αv-整合素超声分子成像可视化评价基质胶血管新生
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摘要
背景和目的：
血管新生(angiogenesis)是指在机体生长发育过程中或创伤修复、缺血缺氧和炎症等情况下,原有微血管内皮细胞(endothelial cell, EC)经过生芽、迁移、增殖与基质重塑等形成新毛细血管的过程。在组织再生、发育和创伤修复过程中,血管形成是一个基本的生理过程,如各种创伤、溃疡、心肌梗塞、慢性感染等愈合过程都需要血管形成的参与,但是在许多病理状态下也存在血管生成失调,例如糖尿病视网膜病、动脉硬化和实体肿瘤等严重危害人类健康的疾病中。因此,高敏感性、无创性、靶向性、早期定量评价血管新生对于心血管疾病(主要是缺血性心脏病和外周动脉闭塞性疾病)和肿瘤性疾病都具有重要的意义。对于心血管疾病来说,通过显示新生血管数量和空间分布来评估微血管对生长因子的早期反应,对指导向缺血局部组织输送促血管合成蛋白或基因是十分有用的。对于肿瘤性疾病来说,及早显影新生血管则能早期发现肿瘤及微小转移灶,有利于病人的早期治疗；而动态性的监测新生血管则可以评估抗肿瘤治疗的效果。
超声分子成像是近年来兴起的一门无创性分子影像技术,它是通过对普通超声微泡表面进行特殊处理,将靶向于病变组织特定分子的特异配体连接于普通超声微泡外壳构建成靶向超声微泡,后者经静脉注入后靶向性黏附、聚集并较长时间滞留于靶组织中,再通过对比超声(contrast-enhanced ultrasound, CEU)检查而产生分子水平的特异的“主动性靶向超声分子显像”(active targeted ultrasound molecular imaging)的一门新兴的超声成像技术。近几年来,运用靶向超声分子成像技术评价血管新生应已日益成为国内外超声领域的研究热点,并已经实现了从分子水平对肿瘤性和缺血性血管新生的初步评价。Ellegala等构建携Echistatin的靶向微泡可以有效评价大鼠恶性神经胶质瘤模型的新生血管。Leong-Poi等通构建了携带抗αv-整合素单抗的靶向超声微泡,体外实验发现该靶向微泡与基质胶新生血管的结合率明显高于正常血管内皮细胞。
基质胶是一种来源于EHS小鼠肿瘤细胞提取的基底膜的复合物。它在4℃的时候为液态,但在室温条件下,可自动聚集产生类似于哺乳动物细胞基底膜的生物活性基质材料,它具有良好的可操作性和可重复性,是公认的血管新生模型,已被广泛应用于血管新生的实验研究。将基质胶注入皮下后,内皮细胞向基质胶中迁移形成新生血管,血管形成过程大约在10天左右完成,当加入适当浓度的促血管生成因子后,可以加快血管的生成。
碱性成纤维细胞生长因子(fibroblast growth factor-2, FGF-2)是体内发现的最为有效的血管形成因子之一。它对新生血管形成过程中多个环节如毛细血管基底膜降解、内皮细胞迁移增生、胶原合成、小血管腔形成均有明显促进作用,并且可以下调基质金属蛋白酶的活性。已证实FGF-2在体内、外均有明显的促新生血管形成作用。FGF-2可促进具有基底膜作用的蛋白激酶释放,从而促进毛细血管基底膜降解,促进小血管的形成。FGF-2能促进αv-整合素的表达,而αv-整合素是良好的血管新生成像靶点。理论上,不同浓度的FGF-2将诱导不同程度的αv-整合素表达。因此,本研究将不同浓度的FGF-2分别加入到基质胶中构建不同程度的血管新生模型。
Leong-Poi等使用αv-整合素靶向超声微泡对基质胶模型进行对比超声研究,靶向超声微泡的声强度高于普通微泡(P<0.05),但未设立封闭组探讨靶向超声微泡评价血管新生的特异性。Stieger等用非靶向微泡评价基质胶模型的血管新生,免疫组化的微血管密度和超声的增强区域正相关(r=0.65,P<0.05)。虽然,超声分子成像评价血管新生已经取得了初步成功,但目前仍没有对靶向超声分子成像评价血管新生的特异性和定量化进行研究的报道。因此,我们通过分别向基质胶中加入不同量的FGF-2,建立基质胶血管新生模型,并通过单抗封闭新生血管αv-整合素分子靶点的方法建立阴性对照模型,旨在探讨携带αv-整合素分子探针的超声分子成像评价不同浓度碱性成纤维细胞生长因子(FGF-2)介导的血管新生,并对血管新生的定量化评价进行初步的研究。
材料和方法
1、超声微泡的制备：各种脂质材料按一定比例75℃水浴溶解于适量蒸馏水中,同时通全氟丙烷(C3F8)气体,超声振荡至形成乳白色液体制备生物素(Biotin)化脂质微泡。生物素化脂质微泡经静置弃下清液并加入等量蒸馏水去除未结合脂质纯化4次后,按一定比例加入抗生蛋白链菌素／亲和素(streptavidin)。继续静置弃下清液并加入等量蒸馏水去除未结合抗生蛋白链菌素纯化微泡2次后得到外壳结合有抗生蛋白链菌素的脂质微泡,再按一定比例加入生物素化鼠抗人αv-整合素单抗(Biotin conjugated mouse Anti-humun avMonoclone Antibody)得到携带抗αv-整合素单抗靶向微泡(MBα),最后静置弃下清液并加入等量蒸馏水去除未结合抗体,纯化携带抗αv-整合素单抗靶向微泡(MBα)2次。用上述方法构建携同型抗体微泡作为对照微泡(MBc)。MBα和MBc均于冰箱中4℃保存备用。采用库尔特计数仪测量MBα及MBc的平均粒径及浓度。
2、MBα的体外鉴定：采用绿色荧光标记的二抗与抗αv-整合素单抗结合,荧光显微镜下观察抗αv-整合素单抗与微泡的连接情况。采用平行板流动腔技术在体外模拟的生理血流条件下评价MBα与αv-整合素Fc段(Recombinant humanαv-integrin/Fc Chimera,αvSFc)的靶向黏附效能。
3、基质胶模型的制备：在昆明小鼠皮下注射基质胶以促进血管新生,分别向基质胶中添加不同量的FGF-2 (sigma公司),配成浓度为1μg/mL和0.5μg/mL的两种基质胶,并加入肝素(64U/mL)。将20只小鼠随机分为2组,分别注射两种浓度的基质胶。20只昆明小鼠经常规麻醉后,在左侧腹部皮下注射0.6ml基质胶,在基质胶注射部位形成椭圆形隆起。
4、CEU检查：在注射基质胶10天后,将小鼠麻醉后进行对比超声检查。将自制水囊放在小鼠的左侧腹部,用自制支架固定超声探头(17L5)于水囊上。调整探头位置获得良好图像后保持探头位置在整个实验过程中不变,仪器的各项参数在整个实验过程中保持不变。CEU采用经尾静脉微泡弹丸式注射法进行,超声造影采用经尾静脉随机(间隔30min)先后注射MBα和MBC(约为5×106个)。经过6min循环时间待血池中循环微泡消失后对实验小鼠进行注射基质胶的部位进行CEU检查,获取显影图像。CEU检查采用二次谐波成像(second harmonic imaging)技术进行,探头发射和接收频率分别为7 MHz和14MHz,机械指数(Mechanical Index, MI)为0.18,超声发射间隔(Pulsing Interval, PI)时间设定为10s,获取第一帧CEU图像后给予高MI(1.9)连续超声发射2-3s以破坏微泡,继续存储本底图像4-5帧,全部声学造影图像存于CD盘,以备脱机分析。采用MCE软件分析CEU图像,测量实验小鼠注射基质胶的部位显影的声强度(video intensity, VI)数值并制作彩色编码图。采用红色、橙色和白色分别代表基质胶显影程度由强到弱。
5、病理学检查和免疫组化检查：完成CEU图像采集后,取出实验小鼠基质胶,10%甲醛固定,行病理学和免疫荧光检查。
6、平均荧光强度(median fluorescence:intensity, MFI)测定和血管计数(micro vessel count, MVC):在荧光照片选取相同面积的50个区域,使用Imagepro plus6.0软件分析平均荧光强度。
①得到灰度图片；
②黑白反相；
③光密度校正；
④在count/size中选择强度阂值10-200,过滤掉明亮的杂质荧光点；
⑤选择面积area与积分光密度(IOD)过滤值30像素；
⑥IOD/area得到该区域像素的平均灰度,即代表平均荧光强度,然后计算这50个区域的平均值。
单位面积血管计数：在上述50个区域,以有管腔结构为准,进行血管计数,计算单位面积血管计数。
结果
1、微泡制备情况：采用库尔特计数仪测得MBc粒径为2.12±1.13μm,浓度约为2.80～4.56×108个／ml；MBα粒径2.08±0.65μm,浓度约为3.07～4.75×108个／ml。
2、MBα体外鉴定结果：采用绿色荧光标记的二抗与抗αv-整合素单抗结合,荧光显微镜下观察显示MBa外壳显明显绿色荧光,表明抗αv-整合素单抗与微泡外壳连接效果良好。平行板流动腔体外评价结果显示,在模拟微血管生理血流条件的剪切应力(<1.0dyn／cm2)下MBα可与包被于平行板流动腔的αvSFc有效结合,显示了很好的主动性靶向黏附效能。
3、超声造影检查：MBα注射后基质胶外周带及周围组织均可见明显的超声显影,而基质胶中心部位未见超声显影,6min后周围组织未见显影,而基质胶外周带仍可见明显的超声显影,MBc注射后显影情况与MBa基本相同,但6min后基质胶未见明显的超声显影。预先用αv-整合素抗体封闭后,MBα及MBc注射后基质胶也可呈明显的超声显影,但6min后MBα和MBc均未见明显的超声显影。
4、图像分析结果：在高浓度组中,封闭前基质胶模型中MBa组呈明显的超声显影,而MBc组仅见轻度的超声显影,两者之间有明显差异(P<0.05)。预先单抗封闭αv-整合素后,MBa和封闭前的MBα之间有明显差异(P<0.05)；而MBc的Ⅵ值较封闭前无明显变化,两者之间无明显差异(P>0.05)。低剂量组同样出现上述结果。
5、基质胶病理及免疫荧光结果：HE染色切片显示,小鼠基质胶外周带可见丰富的新生血管,血管中可见红细胞的存在；高浓度FGF-2组基质胶中新生血管多于低浓度FGF-2组；高浓度组血管密度高,而且更靠近中央区。免疫荧光显示基质胶新生血管内皮细胞有大量的αv-整合素,高浓度FGF-2组基质胶内皮细胞中表达的αv-整合素多于低浓度FGF-2组,封闭后内皮细胞中几乎不表达αv-整合素。
6.在超声造影中,高浓度组封闭前MBα造影Ⅵ值是低浓度组封闭前的1.4倍；高浓度组封闭后MBα造影Ⅵ值是低浓度组封闭前的1.37倍。在免疫荧光强度测定中,高浓度组封闭前单位面积免疫荧光强度是低浓度组封闭前的1.32倍；高浓度组封闭后是低浓度封闭后的1.33倍。在单位面积血管计数中,高浓度组封闭前是封闭后的1.2倍；高浓度组封闭前MBα造影Ⅵ值是低浓度组封闭前的1.16倍。
结论：
1、采用“抗生物素蛋白／生物素复合体”可实现抗αv整合素单抗与脂质微泡外壳的有效连接而成功构建携带抗αv-整合素单抗靶向微泡；
2、应用携带αv-整合素分子探针的超声分子成像可有效特异地评价基质胶血管新生,实现对新生血管的早期评价和诊断；
3、αv-整合素可以作为新生血管分子成像的靶点,使用基质胶模型评价血管新生的研究具有良好的特异性,可用于定量化研究。
Background and Objective:
Angiogenesis is the formation of new capillaries from existent micro vessels by sprouting, migration, proliferation of endothelial cells and matrix remodeling when body is in the process of growth or experiencing wound healing, ischemic hypoxia and inflammation. As a basic physiological procedure, angiogenesis participates in the healing of all kinds of wounds, ulcers, myocardial infarction and chronic infection et al. But there are angiogenic disorders in many pathological states such as diabetic retinopathy, atherosclerosis, and solid tumors and other serious diseases which are hazardous for human beings. Therefore, high sensitive, non-invasive, targeted, quantitative evaluation of angiogenesis has the vital significance to early cardiovascular disease (mainly ischemic heart disease and peripheral arterial occlusive disease) and neoplastic diseases. The number and spatial distribution of new blood vessels can assess the early response of microvasculation reacted to growth factors to guide the delivery pro-angiogenic proteins or genes for cardiovascular disease. The early angiogenesis is able to develop early detection of tumor and micrometastasis conducive to early treatment of patients, and dynamic monitoring of new blood vessels can assess the effect of anti-cancer therapy.
Ultrasonnd molecular imaging is a new online technique of noninvasive molecular imaging technologies. By virtue of connecting special ligands of specific molecules in pathologic tissue to the surface of ultrasound microbubbles, targeted ultrasound microbubbles can be constructed. Via intravenous injection, the targeted ultrasound microbubbles can gather at the target tissue and remain there for a long time, so active targeted ultrasound molecular imaging at molecular lever can be produced by contrast enhanced ultrasound. With extensive perspective and great clinical significance, evaluation of angiogenesis with targeted ultrasound molecular imaging technology has increasingly become a frontline focus in ultrasound research for the past few years. Ellegala built targeted microbubbles with echistatin to effectively evaluate neovascularization of the rat model of malignant glioma. And Leong-Poi constructed microbubbles targeted to av-integrin (MBα) and proofed that the adhesive capacity of the targeted microbubbles in neovascularization was higher than in normal vascular endothelial cells.
Matrigel is a soluble basement membrane matrix extracted from ESH mouse. At room temperature, it can automatically gather and become bioactive materials similar to basement membranes of mammalian cell. Matrigel model has good operability and repeatability which has been widely applied to the experimental study as an acknowledged angiogenesis model. After subcutaneous injection of the Matrigel, endothelial cells migrate to Matrigel to form new blood vessels. Blood vessel formation takes about 10 days to complete and adding appropriate concentration of pro-angiogenic factors can speed up the angiogenesis.
Basic fibroblast growth factor (FGF-2) is one of the most effective pro-angiogenic factors in vivo. It can up-regulate capillary basement membrane degradation, endothelial cell migration and proliferation, collagen synthesis and down-regulation the activity of matrix metalloproteinases. It has proved that FGF-2 plays an important role in vivo as well as in vitro to stimulate newborn blood vessels. FGF-2 can enhance the release of protein kinases which can degrade the basement membrane, thereby promoting angiogenesis, and the formation of small blood vessels.
Leong-Poi et al performed contrast ultrasound study on Matrigel model with MBa and video intensity of MBa is high that of control microbubble(P<0.05). But the specificity of angiogenesis wasn't evaluated because of lack of closed group. Stieger found microvessel density measurements displayed a significant correlation with power Doppler enhanced area (r=0.65, P<0.05) with non-targeted microbubble. Although ultrasound molecular imaging of angiogenesis has achieved initial success, there is no research report about specificity and quantification of angiogenesis. Therefore, we established Matrigel model by adding different levels of FGF-2 and control model by blocking a-integrin in new blood vessels in order to investigate the assessment of angiogenesis mediated by different levels of FGF-2 with ultrasound molecular imaging using microbubbles targeted to endothelialαv-integrins and quantitative evaluation of angiogenesis preliminarily.
Methods:
1. Microbubbles preparation:lipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas (C3H8) with aqueous dispersion of several lipids in determinate ratio. After being washed (4×) to remove excess free unincorporated lipid, streptavidin in determinate ratio were added to the lipid microbubbles with biotin, then washed (2×) to removed excess free unincorporated streptavidin and the biotin conjugated mouse anti-integrin av monoclone antibodies or biotin conjugated isotype control mAb in determinate ratio were added respectively to complete the preparation of MBa and MBc. At last, the MBa and MBc were washed (2×) to remove excess free unincorporated antibodies. Both MBαand MBc were storaged in refrigerator at 4℃. The mean diameter and density in both MBa and MBa were measured by coulter counter.
2. Evaluation of MBa in vitro:Using green fluorescent-labeled antibody for antiαv-integrin monoclonal antibody to identify the linking antibodies on the MBa. To evaluate the combinding efficacy between MBa andαvSFc (Recombinant human av-integrin/Fc Chimera) with a parallel plate flow chamber at a shearing force under physiologic flow conditions.
3. Animal preparation:Matrigel angiogenesis was created by subcutaneous injection of matrigel in Kunming mice. Two different levels of FGF-2 was adding to Matrigel to form matrigel at the FGF-2 concentrations of 1μg/mL and 0.5μg/mL.20 mice were randomly divided into 2 groups injected with two concentrations of Matrigel. Mice were routinely anesthetized and 0.6 ml of enriched Matrigel (4℃) was injected subcutaneously in the left ventral region. Matrigel hardened to form discrete ellipsoid plugs.
4. CEU imaging:CEU of matrigel plugs was performed in 10 anesthetized mice 10 days after matrigel injection. All experimental mice were performed with CEU respectively by using MBa and MBc, the intravenous injection of 4×106 microbubbles were made in random order with 30 minutes interval. After five minutes of intravenous injection, microbubbles in the circulation were eliminated, the ultrasound signal (video intensity, VI) from MBa and MBc were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of 0.18, transmission frequency of 7.0 MHz and receiving frequency of 14.0 MHz. After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background- subtracted VI of tumor was measured. Analysis of VI was performed by MCE software and color-coding for visual analysis was performed.
5. Examination of pathology and immunofluorescence:After CEU imaging, all Matrigel plugs of experimental mice were harvested for the examination of pathology and immunofluorescence.
6. Determination of median fluorescence:intensity and micro vessel count:50 regions with the same area were selected in the fluorescence photograph and median fluorescence:intensity was measured by imagepro software6.0 in these fifty regions.
①Grayscale images acquisition;
②Color reverse;
③Optical density correction;
④Selecting intensity threshold 10-200 in count/size to filter out impurities.
⑤set filter value to30 pixels;
⑥IOD/area representing the median fluorescence intensity and then calculating the average of the 50 regions.
Micro vessels count:Average of the 50 regions was calculated subject to a lumen structure.
Results:
1. Results for microbubble preparation:The density of MBαand MBc is about 3.07～4.75×108/ml and 2.80～4.56×108/ml, the mean sizes for MBa and MBc were about 2.08±0.65μm and 2.12±1.13μm respectively.
2. Results for evaluation of MBa in vitro:The mouse anti-mouse av-integrin monoclonal antibodies linked well to the surface of microbubble, which was observed with fluorescence microscopy. MBa andαvSFc enjoyed a good targeted combination with a shearing force (<1.0dyn/cm2) under physiologic flow conditions, which was observed in the parallel plate flow chamber.
3. Results for CEU imaging:Significant ultrasound imaging was observed in the peripheral zone and the surrounding tissue of the Matrigel with MBa. While there was no CEU imaging in the center of the Matrigel. Six min later no significant CEU imaging was observed in the surrounding tissue, while the peripheral zone was still clearly visible. The CEU imaging with MBc was the same as that with MBa except no obvious CEU imaging was observed in the Matrigel. After blocking with antibody against av-integrin, significant CEU imaging could be observed in the Matrigel with MBa and MBc, but six min later there was no significant CEU imaging.
4. Results for CEU-derived IV:In high lever group, VI of the matrigel was significantly higher for MBa as compared with MBc(P<0.05). After blocking with antibody against av-integrin, a great decrease was observed in the MBa group (P<0.05) while no significant difference was noted for MBc (P>0.05). The trend in low dosage group was similar to that in high lever group.
5. Results for examination of pathology and immunofluorescence:HE showed abundant new vessels in which there were red blood cells in the peripheral zone of the matrigel plugs with a large amount of white blood cells (mononuclear cells) infiltration. Neovessels in matrigel was positive for av-integrin which was observed with fluorescence microscopy. There were more newborn blood vessels in high lever group than that in low lever group and high micro vessel density which is more closed to the central zone could be seen in high lever group. Immunofluorescence showed abundant av-integrin expressed within matrigel in which the amount of av-integrin in high lever group is much than that in low lever group. After blocking with antibody against av-integrin, there was hardlyαv-integrin in endothelial cells.
6. VI of the matrigel was 1.4 times for MBa in high level compared with that in low level. After blocked with antibody againstαv-integrin, VI was 1.37 times for MBa in high level compared with that in low level. The median fluorescence intensity in high level was 0.32 times higher than that in low level. After blocked with antibody, the median fluorescence intensity in high level was 0.32 times higher than that in low level. Micro vessels count per unit was 1.2 times as much as that in low level. After blocked with antibody, micro vessels count per unit was 1.16 times as much as that in low level.
Conclusions:
1. Microbubbles targeted toαv-integrin can be successfully constructed by combining anti av-integrin monoclonal antibodies to lipid microbubbles via "avidin-biotin" bridging chemistry.
2. Ultrasound molecular imaging with microbubbles targeted to av-integrin can be effective and specific in evaluating the angiogenesis in a murine model of subcutaneous matrigel plugs.
3.αv-integrin can be an ideal target for molecular imaging of angiogenesis and assessment of angiogenesis in matrigel has good specificity and a great application prospect in quantify study.
血管新生(angiogenesis)是指在机体生长发育过程中或创伤修复、缺血缺氧和炎症等情况下,原有微血管内皮细胞(endothelial cell, EC)经过生芽、迁移、增殖与基质重塑等形成新毛细血管的过程。在组织再生、发育和创伤修复过程中,血管形成是一个基本的生理过程,如各种创伤、溃疡、心肌梗塞、慢性感染等愈合过程都需要血管形成的参与,但是在许多病理状态下也存在血管生成失调,例如糖尿病视网膜病、动脉硬化和实体肿瘤等严重危害人类健康的疾病中。因此,高敏感性、无创性、靶向性、早期定量评价血管新生对于心血管疾病(主要是缺血性心脏病和外周动脉闭塞性疾病)和肿瘤性疾病都具有重要的意义。对于心血管疾病来说,通过显示新生血管数量和空间分布来评估微血管对生长因子的早期反应,对指导向缺血局部组织输送促血管合成蛋白或基因是十分有用的。对于肿瘤性疾病来说,及早显影新生血管则能早期发现肿瘤及微小转移灶,有利于病人的早期治疗；而动态性的监测新生血管则可以评估抗肿瘤治疗的效果。
超声分子成像是近年来兴起的一门无创性分子影像技术,它是通过对普通超声微泡表面进行特殊处理,将靶向于病变组织特定分子的特异配体连接于普通超声微泡外壳构建成靶向超声微泡,后者经静脉注入后靶向性黏附、聚集并较长时间滞留于靶组织中,再通过对比超声(contrast-enhanced ultrasound, CEU)检查而产生分子水平的特异的“主动性靶向超声分子显像”(active targeted ultrasound molecular imaging)的一门新兴的超声成像技术。近几年来,运用靶向超声分子成像技术评价血管新生应已日益成为国内外超声领域的研究热点,并已经实现了从分子水平对肿瘤性和缺血性血管新生的初步评价。Ellegala等构建携Echistatin的靶向微泡可以有效评价大鼠恶性神经胶质瘤模型的新生血管。Leong-Poi等通构建了携带抗αv-整合素单抗的靶向超声微泡,体外实验发现该靶向微泡与基质胶新生血管的结合率明显高于正常血管内皮细胞。
基质胶是一种来源于EHS小鼠肿瘤细胞提取的基底膜的复合物。它在4℃的时候为液态,但在室温条件下,可自动聚集产生类似于哺乳动物细胞基底膜的生物活性基质材料,它具有良好的可操作性和可重复性,是公认的血管新生模型,已被广泛应用于血管新生的实验研究。将基质胶注入皮下后,内皮细胞向基质胶中迁移形成新生血管,血管形成过程大约在10天左右完成,当加入适当浓度的促血管生成因子后,可以加快血管的生成。
碱性成纤维细胞生长因子(fibroblast growth factor-2, FGF-2)是体内发现的最为有效的血管形成因子之一。它对新生血管形成过程中多个环节如毛细血管基底膜降解、内皮细胞迁移增生、胶原合成、小血管腔形成均有明显促进作用,并且可以下调基质金属蛋白酶的活性。已证实FGF-2在体内、外均有明显的促新生血管形成作用。FGF-2可促进具有基底膜作用的蛋白激酶释放,从而促进毛细血管基底膜降解,促进小血管的形成。FGF-2能促进αv-整合素的表达,而αv-整合素是良好的血管新生成像靶点。理论上,不同浓度的FGF-2将诱导不同程度的αv-整合素表达。因此,本研究将不同浓度的FGF-2分别加入到基质胶中构建不同程度的血管新生模型。
Leong-Poi等使用αv-整合素靶向超声微泡对基质胶模型进行对比超声研究,靶向超声微泡的声强度高于普通微泡(P<0.05),但未设立封闭组探讨靶向超声微泡评价血管新生的特异性。Stieger等用非靶向微泡评价基质胶模型的血管新生,免疫组化的微血管密度和超声的增强区域正相关(r=0.65,P<0.05)。虽然,超声分子成像评价血管新生已经取得了初步成功,但目前仍没有对靶向超声分子成像评价血管新生的特异性和定量化进行研究的报道。因此,我们通过分别向基质胶中加入不同量的FGF-2,建立基质胶血管新生模型,并通过单抗封闭新生血管αv-整合素分子靶点的方法建立阴性对照模型,旨在探讨携带αv-整合素分子探针的超声分子成像评价不同浓度碱性成纤维细胞生长因子(FGF-2)介导的血管新生,并对血管新生的定量化评价进行初步的研究。
材料和方法
1、超声微泡的制备：各种脂质材料按一定比例75℃水浴溶解于适量蒸馏水中,同时通全氟丙烷(C3F8)气体,超声振荡至形成乳白色液体制备生物素(Biotin)化脂质微泡。生物素化脂质微泡经静置弃下清液并加入等量蒸馏水去除未结合脂质纯化4次后,按一定比例加入抗生蛋白链菌素／亲和素(streptavidin)。继续静置弃下清液并加入等量蒸馏水去除未结合抗生蛋白链菌素纯化微泡2次后得到外壳结合有抗生蛋白链菌素的脂质微泡,再按一定比例加入生物素化鼠抗人αv-整合素单抗(Biotin conjugated mouse Anti-humun avMonoclone Antibody)得到携带抗αv-整合素单抗靶向微泡(MBα),最后静置弃下清液并加入等量蒸馏水去除未结合抗体,纯化携带抗αv-整合素单抗靶向微泡(MBα)2次。用上述方法构建携同型抗体微泡作为对照微泡(MBc)。MBα和MBc均于冰箱中4℃保存备用。采用库尔特计数仪测量MBα及MBc的平均粒径及浓度。
2、MBα的体外鉴定：采用绿色荧光标记的二抗与抗αv-整合素单抗结合,荧光显微镜下观察抗αv-整合素单抗与微泡的连接情况。采用平行板流动腔技术在体外模拟的生理血流条件下评价MBα与αv-整合素Fc段(Recombinant humanαv-integrin/Fc Chimera,αvSFc)的靶向黏附效能。
3、基质胶模型的制备：在昆明小鼠皮下注射基质胶以促进血管新生,分别向基质胶中添加不同量的FGF-2 (sigma公司),配成浓度为1μg/mL和0.5μg/mL的两种基质胶,并加入肝素(64U/mL)。将20只小鼠随机分为2组,分别注射两种浓度的基质胶。20只昆明小鼠经常规麻醉后,在左侧腹部皮下注射0.6ml基质胶,在基质胶注射部位形成椭圆形隆起。
4、CEU检查：在注射基质胶10天后,将小鼠麻醉后进行对比超声检查。将自制水囊放在小鼠的左侧腹部,用自制支架固定超声探头(17L5)于水囊上。调整探头位置获得良好图像后保持探头位置在整个实验过程中不变,仪器的各项参数在整个实验过程中保持不变。CEU采用经尾静脉微泡弹丸式注射法进行,超声造影采用经尾静脉随机(间隔30min)先后注射MBα和MBC(约为5×106个)。经过6min循环时间待血池中循环微泡消失后对实验小鼠进行注射基质胶的部位进行CEU检查,获取显影图像。CEU检查采用二次谐波成像(second harmonic imaging)技术进行,探头发射和接收频率分别为7 MHz和14MHz,机械指数(Mechanical Index, MI)为0.18,超声发射间隔(Pulsing Interval, PI)时间设定为10s,获取第一帧CEU图像后给予高MI(1.9)连续超声发射2-3s以破坏微泡,继续存储本底图像4-5帧,全部声学造影图像存于CD盘,以备脱机分析。采用MCE软件分析CEU图像,测量实验小鼠注射基质胶的部位显影的声强度(video intensity, VI)数值并制作彩色编码图。采用红色、橙色和白色分别代表基质胶显影程度由强到弱。
5、病理学检查和免疫组化检查：完成CEU图像采集后,取出实验小鼠基质胶,10%甲醛固定,行病理学和免疫荧光检查。
6、平均荧光强度(median fluorescence:intensity, MFI)测定和血管计数(micro vessel count, MVC):在荧光照片选取相同面积的50个区域,使用Imagepro plus6.0软件分析平均荧光强度。
①得到灰度图片；
②黑白反相；
③光密度校正；
④在count/size中选择强度阂值10-200,过滤掉明亮的杂质荧光点；
⑤选择面积area与积分光密度(IOD)过滤值30像素；
⑥IOD/area得到该区域像素的平均灰度,即代表平均荧光强度,然后计算这50个区域的平均值。
单位面积血管计数：在上述50个区域,以有管腔结构为准,进行血管计数,计算单位面积血管计数。
结果
1、微泡制备情况：采用库尔特计数仪测得MBc粒径为2.12±1.13μm,浓度约为2.80～4.56×108个／ml；MBα粒径2.08±0.65μm,浓度约为3.07～4.75×108个／ml。
2、MBα体外鉴定结果：采用绿色荧光标记的二抗与抗αv-整合素单抗结合,荧光显微镜下观察显示MBa外壳显明显绿色荧光,表明抗αv-整合素单抗与微泡外壳连接效果良好。平行板流动腔体外评价结果显示,在模拟微血管生理血流条件的剪切应力(<1.0dyn／cm2)下MBα可与包被于平行板流动腔的αvSFc有效结合,显示了很好的主动性靶向黏附效能。
3、超声造影检查：MBα注射后基质胶外周带及周围组织均可见明显的超声显影,而基质胶中心部位未见超声显影,6min后周围组织未见显影,而基质胶外周带仍可见明显的超声显影,MBc注射后显影情况与MBa基本相同,但6min后基质胶未见明显的超声显影。预先用αv-整合素抗体封闭后,MBα及MBc注射后基质胶也可呈明显的超声显影,但6min后MBα和MBc均未见明显的超声显影。
4、图像分析结果：在高浓度组中,封闭前基质胶模型中MBa组呈明显的超声显影,而MBc组仅见轻度的超声显影,两者之间有明显差异(P<0.05)。预先单抗封闭αv-整合素后,MBa和封闭前的MBα之间有明显差异(P<0.05)；而MBc的Ⅵ值较封闭前无明显变化,两者之间无明显差异(P>0.05)。低剂量组同样出现上述结果。
5、基质胶病理及免疫荧光结果：HE染色切片显示,小鼠基质胶外周带可见丰富的新生血管,血管中可见红细胞的存在；高浓度FGF-2组基质胶中新生血管多于低浓度FGF-2组；高浓度组血管密度高,而且更靠近中央区。免疫荧光显示基质胶新生血管内皮细胞有大量的αv-整合素,高浓度FGF-2组基质胶内皮细胞中表达的αv-整合素多于低浓度FGF-2组,封闭后内皮细胞中几乎不表达αv-整合素。
6.在超声造影中,高浓度组封闭前MBα造影Ⅵ值是低浓度组封闭前的1.4倍；高浓度组封闭后MBα造影Ⅵ值是低浓度组封闭前的1.37倍。在免疫荧光强度测定中,高浓度组封闭前单位面积免疫荧光强度是低浓度组封闭前的1.32倍；高浓度组封闭后是低浓度封闭后的1.33倍。在单位面积血管计数中,高浓度组封闭前是封闭后的1.2倍；高浓度组封闭前MBα造影Ⅵ值是低浓度组封闭前的1.16倍。
结论：
1、采用“抗生物素蛋白／生物素复合体”可实现抗αv整合素单抗与脂质微泡外壳的有效连接而成功构建携带抗αv-整合素单抗靶向微泡；
2、应用携带αv-整合素分子探针的超声分子成像可有效特异地评价基质胶血管新生,实现对新生血管的早期评价和诊断；
3、αv-整合素可以作为新生血管分子成像的靶点,使用基质胶模型评价血管新生的研究具有良好的特异性,可用于定量化研究。
Background and Objective:
Angiogenesis is the formation of new capillaries from existent micro vessels by sprouting, migration, proliferation of endothelial cells and matrix remodeling when body is in the process of growth or experiencing wound healing, ischemic hypoxia and inflammation. As a basic physiological procedure, angiogenesis participates in the healing of all kinds of wounds, ulcers, myocardial infarction and chronic infection et al. But there are angiogenic disorders in many pathological states such as diabetic retinopathy, atherosclerosis, and solid tumors and other serious diseases which are hazardous for human beings. Therefore, high sensitive, non-invasive, targeted, quantitative evaluation of angiogenesis has the vital significance to early cardiovascular disease (mainly ischemic heart disease and peripheral arterial occlusive disease) and neoplastic diseases. The number and spatial distribution of new blood vessels can assess the early response of microvasculation reacted to growth factors to guide the delivery pro-angiogenic proteins or genes for cardiovascular disease. The early angiogenesis is able to develop early detection of tumor and micrometastasis conducive to early treatment of patients, and dynamic monitoring of new blood vessels can assess the effect of anti-cancer therapy.
Ultrasonnd molecular imaging is a new online technique of noninvasive molecular imaging technologies. By virtue of connecting special ligands of specific molecules in pathologic tissue to the surface of ultrasound microbubbles, targeted ultrasound microbubbles can be constructed. Via intravenous injection, the targeted ultrasound microbubbles can gather at the target tissue and remain there for a long time, so active targeted ultrasound molecular imaging at molecular lever can be produced by contrast enhanced ultrasound. With extensive perspective and great clinical significance, evaluation of angiogenesis with targeted ultrasound molecular imaging technology has increasingly become a frontline focus in ultrasound research for the past few years. Ellegala built targeted microbubbles with echistatin to effectively evaluate neovascularization of the rat model of malignant glioma. And Leong-Poi constructed microbubbles targeted to av-integrin (MBα) and proofed that the adhesive capacity of the targeted microbubbles in neovascularization was higher than in normal vascular endothelial cells.
Matrigel is a soluble basement membrane matrix extracted from ESH mouse. At room temperature, it can automatically gather and become bioactive materials similar to basement membranes of mammalian cell. Matrigel model has good operability and repeatability which has been widely applied to the experimental study as an acknowledged angiogenesis model. After subcutaneous injection of the Matrigel, endothelial cells migrate to Matrigel to form new blood vessels. Blood vessel formation takes about 10 days to complete and adding appropriate concentration of pro-angiogenic factors can speed up the angiogenesis.
Basic fibroblast growth factor (FGF-2) is one of the most effective pro-angiogenic factors in vivo. It can up-regulate capillary basement membrane degradation, endothelial cell migration and proliferation, collagen synthesis and down-regulation the activity of matrix metalloproteinases. It has proved that FGF-2 plays an important role in vivo as well as in vitro to stimulate newborn blood vessels. FGF-2 can enhance the release of protein kinases which can degrade the basement membrane, thereby promoting angiogenesis, and the formation of small blood vessels.
Leong-Poi et al performed contrast ultrasound study on Matrigel model with MBa and video intensity of MBa is high that of control microbubble(P<0.05). But the specificity of angiogenesis wasn't evaluated because of lack of closed group. Stieger found microvessel density measurements displayed a significant correlation with power Doppler enhanced area (r=0.65, P<0.05) with non-targeted microbubble. Although ultrasound molecular imaging of angiogenesis has achieved initial success, there is no research report about specificity and quantification of angiogenesis. Therefore, we established Matrigel model by adding different levels of FGF-2 and control model by blocking a-integrin in new blood vessels in order to investigate the assessment of angiogenesis mediated by different levels of FGF-2 with ultrasound molecular imaging using microbubbles targeted to endothelialαv-integrins and quantitative evaluation of angiogenesis preliminarily.
Methods:
1. Microbubbles preparation:lipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas (C3H8) with aqueous dispersion of several lipids in determinate ratio. After being washed (4×) to remove excess free unincorporated lipid, streptavidin in determinate ratio were added to the lipid microbubbles with biotin, then washed (2×) to removed excess free unincorporated streptavidin and the biotin conjugated mouse anti-integrin av monoclone antibodies or biotin conjugated isotype control mAb in determinate ratio were added respectively to complete the preparation of MBa and MBc. At last, the MBa and MBc were washed (2×) to remove excess free unincorporated antibodies. Both MBαand MBc were storaged in refrigerator at 4℃. The mean diameter and density in both MBa and MBa were measured by coulter counter.
2. Evaluation of MBa in vitro:Using green fluorescent-labeled antibody for antiαv-integrin monoclonal antibody to identify the linking antibodies on the MBa. To evaluate the combinding efficacy between MBa andαvSFc (Recombinant human av-integrin/Fc Chimera) with a parallel plate flow chamber at a shearing force under physiologic flow conditions.
3. Animal preparation:Matrigel angiogenesis was created by subcutaneous injection of matrigel in Kunming mice. Two different levels of FGF-2 was adding to Matrigel to form matrigel at the FGF-2 concentrations of 1μg/mL and 0.5μg/mL.20 mice were randomly divided into 2 groups injected with two concentrations of Matrigel. Mice were routinely anesthetized and 0.6 ml of enriched Matrigel (4℃) was injected subcutaneously in the left ventral region. Matrigel hardened to form discrete ellipsoid plugs.
4. CEU imaging:CEU of matrigel plugs was performed in 10 anesthetized mice 10 days after matrigel injection. All experimental mice were performed with CEU respectively by using MBa and MBc, the intravenous injection of 4×106 microbubbles were made in random order with 30 minutes interval. After five minutes of intravenous injection, microbubbles in the circulation were eliminated, the ultrasound signal (video intensity, VI) from MBa and MBc were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of 0.18, transmission frequency of 7.0 MHz and receiving frequency of 14.0 MHz. After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background- subtracted VI of tumor was measured. Analysis of VI was performed by MCE software and color-coding for visual analysis was performed.
5. Examination of pathology and immunofluorescence:After CEU imaging, all Matrigel plugs of experimental mice were harvested for the examination of pathology and immunofluorescence.
6. Determination of median fluorescence:intensity and micro vessel count:50 regions with the same area were selected in the fluorescence photograph and median fluorescence:intensity was measured by imagepro software6.0 in these fifty regions.
①Grayscale images acquisition;
②Color reverse;
③Optical density correction;
④Selecting intensity threshold 10-200 in count/size to filter out impurities.
⑤set filter value to30 pixels;
⑥IOD/area representing the median fluorescence intensity and then calculating the average of the 50 regions.
Micro vessels count:Average of the 50 regions was calculated subject to a lumen structure.
Results:
1. Results for microbubble preparation:The density of MBαand MBc is about 3.07～4.75×108/ml and 2.80～4.56×108/ml, the mean sizes for MBa and MBc were about 2.08±0.65μm and 2.12±1.13μm respectively.
2. Results for evaluation of MBa in vitro:The mouse anti-mouse av-integrin monoclonal antibodies linked well to the surface of microbubble, which was observed with fluorescence microscopy. MBa andαvSFc enjoyed a good targeted combination with a shearing force (<1.0dyn/cm2) under physiologic flow conditions, which was observed in the parallel plate flow chamber.
3. Results for CEU imaging:Significant ultrasound imaging was observed in the peripheral zone and the surrounding tissue of the Matrigel with MBa. While there was no CEU imaging in the center of the Matrigel. Six min later no significant CEU imaging was observed in the surrounding tissue, while the peripheral zone was still clearly visible. The CEU imaging with MBc was the same as that with MBa except no obvious CEU imaging was observed in the Matrigel. After blocking with antibody against av-integrin, significant CEU imaging could be observed in the Matrigel with MBa and MBc, but six min later there was no significant CEU imaging.
4. Results for CEU-derived IV:In high lever group, VI of the matrigel was significantly higher for MBa as compared with MBc(P<0.05). After blocking with antibody against av-integrin, a great decrease was observed in the MBa group (P<0.05) while no significant difference was noted for MBc (P>0.05). The trend in low dosage group was similar to that in high lever group.
5. Results for examination of pathology and immunofluorescence:HE showed abundant new vessels in which there were red blood cells in the peripheral zone of the matrigel plugs with a large amount of white blood cells (mononuclear cells) infiltration. Neovessels in matrigel was positive for av-integrin which was observed with fluorescence microscopy. There were more newborn blood vessels in high lever group than that in low lever group and high micro vessel density which is more closed to the central zone could be seen in high lever group. Immunofluorescence showed abundant av-integrin expressed within matrigel in which the amount of av-integrin in high lever group is much than that in low lever group. After blocking with antibody against av-integrin, there was hardlyαv-integrin in endothelial cells.
6. VI of the matrigel was 1.4 times for MBa in high level compared with that in low level. After blocked with antibody againstαv-integrin, VI was 1.37 times for MBa in high level compared with that in low level. The median fluorescence intensity in high level was 0.32 times higher than that in low level. After blocked with antibody, the median fluorescence intensity in high level was 0.32 times higher than that in low level. Micro vessels count per unit was 1.2 times as much as that in low level. After blocked with antibody, micro vessels count per unit was 1.16 times as much as that in low level.
Conclusions:
1. Microbubbles targeted toαv-integrin can be successfully constructed by combining anti av-integrin monoclonal antibodies to lipid microbubbles via "avidin-biotin" bridging chemistry.
2. Ultrasound molecular imaging with microbubbles targeted to av-integrin can be effective and specific in evaluating the angiogenesis in a murine model of subcutaneous matrigel plugs.
3.αv-integrin can be an ideal target for molecular imaging of angiogenesis and assessment of angiogenesis in matrigel has good specificity and a great application prospect in quantify study.
引文
[1]Pandya NM, Dhalla NS, Santani DD. Angiogensis—a new target for future therapy. Vascul Pharmacol,2006,44(5):265
[2]Klibanov AL. Microbubble contrast agents:targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Invest Radiol,2006,41:354-362.
[3]Kaufmann BA, Lindner JR. Molecular imaging with targeted ultrasound molecular imaging. Curr Opin Biotechnol,2007,18:11-16.
[4]Avivi Levi S, Gedanken A. The preparation of avidin microspheres using the sonochemical method and the interaction of the microspheres with biotin. Ultrason Sonochem,2005,12:405-409.
[5]Hamilton AJ, Huang SL, Warnick D, et al. Intravascular ultrasound molecular imaging of atheroma components in vivo. J Am Coll Cardiol,2004,43:453-460.
[6]Lindner JR, Song J, Christiansen J, et al. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation, 2001,104:107-112.
[7]Heppner P, Lindner JR. Contrast ultrasound assessment of angiogenesis by perfusion and molecular imaging. Expert Rev Mol Diagn,2005,5(3):447
[8]Leong-PoiH, Christiansen J, Heppner P, et al Assessment of endogenous and therapeutic arteriogenesis by contrast ultrasound molecular imaging of integrin expression. Circulation,2005,111(24):3248～3254.
[9]Ellegala DB, Leong-PoiH, Carpenter J E, et al Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta3. Circulation,2003, 108(3):336～341.
[10]Leong-PoiH, Christiansen J, Klibanov A L, et al Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to alpha(v)-integrins. Circulation,2003,107(3):455～460.
[11]Willmann JK, Paulmurugan R, Chen K, et al US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. Radiology,2008,246(2):508～518.
[12]Alghisi GC, Ruegg C. Vascular intergrins in tumor angiogenesis:Mediators and therapeutic targets. Endothelium,2006,13:113-135.
[13]Ellegala DB, Leong-Poi H, Carpenter JE, et al. Imaging Tumor Angiogenesis With Contrast Ultrasound and Microbubbles Targeted to α v β 3.Circulation 2003,108:336-341.
[14]Ng EW, Adamis AP. Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration. Can J Ophthalmol 2005;40(3):352 368.
[1]Lyshchik A, Fleischer AC, Huamani J, et al. Molecular imaging of vascular endothelial growth factor receptor 2 expression using targeted contrast-enhan- ced high-frequency ultrasonography. J Ultrasound Med,2007,26:1575-1586.
[2]Kaufmann BA, Lindner JR. Molecular imaging with targeted ultrasound molecular imaging. Curr Opin Biotechnol,2007,18:11-16.
[3]Klibanov AL. Molecular imaging with targeted ultrasound contrast microbubbles. Ernst Schering Res Found Workshop,2005,49:171-191.
[4]Lindner JR. Molecular imaging with contrast ultrasound and targeted microbubbles. J Nucl Cardiol,2004,11:215-221.
[5]Behm CZ, Lindner JR. Cellular and molecular imaging with targeted ultrasound molecular imaging. Ultrasound Q,2006,22:67-72.
[6]Klibanov AL. Ligand-carrying gas-filled microbubbles:ultrasound contrast agents for targeted molecular imaging. Bioconjug Chem,2005,16:9-17.
[7]Bloch SH, Dayton PA, Ferrara KW. Targeted imaging using ultrasound contrast agents. Progess and opportunities for clinical and research applications.IEEE Eng Med Biol Mag,2004,23:18-29.
[8]Villanueva FS, Wagner WR, Vannan MA, et al. Targeted ultrasound imaging using microbubbles. Cardiol Clin,2004,22:283-298.
[9]Klibanov AL. Microbubble contrast agents:targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Invest Radiol,2006,41:354-362.
[10]Kondo I, Ohmori K, Oshita A, et al. Leukocyte-targeted myocardial contrast echocardiography can assess the degree of acute allograft rejection in a rat cardiac transplantation model. Circulation,2004,109:1056-1061.
[11]Kaufmann BA, Sanders JM, Davis C, et al. Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation,2007,116:276-284.
[12]Klibanov AL, Rychak JJ, Yang WC, et al. Targeted ultrasound contrast agent for molecular imaging of inflammation in high-shear flow. Contrast Media Mol Imaging,2006,1:259-266.
[13]Weller GE, Villanueva FS, Tom EM, et al. Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewis x. Biotechnol Bioeng,2005,92:780-188.
[14]Weller GE, Villanueva FS, Klibanov AL, et al. Modulating targeted adhesion of an ultrasound contrast agent to dysfunctional endothelium. Ann Biomed Eng, 2002,30:1012-1019.
[15]Kaufmann BA, Lewis C, Xie A, et al. Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography. Eur Heart J,2007,28:2011-2017.
[16]Weller GE, Lu E, Csikari MM, et al.Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation,2003,108:218-224.
[17]Schumann PA, Christiansen JP, Quigley RM, et al. Targeted-microbubble binding selectively to GPⅡb Ⅲa receptors of platelet thrombi. Invest Radiol,2002, 37:587-593.
[18]Martin MJ, Chung EM, Goodall AH, et al.Enhanced detection of thromboemboli with the use of targeted microbubbles. Stroke,2007,38:2726-2732.
[19]Weller GE, Wong MK, Modzelewski RA, et al. Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumor-binding peptide arginine-arginine-leucine. Cancer Res,2005,65:533-539.
[20]Stieger SM, Dayton PA, Borden MA, et al. Imaging of angiogenesis using Cadence contrast pulse sequencing and targeted contrast agents. Contrast Media Mol Imaging,2008,3:9-18.
[21]Willmann JK, Paulmurugan R, Chen K, et al. US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. Radiology,2008,246:508-518.
[22]Tranquart F, Correas JM, Marret H, et al. Recent advances in contrast-enhanced ultrasound for oncology applications. J Radiol,2007,88:1759-1769.
[23]Weller GE, Wong MK, Modzelewski RA, et al. Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumor-binding peptide arginine-arginine-leucine. Cancer Res,2005,65:533-539.
[24]Dayton PA, Pearson D, Clark J, et al. Ultrasonic analysis of peptide-and antibody-targeted microbubble contrast agents for molecular imaging of alphavbeta3-expressing cells. Mol Imaging,2004,3:125-134.
[25]Ellegala DB, Leong-Poi H, Carpenter JE, et al. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta3. Circulation, 2003,108:336-341.
[26]Klibanov AL. Microbubble contrast agents:targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Invest Radiol,2006,41:354-362.
[27]Avivi Levi S, Gedanken A. The preparation of avidin microspheres using the sonochemical method and the interaction of the microspheres with biotin. Ultrason Sonochem,2005,12:405-409.
[28]Hamilton AJ, Huang SL, Warnick D, et al. Intravascular ultrasound molecular imaging of atheroma components in vivo. J Am Coll Cardiol,2004,43:453-460.
[29]Lindner JR, Song J, Christiansen J, et al. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation, 2001,104:107-112.
[30]Ng EW, Adamis AP. Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration. Can J Ophthalmol, 2005,40(3):352-368.
[31]Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin alphav beta 3 for angiogenesis. Science,1994,264(5158):569～571
[32]Takagi J, Springer TA. Integrin activation and structural rearrangement. Immunol Rev,2002,186:141-63.
[33]郑道文,杨莉,宾建平,等.亲和素桥连构建携抗P-选择素单抗靶向超声微泡及体外荧光鉴定.中国医学影像技术,2008,24：1182-1186.
[34]COOK GJ. Oncological molecular imaging:nuclear medicine techniques.B J Radiol,2003,76:152-158.
[35]Klibanov AL. Ultrasound molecular imaging with targeted microbubble contrast agents. J Nucl Cardiol,2007,14(6):876～884.
[36]Lindner JR, Dayton PA, Coggins MP, et al. Noninvasive imaging of Inflammation by ultrasound detection of phagocytosed microbubbles. Circulation, 2000,102(5):531～538.
进行进一步研究对于调控血管新生具有广阔的研究前景,
[1]Folkman J. Angiogenesis:an organizing principle for drug discovery? Nat Rev Drug Discov,2007,6:273-86.
[2]Davis DE, Koh AW, Stratman N. Mechanisms controlling human endothelial lumen formation and tube assembly in three-dimensional extracellular matrices. Birth Defects Res Embryo Today,2007,81:270-85.
[3]Arnaout MA, Mahalingam B, Xiong JP. Integrin structure, allostery, and bidirectional signaling. Annu Rev Cell Dev Biol,2005,21:381-410.
[4]Wegener KL, Campbell ID. Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions. Mol Membr Biol, 2008,25:76-87.
[5]Malinin NL, Zhang L, Choi J, CioceaA,Razorenova O,MaY-Q, et al.Apoint mutationin KINDLIN3 ablates activation of three integrin subfamilies in humans. Nat Med,2009,15:313-8.
[6]Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B. Functional atlas of the integrin adhesome. Nat Cell Biol,2007,9:858-67.
[7]Xu J, Rodriguez D, Petitclerc E, Kim JJ, Hangai M, Moon YS, et al. Proteolytic exposure of a cryptic site within collagen type Ⅳ is required for angiogenesis and tumor growth in vivo. J Cell Biol,2001,154:1069-79.
[8]Margadant C, Raymond K, Kreft M, Sachs N, Janssen H, Sonnenberg A. Integrin a3β1inhibits directional migration and wound re-epithelialization in the skin. J Cell Sci,2009,122:278-88.
[9]Abraham S, Kogata N, Fesler R, Adams RH. Integrin al subunit controls mural cell adhesion, spreading, and blood vessel wall stability. Circ Res, 2008,102:562-70.
[10]Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, et al. Integrin αⅴβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell,1994,79:1157-64.
[11]Brooks PC, Stromblad S, Klemke R, Visscher D, Sarkar FH, Cheresh DA. Antiintegrin αⅴβ3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest,1995,96:1815-22.
[12]Nemeth JA, Nakada MT, Trikha M, Lang Z, Gordon MS, Jayson GC, et al. Alpha-v integrin as therapeutic targets in oncology. Cancer Invest, 2007,25:632-46.
[13]Renolds AR, Renolds LE, Nagel TE, Lively JC, Robinson SD, Hicklin DJ, et al. Elevated Flkl (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis inβ3-integrin-deficient mice. Cancer Res, 2004,64:8643-50.
[14][Kanamori M, Kawaguchi T, Berger MS, Pieper RO. Intracranial microenvironment reveals independent opposing functions of hostαⅴβ3 expression on glioma growth and angiogenesis. J Biol Chem,2006, 281:37256-64.
[15]Chen X. Multimodality imaging of tumor integrinαⅴβ3 expression. Mini-Rev Med Chem,2006,6:227-34.
[16]Xu J, Rodrigue ZD, Kim JJ, Brooks PC. Generation of monoclonal antibody to cryptic collagen sites by using subtractive immunization. Hybridoma, 2000,19:375-85.
[17]Delbaldo C, Raymond E, Vera K, Hammershaimb L, Kaucic K, Lozahic S, et al. Phase I and pharmacokinetic study of etaracizumab (AbegrinTM), a humanized monoclonal antibody againstαⅴβ3 integrin receptor, in patients with advanced solid tumors. Invest New Drugs,2008,26:35-43.
[18]Mullamitha SA, Ton NC, Parker GJM, Jackson A, Julyan PJ, Roberts C, et al. Phase Ⅰ evaluation of a fully human anti-av integrin monoclonal antibody (CNTO 95) in patients with advanced solid tumors. Clin Cancer Res,2007,13: 2128-35.
[19]Pathak A, Zhao R, Huang J, Stouffer GA. Eptifibatide and abciximab inhibit insulin-induced focal adhesion formation and proliferative responses in human aortic smooth muscle cells. Cardiovasc Diabetol,2008,7:36-47.
[20]Beauvais DM, Ell BJ, McWhorter AR, Rapraeger AC. Syndecan-1 regulatesαⅴβ3 andαⅴβ5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor. J Exp Med,2009,206:691-705.
[21]Ricart AD, Tolcher AW, Liu G, Holen K, Schwartz G, Albertini M, et al. Volociximab, a chimeric monoclonal antibody that specifically bindsaα5β1 integrin:a phase I, pharmacokinetic, and biological correlative study. Clin Cancer Res,2008,14:7924-9.
[22]Do~nate F, Parry GC, Shaked Y, Hensley H, Guan X, Beck I, et al. Pharmacology of the novel antiangiogenic peptide ATN-161 (Ac-PHSCN-NH2): observation of a U-shaped dose-response curve in several preclinical models of angiogenesis and tumor growth. Clin Cancer Res,2008,14:2137-44.
[23]Keizer RJ, Zamacona MK, Jansen M, Critchley D, Wanders J, Beijnen JH, et al. Application of population pharmacokinetic modeling in early clinical development of the anticancer agent E7820. Invest New Drugs, 2009,27:140-52.
[2]Klibanov AL. Microbubble contrast agents:targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Invest Radiol,2006,41:354-362.
[3]Kaufmann BA, Lindner JR. Molecular imaging with targeted ultrasound molecular imaging. Curr Opin Biotechnol,2007,18:11-16.
[4]Avivi Levi S, Gedanken A. The preparation of avidin microspheres using the sonochemical method and the interaction of the microspheres with biotin. Ultrason Sonochem,2005,12:405-409.
[5]Hamilton AJ, Huang SL, Warnick D, et al. Intravascular ultrasound molecular imaging of atheroma components in vivo. J Am Coll Cardiol,2004,43:453-460.
[6]Lindner JR, Song J, Christiansen J, et al. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation, 2001,104:107-112.
[7]Heppner P, Lindner JR. Contrast ultrasound assessment of angiogenesis by perfusion and molecular imaging. Expert Rev Mol Diagn,2005,5(3):447
[8]Leong-PoiH, Christiansen J, Heppner P, et al Assessment of endogenous and therapeutic arteriogenesis by contrast ultrasound molecular imaging of integrin expression. Circulation,2005,111(24):3248～3254.
[9]Ellegala DB, Leong-PoiH, Carpenter J E, et al Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta3. Circulation,2003, 108(3):336～341.
[10]Leong-PoiH, Christiansen J, Klibanov A L, et al Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to alpha(v)-integrins. Circulation,2003,107(3):455～460.
[11]Willmann JK, Paulmurugan R, Chen K, et al US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. Radiology,2008,246(2):508～518.
[12]Alghisi GC, Ruegg C. Vascular intergrins in tumor angiogenesis:Mediators and therapeutic targets. Endothelium,2006,13:113-135.
[13]Ellegala DB, Leong-Poi H, Carpenter JE, et al. Imaging Tumor Angiogenesis With Contrast Ultrasound and Microbubbles Targeted to α v β 3.Circulation 2003,108:336-341.
[14]Ng EW, Adamis AP. Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration. Can J Ophthalmol 2005;40(3):352 368.
[1]Lyshchik A, Fleischer AC, Huamani J, et al. Molecular imaging of vascular endothelial growth factor receptor 2 expression using targeted contrast-enhan- ced high-frequency ultrasonography. J Ultrasound Med,2007,26:1575-1586.
[2]Kaufmann BA, Lindner JR. Molecular imaging with targeted ultrasound molecular imaging. Curr Opin Biotechnol,2007,18:11-16.
[3]Klibanov AL. Molecular imaging with targeted ultrasound contrast microbubbles. Ernst Schering Res Found Workshop,2005,49:171-191.
[4]Lindner JR. Molecular imaging with contrast ultrasound and targeted microbubbles. J Nucl Cardiol,2004,11:215-221.
[5]Behm CZ, Lindner JR. Cellular and molecular imaging with targeted ultrasound molecular imaging. Ultrasound Q,2006,22:67-72.
[6]Klibanov AL. Ligand-carrying gas-filled microbubbles:ultrasound contrast agents for targeted molecular imaging. Bioconjug Chem,2005,16:9-17.
[7]Bloch SH, Dayton PA, Ferrara KW. Targeted imaging using ultrasound contrast agents. Progess and opportunities for clinical and research applications.IEEE Eng Med Biol Mag,2004,23:18-29.
[8]Villanueva FS, Wagner WR, Vannan MA, et al. Targeted ultrasound imaging using microbubbles. Cardiol Clin,2004,22:283-298.
[9]Klibanov AL. Microbubble contrast agents:targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Invest Radiol,2006,41:354-362.
[10]Kondo I, Ohmori K, Oshita A, et al. Leukocyte-targeted myocardial contrast echocardiography can assess the degree of acute allograft rejection in a rat cardiac transplantation model. Circulation,2004,109:1056-1061.
[11]Kaufmann BA, Sanders JM, Davis C, et al. Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation,2007,116:276-284.
[12]Klibanov AL, Rychak JJ, Yang WC, et al. Targeted ultrasound contrast agent for molecular imaging of inflammation in high-shear flow. Contrast Media Mol Imaging,2006,1:259-266.
[13]Weller GE, Villanueva FS, Tom EM, et al. Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewis x. Biotechnol Bioeng,2005,92:780-188.
[14]Weller GE, Villanueva FS, Klibanov AL, et al. Modulating targeted adhesion of an ultrasound contrast agent to dysfunctional endothelium. Ann Biomed Eng, 2002,30:1012-1019.
[15]Kaufmann BA, Lewis C, Xie A, et al. Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography. Eur Heart J,2007,28:2011-2017.
[16]Weller GE, Lu E, Csikari MM, et al.Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation,2003,108:218-224.
[17]Schumann PA, Christiansen JP, Quigley RM, et al. Targeted-microbubble binding selectively to GPⅡb Ⅲa receptors of platelet thrombi. Invest Radiol,2002, 37:587-593.
[18]Martin MJ, Chung EM, Goodall AH, et al.Enhanced detection of thromboemboli with the use of targeted microbubbles. Stroke,2007,38:2726-2732.
[19]Weller GE, Wong MK, Modzelewski RA, et al. Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumor-binding peptide arginine-arginine-leucine. Cancer Res,2005,65:533-539.
[20]Stieger SM, Dayton PA, Borden MA, et al. Imaging of angiogenesis using Cadence contrast pulse sequencing and targeted contrast agents. Contrast Media Mol Imaging,2008,3:9-18.
[21]Willmann JK, Paulmurugan R, Chen K, et al. US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. Radiology,2008,246:508-518.
[22]Tranquart F, Correas JM, Marret H, et al. Recent advances in contrast-enhanced ultrasound for oncology applications. J Radiol,2007,88:1759-1769.
[23]Weller GE, Wong MK, Modzelewski RA, et al. Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumor-binding peptide arginine-arginine-leucine. Cancer Res,2005,65:533-539.
[24]Dayton PA, Pearson D, Clark J, et al. Ultrasonic analysis of peptide-and antibody-targeted microbubble contrast agents for molecular imaging of alphavbeta3-expressing cells. Mol Imaging,2004,3:125-134.
[25]Ellegala DB, Leong-Poi H, Carpenter JE, et al. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta3. Circulation, 2003,108:336-341.
[26]Klibanov AL. Microbubble contrast agents:targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Invest Radiol,2006,41:354-362.
[27]Avivi Levi S, Gedanken A. The preparation of avidin microspheres using the sonochemical method and the interaction of the microspheres with biotin. Ultrason Sonochem,2005,12:405-409.
[28]Hamilton AJ, Huang SL, Warnick D, et al. Intravascular ultrasound molecular imaging of atheroma components in vivo. J Am Coll Cardiol,2004,43:453-460.
[29]Lindner JR, Song J, Christiansen J, et al. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation, 2001,104:107-112.
[30]Ng EW, Adamis AP. Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration. Can J Ophthalmol, 2005,40(3):352-368.
[31]Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin alphav beta 3 for angiogenesis. Science,1994,264(5158):569～571
[32]Takagi J, Springer TA. Integrin activation and structural rearrangement. Immunol Rev,2002,186:141-63.
[33]郑道文,杨莉,宾建平,等.亲和素桥连构建携抗P-选择素单抗靶向超声微泡及体外荧光鉴定.中国医学影像技术,2008,24：1182-1186.
[34]COOK GJ. Oncological molecular imaging:nuclear medicine techniques.B J Radiol,2003,76:152-158.
[35]Klibanov AL. Ultrasound molecular imaging with targeted microbubble contrast agents. J Nucl Cardiol,2007,14(6):876～884.
[36]Lindner JR, Dayton PA, Coggins MP, et al. Noninvasive imaging of Inflammation by ultrasound detection of phagocytosed microbubbles. Circulation, 2000,102(5):531～538.
进行进一步研究对于调控血管新生具有广阔的研究前景,
[1]Folkman J. Angiogenesis:an organizing principle for drug discovery? Nat Rev Drug Discov,2007,6:273-86.
[2]Davis DE, Koh AW, Stratman N. Mechanisms controlling human endothelial lumen formation and tube assembly in three-dimensional extracellular matrices. Birth Defects Res Embryo Today,2007,81:270-85.
[3]Arnaout MA, Mahalingam B, Xiong JP. Integrin structure, allostery, and bidirectional signaling. Annu Rev Cell Dev Biol,2005,21:381-410.
[4]Wegener KL, Campbell ID. Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions. Mol Membr Biol, 2008,25:76-87.
[5]Malinin NL, Zhang L, Choi J, CioceaA,Razorenova O,MaY-Q, et al.Apoint mutationin KINDLIN3 ablates activation of three integrin subfamilies in humans. Nat Med,2009,15:313-8.
[6]Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B. Functional atlas of the integrin adhesome. Nat Cell Biol,2007,9:858-67.
[7]Xu J, Rodriguez D, Petitclerc E, Kim JJ, Hangai M, Moon YS, et al. Proteolytic exposure of a cryptic site within collagen type Ⅳ is required for angiogenesis and tumor growth in vivo. J Cell Biol,2001,154:1069-79.
[8]Margadant C, Raymond K, Kreft M, Sachs N, Janssen H, Sonnenberg A. Integrin a3β1inhibits directional migration and wound re-epithelialization in the skin. J Cell Sci,2009,122:278-88.
[9]Abraham S, Kogata N, Fesler R, Adams RH. Integrin al subunit controls mural cell adhesion, spreading, and blood vessel wall stability. Circ Res, 2008,102:562-70.
[10]Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, et al. Integrin αⅴβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell,1994,79:1157-64.
[11]Brooks PC, Stromblad S, Klemke R, Visscher D, Sarkar FH, Cheresh DA. Antiintegrin αⅴβ3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest,1995,96:1815-22.
[12]Nemeth JA, Nakada MT, Trikha M, Lang Z, Gordon MS, Jayson GC, et al. Alpha-v integrin as therapeutic targets in oncology. Cancer Invest, 2007,25:632-46.
[13]Renolds AR, Renolds LE, Nagel TE, Lively JC, Robinson SD, Hicklin DJ, et al. Elevated Flkl (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis inβ3-integrin-deficient mice. Cancer Res, 2004,64:8643-50.
[14][Kanamori M, Kawaguchi T, Berger MS, Pieper RO. Intracranial microenvironment reveals independent opposing functions of hostαⅴβ3 expression on glioma growth and angiogenesis. J Biol Chem,2006, 281:37256-64.
[15]Chen X. Multimodality imaging of tumor integrinαⅴβ3 expression. Mini-Rev Med Chem,2006,6:227-34.
[16]Xu J, Rodrigue ZD, Kim JJ, Brooks PC. Generation of monoclonal antibody to cryptic collagen sites by using subtractive immunization. Hybridoma, 2000,19:375-85.
[17]Delbaldo C, Raymond E, Vera K, Hammershaimb L, Kaucic K, Lozahic S, et al. Phase I and pharmacokinetic study of etaracizumab (AbegrinTM), a humanized monoclonal antibody againstαⅴβ3 integrin receptor, in patients with advanced solid tumors. Invest New Drugs,2008,26:35-43.
[18]Mullamitha SA, Ton NC, Parker GJM, Jackson A, Julyan PJ, Roberts C, et al. Phase Ⅰ evaluation of a fully human anti-av integrin monoclonal antibody (CNTO 95) in patients with advanced solid tumors. Clin Cancer Res,2007,13: 2128-35.
[19]Pathak A, Zhao R, Huang J, Stouffer GA. Eptifibatide and abciximab inhibit insulin-induced focal adhesion formation and proliferative responses in human aortic smooth muscle cells. Cardiovasc Diabetol,2008,7:36-47.
[20]Beauvais DM, Ell BJ, McWhorter AR, Rapraeger AC. Syndecan-1 regulatesαⅴβ3 andαⅴβ5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor. J Exp Med,2009,206:691-705.
[21]Ricart AD, Tolcher AW, Liu G, Holen K, Schwartz G, Albertini M, et al. Volociximab, a chimeric monoclonal antibody that specifically bindsaα5β1 integrin:a phase I, pharmacokinetic, and biological correlative study. Clin Cancer Res,2008,14:7924-9.
[22]Do~nate F, Parry GC, Shaked Y, Hensley H, Guan X, Beck I, et al. Pharmacology of the novel antiangiogenic peptide ATN-161 (Ac-PHSCN-NH2): observation of a U-shaped dose-response curve in several preclinical models of angiogenesis and tumor growth. Clin Cancer Res,2008,14:2137-44.
[23]Keizer RJ, Zamacona MK, Jansen M, Critchley D, Wanders J, Beijnen JH, et al. Application of population pharmacokinetic modeling in early clinical development of the anticancer agent E7820. Invest New Drugs, 2009,27:140-52.