SURVEY OF OPHTHALMOLOGY

CURRENT

VOLUME 36. NUMBER 5 - MARCH-APRIL 1992

RESEARCH

EDWARD COTLIER AND ROBERT WEINREB, EDITORS

Growth Factors in Retinal Diseases: Proliferative Vitreoretinopathy, Proliferative Diabetic Retinopathy, and Retinal Degeneration PETER WIEDEMANN,

M.D.

University Eye Clinic, Kdn,

Gemuny

Abstract. The goal of this review is to present the current knowledge on specific growth factor involvement in posterior segment eye disease. Growth factors can be defined as multifunctional signals which modify cell growth or proliferation, alone or in concert, by binding to specific cell surface receptors. Their biological effects on cells include cell adhesion, migration, survival, differentiation, extracellular man-ix secretion, protease and protease inhibitor release, production of other growth activities, and angiogenesis. Growth factors couple the cell to the microenvironment. As some growth factors are soluble mediators of wound repair and angiogenesis, it seems possible that proliferative vitreoretinopathy and proliferative diabetic retinopathy are caused or aggravated by these factors. Other factors act as survival factors and can possibly prevent retinal degeneration. The multifunctional nature of growth factors makes it probable that practical uses will be found for these agents in the future. (Surv Ophthalmol36:373-384, 1992)

angiogenesis fibroblast growth factor insulin-like growth factor Key words. platelet-derived growth factor proliferative vitreoretinopathy proliferative diabetic retinopathy retinal degeneration transforming growth factor-beta tumor necrosis factor alpha wound repair l

l

l

l

l

l

l

l

l

A growth signal

factor

can be defined

or mediator

proliferation,

that

enhances

alone or in concert,

as a molecular cell growth by binding

to spe-

cific cell surface receptors. However, there other cellular reactions than growth towards position

to a growth

factor.

Growth

cells, especially connective tissue, while being a potent inhibitor of proliferation in others, such as lym-

or

factors

phocytes

are ex-

cells.

from retina

Further,

both

a heat

and a heat stable inhibi-

tor of endothelial cell proliferation have been isolated from human vitreous. 82,87Diabetic neovasculari-

are,

therefore, better defined as a multifunctional and potent set of regulators. ‘I2 Their biological effects on cells include proliferation, chemotaxis, and stimulation of extracellular matrix production. Rather than being merely a stimulatory factor, growth factors shift the delicate balance from a stimulatory to an inhibitory milieu or vice versa. For transforming growth factor (TGF)-beta pleiotropic and can stimulate proliferation

and epithelial

labile mitogen

zation may be explained by a relative concentration change and altered balance of these factors.“* Growth stimulation or inhibition by these factors depends on the state of development and differentiation of the target tissue. The mechanism of local cellular regulation by classical endocrine molecules involves the interface with autocrine (same cell), juxtacrine (neighbor cell), and paracrine (adjacent

example, is highly in some

cells) mechanisms 373

of growth

factor

action.

Peptide

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1992

Abbreviations aFGF BDGF bFGF EDGF EGF ESAF FGF IGF PDECGF PDGF TGF-beta TNF-alpha VEGF PVR

acidic fibroblast growth factor brain derived growth factor basic fibroblast growth factor eye derived growth factor epidermal growth factor endothelial cell-stimulating angiogenic factor fibroblast growth factor insulin-like growth factor platelet derived endothelial cell growth factor platelet derived growth factor transforming growth factor beta tumor necrosis factor alpha vascular endothelial cell growth factor proliferative vitreoretinopathy

TABLE

Growth Factors Involved in Retinal Wound Healing, Neovascularization and Cellular Survival Wound healing: PVR

Angiogenesis: diabetic neovascularization

Survival: retinal degeneration

growth factors are elements of a complex biological language, providing the basis for intercellular communication. The individual cell is not the target but the mediator of growth factor action. Growth factors are coupling the cell to the environmental tissue so that a cell has the plasticity to respond to changes in the microenvironment or in itself. These signaling molecules can be regarded as the “alphabet” of a biological regulatory language; they are the means to convey information from one cell to another, to mediate interaction between cells and to change gene expression. The effect of these multifunctional and pluripotent factors is dependent on the presence or absence of other peptides. Because some peptide growth factors are soluble mediators of wound repair and angiogenesis, it seems possible that proliferative vitreoretinopathy lz4 and diabetic neovascularization36 are caused or aggravated by growth factors. The importance of some of these factors for cell survival is also known. The presence of chemotactic and mitogenic activity for fibroblasts in vitro in vitreous aspirates from patients with proliferative vitreoretinopathy (PVR) has been demonstrated and a good correlation between the presence of chemotactic and mitogenic activity and severity of PVR has been found.” Similarly, vitreous aspirates from human eyes with neovascularization stimulate endothelial cell proliferation in vitro. 24,4’*5gMichelson’s angiogenic factorgo has been searched for by several groups. Meanwhile, the existence of angiogenic components in the retina has been substantiated. A great number of papers have been published, but no further characterization of the(se) mitogenic and angiogenic substance(s) has been provided.” For our discus-

1

Acidic fibroblast growth factor (aFGF) Basic fibroblast growth factor (bFGF) Tumor necrosis factor alpha (TNF-alpha) Transforming growth factor beta (TGF-beta) Platelet derived growth factor (PDGF) Fibroblast growth factors (aFGF and bFGF) Tumor necrosis factor alpha (TNF-alpha) Transforming growth factor beta (TGF-beta) Platelet derived growth factor (PDGF) Insulin-like growth factor Fibroblast growth factors (FGF)

sion, we will consider only peptide growth factors which have been identified, sequenced and cloned, and merit further intensive study (Table 1). The goal of this review is to summarize current knowledge on growth factor involvement in posterior segment eye disease, including a possible role in the prevention and therapy of retinal degeneration.

Inflammation and Re air: Growth Factors and %VR PVR: A WOUND HEALING

RESPONSE

Injury to tissue initiates an orderly but complex series of cellular and biochemical interactions tissue breakdown, coagulation, cellular release that lead to the formation of new tissue and wound repair. Wound healing represents a dynamic physiological process initiated and influenced by many contributory factors. Inflammation is a localized reaction. Its function is to eliminate injured tissue. Inflammatory cells provide the physiological groundwork on which libroblasts and endothelial cells restore damaged tissue. Only the presence of the macrophage appears to be required throughout the tissue repair process. The evolution and progression of tissue repair can be divided into three phases: inflammatory, proliferative, and remodeling. Each phase is involved in the regulation of events that precede and follow. This model parallels the natural course of PVR, which seems to be an

375

GROWTH FACTORS IN RETINAL DISEASES exaggerated

wound healing

response.‘24

CELLS IN WOUND HEALING Cellular components participating in wound healing release a variety of soluble mediators at the site of injury.‘*’ These molecules perform a multitude of functions leading to tissue and matrix biosynthesis, and interactions of cells, such as recruitment of successive cell types. Following connective tissue cell growth, the formation of granulation tissue with its characteristic increase in the biosynthesis of extracellular matrix ensues. Platelets maintain a localized and concentrated chemotactic stimulus at the wound site for neutrophils and macrophages. Essential growth factors released by platelets are platelet-derived growth factor (PDGF), TGF-beta, epidermal growth factor (EGF), and endothelial mitogens. Neutrophils are recruited by chemotactic factors released during platelet aggregation and clot formation. They release additional soluble mediators of cell recruitment, cell activation, and coagulation, which aid in the subsequent recruitment of macrophages.‘04 Irrespective of the initial accumulation of neutrophils, a marked increase occurs in the number of infiltrating mononuclear cells. Monocytes behave in a manner similar to neutrophils by responding to chemoattractants, phagocytosing particulate matter, and releasing various enzymes. However, they can mature into macrophages with unique morphological and functional features. Macrophagederived polypeptide growth factors regulate the inflammatory and tissue repair response@*; they amplify the proliferation of certain cell types and are also chemotactic for inflammatory and matrix producing cells. Thus, macrophages have two critical functions which are required during tissue repair: they are the principal phagocytic cell responsible for clearance of tissue debris, such as dead and damaged cells, fibrin, and matrix components, and they are required for the accumulation and proliferation of connective tissue cells. Expansion of the fibroblast population is the result of both recruitment and proliferation due to locally generated chemotactic and growth factors, respectively.‘*’ Platelets, neutrophils, macrophages, and, possibly, T-cells all contribute a host of chemotactic and proliferative mediators that are not only vital to the inflammatory process, but also to tissue repair and regeneration. PATHOPHYSIOLOGY

OF PVR

Approximately one out of every ten eyes undergoing surgery for retinal detachment develops PVR

that can lead to traction retinal detachment. All the above cells have been documented in PVR membranes.‘26 Macrophages in PVR membranes are probably derived from monocytes, retinal pigment epithelial (RPE) cells and microglial cells.‘** PVR can be considered an “over-healing” disease. Tissue fibrosis is characterized by the accumulation of an excessive number of fibroblasts followed by the unregulated deposition of collagen and other matrix components. Forgetting the “proper blueprints” or templates of the tissue structure during wound healing means susceptibility to fibrosis; the reason is unclear, but attention has been focused on the macrophage and its ability to release inflammatory mediators. Due to constant exposure to growth factors, the cells produce excessive amounts of collagen and other extracellular matrix components. The unbalanced action of growth factors is thought to be a causative element for fibrosis. EXTRACELLULAR MEMBRANES

MATRIX

OF PVR

Matrix proteins may modulate cell responses to soluble growth factors by altering adhesive interactions and tensile forces generated within the cytoskeleton. A dynamic relation between the extracellular matrix and the cytoskeleton is observed. Changes in matrix composition affect fibroblast cell phenotypic features, e.g., the formation of myofibroblasts.37 Fibronectin, one of the macromolecules of the extracellular matrix, is involved in cellular interactions, such as the promotion of cell movement, cell-cell recognition, attachment, spreading, and coagulation. PVR is characterized by a breakdown of the blood-retinal barrier. A provisional extracellular matrix formed by fibrin and plasmatic fibronectin ‘*‘J” is later replaced by a matrix made of collagen and locally synthesized fibronectin.“05”8 The biosynthesis of fibronectin is upregulated by TGFbeta.‘j* The provisional matrix can regulate inflammatory cell immigration. Plasma and cell-derived fibronectin are also potent chemoattractants for fibroblasts. Matrix turnover is controlled by proteolytic enzymes such as plasminogen activator, which catalyzes the conversion of plasminogen to plasmin. Plasmin can convert procollagenase to its active form. This provokes the degradation of collagen and causes destructive proteolysis in pathologic conditions.‘05 Growth factors such as insulin-like growth factor (IGF)l and EGF increase the production of plasminogen activator,45’47 which can liberate basic fibroblast growth factor (bFGF).” On the other hand, TGF-beta increases the secretion of plasminogen activator inhibitor113 (Fig. 1).

376

I

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Ophthalmol

36 (5) March-April

1992

WIEDEMANN TABLE 2

TGF-beta

IGF-I

Synonyms inthe Literature for Growth Factors of the FGF Familv (aFGF and bFGF)

synthesized by

+g-&qw synthesisby endothelialcells

Brain derived growth factor Eye derived growth factor Heparin binding growth factors Retina derived growth factor (RDGF) Retina derived growth promoting substances Growth stimulating activity of the retinal extract

26 26 69 44 21 69

Plasmin activation results in l

proteolysis

l

basement membrane dissolution

l

liberation of bFGF

Fig. 1. Control of plasminogen activator by IGF-1 and

TGF-beta.

SPECIFIC

GROWTH

FACTORS

AND PVR

Retina-derived growth-promoting substances” are probably related to heparin-binding growth factors6g or the growth factors of the fibroblast growth factor (FGF) family, especially bFGF, and stimulate the proliferation of several cells in vitro, including RPE cells2’*78 (Table 2). The mitogenic activity of heparin-binding growth factors for RPE and glial cells is higher in PVR vitreous than in normal vitreous.22’6g On the other hand, RPE cells produce chemotactic and growth stimulating factor(s) for fibroblasts, RPE, and astrocytes, such as PDGF and ftbronectin.16~20 Acidic fibroblast growth factor (aFGF) is expressed in membranes of an experimental PVR model. lo3 It is associated with cells, especially RPE cells and macrophages, in surgically removed human membranes.4,83 In an experimental model, it has been shown that bFGF can act as a potent mitogen on astrocytes and Mtiller cells in viva” and, therefore, might play a role in PVR development. Growth factors like tumor necrosis factor (TNF)alpha (MW = 17000 D), TGF-beta (MW = 25000 D), and PDGF (MW = 30000), which function in the wound repair response and are therefore suspect of being causative of fibrotic disease, including PVR, will now be considered in some detail. The origin of these factors from cells participating in wound repair is depicted in Table 3. Platelets have long been recognized as a major source of mitogenic activity, which was mostly attributed to PDGF. There are three isoforms of PDGF which bind with different affinities and specifities to cell surface receptors.‘25 PDGF has a wide spectrum of chemotactic and mitogenic activities

for mesenchymal and glial cells. Traction retinal detachment occurs in 72% of rabbit eyes within three weeks following intraocular injection of PDGF (and fibronectin). 12’ PDGF stimulates fibroblast collagen deposition and biosynthesis,g2 and is the most important chemotactic and mitogenic agent for glia.30,5’ It also stimulates collagen gel contraction.48 In a very preliminary study, PDGF was found to be elevated in human vitreous of PVR eyes.80 Almost all cells have been shown to synthesize TGF-beta in one of its molecular forms and almost all cells express receptors for TGF-beta. The distribution of three TGF-beta isoforms in the human retina has been described.3 Platelets are the most concentrated source of this growth factor and TGFbeta is released from their alpha granules at sites of tissue injury.5 This process starts a repair cascade. The “master growth factor” TGF-beta (Fig. 2) appears to up-regulate its own gene expression as well as genes that code for other polypeptide growth factors, which are critical for wound healing, e.g., TNF-alpha, interleukin 1, bFGF, and especially all PDGF isoforms,64*‘28 and causes increased expression of EGF receptors on fibroblastsgg TGF-beta induces proliferation of connective tissue cells at low concentrations by stimulating autocrine PDGF secretion.6 The proliferation stimulating effect of TGF-beta is, therefore, possibly an indirect effect mediated by PDGF. TGF-beta is the most potent

TABLE 3 Cellular Sources of Growth Factors Insolved in Wound Repair Platelets

Macrophages

Fibroblasts

PDGF EGF TGF-beta

bFGF TGF-beta PDGF TNF-alpha

PDGF TGF-beta

GROWTH

FACTORS

IN RETINAL

377

DISEASES TABLE

4

Properties of Some Polypeptide Angiogenic Factors, EC = Endothelial Cells

matrix synthesis

-

TGF-

Angiogenesis EC mitogenicity EC chemotaxis EC tube formation EC specificity Secretion

beta

Fig. 2. Control of cells and extracellular beta.

matrix by TGF-

chemotactic agent for monocytes”” and fibroblasts.“’ It participates in the acute inflammatory response, 54 can enhance fibrosis,25 and accelerates tissue repair52 by enhancement of extracellular matrix formation. The net result of these actions is the formation of granulation tissue, ophthalmoscopically visible cellular membranes. TGF-beta is, therefore, used as a “chorioretinal glue” in the rabbit”’ and clinical studies are being performed. A “retinal patch” of fibrous tissue was induced in patients with macular hole by TGF-beta in the treatment of full thickness macular holes.42 TGF-beta stimulates the contraction of collagen gels in vitro,lo2 suggesting that this growth factor is also a regulator of wound contraction in vivo. Therefore the concentration of TGF-beta in pathologic and normal vitreous seems to be particularly important; eyes with retinal detachment have a vitreal concentration of 360291 pM, PVR eyes 1200? 300 pM.25 This study, confirmed by another group,40 demonstrated for the first time a correlation between a clinical setting and the concentration of a growth factor. The fibrotic events may be due to excessive growth factor production or increased sensitivity of the cells. Effects of the factor can be blocked with antibodies against TGF-beta 2, suggesting that it is the active component25 in the vitreous. TNF-alpha, a product of activated macrophages,‘g causes increased expression of EGF receptors on fibroblasts.” EGF-receptor is expressed in human periretinal membranes.54zg3 TNF-alpha stimulates production of collagen and plays a critical role in tissue remodeling.‘28 Several studies support the concept that RPE cells play a prominent role in the pathogenesis of PVR. In vitro experiments suggest that RPE cells can be modulated by several growth

FGF

TGF-beta

TNF-alpha

IGF

+ + + + -

+ Inhibition Not known + +

+ Inhibition + + -

+ + + -

+

factors. Some of them are growth-stimulating (bFGF, PDGF, IGF), others inhibiting (TGF-beta).‘* TNFalpha was the most effective mitogen in human RPE cell cultures of low density, 13,” and elevated concentrations were found in PVR vitreous.*’

Angiogenesis: Diabetic Neovascularization GENERAL ASPECTS OF ANGIOGENESIS Angiogenesis refers to a sequence of cellular events which result in the formation of new blood vessels by sprouting from established vessels to pro-. duce neovascularization. It develops as a series of sequential steps.35 The first step is the degradation of the basement membrane of the parent venule by endothelial cells. This degradation causes gaps in the basement membrane and permits egress of vascular endothelial cells. These migrate from the wall of the existing vessel to form new vascular sprouts. Dissolution of the basement membrane requires production of several enzymes including plasminogen activators (Fig. 1). The next steps are endothelial cell mitosis and vessel assembly, including lumen formation, development of sprouts and loops, then generation of new basement membrane and recruitment of pericytes. Eight angiogenic factors have been purified, sequenced, and cloned,72 which are involved in physiological neovascularization, e.g., in the female reproductive system. The mechanisms of angiogensis factor action are very different and most factors are multipotential; they stimulate proliferation or differentiation of endothelial cells (Table 4). Some angiogenic factors may have an indirect action72 by mobilizing host cells to release endothelial growth factors. Physiological neovascularization, e.g., in the female reproductive system or in wound healing, is brief, tightly regulated, and self-limited. The tight regulation of physiological neovascularization includes storage of growth fac-

378

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1992

tors (FGF), activation of latent forms (TGF-beta), and angiogenic inhibitors. It seems that the process of angiogenesis must be maintained in a constant state of readiness. Thus, the microvascular system remains quiescent, but is capable of responding with rapid capillary growth on short notice toward a positive stimulus. Obviously, retinal neovascularization is not self-limited. To start, it requires both a factor - may it be inflammation and its products, a hypoxic retina, or a specific growth factor - and a diseased retinal vascular bed for its induction.56 However, new vascular fronds tend to regress without a persistent stimulus. Physiologically, the retinal vasculature does not invade the vitreous gel, probably because of the presence of inhibitors.“’ Neovascularization per se is the dominant pathology and the leading cause ofblindness worldwide,3g e.g., diabetes, neovascular glaucoma, and retrolental fbroplasia. PATHOPHYSIOLOGY OF PROLIFERATIVE DIABETIC RETINOPATHY Diabetic retinopathy is a disease of the retinal microvasculature that reflects compromise of metabolic, endocrine, and hematologic systems, but also results from local conditions. The development of retinal neovascularization in diabetic patients poses a major clinical problem, since bleeding from newly formed vessels results both in loss of vitreous integrity, traction detachment, and impaired vision. The evidence is overwhelming that neovascularization follows the interaction of retinal vessels with vitreous, which has enhanced potency for stimulating new vessel development. Clinical studies have shown that a completely detached vitreous is the safest with regard to neovascularization.‘*65,“5 Regulation of angiogenic factors is probably performed by pericytes, hypoxia, and vasodilatation. Vasodilation is the earliest retinovascular change in diabetic retinopathy,74 causing leakage and neovascularization,53 possibly by introduction of blood compounds. The relevance of many angiogenic factors for ocular neovascularization, such as angiogenin, platelet-derived endothelial cell growth factor (PDECGF),55 angiotropin (VEGF or vascular endothelial cell growth factor), and low molecular weight nonpeptide angiogenesis factors, such as prostaglandins,8~*6 is not known or is speculative. Hypoxia may also play an important role in regulating angiogenic factors, and the hypothesisgo that a chemical messenger from the retina provides the stimulus for neovascularization is well known. The angiogenic activity from macrophages varies inversely with tissue oxygen tension.73 In vitro, the production of PDGF, one of the most potent vasoconstrictors, is increased in hypoxia.75 The free and

WIEDEMANN uninhibited flow of growth factors seems to be of importance with regard to iris neovascularization, as (pseudo)phakic eyes are less prone to severe retinal neovascularization than aphakic eyes.“,g6 There are two likely explanations for the increased incidence of neovascularization of the iris after vitrectomy with lensectomy. First, the formed vitreous may inhibit anterior diffusion of vasoproliferative factors which, after vitrectomy, may diffuse into the anterior chamber and onto the iris. Second, vitrectomy, especially if combined with lensectomy, permits anterior chamber oxygen to diffuse posteriorly into the vitreous, the so-called “oxygen steal.” Preservation of the posterior capsule and zonule may hinder both the anterior diffusion of vasoproliferative factors and the posterior diffusion of oxygen.“’ Regulation of angiogenic factors by pericytes possibly controls vessel growth, too. Endothelial cells make frequent contact with mural cells through discontinuities in the basement membrane. The highest ratio of pericytes to endothelial cells is found in the retina where endothelial turnover is lowest.28 Pericyte dropout correlates with the onset of neovascularization. 3’ Pericytes may downregulate endothelial proliferation, possibly by producing inhibitors of angiogenesis.g8 SPECIFIC GROWTH FACTORS AND DIABETIC RETINOPATHY Some specific and quite well characterized factors involved in ocular angiogenesis shall be considered in detail. IGF-I or somatomedin C is a peptide mitogen, structurally related to insulin.g5~‘0gMammalian retina contains both IGF-I and IGF-II receptors, which have a characteristic spatial distribution; inner and outer nuclear layers are most heavily labeled. Receptors for insulin, IGF-I, and IGF-II, are found on retinal capillary endothelial cells and pericytes. 2g,67S70 IGF may be one of the effector molecules by which growth hormone influences the pathogenesis of diabetic retinopathy.‘30 In vitro, retinal endothelial cells respond to IGF-I and IGF-II by an increase in DNA synthesis, IGF-I being more potent than IGF-II.‘* In tissue culture, IGF-I causes a significant increase of tissue type plasminogen activator from human retinal endothelial cells of diabetic origin45 (Fig. 1). I n an experimental model IGF-I induced retinal neovascularization.84 Very high serum levels of IGF-I have been reported in insulin-dependent diabetics with rapidly accelerating retinopathy.88 The concentration of IGF-I in the vitreous of most diabetic patients with severe neovascularization is elevated,46 and is in the range to stimulate cellular differentiation and growth in several assay systems. A positive correlation exists between the concentration of IGF-I and IGF-II in

GROWTH FACTORS IN RETINAL DISEASES vitreous and their concentrations in serum of diabetic subjects, but not in control subjects. This observation is consistent with an alteration of the blood-retinal barrier in these diabetic subjects and the enhanced formation of a potentially angiopathic vitreous. The rise in serum IGF-I concentration during continuous subcutaneous insulin infusion6’ or pump treatment* may enhance new vessel formation in an already ischemic retina. It could be shown that diabetic vitreous contains abnormally high levels of vitreal insulin-like growth factor binding protein,‘*’ which regulates the bioavailability of these potent growth factors.“‘j Fibroblast growth factors also play an important role in neovascularization. Courty et al*’ isolated three growth factor activities from bovine retina designated as eye-derived growth factor (EDGF) I, II, and III.*’ EDGF I and II were later identified as being identical to bFGF (MW = 18000) and aFGF (MW = 16500) and are structurally related. There are multiple molecular forms of the FGF family. The FGF peptides have no signal sequences and are, therefore, released, but not secreted. A high affinity for heparin-like glycosaminoglycans and the binding to basement membranes are characteristic.‘,@ This distinguishes them from EGF, TGF alpha and beta, TNF alpha, and PDECGF. Binding to heparin is critical for its biological action, as heparin protects FGF from denaturation and degradation43 by stabilizing FGF in complexes and limiting its availability to adjacent cells. The association of bFGF with cells, extracellular matrix, and basement membranes”g has led to the speculation that bFGF may be a stored growth factor that can be potentially released by degradative enzymes in tissue injury and repair.14s3* In any case, FGFs are cellular rather than secreted proteins; this is part of a regulating mechanism in which FGF is prevented from stimulating cell proliferation in an autocrine/paracrine manner. Release is a rapid way to mobilize growth factor. Heparin potentiates FGF activity, e.g., based on release of stored bFGF from basement membranes either by heparin displacement or through activation of local collagenases by mast cell proteases. The distribution of FGF is altered in diabetic retinas; minimal bFGF is present in the basement membranes of proliferative diabetic fronds.50 The FGF receptor is expressed in the inner retinal layers of the human retina.4g bFGF is the best characterized macrophage-derived angiogenic polypeptide and is also synthesized by endothelial cells.” The peptide is chemotactic and a potent mitogen for endothelial cells, but it is not clear how FGF mediates angiogenesis in vivo. An intense uptake of bFGF in retinal vascular and capillary endothelial cells is seen. It may be that

379 FGF and endothelial cell-stimulating angiogenic factor (ESAF), another factor found in vitreous and retina, are jointly responsible for the angiogenic potential of the retina and are controlled by endogenous inhibitors of the retina.“‘j In vitro half maximal stimulation of angiogenesis is achieved with 13 pg/ml bFGF.44 Increased levels (>30 ng/ml) of bFGF in vitreous specimens from patients with proliferative diabetic retinopathy particularly those with active proliferative retinopathy”’ were reported. Another group found a correlation between bFGF in the vitreous and the degree of fibrovascular proliferation.” The multifunctional regulator TGF-beta is secreted in a biologically inactive form and activated by proteases. TGF-beta is a mediator of angiogenesis. However, the mechanism of TGF-beta-mediated angiogenesis is not clear, as the “negative” growth factor TGF-beta itself is a potent inhibitor of capillary endothelial cell growth in culture but promotes tube formation.8g TGF-beta is highly chemotactic for cells involved in tissue repair such as fibroblasts and monocytes. These secondary cells may be responsible for producing and releasing angiogenesis factors. As the release of angiogenic factors from macrophages subsides, TGF-beta could then suppress further endothelial proliferation and lead to fibrosis and traction retinal detachment. TGF-beta inhibits the serum and bFGF induced proliferation of bovine retinal endothelial capillary cells in a dose dependent manner. The equilibrium between bFGF and TGF-beta9 seems to have a key role in angiogenesis. In coculture systems, contact between pericytes and endothelial cells was found to be necessary for suppression of endothelial proliferation by pericytes. Only after contact between the two cells did active TGF-beta, a potent inhibitor of endothelial proliferation, appear in the medium. ‘**” The addition of exogenous FGF could overcome the inhibitory effects of pericytes. Capillary growth may be modulated by a balance of FGF derived from endothelial cells and TGF-beta derived from local pericytes. ‘* Increasing evidence suggests that RPE cells, too, may play a central role in the regulation of intraocular neovascularization, e.g., by reducing endothelial cell plasminogen activatorgl (Fig. 1). TNF-alpha, synthesized by activated macrophages, is a secreted protein and pleiotropic mediator of inflammation. It has been claimed to be responsible for the total angiogenic activity of macrophages. ” TNF-alpha and TGF-beta promote angiogenesis in vivo and tube formation in vitro, and inhibit endothelial cell proliferation in vitro. They may be promoters of the differentiation phase of endothelial cells or they cause stimulation of ves-

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WIEDEMANN TABLE

5

Involvement of Different Growth Factors in PVR and Proliferative Diabetic Retinopathy PVR FGF

l l l

Diabetes

Elevated in PVR vitreous Mitogenic Chemotactic

l l l l

TNF-alpha TGF-beta

PDGF

0 Elevated in PVR vitreous l Best mitogen in vitro for RPE l Elevated in PVR vitreous in correlation with disease

state 0 Stimulates contraction l Matrix synthesis l Increased growth factor expression

Elevated in PVR vitreous Used in experimental PVR model 0 Mitogen for glia 0 Stimulates contraction l Weak stimulator of RPE proliferation

l l

In vitro control of endothelial cell proliferation (coculture with pericytes) Produced by RPE cells and pericytes

Angiogenic 0 Vasoconstriction l Increased in hypoxia l

l

l

IGF

l

Elevated in PDR vitreous Correlation with disease state (fibrosis) Release by plasmin Angiogenic not known

in vitro

Elevated in PDR vitreous Elevated in diabetic serum l Mitogen for endothelial cells 0 Plasminogen activator increase

l l

se1 growth by an indirect mechanism. In any case, the mechanism of TNF-alpha mediated angiogenesis is not clear. A strategy for inhibiting pathological angiogenesis could be directed at two possible targets: angiogenie factors or endothelial cells. The first strategy would be based upon blocking expression or production of angiogenic factors, or neutralizing their activity. The second strategy would be to block capillary endothelial cells from responding to angiogenie factors. All angiogenic inhibitors discovered so far, especially angiostatic steroids and derivatives of fumagillin6’ operate on this latter basis.‘*

Retinal Degeneration aFGF and bFGF are the eye-derived growth factors. The terms EDGFl, brain derived growth factor (BDGF) 1 and bFGF are used as synonyms.26 Human RPE cells express bFGF and its receptor; RPE cells are therefore a source of bFGF and may respond to bFGF in an autocrine manner.‘14 An autocrine mechanism may also be involved in photoreceptor cell biology,58~‘” as rod photoreceptors synthesize FGFs, too. 85,94The interphotoreceptor matrix may serve as a depot for FGF sequestration.” The expression pattern of the aFGF gene differs remarkably from that of the bFGF gene in adult rat eyes.g4 A precise mapping of aFGF in normal human ocular structures was performed’: the retina

stains brightly positive in the photoreceptor and plexiform layers. aFGF is associated primarily with the nerve fiber layer and inner and outer segments of photoreceptors. 23 However, aFGF is no trophic factor for chick retinal ganglion cells.76 bFGF, on the other hand, can be described as a survival factor,57 supporting preservation of RPE morphology, longterm viability, and cell division.g4 It is also synthesized by photoreceptors.85,g4 An anterograde axonal transport of bFGFS4 has been described. A lack of bFGF causes anterograde trophic neural cell death; bFGF can even prevent loss of retinal ganglion cells after transsection of the optic nerve. After intraocular injection, bFGF was detected in ganglion cells34 and contralateral lateral geniculate body; only biologically active material was transported. During the past years strategies have been developed to allow us to begin to think about treating various degenerative ocular diseases by promoting repair of the tissue. Photoreceptor degeneration in inherited retinal dystrophy in rats can be prevented by subretinal or intravitreal injection of bFGF. bFGF can therefore act as a survival-promoting factor in a hereditary neuronal degeneration of the central nervous system, 33 thus leading to the same result as the experimental transplantation of RPE cells. It also seems that the survival of photoreceptors after argon laser treatment is improved by an intravitreal bFGF injection.“’

GROWTH FACTORS IN RETINAL DISEASES

Conclusion This review has attempted to briefly outline the knowledge about peptide growth factor involvement in retinal disorders (Table 5), but it is not an in-depth review of each factor. There is an apparent lack of precise data on the interactions between various growth factors causing fibrosis and neovascularization, but further research may provide better understanding of molecular mechanisms, pathogenesis of diseases, and therapeutic interactions. Cell populations, extracellular matrix and soluble factors are closely intertwined. With the advent of recombinant DNA technology and the availability of well characterized factors and specific antibodies, more accurate determinations of the concentrations of these substances in clinical pathology can be expected, and we will see the integration of growth factors into the new generation of ophthalmic pharmaceuticals currently under development. Antagonists can be discovered as naturally occurring, e.g., bFGF versus TGF-beta, or developed as competitive inhibitors of receptor specific analogs. The multifunctional nature provides a definitive note of optimism that further practical uses will be found for these agents in the future. They will become increasingly important for those concerned with the treatment of retinal diseases. Acknowledgment I am indebted to Prof. K. Heimann, MD, K61n, FRG, Prof. J.S. Huang, Ph.D, St. Louis, USA, and M. Weller, MD, Wiirzburg, FRG, for helpful comments.

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