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Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells

^a The University of Iowa Center for Macular Degeneration, Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, Iowa 52242, USA; and

^b Center for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA

	ABSTRACT

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Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.—Hageman, G. S., Mullins, R. F., Russell, S. R., Johnson, L. V., Anderson, D. H. Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.

Key Words: age-related macular degeneration • retinal pigment epithelium • extracellular matrix • heparan sulfate proteoglycan

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
AGE-RELATED MACULAR DEGENERATION (AMD)2 is the leading cause of legal blindness in North America and Western Europe (1 , 2 ). No pharmacologic treatment has been shown to be effective in preventing, arresting, or reversing the loss of vision associated with AMD. Despite its prevalence, the molecular pathogenesis of the disease is not well characterized. It is heritable as an autosomal dominant trait in a significant portion of afflicted individuals 3-5) , although the responsible gene(s) has not been identified conclusively. The visual deficits associated with AMD are due to the dysfunction and progressive degeneration of photoreceptor and retinal pigmented epithelial (RPE) cells, especially notable within the macula.

Histopathologic studies have documented significant and widespread abnormalities in the extracellular matrices associated with the RPE, choroid, and photoreceptors of aged individuals and of those with clinically diagnosed AMD 6-13) . The most prominent of the extracellular matrix (ECM) abnormalities is the formation of drusen, deposits that accumulate between the RPE basal lamina and the inner collagenous layer of Bruch's membrane. Although the presence of drusen per se is not an absolute predictor for the development of AMD, drusen are generally regarded as a strong risk factor for both the atrophic and exudative forms of the disease 13-20) . Few individuals who develop AMD do not possess clinically detectable drusen.

The morphological characteristics of drusen have been described in detail (10 , 21-25 ), but little is known about their molecular composition and origin. Based on the extracellular location of drusen, we screened a battery of antibodies to known ECM components on tissue sections obtained from a large sample of human donor eyes with drusen, including donors with clinically documented AMD. These studies show that vitronectin, a plasma protein and resident ECM protein, is a major constituent of both hard and soft drusen. In addition, epitopes exposed in the conformationally distinct, heparin binding form of human vitronectin characteristic of extracellular matrices 26-28) are present in drusen. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses reveal that vitronectin mRNA is synthesized by cells in the RPE–choroid complex and by cultured RPE cells, suggesting a potential local cellular source of drusen-associated vitronectin.

	MATERIALS AND METHODS

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Tissues
Eighty human donor eyes, ranging in age between 45 and 101 years, were obtained from MidAmerica Transplant Services (St. Louis, Mo.) and the Iowa Lions Eye Bank (Iowa City, Iowa) and were processed within 4 h of death. Seventeen of these donors had a documented clinical diagnosis of AMD (including donors with geographic atrophy (three donors), choroidal neovascularization (six donors), and disciform scars (two donors) in at least one eye) and one donor was diagnosed with cuticular drusen. For the purpose of this study, drusen were categorized as `hard' or `soft' as described previously (29) . Human liver was obtained within 2 h of biopsy. RPE cells were isolated within 5 h of death and were grown in Coon's F-12 media containing 10% fetal bovine serum.

Immunohistochemistry
Posterior poles were fixed in 4.0% (para)formaldehyde in 100 mM cacodylate buffer, pH 7.4. After 2–4 h in fixative, eyes were rinsed in 100 mM sodium cacodylate buffer (3x10 min) prior to embedding. Tissues were infiltrated and embedded in acrylamide and Optimal Cutting Temperature compound (Miles; Elkhart, N.Y.) (30) , snap frozen in liquid nitrogen, and sectioned on a cryostat. Sections spanning from the fovea to the temporal ora serrata were prepared from each donor. The sections were blocked in 10 mM sodium phosphate, pH 7.4, containing 0.85% NaCl, 1 mM each of calcium chloride and magnesium chloride, and 1 mg/ml globulin-free bovine serum albumin (PBS/M/C/BSA). Some sections were pretreated for 10 min with 0.5% trypsin (Sigma, St. Louis, Mo.) or 0.2–0.02 U/ml chondroitinase ABC (Seikagaku; Rockville, Md.) for use in conjunction with antibodies for collagen type IV or various chondroitin sulfate proteoglycans, respectively. Sections were then rinsed and incubated in primary antibody for 1 h. Antibodies were obtained from the sources indicated in Table 1 . Drusen-containing tissues from a minimum of five donor eyes were examined for each antibody. Slides were rinsed in PBS/M/C (2x10 min) and subsequently incubated for 30 min in the appropriate fluorescein isothiocyanate (FITC) -conjugated, human serum adsorbed secondary antibody. Sections were then washed in PBS/M/C (2x10 min) and coverslipped.

For negative controls, sections were exposed to PBS/M/C/BSA containing 1) no primary antibody, 2) 1% (vol/vol) normal serum, and/or c) antibodies to irrelevant proteins. For vitronectin, an additional control included adsorption of primary antibody to purified human vitronectin (Telios, La Jolla, Calif.). Positive controls included reaction of antibodies with the extracellular matrices of sclera, choroid, and vitreous and liver for vitronectin.

Confocal microscopy
Selected specimens of human donor RPE–choroid were fixed by immersion in 4% (para)formaldehyde in 0.1 M sodium cacodylate buffer and processed for laser scanning confocal microscopy, as previously described (31) . Images were captured and displayed using a BioRad 1024 laser scanning confocal microscope equipped with a Nikon inverted microscope.

RNA isolation
Total RNA was isolated from adult human liver, RPE/choroid, and primary cultures of human RPE cells as described by Chirgwin et al. (32) , except that cesium trifluoroacetate was used instead of cesium chloride in the density gradient ultracentrifugation step. The resulting pellet was stored at -80°C. The quality/integrity of RNA obtained was assessed on both agarose gels and Northern blots.

RT-PCR analyses
Total RNA was extracted from the RPE–choroid of four normal adult donor eyes, a primary culture of human RPE cells, and adult human liver. cDNA was synthesized with reverse transcriptase using oligo(dT)₁₆ as a primer. The enzyme was omitted from control reactions. cDNA was amplified using two pairs of hemi-nested vitronectin primers (outside pair: 5'-CGAGGAGAAAAACAATGCCAC-3' and 5'-GAAGCCGTCAGAGATATTTCG-3'; inside pair: 5'-CCTTCACCGACCTCAAGAAC-3' and 5'-GAAGCCGTCAGAGATAT TTCG-3'). PCR amplification products were separated electrophoretically on a 1.8% agarose gel.

	RESULTS

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Antibodies against common ECM constituents including collagens, elastin, entactin/nidogen, fibrillin, fibronectin, hyaluronan, laminin, link protein, various heparan, chondroitin, and keratan sulfate proteoglycans, tenascin, thrombospondin, and von Willebrand factor (Table 1 and Fig. 1 ) did not label either hard or soft drusen. Collagen type IV antibodies, which bound to RPE and vascular basal laminae (especially prominent in the choriocapillaris), occasionally reacted with small, punctate areas along the basal RPE surface, but these were not clearly drusen-related (data not shown). In addition, weak reactivity of some hard drusen with a monoclonal antibody against heparan sulfate glycosaminoglycan (Kimata antibody HK-249) and a polyclonal antibody specific for decorin was observed. Collagen type VI antibodies occasionally labeled the region between the RPE and drusen, along the RPE basal lamina (Fig. 1) . Controls confirm all antibody reactivities to be specific. In addition, the majority of the of the antibodies used bound to the expected regions of sclera, choroid, RPE, retina, vitreous, and/or other `control' tissues (Fig. 1) .

View larger version (50K): [in this window] [in a new window]   Figure 1. Fluorescence light micrographs of sections derived from human donor eyes. Neither hard or soft drusen (asterisks) react with antibodies directed against many common extracellular matrix constituents. The interphotoreceptor martrix (IPM), but not drusen, exhibits chondroitin sulfate immunoreactivity (A); antibodies directed against collagens type I (B), II (C), III (D, F), IV (E), and VI (G), elastin (H), and laminin (I) label various retinal and/or choroidal extracellular matrix regions, but not drusen. Note reactivity of RPE and choriocapillaris basal laminae (arrows) with antibodies to collagen type IV (E). Antibodies to collagen type VI bind weakly to extracellular material located between the RPE and drusen (G), possibly coresponding to basal laminar deposits, and to pericytes (arrows). Yellow autofluorescence of the RPE is visible in all micrographs. 125x.

All drusen phenotypes, in both macular and extramacular regions from donors with and without clinically documented AMD, were bound by antibodies directed against the ECM/serum protein vitronectin (Table 1 and Fig. 2 ). Antibodies directed against the heparin binding epitope of vitronectin also bound both hard and soft drusen (Fig. 2) . Anti-drusen immunoreactivity was eliminated by preadsorption of the antibodies with an excess of purified human vitronectin (data not shown).

View larger version (72K): [in this window] [in a new window]   Figure 2. Fluorescence light micrographs depicting vitronectin antibody reactivity with various classes of drusen (asterisks). Hard (A, C) and soft (B, D) drusen react with polyclonal anti-vitronectin antisera (A, B) and with various monoclonal antibodies (C, D), including 8E6, which binds a conformationally distinct heparin binding epitope (E). Vitronectin immunoreactivity is heterogeneous in some larger, soft drusen (D). A secondary antibody control is depicted (F). Evidence for the relatively specific accumulation of vitronectin in drusen is demonstrated by a double-labeled confocal image showing the binding of monoclonal antibody 16A7, which reacts specifically with multimeric vitronectin (Cy3-conjugated secondary antibody; red), and haptoglobin, a serum protein (FITC-conjugated secondary antibody; green) (G). Areas of overlap (yellow) include the choriocapillaris and portions of Bruch's membrane. Like haptoglobin, serum albumin, whereas abundant in the choroid, is not present in drusen (H). 125x (A, B, E–H); 200x (C–D).

Because drusen are localized at the RPE–choroid interface, we sought (using RT-PCR) to determine whether vitronectin is synthesized by surrounding tissues. Human RPE–choroid, cultured RPE cells, and liver contain vitronectin transcripts of the appropriate sizes for the hemi-nested primer pairs used (Fig. 3 ).

View larger version (62K): [in this window] [in a new window]   Figure 3. Vitronectin mRNA is detected in total RNA extracted from the RPE–choroid (RPE/CHOROID) of four normal adult donor eyes (1–4), primary cultures of human RPE cells (HRPE), and adult human liver (H LIVER). Two nested vitronectin primer pairs were used in PCR reactions. The outside vitronectin primer pair (top panel) amplified two fragments of 832 bp and 502 bp from RPE/CHOROID, HRPE, and H LIVER; these bands correspond to the genomic cDNA and mRNA of human vitronectin, respectively. In the absence of reverse transcriptase, only the 832 bp genomic fragment of vitronectin is evident. The inside vitronectin primer pair (bottom panel) also yields two appropriately sized bands: a 587 bp genomic cDNA fragment and a 257 bp cDNA, which corresponds to human vitronectin mRNA. In the absence of RT, only the 587 bp product is detected.

	DISCUSSION

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AMD is associated with a variety of ocular extracellular matrix abnormalities. Drusen, deposits located between the RPE basal lamina and the inner collagenous layer of Bruch's membrane, are the most prominent of these. Accordingly, we selected a panel of antibodies directed against most major ECM- and basal lamina-associated proteins, and used it to determine whether any of these molecules are components of the drusen present in eyes from donors with and without clinically diagnosed AMD.

The immunohistochemical findings show that vitronectin, a well-characterized ECM molecule and circulating plasma protein (33) , is a constituent of drusen. Vitronectin immunoreactivity was confirmed in drusen of all of the human donor eyes examined whether or not they carried a clinical diagnosis of AMD. Both polyclonal antisera and monoclonal antibodies to vitronectin, as well as several antibodies that preferentially react with the conformationally distinct, heparin binding form of human vitronectin (26 , 27 , 34 , 35 ), showed the same labeling patterns. Moreover, vitronectin immunoreactivity was associated with both hard and soft drusen irrespective of size, phenotype, or location within the fundus. This observation is consistent with our earlier studies of drusen-associated carbohydrates (29) , and suggests that the various drusen phenotypes may be similar in composition and, possibly, origin, as proposed previously (22) . The presence of multimeric vitronectin in drusen could explain the presence of complement proteins (25) and, perhaps, heparan sulfate proteoglycan (36) previously identified as components of these deposits.

In contrast, antibodies to more common ECM components such as laminin, fibronectin, collagens, and proteoglycans displayed no detectable drusen immunoreactivity, although labeling of adjacent extracellular matrices and basal laminae in the choroid, retina, and vitreous was observed. Vitronectin cannot be removed from drusen after extensive rinsing. These data, in addition to the observation that drusen were not labeled with antibodies directed against haptoglobin or serum albumin, indicate that drusen-associated vitronectin is not likely a result of passive sequestration of plasma proteins in drusen during dissection or histochemical processing. Instead, they suggest that the development of drusen involves the selective accumulation of vitronectin within Bruch's membrane, and are inconsistent with the interpretation that drusen are merely composed of the same ECM molecules normally present in Bruch's membrane.

Some previous histochemical and immunohistochemical studies have also implicated ECM components as constituents of drusen, but the collective data are scant and conflicting. For example, Newsome et al. (36) reported that diffuse, soft drusen contained immunologically detectable fibronectin, but that nodular, hard drusen did not. Pauleikhoff and co-workers (37) concluded that phospholipid-containing, but not neutral lipid-containing, drusen were anti-fibronectin antibody reactive. In contrast, van der Schaft and colleagues (38) found no fibronectin immunoreactivity in drusen (38) , a result that is consistent with our own. Although we do find evidence of anti-fibronectin labeling in retinal and choroidal basal lamina, no evidence of anti-fibronectin binding to either hard or soft drusen was noted in any of the donor eyes used in this study. Other ECM molecules, including collagen types I, III, IV, and V, laminin, and heparan sulfate proteoglycan, have also been reported as being components of drusen in `diffuse, mottled or superficial laminar' patterns (36) . Kliffen et al. (39) reported heparan sulfate proteoglycans in basal laminar deposits, but not in drusen. In this study, we did observe weak labeling of some drusen with one heparan sulfate proteoglycan antibody, suggesting that heparan sulfate may be associated with a subset of drusen or that it is always present, but its epitopes are masked.

It has been proposed that age-related deposits in Bruch's membrane may be derived from photoreceptor-associated molecules (12 , 40 ). If this is true, one would expect to detect the presence of photoreceptor- and interphotoreceptor matrix-associated molecules in some drusen. Antibodies that bind interphotoreceptor matrix components (i.e., IPM-1, chondroitin sulfate proteoglycans) do not react with drusen. These results are consistent with previous observations that polyclonal antisera against opsin, the principal photoreceptor membrane protein, also fail to react with drusen (41) .

Vitronectin (serum spreading factor; S-protein) is present in high concentration in plasma and is a common constituent of extracellular matrices (34 , 42 ), where it subserves roles in thrombosis, fibrinolysis, inflammation, and cellular adhesion (33 , 42 ). Until recently, it was thought that vitronectin is synthesized primarily in the liver 43-45) . Recent data show vitronectin mRNA levels in brain, skeletal muscle, lung, uterus, testis, thymus, and kidney range from 1 to 4% of that in liver (43) . Our data indicate that vitronectin mRNA is also expressed in the RPE–choroid complex and by cultured RPE cells, suggesting that a local source(s) of vitronectin might contribute to drusen formation.

The identification of vitronectin as a major constituent of drusen may have important implications for our understanding of drusen biogenesis and the pathogenesis of AMD. Currently, there is no accepted consensus regarding the molecular mechanism(s) of drusen formation or their role in the etiology of AMD (12 , 21 , 23 , 46 , 47 ). One scenerio is that the binding and deposition of vitronectin to Bruch's membrane could compromise the exchange of metabolites between the choriocapillaris and the RPE, eventually leading to RPE and photoreceptor cell dysfunction and degeneration. Others have advanced related hypotheses based on abnormalities of Bruch's membrane (37) . Alternatively, it is conceivable that drusen form as a consequence of RPE dysfunction and deterioration. RPE cells or by-products of abnormal RPE and/or photoreceptor cell metabolism could serve as `nucleation sites' for the deposition of proteins such as vitronectin (see 12, 21, 23, 46, 47 ). In both scenarios, localized degeneration of the RPE would, in the absence of reproliferaton, result in a concomitant degeneration of adjacent photoreceptor cells.

Vitronectin has previously been shown to be a molecular component of abnormal extracellular deposits associated with other age-related disorders, including atherosclerosis (48) , dermal elastosis (49) , and dense deposit disease (50) , and with various amyloidoses (49 , 51 ), including Alzheimer's disease (52) . This is particularly significant in view of clinical studies that have demonstrated correlations between AMD and atherosclerosis (53) and between the incidence of AMD and elastotic degeneration of the dermis (54) . Our current studies are aimed at determining whether drusen share additional compositional similarities to the deposits associated with these diseases and, if so, whether these constituents are derived from local ocular sources. It is hoped that answers to these questions will provide new insight into the process of drusen biogenesis and its role in the pathogenesis of AMD.

	ACKNOWLEDGMENTS

 
Antibodies were kindly provided by Drs. Kermit Carroway, Paul Bishop, Koji Kimata, Nancy Philp, Brian Sires, Randy Bretton, Steven Carlson, Bruce Caterson, John Harper, Alexander Ljubimov, Richard Margolis, Klaus Preissner, and Thomas Wight. The technical assistance of Ms. Bobbie Schneider, Ms. Mary McDonald, Mr. Kaj Anderson, Mr. Ihab Ghaly, Ms. Wen Wu, Mr. Matthew Nelon, Dr. Margaret Kirchoff-Rempe, and Dr. Charles Nye, as well as the secretarial assistance of Ms. Amy Regan and Ms. Linda Koser, is greatly appreciated. Conversations with Mr. Markus Kuehn and Drs. Howard Lazarus, Klaus Preissner, Michael Pierschbacher, and Marilyn Kincaid are appreciated. The authors are especially grateful to Jake Requard, the staff of the Iowa Lions Eye Bank, and the all the tissue donors and their families who gave so unselfishly for the advancement of science.

	FOOTNOTES

 
¹ Correspondence: Department of Ophthalmology and Visual Sciences, The University of Iowa, 11190E PFP, 200 Hawkins Dr., Iowa City, IA 52240, USA. E-mail: gregory-hageman{at}uiowa.edu

² Abbreviations: AMD, age-related macular degeneration; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; PBS/M/C/BSA, 10 mM sodium phosphate, pH 7.4, containing 0.85% NaCl, 1 mM each of calcium chloride and magnesium chloride, and 1 mg/ml globulin-free bovine serum albumin; RPE, retinal pigmented epithelium; RT-PCR, reverse transcriptase-polymerase chain reaction.

Received for publication August 28, 1998. Revision received November 4, 1998.

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