Therapeutic Potential of EPO and its Derivatives for Reducing Blood Pressure

该计划的主要目的是探索rhEPO及其非红细胞生成衍生物的血管舒张潜力，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。 I. EPO的急性血液动力学效应。 一氧化氮（NO）信号的激活被认为是，至少部分地，作为EPO诱导的心脏保护的机制基础。令人惊讶的是，EPO给药后NO激活后的血流动力学反应从未报道过。本研究的目的是评估急性血液动力学和心血管反应EPO管理，以确认其NO的成因，并测试的假设，EPO诱导的心脏保护是通过心血管的变化介导的NO激活。在实验1中，对Wistar大鼠静脉注射3000 U/kg rhEPO后，通过留置导管监测的动脉血压逐渐下降，几乎立即开始，直到注射后90分钟稳定在低于对照水平20%。在实验2中，与对照组相比，在静脉注射3000或150 U/kg的rhEPO后2小时观察到平均血压降低25%。在静脉注射人或大鼠重组EPO（3000 U/kg）后2小时，心脏性能的详细压力体积环分析（实验3）显示收缩功能显著降低（PRSW比对照低33%）。在用一氧化氮合酶非选择性抑制剂（L-NAME）预处理的大鼠中，rhEPO引起的动脉血压降低和心脏收缩功能的降低被阻止。在实验4中，在永久结扎冠状动脉心肌梗死（MI）后24小时，未治疗大鼠的左心室测量为263.5%。冠脉结扎后立即给予3000 U/kg rhEPO的大鼠MI降低了56%。 用L-NAME预处理没有减弱rhEPO对MI大小的有益作用，而单独用L-NAME处理的大鼠的MI大小与对照组没有差异。因此，单次注射rhEPO导致显著的NO介导的全身血压降低和相应的心脏收缩功能降低。然而，EPO诱导的心肌缺血性损伤的保护与NO激活或NO介导的血流动力学反应无关。 二. 促红细胞生成素的非红细胞生成衍生物的抗高血压特性 一种小肽，受阻B表面肽（HBSP），也称为ARA 290，是由Araim Parmartan基于EPO分子合成的，并且不激活EPO受体。这种肽在不同的实验模型中进行了测试，虽然它被证明具有很强的组织保护特性，但不会诱导血细胞产生。最近，我们证明了HBSP具有与rhEPO相似的效力，可将诱导线粒体通透性转换的活性氧（ROS）阈值增加40%，从而保护心肌细胞免受缺血应激。在冠状动脉永久性结扎诱导的MI大鼠模型中，我们也证明了EPO和HBSP单次给药对所产生的MI减少50%的同等效力。在该实验模型中，HBSP的抗凋亡和抗炎潜力与rhEPO相当。 在第一个实验中，我们给一只血压正常的大鼠单次静脉注射60 mcg的HBSP（ARA 290），同时通过插入主动脉的导管监测其动脉血压。我们观察到，收缩压和舒张压在给药后的最初几分钟内开始下降。血压下降约为基线的20%，持续约2小时。其效果与我们以前报道的rhEPO的效果相似。 Dahl盐敏感大鼠是一种广泛应用的盐敏感性高血压实验动物模型。我们将两组盐敏感大鼠置于高盐饮食中。其中一个高盐组通过渗透泵植入腹腔给予HBSP治疗，而另一个高盐组则保持不治疗。采用尾袖法每周测量动脉血压。另一组Dahl大鼠保持正常的低盐饮食，并作为对照。治疗持续9周。HBSP的起始剂量为1 mg/kg/天，但随着动物的生长，逐渐降低至0.4 mg/kg。在9周期间，低盐饮食大鼠的动脉血压随着生长略有增加（收缩压增加12%）。 与此同时，正如该模型所预期的那样，高盐饮食大鼠的动脉血压急剧增加，达到200 mmHg。用HBSP处理的高盐饮食动物的动脉血压保持在低盐动物的水平，即，保持正常。高盐饮食和治疗对心脏和肾脏重塑和功能的影响的超声和组织学比较正在进行中。目前正在启动评估HBSP对不同高血压实验动物模型（自发性高血压大鼠和肾质量减少模型）影响的实验。

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. Activation of nitric oxide (NO) signaling is considered, at least partially, as a mechanistic basis for EPO-induced cardioprotection. 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 genesis, 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, progressively declined, starting almost immediately 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 Parmaceutical on the basis of EPO molecule and does not activate EPO receptors. This peptide was tested in different experimental models and while it proved to possess strong tissue protective properties did not induce blood cell production. Recently we demonstrated that HBSP have similar potency to rhEPO for increasing the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40% and thus protecting cardiac myocytes from ischemic stress. In the rat model of MI induced by a permanent ligation of coronary artery we also demonstrated the equal potency of single administration of EPO and HBSP for 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 we administered a single intravenous dose of 60 mcg of HBSP (ARA290) to a normotensive rat, while monitoring its arterial blood pressure via catheter inserted into the aorta. We observed that systolic and diastolic blood pressures started to fall reduced during the first several minutes following drug administration. The reduction of blood pressure was about 20% of baseline and lasted for about 2 hrs. The effect was similar to the effect of rhEPO that we previously reported. The Dahl salt sensitive rat is a well-known and widely used experimental animal model of salt sensitive hypertension. We placed two groups of salt sensitive rats on the high salt diet. One of the high salt groups was treated with HBSP via osmotic pump implanted into the abdominal cavity, while another high salt group remained untreated. Arterial blood pressure was measured weekly using tail cuff technique. Another group of Dahl rats remained on a normal, low salt diet and served as a control. The treatment continued for 9 weeks. The starting dose of HBSP was 1 mg/kg/day, but with animals' growth it gradually was reduced to 0.4 mg/kg. During 9 weeks arterial blood pressure of low-salt diet rats increased slightly with growth (systolic blood pressure increased by 12%). At the same time, arterial blood pressure in rats on the high salt diet, as expected in this model, increased dramatically, reaching 200 mmHg. The arterial blood pressure of animals on high salt diet treated with HBSP remained at the level of low salt animals, i.e., remained normal. The sonographic and histological comparison of the effects of high salt diet and treatment on cardiac and kidney remodeling and function are being conducted. Experiments assessing the effects on HBSP on different experimental animal models of hypertension (spontaneously hypertensive rats and renal mass reduction model) are being initiated.