# A Role for PPARβ/δ in Ocular Angiogenesis

Authors: David Bishop-Bailey
Journal: PPAR Research
 (2008-02-17)

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## Abstract

The uses of highly selective PPARβ/δ ligands and PPARβ/δ knockout mice have shown a direct ability of PPARβ/δ to regulate angiogenesis in vitro and in vivo in animal models. PPARβ/δ ligands induce the proangiogenic growth factor VEGF in many cells and tissues, though its actions in the eye are not known. However, virtually, all tissue components of the eye express PPARβ/δ. Both angiogenesis and in particular VEGF are not only critical for the development of the retina, but they are also a central component in many common pathologies of the eye, including diabetic retinopathy and age-related macular degeneration, the most common causes of blindness in the Western world. This review, therefore, will discuss the recent evidence of PPARβ/δ-mediated angiogenesis and VEGF release in the context of ocular disorders.


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## Body

## 1. Introduction

Peroxisome proliferator-activated
receptors (PPAR's) belong to the steroid receptor superfamily of
ligand-activated transcription factors [1]. Three PPAR's, PPARα, PPARβ/δ, and PPARγ, have been identified [2]. PPARα is predominantly expressed in liver,
heart, kidney, brown adipose tissue, and stomach mucosa; PPARγ is found primarily in adipose tissue; PPARβ/δ is the most
ubiquitously expressed [3, 4], though its roles in physiological and
pathophysiological processes are far from clear, particularly, in human tissue.
The recent development of PPARβ/δ knockout and transgenic mice has started to implicate
roles for PPARβ/δ in adipose tissue formation, metabolism,
wound healing, brain development, placental function, atherosclerosis,
colorectal carcinogenesis, and skeletal muscle function [5–7]. In this review,
the emerging role of PPARβ/δ in regulating endothelial function and
angiogenesis will be discussed with a particular emphasis to its relevance in
the eye.


## 2. PPARβ/δ Ligands

A number of synthetic PPARβ/δ compounds
have been described including GW0742X, GW2433, GW9578, L-783,483, GW501516,
L-796,449, L-165,461, and compound F [8, 9]. In addition, putative
endogenous PPARβ/δ activators include fatty acids [3, 10], triglycerides
[11], the cyclooxygenase (COX) product, prostacyclin [10], the COX/prostacyclin
synthase derived endocannabinoid metabolites [12], and all-trans retinoic acid (ATRA) [13]. ATRA is derived from vitamin A
(retinol) which is found at its highest levels in the eye and is essential for
its development and function [14]. Retinol is converted to retinaldehyde, a
component of rhodopsin [14] and a functional PPARγ antagonist [15, 16], which in turn is metabolised
to ATRA by retinal dehydrogenases [14]. ATRA has its own family of high-affinity
nuclear receptors, the retinoic acid receptor (RAR)α, -β, and -γ, which like the PPAR's act as heterodimers with RXRα, -β, and -γ, the receptors for the ATRA isomer 9-cis
retinoic acid [17]. Although ATRA can activate PPARβ/δ, it is not
known which, if any, of its actions are mediated by PPARβ/δ. However,
since ATRA is present in such large quantities in ocular tissue, it is
potentially an important site for its actions.


## 3. PPARβ/δ and Endothelial Cells

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. Angiogenesis is the process of new blood
vessel/capillary formation from existing vessels, and hypoxia is a major
signal which drives the process [18]. PPARα, PPARβ/δ, and
PPARγ are
all expressed in endothelial cells [19]. PPARα and PPARγ have well-characterised roles in
endothelial cells, both being in general anti-inflammatory, antiproliferative
[1], and antiangiogenic in a variety of in
vitro and in vivo models, including tumorigenesis [20] and laser-induced
retinal injury [21]. In contrast, the role of PPARβ/δ in
this important cell type has only recent starting to be elucidated. Initial
reports using prostacyclin as a ligand suggested that like PPARα and PPARγ, PPARβ/δ
promoted endothelial cell apoptosis [22]. In contrast, the use of highly
selective synthetic ligands has revealed a contradictory role for PPARβ/δ regulating
endothelial cell survival, proliferation, and angiogenesis.


### 3.1. PPARβ/δ and Endothelial Cell Pproliferation  and Survival

Long- [23] and short-term [24] culture of endothelial
cells with the selective ligand GW501516 induces endothelial cell
proliferation, an effect associated with the induction of the VEGF receptor (Flt-1;
VEGF R1) and VEGF production [23, 24]. In addition to inducing proliferation,
PPARβ/δ activation protects cells from oxidant-induced
apoptosis. Synthetic PPARβ/δ ligands or activation of the COX-prostacyclin
pathway, which signals through PPARβ/δ, induce
the endothelial expression of 14-3-3α protein [25]. 14-3-3 proteins are antiapoptotic and anti-inflammatory
molecules [26]. PPARβ/δ-induced 14-3-3α blocks oxidant- (H2O2-) induced
apoptosis by sequestering the proapoptotic protein Bad, stopping its
translocation to mitochondrial membranes, where it initiates cytochrome c
release and the subsequent activation of the proapoptotic caspase cascade [25].


### 3.2. PPARβ/δ and Angiogenesis

In addition to having effects on endothelial cell
proliferation, PPARβ/δ activation potently induces angiogenesis
of human vascular endothelial cells in tumour extracellular matrix in vitro and in a murine matrigel
plug model in vivo [24]. In addition,
the putative PPARβ/δ ligand prostacyclin analogues [27] and
ATRA [28] also induce angiogenesis, though the latter appears mostly dependent
on its RARα receptor rather than PPARβ/δ [29]. In human endothelial cells, a
major trigger for morphogensis induced by PPARβ/δ
stimulation was the stimulated release of VEGF [24]. In addition to VEGF, mRNA
for the matrix metalloproteinase (MMP)-9, a protease important for cell
migration was also elevated by PPARβ/δ activation [24]; however, whether this was secondary to VEGF release was not
tested. VEGF is expressed as four main splice variants (by amino acid size:
VEGF121, VEGF165, VEGF189, VEGF206)
[29]. VEGF (VEGF-A; VEGF165)
is a well-characterised central mediator of endothelial cell growth and
angiogenesis [29, 30]. Two endothelial VEGF tyrosine kinase receptors have been
identified: VEGFR-1/Flt-1, and VEGFR-2/KDR/Flk1. VEGF R2 appears to be the most
important receptor in VEGF-induced mitogenesis and permeability [29, 30]. In
addition, in two recent studies, the growth of PPARβ/δ wild-type
tumours or angiogenesis in matrigel plugs in PPARβ/δ knockout mice was
tested [31, 32]. The tumours in PPARβ/δ knockout mice compared to wild-type mice were
associated with a diminished blood flow and an immature hyperplastic microvascular
structures. Moreover, the retroviral introduction of PPARβ/δ into matrigel
plugs was able to rescue the knockout phenotype by triggering microvessel
maturation [31]. In the latter of these studies, PPARβ/δ was examined in
tumours from patients who had undergone “angiogenic switch” a proangiogenic state involved
in tumour progression [32]. PPARβ/δ correlated with advanced pathological tumor stage,
increased risk for tumor recurrence, and distant metastasis, and was, therefore,
suggested as a hub node transcription factor regulating tumour angiogenesis
[32].Genomic and proteomic analyses of the PPARβ/δ knockout
endothelial cells isolated from matrigel plugs have also led to the
identification of a number of additional candidate genes to mediate the actions
of PPARβ/δ in angiogenesis. In particular, the Cdkn1c gene which
encodes the cell cycle inhibitor p57Kip2 is a direct PPARβ/δ target gene that
mediates PPARβ/δ effects on cell morphogenesis [31]. In addition, CD36
and thrombospondin were also decreased in matrigel-invading endothelial cells from
PPARβ/δ knockout mice [31]. Thrombospondins by directly
interacting with CD36 inhibit angiogenesis in vivo [33, 34]. Similarly, a proteomic analysis by the same
group [35] on PPARβ/δ knockout endothelial cells has also revealed a
decreased expression of the chloride intracellular channel protein (CLIC)-4 in migrating
endothelial cells from PPARβ/δ knockout mice. In contrast, the expression of
cellular retinol binding protein CRBP1 is increased in migrating endothelial
cells from PPARβ/δ knockout mice [35]. CLIC-4 promotes and plays an essential
role during tubular morphogenesis [36], while CRBP1 inhibits cell survival
pathways by acting as an inhibitor of the AKT signalling pathway [37], an
additional important signalling signal for angiogenesis to occur [38].The combination of these
studies show PPARβ/δ activation induces endothelial cell mitogen and
differentiation signals, including VEGF, 14-3-3α, CD36 and thrombospondin, CLIC4,
CRBP-1, and p57KIP2, all of which may act in a coordinate manner
to bring about the functional morphogenic changes associated with angiogenesis.


### 3.3. PPARβ/δ and VEGF

Although the direct evidence
for a role of PPARβ/δ in angiogenesis is relatively new, there has been an
increasing literature regarding PPARβ/δ regulated tumour cell growth via inducing tumour
cells to release VEGF. PPARβ/δ ligands induce VEGF in bladder cancer cells [39], human
breast (T47D, MCF7), and prostate (LNCaP, PNT1A) cancer cell lines, along with
its receptor flt-1 [22], but not (HT29, colon; HCT116, colon; LS-174T, colon; HepG2,
hepatoma; and HuH7, hepatoma) cell lines [40].In a genetic model of intestinal
polyp development APC/min mouse, deletion of PPARβ/δ decreases intestinal
adenoma growth and inhibits tumour-promoting effects of the PPARβ/δ agonist GW501516
[41]. Moreover, activation of PPARβ/δ upregulated VEGF in colon carcinoma cells, promoting colon
tumour epithelial cell survival through activation of AKT signalling [41]. Angiogenesis
was not studied in this model, however, any substantial tumour growth requires
a blood supply and angiogenesis to allow it to develop. In contrast, in human colon
and liver cancer cell lines [40], PPARβ/δ ligands had no effect on human cancer cell growth,
AKT, VEGF or COX-2 expression in vitro
or on these makers in the liver, colon, and colon polyps in mice treated in vivo [40]. The roles of PPARβ/δ in VEGF-
mediated tumorigenesis
are, therefore, still in need of further clarification.


### 3.4. Expression of PPARβ/δ in the Eye

Angiogenesis regulates both the physiological development and many of the most common
pathophysiology’s of the eye. As yet, there is no direct evidence linking PPARβ/δ and
angiogenesis in the eye, however, PPARβ/δ is
clearly expressed at least in murine ocular tissue. PPARβ/δ is
expressed in the eye ciliary body epithelial cells, cornea epithelial cells, cornea
endothelium, cornea fibroblast, retina inner nuclear layer, and retina ganglion
cell layer [42]. Although one must be cautious interpreting data from nonocular
tissue to the eye [43], as discussed previously and following, pathways that
have direct relevance to ocular angiogenesis are clearly regulated by PPARβ/δ and
are therefore worthy of discussion.


## 3.1. PPARβ/δ and Endothelial Cell Pproliferation  and Survival

Long- [23] and short-term [24] culture of endothelial
cells with the selective ligand GW501516 induces endothelial cell
proliferation, an effect associated with the induction of the VEGF receptor (Flt-1;
VEGF R1) and VEGF production [23, 24]. In addition to inducing proliferation,
PPARβ/δ activation protects cells from oxidant-induced
apoptosis. Synthetic PPARβ/δ ligands or activation of the COX-prostacyclin
pathway, which signals through PPARβ/δ, induce
the endothelial expression of 14-3-3α protein [25]. 14-3-3 proteins are antiapoptotic and anti-inflammatory
molecules [26]. PPARβ/δ-induced 14-3-3α blocks oxidant- (H2O2-) induced
apoptosis by sequestering the proapoptotic protein Bad, stopping its
translocation to mitochondrial membranes, where it initiates cytochrome c
release and the subsequent activation of the proapoptotic caspase cascade [25].


## 3.2. PPARβ/δ and Angiogenesis

In addition to having effects on endothelial cell
proliferation, PPARβ/δ activation potently induces angiogenesis
of human vascular endothelial cells in tumour extracellular matrix in vitro and in a murine matrigel
plug model in vivo [24]. In addition,
the putative PPARβ/δ ligand prostacyclin analogues [27] and
ATRA [28] also induce angiogenesis, though the latter appears mostly dependent
on its RARα receptor rather than PPARβ/δ [29]. In human endothelial cells, a
major trigger for morphogensis induced by PPARβ/δ
stimulation was the stimulated release of VEGF [24]. In addition to VEGF, mRNA
for the matrix metalloproteinase (MMP)-9, a protease important for cell
migration was also elevated by PPARβ/δ activation [24]; however, whether this was secondary to VEGF release was not
tested. VEGF is expressed as four main splice variants (by amino acid size:
VEGF121, VEGF165, VEGF189, VEGF206)
[29]. VEGF (VEGF-A; VEGF165)
is a well-characterised central mediator of endothelial cell growth and
angiogenesis [29, 30]. Two endothelial VEGF tyrosine kinase receptors have been
identified: VEGFR-1/Flt-1, and VEGFR-2/KDR/Flk1. VEGF R2 appears to be the most
important receptor in VEGF-induced mitogenesis and permeability [29, 30]. In
addition, in two recent studies, the growth of PPARβ/δ wild-type
tumours or angiogenesis in matrigel plugs in PPARβ/δ knockout mice was
tested [31, 32]. The tumours in PPARβ/δ knockout mice compared to wild-type mice were
associated with a diminished blood flow and an immature hyperplastic microvascular
structures. Moreover, the retroviral introduction of PPARβ/δ into matrigel
plugs was able to rescue the knockout phenotype by triggering microvessel
maturation [31]. In the latter of these studies, PPARβ/δ was examined in
tumours from patients who had undergone “angiogenic switch” a proangiogenic state involved
in tumour progression [32]. PPARβ/δ correlated with advanced pathological tumor stage,
increased risk for tumor recurrence, and distant metastasis, and was, therefore,
suggested as a hub node transcription factor regulating tumour angiogenesis
[32].Genomic and proteomic analyses of the PPARβ/δ knockout
endothelial cells isolated from matrigel plugs have also led to the
identification of a number of additional candidate genes to mediate the actions
of PPARβ/δ in angiogenesis. In particular, the Cdkn1c gene which
encodes the cell cycle inhibitor p57Kip2 is a direct PPARβ/δ target gene that
mediates PPARβ/δ effects on cell morphogenesis [31]. In addition, CD36
and thrombospondin were also decreased in matrigel-invading endothelial cells from
PPARβ/δ knockout mice [31]. Thrombospondins by directly
interacting with CD36 inhibit angiogenesis in vivo [33, 34]. Similarly, a proteomic analysis by the same
group [35] on PPARβ/δ knockout endothelial cells has also revealed a
decreased expression of the chloride intracellular channel protein (CLIC)-4 in migrating
endothelial cells from PPARβ/δ knockout mice. In contrast, the expression of
cellular retinol binding protein CRBP1 is increased in migrating endothelial
cells from PPARβ/δ knockout mice [35]. CLIC-4 promotes and plays an essential
role during tubular morphogenesis [36], while CRBP1 inhibits cell survival
pathways by acting as an inhibitor of the AKT signalling pathway [37], an
additional important signalling signal for angiogenesis to occur [38].The combination of these
studies show PPARβ/δ activation induces endothelial cell mitogen and
differentiation signals, including VEGF, 14-3-3α, CD36 and thrombospondin, CLIC4,
CRBP-1, and p57KIP2, all of which may act in a coordinate manner
to bring about the functional morphogenic changes associated with angiogenesis.


## 3.3. PPARβ/δ and VEGF

Although the direct evidence
for a role of PPARβ/δ in angiogenesis is relatively new, there has been an
increasing literature regarding PPARβ/δ regulated tumour cell growth via inducing tumour
cells to release VEGF. PPARβ/δ ligands induce VEGF in bladder cancer cells [39], human
breast (T47D, MCF7), and prostate (LNCaP, PNT1A) cancer cell lines, along with
its receptor flt-1 [22], but not (HT29, colon; HCT116, colon; LS-174T, colon; HepG2,
hepatoma; and HuH7, hepatoma) cell lines [40].In a genetic model of intestinal
polyp development APC/min mouse, deletion of PPARβ/δ decreases intestinal
adenoma growth and inhibits tumour-promoting effects of the PPARβ/δ agonist GW501516
[41]. Moreover, activation of PPARβ/δ upregulated VEGF in colon carcinoma cells, promoting colon
tumour epithelial cell survival through activation of AKT signalling [41]. Angiogenesis
was not studied in this model, however, any substantial tumour growth requires
a blood supply and angiogenesis to allow it to develop. In contrast, in human colon
and liver cancer cell lines [40], PPARβ/δ ligands had no effect on human cancer cell growth,
AKT, VEGF or COX-2 expression in vitro
or on these makers in the liver, colon, and colon polyps in mice treated in vivo [40]. The roles of PPARβ/δ in VEGF-
mediated tumorigenesis
are, therefore, still in need of further clarification.


## 3.4. Expression of PPARβ/δ in the Eye

Angiogenesis regulates both the physiological development and many of the most common
pathophysiology’s of the eye. As yet, there is no direct evidence linking PPARβ/δ and
angiogenesis in the eye, however, PPARβ/δ is
clearly expressed at least in murine ocular tissue. PPARβ/δ is
expressed in the eye ciliary body epithelial cells, cornea epithelial cells, cornea
endothelium, cornea fibroblast, retina inner nuclear layer, and retina ganglion
cell layer [42]. Although one must be cautious interpreting data from nonocular
tissue to the eye [43], as discussed previously and following, pathways that
have direct relevance to ocular angiogenesis are clearly regulated by PPARβ/δ and
are therefore worthy of discussion.


## 4. VEGF and Ocular Angiogenesis

VEGF is essential in retinal vasculature development [44]. Initially blood vessels grow
from the optic nerve outwards. As the retinal tissue develops via a complex
interplay between different cellular components such as neurons, glia,
endothelial cells, pericytes, and immune cells, the increased oxygen demand induces
hypoxia, the main stimulant for new vessel growth via angiogenesis. As the
tissue/vasculature develops and gets oxygenated, hypoxia and VEGF decrease
limiting new vessel growth [44].In contrast, neovascularisation of the adult eye via angiogenesis is a critical
component of many disorders of the eye including retinopathy of prematurity, diabetic
retinopathy, and age-related macular degeneration, the latter two being the leading
causes of blindness in the Western world (as reviewed in detail elsewhere [29, 45–48]). Pathological
neovascularisation resulting from tissue damage and hypoxia results in a more
complex “inflammatory” angiogenesis. These new vessels are often fragile and
leaky leading to haemorrhage and vision disturbance and loss. The main trigger
for this new vessel growth still appears to be hypoxia induced VEGF expression
[29, 45–48]. Angiogenesis is a homeostatic repair mechanism that is required for
the reoxygenation of the damaged ischemic tissue [29, 45–48]. The problems that
arise with pathologies such as age-related macular degeneration and diabetic retinopathy
are that this new vessel growth is leaky and has a critical inflammatory
component. VEGF (in particular VEGF A; VEGF165) in addition to
directly stimulating angiogenesis is also a potent vascular permeability factor
and appears to play a role in regulating the local inflammation associated with
pathological neovascularisation [49]. VEGF has become a clear therapeutic
target for the treatment of angiogenesis in the eye. The clinical importance of
VEGF as a target has recently been further demonstrated with the development and
use of two new drugs targeting its actions: Macugen (pegaptanib), an aptamer,
and Lucentis (ranibizumab), a FAB fragment, from a humanised monoclonal
antibody, which both functionally block VEGF. Moreover, Macugen and Lucentis both
show clinical efficacy in patients with age-related macular degeneration [50]; especially
when treated early and a mature neovasculature has yet to form. These therapies
require local delivery by intravitriol injection, which although having the
benefit of overcoming problems such as systemic VEGF blockade, they are clearly
still not ideal, and show that new therapies are still required.


## 5. PPARβ/δ Ocular Angiogenesis,  Inflammation, and Coagulation

Angiogenesis associated with
pathophysiology is often associated with multiple process such as tissue
damage, inflammation, and coagulation. In contrast, developmental angiogenesis
may be a simpler hypoxia driven event. Indeed, an inflammatory response is
induced by VEGF during pathological but not physiological ischemia-induced
retinal angiogenesis [51, 52]. Moreover, specifically blocking inflammatory
cytokines monocyte chemotactic protein-1 and macrophage inflammatory protein-1a
can reduce retinal neovascularisation [53]. Tissue factor is a critical initiator of blood
coagulation, and is associated with tumour aggressiveness and angiogenesis in a
variety of cancer cells [54], as well as in choroidal neovascularisation where
it promotes fibrin formation and the growth of the choroidal angiogenic complex
[55]. One important facet of pathological angiogenesis may
therefore be this involvement additional pathways, and a complex interplay
between processes of tissue damage, hypoxia, inflammation, and coagulation. A
long-term therapeutic aim may therefore be to have revascularisation of hypoxic
tissue similar to development without these additional inflammatory/coagulation processes.PPARβ/δ induces VEGF in a number of cell types and induces angiogenesis. Therefore, one may predict
that a PPARβ/δ antagonist would be useful to treat or at least test
in models of eye disease that involve neovascularisation. However, PPARβ/δ seems consistent
with other PPAR's in that it also has anti-inflammatory and anticoagulation
properties, suggesting that its properties in ocular angiogenesis may be more complex than one
would originally predict.PPARβ/δ activation suppresses endothelial cell tissue factor expression [12].
PPARβ/δ is also expressed in platelets where its ligands reduce platelet aggregation to a
variety of stimuli [56]. Similar to PPARα and PPARγ, PPARβ/δ ligands are anti-inflammatory
in endothelial cells, inhibiting TNFα-induced upregulation of expression of vascular
cell adhesion molecule-1, monocyte chemoattractanct protein-1, and nuclear factor (NF)κB
translocation [57]. In macrophages, PPARβ/δ controls
inflammatory status by its association and disassociation with the
transcriptional repressor BCL-6 [58]; in the absence of ligand, PPARβ/δ physically
associates with and inhibits this anti-inflammatory BCL-6. When a PPARβ/δ ligand is added,
BCL-6 dissociates from PPARβ/δ and represses the inflammation and levels of monocyte
chemoattractanct protein-1, -3, and IL-1β [58].


## 6. Conclusion

PPARβ/δ induces angiogenesis and protects endothelial cells from oxidant damage. A common
signal for PPARβ/δ activation in endothelial cells or surrounding tissue
may be the induction of VEGF. PPARβ/δ is expressed in all tissues in the eye, however its
function has yet to be tested in physiological processes, development, or
pathophysiological disorders. The development of both the eye and common pathological
disorders requires angiogenesis, with VEGF being a primary signalling molecule.
Blocking PPARβ/δ may therefore provide a new therapy to treat
angiogenic eye disorders. The difference between “physiological” and
“pathophysiological” angiogenesis may be additional components of inflammation
and coagulation. PPARβ/δ ligands reduce inflammation and components of the
coagulation cascade. It will be of great interest to test the roles of PPARβ/δ in the eye as a
potential proangiogenic stimulus reliving the hypoxia, while potentially still
capable of reducing the damaging inflammatory/coagulation signals.



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*Source: 825970-2008-03-11.xml*