# Peroxisome Proliferator-Activated Receptors in the Modulation of the

Authors: Ana Z. Fernandez
Journal: PPAR Research
 (2008)

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## Abstract

Inflammation has been recognized as an important hallmark of atherosclerosis. The pharmacological activation of
PPAR-γ  by the thiazolidinediones in diabetes, and of  PPAR-α by the fibrates in hyperlipidemia has been shown to help to reduce inflammatory markers in preclinical and clinical studies. PPARs are known to modulate immune pathways through at least three different mechanisms: by direct binding to PPRE of anti-inflammatory cytokines genes; by transrepression of transcription factors like NF-κB and AP-1; or by corepression. The regulation of the inflammatory pathways by PPARs can be achieved on each one of the cells involved in the atherosclerotic process, that is, monocytes, macrophages, T cells, endothelial cells, and smooth muscle cells. Moreover, as each of these cellular components is interconnected with each other, PPAR activation in one cell type could affect the other ones. As activation of PPARs has clear ant-inflammatory benefits, PPARs ligands should be considered as a new therapeutical approach to ameliorate the exacerbated immune response in atherosclerotic diseases.


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## Body

## 1. Introduction

Cardiovascular
diseases represent the main cause of morbidity and mortality in western societies
since the 20th century and mostly are a consequence of atherosclerosis, a prior
established pathology. Atherosclerosis is defined as a progressive, chronic pathology
characterized by the accumulation of lipids and fibrous elements in the large
arteries. It is classified as an inflammatory disease, since in every phase of
the atherosclerotic process, the immune response has a significant role [1].
Immune cells like monocytes, macrophages, and T-cells are crucial in the
development of the atheroma and the stimulation and activation of endothelial
cells (EC) and smooth muscle cells (SMC), cellular components of the vascular
wall are extremely relevant for the recruitment of the cells responsible for the
immune response.Peroxisome
proliferator-activated receptors (PPAR) and their agonists have been gaining
more attention recently in regard
to the study of the mechanisms involved in
the etiology and pathogenesis of atherosclerosis. The expression of PPAR-α,
-δ/β, and -γ in vascular wall cells and in immune cells, as well as in
atherosclerotic lesions, has
been described [2, 3]. The pharmacological modulations of both PPAR-α and -γ as therapeutic treatments
for diabetes and hyperlipidemia have been linked to an improvement on the low-grade
inflammation associated with these conditions [4, 5]. The inhibition of
function and/or expression of certain genes critical for the initiation or
maintenance of inflammatory cells recruitment, survival, proliferation, and
activation has been shown to alter the progression of atherosclerotic lesions [6].
In this regard, the three PPAR isoforms certainly could play distinctive roles in
modulating the inflammatory response in atherosclerosis.


## 2. PPAR: General Characteristics

The
PPARs belong to a subfamily of the nuclear receptors superfamily and are
ligand-activated transcription factors which heterodimerize with the retinoic X
receptor and recognize PPAR response elements (PPRE) localized in the promoter
region of target genes [2]. In addition to the direct involvement of PPAR in
the gene-specific transcription, PPAR could also repress the transcription of
certain genes brought about by the proinflammatory transcription factors, nuclear  factor (NF)-κB, activation protein-1
(AP-1), and signal transducer and activator of transcription 1 (STAT-1), through
the binding and sequestration of their corresponding cofactors [7].Three
PPAR forms have been described: PPAR-α (NR1C1), PPAR-δ/β (NR1C2), and PPAR-γ
(NR1C3). These different receptors show a similar protein structure in spite of
their different coding genes [3]. PPAR-α and PPAR-γ have been recognized to be
key players in both cellular differentiation processes and anti-inflammatory
regulation and, most recently, PPAR-δ/β has also been implicated in the immune
response [8].PPAR-α
is mainly expressed in tissues characterized by a high rate of fatty acid
catabolism (liver, kidney, heart, and muscle) and is the most abundantly
expressed PPAR isoform in human endothelial cells (EC) [9]. The transcriptional
activity of PPAR-α is stimulated by a variety of compounds (see
Table 1) [2]. PPARα synthetic ligands,
such as clofibrate, fenofibrate, and bezafibrate, were developed as
hypolipidemic agents, through optimization of their lipid-lowering activity in
rodents, even before the discovery of the PPARs [10]. PPAR-α is
involved in the control of lipoproteins metabolism, fatty acid oxidation, and
in the cellular uptake of fatty acids [10]. Studies in vitro suggest that PPAR-α also regulates the
expression of genes that control inflammatory responses in EC, SMC, and
macrophages, cells known to be implicated in the inflammatory response of
vascular EC and in the pathology of atherosclerosis [9, 11].Table 1
Properties and agonists of PPARs.
CharacteristicPPAR-αPPAR-γPPAR-δ/βGeneral distributionLiver, heart, kidney,
muscle.
Endothelial cellsHeart, intestine,
kidney, pancreas, spleen, muscle, adipose tissueLiver, intestine,
kidney, abdominal adipose tissue, skeletal muscleNatural ligandsSaturated and
unsaturated fatty acids; arachidonic acid-derived eicosanoids from
the lipoxygenase pathway: 8-S-hydroxyeicosatetraenoic acid and  leukotriene B4; insulin; oxidized LDLLinoleic acid,
linolenic acid, arachidonic acid, eicosapentenoic acid, 15-deoxyΔ12, 14-prostaglandin
J2;
15-LOX metabolites
(9-HODE and 13-HODE)Saturated and
unsaturated fatty acids;
eicosanoids: PGA1 and PGD2Synthetic agonistsHypolipidemic fibrate
drugs: fenofibrate, genfibrozil; plasticizers, ureidofibrates;
WY14643, JTT-501, GW-2331 and  PD72953Antidiabetic thiazolidinediones: pioglitazone,
troglitazone, rosiglitazone (BRL-49653), MCC-555;
isoxazolidinedione JTT-501; tyrosine-based agonist:
GI2-62570, GW-1929, and GW-7845 from GSK;α-alcoxy-β-phenylpropanoic acid; weak agonist: LTD4 receptor
antagonist LY-171883; COX inhibitors: indomethacin, ibuprofen, fenoprofen,
and flufenamic acid.
Docosohexanoic
derivativesLeukotriene antagonist
L-165041; phenylacetic derivatives L-796449 and L-783483; GW-2433, GW-501516,
GW0742X;
carbaprostacyclinGene/protein expression
affectedLipoprotein lipase, Apo
CIII, Apo AI, Apo AII, fatty acid transporter protein, Acyl-CoA synthetase,
mitochondrial HMG-CoA synthase, mitochondrial uncoupling protein 1Acyl-CoA
synthetase,  fatty acid transporter
protein, CD 36, lipoprotein lipase, TNF-α
mitochondrial
uncoupling protein 1–3; insulin-dependent glucose transporter 4Fatty acid transporter
protein, CD 36, fatty acid translocase, adipocyte lipid-binding protein,
ABCA1,
14-3-3εPPAR-δ/β
is ubiquitously expressed both in vascular SMC as well as in EC, besides liver,
kidney, and abdominal adipose tissues. Several eicosanoids have been reported
to activate PPAR-δ/β, including PGA1 and PGD2, and a
synthetic prostaglandin carbaprostacyclin [10]. The important physiological
roles of PPAR-δ/β are
highlighted in genetically modified mouse models as deletion of PPAR-δ/β in mice leads to incomplete but very high penetration of a lethal
phenotype and PPAR-δ/β heterozygous animals
display abnormal wound healing [12]. Additionally, PPAR-δ/β has been implicated in the maintenance of lipid
homeostasis [13], keratinocyte proliferation in response to injury [12, 14, 15],
hyperplasic development of adipose tissue in animal under a high-fat diet [16],
and was recently shown to have beneficial effects on muscle fat oxidation and
lipid profiles in humans [17, 18].The
best characterized receptor in this group of nuclear factors is the PPAR-γ
which plays a significant role in adipocyte differentiation and fat deposition [10, 19].  This receptor is expressed in
adipose tissue, skeletal, and cardiac muscle and is also expressed in human
peripheral blood monocytes and in monocytic cell lines. The large list of
activators of PPAR-γ includes prostaglandin-derived 15-deoxi-delta 12,14PGJ2 (15d-PGJ2), the thiazolidinediones
(TZD) troglitazone (RezulinR), pioglitazone (ActosR),
and rosiglitazone (AvandiaR), among others (see
Table 1) [2, 10, 20].
The currently marketed TZDs are potent and selective PPAR-γ activators; they are
antidiabetic agents that increase the insulin sensitivity of target tissues in
animal models of non-insulin-dependent diabetes mellitus and in diabetic
patients.One
of the features that characterize the PPAR is the large amounts of natural and
synthetic molecules that can activate them. PPARs are differentially activated
by naturally occurring eicosanoids and related molecules [21, 22]. Nitroalkene
derivatives of fatty acids have also been characterized as endogenous PPAR
ligands. Schopfer et al. [23], using CV-1 reporter cells cotransfected with
plasmids containing the ligand-binding domain for PPAR-α, -δ/β, and -γ, found that
nitrated linoleic acid (1 μM) (LNO2) was capable to induce
significant activation of PPAR-γ (620%), PPAR-α (325%), and PPAR-δ/β (221%), when compared to
control cells. Concomitant works revealed the existence of LNO2 and
other fatty acid nitration products, generated by NO-dependent reactions, in
human red cells, blood, and urine samples [24, 25].The general approach used
to study the effects of PPARs is through PPAR activation by natural or
synthetic agonist. However, PPAR ligands have been shown to have both
PPAR-dependent and -independent actions, which could be addressed by in vivo genetic manipulation, such as
PPAR-knockout animals or in cell-based systems using small interfering RNA [26].


## 3. Inflammation Is Modulated by Ox-ldl through PPAR

A
primary initiating event in atherosclerosis is the accumulation of modified low-density
lipoprotein (LDL) in the subendotelial matrix, such as oxidized-LDL (ox-LDL.
These ox-LDL are taken up by macrophages, inducing the formation of foam cells,
and stimulating the EC to produce a number of proinflammatory molecules, such
as  monocyte chemotactic protein (MCP)-1, whose effects are mediated
by the G protein-coupled receptor CCR2, expressed mainly in monocytes,
basophils, and certain subsets of T cells [27, 28].One
of the most studied factors involved in the atherosclerotic process is ox-LDL [29].
Ox-LDL provides ligands for PPAR-γ and PPAR-α [30, 31]
and also seems to enhance the expression of PPAR-γ in differentiated
macrophages [32]. Ox-LDL, oxidized linoleic acid, and metabolites derived from
it, including 9-hydroxyoctadecaenoic acid (HODE) and 13-HODE, induce PPAR-γ activation
in monocytes and monocytic cellular lines, stimulating the transcription of the
ox-LDL receptor CD36/fatty acid translocase, through a PPRE in the promoter of
CD36/fatty acid translocase gene, which leads to the formation of foam cells [19, 30, 33].The first contact between ox-LDL and monocyte/macrophage cell elicits
reactive oxygen species (ROS) formation, followed by a desensitization of
macrophages via activation of PPAR-γ, which reduces ROS production, giving the
ox-LDL a dual role in the activation/deactivation of macrophages [34]. Ox-LDL
inhibited NF-κB-mediated IL-12 production in LPS-stimulated
mouse macrophages, involving both inhibition of the NF-κB-DNA interactions and physical interactions
between NF-κB and PPAR-γ [35]. Activation of NF-κB is
involved in the pathophysiology of many inflammatory chronic diseases,
including atherosclerosis. Binding sites for NF-κB have been found in
cellular adhesion molecules and chemokines [36, 37]. The NF-κB signaling
pathway is activated by the proinflammatory cytokines TNF-α and IL-1α which are
the major cytokine inducers of gene expression in EC.  In resting macrophages,
PPAR-γ ligands completely blocked the ox-LDL-mediated activation of NF-κB [38].PPAR-α
activation by ox-LDL in the vascular wall components seems to upregulate the
inflammation. The activation of this receptor in human EC by oxidized
components in LDL resulted in an increase in the production of chemotactic
factors for monocytes (MCP-1 and IL-8), conferring it a proinflammatory effect [9].


## 4. Molecular Mechanisms for the Regulation of Inflammatory/Immune Response in Atheroclerosis by PPAR Agonists

The
adhesion of monocytes to the vascular wall is mediated by adhesion molecules
expressed on the surface of the EC, such as vascular cell adhesion molecule-1
(VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P and E selectins  [39], which is a phenotypic hallmark of EC
activation and a critical step of many proinflammatory processes. The
constitutive activation of PPAR-γ in ECs inhibited the expression of VCAM-1,
ICAM-1, and E-selectin, by interference with NF-κB and AP-1 transactivation [40].Many of the immune cells in the atheroma
exhibit signs of activation and produce proinflammatory cytokines. In addition
to monocytes and macrophages, T cells definitely play a significant role in the
lesion, where CD4+ T cells dominate over CD8+ cells. CD4+ T cells differentiate
into several subtypes, being T helper-1 cells (Th1) the predominant pattern in
atherosclerosis. IFN-γ is a major proatherogenic Th1 cytokine, promoting
macrophage and endothelial activation with production of adhesion molecules,
cytokines, chemokines, radicals, proteases, and coagulation factors. In
addition, IFN-γ inhibits cell proliferation, collagen production, and
cholesterol efflux [41]. PPAR-α and PPAR-γ mRNA, and protein are expressed in
isolated human CD4+ T cells, and the activation of each one of them by specific
ligands reduces the secretion of  IFN-γ,
TNF-α, and IL-2  in these lymphocytes
[42]. Furthermore, the effect of PPAR agonist on CD4+ T cells impaired their
action on monocytes and EC, suggesting that PPAR modulation of inflammatory
pathways in T cells may offer clinical benefits in atherosclerosis [43].TNFα
is a catabolic proinflammatory cytokine, produced by Th1 cells and macrophages,
that exerts a wide range of effects on cells and tissues, through the
activation of the transcription factor NF-κB. Gene targeting of TNFα leads to
reduced atherosclerosis [42]. It has been shown that human aortic EC activation
by TNFα could be prevented by incubation with MCC-555, a novel TZD, while
pioglitazone and  rosiglitazone did not [44].PPAR-α has been shown to inhibit transcriptional responses to inflammatory stimuli
by interfering with the activation of NF-κB, leading to the reduction of
VCAM-1 in EC [11]. In vascular SMC, PPAR-α agonists inhibited
IL-1-induced IL-6 expression, cyclooxygenase-2 (COX-2) and prostaglandin
production [45]. The upregulation of antioxidant enzymes activity by the
PPAR-α activators reduced the oxidative stress and, as the result, it might
inhibit the NF-κB activation and subsequent inflammatory response [46].The regulation of chemokine-receptor expression may be a
crucial mechanism to control monocyte responses to chemokines. Monocytic-line
THP-1 cells incubated with rosiglitazone reduced CCR2 surface expression by
about 50–60% (P <
.001) compared with untreated control cells [29]. PPAR-γ agonists suppress
monocyte elaboration of inflammatory cytokines TNFα, IL1, and IL6 [47].
Table 2 summarizes the general effects of PPAR activation on each cell type.Table 2
PPAR-mediated
immune downregulation in monocytes/macrophages, T cells, EC, and SMC.
Monocyte/macrophageT cellECSMCReduced expression of
TNF-α, CCR-2, synthesis of IL-6, IL-1 by  PPAR-γ. Oxidative burst
suppressed by PPAR-γ.Reduced expression of
TNF-α, IFN-γ, IL-2 by activation of PPAR-γ and -α.Reduced expression of
VCAM-1, ICAM-1, E-selectin and MHC-II by PPAR-γ, -α, and -δ/β; impaired TNF-α
activation.Reduced synthesis of
IL6, COX-2, and prostaglandin by PPAR-α.In
murine hypercholesterolemic models, the administration of PPAR-γ ligands
inhibited the development of atherosclerosis, in spite of the high expression
of CD36 in the vascular wall [48]. Downstream
PPARγ-dependent anti-inflammatory effects of 15d-PGJ2 include the
inhibition of transcriptional activation by NF-κB via IκB, which affect gene expression of
inducible nitric oxide synthase (iNOS), TNFα, COX-2, IFN-α, IL-1, IL-6, and LPS-induced
transcription of AP-1 and STAT-1 [49]. Oxidative
burst in macrophages is also attenuated by PPAR-γ activation [50].Other
regulatory mechanism that could be attributed to PPAR is the selective
activation of anti-inflammatory cytokines, like IL-10. IL-10 has potent deactivating
properties in macrophages and T cells and modulates many cellular processes
that may interfere with the development and stability of the atherosclerotic
plaque [51]. Using nanomolar concentrations of rosiglitazone,
Thompson et al. have demonstrated the upregulation of IL-10, likely through a
functional PPRE found in the promoter region of IL-10 gene [52].Major
histocompatibility complex class II molecules (MHC-II) play a critical role in
the induction of immune responses by presenting peptides of foreign antigens to
CD4+ T lymphocytes, which result
in their activation and proliferation.
Human ECs are capable of expressing MHC-II under treatment with IFN-γ
and this induction is repressed by PPAR-γ ligands [53].PPAR-δ/β
seems to have dual effects in regard to inflammation in atherosclerotic models [8, 54–57]. Although results from both PPAR-δ/β–/– and PPAR-δ/β overexpressing
macrophages suggested a proinflammatory role for PPAR-δ/β, treatment of cells with
PPAR-δ/β agonist GW501516 suppressed the expression of MCP-1 and IL-1β in a receptor-dependent
manner, indicating that activation of PPAR-δ/β had an anti-inflammatory effect [54]. The pharmacological
modulation of PPAR-δ/β in atherosclerotic LDLR–/– mice showed decreased expression
of MCP-1, TNFα, and ICAM-1 [55] and similarly, proinflammatory modulators were
suppressed in apoE–/– mice treated with GW501516 [56].
Fan et al. also found an anti-inflammatory effect of
PPAR-δ/β agonists in TNFα-activated EC [8]. In addition, Takata et al. have
found that PPAR-δ/β agonist GW0742 substantially inhibited vascular
proinflammatory gene expression, macrophage content, and atherosclerosis in an
angiotensin II-induced high fat-fed male LDLR–/–   mouse model of accelerated
atherosclerosis [57]. Furthermore, promising results were obtained in a
clinical evaluation of the PPAR-δ/β agonist GW501516 in six obese males [17].
Although inflammatory markers were not considered in this study, the wide range
of beneficial effects by the pharmacological activation of PPAR-δ/β could
suggest an improvement on the inflammatory grade of proatherogenic conditions
and an attractive therapeutic target for drug development to treat
atherosclerosis [56].Post-translational
modifications have been found to modulate transcriptional activity of PPAR-γ [57].
One of these modifications is sumoylation, the covalent attachment of a small
ubiquitin-like proteins (Ubl) called SUMO-1. Pascual et al. [58] proposed a
novel pathway mediating ligand-dependent transrepression of inflammatory
response genes by PPAR-γ in macrophages which involves ligand-dependent
sumoylation of the PPAR-γ ligand-binding domain. This targets PPAR-γ to nuclear
receptor corepressor (NCoR)/histone deacetylase-3 (HDAC3) complexes on
inflammatory gene promoters, which in turn prevents recruitment of the
ubiquitylation/19S proteosome machinery that normally mediates the
signal-dependent removal of corepressor complexes required for gene activation.
As a result, NCoR complexes are not cleared from the promoter and target genes
are maintained in a repressed state. This mechanism provides an explanation for
how an agonist-bound nuclear receptor can be converted from an activator of
transcription to a promoter-specific repressor of NF-κB target genes that
regulate immunity and homeostasis [59].


## 5. Conclusion

Basic and clinical research points
out towards an intrinsic interplay between immune/inflammatory mediators and
PPAR activation in the pathogenesis and development of atherosclerosis. Each
one of PPARs seems to have and share different output in order to reach the
cellular homeostasis. It could be seen that Inflammation, as a disruption of
homeostasis, has not only internal control by its own but an external
regulation through the activation of PPARs.  However, it is still not clear what are the
“real functional in vivo”
natural ligands of PPAR. Meanwhile, the search for the ideal synthetic ligand that
would combine the beneficial effects of PPAR activation is ongoing. Such
“magical” drug would be prescribed either as hypoglycemic, hypolipemic and
anti-inflammatory agent, but would need to be chronically administered. Overall,
pharmacological activation of PPARs might be a better approach to cover all the
underlying inflammatory features of the atherosclerosis.



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