Single dose treatment with PARP-inhibitor INO-1001 improves aging-associated cardiac and vascular dysfunction

https://doi.org/10.1016/j.exger.2007.01.013Get rights and content

Abstract

Overproduction of reactive oxygen species in aging tissues has been implicated in the pathogenesis of aging-associated cardiovascular dysfunction. Oxidant-induced DNA-damage activates the poly(ADP-ribose) polymerase (PARP) pathway, leading to tissue injury. In this study we investigated the acute effects of the PARP inhibitor INO-1001 on aging-associated cardiac and endothelial dysfunction. Using a pressure–volume conductance catheter, left ventricular pressure–volume analysis of young and aging rats was performed before and after a single injection of INO-1001. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Aging animals showed a marked reduction of myocardial contractility and endothelium-dependent relaxant responsiveness of aortic rings. Single dose INO-1001-treatment resulted in acute improvement in their cardiac and endothelial function. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) confirmed enhanced nitro-oxidative stress and PARP-activation in aging animals. Acute treatment with INO-1001 decreased PARP-activation, but did not affect nitrotyrosine-immunoreactivity. Our results demonstrate that the aging-associated chronic cardiovascular dysfunction can be improved, at least, short term, by a single treatment course with a PARP-inhibitor, supporting the role of the nitro-oxidative stress – PARP – pathway in the age-related functional decline of the cardiovascular system. Pharmacological inhibition of PARP may represent a novel therapeutic utility to improve aging-associated cardiovascular dysfunction.

Introduction

There is emerging evidence that aging is an important risk factor in the development of ischemic heart disease. This may be due to an age-associated increase in coronary vascular resistance leading to reduced myocardial blood supply and flow reserve (Hachamovitch et al., 1989). Numerous studies suggest that aging is associated with impaired function of endothelium in laboratory animals (Tschudi et al., 1996) and humans (Egashira et al., 1993) and this endothelial dysfunction predisposes the aging population to cardiovascular complications and micro-thrombus formation. Recent studies demonstrate that the cardiovascular dysfunction associated with advanced aging is related to the local formation of reactive oxygen and nitrogen species in the myocardium and coronary vasculature (Bejma et al., 2000, Van der loo et al., 2000, Csiszar et al., 2002).

Aging organisms are exposed to continuous oxidative injury, due to the higher rate of superoxide and other free-radical production from the mitochondrial electron-transport chain (Sohal and Sohal, 1991). Increases in reactive oxidant species (ROS) and other oxidants at old age can elicit oxidative modifications of various cell components, such as lipid, protein and particularly DNA (de la Asuncion et al., 1996).

Various oxygen and nitrogen species (peroxynitrite, hydrogen peroxide, hydroxyl radical and nitroxyl anion) have been established as pathophysiological relevant endogenous triggers of DNA single-strand breakage and activation of the poly(ADP-ribose) polymerase (PARP) enzyme (Virag and Szabo, 2002). When activated by DNA single strand breaks, PARP initiates an energy-consuming metabolic cycle by transferring ADP-ribose units from NAD+ to nuclear proteins. This process results in the rapid depletion of intracellular ATP-pools and impaired mitochondrial respiration, eventually leading to cellular energetic crisis, dysfunction and death via the necrotic route (Virag and Szabo, 2002).

Pharmacological attempts against nitro-oxidative stress using classic antioxidants, such as vitamin E (which works by scavenging toxic oxidation products), ascorbate or glutathione (which react with peroxynitrite, albeit at a relatively slow rate) resulted in conflicting results in experimental models of disease (Ceriello, 2003). Based on recent studies, pharmacological inhibition of PARP (Szabo et al., 2003, Szabo et al., 2004b, Soriano et al., 2001a, Beller et al., 2006) or decomposition of peroxynitrite (Szabo et al., 2002a, Szabo et al., 2002b, Pacher et al., 2003, Radovits et al., 2007), which block the peroxynitrite DNA injury poly(ADP-ribose) polymerase pathway emerge as potent novel antioxidant therapeutic possibilities in multiple pathophysiological conditions.

Chronic treatment with PARP-inhibitors PJ34 and INO-1001 for 2 months in a rodent model has been demonstrated to improve endothelial and cardiac dysfunction associated with aging (Pacher et al., 2002e, Pacher et al., 2004b) showing the involvement of the nitro-oxidative stress PARP pathway in the pathophysiology of cardiac and vascular aging.

Considering the theoretical possibility of acutely interrupting this pathway by pharmacological inhibition of the PARP enzyme, thereby quickly restoring the ATP-pools and the normal energy supply of the cells, we investigated in this study whether cardiac and vascular dysfunction at old age may be beneficially affected even by a single treatment course with a potent pharmacological inhibitor of PARP.

Section snippets

Animals and treatment protocols

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All procedures and handling of animals during the investigations were reviewed and approved by the Ethical Committee of the Land Baden-Württemberg for Animal Experimentation.

Young adult (3-month-old, 200–250 g) and aging (20-month-old, 450–600 g) male Lewis rats (Charles River, Sulzfeld, Germany) were housed in a room at

Immunohistochemical analysis

Immunohistochemical staining showed increased immunoreactivity for nitrotyrosine and poly(ADP-ribose) indicative of nitrosative stress and enhanced activation of PARP – in the left ventricular myocardium and in the aortic wall (mainly in the endothelium) of aging rats. (Fig. 1, Fig. 2).

Single dose treatment with the potent PARP-inhibitor INO-1001 notably decreased PAR formation both in the myocardium and the aortic wall. Immunoreactivity for nitrotyrosine was not affected by acute

Discussion

In the current study we demonstrate that a single injection of PARP-inhibitor INO-1001 effectively decreases the age-related myocardial and vascular PARP-activation, resulting in acute improvement of left ventricular contractility and enhanced endothelium-dependent vasorelaxation in a rat model of aging-associated cardiovascular dysfunction.

Recent studies elucidated numerous cellular and molecular mechanisms responsible for the functional decline of the cardiovascular system at old age (Csiszar

Acknowledgements

This work was supported by a Grant from the German Research Foundation (SFB 414) to G.S. and by the Hungarian Research Fund (OTKA AT049488) and the National Institutes of Health (R01 GM060915) to C.S. The expert technical assistance of Anne Schuppe and Heike Ziebart are gratefully acknowledged.

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