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The permeability of the endothelium of the capillaries of the rat choriocapillaris to circulating macromolecules was examined during the first post-natal week of development using hemeproteins of different molecular dimensions and ferritin. At this stage of development capillaries and photoreceptor cells in the neural retina are not fully formed, but the choriocapillaris has an adult-like morphology. Thus, through these differences in neural and vascular architectures the possible functional relationships between the retina and choriocapillaris can be explored. High levels of horseradish peroxidase [Einstein-Stokes radius (ESR) 3 nm] activity were localized in Bruch's membrane within 2 minutes after intravenous tracer injection. In contrast, scant levels of hemoglobin (ESR, 3.2 nm) and no catalase (ESR, 5.2 nm) activity were observed here at 75 and 90 minutes, respectively. Only a few ferritin particles (ESR, 6.1 nm) crossed the choriocapillary endothelium after a 90-minute circulation. The results demonstrate that in the neonatal rat the choriocapillaris has the same restrictive properties as have been described for the adult eye (Pino and Essner, 1980, 1981), even in the absence of retinal capillaries and functional photoreceptor cells.

The location and chemical composition of anionic sites on the endothelium of the choriocapillaris was investigated with cationic ferritin and enzyme digestion techniques. Cationic ferritin administered intravenously initially labeled essentially all fenestral diaphragms. Within 30 min after injection, no diaphragms remained labeled, but they could be relabeled by a second cationic ferritin injection. Following perfusion of cationic ferritin, the entire luminal front of the endothelium was labeled: the plasmalemma and fenestral, vesicle, and channel diaphragms. Perfusion of neuraminidase or chondroitinase did not affect subsequent cationic ferritin binding. In contrast, heparitinase removed anionic sites on all structures except fenestral diaphragms. Cationic ferritin did not mark the endothelium following heparinase digestion. All sites were cleaved with pronase E. These results indicate that heparin is the anionic moiety on fenestral diaphragms while the glycocalices of the plasmalemma and vesicle and channel diaphragms are rich in a heparan sulfate proteoglycan. Furthermore, since the heparan sulfate localized to these structures was digested by both heparinase and heparitinase, it is in a form similar to heparin. These findings demonstrate that the endothelium of the choriocapillaris bears cell-surface anionic components that are different than those described for fenestrated endothelia lining other vascular beds.

The photoreceptors of the neural retina require retinol for synthesis of rhodopsin. In the plasma, retinol is bound to retinol binding protein which is carried by transthyretin (TTR; formerly called prealbumin). It is unknown whether, or how, retinol carrier proteins cross the endothelium of the choriocapillaris, the blood supply to the outer neural retina. This was examined in the present study with TTR-gold probes perfused into rats and localized by electron microscopic techniques. TTR-gold, often in clusters, was localized to diaphragmed fenestrae, parajunctional areas, coated pits, transendothelial channels, multivesicular bodies, and to vesicles close to the Golgi apparatus. The probe was also identified at the luminal and abluminal fronts and the interior of transendothelial channels in an apparent sequence of transit. TTR-gold was also found in a series of interconnected vesicles adjacent to the abluminal side of the endothelium. Localizations were not seen when rat albumin fraction V was substituted for TTR and when the rats were perfused with Pronase E before labeling with TTR-gold. These observations indicate that binding and receptor mediated-like transport of TTR by the endothelium of the choriocapillaris is present. This is similar to the processing of heparin-gold by this endothelium.

The choriocapillaris is the fenestrated capillary bed in the choroid of the eye and is the major blood supply to the retinal pigment epithelium (RPE) and photoreceptor cells. Bruch's membrane (BM) is a multilaminated basement membrane that separates the choriocapillaris from the RPE. In a previous study (Pino RM, Essner E; Cell Tissue Res 208:21, 1980) we found that the choriocapillary endothelium restricted the egress of ferritin from the choriocapillaris. In the present study, hemeproteins were used to further establish the permeability characteristics of this capillary bed. Horseradish peroxidase (Einstein-Strokes radius (ESR), 30 A) rapidly crossed the capillary endothelium (less than 5 min) after intravenous administration and after 5 minutes filled BM and the basal infoldings of the RPE. In contrast, hemoglobin (Hg) (ESR, 32 A) and lactoperoxidase (LP) (ESR, approximately 40 A) are markedly restricted at the level of endothelial diaphragmed fenestrae, channels, and intercellular junctions. Little vesicular transport of these proteins was observed. The reaction product of the two hemeprotein activities was not demonstrable in BM for up to 30 min after injection; relatively low levels were detected after 75 min. HG and LP appear to be further restricted by BM, since their reaction products were not demonstrable between the RPE basal infoldings at this time. Catalase (ESR, 52 A) activity was not detected in BM for up to 4 hr after injection. These results indicate that the rat choriocapillary endothelium, unlike the fenestrated endothelia lining other vascular beds, substantially restricts the passage of large tracer molecules.

The endothelium of the choriocapillaris has been shown to restrict molecules with Einstein-Stokes radii greater than or equal to 3.2 nm which correspond to minimal molecular weights of approximately 64,000-68,000 daltons. The present study was undertaken to determine if the endothelium restricts exogenous transthyretin (prealbumin), a 55,000-dalton carrier of retinol-binding protein. Rats were injected with human 125I-transthyretin which was allowed to circulate for 15 and 30 min. Chromatographic analysis demonstrated that the human transthyretin did not bind to rat blood proteins. Eye tissue from injected rats was prepared for light and ultrastructural autoradiographic analysis. Autoradiographic grains were confined to areas over the lumen of the choriocapillaris with few present on Bruch's membrane, the basement membrane common to the endothelium of the choriocapillaris and the retinal pigment epithelium. These findings demonstrate that the choriocapillaris can restrict transthyretin and suggest a possible role of its endothelium in the metabolism of retinol-carrier molecules.

The choriocapillaris is one example of a capillary bed lined by a fenestrated endothelium that is restrictive to exogenous tracers and endogenous plasma proteins. In this study we have examined the distribution of cell-surface monosaccharides utilizing biotinylated lectin-avidin ferritin cytochemistry. Receptors for wheat germ agglutinin were localized to the plasmalemma and diaphragms of some fenestrae, vesicles, and channels at the luminal endothelial front in amounts greater than seen for the other lectins employed. The absence of labeling following inhibition with N-acetylglucosamine and after tissue digestion with N-acetylhexosaminidase, but not after neuraminidase indicated that this lectin marked N-acetylglucosamine residues and not sialic acid. Wheat germ agglutinin receptors were not affected by pronase E or trypsin digestion, but were partially removed by proteinase K. The latter also removed many fenestral diaphragms. Wheat germ agglutinin receptors were cleaved with endoglycosidase D. The combined results indicate that the wheat germ agglutinin receptor is of the low-mannose type and part of a protein with hydrophobic properties. Receptors for concanavalin A (mannose) and Ricinus communis agglutinin (galactose) were also localized to the plasmalemma and endothelial diaphragms. The examination of sections at different tilt angles revealed that these lectins bound to the endothelium in a non-random distribution, encircling diaphragms of fenestrae and channels. Soybean agglutinin (N-acetylgalactosamine) marked endothelial structures sparsely. Following digestion with pronase E or trypsin, receptor sugars for the latter three lectins were completely removed, indicating their presence on protease susceptible glycoproteins. These findings demonstrate that the endothelium of the choriocapillaris bears carbohydrate moieties that are different than those described for permeable fenestrated endothelia.

The choriocapillaris is the fenestrated capillary network that supplies a large portion of the nutrients required by the retinal pigment epithelium, photoreceptor cells, and other cells of the outer neural retina. The permeability of these capillaries was investigated in the rat by the use of ferritin (mol. wt. approx. 480,000; mol. diam. 110A) as a tracer. Ninety minutes after intravascular ferritin administration, a high concentration of tracer particles was distributed uniformly in the capillary lumina but few particles were present in Bruch's membrane, the multilayered basement membrane that separates the choriocapillary endothelium from the retinal pigment epithelium. The bulk of the tracer remained in the capillary lumina with a definate blockage seen at fenestral, channel, and vesicle diaphragms. These results indicate that the ratchoriocapillary endothelium, unlike the fenestrated endothelia lining other capillary beds, constitutes an important barrier to the passage of ferritin and presumably of circulating native molecules of similar size.

The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.

Heparin-gold probes were used to localize regions of heparin binding on the luminal surface of the diaphragmed-fenestrated endothelium of the rat choriocapillaris. Structures of endothelial cells were unlabeled when rats were kept on ice and perfused with solutions at 4 degrees C. When the heparin-gold quantity was doubled, only a few heparin-gold particles marked some diaphragms spanning fenestrae, vesicles and channels, parajunctional regions of the plasmalemma, and coated pits. With solutions at 4 degrees C, but the animals kept at room temperature, all of these structures in the endothelial cells were labeled. This binding was not altered by the perfusion of low levels of unlabeled heparin, but was eliminated following high levels of unlabeled heparin, and by digestion with trypsin and pronase. At 37 degrees C, heparin was localized to the above structures and, in addition, was internalized into coated vesicles, endosomes, and multivesicular bodies, but not other types of lysosomes. Some particles were found in tubules adjacent to the Golgi stacks. Heparin-gold was also transported to the abluminal front of the endothelium by vesicles. A desulfated, non-anticoagulant, fraction of heparin bound to gold was localized to the endothelium in the same manner. These results demonstrate receptors for heparin on the surface of a fenestrated endothelium. Furthermore, they show the pathway of endocytosis and transport of heparin. The possible roles of heparin in the function of the endothelium is discussed.

The organization of anionic (negatively charged) sites in Bruch's membrane of the rabbit eye at various stages of postnatal development was studied using the cationic polymer, polyethyleneimine (PEI). PEI-positive sites were demonstrable as rows of particles (diameter ca. 18 nm) located at intervals along either side of the basal laminae of the retinal pigment epithelium and choriocapillary endothelium. In tangential sections through Bruch's membrane, stained particles appeared to be arranged in a semi-regular, lattice-like pattern in which the sites were separated from each other by an interval of approximately 50 nm. PEI-positive particles were also observed on collagen fibers where they were distributed at regular intervals along the length of the fiber. In tangential sections, collagen fibers formed a loosely packed meshwork in the central zone of Bruch's membrane. In addition, individual fibers were frequently oriented so that one end was located close to or within the substance of the basal laminae, a result suggesting that the anionic sites on these fibers might contribute to the network present in the basal laminae. The findings lend further support to the suggestion that anionic sites in Bruch's membrane may serve as a charge barrier which retards the movement of anionic molecules that are in transit from the choriocapillaris to the retinal pigment epithelium and outer neural retina.