RESUMO
Endothelial cell activation by proinflammatory stimuli drives leukocyte recruitment through enhanced expression of counter-receptors such as vascular cell adhesion molecule-1 (VCAM-1). We previously demonstrated that activation of the receptor tyrosine kinase EphA2 with its ligand ephrin-A1 induces VCAM-1 expression. Here, we sought to characterize the proinflammatory signaling pathways involved. Analysis of over-represented transcription factors in ephrin-A1-induced genes identified multiple potential transcriptional regulators, including the Rel family members nuclear factor-κB (NF-κB/p65) and nuclear factor of activated T-cells (NFAT). While ephrin-A1 failed to induce endothelial NF-κB activation, NF-κB inhibitors prevented ephrin-A1-induced VCAM-1 expression, suggesting basal NF-κB activity is required. In contrast, ephrin-A1 induced a robust EphA2-dependent increase in NFAT activation, and mutation of the NF-κB/NFAT-binding sites in the VCAM-1 promoter blunted ephrin-A1-induced promoter activity. NFAT activation classically occurs through calcium-dependent calcineurin activation, and inhibiting NFAT signaling with calcineurin inhibitors (cyclosporine A, FK506) or direct NFAT inhibitors (A-285222) was sufficient to block ephrin-A1-induced VCAM-1 expression. Consistent with robust NFAT activation, ephrin-A1-induced an EphA2-dependent calcium influx in endothelial cells that was required for ephrin-A1-induced NFAT activation and VCAM-1 expression. This work provides the first data showing EphA2-dependent calcium influx and NFAT activation and identifies NFAT as a novel EphA2-dependent proinflammatory pathway in endothelial activation.
Assuntos
Cálcio/metabolismo , Efrina-A2/metabolismo , Fatores de Transcrição NFATC/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Ciclosporina/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Efrina-A2/antagonistas & inibidores , Efrina-A2/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor EphA2 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-κB activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a protein kinase A-dependent pathway; however, the mechanisms underlying this regulation were unknown. We show that basement membrane proteins limit membrane recruitment of PAK2, the dominant isoform in endothelial cells, by blocking its interaction with the adaptor protein Nck. This uncoupling response requires protein kinase A-dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain. Of importance, shear stress does not stimulate nitric oxide production in endothelial cells on fibronectin, resulting in enhanced PAK activation, NF-κB phosphorylation, ICAM-1 expression, and monocyte adhesion. These data demonstrate that differential flow-induced nitric oxide production regulates matrix-specific PAK signaling and describe a novel mechanism of nitric oxide-dependent NF-κB inhibition.
Assuntos
NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Membrana Basal/metabolismo , Fenômenos Biomecânicos , Bovinos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/patologia , Ativação Enzimática , Matriz Extracelular/metabolismo , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Estresse FisiológicoRESUMO
Despite significant advancements in treatment regimens, cardiovascular disease remains a worldwide leader of morbidity, mortality, and healthcare cost. A large percentage of cardiovascular disease is directly attributable to the process of atherosclerosis, a chronic inflammatory disease of the vessel wall. In the hunt for novel therapeutic targets in cardiovascular disease, neuronal guidance molecules are emerging as significant regulators of cardiovascular remodeling and inflammation. The Eph family of neuronal guidance molecules comprises the largest family of receptor tyrosine kinases in the mammalian genome. While best characterized in embryogenesis and carcinogenesis, Eph receptors and their ephrin ligands are becoming increasingly recognized as important players in chronic inflammatory diseases and immune function. Herein we discuss the current evidence for how Eph/ephrin interactions, particularly EphA2/ephrinA1 and EphB/ephrinB2, affect inflammation and cardiovascular disease.
Assuntos
Efrinas/fisiologia , Receptores da Família Eph/fisiologia , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Células Endoteliais/fisiologia , Humanos , Imunidade , Inflamação/metabolismo , Inflamação/fisiopatologia , Leucócitos/fisiologiaRESUMO
A clear relationship between diabetes and cardiovascular disease has been established for decades. Despite this, the mechanisms by which diabetes contributes to plaque formation remain in question. Some of this confusion derives from studies in type 2 diabetics where multiple components of metabolic syndrome show proatherosclerotic effects independent of underlying diabetes. However, the hyperglycemia that defines the diabetic condition independently affects atherogenesis in cell culture systems, animal models, and human patients. Endothelial cell biology plays a central role in atherosclerotic plaque formation regulating vessel permeability, inflammation, and thrombosis. The current paper highlights the mechanisms by which hyperglycemia affects endothelial cell biology to promote plaque formation.
RESUMO
OBJECTIVE: Endothelial cell activation results in altered cell-cell interactions with adjacent endothelial cells and with infiltrating leukocytes. Eph receptors and their ephrin ligands regulate cell-cell interactions during tissue remodeling, and multiple proinflammatory mediators induce endothelial EphA receptor and ephrinA ligand expression. Therefore, we sought to elucidate the role of EphA receptors and ephrinA ligands in endothelial cell activation and atherosclerosis. METHODS AND RESULTS: Quantitative reverse transcription-polymerase chain reaction screening for EphA/ephrinA expression in atherosclerosis-prone macrovascular endothelium identified EphA2, EphA4, and ephrinA1 as the dominant isoforms. Endothelial activation with oxidized low-density lipoprotein and proinflammatory cytokines induced EphA2 and ephrinA1 expression and sustained EphA2 activation, whereas EphA4 expression was unaffected. Atherosclerotic plaques from mice and humans showed enhanced EphA2 and ephrinA1 expression colocalizing in the endothelial cell layer. EphA2 activation with recombinant Fc-ephrinA1 induced proinflammatory gene expression (eg vascular cell adhesion molecule-1, E-selectin) and stimulated monocyte adhesion, whereas inhibiting EphA2 (small interfering RNA, pharmacological inhibitors) abrogated both ephrinA1-induced and oxidized low-density lipoprotein-induced vascular cell adhesion molecule-1 expression. CONCLUSION: The current data suggest that enhanced EphA2 signaling during endothelial cell activation perpetuates proinflammatory gene expression. Coupled with EphA2 expression in mouse and human atherosclerotic plaques, these data implicate EphA2 as a novel proinflammatory mediator and potential regulator of atherosclerotic plaque development.
Assuntos
Aterosclerose/imunologia , Células Endoteliais/imunologia , Mediadores da Inflamação/metabolismo , Inflamação/imunologia , Receptor EphA2/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Fenótipo , Interferência de RNA , Receptor EphA2/antagonistas & inibidores , Receptor EphA2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
RATIONALE: Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and nuclear factor (NF)-kappaB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-kappaB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-kappaB. OBJECTIVE: To elucidate the mechanisms regulating matrix-specific PAK activation. METHODS AND RESULTS: We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead, basement membrane proteins enhance flow-induced protein kinase (PK)A activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-kappaB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the prostacyclin analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKA inhibitor (PKI) injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. CONCLUSIONS: Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK.