8 Poly(ADP-Ribose) Polymerase Activationand Nitrosative Stress in the Developmentof Cardiovascular Disease in Diabetes

Chapter 8 / PARP Activation 167

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From: Contemporary Cardiology: Diabetes and Cardiovascular Disease, Second Edition Edited by: M. T. Johnstone and A. Veves © Humana Press Inc., Totowa, NJ

8 Poly(ADP-Ribose) Polymerase Activation and Nitrosative Stress in the Development of Cardiovascular Disease in Diabetes

Pál Pacher, ^MD , ^PhD and Csaba Szabó, ^MD , ^PhD

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INTRODUCTION

Macro- and microvascular disease are the most common causes of morbidity and

mortality in patients with diabetes mellitus (DM). Diabetic vascular dysfunction is a

major clinical problem, which underlies the development of various severe complica-

tions including retinopathy, nephropathy, neuropathy, and increase the risk of stroke,

hypertension, and myocardial infarction (MI). Hyperglycemic episodes, which compli-

cate even well-controlled cases of diabetes, are closely associated with oxidative and

nitrosative stress, which can trigger the development of cardiovascular disease. Recently,

emerging experimental and clinical evidence indicates that high-circulating glucose in

DM is able to induce oxidative and nitrosative stress in the cardiovascular system, with

the concomitant activation of an abundant nuclear enzyme, poly(ADP-ribose) poly-

merase-1 (PARP) . This process results in acute loss of the ability of the endothelium to

generate nitric oxide (NO; endothelial dysfunction) and also leads to a severe functional

impairment of the diabetic heart (diabetic cardiomyopathy). Accordingly, neutralization

of peroxynitrite or pharmacological inhibition of PARP protect against diabetic cardio-

vascular dysfunction. The goal of this chapter is to summarize the recently emerging evidence supporting the concept that nitrosative stress and PARP activation play a role in the pathogenesis of diabetic endothelial dysfunction and cardiovascular complications.

POLY(ADP-RIBOSE) POLYMERASE: AN ABUNDANT NUCLEAR ENZYME WITH MULTIPLE REGULATORY FUNCTIONS

PARP-1 (EC 2.4.2.30) [also known as poly(ADP-ribose) synthetase and poly(ADP-

ribose) transferase ] is a member of the PARP enzyme family consisting of PARP-1 and

an increasing number additional, recently identified poly(ADP-ribosylating) enzymes

(minor PARP isoforms). PARP-1, the major PARP isoform, is one of the most abundant

proteins in the nucleus. PARP-1 is a 116 kDa protein which consists of three main

domains: the N-terminal deoxyribonucleic acid (DNA)-binding domain containing two

zinc fingers, the automodification domain, and the C-terminal catalytic domain. The

primary structure of the enzyme is highly conserved in eukaryotes (human and mouse

enzyme have 92% homology at the level of amino acid sequence) with the catalytic

domain showing the highest degree of homology between different species. Many differ-

ences between the various PARP isoenzymes have been demonstrated in domain struc-

ture, subcellular localization, tissue distribution and ability to bind to DNA (1). For the

purpose of this chapter, it is important to note that PARP-1 is considered the major

isoform of PARP in intact cells, and remains commonly termed as “PARP.” This first

isoform of PARP plays a crucial role in the pathophysiology of many diseases. PARP also

has multiple physiological functions, which is a subject of several recent reviews and

monographies (1–6). PARP-1 functions as a DNA damage sensor and signaling molecule

binding to both single- and double-stranded DNA breaks. On binding to damaged DNA

(mainly through the second zinc finger domain), PARP-1 forms homodimers and cata-

lyzes the cleavage of NAD+ into nicotinamide and ADP-ribose and uses the latter to

synthesize branched nucleic acid-like polymers poly(ADP-ribose) covalently attached

to nuclear acceptor proteins. The size of the branched polymer varies from a few to 200

ADP-ribose units. As a result of its high-negative charge, covalently attached ADP-

ribose polymer dramatically affects the function of target proteins. In vivo the most

abundantly poly(ADP-ribosylated) protein is PARP-1 itself and auto-poly(ADP-

ribosylation) represents a major regulatory mechanism for PARP-1 resulting in the

downregulation of the enzyme activity. In addition to PARP-1, histones are also consid-

ered as major acceptors of poly(ADP-ribose). Poly(ADP-ribosylation) confers negative

charge to histones leading to electrostatic repulsion between DNA and histones. This

process has been implicated in chromatin remodeling, DNA repair, and transcriptional

regulation. Several transcription factors, DNA replication factors and signaling mol-

ecules (NFPB, AP-1, Oct-1, YY1, TEF-1, DNA-PK, p53) have also been shown to

become poly(ADP-ribosylated) by PARP-1. The effect of PARP-1 on the function of

these proteins is carried out by noncovalent protein–protein interactions and by covalent

poly(ADP-ribosylation). Poly(ADP-ribosylation) is a dynamic process as indicated by

the short half life of the polymer. Two enzymes—poly(ADP-ribose) glycohydrolase

(PARG) and ADP-ribosyl protein lyase—are involved in the catabolism of poly(ADP-

ribose) with PARG-cleaving ribose–ribose bonds of both linear and branched portions of

poly(ADP-ribose) and the lyase removing the protein proximal ADP-ribose monomer

(7). PARP-1 plays an important role in DNA repair and maintenance of genomic integrity

(8,9) and also regulates the expression of various proteins at the transcriptional level. Of

Chapter 8 / PARP Activation 169

special importance is the regulation by PARP-1 of the production of inflammatory mediators such as inducible NO synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class II (10–14). Nuclear fac- tor-PB (NF-PB) is a key transcription factor in the regulation of this set of proteins and PARP has been shown to act as a co-activator in the NF- PB-mediated transcription.

Poly(ADP-ribosylation) can loosen up the chromatin structure thereby making genes more accessible for the transcriptional machinery (15–20). Additionally, PARP-1 acti- vation has been proposed to represent a cell elimination pathway whereby severely damaged cells are removed from tissues. PARP-1-mediated cell death occurs in the form of necrosis, which is probably the least desirable form of cell death. During necrotic cell death, the cellular content is released into the tissue-posing neighboring cells to harmful attacks by proteases and various proinflammatory intracellular factors. Recently, PARP can also serve as an emergency source of energy used by the base excision machinery to synthesize adenosine triphosphate (ATP) (21). Furthermore, poly(ADP-ribose) may also serve as a signal for protein degradation in oxidatively injured cells (22).

Poly(ADP-Ribose) Polymerase Mediates Oxidant-Induced Cell Dysfunction and Necrosis

Peroxynitrite, a reactive oxidant species, produced from the reaction of NO and super- oxide free radicals, has been established as a pathophysiologically relevant endogenous trigger of DNA single strand breakage and PARP activation (23,24). Additional endog- enous triggers of DNA single strand breakage and PARP activation include hydrogen peroxide, hydroxyl radical, and nitroxyl anion, but not NO, superoxide, or hypochlorous acid (25–29). Peroxynitrite is considered a key trigger of DNA strand breakage because (as opposed to hydroxyl radical, for instance) it can readily travel and cross cell mem- branes. When activated by DNA single-strand breaks, PARP initiates an energy-consum- ing cycle by transferring ADP ribose units from NAD+ to nuclear proteins. This process results in rapid depletion of the intracellular NAD+ and ATP pools, slowing the rate of glycolysis and mitochondrial respiration, eventually leading to cellular dysfunction and death (1).

Poly(ADP-Ribose) Polymerase Regulates Gene Expression and Mononuclear Cell Recruitment

Using pharmacological inhibitors of PARP, it has been demonstrated (as briefly

mentioned earlier) that the activity of PARP is required for the expression of the MHC

class II gene, DNA methyltransferase gene, protein kinase C (PKC), collagenase, ICAM-

1, and (iNOS) (10–14). An oligonucleotide microarray analysis identified multiple genes

that appear to be under the control of PARP-1 in resting cells (14) and even more genes

are affected under conditions of immunostimulation (30). A distinct mode of inhibition

of the expression of pro-inflammatory mediators by inhibition of PARP relates to the

regulation of NF-PB activation. It is unclear whether PARP catalytic activity vs PARP

as a structural protein plays the most important role in its stimulatory role on NF-PB

activation (31,32), it may well be that both mechanisms can be involved under certain

experimental conditions. Pharmacological evidence supports the view that PARP also

regulates the c-fos—AP-1 transcription system and the activation of mitogen-activated

protein kinase (33,34). From the above experimental data it appears that PARP, via a not

yet fully characterized mechanism, regulates the expression of a variety of genes, with

the net result that PARP inhibition or PARP genetic inactivation results in the downregulation of a variety of important pro-inflammatory mediators and pathways.

The PARP-mediated pathway of cell necrosis and the PARP-mediated pathway of inflammatory signal transduction and gene expression may be interrelated in pathophysi- ological conditions. Oxidant stress can generate DNA single-strand breaks. DNA strand breaks then activate PARP, which in turn potentiates NF- PB activation and AP-1 expres- sion, resulting in greater expression of the AP-1 and NF- PB-dependent genes, such as the gene for ICAM-1, and chemokines such as MIP-1F and cytokines such as tumor necrosis factor-F. Chemokine generation, in combination with increased endothelial expression of ICAM-1, recruits more activated leukocytes to inflammatory foci, producing greater oxidant stress. It is possible that a low-level, localized inflammatory response may be beneficial in recruiting mononuclear cells to an inflammatory site. However, in many pathophysiological states the above described feedback cycles amplify themselves beyond control.

Overactivation of PARP represents an important mechanism of tissue damage in various pathological conditions associated with oxidative and nitrosative stress, includ- ing myocardial reperfusion injury (13,35), reperfusion injury after heart transplantation (36), chronic heart failure (37,38), stroke (39), circulatory shock (32,40–42), and the process of autoimmune G-cell destruction associated with DM (43,44). Activation of PARP and beneficial effect of various PARP inhibitors have been demonstrated in vari- ous forms of endothelial dysfunction such as the one associated with circulatory shock, hypertension, atherosclerosis, preeclampsia, and aging (40,45–48). Furthermore, recent evidence demonstrates that activation of PARP importantly contributes to the develop- ment of cardiac and endothelial dysfunction in various experimental models of diabetes and also in humans (49–52). The following chapter will discuss this subject in detail.

THE ROLE OF OXIDATIVE AND NITROSATIVE STRESS IN THE PATHOGENESIS OF DIABETES-INDUCED

CARDIOVASCULAR DYSFUNCTION

Oxidative and Nitrosative Stress in Diabetes-Induced Vascular Dysfunction

Various neurohumoral mediators and mechanical forces acting on the innermost layer

of blood vessels, the endothelium, are involved in the regulation of the vascular tone. The

main pathway of vasoregulation involves the activation of the constitutive, endothelial

isoform of NO synthase (eNOS) resulting in NO production (53). Endothelium-depen-

dent vasodilatation is frequently used as a reproducible and accessible parameter to probe

endothelial function in various pathophysiological conditions. It is well established that

endothelial dysfunction, in many diseases, precedes, predicts, and predisposes for the

subsequent, more severe vascular alterations. Endothelial dysfunction has been docu-

mented in various forms of diabetes, and even in prediabetic individuals (52,54–58). The

pathogenesis of this endothelial dysfunction involves many components including

increased polyol pathway flux, altered cellular redox state, increased formation of

diacylglycerol, and the subsequent activation of specific PKC isoforms, and accelerated

nonenzymatic formation of advanced glycation end-products (AGE) (59–61). Many of

these pathways, in concert, trigger the production of oxygen- and nitrogen-derived oxi-

dants and free radicals, such as superoxide anion and peroxynitrite, which play a signifi-

cant role in the pathogenesis of the diabetes-associated endothelial dysfunction (59,60,62).

Chapter 8 / PARP Activation 171

The cellular sources of reactive oxygen species (ROS) such as superoxide anion are mul- tiple and include AGEs, nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidases, the mitochondrial respiratory chain, xanthine oxidase, the arachidonic acid cas- cade (lipoxygenase and cycloxygenase), and microsomal enzymes (60,63).

Superoxide anion may quench NO, thereby reducing the efficacy of a potent endothe- lium-derived vasodilator system that participates in the homeostatic regulation of the vasculature, and evidence suggests that during hyperglycemia, reduced NO availability exists (64). Hyperglycemia-induced superoxide generation contributes to the increased expression of NAD(P)H oxidase, which in turn generate more superoxide anion. Hyper- glycemia also favors, through the activation of NF-PB an increased expression of iNOS, which may increase the generation of NO (65,66).

Superoxide anion interacts with NO, forming the strong oxidant peroxynitrite (ONOO-), which attacks various biomolecules, leading to—among other processes—the production of a modified amino acid, nitrotyrosine (67). Although nitrotyrosine was initially consid- ered a specific marker of peroxynitrite generation, other pathways can also induce tyrosine nitration. Thus, nitrotyrosine is now generally considered a collective index of reactive nitrogen species, rather than a specific indicator of peroxynitrite formation (68,69). The possibility that diabetes is associated with increased nitrosative stress is supported by the recent detection of increased nitrotyrosine plasma levels in type 2 diabetic patients (70) and iNOS-dependent peroxynitrite production in diabetic platelets (71). Nitrotyrosine formation is detected in the artery wall of monkeys during hypergly- cemia (72) and in diabetic patients during an increase of postprandial hyperglycemia (73). In a recent study we have demonstrated increased nitrotyrosine immunoreactivity in microvasculature of type 2 diabetic patients (52). In the same study significant corre- lations were observed between nitrotyrosine immunostaining intensity and fasting blood glucose, HbA1c, ICAM, and vascular cellular adhesion molecule (VCAM).

The toxic action of nitrotyrosine is supported by the evidence that increased apoptosis of endothelial cells, myocytes and fibroblasts in heart biopsies from diabetic patients (74), in hearts from streptozotocin (STZ)-induced diabetic rats (75), and in working hearts from rats during hyperglycemia (76), and the degree of cell death and/or dysfunc- tion show a correlation with levels of nitrotyrosine found in those cells. There is also evidence that nitrotyrosine can also be directly harmful to endothelial cells (77). Addi- tionally, high glucose-induced oxidative and nitrosative stress alters prostanoid profile in human endothelial cells (78,79). Recent studies have suggested that increased oxida- tive and nitrosative stress is involved in the pathogenesis of diabetic microvascular injury in retinopathy, nephropathy, and neuropathy (80–85).

Oxidative and Nitrosative Stress in Diabetic Cardiomyopathy

The development of myocardial dysfunction independent of coronary artery disease

in DM has been well documented, both in humans and experimental studies in animals

(86–90). Diabetic cardiomyopathy is characterized by complex changes in the mechani-

cal, biochemical, structural, and electrical properties of the heart, which may be respon-

sible for the development of an early diastolic dysfunction and increased incidence of

cardiac arrhythmias in diabetic patients. The mechanism of diastolic dysfunction remains

unknown but it does not appear to be as a result of changes in blood pressure, microvas-

cular complications, or elevated circulating glycated hemoglobin levels (86–90).

There is circumstantial clinical and experimental evidence suggesting that increased sympathetic activity, activated cardiac renin–angiotensin system, myocardial ischemia/

functional hypoxia, and elevated circulating levels of glucose result in oxidative and nitrosative stress in cardiovascular system of diabetic animals and humans. Oxidative stress associated with an impaired antioxidant defense status may play a critical role in subcellular remodeling, calcium-handling abnormalities, and subsequent diabetic cardi- omyopathy (75,89). Oxidative and nitrosative damage may be critical in the early onset of diabetic cardiomyopathy (74,75). Consistent with this idea, significant nitrotyrosine formation was reported in cardiac myocytes from myocardial biopsy samples obtained from diabetic and diabetic-hypertensive patients (74) and in a mouse model of streptozotocin (STZ)-induced diabetes (75). Perfusion of isolated hearts with high glu- cose caused a significant upregulation of iNOS, increased the coronary perfusion pres- sure and both NO and superoxide generation, a condition favoring the production of peroxynitrite, accompanied by the formation of nitrotyrosine and cardiac cell apoptosis (76).

Peroxynitrite Neutralization Improves Cardiovascular Dysfunction in Diabetes

As mentioned above there is circumstantial evidence that nitrosative stress and peroxynitrite formation importantly contribute to the pathogenesis of diabetic cardiomy- opathy both in animals and humans. We have tested a novel metalloporphyrin peroxynitrite decomposition catalyst, FP15, in murine models of diabetic cardiovascular complications (92). We hypothesized that neutralization of peroxynitrite with FP15 would ameliorate the development of cardiovascular dysfunction in a STZ-induced murine model of diabetes. To ensure that the animals received the FP15 treatment at a time when islet cell destruction was already complete and hyperglycemia has stabilized the treat- ment was initiated 6 weeks after the injection of STZ. Although FP15 did not affect blood glucose levels, it provided a marked protection against the loss of endothelium-depen- dent relaxant ability of the blood vessels (Fig. 1A) and improved the depression of both diastolic (Fig. 1B) and systolic function of the heart (92). The mechanism by which FP15 protects diabetic hearts from dysfunction may involve protection against vascular and myocardial tyrosine nitration, PARP activation, lipid peroxidation, and multiple other mechanisms, as all these mechanisms have previously been linked to diabetic cardiomy- opathy and to peroxynitrite-induced cardiac injury. Additional mechanisms of peroxynitrite-mediated diabetic cardiac dysfunction may include inhibition of myofibril- lar creatine kinase (93) and of succinyl-coenzyme A (CoA):3-oxoacid CoA-transferase (94) or activation of metalloproteinases (95).

There are many pathophysiological conditions of the heart that are associated with peroxynitrite formation, including acute MI, chronic ischemic heart failure, doxorubicin- induced and diabetic cardiomyopathy (93,94,96–98). It appears that peroxynitrite decom- position catalysts improve cardiac function and overall outcome in these models. For instance, FP15 reduced myocardial necrosis in our current rat model of acute MI (95) and in a recent porcine study (98). Furthermore, FP15 significantly improved cardiac func- tion in a doxorubicin-induced model of heart failure (95). These observations—coupled with the recently reported protective effect of FP15 against diabetic cardiomyopathy—

support the concept that peroxynitrite is a major mediator of myocardial injury in various

pathophysiological conditions, and its effective neutralization can be of significant thera-

peutic benefit.

Chapter 8 / PARP Activation 173

Fig. 1. (A) Reversal of diabetes-induced endothelial dysfunction by FP15 in vascular rings from STZ-diabetic mice. Acetylcholine (Ach) induced endothelium-dependent relaxation is impaired in rings from diabetic mice, which is markedly improved by FP15 treatment. Each point of the curve represents the mean ± SEM of five to seven pairs of experiments in vascular rings. *p < 0.05 in FP15-treated diabetic mice vs vehicle-treated diabetic mice. (B) Reversal of streptozotocin- evoked diabetes-induced diastolic cardiac dysfunction by FP15 in mice. Effect of diabetes (9–10 weeks) and FP15 treatment in diabetic mice on left ventricular end diastolic pressure (LVEDP) and left ventricular –dp/dt (LV –dp/dt). Results are mean ± SEM of seven experiments in each group.

*p < 0.05 diabetic animals vs control; #p < 0.05 in FP15-treated diabetic mice vs vehicle-treated diabetic mice. (Reproduced with permission from ref. 92.)

THE ROLE OF POLY(ADP-RIBOSE) POLYMERASE ACTIVATION IN THE PATHOGENESIS OF DIABETIC CARDIOVASCULAR

DYSFUNCTION

The Role of Poly(ADP-Ribose) Polymerase Activation in Diabetic Vascular Dysfunction

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We have recently found that high glucose-induced oxidative and nitrosative stress leads to DNA single-strand breakage and PARP activation in murine and human endot- helial cells (49) (Fig. 2). The involvement of oxyradicals and NO-derived reactive spe- cies in PARP activation and the evidence for nitrated tyrosine residues both suggested that peroxynitrite may be one of the final mediators responsible for single-strand break- age, and subsequent PARP activation (49). The role of hyperglycemia-induced oxidative stress in producing DNA damage is supported by the recent findings that increased amounts of 8-hydroxyguanine and 8-hydroxydeoxy guanosine (markers of oxidative damage to DNA) can be found in both the plasma and tissues of streptozotocin diabetic rats (99). Importantly, various forms of oxidant-induced DNA damage (base modifications and DNA strand breaks) have also been demonstrated in diabetic patients (100–104).

Pharmacological inhibition of PARP or genetic inactivation of PARP-1 protects against the development of the high glucose-induced endothelial dysfunction in vitro (49) (Fig. 3) by preventing glucose-induced severe suppression of cellular high-energy phosphate levels and by inhibiting the hyperglycemia-induced suppression of NAD+ and NADPH levels. Because eNOS is an NADPH-dependent enzyme, we proposed that the cellular depletion of NADPH in endothelial cells exposed to high glucose is directly responsible for the suppression of eNOS activity and the reduction in the diabetic vessels’

endothelium-dependent relaxant ability. In support of this hypothesis, we have subse- quently demonstrated that there is a PARP-dependent suppression of vascular NADPH levels in diabetic blood vessels in vivo (50).

Although most of the studies on the role of PARP in the pathogenesis of diabetic

endothelial dysfunction, as discussed above, originated in macrovessels, there is circum-

stantial evidence that similar processes are operative for pathogenesis of diabetic mi-

crovascular injury (retinopathy, nephropathy). In fact, there is now evidence for PARP

activation in the microvessels of the diabetic retina (105). Additionally, a study per-

formed more than a decade ago demonstrated that the presence of glomerular depositions

(mesangial distribution) of IgG was significantly reduced in STZ-diabetic rats treated

with the PARP inhibitor nicotinamide for 6 months (106). In agreement with these

results, we have recently provided evidence that PARP activation is present in the tubuli

of STZ-induced diabetic rats. This PARP activation is attenuated by two unrelated PARP

inhibitors, 3-aminobenzamide and 1,5-isoquinolinediol (ISO), which also counteract the

overexpression of endothelin-1 and endothelin receptors in the renal cortex (107). It has

recently been suggested that PARP activation may also play a key role in the development

diabetic neuropathy: the progressive slowing of sensory and motor neuron conductance

in diabetic rats and mice is preventable by PARP inhibition or PARP deficiency, and this

is associated with maintained neuronal phosphocreatine levels, and improved endoneurial

blood flow (108). Additional studies, utilizing potent and specific inhibitors of PARP are

needed to further delineate the role of PARP in the pathogenesis of diabetic retinopathy,

neuropathy, and nephropathy. It is important to emphasize that, although the above

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Fig. 2. Reactive nitrogen species generation, ssDNA breakage and poly(ADP-ribose) polymerase (PARP) activation in diabetic blood vessels. (A–C) Immunohistochemical staining for nitro- tyrosine in control rings (A), in rings from diabetic mice treated with vehicle at 8 weeks (B), and in rings from diabetic mice treated with PJ34 (C), (D–F) Terminal deoxyribonucleotidyl transferase- mediated dUTP nick-end labeling, an indicator of DNA strand breakage, in control rings (D), in rings from diabetic mice treated with vehicle at 8 weeks (E), and in rings from diabetic mice treated with PJ34 (F), (G–I) Immunohistochemical staining for poly(ADP-ribose), an indicator of PARP activation, in control rings (G), in rings from diabetic mice treated with vehicle at 8 weeks (H), and in rings from diabetic mice treated with PJ34 (I). (Reproduced with permission from ref. 49.)

conditions are generally considered as separate pathophysiological entities, there is good evidence that, at least in part, they all develop on the basis of vascular (endothelial) dysfunction.

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In a mouse model of STZ-induced diabetes the time course of endothelial dysfunction was compared with that of the activation of PARP in the blood vessels. Intravascular PARP activation (seen in endothelial cells, and in vascular smooth muscle cells) was already apparent 2 weeks after the onset of diabetes and thus it slightly preceded the occurrence of the endothelial dysfunction, which developed between the second and the fourth weeks of diabetes (49; Fig. 3). Delayed treatment with the PARP inhibitor—

starting at 1 week after STZ—ameliorated vascular poly(ADP-ribose) accumulation and

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restored normal vascular function without altering systemic glucose levels, plasma- glycated hemoglobin levels, or pancreatic insulin content (49,50). Furthermore, delayed treatment of the animals with the PARP inhibitor restored the established diabetic endot- helial dysfunction (Fig. 4), and even in vitro incubation of diabetic dysfunctional blood vessels with PARP inhibitors of various structural classes (e.g., benzamide-, isoquinoline-, and phenanthridinone-derivatives) significantly enhanced the endothelium-dependent relaxant responsiveness (49,50) (Fig. 5A,B). The development of the endothelial dys- function and its reversibility by pharmacological inhibition of PARP has recently also been demonstrated in an autoimmune model of diabetes, in the nonobese diabetic model in the mouse (51) (Fig. 6). The mode of PARP inhibitors protective action on endothelium likely involves a conservation of energetics, and a prevention of the upregulation of various pro-inflammatory pathways (cytokines, adhesion molecules (ICAM-1, VCAM- 1 and E-selectin), mononuclear cell infiltration) triggered by hyperglycemia (49,109,110).

This latter mechanism may represent an important additional pathway whereby PARP activation can contribute to vascular dysfunction via the upregulation of adhesion mol- ecules. As mentioned earlier, PARP regulates the activation of a variety of signal trans- duction pathways, and some of these pathways regulate the expression of cell surface and soluble adhesion molecules. Recent data indicate that pharmacological inhibition of PARP can suppress this process (110). Intermittent high/low glucose induces a more pronounced expression of adhesion molecules that constant high glucose, and PARP inhibition suppresses NF-PB activation and the expression of adhesion molecules both under constant high glucose and under intermittent high/low glucose conditions in cul- tured endothelial cells in vitro (110).

A recent in vitro study have demonstrated that the pharmacological inhibition of PARP completely blocks hyperglycemia-induced activation of multiple major pathways of vascular damage (111). In cultured endothelial cells placed in high extracellular glucose, the development of DNA single-strand breaks, the activation of PARP and the depletion of intracellular NAD were all blocked by the cellular overexpression of uncoupling protein-1 (UCP-1), indicating that mitochondrial oxidant generation plays a key role in the activation of PARP in endothelial cells placed into high glucose. Incubation of bovine aortic endothelial cells in elevated glucose increased the membrane fraction of intracel- lular PKC activity, an effect which was also blocked by overexpression of UCP-1, consis-

Fig. 3. (opposite page) Reversal of diabetes-induced endothelial dysfunction by pharmacological inhibition of poly(ADP-ribose) polymerase (PARP). Symbols used for the respective groups:

animals which received no streptozotocin injection (䉭), nondiabetic control animals at 8 weeks treated with PJ34 between week 1 and 8 (䉱), diabetic animals at 8 weeks treated with vehicle (䊊), diabetic animals at 8 weeks treated with PJ34 between weeks 1 and 8 (䊉). (A) Blood glucose levels, pancreatic insulin content (ng insulin/mg pancreatic protein) and blood glycosylated he- moglobin (Hb) (expressed as % of total Hb) at 0–8 weeks in nondiabetic, control male BALB/c mice, and at 0–8 weeks after streptozotocin treatment (diabetic) in male BALB/c mice. PARP inhibitor treatment, starting at 1 week after streptozotocin and continuing until the end of week 8, is indicated by the arrow. Pancreatic insulin and Glycated hemoglobin levels content are shown at 8 weeks in vehicle-treated and streptozotocin-treated animals, in the presence or absence of PJ34 treatment. (B) acetylcholine-induced, endothelium-dependent relaxations, phenylephrine-induced contractions, and sodium nitroprusside-induced endothelium-independent relaxations.* p < 0.05 for vehicle-treated diabetic vs PJ34-treated diabetic mice (n = 8 per group). (Reproduced with permission from ref. 49.)

Fig. 4. Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) restores impaired endothelium-dependent relaxant ability of the diabetic vessels. Blood glucose levels and vascular responsiveness. Endothelium-dependent relaxations induced by acetylcholine, contractions in- duced by phenylephrine, and endothelium-independent relaxations induced by sodium nitroprus- side (SNP) in control (nondiabetic) male Balb/c mice and 1, 4, and 8 weeks after streptozotocin (STZ)-induced diabetes. Vehicle or PARP inhibitor (PJ34, 10 mg/kg oral gavage once a day) treatment started at 4 weeks after STZ and continued until 8 weeks (the end of the experimental period). There was a marked and selective impairment of the endothelium-dependent relaxant ability of the vascular rings in diabetes at 4 and 8 weeks. Treatment with the PARP inhibitor between weeks 4 and 8 restored to normal the endothelium-dependent relaxant ability of the diabetic vessels despite the persistence of hyperglycemia. *p < 0.05 for differences between experimental groups, as indicated. n = 8 per group. (Reproduced with permission from ref. 50.)

Chapter 8 / PARP Activation 179

Fig. 5. In vitro treatment with all poly(ADP-ribose) polymerase (PARP) inhibitors improved the endothelium-dependent relaxant ability of the diabetic vessels. (A) Endothelium-dependent relax- ations induced by acetylcholine in control (nondiabetic) male Balb/c mice and 4 weeks after streptozotocin (STZ)-induced diabetes. In a subgroup of the vascular rings, evaluation of vascular responsiveness was preceded by 1-hour incubation with three structurally different PARP inhibi- tors: 3-aminobenzamide (3 mmol/L), 5-iodo-6-amino-1,2-benzopyrone (100 μmol/L), or 1,5- dihydroxyisoquinoline (30 μmol/L). There was a marked and selective impairment of the endothelium-dependent relaxant ability of the vascular rings in diabetes at 4 weeks. In vitro treatment with all PARP inhibitors improved the endothelium-dependent relaxant ability of the diabetic vessels. *p < 0.05 for differences between experimental groups, as indicated. n = 8 per group. (Reproduced with permission from ref. 50.) (B) Endothelium-dependent relaxations in- duced by acetylcholine in control (nondiabetic) male Balb/c mice and 6 weeks after STZ-induced diabetes. In a subgroup of the vascular rings, evaluation of vascular responsiveness was preceded by 1-hour incubation with the novel potent PARP inhibitor, INO1001 (3 μmol/L) (112,113). There was a marked and selective impairment of the endothelium-dependent relaxant ability of the vascular rings in diabetes at 6 weeks. In vitro treatment with all PARP inhibitors improved the endothelium-dependent relaxant ability of the diabetic vessels. *p < 0.05 for differences between experimental groups, as indicated. n = 8 per group.

Fig. 6. Reversal of diabetes-induced endothelial dysfunction by pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) in diabetic NOD mouse vascular rings. Epinephrine- induced contractions (upper panel), acetylcholine-induced endothelium-dependent relaxation (middle panel), and sodium nitroprusside-induced endothelium-independent relaxations (lower panel). Control (䊏); control + PJ34 (䊊); diabetes (䊐); diabetes + PJ34 (䊉). Each point of the curve represents the means ± SE of 5–8 experiments in vascular rings. *p < 0.05 vs C; #p < 0.05 vs D.

(Reproduced with permission from ref. 51.)

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tently with an oxidant-mediated basis of PKC activation in endothelial cells subjected to hyperglycemia. Inhibition of PARP by PJ34 also completely prevented the activation of PKC by 30 mM glucose, indicative that the oxidant-mediated suppression of PKC activ- ity in hyperglycemia involves the activation of PARP. Another key factor in the patho- genesis of hyperglycemia induced endothelial dysfunction is the inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Recent studies demon- strated that this decrease in GAPDH activity is also dependent on PARP, and may be related to direct poly(ADP-ribosyl)ation, and consequent inhibition of GAPDH (111).

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A recent study extended our knowledge on the role of PARP activation in the devel- opment of diabetic endothelial dysfunction into the area of human investigations: in forearm skin biopsies from healthy subjects, healthy individuals with parental history of type 2 diabetes mellitus (T2DM), subjects with impaired glucose tolerance and a group of T2DM patients it was found that the percentage of PARP-positive endothelial nuclei was higher in the group of parental history of T2DM and diabetic patients when compared to the controls (52) (Fig. 7). Additionally, significant correlations were observed between the percentage of PARP-positive endothelial nuclei and fasting blood glucose, resting skin blood flow, maximal skin vasodilatory response to the iontopheresis of acetylcho- line (which indicates endothelium-dependent vasodilation), and sodium nitroprusside (which indicates endothelium-independent vasodilation) and nitrotyrosine immunostaining intensity (52). Nitrotyrosine immunoreactivity (a marker of reactive nitrogen species and peroxynitrite formation) was also higher in the diabetic patients when compared to all other groups (52). Significant correlations were observed between nitrotyrosine immunostaining intensity and fasting blood glucose, HbA1c, ICAM, and VCAM. No differences in the expression of eNOS and receptor of advanced glycation end-products were found among all four groups. The polymorphism of the eNOS gene was also studied and was not found to influence eNOS expression or microvascular functional measure- ments. Thus, in humans, PARP activation is present in healthy subjects at risk of devel- oping diabetes, and in established T2DM patients and it is associated with impairments in the vascular reactivity in the skin microcirculation (52). It remains to be seen whether PARP activation in diabetic or prediabetic humans can be seen as a predictor or early marker for the development of diabetic vascular complications. It also remains to be studied whether various therapeutic interventions, which are known to have vascular protective effects in diabetes (antioxidant therapies, PPAR agonists, etc.), are able to suppress the activation of PARP in the cardiovascular system.

The Role of Poly(ADP-Ribose) Polymerase Activation in the Pathogenesis of Diabetic Cardiomyopathy

P

(ADP-R

) P

A

D

M

The importance of the PARP pathway is well documented in various models of myo-

cardial ischemia-reperfusion injury (a condition in which oxidative and nitrosative stress

plays a key pathogenetic role) (13,35,36). The PARP pathway also plays a role in the

pathogenesis of diabetic cardiomyopathy (51) (Fig. 8). Cardiac dysfunction and PARP

activation in the cardiac myocytes and coronary vasculature were noted both in the STZ-

induced and genetic (nonobese diabetic) models of diabetes mellitus in rats and mice (51)

(Figs. 8 and 9).

Fig. 7. Evidence for poly(ADP-ribose) polymerase (PARP) activation in diabetic skin vessels.

Immunohistochemical staining for PARP formation, an indicator of PARP activation, in skin vessels from healthy subjects (A) and diabetic patients (B). PARP formation is localized in the nuclei of cells (shown in dark). Magnification ×400. (Reproduced with permission from ref. 52.)

P

I

P

(ADP-R

) P

I

D

-I

C

D

Treatment with the phenanthridinone-based PARP inhibitor PJ34, starting 1 week

after the onset of diabetes, restored normal vascular responsiveness and significantly

improved cardiac function in diabetic mice and rats, despite the persistence of severe

hyperglycemia (51) (Fig. 9A,B). The beneficial effect of PARP inhibition persisted even

after several weeks of discontinuation of the PARP inhibitor treatment (51). It is possible

that the diabetic endothelial PARP pathway and the diabetic cardiomyopathy are inter-

related: the impairment of the endothelial function may lead to global or regional myo-

cardial ischemia, which may secondarily impair cardiac performance. The beneficial

Chapter 8 / PARP Activation 183

Fig. 8. Evidence for poly(ADP-ribose) polymerase (PARP) activation in diabetic rat hearts. Im- munohistochemical staining for poly(ADP-ribose) formation, an indicator of PARP activation, in control (A), diabetic (B,D), and PJ34-treated diabetic (C,E) rat hearts. Panels B and D show poly(ADP-ribose) formation localized in the nuclei of myocytes in 5- and 10-week diabetic rat hearts, respectively. The evidence of poly(ADP-ribose) accumulation can be seen as dark, fre- quent, and widespread nuclear staining in panels B and D. Treatment with PJ34 for 4 weeks (C) markedly reduced PARP activation in diabetic (5-week) hearts. Notably, the PARP activation in diabetic hearts (10 weeks) was attenuated even after the discontinuation of the treatment with PJ34 (after 6 weeks) for an additional 3-week period (E). Similar immunohistochemical profiles were seen in n = 4–5 hearts per group. (Reproduced with permission from ref. 51.)

effect of PARP inhibition on myocardial function, however, is not related to an anabolic

effect because PJ34 treatment did not influence the body and heart weight loss in diabetic

animals, although it dramatically improved cardiac function. It is noteworthy that the

protective effect of PARP inhibition against diabetic cardiac dysfunction extended sev-

eral weeks beyond the discontinuation of treatment; this observation may have important

implications for the design of future clinical trials with PARP inhibitors. The prolonged

protective effect may be related to the permanent interruption by the PARP inhibitor of

positive feedback cycles of cardiac injury. Indeed, previous studies in various patho-

physiological conditions have demonstrated that PARP inhibitors suppress positive feed-

back cycles of adhesion receptor expression and mononuclear cell infiltration, and cellular

oxidant generation (13,36,110). The mode of PARP inhibitors’ cardioprotective action

involves a conservation of myocardial energetics, and a prevention of the upregulation

of various proinflammatory pathways (cytokines, adhesion receptors, mononuclear cell

infiltration) triggered by ischemia and reperfusion (13,36). It is conceivable that PARP

inhibition exerts beneficial effects in experimental models of diabetic cardiomyopathy

by affecting both above referenced pathways of injury, and also by suppressing positive

feedback cycles initiated by them. Based on the results of the current study, we conclude

that the ROS/reactive nitrogen species–DNA injury–PARP activation pathway plays a

pathogenetic role in the development of diabetic cardiomyopathy.

184 Pacher and Szabó

Reversal of diabetes-induced cardiac dysfunction by pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) in an autoimmuneic pressure (LVEDP), left; C + PJ34, control treatedp< 0.05 vs C; #p< 0.05 vs D (Reproduced with permission51.)(B)Reversal of streptozotocin-evoked diabetes induced cardiac dysfunction by pharmacological inhibition of PARP in rats. Effect of diabetesrats. C, control; D, diabeticfter 1 week of establishedp < 0.05 vs C; #p < 0.05 vs D. (Reproduced with permission from ref. 51.)

Chapter 8 / PARP Activation 185

Fig. 10. Overview of the role of poly(ADP-ribose) polymerase (PARP) in regulating multiple component of hyperglycemia-induced endothelial dysfunction. High circulating glucose interacts with the vascular endothelium in which it triggers the release of oxidant mediators from the mitochondrial electron transport chain, and from NADH/NADPH oxidase and other sources.

Nitric oxide (NO), in turn, combines with superoxide to yield peroxynitrite. Hydroxyl radical (produced from superoxide via the iron-catalyzed Haber-Weiss reaction) and peroxynitrite or peroxynitrous acid induce the development of DNA single-strand breakage, with consequent activation of PARP. Depletion of the cellular NAD+ leads to inhibition of cellular ATP-generating pathways leading to cellular dysfunction. The PARP-triggered depletion of cellular NADPH directly impairs the endothelium-dependent relaxations. The effects of elevated glucose are also exacerbated by increased aldose reductase activity leading to depletion of NADPH and generation of reactive oxidants. NO alone does not induce DNA single strand breakage, but may combine with superoxide (produced from the mitochondrial chain or from other cellular sources) to yield peroxynitrite. Under conditions of low-cellular L-arginine NOS may produce both superoxide and NO, which then can combine to form peroxynitrite. PARP activation, via a not yet characterized fashion, can promote the activation of nuclear factor kB, AP-1, mitogen-activated protein kinases, and the expression of proinflammatory mediators, adhesion molecules, and of iNOS. PARP ac- tivation contributes to the activation of protein kinase C. PARP activation also leads to the inhibition of cellular GAPDH activity, at least in part via the direct poly(ADP-ribosyl)ation of GAPDH. PARP-independent, parallel pathways of cellular metabolic inhibition can be activated by NO, hydroxyl radical, superoxide, and by peroxynitrite.

CONCLUSIONS AND IMPLICATIONS

The role of PARP activation in diabetes is not limited to the development of various forms of cardiovascular dysfunction. A vast body of evidence supports the role of nitrosative stress and PARP activation in the process of autoimmune islet cell death and in the process of islet regeneration (1). In addition, PARP inhibitors exert beneficial effects in rodent models of diabetic neuropathy and retinopathy (105,108). Taken together, multiple lines of evidence support the view that nitrosative stress and PARP activation play a crucial role in multiple interrelated aspects of the pathogenesis of diabetes and in the development of its complications (Fig. 10). PARP inhibition may emerge as a novel approach for the experimental therapy of diabetes, and for the preven- tion or reversal of its complications.

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