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1.
Gene ; 725: 144143, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31629816

RESUMO

Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. Linc-ROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Autofagia/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/fisiologia , Homocisteína/farmacologia , Humanos , MicroRNAs/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Transdução de Sinais
2.
Nutr Metab Cardiovasc Dis ; 29(8): 856-864, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31272869

RESUMO

BACKGROUND AND AIM: Increased homocysteine (Hcy) is associated with coronary artery disease (CAD). Hcy increases reactive oxygen species (ROS) via NADPH oxidases (Nox), reducing acetylcholine-mediated vasorelaxation. We aimed to determine if putative Nox2 inhibitors prevent Hcy-impaired acetylcholine-mediated vasorelaxation. METHODS AND RESULTS: New Zealand White rabbit and wild-type (C57BL/6) and Nox2-/- (NOX) mice aortic rings were mounted in organ baths. Rabbit rings were incubated with either apocynin (10 µM), gp91ds-tat (GP, 1 µM) or PhoxI2 (1 µM) and mice rings GP (1 µM) only. Some rabbit rings were incubated with 3 mM Hcy, before pre-contraction, followed by dose-response relaxation to acetylcholine (ACh; 0.01µM-10µM). In rabbit rings treated with Hcy and GP, O2‾ donor pyrogallol (1 µM) or Akt activator SC79 (1 µM) was added 5 min before ACh. Mice rings were used to compare Nox2 deletion to normal acetylcholine-mediated relaxation. In rabbits, Hcy reduced acetylcholine-mediated relaxation vs. control (p < 0.0001). Treatment + Hcy reduced relaxation compared with treatment alone (p < 0.0001). Pyrogallol and SC79 reversed the response of GP + Hcy (p = 0.0001). In mice, Nox2 deletion reduced acetylcholine-mediated vasorelaxation. Rabbit tissue analysis revealed that Hcy reduced eNOS phosphorylation at Thr495 and increased eNOS phosphorylation at Ser1177; no further alteration at Thr495 was observed with GP. In contrast, GP prevented increased phosphorylation at Ser1177. CONCLUSIONS: Apocynin, GP and PhoxI2 worsens acetylcholine-mediated vascular relaxation in rabbit aorta, which is supported by results from mouse Nox2 deletion data. These inhibitors worsen Hcy-induced vascular dysfunction, suggesting that current putative Nox2 inhibitors might not be useful in treating HHcy.


Assuntos
Acetilcolina/farmacologia , Aorta/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Homocisteína/farmacologia , NADPH Oxidase 2/antagonistas & inibidores , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Acetofenonas/farmacologia , Animais , Aorta/enzimologia , Glicoproteínas/farmacologia , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Coelhos , Serina , Treonina
3.
Physiol Int ; 106(1): 29-38, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30888218

RESUMO

PURPOSE: We previously found that homocysteine (Hcy)-induced apoptosis in endothelial cells coincided with increased NADPH oxidase (NOX) activity. In addition, in ischemic endothelial cells present in the heart, we showed that loss of serine protease dipeptidyl peptidase IV (DPP4) expression was correlated with induction of tissue factor (TF) expression. Since Hcy can initiate thrombosis through the induction of TF expression, in this study, we evaluated whether the inverse relation of TF and DPP4 is also Hcy-dependent and whether NOX-mediated reactive oxygen species (ROS) is playing a role herein. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with 2.5 mM Hcy for 3 and 6 h. The effects of Hcy on DPP4 and TF expression and NOX2/p47phox-mediated nitrotyrosine (ROS) production were studied using digital-imaging microscopy. RESULTS: In HUVECs, high levels of Hcy showed a significant increase of TF expression and a concomitant loss of DPP4 expression after 6 h. In addition, NOX subunits NOX2 and p47phox were also significantly increased after 6 h of Hcy incubation and coincided with nitrotyrosine (ROS) expression. Interestingly, inhibition of NOX-mediated nitrotyrosine (ROS) with the use of apocynin not only reduced these effects, but also counteracted the effects of Hcy on TF and DPP4 expression. CONCLUSION: These results indicate that the inverse relation of TF and DPP4 in endothelial cells is also Hcy-dependent and related to NOX activity.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Homocisteína/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , NADPH Oxidases/metabolismo , Tromboplastina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
4.
Methods Mol Biol ; 1866: 13-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725404

RESUMO

Many different types of cancer cells have been shown to be methionine (MET) dependent. Cancer cells, unlike normal cells, grow poorly or not at all when MET is restricted. Cancer cells have an elevated requirement for exogenous MET for growth, despite high levels of endogenous synthesis. This requirement reflects increased utilization of MET by cancer cells, analogous to increased utilization glucose by cancer cells (Warburg effect). To answer the critical question of whether MET-dependent cancer cells synthesize normal amounts of MET, we determined the levels of MET, S-adenosylmethionine (AdoMET), and S-adenosylhomocysteine (AdoHCY) that were synthesized by MET-dependent cancer cells under conditions of MET restriction. We demonstrated that MET-dependent cells synthesize a normal amount of endogenously synthesized MET but are still deficient in AdoMET. In contrast, exogenously supplied MET results in normal AdoMET levels. The ratio of AdoMET to AdoHCY is low in MET-dependent cells growing in MET-restricted medium but is normal when MET is supplied. Under conditions of MET restriction, the low AdoMET/AdoHCY ratio probably limits proliferation of MET-dependent cancer cells. The amount of free MET is also low in MET-dependent cancer cells under MET restriction. The elevated MET requirement for cancer cells may be due to enhanced overall rates of transmethylation compared to normal human cells. Thus, MET-dependent cancer cells have low levels of free MET, low levels of AdoMET, and elevated levels of AdoHCY under conditions of MET restriction probably due to overuse of MET for transmethylation reactions ("Hoffman effect"), thereby blocking cellular proliferation.


Assuntos
Metionina/metabolismo , Neoplasias/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Homocisteína/farmacologia , Humanos , Metionina/deficiência , Metilação , Neoplasias/enzimologia , Neoplasias/patologia , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo
5.
Methods Mol Biol ; 1866: 285-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725425

RESUMO

The objective of the proposed clinical interventional trial is to demonstrate the efficacy of a novel therapeutic strategy in subjects with cancer and hyperhomocysteinemia. Following discovery of abnormal homocysteine thiolactone metabolism in cultured malignant cells, thioretinamide, the amide synthesized from retinoic acid and homocysteine thiolactone, and thioretinaco, the complex formed from cobalamin and thioretinamide, were demonstrated to have antineoplastic, anticarcinogenic, and anti-atherogenic properties in animal models. Retinol, ascorbate, and homocysteine thiolactone are necessary for biosynthesis of thioretinamide and thioretinaco by cystathionine synthase and for formation of thioretinaco ozonide from thioretinamide, cobalamin, and ozone. Thioretinaco ozonide is required for prevention of abnormal oxidative metabolism, aerobic glycolysis, suppressed immunity, and hyperhomocysteinemia in cancer.The pancreatic enzyme therapy of cancer promotes catabolism of proteins, nucleic acids, and glycosaminoglycans with excess homocysteinylated amino groups resulting from abnormal accumulation of homocysteine thiolactone in malignant cells. Dietary deficiencies of pyridoxal, folate, cobalamin, and nitriloside contribute to hyperhomocysteinemia in cancer, and in protein energy malnutrition. A deficiency of dietary sulfur amino acids downregulates cystathionine synthase, causing hyperhomocysteinemia.The organic sulfur compound diallyl trisulfide increases hydrogen sulfide production from homocysteine in animal models, inhibits Stat3 signaling in cancer stem cells, and produces apoptosis of malignant cells. The furanonaphthoquinone compound napabucasin inhibits Stat3 signaling and causes mitochondrial dysfunction, decreased oxidative phosphorylation, and apoptosis of malignant cells. The protocol of the proposed clinical trial in subjects with myelodysplasia consists of thioretinamide and cobalamin as precursors of thioretinaco ozonide, combined with pancreatic enzyme extracts, diallyl trisulfide, napabucasin, nutritional modification to minimize processed foods, vitamin supplements, essential amino acids, and beneficial dietary fats and proteins.


Assuntos
Envelhecimento/fisiologia , Homocisteína/análogos & derivados , Homocisteína/uso terapêutico , Neoplasias/tratamento farmacológico , Fosforilação Oxidativa , Vitamina B 12/análogos & derivados , Adulto , Idoso , Drogas em Investigação/farmacologia , Drogas em Investigação/uso terapêutico , Homocisteína/farmacologia , Humanos , Licenciamento , Pessoa de Meia-Idade , Fosforilação Oxidativa/efeitos dos fármacos , Vitamina B 12/uso terapêutico
6.
ACS Chem Biol ; 14(2): 186-191, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30668907

RESUMO

The RhlR quorum sensing (QS) receptor in the pathogen Pseudomonas aeruginosa plays a prominent role in infection, and both antagonism and agonism of RhlR have been shown to negatively regulate important virulence phenotypes. Non-native lactone ligands are known to modulate RhlR activity, but their utility as chemical probes is relatively limited due to hydrolytic instability. Herein, we report our design and biological evaluation of a suite of hybrid AHL analogs with structures merging (1) features of reported lead RhlR ligands and (2) head groups with improved hydrolytic stabilities. The most promising compounds identified were N-acyl l-homocysteine thiolactones, which displayed enhanced stabilities relative to lactones. Moreover, they were highly selective for RhlR over another key QS receptor in P. aeruginosa, LasR. These compounds are among the most potent RhlR modulators known and represent robust chemical tools to dissect the complex roles of RhlR in the P. aeruginosa QS circuitry.


Assuntos
Proteínas de Bactérias/metabolismo , Homocisteína/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Relação Dose-Resposta a Droga , Homocisteína/administração & dosagem , Homocisteína/farmacologia , Pseudomonas aeruginosa/fisiologia
7.
Exp Eye Res ; 178: 228-237, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29608906

RESUMO

This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Müller glial cells. Elevated Hcy has been implicated in retinal diseases including glaucoma and optic neuropathy, which are characterized by retinal ganglion cell (RGC) loss. To understand the mechanisms of Hcy-induced RGC loss, in vitro and in vivo models have been utilized. In vitro isolated RGCs are quite sensitive to elevated Hcy levels, while in vivo murine models of hyperhomocysteinemia (HHcy) demonstrate a more modest RGC loss (∼20%) over a period of many months. This differential response to Hcy between isolated cells and the intact retina suggests that the retinal milieu invokes mechanisms that buffer excess Hcy. Oxidative stress has been implicated as a mechanism of Hcy-induced neuron loss and NRF2 is a transcription factor that plays a major role in regulating cytoprotective responses to oxidative stress. In the present study we investigated whether HHcy upregulates NRF2-mediated stress responses in Müller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Müller cells were exposed to L-Hcy-thiolactone [50µM-10mM] and assessed for viability, reactive oxygen species (ROS), and glutathione (GSH) levels. Gene/protein levels of Nrf2 and levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed in Hcy-exposed Müller cells. Unlike isolated RGCs, isolated Müller cells are viable over a wide range of Hcy concentrations [50 µM - 1 mM]. Moreover, when exposed to elevated Hcy, Müller cells demonstrate decreased oxidative stress and decreased ROS levels. GSH levels increased by ∼20% within 24 h exposure to Hcy. Molecular analyses revealed 2-fold increase in Nrf2 expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly. The consequences of Hcy exposure were evaluated also in Müller cells harvested from Nrf2-/- mice. In contrast to WT Müller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2-/- Müller cells showed a significant increase in oxidative stress. Our data suggest that at least during early stages of Hhcy, a cytoprotective response may be in place, mediated in part by NRF2 in Müller cells.


Assuntos
Células Ependimogliais/efeitos dos fármacos , Homocisteína/análogos & derivados , Fator 2 Relacionado a NF-E2/metabolismo , Protetores contra Radiação/farmacologia , Animais , Elementos de Resposta Antioxidante/fisiologia , Sobrevivência Celular , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Glutationa/metabolismo , Homocisteína/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima
8.
Bioorg Med Chem ; 27(1): 36-42, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30473360

RESUMO

Design and synthesis of LuxS enzyme inhibitors otherwise known as S-ribosylhomocysteine analogues, to target quorum sensing in bacteria, has been considerably developed within the last decade. This review presents which molecules have been synthesized to target LuxS enzyme in other words inhibitors of S-ribosylhomocysteinase. It reports their tested biological activity as LuxS inhibitors when available. A systematic overview has been conducted by searching PubMed, Medline, and The Cochrane Library and data extraction of all synthesized S-ribosylhomocysteine analogues has been collected. This mini-review shows limited data to date on this area and should continue to be studied.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Liases de Carbono-Enxofre/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Homocisteína/análogos & derivados , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/química , Liases de Carbono-Enxofre/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Homocisteína/síntese química , Homocisteína/farmacologia , Percepção de Quorum/efeitos dos fármacos
9.
Biochem Biophys Res Commun ; 506(3): 648-652, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30454701

RESUMO

Homocysteine (HCY) induced neurotoxicity largely depends on interaction of this endogenous amino acid with glutamate NMDA receptors (NMDARs). This receptor type is composed by GluN1 and different GluN2 (A, B, C or D) subunits. However, the receptor activity of HCY in brain regions which differ in relative contribution of GluN2 subunits was not tested so far. In the current study, we explored the action of HCY on cerebellar neurons which natively express GluN2C and GluN2D subunits of NMDARs and compared this with the action of HCY on cortical neurons which are mainly composed by GluN2A and GluN2B subunits. To validate obtained results, we also studied the responses to HCY in recombinant GluN1/2C and GluN1/2D NMDARs expressed in HEK293T cells. Responses to HCY were compared to membrane currents evoked by glutamate or by the specific agonist NMDA. First, we found that on HEK cells expressing GluN1/2C or GluN1/2D NMDARs, HCY was full agonist producing membrane currents similar in amplitude to currents induced by glutamate. The EC50 values for these particular receptor subtype activation were 80 µM and 31 µM, respectively. Then, we found that HCY similarly to NMDA, evoked large slightly desensitizing membrane currents in native NMDARs of cerebellar and cortical neurons. In cortical neurons, the ratio of the respective currents (IHCY/INMDA) was 0.16 and did not significantly change during in vitro maturation. In sharp contrast, in cerebellar neurons, the ratio of currents evoked by HCY and NMDA was dramatically increased from 0.31 to 0.72 from 7 to 21 day in culture. We show that least 75% of HCY-induced currents in cerebellum were mediated by GluN2C- or GluN2D-containing NMDARs. Thus, our data revealed a large population of cerebellar NMDA receptors highly sensitive to HCY which suggest potential vulnerability of this brain region to pathological conditions associated with enhanced levels of this neurotoxic amino acid.


Assuntos
Cerebelo/citologia , Homocisteína/farmacologia , Neurônios/metabolismo , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/agonistas
10.
Nitric Oxide ; 81: 21-27, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30300735

RESUMO

Homocysteine (Hcy) is an independent risk factor for endothelial dysfunction in cardiovascular diseases. We hypothesized that the eNOS transcription enhancer AVE3085 may protect the endothelial function damaged by Hcy in the human internal mammary artery (IMA). Cumulative concentration-relaxation curves to acetylcholine (-10 to -4.5 log mol/L) or sodium nitroprusside were established in IMA from patients undergoing coronary artery surgery precontracted by U46619 (-8 log mol/L) in the absence/presence of Hcy (100 µmol/L) with/without AVE3085 (30 µmol/L) in vitro in a myograph. RT-qPCR and ELISA were used to quantify the mRNA and protein levels of eNOS. Colorimetric assay method was used to detect the production of nitric oxide (NO). Maximal relaxation was significantly attenuated by Hcy in human IMA. Co-incubation with AVE3085 protected endothelium from the impairment by Hcy and increased the production of NO. Exposure to Hcy for 24 h downregulated eNOS protein expression (P < 0.05) whereas it upregulated the expression of eNOS at mRNA levels (P < 0.05). The presence of AVE3085 in addition to Hcy significantly increased the eNOS protein (P < 0.05) and slightly decreased the mRNA level. The study for the first time revealed that in the human blood vessels (IMA) the clinically-relevant high concentration of Hcy directly causes endothelial dysfunction by downregulating eNOS protein that may be reversed by AVE3085. These findings not only provide new direction for protecting endothelium during coronary artery bypass grafting and improving long-term patency of the grafts, but also provide evidence to the use of eNOS enhancer in the patients with endothelial dysfunction in various pathological conditions.


Assuntos
Benzodioxóis/farmacologia , Endotélio Vascular/fisiopatologia , Homocisteína/metabolismo , Indanos/farmacologia , Artéria Torácica Interna/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Acetilcolina/farmacologia , Acetilcisteína/farmacologia , Endotélio Vascular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Homocisteína/farmacologia , Humanos , Artéria Torácica Interna/fisiopatologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Nitroprussiato/farmacologia , Técnicas de Cultura de Órgãos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
11.
Kidney Blood Press Res ; 43(5): 1516-1528, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30336454

RESUMO

BACKGROUND/AIMS: Previous studies have shown that homocysteine (Hcy) is an important intestinal-derived uremic toxin. However, whether Hcy is involved in the epithelial barrier dysfunction observed in uremia remains unclear. This study aimed to investigate the effect of Hcy on intestinal permeability and intestinal barrier structure and function in adenine-induced uremic rats. METHODS: Sprague-Dawley rats were divided into five groups: normal control (group NC), Hcy (group H), uremia (group U), uremia + Hcy (group UH), and uremia + Hcy + VSL#3 (group UHV). Experimental uremia was induced by intragastric adenine administration, and Hcy was injected subcutaneously. The animal models were assessed for renal function and pathological tissue staining. The pathological changes of intestinal tissue were observed by hematoxylin and eosin staining and electron microscopy. The serum and intestinal tissue levels of Hcy, interleukin (IL)-6, tumor necrosis factor (TNF)-α, superoxide dismutase (SOD), and malondialdehyde (MDA) as well as serum endotoxin and intestinal permeability were assessed. The levels of the tight junction proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) were assessed by western blotting. RESULTS: Blood analyses and renal pathology indicated that experimental uremia was induced successfully. Pathological damage to intestinal structure was most obvious in group UH. Serum and tissue Hcy, serum endotoxin, and intestinal permeability were significantly elevated in group UH. The protein levels of claudin-1, occludin, and ZO-1 were decreased to various degrees in group UH compared with groups NC, H, and U. The serum and tissue levels of IL-6, TNF-α, and MDA were significantly increased, while SOD activity was markedly decreased. Supplementation with the probiotic VSL#3 improved these parameters to various degrees and up-regulated the abundance of tight junction proteins, which indicated a role for Hcy in the increase of intestinal permeability and destruction of the epithelial barrier in uremia. CONCLUSION: Hcy aggravates the increase of intestinal permeability and destruction of the epithelial barrier by stimulating inflammatory and oxidative damage. Probiotic administration can ameliorate this damage by reducing the levels of Hcy-induced inflammation and oxidation.


Assuntos
Uremia/patologia , Animais , Endotoxinas/sangue , Epitélio/fisiopatologia , Homocisteína/farmacologia , Inflamação/induzido quimicamente , Mucosa Intestinal/fisiopatologia , Masculino , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
12.
Neurochem Int ; 120: 87-98, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30055195

RESUMO

Elevated homocysteine (Hcy) levels have been implicated in neurodevelopmental and neurodegenerative disorders. Induction of oxidative stress and apoptosis has been reported as major mechanism in Hcy-induced neurotoxicity. Hydrogen sulfide (H2S), as an antioxidant molecule has been reported to exhibit novel protective effect against Hcy-induced cell damage. However, the mechanisms involved in protective effect of H2S against Hcy-induced toxicity in neurons have not been fully elucidated. Herein, effect of sodium hydrogen sulfide (NaHS, a source of H2S) on Hcy-induced neurotoxicity was studied on Neuro-2a (N2a) cells in vitro and in animals subjected to hyperhomocysteinemia. DCFH-DA staining revealed that NaHS effectively attenuated Hcy-induced oxidative damage by reducing intracellular reactive oxygen species (ROS) generation. JC-1 staining and western blot results showed that NaHS pre-treatment prevented Hcy-induced mitochondrial dysfunctions and mitochondria-mediated apoptosis. MTT assay, cell cycle analysis, ethidium bromide/acridine orange (EB/AO) and Hoechst staining results demonstrated that NaHS significantly alleviated Hcy-induced cytotoxicity in N2a cells by preventing oxidative damage. Importantly, the results from agarose gel electrophoresis, comet and TUNEL assay indicated that NaHS also prevented neurodegeneration by reducing DNA damage and apoptotic cell death in animals with hyperhomocysteinemia. Taken together, the results demonstrate that the protective potential of H2S against Hcy-induced neurotoxicity is mediated by preventing oxidative DNA damage and mitochondrial dysfunctions. The findings validate that H2S is a promising therapeutic molecule in neurodegenerative conditions associated with hyperhomocysteinemia.


Assuntos
Encéfalo/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Homocisteína/farmacologia , Masculino , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Síndromes Neurotóxicas/tratamento farmacológico , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
13.
Neuroreport ; 29(12): 1030-1035, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29939872

RESUMO

Homocysteine (HCY), a redox-active metabolite of the methionine cycle, is of particular clinical interest because of its association with various neurodegenerative diseases including amyotrophic lateral sclerosis. It has been previously established that HCY exacerbates damage to motor neurons from reactive oxygen species (ROS) such as hydrogen peroxide. To assess the role of HCY at the mammalian neuromuscular junction, neurotransmission was monitored by electrophysiology at the mouse epitrochleoanconeus muscle. Preparations were preincubated in HCY before inducing ROS and recordings were taken before and after ROS treatment. In this study, HCY was observed to sensitize the neuromuscular junction to ROS-induced depression of spontaneous transmission frequency, an effect we found to be mediated by a N-methyl-D-aspartate receptor (NMDAR) and nitric oxide (NO). The NMDAR antagonist D, L-2-amino-5-phosphonopentanoic acid prevented the HCY-induced sensitization to oxidative stress. Disrupting NO activity with either the nitric oxide synthase I antagonist Nω-nitro-L-arginine methyl ester hydrochloride or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium salt also prevented sensitization. Moreover, replacing HCY with the exogenous NO donor Diethylamine NONOate diethylammonium was sufficient to reconstitute the effects of HCY-induced sensitization to ROS. Interestingly, a novel secondary effect was observed where HCY itself depresses quantal content, an effect found to be mediated by NMDARs independently of nitric oxide and ROS. Collectively, these data present a novel model of two distinct pathways through which HCY alters neurotransmission at the neuromuscular junction. Characterizing HCY's mechanism of action is of particular clinical relevance as many treatments for amyotrophic lateral sclerosis are centered on mitigating HCY-induced pathologies.


Assuntos
Homocisteína/farmacologia , Junção Neuromuscular/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Junção Neuromuscular/efeitos dos fármacos , Óxido Nítrico/agonistas , Óxido Nítrico/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Técnicas de Cultura de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
14.
Mol Med Rep ; 18(2): 1637-1643, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29901193

RESUMO

Homocysteine (Hcy) has been shown to impair the migratory and adhesive activity of endothelial progenitor cells (EPCs). As a peroxisome proliferator­activated receptor γ agonist, pioglitazone (PIO) has been predicted to regulate angiogenesis, and cell adhesion, migration and survival. The aim of the present study was to determine whether PIO could inhibit Hcy­induced EPC dysfunctions such as impairments of cell migration and adhesion. EPC migration and adhesion were assayed using 8.0­µm pore size Transwell membranes and fibronectin­coated culture dishes, respectively. Hcy at a concentration of 200 µM was observed to markedly impair cell migration and adhesiveness, and PIO at a concentration of 10 µM attenuated the Hcy­mediated inhibition of EPC migration and adhesion. The mechanism of these effects may be through the inhibition of protein kinase C (PKC) and reactive oxygen species production. The expression levels of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, NADPH oxidase 2 (Nox2) and p67phox, were upregulated by Hcy, with a peak in levels following treatment with a concentration of 200 µM. PIO downregulated the expression levels of Nox2 and p67phox via the PKC signaling pathway. Furthermore, the mechanism of PIO associated with downregulating the p67phox and Nox2 subunits of NADPH oxidase was verified. Thus, PKC and NADPH oxidase may serve a major role in the protective effects of PIO in EPCs under conditions of high Hcy concentrations.


Assuntos
Células Progenitoras Endoteliais/efeitos dos fármacos , Homocisteína/antagonistas & inibidores , Hipoglicemiantes/farmacologia , NADPH Oxidase 2/genética , Fosfoproteínas/genética , Tiazolidinedionas/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Cultura em Câmaras de Difusão , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica , Homocisteína/farmacologia , Humanos , NADPH Oxidase 2/antagonistas & inibidores , NADPH Oxidase 2/metabolismo , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Pioglitazona , Cultura Primária de Células , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
15.
EBioMedicine ; 31: 202-216, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29735414

RESUMO

The adipose Nod-like receptor protein 3 (NLRP3) inflammasome senses danger-associated molecular patterns (DAMPs) and initiates insulin resistance, but the mechanisms of adipose inflammasome activation remains elusive. In this study, Homocysteine (Hcy) is revealed to be a DAMP that activates adipocyte NLRP3 inflammasomes, participating in insulin resistance. Hcy-induced activation of NLRP3 inflammasomes were observed in both adipocytes and adipose tissue macrophages (ATMs) and mediated insulin resistance. Lysophosphatidylcholine (lyso-PC) acted as a second signal activator, mediating Hcy-induced adipocyte NLRP3 inflammasome activation. Hcy elevated adipocyte lyso-PC generation in a hypoxia-inducible factor 1 (HIF1)-phospholipase A2 group 16 (PLA2G16) axis-dependent manner. Lyso-PC derived from the Hcy-induced adipocyte also activated ATM NLRP3 inflammasomes in a paracrine manner. This study demonstrated that Hcy activates adipose NLRP3 inflammasomes in an adipocyte lyso-PC-dependent manner and highlights the importance of the adipocyte NLRP3 inflammasome in insulin resistance.


Assuntos
Adipócitos/metabolismo , Homocisteína/farmacologia , Inflamassomos/metabolismo , Resistência à Insulina , Lisofosfatidilcolinas/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Adipócitos/química , Adipócitos/citologia , Animais , Feminino , Humanos , Inflamassomos/genética , Lisofosfatidilcolinas/química , Macrófagos/citologia , Masculino , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética
16.
J Cell Biochem ; 119(8): 6684-6694, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29693751

RESUMO

MicroRNAs (miRs) are widely reported to be novel biomarkers involved in the process of coronary atherosclerosis (CAS). Hence, this study aims to explore the function of miR-383-3p targeting IL1R2 on inflammatory injury of coronary artery endothelial cells (CAECs) in CAS. The underlying regulatory mechanisms of miR-383-3p were analyzed in concert with the treatment of miR-383-3p mimics, miR-383-3p inhibitors, and the combination of miR-383-3p inhibitors and siRNA against IL1R2 in homocysteine (HCY)-induced CAECs. MTT, Hoechst 33258 staining, and tube formation assay were employed in order to measure cell viability, apoptosis, and tube formation, respectively. The levels of IL-1ß, IL-6, IL-10, and IL-18 were determined by ELISA. IL1R2 was verified as the target gene of miR-383-3p by dual-luciferase reporter gene assay. MiR-383-3p was down-regulated in myocardial tissues of AS rats while IL1R2 was the reciprocal. The up-regulation of miR-383-3p decreased the levels of IL1R2, caspase-1, IL-1ß, IL-6, and IL-18 expressions, as well as cell apoptosis rate in the HCY-induced CAECs, while IL-10 expression, cell viability, and tube formation ability were increased. These results were contraindicated in the HCY-induced CAECs treated by miR-383-3p inhibitors. In conclusion, miR-383-3p mediating IL1R2 prevents HCY-induced apoptosis and inflammation injury in CAECs through the inhibition of the activation of inflammasome signaling pathway. These findings highly indicate that miR-383-3p may be beneficial in the prevention of CAS and other cardiovascular diseases.


Assuntos
Doença da Artéria Coronariana/metabolismo , Vasos Coronários/metabolismo , Endotélio Vascular/lesões , Homocisteína/efeitos adversos , MicroRNAs/metabolismo , Receptores Tipo II de Interleucina-1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Doença da Artéria Coronariana/induzido quimicamente , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Citocinas/genética , Citocinas/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Homocisteína/farmacologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Receptores Tipo II de Interleucina-1/genética
17.
Ann Clin Lab Sci ; 48(1): 126-131, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29531009

RESUMO

The indoleamine hormone melatonin is synthesized by the pineal gland, controls circadian rhythm, and is dependent upon adenosyl methionine for enzymatic synthesis of melatonin from N-acetyl serotonin. Pineal melatonin secretion declines dramatically with aging and dementia. Elevated plasma homocysteine is a risk factor for atherosclerosis and Alzheimer's disease, and the marked decline in adenosyl methionine with aging leads to dysregulation of methionine metabolism and hyperhomocysteinemia. Thioretinaco ozonide is a disulfonium complex formed from thioretinamide, cobalamin, and ozone, which binds the alpha and gamma-phosphate groups of adenosine triphosphate (ATP) and oxygen in the process of oxidative phosphorylation within mitochondria. Decreased adenosyl methionine concentrations with aging are attributed to the loss of thioretinaco ozonide from mitochondria, impairing adenosyl methionine synthesis from thioretinaco ozonide and ATP. Melatonin is present in mitochondria, where it inhibits the opening of the mitochondrial permeability transition pore, explaining its anti-oxidant and anti-apoptotic effects by reducing oxygen consumption, restoration of membrane potential and reduction of superoxide production. In aging, the enzyme cyclic nucleotide phosphodiesterase is lost from mitochondria by the opening of the permeability transition pore and disruption of the outer mitochondrial membrane, a process that is inhibited by melatonin. Thioretinaco ozonide is progressively lost from dysfunctional mitochondria by disruption of the outer mitochondrial membrane, explaining its depletion during the aging process. Accordingly, the anti-aging effects of diallyl trisulfide and metformin are attributable to inhibition of the opening of the mitochondrial permeability transition pore, preventing loss of thioretinaco ozonide from mitochondria. The hyperhomocysteinemia and suppressed immunity that are observed in atherosclerosis and dementia are attributed to the deficiency of adenosylmethionine caused by increased polyamine synthesis and decreased nitric oxide synthesis by host cells infected with pathogenic microbes. According to this analysis, the critical loss of thioretinaco ozonide from mitochondria through the opening of the permeability transition pore and disruption of the outer mitochondrial membrane by decreased melatonin secretion leads to the impaired oxidative phosphorylation, oxidative stress, calcium influx, apoptosis and mitochondrial dysfunction observed in aging and dementia.


Assuntos
Homocisteína/análogos & derivados , Hiper-Homocisteinemia/fisiopatologia , Melatonina/farmacologia , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , S-Adenosilmetionina/metabolismo , Vitamina B 12/análogos & derivados , Aterosclerose , Demência , Homocisteína/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Vitamina B 12/farmacologia
18.
Microvasc Res ; 119: 13-21, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29601873

RESUMO

The change of autophagy is implicated in cardiovascular diseases (CVDs). Homocysteine (Hcy) up-regulates endothelin type B (ETB) receptors in vascular smooth muscle cells (VSMCs). However, it is unclear whether autophagy is involved in Hcy-induced-up-regulation of ETB receptors in VSMCs. The present study was designed to examine the hypothesis that Hcy up-regulates ETB receptors by inhibiting autophagy in VSMCs. Hcy treated the rat superior mesenteric artery (SMA) without endothelium in the presence and absence of AICAR, rapamycin or MHY1485 for 24 h. The contractile responses to sarafotoxin 6c (S6c) (an ETB receptor agonist) were studied using a sensitive myograph. Levels of protein expression were determined using Western blot analysis. Punctate staining of LC3B was exanimated by immunofluorescence using confocal microscopy. The results showed that Hcy inhibited AMPK, and activated mTOR, followed by impairing autophagy, and increased the levels of ETB receptor protein expression and the ETB receptor-mediated contractile responses to S6c in SMA without endothelium. However, these effects were reversed by AICAR or rapamycin. Additionally, MHY1485 up-regulated the AICAR-inhibited ETB receptor-mediated contractile response and the levels of ETB receptor protein expression in presence of Hcy. In conclusion, this suggested that Hcy up-regulated ETB receptors by inhibiting autophagy in VSMCs via AMPK/mTOR signaling pathway.


Assuntos
Autofagia/efeitos dos fármacos , Homocisteína/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Humanos , Técnicas In Vitro , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/metabolismo , Artéria Mesentérica Superior/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ratos Sprague-Dawley , Receptor de Endotelina B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima , Vasoconstrição/efeitos dos fármacos
19.
Cell Death Dis ; 9(2): 169, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29415998

RESUMO

Elevated homocysteine (Hcy) levels have been reported to be involved in liver injury, and autophagy plays an important role in normal hepatic physiology and pathophysiology, but the mechanism underlying Hcy regulated autophagy is currently unknown. In this study, CBS+/- mice were fed with regular diet for 12 weeks to establish a hyperhomocysteinemia (HHcy) model and HL-7702 cells were treated with Hcy, we found that Hcy increases autophagy and aggravates liver injury by downregulation of cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo and in vitro. Overexpression of CFTR inhibited the formation of autophagosomes and the expression of autophagy-related proteins BECN1, LC3-II/I and Atg12, while the expression of p62 increased in Hcy-treated hepatocytes and CBS+/- mice injected with lentivirus expressing CFTR. Further study showed that CFTR expression is regulated by the interaction of DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2), which, respectively, regulate DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3). In conclusion, our study showed that Hcy activates autophagy by inhibition of CFTR expression via interaction between H3K27me3 and DNA methylation in the mouse liver. These findings provide new insight into the mechanism of Hcy-induced autophagy in liver injury.


Assuntos
Autofagia/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Metilação de DNA/genética , Histonas/metabolismo , Homocisteína/farmacologia , Fígado/metabolismo , Lisina/metabolismo , Animais , Biomarcadores/metabolismo , Cistationina beta-Sintase/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lentivirus/metabolismo , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Camundongos , Regiões Promotoras Genéticas/genética
20.
Mol Med Rep ; 17(4): 5312-5319, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29393420

RESUMO

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in the development of atherosclerosis (AS), and the presence of homocysteine (Hcy) acts as an independent risk factor for AS. However, the underlying mechanisms remain to be elucidated. Phosphatase and tensin homologue on chromosome 10 (PTEN), is endogenously expressed in VSMCs and induces multiple signaling networks involved in cell proliferation, survival and inflammation, however, the specific role of PTEN is still unknown. The present study detected the proliferation ratio of VSMCs following treatment with Hcy and Resveratrol (RSV). In the 100 µM Hcy group, the proliferation ratio increased, and treatment with RSV decreased the proliferation ratio induced by Hcy. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to analyze PTEN expression, RSV treatment was associated with decreased PTEN expression levels in VSMCs. PTEN levels were decreased in Hcy treated cells, and the proliferation ratio of VSMCs were increased following treated with Hcy. To study the mechanism of regulation of PTEN by Hcy, the present study detected PTEN methylation levels in VSMCs, and PTEN DNA methylation levels were demonstrated to be increased in the 100 µM Hcy group, whereas treatment with RSV decreased the methylation status. DNA methyltransferase 1 is important role in the regulation of PTEN methylation. Overall, Hcy impacts the methylation status of PTEN, which is involved in cell proliferation, and induces the proliferation of VSMCs. This effect is alleviated by treatment with RSV, which exhibits an antagonistic mechanism against Hcy.


Assuntos
Metilação de DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Homocisteína/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , PTEN Fosfo-Hidrolase/genética , Estilbenos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Homocisteína/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Resveratrol
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