RESUMO
Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated.
Assuntos
Ácidos Araquidônicos , Ácidos Nucleicos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Araquidonato 12-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Ácidos Araquidônicos/metabolismo , Plaquetas , Glucose/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Lactatos/metabolismo , Ácidos Nucleicos/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease that significantly endangers human health, where metabolism may drive pathogenesis: a shift from mitochondrial oxidation to glycolysis occurs in diseased pulmonary vessels and the right ventricle. An increase in pulmonary vascular resistance in patients with heart failure with a preserved ejection fraction portends a poor prognosis. Luteolin exists in numerous foods and is marketed as a dietary supplement assisting in many disease treatments. However, little is known about the protective effect of luteolin on metabolism disorders in diseased pulmonary vessels. In this study, we found that luteolin apparently reversed the pulmonary vascular remodeling of PAH rats by inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, network pharmacology and metabolomics results revealed that the arachidonic acid pathway, amino acid pathway and TCA cycle were dysregulated in PAH. A total of 14 differential metabolites were significantly changed during the PAH, including DHA, PGE2, PGD2, LTB4, 12-HETE, 15-HETE, PGF2α, and 8-iso-PGF2α metabolites in the arachidonic acid pathway, and L-asparagine, oxaloacetate, N-acetyl-L-ornithine, butane diacid, ornithine, glutamic acid metabolites in amino acid and TCA pathways. However, treatment with luteolin recovered the LTB4, PGE2, PGD2, 12-HETE, 15-HETE, PGF2α and 8-iso-PGF2α levels close to normal. Meanwhile, we showed that luteolin also downregulated the gene and protein levels of COX 1, 5-LOX, 12-LOX, and 15-LOX in the arachidonic acid pathway. Collectively, this work highlighted the metabolic mechanism of luteolin-protected PAH and showed that luteolin would hold great potential in PAH prevention.
Assuntos
Hipertensão Arterial Pulmonar , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Animais , Ácido Araquidônico/metabolismo , Asparagina , Butanos/metabolismo , Butanos/farmacologia , Proliferação de Células , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Dinoprostona/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Leucotrieno B4/metabolismo , Luteolina/farmacologia , Músculo Liso Vascular , Miócitos de Músculo Liso/metabolismo , Farmacologia em Rede , Ornitina/metabolismo , Oxaloacetatos/metabolismo , Oxaloacetatos/farmacologia , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , RatosRESUMO
PURPOSE: Abnormal lipid metabolism has been proved to be implicated in the complex pathogenesis of diabetic retinopathy (DR). 12-lipoxygenase (12-LOX) is a member of lipoxygenase family responsible for the oxygenation of cellular polyunsaturated fatty acids to produce lipid mediators which modulate cell inflammation. This review explores the role of 12-lipoxygenase and its products in the pathogenesis of DR. METHODS: A comprehensive medical literature search was conducted on PubMed till September 2021. RESULTS: Emerging evidence has demonstrated that 12-LOX and its main product 12- hydroxyeicosatetraenoic acid (12-HETE) activate retinal cells, especially retinal vascular endothelial cells, through the activation of NADPH oxidase and the subsequent generation of reactive oxygen species (ROS), mediating multiple pathological changes during DR. Genetic deletion or pharmacological inhibition models of 12-LOX in mice show protection from DR. CONCLUSION: 12-LOX and its product 12-HETE take important part in DR pathogenesis and show their potential as future therapeutic targets for DR. Further studies are needed on the specific mechanism including 12-LOX pathway related molecules, 12-HETE receptors and downstream signaling pathways.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Araquidonato 12-Lipoxigenase , Diabetes Mellitus , Retinopatia Diabética , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Ácidos Hidroxieicosatetraenoicos/uso terapêutico , CamundongosRESUMO
12/15-LO (12/15-lipoxygenase), encoded by Alox15 gene, metabolizes arachidonic acid to 12(S)-HETE (12-hydroxyeicosatetraenoic acid). Macrophages are the major source of 12/15-LO among immune cells, and 12/15-LO plays a crucial role in development of hypertension. Global Alox15- or macrophage-deficient mice are resistant to Ang II (angiotensin II)-induced hypertension. This study tests the hypothesis that macrophage 12(S)-HETE contributes to Ang II-mediated arterial constriction and thus to development of Ang II-induced hypertension. Ang II constricted isolated abdominal aortic and mesenteric arterial rings. 12(S)-HETE (100 nmol/L) alone was without effect; however, it significantly enhanced Ang II-induced constriction. The presence of wild-type macrophages also enhanced the Ang II-induced constriction, while Alox15-/- macrophages did not. Using this model, pretreatment of aortic rings with inhibitors, receptor agonists/antagonists, or removal of the endothelium, systematically uncovered an endothelium-mediated, Ang II receptor-2-mediated and superoxide-mediated enhancing effect of 12(S)-HETE on Ang II constrictions. The role of superoxide was confirmed using aortas from p47phox-/- mice where 12(S)-HETE failed to enhance constriction to Ang II. In cultured arterial endothelial cells, 12(S)-HETE increased the production of superoxide, and 12(S)-HETE or Ang II increased the production of an isothromboxane-like metabolite. A TP (thromboxane receptor) antagonist inhibited 12(S)-HETE enhancement of Ang II constriction. Both Ang II-induced hypertension and the enhancing effect of 12(S)-HETE on Ang II contractions were eliminated by a BLT2 (leukotriene B4 receptor-2) antagonist. These results outline a mechanism where the macrophage 12/15-LO pathway enhances the action of Ang II. 12(S)-HETE, acting on the BLT2, contributes to the hypertensive action of Ang II in part by promoting endothelial synthesis of a superoxide-derived TP agonist.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Angiotensina II/farmacologia , Aorta/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Receptores do Leucotrieno B4/metabolismo , Receptores de Tromboxanos/metabolismo , Animais , Aorta/metabolismo , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Masculino , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Knockout , Superóxidos/metabolismoRESUMO
OBJECTIVE: The enteric nervous system (ENS) plays a key role in controlling the gut-brain axis under normal and pathological conditions, such as type 2 diabetes. The discovery of intestinal actors, such as enterosynes, able to modulate the ENS-induced duodenal contraction is considered an innovative approach. Among all the intestinal factors, the understanding of the role of gut microbes in controlling glycaemia is still developed. We studied whether the modulation of gut microbiota by prebiotics could permit the identification of novel enterosynes. DESIGN: We measured the effects of prebiotics on the production of bioactive lipids in the intestine and tested the identified lipid on ENS-induced contraction and glucose metabolism. Then, we studied the signalling pathways involved and compared the results obtained in mice to human. RESULTS: We found that modulating the gut microbiota with prebiotics modifies the actions of enteric neurons, thereby controlling duodenal contraction and subsequently attenuating hyperglycaemia in diabetic mice. We discovered that the signalling pathway involved in these effects depends on the synthesis of a bioactive lipid 12-hydroxyeicosatetraenoic acid (12-HETE) and the presence of mu-opioid receptors (MOR) on enteric neurons. Using pharmacological approaches, we demonstrated the key role of the MOR receptors and proliferator-activated receptor γ for the effects of 12-HETE. These findings are supported by human data showing a decreased expression of the proenkephalin and MOR messanger RNAs in the duodenum of patients with diabetic. CONCLUSIONS: Using a prebiotic approach, we identified enkephalin and 12-HETE as new enterosynes with potential real beneficial and safety impact in diabetic human.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biossíntese , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Duodeno/fisiologia , Sistema Nervoso Entérico/fisiologia , Prebióticos , Receptores Opioides mu/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Adulto , Idoso , Animais , Eixo Encéfalo-Intestino , Diabetes Mellitus Experimental/fisiopatologia , Duodeno/inervação , Encefalinas/genética , Encefalinas/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Microbioma Gastrointestinal , Teste de Tolerância a Glucose , Humanos , Contração Isotônica/efeitos dos fármacos , Masculino , Camundongos , Pessoa de Meia-Idade , Músculo Liso/fisiologia , Neurônios/fisiologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Oligossacarídeos/farmacologia , PPAR gama/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptores Opioides mu/genética , Transdução de SinaisRESUMO
The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.
Assuntos
Arginase/biossíntese , Citocromo P-450 CYP1A1/fisiologia , Janus Quinase 1/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Peritonite/prevenção & controle , Fator de Transcrição STAT6/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Transferência Adotiva , Animais , Araquidonato 15-Lipoxigenase/fisiologia , Arginase/genética , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Endotoxinas/toxicidade , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Ácidos Hidroxieicosatetraenoicos/genética , Ácidos Hidroxieicosatetraenoicos/farmacologia , Interleucina-4/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/induzido quimicamente , Células RAW 264.7 , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Células THP-1 , Regulação para Cima/efeitos dos fármacosRESUMO
Bioactive lipid mediators derived from n-3 and n-6 fatty acids are known to modulate leukocytes. Metabolic transformation of essential fatty acids to endogenous bioactive molecules plays a major role in human health. Here we tested the potential of substrates; linoleic acid (LA) and docosahexaenoic acid (DHA) and their bioactive products; resolvin D1 (RvD1) and 12- S-hydroxyeicosatetraenoic acids (HETE) to modulate macrophage plasticity and cardiac fibroblast phenotype in presence or absence of lipid metabolizing enzyme 12/15-lipoxygenase (LOX). Peritoneal macrophages and cardiac fibroblasts were isolated from wild-type (C57BL/6J) and 12/15LOX -/- mice and treated with DHA, LA, 12(S)-HETE, and RvD1 for 4, 8, 12, and 24 hr. LA, DHA, 12(S)-HETE, and RvD1 elicited mRNA expression of proinflammatory markers; tumor necrosis factor-α ( Tnf-α), interleukin 6 ( IL-6), chemokine (C-C motif) ligand 2 (Ccl2), and IL-1ß in wild type (WT) and in 12/15LOX -/- macrophages at early time point (4 hr). Bioactive immunoresolvent RvD1 lowered the levels of Tnf-α, IL-6, and IL-1ß at 24 hr time point. Both DHA and RvD1 stimulated the proresolving markers such as arginase 1 ( Arg-1), chitinase-like protein 3 ( Ym-1), and mannose receptor C-type 1 in WT macrophage. RvD1 induced proresolving phenotype Arg-1 expression in both WT 12/15LOX -/- macrophages even in presence of 12(S)-HETE. RvD1 peaked 5LOX expression in both WT and 12/15LOX -/- at 24 hr time point compared with DHA. RvD1 diminished cyclooxygenase-2 but upregulated 5LOX expression in fibroblast compared with DHA. In summary, the feed-forward enzymatic interaction with fatty acids substrates and direct mediators (RvD1 and 12(S)-HETE) are responsive in determining macrophages phenotype and cardiac fibroblast plasticity. Particularly, macrophages and fibroblast phenotypes are responsive to milieu and RvD1 governs the milieu-dependent chemokine signaling in presence or absence of 12/15LOX enzyme to resolve inflammation.
Assuntos
Plasticidade Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Miocárdio/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Animais , Araquidonato 12-Lipoxigenase/deficiência , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/deficiência , Araquidonato 15-Lipoxigenase/genética , Arginase/metabolismo , Células Cultivadas , Microambiente Celular , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Mediadores da Inflamação/metabolismo , Ácido Linoleico/metabolismo , Ácido Linoleico/farmacologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/imunologia , Fenótipo , Transdução de SinaisRESUMO
Dysfunction and injury of endothelial cells play critical roles in pulmonary arterial hypertension, including aberrant proliferation, suppressed apoptosis, and excessive angiogenesis. The 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid pathway, which has been considered as a crucial mediator, elevates pulmonary vascular resistance and pulmonary arterial pressure. However, the mechanisms underlying the bioactivity of 12-hydroxyeicosatetraenoic acid in pulmonary vasculature, especially in endothelial cells, are still elusive. Thus we aim to determine the key role of 12-lipoxygenase/12-hydroxyeicosatetraenoic acid in angiogenesis and survival of pulmonary artery endothelial cells and ascertain the signaling pathways participating in the pathological process. Here we establish that hypoxia increases the formation of endogenous 12-hydroxyeicosatetraenoic acid through stimulation of 12-lipoxygenase. Furthermore, we put forward new information that 12-hydroxyeicosatetraenoic acid promotes endothelial cell migration and tube formation, whereas it inhibits the serum deprivation-induced apoptotic responses under hypoxia. Particularly, the regulatory effects of 12-lipoxygenase/12-hydroxyeicosatetraenoic acid on pulmonary artery endothelial cells, at least in part, depend on phosphatidylinositol 3-kinase (PI3K)/Akt signaling activation. Taken together, these results may have significant implications for understanding of pulmonary hypertension and offer a potential therapeutic concept focusing on the 12-lipoxygenase/12-hydroxyeicosatetraenoic acid signaling system.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Araquidonato 12-Lipoxigenase/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/fisiopatologia , Neovascularização Patológica/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Araquidonato 12-Lipoxigenase/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Masculino , Neovascularização Patológica/metabolismo , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Secretion of 12(S)-HETE by breast cancer emboli provokes "circular chemorepellent induced defects" (CCIDs) in the adjacent lymphatic vasculature facilitating their intravasation and lymph node metastasis which determines prognosis. Therefore, elucidating the mechanism of lymph endothelial cell (LEC) wall disintegration may provide cues for anti-metastatic intervention. The role of intracellular free Ca(2+) for CCID formation was investigated in LECs using MCF-7 or MDA-MB231 breast cancer cell spheroids in a three-dimensional cell co-culture model. 12(S)-HETE elevated the Ca(2+) level in LEC by activating PLC/IP3. Downstream, the Ca(2+)-calmodulin kinase MYLK contributed to the phosphorylation of Ser19-MLC2, LEC contraction and CCID formation. Approved clinical drugs, lidoflazine, ketotifen, epiandrosterone and cyclosporine, which reportedly disturb cellular calcium supply, inhibited 12(S)-HETE-induced Ca(2+) increase, Ser19-MLC2 phosphorylation and CCID formation. This treatment strategy may reduce spreading of breast cancer through lymphatics.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Neoplasias da Mama/patologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Movimento Celular , Células Endoteliais/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Quelantes de Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Miosinas Cardíacas/metabolismo , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Feminino , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Metástase Linfática , Vasos Linfáticos/metabolismo , Células MCF-7 , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Permeabilidade , Fosforilação , Interferência de RNA , Serina , Esferoides Celulares , Fatores de Tempo , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
(1) BACKGROUND: 12-lipoxygenase (12-LO) is involved in the development of diabetic nephropathy (DN). In the present study, we investigated whether 12-LO inhibition may ameliorate type-2 DN (T2DN) by interfering with insulin resistance (IR); (2) METHODS: Rat glomerular mesangial cells, glomeruli and skeletal muscles were isolated and used in this study. Kidney histological changes were confirmed by periodic-acid Schiff staining; mRNA expression was detected by competitive reverse transcription polymerase chain reaction; and the protein level was determined by Western blot and the enzyme-linked immunosorbent assay, respectively; (3) RESULTS: The inhibition of 12-LO attenuated microalbuminuria (MAU) increases in type-2 diabetic rats, but not in type-1 diabetic rats. Infusion of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) significantly increased the expression of angiotensin II (Ang II) and Ang II type 1 receptor (AT1R), but decreased the expression of AT1R-associated protein (ATRAP) in rat glomeruli, compared to the control. An in vitro study revealed that both 12(S)-HETE and insulin upregulated AT1R expression in rat mesangial cells. In the presence of p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, the 12(S)-HETE-induced ATRAP reduction was significantly abolished. Interestingly, 12-LO inhibition did not influence AT1R expression in type-1 diabetic rats, but significantly abolished the increased AT1R and Ang II expression in glomeruli of type-2 diabetic rats. Furthermore, the inhibition of 12-LO significantly corrected impaired insulin sensitivity and fast serum insulin level, as well as the p-AMP-activated protein kinase (AMPK) reduction in skeletal muscle of type-2 diabetic rats; (4) CONCLUSION: The inhibition of 12-LO potentially ameliorated MAU by preventing IR through the downregulation of glomerular AT1R expression in T2DN.
Assuntos
Albuminúria/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Nefropatias Diabéticas/metabolismo , Resistência à Insulina , Receptor Tipo 1 de Angiotensina/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Albuminúria/etiologia , Animais , Células Cultivadas , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Inibidores de Lipoxigenase/farmacologia , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genéticaRESUMO
Embryonic stem (ES) cells can differentiate into various kinds of cells, such as endothelial and hematopoietic cells. In addition, some evidence suggests that inflammatory mediators such as leukotrienes (LTs), which include the 5-lipoxygenase (LOX) family, can regulate endothelial cell differentiation. In the present study, the eicosanoid precursor arachidonic acid (AA) stimulated vasculogenesis of ES cells by increasing the number of fetal liver kinase-1+ vascular progenitor cells as well as vascular structures positive for platelet endothelial cell adhesion protein-1 and vascular endothelial cadherin. The stimulation of vasculogenesis and expression of the rate-limiting enzyme in the LT signaling pathway, 5-LOX-activating protein (FLAP), was blunted upon treatment with the FLAP inhibitors AM643 and REV5901. Vasculogenesis was significantly restored upon exogenous addition of LTs. Downstream of FLAP, the LTB4 receptor (BLT1) blocker U75302, the BLT2 receptor blocker LY255283 as well as the cysteinyl LT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased reactive oxygen species (ROS) generation, which was not affected upon either FLAP or cyclooxygenase-2 inhibition. Prevention of ROS generation by either the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycine or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and blunted the provasculogenic effect of AA. In summary, our data demonstrate that proinflammatory AA stimulates vasculogenesis of ES cells via the LT pathway by mechanisms involving ROS generation.
Assuntos
Ácido Araquidônico/farmacologia , Leucotrienos/farmacologia , Células-Tronco Embrionárias Murinas/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Animais , Vias Biossintéticas/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Leucotrienos/metabolismoRESUMO
We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/antagonistas & inibidores , Retinopatia Diabética/tratamento farmacológico , Proteínas do Olho/farmacologia , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Fatores de Crescimento Neural/farmacologia , Retina/efeitos dos fármacos , Neovascularização Retiniana/tratamento farmacológico , Serpinas/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Acetofenonas/farmacologia , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Regulação da Expressão Gênica , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Injeções Intravítreas , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Increasingly evidence indicates that enzymes, receptors and metabolites of the arachidonic acid (AA) cascade play a role in intestinal epithelial cell proliferation and colorectal tumorigenesis. However, the information available does not provide a complete picture and contains a number of discrepancies. For this reason it might be appropriate a thorough study into the impacts of the AA cascade on intestinal epithelial cell growth. Our data show that non-differentiated Caco-2 cells cultured with 10% fetal bovine serum (FBS) synthesize appreciable amounts of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and 5-, 12 and 15-hydroxyeicosatetraenoic acid (HETE) but not LTD4, 20-HETE and epoxyeicosatrienoic acids. We also found that inhibitors of PGE2, LTB4 and 5-, 12-, 15-HETE synthesis as well as receptor antagonists of PGE2 and LTB4 blocked Caco-2 cell growth and DNA synthesis induced by 10% FBS without cytotoxic or apoptotic activity. Interestingly, PGE2, LTB4 and 5-, 12- and 15-HETE at concentrations reached in 10% FBS Caco-2 cultures (1-10nM) were able to induce Caco-2 cell growth and DNA synthesis. This was due to the interaction of PGE2 with EP1 and EP4 receptors and LTB4 and HETEs with BLT1 and BLT2 receptors. Moreover, we provide evidence that PGE2 stimulates several cell signaling pathways such as ERK, P38α, CREB and GSKß/ß-catenin involved in the regulation of Caco-2 growth. Finally, we provide evidence that the mitogenic effects of LTB4 and HETEs can be dependent, at least in part, on PGE2 synthesis.
Assuntos
Ácido Araquidônico/metabolismo , Diferenciação Celular , Células Epiteliais/citologia , Intestinos/citologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Apoptose/efeitos dos fármacos , Células CACO-2 , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , AMP Cíclico/farmacologia , DNA/biossíntese , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Leucotrieno B4/antagonistas & inibidores , Leucotrieno B4/metabolismo , Lipoxigenase/metabolismo , Espectrometria de Massas , Modelos Biológicos , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores do Leucotrieno B4/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Melanoma Experimental/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Western Blotting , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colágeno/metabolismo , Progressão da Doença , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Considerable evidence has been published demonstrating the importance of lipoxygenase enzymes for vascular smooth muscle cell (VSMC) growth. The current study sets out to determine whether or not 12-lipoxygenase (12LO) is also important for human placental VSMC survival. Both a pharmacological and two 12LO antisense knockdown approaches were applied. The 12LO inhibitor baicalien induced a 2-2.5-fold increase in cell death, which appeared to result from apoptosis, as indicated by DNA fragmentation, activation of procaspase 3 to caspase 3 and cytochrome C release from the mitochondria to the cytosol. This apoptosis could be prevented by treatment with the 12LO product, 12 hydroxyeicosatetraenoic acid (12HETE). Human platelet-type 12LO-antisense knockdown, by either plasmid transfection or adeno-associated virus (AAV) infection also induced substantial VSMC death over controls, which could also be prevented by treatment with 12HETE, but not 5HETE. Hence, biochemical 12LO inhibition or 12LO-antisense knockdown in VSMC can induce programmed cell death. These observations suggest a previously unrecognized association between human VSMC survivability and 12LO.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/enzimologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Apoptose , Araquidonato 12-Lipoxigenase/genética , Transporte Biológico , Caspase 3/metabolismo , Sobrevivência Celular , Células Cultivadas , Citocromos c/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Flavanonas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Inibidores de Lipoxigenase/farmacologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Liso Vascular/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
12/15-Lipoxygenase (12/15-LO) is an enzyme that converts polyunsaturated fatty acids into bioactive lipid derivatives. In this study, we showed that inhibition of 12/15-LO by baicalein (BA) significantly attenuated clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Inhibited migration of autoimmune T cells into the central nervous system (CNS) by BA treatment could be attributed to reduced activation of microglia, which was indicated by suppressed phagocytosis, and decreased production of proinflammatory cytokines and chemokines in the CNS. We further observed that inhibition of 12/15-LO with BA led to increased expression of peroxisome proliferator-activated receptor (PPAR)ß/δ in microglia of EAE mice. This was confirmed in vitro in primary microglia and a microglia cell line, BV2. In addition, we demonstrated that BA did not affect 12/15-LO or 5-lipoxygenase (5-LO) expression in microglia, but significantly decreased 12/15-LO products without influencing the levels of 5-LO metabolites. Moreover, among these compounds only 12/15-LO metabolite 12-hydroxyeicosatetraenoic acid was able to reverse BA-mediated upregulation of PPARß/δ in BV2 cells. We also showed that inhibition of microglia activation by PPARß/δ was associated with repressed NF-κB and MAPK activities. Our findings indicate that inhibition of 12/15-LO induces PPARß/δ, demonstrating important regulatory properties of 12/15-LO in CNS inflammation. This reveals potential therapeutic applications for MS.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Inibidores Enzimáticos/farmacologia , Flavanonas/farmacologia , PPAR delta/agonistas , PPAR beta/agonistas , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Linhagem Celular , Movimento Celular , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/enzimologia , Microglia/patologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , PPAR delta/genética , PPAR delta/metabolismo , PPAR beta/genética , PPAR beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos TRESUMO
The purpose of the current study was to evaluate the effect of 12/15-lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2(Akita) mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1 expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Inibidores Enzimáticos/farmacologia , Flavanonas/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Animais , Transporte Biológico/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dextranos , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Expressão Gênica/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/agonistas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Arachidonic acid (AA) metabolites mediate endothelium-dependent relaxation in many vascular beds. Previously, we identified the major AA 12/15-lipoxygenase (12/15-LO) metabolite of mouse arteries as 12-hydroxyeicosatetraenoic acid (12-HETE). The goal was to determine the stereospecific configuration of mouse vascular 12-HETE and characterize the role of 12-HETE stereoisomers in the regulation of vascular tone. Using normal, reverse phase, and chiral HPLC, the stereospecific configuration was identified as 12(S)-HETE. 12(S)-HETE relaxed U46619-, carbocyclic thromboxane A(2)-, PGF(2α)-, and 8-iso PGF(2α)-preconstricted mesenteric arteries, but not phenylephrine-preconstricted arteries. 12(R)-HETE was more potent than 12(S)-HETE in relaxing U46619-preconstricted mouse arteries (maximum relaxations = 91.4 ± 2.7% and 71.8 ± 5.9%, respectively). Neither 12-HETE isomer caused constriction. Pretreatment with 12(S)- or 12(R)-HETE (1 µM) inhibited constrictions to U46619 but not phenylephrine. To investigate the role of thromboxane A(2) (TP) receptors in 12-HETE vascular actions, [(3)H]SQ29548 radioligand binding studies were performed in mouse platelets. U46619, 12(R)-HETE, and 12(S)-HETE displaced [(3)H]SQ29548 binding with IC(50)s of 0.07, 0.32, and 1.73 µM, respectively. Both 12(S)- and 12(R)-HETE inhibited intracellular calcium increases induced by U46619 (10 nM) in HEK293 cells overexpressing TP(α) receptor (65.5% and 45.1%, respectively) and coexpressing prostacyclin (IP) and TP(α) receptors (58.0% and 27.1%, respectively). The LO inhibitor NDGA (10 µM) reduced AA relaxations in arteries preconstricted with U46619 but not phenylephrine. These results indicate that exogenous and endogenous 12(S)-HETE relax mouse mesenteric arteries that are preconstricted with thromboxane agonists. These 12(S)-HETE relaxations are mediated by TP receptor competitive inhibition and inhibition of TP agonist-induced increases in intracellular calcium.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Endotélio Vascular/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Receptores de Tromboxanos/antagonistas & inibidores , Vasodilatadores/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Ácido Araquidônico/metabolismo , Plaquetas/efeitos dos fármacos , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes , Sinalização do Cálcio/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Insaturados , Células HEK293 , Humanos , Hidrazinas/farmacologia , Contração Isométrica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores do Leucotrieno B4/efeitos dos fármacos , Receptores de Tromboxano A2 e Prostaglandina H2/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Ovarian cancer is the most lethal gynecologic malignancy in women. Despite the fact that the metastatic spread is associated with the majority of deaths from ovarian cancer, the molecular mechanisms regulating the invasive and metastatic phenotypes of ovarian cancer are poorly understood. In this study, we demonstrated that BLT2, a low affinity leukotriene B(4) receptor, is highly expressed in OVCAR-3 and SKOV-3 human ovarian cancer cells, and that this receptor plays a key role in the invasiveness and metastasis of these cells through activation of STAT3 and consequent up-regulation of matrix metalloproteinase 2 (MMP2). In addition, our results suggest that activation of NAD(P)H oxidase-4 (NOX4) and subsequent reactive oxygen species (ROS) generation lie downstream of BLT2, mediating the stimulation of STAT3-MMP2 cascade in this process. For example, knockdown of BLT2 or NOX4 using each specific siRNA suppressed STAT3 stimulation and MMP2 expression. Similarly, inhibition of STAT3 suppressed the expression of MMP2, thus leading to attenuated invasiveness of these ovarian cancer cells. Finally, the metastasis of SKOV-3 cells in nude mice was markedly suppressed by pharmacological inhibition of BLT2. Together, our results implicate a BLT2-NOX4-ROS-STAT3-MMP2 cascade in the invasiveness and metastasis of ovarian cancer cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores do Leucotrieno B4/metabolismo , Fator de Transcrição STAT3/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Ovarianas/patologia , Fenótipo , Interferência de RNA , Espécies Reativas de Oxigênio , Regulação para CimaRESUMO
We test the hypothesis that 12-hydroperoxyeicosatetraenoic acid (12(s)-HPETE) and 12-hydroxyeicosatetraenoic acid (12-HETE) perfused into the renal pelvis increase afferent renal nerve activity (ARNA) and suppress renin release in rats fed a low-salt (LS) diet via activation of the transient receptor potential vanilloid type 1 (TRPV1) expressed in renal sensory nerves. 12(s)-HPETE or 12-HETE given into the left renal pelvis dose-dependently increased ARNA, which was abolished by AMG9810, a selective TRPV1 antagonist, or by RP67580, a selective neurokinin 1 receptor antagonist, in normal salt or LS-treated rats. 12(s)-HPETE, 12-HETE, or substance P perfused into the left renal pelvis suppressed plasma angiotensin I (Ang I) levels in LS rats, which was abolished by AMG9810 or attenuated by ipsilateral renal denervation (RD). 12(s)-HPETE or 12-HETE increased release of substance P and calcitonin gene-related peptide from the ipsilateral kidney, which was abolished by AMG9810 but not RP67580, RD, or RP67580 plus RD. Immunofluorescence staining showed that TRPV1-positive nerve fibers located in the renal cortex, medulla, and pelvis, and that the sympathetic nerve marker, neuropeptide Y, but not neurokinin 1 receptors expressed in the juxtaglomerular region colocalized with renin. Thus, our data show that 12(s)-HPETE and 12-HETE enhance ARNA and substance P/calcitonin gene-related peptide release but suppress renin activity in LS rats, and these effects are abolished when TRPV1 is blocked. These results indicate that TRPV1 mediates 12(s)-HPETE and 12-HETE action in the kidney in such a way that dysfunction in TRPV1 may lead to disintegrated regulation of renin and renal function.