川芎、当归、红花和人参萃取液对低切应力环境下血管内皮细胞凋亡的影响

目的：探讨川芎、当归、红花和人参萃取液对动脉粥样硬化的预防作用及其机制。方法：采用^3H-胸腺嘧啶掺入率分析和DNA片段凝胶电泳测定，研究川芎、当归、红花和人参萃取液抑制体外培养的血管内皮细胞在低切应力环境下的增殖并阻止其凋亡的作用。结果：(1)处理6h后，^3H-胸腺嘧啶掺入率随剂量增加而逐渐下降，0．5％浓度时^3H-胸腺嘧啶掺入率下降已非常明显，达40％，12h后趋于稳定。(2)在低切应力条件下，内皮细胞出现明显的DNA梯形带，在高切应力环境时，内皮细胞凋亡被抑制；给予0．5％的萃取液处理2h后DNA梯形带消失。结论：川芎、当归、红花和人参萃取液可阻止低切应力环境下内皮细胞的凋亡。     

切应力作用下联合培养的血管平滑肌细胞对内皮细胞PDGF-B mRNA表达的影响

目的：探讨切应力作用下联合培养的血管平滑肌细胞（VSMCs）对内皮细胞（ECs)PDGF-BmRNA表达的影响，为预防血管移植物发生再狭窄提供实验资料。方法：用原位杂交和图像分析等技术，以静态条件下单独培养的ECs和联合培养的ECs为两对照组，观察切应力作用下单独培养的ECs和与VSMCs联合培养ECs的PDGF－BmRNA表达变化。结果：静态条件下联合培养ECs和PDGF－BmRNA表达水平比单独培养的ECs下降；切应力作用下，联合培养ECs的PDGF－BmRNA的表达在切应力作用1h左右有瞬时上升，6h后 下降至低于联合培养条件下的静态水平，且瞬时上升的时间点比单独培养的ECs提前。结论：切应力作用下，与VSMCs联合培养ECs的PDGF－BmRNA表达水平下降，这可能有利于抑制VSMCs的增生。     

切应力通过Pim1/eNOS途径调节人脐静脉内皮细胞NO分泌

目的:探讨层流切应力是否可通过Pim1调节内皮型一氧化氮合酶(eNOS)活性,从而调节血管内皮细胞一氧化氮(NO)分泌。方法:体外原代培养人脐静脉内皮细胞(HUVECs),运用平行平板流动腔系统给HUVECs加载层流切应力(15 dyn/cm^2)。采用Western blot法检测Pim1蛋白表达及eNOS-Ser1177磷酸化水平;硝酸还原酶法检测NO分泌量;利用特异性小干扰RNA(siRNA)转染技术沉默Pim1基因后再检测上述指标的变化。结果:切应力作用HUVECs 15 min,可以显著上调Pim1蛋白表达(P<0.05),同时显著增强eNOS-Ser1177磷酸化水平(P<0.05),伴随HUVECs NO分泌显著增多(P<0.05)。转染siPim1可以抑制切应力诱导的Pim1表达(P<0.05),同时抑制eNOS-Ser1177磷酸化(P<0.05),NO分泌随之显著降低(P<0.05)。结论:流体切应力可能通过Pim1/eNOS途径调节血管内皮细胞NO分泌。     

ERK1/2信号转导途径在低切应力上调人脐静脉内皮细胞IL-8基因表达中的作用

血管内皮细胞位于血流和血管壁之间,除受化学因素的调节外还受力学因素的影响。切应力可通过刺激相应的力学感受系统来调节内皮细胞某些基因的表达,其中包括诱导内皮细胞表达IL- 8。为了阐明MAPK信号途径中的ERK1/ 2信号通路是否参与调控低切应力上调人脐静脉内皮细胞IL- 8基因表达,采用Western blot分析了低切应力(4.2 0 dyne/ cm2 )处理不同时间内皮细胞ERK1/ 2的磷酸化水平及TPK抑制剂Genistein,MEK抑制剂PD980 5 9对其磷酸化的影响;采用定量RT- PCR检测内皮细胞经低切应力刺激或给予阻断剂后再行切应力刺激等处理后IL- 8基因的表达。结果显示:(1)低切应力处理可引起内皮细胞ERK1/ 2蛋白磷酸化水平上调,其磷酸化水平与切应力作用时间有关,具有快速、双向性的特点(在刺激10 min时达到高峰,2 h左右降至未刺激水平) ,阻断剂Genistein和PD980 5 9处理后,ERK1/ 2磷酸化水平与低切应力刺激10 min比较明显降低;(2 )阻断剂Genistein,PD980 5 9可显著抑制低切应力所致的内皮细胞IL- 8m RNA上调。结果表明低切应力可通过ERK1/ 2信号途径上调人脐静脉内皮细胞IL- 8基因的表达。     

脂多糖对人脐静脉内皮细胞ET-1、eNOS、iNOS mRNA表达的影响

目的：观察脂多糖（LPS）对人脐静脉内皮细胞ET-1、eNOS、iNOSmRNA表达的影响，在分子水平上进一步探讨LPS影响人脐静脉内皮细胞分泌ET-1、NO的机制。方法：选用体外培养的第3代人脐静脉内皮细胞，以100 μg/L浓度的LPS与之共孵育6 h。提取总RNA，运用半定量RT-RCR的方法，对ET-1、eNOS、iNOS mRNA的表达情况进行分析。结果：ET-1的灰度比值分别为：正常对照组0.82，LPS组1.32；eNOS的灰度比值分别为：正常对照组1.20，LPS组0.65；iNOS组的灰度比值分别为：正常对照组0.20，LPS组0.19。结论：低浓度的LPS对人脐静脉内皮细胞ET-1 mRNA的表达有促进作用，对eNOS mRNA的表达有抑制作用，对iNOS mRNA的表达则无显著作用。     

切应力作用下联合培养的血管平滑肌细胞对内皮细胞F-肌动蛋白构筑的影响

目的　探讨切应力作用下联合培养的血管平滑肌细胞对内皮细胞抗应力和粘附能力的影响 ,为改进血管内皮细胞种植的组织工程学技术提供生物力学基础。　方法　应用荧光标记和激光共聚焦扫描显微镜技术 ,以静态条件下单独培养的内皮细胞、联合培养的内皮细胞以及切应力作用下单独培养的内皮细胞为对照组 ,研究了切应力作用下与血管平滑肌细胞联合培养的内皮细胞的细胞骨架F 肌动蛋白构筑的变化。　结果　静态条件下单独培养的内皮细胞的F 肌动蛋白排列松散 ,不规则 ,微丝较细 ;联合培养的内皮细胞的F 肌动蛋白微丝明显增多增粗。切应力作用下 ,与血管平滑肌细胞联合培养的内皮细胞的F 肌动蛋白发生重排 ,并形成大量沿切应力方向排列的应力纤维 ,且发生重排的时间明显早于单独培养的内皮细胞。　结论　在切应力作用和血管平滑肌细胞的影响下 ,内皮细胞F 肌动蛋白构筑的变化有利于增强内皮细胞的抗应力和粘附能力     

流体切应力强度对内皮细胞IL-8基因表达的影响

内皮细胞位于血流与血管之间，内皮细胞调控的机械力相关反应已成为正常血管反应的一部分。切应力在调节内皮细胞功能上具有重要作用。流体切应力可以直接调节内皮细胞基因的表达，其中包括诱导内皮细胞表达IL－8，而且IL－8的表达量与切应力作用时间有关。 为阐明内皮细胞IL－8基因的表达除了与切应力的作用时间有关外还与切应力的强度有关，我们用不同强度的流体切应力（2．23、4．20、6．08、8．19、9．67、12．15、14．40、16．87、19．29dyne/cm^2）处理培养的人脐静脉内皮细胞，然后采用定量RT－PCR的方法检测内皮细胞IL－8 基因的表达情况。结果显示：未用切应力处理的内皮细胞没有IL－8基因的表达，切应力处理内皮细胞后，低切应力（2．23dyne/cm^2）时IL－8mRNA表达量明显增加为高切应力（19．29dyne/cm^2）时IL－8mRNA表达量的约68（作用1小h）或52倍（作用2h）。IL－8mRNA的表达量与内皮细胞所施加的切应务强度呈反变关系，直线回归方程，1h时为y=7.57-0.11x，相关系数r=7.97;2h时为y=7.92-0.10x，相关系数r＝0．96。式中：y为切应力作用下内皮细胞IL－8mRNA的表达量（拷贝数的对数值）；x为施加于内皮细胞的切应力强度（dyna/cm^2)。不同的切应力作用时间（1h,2h）均表现出相同的IL-8mRNA随切应力强度的变化规律。提示流体应力诱导内皮细胞表达IL－8，不仅与切应力的作用时间有关，而且IL－8的表达量与切应力强度有关。流体切应诱导内皮细胞IL－8mRNA的表达急剧增高，可能在炎症机制和动脉粥样硬化的发生、发展过程中具有重要作用。     

流体低切应力对内皮细胞IL—8mRNA表达的影响

为证实内皮细胞IL-8表达不仅受化学因子的调节,而且还受力学因素的影响。我们用低层流切应力（4.2dyne/cm^2）处理培养的人脐静脉内皮细胞后采用RT-PCR法检测内皮细胞IL-8基因表达,并用免疫细胞化学染色法检测内皮细胞内NF-kB激活的影响,结果发现：①未用切应力处理和切应力作用0.5h后内皮细胞IL-8mRNA表达量很少,切应力作用1h后内皮细胞IL-8 mRNA表达增加,2h进一步增加。②未用切应力处理和切应力作用0.5h内皮细胞核内NF-kBp65染色阴性,切应力作用1h呈弱阳性,2h呈阳性。提示低层流切应力可诱导内皮细胞表达增加,IL-8表达量与切应力作用时间有关。流体切应力可诱导内皮细胞内NF-kB的激活,激活程度与作用时间有关。低切应力诱导内皮细胞表达IL-8,可能在急性炎症和动脉粥样硬化发生、发展过程中具有重要作用。     

流体切应力作用时间对内皮细胞IL-8 基因表达的影响

内皮细胞对力学环境变化敏感，流体切应力可以直接调节内皮细胞基因的表达。为阐明内皮细胞白细胞介素-8（IL-8）基因的表达除受化学因子的调节外还受力学因素的影响，本文用流体切应力（2.23、4.20、6.08dyne/cm^2）处理培养的人脐静脉内皮细胞，然后采用定量RT-PCR的方法检测内皮细胞IL-8基因的表达情况。结果显示：未用切应力处理的内皮细胞没有IL-8基因的表达；切应力处理内皮细胞后，1h IL-8mRNA表达增加，2hIL-8mRNA表达量至最高值，3hIL-8mRNA表达量开始下降，4h后IL-8mRNA持续低表达；各实验组（2.23、4.20、6.08dyne/cm^2）均表现出相同的IL-8mRNA随时间的变化规律。提示流体切应力确可诱导内皮细胞表达IL-8，而且IL-8的表达量与切应力作用时间有关，呈双相性变化。流体切应力诱导内皮细胞表达IL-8，可能在急性炎症和动脉粥样硬化的发生、发展过程中具有重要作用。     

切应力对与内皮细胞联合培养的血管平滑肌细胞粘附的影响及其机制

目的　细胞粘附是细胞迁移的关键性起始步骤。研究切应力和内皮细胞(endothelial cells, ECs)对血管平滑肌细胞(vascular smooth muscle cells, VSMCs)粘附的影响及其可能的信号通路，为探讨流体切应力诱导的血管壁细胞迁移行为的机制提供一些实验依据。方法　应用VSMCs和ECs联合培养流动腔系统，对ECs面施加1.5 Pa切应力，12 h；以静止状态下，单独培养的VSMCs以及与ECs联合培养的VSMCs 为对照，应用细胞粘附实验和Western Blot技术，观察切应力对与EC联合培养的VSMCs粘附的影响及蛋白激酶B(PKB/Akt)磷酸化水平表达变化。结果　静态联合培养12 h，VSMCs的粘附能力明显增强，同时磷酸化Akt的表达平行增高。切应力作用下，明显抑制了联合培养的VSMCs粘附，同时磷酸化Akt的表达平行降低。结论　生理大小切应力明显抑制了ECs诱导的VSMCs粘附，其中Akt信号通路起了关键作用。     

Effects of pulsatile shear stress on signaling mechanisms controlling nitric oxide production, endothelial nitric oxide synthase phosphorylation, and expression in ovine fetoplacental artery endothelial cells.

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm2, then were exposed to 10, 15, or 25 dynes/cm2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p < .05). Production of NO by OFPAE cells exposed to pulsatile shear stress was de novo; i.e., inhibited by L-NMMA (N(G)-monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm2 (8-fold) exceeded (p < .05) that seen for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). Acute rises in NO production with shear stress occurred by eNOS activation, whereas prolonged NO rises were via elevations in both eNOS expression and enzyme activation. The authors therefore used Western analysis to investigate the signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by "flow-adapted" OFPAE cells. Increasing shear stress from 3 to 15 dynes/cm2 very rapidly increased eNOS Ser1177, ERK1/2 (extracellular signal-regulated kinase 1 and 2) and Akt, but not p38 MAPK (p38 mitogen-activated protein kinase) phosphorylation by Western analysis. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by PI-3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and LY294002, but not the MEK (MAPK kinase) inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels induced by 15 dynes/cm2 shear stress. Blocking of either signaling pathways or p38 MAPK did not change the shear stress-induced increase in eNOS protein levels. Therefore, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibiting MEK. Prolonged shear stress-stimulated increases in eNOS protein levels were not affected by inhibition of MEK- or PI-3K-mediated pathways. In conclusion, pulsatile shear stress greatly induces NO production by OFPAE cells through the mechanisms of both PI-3K-mediated eNOS activation and elevations in eNOS protein levels; bFGF does not further stimulate eNOS expression under flow condition.     

Blood pressure control in eNOS knock-out mice: comparison with other species under NO blockade

Changes in arterial blood pressure (ABP) lead to changes in vascular shear stress. This mechanical stimulus increases cytosolic Ca2+ in endothelial cells, which in turn activates the endothelial isoform of the nitric oxide synthase. The subsequently formed NO reaches the adjacent vascular smooth muscle cells, where it reduces vascular resistance in order to maintain ABP at its initial level. Thus, NO may play an important role as a physiological blood pressure buffer. Previous data on the importance of eNOS for blood pressure control are reviewed with special emphasis on the fact that endogenous nitric oxide can buffer blood pressure variability (BPV) in dogs, rats and mice. In previous studies where all isoforms of the nitric oxide synthase were blocked pharmacologically, increases in blood pressure and variability were observed. Thus, we set out to clarify which isoform of the nitric oxide synthase is responsible for this BPV controlling effect. Hence, blood pressure control was studied in knock-out mice lacking specifically the gene for endothelial nitric oxide synthase with their respective wild-type controls. One day after surgery, under resting conditions, blood pressure was increased by 47 mmHg (P < 0.05), heart rate was lower (-77 beats min-1, P < 0.05), and BPV doubled (P < 0.05). Based on these results, we conclude that chronic blood pressure levels are influenced by eNOS and that there is a blood pressure buffering effect of endogenous nitric oxide which is mediated by the endothelial isoform of the nitric oxide synthase.     

Elevated glucose attenuates agonist- and flow-stimulated endothelial nitric oxide synthase activity in microvascular retinal endothelial cells.

Impaired vasoactive release of opposing vasodilator and vasoconstrictor mediators due to endothelial dysfunction is integral to the pathogenesis of diabetic retinopathy. The aim of this study was to determine the effect of hyperglycemia on the expression of endothelial nitric oxide synthase (eNOS) and the release of nitric oxide (NO) in bovine microvascular retinal endothelial cells (BRECs) under both static (basal and acetylcholine stimulated) and flow (laminar shear stress [10 dynes/cm2 and pulsatile flow 0.3 to 23 dynes/cm2) conditions using a laminar shear apparatus and an in vitro perfused transcapillary culture system. The activity and expression of eNOS, measured by nitrate levels and immunoblot, respectively, were determined following exposure of BRECs to varying concentrations of glucose and mannitol (0 to 25 mM). Under static conditions the expression of eNOS decreased significantly following exposure to increasing concentrations of glucose when compared to osmotic mannitol controls and was accompanied by a significant dose-dependent decrease in nitrate levels in conditioned medium. The acetylcholine stimulated increase in NO release (2.0 +/- 0.3-fold) was significantly reduced by 55% +/- 5% and 65% +/- 4.5% following exposure to 16 and 25 mM glucose, respectively, when compared to osmotic controls. In parallel studies, glucose significantly inhibited both laminar shear stress and pulsatile flow-induced activity when compared to mannitol. We conclude that hyperglycemia impairs agonist- and flow-dependent release of NO in retinal microvascular endothelial cells and may thus contribute to the vascular endothelial dysfunction and impaired autoregulation of diabetic retinopathy.     

Blood pressure control in eNOS knock‐out mice: comparison with other species under NO blockade

Changes in arterial blood pressure (ABP) lead to changes in vascular shear stress. This mechanical stimulus increases cytosolic Ca²⁺ in endothelial cells, which in turn activates the endothelial isoform of the nitric oxide synthase. The subsequently formed NO reaches the adjacent vascular smooth muscle cells, where it reduces vascular resistance in order to maintain ABP at its initial level. Thus, NO may play an important role as a physiological blood pressure buffer. Previous data on the importance of eNOS for blood pressure control are reviewed with special emphasis on the fact that endogenous nitric oxide can buffer blood pressure variability (BPV) in dogs, rats and mice. In previous studies where all isoforms of the nitric oxide synthase were blocked pharmacologically, increases in blood pressure and variability were observed. Thus, we set out to clarify which isoform of the nitric oxide synthase is responsible for this BPV controlling effect. Hence, blood pressure control was studied in knock‐out mice lacking specifically the gene for endothelial nitric oxide synthase with their respective wild‐type controls. One day after surgery, under resting conditions, blood pressure was increased by 47 mmHg (P < 0.05), heart rate was lower (?77 beats min^?1, P < 0.05), and BPV doubled (P < 0.05). Based on these results, we conclude that chronic blood pressure levels are influenced by eNOS and that there is a blood pressure buffering effect of endogenous nitric oxide which is mediated by the endothelial isoform of the nitric oxide synthase.     

当归对氧化低密度脂蛋白致内皮细胞分泌一氧化氮下降和细胞间粘附分子-I表达升高的影响

目的：探讨当归对氧化低密度脂蛋白（ＯＬＤＬ）致血管内皮细胞（ＶＥＣ）损伤的细胞保护作用。方法：以人脐静脉内皮细胞９ＨＵＶＥＣ）为实验模型,采用细胞培养,免疫组化等技术观察ＯＫＬＤＬ对ＶＥＣ分泌一氧化氮（ＮＯ）及细胞间粘分子－１（ＩＣＡＭ－１）表达的影响并研究当归的保护作用。结果：ＯＬＤＬ使ＥＣ分泌的ＮＯ显著降低,ＩＣＡＭ－１的表达显著升高。当归明显忱一作用,而阿托品能阻断当归的作用结论：当归能拮抗     

Endothelial nitric oxide synthase transgenic models of endothelial dysfunction

Endothelial production of nitric oxide is critical to the regulation of vascular responses, including vascular tone and regional blood flow, leukocyte–endothelial interactions, platelet adhesion and aggregation, and vascular smooth muscle cell proliferation. A relative deficiency in the amount of bioavailable vascular NO results in endothelial dysfunction, with conditions that are conducive to the development of atherosclerosis: thrombosis, inflammation, neointimal proliferation, and vasoconstriction. This review focuses on mouse models of endothelial dysfunction caused by direct genetic modification of the endothelial nitric oxide synthase (eNOS) gene. We first describe the cardiovascular phenotypes of eNOS knockout mice, which are a model of total eNOS gene deficiency and thus the ultimate model of endothelial dysfunction. We then describe S1177A and S1177D eNOS mutant mice as mouse models with altered eNOS phosphorylation and therefore varying degrees of endothelial dysfunction. These include transgenic mice that carry the eNOS S1177A and S1177D transgenes, as well as knockin mice in which the endogenous eNOS gene has been mutated to carry the S1177A and S1177D mutations. Together, eNOS knockout mice and eNOS S1177 mutant mice are useful tools to study the effects of total genetic deficiency of eNOS as well as varying degrees of endothelial dysfunction caused by eNOS S1177 phosphorylation.     

Molecular mechanisms underlying the activation of eNOS

Endothelial cells situated at the interface between blood and the vessel wall play a crucial role in controlling vascular tone and homeostasis, particularly in determining the expression of pro- and anti-atherosclerotic genes. Many of these effects are mediated by changes in the generation and release of the vasodilator nitric oxide (NO) in response to hemodynamic stimuli exerted on the luminal surface of endothelial cells by the streaming blood (shear stress) and the cyclic strain of the vascular wall. The endothelial NO synthase (eNOS) is activated in response to fluid shear stress and numerous agonists via cellular events such as; increased intracellular Ca²⁺, interaction with substrate and co-factors, as well as adaptor and regulatory proteins, protein phosphorylation, and through shuttling between distinct sub-cellular domains. Dysregulation of these processes leads to attenuated eNOS activity and reduced NO output which is a characteristic feature of numerous patho-physiological disorders such as diabetes and atherosclerosis. This review summarizes some of the recent findings relating to the molecular events regulating eNOS activity.     

Fluid shear stress alters the hemostatic properties of endothelial outgrowth cells

Surface endothelialization is an attractive means to improve the performance of small diameter vascular grafts. While endothelial outgrowth cells (EOCs) are considered a promising source of autologous endothelium, the ability of EOCs to modulate coagulation-related blood activities is not well understood. The goal of this study was to assess the role of arterial flow conditions on the thrombogenic phenotype of EOCs. EOCs derived from baboon peripheral blood, as well as mature arterial endothelial cells from baboons, were seeded onto adsorbed collagen, then exposed to physiologic levels of fluid shear stress. For important hemostatic pathways, cellular responses to shear stress were characterized at the gene and protein level and confirmed with a functional assay for activated protein C (APC) activity. For EOCs, fluid shear stress upregulated gene and protein expression of anticoagulant and platelet inhibitory factors, including thrombomodulin, tissue factor pathway inhibitor, and nitric oxide synthase 3 (eNOS). Fluid shear stress significantly altered the functional activity of EOCs by increasing APC levels. This study demonstrates that fluid shear stress is an important determinant of EOC hemostatic properties. Accordingly, manipulation of EOC phenotype by mechanical forces may be important for the development of thrombo-resistant surfaces on engineered vascular implants.