RESUMEN
Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Células Endoteliales , Neovascularización Fisiológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Animales , Células Endoteliales/metabolismo , Ratones , Morfogénesis , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.
Asunto(s)
Retinopatía Diabética/enzimología , Retinopatía Diabética/prevención & control , Epóxido Hidrolasas/antagonistas & inhibidores , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Proteínas Portadoras/metabolismo , Membrana Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ácidos Docosahexaenoicos/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Ependimogliales , Ácidos Grasos Insaturados/metabolismo , Femenino , Humanos , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Elastasa Pancreática/metabolismo , Pericitos/efectos de los fármacos , Pericitos/patología , Presenilina-1/metabolismo , Retina/efectos de los fármacos , Retina/enzimología , Retina/metabolismo , Retina/patología , Solubilidad , Cuerpo Vítreo/metabolismoRESUMEN
Arachidonic acid epoxides generated by cytochrome P450 (CYP) enzymes have been linked to increased tumor growth and metastasis, largely on the basis of overexpression studies and the application of exogenous epoxides. Here we studied tumor growth and metastasis in Cyp2c44-/- mice crossed onto the polyoma middle T oncogene (PyMT) background. The resulting PyMT2c44 mice developed more primary tumors earlier than PyMT mice, with increased lymph and lung metastasis. Primary tumors from Cyp2c44-deficient mice contained higher numbers of tumor-associated macrophages, as well as more lymphatic endothelial cells than tumors from PyMT mice. While epoxide and diol levels were comparable in tumors from both genotypes, prostaglandin (PG) levels were higher in the PyMTΔ2c44 tumors. This could be accounted for by the finding that Cyp2c44 metabolized the PG precursor, PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), thus effectively reducing levels of effector PGs (including PGE2). Next, proteomic analyses revealed an up-regulation of WD repeating domain FYVE1 (WDFY1) in tumors from PyMTΔ2c44 mice, a phenomenon that was reproduced in Cyp2c44-deficient macrophages as well as by PGE2 Mechanistically, WDFY1 was involved in Toll-like receptor signaling, and its down-regulation in human monocytes attenuated the LPS-induced phosphorylation of IFN regulatory factor 3 and nuclear factor-κB. Taken together, our results indicate that Cyp2c44 protects against tumor growth and metastasis by preventing the synthesis of PGE2 The latter eicosanoid influenced macrophages at least in part by enhancing Toll-like receptor signaling via the up-regulation of WDFY1.
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Neoplasias de la Mama/metabolismo , Familia 2 del Citocromo P450/metabolismo , Linfangiogénesis/fisiología , Prostaglandinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Familia 2 del Citocromo P450/genética , Modelos Animales de Enfermedad , Células Endoteliales/patología , Ácidos Grasos Insaturados/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfangiogénesis/genética , Macrófagos , Ratones , Ratones Noqueados , Monocitos , Procesos Neoplásicos , Proteómica , Transducción de Señal , Receptores Toll-Like , Regulación hacia ArribaRESUMEN
Polyunsaturated fatty acids (PUFAs) are used as traditional remedies to treat hair loss, but the mechanisms underlying their beneficial effects are not well understood. Here, we explored the role of PUFA metabolites generated by the cytochrome P450/soluble epoxide hydrolase (sEH) pathway in the regulation of the hair follicle cycle. Histological analysis of the skin from wild-type and sEH-/- mice revealed that sEH deletion delayed telogen to anagen transition, and the associated activation of hair follicle stem cells. Interestingly, EdU labeling during the late anagen stage revealed that hair matrix cells from sEH-/- mice proliferated at a greater rate which translated into increased hair growth. Similar effects were observed in in vitro studies using hair follicle explants, where a sEH inhibitor was also able to augment whisker growth in follicles from wild-type mice. sEH activity in the dorsal skin was not constant but altered with the cell cycle, having the most prominent effects on levels of the linoleic acid derivatives 12,13-epoxyoctadecenoic acid (12,13-EpOME), and 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Fitting with this, the sEH substrate 12,13-EpOME significantly increased hair shaft growth in isolated anagen stage hair follicles, while its diol; 12,13-DiHOME, had no effect. RNA sequencing of isolated hair matrix cells implicated altered Wnt signaling in the changes associated with sEH deletion. Taken together, our data indicate that the activity of the sEH in hair follicle changes during the hair follicle cycle and impacts on two stem cell populations, i.e., hair follicle stem cells and matrix cells to affect telogen to anagen transition and hair growth.
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Epóxido Hidrolasas , Folículo Piloso , Células Madre , Animales , Epóxido Hidrolasas/metabolismo , Cabello/crecimiento & desarrollo , Folículo Piloso/metabolismo , Homeostasis , Ratones , Ratones Endogámicos C57BL , Células Madre/metabolismoRESUMEN
Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.
Asunto(s)
Neoplasias de la Mama/metabolismo , Epóxido Hidrolasas/metabolismo , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinogénesis , Proliferación Celular/fisiología , Transformación Celular Neoplásica , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Epóxido Hidrolasas/genética , Compuestos Epoxi/metabolismo , Ácidos Grasos Insaturados/metabolismo , Femenino , Eliminación de Gen , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismoRESUMEN
Angiogenesis and inflammation are hallmarks of cancer. Arachidonic acid and other polyunsaturated fatty acids (PUFAs) are primarily metabolized by three distinct enzymatic systems initiated by cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes (CYP) to generate bioactive eicosanoids, including prostanoids, leukotrienes, hydroxyeicosatetraenoic acids, and epoxyeicosatrienoic acids. As some of the PUFA metabolites playing essential roles in inflammatory processes, these pathways have been widely studied as therapeutic targets of inflammation. Because of their anti-inflammatory effects, these pathways were also proposed as anti-cancer targets. However, although the eicosanoids were linked to endothelial cell proliferation and angiogenesis almost two decades ago, it is only recently PUFA metabolites, especially those generated by CYP enzymes and the soluble epoxide hydrolase (sEH), have been recognized as important signaling mediators in physiological and pathological angiogenesis. Despite the fact that tumor growth and invasion are heavily dependent on inner-tumor angiogenesis and influenced by vascular stability, the role played by PUFA metabolites in tumor angiogenesis and vessel integrity has been largely overlooked. This review highlights current knowledge on the function of PUFA metabolites generated by the CYP/sEH pathway in angiogenesis and vascular stability as well as their potential involvement in cancer development.
Asunto(s)
Eicosanoides/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/metabolismo , Animales , Ácido Araquidónico/metabolismo , Biomarcadores , Sistema Enzimático del Citocromo P-450/metabolismo , Metabolismo Energético , Humanos , Metabolismo de los Lípidos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias/etiología , Transducción de SeñalRESUMEN
RATIONALE: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair. OBJECTIVE: To determine the role of the AMPKα2 subunit in vascular repair. METHODS AND RESULTS: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice. CONCLUSIONS: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Apoptosis/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Isquemia/metabolismo , Neutrófilos/metabolismo , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Miembro Posterior/irrigación sanguínea , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Isquemia/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
In addition to being a peptidase, the angiotensin-converting enzyme (ACE) can be phosphorylated and involved in signal transduction. We evaluated the role of ACE in granulocyte-colony-stimulating factor (G-CSF)-induced hematopoietic progenitor cell (HPC) mobilization and detected a significant increase in mice-lacking ACE. Transplantation experiments revealed that the loss of ACE in the HPC microenvironment rather than in the HPCs increased mobilization. Indeed, although ACE was expressed by a small population of bone-marrow cells, it was more strongly expressed by endosteal bone. Interestingly, there was a physical association of ACE with the G-CSF receptor (CD114), and G-CSF elicited ACE phosphorylation on Ser1270 in vivo and in vitro. A transgenic mouse expressing a non-phosphorylatable ACE (ACES/A) mutant demonstrated increased G-CSF-induced HPC mobilization and decreased G-CSF-induced phosphorylation of STAT3 and STAT5. These results indicate that ACE expression/phosphorylation in the bone-marrow niche interface negatively regulates G-CSF-induced signaling and HPC mobilization.
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Células de la Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Peptidil-Dipeptidasa A/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Médula Ósea/enzimología , Células de la Médula Ósea/enzimología , Huesos/enzimología , Proliferación Celular/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Ratones Endogámicos C57BL , Ratones Noqueados , Peptidil-Dipeptidasa A/deficiencia , Peptidil-Dipeptidasa A/genética , Fosforilación , Ramipril/farmacología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Nicho de Células MadreRESUMEN
Polyunsaturated fatty acids (PUFA) and their cytochrome P450 (CYP450) metabolites have been linked to angiogenesis and vessel homeostasis. However, the role of individual CYP isoforms and their endogenous metabolites in those processes are not clear. Here, we focused on the role of Cyp2c44 in postnatal retinal angiogenesis and report that Cyp2c44 is highly expressed in Müller glial cells in the retina. The constitutive as well as inducible postnatal genetic deletion of Cyp2c44 resulted in an increased vessel network density without affecting vessel radial expansion during the first postnatal week. This phenotype was associated with an increased endothelial cell proliferation and attenuated Notch signaling. LC-MS/MS analyses revealed that levels of hydroxydocosahexaenoic acids (HDHA), i.e., 10-, 17- and 20-HDHA were significantly elevated in retinas from 5day old Cyp2c44-/- mice compared to their wild-type littermates. Enzymatic activity assays revealed that HDHAs were potential substrates for Cyp2c44 which could account for the increased levels of HDHAs in retinas from Cyp2c44-/- mice. These data indicate that Cyp2c44 is expressed in the murine retina and, like the soluble epoxide hydrolase, is expressed in Müller glia cells. The enhanced endothelial cell proliferation and Notch inhibition seen in retinas from Cyp2c44-deficient mice indicate a role for Cyp2c44-derived lipid mediators in physiological angiogenesis.
Asunto(s)
Familia 2 del Citocromo P450/metabolismo , Células Ependimogliales/enzimología , Neovascularización Fisiológica , Retina/fisiología , Animales , Proliferación Celular , Familia 2 del Citocromo P450/deficiencia , Familia 2 del Citocromo P450/genética , Ácidos Docosahexaenoicos/metabolismo , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Retina/citologíaRESUMEN
Inhibition or deletion of the soluble epoxide hydrolase (sEH) has been linked to reduced cholesterol and protection against atherosclerosis. This study set out to identify sEH substrate(s) or product(s), altered in livers from sEH(-/-) mice that contribute to these beneficial effects. In livers and isolated hepatocytes, deletion of sEH decreased expression of HMG CoA reductase, fatty acid synthase and low density lipoprotein receptor. Sterol regulatory element binding proteins (SREBPs) regulate the expression of all three enzymes and SREBP activation was attenuated in the absence of sEH. The effect was attributed to the AMPK-activated protein kinase (AMPK) which was activated in the absence of sEH. Livers from wild-type versus sEH(-/-) littermates contained significantly higher levels of the sEH substrate 12,13-epoxyoctadecenoic acid, which elicited AMPK activation, while the corresponding sEH product was inactive. Thus, AMPK activation and subsequent inhibition of SREBP can account for the altered expression of lipid metabolizing enzymes in sEH(-/-) mice.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Colesterol/metabolismo , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Homeostasis , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Activación Enzimática , Epóxido Hidrolasas/antagonistas & inhibidores , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Homeostasis/efectos de los fármacos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Ratones , Ratones Endogámicos C57BL , SolubilidadRESUMEN
RATIONALE: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. OBJECTIVE: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. METHODS AND RESULTS: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223(-/y) animals were enhanced. MiR-223 overexpression had no effect on the growth factor-induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced phosphorylation of their receptors and activation of Akt. ß1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of ß1 integrin into miR-223-ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. CONCLUSIONS: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting ß1 integrin.
Asunto(s)
Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Integrina beta1/metabolismo , MicroARNs/fisiología , Neovascularización Fisiológica/genética , Transducción de Señal/genética , Animales , Células Cultivadas , Sistemas de Liberación de Medicamentos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , PorcinosRESUMEN
Fatty acid epoxides are important lipid signaling molecules involved in the regulation of vascular tone and homeostasis. Tissue and plasma levels of these mediators are determined by the activity of cytochrome P450 epoxygenases and the soluble epoxide hydrolase (sEH), and targeting the latter is an effective way of manipulating epoxide levels in vivo. We investigated the role of the sEH in regulating the mobilization and proliferation of progenitor cells with vasculogenic/reparative potential. Our studies revealed that sEH down-regulation/inhibition impaired the development of the caudal vein plexus in zebrafish, and decreased the numbers of lmo2/cmyb-positive progenitor cells therein. In mice sEH inactivation attenuated progenitor cell proliferation (spleen colony formation), but the sEH products 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) and 11,12- dihydroxyeicosatrienoic acid stimulated canonical Wnt signaling and rescued the effects of sEH inhibition. In murine bone marrow, the epoxide/diol content increased during G-CSF-induced progenitor cell expansion and mobilization, and both mobilization and spleen colony formation were reduced in sEH(-/-) mice. Similarly, sEH(-/-) mice showed impaired functional recovery following hindlimb ischemia, which was rescued following either the restoration of bone marrow sEH activity or treatment with 12,13-DiHOME. Thus, sEH activity is required for optimal progenitor cell proliferation, whereas long-term sEH inhibition is detrimental to progenitor cell proliferation, mobilization, and vascular repair.
Asunto(s)
Epóxido Hidrolasas/metabolismo , Ácidos Grasos/metabolismo , Células Madre Hematopoyéticas/citología , Animales , Epóxido Hidrolasas/genética , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Pez CebraRESUMEN
Cytochrome P450-derived epoxides of arachidonic acid [i.e., the epoxyeicosatrienoic acids (EETs)] are important lipid signaling molecules involved in the regulation of vascular tone and angiogenesis. Because many actions of 11,12-cis-epoxyeicosatrienoic acid (EET) are dependent on the activation of protein kinase A (PKA), the existence of a cell-surface G(s)-coupled receptor has been postulated. To assess whether the responses of endothelial cells to 11,12-EET are enantiomer specific and linked to a potential G protein-coupled receptor, we assessed 11,12-EET-induced, PKA-dependent translocation of transient receptor potential (TRP) C6 channels, as well as angiogenesis. In primary cultures of human endothelial cells, (±)-11,12-EET led to the rapid (30 seconds) translocation a TRPC6-V5 fusion protein, an effect reproduced by 11(R),12(S)-EET, but not by 11(S),12(R)-EET or (±)-14,15-EET. Similarly, endothelial cell migration and tube formation were stimulated by (±)-11,12-EET and 11(R),12(S)-EET, whereas 11(S),12(R)-EET and 11,12-dihydroxyeicosatrienoic acid were without effect. The effects of (±)-11,12-EET on TRP channel translocation and angiogenesis were sensitive to EET antagonists, and TRP channel trafficking was also prevented by a PKA inhibitor. The small interfering RNA-mediated downregulation of G(s) in endothelial cells had no significant effect on responses stimulated by vascular endothelial growth or a PKA activator but abolished responses to (±)-11,12-EET. The downregulation of G(q)/11 failed to prevent 11,12-EET-induced TRPC6 channel translocation or the formation of capillary-like structures. Taken together, our results suggest that a G(s)-coupled receptor in the endothelial cell membrane responds to 11(R),12(S)-EET and mediates the PKA-dependent translocation and activation of TRPC6 channels, as well as angiogenesis.
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Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Canales Catiónicos TRPC/metabolismo , Ácido 8,11,14-Eicosatrienoico/antagonistas & inhibidores , Ácido 8,11,14-Eicosatrienoico/farmacología , Inductores de la Angiogénesis/farmacología , Movimiento Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Humanos , Cultivo Primario de Células , ARN Interferente Pequeño/farmacología , Estereoisomerismo , Canal Catiónico TRPC6 , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Platelets from patients with diabetes are hyperreactive and demonstrate increased adhesiveness, aggregation, degranulation, and thrombus formation, processes that contribute to the accelerated development of vascular disease. Part of the problem seems to be dysregulated platelet Ca(2+) signaling and the activation of calpains, which are Ca(2+)-activated proteases that result in the limited proteolysis of substrate proteins and subsequent alterations in signaling. In the present study, we report that the activation of µ- and m-calpain in patients with type 2 diabetes has profound effects on the platelet proteome and have identified septin-5 and the integrin-linked kinase (ILK) as novel calpain substrates. The calpain-dependent cleavage of septin-5 disturbed its association with syntaxin-4 and promoted the secretion of α-granule contents, including TGF-ß and CCL5. Calpain was also released by platelets and cleaved CCL5 to generate a variant with enhanced activity. Calpain activation also disrupted the ILK-PINCH-Parvin complex and altered platelet adhesion and spreading. In diabetic mice, calpain inhibition reversed the effects of diabetes on platelet protein cleavage, decreased circulating CCL5 levels, reduced platelet-leukocyte aggregate formation, and improved platelet function. The results of the present study indicate that diabetes-induced platelet dysfunction is mediated largely by calpain activation and suggest that calpain inhibition may be an effective way of preserving platelet function and eventually decelerating atherothrombosis development.
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Plaquetas/metabolismo , Calpaína/antagonistas & inhibidores , Calpaína/sangre , Diabetes Mellitus Tipo 2/sangre , Adulto , Anciano , Animales , Plaquetas/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Señalización del Calcio , Calpaína/deficiencia , Calpaína/genética , Estudios de Casos y Controles , Proteínas de Ciclo Celular/sangre , Quimiocina CCL5/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Pioglitazona , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/fisiología , Proteínas Serina-Treonina Quinasas/sangre , Proteómica , Septinas/sangre , Tiazolidinedionas/uso terapéuticoRESUMEN
MicroRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. miR-27 is expressed in endothelial cells, but the specific functions of miR-27b and its family member miR-27a are largely unknown. Here we demonstrate that overexpression of miR-27a and miR-27b significantly increased endothelial cell sprouting. Inhibition of both miR-27a and miR-27b impaired endothelial cell sprout formation and induced endothelial cell repulsion in vitro. In vivo, inhibition of miR-27a/b decreased the number of perfused vessels in Matrigel plugs and impaired embryonic vessel formation in zebrafish. Mechanistically, miR-27 regulated the expression of the angiogenesis inhibitor semaphorin 6A (SEMA6A) in vitro and in vivo and targeted the 3'-untranslated region of SEMA6A. Silencing of SEMA6A partially reversed the inhibition of endothelial cell sprouting and abrogated the repulsion of endothelial cells mediated by miR-27a/b inhibition, indicating that SEMA6A is a functionally relevant miR-27 downstream target regulating endothelial cell repulsion. In summary, we show that miR-27a/b promotes angiogenesis by targeting the angiogenesis inhibitor SEMA6A, which controls repulsion of neighboring endothelial cells.
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Células Endoteliales/metabolismo , MicroARNs/genética , Neovascularización Fisiológica/genética , Semaforinas/genética , Regiones no Traducidas 3'/genética , Animales , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Western Blotting , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Células Endoteliales/fisiología , Expresión Génica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Neovascularización Fisiológica/fisiología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semaforinas/metabolismo , Transfección , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
OBJECTIVE: Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein 3-kinase and an AMP-activated protein kinase (AMPK) kinase in some cell types. Although TAK1(-/-) mice display defects in developmental vasculogenesis, the role of TAK1 in endothelial cells has not been investigated in detail. APPROACH AND RESULTS: TAK1 downregulation (small interfering RNA) in human endothelial cells attenuated proliferation without inducing apoptosis and diminished endothelial cell migration, as well as tube formation. Cytokine- and vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting in a modified spheroid assay were abrogated by TAK1 downregulation. Moreover, VEGF-induced endothelial sprouting was impaired in aortic rings from mice lacking TAK1 in endothelial cells (TAK(ΔEC)). TAK1 inhibition and downregulation also inhibited VEGF-stimulated phosphorylation of several kinases, including AMPK. Proteomic analyses revealed that superoxide dismutase 2 (SOD2) expression was reduced in TAK1-deficient endothelial cells, resulting in attenuated hydrogen peroxide production but increased mitochondrial superoxide production. Endothelial cell SOD2 expression was also attenuated by AMPK inhibition and in endothelial cells from AMPKα1(-/-) mice but was unaffected by inhibitors of c-Jun N-terminal kinase, p38, extracellular signal-regulated kinase 1/2, or phosphatidylinositol 3-kinase/Akt. Moreover, the impaired endothelial sprouting from TAK(ΔEC) aortic rings was abrogated in the presence of polyethylene glycol-SOD, and tube formation was normalized by the overexpression of SOD2. A similar rescue of angiogenesis was observed in polyethylene glycol-SOD-treated aortic rings from mice with endothelial cell-specific deletion of the AMPKα1. CONCLUSIONS: These results establish TAK1 as an AMPKα1 kinase that regulates vascular endothelial growth factor-induced and cytokine-induced angiogenesis by modulating SOD2 expression and the superoxide anion:hydrogen peroxide balance.
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Proteínas Quinasas Activadas por AMP/metabolismo , Células Endoteliales/enzimología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/genética , Animales , Antioxidantes/farmacología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Peróxido de Hidrógeno/metabolismo , Interleucina-1beta/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/deficiencia , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Neovascularización Fisiológica , Oxidación-Reducción , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Factores de Tiempo , Transfección , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The lipid content of skin plays a determinant role in its barrier function with a particularly important role attributed to linoleic acid and its derivatives. Here we explored the consequences of interfering with the soluble epoxide hydrolase (sEH) on skin homeostasis. sEH; which converts fatty acid epoxides generated by cytochrome P450 enzymes to their corresponding diols, was largely restricted to the epidermis which was enriched in sEH-generated diols. Global deletion of the sEH increased levels of epoxides, including the linoleic acid-derived epoxide; 12,13-epoxyoctadecenoic acid (12,13-EpOME), and increased basal keratinocyte proliferation. sEH deletion (sEH-/- mice) resulted in thicker differentiated spinous and corneocyte layers compared to wild-type mice, a hyperkeratosis phenotype that was reproduced in wild-type mice treated with a sEH inhibitor. sEH deletion made the skin sensitive to inflammation and sEH-/- mice developed thicker imiquimod-induced psoriasis plaques than the control group and were more prone to inflammation triggered by mechanical stress with pronounced infiltration and activation of neutrophils as well as vascular leak and increased 12,13-EpOME and leukotriene (LT) B4 levels. Topical treatment of LTB4 antagonist after stripping successfully inhibited inflammation and neutrophil infiltration both in wild type and sEH-/- skin. While 12,13-EpoME had no effect on the trans-endothelial migration of neutrophils, like LTB4, it effectively induced neutrophil adhesion and activation. These observations indicate that while the increased accumulation of neutrophils in sEH-deficient skin could be attributed to the increase in LTB4 levels, both 12,13-EpOME and LTB4 contribute to neutrophil activation. Our observations identify a protective role of the sEH in the skin and should be taken into account when designing future clinical trials with sEH inhibitors.
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Epóxido Hidrolasas , Inflamación , Queratinocitos , Ácido Linoleico , Animales , Ratones , Proliferación Celular , Compuestos Epoxi , Queratinocitos/citología , Queratinocitos/enzimología , Leucotrieno B4 , Ácido Linoleico/metabolismoRESUMEN
Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E(2) is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG(2) and PGH(2) to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE(-/-) mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.
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Sistema Enzimático del Citocromo P-450/fisiología , Ácidos Grasos Insaturados/metabolismo , Macrófagos/enzimología , Monocitos/enzimología , Placa Aterosclerótica/enzimología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Apolipoproteínas E/fisiología , Células Cultivadas , Sistema Enzimático del Citocromo P-450/análisis , Sistema Enzimático del Citocromo P-450/química , Humanos , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , FagocitosisRESUMEN
The cytochrome P450 (CYP) soluble epoxide hydrolase (sEH) pathway generates a large number of biologically active epoxides and diols from a range of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs). While epoxides of arachidonic acid or epoxyeicosatrienoic acids are probably the best studied of these mediators, epoxides of linoleic acid as well as the fish oils; docosahexaenoic acid and eicosapentaenoic acid have also been attributed signaling actions. Cell and tissue levels of the PUFA epoxides are largely determined by the sEH and in many cases inflammation and chronic diseases, e.g., cardiovascular disease, diabetes and Alzheimer's disease, have been associated with increased sEH expression and the accelerated conversion of PUFA epoxides to their corresponding diols. In low concentrations, the diols act to influence stem and progenitor cells as well as brown adipose tissue but in high concentrations, they tend to have pro-inflammatory and cytotoxic effects that promote disease progression. This review outlines some of the actions to the PUFA epoxides and diols in physiology and pathophysiology as well as the beneficial effects associates with sEH inhibition.
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Epóxido Hidrolasas , Ácidos Grasos Omega-3 , Humanos , Epóxido Hidrolasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos Omega-3/metabolismo , Transducción de Señal , Compuestos Epoxi/metabolismoRESUMEN
Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms.