Platelets as a Novel Target for PPARγ Ligands

Biodrugs 2006; 20 (4): 231-241MECHANISMS AND TARGETS 1173-8804/06/0004-0231/$39.95/0

© 2006 Adis Data Information BV. All rights reserved.

Platelets as a Novel Target for PPARγ LigandsImplications for Inflammation, Diabetes, and Cardiovascular Disease

Denise M. Ray,1 Sherry L. Spinelli,2 Jamie J. O’Brien,1 Neil Blumberg2 and Richard P. Phipps1

1 Department of Environmental Medicine and the Lung Biology and Disease Program, University of Rochester, Rochester,New York, USA

2 Department of Pathology and Laboratory Medicine, University of Rochester, Rochester, New York, USA

ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2311. Peroxisome Proliferator-Activated Receptors (PPARs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2312. PPARγ and its Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2323. Platelets as a Target for PPARγ Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2334. Implications for Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2355. Implications for Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2366. Implications for Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2377. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

Peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor for lipid andAbstractglucose metabolism. Currently, the PPARγ ligands rosiglitazone and pioglitazone are used for the treatment oftype 2 diabetes mellitus because they are potent insulin sensitizers. Recently, PPARγ has emerged as animportant anti-inflammatory factor. Platelets, anucleate cells involved in hemostasis, have also been implicatedas key contributors to inflammation, because they produce many pro-inflammatory and pro-atherogenicmediators when activated. Surprisingly, it was discovered recently that platelets contain PPARγ and that PPARγligands, both natural and synthetic, inhibit platelet activation and release of bioactive mediators. In particular,release of soluble CD40 ligand (sCD40L) and thromboxane (TXA2) was inhibited by PPARγ ligands inthrombin-activated platelets. CD40L signaling induces pro-inflammatory processes in many cell types, andincreased blood levels of sCD40L are closely associated with inflammation, diabetes, and cardiovasculardisease. Targeting platelet PPARγ will, therefore, be an important treatment strategy for the attenuation ofchronic inflammatory processes and prevention of thrombus formation.

1. Peroxisome Proliferator-Activated cloned in 1991 and termed PPARα, PPARδ (also called ‘PPARβ’Receptors (PPARs) and will be referred to as ‘PPARδ/β’), and PPARγ.[2] PPARα is

highly expressed in the liver, kidney, and heart. PPARδ/β has aPeroxisome proliferator-activated receptors (PPARs) are broad tissue expression and is considered ubiquitously expressed.

ligand-activated transcription factors and members of the nuclear PPARγ is abundant in adipose tissue, the colon, and in cells of thehormone receptor superfamily. These nuclear receptors were dis- immune system.[3] PPARs function as heterodimers with the reti-covered following the observation that certain carboxylic acids, noid X receptor (RXR; also known as the 9-cis retinoic acidsuch as clofibric acid, induced peroxisome proliferation in the receptor).[4] This heterodimer binds to a cis acting DNA element inliver.[1] The receptors for these peroxisome proliferators were the promoters of target genes called the peroxisome proliferator

232 Ray et al.

response element (PPRE). The physiological functions of PPARα cells, dendritic cells, macrophages, T cells, B cells, and, mostand PPARγ have been well characterized, whereas the function of recently, we identified PPARγ in human platelets.[11-17]

PPARδ/β is poorly understood but has been implicated in control- Soon after the identification of PPARs, it was determined thatling dyslipidemia, possibly contributing to the prevention of ather- the thiazolidinedione (TZD) class of anti-diabetic drugs wereogenesis.[5] A summary of the PPAR subtypes is shown in figure 1. potent agonists of PPARγ.[18] PPARγ has, therefore, receivedIncreasing evidence suggests that PPARs bind to proteins in the much attention, as it is a potential target for the treatment ofcytoplasm of cells, separate from their transcription factor activity. diabetes mellitus and it enhances insulin sensitivity. Since it wasFor example, PPARγ can bind to the pro-inflammatory transcrip- identified, several small molecules have been reported as activa-tion factor nuclear factor-κB (NF-κB) and prevent NF-κB from tors of PPARγ. The natural ligands for PPARγ include platelet-translocating to the nucleus, effectively repressing its transcrip- derived lysophosphatidic acid (LPA), nitrolinoleic acid, and thetional activity.[6,7] This recent addition to the function of PPARs eicosanoid 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2), al-suggests that they are not solely transcription factors. though controversy still exists as to the biological relevance of

15d-PGJ2 in vivo.[19-22]

Synthetic PPARγ agonists include the TZD class of anti-diabet-2. PPARγ and its Ligandsic drugs, which includes ciglitazone, pioglitazone, rosiglitazone,and troglitazone.[18] Two of the TZDs, rosiglitazone and piog-PPARγ was originally described as a transcription factor impor-litazone, are currently used to treat type 2 diabetes. Clinical datatant in adipose tissue for regulating lipid metabolism, but hassupports the theory that these TZDs are potent insulin sensitizers,recently emerged as a significant anti-inflammatory protein, and italthough there are some adverse effects, including weight gain dueis abundantly expressed by cells of the immune system. PPARγto fat accumulation as a result of PPARγ activation, fluid retentionexists as three isoforms, PPARγ1, PPARγ2, and PPARγ3. Thesedue to TZDs targeting the collecting duct of the kidney,[23] and,isoforms share the same gene (PPARG) and are the result ofrarely, liver toxicity. Overall, the insulin-sensitizing benefits anddifferential promoter use and alternative RNA splicing.[8] PPARγ2the possible anti-inflammatory actions of the TZDs outweigh theonly differs from PPARγ1 by an additional 30 amino acids at thepotential adverse effects. However, there is significant interest inN-terminus. Both PPARγ1 and PPARγ2 are highly expressed indeveloping new PPARγ ligands to improve on the insulin-sensi-adipose tissue, but PPARγ2 is not abundantly expressed in othertizing function and reduce the possible adverse effects, particularlytissues, with the exception of the liver.[9] In addition to adiposeweight gain and edema.tissue, PPARγ1 is present in human spleen, liver, intestine, and

One such new class of agents, already in clinical trials for type 2kidney.[9] Little is known regarding the expression and functionaldiabetes, is dual PPARα/γ agonists. Two dual PPARα/γ agonistssignificance of PPARγ3, but PPARG3 mRNA is detectable intested, ragaglitazar and tesaglitazar, appear to be well tolerated butmouse macrophage cells.[10] PPARγ expression has been docu-still cause some edema, as well as anemia and gastrointestinalmented in several cell types, including fibroblasts, endothelialsymptoms.[24,25] In a pre-clinical rat model, ragaglitazar was moreeffective than rosiglitazone for improving insulin resistance.[26]

Human clinical trials have confirmed the insulin-sensitizing actionof ragaglitazar and tesaglitazar.[24,25] A third agent, muraglitazar,which is effective for simultaneous treatment of dyslipidemia andhyperglycemia, has unfortunately had questions raised regardingits safety, with increases in congestive heart failure having beenobserved.[27] The health risks associated with these PPARα/γagonists have prompted recent discontinuation of the further de-velopment of these agents. However, other PPARα/γ are currentlyunder evaluation. It remains possible that other key propertiesindividual to PPARγ and PPARα, such as anti-inflammatory andanti-cancer effects, may also be exploited by the use of PPARα/γdual agonists.[26] Indeed, Liu and colleagues[28] have reported,using a new dual ligand, TZD18, in leukemia cells, that the dual

Fibrates

AdipogenesisLipid metabolismInsulin sensitivity

Inflammation

Insulin sensitivityCholesterol effluxLipid catabolism

Atherosclerosis

PPARα

TZDs

PPARγ

PPARδ/βligands

PPARδ/β

Fig. 1. The physiological importance of peroxisome proliferator-activatedreceptors (PPARs) and their ligands. The PPARs are activated by theirspecific ligands. PPARα and PPARγ control many of the same processes,for example, adipogenesis, lipid metabolism, inflammation, and insulin sen-sitization. PPARδ/β, the least studied of the PPARs, functions in regulatinglipid homeostasis, which may lead to improved insulin sensitivity and re-duced risk of developing atherosclerosis. TZD = thiazolidinedione. ↓ indi-cates decrease; ↑ indicates increase.

© 2006 Adis Data Information BV. All rights reserved. Biodrugs 2006; 20 (4)

Platelets as a Novel Target for PPARγ Ligands 233

PPARα/γ ligand significantly enhanced apoptosis compared with 3. Platelets as a Target for PPARγ Ligandsthe PPARγ ligand pioglitazone alone.

Another promising group of PPARγ ligands are the selective Platelets are anuclear cells derived from megakaryocytes in thePPAR modulators (SPPARMs). The goal with SPPARMs is to bone marrow and contain most of the classical cellular organelles,design partial PPARγ agonists that retain insulin sensitization but including mitochondria, lysosomes, and peroxisomes, but alsolack the fat-accumulating properties of the classical TZD PPARγ have unique structures, such as the surface-connected open cana-ligands.[29] The SPPARMs take advantage of the large ligand licular system, dense granules, and α granules.[41] The content ofbinding pocket of PPARγ, which allows chemically diverse agents platelets is mainly composed of elements of the megakaryocyteto bind and interact with different domains of the transcription parent cell; however, circulating platelets can also uptake compo-factor.[30] Also contributing to the development of SPPARMs is nents of the blood.[42] Interestingly, platelets contain smallthe observation that the insulin-sensitizing ability of PPARγ is amounts of mRNA and retain the spliceosomal components foroptimal when the transcription factor is only partially activated.[31] mRNA processing[43] and the translational machinery for proteinIt is thought that these partial agonists work by differential recruit- synthesis.[44,45] In addition to playing a vital role in hemostasis,ment of co-activators, which could change the gene activation platelets are emerging as key players in inflammation. Uponprofile of PPARγ in a given tissue.[32] For example, the triterpenoid activation, platelets release several pro-inflammatory mediators,2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a partial such as interleukin (IL)-1β, TGFβ, prostaglandin E2 (PGE2),PPARγ agonist currently being tested in clinical trials for leuke- thromboxane (TXA2), and CD40 ligand (CD40L, also known asmia, is less able to recruit cyclic adenosine monophosphate re- CD40LG and formally known as CD154). In particular, the recentsponse element binding protein (CREB)-binding protein discovery that activated platelets express CD40L on their surface(CREBBP), when compared with rosiglitazone.[33] This would and then release soluble CD40L (sCD40L) provides a link be-result in reduced transactivation of PPARγ and further modulation tween platelets and CD40-expressing cells such as macrophagesof downstream effects. As proof that the SPPARMs will succeed and endothelial cells.[46] This CD40/CD40L signaling pathwayin the goal of separating PPARγ activities, the SPPARM PAT5A leads to activation of the CD40-expressing cell, to further enhanceincreased insulin sensitivity, with only weak adipogenic potential inflammatory processes, a topic that will be addressed in morein a pre-clinical mouse model of insulin resistance.[34] Given the detail in the next section.success with SPPARMs in targeting insulin resistance, one can Even though platelets do not have a nucleus, we recentlyspeculate that other properties of PPARγ will be the target of discovered that human platelets contain large amounts of PPARγpartial agonist design in the future. protein.[11] Additionally, human megakaryocytes and a megakary-

An important consideration when designing or utilizing PPARγ oblastic cell line (Meg-01) expressed PPARγ protein. However,ligands is the increasing evidence that PPARγ ligands, including human platelets did not contain mRNA for PPARγ, unlike thethe TZDs, may mediate some of their biological effects through Meg-01 cells. In these studies, human platelets were rigorouslynon-PPARγ-mediated pathways.[35] In particular, the contribution purified and washed, and were proven to contain PPARγ by usingof PPARγ to the anti-inflammatory and anti-cancer effects of a combination of immunohistochemistry, flow cytometry, andPPARγ ligands has been questioned.[35] Macrophages exposed to Western blotting techniques.[11] It is most likely that the PPARγPPARγ ligands have reduced production of pro-inflammatory observed in the platelets was derived from the parent megakaryo-mediators; however, PPARγ knockout macrophages still displayed cyte. Interestingly, we found that the platelet PPARγ had signifi-this reduced pro-inflammatory cytokine production.[36,37] In addi- cant DNA binding ability in the absence of PPARγ ligands, antion, PPARγ agonists are reported to induce apoptosis of several activity that was enhanced by exposure to PPARγ agonists.[11] Wetypes of cancer cells, including breast cancer and colon cancer, next investigated the potential function of PPARγ in platelets andindependently of PPARγ activation.[38,39] However, in human lung postulated that PPARγ agonists may modulate platelet activationcarcinoma, rosiglitazone inhibits cell growth by both PPARγ- and release of granule contents. Surprisingly, we found that lowdependent and -independent mechanisms.[40] Clearly, there is still micromolar concentrations (levels achievable in vivo[47,48]) of bothmuch work to be done in dissecting the PPARγ-dependent versus - the synthetic PPARγ agonist rosiglitazone and the naturally occur-independent properties of PPARγ ligands. Consequently, proving ring ligand 15d-PGJ2 dampened TXA2 release, CD40L surfacePPARγ dependency will be important for future design and thera- expression, and release of sCD40L from thrombin activated plate-peutic use of PPARγ ligands. lets.[11] Moreover, 15d-PGJ2 prevented adenosine triphosphate


234 Ray et al.

(ATP) release and aggregation induced by adenosine diphosphate(ADP). Overall, these novel findings demonstrate that PPARγagonists are powerful inhibitors of platelet activation, which mayhave significant implications for disorders associated with platelethyperactivity. In fact, two reports in animal models of thrombosisdemonstrated that the PPARγ ligand, pioglitazone delayed throm-bus formation, supporting our data that PPARγ ligands bluntplatelet aggregation.[49,50]

A summary of these findings is depicted in figure 2. Of interestis the finding that an endogenous PPARγ ligand, 15d-PGJ2, is avery potent inhibitor of platelet activity. This suggests that PPARγand its natural ligands, such as 15d-PGJ2, LPA, and nitrolinoleicacid, may constitute an endogenous mechanism to reduce plateletactivation. LPA, in addition to being a PPARγ ligand, also actsthrough specific receptors on the platelet surface to induce plateletaggregation.[51] The role of LPA in platelet PPARγ responses hasnot been determined but could be addressed using LPA receptor

Unactivatedplatelet

sCD40L

TXA2

PPARγ ligands+ thrombin

Thrombin

Fig. 2. Human platelet activation is inhibited by peroxisome proliferator-activated receptor (PPAR)γ ligands. Upon activation by thrombin (or otheractivators), platelets release bioactive inflammatory mediators, such assoluble CD40 ligand (sCD40L) and thromboxane (TXA2), and form aggre-gates. If platelets are exposed to PPARγ ligands, such as rosiglitazone or15d-PGJ2, in the presence of platelet activators, the platelets are inhibitedin their ability to aggregate and release sCD40L and TXA2.

antagonists.

The fact that platelets express a transcription factor is very intracellular proteins involved in granule release. This could rangeinteresting and raises questions as to why platelets would have from preventing actin cytoskeletal rearrangement and inhibitingPPARγ and how it functions within platelets to cause such protein kinase C signaling, to the actual exocytosis process, in-profound effects on platelet activity. It is now known that PPARγ cluding docking and fusion of granules to the plasma membrane.is not the only transcription factor expressed by platelets, since it Further studies will be necessary to pinpoint the location ofhas recently been discovered that NF-κB and its inhibitory pro- PPARγ in platelets and the mechanism by which PPARγ ligandsteins are also contained within platelets, although the activation

inhibit platelet activation.status of platelet NF-κB has not been determined.[52] Current

An interesting new direction to explore would be to utilize theresearch on PPARγ suggests that it is not a typical transcriptiondual PPARα/γ agonists or the SPPARMs and determine if thesefactor, in that PPARγ has additional functions independent of itsdrugs are more potent than single agonists as inhibitors of plateletability to initiate transcription of its target genes. For example,activation. Of interest is a recent report that human plateletsPPARγ is now thought to exert some of its anti-inflammatorycontain PPARδ/β.[55] This study investigated ligands for all threeeffects by binding to NF-κB complexes in the nucleus and facili-PPAR subtypes (PPAR α, γ, δ/β) and, in support of our findings,tating its translocation out of the nucleus, thereby sequesteringdiscovered that PPARγ ligands inhibit ADP-induced aggrega-NF-κB in the cytoplasm.[6,7] Whether PPARγ is bound to, or cantion.[55] Interestingly, ligands for both PPARα and PPARδ/β werebind upon ligand activation to, intracellular platelet proteins isalso potent inhibitors of platelet aggregation.[55] This stronglyunknown and is a topic for further research. In addition, PPARγsuggests that dual agonists for PPARα/γ or PPARγ/δ may greatlycan be modified by phosphorylation, which may result in activa-enhance the effects of the individual ligands and may be a viabletion or inactivation, depending on the stimulus and phosphoryl-treatment option for abrogating platelet activity. Additionally,ation site,[53] and by SUMOylation, the covalent addition of theonce future studies have determined the exact mechanism bySUMO protein, similar to ubiquitin, resulting in transrepression ofwhich the PPARs act within platelets, more targeted ligands,inflammatory genes.[54] In this regard, it is unknown whethersimilar to SPPARMs, could be developed to obtain even greaterplatelet PPARγ is modified in response to agonist exposure andactivity.what consequences a modification such as phosphorylation would

have in the platelet. An important consideration in these studies will be to determineif these effects of PPARγ ligands on platelets are indeed actingGiven the findings that platelets abundantly express PPARγthrough PPARγ. This will be a difficult question to answer givenprotein and that PPARγ ligands inhibit release of TXA2, sCD40L,that the traditional methods to inhibit PPARγ, such as antagonistsand ATP, it is possible that PPARγ may interact with and inhibit



and dominant-negative approaches, are focused on preventing mation.[46] Upon activation, platelets transfer CD40L from intra-transcriptional activity and this is not relevant to platelets. The best cellular stores to the cell surface. CD40L is then enzymaticallymethod to study the contribution of PPARγ would be to have cleaved by a poorly understood mechanism, which may involvePPARγ deficient platelets. Unfortunately, knockout of PPARγ in matrix metalloproteinases[61] to release biologically activemice is embryonically lethal because of placental dysfunction.[56] CD40L.[62] In fact, it is estimated that 95% of the sCD40L in bloodPPARγ conditional knockout mice are available, but complete is platelet derived.[63]

knockout is rarely achieved, with the percentage of gene knockout An important target for platelet CD40L in the vasculature is thevarying from tissue to tissue.[57] Many investigators have utilized endothelial cell. CD40 ligation on endothelial cells contributes toPPARγ heterozygous mice to study the function of PPARγ, be- the amplification of inflammatory responses. CD40 signaling in-cause these mice express half the normal level of PPARγ protein duces endothelial cells to produce pro-inflammatory moleculesand exhibit associated changes in phenotype when compared with such as IL-8, IL-6, RANTES (Regulated on Activation, Normal T-wild-type mice.[58] If PPARγ were required for the platelet inhibi- cell Expressed and Secreted), monocyte chemotactic protein-1tory function of PPARγ ligands, one would predict that PPARγ (MCP-1, also known as chemokine ligand CCL2), and TNFα, andligands would be less effective at inhibiting sCD40L and TXA2 upregulates adhesion molecule expression, including that of inter-release in the platelets of the PPARγ heterozygous mice, compared cellular adhesion molecule-1 (ICAM-1) and vascular adhesionwith those of PPARγ-ligand-exposed wild-type mice. Therefore, molecule-1 (VCAM-1).[46,59] All of these signals result in en-further analysis will be necessary to determine the function of hanced inflammation and recruitment of immune cells. PlateletPPARγ in platelets. CD40L can also directly affect immune cells, causing enhance-

ment of B cell antibody production, maturation of antigen present-4. Implications for Inflammation ing cells (dendritic cells), and boosting CD8+ T-cell responses.[64]

Fibroblasts are an additional CD40-expressing cell, which producePlatelets have emerged as key mediators in inflammation. The pro-inflammatory mediators, such as IL-8 and IL-6, and upregu-

prevention of release of sCD40L and possibly other pro-inflam- late expression of cyclo-oxygenase (COX)-2, which results in thematory molecules from platelets is a significant finding and may production of prostaglandins such as PGE2 in response to CD40have considerable implications for the inflammatory process. Al- activation.[62,65,66] A summary of CD40 signaling outcomes isthough platelets produce many bioactive mediators, this review shown in figure 3. Overall, it is clear that platelet-derived CD40L,focuses on the mediators whose release is prevented by PPARγ either surface bound or soluble, is an important contributor toligands. inflammatory responses.

The classical CD40/CD40L signaling pathway has been studiedThe contribution of platelet CD40L to chronic inflammation is

intensively on immune cells, such as T and B lymphocytes andalso an intense area of research. For example, it has been observed

antigen presenting cells. However, as more CD40-expressing cellsthat patients with inflammatory bowel disease (IBD) have a higher

have been identified, it is clear that the CD40/CD40L pathway isnot limited to the primary cells of the immune system. CD40belongs to the tumor necrosis factor (TNF) receptor superfamilyand is a type I transmembrane protein of ≈50 kDa.[59] In addition toimmune cells, CD40 is expressed by endothelial cells, epithelialcells, and fibroblasts, and has been recently identified on thesurface of platelets.[59,60] CD40L is an important platelet agonist,since activation through CD40 results in surface expression ofplatelet α-granule membrane protein selectin P (SELP, alsoknown as CD62P) and granule release.[60] Therefore, targeting theplatelet with PPARγ ligands could prevent activation enhancementcaused by the platelets themselves. CD40L is an ≈39 kDa memberof the TNF superfamily and has both a membrane-bound andsoluble form.[59] The recent discovery that platelets highly expressCD40L has further indicated the platelet as a key player in inflam-

COX-2 PGE2

Activatedplatelet

CD40L

CD40

CD40-expressingvascular cell

Inflammation

Promotion ofdiabetes mellitus

andcardiovascular

disease

IL-8IL-6MCP-1RANTESTissue factor

VCAM-1

ICAM-1

Fig. 3. Effects of platelet-derived CD40 ligand (CD40L). CD40 expressingcells, such as endothelial cells and fibroblasts, can be activated throughCD40 by platelet-derived CD40L, either surface bound or soluble. CD40signaling induces upregulation of the cyclo-oxygenase (COX) pathway andproduction of pro-inflammatory mediators, which leads to recruitment ofimmune cells and induction of inflammation. ICAM-1 = intercellular adhe-sion molecule-1; IL = interleukin; MCP-1 = monocyte chemotactic prote-in-1; PGE2 = prostaglandin E2; RANTES = growth factor (Regulated onActivation, Normal T-cell Expressed and Secreted); VCAM-1 = vascularadhesion molecule-1.


236 Ray et al.

risk of thrombosis.[67] Circulating platelets from IBD patients and inflammation.[81] In addition to the reduced levels of insulin-exhibit a marked increase in activity and have higher surface induced platelet inhibitors, platelets from diabetic patients withCD40L expression compared with healthy individuals.[68] Moreo- vascular disease are less sensitive to the inhibitory effects of PGI2

ver, activated platelets were determined to be the source of en- and NO.[82,83] Platelet hyper-reactivity is further multiplied byhanced sCD40L plasma levels in patients with IBD.[69,70] There is upregulation of the platelet COX pathway resulting in increasedevidence that, in IBD, platelets contribute to the mucosal inflam- TXA2 production, an important molecule for the enhancement ofmation by directly interacting with endothelial cells, resulting in platelet activation.[84] The net result of these alterations is ampli-enhanced ICAM-1 and VCAM-1 expression and IL-8 production fied persistent platelet activation with enhanced granule release inon the mucosal endothelial cells, ultimately augmenting recruit- patients with diabetes. Because PPARγ ligands are potent insulinment of immune cells such as T lymphocytes and neutrophils.[68] sensitizers, part of their effect on platelets may be through insulinDiabetes and cardiovascular disease also have a chronic inflam- signaling mechanisms.matory component and are discussed in sections 5 and 6. Of particular interest is the relationship between CD40L and

Thus, platelet-derived CD40L is an important target for the the pro-inflammatory and pro-thrombotic environment present indevelopment of therapies for inflammatory conditions. The ability type 2 diabetes. In fact, there is increasing evidence supporting aof PPARγ ligands to block platelet surface expression and release role for chronic inflammation in the development of insulin resis-of sCD40L may have important implications for the development tance and related cardiovascular disease.[85] In this regard,of treatments to prevent inflammation. In fact, several recent sCD40L plasma levels are significantly elevated in patients withhuman trials reported that the PPARγ ligands rosiglitazone and diabetes.[86] Additionally, platelet expression of surface CD40Ltroglitazone inhibit NF-κB and reduce serum levels of monocyte- and its receptor CD40 are elevated in both type 1 and type 2derived pro-inflammatory molecules, supporting the in vitro find- diabetes.[86] The increase in CD40L, both soluble and surfaceings that PPARγ ligands are anti-inflammatory.[71,72] forms, is most likely the result of the increased activation status of

the platelets in diabetic patients. However, there is new evidencesuggesting that overall these platelets contain more CD40L and,5. Implications for Diabetes Mellitustherefore, release more sCD40L when activated, compared withplatelets from non-diabetic individuals.[87] The increased releaseIndividuals with type 2 diabetes present with insulin resistanceof CD40L may be the direct result of hyperglycemia.[88] In fact, aand dyslipidemia. Additionally, there is now a strong link betweenrecent study by Varo and colleagues[87] demonstrated that glucosediabetes, platelet activation status, and an increased risk for throm-can directly activate platelets to release CD40L. More importantly,bosis and vascular complications. Recently, there has been muchglucose was shown to target the megakaryocyte and stimulated aninterest in the connection between platelets and the vascular com-increase in CD40L expression in a mouse megakaryocyteplications observed in diabetes. It is now known that platelets frommodel.[87] Therefore, the increase in CD40L levels in diabeticspatients with type 2 diabetes have a more activated phenotype thanmay be a result of glucose stimulation of megakaryocyte CD40Lplatelets from non-diabetics.[73] The changes include increasedproduction, resulting in the creation of CD40L hyper-expressingplatelet volume and number, increased adhesiveness and aggrega-platelets. A further significant finding is that TXA2 may be neces-tion, and enhanced surface expression of the collagen receptorsary for platelet sCD40L release in type 2 diabetes.[89] Since TXA2glycoprotein (GP)VI and the fibrinogen receptor GPIIb-IIIa (in-production is higher in diabetic patients, the end result is increasedtegrin αIIbβ3), just to name a few.[73] Moreover, fibrinogen andsCD40L release, leading to an overall increase in vascular inflam-thrombin levels are increased in patients with insulin resistance,mation. In a small study of diabetic patients, short-term aspirinfurther enhancing the pro-coagulant state.[74,75] Insulin itself hastreatment reduced plasma sCD40L levels, further supporting theanti-platelet activity by stimulating endothelial cell production ofrole of TXB2 in this process.[89]the platelet inhibitors prostacyclin (PGI2) and nitric oxide (NO),

which may account for the decreased levels of these inhibitors It is evident in diabetic patients that CD40L levels are increasedobserved in experimental diabetic models.[76-79] This insulin-in- in plasma and on the platelet surface. This CD40L may contributeduced NO may be an important mechanism for mediating vasodi- to the elevated inflammatory status of both the vasculature andlation.[80] In a human clinical trial, insulin was also reported to other locations in the body. For example, diabetic patients have anhave an anti-inflammatory effect on mononuclear cells by inhib- increased risk for developing periodontal disease, an inflammatoryiting NF-κB, further supporting an important link between insulin condition of the periodontal tissue in the oral cavity.[90] A human



clinical study linked obesity and insulin resistance to the severity The molecular mechanisms that govern the role of platelets inof periodontal disease.[91] Given the increased sCD40L and plate- thrombosis and inflammation are complex in atherogenesis.[102-104]

let-surface-expressed CD40L, it is possible that CD40L contrib- The role of platelets in thrombus formation following plaqueutes to either the development or enhancement of inflammation erosion or rupture is well understood and is a principle factor inassociated with periodontal disease in diabetic individuals. Perio- acute coronary syndromes.[105-107] More recently, platelets havedontal inflammation is associated with increased B-lymphocyte been determined to have an expanded function in atherosclerosis,infiltration and antibody production, and PGE2 synthesis by resi- triggering early events.[108] It is well documented in both humandent cells and infiltrating immune cells.[92-94] Since CD40L plays a studies and animal models that endothelial dysfunction leads tokey role in enhancing B-cell antibody responses and causes platelet adhesion and co-aggregation and recruitment of leuko-upregulation of the COX pathway, platelet-derived CD40L may be cytes to the vascular wall.[108-110] These initial events instigate aan important target for attenuating periodontal inflammation. highly coordinated signaling cascade that amplifies inflammatory

The PPARγ ligands rosiglitazone and pioglitazone are currently responses in a variety of cells, including T cells, dendritic cells,used for the treatment of type 2 diabetes because they are potent and endothelial cells. The result is a profound local inflammatoryinsulin sensitizers. This provides a key population to study the reaction within the vessel, with release of pro-inflammatoryeffects of PPARγ ligands on platelet-related diabetic complica- mediators, including CD40L. Interesting new evidence fromtions. In fact, diabetic patients taking TZDs have significantly mouse studies demonstrates that platelets recruit and mediatelower levels of circulating sCD40L.[95,96] In addition, when admin- differentiation of embryonic endothelial progenitor cells at sites ofistered to non-diabetics with coronary artery disease, rosiglitazone vascular lesions.[111] Thus, platelets have a function in both sus-reduced platelet activity, based on reduction in SELP surface taining vascular integrity and forming new endothelial cells atexpression.[97] Clearly, these observations support our findings sites of vascular injury. This information is exciting in the contextthat PPARγ ligands inhibit platelet activity, and suggest that there that, if the mouse process translates to the human system, thismay be inhibition of additional bioactive mediators not yet evalu- would further argue that aberrant platelet function may contributeated. The current TZDs in clinical use were designed for their to the development and progression of atherosclerosis.insulin-sensitizing abilities. It is now evident that these drugs have

There is compelling evidence for the role of CD40/CD40L inalternative targets in diabetic patients. The development of newatherosclerotic pathogenesis.[46,59,63,112-115] Elevated blood levels ofPPARγ ligands, such as the dual agonists or SPPARMs, to addi-soluble and membrane-bound forms of CD40L occur in individu-tionally target the anti-platelet functions of PPARγ, particularlyals with cardiovascular disease (including unstable angina, coro-inhibition of aggregation and sCD40L release, may have greatnary artery disease, and hypercholesterolemia).[116-119] As statedpotential for the treatment of type 2 diabetes and associatedabove, platelets are central players in the activation of the CD40/complications.CD40L pathway, and this pathway has profound effects for local-

ized vascular inflammation. This system also functions in platelets6. Implications for Cardiovascular Disease

to upregulate the pro-coagulant mediator known as tissue factor

(TF), and to stabilize arterial thrombi formation via the GPIIb-IIIaCardiovascular disease is a broad term that encompasses dys-

receptor.[112,120] Moreover, CD40/CD40L has been implicated infunctional conditions of the heart, arteries, and veins, including

signaling plaque rupture.[115,121,122] Thus, platelet activation signalsatherosclerosis, hypertension, dyslipidemia, myocardial infarc-the upregulation of both pro-coagulative and pro-inflammatorytion, and stroke. Commonly, cardiovascular disease is a result ofpathways in a variety of cells that display CD40.obesity, sedentary lifestyle, smoking, and diabetes. The root of

An important finding is that disruption of the CD40/CD40Lcardiovascular disease is typically atherosclerosis, a chronic in-pathway inhibits the initial phase of arterial plaque formation inflammatory disease that progresses by plaque development inatherosclerosis.[115,121,123] Thus, therapeutics that attenuate thearterial vessels and often evolves to thrombus-induced coronaryCD40/CD40L system would likely provide clinical benefit toevents.[98-100] These plaques or lesions are characterized as stablepatients with atherosclerosis.[124] Our work demonstrated thator unstable, depending on the propensity of a plaque to rupture andPPARγ agonists potently attenuate platelet activation, includingform a thrombus that occludes an artery. Increased platelet activityinhibition of CD40L release.[11] PPARγ agonists are emerging ashas been reported in patients with both stable and unstable cardio-

vascular disease.[101] therapeutics with high potential for the downregulation of the


238 Ray et al.

wanted effects such as fat accumulation, which is an importantconsideration for plaque formation.

7. Conclusion

The evidence reviewed herein strongly supports PPARγ as atarget for anti-platelet therapy. The significant in vitro findingsthat PPARγ ligands inhibit human platelet activity are supportedby both human trials measuring sCD40L and animal models ofplatelet aggregation. Taken together, these important findingsneed to be evaluated in clinical trials for diseases in which plateletactivity is altered. The inhibition of platelet aggregation, sCD40Lrelease, and TXA2 production suggests that PPARγ ligands will

Unactivatedplatelet

sCD40L TXA2

Disease-alteredplatelet

PPARγligands

Fig. 4. Peroxisome proliferator-activated receptors (PPAR)γ ligands for thetreatment of diseases with altered platelet activity. Disease states, such asdiabetes mellitus, cardiovascular disease, and chronic inflammation, areassociated with partially activated platelets and release of soluble CD40ligand (sCD40L) and thromboxane (TXA2). By inhibiting platelet activation,PPARγ ligands represent a novel platelet target for the treatment of suchdiseases.

have important implications for inflammation, diabetes, and car-diovascular disease (see figure 4). With such a broad spectrum of

CD40/CD40L system and are therefore relevant to investigate forbiological effects, PPARγ and its ligands will be valuable targets

the treatment of cardiovascular disease.for many disease conditions. Intense research on the development

There are several reviews that focus on PPARs and theirof new PPARγ ligands to focus activity on the beneficial effects,

regulatory role in atherogenesis.[125,126] The recent report thatfor example, insulin sensitivity and platelet inhibition, without the

platelets contain PPARδ/β, a putative receptor for prostacyclinadverse effects of the current PPARγ ligands, will significantly

(whose activation inhibits platelet aggregation), has lead to specu-enhance treatment options. Therefore, it will be imperative to

lation that this receptor may confer antithrombotic effects.[55]

elucidate the mechanism(s) by which PPARγ ligands inhibit plate-Emerging evidence indicates that the synthetic PPARγ agonists

let functions.TZDs also have anti-atherosclerotic properties affecting both thevascular wall and inflammatory processes.[127] Animal models of Acknowledgementsatherosclerosis demonstrate that the PPARγ ligand pioglitazone

This work was supported by T32-DE07165, T32-ES07026, DE011390,decreases platelet aggregation and delays arterial thrombus forma-and HL078603. The authors declare that no conflicts of interest exist.tion.[128] Clinical investigations have shown that non-diabetic indi-

viduals with coronary artery disease who use rosiglitazone haveReferencesreduced platelet activity.[97] Moreover, inflammatory markers as-

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Platelets as a Novel Target for PPARγ Ligands

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