RESUMO
High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Aterosclerose/terapia , Colesterol/metabolismo , Inflamação/terapia , Lipoproteínas HDL/uso terapêutico , Macrófagos/efeitos dos fármacos , Fator 3 Ativador da Transcrição/genética , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Células Cultivadas , Imunoprecipitação da Cromatina , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lipoproteínas HDL/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Biologia de Sistemas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Atherosclerosis is a chronic inflammatory disease with multiple characteristic facets, including vascular inflammation, endothelial dysfunction, plaque development, impaired blood flow, and cholesterol deposition through dyslipidemia. Toll-like receptors (TLRs) of the innate immune system have been closely linked to the development of atherosclerotic lesions. TLR7 recognizes viral or endogenous single-stranded RNA, which is released during vascular apoptosis and necrosis. The role of TLR7 in vascular disease remains controversial, and therefore, we sought to investigate the effects of TLR7 stimulation in mice.Intravenous injection of a ligand for TLR7 (R848) induced a significant pro-inflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid artery, as measured by Evan's blue staining, and increased numbers of circulating endothelial microparticles (EMPs) and circulating Sca1/Flk1 positive cells as a marker for increased endothelial damage. Chronic subcutaneous stimulation of TLR7 in apolipoprotein E-deficient (ApoE-/-) mice increased aortic production of reactive oxygen species (ROS), the number of circulating EMPs, and most importantly, augmented the formation of atherosclerotic plaque when compared with vehicle-treated animals.Systemic stimulation of TLR7 leads to impaired reendothelialization upon acute vascular injury and is associated with the production of pro-inflammatory cytokines and increased levels of circulating EMPs and Sca1/Flk1 positive cells. Importantly, ApoE-/- mice chronically treated with R848 displayed increased atherosclerotic plaque development and elevated levels of ROS in the aortic tissue. In addition, TLR7-activation-induced apoptosis and impaired migration in human coronary artery endothelial cells and showed significant upregulation of the signaling cascade of IL-1 receptor-associated kinase (IRAK) 2 and IRAK4. Our data highlight the importance of fully understanding the pathomechanisms involved in atherogenesis, and further studies are necessary to identify the ligand-specific effects of TLR7 for possible therapeutic targeting.
Assuntos
Aterosclerose/etiologia , Glicoproteínas de Membrana/metabolismo , Receptor 7 Toll-Like/metabolismo , Animais , Apoptose , Aterosclerose/metabolismo , Movimento Celular , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Endoteliais/fisiologia , Humanos , Imidazóis , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos Knockout para ApoERESUMO
Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA-5) belongs to the intracellular retinoic acid inducible gene-I like receptors and its activation promotes pro-inflammatory mechanisms. Here, we studied the effect of MDA-5 stimulation in vascular biology. To gain insights into MDA-5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA-5 agonist polyIC (long double-stranded RNA). Human coronary endothelial cell expressed MDA-5 and reacted with receptor up-regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro-inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA-5 stimulation. To test these effects in vivo, wild-type mice were transfected with 32.5 µg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC-treated wild-type mice, endothelium-dependent vasodilation and re-endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA-5 deficiency in vivo. Finally, chronic MDA-5 stimulation in Apolipoprotein E/toll-like receptor 3 (TLR3) double(-) deficient (ApoE(-/-) /TLR3(-/-) ) mice-enhanced atherosclerotic plaque formation. This study demonstrates that MDA-5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA-5.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Células Endoteliais/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , RNA de Cadeia Dupla/farmacologia , Animais , Antígenos Ly/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Biomarcadores/metabolismo , Contagem de Células , Vasos Coronários/patologia , Citocinas/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células Endoteliais/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Helicase IFIH1 Induzida por Interferon/deficiência , Espaço Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Poli I-C/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Background: Atherosclerosis has been shown to result from chronic inflammation caused by constitutive activation of the pattern recognition receptors (PRR), which are principle effectors of the innate immune system. PRR are present in the endosome or on the cellular membrane and can sense the aberrant release of nucleic acids, which is often a sign of acute or chronic cellular damage. Absent in melanoma 2 (AIM2) is a PRR that is expressed by vascular cells and specializes in detecting cytoplasmic double-stranded DNA (dsDNA). Activation of AIM2 leads eventually to activation of the inflammasome, but the role of AIM2 in vascular disease and atherosclerosis has not been well-studied. Therefore, in this study we took advantage of acute and chronic models of vascular injury to determine the biological role of AIM2 in atherogenesis. Methods and Results: We were able to induce significant release of proinflammatory cytokines in mice through the intravenous injection of a synthetic ligand for AIM2, double-stranded poly dA:dT. This cytokine release was shown to impair reendothelialization of the carotid artery and increase the number of circulating endothelial microparticles (EMP) after acute denudation, compared to treatment with vehicle. We saw an increase in the production of reactive oxygen species in the aorta, the number of circulating EMP, and, most interestingly, atherosclerotic plaque formation in apolipoprotein E-deficient (ApoE-/-) mice when they received continual subcutaneous poly dA:dT, in contrast to vehicle-treated animals. Finally, treatment with poly dA:dT did not impair vascular reendothelialization in AIM2-/- mice compared to vehicle controls in the carotid artery injury model. Conclusion: Overall, our data suggest that AIM2, as a known regulator of the inflammasome, is an active participant in atherogenesis, and highlight the importance of fully understanding the pathological mechanisms involved. It seems to be worth of further exploration as a therapeutic target, and future studies focusing on the effects of AIM2 activation as well as its pharmacological inhibition may reveal promising new therapeutic concepts for the treatment of atherosclerosis.
RESUMO
BACKGROUND: Toll-like receptors (TLR) of the innate immune system have been closely linked with the development of atherosclerotic lesions. TLR9 is activated by unmethylated CpG motifs within ssDNA, but also by CpG motifs in nucleic acids released during vascular apoptosis and necrosis. The role of TLR9 in vascular disease remains controversial and we sought to investigate the effects of a proinflammatory TLR9 stimulation in mice. METHODS AND FINDINGS: TLR9-stimulation with high dose CpG ODN at concentrations between 6.25 nM to 30 nM induced a significant proinflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid and increased numbers of circulating endothelial microparticles, as a marker for amplified endothelial damage. Chronic TLR9 agonism in apolipoprotein E-deficient (ApoE-/-) mice fed a cholesterol-rich diet increased aortic production of reactive oxygen species, the number of circulating endothelial microparticles, circulating sca-1/flk-1 positive cells, and most importantly augmented atherosclerotic plaque formation when compared to vehicle treated animals. Importantly, high concentrations of CpG ODN are required for these proatherogenic effects. CONCLUSIONS: Systemic stimulation of TLR9 with high dose CpG ODN impaired reendothelialization upon acute vascular injury and increased atherosclerotic plaque development in ApoE-/- mice. Further studies are necessary to fully decipher the contradictory finding of TLR9 agonism in vascular biology.