Therapeutic Potential of EPO and its Derivatives for Reducing Blood Pressure

该计划的主要目标是探索重组人促红细胞生成素及其非红细胞生成物的血管扩张潜力，并对不同的动脉高血压模型进行临床前动物实验，以阐明其作用机制，并评估其转化为人类临床实践的潜力。 1.EPO的急性血流动力学效应。 最近发表的研究有力地表明，促红细胞生成素与一种共同的受体结合，从而激活抗凋亡、抗炎途径和一氧化氮释放。令人惊讶的是，EPO给药后NO激活后的血流动力学反应从未被报道过。本研究的目的是评价EPO对心脏的急性血流动力学和心血管反应，证实它们与NO的相关性，并验证EPO通过与NO激活相关的心血管变化介导心脏保护的假说。实验1给Wistar大鼠静脉注射3000U/kg的重组人促红细胞生成素后，经留置导管监测的动脉血压几乎立即开始逐渐下降，直至90min后趋于平稳，低于对照水平的20%。在实验2中，静脉注射3000或150U/kg的重组人促红细胞生成素后2小时，平均血压比对照组降低25%。静脉注射人或大鼠重组EPO(3000U/kg)2小时后，对心功能进行详细的压力-体积环分析(实验3)，显示心脏收缩功能显著降低(PRSW比对照组减少33%)。非选择性一氧化氮合酶抑制剂(L-NAME)可阻断重组人促红细胞生成素对大鼠动脉血压的降低和心脏收缩功能的影响。在实验4中，在冠状动脉永久性结扎后24小时，心肌梗死(MI)测量了未经处理的大鼠左心室的263.5%。冠脉结扎后即刻给予3000U/kg重组人促红细胞生成素处理的大鼠心肌梗死缩小56%。预先给予L-NAME并不减弱重组人促红细胞生成素对心肌梗死大小的有利作用，而单用L-NAME处理的大鼠心肌梗死大小与对照组无差异。因此，单次注射重组人促红细胞生成素可显著降低全身血压，并相应降低心脏收缩功能。然而，EPO对心肌缺血损伤的保护作用与NO激活或NO介导的血流动力学反应无关。 II.促红细胞生成素非红系衍生物的降压作用 ARA290是由Araim制药公司在部分EPO分子的基础上合成的一种小肽，名为螺旋B表面肽(HBSP)，又称ARA290。这种多肽与一种常见的受体--EPO受体HetheroComplex结合，并不具有促血栓作用。这种多肽在不同的实验模型中进行了测试，虽然它被证明具有强大的组织保护特性，但它不能诱导血细胞的产生。最近，我们证明了HBSP与rhEPO具有相似的效力，可以将诱导线粒体通透性转变的活性氧物种(ROS)阈值提高40%，从而保护心肌细胞免受缺血应激的影响。在冠状动脉永久性结扎诱导的大鼠心肌梗死模型中，我们还证明了单次注射促红细胞生成素和HBSP对所致心肌梗死的减少50%具有同等效力。在实验模型中，HBSP的抗凋亡和抗炎作用与rhEPO相当。 在第一个实验中，在异氟醚吸入麻醉下，通过插入主动脉的导管记录动脉血压后，我们给正常血压的大鼠单次静脉注射60微克的HBSP(ARA290)或生理盐水。两小时后，再次注射异氟醚，并再次测量动脉血压。注射多肽后的血压比注射生理盐水后的血压低约20%。其作用类似于我们先前报道的重组人促红细胞生成素的作用，并被L-NAME预先阻断。 实验二采用3种不同的实验性高血压大鼠模型，观察HBSP的降压作用。在自发性高血压大鼠(SHR)、高盐饮食(HS)盐敏感型Dahl大鼠和切除5/6肾质量(RMR)的Wistar大鼠的腹腔内植入HBSP 0.16~0.09 ug/min的渗透泵，连续8周。WKY组、Dahl盐敏感组和假手术组分别作为3种模型的对照组。在异氟醚麻醉下，采用尾套法测量血压。在所有三种实验模型的未治疗组中，收缩压(SBP)逐渐上升至180-200毫米汞柱。HBSP可使SHR的SBP完全恢复正常，使RMR的SBP降低20%(由190 mm Hg降至150 mm Hg)，并能阻止高盐饮食维持的Dahl盐敏感大鼠的SBP升高。经HBSP治疗后，3组未治疗高血压大鼠均出现明显的心肌纤维化，降至对照组水平。在治疗后的Dahl和SHR中，所有3个未治疗的高血压组中观察到的显著肾硬化也降低到了对照组的水平，但在RMR组中没有。这些结果清楚地表明HBSP具有强大的降压和抗纤维化特性。 为了观察HBSP对非麻醉自由活动动物的长期降压作用，在本报告期间，我们在自发性高血压大鼠(SHR)上使用植入式血压遥测传感器，其中一半用HBSP(0.16-0.09微渗泵)，另一半用生理盐水(微渗泵)治疗8周。在撰写本报告时，数据仍在处理中。

The broad objective of this program is to explore the vasodilative potential of rhEPO and its non-erythropoietic derivatives and to perform preclinical animal experiments on different models of arterial hypertension to elucidate their mechanism of action and to evaluate their potential for translation into clinical practice in humans. I. Acute hemodynamic effects of EPO. Recently published research strongly suggests the erythropoietin binds to a Common receptor and therefore activates anti-apoptotic, anti-inflammatory pathways, and nitric oxide release. Surprisingly, hemodynamic response subsequent to NO activation after EPO administration had never been reported. Objectives of this study were to evaluate the acute hemodynamic and cardiovascular responses to EPO administration, to confirm their NO association, and to test the hypothesis that EPO-induced cardioprotection is mediated through cardiovascular changes related to NO activation. In Experiment 1 after 3000 U/kg of rhEPO was administered intravenously to Wistar rats, arterial blood pressure, monitored via indwelling catheter, almost immediately started progressively to decline, until leveling off 90 minutes after injection at 20% below control level. In Experiment 2 the 25% reduction of mean blood pressure, compared to control group, was observed 2 hrs after intravenous injection of either 3000 or 150 U/kg of rhEPO. Detailed pressure volume loop analyses of cardiac performance (Experiment 3) 2 hrs after intravenous injection of human or rat recombinant EPO (3000U/kg) revealed a significant reduction of systolic function (PRSW was 33% less than control). Reduction of arterial blood pressure and systolic cardiac function in response to rhEPO were blocked in rats pretreated with a non-selective inhibitor of nitric oxide synthase (L-NAME). In Experiment 4, 24 hrs after a permanent ligation of a coronary artery myocardial infarction (MI) measured 263.5% of left ventricle in untreated rats. MI in rats treated with 3000 U/kg of rhEPO immediately after coronary ligation was 56% smaller. Pretreatment with L-NAME did not attenuate the beneficial effect of rhEPO on MI size, while MI size in rats treated with L-NAME alone did not differ from control. Therefore, a single injection of rhEPO resulted in a significant, NO-mediated reduction of systemic blood pressure and corresponding reduction of cardiac systolic function. However, EPO-induced protection of myocardium from ischemic damage is not associated with NO activation or NO-mediated hemodynamic responses. II. Antihypertensive properties of non-erythropoietic derivatives of EPO A small peptide, Helix B Surface Peptide (HBSP), also known as ARA290, was synthesized by Araim Pharmaceutical on the basis of a part of the EPO molecule. This peptide binds to a common receptor, EPO receptor hetherocomplex, and does not have pro-thrombotic effects. This peptide was tested in different experimental models and, while it proved to possess strong tissue protective properties, it did not induce blood cell production. Recently we demonstrated that HBSP has a similar potency to rhEPO for increasing the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40%, thus protecting cardiac myocytes from ischemic stress. In the rat model of MI induced by a permanent ligation of the coronary artery, we also demonstrated the equal potency of a single administration of EPO and HBSP for a 50% reduction of resulting MI. In that experimental model the anti-apoptotic and anti-inflammatory potentials of HBSP were equal to those of rhEPO. In the first experiment, under Isoflurane inhalation narcosis, after recording of arterial blood pressure via catheter inserted into the aorta, we administered either a single intravenous dose of 60 mcg of HBSP (ARA290) or a saline to a normotensive rat. Two hours later the Isoflurane was reintroduced and arterial blood pressure was again measured. The blood pressure after injection of peptide was about 20% lower than after injection of saline. The effect was similar to the effect of rhEPO that we previously reported and was blocked by pretreatment with L-NAME. In the second experiment the arterial blood pressure reducing effect of HBSP was tested in 3 different experimental rat models of hypertension. Osmotic pumps delivering 0.16 - 0.09 ug/min of HBSP for 8 weeks were implanted into the abdominal cavity of spontaneously hypertensive rats (SHR), salt sensitive Dahl rats consuming a high salt diet (HS), and Wistar rats subjected to a resection of 5/6 of the total renal mass (RMR). WKY, Dahl salt sensitive rats maintained on a low salt diet, and sham operated rats, respectively, served as controls for these 3 models. Blood pressure was measured under Isoflurane narcosis using tail cuff technique. In untreated groups of all three experimental models, systolic blood pressure (SBP) gradually increased to 180-200 mmHg. HBSP infusion completely normalized the SBP in SHR, reduced the SBP by 20% (from 190 to 150 mmHg) in RMR, and prevented the elevation of SBP in Dahl salt sensitive rats maintained on high salt diet. Significant myocardial fibrosis occurring in all 3 groups of untreated hypertensive rats was reduced to the level of controls by HBSP. Significant nephrosclerosis observed in all 3 untreated hypertensive groups was also reduced to the level of controls in treated Dahl and SHR, but not in RMR groups. These results clearly demonstrate potent antihypertensive and antifibrotic properties of HBSP. In order to investigate antihypertension effect of HBSP for long-term in un-anesthetized, freely-moving animals, during this reporting period, we used implantable blood pressure telemetry sensors in spontaneously hypertensive rats (SHR), treat one half of them with HBSP (0.16 - 0.09 ug/min via mini-osmotic pump) and other half with saline (via mini-osmotic pump) for 8 weeks. At the time this report is written, data are still in the process.