Caspase 1 Enhances Transport and Golgi Organization Protein 1 Expression to Promote Procollagen Export From the Endoplasmic Reticulum in Systemic Sclerosis Contributing to Fibrosis.

OBJECTIVE: Transport and Golgi Organization protein 1 (TANGO1) is a protein that regulates the export of procollagen from the endoplasmic reticulum and has a role in the organization of exit sites for general protein export. What regulates the expression of TANGO1 and the role of TANGO1 in fibrosis is poorly understood and has never been studied in the setting of systemic sclerosis (SSc). We undertook this study to determine the role of TANGO1 in SSc fibrosis. METHODS: SSc (n = 15) and healthy (n = 12) primary fibroblast lung cell lines were investigated for the expression of TANGO1. Histologic analyses for TANGO1 were performed on lung biopsy samples (n = 12 SSc patient samples and n = 8 healthy control samples). RESULTS: SSc fibroblasts showed increased expression of TANGO1 protein in cultured fibroblasts. TANGO1 colocalizes with α-smooth muscle actin (α-SMA)-positive cells in SSc lung tissue and is highly up-regulated in the neointima of SSc vessels. TANGO1 expression was dependent on the inflammasome activation of caspase 1. It was also dependent on signaling from the interleukin-1 (IL-1) and transforming growth factor ß (TGFß) receptors. The decrease in TANGO1 down-regulated export of larger cargos including collagen and laminin. Reduced TANGO1 protein had no effect on smaller molecular weight cargoes; however, the secretion of elastin was significantly reduced. CONCLUSION: TANGO1 is markedly increased in SSc fibroblasts and was found to be elevated in lung tissue in association with α-SMA-positive cells. TANGO1 expression is driven by inflammasome-dependent caspase 1 activation and is mediated by IL-1 and TGFß downstream signaling. These observations suggest that during fibrosis, caspase 1 promotes the up-regulation of TANGO1 and the organization of endoplasmic reticulum exits sites, ultimately contributing to procollagen export and fibrosis.

TANGO1 Dances to Export of Procollagen from the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) to Golgi secretory pathway is an elegantly complex process whereby protein cargoes are manufactured, folded, and distributed from the ER to the cisternal layers of the Golgi stack before they are delivered to their final destinations. The export of large bulky cargoes such as procollagen and its trafficking to the Golgi is a sophisticated mechanism requiring TANGO1 (Transport ANd Golgi Organization protein 1. It is also called MIA3 (Melanoma Inhibitory Activity protein 3). TANGO1 has two prominent isoforms, TANGO1-Long and TANGO1-Short, and each isoform has specific functions. On the luminal side, TANGO1-Long has an HSP47 recruitment domain and uses this protein to collect collagen. It can also tether its paralog isoforms cTAGE5 and TALI and along with these proteins enlarges the vesicle to accommodate procollagen. Recent studies show that TANGO1-Long combines retrograde membrane flow with anterograde cargo transport. This complex mechanism is highly activated in fibrosis and promotes the excessive deposition of collagen in the tissues. The therapeutic targeting of TANGO1 may prove successful in the control of fibrotic disorders. This review focuses on TANGO1 and its complex interaction with other procollagen export factors that modulate increased vesicle size to accommodate the export of procollagen.

The Mechanism and Regulation of the NLRP3 Inflammasome during Fibrosis.

Fibrosis is often the end result of chronic inflammation. It is characterized by the excessive deposition of extracellular matrix. This leads to structural alterations in the tissue, causing permanent damage and organ dysfunction. Depending on the organ it effects, fibrosis can be a serious threat to human life. The molecular mechanism of fibrosis is still not fully understood, but the NLRP3 (NOD-, LRR- and pyrin-domain-containing protein 3) inflammasome appears to play a significant role in the pathogenesis of fibrotic disease. The NLRP3 inflammasome has been the most extensively studied inflammatory pathway to date. It is a crucial component of the innate immune system, and its activation mediates the secretion of interleukin (IL)-1ß and IL-18. NLRP3 activation has been strongly linked with fibrosis and drives the differentiation of fibroblasts into myofibroblasts by the chronic upregulation of IL-1ß and IL-18 and subsequent autocrine signaling that maintains an activated inflammasome. Both IL-1ß and IL-18 are profibrotic, however IL-1ß can have antifibrotic capabilities. NLRP3 responds to a plethora of different signals that have a common but unidentified unifying trigger. Even after 20 years of extensive investigation, regulation of the NLRP3 inflammasome is still not completely understood. However, what is known about NLRP3 is that its regulation and activation is complex and not only driven by various activators but controlled by numerous post-translational modifications. More recently, there has been an intensive attempt to discover NLRP3 inhibitors to treat chronic diseases. This review addresses the role of the NLRP3 inflammasome in fibrotic disorders across many different tissues. It discusses the relationships of various NLRP3 activators to fibrosis and covers different therapeutics that have been developed, or are currently in development, that directly target NLRP3 or its downstream products as treatments for fibrotic disorders.

Soluble Receptor for Glycation End-products Concentration Increases Following the Treatment of Severe Diabetic Ketoacidosis

Objective: To determine the time relationships of soluble receptor for glycation end-products (sRAGE), [a decoy of the advanced glycation end-products (AGE)-RAGE axis] and D-lactate, (a metabolite of methylglyoxal) in the inflammatory response to diabetic ketoacidosis (DKA). Methods: Sixteen children and adolescents with type 1 diabetes (T1D) had blood samples obtained, 6-12 hours into treatment, at three weeks and three months post start of treatment. sRAGE and D-lactate concentrations at three months were considered baseline. Expression of RAGE was investigated in the myocardium of a newly diagnosed and untreated young person with fatal T1D/DKA. Results: sRAGE 6-12 hours after the start of treatment was 39% lower than the values at two weeks (p=0.0036) and at three months (p=0.0023) post treatment. D-lactate was higher during treatment than at three weeks (p=0.04) and at three months (p=0.035). Conclusion: sRAGE concentration was decreased during treatment, compared to concentrations at two weeks and three months after treatment. The increased D-lactate during treatment was in keeping with the known increase in dicarbonyls at this time. The finding of RAGE expression in a young myocardium prior to DKA treatment suggested cardiovascular inflammation pre-treatment and at a young age.

The MicroRNA miR-155 Is Essential in Fibrosis.

The function of microRNAs (miRNAs) during fibrosis and the downstream regulation of gene expression by these miRNAs have become of great biological interest. miR-155 is consistently upregulated in fibrotic disorders, and its ablation downregulates collagen synthesis. Studies demonstrate the integral role of miR-155 in fibrosis, as it mediates TGF-ß1 signaling to drive collagen synthesis. In this review, we summarize recent findings on the association between miR-155 and fibrotic disorders. We discuss the cross-signaling between macrophages and fibroblasts that orchestrates the upregulation of collagen synthesis mediated by miR-155. As miR-155 is involved in the activation of the innate and adaptive immune systems, specific targeting of miR-155 in pathologic cells that make excessive collagen could be a viable option before the depletion of miR-155 becomes an attractive antifibrotic approach.

Preclinical study of the long-range safety and anti-inflammatory effects of high-dose oral meglumine.

Meglumine is a methylamino derivative of sorbitol that is an approved drug excipient. Recent preclinical studies suggest that administration of high-dose oral meglumine can exert beneficial medicinal effects to treat diabetes, obesity, and fatty liver disease (NAFLD/nonalcoholic steatohepatitis [NASH]). Here we address gaps in knowledge about the pharmacology and toxicology of this substance administered at high concentrations to explore its medicinal potential. We observed that high-dose meglumine limited secretion of proinflammatory cytokines and cell adhesion molecules from activated human THP-1 or murine RAW264.7 monocytes. Preclinical pharmacokinetic analysis in Swiss mice confirmed that meglumine was orally available. Informed by this data, oral doses of 18 to 75 mM meglumine were administered ad libitum in the drinking water of Sprague-Dawley rats and two cohorts of C57BL/6 mice housed in different vivariums. In a 32-week study, urinary isoprostane levels trended lower in subjects consistent with the possibility of anti-inflammatory effects. In full lifespan studies, there was no detrimental effect on longevity. Heart function evaluated in C57BL/6 mice using an established noninvasive cardiac imaging system showed no detrimental effects on ejection fraction, fractional shortening, left ventricle function or volume, and cardiac output in mice up to 15-month old, with a potential positive trend in heart function noted in elderly mice consistent with earlier reported benefits on muscle stamina. Finally, in a transgenic model of inflammation-associated skin carcinogenesis, the incidence, number, and growth of skin tumors trended lower in subjects receiving meglumine. Overall, the evidence obtained illustrating the long-range safety of high-dose oral meglumine support the rationale for its evaluation as a low-cost modality to limit diabetes, hypertriglyceridemia, and NAFLD/NASH.

NADPH oxidase-mediated induction of reactive oxygen species and extracellular matrix deposition by insulin-like growth factor binding protein-5.

Insulin-like growth factor binding protein-5 (IGFBP-5) induces production of the extracellular matrix (ECM) components collagen and fibronectin both in vitro and in vivo and is overexpressed in patients with fibrosing lung diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). However, the mechanism by which IGFBP-5 exerts its fibrotic effect is incompletely understood. Recent reports have shown a substantial role of reactive oxygen species (ROS) in fibrosis; thus we hypothesized that IGFBP-5 induces production of ROS to mediate the profibrotic process. In vitro analyses revealed that ROS production was induced by recombinant and adenoviral vector-mediated IGFBP-5 (AdBP5) in a dose- and time-dependent manner, regulated through MEK/ERK and JNK signaling, and primarily mediated by NADPH oxidase (Nox). Silencing IGFBP-5 in SSc and IPF fibroblasts reduced ROS production. The antioxidants diphenyleneiodonium and N-acetylcysteine blocked IGFBP-5-stimulated ECM production in normal, SSc, and IPF human primary lung fibroblasts. In murine fibroblasts lacking critical components of the Nox machinery, AdBP5-stimulated ROS production and fibronectin expression were reduced compared with wild-type fibroblasts. IGFBP-5 stimulated transcriptional expression of Nox3 in human fibroblasts while selective knockdown of Nox3 reduced ROS production by IGFBP-5. Thus IGFBP-5 mediates fibrosis through production of ROS in a Nox-dependent manner.

The IL-1 family of cytokines. Do they have a role in scleroderma fibrosis?

Systemic sclerosis is a profibrotic autoimmune disease mediated by the dysregulation in collagen synthesis, leading to the increased deposition of collagens, primarily type I and III, and the deposition of other extracellular matrix proteins in the skin and internal organs, in a mechanism that is thought to be an over active wound healing process. These patients experience significant morbidity and the mortality rate in this disease is high. Indeed, scleroderma is the most deadly of diseases in the autoimmune spectrum. Recent evidence has placed the assembly and chronic activation of the inflammasome as a central driver of fibrosis. Once assembled, the inflammasome is a large protein complex that regulates the release of IL-1, IL-18, and IL-33, which are thought to play a role in the fibrotic response. IL-36 also belongs to the IL-1 family of cytokines and is a new comer to this field of research. Recent analyses of the IL-1 family of cytokines have demonstrated that many of them play a role in skin inflammation and fibrosis and their corresponding antagonists (IL-1RA and IL-36RA) can abrogate this pathology. Understanding how these cytokines are regulated and how they contribute to fibrosis will be important to understanding this pathology and may shed light in new areas for therapeutic development.

Mir-155 is overexpressed in systemic sclerosis fibroblasts and is required for NLRP3 inflammasome-mediated collagen synthesis during fibrosis.

BACKGROUND: Despite the important role that microRNAs (miRNAs) play in immunity and inflammation, their involvement in systemic sclerosis (SSc) remains poorly characterized. miRNA-155 (miR-155) plays a role in pulmonary fibrosis and its expression can be induced with interleukin (IL)-1ß. SSc fibroblasts have activated inflammasomes that are integrally involved in mediating the myofibroblast phenotype. In light of this, we investigated whether miR-155 played a role in SSc and if its expression was dependent on inflammasome activation. METHODS: miR-155 expression was confirmed in SSc dermal and lung fibroblasts by quantitative polymerase chain reaction (PCR). Wild-type and NLRP3-deficient murine fibroblasts were utilized to explore the regulation of miR-155 during inflammasome activation. miR-155-deficient fibroblasts and retroviral transductions with a miR-155 expression or control vectors were used to understand the contribution of miR-155 in fibrosis. RESULTS: miR-155 was significantly increased and the highest expressing miRNA in SSc lung fibroblasts. Its expression was dependent on inflammasome activation as miR-155 expression could be blocked when inflammasome signaling was inhibited. In the absence of miR-155, inflammasome-mediated collagen synthesis could not be induced but was restored when miR-155 was expressed in miR-155-deficient fibroblasts. CONCLUSIONS: miR-155 is upregulated in SSc. These results suggest that the inflammasome promotes the expression of miR-155 and that miR-155 is a critical miRNA that drives fibrosis.

Markers of immune-mediated inflammation in the brains of young adults and adolescents with type 1 diabetes and fatal diabetic ketoacidosis. Is there a difference?

Due to the limited data on diabetic ketoacidosis and brain edema (DKA/BE) in children/adolescents and the lack of recent data on adults with type 1 diabetes (T1D), we addressed the question of whether neuroinflammation was present in the fatal DKA of adults. We performed immunohistochemistry (IHC) studies on the brains of two young adults with T1D and fatal DKA and compared them with two teenagers with poorly controlled diabetes and fatal DKA. C5b-9, the membrane attack complex (MAC) had significantly greater deposits in the grey and white matter of the teenagers than the young adults (p=0.03). CD59, a MAC assembly inhibitory protein was absent, possibly suppressed by the hyperglycemia in the teenagers but was expressed in the young adults despite comparable average levels of hyperglycemia. The receptor for advanced glycation end products (RAGE) had an average expression in the young adults significantly greater than in the teenagers (p=0.02). The autophagy marker Light Chain 3 (LC3) A/B was the predominant form of programmed cell death (PCD) in the teenage brains. The young adults had high expressions of both LC3A/B and TUNEL, an apoptotic cell marker for DNA fragmentation. BE was present in the newly diagnosed young adult with hyperglycemic hyperosmolar DKA and also in the two teenagers. Our data indicate that significant differences in neuroinflammatory components, initiated by the dysregulation of DKA and interrelated metabolic and immunologic milieu, are likely present in the brains of fatal DKA of teenagers when compared with young adults.

Transforming growth factor ß1 (TGFß1)-induced CD44V6-NOX4 signaling in pathogenesis of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive clinical syndrome of fatal outcome. The lack of information about the signaling pathways that sustain fibrosis and the myofibroblast phenotype has prevented the development of targeted therapies for IPF. Our previous study showed that isolated fibrogenic lung fibroblasts have high endogenous levels of the hyaluronan receptor, CD44V6 (CD44 variant containing exon 6), which enhances the TGFß1 autocrine signaling and induces fibroblasts to transdifferentiate into myofibroblasts. NADPH oxidase 4 (NOX4) enzyme, which catalyzes the reduction of O2 to hydrogen peroxide (H2O2), has been implicated in the cardiac and lung myofibroblast phenotype. However, whether CD44V6 regulates NOX4 to mediate tissue repair and fibrogenesis is not well-defined. The present study assessed the mechanism of how TGF-ß-1-induced CD44V6 regulates the NOX4/reactive oxygen species (ROS) signaling that mediates the myofibroblast differentiation. Specifically, we found that NOX4/ROS regulates hyaluronan synthesis and the transcription of CD44V6 via an effect upon AP-1 activity. Further, CD44V6 is part of a positive-feedback loop with TGFß1/TGFßRI signaling that acts to increase NOX4/ROS production, which is required for myofibroblast differentiation, myofibroblast differentiation, myofibroblast extracellular matrix production, myofibroblast invasion, and myofibroblast contractility. Both NOX4 and CD44v6 are up-regulated in the lungs of mice subjected to experimental lung injury and in cases of human IPF. Genetic (CD44v6 shRNA) or a small molecule inhibitor (CD44v6 peptide) targeting of CD44v6 abrogates fibrogenesis in murine models of lung injury. These studies support a function for CD44V6 in lung fibrosis and offer proof of concept for therapeutic targeting of CD44V6 in lung fibrosis disorders.

Identification of Optimal Mouse Models of Systemic Sclerosis by Interspecies Comparative Genomics.

OBJECTIVE: Understanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets. METHODS: Gene expression measured in skin from mice with sclerodermatous graft-versus-host disease (GVHD), bleomycin-induced fibrosis, Tsk1/+ or Tsk2/+ mice was mapped to human orthologs and compared to SSc skin biopsy-derived gene expression. Transforming growth factor ß (TGFß) activation was assessed using a responsive signature in mice, and tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression was measured in SSc patient and mouse skin. RESULTS: Gene expression in skin from mice with sclerodermatous GVHD and bleomycin-induced fibrosis corresponded to that in SSc patients in the inflammatory molecular subset. In contrast, Tsk2/+ mice showed gene expression corresponding to the fibroproliferative SSc subset. Enrichment of a TGFß-responsive signature was observed in both Tsk2/+ mice and mice with bleomycin-induced skin fibrosis. Expression of TNFRSF12A (the TWEAK receptor/fibroblast growth factor-inducible 14) was elevated in skin from patients with fibroproliferative SSc and the skin of Tsk2/+ mice. CONCLUSION: This study reveals similarities in cutaneous gene expression between distinct mouse models of SSc and specific molecular subsets of the disease. Different pathways underlie the intrinsic subsets including TGFß, interleukin-13 (IL-13), and IL-4. We identify a novel target, Tnfrsf12a, with elevated expression in skin from patients with fibroproliferative SSc and Tsk2/+ mice. These findings will inform mechanistic and translational preclinical studies in SSc.

Chimeric cells of maternal origin do not appear to be pathogenic in the juvenile idiopathic inflammatory myopathies or muscular dystrophy.

INTRODUCTION: Microchimeric cells have been studied for over a decade, with conflicting reports on their presence and role in autoimmune and other inflammatory diseases. To determine whether microchimeric cells were pathogenic or mediating tissue repair in inflammatory myopathies, we phenotyped and quantified microchimeric cells in juvenile idiopathic inflammatory myopathies (JIIM), muscular dystrophy (MD), and noninflammatory control muscle tissues. METHOD: Fluorescence immunophenotyping for infiltrating cells with sequential fluorescence in situ hybridization was performed on muscle biopsies from ten patients with JIIM, nine with MD and ten controls. RESULTS: Microchimeric cells were significantly increased in MD muscle (0.079 ± 0.024 microchimeric cells/mm(2) tissue) compared to controls (0.019 ± 0.007 cells/mm(2) tissue, p = 0.01), but not elevated in JIIM muscle (0.043 ± 0.015 cells/mm(2)). Significantly more CD4+ and CD8+ microchimeric cells were in the muscle of patients with MD compared with controls (mean 0.053 ± 0.020/mm(2) versus 0 ± 0/mm(2) p = 0.003 and 0.043 ± 0.023/mm(2) versus 0 ± 0/mm(2) p = 0.025, respectively). No differences in microchimeric cells between JIIM, MD, and noninflammatory controls were found for CD3+, Class II+, CD25+, CD45RA+, and CD123+ phenotypes, and no microchimeric cells were detected in CD20, CD83, or CD45RO populations. The locations of microchimeric cells were similar in all three conditions, with MD muscle having more microchimeric cells in perimysial regions than controls, and JIIM having fewer microchimeric muscle nuclei than MD. Microchimeric inflammatory cells were found, in most cases, at significantly lower proportions than autologous cells of the same phenotype. CONCLUSIONS: Microchimeric cells are not specific to autoimmune disease, and may not be important in muscle inflammation or tissue repair in JIIM.

The Tsk2/+ mouse fibrotic phenotype is due to a gain-of-function mutation in the PIIINP segment of the Col3a1 gene.

Systemic sclerosis (SSc) is a polygenic, autoimmune disorder of unknown etiology, characterized by the excessive accumulation of extracellular matrix (ECM) proteins, vascular alterations, and autoantibodies. The tight skin (Tsk)2/+ mouse model of SSc demonstrates signs similar to SSc including tight skin and excessive deposition of dermal ECM proteins. By linkage analysis, we mapped the Tsk2 gene mutation to <3 megabases on chromosome 1. We performed both RNA sequencing of skin transcripts and genome capture DNA sequencing of the region spanning this interval in Tsk2/+ and wild-type littermates. A missense point mutation in the procollagen III amino terminal propeptide segment (PIIINP) of collagen, type III, alpha 1 (Col3a1) was found to be the best candidate for Tsk2; hence, both in vivo and in vitro genetic complementation tests were used to prove that this Col3a1 mutation is the Tsk2 gene. All previously documented mutations in the human Col3a1 gene are associated with the Ehlers-Danlos syndrome, a connective tissue disorder that leads to a defect in type III collagen synthesis. To our knowledge, the Tsk2 point mutation is the first documented gain-of-function mutation associated with Col3a1, which leads instead to fibrosis. This discovery provides insight into the mechanism of skin fibrosis manifested by Tsk2/+ mice.

Molecular activation of the NLRP3 Inflammasome in fibrosis: common threads linking divergent fibrogenic diseases.

SIGNIFICANCE: Over the past 10 years, there has been a plethora of investigations centering on the NLRP3 inflammasome and its role in fibrosis and other disease pathologies. To date, the signaling pathways from the inflammasome to myofibroblast differentiation and chronic collagen synthesis have not been fully elucidated, and many questions are left to be answered. RECENT ADVANCES: Recent studies have demonstrated the significant and critical role of reactive oxygen species (ROS) and calcium signaling in the assembly of the inflammasome, and this may result in autocrine signaling maintaining the myofibroblast phenotype, leading to fibrotic disease. CRITICAL ISSUES: Traditionally, myofibroblasts under tight regulation aid in wound healing and then, once the wound has closed, undergo apoptosis and the collagen in the wound remodels. During fibrosis, however, the myofibroblast maintains an activated state via a chronically activated inflammasome, leading to the continual synthesis of collagens and other extracellular matrix proteins that result in damage to the tissue or organ. The mechanism that is driving this abnormality has not been fully elucidated. FUTURE DIRECTIONS: However, studies have been conducted to suggest that modulating the calcium or the ROS axis may be of therapeutic value in regulating inflammasome activation. A number of novel drugs are currently being developed that may prove beneficial to patients suffering from fibrotic diseases.

Tight Skin 2 Mice Exhibit Delayed Wound Healing Caused by Increased Elastic Fibers in Fibrotic Skin.

Rationale: The Tight Skin 2 (Tsk2) mouse model of systemic sclerosis (SSc) has many features of human disease, including tight skin, excessive collagen deposition, alterations in the extracellular matrix (ECM), increased elastic fibers, and occurrence of antinuclear antibodies with age. A tight skin phenotype is observed by 2 weeks of age, but measurable skin fibrosis is only apparent at 10 weeks. We completed a series of wound healing experiments to determine how fibrosis affects wound healing in Tsk2/+ mice compared with their wild-type (WT) littermates. Method: We performed these experiments by introducing four 4 mm biopsy punched wounds on the back of each mouse, ventral of the midline, and observed wound healing over 10 days. Tsk2/+ mice showed significantly delayed wound healing and increased wound size compared with the WT littermates at both 5 and 10 weeks of age. We explored the potential sources of this response by wounding Tsk2/+ mice that were genetically deficient either for the NLRP3 inflammasome (a known fibrosis mediator), or for elastic fibers in the skin, using a fibulin-5 knockout. Conclusion: We found that the loss of elastic fibers restores normal wound healing in the Tsk2/+ mouse and that the loss of the NLRP3 inflammasome had no effect. We conclude that elastic fiber dysregulation is the primary cause of delayed wound healing in the Tsk2/+ mouse and therapies that promote collagen deposition in the tissue matrix in the absence of elastin deposition might be beneficial in promoting wound healing in SSc and other diseases.

Animal models of systemic sclerosis: their utility and limitations.

Without doubt, animal models have provided significant insights into our understanding of the rheumatological diseases; however, no model has accurately replicated all aspects of any autoimmune disease. Recent years have seen a plethora of knockouts and transgenics that have contributed to our knowledge of the initiating events of systemic sclerosis, an autoimmune disease. In this review, the focus is on models of systemic sclerosis and how they have progressed our understanding of fibrosis and vasculopathy, and whether they are relevant to the pathogenesis of systemic sclerosis.

Tight skin 2 mice exhibit a novel time line of events leading to increased extracellular matrix deposition and dermal fibrosis.

The tight skin 2 (Tsk2) mouse model of systemic sclerosis (SSc) has many features of the human disease including tight skin, fibrosis, extracellular matrix abnormalities, and reported antinuclear antibodies (ANA). Here we report that Tsk2/+ mice develop excess dermal fibrosis with age, as skin is not significantly fibrotic until 10 weeks, a full eight weeks after the development of the physical tight skin phenotype. Concomitantly with the tight skin phenotype at two weeks of age, Tsk2/+ mice demonstrate increased levels of total transforming growth factor beta 1 (TGF-ß1) and excessive accumulation of dermal elastic fibers. The increase in elastic fibers is not responsible for tight skin, however, because Tsk2/+ mice genetically engineered to lack skin elastic fibers nevertheless have tight skin and fibrosis. Finally, about two months after the first measurable increases of total collagen, a portion of Tsk2/+ mice produce ANAs, but at a similar level to wild-type littermates. The timeline of disease development in the Tsk2/+ mouse shows that fibrosis is progressive, with elastic fiber alterations and TGF-ß1 over-production occurring at least two months before bona fide fibrosis, that is not dependent on ANA production.

Targeting the unfolded protein response, XBP1, and the NLRP3 inflammasome in fibrosis and cancer.

Increasing health care costs in the US are due in a large part to the increasing prevalence of chronic diseases in an aging population. Current therapeutic strategies for treating chronic diseases alleviate symptoms allowing patients to live longer with these diseases, but they do little, however, to alter the underlying disease course. Recent advances in molecular biology are revealing new drug targets that may significantly alter the course of these diseases and, as a result, offer economic relief from burgeoning health care costs. Endoplasmic reticulum (ER) stress has been implicated as an underlying pathology in many chronic diseases, and, therefore, the development of therapies designed to ameliorate ER stress may yield novel, effective treatment strategies. Herein, we report that X-box binding protein 1 (XBP1) may be one of the earliest proteins engaged in response to ER stress. We show that a new signaling peptide derived from the ER-embedded transient receptor potential calcium channel protein 1 (TRPC1) engages XBP1 upstream of NLRP3 inflammasome-mediated maturation and secretion of IL-1ß/IL-18. Moreover, we show that a synthetic homolog of this signaling peptide (Naclynamide™) administered intravenously twice weekly over a 4-week treatment course induced suppuration and evoked partial or complete resolution of lesions associated with a fibrotic granuloma, a lymphosarcoma, and a colo-rectal carcinoma in canine patients. The mode of action for Naclynamide™ as a first-in-class anti-cancer drug candidate is discussed.

Two α-dicarbonyls downregulate migration, invasion, and adhesion of liver cancer cells in a p53-dependent manner.

BACKGROUND: Hepatocellular carcinoma accounts for more than 600,000 deaths per year due to it being a highly invasive tumor. The α-dicarbonyl, methylglyoxal demonstrates efficacy at reducing tumor burden, however the anti-cancerous activities of 3-deoxyglucosone, have never been studied. AIMS: To determine the anti-cancerous potential of methylglyoxal and 3-deoxyglucosone on liver tumor cells. METHODS: The in vitro effects of methylglyoxal and 3-deoxyglucosone were studied by investigating migration, invasion, and adhesion of Huh-7, HepG2, and Hep3B cells. RESULTS: 3-Deoxyglucosone inhibited migration of Huh-7 and HepG2 cells. Methylglyoxal decreased migration of HepG2 cells. Additionally, 3-deoxyglucosone and methylglyoxal impaired invasion, and adhesion of Huh-7 and HepG2 cells. In Hep3B cells, a p53 null cell line, 3-deoxyglucosone and methylglyoxal had no effect on migration, invasion, or adhesion. However, both compounds inhibited invasion of wild-type p53 transfected Hep3B cells. Silencing of p53 in Huh-7 and HepG2 cells abrogated the effects of the α-dicarbonyls on cell invasion. 3DG and MG did not alter p53 total protein but promoted nuclear translocation of p53. CONCLUSIONS: These studies suggest that 3-deoxyglucosone and methylglyoxal impair invasion, migration, and adhesion of hepatocellular carcinoma. The effects of both compounds on cell invasion are dependent on p53 and imply that α-dicarbonyls could be efficacious in the treatment of p53-expressing invasive liver tumors.