Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

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established by oxygen-induced retinal ischemia, photodynamically-induced retinal branch vein occlusion and intravitreal implantation of VEGF sustained-release pellets (Campochiaro, 2000; Saito et al., 1997; Ozaki et al., 1997; Tolentino et al., 2002). However, retinal neovascularization in these animal models is either transient or occurs with a delayed onset.

Advanced technology in gene delivery and transgene manipulation has made it possible to generate long-term and reproducible animal models of retinal neovascularization (Wang et al., 2003; Rakoczy et al., 2003; Okamoto et al., 1997; Ruberte et al., 2004). We recently generated a transgenic model of retinal neovascularization by manipulating photoreceptor-specific overexpression of human VEGF (hVEGF) in the eye (Lai et al., in press). A total of four transgenic lines were generated, with one expressing low hVEGF levels and showing correspondingly mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation (Lai et al., in press). By contrast, the other three lines expressed high hVEGF levels accompanied by concomitant severe phenotypes. In this study, several generations of two transgenic lines showing mild or severe vasoproliferative retinopathy (lines 029 and 056, Lai et al., in press) were chosen for further characterization.

2. MATERIALS AND METHODS

2.1. Animals

Transgenic mice produced using a pcDNA.opsin.VEGF165 construct driven by the mouse rhodopsin promoter were used in this study. The offspring were screened for transgenic animals by Southern blot analysis showing the presence of a 2.1 kb fragment containing the truncated mouse rhodopsin promoter and human VEGF165 (hVEGF165) fragments, which was then confirmed by polymerase chain reaction amplification of tail DNA. The heterozygote transgenic offspring of transgenic lines 029 and 056 were used for this investigation (Lai et al., in press).

2.2. Fundus Fluorescein Angiography and Retinal Perfusion with Fluorescein-Labeled Dextran

Five generations of line 029 and two generations of line 056 were examined by fundus fluorescein angiography with a modified portable Kowa Genesis camera after intraperitoneal injection of 0.05 ml of 10% sodium fluorescein as described previously (Zaknich et al., 2002). For retinal perfusion, selected mice were deeply anesthesized and initially perfused with 10 ml of PBS through the left ventricle into the aorta to flush out circulating blood, followed by 2 ml fluorescein-labeled dextran (FITC-dextran, 50 mg/ml, molecular weight, 2.0 ¥ 106; Sigma, St. Louis, MO). The perfused eyes were fixed in 2% paraformaldehyde for 30 min and then flat-mounted for fluorescence microscopy.

2.3. Histology

Eyes were enucleated from transgenic mice showing mild, moderate or severe vasoproliferative retinopathy on angiograms. The enucleated eyes were fixed in 4%

fusion with fluorescein-labeled dextran revealed obvious neovascular proliferation accompanied by the loss of capillaries in the retina (Fig. 28.1E and 28.1F). In contrast to line 029, line 056 transgenic mice predominantly demonstrated severe vasoproliferative retinopathy (Fig. 28.1G-I, Table 28.1). In these eyes, fluorescein angiography showed heavy fluorescein leakage or pooling (Fig. 28.1G) and perfusion with fluorescein-labeled dextran revealed massive neovascular proliferation in the retina (Fig. 28.1H and 28.1I).

Two hundred and one transgenic mice were examined by fluorescein angiography at 6- 8 weeks old. These included 184 transgenic mice from 5 generations of line 029 and 17 transgenic mice from 2 generations of line 056 (Table 28.1). Flourscein angiography showed that 80% of line 029 transgenic mice showed mild/moderate retinopathy while 20% demonstrated severe retinal vascular changes (Table 28.1). In contrast, 76% of the transgenic mice of line 056 showed severe vasoproliferative retinopathy, with only 24% of the transgenic mice developing mild/moderate retinopathy (Table 28.1). Selected animals were examined by fluorescein angiography periodically. The mild and moderate vascular changes of line 029 remained relatively stable for at least 3 months but, in contrast, line 056 transgenic mice with severe vascular proliferation rapidly ceased fluorescein leakage and developed retinal capillary loss with time.

Histologically, eyes with mild retinopathy on angiograms demonstrated scattered vasoproliferation in the subretinal space but without obvious retinal degeneration, although photoreceptors were slightly disturbed (Fig. 28.2A and 28.2B). In eyes showing moderate vasoproliferation, histology revealed a greater number of neovascular lesions. The intraretinal microvascular abnormalities seemed to originate from deep retinal capillaries growing into the subretinal space with an attempt to communicate with choroidal capillaries (Fig. 28.2C and 28.2D). The disturbance to photoreceptors was obvious in these eyes (Fig. 28.2C and 28.2D). In eyes showing heavy fluorescein leakage on angiograms, histology demonstrated massive intraretinal microvascular abnormalities and neovascular proliferation in the subretinal space, accompanied by severe retinal folding and disturbance to photoreceptors (Fig. 28.2E and 28.2F).

C D

E F

Figure 28.2. Histological changes of transgenic mice of lines 029 (A-D) and 056 (E-F) showing intraretinal microvascular abnormalities and vascular proliferation in the subretinal space. The arrows in D and F indicate the growth of new vessels from the outer nuclear layer into the subretinal space. B, D and F are higher magnifications of A, C and E, respectively.

4. DISCUSSION

In this study, we examined five generations of line 029 and two generations of line 056 transgenic mice with a rhodopsin promoter driven hVEGF overexpression in photoreceptors. We found that mice from transgenic line 029 mainly presented mild or moderate proliferative retinopathy with limited disturbance to the neural retina, while mice from line 056 predominantly developed severe vasoproliferative retinopathy with dramatic destruction to the neural retina. In our previous study, both lines 029 and 056 produced increased hVEGF expression but the levels of hVEGF protein in eyes of line 056 were 10-fold higher than those in eyes of line 029, indicating that the pathological phenotypes in the retinae were closely associated with the overexpression of hVEGF in the retina (Lai et al., in press). In this study, the results obtained from five generations of line 029 and two generations of line 056 suggested that the features presented in both transgenic lines remained relatively steady

for as many as five generations. Both transgenic mice lines may prove very useful for testing anti-angiogenic therapies and for investigations of cellular and molecular mechanisms of VEGF-induced retina neovascularization in the eye.

It is very clear that angiogenic factors belonging to the VEGF and angiopoietin protein families play critical roles in retinal neovascularization that occurs in diseases such as diabetic retinopathy, retinopathy of prematurity, retinal branch vein occlusion and retinal central vein occlusion (Campochiaro, 2000; Miller, 1997). Understanding the steps in the angiogenic processes and the angiogenic factors involved in angiogenesis has led to the development of strategies for treatment of ocular angiogenesis. A large number of anti-angiogenic agents have been designed based on strategies by (1) either interfering with VEGF and angiopoietin proteins and their receptors or the downstream signaling or (2) upregulating endogenous angiogenic inhibitors or administration of exogenous inhibitors to counter the angiogenic effect of angiogenic factors (Campochiaro, 2002; Bainbridge et al., 2003). However, evaluation of long-term efficacy of anti-angiogenic therapies has been challenged by transient ocular neovascularization in most animal models. For example, retinal neovascularization in the murine model of ischemia-induced retinopathy occurs as a short-lived response. After post-natal day 21 no further proliferation occurs and the new vessels regress spontaneously (Igarashi et al., 2003; Rota et al., 2004; Bainbridge et al., 2003). In addition, intraocular injection in newly born pups is problematic. In the present study, we have generated a transgenic line (029) with increased expression of VEGF accompanied by mild to moderate vasoproliferation in the retina at an adult age (4 weeks postnatal), and the features of vasoproliferative retinopathy remain stable for at least 8 weeks (12 weeks postnatal). This particular model provides an excellent opportunity to intervene at an early stage in the development of retinal neovascularization in the adult animal.

Although many studies have investigated blood vessel growth in the retina, relatively few studies have addressed the damage to retinal neurons during the processes of retinal neovascularization. In the present model, we also observed disturbance to photoreceptors when new vessels occurred within or adjacent to the outer nuclear layer, and the extent of neural damage was closely associated with the severity of retinal vascular proliferation. There are two basic hypotheses that account for the neural damage accompanied with retinal neovascularization in this model: First, the increased expression of VEGF in photoreceptors result in loss of blood-retinal barrier integrity, which initially manifests as an increase in vascular permeability, causing a failure to control the composition of the extracellular fluid in the retina, which in turn leads to edema and neuronal cell loss. Alternatively, the longterm overexpression of VEGF may initiate apoptosis in the neural retina, leading to gradual loss of neurons. It is not clear which hypothesis will be found to be correct and, in fact, it is likely that vascular permeability and neuronal apoptosis are closely linked components in this model. In a recent study, the retinal neural damage in this model can be remarkably attenuated by recombinant adeno-associated virus mediated transfer of soluble Flt (sFlt) receptor, a heparin-binding protein that complexes VEGF with high affinity, therefore inhibiting the mitogenic response to VEGF by directly sequestering VEGF and in a dominant negative manner via heterodimerizing with the extracellular ligand-binding region spanning FLT-1 and KDR receptors (unpublished data). Since it is now apparent that retinal vascular abnormality and damage to retinal neurons are equally problematic in patients with diabetes (Barber, 1998; Barber et al., 2003), our transgenic models may provide a useful tool for studies on microvascular changes and VEGF-induced retinal neural damage in diabetic retinopathy.

5. ACKNOWLEDGEMENTS

This work was supported by the Juvenile Diabetes Research Foundation International, Australian National Health and Medical Research Council and Westpac (Australia). This work is part of the research effort of the Diabetic Retinopathy Consortium, Perth, Western Australia. We thank the Foundation Fighting Blindness for the Young Investigator Award provided to WYS to attend this meeting.

6. REFERENCES

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Proliferative diabetic retinopathy is characterised by the presence of abnormal vessels arising from the optic disk or retina. New vessels develop as fine, leaky structures and bleed easily into the retina and vitreous, leading to the development of haemorrhages that can cause sudden vision loss. This is accompanied with thickening of the extracellular matrix that instigates contraction of the fibrotic component of vessels, resulting in retinal detachment. Central vision can also be affected as these retinal changes move towards the macula. In severe cases, proliferating vessels obscure normal retinal blood flow and can result in the development of neovascular glaucoma, ultimately leading to severe loss of vision or permanent blindness.

3. LASER PHOTOCOAGULATION THERAPY

Laser photocoagulation of the retina is a non-invasive laser treatment and remains the primary therapy for proliferative retinopathies such as diabetic retinopathy. Current photocoagulation treatment is based on the original developments of ruby, argon and krypton lasers first utilised in the late 1960’s [Reviewed in Petrovic and Bhistkul (1999)]. Tunable dye and diode lasers were subsequently introduced, making available a range of useful techniques for the treatment of proliferative retinopathies.

Laser photocoagulation acts by focusing laser energy on the retinal pigment epithelium (RPE), damaging the outer retinal layers whilst leaving Bruch’s membrane intact. The main site of energy absorption is the melanin within the RPE and choroid as it has an absorption spectrum of between 400-700 nm. The different types of lasers used in photocoagulation include argon (emission at 488 nm, blue/green; 514 nm, green), Nd:YAG (532 nm, green) krypton (647 nm, red) diode (810 nm, infrared) and tuneable dyes such as rhodamine that emit over a selected range of wavelengths. Retinal damage caused by laser photocoagulation can be reduced by decreasing the wavelength, spot size, irradiance and exposure duration (Mainster, 1999). Argon laser is strongly absorbed by melanin and haemoglobin and therefore makes an excellent source for direct targeting of vessels. Krypton laser is less absorbed by melanin and produces deeper and more painful chorioretinal lesions and is therefore less often used in treating retinopathies where induction of choroidal neovascularisation should be avoided.

3.1. Histological Changes

The observed histopathological changes following laser treatment, are a result of the heat transfer out of the absorbing RPE and choroid and subsequent denaturation of the surrounding tissue (Roider et al., 1998). The use of short wavelength lasers such as argon minimises damage to the choroid and therefore is less likely to induce choroidal neovascularisation, detrimental to diabetic retinopathy treatment. The major sites of damage following argon laser photocoagulation are the RPE and outer retinal layers and within the lasered site, the outer segments of the photoreceptors are destroyed (Figure 29.1B). An initial inflammatory response occurs and coagulated cells in the lesion core are removed by phagocytosis. The RPE monolayer can lift to form a gap at the site where photoreceptor outer segments were. This is followed by RPE cell migration and proliferation into multiple layers. Müller cells also proliferate and migrate into areas of damage and interdigitate into RPE cells forming a glial scar (Lewis et al., 1992).