Reduction of myocardial damage during acute ischemia

该项目的主要目的是对心肌缺血动物模型进行临床前实验，以阐明心肌细胞死亡的机制和随后CHF的发展，并评估不同治疗方式的潜力。最终目的是限制心肌损伤的程度，预防或减轻CHF的发展。 促红细胞生成素（Erythropoietin，EPO）是一种刺激红细胞生成的细胞因子，被广泛用于治疗各种原因引起的贫血，近年来研究发现，EPO可抑制脑缺血或创伤后细胞凋亡，减轻脑组织损伤程度。我们曾报道过，与脑组织相似，冠状动脉结扎后单次全身注射重组人EPO可减少危险心肌区域的细胞凋亡，减弱早期左心室重构，8周后，导致梗死面积减少，心脏结构和功能恶化程度降低。我们还报道了rhEPO在大鼠心肌梗死模型中的治疗剂量和治疗窗的研究结果。然而，rhEPO的重复给药明显引起红细胞压积的显著升高。此外，据报道，即使单次注射rhEPO也会导致网织红细胞水平显著升高，这可能代表MI患者的额外风险。因此，具有EPO的组织保护性质而没有其红细胞生成作用的化合物将是偶然的。本研究的目的是研究Warren Pharmaceutical基于EPO结构设计的小分子的可能治疗作用，EPO结构是一种焦谷氨酸螺旋B表面肽（pHBP），其仅包括不与EPO受体结合的EPO分子的一部分，因此不是红细胞生成的，但保留了EPO的组织保护特性。 这项工作是与沃伦制药公司的研究人员以及LCS的Sollott和Boheler博士合作完成的。我们比较了pHBP和EPO在体外保护心肌细胞免受氧化应激和在体内保护心肌组织免受缺血损伤的能力。与EPO类似，HBP将诱导线粒体渗透性转变的活性氧簇（ROS）阈值提高了40%。在大鼠冠状动脉永久结扎诱导的心肌梗死实验模型中，冠状动脉结扎后立即单次推注60 g/kg pHBP（类似于EPO），24小时后检查，风险心肌区域的细胞凋亡减少80%，炎症减少34%。冠脉结扎后24小时测量的心肌梗死（MI）在pHBP和EPO治疗的大鼠中同样减少了50%。手术后两周，通过超声心动图评估的左心室重构和功能下降在pHBP和EPO治疗的大鼠中显著且相似地减弱，MI大小减少了25%。在6周的随访期间，效果得以保留。冠状动脉结扎后立即单次推注pHBP可有效减少MI大小，剂量低至1 g/kg，但如果在MI诱导后24 h给药，则在60 g/kg剂量下无效。我们的结论是pHBP与EPO具有同等的心脏保护作用，值得进一步考虑作为rhEPO的更安全的替代品，以寻找治疗方案，减少冠状动脉循环阻断后的心肌损伤

The broad objective of this program is to perform preclinical experimentation on animal models of myocardial ischemia to elucidate the mechanisms of cellular death in the myocardium and development of the subsequent CHF and to evaluate the potential of different therapeutic modalities. The ultimate goal is to limit the extent of myocardial damage and to prevent or attenuate the development of CHF. Erythropoietin (EPO), a cytokine known to stimulate erythropoiesis and widely used to treat the anemia of different etiology, had been recently reported to suppress apoptosis and to reduce the extent of damage in brain tissue following experimental ischemia or trauma. We had reported that, similar to brain tissue, a single systemic injection of recombinant human EPO following coronary ligation reduce apoptosis in the myocardial area at risk, attenuated the early left ventricular remodeling, and, eight weeks later, resulted in the reduction of the infarct size and the extent of structural and functional deterioration of the heart. We had also reported the results of studies that defined therapeutic doses and therapeutic window of rhEPO in the rat model of MI. However, repeated dosing of rhEPO obviously causes a marked elevation of hematocrit. Moreover, it has been reported that even a single injection of rhEPO resulted in a significant elevation of the level of reticulocytes, which could represent an additional risk for MI patients. Therefore, it would be adventitious to have a compound that would have tissue protective properties of EPO without its erythropoietic effect. The purpose of the current studies was to investigate the possible therapeutic effects of small molecule designed by Warren Pharmaceutical on the basis of EPO structure, a pyroglutamate helix B surface peptide (pHBP) that includes only a part of the EPO molecule that does not bind to EPO receptor and thus, is not erythropoietic, but retains tissue protective properties of EPO. This work had been done in collaboration with researchers from Warren Pharmaceutical and with Drs. Sollott and Boheler of the LCS. We compared the ability of pHBP and EPO to protect cardiac myocytes from oxidative stress in vitro and cardiac tissue from ischemic damage in vivo. HBP, similar to EPO, increased the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40%. In an experimental model of myocardial infarction induced by permanent ligation of a coronary artery in rats, a single bolus injection of 60 g/kg of pHBP immediately after coronary ligation, similar to EPO, reduced apoptosis in the myocardial area at risk, examined 24 h later, by 80% and inflammation by 34%. Myocardial infarction (MI) measured 24 h after coronary ligation was similarly reduced by 50% in both pHBP- and EPO-treated rats. Two wks after surgery, left ventricular remodeling and functional decline assessed via echocardiography were significantly and similarly attenuated in pHBP- and EPO-treated rats, and MI size was reduced by 25%. The effect was retained during the 6-wk follow-up. A single bolus injection of pHBP immediately after coronary ligation was effective in reduction of MI size in a dose as low as 1 g/kg, but was ineffective at a 60 g/kg dose if administered 24 h after MI induction. We conclude that pHBP is equally cardioprotective with EPO and deserves further consideration as a safer alternative to rhEPO in the search for therapeutic options to reduce myocardial damage following blockade of the coronary circulation