# Transcriptional Control of Vascular Smooth Muscle Cell Proliferation by Peroxisome Proliferator-Activated Receptor-γ: Therapeutic Implications for Cardiovascular Diseases

Authors: Florence Gizard; Dennis Bruemmer
Journal: PPAR Research
 (2008)

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## Abstract

Proliferation of vascular smooth muscle cells (SMCs) is a critical process for the development of atherosclerosis and complications of procedures used to treat atherosclerotic diseases, including postangioplasty restenosis, vein graft failure, and transplant vasculopathy. Peroxisome proliferator-activated receptor (PPAR)γ is a member of the nuclear hormone receptor superfamily and the molecular target for the thiazolidinediones (TZD), used clinically to treat insulin resistance in patients with type 2 diabetes. In addition to their efficacy to improve insulin sensitivity, TZD exert a broad spectrum of pleiotropic beneficial effects on vascular gene expression programs. In SMCs, PPARγ is prominently upregulated during neointima formation and suppresses the proliferative response to injury of the arterial wall. Among the molecular target genes regulated by PPARγ in SMCs are genes encoding proteins involved in the regulation of cell-cycle progression, cellular senescence, and apoptosis. This inhibition of SMC proliferation is likely to contribute to the prevention of atherosclerosis and postangioplasty restenosis observed in animal models and proof-of-concept clinical studies. This review will summarize the transcriptional target genes regulated by PPARγ in SMCs and outline the therapeutic implications of PPARγ activation for the treatment and prevention of atherosclerosis and its complications.


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## Body

## 1. Introduction

Most cardiovascular diseases result from complications of
atherosclerosis, which is a multifactorial
process characterized by chronic inflammation, lipid accumulation, and the formation of a complex
atherosclerotic lesion [1]. Recruitment of monocytes,
their differentiation into macrophages, and uptake of LDL-derived cholesterol are the major cellular events
contributing to early fatty streak formation [2, 3].
Continued intracellular cholesterol accumulation results in the generation of
endogenous inducers of inflammatory and proliferative gene expression and a
broad range of cellular and humoral responses contributing to lesion initiation
and progression [4]. The resulting chronic
inflammatory state and the enrichment of lipid-laden macrophages ultimately
lead to the formation of a complex atherosclerotic lesion [5].During the course of
atherosclerotic lesion formation, secreted growth factors and cytokines promote
the migration and proliferation of vascular smooth muscle cells (SMCs) to
contribute to neointima formation [6]. This chronic
proliferative response of SMCs promotes further lesion development through the
production of proinflammatory mediators and the
synthesis of extracellular matrix molecules, which is required for the
retention of lipoproteins and often constitutes the majority of the protein
content of the advanced lesion responsible for luminal obstruction [1]. However, SMC proliferation within
the developing lesion may also exert beneficial effects by forming a
fibrous cap covering the advanced atherosclerotic lesion, an important
mechanism for the stability of the plaque [7]. The result of this chronic
process is the development of an advanced atherosclerotic lesion, which may
ultimately cause luminal obstruction and ischemic complications.Once occlusive atherosclerotic disease has developed, the standard of
care may include angioplasty, coronary artery bypass grafting, or cardiac
transplantation. However, all current treatment approaches are limited by a
varying degree of treatment failure and reocclusion
of the arterial lumen. Among the cellular mechanisms responsible for this failure
of the current interventional procedures used to treat occlusive
atherosclerotic diseases, such as postangioplasty restenosis, transplant
vasculopathy, and coronary artery bypass graft
failure, SMC proliferation constitutes a prime mechanism [6]. In the past decade, elegant progress in interventional
cardiology has provided the introduction of drug-eluting stents delivering
rapamycin or paclitaxel into the vessel wall that target SMC proliferation [8].
However, despite initial enthusiasm, the complete inhibition of the healing response using these approaches may
leave a thrombogenic vessel surface at risk of in-stent thrombosis and vessel
occlusion [9]. Thus, despite these advances, ideal therapy for occlusive vascular
disease is still far from established.In an
era marked by the increasing prevalence of obesity, diabetes, and
cardiovascular disease, members of the nuclear hormone receptor superfamily
have emerged as transcription factors that regulate diverse aspects of
metabolism [10, 11]. In addition to their function to act as
molecular sensors of lipid and carbohydrate homeostasis, several members of the
nuclear hormone receptor family, including the peroxisome proliferator
activated receptor (PPAR) γ, also exert beneficial pleiotropic effects to reduce
atherosclerosis and its complications [12, 13]. PPARγ is
the molecular target for the synthetic thiazolidinediones (TZD), such as
rosiglitazone and pioglitazone, clinically used as insulin sensitizers in
patients with type 2 diabetes [14]. Over the last decade, a wealth of evidence has supported a
beneficial role for TZD PPARγ agonists in the regulation of vascular
gene expression programs [12, 13].
While PPARγ expression itself is increased in
response to vascular injury [15–17],
its activation by TZD suppresses SMC proliferation through several mechanisms
involving the regulation of genes encoding proteins in SMC migration [15],
proliferation [15],
differentiation [18], senescence [19], and apoptosis [16].
In the following review, we will discuss the role of PPARγ in
vascular biology with respect to the control of proliferative gene expression
programs that underlie SMC proliferation and the development of cardiovascular
diseases.


## 2. PPARγ: A Ligand-Activated Transcripton Factor Expressed in Vascular Cells

The detailed structure and molecular biology of PPARγ
have previously been outlined in excellent review articles [11, 20].
Briefly, the PPAR subfamily of nuclear receptors consists of 3 isoforms, that is, PPARα
(NR1C1), PPARβ (also known as δ,
NR1C2), and
PPARγ (NR1C3). PPARs regulate gene expression
upon heterodimerization with the retinoid X receptor (RXR, or NR2B) and
subsequent binding to specific response elements located in the promoter
regions of target genes. Although
presently there are no proven pathways for endogenous ligands in
vivo,  all PPARs are activated in vitro by fatty
acids (FAs). PPARγ is
activated by the prostaglandin D2 derivative 15-deoxy-Δ12, 14-prostaglandin
J2 (15d-PGJ2) [21]
and forms of oxidized linoleic acid, 9- and 13(S)-HODE [22].
Synthetic PPARγ ligands include TZD, such as
troglitazone, rosiglitazone, and pioglitazone, as well as non-TZD derivates. PPARγ is
predominantly expressed in adipose tissue and has been characterized as an
important regulator of adipocyte differentiation and glucose homeostasis [14]. Based on their efficacy to
improve insulin sensitivity, the TZD PPARγ
ligands rosiglitazone and pioglitazone are currently being utilized in clinical
practice to treat insulin resistance in patients with type 2 diabetes [23, 24].In addition to the metabolic effect of PPARγ,
the receptor is expressed in atherosclerotic lesions [15, 25]
and in all vascular cell types including endothelial cells (EC) [26], macrophages [27], T lymphocytes [28],
and SMCs [29].
In EC, PPARγ is activated in response to
atheroprotective laminar flow [30]. Ligand-induced activation of
PPARγ in these cells suppresses the
expression of genes responsible for the adhesion of monocytes to the endothelium
(i.e., VCAM-1 [31, 32],
ICAM-1 [33]) and their transendothelial
migration [34], which are both crucial early
processes for the subsequent development of atherosclerosis. In macrophage
biology, PPARγ has been demonstrated to suppress
inflammatory gene expression and to decrease intracellular lipid accumulation
and foam-cell formation [35, 36].
Finally, increased PPARγ expression has been demonstrated in
neointimal layers during atherosclerotic lesion development [15, 25].
Concomitant with the phenotypic shift from quiescent SMCs resident in the
uninjured vessel wall to proliferating SMCs in the neointima, PPARγ
expression is induced in the neointima following vascular injury [15, 16].
Considering the importance of SMC proliferation during atherosclerosis and its
complications [6], this increased
expression of PPARγ in neointimal SMCs has provided an
important rationale to further exploit the role of PPARγ for
the proliferative response that underlies the development of neointima
formation and atherosclerotic cardiovascular diseases.


## 3. Transcriptional Regulation of Smooth Muscle Cell Proliferation by PPARγ Ligands

The physiological state of the SMCs in the arterial wall is determined by endogenous and exogenous signals, and often the endpoint  that interpretates these signals is gene transcription [37]. Emerging evidence has
implicated PPARγ as a key transcriptional modulator of
SMC function. In the following section, we outline the role of PPARγ in
the regulation of diverse SMC processes including cell proliferation, cell-cycle
progression, senescence, and apoptosis (see Figure 1).Figure 1
Cellular mechanisms
involved in the inhibition of SMC proliferation by PPARγ during cardiovascular diseases. PPARγ
regulates genes encoding proteins involved in diverse SMC processes including
cell proliferation, cell-cycle progression, senescence, and apoptosis.


### 3.1. Regulation of SMC Proliferation and Cell-Cycle Progression by PPARγ aAgonists

Mitogenic growth factors secreted during vascular
injury converge into a final common signaling pathway regulating the
proliferative response of SMCs: the cell-cycle [6] (see Figure 2). While SMCs are in a quiescent state (G0) in the uninjured
artery, they transit in response to mitogenic stimulation through the G1 phase of the cell-cycle
and ultimately enter S phase to undergo replication [38]. Cell-cycle progression is
under the control of cyclins and cyclin-dependent kinases (CDKs), which
phosphorylate the retinoblastoma gene product (pRB) [39]. pRB phosphorylation
represents the critical checkpoint of the G1→S phase transition and increased pRB
phosphorylation correlates with the induction of SMC proliferation
in injured vessels [40, 41].
Consistent with this, maintenance of high levels of phosphorylated pRB is required for the development of
intimal hyperplasia. Upon pRB phosphorylation, sequestered E2F transcription
factors are released to induce the transcription of genes involved in the
regulation of S phase DNA synthesis [42]. Through CDK-inhibitors (CDKI),
including p27Kip1, the activity of cyclin/CDK complexes in quiescent
SMCs is inhibited providing a second layer of regulation [43, 44]. In response to mitogens, p27Kip1 undergoes ubiquitination and degradation through the proteasome pathway
allowing CDK/cyclin complexes to phosphorylate pRB [45]. Therefore, mitogen-induced
degradation of p27Kip1 is an initial requirement for pRB
phosphorylation and subsequent G1→S cell-cycle progression [46].Figure 2
PPARγ
targets cell-cycle progression. Phosphorylation of the retinoblastoma gene
product (pRB) by specific G1 CDKs represents the critical checkpoint
of the G1/S transition of the cell cycle. pRB phosphorylation
releases E2F allowing the expression of genes required for DNA synthesis. By
preventing the degradation of the CDK inhibitor (CDKI) p27Kip1, PPARγ
ligands inhibit mitogen-induced pRB phosphorylation and downstream expression
of key E2F-regulated genes (i.e., MCM genes) responsible for the initiation of DNA replication.PPARγ ligands have been demonstrated in
various studies to prevent mitogen-induced SMC proliferation and the mechanisms
by which this inhibition of proliferation occurs appear to involve an arrest in
the G1 phase of the cell cycle [47–49].
The growth-inhibitory effects of PPARγ
agonists were first associated with their
ability to prevent mitogen-induced degradation of the CDKI cyclin-dependent
kinase inhibitor (CDKI) p27Kip1, which inhibits the activity of cyclin/CDK and consequently
reduces the cellular levels of phosphorylated pRB [47]. Since in
vivo gene transfer of p27Kip1 significantly inhibits neointimal cell proliferation [43], p27Kip1 likely
constitutes an important target for the anti-proliferative effects of PPARγ
activation. Consistent with its
function to suppress the activity of cyclin/CDK-complexes, stabilization of
p27Kip1 by PPARγ ligands has been demonstrated to
inhibit cyclin/CDK activity, an effect that ultimately translates into a
prevention of mitogen-induced pRB phosphorylation [47].DNA microarray analysis further identified that
minichromosome maintenance proteins (MCM) 6 and 7 are inhibited by PPARγ
ligands in SMCs [50].
MCM proteins represent bona fide E2F target genes [51]
and play a central role in the regulation of the initiation of DNA replication
ensuring that DNA replicates only once during cell cycle (for review see [52]). In eukaryotes, MCM2–MCM7
are recruited onto replication origins during the G1 phase of the cell cycle and assembled into a heteromeric hexamer. Formation of this prereplication
complex, a process often referred to as “replication licensing”, establishes
the competence of this origin for the initiation of DNA replication in the
subsequent S phase. Therefore, the inhibition of MCM gene expression by PPARγ
ligands provides evidence that the inhibitory effects of PPARγ
ligands on G1→S
transition are the result of targeting the pRB/E2F/MCM pathway.


### 3.2. PPARγ Activation and Induction of Apoptosis  in SMCs

In addition to the role of TZD in the regulation ofG1→S
cell-cycle progression, several studies have demonstrated that TZD induce
apoptosis in SMCs [16, 53, 54].
Among the regulated target genes mediating PPARγ-induced
apoptosis is the growth-arrest
and DNA damage-inducible gene 45 (GADD45) [53].
Molecular analyses demonstrated that PPARγ-induced GADD45 gene transcription is
mediated through an Oct-1-dependent mechanism [53].
Although the exact function of GADD45 remains unclear, GADD45 has been
implicated in growth suppression [55]
and apoptosis [56, 57].
Through its association with Cdc2, GADD45 disrupts the interactions of Cdc2
with cyclin B1 and, thus, may induce G2/M arrest [58]. The GADD45 gene, therefore,
may represent a unique target for drugs that induce cell-cycle arrest,
apoptosis, and differentiation such as PPARγ
ligands.The second pathway that has been demonstrated to
induce apoptosis by PPARγ ligands involves the induction of
transforming growth factor (TGF)-β by PPARγ [54].
TGF-β is an essential cytokine
involved in the control of the balance between proliferation and
apoptosis in SMCs [59]. Previously, TZD-induced
apoptosis of SMCs has been suggested to depend on the induction of TGF-β and
subsequent downstream nuclear recruitment of phospho-Smad2 [54]. Interestingly, TGF-β-induced
apoptosis is partly mediated by Smad-dependent expression of GADD45 [60].
Therefore, it is possible if not likely that GADD45 constitutes a key
downstream mediator of apoptosis induced by PPARγ
activation.A third mechanism that has been implicated in PPARγ-induced
SMC apoptosis involves the transcriptional induction of the interferon
regulatory factor-1 (IRF-1), a transcriptional factor
with anti-proliferative and proapoptotic properties. Lin et al. recently
demonstrated that both TZD and PPARγ
overexpression upregulate IRF-1 expression in SMCs [61].
Reducing IRF-1 expression by antisense approaches attenuated PPARγ-induced
SMC apoptosis suggesting that the PPARγ-regulated
IRF-1 pathway contributes to the proapoptotic
effects observed with TZD.


### 3.3. Regulation of SMC Telomerase and Senescence by PPARγ Ligands

Telomerase has been linked to multiple developmental
processes including cell proliferation, senescence, and aging [62–64].
Telomeres, the DNA-protein complexes at the ends of chromosomes, are stabilized
by the ribonucleoprotein telomerase reverse transcriptase (TERT) to serve as
protective capping and to prevent cellular senescence [65, 66].
In most adult cells TERT expression and telomerase activity are repressed and
telomeres shorten during tissue renewal [67], and it has been proposed
that this telomere exhaustion is rate limiting for lifespan [68].
Loss of telomere length beyond a critical threshold results in cellular
senescence [59], a state in which cells are
unresponsive to mitogenic stimuli [69]. These molecular features of
telomerase to prevent senescence are highly conserved among eukaryotes and act
on somatic cells as biological clock to ultimately result in permanent growth
arrest and entry into replicative senescence [70].In SMCs, telomerase activity is required
for cell proliferation, and disruption of telomerase activity
reduces atherosclerosis and neointima formation [71–73].
TERT is the limiting factor for telomerase activation in response to mitogenic
stimuli and TERT antisense oligonucleotides inhibit SMC proliferation [71, 72].
This suggests that TERT may play an important role in the regulation of SMC
proliferation and neointima formation. A recent study demonstrated that
mitogen-induced telomerase activity in SMCs is inhibited by ligand-induced and
constitutive PPARγ activation [19]. The transcriptional
mechanisms responsible for the suppression of telomerase activity by PPARγ
ligands involve an inhibition of Ets-1-dependent transactivation
of the TERT promoter [19]. Ets-1 is an early response
gene that mediates a variety of growth signals in neointimal SMC
proliferation [74]; and atherosclerosis [75] and PPARγ ligands have been reported to inhibit
Ets-1 expression [76].
The relevance of telomerase as target for PPARγ was
further demonstrated in SMCs overexpressing telomerase, in which the efficacy
of PPARγ ligand pioglitazone to inhibit cell
proliferation is lost [19]. These studies indicate that
telomerase constitutes an important molecular target for the antiproliferative
effects of PPARγ activation in SMCs.


### 3.4. Ligand-Receptor Relationship and Specificity: Is TZD-Regulated Gene
Expression in SMCs PPARγ-Dependent?

Although the above-described evidence outlines the
ability of TZD to suppress SMC proliferation and induce apoptosis, it remains
controversial whether the cell-cycle-inhibitory effects of TZD occur through a
ligand-dependent activation of PPARγ.
Several experimental approaches have been used by different investigators to
specifically address this question, including PPARγ-deficient
cells [48, 77],
overexpression of either dominant-negative or constitutively-active PPARγ
mutants [19, 50, 53, 78],
or pharmacologic inhibition of PPARγ [16, 53, 54, 61].
In PPARγ-deficient embryonic stem cells, TZD have been demonstrated to inhibit
cell proliferation, which indicated that this effect might occur independent of
their binding and activation of PPARγ [77]. In contrast to these earlier
studies in stem cells, overexpression of a dominant-negative PPARγ mutant has
been demonstrated to increase SMC proliferation in vitro and
neointima formation in vivo (discussed
in Section 4.1) pointing to a role of PPARγ to
function as an endogenous repressor of SMC proliferation [78].
Complementary to these observations, overexpression of a constitutively-active
PPARγ induces SMC apoptosis in the absence of
ligand [53]
while pharmacologic inhibition of PPARγ
prevents rosiglitazone-induced apoptosis of neointimal SMCs [16].
In addition, many of the target genes, thought to be involved in the regulation of SMC proliferation/apoptosis by PPARγ
ligands, have been demonstrated to be either
directly regulated by overexpression of PPARγ or
the ligand effect is reversed following pharmacologic inhibition of PPARγ [16, 19, 48, 50, 53, 54, 61, 78, 79].
These studies in concert support the concept that the antiproliferative
activity of PPARγ ligands against SMC stems at least in part from a
ligand-dependent activation of the receptor. However, further studies including
in particular SMC-specific PPARγ-deficiency or overexpression are
warranted to further support this notion.A second important question that arises from this
discussion relates to ligand specificity and whether the inhibition of SMC
proliferation by agonists for PPARγ is
exclusively mediated through this receptor or whether PPARγ
ligands may also activate PPARα or δ.
Approximately 80% of the 34 residues defining the ligand binding cavity of
PPARγ are conserved across the three receptor
isotypes [11, 20].
In addition, all three isoforms possess unusually large binding pockets,
compared to other nuclear receptors, which accommodate a diverse set of
lipophilic acids as ligands [80]. Furthermore,
anti-proliferative effects of PPARγ ligands are observed at concentrations
considerably higher than their EC50 for transcriptional activation
in cell-based transfection assays or in in vitro binding
assays with isolated ligand-binding domain fragments [15, 81].
Considering this knowledge, at high concentrations spillover of PPARγ-selective
ligands to PPARα and/or PPARδ is
theoretically possible and the antiproliferative activity of TZD observed in
PPARγ-deficient cells could be explained by their binding to and activation of
PPARα or PPARδ.
Indeed, activation of PPARα represses SMC proliferation [82],
while PPARδ activation has been reported to stimulate rather than inhibit
growth of SMCs [83] and keratinocytes [84]. Although very few studies
have directly compared the effects of PPARγ,
PPARα, and PPARδ
ligands on SMC function, Lin et al. recently identified that the
above-described IRF-1-dependent apoptosis induced by PPARγ
ligands is selective and not observed with PPARα or
PPARδ ligands [61].
This study supports ligand selectivity for PPARγ in
SMCs, although detailed studies are required to further address this question.


## 3.1. Regulation of SMC Proliferation and Cell-Cycle Progression by PPARγ aAgonists

Mitogenic growth factors secreted during vascular
injury converge into a final common signaling pathway regulating the
proliferative response of SMCs: the cell-cycle [6] (see Figure 2). While SMCs are in a quiescent state (G0) in the uninjured
artery, they transit in response to mitogenic stimulation through the G1 phase of the cell-cycle
and ultimately enter S phase to undergo replication [38]. Cell-cycle progression is
under the control of cyclins and cyclin-dependent kinases (CDKs), which
phosphorylate the retinoblastoma gene product (pRB) [39]. pRB phosphorylation
represents the critical checkpoint of the G1→S phase transition and increased pRB
phosphorylation correlates with the induction of SMC proliferation
in injured vessels [40, 41].
Consistent with this, maintenance of high levels of phosphorylated pRB is required for the development of
intimal hyperplasia. Upon pRB phosphorylation, sequestered E2F transcription
factors are released to induce the transcription of genes involved in the
regulation of S phase DNA synthesis [42]. Through CDK-inhibitors (CDKI),
including p27Kip1, the activity of cyclin/CDK complexes in quiescent
SMCs is inhibited providing a second layer of regulation [43, 44]. In response to mitogens, p27Kip1 undergoes ubiquitination and degradation through the proteasome pathway
allowing CDK/cyclin complexes to phosphorylate pRB [45]. Therefore, mitogen-induced
degradation of p27Kip1 is an initial requirement for pRB
phosphorylation and subsequent G1→S cell-cycle progression [46].Figure 2
PPARγ
targets cell-cycle progression. Phosphorylation of the retinoblastoma gene
product (pRB) by specific G1 CDKs represents the critical checkpoint
of the G1/S transition of the cell cycle. pRB phosphorylation
releases E2F allowing the expression of genes required for DNA synthesis. By
preventing the degradation of the CDK inhibitor (CDKI) p27Kip1, PPARγ
ligands inhibit mitogen-induced pRB phosphorylation and downstream expression
of key E2F-regulated genes (i.e., MCM genes) responsible for the initiation of DNA replication.PPARγ ligands have been demonstrated in
various studies to prevent mitogen-induced SMC proliferation and the mechanisms
by which this inhibition of proliferation occurs appear to involve an arrest in
the G1 phase of the cell cycle [47–49].
The growth-inhibitory effects of PPARγ
agonists were first associated with their
ability to prevent mitogen-induced degradation of the CDKI cyclin-dependent
kinase inhibitor (CDKI) p27Kip1, which inhibits the activity of cyclin/CDK and consequently
reduces the cellular levels of phosphorylated pRB [47]. Since in
vivo gene transfer of p27Kip1 significantly inhibits neointimal cell proliferation [43], p27Kip1 likely
constitutes an important target for the anti-proliferative effects of PPARγ
activation. Consistent with its
function to suppress the activity of cyclin/CDK-complexes, stabilization of
p27Kip1 by PPARγ ligands has been demonstrated to
inhibit cyclin/CDK activity, an effect that ultimately translates into a
prevention of mitogen-induced pRB phosphorylation [47].DNA microarray analysis further identified that
minichromosome maintenance proteins (MCM) 6 and 7 are inhibited by PPARγ
ligands in SMCs [50].
MCM proteins represent bona fide E2F target genes [51]
and play a central role in the regulation of the initiation of DNA replication
ensuring that DNA replicates only once during cell cycle (for review see [52]). In eukaryotes, MCM2–MCM7
are recruited onto replication origins during the G1 phase of the cell cycle and assembled into a heteromeric hexamer. Formation of this prereplication
complex, a process often referred to as “replication licensing”, establishes
the competence of this origin for the initiation of DNA replication in the
subsequent S phase. Therefore, the inhibition of MCM gene expression by PPARγ
ligands provides evidence that the inhibitory effects of PPARγ
ligands on G1→S
transition are the result of targeting the pRB/E2F/MCM pathway.


## 3.2. PPARγ Activation and Induction of Apoptosis  in SMCs

In addition to the role of TZD in the regulation ofG1→S
cell-cycle progression, several studies have demonstrated that TZD induce
apoptosis in SMCs [16, 53, 54].
Among the regulated target genes mediating PPARγ-induced
apoptosis is the growth-arrest
and DNA damage-inducible gene 45 (GADD45) [53].
Molecular analyses demonstrated that PPARγ-induced GADD45 gene transcription is
mediated through an Oct-1-dependent mechanism [53].
Although the exact function of GADD45 remains unclear, GADD45 has been
implicated in growth suppression [55]
and apoptosis [56, 57].
Through its association with Cdc2, GADD45 disrupts the interactions of Cdc2
with cyclin B1 and, thus, may induce G2/M arrest [58]. The GADD45 gene, therefore,
may represent a unique target for drugs that induce cell-cycle arrest,
apoptosis, and differentiation such as PPARγ
ligands.The second pathway that has been demonstrated to
induce apoptosis by PPARγ ligands involves the induction of
transforming growth factor (TGF)-β by PPARγ [54].
TGF-β is an essential cytokine
involved in the control of the balance between proliferation and
apoptosis in SMCs [59]. Previously, TZD-induced
apoptosis of SMCs has been suggested to depend on the induction of TGF-β and
subsequent downstream nuclear recruitment of phospho-Smad2 [54]. Interestingly, TGF-β-induced
apoptosis is partly mediated by Smad-dependent expression of GADD45 [60].
Therefore, it is possible if not likely that GADD45 constitutes a key
downstream mediator of apoptosis induced by PPARγ
activation.A third mechanism that has been implicated in PPARγ-induced
SMC apoptosis involves the transcriptional induction of the interferon
regulatory factor-1 (IRF-1), a transcriptional factor
with anti-proliferative and proapoptotic properties. Lin et al. recently
demonstrated that both TZD and PPARγ
overexpression upregulate IRF-1 expression in SMCs [61].
Reducing IRF-1 expression by antisense approaches attenuated PPARγ-induced
SMC apoptosis suggesting that the PPARγ-regulated
IRF-1 pathway contributes to the proapoptotic
effects observed with TZD.


## 3.3. Regulation of SMC Telomerase and Senescence by PPARγ Ligands

Telomerase has been linked to multiple developmental
processes including cell proliferation, senescence, and aging [62–64].
Telomeres, the DNA-protein complexes at the ends of chromosomes, are stabilized
by the ribonucleoprotein telomerase reverse transcriptase (TERT) to serve as
protective capping and to prevent cellular senescence [65, 66].
In most adult cells TERT expression and telomerase activity are repressed and
telomeres shorten during tissue renewal [67], and it has been proposed
that this telomere exhaustion is rate limiting for lifespan [68].
Loss of telomere length beyond a critical threshold results in cellular
senescence [59], a state in which cells are
unresponsive to mitogenic stimuli [69]. These molecular features of
telomerase to prevent senescence are highly conserved among eukaryotes and act
on somatic cells as biological clock to ultimately result in permanent growth
arrest and entry into replicative senescence [70].In SMCs, telomerase activity is required
for cell proliferation, and disruption of telomerase activity
reduces atherosclerosis and neointima formation [71–73].
TERT is the limiting factor for telomerase activation in response to mitogenic
stimuli and TERT antisense oligonucleotides inhibit SMC proliferation [71, 72].
This suggests that TERT may play an important role in the regulation of SMC
proliferation and neointima formation. A recent study demonstrated that
mitogen-induced telomerase activity in SMCs is inhibited by ligand-induced and
constitutive PPARγ activation [19]. The transcriptional
mechanisms responsible for the suppression of telomerase activity by PPARγ
ligands involve an inhibition of Ets-1-dependent transactivation
of the TERT promoter [19]. Ets-1 is an early response
gene that mediates a variety of growth signals in neointimal SMC
proliferation [74]; and atherosclerosis [75] and PPARγ ligands have been reported to inhibit
Ets-1 expression [76].
The relevance of telomerase as target for PPARγ was
further demonstrated in SMCs overexpressing telomerase, in which the efficacy
of PPARγ ligand pioglitazone to inhibit cell
proliferation is lost [19]. These studies indicate that
telomerase constitutes an important molecular target for the antiproliferative
effects of PPARγ activation in SMCs.


## 3.4. Ligand-Receptor Relationship and Specificity: Is TZD-Regulated Gene
Expression in SMCs PPARγ-Dependent?

Although the above-described evidence outlines the
ability of TZD to suppress SMC proliferation and induce apoptosis, it remains
controversial whether the cell-cycle-inhibitory effects of TZD occur through a
ligand-dependent activation of PPARγ.
Several experimental approaches have been used by different investigators to
specifically address this question, including PPARγ-deficient
cells [48, 77],
overexpression of either dominant-negative or constitutively-active PPARγ
mutants [19, 50, 53, 78],
or pharmacologic inhibition of PPARγ [16, 53, 54, 61].
In PPARγ-deficient embryonic stem cells, TZD have been demonstrated to inhibit
cell proliferation, which indicated that this effect might occur independent of
their binding and activation of PPARγ [77]. In contrast to these earlier
studies in stem cells, overexpression of a dominant-negative PPARγ mutant has
been demonstrated to increase SMC proliferation in vitro and
neointima formation in vivo (discussed
in Section 4.1) pointing to a role of PPARγ to
function as an endogenous repressor of SMC proliferation [78].
Complementary to these observations, overexpression of a constitutively-active
PPARγ induces SMC apoptosis in the absence of
ligand [53]
while pharmacologic inhibition of PPARγ
prevents rosiglitazone-induced apoptosis of neointimal SMCs [16].
In addition, many of the target genes, thought to be involved in the regulation of SMC proliferation/apoptosis by PPARγ
ligands, have been demonstrated to be either
directly regulated by overexpression of PPARγ or
the ligand effect is reversed following pharmacologic inhibition of PPARγ [16, 19, 48, 50, 53, 54, 61, 78, 79].
These studies in concert support the concept that the antiproliferative
activity of PPARγ ligands against SMC stems at least in part from a
ligand-dependent activation of the receptor. However, further studies including
in particular SMC-specific PPARγ-deficiency or overexpression are
warranted to further support this notion.A second important question that arises from this
discussion relates to ligand specificity and whether the inhibition of SMC
proliferation by agonists for PPARγ is
exclusively mediated through this receptor or whether PPARγ
ligands may also activate PPARα or δ.
Approximately 80% of the 34 residues defining the ligand binding cavity of
PPARγ are conserved across the three receptor
isotypes [11, 20].
In addition, all three isoforms possess unusually large binding pockets,
compared to other nuclear receptors, which accommodate a diverse set of
lipophilic acids as ligands [80]. Furthermore,
anti-proliferative effects of PPARγ ligands are observed at concentrations
considerably higher than their EC50 for transcriptional activation
in cell-based transfection assays or in in vitro binding
assays with isolated ligand-binding domain fragments [15, 81].
Considering this knowledge, at high concentrations spillover of PPARγ-selective
ligands to PPARα and/or PPARδ is
theoretically possible and the antiproliferative activity of TZD observed in
PPARγ-deficient cells could be explained by their binding to and activation of
PPARα or PPARδ.
Indeed, activation of PPARα represses SMC proliferation [82],
while PPARδ activation has been reported to stimulate rather than inhibit
growth of SMCs [83] and keratinocytes [84]. Although very few studies
have directly compared the effects of PPARγ,
PPARα, and PPARδ
ligands on SMC function, Lin et al. recently identified that the
above-described IRF-1-dependent apoptosis induced by PPARγ
ligands is selective and not observed with PPARα or
PPARδ ligands [61].
This study supports ligand selectivity for PPARγ in
SMCs, although detailed studies are required to further address this question.


## 4. TZD in the Treatment of Cardiovascular Disease

### 4.1. Lessons from Animal Models

TZD PPARγ
ligands have been demonstrated to prevent the development of atherosclerosis in
several murine atherosclerosis models including the low-density lipoprotein
receptor-deficient (LDLR−/−) and the apolipoprotein E
deficient mouse model (apoE−/−) [85–88].
This preventive effect on hyperlipidemia-induced atherosclerosis occurs
independently of changes in circulating lipids, blood pressure, glucose,
or insulin, implicating direct pleiotropic effects on the vascular wall.
Inhibition of atherosclerosis by TZD ligands in these models appears to be also
independent of their efficacy to improve insulin sensitivity as the prevention
of atherosclerosis is observed in both insulin-sensitive and insulin-resistant
models⋅ [85–88].
The mechanisms responsible for the prevention of atherosclerosis by TZD in
these murine atherosclerosis models likely involve macrophage-driven processes
contributing to atherosclerosis since conditional deletion of PPARγ in
macrophages accelerates atherosclerosis [89]. In addition, specific
deletion of PPARγ in EC has recently been demonstrated to
increase blood pressure in mice suggesting that PPARγ in EC is an important
regulator of hypertension, which may contribute to the prevention of
atherosclerosis in murine models [90].Consistent
with the observations that TZD PPARγ
ligands limit SMC proliferation in vitro,
Law et al. demonstrated over a decade ago that the TZD ligand troglitazone
reduces intimal hyperplasia in a rat carotid artery balloon injury model [91].
Subsequent studies confirmed these observations and demonstrated that TZD inhibit
intimal hyperplasia in models of restenosis in both insulin-resistant and
insulin-sensitive animals [92–95].
Similarly, Joner et al. recently demonstrated the prevention of in-stent
restenosis by TZD ligands using a hypercholesterolemic rabbit atherosclerosis
model [96].
Additional beneficial effects of TZD in the process of neointima formation
include accelerated reendothelialization,
which is mediated through an enhanced differentiation of angiogenic progenitor cells
into mature endothelial cells [97, 98]. As detailed above, the
question as to whether the prevention of neointima formation by TZD involves a
receptor-dependent pathway has been addressed in a recent study using
overexpression of PPARγ. While in vivo transfer of an
adenoviral vector expressing wild-type PPARγ  inhibited
SMC proliferation and reduced neointima formation after balloon injury, overexpression
of a dominant-negative PPARγ mutant increased neointima formation [78].
These studies have provided the first in vivo evidence
to support a direct role of PPARγ in suppressing the proliferative
response following vascular injury.


### 4.2. Clinical Evidence for Vascular Protection by TZD

#### 4.2.1. Carotid Artery Intima/Media Thickness

Carotid artery intima/media thickness (CIMT) is a
well-described surrogate marker for cardiovascular risk and
correlates not only with the presence of cardiovascular risk factors
but also with the risk of future macrovascular events [99, 100].
The first study that used CIMT to assess whether TZD treatment prevents the
progression of atherosclerosis was performed 10 years ago. In this study 57 patients with type 2 diabetes were treated with 400 mg troglitazone, which
resulted in a significant decline in CIMT after 3 months of treatment [101]. This reduction in CIMT with
troglitazone has been confirmed in a recent cohort of patients with
insulin-requiring type 2 diabetes [102]. A similar decline in CIMT
was observed a few years later in two independent studies performed with
pioglitazone [103, 104].
The recently reported CHICAGO trial (Carotid Intima-Media Thickness in Atherosclerosis
Using Pioglitazone) was a randomized, double-blind, comparator-controlled,
multicenter trial in patients with type 2 diabetes assessing the effect of
pioglitazone versus the sulfonylurea glimepiride on CIMT progression [105].  In this study of 462 patients the primary endpoint
of progression of mean CIMT was less with pioglitazone versus glimepiride after
72 weeks. Notably, the beneficial effect of pioglitazone on mean CIMT was
similar across prespecified subgroups based on age,
sex, systolic blood pressure, duration of type 2 diabetes, body mass index, HbA(1c) value,
and statin use. The fourth CIMT study performed with pioglitazone compared the
effects of pioglitazone (45 mg/d) and glimepiride (2.7 +/− 1.6 mg/d) in a
randomized controlled study of 173 patients with type 2 diabetes [106]. In this study, CIMT was
reduced only in the pioglitazone group and not in patients treated with
glimepiride and this effect was independent of glycemic control.Comparable results on CIMT progression have been
obtained with rosiglitazone. Sidhu et al. analyzed the effect of rosiglitazone
on CIMT progression in a double-blind, placebo-controlled randomized study in
92 non-diabetic patients with documented
coronary artery disease [107].
In this study, rosiglitazone therapy revealed a reduced progression in CIMT
after 48 weeks of treatment. The Rosiglitazone Atherosclerosis Study analyzed
the effect of TZD treatment on CIMT progression in a mixed patient cohort of 555
subjects with type 2 diabetes
or insulin resistance [108].
Although in this study there was no effect of rosiglitazone treatment in the
mixed population of type 2 diabetes and insulin resistance, in the subanalysis of type 2 diabetic patients
there was a reduced progression of CIMT. A third study reported by Stocker et al. analyzed whether rosiglitazone compared to metformin decreased CIMT in 93
subjects with type 2 diabetes [109].
In this study, metformin and rosiglitazone treatment led to similar improvement
in glycemic control; however, CIMT progressed in the metformin group while
regression of maximal CIMT was observed in the rosiglitazone group.


#### 4.2.2. Postangioplasty Restenosis

Takagi
et al. [110–112]
first demonstrated that troglitazone reduced neointimal tissue proliferation
after coronary stent implantation in patients with type 2 diabetes mellitus.
Following the withdrawal of troglitazone from the market, it was subsequently
demonstrated that pioglitazone has similar effects and significantly reduces
neointimal tissue proliferation in patients with type 2 diabetes mellitus [113].
In this study, 44 patients with type 2 diabetes and 44 stented lesions were
randomized to either pioglitazone therapy or control. Intravascular ultrasound
demonstrated that the neointimal index in the pioglitazone group was
significantly smaller than that in the control group. Similarly, Nishio et al.
observed that the late luminal loss and in-stent restenosis were significantly less in patients treated with pioglitazone [114]. A third study performed with
pioglitazone demonstrated in a randomized, placebo-controlled, double-blind
trial that pioglitazone significantly reduced neointima volume after
coronary stent implantation in patients without diabetes [115].Comparable results have been obtained with rosiglitazone
in a prospective, randomized, case-controlled trial involving 95
diabetic patients with coronary artery disease, which demonstrated
that the in-stent restenosis rate was significantly reduced in the
rosiglitazone group compared with the control group [116].
However, a second study of a smaller cohort of sixteen patients did not observe a significant  decrease in in-stent luminal diameter stenosis measured by
quantitative coronary angiography intravascular ultrasound [117]. Finally, the third study
performed by Wang et al. suggested that the occurrence of coronary events
following angioplasty in 71 patients was significantly decreased in the
rosiglitazone group at 6-month follow-up
[118]. These studies in concert
suggest that TZD therapy in patients undergoing coronary stent implantation may
be associated with less in-stent restenosis and repeated revascularization.
This notion is further supported by two recent meta-analyses [119, 120].
However, decisions on clinical use of an adjunctive TZD therapy following
coronary interventions must await larger double-blind clinical trials.


#### 4.2.3. Cardiovascular Outcome Studies

The beneficial vascular effects observed with TZD
provided the rationale for larger cardiovascular trials and the first results
from these studies are beginning to emerge. The Prospective Pioglitazone Clinical
Trial in Macrovascular Events (PROactive) trail is a prospective, randomized
controlled trial in 5238 patients with type 2 diabetes who had evidence of
macrovascular disease [121].
This study tested theeffects of pioglitazone or placebo in addition
to their glucose-lowering drugs and other medications on a combined vascular
endpoint in patients with known vascular disease. The broad primary endpoint
(the composite of all-cause mortality, nonfatal
myocardial infarction (including silent myocardial infarction), stroke, acute
coronary syndrome, endovascular or surgical intervention in the coronary or leg
arteries, and amputation above the ankle) was not statistically different
between the pioglitazone and placebo arm of the study. However, the study
demonstrated a significant 16% reduction of the main cardiovascular
secondary endpoint of all-cause mortality, myocardial infarction,
and stroke in type 2 diabetic patients treated with pioglitazone. A
recently published subanalysis out of this study further
reported the effect of pioglitazone on recurrent myocardial infarction in 2,445
patients with type 2 diabetes and previous myocardial infarction [122]. In this prespecified endpoint, pioglitazone had
a statistically significant beneficial effect on fatal and nonfatal MI (28%
risk reduction) and acute coronary syndrome (37% risk reduction). A second subanalysis from the PROactive trial in
patients with previous stroke (n=486 in the pioglitazone group and n=498 in the
placebo group) further reported that pioglitazone reduced fatal or nonfatal
stroke by 47% [123]. Consistent with the reported
side-effect
profile for TZD, the PROactive trial confirmed an increased rate of edema and
heart failure in patients treated with pioglitazone [121].
However, in this context it is important to note that heart failure was a non-adjudicated event and mortality due
to heart failure was not increased compared to the placebo group.Currently, trials with
rosiglitazone are being performed to determine whether rosiglitazone affects
cardiovascular outcomes. Three clinical trials are currently testing approaches
that use rosiglitazone to reduce cardiovascular disease in patients with
diabetes: the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial [124], the Bypass Angioplasty
Revascularization Investigation 2 Diabetes (BARI 2D) trial [125], and The Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD) trial  [126]. A recent meta-analysis of
trials performed with rosiglitazone reported an association with a significant increase in the risk of myocardial infarction and with a nonsignificant increase of
the risk of death from cardiovascular causes [127]. However, the authors of this meta-analysis
acknowledged considerable limitations of their analysis and the National
Institutes of Health (supporting the ACCORD and BARI 2D trials) found no
evidence in this analysis to require discontinuing the use of rosiglitazone in
the trials or to revise the study protocols [128]. Similarly, an interim
analysis of the RECORD trial did not show a statistically significant
difference between the rosiglitazone group and the control group for
the endpoints acute myocardial infarction and death from cardiovascular
causes, although patients treated with rosiglitazone were at increased risk to
develop heart failure [129].
Therefore, completion of these studies will enable the determination whether
rosiglitazone provides a similar reduction in cardiovascular outcomes as seen
with pioglitazone and will aid to determine the most appropriate combination
therapies for patients with type 2 diabetes.


## 4.1. Lessons from Animal Models

TZD PPARγ
ligands have been demonstrated to prevent the development of atherosclerosis in
several murine atherosclerosis models including the low-density lipoprotein
receptor-deficient (LDLR−/−) and the apolipoprotein E
deficient mouse model (apoE−/−) [85–88].
This preventive effect on hyperlipidemia-induced atherosclerosis occurs
independently of changes in circulating lipids, blood pressure, glucose,
or insulin, implicating direct pleiotropic effects on the vascular wall.
Inhibition of atherosclerosis by TZD ligands in these models appears to be also
independent of their efficacy to improve insulin sensitivity as the prevention
of atherosclerosis is observed in both insulin-sensitive and insulin-resistant
models⋅ [85–88].
The mechanisms responsible for the prevention of atherosclerosis by TZD in
these murine atherosclerosis models likely involve macrophage-driven processes
contributing to atherosclerosis since conditional deletion of PPARγ in
macrophages accelerates atherosclerosis [89]. In addition, specific
deletion of PPARγ in EC has recently been demonstrated to
increase blood pressure in mice suggesting that PPARγ in EC is an important
regulator of hypertension, which may contribute to the prevention of
atherosclerosis in murine models [90].Consistent
with the observations that TZD PPARγ
ligands limit SMC proliferation in vitro,
Law et al. demonstrated over a decade ago that the TZD ligand troglitazone
reduces intimal hyperplasia in a rat carotid artery balloon injury model [91].
Subsequent studies confirmed these observations and demonstrated that TZD inhibit
intimal hyperplasia in models of restenosis in both insulin-resistant and
insulin-sensitive animals [92–95].
Similarly, Joner et al. recently demonstrated the prevention of in-stent
restenosis by TZD ligands using a hypercholesterolemic rabbit atherosclerosis
model [96].
Additional beneficial effects of TZD in the process of neointima formation
include accelerated reendothelialization,
which is mediated through an enhanced differentiation of angiogenic progenitor cells
into mature endothelial cells [97, 98]. As detailed above, the
question as to whether the prevention of neointima formation by TZD involves a
receptor-dependent pathway has been addressed in a recent study using
overexpression of PPARγ. While in vivo transfer of an
adenoviral vector expressing wild-type PPARγ  inhibited
SMC proliferation and reduced neointima formation after balloon injury, overexpression
of a dominant-negative PPARγ mutant increased neointima formation [78].
These studies have provided the first in vivo evidence
to support a direct role of PPARγ in suppressing the proliferative
response following vascular injury.


## 4.2. Clinical Evidence for Vascular Protection by TZD

### 4.2.1. Carotid Artery Intima/Media Thickness

Carotid artery intima/media thickness (CIMT) is a
well-described surrogate marker for cardiovascular risk and
correlates not only with the presence of cardiovascular risk factors
but also with the risk of future macrovascular events [99, 100].
The first study that used CIMT to assess whether TZD treatment prevents the
progression of atherosclerosis was performed 10 years ago. In this study 57 patients with type 2 diabetes were treated with 400 mg troglitazone, which
resulted in a significant decline in CIMT after 3 months of treatment [101]. This reduction in CIMT with
troglitazone has been confirmed in a recent cohort of patients with
insulin-requiring type 2 diabetes [102]. A similar decline in CIMT
was observed a few years later in two independent studies performed with
pioglitazone [103, 104].
The recently reported CHICAGO trial (Carotid Intima-Media Thickness in Atherosclerosis
Using Pioglitazone) was a randomized, double-blind, comparator-controlled,
multicenter trial in patients with type 2 diabetes assessing the effect of
pioglitazone versus the sulfonylurea glimepiride on CIMT progression [105].  In this study of 462 patients the primary endpoint
of progression of mean CIMT was less with pioglitazone versus glimepiride after
72 weeks. Notably, the beneficial effect of pioglitazone on mean CIMT was
similar across prespecified subgroups based on age,
sex, systolic blood pressure, duration of type 2 diabetes, body mass index, HbA(1c) value,
and statin use. The fourth CIMT study performed with pioglitazone compared the
effects of pioglitazone (45 mg/d) and glimepiride (2.7 +/− 1.6 mg/d) in a
randomized controlled study of 173 patients with type 2 diabetes [106]. In this study, CIMT was
reduced only in the pioglitazone group and not in patients treated with
glimepiride and this effect was independent of glycemic control.Comparable results on CIMT progression have been
obtained with rosiglitazone. Sidhu et al. analyzed the effect of rosiglitazone
on CIMT progression in a double-blind, placebo-controlled randomized study in
92 non-diabetic patients with documented
coronary artery disease [107].
In this study, rosiglitazone therapy revealed a reduced progression in CIMT
after 48 weeks of treatment. The Rosiglitazone Atherosclerosis Study analyzed
the effect of TZD treatment on CIMT progression in a mixed patient cohort of 555
subjects with type 2 diabetes
or insulin resistance [108].
Although in this study there was no effect of rosiglitazone treatment in the
mixed population of type 2 diabetes and insulin resistance, in the subanalysis of type 2 diabetic patients
there was a reduced progression of CIMT. A third study reported by Stocker et al. analyzed whether rosiglitazone compared to metformin decreased CIMT in 93
subjects with type 2 diabetes [109].
In this study, metformin and rosiglitazone treatment led to similar improvement
in glycemic control; however, CIMT progressed in the metformin group while
regression of maximal CIMT was observed in the rosiglitazone group.


### 4.2.2. Postangioplasty Restenosis

Takagi
et al. [110–112]
first demonstrated that troglitazone reduced neointimal tissue proliferation
after coronary stent implantation in patients with type 2 diabetes mellitus.
Following the withdrawal of troglitazone from the market, it was subsequently
demonstrated that pioglitazone has similar effects and significantly reduces
neointimal tissue proliferation in patients with type 2 diabetes mellitus [113].
In this study, 44 patients with type 2 diabetes and 44 stented lesions were
randomized to either pioglitazone therapy or control. Intravascular ultrasound
demonstrated that the neointimal index in the pioglitazone group was
significantly smaller than that in the control group. Similarly, Nishio et al.
observed that the late luminal loss and in-stent restenosis were significantly less in patients treated with pioglitazone [114]. A third study performed with
pioglitazone demonstrated in a randomized, placebo-controlled, double-blind
trial that pioglitazone significantly reduced neointima volume after
coronary stent implantation in patients without diabetes [115].Comparable results have been obtained with rosiglitazone
in a prospective, randomized, case-controlled trial involving 95
diabetic patients with coronary artery disease, which demonstrated
that the in-stent restenosis rate was significantly reduced in the
rosiglitazone group compared with the control group [116].
However, a second study of a smaller cohort of sixteen patients did not observe a significant  decrease in in-stent luminal diameter stenosis measured by
quantitative coronary angiography intravascular ultrasound [117]. Finally, the third study
performed by Wang et al. suggested that the occurrence of coronary events
following angioplasty in 71 patients was significantly decreased in the
rosiglitazone group at 6-month follow-up
[118]. These studies in concert
suggest that TZD therapy in patients undergoing coronary stent implantation may
be associated with less in-stent restenosis and repeated revascularization.
This notion is further supported by two recent meta-analyses [119, 120].
However, decisions on clinical use of an adjunctive TZD therapy following
coronary interventions must await larger double-blind clinical trials.


### 4.2.3. Cardiovascular Outcome Studies

The beneficial vascular effects observed with TZD
provided the rationale for larger cardiovascular trials and the first results
from these studies are beginning to emerge. The Prospective Pioglitazone Clinical
Trial in Macrovascular Events (PROactive) trail is a prospective, randomized
controlled trial in 5238 patients with type 2 diabetes who had evidence of
macrovascular disease [121].
This study tested theeffects of pioglitazone or placebo in addition
to their glucose-lowering drugs and other medications on a combined vascular
endpoint in patients with known vascular disease. The broad primary endpoint
(the composite of all-cause mortality, nonfatal
myocardial infarction (including silent myocardial infarction), stroke, acute
coronary syndrome, endovascular or surgical intervention in the coronary or leg
arteries, and amputation above the ankle) was not statistically different
between the pioglitazone and placebo arm of the study. However, the study
demonstrated a significant 16% reduction of the main cardiovascular
secondary endpoint of all-cause mortality, myocardial infarction,
and stroke in type 2 diabetic patients treated with pioglitazone. A
recently published subanalysis out of this study further
reported the effect of pioglitazone on recurrent myocardial infarction in 2,445
patients with type 2 diabetes and previous myocardial infarction [122]. In this prespecified endpoint, pioglitazone had
a statistically significant beneficial effect on fatal and nonfatal MI (28%
risk reduction) and acute coronary syndrome (37% risk reduction). A second subanalysis from the PROactive trial in
patients with previous stroke (n=486 in the pioglitazone group and n=498 in the
placebo group) further reported that pioglitazone reduced fatal or nonfatal
stroke by 47% [123]. Consistent with the reported
side-effect
profile for TZD, the PROactive trial confirmed an increased rate of edema and
heart failure in patients treated with pioglitazone [121].
However, in this context it is important to note that heart failure was a non-adjudicated event and mortality due
to heart failure was not increased compared to the placebo group.Currently, trials with
rosiglitazone are being performed to determine whether rosiglitazone affects
cardiovascular outcomes. Three clinical trials are currently testing approaches
that use rosiglitazone to reduce cardiovascular disease in patients with
diabetes: the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial [124], the Bypass Angioplasty
Revascularization Investigation 2 Diabetes (BARI 2D) trial [125], and The Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD) trial  [126]. A recent meta-analysis of
trials performed with rosiglitazone reported an association with a significant increase in the risk of myocardial infarction and with a nonsignificant increase of
the risk of death from cardiovascular causes [127]. However, the authors of this meta-analysis
acknowledged considerable limitations of their analysis and the National
Institutes of Health (supporting the ACCORD and BARI 2D trials) found no
evidence in this analysis to require discontinuing the use of rosiglitazone in
the trials or to revise the study protocols [128]. Similarly, an interim
analysis of the RECORD trial did not show a statistically significant
difference between the rosiglitazone group and the control group for
the endpoints acute myocardial infarction and death from cardiovascular
causes, although patients treated with rosiglitazone were at increased risk to
develop heart failure [129].
Therefore, completion of these studies will enable the determination whether
rosiglitazone provides a similar reduction in cardiovascular outcomes as seen
with pioglitazone and will aid to determine the most appropriate combination
therapies for patients with type 2 diabetes.


## 4.2.1. Carotid Artery Intima/Media Thickness

Carotid artery intima/media thickness (CIMT) is a
well-described surrogate marker for cardiovascular risk and
correlates not only with the presence of cardiovascular risk factors
but also with the risk of future macrovascular events [99, 100].
The first study that used CIMT to assess whether TZD treatment prevents the
progression of atherosclerosis was performed 10 years ago. In this study 57 patients with type 2 diabetes were treated with 400 mg troglitazone, which
resulted in a significant decline in CIMT after 3 months of treatment [101]. This reduction in CIMT with
troglitazone has been confirmed in a recent cohort of patients with
insulin-requiring type 2 diabetes [102]. A similar decline in CIMT
was observed a few years later in two independent studies performed with
pioglitazone [103, 104].
The recently reported CHICAGO trial (Carotid Intima-Media Thickness in Atherosclerosis
Using Pioglitazone) was a randomized, double-blind, comparator-controlled,
multicenter trial in patients with type 2 diabetes assessing the effect of
pioglitazone versus the sulfonylurea glimepiride on CIMT progression [105].  In this study of 462 patients the primary endpoint
of progression of mean CIMT was less with pioglitazone versus glimepiride after
72 weeks. Notably, the beneficial effect of pioglitazone on mean CIMT was
similar across prespecified subgroups based on age,
sex, systolic blood pressure, duration of type 2 diabetes, body mass index, HbA(1c) value,
and statin use. The fourth CIMT study performed with pioglitazone compared the
effects of pioglitazone (45 mg/d) and glimepiride (2.7 +/− 1.6 mg/d) in a
randomized controlled study of 173 patients with type 2 diabetes [106]. In this study, CIMT was
reduced only in the pioglitazone group and not in patients treated with
glimepiride and this effect was independent of glycemic control.Comparable results on CIMT progression have been
obtained with rosiglitazone. Sidhu et al. analyzed the effect of rosiglitazone
on CIMT progression in a double-blind, placebo-controlled randomized study in
92 non-diabetic patients with documented
coronary artery disease [107].
In this study, rosiglitazone therapy revealed a reduced progression in CIMT
after 48 weeks of treatment. The Rosiglitazone Atherosclerosis Study analyzed
the effect of TZD treatment on CIMT progression in a mixed patient cohort of 555
subjects with type 2 diabetes
or insulin resistance [108].
Although in this study there was no effect of rosiglitazone treatment in the
mixed population of type 2 diabetes and insulin resistance, in the subanalysis of type 2 diabetic patients
there was a reduced progression of CIMT. A third study reported by Stocker et al. analyzed whether rosiglitazone compared to metformin decreased CIMT in 93
subjects with type 2 diabetes [109].
In this study, metformin and rosiglitazone treatment led to similar improvement
in glycemic control; however, CIMT progressed in the metformin group while
regression of maximal CIMT was observed in the rosiglitazone group.


## 4.2.2. Postangioplasty Restenosis

Takagi
et al. [110–112]
first demonstrated that troglitazone reduced neointimal tissue proliferation
after coronary stent implantation in patients with type 2 diabetes mellitus.
Following the withdrawal of troglitazone from the market, it was subsequently
demonstrated that pioglitazone has similar effects and significantly reduces
neointimal tissue proliferation in patients with type 2 diabetes mellitus [113].
In this study, 44 patients with type 2 diabetes and 44 stented lesions were
randomized to either pioglitazone therapy or control. Intravascular ultrasound
demonstrated that the neointimal index in the pioglitazone group was
significantly smaller than that in the control group. Similarly, Nishio et al.
observed that the late luminal loss and in-stent restenosis were significantly less in patients treated with pioglitazone [114]. A third study performed with
pioglitazone demonstrated in a randomized, placebo-controlled, double-blind
trial that pioglitazone significantly reduced neointima volume after
coronary stent implantation in patients without diabetes [115].Comparable results have been obtained with rosiglitazone
in a prospective, randomized, case-controlled trial involving 95
diabetic patients with coronary artery disease, which demonstrated
that the in-stent restenosis rate was significantly reduced in the
rosiglitazone group compared with the control group [116].
However, a second study of a smaller cohort of sixteen patients did not observe a significant  decrease in in-stent luminal diameter stenosis measured by
quantitative coronary angiography intravascular ultrasound [117]. Finally, the third study
performed by Wang et al. suggested that the occurrence of coronary events
following angioplasty in 71 patients was significantly decreased in the
rosiglitazone group at 6-month follow-up
[118]. These studies in concert
suggest that TZD therapy in patients undergoing coronary stent implantation may
be associated with less in-stent restenosis and repeated revascularization.
This notion is further supported by two recent meta-analyses [119, 120].
However, decisions on clinical use of an adjunctive TZD therapy following
coronary interventions must await larger double-blind clinical trials.


## 4.2.3. Cardiovascular Outcome Studies

The beneficial vascular effects observed with TZD
provided the rationale for larger cardiovascular trials and the first results
from these studies are beginning to emerge. The Prospective Pioglitazone Clinical
Trial in Macrovascular Events (PROactive) trail is a prospective, randomized
controlled trial in 5238 patients with type 2 diabetes who had evidence of
macrovascular disease [121].
This study tested theeffects of pioglitazone or placebo in addition
to their glucose-lowering drugs and other medications on a combined vascular
endpoint in patients with known vascular disease. The broad primary endpoint
(the composite of all-cause mortality, nonfatal
myocardial infarction (including silent myocardial infarction), stroke, acute
coronary syndrome, endovascular or surgical intervention in the coronary or leg
arteries, and amputation above the ankle) was not statistically different
between the pioglitazone and placebo arm of the study. However, the study
demonstrated a significant 16% reduction of the main cardiovascular
secondary endpoint of all-cause mortality, myocardial infarction,
and stroke in type 2 diabetic patients treated with pioglitazone. A
recently published subanalysis out of this study further
reported the effect of pioglitazone on recurrent myocardial infarction in 2,445
patients with type 2 diabetes and previous myocardial infarction [122]. In this prespecified endpoint, pioglitazone had
a statistically significant beneficial effect on fatal and nonfatal MI (28%
risk reduction) and acute coronary syndrome (37% risk reduction). A second subanalysis from the PROactive trial in
patients with previous stroke (n=486 in the pioglitazone group and n=498 in the
placebo group) further reported that pioglitazone reduced fatal or nonfatal
stroke by 47% [123]. Consistent with the reported
side-effect
profile for TZD, the PROactive trial confirmed an increased rate of edema and
heart failure in patients treated with pioglitazone [121].
However, in this context it is important to note that heart failure was a non-adjudicated event and mortality due
to heart failure was not increased compared to the placebo group.Currently, trials with
rosiglitazone are being performed to determine whether rosiglitazone affects
cardiovascular outcomes. Three clinical trials are currently testing approaches
that use rosiglitazone to reduce cardiovascular disease in patients with
diabetes: the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial [124], the Bypass Angioplasty
Revascularization Investigation 2 Diabetes (BARI 2D) trial [125], and The Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD) trial  [126]. A recent meta-analysis of
trials performed with rosiglitazone reported an association with a significant increase in the risk of myocardial infarction and with a nonsignificant increase of
the risk of death from cardiovascular causes [127]. However, the authors of this meta-analysis
acknowledged considerable limitations of their analysis and the National
Institutes of Health (supporting the ACCORD and BARI 2D trials) found no
evidence in this analysis to require discontinuing the use of rosiglitazone in
the trials or to revise the study protocols [128]. Similarly, an interim
analysis of the RECORD trial did not show a statistically significant
difference between the rosiglitazone group and the control group for
the endpoints acute myocardial infarction and death from cardiovascular
causes, although patients treated with rosiglitazone were at increased risk to
develop heart failure [129].
Therefore, completion of these studies will enable the determination whether
rosiglitazone provides a similar reduction in cardiovascular outcomes as seen
with pioglitazone and will aid to determine the most appropriate combination
therapies for patients with type 2 diabetes.


## 5. Summary and Conclusions

Research performed over the last decade has
highlighted an important role for TZD-induced PPARγ
activation in vascular cells. TZD exert a broad spectrum of anti-inflammatory
and anti-proliferative on all cell types participating in the
development of cardiovascular diseases. A wealth of evidence from preclinical
and clinical studies supports that these pleiotropic effects of TZD translate
into reduced atherosclerosis and failure of coronary angioplasty as the primary
approach to treat luminal obstruction. The PROactive trial was the first
cardiovascular outcome trial to demonstrate that pioglitazone decreases all-cause
mortality, myocardial infarction, and stroke in patients with type 2
diabetes. Further studies including the ACCORD, RECORD, and BARI 2D trials will determine whether similar effects are seen with
rosiglitazone and outline ideal treatment strategies to reduce cardiovascular
disease in patients with type 2 diabetes.



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*Source: 429123-2007-12-06.xml*