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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
SPECIFIC AIMS
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. AMD is the leading cause of blindness in Western nations. The extent of angiogenesis in vivo depends on the local balance between pro and antiangiogenic molecules. Several inhibitors require apoptotic events to block neovascularization in vivo. FasL induces apoptotic cell death in cells expressing the Fas receptor. In the current study we evaluate the effect of a specific over-expression of FasL in RPE cells on the formation of choroidal neovascular membranes (CNV) in a transgenic mouse model. Our data indicate that specific targeting of the Fas/FasL pathway on RPE cells may prove beneficial in preventing or treating neovascular AMD while leaving the delicate retinal structures unaffected.
PRINCIPAL FINDINGS
1. Transgenic mice express FasL protein specifically in RPE cells
In the RPE layer of wild-type (WT) C57Bl/6J mice, a weak basal expression (without laser) of FasL protein was detected by Western blot analysis. FasL expression in WT mice was not changed two weeks after laser treatment. In contrast, FasL expression was 9,7-fold increased in RPE layer of FasL transgenic mice in comparison to WT. Transgenic mice maintained their concentration of high FasL expression after laser photocoagulation.
2. FasL overexpression in RPE cells reduces vascular leakage and lesion size of laser-induced choroidal neovascularization
To investigate the effect of FasL overexpression in laser-induced choroidal neovascularization, mice were analyzed by fluorescein angiography. Two weeks after laser photocoagulation, pathological leakage resembling CNV formation was observed in most of the WT mice. In FasL transgenic mice, CNV lesions did not exhibit a significant leakage. Most of the evaluated WT mice (68%) had (+++)- and (++++)-grade burns compared with 31.7% of the evaluated FasL mice. Similarly, significantly fewer (++)-grade lesions were found in WT mice when compared with the transgenic FasL mice (16±6% vs. 33.3±10.3, respectively; P=0.03). Two weeks after laser injury, the area of CNV in FasL transgenic mice was up to 70% smaller compared with that of WT mice (0.09±0.04 mm2 vs. 0.025±0.01 mm2, respectively; P=0.034) (Fig. 1
).
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To better quantify the area of leakage, scars on RPE/choroid flatmounts were assessed after immuno-labeling of endothelial cells with a CD31 antibody. CNV lesions corresponded to the laser spots and presented as elevated neovascular complexes attached to the choroid. Clusters of differentiated 31 (CD31)-positive tubular structures, indicating neovascular growth, were observed within the laser scars. This finding is in contrast to the homogeneous structure of the nondamaged choriocapillaris after CD31 staining outside of the laser burns. WT mice demonstrated the expected neovascular response in 72 ± 3.5% of the scars, whereas 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 (2.57±0.45 x 104 µm2 vs. 1.48±0.34 x 104 µm2 in FasL group, P=0.04).
3. FasL overexpression in RPE cells results in a reduced subretinal membrane formation after laser induced choroidal neovascularization
WT and FasL transgenic mice were screened for morphological differences for up to 6 mo of age. No difference was found in the retinal and scleral structure among FasL overexpressing mice and the respective WT mice. Similarly, no differences in the retinal and choroidal vasculature were observed between FasL transgenic and WT mice up to 6 mo of age.
After laser photoculation, a discontinuity in Bruch’s membrane in the area of each laser burn was seen in all mice. At the site of the laser spots—areas of fibrovascular tissue consisting of vessel lumen, but also a large amount of acellular material—were observed. Densely packed RPE cells surrounded the complex and intertwined with factor VIII positive endothelial cells surrounding lumen-like structures. WT mice showed large lesions consisting of fibrovascular tissue, RPE cells and pigment clumping. Attached to the areas of CNV-damaged photoreceptors (loss of outer segments, reduced number of nuclear rows) were observed in some specimens. In contrast, FasL transgenic mice exhibited a smaller amount of vascularized lesions with only a few vessel-like structures (Fig. 2
). Similar to the observation in flat mounts, the number of CNV lesions on the sections was reduced in the FasL transgenic mice. Microglial cells staining for F4/80 were present near or within the laser burns of WT mice but not in FasL transgenic mice. 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.
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4. FasL overexpression in RPE cells results in an increased rate of endothelial cell apoptosis in CNV lesions
To investigate whether endothelial cell apoptosis is involved in the prevention of CNV formation in FasL transgenic mice, TUNEL positivity was assessed. 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. Lesions in FasL transgenic mice showed a higher number of TUNEL-positive cells, although the average lesion size was smaller compared with 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 colocalization of TUNEL- and Factor VIII-positive cells was observed.
FasL-mediated apoptosis is associated with activation of caspase-8. We found a strong expression of the active form of caspase-8 protein in transgenic mice, which was 6.3-fold higher than in WT mice (P<0.05). 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 was increased 4.4-fold in RPE/choroid layer of FasL transgenic mice (P<0.05) compared with the WT mice. Without laser photocoagulation, no difference was found in either caspase 8 or caspase-3 expression between FasL transgenic mice compared with the respective WT mice.
5. The reduction of choroidal neovascularization by FasL overexpression in RPE cells is distinct from vascular endothelial growth factor (VEGF) and PEDF expression
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 the WT and the transgenic groups at any time point.
CONCLUSIONS AND SIGNIFICANCE
RPE cells are considered to regulate between choroidal atrophy and neovascularization, as they secrete growth factors responsible for both survival and proliferation of choroidal endothelial cells. 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. 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. 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.
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 choroids 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 retinal and choroidal vasculature under normal conditions. Therefore, FasL-Fas interactions probably cannot completely abolish choroidal neovascularization but are involved in controlling the extent of pathological subretinal vessels induced by laser photocoagulation.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5653fje
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