RESUMO
MMP12 is mainly secreted by macrophages, is involved in macrophage development, and decomposes the extracellular matrix. Herein, we investigated whether macrophages would change in the intestinal tumor microenvironment after MMP12 knockout. ApcMin/+;MMP12-/-mice were obtained by crossbreeding ApcMin/+ mice with MMP12 knockout mice (MMP12-/- mice). The data showed that the number and volume of intestinal tumors were significantly increased in ApcMin/+;MMP12-/- mice compared with ApcMin/+ mice. Additionally, the tumor biomarkers CA19-9, CEA, and ß-catenin appeared relatively early in intestinal tumors in ApcMin/+;MMP12-/- mice. The results demonstrated that knocking out MMP12 accelerated the tumor growth and pathological process. On further investigation of its mechanism, the proportions of M2 macrophages in the spleen and among peritoneal macrophages were significantly up-regulated in ApcMin/+;MMP12-/- mice. Expression of M2 macrophage-related genes was up-regulated in tumor and peritoneal macrophages. The M2-related cytokine levels of IL-4 and IL-13 were increased in the serum of ApcMin/+;MMP12-/-mice. In vitro, bone marrow-derived M2 macrophages were obtained by treating bone marrow cells with IL-4 and IL-13, and these M2 macrophages secreted cytokines being changed. This finding reveals the crucial role of MMP12 in macrophage development and provides a new target for the control of macrophage polarization. Knocking out MMP12 causes intestinal M2 macrophage accumulation in tumor microenvironment, promoting the growth of intestinal tumors in ApcMin/+ mice.
Assuntos
Neoplasias Intestinais/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Metaloproteinase 12 da Matriz/fisiologia , Microambiente Tumoral/imunologia , Animais , Citocinas/metabolismo , Feminino , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/patologia , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
MMP-12 is a metalloproteinase (MMP) mainly secreted by macrophages and regulating the degradation of the extracellular matrix. MMP-12 is related to several diseases such as emphysema, myocardial infarction and liver fibrosis. However, the functions associated with inflammation of MMP-12 in macrophages have not yet been fully investigated. Therefore, the aim of this study is to elucidate the role of MMP-12 in mouse macrophages during inflammation. Here we show by flow cytometry that MMP-12 was closely associated with the number of F4/80 + macrophages from mouse liver following exposure to LPS. Pro-inflammatory cytokines as well as the proliferation of RAW 264.7 cell line was modulated by MMP-12 knock down as illustrated by qRT-PCR, flow cytometry, CCK-8, Western Blot and EdU staining assays. Furthermore, down-regulation of MMP-12 decreased the expression and the phosphorylation levels of P38 and ERK1/2. Taken together, these data show that MMP-12 contributes to the proliferation of mouse macrophages as well as the secretion of IL-1ß, IL-6, TNF-α, CXCL1 and CXCL3 through the ERK/P38 MAPK signaling pathway.
Assuntos
Inflamação/patologia , Sistema de Sinalização das MAP Quinases , Macrófagos , Metaloproteinase 12 da Matriz/fisiologia , Animais , Proliferação de Células , Citocinas/imunologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Inflamação/induzido quimicamente , Inflamação/metabolismo , Mediadores da Inflamação/imunologia , Lipopolissacarídeos , Fígado/metabolismo , Fígado/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7RESUMO
BACKGROUND: Calcific aortic valve disease (CAVD) is an age-related and slowly progressive valvular disorder. Overexpression of matrix metalloproteinase 12 (MMP-12) has been found in atherosclerosis, stiffed vascular tissue, and calcified aortic valves. We hypothesized that MMP-12 may induce the pro-osteogenic responses in human aortic valve interstitial cells (AVICs). METHODS: Human AVICs were isolated from normal and calcified aortic valves. Cells were treated with MMP-12. The pro-osteogenic marker Runt-related transcription factor 2 (RUNX-2), bone morphogenetic protein 2 (BMP-2), and alkaline phosphatase (ALP), as well as MMP-12-associated signaling molecules, were analyzed. RESULTS: Human calcified aortic valves expressed significantly higher MMP-12 than normal human aortic valves. MMP-12-induced the expression of RUNX-2, BMP-2, ALP, and calcium deposit formation. Suppression of MMP-12 by its inhibitor decreased the expression of RUNX-2, BMP-2, and ALP. MMP-12-induced osteogenic responses were associated with higher levels of phosphorylation of p38 mitogen-activated protein kinases (MAPK), low density lipoprotein-related protein 6 (LRP-6), and ß-catenin signaling molecules. Calcified aortic valves exhibited markedly higher levels of LRP-6 and ß-catenin levels. Inhibition of either p38 MAPK or LRP-6 attenuated MMP-12-induced expression of RUNX-2, BMP-2, and ALP. Suppression of p38 MAPK abrogated MMP-12-induced activation of LRP-6 and ß-catenin signaling pathways. CONCLUSIONS: MMP-12 induces pro-osteogenic responses in AVICs by activation of p38 MAPK-mediated LRP-6 and ß-catenin signaling pathways. The study revealed that the potential role of MMP-12 in the pathogenesis of CAVD and therapeutically targeting MMP-12 may suppress the development of CAVD.
Assuntos
Valva Aórtica/citologia , Metaloproteinase 12 da Matriz/fisiologia , Osteogênese/fisiologia , Idoso , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/metabolismo , Proteína Morfogenética Óssea 2/fisiologia , Calcinose/etiologia , Calcinose/metabolismo , Células Cultivadas , Feminino , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , beta Catenina/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. It has generally been considered a non-Th2-type lung disorder, characterized by progressive airflow limitation with inflammation and emphysema, but its cellular and molecular mechanism remains ill defined, compared with that of asthma characterized by reversible airway obstruction. Here we show a previously unappreciated role for basophils at the initiation phase of emphysema formation in an elastase-induced murine model of COPD in that basophils represent less than 1% of lung-infiltrating cells. Intranasal elastase instillation elicited the recruitment of monocytes to the lung, followed by differentiation into interstitial macrophages (IMs) but rarely alveolar macrophages (AMs). Matrix metalloproteinase-12 (MMP-12) contributing to emphysema formation was highly expressed by IMs rather than AMs, in contrast to the prevailing assumption. Experiments using a series of genetically engineered mice suggested that basophil-derived IL-4, a Th2 cytokine, acted on lung-infiltrating monocytes to promote their differentiation into MMP-12-producing IMs that resulted in the destruction of alveolar walls and led to emphysema development. Indeed, mice deficient for IL-4 only in basophils failed to generate pathogenic MMP-12-producing IMs and hence develop emphysema. Thus, the basophil-derived IL-4/monocyte-derived IM/MMP-12 axis plays a crucial role in emphysema formation and therefore may be a potential target to slow down emphysema progression at the initiation phase of COPD.
Assuntos
Basófilos/patologia , Interleucina-4/metabolismo , Macrófagos Alveolares/patologia , Macrófagos/patologia , Metaloproteinase 12 da Matriz/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/etiologia , Animais , Basófilos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Macrófagos/metabolismo , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologiaRESUMO
Following myocardial infarction (MI), timely resolution of inflammation promotes wound healing and scar formation while limiting excessive tissue damage. Resolution promoting factors (RPFs) are agents that blunt leukocyte trafficking and inflammation, promote necrotic and apoptotic cell clearance, and stimulate scar formation. Previously identified RPFs include mediators derived from lipids (resolvins, lipoxins, protectins, and maresins), proteins (glucocorticoids, annexin A1, galectin 1, and melanocortins), or gases (CO, H2S, and NO). Matrix metalloproteinase-12 (MMP-12; macrophage elastase) has shown promising RPF qualities in a variety of disease states. We review here the evidence that MMP-12 may serve as a novel RPF with potential therapeutic efficacy in the setting of MI.
Assuntos
Metaloproteinase 12 da Matriz/fisiologia , Infarto do Miocárdio/metabolismo , Animais , HumanosRESUMO
Myeloid-derived suppressor cells (MDSCs) are found frequently in patients and mice bearing tumors, which derived from immature myeloid cells. In healthy individuals, immature myeloid cells formed in the bone marrow differentiating to dendritic cells, macrophages and neutrophils. However, it is unclear whether some gene deficiency will lead to MDSCs accumulation in mice without bearing tumor. Here, we observed that MDSCs accumulated in the bone marrow of matrix metalloproteinase 12 knockout mice (MMP12-/- mice) compared with wild type mice (MMP12+/+ mice). And the number of CD4+ cells dramatically decreased, regulatory T cells was up-regulation and MDSCs function were determined. The results suggested that immune surveillance have been impaired in MMP12-/- transgenic mice. After intravenous administration of B16 murine melanoma cells, MMP12-/- mice developed more metastatic pulmonary nodules than MMP12+/+ mice. Meanwhile, more MDSCs appeared in the tumors of MMP12-/- mice compared with those of MMP12+/+ mice. Mechanistically, we performed a MDSC blocking assay, finding that blockade of MDSCs resulted in reducing growth of tumors in MMP12-/- mice. Furthermore, we ascertained that macrophages in MMP12-/- mice abundantly secrete IL-1ß in bone marrow which induce the accumulation of MDSCs in the bone marrow. Together, these results demonstrated that the macrophages in MMP12-/- mice could crosstalk with myeloid cells through IL-1ß, inducing MDSCs accumulation, then contributing to tumor growth. It has revealed that the critical roles of macrophage in myeloid cells differentiation.
Assuntos
Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 12 da Matriz/fisiologia , Células Supressoras Mieloides/metabolismo , Animais , Medula Óssea/fisiologia , Linfócitos T CD4-Positivos , Carcinogênese/metabolismo , Interleucina-1beta , Ativação Linfocitária , Macrófagos , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Células Mieloides/fisiologia , Células Supressoras Mieloides/fisiologia , Linfócitos T Reguladores , Microambiente TumoralRESUMO
Irreversible tissue recession in chronic inflammatory diseases is associated with dysregulated immune activation and production of tissue degradative enzymes. In this study, we identified elevated levels of matrix metalloproteinase (MMP)-12 in gingival tissue of patients with the chronic inflammatory disease periodontitis (PD). The source of MMP12 was cells of monocyte origin as determined by the expression of CD14, CD68, and CD64. These MMP12-producing cells showed reduced surface levels of the coinhibitory molecule CD200R. Similarly, establishing a multicellular three-dimensional model of human oral mucosa with induced inflammation promoted MMP12 production and reduced CD200R surface expression by monocyte-derived cells. MMP12 production by monocyte-derived cells was induced by CSF2 rather than the cyclooxygenase-2 pathway, and treatment of monocyte-derived cells with a CD200R ligand reduced CSF2-induced MMP12 production. Further, MMP12-mediated degradation of the extracellular matrix proteins tropoelastin and fibronectin in the tissue model coincided with a loss of Ki-67, a protein strictly associated with cell proliferation. Reduced amounts of tropoelastin were confirmed in gingival tissue from PD patients. Thus, this novel association of the CD200/CD200R pathway with MMP12 production by monocyte-derived cells may play a key role in PD progression and will be important to take into consideration in the development of future strategies to diagnose, treat, and prevent PD.
Assuntos
Antígenos de Superfície/fisiologia , Gengiva/enzimologia , Metaloproteinase 12 da Matriz/fisiologia , Monócitos/enzimologia , Periodontite/enzimologia , Receptores de Superfície Celular/fisiologia , Adulto , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Inibidores de Ciclo-Oxigenase/farmacologia , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Gengiva/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Inflamação , Queratinócitos/metabolismo , Metaloproteinase 12 da Matriz/biossíntese , Metaloproteinase 12 da Matriz/genética , Monócitos/patologia , Receptores de Orexina , Periodontite/patologia , Pirazóis/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genéticaRESUMO
Persistent activation of macrophages in lungs plays a critical role in the production of matrix metalloproteinases (MMPs) that contributes to the destruction of alveolar walls, a hallmark for pulmonary emphysema. Dysregulated TGF-ß1 signaling has been an essential determinant in the elevation of MMPs during the development of emphysema. Nevertheless, the mechanism for this MMP-dependent pathogenesis has yet to be clearly investigated. Recently, we identified an important role for tyrosine phosphatase Src homology domain-containing protein tyrosine phosphatase 2 (Shp2) in regulating the activation of alveolar macrophages. Over a long-term observation period, mice with Shp2 deletion in macrophages (LysMCre:Shp2fl/fl ) develop spontaneous, progressive emphysema-like injury in the lungs, characterized by massive destruction of alveolar morphology, interstitial extracellular matrix degradation, and elevated levels of MMPs, particularly, significant increases of macrophage elastase (MMP12) in aged mice. Further analysis demonstrated that MMP12 suppression by TGF-ß1 activation was apparently abrogated in LysMCre:Shp2fl/fl mice, whereas the TGF-ß1 concentration in the lungs was relatively the same. Mechanistically, we found that loss of Shp2 resulted in attenuated SMAD2/3 phosphorylation and nuclear translocation in response to TGF-ß activation, thereby upregulating MMP12 expression in macrophages. Together, our findings define a novel physiological function of Shp2 in TGF-ß1/MMP12-dependent emphysema, adding insights into potential etiologies for this chronic lung disorder.
Assuntos
Ativação de Macrófagos , Metaloproteinase 12 da Matriz/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Enfisema Pulmonar/prevenção & controle , Fator de Crescimento Transformador beta1/metabolismo , Envelhecimento , Animais , Pulmão/patologia , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Knockout , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/fisiopatologia , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/genéticaRESUMO
A broad range of immunosuppressive and immunomodulatory effects of heat shock protein 90 (Hsp90) blockade has been described in models of autoimmune bullous diseases, but the direct contribution of this chaperone to neutrophil effector pathways in the context of autoantibody-driven blistering is generally unknown. Therefore, this has been addressed in the current study on the basis of the subepidermal blistering disease epidermolysis bullosa acquisita (EBA) characterized by autoantibodies against type VII collagen, in which a crucial role of neutrophils and both their reactive oxygen species and matrix metalloproteinases in mediating tissue injury has been established. First, the Hsp90 antagonist 17-DMAG dose-dependently inhibited dermal-epidermal separation ex vivo in cryosections of human skin induced by co-incubation of EBA patient autoantibodies with neutrophils from healthy blood donors. Next, 17-DMAG dose-dependently suppressed production and release of reactive oxygen species by human neutrophils induced by both fMLP ± LPS and EBA-specific immune complexes. In addition, co-immunoprecipitation studies revealed that extracellular Hsp90 interacted with secreted matrix metalloproteinases 2 and 12 in sera of EBA patients, suggesting that these basement membrane-degrading proteolytic enzymes are client proteins of Hsp90 and dependent on its chaperone function. Our findings add to the knowledge of the multimodal anti-inflammatory effects of Hsp90 blockade and implicate that Hsp90 is closely involved in the effector mechanisms of neutrophil-driven autoantibody-induced tissue damage, thus being a relevant therapeutic target in patients with neutrophil-mediated autoimmune diseases such as inflammatory types of EBA.
Assuntos
Autoanticorpos/fisiologia , Epidermólise Bolhosa Adquirida/patologia , Epidermólise Bolhosa Adquirida/fisiopatologia , Proteínas de Choque Térmico HSP90/fisiologia , Neutrófilos/patologia , Pele/patologia , Autoanticorpos/imunologia , Benzoquinonas/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Colágeno Tipo VII/imunologia , Relação Dose-Resposta a Droga , Epidermólise Bolhosa Adquirida/imunologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Humanos , Imunoprecipitação , Lactamas Macrocíclicas/farmacologia , Metaloproteinase 12 da Matriz/fisiologia , Metaloproteinase 2 da Matriz/fisiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos dos fármacos , Pele/fisiopatologiaRESUMO
Macrophage elastase (MMP12) is a key mediator of cigarette smoke (CS)-induced emphysema, yet its role in other smoking related pathologies remains unclear. The weight suppressing effects of smoking are a major hindrance to cessation efforts, and MMP12 is known to suppress the vascularization on which adipose tissue growth depends by catalyzing the formation of antiangiogenic peptides endostatin and angiostatin. The goal of this study was to determine the role of MMP12 in adipose tissue growth and smoking-related suppression of weight gain. Whole body weights and white adipose depots from wild-type and Mmp12-deficient mice were collected during early postnatal development and after chronic CS exposure. Adipose tissue specimens were analyzed for angiogenic and adipocytic markers and for content of the antiangiogenic peptides endostatin and angiostatin. Cultured 3T3-L1 adipocytes were treated with adipose tissue homogenate to examine its effects on vascular endothelial growth factor (VEGF) expression and secretion. MMP12 content and activity were increased in the adipose tissue of wild-type mice at 2 weeks of age, leading to elevated endostatin production, inhibition of VEGF secretion, and decreased adipose tissue vascularity. By 8 weeks of age, adipose MMP12 levels subsided, and the protein was no longer detectable. However, chronic CS exposure led to macrophage accumulation and restored adipose MMP12 activity, thereby suppressing adipose tissue mass and vascularity. Our results reveal a novel systemic role for MMP12 in postnatal adipose tissue expansion and smoking-associated weight loss by suppressing vascularity within the white adipose tissue depots.
Assuntos
Tecido Adiposo Branco/enzimologia , Metaloproteinase 12 da Matriz/fisiologia , Fumar/metabolismo , Células 3T3-L1 , Tecido Adiposo Branco/irrigação sanguínea , Tecido Adiposo Branco/imunologia , Adiposidade , Animais , Peso Corporal , Endostatinas/antagonistas & inibidores , Endostatinas/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fumar/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Netrin-1 (Ntn-1) is a potent inducer of neuronal cell migration; however, its molecular mechanism that guides the migratory behavior of stem cells has not been characterized. In this study, we investigate the role of Ntn-1 in promoting the motility of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and its related signaling pathways. Ntn-1 (50 ng/mL) significantly increased motility of UCB-MSCs, which was inhibited by blocking antibodies for deleted in colorectal cancer (DCC) and integrin (IN) α6ß4. Ntn-1 in DCC stimulated protein kinase Cα (PKCα) activation, but not PKCÉ, PKCθ, and PKCζ, while Ntn-1 in INα6ß4 induced the phosphorylation of focal adhesion kinase (FAK) and Fyn. Notably, Ntn-1 induced phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and nuclear factor kappa-B (NF-κB), but they were concurrently downregulated by blocking the activities of PKCα, FAK, and Fyn. Ntn-1 uniquely increased the MMP-12 expression of all the matrix metalloproteinase (MMP) isoforms present in UCB-MSCs, though this was significantly blocked by an NF-κB inhibitor. Finally, Ntn-1 induced the MMP-12-dependent degradation of E-cadherin (E-cad), while Ntn-1 abrogated the interaction between E-cad and p120-catenin. In addition, Ntn-1 has the ability to stimulate cytoskeletal reorganization-related proteins, such as Cdc42, Rac1, Profilin-1, Cofilin-1, α-Actinin-4, and filamentous actin (F-actin) in UCB-MSCs. These results demonstrate that Ntn-1 induces MMP-12-dependent E-cad degradation via the distinct activation of PKCα and FAK/Fyn, which is necessary to govern the activation of ERK, JNK, and NF-κB in promoting motility of UCB-MSCs.
Assuntos
Caderinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Metaloproteinase 12 da Matriz/fisiologia , Células-Tronco Mesenquimais/fisiologia , Fatores de Crescimento Neural/fisiologia , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-fyn/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Antígenos CD , Sinalização do Cálcio , Movimento Celular , Células Cultivadas , Citoesqueleto/metabolismo , Ativação Enzimática , Humanos , Netrina-1 , ProteóliseRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue. METHODS: The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks. RESULTS: MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass. Adipocyte and blood vessel size and density in SC and GON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GON adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GON adipose tissue. CONCLUSIONS: Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS. GENERAL SIGNIFICANCE: MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes.
Assuntos
Tecido Adiposo/patologia , Metaloproteinase 12 da Matriz/fisiologia , Tecido Adiposo/enzimologia , Animais , Peso Corporal , Movimento Celular , Dieta Hiperlipídica , Interferon gama/fisiologia , Macrófagos/fisiologia , Masculino , Camundongos , Obesidade/etiologia , Obesidade/patologiaRESUMO
OBJECTIVE: The purpose of this study was to evaluate potential mechanisms promoting abdominal aortic aneurysm development with tobacco smoke (TS) exposure. METHODS AND RESULTS: Experiments used the elastase perfusion model of abdominal aortic aneurysms with smoke-free controls. The effect of TS exposure was evaluated in C57/Bl6 mice, after broad-spectrum matrix metalloproteinase inhibition with doxycycline and in mice deficient in matrix metalloproteinase-9, matrix metalloproteinase-12, Cathepsin-S, and Neutrophil Elastase. Preparations of washed marrow, spleen, and peripheral blood leukocytes were transferred to smoke-free mice from 6-week TS-exposed mice or smoke-free mice. All mice were euthanized 14 days after elastase perfusion, and the percentage of change in aortic diameter (%Δ aortic diameter) was calculated. Electron microscopy of aortic tissue from animals exposed to TS without elastase exposure did not demonstrate any ultrastructural changes. Neither doxycycline nor any specific elastase deficiency was effective at preventing an increase in %Δ aortic diameter in TS-exposed animals. Smoke exposure for 6 weeks increased the %Δ aortic diameter after a smoke-free interval of up to 6 weeks before elastase perfusion. Leukocyte preparations from TS-exposed mice localized to abdominal aortic aneurysms and increased the %Δ aortic diameter in smoke-free mice. CONCLUSIONS: The effect of TS on the development of abdominal aortic aneurysms is not dependent on the activity of elastolytic enzymes and persists for long periods despite cessation of TS. Alterations in leukocyte response to aortic injury appear to mediate this effect.
Assuntos
Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/fisiopatologia , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/fisiologia , Fumar/efeitos adversos , Animais , Aorta Abdominal/patologia , Aorta Abdominal/fisiopatologia , Aorta Abdominal/ultraestrutura , Aneurisma da Aorta Abdominal/patologia , Catepsinas/deficiência , Catepsinas/genética , Catepsinas/fisiologia , Contagem de Células , Modelos Animais de Doenças , Doxiciclina/farmacologia , Elastase de Leucócito/deficiência , Elastase de Leucócito/genética , Elastase de Leucócito/fisiologia , Masculino , Metaloproteinase 12 da Matriz/deficiência , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/fisiologia , Metaloproteinases da Matriz/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
UNLABELLED: Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP-12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl(4) ) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP-12 elastin degradation was defined mechanistically using CD11b-DTR and MMP-12 knockout mice. In a CCl(4) model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP-12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP-12 was bound in greater quantities to its inhibitor TIMP-1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP-12. Exposure of CCl(4) in MMP-12(-/-) mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP-12(-/-) mice compared with WT. CONCLUSION: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage-derived MMP-12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies.
Assuntos
Elastina/metabolismo , Cirrose Hepática Experimental/metabolismo , Macrófagos/enzimologia , Metaloproteinase 12 da Matriz/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Fígado/metabolismo , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Inibidor Tecidual de Metaloproteinase-1/genética , Tropoelastina/biossínteseRESUMO
How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? Nuclear magnetic resonance suggested soluble elastin covers surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II-III loop and ß-strand I near the back of the catalytic domain. The high degree of exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, the combination of one lesion each at exosites 1 and 2 and the active site decreased the catalytic competence toward soluble elastin by 13-18-fold to the level of MMP-3, homologue and poor elastase. Double-mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) showed they had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 the k(cat) for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced the k(cat) for soluble elastin. In elastin degradation, exosites 1 and 2 contributed in a manner independent of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils.
Assuntos
Domínio Catalítico , Regulação para Baixo , Elastina/metabolismo , Metaloproteinase 12 da Matriz/química , Metaloproteinase 12 da Matriz/fisiologia , Ácido Aspártico/química , Ácido Aspártico/genética , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Regulação para Baixo/genética , Estabilidade Enzimática/genética , Humanos , Hidrólise , Metaloproteinase 12 da Matriz/genética , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Solubilidade , Especificidade por Substrato/genéticaRESUMO
During human pregnancy, the uterine spiral arteries are progressively remodelled to form dilated conduits lacking maternal vasomotor control. This phenomenon ensures that a constant supply of blood is delivered to the materno-fetal interface at an optimal velocity for nutrient exchange. Conversion of a tonic maternal arteriole composed of multiple layers of vascular smooth muscle, elastin and numerous other extracellular matrix components, into a highly dilated yet durable vessel, requires tight regulatory control and the coordinated actions of multiple cell types. Initial disruption of the vascular wall, characterised by foci of endothelial cell loss, and separation and misalignment of vascular smooth muscle cells (VSMC), is coincident with an influx of uterine natural killer (uNK) cells and macrophages. uNK cells are a source of angiogenic growth factors and matrix degrading proteases, thus they possess the capacity to initiate changes in VSMC phenotype and instigate extracellular matrix catabolism. However, complete vascular cell loss, mediated in part by apoptosis and dedifferentiation, is only achieved following colonisation of the arteries by extravillous trophoblast (EVT). EVT produce a variety of chemokines, cytokines and matrix degrading proteases, enabling them to influence the fate of other cells within the placental bed and complete the remodelling process. The complex interplay of cell-cell and cell-matrix interactions required for effective vascular transformation will be examined, with a particular focus on the role of (i) uNK cells and (ii) the enzyme matrix metalloproteinase-12 (MMP-12). Parallels with remodelling events occurring in other vascular beds will also be drawn.
Assuntos
Artérias/fisiologia , Comunicação Celular/fisiologia , Matriz Extracelular/fisiologia , Gravidez/fisiologia , Útero/irrigação sanguínea , Útero/fisiologia , Inibidores da Angiogênese/fisiologia , Angiopoietinas/metabolismo , Animais , Artérias/citologia , Fatores de Coagulação Sanguínea/fisiologia , Movimento Celular/fisiologia , Fatores Quimiotáticos/metabolismo , Quimiotaxia de Leucócito/fisiologia , Meios de Cultivo Condicionados/farmacologia , Decídua/citologia , Elastina/metabolismo , Feminino , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/fisiologia , Leucócitos/citologia , Leucócitos/fisiologia , Metaloproteinase 12 da Matriz/fisiologia , Metaloproteinases da Matriz Secretadas/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Peptídeos/metabolismo , Trofoblastos/citologia , Trofoblastos/fisiologia , Útero/citologia , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Among the large matrix metalloproteinases (MMPs) family, MMP-12, also referred to as macrophage elastase, plays a significant role in chronic pulmonary pathologies characterized by an intense tissue remodeling such as asthma and COPD. This review will summarize knowledge about MMP-12 structure, functions and mechanisms of activation and regulation, including potential MMP-12 modulation by microRNA. As MMP-12 is involved in many tissue remodeling diseases, efforts have been made to develop specific synthetic inhibitors. However, at this time, very few chemical inhibitors have proved to be efficient and specific to a particular MMP. The relevance of silencing MMP-12 by RNA interference is highlighted. The specificity of this approach using siRNA or shRNA and the strategies to deliver these molecules in the lung are discussed.
Assuntos
Inativação Gênica , Pneumopatias/patologia , Pneumopatias/terapia , Metaloproteinase 12 da Matriz/genética , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Pulmão/patologia , Metaloproteinase 12 da Matriz/biossíntese , Metaloproteinase 12 da Matriz/fisiologia , Inibidores de Metaloproteinases de Matriz , Interferência de RNARESUMO
Mice lacking surfactant protein A (SP-A) are susceptible to bacterial infection associated with an excessive inflammatory response in the lung. To determine mechanisms by which SP-A is antiinflammatory in the lung during bacterial infection, SP-A regulation of secretory leukoprotease inhibitor (SLPI), an inhibitor of serine proteases, was assessed. SLPI protein expression and antineutrophil elastase activity were reduced in bronchoalveolar fluid of SP-A(-/-) compared with SP-A(+/+) mice. Intratracheal administration of SP-A to SP-A(-/-) mice enhanced SLPI protein expression and antineutrophil elastase activity in the lung. SLPI mRNA was similar in whole lung and alveolar type II cells; however, it was significantly reduced in alveolar macrophages from SP-A(-/-) compared with SP-A(+/+) mice. In vitro, SP-A enhanced SLPI production by macrophage THP-1 cells but not respiratory epithelial A549 cells. SP-A inhibited LPS induced IkappaB-alpha degradation in THP-1 cells, which was partially reversed with knockdown of SLPI. Matrix metalloproteinase (MMP)-12 cleaved SLPI and incubation with SP-A reduced MMP-12-mediated SLPI cleavage. The collagen-like region of SP-A conferred protection of SLPI against MMP mediated cleavage. SP-A plays an important role in the lung during bacterial infection regulating protease and antiprotease activity.