# PPAR Agonists and Cardiovascular Disease in Diabetes

Authors: Anna C. Calkin; Merlin C. Thomas
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferators activated receptors (PPARs) are ligand-activated nuclear transcription factors that play important roles in lipid and glucose homeostasis. To the extent that PPAR agonists improve diabetic dyslipidaemia and insulin resistance, these agents have been considered to reduce cardiovascular risk. However, data from murine models suggests that PPAR agonists also have independent anti-atherosclerotic actions, including the suppression of vascular inflammation, oxidative stress, and activation of the renin angiotensin system. Many of these potentially anti-atherosclerotic effects are thought to be mediated by transrepression of nuclear factor-kB, STAT, and activator protein-1 dependent pathways. In recent clinical trials, PPARα agonists have been shown to be effective in the primary prevention of cardiovascular events, while their cardiovascular benefit in patients with established cardiovascular disease remains equivocal. However, the use of
PPARγ agonists, and more recently dual PPARα/γ coagonists, has been associated with an excess in cardiovascular events,
possibly reflecting unrecognised fluid retention with potent agonists of the
PPARγ receptor. Newer pan agonists, which retain their anti-atherosclerotic activity without weight gain, may provide one solution to this problem. However, the complex biologic effects of the PPARs may mean that only vascular targeted agents or pure transrepressors will realise the goal of preventing atherosclerotic vascular disease.


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## Body

## 1. Introduction

Cardiovascular complications are the leading cause of premature mortality
in patients with diabetes [1]. While
classical risk factors for cardiovascular disease (CVD), such as smoking,
cholesterol, and hypertension, operate in persons both with and without
diabetes, the absolute risk of death is 2–4 times greater in patients with
diabetes [2] and progressively larger with
each additional risk factor [3]. Moreover, CVD, cerebrovascular
diseases, and peripheral
vascular diseases significantly contribute to the
morbidity in individuals with diabetes [1]. Ultimately, these macrovascular complications will develop in more than half of the
diabetic population [1].
In primary care, over a third of all patients
presenting with type 2 diabetes have an overt history of CVD, with a similar
number again likely to have undiagnosed macrovascular disease [4]. Consequently,
a key component (and some would argue the most important component) in the
management of diabetes is the primary and secondary prevention of
cardiovascular events.Diabetes is said to act as an
amplifier of cardiovascular risk leading to the increased incidence, size, and
complexity of atherosclerotic plaques [5, 6]. A number of components
contribute to accelerated atherosclerosis in diabetes. Diabetic dyslipidaemia
and insulin resistance significantly contribute to the development and
progression of macrovascular disease in diabetes. In addition, inflammation,
oxidative stress, enhanced matrix metalloproteinase activity, activation of the
local renin angiotensin system (RAS), and the accumulation of advanced
glycation end-products (AGEs) in the diabetic vasculature also act to enhance
atherogenesis and impair plaque stability.
Significantly, each of these pathways may be modified by the activity of
peroxisome proliferator-activated
receptors (PPARs), ligand-activated nuclear transcription factors with a
diverse range of metabolic functions [7–11]. This review will examine the
actions of PPARs in diabetes-associated atherosclerosis and explore the recent
controversies surrounding the actions of PPAR agonists on CVD in patients with
diabetes.


## 2. Peroxisome Proliferator-Activated Receptors (PPARs)

PPARs are nuclear transcription
factors that have complex biological effects, resulting from the
transactivation or transrepression of dozens of genes that play
important roles in glucose and lipid homeostasis [12]. Transactivation effects require
ligand-activated dimerisation of PPAR with the retinoid X receptor (RXR),
followed by translocation of the PPAR : RXR heterodimer complex to the nucleus,
whereupon it binds to PPAR response elements of target genes and induces their
expression [12]. Transrepression effects
are mediated via interference with transcription factors such as activator
protein-1 (AP-1) and nuclear factor-κB (NF-κB) [13] (see Figure 1). In addition, conformational
remodelling of the PPAR receptor that follows ligand binding results in the release of co-repressor molecules. The relative importance of activation versus
repression pathways for the in vivo actions of PPAR agonists remains to be
established. Moreover, there is evidence
that all PPAR ligands do not stimulate transactivation and transrepression
pathways to a similar extent, meaning that different agents of the same class
may have potentially disparate effects [14, 15].Figure 1
Transactivation and transrepression effects of peroxisome proliferator-activated receptors.Three different PPAR isoforms
have been identified in humans. These share similar
structural organization and sequence homology. However, these isoforms possess
distinct functions, and vary in their ligand affinity, expression, and activity in different
metabolic pathways.


## 3. Peroxisome Proliferator-Activated Receptor Alpha (PPARα)

PPARα is highly expressed in the vasculature,
including the endothelial cells [16, 17], smooth muscle cells [18], and macrophages [19]. Activation of PPARα leads to modulation of lipid metabolism,
including transcription of apolipoprotein A1 (apoA1) [20] and apolipoprotein AII [21], resulting in increased levels
of “cardioprotective” high-density lipoprotein (HDL) cholesterol. Uptake of HDL
cholesterol is also increased via
the upregulation of CLA-1/SR-B1 [22].
β-oxidation and lipoprotein lipase (LPL) activity are also stimulated following activation of PPARα. This leads to a decrease in triglycerides and free fatty acids, and levels of apolipoprotein CIII, which inhibits LPL-mediated breakdown of triglycerides, further resulting in lower triglyceride levels [23]. Finally, low-density
lipoprotein (LDL) cholesterol particles are shifted from a small, dense to a
large, buoyant form to create particles that are less atherogenic and more
easily cleared [24]. The natural ligands of PPARα include prostaglandins, leukotrienes,
and medium- and long-chain free fatty acids such as eicosapentaenoic acid and
docosahexenoic acid [25, 26]. Synthetic ligands of this
receptor are utilized in the management of dyslipidaemia [27], and include members of the
fibrate drug class (e.g., gemfibrozil, clofibrate,
fenofibrate, and bezafibrate).


## 4. Peroxisome Proliferator-Activated Receptor Gamma (PPARγ)

PPARγ is largely expressed in adipose tissue as well
as in skeletal
muscle, sites where this PPAR isoform exerts much of its metabolic actions [28]. However, PPARγ is also expressed locally in the vasculature,
including the endothelial cells [17], smooth muscle cells [29], and macrophages [13, 19]. Broadly, PPARγ activation results in increased sensitivity to
the metabolic actions of insulin by reversing lipotoxicity-induced insulin
resistance. PPARγ activation
has also been shown to rejuvenate pancreatic β-cell function resulting in their improved
function [30]. In adipose tissue, activation
of  PPARγ leads to
differentiation of adipocytes, making them more able to uptake fatty acids, in
turn, sparing other metabolic tissue such as skeletal muscle and liver [28]. In addition, PPARγ agonists increase the expression and activity
of glucose transporter-4 and phosphatidyl-3-kinase [31, 32]. The natural ligands of PPARγ include prostaglandins, such as
15-deoxy-(12,14)-prostaglandinJ2,
and fatty acids including linoleic and arachidonic acids [32]. Synthetic ligands of PPARγ include the thiazolidinedione drug class (e.g.,
rosiglitazone and pioglitazone). Some other drugs also have partial agonist
activity at the PPARγ receptor,
including the AT1 receptor antagonist, telmisartan [33].


## 5. Peroxisome Proliferator-Activated Receptor Delta (PPARβ/δ)

The PPAR delta isoform (also known as beta) is the most widely distributed of the PPARs, with expression seen in most tissues including the vasculature [34]. Unlike the other PPARs, PPARβ/δ-RXR heterodimers bind
to consensus PPAR DNA response elements in the absence of a ligand, and repress
target gene expression indirectly by recruiting co-repressors [35]. Following ligand activation, the co-repressor complex is disrupted,
leading to enhanced PPARβ/δ target gene expression
by both ligand-induced transcriptional activation and transcriptional
derepression. In addition, repressor molecules, such as BCL-6, are liberated on
ligand binding, leading to the repression of other pathways, such as
inflammation and the transcriptional
activity of PPARα and PPARγ [36, 37]. Because
of its wide tissue expression, it was initially suggested that PPARβ/δ might simply serve a house-keeping role. However, more recent data suggest that PPARβ/δ can play an important
role in wound healing, inflammatory responses,
and lipid metabolism [34, 36]. For example, PPARβ/δ activation has been shown to increase HDL
cholesterol levels like PPARα ligands,
as well as mediate insulin-sensitising and glucose lowering effects like PPARγ [34, 38]. PPARβ/δ deficient
macrophages also show reduced recruitment, which may be particularly important
for plaque stability [36] (see below).
PPARβ/δ is
activated by a large variety of ligands, such as fatty acids and
eiconsanoids including prostaglandin A1, although the major
natural ligand remains to be established [25].
Synthetic agonists with nanomolar affinities for PPARβ/δ have also been generated, although none are currently used in
clinical practice. Interestingly, the physiological action of these agonists
in experimental atherosclerosis is similar to the phenotype observed in PPARβ/δ knockout mice [36, 39], consistent with the
important actions of transcriptional de-repression following activation of PPARβ/δ.


## 6. PPAR Agonists and Diabetic Dyslipidaemia

Diabetic dyslipidaemia
is a major reversible risk factor for the prevention of CVD in individuals with
diabetes [40]. A range of quantitative and
qualitative lipid and lipoprotein abnormalities are observed in patients with
diabetes [41]. The main components of diabetic
dyslipidaemia are excessive postprandial lipaemia associated with increased
plasma triglyceride [42], due to the accumulation of very
low-density lipoprotein (VLDL), chylomicron remnants, and intermediate density
lipoprotein particles in the plasma. This is thought to reflect both the
overproduction of triglyceride-rich VLDL (due to increased flux of free fatty
acids and hepatic resistance to the effects of insulin), together with reduced catabolism
(associated with reduced LPL activity) [43]. HDL cholesterol levels are
invariably reduced in patients with type 2 diabetes, reflecting increased
catabolism of HDL particles [44]. In addition, HDL particles
become enriched with triglyceride, in an attempt to cope with an increased
VLDL burden. Although LDL cholesterol levels in patients with type 2 diabetes
are often within the normal range, there remain significant disturbances in LDL
metabolism in diabetes. For example, LDL production is significantly reduced,
while impaired turnover of LDL particles promotes glycoxidative modification of
lipoprotein particles and cholesterol deposition in the arterial wall [45–47]. Diabetes is also associated
with the accumulation of small dense, triglyceride-rich, LDL particles that
have an increased atherogenic potential [24].As noted above, both PPAR agonists are
able to significantly modify circulating lipid levels, and therein reduce
cardiovascular risk in patients with diabetes [27, 48]. In particular, the use of fibrates
in patients with diabetes increases HDL cholesterol, decreases
triglyceride levels, and shifts LDL cholesterol distribution toward
larger, less atherogenic particles [24, 27, 49]. PPARγ agonists also stimulate reverse cholesterol
transport [50, 51] and have beneficial effects on the
low HDL cholesterol levels and elevated triglyceride levels that characterize
diabetic dyslipidaemia. However, thiazolidinediones can also modestly increase
LDL cholesterol levels in some patients [48]. PPARβ/δ agonists
are able to increase HDL cholesterol levels and
improve postprandial triglyceride clearance [52].


## 7. PPAR Agonists and Insulin Resistance

While glycemic control is important
for the prevention of microvascular complications, its role in the development
of atherosclerotic vascular disease is less clear [53].
For example, in the UKPDS study, macrovascular outcomes were not
correlated with HbA1c. However, CVD was reduced in patients that
received the insulin sensitizer, metformin, when compared to equivalent glycemic
control achieved by sulphonylureas or insulin therapy [54]. This led to the hypothesis that
insulin sensitivity may itself play an important role in the development of
macrovascular disease, and that agents that reduce insulin resistance, such as
metformin and PPARγ agonists,
by extension, may have particular benefits in the management of type 2 diabetes
[55]. Certainly, resistance to the actions
of insulin is strongly associated with CVD in patients with diabetes. To the
extent that insulin resistance is linked to chronic hyperglycaemia,
dyslipidaemia, inflammation, and hypertension as part of the metabolic syndrome,
this association is not surprising. However, it is now clear that insulin also
has direct actions in the vasculature that influence the development and
progression of atherosclerotic disease. For example, in diabetic tissues,
selective insulin resistance in the PI-3-kinase signaling pathway leads to reduced synthesis of nitric oxide, impaired
metabolic control, and compensatory hyperinsulinaemia.
At the same time, insulin signaling, via extracellular signal regulated
kinase-(ERK) dependent pathways, is relatively unaffected in diabetes, meaning
that hyperinsulinaemia is able to stimulate the expression of endothelin and
other pathogenic mediators, tipping the balance of
insulin’s actions in favor of abnormal vasoreactivity, angiogenesis, and other
pathways implicated in atherosclerosis [56, 57]. In addition, preferential impairment
of non-oxidative glucose metabolism in diabetes leads to increased intracellular
formation of AGEs and oxidative stress. Nonetheless, while it is conceivable that
improvements in insulin sensitivity may have beneficial vascular effects in
diabetes, the fact that PPAR agonists retain their anti-atherosclerotic activity
in the absence of insulin [9] suggests that other (direct) actions
may also be important for their anti-atherosclerotic activities.


## 8. The Potential Direct Anti-Atherosclerotic Actions of PPAR Agonists

While improvements in metabolic control and
the lipid profile have important effects on CVD in patients with diabetes, it
is becoming increasingly clear that PPAR agonists have a range of independent
actions on the vascular wall which impact on atherogenesis. In particular, pre-clinical studies
demonstrate ligand-dependent
PPAR activation is able to reduce the development and progression of atherosclerotic lesions in a
range of experimental models, without needing to normalise dyslipidaemia and
hyperglycaemia, or
improve insulin resistance [58]. For example, studies from our
group demonstrated that treatment with the PPARα agonist, gemfibrozil, was able to prevent the accumulation of
atherosclerotic plaque in apolipoprotein E (apoE) knockout (KO) mice, a model in
which PPARα agonists
have no effect on severe dyslipidaemia [8]. Similarly, treatment with the
PPARγ agonist,
rosiglitazone, in insulinopenic diabetic apoE KO mice was also associated with
a reduction in aortic atherosclerosis [9], in the absence of insulin
sensitization or improvement in glucose levels. Finally, treatment with a PPARβ/δ agonist,
GW0742X, has also been shown to attenuate atherosclerosis in LDL receptor
KO mice, in the absence of changes in plasma lipids [39]. Taken together, these studies
point to possible direct effects of PPAR agonists on the vasculature that
impedes pathogenic pathways implicated in the development of atherosclerosis,
including inflammation, oxidative stress, metalloprotease activity, AGE
accumulation, and activation of the RAS.


## 9. PPAR Agonists and Vascular Inflammation

Inflammation
plays a key role in the development and progression of
atherosclerotic vascular disease. Inflammatory cells are a major component of
early atherosclerotic lesions, and inflammatory cytokines and chemokines
accelerate plaque accumulation. Some of the earliest changes involve the
activation of endothelial cells, which then express adhesion molecules such as
vascular-cell adhesion molecule 1 (VCAM-1) [59],
encouraging leucocyte recruitment, the production of chemokines, and further
inflammation. Activation of PPAR
receptors has also been strongly linked to this early inflammatory response.
PPARα,γ
and
β/δ agonists
reduce the expression of adhesion molecules, such as VCAM-1, on the surface of
cytokine-activated endothelial cells, as well as reduce macrophage infiltration
within atherosclerotic plaque [8, 9, 60, 61].
PPAR agonists also reduce the production of inflammatory cytokines including
tumour necrosis factor (TNF)-α, IL-6, and
IL-1β [7, 18].
PPARα activation
indirectly modulates inflammatory components in HDL, such as apoA1, serum
amyloid A, and paraoxonase-1 [62].
Thiazolidinediones are also able to inhibit endothelial cell activation [63]
and indirectly alter systemic inflammation by actions in adipose tissue,
reducing the production of pro-atherogenic adipokines including TNF-α and resistin [64].
PPARβ/δ may also have important anti-inflammatory
actions. For example, in LDLR KO mice treatment with the PPARβ/δ agonist,
GW0742X, was associated with a marked attenuation of atherosclerosis, with a
concomitant decrease in monocyte chemoattractant protein (MCP)-1 and
intercellular adhesion molecule (ICAM)-1 [39].


## 10. PPAR Agonists and Oxidative Stress

Oxidative stress is thought to be a key mediator of atherosclerosis,
contributing to the upregulation of adhesion molecules [65], acceleration of foam cell
formation, and a reduction in plaque stability [66]. PPAR agonists are also able to modulate oxidative stress in vascular
tissues. PPARα activation reduces the expression of the pro-oxidant
NAD(P)H subunit p22phox, and increases endothelial expression of the anti-oxidant, CuZn
superoxide dismutase [67]. PPARγ agonists
also have potent anti-oxidant activity in human endothelial cells [67], hypercholesterolemic rabbits [68], and obese subjects [69]. Studies from our laboratory have shown that treatment of diabetic
animals with either a PPARα or a PPARγ agonist is associated with a reduction in
vascular superoxide production, together with reduced gene expression of the
NAD(P)H oxidase subunits p47phox and gp91phox observed in the aorta of diabetic apoE KO mice [8, 9] (see Figure 3).


## 11. PPAR Agonists and Matrix Metalloproteinases

Atherosclerotic
plaque rupture, with subsequent occlusive thrombosis, is the underlying cause
of sudden cardiac events. Matrix metalloproteinases (MMPs) are thought to
mediate the progression of atherosclerotic lesions to an unstable phenotype
that is more prone to rupture, through the destruction of the overlying fibrous
cap. PPAR agonists may promote plaque stability by reducing the production of
MMPs from monocytes/macrophages and vascular smooth muscle
cells [70]. Our group has recently demonstrated that
gemfibrozil treatment was associated with attenuation of diabetes-associated
MMP-2 and MMP-9 gene expression in aorta of diabetic apoE KO mice [8].
Furthermore, studies in patients with type 2 diabetes and CVD have shown that
treatment with a PPARγ agonist is
associated with a reduction in plasma MMP-9 levels [11].


## 12. PPARs and Advanced Glycation End-Products

The accumulation of  AGEs,
as a result of hyperglycaemia, dyslipidaemia, and oxidative stress in diabetes,
contributes to the development and progression of vascular disease in diabetes [71, 72].
AGEs accumulate in many diabetic tissues [73], including in atherosclerotic
plaques [71]. Their importance as downstream
mediators of hyperglycaemia in diabetes has been amply demonstrated by animal
studies using inhibitors of advanced glycation to retard the development of
atherosclerotic vascular disease without directly influencing plasma glucose
levels [71, 74].
Furthermore, dietary excess of AGEs has been shown to accelerate
atherosclerosis without affecting glycemic control [75].  Recent studies suggest that, in addition to
lowering glucose levels, PPARγ agonists are able to inhibit
the formation of AGEs [76]. The mechanism by which PPARγ
agonists might reduce AGEs
remains to be established, although their anti-oxidant and lipid lowering
activities may be relevant to AGE formation and the advanced glycation pathway [10].


## 13. PPARs and the Renin Angiotensin System

The RAS has an important role in the development and progression of diabetic
atherosclerosis. For example, our group has demonstrated clear anti-atherosclerotic
activity of RAS blockade with an AT1 receptor antagonist or an
angiotensin converting enzyme (ACE) inhibitor in diabetic apoE KO mice [77, 78]. PPAR
activators are known to be negative regulators of the AT1 receptor
gene. For example, our studies with either rosiglitazone or gemfibrozil
resulted in a significant reduction in the vascular expression of the AT1 receptor in diabetic apoE KO mice (see Figure 4). At least in this model, this
repression of AT1 receptor expression by PPAR agonists may function,
in terms of atherogenesis, in an equivalent manner to angiotensin receptor
blockade.


## 14. PPARs and the Diabetic Kidney

Chronic kidney
disease is a major risk factor for cardiovascular disease in patients with
diabetes. For
example, myocardial infarction and stroke are 10 times more common in type 1
diabetic patients with kidney disease than those without renal disease [79]. Below the age of 50 years, the
excess of mortality from cardiovascular disease is almost entirely confined to
patients with diabetic nephropathy [80].
Equally, in patients with type 2 diabetes, the risk of developing
cardiovascular disease is 2-3 times higher in others with microalbuminuria compared to normal
albumin excretion. In patients with
proteinuria, the risk is increased at least 10-fold [81].PPAR agonists have a number of
important actions in the diabetic kidney, which may attenuate renal injury and
therein (indirectly) reduce cardiovascular risk. For example, we have shown
that albuminuria in streptozotocin-diabetic mice is reduced by treatment with
the PPARα agonist,
gemfibrozil, the PPARγ agonist,
rosiglitazone, or the dual PPAR agonist, compound 3q [82]. In addition,
glomerulosclerosis, tubulointerstitial expansion (see Figure 2), and collagen
deposition were significantly attenuated. PPARγ agonists may also have beneficial actions on
renal hypertrophy in models of experimental diabetes [83–85]. Notably, these renoprotective
effects are observed in the absence of changes in glucose or lipid levels,
insulin sensitivity, or a reduction in blood pressure, and taken together
suggest some independent renoprotective action. Moreover, the finding of
similar beneficial effects of PPARα and PPARγ agonists, as well as thiazolidinedione and
non-TZD dual agonist compounds, raises the possibility that neither of these
agents are working through conventional PPARα and γ pathways in this model, but through the
transrepression of other transcription factors implicated in diabetic kidney
disease including AP-1, signal transducers and activators of transcription 1
(STAT-1) and NF-κB, even in the absence of PPAR receptors [86]. Importantly, these renal
benefits have also been observed in clinical trials with PPAR agonists,
including the recently completed FIELD trial where a reduction in
microalbuminuria was observed in patients treated with fenofibrate [87]. Similarly, in the Diabetes
Atherosclerosis Interventional Study, fenofibrate therapy was associated with
reduced progression from normal urinary albumin excretion to microalbuminuria
in patients with type 2 diabetes [88].
Several previous studies have also demonstrated that thiazolidinediones are able to improve
markers of renal structure and function in patients with diabetes [89–91]. However, the cardioprotective
benefits of long-term renoprotection observed in these studies remain to be
established.Figure 2
An increase in renal tubulointerstitial area associated with
streptozotocin diabetes in apoE KO mice is attenuated following treatment with
PPARγ agonist,
rosiglitazone, PPARα agonist,
gemfibrozil, or the dual PPARα/γ agonist, ragaglitazar.Cross-sections of aorta stained for the NAD(P)H oxidase subunit,
p47phox. ApoE KO mouse aorta from (a) control, (b) diabetic, and (c) diabetic +
rosiglitazone treated mice.
(a)(b)(c)Figure 4
Gene expression of the angiotensin II subtype
1 receptor as assessed by real-time RT-PCR in aorta from apoE knockout mice
treated with the PPARγ agonist, rosiglitazone
or the PPARα agonist, gemfibrozil for 20 weeks. P < .05 versus diabetic mice.


## 15. Clinical Trials with PPARα Agonists

A number of clinical studies have
shown that treatment with lipid lowering agents is able to prevent adverse CVD
outcomes in patients with diabetes. Yet while PPARα
agonists are able to reduce lipid levels in patients with diabetes, their
clinical efficacy remains controversial, with a number of both positive and
equivocal results reported in clinical trials.
For example, in the Veterans Affairs High-Density Lipoprotein
Intervention Trial (VA-HIT), patients with diabetes treated with gemfibrozil
had a reduced risk of a composite end point of coronary heart disease (CHD)
death, stroke, or myocardial infarction by 32% and reduced CHD deaths by 41%
compared to those with diabetes receiving standard care [92]. Moreover, the clinical benefit derived
from fibrates exceeded that attributable to changes in the lipid profile. The
Diabetes Atherosclerosis Intervention Study (DAIS) showed that 3 years of
treatment with fenofibrate resulted in significant reductions in angiographic
progression of atherosclerosis and stenosis (P ≤ .03) [27]. Ciprofibrate therapy has also
been associated with an increase in flow-mediated dilation in association with
an improvement in lipid profile in people with type 2 diabetes [49]. However, in the Helsinki Heart
Study, although gemfibrozil reduced the incidence of primary CHD compared with
placebo among patients with diabetes (3.4 versus 10.5%), this difference was not
statistically significant [93]. Similarly, the recently
published Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study
demonstrated a non-significant 11% reduction in the primary end point (coronary
artery disease (CAD) death or non-fatal myocardial infarction (MI); P = .16)
and an 11% reduction in total cardiovascular events (P = .035) [87]. However, a 25% reduction in
total CVD events and coronary heart disease events was observed in patients
without a history of CVD (P = .014). This possibly suggests that early
and primary therapy with PPARα agonists, comparable to the strategy
employed in animal models and shown to be definitively anti-atherosclerotic, may
also be beneficial in the clinical setting. In addition, improvements in
microvascular outcomes, including a reduction in microalbuminuria in the FIELD
study, would be expected to have long-term macrovascular benefits.


## 16. Clinical Trials with PPARγ Agonists

Thiazolidinediones have been
shown to have a range of positive effects on vascular function in clinical
studies. For example, small clinical studies have demonstrated positive effects
of thiazolidinediones on cardiovascular parameters such as
acetylcholine-mediated dilation [94] and pulse wave velocity
[11, 95–97]. Whether such benefits translate
to a reduction in cardiovascular risk has been tested in several recent and
ongoing clinical trials, although these short-term studies may be inadequate to
assess a process like atherosclerosis that takes many decades to evolve.The Prospective Pioglitazone
Clinical Trial in Macrovascular Events (PROACTIVE trial) examined the effect
of pioglitazone, taken in addition to conventional therapy for three years, on
all-cause mortality, non-fatal MI, stroke, acute coronary syndrome, leg
amputation, and coronary or leg revascularisation [98]. While there was a non-significant
10% reduction in this primary outcome P = .09, the main secondary
endpoint (composed of all-cause mortality, non-fatal MI, and stroke) was reduced
by 16% (P = .03). However, heart failure and symptomatic oedema due to
fluid retention due to PPARγ agonists may have masked any benefit from actions on atherogenesis.A recent meta-analysis has also been performed to examine the cardiovascular
effects of the PPARγ agonist, rosiglitazone, which includes outcome data from 35
trials, such as the large Diabetes Reduction Assessment with Ramipiril and Rosiglitazone Medication (DREAM) trial and the A Diabetes
Outcome Prevention Trial (ADOPT) [99–101].
This meta-analysis demonstrated that treatment with rosiglitazone increased the
risk for MI by 43% (P = .03), and death
from cardiovascular causes by 64% (P = .06). Whether this finding also reflects increased fluid retention remains to
be established.


## 17. The Promise of Dualα/γ PPAR Agonists

The apparent efficacy of PPARα
and PPARγ agonists individually on metabolic control,
led to the development of dual PPARα/γ agonists, offering the potential of optimising
the metabolic and anti-atherosclerotic actions arising from activating both
receptors. In general, these agents proved to be more potent agonists of PPARγ than  conventional thiazolidinediones and highly
effective at improving metabolic parameters. For example, ragaglitazar
was more effective at improving glycemic control and attenuating plasma lipid
levels than single agonists such as rosiglitazone [102]. Similarly, treatment with
muraglitazar in db/db mice was more
effective at reducing plasma glucose levels than rosiglitazone [103]. Yet despite improved metabolic
outcomes, the effects on atherogenesis have been less clear. For example, we
found that treatment with the dual PPARα/γ agonist, compound 3q, was associated with a
marked increase in atherosclerosis in control apoE KO mice [104] (see Figure 5), while PPARγ and α agonists
used alone in this model were protective [8, 9]. This increase in
atherosclerotic plaque was observed in control animals despite an improvement
in glycemic control and an improvement in lipid profile [104]. Furthermore, plaque
accumulation in mice treated with the dual PPAR compound was also associated
with a concomitant increase in aortic gene expression of the pro-inflammatory
molecules, P-selectin, CD36, VCAM-1, and MCP-1 and increased macrophage
infiltration, an effect not seen with the single PPAR agonists, rosiglitazone
or gemfibrozil [104]. By contrast, Claudel and
colleagues demonstrated that treatment with the dual PPARα/γ compound,
GW2331, for 11 weeks was more effective at attenuating atherosclerosis in
female apoE KO mice than rosiglitazone alone [105]. Similarly, Zuckerman et al. found that LY465608 reduced atherosclerosis in
male apoE mice fed a high-fat diet in the absence of changes in plasma total
cholesterol levels [106]. More recently, the anti-atherosclerotic
actions of tesaglitazar on vascular disease have also been investigated. In
apoE*Leiden mice fed a high-fat diet, tesaglitazar was associated with a 92%
reduction in aortic atherosclerosis in association with a reduction in
macrophages and collagen in lesions [107]. In high-fat fed LDL receptor KO
mice treatment with tesaglitazar for 12 weeks was associated also with a
decrease in atherosclerosis in female mice in the absence of alterations in
cholesterol or triglyceride levels or a reduction in the inflammatory markers
serum amyloid A and serum amyloid P [108].
The reasons for these conflicting results are unclear. However, it is
possible that the different balance of activation of PPARα and
γ
with each of these agents, as well as
differential effects on transrepression may have contributed to these disparate
findings.Figure 5
Total aortic
plaque area as assessed by an en face
approach in apoE knockout mice treated with the dual PPARα/γ agonist, compound 3q for
20 weeks. P < .05 versus control mice.


## 18. Clinical Studies with Dual PPAR Agonists

Despite
their clear actions as PPARγ and PPARα agonists, and clinical efficacy in terms of
lipid and glycemic control [109–116], which were comparable or better
than achieved by PPAR agonist alone, recent reports have suggested that
dual PPARα/γ agonists may also be associated with
an increased risk of adverse cardiovascular events when used by individuals
with diabetes [116].
In particular, the risk of death, myocardial infarction, stroke, transient ischaemia attack, or CHF was increased by over two-fold (RR
2.62; 95% CI 1.36 to 5.05) in patients with type 2 diabetes receiving the dual agonist,
muraglitazar, compared to those receiving a PPARγ agonist (pioglitazone) alone, despite comparable
effects on glycemic control [116]. Whether this increase in events is
due to an augmentation of atherogenesis, as observed in our pre-clinical models,
or the by-product of augmented fluid retention in patients with a stiff
vasculature, remains to be established. Certainly, the more potent activation
of the PPARγ receptor achieved by dual
agonists may
lead to clinically important fluid retention in some patients, particularly at
high doses or in patients with established congestive heart failure.
Nonetheless, even when patients with NYHA III/IV heart failure were excluded
from these trials, the muraglitazar treated group still had 13
adjudicated cases of heart failure compared with only one patient in
the control group. More recently, the development tesaglitazar has been
discontinued due to concerns about increased serum creatinine and
decreased glomerular filtration rate [117]. Taken together with reports of
toxicity and carcinogenic effects with some of the dual PPAR agonists in pre-clinical
studies [118–120], these finding have meant that
ongoing evaluation of this class of drug has been delayed and largely
superseded by the pan-PPAR agonists (detailed below).


## 19. The Development of Pan-PPAR Agonists

The clinical efficacy of PPAR agonists individually
have led to the development of chemical ligands with activity across all three
receptor isoforms. The potential advantage of such a combination rests in the
finding that these so-called “pan-PPAR” agonists retain their broad metabolic
activity, without the weight gain associated with PPARγ agonists [121, 122]. Cell culture and
pre-clinical studies have also demonstrated the efficacy of pan PPAR agonists in
modulating various pathways linked to the development of atherosclerosis [67, 123, 124].


## 20. Clinical Studies with Pan PPAR Agonists

There are a small
number of pan-PPAR agonists now in the early stages of clinical trials
including GW766954, GW625019, PLX-204, and netoglitazone (MCC-555) [125]. These agents have been shown to improve glycaemic and lipid
control in a range of settings. While such benefits should confer some
cardiovascular benefit, the actions of agents of this class on the development
and progression of atherosclerosis in diabetes remain to be established.
However, some insight into the possible efficacy of pan PPAR agonists may be
inferred from clinical studies using bezafibrate. Although originally classed
as a fibrate, bezafibrate is now considered a pan-PPAR agonist, albeit of low
potency.  Nonetheless, like other PPAR
agonists, treatment with bezafibrate significantly raises HDL cholesterol
levels, reduces triglycerides, and improves insulin sensitivity in patients
with diabetes [126]. In the Bezafibrate Coronary Atherosclerosis Intervention Trial
(BECAIT) of dyslipidemic males under the age of 45 who have experienced a
previous MI, bezafibrate improved dyslipidaemia, reduced the cumulative coronary
event rate (P = .02) and slowed the progression of focal coronary
atherosclerosis. The St Mary’s, Ealing, Northwick Park Diabetes Cardiovascular
Disease Prevention (SENDCAP) trial also demonstrated a reduction in the
combined incidence of ischemic change on resting ECG and documented MI [127]. Despite this, they were unable to see any effect of bezafibrate on
the progression of coronary of femoral atherosclerosis over the 3 years of the
study. By contrast, the Bezafibrate Infarct Prevention (BIP) study demonstrated
no significant effect of bezafibrate on fatal or non-fatal MI in those with
diabetes [128]. Whether newer and more potent pan-PPAR ligands with
differential activation of the various PPAR isoforms will prove to be more
beneficial with respect to cardiovascular outcomes remains to be established. In addition, given the actions of PPAR in the
transcriptional regulation of an enormous range of genes and pathways, the
potential adverse impact of such pan-PPAR activity needs to be carefully
studied.


## 21. Concluding Remarks

Agonists
of the PPAR family have represented the most important development in the
management of diabetes over the last decade. Despite the promise of improved
insulin sensitivity and better lipid control, these agents have not achieved
the cardiovascular benefits expected of them. There is little doubt that in experimental models,
PPAR agonists have clear and independent anti-atherosclerotic actions, including the
suppression of vascular inflammation, oxidative stress, and activation of the
renin angiotensin system. Why this has not translated into clinical benefit
remains to be fully established. It may be that longer-term follow up of
clinical studies will reveal statistically significant results, as the long-term
benefits of improved metabolic control are realized. Equally, the complex
biological effects of the PPARs in a range of organs may mean that any
benefits are offset by unwanted actions that impact on CVD, such as fluid
retention, malignancy, renal impairment, or increases in LDL cholesterol.
Whether more organ-targeted agonists or pan-PPAR agonists will prove more
effective remains to be seen.However, the fact that many of the potentially useful vascular effects are thought to be
mediated by transrepression of pro-atherogenic signalling pathways, should lead
in the future to the development of more selective transrepressors for the
prevention and management of cardiovascular disease in diabetes.



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*Source: 245410-2008-01-02.xml*