Epithelial-mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non-small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA)-related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in NSCLC. miRNA expression profiles were examined before and after transforming growth factor β1 (TGF-β1) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. miRNA array and real-time quantitative reverse transcription PCR (qRT-PCR) revealed that TGF-β1 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW, and PDZ domain-containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN, was diminished in A549 cells with EMT after the TGF-β1 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-β1-induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-β1-induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, in which suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be a new therapeutic target in patients with advanced lung adenocarcinoma, depending on the EMT phenomenon.

Epithelial-mesenchymal transition (EMT) promotes cancer invasion and metastasis, but the integrative mechanisms that coordinate these processes are incompletely understood. In this study, we used a cross-species expression profiling strategy in metastatic cell lines of human and mouse origin to identify 22 up-regulated and 12 down-regulated genes that are part of an essential genetic program in metastasis. In particular, we identified a novel function in metastasis that was not previously known for the transcription factor Forkhead Box Q1 (Foxq1). Ectopic expression of Foxq1 increased cell migration and invasion in vitro, enhanced the lung metastatic capabilities of mammary epithelial cells in vivo, and triggered a marked EMT. In contrast, Foxq1 knockdown elicited converse effects on these phenotypes in vitro and in vivo. Neither ectopic expression nor knockdown of Foxq1 significantly affected cell proliferation or colony formation in vitro. Notably, Foxq1 repressed expression of the core EMT regulator E-cadherin by binding to the E-box in its promoter region. Further mechanistic investigation revealed that Foxq1 expression is regulated by TGF-β1, and that Foxq1 knockdown blocked TGF-β1-induced EMT at both morphological and molecular levels. Our findings highlight the feasibility of cross-species expression profiling as a strategy to identify metastasis-related genes, and they reveal that EMT induction is a likely mechanism underlying a novel metastasis-promoting function of Foxq1 defined here in breast cancer.

The anabolic effects of parathyroid hormone (PTH) on bone formation are impaired by concurrent use of antiresorptive drugs. We found that the release of active transforming growth factor (TGF)-β1 during osteoclastic bone resorption is inhibited by alendronate. We showed that mouse Sca-1-positive (Sca-1(+)) bone marrow stromal cells are a skeletal stem cell subset, which are recruited to bone remodeling sites by active TGF-β1 in response to bone resorption. Alendronate inhibits the release of active TGF-β1 and the recruitment of Sca-1(+) skeletal stem cells for the bone formation. The observation was validated in a Tgfb1(-/-) mouse model, in which the anabolic effects of PTH on bone formation are diminished. The PTH-stimulated recruitment of injected mouse Sca-1(+) cells to the resorptive sites was inhibited by alendronate. Thus, inhibition of active TGF-β1 release by alendronate reduces the recruitment of Sca-1(+) skeletal stem cells and impairs the anabolic action of PTH in bone.

Neuropilin (Nrp)-1 and Nrp-2 are multifunctional proteins frequently expressed by cancer cells and contribute to tumor progression by mechanisms that are not well understood. They are co-receptors for vascular endothelial growth factor and class 3 semaphorins, but recently we found that Nrp1 also binds latent and active transforming growth factor (TGF)-β1, and activates the latent form latency-associated peptide (LAP)-TGF-β1. Here, we report that Nrp1 has affinity for TGF-β receptors TβRI and TβRII, the signaling TGF-β receptors, as well as TβRIII (betaglycan), as determined in binding assays, pull down assays and confocal microscopy. Nrp1 had a higher affinity for TβRI than TβRII and could form a complex with these receptors. In breast cancer cells, Nrp1 and TβRI cointernalized in the presence of TGF-β1. Nrp1 acted as a TGF-β co-receptor by augmenting canonical Smad2/3 signaling. Importantly, Nrp-positive cancer cells, unlike negative cells, were able to activate latent TGF-β1 and respond. We examined two other membrane proteins that bind LAP-TGF-β, i.e. an RGD-binding integrin (αvβ3) and Glycoprotein A repetitions predominant (CLRRC32). RGD-binding integrins are frequently expressed by cancer cells, and glycoprotein A repetitions predominant is expressed by activated regulatory T cells that appear linked to poor tumor immunity. In vitro, these receptors did not activate LAP-TGF-β1, but subsequent addition of Nrp1 activated the cytokine. Thus, Nrp1 might collaborate with other latent TGF-β receptors in TGF-β capture and activation. We also show that Nrp2 has activities similar to Nrp1. We conclude that Nrp1 is a co-receptor for TGF-β1 and augments responses to latent and active TGF-β. Since TGF-β promotes metastasis this is highly relevant to cancer biology.

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC.

Chronic organ injury leads to fibrosis and eventually organ failure. Fibrosis is characterized by excessive synthesis, remodeling, and contraction of extracellular matrix produced by myofibroblasts. Myofibroblasts are the key cells in the pathophysiology of fibrotic disorders and their differentiation can be triggered by multiple stimuli. To develop anti-fibrotic therapies, it is of paramount importance to understand the molecular basis of the signaling pathways contributing to the activation and maintenance of myofibroblasts. Several signal transduction pathways, such as transforming growth factor (TGF)-β, Wingless/Int (WNT), and more recently yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling, have been linked to the pathophysiology of fibrosis. Activation of the TGF-β1-induced SMAD complex results in the upregulation of genes important for myofibroblast function. Similarly, WNT-stabilized β-catenin translocates to the nucleus and initiates transcription of its target genes. YAP and TAZ are two transcriptional co-activators from the Hippo signaling pathway that also rely on nuclear translocation for their functioning. These three signal transduction pathways have little molecular similarity but do share one principle: the cytosolic/nuclear regulation of its transcriptional activators. Past research on these pathways often focused on the isolated cascades without taking other signaling pathways into account. Recent developments show that parts of these pathways converge into an intricate network that governs the activation and maintenance of the myofibroblast phenotype. In this review, we discuss the current understanding on the signal integration between the TGF-β, WNT, and YAP/TAZ pathways in the development of organ fibrosis. Taking a network-wide view on signal transduction will provide a better understanding on the complex and versatile processes that underlie the pathophysiology of fibrotic disorders.

Tumor-derived microvesicles (TD-MVs) are key mediators which are shed by cancer cells and can sensitize neighboring cells in the tumor microenvironment. TD-MVs are extracellular vesicles composed of exosomes and MVs and promote cancer invasion and metastasis. Intratumoral hypoxia is an integral component of all solid tumors. The relationship between hypoxic tumor-shed MVs and NK-mediated cytotoxicity remains unknown. In this paper, we reported that MVs derived from hypoxic tumor cells qualitatively differ from those derived from normoxic tumor cells. Using multiple tumor models, we showed that hypoxic MVs inhibit more NK cell function as compared to normoxic MVs. Hypoxic TD-MVs package two immunosuppressive factors involved in the impairment of natural killer (NK) cell cytotoxicity against different tumor cells in vitro and in vivo. We showed that following their uptake by NK cells, hypoxic TD-MVs transfer TGF-β1 to NK cells, decreasing the cell surface expression of the activating receptor NKG2D, thereby inhibiting NK cell function. MicroRNA profiling revealed the presence of high levels of miR-210 and miR-23a in hypoxic TD-MVs. We demonstrated that miR-23a in hypoxic TD-MVs operates as an additional immunomosuppressive factor, since it directly targets the expression of CD107a in NK cells. To our knowledge, this is the first study to show that hypoxic tumor cells by secreting MVs can educate NK cells and decrease their antitumor immune response. This study highlights the existence of a novel mechanism of immune suppression mediated by hypoxic TD-MVs and further improves our understanding of the immunosuppressive mechanisms prevailing in the hypoxic tumor microenvironment.

Proper activation of macrophages (Mφ) in the inflammatory phase of acute wound healing is essential for physiological tissue repair. However, there is a strong indication that robust Mφ inflammatory responses may be causal for the fibrotic response always accompanying adult wound healing. Using a complementary approach of in vitro and in vivo studies, we here addressed the question of whether mesenchymal stem cells (MSCs)-due to their anti-inflammatory properties-would control Mφ activation and tissue fibrosis in a murine model of full-thickness skin wounds. We have shown that the tumor necrosis factor-α (TNF-α)-stimulated protein 6 (TSG-6) released from MSCs in co-culture with activated Mφ or following injection into wound margins suppressed the release of TNF-α from activated Mφ and concomitantly induced a switch from a high to an anti-fibrotic low transforming growth factor-β1 (TGF-β1)/TGF-β3 ratio. This study provides insight into what we believe to be a previously undescribed multifaceted role of MSC-released TSG-6 in wound healing. MSC-released TSG-6 was identified to improve wound healing by limiting Mφ activation, inflammation, and fibrosis. TSG-6 and MSC-based therapies may thus qualify as promising strategies to enhance tissue repair and to prevent excessive tissue fibrosis.

An emerging family of innate lymphoid cells (termed ILCs) has an essential role in the initiation and regulation of inflammation. However, it is still unclear how ILCs are regulated in the duration of intestinal inflammation. Here, we identify a regulatory subpopulation of ILCs (called ILCregs) that exists in the gut and harbors a unique gene identity that is distinct from that of ILCs or regulatory T cells (Tregs). During inflammatory stimulation, ILCregs can be induced in the intestine and suppress the activation of ILC1s and ILC3s via secretion of IL-10, leading to protection against innate intestinal inflammation. Moreover, TGF-β1 is induced by ILCregs during the innate intestinal inflammation, and autocrine TGF-β1 sustains the maintenance and expansion of ILCregs. Therefore, ILCregs play an inhibitory role in the innate immune response, favoring the resolution of intestinal inflammation.

Transforming growth factor β (TGF-β) has long been implicated in fibrotic diseases, including the multisystem fibrotic disease systemic sclerosis (SSc). Expression of TGF-β-regulated genes in fibrotic skin and lungs of patients with SSc correlates with disease activity, which points to this cytokine as the central mediator of pathogenesis. Patients with SSc often develop pulmonary arterial hypertension (PAH), a particularly lethal complication caused by vascular dysfunction. Several genetic diseases with vascular features related to SSc, such as familial PAH and hereditary haemorrhagic telangiectasia, are caused by mutations in the TGF-β-sensing ALK-1 signalling pathway. These observations suggest that increased TGF-β signalling causes both vascular and fibrotic features of SSc. The question of how latent TGF-β becomes activated in local SSc tissues is, therefore, central to the understanding of SSc. Both TGF-β1 and TGF-β3 can be activated by integrins αvβ6 and αvβ8, whose upregulation in bronchial epithelial cells can activate TGF-β in SSc lungs. Other αv integrins, thrombospondin-1 or altered TGF-β sequestration by matrix proteins might be important in other target tissues. How the immune system triggers this process remains unclear, although links between inflammation and TGF-β activation are emerging. Together, these observations provide an increasingly secure framework for understanding TGF-β in SSc pathogenesis.

Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-β1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

Chronic progressive kidney diseases typically are characterized by active renal fibrosis and inflammation. Transforming growth factor-β1 (TGF-β1) is a key mediator in the development of renal fibrosis and inflammation. TGF-β1 exerts its biological effects by activating Smad2 and Smad3, which is regulated negatively by an inhibitory Smad7. In the context of fibrosis, although Smad3 is pathogenic, Smad2 and Smad7 are protective. Under disease conditions, Smads also interact with other signaling pathways, such as the mitogen-activated protein kinase and nuclear factor-κB pathways. In contrast to the pathogenic role of active TGF-β1, latent TGF-β1 plays a protective role in renal fibrosis and inflammation. Furthermore, recent studies have shown that TGF-β/Smad signaling plays a regulating role in microRNA-mediated renal injury. Thus, targeting TGF-β signaling by gene transfer of either Smad7 or microRNAs into diseased kidneys has been shown to retard progressive renal injury in a number of experimental models. In conclusion, TGF-β/Smad signaling plays a critical role in renal fibrosis and inflammation. Advances in understanding of the mechanisms of TGF-β/Smad signaling in renal fibrosis and inflammation during chronic kidney diseases should provide a better therapeutic strategy to combat kidney diseases.

The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell-specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4⁻CD8⁻TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.

Recent studies have implied that miRNAs act as crucial modulators for epithelial-to-mesenchymal transition (EMT). We found that miR-134 expression correlated with invasive potential and EMT phenotype of NSCLC cells. Functional assays demonstrated that miR-134 inhibited EMT in NSCLC cells. In addition, we showed that Forkhead Box M1 (FOXM1) is a direct target of miR-134. Knockdown of FOXM1 reversed EMT resembling that of miR-134 overexpression. We further found that FOXM1 was involved in TGF-β1-induced EMT in A549 cells. These findings suggest that miR-134 acts as a novel EMT suppressor in NSCLC cells.

OBJECTIVE

Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients have elevated protein and transforming growth factor-beta 1 (TGF-β1) contents and inhibit natural killer (NK) cell cytotoxicity (Haematologica 96, p. 1302, 2011). A potential role of exosomes in predicting responses to chemotherapy (CT) was evaluated in AML patients undergoing treatment.

METHODS

Plasma was obtained from AML patients at diagnosis (n = 16); post-induction CT (n = 9); during consolidation CT (n = 10); in long-term remission (Lt-CR, n = 5); and from healthy volunteers (n = 7). Exosomes were isolated by size-exclusion chromatography and ultracentrifugation. The exosomal protein, soluble TGFβ-1 levels (ELISA), and the TGF-β1 profiles (western blots) were compared among patients' cohorts. The results were correlated with the patients' cytogenetic profile, percentage of leukemic blast, and outcome.

RESULTS

At diagnosis, protein and TGF-β1 levels were higher (p < 0.009 and p < 0.004) in AML than control exosomes. These values decreased after induction CT (p < 0.05 and p < 0.004), increased during consolidation CT (p < 0.02 and p < 0.005), and normalized in Lt-CR. While TGF-β1 and protein levels tracked one another, TGF-β1 pro-peptide, latency-associated peptide (LAP), or mature TGF-β1 differentially decorated exosomes isolated before, during, and after CT. Only TGF-β1 pro-peptide was seen in exosomes of controls or Lt-CR patients. During consolidation CT, exosomes carried TGF-β1 pro-peptide, LAP, and low levels of mature TGF-β1. NK cell co-incubation with AML exosomes carrying all three TGF-β1 forms induced down-regulation of NKG2D expression.

CONCLUSIONS

Changes in exosomal protein and/or TGF-β1 content may reflect responses to CT. The exosomal profile may suggest the presence of residual disease in patients considered to have achieved complete remission.

NK cells that populate the decidua are important regulators of normal placentation. In contrast to peripheral blood NK cells, decidual NK (dNK) cells lack cytotoxicity, secrete proangiogenic factors, and regulate trophoblast invasion. In this study we show that exposure to a combination of hypoxia, TGF-β1, and a demethylating agent results in NK cells that express killer cell Ig-like receptors, the dNK cell markers CD9 and CD49a, and a dNK pattern of chemokine receptors. These cells secrete vascular endothelial growth factor (a potent proangiogenic molecule), display reduced cytotoxicity, and promote invasion of human trophoblast cell lines. These findings have potential therapeutic applications for placental disorders associated with altered NK cell biology.

Cancer-associated fibroblasts (CAFs) provide critical metabolites for tumor growth and undergo metabolic reprogramming to support glycolysis. However, the molecular mechanisms responsible for this change remain unclear. Here, we report that TGF-β1- or PDGF-induced CAFs switch from oxidative phosphorylation to aerobic glycolysis. We identify downregulation of isocitrate dehydrogenase 3α (IDH3α) as a marker for this switch. Furthermore, miR-424 downregulates IDH3α during CAF formation. Downregulation of IDH3α decreases the effective level of α-ketoglutarate (α-KG) by reducing the ratio of α-KG to fumarate and succinate, resulting in PHD2 inhibition and HIF-1α protein stabilization. The accumulation of HIF-1α, in turn, promotes glycolysis by increasing the uptake of glucose, upregulating expression of glycolytic enzymes under normoxic conditions, and inhibiting oxidative phosphorylation by upregulating NDUFA4L2. CAFs from tumor samples exhibit low levels of IDH3α, and overexpression of IDH3α prevents transformation of fibroblasts into CAFs. Our studies reveal IDH3α to be a critical metabolic switch in CAFs.

Assembly of synapses requires proper coordination between pre- and postsynaptic elements. Identification of cellular and molecular events in synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for astrocytes in synapse formation and function has been proposed. Here, we show that transforming growth factor β (TGF-β) signaling is a novel synaptogenic pathway for cortical neurons induced by murine and human astrocytes. By combining gain and loss of function approaches, we show that TGF-β1 induces the formation of functional synapses in mice. Further, TGF-β1-induced synaptogenesis involves neuronal activity and secretion of the co-agonist of the NMDA receptor, D-serine. Manipulation of D-serine signaling, by either genetic or pharmacological inhibition, prevented the TGF-β1 synaptogenic effect. Our data show a novel molecular mechanism that might impact synaptic function and emphasize the evolutionary aspect of the synaptogenic property of astrocytes, thus shedding light on new potential therapeutic targets for synaptic deficit diseases.

BACKGROUND

Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models.

METHODS

The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays.

RESULTS

In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors.

CONCLUSIONS

Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.

Immune thrombocytopenia (ITP) is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. We and others have described impaired regulatory CD4(+)CD25(hi) T cells (Treg) numbers and/or suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients, but information on the immunologic responses of treated patients are lacking. We studied the immunologic profile of chronic ITP patients before (n = 10) and during treatment with thrombopoietin receptor (TPO-R) agonists (n = 9). Treg activity, as measured by suppression of proliferation of autologous CD4(+) CD25(-) cells, was improved in patients on treatment (P < .05), and the improvement correlated with reduction in interleukin-2-producing CD4(+) cells, consistent with dampening of immune responses. There was a concomitant increase in total circulating transforming growth factor-β1 (TGF-β1) levels (P = .002) in patients on treatment, and the levels of TGF-β1 correlated with the degree of improvement in platelet counts (r = .8, P = .0002). This suggests that platelets in patients on TPO-R treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-β1 as a result of greater platelet turnover. In conclusion, our findings suggest that thrombopoietic agents in patients with ITP have profound effects to restore immune tolerance.

During peritoneal dialysis (PD), mesothelial cells undergo mesothelial-to-mesenchymal transition (MMT), a process associated with peritoneal-membrane dysfunction. Because TGF-β1 can induce MMT, we evaluated the efficacy of TGF-β1-blocking peptides in modulating MMT and ameliorating peritoneal damage in a mouse model of PD. Exposure of the peritoneum to PD fluid induced fibrosis, angiogenesis, functional impairment, and the accumulation of fibroblasts. In addition to expressing fibroblast-specific protein-1 (FSP-1), some fibroblasts co-expressed cytokeratin, indicating their mesothelial origin. These intermediate-phenotype (Cyto(+)/FSP-1(+)) fibroblasts had features of myofibroblasts with fibrogenic capacity. PD fluid treatment triggered the appearance of CD31(+)/FSP-1(+) and CD45(+)/FSP-1(+) cells, suggesting that fibroblasts also originate from endothelial cells and from cells recruited from bone marrow. Administration of blocking peptides significantly ameliorated fibrosis and angiogenesis, improved peritoneal function, and reduced the number of FSP-1(+) cells, especially in the Cyto(+)/FSP-1(+) subpopulation. Conversely, overexpression of TGF-β1 in the peritoneum by adenovirus-mediated gene transfer led to a marked accumulation of fibroblasts, most of which derived from the mesothelium. Taken together, these results demonstrate that TGF-β1 drives the peritoneal deterioration induced by dialysis fluid and highlights a role of TGF-β1-mediated MMT in the pathophysiology of peritoneal-membrane dysfunction.

Pancreatic ductal adenocarcinoma is an aggressive cancer that interacts with stromal cells to produce a highly inflammatory tumor microenvironment that promotes tumor growth and invasiveness. The precise interplay between tumor and stroma remains poorly understood. TLRs mediate interactions between environmental stimuli and innate immunity and trigger proinflammatory signaling cascades. Our finding that TLR7 expression is upregulated in both epithelial and stromal compartments in human and murine pancreatic cancer led us to postulate that carcinogenesis is dependent on TLR7 signaling. In a mouse model of pancreatic cancer, TLR7 ligation vigorously accelerated tumor progression and induced loss of expression of PTEN, p16, and cyclin D1 and upregulation of p21, p27, p53, c-Myc, SHPTP1, TGF-β, PPARγ, and cyclin B1. Furthermore, TLR7 ligation induced STAT3 activation and interfaced with Notch as well as canonical NF-κB and MAP kinase pathways, but downregulated expression of Notch target genes. Moreover, blockade of TLR7 protected against carcinogenesis. Since pancreatic tumorigenesis requires stromal expansion, we proposed that TLR7 ligation modulates pancreatic cancer by driving stromal inflammation. Accordingly, we found that mice lacking TLR7 exclusively within their inflammatory cells were protected from neoplasia. These data suggest that targeting TLR7 holds promise for treatment of human pancreatic cancer.

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-β1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate.

Circular RNAs (circRNAs) are a novel type of endogenous noncoding RNA gaining research interest in recent years. Despite this increase in interest, the mechanism of circRNAs in the pathogenesis of multiple cardiovascular diseases, particularly myocardial fibrosis, is rarely reported. In the following study, the expression profiles and potential mechanisms of circRNAs in mice myocardial fibrosis models in vitro are investigated. Previous research examining circRNA expression profiles of diabetic db/db mice myocardium using circRNA microarray found 43 circRNAs were abnormally expressed, including 24 up-regulated circRNAs and 19 down-regulated circRNAs. Furthermore, circRNA_010567 was markedly up-regulated in diabetic mice myocardium and cardiac fibroblasts (CFs) treated with Ang II. Bioinformatics analysis predicted circRNA_010567, sponge miR-141 and miR-141 directly target TGF-β1, which was validated by dual-luciferase assay. Subsequently, functional experiments revealed circRNA_010567 silencing could up-regulate miR-141 and down-regulate TGF-β1 expression, and suppress fibrosis-associated protein resection in CFs, including Col I, Col III and α-SMA. Results demonstrate the circRNA_010567/miR-141/TGF-β1 axis plays an important regulatory role in the diabetic mice myocardial fibrosis model. The present study characterizes a new function of circRNA in the pathogenesis of myocardial fibrosis in a diabetic mouse model, providing novel insight for circRNA-miRNA-mRNA in cardiovascular disease.