RESUMO
Dasatinib is an inhibitor of Src that has anti-tumour effects on many haematological and solid cancers. However, the anti-tumour effects of dasatinib on human oral cancers remain unclear. In this study, we investigated the effects of dasatinib on different types of human oral cancer cells: the non-tumorigenic YD-8 and YD-38 and the tumorigenic YD-10B and HSC-3 cells. Strikingly, dasatinib at 10 µM strongly suppressed the growth and induced apoptosis of YD-38 cells and inhibited the phosphorylation of Src, EGFR, STAT-3, STAT-5, PKB and ERK-1/2. In contrast, knockdown of Src blocked the phosphorylation of EGFR, STAT-5, PKB and ERK-1/2, but not STAT-3, in YD-38 cells. Dasatinib induced activation of the intrinsic caspase pathway, which was inhibited by z-VAD-fmk, a pan-caspase inhibitor. Dasatinib also decreased Mcl-1 expression and S6 phosphorylation while increased GRP78 expression and eIF-2α phosphorylation in YD-38 cells. In addition, to its direct effects on YD-38 cells, dasatinib also exhibited anti-angiogenic properties. Dasatinib-treated YD-38 or HUVEC showed reduced HIF-1α expression and stability. Dasatinib alone or conditioned media from dasatinib-treated YD-38 cells inhibited HUVEC tube formation on Matrigel without affecting HUVEC viability. Importantly, dasatinib's anti-growth, anti-angiogenic and pro-apoptotic effects were additionally seen in tumorigenic HSC-3 cells. Together, these results demonstrate that dasatinib has strong anti-growth, anti-angiogenic and pro-apoptotic effects on human oral cancer cells, which are mediated through the regulation of multiple targets, including Src, EGFR, STAT-3, STAT-5, PKB, ERK-1/2, S6, eIF-2α, GRP78, caspase-9/3, Mcl-1 and HIF-1α.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Dasatinibe/farmacologia , Neoplasias Bucais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , HumanosRESUMO
A number of oxylipins have been described as endogenous PPAR ligands. The very short biological half-lives of oxylipins suggest roles as autocrine or paracrine signaling molecules. While coronary arterial atherosclerosis is the root of myocardial infarction, aortic atherosclerotic plaque formation is a common readout of in vivo atherosclerosis studies in mice. Improved understanding of the compartmentalized sources of oxylipin PPAR ligands will increase our knowledge of the roles of PPAR signaling in diverse vascular tissues. Here, we performed a targeted lipidomic analysis of ex vivo-generated oxylipins from porcine aorta, coronary artery, pulmonary artery and perivascular adipose. Cyclooxygenase (COX)-derived prostanoids were the most abundant detectable oxylipin from all tissues. By contrast, the coronary artery produced significantly higher levels of oxylipins from CYP450 pathways than other tissues. The TLR4 ligand LPS induced prostanoid formation in all vascular tissue tested. The 11-HETE, 15-HETE, and 9-HODE were also induced by LPS from the aorta and pulmonary artery but not coronary artery. Epoxy fatty acid (EpFA) formation was largely unaffected by LPS. The pig CYP2J homologue CYP2J34 was expressed in porcine vascular tissue and primary coronary artery smooth muscle cells (pCASMCs) in culture. Treatment of pCASMCs with LPS induced a robust profile of pro-inflammatory target genes: TNFα, ICAM-1, VCAM-1, MCP-1 and CD40L. The soluble epoxide hydrolase inhibitor TPPU, which prevents the breakdown of endogenous CYP-derived EpFAs, significantly suppressed LPS-induced inflammatory target genes. In conclusion, PPAR-activating oxylipins are produced and regulated in a vascular site-specific manner. The CYP450 pathway is highly active in the coronary artery and capable of providing anti-inflammatory oxylipins that prevent processes of inflammatory vascular disease progression.
Assuntos
Vasos Coronários/efeitos dos fármacos , Ácidos Graxos/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Vasos Coronários/metabolismo , Feminino , Inflamação/induzido quimicamente , Inflamação/metabolismo , Ligantes , Lipidômica/métodos , Lipopolissacarídeos/farmacologia , Miócitos de Músculo Liso/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , SuínosRESUMO
The sequencing of the Crassostrea virginica genome has brought back the interest for gene delivery and editing methodologies. Here, we report the expression in oyster hemocytes of two heterologous expression vectors under the CMV promoter delivered with dendrimers. Expression was monitored using confocal microscopy, flow cytometry, and immunofluorescence assay. C. virginica hemocytes were able to express the green fluorescence protein and Crassostrea gigas vascular endothelial growth factor under CMV viral promoter both in vivo and in vitro. These results provide the bases for interrogating the genome and adapting genome editing methodologies.
Assuntos
Crassostrea/genética , Genômica/métodos , Hemócitos/metabolismo , Fenômica/métodos , Transfecção/métodos , Animais , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Microscopia Confocal , Transfecção/estatística & dados numéricosRESUMO
Meridianin C is a marine natural product with anticancer activity. Several meridianin C derivatives (compounds 7aj) were recently synthesized, and their inhibitory effects on proviral integration site for Moloney murine leukemia virus (PIM) kinases, as well as their antiproliferative effects on human leukemia cells, were reported. However, the antileukemic effects and mechanisms of action of meridianin C and its derivatives remain largely unknown. The aim of the present study was to investigate the effects of meridianin C and its derivatives on MV411 human acute myeloid leukemia cell growth. The parent compound meridianin C did not markedly affect the viability and survival of MV411 cells. By contrast, MV411 cell viability and survival were reduced by meridianin C derivatives, with compound 7a achieving the most prominent reduction. Compound 7a notably inhibited the expression and activity of PIM kinases, as evidenced by reduced Bcell lymphoma2 (Bcl2)associated death promoter phosphorylation at Ser112. However, meridianin C also suppressed PIM kinase expression and activity, and the panPIM kinase inhibitor AZD1208 only slightly suppressed the survival of MV411 cells. Thus, the antisurvival effect of compound 7a on MV411 cells was unrelated to PIM kinase inhibition. Moreover, compound 7a induced apoptosis, caspase9 and 3 activation and poly(ADPribose) polymerase (PARP) cleavage, but did not affect death receptor (DR)4 or DR5 expression in MV411 cells. Compound 7a also induced the generation of cleaved Bcl2, and the downregulation of myeloid cell leukemia (Mcl)1 and Xlinked inhibitor of apoptosis (XIAP) in MV411 cells. Furthermore, compound 7a increased eukaryotic initiation factor (eIF)2α phosphorylation and decreased S6 phosphorylation, whereas GRP78 expression was unaffected. Importantly, treatment with a pancaspase inhibitor (zVADfmk) significantly attenuated compound 7ainduced apoptosis, caspase9 and 3 activation, PARP cleavage, generation of cleaved Bcl2 and downregulation of Mcl1 and XIAP in MV411 cells. Collectively, these findings demonstrated the strong antisurvival and proapoptotic effects of compound 7a on MV411 cells through regulation of caspase9 and 3, Bcl2, Mcl1, XIAP, eIF2α and S6 molecules.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proliferação de Células , Indóis/química , Indóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , Proteínas Reguladoras de Apoptose/genética , Caspase 9/genética , Caspase 9/metabolismo , Chaperona BiP do Retículo Endoplasmático , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fosforilação , Inibidores de Proteínas Quinases/química , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
We are all too familiar with the events that follow a bee sting-heat, redness, swelling, and pain. These are Celsus' four cardinal signs of inflammation that are driven by very well-defined signals and hormones. In fact, targeting the factors that drive this onset phase is the basis upon which most current anti-inflammatory therapies were developed. We are also very well aware that within a few hours, these cardinal signs normally disappear. In other words, inflammation resolves. When it does not, inflammation persists, resulting in damaging chronic conditions. While inflammatory onset is actively driven, so also is its resolution-years of research have identified novel internal counter-regulatory signals that work together to switch off inflammation. Among these signals, lipids are potent signalling molecules that regulate an array of immune responses including vascular hyper reactivity and pain, as well as leukocyte trafficking and clearance, so-called resolution. Here, we collate bioactive lipid research to date and summarize the major pathways involved in their biosynthesis and their role in inflammation, as well as resolution. LINKED ARTICLES: This article is part of a themed section on Eicosanoids 35 years from the 1982 Nobel: where are we now? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.8/issuetoc.
Assuntos
Lipídeos/imunologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Lipídeos/biossíntese , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell differentiation, the ability to fight opportunistic and specific pathogens in the absence of adaptive immunity, as source of alternative drugs, mucosal immunity and, microbiome turnover, toxicology, and cancer research. There is still a long way to go; however, the next time you order a dozen oysters at your favorite raw bar, think about a tasty model organism that will not only please your palate but also help unlock multiple aspects of molluscan biology and improve human health.
Assuntos
Exoesqueleto/fisiologia , Bivalves/imunologia , Microbiota/imunologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Humanos , Imunidade Inata , Modelos Animais , Alimentos MarinhosRESUMO
Meridianin C is a marine natural product known for its anti-cancer activity. At present, the anti-tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD-8, YD-10B, YD-38 and HSC-3. Among the cells tested, meridianin C most strongly reduced the growth of YD-10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD-10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC-dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD-10B cells treated with meridianin C, pointing out that meridianin C-induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf-related protein-3 (DKK-3), a known negative regulator of macropinocytosis. A role for DKK-3 in regulating macropinocytosis in the YD-10B cells was confirmed by siRNA knockdown of endogenous DKK-3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK-3 overexpression, which resulted in a considerable inhibition of the meridianin C-induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti-proliferative effects via macropinocytosis in the highly tumorigenic YD-10B cell line and the effects are mediated in part through down-regulation of DKK-3.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Alcaloides Indólicos/farmacologia , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pinocitose/efeitos dos fármacos , Pirimidinas/farmacologia , Neoplasias da Língua/metabolismo , Neoplasias da Língua/patologia , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocinas , Humanos , Alcaloides Indólicos/química , Indóis/química , Pirimidinas/química , Neoplasias da Língua/ultraestrutura , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
OBJECTIVE: When molecular drivers of healthy adipogenesis are perturbed, this can cause hepatic steatosis. The role of arachidonic acid (AA) and its downstream enzymatic cascades, such as cyclooxygenase, in adipogenesis is well established. The exact contribution of the P450 epoxygenase pathway, however, remains to be established. Enzymes belonging to this pathway are mainly encoded by the CYP2J locus which shows extensive allelic expansion in mice. Here we aimed to establish the role of endogenous epoxygenase during adipogenesis under homeostatic and metabolic stress conditions. METHODS: We took advantage of the simpler genetic architecture of the Cyp2j locus in the rat and used a Cyp2j4 (orthologue of human CYP2J2) knockout rat in two models of metabolic dysfunction: physiological aging and cafeteria diet (CAF). The phenotyping of Cyp2j4-/- rats under CAF was integrated with proteomics (LC-MS/MS) and lipidomics (LC-MS) analyses in the liver and the adipose tissue. RESULTS: We report that Cyp2j4 deletion causes adipocyte dysfunction under metabolic challenges. This is characterized by (i) down-regulation of white adipose tissue (WAT) PPARγ and C/EBPα, (ii) adipocyte hypertrophy, (iii) extracellular matrix remodeling, and (iv) alternative usage of AA pathway. Specifically, in Cyp2j4-/- rats treated with a cafeteria diet, the dysfunctional adipogenesis is accompanied by exacerbated weight gain, hepatic lipid accumulation, and dysregulated gluconeogenesis. CONCLUSION: These results suggest that AA epoxygenases are essential regulators of healthy adipogenesis. Our results uncover their synergistic role in fine-tuning AA pathway in obesity-mediated hepatic steatosis.
Assuntos
Adipogenia , Envelhecimento/metabolismo , Família 2 do Citocromo P450/metabolismo , Dieta da Carga de Carboidratos/efeitos adversos , Gluconeogênese/efeitos dos fármacos , Obesidade/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Ácido Araquidônico/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Família 2 do Citocromo P450/genética , Dieta Hiperlipídica/efeitos adversos , Matriz Extracelular/metabolismo , Metabolismo dos Lipídeos , Masculino , Obesidade/etiologia , PPAR gama/metabolismo , Ratos , Ratos WistarRESUMO
OBJECTIVE: Although initially seemingly paradoxical because of the lack of nucleus, platelets possess many transcription factors that regulate their function through DNA-independent mechanisms. These include the farnesoid X receptor (FXR), a member of the superfamily of ligand-activated transcription factors, that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6α-ethyl-chenodeoxycholic acid, modulate platelet activation nongenomically. APPROACH AND RESULTS: FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization, secretion, fibrinogen binding, and aggregation. Exposure to FXR ligands also reduced integrin αIIbß3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cyclic guanosine monophosphate levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the nongenomic actions of these ligands to the FXR. CONCLUSIONS: This study provides support for the ability of FXR ligands to modulate platelet activation. The atheroprotective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for the prevention of atherothrombotic disease.
Assuntos
Plaquetas/efeitos dos fármacos , Ácido Quenodesoxicólico/análogos & derivados , Hemostasia/efeitos dos fármacos , Isoxazóis/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Trombose/prevenção & controle , Animais , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ácido Quenodesoxicólico/farmacologia , GMP Cíclico/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Genótipo , Humanos , Ligantes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores Citoplasmáticos e Nucleares/sangue , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Trombose/sangue , Fatores de TempoRESUMO
Resolution of inflammation has emerged as an active process in immunobiology, with cells of the mononuclear phagocyte system being critical in mediating efferocytosis and wound debridement and bridging the gap between innate and adaptive immunity. Here we investigated the roles of cytochrome P450 (CYP)-derived epoxy-oxylipins in a well-characterized model of sterile resolving peritonitis in the mouse. Epoxy-oxylipins were produced in a biphasic manner during the peaks of acute (4 h) and resolution phases (24-48 h) of the response. The epoxygenase inhibitor SKF525A (epoxI) given at 24 h selectively inhibited arachidonic acid- and linoleic acid-derived CYP450-epoxy-oxlipins and resulted in a dramatic influx in monocytes. The epoxI-recruited monocytes were strongly GR1(+), Ly6c(hi), CCR2(hi), CCL2(hi), and CX3CR1(lo) In addition, expression of F4/80 and the recruitment of T cells, B cells, and dendritic cells were suppressed. sEH (Ephx2)(-/-) mice, which have elevated epoxy-oxylipins, demonstrated opposing effects to epoxI-treated mice: reduced Ly6c(hi) monocytes and elevated F4/80(hi) macrophages and B, T, and dendritic cells. Ly6c(hi) and Ly6c(lo) monocytes, resident macrophages, and recruited dendritic cells all showed a dramatic change in their resolution signature following in vivo epoxI treatment. Markers of macrophage differentiation CD11b, MerTK, and CD103 were reduced, and monocyte-derived macrophages and resident macrophages ex vivo showed greatly impaired phagocytosis of zymosan and efferocytosis of apoptotic thymocytes following epoxI treatment. These findings demonstrate that epoxy-oxylipins have a critical role in monocyte lineage recruitment and activity to promote inflammatory resolution and represent a previously unidentified internal regulatory system governing the establishment of adaptive immunity.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Monócitos/metabolismo , Oxilipinas/metabolismo , Peritonite/metabolismo , Animais , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , FagocitoseRESUMO
Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.
Assuntos
Antígenos de Plaquetas Humanas/genética , Plaquetas/enzimologia , Dinoprostona/genética , Células Endoteliais/enzimologia , Epoprostenol/genética , Leucócitos/enzimologia , Mutação , Adulto , Plaquetas/patologia , Dinoprostona/biossíntese , Células Endoteliais/patologia , Epoprostenol/biossíntese , Feminino , Humanos , Leucócitos/patologia , Masculino , Ativação Plaquetária/genéticaRESUMO
Vascular pathologies are associated with changes in the presence and expression of morphologically distinct vascular smooth muscle cells. In particular, in complex human vascular lesions and models of disease in pigs and rodents, an intimal smooth muscle cell (iSMC) which exhibits a stable epithelioid or rhomboid phenotype in culture is often found to be present in high numbers, and may represent the reemergence of a distinct developmental vascular smooth muscle cell phenotype. The CYP450-oxylipin - soluble epoxide hydrolase (sEH) pathway is currently of great interest in targeting for cardiovascular disease. sEH inhibitors limit the development of hypertension, diabetes, atherosclerosis and aneurysm formation in animal models. We have investigated the expression of CYP450-oxylipin-sEH pathway enzymes and their metabolites in paired intimal (iSMC) and medial (mSMC) cells isolated from rat aorta. iSMC basally released significantly larger amounts of epoxy-oxylipin CYP450 products from eicosapentaenoic acid > docosahexaenoic acid > arachidonic acid > linoleic acid, and expressed higher levels of CYP2C12, CYP2B1, but not CYP2J mRNA compared to mSMC. When stimulated with the pro-inflammatory TLR4 ligand LPS, epoxy-oxylipin production did not change greatly in iSMC. In contrast, LPS induced epoxy-oxylipin products in mSMC and induced CYP2J4. iSMC and mSMC express sEH which metabolizes primary epoxy-oxylipins to their dihydroxy-counterparts. The sEH inhibitors TPPU or AUDA inhibited LPS-induced NFκB activation and iNOS induction in mSMC, but had no effect on NFκB nuclear localization or inducible nitric oxide synthase in iSMC; effects which were recapitulated in part by addition of authentic epoxy-oxylipins. iSMCs are a rich source but not a sensor of anti-inflammatory epoxy-oxylipins. Complex lesions that contain high levels of iSMCs may be more resistant to the protective effects of sEH inhibitors.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inflamação/prevenção & controle , Músculo Liso Vascular/citologia , Oxilipinas/metabolismo , Túnica Íntima/citologia , Animais , Linhagem Celular , Humanos , Músculo Liso Vascular/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Roedores , Suínos , Túnica Íntima/metabolismoRESUMO
Nuclear receptors sense a wide range of steroids and hormones (estrogens, progesterone, androgens, glucocorticoid, and mineralocorticoid), vitamins (A and D), lipid metabolites, carbohydrates, and xenobiotics. In response to these diverse but critically important mediators, nuclear receptors regulate the homeostatic control of lipids, carbohydrate, cholesterol, and xenobiotic drug metabolism, inflammation, cell differentiation and development, including vascular development. The nuclear receptor family is one of the most important groups of signaling molecules in the body and as such represent some of the most important established and emerging clinical and therapeutic targets. This review will highlight some of the recent trends in nuclear receptor biology related to vascular biology.
Assuntos
Aterosclerose/genética , Vasos Sanguíneos/metabolismo , Homeostase/genética , Receptores Citoplasmáticos e Nucleares/genética , Aterosclerose/metabolismo , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 µl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 µl/cm(2) in 24-well (10 µl) or 96-well (2 µl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.
Assuntos
Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Neovascularização Fisiológica , Células Cultivadas , Técnicas Citológicas/métodos , Proteínas da Matriz Extracelular , Humanos , MitocôndriasRESUMO
The cytochrome P450s (CYPs) represent a highly divergent class of enzymes involved in the oxidation of organic compounds. A subgroup of CYPs metabolize ω3-arachidonic and linoleic acids and ω6-docosahexaenoic and eicosapentaenoic polyunsaturated fatty acids (PUFAs) into a series of related biologically active mediators. Over the past 20 years, increasing evidence has emerged for a role of these PUFA-derived mediators in physiological and pathophysiological processes in the vasculature, during inflammation, and in the regulation of metabolism. With recent technological advances and increased availability of lipid mass spectroscopy, we are now starting to discern the patterns of these CYP-PUFA products in health and disease. These analyses not only are revealing the diverse spectrum of lipid nutrients regulated by CYPs, but also clearly indicate that the balance of these mediators changes with dietary intake of different PUFA classes. These findings suggest that we are only just beginning to understand all of the relevant lipid species produced by CYP pathways. Moreover, we are still a long way from understanding the nature and presence of their receptors, their tissue expression, and the pathophysiological processes they regulate. This review highlights these future issues in the context of lipid-metabolizing CYP enzymes, focusing particularly on the CYP450 family of epoxygenases and the lipid mediators they produce.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Homeostase , Metabolismo dos Lipídeos , Modelos Biológicos , Animais , Sistema Enzimático do Citocromo P-450/genética , Diabetes Mellitus/enzimologia , Diabetes Mellitus/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Obesidade/enzimologia , Obesidade/metabolismo , Especificidade de Órgãos , Especificidade da EspécieRESUMO
The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 µg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC-MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1ß, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1ß and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid>eicosapentaenoic acid > arachidonic acid>docosahexaenoic acid to products with anti-inflammatory activity.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Células Endoteliais/enzimologia , Inflamação/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Linhagem Celular , Citocromo P-450 CYP2J2 , Ativação Enzimática , HumanosRESUMO
The epoxygenase CYP2J2 has an emerging role in inflammation and vascular biology. The role of CYP2J2 in phagocytosis is not known and its regulation in human inflammatory diseases is poorly understood. Here we investigated the role of CYP2J2 in bacterial phagocytosis and its expression in monocytes from healthy controls and Crohns disease patients. CYP2J2 is anti-inflammatory in human peripheral blood monocytes. Bacterial LPS induced CYP2J2 mRNA and protein. The CYP2J2 arachidonic acid products 11,12-EET and 14,15-EET inhibited LPS induced TNFα release. THP-1 monocytes were transformed into macrophages by 48h incubation with phorbol 12-myristate 13-acetate. Epoxygenase inhibition using a non-selective inhibitor SKF525A or a selective CYP2J2 inhibitor Compound 4, inhibited E. coli particle phagocytosis, which could be specifically reversed by 11,12-EET. Moreover, epoxygenase inhibition reduced the expression of phagocytosis receptors CD11b and CD68. CD11b also mediates L. monocytogenes phagocytosis. Similar, to E. coli bioparticle phagocytosis, epoxygenase inhibition also reduced intracellular levels of L. monocytogenes, which could be reversed by co-incubation with 11,12-EET. Disrupted bacterial clearance is a hallmark of Crohn's disease. Unlike macrophages from control donors, macrophages from Crohn's disease patients showed no induction of CYP2J2 in response to E. coli. These results demonstrate that CYP2J2 mediates bacterial phagocytosis in macrophages, and implicates a defect in the CYP2J2 pathway may regulate bacterial clearance in Crohn's disease.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Doença de Crohn/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Escherichia coli/metabolismo , Macrófagos/enzimologia , Monócitos/enzimologia , Fagocitose , Ácido 8,11,14-Eicosatrienoico/metabolismo , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Linhagem Celular , Doença de Crohn/genética , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática/efeitos dos fármacos , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Monócitos/microbiologia , Monócitos/patologiaRESUMO
Cytochrome p450 (CYP)2J2 is an epoxygenase enzyme that metabolises arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are inactivated by soluble epoxide hydrolase (sEH), which converts them in to their corresponding dihydroxyeicosatrienoic acids (DHETs). CYP2J2 is highly expressed in cardiovascular tissue including the heart and vascular endothelial cells. CYP2J2 and the EETs it produces have been shown to have a diverse range of effects on the vasculature, including the regulation of inflammation, vascular tone, cellular proliferation, angiogenesis, and metabolism. This review will examine these established and emerging roles of CYP2J2 in the biology of vascular endothelial cells.