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1.
Artigo em Inglês | MEDLINE | ID: mdl-33444817

RESUMO

BACKGROUND & AIMS: Noroviruses (NoVs) are the leading cause of acute gastroenteritis worldwide and are associated with significant morbidity and mortality. Moreover, an asymptomatic carrier state can persist following acute infection, promoting NoV spread and evolution. Thus, defining immune correlates of NoV protection and persistence is needed to guide the development of future vaccines and limit viral spread. Whereas antibody responses following NoV infection or vaccination have been studied extensively, cellular immunity has received less attention. Data from the mouse NoV model suggest that T cells are critical for preventing persistence and achieving viral clearance, but little is known about NoV-specific T-cell immunity in humans, particularly at mucosal sites. METHODS: We screened peripheral blood mononuclear cells from 3 volunteers with an overlapping NoV peptide library. We then used HLA-peptide tetramers to track virus-specific CD8+ T cells in peripheral, lymphoid, and intestinal tissues. Tetramer+ cells were further characterized using markers for cellular trafficking, exhaustion, cytotoxicity, and proliferation. RESULTS: We defined 7 HLA-restricted immunodominant class I epitopes that were highly conserved across pandemic strains from genogroup II.4. NoV-specific CD8+ T cells with central, effector, or tissue-resident memory phenotypes were present at all sites and were especially abundant in the intestinal lamina propria. The properties and differentiation states of tetramer+ cells varied across donors and epitopes. CONCLUSIONS: Our findings are an important step toward defining the breadth, distribution, and properties of human NoV T-cell immunity. Moreover, the molecular tools we have developed can be used to evaluate future vaccines and engineer novel cellular therapeutics.

2.
Acta Pharmacol Sin ; 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495519

RESUMO

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases and increases mortality in type 2 diabetic patients. HHcy induces endoplasmic reticulum (ER) stress and oxidative stress to impair endothelial function. The glucagon-like peptide 1 (GLP-1) analog exendin-4 attenuates endothelial ER stress, but the detailed vasoprotective mechanism remains elusive. The present study investigated the beneficial effects of exendin-4 against HHcy-induced endothelial dysfunction. Exendin-4 pretreatment reversed homocysteine-induced impairment of endothelium-dependent relaxations in C57BL/6 mouse aortae ex vivo. Four weeks subcutaneous injection of exendin-4 restored the impaired endothelial function in both aortae and mesenteric arteries isolated from mice with diet-induced HHcy. Exendin-4 treatment lowered superoxide anion accumulation in the mouse aortae both ex vivo and in vivo. Exendin-4 decreased the expression of ER stress markers (e.g., ATF4, spliced XBP1, and phosphorylated eIF2α) in human umbilical vein endothelial cells (HUVECs), and this change was reversed by cotreatment with compound C (CC) (AMPK inhibitor). Exendin-4 induced phosphorylation of AMPK and endothelial nitric oxide synthase in HUVECs and arteries. Exendin-4 increased the expression of endoplasmic reticulum oxidoreductase (ERO1α), an important ER chaperone in endothelial cells, and this effect was mediated by AMPK activation. Experiments using siRNA-mediated knockdown or adenoviral overexpression revealed that ERO1α mediated the inhibitory effects of exendin-4 on ER stress and superoxide anion production, thus ameliorating HHcy-induced endothelial dysfunction. The present results demonstrate that exendin-4 reduces HHcy-induced ER stress and improves endothelial function through AMPK-dependent ERO1α upregulation in endothelial cells and arteries. AMPK activation promotes the protein folding machinery in endothelial cells to suppress ER stress.

3.
Int J Colorectal Dis ; 35(6): 1133-1139, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32291508

RESUMO

BACKGROUND: The aim of this study was to evaluate the efficacy of indocyanine green (ICG) fluorescence angiography with respect to the anastomotic leakage rate for patients undergoing colorectal operations. METHODS: This prospective cohort involved patients who underwent colorectal surgery between August 2018 and September 2019. ICG was injected after colonic transection. Vascular perfusion was observed by ICG fluorescence system before completing anastomosis. Data was compared with those by subjective visual evaluation. The primary outcome was anastomotic leakage rate within 30 days from surgery. RESULTS: A total of 131 patients were enrolled, of which ICG was injected in 63 of them. Demographic data were similar between the two groups. There were two (3.23%) and three (4.35%) anastomotic leaks in the ICG and non-ICG group respectively (p = 1.000). Change of resection plane occurred in one patient in the ICG group. There was no ICG related toxicity or adverse events. CONCLUSION: ICG fluorescent imaging is a feasible and safe tool to assess colonic vascularisation for patients undergoing colorectal surgery. However, it did not significantly lower the anastomotic leakage rate. ICG should not be routinely used in colorectal surgery before an available large scale randomised controlled trial to prove any clinical benefits.

4.
Br J Pharmacol ; 177(6): 1258-1277, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31347157

RESUMO

Resveratrol (trans-3,4',5-trihydroxystilbene) belongs to the family of natural phytoalexins. Resveratrol first came to our attention in 1992, following reports of the cardioprotective effects of red wine. Thereafter, resveratrol was shown to exert antioxidant, anti-inflammatory, anti-proliferative, and angio-regulatory effects against atherosclerosis, ischaemia, and cardiomyopathy. This article critically reviews the current findings on the molecular basis of resveratrol-mediated cardiovascular benefits, summarizing the broad effects of resveratrol on longevity regulation, energy metabolism, stress resistance, exercise mimetics, circadian clock, and microbiota composition. In addition, this article also provides an update, both preclinically and clinically, on resveratrol-induced cardiovascular protection and discusses the adverse and inconsistent effects of resveratrol reported in both preclinical and clinical studies. Although resveratrol has been claimed as a master anti-aging agent against several age-associated diseases, further detailed mechanistic investigation is still required to thoroughly unravel the therapeutic value of resveratrol against cardiovascular diseases at different stages of disease development. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.

5.
Cardiovasc Res ; 116(1): 226-236, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30785200

RESUMO

AIMS: Disturbed blood flow at arterial branches and curvatures modulates endothelial function and predisposes the region to endothelial inflammation and subsequent development of atherosclerotic lesions. Activation of the endothelial Toll-like receptors (TLRs), in particular TLR4, contributes to vascular inflammation. Therefore, we investigate whether TLR4 can sense disturbed flow (DF) to mediate the subsequent endothelial inflammation. METHODS AND RESULTS: En face staining of endothelium revealed that TLR4 expression, activation, and its downstream inflammatory markers were elevated in mouse aortic arch compared with thoracic aorta, which were absent in Tlr4mut mice. Similar results were observed in the partial carotid ligation model where TLR4 signalling was activated in response to ligation-induced flow disturbance in mouse carotid arteries, and such effect was attenuated in Tlr4mut mice. DF in vitro increased TLR4 expression and activation in human endothelial cells (ECs) and promoted monocyte-EC adhesion, which were inhibited in TLR4-knockdown ECs. Among endogenous TLR4 ligands examined as candidate mediators of DF-induced TLR4 activation, fibronectin containing the extra domain A (FN-EDA) expressed by ECs was increased by DF and was revealed to directly interact with and activate TLR4. CONCLUSION: Our findings demonstrate the indispensable role of TLR4 in DF-induced endothelial inflammation and pinpoint FN-EDA as the endogenous TLR4 activator in this scenario. This novel mechanism of vascular inflammation under DF condition may serve as a critical initiating step in atherogenesis.


Assuntos
Aterosclerose/metabolismo , Artérias Carótidas/metabolismo , Células Endoteliais/metabolismo , Inflamação/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/fisiopatologia , Velocidade do Fluxo Sanguíneo , Artérias Carótidas/fisiopatologia , Artérias Carótidas/cirurgia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/fisiopatologia , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Inflamação/fisiopatologia , Ligantes , Ligadura , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/metabolismo , Estresse Mecânico , Receptor 4 Toll-Like/genética
6.
Immunity ; 51(5): 840-855.e5, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31606264

RESUMO

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing identified a TCF-1+Ly108+PD-1+ CD8 T cell population that seeds development of mature Tex cells early during chronic infection. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1Hi effectors while fostering KLRG1Lo Tex precursor cells, and PD-1 stabilized this TCF-1+ Tex precursor cell pool. TCF-1 mediated a T-bet-to-Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early-fate-bifurcation-driving Tex precursor cells and also identify PD-1 as a protector of this early TCF-1 subset.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Redes Reguladoras de Genes , Fator 1 de Transcrição de Linfócitos T/metabolismo , Transcrição Genética , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Doença Crônica , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Fator 1 de Transcrição de Linfócitos T/genética , Viroses/genética , Viroses/imunologia , Viroses/virologia
7.
Biochem Pharmacol ; 159: 11-24, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30414390

RESUMO

Augmented endothelium-dependent contractions (EDC) contributes to endothelial dysfunction and vascular disease progression. An early signal in EDC is cytosolic [Ca2+]i rise in endothelial cells, which stimulates the production of contractile prostanoids, leading to vascular contraction. In this study, the molecular identity of Ca2+-permeable channels in endothelial cells and its function were investigated. Vascular tension was measured by wire myograph. EDCs were elicited by acetylcholine (ACH) in the presence of NG-nitro-l-arginine methyl ester (L-NAME). [Ca2+]i was measured using a Ca2+-sensitive fluorescence dye. Enzyme Immunoassay (EIA) was used for prostaglandin measurement. Immunohistochemical staining found the expression of transient receptor potential channel C5 (TRPC5) in endothelial and smooth muscle cells of mouse carotid arteries. ACH-induced EDC in male mouse carotid arteries was found to be substantially reduced in TRPC5 knockout (KO) mice than in wild-type (WT) mice. TRPC5 inhibitors clemizole and ML204 also reduced the EDC. Furthermore, ACH-induced Ca2+ entry in endothelial cells was lower in TRPC5 KO mice than in WT mice. Moreover, the EDC was abolished by a cyclooxygenase-2 (COX-2) inhibitor NS-398, but not affected by a COX-1 inhibitor valeryl salicylate (VAS). Enzyme immunoassay results showed that TRPC5 stimulated the COX-2-linked production of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), and prostaglandin D2 (PGD2). Exogeneous PGF2α, PGE2, and PGD2 could induce contractions in carotid arteries. Our present study demonstrated that TRPC5 in endothelial cells contributes to EDC by stimulating the production of COX-2-linked prostanoids. The finding extends our knowledge about EDC.


Assuntos
Artérias Carótidas/fisiologia , Endotélio Vascular/fisiologia , Contração Muscular/fisiologia , Canais de Cátion TRPC/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Masculino , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Prostaglandinas/metabolismo , Canais de Cátion TRPC/genética
8.
Pharmacol Res ; 139: 384-394, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30503839

RESUMO

Sirtuin 1 (SIRT1) activation reduces oxidative stress, inhibits inflammatory responses, and retards cellular senescence in endothelial cells in mouse models of diabetes. However, whether SIRT1 also plays a protective role in vascular dysfunction of diabetic and obese mice is not fully characterized. Previous work showed that peroxisome proliferator-activated receptor δ (PPARδ) is beneficial in diabetic vascular dysfunction. Whether PPARδ is involved in the beneficial effect of SIRT1 on vascular endothelial function is unknown. We used mice with overexpression of endothelial cell-specific human SIRT1 (SIRT1-Tg) and dominant-negative SIRT1 (SIRT1-mut) fed with normal chow and high fat diet to show that expression of functional SIRT1 in endothelium protects against vascular dysfunction in diet-induced obese mice. Endothelial-specific overexpression of SIRT1 improved endothelium-dependent dilation in aortas treated with risk factors including high glucose, angiotensin II, and lysophosphatidylcholine. Oral treatment with resveratrol treatment improves endothelial function in high fat diet fed wild type Ppard-wt but not in PPARδ knockout Ppard-mut mice. Experiments on isolated arteries also showed that the effect of resveratrol or SIRT1 activator CAY10602 was inhibited by PPARδ antagonist GSK0660. Resveratrol increased PPARδ transcriptional activity in endothelial cells. Results demonstrated here indicated that PPARδ contributes to the beneficial effect of SIRT1 to ameliorate endothelial dysfunction in diabetic and obese mice. These results help to understand SIRT1-based strategy for treating vascular and metabolic dysfunction in the context of obesity and insulin resistance.


Assuntos
Endotélio Vascular/efeitos dos fármacos , PPAR delta/fisiologia , Resveratrol/farmacologia , Sirtuína 1/fisiologia , Animais , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/metabolismo
9.
Proc Natl Acad Sci U S A ; 115(29): E6927-E6936, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29967177

RESUMO

Exosomes, abundant in blood, deliver various molecules to recipient cells. Endothelial cells are directly exposed to circulating substances. However, how endothelial cells respond to serum exosomes (SExos) and the implications in diabetes-associated vasculopathy have never been explored. In the present study, we showed that SExos from diabetic db/db mice (db/db SExos) were taken up by aortic endothelial cells, which severely impaired endothelial function in nondiabetic db/m+ mice. The exosomal proteins, rather than RNAs, mostly account for db/db SExos-induced endothelial dysfunction. Comparative proteomics analysis showed significant increase of arginase 1 in db/db SExos. Silence or overexpression of arginase 1 confirmed its essential role in db/db SExos-induced endothelial dysfunction. This study is a demonstration that SExos deliver arginase 1 protein to endothelial cells, representing a cellular mechanism during development of diabetic endothelial dysfunction. The results expand the scope of blood-borne substances that monitor vascular homeostasis.


Assuntos
Aorta/metabolismo , Arginase/farmacologia , Angiopatias Diabéticas , Endotélio Vascular/metabolismo , Exossomos , Animais , Aorta/patologia , Angiopatias Diabéticas/tratamento farmacológico , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/patologia , Endotélio Vascular/patologia , Camundongos
10.
Antioxid Redox Signal ; 28(5): 358-370, 2018 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28683566

RESUMO

AIMS: Inhibition of microRNA-92a (miR-92a) is reported to suppress endothelial inflammation and delay atherogenesis. We hypothesize that miR-92a inhibition protects endothelial function through suppressing oxidative stress in diabetic db/db mice. RESULTS: In this study, we found elevated expression of miR-92a in aortic endothelium from db/db mice and in renal arteries from diabetic subjects. Endothelial cells (ECs) exposed to advanced glycation end products (AGEs) and oxidized low-density lipoprotein express higher level of miR-92a. Overexpression of miR-92a impairs endothelium-dependent relaxations (EDRs) in C57BL/6 mouse aortas. Overexpression of miR-92a suppresses expression of heme oxygenase-1 (HO-1), a critical cytoprotective enzyme, whereas inhibition of miR-92a increases HO-1 expression in human umbilical vein ECs (HUVECs) and db/db mouse aortas. Importantly, miR-92a inhibition by Ad-anti-miR-92a improved EDRs and reduced reactive oxygen species (ROS) production in db/db mouse aortas. HO-1 inhibition by SnMP or HO-1 knockdown by shHO-1 reversed the suppressive effect of miR-92a inhibition on ROS production induced by AGE treatment in C57BL/6 mouse aortas. In addition, SnMP reversed miR-92a inhibition-induced improvement of EDRs in AGE-treated C57BL/6 mouse aortas and in db/db mouse aortas. INNOVATION: Expression of miR-92a is increased in diabetic aortic endothelium and inhibition of miR-92a exerts vasoprotective effect in diabetic mice through HO-1 upregulation in ECs. CONCLUSION: MiR-92a expression is elevated in diabetic ECs. MiR-92a overexpression impairs endothelial function and suppresses HO-1 expression in ECs. Inhibition of miR-92a attenuates oxidative stress and improves endothelial function through enhancing HO-1 expression and activity in db/db mouse aortas. Antioxid. Redox Signal. 28, 358-370.


Assuntos
Aterosclerose/genética , Heme Oxigenase-1/genética , MicroRNAs/genética , Estresse Oxidativo/genética , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Regulação da Expressão Gênica/genética , Produtos Finais de Glicação Avançada/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/metabolismo , MicroRNAs/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo
11.
Immunity ; 47(4): 723-738.e5, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29031786

RESUMO

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Caliciviridae/imunologia , Diferenciação Celular/imunologia , Gastroenterite/imunologia , Norovirus/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/virologia , Diferenciação Celular/genética , Linhagem Celular , Microambiente Celular/genética , Microambiente Celular/imunologia , Gastroenterite/genética , Gastroenterite/virologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Camundongos Endogâmicos C57BL , Norovirus/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos
12.
Biochem Pharmacol ; 136: 76-85, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28396195

RESUMO

Salvianolic acid B (Sal B) is one of the most abundant phenolic acids derived from the root of Danshen with potent anti-oxidative properties. The present study examined the vasoprotective effect of Sal B in hypertensive mice induced by angiotensin II (Ang II). Sal B (25mg/kg/day) was administered via oral gavage for 11days to Ang II (1.2mg/kg/day)-infused C57BL/6J mice (8-10weeks old). The vascular reactivity (both endothelium-dependent relaxations and contractions) in mouse arteries was examined by wire myography. The production of reactive oxygen species (ROS), protein level and localization of angiotensin AT1 receptors and the proteins involved in ROS formation were evaluated using dihydroethidium (DHE) fluorescence, lucigenin-enhanced chemiluminescence, immunohistochemistry and Western blotting, respectively. The changes of ROS generating proteins were also assessed in vitro in human umbilical vein endothelial cells (HUVECs) exposed to Ang II with and without co-treatment with Sal B (0.1-10nM). Oral administration of Sal B reversed the Ang II-induced elevation of arterial systolic blood pressure in mice, augmented the impaired endothelium-dependent relaxations and attenuated the exaggerated endothelium-dependent contractions in both aortas and renal arteries of Ang II-infused mice. In addition, Sal B treatment normalized the elevated levels of AT1 receptors, NADPH oxidase subunits (NOx-2 and NOx-4) and nitrotyrosine in arteries of Ang II-infused mice or in Ang II-treated HUVECs. In summary, the present study provided additional evidence demonstrating that Sal B treatment for 11days reverses the impaired endothelial function and with a marked inhibition of AT1 receptor-dependent vascular oxidative stress. This vasoprotective and anti-oxidative action of Sal B most likely contributes to the anti-hypertensive action of the plant-derived compound.


Assuntos
Angiotensina II/toxicidade , Benzofuranos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Endotélio Vascular/fisiologia , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Animais , Benzofuranos/farmacologia , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Resultado do Tratamento
13.
Br J Pharmacol ; 174(8): 718-733, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28138957

RESUMO

BACKGROUND AND PURPOSE: Raloxifene can induce both endothelium-dependent and -independent relaxation in different arteries. However, the underlying mechanisms by which raloxifene triggers endothelium-independent relaxation are still incompletely understood. The purpose of present study was to examine the roles of NOSs and Ca2+ channels in the relaxant response to raloxifene in the rat isolated, endothelium-denuded aorta. EXPERIMENTAL APPROACH: Changes in isometric tension, cGMP, nitrite, inducible NOS protein expression and distribution in response to raloxifene in endothelium-denuded aortic rings were studied by organ baths, radioimmunoassay, Griess reaction, western blot and immunohistochemistry respectively. KEY RESULTS: Raloxifene reduced the contraction to CaCl2 in a Ca2+ -free, high K+ -containing solution in intact aortic rings. Raloxifene also acutely relaxed the aorta primarily through an endothelium-independent mechanism involving NO, mostly from inducible NOS (iNOS) in vascular smooth muscle layers. This effect of raloxifene involved the generation of cGMP and nitrite. Also, it was genomic in nature, as it was inhibited by a classical oestrogen receptor antagonist and inhibitors of RNA and protein synthesis. Raloxifene-induced stimulation of iNOS gene expression was partly mediated through activation of the NF-κB pathway. Raloxifene was more potent than 17ß-estradiol or tamoxifen at relaxing endothelium-denuded aortic rings by stimulation of iNOS. CONCLUSIONS AND IMPLICATIONS: Raloxifene-mediated vasorelaxation in rat aorta is independent of a functional endothelium and is mediated by oestrogen receptors and NF-κB. This effect is mainly mediated through an enhanced production of NO, cGMP and nitrite, via the induction of iNOS and inhibition of calcium influx through Ca2+ channels in rat aortic smooth muscle.


Assuntos
Aorta/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Cloridrato de Raloxifeno/farmacologia , Animais , Aorta/metabolismo , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Endotélio/metabolismo , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
14.
Diabetes ; 66(2): 519-528, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27856609

RESUMO

Physical activity has profound benefits on health, especially on cardiometabolic wellness. Experiments in rodents with trained exercise have shown that exercise improves vascular function and reduces vascular inflammation by modulating the balance between nitric oxide (NO) and oxidative stress. However, the upstream regulator of exercise-induced vascular benefits is unclear. We aimed to investigate the involvement of peroxisome proliferator-activated receptor δ (PPARδ) in exercise-induced vascular functional improvement. We show that PPARδ is a crucial mediator for exercise to exert a beneficial effect on the vascular endothelium in diabetic mice. In db/db mice and high-fat diet-induced obese mice, 4 weeks of treadmill exercise restored endothelium-dependent vasodilation of aortas and flow-mediated vasodilation in mesenteric resistance arteries, whereas genetic ablation of Ppard abolished such improvements. Exercise induces AMPK activation and subsequent PPARδ activation, which help to reduce endoplasmic reticulum (ER) and oxidative stress, thus increasing NO bioavailability in endothelial cells and vascular tissues. Chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid decrease ER stress and protect against endothelial dysfunction in diabetic mice. The results demonstrate that PPARδ-mediated inhibition of ER stress contributes to the vascular benefits of exercise and provides potentially effective targets for treating diabetic vasculopathy.


Assuntos
Diabetes Mellitus/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Estresse do Retículo Endoplasmático/genética , Endotélio Vascular/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Condicionamento Físico Animal , Receptores Citoplasmáticos e Nucleares/genética , Vasodilatação/genética , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Miografia , Óxido Nítrico , Obesidade/fisiopatologia , Técnicas de Cultura de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Fenilbutiratos/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Vasodilatação/efeitos dos fármacos
15.
Nature ; 540(7634): 579-582, 2016 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-27926730

RESUMO

The Yorkie homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1), effectors of the Hippo pathway, have been identified as mediators for mechanical stimuli. However, the role of YAP/TAZ in haemodynamics-induced mechanotransduction and pathogenesis of atherosclerosis remains unclear. Here we show that endothelial YAP/TAZ activity is regulated by different patterns of blood flow, and YAP/TAZ inhibition suppresses inflammation and retards atherogenesis. Atheroprone-disturbed flow increases whereas atheroprotective unidirectional shear stress inhibits YAP/TAZ activity. Unidirectional shear stress activates integrin and promotes integrin-Gα13 interaction, leading to RhoA inhibition and YAP phosphorylation and suppression. YAP/TAZ inhibition suppresses JNK signalling and downregulates pro-inflammatory genes expression, thereby reducing monocyte attachment and infiltration. In vivo endothelial-specific YAP overexpression exacerbates, while CRISPR/Cas9-mediated Yap knockdown in endothelium retards, plaque formation in ApoE-/- mice. We also show several existing anti-atherosclerotic agents such as statins inhibit YAP/TAZ transactivation. On the other hand, simvastatin fails to suppress constitutively active YAP/TAZ-induced pro-inflammatory gene expression in endothelial cells, indicating that YAP/TAZ inhibition could contribute to the anti-inflammatory effect of simvastatin. Furthermore, activation of integrin by oral administration of MnCl2 reduces plaque formation. Taken together, our results indicate that integrin-Gα13-RhoA-YAP pathway holds promise as a novel drug target against atherosclerosis.

16.
Chin Med ; 11(1): 38, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27582785

RESUMO

BACKGROUND: Hypertension can be treated effectively by acupuncture; however, the association between acupuncture and endothelial function remains unknown. This study aimed to investigate the effects of acupuncture on endothelial dysfunction and oxidative stress-related parameters in spontaneously hypertensive animals. METHODS: Eighteen-week-old Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were arbitrarily divided into four groups: WKY control (n = 8), SHR control (n = 8), SHR sham-acupuncture (n = 8) and SHR acupuncture (n = 8). The SHR acupuncture group had electroacupuncture for 6 consecutive weeks on acupoints ST36 and LR3. Blood pressure was monitored during the treatment period, and animals were euthanized at the 6th week. Aortas were harvested for determination of angiotensin II levels, NADPH oxidase activity and nitrate/nitrite levels. The level of reactive oxygen species (ROS) was determined by dihydroethidium (DHE) imaging, and functional studies were performed to assess vascular reactivity. Endothelial nitric oxide synthase was measured by Western blot assay. RESULTS: Blood pressure at the end of treatment was significantly lower in the SHR acupuncture group (185.0 ± 5.6 mmHg) compared with the SHR sham-acupuncture and the SHR control groups (201.0 ± 5.4 and 197.4 ± 5.9 mmHg, respectively; P < 0.001). Serum angiotensin II level in the SHR control group was significantly higher than in the WKY control group (P < 0.001), while it was significantly attenuated by acupuncture treatment (P = 0.023). DHE staining showed that ROS level was reduced in the aortas (P = 0.0017) and carotid arteries (P = 0.039) of acupuncture-treated SHRs. Biochemical assays showed that acupuncture inhibited the NADPH oxidase activity (P = 0.022) and enhanced antioxidant capacity (P = 0.0039). In functional studies, endothelium-dependent relaxation of aortic rings (P = 0.018) and carotid arteries (P = 0.022) in response to acetylcholine was improved in the SHR acupuncture group. Aortas of SHRs receiving acupuncture also expressed an elevated level of eNOS (P > 0.001) and p-eNOS (P = 0.012) and a reduced nitrotyrosine level (P = 0.0012). The nitrate/nitrite level in aortic tissue was also attenuated after acupuncture (P = 0.0018). CONCLUSION: The effects of acupuncture in treating hypertension were associated with reduced oxidative stress, increased nitric oxide bioavailability and endothelial function in SHRs.

17.
Biochem Pharmacol ; 116: 51-62, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27449753

RESUMO

Endoplasmic reticulum (ER) stress in endothelial cells often leads to endothelial dysfunction which underlies the pathogenesis of cardiovascular diseases. Paeonol, a major phenolic component extracted from Moutan Cortex, possesses various medicinal benefits which have been used extensively in traditional Chinese medicine. The present study investigated the protective mechanism of paeonol against tunicamycin-induced ER stress in isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). Vascular reactivity in aorta was measured using a wire myograph. The effects of paeonol on protein expression of ER stress markers, reactive oxygen species (ROS) production, nitric oxide (NO) bioavailability and peroxisome proliferator-activated receptor δ (PPARδ) activity in the vascular wall were assessed by Western blot, dihydroethidium fluorescence (DHE) or lucigenin enhanced-chemiluminescence, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA) and dual luciferase reporter assay, respectively. Ex vivo treatment with paeonol (0.1µM) for 16h reversed the impaired endothelium-dependent relaxations in C57BJ/6J and PPARδ wild type (WT) mouse aortas following incubation with tunicamycin (0.5µg/mL). Elevated ER stress markers, oxidative stress and reduction of NO bioavailability induced by tunicamycin in HUVECs, C57BJ/6J and PPARδ WT mouse aortas were reversed by paeonol treatment. These beneficial effects of paeonol were diminished in PPARδ knockout (KO) mouse aortas. Paeonol increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and PPARδ expression and activity while restoring the decreased phosphorylation of eNOS. The present study delineates that paeonol protects against tunicamycin-induced vascular endothelial dysfunction by inhibition of ER stress and oxidative stress, thus elevating NO bioavailability via the AMPK/PPARδ signaling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetofenonas/farmacologia , Antioxidantes/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , PPAR delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Animais , Aorta Torácica , Linhagem Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/agonistas , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , PPAR delta/agonistas , PPAR delta/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnicas de Cultura de Tecidos
18.
Sci Rep ; 6: 28041, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27324576

RESUMO

Melamine incident, linked to nephrotoxicity and kidney stone in infants previously exposed to melamine-contaminated milk products, was unprecedentedly grave in China in 2008 as little was known about the mechanistic process leading to renal dysfunction in affected children. This study investigates whether neonatal ingestion of melamine leads to renal and vascular dysfunction in adulthood; and whether ingestion of melamine in pregnant rats leads to renal dysfunction in their offspring. A combination of approaches employed includes functional studies in rat renal arteries, renal blood flow measurement by functional magnetic resonance imaging, assay for pro-inflammatory and fibrotic biomarkers, immunohistochemistry, and detection of plasma and renal melamine. We provide mechanistic evidence showing for the first time that melamine reduces renal blood flow and impairs renal and vascular function associated with overexpression of inflammatory markers, transforming growth factor-ß1, bone morphogenic protein 4 and cyclooxygenase-2 in kidney and renal vasculature. Melamine also induces renal inflammation and fibrosis. More importantly, melamine causes nephropathies in offsprings from pregnant rat exposed to melamine during pregnancy, as well as in neonatal rat exposed to melamine afterbirth, thus supporting the clinical observations of kidney stone and acute renal failure in infants consuming melamine-contaminated milk products.


Assuntos
Rim/efeitos dos fármacos , Artéria Renal/efeitos dos fármacos , Triazinas/toxicidade , Acetilcolina/farmacologia , Lesão Renal Aguda/etiologia , Animais , Biomarcadores/metabolismo , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Proteína Morfogenética Óssea 4/metabolismo , Ciclo-Oxigenase 2/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/fisiologia , Feminino , Contaminação de Alimentos/análise , Rim/diagnóstico por imagem , Rim/metabolismo , Cálculos Renais/etiologia , Exposição Materna , Gravidez , Ratos , Ratos Sprague-Dawley , Artéria Renal/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Triazinas/sangue
20.
Sci Rep ; 6: 18697, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26739766

RESUMO

Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by N(ω)-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension.


Assuntos
Arginina/metabolismo , Pressão Sanguínea , Endotélio Vascular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Vasodilatação , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Diuréticos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Deleção de Genes , Expressão Gênica , Espaço Intracelular/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Modelos Animais , Ratos , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatação/genética
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