Epigenetic Regulation of EMP/EMT-Dependent Fibrosis.

Fibrosis represents a process characterized by excessive deposition of extracellular matrix (ECM) proteins. It often represents the evolution of pathological conditions, causes organ failure, and can, in extreme cases, compromise the functionality of organs to the point of causing death. In recent years, considerable efforts have been made to understand the molecular mechanisms underlying fibrotic evolution and to identify possible therapeutic strategies. Great interest has been aroused by the discovery of a molecular association between epithelial to mesenchymal plasticity (EMP), in particular epithelial to mesenchymal transition (EMT), and fibrogenesis, which has led to the identification of complex molecular mechanisms closely interconnected with each other, which could explain EMT-dependent fibrosis. However, the result remains unsatisfactory from a therapeutic point of view. In recent years, advances in epigenetics, based on chromatin remodeling through various histone modifications or through the intervention of non-coding RNAs (ncRNAs), have provided more information on the fibrotic process, and this could represent a promising path forward for the identification of innovative therapeutic strategies for organ fibrosis. In this review, we summarize current research on epigenetic mechanisms involved in organ fibrosis, with a focus on epigenetic regulation of EMP/EMT-dependent fibrosis.

Special Issue "Molecular Mechanism in Epithelial-Mesenchymal Transition (EMT) and Fibrosis".

The process known as epithelial-mesenchymal transition (EMT), fundamental for accurate development during embryogenesis, is involved in several pathological mechanisms, such as severe fibrosis and cancer [...].

Towards a Unified Approach in Autoimmune Fibrotic Signalling Pathways.

Autoimmunity is a chronic process resulting in inflammation, tissue damage, and subsequent tissue remodelling and organ fibrosis. In contrast to acute inflammatory reactions, pathogenic fibrosis typically results from the chronic inflammatory reactions characterizing autoimmune diseases. Despite having obvious aetiological and clinical outcome distinctions, most chronic autoimmune fibrotic disorders have in common a persistent and sustained production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements or epithelial to mesenchymal transformation (EMT) that progressively remodels and destroys normal tissue architecture leading to organ failure. Despite its enormous impact on human health, there are currently no approved treatments that directly target the molecular mechanisms of fibrosis. The primary goal of this review is to discuss the most recent identified mechanisms of chronic autoimmune diseases characterized by a fibrotic evolution with the aim to identify possible common and unique mechanisms of fibrogenesis that might be exploited in the development of effective antifibrotic therapies.

Molecular Mechanisms Linking Inflammation to Autoimmunity in Sjögren's Syndrome: Identification of New Targets.

Sjögren's syndrome (SS) is a systemic autoimmune rheumatic disorder characterized by the lymphocytic infiltration of exocrine glands and the production of autoantibodies to self-antigens. The involvement of the exocrine glands drives the pathognomonic manifestations of dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia) that define sicca syndrome. To date, the molecular mechanisms mediating pathological salivary gland dysfunction in SS remain to be elucidated, despite extensive studies investigating the underlying cause of this disease, hampering the development of novel therapeutic strategies. Many researchers have identified a multifactorial pathogenesis of SS, including environmental, genetic, neuroendocrine, and immune factors. In this review, we explore the latest developments in understanding the molecular mechanisms involved in the pathogenesis of SS, which have attracted increasing interest in recent years.

SMADS-Mediate Molecular Mechanisms in Sjögren's Syndrome.

There is considerable interest in delineating the molecular mechanisms of action of transforming growth factor-ß (TGF-ß), considered as central player in a plethora of human conditions, including cancer, fibrosis and autoimmune disease. TGF-ß elicits its biological effects through membrane bound serine/threonine kinase receptors which transmit their signals via downstream signalling molecules, SMADs, which regulate the transcription of target genes in collaboration with various co-activators and co-repressors. Until now, therapeutic strategy for primary Sjögren's syndrome (pSS) has been focused on inflammation, but, recently, the involvement of TGF-ß/SMADs signalling has been demonstrated in pSS salivary glands (SGs) as mediator of the epithelial-mesenchymal transition (EMT) activation. Although EMT seems to cause pSS SG fibrosis, TGF-ß family members have ambiguous effects on the function of pSS SGs. Based on these premises, this review highlights recent advances in unravelling the molecular basis for the multi-faceted functions of TGF-ß in pSS that are dictated by orchestrations of SMADs, and describe TGF-ß/SMADs value as both disease markers and/or therapeutic target for pSS.

Cadherin Signaling in Cancer and Autoimmune Diseases.

Cadherins mediate cell-cell adhesion through a dynamic process that is strongly dependent on the cellular context and signaling. Cadherin regulation reflects the interplay between fundamental cellular processes, including morphogenesis, proliferation, programmed cell death, surface organization of receptors, cytoskeletal organization, and cell trafficking. The variety of molecular mechanisms and cellular functions regulated by cadherins suggests that we have only scratched the surface in terms of clarifying the functions mediated by these versatile proteins. Altered cadherins expression is closely connected with tumorigenesis, epithelial-mesenchymal transition (EMT)-dependent fibrosis, and autoimmunity. We review the current understanding of how cadherins contribute to human health and disease, considering the mechanisms of cadherin involvement in diseases progression, as well as the clinical significance of cadherins as therapeutic targets.

TGFß1-Smad canonical and -Erk noncanonical pathways participate in interleukin-17-induced epithelial-mesenchymal transition in Sjögren's syndrome.

Interleukin-17 (IL-17) is a pleiotropic cytokine that plays a primary role in triggering epithelial-mesenchymal transition (EMT) in many chronic inflammatory diseases. EMT plays a critical role in the progression of salivary gland (SG) fibrosis in primary Sjögren's syndrome (pSS). This study focused on the activation of the canonical TGF-ß1/Smad2/3 and noncanonical TGF-ß1/Erk1/2 pathways in IL-17-dependent TGFß1-induced EMT in human SG epithelial cells (SGEC) derived from healthy subjects. The expression of phosphorylated Smad2/3 and Erk1/2 during IL-17 treatment-stimulated EMT was evaluated in healthy SGEC. Cotreatment with IL-17 and specific TGFß receptor type I kinase inhibitor SB431542, or Erk 1/2 inhibitor U0126, abrogates the corresponding morphological changes and EMT phenotypic markers expression in healthy SGEC. Interestingly, inhibition of canonical TGFß1/Smad2/3 signal transduction had no effect on activation of the noncanonical TGFß1/Erk1/2/EMT pathway, suggesting that the two pathways act independently in activating IL-17-dependent EMT in SGEC.

The role of the epithelial-to-mesenchymal transition (EMT) in diseases of the salivary glands.

The link between inflammatory microenvironment and cancer emerged in the last years as a decisive factor in the induction of the pathological epithelial-mesenchymal transition (EMT). The EMT induces changes of cell states converting the epithelial cells to mesenchymal cells when this program is fully executed and EMT has emerged as a central driver of tumor malignancy. Cellular pathways activated by chronic inflammation brought about by chronic infections, by immune-mediated diseases, or by dysregulated wound healing at sites of repetitive tissue injury, constitute risk factors or initial cell transformation and for cancer progression. EMT and its intermediate states have recently been identified as crucial inducers of organ fibrosis, inflammation and tumor progression. In this review, we discuss the current state-of-the-art and latest findings regarding the link between EMT, inflammation, fibrosis and cancer, highlighting the most recent data on EMT-dependent tissue fibrosis during chronic inflammatory salivary glands conditions and salivary glands tumors.

The TGF-ß1 Signaling Pathway as an Attractive Target in the Fibrosis Pathogenesis of Sjögren's Syndrome.

Transforming growth factor ß1 (TGF-ß1) plays a crucial role in the induction of fibrosis, often associated with chronic phases of inflammatory diseases contributing to marked fibrotic changes that compromise normal organ function. The TGF-ß1 signal exerts its biological effects via the TGF-ß/SMAD/Snail signaling pathway, playing an important pathogenic role in several fibrotic diseases. It has as yet been poorly investigated in the chronic autoimmune disease Sjögren's syndrome (SS). Here, we firstly tested, by immunohistochemistry, whether the TGF-ß1/SMAD/Snail signaling pathway is triggered in human pSS salivary glands (SGs). Next, healthy salivary gland epithelial cell (SGEC) cultures derived from healthy donors were exposed to TGF-ß1 treatment, and the relative gene and protein levels of SMAD2/3/4, Snail, E-cadherin, vimentin, and collagen type I were compared by semiquantitative RT-PCR, quantitative real-time PCR, and Western blot analysis. We observed, both at gene and protein levels, higher expression of SMAD2, 3, and 4 and Snail in the SGEC exposed by TGF-ß1 compared to untreated healthy SGEC. Additionally, in TGF-ß1-treated samples, we found a significant reduction in the epithelial phenotype marker E-cadherin and an increase in the mesenchymal phenotype markers vimentin and collagen type I compared to those in untreated SGEC, indicating that TGF-ß1 induces the EMT via the TGF-ß1/SMAD/Snail signaling pathway. Therefore, by using the specific TGF-ß receptor 1 inhibitor SB-431542 in healthy SGEC treated with TGF-ß1, we showed a significant reduction of the fibrosis markers vimentin and collagen type I while the epithelial marker E-cadherin returns to levels similar to untreated healthy SGEC. These data demonstrate that TGF-ß1 is an important key factor in the transition phase from SG chronic inflammation to fibrotic disease. Characteristic changes in the morphology and function of TGF-ß1-treated healthy SGEC further confirm that TGF-ß1 plays a significant role in EMT-dependent fibrosis.

Co-culture system of human salivary gland epithelial cells and immune cells from primary Sjögren's syndrome patients: an in vitro approach to study the effects of Rituximab on the activation of the Raf-1/ERK1/2 pathway.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder of the exocrine glands with associated lymphocytic infiltrates in the affected glands. Dryness of the mouth and eyes results from involvement of the salivary and lacrimal glands. The efficacy of Rituximab (RTX) in pSS is still open to debate. This study delineates the signaling pathway involved in RTX-mediated down-regulation of pro-inflammatory factors in a co-culture system of pSS salivary gland epithelial cells (SGEC) with syngeneic pSS B-lymphocytes. In addition, the effects of RTX on the activation of the Raf-1/ERK1/2 pathway in pSS SGEC co-cultured with syngeneic pSS T-lymphocytes were also investigated. This study demonstrated that RTX may interfere with the ERK1/2 pathway in a syngeneic co-culture of pSS SGEC with pSS B-lymphocytes, leading to decreased cytokine production by SGEC. These novel findings reveal that syngeneic co-culture of pSS SGEC with pSS B-lymphocytes leads to a down-regulation of Raf-1 in epithelial cells that adversely regulates the activity of the ERK1/2 pathway and determines a subsequent reduction of the release of pro-inflammatory factors.

Rituximab-mediated Raf kinase inhibitor protein induction modulates NF-κB in Sjögren syndrome.

Primary Sjögren syndrome (pSS) is an autoimmune disorder characterized by an epithelial injury surrounded by dense lymphocytic infiltrates. The conditions for the long-term maintenance of human salivary gland epithelial cells from pSS patients and a co-culture system with pSS lymphocytes were used to assess the effect of Rituximab (RTX) on the inflammatory condition and progression in pSS. Quantitative real-time PCR, genes and protein array analysis, Western blot, flow cytometry, small interfering RNA transfection and nuclear factor-κB (NF-κB) DNA binding assays were used as methods. Supporting the benefits of RTX, this study demonstrates that RTX decreases NF-κB activity and interrupts the NF-κB signalling pathway through the up-regulation of the Raf-1 kinase inhibitor protein (RKIP). Over-expression of RKIP down-regulates interleukins, their receptors and the expression of genes encodes proteins that attracted lymphocytes. Silencing of the RKIP gene leads to significantly increased expression and release of pro-inflammatory mediators supporting that RKIP expression could be involved in the suppression of NF-κB activation in pSS salivary gland epithelial cells.

Neovascularization is prominent in the chronic inflammatory lesions of Sjögren's syndrome.

Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in Sjögren's syndrome (SS) remains to be elucidated. Previous SS studies have demonstrated an increase in VEGF-A/VEGFR-2 system expression in minor salivary gland (MSG) biopsies from patients with SS, but differences in the new blood vessel formation between the different grades of disease severity have not been reported. Therefore, experiments were performed to demonstrate angiogenesis during different phases of primary SS (pSS) and to define the relationship between the microvessel density (MVD), macrophage infiltration and histiocyte distribution in SS MSG inflammatory lesions. In this series of experiments, immunohistochemistry was used to examine angiogenesis in serial sections of pSS MSG. Patients with pSS were classified accordingly with the grade of inflammatory lesions as I = low-grade (low focus score of 1 or 2), II = intermediate-grade (focus score of 3­6) and III = extensive inflammation in the MSG (high focus score of 12). Histological examination demonstrated that the MVD increased with the severity of the inflammatory lesions, and in addition, we found an increased infiltration of inflammatory and pro-angiogenic cells.These findings reveal that angiogenesis is intimately involved in the progression of pSS, may be central to the propagation of the chronic immune response observed in pSS and could represent a novel potential biomarker of pSS disease activity.

A potential role of the GRO-α/CXCR2 system in Sjögren's syndrome: regulatory effects of pro-inflammatory cytokines.

Chemokines, small pro-inflammatory cytokines, are involved in migration of inflammatory cells in inflamed tissues and recent studies established their role in angiogenesis, hematopoiesis, cancer and autoimmune conditions. Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXCR2 are involved in the inflammatory processes. Since there is no previous report that supports a possible role of GRO-α/CXCR2 receptor complex during inflammation and neovascularization existing in the autoimmune disease Sjögren's syndrome (SS), in this study, we examined CXCR2 and its ligand GRO-α expression in SS tissues. Immunohistochemistry revealed that GRO-α and its receptor CXCR2 were expressed at high levels in diseased tissues compared to healthy controls. In addition, human salivary gland epithelial cells (SGEC) cultures were submitted to a pro-inflammatory microenvironment using cytokines IL-6 and TNF-α in order to demonstrate that CXCR2 may change its initial expression pattern to another under inflammatory condition. The data show an increased expression of CXCR2 depending on the inflammatory cytokine used in culture in a time-dependent manner. Furthermore, silencing of the pro-angiogenic chemokine GRO-α is proportionally correlated with decreased expression of CXCR2 in pro-inflammatory cytokine-stimulated SGEC indicating the GRO-α/CXCR2 complex as a novel therapeutic target for the chronic inflammatory disease Sjögren's syndrome.

Emerging avenues linking inflammation, angiogenesis and Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease characterized by an inflammatory mononuclear infiltration and the destruction of epithelial cells of the lachrymal and salivary glands. The aetiology is unknown. The expression "autoimmune epithelitis" has been proposed as an alternative to SS, in view of the emerging central role of the epithelial cells in the disease pathogenesis. At the biomolecular level, the epithelial cells play an important role in triggering the autoimmune condition via antigen presentation, apoptosis, and chemokine and cytokines release. Inflammation and angiogenesis are frequently coupled in the pathological conditions associated to autoimmune diseases, and an angiogenic imbalance contributes to the pathogenesis of a number of inflammatory disorders. This work reviews the current knowledge of the molecular and cellular mechanisms underlying the pathogenesis of the inflammatory reactions that characterize SS. The literature and our data on the role of angiogenesis in the pathophysiology of the disease are discussed.

[Paget's disease of bone: new therapeutic strategies]. / La malattia di Paget: a proposito di nuove strategie terapeutiche.

Paget's disease of bone is a chronic disorder of unknown etiology that can result in enlarged and misshapen bones. The excessive breakdown and formation of bone tissue cause affected bones to weaken, resulting in pain, misshapen bones, fractures, and arthritis in the joints. In most cases the diagnosis is achieved casually, as only 5% of patients develop burning pain at the level of affected bones. As regards therapy, the use of anti-reabsorbing drugs, such as bisphosphonates and calcitonin, appears reasonable. Given the disease pathogenesis, the administration of denosumab and tocilizumab may be a valuable alternative to inhibit RANK expression, and thus osteoclast formation, and interleukin-6 production.

Immune and Non-Immune Inflammatory Cells Involved in Autoimmune Fibrosis: New Discoveries.

Fibrosis is an important health problem and its pathogenetic activation is still largely unknown. It can develop either spontaneously or, more frequently, as a consequence of various underlying diseases, such as chronic inflammatory autoimmune diseases. Fibrotic tissue is always characterized by mononuclear immune cells infiltration. The cytokine profile of these cells shows clear proinflammatory and profibrotic characteristics. Furthermore, the production of inflammatory mediators by non-immune cells, in response to several stimuli, can be involved in the fibrotic process. It is now established that defects in the abilities of non-immune cells to mediate immune regulation may be involved in the pathogenicity of a series of inflammatory diseases. The convergence of several, not yet well identified, factors results in the aberrant activation of non-immune cells, such as epithelial cells, endothelial cells, and fibroblasts, that, by producing pro-inflammatory molecules, exacerbate the inflammatory condition leading to the excessive and chaotic secretion of extracellular matrix proteins. However, the precise cellular mechanisms involved in this process have not yet been fully elucidated. In this review, we explore the latest discoveries on the mechanisms that initiate and perpetuate the vicious circle of abnormal communications between immune and non-immune cells, responsible for fibrotic evolution of inflammatory autoimmune diseases.

Interleukin-23 Involved in Fibrotic Autoimmune Diseases: New Discoveries.

Interleukin (IL)-23 is a central pro-inflammatory cytokine with a broad range of effects on immune responses. IL-23 is pathologically linked to the induction of the production of the pro-inflammatory cytokines IL-17 and IL-22, which stimulate the differentiation and proliferation of T helper type 17 (Th17) cells. Recent discoveries suggest a potential pro-fibrotic role for IL-23 in the development of chronic inflammatory autoimmune diseases characterized by intense fibrosis. In this review, we summarized the biological features of IL-23 and gathered recent research on the role of IL-23 in fibrotic autoimmune conditions, which could provide a theoretical basis for clinical targeting and drug development.

Targeting Interleukin-17 as a Novel Treatment Option for Fibrotic Diseases.

Fibrosis is the end result of persistent inflammatory responses induced by a variety of stimuli, including chronic infections, autoimmune reactions, and tissue injury. Fibrotic diseases affect all vital organs and are characterized by a high rate of morbidity and mortality in the developed world. Until recently, there were no approved antifibrotic therapies. In recent years, high levels of interleukin-17 (IL-17) have been associated with chronic inflammatory diseases with fibrotic complications that culminate in organ failure. In this review, we provide an update on the role of IL-17 in fibrotic diseases, with particular attention to the most recent lines of research in the therapeutic field represented by the epigenetic mechanisms that control IL-17 levels in fibrosis. A better knowledge of the IL-17 signaling pathway implications in fibrosis could design new strategies for therapeutic benefits.

Sjögren's syndrome autoantibodies provoke changes in gene expression profiles of inflammatory cytokines triggering a pathway involving TACE/NF-κB.

We explore the association of the inflammatory gene expression profile observed in the chronic inflammatory autoimmune disorder Sjögren's syndrome (SS) with changes in TNF-α converting enzyme (TACE), tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB levels showing that pathways that include TNF-α signaling converge on NF-κB contributing to exacerbate the diseases. The treatment of human salivary gland epithelial cells (SGECs) with SS anti-Ro/SSA autoantibodies (Abs) result in a progressive increase in NF-κB-DNA binding, that includes a marked enhancement in NF-κB subunit p65 protein-DNA binding. A human cytokine multi-analyte array demonstrated that the NF-κB proinflammatory target genes, increased by anti-Ro/SSA Abs treatment, includes CXC chemokines (CXCL1, CXCL6 and CXCL9), CC chemokines (CCL2, CCL13 and CCL20), interleukins (IL-1α, IL-1ß, IL-1F8, IL-6, IL-8, IL-9, IL-13, IL-17 and IL-22) and their receptors (IL-1RN, IL-10Rα, IL-13Rα, CCR1, CCR2, CCR3, CCR4 and CXCR1). Blockade of TACE through the use of the specific inhibitor TAPI-1 regulates proinflammatory cytokines production in SGEC treated with anti-Ro/SSA Abs inhibiting NF-κB nuclear translocation and activation. To further investigate the role of NF-κB on anti-Ro/SSA Abs-determined proinflammatory gene expression, we used the inhibitory protein IκB-α dominant negative super-repressor as inhibitor of NF-κB-DNA binding, demonstrating that transfection with dominant-negative IκB-α in anti-Ro/SSA-treated SGEC determined a marked reduction of proinflammatory cytokines gene expression. Although further studies are needed to clarify the mechanisms underlying SS, our results demonstrate that SS Abs exert their pathogenic effects via triggering the TACE/TNF-α/NF-κB axis.

Neuropilin-1 is upregulated in Sjögren's syndrome and contributes to pathological neovascularization.

Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of Sjögren's syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sjögren's syndrome.