Epithelium-Capillary Interactions in the Eye: The Retinal Pigment Epithelium and the Choriocapillaris

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 114

Epithelium-Capillary Interactions in the Eye: The Retinal Pigment Epithelium and the Choriocapillaris GARYE. KORTE,*MARGARET S. BURNS,?AND ROY W. BELLHORNS *Department of Ophthalmology, Montefiore Medical Center and Albert Einstein College of Medicine, Bronx, New York 10467, fDepartment of Ophthalmology, University of California at Davis, School of Medicine, Davis, California 95616, and $Department of Surgery, University of California at Davis, School of Veterinary Medicine, Davis, California 95616

I. Introduction Closely apposed sheets of epithelia and plexus of capillaries are found in many organs. In this review we survey the evidence for epitheliumcapillary interactions where the two are apposed, emphasizing structural and functional manifestations such as capillary permeability and cell polarity. We then focus on observations derived from human ocular histopathology and experimental animal models in which interactions are evident between (1) the retinal pigment epithelium (RPE) and its apposed capillary plexus, the choriocapillaris, and (2) between RPE and retinal capillaries experimentally brought into apposition with RPE, from which they are normally isolated. These observations are relevant to the pathogenesis of chorioretinal diseases like age-related macular degeneration and retinitis pigmentosa. They are but two of the causes of reduced vision and blindness that probably arise primarily at the RPE and lead to complicating secondary changes in the adjacent choriocapillaris and neural retina (Hogan, 1972; Green and Key, 1977; Gartner and Henkind, 1982; Young, 1987). Finally, possible mechanisms of RPE-choriocapillaris interactions are discussed in light of current work.

11. Histologic Evidence of Epithelium-Capillary Interactions Histologic evidence for an interaction between epithelia and their capillaries is seen in the correlation between (1) the occurrence of fenestrated endothelia near apposed epithelia, and (2) the polarization of epithelial cells toward their proximate capillary beds. 22 1 Copyright 0 1989 by Academic Press. Inc. All righls of reproduction in any form reserved.

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A. CORRELATION BETWEEN CAPILLARY FENESTRATIONS AND EPITHELIUM Qualitatively, there is a conspicuous correlation between the presence of endothelial fenestrae and a proximate epithelium (although it must be noted that fenestrated capillaries do occur without a proximate epithelium, for example, the capillaries of the atrioventricular node of the mammalian heart: Weihe and Kalmbach, 1978). The correlation between fenestrated capillaries and a proximate epithelium is especiaUy striking where fenestrae are present only where the capillary encroaches on the epithelium. For example, peritubular capillaries in the mouse epididymis contain continuous. unfenestrated endothelium except where the capillaries closely appose the epithelium: where this occurs, fenestrae are formed (Abe et al., 1984). Another example comes from pathology, where brain capillaries that normally have a continuous endothelium become fenestrated near metastasized renal carcinoma cells (Hirano and Zimmerman. 1972). Experimental evidence for the capillary-epithelium interactions suggested by these observations is seen in an ultrastructural study of the guinea pig vas deferens and ureter (Campbell and Uehara, 1972). The authors observed fenestrated capillaries only near the epithelium in both organs, and noted that the fenestrae tended to occupy the side of the capillary facing the epithelium. When the mucosa of either organ was transplanted to the anterior chamber of the eye, fenestrated capillaries were seen only in transplants containing epithelium. Capillaries in transplants stripped of their epithelium lost their fenestrae. The epithelium-capillary interactions suggested by these observations are buttressed by the skewing of endothelial fenestrae at these and other sites toward the adjacent epithelium (Cauna and Hinderer, 1969; CasleySmith, 1971: Heriot ef al., 1986; Mancini ef al., 1986). In a morphometric study of rat choriocapillaris, the latter investigators documented the skewing of endothelial fenestrae toward the RPE, offering this as evidence that the RPE influences the polarity of the choriocapillary endothelium.

B. POLARIZATION OF EPITHELIUM TOWARD CAPILLARIES A characteristic of epithelia is their structural and functional polarization. This is striking where the cells form sheets closely apposed to a capillary plexus. At these sites (the W E is a good example: Figs. lA,B) the epithelial cells develop apical and basal specializations, such as the formation of folds and attachment sites on the basal plasma membrane, opposite the choriocapillaris. The epithelium-capillary interaction suggested by this and similar arrangements at other epithelial sheets (kidney tubules and

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FIG.1. (A) Light and (B) transmission electron micrographs of RPE and choriocapillaris of normal rabbit. (A) Outer retina containing somata of photoreceptors in outer nuclear layer (ONL), their inner segments (IS), and outer segments (0s)abutting RPE. Choriocapillaris (C) is also visible. x 800. (B) RPE basal surface (that facing Bruch's membrane, BM) bears numerous folds (F). Endothelium of choriocapillaris ( C ) has extensive plaques of thin cytoplasm (arrows) bearing many fenestrae. x 13,400.

intestines are examples: Casley-Smith, 1971; Rhodin, 1974) gains credence when an epithelium rearranges its structural and functional polarity in relation to changes in the adjacent capillary bed. An example, detailed in Section III,D, is the incorporation of retinal capillaries into the normally avascular RPE. When this occurs the segments of retinal capillaries become inserted between the lateral plasma membranes of RPE cells. This

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normally flat and morphologically undifferentiated membrane develops structural and functional specializations usually restricted to the basal plasma membrane facing the choriocapillaris.

C. EXPLANATIONS FOR EPITHELIUM-CAPILLARY INTERACTIONS Although these observations suggest an interaction between capillaries and epithelium mediated by soluble molecules or the extracellular matrix (ECM; see Section IV), other explanations for observations like those just described have been proposed. Federman (1982) has suggested that altered blood flow dynamics, such as perfusion pressure, could elicit the change in distribution of choriocapillary fenestrae observed at choroidal melanomas. Abe et al. (1984) believe that the fluid contents of the lumen of the epididymis may be responsible for changes in peritubular capillary fenestrations seen after ligation of the efferent duct of the mouse. In neither case is a signaling mechanism proposed, whereas there is abundant evidence that the ECM or soluble molecules from epithelium or endothelium can cause such changes. 111. Epitheiium-Capillary Interactions in the Eye

It has been suggested that the structure of ocular blood vessels “is dictated by the demands of the tissues, and that a change in tissue demand can lead to a change in vessel morphology” (Bellhorn, 1980, p. 328; see also Davson, 1979). This idea has served as a fruitful theme for animal experimentation and the interpretation of human ocular histopathology (e.g., Federman. 1982). It has become an especially attractive idea as evidence documenting dynamic changes in capillary structure and function in response to specific stimuli has accumulated outside the eye and filtered into the field of eye research. Examples are ( I ) the propensity of continuous capillaries to form fenestrae in thrombocytopenia and psoriasis and to lose them upon steroid treatment (Kitchens and Weiss, 1975; Kitchens, 1977; Braverman and Yen, 1977); (2) the increased permeability, due to increased fenestrations, of periovum capillaries prior to ovulation (Okuda et al., 1983); (3) the induction of fenestrae in nonfenestrated capillaries when exposed to a tumor promoter in v i m (Lombard et al., 1986); and (4) the formation of fenestrae by vaginal capillaries in rats treated with estrogen (Wolff and Merker, 1966). These observations bear out the suggestion, made by Bennett et al. (19591, that “capillary endothelial cells may be labile and may change structural characteristics under the influence of circumstances such as anoxia, or under the influence of various reg-

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dating or pharmacological mechanisms” (p. 389). Observations linking numbers of capillary fenestrae with the status of a proximate epithelium, as at guinea pig ureter and vas deferens (Campbell and Uehara, 1972; see Section II,A) or at the RPE and choriocapillaris (see Sections III,C and D), suggest that epithelia may be one of the regulatory mechanisms alluded to by Bennett et al. (1959). A. ORGANIZATION OF THE RPE-CHORIOCAPILLARIS INTERFACE The RPE-choriocapillaris interface consists of a sheet of cuboidal epithelial cells (the RPE) separated from a planar sheet of capillaries (the choriocapillaris)by a connective-tissue lamina called Bruch’s membrane (Fig. lA,B). This composite structure of epithelium, capillary bed, and interposed connective-tissue lamina is coextensive with the neural retina. The RPE, choriocapillaris, and Bruch’s membrane can be considered a structural unit, whose function is to ensure photoreceptor nutrition (Leeson, 1968; Henkind and Gartner, 1983). This concept has been slighted, however, as emphasis in research and pathology has focused on the interface between the RPE and photoreceptors. The impetus for this arose in part from evidence that the RPE serves as an “organizer” of the neural retina during development, aberrations in the RPE resulting in atrophy of the adjacent neural retina, the photoreceptors in particular (Silverstein el al., 1971; Hollyfield and Witkovsky, 1974; Randall et al., 1983). The RPE influence on photoreceptors may work in mature retina as well; the loss of a proximate RPE has been offered as an explanation for the photoreceptor atrophy seen in retinal detachments, where the neural retina splits away from the RPE (Kroll and Machemer, 1968). It has been suggested (among other possibilities) that the influence of RPE on photoreceptors is manifested by an RPE-derived factor, such as a component of the interphotoreceptor matrix (a mucopolysaccharide-richsecretion of both RPE and photoreceptors that occupies the tissue space between them) or an upset in the vitamin A cycle (Kroll and Machemer, 1968; LaVail, 1979; Porrello and LaVail, 1986). Observations from human histopathology and animal experimentation have identified the RPE-choriocapillaris interface as a locus of pathology in some important chorioretinal diseases, such as age-related macular degeneration and retinitis pigmentosa (Gartner and Henkind, 1982; Young. 1987). This has rekindled interest in a suspected trophic influence by RPE on choriocapillaris mentioned as early as 1937 by Mann but never adequately documented. This author later stated (Mann, 1950) that the “choroidal net,” or choriocapillaris, “seems to develop wherever mesoderm is in contact with pigmented epithelium. It appears pari passu with the

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pigment and seems to be in some way related to this, in that if for any reason pigment is absent over an area of the surface of the optic cup, the choroid is absent also” (p. 38). Numerous later histologic observations on the developing eye have documented this correlation in the chick, monkey, human, and rat eye (O’Rahilly, 1%2; Berson, 1%5; Leeson, 1968; Braekevelt and Hollenberg, 1970; Heimann, 1972; Mund et al., 1972; Endo and Hu, 1973; Takei and Ozanics, 1975; Ozanics et al., 1978). In several of these studies the parallel maturation of RPE and choriocapillaris-another manifestation of their proposed interaction (see Section III.C)-is evident in the illustrations, although the authors do not focus on it (Leeson, 1968; Braekevelt and Hollenberg, 1970; Takei and Ozanics, 1975; Ozanics et al., 1978). Other investigators have, however. In a study of rat intestinal capillary development (Milici and Bankston, 1981), a similar tandem maturation of capillary and adjacent intestinal epithelium is described, such that endothelial fenestrae form and become localized toward the epithelium as it matures. The authors note: “Our results may indicate that the maturation of the overlying epithelium is important in the formation of fenestrations” (p. 441). As seen later (Section lll,C), this is true at the RPE-choriocapillaris interface as well.

B. OBSERVATIONS FROM HUMAN HISTOPATHOLOGY Circumstantial evidence that there is an interaction between the RPE and choriocapillaris comes from histopathologic examination of human eyes obtained at autopsy or enucleation. Mann (1937) pointed out that in colobomata (a congenital eye defect in which the choroid fissure fails to close, leaving a gap in the wall of the eye) the atrophy of neural retina and choroid are coextensive with areas where RPE is absent. More definitive observations are those of Sarks (1979, 1980), made on >500 eyes. She observed a correlation between the geographic, or focal, loss of RPE and atrophy of the adjacent choriocapillaris in eyes with age-related macular degeneration. Her conclusions are supported by similar observations on eyes from patients with other conditions, such as retinitis pigmentosa (Gartner and Henkind, 1982; Henkind and Gartner, 1983; for discussions, see also Korte et al., 1984a; Young, 1987) and fundus flavimaculatus (Eagle et 01.. 1980), diseases characterized by progressive RPE degeneration. Similar correlations between RPE and choriocapillaris status have been made in animal models of some ocular diseases, such as a model of gyrate atrophy (a metabolic disease of patients with hyperornithinemia: Takki, 1974) in which rats or monkeys receive intravitreal injections of ornithine hydrochloride (Kuwabara et al., 1981). These studies support a correlation between RPE and choriocapillaris damage-specifically, that choriocap-

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illaris depends on RPE for its survival. Experimental animal models for elucidating this possibility and RPE-choriocapillaris interactions overall were found in rabbits with a sodium iodate-induced retinopathy and in rats that were exposed to excess fluorescent light or dosed with urethane.

c. SODIUM IODATE RETINOPATHYIN RABBITS The retinotoxic effects of iodate were an unfortunate discovery of the 1920s, when intravenous iodine solutions that were used for treating septicemia also caused blindness (Sorsby, 1941). Subsequent studies determined that the cause of the blindness was iodate metabolically derived from the iodine, although the mechanism of iodate’s toxic effect on RPE in particular, and photoreceptors as well, is still not clear (Sorsby, 1941; Potts, 1980).The use of sodium iodate as a research tool began in earnest in the 1950s, when it was noted that intravenous injections of sodium iodate elicit the degeneration of RPE and photoreceptors in cats, rats, rabbits, and other mammals (Noell, I95 1, 1953). Subsequent ultrastructural studies detailed this necrosis, but the choriocapillaris response was not examined other than to note a loss of its fenestrae (Ringvold rt al., 1981). Given the geographic nature of the RPE response, in which areas of RPE can be spared (Flage, 1983; Korte et al., 1984b), it may be expected that only choriocapillaris adjacent to sites where RPE was destroyed would show ultrastructural evidence of atrophy (e.g., loss of fenestrae, thickening of endothelium, necrosis of endothelium)if the relation suggested by ocular histopathology (Section III,B), development (Section III,A), or observations on extraocular tissues (Sections II,A and B) is true. Upon injecting pigmented rabbits and rats intravenously with sodium iodate (sodium iodate is ineffective in albino animals: Sorsby, 1941), a striking geographic correlation was observed between RPE destruction and choriocapillaris atrophy (Korte et al., 1984b, 1986b), illustrated in Fig. 2 and 3. Thin sections taken where RPE was necrotic or destroyed and replaced by scar tissue (Figs. 2B and 3B) showed that choriocapillaris endothelium had thickened and lost its fenestrae. Necrotic endothelial cells were also observed (Korte et al., 1984b).Their removal accounts for the conspicuous atrophy of the choriocapillaris observed in vascular casts examined by scanning electron microscopy (SEM; cf. Fig. 3C and D). Where RPE was spared the adjacent choriocapillaris remained normal in ultrastructure. The sparing of choriocapillaris adjacent to unaffected RPE indicated that the capillary response was not due to a direct effect of iodate on its endothelium, a possible artifactual complication. This was corroborated by observations showing no effect by sodium iodate (at the dosage used in the experiments) on aortic endothelium:

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FIG.2 . ( A ) Light and (B) transmission electron micrographs from area of damaged RPE, 2 days after intravenous injection of sodium iodate into a pigmented rabbit. ( A ) RPE is flattened and depigmented. Choriocapillaris (C) appears normal, but shows ultrastructural changes, as seen in (B). ~ 6 6 0(.B ) Choriocapillaris endothelium adjacent to damaged RPE (cf. normal RPE and choriocapillaris in Fig. 1B) shows early signs of atrophy. such as loss of fenestrae to produce extensive zones of thickened cytoplasm (arrow). Other than some are within normal range separation of disk membrane, photoreceptor outer segments (0.5) of ultrastructural preservation. x 13,400.

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1. Intravenously injected Evans blue (a dye that binds to albumin and acts as a probe of vascular permeability) did not leak into the aortic intima, a test site for endothelial integrity. 2. Interendothelialjunctional complexes were intact and necrotic cells were not observed upon transmission electron microscopic (TEM) examination of the aortic endothelium. 3. SEM of the aortic surface revealed no evidence of endothelial cell loss.

Nor was choriocapillaris atrophy a response to photoreceptor damage (sodium iodate also damages photoreceptors: Noell, 1951, 1953). As gauged by both electrophysiologicand ultrastructural criteria, the RPE response begins within hours of iodate administration (Potts, 1980; Anstadt et af., 1982kpnor to a photoreceptor response (e.g., Fig. 2A and B). Also, when photoreceptors but not RPE are damaged (which does occur in some iodate-dosed rabbits and can be produced purposely in rats by exposing them to fluorescent light or urethane: see Section IlI,D), the choriocapillaris remains normal in appearance. These observations led to a hypothesis that RPE influences the structure and function of choriocapillaris(Henkind and Gartner, 1983; Korte et al., 1984b), and supported the observations of Bellhorn and co-workers on rats in which retinal capillaries were experimentally brought into apposition with RPE (Bellhorn et af., 1980). Their observations (see Section III,D) led them to conclude that “a factor(s) within the retinal pigment epithelial layer determines the morphology of vessels within their environment” (p. 584). The loss of fenestrae by atrophic choriocapillaris suggested that these structures and at least one function-permeability-are influenced by WE. Evidence for this was obtained when rabbits that had received sodium iodate were injected intravenously with the vascular tracer horseradish peroxidase (HRP) prior to euthanasia (Korte et al., 1987). Choriocapillaris profiles adjacent to spared RPE retained their fenestrae, and their normal permeability to HRP. Atrophic choriocapillaris that had lost its fenestrae retained the tracer in its lumen (Fig. 4). Ohkuma and Ryan (1983) also made a correlation between HRP permeability and the degree of endothelial fenestrations at experimentally induced subretinal neovascularizations in the monkey. The influence of RPE on choriocapillaris structure and function suggested by these observations was also seen during their subsequent regeneration (Korte ef al., 1987). Starting - 1 week after administration of iodate in rabbits, the RPE begins to regenerate from the edge of spared RPE. Light-microscopic examination of series of sections of paraffin- and plastic-embedded tissue, augmented by adjacent thin sections, revealed

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that choriocapillaris regeneration paralleled RPE regeneration (Fig. 5A and B). The advancing edges of the regenerating RPE and choriocapillaris were approximately in register in sections, as may be predicted from observations on the developing eye (Mann, 1937, 1950; Heimann, 1972; see Section 111,A). Some regenerating capillary profiles that had advanced beyond the edge of the regenerating RPE showed evidence of secondary atrophy, such as endothelial necrosis. One interpretation of this observation is that the regrown capillaries were dying back where they outranged a trophic influence by the RPE,resulting in the striking geographic match between RPE and choriocapillaris observed in the late stages of the retinopathy (Korte et al., 1984b), and illustrated in Fig. 3A. Since the response of the choriocapillaris endothelium was examined over the course of its atrophy and regeneration, the attendant ultrastructural changes seemed related to loss and subsequent re-formation of cellular polarity in the choriocapillaris, apparently in response to the presence of the adjacent epithelium. Normal choriocapillaris has its fenestrations preferentially located on the side of the endothelial tube facing the RPE, as seen in Fig. 1B. When RPE is destroyed, this polarity disappears as the endothelium thickens and fenestrae are lost (Figs. 2B and 3B). During regeneration of the choriocapillaris, however, this polarity returns once more where the capillaries lie adjacent to newly formed RPE (cf. Fig. 5B and C). Ultrastructural examination of the endothelium of regenerating choriocapillaris showed a discrete series of changes leading to this. First, isolated fenestrae were formed, with no particular localization about the formative endothelial tube. With maturation of the capillary, clusters of

FIG.3. Atrophy of choriocapillaris as seen in sections (A,B) and in vascular casts examined by SEM (C,D). (A) Light micrograph obtained 1 1 weeks after iodate administration, showing border between zone of spared retina (right of arrow) and atrophic retina (left of arrow). RPE stops near arrow and is replaced by retinal scar tissue (S).Choriocapillaris (C) adjacent to scar is atrophic (detailed in B) as compared to that adjacent to spared RPE at right. x390. (B) Transmission electron micrograph of choriocapillaris from area to left of arrow in A, showing advanced capillary atrophy. Endothelium is thickened and bears no fenestrae. RPE has been replaced by retinal scar tissue (S). BM denotes Bruch's membrane. x 13,400. (C) Scanning electron micrograph of vascular cast (viewed from retinal side) of choriocapillaris at far periphery of rabbit that received sodium iodate 6 days prior to euthanasia. Choriocapillaris here is an extensive network of spared capillaries, and corresponds to ones like that to right of arrow in (A). Arrows denote peripheral edge of choriocapillaris. which stops abruptly near base of ciliary body, above. ~ 4 0(D) . Vascular cast more centrally in same specimen as in (C), showing border between spared choriocapillaris (at upper left) and zone of capillary atrophy at lower right; atrophy exposes underlying venules, V. This picture corresponds to zones where spared RPE and choriocapillaris border zones of atrophy or scar formation (e.g., at arrow in A). ~ 6 6 .

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FIG.4. Transmission electron micrograph of atrophic choriocapillaris adjacent to retinal scar (S) of rabbit euthanized 5 weeks after administration of sodium iodate. The animal received HRP prior to euthanasia. Due to thick. unfenestrated endothelium (one manifestation of atrophy). the capillary retains tracer reaction product (black) in its lumen: normally the endothelium is thin and fenestrated, making it permeable to peroxidase (cf. Figs. IB and 6A). Bruch's membrane (BM) is thickened due to deposition of connective tissue, which occurs coincident with capillary atrophy. x 17.000.

FIG.5. Tandem regeneration of RPE and choriocapillaris in rabbits that received sodium iodate at varying times prior to euthanasia. (A) By light microscopy. regenerating RPE consists of flattened cells. with some mitoses evident (arrow). Adjacent choriocapillaris (*) is also . Immature RPE and enregenerating. as seen in (B). Seven days after iodate. ~ 8 5 0 (B) dothelium of choriocapillaris (C) as seen by TEM from area like that seen in ( A ) . KPE as yet lacks basal specializations such as folds (cf. Fig. IB). Choriocapillaris has small plaques of thin. fenestrated cytoplasm (arrows) scattered about its perimeter. BM denotes Bruch's membrane. which contains a portion of a monocyte, M. x 10.000. (C) Regenerated choriocapillaris adjacent to regenerated RPE, in tissue obtained I I weeks after iodate administration. New capillaries are ensheathed in remnant basement membrane (encircled) and bear extensive plaques of thin. fenestrated cytoplasm (arrows) polarized to the side facing the RPE. unlike less mature capillary profiles (cf. part B). x 7000.

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fenestrae became associated with small plaques of thinned cytoplasm, still scattered, however, about the endothelial tube (Fig. 5B). Eventually, as the capillary reached maturity, these plaques of thin, fenestrated cytoplasm enlarged and became concentrated on the side of the endothelial tube facing the regenerated RPE (Fig. 5C). Where regenerating choriocapillaris exceeded the edge of regenerating RPE, the new endothelial tube remained unpolarized in its fenestrations and eventually atrophied (Korte et al., 1987). This contributed to the “end-stage” retinopathy, in which areas where RPE had not regenerated were occupied by a dial scar, the adjacent remnant choriocapillaris consisting of atrophic capillary profiles embedded in a dense, collagenous connective tissue (Korte et al., 1984b, 1986b). A similar correlation between retinal scar formation and choriocapillaris atrophy is seen in rats with photothermal or phototoxic retinopathies (Kuwabara, 1979; Burns et al., 1986). These observations suggest that choriocapillaris responds to the presence of RPE. They obscure, however, an equally important response on the part of the RPE to choriocapillaris. This response was documented in rats that were exposed to fluorescent light or urethane, treatments that selectively destroy the photoreceptors and cause retinal capillaries to become embedded in the RPE-a common response when photoreceptors are lost in the rodent retina (LaVail, 1979). Observations on these intraepithelial capillaries clarify those obtained in iodate rabbits and rats (Korte el d., 1984b. 1986b) by showing that the situation at the RPEchoriocapillaris interface is one of interactions between these components, and not merely the trophic influence of RPE on the capillary.

D. INTRA-RPECAPILLARIES IN RATS When young rats are exposed to fluorescent light or receive subcutaneous injections of urethane, the photoreceptors atrophy and are lost several months later (O’Steen et al., 1972; Bellhorn ef al., 1973, 1980; Shiraki et al., 1982). When and where this occurs, retinal capillaries, normally separated from the RPE by the photoreceptors, become inserted among the RPE cells. These foci of intraepithelial capillaries are excellent sites at which to examine the response of capillaries to an epithelium, and vice versa. The structural and functional characteristics of the intraepithelial capillary segments can be compared to their parent capillaries remaining in the neurosensory retina. These latter are of the continuous type, having a thick, nonfenestrated endothelium that is impermeable to intravenously injected HRP. Bellhorn and co-workers have published a series of articles documenting the structural and functional transformations in these capillaries when they become embedded in the RPE, as well as the response

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of RPE cells to them (Bellhorn ef al., 1973, 1980; Kritzinger and Bellhorn, 1982; Shiraki el al., 1982; Bellhorn and Korte, 1983; Korte et al., 1983, 1984a, 1986a). The endothelium of the intraepithelial capillary segments changes its structure and function (Figs. 6, 7). It thins, develops fenestrae, and becomes permeable to intravenously injected HRP (Korte ef d., 1983, 1984a). These observations buttress the notion that RPE influences choriocapillaris structure and function, for the capillaries are normally fenestrated and permeable to HRP. Retinal capillaries are normally unfenestrated and impermeable to HRP (Fig. 7D). In the course of these investigations it became obvious that the RPE also responded to the presence of the retinal capillary. The RPE cells abutting the segments of capillaries embedded in the epithelium rearranged the structural and functional polarity of the basal surface, which is normally oriented toward the choriocapillaris (Korte et af., 1986a). This was most evident where capillary segments were interposed between the normally flat, undifferentiated lateral plasma membranes of RPE cells (Fig. 7A-C). This lateral membrane developed the attachment sites, infoldings, and tubules normally restricted to the basal plasma membrane facing the choriocapillaris (Miki et al., 1975; Korte, 1984; Korte and Goldberg, 1986). It also assumed two functions it normally does not have: secretion of basement membrane and endocytosis. The latter function was of particular interest, because coated pits are rare on the lateral plasma membrane of rat, rabbit, and human RPE cells, being restricted to the basal and apical plasma membranes (Orzalesi ef al., 1982; Perlman et al., 1984). However, coated pits (where endocytosis occurs) are frequent on the lateral plasma membrane facing an intraepithelial capillary, and numerous HRP-labeled coated vesicles are observed in the adjacent cytoplasm when this tracer is administered (Korte ef af., 1986a). The reorganization of the RPE cell’s polarity toward these intraepithelial capillaries suggests that, in the normal eye, the choriocapillaris exerts a similar influence on the RPE, inducing in it several structural and functional specializations that give the RPE its polarity. This could have important functional implications, since the RPE is the “gate” separating two important tissue spaces: that between the RPE apical plasma membrane and the photoreceptors, and that between the choriocapillaris and the RPE basal plasma membrane. The exchange of ions and molecules such as vitamin A between these compartments is controlled by the RPE. This transport is directional-perhaps due to the polarizing influence of choriocapillaris on RPE plasma membrane constituents or the cytoskeleton. Conversely, transport across choriocapillaris must be polarized due to the influence RPE has on the numbers and distribution of endothelial fenestrae. That proximate cells can affect

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endothelial permeability is seen when glial cells influence the directionality (i.e., polarity) of transport across brain capillaries (Beck et al., 1984), although the effect is on the molecular constitution of the plasma membrane, and not due to changes in endothelial fenestrae. E. RELATEDOBSERVATIONS DERIVEDFROM ANIMAL EXPERIMENTATION Corroborative evidence for the notion that choriocapillaris and RPE interact comes from several quarters. A morphometric study of normal rat RPE cells shows that infoldings of their basal plasma membrane are most extensive opposite a capillary profile; the basal plasma membrane spanning the tissue space between choriocapillaris profiles has less infolding (Heriot et al., 1986). Other morphometric observations in rats with a spontaneous hypertensive retinopathy documented the polarization of choriocapillaris endothelial fenestrae toward the RPE and showed a decrease in fenestrations and increase in endothelial thickness with increasing distance from the RPE (Mancini et al., 1986). Moreover, when RPE cells begin to migrate across the retina (a common phenomenon in retinal disease), they maintain their normal structural polarity where apposing a retinal capillary or the internal limiting membrane (the basement membrane that lines the vitreal surface of the retina and is probably secreted by the Miiller glial cell), as in rats with a hypertensive retinopathy (Frank and Mancini, 1986). This suggests that basement membranes and, more broadly, the ECM, contribute to the control of RPE-choriocapillaris interactions.

FIG.6 . RPE and intraepithelial capillaries from rats exposed to excessive fluorescent light or urethane, which selectively destroy the photoreceptors. The electron micrographs are taken from rats that received HRP I5 minutes prior to euthanasia. (A) When photoreceptors are lost, inner retina (INL denotes inner nuclear layer) encroaches on RPE. RPE appears normal; numerous folds (F) face Bruch’s membrane (BM) and choriocapillaris (C), and apical villi (V) adorn opposite side of the cells. Black reaction product of peroxidase stains Bruch’s membrane and outlines folds due to escape from choriocapillaris (its lumen appears clear due to perfusion fixation). x 5000. (B) Where intraepithelial retinal capillaries (L, lumen) occur. folds (F) form on RPE lateral plasma membrane where it faces the capillary. The capillary endothelium develops fenestrae (encircled) that permit intravenously injected HRP to penetrate into pericapillary space. Parent segments of retinal capillaries in underlying neural retina still retain peroxidase (cf. Fig. 7D). V, apical villi of RPE. BM, Bruch’s membrane. ~ 8 3 0 0 .

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IV. Mechanisms of RPE-Choriocapillaris Interactions The idea that cells of different types influence each other is not new. Consider, for example, the massive literature on epithelium-mesenchyme interactions (see Bissell et al., 1982). The putative inductive influence by W E on ocular mesenchyme (Mann, 1937, 1950; Reinbold, 1%8; Newsome, 1976) probably falls into this category of interaction. There is much more debate, however, on the intermediary of such cell-cell interactions. Abundant evidence indicates that basement membranes, and ECM generally, influence cell structure and function, in part via the cytoskeleton (Bissell et al., 1982; Hay, 1983). The importance of basement membranes in controlling tissue organization at levels beyond just the structural and functional polarity of individual cells has been documented as well (Vracko, 1974; Montesano et al., 1983a,b). In so far as it has been shown that RPE and endothelium (though not choriocapillaris endothelium) are responsive to ECM components (Mandelcorn et al., 1975; Crawford, 1983; Madri et al., 1983; Montesano el al., 1983a,b; Vidaurri-Leal et al., 1984; Milici et al., 1985; Herman, 1987), we may propose the ECM as an intermediary in RPE-choriocapillaris interactions described in Section 111. However, evidence is accumulating that soluble factors released from RPE and choriocapillaris endothelium also contribute to the “status quo” at Bruch’s membrane (Campochiaroand Glaser, 1985b; Glaser er al., 1985); that is, these cells exert paracrine effects on each other in a way similar to the vascularization of corpus luteum in response to the basic fibroblast growth factor of the luteal granulosa cells (Gospodarowicz et al., 1987) or the control of pancreatic islet secretion via local hormonal effects (Bauer, 1983). It is also possible that RPE-choriocapillaris interactions are controlled by the ECM and soluble factors acting in concert-for example, the ability of hematopoietic growth factors (Gordon et al., 1987)

FIG. 7. Details of (A-C) intraepithelial capillary endothelium and RPE lateral plasma membrane facing it; and (D) retinal capillary. (A) RPE lateral plasma membrane facing intraepithelial capillary (E, its endothelium) forms rudimentary folds (arrows) that are outlined by peroxidase reaction product. x 35,000. (B) Fenestrae (arrows) in endothelium of intraepithelial capillary (L, lumen). Lateral RPE plasma membrane (to right) has not formed folds but has formed attachment sites (encircled), which are normally restricted to the basal plasma membrane. x 31,OOO.(C) Fenestrae (encircled) in endothelium of intraepithelial capillary (L, its lumen) permit passage of HRP, contributing the black reaction product in pericapillary space. RPE lateral plasma membrane above bears two coated pits (arrows), which are normally restricted to the basal or apical plasma membranes. ~ 3 1 , 0 0 0 .(D)Capillaries in neural retina are unfenestrated and retain HRP, as seen by restriction of black reaction product to lumen (L). x 17,000.

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or the fibroblast growth factors (Gospodarowicz et al., 1987) to bind to ECM components and maintain their bioactivity. An increase or decrease in the binding of a molecule derived from the RPE, choriocapillaris endothelium, or other cells such as macrophages, by the ECM separating the WE and the choriocapillaris, could create the geographically localized milieu linking secondary changes in RPE or choriocapillaris endothelium to primary changes in one of them.

A. EXTRACELLULAR MATRIX in Section III,D it was noted that W E cells orient themselves in relation to basement membranes (Mandelcom et a / . , 1975; Frank and Mancini, 1986). The ECM components responsible are not known with certainty, although several investigators have attempted to identify them. In one study it was shown that RPE cells in culture could reorient their polarity when a serum-soaked filter was placed on top of them-that is, over their apical surface (Crawford, 1983). The apical surface lost its structural specializations, such as villar projections, and transformed into a basal surface that secreted new basement membrane material. It has been shown that type 11 collagen (the collagen of vitreous) causes cultured RPE cells to lose their polarity and become migratory. They transform into fibrocytelike cells similar to those in the intravitreal “membranes” of human proliferative vitreoretinopathy (Vidaurri-Leal et a / . , 1984). (These membranes are derived in part from RPE cells that migrate across the retina and onto its vitreal surface, or are exposed to vitreous at a retinal tear.) As rudimentary as these observations are, they support the idea that RPE can respond to ECM and basement membrane components. Also, RPE can probably be influenced by a proximate capillary basement membrane, as evidenced by the maintenance of structural polarity when RPE cells arrange themselves along retinal capillaries (Frank and Mancini, 1986; Korte et al., 1986a). There is no information on the involvement of ECM components in the choriocapillaris responses to RPE damage (e.g., the loss of endothelial fenestrae with W E loss), or their re-formation and repolarization adjacent to regenerating RPE (see Sections III,B,C, and D). RPE contains or releases several of the candidate molecules to which endothelium responds, such as type 1V collagen, fibronectin, glycosaminoglycans, and the basic fibroblast growth factor (Turksen et al., 1985; Pino, 1986; D’Amore et ul., 1987; Herman, 1987; Schweigerer et al., 1987; Stramm, 1987). The response of the choriocapil/ary endothelium to these molecules is not known. Other endothelia, however, do interact with these ECM com-

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ponents, as with the formation of tubular structures when rat epididymal fat pad endothelial cells are cultured on basement membrane (as opposed to interstitial) collagen (Madri et al., 1983; see Herman, 1987, for review), and the induction of fenestrae in cultured endothelial cells by the basement membranes of epithelial cells (Milici et a / . , 1985).

B. SOLUBLE FACTORS For years, ill-defined “factors” have been implicated in the biologic economy of the eye and the genesis of ocular disease, especially those involving abnormal blood vessel growth (Henkind, 1978; Glaser et al., 1980; Gamer, 1986). Some sense is being made of these with the realization that disparate factors such as the retina-derived and eye-derived growth factors are both really the acidic fibroblast growth factor (Baird et a / . , 1986; Gospodarowicz et a / . , 1987). The acidic and basic fibroblast growth factors have been characterized (D’Amore et al., 1981 ; Gospodarowicz et a / . , 1986, 1987) and, although their distribution in the retina remains undefined, their influence on blood vessel growth in situ, and thus neovascularization, is suspected. For example, the basic fibroblast growth factor occurs in cultured bovine RPE and stimulates cell division in cultured bovine adrenocortical endothelial cells (Schweigerer et al., 1987). The retina-derived growth factor (i.e., acidic fibroblast growth factor: Gospodarowicz et al., 1987) elicits neovascularization in the cornea (Gospodarowicz et al., 1979) and loss of stress fibers and migration in cultured adrenocortical endothelium (Herman and D’Amore, 1984). Such changes would probably be manifestations of this factor in the retina in situ, which, however, remain undocumented (see Gamer, 1986, for review). Basic fibroblast growth factor, which stimulates the proliferation of endothelial cells, is expressed by bovine RPE cells in culture (Schweigerer et al., 1987). This raises the possibility that RPE cells in situ can produce an autocrine and paracrine factor capable of regulating growth of nearby endothelium and RPE cells. Studies in vitro have suggested other factors working at the RPE-choriocapillaris interface: an inhibitor of endothelial growth released by RPE, and a chemoattractant for RPE that is released by endothelial cells (Glaser et a / . , 1985; Campochiaro and Glaser, 1985b). In the study by Glaser et a / . (1983, culture medium “conditioned” by the growth of human RPE cells caused the regression of new blood vessel growth when applied to chick embryonic yolk sac; it also inhibited mitosis of cultured fetal bovine aortic endothelium exposed to a mitogenic extract of adult bovine retina. The factor(s) involved, as yet uncharacterized or isolated biochemically, could suppress choriocapillaris-derived neovas-

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cularization ( I ) in the normal eye and (2) at sites where neovascularization occurs subsequent to RPE damage but is then suppressed with RPE regeneration. The study by Campochiaro and Glaser (1985b) extends the notion that RPE inhibits capillary growth. It rests on the observation that RPE tends to surround choriocapiUaris-derived neovascularizations that erode through Bruch’s membrane and begin to invade the retina, as if attracted to the new capillaries by their endothelium. They observed that cultured fetal bovine aortic endothelium produces a protein that, in the Boyden chamber assay, acts as a chemoattractant for human RPE cells. Thus, choriocapillaris-derived neovascularizations that cross Bruch’s membrane may attract RPE to them; and the RPE may then inhibit the growth of their endothelial ceUs and suppress the neovascularization. It has been suggested that the RPE that surrounds neovascularizations in situ influences endothelial characteristics other than growth-for example, numbers of fenestrae, and thus permeability (Ohkuma and Ryan, 1983).This possibility is supported by the abundant histopathologic evidence cited earlier (Sections I1,A and II1,C and D). Several considerations complicate interpretation of these in vitro studies. The experiments do not give the results predicted by histopathologic and experimental observations in situ (e.g., see Section 111,C). The studies from animal experimentation and human histopathology show that loss or damage of RPE is followed by choriocapillaris atrophy (see Sections IU,B-D). They are part of a body of observations that supports the idea that RPE is an “organizer” of the chorioretinal interface not only during development (see Section III,B) but in the mature eye as well. Primary damage to RPE by many means, both experimental and during disease, is followed by atrophy of the adjacent choriocapillaris and the adjacent photoreceptors (e.g., Green and Key, 1977; LaVail, 1979; Eagle e l al., 1980; Sarks, 1980; Kuwabara et al., 1981; Ishibashi et a / . , 1986; John et al., 1987). Yet, the in vitro observations cited earlier would lead to the opposite prediction, that is, that loss of RPE would be followed by choriocapillaris growth. [However, one observation has been published that does predict this; in rats whose RPE was selectively damaged by controlled light exposure the adjacent choriocapillaris began sprouting (Heriot et a / ., 1984).] Clearly, additional observations are needed to explain the gamut of RPE-choriocapiIlaris interactions seen in situ and suggested by in vitro observations. A major problem in interpreting the in vitro observations is the technical inability to use choriocapillaris endothelium in culture experiments, although advances are being made in this direction (e.g., Morse et a / . , 1987). This is an important requirement in light of observations that the endo-

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thelium of capillaries and large vessels, or the endothelium of the same class of vessel in different organs or sites, differs in its biochemical and growth characteristics (e.g., the expression of organ-specific antigens: Auerbach et al., 1985). An example of the conflicting observations that can arise in this respect is the observation by Boulton et al. (1987) that human WE-conditioned culture medium has a mitogenic effect on cultured bovine retinal capillary endothelium and on isolated capillaries maintained in vitro. Glaser et al. (1985), however, observed an inhibitory effect by human RPE-conditioned medium on a known mitogen (an extract of adult bovine retina) for cultured bovine fetal aortic endothelium. Other investigators have identified the basic fibroblast growth factor in cultured bovine RPE-a factor with documented mitogenic effects on endothelia (Schweigerer et al., 1987). Repetition of these experiments using RPE and choriocapillaris endothelium from the same species will be most instructive. Further disarray arises from the growing appreciation that either side of the RPE-choriocapillaris equation can be influenced by, or can interact with, the cells of the monocyte-macrophage line (e.g., Penfold et al., 1986; Pollack et al., 1986; Burke and Twining, 1987; Rosenbaum et al., 1987). For example, RPE cells release a chemoattractant for monocytes, which transform into the macrophages seen in the outer retina when RPE or photoreceptors are damaged (Rosenbaum et al., 1987; Penfold et al., 1986; Lai and Rana, 1986). These cells, in turn, may produce a host of factors (e.g., prostaglandins, leukotrienes, platelet-derived growth factor, macrophage-derived growth factor) that could influence RPE and choriocapillaris endothelium (e.g., Campochiaro and Glaser, 1985a; BenEzra, 1978; Folkman and Klagsbrun, 1987). Macrophages, for example, can stimulate capillary growth (Polverini et al., 1977; Werb, 1983). The factor responsible, macrophage growth factor, may really be fibroblast growth factor (Baird et al., 1986). The presence of basic fibroblast growth factor in RPE (Schweigerer et al., 1987) and its ability to cause endothelium in vitro to produce plasminogen activator and organize into capillarylike structures (Montesano et al., 1986)--two counterparts to capillary formation in simsuggests that healing and neovascularization at the RPE-choriocapillaris interface may proceed without the presence of inflammatory cells (i.e., macrophages). The vexing complexity that may characterize the relationship between W E and endothelium is enhanced by observations that RPE can transform into cells with the cytologic characteristics of macrophages and fibrocytes (Machemer and Laqua, 1975; Mandelcorn et al., 1975; Mueller-Jensen et al., 1975; Johnson and Foulds, 1977; Vidaurri-Leal et al., 1984; Lai and Rana, 1986). It has been suggested that capillary segments near these types

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of transformed RPE cells behave differently. Pollack rt al. (1987) observed “macrophagic” RPE near neovascular choriocapillaris, and a cytologically different type of RPE covering capillaries undergoing secondary atrophy, in their investigation of laser-induced subretinal neovascularization in rats. Campochiaro and Glaser ( 1985b) have proposed that transformed RPE cells contribute to the chorioretinal scars that form after laser photocoagulation for treatment of neovascularizations, and are correlated with the regression of neovascularization. Clearly, a biochemical balancing act may control events at the RPEchoriocapillaris interface in a way similar to that proposed between the angiogenic retina-derived (or fibroblast) growth factor and an antiangiogenic vitreal factor. Their imbalance has been offered as one explanation for the neovascularization seen in ischemic retina, and which leads to retrolental fibroplasia and proliferative diabetic retinopathy (Michaelson, 1948; Ashton et al., 1954; Henkind, 1978; Glaser et a/., 1980; Lutty et al., 1983). V. Conclusion Abundant evidence from histology, pathology. and animal experimentation indicates that the RPE and choriocapillaris interact. They probably work as a unit that provides for photoreceptor nutrition. A major challenge to our understanding of the biology of the RPE-choriocapillaris interactions will be to determine the relative contributions of ECM components, soluble factors, and phenotypically different types of RPE cells to observations made in situ. Such information will elucidate the role of the RPE-choriocapillaris interface in retinal, especially photoreceptor, physiology and pathology, as well as in new treatments for diseases resulting from RPE damage or destruction, such as efforts to transplant RPE (Gouras ef al., 1985; Lopez et a/., 1987). The latter are ultimately attempts at restoring the normal interactions between RPE, photoreceptors, and choriocapillaris. ACKNOWLEDGMENT Supported by grants from the National Eye Institute. Research to Prevent Blindness, Inc.. and the National Society for the Prevention of Blindness. REFERENCES Abe. K . . Takano. H . . and Ito. T. (1984). Anar. Rec. 209, 209-218. Anstadt. B.. Blair, N.. Rusin. M . . Cunha-Vaz, J . , and Tso. M . (1982). Exp. Eye Res. 35. 635-662.

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