RESUMO
Insufficient expression of steroidogenic acute regulatory-related lipid transfer protein 5 (StarD5) on liver cholesterol/lipid homeostasis is not clearly defined. The ablation of StarD5 was analyzed in mice on a normal or Western diet (WD) to determine its importance in hepatic lipid accumulation and fibrosis compared with wild-type (WT) mice. Rescue experiments in StarD5-/- mice and hepatocytes were performed. In addition to increased hepatic triglyceride (TG)-cholesterol levels, global StarD5-/- mice fed a normal diet displayed reduced plasma triglycerides and liver very low-density lipoprotein (VLDL) secretion as compared with WT counterparts. Insulin levels and homeostatic model assessment for insulin resistance (HOMA-IR) scoring were elevated, demonstrating developing insulin resistance (IR). WD-fed StarD5-/- mice upregulated WW domain containing transcription regulator 1 (TAZ or WWTR1) expression with accelerated liver fibrosis when compared with WD-fed WT mice. Suppression of oxysterol 7α-hydroxylase (CYP7B1) coupled with chronic accumulation of toxic oxysterol levels correlated with presentation of fibrosis. "Hepatocyte-selective" StarD5 overexpression in StarD5-/- mice restored expression, reduced hepatic triglycerides, and improved HOMA-IR. Observations in two additional mouse and one human metabolic dysfunction-associated steatotic liver disease (MASLD) model were supportive. The downregulation of StarD5 with hepatic lipid excess is a previously unappreciated physiological function appearing to promote lipid storage for future needs. Conversely, lingering downregulation of StarD5 with prolonged lipid-cholesterol excess accelerates fatty liver's transition to fibrosis; mediated via dysregulation in the oxysterol signaling pathway.NEW & NOTEWORTHY We have found that deletion of the cholesterol transport protein StarD5 in mice leads to an increase in insulin resistance and lipid accumulation due to the upregulation of lipid synthesis and decrease VLDL secretion from the liver. In addition, deletion of StarD5 increased fibrosis when mice were fed a Western diet. This represents a novel pathway of fibrosis development in the liver.
Assuntos
Resistência à Insulina , Cirrose Hepática , Fígado , Camundongos Knockout , Animais , Humanos , Masculino , Camundongos , Colesterol/metabolismo , Colesterol/sangue , Dieta Ocidental/efeitos adversos , Progressão da Doença , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Camundongos Endogâmicos C57BL , Triglicerídeos/metabolismoRESUMO
Certain proteoglycans, consisting of a core protein and glycosaminoglycan (GAG) chains, are among the many types of biomolecules that can function as damage-associated molecular pattern molecules (DAMPs). We, therefore, hypothesized that the expression level and structural alteration of GAGs affect inflammation. We have previously reported that the effects on GAG biosynthesis caused by loss of the tumor suppressor gene exostosin-like 2 (Extl2) influence liver injury and regeneration processes. To examine how altered GAG biosynthesis may underscore the relationship between inflammation and tumorigenesis, we assessed its role in non-alcoholic steatohepatitis and hepatocarcinoma (HCC) induced by dietary obesity and insulin-resistance. We demonstrated that GAGs produced in the absence of EXTL2 act as DAMPs and directly input signals into cells via the Toll-like 4 receptor. In addition, the subsequent transcriptional activation of inflammatory and tumor-promoting cytokines by NF-κB contributes to injury- and inflammation-driven tumor promotion. Thus, dysregulated biosynthesis of GAGs is considered to increase the risk of HCC in a background of obesity and diabetes.
Assuntos
Carcinogênese/metabolismo , Glicosaminoglicanos/biossíntese , Inflamação/metabolismo , Fígado/metabolismo , N-Acetilglucosaminiltransferases/deficiência , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Hepatite/metabolismo , Hepatite/patologia , Inflamação/patologia , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Hepatopatia Gordurosa não Alcoólica , Proteoglicanas/metabolismo , Células RAW 264.7RESUMO
NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.
Assuntos
Família 7 do Citocromo P450/metabolismo , Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , Oxisteróis/metabolismoRESUMO
Proteoglycans are a major component of extracellular matrix and contribute to normal embryonic and postnatal development by ensuring tissue stability and signaling functions. We studied five patients with recessive joint dislocations and congenital heart defects, including bicuspid aortic valve (BAV) and aortic root dilatation. We identified linkage to chromosome 11 and detected a mutation (c.830G>A, p.Arg277Gln) in B3GAT3, the gene coding for glucuronosyltransferase-I (GlcAT-I). The enzyme catalyzes an initial step in the synthesis of glycosaminoglycan side chains of proteoglycans. Patients' cells as well as recombinant mutant protein showed reduced glucuronyltransferase activity. Patient fibroblasts demonstrated decreased levels of dermatan sulfate, chondroitin sulfate, and heparan sulfate proteoglycans, indicating that the defect in linker synthesis affected all three lines of O-glycanated proteoglycans. Further studies demonstrated that GlcAT-I resides in the cis and cis-medial Golgi apparatus and is expressed in the affected tissues, i.e., heart, aorta, and bone. The study shows that reduced GlcAT-I activity impairs skeletal as well as heart development and results in variable combinations of heart malformations, including mitral valve prolapse, ventricular septal defect, and bicuspid aortic valve. The described family constitutes a syndrome characterized by heart defects and joint dislocations resulting from altered initiation of proteoglycan synthesis (Larsen-like syndrome, B3GAT3 type).
Assuntos
Glucuronosiltransferase/genética , Cardiopatias Congênitas/patologia , Proteoglicanas/biossíntese , Adolescente , Sequência de Aminoácidos , Valva Aórtica/patologia , Estudos de Casos e Controles , Criança , Sulfatos de Condroitina/análise , Cromossomos Humanos Par 11/genética , Consanguinidade , Dermatan Sulfato/análise , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Imunofluorescência , Proteoglicanas de Heparan Sulfato/análise , Humanos , Immunoblotting , Masculino , Valva Mitral/patologia , Modelos Moleculares , Dados de Sequência Molecular , LinhagemRESUMO
Introduction: Clinical studies have suggested a bidirectional association between non-alcoholic steatohepatitis (NASH) and psoriasis, affecting each other's development and severity. Here, we explored bidirectional causal linkages between NASH and psoriasis using a murine model. Methods: NASH was induced in mice by streptozotocin injection at 2 days of age and by high-fat diet feeding (STAM™ model). Psoriasis was induced by topical application of imiquimod (IMQ) on the ear. The severities of liver damage and psoriatic skin changes were determined using histological analysis. Gene expression in the skin tissues was evaluated using quantitative PCR analysis. Serum cytokine levels were determined using enzyme-linked immunosorbent assay. To examine the innate immune responses of normal human epidermal keratinocytes (NHEKs), the cells were treated with interleukin (IL)-17A, tumor necrosis factor (TNF)-α, and AdipoRon, an adiponectin receptor agonist. Results and Discussion: There were no differences in the degree of liver tissue damage (fat deposition, inflammation, and fibrosis) between NASH mice with and those without psoriasis. Conversely, the co-occurrence of NASH significantly augmented psoriatic skin changes, represented by epidermal hyperplasia, in psoriatic mice. Pro-inflammatory cytokines were expressed in the inflamed skin of psoriatic mice, and the expression of genes, especially Il23a, Il1b, Il36g, and Mip2, was significantly upregulated by the co-occurrence of NASH. The expression of keratinocyte activation marker genes Defb4b and Krt16 was also upregulated by the co-occurrence of NASH. The serum TNF-α and IL-17 levels were increased by the co-occurrence of NASH and psoriasis. The serum adiponectin levels decreased in NASH mice compared with that in non-NASH mice. In NHEK culture, TNF-α and IL-17A synergistically upregulated CXCL1, CXCL8, and IL1B expression. The upregulated pro-inflammatory gene expression was suppressed by AdipoRon treatment, reflecting the anti-inflammatory capacity of adiponectin. Conclusion: The co-occurrence of NASH exacerbated psoriatic skin changes associated with increased serum inflammatory cytokine levels and decreased serum adiponectin levels. Combined with in vitro findings, increased inflammatory cytokine levels and decreased adiponectin levels likely promote innate immune responses in epidermal keratinocytes in psoriatic skin lesions. Overall, therapeutic intervention for co-occurring NASH is essential to achieve a favorable prognosis of psoriasis in clinical practice.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Psoríase , Humanos , Camundongos , Animais , Adiponectina , Fator de Necrose Tumoral alfa , Modelos Animais de Doenças , Citocinas , Interleucina-1RESUMO
Thrombomodulin (TM) is an integral membrane glycoprotein, which occurs as both a chondroitin sulfate (CS) proteoglycan (PG) form (ß-TM) and a non-PG form without a CS chain (α-TM) and hence is a part-time PG. An α-TM preparation isolated from human urine contained the glycosaminoglycan linkage region tetrasaccharide GlcUAß1-3Galß1-3Galß1-4xylose, and the nonreducing terminal GlcUA residue is 3-O-sulfated. Because the human natural killer-1 sulfotransferase (HNK-1ST) transfers a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue in the HNK-1 antigen precursor trisaccharide, GlcUAß1-3Galß1-4GlcNAc, the sulfotransferase activity toward the linkage region was investigated. In fact, the activity of HNK-1ST toward the linkage region was much higher than that toward the glucuronylneolactotetraosylceramide, the precursor of the HNK-1 epitope. HNK-1ST may be responsible for regulating the sorting of α- and ß-TM. Furthermore, HNK-1ST also transferred a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue of a chondroitin chain. Intriguingly, the HNK-1 antibody recognized CS chains and the linkage region if they contained GlcUA(3-O-sulfate), suggesting that HNK-1ST not only synthesizes the HNK-1 epitope but may also be involved in the generation of part-time PGs.
Assuntos
Oligossacarídeos/biossíntese , Sulfotransferases/metabolismo , Trombomodulina/metabolismo , Urina/química , Anticorpos/imunologia , Sulfatos de Condroitina/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Glicosaminoglicanos/metabolismo , Humanos , Cinética , Isoformas de Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Sulfotransferases/imunologiaRESUMO
BACKGROUND: Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix and its sugar backbone consists of repeating disaccharide units: D-glucuronic acid (GlcUA)ß1-3N-acetyl-D-galactosamine (GalNAc). Although CS participates in diverse biological processes such as growth factor signaling and the nervous system's development, the mechanism underlying the functions is not well understood. METHODS: CS was isolated from ray fish cartilage, an industrial waste, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration. RESULTS: The major disaccharide unit in the CS preparation was GlcUA-GalNAc(6-O-sulfate) (61.9%). Minor proportions of GlcUA-GalNAc(4-O-sulfate) (27.0%), GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (8.5%), and GlcUA-GalNAc (2.7%) were also detected. The preparation showed NOP activity in vitro, and this activity was suppressed by antibodies against hepatocyte growth factor (HGF) and its receptor c-Met, suggesting the involvement of the HGF signaling pathway in the expression of the in vitro NOP activity of the CS preparation. The specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy. CONCLUSIONS AND GENERAL SIGNIFICANCE: The NOP activity of CS from ray cartilage was demonstrated to be expressed through the HGF signaling pathway, suggesting that ray cartilage CS may be useful for studying the cooperative function of CS and HGF.
Assuntos
Cartilagem/química , Sulfatos de Condroitina/isolamento & purificação , Sulfatos de Condroitina/farmacologia , Elasmobrânquios/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/efeitos dos fármacos , Animais , Linhagem Celular , Sulfatos de Condroitina/química , Hipocampo/citologia , Camundongos , Neurônios/citologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacosRESUMO
The amniotic membrane (AM) is the innermost layer of fetal membranes and possesses various biological activities. Although the mechanism underlying these biological activities remains unclear, unique components seem to be involved. AM contains various extracellular matrix components such as type I collagen, laminin, fibronectin, hyaluronan, and proteoglycans bearing chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycan side chains. Since CS/DS have been implicated in various biological processes, we hypothesized that CS/DS in AM may play a major role in the biological activities of AM. Therefore, the structure and bioactivity of the CS/DS chains from porcine fetal membranes (FM-CS/DS) were investigated. A compositional analysis using various chondroitinases revealed that the characteristic DS domain comprised of iduronic acid-containing disaccharide units is embedded in FM-CS/DS, along with predominant disaccharide units, GlcA-GalNAc, GlcA-GalNAc(4-O-sulfate), and GlcA-GalNAc(6-O-sulfate), where GlcA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. The average molecular mass of FM-CS/DS chains was unusually large and estimated to be 250 - 300 kDa. The FM-CS/DS chains showed neurite outgrowth-promoting activity, which was eliminated by digestion with chondroitinase ABC of the CS/DS chains. This activity was suppressed by antibodies against growth factors including pleiotrophin, midkine, and fibroblast growth factor-2, suggesting the involvement of these growth factors in the neurite outgrowth-promoting activity. The binding of these growth factors to FM-CS/DS was also demonstrated by surface plasmon resonance spectroscopy.
Assuntos
Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Dermatan Sulfato/análogos & derivados , Membranas Extraembrionárias/química , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Animais , Anticorpos/farmacologia , Forma Celular/efeitos dos fármacos , Fracionamento Químico , Sulfatos de Condroitina/metabolismo , Condroitinases e Condroitina Liases/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Dermatan Sulfato/farmacologia , Dissacarídeos/análise , Glicosaminoglicanos/metabolismo , Ácido Idurônico/análise , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinética , Camundongos , Peso Molecular , Sus scrofaRESUMO
Progression to non-alcoholic steatohepatitis (NASH) manifests as hepatitis, fibrosis, and sometimes carcinoma, resulting in liver failure. Various clinical trials have indicated that several pharmacological agents, including angiotensin II receptor blockers (ARBs) or farnesoid X receptor (FXR) agonists, are effective in NASH treatment. In addition, functional foods are expected to be important alternatives for treating or preventing NASH. Recently, focus has been directed toward microalgae as dietary supplements, mainly for lifestyle-related diseases, because they contain various nutrients and functional ingredients. Specifically, a unicellular microalga Euglena gracilis stores a unique ß-1,3-glucan particle called paramylon that stimulates the immune system. In this study, we evaluated the effects of Euglena and paramylon on NASH in Stelic Animal Model (STAM) mice using Sirius red staining and confirmed that oral administration of Euglena or paramylon inhibits the process of liver fibrosis. Moreover, compared with controls, paramylon decreased non-alcoholic fatty liver disease (NAFLD) activity scores related to inflammation. These results indicate that the oral administration of Euglena and paramylon inhibits fibrosis and ameliorates NASH.
RESUMO
BACKGROUND: Fibrotic diseases result from an exuberant response to chronic inflammation. Myelofibrosis is the end result of inflammation in bone, caused by an inflammatory process triggered by production of abnormal myeloid cells driven by mutations affecting the JAK-STAT pathway. Inflammatory cytokine overproduction leads to increased mesenchymal cell proliferation, culminating in fibrosis. Although JAK2 inhibitors, such as the JAK1/2 inhibitor ruxolitinib and the JAK2/FLT3/CSF1R/IRAK1 inhibitor pacritinib suppress abnormal clone expansion in myelofibrosis, ruxolitinib does not appear to prevent or reverse bone-marrow fibrosis in most patients. In two Phase III clinical trials, pacritinib, however, demonstrated improvements in platelet counts and hemoglobin and reductions in transfusion burden in some patients with baseline cytopenias, suggesting it may improve bone-marrow function. Unlike ruxolitinib, pacritinib suppresses signaling through IRAK1, a key control point for inflammatory and fibrotic signaling. PURPOSE: To investigate potential antifibrotic effects of pacritinib in an animal model of liver fibrosis relevant to the observed course of human disease. METHODS: Pacritinib, negative control (vehicle), and positive control (the angiotensin 2-receptor antagonist and PPARγ partial agonist telmisartan) were assessed in the murine Stelic animal model, which mimics the clinically observed progression from hepatic steatosis to nonalcoholic steatohepatitis, liver fibrosis, and hepatocellular carcinoma. Histopathological analysis used hematoxylin and eosin staining. Body and liver weight changes, nonalcoholic fatty-liver disease activity scores, and plasma cytokeratin 18 fragment levels (a biomarker of hepatic necrosis) were measured. RESULTS: Pacritinib-treated mice had significantly (P<0.01) reduced fibrotic areas in liver compared to vehicle control and significantly (P<0.05) lower levels of CK18. The antifibrotic effect of pacritinib was comparable to that of telmisartan, but without significant effects on fat accumulation. CONCLUSION: These results, the first to demonstrate hepatic antifibrotic effects for pacritinib in an animal model of liver disease, provide preliminary support for potential clinical applications of pacritinib in fibrotic diseases other than myelofibrosis.
RESUMO
BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) can advance, if untreated, to liver fibrosis, cirrhosis, hepatocellular carcinoma, liver failure and liver-related death. In the United States, NASH affects approximately 2-5% of the population and an additional 10-30% have NAFLD. The number of drugs in development for NASH is growing steadily, along with nonclinical models to support prediction of clinical success. Here we evaluate gemcabene, a first-in-class clinical candidate for dyslipidemia, for its potential utility, based on its combined lipid-lowering and anti-inflammatory efficacy in clinical trials, in a preclinical model of NASH. METHODS: Gemcabene was evaluated in the STAM™ murine model of NASH. Gemcabene intervention in mice made diabetic with streptozotocin and fed a high fat high-caloric diet was assessed for changes in plasma, and hepatic histological and mRNA markers of lipid metabolism and inflammation. RESULTS: Gemcabene significantly downregulated hepatic mRNA markers of inflammation (TNF-α, MCP-1, MIP-1ß, CCR5, CCR2, NF-κB), lipogenesis and lipid modulation (ApoC-III, ACC1, ADH-4, Sulf-2), and fibrosis (TIMP-1 and MMP-2). These effects are important for the prevention of steatosis, inflammation, and hepatocyte ballooning (i.e., the components of the NAFLD Activity Score or NAS), and inhibition of fibrosis progression, and were observed following treatment with gemcabene. CONCLUSIONS: These non-clinical findings corroborate with existing clinical data to support the clinical evaluation of gemcabene as a potential new treatment for NASH.
Assuntos
Caproatos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Inflamação/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Biomarcadores/metabolismo , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Progressão da Doença , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Cirrose Hepática/imunologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
BACKGROUND & AIMS: Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC's anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis. METHODS: Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC's antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses. RESULTS: CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight. CONCLUSIONS: CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC's antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).
Assuntos
Antagonistas dos Receptores CCR5/uso terapêutico , Imidazóis/uso terapêutico , Nefropatias/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Antagonistas dos Receptores CCR5/administração & dosagem , Antagonistas dos Receptores CCR5/farmacologia , Quimiotaxia , Fibrose , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Nefropatias/patologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Ratos Sprague-Dawley , Receptores CCR2/antagonistas & inibidores , SulfóxidosRESUMO
Induction of mucosal healing (MH) is an important treatment goal in inflammatory bowel disease (IBD). Although the molecular mechanisms underlying MH in IBD is not fully explored, local fibrosis would contribute to interfere mucosal repair. Carbohydrate sulfotransferase 15 (CHST15), which catalyzes sulfation of chondroitin sulfate to produce rare E-disaccharide units, is a novel mediator to create local fibrosis. Here we have used siRNA-based approach of silencing CHST15 in dextran sulfate sodium (DSS) induced colitis in mice, human colon fibroblasts and cancer cell lines. In a DSS-induced acute colitis model, CHST15 siRNA reduced CHST15 mRNA in the colon, serum IL-6, disease activity index (DAI) and accumulation of F4/80+ macrophages and ER-TR7+ fibroblasts, while increased Ki-67+ epithelial cells. In DSS-induced chronic colitis models, CHST15 siRNA reduced CHST15 mRNA in the colon, DAI, alpha-smooth muscle actin+ fibroblasts and collagen deposition, while enhanced MH as evidenced by reduced histological and endoscopic scores. We also found that endoscopic submucosal injection achieved effective pancolonic delivery of CHST15 siRNA in mice. In human CCD-18 Co cells, CHST15 siRNA inhibited the expression of CHST15 mRNA and selectively reduced E-units, a specific product biosynthesized by CHST15, in the culture supernatant. CHST15 siRNA significantly suppressed vimentin in both TGF-ß-stimulated CCD18-Co cells and HCT116 cells while up-regulated BMP7 and E-cadherin in HCT116 cells. The present study demonstrated that blockade CHST15 represses colonic fibrosis and enhances MH partly though reversing EMT pathway, illustrating a novel therapeutic opportunity to refractory and fibrotic lesions in IBD.
Assuntos
Colite/enzimologia , Colite/patologia , Mucosa Intestinal/patologia , Sulfotransferases/metabolismo , Doença Aguda , Animais , Colite/genética , Colo/patologia , Transição Epitelial-Mesenquimal , Feminino , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Transdução de Sinais , Sulfotransferases/deficiência , Sulfotransferases/genética , Carboidrato SulfotransferasesRESUMO
Chondroitin sulfate E (CS-E), a highly sulfated glycosaminoglycan, is known to promote tumor invasion and metastasis. Because the presence of CS-E is detected in both tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC), multistage involvement of CS-E in the development of PDAC has been considered. However, its involvement in the early stage of PDAC progression is still not fully understood. In this study, to clarify the direct role of CS-E in tumor, but not stromal, cells of PDAC, we focused on carbohydrate sulfotransferase 15 (CHST15), a specific enzyme that biosynthesizes CS-E, and investigated the effects of the CHST15 siRNA on tumor cell proliferation in vitro and growth in vivo. CHST15 mRNA is highly expressed in the human pancreatic cancer cell lines PANC-1, MIA PaCa-2, Capan-1 and Capan-2. CHST15 siRNA significantly inhibited the expression of CHST15 mRNA in these four cells in vitro. Silencing of the CHST15 gene in the cells was associated with significant reduction of proliferation and up-regulation of the cell cycle inhibitor-related gene p21CIP1/WAF1. In a subcutaneous xenograft tumor model of PANC-1 in nude mice, a single intratumoral injection of CHST15 siRNA almost completely suppressed tumor growth. Reduced CHST15 protein signals associated with tumor necrosis were observed with the treatment with CHST15 siRNA. These results provide evidence of the direct action of CHST15 on the proliferation of pancreatic tumor cells partly through the p21CIP1/WAF1 pathway. Thus, CHST15-CS-E axis-mediated tumor cell proliferation could be a novel therapeutic target in the early stage of PDAC progression.
Assuntos
Proliferação de Células/genética , Inativação Gênica/fisiologia , Glicoproteínas de Membrana/genética , Neoplasias Pancreáticas/genética , Sulfotransferases/genética , Adenocarcinoma/genética , Animais , Carcinoma Ductal Pancreático/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The therapeutic strategy against hepatocellular carcinoma (HCC) is determined by tumor stage and liver function. Improvements of stratification contribute to extending the survival of patients. However, stratification has been attributed little attention in animal models largely due to the lack of suitable models. Herein we showed that the recently-reported, non-alcoholic steatohepatitis-derived HCC model (STAM model) is the first murine model in which the concept of human stratification is applicable by demonstrating the following features: (i) at least 4 detectable tumor nodules; (ii) average tumor growth rate of 150 % from 16 to 20 weeks of age; (iii) no visible metastasis; and (iv) relatively preserved liver function. These observations suggested that HCC in STAM mice is equivalent to stages B to C of the Barcelona Clinic Liver Cancer (BCLC) staging system for humans. Application of the stratification concept to experimental animals will create new avenues to establish pharmacological intervention against HCC.
Assuntos
Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/etiologia , Fígado Gorduroso/complicações , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/etiologia , Animais , Carcinoma Hepatocelular/mortalidade , Modelos Animais de Doenças , Humanos , Fígado/metabolismo , Fígado/patologia , Testes de Função Hepática , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos , Estadiamento de Neoplasias , Hepatopatia Gordurosa não Alcoólica , Carga Tumoral , Microtomografia por Raio-XRESUMO
The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.