RESUMEN
Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aorta/citología , Movimiento Celular , Proteínas Ligadas a GPI/metabolismo , Interleucina-17/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Proliferación Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transducción de Señal , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPß and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPß, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Sistema de Señalización de MAP Quinasas , Miocardio/metabolismo , Volumen Sistólico , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Cardiomegalia/genética , Cardiomegalia/patología , Quimiocinas/genética , Quimiocinas/metabolismo , Colágeno/biosíntesis , Colágeno/genética , Modelos Animales de Enfermedad , Femenino , Fibrosis , Regulación de la Expresión Génica , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Masculino , Ratones , Ratones Transgénicos , Miocardio/patología , FN-kappa B/genética , FN-kappa B/metabolismo , Caracteres Sexuales , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Hypertension is the fundamental cause of cardiovascular and cerebrovascular disorders. Several natural and synthetic peptides are being used as antihypertensive agents, which target angiotensin converting enzyme (ACE), the master regulator of angiotensin (Ang) II production. In this study, we have evaluated ACE-inhibitory potential of the tripeptide l-Phenylalanyl-d-Histidyl-l-Leucine (l-Phe-d-His-l-Leu) in vitro and its antihypertensive effect in rat model of dexamethasone-induced hypertension. l-Phe-d-His-l-Leu was custom-designed by changing the configuration of penultimate amino acid residue (histidine) from C-terminal of Ang I, the site at which ACE acts upon and generates Ang II. l-Phe-d-His-l-Leu effectively inhibited ACE activity in a dose-dependent and competitive manner with an IC50 of 53.32 ± 0.13 nmol/L. Both fluorescence spectra and circular dichroism data revealed the direct interaction between l-Phe-d-His-l-Leu and ACE. In addition, molecular docking studies revealed the strong interaction of l-Phe-d-His-l-Leu with the critical active site amino acid residues of ACE. Further, the administration of l-Phe-d-His-l-Leu resulted in decrease in blood pressure (142 ± 3 mmHg) compared to dexamethasone alone group (167 ± 2 mmHg). Besides, l-Phe-d-His-l-Leu decreased the levels of circulating Ang II, and reduced fibrosis in heart and kidney, as evidenced by decreases in collagen deposition. Thus, the strategy of incorporation of d-amino acids in ACE-inhibitory peptides could be valuable in the development of antihypertensive drugs.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Modelos Animales de Enfermedad , Hipertensión/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Oligopéptidos/farmacología , Peptidil-Dipeptidasa A/efectos de los fármacos , Angiotensina II/sangre , Angiotensina II/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Dominio Catalítico , Dexametasona/toxicidad , Hipertensión/inducido químicamente , Hipertensión/enzimología , Hipertensión/fisiopatología , Cinética , Masculino , Oligopéptidos/metabolismo , Oligopéptidos/uso terapéutico , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Wistar , Sistema Renina-AngiotensinaRESUMEN
Sustained activation of the Renin-Angiotensin-Aldosterone System (RAAS) contributes to the pathogenesis of heart failure. Aldosterone (Aldo) is known to induce both myocardial hypertrophy and fibrosis through oxidative stress and proinflammatory pathways. Here we have investigated whether Aldo-mediated cardiomycocyte hypertrophy is dependent on TRAF3IP2, an upstream regulator of IKK and JNK. We also investigated whether the pro-mitogenic and pro-migratory effects of Aldo on cardiac fibroblasts are also mediated by TRAF3IP2. Aldo induced both superoxide and hydrogen peroxide in isolated adult mouse cardiomyocytes (CM), and upregulated TRAF3IP2 expression in part via the mineralocorticoid receptor and oxidative stress. Silencing TRAF3IP2 blunted Aldo-induced IKKß, p65, JNK, and c-Jun activation, IL-18, IL-6 and CT-1 upregulation, and cardiomyocyte hypertrophy. In isolated adult mouse cardiac fibroblasts (CF), Aldo stimulated TRAF3IP2-dependent IL-18 and IL-6 production, CTGF, collagen I and III expression, MMP2 activation, and proliferation and migration. These in vitro results suggest that TRAF3IP2 may play a causal role in Aldo-induced adverse cardiac remodeling in vivo, and identify TRAF3IP2 as a potential therapeutic target in hypertensive heart disease.
Asunto(s)
Aldosterona/fisiología , Movimiento Celular , Proliferación Celular , Fibroblastos/fisiología , Miocitos Cardíacos/fisiología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Células Cultivadas , Receptor gp130 de Citocinas/metabolismo , Hipertrofia/metabolismo , Interleucina-18/fisiología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Oxidación-Reducción , Proteína-Lisina 6-Oxidasa/metabolismo , Transducción de Señal , Remodelación VentricularRESUMEN
Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn(2+)) chelating agents; N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn(2+) and revealed potent inhibition of ECV metalloproteases (ECVMPs) in vitro (IC50: 6.7 µM) compared to DTPA and TTD. The specificity of TPEN towards Zn(2+) was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific Zn(2+) chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage.
Asunto(s)
Quelantes/farmacología , Metaloproteasas/antagonistas & inhibidores , Venenos de Víboras/química , Venenos de Víboras/metabolismo , Viperidae/metabolismo , Zinc/química , Animales , Antivenenos/química , Quelantes/análisis , Quelantes/metabolismo , Disulfiram/metabolismo , Disulfiram/farmacología , Relación Dosis-Respuesta a Droga , Etilenodiaminas/metabolismo , Etilenodiaminas/farmacología , Metaloproteasas/toxicidad , Ratones , Ácido Pentético/metabolismo , Ácido Pentético/farmacología , Espectrofotometría Ultravioleta , Venenos de Víboras/toxicidadRESUMEN
OBJECTIVES: Lipoxygenases (LOX) are the key enzymes involved in the biosynthesis of leukotrienes and reactive oxygen species, which are implicated in pathophysiology of inflammatory disorders. This study was conducted to evaluate the inhibitory effect of water-soluble antioxidant ascorbic acid and its lipophilic derivative, ascorbic acid 6-palmitate (Vcpal) on polymorphonuclear lymphocyte 5-LOX and soybean 15-LOX (sLOX) in vitro. METHODS: LOX activity was determined by measuring the end products, 5-hydroperoxy eicosatetraenoic acid (5-HETE) and lipid hydroperoxides, by spectrophotometric and high performance liquid chromatography methods. The substrate-dependent enzyme kinetics and docking studies were carried out to understand the nature of inhibition. KEY FINDINGS: Vcpal potently inhibited 5-LOX when compared with its inhibitory effect on sLOX (IC50; 2.5 and 10.3 µm respectively, P = 0.003). Further, Vcpal inhibited 5-LOX more strongly than the known synthetic drugs: phenidone and nordihydroguaiaretic acid (P = 0.0007). Enzyme kinetic studies demonstrated Vcpal as a non-competitive reversible inhibitor of 5-LOX. In-silico molecular docking revealed high MolDock and Rerank score for Vcpal than ascorbic acid, complementing in-vitro results. CONCLUSION: Both in-vitro and docking studies demonstrated Vcpal but not ascorbic acid as a non-competitive inhibitor of 5-LOX- and sLOX-induced lipid peroxidation, suggesting a key role for lipophilic nature in bringing about inhibition.