Abstract Peroxynitrite and hydroxyl radicals are potentinitiators of DNA single-strand breakage, which is anobligatory stimulus for the activation of the nuclear en-zyme poly(ADP ribose) polymerase (PARP). In responseto high glucose incubation medium in vitro, or diabetesand hyperglycemia in vivo, reactive nitrogen and oxygenspecies generation occurs. These reactive species triggerDNA single-strand breakage, which induces rapid acti-vation of PARP. PARP in turn depletes the intracellularconcentration of its substrate, NAD+, slowing the rate ofglycolysis, electron transport, and ATP formation. Thisprocess results in acute endothelial dysfunction in dia-betic blood vessels. Accordingly, inhibitors of PARPprotect against endothelial injury under these conditions.In addition to the direct cytotoxic pathway regulated byDNA injury and PARP activation, PARP also appears tomodulate the course of inflammation by regulating theactivation of nuclear factor κB, and the expression of anumber of genes, including the gene for intercellular ad-hesion molecule 1 and the inducible nitric oxide syn-thase. The research into the role of PARP in diabetic vas-cular injury is now supported by novel tools, such asnew classes of potent inhibitors of PARP and geneticallyengineered animals lacking the gene for PARP. Pharma-cological inhibition of PARP emerges as a potential ap-proach for the experimental therapy of diabetic vasculardysfunction.

Keywords Free radicals · Oxidants · Nitric oxide · Diabetes vascular · Cytokine

F.G. Soriano · L. Virág · C. Szabó (✉ )Inotek Corporation, Suite 419E, 100 Cummings Center, Beverly,MA 01915, USAe-mail: szabocsaba@aol.comTel.: +1-978-2329660Fax: +1-978-2328975

F.G. Soriano · C. SzabóDepartment of Surgery, New Jersey Medical School, UMDNJ,Newark, NJ 01703, USA

L. VirágDepartment of Medicinal Chemistry, University of Debrecen,Debrecen, Hungary

J Mol Med (2001) 79:437–448DOI 10.1007/s001090100236

R E V I E W

Francisco Garcia Soriano · László Virág · Csaba Szabó

Diabetic endothelial dysfunction: role of reactive oxygen and nitrogenspecies production and poly(ADP-ribose) polymerase activation

Received: 14 February 2001 / Accepted: 11 April 2001 / Published online: 8 June 2001© Springer-Verlag 2001

FRANCISCO GARCIA SORIANOreceived his M.D. degree fromthe University of Sao Paulo,Brazil, where he remains as apracticing critical care physi-cian. He is currently taking hisFAPESP (Fundação de Am-paro à Pesquisa do Estado deSão Paulo) supported researchfellowship in the Departmentof Surgery, University of Med-icine and Dentistry, N.J., USA.His research interests lay inthe pathophysiological role ofpoly(ADP-ribose) polymeraseand reactive nitrogen speciesin various forms of vascularand organ injury.

CSABA SZABOreceived his M.D. degree atthe Semmelweis UniversityMedical School in Hungary,Ph.D. in physiology and D.S.degree at the Hungarian Acad-emy of Sciences, and Ph.D.degree in pharmacology fromthe University of London. Hehas worked at the WilliamHarvey Research Institute inLondon and the Division ofCritical Care at the Children’sHospital Medical Center inCincinnati, Ohio, USA. He iscurrently Vice President forResearch at Inotek Corp. andholds an adjunct full profes-sorship in surgery at the Uni-versity of Medicine and Den-tistry of New Jersey. His re-search interests are the patho-physiological roles of nitricoxide and poly(ADP-ribose)polymerase activation in vari-ous forms of shock, inflamma-tion, vascular dysfunction, andreperfusion injury.

Abbreviations AGE: Advanced glycation endproduct ·AP: Activator protein · eNOS: Endothelial nitric oxidesynthase · ICAM: Intercellular adhesion molecule ·iNOS: Inducible nitric oxide synthase · MAP: Mitogen-activated protein · NF: Nuclear factor ·NOS: Nitric oxide synthase · PARP: Poly(ADP ribose)polymerase

Free radical and oxidant mediators of the diabetes-associated vascular dysfunction

Diabetes mellitus, even in its most balanced forms, is as-sociated with episodes of high glucose concentration inthe plasma. These hyperglycemic episodes are closelyassociated with the development of vascular failure (evi-denced clinically, among other things, by acceleratedatherosclerosis, dysfunction of the eye, kidney, dimin-ished blood flow to extremities, and increased risk of de-veloping a variety of cardiovascular diseases). It is gen-erally believed that better control of the glucose levelswill lead to a lower incidence of the vascular complica-tions. Nevertheless, it is a fact of life that a significantproportion of diabetic patients will eventually developsome degree of vascular failure (characterized, for exam-ple, by endothelial dysfunction). While microvasculardisease has been strongly related to glycemic control, themajor cause of mortality in diabetes is macrovasculardisease affecting the cardiac and cerebrovascular circula-tion, which appears to have a more complex pathogene-sis. Diabetes is associated with a three- to fivefold in-crease in death from myocardial infarction and similarfigures pertain to stroke. The processes involved inatherothrombotic disease are complex and include varia-tion in lipid metabolism, vascular responses, cell/cell in-teractions, and the fluid and cellular phases of coagula-tion and fibrinolysis. The complex interactions betweenthese processes are crucially altered by the metabolic mi-lieu that characterizes diabetes mellitus, tipping the deli-cate balance towards atheroma formation, platelet aggre-gation and thrombus formation [1, 2, 3, 4, 5].

Endothelial cells actively regulate basal vascular toneand vascular reactivity in physiological and pathologicalconditions by responding to mechanical forces and neu-rohumoral mediators. The main endothelium-derived re-laxing factor has been identified as nitric oxide [6, 7].The activity of the endothelium extends far beyond thecontrol of vascular tone and reactivity; and the release ofNO and other vasodilating mediators clearly reflects on-ly one aspect of the homeostatic and protective role ofthe endothelium. Nevertheless, endothelium-dependentvasodilation is frequently used as a reproducible and ac-cessible parameter to probe endothelial function in vari-ous pathophysiological conditions [6, 7, 8]. Investiga-tions into the mechanism of diabetes-associated vascularfailure and experimental therapeutics aimed at their cor-rection are of great importance. Endothelial damage iswell recognized in diabetes: endothelial cell markers von Willebrand factor, soluble E-selectin, and soluble

thrombomodulin provide further evidence of the rela-tionship between activation and damage to the vascula-ture and clinical disease in this condition. Cell surfacebound adhesion molecules may also have a role in thedevelopment of atherosclerosis in patients with diabetesbut the importance of the soluble forms of these mole-cules, such as intercellular adhesion molecule (ICAM) 1is unclear [9, 10].

Recent studies have established that oxygen- and ni-trogen-derived oxidants and free radicals play a signifi-cant role in diabetes-associated endothelial dysfunction[11, 12, 13, 14]. Hyperglycemia-induced mechanismsthat may induce vascular dysfunction in specific sites ofdiabetic microvascular damage include increased polyolpathway flux, altered cellular redox state, increased for-mation of diacylglycerol and the subsequent activationof specific protein kinase C isoforms, and acceleratednonenzymatic formation of advanced glycation endpro-ducts (AGEs). Several of these mechanisms may be re-sponsible for the potentially damaging overproduction ofreactive oxygen species observed with hyperglycemia.Each of these mechanisms may contribute to the knownpathophysiological features of diabetic complications bya number of mechanisms, including the upregulation ofcytokines and growth factors [9, 10].

Oxyradicals

With respect to oxygen-derived free radicals and oxi-dants, strong evidence has accumulated that the genera-tion of reactive oxygen species (oxidative stress) playsan important role in the etiology of diabetic complica-tions [11, 12]. This hypothesis is supported by evidencethat many biochemical pathways strictly associated withhyperglycemia (glucose auto-oxidation, polyol pathway,prostanoid synthesis, protein glycation) can increase theproduction of free radicals. Furthermore, exposure of en-dothelial cells to high glucose leads to augmented pro-duction of superoxide anion, which may quench nitricoxide, thereby reducing the efficacy of a potent endothe-lium-derived vasodilator system that participates in thegeneral homeostasis of the vasculature. Further, the reac-tion of superoxide and NO not only diminishes the activ-ity of a beneficial vasodilatory pathway but also gener-ates a toxic oxidant species, peroxynitrite (see below) [1,2, 3, 4, 5]. In further support of the consequential injuri-ous role of oxidative stress, many of the adverse effectsof high glucose on endothelial functions, such as reducedendothelial-dependent relaxation and delayed cell repli-cation, are reversed by antioxidants [15, 16]. A rationalextension of this proposed role for oxidative stress is thesuggestion that the different susceptibility of diabetic pa-tients to microvascular and macrovascular complicationsis a function of the endogenous antioxidant status [11].Much attention has been directed towards AGEs formedby the interaction of aldoses with proteins and the subse-quent molecular rearrangements of the covalently linkedsugars, eventuating in a diverse group of fluorescent

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compounds of yellow-brown color. This heterogeneousclass of nonenzymatically glycated proteins or lipids isfound in the plasma. As a consequence of hyperglycemiaAGE formation and deposition are much enhanced in di-abetes, in which their presence has been linked to sec-ondary complications, especially microvascular disease.In the AGE-related dysfunction a central role is playedby a novel receptor for AGE. This receptor mediates thebinding of AGEs to endothelial cells and mononuclearphagocytes, interacts with a lactoferrinlike polypeptide

that also binds AGEs, and appears to activate intracellu-lar signal transduction mechanisms consequent to its in-teraction with the glycated ligand. Since AGEs havebeen shown to generate reactive oxygen intermediates,tethering of AGEs to the cell surface by their receptorsfocuses oxidant stress on cellular targets resulting inchanges in gene expression and the cellular phenotype[8, 9, 10, 11]. There is also increasing evidence for su-peroxide generation by NADH/NADPH oxidases for thedevelopment of vascular (particularly endothelial) dys-function [12]. The effects of elevated glucose are exacer-bated by increased aldose reductase activity leading todepletion of NADPH and generation of reactive oxi-dants. NADPH depletion, due to aldose reductase [andalso due to activation of poly(ADP ribose) polymerase(PARP); see below], directly impairs the endothelium-dependent relaxations (Fig. 1).

Nitric oxide

Recent studies have also implicated the production of ni-trogen-derived free radicals and oxidants in the processof diabetes-associated endothelial dysfunction [13, 14].There is substantial evidence that many forms of vascu-lar dysfunction are associated with profound alterationsin the biosynthesis of the toxic free radical NO from L-arginine. NO is synthesized from the guanidino groupof L-arginine by a family of enzymes termed NO synth-ases (NOS). Three isoforms have been described andcloned: endothelial cell NOS (eNOS), brain NOS, alsoknown as neural NOS (bNOS; nNOS), and an induciblemacrophage type NOS (iNOS). The formation of NO islinked to the incorporation of molecular oxygen into themolecule. All forms of NOS contain four prostheticgroups: flavin-adenine dinucleotide, flavin mononucle-otide, tetrahydrobiopterin, and a heme complex, ironprotoporphyrin IX. NOS has been proposed to form NOand L-citrulline (for reviews see [6, 7, 17, 18]). The NOproduced from eNOS (present in the endothelial cells) isresponsible for many physiological effects, such as themaintenance of a basal vascular tone. The function ofeNOS can be evidenced by endothelium-dependent re-laxant responses (i.e., the ability of isolated blood ves-sels to relax in response to vasoactive substances that in-duce the activation of eNOS and the release of NO fromthe vascular endothelium). Such vasoactive mediatorsare acetylcholine, bradykinin, and ATP, for instance. Onthe other hand, vascular studies utilizing direct NO gen-erators (such as the nitrovasodilators, SNAP or sodiumnitroprusside) can evaluate the ability of the vascularsmooth muscle to react to nitric oxide. There is an over-whelming evidence demonstrating the development ofendothelial dysfunction (the reduced ability of bloodvessels to relax in response to endothelium-dependentrelaxants) in animal models of diabetes [19, 20, 21, 22,23] as well as in human blood vessels from diabetic pa-tients [24]. Paradoxically, the expression of eNOS and itsmRNA appears to be increased in endothelial cells incu-

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Fig. 1 Proposed scheme of PARP-dependent and PARP-indepen-dent cytotoxic pathways involving nitric oxide (NO·), hydroxylradical (OH·) and peroxynitrite (ONOO–) in diabetic endothelialdysfunction. High glucose triggers the release of oxidant media-tors from the mitochondrial electron transport chain, fromNADH/NADPH oxidase and other sources. High glucose may alsoupregulate eNOS expression (eNOS) or trigger the expression ofthe inducible NO synthase (iNOS). NO in turn combines with su-peroxide to yield peroxynitrite. Hydroxyl radical (produced fromsuperoxide via the iron-catalyzed Haber-Weiss reaction) and per-oxynitrite or peroxynitrous acid induce the development of DNAsingle strand breakage, with consequent activation of PARP. De-pletion of the cellular NAD+ leads to inhibition of cellular ATP-generating pathways leading to cellular dysfunction. The PARP-mediated depletion of cellular NADPH directly impairs the endo-thelium-dependent relaxations. The effects of elevated glucose arealso exacerbated by increased aldose reductase activity leading todepletion of NADPH and generation of reactive oxidants. NOalone does not induce DNA single-strand breakage, but may com-bine with superoxide (produced from the mitochondrial chain orfrom other cellular sources) to yield peroxynitrite. Under condi-tions of low cellular L-arginine NOS may produce both superoxideand NO, which then can combine to form peroxynitrite. PARP ac-tivation, via a not yet characterized fashion, promotes the activa-tion of nuclear factor κB, AP-1, MAP kinases, and the expressionof proinflammatory mediators, adhesion molecules, and iNOS.PARP-independent, parallel pathways of cellular metabolic inhibi-tion can be activated by NO, hydroxyl radical, superoxide, andperoxynitrite. By promoting neutrophil recruitment and oxidantgeneration a positive feedback cycle is triggered. PARP activationand mitochondrial oxidant production form another positive feed-back cycle. Circled crosses Positive feedback circles

bated in high glucose [25]. However, in the same workauthors have also reported a threefold elevation in super-oxide production which is probably more relevant in thepathomechanism of hyperglycemia-induced endothelialdysfunction.

Peroxynitrite

The production of superoxide (by activated neutrophils,xanthine oxidase, NADPH oxidases, and other sources)has been previously recognized as an important cyto-toxic factor contributing to vascular damage in variouspathophysiological conditions. From a chemical point ofview, neither NO nor the oxygen radical superoxide alonecan be considered as a strong oxidant towards most typesof biomolecules. Recent evidence suggests that the reac-tion of superoxide with NO yields a toxic oxidant, per-oxynitrite, which plays a central role in the pathophysiol-ogy of inflammation and oxidant stress [26, 27, 28]. Asan important participant in a final common pathway in in-flammatory diabetes, peroxynitrite unites the two previ-ously described cytotoxic pathways: (a) the NO pathwayand (b) the oxyradicals pathway. Importantly, under cer-tain conditions, such as cellular L-arginine depletion,NOS can produce both superoxide and NO, and the re-sulting production of peroxynitrite can exert marked au-tocrine toxicities [29, 30]. Peroxynitrite enhances the cy-totoxic potential of its “precursors.” Much of the NO-related toxicity is in fact due to peroxynitrite formation[26, 28]. A characteristic reaction of peroxynitrite is thenitration of protein tyrosine residues. Antibodies againstnitrated tyrosine can be used to detect a “footprint” ofperoxynitrite [31, 32]. Recent clinical data demonstrateda significantly more intense nitrotyrosine staining in vas-cular endothelium and villous stroma of placentas of dia-betic patients than in normal controls. The localization ofthe nitrotyrosine staining indicates that endothelium-de-rived NO (in the presence of increased oxidant stress)turned into the cytotoxic oxidant peroxynitrite [13]. Fur-thermore, chronic treatment in vivo with NOX-101, a wa-ter-soluble nitric oxide scavenger, prevented endothelialdysfunction in diabetes in Sprague-Dawley rats in a model of streptozotocin-induced diabetes [33]. The latterfinding suggests the possibility that diabetes-induced en-dothelial dysfunction is related to an autocrine, NO-medi-ated cytotoxicity in endothelial cells. Peroxynitrite is apotent reactive oxidant, whereas NO is a weak oxidantchemically. Therefore it is conceivable that – under con-ditions of oxidant stress – the conversion of NO to per-oxynitrite is responsible for the development of this para-doxical NO-dependent endothelial dysfunction.

Poly(ADP-ribose) polymerase: a mediator of diabetic endothelial dysfunction in vitro

PARP, also termed poly(ADP)-ribosyltransferase orpoly(ADP-ribose) synthetase, is an abundant nuclear en-

zyme present throughout the phylogenetic spectrum [1].The precise physiological roles of PARP remain unde-fined; its traditional role as a DNA-repair enzyme hasbeen questioned [2]. PARP appears to play diverseroles, participating in DNA repair [3, 4], chromatin re-laxation [5], cell differentiation [6], DNA replication[7], transcriptional regulation [8], control of cell cycle[9], p53 expression and apoptosis [10], and transforma-tion [11]. In the past decade the novel concept hasemerged that under various pathophysiological condi-tions PARP plays a crucial role in the regulation andgeneration of cell dysfunction and the inflammatory re-sponse [45].

Under basal conditions in vivo, PARP activity is rela-tively quiescent. Under conditions of redox stress the N-terminal domain of PARP recognizes oxidant-inducedDNA single-strand breaks resulting in a conformationalchange that activates its C-terminal catalytic domain[47]. Activated PARP cleaves the substrate NAD+ intoADP-ribose and nicotinamide. During this process PARPcovalently attaches ADP-ribose to various proteins, in-cluding an automodification domain of PARP itself, andthen extends the initial ADP-ribose group into abranched nucleic acid-like homopolymer, poly(ADP ri-bose) [48]. Simultaneously, poly-ADP ribose is degradedby various nuclear enzymes, including poly(ADP-ribose)glycohydrolase. The catalysis of poly ADP-ribose resultsin depletion of NAD+, inhibition of glycolysis and mito-chondrial respiration, and the ultimate reduction of intra-cellular high energy phosphates [22, 38, 39, 41, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58]. The coincident activi-ties of PARP and poly ADP-ribose glycohydrolase maybe regarded functionally as a NADase. Loss of intracel-lular energetic stores has deleterious consequences on abroad range of cellular activities that may be substantial-ly delayed from the time of oxidant exposure.

Recently we investigated the role of PARP in the de-velopment of endothelial dysfunction induced by highglucose and diabetes mellitus. In in vitro studies murineendothelial cells placed into high (30 mM) glucose medi-um served as a model of diabetic endothelial injury [54].Previous work using this in vitro experimental model hasdemonstrated the generation of oxyradicals and identi-fied the mitochondria as a principal site of this oxyradi-cal generation [55]. In response to high glucose, time-dependent DNA single-strand breakage and PARP acti-vation has been observed [54]. The degree of DNA sin-gle-strand breakage was similar in the wild-type(PARP+/+) and the PARP knock-out (PARP–/–) cells.High glucose triggered the activation of PARP in the mu-rine endothelial cells and also in human umbilical veinendothelial cells. The activation of PARP in response tohigh glucose was blocked by PJ34, a novel, potent phar-macological inhibitor of PARP [54, 56]. A small increasein PARP activation was nevertheless also observed in thePARP–/– endothelial cells, which may be related to theadditional role of minor isoforms of PARP. Recent workdemonstrated that there are several enzymes withpoly(ADP-ribos)ylating capabilities: the original, first

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isoform (referred to as “PARP” in the current review) isnow termed PARP-1 [57, 58, 59].

Tyrosine nitration, a marker of peroxynitrite genera-tion, was detected in the endothelial cells exposed tohigh glucose and appeared to localize in the mitochon-dria [54]. This is consistent with the mitochondrial elec-tron chain being the primary source of oxyradical gener-ation in endothelial cells placed in high glucose [55].PARP activation in response to high glucose was attenu-ated by superoxide dismutase or by MnTBAP, a cell-per-meable water-soluble superoxide dismutase mimic, aswell as by carboxy-PTIO, an NO scavenger compound,indicating that the activation of PARP is related to intra-vascular generation of reactive oxygen and nitrogen spe-cies. Similarly, pharmacological inhibition of cellularNO synthesis by NG-methyl-L-arginine or NG-nitro-L-arginine methyl ester (but not aminoguanidine, an inhibi-tor of iNOS) reduced the degree of PARP activation inendothelial cells exposed to high glucose [54].

Similar to the in vivo situation in hyperglycemia anddiabetes, incubation of thoracic aortae from wild-typemice in high glucose concentration for 16 h resulted in asignificant loss of endothelium-dependent function.However, vascular rings from the PARP–/– mice, or wild-type rings incubated in the presence of pharmacologicalPARP inhibitors, were fully resistant against high glu-cose induced endothelial dysfunction [54].

As mentioned above, PARP activation is known totrigger a futile intracellular energetic cycle, leading todepletion of intracellular NAD+ and high energy phos-phate levels. In order to test the relevance of this mech-anism to the high glucose induced cell dysfunction wemeasured cellular pyridine nucleotide concentrations inthe endothelial cells exposed to high glucose. Therewas a severe suppression of cellular high energy phos-phate levels as well as a suppression of NAD+ andNADPH levels in endothelial cells exposed to high glu-cose for 1–2 days. These effects were prevented byPARP inhibition or by PARP–/– phenotype [54]. SinceeNOS is an NADPH-dependent enzyme, it is conceiv-able that the cellular depletion of NADPH in endotheli-al cells exposed to high glucose is directly responsiblefor the suppression of eNOS activity and the reductionin the diabetic vessels’ endothelium-dependent relaxantability.

PARP-dependent loss of intracellular energetics andcellular function has been well demonstrated in multi-ple cell types in vitro [46, 47, 48, 49, 50, 51, 52, 53, 60,61]. Under a continuing, relatively low-level oxidativeand nitrosative stress, cells are likely to persist in astate of PARP-dependent metabolic suppression, whichcannot be classified as “normal,” “necrotic,” or “apo-ptotic” state, although the cells do exhibit certain pat-terns that are characteristic for either of the latter twostates, such as increased DNA strand breakage [46, 47,48, 49, 50, 51, 52, 53, 60, 61], or mitochondrial dys-function [62, 63]. This metabolic suppression state(which may be termed “pre-necrosis”), when main-tained for prolonged periods of time, may culminate in

triggering cell necrosis [46, 47, 48, 49, 50, 51, 52, 53,60, 61, 62, 63]. While cell necrosis itself (once the pro-cess is initiated) is not reversible, it is likely that PARPinhibition can reverse the state of the metabolic sup-pression and therefore improve cell function and pre-vent cell necrosis (Fig. 1).

Poly(ADP-ribose) polymerase: a mediator of diabetic endothelial dysfunction in vivo

In rodent models of diabetes, destruction of pancreaticislet cells can be induced by treatment of mice withhigh-dose streptozotocin. If one initiates a therapeutic in-tervention subsequent to the process of islet destruction,one can prevent the intervention’s interference with theprimary process of islet injury, thereby designing experi-ments aimed at interfering with the secondary processesof diabetes (such as the endothelial dysfunction). Wehave recently completed such studies evaluating the ef-fect of a PARP inhibitor in mice [54] and rats (Fig. 2).The complete loss of pancreatic insulin content triggereda severe hyperglycemia throughout the 8 weeks of theexperiments [54] (Fig. 2). Inhibition of PARP activationwas achieved by chronic treatment with the potent, wa-ter-soluble phenanthridinone derivative PARP inhibitorPJ34, starting 1 week after the initiation of diabetes. In-creases in the immunostaining for nitrotyrosine, an indi-cator of reactive nitrogen species production [54], ele-vated frequency of DNA strand breaks, and markedPARP activation were observed in the diabetic bloodvessels [54] (Figs. 2, 3). The activation of PARP in theblood vessels was already apparent 2 weeks after the on-set of diabetes (Fig. 2) and thus it slightly preceded theoccurrence of the endothelial dysfunction which devel-oped between the 2nd and the 4th week of diabetes in themurine model [54].

Ex vivo experiments demonstrated the loss of endo-thelial function, as measured by the relaxant responsive-ness of precontracted vascular rings to acetylcholine [54](Fig. 3). Delayed treatment with the PARP inhibitorstarting 1 week after streptozotocin, ameliorated vascularpoly(ADP-ribose) activation and restored normal vascu-lar function without altering systemic glucose levels,plasma glycated hemoglobin levels, or pancreatic insulincontent [54]. The endothelium-independent relaxant re-sponsiveness of the blood vessels in response to the NO-releasing compound sodium nitroprusside remainedunchanged, indicating that the ability of the endotheliumto release NO, rather than the ability of the smooth mus-cle to relax to NO is impaired in diabetes. Chronic treat-ment with the PARP inhibitor did not alter blood glu-cose, glycated hemoglobin or vascular responses in con-trol (nondiabetic) animals [54]. Taken together, these da-ta indicate that PARP-dependent mechanisms of diabeticvascular dysfunction are operative in vivo, and PARP in-hibition may be a suitable novel experimental therapeu-tic approach to improve the patency of diabetic bloodvessels.

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PARP as a regulator of proinflammatory signaling pathways

Initial studies of the role of PARP in experimental mod-els of disease focused on its effects on intracellular ener-getics and resultant cellular dysfunction. In the past fewyears, however, a new series of in vivo investigationshave revealed that inhibition of PARP activation, or itsgenetic deficiency, has unexpected actions in diseasesthat are not necessarily associated with oxidant injury.These effects, which are demonstrated in a broad range

of experimental models, appear to be related to the ef-fects of PARP on the expression, activation, and nucleartranslocation of key proinflammatory genes and proteins.The absence of PARP or its pharmacological inhibitionhas been shown to suppress the activation of mitogen-activated protein (MAP) kinase [52], activator protein(AP) 1 complex [65], and nuclear factor (NF) κB [61].Consequently, PARP inhibition interferes with the ex-pression of proinflammatory genes, such as iNOS [62]and ICAM-1, which are dependent upon these signalingpathways [68, 69]. These observations have been con-

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Fig. 2 Evidence for earlyPARP activation in the thoracicaorta of streptozotocin-diabeticmice. Experiments were per-formed, as previously de-scribed [51]. After 2 weeks ofdiabetes thoracic aortae wereremoved embedded in cryo-preservation medium and fro-zen. Sections (10 µm) were cut, and PARP activity was de-tected by PARP enzyme histo-chemistry using biotinylatedNAD+ substrate. Above Control(normoglycemic) animal. Middle Diabetic animal. Notethe increased poly(ADP-ribo-syl)ation in the intima and themedia of the blood vessel. Below Diabetic PARP inhibitorcontrol (staining was carriedout in the presence of 3 µMPJ34)

firmed in a variety of in vitro systems, examining abroad range of cell types, and in various experimentalmodels of inflammation [45].

PARP can regulate transcription by at least two differ-ent mechanisms. PARP can poly-ADP ribosylate tran-scription factors and thereby modify their ability to bindto DNA. Further, poly-ADP ribosylation may initiate therepair of DNA strand breaks which may interfere withtranscription of the damaged genes. PARP may also alterthe activation of proinflammatory pathways via its influ-ence on the expression of AP-1, a heterodimer composedof c-fos and c-jun factors. High levels of transcriptionalactivation of human ICAM-1, C3, and c-fos require AP-1binding to 5′ flanking regulatory regions [60]. In cul-tured cells PARP inhibition blocks oxidant-induced c-fosmRNA expression and AP-1 activation. Since the c-fospromoter contains an AP-1 consensus site, c-fos activa-tion could trigger a positive-feedback cycle of gene ex-pression. Superoxide anion has also been reported to in-duce the posttranslational poly ADP-ribosylation of c-fos[8].

PARP inhibition attenuates iNOS expression in fibro-blasts [60], pancreatic beta islet cells [62] and macro-phages [71]. Similar anti-inflammatory effects have beennoted in vitro in murine macrophages [72] and in vivo inrats [68, 69]. PARP inhibition has been shown, for exam-ple, to suppress endotoxin-induced expression of tumornecrosis factor α, interleukin 6, and iNOS [52] and to el-evate the expression of the anti-inflammatory cytokineinterleukin 10 [52]. These effects are associated with thesuppression of lipopolysaccharide-mediated induction ofNF-κB activation [67] and of MAP kinase activity [52].

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Fig. 3 Role of PARP activation in the development of endothelialdysfunction in streptozotocin-diabetic rats. Experiments in adultSprague-Dawley rats were performed as previously described formice [51] except that the dose of streptozotocin was 65 mg/kg in-travenously. Immunohistochemical staining for poly(ADP-ribose)was performed as described elsewhere [89]. A–C Immunohisto-chemical staining for poly(ADP-ribose), an indicator of PARP ac-tivation. A In control rings. B In rings from diabetic male Wistarrats treated with vehicle at 8 weeks. C In rings from diabetic ratstreated with PJ34. There was a marked increase in PARP activa-tion in diabetic blood vessels (arrow representative PARP-positivenuclei), which was abolished by PJ34 treatment. Similar immuno-histochemical profiles were seen in n=4–5 vascular rings in eachexperimental group. D Acetylcholine (10 µM) induced endotheli-um-dependent relaxant responses of control and diabetic rats inthe presence of vehicle or PJ34 treatment (n=8 per group). Therewas an impairment of the endothelium-dependent relaxations indiabetes, which was restored by PJ34 treatment. *P<0.05 for vehi-cle-treated diabetic vs. PJ34 treated diabetic mice. Chronic PJ34treatment in control (nondiabetic) rats did not affect relaxant re-sponses. In the diabetic rats there was a marked and sustained hy-perglycemia, and increased circulating glycated hemoglobin levelsafter 8 weeks in response to streptozotocin, which were unaffectedby delayed-onset treatment with PJ34. Respective values for bloodglucose: 412±47, 435±39 mg/dl and for glycated hemoglobin:11.2±0.5% and 10.7±0.7% at 8 weeks in vehicle and PJ34 treateddiabetic rats (n=8). There was a slight increase in plasma nitratelevels in the diabetic rats at 8 weeks, which was suppressed byPJ34 treatment. Respective values in control, diabetic and PJ34treated diabetic animals: 8±2, 14±2, and 9±3 µM; n=8, P<0.05

Since MAP kinase plays a major role in the pleiotropictransduction of intracellular inflammatory cascades, theanti-inflammatory effects of PARP inhibition may be ac-counted for at this level of gene regulation. One mayalso expect that PARP-dependent regulation of NF-κBactivation has a pleiotropic effect on the expression ofproinflammatory genes, given the broad role that NF-κBplays in the transcriptional activation of cytokine andchemokine genes. A recently completed microchip anal-ysis study has investigated the changes in the expressionof 15,000 genes in wild-type and PARP deficient fibro-blasts. The study demonstrated that under baseline con-ditions (i.e., in the absence of specific proinflammatorystimuli), that there is a significant alteration in the ex-pression of a whole host of genes [73]. It is expected thateven more significant differences will be found in im-munostimulated or oxidatively stressed cells.

Evidence emerges indicating that the above detailedpathway, the “PARP- proinflammatory mediator produc-tion pathway” may also be related to the pathogenesis ofdiabetic endothelial dysfunction. Adhesion moleculessuch as ICAM-1, iNOS expression, and NF-κB activa-tion have been implicated in the pathogenesis of the en-dothelial dysfunction associated with diabetes [74, 75,76, 77, 78, 79]. Therefore it is conceivable that suppres-sion of these proinflammatory mechanisms is a potentialmode of protection by the absence of functional PARPagainst diabetic endothelial dysfunction in vivo. This hy-pothesis is further supported by our current data demon-strating that PARP inhibitor treatment prevents the dia-betes-induced elevation in circulating nitrite/nitrate lev-els in rodent models of streptozotocin-induced diabetes[54] as well as by the findings demonstrating that PARPdeficiency suppresses NF-κB activation in endothelialcells placed in high glucose [54].

Summary and implications: role of PARP activation in diabetic vascular injury

The following working hypothesis summarizes our cur-rent understanding on the role of PARP activation in thedevelopment of inflammatory injury (Fig. 1). High circu-lating glucose stimulates free radical formation from themitochondrial electron transport chain, aldose reductase,NADH/NADPH oxidase, and a variety of other cellularsources. Also, high glucose may induce the upregulationof the endothelial and inducible isoforms of NO syn-thase, leading to enhanced NO production. (Since super-oxide and not NO is the rate-limiting step in the genera-tion of peroxynitrite in most vascular pathophysiologicalconditions, and endothelial cells constitutively produceNO, we believe that up-regulation of eNOS or iNOS isneither a necessary nor a sufficient factor to induce en-dothelial dysfunction.) At later stages of diabetes, re-cruitment and activation of neutrophils may also occurwhich enhances vascular injury via a variety of mecha-nisms including neutrophil NADPH oxidase. As a conse-quence, the oxidants peroxynitrite, hydrogen peroxide,

and hydroxyl radical are formed in the vascular endothe-lial cells from the interaction of superoxide and NO andby iron-catalyzed oxidation of superoxide. Oxidant stressinduces AP-1 formation and generates DNA single-strand breaks. DNA strand breaks then activate PARP,which in turn potentiates NF-κB activation and AP-1 ex-pression, resulting in greater expression of the AP-1 andNF-κB dependent genes, such as iNOS, ICAM-1, macro-phage inflammatory protein 1α and tumor necrosis fac-tor α, Increased endothelial expression of ICAM-1 mayin turn recruit more activated leukocytes to the diabeticblood vessels, producing greater oxidant stress. The cy-cle is thus renewed as the increase in oxidant stress trig-gers more DNA strand breakage. The proposed cycle ofinflammatory activation would be augmented by PARP-dependent MAP kinase activation and NF-κB transloca-tion and by consequent free radical and oxidant forma-tion and granulocyte recruitment. Thus, PARP occupies acritical position in a positive-feedback loop of diabeticvascular injury. NAD+ depletion induced by PARP acti-vation is likely to accelerate this positive-feedback cycleby preventing the energy-dependent reduction of oxi-dized glutathione, the chief intracellular anti-oxidant andmost abundant thiol in eukaryotic cells. NAD+ is the pre-cursor for NADP, a cofactor that plays a critical role inbioreductive synthetic pathways and the maintenance ofreduced glutathione pools [83]. Depletion of reducedglutathione, as a consequence of intracellular energeticfailure or overwhelming oxidant exposure [84], leavesfurther oxidant stress unopposed, resulting in greaterDNA strand breakage. PARP activation and mitochon-drial injury also form a self-amplifying circle, wherebyPARP-dependent metabolic processes enhance mito-chondrial free radical generation and permeability transi-tion: pharmacological inhibition of PARP or PARP defi-ciency is able to significantly suppress these deleteriousmitochondrial processes [63]. PARP-dependent depletionof NADPH directly contributes to the suppression of en-dothelium-dependent relaxations in diabetic blood ves-sels. If the latter mechanism is operative, one would ex-pect that PARP inhibition is not only able to prevent thedevelopment of diabetic endothelial dysfunction, but itwould also be expected to reverse established endothelialdysfunction. This possibility remains to be investigatedin future studies. It is noteworthy in this respect that re-cent studies in colitis demonstrated that the colitis-relat-ed epithelial dysfunction (hyperpermeability) is rapidlyreversible with pharmacological inhibition of PARP [82].

The studies reviewed above reflect primarily on thepathogenesis of the diabetic macrovascular disease. Al-though oxidant stress is important in the pathogenesis ofboth the macro- and the microvascular complications,these two pathologies are very different entities. There-fore, further work needs to establish whether PARP acti-vation also plays a role in the development of diabeticmicrovascular complications. Our preliminary studies in-dicate that PARP activation may play a role in diabeticmicrovascular complications; we have observed signifi-

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cant poly(ADP-ribose) immunostaining in the microves-sels of diabetic retinae in the rat [83].

As in most previous studies, in our studies dealingwith the role of PARP in the pathogenesis of endothelialdysfunction we have been focusing on the acetylcholine-induced, endothelium-dependent relaxations. Most inves-tigators assume that a particular intervention that im-proves endothelium-dependent vasodilatation is likely toconfer commensurate benefit on other aspects of endothe-lial function. However, this assumption has rarely beenconfirmed by experimental studies. Therefore, it will beimperative to evaluate whether the beneficial effect ofPARP inhibition on endothelium-dependent vasodilata-tion is also manifest in improvements in other functionsof the vascular endothelium (permeability, hemostatic,antiatherogenic, regulation of cell trafficking, etc.).

A variety of experimental therapeutic approacheshave been proposed to prevent or reverse diabetic endo-thelial dysfunction. Many of these approaches (such asthe aldose reductase pathway, the protein kinase C path-way, the advanced glycation end product pathway, theangiotensin-converting enzyme related endothelial dys-function and the folate pathway) are closely related tofree-radical or oxidant-related processes (as reviewed in[1, 2, 3, 4, 5]). As PARP occupies a critical position in aself-amplifying cycle of oxidant-driven damage, it is ap-propriate then to ask whether PARP inhibition is a candi-date for clinical treatment of diabetic vascular dysfunc-tion. Although the activation of PARP has yet to be dem-onstrated in human diabetic vascular tissue, both theproximal triggers of DNA single strand breakage (e.g.,peroxynitrite) [13], and the existence of increased DNAsingle-strand breakage [84] have been demonstrated inhuman clinical samples. Furthermore, DNA single-strand breakage [54, 85] and PARP-dependent endotheli-al cell injury have been demonstrated in human umbili-cal vein endothelial cells incubated with high glucose[54]. Finally, PARP dependent morphological and func-tional injury has been demonstrated in a variety of hu-man cell types exposed to pro-oxidant conditions [49,50, 51, 52, 53, 54]. Therefore it is conceivable thatPARP-dependent processes are being triggered in humandiabetic blood vessels in vivo. A variety of PARP inhibi-tors are in preclinical development, many with potencythat greatly exceeds the prototypic agents used in experi-mental proof-of-concept studies. In general, PARP inhib-itors may be particularly useful in the treatment of acutedisorders, where issues of potential toxicity related tochronic administration are unimportant. Although the ex-act physiological role of PARP is still unclear, and re-mains a matter of dispute, it is logical to suppose it playsan important role since it is one of the most abundantproteins in the nucleus. PARP has been implicated inmany physiological housekeeping functions, such asgene repair, transcription, and cell cycling. Until suchtime as its true physiological functions are more precise-ly defined, there should exist a considerable caution inthe long-term administration of PARP inhibitors to hu-mans. PARP inhibition has also been associated with an

increase in sister chromatid exchange [86], a potentiallyconcerning finding that raises the risk of malignanttransformation. PARP activation clearly leads to celldeath, and some have argued that its physiological role isto eliminate genetically damaged cells, thereby reducingoncogenic potential [87]. PARP inhibition has beenshown to facilitate the rapid ligation of DNA excision-repair patches [88] and to suppress malignant transfor-mation in cells with DNA damage induced by irradiationand chemical carcinogens [89, 90, 91]. Whether chronicPARP inhibition predisposes to malignant transformationis open to question. The PARP deficient mice have notbeen reported to present increased frequency of sponta-neous malignancies although, to our knowledge, this is-sue has not yet been systematically investigated. A cleardistinction must also be made between chronic PARP in-hibition, versus chronic PARP deficiency: the latter con-dition will also affect cellular processes due to the ab-sence of protein-protein interactions in which PARP isknown to participate. Nevertheless, these issues must beadequately addressed prior to considering the develop-ment of PARP inhibitors for chronic administration inorder to treat the vascular dysfunction associated withdiabetes mellitus or other chronic vascular conditions.

Acknowledgements This work was supported by the followinggrant from the National Institutes of Health: R01GM60915 to C.S.L.V. was supported by a Bolyai Fellowship from the HungarianAcademy of Sciences and F.G.S. was supported by a FAPESP(Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil)research fellowship.

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