葡萄子原花青素B2抗内皮细胞糖基化损伤的作用机制研究
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摘要
第一部分GSPB2对内皮细胞糖基化损伤保护作用的研究
研究背景
随着社会经济的发展和人民生活水平的提高,生活方式的改变和社会人口老龄化,糖尿病患病率在世界范围内呈上升趋势,已经成为继心脑血管疾病、肿瘤之后的又一严重危害人类健康的全球公共卫生问题。糖尿病不仅仅以持续性的高血糖为其特点,更重要的是高血糖所带来的血管并发症的损害。血管内皮细胞(vascular endothelial cell, VEC)是人体血管壁的第一道屏障,调节着血管的结构和功能,VEC是最早受到高血糖影响的部位,高血糖可抑制内皮细胞的迁移、增殖和血管发生变化；大量的研究证实：内皮功能障碍包括血管舒张因子与血管收缩因子、抗凝介质与促凝介质或生长抑制因子与生长促进因子之间的不平衡在VR的发生发展过程中起着关键作用。因此,血管早期损伤机制的阐明有助于预防或延缓糖尿病血管重构的发生和发展,并且VEC作为血管重构的干预点具有良好前景,开辟新的糖尿病血管并发症治疗途径具有重要意义。
葡萄子原花青素(grape seed proanthocyanidin extracts, GSPE)是从葡萄子中提取的一种天然多酚类化合物,是迄今为止所发现的最强效的自由基清除剂之一,具有强大的抗氧化、抗非酶糖基化活性,并具有强效的心血管保护作用；其作为一种天然植物成分,无明显的毒性反应,在国内外的应用非常广泛,其中二聚体在各类原花青素中分布最广,抗氧化活性最强。葡萄子原花青素B2 (grape seedprocyanidin B2, GSPB2)是GSPE的主要成分,由两个单体(儿茶素或表儿茶素)C4→C8键合而成的二聚体,在8种异构体中原花青素B2的活性最强。多年来,国内外众多学者一直致力于GSPE的作用机制研究。但是,至今尚未明确GSPB2对糖尿病血管内皮细胞糖基化损伤保护作用的机制研究。在目前的研究中,观察糖基化终末产物对人脐静脉内皮细胞的影响以及探讨GSPB2对其的保护作用,并进一步研究乳凝集素(lactadherin)和糖原合成酶激酶3β(GSK3β)信号通路在GSPB2抗内皮细胞糖基化损伤中的调节机制,将为防治糖尿病血管并发症乃至多种涉及血管重构的疾病开辟新的途径。
研究目的
1.研究GSPB2对血管内皮细胞糖基化损伤的保护作用。
2.研究GSPB2对乳凝集素和GSK3β表达的影响。
研究方法
体外培养人脐静脉内皮细胞(HUVEC),加入不同浓度的GSPB2(0.78、1.56. 3.12.6.25、12.50、25.00、50.00μmol/L)孵育48小时,应用MTT和CCK-8比色法测定HUVEC的细胞生存率,从而确定实验中GSPB2的使用浓度。将GSPB2溶液加入HUVEC中,终浓度为(2.5、5.0、10.0μmol/L),预孵育1小时,然后加入糖基化终末产物(AGE-BSA,200μg/ml)继续共同培养至48h。应用CCK-8比色法测定HUVEC的细胞生存率,流式细胞仪检测细胞凋亡和细胞内活性氧簇(ROS)的形成,实时定量PCR或Western blot技术测定各组细胞lactadherin、cleaved caspase 3、caspase 3、磷酸化GSK3β(ser9)和总GSK3β的表达。
研究结果
1. GSPB2对HUVEC细胞存活率的影响
不同浓度的GSPB2孵育48小时后,HUVEC的细胞生存率在GSPB2浓度≤12.5μmol/L较正常细胞增加,GSPB2在浓度为6.25μmol/L时HUVEC细胞生存率较正常显著增加(P<0.05)。并且当GSPB2浓度超过25.00μmol/L时HUVEC细胞生存率较正常显著降低(P<0.05)。
2. GSPB2对AGEs刺激HUVEC细胞存活率的影响
与正常细胞组相比,AGEs显著降低HUVEC的细胞存活率(P<0.01)。应用不同浓度的GSPB2 (2.5、5.0、10.0μmol/L)和HUVEC预孵育后,能够显著改善AGEs刺激HUVEC的细胞生存率(P<0.05或P<0.01)。
3. GSPB2对AGEs刺激HUVEC细胞凋亡的影响
与正常细胞组相比,AGEs显著增加HUVEC的细胞凋亡(P<0.01)。应用不同浓度的GSPB2 (2.5、5.0、10.0μmol/L)和HUVEC预孵育后,能够显著降低AGEs刺激HUVEC的凋亡(P<0.05)。
4. GSPB2对AGEs刺激HUVEC细胞内ROS的影响
与正常细胞组相比,AGEs刺激HUVEC 0.5小时后,显著增加细胞内ROS的形成(P<0.05)。应用不同浓度的GSPB2 (2.5、5.0、10.0μmol/L)和HUVEC预孵育后,能够呈剂量依赖性的显著降低AGEs刺激HUVEC的ROS形成(P<0.05)。
5. GSPB2对AGEs刺激HUVEC的cleaved caspase 3和lactadherin表达影响
AGEs刺激HUVEC 48小时后,cleaved caspase 3和lactadherin蛋白表达与正常细胞相比显著增加(P<0.05)。经不同浓度GSPB2预孵育后能够显著降低AGEs刺激HUVEC的cleaved caspase 3和lactadherin蛋白的表达,呈剂量依赖性(P<0.05)。同时应用实时定量PCR测定各组细胞lactadherin mRNA的表达,AGEs刺激HUVEC 48小时,lactadherin mRNA的表达也显著增加(P＜0.05), GSPB2能够剂量依赖性的抑制lactadherin mRNA的表达(P<0.05)。
6. GSPB2对AGEs刺激HUVEC的磷酸化GSK3β(ser9)蛋白表达影响
AGEs刺激HUVEC 48小时后,磷酸化GSK3β(ser9)表达显著降低(P<0.05),经不同浓度GSPB2预孵育后能够显著增加AGEs刺激HUVEC的磷酸化GSK3β(ser9)蛋白表达(P<0.05)。
结论
1. AGEs刺激HUVEC引起细胞凋亡,使lactadherin, cleaved caspase 3表达增高,经不同浓度GSPB2预孵育,能够抑制HUVEC损伤,起到保护作用。
2. GSPB2保护内皮细胞糖基化损伤的分子机制：可能通过抑制lactadherin和增加磷酸化GSK3β(ser9)蛋白表达有关。
第二部分Lactadherin介导的GSPB2抗内皮细胞糖基化损伤机制的研究
研究背景
糖基化终末产物(advanced glycation end products, AGEs)是体内蛋白质中赖氨酸的氨基部分、脂类或核酸与还原糖的羰基在无酶的条件下发生反应,形成Schiff碱,经Amadori反应重排后形成的相对稳定的糖基化产物。近来研究发现,糖尿病患者血中AGEs水平明显增高,且随着病程的延长AGEs浓度也逐渐增加。AGEs具有广泛的生物学活性,参与糖尿病血管并发症的发生和发展。在血管壁的聚集直接参与了血管病变的形成。同时,AGEs还与血管壁细胞膜上AGEs受体结合,激活与血管损伤相关的机制,最终导致血管损伤。但是,AGEs导致血管内皮细胞损伤机制尚未阐明。
我们通过第一部分研究发现,AGEs刺激血管内皮细胞导致乳凝集素(lactadherin)表达增高,经GSPB2干预后其表达呈剂量依赖性降低；前期通过蛋白质组学和分子生物学技术发现：在糖尿病大鼠主动脉组织中lactadherin表达显著增加,经GSPE干预后,其在主动脉组织中表达明显降低。乳凝集素又称为乳脂肪球表皮生长因子8 (MFG-E8)是乳汁脂肪小球表面的亲脂性糖蛋白,在机体其他体液中也有表达,发挥多种功能,并与体内多种细胞的整合素受体结合,引发粘附、分化、增殖、凋亡、细胞骨架重排、激酶级联反应等。它主要分布于乳小管顶端的分泌上皮,并在乳腺癌中过度表达；并且也存在于胰腺、主动脉内皮细胞和平滑肌细胞、巨噬细胞及树突状细胞等。本研究首先构建lactadherin过表达慢病毒载体和siRNA,转染HUVEC后,并应用GSPB2干预,测定干预前后内皮细胞凋亡变化,并应用分子生物学技术检测lactadherin、cleaved caspase 3、Bax/Bcl-2和磷酸化GSK3β(ser9)表达变化。
研究目的
1.研究lactadherin siRNA和过表达对内皮细胞糖基化损伤的影响。
2.研究GSPB2抗内皮细胞糖基化损伤的分子调控机制。
研究方法
体外培养HUVEC,构建lactadherin siRNA和过表达载体,成功转染HUVEC后,传代扩增。将转染阴性对照和lactadherin siRNA的HUVEC分别加入糖基化终末产物(AGE-BSA,200μg/ml)继续共同培养至48h,阴性对照同时加入GSPB2 (10.00pmol/L)共孵育；另外,将lactadherin过表达的HUVEC,加入GSPB2 (10.00μmol/L)孵育48小时,应用MTT和CCK-8比色法测定各组细胞生存率,TUNEL法检测细胞凋亡,并收集lactadherin过表达的HUVEC,各组细胞应用电镜观察细胞超微结构变化,应用实时定量PCR或Western blot技术测定各组细胞lactadherin、cleaved caspase 3、磷酸化GSK3P和线粒体凋亡通路Bax/Bcl-2表达变化。
研究结果
1. Lactadherin siRNA和过表达转染HUVEC的有效性
转染的有效性应用荧光显微镜、实时定量PCR和western blot技术评价。转染阴性对照组(NC)、lactadherin siRNA组(LsiRNA)、表达GFP基因组(LV-C)和GFP及lactadherin过表达组(LV),收获各组细胞。转染有效性约为95%,lactadherin siRNA在转染后48小时LsiRNA lactadherin蛋白表达较NC降低60%以上；lactadherin过表达在转染5天后,lactadherin蛋白表达量较LV-C增加超过2倍。
2. Lactadherin对AGEs刺激HUVEC细胞生存率的影响及GSPB2干预作用
HUVEC转染GFP基因或阴性对照并未影响细胞生存率。AGEs (200μg/ml)刺激各组细胞48小时,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞生存率的降低(P<0.05); GSPB2 (10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞生存率的降低(P<0.05)。另外,转染5天后,LV组细胞生存率较LV-C组显著降低(P＜0.05), GSPB2 (10μmol/L)共孵育48小时能够显著改善LV组细胞生存率(P<0.05)。
3. Lactadherin对AGEs刺激HUVEC细胞凋亡的影响及GSPB2干预作用
AGEs (200μg/ml)刺激各组细胞48小时后,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞凋亡(P<0.05); GSPB2 (10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞凋亡(P<0.05)。另外,转染5天后,LV组细胞凋亡较LV-C组显著增加(P<0.05), GSPB2 (10μmol/L)共孵育48小时能够显著改善LV组细胞凋亡(P<0.05)。
4. Lactadherin过表达HUVEC超微结构的改变
透射电镜扫描观察Lactadherin过表达转染5天后及GSPB2共孵育48小时细胞超微结构改变,LV组细胞凋亡显著增加,可见凋亡小体、线粒体偏极化、细胞染色质固缩及染色体聚集于核膜呈境界分明块状等变化,GSPB2共孵育后细胞凋亡显著减轻。
5. Lactadherin对AGEs刺激HUVEC的cleaved caspase-3和Bax/Bcl-2比值影响及GSPB2干预作用
LV-C组、LV组、LsiRNA组和NC组细胞uncleaved caspase-3表达未见显著变化。AGEs (200μg/ml)刺激各组细胞48小时后,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞cleaved caspase-3和Bax/Bcl-2比值的增加(P＜0.05); GSPB2 (10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞cleaved caspase-3和Bax/Bcl-2比值的增加(P<0.05)。另外,转染5天后,LV组细胞cleaved caspase-3和Bax/Bcl-2比值较LV-C组显著增加(P<0.05),GSPB2 (10μmol/L)共孵育48小时也能够显著改善LV组细胞cleaved caspase-3和Bax/Bcl-2比值的增加(P<0.05)。
6. Lactadherin对AGEs刺激HUVEC磷酸化GSK3β(ser9)的影响及GSPB2干预作用
AGEs (200μg/ml)刺激各组细胞48小时后,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞磷酸化GSK3β(ser9)的降低(P<0.05)；GSPB2(10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞磷酸化GSK3β(ser9)的降低(P<0.05)。另外,转染5天后,LV组细胞磷酸化GSK3β(ser9)较LV-C组显著降低(P≤0.05), GSPB2 (10μmol/L)共孵育48小时能够显著改善LV组细胞磷酸化GSK3β(ser9)的降低(P<0.05)。
结论
1. Lactadherin在AGEs诱发的血管内皮细胞凋亡中起着重要的作用。
2. Lactadherin介导的血管内皮细胞凋亡与线粒体凋亡通路和GSK3β(ser9)的磷酸化有关。
3.抗氧化剂GSPB2通过抑制Lactadherin表达起到减轻内皮细胞糖基化损伤。
Part One Protection effects of grape seed proanthocyanidin B2 on endothelial damage caused by AGEs
Background
With the development of social economy, the improvement of people living and population aging, the incidence of diabetes mellitus has increased in the worldwide. Diabetes mellitus have been a globe public health issue next to cardiovascular and cerebrovascular disease and tumor that may seriously harm human healthy. All forms of diabetes are characterized by chronic hyperglycaemia and the development of diabetes-specific vascular complications. Vascular endothelial cells are the first barrier of human body vessel wall. Under normal circumstances, the vascular endothelium influences vessel tone and modulates structure, vessel permeability, adhesion and migration of inflammatory cells, and hemostasis. The endothelium is an early target in diabetes, and dysfunction of vascular endothelial cells plays a critical role in the diabetic vascular disease process.
Grape seed proanthocyanidin extracts (GSPE) derived from grape seeds, have been reported to possess a variety of potent properties including anti-oxidant, anti-nonenzymatic glycation and cardioprotective effect. Dimeric procyanidin B2 is one of the main components of GSPE, composed of two molecules of the flavan-3-ol (-)-epicatechin linked by a 4b→8 bonds. Several studies have shown that procyanidin B2 exerts a more potent anti-tumor-promoting effect greater than other dimers, such as procyanidins B1, B4, and B5. In our present study, we examined the effects of AGEs on the human umbilical vein endothelial cells (HUVEC) to evaluate the efficacy of HUVEC protection by grape seed procyanidin B2 (GSPB2). To delineate the underlying molecular mechanism, we investigated the lactadherin and phosphorylation of glycogen synthase kinase 3β(GSK3β) signaling pathway using quantitative RT-PCR, Western blot.
Objective
1. To study the effects of GSPB2 on the nonenzymatic glycation damage of vascular endothelial cells.
2. To study the effects of GSPB2 on the lactadherin and phosphorylation of glycogen synthase kinase 3β(GSK3β) signaling pathway.
Methods
HUVEC were incubated in complete medium RPMI 1640 containing 10% FBS in Costar flasks. GSPB2 (0.78、1.56、3.12.6.25、12.50、25.00、50.00μmol/L) were added for incubation for 48 hours. To examine the effects of the GSPB2 on HUVEC proliferation, cells were harvested by trypsinization. Cell survival was estimated with MTT and CCK-8 colorimetric assay.2.5μmol/L,5.0μmol/L and 10.0μmol/L of GSPB2 were added for preincubation for 1 hour. After preincubation, the HUVEC were stimulated separately with 200μg/ml of AGEs or unmodified BSA for 48 hours. Cell survival was estimated with CCK-8 colorimetric assay. Apoptosis of cells was analyzed using annexin V-FITC apoptosis detection kit. The production of intracellular reactive oxygen species (ROS) was estimated fluorometrically using 2',7'-dichlorofluorescein diacetate. We determined the lactadherin, cleaved caspase 3, caspase 3, and phosphorylation of GSK-3P in HUVEC by quantitative real-time PCR or western blotting analysis.
Results
1. Effects of GSPB2 on HUVEC viability
The cell viability was increased when HUVEC were exposed to GSPB2 for 48h at the concentration (<12.50μmol/L). GSPB2 increased the cell viability significantly at 6.25μmol/L. Meanwhile, GSPB2 caused approximately 25-35% inhibition of cell viability at 25.00μmol/L or high concentrations.
2. Effects of GSPB2 on viability in HUVEC stimulated by AGEs
Both unmodified BSA and DMSO did not affect cell viability. Figure 2 showed the results of comparisons of cell viability findings every group with CCK-8 assay. The cell viability of AGEs was significantly lower approximately 30%. The pretreatment of HUVEC with different concentrations of GSPB2 significantly improved the AGEs-stimulated cell viability in a dose dependent manner.
3. Effects of GSPB2 on anti-apoptosis in HUVEC stimulated by AGEs
Both unmodified BSA and DMSO did not affect cell apoptosis. It was found that AGEs-stimulated HUVEC significantly increased the percentage of apoptotic cells (P＜0.05). Moreover, pretreatment of HUVEC with different concentrations of GSPB2 significantly improved the AGEs-stimulated cell apoptosis in a dose dependent manner (P<0.05).
4. Effects of GSPB2 on intracellular ROS formation in HUVEC stimulated by AGEs
The levels of ROS were low in the unstimulated HUVEC. AGEs-stimulated HUVEC significantly increased the ROS formation, whereas pretreatment of GSPE in a dose-dependent manner apparently prevented AGEs-induced ROS generation (P<0.05).
5. Effects of GSPB2 on cleaved caspase-3 and lactadherin in HUVEC stimulated by AGEs
Stimulation of HUVEC with AGEs (200μg/mL) resulted in a significant increase in the cleaved caspase-3 and lactadherin by western blotting. The pretreatment with GSPB2 significantly improved the AGEs-stimulated the cleaved caspase-3 and lactadherin for 48h (P＜0.05). By quantitative real-time PCR assay showed that lactadherin mRNA expression in HUVEC exposed to AGEs were significantly upregulated within 48h (P＜0.05). Moreover, pretreatment of HUVEC with different concentrations of GSPB2 significantly inhibited the expression of lactadherin mRNA of HUVEC stimulated by AGEs (P＜0.05).
6. Effects of GSPB2 on the levels of phospho-GSK3βin HUVEC stimulated by AGEs
Stimulation of HUVEC with AGEs (200μg/mL) resulted in a significant decrease in the levels of phospho-GSK3β, whereas pretreatment of HUVEC with different concentrations of GSPB2 significantly attenuated AGEs-stimulated the decreased levels of phospho-GSK3βunder AGEs stimulation for 48h (P<0.05)
Conclusion
1. AGEs induced HUVEC apoptosis and upregulated the expression of caspase-3 activation and lactadherin. Treatment of HEVECs with GSPB2 significantly inhibited the cell apoptosis and the expression of caspase-3 activation and lactadherin induced by AGEs.
2. The regulation of lactadherin and phospho-GSK3βby GSPB2 contributes to the improvement of endothelial dysfunction resulting from the damage from AGEs.
Part Two Investigate the mechanism of grape seed proanthocyanidin B2 on advanced glycation end products induced endothelial cell apoptosis based on gene transfection
Background
Nonenzymatic protein glycation by glucose is a complex cascade of reactions yielding a heterogeneous class of compounds, collectively termed advanced glycation end products (AGEs). The Schiff base can undergo an intramolecular rearrangement to form the Amadori products. Among the many metabolic abnormalities of diabetes mellitus, AGEs are one showing in epidemiological studies the most consistent and significant correlation with diabetic vascular complications. AGEs lead endothelial dysfunction and apoptosis, and endothelial dysfunction play a critical role in the pathophysiology of vascular complications in diabetes mellitus. AGE-modified adducts on long-lived proteins in extracellular matrix alter basement membrane structure by trapping plasma macromolecules and by increasing vessel wall rigidity through formation of cross-links. Meanwhile, interaction of AGEs with receptor for advanced glycation end product results in triggering a range of cellular responses, including transcription factor activation and changes in gene expression. However, the pathophysilogical mechanism of AGEs-related vascular endothelium and clinical complications remains largely elusive.
In the first part, the pretreatment with GSPB2 significantly improved the AGEs-stimulated the expression of lactadherin. Our previous proteomic studies showed that the expression of lactadherin was significantly increased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts (GSPE) significantly inhibited the expression of lactadherin in diabetic rats. Lactadherin is a secreted glycoprotein of milk-fat globule that shares structural domain homology with Del-1. Mouse lactadherin is also known as milk-fat globule-EGF factor 8 (MFG-E8). Lactadherin is expressed in the mammary epithelium, breast cancer, pancreas, aortic endothelial cells and smooth muscle cells, and macrophages and dendritic cells, etc. In our present study, we focused on the molecular mechanisms of human umbilical vein endothelial cells (HUVEC) apoptosis induced by AGEs, particularly 1) the role and molecular mechanism of the lactadherin (overexpression and siRNA) in the AGEs-induced endothelial cells apoptosis,2) the efficacy of HUVEC protection by grape seed procyanidin B2 (GSPB2).
Methods
HUVEC were cultured in six-well plates overnight and transfected with siRNA against lactadherin using Lipofectamine2000. Moreover, HUVEC were transduced with lentiviral vectors of lactadherin overexpression. HUVEC transfected lactadherin genes and siRNA were incubated for 48 h in the presence or absence of GSPB2 (10μmol/L). Meanwhile, HUVEC transfected lactadherin siRNA were stimulated with 200μg/mL of AGEs. Cell survival was estimated with MTT and CCK-8 colorimetric assay. The apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. To evaluate changes in morphology, HUVEC transfected lactadherin genes were visualized by transmission electron microscope (TEM). We determined the lactadherin, cleaved caspase 3, caspase 3, phosphorylation of GSK-3P and Bax/Bcl-2 in HUVEC by quantitative real-time PCR or western blotting analysis.
Results
1. Transduction efficiency with lactadherin siRNA and overexpression plasmids
We transduced the HUVEC with siRNA or lentivirus vector. Transduction conditions were optimized by using different MOI and the transduction efficiency was assessed by fluorescence microscopy, real-time PCR and western blotting. HUVEC carrying negative control siRNA (NC), HUVEC carrying siRNA against lactadherin (LsiRNA) and HUVEC carrying GFP (LV-C), HUVEC carrying both GFP and lactadherin genes (LV) were harvested. The transduction efficiency was about 95% or higher at day 5 or longer. Transgene expression was confirmed by fluorescent microscopic imaging for lactadherin protein expression. The mRNA and protein expression of lactadherin in LsiRNA group decreased to more than 60% the level of the NC group at 48 h after transfection. Lactadherin mRNA and protein expression reached its highest level at day 5 after virus removal.
2. Effects of lactadherin on viability in HUVEC stimulated by AGEs
There were no differences in the cell viability between LsiRNA group and NC group. Stimulation of NC group with AGEs (200μg/mL) resulted in a significant decrease in the cell viability, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the decrease of cell viability compared with NC group under AGEs stimulation for 48h (P＜0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated cell viability for 48h (P<0.05). Both HUVEC transfected GFP and negative control did not affect cell viability. Moreover, the overexpression of lactadherin significantly decreased the cell viability. The cell viability was increased when LV group was exposed to GSPB2 (10μmol/L) for 48h (P<0.05).
3. Effects of lactadherin on apoptosis in HUVEC stimulated by AGEs
Stimulation of NC group with AGEs (200μg/mL) resulted in a significant increase in the cell apoptosis, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the cell apoptosis compared with NC group under AGEs stimulation for 48h (P<0.05). The treatment of NC group with GSPB2 (10μmol/L) also significantly improved the AGEs-stimulated cell apoptosis for 48h (P＜0.05). HUVEC overexpression lactadherin was susceptible to cell apoptosis, while GSPB2 (10μmol/L) significantly attenuated the cell apoptosis for 48 h.
4. The morphological changes of HUVEC overexpression lactadherin
The cell apoptosis significantly increased in the HUVEC overexpression lactadherin, while GSPB2 (10μmol/L) significantly attenuated the cell apoptosis for 48 h.
5. Effects of lactadherin on cleaved caspase-3 and Bax/Bcl-2 ratio
The uncleaved caspase-3 levels were unchanged in LV-C group, LV group, LsiRNA group and NC group. Stimulation of NC group with AGEs (200μg/mL) resulted in a significant increase in the cleaved caspase-3 and Bax/Bcl-2 ratio, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the cleaved caspase-3 and Bax/Bcl-2 ratio compared with NC group under AGEs stimulation for 48h (P＜0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated the cleaved caspase-3 and Bax/Bcl-2 ratio for 48h (P<0.05). The cleaved caspase-3 and Bax/Bcl-2 ratio significantly increased in LV group comparing to LV-C group, while GSPB2 (10μmol/L) significantly inhibited the cleaved caspase-3 and Bax/Bcl-2 ratio in HUVEC overexpression lactadherin for 48 h (P<0.05).
6. Effect of lactadherin on the levels of phospho-GSK3β
Stimulation of NC group with AGEs (200μg/mL) resulted in a significant decrease in the levels of phospho-GSK3β, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the decreased levels of phospho-GSK3βcompared with NC group under AGEs stimulation for 48h (P<0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated the decreased levels of phospho-GSK3P for 48h (P＜0.05). The levels of phospho-GSK3βsignificantly decreased in LV group comparing to LV-C group, while GSPB2 (10μmol/L) significantly improved the phospho-GSK3βin HUVEC overexpression lactadherin for 48 h (P＜0.05).
Conclusion
1. Up-regulation of lactadherin plays a critical role in endothelial cells apoptosis induced by AGEs.
2. Mitochondria apoptosis pathway played a pivotal role in the cell apoptosis by lactadherin.
3. GSPB2 might have benefits in the early stage of diabetic endothelial dysfunction by inhibition of lactadherin.
研究背景
随着社会经济的发展和人民生活水平的提高,生活方式的改变和社会人口老龄化,糖尿病患病率在世界范围内呈上升趋势,已经成为继心脑血管疾病、肿瘤之后的又一严重危害人类健康的全球公共卫生问题。糖尿病不仅仅以持续性的高血糖为其特点,更重要的是高血糖所带来的血管并发症的损害。血管内皮细胞(vascular endothelial cell, VEC)是人体血管壁的第一道屏障,调节着血管的结构和功能,VEC是最早受到高血糖影响的部位,高血糖可抑制内皮细胞的迁移、增殖和血管发生变化；大量的研究证实：内皮功能障碍包括血管舒张因子与血管收缩因子、抗凝介质与促凝介质或生长抑制因子与生长促进因子之间的不平衡在VR的发生发展过程中起着关键作用。因此,血管早期损伤机制的阐明有助于预防或延缓糖尿病血管重构的发生和发展,并且VEC作为血管重构的干预点具有良好前景,开辟新的糖尿病血管并发症治疗途径具有重要意义。
葡萄子原花青素(grape seed proanthocyanidin extracts, GSPE)是从葡萄子中提取的一种天然多酚类化合物,是迄今为止所发现的最强效的自由基清除剂之一,具有强大的抗氧化、抗非酶糖基化活性,并具有强效的心血管保护作用；其作为一种天然植物成分,无明显的毒性反应,在国内外的应用非常广泛,其中二聚体在各类原花青素中分布最广,抗氧化活性最强。葡萄子原花青素B2 (grape seedprocyanidin B2, GSPB2)是GSPE的主要成分,由两个单体(儿茶素或表儿茶素)C4→C8键合而成的二聚体,在8种异构体中原花青素B2的活性最强。多年来,国内外众多学者一直致力于GSPE的作用机制研究。但是,至今尚未明确GSPB2对糖尿病血管内皮细胞糖基化损伤保护作用的机制研究。在目前的研究中,观察糖基化终末产物对人脐静脉内皮细胞的影响以及探讨GSPB2对其的保护作用,并进一步研究乳凝集素(lactadherin)和糖原合成酶激酶3β(GSK3β)信号通路在GSPB2抗内皮细胞糖基化损伤中的调节机制,将为防治糖尿病血管并发症乃至多种涉及血管重构的疾病开辟新的途径。
研究目的
1.研究GSPB2对血管内皮细胞糖基化损伤的保护作用。
2.研究GSPB2对乳凝集素和GSK3β表达的影响。
研究方法
体外培养人脐静脉内皮细胞(HUVEC),加入不同浓度的GSPB2(0.78、1.56. 3.12.6.25、12.50、25.00、50.00μmol/L)孵育48小时,应用MTT和CCK-8比色法测定HUVEC的细胞生存率,从而确定实验中GSPB2的使用浓度。将GSPB2溶液加入HUVEC中,终浓度为(2.5、5.0、10.0μmol/L),预孵育1小时,然后加入糖基化终末产物(AGE-BSA,200μg/ml)继续共同培养至48h。应用CCK-8比色法测定HUVEC的细胞生存率,流式细胞仪检测细胞凋亡和细胞内活性氧簇(ROS)的形成,实时定量PCR或Western blot技术测定各组细胞lactadherin、cleaved caspase 3、caspase 3、磷酸化GSK3β(ser9)和总GSK3β的表达。
研究结果
1. GSPB2对HUVEC细胞存活率的影响
不同浓度的GSPB2孵育48小时后,HUVEC的细胞生存率在GSPB2浓度≤12.5μmol/L较正常细胞增加,GSPB2在浓度为6.25μmol/L时HUVEC细胞生存率较正常显著增加(P<0.05)。并且当GSPB2浓度超过25.00μmol/L时HUVEC细胞生存率较正常显著降低(P<0.05)。
2. GSPB2对AGEs刺激HUVEC细胞存活率的影响
与正常细胞组相比,AGEs显著降低HUVEC的细胞存活率(P<0.01)。应用不同浓度的GSPB2 (2.5、5.0、10.0μmol/L)和HUVEC预孵育后,能够显著改善AGEs刺激HUVEC的细胞生存率(P<0.05或P<0.01)。
3. GSPB2对AGEs刺激HUVEC细胞凋亡的影响
与正常细胞组相比,AGEs显著增加HUVEC的细胞凋亡(P<0.01)。应用不同浓度的GSPB2 (2.5、5.0、10.0μmol/L)和HUVEC预孵育后,能够显著降低AGEs刺激HUVEC的凋亡(P<0.05)。
4. GSPB2对AGEs刺激HUVEC细胞内ROS的影响
与正常细胞组相比,AGEs刺激HUVEC 0.5小时后,显著增加细胞内ROS的形成(P<0.05)。应用不同浓度的GSPB2 (2.5、5.0、10.0μmol/L)和HUVEC预孵育后,能够呈剂量依赖性的显著降低AGEs刺激HUVEC的ROS形成(P<0.05)。
5. GSPB2对AGEs刺激HUVEC的cleaved caspase 3和lactadherin表达影响
AGEs刺激HUVEC 48小时后,cleaved caspase 3和lactadherin蛋白表达与正常细胞相比显著增加(P<0.05)。经不同浓度GSPB2预孵育后能够显著降低AGEs刺激HUVEC的cleaved caspase 3和lactadherin蛋白的表达,呈剂量依赖性(P<0.05)。同时应用实时定量PCR测定各组细胞lactadherin mRNA的表达,AGEs刺激HUVEC 48小时,lactadherin mRNA的表达也显著增加(P＜0.05), GSPB2能够剂量依赖性的抑制lactadherin mRNA的表达(P<0.05)。
6. GSPB2对AGEs刺激HUVEC的磷酸化GSK3β(ser9)蛋白表达影响
AGEs刺激HUVEC 48小时后,磷酸化GSK3β(ser9)表达显著降低(P<0.05),经不同浓度GSPB2预孵育后能够显著增加AGEs刺激HUVEC的磷酸化GSK3β(ser9)蛋白表达(P<0.05)。
结论
1. AGEs刺激HUVEC引起细胞凋亡,使lactadherin, cleaved caspase 3表达增高,经不同浓度GSPB2预孵育,能够抑制HUVEC损伤,起到保护作用。
2. GSPB2保护内皮细胞糖基化损伤的分子机制：可能通过抑制lactadherin和增加磷酸化GSK3β(ser9)蛋白表达有关。
第二部分Lactadherin介导的GSPB2抗内皮细胞糖基化损伤机制的研究
研究背景
糖基化终末产物(advanced glycation end products, AGEs)是体内蛋白质中赖氨酸的氨基部分、脂类或核酸与还原糖的羰基在无酶的条件下发生反应,形成Schiff碱,经Amadori反应重排后形成的相对稳定的糖基化产物。近来研究发现,糖尿病患者血中AGEs水平明显增高,且随着病程的延长AGEs浓度也逐渐增加。AGEs具有广泛的生物学活性,参与糖尿病血管并发症的发生和发展。在血管壁的聚集直接参与了血管病变的形成。同时,AGEs还与血管壁细胞膜上AGEs受体结合,激活与血管损伤相关的机制,最终导致血管损伤。但是,AGEs导致血管内皮细胞损伤机制尚未阐明。
我们通过第一部分研究发现,AGEs刺激血管内皮细胞导致乳凝集素(lactadherin)表达增高,经GSPB2干预后其表达呈剂量依赖性降低；前期通过蛋白质组学和分子生物学技术发现：在糖尿病大鼠主动脉组织中lactadherin表达显著增加,经GSPE干预后,其在主动脉组织中表达明显降低。乳凝集素又称为乳脂肪球表皮生长因子8 (MFG-E8)是乳汁脂肪小球表面的亲脂性糖蛋白,在机体其他体液中也有表达,发挥多种功能,并与体内多种细胞的整合素受体结合,引发粘附、分化、增殖、凋亡、细胞骨架重排、激酶级联反应等。它主要分布于乳小管顶端的分泌上皮,并在乳腺癌中过度表达；并且也存在于胰腺、主动脉内皮细胞和平滑肌细胞、巨噬细胞及树突状细胞等。本研究首先构建lactadherin过表达慢病毒载体和siRNA,转染HUVEC后,并应用GSPB2干预,测定干预前后内皮细胞凋亡变化,并应用分子生物学技术检测lactadherin、cleaved caspase 3、Bax/Bcl-2和磷酸化GSK3β(ser9)表达变化。
研究目的
1.研究lactadherin siRNA和过表达对内皮细胞糖基化损伤的影响。
2.研究GSPB2抗内皮细胞糖基化损伤的分子调控机制。
研究方法
体外培养HUVEC,构建lactadherin siRNA和过表达载体,成功转染HUVEC后,传代扩增。将转染阴性对照和lactadherin siRNA的HUVEC分别加入糖基化终末产物(AGE-BSA,200μg/ml)继续共同培养至48h,阴性对照同时加入GSPB2 (10.00pmol/L)共孵育；另外,将lactadherin过表达的HUVEC,加入GSPB2 (10.00μmol/L)孵育48小时,应用MTT和CCK-8比色法测定各组细胞生存率,TUNEL法检测细胞凋亡,并收集lactadherin过表达的HUVEC,各组细胞应用电镜观察细胞超微结构变化,应用实时定量PCR或Western blot技术测定各组细胞lactadherin、cleaved caspase 3、磷酸化GSK3P和线粒体凋亡通路Bax/Bcl-2表达变化。
研究结果
1. Lactadherin siRNA和过表达转染HUVEC的有效性
转染的有效性应用荧光显微镜、实时定量PCR和western blot技术评价。转染阴性对照组(NC)、lactadherin siRNA组(LsiRNA)、表达GFP基因组(LV-C)和GFP及lactadherin过表达组(LV),收获各组细胞。转染有效性约为95%,lactadherin siRNA在转染后48小时LsiRNA lactadherin蛋白表达较NC降低60%以上；lactadherin过表达在转染5天后,lactadherin蛋白表达量较LV-C增加超过2倍。
2. Lactadherin对AGEs刺激HUVEC细胞生存率的影响及GSPB2干预作用
HUVEC转染GFP基因或阴性对照并未影响细胞生存率。AGEs (200μg/ml)刺激各组细胞48小时,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞生存率的降低(P<0.05); GSPB2 (10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞生存率的降低(P<0.05)。另外,转染5天后,LV组细胞生存率较LV-C组显著降低(P＜0.05), GSPB2 (10μmol/L)共孵育48小时能够显著改善LV组细胞生存率(P<0.05)。
3. Lactadherin对AGEs刺激HUVEC细胞凋亡的影响及GSPB2干预作用
AGEs (200μg/ml)刺激各组细胞48小时后,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞凋亡(P<0.05); GSPB2 (10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞凋亡(P<0.05)。另外,转染5天后,LV组细胞凋亡较LV-C组显著增加(P<0.05), GSPB2 (10μmol/L)共孵育48小时能够显著改善LV组细胞凋亡(P<0.05)。
4. Lactadherin过表达HUVEC超微结构的改变
透射电镜扫描观察Lactadherin过表达转染5天后及GSPB2共孵育48小时细胞超微结构改变,LV组细胞凋亡显著增加,可见凋亡小体、线粒体偏极化、细胞染色质固缩及染色体聚集于核膜呈境界分明块状等变化,GSPB2共孵育后细胞凋亡显著减轻。
5. Lactadherin对AGEs刺激HUVEC的cleaved caspase-3和Bax/Bcl-2比值影响及GSPB2干预作用
LV-C组、LV组、LsiRNA组和NC组细胞uncleaved caspase-3表达未见显著变化。AGEs (200μg/ml)刺激各组细胞48小时后,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞cleaved caspase-3和Bax/Bcl-2比值的增加(P＜0.05); GSPB2 (10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞cleaved caspase-3和Bax/Bcl-2比值的增加(P<0.05)。另外,转染5天后,LV组细胞cleaved caspase-3和Bax/Bcl-2比值较LV-C组显著增加(P<0.05),GSPB2 (10μmol/L)共孵育48小时也能够显著改善LV组细胞cleaved caspase-3和Bax/Bcl-2比值的增加(P<0.05)。
6. Lactadherin对AGEs刺激HUVEC磷酸化GSK3β(ser9)的影响及GSPB2干预作用
AGEs (200μg/ml)刺激各组细胞48小时后,与AGEs刺激NC组细胞相比,LsiRNA能够显著改善AGEs诱发的细胞磷酸化GSK3β(ser9)的降低(P<0.05)；GSPB2(10μmol/L)共孵育48小时也能够显著改善AGEs诱发的细胞磷酸化GSK3β(ser9)的降低(P<0.05)。另外,转染5天后,LV组细胞磷酸化GSK3β(ser9)较LV-C组显著降低(P≤0.05), GSPB2 (10μmol/L)共孵育48小时能够显著改善LV组细胞磷酸化GSK3β(ser9)的降低(P<0.05)。
结论
1. Lactadherin在AGEs诱发的血管内皮细胞凋亡中起着重要的作用。
2. Lactadherin介导的血管内皮细胞凋亡与线粒体凋亡通路和GSK3β(ser9)的磷酸化有关。
3.抗氧化剂GSPB2通过抑制Lactadherin表达起到减轻内皮细胞糖基化损伤。
Part One Protection effects of grape seed proanthocyanidin B2 on endothelial damage caused by AGEs
Background
With the development of social economy, the improvement of people living and population aging, the incidence of diabetes mellitus has increased in the worldwide. Diabetes mellitus have been a globe public health issue next to cardiovascular and cerebrovascular disease and tumor that may seriously harm human healthy. All forms of diabetes are characterized by chronic hyperglycaemia and the development of diabetes-specific vascular complications. Vascular endothelial cells are the first barrier of human body vessel wall. Under normal circumstances, the vascular endothelium influences vessel tone and modulates structure, vessel permeability, adhesion and migration of inflammatory cells, and hemostasis. The endothelium is an early target in diabetes, and dysfunction of vascular endothelial cells plays a critical role in the diabetic vascular disease process.
Grape seed proanthocyanidin extracts (GSPE) derived from grape seeds, have been reported to possess a variety of potent properties including anti-oxidant, anti-nonenzymatic glycation and cardioprotective effect. Dimeric procyanidin B2 is one of the main components of GSPE, composed of two molecules of the flavan-3-ol (-)-epicatechin linked by a 4b→8 bonds. Several studies have shown that procyanidin B2 exerts a more potent anti-tumor-promoting effect greater than other dimers, such as procyanidins B1, B4, and B5. In our present study, we examined the effects of AGEs on the human umbilical vein endothelial cells (HUVEC) to evaluate the efficacy of HUVEC protection by grape seed procyanidin B2 (GSPB2). To delineate the underlying molecular mechanism, we investigated the lactadherin and phosphorylation of glycogen synthase kinase 3β(GSK3β) signaling pathway using quantitative RT-PCR, Western blot.
Objective
1. To study the effects of GSPB2 on the nonenzymatic glycation damage of vascular endothelial cells.
2. To study the effects of GSPB2 on the lactadherin and phosphorylation of glycogen synthase kinase 3β(GSK3β) signaling pathway.
Methods
HUVEC were incubated in complete medium RPMI 1640 containing 10% FBS in Costar flasks. GSPB2 (0.78、1.56、3.12.6.25、12.50、25.00、50.00μmol/L) were added for incubation for 48 hours. To examine the effects of the GSPB2 on HUVEC proliferation, cells were harvested by trypsinization. Cell survival was estimated with MTT and CCK-8 colorimetric assay.2.5μmol/L,5.0μmol/L and 10.0μmol/L of GSPB2 were added for preincubation for 1 hour. After preincubation, the HUVEC were stimulated separately with 200μg/ml of AGEs or unmodified BSA for 48 hours. Cell survival was estimated with CCK-8 colorimetric assay. Apoptosis of cells was analyzed using annexin V-FITC apoptosis detection kit. The production of intracellular reactive oxygen species (ROS) was estimated fluorometrically using 2',7'-dichlorofluorescein diacetate. We determined the lactadherin, cleaved caspase 3, caspase 3, and phosphorylation of GSK-3P in HUVEC by quantitative real-time PCR or western blotting analysis.
Results
1. Effects of GSPB2 on HUVEC viability
The cell viability was increased when HUVEC were exposed to GSPB2 for 48h at the concentration (<12.50μmol/L). GSPB2 increased the cell viability significantly at 6.25μmol/L. Meanwhile, GSPB2 caused approximately 25-35% inhibition of cell viability at 25.00μmol/L or high concentrations.
2. Effects of GSPB2 on viability in HUVEC stimulated by AGEs
Both unmodified BSA and DMSO did not affect cell viability. Figure 2 showed the results of comparisons of cell viability findings every group with CCK-8 assay. The cell viability of AGEs was significantly lower approximately 30%. The pretreatment of HUVEC with different concentrations of GSPB2 significantly improved the AGEs-stimulated cell viability in a dose dependent manner.
3. Effects of GSPB2 on anti-apoptosis in HUVEC stimulated by AGEs
Both unmodified BSA and DMSO did not affect cell apoptosis. It was found that AGEs-stimulated HUVEC significantly increased the percentage of apoptotic cells (P＜0.05). Moreover, pretreatment of HUVEC with different concentrations of GSPB2 significantly improved the AGEs-stimulated cell apoptosis in a dose dependent manner (P<0.05).
4. Effects of GSPB2 on intracellular ROS formation in HUVEC stimulated by AGEs
The levels of ROS were low in the unstimulated HUVEC. AGEs-stimulated HUVEC significantly increased the ROS formation, whereas pretreatment of GSPE in a dose-dependent manner apparently prevented AGEs-induced ROS generation (P<0.05).
5. Effects of GSPB2 on cleaved caspase-3 and lactadherin in HUVEC stimulated by AGEs
Stimulation of HUVEC with AGEs (200μg/mL) resulted in a significant increase in the cleaved caspase-3 and lactadherin by western blotting. The pretreatment with GSPB2 significantly improved the AGEs-stimulated the cleaved caspase-3 and lactadherin for 48h (P＜0.05). By quantitative real-time PCR assay showed that lactadherin mRNA expression in HUVEC exposed to AGEs were significantly upregulated within 48h (P＜0.05). Moreover, pretreatment of HUVEC with different concentrations of GSPB2 significantly inhibited the expression of lactadherin mRNA of HUVEC stimulated by AGEs (P＜0.05).
6. Effects of GSPB2 on the levels of phospho-GSK3βin HUVEC stimulated by AGEs
Stimulation of HUVEC with AGEs (200μg/mL) resulted in a significant decrease in the levels of phospho-GSK3β, whereas pretreatment of HUVEC with different concentrations of GSPB2 significantly attenuated AGEs-stimulated the decreased levels of phospho-GSK3βunder AGEs stimulation for 48h (P<0.05)
Conclusion
1. AGEs induced HUVEC apoptosis and upregulated the expression of caspase-3 activation and lactadherin. Treatment of HEVECs with GSPB2 significantly inhibited the cell apoptosis and the expression of caspase-3 activation and lactadherin induced by AGEs.
2. The regulation of lactadherin and phospho-GSK3βby GSPB2 contributes to the improvement of endothelial dysfunction resulting from the damage from AGEs.
Part Two Investigate the mechanism of grape seed proanthocyanidin B2 on advanced glycation end products induced endothelial cell apoptosis based on gene transfection
Background
Nonenzymatic protein glycation by glucose is a complex cascade of reactions yielding a heterogeneous class of compounds, collectively termed advanced glycation end products (AGEs). The Schiff base can undergo an intramolecular rearrangement to form the Amadori products. Among the many metabolic abnormalities of diabetes mellitus, AGEs are one showing in epidemiological studies the most consistent and significant correlation with diabetic vascular complications. AGEs lead endothelial dysfunction and apoptosis, and endothelial dysfunction play a critical role in the pathophysiology of vascular complications in diabetes mellitus. AGE-modified adducts on long-lived proteins in extracellular matrix alter basement membrane structure by trapping plasma macromolecules and by increasing vessel wall rigidity through formation of cross-links. Meanwhile, interaction of AGEs with receptor for advanced glycation end product results in triggering a range of cellular responses, including transcription factor activation and changes in gene expression. However, the pathophysilogical mechanism of AGEs-related vascular endothelium and clinical complications remains largely elusive.
In the first part, the pretreatment with GSPB2 significantly improved the AGEs-stimulated the expression of lactadherin. Our previous proteomic studies showed that the expression of lactadherin was significantly increased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts (GSPE) significantly inhibited the expression of lactadherin in diabetic rats. Lactadherin is a secreted glycoprotein of milk-fat globule that shares structural domain homology with Del-1. Mouse lactadherin is also known as milk-fat globule-EGF factor 8 (MFG-E8). Lactadherin is expressed in the mammary epithelium, breast cancer, pancreas, aortic endothelial cells and smooth muscle cells, and macrophages and dendritic cells, etc. In our present study, we focused on the molecular mechanisms of human umbilical vein endothelial cells (HUVEC) apoptosis induced by AGEs, particularly 1) the role and molecular mechanism of the lactadherin (overexpression and siRNA) in the AGEs-induced endothelial cells apoptosis,2) the efficacy of HUVEC protection by grape seed procyanidin B2 (GSPB2).
Methods
HUVEC were cultured in six-well plates overnight and transfected with siRNA against lactadherin using Lipofectamine2000. Moreover, HUVEC were transduced with lentiviral vectors of lactadherin overexpression. HUVEC transfected lactadherin genes and siRNA were incubated for 48 h in the presence or absence of GSPB2 (10μmol/L). Meanwhile, HUVEC transfected lactadherin siRNA were stimulated with 200μg/mL of AGEs. Cell survival was estimated with MTT and CCK-8 colorimetric assay. The apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. To evaluate changes in morphology, HUVEC transfected lactadherin genes were visualized by transmission electron microscope (TEM). We determined the lactadherin, cleaved caspase 3, caspase 3, phosphorylation of GSK-3P and Bax/Bcl-2 in HUVEC by quantitative real-time PCR or western blotting analysis.
Results
1. Transduction efficiency with lactadherin siRNA and overexpression plasmids
We transduced the HUVEC with siRNA or lentivirus vector. Transduction conditions were optimized by using different MOI and the transduction efficiency was assessed by fluorescence microscopy, real-time PCR and western blotting. HUVEC carrying negative control siRNA (NC), HUVEC carrying siRNA against lactadherin (LsiRNA) and HUVEC carrying GFP (LV-C), HUVEC carrying both GFP and lactadherin genes (LV) were harvested. The transduction efficiency was about 95% or higher at day 5 or longer. Transgene expression was confirmed by fluorescent microscopic imaging for lactadherin protein expression. The mRNA and protein expression of lactadherin in LsiRNA group decreased to more than 60% the level of the NC group at 48 h after transfection. Lactadherin mRNA and protein expression reached its highest level at day 5 after virus removal.
2. Effects of lactadherin on viability in HUVEC stimulated by AGEs
There were no differences in the cell viability between LsiRNA group and NC group. Stimulation of NC group with AGEs (200μg/mL) resulted in a significant decrease in the cell viability, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the decrease of cell viability compared with NC group under AGEs stimulation for 48h (P＜0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated cell viability for 48h (P<0.05). Both HUVEC transfected GFP and negative control did not affect cell viability. Moreover, the overexpression of lactadherin significantly decreased the cell viability. The cell viability was increased when LV group was exposed to GSPB2 (10μmol/L) for 48h (P<0.05).
3. Effects of lactadherin on apoptosis in HUVEC stimulated by AGEs
Stimulation of NC group with AGEs (200μg/mL) resulted in a significant increase in the cell apoptosis, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the cell apoptosis compared with NC group under AGEs stimulation for 48h (P<0.05). The treatment of NC group with GSPB2 (10μmol/L) also significantly improved the AGEs-stimulated cell apoptosis for 48h (P＜0.05). HUVEC overexpression lactadherin was susceptible to cell apoptosis, while GSPB2 (10μmol/L) significantly attenuated the cell apoptosis for 48 h.
4. The morphological changes of HUVEC overexpression lactadherin
The cell apoptosis significantly increased in the HUVEC overexpression lactadherin, while GSPB2 (10μmol/L) significantly attenuated the cell apoptosis for 48 h.
5. Effects of lactadherin on cleaved caspase-3 and Bax/Bcl-2 ratio
The uncleaved caspase-3 levels were unchanged in LV-C group, LV group, LsiRNA group and NC group. Stimulation of NC group with AGEs (200μg/mL) resulted in a significant increase in the cleaved caspase-3 and Bax/Bcl-2 ratio, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the cleaved caspase-3 and Bax/Bcl-2 ratio compared with NC group under AGEs stimulation for 48h (P＜0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated the cleaved caspase-3 and Bax/Bcl-2 ratio for 48h (P<0.05). The cleaved caspase-3 and Bax/Bcl-2 ratio significantly increased in LV group comparing to LV-C group, while GSPB2 (10μmol/L) significantly inhibited the cleaved caspase-3 and Bax/Bcl-2 ratio in HUVEC overexpression lactadherin for 48 h (P<0.05).
6. Effect of lactadherin on the levels of phospho-GSK3β
Stimulation of NC group with AGEs (200μg/mL) resulted in a significant decrease in the levels of phospho-GSK3β, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the decreased levels of phospho-GSK3βcompared with NC group under AGEs stimulation for 48h (P<0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated the decreased levels of phospho-GSK3P for 48h (P＜0.05). The levels of phospho-GSK3βsignificantly decreased in LV group comparing to LV-C group, while GSPB2 (10μmol/L) significantly improved the phospho-GSK3βin HUVEC overexpression lactadherin for 48 h (P＜0.05).
Conclusion
1. Up-regulation of lactadherin plays a critical role in endothelial cells apoptosis induced by AGEs.
2. Mitochondria apoptosis pathway played a pivotal role in the cell apoptosis by lactadherin.
3. GSPB2 might have benefits in the early stage of diabetic endothelial dysfunction by inhibition of lactadherin.
引文
1. Cull CA, Jensen CC, Retnakaran R, Holman RR. Impact of the metabolic syndrome on macrovascular and microvascular outcomes in type 2 diabetes mellitus: United Kingdom Prospective Diabetes Study 78. Circulation, 2007;116(19): 2119-2126.
2. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract. 2010; 87(1):4-14.
3. Rathmann W, Giani G. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 2004; 27(10):2568-2569.
4. Nieuwdorp M, Holleman F, de Groot E, Vink H, Gort J, Kontush A, Chapman MJ, Hutten BA, Brouwer CB, Hoekstra JB, Kastelein JJ, Stroes ES. Perturbation of hyaluronan metabolism predisposes patients with type 1 diabetes mellitus to atherosclerosis. Diabetologia. 2007; 50(6):1288-1293.
5. Pambianco G, Costacou T, Ellis D, Becker DJ, Klein R, Orchard TJ. The 30-year natural history of type 1 diabetes complications: the Pittsburgh Epidemiology of Diabetes Complications Study experience. Diabetes. 2006; 55(5):1463-1469.
6. Mendez JD, Xie J, Aguilar-Hernandez M, Mendez-Valenzuela V. Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases. Mol Cell Biochem. 2010; 344(1-2): 185-193.
7. Bierhaus A, Nawroth PP. Multiple levels of regulation determine the role of the receptor for AGE (RAGE) as common soil in inflammation, immune responses and diabetes mellitus and its complications. Diabetologia. 2009; 52(11):2251-63.
8.屈琪,马静.晚期糖基化终末产物与动脉粥样硬化的研究进展.国外医学卫生学分册.2008:35(5):300-303.
9. Miyata T, Kurokawa K, Van Ypersele De Strihou C. Advanced glycation and lipoxidation end products: role of reactive carbonyl compounds generated during
10. Naka Y, Bucciarelli LG, Wendt T, Lee LK, Rong LL, Ramasamy R, Yan SF,Schmidt AM. RAGE axis: Animal models and novel insights into the vascular complications of diabetes. Arterioscler Thromb Vase Biol. 2004; 24(8): 1342-1349.
11. Arumugam T, Simeone DM, Schmidt AM, Logsdon CD. SI OOP stimulates cell proliferation and survival via receptor for activated glycation end products (RAGE). J Biol Chem. 2004; 279(7):5059-5065.
12. Sangle GV, Zhao R, Mizuno TM, Shen GX. Involvement of RAGE, NADPH oxidase, and Ras/Raf-1 pathway in glycated LDL-induced expression of heat shock factor-1 and plasminogen activator inhibitor-1 in vascular endothelial cells.Endocrinology. 2010; 151(9):4455-4466.
13. Wautier JL, Schmidt AM. Protein glycation: a firm link to endothelial cell dysfunction. Circ Res. 2004; 95(3):233-238.
14.于佩,于德民,齐建成,王静,张秋梅,张景云,汤云昭,邢秋玲,李明珍.高糖通过PI3K-Akt信号途径抑制内皮细胞迁移和增殖及血管发生改变.中华医学杂志.2006;86(48):3425-3430.
15. Kearney MT, Duncan ER, Kahn M, Wheatcroft SB. Insulin resistance and endothelial cell dysfunction: studies in mammalian models. Exp Physiol. 2008; 93(1): 158-163.
16. Yalcin E, Oruc E, Cavusoglu K, Yapar K. Protective role of grape seed extract against doxorubicin-induced cardiotoxicity and genotoxicity in albino mice. J Med Food. 2010; 13(4):917-925.
17. Houde V, Grenier D, and Chandad F. Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol. 2006; 77(8): 1371-1379.
18. Zi SX, Ma HJ, Li Y, Liu W, Yang QQ, Zhao G, and Lian S. Oligomeric proanthocyanidins from grape seeds effectively inhibit ultraviolet-induced melanogenesis of human melanocytes in vitro. Int J Mol Med. 2009; 23(2):197-204.
19. Yamakoshi J, Kataoka S, Koga T, and Ariga T. Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic arteriosclerosis in cholesterol-fed rabbits. Arteriosclerosis. 1999; 142(1):139-149.
20. Khanna S, Venojarvi M, Roy S, Sharma N, Trikha P, Bagchi D, Bagchi M, Sen CK. Dermal wound healing properties of redox-active grape seed proanthocyanidins. Free Radic Biol Med. 2002; 33(8): 1089-1096.
21. Mackenzie GG, Adamo AM, Decker NP, Oteiza PI. Dimeric procyanidin B2 inhibits constitutively active NF-kappaB in Hodgkin's lymphoma cells independently of the presence of IkappaB mutations. Biochem Pharmacol. 2008;75(7): 1461-1471.
22. Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, Xuan JH. Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol. 2006; 48(2): 47-53.
23. Sliman SM, Eubank TD, Kotha SR, Kuppusamy ML, Sherwani SI, Butler ES, Kuppusamy P, Roy S, Marsh CB, Stern DM, Parinandi NL. Hyperglycemic oxoaldehyde, glyoxal, causes barrier dysfunction, cytoskeletal alterations, and inhibition of angiogenesis in vascular endothelial cells: aminoguanidine protection.Mol Cell Biochem. 2010; 333(l-2):9-26.
24. van Dieren S, Beulens JW, van der Schouw YT, Grobbee DE, Neal B. The global burden of diabetes and its complications: an emerging pandemic. Eur J Cardiovasc Prev Rehabil. 2010; 17 Suppl 1 :S3-8.
25. Ning G, Hong J, Bi Y, Gu W, Zhang Y, Zhang Z, Huang Y, Wang W, Li X. Progress in diabetes research in China. J Diabetes. 2009; 1(3): 163-172.
26. Mendez JD, Xie J, Aguilar-Hernandez M, Mendez-Valenzuela V. Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases. Mol Cell Biochem. 2010; 344(1-2):185-193.
27. 刘萍,何兰杰.氧化应激与糖尿病.医学综述2.005;n(5):435-455.
28. Brownlee M. Biochemistry and molecular cell biology of diabetic complications.
29. Kim NH, Kim YS, Lee YM, Jang DS, Kim JS. Inhibition of aldose reductase and xylose-induced lens opacity iry'puerariafuran from the roots of Pueraria lobata. Biol Pharm Bull. 2010; 33(9): 1605-1609.
30. Das Evcimen N, King GL. The role of protein kinase C activation and the vascular complications of diabetes. Pharmacol Res. 2007; 55(6):498-510.
31. Chen J, Song M, Yu S, Gao P, Yu Y, Wang H, Huang L. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress. Mol Cell Biochem. 2010; 335(1-2):137-146.
32. Gharagozlian S, Henriksen T, Kolset SO. High glucose and Nepsilon-(carboxymethyl) lysine bovine serum albumin modulate release of matrix metalloproteinases in cultured human endothelial cells. Eur J Nutr. 2006; 45(5):283-290.
33. Chen SX, Song T, Zhou SH, Liu YH, Wu SJ, Liu LY. Protective effects of ACE inhibitors on vascular endothelial dysfunction induced by exogenous advanced oxidation protein products in rats. Eur J Pharmacol. 2008; 584(2-3):368-375.
34. Xu B, Chibber R, Ruggiero D, Kohner E, Ritter J, Ferro A. Impairment of vascular endothelial nitric oxide synthase activity by advanced glycation end products. FASEB J. 2003; 17(10): 1289-1291.
35. Potenza MA, Gagliardi S, Nacci C, Carratu' MR, Montagnani M. Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets. Curr Med Chem. 2009; 16(1):94-112.
36. Nagai R, Fujiwara Y, Mera K, Otagiri M. Investigation of pathways of advanced glycation end-products accumulation in macrophages. Mol Nutr Food Res. 2007;51(4):462-467.
37. Kearney MT, Duncan ER, Kahn M, Wheatcroft SB. Insulin resistance and endothelial cell dysfunction: studies in mammalian models. Exp Physiol, 2008; 93(1): 158-163.
38. Marchesi C, Ebrahimian T, Angulo O, Paradis P, Schiffrin EL. Endothelial nitric inflammation contribute to vascular dysfunction in a rodent model of metabolic syndrome. Hypertension, 2009; 54(6): 1384-1392.
39. Sakano K, Mizutani M, Murata M, Oikawa S, Hiraku Y, Kawanishi S. Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage. Free Radic Biol Med. 2005; 39(8):1041-1049.
40. de la Iglesia R, Milagro FI, Campion J, Boque N, Martinez JA. Healthy properties of proanthocyanidins. Biofactors. 2010; 36(3):159-168.
41. Bagchi D, Sen CK, Ray SD, Das DK, Bagchi M, Preuss HG, Vinson JA. Molecular mechanisms of cardioprotection by a novel grape seed proanthocyanidin extract. MutatRes. 2003;523-524:87-97.
43. Nassiri-Asl M, Hosseinzadeh H. Review of the pharmacological effects of Vitis vinifera (Grape) and its bioactive compounds. Phytother Res, 2009; 23(9):1197-1204.
44. Wang L, Zhu LH, Jiang H, Tang QZ, Yan L, Wang D, Liu C, Bian ZY, Li H. Grape seed proanthocyanidins attenuate vascular smooth muscle cell proliferation via blocking phosphatidylinositol 3-kinase-dependent signaling pathways. J Cell Physiol. 2010; 223(3):713-726.
45. Demirkaya E, Avci A, Kesik V, Karslioglu Y, Oztas E, Kismet E, Gokcay E, Durak I, Koseoglu V. Cardioprotective roles of aged garlic extract, grape seed proanthocyanidin, and hazelnut on doxorubicin-induced cardiotoxicity. Can J Physiol Pharmacol. 2009; 87(8):633-640.
46. Bagchi D, Garg A, Krohn RL, Bagchi M, Tran MX, Stohs SJ. Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro. Res Commun Mol Pathol Pharmacol. 1997; 95(2): 179-189.
47. Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, Joshi SS, Pruess HG. Free radicals and grape seed proanthocyanidin extract: importance in human
48. Farrar JL, Hartle DK, Hargrove JL, Greenspan P. Inhibition of protein glycation by skins and seeds of the muscadine grape. Biofactors. 2007; 30(3): 193-200.
49. Ma L, Gao HQ, Li BY, Ma YB, You BA, Zhang FL. Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma. J Cardiovasc Pharmacol. 2007; 49(5): 293-298.
50. Zhang FL, Gao HQ, Shen L. Inhibitory effect of GSPE on RAGE expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol. 2007;50(4):434-440.
51. 周雁,马亚兵,高海青,等.葡萄籽多酚抗糖尿病大鼠非酶糖基化实验研究.中华老年医学杂志.2005:24(1):49-52.
52. Li XL, Li BY, Gao HQ, Cheng M, Xu L, Li XH, Zhang WD, Hu JW. Proteomics approach to study the mechanism of action of grape seed proanthocyanidin extracts on arterial remodeling in diabetic rats. Int J Mol Med. 2010; 25(2):237-248.
53. Li XL, Li BY, Gao HQ, Cheng M, Xu L, Li XH, Ma YB. Effects of grape seed proanthocyanidins extracts on aortic pulse wave velocity in streptozocin induced diabetic rats. Biosci Biotechnol Biochem. 2009; 73(6): 1348-1354.
54. Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, You BA. Back- regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy. J Cell Biochem. 2008; 104(2):668-679.
55. Li M, Ma YB, Gao HQ, Li BY, Cheng M, Xu L, Li XL, Li XH. A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats.Chin Med J (Engl). 2008;121(24):2544-2552.
56. Xu L, Li B, Cheng M, Zhang W, Pan J, Zhang C, Gao H. Oral administration of grape seed proanthocyanidin extracts downregulate RAGE dependant nuclear factor- kappa BP65 expression in the hippocampus of streptozotocin induced
57. Cheng M, Gao HQ, Xu L, Li BY, Zhang H, Li XH. Cardioprotective effects of grape seed proanthocyanidins extracts in streptozocin induced diabetic rats. J Cardiovasc Pharmacol. 2007; 50(5):503-509.
58. Liang Y, Qiu J, Gao HQ, Li BY. Protective effect of grape seed proanthocyanidins extracts on reperfusion arrhythmia in rabbits. J Nutr Sci Vitaminol (Tokyo). 2009;55(3):223-230.
59. Cho ML, Heo YJ, Park MK, Oh HJ, Park JS, Woo YJ, Ju JH, Park SH, Kim HY, Min JK. Grape seed proanthocyanidin extract (GSPE) attenuates collagen-induced arthritis. Immunol Lett. 2009; 124(2): 102-110.
60. Shao ZH, Wojcik KR, Dossumbekova A, Hsu C, Mehendale SR, Li CQ, Qin Y, Sharp WW, Chang WT, Hamann KJ, Yuan CS, Hoek TL. Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt-NOS signaling. J Cell Biochem. 2009; 107(4):697-705.
61. Morin B, Narbonne JF, Ribera D, Badouard C, Ravanat JL. Effect of dietary fat-soluble vitamins A and E and proanthocyanidin-rich extract from grape seeds on oxidative DNA damage in rats. Food Chem Toxicol. 2008; 46(2): 787-796.
62. Bu HF, Zuo XL, Wang X, Ensslin MA, Koti V, Hsueh W, Raymond AS, Shur BD, Tan XD. Milk fat globule-EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium. J Clin hivest. 2007; 117(12):3673-3683.
63. Lin L, Huai Q, Huang M, Furie B, Furie BC. Crystal structure of the bovine lactadherin C2 domain, a membrane binding motif, shows similarity to the C2 domains of factor V and factor VDL J Mol Biol. 2007; 371(3):717-724.
64. Bojsen A, Buesa J, Montava R, Kvistgaard AS, Kongsbak MB, Petersen TE, Heegaard CW, Rasmussen JT. Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo. J Dairy Sci. 2007; 90(1):66-74.
65. Neutzner M, Lopez T, Feng X, Bergmann-Leitner ES, Leitner WW, Udey MC.MFG-E8/lactadherin promotes tumor growth in an angiogenesis-dependent transgenic mouse model of multistage carcinogenesis. Cancer Res. 2007; 67(14):6777-6785.
66. Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing forphospholipid-binding sites. Blood. 2003; 101(7):2628-2636.
67. Hanayama R, Tanaka M, Miwa K, Nagata S. Expression of developmental endothelial locus-1 in a subset of macrophages for engulfinent of apoptotic cells. J Immunol. 2004; 172(6):3876-3882.
68. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, Tamura N, Tamura T, Matsuda T. Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles. Endocrinology. 2007; 148(8):3850-3862.
69. Fu Z, Wang M, Gucek M, Zhang J, Wu J, Jiang L, Monticone RE, Khazan B, Telljohann R, Mattison J, Sheng S, Cole RN, Spinetti G, Pintus G, Liu L, Kolodgie FD, Virmani R, Spurgeon H, Ingram DK, Everett AD, Lakatta EG, Van Eyk JE. Milk fat globule protein epidermal growth factor-8: a pivotal relay element within the angiotensin II and monocyte chemoattractant protein-1 signaling cascade mediating vascular smooth muscle cells invasion. Circ Res. 2009;104(12):1337-1346.
70. Silvestre JS, Thery C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc-Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Levy B, Tedgui A, Amigorena S, Mallat Z. Lactadherin promotes VEGF-dependent neovascularization. Nat Med. 2005; 11(5):499-506.
71. Chen L, Zhang Y, Sun X, Li H, LeSage G, Javer A, Zhang X, Wei X, Jiang Y, Yin D. Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3beta pathway. Bioorg Med Chem. 2009; 17(13):4378-4382.
72. Linseman DA, Butts BD, Precht TA, Phelps RA, Le SS, Laessig TA, Bouchard RJ, Florez-McClure ML, Heidenreich KA. Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci. 2004; 24(44):9993-10002.
1. Lin EH, Rutter CM, Katon W, Heckbert SR, Ciechanowski P, Oliver MM, Ludman EJ, Young BA, Williams LH, McCulloch DK, Von Korff M. Depression and advanced complications of diabetes:a prospective cohort study. Diabetes Care. 2010;33(2):264-269.
2. Wong TY, Mwamburi M, Klein R, Larsen M, Flynn H, Hernandez-Medina M, Ranganathan G, Wirostko B, Pleil A, Mitchell P. Rates of progression in diabetic retinopathy during different time periods:a systematic review and meta-analysis. Diabetes Care.2009; 32(12):2307-2313.
3. Mendez JD, Xie J, Aguilar-Hernandez M, Mendez-Valenzuela V. Trends in advanced glycation end products research in diabetes mellitus and its complications. Mol Cell Biochem.2010; 341(1-2):33-41.
4. Lin EH, Rutter CM, Katon W, Heckbert SR, Ciechanowski P, Oliver MM, Ludman EJ, Young BA, Williams LH, McCulloch DK, Von Korff M. Depression and advanced complications of diabetes:a prospective cohort study. Diabetes Care. 2010; 33(2):264-269.
5. UK Prospective Diabetes Study (UKPDS) Group:Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS33). Lancet.1998; 352(9131):837-853.
6. Brownlee M. The pathobiology of diabetic complications:a unifying mechanism. Diabetes.2005; 54(6):1615-1625.
7. Ramasamy R, Goldberg IJ. Aldose reductase and cardiovascular diseases, creating human-like diabetic complications in an experimental model. Circ Res.2010; 106(9):1449-1458.
8. Ohmura C, Watada H, Azuma K, Shimizu T, Kanazawa A, Ikeda F, Yoshihara T, Fujitani Y, Hirose T, Tanaka Y, Kawamori R. Aldose reductase inhibitor, epalrestat, reduces lipid hydroperoxides in type 2 diabetes. Endocr J.2009; 56(1):149-156.
9. Geraldes P, Hiraoka-Yamamoto J, Matsumoto M, Clermont A, Leitges M, Marette A, Aiello LP, Kern TS, King GL. Activation of PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy. Nat Med. 2009; 15(11):1298-1306.
10.1Kanwar M, Kowluru RA. Role of glyceraldehyde 3-phosphate dehydrogenase in the development and progression of diabetic retinopathy. Diabetes.2009; 58(1):227-234.
11. Hodgkinson CP, Laxton RC, Patel K, Ye S.183.Advanced glycation end-product of low density lipoprotein activates the toll-like 4 receptor pathway implications for diabetic atherosclerosis. Arterioscler Thromb Vase Biol.2008; 28(12):2275-2281.
12. Nevado J, Peiro C, Vallejo S, El-Assar M, Lafuente N, Matesanz N, Azcutia V, Cercas E, Sanchez-Ferrer CF, Rodriguez-Manas L. Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells. Br J Pharmacol.2005; 146(2):268-279.
13. Nin JW, Jorsal A, Ferreira I, Schalkwijk CG, Prins MH, Parving HH, Tarnow L, Rossing P, Stehouwer CD. Higher plasma soluble Receptor for Advanced Glycation End Products (sRAGE) levels are associated with incident cardiovascular disease and all-cause mortality in type 1 diabetes:a 12-year follow-up study. Diabetes.2010; 59(8):2027-2032.
14. Nakamura K, Yamagishi S, Adachi H, Matsui T, Kurita-Nakamura Y, Takeuchi M, Inoue H, Imaizumi T. Circulating advanced glycation end products (AGEs) and soluble form of receptor for AGEs (sRAGE) are independent determinants of serum monocyte chemoattractant protein-1 (MCP-1) levels in patients with type 2 diabetes. Diabetes Metab Res Rev.2008; 24(2):109-114.
15. Mercer N, Ahmed H, Etcheverry SB, Vasta GR, Cortizo AM. Regulation of advanced glycation end product (AGE) receptors and apoptosis by AGEs in osteoblast-like cells. Mol Cell Biochem.2007; 306(1-2):87-94.
16. Cho SJ, Roman G, Yeboah F, Konishi Y. The road to advanced glycation end products:a mechanistic perspective. Curr Med Chem.2007; 14(15):1653-1671.
17. Mahajan N, Malik N, Bahl A, Dhawan V. Receptor for advanced glycation end products (RAGE) and its inflammatory ligand EN-RAGE in non-diabetic subjects with pre-mature coronary artery disease. Atherosclerosis.2009; 207(2):597-602.
18. Hanayama R, Tanaka M, Miwa K, Nagata S. Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells. J Immunol.2004; 172(6):3876-3882.
19. Ait-Oufella H, Kinugawa K, Zoll J, Simon T, Boddaert J, Heeneman S, Blanc-Brude O, Barateau V, Potteaux S, Merval R, Esposito B, Teissier E, Daemen MJ, Leseche G, Boulanger C, Tedgui A, Mallat Z. Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosis in mice. Circulation. 2007; 115(16):2168-2177.
20. Veron P, Segura E, Sugano G, Amigorena S, Thery C. Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells. Blood Cells Mol Dis.2005;35(2):81-88.
21. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, Tamura N, Tamura T, Matsuda T. Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes:redox-and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles. Endocrinology.2007; 148(8):3850-3862.
22.余祖华,丁轲,程相朝,张春杰,李银聚,吴庭才.外源基因转染真核细胞技术的研究进展.安徽农业科学.2008；36(21)：8954-8955.
23. Zhang H, Xie J, Xie Q, Wilson JM, Gao G. Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production. Hum Gene Ther.2009; 20(9):922-929.
24. Hart SL. Multifunctional nanocomplexes for gene transfer and gene therapy. Cell Biol Toxicol.2010; 26(1):69-81.
25. Agarwal C, Veluri R, Kaur M, Chou SC, Thompson JA, Agarwal R. Fractionation of high molecular weight tannins in grape seed extract and identification of procyanidin B2-3,3'-di-O-gallate as a major active constituent causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells. Carcinogenesis.2007; 28(7):1478-1484.
26. Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, Xuan JH. Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol.2006; 48(2):47-53.
27. Ma L, Gao HQ, Li BY, Ma YB, You BA, Zhang FL. Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma. J Cardiovasc Pharmacol.2007; 49(5):293-298.
28. Zhang FL, Gao HQ, Shen L. Inhibitory effect of GSPE on RAGE expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol.2007; 50(4):434-440.
29. McDonald DM, Coleman G, Bhatwadekar A, Gardiner TA, Stitt AW. Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal micro vascular endothelial cell dysfunction. Mol Vis.2009; 15:1509-1520.
30. Sawabe M. Vascular aging:from molecular mechanism to clinical significance. Geriatr Gerontol Int.2010; 10 Suppl 1:S213-220.
31. Xiang M, Yang M, Zhou C, Liu J, Li W, Qian Z. Crocetin prevents AGEs-induced vascular endothelial cell apoptosis. Pharmacol Res.2006; 54(4):268-274.
32.张瑾,李勇.高糖诱导血管内皮细胞凋亡及其调控机制研究进展.国际心血管病杂志.2008；35(6)：380-383.
33.任澎,马业新.动脉粥样硬化和再狭窄中细胞凋亡研究的近况.心血管康复医学杂志.2005；14(6)：597-600.
34.于珮,于德民,齐建成,王静,张秋梅,张景云,汤云昭,邢秋玲,李明珍.高糖通过P13K-Akt信号途径抑制内皮细胞迁移和增殖及血管发生改变.中华医学杂志.2006；86(48)：3425-3430.
35. Schmitt CA, Heiss EH, Dirsch VM. Effect of resveratrol on endothelial cell function:Molecular mechanisms. Biofactors.2010; 36(5):342-349.
36. Malfitano C, Alba Loureiro TC, Rodrigues B, Sirvente R, Salemi VM, Rabechi NB, Lacchini S, Curi R, Irigoyen MC. Hyperglycaemia protects the heart after myocardial infarction:aspects of programmed cell survival and cell death. Eur J Heart Fail.2010; 12(7):659-967.
37.张风雷,高海青,沈琳,吴建敏,由倍安,马亚兵.葡萄籽多酚对糖基化终产物诱导的内皮细胞组织因子表达的影响.中华老年医学杂志,2007；26(1)：29-33.
38. Nakagami H, Kaneda Y, Ogihara T, Morishita R. Endothelial dysfunction in hyperglycemia as a trigger of atherosclerosis. Curr Diabetes Rev.2005; 1(1):59-63.
39. Neutzner M, Lopez T, Feng X, Bergmann-Leitner ES, Leitner WW, Udey MC. MFG-E8/lactadherin promotes tumor growth in an angiogenesis-dependent transgenic mouse model of multistage carcinogenesis. Cancer Res.2007; 67(14):6777-6785.
40. Silvestre JS, Thery C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc-Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Levy B, Tedgui A, Amigorena S, Mallat Z. Lactadherin promotes VEGF-dependent neovascularization. Nat Med.2005; 11(5):499-506.
41. Larsson A, Peng S, Persson H, Rosenbloom J, Abrams WR, Wassberg E, Thelin S, Sletten K, Gerwins P, Westermark P. Lactadherin binds to elastin--a starting point for medin amyloid formation? Amyloid.2006; 13(2):78-85.
42. Morelli AE, Larregina AT, Shufesky WJ, Sullivan ML, Stolz DB, Papworth GD, Zahorchak AF, Logar AJ, Wang Z, Watkins SC, Falo LD Jr, Thomson AW. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood.2004; 104(10):3257-3266.
43. Fu Z, Wang M, Gucek M, Zhang J, Wu J, Jiang L, Monticone RE, Khazan B, Telljohann R, Mattison J, Sheng S, Cole RN, Spinetti G, Pintus G, Liu L, Kolodgie FD, Virmani R, Spurgeon H, Ingram DK, Everett AD, Lakatta EG, Van Eyk JE. Milk fat globule protein epidermal growth factor-8:a pivotal relay element within the angiotensin Ⅱ and monocyte chemoattractant protein-1 signaling cascade mediating vascular smooth muscle cells invasion. Circ Res.2009; 104(12):1337-1346.
44.郭剑英,李英,潘家强,唐兆新.线粒体通透性转换孔及其与细胞凋亡的关系.动物医学进展.2009；30(8)：101-105.
45. Javadov S, Karmazyn M. Mitochondrial permeability transition pore opening as an endpoint to initiate cell death and as a putative target for cardioprotection. Cell Physiol Biochem.2007; 20(1-4):1-22.
46. Xie M, Yang S, Win HL, Xiong L, Huang J, Zhou J. Rabbit annulus fibrosus cell apoptosis induced by mechanical overload via a mitochondrial apoptotic pathway. J Huazhong Univ Sci Technolog Med Sci.2010; 30(3):379-384.
47. Zhao NQ, Yu YR, Tan HW, Deng G, Zhang XX. Role of apoptosis and mitochondrial apoptotic pathway in glucolipotoxicity-induced islet beta-cell dysfunction. Nan Fang Yi Ke Da Xue Xue Bao.2008; 28(11):2009-2013.
48. Jourdain A, Martinou JC. Mitochondrial outer-membrane permeabilization and remodelling in apoptosis. Int J Biochem Cell Biol.2009; 41(10):1884-1889.
49. Gupta S, Kass GE, Szegezdi E, Joseph B. The mitochondrial death pathway:a promising therapeutic target in diseases. J Cell Mol Med.2009; 13(6):1004-1033.
50.王文文,邓毛程.Bcl-2、Bax及Bid与细胞周期阻滞及凋亡调控的关系.广东轻工职业技术学院学报.2009；8(3)：21-24.
51. Janumyan YM, Sansam CG, Chattopadhyay A, Cheng N, Soucie EL, Penn LZ, Andrews D, Knudson CM, Yang E. Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry. EMBO J.2003; 22(20):5459-5470.
52. Cheng N, Janumyan YM, Didion L, Van Hofwegen C, Yang E, Knudson CM. Bcl-2 inhibition of T-cell proliferation is related to prolonged T-cell survival. Oncogene. 2004; 23(21):3770-3780.
53. Zhang Z, Lapolla SM, Annis MG, Truscott M, Roberts GJ, Miao Y, Shao Y, Tan C, Peng J, Johnson AE, Zhang XC, Andrews DW, Lin J. Bcl-2 homodimerization involves two distinct binding surfaces, a topographic arrangement that provides an effective mechanism for Bcl-2 to capture activated Bax. J Biol Chem.2004; 279(42):43920-43928.
54. Tan C, Dlugosz PJ, Peng J, Zhang Z, Lapolla SM, Plafker SM, Andrews DW, Lin J. Auto-activation of the apoptosis protein Bax increases mitochondrial membrane permeability and is inhibited by Bcl-2. J Biol Chem.2006; 281(21):14764-14775.
55. Wang CY, Lin YS, Su WC, Chen CL, Lin CF. Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis. Mol Biol Cell.2009; 20(19):4153-4161.
56. Wang Y, Huang WC, Wang CY, Tsai CC, Chen CL, Chang YT, Kai JI, Lin CF. Inhibiting glycogen synthase kinase-3 reduces endotoxaemic acute renal failure by down-regulating inflammation and renal cell apoptosis. Br J Pharmacol.2009; 157(6):1004-1013.
57. Chen L, Zhang Y, Sun X, Li H, LeSage G, Javer A, Zhang X, Wei X, Jiang Y, Yin D. Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3beta pathway. Bioorg Med Chem.2009; 17(13): 4378-4382.
58. Linseman DA, Butts BD, Precht TA, Phelps RA, Le SS, Laessig TA, Bouchard RJ, Florez-McClure ML, Heidenreich KA. Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci.2004; 24(44):9993-10002.
59. King TD, Bijur GN, Jope RS. Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium. Brain Res.2001; 919(1):106-114.
60. King TD, Jope RS. Inhibition of glycogen synthase kinase-3 protects cells from intrinsic but not extrinsic oxidative stress. Neuroreport.2005; 16(6):597-601.
61. Miura T, Miki T. GSK-3beta, a therapeutic target for cardiomyocyte protection. Circ J.2009; 73(7):1184-1192.
62. Ougolkov AV, Billadeau DD. Inhibition of glycogen synthase kinase-3. Methods Mol Biol.2008; 468:67-75.
63. Gao HK, Yin Z, Zhou N, Feng XY, Gao F, Wang HC. Glycogen synthase kinase 3 inhibition protects the heart from acute ischemia-reperfusion injury via inhibition of inflammation and apoptosis. J Cardiovasc Pharmacol.2008; 52(3):286-292.
64. Ma H, Li SY, Xu P, Babcock SA, Dolence EK, Brownlee M, Li J, Ren J. Advanced glycation endproduct (AGE) accumulation and AGE receptor (RAGE) up-regulation contribute to the onset of diabetic cardiomyopathy. J Cell Mol Med. 2009; 13(8B):1751-1764.
65. Wang Y, Feng W, Xue W, Tan Y, Hein DW, Li XK, Cai L. Inactivation of GSK-3beta by metallothionein prevents diabetes-related changes in cardiac energy metabolism, inflammation, nitrosative damage, and remodeling. Diabetes.2009; 58(6):1391-1402.
66. Zunino S. Type 2 diabetes and glycemic response to grapes or grape products. J Nutr.2009; 139(9):1794S-800S.
67. Houde V, Grenier D, Chandad F. Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol.2006; 77(8):1371-1379.
68. Khanna S, Venojarvi M, Roy S, Sharma N, Trikha P, Bagchi D, Bagchi M, Sen CK. Dermal wound healing properties of redox-active grape seed proanthocyanidins. Free Radic Biol Med.2002; 33(8):1089-1096.
69. Vayalil PK, Mittal A, Katiyar SK. Proanthocyanidins from grape seeds inhibit expression of matrix metalloproteinases in human prostate carcinoma cells, which is associated with the inhibition of activation of MAPK and NF kappa B. Carcinogenesis.2004; 25(6):987-995.
70. Zhang WY, Liu HQ, Xie KQ, Yin LL, Li Y, Kwik-Uribe CL, Zhu XZ. Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells. Biochem Biophys Res Commun.2006; 345(1):508-515.
71. Chen DM, Cai X, Kwik-Uribe CL, Zeng R, Zhu XZ. Inhibitory effects of procyanidin B(2) dimer on lipid-laden macrophage formation. J Cardiovasc Pharmacol.2006; 48(2):54-70.
72. Mackenzie GG, Adamo AM, Decker NP, Oteiza PI. Dimeric procyanidin B2 inhibits constitutively active NF-kappaB in Hodgkin's lymphoma cells independently of the presence of IkappaB mutations. Biochem Pharmacol.2008; 75(7):1461-1471.
73. Li XL, Li BY, Gao HQ, Cheng M, Xu L, Li XH, Ma YB. Effects of grape seed proanthocyanidins extracts on aortic pulse wave velocity in streptozocin induced diabetic rats. Biosci Biotechnol Biochem.2009; 73(6):1348-1354.
74. Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, You BA. Back-regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy. J Cell Biochem.2008; 104(2):668-679.
75. Li M, Ma YB, Gao HQ, Li BY, Cheng M, Xu L, Li XL, Li XH. A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats.Chin Med J (Engl).2008; 121(24):2544-2552.
1) De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, and Vanhoutte PM, Br. J. Pharmacol,130,963-974(2000).
2) Al-Maskari F, El-Sadig M, and Norman JN, Cardiovasc. Diabetol.,6,24(2007).
3) Nieuwdorp M, Holleman F, de Groot E, Vink H, Gort J, Kontush A, Chapman MJ, Hutten BA, Brouwer CB, Hoekstra JB, Kastelein JJ, and Stroes ES, Diabetologia, 50,1288-1293(2007).
4) Rahman S, Rahman T, Ismail AA, and Rashid AR, Diabetes. Obes. Metab.,9,767-780(2007).
5) Barinas-Mitchell E, Kuller LH, Sutton-Tyrrell K, Hegazi R, Harper P, Mancino J, and Kelley DE, Diabetes Care,29,2218-2222(2006).
6) Cameron JD, and Cruickshank JK, Clin. Exp. Pharmacol. Physio.,34,677-682 (2007).
7) Smith A, Karalliedde J, De Angelis L, Goldsmith D, and Viberti G, J. Am. Soc. Nephrol,16,1069-1075(2005).
8) Cruickshank K, Riste L, Anderson SG, Wright JS, Dunn G, and Gosling RG, Circulation,106,2085-2090(2002).
9) Liu XN, Gao HQ, Li BY, Cheng M, Ma YB, Zhang ZM, Gao XM, Liu YP, and Wang M, Hypertens. Res.,30,237-242(2007).
10) Rahman S, Ismail AA, Ismail SB, Naing NN, and Rahman AR, Diabetes. Res. Clin. Pract.,80,253-258(2008).
11) Mulvany MJ, Hypertension,1,7-9(1992).
12) Houde V, Grenier D, and Chandad F, J. Periodontol.,77,1371-1379(2006).
13) Khanna S, Venojarvi M, Roy S, Sharma N, Trikha P, Bagchi D, Bagchi M, and Sen CK, Free. Radic. Biol. Med.,33,1089-1096(2002).
14) Vayalil PK, Mittal A, and Katiyar SK, Carcinogenesis,25,987-995(2004).
15) Cheng M, Gao HQ, Xu L, Li BY, Zhang H, Li XH, J. Cardiovasc. Pharmacol,50, 503-509(2007).
16) Xu L, Li B, Cheng M, Zhang W, Pan J, Zhang C, and Gao H, Exp. Clin. Endocrinol. Diabetes.,116,215-224(2008).
17) Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, and You BA, J. Cell. Biochem.,104,668-679(2008).
18) Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, and Xuan JH, J. Cardiovasc. Pharmacol.,48,47-53(2006).
19) Ma L, Gao HQ, Li BY, Ma YB, You BA, Zhang FL, J. Cardiovasc. Pharmacol.,49, 293-298(2007).
20) Pinent M, Blay M, Blade MC, Salvado MJ, Arola L, and Ardevol A, Endocrinology, 145,4985-4990(2004).
21) Nemoto O, Kawaguchi M, Yaoita H, Miyake K, Maehara K, and Maruyama Y, Circ. J.,70,327-334(2006).
22) Kamata K, Hosokawa M, Matsumoto T, and Kobayashi T, J. Smooth. Muscle. Res., 42,171-187(2006).
23) Gaillard V, Casellas D, Seguin-Devaux C, Schohn H, Dau9a M, Atkinson J, and Lartaud I, Hypertension,46,372-379(2005).
24) Mitchell GF, Pfeffer MA, Finn PV, and Pfeffer JM, J. Appl. Physiol.,82,203-210 (1997).
25) Niederhoffer N, Kieffer P, Desplanches D, Lartaud-Idjouadiene Ⅰ, Somay MH, and Atkinson J, Hypertension,35,919-924(2000).
26) Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, and Jerums G, Diabetes,40, 1328-1334(1991).
27) Candido R, Forbes JM, Thomas MC, Thallas V, Dean RQ Burns WC, Tikellis C, Ritchie RH, Twigg SM, Cooper ME, and Burrell LM, Circ. Res.,92,785-792 (2003).
28) Valitutto M, Osteopath. Med. Prim. Care.,2,4(2008).
29) Brownlee M, Nature,414,813-820(2001).
30) Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, Kislinger T, Stern DM, Schmidt AM, and De Caterina R, Circulation,105,816-822(2002).
31) Chetyrkin SV, Mathis ME, Ham AJ, Hachey DL, Hudson BG, and Voziyan PA, Free. Radic. Biol. Med.,44,1276-1285(2008).
32) San Martin A, Foncea R, Laurindo FR, Ebensperger R, Griendling KK, and Leighton F, Free. Radic. Biol. Med.,42,1671-1679(2007).
33) Hatsuda S, Shoji T, Shinohara K, Kimoto E, Mori K, Fukumoto S, Koyama H, Emoto M, and Nishizaw Y, J.Atheroscler. Thromb.,13,114-121(2006).
34) Vaitkevicius PV, Lane M, Spurgeon H, Ingram DK, Roth GS, Egan JJ, Vasan S, Wagle DR, Ulrich P, Brines M, Wuerth JP, Cerami A, Lakatta EG, Proc. Natl. Acad. Sci.U S A,98,1171-1175(2001).
35) Shiina K, Tomiyama H, Takata Y, Usui Y, Asano K, Hirayama Y, Nakamura T, and Yamashina A, Hypertens. Res.,29,433-441(2006).
36) Naka Y, Bucciarelli LG, Wendt T, Lee LK, Rong LL, Ramasamy R, Yan SF, and Schmidt AM, Arterioscler. Thromb. Vasc. Biol.,24,1342-1349(2004).
37) El-Alfy AT, Ahmed AA, and Fatani AJ, Pharmacol. Res.,52,264-270(2005).
38) Goldin A, Beckman JA, Schmidt AM, and Creager MA, Circulation,114,597-605 (2006).
39) Peppa M, and Vlassara H, Hormones (Athens),4,28-37(2005).
40) Zhang FL, Gao HQ, and Shen L, J. Cardiovasc. Pharmacol.,50,434-440(2007).
41) Ramasamy R, Vannucci SJ, Yan SS, Herold K, Yan SF, and Schmidt AM, Glycobiology,15,16R-28R(2005).
42) Ramasamy R, Yan SF, and Schmidt AM, Trends. Cardiovasc. Med.,15,237-243 (2005).
43) Haslbeck KM, Schleicher E, Bierhaus A, Nawroth P, Haslbeck M, Neundorfer B, and Heuss D, Exp. Clin. Endocrinol. Diabetes.,113,288-291(2005).
44) Cipollone F, Iezzi A, Fazia M, Zucchelli M, Pini B, Cuccurullo C, De Cesare D, De Blasis G, Muraro R, Bei R, Chiarelli F, Schmidt AM, Cuccurullo F, and Mezzetti A, Circulation,108,1070-1077(2003).
45) Fitzpatrick DF, Bing B, Maggi DA, Fleming RC, and O'Malley RM, Ann. N Y Acad. Sci.,957,78-89(2002).
1. Henry RM, Kostense PJ, Spijkerman AM, Dekker JM, Nijpels G, Heine RJ, Kamp O, Westerhof N, Bouter LM, and Stehouwer CD:Arterial stiffness increases with deteriorating glucose tolerance status:the Hoorn Study. Circulation 107: 2089-2095,2003.
2. Terry JG, Tang R, Espeland MA, Davis DH, Vieira JL, Mercuri MF, and Crouse JR: Carotid structure in patients with documented coronary artery disease and disease-free control subjects. Circulation 107:1146-1151,2003.
3. Houde V, Grenier D, and Chandad F:Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol 77:1371-1379,2006.
4. Zi SX, Ma HJ, Li Y, Liu W, Yang QQ, Zhao Q and Lian S:Oligomeric proanthocyanidins from grape seeds effectively inhibit ultraviolet-induced melanogenesis of human melanocytes in vitro. Int J Mol Med 23:197-204,2009.
5. Yamakoshi J, Kataoka S, Koga T, and Ariga T:Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic arteriosclerosis in cholesterol-fed rabbits. Arteriosclerosis 142:139-149,1999.
6. Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, and Xuan JH:Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol 48:47-53,2006.
7. Ma L, Gao HQ, Li BY, Ma YB, You BA, and Zhang FL:Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma. J Cardiovasc Pharmacol 49:293-298, 2007.
8. Pinent M, Blay M, Blade MC, Salvado MJ, Arola L, and Ardevol A:Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines. Endocrinology 145:4985-4990,2004.
9. Holm AM, Andersen CB, Haunsφ S, and Hansen PR:ACE-inhibition promotes apoptosis after balloon injury of rat carotid arteries. Cardiovasc Res 45:777-782, 2000.
10. Buus CL, Pourageaud F, Fazzi GE, Janssen G, Mulvany MJ, and De Mey JG: Smooth muscle cell changes during flow-related remodeling of rat mesenteric resistance arteries. Circ Res 89:180-186,2001.
11. Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, and You BA: Back-regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy. J Cell Biochem 104:668-679,2008.
12. Ji H, Moritz RL, Kim YS, Zhu HJ, and Simpson RJ:Analysis of Ras-induced oncogenic transformation of NIH-3T3 cells using differential-display 2-DE proteomics. Electrophoresis 28:1997-2008,2007.
13. Cimmino F, Spano D, Capasso M, Zambrano N, Russo R, Zollo M, and Iolascon A: Comparative proteomic expression profile in all-trans retinoic acid differentiated neuroblastoma cell line. J Proteome Res 6:2550-2564,2007.
14. Ritorto MS, and Borlak J:A simple and reliable protocol for mouse serum proteome profiling studies by use of two-dimensional electrophoresis and MALDI TOF/TOF mass spectrometry. Proteome Sci 6:25,2008.
15. D'Hertog W, Overbergh L, Lage K, Ferreira GB, Maris M, Gysemans C, Flamez D, Cardozo AK, Van den Bergh G, Schoofs L, Arckens L, Moreau Y, Hansen DA, Eizirik DL, Waelkens E, and Mathieu C:Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-IE cells:new insights into the pathways involved. Mol Cell Proteomics 6:2180-2199,2007.
16. Stenina OI, Krukovets I, Wang K, Zhou Z, Forudi F, Penn MS, Topol EJ, and Plow EF:Increased expression of thrombospondin-1 in vessel wall of diabetic Zucker rat. Circulation 107:3209-3215,2003.
17. Brownlee M:Biochemistry and molecular cell biology of diabetic complications. Nature 414:813-820,2001.
18. Prabakaran S, Wengenroth M, Lockstone HE, Lilley K, Leweke FM, and Bahn S: 2-D DIGE analysis of liver and red blood cells provides further evidence for oxidative stress in schizophrenia. J Proteome Res 6:141-149,2007.
19. Shiota M, Kusakabe H, Hikita Y, Nakao T, Izumi Y, and Iwao H: Pharmacogenomics of cardiovascular pharmacology:molecular network analysis in pleiotropic effects of statin-an experimental elucidation of the pharmacologic action from protein-protein interaction analysis. J Pharmacol Sci 107:15-19,2008.
20. El-Alfy AT, Ahmed AA, and Fatani AJ:Protective effect of red grape seeds proanthocyanidins against induction of diabetes by alloxan in rats. Pharmacol Res 52:264-270,2005.
21. Fitzpatrick DF, Bing B, Maggi DA, Fleming RC, and O'Malley RM:Vasodilating procyanidins derived from grape seeds. Ann N Y Acad Sci 957:78-89,2002.
22. Almofti MR, Huang Z, Yang P, Rui Y, and Yang P:Proteomic analysis of rat aorta during arteriosclerosis induced by high cholesterol diet and injection of vitamin D3. Clin Exp Pharmacol Physiol 33:305-309,2006.
23. Huot J, Houle F, Marceau F, and Landry J:Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80:383-392,1997.
24. Lee CK, Lee HM, Kim HJ, Park HJ, Won KJ, Roh HY, Choi WS, Jeon BH, Park TK, and Kim B:Syk contributes to PDGF-BB-mediated migration of rat aortic smooth muscle cells via MAPK pathways. Cardiovasc Res 74:159-168,2007.
25. Rohrer L, Hersberger M, and von Eckardstein A:High density lipoproteins in the intersection of diabetes mellitus, inflammation and cardiovascular disease. Curr Opin Lipidol 15:269-278,2004.
26. Han R, Lai R, Ding Q, Wang Z, Luo X, Zhang Y, Cui G, He J, Liu W, and Chen Y: Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism. Diabetologia.50:1960-1968,2007.
27. Mantena SK, and Katiyar SK:Grape seed proanthocyanidins inhibit UV-radiation induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes. Free Radic Biol Med 40:1603-1614,2006.
2. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract. 2010; 87(1):4-14.
3. Rathmann W, Giani G. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 2004; 27(10):2568-2569.
4. Nieuwdorp M, Holleman F, de Groot E, Vink H, Gort J, Kontush A, Chapman MJ, Hutten BA, Brouwer CB, Hoekstra JB, Kastelein JJ, Stroes ES. Perturbation of hyaluronan metabolism predisposes patients with type 1 diabetes mellitus to atherosclerosis. Diabetologia. 2007; 50(6):1288-1293.
5. Pambianco G, Costacou T, Ellis D, Becker DJ, Klein R, Orchard TJ. The 30-year natural history of type 1 diabetes complications: the Pittsburgh Epidemiology of Diabetes Complications Study experience. Diabetes. 2006; 55(5):1463-1469.
6. Mendez JD, Xie J, Aguilar-Hernandez M, Mendez-Valenzuela V. Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases. Mol Cell Biochem. 2010; 344(1-2): 185-193.
7. Bierhaus A, Nawroth PP. Multiple levels of regulation determine the role of the receptor for AGE (RAGE) as common soil in inflammation, immune responses and diabetes mellitus and its complications. Diabetologia. 2009; 52(11):2251-63.
8.屈琪,马静.晚期糖基化终末产物与动脉粥样硬化的研究进展.国外医学卫生学分册.2008:35(5):300-303.
9. Miyata T, Kurokawa K, Van Ypersele De Strihou C. Advanced glycation and lipoxidation end products: role of reactive carbonyl compounds generated during
10. Naka Y, Bucciarelli LG, Wendt T, Lee LK, Rong LL, Ramasamy R, Yan SF,Schmidt AM. RAGE axis: Animal models and novel insights into the vascular complications of diabetes. Arterioscler Thromb Vase Biol. 2004; 24(8): 1342-1349.
11. Arumugam T, Simeone DM, Schmidt AM, Logsdon CD. SI OOP stimulates cell proliferation and survival via receptor for activated glycation end products (RAGE). J Biol Chem. 2004; 279(7):5059-5065.
12. Sangle GV, Zhao R, Mizuno TM, Shen GX. Involvement of RAGE, NADPH oxidase, and Ras/Raf-1 pathway in glycated LDL-induced expression of heat shock factor-1 and plasminogen activator inhibitor-1 in vascular endothelial cells.Endocrinology. 2010; 151(9):4455-4466.
13. Wautier JL, Schmidt AM. Protein glycation: a firm link to endothelial cell dysfunction. Circ Res. 2004; 95(3):233-238.
14.于佩,于德民,齐建成,王静,张秋梅,张景云,汤云昭,邢秋玲,李明珍.高糖通过PI3K-Akt信号途径抑制内皮细胞迁移和增殖及血管发生改变.中华医学杂志.2006;86(48):3425-3430.
15. Kearney MT, Duncan ER, Kahn M, Wheatcroft SB. Insulin resistance and endothelial cell dysfunction: studies in mammalian models. Exp Physiol. 2008; 93(1): 158-163.
16. Yalcin E, Oruc E, Cavusoglu K, Yapar K. Protective role of grape seed extract against doxorubicin-induced cardiotoxicity and genotoxicity in albino mice. J Med Food. 2010; 13(4):917-925.
17. Houde V, Grenier D, and Chandad F. Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol. 2006; 77(8): 1371-1379.
18. Zi SX, Ma HJ, Li Y, Liu W, Yang QQ, Zhao G, and Lian S. Oligomeric proanthocyanidins from grape seeds effectively inhibit ultraviolet-induced melanogenesis of human melanocytes in vitro. Int J Mol Med. 2009; 23(2):197-204.
19. Yamakoshi J, Kataoka S, Koga T, and Ariga T. Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic arteriosclerosis in cholesterol-fed rabbits. Arteriosclerosis. 1999; 142(1):139-149.
20. Khanna S, Venojarvi M, Roy S, Sharma N, Trikha P, Bagchi D, Bagchi M, Sen CK. Dermal wound healing properties of redox-active grape seed proanthocyanidins. Free Radic Biol Med. 2002; 33(8): 1089-1096.
21. Mackenzie GG, Adamo AM, Decker NP, Oteiza PI. Dimeric procyanidin B2 inhibits constitutively active NF-kappaB in Hodgkin's lymphoma cells independently of the presence of IkappaB mutations. Biochem Pharmacol. 2008;75(7): 1461-1471.
22. Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, Xuan JH. Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol. 2006; 48(2): 47-53.
23. Sliman SM, Eubank TD, Kotha SR, Kuppusamy ML, Sherwani SI, Butler ES, Kuppusamy P, Roy S, Marsh CB, Stern DM, Parinandi NL. Hyperglycemic oxoaldehyde, glyoxal, causes barrier dysfunction, cytoskeletal alterations, and inhibition of angiogenesis in vascular endothelial cells: aminoguanidine protection.Mol Cell Biochem. 2010; 333(l-2):9-26.
24. van Dieren S, Beulens JW, van der Schouw YT, Grobbee DE, Neal B. The global burden of diabetes and its complications: an emerging pandemic. Eur J Cardiovasc Prev Rehabil. 2010; 17 Suppl 1 :S3-8.
25. Ning G, Hong J, Bi Y, Gu W, Zhang Y, Zhang Z, Huang Y, Wang W, Li X. Progress in diabetes research in China. J Diabetes. 2009; 1(3): 163-172.
26. Mendez JD, Xie J, Aguilar-Hernandez M, Mendez-Valenzuela V. Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases. Mol Cell Biochem. 2010; 344(1-2):185-193.
27. 刘萍,何兰杰.氧化应激与糖尿病.医学综述2.005;n(5):435-455.
28. Brownlee M. Biochemistry and molecular cell biology of diabetic complications.
29. Kim NH, Kim YS, Lee YM, Jang DS, Kim JS. Inhibition of aldose reductase and xylose-induced lens opacity iry'puerariafuran from the roots of Pueraria lobata. Biol Pharm Bull. 2010; 33(9): 1605-1609.
30. Das Evcimen N, King GL. The role of protein kinase C activation and the vascular complications of diabetes. Pharmacol Res. 2007; 55(6):498-510.
31. Chen J, Song M, Yu S, Gao P, Yu Y, Wang H, Huang L. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress. Mol Cell Biochem. 2010; 335(1-2):137-146.
32. Gharagozlian S, Henriksen T, Kolset SO. High glucose and Nepsilon-(carboxymethyl) lysine bovine serum albumin modulate release of matrix metalloproteinases in cultured human endothelial cells. Eur J Nutr. 2006; 45(5):283-290.
33. Chen SX, Song T, Zhou SH, Liu YH, Wu SJ, Liu LY. Protective effects of ACE inhibitors on vascular endothelial dysfunction induced by exogenous advanced oxidation protein products in rats. Eur J Pharmacol. 2008; 584(2-3):368-375.
34. Xu B, Chibber R, Ruggiero D, Kohner E, Ritter J, Ferro A. Impairment of vascular endothelial nitric oxide synthase activity by advanced glycation end products. FASEB J. 2003; 17(10): 1289-1291.
35. Potenza MA, Gagliardi S, Nacci C, Carratu' MR, Montagnani M. Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets. Curr Med Chem. 2009; 16(1):94-112.
36. Nagai R, Fujiwara Y, Mera K, Otagiri M. Investigation of pathways of advanced glycation end-products accumulation in macrophages. Mol Nutr Food Res. 2007;51(4):462-467.
37. Kearney MT, Duncan ER, Kahn M, Wheatcroft SB. Insulin resistance and endothelial cell dysfunction: studies in mammalian models. Exp Physiol, 2008; 93(1): 158-163.
38. Marchesi C, Ebrahimian T, Angulo O, Paradis P, Schiffrin EL. Endothelial nitric inflammation contribute to vascular dysfunction in a rodent model of metabolic syndrome. Hypertension, 2009; 54(6): 1384-1392.
39. Sakano K, Mizutani M, Murata M, Oikawa S, Hiraku Y, Kawanishi S. Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage. Free Radic Biol Med. 2005; 39(8):1041-1049.
40. de la Iglesia R, Milagro FI, Campion J, Boque N, Martinez JA. Healthy properties of proanthocyanidins. Biofactors. 2010; 36(3):159-168.
41. Bagchi D, Sen CK, Ray SD, Das DK, Bagchi M, Preuss HG, Vinson JA. Molecular mechanisms of cardioprotection by a novel grape seed proanthocyanidin extract. MutatRes. 2003;523-524:87-97.
43. Nassiri-Asl M, Hosseinzadeh H. Review of the pharmacological effects of Vitis vinifera (Grape) and its bioactive compounds. Phytother Res, 2009; 23(9):1197-1204.
44. Wang L, Zhu LH, Jiang H, Tang QZ, Yan L, Wang D, Liu C, Bian ZY, Li H. Grape seed proanthocyanidins attenuate vascular smooth muscle cell proliferation via blocking phosphatidylinositol 3-kinase-dependent signaling pathways. J Cell Physiol. 2010; 223(3):713-726.
45. Demirkaya E, Avci A, Kesik V, Karslioglu Y, Oztas E, Kismet E, Gokcay E, Durak I, Koseoglu V. Cardioprotective roles of aged garlic extract, grape seed proanthocyanidin, and hazelnut on doxorubicin-induced cardiotoxicity. Can J Physiol Pharmacol. 2009; 87(8):633-640.
46. Bagchi D, Garg A, Krohn RL, Bagchi M, Tran MX, Stohs SJ. Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro. Res Commun Mol Pathol Pharmacol. 1997; 95(2): 179-189.
47. Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, Joshi SS, Pruess HG. Free radicals and grape seed proanthocyanidin extract: importance in human
48. Farrar JL, Hartle DK, Hargrove JL, Greenspan P. Inhibition of protein glycation by skins and seeds of the muscadine grape. Biofactors. 2007; 30(3): 193-200.
49. Ma L, Gao HQ, Li BY, Ma YB, You BA, Zhang FL. Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma. J Cardiovasc Pharmacol. 2007; 49(5): 293-298.
50. Zhang FL, Gao HQ, Shen L. Inhibitory effect of GSPE on RAGE expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol. 2007;50(4):434-440.
51. 周雁,马亚兵,高海青,等.葡萄籽多酚抗糖尿病大鼠非酶糖基化实验研究.中华老年医学杂志.2005:24(1):49-52.
52. Li XL, Li BY, Gao HQ, Cheng M, Xu L, Li XH, Zhang WD, Hu JW. Proteomics approach to study the mechanism of action of grape seed proanthocyanidin extracts on arterial remodeling in diabetic rats. Int J Mol Med. 2010; 25(2):237-248.
53. Li XL, Li BY, Gao HQ, Cheng M, Xu L, Li XH, Ma YB. Effects of grape seed proanthocyanidins extracts on aortic pulse wave velocity in streptozocin induced diabetic rats. Biosci Biotechnol Biochem. 2009; 73(6): 1348-1354.
54. Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, You BA. Back- regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy. J Cell Biochem. 2008; 104(2):668-679.
55. Li M, Ma YB, Gao HQ, Li BY, Cheng M, Xu L, Li XL, Li XH. A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats.Chin Med J (Engl). 2008;121(24):2544-2552.
56. Xu L, Li B, Cheng M, Zhang W, Pan J, Zhang C, Gao H. Oral administration of grape seed proanthocyanidin extracts downregulate RAGE dependant nuclear factor- kappa BP65 expression in the hippocampus of streptozotocin induced
57. Cheng M, Gao HQ, Xu L, Li BY, Zhang H, Li XH. Cardioprotective effects of grape seed proanthocyanidins extracts in streptozocin induced diabetic rats. J Cardiovasc Pharmacol. 2007; 50(5):503-509.
58. Liang Y, Qiu J, Gao HQ, Li BY. Protective effect of grape seed proanthocyanidins extracts on reperfusion arrhythmia in rabbits. J Nutr Sci Vitaminol (Tokyo). 2009;55(3):223-230.
59. Cho ML, Heo YJ, Park MK, Oh HJ, Park JS, Woo YJ, Ju JH, Park SH, Kim HY, Min JK. Grape seed proanthocyanidin extract (GSPE) attenuates collagen-induced arthritis. Immunol Lett. 2009; 124(2): 102-110.
60. Shao ZH, Wojcik KR, Dossumbekova A, Hsu C, Mehendale SR, Li CQ, Qin Y, Sharp WW, Chang WT, Hamann KJ, Yuan CS, Hoek TL. Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt-NOS signaling. J Cell Biochem. 2009; 107(4):697-705.
61. Morin B, Narbonne JF, Ribera D, Badouard C, Ravanat JL. Effect of dietary fat-soluble vitamins A and E and proanthocyanidin-rich extract from grape seeds on oxidative DNA damage in rats. Food Chem Toxicol. 2008; 46(2): 787-796.
62. Bu HF, Zuo XL, Wang X, Ensslin MA, Koti V, Hsueh W, Raymond AS, Shur BD, Tan XD. Milk fat globule-EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium. J Clin hivest. 2007; 117(12):3673-3683.
63. Lin L, Huai Q, Huang M, Furie B, Furie BC. Crystal structure of the bovine lactadherin C2 domain, a membrane binding motif, shows similarity to the C2 domains of factor V and factor VDL J Mol Biol. 2007; 371(3):717-724.
64. Bojsen A, Buesa J, Montava R, Kvistgaard AS, Kongsbak MB, Petersen TE, Heegaard CW, Rasmussen JT. Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo. J Dairy Sci. 2007; 90(1):66-74.
65. Neutzner M, Lopez T, Feng X, Bergmann-Leitner ES, Leitner WW, Udey MC.MFG-E8/lactadherin promotes tumor growth in an angiogenesis-dependent transgenic mouse model of multistage carcinogenesis. Cancer Res. 2007; 67(14):6777-6785.
66. Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing forphospholipid-binding sites. Blood. 2003; 101(7):2628-2636.
67. Hanayama R, Tanaka M, Miwa K, Nagata S. Expression of developmental endothelial locus-1 in a subset of macrophages for engulfinent of apoptotic cells. J Immunol. 2004; 172(6):3876-3882.
68. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, Tamura N, Tamura T, Matsuda T. Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles. Endocrinology. 2007; 148(8):3850-3862.
69. Fu Z, Wang M, Gucek M, Zhang J, Wu J, Jiang L, Monticone RE, Khazan B, Telljohann R, Mattison J, Sheng S, Cole RN, Spinetti G, Pintus G, Liu L, Kolodgie FD, Virmani R, Spurgeon H, Ingram DK, Everett AD, Lakatta EG, Van Eyk JE. Milk fat globule protein epidermal growth factor-8: a pivotal relay element within the angiotensin II and monocyte chemoattractant protein-1 signaling cascade mediating vascular smooth muscle cells invasion. Circ Res. 2009;104(12):1337-1346.
70. Silvestre JS, Thery C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc-Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Levy B, Tedgui A, Amigorena S, Mallat Z. Lactadherin promotes VEGF-dependent neovascularization. Nat Med. 2005; 11(5):499-506.
71. Chen L, Zhang Y, Sun X, Li H, LeSage G, Javer A, Zhang X, Wei X, Jiang Y, Yin D. Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3beta pathway. Bioorg Med Chem. 2009; 17(13):4378-4382.
72. Linseman DA, Butts BD, Precht TA, Phelps RA, Le SS, Laessig TA, Bouchard RJ, Florez-McClure ML, Heidenreich KA. Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci. 2004; 24(44):9993-10002.
1. Lin EH, Rutter CM, Katon W, Heckbert SR, Ciechanowski P, Oliver MM, Ludman EJ, Young BA, Williams LH, McCulloch DK, Von Korff M. Depression and advanced complications of diabetes:a prospective cohort study. Diabetes Care. 2010;33(2):264-269.
2. Wong TY, Mwamburi M, Klein R, Larsen M, Flynn H, Hernandez-Medina M, Ranganathan G, Wirostko B, Pleil A, Mitchell P. Rates of progression in diabetic retinopathy during different time periods:a systematic review and meta-analysis. Diabetes Care.2009; 32(12):2307-2313.
3. Mendez JD, Xie J, Aguilar-Hernandez M, Mendez-Valenzuela V. Trends in advanced glycation end products research in diabetes mellitus and its complications. Mol Cell Biochem.2010; 341(1-2):33-41.
4. Lin EH, Rutter CM, Katon W, Heckbert SR, Ciechanowski P, Oliver MM, Ludman EJ, Young BA, Williams LH, McCulloch DK, Von Korff M. Depression and advanced complications of diabetes:a prospective cohort study. Diabetes Care. 2010; 33(2):264-269.
5. UK Prospective Diabetes Study (UKPDS) Group:Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS33). Lancet.1998; 352(9131):837-853.
6. Brownlee M. The pathobiology of diabetic complications:a unifying mechanism. Diabetes.2005; 54(6):1615-1625.
7. Ramasamy R, Goldberg IJ. Aldose reductase and cardiovascular diseases, creating human-like diabetic complications in an experimental model. Circ Res.2010; 106(9):1449-1458.
8. Ohmura C, Watada H, Azuma K, Shimizu T, Kanazawa A, Ikeda F, Yoshihara T, Fujitani Y, Hirose T, Tanaka Y, Kawamori R. Aldose reductase inhibitor, epalrestat, reduces lipid hydroperoxides in type 2 diabetes. Endocr J.2009; 56(1):149-156.
9. Geraldes P, Hiraoka-Yamamoto J, Matsumoto M, Clermont A, Leitges M, Marette A, Aiello LP, Kern TS, King GL. Activation of PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy. Nat Med. 2009; 15(11):1298-1306.
10.1Kanwar M, Kowluru RA. Role of glyceraldehyde 3-phosphate dehydrogenase in the development and progression of diabetic retinopathy. Diabetes.2009; 58(1):227-234.
11. Hodgkinson CP, Laxton RC, Patel K, Ye S.183.Advanced glycation end-product of low density lipoprotein activates the toll-like 4 receptor pathway implications for diabetic atherosclerosis. Arterioscler Thromb Vase Biol.2008; 28(12):2275-2281.
12. Nevado J, Peiro C, Vallejo S, El-Assar M, Lafuente N, Matesanz N, Azcutia V, Cercas E, Sanchez-Ferrer CF, Rodriguez-Manas L. Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells. Br J Pharmacol.2005; 146(2):268-279.
13. Nin JW, Jorsal A, Ferreira I, Schalkwijk CG, Prins MH, Parving HH, Tarnow L, Rossing P, Stehouwer CD. Higher plasma soluble Receptor for Advanced Glycation End Products (sRAGE) levels are associated with incident cardiovascular disease and all-cause mortality in type 1 diabetes:a 12-year follow-up study. Diabetes.2010; 59(8):2027-2032.
14. Nakamura K, Yamagishi S, Adachi H, Matsui T, Kurita-Nakamura Y, Takeuchi M, Inoue H, Imaizumi T. Circulating advanced glycation end products (AGEs) and soluble form of receptor for AGEs (sRAGE) are independent determinants of serum monocyte chemoattractant protein-1 (MCP-1) levels in patients with type 2 diabetes. Diabetes Metab Res Rev.2008; 24(2):109-114.
15. Mercer N, Ahmed H, Etcheverry SB, Vasta GR, Cortizo AM. Regulation of advanced glycation end product (AGE) receptors and apoptosis by AGEs in osteoblast-like cells. Mol Cell Biochem.2007; 306(1-2):87-94.
16. Cho SJ, Roman G, Yeboah F, Konishi Y. The road to advanced glycation end products:a mechanistic perspective. Curr Med Chem.2007; 14(15):1653-1671.
17. Mahajan N, Malik N, Bahl A, Dhawan V. Receptor for advanced glycation end products (RAGE) and its inflammatory ligand EN-RAGE in non-diabetic subjects with pre-mature coronary artery disease. Atherosclerosis.2009; 207(2):597-602.
18. Hanayama R, Tanaka M, Miwa K, Nagata S. Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells. J Immunol.2004; 172(6):3876-3882.
19. Ait-Oufella H, Kinugawa K, Zoll J, Simon T, Boddaert J, Heeneman S, Blanc-Brude O, Barateau V, Potteaux S, Merval R, Esposito B, Teissier E, Daemen MJ, Leseche G, Boulanger C, Tedgui A, Mallat Z. Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosis in mice. Circulation. 2007; 115(16):2168-2177.
20. Veron P, Segura E, Sugano G, Amigorena S, Thery C. Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells. Blood Cells Mol Dis.2005;35(2):81-88.
21. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, Tamura N, Tamura T, Matsuda T. Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes:redox-and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles. Endocrinology.2007; 148(8):3850-3862.
22.余祖华,丁轲,程相朝,张春杰,李银聚,吴庭才.外源基因转染真核细胞技术的研究进展.安徽农业科学.2008；36(21)：8954-8955.
23. Zhang H, Xie J, Xie Q, Wilson JM, Gao G. Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production. Hum Gene Ther.2009; 20(9):922-929.
24. Hart SL. Multifunctional nanocomplexes for gene transfer and gene therapy. Cell Biol Toxicol.2010; 26(1):69-81.
25. Agarwal C, Veluri R, Kaur M, Chou SC, Thompson JA, Agarwal R. Fractionation of high molecular weight tannins in grape seed extract and identification of procyanidin B2-3,3'-di-O-gallate as a major active constituent causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells. Carcinogenesis.2007; 28(7):1478-1484.
26. Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, Xuan JH. Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol.2006; 48(2):47-53.
27. Ma L, Gao HQ, Li BY, Ma YB, You BA, Zhang FL. Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma. J Cardiovasc Pharmacol.2007; 49(5):293-298.
28. Zhang FL, Gao HQ, Shen L. Inhibitory effect of GSPE on RAGE expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol.2007; 50(4):434-440.
29. McDonald DM, Coleman G, Bhatwadekar A, Gardiner TA, Stitt AW. Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal micro vascular endothelial cell dysfunction. Mol Vis.2009; 15:1509-1520.
30. Sawabe M. Vascular aging:from molecular mechanism to clinical significance. Geriatr Gerontol Int.2010; 10 Suppl 1:S213-220.
31. Xiang M, Yang M, Zhou C, Liu J, Li W, Qian Z. Crocetin prevents AGEs-induced vascular endothelial cell apoptosis. Pharmacol Res.2006; 54(4):268-274.
32.张瑾,李勇.高糖诱导血管内皮细胞凋亡及其调控机制研究进展.国际心血管病杂志.2008；35(6)：380-383.
33.任澎,马业新.动脉粥样硬化和再狭窄中细胞凋亡研究的近况.心血管康复医学杂志.2005；14(6)：597-600.
34.于珮,于德民,齐建成,王静,张秋梅,张景云,汤云昭,邢秋玲,李明珍.高糖通过P13K-Akt信号途径抑制内皮细胞迁移和增殖及血管发生改变.中华医学杂志.2006；86(48)：3425-3430.
35. Schmitt CA, Heiss EH, Dirsch VM. Effect of resveratrol on endothelial cell function:Molecular mechanisms. Biofactors.2010; 36(5):342-349.
36. Malfitano C, Alba Loureiro TC, Rodrigues B, Sirvente R, Salemi VM, Rabechi NB, Lacchini S, Curi R, Irigoyen MC. Hyperglycaemia protects the heart after myocardial infarction:aspects of programmed cell survival and cell death. Eur J Heart Fail.2010; 12(7):659-967.
37.张风雷,高海青,沈琳,吴建敏,由倍安,马亚兵.葡萄籽多酚对糖基化终产物诱导的内皮细胞组织因子表达的影响.中华老年医学杂志,2007；26(1)：29-33.
38. Nakagami H, Kaneda Y, Ogihara T, Morishita R. Endothelial dysfunction in hyperglycemia as a trigger of atherosclerosis. Curr Diabetes Rev.2005; 1(1):59-63.
39. Neutzner M, Lopez T, Feng X, Bergmann-Leitner ES, Leitner WW, Udey MC. MFG-E8/lactadherin promotes tumor growth in an angiogenesis-dependent transgenic mouse model of multistage carcinogenesis. Cancer Res.2007; 67(14):6777-6785.
40. Silvestre JS, Thery C, Hamard G, Boddaert J, Aguilar B, Delcayre A, Houbron C, Tamarat R, Blanc-Brude O, Heeneman S, Clergue M, Duriez M, Merval R, Levy B, Tedgui A, Amigorena S, Mallat Z. Lactadherin promotes VEGF-dependent neovascularization. Nat Med.2005; 11(5):499-506.
41. Larsson A, Peng S, Persson H, Rosenbloom J, Abrams WR, Wassberg E, Thelin S, Sletten K, Gerwins P, Westermark P. Lactadherin binds to elastin--a starting point for medin amyloid formation? Amyloid.2006; 13(2):78-85.
42. Morelli AE, Larregina AT, Shufesky WJ, Sullivan ML, Stolz DB, Papworth GD, Zahorchak AF, Logar AJ, Wang Z, Watkins SC, Falo LD Jr, Thomson AW. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood.2004; 104(10):3257-3266.
43. Fu Z, Wang M, Gucek M, Zhang J, Wu J, Jiang L, Monticone RE, Khazan B, Telljohann R, Mattison J, Sheng S, Cole RN, Spinetti G, Pintus G, Liu L, Kolodgie FD, Virmani R, Spurgeon H, Ingram DK, Everett AD, Lakatta EG, Van Eyk JE. Milk fat globule protein epidermal growth factor-8:a pivotal relay element within the angiotensin Ⅱ and monocyte chemoattractant protein-1 signaling cascade mediating vascular smooth muscle cells invasion. Circ Res.2009; 104(12):1337-1346.
44.郭剑英,李英,潘家强,唐兆新.线粒体通透性转换孔及其与细胞凋亡的关系.动物医学进展.2009；30(8)：101-105.
45. Javadov S, Karmazyn M. Mitochondrial permeability transition pore opening as an endpoint to initiate cell death and as a putative target for cardioprotection. Cell Physiol Biochem.2007; 20(1-4):1-22.
46. Xie M, Yang S, Win HL, Xiong L, Huang J, Zhou J. Rabbit annulus fibrosus cell apoptosis induced by mechanical overload via a mitochondrial apoptotic pathway. J Huazhong Univ Sci Technolog Med Sci.2010; 30(3):379-384.
47. Zhao NQ, Yu YR, Tan HW, Deng G, Zhang XX. Role of apoptosis and mitochondrial apoptotic pathway in glucolipotoxicity-induced islet beta-cell dysfunction. Nan Fang Yi Ke Da Xue Xue Bao.2008; 28(11):2009-2013.
48. Jourdain A, Martinou JC. Mitochondrial outer-membrane permeabilization and remodelling in apoptosis. Int J Biochem Cell Biol.2009; 41(10):1884-1889.
49. Gupta S, Kass GE, Szegezdi E, Joseph B. The mitochondrial death pathway:a promising therapeutic target in diseases. J Cell Mol Med.2009; 13(6):1004-1033.
50.王文文,邓毛程.Bcl-2、Bax及Bid与细胞周期阻滞及凋亡调控的关系.广东轻工职业技术学院学报.2009；8(3)：21-24.
51. Janumyan YM, Sansam CG, Chattopadhyay A, Cheng N, Soucie EL, Penn LZ, Andrews D, Knudson CM, Yang E. Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry. EMBO J.2003; 22(20):5459-5470.
52. Cheng N, Janumyan YM, Didion L, Van Hofwegen C, Yang E, Knudson CM. Bcl-2 inhibition of T-cell proliferation is related to prolonged T-cell survival. Oncogene. 2004; 23(21):3770-3780.
53. Zhang Z, Lapolla SM, Annis MG, Truscott M, Roberts GJ, Miao Y, Shao Y, Tan C, Peng J, Johnson AE, Zhang XC, Andrews DW, Lin J. Bcl-2 homodimerization involves two distinct binding surfaces, a topographic arrangement that provides an effective mechanism for Bcl-2 to capture activated Bax. J Biol Chem.2004; 279(42):43920-43928.
54. Tan C, Dlugosz PJ, Peng J, Zhang Z, Lapolla SM, Plafker SM, Andrews DW, Lin J. Auto-activation of the apoptosis protein Bax increases mitochondrial membrane permeability and is inhibited by Bcl-2. J Biol Chem.2006; 281(21):14764-14775.
55. Wang CY, Lin YS, Su WC, Chen CL, Lin CF. Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis. Mol Biol Cell.2009; 20(19):4153-4161.
56. Wang Y, Huang WC, Wang CY, Tsai CC, Chen CL, Chang YT, Kai JI, Lin CF. Inhibiting glycogen synthase kinase-3 reduces endotoxaemic acute renal failure by down-regulating inflammation and renal cell apoptosis. Br J Pharmacol.2009; 157(6):1004-1013.
57. Chen L, Zhang Y, Sun X, Li H, LeSage G, Javer A, Zhang X, Wei X, Jiang Y, Yin D. Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3beta pathway. Bioorg Med Chem.2009; 17(13): 4378-4382.
58. Linseman DA, Butts BD, Precht TA, Phelps RA, Le SS, Laessig TA, Bouchard RJ, Florez-McClure ML, Heidenreich KA. Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci.2004; 24(44):9993-10002.
59. King TD, Bijur GN, Jope RS. Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium. Brain Res.2001; 919(1):106-114.
60. King TD, Jope RS. Inhibition of glycogen synthase kinase-3 protects cells from intrinsic but not extrinsic oxidative stress. Neuroreport.2005; 16(6):597-601.
61. Miura T, Miki T. GSK-3beta, a therapeutic target for cardiomyocyte protection. Circ J.2009; 73(7):1184-1192.
62. Ougolkov AV, Billadeau DD. Inhibition of glycogen synthase kinase-3. Methods Mol Biol.2008; 468:67-75.
63. Gao HK, Yin Z, Zhou N, Feng XY, Gao F, Wang HC. Glycogen synthase kinase 3 inhibition protects the heart from acute ischemia-reperfusion injury via inhibition of inflammation and apoptosis. J Cardiovasc Pharmacol.2008; 52(3):286-292.
64. Ma H, Li SY, Xu P, Babcock SA, Dolence EK, Brownlee M, Li J, Ren J. Advanced glycation endproduct (AGE) accumulation and AGE receptor (RAGE) up-regulation contribute to the onset of diabetic cardiomyopathy. J Cell Mol Med. 2009; 13(8B):1751-1764.
65. Wang Y, Feng W, Xue W, Tan Y, Hein DW, Li XK, Cai L. Inactivation of GSK-3beta by metallothionein prevents diabetes-related changes in cardiac energy metabolism, inflammation, nitrosative damage, and remodeling. Diabetes.2009; 58(6):1391-1402.
66. Zunino S. Type 2 diabetes and glycemic response to grapes or grape products. J Nutr.2009; 139(9):1794S-800S.
67. Houde V, Grenier D, Chandad F. Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol.2006; 77(8):1371-1379.
68. Khanna S, Venojarvi M, Roy S, Sharma N, Trikha P, Bagchi D, Bagchi M, Sen CK. Dermal wound healing properties of redox-active grape seed proanthocyanidins. Free Radic Biol Med.2002; 33(8):1089-1096.
69. Vayalil PK, Mittal A, Katiyar SK. Proanthocyanidins from grape seeds inhibit expression of matrix metalloproteinases in human prostate carcinoma cells, which is associated with the inhibition of activation of MAPK and NF kappa B. Carcinogenesis.2004; 25(6):987-995.
70. Zhang WY, Liu HQ, Xie KQ, Yin LL, Li Y, Kwik-Uribe CL, Zhu XZ. Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells. Biochem Biophys Res Commun.2006; 345(1):508-515.
71. Chen DM, Cai X, Kwik-Uribe CL, Zeng R, Zhu XZ. Inhibitory effects of procyanidin B(2) dimer on lipid-laden macrophage formation. J Cardiovasc Pharmacol.2006; 48(2):54-70.
72. Mackenzie GG, Adamo AM, Decker NP, Oteiza PI. Dimeric procyanidin B2 inhibits constitutively active NF-kappaB in Hodgkin's lymphoma cells independently of the presence of IkappaB mutations. Biochem Pharmacol.2008; 75(7):1461-1471.
73. Li XL, Li BY, Gao HQ, Cheng M, Xu L, Li XH, Ma YB. Effects of grape seed proanthocyanidins extracts on aortic pulse wave velocity in streptozocin induced diabetic rats. Biosci Biotechnol Biochem.2009; 73(6):1348-1354.
74. Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, You BA. Back-regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy. J Cell Biochem.2008; 104(2):668-679.
75. Li M, Ma YB, Gao HQ, Li BY, Cheng M, Xu L, Li XL, Li XH. A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats.Chin Med J (Engl).2008; 121(24):2544-2552.
1) De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, and Vanhoutte PM, Br. J. Pharmacol,130,963-974(2000).
2) Al-Maskari F, El-Sadig M, and Norman JN, Cardiovasc. Diabetol.,6,24(2007).
3) Nieuwdorp M, Holleman F, de Groot E, Vink H, Gort J, Kontush A, Chapman MJ, Hutten BA, Brouwer CB, Hoekstra JB, Kastelein JJ, and Stroes ES, Diabetologia, 50,1288-1293(2007).
4) Rahman S, Rahman T, Ismail AA, and Rashid AR, Diabetes. Obes. Metab.,9,767-780(2007).
5) Barinas-Mitchell E, Kuller LH, Sutton-Tyrrell K, Hegazi R, Harper P, Mancino J, and Kelley DE, Diabetes Care,29,2218-2222(2006).
6) Cameron JD, and Cruickshank JK, Clin. Exp. Pharmacol. Physio.,34,677-682 (2007).
7) Smith A, Karalliedde J, De Angelis L, Goldsmith D, and Viberti G, J. Am. Soc. Nephrol,16,1069-1075(2005).
8) Cruickshank K, Riste L, Anderson SG, Wright JS, Dunn G, and Gosling RG, Circulation,106,2085-2090(2002).
9) Liu XN, Gao HQ, Li BY, Cheng M, Ma YB, Zhang ZM, Gao XM, Liu YP, and Wang M, Hypertens. Res.,30,237-242(2007).
10) Rahman S, Ismail AA, Ismail SB, Naing NN, and Rahman AR, Diabetes. Res. Clin. Pract.,80,253-258(2008).
11) Mulvany MJ, Hypertension,1,7-9(1992).
12) Houde V, Grenier D, and Chandad F, J. Periodontol.,77,1371-1379(2006).
13) Khanna S, Venojarvi M, Roy S, Sharma N, Trikha P, Bagchi D, Bagchi M, and Sen CK, Free. Radic. Biol. Med.,33,1089-1096(2002).
14) Vayalil PK, Mittal A, and Katiyar SK, Carcinogenesis,25,987-995(2004).
15) Cheng M, Gao HQ, Xu L, Li BY, Zhang H, Li XH, J. Cardiovasc. Pharmacol,50, 503-509(2007).
16) Xu L, Li B, Cheng M, Zhang W, Pan J, Zhang C, and Gao H, Exp. Clin. Endocrinol. Diabetes.,116,215-224(2008).
17) Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, and You BA, J. Cell. Biochem.,104,668-679(2008).
18) Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, and Xuan JH, J. Cardiovasc. Pharmacol.,48,47-53(2006).
19) Ma L, Gao HQ, Li BY, Ma YB, You BA, Zhang FL, J. Cardiovasc. Pharmacol.,49, 293-298(2007).
20) Pinent M, Blay M, Blade MC, Salvado MJ, Arola L, and Ardevol A, Endocrinology, 145,4985-4990(2004).
21) Nemoto O, Kawaguchi M, Yaoita H, Miyake K, Maehara K, and Maruyama Y, Circ. J.,70,327-334(2006).
22) Kamata K, Hosokawa M, Matsumoto T, and Kobayashi T, J. Smooth. Muscle. Res., 42,171-187(2006).
23) Gaillard V, Casellas D, Seguin-Devaux C, Schohn H, Dau9a M, Atkinson J, and Lartaud I, Hypertension,46,372-379(2005).
24) Mitchell GF, Pfeffer MA, Finn PV, and Pfeffer JM, J. Appl. Physiol.,82,203-210 (1997).
25) Niederhoffer N, Kieffer P, Desplanches D, Lartaud-Idjouadiene Ⅰ, Somay MH, and Atkinson J, Hypertension,35,919-924(2000).
26) Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, and Jerums G, Diabetes,40, 1328-1334(1991).
27) Candido R, Forbes JM, Thomas MC, Thallas V, Dean RQ Burns WC, Tikellis C, Ritchie RH, Twigg SM, Cooper ME, and Burrell LM, Circ. Res.,92,785-792 (2003).
28) Valitutto M, Osteopath. Med. Prim. Care.,2,4(2008).
29) Brownlee M, Nature,414,813-820(2001).
30) Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, Kislinger T, Stern DM, Schmidt AM, and De Caterina R, Circulation,105,816-822(2002).
31) Chetyrkin SV, Mathis ME, Ham AJ, Hachey DL, Hudson BG, and Voziyan PA, Free. Radic. Biol. Med.,44,1276-1285(2008).
32) San Martin A, Foncea R, Laurindo FR, Ebensperger R, Griendling KK, and Leighton F, Free. Radic. Biol. Med.,42,1671-1679(2007).
33) Hatsuda S, Shoji T, Shinohara K, Kimoto E, Mori K, Fukumoto S, Koyama H, Emoto M, and Nishizaw Y, J.Atheroscler. Thromb.,13,114-121(2006).
34) Vaitkevicius PV, Lane M, Spurgeon H, Ingram DK, Roth GS, Egan JJ, Vasan S, Wagle DR, Ulrich P, Brines M, Wuerth JP, Cerami A, Lakatta EG, Proc. Natl. Acad. Sci.U S A,98,1171-1175(2001).
35) Shiina K, Tomiyama H, Takata Y, Usui Y, Asano K, Hirayama Y, Nakamura T, and Yamashina A, Hypertens. Res.,29,433-441(2006).
36) Naka Y, Bucciarelli LG, Wendt T, Lee LK, Rong LL, Ramasamy R, Yan SF, and Schmidt AM, Arterioscler. Thromb. Vasc. Biol.,24,1342-1349(2004).
37) El-Alfy AT, Ahmed AA, and Fatani AJ, Pharmacol. Res.,52,264-270(2005).
38) Goldin A, Beckman JA, Schmidt AM, and Creager MA, Circulation,114,597-605 (2006).
39) Peppa M, and Vlassara H, Hormones (Athens),4,28-37(2005).
40) Zhang FL, Gao HQ, and Shen L, J. Cardiovasc. Pharmacol.,50,434-440(2007).
41) Ramasamy R, Vannucci SJ, Yan SS, Herold K, Yan SF, and Schmidt AM, Glycobiology,15,16R-28R(2005).
42) Ramasamy R, Yan SF, and Schmidt AM, Trends. Cardiovasc. Med.,15,237-243 (2005).
43) Haslbeck KM, Schleicher E, Bierhaus A, Nawroth P, Haslbeck M, Neundorfer B, and Heuss D, Exp. Clin. Endocrinol. Diabetes.,113,288-291(2005).
44) Cipollone F, Iezzi A, Fazia M, Zucchelli M, Pini B, Cuccurullo C, De Cesare D, De Blasis G, Muraro R, Bei R, Chiarelli F, Schmidt AM, Cuccurullo F, and Mezzetti A, Circulation,108,1070-1077(2003).
45) Fitzpatrick DF, Bing B, Maggi DA, Fleming RC, and O'Malley RM, Ann. N Y Acad. Sci.,957,78-89(2002).
1. Henry RM, Kostense PJ, Spijkerman AM, Dekker JM, Nijpels G, Heine RJ, Kamp O, Westerhof N, Bouter LM, and Stehouwer CD:Arterial stiffness increases with deteriorating glucose tolerance status:the Hoorn Study. Circulation 107: 2089-2095,2003.
2. Terry JG, Tang R, Espeland MA, Davis DH, Vieira JL, Mercuri MF, and Crouse JR: Carotid structure in patients with documented coronary artery disease and disease-free control subjects. Circulation 107:1146-1151,2003.
3. Houde V, Grenier D, and Chandad F:Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol 77:1371-1379,2006.
4. Zi SX, Ma HJ, Li Y, Liu W, Yang QQ, Zhao Q and Lian S:Oligomeric proanthocyanidins from grape seeds effectively inhibit ultraviolet-induced melanogenesis of human melanocytes in vitro. Int J Mol Med 23:197-204,2009.
5. Yamakoshi J, Kataoka S, Koga T, and Ariga T:Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic arteriosclerosis in cholesterol-fed rabbits. Arteriosclerosis 142:139-149,1999.
6. Zhang FL, Gao HQ, Wu JM, Ma YB, You BA, Li BY, and Xuan JH:Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol 48:47-53,2006.
7. Ma L, Gao HQ, Li BY, Ma YB, You BA, and Zhang FL:Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma. J Cardiovasc Pharmacol 49:293-298, 2007.
8. Pinent M, Blay M, Blade MC, Salvado MJ, Arola L, and Ardevol A:Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines. Endocrinology 145:4985-4990,2004.
9. Holm AM, Andersen CB, Haunsφ S, and Hansen PR:ACE-inhibition promotes apoptosis after balloon injury of rat carotid arteries. Cardiovasc Res 45:777-782, 2000.
10. Buus CL, Pourageaud F, Fazzi GE, Janssen G, Mulvany MJ, and De Mey JG: Smooth muscle cell changes during flow-related remodeling of rat mesenteric resistance arteries. Circ Res 89:180-186,2001.
11. Li BY, Cheng M, Gao HQ, Ma YB, Xu L, Li XH, Li XL, and You BA: Back-regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy. J Cell Biochem 104:668-679,2008.
12. Ji H, Moritz RL, Kim YS, Zhu HJ, and Simpson RJ:Analysis of Ras-induced oncogenic transformation of NIH-3T3 cells using differential-display 2-DE proteomics. Electrophoresis 28:1997-2008,2007.
13. Cimmino F, Spano D, Capasso M, Zambrano N, Russo R, Zollo M, and Iolascon A: Comparative proteomic expression profile in all-trans retinoic acid differentiated neuroblastoma cell line. J Proteome Res 6:2550-2564,2007.
14. Ritorto MS, and Borlak J:A simple and reliable protocol for mouse serum proteome profiling studies by use of two-dimensional electrophoresis and MALDI TOF/TOF mass spectrometry. Proteome Sci 6:25,2008.
15. D'Hertog W, Overbergh L, Lage K, Ferreira GB, Maris M, Gysemans C, Flamez D, Cardozo AK, Van den Bergh G, Schoofs L, Arckens L, Moreau Y, Hansen DA, Eizirik DL, Waelkens E, and Mathieu C:Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-IE cells:new insights into the pathways involved. Mol Cell Proteomics 6:2180-2199,2007.
16. Stenina OI, Krukovets I, Wang K, Zhou Z, Forudi F, Penn MS, Topol EJ, and Plow EF:Increased expression of thrombospondin-1 in vessel wall of diabetic Zucker rat. Circulation 107:3209-3215,2003.
17. Brownlee M:Biochemistry and molecular cell biology of diabetic complications. Nature 414:813-820,2001.
18. Prabakaran S, Wengenroth M, Lockstone HE, Lilley K, Leweke FM, and Bahn S: 2-D DIGE analysis of liver and red blood cells provides further evidence for oxidative stress in schizophrenia. J Proteome Res 6:141-149,2007.
19. Shiota M, Kusakabe H, Hikita Y, Nakao T, Izumi Y, and Iwao H: Pharmacogenomics of cardiovascular pharmacology:molecular network analysis in pleiotropic effects of statin-an experimental elucidation of the pharmacologic action from protein-protein interaction analysis. J Pharmacol Sci 107:15-19,2008.
20. El-Alfy AT, Ahmed AA, and Fatani AJ:Protective effect of red grape seeds proanthocyanidins against induction of diabetes by alloxan in rats. Pharmacol Res 52:264-270,2005.
21. Fitzpatrick DF, Bing B, Maggi DA, Fleming RC, and O'Malley RM:Vasodilating procyanidins derived from grape seeds. Ann N Y Acad Sci 957:78-89,2002.
22. Almofti MR, Huang Z, Yang P, Rui Y, and Yang P:Proteomic analysis of rat aorta during arteriosclerosis induced by high cholesterol diet and injection of vitamin D3. Clin Exp Pharmacol Physiol 33:305-309,2006.
23. Huot J, Houle F, Marceau F, and Landry J:Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80:383-392,1997.
24. Lee CK, Lee HM, Kim HJ, Park HJ, Won KJ, Roh HY, Choi WS, Jeon BH, Park TK, and Kim B:Syk contributes to PDGF-BB-mediated migration of rat aortic smooth muscle cells via MAPK pathways. Cardiovasc Res 74:159-168,2007.
25. Rohrer L, Hersberger M, and von Eckardstein A:High density lipoproteins in the intersection of diabetes mellitus, inflammation and cardiovascular disease. Curr Opin Lipidol 15:269-278,2004.
26. Han R, Lai R, Ding Q, Wang Z, Luo X, Zhang Y, Cui G, He J, Liu W, and Chen Y: Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism. Diabetologia.50:1960-1968,2007.
27. Mantena SK, and Katiyar SK:Grape seed proanthocyanidins inhibit UV-radiation induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes. Free Radic Biol Med 40:1603-1614,2006.