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1.
J Crohns Colitis ; 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31168612

RESUMO

BACKGROUND AND AIMS: Heat shock protein 90 (Hsp90)-targeted therapy has been proposed as a promising strategy for the treatment of ulcerative colitis (UC) and colitis-associated cancer (CAC). The systemic administration of the Hsp90 inhibitor, 17-AAG, was found to be profoundly protective in preclinical mouse models of inflammatory bowel disease (IBD). However, the therapeutic potential of 17-AAG is limited by potential side effects associated with its systemic exposure and the modest bioavailability afforded by its oral administration. METHODS: In an effort to address these issues, we herein used a versatile single-step surface-functionalizing technique to prepare a 17-AAG oral delivery system using PLGA/PLA-PEG-FA nanoparticles (NP-PEG-FA/17-AAG). RESULTS: NP-PEG-FA could be efficiently taken up by mouse Colon-26 cells and activated Raw 264.7 cells in vitro and by inflamed mouse colitis tissues in vivo. The therapeutic efficacy of orally administrated NP-PEG-FA/17-AAG was evaluated in in vivo models using dextran sulfate sodium (DSS)-induced UC and azoxymethane (AOM)/DSS-induced CAC, and results indicated that NP-PEG-FA/17-AAG significantly alleviated the symptoms of UC and CAC. More importantly, our inflamed colitis-targeted 17-AAG nano-formulation reduced systemic exposure and provided a degree of therapeutic response similar to that obtained by systemic administration (intraperitoneal, IP) of 17-AAG, but at a 10-fold lower dose. CONCLUSIONS: We therefore describe a convenient, orally administrated 17-AAG delivery system that exhibits enhanced efficacy in UC and CAC therapy while reducing systemic exposure. This system may represent a promising therapeutic approach for treating UC and CAC.

2.
Sci Rep ; 8(1): 16220, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385787

RESUMO

CD98 has been implicated in the experimental model of inflammatory bowel disease. We have previously shown that IEC-specific overexpression of CD98 mediates intestinal inflammation and intestinal epithelial barrier dysfunction. Mice overexpressing CD98 exhibited severe colitis and a greater susceptibility to CAC. Here we demonstrated CD98 overexpression to dysregulate homeostatic gradient profile of miRNA and protein expression along the ileal villus-crypt axis. Using miRNA-target gene prediction module, we observed differentially expressed miRNAs to target proteins of villus and crypt profoundly affected by CD98 overexpression. We have utilized online bioinformatics as methods to further scrutinize the biological meanings of miRNA-target data. We identified significant interactions among the differentially regulated proteins targeted by altered miRNAs in Tg mice. The biological processes affected by the predicted targets of miRNAs deviate from the homeostatic functions of the miRNA-gene-protein axis of the wildtype mice. Our results emphasize a dynamic perturbation of miRNA and protein expression in villus-crypt axis contributing to potential biological consequences of altering CD98 expression. Our findings also suggest the need for a consideration of arrays of interacting biological entities (i.e. miRNAs-mRNAs, protein-protein interaction) or a combination comparison for a better understanding of the disease pathology which is necessary for an effective therapeutic target development.

3.
Oncotarget ; 8(55): 94650-94665, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29212256

RESUMO

In colitis associated cancer (CAC), chronic inflammation exposes the epithelial mucosal defensive lining to inflammatory mediators such as cytokines and anti-microbial peptides (AMPs) causing the dysbiosis of microbiota population and the dysregulation of immune response. Matrix Metalloproteinases (MMPs) are zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and carcinogenesis. MMP9 is undetectable in healthy tissue, although highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC opposite to its conventional role of acute inflammation and cancer mediator. In this study, we investigated the mechanistic role of MMP9 in preserving the epithelial mucosal integrity to suppress the progression of tumor microenvironment in CAC. We used transgenic mice constitutively expressing MMP9 in colonic epithelium (TgM9) as an in vivo model and intestinal cell line CaCo2BBE as an in vitro model. We induced CAC with three cycles of dextran sodium sulfate (DSS). We observed that MMP9 expression in colonic epithelium maintains the microbiota. We also observed that MMP9 mediates pro-inflammatory cytokine levels and AMPs but suppresses IL-22 resulting in lower levels of REG3-g and S100A8 AMPs. We also found that MMP9 maintains an efficient barrier function and the integrity of tight junctions. We also observed increased levels of mucin and intestinal trefoil factor among TgM9 mice in CAC. We also found that MMP9 expressing CaCo2BBE cells had increased expressions of EGFR and nuclear transcription factor- specificity protein 1 (Sp1). These data imply that MMP9 acts as a tumor suppressor in CAC by sustaining the epithelial mucosal integrity due to the activation of EGFR-Sp1 signaling pathway.

4.
Oncotarget ; 8(1): 364-378, 2017 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-27861153

RESUMO

Colitis associated cancer (CAC) is chronic inflammation driven colon cancer, prevalent among individuals with Inflammatory Bowel Disease. Matrix-metalloproteinase (MMP9) is one of the essential regulators of extra cellular matrix components. We have shown that MMP9 is protective in CAC contrary to its inflammatory role in acute-colitis. Aim of our study is to identify the mechanism of the protective role of epithelial derived-MMP9 in CAC. We used homozygous transgenic mice constitutively-expressing MMP9 in colonic-epithelium (TgM9) and wild-type (WT) littermates for in vivo experiments. Stably-transfected HCT116 with/without MMP9, and mouse embryonic-fibroblasts (WT and MMP9-/-, MEFs) were used for in vitro experiments. TgM9 mice exhibited less tumor burden, increased apoptosis, and increased expressions of active-Notch1, p53, p21WAF1/Cip1, caspase-3 and cyclin E in CAC compared to WTs. These results were supported by MEFs data. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation, S-phase cell-cycle arrest and less DNA damage compared to vector. MMP9-/- mice showed attenuation of MMP9 was directly associated with p19ARF. Our study identifies the tumor suppressor role of epithelial derived-MMP9 in CAC via novel mechanistic pathway "MMP9-Notch1-ARF-p53 axis" regulating apoptosis, cell-cycle arrest and DNA damage implying, that MMP9 expression might be a natural/biological way to suppress colonic ulceration due to chronic inflammation.


Assuntos
Colite/patologia , Neoplasias do Colo/patologia , Mucosa Intestinal/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Proliferação de Células , Colite/induzido quimicamente , Colite/complicações , Colo/imunologia , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/etiologia , Ciclina E/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Sulfato de Dextrana/toxicidade , Fibroblastos , Células HCT116 , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/patologia , Receptor Notch1/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Transdução de Sinais , Transfecção , Carga Tumoral , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética
5.
Dig Liver Dis ; 49(2): 188-196, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27939923

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid hepatic accumulation. Here, we investigated whether a reduction of CD98 expression mediated by CD98 siRNA-loaded nanoparticles (NPs) could attenuate liver disease markers in a mouse model of NAFLD. NPs were generated using a double emulsion/solvent evaporation technique. Mice fed a high fat diet for 8 weeks to induce fatty liver were treated with vein tail injections of CD98 siRNA-loaded NPs. In vitro, HepG2 treated with CD98 siRNA-loaded NPs showed significant downregulation of CD98 leading to a significant decrease of major pro-inflammatory cytokines and markers. In vivo, CD98 siRNA-loaded NPs strongly decreased all markers of NAFLD, including the blood levels of ALT and lipids accumulation, fibrosis evidence and pro-inflammatory cytokines. In conclusion, our results indicate that CD98 appears to function as a key actor/inducer in NAFLD, and that our NPs approach may offer a new targeted therapeutic for this disease.


Assuntos
Proteína-1 Reguladora de Fusão/genética , Inativação Gênica , Hepatopatia Gordurosa não Alcoólica/terapia , RNA Interferente Pequeno/genética , Animais , Biomarcadores/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Camundongos , Nanopartículas/química
6.
Cell Mol Gastroenterol Hepatol ; 2(3): 340-357, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27458604

RESUMO

BACKGROUND AND AIMS: The human intestinal peptide transporter 1, hPepT1, is expressed in the small intestine at low levels in the healthy colon and upregulated during inflammatory bowel disease. hPepT1 plays a role in mouse colitis and human studies have demonstrated that chronic intestinal inflammation leads to colorectal cancer (colitis-associated cancer; CAC). Hence, we assessed here the role of PepT1 in CAC. METHODS: Mice with hPepT1 overexpression in intestinal epithelial cells (TG) or PepT1 (PepT1-KO) deletion were used and CAC was induced by AOM/DSS. RESULTS: TG mice had larger tumor sizes, increased tumor burdens, and increased intestinal inflammation compared to WT mice. Conversely, tumor number and size and intestinal inflammation were significantly decreased in PepT1-KO mice. Proliferating crypt cells were increased in TG mice and decreased in PepT1-KO mice. Analysis of human colonic biopsies revealed an increased expression of PepT1 in patients with colorectal cancer, suggesting that PepT1 might be targeted for the treatment of CAC. The use of an anti-inflammatory tripeptide KPV (Lys-Pro-Val) transported by PepT1 was able to prevent carcinogenesis in WT mice. When administered to PepT1-KO mice, KPV did not trigger any of the inhibitory effect on tumorigenesis observed in WT mice. CONCLUSIONS: The observations that pepT1 was highly expressed in human colorectal tumor and that its overexpression and deletion in mice increased and decreased colitis associated tumorigenesis, respectively, suggest that PepT1 is a potential therapeutic target for the treatment of colitis associated tumorigenesis.

7.
Sci Rep ; 6: 27119, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27250880

RESUMO

In the jejunum, PepT1 is particularly enriched in the well-differentiated absorptive epithelial cells in the villi. Studies of expression and function of PepT1 along the crypt-villus axis demonstrated that this protein is crucial to the process of di/tripeptide absorption. We recently exhibited that PepT1 plays an important role in multiple biological functions, including the ability to regulate the expression/secretion of specific microRNAs (miRNAs) and the expression levels of multiple proteins. In this study, we observed that PepT1 knockout (KO) mice exhibited reduced body weight and shorten intestinal microvilli. We then examined the expression levels of various miRNAs and their target proteins along the crypt-villi axis in the jejunum of PepT1 KO mice. We found that PepT1 KO altered the distribution of miRNAs along the crypt-villus axis and changed the miRNA profiles of both villi and crypts. Using miRNA-target prediction and 2D-DIGE/mass spectrometry on villi and crypts samples, we found that ablation of PepT1 further directly or indirectly altered expression levels of certain protein targets. Collectively, our results suggest that PepT1 contributes to maintain balance of homeostasis and proper functions in the small intestine, and dysregulated miRNAs and proteins along the crypt-villus axis are highly related to this process.


Assuntos
Intestino Delgado/anormalidades , MicroRNAs/genética , Transportador 1 de Peptídeos/genética , Transportador 1 de Peptídeos/metabolismo , Animais , Tamanho Corporal , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Homeostase , Intestino Delgado/fisiologia , Jejuno/anormalidades , Jejuno/fisiologia , Camundongos
8.
Am J Physiol Gastrointest Liver Physiol ; 304(9): G793-803, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23471340

RESUMO

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease, is a chronic inflammatory disease associated with an increased risk for colon cancer. Matrix metalloproteinases (MMPs) are the predominant proteinases expressed in the gut mucosa during active IBD. Our laboratory has previously demonstrated that epithelial-derived MMP9 is absent in normal colonic tissue but is upregulated during IBD. In this study MMP9 transgenic mice (Tg-villin-MMP9) are generated specifically to overexpress MMP9 in intestinal epithelium to examine the role and underlying mechanism by which it modulates the pathogenesis of acute colitis. Dextran sodium sulfate (3% DSS)- and Salmonella typhimurium (S.T.)-induced colitis models were used to study gut inflammation in Tg-villin-MMP9 and wild-type littermates (WT). Colonic tissue was analyzed via Western blot, histology, myeloperoxidase (MPO) assay, and quantitative PCR. Tg-villin-MMP9 mice expressed significantly increased MMP9 mRNA and protein expression at basal level. There was a significant decrease in the goblet cells, but a significant increase in proliferation and apoptosis were observed among Tg-villin-MMP9 mice compared with WT mice. There was also a significant increase in the proinflammatory chemokine Kc among Tg-villin-MMP9 compared with WT mice. Tg-villin-MMP9 exhibited a severe inflammatory response than WT mice in both DSS- and S.T.-induced colitis models as evident by greater weight loss and higher clinical score, histological score, and MPO activity, which correlated with relative levels of Kc mRNA. MMP9 expressed by intestinal epithelial cells mediates inflammation in colitis with simultaneous increase in proinflammatory cytokine Kc.


Assuntos
Quimiocina CXCL1/metabolismo , Colite/metabolismo , Mucosa Intestinal/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Células HCT116 , Humanos , Doenças Inflamatórias Intestinais/fisiopatologia , Camundongos , Camundongos Transgênicos , Salmonelose Animal/patologia , Salmonelose Animal/fisiopatologia , Salmonella typhimurium
9.
Dig Liver Dis ; 44(10): 819-26, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22721840

RESUMO

BACKGROUND: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis. AIMS: Our goals were to determine if A(2B)AR expression was necessary on immune cells/non-immune cells during colitis and if A(2B)AR was a suitable target for treating intestinal inflammation. METHODS: Wild-type and A(2B)AR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A(2B)AR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A(2B)AR/RAG1 double knockout recipients. Finally, A(2B)AR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. RESULTS: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A(2B)AR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. CONCLUSIONS: Our results suggest that A(2B)AR on non-immune cells plays an important role for the induction of colitis and targeting A(2B)AR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.


Assuntos
Colite/metabolismo , Inflamação/metabolismo , Receptor A2B de Adenosina/metabolismo , Animais , Transplante de Medula Óssea , Colite/induzido quimicamente , Colite/imunologia , Colo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Camundongos , Camundongos Knockout , Nanopartículas , RNA Mensageiro/metabolismo
10.
Gastroenterology ; 141(4): 1381-92, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21723221

RESUMO

BACKGROUND & AIMS: Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice. METHODS: C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9. RESULTS: Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9-/- mice on γ-radiation-induced damage of DNA. CONCLUSIONS: MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.


Assuntos
Colite/enzimologia , Colo/enzimologia , Neoplasias do Colo/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Receptor Notch1/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Apoptose , Azoximetano , Caspase 3/metabolismo , Colite/induzido quimicamente , Colite/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dano ao DNA , Sulfato de Dextrana , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Raios gama , Células HCT116 , Humanos , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptor Notch1/antagonistas & inibidores , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/metabolismo
11.
Am J Physiol Lung Cell Mol Physiol ; 301(1): L79-90, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21531776

RESUMO

Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin. In HMVEC-Ls, EGFR and ErbB2 are expressed at low levels, and both heterodimerize and tyrosine autophosphorylate in response to TSP1. Prior EGFR-selective PTK inhibition with AG1478 or ErbB2-selective PTK inhibition with AG825 protected against TSP1-induced tyrosine phosphorylation of ZA proteins and barrier disruption. Preincubation of HMVEC-Ls with an EGFR ectodomain-blocking antibody also prevented TSP1-induced opening of the paracellular pathway. Therefore, in HMVEC-Ls, TSP1 increases tyrosine phosphorylation of ZA proteins and opens the paracellular pathway, in part, through EGFR/ErbB2 activation. Surprisingly, recombinant TSP1 EGF-like repeats 1-3 and the high-affinity EGFR ligands, EGF, TGF-α, and amphiregulin, each failed to increase paracellular permeability. However, HMVEC-Ls in which EGFR was overexpressed became responsive to the EGF-like repeats of TSP1 as well as to EGF. These studies indicate that TSP1 disrupts the endothelial barrier through EGFR/ErbB2 activation although additional signals are necessary in cells with low receptor expression.


Assuntos
Células Endoteliais/enzimologia , Receptores ErbB/metabolismo , Pulmão/irrigação sanguínea , Microvasos/citologia , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombospondina 1/farmacologia , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Radioisótopos de Carbono , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Multimerização Proteica/efeitos dos fármacos , Receptor ErbB-2/genética , Sequências Repetitivas de Aminoácidos , Soroalbumina Bovina/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Trombospondina 1/química , Fatores de Tempo
12.
Life Sci ; 88(1-2): 74-81, 2011 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-21047522

RESUMO

AIMS: Vascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory bowel disease. Thus, we hypothesized that the potent anti-angiogenic factor endostatin might play a beneficial role in experimental ulcerative colitis (UC). MAIN METHODS: We used three animal models of UC: (1) induced by 6% iodoacetamide (IA) in rats, or (2) by 3% dextran sulfate sodium (DSS) in matrix metalloproteinase-9 (MMP-9) knockout (KO) and wild-type mice, and (3) interleukin-10 (IL-10) KO mice. Groups of MMP-9 KO mice with DSS-induced UC were treated with endostatin or water for 5days. KEY FINDINGS: We found concomitant upregulation of VEGF, PDGF, MMP-9 and endostatin in both rat and mouse models of UC. A positive correlation between the levels of endostatin or VEGF and the sizes of colonic lesions was seen in IA-induced UC. The levels and activities of MMP-9 were also significantly increased during UC induced by IA and IL-10 KO. Deletion of MMP-9 decreased the levels of endostatin in both water- and DSS-treated MMP-9 KO mice. Treatment with endostatin significantly improved DSS-induced UC in MMP-9 KO mice. SIGNIFICANCE: 1) Concomitantly increased endostatin is a defensive response to the increased VEGF in UC, 2) MMP-9 is a key enzyme to generate endostatin which may modulate the balance between VEGF and endostatin during experimental UC, and 3) endostatin treatment plays a beneficial role in UC. Thus, anti-angiogenesis seems to be a new therapeutic option for UC.


Assuntos
Colite Ulcerativa/etiologia , Endostatinas/fisiologia , Animais , Western Blotting , Colite Ulcerativa/metabolismo , Colite Ulcerativa/fisiopatologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/fisiopatologia , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Endostatinas/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Iodoacetamida/farmacologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Camundongos Knockout , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/fisiologia , Ratos , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia
13.
Cancer Res ; 70(2): 792-801, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20068187

RESUMO

There is a well-documented association of matrix metalloproteinase-9 (MMP-9) and receptor Notch-1 overexpression in colon cancer. We recently showed that MMP-9 is also upregulated in colitis, where it modulates tissue damage and goblet cell differentiation via proteolytic cleavage of Notch-1. In this study, we investigated whether MMP-9 is critical for colitis-associated colon cancer (CAC). Mice that are wild type (WT) or MMP-9 nullizygous (MMP-9(-/-)) were used for in vivo studies and the human enterocyte cell line Caco2-BBE was used for in vitro studies. CAC was induced in mice using an established carcinogenesis protocol that involves exposure to azoxymethane followed by treatment with dextran sodium sulfate. MMP-9(-/-) mice exhibited increased susceptibility to CAC relative to WT mice. Elevations in tumor multiplicity, size, and mortality were associated with increased proliferation and decreased apoptosis. Tumors formed in MMP-9(-/-) mice exhibited expression of p21(WAF1/Cip1) and increased expression of beta-catenin relative to WT mice. In vitro studies of MMP-9 overexpression showed increased Notch-1 activation with a reciprocal decrease in beta-catenin. Notch and beta-catenin/Wnt signaling have crucial roles in determining differentiation and carcinogenesis in gut epithelia. Despite being a mediator of proinflammatory responses in colitis, MMP-9 plays a protective role and acts as a tumor suppressor in CAC by modulating Notch-1 activation, thereby resulting in activation of p21(WAF1/Cip1) and suppression of beta-catenin.


Assuntos
Colite/enzimologia , Neoplasias do Colo/enzimologia , Metaloproteinase 9 da Matriz/fisiologia , Animais , Apoptose/fisiologia , Células CACO-2 , Processos de Crescimento Celular/fisiologia , Colite/genética , Colite/metabolismo , Colite/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Ciclo-Oxigenase 2/biossíntese , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Transfecção , beta Catenina/metabolismo
14.
Am J Physiol Gastrointest Liver Physiol ; 296(2): G175-84, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19171847

RESUMO

Matrix metalloproteinases (MMP) play an important role in pathogenesis of inflammatory bowel disease (IBD). Two known gelatinases, MMP-2 and MMP-9, are upregulated during IBD. Epithelial-derived MMP-9 is an important mediator of tissue injury in colitis, whereas MMP-2 protects against tissue damage and maintains gut barrier function. It has been suggested that developing strategies to block MMP-9 activity in the gut might be of benefit to IBD. However, given that MMP-2 and MMP-9 are structurally similar, such approaches would also likely inhibit MMP-2. Thus, to gain insight into outcome of inhibiting both MMP-2 and MMP-9, MMP-2(-/-)/MMP-9(-/-) double knockout mice (dKO) lacking both MMP-2 and MMP-9 were used in this study. Three models of murine colitis were used: dextran sodium sulfate (DSS), Salmonella typhimurium (S.T.), and trinitrobenzene sulfonic acid (TNBS). Our data demonstrate that MMP-2 and MMP-9 activities were highly upregulated in wild-type (WT) mice treated with DSS, S.T., or TNBS whereas dKO mice were resistant to the development of colitis. WT mice had extensive inflammation and tissue damage compared with dKO mice as suggested by histological assessment and myeloperoxidase activity. In conclusion, these results suggest an overriding role of MMP-9 in mediating tissue injury compared with the protective role of MMP-2 in development of colitis. Thus inhibition of MMP-9 may be beneficial in treatment of colitis even if resulting in inhibition of MMP-2.


Assuntos
Colite/prevenção & controle , Colo/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Colite/induzido quimicamente , Colite/enzimologia , Colite/microbiologia , Colite/patologia , Colo/efeitos dos fármacos , Colo/microbiologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Desenho de Drogas , Feminino , Fármacos Gastrointestinais/farmacologia , Mucosa Intestinal/enzimologia , Masculino , Metaloproteinase 2 da Matriz/deficiência , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Proteases/farmacologia , Salmonella typhimurium/patogenicidade , Ácido Trinitrobenzenossulfônico
15.
J Biol Chem ; 284(10): 6389-402, 2009 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-19129184

RESUMO

Thrombospondin (TSP) 1 is a trimeric multidomain protein that contains motifs that recognize distinct host cell receptors coupled to multiple signaling pathways. Selected TSP1-induced cellular responses are tyrosine kinase-dependent, and TSP1 contains epidermal growth factor (EGF)-like repeats. Specific receptor interactions or functions for the EGF-like repeats have not been identified. We asked whether one or more biological responses to TSP1 might be explained through EGF receptor (EGFR) activation. In A431 cells, TSP1 increased autophosphorylation of Tyr-1068 of EGFR in a dose- and time-dependent manner. The ability of TSP1 to activate EGFR was replicated by the tandem EGF-like repeats as a recombinant protein. The three EGF-like repeats alone produced a high level of Tyr-1068 phosphorylation. EGF-like repeats from TSP2 and TSP4 also activated EGFR. Tyr-1068 phosphorylation was less when individual EGF-like repeats were tested or flanking sequences were added to the three EGF-like repeats. TSP1 and its EGF-like repeats also increased phosphorylation of EGFR Tyr-845, Tyr-992, Tyr-1045, Tyr-1086, and Tyr-1173, activated phospholipase Cgamma, and increased cell migration. No evidence was found for binding of the EGF-like repeats to EGFR. Instead, EGFR activation in response to TSP1 or its EGF-like repeats required matrix metalloprotease activity, including activity of matrix metalloprotease 9. Access to the ligand-binding portion of the EGFR ectodomain was also required. These findings suggest release of an endogenous EGFR ligand in response to ligation of a second unknown receptor by the TSPs.


Assuntos
Movimento Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Fosfolipase C gama/metabolismo , Trombospondinas/farmacologia , Motivos de Aminoácidos/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células Epiteliais/citologia , Receptores ErbB/genética , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trombospondinas/metabolismo , Fatores de Tempo
16.
FEMS Microbiol Lett ; 288(2): 196-201, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18811655

RESUMO

By conventional genetic methods, including pulse-field gel electrophoresis and multilocus sequence typing, most pathogenic, cholera toxin-positive O1 and O139 isolates of Vibrio cholerae cannot be distinguished. We evaluated relationships among 173 V. cholerae isolates collected between 1992 and 2007 from different geographic areas in India by analyzing five variable number of tandem repeat (VNTR) loci. Each VNTR locus was highly variable, with between 5 and 19 alleles. eburst analysis revealed four large groups of genetically related isolates. Two groups contained genotypes of isolates with the O139 serogroup (which emerged for the first time in epidemic form in 1992), with the other two groups containing O1 strains. In subsequent analysis, it was possible to track the spread of specific genotypes across time and space. Our data highlight the utility of the methodology as an epidemiologic tool for assessing spread of isolates in both epidemic and endemic settings.


Assuntos
Técnicas de Tipagem Bacteriana , Cólera/epidemiologia , Cólera/microbiologia , Repetições Minissatélites/genética , Vibrio cholerae O139/classificação , Vibrio cholerae O1/classificação , DNA Bacteriano/genética , Estudos Epidemiológicos , Evolução Molecular , Variação Genética , Genótipo , Humanos , Índia/epidemiologia , Vibrio cholerae O1/genética , Vibrio cholerae O1/isolamento & purificação , Vibrio cholerae O139/genética , Vibrio cholerae O139/isolamento & purificação
17.
Gastroenterology ; 132(5): 1877-89, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17484881

RESUMO

BACKGROUND & AIMS: We recently demonstrated that epithelial-derived matrix metalloproteinase (MMP) 9 up-regulated during inflammatory bowel disease is a critical mediator of tissue damage during colitis. MMP-9 null mice (MMP-9(-/-)) develop dramatically reduced inflammatory response to luminally administered colitic agents in the face of intact systemic immune response and inflammatory cell recruitment, suggesting protected epithelial barrier in these mice. In this study, we sought to address the role and mechanism by which MMP-9 influences barrier protective function. METHODS: Wild-type and MMP-9(-/-) mice were used for in vivo studies, and the goblet cell line HT29-cl.16E and the enterocyte cell line Caco2-BBE were used for in vitro studies. RESULTS: Compared with wild-type mice, MMP-9(-/-) mice had an increased number of goblet cells and MUC-2 expression. In addition, KLF-4 and Elf-3, transcription factors involved in terminal differentiation of goblet cells were up-regulated, whereas notch intracellular domain (NICD; activated Notch-1) was down-regulated in MMP-9(-/-) mice. These findings suggest altered epithelial cell differentiation in MMP-9(-/-) mice. Temporal expression of MMP-9 inversely correlated with MUC-2 expression during maturation of goblet cells. MMP-9 over expression inhibited goblet cell differentiation in vitro. Conversely, MMP-9 gene silencing in Caco2-BBE cells resulted in a change in their phenotype toward goblet cells. Finally, MMP-9 over expression or silencing in goblet cells increased or decreased Salmonella typhimurium adherence, respectively. CONCLUSIONS: MMP-9 regulates goblet cell differentiation in colon. The effect of MMP-9 on goblet cells could contribute to alteration in mucosal defense leading to inflammation. Together, our data uncover a novel function of MMP-9 in intestinal epithelial cells.


Assuntos
Diferenciação Celular/fisiologia , Células Caliciformes/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Mucinas/metabolismo , Animais , Aderência Bacteriana/fisiologia , Células CACO-2 , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Suscetibilidade a Doenças , Feminino , Regulação da Expressão Gênica/fisiologia , Células Caliciformes/citologia , Células HT29 , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Knockout , Mucina-2 , Mucinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Infecções por Salmonella/fisiopatologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/patogenicidade , Transfecção
18.
Inflamm Bowel Dis ; 13(1): 97-107, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17206645

RESUMO

Matrix metalloproteinases (MMPs) are a family of Zn(2+)-dependent extracellular matrix (ECM) degrading endopeptidases that share common functional domains, activation mechanisms, and collectively have the capacity to degrade all types of ECM proteins. In addition to playing a central role in ECM turnover, MMPs proteolytically activate or degrade a variety of nonmatrix substrates including chemokines, cytokines, growth factors, and junctional proteins. Thus, they are increasingly recognized as critical players in inflammatory response. Indeed, accumulating data from several studies indicate that they are the predominant proteases involved in the pathogenesis of inflammatory bowel disease (IBD) via their influence on the function and migration of inflammatory cells, mucosal ulceration, as well as matrix deposition and degradation. Some MMPs are constitutively expressed and play a protective role in IBD through their effect on cellular homeostasis, while others are induced during inflammation-mediated tissue damage. This article focuses on the role of the various MMPs in IBD, discussing their physiologic and pathogenetic role in the context of intestinal defense, mucosal inflammatory response, and immune cell-epithelial interaction.


Assuntos
Doenças Inflamatórias Intestinais/fisiopatologia , Metaloproteinases da Matriz/fisiologia , Humanos , Inibidores Teciduais de Metaloproteinases/fisiologia
19.
J Immunol ; 177(6): 4103-12, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16951375

RESUMO

The matrix metalloproteinases (MMPs), MMP-2 and MMP-9, share structural and substrate similarities and are up-regulated during human as well as animal models of inflammatory bowel disease. We recently demonstrated that epithelial-derived MMP-9 is an important mediator of inflammation and tissue damage in colitis. In this study, we examined the role of MMP-2 in acute colitis. Colitis was induced using two models, administration of dextran sodium sulfate (DSS) and Salmonella enterica subsp. serovar Typhimurium (S.T.). Bone marrow chimeras were performed using bone marrow cells from wild-type (WT) and MMP-2(-/-) mice. Colitis was evaluated by clinical symptoms, myeloperoxidase assay, and histology. MMP-2 protein expression and activity were up-regulated in WT mice treated with DSS or S.T. MMP-2(-/-) mice were highly susceptible to the development of colitis induced by DSS (or S.T.) compared with WT. During inflammation, MMP-2 expression was increased in epithelial cells as well as in the infiltrating immune cells. Bone marrow chimera demonstrated that mucosa-derived MMP-2 was required for its protective effects toward colitis. Furthermore, we demonstrate that severe colitis in MMP-2(-/-) is not due to a compensatory increase in MMP-9. Finally, we show that MMP-2 regulates epithelial barrier function. In contrast to MMP-9, mucosa-derived MMP-2 may be a critical host factor that is involved in the prevention or cessation of the host response to luminal pathogens or toxins, an important aspect of healing and tissue resolution. Together, our data suggest that a critical balance between the two gelatinases determines the outcome of inflammatory response during acute colitis.


Assuntos
Colite/enzimologia , Colite/genética , Metaloproteinase 2 da Matriz/deficiência , Metaloproteinase 2 da Matriz/genética , Doença Aguda , Animais , Colite/induzido quimicamente , Colite/microbiologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Salmonella typhimurium/imunologia
20.
Am J Physiol Lung Cell Mol Physiol ; 291(6): L1232-45, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16891393

RESUMO

Tumor necrosis factor (TNF)-alpha is a key mediator of sepsis-associated multiorgan failure, including the acute respiratory distress syndrome. We examined the role of protein tyrosine phosphorylation in TNF-alpha-induced pulmonary vascular permeability. Postconfluent human lung microvascular and pulmonary artery endothelial cell (EC) monolayers exposed to human recombinant TNF-alpha displayed a dose- and time-dependent increase in transendothelial [(14)C]albumin flux in the absence of EC injury. TNF-alpha also increased tyrosine phosphorylation of EC proteins, and several substrates were identified as the zonula adherens proteins vascular endothelial (VE)-cadherin, and beta-catenin, gamma-catenin, and p120 catenin (p120(ctn)). Prior protein tyrosine kinase (PTK) inhibition protected against the TNF-alpha effect. TNF-alpha activated multiple PTKs, including src family PTKs. Prior PTK inhibition with the src-selective agents PP1 and PP2 each protected against approximately 60% of the TNF-alpha-induced increment in [(14)C]albumin flux. PP2 also blocked TNF-alpha-induced tyrosine phosphorylation of VE-cadherin, gamma-catenin, and p120(ctn). To identify which src family kinase(s) was required for TNF-alpha-induced vascular permeability, small interfering RNA (siRNA) targeting each of the three src family PTKs expressed in human EC, c-src, fyn, and yes, were introduced into the barrier function assay. Only fyn siRNA protected against the TNF-alpha effect, whereas the c-src and yes siRNAs did not. These combined data suggest that TNF-alpha regulates the pulmonary vascular endothelial paracellular pathway, in part, through fyn activation.


Assuntos
Caderinas/metabolismo , Endotélio Vascular/fisiologia , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas c-fyn/fisiologia , Mucosa Respiratória/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Caderinas/efeitos dos fármacos , Linhagem Celular , Endotélio Vascular/efeitos dos fármacos , Humanos , Pulmão , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Mucosa Respiratória/efeitos dos fármacos
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