糖尿病大鼠缺血/再灌注后心肌微血管的损伤作用及机制研究
摘要
研究背景:
据国际糖尿病联合会提供的数据,目前全球糖尿病患者超过2.5亿,预计到2025年这个数字将变成3.8亿。由于人口基数大,中国将成为仅次于印度的糖尿病第二大国。因其起病隐匿且造成人体各个器官的血管损害,糖尿病绝对称得上是人体的定时炸弹。目前糖尿病患者死亡的第一位原因就是冠心病。糖尿病合并冠心病患者多为多支血管病变且病变弥漫,PCI术后的无复流发生率和严重程度均明显升高。糖尿病合并冠心病患者无复流现象的预防和治疗已经成为心血管界介入治疗和药物治疗面临的新挑战。
心脏血运重建的过程中出现无复流现象,无复流现象的可能机制为:
①微血管舒张功能障碍及中性粒细胞栓塞;②血小板及中心粒细胞形成微血栓堵塞微血管;③细胞肿胀挤压微血管;④血液粘滞性变化等。可见无复流现象与心肌微血管内皮密切相关,但其发生的具体机制目前尚不清楚。糖尿病以引起多器官的微血管、小血管的病变而著称,糖尿病患者无复流现象的发生很可能与心肌微血管的损伤密切相关。
多项研究证实,糖尿病患者胰岛素受体后信号转导通路受损,引起eNOS表达减少,内皮细胞分泌NO的量及活性降低,可能与内皮依赖性舒张功能障碍、血小板聚集等导致无复流发生的因素有关。而胰岛素参与调节另一途径即分裂原活化蛋白激酶(MAPK)途径则保持完好,甚至加强,产生促进内皮细胞的凋亡效应,这就是近年来有些学者提出的“选择性”胰岛素抵抗。I/R也是引起MAPK信号通路激活的常见刺激因素。在I/R的刺激下,糖尿病患者的心肌微血管内皮细胞会发生怎样的变化,以及这一变化产生的可能机制目前尚不清楚。
因此,本研究的实验目的:
1.明确糖尿病大鼠缺血/再灌注后心肌微血管的损害是否比正常大鼠严重。
2.如果是,糖尿病大鼠心肌缺血/再灌注后PI3K信号转导通路及MAPK通路的影响。这两条信号通路之间是否存在相互关系。
3.如果两条信号通路间的相互关系存在,此种关系在糖尿病引起的心肌微血管损害中起到什么样的作用。
4.阻断两通路间的相互关系是否可以改善糖尿病引起的心肌微血管损害。
实验方法
1.糖尿病大鼠模型的制备:
高脂高糖饮食饲养雄性成年SD大鼠(体重200 g左右)3月后,给予低剂量STZ腹腔注射(50 mg/kg),检测造模前后血糖值,发现造模后大鼠血糖显著增高,达糖尿病诊断标准。大鼠血糖升高1周后用于实验。
2.缺血/再灌注模型的制备:
成年SD雄性大鼠,腹腔注射戊巴比妥麻醉,呼吸机辅助呼吸,于3～4肋间开胸1.5 cm,迅速暴露心脏,冠状动脉左室支下穿线活扣结扎,以标Ⅱ导联心电图改变为阻断血流指标,连续缺血30 min,松线再灌注开始,关胸,制备大鼠心肌I/R模型。I/R术中通过八道生理记录仪持续记录大鼠心电图、动脉压及左室内压等血流动力学指标。再灌注3 h后采用伊文蓝-硫磺素S双染法测定心肌微血管损伤范围,伊文蓝-三苯四唑氯盐(TTC)双染法确定心肌梗死范围。CD31免疫荧光染色确定各组心肌微血管内皮细胞的数量。再灌注后24 h,心脏B超测量各组大鼠心功能。
3.建立心肌微血管内皮细胞(CMECs)H/R模型:
无菌分离成年SD大鼠左心室组织,去除心内外膜及冠脉组织,取心尖部心肌组织剪为1 mm3组织块,经胰蛋白酶、胶原酶消化,条件培养及细胞纯化后获得CMECs。分离培养得到的CMECs置于缺氧细胞培养箱(95% N2 / 5% CO2 , 37℃)1 h后重新置于正常细胞培养箱3 h,硝酸还原法测定各组细胞NO的分泌量,流式细胞仪检测细胞凋亡,western blot方法检测Caspase-3、P-JNK,JNK,PI3K、MEKK1、IRS-1等蛋白的表达及磷酸化水平。
实验结果1.糖尿病(DM)大鼠缺血/再灌注后微血管损伤范围显著大于N + I/R大鼠(1.08±0.07 % vs 3.03±0.11%,P < 0.01)梗死范围显著大于非糖尿病大鼠(57.49±1.25% vs 68.39±2.31%, P < 0.05)。DM大鼠缺血/再灌注后-dp/dtmax低于非糖尿病大鼠(-3613±88.67 vs. -4065.09±99.31,P < 0.05)。DM大鼠缺血/再灌注后左室射血分数较N + I/R组显著降低(32.5±10.0% vs 51.6±9.3%,P < 0.05),也显著低于D组(32.5±10.0% vs 57.3±8.5%,P < 0.05)DM大鼠缺血/再灌注后3 h心肌CD31阳性的细胞密度较N+I/R组显著降低(1600±82/mm vs 2653±41/mm,P < 0.05),较D组也显著降低(1600±82/mm vs 2590±52/mm,P < 0.05)。提示糖尿病大鼠缺血/再灌注后心肌微血管损伤及心功能损伤较非糖尿病大鼠加重。
2.缺氧/复氧(H/R)损伤显著增加糖尿病及正常大鼠CMEC的凋亡率(2.23±0.07% vs. 31±2.32%, P < 0.01和2.33±0.16% vs. 38.17±1.97%, P < 0.01)。Caspase-3的表达也呈现出相同的趋势,另外,H/R处理后糖尿病大鼠CMECs表达Caspase-3的量显著高于非糖尿病大鼠(P < 0.05)。H/R处理后糖尿病及非糖尿病大鼠CMECs分泌NO的能力均显著下降(86.67±2.18% vs. 56.67±1.28%, P < 0.01 and 100% vs. 74.67±0.96%, P < 0.01),H/R后糖尿病大鼠CMECs分泌NO的量显著低于非糖尿病大鼠(74.67±0.96% vs. 56.67±1.28%, P < 0.01)。这些结果提示,H/R对糖尿病大鼠CMECs的分泌功能及凋亡影响更大。
3.DM大鼠CMECs IRS-1的磷酸化水平显著降低(105.33±3.50% vs.56.27±4.60%, P < 0.05)。PI3K的表达在糖尿病大鼠CMECs中的表达也有明显下降(110.00±3.31% vs. 82.22±3.31%, P < 0.01),在糖尿病(82.22±3.31% vs. 35.67±1.78%, P < 0.01)和非糖尿病(110.00±3.31% vs. 89.67±2.91%,P < 0.05)大鼠中均可见到H/R引起的PI3K的表达下降。H/R的刺激正常大鼠的CMECs,MEKK1表达量(101.33±5.18% vs. 299±7.36%, P < 0.05 )JNK的磷酸化水平(97.36±0.76% vs. 162.33±4.49%,P < 0.05)均显著升高,H/R的刺激糖尿病大鼠的CMECs时,JNK的磷酸化水平增加更加显著(148.47±12.27% vs. 399.67±7.77%,P < 0.01)。因此PI3K和JNK很可能是联系糖尿病和缺血/复氧两个病理过程的重要中间分子。
4.H/R处理前1 h给于PI3K的抑制剂LY294002(10μM)可进一步增加H/R导致的DM大鼠CMECs的凋亡(39.07±5.48% vs. 47.55±3.46%, P < 0.01)。LY294002也进一步增加H/R引起的DM大鼠CMECs Caspase-3蛋白的表达(338.00±29.53% vs. 450.00±15.51%, P < 0.05),减少H/R导致的大鼠CMECs分泌NO的量的减少(56.67±3.34% vs. 41.00±5.72%, P < 0.05)。LY294002进一步上调了H/R引起的DM大鼠CMECs JNK磷酸化水平的增加(260.75±8.76% vs. 373.5±10.74%,P < 0.01)。此部分结果说明,PI3K的抑制剂可通过激活JNK进一步恶化H/R导致的DM大鼠CMECs细胞凋亡和分泌功能下降。
5.H/R使DM大鼠CMECs分泌NO的量显著减低(86.67±5.67% vs. 56.67±3.34%,P < 0.01),H/R处理前给于JNK的抑制剂SP600125(10μM)可部分改善DM大鼠CMECs NO分泌的减少(56.67±3.34% vs. 68.75±8.58%,P < 0.05)。SP600125降低H/R引起的糖尿病大鼠CMECs Caspase-3蛋白的表达增加(338.00±29.53% vs. 260.67±26.91%, P < 0.05)。H/R刺激下调DM大鼠PI3K的表达(82.00±9.93% vs. 35.00±5.35%,P < 0.01),JNK的抑制剂可部分上调因H/R导致的PI3K的表达下降(35.00±5.35% vs. 62.00±4.54%,P < 0.05)。此部分结果说明,JNK的抑制剂通过激活PI3K改善H/R导致的DM大鼠CMECs的分泌功能,减少凋亡。
结论
1.糖尿病大鼠缺血/再灌注后较非糖尿病大鼠心肌微血管损伤范围、梗死范围增加,心肌收缩、舒张能力均显著下降,心肌微血管内皮细胞数量减少。
2.DM大鼠CMECs缺氧/复氧处理后凋亡增加,NO分泌量下降,同时伴有JNK磷酸化水平增加及PI3K的表达减少。
3. H/R刺激通过下调PI3K的表达减少DM大鼠CMECs分泌NO的量,而JNK的抑制剂可通过上调PI3K的表达,改善DM大鼠CMECs分泌NO的能力。
4.H/R也激活JNK,引起DM大鼠CMECs的凋亡,PI3K的抑制剂促使JNK进一步活化,增加H/R引起的DM大鼠CMECs的凋亡。
上述结果提示MAPK信号通路与PI3K信号通路的相互关系参与缺血/再灌注引起的糖尿病大鼠心肌微血管的损伤。以JNK和PI3K为连接点的两条信号通路的相互关系的存在为I/R对糖尿病患者造成的心肌微血管损害提供可能的分子机制和治疗靶点。
Background
According to the data provided by International Diabetes Faderation (IDF), the number of the diabetic patients is over 250 million, and the number will become 380 million by 2025. China will be the second biggest diabetic country in the world. The first cause of death in diabetic patients is cardiovascular disease. Multivessel coronary artery disease is always seen in diabetic coronary artery disease patients. The incidence of no-reflow after PCI treatment is much higher in diabetic patients compared with those patients without diabetes. The therapy and prevention of no-reflow penomenon have become a new challenge in the field of cardiovascular disease.
The possible mechanism of no-reflow penomenon in PCI treatment are the following points:①Microvessel vasodialation dysfunction and neutrophilic granulocyte blocking in microvessles;②Thrombocyte occludes in microvessles;③Endothelial cells swelling;④Change of the stagnation of the blood viscosity. It seems that no-reflow phenomenon is associated with microvascular injury, but the accurate mechanism is not clear. Diabetes is famous of causing mulp-organ microvessle injury. The no-reflow phenomenon seen in diabetic patients may be associated with cardia microvessle injury.
Many data reported that the insulin receptor pathway was hurt and the expression of eNOS and secretion of NO were decreased in diabetic patients. These changes were associated with endothelium-dependent vasodialation dysfuncrion and thrombocyte aggragation which were causes of no-reflow phenomenon. But another pathway associated with insulin named MAPK pathway was actived which resulted in endothelial cells apoptosis. This phenomenon was called selective insulin resistance. I/R is another important stimulus for MAPK pathway. What will happen to endothelial cells when stimulated by I/R in diabetic patients? It is not clear now.
Objectives
1. To determine whether cardia microvessle injury in diabetic rats was more severe than in normal rats following the treatment of I/R.
2. If so, to investigate the effect of I/R on PI3K pathway and MAPK pathway, and the possible relationship between the two pathways.
3. If the relationship between the two pathways was exsited, to see the role of it in the cardia microvessle injury caused by I/R in diabetic rats.
4. To elucidate whether inhibition of the relationship between the two pathways was helpful to improve cardia microvessle injury caused by I/R in diabetic rats.
Methods
1. Male Sprague-Dawley rat, 200 g, dieted with high glucose and high cholesterol for 12 weeks, diabetes was induced with a single intraperitoneal injection of streptozotocin (50 mg/kg, Sigma). Random serum glucose greater than 16.7 mmol/L were considered success.
2. Rats were anesthetized and myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk and making a slipknot around the left anterior descending coronary artery. After 30 minutes of ischemia, the slipknot was released and the myocardium was reperfused for 3 hours. Hemodynamic data were continuously monitored on a polygraph (RM-6200C) and simultaneously digitized by using a computer interfaced with an analog-to-digital converter. Even’s blue and TTC staining was used to determine the infarction area, and Even’s blue and Thioflavin-S staining was used to determine the no-reflow area after 30 min ischemia and 3 hours after reperfusion. Immunofluorescence assay of CD31 was used to determine the number of endothelial cells. Echocardiography was performed at 24 hours after reperfusion.
3. Followed by removal of the endocardial endothelium and the epicardial coronaries, the left ventricles were cut into small pieces and incubated in 2 ml 0.2% collagens, isolated and purified cardiac microvascular endothelium cells(CMECs). The isolated primary CMECs were put into hypoxia incubator (95% N2 / 5% CO2 , 37℃) for 1 h and then returned to normal incubator for 3 h. Nitrate reduction kit was used to analyse the secretion of NO. Flow cytometry assay was used to detect the apoptosis of cells, and the expression of PI3K, P-JNK, JNK, MEKK1、IRS-1 were determined by western blot at the end of 3 h reoxygenation.
Results
1. The No-reflow size in diabetic rats significantly larger than that in normal rats after I/R (1.08±0.07 % vs 3.03±0.11%,P < 0.01). The infarct size (IS)/AAR ratio in diabetic rats was significantly larger than in normal rats (57.49±1.25% vs 68.39±2.31%, P < 0.05)too. I/R decreased the -dp/dtmax in both diabetic rats and normal rats(-4326.75±108.90 vs. -3613±88.67, P < 0.01 and -4342.57±87.49 vs. -4065.09±99.31, P < 0.01), and -dp/dtmax was signaificantly lower in diabetic rats compared with normal rats after I/R treatment. The EF and FS value was attenuated in diabetic rats after I/R treatment (32.5±10.0% vs 57.3±8.5%,P < 0.05). The density of CD31 postive cells was lower in in diabetic rats after I/R campared with that in normal rats (1600±82/mm vs 2653±41/mm, P < 0.05).
2. H/R resulted in a significant increased CMECs apoptosis index in both normal and diabetic rats (2.33±0.16% vs. 38.17±1.97%, P < 0.01 and 2.23±0.07% vs. 31±2.32%, P < 0.01). The expression of Caspase-3 showed the same trend. Further more, the expression of Caspase-3 in diabetic CMECs was significantly higher than in normal CMECs (P < 0.05). H/R reduced the secretion of NO in diabetic CMECs compared with normal CMECs after 3 h reoxygenation (74.67±0.96% vs. 56.67±1.28%, P < 0.01).
3. The phosphorylation of IRS-1 and the expression of PI3K were significantly attenuated in diabetic CMECs compared with normal CMECs (P < 0.05). Following 3 h reoxygenation, the expression of PI3K was diminished in both diabetic CMECs and normal CMECs (P < 0.05). H/R increased the expression of MEKK1 and phosphorylation of JNK in both diabetic CMECs and normal CMECs (P < 0.01). The phosphorylation of JNK seems increased more in diabetic CMECs following H/R stimulus compared with in normal CMECs (148.47±12.27% vs. 399.67±7.77%, P < 0.01).
4. PI3K inhibitor LY294002 (10μM) increased the H/R induced apoptosis in diabetic CMECs (39.07±5.48% vs. 47.55±3.46%, P < 0.01) and increased expression of Caspase-3 (338.00±29.53% vs. 450.00±15.51%, P < 0.05). LY294002 attenuated the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 41.00±5.72%, P < 0.05). The phosphorylation of JNK was increased by LY294002 followed by the treatment of H/R in diabetic CMECs (260.75±8.76% vs. 373.5±10.74%, P < 0.01).
5. JNK inhibitor SP600125 (10μM) increased the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 68.75±8.58%, P < 0.05). SP600125 reduced the H/R increased expression of Caspase-3 (338.00±29.53% vs. 260.67±26.91%, P < 0.05). The reduced expression of PI3K induced by H/R was increased partly by SP600125 in diabetic CMECs (35.00±5.35% vs. 62.00±4.54%,P < 0.05).
Conclusions
1. Exaggerated no-reflow areas and infarction areas accompanied by severe impairment of cardiac function and endothelial cell density presented in diabetic rat model after I/R.
2. H/R induced increased apoptosis and decreased NO secretion, accompanied by increased phosphorylation of JNK and attenuated expression of PI3K.
3. JNK inhibitor improved the NO secretion ability of CMECs through increasing the expression of PI3K in diabetic CMECs.
4. PI3K inhibitor increased the H/R induced apoptosis through activating JNK in diabetic CMECs.
据国际糖尿病联合会提供的数据,目前全球糖尿病患者超过2.5亿,预计到2025年这个数字将变成3.8亿。由于人口基数大,中国将成为仅次于印度的糖尿病第二大国。因其起病隐匿且造成人体各个器官的血管损害,糖尿病绝对称得上是人体的定时炸弹。目前糖尿病患者死亡的第一位原因就是冠心病。糖尿病合并冠心病患者多为多支血管病变且病变弥漫,PCI术后的无复流发生率和严重程度均明显升高。糖尿病合并冠心病患者无复流现象的预防和治疗已经成为心血管界介入治疗和药物治疗面临的新挑战。
心脏血运重建的过程中出现无复流现象,无复流现象的可能机制为:
①微血管舒张功能障碍及中性粒细胞栓塞;②血小板及中心粒细胞形成微血栓堵塞微血管;③细胞肿胀挤压微血管;④血液粘滞性变化等。可见无复流现象与心肌微血管内皮密切相关,但其发生的具体机制目前尚不清楚。糖尿病以引起多器官的微血管、小血管的病变而著称,糖尿病患者无复流现象的发生很可能与心肌微血管的损伤密切相关。
多项研究证实,糖尿病患者胰岛素受体后信号转导通路受损,引起eNOS表达减少,内皮细胞分泌NO的量及活性降低,可能与内皮依赖性舒张功能障碍、血小板聚集等导致无复流发生的因素有关。而胰岛素参与调节另一途径即分裂原活化蛋白激酶(MAPK)途径则保持完好,甚至加强,产生促进内皮细胞的凋亡效应,这就是近年来有些学者提出的“选择性”胰岛素抵抗。I/R也是引起MAPK信号通路激活的常见刺激因素。在I/R的刺激下,糖尿病患者的心肌微血管内皮细胞会发生怎样的变化,以及这一变化产生的可能机制目前尚不清楚。
因此,本研究的实验目的:
1.明确糖尿病大鼠缺血/再灌注后心肌微血管的损害是否比正常大鼠严重。
2.如果是,糖尿病大鼠心肌缺血/再灌注后PI3K信号转导通路及MAPK通路的影响。这两条信号通路之间是否存在相互关系。
3.如果两条信号通路间的相互关系存在,此种关系在糖尿病引起的心肌微血管损害中起到什么样的作用。
4.阻断两通路间的相互关系是否可以改善糖尿病引起的心肌微血管损害。
实验方法
1.糖尿病大鼠模型的制备:
高脂高糖饮食饲养雄性成年SD大鼠(体重200 g左右)3月后,给予低剂量STZ腹腔注射(50 mg/kg),检测造模前后血糖值,发现造模后大鼠血糖显著增高,达糖尿病诊断标准。大鼠血糖升高1周后用于实验。
2.缺血/再灌注模型的制备:
成年SD雄性大鼠,腹腔注射戊巴比妥麻醉,呼吸机辅助呼吸,于3～4肋间开胸1.5 cm,迅速暴露心脏,冠状动脉左室支下穿线活扣结扎,以标Ⅱ导联心电图改变为阻断血流指标,连续缺血30 min,松线再灌注开始,关胸,制备大鼠心肌I/R模型。I/R术中通过八道生理记录仪持续记录大鼠心电图、动脉压及左室内压等血流动力学指标。再灌注3 h后采用伊文蓝-硫磺素S双染法测定心肌微血管损伤范围,伊文蓝-三苯四唑氯盐(TTC)双染法确定心肌梗死范围。CD31免疫荧光染色确定各组心肌微血管内皮细胞的数量。再灌注后24 h,心脏B超测量各组大鼠心功能。
3.建立心肌微血管内皮细胞(CMECs)H/R模型:
无菌分离成年SD大鼠左心室组织,去除心内外膜及冠脉组织,取心尖部心肌组织剪为1 mm3组织块,经胰蛋白酶、胶原酶消化,条件培养及细胞纯化后获得CMECs。分离培养得到的CMECs置于缺氧细胞培养箱(95% N2 / 5% CO2 , 37℃)1 h后重新置于正常细胞培养箱3 h,硝酸还原法测定各组细胞NO的分泌量,流式细胞仪检测细胞凋亡,western blot方法检测Caspase-3、P-JNK,JNK,PI3K、MEKK1、IRS-1等蛋白的表达及磷酸化水平。
实验结果1.糖尿病(DM)大鼠缺血/再灌注后微血管损伤范围显著大于N + I/R大鼠(1.08±0.07 % vs 3.03±0.11%,P < 0.01)梗死范围显著大于非糖尿病大鼠(57.49±1.25% vs 68.39±2.31%, P < 0.05)。DM大鼠缺血/再灌注后-dp/dtmax低于非糖尿病大鼠(-3613±88.67 vs. -4065.09±99.31,P < 0.05)。DM大鼠缺血/再灌注后左室射血分数较N + I/R组显著降低(32.5±10.0% vs 51.6±9.3%,P < 0.05),也显著低于D组(32.5±10.0% vs 57.3±8.5%,P < 0.05)DM大鼠缺血/再灌注后3 h心肌CD31阳性的细胞密度较N+I/R组显著降低(1600±82/mm vs 2653±41/mm,P < 0.05),较D组也显著降低(1600±82/mm vs 2590±52/mm,P < 0.05)。提示糖尿病大鼠缺血/再灌注后心肌微血管损伤及心功能损伤较非糖尿病大鼠加重。
2.缺氧/复氧(H/R)损伤显著增加糖尿病及正常大鼠CMEC的凋亡率(2.23±0.07% vs. 31±2.32%, P < 0.01和2.33±0.16% vs. 38.17±1.97%, P < 0.01)。Caspase-3的表达也呈现出相同的趋势,另外,H/R处理后糖尿病大鼠CMECs表达Caspase-3的量显著高于非糖尿病大鼠(P < 0.05)。H/R处理后糖尿病及非糖尿病大鼠CMECs分泌NO的能力均显著下降(86.67±2.18% vs. 56.67±1.28%, P < 0.01 and 100% vs. 74.67±0.96%, P < 0.01),H/R后糖尿病大鼠CMECs分泌NO的量显著低于非糖尿病大鼠(74.67±0.96% vs. 56.67±1.28%, P < 0.01)。这些结果提示,H/R对糖尿病大鼠CMECs的分泌功能及凋亡影响更大。
3.DM大鼠CMECs IRS-1的磷酸化水平显著降低(105.33±3.50% vs.56.27±4.60%, P < 0.05)。PI3K的表达在糖尿病大鼠CMECs中的表达也有明显下降(110.00±3.31% vs. 82.22±3.31%, P < 0.01),在糖尿病(82.22±3.31% vs. 35.67±1.78%, P < 0.01)和非糖尿病(110.00±3.31% vs. 89.67±2.91%,P < 0.05)大鼠中均可见到H/R引起的PI3K的表达下降。H/R的刺激正常大鼠的CMECs,MEKK1表达量(101.33±5.18% vs. 299±7.36%, P < 0.05 )JNK的磷酸化水平(97.36±0.76% vs. 162.33±4.49%,P < 0.05)均显著升高,H/R的刺激糖尿病大鼠的CMECs时,JNK的磷酸化水平增加更加显著(148.47±12.27% vs. 399.67±7.77%,P < 0.01)。因此PI3K和JNK很可能是联系糖尿病和缺血/复氧两个病理过程的重要中间分子。
4.H/R处理前1 h给于PI3K的抑制剂LY294002(10μM)可进一步增加H/R导致的DM大鼠CMECs的凋亡(39.07±5.48% vs. 47.55±3.46%, P < 0.01)。LY294002也进一步增加H/R引起的DM大鼠CMECs Caspase-3蛋白的表达(338.00±29.53% vs. 450.00±15.51%, P < 0.05),减少H/R导致的大鼠CMECs分泌NO的量的减少(56.67±3.34% vs. 41.00±5.72%, P < 0.05)。LY294002进一步上调了H/R引起的DM大鼠CMECs JNK磷酸化水平的增加(260.75±8.76% vs. 373.5±10.74%,P < 0.01)。此部分结果说明,PI3K的抑制剂可通过激活JNK进一步恶化H/R导致的DM大鼠CMECs细胞凋亡和分泌功能下降。
5.H/R使DM大鼠CMECs分泌NO的量显著减低(86.67±5.67% vs. 56.67±3.34%,P < 0.01),H/R处理前给于JNK的抑制剂SP600125(10μM)可部分改善DM大鼠CMECs NO分泌的减少(56.67±3.34% vs. 68.75±8.58%,P < 0.05)。SP600125降低H/R引起的糖尿病大鼠CMECs Caspase-3蛋白的表达增加(338.00±29.53% vs. 260.67±26.91%, P < 0.05)。H/R刺激下调DM大鼠PI3K的表达(82.00±9.93% vs. 35.00±5.35%,P < 0.01),JNK的抑制剂可部分上调因H/R导致的PI3K的表达下降(35.00±5.35% vs. 62.00±4.54%,P < 0.05)。此部分结果说明,JNK的抑制剂通过激活PI3K改善H/R导致的DM大鼠CMECs的分泌功能,减少凋亡。
结论
1.糖尿病大鼠缺血/再灌注后较非糖尿病大鼠心肌微血管损伤范围、梗死范围增加,心肌收缩、舒张能力均显著下降,心肌微血管内皮细胞数量减少。
2.DM大鼠CMECs缺氧/复氧处理后凋亡增加,NO分泌量下降,同时伴有JNK磷酸化水平增加及PI3K的表达减少。
3. H/R刺激通过下调PI3K的表达减少DM大鼠CMECs分泌NO的量,而JNK的抑制剂可通过上调PI3K的表达,改善DM大鼠CMECs分泌NO的能力。
4.H/R也激活JNK,引起DM大鼠CMECs的凋亡,PI3K的抑制剂促使JNK进一步活化,增加H/R引起的DM大鼠CMECs的凋亡。
上述结果提示MAPK信号通路与PI3K信号通路的相互关系参与缺血/再灌注引起的糖尿病大鼠心肌微血管的损伤。以JNK和PI3K为连接点的两条信号通路的相互关系的存在为I/R对糖尿病患者造成的心肌微血管损害提供可能的分子机制和治疗靶点。
Background
According to the data provided by International Diabetes Faderation (IDF), the number of the diabetic patients is over 250 million, and the number will become 380 million by 2025. China will be the second biggest diabetic country in the world. The first cause of death in diabetic patients is cardiovascular disease. Multivessel coronary artery disease is always seen in diabetic coronary artery disease patients. The incidence of no-reflow after PCI treatment is much higher in diabetic patients compared with those patients without diabetes. The therapy and prevention of no-reflow penomenon have become a new challenge in the field of cardiovascular disease.
The possible mechanism of no-reflow penomenon in PCI treatment are the following points:①Microvessel vasodialation dysfunction and neutrophilic granulocyte blocking in microvessles;②Thrombocyte occludes in microvessles;③Endothelial cells swelling;④Change of the stagnation of the blood viscosity. It seems that no-reflow phenomenon is associated with microvascular injury, but the accurate mechanism is not clear. Diabetes is famous of causing mulp-organ microvessle injury. The no-reflow phenomenon seen in diabetic patients may be associated with cardia microvessle injury.
Many data reported that the insulin receptor pathway was hurt and the expression of eNOS and secretion of NO were decreased in diabetic patients. These changes were associated with endothelium-dependent vasodialation dysfuncrion and thrombocyte aggragation which were causes of no-reflow phenomenon. But another pathway associated with insulin named MAPK pathway was actived which resulted in endothelial cells apoptosis. This phenomenon was called selective insulin resistance. I/R is another important stimulus for MAPK pathway. What will happen to endothelial cells when stimulated by I/R in diabetic patients? It is not clear now.
Objectives
1. To determine whether cardia microvessle injury in diabetic rats was more severe than in normal rats following the treatment of I/R.
2. If so, to investigate the effect of I/R on PI3K pathway and MAPK pathway, and the possible relationship between the two pathways.
3. If the relationship between the two pathways was exsited, to see the role of it in the cardia microvessle injury caused by I/R in diabetic rats.
4. To elucidate whether inhibition of the relationship between the two pathways was helpful to improve cardia microvessle injury caused by I/R in diabetic rats.
Methods
1. Male Sprague-Dawley rat, 200 g, dieted with high glucose and high cholesterol for 12 weeks, diabetes was induced with a single intraperitoneal injection of streptozotocin (50 mg/kg, Sigma). Random serum glucose greater than 16.7 mmol/L were considered success.
2. Rats were anesthetized and myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk and making a slipknot around the left anterior descending coronary artery. After 30 minutes of ischemia, the slipknot was released and the myocardium was reperfused for 3 hours. Hemodynamic data were continuously monitored on a polygraph (RM-6200C) and simultaneously digitized by using a computer interfaced with an analog-to-digital converter. Even’s blue and TTC staining was used to determine the infarction area, and Even’s blue and Thioflavin-S staining was used to determine the no-reflow area after 30 min ischemia and 3 hours after reperfusion. Immunofluorescence assay of CD31 was used to determine the number of endothelial cells. Echocardiography was performed at 24 hours after reperfusion.
3. Followed by removal of the endocardial endothelium and the epicardial coronaries, the left ventricles were cut into small pieces and incubated in 2 ml 0.2% collagens, isolated and purified cardiac microvascular endothelium cells(CMECs). The isolated primary CMECs were put into hypoxia incubator (95% N2 / 5% CO2 , 37℃) for 1 h and then returned to normal incubator for 3 h. Nitrate reduction kit was used to analyse the secretion of NO. Flow cytometry assay was used to detect the apoptosis of cells, and the expression of PI3K, P-JNK, JNK, MEKK1、IRS-1 were determined by western blot at the end of 3 h reoxygenation.
Results
1. The No-reflow size in diabetic rats significantly larger than that in normal rats after I/R (1.08±0.07 % vs 3.03±0.11%,P < 0.01). The infarct size (IS)/AAR ratio in diabetic rats was significantly larger than in normal rats (57.49±1.25% vs 68.39±2.31%, P < 0.05)too. I/R decreased the -dp/dtmax in both diabetic rats and normal rats(-4326.75±108.90 vs. -3613±88.67, P < 0.01 and -4342.57±87.49 vs. -4065.09±99.31, P < 0.01), and -dp/dtmax was signaificantly lower in diabetic rats compared with normal rats after I/R treatment. The EF and FS value was attenuated in diabetic rats after I/R treatment (32.5±10.0% vs 57.3±8.5%,P < 0.05). The density of CD31 postive cells was lower in in diabetic rats after I/R campared with that in normal rats (1600±82/mm vs 2653±41/mm, P < 0.05).
2. H/R resulted in a significant increased CMECs apoptosis index in both normal and diabetic rats (2.33±0.16% vs. 38.17±1.97%, P < 0.01 and 2.23±0.07% vs. 31±2.32%, P < 0.01). The expression of Caspase-3 showed the same trend. Further more, the expression of Caspase-3 in diabetic CMECs was significantly higher than in normal CMECs (P < 0.05). H/R reduced the secretion of NO in diabetic CMECs compared with normal CMECs after 3 h reoxygenation (74.67±0.96% vs. 56.67±1.28%, P < 0.01).
3. The phosphorylation of IRS-1 and the expression of PI3K were significantly attenuated in diabetic CMECs compared with normal CMECs (P < 0.05). Following 3 h reoxygenation, the expression of PI3K was diminished in both diabetic CMECs and normal CMECs (P < 0.05). H/R increased the expression of MEKK1 and phosphorylation of JNK in both diabetic CMECs and normal CMECs (P < 0.01). The phosphorylation of JNK seems increased more in diabetic CMECs following H/R stimulus compared with in normal CMECs (148.47±12.27% vs. 399.67±7.77%, P < 0.01).
4. PI3K inhibitor LY294002 (10μM) increased the H/R induced apoptosis in diabetic CMECs (39.07±5.48% vs. 47.55±3.46%, P < 0.01) and increased expression of Caspase-3 (338.00±29.53% vs. 450.00±15.51%, P < 0.05). LY294002 attenuated the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 41.00±5.72%, P < 0.05). The phosphorylation of JNK was increased by LY294002 followed by the treatment of H/R in diabetic CMECs (260.75±8.76% vs. 373.5±10.74%, P < 0.01).
5. JNK inhibitor SP600125 (10μM) increased the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 68.75±8.58%, P < 0.05). SP600125 reduced the H/R increased expression of Caspase-3 (338.00±29.53% vs. 260.67±26.91%, P < 0.05). The reduced expression of PI3K induced by H/R was increased partly by SP600125 in diabetic CMECs (35.00±5.35% vs. 62.00±4.54%,P < 0.05).
Conclusions
1. Exaggerated no-reflow areas and infarction areas accompanied by severe impairment of cardiac function and endothelial cell density presented in diabetic rat model after I/R.
2. H/R induced increased apoptosis and decreased NO secretion, accompanied by increased phosphorylation of JNK and attenuated expression of PI3K.
3. JNK inhibitor improved the NO secretion ability of CMECs through increasing the expression of PI3K in diabetic CMECs.
4. PI3K inhibitor increased the H/R induced apoptosis through activating JNK in diabetic CMECs.
引文
1. Fong DS, Aiello LP, Ferris FL, Klein R: Diabetic retinopathy. Diabetes Care 2004;27:2540-2553.
2. Kunisaki M, Bursell SE, Clermont AC, Ishii H, Ballas LM, Jirousek MR, Umeda F, Nawata H, King GL: Vitamin-e prevents diabetes-induced abnormal retinal blood-flow via the diacylglycerol-protein kinase-c pathway. American Journal of Physiology-Endocrinology and Metabolism 1995;269:E239-E246.
3. Gross JL, de Azevedo MJ, Silveiro SP, Canani LH, Caramori ML, Zelmanovitz T: Diabetic nephropathy: Diagnosis, prevention, and treatment. Diabetes Care 2005;28:164-176.
4. Adler AI, Stevens RJ, Manley SE, Bilous RW, Cull CA, Holman RR: Development and progression of nephropathy in type 2 diabetes: The united kingdom prospective diabetes study (ukpds 64). Kidney International 2003;63:225-232.
5. Standards of medical care in diabetes--2007. Diabetes Care 2007;30 Suppl 1:S4-S41.
6. Boulton AJ, Vinik AI, Arezzo JC, Bril V, Feldman EL, Freeman R, Malik RA, Maser RE, Sosenko JM, Ziegler D: Diabetic neuropathies: A statement by the american diabetes association. Diabetes Care 2005;28:956-962.
7. Maser RE, Mitchell BD, Vinik AI, Freeman R: The association between cardiovascular autonomic neuropathy and mortality in individuals with diabetes: A meta-analysis. Diabetes Care 2003;26:1895-1901.
8. Schofer J, Schluter M, Rau T, Hammer F, Haag N, Mathey DG: Influence of treatment modality on angiographic outcome after coronary stenting in diabetic patients: A controlled study. J Am Coll Cardiol 2000;35:1554- 1559.
9. Creager MA, Luscher TF, Cosentino F, Beckman JA: Diabetes and vascular disease: Pathophysiology, clinical consequences, and medical therapy: Part i. Circulation 2003;108:1527-1532.
10. Luscher TF, Creager MA, Beckman JA, Cosentino F: Diabetes and vascular disease: Pathophysiology, clinical consequences, and medical therapy: Part ii. Circulation 2003;108:1655-1661.
11. Cai H, Harrison DG: Endothelial dysfunction in cardiovascular diseases: The role of oxidant stress. Circ Res 2000;87:840-844.
12. Nitenberg A, Valensi P, Sachs R, Dali M, Aptecar E, Attali JR: Impairment of coronary vascular reserve and ach-induced coronary vasodilation in diabetic patients with angiographically normal coronary arteries and normal left ventricular systolic function. Diabetes 1993;42:1017-1025.
13. Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288: 373-376.
14. Lefer AM, Ma XL: Decreased basal nitric oxide release in hypercholesterolemia increases neutrophil adherence to rabbit coronary artery endothelium. Arterioscler Thromb 1993;13:771-776.
15. Bath PM: The effect of nitric oxide-donating vasodilators on monocyte chemotaxis and intracellular cgmp concentrations in vitro. Eur J Clin Pharmacol 1993;45:53-58.
16. Cannon RO, 3rd, Camici PG, Epstein SE: Pathophysiological dilemma of syndrome x. Circulation 1992;85:883-892.
17. Poornima IG, Parikh P, Shannon RP: Diabetic cardiomyopathy: The search for a unifying hypothesis. Circ Res 2006;98:596-605.
18. Fein FS: Diabetic cardiomyopathy. Diabetes Care 1990;13:1169-1179.
19. Nitenberg A, Ledoux S, Valensi P, Sachs R, Attali JR, Antony I: Impairment of coronary microvascular dilation in response to cold pressor--induced sympathetic stimulation in type 2 diabetic patients with abnormal stress thallium imaging. Diabetes 2001;50:1180-1185.
20. Di Carli MF, Bianco-Batlles D, Landa ME, Kazmers A, Groehn H, Muzik O, Grunberger G: Effects of autonomic neuropathy on coronary blood flow in patients with diabetes mellitus. Circulation 1999;100:813-819.
21. Momose M, Abletshauser C, Neverve J, Nekolla SG, Schnell O, Standl E, Schwaiger M, Bengel FM: Dysregulation of coronary microvascular reactivity in asymptomatic patients with type 2 diabetes mellitus. Eur J Nucl Med Mol Imaging 2002;29:1675-1679.
22. Lusis AJ: Atherosclerosis. Nature 2000;407:233-241.
23. Calles-Escandon J, Cipolla M: Diabetes and endothelial dysfunction: A clinical perspective. Endocr Rev 2001;22:36-52.
24. Zeng G, Quon MJ: Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J Clin Invest 1996;98:894-898.
25. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, Quon MJ: Roles for insulin receptor, pi3-kinase, and akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 2000;101:1539-1545.
26. Kuboki K, Jiang ZY, Takahara N, Ha SW, Igarashi M, Yamauchi T, Feener EP, Herbert TP, Rhodes CJ, King GL: Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo : A specific vascular action of insulin. Circulation 2000;101: 676-681.
27. Montagnani M, Chen H, Barr VA, Quon MJ: Insulin-stimulated activation of enos is independent of ca2+ but requires phosphorylation by akt at ser(1179). J Biol Chem 2001;276:30392-30398.
28. Gogg S, Smith U, Jansson PA: Increased mapk activation and impaired insulin signaling in subcutaneous microvascular endothelial cells in type 2 diabetes: The role of endothelin-1. Diabetes 2009;58:2238-2245.
29. Muller JM, Davis MJ, Chilian WM: Integrated regulation of pressure and flow in the coronary microcirculation. Cardiovasc Res 1996;32:668-678.
30. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA, Jr., Shin WS, Liao JK: Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 1995;96:60-68.
31. Woodman RJ, Chew GT, Watts GF: Mechanisms, significance and treatment of vascular dysfunction in type 2 diabetes mellitus: Focus on lipid-regulating therapy. Drugs 2005;65:31-74.
32. Kamba T, McDonald DM: Mechanisms of adverse effects of anti-vegf therapy for cancer. Br J Cancer 2007;96:1788-1795.
33. Chou E, Suzuma I, Way KJ, Opland D, Clermont AC, Naruse K, Suzuma K, Bowling NL, Vlahos CJ, Aiello LP, King GL: Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic states: A possible explanation for impaired collateral formation in cardiac tissue. Circulation 2002;105:373-379.
34. Miele C, Rochford JJ, Filippa N, Giorgetti-Peraldi S, Van Obberghen E: Insulin and insulin-like growth factor-i induce vascular endothelial growth factor mrna expression via different signaling pathways. J Biol Chem 2000;275:21695-21702.
35. Yoon YS, Uchida S, Masuo O, Cejna M, Park JS, Gwon HC, Kirchmair R, Bahlman F, Walter D, Curry C, Hanley A, Isner JM, Losordo DW: Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: Restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation 2005;111:2073-2085.
36. Jesmin S, Miyauchi T, Goto K, Yamaguchi I: Down-regulated vegf expression in the diabetic heart is normalized by an endothelin eta receptor antagonist. Eur J Pharmacol 2006;542:184-185.
37. Chiarelli F, Spagnoli A, Basciani F, Tumini S, Mezzetti A, Cipollone F, Cuccurullo F, Morgese G, Verrotti A: Vascular endothelial growth factor (vegf) in children, adolescents and young adults with type 1 diabetes mellitus: Relation to glycaemic control and microvascular complications. Diabet Med 2000;17:650-656.
38. Diamant M, Hanemaaijer R, Verheijen JH, Smit JW, Radder JK, Lemkes HH:Elevated matrix metalloproteinase-2 and -9 in urine, but not in serum, are markers of type 1 diabetic nephropathy. Diabet Med 2001;18:423-424.
39. Park CW, Kim HW, Lim JH, Yoo KD, Chung S, Shin SJ, Chung HW, Lee SJ, Chae CB, Kim YS, Chang YS: Vascular endothelial growth factor inhibition by drk6 causes endothelial apoptosis, fibrosis, and inflammation in the heart via the akt/enos axis in db/db mice. Diabetes 2009;58:2666- 2676.
40. Nystrom FH, Quon MJ: Insulin signalling: Metabolic pathways and mechanisms for specificity. Cell Signal 1999;11:563-574.
41. Baron AD, Brechtel-Hook G, Johnson A, Cronin J, Leaming R, Steinberg HO: Effect of perfusion rate on the time course of insulin-mediated skeletal muscle glucose uptake. Am J Physiol 1996;271:E1067-1072.
42. Sung PH, Sun CK, Ko SF, Chang LT, Sheu JJ, Lee FY, Wu CJ, Chua S, Yip HK: Impact of hyperglycemic control on left ventricular myocardium. A molecular and cellular basic study in a diabetic rat model. Int Heart J 2009;50:191-206.
43. Brownlee M: The pathobiology of diabetic complications: A unifying mechanism. Diabetes 2005;54:1615-1625.
44. Ong HL, Cheng KT, Liu X, Bandyopadhyay BC, Paria BC, Soboloff J, Pani B, Gwack Y, Srikanth S, Singh BB, Gill DL, Ambudkar IS: Dynamic assembly of trpc1-stim1-orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem 2007;282:9105-9116.
45. Worthley MI, Kanani RS, Sun YH, Sun Y, Goodhart DM, Curtis MJ, Anderson TJ: Effects of tetrahydrobiopterin on coronary vascular reactivity in atherosclerotic human coronary arteries. Cardiovasc Res 2007;76:539- 546.
46. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH: Proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase i causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation 2007; 116:944-953.
47. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H: High glucose level andfree fatty acid stimulate reactive oxygen species production through protein kinase c--dependent activation of nad(p)h oxidase in cultured vascular cells. Diabetes 2000;49:1939-1945.
48. Ding H, Aljofan M, Triggle CR: Oxidative stress and increased enos and nadph oxidase expression in mouse microvessel endothelial cells. J Cell Physiol 2007;212:682-689.
49. Aljofan M, Ding H: High glucose increases expression of cyclooxygenase-2, increases oxidative stress and decreases the generation of nitric oxide in mouse microvessel endothelial cells. J Cell Physiol 2010;222:669-675.
50. Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M: Reactive oxygen species promote tnfalpha-induced death and sustained jnk activation by inhibiting map kinase phosphatases. Cell 2005;120:649-661.
51. Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, Kamalanathan S, Hattori Y, Ignarro LJ, Iguchi A: Endothelial cellular senescence is inhibited by nitric oxide: Implications in atherosclerosis associated with menopause and diabetes. Proc Natl Acad Sci U S A 2006;103:17018-17023.
52. Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM: Mechanisms of increased vascular superoxide production in human diabetes mellitus: Role of nad(p)h oxidase and endothelial nitric oxide synthase. Circulation 2002;105:1656-1662.
53. Hammes HP, Brownlee M, Lin J, Schleicher E, Bretzel RG: Diabetic retinopathy risk correlates with intracellular concentrations of the glycoxidation product nepsilon-(carboxymethyl) lysine independently of glycohaemoglobin concentrations. Diabetologia 1999;42:603-607.
54. Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813-820.
55. Ono Y, Aoki S, Ohnishi K, Yasuda T, Kawano K, Tsukada Y: Increased serum levels of advanced glycation end products in niddm patients with diabetic complications. Diabetes Care 1998;21:1027.
56. Alikhani M, Maclellan CM, Raptis M, Vora S, Trackman PC, Graves DT:Advanced glycation end products induce apoptosis in fibroblasts through activation of ros, map kinases, and the foxo1 transcription factor. Am J Physiol Cell Physiol 2007;292:C850-856.
57. Zhou YJ, Yang HW, Wang XG, Zhang H: Hepatocyte growth factor prevents advanced glycation end products-induced injury and oxidative stress through a pi3k/akt-dependent pathway in human endothelial cells. Life Sci 2009;85:670-677.
58. Soro-Paavonen A, Zhang WZ, Venardos K, Coughlan MT, Harris E, Tong DC, Brasacchio D, Paavonen K, Chin-Dusting J, Cooper ME, Kaye D, Thomas MC, Forbes JM: Advanced glycation end-products induce vascular dysfunction via resistance to nitric oxide and suppression of endothelial nitric oxide synthase. J Hypertens.2010 [Epub ahead of print]
59. Vlassara H, Palace MR: Diabetes and advanced glycation endproducts. J Intern Med 2002;251:87-101.
60. Nilius B, Viana F, Droogmans G: Ion channels in vascular endothelium. Annu Rev Physiol 1997;59:145-170.
61. Sheng JZ, Wang D, Braun AP: Daf-fm (4-amino-5- methylamino-2',7'- difluorofluorescein) diacetate detects impairment of agonist-stimulated nitric oxide synthesis by elevated glucose in human vascular endothelial cells: Reversal by vitamin c and l-sepiapterin. J Pharmacol Exp Ther 2005;315:931-940.
62. Tamareille S, Mignen O, Capiod T, Rucker-Martin C, Feuvray D: High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells. Cell Calcium 2006;39:47-55.
63. Li Y, Feng Q, Arnold M, Peng T: Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes. Cardiovasc Res 2009;84:100-110.
64. Stalker TJ, Gong Y, Scalia R: The calcium-dependent protease calpain causes endothelial dysfunction in type 2 diabetes. Diabetes 2005;54: 1132-1140.
65. Brechard S, Tschirhart EJ: Regulation of superoxide production in neutrophils:Role of calcium influx. J Leukoc Biol 2008;84:1223-1237.
66. Bishara NB, Ding H: Glucose enhances expression of trpc1 and calcium entry in endothelial cells. Am J Physiol Heart Circ Physiol;298:H171-178.
67. Ding H, Triggle CR: Endothelial dysfunction in diabetes: Multiple targets for treatment. Pflugers Arch.2010 [Epub ahead of print]
68. Kloner RA, Ganote CE, Jennings RB: The "No-reflow" Phenomenon after temporary coronary occlusion in the dog. J Clin Invest 1974;54:1496-1508.
69. Ito H, Tomooka T, Sakai N, Yu H, Higashino Y, Fujii K, Masuyama T, Kitabatake A, Minamino T: Lack of myocardial perfusion immediately after successful thrombolysis. A predictor of poor recovery of left ventricular function in anterior myocardial infarction. Circulation 1992;85:1699-1705.
70. Wu KC, Zerhouni EA, Judd RM, Lugo-Olivieri CH, Barouch LA, Schulman SP, Blumenthal RS, Lima JA: Prognostic significance of microvascular obstruction by magnetic resonance imaging in patients with acute myocardial infarction. Circulation 1998;97:765-772.
71. Gerber BL, Rochitte CE, Melin JA, McVeigh ER, Bluemke DA, Wu KC, Becker LC, Lima JA: Microvascular obstruction and left ventricular remodeling early after acute myocardial infarction. Circulation 2000;101:2734-2741.
72. Bekkers SC, Yazdani SK, Virmani R, Waltenberger J: Microvascular obstruction: Underlying pathophysiology and clinical diagnosis. J Am Coll Cardiol 2010;55:1649-1660.
73. Morishima I, Sone T, Okumura K, Tsuboi H, Kondo J, Mukawa H, Matsui H, Toki Y, Ito T, Hayakawa T: Angiographic no-reflow phenomenon as a predictor of adverse long-term outcome in patients treated with percutaneous transluminal coronary angioplasty for first acute myocardial infarction. J Am Coll Cardiol 2000;36:1202-1209.
74. Gibson CM, Cannon CP, Murphy SA, Marble SJ, Barron HV, Braunwald E: Relationship of the timi myocardial perfusion grades, flow grades, frame count, and percutaneous coronary intervention to long-term outcomes after thrombolytic administration in acute myocardial infarction. Circulation2002;105:1909-1913.
75. Iwakura K, Ito H, Takiuchi S, Taniyama Y, Nakatsuchi Y, Negoro S, Higashino Y, Okamura A, Masuyama T, Hori M, Fujii K, Minamino T: Alternation in the coronary blood flow velocity pattern in patients with no reflow and reperfused acute myocardial infarction. Circulation 1996;94:1269-1275.
76. Lepper W, Hoffmann R, Kamp O, Franke A, de Cock CC, Kuhl HP, Sieswerda GT, Dahl J, Janssens U, Voci P, Visser CA, Hanrath P: Assessment of myocardial reperfusion by intravenous myocardial contrast echocardiography and coronary flow reserve after primary percutaneous transluminal coronary angioplasty [correction of angiography] in patients with acute myocardial infarction. Circulation 2000;101:2368-2374.
77. Gibson CM, Cannon CP, Murphy SA, Ryan KA, Mesley R, Marble SJ, McCabe CH, Van De Werf F, Braunwald E: Relationship of timi myocardial perfusion grade to mortality after administration of thrombolytic drugs. Circulation 2000;101:125-130.
78. van 't Hof AW, Liem A, Suryapranata H, Hoorntje JC, de Boer MJ, Zijlstra F: Angiographic assessment of myocardial reperfusion in patients treated with primary angioplasty for acute myocardial infarction: Myocardial blush grade. Zwolle myocardial infarction study group. Circulation 1998;97: 2302-2306.
79. Ito H, Okamura A, Iwakura K, Masuyama T, Hori M, Takiuchi S, Negoro S, Nakatsuchi Y, Taniyama Y, Higashino Y, Fujii K, Minamino T: Myocardial perfusion patterns related to thrombolysis in myocardial infarction perfusion grades after coronary angioplasty in patients with acute anterior wall myocardial infarction. Circulation 1996;93:1993-1999.
80. Orn S, Manhenke C, Greve OJ, Larsen AI, Bonarjee VV, Edvardsen T, Dickstein K: Microvascular obstruction is a major determinant of infarct healing and subsequent left ventricular remodelling following primary percutaneous coronary intervention. Eur Heart J 2009;30:1978-1985.
81. Lima JA, Judd RM, Bazille A, Schulman SP, Atalar E, Zerhouni EA: Regional heterogeneity of human myocardial infarcts demonstrated by contrast-enhancedmri. Potential mechanisms. Circulation 1995;92: 1117-1125.
82. Nijveldt R, Hofman MB, Hirsch A, Beek AM, Umans VA, Algra PR, Piek JJ, van Rossum AC: Assessment of microvascular obstruction and prediction of short-term remodeling after acute myocardial infarction: Cardiac mr imaging study. Radiology 2009;250:363-370.
83. Yan AT, Gibson CM, Larose E, Anavekar NS, Tsang S, Solomon SD, Reynolds G, Kwong RY: Characterization of microvascular dysfunction after acute myocardial infarction by cardiovascular magnetic resonance first-pass perfusion and late gadolinium enhancement imaging. J Cardiovasc Magn Reson 2006;8:831-837.
84. de Lemos JA, Braunwald E: St segment resolution as a tool for assessing the efficacy of reperfusion therapy. J Am Coll Cardiol 2001;38:1283-1294.
85. Santoro GM, Valenti R, Buonamici P, Bolognese L, Cerisano G, Moschi G, Trapani M, Antoniucci D, Fazzini PF: Relation between st-segment changes and myocardial perfusion evaluated by myocardial contrast echocardiography in patients with acute myocardial infarction treated with direct angioplasty. Am J Cardiol 1998;82:932-937.
86. Cooper HA, de Lemos JA, Morrow DA, Sabatine MS, Murphy SA, McCabe CH, Gibson CM, Antman EM, Braunwald E: Minimal st-segment deviation: A simple, noninvasive method for identifying patients with a patent infarction-related artery after fibrinolytic administration. Am Heart J 2002;144:790-795.
87. Piana RN, Paik GY, Moscucci M, Cohen DJ, Gibson CM, Kugelmass AD, Carrozza JP, Jr., Kuntz RE, Baim DS: Incidence and treatment of 'no-reflow' after percutaneous coronary intervention. Circulation 1994;89:2514-2518.
88. Gregorini L, Marco J, Kozakova M, Palombo C, Anguissola GB, Marco I, Bernies M, Cassagneau B, Distante A, Bossi IM, Fajadet J, Heusch G: Alpha-adrenergic blockade improves recovery of myocardial perfusion and function after coronary stenting in patients with acute myocardial infarction. Circulation 1999;99:482-490.
89. Zhao J, Bolton EM, Bradley JA, Lever AM: Lentiviral-mediated overexpressionof bcl-xl protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis. J Heart Lung Transplant 2009;28:936-943.
90. Otani H: The role of nitric oxide in myocardial repair and remodeling. Antioxid Redox Signal 2009;11:1913-1928.
91. Inamura Y, Miyamae M, Sugioka S, Domae N, Kotani J: Sevoflurane postconditioning prevents activation of caspase 3 and 9 through antiapoptotic signaling after myocardial ischemia-reperfusion. J Anesth 2010;24:215-224.
92. Zhang T, Yang D, Fan Y, Xie P, Li H: Epigallocatechin-3-gallate enhances ischemia/reperfusion-induced apoptosis in human umbilical vein endothelial cells via akt and mapk pathways. Apoptosis 2009;14:1245- 1254.
93. Weyrich AS, Buerke M, Albertine KH, Lefer AM: Time course of coronary vascular endothelial adhesion molecule expression during reperfusion of the ischemic feline myocardium. J Leukoc Biol 1995;57:45-55.
94. Gao F, Yue TL, Shi DW, Christopher TA, Lopez BL, Ohlstein EH, Barone FC, Ma XL: P38 mapk inhibition reduces myocardial reperfusion injury via inhibition of endothelial adhesion molecule expression and blockade of pmn accumulation. Cardiovasc Res 2002;53:414-422.
95. Yang D, Guo S, Zhang T, Li H: Hypothermia attenuates ischemia/reperfusion-induced endothelial cell apoptosis via alterations in apoptotic pathways and jnk signaling. FEBS Lett 2009;583:2500-2506.
96. Hochhauser E, Pappo O, Ribakovsky E, Ravid A, Kurtzwald E, Cheporko Y, Lelchuk S, Ben-Ari Z: Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model. Apoptosis 2008;13:77-86.
97. Lee SR, Lo EH: Interactions between p38 mitogen-activated protein kinase and caspase-3 in cerebral endothelial cell death after hypoxia- reoxygenation. Stroke 2003;34:2704-2709.
98. Zhang CL, Song F, Zhang J, Song QH: Hypoxia-induced bcl-2 expression in endothelial cells via p38 mapk pathway. Biochem Biophys Res Commun 2010; 394:976-980.
99. Montaigne D, Marechal X, Lancel S, Decoster B, Asseman P, Neviere R: The synthetic pentasaccharide fondaparinux prevents coronary microvascular injury and myocardial dysfunction in the ischemic heart. Thromb Haemost 2008;100:912-919.
100. Atmaca A, Dogan S, Dagdelen S, Kabakci G, Kes S, Nazli N, Erbas T: Management and in-hospital outcome of patients with first episode of acute myocardial infarction: Impact of diabetes mellitus. J Natl Med Assoc 2006;98:1752-1757.
101. Segal MS, Shah R, Afzal A, Perrault CM, Chang K, Schuler A, Beem E, Shaw LC, Li Calzi S, Harrison JK, Tran-Son-Tay R, Grant MB: Nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes. Diabetes 2006;55:102-109.
102. Caballero S, Sengupta N, Afzal A, Chang KH, Li Calzi S, Guberski DL, Kern TS, Grant MB: Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells. Diabetes 2007;56:960-967.
103. Tsuruta R, Fujita M, Ono T, Koda Y, Koga Y, Yamamoto T, Nanba M, Shitara M, Kasaoka S, Maruyama I, Yuasa M, Maekawa T: Hyperglycemia enhances excessive superoxide anion radical generation, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats. Brain Res 2010; 1309:155-163.
104. Schneider R, Welt K, Aust W, Loster H, Fitzl G: Cardiac ischemia and reperfusion in spontaneously diabetic rats with and without application of egb 761: Ii. Interstitium and microvasculature. Histol Histopathol 2009;24:587-598.
105. Wang J, Ma H, Tong C, Zhang H, Lawlis GB, Li Y, Zang M, Ren J, Nijland MJ, Ford SP, Nathanielsz PW, Li J: Overnutrition and maternal obesity in sheep pregnancy alter the jnk-irs-1 signaling cascades and cardiac function in the fetal heart. Faseb J. 2010 [Epub ahead of print]
106. Shang L, Ananthakrishnan R, Li Q, Quadri N, Abdillahi M, Zhu Z, Qu W, Rosario R, Toure F, Yan SF, Schmidt AM, Ramasamy R: Rage modulates hypoxia/reoxygenation injury in adult murine cardiomyocytes via jnk andgsk-3beta signaling pathways. PLoS One 2010; 5:e10092.
107. Tanno M, Tsuchida A, Nozawa Y, Matsumoto T, Hasegawa T, Miura T, Shimamoto K: Roles of tyrosine kinase and protein kinase c in infarct size limitation by repetitive ischemic preconditioning in the rat. J Cardiovasc Pharmacol 2000;35:345-352.
108. Talpur N, Echard B, Ingram C, Bagchi D, Preuss H: Effects of a novel formulation of essential oils on glucose-insulin metabolism in diabetic and hypertensive rats: A pilot study. Diabetes Obes Metab 2005;7:193-199.
109. Reffelmann T, Hale SL, Dow JS, Kloner RA: No-reflow phenomenon persists long-term after ischemia/reperfusion in the rat and predicts infarct expansion. Circulation 2003;108:2911-2917.
110. Reffelmann T, Hale SL, Li G, Kloner RA: Relationship between no reflow and infarct size as influenced by the duration of ischemia and reperfusion. Am J Physiol Heart Circ Physiol 2002;282:H766-772.
111. Scarabelli T, Stephanou A, Rayment N, Pasini E, Comini L, Curello S, Ferrari R, Knight R, Latchman D: Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury. Circulation 2001;104:253-256.
112. Scarabelli TM, Stephanou A, Pasini E, Comini L, Raddino R, Knight RA, Latchman DS: Different signaling pathways induce apoptosis in endothelial cells and cardiac myocytes during ischemia/reperfusion injury. Circ Res 2002;90:745-748.
113. Capes SE, Hunt D, Malmberg K, Gerstein HC: Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: A systematic overview. Lancet 2000;355:773-778.
114. Liu X, Wei J, Peng DH, Layne MD, Yet SF: Absence of heme oxygenase-1 exacerbates myocardial ischemia/reperfusion injury in diabetic mice. Diabetes 2005;54:778-784.
115. Marionneau C, Aimond F, Brunet S, Niwa N, Finck B, Kelly DP, Nerbonne JM: Pparalpha-mediated remodeling of repolarizing voltage-gated k+ (kv) channels in a mouse model of metabolic cardiomyopathy. J Mol Cell Cardiol2008;44:1002-1015.
116. Park JH, Okayama N, Gute D, Krsmanovic A, Battarbee H, Alexander JS: Hypoxia/aglycemia increases endothelial permeability: Role of second messengers and cytoskeleton. Am J Physiol 1999;277:C1066-1074.
117. Yuan SY, Breslin JW, Perrin R, Gaudreault N, Guo M, Kargozaran H, Wu MH: Microvascular permeability in diabetes and insulin resistance. Microcirculation 2007;14:363-373.
118. Gao X, Xu X, Belmadani S, Park Y, Tang Z, Feldman AM, Chilian WM, Zhang C: Tnf-alpha contributes to endothelial dysfunction by upregulating arginase in ischemia/reperfusion injury. Arterioscler Thromb Vasc Biol 2007;27:1269-1275.
119. Jaffe R, Charron T, Puley G, Dick A, Strauss BH: Microvascular obstruction and the no-reflow phenomenon after percutaneous coronary intervention. Circulation 2008;117:3152-3156.
120. Zhang C, Xu X, Potter BJ, Wang W, Kuo L, Michael L, Bagby GJ, Chilian WM: Tnf-alpha contributes to endothelial dysfunction in ischemia /reperfusion injury. Arterioscler Thromb Vasc Biol 2006;26:475- 480.
121. Johnson EK, Schelling ME, Quitadamo IJ, Andrew S, Johnson EC: Cultivation and characterization of coronary microvascular endothelial cells: A novel porcine model using micropigs. Microvasc Res 2002;64:278-288.
122. Nishida M, Carley WW, Gerritsen ME, Ellingsen O, Kelly RA, Smith TW: Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am J Physiol 1993;264:H639-652.
123. Grafe M, Auch-Schwelk W, Hertel H, Terbeek D, Steinheider G, Loebe M, Fleck E: Human cardiac microvascular and macrovascular endothelial cells respond differently to oxidatively modified ldl. Atherosclerosis 1998;137: 87-95.
124. Mendoza FJ, Henson ES, Gibson SB: Mekk1-induced apoptosis is mediated by smac/diablo release from the mitochondria. Biochem Biophys Res Commun 2005;331:1089-1098.
125. Huwiler A, Xin C, Brust AK, Briner VA, Pfeilschifter J: Differential binding of ceramide to mekk1 in glomerular endothelial and mesangial cells. BiochimBiophys Acta 2004;1636:159-168.
2. Kunisaki M, Bursell SE, Clermont AC, Ishii H, Ballas LM, Jirousek MR, Umeda F, Nawata H, King GL: Vitamin-e prevents diabetes-induced abnormal retinal blood-flow via the diacylglycerol-protein kinase-c pathway. American Journal of Physiology-Endocrinology and Metabolism 1995;269:E239-E246.
3. Gross JL, de Azevedo MJ, Silveiro SP, Canani LH, Caramori ML, Zelmanovitz T: Diabetic nephropathy: Diagnosis, prevention, and treatment. Diabetes Care 2005;28:164-176.
4. Adler AI, Stevens RJ, Manley SE, Bilous RW, Cull CA, Holman RR: Development and progression of nephropathy in type 2 diabetes: The united kingdom prospective diabetes study (ukpds 64). Kidney International 2003;63:225-232.
5. Standards of medical care in diabetes--2007. Diabetes Care 2007;30 Suppl 1:S4-S41.
6. Boulton AJ, Vinik AI, Arezzo JC, Bril V, Feldman EL, Freeman R, Malik RA, Maser RE, Sosenko JM, Ziegler D: Diabetic neuropathies: A statement by the american diabetes association. Diabetes Care 2005;28:956-962.
7. Maser RE, Mitchell BD, Vinik AI, Freeman R: The association between cardiovascular autonomic neuropathy and mortality in individuals with diabetes: A meta-analysis. Diabetes Care 2003;26:1895-1901.
8. Schofer J, Schluter M, Rau T, Hammer F, Haag N, Mathey DG: Influence of treatment modality on angiographic outcome after coronary stenting in diabetic patients: A controlled study. J Am Coll Cardiol 2000;35:1554- 1559.
9. Creager MA, Luscher TF, Cosentino F, Beckman JA: Diabetes and vascular disease: Pathophysiology, clinical consequences, and medical therapy: Part i. Circulation 2003;108:1527-1532.
10. Luscher TF, Creager MA, Beckman JA, Cosentino F: Diabetes and vascular disease: Pathophysiology, clinical consequences, and medical therapy: Part ii. Circulation 2003;108:1655-1661.
11. Cai H, Harrison DG: Endothelial dysfunction in cardiovascular diseases: The role of oxidant stress. Circ Res 2000;87:840-844.
12. Nitenberg A, Valensi P, Sachs R, Dali M, Aptecar E, Attali JR: Impairment of coronary vascular reserve and ach-induced coronary vasodilation in diabetic patients with angiographically normal coronary arteries and normal left ventricular systolic function. Diabetes 1993;42:1017-1025.
13. Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288: 373-376.
14. Lefer AM, Ma XL: Decreased basal nitric oxide release in hypercholesterolemia increases neutrophil adherence to rabbit coronary artery endothelium. Arterioscler Thromb 1993;13:771-776.
15. Bath PM: The effect of nitric oxide-donating vasodilators on monocyte chemotaxis and intracellular cgmp concentrations in vitro. Eur J Clin Pharmacol 1993;45:53-58.
16. Cannon RO, 3rd, Camici PG, Epstein SE: Pathophysiological dilemma of syndrome x. Circulation 1992;85:883-892.
17. Poornima IG, Parikh P, Shannon RP: Diabetic cardiomyopathy: The search for a unifying hypothesis. Circ Res 2006;98:596-605.
18. Fein FS: Diabetic cardiomyopathy. Diabetes Care 1990;13:1169-1179.
19. Nitenberg A, Ledoux S, Valensi P, Sachs R, Attali JR, Antony I: Impairment of coronary microvascular dilation in response to cold pressor--induced sympathetic stimulation in type 2 diabetic patients with abnormal stress thallium imaging. Diabetes 2001;50:1180-1185.
20. Di Carli MF, Bianco-Batlles D, Landa ME, Kazmers A, Groehn H, Muzik O, Grunberger G: Effects of autonomic neuropathy on coronary blood flow in patients with diabetes mellitus. Circulation 1999;100:813-819.
21. Momose M, Abletshauser C, Neverve J, Nekolla SG, Schnell O, Standl E, Schwaiger M, Bengel FM: Dysregulation of coronary microvascular reactivity in asymptomatic patients with type 2 diabetes mellitus. Eur J Nucl Med Mol Imaging 2002;29:1675-1679.
22. Lusis AJ: Atherosclerosis. Nature 2000;407:233-241.
23. Calles-Escandon J, Cipolla M: Diabetes and endothelial dysfunction: A clinical perspective. Endocr Rev 2001;22:36-52.
24. Zeng G, Quon MJ: Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J Clin Invest 1996;98:894-898.
25. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, Quon MJ: Roles for insulin receptor, pi3-kinase, and akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 2000;101:1539-1545.
26. Kuboki K, Jiang ZY, Takahara N, Ha SW, Igarashi M, Yamauchi T, Feener EP, Herbert TP, Rhodes CJ, King GL: Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo : A specific vascular action of insulin. Circulation 2000;101: 676-681.
27. Montagnani M, Chen H, Barr VA, Quon MJ: Insulin-stimulated activation of enos is independent of ca2+ but requires phosphorylation by akt at ser(1179). J Biol Chem 2001;276:30392-30398.
28. Gogg S, Smith U, Jansson PA: Increased mapk activation and impaired insulin signaling in subcutaneous microvascular endothelial cells in type 2 diabetes: The role of endothelin-1. Diabetes 2009;58:2238-2245.
29. Muller JM, Davis MJ, Chilian WM: Integrated regulation of pressure and flow in the coronary microcirculation. Cardiovasc Res 1996;32:668-678.
30. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA, Jr., Shin WS, Liao JK: Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 1995;96:60-68.
31. Woodman RJ, Chew GT, Watts GF: Mechanisms, significance and treatment of vascular dysfunction in type 2 diabetes mellitus: Focus on lipid-regulating therapy. Drugs 2005;65:31-74.
32. Kamba T, McDonald DM: Mechanisms of adverse effects of anti-vegf therapy for cancer. Br J Cancer 2007;96:1788-1795.
33. Chou E, Suzuma I, Way KJ, Opland D, Clermont AC, Naruse K, Suzuma K, Bowling NL, Vlahos CJ, Aiello LP, King GL: Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic states: A possible explanation for impaired collateral formation in cardiac tissue. Circulation 2002;105:373-379.
34. Miele C, Rochford JJ, Filippa N, Giorgetti-Peraldi S, Van Obberghen E: Insulin and insulin-like growth factor-i induce vascular endothelial growth factor mrna expression via different signaling pathways. J Biol Chem 2000;275:21695-21702.
35. Yoon YS, Uchida S, Masuo O, Cejna M, Park JS, Gwon HC, Kirchmair R, Bahlman F, Walter D, Curry C, Hanley A, Isner JM, Losordo DW: Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: Restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation 2005;111:2073-2085.
36. Jesmin S, Miyauchi T, Goto K, Yamaguchi I: Down-regulated vegf expression in the diabetic heart is normalized by an endothelin eta receptor antagonist. Eur J Pharmacol 2006;542:184-185.
37. Chiarelli F, Spagnoli A, Basciani F, Tumini S, Mezzetti A, Cipollone F, Cuccurullo F, Morgese G, Verrotti A: Vascular endothelial growth factor (vegf) in children, adolescents and young adults with type 1 diabetes mellitus: Relation to glycaemic control and microvascular complications. Diabet Med 2000;17:650-656.
38. Diamant M, Hanemaaijer R, Verheijen JH, Smit JW, Radder JK, Lemkes HH:Elevated matrix metalloproteinase-2 and -9 in urine, but not in serum, are markers of type 1 diabetic nephropathy. Diabet Med 2001;18:423-424.
39. Park CW, Kim HW, Lim JH, Yoo KD, Chung S, Shin SJ, Chung HW, Lee SJ, Chae CB, Kim YS, Chang YS: Vascular endothelial growth factor inhibition by drk6 causes endothelial apoptosis, fibrosis, and inflammation in the heart via the akt/enos axis in db/db mice. Diabetes 2009;58:2666- 2676.
40. Nystrom FH, Quon MJ: Insulin signalling: Metabolic pathways and mechanisms for specificity. Cell Signal 1999;11:563-574.
41. Baron AD, Brechtel-Hook G, Johnson A, Cronin J, Leaming R, Steinberg HO: Effect of perfusion rate on the time course of insulin-mediated skeletal muscle glucose uptake. Am J Physiol 1996;271:E1067-1072.
42. Sung PH, Sun CK, Ko SF, Chang LT, Sheu JJ, Lee FY, Wu CJ, Chua S, Yip HK: Impact of hyperglycemic control on left ventricular myocardium. A molecular and cellular basic study in a diabetic rat model. Int Heart J 2009;50:191-206.
43. Brownlee M: The pathobiology of diabetic complications: A unifying mechanism. Diabetes 2005;54:1615-1625.
44. Ong HL, Cheng KT, Liu X, Bandyopadhyay BC, Paria BC, Soboloff J, Pani B, Gwack Y, Srikanth S, Singh BB, Gill DL, Ambudkar IS: Dynamic assembly of trpc1-stim1-orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem 2007;282:9105-9116.
45. Worthley MI, Kanani RS, Sun YH, Sun Y, Goodhart DM, Curtis MJ, Anderson TJ: Effects of tetrahydrobiopterin on coronary vascular reactivity in atherosclerotic human coronary arteries. Cardiovasc Res 2007;76:539- 546.
46. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH: Proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase i causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation 2007; 116:944-953.
47. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H: High glucose level andfree fatty acid stimulate reactive oxygen species production through protein kinase c--dependent activation of nad(p)h oxidase in cultured vascular cells. Diabetes 2000;49:1939-1945.
48. Ding H, Aljofan M, Triggle CR: Oxidative stress and increased enos and nadph oxidase expression in mouse microvessel endothelial cells. J Cell Physiol 2007;212:682-689.
49. Aljofan M, Ding H: High glucose increases expression of cyclooxygenase-2, increases oxidative stress and decreases the generation of nitric oxide in mouse microvessel endothelial cells. J Cell Physiol 2010;222:669-675.
50. Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M: Reactive oxygen species promote tnfalpha-induced death and sustained jnk activation by inhibiting map kinase phosphatases. Cell 2005;120:649-661.
51. Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, Kamalanathan S, Hattori Y, Ignarro LJ, Iguchi A: Endothelial cellular senescence is inhibited by nitric oxide: Implications in atherosclerosis associated with menopause and diabetes. Proc Natl Acad Sci U S A 2006;103:17018-17023.
52. Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM: Mechanisms of increased vascular superoxide production in human diabetes mellitus: Role of nad(p)h oxidase and endothelial nitric oxide synthase. Circulation 2002;105:1656-1662.
53. Hammes HP, Brownlee M, Lin J, Schleicher E, Bretzel RG: Diabetic retinopathy risk correlates with intracellular concentrations of the glycoxidation product nepsilon-(carboxymethyl) lysine independently of glycohaemoglobin concentrations. Diabetologia 1999;42:603-607.
54. Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813-820.
55. Ono Y, Aoki S, Ohnishi K, Yasuda T, Kawano K, Tsukada Y: Increased serum levels of advanced glycation end products in niddm patients with diabetic complications. Diabetes Care 1998;21:1027.
56. Alikhani M, Maclellan CM, Raptis M, Vora S, Trackman PC, Graves DT:Advanced glycation end products induce apoptosis in fibroblasts through activation of ros, map kinases, and the foxo1 transcription factor. Am J Physiol Cell Physiol 2007;292:C850-856.
57. Zhou YJ, Yang HW, Wang XG, Zhang H: Hepatocyte growth factor prevents advanced glycation end products-induced injury and oxidative stress through a pi3k/akt-dependent pathway in human endothelial cells. Life Sci 2009;85:670-677.
58. Soro-Paavonen A, Zhang WZ, Venardos K, Coughlan MT, Harris E, Tong DC, Brasacchio D, Paavonen K, Chin-Dusting J, Cooper ME, Kaye D, Thomas MC, Forbes JM: Advanced glycation end-products induce vascular dysfunction via resistance to nitric oxide and suppression of endothelial nitric oxide synthase. J Hypertens.2010 [Epub ahead of print]
59. Vlassara H, Palace MR: Diabetes and advanced glycation endproducts. J Intern Med 2002;251:87-101.
60. Nilius B, Viana F, Droogmans G: Ion channels in vascular endothelium. Annu Rev Physiol 1997;59:145-170.
61. Sheng JZ, Wang D, Braun AP: Daf-fm (4-amino-5- methylamino-2',7'- difluorofluorescein) diacetate detects impairment of agonist-stimulated nitric oxide synthesis by elevated glucose in human vascular endothelial cells: Reversal by vitamin c and l-sepiapterin. J Pharmacol Exp Ther 2005;315:931-940.
62. Tamareille S, Mignen O, Capiod T, Rucker-Martin C, Feuvray D: High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells. Cell Calcium 2006;39:47-55.
63. Li Y, Feng Q, Arnold M, Peng T: Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes. Cardiovasc Res 2009;84:100-110.
64. Stalker TJ, Gong Y, Scalia R: The calcium-dependent protease calpain causes endothelial dysfunction in type 2 diabetes. Diabetes 2005;54: 1132-1140.
65. Brechard S, Tschirhart EJ: Regulation of superoxide production in neutrophils:Role of calcium influx. J Leukoc Biol 2008;84:1223-1237.
66. Bishara NB, Ding H: Glucose enhances expression of trpc1 and calcium entry in endothelial cells. Am J Physiol Heart Circ Physiol;298:H171-178.
67. Ding H, Triggle CR: Endothelial dysfunction in diabetes: Multiple targets for treatment. Pflugers Arch.2010 [Epub ahead of print]
68. Kloner RA, Ganote CE, Jennings RB: The "No-reflow" Phenomenon after temporary coronary occlusion in the dog. J Clin Invest 1974;54:1496-1508.
69. Ito H, Tomooka T, Sakai N, Yu H, Higashino Y, Fujii K, Masuyama T, Kitabatake A, Minamino T: Lack of myocardial perfusion immediately after successful thrombolysis. A predictor of poor recovery of left ventricular function in anterior myocardial infarction. Circulation 1992;85:1699-1705.
70. Wu KC, Zerhouni EA, Judd RM, Lugo-Olivieri CH, Barouch LA, Schulman SP, Blumenthal RS, Lima JA: Prognostic significance of microvascular obstruction by magnetic resonance imaging in patients with acute myocardial infarction. Circulation 1998;97:765-772.
71. Gerber BL, Rochitte CE, Melin JA, McVeigh ER, Bluemke DA, Wu KC, Becker LC, Lima JA: Microvascular obstruction and left ventricular remodeling early after acute myocardial infarction. Circulation 2000;101:2734-2741.
72. Bekkers SC, Yazdani SK, Virmani R, Waltenberger J: Microvascular obstruction: Underlying pathophysiology and clinical diagnosis. J Am Coll Cardiol 2010;55:1649-1660.
73. Morishima I, Sone T, Okumura K, Tsuboi H, Kondo J, Mukawa H, Matsui H, Toki Y, Ito T, Hayakawa T: Angiographic no-reflow phenomenon as a predictor of adverse long-term outcome in patients treated with percutaneous transluminal coronary angioplasty for first acute myocardial infarction. J Am Coll Cardiol 2000;36:1202-1209.
74. Gibson CM, Cannon CP, Murphy SA, Marble SJ, Barron HV, Braunwald E: Relationship of the timi myocardial perfusion grades, flow grades, frame count, and percutaneous coronary intervention to long-term outcomes after thrombolytic administration in acute myocardial infarction. Circulation2002;105:1909-1913.
75. Iwakura K, Ito H, Takiuchi S, Taniyama Y, Nakatsuchi Y, Negoro S, Higashino Y, Okamura A, Masuyama T, Hori M, Fujii K, Minamino T: Alternation in the coronary blood flow velocity pattern in patients with no reflow and reperfused acute myocardial infarction. Circulation 1996;94:1269-1275.
76. Lepper W, Hoffmann R, Kamp O, Franke A, de Cock CC, Kuhl HP, Sieswerda GT, Dahl J, Janssens U, Voci P, Visser CA, Hanrath P: Assessment of myocardial reperfusion by intravenous myocardial contrast echocardiography and coronary flow reserve after primary percutaneous transluminal coronary angioplasty [correction of angiography] in patients with acute myocardial infarction. Circulation 2000;101:2368-2374.
77. Gibson CM, Cannon CP, Murphy SA, Ryan KA, Mesley R, Marble SJ, McCabe CH, Van De Werf F, Braunwald E: Relationship of timi myocardial perfusion grade to mortality after administration of thrombolytic drugs. Circulation 2000;101:125-130.
78. van 't Hof AW, Liem A, Suryapranata H, Hoorntje JC, de Boer MJ, Zijlstra F: Angiographic assessment of myocardial reperfusion in patients treated with primary angioplasty for acute myocardial infarction: Myocardial blush grade. Zwolle myocardial infarction study group. Circulation 1998;97: 2302-2306.
79. Ito H, Okamura A, Iwakura K, Masuyama T, Hori M, Takiuchi S, Negoro S, Nakatsuchi Y, Taniyama Y, Higashino Y, Fujii K, Minamino T: Myocardial perfusion patterns related to thrombolysis in myocardial infarction perfusion grades after coronary angioplasty in patients with acute anterior wall myocardial infarction. Circulation 1996;93:1993-1999.
80. Orn S, Manhenke C, Greve OJ, Larsen AI, Bonarjee VV, Edvardsen T, Dickstein K: Microvascular obstruction is a major determinant of infarct healing and subsequent left ventricular remodelling following primary percutaneous coronary intervention. Eur Heart J 2009;30:1978-1985.
81. Lima JA, Judd RM, Bazille A, Schulman SP, Atalar E, Zerhouni EA: Regional heterogeneity of human myocardial infarcts demonstrated by contrast-enhancedmri. Potential mechanisms. Circulation 1995;92: 1117-1125.
82. Nijveldt R, Hofman MB, Hirsch A, Beek AM, Umans VA, Algra PR, Piek JJ, van Rossum AC: Assessment of microvascular obstruction and prediction of short-term remodeling after acute myocardial infarction: Cardiac mr imaging study. Radiology 2009;250:363-370.
83. Yan AT, Gibson CM, Larose E, Anavekar NS, Tsang S, Solomon SD, Reynolds G, Kwong RY: Characterization of microvascular dysfunction after acute myocardial infarction by cardiovascular magnetic resonance first-pass perfusion and late gadolinium enhancement imaging. J Cardiovasc Magn Reson 2006;8:831-837.
84. de Lemos JA, Braunwald E: St segment resolution as a tool for assessing the efficacy of reperfusion therapy. J Am Coll Cardiol 2001;38:1283-1294.
85. Santoro GM, Valenti R, Buonamici P, Bolognese L, Cerisano G, Moschi G, Trapani M, Antoniucci D, Fazzini PF: Relation between st-segment changes and myocardial perfusion evaluated by myocardial contrast echocardiography in patients with acute myocardial infarction treated with direct angioplasty. Am J Cardiol 1998;82:932-937.
86. Cooper HA, de Lemos JA, Morrow DA, Sabatine MS, Murphy SA, McCabe CH, Gibson CM, Antman EM, Braunwald E: Minimal st-segment deviation: A simple, noninvasive method for identifying patients with a patent infarction-related artery after fibrinolytic administration. Am Heart J 2002;144:790-795.
87. Piana RN, Paik GY, Moscucci M, Cohen DJ, Gibson CM, Kugelmass AD, Carrozza JP, Jr., Kuntz RE, Baim DS: Incidence and treatment of 'no-reflow' after percutaneous coronary intervention. Circulation 1994;89:2514-2518.
88. Gregorini L, Marco J, Kozakova M, Palombo C, Anguissola GB, Marco I, Bernies M, Cassagneau B, Distante A, Bossi IM, Fajadet J, Heusch G: Alpha-adrenergic blockade improves recovery of myocardial perfusion and function after coronary stenting in patients with acute myocardial infarction. Circulation 1999;99:482-490.
89. Zhao J, Bolton EM, Bradley JA, Lever AM: Lentiviral-mediated overexpressionof bcl-xl protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis. J Heart Lung Transplant 2009;28:936-943.
90. Otani H: The role of nitric oxide in myocardial repair and remodeling. Antioxid Redox Signal 2009;11:1913-1928.
91. Inamura Y, Miyamae M, Sugioka S, Domae N, Kotani J: Sevoflurane postconditioning prevents activation of caspase 3 and 9 through antiapoptotic signaling after myocardial ischemia-reperfusion. J Anesth 2010;24:215-224.
92. Zhang T, Yang D, Fan Y, Xie P, Li H: Epigallocatechin-3-gallate enhances ischemia/reperfusion-induced apoptosis in human umbilical vein endothelial cells via akt and mapk pathways. Apoptosis 2009;14:1245- 1254.
93. Weyrich AS, Buerke M, Albertine KH, Lefer AM: Time course of coronary vascular endothelial adhesion molecule expression during reperfusion of the ischemic feline myocardium. J Leukoc Biol 1995;57:45-55.
94. Gao F, Yue TL, Shi DW, Christopher TA, Lopez BL, Ohlstein EH, Barone FC, Ma XL: P38 mapk inhibition reduces myocardial reperfusion injury via inhibition of endothelial adhesion molecule expression and blockade of pmn accumulation. Cardiovasc Res 2002;53:414-422.
95. Yang D, Guo S, Zhang T, Li H: Hypothermia attenuates ischemia/reperfusion-induced endothelial cell apoptosis via alterations in apoptotic pathways and jnk signaling. FEBS Lett 2009;583:2500-2506.
96. Hochhauser E, Pappo O, Ribakovsky E, Ravid A, Kurtzwald E, Cheporko Y, Lelchuk S, Ben-Ari Z: Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model. Apoptosis 2008;13:77-86.
97. Lee SR, Lo EH: Interactions between p38 mitogen-activated protein kinase and caspase-3 in cerebral endothelial cell death after hypoxia- reoxygenation. Stroke 2003;34:2704-2709.
98. Zhang CL, Song F, Zhang J, Song QH: Hypoxia-induced bcl-2 expression in endothelial cells via p38 mapk pathway. Biochem Biophys Res Commun 2010; 394:976-980.
99. Montaigne D, Marechal X, Lancel S, Decoster B, Asseman P, Neviere R: The synthetic pentasaccharide fondaparinux prevents coronary microvascular injury and myocardial dysfunction in the ischemic heart. Thromb Haemost 2008;100:912-919.
100. Atmaca A, Dogan S, Dagdelen S, Kabakci G, Kes S, Nazli N, Erbas T: Management and in-hospital outcome of patients with first episode of acute myocardial infarction: Impact of diabetes mellitus. J Natl Med Assoc 2006;98:1752-1757.
101. Segal MS, Shah R, Afzal A, Perrault CM, Chang K, Schuler A, Beem E, Shaw LC, Li Calzi S, Harrison JK, Tran-Son-Tay R, Grant MB: Nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes. Diabetes 2006;55:102-109.
102. Caballero S, Sengupta N, Afzal A, Chang KH, Li Calzi S, Guberski DL, Kern TS, Grant MB: Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells. Diabetes 2007;56:960-967.
103. Tsuruta R, Fujita M, Ono T, Koda Y, Koga Y, Yamamoto T, Nanba M, Shitara M, Kasaoka S, Maruyama I, Yuasa M, Maekawa T: Hyperglycemia enhances excessive superoxide anion radical generation, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats. Brain Res 2010; 1309:155-163.
104. Schneider R, Welt K, Aust W, Loster H, Fitzl G: Cardiac ischemia and reperfusion in spontaneously diabetic rats with and without application of egb 761: Ii. Interstitium and microvasculature. Histol Histopathol 2009;24:587-598.
105. Wang J, Ma H, Tong C, Zhang H, Lawlis GB, Li Y, Zang M, Ren J, Nijland MJ, Ford SP, Nathanielsz PW, Li J: Overnutrition and maternal obesity in sheep pregnancy alter the jnk-irs-1 signaling cascades and cardiac function in the fetal heart. Faseb J. 2010 [Epub ahead of print]
106. Shang L, Ananthakrishnan R, Li Q, Quadri N, Abdillahi M, Zhu Z, Qu W, Rosario R, Toure F, Yan SF, Schmidt AM, Ramasamy R: Rage modulates hypoxia/reoxygenation injury in adult murine cardiomyocytes via jnk andgsk-3beta signaling pathways. PLoS One 2010; 5:e10092.
107. Tanno M, Tsuchida A, Nozawa Y, Matsumoto T, Hasegawa T, Miura T, Shimamoto K: Roles of tyrosine kinase and protein kinase c in infarct size limitation by repetitive ischemic preconditioning in the rat. J Cardiovasc Pharmacol 2000;35:345-352.
108. Talpur N, Echard B, Ingram C, Bagchi D, Preuss H: Effects of a novel formulation of essential oils on glucose-insulin metabolism in diabetic and hypertensive rats: A pilot study. Diabetes Obes Metab 2005;7:193-199.
109. Reffelmann T, Hale SL, Dow JS, Kloner RA: No-reflow phenomenon persists long-term after ischemia/reperfusion in the rat and predicts infarct expansion. Circulation 2003;108:2911-2917.
110. Reffelmann T, Hale SL, Li G, Kloner RA: Relationship between no reflow and infarct size as influenced by the duration of ischemia and reperfusion. Am J Physiol Heart Circ Physiol 2002;282:H766-772.
111. Scarabelli T, Stephanou A, Rayment N, Pasini E, Comini L, Curello S, Ferrari R, Knight R, Latchman D: Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury. Circulation 2001;104:253-256.
112. Scarabelli TM, Stephanou A, Pasini E, Comini L, Raddino R, Knight RA, Latchman DS: Different signaling pathways induce apoptosis in endothelial cells and cardiac myocytes during ischemia/reperfusion injury. Circ Res 2002;90:745-748.
113. Capes SE, Hunt D, Malmberg K, Gerstein HC: Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: A systematic overview. Lancet 2000;355:773-778.
114. Liu X, Wei J, Peng DH, Layne MD, Yet SF: Absence of heme oxygenase-1 exacerbates myocardial ischemia/reperfusion injury in diabetic mice. Diabetes 2005;54:778-784.
115. Marionneau C, Aimond F, Brunet S, Niwa N, Finck B, Kelly DP, Nerbonne JM: Pparalpha-mediated remodeling of repolarizing voltage-gated k+ (kv) channels in a mouse model of metabolic cardiomyopathy. J Mol Cell Cardiol2008;44:1002-1015.
116. Park JH, Okayama N, Gute D, Krsmanovic A, Battarbee H, Alexander JS: Hypoxia/aglycemia increases endothelial permeability: Role of second messengers and cytoskeleton. Am J Physiol 1999;277:C1066-1074.
117. Yuan SY, Breslin JW, Perrin R, Gaudreault N, Guo M, Kargozaran H, Wu MH: Microvascular permeability in diabetes and insulin resistance. Microcirculation 2007;14:363-373.
118. Gao X, Xu X, Belmadani S, Park Y, Tang Z, Feldman AM, Chilian WM, Zhang C: Tnf-alpha contributes to endothelial dysfunction by upregulating arginase in ischemia/reperfusion injury. Arterioscler Thromb Vasc Biol 2007;27:1269-1275.
119. Jaffe R, Charron T, Puley G, Dick A, Strauss BH: Microvascular obstruction and the no-reflow phenomenon after percutaneous coronary intervention. Circulation 2008;117:3152-3156.
120. Zhang C, Xu X, Potter BJ, Wang W, Kuo L, Michael L, Bagby GJ, Chilian WM: Tnf-alpha contributes to endothelial dysfunction in ischemia /reperfusion injury. Arterioscler Thromb Vasc Biol 2006;26:475- 480.
121. Johnson EK, Schelling ME, Quitadamo IJ, Andrew S, Johnson EC: Cultivation and characterization of coronary microvascular endothelial cells: A novel porcine model using micropigs. Microvasc Res 2002;64:278-288.
122. Nishida M, Carley WW, Gerritsen ME, Ellingsen O, Kelly RA, Smith TW: Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am J Physiol 1993;264:H639-652.
123. Grafe M, Auch-Schwelk W, Hertel H, Terbeek D, Steinheider G, Loebe M, Fleck E: Human cardiac microvascular and macrovascular endothelial cells respond differently to oxidatively modified ldl. Atherosclerosis 1998;137: 87-95.
124. Mendoza FJ, Henson ES, Gibson SB: Mekk1-induced apoptosis is mediated by smac/diablo release from the mitochondria. Biochem Biophys Res Commun 2005;331:1089-1098.
125. Huwiler A, Xin C, Brust AK, Briner VA, Pfeilschifter J: Differential binding of ceramide to mekk1 in glomerular endothelial and mesangial cells. BiochimBiophys Acta 2004;1636:159-168.