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Response of Experimental Retinal Neovascularization to Thiazolidinediones
Toshinori Murata, MD;
Yasuaki Hata, MD;
Tatsuro Ishibashi, MD;
Sarah Kim, BS;
Willa A. Hsueh, MD;
Ronald E. Law, PhD;
David R. Hinton, MD
Arch Ophthalmol. 2001;119:709-717.
ABSTRACT
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Objective To determine the effect of thiazolidinediones (TZDs) on experimental
retinal neovascularization.
Methods The ability of the TZDs troglitazone and rosiglitazone maleate (1-20
µmol/L) to inhibit retinal endothelial cell (REC) proliferation, migration,
tube formation, and signaling was determined in response to vascular endothelial
growth factor (VEGF). In vivo studies were performed using the oxygen-induced
ischemia model of retinal neovascularization. Neonatal mice were treated with
intravitreous injection of 0.5 µL of troglitazone (100 µmol/L)
or rosiglitazone maleate (100 µmol/L), or vehicle, and retinal neovascularization
was assayed qualitatively and quantitatively by means of angiography and histological
examination.
Results Expression of the TZD receptor, peroxisome proliferator-activated receptor  ,
was confirmed in RECs by means of Western immunoblotting. Rosiglitazone and
troglitazone inhibited VEGF-induced migration (P<.05),
proliferation (P<.05), and tube formation (P<.01) by RECs in vitro beginning at 10 µmol/L.
Rosiglitazone and troglitazone inhibited phosphorylation of extracellular
signal-regulated mitogen-activated protein kinase 1 in RECs. Intravitreous
injection of rosiglitazone or troglitazone inhibited development of retinal
neovascularization (P<.01) but did not significantly
inhibit VEGF overexpression in the ganglion cell layer of the ischemic retina.
Conclusion The TZDs inhibit experimental retinal neovascularization with an effect
that is primarily downstream of VEGF expression.
Clinical Relevance The TZDs are widely prescribed and should be evaluated for their potential
to inhibit the progression of diabetic retinopathy.
INTRODUCTION
DIABETIC RETINOPATHY is a major cause of blindness in the United States.1 Almost all patients in whom diabetes begins before
30 years of age show some evidence of retinopathy 20 years later, which is
not surprising, given the finding that the risk for retinopathy is directly
related to the degree and duration of hyperglycemia.2
In those patients in whom diabetes develops after 30 years of age, retinopathy
is less frequent but may be the first manifestation of the disease.2 Proliferative diabetic retinopathy (PDR) is the most
sight-threatening form of diabetic retinopathy and is characterized by retinal
neovascularization. The growth of new vessels on the surface of the retina
in PDR can be correlated with the presence of retinal ischemia and increased
local expression of vascular endothelial growth factor (VEGF).3-9
Retinal endothelial cells (RECs) have been shown to express VEGF receptors,
and endothelial cells respond to VEGF by activation of multiple signaling
molecules, including phospholipase C , phosphatidylinositol 3-kinase,
protein kinase C, and the extracellular signal-regulated mitogen-activated
protein kinase (ERK-MAPK) pathway.10-13
A major effort has been made to develop novel therapeutic agents to
inhibit retinal neovascularization. Experimental oxygen-induced retinopathy
is commonly used in these studies as a model for PDR.14-15
In this model, VEGF is up-regulated in the inner retina after neonatal mice
exposed to hyperoxic conditions are returned to room air, resulting in the
development of consistent retinal neovascularization. Several approaches have
met with success in this and other ischemic model systems, including those
that inhibit VEGF by means of soluble receptor or antibody16-17
and those that inhibit VEGF receptor activation18
or its downstream signaling19; however, none
of these have been proven efficacious in humans with PDR.
We considered the possibility that a class of drugs currently in use
for the primary treatment of diabetes may also inhibit neovascular complications
of diabetes independent of their effects on blood glucose level, thus potentially
decreasing the risk for PDR for these patients. Thiazolidinediones (TZDs),
including troglitazone, rosiglitazone maleate, and pioglitazone hydrochloride,
constitute a novel class of drugs that can be used to improve insulin resistance
in type 2 diabetes.20-21 Two members
of this class (rosiglitazone and pioglitazone) are currently approved for
clinical use. The TZDs are synthetic high-affinity ligands for the nuclear
peroxisome proliferator-activated receptor (PPAR-).22-24 Expression of PPAR-
is most abundant in adipose tissue, where it promotes adipocyte differentiation
and regulates the expression of genes involved in fatty acid metabolism; however,
recent studies have shown a more widespread distribution of PPAR-,
suggesting that it may affect other tissues.25
Recently, we and others have demonstrated the expression of PPAR- on
endothelial cells and have shown antiangiogenic effects in corneal and choroidal
experimental systems.26-28
In the present study, we show that RECs express PPAR- protein
and that TZDs inhibit angiogenic responses to VEGF in vitro. We then show
that 2 TZDs inhibit the development of retinal neovascularization in the oxygen-induced
retinopathy model in mice. To further define the mechanism of this effect,
we examined the effect of TZDs on expression of VEGF in vivo and VEGF-induced
ERK-MAPK phosphorylation in vitro.
RESEARCH DESIGN AND METHODS
IN VITRO ASSAYS USING RECs
We isolated bovine RECs using magnetic beads carrying the endothelium-specific
marker Bandeiraea simplicifolia (BS-1; Sigma-Aldrich
Corp, St Louis, Mo), as previously described.29
The cells were confirmed to be vascular endothelial cells by means of positive
findings of immunostaining for von Willebrand factor (vWF) (Dako, Carpinteria,
Calif) and by means of uptake of dil-acetylated low-density lipoprotein (Biomedical
Technologies, Stoughton, Mass). Nuclear and cytosolic fractions of protein
extracts28 were prepared using the method of
Dignam et al.30 Full-length in vitro translated
PPAR-1 was used as a positive control. Gel electrophoresis and Western
blotting were performed as previously described28
using a rabbit polyclonal anti–PPAR- antibody (Santa Cruz Biotechnology,
Santa Cruz, Calif) (1:1000) and a chemoluminescent detection kit (Lumi-GLO;
Kirkegaard & Perry, Gaithersburg, Md). In vitro assays were performed
in response to VEGF (10 ng/mL) after 24 hours of serum starvation. Cell proliferation
was assayed by means of tritiated thymidine uptake.28
Chemotactic migration was examined using transwell cell-culture chambers (Costar,
Cambridge, Mass).28 Capillarylike tube formation
was examined by growing RECs in a collagen mixture as previously described.28 The effect of troglitazone or rosiglitazone (at concentrations
of 0, 0.1, 1, 10, and 20 µmol/L) added with the VEGF was determined
for each of these in vitro assays. Experiments were performed in triplicate
and were repeated at least 3 times.
ANIMAL MODEL OF RETINAL ANGIOGENESIS
The experiments conformed to the Association of Research in Vision and
Ophthalmology Resolution on the Use of Animals in Ophthalmic and Vision Research,
and were approved by the University of Southern California Institutional Care
and Use Committee, Los Angeles. The reproducible murine model of oxygen-induced
retinopathy has been described previously.15
Briefly, litters of 7-day-old C57BL/6N mice were exposed to a mean (±SD)
of 75% ± 2% oxygen for 5 days and then returned to the room air on
postnatal day 12. Twelve mice were included in each group (troglitazone- and
rosiglitazone-treated and control mice). Eight mice were used for quantitation
of retinal angiogenesis or immunohistochemical analysis, and the remaining
4 mice were used for angiography with high-molecular-weight fluorescein dextran.
Intravitreal injections of troglitazone or rosiglitazone were performed as
described below. Mice of the same age that had been kept in room air were
used as controls.
INTRAVITREAL INJECTIONS OF ROSIGLITAZONE OR TROGLITAZONE
Mice were deeply anesthetized by means of intraperitoneal injection
of ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (10 mg/mL).
Intravitreal injections were performed on postnatal days 12 and 14 by delivering
0.5 µL of troglitazone or rosiglitazone maleate (100 µmol/L) diluted
in dimethyl sulfoxide (DMSO) to the left eye and DMSO alone to the right eye
with a 32-gauge Hamilton needle 200 µm posterior to the limbus.
ANGIOGRAPHY WITH HIGH-MOLECULAR-WEIGHT FLUORESCEIN DEXTRAN
Mice were deeply anesthetized as described above, and then 100 µL
of phosphate-buffered saline solution containing 5 mg of fluorescein isothiocynate–dextran
(molecular weight, 2 000 000; Sigma-Aldrich Corp) was injected into
the left ventricle. After 1 minute, the mice were killed using an intraperitoneal
injection of pentobarbital sodium (75 mg/kg). The eyes were enucleated, and
the retinas were dissected and flat-mounted on microscope slides for examination
using a fluorescence microscope (BX50; Olympus, Tokyo, Japan).
HISTOLOGICAL QUANTITATION OF RETINAL ANGIOGENESIS
The mice (postnatal day 17) were killed using an intraperitoneal injection
of pentobarbital sodium (75 mg/kg). Enucleated eyes were fixed with 4% paraformaldehyde
in phosphate-buffered saline solution and embedded in paraffin. Serial axial
sections (3 µm) of the retina were obtained, starting at the optic nerve
head. After staining with hematoxylin-eosin, 10 intact sections of equal length,
each 30 µm apart, were evaluated. All retinal vascular cell nuclei anterior
to the inner limiting membrane were counted in each section; for controls,
this value was 0.
IMMUNOHISTOCHEMICAL ANALYSIS
Immunohistochemical analysis for the endothelium-specific marker vWF
was used to identify intraretinal and preretinal angiogenesis and normal retinal
vascular channels. Immunohistochemical analysis for VEGF (sc-507; Santa Cruz
Biotechnology) was used to detect its overexpression in the hypoxic retina.
Immunoperoxidase detection was performed by the avidin-biotin complex method
with 3-amino 9-ethylcarbazole as the red chromogen. Negative controls included
omission of primary antibody and use of an irrelevant primary antibody of
the same isotype.
p44/p42 ERK-MAPK PHOSPHORYLATION IN RECs
The RECs were seeded into 6 multiwell plates in endothelial growth medium
(Clonetics, Walkerville, Md). Confluent RECs were starved in Dulbecco's Modified
Eagle Medium (DMEM; Cellgro, Herndan, Va) containing 0.1% bovine serum albumin
(Sigma-Aldrich Corp) for 8 hours. The RECs, untreated or treated with 25 ng/mL
recombinant human VEGF 165 (R & D Systems Inc, Minneapolis, Minn) for
5 minutes, were washed once in cold phosphate-buffered saline solution and
lysed in Laemmli buffer (50-mmol/L Tris [pH 6.8], 2% sodium dodecyl sulfate
[SDS], and 10% glycerol; BioRad, Hercules, Calif) containing protease inhibitors.
Cell lysates were heated to 95°C for 2 minutes, and equal volumes of lysate
were subjected to 10% SDS–polyacrylamide gel electrophoresis (PAGE).
The blots were incubated with anti–phosphospecific ERK1/ERK2 (p44/p42)
antibody (New England Biolabs, Beverly, Mass), followed by incubation with
horseradish peroxidase–conjugated secondary antibody (Amersham, Piscataway,
NJ). Visualization was performed using the Amersham enhanced chemiluminescence
detection system. Lane-loading differences were normalized by reblotting with
non–phosphorylation-specific anti-ERK1 antibody (Santa Cruz Biotechnology).
The RECs were then pretreated with vehicle, rosiglitazone maleate (10
µmol/L), or troglitazone (1-20 µmol/L) for 15 minutes before exposure
to vehicle alone or VEGF 165 (25 ng/mL) for 5 minutes. Cell lysates were subjected
to SDS-PAGE, and the evaluation of p44/p42 ERK-MAPK phosphorylation was performed
as already described.
STATISTICAL EVALUATION
Experimental and control groups were compared using independent sample t tests. Statistical significance was defined as  <.05.
RESULTS
EXPRESSION AND SUBCELLULAR LOCALIZATION OF PPAR- IN RECs
To detect the expression of PPAR- protein in bovine RECs, we
performed Western immunoblotting of nuclear and cytosolic proteins. Figure 1 shows a band of 52 kd corresponding
to PPAR-1 that localizes almost exclusively to the nuclear fraction.
IN VITRO EFFECTS OF TZDs ON RECs
Proliferation of RECs was induced using VEGF (10 ng/mL). Rosiglitazone
and troglitazone each significantly inhibited VEGF-induced thymidine incorporation,
beginning at concentrations of 10 µmol/L (P<.05)
and becoming more prominent at concentrations of 20 µmol/L (P<.005) (Figure 2A). Both
TZDs were equally effective at this dose range. Viability, as measured by
means of trypan blue exclusion, was greater than 95% at all drug concentrations
tested.
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Figure 2. Effect of thiazolidinediones on
retinal endothelial cell (REC) thymidine incorporation, migration, and tube
formation in response to vascular endothelial growth factor (VEGF). A, The
proliferation of RECs was induced with VEGF (10 ng/mL) and measured by means
of tritiated thymidine incorporation. Rosiglitazone maleate and troglitazone
each inhibited VEGF-induced thymidine incorporation in a dose-dependent manner
beginning at concentrations of 10 µmol/L (P<.05).
B, Chemotactic migration of RECs to VEGF (10 ng/mL) was measured in a modified
Boyden chamber. Rosiglitazone and troglitazone each inhibited VEGF-induced
REC migration in a dose-dependent manner beginning at concentrations of 10
µmol/L (P<.05). C, Tube formation of RECs
was induced by adding VEGF (10 ng/mL) to collagen gels. Rosiglitazone and
troglitazone prominently inhibited tube formation at concentrations of 10
µmol/L (P<.01) and greater. For each experiment,
the maximal effect was obtained for VEGF-stimulated RECs, and this result
was assigned a value of 100; all subsequent results are presented as an index
relative to this number. Error bars represent SD.
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Rosiglitazone and troglitazone inhibited the chemotactic migration of
RECs to VEGF in a modified Boyden chamber in a dose-dependent manner beginning
at 10 µmol/L (P<.05) (Figure 2B). Viability was maintained at greater than 95% at all
drug concentrations used in this experiment.
Dramatic inhibition of VEGF-induced tube formation by RECs in collagen
gels was seen in the presence of rosiglitazone or troglitazone at a concentration
of 10 µmol/L (P<.01) (Figure 2C). At a concentration of 20 µmol/L, essentially
no tube formation was found. No morphologic evidence of cell death was observed
at any of the TZD drug concentrations tested.
EFFECT OF TZDs ON RETINAL NEOVASCULARIZATION
To evaluate the antiangiogenic effect of TZDs on oxygen-induced retinopathy,
retinas were examined by means of fluorescein-dextran injection angiography.
Retinas of the DMSO-injected eyes of animals at postnatal day 17 (5 days of
relative retinal hypoxia) contained prominent neovascular tufts extending
into the vitreous body at the junction between the perfused and the nonperfused
tissue (Figure 3A-B). In contrast,
in the retinas from experimental animals treated with 0.5 µL of troglitazone
(100 µmol/L) or rosiglitazone maleate (100 µmol/L) and examined
on postnatal day 17, the amount of neovascular tissue was markedly reduced,
despite the presence of comparable pericentral regions of nonperfusion (Figure 3C). The capillary network and nonperfused
areas were well formed, but there was no tuft of neovascularization extending
into the vitreous body (Figure 3D).
Retinas from normoxic controls demonstrated an evenly distributed stained
capillary network (data not shown).
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Figure 3. Flat mounts of retinas infused
with fluorescein isothiocyanate–dextran from control and thiazolidinedione-treated
mice with oxygen-induced retinopathy. Retinas of the dimethyl sulfoxide–injected
eyes of hypoxic animals at postnatal day 17 (A and B). A, Prominent neovascular
tufts extended into the vitreous body at the junction between the perfused
and the nonperfused tissue (original magnification x100). B, The tufts
are shown at higher magnification (original magnification x400). Retinas
from troglitazone-treated eyes at postnatal day 17 (C and D). C, The amount
of neovascular tissue was markedly reduced, despite the presence of comparable
pericentral regions of nonperfusion (original magnification x100). D,
The capillary network and nonperfused areas are well formed, but there are
no tufts of neovascularization extending into the vitreous (original magnification
x400). Images were digitally acquired but not enhanced.
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The neovascularization was quantitated histologically by counting the
endothelial cell nuclei anterior to the inner limiting membrane. Retinas of
DMSO-treated eyes from mice on postnatal day 17 (5 days of relative retinal
hypoxia) contained multiple neovascular tufts extending into the vitreous
(Figure 4A). These tufts originated
from retinal vessels, forming clusters of immature endothelial cells (Figure 4B). Intravitreal injection of troglitazone
(Figure 4C-D) or rosiglitazone reduced
the histologically evident retinal neovascularization in all 8 animals compared
with the DMSO-treated experimental controls (Figure 5). The mean (±SD) number of endothelial nuclei anterior
to the inner limiting membrane was significantly reduced from 474 ±
93 in control eyes (DMSO-injected) with oxygen-induced retinopathy to 267
± 51 in troglitazone-treated eyes (P<.01)
and 299 ± 61 in rosiglitazone-treated eyes (P<.01).
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Figure 4. Histological features of retinal
neovascularization in control and thiazolidinedione-treated mice with oxygen-induced
retinopathy. A, Dimethyl sulfoxide–treated eyes from mice with oxygen-induced
retinopathy contained multiple neovascular tufts extending into the vitreous
(arrows) (hematoxylin-eosin, original magnification x200). B, The tufts
were seen to originate from retinal vessels, forming clusters of immature
endothelial cells (arrows) (hematoxylin-eosin, original magnification x400).
C and D, Intravitreal injection of troglitazone significantly reduced the
retinal neovascularization (arrows) (hematoxylin-eosin, original magnifications
x200 [C] and x400 [D]). Images were digitally acquired but not
enhanced.
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Figure 5. Quantitation of retinal neovascularization
in control and thiazolidinedione-treated mice with oxygen-induced retinopathy.
The number of endothelial nuclei anterior to the inner limiting membrane was
significantly reduced in troglitazone- (P<.001)
and rosiglitazone maleate– (P<.001) treated
eyes. There was no significant statistical difference between rosiglitazone-
and troglitazone-treated groups (P = .27). Error
bars represent SD.
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IMMUNOHISTOCHEMICAL ANALYSIS FOR VEGF AND vWF
Consistent expression of VEGF was seen in all hypoxic retinas, regardless
of the agents injected into the vitreous (Figure 6). In DMSO-injected eyes, VEGF was expressed mainly in the
ganglion cell layer and to a lesser extent in the inner plexiform layer of
the retinas, just beneath the prominent retinal neovascularization (Figure 6A), and adjacent to the intraretinal
neovascularization (Figure 6C).
Although retinal neovascularization was significantly reduced in troglitazone-
or rosiglitazone-injected eyes, VEGF was also prominently expressed in the
ganglion cell layer of these TZD-treated retinas (Figure 6B).
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Figure 6. Immunohistochemical analysis for
vascular endothelial growth factor (VEGF) and the endothelium-specific marker
von Willebrand factor (vWF) in mice with oxygen-induced retinopathy. A, From
dimethyl sulfoxide–injected (control) animals with oxygen-induced retinopathy,
increased VEGF expression was found in the ganglion cell layer (GCL) and inner
plexiform layer (IPL) of the retina in the region of the prominent retinal
neovascularization (arrows) (original magnification x200). B, From troglitazone-injected
eyes, increased VEGF expression was found in the GCL, although retinal neovascularization
is mild (arrow) (original magification x200). C, From control animals
with oxygen-induced retinopathy, endothelial cell labeling with vWF shows
that neovascularization is located not only on the surface of the retina but
also in the inner retina (arrows), adjacent to where VEGF is prominently expressed
(original magnification x20). Images were digitally acquired but not
enhanced (for all, immunohistochemical stain using 3-amino 9-ethylcarbazole
as the red chromogen and hematoxylin as a blue nuclear counterstain).
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VEGF STIMULATES ERK-MAPK PHOSPHORYLATION IN RECs
After 5 minutes of stimulation, VEGF induced p44/p42 ERK-MAPK phosphorylation
(Figure 7). Troglitazone and rosiglitazone
(10 µmol/L) inhibited VEGF-induced ERK-MAPK phosphorylation by greater
than 50% for p44 ERK1. Dose-response analysis demonstrated that 20-mmol/L
troglitazone inhibited ERK-MAPK phosphorylation even more prominently. Troglitazone
also eliminated the weak basal p42 ERK-MAPK phosphorylation without affecting
cellular shape or attachment. In all experiments, blots of total ERK-MAPK
revealed that total ERK-MAPK protein concentration remained unchanged.
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Figure 7. Thiazolidinedione inhibits vascular
endothelial growth factor (VEGF)–induced extracellular signal-regulated
mitogen-activated protein kinase (ERK-MAPK) phosphorylation in retinal endothelial
cells (RECs). Quiescent RECs, untreated or treated with VEGF at a concentration
of 25 ng/mL (5 minutes), were lysed, and the lysates were subjected to 12%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The membrane
was probed with anti–phosphospecific p44/p42 ERK-MAPK antibody (top)
and then reprobed with non–phosphorylation-specific anti–ERK1
antibody (bottom). A, Troglitazone and rosiglitazone maleate (10 µmol/L)
moderately inhibit baseline unstimulated p42 MAPK and VEGF-induced p44/p42
MAPK. B, Dose response for troglitazone shows that the effect is first seen
at 10 µmol/L and becomes even more prominent at 20 µmol/L. Images
were digitally acquired but not enhanced.
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COMMENT
Insulin resistance plays a crucial role in the pathogenesis of type
2 diabetes; however, much of the morbidity of this disease results from its
complications, including microangiopathy and cardiovascular disease. The TZDs
constitute a class of pharmacologic compounds that enhance insulin action
through activation of the PPAR- receptor.22-24
These drugs, when used alone or added to traditional oral hypoglycemic agents,
result in better control of glycemic markers and amelioration of hyperinsulinemia.20-21 Recently, there has been considerable
interest in the additional benefits of these drugs for other metabolic abnormalities
in diabetes, including improvements in lipid profile and blood pressure control.20 Moreover, it has recently been suggested that TZDs
independently delay certain diabetic complications through direct activation
of PPAR- in vascular cells.31 Troglitazone
inhibits endothelial cell activation26-27
and platelet aggregation32 and delays development
of atherosclerosis in animal models.33 Beneficial
effects on other diabetic complications have also been reported. The TZDs
may inhibit microalbuminuria in patients with incipient diabetic nephropathy34 and may protect against nephropathy in Zucker fatty
rats.35 Inhibitory effects of TZDs on diabetic
neuropathy in streptozocin-induced diabetic rats have also been described.36
In the present report, we provide evidence that TZDs may reduce the
complications of diabetic retinopathy. Diabetic retinopathy encompasses the
following 5 basic abnormalities: formation of retinal capillary microaneurysms,
development of excessive vascular permeability, vascular occlusion, proliferation
of new blood vessels, and contraction of accompanying fibrous tissue on the
surface of the retina.2 Proliferative diabetic
retinopathy is particularly devastating and is characterized by the growth
of new vessels on the surface of the retina. At the present time, the risk
for blindness with untreated PDR is greater than 50% at 5 years, but with
appropriate current therapy, it can be reduced to less than 5%.37
Despite these improvements, it remains a very common cause of blindness that
would be treated more appropriately by prevention.
Neovascularization is a multistep process that includes degradation
of basement membrane and proliferation, migration, and tube formation by endothelial
cells.38 The neovascularization process is
stimulated by a variety of growth factors and cytokines; however, VEGF has
been shown to be central to this process in PDR.3-8
It has been reported previously that endothelial cells from several sources,
including the umbilical vein, aorta, and choroid, express the TZD receptor
PPAR-; nevertheless, it is important to confirm this finding in the
specific endothelial populations being studied, since endothelial cells are
heterogeneous in their phenotype and function.26-28
We show herein that RECs strongly express PPAR-1 protein. The PPAR- gene produces 2 messenger RNA species by means
of alternative promoter use, each containing different 5' exons that
are spliced onto common downstream exons. The PPAR-2 protein differs
from PPAR-1 by the presence of 30 additional amino acids at its N-terminal.39 Although both isoforms of this nuclear receptor are
expressed in abundant levels in human adipose tissues, PPAR-1 expression
is typically much higher than PPAR-2 expression in nonadipose tissues.25, 39 Consistent with these findings, RECs
express PPAR-1 protein that is localized almost exclusively to the
nuclear fraction.
The antidiabetic action of the TZDs appears to be mediated primarily
through activation of PPAR-; however, troglitazone is distinguishable
from other TZD PPAR- ligands because it also contains an -tocopherol
moiety, which could have effects due to its antioxidant activity40
or its ability to inhibit protein kinase C.41-42
Both TZDs studied in this report inhibited the proliferation, migration, and
tube formation of RECs in response to VEGF. There was no apparent difference
in the sensitivity of RECs to troglitazone and rosiglitazone, providing strong
support for the notion that both drugs have effects on RECs by binding to
PPAR-.
Previous in vivo studies on the antiangiogenic effects of PPAR-
ligands have been limited to a VEGF-containing corneal pocket assay27 and a laser injury model of choroidal neovascularization.28 The present study uses a much more pathologically
relevant model in which the neovascularization is stimulated by increased
VEGF expression in the inner retina. Although the model lacks other metabolic
abnormalities found in diabetes, it isolates the VEGF-driven process to the
inner retina and allows more precise examination of the mechanism within a
clinically relevant microenvironment. By use of qualitative and quantitative
analyses, we demonstrated a decrease in the number of microvascular tufts
that were induced on the retinal surface, suggesting that the TZDs are inhibiting
an early aspect of neovascularization. Again, it was important to demonstrate
similar degrees of inhibition of in vivo angiogenesis by 2 PPAR- ligands,
implying that the drugs are acting similarly through PPAR- activation.
Although rosiglitazone binds to PPAR- with higher avidity than to troglitazone,
the TZDs were equally potent in inhibiting VEGF-mediated growth and REC migration.
These observations are similar to our previous findings for their antiproliferative
and antimigration activity in vascular smooth muscle cells.43
When VEGF expression was examined immunohistochemically, we found that approximately
equal amounts of VEGF were expressed in the ganglion cell layer of positive
control animals with neovascularization as were found in animals in which
retinal neovascularization was inhibited by TZDs, supporting the contention
that the TZDs do not interfere with VEGF expression in the region where neovascularization
occurs. This contention is further supported by in vitro data demonstrating
that hypoxia-induced VEGF secretion by viable retinal pericytes and astrocytes
is not significantly reduced by treatment of cells with troglitazone (results
not shown).
Previous studies in vascular smooth muscle cells show that PPAR-
ligands may be acting, at least in part, downstream of growth factor receptor
tyrosine kinase activation through inhibition of the ERK-MAPK pathway.44-45 Although in those studies it was
suggested that the effect may be downstream of ERK1/2 activation, we demonstrate
here in RECs the significant inhibition of VEGF-induced ERK1/2 phosphorylation
by a TZD. Troglitazone has also been shown to inhibit angiotensin II–induced
ERK-MAPK activity in vascular smooth muscle cells.46
Since the ERK-MAPK pathway plays an important role in cellular proliferation
and migration, inhibition of this pathway could explain the effects that we
found in RECs in vitro.45-46
The TZDs may also have more widespread effects that might improve their
effectiveness as inhibitors of neovascularization in PDR. Although VEGF is
thought to be the primary effector of PDR, neovascularization occurs in a
complex environment of multiple growth factors.3
The ERK-MAPK pathway is known to be a common pathway of activation by multiple
growth factors, and PPAR- ligands have been shown to inhibit migration
mediated by multiple chemoattractants in vascular smooth muscle cells, including
insulin-like growth factor I and platelet-derived growth factor; therefore,
effects of other growth factors in endothelial cells may be inhibited.45 Other possible mechanisms by which PPAR- ligands
may inhibit retinal vascular dysfunction include inhibition of macrophage
activation and inflammation,47-48
decrease in VEGF receptor expression,27 decrease
in matrix metalloproteinase 9 secretion,49
decrease in platelet aggregation,32 increase
in plasminogen activator inhibitor 1 expression,50
induction of endothelial cell apoptosis,51
and suppression of endothelin 1 secretion from endothelial cells.52
The results of the present study further support the idea that TZDs
may have beneficial effects on the diabetic patient beyond those of improving
insulin resistance by reducing or delaying the onset of complications such
as PDR. The common effect of both agents studied herein suggests that the
effect on experimental retinal neovascularization is mediated through PPAR-,
and our results suggest that much of the effect appears to be mediated downstream
of VEGF expression, possibly through inhibition of ERK1 in the ERK-MAPK pathway.
Carefully designed clinical studies should be initiated to determine whether
diabetic patients currently being treated with PPAR- ligands demonstrate
inhibition in the development or progression of PDR.
AUTHOR INFORMATION
Accepted for publication July 10, 2000.
This study was supported by grants EY01545 (Dr Hinton), EY03040 (Dr
Hinton), and HL58328 (Dr Hsueh) from the National Institutes of Health, Bethesda,
Md; and by the American Diabetes Association, Alexandria, Va (Dr Law).
The authors would like to thank Laurie LaBree, MS, for assistance in
statistical analysis; Christine Spee for culture of retinal endothelial cells;
Ernesto Barron for preparation of figures; and Susan Clarke for editorial
review.
Corresponding author: David R. Hinton, MD, Department of Pathology,
Keck School of Medicine of the University of Southern California, Los Angeles,
CA 90033 (e-mail: dhinton{at}hsc.usc.edu).
From the Departments of Ophthalmology (Dr Murata) and Pathology (Dr
Hinton), Keck School of Medicine of the University of Southern California,
Doheny Eye Institute (Dr Hinton), and the Division of Endocrinology, Diabetes
and Hypertension, University of California–Los Angeles (Ms Kim and Drs
Hsueh and Law), Los Angeles, Calif; and the Department of Ophthalmology, Kyushu
University, Fukuoka, Japan (Drs Murata, Hata, and Ishibashi). Dr Hsueh has
received honoraria from SmithKline Beecham and Dr Law, from Warner-Lambert/Parke-Davis.
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