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* Laboratory of Tumor and Development Biology, University of Liège, B-4000 Liège, Belgium;
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Campus Gasthuisberg, University of Leuven, Belgium;
Laboratory of Cytology and Histolgy, University of Liège, B-4000 Liège, Belgium; and
Department of Ophthalmology, University Hospital, Sart-Tilman, B-4000 Liège, Belgium
1Correspondence: Laboratory of Tumor and Development Biology, University of Liège, Pathology Tower (B23), Sart-Tilman, B-4000 Liège, Belgium. E-mail: vincent.lambert{at}ulg.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: angiogenesis • retinal disease • proteases • viral vector • macular degeneration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Molecular signals involved in the development of choroidal
neovascularization are not well defined. Alpha-v integrins show a
specific expression pattern during retinal angiogenesis
(1)
and antibodies to this integrin or its ligands such as
vitronectin inhibit neovascularization in different retinal models
(2
3
4)
. Among growth factors, several lines of evidence
suggest that vascular endothelial growth factor (VEGF) could be
implicated in the pathogenesis of AMD since this cytokine is present in
pathological specimens (5
, 6)
and is expressed in several
models of hypoxia-related retinal neovascularization (7)
.
However, VEGF retinal overexpression alone was not able to induce
choroidal neovascularization in a transgenic mouse model
(8)
, suggesting either a retinal specificity in the
mechanisms controlling angiogenesis or a requirement for additional
angiogenic molecules in AMD. In the intact choroid, polarized secretion
of VEGF by retinal pigment epithelium could play an important role in
the maintenance of a normal choriocapillaris (9)
. Recent
studies suggest also a role for Fas ligand in the control of choroidal
neovascularization (10)
.
Angiogenesis is an invasive process that requires proteolysis of the
extracellular matrix, proliferation, and migration of endothelial cells
with simultaneous synthesis of new matrix components. Such migratory
and tissue remodeling events are regulated by different proteolytic
systems including matrix metalloproteases (MMPs) and serine proteases
of the plasminogen/plasminogen activator system. The specific roles of
MMPs, plasminogen activators, and their inhibitors in neovascular
chorioretinopathies remain largely unexplored. In retinal pathology, a
mutation in a tissue inhibitor of MMPs (TIMP3) is associated with a
rare form of macular dystrophy (11)
, and the expression of
several MMPs has been demonstrated in human choroidal neovascular
membranes (12)
. Urokinase-type (uPA) and tissue-type (tPA)
plasminogen activators are serine proteases, both able to activate the
zymogen plasminogen into plasmin. Plasmin is a broadly acting enzyme
that degrades extracellular matrix proteins and activates pro-MMPs and
growth factors (13)
. Plasminogen activator inhibitor
type-1 (PAI-1) is the main physiological inhibitor of uPA and tPA. It
not only regulates the proteolytic activity of uPA, but also determines
the level of uPA bound to its cell surface receptor (uPAR) by promoting
the rapid endocytosis of the trimolecular uPA-PAI-1–uPAR complex
(14)
. The importance of PAI-1 for tumoral angiogenesis has
recently been demonstrated in vivo in experimental squamous
cell carcinomas (15)
. Elevated PAI-1 levels have been
correlated clinically not only with a poor prognosis in patients
suffering from a variety of cancers (16)
, but also with
various chorioretinal pathologies (17
, 18)
.
To evaluate the biological relevance of PAI-1 in subretinal
angiogenesis, we induced choroidal neovascularization in
vivo with argon laser burns (19)
into
PAI-1-/- and wild-type (WT) mice. A choroidal
neovascular membrane with leakage on fluorescein angiograms was
produced at laser impacts in WT mice but not in PAI-1-deficient mice.
In these PAI-1-/- mice, choroidal
neovascularization similar to that occurring in WT mice was restored
when PAI-1 expression was achieved by injecting a recombinant
adenoviral vector bearing PAI-1 cDNA. These observations highlight the
proangiogenic activity of PAI-1 in choroidal neoangiogenesis and
identify PAI-1 as a potential therapeutic target against AMD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. There were five or
more mice in each group. The animals were maintained with a 12 h
light/12 h dark cycle and had free access to food and water.
Laser photocoagulation and fluorescein angiograms
Choroidal neovascularization was induced in mice by laser burns
as described previously (19)
. Briefly, mice were
anesthetized with intraperitoneal (i.p.) injection of Avertin. Both
pupils were dilated with 1% topical tropicamide; three burns were
delivered (usually at the 9, 12, and 3 o’clock positions around the
optic disc) using a green argon laser (532 nm; 50 µm diameter spot
size; 0.05 s duration; 400 mW) and a cover slide as a contact
lens. Fluorescein angiograms were performed 14 days later by taking
serial fundus photographs (Canon) after i.p. injection of 0.3 ml of 1%
fluorescein sodium (Ciba, Summit, N.J.). The percentage of burns
developing late-phase hyperfluorescent spots (evaluated in comparison
with the retinal normal vasculature) corresponding to the leakage of
fluorescein from newly formed hyperpermeable vessels was evaluated.
Animals were then killed, eyes were enucleated and either fixed in
buffered 3.5% formalin solution for routine histology or embedded in
Tissue TeK (Miles Laboratories, Naperville, Ill.), and frozen in liquid
nitrogen for cryostat sectioning.
Quantitation of choroidal neovascularization
A quantitative morphometric assessment of thickness of choroidal
new vessels was carried out using a computer-assisted image analysis
system (Olympus Micro Image version 3.0 for Windows 95/NT, Olympus
Optical Co. Europe GmBH). Microscopic images (working magnification of
x200) of hematoxylin-stained eye section were acquired via a video
camera, digitalized, and analyzed. Frozen serial sections were cut
throughout the entire extent of each burn and the thickest lesions (at
least 5 sections per lesion) was used for the quantitation studies (96
sections studied). Neovascularization was estimated by the ratio (B/C)
of the thickness from the bottom of the pigmented choroidal layer to
the top of the neovascular membrane (B) to the thickness of the intact
pigmented choroid adjacent to the lesion (C). The advantage of this
method of quantification (over surface estimation) was its independence
in relation to oblique sections (see example in Fig. 2c
).
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Immunofluorescence
Cryostat sections (5 µm in thickness) were fixed first in
acetone at -20°C and then in methanol at 4°C before incubation
with the primary antibodies. Antibodies raised against mouse PECAM (rat
monoclonal antibody; PharMingen, San Diego, Calif.; diluted 1/20)
or mouse type IV collagen (guinea pig polyclonal antibody produced in
our laboratory; diluted 1/100) were incubated for 1 h at room
temperature. Antibodies to mouse PAI-1 (rabbit polyclonal antibody
produced in our laboratory, 10 µg/ml) were incubated overnight at
4°C. The sections were washed three times for 10 min in phosphate
buffered saline (PBS) before the appropriate secondary antibodies
conjugated to fluorescein-isothiocyanate (FITC) or Texas red were
added. Swine anti-rabbit (Dakopat, Glostrup, Denmark; diluted 1/40) or
rabbit anti-rat (Sigma, St. Louis, Mo.; diluted 1/40) were applied for
30 min. For double immunofluorescence-labeling studies, sections were
first incubated with the two primary antibodies and then with FITC- and
Texas red-conjugated secondary antibodies. After three washes in PBS
for 10 min each and a final rinse in 10 mM Tris-HCl buffer, pH 8.8,
coverslips were mounted and labeling was analyzed under an inverted
microscope equipped with epifluorescence optics. Staining for
ß-galactosidase activity was performed with
5-bromo-4-chloro-3-indolyl-ß-galactopyronoside (X-gal) as described
(21)
. In some assays, primary anti-PAI-1 antibodies were
preabsorbed for 15 min with recombinant PAI-1 (1 µg/ml, a generous
gift from P. Declerck, Katholieke Universiteit Leuven, Belgium).
Adenovirus-mediated PAI-1 gene transfer
Recombinant adenovirus bearing human PAI-1
(AdCMVPAI1), Escherichia coli ß-galactosidase
(AdCMVlacz) and control adenovirus (AdRR5) were generated as
described (22)
. Twenty-four hours after laser spot
production, mice were intravenously (i.v.) injected with 200 µl of
control or recombinant adenovirus (7x108 PFU).
After 5 days, blood was sampled from the right retro-orbital sinus and
PAI-1 antigen was measured by ELISA as reported (22)
. On
day 14, mice were killed and eyes were excised and processed as
described above. According to regulatory constraints, the virally
infected animals were permanently housed under BL3 containment and,
consequently, fluorescein angiograms could not be performed.
RT-PCR for PAI-1 expression
Total RNA from eyes were extracted using RNeasy Mini Kit
(Qiagen, Chatsworth, Calif.) as described by the manufacturer. PAI-1
mRNA and 28S rRNA were measured in 10 ng aliquots of total RNA using
the GeneAmp Thermostable rTth reverse transcriptase RNA PCR kit
(Perkin Elmer, Norwalk, Conn.) and two pairs of primers (Gibco BRL-Life
Technologies, Grand Island, N.Y.): 5'-AGGGCTTCATGCCCCACTTCTTCA-3'
(sense primer) and 5'-AGTAGAGGGCATTCACCAGCACCA-3' (antisense
primer) for PAI-1 and 5'-GTTCACCCACTAATAGGGAACGTGA-3' (sense primer)
and 5'-GGATTCTGACTTAGAGGCGTTCAGT-3' (antisense primer) for 28S. Reverse
transcription was performed at 70°C for 15 min, followed by 2 min
incubation at 95°C for denaturation of RNA–DNA heteroduplexes.
Amplification started by 15 s at 94°C, 20 s at 68°C, and
10 s at 72°C (35 cycles for PAI-1 and 19 cycles for 28S) and
terminated by 2 min at 72°C. RT-PCR products were resolved on 10%
acrylamide gels and analyzed using a Fluor-S MultiImager (Bio-Rad,
Hercules, Calif.) after staining with Gelstar (FMC BioProducts,
Rockland, Maine) dye. The expected size is 191 bp for PAI-1 and 212 bp
for 28S.
Statistical analysis
Data were analyzed with GraphPad Prism 3.0 (San Diego, Calif.).
The 
2 test, Student’s t test,
one-way ANOVA, and Newman-Keuls post tests were used to determine
whether there were significant (P<0.01) differences between
WT and PAI-1-/- mice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, b
) showed the appearance of newly formed microvessels with
significant leakage of fluorescein in 72% (32/44) of the laser-induced
lesions. The incidence of leaking spots was reduced to 21% (5/24) in
PAI-1-/- mice (Fig. 1c
, P<0.001). Histological analysis and immunostaining with
anti-PECAM and anti-collagen type IV antibodies confirmed the presence
of newly formed capillaries in lesions identified by fluorescein
angiography (Fig. 2
). WT mice showed typically large ‘mushroom-like’ areas of choroidal
neovascularization at the site of laser-induced trauma, with migration
of retinal pigmented epithelial cells along newly formed microvessels
(Fig. 2a
). The wound was usually not covered by a continuous
layer of retinal pigmented epithelial cells. In
PAI-1-/- mice, the choroidal neovascular
reaction was much more restricted and consisted of only a diffuse and
moderate thickening covered by confluent retinal pigment epithelium
(Fig. 2b
). In most instances, these lesions were only
indirectly identified by examination of the retinal photoreceptor
damage. Immunostaining with anti-type IV collagen or anti-PECAM
antibodies failed to show any significant angiogenesis above the
scarring tissue (Fig. 2d
). Neovascularization was estimated
by measuring, on serial sections, the maximal height of the lesion
above the choroidal layer observed in neighboring intact zones. This
was performed by determining the B/C ratio between total thickness of
lesions (‘B’ from the bottom of the choroid to the top of the
neovascular area) to the thickness of adjacent normal choroid (‘C’).
A 45% reduction of the B/C ratio was observed in PAI-deficient mice
(P<0.001) as compared to WT mice.
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PAI-1 is present in the neovascular area
Immunohistochemical staining demonstrated the presence of
PAI-1 exclusively within choroidal neovascular membrane, but not in
normal intact zones of WT mice (Fig. 3a
). Controls in which the primary antibody had been
preabsorbed with recombinant PAI-1 were negative (Fig. 3b
).
No staining was observed at any location in
PAI-1-/- mice (Fig. 3c
). RT-PCR
applied on the eyes (Fig. 3d
) demonstrated, 14 days after
injection with adenoviral vector carrying AdCMVPAI1, a weak
human PAI-1 mRNA expression in PAI-1-deficient mice.
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PAI-1 adenovirus injection restores choroidal angiogenesis
To further confirm the role of PAI-1 in choroidal
angiogenesis in vivo, WT and
PAI-1-/- mice were i.v. injected 1 day
after laser burns with either a recombinant adenovirus
(AdCMVPAI1) carrying human PAI-1 cDNA, a control virus
(AdRR5), or a vector carrying LacZ cDNA (AdCMVLacZ).
Immunostaining with anti-type IV collagen and anti-PECAM antibodies
demonstrated a significant angiogenesis in
PAI-1-/- mice injected with
AdCMVPAI1 (Fig. 4a
) but not in PAI-1-/- mice injected
with a control virus (Fig. 4b
). The injection of the
virus carrying the LacZ cDNA resulted in the expression of
ß-galactosidase in the retinal pigmented epithelium (Fig. 4c
). This demonstrates that the recombinant adenoviruses
were able to transduce the PAI-1 cDNA into cells in close proximity to
the burn. Four days after the injection of AdCMVPAI1,
the measured plasma levels of human PAI-1 were higher in
PAI-1-/- mice (mean 7340 ng/ml, range
1850–12600) than the normal murine PAI-1 value in WT mice (2 ng/ml).
PAI-1 was undetectable after 2 wk. Quantitation of the
neovascularization on frozen sections (Fig. 4d
)
showed that the human gene expression in PAI-1-deficient mice resulted
in a neovascular thickness ratio (B/C) 90% of that observed in WT
animals. In contrast, neovascularization estimated by the B/C ratio in
PAI-1-deficient mice injected with AdRR5 was similar to that observed
in PAI-1-deficient animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. MMPs and serine proteases have been
implicated in the extracellular matrix remodeling associated with
neo-angiogenesis. Since most MMPs are secreted as zymogens, their
activation requires a limited proteolysis by plasmin.
Neovascularization requires therefore a delicate balance between the
activation of the serine and metalloproteases and their inhibition by
specific inhibitors. The specific roles of MMPs, plasminogen
activators, and inhibitors in neovascular chorioretinopathies remain
largely unexplored.
Previous clinical studies detected measurable concentrations of tPA and
PAI in the aqueous humor of normal eyes (24
, 25)
. An
increase in Bruch’s membrane TIMP-3 (26)
and
interphotoreceptor matrix MMP-2 (27)
has been associated
with age-related macular degeneration. In the vitreous fluid of
diabetic patients suffering from proliferative retinopathy, elevated
concentrations of ProMMP-9 (28)
, tPA, and PAI-1
(29)
were recently reported to be associated with high
VEGF levels. As PAI-1 and TIMP-3 inhibit MMPs activation, it might have
been anticipated that they reduce choroidal angiogenesis. The apparent
paradox of increased tissue levels of PAI and TIMP-3 in choroidal
pathology associated with neovascularization suggests that their
contribution to the angiogenic ocular disorders could be different from
that anticipated. We reported recently that PAI-1 is a key
proangiogenic molecule during tumorigenesis and that PAI-1 deletion
results in the absence of tumor formation in an animal model of
squamous cells carcinoma (15)
.
We demonstrate here that PAI-1 plays an important role in choroidal neovascularization. In a model of laser-induced choroidal neovascularization, angiogenesis detected by fluorescein angiography and neovascular volume appreciated by immunohistochemistry and quantitative histology were reduced in PAI-1-deficient animals. Furthermore, restoration of PAI-1 expression in these mice by injection with recombinant adenoviruses bearing human PAI-1 cDNA led to a choroidal neovascularization identical to that observed in WT animals. It could be argued that the inflammatory reaction may play a role in the results involving adenoviral delivery. This is unlikely, as control and lacZ viruses were inefficient. Quantification of inflammatory cells in the lesions failed to demonstrate any significant difference (data not shown).
Although it has been suggested in a model of retinopathy of prematurity
that up-regulation of endogenous PAI-1 could protect from retinal and
choroidal neovascularization (30)
, in accordance with
clinical observations our results suggest, paradoxically, the opposite
effect and show that PAI-1 expression is necessary for choroidal
angiogenesis.
Taken together with results observed previously in tumoral models, our observations confirm the role of PAI-1 in the development of pathological angiogenesis. The effect of PAI-1 is indeed restricted to tumoral and ocular pathological neovascularization but is not observed in placentation, embryo-development, and wound-healing reaction (T. Frandsen, unpublished observations), which are essentially normal in PAI-1-deficient mice.
Although the exact mechanism of action of PAI-1 remains to be
elucidated, at least three different hypothesis can be formulated.
PAI-1 could prevent excessive matrix degradation against uPA-mediated
degradation, thereby providing a cell adhesion substrate for
endothelial cell migration. It has been shown in vitro that
excessive proteolysis prevents the coordinated assembly of endothelial
cells into capillary shoots (31)
. PAI-1 could also be
considered as the molecular switch that governs uPAR- and/or
integrin-mediated cell adhesion and release (32)
. Finally,
through the inhibition of plasmin, uPA, and tPA, PAI-1 could promote
angiogenesis by reducing the angiostatin generation from plasminogen
(33)
. The use of adenovirus with mutated PAI-1 forms
modulating these different pathways is one of the strategies that could
give new insight into the mechanisms of PAI-1 action in angiogenesis.
Laser-induced choroidal neovascularization in mice is useful for basic investigation of choroidal angiogenesis, although it probably differs from that occurring naturally in human AMD. Our observations nevertheless emphasize the essential role of PAI-1 in the development of subretinal neovascularization and identify PAI-1 as a potential target for therapeutic retinal anti-angiogenic strategies.
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ACKNOWLEDGMENTS |
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Received for publication June 22, 2000.
Revision received September 21, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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