诊断超声联合微泡介导经静脉移植内皮祖细胞治疗心肌梗死的初步研究
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摘要
背景和目的:
缺血性心脏病(Ischemic heart disease,IHD)是由于冠状动脉循环改变引起冠状动脉血流和心肌需求之间不平衡而导致的心肌损害,其中最常见的原因是冠心病,约占90%。传统的治疗手段主要是重建血供,而不能修复损伤和坏死的心肌。近几年兴起的干细胞移植法通过再生血管与心肌组织,使人们看到了治疗IHD的希望。
血管内皮祖细胞(Endothelial progenitor cells, EPCs)是指具有特异性归巢于血管新生组织并能分化增殖为成熟内皮细胞(Endothelial cells, ECs)的一群干/祖细胞。研究发现EPCs参与出生后的内皮修复和血管新生过程,提示其在IHD中重要的治疗作用以及广阔的临床应用前景。高增殖潜能和缺血区定向归巢特性使EPCs成为缺血性心血管疾病治疗的良好种子细胞。正常情况下,骨髓中EPCs处于休眠状态,在损伤、缺血等因素作用下EPCs从骨髓释放到外周血循环或经人为移植到体内,归巢于特定器官或组织,并在局部分化、增殖形成内皮细胞和新生血管。研究显示,缺血性心脏病患者血液循环中的EPCs数量下降了近50%,迁移能力受损。因此移植高增殖活性的EPCs是理想的细胞补充治疗方法。
目前,EPCs治疗IHD常用的移植途径主要有经皮冠状动脉内注射、心肌内注射及经心内膜注射等途径,以上途径创伤性较大,因此极大地限制了EPCs在IHD中的应用。此前研究认为,只有通过介入及有创的方法方能将细胞准确定位并高浓度聚集于缺血心肌,从而实现局部治疗。然而有研究报道,大部分心肌内注射的骨髓源性基质干细胞在4d后发生死亡,约60%经冠脉内注射的骨髓单核细胞(Marrow mononuclear cell, MMNC)被脾脏、肝脏摄取,心脏残留的细胞数量仅约10%。那么如何解决EPCs移植有创且效率低下的问题呢?这是能否将这种血管新生及细胞再生治疗方法广泛应用的一个关键问题。因此,急需寻找一种无创、有效且定向性好的EPCs移植方法。
最近Zen等利用脉冲式治疗超声联合白蛋白微泡(Microbubbles) Optison,将骨髓单核细胞经外周静脉输入充血性心肌病(Congestive cardiomyopathy, CC)动物模型,结果显示超声辐照+微泡+细胞移植组实验动物心肌组织血管细胞粘附分子-1(VCAM-1)和细胞间粘附分子-1(ICAM-1)表达水平比单纯静脉注射细胞组更高,其靶向性粘附能力显著高于单纯静脉移植细胞组,说明在超声波作用下微泡破裂能提高细胞移植的靶向能力进而可能更有效地实现细胞的靶向传输。
本研究尝试利用超声联合超声造影剂(微泡)介导EPCs的无创、定向移植探索一种新方法,探讨在该传递系统介导下能否有效促进细胞靶向归巢,初步探索其治疗心肌梗死的可行性、有效性。
方法:
1.大鼠骨髓源性EPCs分离、诱导培养及鉴定:
Ficoll密度梯度离心法分离SD大鼠骨髓源性单个核细胞,接种于包被纤维连接蛋白的培养板中,在添加了一定浓度的VEGF(50ng/mL)、bFGF(5ng/mL)和ECF(10ng/mL)等生长因子的培养液中常规培养;倒置显微镜下动态观察细胞生长特性及形态学变化,绘制细胞生长曲线;流式细胞技术检测细胞表面抗原CD34和VEGFR-2的表达;免疫荧光染色法检测内皮细胞特异性分子标志vWF以及摄取乙酰化低密度脂蛋白(acLDL)和结合荆豆凝集素-I(UEA-I)的特性。
2.大鼠心肌梗死(Myocardial infarction, MI)模型的建立与评价:
40只健康SD大鼠,结扎冠状动脉左前降支(Left anterior descending coronary artery, LAD)建立MI模型。建立模型之前和建模后1w,二维超声与M型超声观察梗死区室壁厚度、搏动幅度,M型超声与左心室Tei指数检测左心室功能。采用HE染色与Masson’s染色法评价模型的建立。
3.诊断超声联合微泡经静脉移植EPCs治疗MI的有效性评价:
将建模成功的42只模型大鼠随机分为三组:单纯EPCs组(n=15)、超声辐照(Ultrasound, US)+微泡(Microbubbles, MB)+EPCs组(n=15)、对照组(Normal sodium, NS) (n=12)。超声条件设置:Vivid 7超声诊断仪,3S探头,频率设定为5MHz,机械指数(Mechanical index, MI)1.3,作用时间8 min,采用触发条件,触发间期为1s。模型建立1w后分组治疗,对照组经尾静脉给予1ml NS,单纯EPCs组直接将细胞(1×106个混悬于1mlNS)经尾静脉缓慢注入,US+MB+EPCs组则先经尾静脉注入本科自制的超声造影剂“脂氟显”(稀释10倍后按1ml/kg剂量),然后在1min内缓慢注入EPCs,注入微泡时启动超声。细胞移植治疗4w后,处死动物、取材,采用HE染色,观测并计数梗死区及周围区毛细血管密度(Capillary density, CD)、心肌组织与血管通透性改变;采用Masson’s染色测量计算心肌梗死面积;免疫组织化学检测梗死区CD34表达。M型超声心动图与Tei指数评价各组左心室功能。
所有实验数据以均数±标准差( x±s)表示,统计学处理采用单因素方差分析和配对t检验。所有统计计算由SPSS13.0软件完成,统计结果以P<0.05为差异有统计学意义。结果:
1.大鼠骨髓源性EPCs培养及鉴定:
细胞培养3d后可以观察到细胞增多增大,呈纺锤形或短梭形细胞,逐渐伸出伪足样突起。5d后细胞较前明显增多,7d后细胞数显著增加,呈内皮细胞特异性的条索样结构,呈集落样生长。随着培养时间的延长,细胞相互融合,首尾相连呈“铺路石”状。流式细胞技术检测细胞表面抗原CD34、VEGFR-2表达率分别为86.02 %和81.37%;免疫荧光染色检测显示所培养的细胞表达vWF,并具有摄取acLDL和结合UEA-I的能力。
2.大鼠MI模型的建立与评价:
成功建立MI模型29只,其余11只大鼠分别死于麻醉意外、呼吸衰竭和心力衰竭等原因。建模成功大鼠二维超声检查可以看到其左心室前壁变薄、运动减弱或消失;超声检测建模前后大鼠左心功能,差异有十分显著的统计学意义(P<0.01)。HE染色显示大量心肌细胞坏死,疤痕形成;Masson’s染色显示左心室前壁心肌梗死区被胶原纤维取代,染色呈蓝色。
3.诊断超声联合微泡经静脉移植EPCs治疗MI的有效性评价:
HE染色计数心肌内CD:US+MB+EPCs组CD为(25.3±12.2)个/视野,明显高于对照组(12.6±4.5)个/视野和单纯EPCs组(19.2±6.2)个/视野(两者P<0.01)。大鼠心肌梗死面积的测定结果显示对照组的梗死面积为(41.9±4.3)%,EPCs组梗死面积为(36.7±3.8)%,而US+MB+EPCs组梗死面积为(25.3±4.5)%,明显低于前两者(两者P<0.01)。
免疫组织化学检测采用CD34显示各组血管,阳性呈棕黄色。US+MB+EPCs组阳性血管最多,分布密集,单纯EPCs组也可见较丰富的血管分布,而对照组的阳性血管最少。
US+MB+EPCs组的左心室收缩功能FS测值(50.27±5.98)% ,高于对照组(30.24±9.35) %(P<0.01)和单纯EPCs组(41.60±8.0) %(P<0.05)。EF测值比较,US+MB+EPCs组(85.9±2.18)%高于对照组(61.94±14.06)%(P<0.01),高于单纯EPCs组(76.84±9.45)% (P<0.05)。左心室Tei指数检测:US+MB+EPCs组(0.57±0.10)分别低于单纯EPCs组(0.66±0.08)(P<0.05)和对照组(0.83±0.13)(P<0.01)。
结论:
1.大鼠骨髓中单个核细胞可诱导分化为EPCs。VEGF、bFGF和EGF可促进EPCs的增殖分化。
2.通过结扎大鼠LAD成功建立了稳定的MI模型。左心室Tei指数可作为评价心肌梗死后心功能的指标。
3.诊断超声联合微泡介导经静脉移植EPCs可改善MI大鼠心功能。其机制可能与超声空化效应与机械效应通过增加局部血管通透性促使EPCs归巢心肌缺血区并分化形成血管内皮,促进血管新生有关。
Backgrounds and objectives:
Ischemic heart disease (IHD) is a kind of myocardial injury caused by the imbalance between coronary perfusion and requirement of myocardium due to the change of coronary circulation, about 90% of which consists of coronary artery disease. The conventional therapy is to reconstruct the blood supply, but it can not repair the injuried and necrotic myocardium. The stem cells transplantation might regenerate the vessel and myocardium, which lights the hope of treating IHD completely in the future.
Recent studies showed that endothelial progenitor cells (EPCs) contributed to angiogenesis involved in the repair of ischemic tissues after birth and had potential to be a kind of therapeutic method for myocardial ischemia (MI). A number of experimental and clinical trials have proved that transplantation of EPCs in ischemic heart disease made an effect. But there were some problems yet limiting the application of EPCs in IHC, such as the invasive operation of implantation, the low efficiency of cell therapy and the targeted homing ability of stem cells needing to improve.
The possible mechanisms of the transplantation of EPCs by the ultrasound-mediated microbubble destruction in prompting the homing and the treatment of EPCs in ischemic heart are as following. Firstly, the sonoporation induced by ultrasound-mediated microbubbles destruction leads to the increased permeability of blood vessels in myocardium, which is helpful to the homing of EPCs into ischemic heart, and the shock wave and microstream produced during bubbles’blowing up prompts the EPCs to come into ischemic heart and helps the neovascularization and cell proliferation. Secondly, the ultrasound probe was positioned on the chest, so the regional part of ishemic anterior wall was improved in permeability and the intravenously injected EPCs were easy to home to ischemic heart. What’s more, studies showed that cavitation induced by ultrasound and microbubbles might stimulate vessel formation.
This research tried to explore a non-invasive and targeted cell transplantation method by applying ultrasound-mediated microbubbles destruction system, find out whether the targeted homing of EPCs would be improved, and evaluate the feasibility and the efficiency of EPCs in the treatment of myocardial infarction.
Methods:
1. Isolation, culture and identification of bone marrow-derived EPCs:
EPCs derived from bone marrow mononuclear cells of rats were isolated by Ficoll gradient centrifugation. Cells were planted on culture dishes coated with human fibronectin and maintained in M199 supplemented with VEGF(50ng/mL)、bFGF(5ng/mL) and EGF(10ng/mL) . Cell growth and morphology were observed. The cell specific surface mark CD34 and VEGFR-2 were assessed by fluorescence activated cell sorter ( FACS) analysis.The cell phaenotype vWF, the function of EPCs taking in ac-LDL and binding UEA-I were detected by immunofluorescence.
2. Myocardial infarction model building and assessment:
Model of myocardial infarction of 40 adult SD rats builded by ligating the left anterior descending coronary artery. Before building and one week after modeling, two-dimensional ultrasound was performed to observe the thickness and motion of infarcted wall; left ventricular function was analyzed with M-mode echocardiography; Tei index was also detected to evaluated the left heart function. HE and Masson’s dye were applied to prove the successful building of MI models in pathology.
3. Efficacy of intravenous injection of EPCs mediated by the diagnostic ultrasound combined with microbubbles in MI rats:
Fourty two MI rats were randomly divided into three groups: EPCs infusion group (n=15), ultrasound (US) + microbubbles (MB) + EPCs group ( n =15) and the control group (NS only) (n=12),. The ultrasound parameters were set as following: VIVID 7 ultrasound system, 3S probe with frequency 5 MHz, Mechanical Index (MI) set as 1.3 for eight-minute irradiation with a time trigger at 1 second. After the model had been builded for 1 week, 1 ml saline was injected, 1×106 cells were infused within 1 ml saline and injected through tail vena caudalis slowly in EPCs infusion group, and lipid microbubbles of 1ml/kg were injected, followed by intravenous injection of EPCs combined with the diagnostic ultrasound irradiation for 8 minutes. Four weeks after cell therapy, capillary density (CD) was counted with HE dye slices in each group, and infarct size was determined;CD34 expression in ischemic myocardium was assessed with immunohistochemistry, and left cardiac function were detected by M-mode and Tei index and compared in three groups.
All the data were presented as Mean±SD ( x±s) and one-way ANOVA and paired t test were applied in the statistic analysis, which was accomplished by SPSS 13.0 software in computer, P<0.05 was regarded as having statistical significance.
Results:
1. Isolation,culture and identification of bone marrow-derived EPCs:
Three days later, cells number increased and cell body stretched. Fusiform cells appeared accompanied with some cell processes. Five days later, the number of fusiform cells augmented obviously and cell cluster appeared. Seven days later, cells connected with each other and led to chord, reticulate, or blood capillary-shaped structure. With the culture time lasting, cells mixed together, resulting in the cobble-stone morphology. CD34 and VEGFR-2 and positive cells were 86.02% and 81.37% respectively, assessed by fluorescence activated cell sorter ( FACS) analysis. Cells cultivating expressed vWF phaenotype, capable of intaking acLDL and binding UEA-I.
2. Myocardial infarction model building and assessment:
MI model was builded successfully in 29 rats and the other 11 rats died of anesthetic accident, respiratory failure, cardia failure and etc. The anterior wall of left ventricle thinningzed and asynersised, observed by 2D ultrasound. Left ventricular function(LVF) detected with ultrasound before and after model establishment showed that there was distinguished statistical significance (P<0.01). HE dye showed that there was a great quantity of cardiac muscle cellular necrosis. Masson’s dye showed that the MI area of left ventricle’anterior wall was dyed into blue.
3. Efficacy of intravenous injection of EPCs mediated by the diagnostic ultrasound combined with microbubbles in MI rats:
There was significant differences of CD between US+MB+EPCs group(25.3±12.2)and control group(12.6±4.5)and EPCs infusion group (19.2±6.2) respectively, both P<0.01. There were erythrocytes among the myocardial fibers in US+MB+EPCs group, which leaked out of the vessels, while there was nearly none in the other two groups. The result of the infarct size showed that US+MB+EPCs group(25.3±4.5)% was obviously lower than control group (41.9±4.3)% and EPCs infusion group(36.7±3.8)%( P<0.01). CD34 was expressed most in US+MB+EPCs group, with the brown positive blood vessels distributing intensively, while it was less in EPCS and the lest in control. There were significant differences in FS, EF and Tei index between US+MB+EPCs group and the other two groups. FS in US+MB+EPCs group was much higher than those in EPCs infusion group and control group (P<0.05). So were the EF and Tei index (P<0.05, P<0.01).
All the data were presented as Mean±SD ( x±s) and one-way ANOVA and paired t test applied in the statistic analysis, which was accomplished by SPSS 13.0 software in computer, P<0.05 was regarded as having statistical significance.
Conclusions:
1. The mononuclear cells from the bone marrow of rats could be induced into endothelial progenitor cells. The VEGF, bFGF and the EGF can make up the optimal induction condition.
2. MI model of rat built with LAD ligation is successful and stable. Tei index is a good parameter to appraise cardiac function after myocardial infarction.
3. Intravenous implantation of EPCs mediated by diagnostic ultrasound and microbubbles can improve the heart function of MI rat. It relates to the the effect of cavitation and mechanical effect that ultrasound combined with microbubbles can increase the permeability of the ischemic heart, which facilitates the homing and gathering of EPCs and results in the enhanced neovascularization.
缺血性心脏病(Ischemic heart disease,IHD)是由于冠状动脉循环改变引起冠状动脉血流和心肌需求之间不平衡而导致的心肌损害,其中最常见的原因是冠心病,约占90%。传统的治疗手段主要是重建血供,而不能修复损伤和坏死的心肌。近几年兴起的干细胞移植法通过再生血管与心肌组织,使人们看到了治疗IHD的希望。
血管内皮祖细胞(Endothelial progenitor cells, EPCs)是指具有特异性归巢于血管新生组织并能分化增殖为成熟内皮细胞(Endothelial cells, ECs)的一群干/祖细胞。研究发现EPCs参与出生后的内皮修复和血管新生过程,提示其在IHD中重要的治疗作用以及广阔的临床应用前景。高增殖潜能和缺血区定向归巢特性使EPCs成为缺血性心血管疾病治疗的良好种子细胞。正常情况下,骨髓中EPCs处于休眠状态,在损伤、缺血等因素作用下EPCs从骨髓释放到外周血循环或经人为移植到体内,归巢于特定器官或组织,并在局部分化、增殖形成内皮细胞和新生血管。研究显示,缺血性心脏病患者血液循环中的EPCs数量下降了近50%,迁移能力受损。因此移植高增殖活性的EPCs是理想的细胞补充治疗方法。
目前,EPCs治疗IHD常用的移植途径主要有经皮冠状动脉内注射、心肌内注射及经心内膜注射等途径,以上途径创伤性较大,因此极大地限制了EPCs在IHD中的应用。此前研究认为,只有通过介入及有创的方法方能将细胞准确定位并高浓度聚集于缺血心肌,从而实现局部治疗。然而有研究报道,大部分心肌内注射的骨髓源性基质干细胞在4d后发生死亡,约60%经冠脉内注射的骨髓单核细胞(Marrow mononuclear cell, MMNC)被脾脏、肝脏摄取,心脏残留的细胞数量仅约10%。那么如何解决EPCs移植有创且效率低下的问题呢?这是能否将这种血管新生及细胞再生治疗方法广泛应用的一个关键问题。因此,急需寻找一种无创、有效且定向性好的EPCs移植方法。
最近Zen等利用脉冲式治疗超声联合白蛋白微泡(Microbubbles) Optison,将骨髓单核细胞经外周静脉输入充血性心肌病(Congestive cardiomyopathy, CC)动物模型,结果显示超声辐照+微泡+细胞移植组实验动物心肌组织血管细胞粘附分子-1(VCAM-1)和细胞间粘附分子-1(ICAM-1)表达水平比单纯静脉注射细胞组更高,其靶向性粘附能力显著高于单纯静脉移植细胞组,说明在超声波作用下微泡破裂能提高细胞移植的靶向能力进而可能更有效地实现细胞的靶向传输。
本研究尝试利用超声联合超声造影剂(微泡)介导EPCs的无创、定向移植探索一种新方法,探讨在该传递系统介导下能否有效促进细胞靶向归巢,初步探索其治疗心肌梗死的可行性、有效性。
方法:
1.大鼠骨髓源性EPCs分离、诱导培养及鉴定:
Ficoll密度梯度离心法分离SD大鼠骨髓源性单个核细胞,接种于包被纤维连接蛋白的培养板中,在添加了一定浓度的VEGF(50ng/mL)、bFGF(5ng/mL)和ECF(10ng/mL)等生长因子的培养液中常规培养;倒置显微镜下动态观察细胞生长特性及形态学变化,绘制细胞生长曲线;流式细胞技术检测细胞表面抗原CD34和VEGFR-2的表达;免疫荧光染色法检测内皮细胞特异性分子标志vWF以及摄取乙酰化低密度脂蛋白(acLDL)和结合荆豆凝集素-I(UEA-I)的特性。
2.大鼠心肌梗死(Myocardial infarction, MI)模型的建立与评价:
40只健康SD大鼠,结扎冠状动脉左前降支(Left anterior descending coronary artery, LAD)建立MI模型。建立模型之前和建模后1w,二维超声与M型超声观察梗死区室壁厚度、搏动幅度,M型超声与左心室Tei指数检测左心室功能。采用HE染色与Masson’s染色法评价模型的建立。
3.诊断超声联合微泡经静脉移植EPCs治疗MI的有效性评价:
将建模成功的42只模型大鼠随机分为三组:单纯EPCs组(n=15)、超声辐照(Ultrasound, US)+微泡(Microbubbles, MB)+EPCs组(n=15)、对照组(Normal sodium, NS) (n=12)。超声条件设置:Vivid 7超声诊断仪,3S探头,频率设定为5MHz,机械指数(Mechanical index, MI)1.3,作用时间8 min,采用触发条件,触发间期为1s。模型建立1w后分组治疗,对照组经尾静脉给予1ml NS,单纯EPCs组直接将细胞(1×106个混悬于1mlNS)经尾静脉缓慢注入,US+MB+EPCs组则先经尾静脉注入本科自制的超声造影剂“脂氟显”(稀释10倍后按1ml/kg剂量),然后在1min内缓慢注入EPCs,注入微泡时启动超声。细胞移植治疗4w后,处死动物、取材,采用HE染色,观测并计数梗死区及周围区毛细血管密度(Capillary density, CD)、心肌组织与血管通透性改变;采用Masson’s染色测量计算心肌梗死面积;免疫组织化学检测梗死区CD34表达。M型超声心动图与Tei指数评价各组左心室功能。
所有实验数据以均数±标准差( x±s)表示,统计学处理采用单因素方差分析和配对t检验。所有统计计算由SPSS13.0软件完成,统计结果以P<0.05为差异有统计学意义。结果:
1.大鼠骨髓源性EPCs培养及鉴定:
细胞培养3d后可以观察到细胞增多增大,呈纺锤形或短梭形细胞,逐渐伸出伪足样突起。5d后细胞较前明显增多,7d后细胞数显著增加,呈内皮细胞特异性的条索样结构,呈集落样生长。随着培养时间的延长,细胞相互融合,首尾相连呈“铺路石”状。流式细胞技术检测细胞表面抗原CD34、VEGFR-2表达率分别为86.02 %和81.37%;免疫荧光染色检测显示所培养的细胞表达vWF,并具有摄取acLDL和结合UEA-I的能力。
2.大鼠MI模型的建立与评价:
成功建立MI模型29只,其余11只大鼠分别死于麻醉意外、呼吸衰竭和心力衰竭等原因。建模成功大鼠二维超声检查可以看到其左心室前壁变薄、运动减弱或消失;超声检测建模前后大鼠左心功能,差异有十分显著的统计学意义(P<0.01)。HE染色显示大量心肌细胞坏死,疤痕形成;Masson’s染色显示左心室前壁心肌梗死区被胶原纤维取代,染色呈蓝色。
3.诊断超声联合微泡经静脉移植EPCs治疗MI的有效性评价:
HE染色计数心肌内CD:US+MB+EPCs组CD为(25.3±12.2)个/视野,明显高于对照组(12.6±4.5)个/视野和单纯EPCs组(19.2±6.2)个/视野(两者P<0.01)。大鼠心肌梗死面积的测定结果显示对照组的梗死面积为(41.9±4.3)%,EPCs组梗死面积为(36.7±3.8)%,而US+MB+EPCs组梗死面积为(25.3±4.5)%,明显低于前两者(两者P<0.01)。
免疫组织化学检测采用CD34显示各组血管,阳性呈棕黄色。US+MB+EPCs组阳性血管最多,分布密集,单纯EPCs组也可见较丰富的血管分布,而对照组的阳性血管最少。
US+MB+EPCs组的左心室收缩功能FS测值(50.27±5.98)% ,高于对照组(30.24±9.35) %(P<0.01)和单纯EPCs组(41.60±8.0) %(P<0.05)。EF测值比较,US+MB+EPCs组(85.9±2.18)%高于对照组(61.94±14.06)%(P<0.01),高于单纯EPCs组(76.84±9.45)% (P<0.05)。左心室Tei指数检测:US+MB+EPCs组(0.57±0.10)分别低于单纯EPCs组(0.66±0.08)(P<0.05)和对照组(0.83±0.13)(P<0.01)。
结论:
1.大鼠骨髓中单个核细胞可诱导分化为EPCs。VEGF、bFGF和EGF可促进EPCs的增殖分化。
2.通过结扎大鼠LAD成功建立了稳定的MI模型。左心室Tei指数可作为评价心肌梗死后心功能的指标。
3.诊断超声联合微泡介导经静脉移植EPCs可改善MI大鼠心功能。其机制可能与超声空化效应与机械效应通过增加局部血管通透性促使EPCs归巢心肌缺血区并分化形成血管内皮,促进血管新生有关。
Backgrounds and objectives:
Ischemic heart disease (IHD) is a kind of myocardial injury caused by the imbalance between coronary perfusion and requirement of myocardium due to the change of coronary circulation, about 90% of which consists of coronary artery disease. The conventional therapy is to reconstruct the blood supply, but it can not repair the injuried and necrotic myocardium. The stem cells transplantation might regenerate the vessel and myocardium, which lights the hope of treating IHD completely in the future.
Recent studies showed that endothelial progenitor cells (EPCs) contributed to angiogenesis involved in the repair of ischemic tissues after birth and had potential to be a kind of therapeutic method for myocardial ischemia (MI). A number of experimental and clinical trials have proved that transplantation of EPCs in ischemic heart disease made an effect. But there were some problems yet limiting the application of EPCs in IHC, such as the invasive operation of implantation, the low efficiency of cell therapy and the targeted homing ability of stem cells needing to improve.
The possible mechanisms of the transplantation of EPCs by the ultrasound-mediated microbubble destruction in prompting the homing and the treatment of EPCs in ischemic heart are as following. Firstly, the sonoporation induced by ultrasound-mediated microbubbles destruction leads to the increased permeability of blood vessels in myocardium, which is helpful to the homing of EPCs into ischemic heart, and the shock wave and microstream produced during bubbles’blowing up prompts the EPCs to come into ischemic heart and helps the neovascularization and cell proliferation. Secondly, the ultrasound probe was positioned on the chest, so the regional part of ishemic anterior wall was improved in permeability and the intravenously injected EPCs were easy to home to ischemic heart. What’s more, studies showed that cavitation induced by ultrasound and microbubbles might stimulate vessel formation.
This research tried to explore a non-invasive and targeted cell transplantation method by applying ultrasound-mediated microbubbles destruction system, find out whether the targeted homing of EPCs would be improved, and evaluate the feasibility and the efficiency of EPCs in the treatment of myocardial infarction.
Methods:
1. Isolation, culture and identification of bone marrow-derived EPCs:
EPCs derived from bone marrow mononuclear cells of rats were isolated by Ficoll gradient centrifugation. Cells were planted on culture dishes coated with human fibronectin and maintained in M199 supplemented with VEGF(50ng/mL)、bFGF(5ng/mL) and EGF(10ng/mL) . Cell growth and morphology were observed. The cell specific surface mark CD34 and VEGFR-2 were assessed by fluorescence activated cell sorter ( FACS) analysis.The cell phaenotype vWF, the function of EPCs taking in ac-LDL and binding UEA-I were detected by immunofluorescence.
2. Myocardial infarction model building and assessment:
Model of myocardial infarction of 40 adult SD rats builded by ligating the left anterior descending coronary artery. Before building and one week after modeling, two-dimensional ultrasound was performed to observe the thickness and motion of infarcted wall; left ventricular function was analyzed with M-mode echocardiography; Tei index was also detected to evaluated the left heart function. HE and Masson’s dye were applied to prove the successful building of MI models in pathology.
3. Efficacy of intravenous injection of EPCs mediated by the diagnostic ultrasound combined with microbubbles in MI rats:
Fourty two MI rats were randomly divided into three groups: EPCs infusion group (n=15), ultrasound (US) + microbubbles (MB) + EPCs group ( n =15) and the control group (NS only) (n=12),. The ultrasound parameters were set as following: VIVID 7 ultrasound system, 3S probe with frequency 5 MHz, Mechanical Index (MI) set as 1.3 for eight-minute irradiation with a time trigger at 1 second. After the model had been builded for 1 week, 1 ml saline was injected, 1×106 cells were infused within 1 ml saline and injected through tail vena caudalis slowly in EPCs infusion group, and lipid microbubbles of 1ml/kg were injected, followed by intravenous injection of EPCs combined with the diagnostic ultrasound irradiation for 8 minutes. Four weeks after cell therapy, capillary density (CD) was counted with HE dye slices in each group, and infarct size was determined;CD34 expression in ischemic myocardium was assessed with immunohistochemistry, and left cardiac function were detected by M-mode and Tei index and compared in three groups.
All the data were presented as Mean±SD ( x±s) and one-way ANOVA and paired t test were applied in the statistic analysis, which was accomplished by SPSS 13.0 software in computer, P<0.05 was regarded as having statistical significance.
Results:
1. Isolation,culture and identification of bone marrow-derived EPCs:
Three days later, cells number increased and cell body stretched. Fusiform cells appeared accompanied with some cell processes. Five days later, the number of fusiform cells augmented obviously and cell cluster appeared. Seven days later, cells connected with each other and led to chord, reticulate, or blood capillary-shaped structure. With the culture time lasting, cells mixed together, resulting in the cobble-stone morphology. CD34 and VEGFR-2 and positive cells were 86.02% and 81.37% respectively, assessed by fluorescence activated cell sorter ( FACS) analysis. Cells cultivating expressed vWF phaenotype, capable of intaking acLDL and binding UEA-I.
2. Myocardial infarction model building and assessment:
MI model was builded successfully in 29 rats and the other 11 rats died of anesthetic accident, respiratory failure, cardia failure and etc. The anterior wall of left ventricle thinningzed and asynersised, observed by 2D ultrasound. Left ventricular function(LVF) detected with ultrasound before and after model establishment showed that there was distinguished statistical significance (P<0.01). HE dye showed that there was a great quantity of cardiac muscle cellular necrosis. Masson’s dye showed that the MI area of left ventricle’anterior wall was dyed into blue.
3. Efficacy of intravenous injection of EPCs mediated by the diagnostic ultrasound combined with microbubbles in MI rats:
There was significant differences of CD between US+MB+EPCs group(25.3±12.2)and control group(12.6±4.5)and EPCs infusion group (19.2±6.2) respectively, both P<0.01. There were erythrocytes among the myocardial fibers in US+MB+EPCs group, which leaked out of the vessels, while there was nearly none in the other two groups. The result of the infarct size showed that US+MB+EPCs group(25.3±4.5)% was obviously lower than control group (41.9±4.3)% and EPCs infusion group(36.7±3.8)%( P<0.01). CD34 was expressed most in US+MB+EPCs group, with the brown positive blood vessels distributing intensively, while it was less in EPCS and the lest in control. There were significant differences in FS, EF and Tei index between US+MB+EPCs group and the other two groups. FS in US+MB+EPCs group was much higher than those in EPCs infusion group and control group (P<0.05). So were the EF and Tei index (P<0.05, P<0.01).
All the data were presented as Mean±SD ( x±s) and one-way ANOVA and paired t test applied in the statistic analysis, which was accomplished by SPSS 13.0 software in computer, P<0.05 was regarded as having statistical significance.
Conclusions:
1. The mononuclear cells from the bone marrow of rats could be induced into endothelial progenitor cells. The VEGF, bFGF and the EGF can make up the optimal induction condition.
2. MI model of rat built with LAD ligation is successful and stable. Tei index is a good parameter to appraise cardiac function after myocardial infarction.
3. Intravenous implantation of EPCs mediated by diagnostic ultrasound and microbubbles can improve the heart function of MI rat. It relates to the the effect of cavitation and mechanical effect that ultrasound combined with microbubbles can increase the permeability of the ischemic heart, which facilitates the homing and gathering of EPCs and results in the enhanced neovascularization.
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2. Caprioli A, Jaffredo T, Gautier R, et al. Bolld borneseeding by hematopoietic and endothelial precursors fromthe allontois [J]. Proc Natl Acad Sci USA, 1998, 95: 1641～1646.
3. Schuh A, Liehn EA, Sasse A, et al. Transplantation of endothelial progenitor cells improves neovascularization and left ventricular function after myocardial infarction in a rat model. Basic Res Cardiol, 2008, 103(1): 69-77.
4. Fox A, Smythe J, Fisher N, et al. Mobilization of endothelial progenitor cells into the circulation in burned patients. Br J Surg, 2008, 95(2): 244-251.
5. Murohara T, Therapeutic vasculogenesis using human coed blood-derived endtothelial progenitors. Trends Cardiovasc Med, 2001, 11: 303-307.
6. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000, 105: 1527-1536.
7. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow [J ]. Clin Invest, 2002, 109 (3): 337-346.
8. Sainz J, Al Haj Zen A, Caligiuri G, et al. Isolation of "side population" progenitor cells from healthy arteries of adult mice. Arterioscler Thromb Vasc Biol, 2006, 26(2): 281-286.
9. Harraz M, Jiao C, Hanlon HD, et al. CD34-blood derived human endothelial cell progenitors. Stem Cells, 2001,19(4): 304.
10. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow [J ]. Clin Invest, 2002, 109 (3): 337-346.
11. Li B,Sharpe EE,Maupin AB,et al.1 VEGF and PGF promote adult vasculogenesis by enhancing EPC recruitment and vessel formation at the site of tumor neovascularization. FASEB [J ], 2006, 20(9):1495.
12. Verama S,Kuliszewski MA,Li SH,et al. C-reactive protein attenuates endothelial progenitor cell survival,differentiation,and function:further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation, 2004, 109(17):2058.
13. Murayama T,T epper OM,Silver M,et al. Determination of bone marrow derived endothelial progenitor cell significance in angiogenic growth factor induced neovascularization in vivo 1 Exp Hematol,2002,30(8):967.
14. Walter DH,Rittig K,Bahhnann FH,et al. Statin therapy accelerates endothelialization:a novel efect involving mobilization and incorporation of bone marrow derived endothelial progenitor cells Circulation, 2002,105(25): 3017.
15. Landmeser U, Eug berdiug N, Bahlmann FH,et al. Statin induced improvement ofendothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function and survival after experimental myocardial infarction requires endothelial nitricoxide synthase. Circulation, 2004,110(14):1933.
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18. Ribatti D. The discovery of endothelial progenitor cells: An historical review. Leuk Res, 2007, 31(4): 439.
19. Ni H, Yuen P S, Papalia J M, et al. Plasma fibronectin promotes thrombus growth and stability in injured arterioles[J]. Proc Natl Acad Sci USA, 2003, 100 (5): 2415-2419.
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22. Schachinger V, Assmus B, Britten MB, et al. T ransplantation of progenitor cells and regeneration enhancement in acute myocardial infarction:final one year results of the TOPCARE AMI T rial1 J Am Coll Cardiol, 2004, 44(8): l690.
23. Kalka C, Masuda H, T akahashi T, et al. Transplantation of exvivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA, 2000, 97(7): 3422.
24. Ikenaga S, Hamano K, Nishida M, et al. Autologous bone marrow implantation induced angiogenes is and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res, 2001, 96(2): 277.
25. Taniguchi E, K in M, T orimur TA, et al. Endothelial progenitor cell transplantationimproves the survival following liver injury in mice. Gastro enterology, 2006, 130(2): 521.
26. Schmidt D, Breymann C, Weber A, et al. Umbilical cord blood derived endothelial progenitor cells for tissue engineering of vascular grafts1 Ann Thorac Surg, 2004, 78(6): 2094.
27. Kaushal S, Amiel GE, Guleserian K J, et al. Functional small diameter neovessels created using endothelial progenitor cells expanded exvivo. Nat Med, 2001, 7(9): 1035.
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9. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000, 105: 1527-1536.
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11. Sainz J, Al Haj Zen A, Caligiuri G, et al. Isolation of "side population" progenitor cells from healthy arteries of adult mice. Arterioscler Thromb Vasc Biol, 2006, 26(2): 281-286.
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1. Asahara T, Mumhara T, Suilivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science,1997, 275 (5302): 964.
2. Caprioli A, Jaffredo T, Gautier R, et al. Bolld borneseeding by hematopoietic and endothelial precursors fromthe allontois [J]. Proc Natl Acad Sci USA, 1998, 95: 1641～1646.
3. Schuh A, Liehn EA, Sasse A, et al. Transplantation of endothelial progenitor cells improves neovascularization and left ventricular function after myocardial infarction in a rat model. Basic Res Cardiol, 2008, 103(1): 69-77.
4. Fox A, Smythe J, Fisher N, et al. Mobilization of endothelial progenitor cells into the circulation in burned patients. Br J Surg, 2008, 95(2): 244-251.
5. Murohara T, Therapeutic vasculogenesis using human coed blood-derived endtothelial progenitors. Trends Cardiovasc Med, 2001, 11: 303-307.
6. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000, 105: 1527-1536.
7. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow [J ]. Clin Invest, 2002, 109 (3): 337-346.
8. Sainz J, Al Haj Zen A, Caligiuri G, et al. Isolation of "side population" progenitor cells from healthy arteries of adult mice. Arterioscler Thromb Vasc Biol, 2006, 26(2): 281-286.
9. Harraz M, Jiao C, Hanlon HD, et al. CD34-blood derived human endothelial cell progenitors. Stem Cells, 2001,19(4): 304.
10. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow [J ]. Clin Invest, 2002, 109 (3): 337-346.
11. Li B,Sharpe EE,Maupin AB,et al.1 VEGF and PGF promote adult vasculogenesis by enhancing EPC recruitment and vessel formation at the site of tumor neovascularization. FASEB [J ], 2006, 20(9):1495.
12. Verama S,Kuliszewski MA,Li SH,et al. C-reactive protein attenuates endothelial progenitor cell survival,differentiation,and function:further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation, 2004, 109(17):2058.
13. Murayama T,T epper OM,Silver M,et al. Determination of bone marrow derived endothelial progenitor cell significance in angiogenic growth factor induced neovascularization in vivo 1 Exp Hematol,2002,30(8):967.
14. Walter DH,Rittig K,Bahhnann FH,et al. Statin therapy accelerates endothelialization:a novel efect involving mobilization and incorporation of bone marrow derived endothelial progenitor cells Circulation, 2002,105(25): 3017.
15. Landmeser U, Eug berdiug N, Bahlmann FH,et al. Statin induced improvement ofendothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function and survival after experimental myocardial infarction requires endothelial nitricoxide synthase. Circulation, 2004,110(14):1933.
16. Iwaguro H, Yamaguchi J, Kalka C, et al. Endothelial p rogenitor cell vascular endothelial growth factor gene transfer for vascular regeneration[J]. Circulation, 2002, 105(6): 732-738.
17. Kalka C, Masuda H, Takahashi T, et al. Transplantation of exviov expanded endothelial progenitor cells for therapeutic neovascularization [J] . Proc Natl Acad Sci USA, 2000, 97 (7): 3422-3427.
18. Ribatti D. The discovery of endothelial progenitor cells: An historical review. Leuk Res, 2007, 31(4): 439.
19. Ni H, Yuen P S, Papalia J M, et al. Plasma fibronectin promotes thrombus growth and stability in injured arterioles[J]. Proc Natl Acad Sci USA, 2003, 100 (5): 2415-2419.
20. Gerhard M, Roddy M A, Greager S J, et al. Ageing progressively impairs endothelium-dependent vasodilation in forearm resistance vessels of human[J]. Human, 1996, 27(4): 849-853.
21. Cosentino F, Luscher T F. Endothelial dysfunction in diabetes mellitus [J]. J Cardio Vasc Pharmocol, 1998, 32 (Supp l3):54-56.
22. Schachinger V, Assmus B, Britten MB, et al. T ransplantation of progenitor cells and regeneration enhancement in acute myocardial infarction:final one year results of the TOPCARE AMI T rial1 J Am Coll Cardiol, 2004, 44(8): l690.
23. Kalka C, Masuda H, T akahashi T, et al. Transplantation of exvivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA, 2000, 97(7): 3422.
24. Ikenaga S, Hamano K, Nishida M, et al. Autologous bone marrow implantation induced angiogenes is and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res, 2001, 96(2): 277.
25. Taniguchi E, K in M, T orimur TA, et al. Endothelial progenitor cell transplantationimproves the survival following liver injury in mice. Gastro enterology, 2006, 130(2): 521.
26. Schmidt D, Breymann C, Weber A, et al. Umbilical cord blood derived endothelial progenitor cells for tissue engineering of vascular grafts1 Ann Thorac Surg, 2004, 78(6): 2094.
27. Kaushal S, Amiel GE, Guleserian K J, et al. Functional small diameter neovessels created using endothelial progenitor cells expanded exvivo. Nat Med, 2001, 7(9): 1035.