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,1,1,
,
,
* Department of Vitreoretinal Surgery, Center for Ophthalmology, and
Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany;
Center for Biochemistry,
School of Biological Sciences, University of Southhampton, Southampton, UK; and
|| Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
2Correspondence: Department of Vitreoretinal Surgery, Center for Ophthalmology, Joseph Stelzmannstr. 9, Köln 50931, Germany. E-mail: joussena{at}googlemail.com
ABSTRACT
Choroidal neovascularization (CNV) is responsible for the severe visual loss in age-related macular degeneration. CNV formation is considered to be due to an imbalance between pro- and antiangiogenic factors that lead to neovascular growth from the choriocapillaris into the subretinal space. To define whether FasL overexpression in retinal pigment epithelial cells (RPE) can inhibit choroidal neovascularization through Fas-FasL-mediated apoptosis, we examined the role of this pathway in a mouse model of laser-induced choroidal neovascularization. FasL was expressed in the retinal pigment epithelium of transgenic mice. Polymerase chain reaction (PCR), immunoblot, and immunohistochemistry confirmed that the transgene FasL was specifically expressed in RPE. The established laser model was used to induce choroidal neovascularization (CNV) in wild-type (WT) and transgenic mice. CNV formation was compared with respect to fluorescein angiographic leakage (at days 0 and 14 after laser injury) and histological appearance. The lesions were assessed on RPE-choroidal flatmounts after CD31-labeling and with confocal microscopy after perfusion with rhodamine-labeled concanavalin A (Con A). Apoptosis was quantified by TUNEL positivity and caspase activation. FasL mRNA and protein were highly expressed in the RPE of the transgenic mice before and after laser photocoagulation. In contrast, FasL was only weakly expressed in the RPE layer of WT C57BL/6J mice. While ruptures of Bruch’s membrane and CNV formation were observed histologically two weeks after laser photocoagulation in transgenic as well as control eyes, the shape and size of CNV lesions were reduced in the transgenic mice. The area of leakage was decreased by 70% in FasL transgenic mice compared with WT mice (P<0.005). The number of TUNEL-positive cells was greater in FasL-overexpressing mice and correlated with the expression of activated caspases. Th expression of other antiangiogenic factors such as PEDF remained unchanged. The specific overexpression of FasL in RPE layer reduced CNV formation in our laser model. Our results strongly point to the FasL-Fas pathway as a potential therapeutic target in controlling pathological choroidal neovascularization.—Semkova, I., Fauser, S., Lappas, A., Smyth, N., Kociok, N., Kirchhof, B., Paulsson, M., Poulaki, V., Joussen, A. M. Overexpression of FASL in retinal pigment epithelial cells reduces choroidal neovascularization.
Key Words: choroidal neovascularization • apoptosis • inflammation
IN EXSUDATIVE OR NEOVASCULAR age-related macular degeneration (AMD), newly developed capillaries bud from the choroid through Bruch’s membrane under and into the neurosensory retina (1
2
3
4
5)
. Submacular neovascularization may initially be localized outside the fovea but will with time extend under the fovea. In its natural course, AMD results in a submacular fibrotic scar, causing degeneration of the sensory retina and photoreceptor loss in the macula (6
7
8)
. AMD is the leading cause of blindness in Western nations (9)
.
Histopathological studies of choroidal neovascular membranes from patients with AMD have demonstrated the presence of various angiogenic and growth factors, including vascular endothelial growth factor (VEGF) (10
11
12)
, fibroblast growth factor (FGF-2) (10)
, and transforming growth factor-ß (TGF-ß) (13)
. VEGF, normally expressed by the retinal pigment epithelium (RPE), ganglion cells, and the inner nuclear layer, modulates vascular permeability (14)
, vasculogenesis (15)
, and neovascular proliferation (16)
.
The extent of angiogenesis in vivo depends on the local balance between pro and antiangiogenic molecules. Many natural inhibitors, including thrombospondin-1 (TSP1) (17)
and pigment epithelium-derived factor (PEDF) (18)
, as well as angiostatin (19)
and endostatin (20)
, induce apoptosis in cultured endothelial cells. In contrast, in vitro studies have shown that proangiogenic molecules, including vascular VEGF, basic fibroblast growth factor (bFGF), and interleukin (IL)-8 (IL-8), promote endothelial cell survival. The outcome of these opposing influences determines whether endothelial cells live or die, and regulate the formation and regression of blood vessels.
Several inhibitors require apoptotic events to block neovascularization in vivo. In case of PEDF and TSP1, apoptosis is mediated by the Fas/FasL pathway (21)
. Fas and FasL are membrane proteins found at numerous sites throughout the body. FasL induces apoptotic cell death in cells expressing the Fas receptor. Although they were originally thought to function mainly in the peripheral deletion of T cells (22)
, it is now known that Fas-FasL interactions are important in other contexts, such as T cell cytotoxicity (23)
, tumorogenesis (24)
, and liver disease (25)
. The Fas receptor (CD95, APO-1) is a type I membrane protein of 45 kDa that belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family (26
27)
. FasL is a type II membrane protein of 40 kDa that also belongs to the TNF family. The binding of FasL to Fas receptor triggers apoptosis (28)
. FasL is particularly important in immune-privileged sites, such as the eye (29)
controlling the entry of potentially damaging lymphoid cells into this organ. FasL is expressed throughout the retina (29
30
31
32)
in particular on RPE cells (30
31
32
33)
. Evidence for the Fas-FasL-mediated inhibition of vessel growth comes from experiments with a murine carcinoma, where FasL+ T lymphocytes suppress tumor vessel growth (34)
. In the eye, Kaplan and co-workers (35)
investigated choroidal vessel growth after laser-induced neovascularization and showed that RPE cells inhibit the growth of newly formed choroidal endothelial vessels through Fas-FasL-mediated apoptosis. These results clearly show that FasL on RPE cells may be well positioned to control the growth of new vessels from the vascularized choroid. In the current study we evaluate the effect of specific over-expression of FasL on RPE cells on the formation of CNV membranes in a transgenic mouse model.
MATERIALS AND METHODS
Generation of transgenic mice
A full-length FasL cDNA (839 bp), generated from mouse cDNA by PCR, was cloned directionally after the murine RPE 65 promoter (36)
into a pGEM plasmid (Promega, Mannheim, Germany). The poly(A) signal of human growth factor was directionally cloned at the 3' end of the FasL-coding sequence (Fig. 1
). The sequence of the final construct was confirmed (2058 bp) by DNA sequencing. The construct was excised and used to generate three founder mice as described previously (37)
. The mice were screened for the integrity of the transgene by sequencing through the entire construct. To expand the transgenic lines, the founder mice were crossed into a C57BL/6J-Tyr C-2J background (Jackson Lab, ME, USA). All animals investigated were derived from founder no. 2 and were heterozygous for the transgene, as they were generated by mating a transgenic parent with a C57BL/6J-Tyr C-2J mouse. From our previous studies investigating mice overexpressing VEGF 164 under the same promotor (36)
, we know that transgenic VEGF 164 mRNA increased toward 4 mo of age and afterward again decreased. Thus, we used 7–8 wk old mice, exhibiting a stable expression of FasL, for the current experiments.
|
PCR
Mice were screened for the presence of the transgene by PCR on genomic DNA. Tail pieces were digested overnight at 56°C in lysis buffer (DNeasy tissue kit, Qiagen, Hilden, Germany), supplemented with 20 mg/ml proteinase K. DNA was isolated and quantified according to the manufacturer’s instructions (Qiagen). For the amplification of the transgene-specific sequence (130 bp) at 56°C, a 5'-primer (5'-ATG ACT GAG GTC automatic gain control TCA GGA CTG C-3'), and a 3'-primer (5'-CAA GAT GAA GTG GCA CTG CTG TCT acetyl-coenzyme A carboxylase-3') were used to produce a 130 bp product. The amplicons were separated by agarose gel electrophoresis and detected by ethidium bromide staining.
Cultivation of primary rat RPE cells
RPE cells were obtained from Long Evans rats weighing 150–250 g. The cells were prepared and cultured as described previously by Chang and coworkers (38)
. Cultures were routinely screened for endothelial cell contamination, and immunohistochemistry demonstrated a 99% purity of cytokeratin-positive RPE cells. Cells were cultured at 37° in a constant atmosphere of 5% CO2 and 95% air. RPE cells of passage two, grown to 75–80% confluence, were used for the experiments. The epithelial origin was routinely confirmed by immunohistochemical staining for cytokeratin subtypes with a monoclonal antipan cytokeratin antibody (Ab) (Sigma-Aldrich).
Transduction of the RPE cells
RPE cells were transfected with pRPE65FasL plasmid (5056 bp) using the FuGene6 transfection reagent according to the manufacturer’s instructions (Roche, Mannheim, Germany). The ratio of FuGene: plasmid DNA was 3:1 (3 µl FuGene: 1 µg DNA). The pRPE65FasL plasmid was concentrated to 1 µg/µl. A reporter plasmid expressing the cycle 3-GFP gene under a CMV2 promoter (39)
at a concentration of 1 µg/µl (pTracerTM – CMV2, Invitrogen, Karlsruhe, Germany) served to control the transfection efficiency. The size of the reporter plasmid (6049 bp) was similar to the size of pRPE65FasL plasmid. DNA (2 µg) was used to transfect a 25 cm2 flask of 70–80% confluent RPE cells (approx. 1x106 cells). After 48 h, the medium was removed and the cells were harvested in lysis buffer supplemented with protease inhibitors for protein extraction (see below). Three different transfection groups (three flasks per group) were prepared: pRPE65FasL; GFP reporter plasmid; and control cells treated only with FuGene transfection reagent. For imaging, the cells were fixed in 2% paraformaldehyde for 1 h at room temperature and then washed three times with PBS.
Western blot analysis of cultured RPE cells and of murine RPE
The expression of FasL, caspase-3, caspase-8, PEDF, and VEGF in the RPE layer of transgenic and WT mice was evaluated by immunoblotting. The expression of FasL protein was also investigated in cultured RPE cells. Briefly, the vitreous and retina were removed and RPE layers were lysed for 30 min on ice in lysis buffer [1% Nonidet P-40, 0.5% deoxycholate, 1% SDS, 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH=8)] supplemented with a mixture of protease inhibitors (Sigma-Aldrich). Cultured cells were harvested with 50 µl lysis buffer/flask (approx. 1x106 cells). The samples were cleared by microcentrifugation and assayed for protein concentration (Bradford assay, Bio-Rad Laboratories, München, Germany). Protein (20 µg) was electrophoresed in a 10% Tris-glycin gel (Novex, Invitrogen). Proteins were electrophoretically transferred to a nitrocellulose membrane followed by treatment with blocking buffer (Starting BlockTM T120-TBS, Perbio Science). Equal loading was ascertained by Ponceau S staining. Blots were probed with antibodies to FasL (1:300, rabbit polyclonal, Santa Cruz Biotechnology, Heidelberg, Germany); caspase-3 (recognizes the cleaved p17 active fragment of caspase-3; rabbit polyclonal, 1:200 dilution, Chemicon International, Hofheim, Germany); caspase-8 [rat monoclonal, detects both full-length (
55 kDa) and cleaved (18 kDa) form, 1:200 dilution, Alexis Biochemicals, Grünberg, Germany]; PEDF (rabbit polyclonal, 1:100 dilution, Upstate Biotechnology, Lake Placid, NY), VEGF (rabbit polyclonal, 1:400 dilution, Chemicon International), ß-actin (mouse monoclonal, 1: 10000 dilution, Sigma-Aldrich). After washing, the respective secondary peroxidase-labeled Ab was applied at 1:1000–1:5000 dilution for 1 h at room temperature. Immunoreactive detection was by chemiluminescence (SuperSignalTM West Pico Chemiluminiscent Substrate, Perbio Science). Density of the immunoreactive bands was measured with the Scion Image software (Scion Corp., Frederick, MD, USA). Protein expression values in transgenic and WT mice were normalized by comparison to the expression concentration of ß-actin.
Induction of experimental CNV via laser-photocoagulation
All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CNV was generated as described previously (40)
by laser-induced rupture of Bruch’s membrane in anesthetized mice. Briefly, the pupils were dilated with phenylephrine HCl (0.25%)-tropicamide (0.05%). With a coverslip used as a contact lens, 6 argon laser spots (150 mW intensity, 100 ms duration, 50 µm size; Coherent Novus 2000; Carl Zeiss Meditec, Oberkochen, Germany) were delivered to the retina and choroid of both eyes. Laser spots were applied in a standardized fashion around the optic nerve. Production of a bubble at the time of laser exposure, which indicates rupture of Bruch’s membrane, is an important factor in inducing CNV (40)
. Thus, only mice in which a bubble was observed were included in the study. The development of CNV in the laser lesions was confirmed by fluorescein angiography.
Fluorescein angiography
Fluorescein angiography was performed in anesthetized mice immediately (day 0) and 14 d after laser photocoagulation using a digital imaging system (Heidelberg Retina Angiograph II, Heidelberg Engineering, Dossenheim, Germany). To compensate for the dimensions of the mouse eye, a 30-D lens was attached to the objective of the camera. Angiography was performed immediately after intraperitoneal (i.p.) injection of 0.1 ml of 2.5% fluorescein sodium (Alcon, Freiburg, Germany). Each analysis included early-phase (1–3 min after injection) and late-phase (6–8 min after injection) images.
Fluorescein angiograms were evaluated qualitatively and quantitatively by a blinded observer. The degree of laser-induced CNV was estimated by angiographic leakage. The laser-induced lesions were graded according to the increase in fluorescein leakage between early and late phase into four different groups as shown in Fig. 2
and Table 1
.
|
|
CNV lesions were also quantitatively assessed. The fluorescein leakage area (determined as the area of hyperfluorescence in which no normal retinal blood vessels were observed) was measured for each burn using the Scion Image software. Angiograms taken immediately after laser photocoagulation on day 0 (early and late phase) served as controls and were compared with the angiographic appearance (also early and late phase) of the respective areas on day 14 after photocoagulation. The development of the lesions was characterized by the difference of the leakage areas for each burn. Five control eyes isolated from WT mice and four eyes from transgenic FasL mice that had burns with clearly distinguishable borders were chosen for the quantification.
Evaluation of choroidal neovascularization on flatmounts
The eyes were enucleated and fixed in 2% paraformaldehyde for 1 h at room temperature. Eyecups were washed three times with PBS, and the RPE-choroid-sclera complex was flatmounted. After blocking with PBS containing 5% normal goat serum (Dako, Hamburg, Germany) and 0.5% Triton X-100 (Sigma-Aldrich) for 1 h at room temperature, the flatmounts were incubated overnight at 4°C with rat antimouse CD31 monoclonal antibody (1:100; BD Biosciences Pharmingen, Heidelberg, Germany), which binds to the surface of endothelial cells. After washing a Cy3-conjugated goat antirat IgG serum; (1:500, Dianova, Hamburg, Germany) was used for detection. The flatmounts were examined by a fluorescence microscope (Zeiss Axioplan; Carl Zeiss Meditec) with digital images taken under the same conditions and analyzed with Openlab software (ImproVision Inc., Lexington, MA, USA).
The areas of CNV growth (red hyperfluorescent scars with vessel morphology) were measured with the Scion Image software (see above) via setting a threshold concentration of intensity, above which only these intensively red fluorescent scars were captured (density slicing). The imaging and quantification was performed in a masked manner.
To verify CNV evaluation, animals were perfused with rhodamine-labeled Con A lectin (20 µg/ml, in PBS; Alexis). The eyes were enucleated and fixed in 2% paraformaldehyde and the posterior cup was flatmounted with the retina facing up. The eyes were studied using confocal microscopy (Leica, Bensheim, Germany). The red hyperfluorescence areas at the concentration of choroid were compared with the CNV areas after staining with CD31 Ab.
Histology and immunohistochemistry
For histology, eyes were fixed in 2% paraformaldehyde and routinely processed for paraffin embedding to screen morphological differences between transgene and WT mice aged 1 to 6 mo. Sections (5 µm) were used for hematoxylin and eosin staining, as well as immunohistological and TUNEL analyses.
Blood vessels were visualized by immunohistochemistry for the endothelial cell marker von Willebrandt factor. Paraffin sections were dewaxed and treated with Target Retrieval Solution (Dako). After blocking (blocking reagent, Histar detection kit; Star 4000; Serotec, Eching, Germany), sections were stained for von Willebrand factor (polyclonal rabbit Ab 1:400 in 1% BSA, Dako). The labeling was detected with the appropriate secondary Ab, a Cy3 conjugated goat anti-rabbit IgG (1:500, Dianova). After washing, the staining was evaluated by fluorescence microscopy.
Additionally, macrophages were labeled with F4/80 Ab on paraffin sections 3 d after laser injury. After dewaxing and blocking, sections were stained for F4/80 (monoclonal, rat-antimouse, 1:50 in PBS; Serotec). The cytoplasmic labeling was detected with Serotec histar detection system and visualized by a chromogenic substrate that develops red color. The cell nuclei were stained with Mayer’s Hemalaun solution.
Apoptotic cell death was detected by terminal deoxy nucleotide transferase nick end labeling (In Situ Cell Death Detection Kit, Roche Diagnostics), according to the manufacturer’s instructions and analyzed by fluorescence microscopy. The sections for TUNEL staining were obtained from eyes, enucleated 14 d after laser photocoagulation, which is within the period of active growth and formation of CNV lesions after laser injury. To test the specificity of the TUNEL assay, slides were stained with label solution without terminal transferase (negative control). As expected, in this case no apoptotic nuclei were observed. In addition, a positive control was prepared by treatment of the slices with DNase. Here TUNEL-positive cells were observed in all retinal nuclear layers as well as in the choroid and sclera.
Statistical analysis
All results are expressed as the mean and SD. Student’st test was used for populations with normal distribution and equal variance. The in vivo data were analyzed by Whitney-Mann-U test with post hoc comparisons tested using Fisher’s protected least significant difference procedure. Differences were considered statistically significant when P-values were <0.05.
RESULTS
Generation of transgenic mice and PCR evaluation of transgene expression
Offspring from line no. 2 mice were generated by mating a transgenic parent with a C57BL/6J-Tyr C-2J mouse and expanded by at least six back-crosses. Animals were kept heterozygous for the transgene. Sequencing of the transgene showed the correct sequence of the complete RPE65/FasL construct.
The expression of the transgenic construct was assessed by PCR on genomic DNA. Transgene-specific primers amplified a transgene cDNA-specific 130-bp sequence in transgenic, but not in the WT, mice (Fig. 3
).
|
Expression of transgenic FasL protein in cultured RPE cells and in the RPE layer of transgenic mice
Primary rat RPE cultures were used to confirm transgene expression in RPE cells. Transfection of cultured RPE cells by use of FuGene6 was almost 80% efficient, as determined from the expression of the reporter GFP plasmid (Fig. 4
A). The control RPE cells, transfeced with reporter plasmid (Fig. 4B
, pGFP, lane 2) or exposed to transfected reagent only (Fig. 4B, C
, lane 1), expressed relatively low levels of intracellular FasL protein. Western blotting demonstrated that intracellular FasL protein was highly expressed in lysates of pRPE65FasL-transduced RPE cells (Fig. 4B
, lane 3). Densitometric analysis of the FasL protein expression (Fig. 4C
) showed an almost 17.4-fold increase after transfection of the RPE cells with the pRPE65FasL plasmid. Relative protein expression values are represented as a normalized to the ß-actin expression value and compared with the nontransfected cells (control) set to 1.
|
Similarly, in the RPE layer of WT C57Bl/6J mice, we found a weak basal expression (without laser) of FasL protein (Fig. 5
A) as detected by Western blot analysis. FasL expression in WT mice was not changed two weeks after laser treatment (Fig. 5A
). In contrast, FasL expression was 9.7-fold increased in the RPE layer of FasL transgenic mice (Fig. 5A
, T=transgene, and B, densitometric analysis) when compared with WT mice. Transgenic mice maintained their high FasL expression after laser photocoagulation (Fig. 5A, B
).
|
Eye morphology of transgenic mice
WT and FasL transgenic mice were screened for morphological differences up to 6 mo of age. H&E stained paraffin sections demonstrated a normal choroid and RPE/Bruch’s membrane (Fig. 6
A, C). No difference was found in the retinal and scleral structure between FasL overexpressing mice and the respective WT mice. To evaluate differences in vascularization, immunohistochemistry was performed for the endothelial cell marker von Willebrandt factor (Fig. 6B, D
). No differences in the retinal and choroidal vasculature were observed between FasL transgenic and WT mice up to 6 mo of age.
|
Quantitative assessment of fluorescein angiographic features of laser-induced CNV
Experimental CNV was induced by laser photocoagulation of the RPE-choroid and analyzed by in vivo angiography before eyes were removed for histological evaluation. Two weeks after laser photocoagulation, pathological leakage resembling CNV formation had occurred in most of the WT mice as seen in representative angiograms taken from the early (1–3 min) and late phase (6–8 min) after fluorescein injection (Fig. 7
A). Large and diffuse areas of leakage were observed in WT mice, while in FasL transgenic mice CNV lesions did not exhibit significant leakage (Fig. 7A
). Most of the evaluated WT mice (68%) had (+++)- and (++++)-grade burns, as compared to 31.7% of FasL transgenic mice. Similarly, significantly fewer (++) -grade lesions were found in WT mice when compared to the transgenic FasL mice (16±6% vs. 33.3±10.3, respectively; P=0.03) (Table 2
). To quantitatively assess vascular leakage and the size of membranes, the area of leakage representing the area of CNV was measured in both groups. Two weeks after laser injury, the area of CNV in FasL transgenic mice was up to 70% smaller as compared with WT mice (0.025±0.01 mm2 vs. 0.09±0.04 mm2, respectively; P=0.034) (Fig. 7B
).
|
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Reduction of CNV lesions on RPE/choroid flatmounts from FasL transgenic mice
Two weeks after laser injury, scars on RPE/choroid flatmounts were identified in bright field images by the dark spots of melanin clumping. Subsequent visualization of CNV lesions was achieved by immunolabeling of endothelial cells with a CD31 Ab on RPE/choroid flatmounts (Fig. 8
). We observed CD31-positive areas resembling CNV lesions surrounding the optic nerve (Fig. 8A, B
). These lesions corresponded to the laser spots and presented as elevated neovascular complexes attached to the choroid. We observed cluster of differentiation 31-positive tubular structures, indicating neovascular growth within the laser scars. On higher magnification, these were identified as specifically cell-surface stained endothelial cells (Fig. 8C
). Where present, these structures were found both at the margin and in the central area of the scars. This is in contrast to the homogeneous structure of the nondamaged choriocapillaris stained outside of the region of the laser burns (Fig. 8D
). On flatmounts obtained from the WT mice, large areas of four to five laser scars around the optic nerve were detected, occupied by contained proliferating flat and vessel-forming, tube-like endothelial cells (Fig. 8B, C
). The number of the scars, as well as their size and fluorescence intensity, was reduced on flatmounts obtained from FasL transgenic mice. WT mice demonstrated the expected neovascular response in 72 ± 3.5% of the scars, while only 37.8 ± 7.9% (P<0.05) of the scars from FasL transgenic animals had pathological vessels or proliferating endothelial cells, although dark nonstained areas within the burns were observed as an indicator of laser-induced RPE proliferation and melanin clumping. The relative area of CD31 staining per lesion was significantly smaller in the FasL group than in control mice (1.48±0.34x104 µm2 in the FasL group vs. 2.57±0.45x104 µm2 in the control group P=0.04) (Fig. 8E
).
|
In WT animals, the retina was frequently attached to the choroid at the area of the laser burn, suggesting the presence of vascular communication as well as penetrating and proliferating RPE cells lying between the subretinal neovascular complex and the retina. Confocal microscopy was performed to further investigate the CNV in relation to the retina on retina/RPE/choroidal flatmounts after perfusion with rhodamine-labeled Con A. Figure 9
shows a confocal image (Z-projection) taken from a laser scar two weeks after laser treatment. The laser scar can be seen under the deeper layer of retinal vessels.
|
Histological evaluation of laser burns
Histopathological evaluation of hematoxylin and eosin stained paraffin cross sections showed in all mice a discontinuity in Bruch’s membrane at the area of each laser burn (Fig. 10
C–H). At the site of the laser spots, areas of fibrovascular tissue consisting of vessel lumen, and also large amounts of acellular material, were observed. Densely packed RPE cells surrounded the complex and intertwined with factor VIII positive endothelial cells surrounding lumen-like structures (Fig. 10)
.
|
A gradual difference of the neovascular response was found between WT and transgenic mice. WT mice showed large lesions consisting of fibrovascular tissue, RPE cells, and pigment clumping, while vWF staining demonstrated subretinal extension of choroidal vessels through Bruch’s membrane into the subretinal space (Fig. 10D, F
). In some specimens, damaged photoreceptors (loss of outer segments, reduced number of nuclear rows) were observed attached to the areas of CNV. In contrast, FasL transgenic mice exhibited sparse vascularized lesions with rare vessel-like structures (Fig. 10G, H
).
Microglial cells staining for F4/80 were present near or within the laser burns of WT mice but not of FasL transgenic mice (Fig. 11
A, B). F4/80-labeled cells had an amoeboid appearance. Further, F4/80 immunoreactivity was found within the upper area of the outer nuclear layer in sections derived from both groups.
|
Apoptotic activity in FasL transgenic mice after laser photocoagulation
We further investigated whether endothelial cell apoptosis is involved in the prevention of CNV formation in FasL transgenic mice. Without laser treatment, no apoptotic cells were observed in the retina and choroid of both the WT and the transgenic group. In contrast, TUNEL-positive nuclei were observed in the laser scars of the WT and transgenic group after laser treatment (Fig. 12
A–C). Lesions in FasL transgenic showed a higher number of these TUNEL-positive cells, although the average lesion size was smaller than in WT animals (14.8±3.01 in FasL mice vs. 5.1±1.5 in WT mice; TUNEL-positive cells per lesion; P<0.02). Apoptotic cells were localized around the vessel lumen but limited to the CNV lesions in both FasL transgenic mice and WT animals. To prove the endothelial origin of TUNEL-labeled cells, we stained corresponding slices with Factor VIII. In both groups costaining of TUNEL- and Factor VIII-positive cells was observed.
|
FasL-mediated apoptosis is associated with activation of caspase-8. Therefore, we investigated the expression of this enzyme in protein lysates from RPE/choroid layers taken from WT and transgenic mice one week after laser photocoagulation. We found a strong expression of the active cleaved (18 kDa) form of caspase-8 protein in transgenic mice (Fig. 13
A), which was 6.3-fold higher than in WT mice (P<0.05). There was only weak basal expression of the 18 kD caspase-8 protein in WT mice. Without laser treatment, the expression of activated form of caspase-8 was not enhanced in either group. Fas-activated caspase-8 is sufficient to trigger efficient activation of executional caspases, especially caspase-3, resulting in the final step of apoptosis. After laser treatment the expression of caspase-3 (17 kDa) was increased 4.4-fold in RPE/choroid layer of FasL transgenic mice (P<0.05; Fig. 13B
, T=transgene) compared to the WT mice. Without laser photocoagulation, no difference was found in caspase-3 expression between FasL transgenic and the respective WT mice.
|
VEGF and PEDF expression in FasL transgenic mice
The expression of VEGF and PEDF was investigated by Western blot of lysates of the RPE/choroid and retina isolated before and two weeks after laser photocoagulation in both WT and transgenic mice. No difference was seen in their expression between either groups at any time point.
DISCUSSION
We demonstrate here that mice specifically overexpressing FasL in RPE cells exhibit a reduced response to laser-induced ruptures in Bruch’s membrane while maintaining an otherwise unaffected retina and choroid. Our data support the primary role of RPE cells in the pathogenesis of choroidal neovascularization.
Non-neovascular AMD is generally characterized by a mostly gradual loss of vision that is associated with drusen, pigment changes, and the development of geographic atrophy. In contrast, the more acute form of AMD is characterized by choroidal neovascularization. The time course of the neovascularization depends on the growth pattern of endothelial cells and the interaction with Bruch’s membrane. Nevertheless, the activity of the neovascular disease is finally limited by subretinal scar formation. Altered Fas-FasL interactions are likely to be effective during the active phase of neovascularization, when the expression of VEGF and other molecules is increased and probably alters the normal expression levels of FasL and Fas. This active neovascular phase is adopted in our model of laser-induced neovascularization.
RPE cells are considered to regulate choroidal atrophy as well as neovascularization, as they secrete growth factors responsible for both survival and proliferation of choroidal endothelial cells (41)
. According to current knowledge, defects in Bruch’s membrane associated with excess growth factor expression lead to the formation of CNV that evolve by the migration of choroidal endothelial cells through Bruch’s membrane into the subretinal space (42
43
44)
. The assembly of new vessels continues with the recruitment of other cell types such as RPE cells, which migrate from the RPE monolayer, often become positive for smooth muscle actin (SMA) and express bFGF and VEGF (45)
that in turn further stimulate neovascular growth. To investigate the mechanisms controlling pathological blood vessel growth into the subretinal space, we generated transgenic mice expressing FasL protein in the RPE layer. These FasL transgenic mice showed a strong expression of FasL protein in RPE layer, while the expression of FasL in WT mice was comparatively weak.
The Fas-FasL pathway may be an important mechanism for the maintenance of immune privilege (46)
and controls the entry of potentially damaging lymphoid cells into the eye. Signaling through Fas requires the presence of FasL, which is predominantly expressed on activated T cells, but also throughout the retina (29
30
31)
and cornea (29
, 47)
. FasL is also expressed on the RPE (29
30
31)
and may be involved in regulation of pathological subretinal neovascularization. In this way Fas-FasL interactions may inhibit inflammatory mechanisms that contribute to CNV formation. Our model of laser-induced choroidal neovascularization is well known to be associated with inflammation. In fact, we found an increased number of F4/80-positive cells within the laser burns of WT mice in comparison with transgenic animals. Excised neovascular membranes from patients with AMD demonstrate a pattern of Fas positive new vessels in the center of vascular complex, surrounded by FasL-positive RPE cells (32
, 35)
. Apoptosis in highly vascularized CNV membranes is associated with prominent Fas and FasL expression in stromal RPE cells, endothelial cells, and occasionally in macrophages.
Interestingly, our transgenic mice specifically overexpressing FasL in the RPE layer did not show any pathological changes in blood vessel growth within the retina and choroid. In contrast, increased retinal neovascularization was found in FasL-deficient mice (48)
. Similar to mice overexpressing VEGF in the RPE cells under the RPE 65 promotor (36)
, our mice overexpressing FasL showed normal levels in the first weeks of life (results not shown), and only later did overexpression become evident. This ensures a normal development of the vasculature. Thus, FasL-mediated apoptosis could be restricted to pathological neovascularization. In contrast, in a physiological situation the integrity of Bruch’s membrane may be sufficient to prevent FasL from interacting with endothelial cells.
Therefore, Fas-FasL interactions appear to be important for the growth of pathological vessels in diseases, such as diabetic and ischemic retinopathy, but not for preexisting vessels and the adult vasculature. Indeed, the pathological vaso-obliteration and regressed new vessel growth seen in a mouse model of oxygen-induced ischemia was significantly suppressed by FasL blockade with a FasL-neutralizing Ab (49)
. Similarly, Davies and coworkers (48)
showed that the absence of functional FasL and FasL-Fas interactions (gld mice) leads to an increase in preretinal neovascular nuclei and decreased retinal apoptosis in the same model of oxygen-induced retinopathy. Oxygen-induced retinopathy was further significantly increased in FasL-defective gld and Fas-defective lpr mice (50)
.
To determine whether FasL overexpression influences the growth of choroidal neovascularization, we induced CNV lesions in our transgenic model by laser photocoagulation as described previously (40)
. This results in the focal destruction of the RPE layer and the underlying Bruch’s membrane. Although this model does not exactly mimic all the aspects of CNV that occur in association with AMD, it shares two critical features: abnormalities in Bruch’s membrane are similar to those in AMD; the new vessels develop from the choroid, grow along the edge of the laser burn, and proliferate into the subretinal space (51)
. In humans, defects in the integrity of Bruch’s membrane, either in connective tissue disorders or in high myopia or those iatrogenically produced after intense laser coagulation, are also associated with CNV (52)
.
The role of Fas-FasL in macrophage-related inflammatory reactions was recently discussed in other systems (53)
, and, interestingly, several reports demonstrated that inflammatory cells such as macrophages play an important role in laser-induced CNV (54
55)
. Our current results, however, do not directly support the inflammatory hypothesis but merely suggest a role for RPE cells in the modulation of choroidal neovascularization. This finding is in agreement with Kaplan and coworkers (35)
, who showed that Fas (CD95)-deficient (lpr) and FasL-defective (gld) mice had an increased incidence of neovascularization compared with normal mice in a model of laser-induced damage. Here, the massive subretinal neovascularization with uncontrolled growth of vessels was not due to inflammatory cell defects since chimeric mice with FasL- (gld) RPE cells and normal lymphoid (inflammatory) cells developed neovascularization at the same rate and severity as did nonchimeric gld mice (35)
. Thus, although our model is closely related to inflammation, it is more likely that the Fas-mediated effect on choroidal neovascularization is mediated by endothelial apoptosis.
In our model, we have investigated the pathological changes and blood vessel growth by fluorescein angiography and histology two weeks after laser treatment, the time of peak of CNV extension in rodents (40
, 56
, 57)
. Severe damage with large areas of fluorescein leakage and advanced histological changes was observed in WT mice. In general, in FasL transgenic mice CNV membranes had a reduced size of lesion and a decreased fluorescein leakage when compared with WT mice, a finding that was confirmed by paraffin sections. New blood vessels, as visualized by vWF immunoreactivity, grew from the choroid into the subretinal space. While in WT animals CNV formation is not inhibited by Fas/FasL mediated apoptosis, mice overexpressing FasL possess a mechanism to specifically limit this neovascular growth. This indicates that RPE cells over-expressing FasL can in part control the spread of new Fas-positive endothelial cells as they penetrate Bruch’s membrane and grow into the retina.
It is now believed that RPE senescence is an important event in the development of AMD. Under normal conditions, FasL, expressed in the RPE, inhibits any abnormal growth of new vessels from the choroid into the retina. With age, the expression on FasL may be altered or reduced and subretinal neovascularization associated with vision loss occurs. Our study indicates the importance of the FasL-Fas pathway in controlling pathological vessel growth. Besides its relevance as a potential therapeutic target, these results suggest that subretinal neovascularization in AMD patients may result from a lack of an inhibitory function of FasL-positive RPE on angiogenesis.
Endothelial cells have been shown to express both Fas and FasL but are resistant to FasL-induced apoptosis under normal conditions because cell death is prevented by the cellular caspase inhibitory protein FLIP (58
59
60)
. In fact, we did not find apoptotic cells within the choroid and retina taken from FasL transgenic and WT mice. In some sections few TUNEL-positive cells were found within the retinae of both groups, probably due to damage during tissue preparation.
In contrast, under pathological conditions, for example LDL-induced toxicity, endothelial cell death has been demonstrated to occur through the Fas pathway via reduction of FLIP activity (58)
. Two weeks after laser photocoagulation, TUNEL-positive cells were observed on paraffin cross sections within the areas of laser burns in both groups. The total number of apoptotic cells within the laser burns was higher in FasL transgenic mice, with most apoptotic cells localized in the subretinal space. Only a few TUNEL-positive cells were found within the photoreceptor nuclear layer, probably associated with the CNV-induced damage in this region. Staining for a specific endothelial cell marker (vWF) showed localization of TUNEL-positive cells within the vascular structures. We suggest that the reduction of the CNV lesions in FasL transgenic mice might be a direct result of increased endothelial cell apoptosis in these mice, due to contact of Fas-positive proliferating endothelial cells to the FasL-positive RPE cells. This hypothesis is supported by the observation by Kaplan and coworkers (35)
that human choroidal endothelial cells undergo apoptotic cell death through the FasL-Fas pathway when cultured together with FasL-positive RPE cells.
Apoptosis through the Fas receptor is a tightly controlled process. Membrane FasL and secondarily aggregated soluble FasL induce reorganization of inactive Fas complexes to supramolecular clusters that have the capacity to signal apoptosis. On formation of apoptosis-competent signaling clusters, Fas can recruit the cytoplasmic death domain-containing adaptor protein FADD (Fas-associated death domain protein), which in turn recruits procaspase-8. As a result, procaspase-8 is activated by dimerization (61
, 62)
, which leads to autoproteolytic processing and release of the mature and active heterotetrameric form of enzyme. Furthermore, Fas-activated caspase-8 is sufficient to trigger efficient activation of effector caspases, especially caspase-3, resulting in execution of the final steps of apoptosis (63)
. In RPE/choroid lysates taken from FasL transgenic mice, we found an increased expression of the active, cleaved form of caspase-8 and caspase-3 indicating activation of FasL-Fas pathway after laser photocoagulation.
It has been shown that both TSP1 and PEDF require FasL to block angiogenesis in vitro and in vivo (21)
. Fas is induced by angiogenic stimuli, marking newly forming vessels for destruction by the FasL, induced by TSP1 and PEDF. In our study, we found that FasL overexpression in RPE layer did not influence the concentration of PEDF and VEGF protein within RPE/choroid.
In conclusion, we demonstrate here that the expression of FasL on RPE cells can modulate the formation of new vessels induced by laser injury and that FasL-Fas interactions control the extent of neovascularization in the choroid via induction of apoptosis in newly forming vessels. Nevertheless, it should be stressed that this interaction does not form an absolute barrier but is merely a mechanism to regulate the extent of the disease. In relation to this, we observed that FasL transgenic mice have no obvious changes in with retinal and choroidal vasculature under normal conditions. Therefore, FasL-Fas interactions cannot completely abolish choroidal neovascularization but are involved in controlling the extent of pathological subretinal vessels induced by laser photocoagulation.
ACKNOWLEDGMENTS
This study was funded by the grants ZMMK TV 76, DFG Jo 324 /4–1, DFG Jo 324/6–1 (Emmy-Noether), and DFG Ki 743/6–1 (DFG Priority Program Macular Degeneration) and by the Kämpgen Stiftung, RetinoVit Stiftung Köln, and the Köln Fortune program of the Medical Faculty, University of Cologne. The authors thank Frank Lacina, Claudia Gavranic, and Martina Becker for excellent technical assistance. Julia Schiekel, BSc, was responsible for the mouse breeding.
FOOTNOTES
1 These authors contributed equally to this work. 
Received for publication January 12, 2006. Accepted for publication March 20, 2006.
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