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