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1.
Int J Mol Sci ; 20(13)2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31277498

RESUMO

The maintenance of physiological levels of nitric oxide (NO) produced by eNOS represents a key element for vascular endothelial homeostasis. On the other hand, NO overproduction, due to the activation of iNOS under different stress conditions, leads to endothelial dysfunction and, in the late stages, to the development of atherosclerosis. Oxidized LDLs (oxLDLs) represent the major candidates to trigger biomolecular processes accompanying endothelial dysfunction and vascular inflammation leading to atherosclerosis, though the pathophysiological mechanism still remains to be elucidated. Here, we summarize recent evidence suggesting that oxLDLs produce significant impairment in the modulation of the eNOS/iNOS machinery, downregulating eNOS via the HMGB1-TLR4-Caveolin-1 pathway. On the other hand, increased oxLDLs lead to sustained activation of the scavenger receptor LOX-1 and, subsequently, to NFkB activation, which, in turn, increases iNOS, leading to EC oxidative stress. Finally, these events are associated with reduced protective autophagic response and accelerated apoptotic EC death, which activates atherosclerotic development. Taken together, this information sheds new light on the pathophysiological mechanisms of oxLDL-related impairment of EC functionality and opens new perspectives in atherothrombosis prevention.


Assuntos
Aterosclerose/enzimologia , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Inflamação/enzimologia , Lipoproteínas LDL/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Humanos , Inflamação/patologia , Óxido Nítrico/metabolismo
2.
PLoS One ; 14(5): e0216569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31067276

RESUMO

Although accepted agents in chorioamnionitis and preterm birth, the role of Ureaplasma species (spp.) in inflammation-driven morbidities of prematurity, including the development of bronchopulmonary dysplasia, remains controversial. To add to scarce in vitro data addressing the pro-inflammatory capacity of Ureaplasma spp., pulmonary epithelial-like A549 cells and human pulmonary microvascular endothelial cells (HPMEC) were incubated with Ureaplasma (U.) urealyticum, U. parvum, and Escherichia coli lipopolysaccharide (LPS). Ureaplasma isolates down-regulated caspase mRNA levels in A549 cells (caspase 8: p<0.001, 9: p<0.001, vs. broth), while increasing caspase protein expression, enzyme activity, and cell death in HPMEC (active caspase 3: p<0.05, caspase 8: p<0.05, active caspase 9: p<0.05, viability: p<0.05). LPS, contrarily, induced caspase mRNA expression in HPMEC (caspase 3: p<0.01, 4: p<0.001, 5: p<0.001, 8: p<0.001, vs. control), but not in A549 cells, and did not affect enzyme activity or protein levels in either cell line. LPS, but neither Ureaplasma isolate, enhanced mRNA expression of pro-inflammatory interleukin (IL)-6 in both A549 (p<0.05, vs. control) and HPMEC (p<0.001) as well as tumor necrosis factor-α (p<0.01), IL-1ß (p<0.001), and IL-8 (p<0.05) in HPMEC. We are therefore the first to demonstrate a differential modulation of pulmonary caspases by Ureaplasma spp. in vitro. Ureaplasma-driven enhanced protein expression and activity of caspases in pulmonary endothelial cells result in cell death and may cause structural damage. Down-regulated caspase mRNA in pulmonary epithelial cells, contrarily, may indicate Ureaplasma-induced inhibition of apoptosis and prevent effective immune responses. Both may ultimately contribute to chronic Ureaplasma colonization and long-term pulmonary inflammation.


Assuntos
Apoptose , Caspases/metabolismo , Citocinas/metabolismo , Endotélio Vascular/enzimologia , Células Epiteliais/enzimologia , Pneumonia/etiologia , Infecções por Ureaplasma/complicações , Células A549 , Células Cultivadas , Humanos , Pneumonia/enzimologia , Pneumonia/patologia , Ureaplasma/isolamento & purificação
3.
Amino Acids ; 51(7): 983-990, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31062169

RESUMO

Rheumatoid Arthritis (RA) confers an increased cardiovascular disease (CVD) risk which accounts for much of the premature morbidity and mortality observed in this population. Alterations in vascular function and morphology leading to increased atherosclerotic burden are considered the main drivers of CVD in RA individuals with systemic inflammation playing a key role in the dysregulation of endothelial homeostasis and initiation of vascular injury. Dimethylarginines are endogenous inhibitors of nitric oxide (NO) synthase and have emerged as novel, independent biomarkers of CVD in a wide range of conditions associated with vascular pathology. In RA several reports have demonstrated abnormal dimethylarginine metabolism attributable to various factors such as systemic inflammation, decreased degradation or upregulated synthesis. Although a causal relationship between dimethylarginines and vascular damage in RA has not been established, the tight interrelations between inflammation, dimethylarginines and endothelial dysfunction suggest that determination of dimethylarginine regulators may shed more light in the pathophysiology of the atherosclerotic process in RA and may also provide new therapeutic targets. The Alanine-Glyoxylate Aminotransferase 2 (AGTX2)-dependent pathway is a relatively recently discovered alternative pathway of dimethylarginine catabolism and its role on RA-related atherosclerotic disease is yet to be established. As factors affecting dimethylarginine concentrations linked to CVD risk and endothelial dysfunction are of prominent clinical relevance in RA, we present preliminary evidence that gene variants of AGTX-2 may influence dimethylarginine levels in RA patients and provide the rationale for larger studies in this field.


Assuntos
Arginina/análogos & derivados , Artrite Reumatoide/metabolismo , Doenças Cardiovasculares/genética , Transaminases/genética , Arginina/metabolismo , Artrite Reumatoide/complicações , Artrite Reumatoide/genética , Artrite Reumatoide/fisiopatologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Biomarcadores/sangue , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/mortalidade , Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Polimorfismo de Nucleotídeo Único , Transaminases/metabolismo
4.
Cell Mol Biol (Noisy-le-grand) ; 65(3): 119-124, 2019 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-30942165

RESUMO

To investigate the effect of piperazine ferulate (PF) on hypertension and endothelial function, and to assess the possible underlying mechanism. Human umbilical vein endothelial cells (HUVEC), adult male Wistar Kyoto (WKY) rats aged 12 to 14 weeks, and spontaneously hypertensive (SH) and Sprague Dawley (SD) rats were used for this study. Cell viability, activities of angiotensin-converting enzyme (ACE) and heme oxygenase-1 (HO-1), in vivo NO synthesis, arterial systolic blood pressure, vascular function, expressions of endothelial NO synthase (eNOS) and phosphorylated-eNOS (p-eNOS) were determined or assessed as appropriate. The results of MTT assay showed the number of viable cells were significantly increased with increase in PF concentration (p < 0.05). The level of expression of ACE was significantly reduced with increase in PF concentration (p < 0.05), while the level of HO-1 expression significantly increased (p < 0.05). Results of DAF-FM fluorescent staining showed that the amounts of NO synthesized in vivo was significantly higher in aortic rings of SH and SD rats treated with PF than in the corresponding control groups (p < 0.05). Treatment with PF in vivo significantly improved impaired acetylcholine-induced aortic relaxation in SH rats. Total eNOS expression was significantly increased after treatment with PF (p < 0.05). The expressions of total eNOS and p-eNOS in both groups were not affected by PF when compared to the control group. These results indicate that PF exerts antihypertensive effect and improves endothelial function in vitro and in vivo via the activation of eNOS.


Assuntos
Anti-Hipertensivos/farmacologia , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo , Piperazina/farmacologia , Animais , Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Óxido Nítrico/metabolismo , Peptidil Dipeptidase A/metabolismo , Fosforilação , Piperazina/química , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Sístole/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
5.
Hypertens Pregnancy ; 38(2): 96-104, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30821524

RESUMO

OBJECTIVE: We examined whether trimethylamine-N-oxide (TMAO) plays a role in endothelial dysfunction and hypertension in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia (PE).  Methods: Normal pregnant rats and RUPP rats were treated without or with 3,3-Dimethyl-1-butanol (DMB, a TMAO inhibitor) from gestational day 14.  Results: On day 19 of gestation, RUPP rats had higher plasma TMAO, impaired vasodilation and hypertension, decreased interleukin (IL)-10, increased superoxide production and proinflammatory cytokines in the aorta. All of which were reversed by DMD.  Conclusion: Increased circulating TMAO downregulates IL-10 and promotes vascular inflammation and oxidative stress, contributing to endothelial dysfunction and hypertension in PE.


Assuntos
Endotélio Vascular/fisiopatologia , Metilaminas/sangue , Pré-Eclâmpsia/sangue , Animais , Modelos Animais de Doenças , Endotélio Vascular/enzimologia , Feminino , Hexanóis , Inflamação/sangue , Interleucina-10/sangue , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pré-Eclâmpsia/enzimologia , Pré-Eclâmpsia/fisiopatologia , Gravidez , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
6.
Radiat Oncol ; 14(1): 25, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717801

RESUMO

BACKGROUND: Radiotherapy is administered in more than 60% of all solid tumors. Most patients are cured but a significant number develops local recurrences or distant metastases. The question arises if irradiation might influence the metastatic process. In the present study we examined whether the adhesion of glioblastoma or breast cancer cells to endothelial cells, an important step in metastasis, is affected by photon irradiation. METHODS: U-87 MG, U-373 MG and MDA-MB-231 cancer cells as well as primary human endothelial cells were irradiated with 0, 2, 4, or 8 Gy photons at a dose rate of 5 Gy/min. The adhesion of cancer cells to endothelial cells was tested either with the Vybrant based assay via fluorescent labelling or with an ibidi pump system able to mimic the physiological blood flow in vitro. In addition, the impact of FAK (focal adhesion kinase) inhibitor PF-573, 228 on the adhesion of non-irradiated and irradiated tumor cells was analyzed. Adhesion related and regulated proteins were analyzed by Western blotting. RESULTS: The cellular adhesion was increased after irradiation regardless of which cell type was irradiated. The FAK-inhibitor was able to reduce the adhesion of non-irradiated cells but also the irradiation-induced increase in adhesion of tumor cells to endothelium. Adhesion related proteins were enhanced after irradiation with 4 Gy or 8 Gy in both cells types. The increased adhesion after irradiation is accompanied by the phosphorylation of src (Y416), FAK (Y397) and increased expression of paxillin. CONCLUSION: Irradiation with photons in therapeutic doses is able to enhance the interaction between tumor cells and endothelial cells and by that might influence important steps of the metastatic process.


Assuntos
Neoplasias da Mama/patologia , Adesão Celular , Endotélio Vascular/patologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Raios gama , Glioblastoma/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/radioterapia , Proliferação de Células , Endotélio Vascular/enzimologia , Endotélio Vascular/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Feminino , Glioblastoma/enzimologia , Glioblastoma/radioterapia , Humanos , Células Tumorais Cultivadas
7.
Int J Food Sci Nutr ; 70(3): 267-284, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30185085

RESUMO

The main phenol in mate and coffee, 5-caffeoylquinic-acid (5-CQA), and its relevant microbial metabolites, dihydrocaffeic (DHCA) and dihydroferulic (DHFA) acids, have shown oxidative-stress protective effects in HepG2 cells. To evaluate possible endothelial-protective effects of the extracts and compounds, endothelial EA.hy926 cells were pre-treated with yerba mate (YME) and green coffee bean (GCBE) phenolic extracts, 5-CQA, DHCA and DHFA and afterwards stressed with tumour-necrosis-factor-alpha (TNF-α). Then oxidative-stress markers and endothelial-nitric-oxide-synthase levels were studied. TNF-α (10 ng/mL, 24 h) depleted reduced glutathione (GSH) and eNOS levels, increased reactive oxygen species (ROS) production, glutathione peroxidase (GPx) and reductase (GR) activities, and protein oxidation (carbonyl groups, CG) in EA.hy926 cells. Pre-treatment with YME, GCBE, 5-CQA, DHCA at certain physiological concentrations, lowered ROS production, recovered depleted GSH, reduced GR and GPx activities, and CG levels, and enhanced eNOS concentration.. YME, GCBE and 5-CQA show antioxidant effects in endothelial cells playing DHCA an important role in such protection; moreover, the extracts, 5-CQA, DHCA and DHFA increased eNOS levels.


Assuntos
Ácidos Cafeicos/farmacologia , Café/química , Endotélio Vascular/efeitos dos fármacos , Ilex paraguariensis/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ácido Quínico/análogos & derivados , Fator de Necrose Tumoral alfa/farmacologia , Antioxidantes/farmacologia , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Quínico/farmacologia , Espécies Reativas de Oxigênio/metabolismo
8.
Am J Physiol Renal Physiol ; 316(1): F32-F41, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30303710

RESUMO

Extracellular superoxide dismutase 3 (SOD3), one member of the antioxidant defense system and a superoxide scavenger, has been noted to be downregulated in the kidneys of diabetic mice and is characterized by a heparin-binding domain that can anchor the protein to the endothelium and extracellular matrix. The association of the serum and urinary SOD3 levels with diabetic nephropathy in different stages has never been evaluated. It remains unclear how urinary SOD3 changes in different renal diseases. We recruited 98 Taiwanese patients with type 2 diabetes and 10 patients with early chronic kidney disease (CKD) into this study. Biochemical analyses were performed, including evaluation of the serum SOD3, urinary SOD3, urinary albumin, urinary vascular endothelial growth factor (VEGF), and urinary angiotensinogen (ANG). The Kruskal-Wallis rank sum test was used to compare various parameters among the three groups of patients: early CKD, diabetes alone, and diabetes with CKD. Results showed that lower serum and urinary SOD3 levels were observed in the group of patients with diabetes alone. Higher serum and urinary SOD3 levels were observed in the group of patients with diabetes and CKD, which had higher albuminuria and serum creatinine levels. The serum SOD3 levels were significantly positively correlated with renal function, according to the serum creatinine level. The urinary levels of SOD3 were significantly correlated with other urinary biomarkers such as urinary ANG and VEGF. Furthermore, albuminuria can positively predict the serum SOD3 level for the ratio of urinary albumin to urinary creatinine (ACR) >1,190.769 mg/g and the urinary SOD3 level for ACR ≥300 mg/g.


Assuntos
Diabetes Mellitus Tipo 2/enzimologia , Nefropatias Diabéticas/etiologia , Rim/enzimologia , Insuficiência Renal Crônica/enzimologia , Superóxido Dismutase/sangue , Superóxido Dismutase/urina , Adulto , Idoso , Albuminúria/sangue , Albuminúria/enzimologia , Albuminúria/urina , Biomarcadores/sangue , Biomarcadores/urina , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/urina , Endotélio Vascular/enzimologia , Feminino , Humanos , Rim/patologia , Túbulos Renais/enzimologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/urina , Fatores de Risco , Ácido Úrico/sangue
9.
Mol Genet Metab ; 125(4): 338-344, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30413389

RESUMO

Fabry disease, a rare, X-linked lysosomal storage disease, arises from deficiency of the lysosomal hydrolase, α-galactosidase A (GLA) which disrupts the catabolism of globo- series glycosphingolipids (GSLs). One potential link between GLA deficiency and vascular dysfunction may be changes in endothelial nitric oxide synthase (eNOS) function. GLA-deficient EA.hy926 cells were obtained by siRNA knockdown of GLA expression and by mutation of GLA with CRISPR/Cas9 gene editing to investigate the effects of GLA deficiency on eNOS. As previously observed with siRNA knockdown of GLA, globotriaosylceramide (Gb3) accumulated in EA.hy926 cells. In contrast, Gb3 did not accumulate in CRISPR/Cas9 gene edited GLA-deficient cells, but instead, globotetraosylceramide (Gb4). However, in both the siRNA and CRISPR/Cas9 models globotriaosylsphingosine (lyso-Gb3) was elevated. As was previously observed with siRNA knockdown of GLA expression, CRISPR/Cas9 GLA-deficient cells had lower eNOS activity. Restoring GLA activity in GLA-deficient cells with exogenous GLA treatment improved eNOS activity. In contrast, treating cells with the glucosylceramide synthase inhibitor, eliglustat, decreased NOS activity. These results suggest that eNOS uncoupling is due to GLA deficiency, and not necessarily due to elevated Gb3 per se. It was observed that lyso-Gb3 inhibits eNOS activity.


Assuntos
Endotélio Vascular/patologia , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Óxido Nítrico Sintase Tipo III/metabolismo , Triexosilceramidas/metabolismo , alfa-Galactosidase/antagonistas & inibidores , Células Cultivadas , Endotélio Vascular/enzimologia , Humanos , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
10.
Arterioscler Thromb Vasc Biol ; 38(11): 2665-2677, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30354240

RESUMO

Objective- Mitochondria are the important yet most underutilized target for cardio-cerebrovascular function integrity and disorders. The Tom (translocases of outer membrane) complex are the critical determinant of mitochondrial homeostasis for making organs acclimate physiological and pathological insults; however, their roles in the vascular system remain unknown. Approach and Results- A combination of studies in the vascular-specific transgenic zebrafish and genetically engineered mice was conducted. Vascular casting and imaging, endothelial angiogenesis, and mitochondrial protein import were performed to dissect potential mechanisms. A loss-of-function genetic screening in zebrafish identified that selective inactivation of the tomm7 (translocase of outer mitochondrial membrane 7) gene, which encodes a small subunit of the Tom complex, specially impaired cerebrovascular network formation. Ablation of the ortholog Tomm7 in mice recapitulated cerebrovascular abnormalities. Restoration of the cerebrovascular anomaly by an endothelial-specific transgenesis of tomm7 further indicated a defect in endothelial function. Mechanistically, Tomm7 deficit in endothelial cells induced an increased import of Rac1 (Ras-related C3 botulinum toxin substrate 1) protein into mitochondria and facilitated the mitochondrial Rac1-coupled redox signaling, which incurred angiogenic impairment that underlies cerebrovascular network malformation. Conclusions- Tomm7 drives brain angiogenesis and cerebrovascular network formation through modulating mitochondrial Rac1 signaling within the endothelium.


Assuntos
Encéfalo/irrigação sanguínea , Proteínas de Transporte/metabolismo , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Neovascularização Fisiológica , Neuropeptídeos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Transtornos Cerebrovasculares/enzimologia , Transtornos Cerebrovasculares/genética , Endotélio Vascular/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos Knockout , Proteínas Mitocondriais/genética , Neovascularização Fisiológica/genética , Neuropeptídeos/genética , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas rac1 de Ligação ao GTP/genética
11.
Life Sci ; 212: 168-175, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30292829

RESUMO

Liver cirrhosis is associated with a wide range of cardiovascular abnormalities including hyperdynamic circulation and cirrhotic cardiomyopathy. The pathogenic mechanisms of these cardiovascular changes are multifactorial and include vascular dysregulations. AIM: The present study tested the hypothesis that the systemic vascular hyporesponsiveness in thioacetamide (TAA)-induced liver injury model is dependent on nitric oxide (NO) and cyclooxygenase (COX) derivatives. MAIN METHODS: Wistar rats were treated with TAA for eight weeks to induce liver injury. KEY FINDINGS: The maximal contractile response in concentration-effect curves to phenylephrine was decreased in aorta from TAA-treated rats, but no differences were found in aorta without endothelium, suggesting an endothelium-dependent mechanism in decreased contractile response. There was no difference in the contractile response with and without L-NAME (N(ω)-nitro-l-arginine methyl ester) in rats with liver injury, showing that the TAA treatment impairs NO synthesis. Pre-incubation of the aorta with indomethacin, a COX-inhibitor, normalized the reduced contractile response to phenylephrine in arteries from TAA group. Also, COX-2 and iNOS (inducible nitric oxide syntase) protein expression was increased in aorta from TAA group compared to control group. Animals submitted to TAA treatment had a reduction in systolic blood pressure. Our findings demonstrated that liver injury induced by TAA caused a decrease in aortic contractile response by a COX-dependent mechanism but not by NO release. Also, it was demonstrated an inflammatory process in the aorta of TAA-treated rats by increased expression of COX-2 and iNOS. SIGNIFICANCE: Therefore, there is an essential contribution of COX-2 activation in extra-hepatic vascular dysfunction and inflammation present in cirrhosis induced by TAA.


Assuntos
Aorta Torácica/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Ciclo-Oxigenase 2/metabolismo , Endotélio Vascular/patologia , Tioacetamida/toxicidade , Doenças Vasculares/etiologia , Animais , Aorta Torácica/enzimologia , Pressão Sanguínea , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Endotélio Vascular/enzimologia , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Doenças Vasculares/enzimologia
12.
Exp Cell Res ; 373(1-2): 99-111, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30342992

RESUMO

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the ß1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether ß1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1ß (IL-1ß)-induced inflammatory in vitro BBB model. We demonstrate that IL-1ß increases cell-matrix adhesion and induces a redistribution of active ß1 integrins to the basal surface. In particular, binding of α5ß1 integrin to its ligand fibronectin is enhanced and α5ß1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5ß1 integrin reduces the IL-1ß-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1ß-induced inflammation not only destabilizes tight junctions but also increases α5ß1 integrin-dependent cell-matrix adhesion to fibronectin.


Assuntos
Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Integrina alfa5beta1/metabolismo , Interleucina-1beta/farmacologia , Leucócitos Mononucleares/fisiologia , Migração Transendotelial e Transepitelial , Barreira Hematoencefálica , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfa5/metabolismo , Integrina alfa5beta1/antagonistas & inibidores , Integrina beta1/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
13.
J Am Coll Cardiol ; 72(7): 769-780, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30092954

RESUMO

BACKGROUND: Cardiovascular complications are major clinical problems in type 2 diabetes mellitus (T2DM). The authors previously demonstrated a crucial role of red blood cells (RBCs) in control of cardiac function through arginase-dependent regulation of nitric oxide export from RBCs. There is alteration of RBC function, as well as an increase in arginase activity, in T2DM. OBJECTIVES: The authors hypothesized that RBCs from patients with T2DM induce endothelial dysfunction by up-regulation of arginase. METHODS: RBCs were isolated from patients with T2DM and age-matched healthy subjects and were incubated with rat aortas or human internal mammary arteries from nondiabetic patients for vascular reactivity and biochemical studies. RESULTS: Arginase activity and arginase I protein expression were elevated in RBCs from patients with T2DM (T2DM RBCs) through an effect induced by reactive oxygen species (ROS). Co-incubation of arterial segments with T2DM RBCs, but not RBCs from age-matched healthy subjects, significantly impaired endothelial function but not smooth muscle cell function in both healthy rat aortas and human internal mammary arteries. Endothelial dysfunction induced by T2DM RBCs was prevented by inhibition of arginase and ROS both at the RBC and vascular levels. T2DM RBCs induced increased vascular arginase I expression and activity through an ROS-dependent mechanism. CONCLUSIONS: This study demonstrates a novel mechanism behind endothelial dysfunction in T2DM that is induced by RBC arginase I and ROS. Targeting arginase I in RBCs may serve as a novel therapeutic tool for the treatment of endothelial dysfunction in T2DM.


Assuntos
Arginase/biossíntese , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/enzimologia , Endotélio Vascular/enzimologia , Eritrócitos/enzimologia , Idoso , Animais , Arginase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologia
14.
Bull Exp Biol Med ; 165(3): 360-363, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30003420

RESUMO

The time course of angiotensin-converting enzyme activity in the rat aorta after fractionated exposure to ionizing radiation and the effects of dihydroquercetin and fucoidin on this parameter were studied. Male Wistar rats were exposed to single or repeated (fractionated) X-ray radiation in a dose of 2.5 Gy at 200 kV. Activity of angiotensin-converting enzyme in aorta segments was evaluated 2 h after the last exposure by hydrolysis of hippuryl-histidineleucin substrate. Enzyme activity in the rat aorta was higher than normally after all the studied doses of fractionated exposure (2.5 Gy per fraction) with the maximum effect after the total dose of 7.5 Gy (3 fractions). Fucoidin, a blocker of endothelium receptors realizing the leukocyte adhesion to the endothelium, and flavonoid dihydroquercetin inhibiting expression of adhesion molecules in the endothelium abolished the increase in activity of angiotensinconverting enzyme in the rat aorta after single exposure; moreover, dihydroquercetin reduced significantly the effect of fractionated exposure. These data indicate that leukocyte adhesion to the endothelium is an important factor contributing to the increase of angiotensin-converting enzyme activity in the aorta.


Assuntos
Aorta/efeitos da radiação , Endotélio Vascular/efeitos da radiação , Peptidil Dipeptidase A/genética , Polissacarídeos/farmacologia , Quercetina/análogos & derivados , Administração Oral , Animais , Aorta/enzimologia , Relação Dose-Resposta à Radiação , Endotélio Vascular/enzimologia , Expressão Gênica , Injeções Intravenosas , Masculino , Peptidil Dipeptidase A/metabolismo , Quercetina/farmacologia , Ratos , Ratos Wistar , Irradiação Corporal Total/métodos , Raios X
15.
Arterioscler Thromb Vasc Biol ; 38(10): 2345-2357, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29976767

RESUMO

Objective- Members of the microRNA (miR)-199a family, namely miR-199a-5p and miR-199a-3p, have been recently identified as potential regulators of cardiac homeostasis. Also, upregulation of miR-199a expression in cardiomyocytes was reported to influence endothelial cells. Whether miR-199a is expressed by endothelial cells and, if so, whether it directly regulates endothelial function remains unknown. We investigate the implication of miR-199a products on endothelial function by focusing on the NOS (nitric oxide synthase)/NO pathway. Approach and Results- Bovine aortic endothelial cells were transfected with specific miRNA inhibitors (locked-nucleic acids), and potential molecular targets identified with prediction algorithms were evaluated by Western blot or immunofluorescence. Ex vivo experiments were performed with mice treated with antagomiRs targeting miR-199a-3p or -5p. Isolated vessels and blood were used for electron paramagnetic resonance or myograph experiments. eNOS (endothelial NO synthase) activity (through phosphorylations Ser1177/Thr495) is increased by miR-199a-3p/-5p inhibition through an upregulation of the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) and calcineurin pathways. SOD1 (superoxide dismutase 1) and PRDX1 (peroxiredoxin 1) upregulation was also observed in locked-nucleic acid-treated cells. Moreover, miR-199a-5p controls angiogenesis and VEGFA (vascular endothelial growth factor A) production and upregulation of NO-dependent relaxation were observed in vessels from antagomiR-treated mice. This was correlated with increased circulated hemoglobin-NO levels and decreased superoxide production. Angiotensin infusion for 2 weeks also revealed an upregulation of miR-199a-3p/-5p in vascular tissues. Conclusions- Our study reveals that miR-199a-3p and miR-199a-5p participate in a redundant network of regulation of the NOS/NO pathway in the endothelium. We highlighted that inhibition of miR-199a-3p and -5p independently increases NO bioavailability by promoting eNOS activity and reducing its degradation, thereby supporting VEGF-induced endothelial tubulogenesis and modulating vessel contractile tone.


Assuntos
Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , MicroRNAs/metabolismo , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Vasodilatação , Inibidores da Angiogênese/farmacologia , Animais , Antagomirs/genética , Antagomirs/metabolismo , Bovinos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Estabilidade Enzimática , Regulação Neoplásica da Expressão Gênica , Hipertensão/enzimologia , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/genética , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Peroxirredoxinas/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vasodilatação/efeitos dos fármacos
16.
Arch Biochem Biophys ; 654: 55-69, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30016634

RESUMO

Myeloperoxidase is a member of the mammalian peroxidase family, mainly expressed in the myeloblastic cell lineage. It is considered a major bactericidal agent as it is released in the phagosome where it catalyzes the formation of reactive oxygen species. It is also released in the extracellular spaces including blood where it is absorbed on (lipo)proteins and endothelial cell surface, interfering with endothelial function. We performed RNA sequencing on MPO-treated endothelial cells, analyzed their transcriptome and validated the profile of gene expression by individual qRT-PCR. Some of the induced genes could be grouped in several functional networks, including tubulogenesis, angiogenesis, and blood vessel morphogenesis and development as well as signal transduction pathways associated to these mechanisms. MPO treatment mimicked the effects of VEGF on several signal transduction pathways, such as Akt, ERK or FAK involved in angiogenesis. Accordingly MPO, independently of its enzymatic activity, stimulated tube formation by endothelial cells. RNA interference also pointed at a role of endogenous MPO in tubulogenesis and endothelium wound repair in vitro. These data suggest that MPO, whether from endogenous or exogenous sources, could play a role in angiogenesis and vascular repair in vivo.


Assuntos
Endotélio Vascular/enzimologia , Sistema de Sinalização das MAP Quinases , Peroxidase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Transformada , Humanos , Neovascularização Patológica/metabolismo , Processamento de Proteína Pós-Traducional , Transcriptoma
17.
Sci Rep ; 8(1): 10581, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002415

RESUMO

Nucleoside diphosphate kinase B (NDPK-B) acts as a protective factor in the retinal vasculature. NDPK-B deficiency leads to retinal vasoregression mimicking diabetic retinopathy (DR). Angiopoetin 2 (Ang-2), an initiator of retinal vasoregression in DR, is upregulated in NDPK-B deficient retinas and in NDPK-B depleted endothelial cells (ECs) in vitro. We therefore investigated the importance of Ang-2 in NDPK-B deficient retinas and characterized the mechanisms of Ang-2 upregulation upon NDPK-B depletion in cultured ECs. The crucial role of retinal Ang-2 in the initiation of vasoregression was verified by crossing NDPK-B deficient with Ang-2 haplodeficient mice. On the molecular level, FoxO1, a transcription factor regulating Ang-2, was upregulated in NDPK-B depleted ECs. Knockdown of FoxO1 abolished the elevation of Ang-2 induced by NDPK-B depletion. Furthermore O-GlcNAcylated FoxO1 was found preferentially in the nucleus. An increased O-GlcNAcylation of FoxO1 was revealed upon NDPK-B depletion. In accordance, the inhibition of protein O-GlcNAcylation normalized NDPK-B depletion induced Ang-2 upregulation. In summary, we demonstrated that the upregulation of Ang-2 upon NDPK-B deficiency is driven by O-GlcNAcylation of FoxO1. Our data provide evidence for a central role of protein O-GlcNAcylation in NDPK-B associated vascular damage and point to the hexosamine pathway as an important target in retinal vasoregression.


Assuntos
Angiopoietina-2/genética , Retinopatia Diabética/patologia , Proteína Forkhead Box O1/metabolismo , Nucleosídeo NM23 Difosfato Quinases/deficiência , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Retina/patologia , Acetilglucosamina/metabolismo , Angiopoietina-2/metabolismo , Animais , Núcleo Celular/metabolismo , Retinopatia Diabética/genética , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Proteína Forkhead Box O1/genética , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Knockout , Nucleosídeo NM23 Difosfato Quinases/genética , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Retina/citologia , Retina/enzimologia , Vasos Retinianos/citologia , Vasos Retinianos/enzimologia , Vasos Retinianos/patologia , Regulação para Cima
18.
Drug Des Devel Ther ; 12: 1743-1751, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29942117

RESUMO

Aim: The aim of the study was to investigate whether miR-126, a regulator of MAPK signaling via targeting sprouty-related EVH1 domain-containing protein 1 (SPRED1) mRNA, is involved in the process by which icariside II (ICA II) ameliorates endothelial dysfunction in human cavernous endothelial cells (hCECs) exposed to a diabetic-like environment. Materials and methods: Primary hCECs were isolated and divided into three groups, normal control, diabetes mellitus (DM), and DM treated with ICA II. The cell proliferation and migration abilities of the hCECs were examined. The expression levels of endothelial-related microRNAs and relative target mRNAs (SPRED1, phosphoinositol-3 kinase regulatory subunit 2, and vascular cell adhesion molecule 1) of miR-126 were determined by real-time PCR. The protein expression of endothelial nitric oxide synthase, receptor for advanced glycation end products, and SPRED1, and MAPK signaling activities was determined by Western blot analysis. In addition, miR-126 agomir and antagomir were used for transfection into hCECs to further testify the association between miR-126 and its targeting mRNA SPRED1. Results: hCECs induced with glucose plus advanced glycation end product-BSA showed a significant decrease in endothelial nitric oxide synthase, Ki-67, and miR-126 expression; a downregulated cell migration ability and an increased receptor for advanced glycation end products level. ICA II could partially reverse these changes. SPRED1 mRNA showed a contrary tendency with the miR-126-3p changes. The level of SPRED1 protein increased after the hCECs were induced with glucose plus advanced glycation end product-BSA, and ICA II could rescue its aberrant expression. In addition, the MAPK pathway was downregulated in the hCECs under diabetic conditions, and ICA II could partially enhance its signaling activities. miR-126 was obviously downregulated, and SPRED1 was accordingly upregulated after miR-126 antagomir transfection, while ICA II treatment could recover the expressions of both miR-126 and SPRED1. Moreover, the upregulation of miR-126 and the inhibition of SPRED1 were noticed in the diabetic hCECs by further transfection with miR-126 agomir. Conclusion: ICA II could ameliorate endothelial dysfunction by regulating the MAPK pathway via miR-126/SPRED1 in hCECs exposed to a diabetic-like environment, and ICA II might be a protective agent for endothelial function in diabetic ED.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Flavonoides/farmacologia , Hipoglicemiantes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Microambiente Celular , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Glucose/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , MicroRNAs/genética , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Soroalbumina Bovina/farmacologia , Transdução de Sinais/efeitos dos fármacos
19.
Br J Pharmacol ; 175(17): 3453-3469, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29859010

RESUMO

BACKGROUND AND PURPOSE: NO-mediated, endothelium-dependent relaxations of isolated arteries are blunted by ageing and high-fat diets, as well as by apolipoprotein E deletion. The present study was designed to test the hypothesis that apolipoprotein E deletion impairs endothelium-dependent responses to prostacyclin (IP) receptor activation. EXPERIMENTAL APPROACH: Five-week-old ApoE+/+ and ApoE-/- mice were fed normal chow or high-fat diet for 29 weeks. The aortae were isolated for the measurements of isometric tension in Halpern-Mulvany myographs. Levels of proteins were assessed by Western blotting and immunofluorescence, and cyclic nucleotide levels by elisa. KEY RESULTS: The IP receptor agonist, iloprost, induced endothelium-, NO-synthase- and IP-dependent relaxations in aortae of young ApoE+/+ mice. High-fat diet favoured activation of thromboxane receptors by iloprost, causing contraction. Apolipoprotein E was present in aortae of ApoE+/+ mice, especially in endothelium. Its presence was augmented by high-fat diet. Its deletion potentiated iloprost-induced relaxations in aortae of young mice and prevented the blunting of this response by high-fat diet. Levels of cAMP were higher, but those of cGMP were lower in the aorta of ApoE-/- than in ApoE+/+ mice of the same age. The levels of IP receptor protein were not different between ApoE+/+ and ApoE-/- mice. CONCLUSIONS AND IMPLICATIONS: Iloprost induced an endothelium-dependent relaxation in the aorta of young healthy mice which involved both the cGMP and cAMP pathways. This response was blunted by prolonged exposure to a high-fat diet. Apolipoprotein E deletion potentiated relaxations to IP receptor activation, independently of age and diet.


Assuntos
Aorta/metabolismo , Apolipoproteínas E/fisiologia , Receptores de Epoprostenol/agonistas , Acetilcolina/farmacologia , Animais , Aorta/enzimologia , Apolipoproteínas E/genética , Western Blotting , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Dieta Hiperlipídica , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Ensaio de Imunoadsorção Enzimática , Epoprostenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/metabolismo
20.
Vascul Pharmacol ; 109: 36-44, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29894845

RESUMO

Protein tyrosine phosphatase 1B (PTP1B) impairs nitric oxide (NO) production and induces endothelial dysfunction in various diseases, including diabetes, septic shock and heart failure. In non-cardiovascular tissues, PTP1B modulates endoplasmic reticulum stress (ERS) however this role has never been assessed in endothelial cells. We evaluated the link between PTP1B, ERS and endothelial dysfunction in mice. Induction of ERS (Tunicamycin) in vivo in mice or ex vivo in mouse arteries led to severe arterial endothelial dysfunction (i.e. reduced flow-dependent, NO mediated dilatation in isolated small mesenteric arteries), and this was prevented by the PTP1B inhibitor trodusquemine and absent in PTP1B-/- mice. Trodusquemine also prevented the Tunicamycin -induced increased arterial levels of the molecular ERS actors 78 kDa glucose-regulated protein (GRP78) and Activating Transcription Factor 6 (ATF6α). Tunicamycin strongly increased the interactions of PTP1B with GRP78 and the activated forms of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and IRE1α (proximity Ligation Assay). Thus, PTP1B plays a central role in the regulation of ERS in the endothelium, and the endothelial protective effect of PTP1B inhibition appears likely due at least in part to reduction of endothelial ERS, notably by promoting PERK protective pathway. Modulation of ER stress via PTP1B inhibitors may be a promising approach to protect the endothelium in cardiovascular diseases.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Tunicamicina/farmacologia , Vasodilatação/efeitos dos fármacos , Fator 6 Ativador da Transcrição/metabolismo , Animais , Colestanos/farmacologia , Endorribonucleases/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Óxido Nítrico/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermina/análogos & derivados , Espermina/farmacologia , eIF-2 Quinase/metabolismo
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