# The Role of PPARs in the Endothelium: Implications  for Cancer Therapy

Authors: David Bishop-Bailey; Karen E. Swales
Journal: PPAR Research
 (2008)

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## Abstract

The growth and metastasis of cancers intimately involve the vasculature and in particular the endothelial cell layer. Tumours require new blood vessel formation via angiogenesis to support growth. In addition, inflammation, coagulation, and platelet activation are common signals in the growth and metastasis of tumour cells. The endothelium plays a central role in the homeostatic control of inflammatory cell recruitment, regulating platelet activation and coagulation pathways. PPARα, -β/δ, and -γ are all expressed in endothelial cells. This review will discuss the roles of PPARs in endothelial cells in relation to angiogenesis, inflammation, coagulation, and platelet control pathways. In particular, we will discuss the recent evidence that supports the hypothesis that PPARα and PPARγ are antiangiogenic receptors, while PPARβ/δ is proangiogenic.


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## Body

## 1. Importance of The Endothelial Cell in Cancer

Endothelial cells
play critical roles in vascular biology, being both the protective inner lining
of vessels and the local site for delivery of oxygen to all tissues. It has
become clear, particularly from the seminal work of Professor Judah Folkman, whom
this issue is dedicated to, that the endothelium plays a critical role in the
growth and spread of cancer [1–4]. The growth of
tumours, or indeed any tissue growth requires new blood vessel formation to
sustain it. This process of angiogenesis as a target for modulating cancer
growth has been a major research theme. The critical initial stimulus for
angiogenesis appears to be hypoxia in the growing tumour. The hypoxia leads to
upregulation of hypoxia-induced transcription factors, for example, hypoxia
inducible factor (HIF)-1α and HIF-2α [5–8], which stimulate
the expressions of genes involved in oxygen homeostasis, and secretion of proangiogenic
mediators such as vascular endothelial growth factor (VEGF) and basic
fibroblast growth factor (bFGF) [4, 9, 10]. Although these
are key growth factors for endothelial cell growth and morphogenesis, it is
clear that there are an increasing number of endogenous proangiogenic factors (PGDF,
IL-8, angiopoietin-1, leptin, matrix metalloproteinases, thrombin, plasminogen activators) and
antiangiogenic factors (endostatin, angiostatin, thrombospondin-1,
angiopoietin-2, IL-4, IL-12, IL-18, tissue inhibitor of MMPs, TGF-β, IFNα, -β, and -γ) [1, 4, 10, 11]. When the
cumulative actions of the proangiogenic mediators outweigh their antiangiogenic
counterparts an “angiogenic switch” occurs [12]. In particular, VEGF
(VEGF-A; VEGF165) is a central mediator of endothelial cell growth
and angiogenesis [13]. Two endothelial VEGF tyrosine
kinase receptors have been identified: VEGFR-1/Flt-1, and VEGFR-2/KDR/Flk1,
with the latter being the most important in VEGF-induced mitogenesis and
permeability [13]. The lymphatic system and in
particular lymphangiogenesis also contributes significantly to tumour
metastasis. Unlike angiogenesis, where VEGF-(A) and VEGFR1/2 are key
regulators, lymphangiogenesis is regulated by VEGFR-3 and VEGF-C/D isoforms
(along with PROX1, podoplanin, LYVE-1, ephrinB2, and FOXC2) [14, 15]. Once stimulated
by VEGF, the receptors initiate a signal transduction cascade, activating
kinases such as ERK1/2 and Akt, which phosphorylate and activate further
mediators of endothelial cell proliferation, apoptosis, and angiogenesis, such
as eNOS [16].The endothelium
local to the tumour itself also contributes to tumour growth and metastasis via
mechanisms independent of angiogenesis. Of increasing importance is the role of
chronic inflammation in tumour progression. Chronic inflammation, in particular
the presence of neutrophils, macrophages, and mast cells, correlates with poor
prognosis and the angiogenic state of the tumour [17, 18]. The activation
of the endothelium and its subsequent expression of adhesion molecules and
chemokines is the interface for local inflammatory cell recruitment and
extravasation. Central to these processes are proinflammatory transcription
factors such as NFκB. NFκB regulates many inflammatory processes including
inducible cytokine/chemokine and adhesion molecule expressions that are central to inflammatory cell recruitment, as
well acting as a potent prosurvival signal within the cell
[19].In addition to
angiogenesis and inflammation, cancer progression and metastasis is also facilitated
by circulating cells and mediators regulated by the endothelium. The
endothelium  provides an antithrombotic
surface and produces powerful antiplatelet and anticoagulant mediators such as
prostacyclin, nitric oxide, and tissue- and urokinase-plasminogen activators [20]. Under physiological conditions,
the endothelial surface is antithrombotic. Activated endothelial cells, however,
are able to release prothrombotic/procoagulation mediators such as prostaglandin
PGE2 [21, 22], plasminogen
activator inhibitor (PAI)-1 [23], and tissue factor [23]. In cancer, thrombocytosis is
common [24], suggesting that
the physiological protective system usually provided by endothelial cells may
be dysfunctional or overpowered by prothrombotic pathways. Driving this
thrombosis may be tumour-derived thrombopoietin, and tumour- and platelet-derived
growth factors and microparticles [24]. The consequence
of activation of the coagulation cascade in cancer progression can be seen
using thrombin as an example. Thrombin activates tumour cell adhesion to
platelets and endothelial cells, and induces tumour cell growth, metastasis,
and angiogenesis [25].The movement of
tumour cells into and out of the circulation (or the lymphatics) involves
interaction with, and crossing of, the endothelial barrier. Although tumour
endothelial cells are generally highly permeable (induced by factors such as
VEGF), it is still unlikely that tumour cell movement is a passive process [26]. Within the circulation, transit
of tumour cells is facilitated by their interactions with activated platelets [26]. The platelets are believed to
act as a shield, protecting tumour cells from both physical forces and
immune-mediated killing [26].In summary, along
with angiogenesis and lymphangiogenesis, endothelial cells regulate
tumour progression not only by directly interacting with tumour cells, but also by regulating local inflammatory cell recruitment, the
coagulation cascade, and platelet activity. When discussing the actions of
PPARs in endothelial cells it is, therefore, important  to consider all these
properties.


## 2. PPARs and Endothelial Cells

PPARα, PPARβ/δ, and PPARγ are expressed in endothelial cells [27, 28], where they regulate cell
proliferation, angiogenesis, inflammation, thrombosis, and coagulation (Figure 1). PPARα is expressed in human aortic endothelial
cells, carotid artery endothelial cells, and human umbilical vein endothelial
cells [27, 29–31]. PPARγ is similarly
expressed in human endothelial cells both in vitro and in vivo [27, 28, 31, 32], while
PPARβ is ubiquitously expressed. The role of PPARγ has been well characterised in
endothelial cell inflammation and angiogenesis [33, 34].  In contrast, the functions of PPARα and PPARβ/δ in endothelial cells, especially in terms of angiogenesis,
are only just beginning to be understood. Indeed, although the role of
PPARγ will be discussed in this
review, since there is considerable information on PPARγ in cancer [35] and an article on PPARγ regulation of the angiogenic
switch in this review series [36], this manuscript will
focus more on recent observations highlighting novel roles for PPARα and PPARβ/δ in endothelial
cell function and in particular on the regulation of angiogenesis. The focus of
this review is the endothelial cell, but it is important to note that PPARα, β/δ, and γ expression and activity have been demonstrated
in a variety of cancers, inflammatory cells [34], and in platelets [37–39].
Therefore, any effects of PPAR ligands on the development of cancer may be
influenced by responses in these nonendothelial cell types as well.Figure 1
The endothelial cell is the interface between the circulation and underlying tissue, and as
such plays an important homeostatic role both producing and responding to a
variety of pro- and antiangiogenic, inflammatory, and coagulation factors. The
balance between these opposing pathways is critical in the growth, development,
spread, and metastasis of tumours.


## 3. PPARα and PPARγ: Anticancer Targets  in The Endothelium

### 3.1. PPARα and PPARγ Ligands

When discussing
the roles of PPARs it is important to note the types of ligands potentially
used in studies. Activators of PPARα include a variety of eicosanoids, fatty acids, and synthetic
compounds including the clinically used dyslipidemic drugs, the fibrates (gemfibrozil,
fenofibrate, bezafibrate, ciprofibrate) [40, 41]. Similarly, PPARγ activators also include a variety of eicosanoids, fatty acids, and synthetic compounds
including the clinically used insulin sensitising thiazolidinedione drugs (rosiglitazone, pioglitizone, troglitizone (now withdrawn)
[40, 41]. (See Figures 2 and 3.)Figure 2
Endothelial PPARα has predominantly inhibitory actions on
endothelial cell activation. The majority of studies so far indicate that PPARα activation induces (solid line)
antiangiogenic factors, while reduces (broken line) proangiogenic factors,
proinflammatory pathways, and procoagulant mediator release.Figure 3
Endothelial PPARγ has predominantly inhibitory actions on
endothelial cell activation. The majority of studies so far indicate that PPARγ activation inhibits (broken line)
proangiogenic factors, proinflammatory pathways, and procoagulant mediator
release, while inducing (solid line) antiangiogenic factors.


### 3.2. PPARα and PPARγ in Cancer

One early observation regarding PPARα activation by
peroxisome proliferators was the induction of hepatocarcinogenesis in rodents; an
effect absent in PPARα (−/−) knockout
mice [42, 43]. Although there
has been a considerable amount of interest in the field, especially as the PPARα activating fibrates are in clinical use,
there is no evidence that long-term activation of PPARα in nonrodent species including man is
linked to hepatocarcinogenesis [42, 43].In extrahepatic tissues, there have been fewer studies regarding PPARα and cancer.
Initially, it was suggested that PPARα may prevent
skin cancer [44, 45]. However,
topical PPARα agonists were only moderately protective against tumour promotion in
mouse skin, despite the upregulation of PPARα in tumours compared
to normal epidermis [46]. Recent
studies have revealed that PPARα is commonly expressed in tumour cell lines, including lung, liver,
leukaemia, prostate, pancreas, bladder, colon, glioblastoma, hemangioma,
melanoma, ovarian, and breast [47–49]. PPARα ligands inhibit
the growth of colon, breast, endometrial, and skin cells in vitro [46, 48, 50–52] and human
ovarian cancer [53],
melanoma, lung carcinoma, glioblastoma, and fibrosarcoma [48]. PPARα ligands also decrease
tumour development in colon carcinogenesis [52] and inhibit
melanoma cell metastasis in vitro and
in vivo [50, 54].PPARγ is expressed in prostate, thyroid, colon, breast and hepatocellular carcinoma,
gastric, pancreatic and lung cancer, neuroblastoma, astrocytoma, and glioma,
where the receptors' ligands are antiproliferative and proapoptotic [35]. It is
beyond the scope of this review to discuss all the findings of PPARγ in cancer,
and there are a number of excellent reviews in the field [33, 35, 55, 56]
including one on PPARγ and angiogenesis in this series [36].The majority of the evidence points towards PPARγ ligands
suppressing tumourgenesis, for example, the receptors' ligands inhibit the
growth of xenografts of many of the aforementioned tumours in vivo [35].
However, in colon cancer, the beneficial role for PPARγ agonists is
controversial [57]. In
the APCmin/+ mouse, PPARγ ligands increased
precancerous polyp formation and the frequency and size of tumours in the colon
[58, 59]. In contrast,
heterozygous loss of PPARγ increases colon cancer incidence in mice [60]. This
latter study corresponds with most of the available data, suggesting that PPARγ has
antineoplastic effects in colon cancer; a point further supported in colon
cancer patient studies by the detection of mutations causing loss of function
or impaired ligand binding of PPARγ  [61] and
polymorphisms of the PPARγ gene [62].There have been positive results using PPARγ ligands to
treat tumours experimentally both in
vitro and in vivo, but so far this has not been successfully translated
into a beneficial anticancer therapy in man. There have been a number of small
scale clinical trials testing PPARγ ligands in
cancer in man with varying success [63]. The
most promising results were from small phase II studies treating prostate
cancer [64] and
liposarcoma patients [65] with
troglitazone. In contrast, a phase II study treating liposarcoma patients with
rosiglitazone did not significantly improve clinical outcome [66] and so
far no beneficial effects of PPARγ ligands have
been observed in trials for breast or colon cancer patients [35].


### 3.3. PPARα and PPARγ Regulation of Angiogenesis

Early studies showed no effect of the selective PPARα ligand WY-14643 on endothelial cell proliferation [27],
however, recent studies using immortalised human dermal microvascular
endothelial cells show that the PPARα ligand fenofibrate
inhibits endothelial cell proliferation, migration, and tube formation (on a
fibrin matrix) in vitro and
angiogenesis in vivo [67]. Fenofibrate
acts by disrupting the formation of the actin cytoskeleton and inhibits bFGF-induced
Akt activation and cyclooxygenase 2 (COX-2) gene expression [67].
Similar results were found in a porcine model of vascular remodelling after
coronary artery angioplasty where fenofibrate increased lumen size and vessel
area and inhibited constrictive remodelling and inflammatory cell infiltration [68]. Importantly,
adventitial angiogenesis was significantly reduced by fenofibrate in the injured
vessels 3 days after angioplasty [68].In contrast to this vascular study, the investigation of PPARα regulation of
tumour angiogenesis has only just begun. In a recent report, Panigraphy et al.
provide compelling evidence for PPARα inhibition of
tumour growth by targeting angiogenesis [48].
Similar to previous findings, PPARα activation
had direct effects on endothelial cells, inhibiting VEGF-induced endothelial
cell migration in vitro and FGF2 induced corneal angiogenesis in
vivo [48].
Tumour cell synthesis of VEGF and FGF2 was also suppressed by PPARα activation in
conjunction with an increased expression of antiangiogenic thrombospondin-1
(TSP-1) [48]. In
subcutaneously implanted human pancreatic cancer cells grown in mice, as well
as in human prostate cancer, PPARα expression
was detected not only in the tumour cells, but also in the new invading
microvessels [48]. Systemic
treatment of mice with PPARα ligands inhibited the growth of melanoma, glioblastoma, and fibrosarcoma
tumours implanted in vivo,
which was associated with a reduction in vessel density and inflammation [48]. To
dissect the mechanism by which PPARα suppressed
tumour growth (i.e., direct effects on the tumour and/or angiogenesis), embryonic
fibroblasts from PPARα (−/−) knockout mice were transformed with SV40 large T antigen and
H-ras oncogenes then implanted into wild-type and PPARα −/− mice. The
growth of these cells into tumours could be suppressed by PPARα ligands in
wild-type mice only, indicating that tumour
suppression by PPARα ligands was completely dependent on the expression of PPARα in the host
but not in the tumour cells [48].
Fenofibrate strongly induced the antiangiogenic factors TSP-1 and endostatin in
wild-type, but not PPARα −/− mice, supporting the role of PPARα as an antiangiogenic
regulator [48]. Angiogenesis
and inflammation are central processes through which the tumour interacts with
its surroundings to influence tumour growth. Although this study does not rule
out an anti-inflammatory effect of the PPARα ligands, it
is highly unlikely that the antitumour host-derived effects are due to
suppression of inflammation because mice deficient in PPARα generally
exhibit enhanced inflammation [64].TSP-1 is a potent angiogenesis inhibitor that targets endothelial cells
for apoptosis by initiating a signalling cascade through the CD36 receptor.
PPARα directly induces TSP-1 and can enhance TSP-1 signalling indirectly by
upregulating CD36 in the endothelium. PPARα activation
upregulates CD36 expression in the liver [69] and in
macrophages [70].  Moreover, coadministration of PPARγ ligands with
exogenous TSP-1 or the TSP-1 peptide derivative ABT510 synergises to suppress
angiogenesis and induce endothelial cell apoptosis [71]. The
improvement of the antiangiogenic efficacy of TSP-1 was attributed to PPARγ-induced CD36 expression via a PPAR response element in the CD36 promoter
[69, 71].The vast majority of studies have indicated an antiangiogenic role for
PPARα and PPARγ in a variety of models. However, it is important to note that the VEGF
promoter contains a PPAR response element and PPARα and -γ ligands can
induce VEGF in certain cell types [72–75].
Moreover, in contrast to the majority of findings, a recent study suggests that
both PPARα and PPARγ ligands may also have proangiogenic properties in vitro in an endothelial/interstitial cell coculture assay and in a
murine corneal angiogenesis model in vivo [72]. The angiogenesis induced by PPARα and PPARγ ligands was
associated with the induction of VEGF, accompanied by increased activation of AKT
and eNOS (by phosphorylation) [72]. How
the levels of PPARα- or PPARγ-mediated angiogenesis are compared to traditional growth factor-induced angiogenesis is
not known? Indeed, these results are controversial,
as previous corneal angiogenesis models clearly demonstrate antiangiogenic
effects of PPARα and PPARγ ligands [28, 48, 76].Multiple mechanisms have been proposed by which PPARα and PPARγ regulate the
changes in pro- and antiangiogenic factors. Here, we will focus on the central
target for PPAR regulation of angiogenesis, the proangiogenic VEGF/VEGFR
signalling pathway. PPARγ can downregulate VEGF either directly through a PPAR response element
within the VEGF promoter [77] or by
decreasing PGE2, an endogenous stimulator of angiogenesis [78]. PPARγ can also
decrease VEGF responses by suppressing transcription of its receptor VEGFR2, by
interacting with and preventing Sp1 binding to DNA [79].In colorectal cancer cell lines, PPARα also inhibits
the transcription factor AP-1, impairing its binding to response elements in
the VEGF and COX-2 genes and inhibiting c-jun transactivation activity, thus
downregulating VEGF and COX-2 expression [80]. It
is, therefore, clear that the regulation of angiogenic factors by PPARα and PPARγ may be
determined by cell and cancer type and the experimental models used. Much more
research is required to fully understand whether PPAR activation will be pro-
or antiangiogenic in specific human cancers.


### 3.4. The Effects of PPARα and PPARγ on Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) present in peripheral blood promote angiogenesis and
improve endothelial function. The research on the effects of PPARs on EPCs has
focused on PPARγ. Despite PPARγ generally being considered
antiangiogenic, the PPARγ ligands
rosiglitazone and pioglitazone in diabetic patients increase endothelial progenitor
cell (EPC) number and migratory activity [81, 82]. Pioglitazone
and rosiglitazone also improve the adhesive capacity of EPCs to fibronectin and
collagen [82] and promote EPC
colony formation, [83, 84]. In vitro, pioglitazone increased EPC
proliferation, colony formation, and attenuated apoptosis [85]. Similarly, in
mice pioglitazone induced the number and migratory activity of EPCs while
decreasing their apoptosis, resulting in increased in vivo neoangiogenesis [86]. From these
results, it has been proposed that PPARγ ligands may have
a double-edged role in angiogenesis, with proangiogenic effects on EPCs at
low-systemic concentrations and antiangiogenic effects at higher local
concentrations [86]. Indeed,
biphasic effects of pioglitazone were observed on EPCs in culture, when the
number of EPC colonies and amount of adhesion were increased by 1 μM but not 10 μM [87]. This higher
concentration of pioglitazone induced TGF-β1 and its
receptor endogolin, which suppress EPC function [87]. These findings
have important clinical implications suggesting that the pro-/antiangiogenic
properties of PPARγ ligands may be
largely dose-driven. Moreover, understanding this mechanism by which PPARγ may regulate both pro- and
antiangiogenic pathways at least in EPCs may help to explain some of the
contradictions in the studies examining the role of PPARγ in angiogenesis.


### 3.5. Effects of PPARα and PPARγ on Endothelial  Cell Inflammation

The role of PPARα in inflammation has been studied in animal
models, particularly in wound healing and cardiovascular disease models (atherosclerosis
and restenosis) [55, 56]. PPARα is a negative regulator of inflammation [34] in inflammatory models. Supporting this, PPARα −/− mice exhibit
enhanced inflammation [88], although this
may be due in part to deceased β-oxidation and
accumulation of biologically active lipid mediators.In addition to
these experimental models, PPARα agonists
decrease the expression of inflammatory markers both in
human cells and patients treated with fibrates [89, 90]. In
human endothelial cells in culture, PPARα
ligands inhibit the cytokine/LPS induction of COX-2 [38, 69], ICAM-1 [91], VCAM-1
[29, 31], endothelin-1
[92], IL-6,
and prostaglandin E2 [32, 93]. Similarly,
PPARα ligands
repress thrombin-induced expression of endothelin-1 [32]. The
PPARα ligand fenofibrate, but not the PPARγ ligand rosiglitazone, also reduces the
induction of tissue factor in human endothelial cells [94], while PAI-1 levels
remain unchanged [31]. PPARα inhibits proinflammatory mediators by
interfering with the transactivation activity of NFκB and AP-1, the main transcription
factors mediating inflammatory and growth factor responses. PPARα via direct protein-protein interactions
can bind and inhibit the actions p65 and c-jun subunits, respectively [95, 96].Although the weight of evidence points towards an anti-inflammatory role
for PPARα, oxidised lipids that can activate PPARα have
been shown to increase the release of neutrophil chemoattractant IL-8 and MCP-1
from endothelial cells [30].
Similarly, PPARα ligands induce COX-2 in human breast and colon cancer cells [97, 98].PPARγ, similarly, is a well-established negative regulator of the inflammatory
response in vitro and in vivo [34]. PPARγ agonists have been shown to mediate effects on cell survival,
surface-protein expression, and cytokine and chemokine production. In
endothelial cells, PPARγ ligands can induce
apoptosis [27] and decrease
inflammatory cell recruitment by inhibiting the production of chemokines IL-8, MCP-1
[30, 99],
IP-10, Mig, and I-TAC [100] and reducing
ICAM-1 expression [101].
Similar to PPAR-α, PPARγ ligands repress thrombin-induced expression of endothelin-1 [32].


## 3.1. PPARα and PPARγ Ligands

When discussing
the roles of PPARs it is important to note the types of ligands potentially
used in studies. Activators of PPARα include a variety of eicosanoids, fatty acids, and synthetic
compounds including the clinically used dyslipidemic drugs, the fibrates (gemfibrozil,
fenofibrate, bezafibrate, ciprofibrate) [40, 41]. Similarly, PPARγ activators also include a variety of eicosanoids, fatty acids, and synthetic compounds
including the clinically used insulin sensitising thiazolidinedione drugs (rosiglitazone, pioglitizone, troglitizone (now withdrawn)
[40, 41]. (See Figures 2 and 3.)Figure 2
Endothelial PPARα has predominantly inhibitory actions on
endothelial cell activation. The majority of studies so far indicate that PPARα activation induces (solid line)
antiangiogenic factors, while reduces (broken line) proangiogenic factors,
proinflammatory pathways, and procoagulant mediator release.Figure 3
Endothelial PPARγ has predominantly inhibitory actions on
endothelial cell activation. The majority of studies so far indicate that PPARγ activation inhibits (broken line)
proangiogenic factors, proinflammatory pathways, and procoagulant mediator
release, while inducing (solid line) antiangiogenic factors.


## 3.2. PPARα and PPARγ in Cancer

One early observation regarding PPARα activation by
peroxisome proliferators was the induction of hepatocarcinogenesis in rodents; an
effect absent in PPARα (−/−) knockout
mice [42, 43]. Although there
has been a considerable amount of interest in the field, especially as the PPARα activating fibrates are in clinical use,
there is no evidence that long-term activation of PPARα in nonrodent species including man is
linked to hepatocarcinogenesis [42, 43].In extrahepatic tissues, there have been fewer studies regarding PPARα and cancer.
Initially, it was suggested that PPARα may prevent
skin cancer [44, 45]. However,
topical PPARα agonists were only moderately protective against tumour promotion in
mouse skin, despite the upregulation of PPARα in tumours compared
to normal epidermis [46]. Recent
studies have revealed that PPARα is commonly expressed in tumour cell lines, including lung, liver,
leukaemia, prostate, pancreas, bladder, colon, glioblastoma, hemangioma,
melanoma, ovarian, and breast [47–49]. PPARα ligands inhibit
the growth of colon, breast, endometrial, and skin cells in vitro [46, 48, 50–52] and human
ovarian cancer [53],
melanoma, lung carcinoma, glioblastoma, and fibrosarcoma [48]. PPARα ligands also decrease
tumour development in colon carcinogenesis [52] and inhibit
melanoma cell metastasis in vitro and
in vivo [50, 54].PPARγ is expressed in prostate, thyroid, colon, breast and hepatocellular carcinoma,
gastric, pancreatic and lung cancer, neuroblastoma, astrocytoma, and glioma,
where the receptors' ligands are antiproliferative and proapoptotic [35]. It is
beyond the scope of this review to discuss all the findings of PPARγ in cancer,
and there are a number of excellent reviews in the field [33, 35, 55, 56]
including one on PPARγ and angiogenesis in this series [36].The majority of the evidence points towards PPARγ ligands
suppressing tumourgenesis, for example, the receptors' ligands inhibit the
growth of xenografts of many of the aforementioned tumours in vivo [35].
However, in colon cancer, the beneficial role for PPARγ agonists is
controversial [57]. In
the APCmin/+ mouse, PPARγ ligands increased
precancerous polyp formation and the frequency and size of tumours in the colon
[58, 59]. In contrast,
heterozygous loss of PPARγ increases colon cancer incidence in mice [60]. This
latter study corresponds with most of the available data, suggesting that PPARγ has
antineoplastic effects in colon cancer; a point further supported in colon
cancer patient studies by the detection of mutations causing loss of function
or impaired ligand binding of PPARγ  [61] and
polymorphisms of the PPARγ gene [62].There have been positive results using PPARγ ligands to
treat tumours experimentally both in
vitro and in vivo, but so far this has not been successfully translated
into a beneficial anticancer therapy in man. There have been a number of small
scale clinical trials testing PPARγ ligands in
cancer in man with varying success [63]. The
most promising results were from small phase II studies treating prostate
cancer [64] and
liposarcoma patients [65] with
troglitazone. In contrast, a phase II study treating liposarcoma patients with
rosiglitazone did not significantly improve clinical outcome [66] and so
far no beneficial effects of PPARγ ligands have
been observed in trials for breast or colon cancer patients [35].


## 3.3. PPARα and PPARγ Regulation of Angiogenesis

Early studies showed no effect of the selective PPARα ligand WY-14643 on endothelial cell proliferation [27],
however, recent studies using immortalised human dermal microvascular
endothelial cells show that the PPARα ligand fenofibrate
inhibits endothelial cell proliferation, migration, and tube formation (on a
fibrin matrix) in vitro and
angiogenesis in vivo [67]. Fenofibrate
acts by disrupting the formation of the actin cytoskeleton and inhibits bFGF-induced
Akt activation and cyclooxygenase 2 (COX-2) gene expression [67].
Similar results were found in a porcine model of vascular remodelling after
coronary artery angioplasty where fenofibrate increased lumen size and vessel
area and inhibited constrictive remodelling and inflammatory cell infiltration [68]. Importantly,
adventitial angiogenesis was significantly reduced by fenofibrate in the injured
vessels 3 days after angioplasty [68].In contrast to this vascular study, the investigation of PPARα regulation of
tumour angiogenesis has only just begun. In a recent report, Panigraphy et al.
provide compelling evidence for PPARα inhibition of
tumour growth by targeting angiogenesis [48].
Similar to previous findings, PPARα activation
had direct effects on endothelial cells, inhibiting VEGF-induced endothelial
cell migration in vitro and FGF2 induced corneal angiogenesis in
vivo [48].
Tumour cell synthesis of VEGF and FGF2 was also suppressed by PPARα activation in
conjunction with an increased expression of antiangiogenic thrombospondin-1
(TSP-1) [48]. In
subcutaneously implanted human pancreatic cancer cells grown in mice, as well
as in human prostate cancer, PPARα expression
was detected not only in the tumour cells, but also in the new invading
microvessels [48]. Systemic
treatment of mice with PPARα ligands inhibited the growth of melanoma, glioblastoma, and fibrosarcoma
tumours implanted in vivo,
which was associated with a reduction in vessel density and inflammation [48]. To
dissect the mechanism by which PPARα suppressed
tumour growth (i.e., direct effects on the tumour and/or angiogenesis), embryonic
fibroblasts from PPARα (−/−) knockout mice were transformed with SV40 large T antigen and
H-ras oncogenes then implanted into wild-type and PPARα −/− mice. The
growth of these cells into tumours could be suppressed by PPARα ligands in
wild-type mice only, indicating that tumour
suppression by PPARα ligands was completely dependent on the expression of PPARα in the host
but not in the tumour cells [48].
Fenofibrate strongly induced the antiangiogenic factors TSP-1 and endostatin in
wild-type, but not PPARα −/− mice, supporting the role of PPARα as an antiangiogenic
regulator [48]. Angiogenesis
and inflammation are central processes through which the tumour interacts with
its surroundings to influence tumour growth. Although this study does not rule
out an anti-inflammatory effect of the PPARα ligands, it
is highly unlikely that the antitumour host-derived effects are due to
suppression of inflammation because mice deficient in PPARα generally
exhibit enhanced inflammation [64].TSP-1 is a potent angiogenesis inhibitor that targets endothelial cells
for apoptosis by initiating a signalling cascade through the CD36 receptor.
PPARα directly induces TSP-1 and can enhance TSP-1 signalling indirectly by
upregulating CD36 in the endothelium. PPARα activation
upregulates CD36 expression in the liver [69] and in
macrophages [70].  Moreover, coadministration of PPARγ ligands with
exogenous TSP-1 or the TSP-1 peptide derivative ABT510 synergises to suppress
angiogenesis and induce endothelial cell apoptosis [71]. The
improvement of the antiangiogenic efficacy of TSP-1 was attributed to PPARγ-induced CD36 expression via a PPAR response element in the CD36 promoter
[69, 71].The vast majority of studies have indicated an antiangiogenic role for
PPARα and PPARγ in a variety of models. However, it is important to note that the VEGF
promoter contains a PPAR response element and PPARα and -γ ligands can
induce VEGF in certain cell types [72–75].
Moreover, in contrast to the majority of findings, a recent study suggests that
both PPARα and PPARγ ligands may also have proangiogenic properties in vitro in an endothelial/interstitial cell coculture assay and in a
murine corneal angiogenesis model in vivo [72]. The angiogenesis induced by PPARα and PPARγ ligands was
associated with the induction of VEGF, accompanied by increased activation of AKT
and eNOS (by phosphorylation) [72]. How
the levels of PPARα- or PPARγ-mediated angiogenesis are compared to traditional growth factor-induced angiogenesis is
not known? Indeed, these results are controversial,
as previous corneal angiogenesis models clearly demonstrate antiangiogenic
effects of PPARα and PPARγ ligands [28, 48, 76].Multiple mechanisms have been proposed by which PPARα and PPARγ regulate the
changes in pro- and antiangiogenic factors. Here, we will focus on the central
target for PPAR regulation of angiogenesis, the proangiogenic VEGF/VEGFR
signalling pathway. PPARγ can downregulate VEGF either directly through a PPAR response element
within the VEGF promoter [77] or by
decreasing PGE2, an endogenous stimulator of angiogenesis [78]. PPARγ can also
decrease VEGF responses by suppressing transcription of its receptor VEGFR2, by
interacting with and preventing Sp1 binding to DNA [79].In colorectal cancer cell lines, PPARα also inhibits
the transcription factor AP-1, impairing its binding to response elements in
the VEGF and COX-2 genes and inhibiting c-jun transactivation activity, thus
downregulating VEGF and COX-2 expression [80]. It
is, therefore, clear that the regulation of angiogenic factors by PPARα and PPARγ may be
determined by cell and cancer type and the experimental models used. Much more
research is required to fully understand whether PPAR activation will be pro-
or antiangiogenic in specific human cancers.


## 3.4. The Effects of PPARα and PPARγ on Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) present in peripheral blood promote angiogenesis and
improve endothelial function. The research on the effects of PPARs on EPCs has
focused on PPARγ. Despite PPARγ generally being considered
antiangiogenic, the PPARγ ligands
rosiglitazone and pioglitazone in diabetic patients increase endothelial progenitor
cell (EPC) number and migratory activity [81, 82]. Pioglitazone
and rosiglitazone also improve the adhesive capacity of EPCs to fibronectin and
collagen [82] and promote EPC
colony formation, [83, 84]. In vitro, pioglitazone increased EPC
proliferation, colony formation, and attenuated apoptosis [85]. Similarly, in
mice pioglitazone induced the number and migratory activity of EPCs while
decreasing their apoptosis, resulting in increased in vivo neoangiogenesis [86]. From these
results, it has been proposed that PPARγ ligands may have
a double-edged role in angiogenesis, with proangiogenic effects on EPCs at
low-systemic concentrations and antiangiogenic effects at higher local
concentrations [86]. Indeed,
biphasic effects of pioglitazone were observed on EPCs in culture, when the
number of EPC colonies and amount of adhesion were increased by 1 μM but not 10 μM [87]. This higher
concentration of pioglitazone induced TGF-β1 and its
receptor endogolin, which suppress EPC function [87]. These findings
have important clinical implications suggesting that the pro-/antiangiogenic
properties of PPARγ ligands may be
largely dose-driven. Moreover, understanding this mechanism by which PPARγ may regulate both pro- and
antiangiogenic pathways at least in EPCs may help to explain some of the
contradictions in the studies examining the role of PPARγ in angiogenesis.


## 3.5. Effects of PPARα and PPARγ on Endothelial  Cell Inflammation

The role of PPARα in inflammation has been studied in animal
models, particularly in wound healing and cardiovascular disease models (atherosclerosis
and restenosis) [55, 56]. PPARα is a negative regulator of inflammation [34] in inflammatory models. Supporting this, PPARα −/− mice exhibit
enhanced inflammation [88], although this
may be due in part to deceased β-oxidation and
accumulation of biologically active lipid mediators.In addition to
these experimental models, PPARα agonists
decrease the expression of inflammatory markers both in
human cells and patients treated with fibrates [89, 90]. In
human endothelial cells in culture, PPARα
ligands inhibit the cytokine/LPS induction of COX-2 [38, 69], ICAM-1 [91], VCAM-1
[29, 31], endothelin-1
[92], IL-6,
and prostaglandin E2 [32, 93]. Similarly,
PPARα ligands
repress thrombin-induced expression of endothelin-1 [32]. The
PPARα ligand fenofibrate, but not the PPARγ ligand rosiglitazone, also reduces the
induction of tissue factor in human endothelial cells [94], while PAI-1 levels
remain unchanged [31]. PPARα inhibits proinflammatory mediators by
interfering with the transactivation activity of NFκB and AP-1, the main transcription
factors mediating inflammatory and growth factor responses. PPARα via direct protein-protein interactions
can bind and inhibit the actions p65 and c-jun subunits, respectively [95, 96].Although the weight of evidence points towards an anti-inflammatory role
for PPARα, oxidised lipids that can activate PPARα have
been shown to increase the release of neutrophil chemoattractant IL-8 and MCP-1
from endothelial cells [30].
Similarly, PPARα ligands induce COX-2 in human breast and colon cancer cells [97, 98].PPARγ, similarly, is a well-established negative regulator of the inflammatory
response in vitro and in vivo [34]. PPARγ agonists have been shown to mediate effects on cell survival,
surface-protein expression, and cytokine and chemokine production. In
endothelial cells, PPARγ ligands can induce
apoptosis [27] and decrease
inflammatory cell recruitment by inhibiting the production of chemokines IL-8, MCP-1
[30, 99],
IP-10, Mig, and I-TAC [100] and reducing
ICAM-1 expression [101].
Similar to PPAR-α, PPARγ ligands repress thrombin-induced expression of endothelin-1 [32].


## 4. PPARβ/δ

### 4.1. PPARβ/δ Ligands

PPARβ/δ (Figure 4) is almost ubiquitously expressed [102], although
compared to PPARα and -γ, less is known regarding its role in the
body. However, like PPARα and -γ, it appears able to regulate lipid
metabolism, cellular proliferation, and the inflammatory response [55, 56]. Activators of PPARβ/δ include a variety of eicosanoids (the COX product prostacyclin [40, 41], COX/prostacyclin synthase-derived
endocannabinoid metabolites [103]); fatty acids and synthetic compounds including
GW0742X, GW501516, L-165,461, and compound F [40, 41].Figure 4
Endothelial PPARβ/δ has
predominantly proangiogenic actions on endothelial cells. The majority of
studies so far indicate that PPARβ/δ activation induces (solid line)
proangiogenic factors, while reduces (broken line) antiangiogenic factors.
Similar to PPARα and PPARγ, PPARβ/δ also appears to be anti-inflammatory by
reducing proinflammatory pathways and potentially anticoagulant by reducing
tissue factor release.


### 4.2. PPARβ/δ and Cancer

There has recently been an increasing amount of contradictory
literature published regarding PPARβ/δ regulation
of tumour cell growth and tumour cell release of VEGF. PPARβ/δ ligands
induce VEGF in bladder cancer [104], human breast
(T47D, MCF7) and prostate (LNCaP, PNT1A) cancer cell lines, along with its
receptor VEGFR1 [105], but not in colon
(HT29, HCT116, LS-174T) and hepatoma (HepG2, HuH7) cell lines [106].Much of the research into PPARβ/δ in cancer has focused on gastrointestinal cancer. PPARβ/δ expression is
enhanced in human and rodent colorectal tumours, as well as preneoplastic
colonic mucosa [107, 108]. PPARβ/δ is
transcriptionally regulated by β-catenin/Tcf-4,
which can be suppressed APC. Therefore, in colorectal cancer cells that commonly
carry an APC mutation, PPARβ/δ is upregulated [108]. Interestingly, PPARβ/δ accumulation
was localised to human colorectal carcinoma cells with a highly malignant
morphology [109],
suggesting PPARβ/δ promotes tumourogenesis. Supporting this theory, the growth of PPARβ/δ −/− HCT-116
human colon carcinoma cell xenografts was reduced compared to wild-type PPARβ/δ expressing cells [83].Using animal models, a positive
link has been made between PPARβ/δ and colon cancer development, especially using the intestinal polyp
model, APCmin/+ mice. In this model, deletion of PPARβ/δ decreases intestinal adenoma growth and
inhibits the tumour-promoting effects of the PPARβ/δ agonist GW501516 [85, 110]. PPARβ/δ activation induces VEGF in colon carcinoma cells, promoting cell survival by
activation of Akt signalling [85]. Angiogenesis was not studied in these
experiments, however, for a tumour to grow greater than 2 mm in diameter a functional
vessel network is required [111]. Indeed, the
most prominent effect of PPARβ/δ activation in APCmin/+ mice,
observed by Gupta et al., was a significant increase in the number of polyps
greater than 2 mm in diameter [110]. Whereas there
was a significant decrease in the growth of polyps greater than 2 mm in
diameter in PPARβ/δ −/− APCmin/+ mice, despite a
lack of effect on overall polyp incidence [112]; indicating that
PPARβ/δ promotes tumour growth via angiogenesis.In contrast, deletion of PPARβ/δ in APCmin/+
mice enhanced colon polyp formation in untreated mice and in mice with
chemically induced colon carcinogenesis [113, 114]. The PPARβ/δ ligand GW0742 inhibited chemically induced colon carcinogenesis in PPARβ/δ wild-type but
not PPARβ/δ −/− mice [115]. The differences between these contrasting results
have been suggested to be due to differences in genetic background, breeding,
or the PPARβ/δ knockout
strategy of the APCmin/+ mouse models [116]. However, this would not explain why in human colon and liver cancer
cell lines, PPARβ/δ ligands had no effect on cell growth, Akt
phosphorylation, or VEGF and COX-2 expression in vitro or on these markers in the liver, colon and colon polyps in mice treated
in vivo [106]. The role of PPARβ/δ in VEGF-mediated
tumourgenesis, therefore, still requires further study and clarification.


### 4.3. PPARβ/δ and Angiogenesis

Initial reports using
prostacyclin as a ligand suggested that similar to PPARα and PPARγ, PPARβ/δ promoted endothelial cell apoptosis [117], and potentially decreased angiogenesis. In contrast, with the development
of highly selective synthetic ligands, there is an increasing evidence to
propose a role for PPARβ/δ in regulating
endothelial cell survival, proliferation, and angiogenesis. Indeed, treating endothelial
cells with the selective PPARβ/δ ligand GW501516 induces proliferation, VEGF receptor (Flt-1; VEGF R1) expression,
and VEGF production [105, 118]. In addition to inducing proliferation, PPARβ/δ also protects the endothelial
cell from oxidant injury via induction of the antiapoptotic and anti-inflammatory protein
14-3-3α [119].PPARβ/δ potently induces angiogenesis by human
and murine vascular endothelial cells in tumour extracellular matrix in vitro and in a murine matrigel plug model
in vivo [118]. The stimulated release of VEGF from human endothelial cells was a major
trigger for morphogenesis, although mRNA for the matrix metalloproteinase (MMP)-9, a protease important for cell migration,
was also elevated [118]. In addition to VEGF, genomic and proteomic analysis of
PPARβ/δ −/− endothelial cells isolated from matrigel plugs identified
a number of additional candidate genes that may mediate the angiogenic actions
of PPARβ/δ. Cdkn1c, which encodes
the cell cycle inhibitor p57Kip2, is induced by PPARβ/δ [120]. The chloride intracellular channel protein (CLIC)-4 is decreased in
migrating endothelial cells from PPARβ/δ knockout mice, whereas the expression of cellular retinol binding
protein CRBP1 is increased [121]. CLIC-4 plays an
essential role during tubular morphogenesis [122], while CRBP1 inhibits cell survival pathways by blocking
the Akt signalling pathway [123]. The combination of these studies indicates that PPARβ/δ may induce endothelial cell
mitogenesis and differentiation signals, including VEGF, 14-3-3α, CLIC4, CRBP-1, and p57KIP2, which
may combine to bring about the functional morphogenic changes associated with the
angiogenic switch.Two recent studies in
particular have addressed the regulation of angiogenesis by PPARβ/δ in matrigel plugs in PPARβ/δ wild-type and knockout
mice [120, 124]. Xenograft tumours in PPARβ/δ −/− mice exhibited a diminished blood flow and immature hyperplastic
microvascular structures when compared to wild-type mice. Moreover, the reintroduction
of PPARβ/δ into the matrigel plugs
was able to rescue the knockout phenotype
by triggering microvessel maturation [120]. In addition, tumour angiogenesis and growth are
markedly inhibited in PPARβ/δ −/− mouse models of subcutaneous Lewis lung carcinoma
and B16 melanoma. PPARβ/δ expression correlated with advanced
pathological tumour stage and increased risk for tumour recurrence and distant
metastasis in pancreatic
tumours from patients who had undergone the “angiogenic switch” [124]. PPARβ/δ has, therefore, been suggested as a “hub node” transcription factor,
regulating the tumour angiogenic switch [124].


### 4.4. The Effects of PPARγβ/δ on Endothelial  Progenitor Cells

Little is known
about the effects of PPARβ/δ on EPCs, but there is one study that
shows that PPARβ/δ is a key regulator of EPC proangiogenic
functions. Prostacyclin is a putative PPARβ/δ ligand and proangiogenic factor,
produced by COX and PGI2 synthase in the endothelium. EPC tube
formation and proliferation are induced by the selective PPARβ/δ  ligand GW510516.  EPCs treated with an inhibitor of COX or
COX-1, prostacyclin synthase, or PPARβ/δ specific siRNA, exhibit
decreased cell proliferation and tube formation [125]. Thus the proangiogenic effects
of human EPCs appear in part dependent on the biosynthesis of prostacyclin and
the subsequent activation of PPARβ/δ.


### 4.5. The Effect of PPARβ/δ on Endothelial  Cell Inflammation

Little is known regarding the role of PPARβ/δ in endothelial cell inflammation and mediator secretion. PPARβ/δ ligands, similar to PPARα and PPARγ ligands, inhibit cytokine-stimulated upregulation of adhesion molecules
ICAM-1, VCAM-1, and e-selectin and NFκB translocation [126, 127]. These anti-inflammatory
effects of PPARβ/δ in endothelial cells
occur when the complex between PPARβ/δ and the transcriptional repressor BCL6 is removed by ligand activation,
identical to the mechanism identified in monocytes [128]. PPARβ/δ and BCL6 are then free to act on PPARβ/δ targets (including SOD and catalase) and BCL6 targets which importantly
include the repression of NFκB. In addition to anti-inflammatory effects, endogenous PPARβ/δ ligands are continuously produced in endothelial cells to suppress the
release of tissue factor, the primary initiator of coagulation [103].


## 4.1. PPARβ/δ Ligands

PPARβ/δ (Figure 4) is almost ubiquitously expressed [102], although
compared to PPARα and -γ, less is known regarding its role in the
body. However, like PPARα and -γ, it appears able to regulate lipid
metabolism, cellular proliferation, and the inflammatory response [55, 56]. Activators of PPARβ/δ include a variety of eicosanoids (the COX product prostacyclin [40, 41], COX/prostacyclin synthase-derived
endocannabinoid metabolites [103]); fatty acids and synthetic compounds including
GW0742X, GW501516, L-165,461, and compound F [40, 41].Figure 4
Endothelial PPARβ/δ has
predominantly proangiogenic actions on endothelial cells. The majority of
studies so far indicate that PPARβ/δ activation induces (solid line)
proangiogenic factors, while reduces (broken line) antiangiogenic factors.
Similar to PPARα and PPARγ, PPARβ/δ also appears to be anti-inflammatory by
reducing proinflammatory pathways and potentially anticoagulant by reducing
tissue factor release.


## 4.2. PPARβ/δ and Cancer

There has recently been an increasing amount of contradictory
literature published regarding PPARβ/δ regulation
of tumour cell growth and tumour cell release of VEGF. PPARβ/δ ligands
induce VEGF in bladder cancer [104], human breast
(T47D, MCF7) and prostate (LNCaP, PNT1A) cancer cell lines, along with its
receptor VEGFR1 [105], but not in colon
(HT29, HCT116, LS-174T) and hepatoma (HepG2, HuH7) cell lines [106].Much of the research into PPARβ/δ in cancer has focused on gastrointestinal cancer. PPARβ/δ expression is
enhanced in human and rodent colorectal tumours, as well as preneoplastic
colonic mucosa [107, 108]. PPARβ/δ is
transcriptionally regulated by β-catenin/Tcf-4,
which can be suppressed APC. Therefore, in colorectal cancer cells that commonly
carry an APC mutation, PPARβ/δ is upregulated [108]. Interestingly, PPARβ/δ accumulation
was localised to human colorectal carcinoma cells with a highly malignant
morphology [109],
suggesting PPARβ/δ promotes tumourogenesis. Supporting this theory, the growth of PPARβ/δ −/− HCT-116
human colon carcinoma cell xenografts was reduced compared to wild-type PPARβ/δ expressing cells [83].Using animal models, a positive
link has been made between PPARβ/δ and colon cancer development, especially using the intestinal polyp
model, APCmin/+ mice. In this model, deletion of PPARβ/δ decreases intestinal adenoma growth and
inhibits the tumour-promoting effects of the PPARβ/δ agonist GW501516 [85, 110]. PPARβ/δ activation induces VEGF in colon carcinoma cells, promoting cell survival by
activation of Akt signalling [85]. Angiogenesis was not studied in these
experiments, however, for a tumour to grow greater than 2 mm in diameter a functional
vessel network is required [111]. Indeed, the
most prominent effect of PPARβ/δ activation in APCmin/+ mice,
observed by Gupta et al., was a significant increase in the number of polyps
greater than 2 mm in diameter [110]. Whereas there
was a significant decrease in the growth of polyps greater than 2 mm in
diameter in PPARβ/δ −/− APCmin/+ mice, despite a
lack of effect on overall polyp incidence [112]; indicating that
PPARβ/δ promotes tumour growth via angiogenesis.In contrast, deletion of PPARβ/δ in APCmin/+
mice enhanced colon polyp formation in untreated mice and in mice with
chemically induced colon carcinogenesis [113, 114]. The PPARβ/δ ligand GW0742 inhibited chemically induced colon carcinogenesis in PPARβ/δ wild-type but
not PPARβ/δ −/− mice [115]. The differences between these contrasting results
have been suggested to be due to differences in genetic background, breeding,
or the PPARβ/δ knockout
strategy of the APCmin/+ mouse models [116]. However, this would not explain why in human colon and liver cancer
cell lines, PPARβ/δ ligands had no effect on cell growth, Akt
phosphorylation, or VEGF and COX-2 expression in vitro or on these markers in the liver, colon and colon polyps in mice treated
in vivo [106]. The role of PPARβ/δ in VEGF-mediated
tumourgenesis, therefore, still requires further study and clarification.


## 4.3. PPARβ/δ and Angiogenesis

Initial reports using
prostacyclin as a ligand suggested that similar to PPARα and PPARγ, PPARβ/δ promoted endothelial cell apoptosis [117], and potentially decreased angiogenesis. In contrast, with the development
of highly selective synthetic ligands, there is an increasing evidence to
propose a role for PPARβ/δ in regulating
endothelial cell survival, proliferation, and angiogenesis. Indeed, treating endothelial
cells with the selective PPARβ/δ ligand GW501516 induces proliferation, VEGF receptor (Flt-1; VEGF R1) expression,
and VEGF production [105, 118]. In addition to inducing proliferation, PPARβ/δ also protects the endothelial
cell from oxidant injury via induction of the antiapoptotic and anti-inflammatory protein
14-3-3α [119].PPARβ/δ potently induces angiogenesis by human
and murine vascular endothelial cells in tumour extracellular matrix in vitro and in a murine matrigel plug model
in vivo [118]. The stimulated release of VEGF from human endothelial cells was a major
trigger for morphogenesis, although mRNA for the matrix metalloproteinase (MMP)-9, a protease important for cell migration,
was also elevated [118]. In addition to VEGF, genomic and proteomic analysis of
PPARβ/δ −/− endothelial cells isolated from matrigel plugs identified
a number of additional candidate genes that may mediate the angiogenic actions
of PPARβ/δ. Cdkn1c, which encodes
the cell cycle inhibitor p57Kip2, is induced by PPARβ/δ [120]. The chloride intracellular channel protein (CLIC)-4 is decreased in
migrating endothelial cells from PPARβ/δ knockout mice, whereas the expression of cellular retinol binding
protein CRBP1 is increased [121]. CLIC-4 plays an
essential role during tubular morphogenesis [122], while CRBP1 inhibits cell survival pathways by blocking
the Akt signalling pathway [123]. The combination of these studies indicates that PPARβ/δ may induce endothelial cell
mitogenesis and differentiation signals, including VEGF, 14-3-3α, CLIC4, CRBP-1, and p57KIP2, which
may combine to bring about the functional morphogenic changes associated with the
angiogenic switch.Two recent studies in
particular have addressed the regulation of angiogenesis by PPARβ/δ in matrigel plugs in PPARβ/δ wild-type and knockout
mice [120, 124]. Xenograft tumours in PPARβ/δ −/− mice exhibited a diminished blood flow and immature hyperplastic
microvascular structures when compared to wild-type mice. Moreover, the reintroduction
of PPARβ/δ into the matrigel plugs
was able to rescue the knockout phenotype
by triggering microvessel maturation [120]. In addition, tumour angiogenesis and growth are
markedly inhibited in PPARβ/δ −/− mouse models of subcutaneous Lewis lung carcinoma
and B16 melanoma. PPARβ/δ expression correlated with advanced
pathological tumour stage and increased risk for tumour recurrence and distant
metastasis in pancreatic
tumours from patients who had undergone the “angiogenic switch” [124]. PPARβ/δ has, therefore, been suggested as a “hub node” transcription factor,
regulating the tumour angiogenic switch [124].


## 4.4. The Effects of PPARγβ/δ on Endothelial  Progenitor Cells

Little is known
about the effects of PPARβ/δ on EPCs, but there is one study that
shows that PPARβ/δ is a key regulator of EPC proangiogenic
functions. Prostacyclin is a putative PPARβ/δ ligand and proangiogenic factor,
produced by COX and PGI2 synthase in the endothelium. EPC tube
formation and proliferation are induced by the selective PPARβ/δ  ligand GW510516.  EPCs treated with an inhibitor of COX or
COX-1, prostacyclin synthase, or PPARβ/δ specific siRNA, exhibit
decreased cell proliferation and tube formation [125]. Thus the proangiogenic effects
of human EPCs appear in part dependent on the biosynthesis of prostacyclin and
the subsequent activation of PPARβ/δ.


## 4.5. The Effect of PPARβ/δ on Endothelial  Cell Inflammation

Little is known regarding the role of PPARβ/δ in endothelial cell inflammation and mediator secretion. PPARβ/δ ligands, similar to PPARα and PPARγ ligands, inhibit cytokine-stimulated upregulation of adhesion molecules
ICAM-1, VCAM-1, and e-selectin and NFκB translocation [126, 127]. These anti-inflammatory
effects of PPARβ/δ in endothelial cells
occur when the complex between PPARβ/δ and the transcriptional repressor BCL6 is removed by ligand activation,
identical to the mechanism identified in monocytes [128]. PPARβ/δ and BCL6 are then free to act on PPARβ/δ targets (including SOD and catalase) and BCL6 targets which importantly
include the repression of NFκB. In addition to anti-inflammatory effects, endogenous PPARβ/δ ligands are continuously produced in endothelial cells to suppress the
release of tissue factor, the primary initiator of coagulation [103].


## 5. PPAR Therapy for Cancer

The PPARs have pleiotrophic actions on nonvascular and vascular cells.
PPARα and PPARγ ligands (although there are well-detailed current concerns for
rosiglitazone) are in clinical use, are considered safe, and have high
tolerability with chronic use. There is considerable evidence that PPARγ and
increasing evidence that PPARα are vascular protective and reduce angiogenesis. Unfortunately, as yet,
there is a little clinical evidence to support these actions, apart from the
promising results with the PPARγ ligand troglitazone in liposarcoma and prostate cancer previously
mentioned [64, 65].
Clinically, PPARα and γ ligands do not appear to be
strong antiangiogenic drugs. However, since PPARα and PPARγ ligands are
in clinical use and lack severe side effects, the potential for their use to
complement or augment current and new therapies to treat a variety of cancers
is currently being tested in small scale trials. For example, a phase II trial
combining anti-inflammatory and angiostatic therapy (PPARγ ligand
pioglitazone and COX-2 inhibitor, rofecoxib) with metronomic low-dose
chemotherapy (trofosamide) found that the progression-free survival rates of
advanced melanoma patients were longer with the combination treatment than with
metronomic chemotherapy alone [129]. This
combination therapy was also successful in achieving disease stabilization or
remission in patients with advanced progressive malignant vascular tumours [130] and
partial remission in a single patient with endemic Kaposi sarcoma [131].
However, a similar phase II study on high-grade glioma patients, showed disease
stabilisation in only 4 out of 14 patients, suggesting that this combined
therapy may only be suitable for a subset of patients [132]. The
COX-2 inhibitor rofecoxib was included in the trial because COX-2 plays a role
in endothelial tube formation, pericyte recruitment, and endothelial cell
survival during early angiogenesis [133].  As PPARα and γ ligands have been shown to inhibit COX-2
induction in endothelial cells, it would be interesting to test the combined
effects of PPARα or −γ ligands with metronomic
chemotherapy alone.In contrast to PPARα and PPARγ, there is
increasing evidence that PPARβ/δ is proangiogenic and an important
transcription factor in the angiogenic switch. PPARβ/δ has an
interesting activity profile in that like the other PPARs it also appears to
have anti-inflammatory properties. As PPARβ/δ is considered a target to treat
dyslipidaemia, its proangiogenic properties should, therefore, be considered in
the long-term use of PPARβ/δ ligands to treat chronic metabolic
diseases. The development of selective antagonists for PPARβ/δ offers great
potential for cancer treatment. One such antagonist has recently been
identified, GSK0660, which can compete with agonist in a cellular context and
by itself exhibits inverse agonist activity [134]. This antagonist
appears to act by promoting PPARβ/δ-mediated repression of gene expression.
Unfortunately, this compound lacks in
vivo bioavailability, but will be a valuable tool for elucidating the
role of PPARβ/δ in cancer and
angiogenesis in vitro and a basis for further development of
a selective bioavailable PPARβ/δ antagonist [134]. Selective
modulators of PPARβ/δ, which maintain the beneficial metabolic
(and anti-inflammatory) effects while exerting no proangiogenic effects would
also be beneficial. Interestingly, there is a newly
developed PPAR-α agonist (R)-K-13675, which inhibits the secretion of inflammatory
markers without affecting cell proliferation or endothelial tube formation [135], which
suggests that selective modulators for the other PPARs may soon be available.



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*Source: 904251-2008-11-24.xml*