BACKGROUND

Crohn's disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7.

METHODS

In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohn's disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohn's Disease Activity Index (CDAI) score of less than 150, with maintenance of remission for at least 2 weeks, and the safety of mongersen treatment. A secondary outcome was clinical response (defined as a reduction of 100 points or more in the CDAI score) at day 28.

RESULTS

The proportions of patients who reached the primary end point were 55% and 65% for the 40-mg and 160-mg mongersen groups, respectively, as compared with 10% for the placebo group (P<0.001). There was no significant difference in the percentage of participants reaching clinical remission between the 10-mg group (12%) and the placebo group. The rate of clinical response was significantly greater among patients receiving 10 mg (37%), 40 mg (58%), or 160 mg (72%) of mongersen than among those receiving placebo (17%) (P=0.04, P<0.001, and P<0.001, respectively). Most adverse events were related to complications and symptoms of Crohn's disease.

CONCLUSIONS

We found that study participants with Crohn's disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo. (Funded by Giuliani; EudraCT number, 2011-002640-27.).

During development of TGF-β1-initiated fibroproliferative disorders, NADPH oxidases (NOX family members) generate reactive oxygen species (ROS) resulting in downstream transcription of a subset genes encoding matrix structural elements and profibrotic factors. Prominent among the repertoire of disease-implicated genes is the TGF-β1 target gene encoding the potent profibrotic matricellular protein plasminogen activator inhibitor-1 (PAI-1 or SERPINE1). PAI-1 is the major physiologic inhibitor of the plasmin-based pericellular cascade and a causative factor in the development of vascular thrombotic and fibroproliferative disorders. ROS generation in response to TGF-β1 stimulation is rapid and precedes PAI-1 induction; engagement of non-SMAD (e.g., EGFR, Src kinase, MAP kinases, p53) and SMAD2/3 pathways are both required for PAI-1 expression and are ROS-dependent. Recent findings suggest a novel role for p53 in TGF-β1-induced PAI-1 transcription that involves ROS generation and p53/SMAD interactions. Targeting ROS and ROS-activated cellular events is likely to have therapeutic implications in the management of fibrotic disorders, particularly in the context of prolonged TGF-β1 signaling.

Hepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6-independent hepcidin up-regulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-β1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6-dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6-independent, but partially TGF-β-dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis.

OBJECTIVE

To elucidate molecular pathways contributing to metastatic cancer progression and poor clinical outcome in serous ovarian cancer.

METHODS

Poor survival signatures from three different serous ovarian cancer datasets were compared and a common set of genes was identified. The predictive value of this gene signature was validated in independent datasets. The expression of the signature genes was evaluated in primary, metastatic, and/or recurrent cancers using quantitative PCR and in situ hybridization. Alterations in gene expression by TGF-β1 and functional consequences of loss of COL11A1 were evaluated using pharmacologic and knockdown approaches, respectively.

RESULTS

We identified and validated a 10-gene signature (AEBP1, COL11A1, COL5A1, COL6A2, LOX, POSTN, SNAI2, THBS2, TIMP3, and VCAN) that is associated with poor overall survival (OS) in patients with high-grade serous ovarian cancer. The signature genes encode extracellular matrix proteins involved in collagen remodeling. Expression of the signature genes is regulated by TGF-β1 signaling and is enriched in metastases in comparison with primary ovarian tumors. We demonstrate that levels of COL11A1, one of the signature genes, continuously increase during ovarian cancer disease progression, with the highest expression in recurrent metastases. Knockdown of COL11A1 decreases in vitro cell migration, invasion, and tumor progression in mice.

CONCLUSIONS

Our findings suggest that collagen-remodeling genes regulated by TGF-β1 signaling promote metastasis and contribute to poor OS in patients with serous ovarian cancer. Our 10-gene signature has both predictive value and biologic relevance and thus may be useful as a therapeutic target.

Transforming growth factor-β1 (TGF-β) was first implicated in mammary epithelial development by Daniel and Silberstein in 1987 and in breast cancer cells and hormone resistance by Lippman and colleagues in 1988. TGF-β is critically important for mammary morphogenesis and secretory function through specific regulation of epithelial proliferation, apoptosis, and extracellular matrix. Differential TGF-β effects on distinct cell types are compounded by regulation at multiple levels and the influence of context on cellular responses. Studies using controlled expression and conditional-deletion mouse models underscore the complexity of TGF-β biology across the cycle of mammary development and differentiation. Early loss of TGF-β growth regulation in breast cancer evolves into fundamental deregulation that mediates cell interactions and phenotypes driving invasive disease. Two outstanding issues are to understand the mechanisms of biological control in situ and the circumstances by which TGF-β regulation is subverted in neoplastic progression.

Activation of the PI3 kinase pathway can induce skeletal muscle hypertrophy, defined as an increase in skeletal muscle mass. In mammals, skeletal muscle hypertrophy occurs as a result of an increase in the size, as opposed to the number, of pre-existing skeletal muscle fibers. This pathway's effects on skeletal muscle have been implicated most prominently downstream of Insulin-like growth factor 1 signaling. IGF-1's pro-hypertrophy activity comes predominantly through its ability to activate the Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Akt is a serine-threonine protein kinase that can induce protein synthesis and block the transcriptional upregulation of key mediators of skeletal muscle atrophy, the E3 ubiquitin ligases MuRF1 and MAFbx (also called Atrogin-1), by phosphorylating and thereby inhibiting the nuclear translocation of the FOXO (also called "forkhead") family of transcription factors. Once phosphorylated by Akt, the FOXOs are excluded from the nucleus, and upregulation of MuRF1 and MAFbx is blocked. MuRF1 and MAFbx mediate atrophy by ubiquitinating particular protein substrates, causing them to undergo degradation by the proteasome. MuRF1's substrates include several components of the sarcomeric thick filament, including Myosin Heavy Chain (MyHC). Thus, by blocking MuRF1 activation, IGF-1 helps prevent the breakdown of the thick filament under atrophy conditions.IGF1/PI3K/Akt signaling also can dominantly inhibit the effects of a secreted protein called "myostatin," which is a member of the TGFβ family of proteins. Deletion or inhibition of myostatin causes an increase in skeletal muscle size, because myostatin acts both to inhibit myoblast differentiation and to block the Akt pathway. Thus by blocking myostatin, PI3K/Akt activation stimulates differentiation and protein synthesis by this distinct mechanism. Myostatin induces the phosphorylation and activation of the transcription factors of Smad2 and Smad3, downstream of the ActRII (Activin Receptor type II)/Alk (Activin Receptor-like kinase) receptor complex. Other TGFβ-like molecules can also block differentiation, including TGF-b1, GDF-11, activinA, BMP-2 and BMP-7. As mentioned, myostatin also downregulates the Akt/mTOR/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using siRNA to RAPTOR, a component of "TORC1" (TOR signaling Complex 1), increases myostatin-induced phosphorylation of Smad2; this establishes a "feed-forward mechanism," because myostatin can downregulates TORC1, and this downregulation in turn amplifies myostatin signaling. Blockade of RAPTOR also facilitates myostatin's inhibition of muscle differentiation. When added to post-differentiated myotubes, myostatin causes a decrease in their diameter - however, this does not happen through the normal "atrophy pathway." Rather than causing upregulation of the E3 ubiquitin ligases MuRF1 and MAFbx, previously shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation, such as MyoD and myogenin. These findings show that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct "atrophy program."

Because fibrotic kidneys exhibit aberrant activation of β-catenin signaling, this pathway may be a potential target for antifibrotic therapy. In this study, we examined the effects of β-catenin activation on tubular epithelial-mesenchymal transition (EMT) in vitro and evaluated the therapeutic efficacy of the peptidomimetic small molecule ICG-001, which specifically disrupts β-catenin-mediated gene transcription, in obstructive nephropathy. In vitro, ectopic expression of stabilized β-catenin in tubular epithelial (HKC-8) cells suppressed E-cadherin and induced Snail1, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) expression. ICG-001 suppressed β-catenin-driven gene transcription in a dose-dependent manner and abolished TGF-β1-induced expression of Snail1, PAI-1, collagen I, fibronectin, and α-smooth muscle actin (α-SMA). This antifibrotic effect of ICG-001 did not involve disruption of Smad signaling. In the unilateral ureteral obstruction model, ICG-001 ameliorated renal interstitial fibrosis and suppressed renal expression of fibronectin, collagen I, collagen III, α-SMA, PAI-1, fibroblast-specific protein-1, Snail1, and Snail2. Late administration of ICG-001 also effectively attenuated fibrotic lesions in obstructive nephropathy. In conclusion, inhibiting β-catenin signaling may be an effective approach to the treatment of fibrotic kidney diseases.

In addition to mesenchymal cells, endothelial cells may contribute to fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). We investigated whether human intestinal microvascular endothelial cells (HIMEC) undergo EndoMT and contribute to fibrosis in human and experimental inflammatory bowel disease (IBD). HIMEC were exposed to TGF-β1, IL-1β, and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) and evaluated for morphological, phenotypic, and functional changes compatible with EndoMT. Genomic analysis was used to identify transcription factors involved in the transformation process. Evidence of in situ and in vivo EndoMT was sought in inflamed human and murine intestine. The combination of TGF-β1, IL-1β and TNF-α, or activated LPMC supernatants induced morphological and phenotypic changes consistent with EndoMT with a dominant effect by IL-1. These changes persisted after removal of the inducing agents and were accompanied by functional loss of acetylated LDL-uptake and migratory capacity, and acquisition of de novo collagen synthesis capacity. Sp1 appeared to be the main transcriptional regulator of EndoMT. EndoMT was detected in microvessels of inflammatory bowel disease (IBD) mucosa and experimental colonic fibrosis of Tie2-green fluorescent protein (GFP) reporter-expressing mice. In conclusion, chronic inflammation induces transdifferentiation of intestinal mucosal microvascular cells into mesenchymal cells, suggesting that the intestinal microvasculature contributes to IBD-associated fibrosis through the novel process of EndoMT.

Aberrant neovascularization contributes to diseases such as cancer, blindness and atherosclerosis, and is the consequence of inappropriate angiogenic signalling. Although many regulators of pathogenic angiogenesis have been identified, our understanding of this process is incomplete. Here we explore the transcriptome of retinal microvessels isolated from mouse models of retinal disease that exhibit vascular pathology, and uncover an upregulated gene, leucine-rich alpha-2-glycoprotein 1 (Lrg1), of previously unknown function. We show that in the presence of transforming growth factor-β1 (TGF-β1), LRG1 is mitogenic to endothelial cells and promotes angiogenesis. Mice lacking Lrg1 develop a mild retinal vascular phenotype but exhibit a significant reduction in pathological ocular angiogenesis. LRG1 binds directly to the TGF-β accessory receptor endoglin, which, in the presence of TGF-β1, results in promotion of the pro-angiogenic Smad1/5/8 signalling pathway. LRG1 antibody blockade inhibits this switch and attenuates angiogenesis. These studies reveal a new regulator of angiogenesis that mediates its effect by modulating TGF-β signalling.

Telomerase activation through induction of telomerase reverse transcriptase (hTERT) contributes to malignant transformation by stabilizing telomeres. Clinical studies demonstrate that higher hTERT expression is associated with cancer progression and poor outcomes, but the underlying mechanism is unclear. Because epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are key factors in cancer metastasis and relapse, and hTERT has been shown to exhibit multiple biological activities independently of its telomere-lengthening function, we address a potential role of hTERT in EMT and CSCs using gastric cancer (GC) as a model. hTERT overexpression promotes, whereas its inhibition suppresses, EMT and stemness of GC cells, respectively. Transforming growth factor (TGF)-β1 and β-catenin-mediated EMT was abolished by small interfering RNA depletion of hTERT expression. hTERT interacts with β-catenin, enhances its nuclear localization and transcriptional activity, and occupies the β-catenin target vimentin promoter. All these hTERT effects were independent of its telomere-lengthening function or telomerase activity. hTERT and EMT marker expression correlates positively in GC samples. Mouse experiments demonstrate the in vivo stimulation of hTERT on cancer cell colonization. Collectively, hTERT stimulates EMT and induces stemness of cancer cells, thereby promoting cancer metastasis and recurrence. Thus, targeting hTERT may prevent cancer progression by inhibiting EMT and CSCs.

Most chronic kidney injuries inevitably progress to irreversible renal fibrosis. Tubular epithelial-to-mesenchymal transition (EMT) is recognized to play pivotal roles in the process of renal fibrosis. However, a comprehensive understanding of the pathogenesis of renal scar formation and progression remains an urgent task for renal researchers. The endogenously produced microRNAs (miRNAs), proved to play important roles in gene regulation, probably regulate most genes involved in EMT. In this study, we applied microarray analysis to investigate the expression profiles of miRNA in murine interstitial fibrotic kidneys induced by unilateral ureteral obstruction (UUO). It was found that miR-200a and miR-141, two members of the miR-200 family, were downregulated at the early phase of UUO. In TGF-β1-induced tubular EMT in vitro, it was also found that the members of the miR-200 family were downregulated in a Smad signaling-dependent manner. It was demonstrated that the miR-200 family was responsible for protecting tubular epithelial cells from mesenchymal transition by target suppression of zinc finger E-box-binding homeobox (ZEB) 1 and ZEB2, which are E-cadherin transcriptional repressors. The results suggest that downregulation of the miR-200 family initiates the dedifferentiation of renal tubules and progression of renal fibrosis, which might provide important targets for novel therapeutic strategies.

BACKGROUND

Previous studies of bioactive molecules in platelet-rich plasma (PRP) have documented growth factor concentrations that promote tissue healing. However, the effects of leukocytes and inflammatory molecules in PRP have not been defined.

OBJECTIVE

The hypothesis for this study was that the concentration of growth factors and catabolic cytokines would be dependent on the cellular composition of PRP.

METHODS

Controlled laboratory study.

METHODS

Platelet-rich plasma was made from 11 human volunteers using 2 commercial systems: Arthrex ACP (Autologous Conditioned Plasma) Double Syringe System (PRP-1), which concentrates platelets and minimizes leukocytes, and Biomet GPS III Mini Platelet Concentrate System (PRP-2), which concentrates both platelets and leukocytes. Transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-AB (PDGF-AB), matrix metalloproteinase-9 (MMP-9), and interleukin-1β (IL-1β) were measured with enzyme-linked immunosorbent assay (ELISA).

RESULTS

The PRP-1 system consisted of concentrated platelets (1.99×) and diminished leukocytes (0.13×) compared with blood, while PRP-2 contained concentrated platelets (4.69×) and leukocytes (4.26×) compared with blood. Growth factors were significantly increased in PRP-2 compared with PRP-1 (TGF-β1: PRP-2 = 89 ng/mL, PRP-1 = 20 ng/mL, P < .05; PDGF-AB: PRP-2 = 22 ng/mL, PRP-1 = 6.4 ng/mL, P < .05). The PRP-1 system did not have a higher concentration of PDGF-AB compared with whole blood. Catabolic cytokines were significantly increased in PRP-2 compared with PRP-1 (MMP-9: PRP-2 = 222 ng/mL, PRP-1 = 40 ng/mL, P < .05; IL-1β: PRP-2 = 3.67 pg/mL, PRP-1 = 0.31 pg/mL, P < .05). Significant, positive correlations were found between TGF-β1 and platelets (r(2) = .75, P < .001), PDGF-AB and platelets (r(2) = .60, P < .001), MMP-9 and neutrophils (r(2) = .37, P < .001), IL-1β and neutrophils (r(2) = .73, P < .001), and IL-1β and monocytes (r(2) = .75, P < .001).

CONCLUSIONS

Growth factor and catabolic cytokine concentrations were influenced by the cellular composition of PRP. Platelets increased anabolic signaling and, in contrast, leukocytes increased catabolic signaling molecules. Platelet-rich plasma products should be analyzed for content of platelets and leukocytes as both can influence the biologic effects of PRP.

CONCLUSIONS

Depending on the clinical application, preparations of PRP should be considered based on their ability to concentrate platelets and leukocytes with sensitivity to pathologic conditions that will benefit most from increased platelet or reduced leukocyte concentration.

TGF-β1 is a regulatory cytokine that has an important role in controlling T cell differentiation. T cell-produced TGF-β1 acts on T cells to promote Th17 cell differentiation and the development of experimental autoimmune encephalomyelitis (EAE). However, the exact TGF-β1-producing T cell subset required for Th17 cell generation and its cellular mechanism of action remain unknown. Here we showed that deletion of the Tgfb1 gene from activated T cells and Treg cells, but not Treg cells alone, abrogated Th17 cell differentiation, resulting in almost complete protection from EAE. Furthermore, differentiation of T cells both in vitro and in vivo demonstrated that TGF-β1 was highly expressed by Th17 cells and acted in a predominantly autocrine manner to maintain Th17 cells in vivo. These findings reveal an essential role for activated T cell-produced TGF-β1 in promoting the differentiation of Th17 cells and controlling inflammatory diseases.

Myeloid-derived suppressor cells (MDSCs) potently suppress the anti-tumor immune responses and also orchestrate the tumor microenvironment that favors tumor angiogenesis and metastasis. The molecular networks regulating the accumulation and functions of tumor-expanded MDSCs are largely unknown. In this study, we identified microRNA-494 (miR-494), whose expression was dramatically induced by tumor-derived factors, as an essential player in regulating the accumulation and activity of MDSCs by targeting of phosphatase and tensin homolog (PTEN) and activation of the Akt pathway. TGF-β1 was found to be the main tumor-derived factor responsible for the upregulation of miR-494 in MDSCs. Expression of miR-494 not only enhanced CXCR4-mediated MDSC chemotaxis but also altered the intrinsic apoptotic/survival signal by targeting of PTEN, thus contributing to the accumulation of MDSCs in tumor tissues. Consequently, downregulation of PTEN resulted in increased activity of the Akt pathway and the subsequent upregulation of MMPs for facilitation of tumor cell invasion and metastasis. Knockdown of miR-494 significantly reversed the activity of MDSCs and inhibited the tumor growth and metastasis of 4T1 murine breast cancer in vivo. Collectively, our findings reveal that TGF-β1-induced miR-494 expression in MDSCs plays a critical role in the molecular events governing the accumulation and functions of tumor-expanded MDSCs and might be identified as a potential target in cancer therapy.

We studied the expression and regulatory effects of ESC self-renewal molecule Nanog in colorectal cancer (CRC). Immunohistochemical analysis of 175 colorectal tumor samples showed that overexpression of Nanog was strongly correlated with poor prognosis, lymph node metastasis and Dukes classification for CRC. Univariate and multivariate survival analyses further indicated that Nanog expression was a potential prognostic factor for CRC. Gain-of-function analysis revealed that lentivirus-mediated Nanog overexpression promoted proliferation, motility and migration of human CRC cells. Interestingly, we found that Nanog played as both an inducer and a receipt of epithelial-mesenchymal transition (EMT) related signals. Nanog induced expression of Slug and Snail, two major regulator of EMT. Meanwhile, Nanog could also be regulated by Snail and initiated by TGF-β1. Our data demonstrate self-renewal gene Nanog has a prognostic role in CRC, which functions in progression of CRC by promoting proliferation, invasion, and motility of human CRC cells, and participates EMT process during CRC progression.

BACKGROUND

Clinical studies claim that platelet-rich plasma (PRP) shortens recovery times because of its high concentration of growth factors that may enhance the tissue repair process. Most of these studies obtained PRP using different separation systems, and few analyzed the content of the PRP used as treatment.

OBJECTIVE

This study characterized the composition of single-donor PRP produced by 3 commercially available PRP separation systems.

METHODS

Controlled laboratory study.

METHODS

Five healthy humans donated 100 mL of blood, which was processed to produce PRP using 3 PRP concentration systems (MTF Cascade, Arteriocyte Magellan, Biomet GPS III). Platelet, white blood cell (WBC), red blood cell, and fibrinogen concentrations were analyzed by automated systems in a clinical laboratory, whereas ELISA determined the concentrations of platelet-derived growth factor αβ and ββ (PDGF-αβ, PDGF-ββ), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF).

RESULTS

There was no significant difference in mean PRP platelet, red blood cell, active TGF-β1, or fibrinogen concentrations among PRP separation systems. There was a significant difference in platelet capture efficiency. The highest platelet capture efficiency was obtained with Cascade, which was comparable with Magellan but significantly higher than GPS III. There was a significant difference among all systems in the concentrations of WBC, PDGF-αβ, PDGF-ββ, and VEGF. The Cascade system concentrated leukocyte-poor PRP, compared with leukocyte-rich PRP from the GPS III and Magellan systems.

CONCLUSIONS

The GPS III and Magellan concentrate leukocyte-rich PRP, which results in increased concentrations of WBCs, PDGF-αβ, PDGF-ββ, and VEGF as compared with the leukocyte-poor PRP from Cascade. Overall, there was no significant difference among systems in the platelet concentration, red blood cell, active TGF-β1, or fibrinogen levels.

CONCLUSIONS

Products from commercially available PRP separation systems produce differing concentrations of growth factors and WBCs. Further research is necessary to determine the clinical relevance of these findings.

Angiotensin II (Ang II) stimulates pathologic myocardial fibrosis. Cardiac fibroblasts (CFb) and myofibroblasts mediate this response, perhaps in part by indirect production of specific cytokines. We sought to determine if Ang II could stimulate transforming growth factor-beta1 (TGF-beta1) gene expression and protein production in adult rat CFb and two cardiac myofibroblast cell types, scar myofibroblasts (MyoFb) and valvular interstitial cells (VIC). Confluent CFb, MyoFb, and VIC in serum-deprived (0.4% FCS) media were treated with Ang II (10(-7) m for CFb; 10(-9) m for MyoFb, VIC) for 24 h. Untreated cells served as controls. Culture media was collected and TGF-beta1 levels determined in triplicate using a sandwich ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to determine TGF-beta1 mRNA expression. Ang II increased CFb (P<0.02) and VIC (P<0.04) TGF-beta1 mRNA expression, while the increase in MyoFb was not statistically significant. MyoFb produced the highest TGF-beta1 levels under control conditions compared to VIC and CFb. Ang II stimulated further TGF-beta1 secretion in VIC and CFb, but not MyoFb. The AT1 receptor antagonist Losartan (10(-7) m) greatly attenuated Ang II-stimulated TGF-B1 secretion and decreased TGF-beta1 immunostaining in VIC. The AT2 receptor antagonist PD123177 (10(-7) m) also decreased secretion and immunostaining of TGF-beta1 in VIC, but to a lesser extent than Losartan. TGF-beta1 secretion by MyoFb was unaffected by Losartan and PD123177, although TGF-B1 immunostaining was absent or greatly decreased, respectively, compared to Ang II-treated MyoFb. Ang II stimulates TGF-beta1 gene expression and/or protein production in cardiac fibroblast-like cells which may act as an autocrine/paracrine stimulus to collagen formation. Furthermore, TGF-beta1 production and secretion in these cells can be modulated by specific Ang II receptor antagonists, suggesting a potential benefit in preventing/attenuating pathologic myocardial fibrosis.

BACKGROUND

The initiation and regulation of pulmonary fibrosis are not well understood. IL-33, an important cytokine for respiratory diseases, is overexpressed in the lungs of patients with idiopathic pulmonary fibrosis.

OBJECTIVE

We aimed to determine the effects and mechanism of IL-33 on the development and severity of pulmonary fibrosis in murine bleomycin-induced fibrosis.

METHODS

Lung fibrosis was induced by bleomycin in wild-type or Il33r (St2)(-/-) C57BL/6 mice treated with the recombinant mature form of IL-33 or anti-IL-33 antibody or transferred with type 2 innate lymphoid cells (ILC2s). The development and severity of fibrosis was evaluated based on lung histology, collagen levels, and lavage cytology. Cytokine and chemokine levels were quantified by using quantitative PCR, ELISA, and cytometry.

RESULTS

IL-33 is constitutively expressed in lung epithelial cells but is induced in macrophages by bleomycin. Bleomycin enhanced the production of the mature but reduced full-length form of IL-33 in lung tissue. ST2 deficiency, anti-IL-33 antibody treatment, or alveolar macrophage depletion attenuated and exogenous IL-33 or adoptive transfer of ILC2s enhanced bleomycin-induced lung inflammation and fibrosis. These pathologic changes were accompanied, respectively, by reduced or increased IL-33, IL-13, TGF-β1, and inflammatory chemokine production in the lung. Furthermore, IL-33 polarized M2 macrophages to produce IL-13 and TGF-β1 and induced the expansion of ILC2s to produce IL-13 in vitro and in vivo.

CONCLUSIONS

IL-33 is a novel profibrogenic cytokine that signals through ST2 to promote the initiation and progression of pulmonary fibrosis by recruiting and directing inflammatory cell function and enhancing profibrogenic cytokine production in an ST2- and macrophage-dependent manner.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.

OBJECTIVE

To examine the role of galectin-3 in pulmonary fibrosis.

METHODS

We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.

RESULTS

Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1-induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β-induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.

CONCLUSIONS

This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.

Invasion of hepatocellular carcinoma (HCC) cells is a leading cause of intrahepatic dissemination and metastasis. Autophagy is considered to be an important mediator in the invasion of cancer cells. However, the precise contribution of autophagy to cancer cell invasion and underlying mechanisms remain unclear. Autophagy was induced in HepG2 and BEL7402 cells by starvation in Hank's balanced salt solution. Induction of autophagy inhibited the expression of epithelial markers and induced expression of mesenchymal markers as well as matrix metalloproteinase-9 stimulating cell invasion. Starvation-induced autophagy promoted the expression of epithelial-mesenchymal transition (EMT) markers and invasion in HepG2 and BEL7402 cells through a transforming growth factor-beta (TGF-β)/Smad3 signaling-dependent manner. The small interfering RNAs (siRNAs) for Atg3 or Atg7 and chloroquine inhibited autophagy of HepG2 and BEL7402 cells during starvation, resulting in suppression of EMT and diminished invasiveness of HCC cells. Administration of SIS3 also attenuated EMT and invasion of HepG2 and BEL7402 cells during starvation. Recombinant TGF-β1 was capable of rescuing EMT and invasion that was inhibited by siRNA for Atg3 and 7 in HepG2 and BEL7402 cells under starvation. These findings suggest that autophagy is critical for the invasion of HCC cells through the induction of EMT and that activation of TGF-β/Smad3-dependent signaling plays a key role in regulating autophagy-induced EMT. Inhibition of autophagy may represent a novel target for therapeutic interventions.

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ~2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2 × 10(-8)) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0 × 10(-9)). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1 × 10(-7)), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.

Renal fibrosis is a final stage of many forms of kidney disease and leads to impairment of kidney function. The molecular pathogenesis of renal fibrosis is currently not well-understood. microRNAs (miRNAs) are important players in initiation and progression of many pathologic processes including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in kidney injury and repair is not well-characterized. In the present study, we found a unique miRNA signature associated with unilateral ureteral obstruction (UUO)-induced renal fibrosis. We found altered expression in UUO kidneys of miRNAs that have been shown to be responsive to stimulation by transforming growth factor (TGF)-β1 or TNF-α. Among these miRNAs, miR-21 demonstrated the greatest increase in UUO kidneys. The enhanced expression of miR-21 was located mainly in distal tubular epithelial cells. miR-21 expression was upregulated in response to treatment with TGF-β1 or TNF-α in human renal tubular epithelial cells in vitro. Furthermore, we found that blocking miR-21 in vivo attenuated UUO-induced renal fibrosis, presumably through diminishing the expression of profibrotic proteins and reducing infiltration of inflammatory macrophages in UUO kidneys. Our data suggest that targeting specific miRNAs could be a novel therapeutic approach to treat renal fibrosis.

Worldwide, approximately 405 000 cases of oral cancer (OSCC) are diagnosed each year, with a rising incidence in many countries. Despite advances in surgery and radiotherapy, which remain the standard treatment options, the mortality rate has remained largely unchanged for decades, with a 5-year survival rate of around 50%. OSCC is a heterogeneous disease, staged currently using the TNM classification, supplemented with pathological information from the primary tumour and loco-regional lymph nodes. Although patients with advanced disease show reduced survival, there is no single pathological or molecular feature that identifies aggressive, early-stage tumours. We retrospectively analysed 282 OSCC patients for disease mortality, related to clinical, pathological, and molecular features based on our previous functional studies [EGFR, αvβ6 integrin, smooth muscle actin (SMA), p53, p16, EP4]. We found that the strongest independent risk factor of early OSCC death was a feature of stroma rather than tumour cells. After adjusting for all factors, high stromal SMA expression, indicating myofibroblast transdifferentiation, produced the highest hazard ratio (3.06, 95% CI 1.65-5.66) and likelihood ratio (3.6; detection rate: false positive rate) of any feature examined, and was strongly associated with mortality, regardless of disease stage. Functional assays showed that OSCC cells can modulate myofibroblast transdifferentiation through αvβ6-dependent TGF-β1 activation and that myofibroblasts promote OSCC invasion. Finally, we developed a prognostic model using Cox regression with backward elimination; only SMA expression, metastasis, cohesion, and age were significant. This model was independently validated on a patient subset (detection rate 70%; false positive rate 20%; ROC analysis 77%, p < 0.001). Our study highlights the limited prognostic value of TNM staging and suggests that an SMA-positive, myofibroblastic stroma is the strongest predictor of OSCC mortality. Whether used independently or as part of a prognostic model, SMA identifies a significant group of patients with aggressive tumours, regardless of disease stage.

Loss of microRNA-29 (miR-29) is known to be a mechanism of transforming growth factor-β (TGF-β)-mediated pulmonary fibrosis, but the therapeutic implication of miR-29 for pulmonary fibrosis remains unexplored. The present study investigated whether miR-29 had therapeutic potential for lung disease induced by bleomycin in mice. In addition, the signaling mechanisms that regulated miR-29 expression were investigated in vivo and in vitro. We found that miR-29 was a downstream target gene of Smad3 and negatively regulated by TGF-β/Smad signaling in fibrosis. This was evidenced by the findings that mice or pulmonary fibroblasts null for Smad3 were protected against bleomycin or TGF-β1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulated TGF-β and connective tissue growth factor (CTGF) expression and Smad3 signaling. Therefore, Sleeping Beauty (SB)-mediated miR-29 gene transfer into normal and diseased lung tissues was capable of preventing and treating pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice. In conclusion, miR-29 is negatively regulated by TGF-β/Smad3 and has a therapeutic potential for pulmonary fibrosis. SB-mediated miR-29 gene therapy is a non-invasive therapeutic strategy for lung disease associated with fibrosis.