VEGF及EPCs结合胶促进大鼠梗死心肌血管再生的可行性研究
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摘要
目的:通过相应的组织工程方法使得心梗后心肌血管再生。本研究包括:1,研究内皮细胞生长因子通过纤维蛋白胶释放到心肌从而刺激心肌血管的再生的可能性并观察心功能改善情况;2,通过可降解材料纤维蛋白胶将内皮祖细胞移植到大鼠心肌观察心肌血管再生情况和心功能改善情况。方法:1,本课题第一部分选择了28只9周龄SD大鼠,其中13只进行了左前降支(LAD)结扎术导致左室急性心肌梗死为心梗组,15只大鼠未行结扎术为非心梗组。首先制作纤维蛋白胶以及构成重组体α2-PI1–8-VEGF121,然后使二者结合构成VEGF结合胶用于移植。将7只心梗大鼠和6只非心梗大鼠心脏移植了VEGF结合胶;作为对照对6只心梗大鼠和6只非心梗大鼠移植了空白的纤维蛋白胶;另外3只为正常大鼠未作任何处理来作为组织学对照。在心梗大鼠中,移植在心梗后即刻进行。所有大鼠保存4周,处死前行心功能的超声检查。处死后对大鼠心肌进行组织学与免疫组织化学研究,即行CD31抗体染色和α-平滑肌肌动蛋白抗体染色(SMA)来探测血管。对非心梗心脏,首先定义两个区域,即胶下和间隔区(远离胶);而对心梗心脏定义为缺血区和间隔区。每个区域在放大200倍条件下各采3张相片,每张共6张相片。应用生物通用分析处理软件做定量的血管形态分析(为盲法)。通过3个参数来测量血管,即:①总的血管密度(在总的组织中的血管数量/mm2);②总的血管数在心肌区域中的比例(心肌区域中的总血管数量/mm2);③总血管区的百分比(总组织中血管区域的百分比)。总的组织部分要去除白色空白区域来除外因不同原因所致的空白区域(如切片制作过程中的破口等)所造成的偏倚,然后从总组织区域中去除血管区域来确定心肌区域。生物通用处理软件直接将血管分成3组,即:毛细血管组(血管直径<10μm);微血管组(10μm≤血管直径≤20μm);大血管组(血管直径>20μm)。动脉数量通过肉眼确定,即将组织切片放大100倍条件下通过肉眼对其组织学特性的标准来区别计数动脉数量。检测整个左室的动脉,动脉密度以每平方毫米总组织区域中动脉血管的数量来表达;2,第二部分选择了27只9周龄SD大鼠,体重300～350g。其中18只进行了左前降支(LAD)结扎术导致左室急性心肌梗死,其余9只未行结扎术。首先制作纤维蛋白胶以及从人脐带血中用磁珠法分离内皮祖细胞,然后将内皮祖细胞种植于纤维蛋白胶构成载有内皮祖细胞的纤维蛋白材料用于移植。将大鼠分为3个组:第一组为9只无心梗大鼠;第二组为9只心梗后立即进行移植的大鼠;第三组为9只心梗后1周进行移植的大鼠。每个组在移植后3周或8周处死。为了抑制大鼠对EPCs胶的免疫排斥反应本研究使用了微量渗透泵持续给予环保菌素A。大鼠处死前行经胸腔超声心动图检查以测定左室功能。处死后行组织学与免疫组织化学研究,即经股静脉注射15%KCI 2毫升处死大鼠使大鼠心跳停止在舒张期,取心脏进行福尔马林固定,蜡块包埋后做成3μm组织切片,行苏木精-伊红(HE)和免疫组化染色,人CD34抗体和Tra-1-85抗体染色来探测移植的人类细胞。心脏血管测量的方法与VEGF相同,应用同样的采样方法。即3个CD34抗体染色切片,用同样的方法在每个切片选择1个区域(非心梗心脏在胶下,心梗心脏在缺血区),且每个区域取6张相片。计算机评估血管的形态同第一部分。结果:1, VEGF纤维蛋白结合胶的血管生成效应:①首先为了除外因VEGF浓度过高使得血管的渗透性增高导致渗漏并使得心包或胸膜渗出,肉眼检查分析了VEGF胶移植在大鼠左室壁的效应,结果胸腔和心包均未发现异常渗出。在心脏和胸腔之间见到疏松的结缔组织,与空白胶组比较没有统计学差异。组织学和免疫组化研究,HE染色的心脏组织切片无移植物的心脏和有移植物4周后的心脏,可见一些疏松的结缔组织,但其在非心梗心脏中移植纤维蛋白胶组和未移植纤维蛋白胶组之间没有统计学差异可能因手术或缝线所致。心脏结构为相对正常形态,无血管瘤或血管畸形等发现。结果显示,与非心梗相比,在梗死心肌附近观察到含有新生血管的疏松结缔组织,但其在VEGF胶组和空白胶组之间没有统计学差异。②血管检测平滑肌细胞层的动脉和静脉均由α-平滑肌肌动蛋白抗体(SMA)染色,呈现棕色,但是,α-平滑肌肌动蛋白抗体(SMA)不能识别没有平滑肌层的毛细血管,故本研究使用CD31抗体染色内皮细胞识别任何一种血管。总的血管数量在心肌区域的比例和三个采样区域(即胶下区、胶边缘区和远离胶区)占总血管区域的百分比。非心梗大鼠15只(VEGF胶组6只,空胶组6只),在VEGF胶组或空白胶组中三个采样区域之间的三个参数测量结果中无统计学差异。然后比较了三个采样区域的VEGF胶组和空白胶组之间的相同的参数。结果发现,VEGF胶组的血管的数量有增多的趋势,但无统计学意义。然而,当将这三个采样区域联合在一起,观察所处理动物之间其占血管总数的百分比时,结果有统计学差异(ANOVA F(2,59)=3.46;P<0.05)。即VEGF胶组大鼠总血管区域的百分比明显高于空白胶组(P<0.05)。而且总血管的百分比的差异和总血管密度的差异很接近,也有统计学意义(ANOVA F(2,59)=6;P<0.005),即VEGF胶组在心肌区域总血管的比例与空白胶组相比有显著性差异(P<0.005)。而自从在三个采样区域之间参数没有显著性差异。本研究将三个采样区域改为两个区域,即胶缘区和远离胶区(间隔区),在每个区域采像6张。因三个参数的结果非常相近,故本研究采用了血管密度作为测量所有血管的参数。在非心梗组中,总血管密度首先在空白胶组与无胶组之间没有统计学差异,而且在VEGF胶组、空白胶组和无胶组中不同区域间(胶附近区和间隔区)也无统计学差异。在心梗组中,VEGF胶组总血管密度显著高于空白胶组,并且胶附近区也显著高于间隔区,而在空白胶组中两个采样区域之间未发现显著性差异。在心梗组和非心梗组之间的VEGF组或空白胶比较,结果没有统计学意义。因总的血管密度不是正态分布,本研究通过平方根转换使其正态化。对每个大鼠有36个观察点(3张切片2个部位,每个部位6张相片)。运用回归分析和重复测量设计的方差分析来评估观察组。这两种方法评估在同样的大鼠不独立。这种运算结果的模式说明组间没有相互影响的总血管密度平方根中的变量的25%为有意义。结果示VEGF胶组的总血管密度明显高于空胶组(P=0.004)和未处理组(P=0.010)。本研究又用方差分析比较了各亚组间的总血管密度平方根,将同一大鼠重复观察的数据带入计算,结果显示,只在心梗大鼠组中VEGF胶组与空白胶组之间就胶附近区有统计学差异。将非心梗组的6个亚组用方差分析对其总血管密度平方根进行分析,结果显示组间有差异(P=0.0057),VEGF胶组明显高于空白胶组(P=0.0206)或未处理组(P=0.0026)。在空白胶组和未处理组之间仍无统计学差异。而在不同区域之间也无统计学差异。毛细血管的密度在心梗和非心梗组中,VEGF胶组均明显高于空胶组。在心梗组中VEGF胶组缺血区明显高于间隔区。在非心梗组中两个区域之间无显著性差异。在心梗组和非心梗组之间比较,无论VEGF胶组或是空胶组均无统计学差异。通过方差分析比较了亚组中毛细血管平方根,对同样的大鼠进行重复观察计算。结果显示,在心梗组中,就胶附近区在VEGF胶组和空白胶组之间有显著性差异。在非心梗大鼠心脏的胶附近,无论是VEGF胶组还是空白胶组,使用多元回归分析,结果未发现有统计学差异。重新对非心梗大鼠组中6个亚组的毛细血管密度平方根进行方差分析,结果显示VEGF胶组显著高于空白胶组(P=0.0228)。而不同的采样区域之间没有统计学差异,并且区域间无相互作用。微血管和大血管密度的评价:微血管和大血管密度不是正态分布。使用多元回归分析根据3个独立的变量:心梗(是与否)、采样部位(间隔:是与否)和处理(VEGF胶和空胶)以及它们之间的相互作用来测定微血管的密度。结果解释为:组间无相互影响的微小血管密度变量中的22%为有意义。结果显示:VEGF胶组和空白胶组之间无显著性差异(P=0.068)。再用方差分析计算结果也显示为:在心梗组(P=0.1332)和非心梗组(P=0.6217)中,无论在VEGF胶组还是在空白胶组均无统计学差异。对大血管密度同样采用了多元回归分析,结果解释为组间无相互影响的大血管密度变量的18%为有意义。结果显示为,VEGF胶组大血管密度显著高于空白胶组(P=0.021)。而对大血管密度亚组进行方差分析,结果发现无论在非心梗组(P=0.0586)或心梗组(P=0.2481)中,VEGF胶组和空白胶组之间均无统计学差异。动脉密度的评价:因为我们所要测得的大血管包括动脉和静脉,每个采样区域中的6张相片不足以防止在结果中产生偏倚。故对每个心脏左室的60张切片中的3张切片中的动脉数量进行记录。结果显示,在非心梗和心梗大鼠心脏中VEGF胶组和空白胶组之间无统计学差异。③在处死大鼠之前根据M-超声波检查测量的参数来评估大鼠的心功能:显示非心梗大鼠和心梗大鼠组中VEGF胶组和空白胶组中的不同心脏参数如:左室收缩末直径(LVDs)、舒张末直径(LVDd)和左室短轴缩短率(FS)的表现。结果显示,在非心梗大鼠组中,VEGF胶组与空白胶组之间无显著性差异。而在心梗组中,VEGF胶组有心功能改善的趋势,但无统计学意义,可能需要更多的样本例数;2, EPCs移植纤维蛋白胶的移植:①肉眼观察,将20只移植了EPC胶的大鼠心脏中的2只在显微镜下进行观察。观察到在心脏和胸部之间有一些疏松的结缔组织,而其在EPCs胶组和空白胶组之间无明显差异。组织学和免疫组化研究,被移植EPCs胶和空白胶的心脏结构不易区分并且相对正常,未发现血管瘤、血管畸形和肿瘤。②血管检测为了检测大鼠心脏中的被移植的EPC细胞和其产生的效果,使用了CD34抗体标记,CD34标记物在人胎盘中为阳性切片。然而对非心梗和心梗大鼠使用CD34抗体的免疫组化染色中,结果无法取得EPC胶移植到大鼠心脏后的CD34阳性的细胞。曾经有报道在被移植人类CD34阳性细胞大鼠被检测到。而本研究用该抗体检测了EPC移植胶上的阳性表达,但是移植到大鼠心肌后的EPC细胞对CD34抗体为阴性表达。故本研究用Tra-1-85抗体染色来探测移植EPC胶大鼠心肌的EPCs或它们的分化产物即EPCs源的Ecs,结果未探测到Tra-1-85抗体阳性表达的细胞。因通过免疫组化检查观察未发现阳性结果,故本研究尝试初步运用ISH来鉴别人类细胞。ISH可探测到人类细胞核的重复(Alu)序列(蓝染)。结果未发现任何人类细胞。鉴于以上的结果,本研究评估了EPCs对大鼠心肌血管再生的效应。对所有组织区域内所有的血管和毛细血管数量及密度进行了测量,结果显示,EPC胶组和空胶组之间,以及在不同的EPC胶组之间均无显著性差异。本研究没有足够的非心梗大鼠作统计学分析,只比较了大鼠心梗心肌移植空白胶和EPCs胶之间的差异。③超声心动图评价移植EPCs后的左心功能:采用了方差分析,各组之间无显著性差异(P=0.612)。结论:1,VEGF纤维蛋白胶可以通过增加心梗和非心梗的毛细血管而促进血管生成,并且在梗死周围缺血区域更有意义。可以通过控制并维持低剂量的释放VEGF来预防血管结构的异常。移植VEGF胶的心梗大鼠的心功能较使用空白胶组有好转的趋势,但为显示有意义的结果需要更多数量的大鼠;2,尽管EPCs结果没有阳性表现,本研究将修改并提高细胞通过纤维蛋白基质传递的方法策略,确信细胞传递系统提供的有益和有效性将会进一步得到证实。如果这种结果被证实有效,纤维蛋白胶将来可作为传递血管再生因子到心脏的有效手段。
Objective:The aim of this thesis has been to investigate the relevance of tissue engineering strategies for the regeneration of the heart following MI. The aim of this thesis has been to investigate the relevance of tissue engineering strategies for the regeneration of the heart following MI. We have assessed the use of fibrin-based hydrogels to provide either VEGF or CD133-selected putative EPCs to the heart in order to stimulate neoangiogenesis. Specifically, we investigated 2 major questions: 1, Angiogenic effects of VEGF121 covalently bound to fibrin gel in non-infarcted and infarcted rat hearts, after 4 weeks of implantation. In particular we tested: 1) If VEGF121 covalently bound to fibrin gel could promote angiogenesis in the non-infarcted and infarcted rat hearts; 2) If this cell-demanded release of VEGF121 from fibrin gel could induce proper angiogenesis, without leading to hemangioma formation; 3) How far the angiogenic effect of VEGF121 can reach; 4) Which kind of vessel VEGF can promote; 5) Whether the angiogenic effect of VEGF121 can improve the heart function; 6) Whether the fibrin gel per se has some angiogenic influences to the rat hearts. 2, Possible vasculogenic or angiogenic effects of EPCs incorporated onto fibrin gels in the non-infarcted or infarcted rat hearts after 3 or 8 weeks of transplantation: 1) Whether EPCs delivered by fibrin could proliferate in the non-infarcted and infarcted rat hearts, incorporate to the neovessels and thus promote neovascularization; 2) Whether EPCs delivery to the heart can improve heart function.
Methods: First of all that are Preparation of fibrin gel matrices, preparation of recombinantα2-PI1–8-VEGF121and Fibrin gel matrices formulated withα2-PI1–8- VEGF121. 1, A total of 28 male Sprague-Dawley (SD) rats(9 week-old, weight 300-350 g) were included in this experiment, 13 of them received left anterior descending artery (LAD) ligation to induce the left ventricular myocardial infarction (MI).15 non-infarcted and 7 infarcted and 6 non-infarcted rats were implanted with fibrin gels covalently conjugated with VEGF. As a control, 6 non-infarcted rats and 6 infarcted rats received empty fibrin gels; other 3 normal rats with no treatment at all were use as control for the histology studies. In the infarcted rats, implantations were performed immediately after the ligation. All rats were kept for 4 weeks and before sacrifice, the heart function was evaluated by echocardiography. Histological and immunohistochemi- cal study:Rats were sacrificed with intravenous injection of potassium chloride (2 ml KCl 15%) at the level of the femoral vein, in order to stop the heart in diastole. Hearts were rapidly excised, the cardiac cavities were rinsed with PBS to remove blood and thrombus, then the hearts were fixed with 10% formalin for 24 hours. Afterwards, the hearts were cut into 3 parts parallel to the atrioventricular groove before being dehydrated and embedded in paraffin. Blocks containing the sections in contact with the cardiac patch were cut into 3μm thickness slices and stained with hematoxylin and eosin. Consecutive sections were used for the immunohistochemical study. For the VEGF conditions, 3 in 60 sections were stained with anti-CD31 and 1 in 60 sections was stained with anti-α?smooth muscle actin (SMA) to detect the vessels. 3 in 60 sections per heart were stained with anti-CD31 and 9 or 12 photos were taken per section at a magnification of 200X. For the non-infarcted hearts, 2 zones picture by taken: under and border of the gel, i.e. near the gel and far from the gel, i.e. in the septum. And took six photos per zone. Similarly, for the infarcted hearts, 6 pictures were randomly taken in two newly-defined zones, i.e. in the ischemic border zone of the infarct (near the gel) and (in the septum). For vascular measurements, three parameters were determined: 1) total vessel density (total vessel number per mm2 of total tissue area), 2) ratio of total vessel number to myocardial area (total vessel number per mm2 of myocardial area) and 3) percentage of total vessel area (total vessel area per total tissue area, %). Total tissue area was defined as the total image area minus the interstitial space (white empty space), to exclude a bias due to differences in the occurrence of interstitial space (either natural or artifactual, for instance tissue laceration during processing). Then, myocardial area was defined as total tissue area minus total vessel area. To assess which kind of vessel VEGF could promote, vessels were directly assigned by the Metamorph Software to one of three groups:①capillaries (vessel diameter<10μm),②microvessels (10μm≤vessel diameter≤20μm) and③macrovessels (vessel diameter>20μm). 2, 27 male Sprague-Dawley (SD) rats (9 week-old, weight 300-350 g) were included in this experiment. The animals were divided into 3 main groups: group 1: non-infarcted rats (n=9); group 2: rats engrafted immediately after MI (n=9); and group 3: rats engrafted 1 week after MI (n=9). For each group, the rats were sacrificed either 3 weeks or 8 weeks after implantation. To prevent rejection of EPC-seeded gels, the rats were immunosuppressed by a cyclosporin A (CsA) delivered continuously via an osmotic minipump. For the evaluation of left ventricular function, transthoracic echocardiography was performed on the rats before sacrifice. Process of sacrificed and qictures taken of rat same with VEGF. The EPCs-gels transplanted hearts, 3 in 180 sections were stained with anti-CD34, every tenth of 180 sections were stained with anti-human CD34 and Tra-1-85 to detect the transplanted human cells. The EPCs study used the same sampling, i.e. 3 CD31- stained sections per heart and chose the simplified method: 1 zone per section was chosen (near the gel in the non-infarcted hearts and in the ischemic border zone of the infarct in the infarcted hearts) and 6 pictures per zone were taken. The method of vascular measurements is same with VEGF.
Results: 1, At the first analyzed the effect of VEGF-engineered gels engrafted on rat left ventricles.To rule out the possibility that VEGF administrated at too high concentrations could increase the vascular permeability leading to leakiness and causing pericardial or pleural effusion, to examine the chest cavity surrounding the hearts upon their excision and did not see any sign of effusion neither in the chest nor in the pericardium. Some loose connective tissue was usually present between the heart and the chest, but no difference was found between the rats with VEGF gels and the rats with empty fibrin gels. Observed by H&E staining had a relatively normal morphology, and no hemangioma or vessel malformations were found. Observed slightly more loose connective tissue containing signs of an active neovascularization near the infarct area as compared to non-infarcted hearts, but this was not apparently different in rats receiving VEGF gels as compared to rats with empty gels.Then compared the total vessel density, the ratio of total vessel number to myocardial area, and the percentage of total vessel area in the three sampling areas (under the gel, at the border of the gel and far from the gel). In non-infarcted rats, no significant difference was found for these three parameters measured in the three sampling areas, and this was the case for either the VEGF-gel group or the empty-gel group.And then compared the same parameters between the VEGF-gel group and the empty-gel group respectively in the three sampling areas (under the gel, at the border of the gel and far from the gel). We observed a trend for an increased number of vessels in the VEGF-gel group but the difference was not statistically significant.However, when we combined the three sampling areas together, significant discrepancies in percentage of total vessel area (ANOVA F(2,59)=3.46; p<0.05) were observed between treated animals. Indeed, animals that received VEGF-fibrin gels had a significantly higher percentage of total vessel area than those with empty gels (p<0.05). The differences in percentage of total vessel area were closely correlated to differences in total vessel density: here the statistical significance was even higher (ANOVA F(2,59)=6; p<0.005) and animals exposed to VEGF also had a significantly higher ratio of total vessel number to myocardial area than those receiving empty gels (p<0.005). Since we did not see any significant difference among the three sampling areas (under the gel, at the border of the gel and far from the gel), we changed the sampling method to two zones (near the gel and in the septum), and took 6 instead of 3 images per zone. Given that the three parameters (vessel density, ratio of vessel number to myocardial area, and percentage of vessel area) gave very similar results, we used vessel density to perform all the vascular measurements. Since the total vessel density was not normally distributed, we were able to normalize it using the square root transform, namely getting a Gaussian distribution. As 36 observations were performed in each rat (3 sections, 2 zones, 6 pictures by zone), we took into account the clustering of the observation during the subsequent regression analysis and applied an ANOVA with a repeated measure design, both techniques taking into account the fact that the observations are not independent within the same rat.Considering all the groups together and using multiple regression, we predicted the square root of total vessel density from 3 independent variables: infarct (no or yes), localization of the sample (septum: no or yes) and treatment group (without treatment, empty-gel, VEGF-gel) and their respective interactions. The resulting model explained 25% of the variance in the square root of total vessel density, with none of the interaction terms being significant, and only one main effect remaining significant: the total vessel density in the VEGF-gel group being significantly higher than in both empty gel group (P=0.004) and without treatment group (P=0.010).We then compared the square root of total vessel density among the subgroups by ANOVA, taking into account that repeated observations were performed on the same rats. However, statistical significance was found in the differences among the VEGF-gel, empty gel and without treatment groups in the area near the gel for the non-infarcted rats when using multiple regression analysis (P=0.030). Considering all the 6 subgroups together for the non-infarcted hearts, square root of total vessel density analyzed by ANOVA shows a significant group effect (P=0.0057), but no zone effect and no interaction of zone with group. It was significantly higher in the VEGF-gel group than in the empty gel group (P=0.0206) or the group without treatments (P=0.0026). Again, the empty-gel group and the group without treatment were not different.To investigate which kind(s) of vessel(s) VEGF could promote, we classified vessels by Metamorph Software analysis into the following three groups: (1) capillaries (vessel diameter < 10μm), (2) microvessels (10μm≤vessel diameter≤20μm) and (3) macrovessels (vessel diameter>20μm). We calculated the following parameters: capillary density, microvessel density and macrovessel density. The capillary density in non-infarcted and infarcted rat hearts was significantly higher in the VEGF-gel group than in the empty gel group. While in non-infarcted hearts, no difference was observed between the areas near the gel and in the septum, regardless of the treatment, in infarcted rats, the capillary density in the VEGF-gel group was significantly higher near the gel as compared to the septum. Differences between non-infarcted and infarcted heart were not significant, neither in the VEGF-gel group nor in empty gel group. Then normalized the capillary density by calculating its square root (sqrt) (as described above). Using multiple regression (with rats clustering), we predicted the square root of capillary density from 3 independent variables: infarct (no or yes), localization of the sample (septum: no or yes) and treatment group (empty gel or VEGF-gel) and their respective interactions. The resulting model explained 24% of the variance in the square root of capillary density, with none of the interaction terms being significant and only one main effect being significant: sqrt of capillary density in the VEGF-gel group being significantly higher than in empty gel group (P=0.003).Compared the sqrt of capillary density among the subgroups by ANOVA, taking into account that repeated observations were performed on the same rats. Only the difference between the VEGF-gel group and empty gel group in the area near the gel was significant in infarcted rat hearts. Multiple regression analysis did not detect significant difference between the VEGF-gel group and the empty gel group in the area near the gel in the non-infarcted rat hearts.Again considering all the 6 subgroups together for the non-infarcted hearts, square root of capillary density analyzed by ANOVA was significant higher in the VEGF-gel group than in the empty gel group (P=0.0228,) but no zone effect and no interaction of zone with group were observed. For the macrovessel density, we used the same multiple regression model. The model explained 18% of the variance, with none of the interaction terms being significant except one main effect: macrovessel density in the VEGF-gel group was significantly higher than empty gel group (P=0.021). When the macrovessel density among the subgroups was analyzed by ANOVA, no significant difference was found between VEGF-gel group and empty gel group either in the non-infarcted rats (P=0.0586) or in the infarcted rats (P=0.2481). Measured macrovessels included both arteries and veins and we judged the sampling of 6 images per zone not sufficient to prevent a bias on the result interpretation, we scored the number of arteries in the whole left ventricle in 3 of 60 sections per heart shows no significant difference in the artery density between the VEGF-gel group and the empty gel group either in the non-infarcted or in the infarcted rat hearts. The echocardiography was performed before animal sacrifice and cardiac function was assessed according to the parameters measured on the M-mode tracing showed the difference of cardiac parameters such as LVDs, LVDd and FS between the VEGF-gel group and the empty gel group in non-infarcted and infarcted rat hearts. No significant difference was found between the VEGF-gel group and empty-gel group in the non-infarcted rats, but in the infarcted rats, a pronounced trend to have better heart function in the VEGF-group was detected even though it was not statistically significant. To get the significance, more rats are needed. 2, Transplantation of EPCs-seeded fibrin gels. Macroscopic views of 2 of 20 rat hearts implanted with EPC-seeded gels. Performed 180 sections in the proximity of the gel area and 1 every 10 were stained by H&E. Heart structure engrafted with either EPCs- gels or empty-gels were indistinguishable and relatively normal, with no visible hemangioma, vessel malformations or tumors.To detect the transplanted human EPCs or their progenies in the rat hearts and used anti-human CD34 antibody.CD34 marker worked nicely as positive control in human placenta sections. However, we were not able to retrieve CD34-positive cells in the rat hearts transplanted with EPCs-gels.It shows the immunohistochemical staining using anti-human CD34 antibody in non-infarcted as well as infarcted hearts. Given that no human CD34 (+) cells were ever found in any of the transplanted rats, we checked this antibody on EPCs-gels embedded for immunohistochemical examination. Surprisingly, EPCs were negative for anti-human CD34 antibody. Then chose another marker, the Tra-1-85 which is present on the surface of all human cells. Tested the anti-Tra-1-85 antibody on human placenta and on EPC-seeded fibrin gels It shown both placenta and EPCs in vitro were positively stained. Then used Tra-1-85 to detect the transplanted human EPCs or their progenies (EPC-derived ECs) in the EPCs-gel transplanted rat hearts. Unfortunately, no Tra-1-85-positive cells were detected in 1 every 10 sections examined over a total 180 sections. Given that no positive result was obtained by immunohistochemical examination, we tried preliminarily a technique of in situ hybridization (ISH) to identify human cells. ISH can detect Alu sequences in the human nuclei (stained in blue). In a first trial did not find any human cells in the 3-4 sections examined. Despite the lack of human EPC retrieval in the engrafted hearts, then decided to assess whether the transplanted EPCs had somehow an angiogenesis effect in the rat hearts. Total tissue area, total vessel and capillary number as well as their density were measured. No significant differences were found either between the rats with EPCs-gels and the rats with empty gels or among the different EPCs-gel groups.Evaluation of left ventricular function after EPC engraftment, there were not enough non-infarcted rats for the statistical analysis,so we compared only the infarcted rats engrafted with empty or EPC-seeded gels. Using ANOVA analysis, there was no significant difference among all the groups.
Conclusions: 1, VEGF covalently immobilized in the fibrin gel could promote angiogenesis by increasing capillary formation in the non-infarcted and infarcted rat hearts, an effect more pronounced in the ischemic area of infarcted rat hearts. The study controlled sustained low dose release of VEGF prevented the formation of aberrant vessels. Infarcted rats with VEGF-gels have a trend to have better cardiac function compared to the rats with empty gels. Further studies with a higher number of rats are in process in order to show a significant impact of such strategy on the heart function. If the results presented in this work will be confirmed, fibrin gel matrices could constitute a future efficient vehicle for delivering angiogenic factors to the heart. 2, Although our EPCs results were not positive at this point, we will modify and improve our strategy of cell delivery through fibrin matrices and we are confident that such a cell delivery system will prove beneficial and effective in the future.
Objective:The aim of this thesis has been to investigate the relevance of tissue engineering strategies for the regeneration of the heart following MI. The aim of this thesis has been to investigate the relevance of tissue engineering strategies for the regeneration of the heart following MI. We have assessed the use of fibrin-based hydrogels to provide either VEGF or CD133-selected putative EPCs to the heart in order to stimulate neoangiogenesis. Specifically, we investigated 2 major questions: 1, Angiogenic effects of VEGF121 covalently bound to fibrin gel in non-infarcted and infarcted rat hearts, after 4 weeks of implantation. In particular we tested: 1) If VEGF121 covalently bound to fibrin gel could promote angiogenesis in the non-infarcted and infarcted rat hearts; 2) If this cell-demanded release of VEGF121 from fibrin gel could induce proper angiogenesis, without leading to hemangioma formation; 3) How far the angiogenic effect of VEGF121 can reach; 4) Which kind of vessel VEGF can promote; 5) Whether the angiogenic effect of VEGF121 can improve the heart function; 6) Whether the fibrin gel per se has some angiogenic influences to the rat hearts. 2, Possible vasculogenic or angiogenic effects of EPCs incorporated onto fibrin gels in the non-infarcted or infarcted rat hearts after 3 or 8 weeks of transplantation: 1) Whether EPCs delivered by fibrin could proliferate in the non-infarcted and infarcted rat hearts, incorporate to the neovessels and thus promote neovascularization; 2) Whether EPCs delivery to the heart can improve heart function.
Methods: First of all that are Preparation of fibrin gel matrices, preparation of recombinantα2-PI1–8-VEGF121and Fibrin gel matrices formulated withα2-PI1–8- VEGF121. 1, A total of 28 male Sprague-Dawley (SD) rats(9 week-old, weight 300-350 g) were included in this experiment, 13 of them received left anterior descending artery (LAD) ligation to induce the left ventricular myocardial infarction (MI).15 non-infarcted and 7 infarcted and 6 non-infarcted rats were implanted with fibrin gels covalently conjugated with VEGF. As a control, 6 non-infarcted rats and 6 infarcted rats received empty fibrin gels; other 3 normal rats with no treatment at all were use as control for the histology studies. In the infarcted rats, implantations were performed immediately after the ligation. All rats were kept for 4 weeks and before sacrifice, the heart function was evaluated by echocardiography. Histological and immunohistochemi- cal study:Rats were sacrificed with intravenous injection of potassium chloride (2 ml KCl 15%) at the level of the femoral vein, in order to stop the heart in diastole. Hearts were rapidly excised, the cardiac cavities were rinsed with PBS to remove blood and thrombus, then the hearts were fixed with 10% formalin for 24 hours. Afterwards, the hearts were cut into 3 parts parallel to the atrioventricular groove before being dehydrated and embedded in paraffin. Blocks containing the sections in contact with the cardiac patch were cut into 3μm thickness slices and stained with hematoxylin and eosin. Consecutive sections were used for the immunohistochemical study. For the VEGF conditions, 3 in 60 sections were stained with anti-CD31 and 1 in 60 sections was stained with anti-α?smooth muscle actin (SMA) to detect the vessels. 3 in 60 sections per heart were stained with anti-CD31 and 9 or 12 photos were taken per section at a magnification of 200X. For the non-infarcted hearts, 2 zones picture by taken: under and border of the gel, i.e. near the gel and far from the gel, i.e. in the septum. And took six photos per zone. Similarly, for the infarcted hearts, 6 pictures were randomly taken in two newly-defined zones, i.e. in the ischemic border zone of the infarct (near the gel) and (in the septum). For vascular measurements, three parameters were determined: 1) total vessel density (total vessel number per mm2 of total tissue area), 2) ratio of total vessel number to myocardial area (total vessel number per mm2 of myocardial area) and 3) percentage of total vessel area (total vessel area per total tissue area, %). Total tissue area was defined as the total image area minus the interstitial space (white empty space), to exclude a bias due to differences in the occurrence of interstitial space (either natural or artifactual, for instance tissue laceration during processing). Then, myocardial area was defined as total tissue area minus total vessel area. To assess which kind of vessel VEGF could promote, vessels were directly assigned by the Metamorph Software to one of three groups:①capillaries (vessel diameter<10μm),②microvessels (10μm≤vessel diameter≤20μm) and③macrovessels (vessel diameter>20μm). 2, 27 male Sprague-Dawley (SD) rats (9 week-old, weight 300-350 g) were included in this experiment. The animals were divided into 3 main groups: group 1: non-infarcted rats (n=9); group 2: rats engrafted immediately after MI (n=9); and group 3: rats engrafted 1 week after MI (n=9). For each group, the rats were sacrificed either 3 weeks or 8 weeks after implantation. To prevent rejection of EPC-seeded gels, the rats were immunosuppressed by a cyclosporin A (CsA) delivered continuously via an osmotic minipump. For the evaluation of left ventricular function, transthoracic echocardiography was performed on the rats before sacrifice. Process of sacrificed and qictures taken of rat same with VEGF. The EPCs-gels transplanted hearts, 3 in 180 sections were stained with anti-CD34, every tenth of 180 sections were stained with anti-human CD34 and Tra-1-85 to detect the transplanted human cells. The EPCs study used the same sampling, i.e. 3 CD31- stained sections per heart and chose the simplified method: 1 zone per section was chosen (near the gel in the non-infarcted hearts and in the ischemic border zone of the infarct in the infarcted hearts) and 6 pictures per zone were taken. The method of vascular measurements is same with VEGF.
Results: 1, At the first analyzed the effect of VEGF-engineered gels engrafted on rat left ventricles.To rule out the possibility that VEGF administrated at too high concentrations could increase the vascular permeability leading to leakiness and causing pericardial or pleural effusion, to examine the chest cavity surrounding the hearts upon their excision and did not see any sign of effusion neither in the chest nor in the pericardium. Some loose connective tissue was usually present between the heart and the chest, but no difference was found between the rats with VEGF gels and the rats with empty fibrin gels. Observed by H&E staining had a relatively normal morphology, and no hemangioma or vessel malformations were found. Observed slightly more loose connective tissue containing signs of an active neovascularization near the infarct area as compared to non-infarcted hearts, but this was not apparently different in rats receiving VEGF gels as compared to rats with empty gels.Then compared the total vessel density, the ratio of total vessel number to myocardial area, and the percentage of total vessel area in the three sampling areas (under the gel, at the border of the gel and far from the gel). In non-infarcted rats, no significant difference was found for these three parameters measured in the three sampling areas, and this was the case for either the VEGF-gel group or the empty-gel group.And then compared the same parameters between the VEGF-gel group and the empty-gel group respectively in the three sampling areas (under the gel, at the border of the gel and far from the gel). We observed a trend for an increased number of vessels in the VEGF-gel group but the difference was not statistically significant.However, when we combined the three sampling areas together, significant discrepancies in percentage of total vessel area (ANOVA F(2,59)=3.46; p<0.05) were observed between treated animals. Indeed, animals that received VEGF-fibrin gels had a significantly higher percentage of total vessel area than those with empty gels (p<0.05). The differences in percentage of total vessel area were closely correlated to differences in total vessel density: here the statistical significance was even higher (ANOVA F(2,59)=6; p<0.005) and animals exposed to VEGF also had a significantly higher ratio of total vessel number to myocardial area than those receiving empty gels (p<0.005). Since we did not see any significant difference among the three sampling areas (under the gel, at the border of the gel and far from the gel), we changed the sampling method to two zones (near the gel and in the septum), and took 6 instead of 3 images per zone. Given that the three parameters (vessel density, ratio of vessel number to myocardial area, and percentage of vessel area) gave very similar results, we used vessel density to perform all the vascular measurements. Since the total vessel density was not normally distributed, we were able to normalize it using the square root transform, namely getting a Gaussian distribution. As 36 observations were performed in each rat (3 sections, 2 zones, 6 pictures by zone), we took into account the clustering of the observation during the subsequent regression analysis and applied an ANOVA with a repeated measure design, both techniques taking into account the fact that the observations are not independent within the same rat.Considering all the groups together and using multiple regression, we predicted the square root of total vessel density from 3 independent variables: infarct (no or yes), localization of the sample (septum: no or yes) and treatment group (without treatment, empty-gel, VEGF-gel) and their respective interactions. The resulting model explained 25% of the variance in the square root of total vessel density, with none of the interaction terms being significant, and only one main effect remaining significant: the total vessel density in the VEGF-gel group being significantly higher than in both empty gel group (P=0.004) and without treatment group (P=0.010).We then compared the square root of total vessel density among the subgroups by ANOVA, taking into account that repeated observations were performed on the same rats. However, statistical significance was found in the differences among the VEGF-gel, empty gel and without treatment groups in the area near the gel for the non-infarcted rats when using multiple regression analysis (P=0.030). Considering all the 6 subgroups together for the non-infarcted hearts, square root of total vessel density analyzed by ANOVA shows a significant group effect (P=0.0057), but no zone effect and no interaction of zone with group. It was significantly higher in the VEGF-gel group than in the empty gel group (P=0.0206) or the group without treatments (P=0.0026). Again, the empty-gel group and the group without treatment were not different.To investigate which kind(s) of vessel(s) VEGF could promote, we classified vessels by Metamorph Software analysis into the following three groups: (1) capillaries (vessel diameter < 10μm), (2) microvessels (10μm≤vessel diameter≤20μm) and (3) macrovessels (vessel diameter>20μm). We calculated the following parameters: capillary density, microvessel density and macrovessel density. The capillary density in non-infarcted and infarcted rat hearts was significantly higher in the VEGF-gel group than in the empty gel group. While in non-infarcted hearts, no difference was observed between the areas near the gel and in the septum, regardless of the treatment, in infarcted rats, the capillary density in the VEGF-gel group was significantly higher near the gel as compared to the septum. Differences between non-infarcted and infarcted heart were not significant, neither in the VEGF-gel group nor in empty gel group. Then normalized the capillary density by calculating its square root (sqrt) (as described above). Using multiple regression (with rats clustering), we predicted the square root of capillary density from 3 independent variables: infarct (no or yes), localization of the sample (septum: no or yes) and treatment group (empty gel or VEGF-gel) and their respective interactions. The resulting model explained 24% of the variance in the square root of capillary density, with none of the interaction terms being significant and only one main effect being significant: sqrt of capillary density in the VEGF-gel group being significantly higher than in empty gel group (P=0.003).Compared the sqrt of capillary density among the subgroups by ANOVA, taking into account that repeated observations were performed on the same rats. Only the difference between the VEGF-gel group and empty gel group in the area near the gel was significant in infarcted rat hearts. Multiple regression analysis did not detect significant difference between the VEGF-gel group and the empty gel group in the area near the gel in the non-infarcted rat hearts.Again considering all the 6 subgroups together for the non-infarcted hearts, square root of capillary density analyzed by ANOVA was significant higher in the VEGF-gel group than in the empty gel group (P=0.0228,) but no zone effect and no interaction of zone with group were observed. For the macrovessel density, we used the same multiple regression model. The model explained 18% of the variance, with none of the interaction terms being significant except one main effect: macrovessel density in the VEGF-gel group was significantly higher than empty gel group (P=0.021). When the macrovessel density among the subgroups was analyzed by ANOVA, no significant difference was found between VEGF-gel group and empty gel group either in the non-infarcted rats (P=0.0586) or in the infarcted rats (P=0.2481). Measured macrovessels included both arteries and veins and we judged the sampling of 6 images per zone not sufficient to prevent a bias on the result interpretation, we scored the number of arteries in the whole left ventricle in 3 of 60 sections per heart shows no significant difference in the artery density between the VEGF-gel group and the empty gel group either in the non-infarcted or in the infarcted rat hearts. The echocardiography was performed before animal sacrifice and cardiac function was assessed according to the parameters measured on the M-mode tracing showed the difference of cardiac parameters such as LVDs, LVDd and FS between the VEGF-gel group and the empty gel group in non-infarcted and infarcted rat hearts. No significant difference was found between the VEGF-gel group and empty-gel group in the non-infarcted rats, but in the infarcted rats, a pronounced trend to have better heart function in the VEGF-group was detected even though it was not statistically significant. To get the significance, more rats are needed. 2, Transplantation of EPCs-seeded fibrin gels. Macroscopic views of 2 of 20 rat hearts implanted with EPC-seeded gels. Performed 180 sections in the proximity of the gel area and 1 every 10 were stained by H&E. Heart structure engrafted with either EPCs- gels or empty-gels were indistinguishable and relatively normal, with no visible hemangioma, vessel malformations or tumors.To detect the transplanted human EPCs or their progenies in the rat hearts and used anti-human CD34 antibody.CD34 marker worked nicely as positive control in human placenta sections. However, we were not able to retrieve CD34-positive cells in the rat hearts transplanted with EPCs-gels.It shows the immunohistochemical staining using anti-human CD34 antibody in non-infarcted as well as infarcted hearts. Given that no human CD34 (+) cells were ever found in any of the transplanted rats, we checked this antibody on EPCs-gels embedded for immunohistochemical examination. Surprisingly, EPCs were negative for anti-human CD34 antibody. Then chose another marker, the Tra-1-85 which is present on the surface of all human cells. Tested the anti-Tra-1-85 antibody on human placenta and on EPC-seeded fibrin gels It shown both placenta and EPCs in vitro were positively stained. Then used Tra-1-85 to detect the transplanted human EPCs or their progenies (EPC-derived ECs) in the EPCs-gel transplanted rat hearts. Unfortunately, no Tra-1-85-positive cells were detected in 1 every 10 sections examined over a total 180 sections. Given that no positive result was obtained by immunohistochemical examination, we tried preliminarily a technique of in situ hybridization (ISH) to identify human cells. ISH can detect Alu sequences in the human nuclei (stained in blue). In a first trial did not find any human cells in the 3-4 sections examined. Despite the lack of human EPC retrieval in the engrafted hearts, then decided to assess whether the transplanted EPCs had somehow an angiogenesis effect in the rat hearts. Total tissue area, total vessel and capillary number as well as their density were measured. No significant differences were found either between the rats with EPCs-gels and the rats with empty gels or among the different EPCs-gel groups.Evaluation of left ventricular function after EPC engraftment, there were not enough non-infarcted rats for the statistical analysis,so we compared only the infarcted rats engrafted with empty or EPC-seeded gels. Using ANOVA analysis, there was no significant difference among all the groups.
Conclusions: 1, VEGF covalently immobilized in the fibrin gel could promote angiogenesis by increasing capillary formation in the non-infarcted and infarcted rat hearts, an effect more pronounced in the ischemic area of infarcted rat hearts. The study controlled sustained low dose release of VEGF prevented the formation of aberrant vessels. Infarcted rats with VEGF-gels have a trend to have better cardiac function compared to the rats with empty gels. Further studies with a higher number of rats are in process in order to show a significant impact of such strategy on the heart function. If the results presented in this work will be confirmed, fibrin gel matrices could constitute a future efficient vehicle for delivering angiogenic factors to the heart. 2, Although our EPCs results were not positive at this point, we will modify and improve our strategy of cell delivery through fibrin matrices and we are confident that such a cell delivery system will prove beneficial and effective in the future.
引文
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