RESUMO
Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis and other vascular diseases. Fragile-X related protein-1 (FXR1) is a muscle-enhanced RNA binding protein whose expression is increased in injured arteries. Previous studies suggest that FXR1 negatively regulates inflammation, but its causality in vascular disease is unknown. In the current study, RNA-sequencing of FXR1-depleted VSMC identified many transcripts with decreased abundance, most of which were associated with proliferation and cell division. mRNA abundance and stability of a number of these transcripts were decreased in FXR1-depleted hVSMC, as was proliferation (P < 0.05); however, increases in beta-galactosidase (P < 0.05) and γH2AX (P < 0.01), indicative of senescence, were noted. Further analysis showed increased abundance of senescence-associated genes with FXR1 depletion. A novel SMC-specific conditional knockout mouse (FXR1SMC/SMC) was developed for further analysis. In a carotid artery ligation model of intimal hyperplasia, FXR1SMC/SMC mice had significantly reduced neointima formation (P < 0.001) after ligation, as well as increases in senescence drivers p16, p21, and p53 compared with several controls. These results suggest that in addition to destabilization of inflammatory transcripts, FXR1 stabilized cell cycle-related genes in VSMC, and absence of FXR1 led to induction of a senescent phenotype, supporting the hypothesis that FXR1 may mediate vascular disease by regulating stability of proliferative mRNA in VSMC.
Assuntos
Músculo Liso Vascular , Doenças Vasculares , Animais , Camundongos , Artérias Carótidas/metabolismo , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Hiperplasia/patologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , RNA Mensageiro/metabolismo , Doenças Vasculares/patologiaRESUMO
Interleukin enhancer-binding factor 3 (ILF3), an RNA-binding protein, is best known for its role in innate immunity by participation in cellular antiviral responses. A role for ILF3 in angiogenesis is unreported. ILF3 expression in CD31+ capillaries of hypoxic cardiac tissue was detected by immunohistochemistry. Proangiogenic stimuli induce ILF3 mRNA and protein expression in cultured human coronary artery endothelial cells (hCAECs). Angiogenic indices, including proliferation, migration, and tube formation, are all significantly reduced in hCAECs when ILF3 is knocked down using small interfering RNA (siRNA), but are significantly increased when ILF3 is overexpressed using adenovirus. Protein and mRNA abundance of several angiogenic factors including CXCL1, VEGF, and IL-8 are decreased when ILF3 is knocked down by siRNA. These factors are increased when ILF3 is overexpressed by adenovirus. ILF3 is phosphorylated and translocates from the nucleus to the cytoplasm in response to angiogenic stimuli. Proangiogenic transcripts containing adenine and uridine-rich elements were bound to ILF3 through RNA immunoprecipitation. ILF3 stabilizes proangiogenic transcripts including VEGF, CXCL1, and IL-8 in hCAECs. Together these data suggest that in endothelial cells, the RNA stability protein, ILF3, plays a novel and central role in angiogenesis. Our working hypothesis is that ILF3 promotes angiogenesis through cytokine-inducible mRNA stabilization of proangiogenic transcripts.-Vrakas, C. N., Herman, A. B., Ray, M., Kelemen, S. E., Scalia, R., Autieri, M. V. RNA stability protein ILF3 mediates cytokine-induced angiogenesis.
Assuntos
Neovascularização Fisiológica , Proteínas do Fator Nuclear 90/metabolismo , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas do Fator Nuclear 90/antagonistas & inibidores , Proteínas do Fator Nuclear 90/genética , Fosforilação , Transporte Proteico , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Regulação para CimaRESUMO
OBJECTIVE: Stress granules (SGs) are dynamic cytoplasmic aggregates containing mRNA, RNA-binding proteins, and translation factors that form in response to cellular stress. SGs have been shown to contribute to the pathogenesis of several human diseases, but their role in vascular diseases is unknown. This study shows that SGs accumulate in vascular smooth muscle cells (VSMCs) and macrophages during atherosclerosis. Approach and Results: Immunohistochemical analysis of atherosclerotic plaques from LDLR-/- mice revealed an increase in the stress granule-specific markers Ras-G3BP1 (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP1+ SGs were rapidly induced in VSMC and bone marrow-derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α (eukaryotic translation initiation factor 2-alpha) phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes. CONCLUSIONS: These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.
Assuntos
Aterosclerose/metabolismo , Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Regulação da Expressão Gênica , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Lesões do Sistema Vascular/metabolismo , Animais , Aterosclerose/patologia , Biópsia por Agulha , Células Cultivadas , Colesterol/farmacologia , DNA Helicases/metabolismo , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Estresse Oxidativo , RNA Helicases/metabolismo , Distribuição Aleatória , Sensibilidade e Especificidade , Lesões do Sistema Vascular/patologiaRESUMO
OBJECTIVE: To test the hypothesis that loss of IL-19 (interleukin-19) exacerbates atherosclerosis. APPROACH AND RESULTS: Il19-/- mice were crossed into Ldlr-/- (low-density lipoprotein receptor knock out) mice. Double knockout (dKO) mice had increased plaque burden in aortic arch and root compared with Ldlr-/- controls after 14 weeks of high-fat diet (HFD). dKO mice injected with 10 ng/g per day rmIL-19 had significantly less plaque compared with controls. qRT-PCR and Western blot analysis revealed dKO mice had increased systemic and intraplaque polarization of T cells and macrophages to proinflammatory Th1 and M1 phenotypes, and also significantly increased TNF (tumor necrosis factor)-α expression in spleen and aortic arch compared with Ldlr-/- controls. Bone marrow transplantation suggests that immune cells participate in IL-19 protection. Bone marrow-derived macrophages and vascular smooth muscle cells isolated from dKO mice had a significantly greater expression of inflammatory cytokine mRNA and protein compared with controls. Spleen and aortic arch from dKO mice had significantly increased expression of the mRNA stability protein HuR (human antigen R). Bone marrow-derived macrophage and vascular smooth muscle cell isolated from dKO mice also had greater HuR abundance. HuR stabilizes proinflammatory transcripts by binding AU-rich elements in the 3' untranslated region. Cytokine and HuR mRNA stability were increased in dKO bone marrow-derived macrophage and vascular smooth muscle cell, which was rescued by addition of IL-19 to these cells. IL-19-induced expression of miR133a, which targets and reduced HuR abundance; miR133a levels were lower in dKO mice compared with controls. CONCLUSIONS: These data indicate that IL-19 is an atheroprotective cytokine which decreases the abundance of HuR, leading to reduced inflammatory mRNA stability.
Assuntos
Aorta Torácica/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Deleção de Genes , Interleucina-10/deficiência , Estabilidade de RNA , RNA Mensageiro/metabolismo , Receptores de LDL/deficiência , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Proteína Semelhante a ELAV 1/genética , Feminino , Predisposição Genética para Doença , Interleucina-10/administração & dosagem , Interleucina-10/genética , Interleucinas , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fenótipo , Placa Aterosclerótica , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , Receptores de LDL/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The transformation of vascular smooth muscle cells [VSMC] into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we report that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. Herein we show that IL-19 reduces lipid accumulation in VSMC, and LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Endoteliais/metabolismo , Interleucinas/metabolismo , Lipoproteínas LDL/metabolismo , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Colesterol/metabolismo , Regulação da Expressão Gênica , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Camundongos , Interferência de RNA , RNA Mensageiro/genéticaRESUMO
Neovascularization and inflammation are independent biological processes but are linked in response to injury. The role of inflammation-dampening cytokines in the regulation of angiogenesis remains to be clarified. The purpose of this work was to test the hypothesis that IL-19 can induce angiogenesis in the absence of tissue hypoxia and to identify potential mechanisms. Using the aortic ring model of angiogenesis, we found significantly reduced sprouting capacity in aortic rings from IL-19(-/-) compared with wild-type mice. Using an in vivo assay, we found that IL-19(-/-) mice respond to vascular endothelial growth factor (VEGF) significantly less than wild-type mice and demonstrate decreased capillary formation in Matrigel plugs. IL-19 signals through the IL-20 receptor complex, and IL-19 induces IL-20 receptor subunit expression in aortic rings and cultured human vascular smooth muscle cells, but not endothelial cells, in a peroxisome proliferator-activated receptor-γ-dependent mechanism. IL-19 activates STAT3, and IL-19 angiogenic activity in aortic rings is STAT3-dependent. Using a quantitative RT-PCR screening assay, we determined that IL-19 has direct proangiogenic effects on aortic rings by inducing angiogenic gene expression. M2 macrophages participate in angiogenesis, and IL-19 has indirect angiogenic effects, as IL-19-stimulated bone marrow-derived macrophages secrete proangiogenic factors that induce greater sprouting of aortic rings than unstimulated controls. Using a quantitative RT-PCR screen, we determined that IL-19 induces expression of angiogenic cytokines in bone marrow-derived macrophages. Together, these data suggest that IL-19 can promote angiogenesis in the absence of hypoxia by at least two distinct mechanisms: 1) direct effects on vascular cells and 2) indirect effects by stimulation of macrophages.
Assuntos
Aorta Torácica/metabolismo , Interleucina-10/metabolismo , Neovascularização Fisiológica , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/imunologia , Células Cultivadas , Colágeno/farmacologia , Meios de Cultivo Condicionados/metabolismo , Combinação de Medicamentos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Genótipo , Humanos , Interleucina-10/deficiência , Interleucina-10/genética , Interleucinas , Laminina/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Fenótipo , Proteoglicanas/farmacologia , Interferência de RNA , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Tempo , Técnicas de Cultura de Tecidos , Transfecção , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Hypoxia in ischemic limbs typically initiates angiogenic and inflammatory factors to promote angiogenesis in attempt to restore perfusion. There is a gap in our knowledge concerning the role of anti-inflammatory interleukins in angiogenesis, macrophage polarization, and endothelial cell activation. Interleukin-19 is a unique anti-inflammatory Th2 cytokine that promotes angiogenic effects in cultured endothelial cells (EC); the purpose of this study was to characterize a role for IL-19 in restoration of blood flow in hind-limb ischemia, and define potential mechanisms. Hind limb ischemia was induced by femoral artery ligation, and perfusion quantitated using Laser Doppler Perfusion Imaging (LDPI). Wild type mice which received i.p. injections of rIL-19 (10ng/g/day) showed significantly increased levels of perfusion compared to PBS controls. LDPI values were significantly decreased in IL-19(-/-) mice when compared to wild type mice. IL-19(-/-) mice injected with rIL-19 had significantly increased LDPI compared with PBS control mice. Significantly increased capillary density was quantitated in rIL-19 treated mice, and significantly less capillary density in IL-19(-/-) mice. Multiple cell types participate in IL-19 induced angiogenesis. IL-19 treatment of human microvascular EC induced expression of angiogenic cytokines. M2 macrophage marker and VEGF-A expression were significantly increased in macrophage and the spleen from rIL-19 injected mice, and M1 marker expression was significantly increased in the spleen from IL-19(-/-) compared with controls. Plasma VEGF-A levels are higher in rIL-19 injected mice. IL-19 decreased the expression of anti-angiogenic IL-12 in the spleen and macrophage. This study is the first to implicate IL-19 as a novel pro-angiogenic interleukin and suggests therapeutic potential for this cytokine.
Assuntos
Polaridade Celular , Células Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Interleucina-10/metabolismo , Isquemia/patologia , Macrófagos/citologia , Neovascularização Fisiológica , Animais , Capilares/metabolismo , Capilares/patologia , Regulação da Expressão Gênica , Membro Posterior/patologia , Humanos , Interleucina-10/deficiência , Subunidade p40 da Interleucina-12/metabolismo , Interleucinas , Isquemia/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/genética , Fenótipo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We tested the hypothesis that IL-19, a putative member of the type 2 helper T-cell family of anti-inflammatory interleukins, can attenuate intimal hyperplasia and modulate the vascular smooth muscle cell (VSMC) response to injury. Ligated carotid artery of IL-19 knockout (KO) mice demonstrated a significantly higher neointima/intima ratio compared with wild-type (WT) mice (P = 0.04). More important, the increased neointima/intima ratio in the KO could be reversed by injection of 10 ng/g per day recombinant IL-19 into the KO mouse (P = 0.04). VSMCs explanted from IL-19 KO mice proliferated significantly more rapidly than WT. This could be inhibited by addition of IL-19 to KO VSMCs (P = 0.04 and P < 0.01). IL-19 KO VSMCs migrated more rapidly compared with WT (P < 0.01). Interestingly, there was no type 1 helper T-cell polarization in the KO mouse, but there was significantly greater leukocyte infiltrate in the ligated artery in these mice compared with WT. IL-19 KO VSMCs expressed significantly greater levels of inflammatory mRNA, including IL-1ß, tumor necrosis factor α, and monocyte chemoattractant protein-1 in response to tumor necrosis factor α stimulation (P < 0.01 for all). KO VSMCs expressed greater adhesion molecule expression and adherence to monocytes. Together, these data indicate that IL-19 is a previously unrecognized counterregulatory factor for VSMCs, and its expression is an important protective mechanism in regulation of vascular restenosis.
Assuntos
Interleucina-10/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/patologia , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Hiperplasia/patologia , Interleucinas , Ligadura , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/citologia , Proteínas Recombinantes/metabolismo , Túnica Íntima/patologiaRESUMO
OBJECTIVE: Interleukin-19 (IL-19) is a putative Th2, anti-inflammatory interleukin. Its expression and potential role in atherogenesis are unknown. IL-19 is not detected in normal artery and is expressed to a greater degree in plaque from symptomatic versus asymptomatic patients, suggesting a compensatory counter-regulatory function. We tested whether IL-19 could reduce atherosclerosis in susceptible mice and identified plausible mechanisms. APPROACH AND RESULTS: LDLR(-/-) mice fed an atherogenic diet and injected with either 1.0 or 10.0 ng/g per day recombinant mouse IL-19 had significantly less plaque area in the aortic arch compared with controls (P<0.0001). Weight gain, cholesterol, and triglyceride levels were not significantly different. Gene expression in splenocytes from IL-19-treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, interferon-γ, interleukin-1ß, and interleukin-12ß and increased expression of GATA3 and FoxP3 mRNA. A greater percentage of lymphocytes were Th2 polarized in IL-19-treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated that IL-19-treated mice have significantly less macrophage infiltrate compared with controls (P<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial cells, vascular smooth muscle cells, and bone marrow-derived macrophages with IL-19 resulted in a significant decrease in chemokine mRNA and mRNA stability protein human antigen R. CONCLUSIONS: These data suggest that IL-19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. This may identify IL-19 as a novel therapeutic to limit vascular inflammation.
Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Interleucina-10/farmacologia , Idoso , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Biomarcadores/sangue , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Colesterol/sangue , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucinas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/imunologia , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteínas Recombinantes/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Fatores de Tempo , Triglicerídeos/sangueRESUMO
Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Heme Oxigenase-1/biossíntese , Interleucinas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Heme Oxigenase-1/imunologia , Humanos , Interleucinas/genética , Interleucinas/imunologia , Camundongos , Músculo Liso Vascular/imunologia , Miócitos de Músculo Liso/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Espécies Reativas de Oxigênio/imunologia , Elementos de Resposta/fisiologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/genética , Vasculite/imunologia , Vasculite/metabolismoRESUMO
OBJECTIVE: To characterize the expression and function of interleukin (IL) 19, a recently described T-helper 2 anti-inflammatory IL, on endothelial cell (EC) pathophysiological features. METHODS AND RESULTS: The expression and effects of anti-inflammatory ILs on EC activation and development of angiogenesis are uncharacterized. We demonstrate by immunohistochemistry and immunoblot that IL-19 is expressed in inflamed, but not normal, human coronary endothelium and can be induced in cultured human ECs by serum and basic fibroblast growth factor. IL-19 is mitogenic and chemotactic, and it promotes EC spreading. IL-19 activates the signaling proteins STAT3, p44/42, and Rac1. In functional ex vivo studies, IL-19 promotes cordlike structure formation of cultured ECs and enhances microvessel sprouting in the mouse aortic ring assay. IL-19 induces tube formation in gelatinous protein (Matrigel) plugs in vivo. CONCLUSIONS: To our knowledge, these data are the first to report expression of the anti-inflammatory agent, IL-19, in ECs; and the first to indicate that IL-19 is mitogenic and chemotactic for ECs and can induce the angiogenic potential of ECs.
Assuntos
Células Endoteliais/metabolismo , Inflamação/prevenção & controle , Interleucinas/metabolismo , Neovascularização Fisiológica , Animais , Western Blotting , Proliferação de Células , Forma Celular , Células Cultivadas , Quimiotaxia , Células Endoteliais/imunologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Tempo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Endothelial cell (EC) activation plays a key role in vascular inflammation, thrombosis, and angiogenesis. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein that has been implicated in the regulation of inflammation. The expression and function of AIF-1 in EC is uncharacterized, and the purpose of this study was to characterize AIF-1 expression and function in ECs. AIF-1 expression colocalized with CD31-positive ECs in neointima of inflamed human arteries but not normal arteries. AIF-1 is detected at low levels in unstimulated EC, but expression can be increased in response to serum and soluble factors. Stable transfection of AIF-1 small interfering RNA (siRNA) in ECs reduced AIF-1 protein expression by 73% and significantly reduced EC proliferation and migration (P < 0.05 and 0.001). Rescue of AIF-1 expression restored both proliferation and migration of siRNA-expressing ECs, and AIF-1 overexpression enhanced both of these activities, suggesting a strong association between AIF-1 expression and EC activation. Activation of mitogen-activated protein kinase p44/42 and PAK1 was significantly reduced in siRNA ECs challenged with inflammatory stimuli. Reduction of AIF-1 expression did not decrease EC tube-like structure or microvessel formation from aortic rings, but overexpression of AIF-1 did significantly increase the number and complexity of these structures. These data indicate that AIF-1 expression plays an important role in signal transduction and activation of ECs and may also participate in new vessel formation.
Assuntos
Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Animais , Aorta Torácica/metabolismo , Proteínas de Ligação ao Cálcio , Bovinos , Células Cultivadas , Vasos Coronários/metabolismo , Células Endoteliais/enzimologia , Ativação Enzimática , Feminino , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Interferência de RNA , Fatores de Tempo , Técnicas de Cultura de Tecidos , Transfecção , Quinases Ativadas por p21/metabolismoRESUMO
Anti-inflammatory cytokines may play a protective role in the progression of vascular disease. The purpose of this study was to characterize interleukin (IL)-19 expression and function in the development of intimal hyperplasia, and discern a potential mechanism of its direct effects on vascular smooth muscle cells (VSMCs). IL-19 is an immunomodulatory cytokine, the expression of which is reported to be restricted to inflammatory cells. In the present study, we found that IL-19 is not expressed in quiescent VSMCs or normal arteries but is induced in human arteries by injury and in cultured human VSMCs by inflammatory cytokines. Recombinant IL-19 significantly reduced VSMC proliferation (37.1 +/- 4.8 x 10(3) versus 72.2 +/- 6.1 x 10(3) cells/cm(2)) in a dose-dependent manner. IL-19 adenoviral gene transfer significantly reduced proliferation and neointimal formation in balloon angioplasty-injured rat carotid arteries (0.172 +/- 29.9, versus 0.333 +/- 71.9, and 0.309 +/- 56.6 microm(2)). IL-19 induced activation of STAT3 as well as the expression of the suppressor of cytokine signaling 5 (SOCS5) in VSMCs. IL-19 treatment significantly reduced the activation of p44/42 and p38 MAPKs in stimulated VSMCs. Additionally, SOCS5 was found to interact with both p44/42 and p38 MAPKs in IL-19-treated human VSMCs. This is the first description of the expression of both IL-19 and SOCS5 in VSMCs and of the functional interaction between SOCS5 and MAPKs. We propose that through induction of SOCS5 and inhibition of signal transduction, IL-19 expression in VSMCs may represent a novel, protective, autocrine response of VSMCs to inflammatory stimuli.
Assuntos
Interleucinas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais/fisiologia , Túnica Íntima/patologia , Animais , Western Blotting , Lesões das Artérias Carótidas/metabolismo , Proliferação de Células , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Hiperplasia , Imuno-Histoquímica , Imunoprecipitação , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Allograft Inflammatory Factor-1 (AIF-1) is a calcium binding scaffold protein which is rapidly induced in vascular smooth muscle cells (VSMCs) in response to injury and inflammation. A transgenic mouse in which AIF-1 expression was driven by a VSMC-specific SM22alpha promoter was generated to establish a direct relationship between AIF-1 expression and intimal hyperplasia. METHODS AND RESULTS: Morphological analysis of partially ligated carotid artery demonstrate a significant increase in neointimal area of AIF-1 Tg versus wild-type mice (569+/-64 um versus 256+/-49 um, P=0.004). Immunohistochemistry using antibody to the proliferation marker Ki-67 show a significantly greater number of proliferating cells in the AIF-1 Tg lesion compared with wild-type arteries (10.6%+/-1.0 versus 3.6%+/-.9, P=0.0007). AIF-1 Tg arteries also had a greater number of cells with activated signal transduction kinase p38 (55.4%+/-7.0 versus 22.6%+/-5.4, P=0.002) and PAK1 (67.5%+/-6.7 versus 35.3%+/-10.2, P=0.02) compared with wild-type. Cultured VSMCs explanted from AIF-1 Tg proliferate (55.5+/-3.6x10(3) versus 37.2+/-2.0x10(3) cells/mL, P=0.0001) and migrate more rapidly (39.2+/-3.2 versus 17.1+/-1.5 VSMCs per HPF, P=0.0003) than wild-type, and have significantly greater levels of activated p38 and PAK1 than did VSMCs from wild-type littermates (P<0.05). CONCLUSIONS: These data indicate that AIF-1 expression results in increased signal transduction, neointimal formation, and VSMC proliferation in injured mouse carotid arteries.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Doenças das Artérias Carótidas/fisiopatologia , Músculo Liso Vascular , Miócitos de Músculo Liso/fisiologia , Túnica Íntima/patologia , Animais , Proteínas de Ligação ao Cálcio/genética , Artérias Carótidas/fisiopatologia , Proliferação de Células , Modelos Animais de Doenças , Hiperplasia , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Regiões Promotoras Genéticas , Transdução de Sinais , Túnica Íntima/lesões , Túnica Íntima/fisiopatologiaRESUMO
Mucous metaplasia is an important determinant of small airway obstruction in COPD. Its relationship to small airway inflammation is poorly defined. We analyzed 4 to 6 small airways in 19 COPD patients, GOLD stages 0-4, from lobectomy or lung volume reduction surgery tissue samples. To identify intracellular mucin, periodic acid fluorescent Schiff's (PAFS) stained slides were imaged by fluorescence microscopy. PAFS+ staining area, basement membrane length (L(BM)), epithelial height and area were measured. Mucin was expressed as a percentage of epithelial area. Mucin volume density (MVD) was calculated as PAFS+ area divided by the product of L(BM) and 4/pi. Airways were Giemsa stained for eosinophils and immunostained with antibodies against CD3, CD4, CD8, CD68, and neutrophil elastase (NE), and the number of positively stained cells/mm(2) was quantified in the airway wall. Mucin percent correlated with CD3(+) cell density (r = 0.553, P < 0.0001), and MVD correlated with CD3(+) (r = 0.570, P < 0.0001) and CD8(+) cell density (r = 0.279, P = 0.016). There were weak negative correlations between mucin percent as well as MVD and CD68(+) cell density (r = -0.270, P = 0.02 and r = -0.245, P = 0.036). There was no relationship between epithelial mucin content and CD4(+), NE(+), or eosinophil cell density. CD3(+) and CD8(+) lymphocytic inflammation is related to small airway mucous metaplasia in COPD and may play a causative role in its development.
Assuntos
Bronquíolos/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Idoso , Bronquíolos/metabolismo , Feminino , Humanos , Inflamação , Masculino , Metaplasia , Pessoa de Meia-Idade , Mucinas , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/metabolismoRESUMO
This work identifies the fragile-X-related protein (FXR1) as a reciprocal regulator of HuR target transcripts in vascular smooth muscle cells (VSMCs). FXR1 was identified as an HuR-interacting protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HuR-FXR1 interaction is abrogated in RNase-treated extracts, indicating that their association is tethered by mRNAs. FXR1 expression is induced in diseased but not normal arteries. siRNA knockdown of FXR1 increases the abundance and stability of inflammatory mRNAs, while overexpression of FXR1 reduces their abundance and stability. Conditioned media from FXR1 siRNA-treated VSMCs enhance activation of naive VSMCs. RNA EMSA and RIP demonstrate that FXR1 interacts with an ARE and an element in the 3' UTR of TNFα. FXR1 expression is increased in VSMCs challenged with the anti-inflammatory cytokine IL-19, and FXR1 is required for IL-19 reduction of HuR. This suggests that FXR1 is an anti-inflammation responsive, HuR counter-regulatory protein that reduces abundance of pro-inflammatory transcripts.
Assuntos
Proteína Semelhante a ELAV 1/genética , Músculo Liso Vascular/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Células Cultivadas , Proteína Semelhante a ELAV 1/metabolismo , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Ligação Proteica , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Development of vascular restenosis is a multifaceted process characterized by migration and proliferation of vascular smooth muscle cells (VSMCs), resulting in loss of lumen diameter. Characterization of proteins that mediate this process is essential in our understanding of the pathogenesis of arterial injury. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding protein that is expressed in VSMCs by allograft and balloon angioplasty injury. AIF-1 is not present in cultured human VSMCs but is induced by cytokines, and overexpression of AIF-1 results in increased VSMC growth and cell-cycle gene expression. To characterize AIF-1 modulatory effects in primary human VSMCs, AIF-1-interacting proteins were identified by an AIF-1/glutathione S transferase fusion protein affinity assay. MALDI-TOF mass spectrophotometric amino analysis identified actin as an AIF-1 interacting protein. This interaction was verified by coimmunoprecipitation. This is a functional interaction, because AIF-1 binds to and polymerizes F-actin in vitro. In unstimulated VSMCs, AIF-1 colocalizes with F-actin but translocates to lamellipodia on stimulation with platelet-derived growth factor. VSMCs stably transduced with AIF-1 retrovirus migrate 2.6-fold more rapidly (85.1+/-2.9 versus 225.5+/-16.6; P<0.001) in response to platelet-derived growth factor versus control cells. AIF-1 colocalizes with Rac1, and AIF-1-transduced VSMCs show a constitutive and enhanced activation of Rac1, providing a mechanism for the increased migration. These data indicate that AIF-1 binds and polymerizes F actin and also regulates Rac1 activity and VSMC migration. Considering the AIF-1 expression pattern in injured arteries, this suggests that AIF-1 may be involved in the cytoskeletal signaling network leading to vascular remodeling.
Assuntos
Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/fisiologia , Músculo Liso Vascular/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Proteínas dos Microfilamentos , Músculo Liso Vascular/citologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: Allograft inflammatory factor-1 (AIF-1) is associated with vascular smooth muscle cell (VSMC) activation and vascular injury. The purpose of this study was to characterize the molecular mechanism of AIF-1 growth-enhancing effects in human VSMC. METHODS AND RESULTS: Primary human VSMCs were stably transduced with AIF-1 retrovirus (RV). Impact on cell growth was evaluated by the increase in cell number, and the effects on gene expression were determined by cDNA microarray analysis. AIF-RV overexpressing cells grew significantly more rapidly than empty-RV control cells in growth medium and serum-reduced medium (P<0.01 and 0.02, respectively). cDNA microarray analysis and Western blotting on serum-starved AIF-1-transduced VSMCs identified increased mRNA expression of several cell cycle proteins and, surprisingly, the cytokine G-CSF. Addition of G-CSF caused a 75% increase in proliferation of VSMCs in the absence of serum growth factors. The proliferative effects of AIF-1 were abrogated by neutralizing antibodies to G-CSF (P<0.05), and AIF-1-transduced VSMCs are chemotactic for human monocytes. Increased expression of G-CSF and colocalization with AIF-1 positive cells were seen in diseased, not normal human coronary arteries. CONCLUSIONS: This study indicates that AIF-1 enhances VSMC growth by autocrine production of G-CSF, and AIF-1 expression may influence VSMC-inflammatory cell communication.
Assuntos
Comunicação Autócrina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Miócitos de Músculo Liso/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Quimiotaxia/efeitos dos fármacos , Vasos Coronários/citologia , Vasos Coronários/lesões , Vasos Coronários/patologia , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Proteínas dos Microfilamentos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Miócitos de Músculo Liso/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes de Fusão/fisiologia , Transdução Genética , Túnica Íntima/patologiaRESUMO
BACKGROUND: Early growth response factor (Egr)-1 is a transcription factor induced by inflammatory cytokines that regulates the expression of cytokines, adhesion molecules, other genes pertinent to inflammatory, and proliferative pathologies. Its expression in allografted tissue and role in the pathogenesis of graft rejection has not been explored. The goal of this work is to determine whether Egr-1 expression could be used as a surrogate marker of cardiac allograft rejection. METHODS: Egr-1 protein expression was analyzed in endomyocardial biopsies of different rejection grades by immunohistochemistry. Egr-1 mRNA expression was analyzed in 106 biopsies from 11 transplant patients by semiquantitative reverse-transcriptase polymerase chain reaction. Egr-1 was also analyzed in coronary arteries from patients with coronary artery vasculopathy (CAV) by Western blot and immunohistochemistry. RESULTS: No expression of Egr-1 protein was observed in grade 0 biopsies by immunohistochemistry. Strong nuclear Egr-1 was noted in leukocytes and cardiac myocytes in grade 3 biopsies. A clear pattern emerged where 20% (6/30), 34% (20/58), 22% (2/9), and 89%(8/9) of International Society For Heart and Lung Transplantation grade 0, 1, 2, and 3 biopsies were positive for Egr-1 mRNA. There was a significant (P<0.005) relationship between Egr-1 mRNA expression and rejection grade in endomyocardial biopsies. The calculated odds ratio indicates that a biopsy has a 2.18% greater probability of Egr-1 expression per increasing grade of rejection. Egr-1 was also up-regulated in vascular cells in coronary arteries from patients with CAV. CONCLUSIONS: In consideration of its role as a transcription factor for genes involved in pathologic processes, Egr-1 expression in endomyocardial biopsies may act as a surrogate marker of cardiac allograft rejection.
Assuntos
Proteínas de Ligação a DNA/genética , Rejeição de Enxerto/patologia , Rejeição de Enxerto/fisiopatologia , Transplante de Coração , Proteínas Imediatamente Precoces/genética , Fatores de Transcrição/genética , Biomarcadores , Biópsia , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Proteína 1 de Resposta de Crescimento Precoce , Expressão Gênica , Humanos , RNA Mensageiro/análise , Transplante HomólogoRESUMO
Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, scaffold signal transduction protein constitutively expressed in inflammatory cells, but inducible in vascular smooth muscle cells (VSMCs) in response to injury or inflammatory stimuli. Although several basic science and population studies have reported increased AIF-1 expression in human and experimental atherosclerosis, a direct causal effect of AIF-1 expression on development of atherosclerosis has not been reported. The purpose of this study is to establish a direct relationship between AIF-1 expression and development of atherosclerosis. AIF-1 expression is detected VSMC in atherosclerotic lesions from ApoE(-/-) mice, but not normal arteries from wild-type mice. AIF-1 expression can be induced in cultured VSMC by stimulation with oxidized LDL (ox-LDL). Transgenic mice in which AIF-1 expression is driven by the G/C modified SM22 alpha promoter to restrict AIF-1 expression to VSMC develop significantly increased atherosclerosis compared with wild-type control mice when fed a high-fat diet (P=0.022). Cultured VSMC isolated from Tg mice demonstrated significantly increased migration in response to ox-LDL compared with matched controls (P<0.001). VSMC isolated from Tg mice and cultured human VSMC which over express AIF-1 demonstrated increased expression of MMP-2 and MMP-9 mRNA and protein and increased NF-κB activation in response to ox-LDL as compared with wild-type control mice. VSMC which over express AIF-1 have significantly increased uptake of ox-LDL, and increased CD36 expression. Together, these data suggest a strong association between AIF-1 expression, NF-κB activation, and development of experimental atherosclerosis.