RESUMO
Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Proteoglicanas de Heparan Sulfato/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Antígeno de Maturação de Linfócitos B/metabolismo , Sítios de Ligação , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/mortalidade , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangue , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/deficiênciaRESUMO
RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
Assuntos
Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Interferon Tipo I/biossíntese , Mitocôndrias/metabolismo , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipopolissacarídeos/toxicidade , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Adulto JovemRESUMO
Receptor tyrosine kinase c-Kit and its ligand stem cell factor (SCF) regulate resident vascular wall cells and recruit circulating progenitors. We tested whether SCF may be induced by oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) known to accumulate in atherosclerotic vessels. Gene expression analysis demonstrated OxPAPC-induced upregulation of SCF mRNA and protein in different types of endothelial cells (ECs). Elevated levels of SCF mRNA were observed in aortas of ApoE-/- knockout mice. ECs produced biologically active SCF because conditioned medium from OxPAPC-treated cells stimulated activation (phosphorylation) of c-Kit in naïve ECs. Induction of SCF by OxPAPC was inhibited by knocking down transcription factor NRF2. Inhibition or stimulation of NRF2 by pharmacological or molecular tools induced corresponding changes in SCF expression. Finally, we observed decreased levels of SCF mRNA in aortas of NRF2 knockout mice. We characterize OxPLs as a novel pathology-associated stimulus inducing expression of SCF in endothelial cells. Furthermore, our data point to transcription factor NRF2 as a major mediator of OxPL-induced upregulation of SCF. This mechanism may represent one of the facets of pleiotropic action of NRF2 in vascular wall.
Assuntos
Aorta/metabolismo , Regulação da Expressão Gênica , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilcolinas/metabolismo , Fator de Células-Tronco/biossíntese , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout para ApoE , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Fosfatidilcolinas/genética , Fator de Células-Tronco/genéticaAssuntos
Vesículas Extracelulares , MicroRNAs , Aterosclerose , Movimento Celular , Humanos , MacrófagosRESUMO
RATIONALE: Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) generates a group of bioactive oxidized phospholipid products with a broad range of biological activities. Barrier-enhancing and anti-inflammatory effects of OxPAPC on pulmonary endothelial cells are critical for prevention of acute lung injury caused by bacterial pathogens or excessive mechanical ventilation. Anti-inflammatory properties of OxPAPC are associated with its antagonistic effects on Toll-like receptors and suppression of RhoA GTPase signaling. OBJECTIVE: Because OxPAPC exhibits long-lasting anti-inflammatory and lung-protective effects even after single administration in vivo, we tested the hypothesis that these effects may be mediated by additional mechanisms, such as OxPAPC-dependent production of anti-inflammatory and proresolving lipid mediator, lipoxin A4 (LXA4). METHODS AND RESULTS: Mass spectrometry and ELISA assays detected significant accumulation of LXA4 in the lungs of OxPAPC-treated mice and in conditioned medium of OxPAPC-exposed pulmonary endothelial cells. Administration of LXA4 reproduced anti-inflammatory effect of OxPAPC against tumor necrosis factor-α in vitro and in the animal model of lipopolysaccharide-induced lung injury. The potent barrier-protective and anti-inflammatory effects of OxPAPC against tumor necrosis factor-α and lipopolysaccharide challenge were suppressed in human pulmonary endothelial cells with small interfering RNA-induced knockdown of LXA4 formyl peptide receptor-2 (FPR2/ALX) and in mFPR2-/- (mouse formyl peptide receptor 2) mice lacking the mouse homolog of human FPR2/ALX. CONCLUSIONS: This is the first demonstration that inflammation- and injury-associated phospholipid oxidation triggers production of anti-inflammatory and proresolution molecules, such as LXA4. This lipid mediator switch represents a novel mechanism of OxPAPC-assisted recovery of inflamed lung endothelium.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Lipoxinas/metabolismo , Fosfatidilcolinas/uso terapêutico , Lesão Pulmonar Aguda/prevenção & controle , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Células Cultivadas , Humanos , Lipoxinas/farmacologia , Lipoxinas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilcolinas/farmacologia , Resultado do TratamentoRESUMO
Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents.
Assuntos
Lesão Pulmonar/microbiologia , Pulmão/microbiologia , Microbiota/efeitos dos fármacos , Síndrome do Desconforto Respiratório/microbiologia , Animais , Sequência de Bases , Líquido da Lavagem Broncoalveolar/microbiologia , Brucellaceae/genética , Brucellaceae/isolamento & purificação , DNA Bacteriano/genética , Modelos Animais de Doenças , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Xanthomonadaceae/genética , Xanthomonadaceae/isolamento & purificaçãoRESUMO
Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA(+) MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE(+) MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.
Assuntos
Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Epitopos/imunologia , Imunoglobulina M/imunologia , Síndrome Coronariana Aguda/imunologia , Síndrome Coronariana Aguda/metabolismo , Adulto , Feminino , Humanos , Masculino , Malondialdeído/metabolismo , OxirreduçãoRESUMO
OBJECTIVE: Oxidized phospholipids (OxPLs), which are highly abundant in atherosclerotic lesions, are known to induce electrophilic stress response (ESR). ESR induces cytoprotective genes via the NF-E2-related factor 2 (NRF2) transcription factor. In order to get further insight into the mechanisms of ESR, we studied the role of microRNA (miR)-320a in induction of NRF2-dependent genes by OxPLs. METHODS: Microarray profiling and qRT-PCR methods were used for measurements of mRNA and miRNA levels. miR-320a levels were changed by transfection with synthetic oligonucleotides. Protein analysis was performed by Western blotting. The functional activity of NRF2 was measured by DNA-binding ELISA. RESULTS: Oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) induced miR-320a in endothelial cells. Induction of HO-1, OKL38 and GCLM mRNAs by OxPAPC and sulforaphane was attenuated upon knockdown of miR-320a. In contrast, transfection of ECs with miR-320a mimic oligonucleotide potentiated the effects of OxPAPC and sulforaphane on induction of HO-1, OKL38 and GCLM mRNAs. OxPAPC-induced p38 activation, levels of NRF2 protein and its ability to bind to consensus NRF2 DNA binding site were elevated in ECs transfected with miR-320a mimic. miR-320a positively regulated induction of VEGF mRNA by OxPAPC. Levels of miR-320a and HO-1 and OKL38 mRNAs were elevated in aortas of ApoE knockout mice fed with high fat diet. Manipulation of miR-320a level in ECs did not affect ability of OxPAPC to induce IL-8, COX-2 and MCP-1. CONCLUSION: miR-320a plays important role in induction of expression of HO-1, GCLM and OKL38 upon ESR induced either by OxPAPC or sulforaphane. These observations propose a general role of miR-320a in control of ESR induced by different electrophilic agents.
Assuntos
Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/metabolismo , Isotiocianatos/química , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosfolipídeos/química , Proteínas/metabolismo , Animais , Apolipoproteínas E/genética , Proteínas Reguladoras de Apoptose , Ensaio de Imunoadsorção Enzimática , Feminino , Glutamato-Cisteína Ligase/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Estresse Oxidativo , Oxigênio/química , RNA Mensageiro/metabolismo , Sulfóxidos , TransfecçãoRESUMO
Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.
Assuntos
Fator 4 Ativador da Transcrição/genética , Caseína Quinase II/genética , Fosfolipídeos/metabolismo , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator 4 Ativador da Transcrição/biossíntese , Western Blotting , Caseína Quinase II/biossíntese , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Espectrometria de Massas , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
OBJECTIVE: Atherosclerotic lesions contain high concentrations of oxidized phospholipids (OxPLs) known to induce VEGF via the ATF4 arm of unfolded protein response (UPR), and to promote angiogenic reactions thus potentially contributing to the progression and destabilization of atherosclerotic plaques. In order to get further insights into the mechanisms of cellular stress-induced angiogenesis we studied the role of a specific microRNA (miR-663) in the mechanisms of VEGF induction by OxPLs and inducers of UPR. METHODS: miRNA and mRNA levels were determined using microarray profiling and qRT-PCR methods. Proteins were analyzed by Western blotting. miR-663 levels were changed by transfecting cells with synthetic oligonucleotides. RESULTS: OxPAPC elevated miR-663 in two types of human endothelial cells (ECs). Knockdown of miR-663 inhibited upregulation of VEGF mRNA in ECs treated by OxPAPC, OxPAPS or OxPAPA. In addition, silencing of miR-663 suppressed upregulation by OxPAPC of ATF4 mRNA and protein, as well as a downstream gene TRIB. Similarly to the inhibition of OxPAPC effects, knockdown of miR-663 suppressed elevation of ATF4, VEGF and TRIB in response to another inducer of UPR, tunicamycin. Overexpression of miR-663 reversed the inhibition of VEGF induction by miR-663 inhibitor. CONCLUSION: miR-663 is critically important for 2 key events induced in ECs by stress agents and oxidized lipids, namely induction of transcription factor ATF4 and its downstream gene VEGF. These findings allow hypothesizing that miR-663 plays a general role in control of the ATF4 branch of UPR induced by different agents.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , MicroRNAs/fisiologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/antagonistas & inibidores , Oxirredução , Fosfatidilcolinas , Fosfolipídeos/metabolismo , Tunicamicina/farmacologiaRESUMO
The farnesoid X receptor (FXR) belonging to the metabolic subfamily of nuclear receptors is a ligand-induced transcriptional activator. Its central function is the physiological maintenance of bile acid homeostasis including the regulation of glucose and lipid metabolism. Accessible structural information about its ligand-binding domain renders FXR an attractive target for in silico approaches. Integrated to natural product research these computational tools assist to find novel bioactive compounds showing beneficial effects in prevention and treatment of, for example, the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. Virtual screening experiments of our in-house Chinese Herbal Medicine database with structure-based pharmacophore models, previously generated and validated, revealed mainly lanostane-type triterpenes of the TCM fungus Ganoderma lucidum Karst. as putative FXR ligands. To verify the prediction of the in silico approach, two Ganoderma fruit body extracts and compounds isolated thereof were pharmacologically investigated. Pronounced FXR-inducing effects were observed for the extracts at a concentration of 100 µg/mL. Intriguingly, five lanostanes out of 25 secondary metabolites from G. lucidum, that is, ergosterol peroxide (2), lucidumol A (11), ganoderic acid TR (12), ganodermanontriol (13), and ganoderiol F (14), dose-dependently induced FXR in the low micromolar range in a reporter gene assay. To rationalize the binding interactions, additional pharmacophore profiling and molecular docking studies were performed, which allowed establishing a first structure-activity relationship of the investigated triterpenes.
Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Reishi/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Animais , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood. Here we show that upregulation of NRF2 by OxPLs depends on the activity of the CK2 protein kinase. Inactivation of CK2 by chemical inhibitors or gene silencing resulted in diminished accumulation of NRF2 and its target genes, GCLM, HMOX1, and NQO1, downstream in response to OxPLs. Furthermore, inhibition of CK2 suppressed NRF2-dependent induction of ATF4 and its downstream gene VEGF. Thus, inactivation of CK2 in OxPL-treated endothelial cells results in inhibition of the NRF2-ATF4-VEGF axis and is likely to produce antiangiogenic effects. This work characterizes novel cross-talk between CK2 and cellular stress pathways, which may provide additional insights into the mechanisms of beneficial action and side-effects of CK2 inhibitors.
Assuntos
Caseína Quinase II/fisiologia , Células Endoteliais/enzimologia , Fosfolipídeos/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Inativação Gênica , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Oxigênio/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The pleiotropic cytokine oncostatin M (OSM), a member of the glycoprotein (gp)130 ligand family, plays a key role in inflammation and cardiovascular disease. As inflammation precedes and accompanies pathological angiogenesis, we investigated the effect of OSM and other gp130 ligands on vascular endothelial growth factor (VEGF) production in human vascular smooth muscle cells (SMC). Human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) were treated with different gp130 ligands. VEGF protein was determined by ELISA. Specific mRNA was detected by RT-PCR. Western blotting was performed for signal transducers and activators of transcription1 (STAT1), STAT3, Akt and p38 mitogen-activated protein kinase (p38 MAPK). OSM mRNA and VEGF mRNA expression was analyzed in human carotid endaterectomy specimens from 15 patients. OSM increased VEGF production in both HCASMC and HASMC derived from different donors. OSM upregulated VEGF and OSM receptor-specific mRNA in these cells. STAT3 inhibitor WP1066, p38 MAPK inhibitors SB-202190 and BIRB 0796, extracellular signal-regulated kinase1/2 (Erk1/2) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitors LY-294002 and PI-103 reduced OSM-induced VEGF synthesis. We found OSM expression in human atherosclerotic lesions where OSM mRNA correlated with VEGF mRNA expression. Interferon-γ (IFN-γ), but not IL-4 or IL-10, reduced OSM-induced VEGF production in vascular SMC. Our findings that OSM, which is present in human atherosclerotic lesions and correlates with VEGF expression, stimulates production of VEGF by human coronary artery and aortic SMC indicate that OSM could contribute to plaque angiogenesis and destabilization. IFN-γ reduced OSM-induced VEGF production by vascular SMC.
Assuntos
Interferon gama/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Oncostatina M/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Aterosclerose/metabolismo , Células Cultivadas , Vasos Coronários/metabolismo , Feminino , Humanos , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response. METHODS AND RESULTS: Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation. CONCLUSION: Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Células Endoteliais/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neovascularização Fisiológica , Fosfolipídeos/metabolismo , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 4 Ativador da Transcrição/genética , Sítios de Ligação , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Humanos , Lipoproteínas LDL/metabolismo , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Regiões Promotoras Genéticas , Prostaglandinas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ativação Transcricional , Regulação para CimaRESUMO
Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N-acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.
Assuntos
Antioxidantes/metabolismo , Artérias Carótidas/enzimologia , Núcleo Celular/metabolismo , Células Endoteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilcolinas/farmacocinética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Artérias Carótidas/citologia , Núcleo Celular/genética , Células Endoteliais/citologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutamato-Cisteína Ligase/biossíntese , Glutamato-Cisteína Ligase/genética , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Camundongos , Camundongos Mutantes , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/biossíntese , NADPH Desidrogenase/genética , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Oxirredução/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Fosfolipídeos/fisiologia , Desnaturação Proteica , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/genética , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Oxirredução , Regulação para CimaRESUMO
There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor alpha1 subunit, integrin beta3, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin beta3 with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone.
Assuntos
Caderinas/metabolismo , Células Endoteliais/citologia , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Biotinilação , Membrana Celular , Sobrevivência Celular , Chaperona BiP do Retículo Endoplasmático , Glicosilfosfatidilinositóis/metabolismo , Humanos , Integrina beta3/metabolismo , Espectrometria de Massas , Microdomínios da Membrana/metabolismo , Microscopia Confocal , Modelos Biológicos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Macrophages produce the cytokine oncostatin M (OSM), which beside other functions is also involved in inflammation. The complement component C5a mobilizes and activates these cells at inflammatory sites. We examined the effect of C5a on OSM production in human monocytes and in human monocyte-derived macrophages. METHODS AND RESULTS: For macrophage transformation peripheral blood monocytes were cultivated for 8 to 10 days in the presence of human serum. C5a significantly increased in these cells OSM antigen as determined by specific ELISA and mRNA as quantitated by real-time polymerase chain reaction in these cells as well as in plaque macrophages. This effect was blocked by antibodies against the receptor C5aR/CD88 and by pertussis toxin. The C5a-induced phosphorylation of p38 and JNK and the C5a-induced increase in OSM production in macrophages was abolished by 2 p38 inhibitors and by a JNK inhibitor. Furthermore C5a increased the nuclear translocation of c-fos and c-jun. Using different OSM promoter deletion mutant constructs we show that the putative AP-1 element is responsible for activation of OSM promoter activity by C5a. CONCLUSIONS: Our data establish a link between the complement system and the gp130 receptor cytokine family with possible implications for the pathology of inflammatory diseases.
Assuntos
Aterosclerose/metabolismo , Complemento C5a/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Oncostatina M/metabolismo , Fator de Transcrição AP-1/metabolismo , Análise de Variância , Aterosclerose/complicações , Western Blotting , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Inflamação/complicações , Mediadores da Inflamação/metabolismo , Macrófagos/citologia , Oncostatina M/genética , Probabilidade , Ligação Proteica , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fator de Transcrição AP-1/genética , Regulação para CimaRESUMO
Plasminogen activator inhibitor-1 (PAI-1) plays a pivotal role in the regulation of the fibrinolytic system and in the modulation of extracellular proteolysis. Increased PAI-1 was found in atherosclerotic lesions, and high PAI-1 plasma levels were associated with coronary heart disease. Smooth muscle cells (SMC) are a major source of PAI-1 within the vascular wall, and PAI-1 was implicated in SMC migration, proliferation, and apoptosis. We treated human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) with the glycoprotein 130 (gp130) ligands cardiotrophin-1, interleukin-6 (IL-6), leukemia inhibitory factor (LIF), or oncostatin M (OSM). Only OSM increased PAI-1 antigen and activity production significantly in these cells up to 20-fold. OSM upregulated mRNA specific for PAI-1 up to 4.5-fold in these cells. HCASMC and HASMC express gp130, OSM receptor, IL-6 receptor, and LIF receptor. OSM induced extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylations in HASMC. A phosphatidylinositol 3-kinase inhibitor and a mitogen-activated protein/extracellular signal-regulated kinase inhibitor reduced Akt and ERK1/2 phosphorylation, respectively, and abolished OSM-induced PAI-1 upregulation. A janus kinase/signal transducer and activator of transcription inhibitor, a p38 mitogen-activated protein kinase inhibitor, or c-Jun NH(2)-terminal kinase inhibitor I did not inhibit the OSM-dependent PAI-1 induction. OSM enhanced proliferation of both HCASMC and HASMC by 77 and 90%, respectively. We hypothesize that, if the effect of OSM on PAI-1 expression in smooth muscle cells is operative in vivo, it could, via modulation of fibrinolysis and extracellular proteolysis, be involved in the development of vascular pathologies such as plaque progression, destabilization and subsequent thrombus formation, and restenosis and neointima formation.
Assuntos
Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/metabolismo , Oncostatina M/farmacologia , Oncostatina M/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Transdução de Sinais/fisiologia , Aorta/citologia , Aorta/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Músculo Liso Vascular/citologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2-generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.