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1.
DNA Cell Biol ; 40(2): 393-404, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33539267

RESUMO

The objective of this study was to investigate the role of endothelial nitric oxide synthase (eNOS), matrix metalloproteinases (MMP)-9, and Bcl2-associated oncogene 6 (BAG-6) gene polymorphisms, as well as the combined role of single nucleotide polymorphisms (SNPs) in hypertensive disorders of pregnancy (HDP) patients. This case-control study consisted of women with 326 HDP and 312 healthy pregnant controls. Multiplex PCR combined with next-generation sequencing method was used for determination of gene polymorphisms. Nine SNPs were analyzed, and we classified these case samples in depth by preeclampsia (PE) non-PE or blood pressure stages. We undertook allele and genotype haplotype association studies in all the cases and in the subgroups, as well as adjust age by binary logistic regression. Furthermore, the distribution of the haplotypes formed by the nine SNPs mentioned between the HDP patients and healthy pregnant controls were analyzed. There were no statistically significant differences in the nine SNPs of eNOS, MMP-9, and BAG-6 gene allele and genotype frequencies between HDP or subtypes and controls. However, for haplotype analyses, we found that the frequencies of AGACGCCGA (p = 3.67e-005), AGACGCGCA (p = 0.03127), and GAACACCGA (p = 0.02449) were significantly lower in the cases than in the controls. However, the haplotype of GGACGCCGA (p = 0.000686) was higher in the cases than in the controls. Our results suggested protective effect of the haplotypes AGACGCCGA, AGACGCGCA, and GAACACCGA against the development of HDP, and the haplotype GGACGCCGA was the risk factor of HDP.


Assuntos
Predisposição Genética para Doença/genética , Hipertensão Induzida pela Gravidez/genética , Metaloproteinase 9 da Matriz/genética , Chaperonas Moleculares/genética , Óxido Nítrico Sintase Tipo III/genética , Polimorfismo de Nucleotídeo Único , Adulto , Estudos de Casos e Controles , China , Feminino , Haplótipos , Humanos , Pré-Eclâmpsia/genética , Gravidez
2.
J Surg Res ; 257: 178-188, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32835951

RESUMO

Cardiac surgery, including cardioplegic arrest and extracorporeal circulation, causes endothelial dysfunction, which can lead to no-reflow phenomenon and reduction of myocardial pump function. Nitric oxide (NO) deficiency is involved in this pathologic process, thereby providing a fundamental basis for the use of NO replacement therapy. Presently used drugs and additives to cardioplegic and heart preservation solutions are not able to reliably protect endothelial cells and cardiomyocytes from ischemia-reperfusion injury. This review discusses promising NO-releasing compounds of various chemical classes for cardioplegia and reperfusion, which effectively maintain NO homeostasis under experimental conditions, and presents the mechanisms of their action on the cardiovascular system. Incomplete preclinical studies and a lack of toxicity assessment, however, hinder translation of these drug candidates into the clinic. Perspectives for modulation of endothelial function using NO-mediated mechanisms are discussed. They are based on the cardioprotective potential of targeting vascular gap junctions and endothelial ion channels, intracoronary administration of progenitor cells, and endothelial-specific microRNAs. Some of these strategies may provide important therapeutic benefits for human cardiovascular interventions.


Assuntos
Cardiotônicos/farmacologia , Parada Cardíaca Induzida/efeitos adversos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Óxido Nítrico/metabolismo , Animais , Cardiotônicos/uso terapêutico , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Junções Comunicantes/efeitos dos fármacos , Coração/efeitos dos fármacos , Humanos , Canais Iônicos/metabolismo , MicroRNAs/uso terapêutico , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Óxido Nítrico/agonistas , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Transplante de Células-Tronco/métodos
3.
Am J Physiol Heart Circ Physiol ; 319(4): H824-H834, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32822216

RESUMO

This study used an integrative experimental model in humans to investigate whether muscle angiogenic factors are differentially modulated by exercise stimuli eliciting different degrees of mechanical and metabolic stress. In a randomized crossover design, 12 men performed two low-volume high-intensity exercise regimens, including short sprint intervals (SSI) or long sprint intervals (LSI) inducing pronounced mechanical/metabolic stress, and a high-volume moderate-intensity continuous exercise protocol (MIC) inducing mild but prolonged mechanical/metabolic stress. Gene and protein expression of angiogenic factors was determined in vastus lateralis muscle samples obtained before and after exercise. Exercise upregulated muscle VEGF mRNA to a greater extent in LSI and MIC compared with SSI. Analysis of angiogenic factors sensitive to shear stress revealed more marked exercise-induced VEGF receptor 2 (VEGF-R2) mRNA responses in MIC than SSI, as well as greater platelet endothelial cell adhesion molecule (PECAM-1) and endothelial nitric oxide synthase (eNOS) mRNA responses in LSI than SSI. No apparent exercise-induced phosphorylation of shear stress-sensory proteins VEGF-R2Tyr1175, PECAM-1Tyr713, and eNOSSer1177 was observed despite robust elevations in femoral artery shear stress. Exercise evoked greater mRNA responses of the mechanical stretch sensor matrix metalloproteinase-9 (MMP9) in SSI than MIC. Exercise-induced mRNA responses of the metabolic stress sensor hypoxia-inducible factor-1α (HIF-1α) were more profound in LSI than SSI. These results suggest that low-volume high-intensity exercise transcriptionally activates angiogenic factors in a mechanical/metabolic stress-dependent manner. Furthermore, the angiogenic potency of low-volume high-intensity exercise appears similar to that of high-volume moderate-intensity exercise, but only on condition of eliciting severe mechanical/metabolic stress. We conclude that the angiogenic stimulus produced by exercise depends on both magnitude and protraction of myocellular homeostatic perturbations.NEW & NOTEWORTHY Skeletal muscle capillary growth is orchestrated by angiogenic factors sensitive to mechanical and metabolic signals. In this study, we employed an integrative exercise model to synergistically target, yet to different extents and for different durations, the mechanical and metabolic components of muscle activity that promote angiogenesis. Our results suggest that the magnitude of the myocellular perturbations incurred during exercise determines the amplitude of the angiogenic molecular signals, implying hormetic modulation of skeletal muscle angiogenesis by exercise-induced mechanical and metabolic stress.


Assuntos
Proteínas Angiogênicas/metabolismo , Metabolismo Energético , Hormese , Contração Muscular , Músculo Quadríceps/irrigação sanguínea , Músculo Quadríceps/metabolismo , Estresse Fisiológico , Adulto , Proteínas Angiogênicas/genética , Ciclismo , Estudos Cross-Over , Hemodinâmica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Consumo de Oxigênio , Resistência Física , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Distribuição Aleatória , Fatores de Tempo , Ativação Transcricional , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(6): 520-526, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-32696742

RESUMO

Objective To investigate the roles of protein phosphatase 2C (PP2C) activated by palmitic acid (PA) in the phosphorylation modulation of endothelial nitric oxide (eNOS) at the site of serine 1177 (eNOS Ser1177) in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were randomly divided into control group, PA group, specific PP2C inhibitor sanguinarine (San) combined with PA group and PP2Cα specific small interference RNA (siRNA) transfection group. The protein expression of total eNOS, phosphorylated eNOS Ser1177 and PP2Cα were detected by Western blotting. The intracellular NO content was measured by DAF-FM DA. The co-localization of eNOS protein and PP2C protein was observed by co-immunoprecipitation. Results Compared with the control group, the phosphorylation levels of eNOS Ser1177 and NO content decreased significantly in the PA group. The PP2C inhibitor sanguinarine reversed PA-induced decrease of eNOS Ser1177 phosphorylation level and NO content. The phosphorylation levels of eNOS Ser1177 were enhanced dramatically after PP2Cα protein was knocked down by specific siRNA. The eNOS protein and PP2C protein co-localized in endothelial cells. Conclusion PA reduces the phosphorylation level of endothelial eNOS Ser1177 in HUVECs by activating PP2C.


Assuntos
Células Endoteliais da Veia Umbilical Humana , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Óxido Nítrico , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Palmítico/farmacologia , Fosforilação , Proteína Fosfatase 2C , Veias Umbilicais/metabolismo
5.
Arterioscler Thromb Vasc Biol ; 40(9): 2244-2264, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640900

RESUMO

OBJECTIVE: Nanog is expressed in adult endothelial cells (ECs) at a low-level, however, its functional significance is not known. The goal of our study was to elucidate the role of Nanog in adult ECs using a genetically engineered mouse model system. Approach and Results: Biochemical analyses showed that Nanog is expressed in both adult human and mouse tissues. Primary ECs isolated from adult mice showed detectable levels of Nanog, Tert (telomerase reverse transcriptase), and eNos (endothelial nitric oxide synthase). Wnt3a (Wnt family member 3A) increased the expression of Nanog and hTERT (human telomerase reverse transcriptase) in ECs and increased telomerase activity in these cells. In a chromatin immunoprecipitation experiment, Nanog directly bound to the hTERT and eNOS promoter/enhancer DNA elements, thereby regulating their transcription. Administration of low-dose tamoxifen to ROSAmT/mG::Nanogfl/+::Cdh5CreERT2 mice induced deletion of a single Nanog allele, simultaneously labeling ECs with green fluorescent protein and resulting in decreased Tert and eNos levels. Histological and morphometric analyses of heart tissue sections prepared from these mice revealed cell death, microvascular rarefaction, and increased fibrosis in cardiac vessels. Accordingly, EC-specific Nanog-haploinsufficiency resulted in impaired EC homeostasis and angiogenesis. Conversely, re-expression of cDNA encoding the hTERT in Nanog-depleted ECs, in part, restored the effect of loss of Nanog. CONCLUSIONS: We showed that low-level Nanog expression is required for normal EC homeostasis and angiogenesis in adulthood.


Assuntos
Proliferação de Células , Senescência Celular , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Proteína Homeobox Nanog/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Fibrose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Homeobox Nanog/deficiência , Proteína Homeobox Nanog/genética , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Telomerase/genética , Telomerase/metabolismo , Ativação Transcricional , Via de Sinalização Wnt , Proteína Wnt3A/farmacologia
6.
Am J Physiol Heart Circ Physiol ; 319(2): H341-H348, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32618512

RESUMO

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.


Assuntos
Núcleo Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/biossíntese , Progesterona/farmacologia , Receptores de Progesterona/agonistas , Fator de Transcrição Sp1/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Sítios de Ligação , Núcleo Celular/metabolismo , Células Cultivadas , Indução Enzimática , Feminino , Antagonistas de Hormônios/farmacologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/enzimologia , Óxido Nítrico Sintase Tipo III/genética , Regiões Promotoras Genéticas , Ratos Sprague-Dawley , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Transdução de Sinais , Vasodilatação/efeitos dos fármacos
7.
J Oral Sci ; 62(3): 322-326, 2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32493866

RESUMO

Oral carcinoma is the sixth most common malignancy worldwide, with survival rates of approximately 50%. The major type of oral cancer, present in 90% of the cases, is oral squamous cell carcinoma (OSCC). The genetic background predisposing an individual to OSCC is complex and largely unknown. Studies have suggested that endothelial nitric oxide synthase (eNOS) gene polymorphisms modulate the cancer risk, prompting us to assess the impact of three functional eNOS gene polymorphisms on OSCC risk. The present study included 50 patients with OSCC and 110 controls. Polymerase chain reaction and restriction fragment length polymorphism analysis were used for genotyping of single-nucleotide polymorphisms -786 T/C (rs2070744) and 894 G/T (rs1799983) and variable number of tandem repeats (VNTR) intron 4b/a polymorphism. Homozygous carriers of -786 T/C and intron 4b/a VNTR variant alleles paired with a significant increase of oral cancer risk [odds ratio (OR): 3.63, 95% confidence interval (CI): 1.08-12.21; P = 0.045 and OR: 11.29, 95% CI: 2.71-47.11; P < 0.001, respectively]. When combined, CC and 4b4a genotypes together led to a 21-fold OSCC risk increase (OR: 21, 95% CI: 2.07-213.29; P = 0.006). Haplotype analysis showed that the C-G-4b haplotype conferred an 11-fold increase in OSCC risk. In conclusion, eNOS polymorphisms considerably influence levels of OSCC risk in the Serbian population.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Bucais , Estudos de Casos e Controles , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Humanos , Óxido Nítrico Sintase Tipo III/genética , Polimorfismo de Nucleotídeo Único
8.
Sci Rep ; 10(1): 7734, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382091

RESUMO

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation's effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation-induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Fatores de Transcrição Kruppel-Like/genética , Óxido Nítrico Sintase Tipo III/genética , Trombomodulina/genética , Fracionamento da Dose de Radiação , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Fatores de Transcrição MEF2/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/radioterapia , Radiação , Transdução de Sinais/efeitos da radiação
9.
Proc Natl Acad Sci U S A ; 117(21): 11483-11492, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404425

RESUMO

Endothelial cell nitric oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in endothelial cells, is regulated by complex posttranslational mechanisms. Sinusoidal portal hypertension, a disorder characterized by liver sinusoidal endothelial cell (SEC) injury with resultant reduced eNOS activity and NO production within the liver, has been associated with defects in eNOS protein-protein interactions and posttranslational modifications. We and others have previously identified novel eNOS interactors, including G protein-coupled receptor (GPCR) kinase interactor 1 (GIT1), which we found to play an unexpected stimulatory role in GPCR-mediated eNOS signaling. Here we report that ß-arrestin 2 (ß-Arr2), a canonical GPCR signaling partner, localizes in SECs with eNOS in a GIT1/eNOS/NO signaling module. Most importantly, we show that ß-Arr2 stimulates eNOS activity, and that ß-Arr2 expression is reduced and formation of the GIT1/eNOS/NO signaling module is interrupted during liver injury. In ß-Arr2-deficient mice, bile duct ligation injury (BDL) led to significantly reduced eNOS activity and to a dramatic increase in portal hypertension compared to BDL in wild-type mice. Overexpression of ß-Arr2 in injured or ß-Arr2-deficient SECs rescued eNOS function by increasing eNOS complex formation and NO production. We also found that ß-Arr2-mediated GIT1/eNOS complex formation is dependent on Erk1/2 and Src, two kinases known to interact with and be activated by ß-Arr2 in response to GCPR activation. Our data emphasize that ß-Arr2 is an integral component of the GIT1/eNOS/NO signaling pathway and have implications for the pathogenesis of sinusoidal portal hypertension.


Assuntos
Óxido Nítrico Sintase Tipo III/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestina 2/metabolismo , Animais , Células Cultivadas , Células Estreladas do Fígado/metabolismo , Hipertensão Portal/metabolismo , Fígado/citologia , Fígado/metabolismo , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/genética , beta-Arrestina 2/genética
10.
Braz J Med Biol Res ; 53(6): e9113, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401924

RESUMO

Chemerin is an adipokine that has been associated with components of metabolic syndrome. It has been described to affect adipocyte metabolism and inflammatory responses in adipose tissue, as well as the systemic metabolism of lipids and glucose. Few epidemiological studies have evaluated classical and genetics cardiovascular risk factors (CVRFs) in the mixed adult rural population in Brazil. Therefore, the present study explored possible associations between CVRFs and chemerin. This cross-sectional study included 508 adults from the rural localities of Lavras Novas, Chapada, and Santo Antônio do Salto in Ouro Preto, Minas Gerais, Southeast Brazil. Demographic, behavioral, clinical, biochemical, anthropometric variables, and 12 single nucleotide polymorphisms (SNPs) linked with metabolic syndrome phenotypes were evaluated for associations with chemerin level. There was a significant association of high triglyceride levels [odds ratio (OR)=1.91, 95%CI: 1.23-2.98], insulin resistance (OR=1.82, 95%CI: 1.03-3.22), age (OR=1.64, 95%CI: 1.08-2.49), and sex (OR=1.99, 95%CI: 1.35-2.95) with high levels of chemerin. High chemerin levels were significantly associated with the genetic polymorphisms rs693 in the APOB gene (OR=1.50, 95%CI: 1.03-2.19) and rs1799983 in the NOS3 gene (OR=1.46, 95%CI: 1.01-2.12) for the AA and GT+TT genotypes, respectively. In the concomitant presence of genotypes AA of rs693 and GT+TT of rs1799983, the chance of presenting high levels of chemerin showed a 2.21-fold increase (95%CI: 1.25-3.88) compared to the reference genotype. The development of classical CVRFs in this population may be influenced by chemerin and by two risk genotypes characteristic of variants in well-studied genes for hypertension and dyslipidemia.


Assuntos
Apolipoproteínas B/genética , Doenças Cardiovasculares/genética , Quimiocinas/sangue , Óxido Nítrico Sintase Tipo III/genética , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Doenças Cardiovasculares/metabolismo , Quimiocinas/genética , Estudos Transversais , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , População Rural , Adulto Jovem
11.
Sci Rep ; 10(1): 8115, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415164

RESUMO

Germinal matrix hemorrhage (GMH) is a detrimental form of neonatal CNS injury. Following GMH-mediated eNOS inhibition, inflammation arises, contributing to GMH-induced brain injury. We investigated the beneficial effects of Serelaxin, a clinical tested recombinant Relaxin-2 protein, on brain injury after GMH in rats. We investigated whether effects of Serelaxin are mediated by its ability to activate the GMH-suppressed eNOS pathway resulting in attenuation of inflammatory marker overproduction. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U). Seven day old Sprague-Dawley rat pups (P7) were used (n = 63). GMH animals were divided in vehicle or serelaxin treated (3 µg once, 30 µg once, 30 µg multiple, i.p., starting 30 after GMH and then daily). Sham operated animals were used. We monitored the developmental profile working memory and spatial function (T-maze and open field test respectively). At day 28, all rats underwent MRI-scans for assessment of changes in cortical thickness and white matter loss. Effects of Serelaxin on eNOS pathway activation and post-GMH inflammation were evaluated. We demonstrated that Serelaxin dose-dependently attenuated GMH-induced developmental delay, protected brain and improved cognitive functions of rats after GMH. That was associated with the decreased post-GMH inflammation, mediated at least partly by amelioration of GMH-induced eNOS inhibition.


Assuntos
Hemorragia Cerebral/complicações , Disfunção Cognitiva/prevenção & controle , Deficiências do Desenvolvimento/prevenção & controle , Inflamação/prevenção & controle , Óxido Nítrico Sintase Tipo III/metabolismo , Relaxina/administração & dosagem , Relaxina/metabolismo , Animais , Animais Recém-Nascidos , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/patologia , Deficiências do Desenvolvimento/etiologia , Deficiências do Desenvolvimento/patologia , Feminino , Inflamação/etiologia , Inflamação/patologia , Masculino , Óxido Nítrico Sintase Tipo III/genética , Ratos , Ratos Sprague-Dawley , Relaxina/genética
12.
Sci Rep ; 10(1): 5804, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242066

RESUMO

Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.


Assuntos
Humor Aquoso/metabolismo , Pressão Intraocular , Mecanotransdução Celular , Malha Trabecular/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Idoso , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/metabolismo , Regiões Promotoras Genéticas , Estresse Mecânico
13.
Lab Invest ; 100(8): 1068-1079, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32341517

RESUMO

Increased permeability and growth (angiogenesis) of blood vessels play a key role in joint swelling and pannus formation in inflammatory arthritis, a family of diseases influenced by reproductive hormones. The hormone prolactin (PRL) protects against joint inflammation, pannus formation, and bone destruction in adjuvant-induced arthritis and these effects may involve its proteolytic conversion to vasoinhibin, a PRL fragment that inhibits angiogenesis and vasopermeability. Here, we show that the intra-articular injection of an adeno-associated virus type-2 (AAV2) vector encoding vasoinhibin reduced joint inflammation, the hyperplasia, vascular density, and vasopermeability of the pannus, and the loss of bone in mice subjected to antigen-induced arthritis. In agreement, the AAV2 vasoinhibin vector reduced the expression of proinflammatory cytokines (interleukin-1ß, interleukin-6), an endothelial cell marker (platelet endothelial cell-adhesion molecule 1), and proangiogenic molecules [vascular endothelial growth factor (VEGF), VEGF receptor 2, and hypoxia-inducible factor 1α] in the arthritic joint. Also, vasoinhibin reduced the synovial vasopermeability induced by the intra-articular injection of VEGF in healthy mice. Finally, vasoinhibin signals by blocking the phosphorylation/activation of endothelial nitric oxide synthase (eNOS) at Ser1179 and the AAV2 vasoinhibin vector inhibited the enhanced phosphorylation of eNOS Ser1179 in the arthritic joint. We conclude that vasoinhibin reduces joint inflammation and bone loss in arthritis by inhibiting pannus angiogenesis and vasopermeability via the blockage of VEGF-induced eNOS activation. These findings suggest the potential therapeutic benefit of AAV2-mediated vasoinhibin gene delivery in arthritis.


Assuntos
Artrite Experimental/terapia , Permeabilidade Capilar/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Osteíte/prevenção & controle , Osteoporose/prevenção & controle , Prolactina/farmacologia , Animais , Antígenos , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Proteínas de Ciclo Celular/genética , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Articulações/patologia , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Osteíte/genética , Osteíte/terapia , Osteoporose/genética , Osteoporose/terapia
14.
Sci Rep ; 10(1): 6652, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313043

RESUMO

The role of Mesenchymal-endothelial transition (MEndoT) in cardiac hypertrophy is unclear. To determine the difference between MEndoT-derived and coronary endothelial cells is essential for understanding the revascularizing strategy in cardiac repair. Using lineage tracing we demonstrated that MEndoT-derived cells exhibit highly heterogeneous which were characterized with highly expression of endothelial markers such as vascular endothelial cadherin(VECAD) and occludin but low expression of Tek receptor tyrosine kinase(Tek), isolectin B4, endothelial nitric oxide synthase(eNOS), von Willebrand factor(vWF), and CD31 after cardiac hypertrophy. RNA-sequencing showed altered expression of fibroblast lineage commitment genes in fibroblasts undergoing MEndoT. Compared with fibroblasts, the expression of p53 and most endothelial lineage commitment genes were upregulated in MEndoT-derived cells; however, the further analysis indicated that MEndoT-derived cells may represent an endothelial-like cell sub-population. Loss and gain function study demonstrated that MEndoT-derived cells are substantial sources of neovascularization, which can be manipulated to attenuate cardiac hypertrophy and preserve cardiac function by improving the expression of endothelial markers in MEndoT-derived cells. Moreover, fibroblasts undergoing MEndoT showed significantly upregulated anti-hypertrophic factors and downregulated pro-hypertrophic factors. Therefore MEndoT-derived cells are an endothelial-like cell population that can be regulated to treat cardiac hypertrophy by improving neovascularization and altering the paracrine effect of fibroblasts.


Assuntos
Cardiomegalia/genética , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem da Célula/genética , Rastreamento de Células , Transdiferenciação Celular/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Fibroblastos/patologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ocludina/genética , Ocludina/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Transdução de Sinais , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
15.
Environ Sci Pollut Res Int ; 27(18): 22434-22440, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32314287

RESUMO

Recent studies have demonstrated that making the sorts of oxygen reactive, such as nitric oxide, can cause oxidative lipid damage, protein damage, and damage to the DNA of cells. Sperm DNA damage effect on the reduction of sperm mobility and damage of acrosome membrane lead to the inability of sperm to fertilize the oocyte. Increasing expression of endothelial nitric oxide synthase (eNOS) gene is seen in various diseases such as cardiovascular and infertility diseases. This study aimed to assess the association between eNOS gene single nucleotide polymorphism/SNP (rs2070744, T786C) and risk of male infertility and the quality of sperm parameters in an Iranian population. In this case-control study, 100 infertile men were enrolled as a patient group. Control groups consisted of 100 fertile men. T786C genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results showed that T786C SNP, contained frequent genotype TC (p = 0.000; OR = 0.000; 95% CI = 0.000-0.015), TC + CC genotypes (p = 0.000; OR = 0.000; 95% CI = 0.000-0.015), and C allele (p = 0.000; OR = 0.00; 95% CI = 0.000-0.007), revealed a significant with male infertility. Based on the findings of this study suggested that although T786C SNP could not be applied as an appropriate genetic risk factor for male infertility, it probably may be considered a protective marker for other researchers. However, more comprehensive studies in different populations are required to confirm our data.


Assuntos
Predisposição Genética para Doença , Infertilidade Masculina , Óxido Nítrico Sintase Tipo III/genética , Estudos de Casos e Controles , Genótipo , Humanos , Irã (Geográfico) , Masculino
16.
Proc Natl Acad Sci U S A ; 117(17): 9497-9507, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32300005

RESUMO

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein-protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein-protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS-PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Disponibilidade Biológica , Linhagem Celular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Óxido Nítrico , Óxido Nítrico Sintase Tipo III/genética , Piperazinas/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Ligação Proteica , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , para-Aminobenzoatos/farmacologia
17.
Clin Sci (Lond) ; 134(8): 985-999, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32297634

RESUMO

Small extracellular vesicles (sEVs) as natural membranous vesicles are on the frontiers of nanomedical research, due to their ability to deliver therapeutic molecules such as microRNAs (miRNAs). The miRNA-21 (miR-21) is thought to be involved in the initiation and development of myocardial infarction (MI). Here, we examined whether miR-21 regulation using human peripheral blood-derived sEVs (PB-sEVs) could serve as a potential therapeutic strategy for MI. First, we examined miR-21 levels in hypoxic conditions and validated the ability of PB-sEVs to serve as a potential delivery system for miRNAs. Further, bioinformatics analysis and luciferase assay were performed to identify target genes of miR-21 mechanistically. Among numerous target pathways, we focused on nitrogen metabolism, which remains relatively unexplored compared with other possible miR-21-mediated pathways; hence, we aimed to determine novel target genes of miR-21 related to nitrogen metabolism. In hypoxic conditions, the expression of miR-21 was significantly up-regulated and correlated with nitric oxide synthase 3 (NOS3) levels, which in turn influences cardiac function. The down-regulation of miR-21 expression by PB-sEVs loaded with anti-miR-21 significantly improved survival rates, consistent with the augmentation of cardiac function. However, the up-regulation of miR-21 expression by PB-sEVs loaded with miR-21 reversed these effects. Mechanistically, miR-21 targeted and down-regulated the mRNA and protein expression of striatin (STRN), which could regulate NOS3 expression. In conclusion, we identified a novel therapeutic strategy to improve cardiac function by regulating the expression of miR-21 with PB-sEVs as an miR-21 or anti-miR-21 delivery vehicle and confirmed the miR-21-associated nitrogen metabolic disorders in MI.


Assuntos
Vesículas Extracelulares/química , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/terapia , Animais , Análise Química do Sangue , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Feminino , Terapia Genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo
18.
Am J Physiol Heart Circ Physiol ; 318(5): H1233-H1244, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275471

RESUMO

An important physiological role of the aorta is to convert the pulsatile blood flow that originates in the heart to a nearly continuous flow in the peripheral vessels. Previously, we demonstrated that basal, unstimulated nitric oxide (NO) production is more abundant in large as compared with muscular arteries and that it is an important regulator of arterial (aortic) stiffness. Hence, endothelial function and NO bioavailability are important determinants of aortic biomechanics, and mouse models with altered NO signaling might be of interest to investigate the (patho)physiological role of the NO signaling as a dynamic regulator of arterial stiffness. We aimed to characterize the ex vivo biomechanical properties of aortic segments from mice with no (eNOS-/-), normal [wild type (WT)], or high (eNOS-tg) endothelial NO synthase (eNOS) expression. Isobaric aortic diameter and compliance were lower in eNOS-/- mice and increased in eNOS-tg mice as compared with WT mice. Interestingly, these differences remained when NO levels were pharmacologically restored ex vivo, suggesting that they were not merely the result of a lack or excess of the vasodilator effects of NO. Analysis of basal vascular smooth muscle cell tone and the phasic as well as the tonic contraction in response to α1-adrenergic stimulation with phenylephrine revealed that the chronic lack of eNOS expression affected aortic reactivity similarly but with different magnitude as compared with acute eNOS blockade using Nω-nitro-l-arginine methyl ester in WT and eNOS-tg mice, suggesting that chronical distortion of NO signaling triggered several compensatory mechanisms that reflect the organism's attempt to restore the contractile imbalance and maintain optimal central hemodynamics.NEW & NOTEWORTHY Endothelial function and NO bioavailability are important determinants of aortic biomechanics and function. With a new technique we investigated the ex vivo aortic segment biomechanics of different mouse models with altered NO signaling. Our experiments clearly show that chronic distortion of NO signaling triggered several compensatory mechanisms that reflect the organism's attempt to maintain optimal central hemodynamics.


Assuntos
Aorta/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Rigidez Vascular , Animais , Aorta/metabolismo , Fenômenos Biomecânicos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tono Muscular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Transdução de Sinais , Vasoconstrição
19.
J Vasc Res ; 57(3): 136-142, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32224624

RESUMO

Acute coronary syndrome occurs when the heart muscle does not receive adequate oxygen and nutrients in a timely manner. Acute coronary syndromes are primarily due to atherosclerosis of the coronary arteries, i.e., coronary heart disease. Nitric oxide (NO) is synthesised from L-arginine in endothelial cells by the constitutive calcium-calmodulin-dependent enzyme, nitric oxide synthase (NOS), which mediates endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) is predominantly expressed in endothelial cells. Three NOS isoforms have been detected in different tissue: (1) neuronal NOS (nNOS) (NOS1), (2) eNOS (NOS2), and (3) inducible NOS (iNOS) (NOS3). These isoforms are encoded by three different genes. NOS3 is located on chromosome 7q35-36 and contains 26 exons. Previous studies have suggested that NOS3 polymorphisms may be associated with acute coronary syndromes. Therefore, the aim of the study was to examine the associations between NOS3 rs1799983 (894G/T)andrs2070744 (-786T/C) polymorphisms and unstable angina. This study included 246 patients with unstable angina, as confirmed by coronary angiography. We also included 189 healthy controls who were also assessed by this technique. There were no significant differences in genotype distributions of NOS3 rs1799983and rs2070744 polymorphisms in patients with unstable angina and healthy controls in both univariate and multivariate analyses. In patients with the NOS3 rs1799983 TT genotype, we observed a higher BMI (TT vs. GT + GG, p = 0.068), and in patients with the NOS3 rs2070744 TT genotype, we observed a higher waist circumference (TT vs. TC + CC, p = 0.023; TT vs. CC, p = 0.0053). These data suggest a lack of association between the NOS3 rs1799983andrs2070744 polymorphisms and unstable angina in our patient population. However, these polymorphisms may be associated with some obesity parameters, rs1799983 in females and rs2070744 in males.


Assuntos
Angina Instável/genética , Óxido Nítrico Sintase Tipo III/genética , Polimorfismo de Nucleotídeo Único , Idoso , Angina Instável/diagnóstico , Angina Instável/enzimologia , Estudos de Casos e Controles , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/diagnóstico , Obesidade/enzimologia , Obesidade/genética , Fenótipo , Fatores de Risco , Circunferência da Cintura/genética
20.
PLoS One ; 15(4): e0230665, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251485

RESUMO

Phagocytes in patients with chronic granulomatous disease (CGD) do not generate reactive oxidative species (ROS), whereas nitric oxide (NO) production is increased in response to the calcium ionophore A23187 in CGD phagocytes compared with healthy phagocytes. Recently, patients with X-linked CGD (X-CGD) have been reported to show higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production from phagocytes. We studied NOS3 and EDN1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs) in a co-culture system with neutrophils from X-CGD patients. HUVECs were co-cultured for 30 minutes with human neutrophils from X-CGD or healthy participants in response to A23187 without cell-to-cell contact. The expression of NOS3 and EDN1 mRNA in HUVECs was quantified by real-time polymerase chain reaction. Moreover, we demonstrated the protein expression of eNOS, ET-1, and NFκB p65, including phosphorylation at Ser1177 of eNOS and Ser536 of NFκB p65. Neutrophils from X-CGD patients showed significantly higher NO and lower H2O2 production in response to A23187 than healthy neutrophils in vitro. Compared with healthy neutrophils, X-CGD neutrophils under A23187 stimulation exhibited significantly increased NO and decreased H2O2, and promoted downregulated NOS3 and EDN1 expression in HUVECs. The total expression and phosphorylation at Ser1177 of eNOS and ET-1 expression were significantly decreased in HUVECs co-cultures with stimulated X-CGD neutrophils. Also, phosphorylation at Ser536 of NFκB p65 were significantly decreased. In conclusions, eNOS and ET-1 significantly down-regulated in co-culture with stimulated X-CGD neutrophils through their excessive NO and the lack of ROS production. These findings suggest that ROS generated from neutrophils may mediate arterial tone affecting eNOS and ET-1 expression via their NO and ROS production.


Assuntos
Técnicas de Cocultura , Regulação para Baixo , Endotelina-1/metabolismo , Doença Granulomatosa Crônica/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Adolescente , Estudos de Casos e Controles , Endotelina-1/genética , Feminino , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , RNA Mensageiro/genética , Fator de Transcrição RelA/metabolismo
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