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1.
Pharmaceutics ; 15(2)2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36839820

RESUMO

The progressive loss of beta-cell mass is a hallmark of diabetes and has been suggested as a complementary approach to studying the progression of diabetes in contrast to the beta-cell function. Non-invasive nuclear medicinal imaging techniques such as Positron Emission Tomography using radiation emitting tracers have thus been suggested as more viable methodologies to visualize and quantify the beta-cell mass with sufficient sensitivity. The transmembrane G protein-coupled receptor GPR44 has been identified as a biomarker for monitoring beta-cell mass. MK-7246 is a GPR44 antagonist that selectively binds to GPR44 with high affinity and good pharmacokinetic properties. Here, we present the synthesis of MK-7246, radiolabeled with the positron emitter fluorine-18 for preclinical evaluation using cell lines, mice, rats and human pancreatic cells. Here, we have described a synthesis and radiolabeling method for producing [18F]MK-7246 and its precursor compound. Preclinical assessments demonstrated the strong affinity and selectivity of [18F]MK-7246 towards GPR44. Additionally, [18F]MK-7246 exhibited excellent metabolic stability, a fast clearance profile from blood and tissues, qualifying it as a promising radioactive probe for GPR44-directed PET imaging.

2.
Biomedicines ; 9(4)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923731

RESUMO

A validated imaging marker for beta-cell mass would improve understanding of diabetes etiology and enable new strategies in therapy development. We previously identified the membrane-spanning protein GPR44 as highly expressed and specific to the beta cells of the pancreas. The selective GPR44 antagonist MK-7246 was radiolabeled with carbon-11 and the resulting positron-emission tomography (PET) tracer [11C]MK-7246 was evaluated in a pig model and in vitro cell lines. The [11C]MK-7246 compound demonstrated mainly hepatobiliary excretion with a clearly defined pancreas, no spillover from adjacent tissues, and pancreatic binding similar in magnitude to the previously evaluated GPR44 radioligand [11C]AZ12204657. The binding could be blocked by preadministration of nonradioactive MK-7246, indicating a receptor-binding mechanism. [11C]MK-7246 showed strong potential as a PET ligand candidate for visualization of beta-cell mass (BCM) and clinical translation of this methodology is ongoing.

3.
RSC Adv ; 11(1): 349-353, 2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-35423044

RESUMO

The 3,4-dihydroquinazolinone (DHQ) moiety is a highly valued scaffold in medicinal chemistry due to the vast number of biologically-active compounds based on this core structure. Current synthetic methods to access these compounds are limited in terms of diversity and flexibility and often require the use of toxic reagents or expensive transition-metal catalysts. Herein, we describe the discovery and development of a novel cascade cyclization/Leuckart-Wallach type strategy to prepare substituted DHQs in a modular and efficient process using readily-available starting materials. Notably, the reaction requires only the addition of formic acid or acetic acid/formic acid and produces H2O, CO2 and methanol as the sole reaction byproducts. Overall, the reaction provides an attractive entry point into this important class of compounds and could even be extended to isotopic labelling via the site-selective incorporation of a deuterium atom.

4.
J Immunol ; 204(1): 3-11, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31848300

RESUMO

CD13/aminopeptidase N is a widely expressed ectoenzyme with multiple functions. As an enzyme, CD13 regulates activities of numerous cytokines by cleaving their N-terminals and is involved in Ag processing by trimming the peptides bound to MHC class II. Independent of its enzymatic activity, cell membrane CD13 functions by cross-linking-induced signal transduction, regulation of receptor recycling, enhancement of FcγR-mediated phagocytosis, and acting as a receptor for cytokines. Moreover, soluble CD13 has multiple proinflammatory roles mediated by binding to G-protein-coupled receptors. CD13 not only modulates development and activities of immune-related cells, but also regulates functions of inflammatory mediators. Therefore, CD13 is important in the pathogenesis of various inflammatory disorders. Inhibitors of CD13 have shown impressive anti-inflammatory effects, but none of them has yet been used for clinical therapy of human inflammatory diseases. We reevaluate CD13's regulatory role in inflammation and suggest that CD13 could be a potential therapeutic target for inflammatory disorders.


Assuntos
Anti-Inflamatórios/farmacologia , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Apresentação de Antígeno/imunologia , Citocinas/metabolismo , Humanos , Fagocitose/imunologia , Receptores de Citocinas/metabolismo , Transdução de Sinais/imunologia
5.
J Immunol ; 203(2): 360-369, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31189572

RESUMO

Aminopeptidase N/CD13 is expressed by fibroblast-like synoviocytes (FLS) and monocytes (MNs) in inflamed human synovial tissue (ST). This study examined the role of soluble CD13 (sCD13) in angiogenesis, MN migration, phosphorylation of signaling molecules, and induction of arthritis. The contribution of sCD13 was examined in angiogenesis and MN migration using sCD13 and CD13-depleted rheumatoid arthritis (RA) synovial fluids (SFs). An enzymatically inactive mutant CD13 and intact wild-type (WT) CD13 were used to determine whether its enzymatic activity contributes to the arthritis-related functions. CD13-induced phosphorylation of signaling molecules was determined by Western blotting. The effect of sCD13 on cytokine secretion from RA ST and RA FLS was evaluated. sCD13 was injected into C57BL/6 mouse knees to assess its arthritogenicity. sCD13 induced angiogenesis and was a potent chemoattractant for MNs and U937 cells. Inhibitors of Erk1/2, Src, NF-κB, Jnk, and pertussis toxin, a G protein-coupled receptor inhibitor, decreased sCD13-stimulated chemotaxis. CD13-depleted RA SF induced significantly less MN migration than sham-depleted SF, and addition of mutant or WT CD13 to CD13-depleted RA SF equally restored MN migration. sCD13 and recombinant WT or mutant CD13 had similar effects on signaling molecule phosphorylation, indicating that the enzymatic activity of CD13 had no role in these functions. CD13 increased the expression of proinflammatory cytokines by RA FLS, and a CD13 neutralizing Ab inhibited cytokine secretion from RA ST organ culture. Mouse knee joints injected with CD13 exhibited increased circumference and proinflammatory mediator expression. These data support the concept that sCD13 plays a pivotal role in RA and acute inflammatory arthritis.


Assuntos
Indutores da Angiogênese/metabolismo , Artrite Reumatoide/metabolismo , Antígenos CD13/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais/fisiologia , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Sinoviócitos/metabolismo , Células U937
6.
Cell Rep ; 17(10): 2620-2631, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27926866

RESUMO

Approximately 50% of prostate cancers are associated with gene fusions of the androgen-regulated gene TMPRSS2 to the oncogenic erythroblast transformation-specific (ETS) transcription factor ERG. The three-dimensional proximity of TMPRSS2 and ERG genes, in combination with DNA breaks, facilitates the formation of TMPRSS2-ERG gene fusions. However, the origins of DNA breaks that underlie gene fusion formation in prostate cancers are far from clear. We demonstrate a role for inflammation-induced oxidative stress in the formation of DNA breaks leading to recurrent TMPRSS2-ERG gene fusions. The transcriptional status and epigenetic features of the target genes influence this effect. Importantly, inflammation-induced de novo genomic rearrangements are blocked by homologous recombination (HR) and promoted by non-homologous end-joining (NHEJ) pathways. In conjunction with the association of proliferative inflammatory atrophy (PIA) with human prostate cancer, our results support a working model in which recurrent genomic rearrangements induced by inflammatory stimuli lead to the development of prostate cancer.


Assuntos
Inflamação/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Serina Endopeptidases/genética , Androgênios/genética , Linhagem Celular Tumoral , Quebras de DNA , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , Humanos , Inflamação/complicações , Inflamação/patologia , Masculino , Estresse Oxidativo/genética , Neoplasias da Próstata/complicações , Neoplasias da Próstata/patologia , Regulador Transcricional ERG/genética
7.
Am J Physiol Gastrointest Liver Physiol ; 311(4): G587-G598, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27514479

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease and is increasing with the rising rate of obesity in the developed world. Signaling pathways known to influence the rate of lipid deposition in liver, known as hepatic steatosis, include the transforming growth factor (TGF) superfamily, which function through the SMAD second messengers. The kielin/chordin-like protein (KCP) is a large secreted protein that can enhance bone morphogenetic protein signaling while suppressing TGF-ß signaling in cells and in genetically modified mice. In this report, we show that aging KCP mutant (Kcp-/-) mice are increasingly susceptible to developing hepatic steatosis and liver fibrosis. When young mice are put on a high-fat diet, Kcp-/- mice are also more susceptible to developing liver pathology, compared with their wild-type littermates. Furthermore, mice that express a Pepck-KCP transgene (KcpTg) in the liver are resistant to developing liver pathology even when fed a high-fat diet. Analyses of liver tissues reveal a significant reduction of P-Smad3, consistent with a role for KCP in suppressing TGF-ß signaling. Transcriptome analyses show that livers from Kcp-/- mice fed a normal diet are more like wild-type livers from mice fed a high-fat diet. However, the KCP transgene can suppress many of the changes in liver gene expression that are due to a high-fat diet. These data demonstrate that shifting the TGF-ß signaling paradigm with the secreted regulatory protein KCP can significantly alter the liver pathology in aging mice and in diet-induced NAFLD.


Assuntos
Proteínas de Transporte/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Transdução de Sinais/fisiologia , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Proteínas de Transporte/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Fígado/patologia , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Fosforilação , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo
8.
Ann Rheum Dis ; 74(7): 1459-66, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24665114

RESUMO

OBJECTIVES: Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. METHODS: We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1(-/-)), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1(-/-) and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1(-/-) and wt ECs for adhesion molecule expression. RESULTS: Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1(-/-) ECs formed significantly fewer tubes on Matrigel. Fut1(-/-) mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1(-/-) mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1(-/-) ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1(-/-) arthritic ankle cryosections compared with wt ankles. CONCLUSIONS: Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.


Assuntos
Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Fucosiltransferases/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Neovascularização Patológica/fisiopatologia , Animais , Artrite Experimental/patologia , Adesão Celular/fisiologia , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Galactosídeo 2-alfa-L-Fucosiltransferase
9.
J Pharmacol Exp Ther ; 349(3): 480-6, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24706986

RESUMO

Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)-and serum response factor (SRF)-regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)-and transforming growth factor ß (TGFß)-stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders.


Assuntos
Proteínas de Ligação a DNA/genética , Miofibroblastos/efeitos dos fármacos , Ácidos Nipecóticos/uso terapêutico , Proteínas de Fusão Oncogênica/genética , Escleroderma Sistêmico/tratamento farmacológico , Fator de Resposta Sérica/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Modelos Animais de Doenças , Feminino , Humanos , Lisofosfolipídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células NIH 3T3 , Ácidos Nipecóticos/administração & dosagem , Ácidos Nipecóticos/farmacologia , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Transativadores , Fator de Crescimento Transformador beta/farmacologia
10.
Arthritis Rheumatol ; 66(8): 2047-58, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24692243

RESUMO

OBJECTIVE: To determine the role of α(1,2)-linked fucosylation of proteins by fucosyltransferase 1 (FUT1) in rheumatoid arthritis (RA) angiogenesis. METHODS: Analysis of α(1,2)-linked fucosylated proteins in synovial tissue (ST) samples was performed by immunohistologic staining. Expression of α(1,2)-linked fucosylated angiogenic chemokine in synovial fluid (SF) was determined by immunoprecipitation and lectin blotting. To determine the angiogenic role of α(1,2)-linked fucosylated proteins in RA, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis and Matrigel assays using sham-depleted and α(1,2)-linked fucosylated protein-depleted RA SF samples. To examine the production of proangiogenic chemokines by FUT1 in HMVECs, cells were transfected with FUT1 sense or antisense oligonucleotides, and enzyme-linked immunosorbent assay was performed. We then studied mouse lung endothelial cell (EC) chemotaxis using wild-type and FUT1 gene-deficient mouse lung ECs. RESULTS: RA ST endothelial cells showed high expression of α(1,2)-linked fucosylated proteins compared to normal ST. The expression of α(1,2)-linked fucosylated monocyte chemoattractant protein 1 (MCP-1)/CCL2 was significantly elevated in RA SF compared with osteoarthritis SF. Depletion of α(1,2)-linked fucosylated proteins in RA SF induced less HMVEC migration and tube formation than occurred in sham-depleted RA SF. We found that blocking FUT1 expression in ECs resulted in decreased MCP-1/CCL2 and RANTES/CCL5 production. Finally, we showed that FUT1 regulates EC migration in response to vascular endothelial cell growth factor. CONCLUSION: Our findings indicate that α(1,2)-linked fucosylation by FUT1 may be an important new target for angiogenic diseases such as RA.


Assuntos
Artrite Reumatoide/complicações , Fucosiltransferases/fisiologia , Neovascularização Patológica/etiologia , Animais , Movimento Celular , Células Cultivadas , Células Endoteliais/fisiologia , Humanos , Camundongos , Galactosídeo 2-alfa-L-Fucosiltransferase
11.
Arthritis Res Ther ; 16(1): R28, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24467809

RESUMO

INTRODUCTION: We previously reported that sialyl Lewis(y), synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewis(y) antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined. METHODS: Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed. RESULTS: Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation. CONCLUSIONS: These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.


Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Fucosiltransferases/metabolismo , Neovascularização Patológica/metabolismo , Membrana Sinovial/metabolismo , Artrite Reumatoide/patologia , Adesão Celular/fisiologia , Proliferação de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fibroblastos/patologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Regulação para Cima , Galactosídeo 2-alfa-L-Fucosiltransferase
12.
Angiogenesis ; 16(1): 195-205, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23065099

RESUMO

RATIONALE: Angiogenesis plays an important role in wound healing and tumor growth. Fucosyltransferases synthesize fucosylated glycans and may play a major role in vascular biology. OBJECTIVE: To examine the role of an alpha(1,2) fucosyltransferase (Fut2) in angiogenesis. METHODS AND RESULTS: We found that Fut2 mRNA and protein expression is inducible in human dermal microvascular endothelial cells (HMVECs). After finding that Fut2 is inducible in HMVECs, we examined if Fut2 contributes to angiogenesis. We found that Fut2 null endothelial cell (EC) migration and tube formation were significantly less compared to wild type (wt) ECs. Angiogenesis was impaired in Fut2 null compared to wt mice in the mouse Matrigel plug and the sponge granuloma angiogenesis assays. To assess the characteristics of Fut2 null ECs in vivo, we performed Matrigel plug angiogenesis assays in wt mice using Fut2 null and wt mouse ECs. We found a significant decrease in Fut2 null EC incorporation in neoangiogenesis compared to wt ECs. ERK1/2 activation, fibroblast growth factor receptor2, and vascular endothelial growth factor expression were less in Fut2 null ECs, suggesting a possible mechanism of impaired angiogenesis when Fut2 is lacking. CONCLUSIONS: These data suggest a novel role for Fut2 as a regulator of angiogenesis.


Assuntos
Fucosiltransferases/biossíntese , Neovascularização Fisiológica , Animais , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Derme/irrigação sanguínea , Modelos Animais de Doenças , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Indução Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemoglobinas/metabolismo , Humanos , Técnicas In Vitro , Interleucina-1beta/farmacologia , Laminina/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de von Willebrand/metabolismo , Galactosídeo 2-alfa-L-Fucosiltransferase
13.
Open Access Rheumatol ; 4: 93-98, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-27790017

RESUMO

OBJECTIVE: Monocyte (MN) recruitment is an essential inflammatory component of many autoimmune diseases, including rheumatoid arthritis (RA). In this study we investigated the ability of 2-fucosyllactose (H-2g), a glucose analog of blood group H antigen to induce MN migration in vivo and determined if H-2g-induced interleukin-8 (IL-8/CXCL8) plays a role in MN ingress in RA. METHODS: Sponge granuloma and intravital microscopy assays were performed to examine H-2g-induced in vivo MN migration and rolling, respectively. MNs were stimulated with H-2g, and the production of IL-8/CXCL8 was assessed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Lastly, in vitro MN migration assays and an in vivo RA synovial tissue severe combined immunodeficiency mouse model were used to determine the role of IL-8/CXCL8 in H-2g-induced MN migration. RESULTS: In vivo, H-2g induced significantly greater MN migration compared to phosphate buffered saline. Intravital microscopy revealed that H-2g mediates MN migration in vivo by inducing MN rolling. In addition, H-2g induced MN production of IL-8/CXCL8, a process that was dependent on Src kinase. Moreover, we found that H-2g mediated MN migration in vitro, and in vivo migration was inhibited by a neutralizing anti-IL-8/CXCL8 antibody. CONCLUSION: These findings suggest that H-2g mediates MN recruitment in vitro and in vivo (in part) via IL-8/CXCL8.

14.
Arthritis Res Ther ; 14(2): R88, 2012 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-22534470

RESUMO

INTRODUCTION: TNFα is a proinflammatory cytokine that plays a central role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of certolizumab pegol, a TNFα blocker, on endothelial cell function and angiogenesis. METHODS: Human dermal microvascular endothelial cells (HMVECs) were stimulated with TNFα with or without certolizumab pegol. TNFα-induced adhesion molecule expression and angiogenic chemokine secretion were measured by cell surface ELISA and angiogenic chemokine ELISA, respectively. We also examined the effect of certolizumab pegol on TNFα-induced myeloid human promyelocytic leukemia (HL-60) cell adhesion to HMVECs, as well as blood vessels in RA synovial tissue using the Stamper-Woodruff assay. Lastly, we performed HMVEC chemotaxis, and tube formation. RESULTS: Certolizumab pegol significantly blocked TNFα-induced HMVEC cell surface angiogenic E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression and angiogenic chemokine secretion (P < 0.05). We found that certolizumab pegol significantly inhibited TNFα-induced HL-60 cell adhesion to HMVECs (P < 0.05), and blocked HL-60 cell adhesion to RA synovial tissue vasculature (P < 0.05). TNFα also enhanced HMVEC chemotaxis compared with the negative control group (P < 0.05) and this chemotactic response was significantly reduced by certolizumab pegol (P < 0.05). Certolizumab pegol inhibited TNFα-induced HMVEC tube formation on Matrigel (P < 0.05). CONCLUSION: Our data support the hypothesis that certolizumab pegol inhibits TNFα-dependent leukocyte adhesion and angiogenesis, probably via inhibition of angiogenic adhesion molecule expression and angiogenic chemokine secretion.


Assuntos
Anticorpos Monoclonais Humanizados/fisiologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Fragmentos Fab das Imunoglobulinas/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Células Cultivadas , Certolizumab Pegol , Quimiocinas/antagonistas & inibidores , Quimiocinas/metabolismo , Endotélio Vascular/patologia , Células HL-60 , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Fisiológica/imunologia , Polietilenoglicóis , Fator de Necrose Tumoral alfa/fisiologia
15.
J Immunol ; 185(3): 1777-85, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20592283

RESUMO

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed by endothelial cells (ECs) that plays a role in tight junction formation, leukocyte adhesion, and transendothelial migration. In the current study, we investigated whether JAM-C is found in soluble form and whether soluble JAM-C (sJAM-C) mediates angiogenesis. We found that JAM-C is present in soluble form in normal serum and elevated in rheumatoid arthritis (RA) serum. The concentration of sJAM-C is also elevated locally in RA synovial fluid compared with RA serum or osteoarthritis synovial fluid. sJAM-C was also present in the culture supernatant of human microvascular ECs (HMVECs) and immortalized human dermal microvascular ECs, and its concentration was increased following cytokine stimulation. In addition, sJAM-C cleavage from the cell surface was mediated in part by a disintegrin and metalloproteinases 10 and 17. In functional assays, sJAM-C was both chemotactic and chemokinetic for HMVECs and induced HMVEC tube formation on Matrigel in vitro. Neutralizing anti-JAM-C Abs inhibited RA synovial fluid-induced HMVEC chemotaxis and sJAM-C-induced HMVEC tube formation on Matrigel. sJAM-C also induced angiogenesis in vivo in the Matrigel plug and sponge granuloma models. Moreover, sJAM-C-mediated HMVEC chemotaxis was dependent on Src, p38, and PI3K. Our results show that JAM-C exists in soluble form and suggest that modulation of sJAM-C may provide a novel route for controlling pathological angiogenesis.


Assuntos
Moléculas de Adesão Celular/fisiologia , Imunoglobulinas/fisiologia , Neovascularização Fisiológica/imunologia , Animais , Artrite Reumatoide/sangue , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , Moléculas de Adesão Celular/sangue , Moléculas de Adesão Celular/uso terapêutico , Linhagem Celular Transformada , Movimento Celular/imunologia , Células Cultivadas , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/uso terapêutico , Mediadores da Inflamação/sangue , Mediadores da Inflamação/fisiologia , Mediadores da Inflamação/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular , Solubilidade , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo
16.
Am J Respir Crit Care Med ; 182(8): 1030-7, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20581170

RESUMO

RATIONALE: Tissue injury and repair involve highly conserved processes governed by mechanisms that can be co-opted in tumors. We hypothesized that soluble factors released during the repair response to lung injury would promote orthotopic tumor growth. OBJECTIVES: To determine whether lung injury promoted growth of orthotopic lung tumors and to study the molecular mechanisms. METHODS: We initiated lung injury in C57Bl6 mice using different stimuli, then injected Lewis lung carcinoma cells during the repair phase. We assessed tumor growth 14 days later. We measured tumor angiogenesis, cytokine expression, proliferation, and apoptosis. MEASUREMENTS AND MAIN RESULTS: Regardless of the mechanism, injured lungs contained more numerous and larger tumors than sham-injured lungs. Tumors from injured lungs were no more vascular, but had higher levels of proliferation and reduced rates of apoptosis. The cytokine macrophage migration inhibitory factor (MIF) was highly expressed in both models of tissue injury. We observed no increase in tumor growth after lung injury in MIF knockout mice. We induced lung-specific overexpression of MIF in a double-transgenic mouse, and observed that MIF overexpression by itself was sufficient to accelerate the growth of orthotopic Lewis lung carcinoma tumors. CONCLUSIONS: Lung injury leads to increased expression of the cytokine MIF, which results in protection from apoptosis and increased proliferation in orthotopic tumors injected after the acute phase of injury.


Assuntos
Lesão Pulmonar/imunologia , Neoplasias Pulmonares/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Lewis , Proliferação de Células , Doença Crônica , Citocinas/sangue , Modelos Animais de Doenças , Imuno-Histoquímica , Lesão Pulmonar/patologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Células Tumorais Cultivadas
17.
Arthritis Rheum ; 56(6): 1787-97, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17530707

RESUMO

OBJECTIVE: Interleukin-18 (IL-18) is a proinflammatory cytokine implicated in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to examine the role of IL-18 in up-regulating secretion of the angiogenic factors stromal cell-derived factor 1alpha (SDF-1alpha)/CXCL12, monocyte chemoattractant protein 1 (MCP-1)/CCL2, and vascular endothelial growth factor (VEGF) in RA synovial tissue (ST) fibroblasts, and the underlying signaling mechanisms involved. METHODS: We used enzyme-linked immunosorbent assays, Western blotting, and chemical inhibitors/antisense oligodeoxynucleotides to signaling intermediates to assess the role of IL-18. RESULTS: IL-18 significantly enhanced the production of SDF-1alpha/CXCL12, MCP-1/CCL2, and VEGF in RA ST fibroblasts, in a time- and concentration-dependent manner. IL-18-induced SDF-1alpha/CXCL12 up-regulation was dependent on JNK, p38 MAPK, phosphatidylinositol 3-kinase (PI3K), and NFkappaB. While IL-18-induced production of SDF-1alpha/CXCL12 was also dependent on protein kinase Cdelta (PKCdelta), production of MCP-1/CCL2 was dependent on PKCalpha, not PKCdelta. Additionally, RA ST fibroblast IL-18-induced MCP-1/CCL2 production was mediated by JNK, PI3K, and NFkappaB. In contrast, IL-18 did not induce secretion of RA ST fibroblast MCP-1/CCL2 or VEGF via p38 MAPK. IL-18-induced RA ST fibroblast production of VEGF was mediated mainly by JNK-2, PKCalpha, and NFkappaB. IL-18 induced phosphorylation of JNK, PKCdelta, p38 MAPK, and activating transcription factor 2 (ATF-2) in RA ST fibroblasts in a time-dependent manner, with JNK-2 being upstream of PKCdelta, ATF-2, and NFkappaB. CONCLUSION: These data support the notion that IL-18 has a unique role in inducing the secretion of angiogenic SDF-1alpha/CXCL12, MCP-1/CCL2, and VEGF in RA ST fibroblasts, via distinct signaling intermediates.


Assuntos
Indutores da Angiogênese/metabolismo , Artrite Reumatoide/metabolismo , Quimiocina CCL2/metabolismo , Quimiocinas CXC/metabolismo , Fibroblastos/metabolismo , Interleucina-18/fisiologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Artrite Reumatoide/patologia , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CXCL12 , Quimiocinas CXC/genética , Fibroblastos/patologia , Humanos , MAP Quinase Quinase 7/metabolismo , NF-kappa B/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-delta/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Arthritis Res Ther ; 8(4): R132, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16872482

RESUMO

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis. In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCdelta inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCdelta, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.


Assuntos
Artrite Reumatoide/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Animais , Artrite Experimental/induzido quimicamente , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Articulações/metabolismo , Fatores Inibidores da Migração de Macrófagos/deficiência , Fatores Inibidores da Migração de Macrófagos/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fatores de Tempo , Regulação para Cima , Zimosan
19.
Arthritis Rheum ; 48(12): 3568-83, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14674010

RESUMO

OBJECTIVE: This study was undertaken to characterize the role of CC chemokines and their receptors in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis (RA). Furthermore, we investigated the signaling pathways associated with CC receptors as well as the cell type distribution of the receptors. METHODS: Using TaqMan real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we defined chemokine and chemokine receptor messenger RNA (mRNA) expression, CC chemokine receptor (CCR) protein activation during the disease course, CCR-associated signaling pathways, and immunopositive CCR5, phosphorylated signal transducer and activator of transcription 1 (p-STAT-1), and p-STAT-3 cells in rat AIA versus control joints. RESULTS: We showed significant up-regulation of CCR1, CCR2, CCR5, and macrophage inflammatory protein 1beta/CCL4 mRNA in AIA on post-adjuvant injection day 18, coincident with peak inflammation. Additionally, increases in tyrosine phosphorylation of CCR1 (days 14, 18, 21, and 24), CCR2 (days 14 and 18), and CCR5 (days 14, 18, and 21) were detected in AIA rats compared with control (nonarthritic) rats. JAK-1, STAT-1, and STAT-3 were associated with CCR1 and were highly tyrosine phosphorylated on days 14 and 18. Moreover, CCR2 was associated with JAK-2, STAT-1, and STAT-3 on day 18. The association of STAT-1 and STAT-3 with CCR5 on days 18 and 21 correlated with JAK-1 phosphorylation and binding on day 18. However, the activation of JNK was not associated with CCR5 activation in rat AIA. Immunohistochemical analysis demonstrated that the expression of CCR5, p-STAT-1, and p-STAT-3 was detected on synovial lining cells, macrophages, and endothelial cells in arthritic rat ankles on post-adjuvant injection day 18. While the majority of the CCR5 and p-STAT-1 immunostaining was on synovial lining cells and macrophages, p-STAT-3 was predominantly expressed on endothelial cells. CONCLUSION: CCR1, CCR2, and CCR5 mRNA expression and tyrosine phosphorylation increased with peak inflammation in the AIA model. CCR1, CCR2, and CCR5 tyrosine phosphorylation are associated with the JAK/STAT-1/STAT-3 pathway at different stages of rat AIA, as well as with macrophage and endothelial cell infiltration. However, their signaling activation overlaps with peak inflammation. Up-regulation and activation of CCRs may play a role in macrophage and endothelial cell infiltration in rat AIA joints in addition to activating the associated signaling pathways. The downstream intermediate signaling proteins associated with CC receptors may be used as potential tools to control inflammation in RA.


Assuntos
Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Receptores de Quimiocinas/genética , Transdução de Sinais/imunologia , Animais , Artrite Experimental/imunologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica/imunologia , Janus Quinase 1 , Fosforilação , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos Lew , Receptores CCR1 , Receptores CCR2 , Receptores CCR5/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Taq Polimerase , Transativadores/metabolismo
20.
Hosp Q ; 6(3): 47-4, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12846144

RESUMO

The Canadian blood system is a critical interface between public health and the delivery of patient care. The organization of the blood system plays a vital role in ensuring that its functions are effectively fulfilled. Previous structural problems in the blood system led to serious health consequences by contributing to the blood transmission of hepatitis C and HIV in the 1980s. To address these problems, the Canadian blood system has recently undergone considerable organizational reform. However, policy-makers, particularly in Ontario, are considering further structural reform specifically focusing on how the blood system is financed. This move for reform is partially motivated by the rising cost of blood products and the perception that the current system has failed to provide incentives for the efficient use of these products. The suggested payment mechanism, a "chargeback system," involves the provincial ministries of health funding hospitals so that hospitals can directly purchase blood products from the Canadian Blood Services. This approach would replace the current system in which the provinces directly fund the Canadian Blood Services, which in turn provides blood products free of charge to hospitals. Based on a review of documents and stakeholder interviews, we report the potential advantages and disadvantages of a change to a new system of funding and make recommendations on how provinces should proceed.


Assuntos
Pessoal Administrativo/psicologia , Bancos de Sangue/organização & administração , Reforma dos Serviços de Saúde/organização & administração , Programas Nacionais de Saúde/organização & administração , Mecanismo de Reembolso , Pessoal Administrativo/estatística & dados numéricos , Atitude do Pessoal de Saúde , Bancos de Sangue/economia , Bancos de Sangue/normas , Canadá , Humanos , Entrevistas como Assunto , Programas Nacionais de Saúde/economia , Programas Nacionais de Saúde/normas , Inovação Organizacional , Responsabilidade Social
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