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1.
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 Gênica , 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
2.
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
3.
Proc Natl Acad Sci U S A ; 119(17): e2106083119, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35446623

RESUMO

CD8 T cells mediate protection against intracellular pathogens and tumors. However, persistent antigen during chronic infections or cancer leads to T cell exhaustion, suboptimal functionality, and reduced protective capacity. Despite considerable work interrogating the transcriptional regulation of exhausted CD8 T cells (TEX), the posttranscriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses during chronic viral infection and antagonized exhaustion. miR-29a inhibited exhaustion-driving transcriptional pathways, including inflammatory and T cell receptor signaling, and regulated ribosomal biogenesis. As a result, miR-29a fostered a memory-like CD8 T cell differentiation state during chronic infection. Thus, we identify miR-29a as a key regulator of TEX and define mechanisms by which miR-29a can divert exhaustion toward a more beneficial memory-like CD8 T cell differentiation state.


Assuntos
MicroRNAs , Neoplasias , Linfócitos T CD8-Positivos , Humanos , Imunoterapia/métodos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/metabolismo , Infecção Persistente
4.
J Lipid Res ; 65(1): 100472, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37949368

RESUMO

Liver steatosis is a common metabolic disorder resulting from imbalanced lipid metabolism, which involves various processes such as de novo lipogenesis, fatty acid uptake, fatty acid oxidation, and VLDL secretion. In this study, we discovered that KLF2, a transcription factor, plays a crucial role in regulating lipid metabolism in the liver. Overexpression of KLF2 in the liver of db/db mice, C57BL/6J mice, and Cd36-/- mice fed on a normal diet resulted in increased lipid content in the liver. Additionally, transgenic mice (ALB-Klf2) that overexpressed Klf2 in the liver developed liver steatosis after being fed a normal diet. We found that KLF2 promotes lipogenesis by increasing the expression of SCAP, a chaperone that facilitates the activation of SREBP, the master transcription factor for lipogenic gene expression. Our mechanism studies revealed that KLF2 enhances lipogenesis in the liver by binding to the promoter of SCAP and increasing the expression of genes involved in fatty acid synthesis. Reduction of KLF2 expression led to a decrease in SCAP expression and a reduction in the expression of SREBP1 target genes involved in lipogenesis. Overexpression of KLF2 also increased the activation of SREBP2 and the mRNA levels of its downstream target SOAT1. In C57BL/6J mice fed a high-fat diet, overexpression of Klf2 increased blood VLDL secretion, while reducing its expression decreased blood cholesterol levels. Our study emphasizes the novelty that hepatic KLF2 plays a critical role in regulating lipid metabolism through the KLF2/SCAP/SREBPs pathway, which is essential for hepatic lipogenesis and maintaining blood cholesterol homeostasis.


Assuntos
Fígado Gorduroso , Lipogênese , Camundongos , Animais , Lipogênese/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos/genética , Ácidos Graxos/metabolismo , Colesterol/metabolismo , Homeostase
5.
Circ Res ; 131(5): 424-441, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35899624

RESUMO

BACKGROUND: Inflamed endothelial cells (ECs) trigger atherogenesis, especially at arterial regions experiencing disturbed blood flow. UCP2 (Uncoupling protein 2), a key mitochondrial antioxidant protein, improves endothelium-dependent relaxation in obese mice. However, whether UCP2 can be regulated by shear flow is unknown, and the role of endothelial UCP2 in regulating inflammation and atherosclerosis remains unclear. This study aims to investigate the mechanoregulation of UCP2 expression in ECs and the effect of UCP2 on endothelial inflammation and atherogenesis. METHODS: In vitro shear stress simulation system was used to investigate the regulation of UCP2 expression by shear flow. EC-specific Ucp2 knockout mice were used to investigate the role of UCP2 in flow-associated atherosclerosis. RESULTS: Shear stress experiments showed that KLF2 (Krüppel-like factor 2) mediates fluid shear stress-dependent regulation of UCP2 expression in human aortic and human umbilical vein ECs. Unidirectional shear stress, statins, and resveratrol upregulate whereas oscillatory shear stress and proinflammatory stimuli inhibit UCP2 expression through altered KLF2 expression. KLF2 directly binds to UCP2 promoter to upregulate its transcription in human umbilical vein ECs. UCP2 knockdown induced expression of genes involved in proinflammatory and profibrotic signaling, resulting in a proatherogenic endothelial phenotype. EC-specific Ucp2 deletion promotes atherogenesis and collagen production. Additionally, we found endothelial Ucp2 deficiency aggravates whereas adeno-associated virus-mediated EC-Ucp2 overexpression inhibits carotid atherosclerotic plaque formation in disturbed flow-enhanced atherosclerosis mouse model. RNA-sequencing analysis revealed FoxO1 (forkhead box protein O1) as the major proinflammatory transcriptional regulator activated by UCP2 knockdown, and FoxO1 inhibition reduced vascular inflammation and disturbed flow-enhanced atherosclerosis. We showed further that UCP2 level is critical for phosphorylation of AMPK (AMP-activated protein kinase), which is required for UCP2-induced inhibition of FoxO1. CONCLUSIONS: Altogether, our studies uncover that UCP2 is novel mechanosensitive gene under the control of fluid shear stress and KLF2 in ECs. UCP2 expression is critical for endothelial proinflammatory response and atherogenesis. Therapeutic strategies enhancing UCP2 level may have therapeutic potential against atherosclerosis.


Assuntos
Aterosclerose , Placa Aterosclerótica , Proteína Desacopladora 2/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Células Cultivadas , Endotélio/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Placa Aterosclerótica/metabolismo , Estresse Mecânico
6.
Acta Pharmacol Sin ; 45(5): 975-987, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38279042

RESUMO

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, and EDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 µM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 µM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.


Assuntos
Artérias Carótidas , Células Endoteliais , Endotélio Vascular , Hipertensão , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína ORAI1 , Canais de Cátion TRPC , Animais , Proteína ORAI1/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Canais de Cátion TRPC/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Acetilcolina/farmacologia , Angiotensina II/farmacologia , Vasoconstrição/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo
8.
Acta Pharmacol Sin ; 44(10): 1977-1988, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37217602

RESUMO

Atherosclerotic diseases remain the leading cause of adult mortality and impose heavy burdens on health systems globally. Our previous study found that disturbed flow enhanced YAP activity to provoke endothelial activation and atherosclerosis, and targeting YAP alleviated endothelial inflammation and atherogenesis. Therefore, we established a luciferase reporter assay-based drug screening platform to seek out new YAP inhibitors for anti-atherosclerotic treatment. By screening the FDA-approved drug library, we identified that an anti-psychotic drug thioridazine markedly suppressed YAP activity in human endothelial cells. Thioridazine inhibited disturbed flow-induced endothelial inflammatory response in vivo and in vitro. We verified that the anti-inflammatory effects of thioridazine were mediated by inhibition of YAP. Thioridazine regulated YAP activity via restraining RhoA. Moreover, administration of thioridazine attenuated partial carotid ligation- and western diet-induced atherosclerosis in two mouse models. Overall, this work opens up the possibility of repurposing thioridazine for intervention of atherosclerotic diseases. This study also shed light on the underlying mechanisms that thioridazine inhibited endothelial activation and atherogenesis via repression of RhoA-YAP axis. As a new YAP inhibitor, thioridazine might need further investigation and development for the treatment of atherosclerotic diseases in clinical practice.


Assuntos
Aterosclerose , Células Endoteliais , Tioridazina , Animais , Humanos , Camundongos , Aterosclerose/tratamento farmacológico , Aterosclerose/prevenção & controle , Inflamação/etiologia , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Tioridazina/uso terapêutico , Proteínas de Sinalização YAP/efeitos dos fármacos
9.
Prostate ; 82(1): 13-25, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34570375

RESUMO

INTRODUCTION: Androgen deprivation therapy (ADT) is a key treatment modality in the management of prostate cancer (PCa), especially for patients with metastatic disease. Increasing evidences suggest that patients who received ADT have increased incidence of diabetes, myocardial infarction, stroke, and even mortality. It is important to understand the pathophysiological mechanisms on how ADT increases cardiovascular risk and induces cardiovascular events, which would provide important information for potential implementation of preventive measures. METHODS: Twenty-six 12-week-old male SD rats were divided into four groups for different types of ADTs including: the bilateral orchidectomy group (Orx), LHRH agonist group (leuprolide), LHRH antagonist group (degarelix), and control group. After treated with drug or adjuvant injection every 3 weeks for 24 weeks, all rats were sacrificed and total blood were collected. Aorta, renal arteries, and kidney were preserved for functional assay, immunohistochemistry, western blot, and quantitative reverse-transcription polymerase chain reaction. RESULTS: In vascular reactivity assays, aorta, intrarenal, and coronary arteries of all three ADT groups showed endothelial dysfunction. AT1R and related molecules at protein and messenger RNA (mRNA) level were tested, and AT1R pathway was shown to be activated and played a role in endothelial dysfunction. Both ACE and AT1R mRNA levels were doubled in the aorta in the leuprolide group while Orx and degarelix groups showed upregulation of AT1R in the kidney tissues. By immunohistochemistry, our result showed higher expression of AT1R in the intrarenal arteries of leuprolide and degarelix groups. The role of reactive oxygen species in endothelial dysfunction was confirmed by DHE fluorescence, nitrotyrosine overexpression, and upregulation of NOX2 in the different ADT treatment groups. CONCLUSION: ADT causes endothelial dysfunction in male rats. GnRH receptor agonist compared to GnRH receptor antagonist, showed more impairment of endothelial function in the aorta and intrarenal arteries. Such change might be associated with upregulation and activation of AngII-AT1R-NOX2 induced oxidative stress in the vasculature. These results help to explain the different cardiovascular risks and outcomes related to different modalities of ADT treatment.


Assuntos
Antagonistas de Androgênios , Artérias , Endotélio Vascular , Leuprolida , Oligopeptídeos , Orquiectomia/métodos , Antagonistas de Androgênios/efeitos adversos , Antagonistas de Androgênios/análise , Antagonistas de Androgênios/metabolismo , Animais , Artérias/efeitos dos fármacos , Artérias/metabolismo , Artérias/patologia , Correlação de Dados , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Hormônio Liberador de Gonadotropina/agonistas , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Fatores de Risco de Doenças Cardíacas , Imuno-Histoquímica , Leuprolida/administração & dosagem , Leuprolida/efeitos adversos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/efeitos adversos , Ratos , Espécies Reativas de Oxigênio/análise , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/metabolismo
10.
Cardiovasc Drugs Ther ; 36(4): 633-643, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-34319490

RESUMO

PURPOSE: The aim of this study was to evaluate the effects of berberine on nitroglycerin (NTG) tolerance and explore the underlying mechanism involved. METHODS: NTG tolerance was induced by pre-exposure of Sprague-Dawley rat aortas to NTG in vitro or by pretreating Sprague-Dawley rats with an NTG patch in vivo. The aortas were pre-treated with berberine or PKC inhibitors for different durations of time before induction of NTG tolerance. NTG-induced vasorelaxations was measured on wire myograph. Primary vascular smooth cells (VSMCs) were used to dissect the underlying mechanism of berberine-induced inhibition of NTG tolerance. RESULTS: NTG tolerance induced by either prior exposure of rat aortas to NTG in vitro or pretreatment with an NTG patch in vivo was reversed by co-treatment with berberine, as well as the inhibitors of protein kinase C (PKC) and protein kinase C alpha (PKCα). The mechanistic study revealed that PKCα participated in the development of NTG tolerance as NTG increased the activity of PKCα with enriched PKCα membrane localization and elevated phosphorylation of PKCα in VSMCs, which was reversed by berberine or PKCα inhibitors. CONCLUSION: This study is probably the first demonstration that berberine reverses NTG tolerance through inhibiting PKCα activity in VSMCs and PKCα is an important contributor to the development of NTG tolerance. These new findings suggest that berberine could become a promising drug for prevention of NTG tolerance and that targeting PKCα in VSMCs is likely to be a potential therapeutic strategy for reversal of NTG tolerance in blood vessels.


Assuntos
Berberina , Nitroglicerina , Animais , Berberina/farmacologia , Músculo Liso Vascular , Nitroglicerina/farmacologia , Fosforilação , Proteína Quinase C-alfa , Ratos , Ratos Sprague-Dawley
11.
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.

12.
Acta Pharmacol Sin ; 42(10): 1598-1609, 2021 Oct.
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.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Exenatida/farmacologia , Homocisteína/efeitos adversos , Dobramento de Proteína/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
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
14.
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.


Assuntos
Fístula Anastomótica/etiologia , Colo/irrigação sanguínea , Colo/cirurgia , Angiofluoresceinografia , Idoso , Anastomose Cirúrgica , Corantes , Feminino , Humanos , Verde de Indocianina , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos
15.
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
16.
Arterioscler Thromb Vasc Biol ; 36(3): 553-60, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26769046

RESUMO

OBJECTIVE: Bone morphogenic protein 4 (BMP4) is an important mediator of endothelial dysfunction in cardio-metabolic diseases, whereas platelet-derived growth factors (PDGFs) are major angiogenic and proinflammatory mediator, although the functional link between these 2 factors is unknown. The present study investigated whether PDGF mediates BMP4-induced endothelial dysfunction in diabetes mellitus. APPROACH AND RESULTS: We generated Ad-Bmp4 to overexpress Bmp4 and Ad-Pdgfa-shRNA to knockdown Pdgfa in mice through tail intravenous injection. SMAD4-shRNA lentivirus, SMAD1-shRNA, and SMAD5 shRNA adenovirus were used for knockdown in human and mouse endothelial cells. We found that PDGF-AA impaired endothelium-dependent vasodilation in aortas and mesenteric resistance arteries. BMP4 upregulated PDGF-AA in human and mouse endothelial cells, which was abolished by BMP4 antagonist noggin or knockdown of SMAD1/5 or SMAD4. BMP4-impared relaxation in mouse aorta was also ameliorated by PDGF-AA neutralizing antibody. Tail injection of Ad-Pdgfa-shRNA ameliorates endothelial dysfunction induced by Bmp4 overexpression (Ad-Bmp4) in vivo. Serum PDGF-AA was elevated in both diabetic patients and diabetic db/db mice compared with nondiabetic controls. Pdgfa-shRNA or Bmp4-shRNA adenovirus reduced serum PDGF-AA concentration in db/db mice. PDGF-AA neutralizing antibody or tail injection with Pdgfa-shRNA adenovirus improved endothelial function in aortas and mesenteric resistance arteries from db/db mice. The effect of PDGF-AA on endothelial function in mouse aorta was also inhibited by Ad-Pdgfra-shRNA to inhibit PDGFRα. CONCLUSIONS: The present study provides novel evidences to show that PDGF-AA impairs endothelium-dependent vasodilation and PDGF-AA mediates BMP4-induced adverse effect on endothelial cell function through SMAD1/5- and SMAD4-dependent mechanisms. Inhibition of PGDF-AA ameliorates vascular dysfunction in diabetic mice.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Diabetes Mellitus/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Smad Reguladas por Receptor/metabolismo , Vasodilatação , Adulto , Idoso , Animais , Anticorpos Neutralizantes/farmacologia , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Interferência de RNA , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad Reguladas por Receptor/genética , Fatores de Tempo , Técnicas de Cultura de Tecidos , Transfecção , Regulação para Cima , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
17.
Eur J Nutr ; 55(5): 1963-72, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26276555

RESUMO

PURPOSE: Menopause escalates the risk of cardiovascular diseases in women. There is an unmet need for better treatment strategy for estrogen-deficiency-related cardiovascular complications. Here we investigated the impact of chronic black tea extract (BT) consumption on cardiovascular function and lipid metabolism using a rat model of estrogen deficiency. METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) and treated with BT (15 mg/kg/day, 4 weeks; active ingredients: theaflavins) or estrogen (E2) treatment for 4 weeks. Serum was collected for measuring cholesterol, triacylglycerol and estradiol levels. Changes in vascular reactivity were examined. The protein levels of NADPH oxidases were assessed by Western blotting. Reactive oxygen species (ROS) level was measured using dihydroethidium fluorescence imaging. The concentrations of cGMP were measured using ELISA kit. RESULTS: Aortic rings from control, BT-treated and E2-treated OVX rats exhibited a greater increase in Phe-induced contraction after inhibition of NO synthase compared with those from OVX rats. ACh-induced endothelium-dependent relaxations were augmented in aortae and renal arteries in BT/E2-treated OVX rats than in OVX rats. BT/E2 treatment improved flow-mediated dilatation in small mesenteric resistance arteries of OVX rats. BT/E2 treatment restored the eNOS phosphorylation level and reversed the up-regulation of NADPH oxidases and ROS overproduction in OVX rat aortae. ACh-stimulated cGMP production was significantly elevated in the aortae from BT- and E2-treated rats compared with those from OVX rats. BT/E2 treatment reduced circulating levels of total cholesterol. CONCLUSIONS: The present study reveals the novel benefits of chronic BT consumption to reverse endothelial dysfunction and favorably modifying cholesterol profile in a rat model of estrogen deficiency and provides insights into developing BT as beneficial dietary supplements for postmenopausal women.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Ovariectomia , Extratos Vegetais/farmacologia , Chá/química , Animais , Antioxidantes/farmacologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Biflavonoides/farmacologia , Catequina/farmacologia , Endotélio Vascular/metabolismo , Estrogênios/farmacologia , Feminino , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
18.
Arterioscler Thromb Vasc Biol ; 34(4): 830-6, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24482374

RESUMO

OBJECTIVE: 5' Adenosine monophosphate-activated protein kinase (AMPK) interacts with peroxisome proliferator-activated receptor δ (PPARδ) to induce gene expression synergistically, whereas the activation of AMPK inhibits endoplasmic reticulum (ER) stress. Whether the vascular benefits of antidiabetic drug metformin (AMPK activator) in diabetes mellitus and obesity is mediated by PPARδ remains unknown. We aim to investigate whether PPARδ is crucial for metformin in ameliorating ER stress and endothelial dysfunction induced by high-fat diet. APPROACH AND RESULTS: Acetylcholine-induced endothelium-dependent relaxation in aortae was measured on wire myograph. ER stress markers were determined by Western blotting. Superoxide production in mouse aortae and NO generation in mouse aortic endothelial cells were assessed by fluorescence imaging. Endothelium-dependent relaxation was impaired and ER stress markers and superoxide level were elevated in aortae from high-fat diet-induced obese mice compared with lean mice. These effects of high-fat diet were reversed by oral treatment with metformin in diet-induced obese PPARδ wild-type mice but not in diet-induced obese PPARδ knockout littermates. Metformin and PPARδ agonist GW1516 reversed tunicamycin (ER stress inducer)-induced ER stress, oxidative stress, and impairment of endothelium-dependent relaxation in mouse aortae as well as NO production in mouse aortic endothelial cells. Effects of metformin were abolished by cotreatment of GSK0660 (PPARδ antagonist), whereas effects of GW1516 were unaffected by compound C (AMPK inhibitor). CONCLUSIONS: Metformin restores endothelial function through inhibiting ER stress and oxidative stress and increasing NO bioavailability on activation of AMPK/PPARδ pathway in obese diabetic mice.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus/tratamento farmacológico , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Obesidade/tratamento farmacológico , PPAR gama/agonistas , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Obesidade/enzimologia , Obesidade/genética , Obesidade/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/deficiência , PPAR gama/genética , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
19.
Arterioscler Thromb Vasc Biol ; 34(1): 152-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24202302

RESUMO

OBJECTIVE: Bone morphogenic protein 4 (BMP4) is involved in the development of endothelial dysfunction in hypertension. This study investigated whether the inhibition of BMP4 signaling improves endothelial function in db/db diabetic mice. APPROACH AND RESULTS: Male db/db mice were treated with noggin via osmotic pump infusion (1 µg/[h·kg(-1)]) for 2 weeks. Adenovirus BMP4-short hairpin RNA was introduced via tail vein injection at a dosage of 10(9) pfu/mouse and its effects were examined 7 days after. Vasoreactivity was studied on wire and pressure myograph. Both noggin treatment and adenovirus BMP4-short hairpin RNA transduction improved endothelium-dependent relaxations in aortae and flow-mediated dilatation in mesenteric arteries of db/db mice. Ex vivo treatment with BMP4 inhibitors and adenovirus BMP4-short hairpin RNA rescued the impaired endothelium-dependent relaxations in db/db mouse aortae and reduced reactive oxygen species overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortae, and also in ex vivo cultured C57BL/6 mouse aortae or primary mouse aortic endothelial cells treated with high glucose. Likewise, activin receptor-like kinase 3 silencing by short hairpin RNA lentivirus improved endothelium-dependent relaxations in db/db mouse aortae accompanied by reactive oxygen species inhibition in endothelial cells. In addition, noggin reduced BMP4 upregulation in high-glucose-treated endothelial cells and in C57BL/6 mouse aortae and in aortae from db/db mice. CONCLUSIONS: Inhibition of BMP4/activin receptor-like kinase 3/reactive oxygen species signaling improved endothelial function in diabetic mice through limiting oxidative stress in endothelium. Inhibiting BMP4 cascade can become another potential therapeutic strategy against diabetic vascular dysfunction.


Assuntos
Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteínas de Transporte/farmacologia , Angiopatias Diabéticas/terapia , Endotélio Vascular/efeitos dos fármacos , Terapia Genética/métodos , Interferência de RNA , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Adenoviridae/genética , Animais , Glicemia/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Células Cultivadas , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Vetores Genéticos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Vasodilatadores/farmacologia
20.
Circ Res ; 111(3): e55-66, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22679141

RESUMO

RATIONALE: The expression of osteocalcin is augmented in human atherosclerotic lesions. How osteocalcin triggers vascular pathogenesis and remodeling is unclear. OBJECTIVE: To investigate whether osteocalcin promotes transformation of adventitial fibroblast to myofibroblasts and the molecular mechanism involved. METHODS AND RESULTS: Immunohistochemistry indicated that osteocalcin was expressed in the neointima of renal arteries from diabetic patients. Western blotting and wound-healing assay showed that osteocalcin induced fibroblast transformation and migration, which were attenuated by blockers of the renin-angiotensin system and protein kinase Cδ (PKCδ), toll-like receptor 4 (TLR4) neutralizing antibody, and antagonist and inhibitors of free radical production and cyclooxygenase-2. Small interfering RNA silencing of TLR4 and PKCδ abolished fibroblast transformation. Angiotensin II level in the conditioned medium from the osteocalcin-treated fibroblasts was found elevated using enzyme immunoassay. Culturing of fibroblasts in conditioned medium collected from differentiated osteoblasts promoted fibroblast transformation. The expression of fibronectin, TLR4, and cyclooxygenase-2 is augmented in human mesenteric arteries after 5-day in vitro exposure to osteocalcin. CONCLUSIONS: Osteocalcin transforms adventitial fibroblasts to myofibroblasts through stimulating angiotensin II release and subsequent activation of PKCδ/TLR4/reactive oxygen species/cyclooxygenase-2 signaling cascade. This study reveals that the skeletal hormone osteocalcin cross-talks with vascular system and contributes to vascular remodeling.


Assuntos
Angiotensina II/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Miofibroblastos/metabolismo , Osteocalcina/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Ciclo-Oxigenase 2/fisiologia , Citoesqueleto/enzimologia , Citoesqueleto/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Miofibroblastos/citologia , Miofibroblastos/enzimologia , Ratos , Transdução de Sinais/fisiologia
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