# Protective Actions of PPAR-γ Activation in Renal Endothelium

Authors: Peter E. Westerweel; Marianne C. Verhaar
Journal: PPAR Research
 (2008)

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## Abstract

Renal endothelial damage is pivotal in the initiation and progression of renal disease. Damaged
renal endothelium may be regenerated through proliferation of local endothelium and circulation-derived
endothelial progenitor cells. Activation of the PPAR-γ-receptors present on endothelial cells affects their
cellular behavior. Proliferation, apoptosis, migration, and angiogenesis by endothelial cells are modulated,
but may involve both stimulation and inhibition depending on the specific circumstances.  PPAR-γ-receptor
activation stimulates the production of nitric oxide, C-type natriuretic peptide, and superoxide dismutase,
while endothelin-1 production is inhibited. Together, they augment endothelial function, resulting in blood
pressure lowering and direct renoprotective effects. The presentation of adhesion molecules and release
of cytokines recruiting inflammatory cells are inhibited by PPAR-γ-agonism. Finally, PPAR-γ-receptors are
also found on endothelial progenitor cells and PPAR-γ-agonists stimulate progenitor-mediated endothelial repair.
Together, the stimulatory effects of PPAR-γ-agonism on endothelium make an important contribution to the
beneficial actions of PPAR-γ-agonists on
renal disease.


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## Body

## 1. Introduction

PPAR-γ-agonists are widely used for their insulin-sensitizing
actions in the treatment of type 2 diabetes mellitus, but have additional
therapeutic potential beyond the metabolic effects. Currently, the clinically
most used PPAR-γ-agonistic drugs are the thiazolidinediones (TZDs). PPAR-γ-agonists may favorably affect the course of renal
disease in both diabetic and nondiabetic conditions [1, 2]. In
nondiabetic animals, beneficial effects have been shown for anti-GBM
antibody-induced crescentic glomerulonephritis [3],
passive Heymann nephritis [4],
the development of glomerulosclerosis after 5/6 nephrectomy [5],
renal ischemia-reperfusion induced damage [6], and anti-Thy-1-glomerulonephritis [7]. One
potential mechanism is the modulation of endothelial cell function through
activation of PPAR-γ receptors, which are expressed on glomerular
endothelium [8, 9]. In
response to injury, the endothelial expression of PPAR-γ-receptors may be
increased, for example, as transiently occurs after ischemia- reperfusion [8, 9]. Treatment with PPAR-γ-agonists also increases the expression of PPAR-γ-receptors
on renal endothelium in both the glomerulus and the capillary endothelium of
the medullary vasa recta [10]. The
relevance for renal disease of activating the PPAR-γ-receptors on renal endothelium
is becoming increasingly clear and is the focus of this review.


## 2. The Role of Endothelium in Renal Disease

Renal microvascular endothelial injury is a pivotal pathogenic factor for
various renal diseases. Renal disease conditions involving prominent
endothelial damage include ischemic nephropathy, glomerulonephritis,
interstitial nephritis, and allograft rejection [11, 12]. Endothelial dysfunction and attenuated angiogenesis contribute to declining
renal function with ageing [13] and the pathogenesis and progression of chronic kidney disease [11]. The
microvascular endothelium, by the release of endothelium-derived factors such
as nitric oxide (NO) and as a critical component of the glomerular filtration
barrier, exerts important protection against progressive renal damage. Endothelial
dysfunction results in increased permeability causing passage of macromolecules
(microalbuminuria), which is considered to be the earliest renal sign of
vascular dysfunction [14, 15]. Endothelial dysfunction has been shown to predict susceptibility to renal
damage in a rat renal injury model [16].Progression of renal
disease does not only depend on the degree of microvascular endothelial injury,
but also on the effectiveness of endothelial repair. Impaired glomerular capillary repair was found
to be associated with the development of glomerulosclerosis and renal failure [17]. During experimental
glomerulonephritis, angiogenic factors such as VEGF and bFGF are released,
which stimulate endothelial regeneration [18–20]. Blocking the VEGF-induced endothelial repair with a VEGF-antagonist interferes
with renal recovery and results in progressive renal impairment [21]. Consistently, progressive renal disease is
associated with reduced expression of angiogenic growth factors and enhanced
expression of antiangiogenic factors [22, 23]. The glomerular endothelium can recover from injury by replacing lost or
damaged endothelial cells, in part through proliferation of local endothelium,
stimulated by the release of angiogenic growth factors [11, 12]. We [24–26] and others [27] have observed in both human and experimental animal studies that damaged
glomerular endothelium may also be regenerated from circulating bone marrow-derived
endothelial progenitor cells. Endothelial progenitor cells incorporate into the
damaged glomerulus, differentiate into mature endothelial cells, and eventually
fully integrate into the resident endothelium [26].Enhancing renal endothelial
repair offers therapeutic potential. Stimulating angiogenesis with
VEGF-treatment augments capillary repair and renal recovery after
glomerulonephritis [19]. Enhancing NO production by supplementing the substrate L-arginine improves the
clinical course of anti-Thy-1-glomerulonephritis [28]. Infusion of unselected bone marrow cells ameliorates experimental progressive
glomerulosclerosis [29]. Intrarenal administration of endothelial
progenitor cells attenuates endothelial injury and mesangial activation in
experimental glomerulonephritis [30].


## 3. The Role of PPAR-γ-Receptors in Vascular Development and Remodeling

Phenotypical
studies in humans and animals with genetic mutations in the PPAR-γ-receptor
imply a role in the regulation of vascular function and remodeling. In humans
with dominant-negative heterozygous mutations in the PPAR-γ-receptor causing an
impaired capacity for transcriptional activation, diabetes and also
hypertension occur at an unusually young age [31]. Heterozygous knockout mice have
increased insulin sensitivity [32] and decreased fat mass [33], but do not have a vascular phenotype [34]. However, but PPAR-γ-null mice are
embryonically lethal due to placental dysfunction, characterized by defective
trophoblast differentiation and markedly impaired placental vascularization [34]. A surviving PPAR-γ-knockout
mouse that was supplemented
with a wild-type placenta developed an apparently normal vascular system during
further embryogenesis, but died some days after birth due to a combination of
pathologies, including severe lipodystrophic changes and hemorrhages [34]. In a conditional knockout
model using a cre-lox system to save floxed PPAR-γ-knockout mice from embryonic
lethality by preserving PPAR-γ-function in the trophoblast marked lipodystrophy
was also observed, together with insulin resistance. Surprisingly, these mice
were hypotensive and showed increased endothelium-dependent relaxation in
response to acetylcholine [35]. In the latter study, PPAR-γ-function
was deficient in all cell types. Mice in which PPAR-γ-function was selectively
knocked out in endothelial cells only using again a cre-lox system were found
to be hypertensive when fed a high-fat diet [36]. Also, in mice with a nonlethal-dominant
negative mutation in the PPAR-γ-receptor, endothelium-dependent blood vessel dilatation
is impaired, while the endothelium is more sensitive to endothelin-1-induced
vasoconstriction [37]. Superoxide levels in these
mice are elevated and treatment with a superoxide scavenger can reverse the
impaired endothelial vasodilation, highlighting the pivotal role of increased
radical formation [37]. Cerebral arterioles were
hypertrophied with a decrease in luminal diameters, indicative of adverse
inward vascular remodeling [37]. Of note, dominant negative
PPAR-γ-mutations were found not to be fully selective, therefore, it cannot be
excluded that some of the effects observed in the knockout systems are
attributable to PPAR-γ-receptor-independent signaling, in particular through
the alternative PPAR-receptors [38].


## 4. Effects of PPAR-γ-Activation on Endothelial Proliferation, Migration, Angiogenesis, and Apoptosis

Reports on the direct
effects of PPAR-γ-agonist treatment on proliferation, migration, and angiogenic
network formation of cultured endothelial cells showed variable results. Fukunaga
et al. demonstrated increased proliferation with both troglitazone and
pioglitazone in four different endothelial cell lines [39]. In
contrast, two other studies found troglitazone to inhibit proliferation of
macrovascular endothelial cells [40] and human umbilical vein endothelial cells [41]. Rosiglitazone
was shown also to inhibit proliferation in one study [42], while
another study found no effect at all [40].VEGF-induced endothelial
migration was found to be inhibited by PPAR-γ-agonists troglitazone and
ciglitazone, which was mediated by inhibition of Akt [43]. This
is in line with the reduced migration and inhibition of angiogenic network formation
by PPAR-γ-agonism observed by Xin et al [44]. However,
in the study by Fukunaga et al., troglitazone stimulated both endothelial cell
migration and proliferation, resulting in accelerated coverage of a disrupted
endothelial monolayer in a wound healing assay [39]. Also,
Biscetti et al. found stimulation of endothelial network formation with PPAR-γ
activation using the GW1929 compound, mainly through a VEGF-dependent mechanism [45].In vitro effects of PPAR-γ-agonism
on endothelial cell apoptosis have been similarly variable. Both spontaneous
and TNF-alpha-induced endothelial apoptosis were shown to be inhibited by
various thiazolidinedione PPAR-γ-agonists [46] and troglitazone was found to markedly reduce apoptosis in serum-starved
endothelial cells [47], while another study observed induction of apoptosis using both
ciglitazone and PPAR-γ-receptor overexpression [48].
In several studies, the endogenous PPAR-γ ligand 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2)-induced endothelial
cell apoptosis [46, 48, 49], but it is important to note that in vascular endothelial cells, the
effects of 15d-PGJ2 may be independent
of PPAR-γ-receptor activation [49, 50].Taken together, it is clear
that PPAR-γ-agonists may modulate endothelial proliferation, migration,
angiogenesis, and apoptosis in vitro, but that this may result in both
stimulation and inhibition. The factors that determine whether treatment with a PPAR-γ-agonist results in a stimulatory or inhibitory effect on endothelial cells remain largely unidentified. This may involve PPAR-γ-receptor-independent effects as
reported for 15d-PGJ2 [49, 50] and also with
the various TZDs [45].
Fukunaga et al. posed that a concentration dependency may explain some of the
observed discrepancies, as they observed stimulation of DNA-synthesis at low PPAR-γ-agonist
dosages and inhibition at higher dosages [39]. However, this cannot explain all of the divergent findings.In
vivo studies in diabetic animals with impaired angiogenesis in response to
peripheral ischemia showed that treatment with PPAR-γ-agonist pioglitazone
augmented the angiogenic response and increased blood flow recovery [51]. In rats with experimental
focal cerebral ischemia, PPAR-γ-agonist treatment stimulated local angiogenesis
and improved functional neurological recovery [52]. In contrast, PPAR-γ-agonist
treatment inhibited pathological choroidial and retinal neovascularization [53] and suppressed tumor growth
and metastasis by inhibiting tumor angiogenesis in several primary tumors, in
part through decreasing VEGF-production by tumor cells and blocking the production
of angiogenic ELR+CXC-chemokines, mediated through antagonizing NF-kappaB
activation [54, 55]. These findings suggest that PPAR-γ-agonism may differentially affect
neovascularization with inhibition of pathological neovascularization and
augmentation of physiological neovascularization. This is in line with the in
vitro observations of both stimulatory and inhibitory actions.


## 5. Effects of PPAR-γ-Activation on Endothelial
Dysfunction

Endothelial integrity does
not only rely on the number of cells, but also on their function. NO-production
is a key component of endothelial function. NO stimulates vasodilation, inhibits
inflammation, prevents platelet activation, and scavenges radicals [56]. In
vitro studies on the effect of PPAR-γ-activation have consistently shown a
stimulating effect on NO production by endothelial cells [57–60]. The observation that siRNA against PPAR-γ blocked the increase in
NO-production confirmed that this effect
is PPAR-γ-receptor mediated [59]. In
both experimental animal studies and in humans, PPAR-γ-activation has been
shown to augment systemic NO-production and endothelial function. In diabetic
rats, PPAR-γ-agonist treatment restored
impaired endothelium-dependent arterial relaxation by increasing NO-production
while reducing oxidative stress [61]. In
healthy human subjects, a single dose of troglitazone increased NO-dependent
endothelial function measured by venous occlusion plethysmography of the
forearm and nitrite levels [62]. In
nondiabetic patients with hypertension or hypercholesterolemia, endothelial
function was improved with pioglitazone [63]. In
type 2 diabetic patients, vascular resistance was shown to be reduced with
troglitazone treatment [64, 65] and in type 2 diabetic patients with angina pectoris, troglitazone reduced the frequency of angina
pectoris with improving endothelial function [66]. Rosiglitazone attenuated the detrimental effects
of the presence of diabetes on NO-production measured directly using an
intravital probe and by assessing blood flow in the peripheral skin [67]. Long-term
treatment with pioglitazone resulted in a reduced pulse wave velocity,
indicative of reduced vascular stiffness [68]. In renal transplant recipients, PPAR-γ-agonist
treatment enhanced endothelial function [69].Importantly, the observed
beneficial effects on systemic endothelial function were found to extend to improving intrarenal
NO-production. In human type 2 diabetic patients, rosiglitazone treatment
increased intrarenal NO levels, which was associated with improvement of renal
hemodynamics and reduction of proteinuria [70]. This is in line with animal studies. In obese Zucker rats, PPAR-γ-agonist
treatment lowered blood pressure and ameliorated abnormal pressure natriuresis in association with
increased renal NO-metabolite nitrite/nitrate
production [71]. In
obese hypertensive Sprague-Dawley rats, PPAR-γ-agonist treatment also reduced blood
pressure, increased NO-metabolite nitrite/nitrate excretion, and reduced
excretion of oxidative-stress associated urinary isoprostanes and lipid
peroxides [72]. In
the kidneys of these rats, eNOS expression was increased while the pathological
increase in p47phox and gp91phox associated with obesity was attenuated [72].Differences have been
observed between the various PPAR-γ-activating compounds in the signaling level
at which NO-production is stimulated. The NO producing enzyme endothelial
nitric oxide synthase (eNOS) is not only regulated at the level of
transcription and translation, but also has multiple phosphorylation sites for
activation and deactivation of the enzyme and requires translocation to the
caveolae to associate with its cofactors and effectively produce NO [73, 74]. Troglitazone
has been shown to enhance NO-production by increasing eNOS transcription and
eNOS protein translation in
isolated endothelial cells [58],
while no effect was found at the transcriptional level for 15d-PGJ2 [57, 58], pioglitazone [58], and ciglitazone [57]. 15d-PGJ2 and rosiglitazone, but not
ciglitazone, were found to stimulate eNOS-phophorylation at activation site ser-1177,
thought to be mediated through increasing heat shock protein (hsp)-90
association with eNOS [59]. Interestingly, there is a cross-talk between
NO and PPAR-γ pathways as NO has been shown to rapidly and dose-dependently
increase PPAR-γ-binding, mediated by p38 MAPK activation [60].In
vivo, the stimulatory effect of PPAR-γ-agonists rosiglitazone and pioglitazone
on endothelial NO-production was associated with increased
eNOS-phosphorylation, while eNOS mRNA and total protein levels were not
affected [51, 75]. Rosiglitazone treatment has been shown to
enhance NO-production through enhancement of cellular transport of arginine,
the substrate for NO [76]. This is particularly relevant for renal
disease, as arginine transport was found to be markedly impaired in uremic
conditions [77–79].PPAR-γ-agonism favorably
affects other endothelium-derived factors that act in conjunction with NO to maintain
endothelial homeostasis. The release of C-type natriuretic peptide, another
vasodilatory peptide, by endothelial cells is increased by PPAR-γ-agonist
treatment [39]. In addition, PPAR-γ-agonist treatment increased Cu2+ and  Zn2+-superoxide
dismutase expression in cultured endothelial cells, thereby increasing their potential
for oxygen radical scavenging [80, 81]. Also, PPAR-γ-agonist treatment decreases
the production of radical oxygen species in endothelial cells [81, 82], in
part through decreasing the expression of subunits of NADPH-oxidase [80, 81].


## 6. Antihypertensive Effects of PPAR-γ-Agonism Mediated by the Endothelium

Improving NO-availability
is thought to be a major mechanism mediating the blood pressure lowering effect
of PPAR-γ-agonist treatment. Pioglitazone treatment prevented hypertension and renal oxidative stress
both by reducing free-radical production and by increasing nitric oxide
production [72]. No blood
pressure reduction was seen with PPAR-γ-agonist treatment in rats also
receiving NO-inhibitor L-NAME [83]. However, other mechanisms may play a role in the anti-hypertensive effect of PPAR-γ-agonist
treatment, such as effects on the contractility and proliferation of smooth
muscle cells [84].A naturally occurring
antagonist of NO is the vasoconstrictor endothelin-1, which is involved in
atherosclerosis and hypertension [85]. PPAR-γ-activation inhibits the production of
endothelin-1 from endothelial cells in vitro [39, 86–88]. In
type 2 diabetic patients, pioglitazone treatment reduced urinary endothelin-1
secretion, along with decreasing microalbuminuria [89]. Interestingly, in this
study, serum endothelin-1 levels were not affected, suggesting a specific
pathogenic role for endothelin-1 secretion in the kidney [89]. In DOCA-salt hypertensive rats, TZD treatment
reduced endothelin-1 production and blunted radical oxygen species production
with diminished hypertension progression and vascular remodeling [90]. Several studies show that the
inhibitory effect of PPAR-γ-activation on endothelin-1 secretion takes place at
the transcriptional level [86, 87] and
it was found to be NO-dependent [87]. Delerive et al. demonstrated
that PPAR-γ-activation negatively interferes with the activator protein-1 signaling
cascade, resulting in inhibition of thrombin-induced transcription of
endothelin-1 [88].In
hypertension, increased production and secondary effects of angiotensin-II play
a major role. In endothelial cells, angiotensin-II is a strong inducer of
NADPH-oxidase, resulting in the production of radical oxygen species [91]. Interestingly, there is a
cross-talk between the Angiotensin-II and PPAR-γ signaling pathways. Infusion
of angiotensin-II downregulates PPAR-γ-receptor expression in the vascular wall [92] and treatment with PPAR-γ-agonists
abrogates many of the angiotensin-II pathophysiological effects [93]. Of note, angiotensin-II-receptor-1
antagonists have been shown to act as partial PPAR-γ-agonists [94].


## 7. Effects of PPAR-γ-Activation on Inflammatory
Cell Recruitment to the Endothelium

Endothelial cells form an
important barrier between the blood and peripheral tissues and regulate homing,
adhesion, and transmigration of inflammatory cells. Upon activation,
endothelial cells may further release inflammatory cytokines and express
adhesion molecules to attract inflammatory cells. PPAR-γ-agonist treatment
inhibits the increased expression of adhesion molecules such as VCAM-1, ICAM-1,
and E-selectin and release of inflammatory cytokines upon stimulation of
endothelial cells with PMA [95, 96], TNF-α [95, 97], IFN-γ [98], IL-1β [99], LPS [96], microparticles [100], and high glucose [101]. In vitro studies confirmed that this resulted in decreased
adhesion of inflammatory cells to the endothelium [96, 97, 101, 102]. In addition, a recent study showed that in activated primary
human brain endothelial cells, PPAR-γ-activation resulted in a marked reduction
of monocyte adhesion and in vitro transendothelial migration, mediated by
inhibition of Rac1 and RhoA GTPases [102].  In vivo, troglitazone and 15d-PGJ2 reduced
ICAM-1 and VCAM-1 expression
on endothelial cells and reduced inflammatory cell homing to atherosclerotic
plaques in a mouse model [103].


## 8. Effects of PPAR-γ-Activation on Endothelial
Progenitor Cells

A recently uncovered effect
of PPAR-γ-agonists is the capacity to augment the level and function of endothelial
progenitor cells. PPAR-γ-agonist treatment increases endothelial
progenitor cell levels in mice [104, 105] and
humans [104, 106, 107]. A stimulatory effect on endothelial
progenitor cell outgrowth was also observed when cultured peripheral blood
mononuclear cells were exposed to PPAR-γ-agonist treatment ex vivo [104, 105, 108, 109], suggesting an effect on endothelial progenitor cell survival, adhesion,
or differentiation. PPAR-γ-agonist treatment largely prevented apoptosis of endothelial progenitor
cells induced by CRP [109] and H2O2
[105]. Based on marker expression patterns, the
differentiation of endothelial progenitor cells toward the endothelial lineage appears
to be stimulated [104, 108], while expression of smooth muscle cell markers
is inhibited [104]. Pioglitazone inhibited detrimental effects of
angiotensin-II on endothelial progenitor cells, including inhibiting the
induction of cellular senescence of endothelial progenitor cells via
downregulation of the expression of angiotensin-II-receptor-1 and limiting the angiotensin-II-induced
increased generation of peroxynitrate and superoxide by the NADPH-oxidase
subunit gp91phox [110].Functionally,
PPAR-γ-agonist treatment was shown to stimulate angiogenic network formation by
endothelial progenitor cells in vitro [109] and in vivo [105]. The adhesion capacity may be increased upon
TZD-treatment [108, 109]. In addition,
endothelial progenitor cell-mediated reendothelialization of a denuded segment
of the femoral artery in mice was accelerated in PPAR-γ-agonist treated animals [104]. As
endothelial progenitor cells may also participate in regeneration of damaged renal
endothelium [26],
we investigated a potential role for enhanced endothelial progenitor cell homing
and glomerular incorporation by PPAR-γ-agonist treatment [7]. However,
using a rat allogenic bone marrow transplantation model, we could not detect an
effect on the number of incorporated circulation-derived glomerular endothelial
cells in the recovering glomerulus with rosiglitazone treatment, although
rosiglitazone did attenuate the clinical course of glomerulonephritis.


## 9. Conclusions and Perspectives

Activation of the PPAR-γ-receptors present on renal endothelial cells
affects their cellular behavior. Proliferation, apoptosis, migration, and
angiogenesis by endothelial cells are modulated, but may involve both stimulation and
inhibition depending on the specific circumstances. It remains unclear how this
bimodal potential of PPAR-γ-agonists is regulated. An important consequence of PPAR-γ-receptor
activation is the enhanced production of nitric oxide and C-type natriuretic
peptide and superoxide dismutase, while endothelin-1 production is inhibited.
Together, this improves the capacity of the endothelium to exert vasodilatory,
anti-inflammatory, and antioxidative actions, resulting in blood pressure
lowering and direct renoprotective effects. In addition, the presentation of adhesion
molecules and release of cytokines aimed at recruiting inflammatory cells to
activated endothelium is inhibited by PPAR-γ-agonism. This may also in part
account for the anti-inflammatory effects of PPAR-γ-agonists, supplementary to
direct effects on the inflammatory cells themselves. Finally, PPAR-γ-receptors
are also found on endothelial progenitor cells and PPAR-γ-agonist stimulate
progenitor-mediated endothelial repair, although definitive evidence that this
occurs in the kidney is currently lacking.Activation of other PPAR-receptors besides PPAR-γ may also have
beneficial effects on the endothelium. Recently, selective agonists for PPAR-β/δ
were developed [111]. PPAR-β/δ receptors are present on endothelium and stimulation with a
pharmacological PPAR-β/δ-agonist was found to increase endothelial cell
proliferation and angiogenesis in vitro and in vivo, mainly mediated through
VEGF [112]. Like PPAR-γ-agonists, PPAR-β/δ-agonists increase circulating endothelial
progenitor cell levels, augment endothelial progenitor cell function in vitro,
and improve endothelial progenitor cell-mediated neovascularization of ischemic
tissue [113]. Also, much like PPAR-γ-agonists, PPAR-β/δ-agonist treatment reduced the
expression of adhesion molecules and monocyte binding to activated endothelial
cells [114].Currently, PPAR-γ-agonist treatment is not standard clinical practice in
nondiabetic renal patients. As shown in this review, experimental studies
indicate that stimulatory effects of PPAR-γ-agonism on endothelium may provide
additional benefit in nondiabetic renal disease. Together with other
potentially therapeutic effects independent of the insulin-sensitizing action
such as the anti-inflammatory actions, this provides a rationale for further
clinical evaluation in nondiabetic renal patients. For patients with diabetic
kidney disease, a pressing question is whether glycemic control with a TZD is
superior to other antidiabetic drugs for preventing the decline of renal
function. To date, only retrospective studies, post hoc analyses, and pilot studies are available
to help answer this question. Trials designed to specifically evaluate this
question have yet to be performed.



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