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1.
BMC Nephrol ; 20(1): 135, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30999892

RESUMO

AIMS: Sphingosine-1-phosphate receptor 2 (S1PR2) is a G-protein-coupled receptor that regulates sphingosine-1-phosphate-triggered cellular response. However, the role of S1PR2 in diabetes-induced glomerular endothelial cell dysfunction remains unclear. This study aims to investigate the effect of S1PR2 blockade on the morphology and function of mitochondria in human renal glomerular endothelial cells (HRGECs). METHODS: HRGECs were pretreated with a S1PR2 antagonist (JTE-013) or a Rho-associated coiled coil-containing protein kinase 1 (ROCK1) inhibitor (Y27632) for 30 min and then cultured with normal glucose (5.5 mM) or high glucose (30 mM) for 72 h. The protein expression levels of RhoA, ROCK1, and Dynmin-related protein-1(Drp1) were evaluated by immunoblotting; mitochondrial morphology was observed by electron microscopy; intracellular levels of ATP, ROS, and Ca2+ were measured by ATPlite, DCF-DA, and Rhod-2 AM assays, respectively. Additionally, the permeability, apoptosis, and migration of cells were determined to evaluate the effects of S1PR2 and ROCK1 inhibition on high glucose-induced endothelial dysfunction. RESULTS: High glucose induced mitochondrial fission and dysfunction, indicated by increased mitochondrial fragmentation, ROS generation, and calcium overload but decreased ATP production. High glucose also induced endothelial cell dysfunction, indicated by increased permeability and apoptosis but decreased migration. However, inhibition of either S1PR2 or ROCK1 almost completely blocked these high glucose-mediated cellular responses. Furthermore, inhibiting S1PR2 resulted in the deceased expression of RhoA, ROCK1, and Drp1 while inhibiting ROCK1 led to the downregulated expression of Drp1. CONCLUSIONS: S1PR2 antagonist modulates the morphology and function of mitochondria in HRGECs via the positive regulation of the RhoA/ROCK1/Drp1 signaling pathway, suggesting that the S1PR2/ROCK1 pathway may play a crucial role in high glucose milieu.

2.
Immunobiology ; 224(3): 339-346, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30975435

RESUMO

A deficiency of complement factor H may lead to excessive consumption of C3 and an increase in C3b deposition, which are important pathological characteristics of lupus nephritis. Complement factor H-related proteins (CFHRs), comprising CFHR1 to CFHR5 (CFHR1-5), are members of the wider factor H/CFHR family. Their role in lupus nephritis remains unclear. In this study, we compared circulating levels of CFHR1-5 in 152 patients diagnosed with lupus nephritis and 20 unrelated healthy individuals to explore the relationship between the expression of CFHR1-5 and development of the disease. We found that plasma levels of CFHR3 and CFHR5 were higher in patients with lupus nephritis than in healthy individuals; also, CFHR3 and CFHR5 concentrations increased with increasing systemic lupus erythematosus disease activity index (SLEDAI) values (P < 0.05). Pearson's and Spearman's correlation test results confirmed that plasma CFHR3 and CFHR5 levels in lupus nephritis patients were positively correlated with proteinuria and levels of creatinine (Cr) and anti-dsDNA (correlation coefficients = 0.491-0.717, P < 0.05), while they were negatively correlated with plasma C3 levels and eGFR [correlation coefficients = -(0.706-0.788), P < 0.05]. Receiver operating characteristic (ROC) curve analysis results confirmed that plasma CFHR3 and CFHR5 levels were predictive of SLEDAI values and disease end points (area under the curve = 0.664-0.884, P < 0.05), with patients with both high CFHR3 and high CFHR5 exhibiting the shortest progression-free survival. Thus, both CFHR3 and CFHR5 are of prognostic value in lupus nephritis status.

3.
Exp Ther Med ; 17(4): 2425-2432, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30906429

RESUMO

It is well known that the lipotoxic mechanism of palmitic acid (PA), a main constituent of triglyceride, is dependent on reactive oxygen species (ROS). Recently, it has also been reported that PA is an autophagy inducer. However, the causal association and underlying mechanism of induced autophagy and ROS in PA toxicity remain unclear. The present study demonstrates for the first time that PA-induced autophagy enhances ROS generation via activating the calcium ion/protein kinase Cα/nicotinamide adenine dinucleotide phosphate oxidase 4 (Ca2+/PKCα/NOX4) pathway in human umbilical vein endothelial cells (HUVECs). It was revealed that PA treatment resulted in a significant increase in ROS generation and autophagic activity, leading to endothelial dysfunction as indicated by downregulated nitric oxide synthesis, decreased capillary-like structure formation and damaged cell repair capability. Furthermore, PA effectively activated the Ca2+/PKCα/NOX4 pathway, which is indicative of upregulated cytosolic Ca2+ levels, activated PKCα and increased NOX4 protein expression. 3-Methyladenine was then used to inhibit autophagy, which significantly reduced PA-induced ROS generation and blocked the Ca2+/PKCα/NOX4 pathway. The endothelial dysfunction caused by PA was ameliorated by downregulating ROS generation using a NOX4 inhibitor. In conclusion, PA-induced autophagy contributes to endothelial dysfunction by increasing oxidative stress via the Ca2+/PKCα/NOX4 pathway in HUVECs.

4.
J Cell Mol Med ; 23(2): 798-810, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30444033

RESUMO

The dipeptidyl peptidase 4 inhibitor vildagliptin (VLD), a widely used anti-diabetic drug, exerts favourable effects on vascular endothelium in diabetes. We determined for the first time the improving effects of VLD on mitochondrial dysfunction in diabetic mice and human umbilical vein endothelial cells (HUVECs) cultured under hyperglycaemic conditions, and further explored the mechanism behind the anti-diabetic activity. Mitochondrial ROS (mtROS) production was detected by fluorescent microscope and flow cytometry. Mitochondrial DNA damage and ATP synthesis were analysed by real time PCR and ATPlite assay, respectively. Mitochondrial network stained with MitoTracker Red to identify mitochondrial fragmentation was visualized under confocal microscopy. The expression levels of dynamin-related proteins (Drp1 and Fis1) were determined by immunoblotting. We found that VLD significantly reduced mtROS production and mitochondrial DNA damage, but enhanced ATP synthesis in endothelium under diabetic conditions. Moreover, VLD reduced the expression of Drp1 and Fis1, blocked Drp1 translocation into mitochondria, and blunted mitochondrial fragmentation induced by hyperglycaemia. As a result, mitochondrial dysfunction was alleviated and mitochondrial morphology was restored by VLD. Additionally, VLD promoted the phosphorylation of AMPK and its target acetyl-CoA carboxylase in the setting of high glucose, and AMPK activation led to a decreased expression and activation of Drp1. In conclusion, VLD improves endothelial mitochondrial dysfunction in diabetes, possibly through inhibiting Drp1-mediated mitochondrial fission in an AMPK-dependent manner.

5.
Eur J Pharmacol ; 845: 91-98, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30287151

RESUMO

Vitamin D has been suggested to harbor multiple biological activities, among them the potential of vitamin D in the protection of diabetic nephropathy (DN) has attracted special attention. Both animal studies and clinical trials have documented an inverse correlation between low vitamin D levels and DN risk, and supplementation with vitamin D or its active derivatives has been demonstrated to improve endothelial cell injury, reduce proteinuria, attenuate renal fibrosis, and resultantly retard DN progression. Vitamin D exerts its pharmacological effects primarily via vitamin D receptor, whose activation inhibits the renin-angiotensin system, a key culprit for DN under hyperglycemia. The anti-DN benefit of vitamin D can be enhanced when administrated in combination with angiotensin converting enzyme inhibitors or angiotensin II receptor blockers. Mechanistic studies reveal that pathways relevant to inflammation participate in the pathogenesis of DN, however, consumption of vitamin D-related products negatively regulates inflammatory response at multiple levels, indicated by inhibiting macrophage infiltration, nuclear factor-kappa B (NF-κB) activation, and production of such inflammatory mediators as transforming growth factor-ß(TGF-ß), monocyte chemoattractant protein 1(MCP-1), and regulated upon activation normal T cell expressed and secreted protein(RANTES). The robust anti-inflammatory property of vitamin D-related products allows them with a promising renoprotective therapeutic option for DN. This review summarizes new advances in our understanding of vitamin D-related products in the DN management.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Proteinúria/tratamento farmacológico , Receptores de Calcitriol/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Vitamina D/uso terapêutico , Antagonistas de Receptores de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CCL5/efeitos dos fármacos , Quimioterapia Combinada , Células Endoteliais/patologia , Humanos , NF-kappa B/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Fator de Crescimento Transformador beta/efeitos dos fármacos , Vitamina D/farmacologia
6.
Mol Med Rep ; 17(1): 1998-2004, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29257217

RESUMO

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) contributes to dysfunction of endothelial cells via its receptor, Fn14. However, its role in the production of reactive oxygen species (ROS), particularly mitochondrial ROS (mtROS) and the subsequent decrease in nitric oxide (NO) in endothelial cells remains unclear. In this study, the effect of TWEAK/Fn14 on generation of ROS, mtROS and NO in endothelial cells and its potential mechanism was investigated. Human umbilical vein endothelial cells (HUVECs) were treated with TWEAK with Fn14 small interfering (si)RNA or negative control RNA. It was demonstrated that TWEAK induced the production of ROS and mtROS in HUVECs, which were detected by fluorescent microscope, and flow cytometry. In addition, TWEAK decreased the generation of NO as indicated using the Nitric Oxide Assay kit. Furthermore, TWEAK aggravated mtDNA damage as measured by quantitative polymerase chain reaction analysis. Inhibition of Fn14 by Fn14 siRNA decreased TWEAK­induced ROS and mtROS production, as well as mtDNA damage, while it increased the production of NO in endothelial cells. In addition, TWEAK inhibited the expression of active AMP­activated protein kinase (AMPK) and its downstream protein peroxisome proliferator­activated receptor­Î³ coactivator-1α (PGC­1α) and manganese superoxide dismutase (MnSOD). Notably, Fn14 siRNA enhanced the expression of the aforementioned proteins. Taken together, TWEAK/Fn14 contributes to endothelial dysfunction through modulation of ROS and mtROS. In addition, the underlying mechanism is implicated in the AMPK/PGC­1α/MnSOD signaling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Citocina TWEAK/metabolismo , Células Endoteliais/metabolismo , Estresse Oxidativo , Transdução de Sinais , Receptor de TWEAK/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
7.
Exp Ther Med ; 14(2): 891-897, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28781615

RESUMO

At present, it is commonly accepted that atherosclerosis is a chronic inflammatory disease characterized by disorder of the arterial wall. As one of the inflammatory cytokines of the tumor necrosis factor superfamily, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) participates in the formation and progression of atherosclerosis. TWEAK, when binding to its initial receptor, fibroblast growth factor inducible molecule 14 (Fn14), exerts adverse biological functions in atherosclerosis, including dysfunction of endothelial cells, phenotypic change of smooth muscle cells and inflammatory responses of monocytes/macrophages. However, accumulating data supports that, besides Fn14, TWEAK also binds to cluster of differentiation (CD)163, an anti-inflammatory cytokine and a scavenger receptor exclusively expressed by monocytes and macrophages. Furthermore, it has been demonstrated that CD163 is able to internalize TWEAK and likely elicits protective effects in atherosclerosis by terminating inflammation induced by TWEAK. In the present study, the role of TWEAK in atherosclerosis was reviewed, with a predominant focus on CD163 and Fn14 receptors.

8.
Diab Vasc Dis Res ; 14(6): 494-501, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28777009

RESUMO

OBJECTIVE: Mitochondrial Ca2+ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment. METHODS: Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2',7'-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model. RESULTS: High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p < 0.05). These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively. CONCLUSION: Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rutênio Vermelho/farmacologia , Espermina/farmacologia , Fatores de Tempo , Regulação para Cima
9.
Biochem Biophys Res Commun ; 490(3): 1119-1124, 2017 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-28676392

RESUMO

Vascular complications are the main cause of morbidity and mortality associated with type 2 diabetes mellitus. An early hallmark of the onset of vascular complications is endothelial dysfunction and apoptosis. We aimed to explore the role of sphingosine-1-phosphatereceptor 2 (S1PR2) in high glucose-induced endothelial cells apoptosis and to elaborate the underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were cultured in a high glucose with or without S1PR2 antagonist. The apoptosis of the cells was measured by flow cytometry and mitochondrial membrane permeability was detected by the fluorescent probe JC-1. The expression of the related protein was determined by western blot. Cell apoptosis and the loss of mitochondrial membrane permeability were induced under high glucose conditions in HUVECs. The expression of mitochondrial apoptosis related protein bax increased and bcl-2 decreased in high glucose-induced HUVECs. The level of cytochrome c released into the cytoplasm increased when cells were exposed to high glucose. In addition, the expression of p-AKT and p-GSK3ß was reduced when HUVECs were treated with high glucose. However, these effects were reversed in HUVECs when cells treated with S1PR2 antagonist. In conclusion, S1PR2 antagonist protects endothelial cells against high glucose-induced mitochondrial apoptosis through the Akt/GSK-3ß signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Glucose/metabolismo , Substâncias Protetoras/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo
10.
Int J Clin Exp Med ; 7(4): 879-85, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24955157

RESUMO

OBJECTIVE: This study aimed to investigate the molecular mechanism underlying the myeloperoxidase (MPO) induced apoptosis of human umbilical vein endothelial cells (HUVECs). METHODS: HUVEC-12 cells were treated with myeloperoxidase at different concentrations (0.1 µ/ml, 0.2 µ/ml, 0.4 µ/ml and 0.6 µ/ml) and apoptotic cells were detected by flow cytometry. Then, cells were harvested for the detection of mRNA and protein expression of caspase-3 and Bax by reverse transcription PCR and Western blot assay, respectively. RESULTS: When compared with negative control group, the apoptosis index in 0.2 µ/ml MPO group, 0.4 µ/ml MPO group and 0.6 µ/ml MPO group increased markedly (P<0.05). When compared with negative control group, the mRNA expression of caspase-3 in 0.6 µ/ml MPO group and positive control group increased dramatically (P<0.05). When compared with negative control group, the protein expression of pre-caspase-3 and activated caspase-3 elevated significantly in 0.4 µ/ml MPO group, 0.6 µ/ml MPO group and positive control group (P<0.05). When compared with negative control group, the mRNA and protein expression of Bax elevated dramatically in 0.4 µ/ml MPO group, 0.6 µ/ml MPO group and positive control group (P<0.05). CONCLUSION: MPO at certain extents may induce the apoptosis of HUVEC-12. The MPO induced apoptosis of HUVEC-12 may be dependent on capase-3 signaling pathway, and Bax is also involved in the MPO induced apoptosis of HUVEC-12.

11.
Exp Ther Med ; 7(5): 1177-1180, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24940407

RESUMO

The aim of this study was to investigate the correlation between the severity of coronary artery lesions in patients with acute coronary syndromes (ACS) and levels of estrogen, high-sensitivity C-reactive protein (hs-CRP) and matrix metalloproteinase-9 (MMP-9). A total of 65 patients with ACS, 33 patients with stable angina pectoris (SAP) and 36 healthy controls were randomly enrolled. Patients with ACS were subdivided into two groups: Acute myocardial infarction (AMI; n=30) and unstable angina pectoris (UAP; n=35). Serum levels of estrogen, hs-CRP and MMP-9 were detected in the four groups of subjects. Serum estrogen levels in patients with AMI, UAP and SAP were significantly lower than those in the control group (P<0.05). Estrogen levels were also significantly different among the AMI, UAP and SAP groups (P<0.05), with a progressive increase across the three respective groups. Compared with healthy subjects, patients with AMI had the highest levels of hs-CRP and MMP-9, followed in descending order by those with UAP and SAP (P<0.05). Levels of hs-CRP and MMP-9 were also significantly different among the AMI, UAP and SAP groups (P<0.05). Serum estrogen levels were negatively correlated with hs-CRP and MMP-9 levels (r=-0.6634 and -0.6878, respectively; both P<0.05). hs-CRP and MMP-9 levels correlated positively (r=0.7208, P<0.05). The number of stenosed coronary vessels was negatively correlated with estrogen levels (r=-0.6467, P<0.05), and positively correlated with hs-CRP and MMP-9 levels (r=0.6519 and 0.6835, respectively; both P<0.05). In conclusion, serum estrogen, hs-CRP and MMP-9 levels were significantly correlated with the severity of coronary artery lesions. There was also a significant correlation between serum estrogen, hs-CRP and MMP-9 levels. These data indicate that serum estrogen, hs-CRP and MMP-9 have the potential to be used as biomarkers for evaluating the severity of coronary artery lesions and the stability of coronary artery plaques.

12.
Genet Mol Biol ; 36(2): 177-82, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23885198

RESUMO

Osteoprotegerin (OPG) gene polymorphisms (T245G, T950C and G1181C) have been associated with osteoporosis and early predictors of cardiovascular disease. The aim of this study was to evaluate whether these polymorphisms contribute to cardiovascular disease (CVD) in type 2 diabetic patients. We performed a case-control study with 178 CVD subjects with diabetes and 312 diabetic patients without CVD to assess the impact of variants of the OPG gene on the risk of CVD. The OPG gene polymorphisms were analyzed by using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). There was no significant association between the T245G and G1181C polymorphisms and CVD in the additive genetic model (OR = 0.96, 95% CI 0.64-1.45, p = 0.79; OR = 1.06, 95% CI 0.81-1.39, p = 0.65, respectively). However, the C allele of the T950C polymorphism was independently associated with a risk of CVD in type 2 diabetic patients in this genetic model (OR = 1.38, 95% CI 1.07-1.80, p = 0.01). This study provides evidence that the C allele of the T950C polymorphism is associated with increased risk of CVD in diabetic patients. However, well-designed prospective studies with a larger sample size are needed to validate these results.

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