摘要
背景:正常骨骼肌生理性缺血训练可促进远隔病理性缺血部位侧支循环生成,但其作用的具体机制尚不清楚。内皮祖细胞(EPCs)是侧支循环形成的细胞学基础,骨髓的EPCs可通过归巢机制至缺血靶组织,分化为血管内皮。EPCs迁移受血管内皮生长因子(VEGF),一氧化氮(NO)等多种细胞因子调节,而VEGF/NO对生理性缺血训练促进远隔侧支循环过程中EPCs归巢的调控作用尚未见报道。本研究假说为:适宜的正常骨骼肌生理性缺血训练可以促进内源性EPCs释放,通过VEGF-NO介导的EPCs归巢机制,促进远隔病理性缺血心肌侧支循环形成,实现“生物搭桥”。
目的:探索VEGF/NO对正常骨骼肌生理性缺血训练促进远隔缺血心肌侧支循环过程中EPCs归巢的调控机制。
方法:54只新西兰兔冠状动脉左室支(LVB)植入水囊缩窄器制作可控性心肌缺血模型。造模成功后随机分为6组,休息一周后进行相应缺血干预:①假手术组(Sham),手术植入水囊,但不进行任何缺血刺激,安静笼养4周;②单纯心肌缺血组(MI),模拟心肌病理性缺血,水囊充水阻断冠状动脉血流2分钟/次,间隔10分钟,2次/天,5天/周,共4周;③单纯生理性缺血训练组(PIT),心肌缺血与MI组相同,双后肢同时进行生理性缺血训练即通过双后肢气压止血带环扎以阻断局部血流,3分钟/次,间隔5分钟,3次/天,5次/周,共4周;④NO抑制的PIT组,造模手术后该组兔采用160μg/ml浓度的NO合成酶抑制剂L-nitroarginine methyl ester(L-NAME)溶于兔饮用水中,饲养7天后达NO阻断后稳态,称为(L-NAME)组,此组同时进行心脏病理性缺血和生理性缺血训练;⑤VEGF阻断的PIT组(Anti-VEGF),心肌缺血和后肢生理性缺血训练同PIT组。VEGF阻断采用Anti-VEGF(Bevacizumab,安维汀)10mg/kg,术后休息1周并于训练前24小时静脉注射,每2周注射一次。⑥VEGF与NO双阻断组(L-NAME+Anti-VEGF),同时接受以上L-NAME及VEGF抗体处理,心肌缺血及生理性缺血训练亦PIT组。实验终点时超声心电图检测心功能,后处死取材,进行实验室检测:微球技术检测局部心肌侧支循环血流(CCBF);免疫组化染色检测缺血心肌毛细血管密度(CD);采用流式细胞仪进行外周血循环EPCs(CD34~+/Flk-1~+)计数;ELISA检测外周血VEGF含量;硝酸还原酶法检测外周血中NO含量;采用定量RT-PCR及Western blotting法检测局部缺血心肌eNOS及VEGF的mRNA及蛋白含量。
结果:①实验过程中,11只兔最终退出实验,其中5只因各种感染死亡,6只因心室颤动死亡。Sham, MI, L-NAME,Anti-VEGF, L-NAME+Anti-VEGF组各有1只死于感染,Sham, MI, L-NAME, Anti-VEGF组各有1只死于室颤,L-NAME+Anti-VEGF组有2只死于室颤。②MI组循环中EPCs数目(CD34+/Flk-1+)训练前后增高的倍数显著高于Sham组(P<0.05);EPCs数增高倍数在PIT组显著高于MI组(P<0.001);VEGF和/或NO阻断后,PIT诱导的EPCs增高效应均被相应阻断,且导致兔心脏左室射血率降低。③在200倍光镜下,MI组心肌毛细血管密度显著高于Sham组(P<0.05)。心肌毛细血管密度在PIT组显著高于MI组(P<0.01)。L-NAME(P<0.01),Anti-VEGF(P<0.01),L-NAME+Anti-VEGF(P<0.001)组心肌毛细血管密度,与PIT组相比,均显著下降。④实验开始时,六组之间的局部冠状动脉阻断后即侧支循环血流量(CCBF)无统计学差异(P>0.05);经四周训练至实验终点时,CCBF在PIT组(2643±183)显著高于Sham(1739±190, P<0.05)及M(I1840±122, P<0.05)组; CCBF在L-NAME (1975±194), Anti-VEGF (1837±150),L-NAME+Anti-VEGF(1675±71)组均低于PIT组,差异均有统计学意义(P<0.05)。⑤PIT组eNOS mRNA表达水平显著高于Sham和MI组(P<0.05);PIT,L-NAME,Anti-VEGF三组之间eNOS mRNA表达水平无显著性差异(P>0.05);MI组VEGF mRNA表达水平显著高于Sham组(P<0.05);VEGFmRNA表达水平在PIT组较MI组明显增加(P<0.01);L-NAME+Anti-VEGF组VEGF mRNA表达水平显著低于PIT(P<0.05),L-NAME(P<0.01),Anti-VEGF组(P<0.01)。VEGFR-1mRNA表达水平在PIT组显著高于Sham组(P<0.05);PIT组VEGFR-1mRNA表达水平显著低于L-NAME(P<0.001),Anti-VEGF(P<0.05),L-NAME+Anti-VEGF组(P<0.001);VEGFR-2mRNA表达水平在PIT组显著高于Sham,MI,Anti-VEGF及L-NAME+Anti-VEGF组(P<0.001);PIT组sVEGFR-1mRNA表达水平显著高于Sham及MI组(P<0.05);sVEGFR-1mRNA表达水平在L-NAME+Anti-VEGF组显著高于PIT及Anti-VEGF组(P<0.05)。⑥VEGF蛋白表达水平在PIT组(2.03±0.291)显著高于Sham(0.76±0.097, P<0.001)及MI组(1.26±0.149, P<0.05);而VEGF抗体可阻断此效应(P<0.001);六组之间的eNOS蛋白表达量无统计学差异(P>0.05);PIT组p-eNOS蛋白表达量(0.87±0.151)较Sham(0.24±0.015, P<0.01)及MI组(0.6±0.042, P<0.05)明显增加,而此效应可被VEGF及L-NAME预处理阻断(P <0.001)。外周血VEGF及NO含量与局部缺血心肌VEGF及p-eNOS趋势基本一致。⑦循环EPCs升高的倍数分别与局部心肌毛细血管密度(r=0.893,P=0.000)和局部缺血心肌侧支血流量(r=0.877,P=0.000)成正相关。结论:在兔的可控性心肌缺血模型上,PIT可通过VEGF/eNOS相关途径提高EPCs迁移及血管新生,促进局部心肌侧支循环生成。
Objective: Ischemia-induced angiogenesis have been promising which aims toimprove neovascularization by delivery of angiogenic factors or endothelialprogenitor cells (EPCs) to cardiac ischemic area. In order to avoid the risk ofexcessive myocardial ischemia, therefore, we hypothesized that physiologicalischemic training (PIT) of normal skeletal muscle might contribute to myocardialangiogenesis via mobilization of EPCs from the bone marrow in the establishedrabbit model of controllable myocardial ischemia.
Methods: The rabbits were grouped by sham-operation (Sham), myocardialischemia without PIT (MI), PIT, PIT with the pretreatment with the endothelial nitricoxide synthase (eNOS) inhibitor L-nitroarginine methyl ester (L-NAME), PIT withanti-vascular endothelial growth factor antibody (Anti-VEGF), or both L-NAME andAnti-VEGF antibody (L-NAME+Anti-VEGF). Controlled myocardial ischemia wasmodeled by a water balloon constrictor implanted on their left ventricular branch PITprocedure included three cycles of3minutes of cuff inflation followed by5minutesof deflation on hind limbs of the rabbits for4weeks. At the endpoints, circulatingEPCs (CD34+/Flk-1+) were measured by fluorescence-activated cell sorter; capillarydensity, by immunohistochemistry; blood flow, by a microspheres technique; leftventricular ejection fraction, by echocardiogram; plasma VEGF and nitric oxide, by enzyme linked immunosorbent assay (ELISA) and nitrate reductase method; themRNA and protein expression of VEGF and eNOS, by real-time RT (reversetranscription)–PCR and Western blotting respectively.
Results: Compared with Sham and MI groups, the PIT group had the highest EPCcount (P <0.001), and the increase of capillary density (P <0.01) and collateralblood flow (P <0.05) in the ischemic myocardium were consistent with the findingof EPC count. Blockade of VEGF or eNOS prevented all such PIT–induced effectsand caused the decrease of left ventricular ejection fraction (P <0.05). The mRNAlevels of eNOS and VEGF were significantly higher in the groups received PITcompared with the Sham group (P <0.05). VEGF protein expression was higher inthe groups received PIT than the Sham and MI groups (P <0.05), and the effects wasinhibited by Anti-VEGF pretreatment (P <0.05). Phospho-eNOS protein expressionwas higher in the PIT group than the Sham and MI groups (P <0.05), and the effectwas inhibited by L-NAME and Anti-VEGF antibody pretreatment (P <0.05). Theplasma VEGF and nitric oxide were consistent with the myocardial VEGF and eNOSprotein levels respectively. The capillary density and collateral blood flow werehighly correlated with the increase of EPC count (r=0.893and r=0.877,respectively, P=0.000).
Conclusion: PIT improved EPC mobilization and myocardial angiogenesis in vivothough VEGF/eNOS-related pathway. Consequently, these results might support thefuture development of strategies for therapeutic neovascularization.
引文
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