Elsevier

Experimental Eye Research

Volume 77, Issue 4, October 2003, Pages 433-445
Experimental Eye Research

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid

https://doi.org/10.1016/S0014-4835(03)00174-XGet rights and content

Abstract

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy.

Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis.

The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE–Bruch's membrane–choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1·47 vs. 1·44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2·24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1·0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures.

In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease (p<0·05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.

Introduction

Endothelial cell proliferation is rare in normal retinal vessels (Engerman et al., 1967). Angiogenesis, proliferation and migration of endothelial cells to make new blood vessels, is under tight regulation in most healthy tissues and is most probably controlled by the balance between angiogenic and anti-angiogenic factors (D'Amore, 1994). The disruption of such a balance has been thought to play an essential role in the development of a variety of retinal vascular diseases (King and Suzuma, 2000, Gao et al., 2001). It is possible that retinal ischemia may not only stimulate the expression of angiogenic growth factors (Michaelson, 1948) but also inhibit the release of anti-angiogenic factors.

Recent studies on retinal angiogenesis have focused on the balance of two growth factors: pigment epithelium-derived factor (PEDF) as an angiogenesis inhibitor or anti-angiogenic factor and vascular endothelial growth factor (VEGF) as an angiogenic growth factor (Gao et al., 2001, Ohno-Matsui et al., 2001). PEDF was first purified from the conditioned media of human retinal pigment epithelial cells and was found to be a neurotrophic factor (Tombran-Tink et al., 1991). Recently, PEDF was found to be anti-angiogenic as well (Dawson et al., 1999). VEGF is produced in hypoxic retina and is thought to be Factor X that Michaelson (1948) hypothesized was the soluble angiogenic growth factor responsible for retinal neovascularization (D'Amore, 1994, Aiello, 1997). Less PEDF is produced and VEGF levels increase in oxygen-induced retinopathy (OIR) correlating with the development of neovascularization (Dawson et al., 1999). When oxygen is sufficient, the VEGF level decreases and the PEDF level is upregulated to possibly prevent further growth of new blood vessels (Gao et al., 2001).

PEDF administered systemically or intravitreally inhibits aberrant blood vessel growth in a murine model of OIR or in VEGF-induced neovascularization (Stellmach et al., 2001, Duh et al., 2002). In vitro, PEDF inhibits endothelial cell migration in a dose-dependent manner and is more active than other recently discovered anti-angiogenic agents: angiostatin, thrombospodin-I, and endostatin (Dawson et al., 1999).

In sickle cell disease, sickle erythrocyte-mediated vaso-occlusions in retina induce ischemia in the peripheral retina. Presumably in response to angiogenic factors made in ischemic peripheral retina (Cao et al., 1999), extraretinal neovascularization (sea fan formations) develops at the border of perfused and non-perfused peripheral retina (Romayananda et al., 1973, Goldberg, 1976, McLeod et al., 1997). Each sea fan formation appears to result from several simultaneous neovascular events (McLeod et al., 1997). Autoinfarction of sea fans is a well-documented naturally occurring event (Nagpal et al., 1975, Condon and Serjeant, 1980).

Changes in PEDF level in vitreous or whole retina have been observed in several ocular diseases including proliferative diabetic retinopathy (Ogata et al., 2001a, Ogata et al., 2001b, Ogata et al., 2001, Spranger et al., 2001) and other ischemic retinopathies (Gao et al., 2001). Changes in PEDF in sickle cell retinopathy still are unknown. The purpose of this present study is to determine the localization and relative levels of PEDF and VEGF in human retinas and choroids using immunohistochemical analysis and evaluate the changes in localization and the relationship between PEDF and VEGF with progression of proliferative sickle cell retinopathy.

Section snippets

Materials and methods

Four control eyes, three eyes with non-proliferative sickle cell retinopathy (two with sickle cell anemia (SS) and one with SC disease (SC)), and four with proliferative sickle cell retinopathy (one SS, three SC) were evaluated (Table 1). The diagnosis of proliferative or non-proliferative retinopathy was made by reviewing the systemic and ocular medical history and the postmortem fundus examination, using a Zeiss dissecting microscope. Our criterion for proliferative sickle retinopathy was

Immunolocalization of PEDF in normal retina and choroid

The most prominent sites of PEDF immunoreactivity in the normal eye were the vitreous condensed at the internal limiting membrane and RPE–Bruch's membrane–choriocapillaris complex including RPE basal lamina, intercapillary septa, and choroidal stroma (Fig. 1, Fig. 2). There was also immunoreactivity in some cells in the inner nuclear and ganglion cell layers, in some blood vessels, and in the interphotoreceptor matrix (IPM, Fig. 1, Fig. 2).

The validity of binding of the polyclonal anti-human

Discussion

PEDF is a multifunctional factor that has both neurotrophic and anti-angiogenic activities (Chader, 2001). In the present study, PEDF immunoreactivity was associated with both neuronal and vascular structures as well as various extracellular matrices. Immunoreactivity for PEDF was the most prominent in condensed vitreous, the internal limiting membrane, some large retinal blood vessels, RPE–Bruch's membrane–choriocapillaris complex, and choroidal stroma of the normal eye. Some cells in the

Acknowledgements

This work was supported by grants from the Panitch Fund, the Reginald F. Lewis Foundation, the Foundation Fighting Blindness, and NIH grants HL45922 (G.L.), KO8-00362 (P.T.), and EY01765 (Wilmer). The authors thank Alex Ljubimov for graciously providing the antibody against HSPG. The authors also thank the eye donors and their relatives for their generosity.

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