小型猪急性心肌梗死模型血管再生的实验研究
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摘要
前言
动脉粥样硬化导致的冠心病是西方国家发病率和死亡率最高的疾病,在发展中国家的发病率和死亡率也以惊人的速度在增长。近几十年来,冠心病的治疗取得了很大的发展,经皮冠状动脉内血管成形术(PTCA)、冠状动脉内支架置入术、冠状动脉旁路移植术(CABG)等都已成为临床上成熟的治疗手段,挽救了数百万患者的生命。但是,随着世界人口的老龄化,冠心病发病率仍在持续增高,越来越多的患者的病情复杂化,不适合这些传统方法的治疗。这些复杂病变主要包括弥漫性小血管病变、慢性完全性堵塞病变、多支血管病变、多次介入治疗或手术后血管闭塞等,近年来发展起来的治疗性血管生成(therapeutic angiogenesis)为解决这一问题提供了新的思路和方法。
治疗性血管生成指的是将外源性促血管生长因子导入缺血心肌以刺激侧支血管生成、改善心肌灌注和功能的方法。自从该思路提出30多年以来,受到研究者的广泛关注,动物实验取得了令人鼓舞的结果,但是单一促血管生长因子的临床研究尚未达到理想的预期目的。药物公司资助的临床试验常常选用公司具有知识产权或FDA比较容易批准的促血管生长因子,目前大多数临床研究主要集中于血管内皮生长因子VEGF-A,而VEGF-A作用的主要靶细胞是内皮细胞。血管生成是一个需要多种因素参与的复杂过程,单一促血管生长因子的作用无法激活和完成血管新生和成熟的整个调控过程,这可能是临床试验失败的原因之一。
成人体内的血管新生(neovacularization)过程首先是从原有的血管通过芽生的方式长出原始的血管网络,包括内皮细胞的增殖、迁移、管状结构形成以及细胞外基质的重构,这一过程称为血管生成(angiogenesis);随后这一初级的毛细血管网被平滑肌细胞和周细胞等血管支持细胞包裹,逐步形成具有完整结构且通过血管造影可显影的动脉血管网络,此过程称为动脉生成(arteriogenesis)。在血管生成(angiogenesis)阶段起主要作用的因子称为促血管生成因子(angiogenic factor),如FGF-2和VEGF。在动脉生成(arteriogenesis)阶段发挥主要作用的血管生长因子称为促动脉生成因子(arteriogenic factor),如PDGF-AB、PDGF-BB、血管生成素-1(Ang-1)、HGF和TGF-β等。
目前,促血管生成因子VEGF和FGF-2的研究最为深入和广泛,也是目前已进入临床试验阶段的因子。由于VEGF具有很强的促进血管通透性的作用,因此也被称为血管通透因子(Vascularpermeability factor,VPF)。FGF-2虽然缺乏向细胞外分泌的信号序列,但它能够调节靶细胞包括内皮细胞的生长和分化。尤为重要的是,FGF-2体内注射后可选择性地刺激血管生成但不影响其他组织的生长。但是,通过单一促血管生成因子诱导产生的新生血管结构以内皮细胞为主,没有血管平滑肌细胞和周细胞包被形成完整的动脉中膜结构,因此血管容易出血渗出,亦无法输送血流,最终会导致新生血管退化。而PDGF家族成员尤其是PDGF-BB是作用很强的促动脉生成因子,主要作用于血管壁细胞包括周细胞和血管平滑肌细胞。PDGF-BB与细胞表面的酪氨酸蛋白激酶受体PDGFR-α和PDGFR-β的同型二聚体和异二聚体结合,募集周细胞和血管平滑肌细胞包绕新生血管网络形成完整的血管结构从而发挥稳定血管的作用。
为了提高缺血区血流的灌注,必须重建长期稳定的功能性动脉网络,因为只有肌性动脉才能提供足够的血流量改善缺血心肌的功能以达到治疗目的。目前,在治疗性血管生成的研究中,仍有许多重要的问题尚待解决:既然单一因子无法诱导有功能的动脉血管形成,那么,不同的促血管生长因子与促动脉生长因子组合是否可以产生理想的效果?哪些血管生长因子之间的“搭档”能建立有效的侧支循环、改善缺血组织的血流灌注和功能?客观、准确地评价动脉生成的指标是什么?如何选择适宜的心肌梗死动物模型进行临床前评估?
基于血管生成的研究现状,我们提出如下假说:不同的促血管生成因子和促动脉生成因子均有各自特定的“搭档”,在血管新生的过程中互相影响并发挥协同作用,逐步激活和完成血管新生的整个过程;联合应用特定的促血管生成因子和促动脉生成因子能够诱导成熟、稳定的动脉血管网络的形成,并改善缺血心肌的灌注和功能。
目的
1.在小鼠角膜模型中,将VEGF、FGF-2和PDGF家族成员(PDGF-AA,PDGF-AB,PDGF-BB)两两组合进行初步筛选,确定有协同促血管生成效应的不同生长因子之间的组合。
2.根据筛选结果进一步探讨最佳组合因子在小型猪心肌梗死模型中的治疗效果及其可能的机制。
1.方法
1.1生长因子组合的筛选
角膜是没有血管的组织,因此它是研究血管生成的理想模型。我们以hydron聚合体包被蔗糖硫酸铝制作缓释系统,利用该系统包载不同血管生长因子使其成为缓释剂,置入小鼠角膜。不同生长因子单独或联合应用的剂量如下:FGF-2(80 ng),VEGF(160 ng),PDGF-AA(160 ng),PDGF-AB(160 ng),PDGF-BB(160 ng),PDGF-AA(160 ng)/FGF-2(80 ng),PDGF-AB(160 ng)/FGF-2(80 ng),PDGF-BB(160 ng)/FGF-2(40 ng),FGF-2(80 ng)/VEGF(160 ng),PDGF-BB(160 ng)/VEGF(160 ng)。在置入因子后5天后测定每一组动物角膜新生血管的长度和面积。为了观察血管新生的稳定性,在置入因子后第12,24,70天时分别再次测定血管长度和面积,并观察新生血管外形和轮廓的变化,确定有协同促血管生成作用的生长因子的组合,并根据筛选结果在小型猪急性心肌梗死模型中做进一步研究。
1.2小型猪急性心肌梗死模型的建立以及血管生长因子的导入
中国实验用小型猪40只,随机分为4组,每组10只。①PBS对照组,②FGF-2(5μg)治疗组,③PDGF-BB(10μg)治疗组,④FGF-2(5μg)/PDGF-BB(10μg)联合治疗组。所有实验动物的管理均遵循中华人民共和国卫生部动物实验管理条例(No.55,2001)和山东大学齐鲁医院实验动物管理条例。
动物经过诱导麻醉后建立静脉通路,并静脉注射3%戊巴比妥钠维持麻醉。然后行气管插管,呼吸机机械通气。经前正中线开胸,显露冠状动脉左前降支(LAD)后,于左前降支中远端1/3处用无创针线预结扎(不全部阻断血流)10min,然后完全结扎。
冠状动脉结扎后监测心率和血压的变化,急性心肌梗死模型构建成功的判定指标为:心电监护显示ST段弓背向上抬高持续30min以上,结扎动脉支配区域的心肌变暗变紫,运动减弱。以hydron聚合体包被蔗糖硫酸铝制作缓释系统,利用该系统包载不同血管生长因子使其成为缓释剂,包埋于心包下心肌梗死边缘的缺血区域。
1.3选择性冠状动脉造影术及评价侧支循环
麻醉动物股动脉穿刺成功后,经股动脉将6F导管送入左右冠状动脉行冠状动脉造影,用以证实观察左前降支结扎部位血流完全阻断,并评价LAD分布区的侧支循环。侧支循环指数按Rentrop分级分为0～3级:0级为冠状动脉系统无侧支显影;1级为心外膜下冠状动脉细小分支内有很弱的充盈,但是心外膜主要分支内无显影;2级为心外膜下冠状动脉主要分支部分充盈;3级为心外膜下冠状动脉主要分支完全充盈。
1.4应用彩色微粒测定局部心肌血流量
在治疗前和治疗后第6周和14周分别应用红色、黄色和蓝色三种不同颜色的彩色微粒(直径10±2μm,E-Z Trac,LosAngeles,CA)来评估这三个时间点的心肌局部血流量。将5F猪尾导管经股动脉进入左心室,经导管向左心室内注射入5×10~6个彩色微粒(将彩色微粒稀释于10ml生理盐水中),注射时间为30s,然后用10ml生理盐水冲洗导管。在注射彩色微粒前10s,使用定速抽血泵由股动脉抽取参考血液样本,抽取速率为10ml/min,共90s。血样-20℃保存。根据公司提供的操作步骤回收并计数组织和参考血样中的彩色微粒。根据公式计算心肌局部血流量:Q_m=(C_m×Q_r)/C_r。其中,Q_m是指每克心肌组织的血流量(ml/min/g),C_m是指每克心肌组织的彩色微粒数,Q_r是指参考血样的抽取速度(ml/min),C_r是指参考血样中的彩色微粒数。
1.5超声心动图测定左心室整体和局部收缩功能
将动物于开胸状态下行心外膜超声检测。在心尖两腔和四腔心切面采以校正的Simpson's方法测定左心室射血分数(LVEF)。在二维灰阶图像上分别测定梗死边缘的缺血区左心室舒张末期(心电图R波峰值处)和收缩末期(心电图T波末端)的室壁厚度,按以下公式计算缺血心肌局部室壁增厚率(wall thickening,WT):WT=(收缩末室壁厚度-舒张末室壁厚度)/舒张末室壁厚度×100%
1.6治疗效果评价
心脏缺血区导入生长因子6周后,进行冠状动脉造影和彩色微粒注射分别评价侧支循环和局部心肌血流量;导入生长因子14周后,再次行冠状动脉造影、彩色微粒注射和超声心动图检查来评价侧支循环、心肌灌注以及左室整体和缺血区局部功能。
1.7免疫组化
于第14周实验结束后,麻醉状态下静脉注射氯化钾处死所有实验动物,取出心脏,将左心室LAD结扎远端的缺血心肌分别置于多聚甲醛和液氮中备用,行相关组织学和分子生物学检测。
新生血管的密度测定方法:缺血区组织以多聚甲醛浸泡固定、石蜡包埋,切片厚度5μm。通过兔多克隆抗体von Willebrand factor(vWF)和小鼠单克隆抗体α-smooth muscle actin(α-SMA)免疫组化标记缺血区心肌的新生毛细血管和小动脉,并用荧光双标来评价新生血管的成熟程度和稳定性。这里我们引入了一个参数:成熟指数(maturation index),即被平滑肌包被的新生血管所占所有血管的百分比。
1.8统计学处理
定量数据均以均数士标准误表示,用SPSS 11.5统计软件进行统计分析。在做分析之前,对所有的数据进行正态分布检验。连续变量应用2×2析因设计和重复测量的方差分析。不同时间点连续变量应用重复测量的方差分析,进一步组间两两比较应用LSD或Dunnutt T3(方差不齐时)以及多元方差分析进行。等级资料用非参数检验分析,多组之间的比较用Kruskal-Wallis检验,组间两两比较Mann-Whitney检验。设P＜0.05为有统计学意义。
2.结果
2.1小鼠角膜模型血管生长因子的筛选
将不同生长因子缓释剂置入角膜第5天时检测结果表明,无论单独应用一种还是联合两种生长因子均可诱导角膜新生血管的形成。与单一因子比较,VEGF/FGF-2和VEGF/PDGF-BB这两种因子组合无显著协同作用。而FGF-2和PDGF家族成员PDGF-AA,PDGF-AB,PDGF-BB联合应用均有显著的协同促血管生成效应。但是到了第70天,联合应用FGF-2和PDGF-AA组诱导的血管几乎完全退化,而FGF-2与PDGF-AB或PDGF-BB协同诱导的新生血管得以维持。因此,我们选择了协同促血管生成作用最强的生长因子组合(FGF-2和PDGF-BB)在小型猪急性心肌梗死模型中作进一步研究。
40只实验用小型猪,第一次手术时死亡5只,其中2只死于麻醉意外,3只于结扎LAD时死于心室颤动。第二次冠状动脉造影时1只动物死于麻醉意外。共有34只小型猪完成了整个实验。
2.2冠状动脉造影评估侧支指数的变化
第一次手术时四组动物的侧支指数均为0,第6周时侧支指数在四组之间的差别有统计学意义(Kruskal-Wallis检验,P=0.026)。进一步组间比较发现,联合应用FGF-2/PDGF-BB组的侧支指数较单独应用FGF-2、PDGF-BB或PBS对照组均显著增高(P值分别为0.03 7,0.021和0.002),在结扎的冠状动脉远端形成可显影的侧支血管网络。与基础值相比,单独应用FGF-2治疗也可使缺血区侧支指数增加(P=0.018)。单独应用PDGF-BB治疗后虽然侧支指数有增加趋势,但差异未达到统计学意义(P=0.058)。而PBS对照组无明显侧支血管生成。
为了进一步观察这些侧支血管网是否稳定,在置入因子第14周后再次行冠状动脉造影检查,结果发现,联合应用FGF-2/PDGF-BB组的血管网络依然稳定存在,第14周的结果较第6周时的结果无显著差异。
2.3彩色微粒测定局部心肌血流量的变化
治疗前各组心肌血流量无差别,治疗后第6周和第14周,各组间差别有统计学意义(P＜0.05)。组间比较表明,FGF-2/PDGF-BB联合应用组较单独应用一种生长因子及PBS对照组心肌血流量显著增加(6周,P＜0.01;14周,P＜0.01)。析因设计的方差分析发现FGF-2和PDGF-BB之间有正交互作用(6周,F=7.317,P=0.011;14周,F=4.930,P=0.034)。第14周的结果较第6周时的结果无显著差异。
2.4左心室整体和局部功能的变化
治疗后第14周,FGF-2/PDGF-BB联合治疗组的左室射血分数(LVEF)较对照组显著增高(P=0.034),但是与单独应用FGF-2或PDGF-BB比较,差别无统计学意义(P值分别为0.10和0.082)。单独应用FGF-2或PDGF-BB较对照组LVEF亦无显著性改变。
治疗前各组间缺血区室壁增厚率无差别。第14周后单独应用FGF-2或PDGF-BB组较对照组WT差别无统计学意义,FGF-2/PDGF-BB联合治疗组较单独应用一种生长因子组的WT显著增高(P＜0.01)。
2.5联合治疗对于促血管生成的协同作用
通过免疫组化方法以血管性血友病因子(vWF,von Willebrandfactor)和α平滑肌肌动蛋白(α-SMA,α-smooth muscle actin)标记新生毛细血管和小动脉,并用荧光双标测定新生血管的成熟指数(maturation index),即被平滑肌包被的新生血管所占所有血管的百分比,以此来评价新生血管的成熟程度和稳定性。FGF-2组和FGF-2/PDGF-BB联合应用组的毛细血管密度较PDGF-BB组和对照组明显增高。FGF-2/PDGF-BB联合应用组与FGF-2组之间差别没有统计学意义。生长因子治疗的三个组的小动脉密度均较对照组明显增高,其中FGF-2/PDGF-BB联合治疗组的小动脉密度显著高于FGF-2或PDGF-BB单独治疗组(P＜0.01)及PBS对照组(P＜0.01)。FGF-2/PDGF-BB联合治疗组的血管成熟指数也明显高于FGF-2或PDGF-BB单独治疗组及PBS对照组(P＜0.01)。
2.6血管生长因子受体表达增高
FGF-2在心脏的受体主要是FGFR-1。PDGF-BB的两个受体是PDGFR-α和PDGFR-β。我们通过免疫组化检测了这三个受体的表达水平,结果发现,联合应用FGF-2/PDGF-BB组心肌微血管的FGFR-1、PDGFR-α和PDGFR-β的表达明显高于其他组。
同时用免疫组化和实时定量PCR的方法检测了VEGF和它的受体flk的表达,结果发现,在蛋白和mRNA水平,VEGF和flk在这四组中的表达均无显著性差异。
3.结论
(1)联合应用“促血管生成因子”FGF-2和“促动脉生成因子”PDGF-BB有协同的促血管生成作用,能够建立稳定的侧支血管网络,增加心肌血流量,明显改善心功能。
(2)FGF-2和PDGF-BB协同促血管生成机制与血管生成过程中FGFR-1,PDGFR-α和PDGFR-β在心肌微血管中表达上调以及PDGFR和FGFR之间的交互作用有关。这方面的深入研究为将来临床心肌梗死患者的促血管生成治疗奠定基础。
前言
既往研究表明左心室收缩期室壁厚度的增加主要来自心内膜下层心肌纤维的收缩,约占整个室壁增厚率的2/3,而心外膜肌纤维的收缩仅仅占室壁增厚率的1/3。因此,心内膜下心肌承受的收缩期压力更大,需氧量也高于心外膜下心肌。当心外膜下冠状动脉狭窄导致冠状动脉驱动压降低时,心内膜下层心肌血流量首先降低,导致心肌血流量在心内膜下层与心外膜下层的不均匀分布,心内膜下层与心外膜下层的跨壁梯度(transmural gradient)降低。收缩功能和心肌灌注的跨壁梯度在正常生理状态下可以忽略不计,但在定量评价冠状动脉疾病方面却有重要意义。理想的评价左心室功能的影像技术应该是既能测定心内外膜下层心肌的收缩功能也能评估心内外膜下层的血流灌注。然而,多数传统的影像技术包括二维超声心动图、核素心室造影和磁共振显像等仅仅能评估左心室整体功能而不能分层评估,因此这些技术对临床检测心肌缺血均有其局限性。虽然实时心肌声学造影技术能够测定心肌灌注的跨壁梯度,但是由于缺乏理想的造影剂,且存在声波衰减所致的假性无灌注区,该项技术仍未获得实际应用。
新近发展起来的应变和应变率显像技术(strain and strain rateimaging,SRI)是通过测定局部心肌形变和形变的速度来评价心肌局部收缩功能的一种方法。随着该技术的空间分辨率和数字处理能力的提高,现在已经能够实时测量心肌应变和应变率跨壁梯度分布。左心室不同层次的心肌纤维走行方向不同,心内膜下层与心外膜下层接近纵向,而中层心肌纤维接近横向走行,心肌纤维的这种三维立体走向特点决定了左心室的收缩运动包括三个方向,即径向收缩、纵向收缩和周向收缩。尽管心肌纵行纤维在左心室质量中所占比重较小,但由于心内膜下心肌为纵行纤维,更容易受到缺血的影响,因此局部心肌缺血对长轴方向收缩功能的损害早于短轴方向。从理论上推测,左室长轴方向收缩功能的变化能更敏感地反映心肌缺血导致的局部心功能的变化,定量评价左心室节段性纵向收缩功能可能具有重要的临床意义。然而,不同病理生理状态下心肌纵向应变和应变率的分层分布特点及其与心肌灌注跨壁梯度的关系目前尚不清楚。因此,本研究拟检验的假说为:应变率显像技术能够通过定量评价心室肌不同层次的纵向收缩功能,无创性估测局部的心肌灌注。
目的
1)在小型猪心肌缺血梗死模型中,观察不同状态下(正常、缺血和梗死节段)心肌的内、外膜层纵向应变和应变率的变化特点。
2)探讨心肌纵向应变和应变率的跨壁梯度与心肌跨壁血流的关系。
1方法
1.1动物模型的建立
12只雄性中国实验用小型猪,体重25～35kg(平均28.20±4.77kg),购自中国农业大学(北京)。所有动物饲养和实验程序都遵守中国卫生部实验动物管理条例(No 55,2001)和山东大学齐鲁医院实验动物管理条例。
肌肉注射安定50mg(2mg/kg)、阿托品1～2mg(0.06～0.08mg/kg)和氯胺酮500mg(18～20mg/kg),经3～5min后猪站立不稳而卧倒。诱导麻醉后将猪仰卧固定于操作台,气管插管,呼吸机机械通气,呼吸频率16～20次/分。心电监护仪进行心电监护。静注3%戊巴比妥钠3～5mL麻醉后实施手术。
实验动物平卧,备皮,常规碘伏消毒,铺巾。开胸之前,经静脉注射肌松药琥珀胆碱0.1g,用20ml生理盐水稀释。经前正中切开皮肤,高频电刀切开肌肉,并对出血点进行灼烧止血。用胸骨牵开器撑开胸腔,暴露心包和肺脏。纵形切开心包,并用7号缝线固定心包。显露冠状动脉左前降支,于左前降支中远端1/3处用无创针线预结扎(不全部阻断血流)10分钟,然后结扎。冠状动脉结扎后如心电监护出现ST段弓背抬高,结扎动脉区域心肌变暗变紫,然后行股动脉穿刺,经股动脉将6F造影导管送入左右冠状动脉行冠状动脉造影术,如显示左前降支结扎部位完全梗阻,证明模型建立成功。结扎冠脉1小时以后行心外膜超声心动图检查。
1.2应变和应变率显像
所用仪器为Vivid 7~(TM)彩色多普勒超声显像系统(Vivid 7,GEVingmed,Horten,N0rway),M3S探头,频率1.5～4.0 MHz。该系统装有应变和应变率的图像分析与后处理软件,可自动定量分析心内膜和心外膜下心肌的同步应变和应变率曲线。动物仰卧位,获取心尖四腔切面标准图像,室间隔尽可能与超声束方向平行,帧频大于100帧/s,在应变和应变率模式下6个心动周期的动态图像存储于硬盘中,待脱机后处理和分析。
在本研究中,我们将心肌明显变薄运动减低甚至矛盾运动的室间隔心尖段界定为梗死区,紧邻着梗死区域的室问隔中间段界定为缺血区,由右冠状动脉供血的室间隔基底段心肌界定为正常节段。进入TVI定量分析软件系统后将应变长度(strain length)设为4mm,将两个大小为5mm×1mm的椭圆形的取样容积分别平行置于心内膜下和心外膜下心肌层,分别测定正常、缺血和梗死节段心内外膜下层的应变和应变率。测量中注意调整收缩提取样容积的角度和位置,以保证两个取样容积在整个心动周期中保持在同一位置。系统将显示出该部位的应变曲线和应变率曲线,分别测量收缩期最大应变率(SRp)和收缩期最大应变(Sp)2项指标,然后计算心内外膜下层心肌测值的比值,作为内外膜应变和应变率的跨壁梯度。每个指标连续测量三个心动周期取其均值。
1.3局部心肌血流量的测定
应用彩色微粒(直径10±2μm,E-Z Trac,Los Angeles,CA)测量心肌局部血流量。将5F猪尾导管经股动脉送入左心室,经导管向左心室内注射入5×10~6个彩色微粒(将彩色微粒稀释于10ml生理盐水中),注射时间为30s,然后用10ml生理盐水冲洗导管。在注射彩色微粒前10s,使用定速抽血泵由股动脉抽取参考血液样本,抽取速率为10ml/min,共90s。血样-20℃保存。动物麻醉处死后,根据超声图像将室间隔分为正常、缺血和梗死三部分,每一部分分为心内膜下层和心外膜下层分别称重。根据产品提供的说明步骤对组织和参考血样中的彩色微粒进行回收。根据下面的公式计算心肌局部血流量:Q_m=(C_m×Q_r)/C_r。其中,Q_m是指每克心肌组织的血流量(ml/min/g),C_m是指每克心肌组织的彩色微粒数,Q_r是指参考血样的抽取速度(ml/min),C_r是指参考血样中的彩色微粒数。
1.4重复性检验
我们以缺血区域的心内膜和心外膜下层的TVI图像进行重复性分析,测量室间隔缺血区的Sp和SRp,检验这两个定量指标在观察者内部与观察者之间的差异。以同一观察者前后或不同观察者之间两次测值的绝对差值占两次测值均数的百分数表示。
1.5统计分析:
采用SPSS 11.5统计分析软件进行分析。计数资料用均数士标准差表示。不同节段的MBF,Sp and SRp应用one-way ANOVA比较,组间两两比较进一步用LSD分析。同一节段心内外膜下层参数用配对t检验比较。采用相关回归分析检验Sp和SRp与相应部位的MBF的相关性。设P＜0.05为有统计学差异。
2.结果
2.1正常、缺血和梗死节段的心肌应变和应变率
在心肌正常节段,心内膜下层Sp明显高于心外膜下层Sp(-11.39±2.30 vs-9.48+1.66,P=0.001),心内外膜跨壁梯度大于1。与正常节段心肌相比,缺血区的心内膜下层和心外膜下层Sp均显著降低(P均＜0.001),但是心内膜下层心肌Sp降低程度更重,心内膜下层Sp明显低于心外膜下层Sp(-5.99±1.59 vs-7.23±1.29,P=0.002),使得内外膜跨壁梯度发生逆转,Sp-EER小于1(Sp-EER:0.82±0.14)。在梗死节段,内外膜的Sp进一步显著降低接近于0,使得跨壁梯度消失。无论是心内膜下层还是心外膜下层的正常、缺血和梗死节段Sp两两比较,三者之间均存在显著差异(P＜0.001)。
在正常心肌节段,心内膜下层的SRp明显高于心外膜下层的SRp(-2.78±1.05 vs-2.34±0.87,P=0.007),心内外膜下层的SRp跨壁梯度大于1。与正常节段心肌相比,缺血区的心内膜下层和心外膜下层的SRp均显著降低(P均＜0.001),但是心内膜下层心肌的SRp降低程度更重,心内膜下层SRp明显低于心外膜下层SRp(-1.07±0.37 vs-1.47±0.57,P=0.006),使得心内外膜下层的跨壁梯度发生逆转,SRp-EER小于1(SRp-EER:0.76±0.15)。在梗死节段,心内外膜下层的SRp进一步显著降低(-0.47±0.22和-0.45+0.19,P＜0.001),接近于0,使得跨壁梯度消失。无论是心内膜下层还是心外膜下层的正常、缺血和梗死节段SRp两两比较,三者之间均存在显著差异(P均＜0.001)。
2.2正常、缺血和梗死节段的心肌血流量
在正常心肌节段,心内外膜下层的心肌血流跨壁分布一致(1.50±0.13 vs 1.46±0.16,P＞0.05)。在缺血区,心内外膜下层血流均降低(P均＜0.001),但是心内膜下层血流降低更显著使得心内膜下层血流明显低于心外膜下层血流(0.70±0.15 vs 0.87±0.08,P＜0.01),出现心内外膜下层跨壁梯度小于1(MBF-EER:0.81±0.12)。在梗死节段,心内外膜下层血流进一步显著降低,跨壁梯度消失,心内外膜下层血流比值等于1(0.26±0.08 vs 0.26±0.07,P＞0.05)。
2.3心肌节段血流和应变的相关性
线性相关回归分析表明,心内膜下层的Sp与MBF显著相关(r=-0.84,P＜0.001);心外下层膜的Sp与MBF显著相关(r=-0.81,P＜0.001)。心内膜下层的SRp与MBF也有很好的相关性(r=-0.78,P＜0.001);心外膜下层的SRp与MBF也有很好的相关性(r=-0.75,P＜0.001)。
2.4重复性检验
各项指标在观察者内部的差异分别为:心内膜下层Sp,10.2±3.6%;心外膜下层Sp,10.3±4.2%;心内膜下层SRp,12.9±3.8%和心外膜下层SRp,13.3±4.1%。观察者之间的差异分别是:心内膜下层Sp,11.5±3.9%;心外膜下层Sp,11.9±3.7%;心内膜下层SRp,13.8±4.1%和心外膜下层SRp,14.2±4.3%。
3结论
(1)本研究在小型猪心肌缺血和梗死模型中发现,在正常、缺血和梗死不同状态下心内外膜下层心肌纵向应变和应变率及其跨壁梯度明显不同。
(2)定量分析心肌纵向跨壁应变和应变率的改变能够区分正常、缺血和梗死区域,评估心肌缺血的严重性,表明纵向心肌形变的跨壁分布测定对评价患者缺血心肌局部灌注可能是一个很有价值的指标。
Introdnction
Atherosclerosis-induced coronary artery disease(CAD)is the leading cause of morbidity and mortality in Western societies and increases at an alarming speed in developing countries,despite continued advances in the treatment of CAD such as PTCA (Percutaneous Transluminal Coronary Angioplasty),CABG(Coronary Artery Bypass Graft)and intracoronary stent placement.Except for surgical interventions,an effective therapeutic method for treatment has been lacking.An increasing number of patients are not suitable for conventional revascularization strategies because of diffuse small vessel disease,multi-vessel disease,long lesions,occluded vessels without collateral recanalization,or other comorbidities such as diabetes mellitus and obesity.Therapeutic angiogenesis induced by angiogenic growth factors may provide an alternative approach to the treatment of myocardial ischemia for these patients.
Therapeutic angiogenesis is a process that seeks to stimulate collaterogenesis and to improve myocardial perfusion and function by delivery of proangiogenic factors to the ischemic myocardium. Numerous experimental and clinical studies have evaluated therapeutic angiogenesis as a treatment for ischemic heart disease. Despite the promising results of therapeutic angiogenesis in preclinical studies,clinical evaluation of these individual proangiogenic molecules has produced unfulfilled promises since this idea has been proposed for more than 30 years.After more than a decade of clinical practice with different single proangiogenic factors for the treatment of ischemic disorders,almost all large randomized,double-blinded,and placebo-controlled human trials have proven to be non-beneficial.Pharmaceutical companies that sponsor clinical trials often choose an angiogenic agent based on the intellectual properties owned by the company and the possible easy approval by the Food and Drug Administration(FDA).
Most previous preclinical and clinical studies on the development of proangiogenic therapies for treating ischemic myocardium have been based on mono-therapeutic approaches.In addition,most studies have focused on evaluation of the therapeutic efficacy of vascular endothelial cell growth factor(VEGF)-A,which primarily targets the endothelial cell compartment.The establishment of stable and functional blood vessel networks,however,is a complex process that requires several angiogenic factors targeted different cell populations in the vasculature.The reason for this discrepancy is unclear,and in part that current angiogenic therapies in clinical trials are based on a single angiogenic factor delivered, which is insufficient to initiate the entire cascade of events leading to a mature,functional and stable vascular network.
Neovascularization in the ischemic adult heart is a combination of several processes including angiogenesis,arteriogenesis and potentially vasculogenesis.Angiogenesis is defined as the sprouting of new blood vessels from pre-existing vasculature and consists of distinct stages including cell migration,proliferation and differentiation into capillaries and blood vessels.Arteriogenesis refers to the process of maturation or de novo growth of collateral conduits and produce vessels capable of carrying significant blood flow.These vessels are of a sufficient diameter to be visualized with angiography.
The angiogenic factors,VEGF and FGF-2,are perhaps the most extensively studied angiogenic growth factors,which has been used in clinical trials.VEGF exerts its effects on endothelial cells that include enhanced survival,increased permeability,enhanced migration and proliferation,all of which contribute to angiogenesis. VEGF is also named VPF(vascular permeability factor)due to its high permeability of vessel.
Like VEGF,FGF-2 also stimulates endothelial cell proliferation and migration,which plays a critical role in triggering an initial robust angiogenic response.Although the release of the signal peptide-less FGF-2 from its synthetic cells remains enigmatic,this growth factor plays a crucial role extracellularly in the regulation of the growth of its target cells including endothelial cells.Especially important,administration of FGF-2 in vivo selectively stimulates angiogenesis but not other tissue growth.
However,endothelia cells channels,induced by single angiogenic factor,are naked,leaky,and fragile,which are easily ruptured and bleed.Insufficient perfusion eventually leads to vessel regression.
In contrast to FGF-2 and VEGF,PDGF-BB acts mainly on vascular mural cells including pericytes and vascular smooth muscle cells(VSMCs).PDGF-BB interacts with both homodimeric and heterodimeric PDGFR-αand PDGFR-βcomplexes,especially PDGFR-β,which plays an essential role in stabilization of blood vessels via the recruitment of pericytes and VSMCs onto the nascent vasculature.
For improvement of perfusion of high-oxygenated blood in ischemic tissues,it is essential to re-establish functional arterial vascular networks,which should remain stable for long term.The clinical failures with this attractive approach have raised several unresolved fundamental issues regarding the basic mechanisms of neovasculorazation.These include the underlying mechanisms of angiogenesis versus arteriogenesis,choice of proangiogenic agents, monotherapy versus combinatorial therapy,selection of optimal combination and drug release systems,evaluation standard for functional arterial networks and appropriate animal models for preclinical evaluation.
Based on these conditions,we propose a hypothesis as following: the interplay and synergy between various angiogenic and arteriogenic factors for promoting arteriogenesis is specifically limited to certain combinations,and that each factor has its own specific partners.The optimal combination of angiogenic and arteriogenic factors could induce stable arterial networks and improve collateral growth and functional recovery.
The purpose of this study was,based on the selection of optimal combination of angiogenic and arteriogenic factors in mouse cornea, to explore therapeutic evaluation of the combination in a pig myocardial infarction model.
1.Methods
1.1 Selection of optimal combination of angiogenic and arteriogenic factors
The avascular feature of the corneal tissue made it an ideal model for assessing vascular network formation.A micropellet(0.35×0.35 mm)of sucrose aluminum sulfate coated with hydron polymer type NCC containing 160 ng of VEGF,PDGF-AA,-AB,-BB,or 80 ng of FGF-2,or 160 ng PDGF-AA/80 ng FGF-2,or 160 ng PDGF-AB/80 ng FGF-2,or 160 ng PDGF-BB/40 ng FGF-2,or 160 ng PDGF-BB/160 ng VEGF was surgically implanted into a micropocket in the mouse cornea(one pellet/eye,one eye implanted/mouse).The corneal neovascularization was examined and quantified on day 5 after pellet implantation.Vascularization areas were calculated by measuring vessel lengths and clock-hours(the circumferential area of neovascularization if the eye is considered as a clock).For vascular stability analysis,the corneal neovascularization pattern/profile was examined on days 5,12,24,and 70 after pellet implantation.Finally, we thus chose an optimal combination for the therapeutic evaluation and further studies in a pig model of myocardial infarction.
1.2 Pig myocardial infarction model and growth factor implantation
40 Chinese experimental mini-pigs weighing between 25-30 kg/each were used for the myocardial infarction model(China Agricultural University,Beijing).All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001)and the guidelines for the Care and Use of Laboratory Animals of Qi Lu Hospital,Shandong University,China.Animals were randomly divided into four groups(n=10 pigs/group):(A)PBS, (B)FGF-2(5μg),(C)PDGF-BB(10μg),and(D)FGF-2 and PDGF-BB(5μg and 10μg,respectively).
Before all experimental procedures,all animals were anesthetized by intramuscular administration of ketamine hydrochloride(20 mg/kg,Heng Rui Medicine Co.LTD.Jiang Su, China)together with intravenous injection of sodium pentobarbital (30 mg/kg,Beijing Chemical Reagent Company,Beijing,China)and were mechanically ventilated with a volume respirator(Newport E100m Ventilator,Newport Medical Instruments,CA,USA).Median sternotomy was performed to expose the heart followed by incision of the pericardium.Acute myocardial infarction was produced by ligation of the LAD coronary artery distal to its third diagonal branch using a 7.0-prolene suture.Selective left and right angiography, performed through a standard procedure,was made to confirm complete occlusion of the LAD and to assess baseline levels of collaterals to assess the collateral index.
A slow-releasing pellet of sucrose aluminum sulfate coated with hydron polymer(0.2×0.2 cm)containing PBS,5μg FGF-2,10μg PDGF-BB,or 5μg FGF-2 and 10μg PDGF-BB was attached to an aseptic application(0.5×0.5 cm),which was sutured(7.0-prolene) onto the border zone adjacent to the mid and distal LAD.
1.3 Coronary arteriography
Six and fourteen weeks after treatments,repeated,selective angiography was performed.Under general anesthesia,the animals received a contrast agent,meglucamine diatrizoate,through a standard femoral puncture using the digital subtraction angiography (CGO-2100,Wandong Medical Equipment Co.LTD,China).The angiographic index was assessed using a standard protocol based on Rentrop's grading scales from 0 to 3:0,none;1,filling of side branches of the LAD;2,partial filling of the LAD main artery via collateral channels;3,complete filling of the LAD.
1.4 Regional myocardial blood flow
For determination of regional myocardial blood flow at different time points,colored microspheres(10±2μm diameter;E-Z Trac,Los Angeles,CA)labeled with red,yellow and green were used prior to treatment,and at the end of week 6 and 14.5×10~6 microspheres were injected into the left ventricle by a 5F pigtail catheter(Cordis,USA)with 10 ml of saline.After completing the injection,the catheter was flushed with 10 ml of saline.An arterial Reference Blood Sample was collected for each colored microsphere injection.Starting 10 seconds before injection,reference blood samples were withdrawn by a withdrawal pump at a constant rate of 10 ml/min for a period of 90 seconds.Recovery of microspheres from tissue and blood was performed according to the manufacturer's instruction.Blood flow values are calculated from the following equation:Q_m=(C_m×Q_r)/C_r.Where Q_m is the myocardial blood flow per gram(ml/min/g),C_m is the microsphere count per gram of tissue, Q_r is the withdrawal rate of the reference blood sample(ml/min),and C_r is the microsphere count in the reference blood sample.
1.5 Echocardiography
Two-dimensional echocardiography was used to measure global and regional left ventricular function in all animals with open chest following angiographic assessment before and 14 weeks after treatment using an ultrasound scanner(SONOS 7500,Philips Medical Systems Inc.,Andorver,MA).Analyses of LVEF and systolic wall thickening(WT%)were performed to determine the global function of the left ventricle and the regional function of the myocardium, respectively.LVEF was determined from the apical two-chamber and four-chamber views using a modified Simpson's algorithm. Regional wall thickness was measured at end-diastole(the peak of R wave of the ECG)and end-systole(the end of T wave of the ECG) individually on two-dimensional echocardiograms.
Left ventricular systolic wall thickening(WT%)was calculated as: WT%=(SWT-DWT)/DWT×100%.
1.6 Immunohistochemistry
The treated areas of the left ventricle myocardium in the LAD territory were fixed with paraformaldehyde or were immediately frozen in liquid nitrogen.To determine vessel density, paraffin-embedded 5μm sections were incubated with a rabbit anti-von Willebrand factor(vWF)antibody or a mouse anti-alpha smooth muscle actin(αSMA)antibody,followed by incubation with secondary antibodies labeled with horseradishperoxidase.Ischemic myocardial sections were also used for detection of vWF/αSMA double positivity using immunofluorescent analysis.A maturation index(percentage of smooth muscle cell positive vessels vs.total vessel numbers)was calculated.
1.7 Data analysis
Data are presented as the mean±standard errors of the mean and were analyzed with SPSS 11.5 software.A 2×2 factorial analysis of variance was used to examine the interaction between FGF-2 and PDGF-BB.Continuous variables were compared by using repeated measures analysis of variance(RM ANOVA)followed by the LSD or Dunnutt T3 corrected post hoc analysis for multiple comparison procedure.Nonparametric variables were compared between groups by using two-sided Kruskal-Wallis(multiple group comparison)and Mann-Whitney(two group comparisons)tests.Collateral index was compared within groups before and after treatment using Wilcoxon test.All reported P values were two-tailed,and a P value＜0.05 was considered statistically significant.
2.Results
2.1 Selection of optimal combination of angiogenic and arteriogenic factors in mouse cornea
Combination of PDGF-AA/VEGF,PDGF-AB/VEGF or PDGFBB/VEGF didn't result in synergistic angiogenesis. PDGF-AA/FGF-2,PDGF-AB/FGF-2,or PDGF-BB/FGF-2 combinations synergistically induced angiogenesis as compared with single growth factor-induced angiogenesis.At day 70 after implantation,PDGF-AA/FGF-2-induced vessels were almost completely regressed,suggesting that this combination is unable to stabilize the newly formed vasculature.Interestingly,both PDGF-AB/FGF-2- and PDGF-BB/FGF-2-induced corneal vascular networks remained stable at this and later time points.These findings demonstrate that only PDGF-AB/FGF-2 or PDGFBB/FGF-2,but not PDGF-AA/FGF-2,are able to stabilize the newly formed vasculature although all three combinations promote angiogenic synergisms.We thus chose a combination of PDGF-BB/FGF-2 for the therapeutic evaluation and further studies.
Forty animals received surgery and five animals died in the initial operation,of which two animals died of anesthesia accident and three animals died of ventricular fibrillation during ligation of the left anterior descending(LAD)coronary artery to make myocardial infarction model.One animal(PBS control)died of anesthesia accident in the mid-study.Thus,the remaining thirty-four animals(PBS,n=7;FGF-2,n=10;PDGF-BB,n=8;FGF-2+PDGF-BB, n=9)completed the entire study.No abnormal adverse events were noted during the entire course of these experiments in those animals that survived to the 14-week end point.
2.2 Angiographic analysis of collateral formation
Analysis of the collateral index demonstrated a difference among the four groups at week 6(Kruskal-Wallis test,P=0.026), whereas baseline collateral index in all groups was similar.Further multiple comparisons showed that PDGF-BB/FGF-2 significantly induced myocardial collateral growth as compared with various individual FGF-2 or PDGF-BB or PBS -treated myocardium(P=0.037, P=0.021 and P=0.002,respectively).These newly established collaterals formed vascular networks proximally and distally from the ligation site.Compared with the baseline,collateral index had increased significantly in the FGF-2 group and in PDGF-BB/FGF-2 groups at week 6(P=0.018 and P=0.001,respectively).Despite a trend toward increase in collateral index in the PDGF-BB group compared with the baseline,the difference did not reach statistical significance(P=0.058).
To study the stability of these newly established collateral networks,coronary angiography was again performed on week 14 after treatment.Remarkably,the PDGF-BB/FGF-2-induced collaterals had developed into well-established coronary arterials, indicating the re-established collaterals remained stable.
2.3 Assessment of regional myocardial blood flow(MBF)
The myocardial blood flow was assessed by a colored microsphere method.Prior to treatment,the basal levels of myocardial blood flow in the LAD territory were indistinguishable among all groups.At week 6,the PDGF-BB/FGF-2 together-treated group exhibited remarkably higher MBF than either factor alone-treated myocardium(P＜0.05).The high level of MBF persisted for long term in the PDGF-BB/FGF-2-treated group and no reduction of MBF was observed at the end point of the experiment(14 weeks). Statistical analysis of these data using factorial analysis of variance showed that MBF in the PDGF-BB/FGF-2 together-treated group was markedly greater than the sum effects obtained from two factors alone-treated myocardium(F=7.317,P=0.011,at week 6;and F=4.930,and P=0.034,at week 14).
2.4 Echocardiographic analysis of global and regional myocardial function
Echocardiography was performed to monitor global and regional myocardial function.Measurement of left ventricular ejection fraction(LVEF),a sfandard parameter to monitor global myocardial contractile function,analysis showed that the basal cardiac dysfunction was indistinguishable among all groups before treatment. At week 14 after treatment,a significant improvement of LVEF was observed in the PDGF-BB/FGF-2-treated group as compared with the buffer-treated group,however,there were no significant differences between the combination treatment group and groups treated with either FGF-2(P=0.10)or PDGF-BB(P=0.082)alone.Neither single growth factor-treated group showed significant improvements of LVEF.
To assess regional wall motion,left ventricular systolic wall thickening(WT%)was calculated.There were also no significant differences in the baseline regional function among groups.Similar to LVEF results,PFGF-BB- and FGF-2-single treated groups did not show any significant improvement in WT%as compared with buffer-controls.In contrast,the PDGF-BB/FGF-2-treated group significantly improved the WT%as compared with either buffer- or single factor-treated groups(P＜0.01).
2.5 Vessel density and maturation index
Collateral micro-vessel formation was further examined by measuring the number of capillaries and arterioles by von Willebrand factor(vWF)andα-smooth muscle actin(α-SMA) immunohistochemistry in light microscopic sections taken from ischemic myocardium.Myocardial capillary density in the border zone in FGF-2 and the combination group was significantly higher compared with PBS and PDGF-BB group(P＜0.05).No apparent difference was observed between the FGF-2 and the combination group.Arteriolar density was significantly higher in growth factors treated animals than in PBS controls.Consistent with the coronary angiographic analysis,high numbers of arterial vessels were detected in the PDGF-BB/FGF-2-treated myocardium at week 14 post-treatment as compared with those of single factor-treated or control samples.
In addition,for assessing the maturity and stability of myocardial newly vessels,a maturation index defined as proportion of vessels surrounded by smooth muscle cells was determined by vWF andα-SMA double-labeled immunofluorescence.At week 14 after treatment,α-SMA positive vessels in the PFGF-BB/FGF-2 together-treated group was significantly higher than those detected in the PFGF-BB,FGF-2,or buffer alone-treated myocardium(P＜0.05).
2.6 Upregulation of FGFR-1,PDGFR-αand PDGFR-β
The proangiogenic factors exert their cellular effects by binding to and activating protein tyrosine kinase receptors expressed on target cells.FGFR-1,PDGFR-αand PDGFR-βwere examined by immunohistochemistry.FGFR-1,PDGFR-αand PDGFR-βexpression significantly higher in the PFGF-BB/FGF-2 group than others.
To further identify whether VEGF may in part involve in vascular stability by activating flk,we also examined the expression of VEGF mRNA and protein by realtime PCR and immunohistochemistry,respectively.There were no significant differences at both mRNA and protein level in VEGF and flk among groups.
Conclusion
(1)A protein-based angiotherapeutic approach,based on dual delivery of arteriogenic and angiogenic factors,significantly improves myocardial collateral growth,blood perfusion,and cardiac function in a pig ischemic myocardial model.
(2)A possible mechanism of angiogenic synergism involves upregulation of the expression of FGFR-1,PDGFR-αand PDGFR-βin angiogenesis,and the crosstalk between PDGFR and FGFR.
INTRODUCTION
Experimental studies have demonstrated that left ventricular systolic wall thickening stems mainly from the contraction of the subendocardial myocardial fibers with the inner half layer of the ventricular wall contributing to about two-thirds and the outer half layer contributing to about one-thirds of systolic wall thickening. Therefore,the subendocardium sustains a higher systolic stress and demands more oxygen consumption and blood supply than the subepicardium.Thus,the subendocardium is more susceptible to ischemia than the subepicardium.The transmural gradient of myocardial perfusion is of negligible significance in physiological conditions but becomes extremely important in the presence of epicardial coronary artery stenosis because an accurate assessment of the transmural perfusion gradient may significantly improve the sensitivity and specificity of diagnostic techniques in the early detection of coronary artery disease.Therefore,an ideal imaging technique for detecting coronary artery disease should be able to measure subendocardial and subepicardial myocardial perfusion separately.Most conventional imaging modalities,however,can only assess myocardial perfusion across the entire ventricular wall rather than in different layers due to the lack of a high spatial resolution, and thus,the clinical value of these techniques in detecting myocardial ischemia is rather limited.Recently real-time myocardial contrast echocardiography has shown the potential to delineate transmural distribution of myocardial perfusion but the lack of an ideal myocardial contrast has significantly hindered development of this technique.
Strain and strain rate imaging is a new modality of echocardiographic techniques designed for measuring the regional myocardial deformation or the velocity of deformation.With a high spatial resolution and frame rate as well as digital processing capability of these techniques,it has been possible to measure the transmural gradient of myocardial strain and strain rate in real time. As myocardial longitudinal shortening plays an important role in cardiac contraction,and longitudinal myocardial fibers contract earlier and are likely more vulnerable to ischemia than circular fibers, measurement of the transmural gradient of the longitudinal myocardial strain and strain rate may provide important information on the transmural distribution of myocardial contraction in physiological conditions and myocardial ischemia.It is still unclear whether the transmural systolic strain and strain rate could predict the transmural myocardial blood flow at different perfusion levels. Therefore,we propose the hypothesis that the transmural gradient of the longitudinal myocardial contraction can be assessed by strain and strain rate imaging,which can in turn be used to predict the transmural gradient of myocardial blood flow(MBF)measured by colored microspheres in the normal,ischemic and infarct segments in a pig model with acute myocardial infarction.
OBJECTIVE:
To study the contraction patterns in subendocardium and subepicardium across the normal,ischemic and infarct segments by SRI and to explore the relationship between the regional myocardial blood flow and strain and strain rate in the subendocardium and subepicardium at different perfusion levels.
1.METHODS
1.1 Animal preparation
Twelve Chinese experimental mini-pigs(males,25-30kg,China Agricultural University,Beijing)were included in this study.All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001)and the guidelines for the Care and Use of Laboratory Animals of Shandong University Qilu Hospital, China.
General anesthesia was achieved in all pigs by intramuscular injection of ketamine hydrochloride(20 mg/kg,Heng Rui Medicine Co.LTD.Jiang Su,China)together with intravenous injection of sodium pentobarbital(30 mg/kg,Beijing Chemical Reagent Company, Beijing,China).Pigs were placed in the supine position,intubated and mechanically ventilated with a volume respirator(Newport E100m Ventilator,Newport Medical Instruments,Inc,Newport Beach, CA)at a rate of 16-20 breaths per minute.All animals underwent continuous electrocardiographic(ECG)monitoring throughout the experiment.The heart was exposed through a median sternotomy and supported by a pericardial cradle.A 6F Judkins right catheter was introduced via the femoral artery for the left coronary angiography. Distal to the second diagonal branch,the left anterior descending (LAD)coronary artery was completely occluded for 60 minutes with a 7.0-prolene suture.Successful construction of an acute myocardial infarction model was confirmed by left coronary angiography and by the presence of typical ST segment elevation on an electrocardiogram.
1.2 Strain and Strain Rate Imaging
Echocardiographic imaging was performed 1 hour after LAD ligation with a Vivid 7~(TM)ultrasound system(Vivid 7,GE Vingmed, Horten,Norway)with a M3S transducer(1.5 to 4.0MHZ).Strain and strain rate imaging were derived from the apical four-chamber view with the interventricular septum as parallel as possible to the direction of the ultrasound beam.A high frame rate(＞100 frames/s) was used to obtain as much data as possible in a given period of time and a latex bag filled with degassed saline served as an acoustic interface between the epicardium and the transducer.A cine-loop comprising least 6 cardiac cycles was stored digitally on a magneto optic disk for later offline analysis.
The ventricular septum was divided into three segments based on their two-dimensional images:the dramatically thinned and akinetic or dyskinetic region,usually localized in the distal septum in the present model,was defined as an infarct segment.The hypokinetic region which was close to the infarct segment and usually located in the middle septum was defined as an ischemic segment.The basal septum,whose blood supply stems from the right coronary artery, was defined as a normal segment.A myocardial length of 4 mm was used for strain analysis and two distinct and parallel sample volumes with an elliptical shape and a size of 5 mm×1 mm were placed in the subendocardial and subepicardial layers in the middle of the normal, ischemic and infarct segments,respectively.The strain curves from both subendocardial and subepicardial layers were simultaneously displayed in the normal,ischemic and infarct segments and the peak systolic strain from the subendocardium(Sp-endo)and subepicardium(Sp-epi)was measured from the two curves.The transmural gradient of myocardial strain was calculated as the ratio of Sp-endo/ Sp-epi(SpoEER)in the three segments,respectively. Similarly,The strain rate curves from both subendocardial and subepicardial layers were simultaneously displayed in the normal, ischemic and infarct segments,respectively and the peak systolic strain rate from the subendocardium(SRp-endo)and subepicardium (SRp-epi)was measured from the two curves.The transmural gradient of myocardial strain rate was calculated as the endocardial to epicardial ratio of strain(SRp-EER),i.e.,SRp-endo/SRp-epi in the three segments.Three cardiac cycles were measured and the values averaged.To ensure sample volumes to be kept in the same desired position,mild manual readjustment of the position of sample volumes during systole was necessary.
1.3 Regional Myocardial Blood Flow Measurement
Regional MBF was measured by injection of 5×10~6 colored microspheres(10±2μm in diameter,E-Z Trac,Los Angeles,CA) into the left ventricle by a 5F pigtail catheter(Cordis,USA)which was followed by a flash injection of 10 ml saline.Starting 10 seconds before each colored microspheres injection,an arterial reference blood sample was withdrawn by a withdrawal pump at a constant rate of 10 ml/min for a period of 90 seconds.After the animal was euthanized,the cardiac sections were cut in accordance with the echocardiaographic apical four chamber views and the transmural tissue slices from normal,ischemic and infarct zones in the ventricular septum according to sample volume positions were derived,each of which was divided into subendocardial and subepicardial layers and weighed individually.Microspheres in the myocardial tissue and blood sample were recovered according to the manufacturer's instruction.MBF values in the subendocardial (MBF-endo)and subepicardial(MBF-epi)layers were calculated separately from the following equation:Q_m=(C_m×Q_r)/C_r,where Q_m is the myocardial blood flow per gram(ml/min/g),C_m is the microspheres count per gram of tissue,Q_r is the withdrawal rate of the reference blood sample(ml/min),and C_r is the microspheres count in the reference blood sample.The transmural gradient of myocardial perfusion(MBF-EER)was calculated as the ratio of MBF-endo/MBF-epi in the three segments.
1.4 Inter-observer and intra-observer variability
In order to assess the reproducibility of strain and strain rate measurement,Sp and SRp in the subendocardium and subepicardium in all animals were measured in the ischemic segment.The inter-observer variability was calculated from the repeated measurements performed by two independent observers and the intra-observer variability was calculated from the repeated measurements performed by one observer who measured twice one week apart.Variability was expressed as the percentage of the absolute difference between two measurements divided by the mean value of the two measurements.
1.5 Statistical Analysis
Data analysis was performed using SPSS 11.5 statistical software (SPSS Inc.,Chicago,USA).Values were expressed as mean±SD. Comparisons of MBF,Sp and SRp among all segments were performed by one-way ANOVA,and LSD test was used to assess the difference between multiple comparisons.Paired student's t-test was used to analyze the differences between parameters measured from the subendocardium and subepicardium in the same segment. Correlations between MBF and Sp or SRp from all three segments were performed by linear regression analysis.P value＜0.05 was considered statistically significant.
2.RESULTS
2.1 Strain and Strain Rate Imaging Measurements
Two pigs died of ventricular fibrillation after LAD occlusion and 10 pigs entered the final data analysis.The peak systolic strain in both subendocardium(Sp-endo)and subepicardium(Sp-epi) decreased progressively from the normal to the ischemic and the infarct segments and there were significant differences among the three segments(all p＜0.001).Sp-endo was significantly higher than Sp-epi in the normal segment(p=0.001)whereas significantly lower than Sp-epi in the ischemic segment(p=0.002).On the other hand, there was no significant difference between Sp-endo and Sp-epi in the infarct segment(p＞0.05).Consequently,Sp-EER calculated was the highest in the normal segment and the lowest in the ischemic segment and there was a significant difference in Sp-EER between normal and ischemic segments(p＜0.001)and between normal and infarct segments(p=0.013).Moreover,Sp-EER was significantly lower in the ischemic than in the infarct segment(p=0.004).
Peak systolic strain rate showed similar changes as peak systolic strain and exhibited a progressive decline from the normal to the ischemic and the infarct segments.(all p＜0.001).SR-endo was significantly higher than SR-epi in the normal segment(p=0.007)but significantly lower than SR-epi in the ischemic segment(p=0.006).In contrast,there was no significant difference between SR-endo and SR-epi in the infarct segment.Compared with the normal segment, SR-EER was significantly reduced in the ischemic(p＜0.001)and infarct segments(p=0.028).Also SR-EER was significantly lower in the ischemic than in the infarct segment(p=0.001).
2.2 Regional Myocardial Blood Flow
The regional MBF was similar in the subendocardium and the subepicardium in the normal segment with a calculated MBF-EER of 1.03±0.07.Compared with MBF in the normal segment,MBF in both subendocardium and subepicardium in the ischemic segment decreased significantly(both P＜0.001)with a more remarkable decline in MBF-endo(p?
动脉粥样硬化导致的冠心病是西方国家发病率和死亡率最高的疾病,在发展中国家的发病率和死亡率也以惊人的速度在增长。近几十年来,冠心病的治疗取得了很大的发展,经皮冠状动脉内血管成形术(PTCA)、冠状动脉内支架置入术、冠状动脉旁路移植术(CABG)等都已成为临床上成熟的治疗手段,挽救了数百万患者的生命。但是,随着世界人口的老龄化,冠心病发病率仍在持续增高,越来越多的患者的病情复杂化,不适合这些传统方法的治疗。这些复杂病变主要包括弥漫性小血管病变、慢性完全性堵塞病变、多支血管病变、多次介入治疗或手术后血管闭塞等,近年来发展起来的治疗性血管生成(therapeutic angiogenesis)为解决这一问题提供了新的思路和方法。
治疗性血管生成指的是将外源性促血管生长因子导入缺血心肌以刺激侧支血管生成、改善心肌灌注和功能的方法。自从该思路提出30多年以来,受到研究者的广泛关注,动物实验取得了令人鼓舞的结果,但是单一促血管生长因子的临床研究尚未达到理想的预期目的。药物公司资助的临床试验常常选用公司具有知识产权或FDA比较容易批准的促血管生长因子,目前大多数临床研究主要集中于血管内皮生长因子VEGF-A,而VEGF-A作用的主要靶细胞是内皮细胞。血管生成是一个需要多种因素参与的复杂过程,单一促血管生长因子的作用无法激活和完成血管新生和成熟的整个调控过程,这可能是临床试验失败的原因之一。
成人体内的血管新生(neovacularization)过程首先是从原有的血管通过芽生的方式长出原始的血管网络,包括内皮细胞的增殖、迁移、管状结构形成以及细胞外基质的重构,这一过程称为血管生成(angiogenesis);随后这一初级的毛细血管网被平滑肌细胞和周细胞等血管支持细胞包裹,逐步形成具有完整结构且通过血管造影可显影的动脉血管网络,此过程称为动脉生成(arteriogenesis)。在血管生成(angiogenesis)阶段起主要作用的因子称为促血管生成因子(angiogenic factor),如FGF-2和VEGF。在动脉生成(arteriogenesis)阶段发挥主要作用的血管生长因子称为促动脉生成因子(arteriogenic factor),如PDGF-AB、PDGF-BB、血管生成素-1(Ang-1)、HGF和TGF-β等。
目前,促血管生成因子VEGF和FGF-2的研究最为深入和广泛,也是目前已进入临床试验阶段的因子。由于VEGF具有很强的促进血管通透性的作用,因此也被称为血管通透因子(Vascularpermeability factor,VPF)。FGF-2虽然缺乏向细胞外分泌的信号序列,但它能够调节靶细胞包括内皮细胞的生长和分化。尤为重要的是,FGF-2体内注射后可选择性地刺激血管生成但不影响其他组织的生长。但是,通过单一促血管生成因子诱导产生的新生血管结构以内皮细胞为主,没有血管平滑肌细胞和周细胞包被形成完整的动脉中膜结构,因此血管容易出血渗出,亦无法输送血流,最终会导致新生血管退化。而PDGF家族成员尤其是PDGF-BB是作用很强的促动脉生成因子,主要作用于血管壁细胞包括周细胞和血管平滑肌细胞。PDGF-BB与细胞表面的酪氨酸蛋白激酶受体PDGFR-α和PDGFR-β的同型二聚体和异二聚体结合,募集周细胞和血管平滑肌细胞包绕新生血管网络形成完整的血管结构从而发挥稳定血管的作用。
为了提高缺血区血流的灌注,必须重建长期稳定的功能性动脉网络,因为只有肌性动脉才能提供足够的血流量改善缺血心肌的功能以达到治疗目的。目前,在治疗性血管生成的研究中,仍有许多重要的问题尚待解决:既然单一因子无法诱导有功能的动脉血管形成,那么,不同的促血管生长因子与促动脉生长因子组合是否可以产生理想的效果?哪些血管生长因子之间的“搭档”能建立有效的侧支循环、改善缺血组织的血流灌注和功能?客观、准确地评价动脉生成的指标是什么?如何选择适宜的心肌梗死动物模型进行临床前评估?
基于血管生成的研究现状,我们提出如下假说:不同的促血管生成因子和促动脉生成因子均有各自特定的“搭档”,在血管新生的过程中互相影响并发挥协同作用,逐步激活和完成血管新生的整个过程;联合应用特定的促血管生成因子和促动脉生成因子能够诱导成熟、稳定的动脉血管网络的形成,并改善缺血心肌的灌注和功能。
目的
1.在小鼠角膜模型中,将VEGF、FGF-2和PDGF家族成员(PDGF-AA,PDGF-AB,PDGF-BB)两两组合进行初步筛选,确定有协同促血管生成效应的不同生长因子之间的组合。
2.根据筛选结果进一步探讨最佳组合因子在小型猪心肌梗死模型中的治疗效果及其可能的机制。
1.方法
1.1生长因子组合的筛选
角膜是没有血管的组织,因此它是研究血管生成的理想模型。我们以hydron聚合体包被蔗糖硫酸铝制作缓释系统,利用该系统包载不同血管生长因子使其成为缓释剂,置入小鼠角膜。不同生长因子单独或联合应用的剂量如下:FGF-2(80 ng),VEGF(160 ng),PDGF-AA(160 ng),PDGF-AB(160 ng),PDGF-BB(160 ng),PDGF-AA(160 ng)/FGF-2(80 ng),PDGF-AB(160 ng)/FGF-2(80 ng),PDGF-BB(160 ng)/FGF-2(40 ng),FGF-2(80 ng)/VEGF(160 ng),PDGF-BB(160 ng)/VEGF(160 ng)。在置入因子后5天后测定每一组动物角膜新生血管的长度和面积。为了观察血管新生的稳定性,在置入因子后第12,24,70天时分别再次测定血管长度和面积,并观察新生血管外形和轮廓的变化,确定有协同促血管生成作用的生长因子的组合,并根据筛选结果在小型猪急性心肌梗死模型中做进一步研究。
1.2小型猪急性心肌梗死模型的建立以及血管生长因子的导入
中国实验用小型猪40只,随机分为4组,每组10只。①PBS对照组,②FGF-2(5μg)治疗组,③PDGF-BB(10μg)治疗组,④FGF-2(5μg)/PDGF-BB(10μg)联合治疗组。所有实验动物的管理均遵循中华人民共和国卫生部动物实验管理条例(No.55,2001)和山东大学齐鲁医院实验动物管理条例。
动物经过诱导麻醉后建立静脉通路,并静脉注射3%戊巴比妥钠维持麻醉。然后行气管插管,呼吸机机械通气。经前正中线开胸,显露冠状动脉左前降支(LAD)后,于左前降支中远端1/3处用无创针线预结扎(不全部阻断血流)10min,然后完全结扎。
冠状动脉结扎后监测心率和血压的变化,急性心肌梗死模型构建成功的判定指标为:心电监护显示ST段弓背向上抬高持续30min以上,结扎动脉支配区域的心肌变暗变紫,运动减弱。以hydron聚合体包被蔗糖硫酸铝制作缓释系统,利用该系统包载不同血管生长因子使其成为缓释剂,包埋于心包下心肌梗死边缘的缺血区域。
1.3选择性冠状动脉造影术及评价侧支循环
麻醉动物股动脉穿刺成功后,经股动脉将6F导管送入左右冠状动脉行冠状动脉造影,用以证实观察左前降支结扎部位血流完全阻断,并评价LAD分布区的侧支循环。侧支循环指数按Rentrop分级分为0～3级:0级为冠状动脉系统无侧支显影;1级为心外膜下冠状动脉细小分支内有很弱的充盈,但是心外膜主要分支内无显影;2级为心外膜下冠状动脉主要分支部分充盈;3级为心外膜下冠状动脉主要分支完全充盈。
1.4应用彩色微粒测定局部心肌血流量
在治疗前和治疗后第6周和14周分别应用红色、黄色和蓝色三种不同颜色的彩色微粒(直径10±2μm,E-Z Trac,LosAngeles,CA)来评估这三个时间点的心肌局部血流量。将5F猪尾导管经股动脉进入左心室,经导管向左心室内注射入5×10~6个彩色微粒(将彩色微粒稀释于10ml生理盐水中),注射时间为30s,然后用10ml生理盐水冲洗导管。在注射彩色微粒前10s,使用定速抽血泵由股动脉抽取参考血液样本,抽取速率为10ml/min,共90s。血样-20℃保存。根据公司提供的操作步骤回收并计数组织和参考血样中的彩色微粒。根据公式计算心肌局部血流量:Q_m=(C_m×Q_r)/C_r。其中,Q_m是指每克心肌组织的血流量(ml/min/g),C_m是指每克心肌组织的彩色微粒数,Q_r是指参考血样的抽取速度(ml/min),C_r是指参考血样中的彩色微粒数。
1.5超声心动图测定左心室整体和局部收缩功能
将动物于开胸状态下行心外膜超声检测。在心尖两腔和四腔心切面采以校正的Simpson's方法测定左心室射血分数(LVEF)。在二维灰阶图像上分别测定梗死边缘的缺血区左心室舒张末期(心电图R波峰值处)和收缩末期(心电图T波末端)的室壁厚度,按以下公式计算缺血心肌局部室壁增厚率(wall thickening,WT):WT=(收缩末室壁厚度-舒张末室壁厚度)/舒张末室壁厚度×100%
1.6治疗效果评价
心脏缺血区导入生长因子6周后,进行冠状动脉造影和彩色微粒注射分别评价侧支循环和局部心肌血流量;导入生长因子14周后,再次行冠状动脉造影、彩色微粒注射和超声心动图检查来评价侧支循环、心肌灌注以及左室整体和缺血区局部功能。
1.7免疫组化
于第14周实验结束后,麻醉状态下静脉注射氯化钾处死所有实验动物,取出心脏,将左心室LAD结扎远端的缺血心肌分别置于多聚甲醛和液氮中备用,行相关组织学和分子生物学检测。
新生血管的密度测定方法:缺血区组织以多聚甲醛浸泡固定、石蜡包埋,切片厚度5μm。通过兔多克隆抗体von Willebrand factor(vWF)和小鼠单克隆抗体α-smooth muscle actin(α-SMA)免疫组化标记缺血区心肌的新生毛细血管和小动脉,并用荧光双标来评价新生血管的成熟程度和稳定性。这里我们引入了一个参数:成熟指数(maturation index),即被平滑肌包被的新生血管所占所有血管的百分比。
1.8统计学处理
定量数据均以均数士标准误表示,用SPSS 11.5统计软件进行统计分析。在做分析之前,对所有的数据进行正态分布检验。连续变量应用2×2析因设计和重复测量的方差分析。不同时间点连续变量应用重复测量的方差分析,进一步组间两两比较应用LSD或Dunnutt T3(方差不齐时)以及多元方差分析进行。等级资料用非参数检验分析,多组之间的比较用Kruskal-Wallis检验,组间两两比较Mann-Whitney检验。设P＜0.05为有统计学意义。
2.结果
2.1小鼠角膜模型血管生长因子的筛选
将不同生长因子缓释剂置入角膜第5天时检测结果表明,无论单独应用一种还是联合两种生长因子均可诱导角膜新生血管的形成。与单一因子比较,VEGF/FGF-2和VEGF/PDGF-BB这两种因子组合无显著协同作用。而FGF-2和PDGF家族成员PDGF-AA,PDGF-AB,PDGF-BB联合应用均有显著的协同促血管生成效应。但是到了第70天,联合应用FGF-2和PDGF-AA组诱导的血管几乎完全退化,而FGF-2与PDGF-AB或PDGF-BB协同诱导的新生血管得以维持。因此,我们选择了协同促血管生成作用最强的生长因子组合(FGF-2和PDGF-BB)在小型猪急性心肌梗死模型中作进一步研究。
40只实验用小型猪,第一次手术时死亡5只,其中2只死于麻醉意外,3只于结扎LAD时死于心室颤动。第二次冠状动脉造影时1只动物死于麻醉意外。共有34只小型猪完成了整个实验。
2.2冠状动脉造影评估侧支指数的变化
第一次手术时四组动物的侧支指数均为0,第6周时侧支指数在四组之间的差别有统计学意义(Kruskal-Wallis检验,P=0.026)。进一步组间比较发现,联合应用FGF-2/PDGF-BB组的侧支指数较单独应用FGF-2、PDGF-BB或PBS对照组均显著增高(P值分别为0.03 7,0.021和0.002),在结扎的冠状动脉远端形成可显影的侧支血管网络。与基础值相比,单独应用FGF-2治疗也可使缺血区侧支指数增加(P=0.018)。单独应用PDGF-BB治疗后虽然侧支指数有增加趋势,但差异未达到统计学意义(P=0.058)。而PBS对照组无明显侧支血管生成。
为了进一步观察这些侧支血管网是否稳定,在置入因子第14周后再次行冠状动脉造影检查,结果发现,联合应用FGF-2/PDGF-BB组的血管网络依然稳定存在,第14周的结果较第6周时的结果无显著差异。
2.3彩色微粒测定局部心肌血流量的变化
治疗前各组心肌血流量无差别,治疗后第6周和第14周,各组间差别有统计学意义(P＜0.05)。组间比较表明,FGF-2/PDGF-BB联合应用组较单独应用一种生长因子及PBS对照组心肌血流量显著增加(6周,P＜0.01;14周,P＜0.01)。析因设计的方差分析发现FGF-2和PDGF-BB之间有正交互作用(6周,F=7.317,P=0.011;14周,F=4.930,P=0.034)。第14周的结果较第6周时的结果无显著差异。
2.4左心室整体和局部功能的变化
治疗后第14周,FGF-2/PDGF-BB联合治疗组的左室射血分数(LVEF)较对照组显著增高(P=0.034),但是与单独应用FGF-2或PDGF-BB比较,差别无统计学意义(P值分别为0.10和0.082)。单独应用FGF-2或PDGF-BB较对照组LVEF亦无显著性改变。
治疗前各组间缺血区室壁增厚率无差别。第14周后单独应用FGF-2或PDGF-BB组较对照组WT差别无统计学意义,FGF-2/PDGF-BB联合治疗组较单独应用一种生长因子组的WT显著增高(P＜0.01)。
2.5联合治疗对于促血管生成的协同作用
通过免疫组化方法以血管性血友病因子(vWF,von Willebrandfactor)和α平滑肌肌动蛋白(α-SMA,α-smooth muscle actin)标记新生毛细血管和小动脉,并用荧光双标测定新生血管的成熟指数(maturation index),即被平滑肌包被的新生血管所占所有血管的百分比,以此来评价新生血管的成熟程度和稳定性。FGF-2组和FGF-2/PDGF-BB联合应用组的毛细血管密度较PDGF-BB组和对照组明显增高。FGF-2/PDGF-BB联合应用组与FGF-2组之间差别没有统计学意义。生长因子治疗的三个组的小动脉密度均较对照组明显增高,其中FGF-2/PDGF-BB联合治疗组的小动脉密度显著高于FGF-2或PDGF-BB单独治疗组(P＜0.01)及PBS对照组(P＜0.01)。FGF-2/PDGF-BB联合治疗组的血管成熟指数也明显高于FGF-2或PDGF-BB单独治疗组及PBS对照组(P＜0.01)。
2.6血管生长因子受体表达增高
FGF-2在心脏的受体主要是FGFR-1。PDGF-BB的两个受体是PDGFR-α和PDGFR-β。我们通过免疫组化检测了这三个受体的表达水平,结果发现,联合应用FGF-2/PDGF-BB组心肌微血管的FGFR-1、PDGFR-α和PDGFR-β的表达明显高于其他组。
同时用免疫组化和实时定量PCR的方法检测了VEGF和它的受体flk的表达,结果发现,在蛋白和mRNA水平,VEGF和flk在这四组中的表达均无显著性差异。
3.结论
(1)联合应用“促血管生成因子”FGF-2和“促动脉生成因子”PDGF-BB有协同的促血管生成作用,能够建立稳定的侧支血管网络,增加心肌血流量,明显改善心功能。
(2)FGF-2和PDGF-BB协同促血管生成机制与血管生成过程中FGFR-1,PDGFR-α和PDGFR-β在心肌微血管中表达上调以及PDGFR和FGFR之间的交互作用有关。这方面的深入研究为将来临床心肌梗死患者的促血管生成治疗奠定基础。
前言
既往研究表明左心室收缩期室壁厚度的增加主要来自心内膜下层心肌纤维的收缩,约占整个室壁增厚率的2/3,而心外膜肌纤维的收缩仅仅占室壁增厚率的1/3。因此,心内膜下心肌承受的收缩期压力更大,需氧量也高于心外膜下心肌。当心外膜下冠状动脉狭窄导致冠状动脉驱动压降低时,心内膜下层心肌血流量首先降低,导致心肌血流量在心内膜下层与心外膜下层的不均匀分布,心内膜下层与心外膜下层的跨壁梯度(transmural gradient)降低。收缩功能和心肌灌注的跨壁梯度在正常生理状态下可以忽略不计,但在定量评价冠状动脉疾病方面却有重要意义。理想的评价左心室功能的影像技术应该是既能测定心内外膜下层心肌的收缩功能也能评估心内外膜下层的血流灌注。然而,多数传统的影像技术包括二维超声心动图、核素心室造影和磁共振显像等仅仅能评估左心室整体功能而不能分层评估,因此这些技术对临床检测心肌缺血均有其局限性。虽然实时心肌声学造影技术能够测定心肌灌注的跨壁梯度,但是由于缺乏理想的造影剂,且存在声波衰减所致的假性无灌注区,该项技术仍未获得实际应用。
新近发展起来的应变和应变率显像技术(strain and strain rateimaging,SRI)是通过测定局部心肌形变和形变的速度来评价心肌局部收缩功能的一种方法。随着该技术的空间分辨率和数字处理能力的提高,现在已经能够实时测量心肌应变和应变率跨壁梯度分布。左心室不同层次的心肌纤维走行方向不同,心内膜下层与心外膜下层接近纵向,而中层心肌纤维接近横向走行,心肌纤维的这种三维立体走向特点决定了左心室的收缩运动包括三个方向,即径向收缩、纵向收缩和周向收缩。尽管心肌纵行纤维在左心室质量中所占比重较小,但由于心内膜下心肌为纵行纤维,更容易受到缺血的影响,因此局部心肌缺血对长轴方向收缩功能的损害早于短轴方向。从理论上推测,左室长轴方向收缩功能的变化能更敏感地反映心肌缺血导致的局部心功能的变化,定量评价左心室节段性纵向收缩功能可能具有重要的临床意义。然而,不同病理生理状态下心肌纵向应变和应变率的分层分布特点及其与心肌灌注跨壁梯度的关系目前尚不清楚。因此,本研究拟检验的假说为:应变率显像技术能够通过定量评价心室肌不同层次的纵向收缩功能,无创性估测局部的心肌灌注。
目的
1)在小型猪心肌缺血梗死模型中,观察不同状态下(正常、缺血和梗死节段)心肌的内、外膜层纵向应变和应变率的变化特点。
2)探讨心肌纵向应变和应变率的跨壁梯度与心肌跨壁血流的关系。
1方法
1.1动物模型的建立
12只雄性中国实验用小型猪,体重25～35kg(平均28.20±4.77kg),购自中国农业大学(北京)。所有动物饲养和实验程序都遵守中国卫生部实验动物管理条例(No 55,2001)和山东大学齐鲁医院实验动物管理条例。
肌肉注射安定50mg(2mg/kg)、阿托品1～2mg(0.06～0.08mg/kg)和氯胺酮500mg(18～20mg/kg),经3～5min后猪站立不稳而卧倒。诱导麻醉后将猪仰卧固定于操作台,气管插管,呼吸机机械通气,呼吸频率16～20次/分。心电监护仪进行心电监护。静注3%戊巴比妥钠3～5mL麻醉后实施手术。
实验动物平卧,备皮,常规碘伏消毒,铺巾。开胸之前,经静脉注射肌松药琥珀胆碱0.1g,用20ml生理盐水稀释。经前正中切开皮肤,高频电刀切开肌肉,并对出血点进行灼烧止血。用胸骨牵开器撑开胸腔,暴露心包和肺脏。纵形切开心包,并用7号缝线固定心包。显露冠状动脉左前降支,于左前降支中远端1/3处用无创针线预结扎(不全部阻断血流)10分钟,然后结扎。冠状动脉结扎后如心电监护出现ST段弓背抬高,结扎动脉区域心肌变暗变紫,然后行股动脉穿刺,经股动脉将6F造影导管送入左右冠状动脉行冠状动脉造影术,如显示左前降支结扎部位完全梗阻,证明模型建立成功。结扎冠脉1小时以后行心外膜超声心动图检查。
1.2应变和应变率显像
所用仪器为Vivid 7~(TM)彩色多普勒超声显像系统(Vivid 7,GEVingmed,Horten,N0rway),M3S探头,频率1.5～4.0 MHz。该系统装有应变和应变率的图像分析与后处理软件,可自动定量分析心内膜和心外膜下心肌的同步应变和应变率曲线。动物仰卧位,获取心尖四腔切面标准图像,室间隔尽可能与超声束方向平行,帧频大于100帧/s,在应变和应变率模式下6个心动周期的动态图像存储于硬盘中,待脱机后处理和分析。
在本研究中,我们将心肌明显变薄运动减低甚至矛盾运动的室间隔心尖段界定为梗死区,紧邻着梗死区域的室问隔中间段界定为缺血区,由右冠状动脉供血的室间隔基底段心肌界定为正常节段。进入TVI定量分析软件系统后将应变长度(strain length)设为4mm,将两个大小为5mm×1mm的椭圆形的取样容积分别平行置于心内膜下和心外膜下心肌层,分别测定正常、缺血和梗死节段心内外膜下层的应变和应变率。测量中注意调整收缩提取样容积的角度和位置,以保证两个取样容积在整个心动周期中保持在同一位置。系统将显示出该部位的应变曲线和应变率曲线,分别测量收缩期最大应变率(SRp)和收缩期最大应变(Sp)2项指标,然后计算心内外膜下层心肌测值的比值,作为内外膜应变和应变率的跨壁梯度。每个指标连续测量三个心动周期取其均值。
1.3局部心肌血流量的测定
应用彩色微粒(直径10±2μm,E-Z Trac,Los Angeles,CA)测量心肌局部血流量。将5F猪尾导管经股动脉送入左心室,经导管向左心室内注射入5×10~6个彩色微粒(将彩色微粒稀释于10ml生理盐水中),注射时间为30s,然后用10ml生理盐水冲洗导管。在注射彩色微粒前10s,使用定速抽血泵由股动脉抽取参考血液样本,抽取速率为10ml/min,共90s。血样-20℃保存。动物麻醉处死后,根据超声图像将室间隔分为正常、缺血和梗死三部分,每一部分分为心内膜下层和心外膜下层分别称重。根据产品提供的说明步骤对组织和参考血样中的彩色微粒进行回收。根据下面的公式计算心肌局部血流量:Q_m=(C_m×Q_r)/C_r。其中,Q_m是指每克心肌组织的血流量(ml/min/g),C_m是指每克心肌组织的彩色微粒数,Q_r是指参考血样的抽取速度(ml/min),C_r是指参考血样中的彩色微粒数。
1.4重复性检验
我们以缺血区域的心内膜和心外膜下层的TVI图像进行重复性分析,测量室间隔缺血区的Sp和SRp,检验这两个定量指标在观察者内部与观察者之间的差异。以同一观察者前后或不同观察者之间两次测值的绝对差值占两次测值均数的百分数表示。
1.5统计分析:
采用SPSS 11.5统计分析软件进行分析。计数资料用均数士标准差表示。不同节段的MBF,Sp and SRp应用one-way ANOVA比较,组间两两比较进一步用LSD分析。同一节段心内外膜下层参数用配对t检验比较。采用相关回归分析检验Sp和SRp与相应部位的MBF的相关性。设P＜0.05为有统计学差异。
2.结果
2.1正常、缺血和梗死节段的心肌应变和应变率
在心肌正常节段,心内膜下层Sp明显高于心外膜下层Sp(-11.39±2.30 vs-9.48+1.66,P=0.001),心内外膜跨壁梯度大于1。与正常节段心肌相比,缺血区的心内膜下层和心外膜下层Sp均显著降低(P均＜0.001),但是心内膜下层心肌Sp降低程度更重,心内膜下层Sp明显低于心外膜下层Sp(-5.99±1.59 vs-7.23±1.29,P=0.002),使得内外膜跨壁梯度发生逆转,Sp-EER小于1(Sp-EER:0.82±0.14)。在梗死节段,内外膜的Sp进一步显著降低接近于0,使得跨壁梯度消失。无论是心内膜下层还是心外膜下层的正常、缺血和梗死节段Sp两两比较,三者之间均存在显著差异(P＜0.001)。
在正常心肌节段,心内膜下层的SRp明显高于心外膜下层的SRp(-2.78±1.05 vs-2.34±0.87,P=0.007),心内外膜下层的SRp跨壁梯度大于1。与正常节段心肌相比,缺血区的心内膜下层和心外膜下层的SRp均显著降低(P均＜0.001),但是心内膜下层心肌的SRp降低程度更重,心内膜下层SRp明显低于心外膜下层SRp(-1.07±0.37 vs-1.47±0.57,P=0.006),使得心内外膜下层的跨壁梯度发生逆转,SRp-EER小于1(SRp-EER:0.76±0.15)。在梗死节段,心内外膜下层的SRp进一步显著降低(-0.47±0.22和-0.45+0.19,P＜0.001),接近于0,使得跨壁梯度消失。无论是心内膜下层还是心外膜下层的正常、缺血和梗死节段SRp两两比较,三者之间均存在显著差异(P均＜0.001)。
2.2正常、缺血和梗死节段的心肌血流量
在正常心肌节段,心内外膜下层的心肌血流跨壁分布一致(1.50±0.13 vs 1.46±0.16,P＞0.05)。在缺血区,心内外膜下层血流均降低(P均＜0.001),但是心内膜下层血流降低更显著使得心内膜下层血流明显低于心外膜下层血流(0.70±0.15 vs 0.87±0.08,P＜0.01),出现心内外膜下层跨壁梯度小于1(MBF-EER:0.81±0.12)。在梗死节段,心内外膜下层血流进一步显著降低,跨壁梯度消失,心内外膜下层血流比值等于1(0.26±0.08 vs 0.26±0.07,P＞0.05)。
2.3心肌节段血流和应变的相关性
线性相关回归分析表明,心内膜下层的Sp与MBF显著相关(r=-0.84,P＜0.001);心外下层膜的Sp与MBF显著相关(r=-0.81,P＜0.001)。心内膜下层的SRp与MBF也有很好的相关性(r=-0.78,P＜0.001);心外膜下层的SRp与MBF也有很好的相关性(r=-0.75,P＜0.001)。
2.4重复性检验
各项指标在观察者内部的差异分别为:心内膜下层Sp,10.2±3.6%;心外膜下层Sp,10.3±4.2%;心内膜下层SRp,12.9±3.8%和心外膜下层SRp,13.3±4.1%。观察者之间的差异分别是:心内膜下层Sp,11.5±3.9%;心外膜下层Sp,11.9±3.7%;心内膜下层SRp,13.8±4.1%和心外膜下层SRp,14.2±4.3%。
3结论
(1)本研究在小型猪心肌缺血和梗死模型中发现,在正常、缺血和梗死不同状态下心内外膜下层心肌纵向应变和应变率及其跨壁梯度明显不同。
(2)定量分析心肌纵向跨壁应变和应变率的改变能够区分正常、缺血和梗死区域,评估心肌缺血的严重性,表明纵向心肌形变的跨壁分布测定对评价患者缺血心肌局部灌注可能是一个很有价值的指标。
Introdnction
Atherosclerosis-induced coronary artery disease(CAD)is the leading cause of morbidity and mortality in Western societies and increases at an alarming speed in developing countries,despite continued advances in the treatment of CAD such as PTCA (Percutaneous Transluminal Coronary Angioplasty),CABG(Coronary Artery Bypass Graft)and intracoronary stent placement.Except for surgical interventions,an effective therapeutic method for treatment has been lacking.An increasing number of patients are not suitable for conventional revascularization strategies because of diffuse small vessel disease,multi-vessel disease,long lesions,occluded vessels without collateral recanalization,or other comorbidities such as diabetes mellitus and obesity.Therapeutic angiogenesis induced by angiogenic growth factors may provide an alternative approach to the treatment of myocardial ischemia for these patients.
Therapeutic angiogenesis is a process that seeks to stimulate collaterogenesis and to improve myocardial perfusion and function by delivery of proangiogenic factors to the ischemic myocardium. Numerous experimental and clinical studies have evaluated therapeutic angiogenesis as a treatment for ischemic heart disease. Despite the promising results of therapeutic angiogenesis in preclinical studies,clinical evaluation of these individual proangiogenic molecules has produced unfulfilled promises since this idea has been proposed for more than 30 years.After more than a decade of clinical practice with different single proangiogenic factors for the treatment of ischemic disorders,almost all large randomized,double-blinded,and placebo-controlled human trials have proven to be non-beneficial.Pharmaceutical companies that sponsor clinical trials often choose an angiogenic agent based on the intellectual properties owned by the company and the possible easy approval by the Food and Drug Administration(FDA).
Most previous preclinical and clinical studies on the development of proangiogenic therapies for treating ischemic myocardium have been based on mono-therapeutic approaches.In addition,most studies have focused on evaluation of the therapeutic efficacy of vascular endothelial cell growth factor(VEGF)-A,which primarily targets the endothelial cell compartment.The establishment of stable and functional blood vessel networks,however,is a complex process that requires several angiogenic factors targeted different cell populations in the vasculature.The reason for this discrepancy is unclear,and in part that current angiogenic therapies in clinical trials are based on a single angiogenic factor delivered, which is insufficient to initiate the entire cascade of events leading to a mature,functional and stable vascular network.
Neovascularization in the ischemic adult heart is a combination of several processes including angiogenesis,arteriogenesis and potentially vasculogenesis.Angiogenesis is defined as the sprouting of new blood vessels from pre-existing vasculature and consists of distinct stages including cell migration,proliferation and differentiation into capillaries and blood vessels.Arteriogenesis refers to the process of maturation or de novo growth of collateral conduits and produce vessels capable of carrying significant blood flow.These vessels are of a sufficient diameter to be visualized with angiography.
The angiogenic factors,VEGF and FGF-2,are perhaps the most extensively studied angiogenic growth factors,which has been used in clinical trials.VEGF exerts its effects on endothelial cells that include enhanced survival,increased permeability,enhanced migration and proliferation,all of which contribute to angiogenesis. VEGF is also named VPF(vascular permeability factor)due to its high permeability of vessel.
Like VEGF,FGF-2 also stimulates endothelial cell proliferation and migration,which plays a critical role in triggering an initial robust angiogenic response.Although the release of the signal peptide-less FGF-2 from its synthetic cells remains enigmatic,this growth factor plays a crucial role extracellularly in the regulation of the growth of its target cells including endothelial cells.Especially important,administration of FGF-2 in vivo selectively stimulates angiogenesis but not other tissue growth.
However,endothelia cells channels,induced by single angiogenic factor,are naked,leaky,and fragile,which are easily ruptured and bleed.Insufficient perfusion eventually leads to vessel regression.
In contrast to FGF-2 and VEGF,PDGF-BB acts mainly on vascular mural cells including pericytes and vascular smooth muscle cells(VSMCs).PDGF-BB interacts with both homodimeric and heterodimeric PDGFR-αand PDGFR-βcomplexes,especially PDGFR-β,which plays an essential role in stabilization of blood vessels via the recruitment of pericytes and VSMCs onto the nascent vasculature.
For improvement of perfusion of high-oxygenated blood in ischemic tissues,it is essential to re-establish functional arterial vascular networks,which should remain stable for long term.The clinical failures with this attractive approach have raised several unresolved fundamental issues regarding the basic mechanisms of neovasculorazation.These include the underlying mechanisms of angiogenesis versus arteriogenesis,choice of proangiogenic agents, monotherapy versus combinatorial therapy,selection of optimal combination and drug release systems,evaluation standard for functional arterial networks and appropriate animal models for preclinical evaluation.
Based on these conditions,we propose a hypothesis as following: the interplay and synergy between various angiogenic and arteriogenic factors for promoting arteriogenesis is specifically limited to certain combinations,and that each factor has its own specific partners.The optimal combination of angiogenic and arteriogenic factors could induce stable arterial networks and improve collateral growth and functional recovery.
The purpose of this study was,based on the selection of optimal combination of angiogenic and arteriogenic factors in mouse cornea, to explore therapeutic evaluation of the combination in a pig myocardial infarction model.
1.Methods
1.1 Selection of optimal combination of angiogenic and arteriogenic factors
The avascular feature of the corneal tissue made it an ideal model for assessing vascular network formation.A micropellet(0.35×0.35 mm)of sucrose aluminum sulfate coated with hydron polymer type NCC containing 160 ng of VEGF,PDGF-AA,-AB,-BB,or 80 ng of FGF-2,or 160 ng PDGF-AA/80 ng FGF-2,or 160 ng PDGF-AB/80 ng FGF-2,or 160 ng PDGF-BB/40 ng FGF-2,or 160 ng PDGF-BB/160 ng VEGF was surgically implanted into a micropocket in the mouse cornea(one pellet/eye,one eye implanted/mouse).The corneal neovascularization was examined and quantified on day 5 after pellet implantation.Vascularization areas were calculated by measuring vessel lengths and clock-hours(the circumferential area of neovascularization if the eye is considered as a clock).For vascular stability analysis,the corneal neovascularization pattern/profile was examined on days 5,12,24,and 70 after pellet implantation.Finally, we thus chose an optimal combination for the therapeutic evaluation and further studies in a pig model of myocardial infarction.
1.2 Pig myocardial infarction model and growth factor implantation
40 Chinese experimental mini-pigs weighing between 25-30 kg/each were used for the myocardial infarction model(China Agricultural University,Beijing).All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001)and the guidelines for the Care and Use of Laboratory Animals of Qi Lu Hospital,Shandong University,China.Animals were randomly divided into four groups(n=10 pigs/group):(A)PBS, (B)FGF-2(5μg),(C)PDGF-BB(10μg),and(D)FGF-2 and PDGF-BB(5μg and 10μg,respectively).
Before all experimental procedures,all animals were anesthetized by intramuscular administration of ketamine hydrochloride(20 mg/kg,Heng Rui Medicine Co.LTD.Jiang Su, China)together with intravenous injection of sodium pentobarbital (30 mg/kg,Beijing Chemical Reagent Company,Beijing,China)and were mechanically ventilated with a volume respirator(Newport E100m Ventilator,Newport Medical Instruments,CA,USA).Median sternotomy was performed to expose the heart followed by incision of the pericardium.Acute myocardial infarction was produced by ligation of the LAD coronary artery distal to its third diagonal branch using a 7.0-prolene suture.Selective left and right angiography, performed through a standard procedure,was made to confirm complete occlusion of the LAD and to assess baseline levels of collaterals to assess the collateral index.
A slow-releasing pellet of sucrose aluminum sulfate coated with hydron polymer(0.2×0.2 cm)containing PBS,5μg FGF-2,10μg PDGF-BB,or 5μg FGF-2 and 10μg PDGF-BB was attached to an aseptic application(0.5×0.5 cm),which was sutured(7.0-prolene) onto the border zone adjacent to the mid and distal LAD.
1.3 Coronary arteriography
Six and fourteen weeks after treatments,repeated,selective angiography was performed.Under general anesthesia,the animals received a contrast agent,meglucamine diatrizoate,through a standard femoral puncture using the digital subtraction angiography (CGO-2100,Wandong Medical Equipment Co.LTD,China).The angiographic index was assessed using a standard protocol based on Rentrop's grading scales from 0 to 3:0,none;1,filling of side branches of the LAD;2,partial filling of the LAD main artery via collateral channels;3,complete filling of the LAD.
1.4 Regional myocardial blood flow
For determination of regional myocardial blood flow at different time points,colored microspheres(10±2μm diameter;E-Z Trac,Los Angeles,CA)labeled with red,yellow and green were used prior to treatment,and at the end of week 6 and 14.5×10~6 microspheres were injected into the left ventricle by a 5F pigtail catheter(Cordis,USA)with 10 ml of saline.After completing the injection,the catheter was flushed with 10 ml of saline.An arterial Reference Blood Sample was collected for each colored microsphere injection.Starting 10 seconds before injection,reference blood samples were withdrawn by a withdrawal pump at a constant rate of 10 ml/min for a period of 90 seconds.Recovery of microspheres from tissue and blood was performed according to the manufacturer's instruction.Blood flow values are calculated from the following equation:Q_m=(C_m×Q_r)/C_r.Where Q_m is the myocardial blood flow per gram(ml/min/g),C_m is the microsphere count per gram of tissue, Q_r is the withdrawal rate of the reference blood sample(ml/min),and C_r is the microsphere count in the reference blood sample.
1.5 Echocardiography
Two-dimensional echocardiography was used to measure global and regional left ventricular function in all animals with open chest following angiographic assessment before and 14 weeks after treatment using an ultrasound scanner(SONOS 7500,Philips Medical Systems Inc.,Andorver,MA).Analyses of LVEF and systolic wall thickening(WT%)were performed to determine the global function of the left ventricle and the regional function of the myocardium, respectively.LVEF was determined from the apical two-chamber and four-chamber views using a modified Simpson's algorithm. Regional wall thickness was measured at end-diastole(the peak of R wave of the ECG)and end-systole(the end of T wave of the ECG) individually on two-dimensional echocardiograms.
Left ventricular systolic wall thickening(WT%)was calculated as: WT%=(SWT-DWT)/DWT×100%.
1.6 Immunohistochemistry
The treated areas of the left ventricle myocardium in the LAD territory were fixed with paraformaldehyde or were immediately frozen in liquid nitrogen.To determine vessel density, paraffin-embedded 5μm sections were incubated with a rabbit anti-von Willebrand factor(vWF)antibody or a mouse anti-alpha smooth muscle actin(αSMA)antibody,followed by incubation with secondary antibodies labeled with horseradishperoxidase.Ischemic myocardial sections were also used for detection of vWF/αSMA double positivity using immunofluorescent analysis.A maturation index(percentage of smooth muscle cell positive vessels vs.total vessel numbers)was calculated.
1.7 Data analysis
Data are presented as the mean±standard errors of the mean and were analyzed with SPSS 11.5 software.A 2×2 factorial analysis of variance was used to examine the interaction between FGF-2 and PDGF-BB.Continuous variables were compared by using repeated measures analysis of variance(RM ANOVA)followed by the LSD or Dunnutt T3 corrected post hoc analysis for multiple comparison procedure.Nonparametric variables were compared between groups by using two-sided Kruskal-Wallis(multiple group comparison)and Mann-Whitney(two group comparisons)tests.Collateral index was compared within groups before and after treatment using Wilcoxon test.All reported P values were two-tailed,and a P value＜0.05 was considered statistically significant.
2.Results
2.1 Selection of optimal combination of angiogenic and arteriogenic factors in mouse cornea
Combination of PDGF-AA/VEGF,PDGF-AB/VEGF or PDGFBB/VEGF didn't result in synergistic angiogenesis. PDGF-AA/FGF-2,PDGF-AB/FGF-2,or PDGF-BB/FGF-2 combinations synergistically induced angiogenesis as compared with single growth factor-induced angiogenesis.At day 70 after implantation,PDGF-AA/FGF-2-induced vessels were almost completely regressed,suggesting that this combination is unable to stabilize the newly formed vasculature.Interestingly,both PDGF-AB/FGF-2- and PDGF-BB/FGF-2-induced corneal vascular networks remained stable at this and later time points.These findings demonstrate that only PDGF-AB/FGF-2 or PDGFBB/FGF-2,but not PDGF-AA/FGF-2,are able to stabilize the newly formed vasculature although all three combinations promote angiogenic synergisms.We thus chose a combination of PDGF-BB/FGF-2 for the therapeutic evaluation and further studies.
Forty animals received surgery and five animals died in the initial operation,of which two animals died of anesthesia accident and three animals died of ventricular fibrillation during ligation of the left anterior descending(LAD)coronary artery to make myocardial infarction model.One animal(PBS control)died of anesthesia accident in the mid-study.Thus,the remaining thirty-four animals(PBS,n=7;FGF-2,n=10;PDGF-BB,n=8;FGF-2+PDGF-BB, n=9)completed the entire study.No abnormal adverse events were noted during the entire course of these experiments in those animals that survived to the 14-week end point.
2.2 Angiographic analysis of collateral formation
Analysis of the collateral index demonstrated a difference among the four groups at week 6(Kruskal-Wallis test,P=0.026), whereas baseline collateral index in all groups was similar.Further multiple comparisons showed that PDGF-BB/FGF-2 significantly induced myocardial collateral growth as compared with various individual FGF-2 or PDGF-BB or PBS -treated myocardium(P=0.037, P=0.021 and P=0.002,respectively).These newly established collaterals formed vascular networks proximally and distally from the ligation site.Compared with the baseline,collateral index had increased significantly in the FGF-2 group and in PDGF-BB/FGF-2 groups at week 6(P=0.018 and P=0.001,respectively).Despite a trend toward increase in collateral index in the PDGF-BB group compared with the baseline,the difference did not reach statistical significance(P=0.058).
To study the stability of these newly established collateral networks,coronary angiography was again performed on week 14 after treatment.Remarkably,the PDGF-BB/FGF-2-induced collaterals had developed into well-established coronary arterials, indicating the re-established collaterals remained stable.
2.3 Assessment of regional myocardial blood flow(MBF)
The myocardial blood flow was assessed by a colored microsphere method.Prior to treatment,the basal levels of myocardial blood flow in the LAD territory were indistinguishable among all groups.At week 6,the PDGF-BB/FGF-2 together-treated group exhibited remarkably higher MBF than either factor alone-treated myocardium(P＜0.05).The high level of MBF persisted for long term in the PDGF-BB/FGF-2-treated group and no reduction of MBF was observed at the end point of the experiment(14 weeks). Statistical analysis of these data using factorial analysis of variance showed that MBF in the PDGF-BB/FGF-2 together-treated group was markedly greater than the sum effects obtained from two factors alone-treated myocardium(F=7.317,P=0.011,at week 6;and F=4.930,and P=0.034,at week 14).
2.4 Echocardiographic analysis of global and regional myocardial function
Echocardiography was performed to monitor global and regional myocardial function.Measurement of left ventricular ejection fraction(LVEF),a sfandard parameter to monitor global myocardial contractile function,analysis showed that the basal cardiac dysfunction was indistinguishable among all groups before treatment. At week 14 after treatment,a significant improvement of LVEF was observed in the PDGF-BB/FGF-2-treated group as compared with the buffer-treated group,however,there were no significant differences between the combination treatment group and groups treated with either FGF-2(P=0.10)or PDGF-BB(P=0.082)alone.Neither single growth factor-treated group showed significant improvements of LVEF.
To assess regional wall motion,left ventricular systolic wall thickening(WT%)was calculated.There were also no significant differences in the baseline regional function among groups.Similar to LVEF results,PFGF-BB- and FGF-2-single treated groups did not show any significant improvement in WT%as compared with buffer-controls.In contrast,the PDGF-BB/FGF-2-treated group significantly improved the WT%as compared with either buffer- or single factor-treated groups(P＜0.01).
2.5 Vessel density and maturation index
Collateral micro-vessel formation was further examined by measuring the number of capillaries and arterioles by von Willebrand factor(vWF)andα-smooth muscle actin(α-SMA) immunohistochemistry in light microscopic sections taken from ischemic myocardium.Myocardial capillary density in the border zone in FGF-2 and the combination group was significantly higher compared with PBS and PDGF-BB group(P＜0.05).No apparent difference was observed between the FGF-2 and the combination group.Arteriolar density was significantly higher in growth factors treated animals than in PBS controls.Consistent with the coronary angiographic analysis,high numbers of arterial vessels were detected in the PDGF-BB/FGF-2-treated myocardium at week 14 post-treatment as compared with those of single factor-treated or control samples.
In addition,for assessing the maturity and stability of myocardial newly vessels,a maturation index defined as proportion of vessels surrounded by smooth muscle cells was determined by vWF andα-SMA double-labeled immunofluorescence.At week 14 after treatment,α-SMA positive vessels in the PFGF-BB/FGF-2 together-treated group was significantly higher than those detected in the PFGF-BB,FGF-2,or buffer alone-treated myocardium(P＜0.05).
2.6 Upregulation of FGFR-1,PDGFR-αand PDGFR-β
The proangiogenic factors exert their cellular effects by binding to and activating protein tyrosine kinase receptors expressed on target cells.FGFR-1,PDGFR-αand PDGFR-βwere examined by immunohistochemistry.FGFR-1,PDGFR-αand PDGFR-βexpression significantly higher in the PFGF-BB/FGF-2 group than others.
To further identify whether VEGF may in part involve in vascular stability by activating flk,we also examined the expression of VEGF mRNA and protein by realtime PCR and immunohistochemistry,respectively.There were no significant differences at both mRNA and protein level in VEGF and flk among groups.
Conclusion
(1)A protein-based angiotherapeutic approach,based on dual delivery of arteriogenic and angiogenic factors,significantly improves myocardial collateral growth,blood perfusion,and cardiac function in a pig ischemic myocardial model.
(2)A possible mechanism of angiogenic synergism involves upregulation of the expression of FGFR-1,PDGFR-αand PDGFR-βin angiogenesis,and the crosstalk between PDGFR and FGFR.
INTRODUCTION
Experimental studies have demonstrated that left ventricular systolic wall thickening stems mainly from the contraction of the subendocardial myocardial fibers with the inner half layer of the ventricular wall contributing to about two-thirds and the outer half layer contributing to about one-thirds of systolic wall thickening. Therefore,the subendocardium sustains a higher systolic stress and demands more oxygen consumption and blood supply than the subepicardium.Thus,the subendocardium is more susceptible to ischemia than the subepicardium.The transmural gradient of myocardial perfusion is of negligible significance in physiological conditions but becomes extremely important in the presence of epicardial coronary artery stenosis because an accurate assessment of the transmural perfusion gradient may significantly improve the sensitivity and specificity of diagnostic techniques in the early detection of coronary artery disease.Therefore,an ideal imaging technique for detecting coronary artery disease should be able to measure subendocardial and subepicardial myocardial perfusion separately.Most conventional imaging modalities,however,can only assess myocardial perfusion across the entire ventricular wall rather than in different layers due to the lack of a high spatial resolution, and thus,the clinical value of these techniques in detecting myocardial ischemia is rather limited.Recently real-time myocardial contrast echocardiography has shown the potential to delineate transmural distribution of myocardial perfusion but the lack of an ideal myocardial contrast has significantly hindered development of this technique.
Strain and strain rate imaging is a new modality of echocardiographic techniques designed for measuring the regional myocardial deformation or the velocity of deformation.With a high spatial resolution and frame rate as well as digital processing capability of these techniques,it has been possible to measure the transmural gradient of myocardial strain and strain rate in real time. As myocardial longitudinal shortening plays an important role in cardiac contraction,and longitudinal myocardial fibers contract earlier and are likely more vulnerable to ischemia than circular fibers, measurement of the transmural gradient of the longitudinal myocardial strain and strain rate may provide important information on the transmural distribution of myocardial contraction in physiological conditions and myocardial ischemia.It is still unclear whether the transmural systolic strain and strain rate could predict the transmural myocardial blood flow at different perfusion levels. Therefore,we propose the hypothesis that the transmural gradient of the longitudinal myocardial contraction can be assessed by strain and strain rate imaging,which can in turn be used to predict the transmural gradient of myocardial blood flow(MBF)measured by colored microspheres in the normal,ischemic and infarct segments in a pig model with acute myocardial infarction.
OBJECTIVE:
To study the contraction patterns in subendocardium and subepicardium across the normal,ischemic and infarct segments by SRI and to explore the relationship between the regional myocardial blood flow and strain and strain rate in the subendocardium and subepicardium at different perfusion levels.
1.METHODS
1.1 Animal preparation
Twelve Chinese experimental mini-pigs(males,25-30kg,China Agricultural University,Beijing)were included in this study.All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001)and the guidelines for the Care and Use of Laboratory Animals of Shandong University Qilu Hospital, China.
General anesthesia was achieved in all pigs by intramuscular injection of ketamine hydrochloride(20 mg/kg,Heng Rui Medicine Co.LTD.Jiang Su,China)together with intravenous injection of sodium pentobarbital(30 mg/kg,Beijing Chemical Reagent Company, Beijing,China).Pigs were placed in the supine position,intubated and mechanically ventilated with a volume respirator(Newport E100m Ventilator,Newport Medical Instruments,Inc,Newport Beach, CA)at a rate of 16-20 breaths per minute.All animals underwent continuous electrocardiographic(ECG)monitoring throughout the experiment.The heart was exposed through a median sternotomy and supported by a pericardial cradle.A 6F Judkins right catheter was introduced via the femoral artery for the left coronary angiography. Distal to the second diagonal branch,the left anterior descending (LAD)coronary artery was completely occluded for 60 minutes with a 7.0-prolene suture.Successful construction of an acute myocardial infarction model was confirmed by left coronary angiography and by the presence of typical ST segment elevation on an electrocardiogram.
1.2 Strain and Strain Rate Imaging
Echocardiographic imaging was performed 1 hour after LAD ligation with a Vivid 7~(TM)ultrasound system(Vivid 7,GE Vingmed, Horten,Norway)with a M3S transducer(1.5 to 4.0MHZ).Strain and strain rate imaging were derived from the apical four-chamber view with the interventricular septum as parallel as possible to the direction of the ultrasound beam.A high frame rate(＞100 frames/s) was used to obtain as much data as possible in a given period of time and a latex bag filled with degassed saline served as an acoustic interface between the epicardium and the transducer.A cine-loop comprising least 6 cardiac cycles was stored digitally on a magneto optic disk for later offline analysis.
The ventricular septum was divided into three segments based on their two-dimensional images:the dramatically thinned and akinetic or dyskinetic region,usually localized in the distal septum in the present model,was defined as an infarct segment.The hypokinetic region which was close to the infarct segment and usually located in the middle septum was defined as an ischemic segment.The basal septum,whose blood supply stems from the right coronary artery, was defined as a normal segment.A myocardial length of 4 mm was used for strain analysis and two distinct and parallel sample volumes with an elliptical shape and a size of 5 mm×1 mm were placed in the subendocardial and subepicardial layers in the middle of the normal, ischemic and infarct segments,respectively.The strain curves from both subendocardial and subepicardial layers were simultaneously displayed in the normal,ischemic and infarct segments and the peak systolic strain from the subendocardium(Sp-endo)and subepicardium(Sp-epi)was measured from the two curves.The transmural gradient of myocardial strain was calculated as the ratio of Sp-endo/ Sp-epi(SpoEER)in the three segments,respectively. Similarly,The strain rate curves from both subendocardial and subepicardial layers were simultaneously displayed in the normal, ischemic and infarct segments,respectively and the peak systolic strain rate from the subendocardium(SRp-endo)and subepicardium (SRp-epi)was measured from the two curves.The transmural gradient of myocardial strain rate was calculated as the endocardial to epicardial ratio of strain(SRp-EER),i.e.,SRp-endo/SRp-epi in the three segments.Three cardiac cycles were measured and the values averaged.To ensure sample volumes to be kept in the same desired position,mild manual readjustment of the position of sample volumes during systole was necessary.
1.3 Regional Myocardial Blood Flow Measurement
Regional MBF was measured by injection of 5×10~6 colored microspheres(10±2μm in diameter,E-Z Trac,Los Angeles,CA) into the left ventricle by a 5F pigtail catheter(Cordis,USA)which was followed by a flash injection of 10 ml saline.Starting 10 seconds before each colored microspheres injection,an arterial reference blood sample was withdrawn by a withdrawal pump at a constant rate of 10 ml/min for a period of 90 seconds.After the animal was euthanized,the cardiac sections were cut in accordance with the echocardiaographic apical four chamber views and the transmural tissue slices from normal,ischemic and infarct zones in the ventricular septum according to sample volume positions were derived,each of which was divided into subendocardial and subepicardial layers and weighed individually.Microspheres in the myocardial tissue and blood sample were recovered according to the manufacturer's instruction.MBF values in the subendocardial (MBF-endo)and subepicardial(MBF-epi)layers were calculated separately from the following equation:Q_m=(C_m×Q_r)/C_r,where Q_m is the myocardial blood flow per gram(ml/min/g),C_m is the microspheres count per gram of tissue,Q_r is the withdrawal rate of the reference blood sample(ml/min),and C_r is the microspheres count in the reference blood sample.The transmural gradient of myocardial perfusion(MBF-EER)was calculated as the ratio of MBF-endo/MBF-epi in the three segments.
1.4 Inter-observer and intra-observer variability
In order to assess the reproducibility of strain and strain rate measurement,Sp and SRp in the subendocardium and subepicardium in all animals were measured in the ischemic segment.The inter-observer variability was calculated from the repeated measurements performed by two independent observers and the intra-observer variability was calculated from the repeated measurements performed by one observer who measured twice one week apart.Variability was expressed as the percentage of the absolute difference between two measurements divided by the mean value of the two measurements.
1.5 Statistical Analysis
Data analysis was performed using SPSS 11.5 statistical software (SPSS Inc.,Chicago,USA).Values were expressed as mean±SD. Comparisons of MBF,Sp and SRp among all segments were performed by one-way ANOVA,and LSD test was used to assess the difference between multiple comparisons.Paired student's t-test was used to analyze the differences between parameters measured from the subendocardium and subepicardium in the same segment. Correlations between MBF and Sp or SRp from all three segments were performed by linear regression analysis.P value＜0.05 was considered statistically significant.
2.RESULTS
2.1 Strain and Strain Rate Imaging Measurements
Two pigs died of ventricular fibrillation after LAD occlusion and 10 pigs entered the final data analysis.The peak systolic strain in both subendocardium(Sp-endo)and subepicardium(Sp-epi) decreased progressively from the normal to the ischemic and the infarct segments and there were significant differences among the three segments(all p＜0.001).Sp-endo was significantly higher than Sp-epi in the normal segment(p=0.001)whereas significantly lower than Sp-epi in the ischemic segment(p=0.002).On the other hand, there was no significant difference between Sp-endo and Sp-epi in the infarct segment(p＞0.05).Consequently,Sp-EER calculated was the highest in the normal segment and the lowest in the ischemic segment and there was a significant difference in Sp-EER between normal and ischemic segments(p＜0.001)and between normal and infarct segments(p=0.013).Moreover,Sp-EER was significantly lower in the ischemic than in the infarct segment(p=0.004).
Peak systolic strain rate showed similar changes as peak systolic strain and exhibited a progressive decline from the normal to the ischemic and the infarct segments.(all p＜0.001).SR-endo was significantly higher than SR-epi in the normal segment(p=0.007)but significantly lower than SR-epi in the ischemic segment(p=0.006).In contrast,there was no significant difference between SR-endo and SR-epi in the infarct segment.Compared with the normal segment, SR-EER was significantly reduced in the ischemic(p＜0.001)and infarct segments(p=0.028).Also SR-EER was significantly lower in the ischemic than in the infarct segment(p=0.001).
2.2 Regional Myocardial Blood Flow
The regional MBF was similar in the subendocardium and the subepicardium in the normal segment with a calculated MBF-EER of 1.03±0.07.Compared with MBF in the normal segment,MBF in both subendocardium and subepicardium in the ischemic segment decreased significantly(both P＜0.001)with a more remarkable decline in MBF-endo(p?
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