Increased vascular endothelial growth factor (VEGF) and transforming growth factorβ (TGFβ) in experimental autoimmune uveoretinitis: upregulation of VEGF without neovascularization

Journal of Neuroimmunology 89 Ž1998. 43–50

Increased vascular endothelial growth factor ž VEGF/ and transforming growth factor b ž TGFb / in experimental autoimmune uveoretinitis: upregulation of VEGF without neovascularization S.A. Vinores a

a,)

, C.-C. Chan b, M.A. Vinores a , D.M. Matteson b, Y.-S. Chen A. Shi a , H. Ozaki a , P.A. Campochiaro a

a,c

, D.A. Klein a ,

825 Maumenee Building, The Wilmer Ophthalmologic Institute, Johns Hopkins UniÕersity School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287-9289, USA b Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD, USA c Cathay General Hospital, Taipei, Taiwan Received 25 August 1997; revised 5 March 1998; accepted 6 March 1998

Abstract Experimental autoimmune uveoretinitis ŽEAU. was induced in Lewis rats and B10.A mice by immunization with S-antigen ŽS-Ag. to study the potential roles of vascular endothelial growth factor ŽVEGF. and the b 1 and b 2 isoforms of transforming growth factor ŽTGFb 1 and TGFb 2 . during the progression of the disease. VEGF has been implicated as an angiogenic factor in ischemic retinopathies; however, Lewis rats developing EAU have high levels of VEGF in the retina, but no neovascularization. In the present study, immunohistochemical staining for VEGF, TGFb 1 and TGFb 2 was performed on the retinas of Lewis rats developing EAU or with oxygen-induced ischemic retinopathy. In rats immunized with S-antigen, a marked upregulation of VEGF was immunohistochemically visualized from the inner nuclear layer to the inner limiting membrane prior to blood-retinal barrier ŽBRB. failure and lymphocytic infiltration. VEGF is normally induced by hypoxia and its induction leads to neovascularization. Coincident with the increase in VEGF, there was increased immunoreactivity for TGFb 1 and TGFb 2 within the same layers of the retina. In contrast, rats with ischemic retinopathy and retinal neovascularization showed only a modest increase in VEGF immunoreactivity, which is largely confined to retinal ganglion cells and inner retinal vessels, and little or no increase in TGFb 1 or TGFb 2 . In addition, in mice developing EAU, which does not have an abrupt onset as it does in rats and may involve neovascularization, a comparable upregulation of VEGF in the inner retina to that seen in rats developing EAU occurs with no increase in TGFb 1 or TGFb 2 . Since TGFb can inhibit endothelial cell proliferation, it is likely that an increase in TGFb may prevent VEGF from exerting its endothelial growth activity in the rat EAU model, but VEGF may be operative in inducing BRB failure. These data suggest that there is a complex interaction among growth factors in the retina and that retinal neovascularization may require an imbalance between stimulatory and inhibitory factors. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Vascular endothelial growth factor; Transforming growth factor beta; Experimental autoimmune uveoretinitis; Retina; Blood-retinal barrier

1. Introduction Vascular endothelial growth factorrvascular permeability factor ŽVEGFrVPF. is an angiogenic factor that is also a potent mediator of increased vascular permeability ŽSenger et al., 1983, 1986, 1993; Dvorak et al., 1995.. VEGF is induced by hypoxia ŽPlate et al., 1993; Goldberg

)

Corresponding author. Tel.: q1 410 9554103; fax: q1 410 5025382.

0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 0 7 5 - 7

and Schneider, 1994; Hashimoto et al., 1994; Minchenko et al., 1994a,b; Shima and D’Amore, 1995. and is upregulated in ischemic retinopathies where it appears to promote retinal neovascularization ŽAdamis et al., 1993, 1994; Aiello et al., 1994; Malecaze et al., 1994; Miller et al., 1994; Dastgheib et al., 1995; Pierce et al., 1995; Vinores et al., 1997.. However, there are disease processes in which there are high levels of VEGF, but no retinal neovascularization. Examples of these disease processes in humans include ocular melanoma ŽVinores et al., 1995., nonprolif-

44

S.A. Vinores et al.r Journal of Neuroimmunology 89 (1998) 43–50

erative diabetic retinopathy ŽLutty et al., 1996., and a variety of non-ischemic retinal disorders ŽVinores et al., 1997.. The same is shown in experimental autoimmune uveoretinitis ŽEAU., a T-cell mediated autoimmune disease which serves as a model for endogenous uveitis in humans ŽForrester et al., 1990, 1992; Nussenblatt and Gery, 1996; Rajasingh et al., 1966.. It is induced by immunization of Lewis rats with S-antigen ŽS-Ag. and is characterized by mainly lymphocytic infiltration beginning 10 days after immunization with concommitant BRB breakdown, followed by immune-mediated retinal destruction, initially directed against the rod outer segments and then rapidly spreading to the other layers of the retina ŽGreenwood, 1992; McMenamin et al., 1993a,b.. EAU can also be induced in mice ŽCaspi et al., 1988., but the disease differs from that seen in rats in that the course of the disease is longer, the onset is not abrupt, recurrence is common, and neovascularization may occur. There are several possible reasons that could account for the absence of neovascularization despite VEGF upregulation in various disease processes. Ž1. It is possible that one or more additional angiogenic growth factors are necessary for retinal neovascularization to occur and are missing in these disease processes. Ž2. There may not be upregulation of VEGF receptors as occurs in ischemic retinopathies. Ž3. There may be greater expression of an inhibitor of neovascularization than in ischemic retinopathies in which retinal neovascularization occurs. TGFb is one of the possible inhibitors of neovascularization as both TGFb 1 and TGFb 2 suppress vascular endothelial cell proliferation ŽJennings et al., 1988; McAvoy and Chamberlain, 1990; Chakravarthy and Archer, 1992; Pertovaara et al., 1994; Behzadian et al., 1995; Kulkarni et al., 1995; Yoshimura et al., 1995.. The present study investigates the hypothesis that altered levels of TGFb in the retina could account for the differential influence of VEGF on retinal vascular endothelial proliferation seen when comparing EAU with ischemic retinopathies.

2. Materials and methods EAU was induced in 6–8 week old female Lewis rats weighing 175–200 g by injecting one hind footpad with 30 m g S-antigen mixed with an equal amount of complete Freund’s adjuvant ŽCFA. enriched with mycobacterium tuberculosis strain H37Ra ŽDIFCO, Detroit, MI. in a total volume of 0.1 ml. Normal-unimmunized rats were used for comparison. For light microscopic immunohistochemistry, 18 rats 8 days after immunization with S-antigen Žprior to lymphocytic infiltration., 16 rats 11 days after immunization Žat the onset of lymphocyte infiltration., and 7 normal rats were used. One eye from each animal was used for immunohistochemistry. EAU was induced in 6–8 week old female B10.A mice by immunizing them with 50 m g of the retinal antigen,

IRBP, in CFA as previously described ŽSun et al., 1997.. Mice were sacrificed on days 0 Ž n s 2., 11 Ž n s 4., 16 Ž n s 4., and 21 Ž n s 5.. The severity of EAU was graded as previously described ŽChan et al., 1990., taking into consideration inflammatory cell infiltration, vasculitis, granuloma formation, retinal folding andror detachment, photoreceptor damage, and the size and number of lesions. One eye from each animal was fixed in formalin and embedded in paraffin. A hematoxylin and eosin-stained section from each specimen was independently graded in increments of 0.5 for the severity of disease by 2 separate investigators who were masked to the identity of the specimen. The mean of the grades was used for statistical analysis. Ischemic retinopathy, which leads to neovascularization, was induced in 28 newborn Sprague–Dawley rats as previously described ŽPenn et al., 1993. by maintaining rats in hyperoxic conditions in which the oxygen level was alternated between 40 and 80% every 12 h for 14 days and then returning them to the relative hypoxia of room air for 5 days. Ten newborn Sprague–Dawley rats maintained in room air for the duration of the experiment served as controls. One eye from each of these Sprague–Dawley rats was fixed in paraformaldehyde and frozen in O.C.T. compound. A modification of this technique ŽPenn et al., 1994., in which newborn rats were immediately placed in an environment of 40% oxygen alternating every 48 h with 80% oxygen for 14 days, was found to induce even greater neovascularization in Sprague–Dawley rats. It was not known whether Lewis rats would respond in the same way to this treatment, but since Lewis rats were used for the induction of EAU, they were subjected to these conditions to compare the autoimmune and ischemic models in the same strain. Four Lewis rats and a Sprague–Dawley rat Žfor comparison. were treated in this fashion with 3 Lewis and 6 Sprague–Dawley rats from the same respective litters that were maintained in room air, 2 normal adult Lewis rats, and 3 normal adult Sprague–Dawley rats to serve as controls. One eye from each of these animals was fixed in formalin and embedded in paraffin. Formalin-fixed, paraffin-embedded sections or paraformaldehyde-fixed frozen sections ŽSprague–Dawley rats only. were used for immunohistochemistry. Paraffin sections were deparaffinized with xylene, re-hydrated through a series of graded alcohols, and immunoreacted with affinity-purified rabbit polyclonal antibodies ŽSanta Cruz Biotechnology, Santa Cruz, CA. directed against VEGF Ž1:20–1:40., TGFb 1 Ža 1:500 dilution was used with or without control peptide to determine specificity; a 1:100 dilution was used for photographing sections where a comparison with control peptide was not done., or TGFb 2 Ž1:100.. Primary antibodies were diluted with 1% normal goat serum ŽNGS. in 0.05 M Tris-buffered saline, ph 7.6, ŽTBS. and blocking was achieved with 10% NGS in TBS. For controls, non-immune serum was substituted for primary antibody or the primary antibodies were pre-in-

S.A. Vinores et al.r Journal of Neuroimmunology 89 (1998) 43–50 Table 1 Mean grading of staining intensity for VEGF Treatment group

Mean staining intensity" standard error

Normal Lewis rats Lewis rats, 8 days after immunization with S-antigen Lewis rats, 11 days after immunization with S-antigen Normal B10.A. mice B10.A mice 11 days after immunization with IRBP B10.A mice 16 days after immunization with IRBP B10.A mice 21 days after immunization with IRBP Lewis rats with oxygen-induced ischemic retinopathy

0.14"0.06 2.75"0.25 3.00"0.00 1.00"0.00 2.37"0.12 2.87"0.12

45

ŽChen et al., 1997., and the slides were counterstained with Meyer’s hemotoxylin. For VEGF, TGFb 1 , and TGFb 2 , the immunohistochemically stained slides were graded in increments of 0.5 by a masked investigator for intensity of stain with 0 being negative, 1 s weak, 2 s moderate, and 3 s intense staining. These numerical values were analyzed by the Mann– Whitney U-test and statistical significance was determined by comparing the groups. For mice developing EAU, the grade for the stage of the disease was compared to the numerical score for the intensity of VEGF staining by the Wilcoxon Signed Rank Test.

1.8"0.20 0.5"0.29

3. Results 3.1. VEGF

cubated for 2 h at room temperature or overnight at 48C with a tenfold excess of their respective control peptides ŽSanta Cruz Biotechnology.. Immunoreactivity was visualized with HistoMark Red and sections were counterstained with Contrast Blue according to the manufacturer’s instructions ŽKirkegaard and Perry, Gaithersburg, MD.. Immunoreactivity on some of the sections from rats with oxygen-induced ischemic retinopathy were visualized with 3-amino-9-ethylcarbazole ŽAEC., as previously described

Normal Lewis rats were entirely negative for VEGF or had very weak staining in the inner plexiform layer ŽTable 1; Fig. 2A.. Eight days after immunization, a significant upregulation of VEGF positivity was demonstrated in all animals in the ganglion cells, the nerve fiber layer, the inner plexiform layer, and in some cells located in the inner nuclear layer Ž p s 0.003; Fig. 1A.. By 11 days after immunization, VEGF immunoreactivity became even more intense and was seen in more cells in the inner nuclear layer and in the outer retina Ž p s 0.003; Fig. 1B; Fig. 2B..

Fig. 1. ŽA. Eight days after immunization with S-antigen, VEGF is demonstrated Žred. throughout the inner retina from the inner nuclear layer to the inner limiting membrane Žtop.. ŽB. Eleven days after immunization with S-antigen, intense VEGF positivity is clearly seen in the inner retina and weaker staining is also demonstrated in the outer retina Žbottom.. ŽC. Immunohistochemical staining for TGFb 1 in normal Lewis rat retina showed weak staining of ganglion cells Žarrows.; otherwise, the retina was negative. ŽD. Eleven days after immunization with S-antigen, the inner retina was conspicuously stained for TGFb 1 . ŽE. Pre-incubation of the TGFb 1 primary antibody with control peptide resulted in a marked reduction in staining on a concurrently run serial section from the same eye.

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S.A. Vinores et al.r Journal of Neuroimmunology 89 (1998) 43–50

Fig. 2. ŽA. VEGF staining in the retina of a normal Lewis rat is negative. ŽB. Higher magnification of the inner retina of a Lewis rat 11 days after immunization with S-antigen Žcompare to Fig. 1B., which has been stained for VEGF Žred.. Some cells in the inner nuclear layer are positive and staining extends from the inner nuclear layer to the inner limiting membrane Žtop.. Note intense positivity in retinal ganglion cells Žarrow.. ŽC. VEGF is demonstrated in retinal ganglion cells and in vessels in the inner retina of a Lewis rat with oxygen-induced ischemic retinopathy. Immunoreactive sites are visualized with AEC rather than HistoMark Red.

Normal B10.A mice showed weak staining in the inner retina and demonstrated an upregulation of VEGF in the inner retina at 11 Ž p s 0.02., 16 Ž p s 0.02., and 21 Ž p s 0.05. days after immunization with IRBP ŽTable 1.. Since each mouse at a given time point during the onset might not be affected exactly the same by the disease, a comparison was made between VEGF staining intensity and severity of EAU. The Wilcoxon Signed Rank Test showed a direct correlation between VEGF upregulation in the inner retina and the grading of EAU in mice Ž p s 0.001.. Sprague–Dawley rats with oxygen-induced ischemic retinopathy resulting from a return from hyperoxic conditions to the relative hypoxia of room air for 5 days following alternation of the oxygen levels between 40 and 80% every 12 or 48 h for 14 days developed retinal neovascularization ŽPenn et al., 1993, 1994. and showed increased VEGF staining, which was largely confined to the retinal ganglion cells, as was previously reported ŽVinores et al., 1997.. Immunostaining for VEGF was eliminated by pre-incubation of the primary antibody with the purified antigen, as reported elsewhere ŽVinores et al., 1997.. Lewis rats with oxygen-induced ischemic retinopathy also developed neovascularization and showed similar upregulation of VEGF in retinal ganglion cells with occa-

sional staining of inner retinal vessels ŽFig. 2C.. Nonischemic control rats of both strains were negative for VEGF. 3.2. TGFb 1 Normal Lewis rat retinas were either negative or weakly stained for TGFb 1 in the inner retina, primarily in the ganglion cells ŽFig. 1C., but occasionally extending into the inner and outer nuclear layers. When TGFb 1-positivity was demonstrated, the same cells that stained for VEGF, namely the ganglion cells, the nerve fiber layer, the inner plexiform layer, and some cells in the inner nuclear layer were positive for TGFb 1. Eight days after immunization with S-antigen, the intensity of staining ŽTable 2. in Lewis rats was not significantly increased in either the peripheral or the posterior retina, but by 11 days it was enhanced Ž p s 0.014.. Normal B10.A mice showed weak to very weak staining for TGFb 1 that extended from the inner plexiform layer to the inner limiting membrane. There was no change in TGFb 1 staining at 11, 16, or 21 days after immunization with IRBP. Neither Lewis nor Sprague– Dawley rats with oxygen-induced ischemic retinopathy

Table 2 Mean grading of staining intensity for TGFb 1 Treatment group

Mean staining intensity in peripheral retina " standard error

Mean staining intensity in posterior retina" standard error

Normal Lewis rats Lewis rats, 8 days after immunization with S-antigen Lewis rats, 11 days after immunization with S-antigen Normal B10.A mice B10.A mice, 11 days after immunization with IRBP B10.A mice, 16 days after immunization with IRBP B10.A mice, 21 days after immunization with IRBP Lewis rats with oxygen-induced ischemic retinopathy

0.57 " 0.20 1.50 " 0.65 1.75 " 0.25 0.75 " 0.25 0.50 " 0.00 0.87 " 0.12 0.80 " 0.12 0.00 " 0.00

0.57 " 0.20 0.75 " 0.48 1.75 " 0.25 0.75 " 0.25 0.50 " 0.00 0.87 " 0.12 0.80 " 0.12 0.00 " 0.00

S.A. Vinores et al.r Journal of Neuroimmunology 89 (1998) 43–50

47

Fig. 3. ŽA. The posterior retina of most normal Lewis rats is negative for TGFb 2 . ŽB. The peripheral retina from the same eye demonstrates weak positivity in the inner retina Žtop.. ŽC. Increased TGFb 2 immunostaining Žred. is seen in the inner portion of the peripheral retina 8 days after immunization with S-antigen. Staining is localized to the nerve fiber layer, ganglion cells, inner plexiform layer, and some cells in the inner nuclear layer with some weak staining in the outer retina. ŽD. Staining for TGFb 2 in the posterior retina of the same eye is not increased. ŽE. Pre-incubation of the primary anti-TGFb 2 antibodies with control peptide eliminated the staining in the mid-peripheral retina from a rat 8 days after immunization with S-antigen.

induced by either protocol showed any significant change in TGFb 1 immunostaining and most were negative. Pre-incubation of the TGFb 1 primary antibodies with control peptide markedly reduced staining Žcompare Fig. 1D,E.. Non-ischemic neonatal rats and normal adult rat controls from both strains were negative for TGFb 1. 3.3. TGFb 2 Normal Lewis rat retinas were negative or weakly positive for TGFb 2 ŽFig. 3A,B.. Staining, when it occurred, was localized primarily to the inner retina. All rats immunized with S-antigen showed TGFb 2-positivity, which was localized to the same cells in the inner retina as VEGF and

TGFb 1. The intensity of TGFb 2 staining ŽTable 3. in the inner retina was not significantly increased at 8 days, but it was by 11 days after immunization with S-antigen Ž p s 0.05.. Increased TGFb 2 immunostaining, when it occurred, was predominantly seen in the anterior retina ŽFig. 3C. with little or no increase in the posterior retina in most cases ŽFig. 3D.. Normal B10.A mice demonstrated very weak staining for TGFb 2 from the inner plexiform layer to the inner limiting membrane, but the localization and extent of staining was unchanged at 11, 16, and 21 days after immunization with IRBP. Sprague–Dawley rats with oxygen-induced ischemic retinopathy showed occasional weak staining of the ganglion cells, astrocytes, outer nuclear layer, and RPE and both Lewis and Sprague–Dawley

Table 3 Mean grading of staining intensity for TGFb 2 Treatment group

Mean staining intensity in peripheral retina " standard error

Mean staining intensity in posterior retina" standard error

Normal Lewis rats Lewis rats, 8 days after immunization with S-antigen Lewis rats, 11 days after immunization with S-antigen Normal B10.A mice B10.A mice, 11 days after immunization with IRBP B10.A mice, 16 days after immunization with IRBP B10.A mice, 21 days after immunization with IRBP Lewis rats with oxygen-induced ischemic retinopathy

0.56 " 0.18 1.12 " 0.31 1.50 " 0.29 0.50 " 0.00 0.25 " 0.14 0.50 " 0.00 0.50 " 0.00 0.87 " 0.12

0.56 " 0.18 0.62 " 0.24 1.50 " 0.29 0.50 " 0.00 0.25 " 0.14 0.50 " 0.00 0.50 " 0.00 0.87 " 0.12

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S.A. Vinores et al.r Journal of Neuroimmunology 89 (1998) 43–50

rats demonstrated staining of the outer segments of the photoreceptors; however, the intensity of staining was comparable in ischemic and non-ischemic rats of both strains. Pre-incubation of the TGFb 2 primary antibodies with control peptide eliminated the staining ŽFig. 3E..

4. Discussion EAU is characterized by lymphocytic and monocytic infiltration into the retina beginning 10 days after immunization with S-antigen, followed by destruction of the retina that is initially directed against the photoreceptors and later spreads to other layers of the retina in the rat ŽGreenwood, 1992; Lightman and Greenwood, 1992; McMenamin et al., 1993a,b. and focal lesions in the mouse ŽCaspi et al., 1988; Chan et al., 1990; Sun et al., 1997.. Subretinal exudation and focal retinal edema may occur in EAU and suggest an early disruption of the BRB ŽDeKozak et al., 1981.. Since BRB dysfunction occurs before any structural damage is evident, it is likely to be mediated by soluble factors. One possible contributor is VEGF since it is markedly upregulated in the inner retina of animals developing EAU and it is a potent inducer of vascular permeability ŽSenger et al., 1983, 1986.. Experiments showing that VEGF injected intravitreally ŽLuna et al., 1997; Tolentino et al., 1996. or slowly released by intravitreal implants ŽOzaki et al., 1997. causes retinal vascular leakage, support this hypothesis. VEGF and its receptors are induced under ischemic conditions ŽAdamis et al., 1993, 1994; Aiello et al., 1994; Malecaze et al., 1994; Miller et al., 1994; Dastgheib et al., 1995; Pierce et al., 1995; Vinores et al., 1997., but there is no pathological evidence of ischemia in the early stages of EAU ŽMcMenamin et al., 1993a,b.. Soluble factors rather than hypoxia may stimulate VEGF upregulation. A variety of inflammatory mediators and lymphokines are released as EAU develops ŽMahalak et al., 1991; DeKozak et al., 1994; Barton et al., 1995; Ramanthan et al., 1996. and some of these factors, such as interleukin-1b ŽLehrmann et al., 1995., interleukin-6 ŽCohen et al., 1996., and prostaglandins E 1 and E 2 ŽHarada et al., 1994., have been shown to induce VEGF in other systems and these may operate in a similar fashion in EAU. VEGF is a potent angiogenic agent ŽSenger et al., 1983, 1986, 1993; Dvorak et al., 1995., but, despite its upregulation, neovascularization does not occur in the early stages of EAU in the rat, although choroidal neovascularization may occur in mice developing EAU ŽChan et al., 1990.. TGFb 1 and TGFb 2 are also upregulated in neurons and glia in the same regions of the retina in the early stages of EAU in rats and TGFb has previously been found to be elevated in retinal vessels of rats with EAU during the peak of inflammation Ždays 13–16 post-immunization. ŽMahalak et al., 1991., but TGFb 1 and TGFb 2 are not increased in the retinas of mice developing EAU. There is

a much more modest upregulation of VEGF in oxygen-induced ischemic retinopathy in which retinal neovascularization does occur, but there is not a concurrent upregulation of TGFb 1 or TGFb 2 . These findings are consistent with the hypothesis that VEGF is capable of inducing an angiogenic response Žas in ischemic conditions., unless an inhibitor such as TGFb is present, as in rats developing EAU. TGFb 1 , and to a lesser extent TGFb 2 , can inhibit endothelial cell growth directly or by modulating receptors to other growth factors ŽHaddow, 1972; Glaser et al., 1985; Jennings et al., 1988; Sato and Rifkin, 1989; Roberts and Sporn, 1990; Pertovaara et al., 1994; Behzadian et al., 1995; Yoshimura et al., 1995.. For example, TGFb 1 has recently been shown to downregulate the VEGF receptor, flk-1 ŽMandriota et al., 1996.. TGFb can also inhibit bFGF-induced proliferation of RVE cells ŽBensaid et al., 1989; Roberts and Sporn, 1990. and may similarly prevent VEGF-mediated RVE cell proliferation without interfering with its vascular permeability-promoting function. TGFb stimulates its own production ŽBorder and Noble, 1994. and induces VEGF ŽPertovaara et al., 1994., which might further augment VEGF levels in S-antigen immunized rats. A better understanding of the complex interaction of the cytokine network and inflammatory mediators and their relationship to BRB integrity, in particular, to the development of macular edema, will foster the investigation of more optimal therapeutic regimens for the treatment of patients with uveitis. This study suggests that the presence of one growth factor may significantly alter the effects of another and that pathologic states such as retinal neovascularization or macular edema may result from particular combinations of growth factors. By gaining a greater understanding of interactions between growth factors, it may be possible to develop new strategies for the treatment of these conditions.

Acknowledgements This study was supported, in part, by NIH grants EY10017 and EY05951 from the Public Health Service, US Department of Health and Human Services, Bethesda, MD, USA. HistoMark Red kits were provided by T. Woerner ŽKirkegaard and Perry..

References Adamis, A.P., Shima, D.T., Yeo, K.-T., Yeo, T.-K., Brown, L.F., Berse, B., D’Amore, P.A., Folkman, J., 1993. Synthesis and secretion of vascular permeability factorrvascular endothelial growth factor by human retinal pigment epithelial cells. Biochem. Biophys. Res. Commun. 193, 631–638. Adamis, A.P., Miller, J.W., Bernal, M.-T., D’Amico, D.J., Folkman, J., Yeo, T.-K., Yeo, K.-T., 1994. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am. J. Ophthalmol. 118, 445–450.

S.A. Vinores et al.r Journal of Neuroimmunology 89 (1998) 43–50 Aiello, L.P., Avery, R.L., Arrigg, P.G., Keyt, B.A., Jampel, H.D., Shah, S.T., Pasquale, L.R., Thieme, H., Iwamoto, M.A., Park, J.E., Nguyen, H.V., Aiello, L.M., Ferrara, N., King, G.L., 1994. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. New Engl. J. Med. 331, 1480–1487. Barton, K., McLauchlan, M.T., Calder, V.L., Lightman, S., 1995. The kinetics of cytokine mRNA expression in the retina during experimental autoimmune uveoretinitis. Cell. Immunol. 164, 133–140. Behzadian, M.A., Wang, X.-L., Jiang, B., Caldwell, R.B., 1995. Angiostatic role of astrocytes: suppression of vascular endothelial cell growth by TGF-b and other inhibitory factorŽs.. Glia 15, 480–490. Bensaid, M., Malecaze, F., Bayard, F., Tauber, J.P., 1989. Opposing effects of basic fibroblast growth factor and transforming growth factor-b on the proliferation of cultured bovine retinal capillary endothelial ŽBREC. cells. Exp. Eye Res. 48, 791–799. Border, W.A., Noble, N.A., 1994. Transforming growth factor b in tissue fibrosis. New Engl. J. Med. 331, 1286–1292. Caspi, R.R., Roberge, F.G., Chan, C.-C., Wiggert, B., Chader, G.J., Rosenszajn, L.A., Cando, Z., Nussenblatt, R.B., 1988. A new model of autoimmune disease: experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J. Immunol. 140, 1490–1495. Chakravarthy, U., Archer, D.B., 1992. Endothelin: a new vasoactive ocular peptide. Br. J. Ophthalmol. 76, 107–108. Chan, C.-C., Caspi, R.R., Ni, M., Leake, W.C., Wiggert, B., Chader, G.J., Nussenblatt, R.B., 1990. Pathology of experimental autoimmune uveoretinitis in mice. J. Autoimmun. 3, 247–255. Chen, Y.-S., Hackett, S.F., Schoenfeld, C.-L., Vinores, M.A., Vinores, S.A., Campochiaro, P.A., 1997. Localization of vascular endothelial growth factor and its receptors to cells of vascular and avascular epiretinal membranes. Br. J. Ophthalmol. 81, 919–926. Cohen, T., Nahari, D., Cerem, L.W., Neufeld, G., Levi, B.Z., 1996. Interleukin 6 induces the expression of vascular endothelial growth factor. J. Biol. Chem. 271, 736–741. Dastgheib, K., Li, Q., Chan, C.-C., Roberge, F.G., Csaky, K., Green, W.R., 1995. Vascular endothelial growth factor ŽVEGF. in neovascular age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 36, S102. DeKozak, Y., Sakai, J., Thillaye, B., Faure, J.P., 1981. S-antigen-induced experimental autoimmune uveoretinitis in rats. Curr. Eye Res. 1, 327–337. DeKozak, Y., Naud, M.-C., Bellot, J., Faure, J.-P., Hicks, D., 1994. Differential tumor necrosis factor expression by resident retinal cells from experimental uveitis-susceptible and -resistant rat strains. J. Neuroimmunol. 55, 1–9. Dvorak, H.F., Brown, L.F., Detmar, M., Dvorak, A.M., 1995. Vascular permeability factorrvascular endothelial growth factor, microvascular hyperpermeability and angiogenesis. Am. J. Pathol. 146, 1029–1039. Forrester, J.V., Liversidge, J., Towler, H., McMenamin, P.G., 1990. Comparison of clinical and experimental uveitis. Curr. Eye Res. 9, 75–84, Suppl. Forrester, J.V., Liversidge, J., Dua, H.S., Dick, A., Harper, F., McMenamin, P.G., 1992. Experimental autoimmune uveoretinitis: a model system for immunomodulation. Curr. Eye Res. 11, 33–40, Suppl. Glaser, B.M., Campochiaro, P.A., Davis, J.L., Sato, M., 1985. Retinal pigment epithelial cells release an inhibitor of neovascularization. Arch. Ophthalmol. 103, 1870–1875. Goldberg, M.A., Schneider, T.J., 1994. Similarities between the oxygensensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J. Biol. Chem. 269, 4355–4359. Greenwood, J., 1992. The blood-retinal barrier in experimental autoimmune uveoretinitis ŽEAU.: a review. Curr. Eye Res. 11, 25–32. Haddow, A., 1972. Molecular repair, wound healing, and carcinogenesis: tumor production a possible overhealing?. Adv. Cancer Res. 16, 181–234. Harada, S., Nagy, J.A., Sullivan, K.A., Thomas, K.A., Endo, N., Rodan, G.A., Rodan, S.B., 1994. Induction of vascular endothelial growth

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factor expression by prostaglandin E 2 and E 1 in osteoblasts. J. Clin. Invest. 93, 2490–2496. Hashimoto, E., Kage, K., Ogita, T., Nakaoka, T., Matsuoka, R., Kira, Y., 1994. Adenosine as an endogenous mediator of hypoxia for induction of vascular endothelial growth factor mRNA in U-937 cells. Biochem. Biophys. Res. Commun. 204, 318–324. Jennings, J.C., Mohan, S., Linkhart, T.A., Widstrom, R., Bayling, K.J., 1988. Comparison of the biological activities of TGF-beta 1 and TGF-beta 2: differential activity in endothelial cells. J. Cell Physiol. 137, 167–172. Kulkarni, A.B., Ward, J.M., Yaswen, L., Mackall, C.L., Bauer, S.R., Huh, C.-G., Gress, R.E., Karlsson, S., 1995. Transforming growth factor-b 1 null mice. An animal model for inflammatory disorders. Am. J. Pathol. 146, 264–275. Lehrmann, E., Kiefer, R., Finsen, B., Diemer, N.H., Zimmer, J., Hartung, H.-P., 1995. Cytokines in cerebral ischemia: expression of transforming growth factor beta-1 ŽTGF-b 1. mRNA in the postischemic adult rat hippocampus. Exp. Neurol. 131, 114–123. Lightman, S., Greenwood, J., 1992. Effect of lymphocytic infiltration on the blood-retinal barrier in experimental autoimmune uveoretinitis. Clin. Exp. Immunol. 88, 473–477. Luna, J.D., Chan, C.-C., Derevjanik, N.L., Mahlow, J., Chiu, C., Peng, B., Tobe, T., Campochiaro, P.A., Vinores, S.A., 1997. Blood-retinal barrier ŽBRB. breakdown in experimental autoimmune uveoretinitis: Comparison with vascular endothelial growth factor, tumor necrosis factor a , and interleukin-1 b-mediated breakdown. J. Neurosci. Res. 49, 268–280. Lutty, G.A., McLeod, D.S., Merges, C., Diggs, A., Plouet, J., 1996. Localization of vascular endothelial growth factor in human retina and choroid. Arch. Ophthalmol. 114, 971–977. Mahalak, S.M., Lin, W.-L., Essner, E., Shichi, H., 1991. Increased immunoreactivity of collagen types I, III and V, fibronectin- and TGF-b in retinal vessels of rats with experimental autoimmune uveoretinitis. Curr. Eye Res. 10, 1059–1063. Malecaze, F., Clamens, S., Simorre-Pinatel, V., Mathis, A., Chollet, P., Favard, C., Bayard, F., Plouet, J., 1994. Detection of vascular endothelial growth factor messenger RNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy. Arch. Ophthalmol. 112, 1476–1482. Mandriota, S.J., Menoud, P.A., Pepper, M.S., 1996. Transforming growth factor b 1 down-regulates vascular endothelial growth factor receptor 2rflk-1 expression in vascular endothelial cells. J. Biol. Chem. 271, 11500–11505. McAvoy, J., Chamberlain, C.G. Growth factors in the eye. In: Progress in Growth Factor Research, Vol. 2. Pergamon, 1990, pp. 29–43. McMenamin, P.G., Broekhuyse, R.M., Forrester, J.V., 1993a. Ultrastructural pathology of experimental autoimmune uveitis: a review. Micron 24, 521–546. McMenamin, P.G., Forrester, J.V., Steptoe, R., Dua, H.S., 1993b. Ultrastructural pathology of experimental autoimmune uveitis in the rat. Autoimmunity 16, 83–93. Miller, J.W., Adamis, A.P., Shima, D.T., D’Amore, P.A., Moulton, R.S., O’Reilly, M.S., Folkman, J., Dvorak, H.F., Brown, L.F., Berse, B., Yeo, T.-K., Yeo, K.-T., 1994. Vascular endothelial growth factorrvascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am. J. Pathol. 145, 574–584. Minchenko, A., Bauer, T., Salceda, S., Caro, J., 1994a. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab. Invest. 71, 374–379. Minchenko, A., Salceda, S., Bauer, T., Caro, J., 1994b. Hypoxia regulatory elements of the human vascular endothelial growth factor gene. Cell. Mol. Biol. Res. 40, 35–39. Nussenblatt, R.B., Gery, I., 1996. Experimental autoimmune uveitis and its relationship to clinical ocular inflammatory disease. J. Autoimmun. 9, 575–585. Ozaki, H., Hayashi, H., Vinores, S.A., Moromisato, Y., Campochiaro,

50

S.A. Vinores et al.r Journal of Neuroimmunology 89 (1998) 43–50

P.A., Ohshima, K., 1997. Intravitreal sustained release of VEGF causes retinal neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and primates. Exp. Eye Res. 64, 505–517. Penn, J.S., Tolman, B.L., Lowery, L.A., 1993. Variable oxygen exposure causes preretinal neovascularization in the newborn rat. Invest. Ophthalmol. Vis. Sci. 34, 576–585. Penn, J.S., Tolman, B.L., Henry, M.M., 1994. Oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization. Invest. Ophthalmol. Vis. Sci. 35, 3429–3435. Pertovaara, L., Kaipainen, A., Mustonen, T., Orpana, A., Ferrara, N., Saksela, O., Alitalo, K., 1994. Vascular endothelial growth factor is induced in response to transforming growth factor-b in fibroblastic and epithelial cells. J. Biol. Chem. 269, 6271–6274. Pierce, E.A., Avery, R.L., Foley, E.D., Aiello, L.P., Smith, L.E.H., 1995. Vascular endothelial growth factorrvascular permeability factor expression in a mouse model of retinal neovascularization. Proc. Natl. Acad. Sci. U.S.A. 92, 905–909. Plate, K.H., Breier, G., Millauer, B., Ullrich, A., Risau, W., 1993. Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res. 53, 5822–5827. Rajasingh, J., Singh, V.K., Singh, V., Sharma, K., Agarwal, S.S., 1966. Cellular immune response to retinal S-antigen and interphotoreceptor retinoid binding protein fragments in idiopathic human uveitis. Indian J. Med. Res. 103, 222–226. Ramanthan, S., DeKozak, Y., Saoudi, A., Goureau, O., van der Meide, P.H., Druet, P., Bellow, B., 1996. Recombinant IL-4 aggravates experimental autoimmune uveoretinitis. J. Immunol. 157, 2209–2215. Roberts, A.B., Sporn, M.B. The transforming growth factor-b s. In: Sporn, M.B., Roberts, A.B. ŽEds.., Peptide Growth Factors and their Receptors I. Springer-Verlag, New York, 1990, pp. 419–472. Sato, Y., Rifkin, D.B., 1989. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor b 1 like molecule by plasmin during co-culture. J. Cell. Biol. 109, 309–315.

Senger, D.R., Galli, S.J., Dvorak, A.M., Perruzzi, C.A., Harvey, V.S., Dvorak, H.F., 1983. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219, 983–985. Senger, D.R., Perruzzi, C.A., Feder, J., Dvorak, H.F., 1986. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res. 46, 5629–5632. Senger, D.R., van de Water, L., Brown, L.F., Nagy, J.A., Yeo, K.-T., Yeo, T.-K., Berse, B., Jackman, R.W., Dvorak, A.M., Dvorak, H.F., 1993. Vascular permeability factor ŽVPF, VEGF. in tumor biology. Cancer Metastasis Rev. 12, 303–324. Shima, D.T., D’Amore, P.A., 1995. Molecular mechanisms of hypoxic regulation of vascular endothelial growth factor ŽVEGF.. Invest. Ophthalmol. Vis. Sci. 36, S870. Sun, B., Rizzo, L.V., Sun, S.-H., Chan, C.-C., Wiggert, B., Wilder, R.L., Caspi, R.R., 1997. Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a T helper-1like or a T helper-2-like response. J. Immunol. 159, 1004–1011. Tolentino, M.J., Miller, J.W., Gragoudas, E.S., Jakobiec, F.A., Flynn, E., Chatzistefanou, K., Ferrara, N., Adamis, A.P., 1996. Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate. Ophthalmology 103, 1820–1828. Vinores, S.A., Kuchle, M., Mahlow, J., Chiu, C., Green, W.R., Cam¨ pochiaro, P.A., 1995. Blood-ocular barrier breakdown in eyes with ocular melanoma: a potential role for vascular endothelial growth factorrvascular permeability factor. Am. J. Pathol. 147, 1289–1297. Vinores, S.A., Youssri, A.I., Luna, J.D., Chen, Y.-S., Bhargave, S., Vinores, M.A., Schoenfeld, C.-L., Peng, B., Chan, C.-C., LaRochelle, W., Green, W.R., Campochiaro, P.A., 1997. Upregulation of vascular endothelial growth factor in ischemic and non-ischemic human and experimental retinal disease. Histol. Histopathol. 12, 99–109. Yoshimura, N., Matsumoto, M., Shimizu, H., Mandai, M., Hata, Y., Ishibashi, T., 1995. Photocoagulated human retinal pigment epithelial cells produce an inhibitor of vascular endothelial cell proliferation. Invest. Ophthalmol. Vis. Sci. 36, 1686–1691.