# Peroxisome Proliferator-Activated

Authors: Costas Giaginis; Anna Tsantili-Kakoulidou; Stamatios Theocharis
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has currently been considered as molecular target for the treatment of human metabolic disorders. Experimental data from in vitro cultures, animal models, and clinical trials have shown that PPAR-γ ligand activation regulates differentiation and induces cell growth arrest and apoptosis in a variety of cancer types. Tumor angiogenesis constitutes a multifaceted process implicated in complex downstream signaling pathways that triggers tumor growth, invasion, and metastasis. In this aspect, accumulating in vitro and in vivo studies have provided extensive evidence that PPAR-γ ligands can function as modulators of the angiogenic signaling cascade. In the current review, the crucial role of PPAR-γ ligands and the underlying mechanisms participating in tumor angiogenesis are summarized. Targeting PPAR-γ may prove to be a potential therapeutic strategy in combined treatments with conventional chemotherapy; however, special attention should be taken as there is also substantial evidence to support that PPAR-γ ligands can enhance angiogenic phenotype in tumoral cells.


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## Body

## 1. Introduction

Angiogenesis, the development of new capillaries from preexisting
microvessels, plays a crucial role in several normal physiological processes,
such as embryonic development, ovulation, wound healing, as well as tissue and
organ regeneration. Angiogenesis also constitutes a crucial step in the aetiology
of diverse pathological states, including cancer, diabetic retinopathy,
age-related macular degeneration, psoriasis, and rheumatoid arthritis [1, 2]. In the last few years, the complicated
biochemical mechanisms governing neovessel formation have been well
established. These include the proliferation of endothelial cells (ECs) from
preexisting capillaries, the breakdown and reassembly of the extracellular
matrix (ECM) and the morphogenic process of endothelial tube formation [2, 3]. Numerous growth factors,
including vascular endothelial growth factor (VEGF) family, basic fibroblast
growth factors (bFGFs), platelet-derived growth factor (PDGF), hepatocyte
growth factor (HGF), placenta growth factor (PGF), matrix metalloproteinases
(MMPs), ephrin family, angiopoetin-1 (Ang-1), interleukins (IL-2, -6, -8), as
well as various endothelial surface molecules such CD31, CD34, CD36, CD144, and
avb3 integrins, have been found to control essential
steps within angiogenesis process [1–3].
The generation and release of antiangiogenic factors, such as interferon (INF)
-α, -β, -γ, platelet factor 4 (PF4), and tissue
inhibitors of MMPs (TIMPs) contribute to the coordinated downregulation of the
angiogenic process within physiologic angiogenesis [4].Peroxisome proliferator-activated receptors
(PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated
transcription factors and include three different isotypes: PPAR-α, PPAR-β/δ, and PPAR-γ [5, 6]. PPAR-γ, the most extensively studied amongst them,
functions as ligand-activated transcription factor by binding to specific DNA
sequences, termed to as peroxisome proliferator response elements (PPREs), in
the promoter of the target genes only as a heterodimer with the retinoid X receptor
(RXR) [7–9]. PPRE has been
mainly identified in the upstream regulatory sequences of genes related to
metabolic pathways [7–9]. In addition, recent studies have
revealed that PPAR-γ can regulate gene expression independently of PPRE, either
by suppressing growth hormone protein-1 (GHP-1), a transcription factor
involved in pituitary specific gene expression, or by interfering with the
function of activator protein-1 (AP-1), signal transducer and activator of
transcription-1 (STAT-1) and nuclear factor-κB (NF-κB) [7, 10–12]. In this context, the
identification of a sumoylation-dependent pathway by which PPAR-γ represses transcriptional activation of
inflammatory response genes has recently been reported [13]. This mechanism
provides a possible explanation for how ligand-bound PPAR-γ activation can be converted from an activator
of transcription to a promoter-specific repressor of NF-κB target genes [13].A wide range of natural and synthetic structurally diverse compounds has
been reported as potent PPAR-γ ligands. The long chain polyunsaturated fatty acids and their
derivatives, such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), as
well as nitrolinoleic
acids are known natural occurring PPAR-γ ligands [14, 15]. Recently, curcumin, a well-documented anticancer
phytochemical component of turmeric, has been shown to exert anti-inflammatory
functions via upregulation of PPAR-γ activation [16]. Thiazolidinediones
(TZDs) and tyrosine-based derivatives, such as glitazars (tesaglitazar,
farglitazar), constitute the most well-known synthetic ligands [17, 18], while relatively lower binding affinity for PPAR-γ has also been reported for some nonsteroidal anti-inflammatory drugs (NSAIDs) [19]. TZDs represent a promising class of oral antidiabetic agents,
some of which are already marketed drugs (pioglitazone-PGZ and rosiglitazone-RGZ)
for the treatment of type II diabetes mellitus [20].
Interestingly, a wide spectrum of action for TZDs beyond the treatment of
diabetes, including anti-inflammatory and antineoplastic properties, as well as targeting signaling pathways
implicated in atherosclerosis and osteoporosis has been reported [21–23]. In the last decade, more than 1000 PPAR-γ ligands belonged to several distinct chemical classes have
been synthesized and evaluated for their binding and transactivation to their
receptor. In this aspect, screening drug-like characteristics in the chemical space of PPAR-γ
ligands have currently been considered as an emerging demand in the aim to
discover more potent compounds with improved absorption, distribution,
metabolism, excretion/toxicity (ADME/Tox) properties, avoiding potential toxic
side effects, as well as pharmacokinetic and pharmacodynamic problems [24, 25].To date, there has been a substantial accumulation of evidence that PPAR-γ ligands exert
regulatory effects on angiogenesis process related to diverse disease states,
including cancer and diabetes [26–28]. It is also well documented that they directly affect tumor cells by inhibiting cell growth and inducing
cell differentiation and apoptosis in various cancer types [21, 29, 30]. In view of the fact that
angiogenesis is implicated in tumor development and metastasis and its
inhibition could serve as potent antitumor side-therapeutic approach, the current
review summarizes the latest knowledge of the role of PPAR-γ ligands in angiogenesis related to cancer, highlighting in
the underlying mechanisms.


## 2. Angiogenesis in Cancer

Tumor angiogenesis constitutes an
essential component of tumor growth, invasion, and metastasis that depends on a
net balance of angiogenic and antiangiogenic mediators, which are secreted by
both tumor and host infiltrating cells [31]. Currently, it is well established that
this dynamic balance between angiogenic stimulators and inhibitors, controls
the angiogenic signaling cascade governing the transformation of a tumor from a
nonangiogenic to an angiogenic phenotype [32]. The acquisition of angiogenic phenotype has been considered
as a rate-limiting step in tumor progression, which allows the tumor to
transform from a small lesion to a rapidly expanding mass with metastatic
potency [33]. On the other hand, human tumors arise in the absence of
angiogenic activity and may exist in a microscopic dormant state for months to
years without neovascularization [34]. In this context, hypoxia, developed
within rapidly proliferating tissues or as a result of the occlusion of blood
vessels, has been considered as a primary physiological regulator of the
angiogenic switch [35]. The key mediators of this response are members of the
hypoxia-inducible factor (HIF) family of proteins that function as
transcriptional regulators, stimulating the expression of a multitude of genes
important for oxygen homeostasis [36, 37]. In addition, HIF has been found to
enhance the expression of several angiogenic mediators, including VEGF-R1,
VEGF-R2, Ang-1, Ang-2, MMP-2, and MMP-9 in malignant tumors [36, 38].In response to hypoxia, tumor cells
turn on the angiogenic signaling cascade by secreting various potent angiogenic
mediators, such as VEGF, PDGF, bFGFs, angiopoetins, HGF, fibronectin, and
heparanase that in turn activate endothelial cells of preexisting capillaries
to produce MMPs for the collapse of ECM [39]. Degradation of ECM by MMPs allows
endothelial cells to migrate in response to chemotactic growth factors, including
VEGF, PDGF, and bFGFs [33, 39]. Members of CXC chemokine family, such as IL-2,
-6, -8, and integrins αvβ3,  are also involved in the angiogenic cascade. It should be noted that
in the case of high progressive tumors, the release of endogenous
antiangiogenic factors are insufficient to counteract the net effect of
angiogenic ones. Thus, the formation of new blood vessel is formed after
attracting accessory cells, mainly pericytes and smooth muscle cells, producing
a new basement membrane and a firm ECM [39, 40]. The above-mentioned angiogenic
mediators have been joined by others including Notch/Delta, semaphorin, ephrin,
and roundabout/slit families of proteins [40]. Besides this, blockage of NF-κB activity has been shown to reduce VEGF gene
expression in highly malignant tumor cells, since a binding site for this
transcription factor has been identified within the VEGF promoter [41]. Each
of the sequential steps within angiogenic cascade could be considered as a
potential single target for the development of new drug candidates against
tumor vasculogenesis.Currently,numerous therapeutic approaches have been
designed in the aim to control tumor angiogenic cascade by targeting the above-mentioned
angiogenic mediators [40, 42]. In this context, more than a few angiogenesis inhibitors have
already been approved for the treatment of cancer, while several compounds are
in the late stage of clinical trials. The main category of the antiangiogenic
compounds exerts its   action indirectly either by neutralization of
tumor-derived angiogenic factors or preventing the receptors/signaling pathways
of these growth factors. In this regard, VEGF isoforms and their tyrosine
kinase receptors VEGFRs, as well as epidermal growth factor (EGF) and its
receptor (EGFR) are currently explored in clinical trials as drug candidates
against cancer [43–45].With
respect to angiogenesis inhibitors, several angiostatic compounds, such as
endostatin, thrombospodin-1 (TSP-1), tumstatin, angiostatin, and 16-kDa
N-terminal fragment of human prolactin (16K hPRL) have been reported to directly
and selectively suppress endothelial cell migration inducing EC apoptosis and
cell cycle arrest within tumor neovascularization [46, 47]. It should be
mentioned that most of these angiostatic compounds are also naturally occurring
molecules that compensate with angiogenic factors in order to control
angiogenic cascade in normal physiologic conditions. In addition, targeting MMPs
by such agents has been reported, underlining the importance of ECM remodeling
during angiogenesis process. Activation of NF-κB may also be a possible mechanism of such
angiostatic agents to induce EC apoptosis and to improve immune response within
angiogenesis process [46, 47].


## 3. Inhibition of Angiogenesis by PPAR-γ Ligands

PPAR-γ ligands can regulate
tumor angiogenesis via direct effects on ECs proliferation and migration and/or
through indirect mode of action by affecting the counterbalance between
angiogenic and antiangiogenic mediators (Figure 1, Table 1).Table 1
Effects of PPAR-γ ligands on tumor angiogenesis.
PPAR-γ ligandsType
of cells/organismsEffectsRef.RGZIn vitroGlioblastoma U87VEGF↓[57]Lewis lung carcinomaVEGF↓[57]Pancreatic tumor AsPC-1 cellstPA↓[58]Human breast cancer cell line MDA-MB-TIMP-1↑[59]231gelatinases↓Transformed human endometrial cellsVEGF↓[60](transiently transfected Ishikawa
cells)Human anaplastic thyroid carcinoma cellsinvasive potential↓[61]MSA, IAA, ROA, K119, KOA-2In vivoChick chorioallantoic membraneChoroidal[57]neovascularization↓C57/BL6 xenografted with 253J B-vNeovascularization↓[62]bladder tumor cellsEC apoptosis↑TGZIn vitroHuman non small cell lungELR + CXC[63]cancer cells A459chemokines↓In vivoC57/BL6 xenografted with 253J B-vNeovascularization↓[62]bladder tumor cellsEC apoptosis↑CGZIn vitroHuman non-small-cell lung carcinomaPGE2, COX-2↓[64]A427 and A549 cellHuman ovarian cancer cells OVCAR-2,VEGF, PGE2↓[65]DISSIn vivoBALB/c nu/nu mice xenografted withVEGF, PGE2↓[65]OVCAR-2 or DISSPGZIn vitroRenal cell carcinoma cells SMKT-R-1, -2,VEGF, bFGF↓[66]-3, -4Human non small cell lungELR + CXC[63]cancer cells A459chemokines↓Human anaplastic thyroid carcinoma cellsinvasive potential↓[61]MSA, IAA, ROA, K119 and KOA-215d-PGJ2In vitroRenal cell carcinoma SMKT-R-1, -2, -3, -4VEGF, bFGF↓[66]Human gastric cancerAng-1↓[67]cells MKN45Human PC-3 cellsVEGF↑[68]Human 5637 urinary bladder cellsVEGF↑[68]Human breast MCF-7 cellsVEGF↑[69]Human anaplastic thyroid carcinoma cellsinvasive potential↓[61]MSA, IAA, ROA, K119, KOA-2Human pancreatic cancer cells BxPC-3MMP-2, -9↓[70]Transformed human endometrial cellsVEGF↓[60](transiently transfected Ishikawa
cells)In vivoC57/BL6 xenografted with 253J B-vNeovascularization↓[62]bladder tumor cellsEC apoptosis↑RS5444In vitroHuman anaplastic thyroid carcinoma cellsCD31↓[71]DRO90-1, ARO81In vivoNude mice xenografted with DRO90-1 orCD31↓[71]ARO81 tumor cellsRS1303In vitroHuman anaplastic thyroid carcinoma cellsInvasive potential↓[61]MSA, IAA, ROA, K119, KOA-2NimesulideIn vitroHuman pancreatic cancer cells BxPC-3VEGF↑[72]and MIA PaCa-2Figure 1
The network of the components implicated in the angiogenesis process in cancer and the impact of PPAR-γ ligands illustrated by blue color.


### 3.1. Direct Effects on Endothelium

PPAR-γ has been reported to be expressed in endothelial cells and PPAR-γ ligands are well established to exert direct effects on them [48, 49]. PPAR-γ activation by either naturally occurring or
synthetic ligands resulted in potent inhibition of growth factor-induced differentiation
and proliferation in human
umbilical vein endothelial cells (HUVECs) and choroidal
endothelial cells (CECs) [48, 49]. In this regard, PPAR-γ dependent mode of action has been shown to stimulate caspase-mediated
ECs apoptosis [50]. Importantly,
RGZ levels able to inhibit ECs proliferation are readily achieved in patients
undergoing standard antidiabetic RGZ treatment [51]. Moreover, both RGZ and PGZ, at relative pharmacological
concentrations, resulted in a strong prevention of VEGF-induced tube formation
and ECs migration [52, 53]. Mechanistically, it has been supported that angiogenesis inhibition by RGZ in HUVECs
involves a proapoptotic mechanism which includes the implication of the PPAR-γ-mediated NO production and the maxi-K channel activation [54]. Maxi-K
channels, essential mediators of vascular remodeling and angiogenesis, are
synergically regulated by various intracellular second messengers including NO
[54]. Hence, a possible proapoptotic mechanism for the PPAR-γ-mediated NO production has been
suggested [55]. Recently, pigment epithelium-derived factor
(PEDF), a potent antiangiogenic glycoprotein, has been shown to stimulate
HUVECs apoptosis through sequential induction in the expression and
transcriptional activity of PPAR-γ. PEDF upregulated
p53 expression via PPAR-γ, supporting evidence that p53 may be a major
target in PPAR-γ mediated ECs apoptosis [56].PPAR-γ has
also been reported to be expressed in tumor ECs, presenting a relative
overexpression in tumor-induced endothelial sprouts compared to normal endothelium.
In this case, endothelial and tumoral cells have been shown to display
inhibition even at low TZDs doses [57]. Importantly, TZDs inhibited tumor cell
invasion across blood vessel endothelium. In fact, RGZ at concentrations close
to the range of its binding affinity for PPAR-γ
[8] exerted inhibitory effects on tumor angiogenesis in malignant cell lines
and in immunodeficient mice with transplanted tumors [57]. In this regard, it
should be mentioned that RGZ concentrations of 5 μM and higher led to the phosphorylation of
eIF-2α
in HUVECs, supporting
evidence that the inhibition of ECs proliferation could also be mediated
through a PPAR-γ
independent pathway. However, at even lower concentration range (0.1–1 μM), at which PPAR-γ is activated, RGZ was capable of exerting even stronger
antiproliferative effects on ECs in vitro [57]. In this
context, the concentration range of PPAR-γ ligands should be taken into
careful consideration, because over a concentration limit, which may be varied
amongst the different types of cells, in vitro, as well as amongst different
species, in vivo, receptor-independent actions could be elicited. Such PPAR-γ mode of action has recently been reviewed by
Feinstein et al., who suggested an alternative mitochondrial target for TZDs,
termed as mitoNEET  [12]. To this point, it should be noted that higher doses of RGZ were less
effective in inhibiting angiogenesis and hence lung metastasis than lower doses
that are actually comparable to the serum levels of RGZ in diabetic patients [27, 51]. Overall, although PPAR-γ ligands can also induce EC apoptosis as mentioned in the previous
paragraph, it is unlikely that they do this  under physiological conditions as
this may result in a severe thrombosis. Thus, it should be emphasized the fact PPAR-γ ligands may target better EC proliferation as shown by Panigrahy et
al. [27] and Freed
et al. [51].Orthotopic
implantation of H2122 nonsmall cell lung adenocarcinoma cells overexpressing
PPAR-γ into the lungs of nude mice attenuated tumor
growth and metastasis by selective inhibition of invasive metastasis, and
activation of pathways that promote a more differentiated epithelial phenotype
[73]. This evidence deserves special attention since both
angiogenesis and invasion are crucial for the formation of metastasis and the recurrence
of tumors. Moreover, reintroduction of exogenous TSP1 or its peptide
derivative ABT510 can reverse the angiogenic switch, and thus blocking tumor
expansion. TSP-1 is a well-known potent angiogenesis inhibitor that targets ECs
for apoptosis through signaling cascade at its receptor CD36. In tumor
xenografts, TGZ, RGZ, and 15d-PGJ2 coupled to ABT510 suppressed angiogenesis
and induced ECs apoptosis in a CD36 dependent manner [62]. In this context, 15d-PGJ2
treatment upregulated CD36 surface expression in human monocytic cell line THP-1
by enhancement of CD36 gene transcription [74]. Thus, PPAR-γ
could be considered as a critical regulator of CD36 expression, as both natural
and synthetic PPAR-γ ligands are capable of increasing CD36
expression [75].Receptor-mediated effects for PPAR-γ ligands
in inhibiting angiogenesis through direct mode of action on endothelium seem to
be dominated [28, 57]. In this regard, PPAR-γ knockout mice embryos died on day 10 of life because of interference
with the terminal differentiation pattern of trophoblasts, as well as the loss
of vascular development in the placenta [76, 77]. It has also been suggested
that PPAR-binding protein (PBP), a coactivator of PPAR-γ, may
function as a negative modulator of ECs proliferation [77]. Such genetic data
provides additional evidence that PPAR-γ functions as modulator of angiogenesis; however, receptor-independent action should
not be excluded. In this aspect, Artwohl et al. showed PPAR-γ-independent
antiproliferative effects on HUVECs associated with lactate release, possibly
due to inhibition of mitochondrial function [78].


### 3.2. Indirect Effects on the Net Balance between Angiogenic and Antiangiogenic Factors

Beyond the direct mode of action on
the endothelium, PPAR-γ ligands have been reported to downregulate
angiogenesis process via indirect mechanisms by modulating the levels of the endogenous angiogenesis mediators (Figure 1, Table 1). In this context, VEGF/VEGFR signaling pathway seems to be a key target for PPAR-γ
ligands in inhibiting angiogenesis. Xin et al. provided the first evidence that 15d-PGJ2 reduced VEGFRs
m-RNA levels in HUVECs [48]. It
has also been supported that PPAR-γ ligands may have bifunctional properties in
KDR gene expression that involve the enhancement of Sp1-DNA binding in absence
of ligand by PPAR-γ itself and the suppression of Sp1-DNA-binding in presence
of PPAR-γ ligands [79]. Moreover, PPAR-γ activation has been shown
to downregulate leptin and tumor necrosis factor (TNF-α), two well-known angiogenesis-inducing factors
[80, 81]. In fact, PPAR-γ activation by TZDs attenuated leptin gene
expression both in vivo and in vitro [82, 83] and blocked
leptin-induced ECs migration through inhibition of Akt and eNOS signaling [84].
This evidence suggests that endothelial phosphatase and tensin homologue
mutated on chromosome ten (PTEN), a negative regulator of PI3K → Akt signaling,
may play a crucial role in the ECs antimigratory actions of TZDs [84].Tumor-associated angiogenesis has
been reported to be indirectly suppressed by blocking the expression of
angiogenic stimulators in response to PPAR-γ
ligand activation. In this regard, PPAR-γ
activation by TGZ or PGZ diminished the production of the angiogenic ELR + CXC
chemokines IL-8 (CXCL8), ENA-78 (CXCL5), and Gro-α (CXCL1) in human non-small-cell lung cancer
cell line A459 [63]. This effect was ascribed to the negative modulation of NF-κB activation [63]. In
addition, CGZ was found to decrease PGE2 production through
downregulation of cyclooxygenase-2 (COX-2) expression in human non-small-cell
lung carcinoma A427 and A549 cell lines [64]. Interestingly, utilization of a
dominant negative PPAR-γ construct
revealed that the effect of CGZ on both COX-2 and PGE2 was mediated through
PPAR-γ
independent pathways [64]. Another study demonstrated that 15d-PGJ2 attenuated
the expression of Ang-1 and hence the angiogenic process through the
angiopoietin-Tie2 system in the gastric cancer cell line MKN45 [67]. Ang-1 is
involved in the regulation of maturation and stabilization of the vascular wall,
and thus it might be a potential target for inhibiting tumor angiogenesis. Moreover,
in a model of human anaplastic thyroid carcinoma, RS5444, a novel high-affinity
PPAR-γ agonist exerted
potent antiangiogenic action, in vivo, by decreasing CD31, a specific molecular
marker of blood vessels [71]. In this regard, PPAR-γ ligand treatment (TZDs,
15d-PGJ2, and RS1303) dose-dependently suppressed cell proliferation by
inducing apoptosis instead of differentiation in five human anaplastic
carcinoma cell lines (MSA, IAA, ROA, K119, and KOA-2) [61]. Recently, CGZ has also
been shown to produce antitumor effects against ovarian cancer, in vitro and in
vivo, in conjunction with reduced angiogenesis and induction of apoptosis [65].
In this case, CGZ induced antitumor effects were comparable to that of
cisplatin and were ascribed to inhibition of VEGF production in relation to PGE2 reduction, an endogenous stimulator of angiogenesis and invasiveness [65]. PPAR-γ ligands have also been shown to repress VEGF
gene expression via a PPAR-γ-responsive
element (PPRE) in the VEGF gene promoter in both primary and transformed human
endometrial cell cultures [60]. This study provided substantial evidence that
PPAR-γ ligands may be exploited pharmacologically to
inhibit pathological vascularization in complications of pregnancy,
endometriosis, and endometrial adenocarcinoma [60].As mentioned in Section3.1, RGZ
suppressed tumor angiogenesis by direct mode of action in endothelium; however,
indirect antiangiogenic effects have also been reported [57]. More to the
point, RGZ, at low doses, in vitro,
inhibited bovine capillary ECs and reduced VEGF production by tumor cells [57].
RGZ also suppressed angiogenesis in the chick chorioallantoic membrane, in the
avascular cornea, in vivo, as
well as in a variety of primary tumors, such as glioblastoma U87 and Lewis lung
carcinoma cells, in vitro [57].
Likewise, both PGZ and 15d-PGJ2 have been shown to inhibit, dose- and
time-dependently, VEGF and bFGF secretion in human renal cell carcinoma cells [66].
Importantly, antiangiogenic effects were observed at the dose of 5 μM PGZ, a level that is
also obtained in diabetic patients after standard PGZ treatment [66]. On the
other hand, there is nonavailable data so far concerning the effect of PPAR-γ ligand treatment on the expression
and/or secretion of antiangiogenic mediators. In this regard, future studies focused
on the impact of PPAR-γ ligands in mediators, such as endostatin,
TSP-1, tumstatin, angiostatin, and 16K hPRL are strongly recommended.Angiogenesis constitutes a crucial
step for tumor invasion and formation of metastasis. In this aspect, PPAR-γ ligand treatment attenuated
the invasiveness of pancreatic tumor cells, reducing MMP-2 and -9 protein
levels and activity [70]. Moreover, the secretion of the invasive factor tissue
plasminogen activator (tPA) was decreased by RGZ treatment in pancreatic tumor
AsPC-1 cells through receptor mediated mechanisms [58]. Treatment of the highly
aggressive human breast cancer cell line MDA-MB-231 with synthetic and natural
PPAR-γ
ligands, at
noncytotoxic concentrations, also resulted in a
significant inhibition of the invasive capacity [59]. In fact, TIMP-1 was
upregulated by PPAR-γ ligand
treatment, while the gelatinolytic activities of gelatinases in the conditioned
media were decreased [59]. Moreover, PPAR-γ
ligands downregulated the invasive potential of anaplastic thyroid carcinoma
cells, and this effect was prominent in 3 cell lines, which exhibited higher
expression level of the PPAR-γ gene or protein [61].Clinical evidence from a pilot study enrolled 6 patients
with angiosarcoma and hemangioendothelioma, revealed that the angiostatic
triple combination of PGZ, rofecoxib, and metronomic trofosfamide exhibited
high efficacy in the palliative care of patients [85]. Until this study, antiangiogenic
drugs such PGZ and rofecoxib had not been considered for the treatment of human
angiosarcoma. In support of this view, a case report study has demonstrated
that this novel antiangiogenic therapy was effective in a patient with endemic Kaposi
sarcoma and led to partial remission that was stable for 18 months without
significant side effects [86]. Hence, targeting PPAR-γ may prove to be a potential therapeutic strategy in combined treatments
with conventional chemotherapy for patients with vascular disorders [87].


## 3.1. Direct Effects on Endothelium

PPAR-γ has been reported to be expressed in endothelial cells and PPAR-γ ligands are well established to exert direct effects on them [48, 49]. PPAR-γ activation by either naturally occurring or
synthetic ligands resulted in potent inhibition of growth factor-induced differentiation
and proliferation in human
umbilical vein endothelial cells (HUVECs) and choroidal
endothelial cells (CECs) [48, 49]. In this regard, PPAR-γ dependent mode of action has been shown to stimulate caspase-mediated
ECs apoptosis [50]. Importantly,
RGZ levels able to inhibit ECs proliferation are readily achieved in patients
undergoing standard antidiabetic RGZ treatment [51]. Moreover, both RGZ and PGZ, at relative pharmacological
concentrations, resulted in a strong prevention of VEGF-induced tube formation
and ECs migration [52, 53]. Mechanistically, it has been supported that angiogenesis inhibition by RGZ in HUVECs
involves a proapoptotic mechanism which includes the implication of the PPAR-γ-mediated NO production and the maxi-K channel activation [54]. Maxi-K
channels, essential mediators of vascular remodeling and angiogenesis, are
synergically regulated by various intracellular second messengers including NO
[54]. Hence, a possible proapoptotic mechanism for the PPAR-γ-mediated NO production has been
suggested [55]. Recently, pigment epithelium-derived factor
(PEDF), a potent antiangiogenic glycoprotein, has been shown to stimulate
HUVECs apoptosis through sequential induction in the expression and
transcriptional activity of PPAR-γ. PEDF upregulated
p53 expression via PPAR-γ, supporting evidence that p53 may be a major
target in PPAR-γ mediated ECs apoptosis [56].PPAR-γ has
also been reported to be expressed in tumor ECs, presenting a relative
overexpression in tumor-induced endothelial sprouts compared to normal endothelium.
In this case, endothelial and tumoral cells have been shown to display
inhibition even at low TZDs doses [57]. Importantly, TZDs inhibited tumor cell
invasion across blood vessel endothelium. In fact, RGZ at concentrations close
to the range of its binding affinity for PPAR-γ
[8] exerted inhibitory effects on tumor angiogenesis in malignant cell lines
and in immunodeficient mice with transplanted tumors [57]. In this regard, it
should be mentioned that RGZ concentrations of 5 μM and higher led to the phosphorylation of
eIF-2α
in HUVECs, supporting
evidence that the inhibition of ECs proliferation could also be mediated
through a PPAR-γ
independent pathway. However, at even lower concentration range (0.1–1 μM), at which PPAR-γ is activated, RGZ was capable of exerting even stronger
antiproliferative effects on ECs in vitro [57]. In this
context, the concentration range of PPAR-γ ligands should be taken into
careful consideration, because over a concentration limit, which may be varied
amongst the different types of cells, in vitro, as well as amongst different
species, in vivo, receptor-independent actions could be elicited. Such PPAR-γ mode of action has recently been reviewed by
Feinstein et al., who suggested an alternative mitochondrial target for TZDs,
termed as mitoNEET  [12]. To this point, it should be noted that higher doses of RGZ were less
effective in inhibiting angiogenesis and hence lung metastasis than lower doses
that are actually comparable to the serum levels of RGZ in diabetic patients [27, 51]. Overall, although PPAR-γ ligands can also induce EC apoptosis as mentioned in the previous
paragraph, it is unlikely that they do this  under physiological conditions as
this may result in a severe thrombosis. Thus, it should be emphasized the fact PPAR-γ ligands may target better EC proliferation as shown by Panigrahy et
al. [27] and Freed
et al. [51].Orthotopic
implantation of H2122 nonsmall cell lung adenocarcinoma cells overexpressing
PPAR-γ into the lungs of nude mice attenuated tumor
growth and metastasis by selective inhibition of invasive metastasis, and
activation of pathways that promote a more differentiated epithelial phenotype
[73]. This evidence deserves special attention since both
angiogenesis and invasion are crucial for the formation of metastasis and the recurrence
of tumors. Moreover, reintroduction of exogenous TSP1 or its peptide
derivative ABT510 can reverse the angiogenic switch, and thus blocking tumor
expansion. TSP-1 is a well-known potent angiogenesis inhibitor that targets ECs
for apoptosis through signaling cascade at its receptor CD36. In tumor
xenografts, TGZ, RGZ, and 15d-PGJ2 coupled to ABT510 suppressed angiogenesis
and induced ECs apoptosis in a CD36 dependent manner [62]. In this context, 15d-PGJ2
treatment upregulated CD36 surface expression in human monocytic cell line THP-1
by enhancement of CD36 gene transcription [74]. Thus, PPAR-γ
could be considered as a critical regulator of CD36 expression, as both natural
and synthetic PPAR-γ ligands are capable of increasing CD36
expression [75].Receptor-mediated effects for PPAR-γ ligands
in inhibiting angiogenesis through direct mode of action on endothelium seem to
be dominated [28, 57]. In this regard, PPAR-γ knockout mice embryos died on day 10 of life because of interference
with the terminal differentiation pattern of trophoblasts, as well as the loss
of vascular development in the placenta [76, 77]. It has also been suggested
that PPAR-binding protein (PBP), a coactivator of PPAR-γ, may
function as a negative modulator of ECs proliferation [77]. Such genetic data
provides additional evidence that PPAR-γ functions as modulator of angiogenesis; however, receptor-independent action should
not be excluded. In this aspect, Artwohl et al. showed PPAR-γ-independent
antiproliferative effects on HUVECs associated with lactate release, possibly
due to inhibition of mitochondrial function [78].


## 3.2. Indirect Effects on the Net Balance between Angiogenic and Antiangiogenic Factors

Beyond the direct mode of action on
the endothelium, PPAR-γ ligands have been reported to downregulate
angiogenesis process via indirect mechanisms by modulating the levels of the endogenous angiogenesis mediators (Figure 1, Table 1). In this context, VEGF/VEGFR signaling pathway seems to be a key target for PPAR-γ
ligands in inhibiting angiogenesis. Xin et al. provided the first evidence that 15d-PGJ2 reduced VEGFRs
m-RNA levels in HUVECs [48]. It
has also been supported that PPAR-γ ligands may have bifunctional properties in
KDR gene expression that involve the enhancement of Sp1-DNA binding in absence
of ligand by PPAR-γ itself and the suppression of Sp1-DNA-binding in presence
of PPAR-γ ligands [79]. Moreover, PPAR-γ activation has been shown
to downregulate leptin and tumor necrosis factor (TNF-α), two well-known angiogenesis-inducing factors
[80, 81]. In fact, PPAR-γ activation by TZDs attenuated leptin gene
expression both in vivo and in vitro [82, 83] and blocked
leptin-induced ECs migration through inhibition of Akt and eNOS signaling [84].
This evidence suggests that endothelial phosphatase and tensin homologue
mutated on chromosome ten (PTEN), a negative regulator of PI3K → Akt signaling,
may play a crucial role in the ECs antimigratory actions of TZDs [84].Tumor-associated angiogenesis has
been reported to be indirectly suppressed by blocking the expression of
angiogenic stimulators in response to PPAR-γ
ligand activation. In this regard, PPAR-γ
activation by TGZ or PGZ diminished the production of the angiogenic ELR + CXC
chemokines IL-8 (CXCL8), ENA-78 (CXCL5), and Gro-α (CXCL1) in human non-small-cell lung cancer
cell line A459 [63]. This effect was ascribed to the negative modulation of NF-κB activation [63]. In
addition, CGZ was found to decrease PGE2 production through
downregulation of cyclooxygenase-2 (COX-2) expression in human non-small-cell
lung carcinoma A427 and A549 cell lines [64]. Interestingly, utilization of a
dominant negative PPAR-γ construct
revealed that the effect of CGZ on both COX-2 and PGE2 was mediated through
PPAR-γ
independent pathways [64]. Another study demonstrated that 15d-PGJ2 attenuated
the expression of Ang-1 and hence the angiogenic process through the
angiopoietin-Tie2 system in the gastric cancer cell line MKN45 [67]. Ang-1 is
involved in the regulation of maturation and stabilization of the vascular wall,
and thus it might be a potential target for inhibiting tumor angiogenesis. Moreover,
in a model of human anaplastic thyroid carcinoma, RS5444, a novel high-affinity
PPAR-γ agonist exerted
potent antiangiogenic action, in vivo, by decreasing CD31, a specific molecular
marker of blood vessels [71]. In this regard, PPAR-γ ligand treatment (TZDs,
15d-PGJ2, and RS1303) dose-dependently suppressed cell proliferation by
inducing apoptosis instead of differentiation in five human anaplastic
carcinoma cell lines (MSA, IAA, ROA, K119, and KOA-2) [61]. Recently, CGZ has also
been shown to produce antitumor effects against ovarian cancer, in vitro and in
vivo, in conjunction with reduced angiogenesis and induction of apoptosis [65].
In this case, CGZ induced antitumor effects were comparable to that of
cisplatin and were ascribed to inhibition of VEGF production in relation to PGE2 reduction, an endogenous stimulator of angiogenesis and invasiveness [65]. PPAR-γ ligands have also been shown to repress VEGF
gene expression via a PPAR-γ-responsive
element (PPRE) in the VEGF gene promoter in both primary and transformed human
endometrial cell cultures [60]. This study provided substantial evidence that
PPAR-γ ligands may be exploited pharmacologically to
inhibit pathological vascularization in complications of pregnancy,
endometriosis, and endometrial adenocarcinoma [60].As mentioned in Section3.1, RGZ
suppressed tumor angiogenesis by direct mode of action in endothelium; however,
indirect antiangiogenic effects have also been reported [57]. More to the
point, RGZ, at low doses, in vitro,
inhibited bovine capillary ECs and reduced VEGF production by tumor cells [57].
RGZ also suppressed angiogenesis in the chick chorioallantoic membrane, in the
avascular cornea, in vivo, as
well as in a variety of primary tumors, such as glioblastoma U87 and Lewis lung
carcinoma cells, in vitro [57].
Likewise, both PGZ and 15d-PGJ2 have been shown to inhibit, dose- and
time-dependently, VEGF and bFGF secretion in human renal cell carcinoma cells [66].
Importantly, antiangiogenic effects were observed at the dose of 5 μM PGZ, a level that is
also obtained in diabetic patients after standard PGZ treatment [66]. On the
other hand, there is nonavailable data so far concerning the effect of PPAR-γ ligand treatment on the expression
and/or secretion of antiangiogenic mediators. In this regard, future studies focused
on the impact of PPAR-γ ligands in mediators, such as endostatin,
TSP-1, tumstatin, angiostatin, and 16K hPRL are strongly recommended.Angiogenesis constitutes a crucial
step for tumor invasion and formation of metastasis. In this aspect, PPAR-γ ligand treatment attenuated
the invasiveness of pancreatic tumor cells, reducing MMP-2 and -9 protein
levels and activity [70]. Moreover, the secretion of the invasive factor tissue
plasminogen activator (tPA) was decreased by RGZ treatment in pancreatic tumor
AsPC-1 cells through receptor mediated mechanisms [58]. Treatment of the highly
aggressive human breast cancer cell line MDA-MB-231 with synthetic and natural
PPAR-γ
ligands, at
noncytotoxic concentrations, also resulted in a
significant inhibition of the invasive capacity [59]. In fact, TIMP-1 was
upregulated by PPAR-γ ligand
treatment, while the gelatinolytic activities of gelatinases in the conditioned
media were decreased [59]. Moreover, PPAR-γ
ligands downregulated the invasive potential of anaplastic thyroid carcinoma
cells, and this effect was prominent in 3 cell lines, which exhibited higher
expression level of the PPAR-γ gene or protein [61].Clinical evidence from a pilot study enrolled 6 patients
with angiosarcoma and hemangioendothelioma, revealed that the angiostatic
triple combination of PGZ, rofecoxib, and metronomic trofosfamide exhibited
high efficacy in the palliative care of patients [85]. Until this study, antiangiogenic
drugs such PGZ and rofecoxib had not been considered for the treatment of human
angiosarcoma. In support of this view, a case report study has demonstrated
that this novel antiangiogenic therapy was effective in a patient with endemic Kaposi
sarcoma and led to partial remission that was stable for 18 months without
significant side effects [86]. Hence, targeting PPAR-γ may prove to be a potential therapeutic strategy in combined treatments
with conventional chemotherapy for patients with vascular disorders [87].


## 4. Induction of Angiogenesis by PPAR-γ Ligands

The most comprehensive data so far render
PPAR-γ ligands as potent inhibitors of angiogenesis;
however, there are several lines of evidence to support that PPAR-γ ligand activation can also trigger
angiogenic cascade (Table 1). In fact, increased VEGF mRNA levels and induction of angiogenesis in response to
PPAR-γ ligands treatment have been reported both in vitro and in vivo [88–91]. Interestingly, TZDs have been considered as potential
pharmacological agents for angiogenesis induction in the treatment of ischemic
artery disease [89]. Recent clinical evidence has also demonstrated
that RGZ treatment improved endothelial progenitor cell (EPC) number and
migratory activity in diabetic patients [92, 93].
In addition, PGZ treatment was found to improve endothelial function by
increasing the number and the migratory capacity of EPCs in animal and human studies
[94, 95]. Another study has revealed that eNOS upregulation induced by RGZ may
be the dominant mechanism through which RGZ enhanced angiogenesis [91].
However, Gensh et al. did not observe upregulation of vascular eNOS mRNA
expression or setback of the PGZ-induced increase of EPCs in the presence of
1-NAME, a NOS inhibitor [94]. These authors suggested that TZDs may regulate
EPCs by a mechanism independent of eNOS [94]; however, further studies based on
pharmacologic blocking or knockout modeling of eNOS are strongly recommended in
order for precise conclusion to be drawn. Importantly, taking into account the
discrepancy in literature, Gensh et al. assumed that TZDs may play a
double-edged role in angiogenesis signaling by promoting the number and
migration of EPCs at lower tissue concentrations obtained by systematic
treatment, whereas the antiangiogenic effects are elicited at higher local
concentrations [94]. This major remark has also been reported in the case of
breast cancer cells where low concentration of PPAR-γ ligands increase cell proliferation in
contrast to the higher concentrations that suppress cell growth [96]. The urgent demand to define and monitor the dosage
of PPAR-γ ligands in clinical trials for cancer therapy
is thoroughly discussed by Panigrahy et al. [27]. In this aspect, special
attention deserves the fact that atorvastatin, a 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase inhibitor, and PGZ increased myocardial 15d-PGJ2 levels in the rat myocardium and HUVECs [97]. 15d-PGJ2 was produced mainly via COX-2 and activated PPAR-γ. Interestingly, it was
supported that PPAR-γ activation was exclusively mediated by 15d-PGJ2 in the case of atorvastatin, whereas PGZ activated directly PPAR-γ or indirectly via 15d-PGJ2 [97]. Thus, these recent findings raise the question
whether the final effect of PPAR-γ ligands is completely ascribed to the dose of
PPAR-γ ligand
treatment or in addition to the induction of endogenous PPAR-γ activators, such as 15d-PGJ2. It should also be taken into account the fact
that endogenous nitrated fatty acids that comprise a class of nitric
oxide-derived, PPAR-γ dependent and cell signaling mediators can modulate systematic
inflammatory responses within physiological concentration ranges [98].There
is also substantial evidence, which suggests that PPAR-γ ligands stimulate tumor angiogenesis. In this
context, 15d-PGJ2 treatment was
found to dose-dependently increase the VEGF mRNA expression in both human
androgen-independent PC-3 prostate and 5637 urinary bladder carcinoma cells [68].
In addition, 15d-PGJ2 resulted in upregulation of VEGF expression through the
induction of heme oxygenase (OH)-1 ERK1/2 phosphorylation in human breast
cancer MCF-7 cells, thus contributing to increased angiogenesis in this type of
tumor cells [69]. Nimesulide, a selective COX-2 inhibitor, although at
relatively high concentrations, enhanced VEGF secretion from pancreatic cancer
cells in vitro, as well as from
both COX-2-positive and COX-2-negative pancreatic tumors through PPAR-γ activation [72]. Importantly, in the case of COX-2-negative pancreatic tumors,
nimesulide-stimulated VEGF production was considerably associated with enhanced
angiogenesis and tumor growth [72]. Besides this, VEGF was differentially
increased, according to the differentiation state of the cells, by the three
PPAR isotypes, -α, -β/δ, and -γ, in two different human
urinary bladder cancer cell lines, RT4 and T24, derived from grade-I and
grade-III tumors, respectively [99]. The PPAR ligand-induced VEGF expression
seemed to be PPAR-specific and involved an indirect mechanism requiring an
intermediary regulatory protein through the MAP (ERK1/2) kinase pathway,
probably by a modulation of the phosphorylation state of PPARs [99]. Immunohistochemical
analysis in human bladder tumor specimens also revealed statistically
significant associations between PPAR-γ and several angiogenic factors, such as VEGF,
bFGF, platelet-derived endothelial cell growth factor (PDECGF), and EGFR in
respect to the incidence of tumor recurrence or progression [100]. On the other
hand, no statistically significant differences were observed between PPAR-γ immunoreactivity and angiogenesis parameters in skin cancer, whereas
the microvessel density was significantly higher in actin keratosis and
squamous cell carcinoma that expressed PPAR-β/δ [101]. These clinical data on PPAR-γ-induced signaling implicated in the expression of crucial angiogenic
factors in human neoplasia may unfold the development of new therapeutic
approaches in those types of cancer in which excessive angiogenesis represents
a negative prognostic factor.


## 5. The Impact of PPAR-γ Ligands in Hypoxia-Associated Signaling Pathways

As hypoxia is a key regulator of the
angiogenic switch, hypoxia-induced angiogenesis is gaining gradually increasing
interest as a potential target for cancer therapy. In human bladder tumors and
cell lines, several components of the hypoxia response pathway, including HIF-1α and HIF-2α have been considered as
important cofactors of the regulation of VEGF [102]. Recent findings have
revealed that PPAR-γ can modulate arterial remodeling associated with hypoxic hypertension [103].
In fact, RGZ was found to attenuate and reverse pulmonary arterial remodeling
and neomuscularization in rats subjected to chronic hypoxia [104]. Decreased
pulmonary arterial (PA) remodeling in RGZ-treated animals was associated with
decreased smooth muscle cell proliferation, decreased collagen and elastin
deposition, and increased matrix MMP-2 activity in the PA wall [104]. In this
aspect, PPAR-γ
mRNA levels were found significantly lower in human adhesion fibroblasts
compared to normal ones in response to hypoxia [105]. Moreover, hypoxia has
demonstrated to reduce the mRNA levels of PPAR-γ protein in human proximal renal tubular
epithelial cells (HPTECs). However, knockout of HIF-1α with its dominant negative form did not block
the hypoxia-induced reduction in PPAR-γ expression [106]. In this regard, substantial
evidence has revealed that 15d-PGJ2 can modulate the activities of several
transcriptional factors, such as NF-κB and AP-1, including also HIF-1 [107]. The
regulation of the aforementioned redox-sensitive transcription factors by
15d-PGJ2 was not necessarily mediated via PPAR-γ activation, but rather involves covalent
modification or oxidation of their critical cysteine residues acting as a redox
sensor [107]. Overall, targeting hypoxia-induced angiogenesis by PPAR-γ ligands may prove to be a promising therapy
for the treatment of cancer; however, the precise mechanisms involved in
hypoxia-induced angiogenesis process remain to be clarified.


## 6. Conclusion

At the present, there is quite a
lot of evidence to support that PPAR-γ may be considered as therapeutic
target for diverse disease states in which excessive angiogenesis is
implicated, including cancer. The most comprehensive data so far have revealed that PPAR-γ ligands are capable of inhibiting
angiogenesis implicated in tumor malignant transformation and expansion. Targeting
ECs proliferation and migration seems to be a dominant effect of PPAR-γ ligands on tumor angiogenesis.
Indirect mechanisms that involve the counterbalance between a multitude of endogenous
angiogenic and antiangiogenic factors further account for the inhibitory
effects of PPAR-γ ligands on
tumor angiogenesis. According
to these data, PPAR-γ ligands may unfold new perspectives in clinical use against primary
tumor growth and metastasis, since tumors that exhibit multidrug resistance are
effectively targeted by antiangiogenic chemotherapy. Such perspectives could be
clinically relevant, as PGZ and RGZ are orally administered FDA-approved drugs,
already been used by million patients undergoing standard antidiabetic
treatment.On the other hand, there are
several lines of evidence that PPAR-γ ligands can also enhance tumor angiogenesis progression under certain
conditions. This controversy could
be attributed to the pleiotropic action of PPAR-γ
ligands, possibly via cofactors, either coactivators or corepressors. Such
discrepancies may also be ascribed either to differences in time and dose of
PPAR-γ ligand
treatment, or to differences among the various organisms and types of cells
that have been studied. It should be taken into account that angiogenesis is a
multifaceted process that involves a wide range of mediators capable of
inducing or suppressing angiogenesis in addition to the degree of tissue
hypoxia. Consequently, the final outcome is difficult to be assessed accurately
and depends significantly on experimental models and/or treatment conditions. Moreover,
each type of cancer in humans presents individual and distinct vascular pattern
on the microenvironment in which it is located. Thus, it should be taken into careful
consideration the type of cancer being treated when deciding an appropriate
therapeutic strategy.In this aspect, the use of
different cancer models, in vitro
and in vivo, are strongly recommended
to further define the molecular interactions amongst PPAR-γ, angiogenic/antiangiogenic factors, and tumor progression markers
within the distinct cancer types. Future research effort should also be
orientated to the clinical evaluation of PPAR-γ expression in aggressive tumor
cancers in which various angiogenic/antiangiogenic factors exhibit high
prognostic value. Such studies could delineate the potential of PPAR-γ ligands in future anticancer therapeutic strategies, either alone or combined
with conventional chemotherapy.



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*Source: 431763-2008-04-23.xml*