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1.
Am J Pathol ; 186(1): 87-100, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26610869

RESUMO

Cardiac fibrosis is observed across diverse etiologies of heart failure. Granzyme B (GzmB) is a serine protease involved in cell-mediated cytotoxicity in conjunction with the pore-forming protein, perforin. Recent evidence suggests that GzmB also contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. However, the role of GzmB in the onset and progression of cardiac fibrosis remains elusive. The present study investigated the role of GzmB in the pathogenesis of cardiac fibrosis. GzmB was elevated in fibrotic human hearts and in angiotensin II-induced murine cardiac fibrosis. Genetic deficiency of GzmB reduced angiotensin II-induced cardiac hypertrophy and fibrosis, independently of perforin. GzmB deficiency also reduced microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro, GzmB cleaved the endothelial junction protein, vascular endothelial (VE)-cadherin, resulting in the disruption of endothelial barrier function. Together, these results suggest a perforin-independent, extracellular role for GzmB in the pathogenesis of cardiac fibrosis.


Assuntos
Granzimas/metabolismo , Cardiopatias/enzimologia , Cardiopatias/patologia , Adulto , Idoso , Animais , Modelos Animais de Doenças , Feminino , Fibrose/enzimologia , Fibrose/patologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
2.
Lab Invest ; 94(7): 716-25, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24791744

RESUMO

The formation of unstable, leaky neovessels underlies the pathogenesis of many chronic inflammatory diseases. Granzyme B (GZMB) is an immune-derived serine protease that accumulates in the extracellular matrix (ECM) during chronic inflammation and is capable of cleaving fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM through its interaction with FN. As GZMB levels are elevated in chronic inflammatory diseases that are associated with increased vascular permeability, the role of GZMB in the regulation of VEGF bioavailability and vascular permeability were assessed. GZMB was added to either VEGF bound to FN or VEGF bound to endothelial cell (EC)-derived ECM. Supernatants containing released VEGF were assessed to determine VEGF activity by treating EC and evaluating VEGF receptor-2 (VEGFR2) phosphorylation. GZMB released VEGF from both FN and from EC-derived matrix, whereas GZMB inhibition prevented FN cleavage and VEGF release. GZMB-mediated VEGF release resulted in significant phosphorylation of VEGFR2. The role of GZMB-mediated VEGF release in altering vascular permeability was also assessed in vivo using Miles/Evans blue permeability assay. GZMB induced a significant VEGF-dependent increase in vascular permeability in vivo that was reduced in the presence of an anti-VEGF-neutralizing antibody. Inflammatory-mediated vascular leakage was also assessed in GZMB-KO mice using a delayed-type hypersensitivity model. GZMB-KO mice exhibited reduced microvascular leakage compared with C57\B6 controls. GZMB increases vascular permeability in part through the proteolytic release of ECM-sequestered VEGF, leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.


Assuntos
Permeabilidade Capilar/fisiologia , Matriz Extracelular/metabolismo , Granzimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Western Blotting , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/genética , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/sangue , Fibronectinas/metabolismo , Granzimas/genética , Granzimas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipersensibilidade Tardia/genética , Hipersensibilidade Tardia/metabolismo , Hipersensibilidade Tardia/fisiopatologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Environ Mol Mutagen ; 53(5): 350-7, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22508569

RESUMO

One model for cancer initiation by 4-aminobiphenyl (ABP) involves N-oxidation by cytochrome P450 CYP1A2 followed by O-conjugation by N-acetyltransferase(s) NAT1 and/or NAT2 and decomposition to a DNA-binding nitrenium ion. We recently observed that neonatal ABP exposure produced liver tumors in male but not in female mice, and that NAT deficiency reduced liver tumor incidence. However, ABP-induced liver tumor incidence did not correlate with liver levels of N-(deoxyguanosin-8-yl)-ABP adducts 24 hr after exposure. In this study, we compared in vivo ABP-induced DNA mutant frequencies and spectra between male and female wild-type and NAT-deficient Muta™Mouse using both the tumor-inducing neonatal exposure protocol and a 28-day repetitive dosing adult exposure protocol. ABP produced an increase in liver DNA mutant frequencies in both neonates and adults. However, we observed no sex or strain differences in mutant frequencies in neonatally exposed mice, and higher frequencies in adult females than males. Neonatal ABP exposure of wild-type mice increased the proportion of G-T transversions in both males and females, while exposure of Nat1/2(-/-) mice produced increased G-T transversions in males and a decrease in females, even though females had higher levels of N-(deoxyguanosin-8-yl)-4-ABP adducts. There was no correlation of mutant frequencies or spectra between mice dosed as neonates or as adults. These results suggest that observed sex- and NAT-dependent differences in ABP-induced liver tumor incidence in mice are not due to differences in either mutation rates or mutational spectra, and that mechanisms independent of carcinogen bioactivation, covalent DNA binding and mutation may be responsible for these differences.


Assuntos
Compostos de Aminobifenil/toxicidade , Arilamina N-Acetiltransferase/metabolismo , Fígado/efeitos dos fármacos , Mutagênicos/toxicidade , Animais , Sequência de Bases , Primers do DNA , Feminino , Frequência do Gene , Masculino , Camundongos , Reação em Cadeia da Polimerase
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