Mapping the differential distribution of glycosaminoglycans in the adult human retina, choroid, and sclera

Abstract

PURPOSE. To map the distribution of different classes of glycosaminoglycans (GAGs) in the healthy human retina, choroid, and sclera. METHODS. Frozen tissue sections were made from adult human donor eyes. The GAG chains of proteoglycans (PGs) were detected with antibodies directed against various GAG structures (either directly or after pretreatment with GAG-degrading enzymes); hyaluronan (HA) was detected using biotinylated recombinant G1-domain of human versican. The primary detection reagents were identified with FITC-labeled probes and analyzed by fluorescence microscopy. RESULTS. Heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and HA were present throughout the retina and choroid, but keratan sulfate (KS) was detected only in the sclera. HS labeling was particularly strong in basement membrane-containing structures, the nerve fiber layer (NFL), and retinal pigment epithelium (RPE)-for example, intense staining was seen with an antibody that binds strongly to sequences containing 3-O-sulfation in the internal limiting membrane (ILM) and in the basement membrane of blood vessels. Unsulfated CS was seen throughout the retina, particularly in the ILM and interphotoreceptor matrix (IPM) with 6-O-sulfated CS also prominent in the IPM. There was labeling for DS throughout the retina and choroid, especially in the NFL, ganglion cell layer, and blood vessels. CONCLUSIONS. The detection of GAG chains with specific probes and fluorescence microscopy provides for the first time a detailed analysis of their compartmentalization in the human retina, by both GAG chain type and sulfation pattern. This reference map provides a basis for understanding the functional regulation of GAG-binding proteins in health and disease processes.

Figure 1.

Localization of HS in retina, choroid, and sclera. Human tissue sections were stained using the pan HS antibody 10E4 (green) either without enzymatic pretreatment (left side) or after treatment with heparinases (right side). Top: the neurosensory retina; bottom: RPE, Bruch's membrane, choroid, and sclera. Here, and in all other figures, the images shown are representative of three individual donors (summarized in Table 2). Blue: DAPI staining of cell nuclei. Scale bar, 100 μm.

Figure 2.

Detection of HS with sulfate-specific antibodies. Phage-display antibodies (Table 1) were used to map HS epitopes in the sclera and chorioretinal complex in human eye tissues (green). (A, left) LKIV69 recognizes N- and 2-O-sulfated regions of HS GAG chains; (middle) RB4EA-12 binds to sequences comprising iduronic acid followed by a glucosamine sulfated at the N- and 6-O positions; (right) AO4B08 recognizes N-sulfated octa-saccharide epitopes with three consecutive 6-O sulfates and an internal 2-O sulfate. (B) Close-up images of HS antibody staining in the inner retina; arrows: staining of retinal blood vessels with the RB4EA-12 and AO4B08 antibodies. Scale bar, 100 μm.

Figure 3.

Distribution of 3-O-sulfated HS epitopes in retina and choroid. (A) Staining of retina and choroid with HS4C3 (green), which recognizes 3-O-sulfated HS containing the antithrombin binding sequence (see Table 1); arrows: blood vessels in the retina (i) and choroid (ii), which stain strongly with this antibody. (B) Detail of a retinal blood vessel (from the same donor) lying underneath the ILM (scale bar, 50 μm). (C) Three-dimensional reconstruction by confocal microscopy of blood vessel in (B) in which the ILM is pseudocolored blue for clarity. Scale bar: (A, C) 100 μm; (B) 50 μm.

Figure 4.

Localization of CS subtypes in retina, choroid, and sclera. The presence of CS was detected using either (A) anti-stub antibodies (1B5, 2B6, and 3B3; Table 1) after chondroitin AC lyase digestion or (B) the LY111 antibody raised against a whole C4S chain. Top: the neurosensory retina; bottom: the RPE, choroid, and sclera. (B) Digestion with chondroitin AC lyase removed ∼90% of the green fluorescent signal associated with LY111, although some residual labeling of the RPE and sclera remains. Scale bar, 100 μm.

Figure 5.

Distribution of DS determined with 2B6 antibody. DS stubs were detected with 2B6 antibody (green) after predigestion of sections with chondroitin B lyase. Scale bar, 100 μm.

Figure 6.

KS is present only in sclera of the adult human eye. KS GAG chains (green) were detected with the pan-specific antibody 5D4. Scale bar, 100 μm.

Figure 7.

Expression, purification, and HA-binding activity of VG1. (A) Protein expression was induced with IPTG and cultures analyzed after 20 hours by SDS-PAGE (under reducing conditions), revealing a band at ∼40 kDa (arrow) that had the expected N-terminal sequence (data not shown). (B) After inclusion body preparation and refolding, VG1 was purified by ion exchange chromatography (Q Sepharose); horizontal bar: pooled eluents. (C) The VG1 preparation was analyzed by SDS-PAGE after refolding (lane 1) and ion exchange chromatography (lane 2), revealing the purity of the recombinant protein. (D) The HA-binding activity of the VG1 was demonstrated using biotinylated-HA binding to VG1 immobilized on microtiter plates at a range of concentrations (0–11 pmol/well). Values are plotted as the mean absorbance (A_405nm) ±SE (n = 8).

Figure 8.

Localization of HA in the retina and choroid. HA (green) was detected with our bVG1 (top) and a commercially available HA-binding protein (bHABP; bottom) in the absence (A, C) and presence (B, D) of hyaluronidase pretreatment. (A) Strong labeling was seen, for example, in choroidal blood vessels and on the RPE, with intense staining on its apical side (arrow). (B) The staining of a few individual cells remaining after hyaluronidase digestion, which are mostly in the lumen of blood vessels, probably resulted from the interaction of the VG1 protein with other versican ligands. (C) A similar pattern of HA localization was observed with bHABP, except that the staining seen in the IPM is likely to be nonspecific, given that it was not fully removed by hyaluronidase treatment (D). Scale bar, 100 μm.