Effects Of TGF-beta Research Articles

Abstract 710: Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5

Abstract Radiation pneumonitis is one of the most common but challenging toxicities seen in patients that are receiving ionizing radiotherapy for thoracic malignancies. While radiation effectively eliminates cancer cells, the damage it causes to surrounding healthy lung tissue leads to an immune response that can impact treatment regimen and lead to long term fibrotic tissue formation. The mechanism of radiation pneumonitis and the inflammatory response in the lungs is poorly understood. However, it is known that the TRAIL pathway plays an important role in inflammatory and fibrotic response. Through our previous work, it was discovered that modulating the TRAIL pathway via pegylated recombinant long-acting TRAIL (TLY012) in both WT and TRAIL-/- C57Bl/6 could suppress radiation pneumonitis in mice that were exposed to 20 Gy of thoracic radiation with shielding of other organs as early two weeks post exposure. In addition to the TRAIL pathway, transforming growth factor-beta (TGF-β) is a cytokine that is known to play a key role in fibrosis. Broad spectrum integrin inhibitor GLPG0187 is known to inhibit the activation of TGF-β via binding to specific integrin receptors. In order to determine if the inhibition of TGF-β could suppress radiation pneumonitis without stimulation of the TRAIL pathway, DR5 null mice were utilized for the experiment. Male DR5-/- C57Bl/6 mice received a single 20 Gy thoracic radiation dose with shielding of other organs and were treated with 100 mg/kg of GLPG-0187 or control twice a week via IP injection with the first dose being administered 1 hour before radiation (n = 4/treatment/group). 13 days post-irradiation, lungs, sternal bone marrow, and peripheral serum were collected. Upon histological analysis, it was observed that there were thinner alveolar borders and lessened inflammation compared to the control mice. Mice were weighed every three days, and it was discovered that mice treated with GLPG-0187 maintained steady weight compared to the control group. Additional analysis including immunohistochemistry, cytokine profiling, and quantification needs to be conducted. As the TRAIL pathway could not be modulated in the DR5 null mice, these findings suggest that GLPG-0187 was able to suppress radiation pneumonitis via inhibition of TGF-β. While some rescue from radiation pneumonitis was observed via GLPG0187, it was to a lesser extent compared to treatment of WT and TRAIL-/- C57Bl/6 mice with TLY012. Future directions include investigating the synergistic effects between TLY012 and GLPG0187 in suppressing radiation pneumonitis and decreasing fibrotic response to radiation. Citation Format: Jillian Strandberg, Anna Louie, William MacDonald, Praveen Srinivasan, Leiqing Zhang, Lanlan Zhou, Seulki Lee, Wafik El-Deiry. Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 710.

Role of immunotherapy in treatment refractory chordomas: review of current evidence.

Chordomas are aggressive tumors that are thought to arise from remnants of the embryological notochord. They can arise along the ventromedial aspect of the sacrum, mobile spine, and clivus-with most cases occurring in the sacrum or skull base. Despite surgery and radiation, chordomas often progress and become refractory to further treatment. The high recurrence rate of chordomas has created an urgent need to develop new systemic treatment options. Recent case reports and clinical trials have highlighted the use of immunotherapy for refractory chordomas. In this review, we summarize the results of these studies and discuss the potential role of immunotherapy for chordomas. The PUBMED database was queried for studies mentioning both "Chordoma" and "Immunotherapy." All case series and case reports that involved administration of an immunotherapy for chordoma were included. Additional studies that were found during literature review were added. ClinicalTrials.Gov was queried for studies mentioning both "Chordoma" and "Immunotherapy." The final cohort consisted of all clinical trials that utilized immunotherapy for chordomas of any location. Eight case reports and series detailing the use of immunotherapy for treatment refractory chordoma were identified. Most patients received immunotherapy targeting the PD-1/PD-L1 interaction, and two patients received therapy targeting this interaction along with the tyrosine kinase inhibitor pazopanib. One patient received a vaccine derived from autologous tumor cells, and one patient received a viral vector that downregulated the effect of TGF-beta. One clinical trial utilized a brachyury vaccine in conjunction with standard of care radiotherapy. Immunotherapy for chordoma is a promising area of investigation with increasing, but small, numbers of case series and clinical trials. Despite challenges in patient accrual, future directions in chordoma immunotherapy may lie in vaccine-based therapies and immune checkpoint inhibitors. Understanding chordoma heterogeneity and microenvironment will likely elucidate important chordoma features that will inform future clinical trial design.

Cigarette Smoke Impairs Airway Epithelial Wound Repair: Role of Modulation of Epithelial-Mesenchymal Transition Processes and Notch-1 Signaling.

Cigarette smoke (CS) induces oxidative stress and chronic inflammation in airway epithelium. It is a major risk factor for respiratory diseases, characterized by epithelial injury. The impact of CS on airway epithelial repair, which involves epithelial-mesenchymal transition (EMT) and the Notch-1 pathway, is incompletely understood. In this study, we used primary bronchial epithelial cells (PBECs) to evaluate the effect of CS on epithelial repair and these mechanisms. The effect of CS and/or TGF-beta1 on wound repair, various EMT and Notch-1 pathway markers and epithelial cell markers (TP63, SCGB1A) was assessed in PBECs cultured submerged, at the air–liquid interface (ALI) alone and in co-culture with fibroblasts. TGF-beta1 increased epithelial wound repair, activated EMT (shown by decrease in E-cadherin, and increases in vimentin, SNAIL1/SNAIL2/ZEB1), and increased Notch-1 pathway markers (NOTCH1/JAGGED1/HES1), MMP9, TP63, SCGB1A1. In contrast, CS decreased wound repair and vimentin, NOTCH1/JAGGED1/HES1, MMP9, TP63, SCGB1A1, whereas it activated the initial steps of the EMT (decrease in E-cadherin and increases in SNAIL1/SNAIL2/ZEB1). Using combined exposures, we observed that CS counteracted the effects of TGF-beta1. Furthermore, Notch signaling inhibition decreased wound repair. These data suggest that CS inhibits the physiological epithelial wound repair by interfering with the normal EMT process and the Notch-1 pathway.

Local TGF-beta sequestration by fibrillin-1 regulates vascular wall homeostasis in the thoracic aorta

Abstract Background Aortic dissection and rupture is the main cause of early cardiovascular mortality in patients with Marfan syndrome (MFS). MFS is caused by a defect in fibrillin-1, a building block of microfibrils in the extracellular matrix which binds transforming growth factor beta (TGF-beta) via interaction with latent TGF-beta binding proteins (LTBPs). Multiple mouse models, both pharmaceutically induced and genetically manipulated, have been used to investigate the pathophysiology and biomechanical aspects of thoracic aortic aneurysms and dissections. However, the role of TGF-beta in MFS has been controversial, with earlier studies suggesting that excess release of TGF-beta due to decreased interaction with dysfunctional fibrillin-1 leads to aortic dilation and vascular damage, while other studies have shown an important protective effect of TGF-beta. Studying dedicated mouse models for MFS, with defects interfering with TGF-beta binding and -function may help resolve these discrepancies. Purpose This study aimed to reveal insights in the role of TGF-beta signaling in aneurysm formation and dissection in MFS. Methods Mice lacking the fibrillin-1 binding site for LTBPs (Fbn1H1Δ/+ and Fbn1H1Δ/H1Δ), mice with a truncated fibrillin-1 (Fbn1GT-8/+), and mice with a combination of both alleles (Fbn1GT-8/ H1Δ) were subjected to in vivo cardiac ultrasound analysis. Ex vivo phase-contrast synchrotron X-ray imaging was performed at the Paul Scherrer Institute to visualize the elastic lamellae architecture in the vascular wall of the entire excised thoracic aorta in a subset of mice from each group. Results Fbn1GT-8/+, Fbn1 H1Δ/+ and Fbn1H1Δ/H1Δ mice had a normal life span, but Fbn1GT-8/ H1Δ mice showed increased mortality due to aortic rupture starting at 4–5 months of age. The aortic root was dilated both in Fbn1GT-8/+ and Fbn1GT-8/ H1Δ mice at 6 months of age, but not in Fbn1H1D/+ or Fbn1H1Δ/H1Δ mice. Synchrotron images showed significant elastic lamellae fragmentation in the thoracic aortic wall of Fbn1GT-8/+ mice, and to a larger extent in Fbn1GT-8/ H1Δ mice. Surprisingly, localized elastin fragmentation was also found in the ascending thoracic aorta of Fbn1 H1Δ/+ and Fbn1H1Δ/H1Δ mice, despite a lack of aortic aneurysm formation. Moreover, Fbn1H1Δ/H1Δ mice displayed more severe aortic wall damage. The localized microdissections found in these mouse models were characterized by a severe alteration of the elastic fiber organization, cellular influx and increased collagen deposition, as confirmed by histological analysis. Conclusions Our data suggest that loss of LTBP binding to fibrillin-1 leads to the development of localized microdissections in the aorta in the absence of aortic aneurysm, and exacerbates the aortic wall morphology abnormalities in mice with truncated fibrillin-1. We therefore hypothesize that local TGF-beta sequestration is required to maintain aortic homeostasis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Foundation for Cardiac Surgery (“VZW Fonds voor Hartchirurgie”), grant No. 489644Baillet-Latour Grant for Medical Research

Pre-Transplant Gene Expression May Predict Risk of Graft Versus Host Disease

Background Allogeneic Hematopoietic Cell Transplantation (allo HCT) is currently the only curative therapy for high-risk hematologic malignancies due to graft versus tumor effect (GVT), but with the cost of Graft Versus Host Disease (GVHD). Despite extensive research, very few predictors of GVHD have been identified to date. To study the mechanisms of GVHD and GVT and to identify potential GVHD markers we applied a novel approach, called Transcriptome Fingerprint Assay (TFA), relying on high frequency sampling and blood transcriptome profiling. The TFA is a multiplex microfluidics q-PCR based assay, linked to a computational model for functional analyses, uniquely tailored to answer complex questions on immune perturbations through frequent profiling of gene expression signatures (Chaussabel et a 2014, Speake et a 2017). This approach has been successfully applied to stratify patients' prognosis in autoimmune and infectious diseases. Herein we report the discovery of an association between the expression abundance of a set of genes pre-transplant and the development of acute or chronic GVHD. Methods Seventy-eight patients candidate to allo-HCT were enrolled on the study. Patients donated micro-quantities of blood (50-600 microliters) prior to transplant conditioning, weekly until day 100 post-transplant and every 2 weeks thereafter until 2 years after transplant. Detailed clinical, laboratory and therapy annotations are captured during the follow-up. Gene expression of 264 immune-related genes for each sample are measured through Fluidigm BioMark high-throughput qPCR system. Data interpretation is performed through TFA modular analyses and correlated with the clinical annotations. Here we report the univariate analysis of gene expression data and its association with GVHD in 31 patients for whom data are available until at least day 169 post-transplant (169-672, median 513). SIgnificant genes were tested in a longitudinal analysis with 4 combined linear-mixed models, measuring the parameters: 1) groups (GVHD NO-GVHD) 2) time 3) interaction of groups over time. Results Patients' characteristics are summarized in Fig 1; 26 patients developed GVHD (12 acute GVHD only, 3 late acute GVHD, 1 overlap syndrome, 5 acute and chronic GVHD and 5 chronic GVHD only). By TFA analysis of pre-transplant samples, we identified 3 genes significantly downregulated among patients who developed GVHD (GPSM3, LRG1 and EPHX4), and 2 genes borderline (<1.5 folds) upregulated (IFI35, PLK3). Among these, GPSM3 (an MHC regulated gene) has shown to have alleles protective against severe autoimmune diseases as RA, and LRG1 to facilitate CD34 and myeloid progenitors growth by antagonizing TGF-beta effects, but also to be upregulated in GVHD patients. Several other genes (111) were found to be >1.5 fold upregulated in the GVHD group, but with a non-significant p-value (Volcano-plot Fig.1). Of note, 12 of them were >4 fold upregulated, and their transcriptome pertains to B cells, T lymphocytes, monocytes, IFN, TNF, other cytokines and neutrophil activation. Moreover, among these genes IFN-genes were consistently represented by multiple IFN-modules, emerging as promising candidates. Longitudinal analyses confirmed that EPHX4 and GPSM3 distinguished the cohorts for all the measured parameters in the post-transplant time. EPHX4 increased over time in both groups but significantly more in the NO-GVHD group (with a fixed effect estimate of 2.05); GPSM3 instead decreased over time, again significantly more in the NO-GVHD group (with a fixed effect estimate of -0.95). LRG3 showed significance only for the parameter "time", resulting after transplant more abundant in the GVHD group, thus confirming previous literature data. IFN modules were also tested longitudinally, showing very distinct patterns in the cohorts, and several significant genes (IFI27, NT5C3, CCR1, LBA1, Fig.1) Conclusion Our data demonstrate that TFA may be used to assess expression of immune related genes in transplant patients and to identify specific pathways that may be predictive for GVHD development. Additional work is required to validate our data in a larger cohort and for better understanding of the role of those genes in triggering or protecting against GVHD. Upon completion of the study and the longitudinal analyses, we aim to define in depth transcriptome signatures of GVHD and relapse. Disclosures No relevant conflicts of interest to declare.

P0027METFORMIN INHIBITS TGF-BETA1-INDUCED FIBROTIC RESPONSE IN MOUSE INNER MEDULLARY COLLECTING DUCT CELLS IN ASSOCIATION WITH ACTIVATION OF PPARA SIGNALLING

Abstract Background and Aims Metformin has been recognized to inhibit renal fibrosis through protection of proximal tubular epithelial cells from inflammation and fibrotic response. However, the role of other segments of renal tubular epithelial cells in the development of renal fibrosis remains unclear. This study aimed to investigate the profibrotic effects of TGF-beta1 and the anti-fibrotic effects of metformin in mouse inner medullary collecting duct cells using mIMCD-3 cell line. Method Cultured mIMCD-3 cells were treated with TGF-beta 1 and metformin either alone or in combination. The morphological change of cells was inspected under an inverted microscope. The mRNA of genes associated with renal fibrosis was evaluated by real-time quantitative PCR. The protein expression of E-cadherin, Vimentin, alpha-SMA were evaluated by Western blot. Results The shape of mIMCD-3 cells changed from cobblestone to spindle-like after exposure to TGF-beta 1 at 10ng/mL for 72 hours, which can be inhibited by co-treatment with metformin. TGF-beta 1 remarkably increased the expression of alpha-SMA and PAI-1, while decreased E-cadherin mRNA in mIMCD-3 cells. Metformin inhibited TGF-beta 1-induced reduction of E-cadherin and upregulation of PAI-1. The protein level of E-cadherin was also decreased and alpha-SMA increased by TGF-beta 1. Metformin inhibited TGF-beta 1-induced protein expression of alpha-SMA and vimentin, and downregulation of E-cadherin. It has been reported that impaired renal PPARα signaling promoted renal fibrosis. In this study, we found that metformin upregulated basal level of PPARα mRNA. PPARα target genes including Acadvl and Cpt1a were downregulated by TGF-beta 1 in mIMCD-3 cells, which could be prevented by metformin. Conclusion Metformin inhibits the profibrotic effects of TGF-beta 1 probably through suppression of EMT and maintaining PPARα signaling in mouse inner medullary collecting duct cells.

Rapid and Efficient Conversion of Human Fibroblasts into Functional Neurons by Small Molecules.

SummaryRecent studies have demonstrated that human astrocytes and fibroblasts can be directly converted into functional neurons by small molecules. However, fibroblasts, as a potentially better cell resource for transplantation, are not as easy to reprogram as astrocytes regarding their fate to neurons, and chemically induced neurons (iNs) with low efficiency from fibroblasts resulted in limited application for the treatment of neurological disorders, including depression. Here, we report that human fibroblasts can be efficiently and directly reprogrammed into glutamatergic neuron-like cells by serially exposing cells to a combination of small molecules. These iNs displayed neuronal transcriptional networks, and also exhibited mature firing patterns and formed functional synapses. Importantly, iNs could integrate into local circuits after transplantation into postnatal mouse brain. Our study provides a rapid and efficient transgene-free approach for chemically generating neuron-like cells from human fibroblasts. Furthermore, our approach offers strategies for disease modeling and drug discovery in central nervous system disorders.

SUN-151 TGF-BETA1 MODIFIES HISTONE ACETYLATION AND ACETYL-COENZYME A METABOLISM IN RENAL MYOFIBROBLASTS

Histone acetylation is an important modulator of gene expression in fibrosis. This study examined the effect of the pre-eminent fibrogenic cytokine TGF-beta1 on histone 3 (H3) acetylation and its regulatory kinetics in renal myofibroblasts. Primary cultures of fibroblasts propagated from rat kidneys 3-days after ureteric obstruction were treated with 1ng/ml recombinant TGF-beta1 or vehicle for 48 hours. Flow cytometry (FC) was used to compare expression of alpha smooth muscle actin (SMA) and relative enrichment of H3 marks, while whole transcriptome sequencing (RNA-seq), FC and kinetic assays were used to examine the role of enzymes regulating acetylation. The effect of TGF-beta1 on acetyl-CoA (substrate) availability and synthesis were studied kinetically. TGF-beta1 -induced myofibroblast activation (increased SMA expression) (p<0.001 vs control) was accompanied by a net decrease in total H3 acetylation (p<0.001 vs control), although changes in individual marks were variable. This was paralleled by a generalised reduction in histone acetyltransferases (HAT), and divergent changes in histone deacetylase (HDAC) enzymes at both transcript and protein levels. Globally this was manifest in a reduction in total HAT activity (p<0.001) and increase in HDAC activity (p<0.01). TGF-beta1 induced a shift in cellular metabolism from oxidative respiration to aerobic glycolysis resulting in reduced acetyl-CoA. The reduction in total H3 acetylation could be rescued by providing exogenous citrate (p=ns vs control), a source of acetyl-CoA, without ameliorating changes in HAT/HDAC activity. TGF-beta1 produces a metabolic reprogramming in renal fibroblasts, with less H3 acetylation through reduced acetyltransferase activity, increased deacetylation and changes in carbon availability. Our results suggest that acetyl-CoA availability predominates over HAT and HDAC activity as a key determinant of H3 acetylation in response to TGF-beta1

Yin/Yang expression of CCN family members: Transforming growth factor beta 1, via ALK5/FAK/MEK, induces CCN1 and CCN2, yet suppresses CCN3, expression in human dermal fibroblasts

The role of the microenvironment in driving connective tissue disease is being increasingly appreciated. Matricellular proteins of the CCN family are signaling modifiers that are secreted by cells into the extracellular matrix microenvironment where they have profound, context-dependent effects on organ development, homeostasis and disease. Indeed, CCN proteins are emergent targets for therapeutic intervention. Recent evidence suggests that, in vivo, CCN3 has effects opposing CCN2. Moreover, when CCN3 expression is high, CCN2 expression is low. That is, they appear to be regulated in a yin/yang fashion, leading to the hypothesis that the CCN2:CCN3 ratio is important to control tissue homeostasis. To begin to test the hypothesis that alterations in CCN2:CCN3 expression might be important in skin biology in vivo, we evaluated the relative ex vivo effects of the profibrotic protein TGFbeta1 on dermal fibroblasts on protein and RNA expression of CCN3 and CCN2, as well as the related protein CCN1. We also used signal transduction inhibitors to begin to identify the signal transduction pathways controlling the ability of fibroblasts to respond to TGFbeta1. As anticipated, CCN1 and CCN2 protein and mRNA were induced by TGFbeta1 in human dermal fibroblasts. This induction was blocked by TAK1, FAK, YAP1 and MEK inhibition. Conversely, TGFbeta1 suppressed CCN3 mRNA expression in a fashion insensitive to FAK, MEK, TAK1 or YAP1 inhibition. Unexpectedly, CCN3 protein was not detected in human dermal fibroblasts basally. These data suggest that, in dermal fibroblasts, the profibrotic protein TGFbeta1 has a divergent effect on CCN3 relative to CCN2 and CCN1, both at the mRNA and protein level. Given that the major source in skin in vivo of CCN proteins are fibroblasts, our data are consistent that alterations in CCN2/CCN1: CCN3 ratios in response to profibrotic agents such as TGFbeta1 may play a role in connective tissue pathologies including fibrosis.

Effect of TGF-beta1 on long-term synaptic plasticity and distribution of AMPA receptors in the CA1 field of the hippocampus

Using the methods of electrophysiology and immunohistochemistry, the effect of the transforming factor beta-1(TGF-β1), an anti-inflammatory cytokine, on the long-term post-tetanic potentiation (LTP) in CA1 field hippocampal slices and the distribution of the GluR1 subunit of the AMPA receptor has been studied.It was shown that TGF-β1 at a concentration of 10 ng/ml did not significantly affect the initial stage of LTP and substantially changed the distribution of synaptic AMPA receptors in response to tetanic stimulation. Twenty five minutes after the tetanization, the main pool of AMPA receptors (90%) was due to the postsynaptic density (PSD). By contrast, LTP in the presence of TGF-β1 was accompanied by less pronounced changes in the distribution of AMPA receptors. Their localization in both pre- and postsynaptic regions remained nearly the same as that in the control. It may be suggested that the normal distribution of AMPA receptors in spinous synapses promotes the stabilization of potentiated synapses, thereby retaining LTP for longer terms.

3123 TGFbeta, Early Cytokine Dysregulation, and Airway Smooth Muscle Dysfunction in Cystic Fibrosis

OBJECTIVES/SPECIFIC AIMS: This study aims to first describe the unique cytokine profile and TGFbeta levels of young children with CF, then understand the pathologic effects of TGFbeta on lung function in a CF animal model. These powerful translational studies linking observations in clinical disease with a transgenic mouse model allow us a unique opportunity to investigate the role of TGFbeta in early CF lung disease. METHODS/STUDY POPULATION: Cytokine levels (TGFbeta, TNFalpha, IL-8, IL-6, HNE, and IL-1beta) in bronchoalveolar lavage fluid (BALF) from CF patients (n = 15) and non-CF control patients (n = 21) under 6 years old were determined by ELISA and Luminex assay. Tracheotomized patients without significant underlying lung disease were chosen as non-CF inflamed control patients, as they had similar levels of neutrophilic inflammation and infection as CF patients. The percentage of BAL neutrophils (% PMNs) in each sample was assessed. The relationships between cytokines were analyzed using linear regression and principal components analysis. In animal studies, CF and non-CF mice (n = 4-5 per group) were treated with intratracheal adenoviral TGFbeta1 vector, an empty vector control, or PBS. After one week, animals were collected; lung function, response to the bronchoconstrictor methacholine, and rescue with albuterol were measured utilizing a FlexiVent machine. Lungs were collected for histology. Immunohistochemistry for alpha-SMA was performed and pictures of all cross-sectional airways were obtained. Burden of ASM was assessed by dividing the square root of alpha-SMA stained airway smooth muscle by the basement membrane perimeter length of each airway. RESULTS/ANTICIPATED RESULTS: Patient characteristics of CF and non-CF inflamed control patients were similar in terms of age (3.6 yrs vs 3.3 yrs respectively, p = 0.49), positive BAL culture (13% vs 14%, p = 0.94), and % PMNs (65% vs 56%, p = 0.64). Despite these similarities, TGFbeta levels were 2-fold higher in CF versus non-CF BAL (p = 0.034). Analysis of BAL cytokines from both patient groups showed that three principal components describe 86% of total variance across the cytokine variables. These components represent different contributions from the cytokines, with TGFbeta, IL6, and % PMNs comprising one component of similarly regulated inflammatory markers. These components can distinguish CF versus non-CF patients with 77% accuracy (area under the curve: 0.77). TGFbeta concentrations were uniquely associated with increased IL-6 in CF samples (r = 0.74; p = 0.0015) but did not demonstrate association with other cytokines. After TGFbeta exposure, CF mice demonstrated greater abnormalities in airway resistance than non-CF mice, with heightened response to methacholine. Importantly, this increase in airway obstruction in CF mice was reversible with albuterol treatment, indicating airway smooth muscle dysfunction as a principal driver of lung function abnormalities. Furthermore, TGFbeta induced an increased ASM burden on lung histology in both CF and non-CF mice (p&lt;0.05). IL-6 levels in the BAL of CF mice showed greater increases after TGFbeta treatment compared to non-CF mice (p&lt;0.05). Empty vector control treatment did not cause lung pathology. DISCUSSION/SIGNIFICANCE OF IMPACT: Young children with CF have a unique pattern of pulmonary inflammation compared to inflamed non-CF control patients. In CF, TGFbeta pulmonary levels are uniquely associated with IL-6, a driver of ASM dysfunction in other pulmonary diseases. We followed up this clinical observation study by investigating the effect of TGFbeta on pulmonary disease in a mouse model. CF mice demonstrate increased pulmonary IL-6, airway obstruction, and ASM dysfunction after TGFbeta exposure. This study provides evidence that TGFbeta is associated with a distinct cytokine pattern that may promote ASM dysfunction in early CF lung disease. Understanding the mechanism of early CF pathophysiology will be critical in developing targeted therapeutics that can prevent early lung damage.

TGF beta inhibits HGF, FGF7, and FGF10 expression in normal and IPF lung fibroblasts.

TGF beta is a multifunctional cytokine that is important in the pathogenesis of pulmonary fibrosis. The ability of TGF beta to stimulate smooth muscle actin and extracellular matrix gene expression in fibroblasts is well established. In this report, we evaluated the effect of TGF beta on the expression of HGF, FGF7 (KGF), and FGF10, important growth and survival factors for the alveolar epithelium. These growth factors are important for maintaining type II cells and for restoration of the epithelium after lung injury. Under conditions of normal serum supplementation or serum withdrawal TGF beta inhibited fibroblast expression of HGF, FGF7, and FGF10. We confirmed these observations with genome wide RNA sequencing of the response of control and IPF fibroblasts to TGF beta. In general, gene expression in IPF fibroblasts was similar to control fibroblasts. Reduced expression of HGF, FGF7, and FGF10 is another means whereby TGF beta impairs epithelial healing and promotes fibrosis after lung injury.

TGF beta inhibits expression of SP-A, SP-B, SP-C, but not SP-D in human alveolar type II cells

TGF beta is a multifunctional cytokine that regulates alveolar epithelial cells as well as immune cells and fibroblasts. TGF beta inhibits surfactant protein A, B and C expression in fetal human lung and can inhibit type II cell proliferation induced by FGF7 (KGF). However, little is known about direct effects of TGF beta on adult human type II cells. We cultured alveolar type II cells under air/liquid interface conditions to maintain their state of differentiation with or without TGF beta. TGF beta markedly decreased expression of SP-A, SP-B, SP-C, fatty acid synthase, and the phospholipid transporter ABCA3. However, TGF beta increased protein levels of SP-D with little change in mRNA levels, indicating that it is regulated independently from other components of surfactant. TGF beta is a negative regulator of both the protein and the phospholipid components of surfactant. TGF beta did not induce EMT changes in highly differentiated human type II cells. SP-D is an important host defense molecule and regulated independently from the other surfactant proteins.Taken together these data are the first report of the effect of TGF beta on highly differentiated adult human type II cells. The effects on the surfactant system are likely important in the development of fibrotic lung diseases.

Antioxidants and NOX1/NOX4 inhibition blocks TGFβ1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts.

TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents.

Abstract P107: Hypertension Enhances the Differentiation of Cardiac Fibroblasts Into Myofibroblasts After TGF-beta1 Treatment

Objectives: In cardiac fibrosis associated with hypertension, TGF-beta1 plays a key role by acting on differentiation of cardiac fibroblasts (CF) into alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts. In this study, we tested the effect of TGF-beta1 during the myofibroblast differentiation process of CF from normotensive and hypertensive rats. Methods: CF were obtained by enzymatic digestion of hearts isolated from Spontaneously Hypertensive (hCF) and normotensive Wistar Kyoto (nCF) rats (n=5 rat/group). Gene and protein expression in CF was evaluated by Western blot and qRT-PCR analyses, respectively. Immunohistochemistry analysis for integrin alpha-v beta-5 was performed on rat cardiac tissue (n=5 rat/group). Results: Cultured hCF showed an enhanced SMAD2/3 activation and alpha-SMA protein expression after treatment with TGF-beta1 (5 ng/ml) in comparison with nCF. Alpha-SMA up-regulation was further confirmed by qRT-PCR analysis that showed a significant increase in alpha-SMA gene expression in hCF after TGF-beta1 treatment (2.78±0.25 vs 2.01±0.21 fold increase, p &lt;0.05). Moreover, immunostaining on cardiac tissues revealed a higher expression of integrin alpha-v beta-5 in hypertensive vs normotensive rat hearts (345.3±170.0 vs 48.2±22.3 mm 2 of integrin-positive area, p &lt;0.05). This result was also confirmed in vitro ; indeed, integrin alpha-v beta-5 gene expression in hCF increased 2.8-fold in basal condition and 5.12-fold after TGF-beta1 treatment when compared to untreated nCF. Conclusions: Taken together, these results suggest that hCF are more prone to upregulate integrin alpha-v beta-5 and consequently differentiate into myofibroblasts in vitro under TGF-beta1 treatment. Thus, targeting alpha-v beta-5 might open a novel prospective for the treatment of fibrosis in hypertensive hearts likely reducing integrin-mediated TGF-beta1 activation.

Effect of TGF-beta1 on MMP/TIMP and TGF-beta1 receptors in great saphenous veins and its significance on chronic venous insufficiency.

Objectives Transforming growth factor-beta1 (TGF-β1) may participate in local chronic inflammatory processes in varicose veins and in venous wall structure modifications through regulation of matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitor of metalloproteinase (TIMP)). The aim of this study was to analyze the effect of TGF-β1 in the vein wall, namely on the gene expression of selected MMP, TIMP and TGF-β1 receptors. Methods Healthy vein samples were harvested from eight subjects who underwent coronary bypass graft surgery with great saphenous vein. Each vein sample was divided into two segments, which were cultivated separately invitro (one of the segments had TGF-β1 added) and then submitted to gene expression analysis. Results In the TGF-β1 supplemented group, there was a general increase in the mean gene expression. Specifically, expression of MMP9, MMP12, TIMP1 and TIMP2 were statistically significant. Conclusion The results of this study demonstrate that the gene expression of MMP9, MMP12, TIMP1 and TIMP2 was influenced by the addition of TGF-β1. These results may be translated to chronic venous insufficiency framework and suggest involvement of TGF-β1 in the vein wall pathology.

P-127 GPR15 Expression in Mouse and Human

We have shown previously that GPR15, an orphan G-protein coupled receptor known as a SIV/HIV co-receptor, controls the specific homing of T cells, preferentially FOXP3+ regulatory T cells (Tregs), to the large intestine lamina propria (LILP). GPR15-deficient mice had fewer Tregs in LILP and were prone to develop more severe large intestine inflammation, which was rescued by the transfer of GPR15-sufficient Tregs. In the current studies, we examined the factors affecting GPR15 expression in vitro and in vivo, and also the expression pattern of GPR15 in human T cells. For mouse T cells, we used Gpr15gfp/+Foxp3mrfp mice and sorted naive CD4+ T cells, Tregs with naive phenotype (CD62LhiCD44lo), or cells with memory phenotype (CD62LloCD44hi). For human T cells, CD45RAhiCD25lo cells were sorted as naive T cells. These cells were stimulated with anti-CD3/CD28 in the presence of various cytokines or short-chain fatty acids. Later, GPR15 expression was determined by GFP (in mouse T cells) and by monoclonal antibody staining (in human T cells). In mouse T cells, a combination of IL-21 (or IL-6) and TGFbeta induced GPR15 expression preferentially in Tregs in vitro. The effect of TGFbeta in vivo was confirmed in vivo, but IL-21RKOxIL-6KO mice still showed GPR15 expression in vivo. Antibiotic treatment in mice severely reduced GPR15 expression level in vivo, suggesting that the presence of gut microbiota affects GPR15 expression. However, in vitro treatment of short-chain fatty acids did not induce GPR15 expression in T cells. In human T cells, iTregs and Th1 cells showed a significant expression of GPR15 in vitro. GPR15 is preferentially expressed in Tregs. In mice, GPR15 is induced by gut microbiota in vivo or by a combination of Stat3-dependent cytokines and TGFbeta in vitro.

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 5066: Reprogramming the TGF-beta signaling in cancer-associated fibroblasts inhibits ovarian cancer progression

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. While major research efforts have been dedicated to identify disease related genes and mutations in cancer cells, the role of the stromal tumor microenvironment in disease progression is relatively unexplored. We previously reported that while ovarian cancer cells have minimal response to TGF-beta, TGF-beta induced secretory proteins from cancer associated fibroblasts (CAFs) promote ovarian cancer motility and invasion potential. The present study seeks to further evaluate the involvement of TGF-beta signaling in CAFs in the progression of ovarian cancer and the therapeutic potential of targeting such signaling in cancer treatment. By comparing the gene expression profiles of laser-microdissected ovarian tumor stromal tissues from high-grade serous ovarian cancer (HGSOC) patients with short and long survival, a differentially expressed gene signature was identified. Using the IPA platform, signaling pathways and upstream regulators activated in CAFs from patients with short survival were identified. TGF-beta signaling was found to be significantly activated (p = 2.47E-03), while the ligand TGF-beta 1 was identified as the most significant upstream regulator (Z-score = 5.189, p = 8.85E-29), suggested the involvement of CAF TGF-beta signaling in poor patient survival. Further immunohistochemistry and survival analyses revealed the clinical relevance of multiple TGF-beta regulated stromal genes, including VCAN, MFAP5 and COMP in poor disease prognosis. Subsequently, cell culture studies were performed to evaluate the effects of TGF-beta in ovarian cancer progression. While exogenous TGF-beta did not affect proliferation, motility and invasion potential of ovarian cancer cells directly, TGF-beta promoted ovarian cancer cells motility and invasion potential in a co-culture system of cancer cells and CAFs. Similarly, treatment of exogenous TGF-beta-induced CAF secretory proteins promoted ovarian cancer progression, suggested the presence of CAFs is required for TGF-beta mediated ovarian cancer progression and targeting such TGF-beta signaling in CAFs could be a novel modality of ovarian cancer treatment. To reprogram CAFs by targeting its TGF-beta signaling, multiple approaches were tested in co-culture systems. Using either siRNAs or small molecule inhibitors, TGF-beta signaling in CAFs was effectively suppressed and inhibited ovarian cancer progression was observed. Based on our current findings, animal studies will be performed to further evaluate the efficacy of targeting the CAF TGF-beta signaling pathway in ovarian cancer treatment. This study presents the paradigm which activation of TGF-beta signaling in ovarian CAFs is significantly associated to the poor clinical outcomes. Targeting such pathway activation in CAFs could effectively suppress tumor progression and thereby presented a novel approach in cancer treatment. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Reprogramming the TGF-beta signaling in cancer-associated fibroblasts inhibits ovarian cancer progression. [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 5066. doi:10.1158/1538-7445.AM2015-5066

Fibulin-2 is a key mediator of the pro-neurogenic effect of TGF-beta1 on adult neural stem cells

Transforming growth factor beta 1 (TGF-beta1), an anti-inflammatory cytokine, has been shown to have pro-neurogenic effects on adult Neural Stem Cells (aNSC) from the dentate gyrus and in vivo models. Here, we expanded the observation of the pro-neurogenic effect of TGF-beta1 on aNSC from the subventricular zone (SVZ) of adult rats and performed a functional genomic analysis to identify candidate genes to mediate its effect. 10 candidate genes were identified by microarray analysis and further validated by qRT-PCR. Of these, Fibulin-2 was increased 477-fold and its inhibition by siRNA blocks TGF-beta1 pro-neurogenic effect. Curiously, Fibulin-2 was not expressed by aNSC but by a GFAP-positive population in the culture, suggesting an indirect mechanism of action. TGF-beta1 also induced Fibulin-2 in the SVZ in vivo. Interestingly, 5 out of the 10 candidate genes identified are known to interact with integrins, paving the way for exploring their functional role in adult neurogenesis. In conclusion, we have identified 10 genes with putative pro-neurogenic effects, 5 of them related to integrins and provided proof that Fibulin-2 is a major mediator of the pro-neurogenic effects of TGF-beta1. These data should contribute to further exploring the molecular mechanism of adult neurogenesis of the genes identified and the involvement of the integrin pathway on adult neurogenesis.