利用吡唑类衍生物研究体外缺血模型下血管内皮细胞的血管生成机制
摘要
研究背景和研究目的
缺血性疾病是动脉供血不足或障碍引起来的一系列疾病,主要包括周围动脉闭塞性疾病(PAOD)、缺血性心脏疾病以及缺血性脑血管疾病等。PAOD又包括动脉硬化闭塞症、血栓闭塞性脉管炎、动脉血栓形成、动脉栓塞等疾病。PAOD作为血管外科的常见病、多发病,具有较高的发病率、致残率和致死率。缺血也常发生在心脏或脑,危险性就更大,可引起患者猝死。对许多缺血性疾病的患者来说,利用腔内修复技术或外科开放手术进行血管的重建,并同时将各种危险因素降到最低,有时并不能完全拯救肢体、脏器并缓解缺血性静息痛。因此,在过去的20年里,以促进缺血部位新生血管形成及侧支重建为目的的治疗性策略及方法得到了长足的发展。换言之,治疗性血管生成对这些缺血性患者具有十分重要的价值。促进血管生成确实能够减轻缺血对组织及器官的损害,促进缺血状态下的血管生成对一些介入或手术难以解决的患者来说具有极其重要的临床意义。许多缺血性疾病如冠状动脉缺血性疾病、脑梗塞及肢体缺血性疾病都可以从治疗性血管生成中获益。但是,异常或过多的血管形成能促进肿瘤的血管形成及肿瘤细胞的转移。因此,对缺血状态下的血管生成调控机制的更深层次的探索对我们的临床实践是十分必要的。如果我们对血管生成的分子机制有了更好的了解,就可以根据人类自身的需要在不同的情况下通过调控这些机制来促进或抑制血管生成。
利用细胞渗透性小分子干扰生物现象进行的研究称之为化学遗传学,其在生物医学的各个领域均发挥了非常重要之作用。有很多研究是利用化学遗传学来寻找新的小分子促血管生成剂；另外,这些小分子化合物也可作为阐明血管生成机制的工具。而且,它们也可以作为治疗性血管形成的潜在药物。有大量的证据显示血管内皮细胞(VEC)与疾病过程中的血管生成密切相关,所以血管内皮细胞是治疗性血管生成非常有吸引力的靶点。血管生成的启动与血管内皮细胞的增殖及迁移密切相关,因此找到能够促进血管内皮细胞迁移并诱导血管生成的小分子化合物是至关重要的。
体外生长的血管内皮细胞在去除血清和生长因子的状态下,将会逐渐脱离培养皿并走向凋亡,这个体外模型常用来模拟体内缺血状态并且已得到了广泛的应用。乙烷基3-(0-氯苯基)-5-甲基-1-苯基-1H-吡唑-羧酸酯(MPD)是吡唑类的衍生物,前期研究表明MPD在20和25 gM时可以抑制这个体外缺血模型下的血管内皮细胞的凋亡,而且在5和10μM时并不影响该模型下血管内皮细胞的生存能力。所以我们进一步研究在体外缺血模型下MPD能否在这四个浓度时促进血管生成。
在体内,一氧化氮(NO)由一氧化氮合成酶(NOS)催化L-精氨酸合成,NOS是一种钙离子依赖性酶并且与血管生成密切相关。NO作为一种血管扩张剂可以促进小动脉的生成(形成肌肉动脉)和血管生成(形成新的血管主要是毛细血管)。NO的生物活性的降低是内皮功能紊乱的一个标记并且与一系列的血管功能紊乱有关如受损的血管生成、痉挛、高血压、动脉粥样硬化及PAOD有关。在体外NO可以促进血管内皮细胞血管生成相关的特性包括迁移、生长和形成管状结构。增强的NO生物活性可增强血管内皮细胞的存活力进而促进缺血介导的血管生成。NO在血管生成中发挥着至关重要的作用。所以,我们利用MPD进一步研究NO在血管生成中的调控作用。
活性氧(ROS)是普遍存在并且广泛分布的活性分子并由氧分子的降解生成。它们通常在吞噬细胞针对抗原的防御机制的呼吸链中产生。最近,研究者发现血管内皮细胞亦可产生ROS,并且它们与内皮的功能紊乱密切相关,如动脉粥样硬化及缺血-再灌注损伤等。ROS参与凋亡并证明是血管生成的重要调节因子。在体外,ROS促进血管生成。在体内,ROS水平的提高与动脉粥样硬化模型主动脉斑块中的新生血管形成及血管内皮生长因子(VEGF)表达密切相关。因此,ROS无论是对体内还是体外血管生成过程中都具有重要的作用。ROS是在受到低氧、缺血及促血管生长因子如VEGF和血管生成素等的刺激而产生的。但是,ROS是否参与MPD调节的血管生成过程尚不明了。
干扰素诱导蛋白-10(CXCL10/IP-10)是CXC趋化因子家族中的重要成员,可由活化的内皮细胞、单核细胞、纤维母细胞及角化细胞等多种细胞生成。IP-10是一种强有力的血管生成抑制剂。IP-10在体外可以抑制内皮细胞增殖,并且IP-10可抑制血管内皮细胞的生长与VEGF的生成呈负相关关系。如果将IP-10作用于在Matrigel基质上培养的内皮细胞,它将以浓度依赖模式抑制内皮细胞向管状结构分化并且减少新生血管网状结构的形成。IP-10在动物模型中也可抑制新生血管生成。总而言之,IP-10不论在体内还是体外均具有明显的抗血管生成作用。另有证据显示,这种趋化因子与心血管疾病及冠脉综合征的发病相关。越来越多的证据显示这种趋化因子在感染性及非感染性疾病中均可导致损伤；并且IP-10水平与疾病的严重性相关。我们的模型是模仿体内缺血状态。然而,IP-10是否与缺血状态相关,并且是否在调节血管生成的过程中发挥作用,目前仍不清楚。
从化学遗传学的角度,利用小分子化合物可以发现参与血管生成的关键因子,因此可为阐明血管生成的分子机制提供实验证据。然而,血管内皮细胞在体外缺血模型下参与血管生成的调控机理还没有得到很好的阐明,仍然有待于进一步研究。本论文旨在利用MPD作为研究工具回答上述科学问题,明确NO,ROS和IP-10在血管内皮细胞介导的血管生成调控过程中的作用及其作用机理,为阐明血管生成的调控机制提供实验证据,为缺血性疾病的治疗性血管生成提供潜在的药物,同时为缺血性疾病的治疗提供新的线索和靶点。
研究内容
1.研究MPD能否在体外缺血模型下(去除血清与生长因子的条件下)抑制VEC凋亡
2.研究MPD能否在体外缺血模型下促进血管生成
3. MPD抑制血管内皮细胞凋亡及促进血管生成的分子机制研究
研究方法
1.血管内皮细胞培养：人脐静脉内皮细胞的提取和培养参考Jaffe et al的方法[Jaffe EA et al,1973]
2.细胞凋亡检测：1)利用倒置相差显微镜观察细胞的形态变化2)MTT方法检测细胞的存活率3)吖啶橙染色结合(激光扫描)共聚焦显微镜观察,检测细胞核凝集及片断化
3.体外血管形成检测：利用Matrigel方法,参考[Kureishi Y et al,2000]
4.体外细胞迁移(划伤)检测：即平面单层细胞损伤愈合实验,并结合倒置相差显微镜观察,参考[Burk,1973; Vasvari et al,2007]
5.NO含量的检测：利用NO检测试剂盒
6.ROS检测：利用荧光探针(DCHF)并结合(激光扫描)共聚焦显微术检测
7.细胞内蛋白分布及表达水平检测：
1)免疫细胞化学法结合(激光扫描)共聚焦显微监测技术,检测Alix蛋白的表达水平及分布
2) Western blot方法,检测Alix, Ets-1和IP-10蛋白的表达水平
研究结果：
1.MPD抑制去除血清和生长因子诱导血管内皮细胞凋亡MPD(20和25μM)处理血管内皮细胞24 h,倒置相差显微镜下,能显著抑制体外缺血模型诱导的HUVEC的凋亡。MPD(5和10μM)处理细胞24 h,相差显微镜下观察,对体外缺血模型诱导的HUVEC的凋亡无影响。倒置相差显微镜下观察,可见凋亡小体明显变少；同样吖啶橙染色显示,MPD可抑制染色质凝集和细胞核片断化。
2.MPD在体外缺血模型下促进血管生成
2.1体外Matrigel毛细血管样结构形成实验显示,去除血清和FGF-2条件下,MPD(5和10μM)在6,12和24 h时能显著促进血管生成即促进VEC在Matrigel基质上形成管状结构(*p<0.05 or** p<0.01)。MPD(20和25μM)在6,12和24 h时不能在Matrigel基质上很好地形成管状结构。
2.2单层细胞损伤愈合实验显示,MPD(5和10μM)在6,12和24 h显著促进细胞迁移(*p<0.05 or**p<0.01),并且在24 h,这个间隙几乎愈合。MPD(20和25μM)在6,12和24 h并不能明显促进细胞迁移。
3.MPD抑制血管内皮细胞凋亡和促进血管生成的分子机制
3.1 MPD(5,10和25μM)处理细胞6,12和24 h,MPD在5和10μM时可以在6和12h时显著升高细胞外液中NO水平(*p<0.05),并且MPD在25μM时可以在6和12h时极其显著升高细胞外液中NO水平(**p<0.01)。而MPD在5,10和25μM时在24 h均不引起细胞外液中NO水平的变化(p>0.05)。
3.2 MPD(5,10和25μM)处理细胞6,12和24 h,与对照组相比,MPD在5和10μM时可以在三个时间点显著升高细胞内ROS水平(p<0.05),而MPD在25pM时可以在三个时间点显著降低细胞内ROS水平(p<0.05)。
3.3 Western Blot检测发现,MPD(5,10和25μM)处理细胞6,12和24 h,MPD在5,10和25μM时在24 h均显著降低细胞内IP-10水平(*p<0.05)。而MPD在5,10和25μM时在6和12 h均不引起细胞内IP-10水平的变化(p<0.05)。
结论：
1.MPD在体外缺血模型下能够抑制血管内皮细胞的凋亡并有效促进血管生成。
2.在体外缺血模型下,MPD在5和10μM时通过上调NO水平、ROS水平和抑制IP-10水平而促进血管内皮细胞迁移和血管生成。MPD在25μM时通过极其显著上调NO水平、抑制ROS水平和IP-10水平而抑制细胞凋亡
4.ROS和NO之间有密切联系且均与细胞凋亡和血管生成相关。MPD很可能通过影响这一条信号转导途径中的相关因子,即控制ROS和NO的水平,在不同浓度抑制了去除血清和生长因子诱导的VEC凋亡并促进了血管形成。
4.MPD能够抑制IP-10的表达,而且IP-10能够抑制血管形成并与疾病的严重程度有关,我们的实验模型是一种体外缺血疾病模型。所以,理论上讲,MPD能够抑制缺血对机体的损害,可作为缺血性疾病的一个治疗的靶点。
5.MPD可作为一种有效的工具更深入的研究细胞分化与凋亡之间的差别、细胞凋亡和血管生成的分子机制,同时可为作为缺血性疾病的治疗潜在的药物,并为缺血性疾病的治疗提供新的线索和靶点。
BACKGROUND AND OBJECTIVE
Ischemic diseases, a series of diseases caused by insufficient artery blood supply mainly include peripheral arterial occlusive disease (PAOD), ischemic heart disease, and ischemic cerebrovascular disease. PAOD includes atherosclerosis obliterans, thromboangitis obliterans, arterial thrombosis and arterial embolism. PAOD is commen in vascular surgery. It is characterized by high disease, disability and death rates. In ischemic diseases, ischemia is more dangerous in the heart and brain. In many patients with ischemic diseases, revascularization by endovascular means or by open surgery, combined with best possible risk factor modification, does not salvage limbs or viscera or relieve ischemic rest pain. As a consequence, novel therapeutic strategies developed over the last two decades have aimed to promote neovascularization and remodelling of collaterals. In other words, therapeutic angiogenesis is important to these patients with ischemia. Indeed, stimulating angiogenesis could help reduce the damage to ischemic tissues. Many ischemic diseases such as ischemic coronary artery disease, critical limb ischemia and brain infarction may benefit from the induction of angiogenesis. However, aberrant or excessive angiogenesis allows for vascularization of solid tumors and provides routes for metatasis of cancer cells. A better understanding of the steps controlling angiogenesis under the ischemic state should further advance our attempts to stimulate or inhibit angiogenesis when warranted.
The use of small cell-permeable molecules to effect biological phenomena, also known as chemical genetics, has made a significant impact in diverse areas of biological medicine. Our recent studies have used chemical biology to discover novel small-molecule angiogenic promoters. These chemical molecules could be used as tools to clarify the mechanism of angiogenesis. Furthermore, they can be used as potential drugs for therapeutic angiogenesis. Vascular endothelial cells (VECs) are an attractive target for therapeutic angiogenesis because they are intimately involved in disease processes associated with angiogenesis. Initiation of angiogenesis involves migration and proliferation of VECs. Finding small molecules that could promote VEC migration and induce angiogenesis is crucial for patients and for researchers.
When deprived of serum and FGF-2, in vitro-grown HUVECs gradually detach from the Petri dish and undergo apoptosis. This in vitro model is often used to mimic the in vivo ischemic state and has been widely used. Our previous study showed that a novel pyrazole derivative, ethyl3-(o-chlorophenyl)-5-methyl-l-phenyl-lH-pyrazole-4-carboxylate (MPD) at 20 and 25μM increased VEC viability and inhibited VEC apoptosis induced by deprivation of serum and fibroblast growth factor-2 (FGF-2). Moreover, MPD at 5 and 10μM did not affect VEC viability or apoptosis induced by deprivation of serum and FGF-2. In this study, we used this model to investigate whether MPD could promote angiogenesis under the ischemic state at these four concentrations.
In vivo, NO is produced by catalysis of 1-arginine by endothelial nitric oxide synthase (NOS), a calcium-dependent enzyme involved in angiogenesis. NO presumably acts as a vasodilator to result in arteriolar genesis (formation of muscular arterioles) and angiogenesis (formation of new blood vessels, mainly capillaries). Diminished bioavailability of NO is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis, vasoconstriction, hypertension, atherosclerosis, and PAOD. In vitro, NO promotes most angiogenesis-related properties of endothelial cells, including migration and growth, as well as formation of capillary-like structures. Increased NO bioavailability can enhance endothelial cell survival, thus facilitating ischemia-mediated angiogenesis. NO plays a central role in angiogenesis. Therefore, we use MPD to investigate the roles of NO in angiogenesis.
ROS are ubiquitous, highly diffusible and reactive molecules produced by the reduction of molecular oxygen. They are normally produced during the respiratory burst of phagocytes as a defense mechanism against pathogens. More recently, vascular cells have been found to produce ROS, and these molecules are implicated in endothelial dysfunction associated with atherosclerosis and ischemia-reperfusion injury. ROS participates in the apoptosis of VECs and have been suggested as important mediators for angiogenesis. ROS stimulates angiogenesis in vitro. In vivo, elevated oxidative stress is directly associated with neovascularization and vascular endothelial growth factor (VEGF) expression in aortic plaque of models of atherosclerosis. ROS play an important role in angiogenesis both in vitro and in vivo. They are produced in response to hypoxia, ischemia, and angiogenic growth factors such as VEGF and angiopoietin-1, thereby stimulating VEC proliferation and migration. However, whether MPD modulates angiogenesis through ROS is unknown.
Interferon-inducible protein 10 (CXCL10/IP-10) is a CXC chemokine produced by certain types of cells, including activated endothelial cells, monocytes, fibroblasts, and keratinocytes. IP-10 is a potent inhibitor of angiogenesis. IP-10 inhibits proliferation of endothelial cells in vitro. IP-10 inhibits endothelial cell growth and is inversely correlated with VEGF production. IP-10, added to HUVECs, cultured on a Matrigel substrate, inhibits their differentiation into tube-like structures in a dose-dependent fashion and reduces the extent of the neo-vascular network; IP-10 neovascularization in animal models. In summary, IP-10 plays an import role in angiogenesis both in vitro and in vivo. It has been proposed that this chemokine is also involved in the pathogenesis of cardiovascular diseases and coronary syndromes. There is growing evidence implicating this chemokine in both infectious and noninfectious causes of injury. The level of IP-10 increased as disease severity increased. Our model is used to mimic the in vivo ischemic state. However, whether IP-10 is associated with ischemic state and modulates angiogenesis is not clear.
The utilization of chemical genetics may discover novel key factors involved in angiogenesis. Therefore, it can provide experimental evidences to illustrate issues mentioned above. However, the molecular mechanisms of VEC angiogensis remain unclear. They need further study. Based on the backgrounds mentioned above, the objective of this study was as follows:To investigate the roles of NO, ROS and IP-10 in VEC angiogenesis by using MPD, and the molecular mechanisms mediated by these three elements. The researches would provide a theoretical basis for studying the molecular mechanisms of VEC angiogenesis. Our study provides potential drug for therapeutic angiogenesis of ischemic diseases. Moreover, our data would provide provided new clues and targets for ischemic diseases therapy in clinic.
STUDY CONTENTS
1. Study of whether MPD could inhibit VEC apoptosis in vitro ischemic model (HUVECs cultured without FGF-2 and serum).
2. Study of whether MPD could promote angiogenesis in vitro ischemic model
3. Study of the molecular mechanisms of inhibiting VEC apoptosis and promoting angiogensis by MPD
METHODS:
1. Vascular endothelial cell culture:HUVECs were obtained as described before by [Jaffe et al.,1973].
2. Cell apoptosis analysis:
1) observation of cell morphological changes by phase contrast microscope
2) cell viability was determined by MTT-assay
3) analysis of nuclear fragmentation and chromatin condensation by the acridine orange staining combined with (laser scan) confocol microscope
3. Angiogenesis assay in vitro:capillary-like tube formation on Matrigel as described previously [Kureishi Y et al,2000]
4. Cell migration assay in vitro:monolayer cell wound healing assay as described previously [Burk,1973; Vasvari et al,2007]
5. NO production assay:NO detection kit
6. Analysis ROS level by the fluorescence probe (DCHF) combined with laser scan confocol microscope.
7. Analysis of expression and distribution of proteins:
1) examination of the changes in Alix protein level and distribution by immunocytochemistry combined with laser scan confocol microscope
2)Analysis the levels of Alix, Ets-1 and IP-10 by Western blot assay.
RESULTS:
1. MPD inhibited VEC apoptosis induced by deprivation of serum and FGF-2. Morphological changes of VECs were observed with phasecontrast microscopy. MPD at 20 and 25μM could inhibit VEC apoptosis induced by deprivation of serum and FGF-2 at 24 h. On the contrast, MPD at 5 and 10μM did not affect VEC viability and apoptosis induced by deprivation of serum and FGF-2 at 24 h., The number of apoptotic bodies decreased obviously with treatment of MPD (25μM) under phase microscope. AO staining showed that MPD (25μM) inhibited nuclear fragmentation and chromatin condensation.
2. MPD promoted angiogenesis in vitroischemic model.
2.1 To demonstrate the angiogenic-inducing function of MPD in HUVECs, we assayed capillary-like tube formation on Matrigel. Capillary-like tubes developed well in HUVECs treated with MPD (5 and 10μM) on Matrigel-coated 24-well plates with basal M199 medium at 6,12 and 24 h. HUVECs treated with MPD at 5 and 10μM could form tubes (* p<0.05 or** p<0.01). However, HUVECs treated with MPD at 20 and 25μM could not form tubes well.
2.2 We performed monolayer cell-wound healing assay to determine whether MPD affects HUVEC migration. MPD at 5 and 10μM promoted cell migration(*p<0.05 or** p<0.01), with the gap nearly closed at 24 h. MPD at 20 and 25μM could not promote cell migration obviously.
3. The molecular mechanisms of inhibiting VEC apoptosis and promoting angiogensis by MPD
3.1 We determined the level of NO in MPD-induced angiogenesis after HUVECs were treated with MPD for 6,12 and 24 h. NO production was moderately elevated in HUVECs treated with 5 and 10μM (* p<0.05) MPD and extremely elevated with 25μM MPD (** p<0.01) at 6 and 12 h. MPD at 5,10 and 25μM did not affect the level of NO at 24 h(p>0.05).
3.2 We detected the levels of intracellular ROS in HUVECs treated for 6,12 and 24 h. The level of ROS was increased in cells treated with 5 and 10μM MPD (* p <0.05) but was decreased in cells treated with 25μM MPD (* p<0.05) as compared with controls.
3.3 We investigated the effects of MPD on IP-10 in VECs. When VECs were treated with 5,10 and 25μM MPD for 24 h, the level of IP-10 was much lower than that of the control group (p<0.05); MPD at 5,10 and 25μM did not affect the level of IP-10 at 6 and 12 h compared to the control group (p>0.05)
CONCLUSIONS:
1. MPD could both inhibit VEC apoptosis and promote VEC angiogenesis in vitro ischemic model.
2. In vitro ischemic model, MPD at 5 and 10μM promoted VEC migration and angiogenesis by upregulating NO and ROS levels and depressing IP-10 level. MPD at 25μM inhibited VEC apoptosis by greatly upregulating NO level and depressing ROS and IP-10 levels.
3. ROS had close relationship with NO and both of them were associated with apoptosis and angiogenesis. MPD at different concentrations probably effected related factors involved in this signal transduction pathway, that is, controlling ROS and NO levels, to inhibit VEC apoptosis induced by deprived of serum and FGF-2 and promote angiogenesis.
4. MPD could inhibit the expression of IP-10. The level of IP-10 increased as disease severity increased. Our model is used to mimic the in vivo ischemic state. Therefore, our data indicated that MPD could reduce damage in ischemic state by depressing the level of IP-10. IP-10 may become a new target for future clinical treatment of ischemic diseases.
5. MPD is a good tool for investigating the difference of cell differentiation and apoptosis and for investigating the mechanisms of VEC apoptosis and angiogenesis, and MPD might be useful in the development of new drugs in therapy of ischemic diseases. Moreover, it also provides new clues and target for clinical treatment of ischemic diseases.
缺血性疾病是动脉供血不足或障碍引起来的一系列疾病,主要包括周围动脉闭塞性疾病(PAOD)、缺血性心脏疾病以及缺血性脑血管疾病等。PAOD又包括动脉硬化闭塞症、血栓闭塞性脉管炎、动脉血栓形成、动脉栓塞等疾病。PAOD作为血管外科的常见病、多发病,具有较高的发病率、致残率和致死率。缺血也常发生在心脏或脑,危险性就更大,可引起患者猝死。对许多缺血性疾病的患者来说,利用腔内修复技术或外科开放手术进行血管的重建,并同时将各种危险因素降到最低,有时并不能完全拯救肢体、脏器并缓解缺血性静息痛。因此,在过去的20年里,以促进缺血部位新生血管形成及侧支重建为目的的治疗性策略及方法得到了长足的发展。换言之,治疗性血管生成对这些缺血性患者具有十分重要的价值。促进血管生成确实能够减轻缺血对组织及器官的损害,促进缺血状态下的血管生成对一些介入或手术难以解决的患者来说具有极其重要的临床意义。许多缺血性疾病如冠状动脉缺血性疾病、脑梗塞及肢体缺血性疾病都可以从治疗性血管生成中获益。但是,异常或过多的血管形成能促进肿瘤的血管形成及肿瘤细胞的转移。因此,对缺血状态下的血管生成调控机制的更深层次的探索对我们的临床实践是十分必要的。如果我们对血管生成的分子机制有了更好的了解,就可以根据人类自身的需要在不同的情况下通过调控这些机制来促进或抑制血管生成。
利用细胞渗透性小分子干扰生物现象进行的研究称之为化学遗传学,其在生物医学的各个领域均发挥了非常重要之作用。有很多研究是利用化学遗传学来寻找新的小分子促血管生成剂；另外,这些小分子化合物也可作为阐明血管生成机制的工具。而且,它们也可以作为治疗性血管形成的潜在药物。有大量的证据显示血管内皮细胞(VEC)与疾病过程中的血管生成密切相关,所以血管内皮细胞是治疗性血管生成非常有吸引力的靶点。血管生成的启动与血管内皮细胞的增殖及迁移密切相关,因此找到能够促进血管内皮细胞迁移并诱导血管生成的小分子化合物是至关重要的。
体外生长的血管内皮细胞在去除血清和生长因子的状态下,将会逐渐脱离培养皿并走向凋亡,这个体外模型常用来模拟体内缺血状态并且已得到了广泛的应用。乙烷基3-(0-氯苯基)-5-甲基-1-苯基-1H-吡唑-羧酸酯(MPD)是吡唑类的衍生物,前期研究表明MPD在20和25 gM时可以抑制这个体外缺血模型下的血管内皮细胞的凋亡,而且在5和10μM时并不影响该模型下血管内皮细胞的生存能力。所以我们进一步研究在体外缺血模型下MPD能否在这四个浓度时促进血管生成。
在体内,一氧化氮(NO)由一氧化氮合成酶(NOS)催化L-精氨酸合成,NOS是一种钙离子依赖性酶并且与血管生成密切相关。NO作为一种血管扩张剂可以促进小动脉的生成(形成肌肉动脉)和血管生成(形成新的血管主要是毛细血管)。NO的生物活性的降低是内皮功能紊乱的一个标记并且与一系列的血管功能紊乱有关如受损的血管生成、痉挛、高血压、动脉粥样硬化及PAOD有关。在体外NO可以促进血管内皮细胞血管生成相关的特性包括迁移、生长和形成管状结构。增强的NO生物活性可增强血管内皮细胞的存活力进而促进缺血介导的血管生成。NO在血管生成中发挥着至关重要的作用。所以,我们利用MPD进一步研究NO在血管生成中的调控作用。
活性氧(ROS)是普遍存在并且广泛分布的活性分子并由氧分子的降解生成。它们通常在吞噬细胞针对抗原的防御机制的呼吸链中产生。最近,研究者发现血管内皮细胞亦可产生ROS,并且它们与内皮的功能紊乱密切相关,如动脉粥样硬化及缺血-再灌注损伤等。ROS参与凋亡并证明是血管生成的重要调节因子。在体外,ROS促进血管生成。在体内,ROS水平的提高与动脉粥样硬化模型主动脉斑块中的新生血管形成及血管内皮生长因子(VEGF)表达密切相关。因此,ROS无论是对体内还是体外血管生成过程中都具有重要的作用。ROS是在受到低氧、缺血及促血管生长因子如VEGF和血管生成素等的刺激而产生的。但是,ROS是否参与MPD调节的血管生成过程尚不明了。
干扰素诱导蛋白-10(CXCL10/IP-10)是CXC趋化因子家族中的重要成员,可由活化的内皮细胞、单核细胞、纤维母细胞及角化细胞等多种细胞生成。IP-10是一种强有力的血管生成抑制剂。IP-10在体外可以抑制内皮细胞增殖,并且IP-10可抑制血管内皮细胞的生长与VEGF的生成呈负相关关系。如果将IP-10作用于在Matrigel基质上培养的内皮细胞,它将以浓度依赖模式抑制内皮细胞向管状结构分化并且减少新生血管网状结构的形成。IP-10在动物模型中也可抑制新生血管生成。总而言之,IP-10不论在体内还是体外均具有明显的抗血管生成作用。另有证据显示,这种趋化因子与心血管疾病及冠脉综合征的发病相关。越来越多的证据显示这种趋化因子在感染性及非感染性疾病中均可导致损伤；并且IP-10水平与疾病的严重性相关。我们的模型是模仿体内缺血状态。然而,IP-10是否与缺血状态相关,并且是否在调节血管生成的过程中发挥作用,目前仍不清楚。
从化学遗传学的角度,利用小分子化合物可以发现参与血管生成的关键因子,因此可为阐明血管生成的分子机制提供实验证据。然而,血管内皮细胞在体外缺血模型下参与血管生成的调控机理还没有得到很好的阐明,仍然有待于进一步研究。本论文旨在利用MPD作为研究工具回答上述科学问题,明确NO,ROS和IP-10在血管内皮细胞介导的血管生成调控过程中的作用及其作用机理,为阐明血管生成的调控机制提供实验证据,为缺血性疾病的治疗性血管生成提供潜在的药物,同时为缺血性疾病的治疗提供新的线索和靶点。
研究内容
1.研究MPD能否在体外缺血模型下(去除血清与生长因子的条件下)抑制VEC凋亡
2.研究MPD能否在体外缺血模型下促进血管生成
3. MPD抑制血管内皮细胞凋亡及促进血管生成的分子机制研究
研究方法
1.血管内皮细胞培养：人脐静脉内皮细胞的提取和培养参考Jaffe et al的方法[Jaffe EA et al,1973]
2.细胞凋亡检测：1)利用倒置相差显微镜观察细胞的形态变化2)MTT方法检测细胞的存活率3)吖啶橙染色结合(激光扫描)共聚焦显微镜观察,检测细胞核凝集及片断化
3.体外血管形成检测：利用Matrigel方法,参考[Kureishi Y et al,2000]
4.体外细胞迁移(划伤)检测：即平面单层细胞损伤愈合实验,并结合倒置相差显微镜观察,参考[Burk,1973; Vasvari et al,2007]
5.NO含量的检测：利用NO检测试剂盒
6.ROS检测：利用荧光探针(DCHF)并结合(激光扫描)共聚焦显微术检测
7.细胞内蛋白分布及表达水平检测：
1)免疫细胞化学法结合(激光扫描)共聚焦显微监测技术,检测Alix蛋白的表达水平及分布
2) Western blot方法,检测Alix, Ets-1和IP-10蛋白的表达水平
研究结果：
1.MPD抑制去除血清和生长因子诱导血管内皮细胞凋亡MPD(20和25μM)处理血管内皮细胞24 h,倒置相差显微镜下,能显著抑制体外缺血模型诱导的HUVEC的凋亡。MPD(5和10μM)处理细胞24 h,相差显微镜下观察,对体外缺血模型诱导的HUVEC的凋亡无影响。倒置相差显微镜下观察,可见凋亡小体明显变少；同样吖啶橙染色显示,MPD可抑制染色质凝集和细胞核片断化。
2.MPD在体外缺血模型下促进血管生成
2.1体外Matrigel毛细血管样结构形成实验显示,去除血清和FGF-2条件下,MPD(5和10μM)在6,12和24 h时能显著促进血管生成即促进VEC在Matrigel基质上形成管状结构(*p<0.05 or** p<0.01)。MPD(20和25μM)在6,12和24 h时不能在Matrigel基质上很好地形成管状结构。
2.2单层细胞损伤愈合实验显示,MPD(5和10μM)在6,12和24 h显著促进细胞迁移(*p<0.05 or**p<0.01),并且在24 h,这个间隙几乎愈合。MPD(20和25μM)在6,12和24 h并不能明显促进细胞迁移。
3.MPD抑制血管内皮细胞凋亡和促进血管生成的分子机制
3.1 MPD(5,10和25μM)处理细胞6,12和24 h,MPD在5和10μM时可以在6和12h时显著升高细胞外液中NO水平(*p<0.05),并且MPD在25μM时可以在6和12h时极其显著升高细胞外液中NO水平(**p<0.01)。而MPD在5,10和25μM时在24 h均不引起细胞外液中NO水平的变化(p>0.05)。
3.2 MPD(5,10和25μM)处理细胞6,12和24 h,与对照组相比,MPD在5和10μM时可以在三个时间点显著升高细胞内ROS水平(p<0.05),而MPD在25pM时可以在三个时间点显著降低细胞内ROS水平(p<0.05)。
3.3 Western Blot检测发现,MPD(5,10和25μM)处理细胞6,12和24 h,MPD在5,10和25μM时在24 h均显著降低细胞内IP-10水平(*p<0.05)。而MPD在5,10和25μM时在6和12 h均不引起细胞内IP-10水平的变化(p<0.05)。
结论：
1.MPD在体外缺血模型下能够抑制血管内皮细胞的凋亡并有效促进血管生成。
2.在体外缺血模型下,MPD在5和10μM时通过上调NO水平、ROS水平和抑制IP-10水平而促进血管内皮细胞迁移和血管生成。MPD在25μM时通过极其显著上调NO水平、抑制ROS水平和IP-10水平而抑制细胞凋亡
4.ROS和NO之间有密切联系且均与细胞凋亡和血管生成相关。MPD很可能通过影响这一条信号转导途径中的相关因子,即控制ROS和NO的水平,在不同浓度抑制了去除血清和生长因子诱导的VEC凋亡并促进了血管形成。
4.MPD能够抑制IP-10的表达,而且IP-10能够抑制血管形成并与疾病的严重程度有关,我们的实验模型是一种体外缺血疾病模型。所以,理论上讲,MPD能够抑制缺血对机体的损害,可作为缺血性疾病的一个治疗的靶点。
5.MPD可作为一种有效的工具更深入的研究细胞分化与凋亡之间的差别、细胞凋亡和血管生成的分子机制,同时可为作为缺血性疾病的治疗潜在的药物,并为缺血性疾病的治疗提供新的线索和靶点。
BACKGROUND AND OBJECTIVE
Ischemic diseases, a series of diseases caused by insufficient artery blood supply mainly include peripheral arterial occlusive disease (PAOD), ischemic heart disease, and ischemic cerebrovascular disease. PAOD includes atherosclerosis obliterans, thromboangitis obliterans, arterial thrombosis and arterial embolism. PAOD is commen in vascular surgery. It is characterized by high disease, disability and death rates. In ischemic diseases, ischemia is more dangerous in the heart and brain. In many patients with ischemic diseases, revascularization by endovascular means or by open surgery, combined with best possible risk factor modification, does not salvage limbs or viscera or relieve ischemic rest pain. As a consequence, novel therapeutic strategies developed over the last two decades have aimed to promote neovascularization and remodelling of collaterals. In other words, therapeutic angiogenesis is important to these patients with ischemia. Indeed, stimulating angiogenesis could help reduce the damage to ischemic tissues. Many ischemic diseases such as ischemic coronary artery disease, critical limb ischemia and brain infarction may benefit from the induction of angiogenesis. However, aberrant or excessive angiogenesis allows for vascularization of solid tumors and provides routes for metatasis of cancer cells. A better understanding of the steps controlling angiogenesis under the ischemic state should further advance our attempts to stimulate or inhibit angiogenesis when warranted.
The use of small cell-permeable molecules to effect biological phenomena, also known as chemical genetics, has made a significant impact in diverse areas of biological medicine. Our recent studies have used chemical biology to discover novel small-molecule angiogenic promoters. These chemical molecules could be used as tools to clarify the mechanism of angiogenesis. Furthermore, they can be used as potential drugs for therapeutic angiogenesis. Vascular endothelial cells (VECs) are an attractive target for therapeutic angiogenesis because they are intimately involved in disease processes associated with angiogenesis. Initiation of angiogenesis involves migration and proliferation of VECs. Finding small molecules that could promote VEC migration and induce angiogenesis is crucial for patients and for researchers.
When deprived of serum and FGF-2, in vitro-grown HUVECs gradually detach from the Petri dish and undergo apoptosis. This in vitro model is often used to mimic the in vivo ischemic state and has been widely used. Our previous study showed that a novel pyrazole derivative, ethyl3-(o-chlorophenyl)-5-methyl-l-phenyl-lH-pyrazole-4-carboxylate (MPD) at 20 and 25μM increased VEC viability and inhibited VEC apoptosis induced by deprivation of serum and fibroblast growth factor-2 (FGF-2). Moreover, MPD at 5 and 10μM did not affect VEC viability or apoptosis induced by deprivation of serum and FGF-2. In this study, we used this model to investigate whether MPD could promote angiogenesis under the ischemic state at these four concentrations.
In vivo, NO is produced by catalysis of 1-arginine by endothelial nitric oxide synthase (NOS), a calcium-dependent enzyme involved in angiogenesis. NO presumably acts as a vasodilator to result in arteriolar genesis (formation of muscular arterioles) and angiogenesis (formation of new blood vessels, mainly capillaries). Diminished bioavailability of NO is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis, vasoconstriction, hypertension, atherosclerosis, and PAOD. In vitro, NO promotes most angiogenesis-related properties of endothelial cells, including migration and growth, as well as formation of capillary-like structures. Increased NO bioavailability can enhance endothelial cell survival, thus facilitating ischemia-mediated angiogenesis. NO plays a central role in angiogenesis. Therefore, we use MPD to investigate the roles of NO in angiogenesis.
ROS are ubiquitous, highly diffusible and reactive molecules produced by the reduction of molecular oxygen. They are normally produced during the respiratory burst of phagocytes as a defense mechanism against pathogens. More recently, vascular cells have been found to produce ROS, and these molecules are implicated in endothelial dysfunction associated with atherosclerosis and ischemia-reperfusion injury. ROS participates in the apoptosis of VECs and have been suggested as important mediators for angiogenesis. ROS stimulates angiogenesis in vitro. In vivo, elevated oxidative stress is directly associated with neovascularization and vascular endothelial growth factor (VEGF) expression in aortic plaque of models of atherosclerosis. ROS play an important role in angiogenesis both in vitro and in vivo. They are produced in response to hypoxia, ischemia, and angiogenic growth factors such as VEGF and angiopoietin-1, thereby stimulating VEC proliferation and migration. However, whether MPD modulates angiogenesis through ROS is unknown.
Interferon-inducible protein 10 (CXCL10/IP-10) is a CXC chemokine produced by certain types of cells, including activated endothelial cells, monocytes, fibroblasts, and keratinocytes. IP-10 is a potent inhibitor of angiogenesis. IP-10 inhibits proliferation of endothelial cells in vitro. IP-10 inhibits endothelial cell growth and is inversely correlated with VEGF production. IP-10, added to HUVECs, cultured on a Matrigel substrate, inhibits their differentiation into tube-like structures in a dose-dependent fashion and reduces the extent of the neo-vascular network; IP-10 neovascularization in animal models. In summary, IP-10 plays an import role in angiogenesis both in vitro and in vivo. It has been proposed that this chemokine is also involved in the pathogenesis of cardiovascular diseases and coronary syndromes. There is growing evidence implicating this chemokine in both infectious and noninfectious causes of injury. The level of IP-10 increased as disease severity increased. Our model is used to mimic the in vivo ischemic state. However, whether IP-10 is associated with ischemic state and modulates angiogenesis is not clear.
The utilization of chemical genetics may discover novel key factors involved in angiogenesis. Therefore, it can provide experimental evidences to illustrate issues mentioned above. However, the molecular mechanisms of VEC angiogensis remain unclear. They need further study. Based on the backgrounds mentioned above, the objective of this study was as follows:To investigate the roles of NO, ROS and IP-10 in VEC angiogenesis by using MPD, and the molecular mechanisms mediated by these three elements. The researches would provide a theoretical basis for studying the molecular mechanisms of VEC angiogenesis. Our study provides potential drug for therapeutic angiogenesis of ischemic diseases. Moreover, our data would provide provided new clues and targets for ischemic diseases therapy in clinic.
STUDY CONTENTS
1. Study of whether MPD could inhibit VEC apoptosis in vitro ischemic model (HUVECs cultured without FGF-2 and serum).
2. Study of whether MPD could promote angiogenesis in vitro ischemic model
3. Study of the molecular mechanisms of inhibiting VEC apoptosis and promoting angiogensis by MPD
METHODS:
1. Vascular endothelial cell culture:HUVECs were obtained as described before by [Jaffe et al.,1973].
2. Cell apoptosis analysis:
1) observation of cell morphological changes by phase contrast microscope
2) cell viability was determined by MTT-assay
3) analysis of nuclear fragmentation and chromatin condensation by the acridine orange staining combined with (laser scan) confocol microscope
3. Angiogenesis assay in vitro:capillary-like tube formation on Matrigel as described previously [Kureishi Y et al,2000]
4. Cell migration assay in vitro:monolayer cell wound healing assay as described previously [Burk,1973; Vasvari et al,2007]
5. NO production assay:NO detection kit
6. Analysis ROS level by the fluorescence probe (DCHF) combined with laser scan confocol microscope.
7. Analysis of expression and distribution of proteins:
1) examination of the changes in Alix protein level and distribution by immunocytochemistry combined with laser scan confocol microscope
2)Analysis the levels of Alix, Ets-1 and IP-10 by Western blot assay.
RESULTS:
1. MPD inhibited VEC apoptosis induced by deprivation of serum and FGF-2. Morphological changes of VECs were observed with phasecontrast microscopy. MPD at 20 and 25μM could inhibit VEC apoptosis induced by deprivation of serum and FGF-2 at 24 h. On the contrast, MPD at 5 and 10μM did not affect VEC viability and apoptosis induced by deprivation of serum and FGF-2 at 24 h., The number of apoptotic bodies decreased obviously with treatment of MPD (25μM) under phase microscope. AO staining showed that MPD (25μM) inhibited nuclear fragmentation and chromatin condensation.
2. MPD promoted angiogenesis in vitroischemic model.
2.1 To demonstrate the angiogenic-inducing function of MPD in HUVECs, we assayed capillary-like tube formation on Matrigel. Capillary-like tubes developed well in HUVECs treated with MPD (5 and 10μM) on Matrigel-coated 24-well plates with basal M199 medium at 6,12 and 24 h. HUVECs treated with MPD at 5 and 10μM could form tubes (* p<0.05 or** p<0.01). However, HUVECs treated with MPD at 20 and 25μM could not form tubes well.
2.2 We performed monolayer cell-wound healing assay to determine whether MPD affects HUVEC migration. MPD at 5 and 10μM promoted cell migration(*p<0.05 or** p<0.01), with the gap nearly closed at 24 h. MPD at 20 and 25μM could not promote cell migration obviously.
3. The molecular mechanisms of inhibiting VEC apoptosis and promoting angiogensis by MPD
3.1 We determined the level of NO in MPD-induced angiogenesis after HUVECs were treated with MPD for 6,12 and 24 h. NO production was moderately elevated in HUVECs treated with 5 and 10μM (* p<0.05) MPD and extremely elevated with 25μM MPD (** p<0.01) at 6 and 12 h. MPD at 5,10 and 25μM did not affect the level of NO at 24 h(p>0.05).
3.2 We detected the levels of intracellular ROS in HUVECs treated for 6,12 and 24 h. The level of ROS was increased in cells treated with 5 and 10μM MPD (* p <0.05) but was decreased in cells treated with 25μM MPD (* p<0.05) as compared with controls.
3.3 We investigated the effects of MPD on IP-10 in VECs. When VECs were treated with 5,10 and 25μM MPD for 24 h, the level of IP-10 was much lower than that of the control group (p<0.05); MPD at 5,10 and 25μM did not affect the level of IP-10 at 6 and 12 h compared to the control group (p>0.05)
CONCLUSIONS:
1. MPD could both inhibit VEC apoptosis and promote VEC angiogenesis in vitro ischemic model.
2. In vitro ischemic model, MPD at 5 and 10μM promoted VEC migration and angiogenesis by upregulating NO and ROS levels and depressing IP-10 level. MPD at 25μM inhibited VEC apoptosis by greatly upregulating NO level and depressing ROS and IP-10 levels.
3. ROS had close relationship with NO and both of them were associated with apoptosis and angiogenesis. MPD at different concentrations probably effected related factors involved in this signal transduction pathway, that is, controlling ROS and NO levels, to inhibit VEC apoptosis induced by deprived of serum and FGF-2 and promote angiogenesis.
4. MPD could inhibit the expression of IP-10. The level of IP-10 increased as disease severity increased. Our model is used to mimic the in vivo ischemic state. Therefore, our data indicated that MPD could reduce damage in ischemic state by depressing the level of IP-10. IP-10 may become a new target for future clinical treatment of ischemic diseases.
5. MPD is a good tool for investigating the difference of cell differentiation and apoptosis and for investigating the mechanisms of VEC apoptosis and angiogenesis, and MPD might be useful in the development of new drugs in therapy of ischemic diseases. Moreover, it also provides new clues and target for clinical treatment of ischemic diseases.
引文
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