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Paraquat (PQ) causes fatal poisoning that leads to systemic multiple organ fibrosis, and transforming growth factor (TGF)-β1 plays a critical role in this process. In this study, we aimed to investigate the effects of AZ12601011 (a small molecular inhibitor of TGFβRI) on PQ-induced multiple organ fibrosis. We established a mouse model of PQ in vivo and used PQ-treated lung epithelial cell (A549) and renal tubular epithelial cells (TECs) in vitro. Haematoxylin-eosin and Masson staining revealed that AZ12601011 ameliorated pulmonary, hepatic, and renal fibrosis, consistent with the decrease in the levels of fibrotic indicators, alpha-smooth muscle actin (α-SMA) and collagen-1, in the lungs and kidneys of PQ-treated mice. In vitro data showed that AZ12601011 suppressed the induction of α-SMA and collagen-1 in PQ-treated A549 cells and TECs. In addition, AZ12601011 inhibited the release of inflammatory factors, interleukin (IL)-1β, IL-6, and tumour necrosis factor-α. Mechanistically, TGF-β and TGFβRI levels were significantly upregulated in the lungs and kidneys of PQ-treated mice. Cellular thermal shift assay and western blotting revealed that AZ12601011 directly bound with TGFβRI and blocked the activation of Smad3 downstream. In conclusion, our findings revealed that AZ12601011 attenuated PQ-induced multiple organ fibrosis by blocking the TGF-β/Smad3 signalling pathway, suggesting its potential for PQ poisoning treatment., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

The cyclin D-CDK4/6 complex has two distinct functions. Its kinase-dependent role involves its ability to act as serine/threonine kinase, responsible for phosphorylation of substrates required for cell cycle transitions, while its kinase-independent function involves its ability to act as a reservoir for p27 Kip1 . This association sequesters p27 from cyclin E-CDK2 complexes, allowing them to remain active. The aim of this current study is two-fold: to understand the contribution of the kinase-dependent and kinase-independent functions of CDK4 and CDK6 in epithelial cells and to directly compare CDK4 and CDK6 in a simple model system, TGF-β treatment, where arrest is initiated by the expression of p15 Ink4b . Cells that overexpressed a catalytically inactive, p15-insensitive CDK6 variant (p27 sequestration only mutant) were able to overcome TGF-β-mediated arrest by maintaining CDK2 activity, while cells expressing the identical mutations in CDK4 were not. This result can be partially explained by the presence of a previously unidentified cyclin D-CDK6 dimer, which serves as a sink for free p27 during TGF-β treatment, enabling CDK2 to remain inhibitor free. The use of the TGF-β model system and the characterization of CDK pool dynamics and p27 switching is relevant to the CDK4/6 specific inhibitors, such as palbociclib, whose mechanism of action may resemble that of p15.

Abnormal activation of transforming growth factor (TGF)-β is a common cause of fibroblast activation and fibrosis. In bleomycin (BLM)-induced lung fibrosis, the marked expression of phospho-Src homology-2 domain-containing phosphatase (SHP) 2, phospho-signal transducer and activator of transcription (STAT) 3, and suppressor of cytokine signaling (SOCS) 3 was highly associated with pulmonary parenchymal lesions and collagen deposition. Human pulmonary fibroblasts differentiated into myofibroblasts exhibited activation of SHP2, SOCS3, protein inhibitor of activated STAT1, STAT3, interleukin (IL)-6, and IL-10. The significant retardation of interferon (IFN)-γ signaling in myofibroblasts was revealed by the decreased expression of phospho-STAT1, IFN-γ-associated genes, and IFN-γ-inducible protein (IP) 10. Microarray analysis showed an induction of fibrotic genes in TGF-β1-differentiated myofibroblasts, whereas IFN-γ-regulated anti-fibrotic genes were suppressed. Interestingly, BIBF 1120 treatment effectively inhibited both STAT3 and SHP2 phosphorylation in TGF-β1-differentiated myofibroblasts and BLM fibrotic lung tissues, which was accompanied by suppression of fibroblast-myofibroblast transition. Moreover, the combined treatment of BIBF 1120 plus IFN-γ or SHP2 inhibitor PHPS1 plus IFN-γ markedly reduced TGF-β1-induced α-smooth muscle actin and further ameliorated BLM lung fibrosis. Accordingly, myofibroblasts were hyporesponsiveness to IFN-γ, while blockade of SHP2 contributed to the anti-fibrotic efficacy of IFN-γ., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Renal fibrosis is one of the characteristic features of chronic kidney disease (CKD). Fibrotic change not only impairs the filtration function of the kidney but is also recognized as a marker of end-stage renal disease (ESRD). The epithelial to mesenchymal transition (EMT) is known to play a role in embryonic development and organ formation, but it is getting much attention for its pathological role in the invasion and metastasis of carcinoma. Recently, it has also been reported that EMT plays a role in the formation of fibrosis during chronic inflammation. EMT contribute to the development of the fibrosis in CKD. Moreover, glomerular podocytes and tubular epithelial cells can also undergo mesenchymal transition in CKD. Hesperetin is a flavonoid present in citrus and is well known for its antioxidant and anti-inflammatory properties. In this study, we investigated the effects of hesperetin on the EMT-elicited podocytes. First, we generated an EMT model by treating transforming growth factor (TGF)-β1, a potent inducer of EMT to the podocytes. TGF-β1 decreased the expression of epithelial markers such as nephrin, zonula occludens-1 (ZO-1), while it increased the mesenchymal markers, including fibronectin (FN), vimentin, and α-smooth muscle actin (α-SMA) in the podocytes. Hesperetin suppressed EMT-like changes elicited by TGF-β1. Interestingly, hesperetin did not interfere with the Smad signaling-the classical TGF-β signaling-pathway, which was confirmed by the experiment with smad 2/3 -/- podocytes. Instead, hesperetin suppressed EMT-like changes by inhibiting the mTOR pathway-one of the alternative TGF-β signaling pathways. In conclusion, hesperetin has a protective effect on the TGF-β1 elicited EMT-like changes of podocytes through regulation of mTOR pathway. It could be a good candidate for the suppression of kidney fibrosis in various CKD., Competing Interests: Declaration of competing interest The authors claim no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Small-molecule inhibitors of transforming growth factor beta receptor 1 (TGFβRI) have a history of significant class-based toxicities (eg, cardiac valvulopathy) in preclinical species that have limited their development as new medicines. Nevertheless, some TGFβRI inhibitors have entered into clinical trials using intermittent-dosing schedules and exposure limits in an attempt to avoid these toxicities. This report describes the toxicity profile of the small-molecule TGFβRI inhibitor, BMS-986260, in rats and dogs. Daily oral dosing for 10 days resulted in valvulopathy and/or aortic pathology at systemic exposures that would have been targeted clinically, preventing further development with this dosing schedule. These toxicities were not observed in either species in 1-month studies using the same doses on an intermittent-dosing schedule of 3 days on and 4 days off (QDx3 once weekly). Subsequently, 3-month studies were conducted (QDx3 once weekly), and while there were no cardiovascular findings in dogs, valvulopathy and mortality occurred early in rats. The only difference compared to the 1-month study was that the rats in the 3-month study were 2 weeks younger at the start of dosing. Therefore, a follow-up 1-month study was conducted to evaluate whether the age of rats influences sensitivity to target-mediated toxicity. Using the same dosing schedule and similar doses as in the 3-month study, there was no difference in the toxicity of BMS-986260 in young (8 weeks) or adult (8 months) rats. In summary, an intermittent-dosing schedule mitigated target-based cardiovascular toxicity in dogs but did not prevent valvulopathy in rats, and thus the development of BMS-986260 was terminated., (© 2020 John Wiley & Sons, Ltd.)

In the current work we show that the profibrotic actions of TGF-β are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF-β required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF-β. We further demonstrated that the NAD-dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF-β. Lastly, administration of the GLS1 inhibitor CB-839 attenuated bleomycin-induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF-β-dependent manner., (© 2020 Federation of American Societies for Experimental Biology.)

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible disease characterized by excessive fibroblast to myofibroblast differentiation with limited therapeutic options. Curdione, a sesquiterpene compound extracted from the essential oil of Curcuma aromatica Salisb, has anti-inflammatory and anti-tumor effects. However, the role of curdione in IPF is still unclear., Methods: The effects of curdione were evaluated in a bleomycin (BLM)-induced pulmonary fibrosis mouse model. C57BL/6 mice were treated with BLM on day 0 by intratracheal injection and intraperitoneal administered curdione or vehicle. In vitro study, expression of fibrotic protein was examined and the transforming growth factor (TGF)-β-related signaling was evaluated in human pulmonary fibroblasts (HPFs) treated with curdione following TGF-β1 stimulation., Results: Histological and immunofluorescent examination showed that curdione alleviated BLM-induced lung injury and fibrosis. Specifically, curdione significantly attenuated fibroblast to myofibroblast differentiation in the lung in BLM induced mice. Furthermore, curdione also decreased TGF-β1 induced fibroblast to myofibroblast differentiation in vitro, as evidenced by low expression of α-SMA, collagen 1 and fibronectin in a dose dependent manner. Mechanistically, curdione suppressed the phosphorylation of Smad3 following TGF-β1 treatment, thereby inhibiting fibroblast differentiation., Conclusions: Overall, curdione exerted therapeutic effects against pulmonary fibrosis via attenuating fibroblast to myofibroblast differentiation. As curdione had been shown to be safe and well-tolerated in BLM-induced mouse model, curdione might be useful for developing novel therapeutics for IPF.

Peritoneal fibrosis (PF) is an intractable complication of peritoneal dialysis (PD) that leads to peritoneal membrane failure. This study investigated the role of suppression of tumorigenicity (ST)2 in PF using patient samples along with mouse and cell-based models. Baseline dialysate soluble (s)ST2 level in patients measured 1 month after PD initiation was 2063.4 ± 2457.8 pg/mL; patients who switched to haemodialysis had elevated sST2 levels in peritoneal effluent (1576.2 ± 199.9 pg/mL, P = .03), which was associated with PD failure (P = .04). Baseline sST2 showed good performance in predicting PD failure (area under the receiver operating characteristic curve = 0.780, P = .001). In mice with chlorhexidine gluconate-induced PF, ST2 was expressed in fibroblasts and mesothelial cells within submesothelial zones. In primary cultured human peritoneal mesothelial cells (HPMCs), transforming growth factor-β treatment increased ST2, fibronectin, β-galactosidase and Snail protein levels and decreased E-cadherin level. Anti-ST2 antibody administration reversed the up-regulation of ST2 and fibronectin expression; it also reduced fibrosis induced by high glucose (100 mmol/L) in HPMCs. Thus, high ST2 level in dialysate is a marker for fibrosis and inflammation during peritoneal injury, and blocking ST2 may be an effective therapeutic strategy for renal preservation., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: It has been proposed that peroxisome proliferator-activated receptor-γ (PPARγ) agonists might reduce renal fibrosis, however, several studies had contradictory results. Moreover, the possible interaction of TGF-β 1 , PPARγ, and transcription factors in renal fibrosis have not been investigated. We hypothesized that oral pioglitazone treatment would inhibit TGF-β-driven renal fibrosis and its progression, by modulating profibrotic transcription factors in TGF-β 1 transgenic mice., Methods: Male C57Bl/6 J mice (control, CTL, n = 14) and TGF-β overexpressing transgenic mice (TGFβ, n = 14, having elevated plasma TGF-β 1 level) were divided in two sets at 10 weeks of age. Mice in the first set were fed with regular rodent chow (CTL and TGFβ, n = 7/group). Mice in the second set were fed with chow containing pioglitazone (at a dose of 20 mg/kg/day, CTL + Pio and TGFβ+Pio, n = 7/group). After 5 weeks of treatment, blood pressure was assessed and urine samples were collected, and the kidneys were analyzed for histology, mRNA and protein expression., Results: TGF-β 1 induced glomerulosclerosis and tubulointerstitial damage were significantly reduced by pioglitazone. Pioglitazone inhibited renal mRNA expression of all the profibrotic effectors: type-III collagen, TGF-β 1 , CTGF and TIMP-1, and alike transcription factors cFos/cJun and protein expression of EGR-1, and STAT3 protein phosphorylation., Conclusions: Oral administration of PPARγ agonist pioglitazone significantly reduces TGF-β 1 -driven renal fibrosis, via the attenuation of EGR-1, STAT3 and AP-1. This implies that PPARγ agonists might be effective in the treatment of chronic kidney disease patients.

Background: Renal fibrosis is the pathological feature of chronic kidney disease (CKD) which leads to end-stage renal disease (ESRD) and renal failure. Resveratrol [3,5,4'-trihydroxy-trans-stilbene (RSV)] has shown benefits for metabolic diseases and anti-cancer therapy, but its potential risk on renal health has not been fully evaluated., Purpose: To investigate the global effects of RSV on renal fibrosis in human tubular epithelial cell (TEC) line HK-2, and in mice with unilateral ureteral obstruction (UUO)., Methods: A TGF-β-induced in vitro model of epithelial-mesenchymal transition (EMT) in TEC was established. The effects of RSV on cell viability, pro-fibrotic factors, oxidative stress, mitochondria function, and underlying pathway proteins were analyzed. In vivo, the effects of RSV on renal function and fibrosis were assayed in UUO mice., Results: Our results showed that low concentrations of RSV (5-20 μM) decreased TGF-β-induced EMT via Sirt1-dependent deacetylation of Smad3/Smad4 mechanism. By contrast, long-term (72 h) exposure to high concentrations of RSV (≥ 40 μM) promoted EMT in HK-2 cells via mitochondrial oxidative stress and ROCK1-mediated disordered cytoskeleton remodeling. In vivo, low-dose treatment of RSV (≤ 25 mg/kg) partly improved renal function, whereas high-dose treatment of RSV (≥ 50 mg/kg) lost its anti-fibrotic role and even aggravated renal fibrosis. However, mice with UUO were more susceptible to high RSV-induced renal injury than normal mice., Conclusion: Dependent on dose, RSV activated either anti-fibrotic or pro-fibrotic effects in kidneys. The risk of RSV consumption in individuals with impaired kidney function should be carefully considered., (Copyright © 2018. Published by Elsevier GmbH.)

Objective: This study aims to explore the protective effect of cyclosporine on inflammation-induced renal tubular epithelial cells and its potential mechanism., Materials and Methods: Human kidney-2 (HK-2) cells were induced by transforming growth factor-β (TGF-β) for constructing an inflammatory injury model. Cells were then treated with different concentrations of cyclosporine for further investigating the biological functions. Cell viability was detected via cell counting kit-8 assay (CCK-8). The cytotoxicity was detected via lactate dehydrogenase (LDH) release assay. Expression levels of cell damage factors and mammalian target of rapamycin (mTOR) pathway-related genes were detected via polymerase chain reaction (PCR), immunofluorescence and Western blotting, respectively., Results: TGF-β inhibited the viability of HK-2 cells, increased expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and apoptosis-related genes. Cyclosporine treatment greatly reversed the cell damage on HK-2 cells induced by TGF-β. Expression levels of mTOR pathway-related genes were downregulated after cyclosporine treatment., Conclusions: Cyclosporine protects HK-2 cells from inflammatory injury via regulating mTOR pathway.

Pulmonary fibrosis is a progressive fibrotic lung disease of persisting lung injury and ineffective wound repair, with poor prognosis. Epithelial-mesenchymal transition (EMT) of alveolar epithelia cells is an early event in the development of pulmonary fibrosis, and transforming growth factor β (TGF-β) is an acknowledged inducer of EMT. Epidemiological studies demonstrated that serum levels of 25-hydroxy-vitamin D were associated with the presence of fibrosis diseases. We investigated whether vitamin D attenuated TGF-β-induced pro-fibrotic effects through inhibiting EMT in human alveolar epithelia A549 cells. A549 cells were cultured with TGF-β alone or in combination with 1α,25-dihydroxyvitamin D3 (1α,25(OH)₂D₃). TGF-β increased the expression of the mesenchymal markers (N-cadherin and Vimentin), and decreased the expression of epithelial markers (E-cadherin). 1α,25(OH)₂D₃ attenuated these TGF-β-induced alterations. Furthermore, the EMT-related transcription factors (Snail and β-catenin) and the extracellular matrix genes (Collagen I and fibronectin) were inhibited by 1α,25(OH)₂D₃, while the expression of vitamin D receptor (VDR) was elevated. In addition, 1α,25(OH)₂D₃ alleviated the cell migration and the invasion abilities in TGF-β-stimulated A549 cells, determined by the scratch wound healing and transwell assays. Our findings suggested that 1α,25(OH)₂D₃ inhibited the pro-fibrotic phenotype of lung epithelial cells under TGF-β stimulation and provided new clues in the clinical management of pulmonary fibrosis., Competing Interests: The authors declare no conflict of interest.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) governs antioxidant, innate immune and cytoprotective responses and its deregulation is prominent in chronic inflammatory conditions. To examine the hypothesis that Nrf2 might be implicated in systemic sclerosis (SSc), we investigated its expression, activity, and mechanism of action in SSc patient samples and mouse models of fibrosis and evaluated the effects of a novel pharmacologic Nrf2 agonist. We found that both expression and activity of Nrf2 were significantly reduced in SSc patient skin biopsies and showed negative correlation with inflammatory gene expression. In skin fibroblasts, Nrf2 mitigated fibrotic responses by blocking canonical transforming growth factor-β (TGF-β)-Smad signaling, whereas silencing Nrf2 resulted in constitutively elevated collagen synthesis, spontaneous myofibroblast differentiation, and enhanced TGF-ß responses. Bleomycin treatment of Nrf2-null mice resulted in exaggerated fibrosis. In wild-type mice, treatment with a novel pharmacologic Nrf2 agonist 2-trifluoromethyl-2'-methoxychalcone prevented dermal fibrosis induced by TGF-β. These findings are the first to identify Nrf2 as a cell-intrinsic antifibrotic factor with key roles in maintaining extracellular matrix homeostasis and a pathogenic role in SSc. Pharmacologic reactivation of Nrf2, therefore, represents a novel therapeutic strategy toward effective treatment of fibrosis in SSc., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4(th)_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4(th)_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

Background: NEDD9 is one of the Crk-associated substrate (Cas) family proteins that mediate downstream signaling processes including cytoskeletal organization, cell-cycle and tumorigenesis. While NEDD9 plays a crucial role in epithelial-mesenchymal transition (EMT), the functional mechanism underlying NEDD9-mediated EMT in prostate cancer (PCa) remains uncertain., Methods: The expression levels of NEDD9 and its downstream molecules in PC-3, LNCaP, and VCaP cells exposed to transforming growth factor-β (TGF-β) were determined by western blotting. The invasion of these cells with ectopic overexpression of NEDD9 or silencing of NEDD9 expression was measured by transwell invasion assay. Human tissue samples comprising 45 PCa specimens and ten specimens of normal prostatic tissue were used for immunohistochemical (IHC) analysis of NEDD9 expression., Results: Both NEDD9 and its downstream signaling molecules associated with EMT were strongly induced by TGF-β in PCa cells. PC-3 cells with stable overexpression of NEDD9 had a mesenchymal phenotype and significantly enhanced cell invasion, despite their decreased cell proliferation. Knockdown of endogenous NEDD9 expression completely diminished TGF-β-triggered tumor invasion in several PCa cell lines. The IHC data revealed a significant positive correlation between the NEDD9 staining score and tumor aggressiveness (e.g., Gleason grade, serum PSA level). The NEDD9 staining score in primary PCa with bone metastasis was significantly higher than that in PCa without metastasis., Conclusions: NEDD9 may be a key mediator involved in TGF-β-mediated EMT and cell motility in PCa cells and a novel target in the treatment of metastatic PCa and prevention of spread of localized PCa cells to other organs., (© 2014 Wiley Periodicals, Inc.)

Escherichia coli-derived recombinant human (rh)BMP-2 (E.BMP-2) can be used as a bone graft substitute because to its high osteoinductivity, but its toxicity is not well understood. Thus, we report on the toxicity of E.BMP-2 in Sprague-Dawley rats under the condition of repetitive injection for 2 weeks. Randomly selected 10 male and female rats were administered with E.BMP-2 at a dose of 0.05, 0.18 or 0.5 mg/kg as an experimental group. A control group with another 10 rats was given E.BMP-2 carrier. Both E.BMP-2 and E.BMP-2 carrier were administered through intravenous injection for 2 weeks. For toxicokinetics study, 3 male and female rats were randomly selected from each group. During the observation period, general symptom, weight and food intakes were monitored, and ophthalmic and urine tests were performed as well. After the observation period, all animals were subjected to blood test, biochemical analysis and organ-weight measurement. During autopsy, visual inspections and histopathological examinations were done. Toxicokinetics study confirmed systemic exposure of the test material. No death or abnormal clinical sign was found during the injection period. Toxicity changes induced by the injection were not detected in autopsy or the tests for weight, food intakes, ophthalmology, hematology and serum biochemistry. The female groups administered with 0.18 and 0.5 mg/kg (the female 0.18-mg/kg group and the female 0.5-mg/kg group) showed absolute and relative weight loss in ovaries and reduced corpora lutea. It was the expected pharmacologic activity, rather than toxicity. The histopathological test revealed cartilage formation and increased fibroblast around the tail vein, but these were thought to be the result of osteoinductivity of the test material. In the male group with 0.5 mg/kg of E.BMP-2 (the male 0.5-mg/kg group), local appearance of multinucleated cells in lung parenchyma was observed, but it was considered as the natural reaction to remove E.BMP-2, which is a recombinant protein. In toxicokinetics study, systemic exposure (area under the serum concentration-time curve and maximum observed serum concentration) increased as the injection dose was increased in both male and female rats, and no clear difference was noticed between the sexes. Blood drug content did not change during the injection period, but the half-life was shortened as the injection dose was increased. Under the condition of this study, the no observed adverse effects level of E.BMP-2 was over 0.5 mg/kg in both male and female rats.

Introduction: The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA., Methods: The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry., Results: Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining., Conclusions: The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression.

Introduction: Transforming growth factor-beta (TGF-β) is a well-known regulator of fibrosis and inflammation in many tissues. During embryonic development, TGF-β signaling induces expression of the transcription factor scleraxis, which promotes fibroblast proliferation and collagen synthesis in tendons. In skeletal muscle, TGF-β has been shown to induce atrophy and fibrosis, but the effect of TGF-β on muscle contractility and the expression of scleraxis and atrogin-1, an important regulator of muscle atrophy, were not known., Methods: We treated muscles from mice with TGF-β and measured force production, scleraxis, procollagen Iα2, and atrogin-1 protein levels., Results: TGF-β decreased muscle fiber size and dramatically reduced maximum isometric force production. TGF-β also induced scleraxis expression in muscle fibroblasts, and increased procollagen Iα2 and atrogin-1 levels in muscles., Conclusion: These results provide new insight into the effect of TGF-β on muscle contractility and the molecular mechanisms behind TGF-β-mediated muscle atrophy and fibrosis., (Copyright © 2011 Wiley Periodicals, Inc.)

Reactive oxygen species (ROS) have been implicated in hepatic stellate cell activation and liver fibrosis. We previously reported that α-lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) inhibited toxicant-induced inflammation and ROS generation. In the present study, we further examined the effects of LA/DHLA on thioacetamide (TAA)-induced liver fibrosis in rats and the possible underlying mechanisms in hepatic stellate cells in vitro. We found that co-administration of LA to rats chronically treated with TAA inhibited the development of liver cirrhosis, as indicated by reductions in cirrhosis incidence, hepatic fibrosis, and AST/ALT activities. We also found that DHLA inhibited TGF-β/PDGF-stimulated HSC-T6 activation and ROS generation. These effects could be mediated by the MAPK and PI3K/Akt pathways. According to our current results, LA may have a beneficial role in the treatment of chronic liver diseases caused by ongoing hepatic damage., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Study Design: In vivo and in vitro model., Objective: Investigate soft-tissue inflammation caused by rhBMP-2., Summary of Background Data: Although rhBMP-2 produces excellent rates of fusion in the spine, dysphagia and respiratory compromise have occurred when used in the neck. The mechanism of the swelling and inflammatory response has yet to be fully elucidated., Methods: ELISA kits (IL-6, IL-10, TNF-α) were used to measure cytokine levels at different concentrations of rhBMP-2. Absorbable collagen sponges were implanted with or without different concentrations of rhBMP-2 into the backs of rats subcutaneously (SC) and intramuscularly (IM). Magnetic resonance imaging was used to measure inflammation at 3 hours and 2, 4, and 7 days. The inflammatory volumes were measured and compared using MIPAV software. Rats were killed after 7 days and studied., Results: IL-6, IL-10, and TNF-α release was dose-dependent. Soft-tissue edema after rhBMP-2 implantation was also dose-dependent, peaking at 3 hours SC, after SC and IM implantations, and on day 2 IM after IM implantation. All formed a granuloma-type mass after SC insertion. The mass was much larger in the 10 and 20 μg/10 μL (high-concentration) groups. The inflammatory response did not diffuse across physiologic barriers (subcutaneous fascia). Both high-dose groups were associated with encapsulated hematomas and a significant increase in the inflammatory zone., Conclusion: Swelling and inflammation after rhBMP-2 use are dose-dependent. Swelling may be due to direct contact as well as spread in the plane of access. The causes are a robust inflammatory reaction as well as sterile seroma and encapsulated hematoma formation.

Background/aims: Alcoholic liver disease is continuously increasing in developed countries being a leading cause of death worldwide. Chronic ethanol consumption induces oxidative stress by accumulation of reactive oxygen intermediates (ROI) while reducing the cellular antioxidant defense. Induction of heme oxygenase-1 (HO-1) may protect primary human hepatocytes (hHeps) from such damage. Thus, the aim of this study was to investigate the potential of polyphenols to protect hHeps from ethanol-dependent oxidative damage., Methods: hHeps were isolated by collagenase perfusion. ROI and cellular glutathione (GSH) were measured by fluorescent-based assays. Cellular damage was determined by lactate dehydrogenase (LDH) leakage and staining for apoptosis and necrosis. Nuclear translocation of Nrf2 and HO-1 expression were analyzed by Western blot., Results: Ethanol and TGF-β rapidly increase ROI and reduce GSH in hHeps, causing apoptosis with a release of approximately 40% total LDH after 72 h. Similar to incubation with hemin preincubation and co-incubation of cells with nifedipine, verapamil and quercetin significantly reduce oxidative stress and resulting cellular damage, in a dose-dependent manner, by initiating nuclear translocation of Nrf2 which in turn induces HO-1 under the control of p38 and ERK. Blocking of HO-1 activity with ZNPP9 reverses the protective effect of all three substances., Conclusion: Our results suggest that increasing HO-1 activity in hHeps protects them from oxidative stress-dependent damage. As polyphenols have great potential to induce HO-1 expression, they may play an important role for future therapeutic strategies to protect liver from oxidative stress-dependent damage observed during chronic alcohol consumption., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: Trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to suppress TGF-beta-induced fibrogenesis in many nonocular tissues. The authors evaluated TSA cytotoxicity and its antifibrogenic activity on TGF-beta-driven fibrosis in the cornea with the use of in vitro and in vivo models., Methods: Human corneal fibroblasts (HSFs) were used for in vitro studies, and New Zealand White rabbits were used for in vivo studies. Haze in the rabbit cornea was produced with photorefractive keratectomy (PRK) using excimer laser. Trypan blue exclusion and MTT assays evaluated TSA cytotoxicity to the cornea. Density of haze in the rabbit eye was graded with slit lamp biomicroscopy. Real-time PCR, immunoblotting, or immunocytochemistry was used to measure alpha-smooth muscle actin (SMA), fibronectin, and collagen type IV mRNA or protein levels. TUNEL assay was used to detect cell death., Results: TSA concentrations of 250 nM or less were noncytotoxic and did not alter normal HSF morphology or proliferation. TGF-beta1 treatment of HSF significantly increased mRNA and protein levels of SMA (9-fold), fibronectin (2.5-fold), and collagen type IV (2-fold). TSA treatment showed 60% to 75% decreases in TGF-beta1-induced SMA and fibronectin mRNA levels and 1.5- to 3.0-fold decreases in protein levels but had no effect on collagen type IV mRNA or protein levels in vitro. Two-minute topical treatment of TSA on rabbit corneas subjected to -9 D PRK significantly decreased corneal haze in vivo., Conclusions: TSA inhibits TGF-beta1-induced accumulation of extracellular matrix and myofibroblast formation in the human cornea in vitro and markedly decreases haze in rabbit cornea in vivo.

Between 2 and 10 years of age, the developing craniofacial skeleton poses a significant reconstructive challenge. Local autogenous bone is largely unavailable, distant bone grafts are fraught with significant morbidity and limited yield, and alloplastic materials are incompatible with the growing calvarium and facial skeleton. Bone morphogenetic protein (BMP) 2, a member of a class of proteins first noticed in the 1960s to promote bone deposition in soft tissues, offers a potential solution to the bone shortage historically faced by the pediatric craniofacial surgeon. A review of English language literature was conducted from the 1960s to the present.Attention was focused on BMP-2's osteoinductive mechanism, basic science and translational laboratory findings, and multidisciplinary clinical experiences. Bone morphogenetic protein 2 has been embraced by spine surgeons, is gaining in popularity for long-bone repair, and is making its way into the plastic surgery literature. Bone morphogenetic protein 2 may provide a basis for an off-the-shelf tissue-engineered bone construct that is compatible with the growing craniofacial skeleton while free from the morbidities of distant graft harvest. Questions remain, however, regarding the safety and efficacy of this compound in the context of pediatric craniofacial surgery. In an effort to facilitate the clinician's risk-benefit analysis of this emerging technology, we present a primer on the basic science of BMP-2, a discussion of possible morbidities associated with its use, a review of laboratory and clinical trials with this substance to date, and an analysis of strategies to maximize its efficacy in craniofacial surgery.

Purpose: The purpose of this study was to evaluate radiographically the pulpal and periapical response of dogs' teeth after pulpotomy and the use of recombinant human bone morphogenetic protein-7 (rHuBMP-7)., Methods: Pulpotomies were performed in 60 teeth of 6 dogs, and the remaining radicular pulp tissue was capped with the following materials: (a) groups 1 and 5-recombinant human bone morphogenetic protein-7 associated with recombinant human-like collagen; (b) groups 2 and 6-recombinant human-like collagen; (c) groups 3 and 7-calcium hydroxide; and (d) groups 4 and 8-zinc oxide and eugenol cement. After 7 days (groups 1-4) and 70 days (groups 5-8), standardized periapical radiographs were taken and the integrity of the lamina dura, presence of areas of periapical bone rarefaction, internal/external root resorption, and dentin bridge formation were evaluated. The results were analyzed statistically by Fisher's exact test and Bonferroni correction. The radiolucent areas suggestive of periapical lesions associated with the roots were measured in mm2, and the results were compared by Kruskal-Wallis test., Results: In the 7-day period, all specimens in groups 1 to 4 presented intact lamina dura and absence of periapical bone rarefaction, internal/external root resorption or dentin bridge formation. In the 70-day period, no specimen in groups 5, 6, and 8 presented dentin bridge formation. Periapical bone rarefaction areas were observed to be associated with 100%, 60%, and 40% of the roots in group 6, 8, and 5, respectively. The largest lesions were found in group 6, followed by groups 5 and 8 (P<.05). In group 7, there was dentin bridge formation in 60% of the cases and intact lamina dura and no periapical bone rarefaction in all specimens., Conclusion: The use of rHuBMP-7/rHuCollagen as a capping material after pulpotomy did not induce mineralized tissue deposition, leading to the formation of radiographically visible periapical lesions.

Progressive fibrosis is a cause of progressive organ dysfunction. Lack of quantitative in vitro models of fibrosis accounts, at least partially, for the slow progress in developing effective antifibrotic drugs. Here, we report two complementary in vitro models of fibrosis suitable for high-throughput screening. We found that, in mesangial cells and renal fibroblasts grown in eight-well chamber slides, transforming growth factor-beta1 (TGF-beta1) disrupted the cell monolayer and induced cell migration into nodules in a dose-, time- and Smad3-dependent manner. The nodules contained increased interstitial collagens and showed an increased collagen I:IV ratio. Nodules are likely a biological consequence of TGF-beta1-induced matrix overexpression since they were mimicked by addition of collagen I to the cell culture medium. TGF-beta1-induced nodule formation was inhibited by vacuum ionized gas treatment of the plate surface. This blockage was further enhanced by precoating plates with matrix proteins but was prevented, at least in part, by poly-l-lysine (PLL). We have established two cell-based models of TGF-beta-induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL-coated 96-well plates, on which TGF-beta1-induced two-dimensional matrix accumulation, three-dimensional nodule formation, and monolayer disruption can be quantitated either spectrophotometrically or by using a colony counter, respectively. As a proof of principle, chemical inhibitors of Alk5 and the antifibrotic compound tranilast were shown to have inhibitory activities in both assays.

Ethanol treatment causes an increase in expression of TGF-beta1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-beta1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-beta1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-beta1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-beta1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-beta1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-beta1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-beta1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-beta1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease.

Here, we provide first evidence that long-term continuous infusion of highly purified antisense phosphorothioate oligodeoxynucleotides (S-ODN) into brain parenchyma is well tolerated and thus highly suitable for in vivo application. AP 12009 is an S-ODN for the therapy of malignant glioma. It is directed against human transforming growth factor-beta (TGF-beta2) mRNA. In the clinical setting, AP 12009 is administered intratumorally by continuous infusion directly into the brain tumor. In view of this clinical application, the focus of our data is on local toxicology studies in rabbits and monkeys to evaluate the safety of AP 12009. AP 12009 was administered either by intrathecal bolus injection into the subarachnoidal space of the lumbar region of both cynomolgus monkeys and rabbits or by continuous intraparenchymatous infusion directly into the brain tissue of rabbits. Intrathecal bolus administration of 0.1 ml of 500 microM AP 12009 showed neither clinical signs of toxicity nor macroscopically visible or histomorphologic changes. After a 7-day intraparenchymatous continuous infusion of 500 microM AP 12009 at 1 microl/h in rabbits, there was no evidence of toxicity except for local mild to moderate lymphocytic leptomeningoencephalitis. Additionally, AP 12009 showed good tolerability in safety pharmacology as well as in acute toxicity studies and 4-week subchronic toxicity studies in mice, rats, and monkeys. This favorable safety profile proves the suitability of AP 12009 for local administration in brain tumor patients from the point of view of toxicology.

Fibrosis and apoptosis are juxtaposed in pulmonary disorders such as asthma and the interstitial diseases, and transforming growth factor (TGF)-beta(1) has been implicated in the pathogenesis of these responses. However, the in vivo effector functions of TGF-beta(1) in the lung and its roles in the pathogenesis of these responses are not completely understood. In addition, the relationships between apoptosis and other TGF-beta(1)-induced responses have not been defined. To address these issues, we targeted bioactive TGF-beta(1) to the murine lung using a novel externally regulatable, triple transgenic system. TGF-beta(1) produced a transient wave of epithelial apoptosis that was followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast and myocyte hyperplasia, and septal rupture with honeycombing. Studies of these mice highlighted the reversibility of this fibrotic response. They also demonstrated that a null mutation of early growth response gene (Egr)-1 or caspase inhibition blocked TGF-beta(1)-induced apoptosis. Interestingly, both interventions markedly ameliorated TGF-beta(1)-induced fibrosis and alveolar remodeling. These studies illustrate the complex effects of TGF-beta(1) in vivo and define the critical role of Egr-1 in the TGF-beta(1) phenotype. They also demonstrate that Egr-1-mediated apoptosis is a prerequisite for TGF-beta(1)-induced fibrosis and remodeling.

Background: Recent studies have demonstrated that transforming growth factor beta 1 (TGF-beta1) expression is markedly enhanced in invasive ductal pancreatic adenocarcinomas, although the precise role of TGF-beta1 in pancreatic carcinogenesis remains unclear. We analyzed TGF-beta1 expression in pancreatic intraepithelial neoplasias (PanINs) and the effects of chronic TGF-beta1 exposure on conditionally immortalized pancreatic epithelial (IMPE) cells., Methods: Sixty-one PanIN lesions were immunohistochemically stained with a polyclonal rabbit antibody against human TGF-beta1. Growth-inhibitory effects of short-term exposure to TGF-beta1 were examined in IMPE cells. IMPE cells resistant to TGF-beta1 (IMPE-Tr cells) were generated by continuous exposure to 1 ng/mL of TGF-beta1 for more than 50 days. Phenotypic alterations of IMPE-Tr cells were examined by soft agar and Matrigel assay and Western blot analysis. IMPE and IMPE-Tr cells were injected subcutaneously into nude mice for an in vivo tumorigenicity assay., Results: Forty-six percent of PanINs (28/61) were positive for TGF-beta1 expression, whereas all the epithelia of normal pancreatic ducts were negative. TGF-beta1 treatment showed the marked growth-inhibitory effects (>75%) in IMPE cells, whereas its effects were not observed in IMPE-Tr cells. IMPE-Tr cells were more spindle shaped compared with IMPE cells. In soft agar and Matrigel, formations of many colonies were observed in IMPE-Tr cells, but not in IMPE cells. Interestingly, the expression of p21(WAF1/CIP1) was induced by short-term exposure to TGF-beta1 in IMPE cells, whereas the induction was decreased in IMPE-Tr cells. All of the IMPE-Tr cell-injected mice (5/5) had subcutaneous tumors, although no tumor was found in the IMPE cell-injected mice., Conclusions: TGF-beta1 expression in PanINs and neoplastic transformation of IMPE cells by long-term exposure to TGF-beta1 suggest that TGF-beta1 may act as a tumor promoter in the early stage of pancreatic carcinogenesis., (Copyright 2004 Elsevier Inc.)

Purpose: TGFbeta can induce development in lenses of opaque subcapsular fibrotic plaques that have many features of human subcapsular cataracts. To understand further the events associated with the onset and progression of TGFbeta-induced cataract, several different models for anterior subcapsular cataract (ASC) were used and characterized., Methods: Anterior subcapsular plaques were induced in rat lenses cultured with TGFbeta and in transgenic mice overexpressing TGFbeta in the lens. ASC was also examined in lenses of mice haploinsufficient for Pax6, as well as in human biopsy specimens. Immunofluorescence and in situ hybridization labeling were used to examine changes in patterns of gene expression associated with cataract formation in these models., Results: Examination of TGFbeta-induced cataract in transgenic mice established that the subcapsular plaques are composed of a heterogenous cell population: a population of myofibroblastic cells as well as a population of lens-fiber-like cells. Further support for phenotypic change comes from the observation that the cells in these plaques no longer expressed lens epithelial markers, such as Pax6 and Connexin43. Subsequent examination of human biopsy specimens of ASC, as well as lenses from Pax6-deficient mice, showed that the anterior subcapsular plaques in both cases were also composed of a heterogenous population of cells. In contrast, anterior subcapsular plaques that developed in vitro in response to TGFbeta did not have this same cellular heterogeneity, as no fiber-like cells were present., Conclusions: These findings suggest that in vivo, during TGFbeta-induced cataract formation, some lens epithelial cells transform into myofibroblastic cells, whereas others differentiate into fiber cells. As this pathologic change is accompanied by altered expression of genes characteristic of the normal lens epithelial cell phenotype and as lenses from Pax6-deficient mice exhibit development of anterior subcapsular plaques closely resembling those induced by TGFbeta in transgenic mice, the authors propose that a reduction in Pax6 levels may be essential for this pathologic process to progress. Furthermore, it is clear from these in vitro studies that TGFbeta alone cannot reproduce the same morphologic and molecular changes associated with ASC formation in vivo, indicating that additional molecule(s) in the eye are important in this process.

Precision-cut rat lung slices have been employed in combination with an extensive immunohistochemistry of paraffin-embedded slices for monitoring of early pathohistological changes after exposure to CdCl(2)/TGF-beta(1). Three days of CdCl(2) exposure in combination with TGF-beta(1) seem to be sufficient to induce lung injury with alterations similar to changes observed in early lung fibrogenesis: (1) extracellular matrix accumulation and myofibroblast transdifferentiation (Sirius red staining, collagen type IV, alpha-smooth muscle actin), (2) type I cell injury with loss of type I cell antigens (T1alpha antigen, aquaporin-5, RAGE), (3) increased apoptosis of pulmonary cells (active caspase-3, vimentin cleavage product V1 of caspase-9), and (4) activation of microvascular endothelial cells (podocalyxin, caveolin-1). Western blot analysis confirmed the increasing amount of alpha-smooth muscle actin, the loss of T1alpha antigen, and the increase in caveolin-1 immunoreactivity. The explant culture using CdCl(2)/TGF-beta(1) provides a suitable tool for the study of other factors involved in pulmonary pathology including transcription factors, cytokines, and other metabolites involved in early stages of fibrogenesis.

Transforming growth factor-beta2 (TGF-beta2) induces anterior subcapsular cataracts, with a marked increase in cytoskeletal and extracellular matrix proteins, such as alpha-smooth muscle actin (alphaSMA). It has been shown that 17beta-estradiol (E2) can prevent TGF-beta2-induced cataracts in lenses from ovariectomized female rats. The purpose of the current study was to extend this finding by testing whether E2 can prevent TGF-beta2-induced cataracts and inhibit the induction of alphaSMA gene expression in normal male and normal, non-ovariectomized female rats.Sex-specific differences were observed in 17-week-old rat lenses incubated in 0.15 ng ml(-1) TGF-beta2 and in 10(-8)M E2 plus TGF-beta2. TGF-beta2 induced approximately twice as many anterior subcapsular plaques and 1.5 times the level of alphaSMA transcripts in male lenses compared to female lenses. Notably, E2 inhibited plaque formation and the induction of alphaSMA transcripts in female rat lenses but not in male rat lenses. E2 also inhibited the induction of alphaSMA in TGF-beta2-incubated lenses from ovariectomized female rats.E2 prevented lens opacification and the induction of alphaSMA gene expression in female, but not male, lenses. This sex-specific difference may have implications for studies on the therapeutic use of estradiol for treatment of secondary cataract.

Human bone marrow stroma contains pluripotent mesenchymal progenitor cells that can give rise to many mesenchymal lineages, including chondroblasts, adipocytes or osteoblasts. The differentiation of these cells towards a specific lineage is dependent on hormonal and local factors activating specific transcription factors. Attempts have been recently made to identify osteoprogenitor cells in the human bone marrow and to identify the molecular mechanisms responsible for lineage-specific differentiation of human bone marrow stromal cells. Using a clonal pluripotent human bone marrow stromal cell line with tri-potential characteristics, we have provided evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of human bone marrow stromal cells. We have also shown that administration of TGFss that regulates the expression of specific osteoblast and adipocyte transcription factors can promote osteoblast differentiation and inhibit adipocyte conversion of rat marrow stromal cells in vivo. This indicates that the reciprocal relationship between osteoblastogenesis and adipogenesis can be manipulated in vivo in order to improve bone formation. Future studies will have to identify key signals for lineage-specific differentiation of human marrow stromal cells. This may result in the development of therapeutic strategies to promote the differentiation of these cells towards the osteoblast lineage and to inhibit excessive bone marrow adipogenesis associated with aging.

In a previous study, we showed that BALB/c mice demonstrate significant increases in accumulation of airway collagen after 4 weeks of exposure to ovalbumin aerosol. In the current study we examined the response to ovalbumin aerosol of a different strain of mice, C57BL/6, and compared this response to an otherwise isogenic C57BL strain (iNOS(-/-)) in which the gene for inducible nitric oxide synthetase (iNOS) had been knocked out. We hypothesized that C57BL mice, a Th-1-responsive strain, would be relatively resistant to ovalbumin exposure compared with our previous observations in the BALB/c strain, a Th-2 responder. Our results are consistent with this hypothesis, especially with respect to the accumulation of collagen in the airways of the mice exposed to ovalbumin and increased airway reactivity to challenge with methacholine, as measured by the Penh response. Since NO participates in multiple signal transduction pathways, there was no a priori reason to predict whether iNOS(-/-) mice would be more or less susceptible to allergen-induced airway inflammation than their parental wild-type strain. Responses to ovalbumin exposure of the Th-1-responsive C57BL animals were significantly less (or slower) than those we observed with the iNOS(-/-) mice. Significant increases in airway collagen content were seen only after 6 weeks of exposure of the C57BL mice, as contrasted with 4 weeks in the iNOS(-/-) animals. At each time point examined, Penh values for the iNOS(-/-) mice were significantly increased, while no increases were observed with the C57BL strain. Thus, the iNOS(-/-) mice are more susceptible to ovalbumin-induced airway inflammation and fibrosis than the C57BL strain, giving results intermediate between the previous observations in BALB/c mice and our current findings in C57BL animals with the various assays performed. We also asked whether the effects of knocking out the iNOS gene were exerted before or after the release of TGF-beta(1) by eosinophils and other effector cells in the lung. We measured the response of C57BL and iNOS(-/-) mice to direct intratracheal challenge with TGF-beta(1). There was no apparent response of C57BL mice to TGF-beta(1) at 4 or 11 days after TGF-beta(1) challenge, as evaluated by bronchoprovocation testing. On the other hand, the observed Penh values were significantly greater in iNOS(-/-) mice that had also received TGF-beta(1) 4 days previously. These results strongly support the hypothesis that the increased sensitivity of iNOS(-/-) mice to ovalbumin is at least partially dependent on pathways that come into play subsequent to the release of TGF-beta(1) by effector cells in the lungs of mice exposed to ovalbumin aerosol.

Vasoactive peptides are implied in the development of renal sclerosis as evidenced by the efficiency of their antagonists in preventing glomerulosclerosis of experimental and human nephropathies. Genetically engineered models provide a new approach to investigate the mechanisms of the renal profibrotic actions of angiotensin II and endothelin. Overexpression of the human angiotensinogen and renin genes in rats induces renal sclerosis independently of changes in systemic hemodynamics. The same results are observed when the endothelin-1 gene is overexpressed in mice. Transgenic mice harboring the luciferase gene under the control of the collagen I-alpha 2 chain promoter (procol alpha 2[1]) and made hypertensive by induction of nitric oxide (NO) deficiency were used to study the renal profibrotic actions of vasoactive peptides. In this strain of mice, luciferase activity is an early index of renal fibrosis. Luciferase activity was increased in preglomerular arterioles and glomeruli when mice were deficient in NO. The pharmacological blockade of angiotensin II and endothelin prevented the development of renal sclerosis without modifying blood pressure. Moreover, when the endothelin receptor antagonist was administered after the development of renal fibrosis, preformed glomerulosclerosis partially regressed. Acute administration of vasoactive peptides and TGF-beta in transgenic procol alpha 2[1] mice showed that the angiotensin II activation of collagen I gene requires participation and/or cooperation of endothelin and TGF-beta. Recent data suggest that the profibrotic actions of vasoactive peptides also need the activation of EGF receptor, ERK and rho kinase pathways in renal and vascular cells.

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.

Background: The biologic responses to transforming growth factor-beta (TGF-beta) suggest many potential therapeutic applications; however, in the only clinical trial to examine the effect of the systemic administration of a TGF-beta isoform, patients experienced significant but reversible declines in renal function. We studied the effects of administering human recombinant TGF-beta2 to adult mice., Methods: The effect of daily administration of TGF-beta2 on tissue vasoconstriction, tissue levels of endothelin and angiotensin II, tissue hypoxia, and renal fibrosis were examined., Results: Daily administration of TGF-beta2 at 10 or 100 microg/kg caused apparent tissue vasoconstriction that was visualized by vascular casting, with the largest impact seen in the kidney. Tissue levels of endothelin 1 and angiotensin II were significantly elevated in kidneys of treated mice, as was urinary thromboxane beta2. Renal fibrosis was observed in the cortical tubular interstitium and vasculature, particularly at the cortical-medullary junction and medullary vasa recta; however, glomerular sclerosis was not observed. Fibrosis was correlated to focal tissue hypoxia as determined by immunohistochemical detection of tissue bound pimondazole., Conclusion: We conclude that there are significant histopathologic consequences, focused in the kidney, resulting from the daily administration of high doses of human recombinant TGF-beta2, and we propose that selective vascular constriction with consequent tissue hypoxia is a contributing factor.

Purpose: In a previous study, it was determined that TGFbeta induces cataractous changes in the rat lens in vitro. The purpose of the present study was to determine whether the introduction of biologically active TGFbeta into the vitreous stimulates cataractous changes in the rat lens in situ., Methods: TGFbeta was injected into the vitreous of the left eye of anesthetized adult male Wistar rats. The right eye received sterile vehicle as a control. Three to four months after injection, animals were killed, and lenses were enucleated and examined for cataractous changes., Results: All lenses from control eyes remained transparent and maintained normal cellular architecture throughout. In contrast, lenses from TGFbeta-injected eyes displayed cloudiness in the cortex. In some lenses, distinct opacities were also apparent at the equator and extending some distance toward the anterior and posterior poles. Histologically, the opacities corresponded to subcapsular plaques containing aberrant cells and accumulations of extracellular matrix. In addition, cortical fibers in the anterior and posterior of all lenses displayed variable degrees of swelling, and many retained their nuclei. In some regions, the fiber cells appeared to have degenerated to form large homogeneous areas. The cellular architecture of the equator of these lenses was also disrupted and, in the most severe case, no bow zone was apparent with nucleated cells extending to the posterior pole., Conclusion: The introduction of active TGFbeta into the vitreous induced lenses to undergo cataractous changes. In addition to the TGFbeta-induced changes in the epithelium that were reported previously, cataractous changes observed in this study also involved the lens fiber cells and resembled changes observed in human posterior subcapsular and cortical cataracts.

Transforming growth factor-beta1 (TGF-beta1) has been shown to induce apoptosis in normal or transformed hepatocytes. To elucidate the biochemical pathways leading to apoptosis induced by TGF-beta1 in human hepatoma cells (HuH-7), we examined the expression of Bcl-2-related proteins and X-chromosome-linked inhibitor of apoptosis (XIAP), and activation of the caspase cascade following TGF-beta1 treatment. Bcl-xL expression began to decline at 12 hours after TGF-beta1 treatment and progressively decreased to very low levels in a time-dependent manner. Bax expression showed a little change throughout the experiment. On the other hand, activation of caspase-8 was clearly observed at 36 hours after TGF-beta1 treatment, followed by activation of caspase-9, and caspase-3 was activated at 48 hours after treatment at which time apoptosis of HuH-7 cells was observed. TGF-beta1 significantly decreased XIAP expression in HuH-7 cells. Addition of an inhibitor of caspase-8 or caspase-3 (IETD-FMK or DEVD-CHO) markedly inhibited TGF-beta1-induced apoptosis of HuH-7 cells. Fas/Fas ligand (FasL) interactions in HuH-7 cells were not involved in the apoptotic process. Furthermore, epidermal growth factor (EGF) also completely inhibited TGF-beta1-induced apoptosis of HuH-7 cells by inhibiting activation of the caspase cascade. Our results suggested that activation of caspase-3 initiated through caspase-8 activation is involved in the apoptotic process induced by TGF-beta1 in HuH-7 cells. Our results also showed that down-regulation of the expression of Bcl-xL and XIAP by TGF-beta1 may facilitate activation of caspase-3 in these cells.

Transforming growth factor betas (TGF-betas) are a growth factor family with negative autocrine growth functions for most epithelial cells including colon carcinoma cell lines. Both type I (RI) and type II (RII) transmembrane TGF-beta receptors have been shown to be indispensable for TGF-beta-mediated cell growth regulation. Previous studies using different model systems have shown that both overexpression of TGF-beta1 and transfection of antisense TGF-beta1 to reduce TGF-beta1 expression could lead to increased tumorigenicity. These results are seemingly contradictory and suggest that effects of TGF-beta modulation on malignant properties of cancer cells may be contextual. This study addresses this issue using human colon carcinoma cells (CBS and FET) to determine the effects of modulation of the various components of the TGF-beta system on in vitro and in vivo growth properties in two independent isogenic models of colon carcinoma. Cells were stably transfected with a tetracycline-repressible RII expression vector (CBS4-RII), a tetracycline-repressible expression vector containing a truncated RII cDNA lacking the serine/threonine kinase domain (CBS4-deltaRII and FET6-deltaRII), or with a vector containing the TGF-beta1 cDNA (CBS4-beta1S and FET-beta1S). Expression of the truncated RII reduced TGF-beta sensitivity, whereas overexpression of RII increased TGF-beta sensitivity. TGF-beta overexpression did not affect TGF-beta response. In vivo tumorigenicity assays revealed that CBS4-RII cells had lower tumorigenicity than control cells, whereas CBS4-deltaRII and CBS4-beta1S had higher tumorigenicity than controls. The CBS4 cells are poorly tumorigenic in athymic mice, and the wild-type FET6 cells are nontumorigenic. FET6-deltaRII cells formed rapidly growing tumors, and FET-beta1S cells also formed tumors. These data illustrate the paradoxical tumor-promoting and -suppressing effects of TGF-beta signaling activity in two isogenic model systems from human colon carcinomas, thus demonstrating that the effects of modulation of TGF-beta expression or TGF-beta signaling capability affects malignancy in a contextual manner.

Transforming growth factor beta1 (TGF-beta1) has been implicated in formation of astrocyte scars, which prevents axonal regeneration. A coculture system of astrocytes and cerebellar cells was used to investigate possible neurotoxic effects of TGF-beta1. Although not directly neurotoxic, TGF-beta1 was toxic to cerebellar cells in the presence of astrocytes. This toxicity is based on an effect of the cytokine on astrocytes, as conditioned medium from astrocyte cultures treated with TGF-beta1 was more toxic by a similar mechanism. This neurotoxicity was mediated by glutamate present in the culture medium as demonstrated by inhibition by MK-801. Astrocytic ability to metabolise glutamate was compromised by TGF-beta1, as this cytokine increased glutamate concentration. The astrocytes in the coculture system responded to the presence of neurones by secreting neuroprotective interleukin-6, which was partly protective against the TGF-beta1-induced toxicity. In the coculture system, neurones responded to the presence of astrocytes by a reduction in resistance to glutamate toxicity. On addition of TGF-beta1, which compromised astrocytic clearance of glutamate, this reduction in resistance to glutamate toxicity led to a reduction in neuronal survival. These results suggest that when neurones are cocultured with astrocytes they become dependent on astrocytes for survival. This dependence makes neurones susceptible to damage when astrocytes are activated by substances such as TGF-beta1.

Transforming growth factor (TGF)-beta2 is a pleiotropic cytokine associated with remissions in multiple sclerosis (MS) and amelioration of allergic encephalomyelitis. We assessed the safety of TGF-beta2 in an open-label trial of 11 patients with secondary progressive (SP) MS. Five patients had a reversible decline in the glomerular filtration rate. There was no change in expanded disability status scale or MRI lesions during treatment. Systemic TGF-beta2 may be associated with reversible nephrotoxicity, and further investigation of its therapeutic potential in MS should be performed with caution.

The hydrophilic bile salt ursodeoxycholic acid (UDCA) protects against the membrane-damaging effects associated with hydrophobic bile acids. This study was undertaken to (a) determine if UDCA inhibits apoptosis from deoxycholic acid (DCA), as well as from ethanol, TGF-beta1, Fas ligand, and okadaic acid; and to (b) determine whether mitochondrial membrane perturbation is modulated by UDCA. DCA induced significant hepatocyte apoptosis in vivo and in isolated hepatocytes determined by terminal transferase-mediated dUTP-digoxigenin nick end-labeling assay and nuclear staining, respectively (P < 0.001). Apoptosis in isolated rat hepatocytes increased 12-fold after incubation with 0.5% ethanol (P < 0.001). HuH-7 cells exhibited increased apoptosis with 1 nM TGF-beta1 (P < 0. 001) or DCA at >/= 100 microM (P < 0.001), as did Hep G2 cells after incubation with anti-Fas antibody (P < 0.001). Finally, incubation with okadaic acid induced significant apoptosis in HuH-7, Saos-2, Cos-7, and HeLa cells. Coadministration of UDCA with each of the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes (P < 0.001) in all the cell types. Also, UDCA reduced the mitochondrial membrane permeability transition (MPT) in isolated mitochondria associated with both DCA and phenylarsine oxide by > 40 and 50%, respectively (P < 0.001). FACS(R) analysis revealed that the apoptosis-inducing agents decreased the mitochondrial transmembrane potential and increased reactive oxygen species production (P < 0.05). Coadministration of UDCA was associated with significant prevention of mitochondrial membrane alterations in all cell types. The results suggest that UDCA plays a central role in modulating the apoptotic threshold in both hepatocytes and nonliver cells, and inhibition of MPT is at least one pathway by which UDCA protects against apoptosis.

Cadmium chloride (CdCl2) exposure has been reported to induce pulmonary fibrosis in rats. Accumulating evidence has shown that cytokines play a pivotal role in the excessive production of connective tissue components in pulmonary fibrosis. In this report, rat lung slice cultures were used to study the synergistic involvement of transforming growth factor-beta1 (TGF-beta1) in CdCl2-induced alveolar fibrosis. Rat lung slices were maintained at the interphase of air and medium on a polyester mesh stretched on a plastic scaffold. Treatment of lung slices with 2.5, 5 or 10 microM CdCl2 for 7 days resulted in 85, 40 and 6% respectively for relative survival. Under these culture conditions, CdCl2 alone did not induce alveolar fibrosis in rat lung slices. However, in the presence of 0.5 ng/ml TGF-beta1, CdCl2 at a dose ranging from 1 to 5 microM increased the thickness of alveolar septa. Furthermore, the thickness of alveolar septa in lung slices treated with CdCl2 was dose-dependently increased by the presence of TGF-beta1. The thickened alveolar septa were apparently due to the deposition of excessive extracellular matrix, as revealed by trichrome stain and ultrastructural examination. Our results also show that fibrogenic activity induced by the combined treatment with CdCl2 and TGF-beta1 can be reduced by co-treatment with 200 microg/ml lambda-carrageenan, a TGF-beta1 inhibitor. Therefore, the present results indicate that TGF-beta1 can synergistically stimulate the fibrogenic activity in lung tissue subsequent to CdCl2 injury.

Renal proximal tubular cell (RPTC) monolayers exposed to the model oxidant tert-butylhydroperoxide (TBHP; 0.8 mM) for 1.5 hrs were 33 and 31% confluent after 1 and 4 days, respectively. Control monolayers remained 100% confluent throughout the experiment. Exogenous TGF-beta1 promoted monolayer deterioration by potentiating cellular death and suppressed EGF-stimulated regeneration of the RPTC monolayer. Net TGF-beta1 production in injured RPTC increased 1.7- and 3.2-fold on Days 1 and 2, respectively, and returned to control levels 4 days following TBHP treatment. An anti-TGF-beta antibody increased monolayer confluence to 50% and DNA content 1.3-fold 4 days after TBHP exposure. L-Ascorbic acid 2-phosphate (AscP) present only during the recovery period increased monolayer confluence to 67% but had no effect on RPTC proliferation, suggesting that AscP promoted monolayer regeneration by cellular migration/spreading. AscP present continuously had no effect on the extent of TBHP-induced injury but promoted regeneration of RPTC with increased monolayer confluence (1.8-fold) and DNA content (1.8-fold) and decreased cellular lysis by 52% 4 days following TBHP exposure. The results demonstrate that TBHP-induced injury increases net TGF-beta1 production in RPTC and that autocrine TGF-beta1 inhibits regeneration of the monolayer by potentiating cellular injury and monolayer deterioration. The data also show that AscP is not cytoprotective during TBHP exposure but promotes RPTC regeneration by stimulating proliferation and migration/spreading and decreasing cellular death during the recovery period.

Induction of apoptosis in transformed fibroblasts by surrounding normal cells has been discussed as a potent early control step in carcinogenesis. According to this hypothesis, tumor progression should require resistance of transformed cells against this TGF-beta-triggered control mechanism. Here we show that Bcl-2, a protein involved in inhibition of apoptosis, can protect transformed cells from induction of apoptosis by surrounding cells. Rather than acting on the transformation process itself, Bcl-2 may thus represent an efficient modulator of carcinogenesis at an intercellular level.

Cataract, already a major cause of visual impairment and blindness, is likely to become an increasing problem as the world population ages. In a previous study, we showed that transforming growth factor-beta (TGFP) induces rat lenses in culture to develop opacities and other changes that have many features of human subcapsular cataracts. Here we show that estrogen protects against cataract. Lenses from female rats are more resistant to TGFbeta-induced cataract than those from males. Furthermore, lenses from ovariectomized females show increased sensitivity to the damaging effects of TGFbeta and estrogen replacement in vivo, or exposure to estrogen in vitro, restores resistance. Sex-dependent and estrogen-related differences in susceptibility to cataract formation, consistent with a protective role for estrogen, have been noted in some epidemiological studies. The present study in the rat indicates that estrogen provides protection against cataract by countering the damaging effects of TGFbeP. It also adds to an increasing body of evidence that hormone replacement therapy protects postmenopausal women against various diseases.

Transforming growth factor-beta 1 (TGF-beta 1) and androgen are potential physiological regulators of prostate cancer cells. In the present study, we have used LNCaP cells as a model of androgen-responsive prostate cancer to investigate the effects of dihydrotestosterone (DHT) on the sensitivity to TGF-beta 1. The ability of LNCaP cells to respond to TGF-beta has been controversial. In some studies, LNCaP cells were insensitive to TGF-beta 1 while, in others, they were sensitive to the growth inhibitory effect of TGF-beta 1. The present study was carried out to establish androgenic conditions that rendered LNCaP cells sensitive to TGF-beta 1. Cells were cultured in phenol-red-free RPMI 1640 medium supplemented with 10% charcoal-stripped fetal bovine serum. DHT was added at the following concentrations: 0, 10(-12), 10(-10), and 10(-7) M. These concentrations were selected because they represent the zero DHT control, the low-proliferative dose, the high-proliferative dose, and the growth-arrest dose, respectively. The effects of TGF-beta 1 observed on LNCaP cells included inhibition of cell proliferation, decrease in cell viability, alteration in cell morphology, and enhancement of gene transcriptional activity through activation of a TGF-beta responsive promoter. Of the various DHT concentrations investigated in this study, these effects of TGF-beta 1 on LNCaP cells were consistently demonstrated only at 10(-10) M. At other concentrations, the effects of TGF-beta 1 were either minimal or undetectable. Accompanying these effects of TGF-beta 1, a low but statistically significant level of TGF-beta 1-specific binding and an increased protein level of TGF-beta receptor type II were detected by a competitive binding assay and Western blot analysis respectively. These results indicate that LNCaP cells can be induced by DHT to respond to TGF-beta 1 and that DHT modulates the sensitivity to TGF-beta 1 and the level of TGF-beta receptor type II in these cells.

Purpose: To evaluate the ocular toxicity of intravitreous transforming growth factor-beta 1 (TGF-beta(1))., Methods: Eyes of New Zealand white rabbits were injected intravitreally with TGF-beta(1) in doses of 20, 10, 5, 1 or 0.5 micrograms. Electroretinography and clinical examination were performed before and after the injection. At either 10 or 30 days after injection, the eyes were enucleated and examined histologically. The endothelium of fresh corneas, which had received 20 micrograms TGF-beta(1) or nothing, were checked 5 days after injection., Results: Corneal opacity was caused by doses > or = 10 micrograms in 7 of 7 eyes; by doses of 5 micrograms in 3 of 4 eyes; and by doses of 1 microgram in 1 of 4 eyes. Doses > or = 5 micrograms also caused pannus formation in 15 of 16 eyes. Ten days after injection there was some decrease in B-wave amplitude on electroretinography. Thirty days after injection the electroretinographic responses had recovered, except in the eyes injected with 20 micrograms TGF-beta(1), which still showed a decrease in step 1. Histological examination of paraffin sections did not demonstrate significant change. Corneal thickness of the eyes receiving 20 micrograms TGF-beta(1) was twice that of the normal cornea and some corneal endothelial cells lost their hexagonal shape., Conclusion: TGF-beta(1) injected intravitreally at doses > or = 1 micrograms is toxic to the cornea. Retinal function is only slightly disturbed by 20 micrograms of TGF-beta(1).