Associate editor: B.J. McDermott

Signicance of peroxisome proliferator-activated receptors in the cardiovascularsystem in health and disease

Emma Robinson, David J. Grieve Centre for Vision and Vascular Science, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, 3rd Floor, Medical Biology Centre, 97 Lisburn Road,

a r t i c l e

Keywords:Peroxisome prolifer(PPAR)Cardiovascular disea

tion factors thatand , which playibution. PPAR isoxidation. PPARsulin sensitivity.

PPAR is abundantly and ubiquitously expressed, but as yet its function has not been clearly dened. Activators

. . .ardiova. . .

Pharmacology & Therapeutics 122 (2009) 246263

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

j ourna l homepage: www.e lsev ie r.com/ locate /pharmtheraAbbreviations: ACE, Angiotensin-converting enzyme; AF, Activation function; ANF, Atrial natriuretic factor; Ang II, Angiotensin; AP-1, Activator protein-1; BNP, Brain natriureticpeptide; COX, Cyclooxygenase; CVD, Cardiovascular disease; DBD, DNA binding domain; DHA, Docosahexaenoic acid; DOCA, Deoxycorticosterone acetate; ERK, Extracellular

regulated kinase; ET, Endothelin; FATP, Fatty acid transdomain; LDL, Low density lipoprotein; L-NAME, N-nitrprotein-1; MI, Myocardial infarction; MMP, Matrix meNuclear factor-kappa B; NO, Nitric oxide; NOS, Nitric oactivated receptor; PPRE, Peroxisome proliferator responmediator for retinoid and thyroid hormone receptor; STA Corresponding author. Tel.: +44 2890972097; fax:

E-mail address: d.grieve@qub.ac.uk (D.J. Grieve).

0163-7258/$ see front matter 2009 Elsevier Inc. Aldoi:10.1016/j.pharmthera.2009.03.003. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

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258258258Acknowledgment . . . . .References . . . . . . . .2. PPARs and the c3. Conclusions . .Contents

1. Introduction . .found to confer additional benets on endothelial function, inammation and thrombosis, suggesting thatPPAR agonists may be good candidates for the treatment of cardiovascular disease. In this regard, it has beendemonstrated that PPAR activators are capable of reducing blood pressure and attenuating the development ofatherosclerosis and cardiac hypertrophy. This review will provide a detailed discussion of the currentunderstanding of basic PPAR physiology, with particular reference to the cardiovascular system. It will alsoexamine the evidence supporting the involvement of the different PPAR isoforms in cardiovascular disease anddiscuss the current and potential future clinical applications of PPAR activators.

2009 Elsevier Inc. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246scular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250Hypertrophyof hyperlipidaemia and to improve insulin sensitivity in diabetes. More recently, PPAR activation has beenHypertension

of PPAR (brates) and (thiazolidinediones) have been used clinically for a number of years in the treatmentHeartAtherosclerosis

expressed predominantly inis mainly associated with aa b s t r a c ti n f o

ator-activated receptor

se (CVD)

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcripbelong to the nuclear receptor superfamily. Three isoforms of PPAR have been identied,, distinct roles in the regulation of key metabolic processes, such as glucose and lipid redistr

the liver, kidney and heart, and is primarily involved in fatty aciddipose tissue, where it controls adipocyte differentiation and inBelfast, BT9 7BL UKport protein; HDL, High density lipoprotein; ICAM, Intercellular adhesion molecule; IL, Interleukin; LBD, Ligand bindingo-L-arginine methyl ester; LV, Left ventricular; MAPK, Mitogen activated protein kinase; MCP-1, Monocytic chemotactictalloproteinases; NADPH, Nicotinamide adenosine dinucleotide phosphate; NcoR, Nuclear receptor co-repressor; NF-B,xide synthase; PGC-1, Peroxisome proliferator-activated receptor gamma co-activator-1; PPAR, Peroxisome proliferator-se element; ROS, Reactive oxygen species; RXR, Retinoid X receptor; SHR, Spontaneously hypertensive rats; SMRT, SilencingT, Signal transducers and activator of transcription; TNF, Tumour necrosis factor; VCAM, Vascular cell adhesionmolecule.+44 2890975775.

l rights reserved.

1. Introduction

Despite major therapeutic advances, cardiovascular disease (CVD)remains one of the leading causes of morbidity and mortality in thewestern world. Traditional risk factors for CVD include hypertension,hyperglycaemia and dyslipidaemia (Kannel, 1997). However therapidly increasing incidence of metabolic disorders such as diabetes,obesity and metabolic syndrome combined with more aggressivetreatment of hypertension is shifting the underlying aetiology towardshyperglycaemia and dyslipidaemia. CVD is frequently characterised by

peroxisome size or number (Kliewer et al., 2001). The group ofstructurally disparate compounds, which were initially found tostimulate peroxisome proliferation in rats, were assigned as peroxi-some proliferators although the mechanism of action of thesecompounds was unknown at that time.

In 1990, the cloning of a mouse gene linked to peroxisomeproliferation was rst described (Isseman & Green 1990). It was subse-quently discovered that peroxisome proliferators acted via stimulationof an orphan nuclear hormone receptor, which was named theperoxisome proliferator-activated receptor or PPAR; the original receptor

domC-tdin

247E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263dysregulation of fatty acids, particularly in patients with metabolicdisease (Abate, 2000; Grundy, 2004). In healthy individuals the ratesof uptake and utilisation of fatty acids are tightly controlled in order tomatch tissue energy demands and maintain lipid balance. However,when this balance is upset the subsequent increase in circulating fattyacids becomes a primary risk factor for the development of hyperten-sion and atherosclerosis (Egan et al., 2001). Several classes of drugs,including bile acid sequestrants, brates and statins, have been usedhistorically to reduce cholesterol levels and maintain physiologicalplasma concentrations (Knopp, 1999). Of these, statins have nowbecome the primary therapeutic choice for CVD prevention, subse-quent to several large-scale primary and secondary clinical trialswhich demonstrated that chronic treatment caused a signicantreduction in the incidence of coronary heart disease (ScandinavianStudy Simvastatin Survival Group Study 1994; Shepherd et al., 1995).Nevertheless, there remains a substantial incidence of CVD inoptimally-treated patients, especially thosewith additional underlyingpathologies, such as diabetes. In the ongoing search for amore effectivealternative to statins, recent attention has begun to focus on bratesand thiazoldidenodiones, due to their therapeutic value in the specictreatment of hyperlipidaemia and diabetes, respectively. These drugsexert their effects via activation of peroxisome proliferator-activatedreceptors (PPARs), which belong to the nuclear hormone receptorsuperfamily. This review will discuss the current understandingof PPAR physiology and pharmacology and how activation of thePPAR pathway may modify the development of several diseases of thecardiovascular system.

1.1. PPAR discovery and classication

Peroxisomes are subcellular organelles whose classical role is toremove hydrogen through the use of molecular oxygen via a seriesof oxidase and catalase enzymes. They also play a crucial role inseveral cellular metabolic processes, including the catabolism ofcholesterol to bile and the -oxidation of fatty acids. Under normalphysiological conditions, peroxisomemetabolism occurs secondary tothat in the mitochondrial system (Vamecq & Drey, 1989), andprimarily involves oxidation of very long-chain fatty acids that cannotbe otherwise metabolised. In rat liver cells activation of peroxisomesby various pharmacological stimuli has been shown to induceperoxisomes to increase in size and number (Reddy & Krishnakantha,1975; Lock et al., 1989), and is associated with an increased expressionof genes involved in fatty acid oxidation. In humans, pharmacologicalagents may activate peroxisomal gene transcription in a similarmanner but in contrast to the rat there is no corresponding increase in

Fig. 1. Schematic Structure of PPAR. PPARs have six structural regions and four functionalactivation function (AF-1). The C domain promotes PPAR binding to a DNA sequence. Thereceptor. The D domain or hinge domain links the DNA binding domain to the ligand bin

to assist the transcription process via the ligand-dependent transactivation function (AF-2)is now known as PPAR. Together, PPARs form group C in subfamily 1of the superfamily of nuclear hormone receptors, i.e., NR1C. The largenuclear receptor superfamily comprises ligand-activated transcriptionalfactors that regulate the expression of a large number of genes involvedin carbohydrate and lipid metabolism (Giguere, 1999). Also includedin this group are the steroid, thyroid, vitamin D and retinoic acidreceptors, as well as several other orphan receptors for which aligand/activator has yet to be identied (Manglesdorf et al., 1995;Blumberg & Evans 1998). Leading on from the discovery of theprototypic mouse PPAR, (PPAR, NR1C1), the cDNA of two othermajor isotypes of this nuclear receptor subfamily, PPAR (NR1C2)and PPAR (NR1C3), were identied, although neither of these PPARsubtypes were found to be associated with peroxisome prolifera-tion. All three PPARs are encoded by separate genes and have beenfound to be expressed in amphibians (Dreyer et al., 1992), rodents(Gottlichter et al., 1992; Kliewer et al., 1994) and humans (Schmidtet al., 1992; Sher et al., 1993; Greene et al., 1995). PPAR and PPARappear to be highly conserved across species, whereas PPAR isconsiderably divergent (Kliewer et al., 1994).

1.2. PPAR structure and mechanism of action

PPARs are compact ligand-activated transcription factors thatcontrol gene expression and have a structural organisation compar-able to the other members of the nuclear hormone receptor family.PPARs have ve or six structural regions within four functionaldomains, known as A/B, C, D and E/F, as depicted in Fig. 1. The amino-terminal A/B domain, which is poorly conserved between the threePPAR isotypes, contains a ligand-independent activation function-1(AF-1) (Werman et al., 1997). Phosphorylation in this region modulatesreceptor activity in an isotype-dependent manner; for example,insulin can stimulate PPAR-mediated transcription via mitogen-activated protein kinase (MAPK)-induced phosphorylation at Ser12and Ser21 (Shalev et al., 1996; Juge-Aubry et al., 1999), whereas MAPK-mediated phosphorylation of Ser112 has an inhibitory action on PPAR(Adams et al., 1997; Camp & Tafuri, 1997). The 70 amino acid longDNA binding domain (DBD) or C domain is comprised of two highlyconserved zinc nger-like structures and promotes binding of thereceptor to a DNA sequence in the promoter region of target genes,known as the peroxisome proliferator response element (PPRE)(Kliewer et al., 1992). The C-terminal, E/F domain or ligand bindingdomain (LBD) is responsible for ligand specicity and activation of PPARbinding to the PPRE of target genes. The D domain or hinge domainlinks the DBD to the LBD and acts as a docking site for co-factors. The Nterminal or E/F domain recruits co-factors to assist the transcription

ains A/B, C, D and E/F. The amino-terminal A/B domain contains a ligand-independenterminal, E/F domain or ligand binding domain is responsible for ligand specicity of theg domain and acts as a docking site for co-factors. The E/F domain also recruits co-factors

.

248 E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263process via the ligand-dependent transactivation function (AF-2)(Berger & Moller, 2002).

Upon activation by endogenous or synthetic ligands, PPARs, likeother nuclear hormone receptors, form obligate heterodimers withthe 9-cis retinoic acid receptors (retinoid X receptor, RXR), a processfacilitated by the LBD. The resulting complex undergoes a conforma-tional change which allows binding of the heterodimer to the PPRE(consensus sequence: 5-AGGTCA n AGGTCA-3), which is located inthe promoter region of the target gene (Torra et al., 2001). The PPAR/RXR heterodimer binds to the PPRE with PPAR occupying the 5 halfsite, whilst RXR occupies the 3 half site. The PPRE consensus sequence(5-AGGTCA n AGGTCA-3) ts a pattern of two direct repeats spacedby one nucleotide (DR1), and is specic for the PPAR/RXR hetero-dimer, setting it apart from other nuclear receptors, such as thethyroid or vitamin D receptors (Ijpenberg et al., 1997; Kliewer et al.,2001). Binding of the heterodimer complex to the PPRE is diagram-matically represented in Fig. 2. Ligands of either of the heterodimeric

Fig. 2. Basic mechanism of action of PPARs. PPARs bind to a specic sequence in thepromoter of target genes (peroxisome proliferator response element; PPRE) andactivate transcription. The PPAR/retinoid X receptor (RXR) heterodimer binds to thePPRE with PPAR occupying the 5half site whilst RXR occupies the 3 half site. The PPREconsensus sequence, 5-AGGTCA n AGGTCA-3, ts a DR1 pattern of two direct repeatsspaced by one nucleotide, and is specic for the PPAR/RXR heterodimer, setting it apartfrom other nuclear receptors.receptors are able to independently induce transcription of targetgenes, but when both PPAR and RXR are activated simultaneously, itresults in signicant synergistic enhancement of gene transcription(Kleiwer et al., 1992; Mangelsdorf & Evans, 1995).

1.3. Co-activators/repressors of PPARs

Many proteins act as co-activators or co-repressors that regulatethe ability of nuclear hormone receptors, such as PPARs, to eitherstimulate or repress gene transcription. PPARs do not appear to beactive under basal conditions. In the unbound state, PPAR/RXRheterodimers are associated with co-repressors, such as silencingmediator for retinoid and thyroid hormone receptor (SMRT) andnuclear receptor co-repressor (NcoR), which prevent gene transcrip-tion through their histone deacetylase activity (Chen & Evans, 1995;Horlein et al.,1995; Xu et al.,1999). However, once a ligand binds to thereceptor a conformational change occurs that not only facilitates co-repressor dissociation, but also the recruitment of several positive co-activators, including PBP, PPAR binding protein and steroid receptorco-activator 1 (SRC-1) (Zhu et al., 1997; Nolte et al., 1998). Morerecently identied co-activators include the PPAR co-activator-1(PGC-1) proteins, PGC-1 (Puigserver et al., 1998) and PGC-1 (Lin etal., 2002), both of which are found in tissues which exhibit a high rateof mitochondrial metabolism. Once a ligand becomes bound to thereceptor, the histone acetylase activity intrinsic to these co-activatorsinitiates a sequence of events which ultimately lead to genetranscription (Soutoglou et al., 2001). Although co-activators and co-repressors appear to be the major factors responsible for regulation ofPPAR activity, these receptors can also bemodulated byMAPK-inducedphosphorylation, adding a further dimension to an already intricatesystem of control. For example, phosphorylation by extracellularregulated kinases (ERK) has been found to repress PPAR activity(Barger & Kelly, 2000), whereas that induced by p38 MAPK activationenhances PPAR-mediated gene expression (Barger et al., 2001).

1.4. PPAR ligands

PPARs have a remarkable ability to be activated by a wide range ofstructurally diverse endogenous and synthetic ligands. However,heterogeneity of the LBD between the three PPAR isotypes is suchthat there is a degree of ligand specicity. Among the syntheticligands, brates (e.g. Wy 14,643, clobrate, gembrozil, fenobrate,bezabrate) are a class of hypolipidemic drugs which are commonlyused to reduce plasma triglycerides, an established risk factor for thedevelopment of CVD. Although the majority of brates preferentiallyactivate PPAR, few are specic. Wy 14,643 and clobrate were therst reported activators of PPARs; they are selective for PPAR atconcentrations up to 10 M, but at higher concentrations also activatePPAR (Lehmann et al., 1997). Other PPAR agonists appear to have ahigher afnity for PPAR, such as GW2331, which has a Kd of 140 nM(Kliewer et al., 1997). Glitazones are thiazolidinedione-based anti-diabetic compounds which are preferential agonists of PPAR. Ciglita-zone was originally derived from clobrate by optimisation of its weakglucose-lowering properties. In addition to these anti-hyperglycaemicactions, ciglitazone was also found to reduce glucose levels whilstretaining some of the lipid-lowering properties of brates. Since theirinitial discovery, more potent derivatives have been developed, such asrosiglitazone, pioglitazone and troglitazone (Kliewer et al., 2001). Ingeneral, PPAR agonists show greater selectivity than those of PPAR;for example, rosiglitazone, the rst high afnity PPAR ligand to beidentied, has a Kd of 43 nM as compared to the micromolar afnitiescommonly associated with brates.

In addition to agonists of PPAR and PPAR which have wideclinical applications, synthetic ligands for PPAR have also beendeveloped (Berger et al., 1999). L165041, a phenoxyacetic acidderivative and GW0742X (Michalik et al., 2006) act as specicPPAR agonists, which may have benecial effects on lipid andglucose metabolism, in addition to playing a role in fertility andcancer. Despite concerted efforts to develop high afnity, isotype-specic PPAR agonists, it is becoming increasingly apparent that manysynthetic PPAR ligands also exert PPAR-independent effects. Whilstmuch of the PPAR research to date employing such synthetic agonistshas provided tremendous insight into PPAR biology, the results ofthese studies must be interpreted with care. It is clear that theexistence of a specic endogenous ligand would enable a morefocussed interrogation and detailed understanding of PPAR function,and considerable effort has been invested in this direction. The maincandidate for an endogenous PPAR activator appears to be fatty acids,which are known to possess similar characteristics to brates;however, whether or not they are true natural PPAR ligands is stillopen to debate. An early investigation revealed that PPAR isactivated by long-chain fatty acids (Gottlicher et al., 1992), andsubsequently the ability of individual fatty acids of variable chainlength and degree of saturation to act as ligands for the three PPARisotypes has become the focus of many in vitro studies. PPAR subtypeshave varying afnities for different fatty acids. For example, PPARand PPAR have comparable afnities for long-chain saturated, mono-and poly-unsaturated fatty acids, whereas PPAR has a very lowafnity for saturated fatty acids (Forman et al., 1997; Johnson et al.,1997; Kliewer et al., 1997; Xu et al., 1999). However, the in vitro

afnities of the these fatty acids for their respective PPARs are in the

micromolar to submillimolar range; if they were to be true selectiveendogenous ligands these afnities should be at much lowerconcentrations, within the nanomolar range.

Another factor in consideration of fatty acids as potential endogen-ous PPAR ligands is the mechanism by which these labile moleculescould become sufciently concentrated within the nucleus so as toactivate PPAR. In this regard, it has recently been shown that both localgenerationwithin thenucleus via the action of phospholipases and fattyacid transport may be involved in fatty acid-mediated PPAR activation(Han et al., 2002; Tan et al., 2002; Gilde & Van Bilsen, 2003). Certainderivatives of fatty acids are also capable of binding to PPARs, withproducts of lipooxygenase and cyclooxygenase metabolism appearingto bemore specic and potent than their fatty acid precursors. Indeed, ametabolite of the prostaglandin J2 group, 15d-PGJ2 and prostacyclinhave previously been suggested to be natural ligands for PPAR andPPAR, respectively, due to their high afnities for each receptor(Forman et al., 1995; Kliewer et al., 1995; Lim et al., 1999). Despite this,recent evidence has demonstrated that 15d-PGJ2 exerts PPAR-independent effects (Kaplan et al., 2007) which imply that it is not anendogenous PPAR ligand. Details of the endogenous and syntheticagonists of PPARs and their typical EC50 values are given in Fig. 3.

1.5. Selective PPAR modulators (SPPARM)

Upon binding to PPARs, different ligands can induce differentstimulatory or inhibitory responses depending on the nature of thespecic target gene and its cellular location, a principle which hasbeen termed the selective PPAR modulator (SPPARM) theory. Itsuggests that once ligands become bound to the receptor, they caneach induce unique and distinct conformational changes, leading todifferential co-activator/co-repressor interactions, enabling subtledifferences in transcriptional activation of target genes. Therefore,

different physiological responses depending on, for example, cell type.The SPPARM theory is currently being employed to aid the develop-ment of new generation PPAR agonists, with the hope that speciccompounds may be identied that are capable of activating thetranscription of desirable target genes whilst minimising/repressingthe transcription of those which are detrimental (Chinetti-Gbaguidiet al., 2005a; Fruchart, 2007).

1.6. Tissue distribution of PPARs

In order to begin to understand the complex physiological role ofthe different PPARs, it is essential to examine their tissue expression.In both rodents and humans, PPAR is predominantly expressed incells with high rates of fatty acid catabolism and peroxisome-dependent oxidation, such as those found in liver, heart, kidney,skeletal muscle, pancreas and intestinal mucosa (Braissant et al., 1996;Schoonjans et al., 1996a). PPAR is mainly associated with adiposetissue, with a low level of more ubiquitous expression in liver, heart,skeletal muscle and bone marrow (Escher & Wahli, 2000). Interest-ingly, splice variants of the human PPAR (PPAR1 and PPAR2) andmore recently, PPAR have been identied (Fajas et al., 1997; Gervoiset al., 1999). PPAR2 has been found to be exclusively and abundantlyexpressed in fat tissue, whereas PPAR1 has a more ubiquitousexpression prole (Schoonjans et al., 1996b). PPAR is abundantly andubiquitously expressed at much higher levels than PPAR and PPAR(Kliewer et al., 1994). It is important to note that tissue expression ofall three PPAR isotypes is likely to vary under differing physiologicaland/or pathological conditions.

1.7. Physiological function of PPARs

Activation of PPARs is a multifaceted process that relies on efcient

PPAs wh

249E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263distinct ligands for one common receptor are capable of inducing

Fig. 3. Table of typical EC50 values for endogenous and synthetic PPAR ligands at murinePPAR, and the glitazones, a thiazolidinedione-based group of anti-diabetic compound

fatty acids or their derivatives.receptor dimerisation, co-factor recruitment, phosphorylation and

R receptors. Synthetic PPAR agonists include the brates, which preferentially activateich are preferential agonists of PPAR. Endogenous PPAR activators are predominantly

250 E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263ligand binding. The diverse physiological responses elicited by PPARactivation are made possible by the complexities of the receptoractivation pathway and the disparate tissue expression of eachreceptor subtype.

1.7.1. PPARIdentication of PPAR target genes rst illustrated that PPAR is a

major regulator of fatty acid homeostasis. PPAR controls theexpression of a wide range of proteins involved in both the transportand -oxidation of free fatty acids. Specically, PPAR plays a criticalrole in the regulation of fatty acid transport protein (FATP), whichfacilitates the uptake of long-chain fatty acids across the plasmamembrane, and several key enzymes involved in their subsequentcatabolism within the cell. PPAR has been shown to induceactivation of acyl-CoA oxidase, thiolase, acyl-CoA dehydrogenaseand cytochrome P450 -hydroxylase, which are all essential to the-oxidation of fatty acids within peroxisomes, mitochondria andmicrosomes (Schoonjans et al., 1996b). It has been suggested thatPPARmay also act as a cellular sensor capable of detecting changesin circulating free fatty acids or their associated metabolites andintermediates. PPAR-stimulated expression of lipoprotein lipaseis known to promote the release of fatty acids from lipoproteinparticles and their subsequent uptake (Schoonjans et al., 1996b).PPAR expression has also been found to be signicantly increasedin situations of metabolic stress, such as fasting or severe cold, whenincreased energy production requires the release of fatty acids fromadipose tissue (Lemberger et al., 1996). Denitive evidence tosupport the critical requirement of PPAR for effective lipid handlingcomes from a study in PPAR null mice which examined the effectof pharmacologic inhibition (etomoxir) of mitochondrial fatty acidimport on lipid metabolism.Whereas wild-type animals were able totolerate etomoxir treatment, PPAR null mice exhibited preferentiallipid accumulation in tissues with a high rate of lipid oxidation, suchas liver, heart and kidney, which may ultimately lead to lipotoxicityand death (Djouadi et al., 1998). Indeed, PPAR agonists are widelyused in the treatment of disorders characterised by elevated levels ofplasma lipids, i.e. dyslipidaemias. Fibrates exert their positive effectson lipid handling by inducing hepatic uptake and -oxidation of fattyacids and increasing lipoprotein lysis, whilst also conferring benecialeffects on the high density lipoprotein (HDL) to low density lipoprotein(LDL) ratio.

PPAR activation has also been shown to have anti-inammatoryeffects. In gene-modied mice lacking PPAR, a considerablyprolonged inammatory response was observed, and this wassuggested to be due to degradation of the chemotatic inammatorymediator, leukotriene B4 (Devchand et al., 1996). Increasing evidencesuggests that PPAR may mediate its anti-inammatory actionsthrough reduced generation of cytokines. This may occur secondary todownregulation of the activity of nuclear factor-kappa B (NF-B) andinducible cyclooxygenase-2 (COX-2) (Poynter & Daynes, 1998; Staelset al., 1998).

PPAR-induced peroxisome proliferation has been associated withthe development of hepatic carcinomas in rodents, possibly due toincreased production of H2O2 in the absence of a compensatory rise incatalase activity (Gonzalez et al., 1998). However, this is likely to be oflittle clinical relevance due to the absence of peroxisome proliferationin humans upon PPAR activation. Furthermore, the concentration ofPPAR agonist required to stimulate lipidmetabolism (as desiredwithbrate treatment) would be too low to stimulate the transcriptionalinduction of genes involved in peroxisome proliferation (Chevalier &Roberts, 1998).

1.7.2. PPARPPAR acts as a primary regulator of adipocyte differentiation in

the process of adipogenesis. Once fully matured, adipocytes are

capable of producing various hormones and cytokines, in addition tothe uptake and storage of lipids. Activation of PPAR upregulates theexpression of genes involved in fatty acid uptake and lipogenesis aswell as glucose transporters (Shimaya et al., 1998). PPAR activationalso promotes apoptosis inmature adipocytes, resulting in stimulationof adipogenesis and the formation of small insulin-sensitive adipo-cytes (Okuno et al., 1998). This is one potential mechanism by whichthiazolidinediones may improve insulin sensitivity in diabetes. PPARis known to regulate many genes involved in insulin signalling, such asthose that control the expression of the pro-inammatory cytokine,tumour necrosis factor (TNF). PPAR activation signicantlyreduces the production of TNF by adipocytes, which plays anestablished role in the development of insulin resistance (Moller,2000). In diabetes, PPAR improves overall glucose homeostasis byincreasing glucose transport in adipocytes, regulating adipocyte-derived hormone release, decreasing glucose formation and increas-ing glucose disposal in skeletal muscle (Kota et al., 2005). A recentstudy by Odegaard et al. (2007) has suggested that macrophage-specic PPAR activation also reduces insulin resistance in adiposetissue via differentiation of alternatively activated monocytes with ananti-inammatory phenotype. Indeed, PPAR, like PPAR, appears toexert signicant anti-inammatory effects. PPAR expression ismarkedly increased in activated macrophages, and stimulation ofthese upregulated receptors by the PPAR activator PGJ2, has beenshown to inhibit the activity of NF-B, STAT and activator protein-1(AP-1). These transcription factors are all known to increase theexpression of genes encoding pro-inammatory cytokines (Ricoteet al., 1998a), production of which has also been demonstrated to bereduced by PPAR activation in human monocytes (Jiang et al., 1998).Non-steroidal anti-inammatory drugs, such as ibuprofen anddiclofenac, can activate PPAR at concentrations higher than thoserequired for their characteristic COX activity. This activation has alsobeen associated with the inhibition of cytokine production frommonocytes, suggesting that endogenous intermediates which nor-mally activate the COX pathway may potentially exert an additionalopposing anti-inammatory role through activation of PPAR.

The fact that PPAR activation stimulates cellular differentiationand apoptosis suggests that this approach may be benecial in thetreatment of cancers. Indeed, PPAR agonists have been demonstratedto have potent anti-tumour effects in breast (Mueller et al., 1998),prostate (Kubota et al., 1998) colon (Jackson et al., 2003) and gastric(Sato et al., 2000) malignancies in both isolated cell and whole animalstudies. However, it is not yet knownwhether these benets extend tothe clinical arena.

1.7.3. PPARDue to the ubiquitous nature of the expression of PPAR, it has

been implicated in a variety of physiological and pathophysiologicalprocesses. Like PPAR and , PPAR plays a role in the regulation ofcirculating lipid and glucose levels (Berger et al., 1999). PPAR has alsobeen suggested to modulate insulin resistance through activation ofalternatively activated macrophages which inhibit inammation inboth adipose tissue and liver (Kang et al., 2008; Odegaard et al., 2008).Furthermore, it appears likely that PPAR activation may be involvedin fertility and pregnancy, as PPAR is highly expressed in implanta-tion sites within the uterus (Lim et al., 1999; Ding et al., 2003). PPARwas initially implicated in tumour development (Gupta et al., 2000),but more recent work suggests that activation of PPAR may in factattenuate carcinogenesis (Harman et al., 2004). The fact that PPAR ishighly expressed in the central nervous system has generated muchinterest in a potential role in neural pathologies, and this is the subjectof ongoing research.

2. PPARs and the cardiovascular system

PPARs are widely expressed in both the vasculature and the

myocardium, as well as in immune cells such as monocytes and

251E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263macrophages. In addition to their role in transcriptional activation, inthe unbound state, PPAR-RXR heterodimers can also repress targetgene expression. Hence, PPARs can exert positive and negativeregulatory control over a range of genes involved in metabolism andinammation (Bensinger & Tontonoz, 2008). As a result, their impacton cardiovascular (patho)physiology stretches far beyond theirestablished effects on carbohydrate and lipid metabolism. This sectionwill discuss the involvement of different PPAR isoforms in variouscardiovascular pathologies and highlight how therapeutic activationof PPARs may prove benecial in preventing the development andprogression of CVD.

2.1. Atherosclerosis

Atherosclerotic vascular disease is one of the leading causes ofmortality in thewesternworld. It is principally an inammatory diseasecharacterised by high plasma concentrations of cholesterol, particularlyin the form of LDL. As elevated circulating lipids have long beenestablished as the principal risk factor for the development ofatherosclerosis, it was originally thought to be a process mainlyconsisting of the accumulation of lipidswithin the arterywall. However,it is now known to be a much more complex and multifaceted disease.

Atherosclerotic lesions occur in large andmedium-sized arteries asa result of increased lipid levels, hypertension, increased free radicals(e.g. from smoking) and diabetes. Their formation is triggered byendothelial cell activation and dysfunction causing the release ofvasoactive molecules and cytokines, which stimulate an inammatoryresponse and recruitment/migration of leukocytes into the arterialwall (Ross, 1999). Leukocyte migration relies on the interactionbetween endothelial cell adhesion molecules, such as vascular celladhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1(ICAM-1), E-selectin and P-selectin, and their cognate ligands oncirculating monocytes (Faggiotto et al., 1984; Springer, 1994).Increased expression of adhesionmolecules within the atheroscleroticlesion stimulates monocyte recruitment and transmigration into thearterial intima, and accumulation of lipids and extracellular matrixmay further amplify the local inammatory response (Van der Walet al., 1992).

Once within the subendothelial space, monocytes rapidly matureinto tissue macrophages which take up oxidised lipoproteins viascavenger receptors (Goldstein et al., 1979; Brown & Goldstein, 1990).Intracellular accumulation of cholesterol results in the characteristicformation of foam cells and stimulates macrophages to secretecytokines, growth factors and other mediators that promote smoothmuscle cell proliferation and potentiate the inammatory response,leading to arterial remodeling (Brown & Goldstein,1983). Apoptosis ofmacrophages and smooth muscle cells then occurs, which furtherenhances cytokine release. A continuing cycle of inammation andcell inltration causes progressive enlargement of the plaque, whichprotrudes into the arterial lumen blocking normal blood ow.Eventually, the plaque ruptures due to degradation by macrophage-induced matrix metalloproteinases (MMPs) and hydrolytic enzymes,resulting in thrombus formation and tissue infarction (Libby et al.,1996; Ross, 1999). The combined inuence of lipid dysregulation andinammation in atherogenesis strongly implies that PPARs, whichpositively inuence both processes, may play a benecial role inattenuating the development of this disease.

2.1.1. PPAR in atherosclerosisEndothelial cells (Inoue et al., 1998), vascular smooth muscle cells

(Staels et al., 1998) and monocytes/macrophages (Chinetti et al.,1998) are all known to express PPAR. In atherosclerosis, activation ofPPAR in these cells acts to reduce leukocyte recruitment, celladhesion, inammation and injury. Endothelial activation of PPARhas been demonstrated to downregulate cytokine-induced genes,

such as VCAM-1 and tissue factor, and to inhibit the release ofmonocytic chemotactic protein-1 (MCP-1), resulting in reducedinammatory cell adhesion and attenuation of atheroma develop-ment (Marx et al., 1999; Marx et al., 2000). A conicting studyreported that PPAR activation increased production of MCP-1 fromendothelial cells, suggesting a pro-inammatory role for PPAR (Leeet al., 2000). However, the consensus from the majority of studiesexamining the endothelial actions of PPAR agonists appears to bethat PPAR stimulation exerts positive anti-inammatory effects.

In addition to inhibiting the inammatory response, PPARagonists can also modify the release of vasoactive mediators, such asendothelin-1 (ET-1) (Delerive et al., 1999a) and nitric oxide (NO)(Goya et al., 2004) from the endothelium, to favour vasodilatation. It islikely that ET-1 is involved in atherogenesis as it is capable of inducingboth vascular smooth muscle cell proliferation and endothelial celladhesion molecules (McCarron et al., 1993) and also exerts chemo-tactic properties on monocytes (Achmad & Rao, 1992). Indeed, ET-1 ishighly expressed in atherosclerotic lesions and inhibition of ET-1 byPPAR agonists not only improves endothelial function but alsoreduces inammation (Jones et al., 1996).

In vascular smooth muscle cells the anti-inammatory actions ofPPAR can be demonstrated by PPAR agonist-induced inhibition ofinterleukin-1 (IL-1)-stimulated IL-6 production, prostaglandin synth-esis and COX-2 induction, effects which have been shown to occursecondary to downregulation of NF-B and induction of p38MAPK-dependent apoptosis (Chinetti et al., 1998; Staels et al., 1998; Diepet al., 2000). PPAR activators have also been found to attenuateatherosclerotic vascular remodeling by inhibiting smooth muscle cellproliferation and migration (Delerive et al., 1999b). Furthermore,vascular smooth muscle cell migration and atherogenic plaquedestabilisation may be prevented by PPAR-dependent inhibition ofmacrophage MMP-9 expression (Marx et al., 1998; Shu et al., 2000).

Activated PPAR not only inuences vascular inammation, reactiv-ity and remodeling, but can also positively alter macrophage lipidhandling within the plaque itself. PPAR activation has been shown toreduce macrophage triglyceride accumulation and to promote redis-tribution of cholesterol from intracellular stores to the plasmamembrane, making it available for HDL-dependent efux and reversetransport (Chinetti et al., 2001;Haraguchi et al., 2003; Chinetti-Gbaguidiet al., 2005b). In human macrophages, PPAR-mediated inhibition oflipoprotein lipase secretion results in reduced uptake of glycatedlipoprotein by these cells (Gbaguidi et al., 2002). In addition tomodifying macrophage cholesterol transport, PPAR can also inducenicotinamide adenosine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) production, stimulatingmodication of LDL and enabling it to act as a PPAR ligand to furtherinhibit the induction of inammatory mediators (Teissier et al., 2004).

Despite the overwhelming evidence supporting a benecial rolefor PPAR in atherosclerosis, preliminary studies in PPAR null micesuggest that these animals are actually protected against diseasedevelopment (Tordjman et al., 2001; Tordjman et al., 2007). However,it is possible that these conicting ndings may not have been relatedto the absence of PPAR, but to unintended disruption of one or moreother genes (Yagil & Yagil, 2007). Further research is needed to resolvethis issue and conrm the protective role of PPAR in atherosclerosis.

2.1.2. PPAR and atherosclerosisPPAR has a similar vascular prole to PPAR, with signicant

expression in endothelial cells (Satoh et al., 1999), smooth musclecells (Law et al., 2000) and monocytes/macrophages (Ricote et al.,1998b), and has many similar actions. Signicant levels of PPAR havebeen found in atherosclerotic lesions and its activation reducesmonocyte recruitment by the plaque (Marx et al., 1998). In endothelialcells, PPAR activation has several benecial actions on the inam-matory response, including inhibition of TNF and MCP-1 andattenuation of TNF-induced expression of VCAM-1 and ICAM-1

(Lee et al., 2000; Pasceri et al., 2000; Bruemmer et al., 2005). Like

252 E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263PPAR, PPAR also inhibits endothelial ET-1 production (Deleriveet al., 1999a) and stimulates NO release (Calnek et al., 2003), resultingin both vasodilatation and improved endothelial cell function. Inmacrophages, PPAR agonists have been demonstrated to bothincrease PPAR expression and inhibit MMP synthesis (Ricote et al.,1999), which contributes to their inhibitory effects on smooth musclecell proliferation. In addition, PPAR ligands are known to inhibitmonocyte/macrophage production of other inammatory cytokines,such as IL-6, IL-1 and TNF, by decreasing the activity oftranscription factors, such as NF-B (Jiang et al., 1998). In vascularsmooth muscle, PPAR agonists also attenuate cell migration andproliferation (Law et al., 2000). Supportive evidence for a benecialanti-inammatory role of PPAR in atherosclerosis is provided by astudy using an experimental mouse model, in which PPAR agonistswere found to limit intimal hyperplasia and reduce lesion size andinammation (Law et al., 1996).

Although the majority of studies support a benecial effect ofPPAR in atherogenesis, some investigators have suggested thatPPAR may be deleterious in this situation. PPAR has been found tostimulate genes involved in cholesterol uptake by macrophages, suchas CD36, and this process may be accentuated by further activation ofPPAR by oxidised LDL (Nagy et al., 1998; Han et al., 2000). Thisimplies that PPAR may promote foam cell formation; however,macrophage cholesterol content is not increased by PPAR agonists inthe presence of acelytated LDL (Chinetti et al., 2001). Furthermore, ithas been demonstrated that PPAR ligands can signicantly inhibitthe development of atherosclerosis, in spite of elevated levels of CD36in the arterial wall (Li et al., 2000). Activated PPAR has been shown toreduce macrophage triglyceride accumulation, lipoprotein secretionand glycated lipoprotein uptake, whilst enhancing HDL-dependentcholesterol efux and reverse cholesterol transport (Akiyama et al.,2002; Gbaguidi et al., 2002; Haraguchi et al., 2003). These studies addfurther complexity to an already complicated picture; how can PPARactivation increase CD36 in the absence of a concomitant increase inmacrophage LDL? The unied model (Zhang & Chawla, 2004) linksthese two conicting arguments by suggesting that PPAR reducesaccumulation of atherogenic oxidised LDL in the vessel wall byincreasing both macrophage uptake and efux via upregulation ofCD36. In light of all available evidence, this theory seems to be themost plausible and supports the benecial effects of PPAR inatherosclerosis. A recent study by Babaev et al. (2005) employinggene-modied mice provides additional evidence to support thesuggestion that PPAR-mediated immune cell modulation is bene-cial in atherosclerosis. In this study macrophage-specic deletion ofPPAR resulted in a marked increase in atherosclerosis developmentsuggesting that PPAR-mediated macrophage activation is protectivein this pathology. Recent studies have subsequently revealed theimportance of the PPAR as a key regulator of macrophage activityand function (Majai et al., 2007; Odegaard et al., 2007). Furthermore,in human atherosclerotic lesions PPAR activation has been reportedto promote differentiation of proatherogenic M1 macrophages into analternative anti-inammatory phenotype, M2, which could protectagainst the development of atherosclerosis (Bouhlel et al., 2007). Adenitive role for PPAR in atherogenesis could potentially beestablished with further studies in gene-modied mice such as theglobal PPAR/ or the endothelial cell-specic PPAR/. Devel-opment of a vascular smooth muscle cell-specic PPAR/ mousewould also signicantly aid the identication of the role of PPAR inatherosclerosis (Duan et al., 2008).

2.1.3. PPAR and atherosclerosisThe ubiquitous nature of PPAR expression suggests that its

activation may play a role in the development of atherosclerosis.Indeed, recent research has extended beyond the more establishedeffects of PPAR and to begin to focus on PPAR, which also appears

to exert benecial actions on atherogenesis. In a mouse model ofatherosclerosis, PPAR activation has been found to decrease expres-sion of MCP-1, ICAM-1 and inammatory cytokines and to attenuatedisease development (Li et al., 2004; Graham et al., 2005). In themacrophage, PPAR activation reduces circulating levels of pro-inammatory cytokines and TNF expression and has a positive effecton lipid handling by promoting cholesterol efux, reverse cholesteroltransport and fatty acid catabolism (Oliver et al., 2001; Graham et al.,2005; Lee et al., 2006). Early work therefore suggests that activation ofPPAR may be of potential therapeutic benet in the treatment ofatherosclerosis. However, more detailed studies are required in orderto dene its precise role in disease development and progression.

2.2. Hypertension

Chronic hypertension is a primary risk factor for CVD, particularlyatherosclerosis and heart failure (Zahradka, 2007). Despite improvedmedical treatments, its incidence is rapidly increasing and is set to reachepidemic proportions. Several factors inuence the development ofhypertension, including age, gender, the existence of associatedconditions such as diabetes and obesity, and lifestyle factors such asalcohol consumption and smoking. The development of pathologicalhypertension usually occurs secondary to endothelial dysfunction andan imbalance between vasoconstrictors, such as angiotensin II (Ang II)and ET-1, and vasodilators, such as NO (Schulman et al., 2006).Atherosclerosis itself can also cause hypertension due to protrusion ofthe lesion into the lumen, resulting in narrowing of the blood vessel,which cannot be counteracted by local vasorelaxant mechanisms (Ross,1999).

To date the role of the different PPAR isoforms in hypertensionhave been examined in a number of commonly used experimentalmodels. These include (1) the deoxyycorticosterone acetate (DOCA)-salt model, which is associated with enhanced expression ofpreproET-1; (2) the spontaneously hypertensive rat (SHR), in whicha genetic mutation results in alteration of the reninangiotensinaldosterone system, causing sodium retention and elevated bloodpressure in the absence of concomitant obesity and diabetes (Koboriet al., 2005); (3) chronic Ang II infusion. Interestingly, PPAR agonistshave been demonstrated to have direct vasorelaxant effects (Goyaet al., 2004), suggesting that they may hold therapeutic potential aseffective anti-hypertensive agents.

2.2.1. PPAR and hypertensionPPAR is widely expressed in all vascular cell types suggesting that

its activation may be involved in the control of blood pressure bymodulation of vascular tone. Indeed, PPAR-dependent regulation ofblood pressure has been demonstrated in each of the aforementionedexperimental models of hypertension. In Ang II-infused rats, thePPAR agonist, docosahexaenoic acid (DHA) was found to reduceblood pressure and attenuate vascular remodeling, possibly byinhibiting the development of NADPH oxidase-induced endothelialdysfunction (Diep et al., 2002a). In the same experimental model, thePPAR agonist, fenobrate-induced a signicant decrease in bloodpressure which was associated with reduced expression of vascularinammatory mediators (Diep et al., 2004). However, in the DOCA-salt model of hypertension, fenobrate failed to normalise meanarterial blood pressure, despite PPAR activation causing a signicantreduction in NADPH oxidase-dependent superoxide generation(Iglarz et al., 2003). Interestingly, clobrate was also found to inhibitNADPH oxidase activity in this model, but in contrast to fenobrate,signicantly reduced blood pressure, an effect whichwas attributed toinhibition of endothelial ET-1 production (Delerive et al., 1999a;Newaz et al., 2005). In the SHR, increased PPAR expression has beenfound in both whole blood vessels and cultured vascular smoothmuscle cells from these animals (Diep & Schiffrin, 2001). In thismodel, chronic PPAR activation with fenobrate has also been

demonstrated to be associated with a reduction in systolic blood

253E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263pressure (Li et al., 2008), although other investigators have failed toreport anti-hypertensive effects (Wu et al., 2004).

Studies using PPAR/mice have produced conicting ndings inregard to its role in blood pressure regulation. Deletion of PPARhas been shown to signicantly increase (Newaz et al., 2005), decrease(Guellich et al., 2007) andhaveno signicant effect (Loichot et al., 2006)on systolic blood pressure compared with wild-type controls. Interest-ingly, Newaz et al. (2005) found that the increased blood pressure intheir PPAR/ mice could be abolished by the NO synthase (NOS)inhibitor, N-nitro-L-arginine methyl ester (L-NAME), suggesting thatPPARmay modulate endothelial NO production (Goya et al., 2004).

2.2.2. PPAR and hypertensionPPAR is expressed in vascular smooth muscle and endothelial

cells, and glitazone-induced activation has been found to exert bloodpressure-lowering effects in insulin-resistant fatty Zucker rats,although the mechanisms of action were unclear (Walker et al.,1999). In Ang II-induced hypertension, the PPAR agonists, pioglita-zone and rosiglitazone caused a signicant decrease in blood pressure,in addition to benecial actions on cell growth, endothelial functionand vascular inammation. These effects were found to be mediatedvia inhibition of DNA synthesis, NF-B activity, and endothelial/platelet adhesion molecule expression (Diep et al., 2002b). It has alsobeen suggested that the apparent hypotensive effects of PPARagonists in this model may result from downregulation of the Ang IItype 1 receptor (Sugawara et al., 2001). More recently, investigatorshave begun to examine how PPAR agonists directly affect the reninangiotensin system, with initial studies suggesting that PPARactivation stimulates renin gene expression (Todorov et al., 2007).This could potentially lead to increased Ang II production with aresultant increase in blood pressure, but is contradictory to thebenecial hypotensive effects previously observed with PPARagonists. One possible explanation could be that a delicate balanceexists in the vasculature between the benecial effects of PPAR andthe deleterious inuence of the reninangiotensin system, so tippingthe balance in favour of the latter could potentially be a key factorunderlying the development of hypertension (Weatherford et al.,2007).

In DOCA-salt hypertensive rats, mean arterial blood pressure wasalso normalised by administration of rosiglitazone, an effect whichwas not seen with the PPAR agonist, fenobrate (Iglarz et al., 2003).However, both PPAR activators were found to attenuate the increasedpreproET-1 expression and concentric hypertrophy which are char-acteristic of this model. Although only the PPAR agonist preventedhypertensive endothelial dysfunction, both agonists were found tosignicantly reduce ROS production, implying that this is not theprimary mechanism underlying impaired vascular function in thismodel. PPAR has also been shown to antagonise endothelial ET-1secretion, so its anti-hypertensive effects in this ET-1-dependentmodel of hypertension are maybe not surprising (Satoh et al., 1999).

In the SHR, the PPAR agonist, pioglitazone has been demonstratedto lower blood pressure (Verma et al., 1998; Grinsell et al., 2000).However, when these animals were treated with a NOS inhibitor, L-NAME, effects on blood pressure, metabolism and serum NO levelswere no longer evident, although pioglitazone did prevent vascularinammation and the development of atherosclerosis (Ishibashi et al.,2002). Furthermore, administration of ciglitazone to SHRs has beenfound to ameliorate vascular endothelial dysfunction via increasedNOS activity (Smiley et al., 2004), supporting the suggestion thatstimulation of NO production is essential to this PPAR-mediatedreduction of blood pressure.

It has previously been suggested that downregulation of PPARmay be responsible for the vascular proliferation, migration, inam-mation and brosis found in the SHR (Schiffrin, 2005). Indeed,vascular expression of PPAR proteins was found to be reduced at

21 weeks in this model, compared to age-matched wild-type animals,although at a younger age (513 weeks) no signicant difference wasdetected (Wu et al., 2004). Treatment with the PPAR agonist,rosiglitazone from 513 weeks, attenuated the subsequent develop-ment of, hypertension, although PPAR activation had no signicanteffect (Wu et al., 2004). However, PPAR activation in the SHR, whilstconferring benecial effects on blood pressure, was also found to beassociated with left ventricular (LV) hypertrophy. In contrast to thisstudy, there are previous reports of increased vascular expression ofboth PPAR and in SHR's at 16 weeks (Diep & Schiffrin, 2001), so theprecise role of PPAR in this model is far from certain. Taken together,it appears that PPAR attenuates the development of hypertension viaa mechanism involving increased NO production, reduced ET-1secretion and inhibition of NADPH oxidase. However, the potentialclinical benet may be limited by side effects such as weight gain,oedema, headache and visual disturbances which are commonlyassociated with PPAR receptor agonists.

The effect of PPAR deletion on blood pressure has recently beenexamined in the newly developed generalised PPAR/ mouse, inwhich the embryonic lethality of global PPAR deletion was rescuedby preserving trophoblastic PPAR expression (Duan et al., 2007).These mice were found to exhibit hypotension, which was resistant tocorrection by high salt loading. Further ex vivo studies revealed thatthe vasculature of these animals was more sensitive to endothelium-dependent relaxation caused by muscarinic stimulation (in theabsence of changes in endothelial NOS expression or phosphoryla-tion) and less responsive to -adrenergic contractile agents, leadingthe authors to conclude that the hypotension observed in thesePPAR/ mice occurred via a mechanism involving increasedvascular relaxation. Whilst previous studies have reported PPARactivation to also cause hypotension (Grinsell et al., 2000; Duan et al.,2007), this was found to occur in the presence of increased endothelialNOS expression which was not observed in vessels from generalisedPPAR/mice. Interestingly, this may suggest that PPAR is capable ofmodulating blood pressure via divergent mechanisms, although moredetailed studies in the generalised PPAR/ are clearly required tofurther investigate this possibility.

2.2.3. PPAR and hypertensionDespite the recent discovery of specic PPAR ligands, such as

L165041 and GW0742X, the role of PPAR in the regulation of vasculartone and development of hypertension remains unknown.

2.3. Cardiac ischaemiareperfusion

Chronic heart failure affects up to 2% of the adult population in thewestern world, the most common cause of which is myocardialinfarction (MI). MI results from coronary artery occlusion, usuallyoccurring after atherosclerotic plaque rupture. This causes ischaemiaof the cardiac muscle, the severity of which varies depending on thelocation of the occlusion within the coronary vasculature. Prolongedischaemia leads to cardiomyocyte death which is followed by a seriesof structural and functional alterations in the viable myocardium,known as cardiac remodeling. In particular, adaptive changes in theextracellular matrix and in cardiomyocyte biology occur, which areinitially able to maintain contractile function. However, progressivecardiac remodeling leads to chamber dilatation, contractile dysfunc-tion and ultimately heart failure (Snghedauw, 1999). The resultantmetabolic changes may also lead to the development of potentiallylife-threatening arrhythmias. Myocardial reperfusion is the standardrst-line treatment in acute MI, aiming to restore blood ow to theischaemic myocardium and promote tissue survival. However,reperfusion itself has been found to exacerbate ischaemic damage,causing further depression of cardiac function (Piper et al., 2003).Various experimental models have been employed to study eithermyocardial ischaemia with subsequent reperfusion or cardiac remo-

deling associated with chronic MI.

254 E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263Inltration of neutrophils and macrophages is known to enhancethe inammatory response to myocardial ischaemia (Frangogianniset al., 2002) and in combination with rapid accumulation of ROSwithin the ischaemic zone can lead to tissue necrosis upon reperfusion(Li et al., 1999). This is further amplied by activation of redox-sensitive transcription factors, such as NF-B and AP-1, which controlthe expression of pro-inammatory mediators, such as IL-12 andTNF. Indeed, in an experimental rat model, inhibition of NF-B hasbeen demonstrated to reduce reperfusion injury after a brief period ofischaemia (Onai et al., 2004). Furthermore, upregulation of AP-1 hasbeen observed in cardiomyocytes in the presence of increased levels ofROS (Aggeli et al., 2006), such as those observed during ischaemia andreperfusion, suggesting that this transcription factor may be involvedin the pathogenesis of ischaemia and subsequent reperfusion.Interestingly, PPARs have been shown to exert their anti-inammatoryeffects via NF-B and AP-1 inhibition, suggesting a potential mechan-ism by which agonists of these receptors may be benecial inattenuating ischaemiareperfusion injury (Chinetti et al., 1998;Dragomir et al., 2006; Smeets et al., 2007).

2.3.1. PPAR and cardiac ischaemiareperfusionPPAR is abundantly expressed in the heart, where it plays an

important role in the regulation of fatty acid oxidation by inducing theexpression of genes encoding proteins involved in fatty acid uptakeand metabolism. Under normal physiological conditions, fatty acidsact as the primary energy source for adult cardiomyocytes. In theischaemic myocardium and after reperfusion PPAR has been foundto be downregulated, although it is unclear whether this is a benecialor detrimental adaptation (Dewald et al., 2005). In an attempt toclarify the precise role of PPAR in ischaemiareperfusion andestablish whether PPAR ligands may be benecial in its treatment,many studies have examined their effects in various experimentalmodels of ischaemia and/or reperfusion.

Overall, PPAR appears to have a benecial effect in ischaemiareperfusion. In an in vivo rat model of ischaemiareperfusion, infusionof the PPAR agonist, Wy 14,643 prior to coronary artery occlusionresulted in a signicant decrease in infarct size, which was associatedwith increased myocardial contractility (Wayman et al., 2002a;Bulhak et al., 2006), suggesting that PPAR activation may attenuatethe depression of cardiac function that typically occurs duringreperfusion (Yeh et al., 2006). The cardioprotective effect of PPARactivation has been linked to inhibition of the NF-B pathway (Yueet al., 2003; Yeh et al., 2006) and the associated decrease in expressionof inammatory mediators. Another PPAR agonist, GW7647 has alsobeen demonstrated to signicantly reduce infarct size in a mousemodel of ischaemiareperfusion (Yue et al., 2003). This positive effecton ischaemiareperfusion was abolished in PPAR/mice, suggest-ing that it occurs through direct activation of the receptor. Adminis-tration of the brates, clobrate (Wayman et al., 2002b; Tian et al.,2006) and fenobrate (Tabernero et al., 2002) have also been shownto have benecial effects in ischaemiareperfusion both ex vivo and invivo, adding further support to the idea that PPAR activation may becardioprotective.

Despite the widely reported benecial effects of Wy 14,643 inischaemiareperfusion, a conicting study has demonstrated that it isassociated with negative effects in an in vivo mouse model ofrepetitive ischaemiareperfusion (Dewald et al., 2005). However,signicant differences between the time course, metabolic effects andmechanisms underlying ischaemic injury in this model comparedwith the single ischaemic insult employed by most studies, mayaccount for the lack of effect of the PPAR agonist. Findings fromPPAR gene-modied mice are also somewhat contradictory. Forexample, whereas the presence of PPAR has been found to bebenecial in preserving cardiac function in ischaemiareperfusion(Tabernero et al., 2002), isolated perfused hearts from PPAR/mice demonstrated improved function after ischaemia (Panagia et al.,2005; Sambandam et al., 2006) and those from animals overexpres-sing PPAR exhibited reduced recovery (Sambandam et al., 2006).Furthermore, some investigators have demonstrated a positive effectof PPAR ligands against hypoxic damage (Wayman et al., 2002a,b;Bulhak et al., 2006), whilst others have reported that they have nosignicant effect on (Aasum et al., 2003) or are deleterious to(Sambandam et al., 2006) cardiac functional recovery after ischaemia.A likely explanation for these conicting ndings appears to be thechoice of experimental model, as in general, in vivo studies havetended to show PPAR agonists to exert cardioprotective effects,whereas ex vivo studies have produced varying results. The isolatedheart preparation, for example, is not subject to the neurohumoralinuences experienced in vivo and the use of pharmacological PPARagonists versus PPAR gene-modied animals may also contribute tothe diversity of experimental ndings.

2.3.2. PPAR and cardiac ischaemiareperfusionAs PPAR is predominantly expressed in adipocytes, the majority

of studies have tended to focus on its predominant role in lipidmetabolism. As such, the precise physiological role of PPAR in themyocardium has yet to be fully established. PPAR has, however, beenfound to be expressed in rat heart and PPAR agonists have beenshown to reduce myocardial infarct size (Wayman et al., 2002b).Overall, the ndings from studies on the effects of PPAR activation inischaemiareperfusion are much more consistent than those foundwith PPAR, suggesting that PPAR agonists may be of greaterpotential therapeutic benet in this setting.

Studies employing chemically different ligands to activate PPAR,such as the thiazolidinediones (rosiglitazone, troglitazone, pioglita-zone and ciglitazone) and prostaglandins (15d-PGJ2 and PGA1) havedemonstrated signicant reduction in infarct size and improvement ofmyocardial function. Rosiglitazone has been shown to reduce infarctsize in various in vivo (Yue et al., 2001; Wayman et al., 2002b; Liuet al., 2004; Molavi et al., 2006) and ex vivo (Khandoudi et al., 2002;Sidell et al., 2002) experimental models of ischaemiareperfusion.Furthermore, signicant recovery of LV developed pressure has beenobserved during post-ischaemic reperfusion after rosiglitazone treat-ment (Gonon et al., 2007). Pioglitazone and ciglitazone have beenfound to exert similar positive effects to rosiglitazone in all models ofischaemiareperfusion, causing signicant infarct size reduction andimproved cardiac function (Wayman et al., 2002b; Ito et al., 2003;Sivarajah et al., 2005; Wynne et al., 2005; Zingarelli et al., 2007). In anin vivo pig model of myocardial ischaemiareperfusion, the PPARagonist troglitazone was found to improve recovery of LV systolic anddiastolic function (Zhu et al., 2000), a nding which is supported bysimilar studies using other animal models (Shimabukuro et al., 1996;Lee & Chou, 2003). In addition, the high afnity endogenous ligand forPPAR, 15d-PGJ2 (which also has PPAR-independent actions; Kaplanet al., 2007), has been found to reduce infarct size and improve cardiacfunctional recovery after ischaemia, which suggests that the effects ofPPAR agonists are mediated via a direct action on the receptor.Indeed, inhibition of PPAR with the specic receptor antagonist,GW9662, results in a signicant increase in infarct size followingischaemiareperfusion (Sivarajah et al., 2005). However, in contrast tothe majority of evidence implying that thiazolidinediones arebenecial in ischaemiareperfusion, troglitazone was shown tosignicantly increase the incidence of ventricular arrhythmias duringa period of 90 min ischaemia followed by the same duration ofreperfusion in an in vivo pig model (Xu et al., 2003).

Several different mechanisms have been suggested to mediate thecardioprotective effects of PPAR, such as inhibition of NF-B,(Wayman et al., 2002b), reduced inltration of leukocytes (Yueet al, 2001; Ito et al., 2003) and inhibition of apoptosis (Liu et al.,2004). PPAR agonists have been demonstrated to rapidly inducetheir cardioprotective effects, within minutes of administration, and

a bolus dose of ligand prior to ischaemia is just as effective in

255E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263attenuating myocardial injury as continual infusion. These observa-tions suggest that the benecial effects of PPAR activation inischaemiareperfusion are not mediated through longer-term altera-tions in gene expression, although they may occur secondary to theiranti-inammatory actions.

Few studies have examined the effects of PPAR ( or ) activationin experimental models of chronic MI, in which permanent coronaryartery occlusion leads to cardiomyocyte death within the ischemiczone and remodeling of the viable myocardium. Those which havedone have produced conicting ndings. For example, the PPARagonist, fenobrate has been found to accelerate the development ofLV hypertrophy (Morgan et al., 2006), whereas the PPAR agonist,rosiglitazone has been reported to both increase mortality (Lygateet al., 2003) and improvemyocardial remodeling (Geng et al., 2006). Itis possible that these inconsistent results may be due to differences inexperimental study design. Nonetheless, signicant further research isrequired in order to delineate the precise role of PPARs in chronicischaemic remodeling.

2.3.3. PPAR and cardiac ischaemiareperfusionDespite the preponderance of evidence suggesting that activation

of PPAR and is cardioprotective in ischaemiareperfusion, the roleof PPAR has yet to be established.

2.4. Cardiac hypertrophy

LV hypertrophy is an independent risk factor for heart failure,arrhythmia and sudden death and is one of themost potent predictorsof adverse cardiovascular outcomes in hypertensive patients (Healey &Connolly, 2003; Gradman & Alfayoumi, 2006). It is characterised bymaladaptive changes in myocardial structure and function, which arecollectively known as cardiac remodeling. The heart initially compen-sates for the increasedwall stress by undergoing signicant alterationsin cardiomyocyte biology and in the extracellular matrix. However,progressive LV hypertrophy combined with loss of collagen cross-linking and myocyte slippage causes increased wall stress leading tocardiac chamber dilatation, contractile dysfunction and ultimatelydecompensated congestive heart failure (Snghedauw, 1999; Frey &Olsen, 2003). Despite improved clinical management of hypertensionwith agents such as angiotensin-converting enzyme (ACE) inhibitorsand -blockers, which attenuate cardiac remodeling and havemorbidity/mortality benets, there remains a substantial incidenceof heart failure even in optimally-treated patients (Francis & Young,2001). A detailed understanding of the complex regulatory mechan-isms underlying the pathogenesis of cardiac remodeling is thereforeessential to inform the development of more effective treatments.

The development of cardiac hypertrophy is inuenced by severalfactors such as age, weight and obesity. It may also develop in theabsence of increases in blood pressure in patients with obesity anddiabetes, where the heart acts to compensate for the loss of functionalheart muscle through the effects of disease and injury (Otto et al.,2004). Changes in structure and function of the heart are mediated bya variety of mechanical, neuronal and hormonal factors.

2.4.1. PPAR and cardiac hypertrophyIn the normal adult heart, fatty acids serve as the chief energy

substrate (Taegtmeyer, 1994) as they are more efcient, providing agreater yield of ATP compared to either glucose or lactate. Circulatingfatty acids are transported into the cardiomyocyte, where they aremetabolised via mitochondrial -oxidation. In contrast, the fetal heartrelies primarily on glucose and lactate due to its relatively hypoxicenvironment, as glycolytic production of ATP is more oxygen efcientthan fatty acidmetabolism. In the neonatal heart, the capacity for fattyacid oxidation rapidly increases in parallel with mitochondrialproliferation within the cardiomyocyte, establishing fatty acids as

the primary source of ATP.PPAR activation in the heart stimulates upregulation of genescontrollingmitochondrial fatty acid uptake, which results in increasedfatty acid metabolism and generation of ATP (Brandt et al., 1998; vander Lee et al., 2000a,b; Vosper et al., 2002). In the hypertensive heart,the increased demands on the myocardium trigger signicantalterations in energy metabolism. Both clinical and experimentalstudies in hypertrophied and failing hearts demonstrate a decrease inthe expression of genes involved in fatty acid oxidation, with anincrease in glucose oxidation, although in this setting the energyprovided by this alternative source does not correspond to thatpreviously supplied via fatty acid metabolism (Bishop & Altschuld,1970; Christie & Rodgers, 1994; Takeyama et al., 1995; Sack et al.,1996). This reversion to the fetal genotype is a characteristic feature ofcardiac hypertrophy, and occurs not only in genes involved inoxidative metabolism, but also in those controlling other aspects ofcardiomyocyte biology. As PPAR is the principal regulator ofmyocardial fatty acid oxidation, it seems likely that the decreasedexpression of genes involved in fatty acid oxidation as observed incardiac hypertrophy may be a consequence of altered PPAR activityor expression.

PPAR was initially implicated in cardiac hypertrophy whenchildren with congenital defects in fatty acid oxidation were foundto develop the disease (Kelly & Strauss, 1994). In adults, variations inthe genes encoding for PPAR have also been shown to inuence bothphysiological and pathological hypertrophic growth of the myocar-dium (Jamshidi et al., 2002). In a mouse model of pressure overload-induced by transverse aortic constriction, Barger et al. (2000)reported downregulation of PPAR and several of its target genes inparallel with signicant increases in morphometric hypertrophy andatrial natriuretic factor (ANF) expression, suggesting that down-regulation of PPAR may be responsible for reversion to the fetalmetabolic genotype in the hypertrophied myocardium.

Whether the altered activity of PPAR in hypertrophy is adaptiveor indeed related to myocardial pathology has been the subject ofintense debate. In pressure overload-induced cardiac hypertrophy,reversion to the fetal genotype is initially thought to be an adaptiveand benecial response (Frey & Olsen, 2002), as glucose oxidationsubstitutes for fatty acid metabolism to reduce oxygen consumption.However, with progressive hypertrophy, reduced ATP production viathe glucose oxidation pathway, results in cardiac contractile dysfunc-tion and myocardial lipid accumulation and toxicity (Barger & Kelly,2000). This suggests that although downregulation of PPAR may beinitially benecial, it may also be maladaptive during the decom-pensated stage of cardiac hypertrophy.

Many studies have attempted to clarify the precise role of PPARin cardiac hypertrophy, but conicting ndings have only served tofurther confuse the issue. An early study employing a rat model ofascending aortic constriction for 7 days found that pharmacologicalreactivation of PPAR with Wy 14,643 had no signicant effect onmorphological hypertrophy or induction of ANF (Young et al., 2001).Surprisingly, reactivation of PPAR in the hypertrophied heart alsoled to severe depression of cardiac power and efciency assessed exvivo, suggesting that downregulation of PPAR may be a necessaryadaptation to maintain myocardial function. Indeed, administrationof Wy 14,643 was also found to cause a substantial decline inglucose oxidation, rather than the anticipated increase in fatty acidoxidation, which would result in myocardial energy starvation, thusaccounting for the observed cardiac dysfunction (Young et al.,2001). In another study, normal rats fed with Wy 14,643 for26 weeks were found to develop signicant morphometric cardiachypertrophy, indicating that PPAR activation alone can act as ahypertrophic stimulus (Hamano et al., 2001). Furthermore, the sameauthors reported that co-application of Wy 14,643 and clobrate to arat cardiomyocyte cell line signicantly increased transcription ofthe pro-hypertropic marker gene, myosin light chain-2, supporting

their in vivo observation (Hamano et al., 2001). Cardiac-specic

overexpression of PPAR in mice has also been demonstrated to bedeleterious, resulting in signicantly reduced myocardial glucoseoxidation. Interestingly, these mice, unlike the previously describedrat models, exhibited increased fatty acid oxidation, althoughthis was not sufcient to prevent cardiac dysfunction (Finck et al.,2002). This suggests that PPAR downregulation is benecial, butthat overexpression of PPAR induces a complete reliance on fattyacid oxidation, leading to oxygen depletion and loss of normalfunction. However, the function of PPAR in this rather articialsituation may not be indicative of its role at physiological expressionlevels.

Although many studies have demonstrated a negative inuence ofPPAR in cardiac hypertrophy, there are an increasing number ofconicting reports indicating that PPAR activation may actually bebenecial in this setting. In vivo activation of PPAR by fenobrate hasbeen found to inhibit the development of myocardial brosis in ratssubjected to pressure overload via inhibition of ET-1-mediatedbroblast proliferation (Ogata et al., 2002). Fenobrate treatmenthas also been found to attenuate the development of myocardialhypertrophy and brosis and to preserve in vivo contractile function inDahl salt-sensitive rats, through inhibition of NF-B-mediatedinammation (Ichihara et al., 2006). These effects were thought tooccur via inhibition of the inammatory response through down-regulation of NF-B, and deactivation of redox-regulated transcriptionfactors and the subsequent reduction in ROS production. In DOCA-salthypertensive rats overexpressing ET-1, Iglarz et al. (2003) conrmedthat fenobrate administration signicantly attenuated cardiac bro-sis and remodeling, which was associated with decreased myocardial

Studies using PPAR/ mice have predominantly shown thatabsence of the receptor is detrimental to cardiac function. A recentstudy by Smeets et al. (2008b) demonstrated that PPAR/ micesubjected to chronic pressure overload developed signicantly morepronounced cardiac hypertrophy and contractile dysfunction com-pared to wild-type controls. Other groups using the same micehave demonstrated that the absence of PPAR results in reducedmyocardial brosis and ex vivo contractile dysfunction in isolatedhearts, which was more susceptible to further deterioration but couldbe rescued by increasing cardiac glucose utilisation (Luptak et al.,2005; Loichot et al., 2006). It has also been reported that fenobratetreatment further exacerbates cardiac hypertrophy, brosis andremodeling in PPAR/ mice subjected to chronic pressure over-load, whilst reducing remodeling inwild-type animals, indicating thatPPAR agonists may exert deleterious effects which are independentof receptor activation (Duhaney et al., 2007). This may also explain thenegative ndings in respect to pharmacological PPAR reactivationwithWy 14,643 in the hypertrophiedmyocardium. As all studies usingfenobrate in wild-type animals have reported positive effects oncardiac hypertrophy, this suggests that the benecial actions of PPARactivation may outweigh any negative receptor-independent effects.

The link between PPAR activation and inhibition of ROS whichhas been borne out by many of the pharmacological studies issupported by the ndings of another study in PPAR/ mice, inwhich myocardial expression of the ROS-scavenging enzyme, super-oxide dismutase, was found to be signicantly reduced and associatedwith oxidative damage to cardiac myosin. The authors suggested thatdecreased superoxide dismutase activity may lead to elevated

ionphy

256 E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263inammation. Activation of PPAR by fenobrate was also shown toinhibit ET-1-induced cardiac hypertrophy in isolated rat cardiomyo-cytes (Li et al., 2007), adding further weight to the suggestion thatfenobrate-induced PPAR activation inhibits cardiac hypertrophy.Furthermore, in vitro studies using rat neonatal cardiomyocytes havereported fenobrate to inhibit ET-1-induced hypertrophy and proteinsynthesis (Liang et al., 2003; Irukayama-Tomobe et al., 2004) and Wy14,643 to inhibit hypertrophy via inhibition of NF-B (Smeets et al.,2008a).

Fig. 4. Role of ROS and PPAR in the development of cardiac hypertrophy. The productintracellular responses which eventually lead to the development of myocardial hypertro

of PPAR, leading to decreased fatty acid oxidation and glucose metabolism which is charamyocardial ROS production, which could be responsible for theimpaired cardiac contractile function observed in PPAR/ mice(Guellich et al., 2007). The role of ROS and the downregulation ofPPAR in hypertrophy development are illustrated in Fig. 4.

Taken together, it appears that PPAR downregulation or absenceof the receptor is detrimental in cardiac hypertrophy. Pharmacologicalreactivation of PPAR may attenuate the progression of hypertrophyand associated contractile dysfunction, although a more completeunderstanding of the receptor-independent actions of PPAR agonists

of ROS and downregulation of PPAR (as a result of hypertension) initiates a series ofand remodeling. It has been suggested that ROS may also stimulate the downregulation

cteristic of cardiac hypertrophy.

257E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263is required before their potential therapeutic benet can be accuratelyassessed.

2.4.2. PPAR and cardiac hypertrophySimilar to PPAR, the precise involvement of PPAR in cardiac

hypertrophy remains somewhat controversial. PPAR is known to beexpressed at a low level in the heart (Escher & Wahli et al., 2000;Wayman et al., 2002b). Nonetheless, the majority of studies investi-gating the effects of PPAR agonists in cardiac hypertrophy suggestthat they may play a protective role, although the mechanismsunderlying these actions are unclear. Of note, agonists for PPARhave been found to have no signicant effect on cardiomyocyteexpression of target genes involved in fatty acid oxidation (Barger &Kelly, 2000). Consistent with this nding, a study by Gilde et al. (2003)in rat neonatal cardiomyocytes revealed that the rate of fatty acidoxidation signicantly increased after exposure to PPAR and PPARligands, but not to PPAR ligands. Similarly, the fatty acid-mediatedexpression of fatty acid-handling proteins was mimicked by PPARand PPAR but not by PPAR ligands. Furthermore, in adult ratcardiomyocytes activation of PPAR had no signicant effect on fattyacid metabolism but prevented cell hypertrophy, suggesting thatinactivation of PPAR may enable the development of hypertrophy(Pellieux et al., 2007). Taken together, it appears that PPAR may notexert its cardiac effects via regulation of myocardial metabolism.

Several studies employing various in vitro and in vivo experimentalmodels have demonstrated that PPAR activation signicantlyreduces the development of cardiac hypertrophy and brosis. Boththe synthetic agonists of PPAR, the thiazolidinediones, and theendogenous activator, 15d-PGJ2, have been found to attenuatemechanical strain and ET-1-induced hypertrophy in neonatal cardi-omyocytes, respectively (Yamamoto et al., 2001; Liang et al., 2003).This effect was shown to bemediated via PPAR-dependent inhibitionof the NF-B pathway (Yamamoto et al., 2001) and could be reversedwith a specic PPAR antagonist, leading to induction of the pro-hypertrophic marker gene, brain natriuretic peptide (BNP) (Liang etal., 2003). This latter observation implies that the benecial effects ofPPAR agonists in cardiac hypertrophy could be due to direct receptoractivation. Indeed, the heterozygous PPAR+/mouse has been foundto exhibit cardiac hypertrophy, which is further accentuated uponimposition of chronic pressure overload (Asakawa et al., 2002).Rosiglitazone has also been found to signicantly reduce cardiacremodeling and brosis in DOCA-salt hypertensive rats (Iglarz et al.,2003), as has ciglitazone in a mouse model of chronic pressureoverload-induced by abdominal aortic constriction (Henderson et al.,2007). Ciglitazone also attenuated pressure overload-inducedincreases in the expression of the NADPH oxidase subunit, Nox4,suggesting a potential role for ROS in its cardioprotective actions(Henderson et al., 2007).

Despite the large amount of evidence supporting a benecial rolefor PPAR in cardiac hypertrophy, several studies have reportedcontradictory ndings. In vivo administration of rosiglitazone wasshown to accentuate the development of cardiac hypertrophy in SHRs(Wu et al., 2004). Furthermore, chronic treatment of normal rats withboth the selective PPAR agonist, X334, and the novel thiazolidine-dione, T-174, were found to induce cardiac hypertrophy (Arakawa etal., 2004; Edgley et al., 2006). However, in the latter study, this effectwas attributed to an increase in blood volume following T-174treatment, rather than a direct effect on the myocardium. Indeed,hypervolaemia and oedema are known to be common side effects ofthiazolidinedione treatment (Edrmann & Wilcox, 2008), which mayalso account for the hypertrophic effects observed in the other twostudies. Interestingly, rosiglitazone has been reported to inducecardiac hypertrophy in both cardiomyocyte-specic PPAR/ miceand wild-type littermate controls via activation of distinctly differentpathways (Duan et al., 2005), indicating that the presence of PPAR in

the myocardium may suppress the actions of trophic stimuli, and thatPPAR ligands could mediate their effects independently of PPAR.This suggestion is supported by an elegant study which employed bothcardiomyocyte and macrophage-specic PPAR/mice to investigatethe effect of PPAR ligands on the development of angiotensin II(Ang II)-induced cardiac hypertrophy and brosis (Caglayan et al.,2008). Administration of the PPAR agonist, pioglitazone was found topromote Ang II-induced cardiac hypertrophy in both cardiomyocyte-specic PPAR/ mice and wild-type littermate controls, whilstattenuating parallel increases in myocardial brosis, again indicatingthat PPAR ligands may exert their cardiac effects independently ofPPAR. Furthermore, the benecial actions of pioglitazone on Ang II-induced brosis were found to be absent in macrophage-specicPPAR/mice, suggesting thatmacrophage PPAR-induced inhibitionof myocardial macrophage inltration is critical to this effect. Previousstudies relating to the effects of PPAR agonists on cardiac hypertrophymust therefore be interpreted with care given these PPAR-independenteffects. Nonetheless, ndings from gene-modiedmice are encouragingand suggest that PPAR may be benecial in preventing myocardialhypertrophy. It is possible that the future development of more specicPPAR agonists may reveal the true role of PPAR and unlock theirtherapeutic potential.

2.4.3. PPAR and cardiac hypertrophyPPAR is signicantly expressed in the myocardium (Gilde et al.,

2003; Cheng et al., 2004a), where it has been reported to be involvedin the transcriptional regulation of lipid metabolism (Barger & Kelly,2000). However, to date, few studies have examined the role of PPARin cardiac hypertrophy. Cardiomyocyte-specic PPAR/ mice havebeen shown to exhibit myocardial lipid accumulation, hypertrophyand heart failure with reduced survival (Cheng et al., 2004b),suggesting that PPAR may play a crucial role in maintaining normalcardiac function. Furthermore, in vitro activation of PPAR with bothL-165041 and GW501516 was found to inhibit phenylephrine-inducedhypertrophy of neonatal rat cardiomyocytes via inhibition of NF-B(Planavila et al., 2005; Smeets et al., 2008a). The positive ndingsfrom these preliminary studies are encouraging, but signicantfurther investigation is required in order to identify the precise roleof PPAR activation in cardiac hypertrophy.

2.5. Clinical implications

Fibrates and thiazolidinediones arewidely used in the treatment ofhyperlipidaemia and type II diabetes, respectively. Although these aretheir primary indications due to positive effects on glucose home-ostasis, lipid metabolism, atherogenic proteins, endothelial function,inammation and thrombosis, these compounds may also be ofbenet in other related pathologies, such as CVD (Verges, 2004).Indeed, recent attention has focussed on the potential use of PPARagonists in the treatment of CVD, and how improvements in lipidbalance, through continued treatment with these compounds, maybenecially affect cardiovascular morbidity and mortality. Firstgeneration brates, such as clobrate, are known to be effective inlowering blood lipids with some studies revealing an associatedreduction in the incidence of cardiovascular events (Vosper et al.,2002). This observation is supported by both the Helsinki Heart Study(Frick et al., 1987) and the Veterans Affairs HDL Intervention Trial(Rubins et al., 1999), in which treatment with the second generationbrate, gembrizil, for 5 years was found to result in a 34% reductionin the cardiovascular event rate and a 22% decrease in mortality,respectively. Interestingly, subjects who had elevated circulatingglucose levels and/or were overweight were found to benet mostfrom gembrizil treatment (Tenkanen et al., 1995). More recently, theFenobrate Intervention and Event Lowering in Diabetes (FIELD)study found that the incidence of non-fatal MI and cardiovascularmortality in patients with type II diabetes was not signicantly

decreased by fenobrate treatment (Keech et al., 2005). However,

258 E. Robinson, D.J. Grieve / Pharmacology & Therapeutics 122 (2009) 246263secondary end points, such as stroke and vascular disease, weresignicantly reduced, although the fact that this was not associatedwith a concomitant decrease in mortality brings the overallcardiovascular benet of long-term fenobrate therapy into question.

PPAR agonists have also been employed in clinical trials to examinewhether their known insulin-sensitising and anti-inammatory actionsare benecial in CVD. In diabetic patients, pioglitazone therapy wasfound to cause a signicant reduction in all cause mortality, non-fatalMI and stroke (Dormandyet al., 2005) and to signicantly reduce carotidintima/media thickness, which is used as a measure of atheroscleroticvascular remodeling (Mazzone et al., 2006). However, the potential useof PPAR agonists in the treatment of CVD may be limited by profoundside effects. Glitazone treatment has been associated with weight gainand peripheral oedema,whichmay precipitate an increased risk of heartfailure (Lindberg & Astrup, 2007). More worryingly, a recent meta-analysis of clinical trials conducted in diabetic patients concluded thatrosiglitazone treatment conferred an elevated risk of MI and cardiovas-cular mortality (Nissen & Wolski, 2007).

At present, statins remain the drug of choice for the treatment andprevention of CVD, due to their benecial effects on cardiovascularmortality. Nonetheless, it is clear that PPAR agonists, such as bratesand glitazones confer some favourable cardiovascular effects, espe-cially in diabetic and/or obese individuals, and therefore holdtherapeutic potential for the treatment of CVD. However, signicantfurther research is required in order to develop more specic drugswith reduced side effects which are able to compete with statins interms of clinical outcome.

2.6. Dual PPAR agonists

In light of the encouraging ndings obtained from clinical trialsemploying PPAR agonists, recent attention has begun to focus oncompounds that are capable of targeting more than one PPAR isotype.Currently, these include PPAR/, PPAR/ and PPAR/ dualagonists, together with PPAR// pan agonists.

PPAR/ dual agonists, such as tesaglitazar andmuraglitazar werecreated in order to elicit synergistic anti-diabetic and cardioprotectiveeffects (Chaput et al., 2000). Tesaglitazar has been demonstrated toreduce the development of atherosclerosis in an experimental mousemodel (Chira et al., 2007) and to improve atherogenic dyslipidaemiain non-diabetic patients with insulin resistance (Schuster et al., 2008).Furthermore, a newly developed PPAR/ dual agonist has beenfound to not only improve insulin sensitivity, but also to prevent LVdysfunction in micewith combined leptin and LDL receptor deciency(Verreth et al., 2006). Dual activation of both PPAR and PPAR alsoappeared to reduce the side effe