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Heme oxygenase-1 (HO-1) has been reported to protect against oxidation and inflammation in atherosclerosis. It remains unclear how the immune system participates in the cytoprotective function of HO-1 in the context of atherosclerosis. In this study, we attempted to investigate the potential effect of a HO-1 inducer, hemin, and a HO-1 inhibitor, Tin-protoporphyrin IX (SnPP), on the progression of atherosclerosis in ApoE deficient mice. Using mass cytometry, 15 immune cell populations and 29 T cell sub-clusters in spleen and peripheral blood were thoroughly analyzed after hemin or SnPP treatment. SnPP elevated risk factors of atherosclerosis, whereas hemin reduced them. In-depth analysis showed that hemin significantly modified the immune system in both spleen and peripheral blood. Hemin increased dendritic (DC) and myeloid-derived suppressor cells (MDSCs), but decreased natural killer (NK) cells. An opposite effect was observed with SnPP treatment in terms of NK cells. NK cells and MDSCs were positively and negatively correlated with total cholesterol and low-density lipoprotein, respectively. Moreover, the T cell profiles were significantly reshaped by hemin, whereas only minor changes were observed with SnPP. Several hemin-modulated T cell clusters associated with atherosclerosis were also identified. In summary, we have unraveled an important regulatory role for HO-1 pathway in immune cell regulation and atherosclerosis. Our finding suggests that modulating HO-1 signaling represents a potential therapeutic strategy against atherosclerosis.
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Atherosclerosis is a chronic inflammatory disease caused mainly by lipid accumulation and excessive inflammatory immune response. Although the lipid-lowering and cardioprotective properties of bilirubin, as well as the negative relationship between bilirubin and atherosclerosis, were well documented, it is not yet clear whether bilirubin can attenuate atherosclerosis in vivo. In this study, we investigated the role of bilirubin in improving atherosclerosis. We found that mildly elevated bilirubin significantly reduced the risk factors of atherosclerosis, such as plasma glucose, total cholesterol, and low-density lipoprotein cholesterol, and the formation of atherosclerotic plaques, liver total cholesterol, and cholesterol ester concentration in apolipoprotein E-deficient (ApoE-/-) mice fed a western-type (high fat) diet. It was further found that bilirubin could promote the degradation of 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), a rate-limiting enzyme for endogenous cholesterol synthesis. Using mass cytometry-based high dimensional single cell analysis, we observed a decrease of natural killer cells and an increase of dendritic cells and myeloid-derived suppressor cells, which all are closely associated with atherosclerosis risk factors and contribute to the improvement of atherosclerosis, in ApoE-/- mice treated with bilirubin. By in-depth analysis, modulation of multiple spleen or peripheral blood T cell clusters exhibiting either positive or negative correlations with total cholesterol or low-density lipoprotein cholesterol was detected after bilirubin treatment. In this study, we demonstrate that bilirubin serves as a negative regulator of atherosclerosis and reduces atherosclerosis by inhibiting cholesterol synthesis and modulating the immune system.
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Aterosclerose , Placa Aterosclerótica , Animais , Apolipoproteínas E , Bilirrubina , LDL-Colesterol , Sistema Imunitário/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Background and Aim: Hepatic encephalopathy (HE) is a neurological disease caused by severe liver disease. Early identification of the risk factor is beneficial to the prevention and treatment of HE. Free bilirubin has always been considered to be the culprit of neonatal kernicterus, but there is no research to explore its role in HE. In this study, we aim to study the clinical significance of the indirect bilirubin-albumin ratio in HE. Methods: A retrospective case-control study of 204 patients with liver failure was conducted. Human serum albumin (HSA) or heme oxygenase-1 (HO-1) inhibitor SnPP (Tin protoporphyrin IX dichloride) was injected intraperitoneally into Ugt1 -/- mice to establish a treatment model for endogenous hyperbilirubinemia. Results: IBil/albumin ratio (OR = 1.626, 95% CI1.323-2.000, P < 0.001), white blood cell (WBC) (OR = 1.128, 95% CI 1.009-1.262, P = 0.035), ammonia (OR = 1.010, 95% CI 1.001-1.019, P = 0.027), platelet (OR=1.008, 95% CI 1.001-1.016, P = 0.022), Hb (OR = 0.977, 95% CI 0.961-0.994, P = 0.007), and PTA (OR = 0.960, 95% CI 0.933-0.987, P = 0.005) were independent factors of HE. Patients with a history of liver cirrhosis and severe HE (OR = 12.323, 95% CI 3.278-47.076, P < 0.001) were more likely to die during hospitalization. HSA or SnPP treatment improved cerebellum development and reduced apoptosis of cerebellum cells. Conclusion: The IBil/albumin ratio constitutes the most powerful risk factor in the occurrence of HE, and reducing free bilirubin may be a new strategy for HE treatment.
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Melanoma, the most threatening cancer in the skin, has been considered to be driven by the carcinogenic RAF-MEK1/2-ERK1/2 signaling pathway. This signaling pathway is usually mainly dysregulated by mutations in BRAF or RAS in skin melanomas. Although inhibitors targeting mutant BRAF, such as vemurafenib, have improved the clinical outcome of melanoma patients with BRAF mutations, the efficiency of vemurafenib is limited in many patients. Here, we show that blood bilirubin in patients with BRAF-mutant melanoma treated with vemurafenib is negatively correlated with clinical outcomes. In vitro and animal experiments show that bilirubin can abrogate vemurafenib-induced growth suppression of BRAF-mutant melanoma cells. Moreover, bilirubin can remarkably rescue vemurafenib-induced apoptosis. Mechanically, the activation of ERK-MNK1 axis is required for bilirubin-induced reversal effects post vemurafenib treatment. Our findings not only demonstrate that bilirubin is an unfavorable for patients with BRAF-mutant melanoma who received vemurafenib treatment, but also uncover the underlying mechanism by which bilirubin restrains the anticancer effect of vemurafenib on BRAF-mutant melanoma cells.
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BACKGROUND AND PURPOSE: Neointimal hyperplasia (NIH) is the fundamental cause for vascular diseases and vascular smooth muscle cell (VSMC) dysregulation has been widely implicated in NIH. Neutrophil elastase is a potential therapeutic target for multiple diseases. We investigated the role of neutrophil elastase in VSMC functions and injury-induced NIH and explored the therapeutic potential of targeting neutrophil elastase in NIH. EXPERIMENTAL APPROACH: VSMCs were used to analyse the effects of neutrophil elastase. Proteomic analysis was used to identify potential neutrophil elastase targets. Artery injury model and neutrophil elastase inhibitor GW311616A were used to investigate the role of neutrophil elastase in NIH. KEY RESULTS: TNF-α up-regulated neutrophil elastase in VSMCs through modulating GAPBα/Runx1/CEBPα/c-Myb signalling. Up-regulated neutrophil elastase promoted VSMC migration, proliferation and inflammation. Toll-like receptor 4 (TLR4) was identified as a target protein for neutrophil elastase in VSMCs and the TLR4/MyD88/IRAK1/TRAF6/NF-κB regulatory axis was shown to be the signalling pathway for neutrophil elastase in VSMC pathology. Importantly, TLR4 inhibition abolished neutrophil elastase-mediated VSMC dysregulation. Injury-induced NIH was significantly reduced in both neutrophil elastase-deficient mice and mice treated with GW311616A. The formation of neutrophil extracellular traps was impaired in injured arteries from neutrophil elastase-deficient mice. Finally, a similar role for neutrophil elastase in human VSMC pathology was confirmed and we observed higher expression levels of neutrophil elastase but lower expression levels of TLR4 in human atherosclerotic lesions. CONCLUSION AND IMPLICATIONS: We provide new insight into the molecular mechanisms underlying NIH and identify neutrophil elastase as a potential therapeutic target for vascular disease.
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NF-kappa B , Receptor 4 Toll-Like , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Hiperplasia/patologia , Elastase de Leucócito , Camundongos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima/patologia , ProteômicaRESUMO
BACKGROUND: To investigate whether neutrophil elastase (NE) plays a causal role in atherosclerosis, and the molecular mechanisms involved. METHODS AND RESULTS: NE genetic-deficient mice (Apolipoprotein E-/-/NE-/- mice), bone marrow transplantation, and a specific NE inhibitor (GW311616A) were employed in this study to establish the causal role of NE in atherosclerosis. Aortic expression of NE mRNA and plasma NE activity was significantly increased in high-fat diet (HFD)-fed wild-type (WT) (Apolipoprotein E-/-) mice but, as expected, not in NE-deficient mice. Selective NE knockout markedly reduced HFD-induced atherosclerosis and significantly increased indicators of atherosclerotic plaque stability. While plasma lipid profiles were not affected by NE deficiency, decreased levels of circulating proinflammatory cytokines and inflammatory monocytes (Ly6Chi/CD11b+) were observed in NE-deficient mice fed with an HFD for 12 weeks as compared with WT. Bone marrow reconstitution of WT mice with NE-/- bone marrow cells significantly reduced HFD-induced atherosclerosis, while bone marrow reconstitution of NE-/- mice with WT bone marrow cells restored the pathological features of atherosclerotic plaques induced by HFD in NE-deficient mice. In line with these findings, pharmacological inhibition of NE in WT mice through oral administration of NE inhibitor GW311616A also significantly reduced atherosclerosis. Mechanistically, we demonstrated that NE promotes foam cell formation by increasing ATP-binding cassette transporter ABCA1 protein degradation and inhibiting macrophage cholesterol efflux. CONCLUSIONS: We outlined a pathogenic role for NE in foam cell formation and atherosclerosis development. Consequently, inhibition of NE may represent a potential therapeutic approach to treating cardiovascular disease.
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Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/deficiência , Neutrófilos/efeitos dos fármacos , Piperidinas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/enzimologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Colesterol/sangue , Citocinas/sangue , Modelos Animais de Doenças , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Células Espumosas/patologia , Mediadores da Inflamação/sangue , Elastase de Leucócito/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Neutrófilos/enzimologia , Placa Aterosclerótica , ProteóliseRESUMO
OBJECTIVE: hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1) plays a variety of roles in gene expression. However, little is known about the functional involvement of hnRNPA1 in vascular smooth muscle cell (VSMC) function and neointima hyperplasia. In this study, we have attempted to investigate the functional roles of hnRNPA1 in the contexts of VSMC function, injury-induced vessel remodeling, and human atherosclerotic lesions, as well as discern the molecular mechanisms involved. APPROACH AND RESULTS: hnRNPA1 expression levels were consistently modulated during VSMC phenotype switching and neointimal lesion formation induced by wire injury. Functional studies showed that VSMC-specific gene expression, proliferation, and migration were regulated by hnRNPA1. Our data show that hnRNPA1 exerts its effects on VSMC functions through modulation of IQGAP1 (IQ motif containing GTPase activating protein 1). Mechanistically, hnRNPA1 regulates IQGAP1 mRNA degradation through 2 mechanisms: upregulating microRNA-124 (miR-124) and binding to AU-rich element of IQGAP1 gene. Further evidence suggests that hnRNPA1 upregulates miR-124 by modulating miR-124 biogenesis and that IQGAP1 is the authentic target gene of miR-124. Importantly, ectopic overexpression of hnRNPA1 greatly reduced VSMC proliferation and inhibited neointima formation in wire-injured carotid arteries. Finally, lower expression levels of hnRNPA1 and miR-124, while higher expression levels of IQGAP1, were observed in human atherosclerotic lesions. CONCLUSIONS: Our data show that hnRNPA1 is a critical regulator of VSMC function and behavior in the context of neointima hyperplasia, and the hnRNPA1/miR-124/IQGAP1 regulatory axis represents a novel therapeutic target for the prevention of cardiovascular diseases.
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Lesões das Artérias Carótidas/metabolismo , Proliferação de Células , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Regiões 3' não Traduzidas , Animais , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Hiperplasia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
AIMS: We have recently reported that microRNA-34a (miR-34a) regulates vascular smooth muscle cell (VSMC) differentiation from stem cells in vitro and in vivo. However, little is known about the functional involvements of miR-34a in VSMC functions and vessel injury-induced neointima formation. In the current study, we aimed to establish the causal role of miR-34a and its target genes in VSMC proliferation, migration and neointima lesion formation. METHODS AND RESULTS: Various pathological stimuli regulate miR-34a expression in VSMCs through a transcriptional mechanism, and the P53 binding site is required for miR-34a gene regulation by these stimuli. miR-34a over-expression in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miR-34a dramatically promoted VSMC proliferation and migration, respectively. Notch homolog 1 (Notch1), a well-reported regulator in VSMC functions and arterial remodeling, was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools, and was negatively regulated by miR-34a in VSMCs. Luciferase assay showed miR-34a substantially repressed wild type Notch1-3'-UTR-luciferase activity in VSMCs, but not mutant Notch1-3'-UTR-luciferease reporter, confirming the Notch1 is the functional target of miR-34a in VSMCs. Data from co-transfection experiments also revealed that miR-34a inhibited VSMC proliferation and migration through modulating Notch gene expression levels. Importantly, the expression level of miR-34a was significantly down-regulated in injured arteries, and miR-34a perivascular over-expression significantly reduced Notch1 expression levels, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries. CONCLUSION: Our data have demonstrated that miR-34a is an important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for vascular diseases.
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Movimento Celular , MicroRNAs/metabolismo , Miócitos de Músculo Liso/patologia , Neointima/genética , Neointima/patologia , Animais , Apoptose , Sequência de Bases , Movimento Celular/genética , Proliferação de Células , Artéria Femoral/lesões , Artéria Femoral/patologia , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência Molecular , Músculo Liso Vascular/patologia , Fenótipo , Receptores Notch/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
Matrix metalloproteinase-8 (MMP8) has been shown to influence various cellular functions. As monocytes and macrophages (Mφ) express MMP8, we investigated if MMP8 played a role in macrophage differentiation and polarization. MMP8 expression was significantly increased during monocyte differentiation into Mφ. Monocyte-derived Mφ from MMP8-deficient mice expressed higher levels of M1-Mφ markers but lower levels of M2-Mφ markers than monocyte-derived Mφ from wild-type mice. Although Mφ from either MMP8-deficient or wild-type mice were inducible by interferon-γ into M1-Mφ, only wild-type Mφ but not MMP8-deficient Mφ could be induced into M2-Mφ by interleukin-4. However, MMP8-deficient Mφ exposed to conditioned culture media of wild-type Mφ developed a M2-Mφ phenotype. Compared with conditioned culture media of wild-type Mφ, conditioned culture media of MMP8-deficient Mφ contained a lower concentration of active transforming growth factor-ß (TGF-ß), an M2-Mφ inducer. Moreover, evidence also showed that the degradation of the TGF-ß sequester, fibromodulin, was modulated by MMP8. The data indicate a previously unknown role of MMP8 in M2-Mφ polarization by cleaving fibromodulin and therefore increasing the bioavailability of the M2-Mφ inducer TGF-ß.
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Diferenciação Celular , Polaridade Celular , Macrófagos/fisiologia , Metaloproteinase 8 da Matriz/genética , Animais , Linhagem Celular , Proteínas da Matriz Extracelular/metabolismo , Fibromodulina , Regulação Enzimológica da Expressão Gênica , Interleucina-4/fisiologia , Metaloproteinase 8 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteoglicanas/metabolismo , Proteólise , Transdução de Sinais , Fator de Crescimento Transformador beta1/fisiologia , Regulação para CimaRESUMO
OBJECTIVE: In this study, we attempted to uncover the functional impact of microRNA-22 (miR-22) and its target gene in smooth muscle cell (SMC) differentiation and delineate the molecular mechanism involved. APPROACH AND RESULTS: miR-22 was found to be significantly upregulated during SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells. Enforced expression of miR-22 by its mimic, while knockdown of miR-22 by its antagomiR, promotes or inhibits SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells, respectively. Expectedly, miR-22 overexpression in stem cells promoted SMC differentiation in vivo. Methyl CpG-binding protein 2 (MECP2) was predicted as one of the top targets of miR-22. Interestingly, the gene expression levels of MECP2 were significantly decreased during SMC differentiation, and MECP2 was dramatically decreased in miR-22 overexpressing cells but significantly increased when miR-22 was knockdown in the differentiating stem cells. Importantly, luciferase assay showed that miR-22 substantially inhibited wild-type, but not mutant MECP2-3' untranslated region-luciferase activity. In addition, modulation of MECP2 expression levels affects multiple SMC-specific gene expression in differentiated embryonic stem cells. Mechanistically, our data showed that MECP2 could transcriptionally repress SMC gene expression through modulating various SMC transcription factors, as well as several proven SMC differentiation regulators. Evidence also revealed that enrichment of H3K9 trimethylation around the promoter regions of the SMC differentiation regulators genes were significantly increased by MECP2 overexpression. Finally, miR-22 was upregulated by platelet-derived growth factor-BB and transforming growth factor-ß through a transcriptional mechanism during SMC differentiation. CONCLUSIONS: miR-22 plays an important role in SMC differentiation, and epigenetic regulation through MECP2 is required for miR-22 mediated SMC differentiation.
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Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Regiões 3' não Traduzidas , Animais , Becaplermina , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Epigênese Genética , Regulação da Expressão Gênica , Histonas/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Metilação , Camundongos , MicroRNAs/genética , Mutação , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Interferência de RNA , Elemento de Resposta Sérica , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/farmacologiaRESUMO
To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigel-coated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation. CD146(+) cells were isolated from differentiating hES cells and expanded in vitro. The in vitro expanded CD146(+) cells were positive for EC markers, capable of Ac-LDL uptake, lectin binding, and the formation of vascular structures in vitro and in vivo. miRNA gain/loss-of-function analyses revealed that miR-150 and miR-200c were crucial in EC differentiation. Transcriptional repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the communal target gene of miRNA-200C and -150, and inhibition of ZEB1 was required for miRNA-200C or -150 mediated EC gene expressions. Moreover, we demonstrated that ZEB1 could transcriptionally repress EC gene expression through direct binding to promoters of EC genes. Finally, we also demonstrated that miRNA-200c and -150 played an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or -150 in developing chick embryos, and blocking ZEB1 signaling in CD146-positive cells could rescue the inhibitory effects of miR-200c inhibiton in in vivo vasculogenesis. Our findings revealed that miR-150 and miR-200c play an important role in human endothelial lineage specification and chick embryonic vasculogenesis by targeting ZEB1.
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Vasos Sanguíneos/crescimento & desenvolvimento , Diferenciação Celular/genética , Células Endoteliais/citologia , MicroRNAs/metabolismo , Neovascularização Fisiológica/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Biomarcadores/metabolismo , Antígeno CD146/metabolismo , Linhagem da Célula/genética , Embrião de Galinha , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , MicroRNAs/genética , Morfogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
AIMS: Growing evidence suggests a close association of plaque angiogenesis with atherosclerotic plaque formation and progression, and an important role of matrix metalloproteinase (MMP) in angiogenesis and atherosclerosis. We attempted to investigate the functional involvements of MMP8 in angiogenesis. METHODS AND RESULTS: Knockdown of MMP8 in human umbilical vein endothelial cells (HuVECs) with MMP-8 shRNA lentivirus resulted in a decrease in in vitro capillary-like network formation, cell proliferation and migration, and impaired its capacity of in vivo angiogenesis. Less nuclear accumulation of ß-catenin and lower ß-catenin target gene expression levels was observed in the HuVECs expressing lower levels of endogenous MMP8. Knockdown of endogenous MMP8 in HuVECs down-regulated platelet/endothelial cell adhesion molecule-1 (PECAM-1) expression by converting less angiotensin I to II, which is an inducer for PECAM-1 gene expression. Aortic rings isolated from MMP8(-/-)/apoE(-/-) mice had less endothelial cell sprouting, and endothelial cells in MMP8(-/-)/apoE(-/-) mice had a lower ability to migrate into Matrigel plugs and less capacity of proliferation and angiogenesis. Moreover, immunohistochemical analyses revealed that MMP8 was expressed in microvessels within human atherosclerotic plaques and aneurysm. Finally, analyses of MMP8(-/-)/apoE(-/-) and MMP8(+/+)/apoE(-/-) mice fed a Western diet for 12 weeks showed that MMP8-deficient mice had small lesion size and less endothelial cells within atherosclerotic lesions. CONCLUSION: We demonstrated for the first time that MMP8 plays an important role in angiogenesis in vitro and in vivo. Our findings provide new insights into the molecular mechanisms of plaque angiogenesis and suggest that MMP8 is a potential therapeutic target of cardiovascular diseases.
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Células Endoteliais da Veia Umbilical Humana/enzimologia , Metaloproteinase 8 da Matriz/metabolismo , Neovascularização Fisiológica , Aneurisma/enzimologia , Aneurisma/patologia , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/enzimologia , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Metaloproteinase 8 da Matriz/deficiência , Metaloproteinase 8 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Placa Aterosclerótica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Interferência de RNA , Fatores de Tempo , Transfecção , beta Catenina/metabolismoRESUMO
To investigate the functional involvements of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) in smooth muscle cell (SMC) differentiation from stem cells, embryonic stem cells were cultivated on collagen IV-coated plates to allow for SMC differentiation. We found that hnRNPA1 gene and protein expression was upregulated significantly during differentiation and coexpressed with SMC differentiation markers in the stem cell-derived SMCs as well as embryonic SMCs of 12.5 days of mouse embryos. hnRNPA1 knockdown resulted in downregulation of smooth muscle markers and transcription factors, while enforced expression of hnRNPA1 enhanced the expression of these genes. Importantly, knockdown of hnRNPA1 also resulted in impairment of SMC differentiation in vivo. Moreover, we demonstrated that hnRNPA1 could transcriptionally regulate SMC gene expression through direct binding to promoters of Acta2 and Tagln genes using luciferase and chromatin immunoprecipitation assays. We further demonstrated that the binding sites for serum response factor (SRF), a well-investigated SMC transcription factor, within the promoter region of the Acta2 and Tagln genes were responsible for hnRNPA1-mediated Acta2 and Tagln gene expression using in vitro site-specific mutagenesis and luciferase activity analyses. Finally, we also demonstrated that hnRNPA1 upregulated the expression of SRF, myocyte-specific enhancer factor 2c (MEF2c), and myocardin through transcriptional activation and direct binding to promoters of the SRF, MEF2c, and Myocd genes. Our findings demonstrated that hnRNPA1 plays a functional role in SMC differentiation from stem cells in vitro and in vivo. This indicates that hnRNPA1 is a potential modulating target for deriving SMCs from stem cells and cardiovascular regenerative medicine.
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Células-Tronco Embrionárias/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Miócitos de Músculo Liso/fisiologia , Animais , Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/biossíntese , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Camundongos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
RATIONALE: Accumulating evidence indicates that stem/progenitor cells (SPCs) represent an important source of cells in atheromas and contribute to lesion formation and progression. OBJECTIVE: We investigated whether matrix metalloproteinase-8 (MMP8) played a role in SPC migration and their recruitment into atheromas. METHODS AND RESULTS: We found that SPCs in atheromas expressed MMP8 and that MMP8 knockout significantly reduced SPC numbers in atherosclerotic lesions in apolipoprotein E (ApoE)-deficient mice fed a Western diet. Further in vivo experiments showed that ApoE(-/-)/MMP8(-/-) mice injected with stem cells isolated from bone marrows of ApoE(-/-)/MMP8(-/-) mice had fewer SPCs in atheromas and smaller lesions than ApoE(-/-)/MMP8(-/-) mice injected with stem cells isolated from bone marrows of ApoE(-/-)/MMP8(+/+) mice. Ex vivo experiments showed that MMP8 deficiency inhibited the ability of SPCs to migrate from the arterial lumen and the adventitia into atherosclerotic lesions. In vitro assays indicated that MMP8 facilitated SPC migration across endothelial cells and through Matrigel or collagen I. We also found that MMP8 cleaved a-disintegrin-and-metalloproteinase-domain-10 and that MMP8 deficiency reduced mature a-disintegrin-and-metalloproteinase-domain-10 on SPCs. Knockdown of MMP8 or incubation with the a-disintegrin-and-metalloproteinase-domain-10 inhibitor GI254023X decreased E-cadherin shedding on SPCs. The decrease in migratory ability of SPCs with MMP8 knockdown was reduced by incubation of such cells with culture supernatant from SPCs without MMP8 knockdown, and this compensatory effect was abolished by an antibody against soluble E-cadherin. CONCLUSIONS: MMP8 plays an important role in SPC migration and their recruitment into atherosclerotic lesions.
Assuntos
Aorta/enzimologia , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Movimento Celular , Metaloproteinase 8 da Matriz/metabolismo , Células-Tronco/enzimologia , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Animais , Antígenos Ly/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Biomarcadores/metabolismo , Caderinas/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ácidos Hidroxâmicos/farmacologia , Masculino , Metaloproteinase 8 da Matriz/deficiência , Metaloproteinase 8 da Matriz/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Placa Aterosclerótica , Inibidores de Proteases/farmacologia , Interferência de RNA , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Fatores de Tempo , TransfecçãoRESUMO
Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.
Assuntos
Especificidade de Órgãos/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Xantonas/farmacologia , Animais , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Células K562 , Leucemia/metabolismo , Leucemia/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Camundongos , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Tripsina/metabolismo , Ubiquitinação/efeitos dos fármacos , Xantonas/químicaRESUMO
Several flavonoids have been reported to be proteasome inhibitors, but whether prenylated flavonoids are able to inhibit proteasome function remains unknown. We report for the first time that Sanggenon C, a natural prenylated flavonoid, inhibits tumor cellular proteasomal activity and cell viability. We found that (1) Sanggenon C inhibited tumor cell viability and induced cell cycle arrest at G0/G1 phase; (2) Sanggenon C inhibited the chymotrypsin-like activity of purified human 20S proteasome and 26S proteasome in H22 cell lysate, and Sanggenon C was able to dose-dependently accumulate ubiquitinated proteins and proteasome substrate protein p27; (3) Sanggenon C-induced proteasome inhibition occurred prior to cell death in murine H22 and P388 cell lines; (4) Sanggenon C induced death of human K562 cancer cells and primary cells isolated from leukemic patients. We conclude that Sanggenon C inhibits tumor cell viability via induction of cell cycle arrest and cell death, which is associated with its ability to inhibit the proteasome function and that proteasome inhibition by Sanggenon C at least partially contributes to the observed tumor cell growth-inhibitory activity.
Assuntos
Benzofuranos/farmacologia , Cromonas/farmacologia , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , CamundongosRESUMO
Shikonin, extracted from medicinal Chinese herb (Lithospermum erythrorhizo), was reported to exert anti-inflammatory and anti-cancer effects both in vitro and in vivo. We have found that proteasome was a molecular target of shikonin in tumor cells, but whether shikonin targets macrophage proteasome needs to be investigated. In the current study, we report that shikonin inhibited inflammation in mouse models as efficiently as dexamethasone. Shikonin at 4 µM reduced the Lipopolysaccharides (LPS)-mediated TNFα release in rat primary macrophage cultures, and blocked the translocation of p65-NF-κB from the cytoplasm to the nucleus, associated with decreased proteasomal activity. Consistently, shikonin accumulated IκB-α, an inhibitor of NF-κB, and ubiquitinated proteins in rat primary macrophage cultures, demonstrating that the proteasome is a target of shikonin under inflammatory conditions. Shikonin also induced macrophage cell apoptosis and cell death. These results demonstrate for the first time that proteasome inhibition by shikonin contributes to its anti-inflammatory effect. The novel finding about macrophage proteasome as a target of shikonin suggests that this medicinal compound has great potential to be developed into an anti-inflammatory agent.
Assuntos
Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Naftoquinonas/isolamento & purificação , Naftoquinonas/farmacologia , Inibidores de Proteassoma , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Permeabilidade Capilar/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Medicamentos de Ervas Chinesas/uso terapêutico , Pavilhão Auricular/efeitos dos fármacos , Pavilhão Auricular/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Lithospermum/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , NF-kappa B/metabolismo , Naftoquinonas/uso terapêutico , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismoRESUMO
UNLABELLED: ADAM12-S transgenic mice exhibit a pronounced increase in the length of bones, such as femur, tibia, and vertebrae. The effect of ADAM12-S on longitudinal bone growth involves the modulation of chondrocyte proliferation and maturation, likely through proteolytic activities and altered cell-extracellular matrix interactions in the growth plate. INTRODUCTION: The disintegrin and metalloprotease ADAM12 is expressed in both osteoblasts and osteoclasts, suggesting a regulatory role of ADAM12 in bone. However, thus far, no in vivo function of ADAM12 in the skeleton has been reported. MATERIALS AND METHODS: Transgenic mice expressing the secreted form of human ADAM12, ADAM12-S, or a truncated metalloprotease-deficient form of ADAM12-S in the circulation were used to study the effects of ADAM12 on the skeleton. In addition, murine chondrocyte cultures were used to study the effect of ADAM12-S on cell-extracellular matrix interactions. RESULTS: ADAM12-S transgenic mice exhibit increased longitudinal bone growth. The increased bone length is progressive and age dependent, with a maximum increase of 17% seen in the femur from 6-month-old transgenic mice. The effect is gene dose dependent, being more pronounced in mice expressing higher levels of the transgene than in a lower-expressing line. Histological analysis revealed no alterations in the growth plate organization, but mean growth plate width was increased. Both the cellular incorporation of bromodeoxyuridine and the width of the collagen type X-positive hypertrophic zone were increased in the growth plate of ADAM12-S transgenic mice. Importantly, mice expressing a truncated form of ADAM12-S that lacked the pro- and metalloprotease domains showed no alterations in bone length, suggesting that protease activity is required for the ADAM12-S effect. In vitro studies showed that ADAM12-S inhibits chondrocyte adhesion to fibronectin and collagen type II. CONCLUSIONS: ADAM12-S stimulates bone growth in mice by modulating chondrocyte proliferation and maturation through mechanisms probably involving both metalloprotease and adhesion activities.
Assuntos
Proteínas ADAM/metabolismo , Desenvolvimento Ósseo , Osso e Ossos/anatomia & histologia , Diferenciação Celular , Condrócitos/citologia , Proteínas de Membrana/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAM12 , Animais , Desenvolvimento Ósseo/genética , Osso e Ossos/química , Osso e Ossos/metabolismo , Adesão Celular , Proliferação de Células , Condrócitos/fisiologia , Humanos , Integrinas/análise , Integrinas/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Deleção de SequênciaRESUMO
To investigate effects of rat bone marrow mesenchymal stem cells (rBMMSC) on hematopoiesis after allo-hematopoietic stem cell transplantation (HSCT), allogeneic BMT model from Fischer 344 rats (RT-1Al) to Wistar rats (RT-1Au) was established; effects of MSCs on hematopoietic reconstitution were studied by survival rate, peripheral blood counts, histological analysis and FACS at day 30 after transplantation. The results showed that (1) MSCs from donor Fisher344 could survive in recipient irradiated by lethal dose and could be found in the thymus, spleen and bone marrow of the recipient at 30 days after cotransplantation with BM by measuring EGFP gene. (2) Cotransplanation of MSCs and BM improved hematopoietic reconstitution. Lymphocyte and platelet counts of peripheral blood in cotransplantation group were higher than those in the control group. Active hematopoiesis and increase of bone marrow nucleated cells were observed in cotransplantation group. MSCs significantly enhanced hematopoiesis of B lymphocyte and megakaryocytopoietic lineages by FACS analysis. It is concluded that (1) MSCs of Fisher344 can be found in the thymus, spleen, bone marrow of the recipients at 30 days after cotransplantion by measuring EGFP gene. (2) hematopoietic reconstitution is significantly enhanced by MSCs cotransplanted with BM.
Assuntos
Transplante de Medula Óssea/métodos , Hematopoese/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Animais , Diferenciação Celular/fisiologia , Citometria de Fluxo , Contagem de Linfócitos , Masculino , Células-Tronco Mesenquimais/citologia , Modelos Animais , Contagem de Plaquetas , Ratos , Ratos Endogâmicos F344 , Ratos WistarRESUMO
OBJECTIVE: To investigate the effect and its mechanism of reducing graft-versus-host disease (GVHD) by in vitro blockade of CD(40)-CD(40)L pathway in vitro, the donor T lymphocytes cultured in vitro with anti-CD(40)L mAb were transfused in bone marrow transplantation (BMT) GVHD mouse model. METHODS: C57BL/6(H-2b) spleen T cells were isolated as responder cells, and BALB/c(H-2d) spleen cells as stimulator cells. They were cocultured with or without Anti-CD(40)L mAb as anti-CD(40)L mAb group and control group, respectively. At day 5, the mixed lymphocyte response (MLR)-culture cells mixed with bone marrow cells and transfused respectively into the TBI conditioned recipient mice. The mice were divided into two groups: group A, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR control group; group B, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR anti-CD(40)L mAb group. The GVHD incidence and hematopoietic reconstitution were observed. Peripheral blood sera and spleen cells of the recipients mice were harvested at scheduled time points for the measurement of cytokines and T cell immunophenotyping with flow cytometry. RESULTS: The incidence of GVHD in group A was 100% (10/10), and in group B was 20% (2/10). The percentage of H-2D(b) positive cells in group B (n = 8) was (93.54 +/- 2.32)% at day 40 after transplantation. The levels of cytokines in serum from group B were significantly lower than those from group A (P < 0.05). The expressions of CD(4)(+), CD(8)(+), CD(4)(+)CD(25)(+), CD(8)(+)CD(25)(+), CD(4)(+)CD(69)(+), CD(8)(+)CD(69)(+) and CD(4)(+)CD(40)L(+) were lower in group B than in group A (P < 0.05). The expressions of CD(8)(+)CD(40)L(+) and CD(4)(+)CD(45)RA(+) were similar in the two groups (P > 0.05). CONCLUSION: Blockade of CD(40)-CD(40)L interaction in vitro could induce immune tolerance in vivo, reduce aGVHD in aGVHD mice model and form chimerism, which was mediated by inhibiting the Th1 and Th2 cytokines production, inducing tolerance of CD(4)(+) and CD(8)(+) cells to alloantigens. The obstruction of T cells activation after tolerance happened mainly at the early and mature phase of T cells activation. These provided the experimental basis for the use of anti-CD(40)L mAb in the clinical transplantation to prevent aGVHD.