Pathophysiology and therapeutic strategy of coronary vasospasm of microvessels using a novel porcine model.

12只猪每隔2周行冠状动脉左前降支(LAD)球囊内皮剥脱术(ED)，共4次。在每次剥脱前和8周时，评估乙酰胆碱(ACh)或5-羟色胺引起的剥脱部位直径和LAD血流量的变化。连续监测清醒和自由状态猪的血压、左前降支部位的心电图和左前降支血流量。2周左右出现自发性心电图ST段压低伴左前降支血流减少。因此，在8周时，0.05μg/kg ACh可引起类似的心电和LAD血流改变，但未见裸露部位狭窄，提示微血管痉挛(Coron ARA Dis 2004；15：137145)。此外，与对照组相比，重复内皮剥离8周时，冠状静脉窦血浆内皮素-1(ET-1)的基础水平显著升高(P<0.01)。注射ACh后1min，ED组冠状静脉窦内ET-1浓度在第4、6、8周较基础值进一步升高(P均<0.01)。经抗坏血酸自由基(AFR)和二氢乙锭染色检测，反复电刺激可增加ROS的产生，而ET-1A型受体(ET_A)拮抗剂几乎可阻止其产生。ED+ET_A组eNOS在微血管中的表达减少，下调作用减弱。组织学分析显示，ET_A拮抗剂可抑制反复ED所致的下游血管重构。ET-1在冠脉微血管痉挛中起重要作用，阻断ET-1可能有利于冠脉血管痉挛的治疗。此外，我们发现了RhoA的快速激活途径，它在内皮功能障碍中起着重要作用(J Biol Chem 2005；280：10182-10188)。这种快速的RhoA激活是否与冠状动脉血管痉挛的内皮功能障碍有关还有待阐明。

Balloon endothelial denudation (ED) was carried out in the epicardial left anterior descending coronary artery (LAD) every 2 weeks, for a total of four times, in 12 pigs. Changes in the denuded site diameter and LAD blood flow caused by acetylcholine (ACh) or serotonin were assessed before each denudation and at 8 weeks. Blood pressure, electrocardiogram (ECG) from the LAD area and LAD blood flow were monitored continuously in conscious and unrestrained pigs. Spontaneous ECG ST depression with a decrease in LAD blood flow appeared at around 2 weeks. In accordance with this, 0.05 μg/kg ACh induced similar ECG and LAD blood flow changes without denuded site narrowing at 8 weeks, suggesting microvascular spasm (Coron Artery Dis 2004 ; 15 : 137-145). In addition, repeated endothelial denudation significantly increased the basal level of plasma concentration of endothelin-1 (ET-1) in coronary sinus at 8weeks compared with control group p<0.01). ET-1 concentration in coronary sinus 1 minute after ACh injection was further augmented in ED group at 4, 6 and 8 weeks compared with the basal level (p<0.01, each). Repeated ED increased the production of reactive oxygen species (ROS) detected by ascorbyl free radical (AFR) and dihydroethidium staining, whereas it was almost prevented by ET-1 type A receptor (ET_A) antagonist. ED induced the decrease of eNOS expression in the microvessels and the downregulation was attenuated in ED+ET_A group. Histological analysis revealed that ET_A antagonist suppressed the downstream vascular remodeling induced by repeated ED. ET-1 plays a crucial role for coronary microvascular spasm and the blockade of ET-1 may be beneficial for treating coronary vasospasm. Furthermore, we found a rapid activation pathway of RhoA which plays an important role in endothelial dysfunction (J Biol Chem 2005 ; 280 : 10182-10188). It remains to elucidate whether this rapid RhoA activation associates with endothelial dysfunction of coronary vasospasm.