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Ligands Inhibit Choroidal Neovascularization
From the Departments of 1 Ophthalmology and 2 Pathology, Keck School of Medicine of the University of Southern California and 3 Doheny Eye Institute, Los Angeles; Department of 4 Ophthalmology, Kyushu University, Fukuoka, Japan; 5 Division of Endocrinology, Diabetes and Hypertension, University of California, Los Angeles, School of Medicine.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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agonists on ocular cells
involved in the pathogenesis of choroidal neovascularization (CNV) in
vitro and on experimental laser photocoagulation–induced CNV in vivo.
METHODS. PPAR- expression in human retinal pigment epithelial (RPE) cells and
bovine choroidal endothelial cells (CECs) was determined using an RNase
protection assay and Western blot analysis. Two PPAR- ligands,
troglitazone (TRO) and rosiglitazone (RSG; 0.1–20 µM), were used to
assess effects on RPE and CEC proliferation and migration and CEC tube
formation in response to vascular endothelial growth factor (VEGF). The
effects of intravitreal injection of TRO on laser
photocoagulation–induced CNV lesions in rat eyes (15 experimental, 15
control, nine burns per eye) and cynomolgus monkey eyes (two
experimental, two control, seven paramacular burns per eye) was
assessed by fluorescein angiography and histologic evaluation.
RESULTS. PPAR-1 was expressed in both RPE and CEC. PPAR- ligands
significantly inhibited VEGF-induced migration and proliferation in
both cell types and tube formation of CEC in a dose–response manner.
CNV in rats was markedly inhibited by intravitreous injection of TRO
(P < 0.001). Lesions showed significantly less
fluorescein leakage and were histologically thinner in the TRO-treated
animals. Similar findings were present in the TRO-treated lesions in
two monkey eyes. The drug showed no apparent adverse effects in the
adjacent retina or in control eyes.
CONCLUSIONS. The inhibition of VEGF-induced choroidal angiogenesis in vitro, and CNV
in vivo by PPAR- ligands suggests the potential application of these
agents in the large group of patients with age-related macular
degeneration complicated by CNV.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Thiazolidinediones (TZDs) are a novel class of drugs that can be used
to improve insulin resistance in non–insulin-dependent diabetes
mellitus (NIDDM).9
10
Several members of the TZD class,
including troglitazone (TRO) and rosiglitazone (RSG, formerly BRL
49653), have been or are currently available for clinical use in the
treatment of NIDDM.11
12
TRO and RSG are synthetic
high-affinity ligands for peroxisome proliferator–activated receptor
(PPAR)-, a nuclear hormone receptor that is a member of the PPAR
family.13
14
15
PPAR- acts by forming a heterodimer with
the retinoid X receptor and binding to direct repeats of hormone
response elements.10
16
PPAR- is expressed most
abundantly in adipose tissue, where it promotes adipocyte
differentiation and regulates the expression of genes involved in fatty
acid metabolism.10
17
Recent studies have shown a more
widespread distribution of PPAR- receptors, suggesting that PPAR-
ligands may have effects in other tissues.17
18
19
These
actions include inhibition of inflammatory processes in
macrophages,20
21
22
neointima formation after vascular
balloon injury,23
and growth of cancer
cells.24
25
26
27
28
The localization of PPAR- in specific
retinal cell types has not been previously determined.
Increasing evidence suggests that PPAR- ligands have direct effects
on endothelial cells (ECs). ECs express PPAR- mRNA and
protein,29
30
and PPAR- ligands inhibit growth
factor-induced proliferation of ECs,31
increase
plasminogen activator inhibitor (PAI)-1 expression,30
and
suppress endothelin-1 secretion by ECs.32
PPAR- ligands
inhibit growth factor–induced tube formation in human umbilical vein
endothelial cells (HUVECs) and reduce VEGF receptor (VEGF-R)
expression.29
The mitogen-activated protein kinase (MAPK)
pathway is activated in ECs after stimulation by VEGF33
34
and may play an important role in cellular migration35
;
PPAR- ligands inhibit MAPK-dependent migration of smooth muscle
cells36
and may similarly inhibit this pathway in ECs.
Most recently, data supporting an antiangiogenic effect of a PPAR-
ligand (15-deoxy-Delta12,14-prostaglandin J2
[15d-PGJ2]) in vivo were provided by
using a short-term VEGF-pellet corneal micropocket
assay.29
Laser photocoagulation has been used successfully in animals to produce
retinal lesions similar to those of human CNV.37
38
In
both human and experimental CNV, a subretinal membrane forms that is
composed of choroidal ECs (CECs) and RPE cells, is associated with
local endogenous expression of VEGF, and leaks fluorescein for an
extended period.4
37
38
39
40
In this study, both CECs and RPE
cells expressed PPAR- and were inhibited in their response
to VEGF by PPAR- ligands. In this well-established model of a common
chronic neovascular disease, intraocular administration of the PPAR-
ligand TRO had a prominent antiangiogenic effect on the development of
CNV, without apparent toxicity to the adjacent retina.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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CECs were isolated from bovine eyes for functional assays and Western Blot and from human fetal eyes for RNase protection assays (RPA)s. Cells were confirmed to be vascular ECs by positive immunostaining for von Willebrand factor and by binding of dil-acetylated low-density lipoprotein (LDL).42 Epithelial contamination was excluded by staining for cytokeratin. The CECs were isolated using magnetic beads carrying specific endothelial markers, as previously described.42 Lycopersicon esculentum (Sigma) was used for isolation of bovine CECs, and anti-CD31 antibody (Dako, Carpinteria, CA) was used for isolation of human CECs.
RNase Protection Assays
Antisense RNA probes were prepared from pGEM-3 cloning vectors
containing partial cDNA probes for PPAR- (kindly provided by
Jeffrey Flier, Harvard University, Cambridge, MA). RPA vectors
were linearized with either HindIII or EcoRI to
prepare antisense probe and sense RNA reference, respectively.
Antisense probes and sense RNAs were transcribed in vitro from
linearized template DNA using bacteriophage T7, Sp6 (Promega, Madison
WI), or T3 (Stratagene, La Jolla, CA) RNA polymerases. Antisense probes
were used in an RPA to detect PPAR- and -
based on the sizes of
protected bands: human PPAR-1, 258 bases; human PPAR-2, 348
bases; human retinoid X receptor (RXR)-, 101 bases; and
human reduced glyceraldehyde-phosphate dehydrogenase (GAPDH), 96 bases.
GAPDH was included as a housekeeping mRNA in RPA to confirm the
integrity of RNA in different samples.
For RPA, [32P] UTP-labeled RNA probes were incubated with 10 µg RNA in a final volume of 25 µl of hybridization solution for 12 to 16 hours at 56°C. Samples were then incubated for 1 hour at 45°C with a mixture of RNase A (90 µg/ml) and RNase T1 (600 U/ml). Nucleases were inactivated by treating samples with 10 mg/ml proteinase K for 1 to 2 hours at 37°C. After precipitation with ethanol, samples were resuspended in 7 µl gel-loading buffer (25% Ficoll, 0.2 M EDTA [pH 8], 0.25% bromophenol blue, 0.25% xylene cyanol). Samples were then electrophoresed on 6% nondenaturing polyacrylamide gels at 200 to 250 V for 2 hours. After electrophoresis, gels were dried and protected bands visualized by autoradiography.
Western Immunoblot Analysis
Protein extracts from CECs and RPE cells grown in vitro were lysed
in a buffer containing 50 mM sodium pyrophosphate (pH 7.4), 50 mM NaCl,
50 mM NaF, 5 mM EDTA, 5 mM EGTA, and 100 µg/ml leupeptin before they
were flash frozen in a dry ice and ethanol bath. After thawing, cells
were scraped off the dish and centrifuged at 14,000 rpm. Nuclear and
cytosolic fractions were prepared by the method of Dignam et
al.43
Protein concentrations were determined using the
Bradford assay. Full-length in vitro translated PPAR-1 was used as a
positive control. Equal amounts of proteins were separated by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes using a transblotter (Bio-Rad, Richmond, CA).
Nonspecific binding was blocked by using 5% fat-free milk powder and
0.1% Tween 20 in Tris-buffered saline. Membranes were incubated with a
rabbit polyclonal anti-PPAR- antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) at a concentration of 1:1000 for 2 hours in 0.2 M
Tris-HCl (pH 7.5), 0.5 M NaCl buffer containing 5% fat-free milk
powder and 0.1% Tween 20. Blots were washed and incubated for another
hour with a goat anti-rabbit horseradish peroxidase–conjugated
antibody (1:500) before final development using the chemoluminescent
detection (Lumi-Glo; Kirkegaard & Perry, Gaithersburg, MD).
Effect of TRO and RSG on Cell Proliferation
To determine the effect of PPAR- ligands on proliferation of
CECs, a [3H]thymidine uptake
assay23
was performed in the presence or absence of TRO or
RSG (0, 0.1, 1, 10, and 20 µM). Each experiment was performed in
quadruplicate and repeated three times.
Effects of TRO and RSG on Migration
CEC and RPE cell migration was examined in cell culture chambers
(Transwell; Costar Cambridge, MA). The polycarbonate membrane insert
(with 8-µm pores) was coated with fibronectin (5 µg/well). Fifty
thousand cells were added to the upper compartment of each well, and
cells were then incubated at 37°C (95% air-5%
CO2). Chemotaxis was induced by the addition of
VEGF (10 ng/ml) to the lower compartment in the presence or absence of
TRO or RSG (0, 0.1, 1, 10, and 20 µM). After 6 hours, the filters
were washed with PBS and fixed with methanol (10 minutes at 4°C),
followed by counterstaining with hematoxylin. The number of CECs and
RPE cells per field that migrated to the lower surface of the filters
was determined microscopically (four fields per filter). Experiments
were performed in triplicate and were repeated at least three times.
Effect of TRO on Capillary-Like Tube Formation of CECs
Three-dimensional tube formation was measured as previously
described.41
A collagen mixture was placed in 24-well
plates and incubated at 37°C for 3 hours to form gels. After
polymerization of the gels, 1.0 x 105 CECs
were seeded on each gel and incubated with endothelial basal medium
(EBM) with 2% FBS overnight. The medium was then aspirated,
and the remaining cells were overlaid with more gel (120 µl/well).
EBM (1.2 ml) with 2% FBS, VEGF (10 ng/ml), and TRO (0–20 µM) was
added to each well, and the medium was changed every 2 days. To
evaluate the lengths of the capillary-like tubes in the gels,
photographs were taken on day 5 with a phase-contrast microscope (nine
random x10 fields). The lengths of the tubes were measured with an
image analyzer (Cosmozone 1S; Nikon, Tokyo, Japan) and were expressed
as an average length ± SD per well.
Effect of Intravitreal Injection of TRO on Development of CNV
Induced by Photocoagulation
Thirty male pigmented rats (Brown Norway) and four cynomolgus
monkeys were used. All procedures were conducted in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic and Vision Research
and were approved by the University of Southern California
Institutional Care and Use Committee. The rats were anesthetized with
ketamine hydrochloride (45 mg/kg) and xylazine hydrochloride (10
mg/kg). The monkeys were anesthetized with ketamine hydrochloride (24
mg/kg), acepromazine maleate (0.005 mg/kg), and atropine sulfate (0.03
mg/kg). In rats, nine burns of diode laser photocoagulation (75-µm
spot size, 0.1-second duration, 100 mW) were delivered to the retina,
lateral to the optic disc through a slit lamp using a coverglass as a
contact lens. Only lesions in which subretinal bubble or focal serous
detachment of the retina developed were used for experiments. Fifteen
rats received intravitreal injection of TRO (25 µM in 20 µl
dimethyl sulfoxide [DMSO]), on days 1, 3, and 7, whereas the
remaining 15 rats received only vehicle. In monkeys, seven
photocoagulation burns (75 µm spot size, 0.1-second duration, 300 mW)
were delivered in the paramacular region. Two eyes received
intravitreous injection of TRO (250 µM in 100 µl DMSO) immediately
after photocoagulation, whereas two other eyes received only vehicle.
In the monkeys, a single injection of TRO was given to decrease the
chance of traumatic cataract formation. CNV formation was detected as
fluorescein leakage from the photocoagulation lesions in fluorescein
angiography at 2 weeks (rats) and at 3 months (primates).
Quantitation of Fluorescein Angiograms
The effect of drug treatment was evaluated by semiquantitative
assessment of late-phase (100–140 seconds after dye injection in rats
and 5–5.5 minutes after dye injection in monkeys) fluorescein
angiography, as previously described.44
Leakage was
defined as the presence of a hyperfluorescent lesion that increased in
size with time in the late-phase angiogram. Angiography was graded in a
masked fashion by two examiners using reference
angiograms.44
When the two scores for a lesion did not
coincide, the higher score was used. Discrepant scoring was observed on
only 12 of 142 lesions and was never greater than one grade. Angiograms
were graded as follows: 0, no leakage; 1, slight leakage; 2, moderate
leakage; 3, prominent leakage.
Histopathologic Study of the Retina
Fourteen days after photocoagulation, the rats were killed and
eyes were enucleated and fixed in 0.2% glutaraldehyde and 2%
paraformaldehyde in 0.1 M PBS (pH 7.4). Tissue samples were dehydrated
and embedded in paraffin. Ten 3-µm serial sections were cut
perpendicular to the retina from the central part of the lesion
exhibiting the thickest laser-induced retinal
destruction.44
The maximal thickness of the lesions from
the disrupted RPE layer to the innermost aspect of the lesion was
measured using an objective micrometer. In primates, 3 months after
laser photocoagulation, one eye was fixed with 0.2% glutaraldehyde and
2% paraformaldehyde in 0.1 M PBS (pH 7.4) immediately after
enucleation. The dissected macular regions were dehydrated in a series
of graded alcohols and embedded in glycol methacrylate. Two- to
three-micrometer sections were stained with periodic acid–Schiff.
Electroretinograms
Electroretinograms (ERG) were obtained for all primate eyes at
baseline, and soon after photocoagulation and TRO injection. The pupils
were dilated with topical 1% tropicamide sodium and 2.5%
phenylephrine hydrochloride followed by dark adaptation for 30 minutes.
Scotopic and photopic ERG were then obtained.
Statistical Evaluation
In Vitro Studies.
In all analyses the normality for each group of numbers was tested
using the Shapiro–Wilk statistic. Independent-sample t-test
P was used to compare the control group and each of the TRO-
and RSG-treated groups with the VEGF treatment group. Because four
treatment groups were compared with the VEGF group, the accepted level
of significance for all tests was = 0.05/4 = 0.0125.
In Vivo Studies.
The score per lesion and the percentage of lesions with leakage were
calculated for each animal. Independent-sample t-tests were
used to compare mean score per lesion, percentage leakage, and
thickness between the TRO and control groups. Statistical significance
was defined for < 0.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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in Retinal Cells Involved in CNV is expressed by cultured human RPE
cells and CECs, we analyzed RNA from these sources using a sensitive
RPA. As shown in Figure 1
, human CECs and RPE cells exclusively express PPAR-1 mRNA as is
evidenced by the single protected band of 258 bases detected by RPA. In
contrast, both PPAR-1 and -2 (348-base protected band) mRNAs were
expressed in untreated and phorbol 12-myristate 13-acetate
(PMA)-stimulated human monocytes. Significantly longer exposures failed
to reveal the expression of PPAR-2 in CEC or RPE. Both RPE cells and
CECs expressed mRNA for RXR (101-base protected band), a
heterodimeric partner of PPAR-.
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protein in bovine CECs and
human RPE cells, we performed Western immunoblot analysis of nuclear
and cytosolic proteins. The data in Figure 2
reveal a band of 52 kDa corresponding to PPAR-1 that localizes
almost exclusively to the nuclear fraction.
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Ligands on Thymidine Incorporation
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Activation Inhibits VEGF-Induced Migration of Retinal
Cells
PPAR- Activation Inhibits VEGF-Induced Tube Formation of ECs
Assembly of ECs into tubes is required for the formation of new
vessels. VEGF (10 ng/ml) induced tube formation by CECs in a
three-dimensional assay (P < 0.001; Fig. 5
). Histologic confirmation of tube formation was obtained by microscopic
examination of frozen sections of the gels (results not shown). Length
of the tubes was measured with and without TRO in the presence of VEGF
after 5 days of growth. TRO prominently inhibited tube formation at 10
µM (P < 0.001). At 20 µM, essentially no tube
formation occurred. No morphologic evidence of cell death was observed.
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When the retina and choroid of pigmented rats was exposed to intense laser photocoagulation, CNV developed in approximately 50% (34/64) of laser spots, determined by the presence 2 weeks later of well-defined lesions showing late-phase angiographic leakage of fluorescein dye (Fig. 6) . Rats treated with intravitreous injection of TRO (20 µM) on days 1, 3, and 7 showed approximately 70% inhibition in the number of leaking lesions that developed (P < 0.0001; Table 1 ). The average leakage score per lesion was also significantly less in the TRO-treated animals (P < 0.0001).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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PPAR- has very recently been recognized as a potential target for
the pharmacologic therapy of pathologic
neovascularization.29
PPAR- mRNA and protein are
expressed by HUVECs, and their activation by PPAR- ligands
inhibits the differentiation of HUVECs into tubelike
structures.29
30
Furthermore, PPAR- ligands inhibit
VEGF-induced angiogenesis in rat cornea.29
We demonstrated the expression of PPAR- and its heterodimeric
partner RXR in RPE and in ECs derived from the choroid. The PPAR-
gene produces two mRNA species by alternative promoter usage, each
containing different 5' exons that are spliced onto common downstream
exons. PPAR-2 differs from -1 by the presence of 30 additional
amino acids at its N terminus.46
Although both isoforms
are expressed in abundant levels in human adipose tissues, PPAR-1
expression is typically much higher than PPAR-2 in nonadipose
tissues. Consistent with these findings, both CECs and RPE cells
express only PPAR-1 mRNA. At this time, the functional differences
between the two isoforms are unknown.10
PPAR- can be activated by poorly defined, naturally occurring
ligands, or by members of the TZD class of drugs.10
The
antidiabetic action of TZDs appears to be primarily mediated through
activation of PPAR-; however, TRO is distinguishable from other TZD
PPAR- ligands, because it also contains an -tocopherol moiety
which could have effects due to its antioxidant
properties.47
Therefore in this study, in vitro
experiments were also performed using RSG, a TZD that has no
-tocopherol moiety.
Neovascularization is a multistep process that includes degradation of
basement membrane and proliferation, migration, and tube formation by
ECs; the process is stimulated by a variety of growth factors and
cytokines.48
VEGF plays a prominent role in the induction
of ocular neovascularization and has been identified, and localized to
RPE cells in surgically excised CNV membranes.4
In the
retina, high-affinity VEGF-Rs are predominantly localized on ECs.
Recently, functional KDR and flt-1 VEGF-Rs have also been identified on
RPE cells, and because RPE cells are a major source of VEGF in
exudative AMD, it has been suggested that VEGF may also be an autocrine
growth factor for RPE cells in AMD, as well as a paracrine factor for
the choroidal endothelium.45
TRO and RSG both induced a
strong dose-dependent inhibition of endothelial cell growth and
migration in response to VEGF, providing strong support for the
contention that this effect is mediated through PPAR-. Similar to
previous studies concerning the effects of basic fibroblast growth
factor (bFGF) on smooth muscle cell proliferation, RSG demonstrated
stronger antiproliferative effect than TRO at lower concentrations (1
µm), consistent with its stronger binding affinity to
PPAR-.49
The additional benefit of TRO over RSG, seen
at concentrations of 10 µm and above, could be due to other
non-PPAR-–mediated mechanisms that have been observed recently in
other cell types. TRO contains a vitamin E (-tocopherol)
moiety,47
and we have previously shown that vitamin E
(-tocopherol) succinate inhibits both the proliferation and
migration of RPE cells in vitro.50
The effect of higher
concentrations of TRO on tube formation was particularly strong,
suggesting that TRO may be acting at multiple levels to inhibit this
complex process.
It is important to note that RPE cells were similarly inhibited by the
PPAR- ligands in their response to VEGF. RPE cells are one of the
few normal cell types, other than ECs, that express high-affinity
VEGF-Rs and respond to VEGF as a mitogen and chemotactic
agent.45
The RPE cells occupy a critical position at the
interface between the neural retina and the choroid. In neovascular AMD
they proliferate, migrate from the monolayer, and secrete VEGF, making
them a central regulator of the neovascular process.4
39
Thus, inhibiting their growth can help to prevent a potentially
initiating and amplifying event in the neovascularization process.
The molecular basis for the inhibition of CEC and RPE cell
proliferation and migration and CEC tube formation is currently
unclear. It is likely that PPAR- ligands act by inhibition of
transcription factor function and that this effect is mediated by their
central DNA-binding domain that recognizes response elements in
promoters of specific target genes.10
We have previously
shown that TRO inhibits the activity of ELK-1, an ets-family
transcription factor, after mitogenic stimulation of vascular smooth
muscle cells by bFGF. Similarly, PPAR- ligands regulate certain
macrophage functions through transrepression of several transcription
factors, including nuclear factor (NF)-
B and AP-1.21
Only one previous study has reported the effect of PPAR- ligands on
angiogenesis in vivo. In that study of experimental corneal
angiogenesis, hydron pellets containing VEGF with or without a PPAR-
ligand were implanted in a corneal pocket and neovascular area measured
after 6 days. 15d-PGJ2, a putative physiologic
PPAR- ligand significantly inhibited VEGF-induced neovascular area
in this model. Although such an assay provides support for the
potential use of these ligands in modulating angiogenesis, a more
stringent test of their efficacy is provided by use of an animal model
in which angiogenesis is generated by naturally occurring wound healing
responses. The laser photocoagulation–induced CNV model, originally
described in the monkey37
and more recently translated to
the rat,38
44
induces a localized wound-healing response
in the outer retina. Pathologic studies of these lesions reveal
evidence of migration and proliferation of RPE cells, infiltration by
macrophages, increased local expression of VEGF, and growth of new
vessels into the subretinal space.37
38
39
40
These membranes
have similar complex pathologic features in common with human CNV
membranes,4
and they exhibit leakage of fluorescein dye, a
dysfunction critically associated with active human CNV
membranes.40
Our demonstration that intravitreal injection
of TRO (days 1, 3, and 7) in rats dramatically inhibited both the
percentage of lesions that show leakage as well as the amount of
leakage per lesion makes a strong case for the functional value of this
drug in the inhibition of pathologic neovascularization in the complex
environment of the damaged outer retina. The functional study is
supported by the histologic study demonstrating that the lesions were
also thinner than those from untreated control eyes and contained
reduced populations of RPE cells and CECs. The mechanism by which TRO
inhibited CNV formation in this model is likely to be multifactorial.
TRO inhibited VEGF-induced CEC and RPE cell migration and
proliferation, which are critical features of the pathogenesis of CNV.
In vivo this direct effect may be augmented by inhibition of VEGF-R
expression; HUVECs treated with 15d-PGJ2 showed
decreased expression of both VEGF-R1 and -R2.28
TRO may
also inhibit extracellular matrix degradation, a process critical to
neovascularization; PPAR- ligands are strong inhibitors of
macrophage activation and have been shown to reduce matrix
metalloproteinase (MMP)-9 activity in these cells.22
They
also have been shown to inhibit proteolysis by inhibiting expression of
urokinase plasminogen activator and stimulating expression of
plasminogen activator inhibitor-1 in HUVECs.29
It is
likely that a combination of effects resulting from inhibition of
macrophage, RPE, and endothelial cell activation leads to the
antiangiogenic response of PPAR- ligands.
The significant differences between the retinal structure of the rat
and primates led us to assess the effects of PPAR- ligand treatment
in a primate model. In particular, the macula, a region specialized for
color and detailed vision in the primate is not found in the rat eye.
In humans, CNV membranes in exudative AMD occur predominantly in the
macular region; therefore, our primate studies were performed using
paramacular laser photocoagulation. No evidence of acute toxicity was
found by electrophysiology (ERG) in either the rat or primate models.
Electrophysiologic studies were not performed at later time points, and
late functional abnormalities therefore cannot be ruled out; however,
light microscopic histologic studies at day 14 in the rats and at 3
months in the primates showed no abnormalities in retinal regions away
from the laser burns, including the macula in the primate eyes. Of
interest, TRO treatment of laser-induced CNV lesions in the monkey
resulted in a similar trend of inhibition of fluorescein leakage,
although the number of animals required to demonstrate a statistical
effect could not be studied. The apparent decrease in effectiveness of
the drug in the monkey may have been a result of the single injection
of TRO compared with three injections in the rat. The intraocular route
of administration of TRO is practical for model systems, because the
drug is localized to the site of disease; however, systemic
administration is more appropriate for AMD patients.44
We have demonstrated the efficacy of PPAR- ligands in the inhibition
of cells critically involved in the pathogenesis of CNV in vitro and
the dramatic effect of one of these ligands (TRO) on CNV formation and
function in vivo. Together, these studies implicate PPAR- as an
important potential target for therapy in exudative AMD. The
significance of this study may have widespread implication in other
neovascular disorders.2
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Acknowledgements |
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Footnotes |
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Submitted for publication September 23, 1999; revised January 13, 2000; accepted February 10, 2000.
Commercial relationships policy: C3 (REL, WAH).
Corresponding author: David R. Hinton, Department of Pathology, Keck School of Medicine of the University of Southern California, 2011 Zonal Avenue, HMR 209, Los Angeles, CA 90033. dhinton{at}hsc.usc.edu
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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in rat and human vascular smooth muscle cells Circulation 101,1311-1318This article has been cited by other articles:
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  L. Q. Shen, A. Child, G. M. Weber, J. Folkman, and L. P. Aiello Rosiglitazone and Delayed Onset of Proliferative Diabetic Retinopathy Arch Ophthalmol, June 1, 2008; 126(6): 793 - 799. [Abstract] [Full Text] [PDF]
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 F. Biscetti, E. Gaetani, A. Flex, T. Aprahamian, T. Hopkins, G. Straface, G. Pecorini, E. Stigliano, R. C. Smith, F. Angelini, et al. Selective Activation of Peroxisome Proliferator-Activated Receptor (PPAR){alpha} and PPAR{gamma} Induces Neoangiogenesis Through a Vascular Endothelial Growth Factor-Dependent Mechanism Diabetes, May 1, 2008; 57(5): 1394 - 1404. [Abstract] [Full Text] [PDF]
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 S. Z. Duan, M. G. Usher, and R. M. Mortensen Peroxisome Proliferator-Activated Receptor-{gamma}-Mediated Effects in the Vasculature Circ. Res., February 15, 2008; 102(3): 283 - 294. [Abstract] [Full Text] [PDF]
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N. Mochizuki and Y.-G. Kwon 15-Lipoxygenase-1 in the Vasculature: Expanding Roles in Angiogenesis Circ. Res., February 1, 2008; 102(2): 143 - 145. [Full Text] [PDF]
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