Abnormal Vessel Formation in the Choroid of Mice
Lacking Tissue Inhibitor of Metalloprotease-3
Andreas Janssen,1 Julia Hoellenriegel,1 Marton Fogarasi,1 Heinrich Schrewe,2,3
Mathias Seeliger,4 Ernst Tamm,5 Andreas Ohlmann,5 Christian Albrecht May,6
Bernhard H. F. Weber,1 and Heidi Stöhr1
PURPOSE. Tissue inhibitor of metalloprotease (TIMP)-3 is an
inhibitor of matrix metalloprotease (MMP) and regulates angiogenesis. In the eye, TIMP3 is tightly associated with Bruch’s
membrane. In this study, the authors analyzed mice lacking
TIMP3 for retinal abnormalities.
METHODS. Mice with targeted disruption of the Timp3 gene
were generated (Timp3⫺/⫺) and bred into C57/Bl6 and CD1
backgrounds. Eyes were analyzed by light and electron microscopy. Vasculature was examined by scanning laser ophthalmoscopy, corrosion casts, and whole mount preparations.
MMP activity was assessed by in situ zymography, angiogenic
potential was evaluated by tube formation, and aortic ring
assays and signaling pathways were studied by immunoblotting.
RESULTS. TIMP3-deficient mice develop abnormal vessels with
dilated capillaries throughout the choroid. Enhanced MMP
activity in the choroid region of Timp3⫺/⫺ eyes was detected
when compared with controls. Timp3⫺/⫺-derived tissue
showed an increased angiogenic activity over wild-type, an
effect that could specifically be inhibited by recombinant
TIMP3. Moreover, the antiangiogenic property of TIMP3 was
demonstrated to reside within the C-terminal domain. When
VEGFR2 inhibitor was added to Timp3⫺/⫺ aortic explants,
endothelial sprout formation was markedly reduced, which
provided evidence for an unbalanced VEGF-mediated angiogenesis in Timp3⫺/⫺ animals. Finally, angiogenic signaling
pathways are activated in Timp3⫺/⫺-derived cells.
CONCLUSIONS. These findings suggest that the distinct choroidal
phenotype in mice lacking TIMP3 may be the result of a local
disruption of extracellular matrix and angiogenic homeostasis,
and they support an important role of TIMP3 in the regulation
From the 1Institute of Human Genetics and the 5Department of
Anatomy, University of Regensburg, Regensburg, Germany; 2MaxPlanck Institute for Molecular Genetics, Berlin, Germany; 3Institute of
Medical Genetics, Charité-University Medicine Berlin, Berlin, Germany;
4
Retinal Electrodiagnostics Research Group, Department of Pathophysiology of Vision and Neuroophthalmology, University of Tübingen,
Tübingen, Germany; and the 6Institute of Anatomy, Technical University of Dresden, Dresden, Germany.
Supported by grants from the Deutsche Forschungsgemeinschaft
(SFB581/TP13).
Submitted for publication November 9, 2007; revised February 22,
2008; accepted May 19, 2008.
Disclosure: A. Janssen, None; J. Hoellenriegel, None; M. Fogarasi, None; H. Schrewe, None; M. Seeliger, None; E. Tamm, None;
A. Ohlmann, None; C.A. May, None; B.H.F. Weber, None; H. Stöhr,
None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Heidi Stöhr, Institute of Human Genetics,
University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany; [email protected].
2812
of choroidal vascularization. (Invest Ophthalmol Vis Sci. 2008;
49:2812–2822) DOI:10.1167/iovs.07-1444
T
issue inhibitor of metalloprotease (TIMP)-3 was initially
identified as a 21-kDa protein that strongly binds to the cell
substratum of cultured chicken embryo fibroblasts infected
with Rous sarcoma virus.1 Further studies revealed complex
and multifunctional properties of this molecule. TIMP3 is the
only member of the TIMP protein family that exclusively localizes to the extracellular matrix (ECM), a dynamic fibrillar structure that surrounds cells and exhibits scaffolding and signaling
functions. Within the ECM, TIMP3 forms noncovalent complexes with numerous secreted and membrane-type matrix
metalloproteinases (MMPs; e.g., MMP-1, -2, -3, -9, MT1MMP),2,3 a large group of zinc-dependent endopeptidases. The
N-terminal domain of TIMP3 binds the active site of MMPs to
inhibit their proteolytic activity.4 In addition, the C-terminal
domain of TIMP3 interacts with MMP2 and MMP9 proenzymes
to facilitate their activation.5,6 MMPs are responsible for the
enzymatic digestion of ECM components and the liberation of
signaling molecules from the ECM. Coordinating local MMP
and TIMP levels is crucial for normal ECM remodeling, which
is involved in various physiological processes including development, wound healing, angiogenesis, apoptosis, migration,
and ovulation. As a consequence, disturbances in the delicate
MMP/TIMP balance underlie a number of pathologic conditions such as arthritis, cardiovascular disease, and tumor invasion. Besides MMPs, TIMP3 inhibits selective members of the
ADAM family (a disintegrin and a metalloproteinase domain)
and aggrecanases (ADAM with thrombospondin-like repeats).7
Notably, TIMP3 binding of ADAM17 (alias TNF-␣– cleaving enzyme [TACE]) prevents the shedding of TNF-␣ from its receptor and, thus, suggests a role for TIMP3 in inflammation.8
Recently, mice carrying a targeted deletion of the Timp3
gene have been generated to examine the role of this protein
in vivo.9 TIMP3-deficient animals develop spontaneous pulmonary air space enlargement accompanied by an enhanced degradation of collagen in the peribronchiolar space.9 In addition,
a reduced number of bronchioles and dilated tubes indicates an
impaired bronchiole branching morphogenesis in the null
mouse.10 TIMP3 ablation in the mouse also causes spontaneous
left ventricular dilation, cardiomyocyte hypertrophy, and contractile dysfunction reminiscent of human dilated cardiomyopathy.11 The pathogenic changes observed in lung and heart are
believed to be caused by enhanced matrix degradation by
MMPs and uncontrolled cleavage of the cytokine TNF-␣ by
TACE in the absence of TIMP3. Elevated TNF-␣ levels in TIMP3null mice may also be responsible for chronic hepatic inflammation and impaired liver regeneration after partial hepatectomy12 and enhanced inflammatory response in antigen-induced
arthritis.13 TIMP3-deficient mice have also been used to investigate the role of TIMP3 in tumorigenesis. The absence of
TIMP3 in host stroma was demonstrated to promote tumor
growth with increased angiogenesis and extensive vessel dilaInvestigative Ophthalmology & Visual Science, July 2008, Vol. 49, No. 7
Copyright © Association for Research in Vision and Ophthalmology
IOVS, July 2008, Vol. 49, No. 7
Choroidal Abnormalities in TIMP3-Deficient Mice
2813
FIGURE 1. Targeted disruption of the
mouse Timp3 gene. (A) The targeting construct contains a neomycin
resistance cassette (PGKneobpA) inserted into the HincII (H) site of
exon 3 (E3) of the Timp3 gene and a
HSV-TK expression cassette at the 3⬘
end for negative selection. Positions
of the 5⬘ and 3⬘ primer pairs used to
PCR screen for correctly targeted alleles in ES cells and offspring of chimeric animals, as well as the sizes of
the PCR products, are indicated. (B)
Exemplary PCR amplification of
genomic DNA from tails of Timp3⫹/⫹
and Timp3⫺/⫺ mice confirm the correct targeting of Timp3 in the knockout animals. (C) Western blot analysis of ECM extracts obtained from
primary fibroblast cultures of wildtype and Timp3⫺/⫺ mice. TIMP3 protein is absent in Timp3⫺/⫺ ECM. Immunoblot with laminin B2 antibodies
served as a control.
tion within the tumor.14 Furthermore, TIMP3-null mice exhibit
enhanced metastatic colonization by tumor cells.15 The promotion of different stages of tumor development has been
attributed to the loss of TIMP3 protease inhibitory activity.
Earlier studies have shown that overexpression of recombinant
TIMP3 in cancer cells inhibits tumor growth in vivo16 and that
TIMP3 gene expression is reduced in several human tumors
because of promoter hypermethylation.17 Taken together,
these findings indicate an important but complex role of
TIMP3 in cancer development and progression.
One of the few recognized cellular activities of TIMP3 that
seems to be independent of its inhibitory activity on proteolytic enzymes is its ability to specifically bind VEGFR2 in vitro,
thereby blocking the interaction between VEGFR2 and its
ligand VEGF.18 This feature of TIMP3 is considered to be
responsible for the suppression of VEGF-mediated angiogenesis.19 A direct link between the antiangiogenic effect of TIMP3
and the inhibition of VEGF/VEGFR2 interaction in vivo, however, still requires confirmation.
The RPE in the outer eye wall is one of the major sites of
TIMP3 expression.20 After secretion from RPE cells, TIMP3
accumulates in Bruch’s membrane,21 a five-layered sheet of
connective ECM material located between the RPE and the
choroid. Here, TIMP3 is assumed to be one of the crucial
regulators of Bruch’s membrane turnover. An excessive
amount of TIMP3 protein in abnormal Bruch’s membrane deposits was found in a human donor eye with Sorsby fundus
dystrophy (SFD),22 a late-onset autosomal dominant macular
disease caused by mutations in TIMP3.23 In addition, increased
thickening of Bruch’s membrane and breakdown of the RPE
microvilli labyrinth has been observed in mice carrying an
SFD-related mutation (S156C) in the murine Timp3 gene.21 In
SFD patients but not in Timp3S156C knock-in mice, new thinwalled blood vessels frequently grow into the subretinal space,
causing severe macular damage and blindness. To further understand the physiological role of TIMP3 at the level of the
RPE/Bruch’s membrane/choroid interface, we have generated
mice with a targeted disruption of the Timp3 gene independently of a previous model9 and the eye phenotype analyzed
for pathologic abnormalities.
MATERIALS
AND
METHODS
Generation of Timp3ⴚ/ⴚ Mice
The Timp3 gene locus was isolated and cloned from a 129/SvJ mouse
genomic DNA library, as described earlier.21 The neor expression
cassette of plasmid PGKneobpA was inserted into the HincII site of
exon 3 (Fig. 1A). To permit negative selection against random integration, the HSV-TK expression cassette was introduced at the 3⬘ end of
the targeting construct. After linearization, the construct was transfected into CJ7 embryonic stem (ES) cells. Clones positive for correct
homologous recombination events were identified by PCR screening
of ES cell DNA using primer pair mTimp3-IVS-F2 (5⬘-CAG GAG GCT
TTA AGG ACA TTG-3⬘)/PGKneoR (5⬘-ATG TGG AAT GTG TGC GAG
G-3⬘) and primer pair PGKneoF1 (5⬘-CGC ATT GTC TGA GTA GGT
GTC A-3⬘)/mTimp3-UTR-R1 (5⬘-TTG CCA GCT CAG AAG TTC TG-3⬘) to
amplify 5⬘ and 3⬘ fragments, respectively, external to the targeting
2814
Janssen et al.
construct (Figs. 1A, 1B). The injection of mutant ES cells into C57BL/6
blastocysts, the generation of chimeras, and the breeding to C57BL/6
mice was performed as described elsewhere.21 Genotyping of animals
was performed by PCR amplification of DNA extracted from mouse
tails, as described. Expression of TIMP3 and laminin B2 in ECM extracts
from murine fibroblasts was monitored by Western blotting using
rabbit anti–mouse TIMP3 and mouse anti–mouse laminin B2 (Upstate
Biotechnology, Lake Placid, NY) antibodies, as described.24 Before
analysis, mice carrying the mutant allele were backcrossed more than
six times into the C57BL/6 and the CD1 strains. Mice were housed
under specific pathogen-free conditions at the Central Animal Facility
of Regensburg University and maintained under the guidelines established by the institution for their use. All experiments were performed
in accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
Light and Electron Microscopy and Scanning
Laser Ophthalmoscopy
Enucleated mouse eyes were pierced with a fine needle and placed in
Ito fixative25 for 24 hours. Subsequently, the eyes were washed overnight in cacodylate buffer, postfixed with OsO4, dehydrated, and
embedded in Epon (Roth, Karlsruhe, Germany). Semithin sections (1
␮m) were stained with methylene blue for histologic analysis. Ultrathin
sections were stained with uranyl acetate and lead citrate and were
viewed with an electron microscope (EM 902; Zeiss, Göttingen, Germany). Indocyanine green angiography (ICGA) was performed with a
confocal scanning laser ophthalmoscope (Heidelberg Engineering
GmbH, Heidelberg, Germany) using an infrared wavelength of 795 nm.
Vascular Corrosion Casts
Mice (n ⫽ 4) were perfused through the heart with liquid plastic
containing araldite CY 223 (45%), hardener HY (25%), and acetone
(30%). After 24 hours, the eyes were enucleated and macerated in
several changes of concentrated KOH. The eyes were then fixed on a
holder and sputtered with gold. Vascular corrosion casts were examined with a scanning electron microscope (Stereoscan 90; Cambridge
Instruments, Cambridge, UK).
Fluorescein-Dextran Angiography and Whole
Mount Lectin Staining
For fluorescein-dextran angiography, mice were deeply anesthetized
intramuscularly with ketamine (120 mg/kg) and xylazine (8 mg/kg)
and perfused through the left ventricle with 1 mL PBS containing 50
mg 2 million molecular weight fluorescein-conjugated dextran (Sigma
Chemical Co., St. Louis, MO). Eyes were enucleated and fixed in 4%
paraformaldehyde for 4 hours. Retinas were dissected and flat mounted
using fluorescence mounting medium (Dako, Hamburg, Germany). For
whole mount staining, fixed retinal tissue was passed through a graded
series of ethanol to xylene and returned through alcohol to PBS. Before
blocking in 1% milk and 0.05% Tween 20 in PBS for 30 minutes at room
temperature, retinal tissues were incubated in 25% Triton-X-100, 25%
Tween 20, and 50% 1⫻ PBS overnight at 4°C. FITC-conjugated isolectin
B4 (1:200; Vector Laboratories, Burlingame, CA) was applied for 4
hours at 37°C. The retinas were then washed with PBS and labeled
with polyclonal rabbit antibodies against pericyte marker NG2 (1:200;
Chemicon, Temecula, CA; a gift from Ludwig Aigner, University of
Regensburg, Germany), and secondary goat anti–rabbit IgG conjugated
to Alexa 594 (1:1000; Molecular Probes, Eugene, OR).
In Situ Zymography and Immunohistochemistry
DQ gelatin from pig skin, fluorescein conjugated (Molecular Probes),
was reconstituted in ISZ buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5
mM CaCl2, 0.2 mM sodium azide) at a concentration of 80 ␮g/mL.
Before addition to unfixed 10-␮m retinal cryosections, the gelatin was
diluted 1:1 in 2% agarose in ISZ buffer. The sections were incubated at
IOVS, July 2008, Vol. 49, No. 7
37°C for 12 hours. As a control, 15 mM EDTA was added to ISZ buffer.
Immunohistochemistry was performed on 4% paraformaldehyde-fixed
retinal cryosections, as described,21 using rabbit collagen XVIII antibodies diluted 1:100 (a generous gift from Ritva Heljasvaara, University
of Oulu, Finland), and secondary goat anti–rabbit IgG conjugated to
Alexa 488 (Molecular Probes). Sections and whole mount preparations
were analyzed under a fluorescence microscope (Axioskop-2; Zeiss).
Aortic Ring Assay
Mice were humanely killed by CO2 asphyxiation. A midline incision
that included splitting the sternum was made into the abdominal and
thoracic cavities. Thoracic aortas were excised and placed in ice-cold
PBS. After thorough removal of blood and fibroadipose tissue, the
aortas were cross-sectioned into 1 mm–long rings (approximately
12–16 rings per mouse aorta). The rings were placed in a 48-well tissue
culture dish containing 400 ␮L endothelial basal medium-2 (Lonza
GmbH, Wuppertal, Germany) supplemented with 0.25% fibrinogen
and 0.5% ␧-amino-n-caproic acid (Sigma Chemical Co.). Per well, 0.05
U thrombin (Calbiochem, La Jolla, CA) was added, and the gel was
allowed to clot at 37°C, 5% CO2, for 30 minutes. An equal volume of
EBM medium (Lonza GmbH, Wuppertal, Germany) was added. In some
assays, EBM was supplemented with purchasable recombinant VEGF
(rVEGF; R&D Systems GmbH, Wiesbaden, Germany) or rTIMP3 (EMD
Chemicals, San Diego, CA). In addition, rTIMP1, rTIMP2, rTIMP3,
N-terminal rTIMP3 (aa 1–121), C-terminal rTIMP3 (aa 122–188), and
rTIMP4 were expressed in Escherichia coli with a C-terminal hexahistidine tag and purified from inclusion bodies by a Ni-NTA agarose
column. Protein refolding was achieved by dropwise addition of 1 mL
denatured protein to 100 mL refolding buffer (100 mM Tris-HCl, 1 M
arginine, 100 mM NaCl, 5 mM reduced glutathione, 0.5 mM oxidized
glutathione, pH 8.0) at 4°C. Recombinant proteins were used in angiogenesis assays at a concentration of 2 ␮g/mL. VEGFR2 inhibitor
ZM323881 was obtained from Merck Biosciences (Darmstadt, Germany). Aortic rings were kept at 37°C and 5% CO2 for 6 days with a
medium change once at day 3. Quantification of angiogenesis was
performed on day 6. Aortic rings were photographed, and the number
of microvessels, including branches, was counted as described.26 Each
set of experiments was repeated three times.
Isolation of Aortic Endothelial Cells
Isolated thoracic aortas were cut into pieces of approximately 3 mm,
placed in a 6-cm gelatin-coated cell culture well, and moistened with
EBM. Explants were cultured at 37°C and 5% CO2; medium was
routinely changed every other day. Aortic explants were removed
when cells covered 80% of the cell culture dish. Confluent cells were
transferred to 10-cm dishes without gelatin coating (passage 1) and
were propagated. To purify aortic endothelial cells (AECs), cells from
passage 4 were labeled with 5 ␮g/mL di-I-acetylated-LDL (Molecular
Probes, Leiden, Netherlands) in EBM at 37°C, 5% CO2, overnight. Cells
that underwent sorting (FACSAriacell; Becton Dickinson, Franklin
Lakes, NJ) were subsequently cultured in EBM containing 20% FCS and
termed passage 1. The MHEC5-T transformed murine endothelial cell
line served as a positive control. FACS-sorted AECs expressed endothelial cell surface markers CD31 and VE-cadherin and formed tube-like
structures on basement membrane matrix (Matrigel; BD Biosciences,
San Jose, CA).
Fibrin Bead Assay
FACS-sorted AECs were mixed with microcarriers (Cytodex 3; GE
Healthcare Europe GmbH, Munich, Germany) at a concentration of
400 AECs per bead in 10 mL EBM-2. Beads were incubated for 4 hours
at 37°C and 5% CO2 with occasional shaking. Cell-coated beads were
transferred to a 25-cm2 tissue culture flask and left overnight in 5 mL
EBM-2. After washing with EBM-2, the cell-coated beads were resuspended in fibrinogen solution at a concentration of 100 beads/mL.
Then 500 ␮L fibrinogen/bead solution was added to 0.05 U thrombin
IOVS, July 2008, Vol. 49, No. 7
Choroidal Abnormalities in TIMP3-Deficient Mice
2815
FIGURE 2. Light and transmission
electron microscopy of wild-type
and TIMP3-deficient mice. (A, B)
Semithin (1 ␮m) retinal sections of
1.5-year-old Timp3⫹/⫹ and Timp3⫺/⫺
mice were investigated by light microscopy and revealed no morphologic abnormalities. Similar images
were obtained for younger age
groups (data not shown). (C, D) Ultrathin (50-nm) sections showing the
RPE/Bruch’s membrane/choroid junction of 6-month-old Timp3⫹/⫹ and
Timp3⫺/⫺ mice with a 20,000 foldmagnification. CH, choroid; OS, outer
segments; IS, inner segments; ONL,
outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer;
IPL, inner plexiform layer; GCL,
ganglion cell layer; BM, Bruch’s
membrane.
in one well of a 24-well tissue culture plate. The fibrinogen/bead
solution was allowed to clot for 20 minutes at 37°C, 5% CO2. Lung
fibroblasts were layered on top of the clot at a concentration of 20,000
cells/well. After attachment of the cells, 1 mL fresh EBM supplemented
with various recombinant TIMPs was added as described. Plates were
kept in a humidified incubator at 37°C for 6 days. To quantify vessel
outgrowth, in vitro high-resolution images of cell-coated beads were
taken, and the number of sprouts per bead was counted. Sprouts larger
than half a length of particle diameter were included. Each set of
experiments was repeated at least three times.
Western Blotting
mRNA. Successful germ line transmission of the correctly targeted allele was confirmed by two locus-specific genomic PCR
reactions. With tail DNA from mice carrying the recombinant
allele, 2.8- and 2.9-kb fragments were amplified, representing
sequences 5⬘ and 3⬘ from the PGKneobpA cassette, respectively (Figs. 1A, 1B). Absence of TIMP3 protein in fibroblast
ECM extracts derived from mice homozygous for the disrupted
Timp3 gene (Fig. 1C) indicated that the targeted allele indeed
represented a true null allele.
Regular Appearance of the Retina, RPE, and
Bruch’s Membrane in TIMP3-Deficient Mice
FACS-sorted AECs (passage 3) were resuspended and sonicated in
buffer (Phosphosafe Buffer; Novagen, Madison, WI) and were centrifuged for 20 minutes at 14,000g, 4°C. Loading buffer (125 mM Tris-HCl
[pH 6.8], 10% sucrose, 2% SDS, 5% ␤-mercaptoethanol, 0.05% bromphenol blue) was added, and the extracts were electrophoretically
separated on polyacrylamide gels and transferred to polyvinylidene
fluoride membranes. Immunoblotting was performed with primary
antibodies toward ERK1/2, phospho ERK1/2, p38, phospho p38 (all
from Sigma Chemical Co.), VEGFR2 (Abcam, Cambridge, MA), and
phospho VEGFR2 (Upstate Biotechnology, Lake Placid, NY) and secondary goat anti–mouse antibodies conjugated to horseradish peroxidase (EMD Chemicals, San Diego, CA). Blots were developed using
enhanced chemiluminescence substrate (GE Healthcare Europe
GmbH).
Histologic examination of semithin retinal sections from
Timp3⫺/⫺ eyes of different ages (2–18 months) revealed no
morphologic abnormalities in the structure of the different
retinal layers, Bruch’s membrane, or the RPE compared with
wild-type littermates at comparable ages (Figs. 2A, 2B). Similarly, transmission electron microscopy of ultrathin sections of
eyes from TIMP3-null mice (ages 2–18 months) showed a
regular appearance of RPE cells and Bruch’s membrane, with
its characteristic five-layered structure composed of a central
elastic layer flanked by an inner and an outer collagenous layer
and two basal membranes derived from the RPE and the choriocapillaris, respectively (Figs. 2C, 2D). There was no significant difference in the thickness of Bruch’s membrane between
Timp3⫺/⫺ and control mice.
RESULTS
Choroidal Vascular Abnormalities in
TIMP3-Null Mice
Generation of TIMP3-Deficient Mice
Disruption of the mouse Timp3 gene was achieved by inserting
a neomycin-resistance gene expression cassette (PGKneobpA)
into exon 3 of Timp3 (Fig. 1A). This leads to a frameshift and
the generation of a premature stop codon. Thus, the sequence
of the targeted allele after homologous recombination should
direct expression of a truncated and nonfunctional TIMP3
To detect retinal defects that might not be obvious by analyzing retinal cross-sections, we performed ICGA using in vivo
scanning laser ophthalmoscopy. This technique provides detailed information on the structural integrity of the fundus by
sequential viewing of the different planes of the posterior pole
from the surface of the retina down to the choroid. Importantly, ICGA uses the near-infrared light and highly protein-
2816
Janssen et al.
IOVS, July 2008, Vol. 49, No. 7
bound dye ICG to effectively visualize the choroidal vasculature. In ICG angiograms, normal development of the retinal
vasculature was observed in mice lacking TIMP3. In contrast,
irregularities at the level of the choroid were seen in TIMP3deficient C57BL/6 mice that were absent in control animals.
This includes hyperfluorescent larger caliber vessels and prominent balloon-like spots associated with the vascular layer of
the choroid (Figs. 3A, 3B). Because ICG fluorescence is partially obscured by the RPE in pigmented mice, the Timp3⫺/⫺
genotype was backcrossed onto the CD1 albino outbred background. Subsequently, scanning laser ophthalmoscopic images
of CD1-TIMP3-null mice showed more prominent changes of
the deep choroid with numerous dilated vessels clearly not
present in control littermates (Figs. 3C–F). The choroidal phenotype was observed in all Timp3⫺/⫺ mice tested (n ⫽ 9)
without an apparent progression of the vascular lesions. To
further examine the nature of the vessel abnormalities in
Timp3⫺/⫺ mice, vascular corrosion casts of Timp3⫺/⫺
(C57BL/6) and wild-type eyes were analyzed by scanning laser
electron microscopy. Although wild-type animals exhibited a
regular network of choroidal blood vessels (Fig. 3G), the choroid of Timp3⫺/⫺ mice revealed strongly dilated or fused
vessel formations (Fig. 3H) that likely corresponded to the
abnormalities seen by scanning laser ophthalmoscopy (Fig.
3B).
Close examination of whole mounted Timp3⫺/⫺ and control retinas after fluorescein-dextran perfusion (Figs. 4A–D, F–I)
or isolectin B4-staining (data not shown) revealed regular morphologies of the retinal vascular plexi in Timp3⫺/⫺ mice without any signs of neovascular pathology (Figs. 4F–I). These
observations confirm the ICGA findings. In addition, the localization and morphology of pericytes accompanying endothelial
sprouts in deep retinal capillary layers were similar in
Timp3⫺/⫺ and wild-type retinas (Figs. 4E, 4J). Taken together,
these data suggest that normal retinal vascularization in mice is
apparently not influenced by the lack of TIMP3 in mice.
Enhanced MMP Activity in TIMP3-Deficient Eyes
To dissect the molecular pathway leading to choroidal abnormalities in TIMP3-null mice, we first performed in situ zymographic analyses of cryosections derived from wild-type and
Timp3⫺/⫺ eyes. Fluorescence signals in this assay indicate
gelatin degradation by active MMPs (MMP2 and MMP9). For
both genotypes, we observed fluorescence of similar intensity
in the inner and outer nuclear layers and the ganglion cells
(Figs. 5A, 5E). In contrast, a significant enhancement of fluorescent signal along the basal aspect of the RPE monolayer was
repeatedly observed in Timp3⫺/⫺ sections (Figs. 5E, 5F) compared with wild-type (Figs. 5A, 5B), indicating locally elevated
gelatinase (MMP2 and MMP9) activity in the knock-out animals.
The fluorescence pattern resulting from gelatinolytic activity at
this site differed from the immunolabeling with collagen XVIII
antibodies in consecutive cryosections (Figs. 5D, 5H) in that
the latter explicitly stained Bruch’s membrane in Timp3⫺/⫺
and control eyes whereas MMP activity was found more externally toward the choroid and in ECM domains that interdigitate
between individual choriocapillaris lobules. In addition, the
intensity of collagen XVIII staining of Bruch’s membrane was
similar for both genotypes, demonstrating that the enhanced
fluorescence produced by increased MMP2 and MMP9 activity
in Timp3⫺/⫺ was not caused by artifacts in section preparations but indeed reflected functional differences in TIMP3
activities. Together these findings suggest that the lack of
TIMP3 causes a marked disruption of the TIMP/MMP balance in
the choroid region. In situ zymography in the presence of
FIGURE 3. In vivo imaging and scanning laser microscopy of wild-type
and TIMP3-deficient mice. (A, B) Panoramic ICG angiograms of
3-month-old C57/Bl6 mice carrying homozygote wild-type (⫹/⫹) or
mutant (⫺/⫺) Timp3 alleles display the choroidal vascular network
with overlying retinal vessels. Mutant animals exhibit abnormally enlarged choroidal vessels that occasionally form balloon-like structures
(white arrows). Scanning laser microscopic images of 4-week-old (C,
D) and 8-month-old (E, F) Timp3⫹/⫹ and Timp3⫺/⫺ CD1 albino mice.
TIMP3-deficient mice exhibit numerous dilated vessels. (G, H) Scleral
view of scanning electron micrographs of corrosion casts showing
approximately one fourth of the choroidal vasculature of 2-month-old
Timp3⫹/⫹ and Timp3⫺/⫺ C57/Bl6 mice. Timp3⫺/⫺ mice display enlarged or fused vascular structures.
metalloprotease inhibitor (EDTA) abolished the fluorescence
signals (Figs. 5C, 5G), confirming that gelatin cleavage in the
assays is mediated by MMPs.
IOVS, July 2008, Vol. 49, No. 7
Choroidal Abnormalities in TIMP3-Deficient Mice
2817
FIGURE 4. Whole mount vessel staining
of wild-type and TIMP3-deficient retinas. Fluorescein-dextran flat mounts
of retinas from 4-month-old wild-type
(A–D) and Timp3⫺/⫺ (F–I) CD1 mice.
Fluorescence microscopy of the retinal vasculature was performed at ⫻4
(A, F) magnification. Higher magnification images (⫻40) were collected
from the superficial (B, G), intermediate (C, H), and deep (D, I) retinal
plexus. Retinal vascular network architecture is similar between control
and Timp3⫺/⫺ animals. (I, J) Localization of pericytes on isolectin B4stained capillaries (green) in deep
capillary plexus. Pericytes were immunolabeled with NG2 antibodies
(red). Scale bars, 50 ␮m.
Enhanced Capillary Sprouting of Aortic Explants
in TIMP3-Deficient Mice
Antiangiogenic Activity Resides within the
C-Terminal Domain of TIMP3
VEGF overstimulation is known to produce abnormally enlarged blood vessels and vascular hyperfusion.27,28 Dysregulation of VEGF-induced angiogenesis may therefore be involved
in the development of the choroidal vascular abnormalities
observed in TIMP3-null mice. We examined the consequences
of TIMP3 deficiency on VEGF-mediated angiogenesis using the
ex vivo aortic ring angiogenesis assay. Aortic explants from
Timp3⫺/⫺ mice reproducibly showed a significantly enhanced
spontaneous microvessel outgrowth when compared with explants from wild-type littermates (Figs. 6A, 6Aa). The addition
of recombinant TIMP3 (Fig. 6Bb) reduced capillary sprouting
of Timp3⫺/⫺ aortic rings to the level seen in wild-type (Fig.
6B). Treatment with recombinant VEGF greatly stimulated vessel formation in wild-type aortas to a level comparable to
spontaneous vessel outgrowth in Timp3⫺/⫺ explants (Figs. 6C,
6Cc). Furthermore, addition of VEGFR2 inhibitor to Timp3⫺/⫺
aortic rings dramatically reduced capillary sprouting by more
than 80% (Figs. 6D, 6Dd). Supplementation of control and
Timp3⫺/⫺ aortic explants with recombinant TIMP3, but not
with recombinant TIMP2, decreased vessel outgrowth, indicating that the angio-inhibitory effect is specific to TIMP3 (Figs.
6E, 6F). Taken together, these data suggest that a lack of
competitive interaction between TIMP3 and VEGF for binding
to VEGFR2 leads to the strong sprouting activity in TIMP3deficient aortic explants.
To define the region of the TIMP3 protein responsible for the
antiangiogenic activity, we compared the capillary vessel outgrowth of Timp3⫺/⫺ aortic rings supplemented with recombinant proteins representing full-length TIMP3 (aa 1–188), the
N-terminal part (aa 1–121) containing the MMP inhibitory domain, or the C terminus (aa 122–188; Figs. 7B–E). In contrast to
N-terminal TIMP3, the addition of recombinant full-length
TIMP3 or C-terminal TIMP3 drastically diminished capillary
sprouting in aortic rings from Timp3⫺/⫺ mice to a level lower
than the spontaneous microvessel outgrowth from wild-type
explants (Fig. 7A). The assessment of angiogenesis by fibrin
bead assays (Figs. 7F–J) showed that isolated Timp3⫺/⫺ AECs
significantly promote the formation of capillary-like vessels
when compared with AECs from wild-type animals (Figs. 7F,
7G). Similar to the capillary sprouting, enhanced tube formation of Timp3⫺/⫺ AECs was specifically blocked by the addition of recombinant full-length TIMP3 or C-terminal TIMP3
(Figs. 7H–J). These independent data demonstrate that the
C-terminal domain of TIMP3 is sufficient to exert the antiangiogenic properties of TIMP3.
Activation of Angiogenic Signaling Pathways in
the Absence of TIMP3
VEGF induces several endothelial cell functions through activation of its cell surface receptor, VEGFR2, and downstream
2818
Janssen et al.
IOVS, July 2008, Vol. 49, No. 7
FIGURE 5. Gelatinolytic activity in the retina of wild-type and TIMP3-deficient mice. In situ zymography
was performed with retinal cryosections from (A–C) wild-type and (E–G) Timp3⫺/⫺ mice. MMP-mediated
degradation of the DQ gelatin substrate yields highly fluorescent peptides (green). Fluorescent signals in
the ONL, INL, and GCL were similar in control (A) and TIMP3 knock-out (E) retinas. In contrast, enhanced
gelatinolytic activity at the level of the choroid in Timp3⫺/⫺ when compared to wild-type is visualized in
the overview micrographs of the retinal cryosections (A, E) and at higher magnification (B, F; white
arrows). MMP activity was successfully blocked by the addition of 15 mM EDTA (C, G). Immunofluorescence micrographs of (D) wild-type and (H) Timp3⫺/⫺ retina showing Bruch’s membrane labeled with
collagen XVIII antiserum (green). Nuclei were counterstained with DAPI (blue). GCL, ganglion cell layer;
IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer;
OS, outer segments; RPE, retinal pigment epithelium; CH, choroid.
signaling molecules. To further understand how the lack of
TIMP3 promotes VEGF-mediated angiogenesis, we evaluated
the effect of TIMP3 deficiency in AECs of Timp3⫺/⫺ mice on
the activation of VEGF/VEGFR2 signaling pathways. Immunoblot analyses show that the autophosphorylation of VEGFR2
and the phosphorylation of ERK1/2 and p38 mitogen-activated
protein kinases (MAPKs), which are downstream signaling molecules of VEGFR2, were enhanced in AECs of Timp3⫺/⫺ animals when compared with wild-type (Fig. 8). This suggests that
the absence of TIMP3 leads to a constitutive activation of
VEGF/VEGFR2 signaling pathways. Lack of TIMP3 had no influence on the levels of dephosphorylated VEGFR2, ERK1/2,
and p38 proteins.
DISCUSSION
We demonstrated that TIMP3 deficiency in the mouse causes a
distinct choroidal, but not retinal, phenotype characterized by
prominently enlarged choroidal blood vessels. Thus, this study
provides the first in vivo evidence in support of the hypothesized role of TIMP3 in the regulation of choroidal vascularization of the retina.
The choroidal vascular system starts to form early during
mouse eye development, namely at the time of optic vesicle
invagination.29 Primitive periocular blood vessels are generated from a perineural vascular plexus at embryonic day 11.5
and extend around the outer layer of the optic cup. This initial
network develops into a complex vasculature by a process that
involves sprouting and nonsprouting angiogenesis accompanied by extensive remodeling and maturation of blood vessels.30 The persistent interaction between blood vessels and
the adjacent RPE is thereby crucial for the formation and
maintenance of an intact choroidal vasculature. It has been
shown that the suppression of RPE differentiation inhibits
choroidal development,31 whereas biochemical or mechanical
destruction of the RPE monolayer causes loss of endothelial
fenestration, atrophy of the choriocapillaris and underlying
choroidal vessels, and abnormal collagen deposition in the
choroid.32–34 Growth factors released by the RPE play an
important role in conducting the modulation and survival effect of the RPE on the choroidal vascular system.35,36 This
includes VEGF, a crucial vascular permeability and angiogenic
factor involved in all stages of blood vessel formation in the
course of vasculogenesis and angiogenesis. RPE cells express
IOVS, July 2008, Vol. 49, No. 7
Choroidal Abnormalities in TIMP3-Deficient Mice
FIGURE 6. Enhanced vessel sprouting in aortic explants from TIMP3-deficient mice is mediated by
unbalanced VEGF-induced angiogenesis. Aortic ring assays showing spontaneous vessel outgrowth in
Timp3⫹/⫹ (A) versus Timp3⫺/⫺ (Aa) aortic rings, spontaneous vessel outgrowth in Timp3⫹/⫹ (B) versus
vessel sprouting in Timp3⫺/⫺ aortic rings supplemented with recombinant TIMP3 (Bb), vessel sprouting
in Timp3⫹/⫹ supplemented with recombinant VEGF (C) versus spontaneous vessel outgrowth in
Timp3⫺/⫺ (Cc) aortic rings, and spontaneous vessel outgrowth in Timp3⫺/⫺ (D) versus vessel sprouting
in Timp3⫺/⫺ aortic rings supplemented with VEGFR2 inhibitor (Dd). Quantification of sprouting at days
2, 4, and 6 is depicted by light- and dark-shaded columns in charts beside the respective photographs
(taken at day 6 of the assay); d, day. Capillary sprouting of aortic explants from (E) Timp3⫹/⫹ or (F)
Timp3⫺/⫺ aortic rings supplemented with recombinant TIMP2 and TIMP3. Light-shaded columns represent vessel outgrowth at day 2, dark-shaded columns at day 4, and black columns at day 6. All data are
presented as mean (n ⫽ 3; bars) ⫾ SD (y-axis; sprouts/aorta).
2819
2820
Janssen et al.
IOVS, July 2008, Vol. 49, No. 7
FIGURE 7. C-terminal domain of TIMP3 inhibits capillary sprouting. (A–E) Aortic ring and (F–J) fibrin bead assays were used to define the region
of the TIMP3 protein responsible for the antiangiogenic activity. Angiogenesis in Timp3⫺/⫺ versus Timp3⫹/⫹ mice as measured by increased (A,
B) vessel sprouting and (F, G) tube formation. Increased angiogenic activity in Timp3⫺/⫺ mice was significantly inhibited by the addition of
recombinant full-length TIMP3 (aa 1–188) (C, H) or the C-terminal part of TIMP3 (aa 122–188) (E, J). Quantification of sprouting (y-axis,
sprouts/aorta) and tube formation (y-axis, sprouts/bead) is shown in graphs beside the photographs. Data are presented as mean (n ⫽ 3; bars) ⫾ SD.
VEGF during choroidal development and continue to secrete
VEGF toward the basal choroidal side throughout adulthood.37–39 In support of a VEGF-mediated paracrine relation
between the RPE and the choroid, VEGFR2 was shown to
localize at the inner choriocapillaris.38 In coculture experiments, RPE-derived VEGF induces tube formation of underlying
choroidal endothelial cells, a process that can be inhibited by
VEGF antibodies.40 The observation that mice with a conditional inactivation of endogenous Vegf gene expression in the
RPE are characterized by an absence of choroidal tissue and
abnormalities in RPE and Bruch’s membrane morphology eventually confirms the fundamental role of VEGF in choroid formation.38
TIMP3 was shown to inhibit VEGF-induced angiogenesis in
a chick embryo chorioallantoic membrane assay, with recombinant TIMP3 interfering with VEGF-induced endothelial cell
migration and proliferation in vitro.18 The antiangiogenic properties of TIMP3 were attributed to its ability to bind VEGFR2,
thereby blocking the activation of intracellular downstream
signaling molecules required to stimulate various endothelial
cell functions. In accordance with this hypothesis, we observed an increased angiogenic response in ex vivo angiogenesis assays using organ explants from TIMP3-deficient mice, an
effect that could be reversed by the addition of VEGFR2 inhibitor. It is therefore reasonable to assume that under physiological conditions, the absence of TIMP3 protein in Bruch’s membrane perturbs VEGF-mediated paracrine signaling between
the RPE and the choroid. As a consequence, enhanced stimulation of VEGFR2 signaling pathways by unopposed VEGF
binding to endothelial cells may be responsible for increased
cell proliferation and migration, leading to the formation of
enlarged choroidal vessels. Consistent with this model are data
demonstrating that transgenic mice with RPE cells overexpressing VEGF under the RPE65 promoter exhibit a thickened
choroid with dilated vessels and wide lumina.41 In addition,
exogenous VEGF stimulation is known to induce malformed,
fused vessels with enlarged lumens,27 and elevated VEGF concentrations enhance vessel diameter through VEGFR2 pathways in vitro.42
Our immunoblot experiments with whole AEC cell extracts
show differences in the activation of VEGFR2 and downstream
MAPK signaling between Timp3⫺/⫺ and wild-type mice. Signal
transmission of several growth factors occurs through activated MAPK pathways, and it is likely that factors other than
VEGF added to the enhanced phosphorylation of ERK1/2 and
p38. One such candidate would be FGF-2, which has recently
been shown in extracellular matrix plug and gel foam assays to
be a stronger angiogenic stimulus than VEGF in Timp3⫺/⫺
mice.14 Assuming that more than one angiogenic pathway is
affected by TIMP3 deficiency, the analysis of the role of specific angiogenic factors in the development of choroidal abnormalities represents an important next step to further understand TIMP3 function in choroidal vascularization.
Higher levels of active and inactive forms of MMP-2 and
MMP-9 were previously found in primary fibroblasts isolated
from the lungs of our TIMP3 knock-out mice,24 correlating
with heightened metalloprotease levels detected in lung and
heart of an independently generated TIMP3-null mouse
line.9 –11 Although a notable local increase of gelatinase activity
in the choroid region of Timp3⫺/⫺ eyes was easily detected by
our in situ zymography experiments, MMP activity within
Bruch’s membrane is difficult to assess. The observation that
TIMP3-deficient mice do not show alterations in thickness and
integrity of the individual layers of Bruch’s membrane, however, argues against a profound imbalance between MMPs and
TIMPs within this specific matrix. MMPs are known to regulate
vascular morphogenesis in multiple ways. Besides their prominent role in ECM degradation to facilitate the migration and
proliferation of endothelial cells, MMPs are required to activate, liberate, and modify proangiogenic, but also antiangiogenic, factors sequestered in the pericellular ECM.43 For example, MMPs have been implicated in the coordination of VEGF
release from extracellular stores by matrix breakdown and
directly through intramolecular processing.44 Enhanced MMP
activity in the absence of TIMP3 might therefore trigger a
cascade of events contributing to the vascular abnormalities
seen in TIMP3-deficient eyes.
Vessel abnormalities have not been reported in other organs
of mice with a targeted TIMP3 deficiency. Injection of melanoma cells into TIMP3-null mice, however, was shown to
result in increased tumor growth and angiogenesis characterized by an enhanced formation of blood vessels with larger
diameters than those in wild-type littermates.14 This may indicate that the impact of TIMP3 ablation on vascular morphogenesis is subtle and dependent on the cellular microenvironment. In addition, phenotypic variability in TIMP3-null mice
with different genetic backgrounds was observed in this and a
previous study.11 Such an effect is generally attributable to
modifier genes that act in concert with the causative gene.
Further studies are needed to evaluate the influence of TIMP3
IOVS, July 2008, Vol. 49, No. 7
Choroidal Abnormalities in TIMP3-Deficient Mice
2821
giogenic properties are retained in mutant TIMP3 proteins.
This should contribute to unraveling the still unknown molecular mechanism of TIMP3-related pathology in SFD.
Acknowledgments
The authors thank Barbara Bathke for help with breeding and genotyping of the mouse lines.
References
FIGURE 8. Analysis of VEGF-induced signaling pathways. Total protein
extracts from FACS-selected AECs subcultured from Timp3⫹/⫹ and
Timp3⫺/⫺ aortic explants were immunoblotted with antibodies toward VEGF downstream signaling molecules. Immunoblotting with
antibodies against ␤-actin served as a control for equal loading.
deficiency on vasculogenesis and angiogenesis in different
mouse strains.
Modulation of VEGF-induced angiogenesis has been demonstrated to be independent of the MMP-inhibitory properties of
TIMP3.18 Consistent with this, we mapped the region of the
TIMP3 protein responsible for its antiangiogenic activity to its
C-terminal domain. Interestingly, all 10 TIMP3 mutations underlying SFD are located in or affect the C-terminal part of
TIMP3. A next step will be to determine whether the antian-
1. Blenis J, Hawkes SP. Transformation-sensitive protein associated
with the cell substratum of chicken embryo fibroblasts. Proc Natl
Acad Sci U S A. 1983;80:770 –774.
2. Apte SS, Olsen BR, Murphy G. The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities
define the distinct TIMP gene family. J Biol Chem. 1995;270:
14313–14318.
3. Saunders WB, Bohnsack BL, Faske JB, et al. Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte
TIMP-3. J Cell Biol. 2006;175:179 –191.
4. Bond M, Murphy G, Bennett MR, et al. Localization of the death
domain of tissue inhibitor of metalloproteinase-3 to the N
terminus: metalloproteinase inhibition is associated with proapoptotic activity. J Biol Chem. 2000;275:41358 – 41363.
5. Butler GS, Apte SS, Willenbrock F, Murphy G. Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and Cterminal domains of gelatinases A and B. Regulation by polyanions.
J Biol Chem. 1999;274:10846 –10851.
6. Zhao H, Bernardo MM, Osenkowski P, et al. Differential inhibition
of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and
MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and
TIMP-3 regulates pro-MMP-2 activation. J Biol Chem. 2004;279:
8592– 8601.
7. Lambert E, Dasse E, Haye B, Petitfrere E. TIMPs as multifacial
proteins. Crit Rev Oncol Hematol. 2004;49:187–198.
8. Amour A, Slocombe PM, Webster A, et al. TNF-alpha converting
enzyme (TACE) is inhibited by TIMP-3. FEBS Lett. 1998;435:39 –
44.
9. Leco KJ, Waterhouse P, Sanchez OH, et al. Spontaneous air space
enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3). J Clin Invest. 2001;108:817– 829.
10. Gill SE, Pape MC, Khokha R, Watson AJ, Leco KJ. A null mutation
for tissue inhibitor of metalloproteinases-3 (Timp-3) impairs murine bronchiole branching morphogenesis. Dev Biol. 2003;261:
313–323.
11. Fedak PW, Smookler DS, Kassiri Z, et al. TIMP-3 deficiency leads
to dilated cardiomyopathy. Circulation. 2004;110:2401–2409.
12. Mohammed FF, Smookler DS, Taylor SE, et al. Abnormal TNF
activity in Timp3⫺/⫺ mice leads to chronic hepatic inflammation
and failure of liver regeneration. Nat Genet. 2004;36:969 –977.
13. Mahmoodi M, Sahebjam S, Smookler D, Khokha R, Mort JS. Links
Lack of tissue inhibitor of metalloproteinases-3 results in an enhanced inflammatory response in antigen-induced arthritis. Am J
Pathol. 2005;166:1733–1740.
14. Cruz-Munoz W, Kim I, Khokha R. TIMP-3 deficiency in the host,
but not in the tumor, enhances tumor growth and angiogenesis.
Oncogene. 2006;25:650 – 655.
15. Cruz-Munoz W, Sanchez OH, Di Grappa M, English JL, Hill RP,
Khokha R. Enhanced metastatic dissemination to multiple organs
by melanoma and lymphoma cells in Timp-3⫺/⫺ mice. Oncogene.
2006;25:6489 – 6496.
16. Anand-Apte B, Bao L, Smith R, et al. A review of tissue inhibitor of
metalloproteinases-3 (TIMP-3) and experimental analysis of its effect on primary tumor growth. Biochem Cell Biol. 1996;74:853–
862.
17. Wild A, Ramaswamy A, Langer P, et al. Frequent methylationassociated silencing of the tissue inhibitor of metalloproteinase-3
gene in pancreatic endocrine tumors. J Clin Endocrinol Metab.
2003;88:1367–1373.
18. Qi JH, Ebrahem Q, Moore N, et al. A novel function for tissue
inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogen-
2822
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Janssen et al.
esis by blockage of VEGF binding to VEGF receptor-2. Nat Med.
2003;9:407– 415.
Anand-Apte B, Pepper MS, Voest E, et al. Inhibition of angiogenesis
by tissue inhibitor of metalloproteinase-3. Invest Ophthalmol Vis
Sci. 1997;38:817– 823.
Della NG, Campochiaro PA, Zack DJ. Localization of TIMP-3 mRNA
expression to the retinal pigment epithelium Invest Ophthalmol
Vis Sci. 1996;37:1921–1924.
Weber BH, Lin B, White K, et al. A mouse model for Sorsby fundus
dystrophy. Invest Ophthalmol Vis Sci. 2002;43:2732–2740.
Fariss RN, Apte SS, Luthert PJ, Bird AC, Milam AH. Accumulation of
tissue inhibitor of metalloproteinases-3 in human eyes with Sorsby’s fundus dystrophy or retinitis pigmentosa. Br J Ophthalmol.
1998;82:1329 –1334.
Weber BH, Vogt G, Pruett RC, Stohr H, Felbor U. Mutations in the
tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with
Sorsby’s fundus dystrophy. Nat Genet. 1994;8:352–356.
Soboleva G, Geis B, Schrewe H, Weber BH. Sorsby fundus dystrophy mutation Timp3(S156C) affects the morphological and biochemical phenotype but not metalloproteinase homeostasis. J Cell
Physiol. 2003;197:149 –156.
Karnovsky MJ. The ultrastructural basis of capillary permeability
studied with peroxidase as a tracer. J Cell Biol. 1967;35:213–236.
Nicosia RF, Ottinetti A. Growth of microvessels in serum-free
matrix culture of rat aorta: a quantitative assay of angiogenesis in
vitro. Lab Invest. 1990;63:115–122.
Drake CJ, Hungerford JE, Little CD. Morphogenesis of the first
blood vessels. Ann N Y Acad Sci. 1998;857:155–179.
Springer ML, Chen AS, Kraft PE, Bednarski M, Blau HM. VEGF gene
delivery to muscle: potential role for vasculogenesis in adults. Mol
Cell. 1998;2:549 –558.
Saint-Geniez M, D’Amore PA. Development and pathology of the
hyaloid, choroidal and retinal vasculature. Int J Dev Biol. 2004;48:
1045–1058.
Risau W. Mechanisms of angiogenesis. Nature . 1997;386:671–
674.
Zhao S, Overbeek PA. Regulation of choroid development by the
retinal pigment epithelium. Mol Vis. 2004;7:277–282.
Korte GE, Reppucci V, Henkind P. RPE destruction causes choriocapillary atrophy. Invest Ophthalmol Vis Sci. 1984;25:1135–1145.
IOVS, July 2008, Vol. 49, No. 7
33. Del Priore LV, Hornbeck R, Kaplan HJ, et al. Debridement of the
pig retinal pigment epithelium in vivo. Arch Ophthalmol. 1995;
113:939 –944.
34. Leonard DS, Zhang XG, Panozzo G, Sugino IK, Zarbin MA. Clinicopathologic correlation of localized retinal pigment epithelium
debridement. Invest Ophthalmol Vis Sci. 1997;38:1094 –1109.
35. Campochiaro PA, Hackett SF, Vinores SA. Growth factors in the
retina and retinal pigmented epithelium. Prog Ret Eye Res. 1996;
15:547–567.
36. Liu X, Ye X, Yanoff M, Li W. Regulatory effects of soluble growth
factors on choriocapillaris endothelial growth and survival. Ophthalmic Res. 1998;30:302–313.
37. Blaauwgeers HG, Holtkamp GM, Rutten H, et al. Polarized vascular
endothelial growth factor secretion by human retinal pigment
epithelium and localization of vascular endothelial growth factor
receptors on the inner choriocapillaris: evidence for a trophic
paracrine relation. Am J Pathol. 1999;155:421– 428.
38. Marneros AG, Fan J, Yokoyama Y, et al. Vascular endothelial
growth factor expression in the retinal pigment epithelium is
essential for choriocapillaris development and visual function.
Am J Pathol. 2005;167:1451–1459.
39. Saint-Geniez M, Maldonado A, D’Amore PA. VEGF expression and
potential function in the choroid during development and in the
adult. Invest Ophthalmol Vis Sci. 2006;47:3135–3142.
40. Sakamoto T, Sakamoto H, Murphy TL, et al. Vessel formation by
choroidal endothelial cells in vitro is modulated by retinal pigment
epithelial cells. Arch Ophthalmol. 1995;113:512–520.
41. Schwesinger C, Yee C, Rohan RM, et al. Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial
growth factor in the retinal pigment epithelium. Am J Pathol.
2001;158:1161–1172.
42. Nakatsu MN, Sainson RC, Perez-del-Pulgar S, et al. VEGF(121) and
VEGF(165) regulate blood vessel diameter through vascular endothelial growth factor receptor 2 in an in vitro angiogenesis model.
Lab Invest. 2003;83:1873–1885.
43. Rundhaug JE. Matrix metalloproteinases and angiogenesis. J Cell
Mol Med. 2005;9:267–285.
44. Lee S, Jilani SM, Nikolova GV, Carpizo D, Iruela-Arispe ML. Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. J Cell Biol. 2005;169:
681–191.