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 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 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 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 g/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. 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 1905 to 1502 mmHg) in RMR, and prevented the elevation of SBP in HS. 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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000078-02
Application #
8736478
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$265,771
Indirect Cost
Name
National Institute on Aging
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