast, TNFR2-/- ISO mice exhibited augmented NF-κB and AP-1 activation, increased IL-1β and diminished IL-10 expression at 1 day, and significant exaggeration of hypertrophy and less contractile augmentation at 7 days. Moreover, TNFR2-/- mice exposed to tenfold higher ISO doses displayed significant mortality. TNF signaling contributes to β-AR-mediated cardiac remodeling in vivo in a receptor-specific manner. Unopposed TNFR1 activation is pro-inflammatory, pro-hypertrophic and promotes functional decline. However, co-activation of TNFR2 during β-AR stress is anti-inflammatory and counterbalances these deleterious effects. TNF modulatory strategies that maintain TNFR2 signaling may help prevent the detrimental long-term effects of β-AR stimulation in the heart.     

IL-1β-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

Aims/hypothesis IL-1β released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1β-induced pro-apoptotic signalling in beta cells. Pancreatic beta cells express NCAM, but its biological effects in these cells are unclear. The aim of this study was to investigate whether there is cross-talk between NCAM signalling and cytokine-induced pro-apoptotic signalling.Materials and methods Western blotting was used to investigate levels of NCAM and inducible nitric oxide synthase, phosphorylation of Src and MAPKs, and cleavage of caspase-3. MAPK activity was investigated with an in vitro kinase assay. Apoptosis was detected by cleaved caspase-3 and a Cell Death Detection ELISA^plus assay. NCAM-induced fibroblast growth factor receptor (FGFR) activation was investigated in NCAM^−/− Trex293 cells where FGFR phosphorylation was measured by Western blotting after NCAM transfection.Results Pre-exposure of INS-1E cells to the FGFR-inhibitor SU5402, but not to the Src-inhibitor PP2, dose-dependently inhibited IL-1β-mediated MAPK activity. A synthetic peptide, C3d, reported to bind NCAM, did not activate MAPK or Akt as reported in neurons but inhibited IL-1β-induced MAPK activity, thereby mimicking the effect of SU5402. Furthermore, C3d inhibited NCAM-induced FGFR phosphorylation and apoptosis induced by IL-1β plus IFN-γ, but did not affect IL-1β-induced NF-κB signalling.Conclusions/interpretation We suggest that NCAM signalling through FGFR is required for efficient IL-1β pro-apoptotic signalling by facilitating IL-1β-induced MAPK activation downstream of the NF-κB-MAPK branching point. Further, these data identify a novel function of C3d as an inhibitor of NCAM-induced FGFR activity and of IL-1β-induced MAPK activation in beta cells.     

Inhibition of Immune-Mediated Concanavalin A-Induced Liver Damage by Free-Radical Scavengers

Background/Aims The aims of the present study were to elucidate whether oxidative stress has a role in Con A-induced hepatitis and to examine if antioxidants may protect against liver damage in this model. Methods Hepatitis was induced in Balb/c mice by administration of Con A (18 mg/kg) to the tail vein. Liver enzymes and histology were determined 24 h after Con A injection. Tumor necrosis factor alpha (TNFα) and interleukin-10 (IL-10) levels were assayed 2 h after Con A injection. Hepatic malondialdehyde levels were measured at 1, 3, 8, 12, 18, and 24 h after Con A injection in order to examine the timing of free-radicals formation. Nuclear factor kappa B (NF-κβ) activation was determined by electrophoresis mobility shift assay (EMSA) 1 and 2 h after Con A injection. In separate experiments, mice were pretreated with either dimethylsulfoxide or dimethylthiourea before Con A inoculation. The antioxidant and NF-κβ inhibitor pyrrolidine dithiocarbamate (PDTC) was used as positive control. Results Hepatic malondialdehyde levels increased 12, 18, and 24 h after Con A inoculation but not earlier. Serum levels of liver enzymes and TNFα, hepatic malondialdehyde, and protein carbonyls and the histologic necroinflammatory score were significantly reduced in the antioxidants-treated mice, while IL-10 levels were increased. Dimethylsulfoxide, dimethylthiourea, and PDTC inhibited oxidative stress, but only PDTC inhibited Con A-induced NF-κB activation. Conclusions Reactive oxygen species play a role in immune-mediated Con A-induced hepatitis probably secondary to immune-mediated liver damage. Scavenging of reactive oxygen species by antioxidants prevents hepatitis independently of NF-κB inhibition and may be a new therapeutic target in this experimental model.     

Modification of TGF-β1 signaling pathway during NB4 cells differentiation by all-trans retinoid acid induction

The aim of the study was to present the possible mechanisms of transforming growth factor beta 1(TGF-β1) signal pathway during cell differentiation by studying the expression levels of six components of TGF-β1 pathway (TGF-β1, two TGF-β1 receptors and three Smad proteins). The morphology change, the CD11 expression levels, and the mRNA and protein expression levels of TGF-β1, TGF-β ReceptorI (TβRI), TGF-β ReceptorII (TβRII), Smad2, Smad4 and Smad7 were assessed by exposing NB4 cells to all-trans retinoid acid (ATRA) using Wright’s stain, flow cytometry, real-time PCR assay and Western blot analysis. The mRNA and protein expression levels of all six components increased during NB4 cells differentiation induced by ATRA. They were most significantly increased after 24–72 h individually when cells were induced by ATRA (the mRNA and protein expression levels of TGF-β1, TβRI, TβRII and Smad2 reached their peaks at 48 and 48 h individually after the treatment, Smad4 at 48 and 72 h, and Smad7 at 72 and 72 h). The change in mRNA expression levels was earlier than the change in the same gene controlling protein. These results indicate that the upregulation of TGF-β1 pathway plays an important role in NB4 cells differentiation induced by ATRA.     

Transforming growth factor beta-1 in rectal tumour,mucosa and plasma in relation to radiotherapy and clinical outcome in rectal cancer patients

Background Rectal cancer patients are treated with surgery and sometimes radiotherapy. Transforming growth factor-β1 (TGF-β1) acts both as an inhibitor of tumour growth and as a promoter of tumour progression. The aim of this study was to determine the levels of TGF-β1 in tumour tissue, adjacent mucosa and plasma in rectal cancer patients and relate these to the effect of radiotherapy and clinical outcome. Materials and methods One hundred and ten patients scheduled for rectal cancer surgery were included, 49% received pre-operative radiotherapy three-field treatment 5 × 5 Gy. Blood samples and biopsies were taken during surgery and later assayed with enzyme-linked immunosorbent assay for total TGF-β1 and active TGF-β1. Patients were then followed for 3 years. Results Total and active TGF-β1 was higher in tumour tissue compared with rectal mucosa (p < 0.0001). Active TGF-β1 in tumour tissue and rectal mucosa was lower in the irradiated group (p = 0.007; p < 0.0001). Total TGF-β1 was higher in patients with metastases at primary diagnosis (p = 0.005) compared to patients without. In patients who later developed metastases, the levels of active TGF-β1 in plasma were lower (p = 0.004). Local recurrence was associated with lower levels of total TGF-β1 in the rectal mucosa (p = 0.038). Conclusions Higher levels of total TGF-β1 in tumour tissue at surgery may be indicative of distant metastases, and low levels of active TGF-β1 in plasma may indicate a risk of developing secondary metastases. Lower levels of total TGF-β1 in rectal mucosa may influence risk of local recurrence. Measurement of TGF-β1 in rectal cancer patients may be of clinical use in the future.     

Effect of Angiotensin-converting Enzyme Inhibition on Experimental Hepatic Fibrogenesis

The renin-angiotensin system is suggested to be important in liver fibrogenesis. It induces hepatic stellate cell proliferation and up-regulates transforming growth factor beta-1 (TGF-β1) expression. Matrix metalloproteinase-2 (MMP-2) is involved in extracellular matrix remodelling. Fibrosis, a consequence of most chronic liver diseases, may be the result of a disturbed balance between fibrogenesis and fibrolysis. The aim of this study was to investigate the effect of enalapril on liver fibrogenesis induced in rats by bile-duct ligation. Forty-seven rats were divided into two groups: bile-duct ligated (BDL) (n = 24) and BDL + enalapril (n = 23). Fibrosis was evaluated by the Knodell scoring system, and TGF-β1 and MMP-2 were assessed with immunohistochemistry at the second, fourth and sixth weeks after bile-duct ligation. In the BDL group, TGF-β1 increased by the second week and this increase continued through weeks 4 and 6. In the BDL + enalapril group, TGF-β1 was significantly lower than the other group (P < 0.05). MMP-2 progressively decreased after week 2 in the BDL group. In the BDL + enalapril group, MMP-2 was significantly higher than the BDL group at the fourth and sixth weeks. These results suggest that enalapril reduces the liver tissue TGF-β1 and has an ameliorating effect on the fibrosis markers TGF-β1 and MMP-2.     

Amelioration of glucose tolerance by hepatic inhibition of nuclear factor κB in db/db mice

Aims/hypothesis Recent studies have identified the involvement of inhibitor IκB kinase (IKK) in the pathogenesis of insulin resistance. To investigate the mechanism involved, we examined the role of nuclear factor κB (NF-κB), the distal target of IKK, in hepatic glucose metabolism. Methods To inhibit NF-κB activity, db/db mice were infected with adenovirus expressing the IκBα super-repressor. Results The IκBα super-repressor adenovirus infection caused a moderate reduction of NF-κB activity in liver. The treatment was associated with improved glucose tolerance, reduction in the serum insulin level, and increased hepatic triacylglycerol and glycogen contents, but had no effect on insulin-stimulated phosphorylation of Akt. On the other hand, quantification of mRNA in the liver revealed marked reduction of expression of gluconeogenic genes, such as those encoding phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, concurrent with reduced expression of gene encoding peroxisome proliferator-activated receptor gamma coactivator-1α (PPARGC1A, also known as PGC-1α). Furthermore, the production of super-repressor IκBα suppressed the increase in blood glucose level after pyruvate injection. Conclusions/interpretation Our results indicate that moderate inhibition of NF-κB improved glucose tolerance through decreased gluconeogenesis associated with reduced PGC-1α gene expression in db/db mice, and suggest that inhibition of NF-κB activity in liver is a potentially suitable strategy for the normalisation of blood glucose concentration in type 2 diabetes. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users.     

LR-90 prevents dyslipidaemia and diabetic nephropathy in the Zucker diabetic fatty rat

Aims/hypothesis Previous studies have shown that LR-90, a new inhibitor of AGE formation, prevented the development of experimental type 1 diabetic nephropathy. In this study, we examined the effects of LR-90 in the Zucker diabetic fatty (ZDF) rat, a model of type 2 diabetes and metabolic syndrome, and investigated the mechanisms by which it may protect against renal injury. Methods Male ZDF rats were treated without or with LR-90 from age 13 to 40 weeks. Metabolic and kidney functions and renal histology were evaluated. AGE accumulation and the production of the receptor for AGE (AGER) were measured. Profibrotic growth factors, extracellular matrix proteins and intracellular signalling pathways associated with glomerular and tubular damage were also analysed. Results LR-90 dramatically reduced plasma lipids in ZDF rats, with only modest effects on hyperglycaemia. Renal AGE, AGER and lipid peroxidation were all attenuated by LR-90. LR-90 significantly retarded the increase in albuminuria and proteinuria. This was associated with reduction in glomerulosclerosis and tubulointerstitial fibrosis, concomitant with marked inhibition of renal overproduction of TGF-β1, connective tissue growth factor, fibronectin and collagen IV. Additionally, LR-90 downregulated the activation of key mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) in the renal cortex. Conclusions/interpretation These results support our earlier studies on the renoprotective effects of LR-90 on type 1 diabetic nephropathy and provide further evidence that LR-90, an AGE inhibitor with pleiotrophic effects, may also be beneficial for the prevention of type 2 diabetic nephropathy, where multiple risk factors, such as hyperglycaemia, dyslipidaemia, obesity, insulin resistance and hypertension, contribute to renal injury. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

EDB fibronectin and angiogenesis – a novel mechanistic pathway

Extra domain-B containing fibronectin (EDB⁺ FN), a recently proposed marker of angiogenesis, has been shown to be expressed in a number of human cancers and in ocular neovascularization in patients with proliferative diabetic retinopathy. To gain molecular understanding of the functional significance of EDB⁺ FN, we have investigated possible regulatory mechanisms of induction and its role in endothelial cell proliferation and angiogenesis. Human vascular endothelial cells were cultured in high levels of glucose, and fibrogenic growth factors, transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). Our results show that high glucose levels, TGF-β1, and ET-1 upregulated EDB⁺ FN expression. Treatment of cells exposed to high glucose with TGF-β1 neutralizing antibody and ET receptor antagonist prevented high glucose-induced EDB⁺ FN expression. In order to elucidate the functional significance of EDB⁺ FN upregulation, cells were subjected to in vitro proliferation and angiogenesis assays following EDB peptide treatment and specific EDB⁺ FN gene silencing. Our results show that exposure of cells to EDB peptide increased vascular endothelial growth factor (VEGF) expression, endothelial proliferation, and tube formation. Furthermore, specific EDB⁺ FN gene silencing prevented both basal and high glucose-induced VEGF expression and reduced the proliferative capacity of endothelial cells. In conclusion, these results indicate that EDB⁺ FN is involved in endothelial cell proliferation and vascular morphogenesis, findings which may provide novel avenues for the development of anti-angiogenic therapies.     

Insulin-like growth factor binding protein-3 and-5 are regulated by transforming growth factor-β and retinoic acid in the human prostate adenocarcinoma cell line PC-3

The family of insulin-like growth factor binding proteins (IGFBPs) can affect cell proliferation by modulating the availability and bioactivity of insulin-like growth factors (IGFs), or by mechanisms independent of IGFs. To understand better the role(s) of IGFBPs in prostate growth and malignancy, we examined the regulation of IGFBPs in PC-3 cells, a human prostatic adenocarcinoma epithelial cell line that is androgeninsensitive. Both transforming growth factor-β (TGF-β) and retinoic acid (RA), known inhibitors of cellular proliferation, significantly changed the IGFBP profile in PC-3 cells. In cells that were treated with transforming growth factor β-2 (TGF-β2) (0.5–10 ng/mL), IGFBP-3, and IGFBP-5 protein and mRNA increased in a time-and dose-dependent manner. At 10 ng/mL TGF-β, IGFBP-3, and IGFBP-5 protein concentrations were 14- and 9-fold, respectively, over that of controls. Cells treated with Ra (0–1 μM) also showed a time- and dose-dependent increase in IGFBP-3 protein and mRNA levels. However, in contrast to TGF-β2, high concentrations of RA (1 μM) negatively regulated IGFBP-5 expression, with IGFBP-5 mRNA levels downregulated to 20% of that of the control, and protein levels were decreased by 50%. Since both TGF-β and RA increased IGFBP-3 expression and both are known to inhibit prostate cell growth, we speculate that the inhibition of growth is mediated, at least in part, by IGFBP-3.