银杏叶提取物对脂多糖介导的血管内皮细胞低密度脂蛋白受体LOX-1表达的影响

[目的]观察银杏叶提取物（GBE50）对脂多糖（LPS）诱导的人脐静脉内皮细胞（HUVECs）氧化低密度脂蛋白受体（lectin like oxidized low density lipoprotein receptor,LOX-1）表达的影响.[方法]应用LPS刺激体外培养的HUVECs 24h.RT-PCR方法检测（lectin like oxidized low density lipoprotein receptor mRNA,LOX-1 mRNA）表达水平;采用蛋白质印迹分析检测LOX-1蛋白表达水平.[结果]LPS （1 μg/ mL）上调LOX-1 mRNA和蛋白表达水平,GBE50（40 μg/mL）轻度抑制LPS介导的LOX-1 mRNA（P＜0.005）和蛋白表达增加（但没有统计学意义,P＞0.05）,GBE50（80 μg/mL）及NF-κB抑制剂CAPE（20 μg/mL）明显抑制LOX-1 mRNA和蛋白表达上调（P＜0.01）.[结论] GBE50可能通过抑制LOX-1表达,在防治动脉粥样硬化中发挥作用.     

重组人血管内皮抑制素联合顺铂抗血管形成作用的实验研究

目的:揭示新型重组人血管内皮抑制素(恩度,YH-16)与顺铂(CDDP)联合使用的抗血管形成作用.方法:首先以细胞增殖抑制、克隆形成实验和细胞凋亡分析,考察YH-16联合CDDP对人脐静脉内皮细胞(HUVECs)、肝癌细胞系QGY-7701和SMMC-7721生物学活性的影响;其次,在HUVECs体外培养体系上,评价两药联合使用对细胞迁徙/侵袭和管道形成的抑制作用.结果:YH-16与CDDP联合使用能协同地抑制HUVECs增殖和克隆形成,并增强了诱导细胞凋亡,上述作用具有血管内皮细胞特异性.两药联合使用对HUVECs的迁徙/侵袭和管道形成也有协同的抑制作用.结论:在血管形成的多个环节上,YH-16与CDDP联合使用提高了抗血管形成的效能,联合用药模式具有临床推广价值.     

Dietary Nucleotides Retard Oxidative Stress-Induced Senescence of Human Umbilical Vein Endothelial Cells

Several lines of evidence suggest an inhibitory role of dietary nucleotides (NTs) against oxidative stress and inflammation, which promote senescence in age-associated cardiovascular diseases. We sought to test whether the dietary NTs could retard the hydrogen peroxide (H₂O₂)-induced senescence of human umbilical vein endothelial cells (HUVECs) and to elucidate the efficiency of different NTs as well as the potential mechanism. Senescence was induced in HUVECs by 4 h exposure to 200 µM H₂O₂ and was confirmed using senescence-associated-β-galactosidase staining (SA-β-gal), cell viability, and Western blot analyses of p16^INK4A and p21^Waf1/Cip1 after 24 h administration of growth medium. We find that NTs retards oxidative stress-induced HUVECs senescence, as shown by a lower percentage of SA-β-gal-positive cells, lower expression of p16^INK4A, and p21^Waf1/Cip1 as well as higher cell viability. GMP100 was the most excellent in delaying HUVECs senescence, which was followed by the NTs mixture, NMN, CMP50, and UMP50/100, while AMP retards HUVECs senescence by specifically reducing p15^INK4b expression. NTs all have significant anti-inflammatory effects; AMP and CMP were more prominent in restoring mitochondrial function, GMP and CMP were more competent at eliminating ROS and MDA, while AMP and UMP were more efficient at enhancing antioxidant enzyme activity. The role of the NTs mixture in retarding HUVECs senescence is full-scaled. These results stated that the mechanisms of NTs retarding HUVECs senescence could be related to its antioxidant and anti-inflammation properties promoting cell proliferation and protecting mitochondrial function activities.     

甜橙黄酮对斑马鱼及人脐静脉内皮细胞抗血管新生活性的作用

目的:观察甜橙黄酮在斑马鱼及人脐静脉内皮细胞(HUVECs)上的抗血管新生能力,并探讨其相关作用机制。方法:以150 nmol/L内皮细胞生长因子受体酪氨酸激酶抑制剂(VRI)为阳性对照组、以相应浓度DMSO为空白对照组,观察不同浓度(3,10,30μmol/L)甜橙黄酮对转基因斑马鱼血管系统的影响,并通过实时荧光定量PCR检测相关血管新生基因表达的影响;分别采用XTT与流式细胞术检测3～100μmol/L甜橙黄酮对20 ng/ml内皮细胞生长因子(VEGF)诱导的HUVECs增殖和细胞周期的影响。结果:甜橙黄酮可以剂量依赖性地抑制斑马鱼节间血管(ISV)的形成,下调血管新生相关基因hras、kdrl和vegfaa的表达;可剂量依赖性抑制VEGF诱导的HUVECs增殖,并诱导HUVECs停滞于细胞周期G0/G1期。结论:甜橙黄酮可能通过阻滞细胞周期和影响相关基因的表达达到在斑马鱼体内模型和HUVECs体外模型的抗血管新生作用。     

GLP-1 promotes angiogenesis in human endothelial cells in a dose-dependent manner,through the Akt,Src and PKC pathways

Introduction

Novel anti-diabetic medications that mimic or augment the physiological actions of GLP-1 improve cardiovascular risk factors in diabetics and GLP-1 has been proposed to have a beneficial role in the cardiovascular system. GLP-1 may have a direct cardioprotective role by decreasing infarct size and protecting from ischemia–reperfusion injury while prolonging survival in rodent models. The mechanisms underlying these observations remain largely unknown. In vitro studies suggest that GLP-1 may promote endothelial cell proliferation, but no study to date has evaluated a potential direct effect of GLP-1 on angiogenesis.

Specific Aim

To evaluate whether GLP-1 affects angiogenesis in humans and to elucidate underlying molecular mechanisms.

Material and Methods

We utilized a 3D culture system where spherules of human umbilical vein endothelial cells (HUVECs) embedded in a collagen scaffold were treated with escalating doses of human recombinant GLP-1 (50–2000 nmol/L) and the formation of new vessels was observed and quantified. Signaling inhibitors were utilized to identify molecular pathways through which GLP-1 promotes angiogenesis.

Results

We demonstrate that GLP-1 promotes angiogenesis in a dose-dependent manner. The maximum effect on angiogenesis was observed at a GLP-1 dose of 500 nmol/L, while increased angiogenesis occurred in response to doses ranging from 200 nmol/L to 1000 nmol/L. Pre-treatment of the system with Akt inhibitor IV, Bisindolylmaleimide (PKC inhibitor) and src inhibitor I resulted in a significant decrease of the GLP-1 induced angiogenesis.

Conclusions

This is the first study to demonstrate that GLP-1 promotes angiogenesis in a HUVEC three dimensional in vitro model. This effect requires pharmacological doses and is mediated through the Akt, PKC and src pathways.     

A key mediator,PTX3, of IKK/IκB/NF-κB exacerbates human umbilical vein endothelial cell injury and dysfunction

Objective: This study was performed to investigate PTX3-mediated iNOS expression and IKK/IκB/NF-κB activation in PA-induced atherosclerotic HUVECs injury model. Methods: The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining. Expression of genes and proteins were analyzed by real-time PCR and western blotting respectively. Cells were transfected with siRNAs as a gene silencing methods. Results: PA induced cell apoptosis in human umbilical vein endothelial cells in a time and dose-dependent manner. PA also induced upregulation expression of PTX3. TPCA-1, an inhibitor of IKK-2, could suppress the expression of PTX3 and phospho-IκB-α in PA-induced endothelial dysfunction cell model. We also found that transfection of cells with PTX3 siRNA reduced the expression of iNOS and NO, and protected PA-induced cell apoptosis in HUVECs. Conclusions: PTX3 could exacerbate endothelial dysfunction, at least partially, through IKK/IκB/NF-κB activation and overexpression of iNOS and NO, and advance the development of atherosclerosis.     

Crosstalk between oxidative stress,autophagy and apoptosis in hemoporfin photodynamic therapy treated human umbilical vein endothelial cells

BackgroundPhotodynamic therapy (PDT) provides a treatment for port-wine stain (PWS) using hemoporfin (hematoporphyrin monomethyl ether, HMME), a novel photosensitizer, reporting better efficacy and lower recurrence rate. This study investigated the effects of HMME-PDT on human umbilical vein endothelial cells (HUVECs) as well as underlying mechanisms.MethodsCell proliferation ability was measured by CCK8 assay and cell apoptosis was determined by TUNEL assay and Western blot analysis. Confocal fluorescence microscopy monitoring RFP-GFP-LC3 transfected HUVECs and Western blot analysis were used to evaluate autophagy. 3-Methyladenine (3-MA), Z-VAD-FMK, N-acetylcysteine (NAC) were used for inhibitor studies.ResultsHMME-PDT decreased cell proliferation ability in an HMME concentration and light dose-dependent manner. Oxidative stress played an important role in HMME-PDT induced cell apoptosis and autophagy in HUVECs. Pretreatment with Z-VAD-FMK, the inhibitor of apoptosis, enhanced HMME-PDT induced autophagy. 3-MA, the suppressor of autophagy, significantly increased HMME-PDT induced apoptosis rates.ConclusionsOur study demonstrated that HMME-PDT induced both apoptosis and autophagy in HUVECs via oxidative stress. Our data suggested that HMME-PDT- induced autophagy was able to prevent apoptotic cell death of HUVECs and rendered them more resistant to HMME-PDT induced toxicity.     

Effect of Brazilian propolis on human umbilical vein endothelial cell apoptosis

Brazilian propolis has been widely studied in recent years. Considering the lack of data concerning the effects of Brazilian propolis on human umbilical vein endothelial cells (HUVECs), we examined the effects of ethanol-extracted Brazilian propolis (EEBP) at 12.5, 25 and 50 μg/ml on apoptosis of HUVECs deprived of basic fibroblast growth factor (FGF-2) and serum. A high concentration of the extract induced HUVEC apoptosis at 24 h. Furthermore, we investigated the molecular mechanisms of HUVEC apoptosis induced by EEBP by testing the levels of integrin β4, p53, reactive oxygen species (ROS) and mitochondrial membrane potential. A low concentration of EEBP (12.5 μg/ml) could decrease the expression of integrin β4, p53 and ROS levels, whereas high concentrations (25 and 50 μg/ml) could increase the levels of integrin β4, p53 and ROS at 24 h and depress mitochondrial membrane potential level at all times. Considering the doses and the results obtained in this study, Brazilian propolis at high concentrations may be an apoptosis-inducing agent associated with the signal pathway mediated by integrin β4, p53, ROS and mitochondrial membrane potential, thus, propolis should be used in safer levels for human health.     

Toona sinensis (leaf extracts) inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells

Aim of the study

Toona sinensis is well known as a traditional Chinese medicine; also, it has been shown to exhibit anticancer and anti-inflammatory effects. This study was aimed at evaluating the anti-angiogenesis effect of the aqueous extracts of Toona sinensis (TS extracts) or gallic acid, a major component of TS extracts, against both VEGF-induced EA.hy 926 and human umbilical vein endothelial cells (HUVECs).

Materials and methods

Anti-proliferative activity of TS extracts or gallic acid, was determined against EA.hy 926 and HUVECs by trypan blue exclusion method. Invasion, tube formation and chick chorioallantoic membrane assay were carried out to determine the in vitro and in vivo anti-angiogenic effects.

Results

Non-cytotoxic concentration of TS extracts (50-100 μg/mL) and gallic acid (5 μg/mL) inhibited the proliferation of VEGF-stimulated EA.hy 926 and HUVECs. Inhibitory effects of TS extracts and gallic acid on angiogenesis were assessed by VEGF-induced migration/invasion and capillary-like tube formation by EA.hy 926 and HUVECs. Additionally, gelatin zymography assays showed that TS extracts and gallic acid suppressed the activity of metalloproteinase (MMP)-9 and MMP-2 activated by VEGF. In vivo, TS extracts and gallic acid strongly suppressed neovessel formation in the chorioallantoic membrane of chick embryos. Flow cytometry analyses and Western blot demonstrated that treatment with TS extracts and gallic acid induced G₀/G₁ arrest in VEGF-stimulated EA.hy 926 cells via a reduction in the amounts of cyclin D1, cyclin E, CDK4, hyperphosphorylated retinoblastoma protein (pRb), VEGFR-2, and eNOS.

Conclusions

These results support an anti-angiogenic activity of Toona sinensis that may contribute critically to its cancer and inflammation chemopreventive potentials.