TGF-beta Isoforms Research Articles

Salivary transforming growth factor beta in oral submucous fibrosis: A diagnostic and predictive marker.

Growth factors and cytokines like transforming growth factor beta (TGF-β) play a key role in the pathogenesis of oral submucous fibrosis. To elucidate the role of Salivary TGF-β isoforms as a predictive and diagnostic marker for oral submucous fibrosis. A total of 30 OSMF and 10 control patients were included in this study, and their clinic-epidemiological data was recorded. The expression of TGF-β genes-TGF-β1, TGF-β2, TGF-β3-was studied by a real-time polymerase chain reaction in tissue and saliva. Patients were given medicinal intervention for 12 weeks along with jaw-opening exercises. Expression of salivary TGF-β genes was studied at 12 weeks. SPSS software version 20. Expression of salivary TGF beta isoforms in OSMF was more than in the control group. There was an increase in salivary TGF-β1, β2, β3 expressions with increasing clinical grades of OSMF and advancing the stage of the disease. Expression of all the TGF beta isoforms was decreased after treatment with statistically significant results. Statistically significant correlations were found between the mean difference of TGF-β1 and the mean difference between mouth opening and tongue protrusion. Salivary TGF-β isoforms may be used in diagnosis, risk assessment, and screening of the entire population at risk of OSMF after its clinical validation. However, adequate sample size and segmental assessment of the expression of TGF-β isoforms are needed for further evaluation.

Abstract 6710: AVID200, a first-in-class selective TGF-beta 1 and -beta 3 inhibitor, sensitizes tumors to immune checkpoint blockade therapies

Abstract AVID200 is a first-in-class rationally designed receptor ectodomain trap that selectively neutralizes TGF-beta 1 and -beta 3 with pM potency while sparing TGF-beta 2, thereby reducing the potential for toxicities associated with neutralization of this isoform. A Phase 1 dose-escalation clinical study of AVID200 monotherapy has recently been completed and established that the agent was well tolerated and exhibited target engagement at all dose levels tested without reaching a maximally tolerated dose. To explore the potential of AVID200 as a combination agent with immunotherapy, expression analysis of TGF-beta isoforms across various solid tumors has been carried out and the ability of AVID200 to increase anti-tumor immunity as a single agent and in combination has been explored in vivo. Analysis of >10,000 tumor samples from TCGA revealed that TGF-beta1 and -beta3 are the predominant isoforms expressed in solid tumors, with TGF-beta2 showing only minimal expression. This corroborates the attractiveness of TGF-beta1 and -beta3 as promising anti-cancer targets and emphasizes the need to target both isoforms simultaneously. Furthermore, AVID200 increased T-cell-mediated cytotoxicity as a single agent as well as potentiating the efficacy of immune checkpoint inhibitors (ICI) in syngeneic mouse tumor models. In line with these observations, we have found that AVID200 increases the infiltration of T-cells in the tumor microenvironment. In conclusion, AVID200, a first-in-class, potent and selective TGF-beta trap, enhances the activity of ICI agents in vivo and holds promise as a potential novel immunotherapy combination regimen which warrants exploration in patients with solid tumor malignancies. Phase 1 clinical trials are underway in various indications. Citation Format: Gilles Tremblay, Tina Gruosso, Jean-François Denis, Rene Figueredo, Jim Koropatnick, Maureen O'Connor-McCourt. AVID200, a first-in-class selective TGF-beta 1 and -beta 3 inhibitor, sensitizes tumors to immune checkpoint blockade therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6710.

504P - AVID200 neutralizes TGF-beta1 and -beta3, the principal immunosuppressive TGF-beta isoforms overexpressed by tumours, and sensitizes tumours to immune checkpoint inhibitors

BackgroundResistance to immune checkpoint inhibitors (ICIs) is a major unmet medical need. The TGF-beta pathway is a key immunosuppressive mechanism that correlates with resistance to PD-1/PD-L1 inhibitors. TGF-beta acts by inhibiting the recruitment and activation of anti-tumor T-cells, either directly, or indirectly through its action on cancer-associated fibroblasts. AVID200 is a receptor ectodomain trap that was rationally-designed to inhibit with pM potency TGF-beta1 and -beta3, the principal oncogenic TGF-beta isoforms. Targeting these two isoforms with AVID200 has the potential to increase the number of patients that benefit from ICIs. MethodsThe percent of patients exhibiting tumor over-expression (>30FPKM) of TGF-beta isoforms was analyzed in>10,000 samples from the CGA RNAseq data. The potency and selectivity of AVID200 and other clinical stage TGF-beta inhibitors was assessed using an A549 cell-based. The ability of AVID200 to enhance the tumor-cell killing activity of T-cells was evaluated in vivo in a syngeneic 4T1 cancer model. The ability of AVID200 to enhance T-cell infiltration and the efficacy of ICIs was assessed in EMT-6 and MC-38 cancer models. ResultsGene expression analysis revealed that TGF-beta1 is the predominant isoform expressed in solid tumors, with TGF-beta3 also being expressed in multiple tumor types. This indicates that it is important to target both ligand isoforms for maximal efficacy, particularly because TGF-beta 1 and 3 can functionally substitute for each other. In vitro, AVID200 exhibited the best potency (low pM) and selectivity for TGF-beta 1 & 3 vs TGF-beta 2 amongst the TGF-beta inhibitors tested. Having minimal activity on TGF-beta2 is desirable because TGF-beta2 is involved in cardiac homeostasis and the positive regulation of hematopoiesis. In vivo, AVID200 was shown to promote T-cell infiltration and to increase the anti-tumor activity of ICIs. ConclusionsIn conclusion, AVID200 efficiently neutralizes TGF-beta 1 and 3, the main oncogenic isoforms, with best-in-class potency and sensitizes tumors to immune checkpoint blockade. The AVID200 Phase 1 study in patients with solid tumors is ongoing. Legal entity responsible for the studyForbius (Formation Biologics). FundingForbius (Formation Biologics). DisclosureT. Gruosso: Full / Part-time employment: Forbius (Formation Biologics). M. O’Connor: Leadership role, Licensing / Royalties, Full / Part-time employment, Officer / Board of Directors: Forbius (Formation Biologics). J. Denis: Full / Part-time employment: Forbius (Formation Biologics). R. Figueredo: Research grant / Funding (self): Forbius (Formation Biologics); Research grant / Funding (self): Trillium Therapeutics, Inc.; Research grant / Funding (self): Critical Outcome Technologies, Inc. J. Koropatnick: Licensing / Royalties: Sarissa Inc; Research grant / Funding (self): Forbius (Formation Biologics); Research grant / Funding (self): Trillium Therapeutics, Inc; Research grant / Funding (self): Critical Outcome Technologies, Inc. G. Tremblay: Licensing / Royalties: Alethia Biotherapeutics; Full / Part-time employment: Forbius (Formation Biologics). I.A. Tikhomirov: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Full / Part-time employment, Officer / Board of Directors: Forbius (Formation Biologics).

Abstract 4688: AVID200: a novel computationally-designed TGF beta trap promoting anti-tumor T cell activity

Abstract AVID200 is a computationally-designed, avidity-enhanced, receptor ectodomain-based trap that binds and neutralizes TGF-beta 1 and 3 with low pM potency. Of the three TGF-beta isoforms, blockade of TGF-beta 2 has been shown to promote cancer metastasis. Therefore, AVID200 is designed to inhibit TGF-beta 1 and 3 only. TGF-beta is a strongly immunosuppressive molecule that, when expressed by cancers, mediates escape from immune surveillance. Increased TGF-beta ligand promotes cancer progression by suppressing anti-tumor immunity, predominantly by suppressing recruitment and activation of anti-tumor T cells. Consequently, TGF-beta is an important therapeutic target in cancer.AVID200 immunotherapy blocks the capacity of TGF-beta 1 and 3 ligands to interact with their receptors. That blockage induces T cell infiltration into tumors, thereby promoting a "T cell-inflamed" tumor state. Multiple trap formats with differing in vitro blocking potency, biophysical traits, and circulating half-lives in rodents have been designed, synthesized, and tested. On the basis of favorable characteristics, the AVID200 molecule was selected for in vivo assessment of efficacy in inhibiting growth of syngeneic 4T1 triple negative breast cancer (TNBC) homografts in immunocompetent host mice. In addition, CD4+ and CD8+ T cells isolated from draining lymph nodes of 4T1 tumor-bearing mice, treated and untreated with AVID200 and other candidate TGF-beta traps, were assessed for activities important in anti-tumor immune activity. We report that AVID200 immunotherapy decreased T-cell apoptosis, stimulated T-cell proliferation in response to tumor cell lysate-loaded dendritic cells, and enhanced the capacity of T-cells to specifically recognize and kill 4T1 tumor cells in ex vivo 2D cultures. The effect of combining AVID200 with immune checkpoint inhibitors in the treatment of syngeneic homografts in immune-competent mice is being assessed. AVID200 is a promising new immunotherapy to selectively inhibit the TGF-beta 1 and 3 isoforms, thereby enhancing desirable anti-tumor immunity while avoiding the tumor-promoting effects resulting from TGF-beta 2 neutralization. Citation Format: Maureen D. O'Connor-McCourt, Anne E. Lenferink, John Zwaagstra, Traian Sulea, Catherine Collins, Renu Singh, Yves Durocher, Christiane Cantin, James Koropatnick. AVID200: a novel computationally-designed TGF beta trap promoting anti-tumor T cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4688. doi:10.1158/1538-7445.AM2017-4688

Abstract A054: Inhibition of TGF-beta isoforms 1 and 2 enhances therapeutic tumor vaccine efficacy

Abstract Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine comprised of three isoforms, TGF-beta1, 2, and 3, that exhibits immunosuppressive functions. In the context of immunosuppression, TGF-beta dampens activation and maturation of both innate and adaptive immune cells, including natural killer cells, macrophages, and dendritic cells, as well as CD4+ and CD8+ T cells. Thus, TGF-beta inhibition, like blockade of other negative regulators of immunity, provides a potential modality of cancer treatment that can be used in combination therapy. Although we have previously demonstrated that blockade of all three isoforms of TGF-beta using a monoclonal antibody increases tumor vaccine efficacy, as measured by the reduction in tumor growth and increase in the number of antigen-specific CTLs, the function of individual isoforms of TGF-beta in the framework of tumor immunity is not well understood. In this study, we examined whether it is necessary to block TGF-beta3, in addition to the two other isoforms of TGF-beta, to improve vaccine-induced tumor immunity against the syngeneic TC1 tumor model, a C57BL/6 lung epithelial cell line transfected with HPV-16 E6 and E7 genes. When the subcutaneous tumors became approximately 5 mm in diameter, the mice were immunized with a peptide-based vaccine that targets the E7 oncogene with or without the simultaneous administration of anti-TGF-beta antibodies with unique specificities for the three isoforms. Mice immunized with the vaccine alone exhibited slightly delayed tumor growth relative to the untreated control. This effect could be enhanced by the combination of anti-TGF-beta antibodies that neutralize all three isoforms of TGF-beta. Interestingly, blockade of only TGF-beta1 & 2 also improved vaccine efficacy to a similar degree as the blockade of all three isoforms, though neutralization of TGF-beta alone could not suppress tumor growth. Therefore, it is not necessary to inhibit TGF-beta3 to generate significant vaccine-induced anti-tumor immunity. Systemic depletion of CD8+ T cells using anti-CD8-depleting antibodies completely abrogated the tumor protection observed in mice immunized with vaccine alone or vaccine in combination with TGF-beta blockade, suggesting that the mechanism of anti-tumor immunity in these conditions relies entirely on CD8+ T cells. In vivo CTL assays revealed that anti-TGF-beta antibodies tended to increase the quality of CTL activity induced by the vaccine. To better understand the effect of treatment on the tumor microenvironment, we used flow cytometric analysis to examine T cells in the tumor and tumor draining lymph nodes. While the vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in both lymph nodes and tumors, TGF-beta inhibition at two weeks, but not one week, after vaccination further augmented the increase in the number of T cells infiltrating tumors. Vaccine in combination with blockade of TGF-beta1 & 2, regardless of TGF-beta3 inhibition, also led to the greatest increase in the number of T-bet-expressing CD4+ and CD8+ T cells in tumors. This observation implies that blockade of TGF-beta1 & 2 alone may be sufficient to improve therapeutic tumor vaccines by facilitating the infiltration of more Th1/Tc1-type T cells into tumors. Because several studies suggest that TGF-beta3 actually plays a protective role against tumorigenesis in a range of tissues, developing TGF-beta blockade strategies that do not inhibit this isoform could be beneficial in certain settings. Citation Format: Katharine Clark, Faith C. Robertson, Emma De Ravin, Shingo Kato, Anja Bloom, Amer Mirza, Jay A. Berzofsky, Masaki Terabe. Inhibition of TGF-beta isoforms 1 and 2 enhances therapeutic tumor vaccine efficacy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A054.

Abstract B137: Inhibition of TGF-beta1 and 2 facilitates therapeutic tumor vaccine efficacy

Abstract Targeting cell surface inhibitory receptors (checkpoint inhibitors) has established immunotherapy as a major modality of cancer treatment. However, the success of cancer immunotherapy is still limited as immune regulation of tumor immunity is very complicated and mechanisms of the regulation involved may also differ among different cancer types. In addition to checkpoints, other good candidates for immunotherapy are immunosuppressive cytokines. TGF-beta is a very potent immunosuppressive cytokine which has been shown to be involved in suppression of tumor immunity. TGF-beta has three isoforms, TGF-beta1, 2 and 3. It has been demonstrated in multiple mouse tumor models that inhibition of all three isoforms of TGF-beta facilitates natural tumor immunosurveillance as well as tumor vaccine efficacy. It also has been demonstrated that TGF-beta1 plays a critical role in the induction of Treg cells, IL-17-producing T cells and MDSCs. However, roles of other isotypes of TGF-beta are not well studied yet. In this study we asked whether it is necessary to inhibit TGF-beta3 to enhance tumor immunity induced by a tumor vaccine in a syngeneic TC1 tumor model. When the tumor reached at least 5 mm in diameter, the mice were given a peptide-based vaccine targeting HPV16 E7, which is an oncogene expressed in TC1 cells. Although the vaccine alone had minimal effects on tumor growth, combination with an anti-TGF-beta antibody that neutralizes all three isoforms significantly delayed tumor growth. A similar effect was obtained with an antibody that neutralizes only TGF-beta1 and 2 but not TGF-beta3. Thus, it is not necessary to block TGF-beta3 to overcome immune suppression. Flow cytometric analysis of immune cells in tumor draining lymph nodes and tumors showed that there was no difference in the number of Treg and MDSCs between mice with/without treatment. The vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in tumors. The TGF-beta inhibitor together with the vaccine increased the number of T cells infiltrating tumors two weeks after the vaccination, but not just one week after vaccination. The vaccine also induced a significant number of T-bet-expressing T cells (both CD4 and CD8) in tumors, and anti-TGF-beta, regardless of TGF-beta3 blockade, further increased the number of these cells. Together the results suggested that blockade of TGF-beta1 and 2 alone is sufficient to enhance therapeutic tumor vaccine efficacy by facilitating the induction of Th1/Tc1 type T cells. With the notion that TGF-beta3 might be beneficial for patients in some cancers, developing TGF-beta antagonists that do not inhibit TGF-beta3 may be lead to better outcomes. Citation Format: Masaki Terabe, Faith C. Robertson, Shingo Kato, Emma De Ravin, Katharine Clark, Anja Bloom, Amer M. Mirza, Jay A. Berzofsky. Inhibition of TGF-beta1 and 2 facilitates therapeutic tumor vaccine efficacy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B137.

Abstract B024: Single-chain traps targeting transforming growth factor-beta (TGF-beta) home to tumors and reduce tumor growth and metastasis by counteracting TGF-beta-mediated immunosuppression

Abstract The TGF-beta superfamily includes a large number of ligands with physiological and pathological significance. In particular, elevated TGF-beta in the tumor microenvironment markedly augments cancer progression by inducing metastasis and angiogenesis and by suppressing the immune system. Therapeutic agents targeting TGF-beta, such as antibodies or IgG-Fc-fused receptor ectodomains, have been developed, however these agents are relatively large molecules, which may restrict tumor penetration. We have computationally designed single-chain traps that are comprised of tandemly-fused TGF-beta receptor domains. These traps are approximately one third the size of monoclonal antibodies which would potentially facilitate a better tumor penetration. The homobivalent TGF-beta RII-RII traps (i.e. T22d35 and T22d60) neutralize TGF-beta isoforms 1 and 3 and the heterovalent TGF-beta RI-RII based traps (i.e. T12d and T122bt) are pan-specific and neutralize all three isoforms (TGF-beta 1, 2 and 3). Both the T22d35 and T12d trap blocked TGF-beta 1 and 3, induced epithelial mesenchymal transition (EMT) and motility of mouse mammary tumor cells (JM01). Heterovalent T12d also blocked TGF-beta2 effects on these phenotypes. In vivo comparison of T22d35 trap and the pan-neutralizing TGF-beta antibody 1D11 indicated that T22d35 treatment but not 1D11 reduced the growth of established primary 4T1 mammary tumors, suggesting better neutralization of TGF-beta due to a better tumor penetration of the T22d35 trap (Mol. Cancer Ther. 11:1477, 2012). Trap treatment significantly increased T lymphocyte infiltration and cytotoxic activity within the tumors. Biodistribution studies demonstrated that the T22d35 trap, although eliminated rapidly from the circulating blood and other tissues (within 24 hours), localized and was retained within primary 4T1 tumors. Furthermore, a single injection of trap per week reduced 4T1 metastatic lesions by more than 80%, relative to saline controls, indicating that a short-term trap exposure in the host is sufficient to trigger long-lasting effects. Together, our results indicate that the TGF-beta traps readily home to tumors, antagonize immunosuppression and reduce metastatic spread. Citation Format: John Zwaagstra, Traian Sulea, Anne E.G. Lenferink, Jason Baardsnes, Catherine Collins, Christiane Cantin, Lucie Couture, Limei Tao, Yves Durocher, Maureen O'Connor-McCourt. Single-chain traps targeting transforming growth factor-beta (TGF-beta) home to tumors and reduce tumor growth and metastasis by counteracting TGF-beta-mediated immunosuppression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B024.

Abstract LB-233: Blockade of TGF-beta1 and 2 without TGF-beta3 blockade is sufficient to facilitate tumor vaccine efficacy

Abstract TGF-beta is one of the most potent immunosuppressive cytokines involved in the regulation of tumor immunity. TGF-beta has three isoforms, TGF-beta1, 2 and 3. It has been demonstrated in multiple mouse tumor models that blockade of all three isoforms of TGF-beta facilitates natural tumor immunosurveillance as well as tumor vaccine efficacy. Most studies on the roles of TGF-beta in immunology are on TGF-beta1, which has been demonstrated to induce Treg cells, IL-17-producing T cells and MDSCs. Although some studies reported immunosuppressive activities of TGF-beta2, the role of TGF-beta3 in immune regulation is not well understood. In this study we asked whether it is necessary to inhibit TGF-beta3 to enhance tumor immunity induced by a tumor vaccine in a syngeneic TC1 tumor model. When the tumor reached at least 5 mm in diameter, the mice were given a peptide-based vaccine targeting HPV16 E7, which is an oncogene expressed in TC1 cells. Although the vaccine alone had minimal effects on tumor growth, combination with an anti-TGF-beta antibody that neutralizes all three isoforms significantly delayed tumor growth. A similar effect was obtained with an antibody that neutralizes only TGF-beta1 and 2 but not TGF-beta3. Thus, it is not necessary to block TGF-beta3 to overcome immune suppression. Flow cytometric analysis of immune cells in tumor draining lymph nodes and tumors showed that there was no difference in the number of Treg and MDSCs between mice with/without treatment. The vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in both lymph nodes and tumors, but there was no further increase in combination with anti-TGF-beta. The vaccine also induced a significant number of T-bet-expressing T cells (both CD4 and CD8) in both tumor draining lymph nodes and tumors, and anti-TGF-beta, regardless of TGF-beta3 blockade, further increased the number of these cells. Together the results suggested that blockade of TGF-beta1 and 2 alone is sufficient to enhance therapeutic tumor vaccine efficacy by facilitating the induction of Th1 type T cells. With the notion that TGF-beta3 might be beneficial for patients in some cancers, developing TGF-beta antagonists that do not inhibit TGF-beta3 may be lead to better outcomes. Citation Format: Masaki Terabe, Faith Robertson, Shingo Kato, Emma De Ravin, Katharine Clark, Amer M. Mizra, Jay A. Berzofsky. Blockade of TGF-beta1 and 2 without TGF-beta3 blockade is sufficient to facilitate tumor vaccine efficacy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-233. doi:10.1158/1538-7445.AM2015-LB-233

IL-10 Regulation Of Hyaluronan Synthesis Is Mediated Via TGF-Beta

The fetal scarless phenotype is characterized by an attenuated inflammatory response, an extracellular matrix (ECM) comprised of increased levels of hyaluronan (HA), and elevated levels of TGFB3 compared to TGFB1. We have reported a novel role for the anti-inflammatory cytokine, IL-10, in the fetal regenerative phenotype and its ability to induce scarless healing in adult wounds. Further, we have demonstrated a new non- immunologic mechanism for IL-10, which is regulation of HA. The TGF beta isoforms have also been implicated in HA regulation.

The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation

Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent.

Angiotensin II increases mRNA levels of all TGF‐β isoforms in quiescent and activated rat hepatic stellate cells

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF-beta (transforming growth factor-beta). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF-beta actions. Here, we studied the effect of AII on the mRNA levels of TGF-beta isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF-beta1, TGF-beta2 and TGF-beta3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF-beta isoforms, particularly TGF-beta2and TGF-beta3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF-beta protein after AII treatment. The mRNA expression of TGF-beta isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF-beta expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.

Role of transforming growth factor Beta in corneal function, biology and pathology.

Transforming growth factor-beta (TGFbeta) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFbeta are known in mammals and the human cornea expresses all of them. TGFbeta1 has been shown to play a central role in scar formation in adult corneas whereas TGFbeta2 and TGFbeta3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFbeta in the cornea have been shown to follow Smad dependent as well as Smad-independent signaling pathways depending upon cellular responses and microenvironment. Corneal TGFbeta expression is necessary for maintaining corneal integrity and corneal wound healing. On the other hand, TGFbeta is perhaps the most important cytokine in the pathogenesis of fibrotic disease in the cornea. Although the transformation of keratocytes to myofibroblasts induced by TGFbeta is largely believed to cause corneal fibrosis or scarring, the precise molecular mechanism(s) involved in this process is still unknown. Currently no drugs are available to treat corneal scarring effectively without causing significant side effects. Many approaches to treat TGFbeta-mediated corneal scarring are under investigation. These include blocking of TGFbeta, TGFbeta receptor, TGFbeta function and/or TGFbeta maturation. Other strategies such as modulating keratocyte proliferation, apoptosis, transcription and DNA condensation are also being investigated. The potential of gene therapy to neutralize the pathologic effects of TGFbeta has also been demonstrated recently.

Fra-2 mediates oxygen-sensitive induction of transforming growth factor β in cardiac fibroblasts

In the ischaemia-reperfused heart, transforming growth factor beta (TGFbeta) proteins trigger the differentiation of cardiac fibroblasts (CFs) contributing to fibrosis. Reoxygenation of the heart, in addition to being a trigger for reperfusion injury, induces tissue remodelling by hyperoxia-sensitive signalling processes involving TGFbeta. Here, we sought to characterize the molecular mechanisms responsible for the O(2)-sensitive transcriptional induction of TGFbeta in murine CF and to test the significance of such findings in the infarcted myocardium in vivo using laser capture microdissection. All three isoforms of TGFbeta were induced in the CF-rich peri-infarct tissue as well as in CF exposed to hyperoxic challenge. Reporter studies demonstrated that TGFbeta transcription is hyperoxia inducible. Deletion of any one or both of the activating protein-1 (AP-1) binding sites in the TGFbeta reporter construct resulted in loss of O(2) sensitivity, demonstrating that AP-1 confers O(2) sensitivity to TGFbeta transcription. Fos-related AP-1 transcription factor (Fra-2) and Ask-1 (apoptosis signal-regulating kinase-1) were identified as key mediators of AP-1-dependent O(2)-sensitive TGFbeta transcription. Knockdown of Fra-2 significantly blunted O(2)-induced expression of TGFbeta1 as well as TGFbeta3 in CF. Knockdown of Ask-1 blunted hyperoxia-induced Fra-2 gene expression and nuclear localization in CF. Collectively, these observations point towards a central role of Ask-1 and Fra-2 in O(2)-inducible AP-1 activation and induction of TGFbeta. Taken together with the observation that Fra-2-regulated genes are implicated in fibrosis, identification of Fra-2 as an O(2)-sensitive transcriptional regulator of inducible TGFbeta expression positions Fra-2 as an important player in reoxygenation-induced fibrosis.

Effect of the abrogation of TGF-β1 by antisense oligonucleotides on the expression of TGF-β-isoforms and their receptors I and II in isolated fibroblasts from keloid scars

Disequilibrium of dermal wound repair can result in continued accumulation of ECM and excessive scar formation. In susceptible genetically predisposed individuals, keloid formation can be observed. Keloid disease represents a benign dermal fibroproliferative tumor that is unique to humans. TGF-beta is known to play a key role in the pathogenesis of this disease which is still not fully understood. The isoforms TGF-beta1 and TGF-beta2 have profibrotic properties, whereas TGF-beta3 may have antifibrotic functions. TGF-beta exerts its influence by binding to type I and type II TGF-beta receptors, thereby forming a complex and activating specific downstream effector molecules. The aim of this study was to investigate the effect of TGF-beta1 targeting by antisense oligonucleotides on the RNA synthesis and protein expression of TGF-beta isoforms and their receptors in keloid-derived fibroblasts. In tissue samples with normal fibroblasts (NFs) serving as control samples, expression of TGF-beta1 and -beta2 was decreased when compared to keloid fibroblasts (KFs), while expression of TGF-beta3 and of TGF-betaRII was significantly higher in NFs. In the ELISA assay, abrogation of TGF-beta1 led to a significant decrease in TGF-beta1 and -beta2 (p<0.05). Expression of TGF-beta2 mRNA was reduced. Expression of TGF-beta3 mRNA revealed contrary patterns in KFs from different patients while expression of TGF-betaRI was found to be equal during the measurement period. TGF-betaRII mRNA expression was increased after 48 and 72 h respectively. There is growing evidence for a regulatory mechanism between TGF-beta1 and its receptors. Our findings support this theory by suggesting interrelations between the different TGF-beta isoforms and their receptors. Abnormal response of KFs to TGF-betamight reflect a modification in the regulatory pathway that occurs at the receptor level or during intracellular trans-duction. Improving the understanding of TGF-beta in keloid disease could lead to the development of clinically useful therapeutic modalities for treatment of keloid disease or even allow identification of preventive strategies.

TGF-β Isoform Specific Regulation of Airway Inflammation and Remodelling in a Murine Model of Asthma

The TGF-β family of mediators are thought to play important roles in the regulation of inflammation and airway remodelling in asthma. All three mammalian isoforms of TGF-β, TGF-β1–3, are expressed in the airways and TGF-β1 and -β2 are increased in asthma. However, there is little information on the specific roles of individual TGF-β isoforms. In this study we assess the roles of TGF-β1 and TGF-β2 in the regulation of allergen-induced airway inflammation and remodelling associated with asthma, using a validated murine model of ovalbumin sensitization and challenge, and isoform specific TGF-β neutralising antibodies. Antibodies to both isoforms inhibited TGF-β mediated Smad signalling. Anti-TGF-β1 and anti-TGF-β2 inhibited ovalbumin-induced sub-epithelial collagen deposition but anti-TGF-β1 also specifically regulated airway and fibroblast decorin deposition by TGF-β1. Neither antibody affected the allergen-induced increase in sub-epithelial fibroblast-like cells. Anti- TGF-β1 also specifically inhibited ovalbumin-induced increases in monocyte/macrophage recruitment. Whereas, both TGF-β1 and TGF-β2 were involved in regulating allergen-induced increases in eosinophil and lymphocyte numbers. These data show that TGF-β1 and TGF-β2 exhibit a combination of specific and shared roles in the regulation of allergen-induced airway inflammation and remodelling. They also provide evidence in support of the potential for therapeutic regulation of specific subsets of cells and extracellular matrix proteins associated with inflammation and remodelling in airway diseases such as asthma and COPD, as well as other fibroproliferative diseases.

Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

BackgroundTo test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC).Patients and MethodsWe analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFβ1, TGFβ2, TGFβ3 in oral SCC.ResultsOur results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFβ isoforms, we found that Smad2 mRNA and TGFβ1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFβ1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population.ConclusionSmad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFβ signaling should be better clarified in the future.

Mechanisms of Immune Suppression for CD8+T Cells by Human Corneal Endothelial Cells via Membrane-Bound TGFβ

To determine whether CD8(+) T cells in vitro are suppressed or converted into regulators (Treg cells) by human corneal endothelial (HCE) cells. HCE cell lines were established from primary HCE cells, and allogeneic CD8(+) T cells were isolated from peripheral blood mononuclear cells of healthy donors. T-cell activation was assessed for proliferation by [(3)H]-thymidine incorporation. Expression of TGFbeta on HCE cells was evaluated by flow cytometry, RT-PCR, and immunohistochemistry. Anti-TGFbeta-neutralizing antibodies or TGFbeta siRNA was used to abolish the HCE-mediated inhibitory function. CD8(+) T cells exposed to HCE cells were used as Treg cells, to induce Treg cells in vitro by exposure to HCE cells. Expression of CD25 or Foxp3 on Treg cells was evaluated by flow cytometry. Cultured HCE cells produced the membrane-bound active TGFbeta isoform 2. HCE surface TGFbeta2 was necessary, to inhibit CD8(+) T-cell activation via direct cell contact. In addition, although HCE cells were found to constitutively express costimulatory molecules, such as programmed death-ligand 1 (PD-L1) and PD-L2, and secreted thrombosponsin-1, only membrane-bound TGFbeta2 was actually delivered to the CD8(+) T cells. HCE cells were also found to convert some CD8(+) T-cell populations into Treg cells via their membrane-bound TGFbeta. These HCE-induced Treg cells produced soluble forms of TGFbeta1, but not of TGFbeta2, and they also acquired a regulatory phenotype that expressed CD25(high) and Foxp3. Ocular resident tissue-exposed T cells can be suppressed or induced to become regulators within the human peripheral microenvironment.

Transforming Growth Factor- Signaling across Ages: From Distorted Lung Development to Chronic Obstructive Pulmonary Disease

The transforming growth factor (TGF)-beta superfamily of secreted growth factors consists of more than 40 members, including the TGF-beta isoforms themselves, bone morphogenetic proteins, and activins. Most of these factors have been shown to be essential for proper organ development, a process often recapitulated in chronic diseases. Importantly, TGF-beta superfamily members are key regulators of extracellular matrix composition and alveolar epithelial cell and fibroblast function in the lung. Both during lung development and disease, TGF-betas therefore control lung homeostasis by providing the structural requirements and functional micromilieu needed for physiological epithelial cell function and proper gas exchange. Prolonged alterations of TGF-beta signaling have been shown to result in structural changes in the lung that compromise gas exchange and lung function, as seen in arrested lung development, a feature of bronchopulmonary dysplasia, lung fibrosis, and chronic obstructive pulmonary disease. All these syndromes share a loss of functional alveolar structures, which ultimately leads to a decreased life expectancy. In this review, we cover our current understanding of the impact of TGF-beta signaling on chronic lung disease. We focus on distorted TGF-beta signaling in bronchopulmonary dysplasia and chronic obstructive pulmonary disease as prototype diseases of the premature and matured lung, respectively, which are both characterized by functional and structural loss of alveolar units.

Temporal and spatial distribution of TGF-beta isoforms and signaling intermediates in corneal regenerative wound repair.

The present study analyzed the temporal and spatial expression of TGF-beta isoforms and activated pSmad2 and p38MAPK during epithelial debridement wound repair, using chick cornea by immunohistochemistry. Normal corneas showed low-level TGF-betas staining. Following wounding, TGF-beta1 expression was strong in the Bowman's layer (BL). TGF-beta3 expression was confined to basal cells in the regenerating and unwounded regions, and was not detected in migrating epithelial, stromal or endothelial cells. In addition, TGF-beta3 treatment stimulated the proliferation of cultured epithelial cells. Our present findings seem to suggest that the TGF-beta3 signal may be required for epithelial cell proliferation. TGF-beta2 expression was strong in migrating and proliferating epithelial cells, many active migrating fibroblasts at the wound edge, endothelial cells and Descemet's membrane (DM). Although both nuclear pSmad2 and p38MAPK staining was observed in many basal epithelial cells, pSmad2 positive cells were co-localized with PCNA positive cells. Therefore, it seems likely that the pSmad2 signal may affect epithelial cell proliferation in healing corneas. Both pSmad2 and p38MAPK expression were also observed in endothelial cells. Interestingly, many active fibroblasts over the whole stroma in early wound healing at day 2 expressed nuclear pSmad2, but little if any cytoplasmic p38MAPK. Collectively, temporal/spatial up-regulation and distribution of the three TGF-beta isoforms, as well as concerted activation of both Smad2 and p38MAPK, appears to be a key aspect of regenerative corneal wound healing in the chick.

Transforming Growth Factor-β Isoforms and the Induction of Bone Formation

Craniofacial skeletal reconstruction remains a challenging problem despite major molecular and surgical developments in the understanding of bone formation by induction. The induction of bone formation has been a critical topic of research across the planet. The bone induction principle identified important cues for tissue engineering of bone, namely, osteogenic soluble molecular signals, the bone morphogenetic and osteogenic proteins, and insoluble signals or substrata including biomimetic bioactive matrices and responding stem cells. In primates, and in primates only, the osteogenic soluble molecular signals that initiate the induction of bone formation additionally include the 3 mammalian transforming growth factor-beta (TGF-beta) isoforms, members of the TGF-beta supergene family. The mammalian TGF-beta isoforms, when implanted in the rectus abdominis muscle of the nonhuman primate Papio ursinus, induce rapid and substantial endochondral bone formation resulting in large corticalized ossicles by day 30 after heterotopic implantation; in calvarial defects of the same nonhuman primates, identical or higher doses of the TGF-beta protein do not induce bone formation because of the overexpression of Smad-6 and Smad-7, gene product inhibitors of the TGF-beta signaling pathway. The addition of minced fragments of autogenous rectus abdominis muscle partially restores the osteoinductive activity of the human TGF-beta3 isoform resulting in the induction of bone formation in the treated calvarial defects. Recombinant human TGF-beta3 delivered by Matrigel matrix and implanted in class II and III furcation defects of mandibular molars of P. ursinus induce periodontal tissue regeneration. The addition of minced fragments of autogenous rectus abdominis muscle significantly enhances cementogenesis. This review highlights the induction of bone formation by the osteogenic proteins of the TGF-beta superfamily in the nonhuman primate P. ursinus and reviews combinatorial applications of myoblastic/myogenic stem cell-based therapeutics for bone induction and morphogenesis. The recruitment of myoendothelial cells is also discussed in the light of the intrinsic and spontaneous induction of bone formation by smart biomaterial matrices that induce bone differentiation in heterotopic extraskeletal sites of P. ursinus without the exogenous application of the osteogenic soluble molecular signals of the TGF-beta superfamily.