Evidence of hyaluronic acid and hyaluronic acid binding sites on human corneal endothelium Anders Harfstrand, M.D., Nils Molander, M.D., Ulf Stenevi, M.D., David Apple, M.D., Mona Schenholm, Kjell Madsen, M.D.
Sodium hyaluronate (NaHA) is synthesized in the cell membrane by the sequential addition of N-acetyl D-glucosamine and D-glucuronic acid to form a long molecule. 1 It is widely distributed in connective tissue as a high molecular weight voluminous coil structure. 2,3 The substance is associated with almost all cells of the body and high tissue concentrations are found in the umbilical cord (4,100 mg/I),4 in the human vitreous body (1,200 mg/I),5 and in the human aqueous humor (1.4 mg/l). 6 Sodium hyaluronate has been reported to be involved in cell protection, control of cell migration, growth control, cellular differentiation, and tissue morphogenesis. 7 It is also an important matrix component of the extracellular compartment. Specific NaHA receptors have been identi-
fied on various cells. 8- 10 Hyaluronate-binding sites have also· been found on the corneal endothelium. 11 Previous studies on the visualization of glycosaminoglycans (GAGs) in mammalian tissues have used various histochemical methods such as toluidine blue, 12 cresyl violet, 13 colloidal iron, 14 alcian blue,15,16 or ruthenium red. 17 However, these methods have poor specificity for different GAGs. Thus, to demonstrate hyaluronic acid (HA) in tissues unequivocally, another approach was needed. Extensive studies of proteoglycan aggregates from cartilage and other tissues have shown that they consist of an HA backbone to which the proteoglycan monomers are noncovalently bound. 18 Thus, the core protein of aggregating proteogly-
From the Department 0/ Preclinical Sciences, Kabi Pharmacia Ophthalmics AB, Uppsala, Sweden (Har/strand, Schenholm) , the Department o/Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden (Har/strand) , the Department o/Ophthalmology, Hospital 0/ Lund, Lund, Sweden (Molander, Stenevi), and Storm Eye Institute, Charleston, South Carolina (Apple). Reprint requests to Anders Har/strand, M.D., Kabi Pharmacia Ophthalmics AB, S-751 82 Uppsala, Sweden.
J CATARACf REFRACf SURG-VOL 18, MAY
1992
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cans contains an HA binding domain with high affinity and specificity. 19,20 In this study, we used purified HA-binding domains from cartilage proteoglycans as specific high affinity probes for both biochemical quantification and histochemical visualization of HA. Hyaluronic acid is likely to be important to the endothelium. Several surgical techniques use irrigation. To test the resistance to such physical forces on the HA layer, a separate experiment in rabbits was included. MATERIALS AND METHODS
Human Corneas Corneal buttons from transplantation patients were directly immersed in 4 % buffered formaldehyde containing 1 % (w/v) cetylpyridine chloride (CPC). Pilot experiments showed that CPC, which precipitates HA in the tissue, reduced loss during preparation and staining (data not shown). The corneas were subsequently dehydrated in a graded ethanol series and embedded in paraffin. Then 5 Jlm sections were cut on a microtome. Corneal buttons for biochemical quantification of HA were frozen and kept at -20°C. On some corneas the endothelium was scraped away before freezing. Postmortem eyes were used for the binding experiment. The eyes were enucleated from patients without eye disease and maintained intact on ice until use. Total postmortem time never exceeded 24 hours.
and then removed. After incubation with HABR biotin for two hours at room temperature, the sections were rinsed in phosphate-buffered saline (PBS) and incubated for one hour with ABC Vectastain reagent (Vector Laboratories Inc., Burlingham, CA). The sections that now were labeled with peroxidase were visualized with ethyl carbazole. Control sections were incubated for two hours in a humidified chamber at 37°C with 50 units/ml of Streptomyces hyaluronidase (Seikagaku Fine Biochemicals, Tokyo, Japan). The sections were mounted under cover glass in Kaiser's glyceringelatin and examined by light microscopy. TN
Biochemical Analysis The concentration of HA in cornea extract was determined in duplicate with a radiometric assay (HA 50 test, Pharmacia Diagnostics, Uppsala, Sweden) according to principles previously outlined. 21 The assay was modified for analysis of tissue extracts 23 and for serum samples. 24 The assay is specific for HA determination. 24 Briefly, the corneal buttons were extracted with 0.5 ml of 0.5 M NaCI, 200 JlI 125I-Iabeled HABR were added, and the samples were incubated for 60 minutes at 4° to 7°C. A small amount of HA Sepharose in 100 JlI buffer was added and the tubes incubated for 45 minutes at the same temperature. Finally, 2 ml of washing solution were added and Sepharose was recovered after centrifugation at 200 g for ten minutes. Bound radioactivity in the pellet was measured in a gamma counter. Known amounts of HA were used to construct a standard curve. 25
Animals Six- to eight-week-old New Zealand white rabbits and guinea pigs weighing about 300 g were obtained from local breeders. The animals were subjected to a routine two weeks' quarantine before use. They were killed with an overdose of pentobarbital and the corneas were excised at the limbus. The corneas were subsequently frozen and kept at -200C until biochemical analysis. All animal handling was in compliance with the ARVO Resolution on the Use of Animals in Research. The experimental protocols were approved by the regional ethical committee.
Irrigation Experiment Rabbits were anesthetized by intramuscular injections of ketamine (Ketalar®), 35 mg/kg, and xylazine chloride (Rompun®), 5 mg/kg, supplemented with Tetrakain® eye drops (Alcon-Couvreur). A 3-mm incision into the anterior chamber was made in the cornea 1 mm from the limbus. The corneas were irrigated with 25 ml balanced salt solution. The animals were then sacrificed and the corneas taken for biochemical analysis as described.
Histochemical Experiments The HA-binding region of cartilage proteoglycans (HABR) used in the staining technique was prepared by affinity chromatography on HA Sepharose as described earlier.21 The purified protein was then linked to biotin according to the principles outlined by Ripellino and coauthors.22 Hyaluronic acid was used during the biotinylation to protect the HA-binding site on the HABR
Binding Experiment Specific HA binding was determined with tritiated high molecular weight HA as described. 11 The labeled HA (Mr 3 X 10 6 ,2.02 X 105 dpm/Jlg) was provided by Dr. John R. E. Fraser (Melbourne University, Australia). Briefly, the corneas were cut out with a small scleral rim to minimize diffusion into the stroma. Endogenous HA was removed by digestion with
266
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hyaluronidase for one hour at 37°C. After rinsing to remove the enzyme, 1 flg/ml of tritiated HA was added and the cornea was incubated for 90 minutes at room temperature. One cornea from each pair of eyes was incubated in the presence of 100 flg/ml of unlabeled HA (Healon®) to give an estimate of unspecific binding. The corneas were dissolved in 1 M KOH. Radioactivity was determined by liquid scintillation counting.
Table 1. Amount of HA on explanted corneas. HA (~g/g)
Number
Human
1.20 ± 0.85*
12
Human endothelium removed
0.28 ± 0.09
3
Rabbit
0.69 ± 0.07
14
Sodium hyaluronate (NaHA) is synthesized in the cell membrane by the sequential addition of N-acetyl D-glucosamine and D-glucuronic acid to form a long molecule. 1 It is widely distributed in connective tissue as a high molecular weight voluminous coil structure. 2,3 The substance is associated with almost all cells of the body and high tissue concentrations are found in the umbilical cord (4,100 mg/I),4 in the human vitreous body (1,200 mg/I),5 and in the human aqueous humor (1.4 mg/l). 6 Sodium hyaluronate has been reported to be involved in cell protection, control of cell migration, growth control, cellular differentiation, and tissue morphogenesis. 7 It is also an important matrix component of the extracellular compartment. Specific NaHA receptors have been identi-
fied on various cells. 8- 10 Hyaluronate-binding sites have also· been found on the corneal endothelium. 11 Previous studies on the visualization of glycosaminoglycans (GAGs) in mammalian tissues have used various histochemical methods such as toluidine blue, 12 cresyl violet, 13 colloidal iron, 14 alcian blue,15,16 or ruthenium red. 17 However, these methods have poor specificity for different GAGs. Thus, to demonstrate hyaluronic acid (HA) in tissues unequivocally, another approach was needed. Extensive studies of proteoglycan aggregates from cartilage and other tissues have shown that they consist of an HA backbone to which the proteoglycan monomers are noncovalently bound. 18 Thus, the core protein of aggregating proteogly-
From the Department 0/ Preclinical Sciences, Kabi Pharmacia Ophthalmics AB, Uppsala, Sweden (Har/strand, Schenholm) , the Department o/Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden (Har/strand) , the Department o/Ophthalmology, Hospital 0/ Lund, Lund, Sweden (Molander, Stenevi), and Storm Eye Institute, Charleston, South Carolina (Apple). Reprint requests to Anders Har/strand, M.D., Kabi Pharmacia Ophthalmics AB, S-751 82 Uppsala, Sweden.
J CATARACf REFRACf SURG-VOL 18, MAY
1992
265
cans contains an HA binding domain with high affinity and specificity. 19,20 In this study, we used purified HA-binding domains from cartilage proteoglycans as specific high affinity probes for both biochemical quantification and histochemical visualization of HA. Hyaluronic acid is likely to be important to the endothelium. Several surgical techniques use irrigation. To test the resistance to such physical forces on the HA layer, a separate experiment in rabbits was included. MATERIALS AND METHODS
Human Corneas Corneal buttons from transplantation patients were directly immersed in 4 % buffered formaldehyde containing 1 % (w/v) cetylpyridine chloride (CPC). Pilot experiments showed that CPC, which precipitates HA in the tissue, reduced loss during preparation and staining (data not shown). The corneas were subsequently dehydrated in a graded ethanol series and embedded in paraffin. Then 5 Jlm sections were cut on a microtome. Corneal buttons for biochemical quantification of HA were frozen and kept at -20°C. On some corneas the endothelium was scraped away before freezing. Postmortem eyes were used for the binding experiment. The eyes were enucleated from patients without eye disease and maintained intact on ice until use. Total postmortem time never exceeded 24 hours.
and then removed. After incubation with HABR biotin for two hours at room temperature, the sections were rinsed in phosphate-buffered saline (PBS) and incubated for one hour with ABC Vectastain reagent (Vector Laboratories Inc., Burlingham, CA). The sections that now were labeled with peroxidase were visualized with ethyl carbazole. Control sections were incubated for two hours in a humidified chamber at 37°C with 50 units/ml of Streptomyces hyaluronidase (Seikagaku Fine Biochemicals, Tokyo, Japan). The sections were mounted under cover glass in Kaiser's glyceringelatin and examined by light microscopy. TN
Biochemical Analysis The concentration of HA in cornea extract was determined in duplicate with a radiometric assay (HA 50 test, Pharmacia Diagnostics, Uppsala, Sweden) according to principles previously outlined. 21 The assay was modified for analysis of tissue extracts 23 and for serum samples. 24 The assay is specific for HA determination. 24 Briefly, the corneal buttons were extracted with 0.5 ml of 0.5 M NaCI, 200 JlI 125I-Iabeled HABR were added, and the samples were incubated for 60 minutes at 4° to 7°C. A small amount of HA Sepharose in 100 JlI buffer was added and the tubes incubated for 45 minutes at the same temperature. Finally, 2 ml of washing solution were added and Sepharose was recovered after centrifugation at 200 g for ten minutes. Bound radioactivity in the pellet was measured in a gamma counter. Known amounts of HA were used to construct a standard curve. 25
Animals Six- to eight-week-old New Zealand white rabbits and guinea pigs weighing about 300 g were obtained from local breeders. The animals were subjected to a routine two weeks' quarantine before use. They were killed with an overdose of pentobarbital and the corneas were excised at the limbus. The corneas were subsequently frozen and kept at -200C until biochemical analysis. All animal handling was in compliance with the ARVO Resolution on the Use of Animals in Research. The experimental protocols were approved by the regional ethical committee.
Irrigation Experiment Rabbits were anesthetized by intramuscular injections of ketamine (Ketalar®), 35 mg/kg, and xylazine chloride (Rompun®), 5 mg/kg, supplemented with Tetrakain® eye drops (Alcon-Couvreur). A 3-mm incision into the anterior chamber was made in the cornea 1 mm from the limbus. The corneas were irrigated with 25 ml balanced salt solution. The animals were then sacrificed and the corneas taken for biochemical analysis as described.
Histochemical Experiments The HA-binding region of cartilage proteoglycans (HABR) used in the staining technique was prepared by affinity chromatography on HA Sepharose as described earlier.21 The purified protein was then linked to biotin according to the principles outlined by Ripellino and coauthors.22 Hyaluronic acid was used during the biotinylation to protect the HA-binding site on the HABR
Binding Experiment Specific HA binding was determined with tritiated high molecular weight HA as described. 11 The labeled HA (Mr 3 X 10 6 ,2.02 X 105 dpm/Jlg) was provided by Dr. John R. E. Fraser (Melbourne University, Australia). Briefly, the corneas were cut out with a small scleral rim to minimize diffusion into the stroma. Endogenous HA was removed by digestion with
266
J CATARACT REFRACT SURG-VOL 18, MAY 1992
hyaluronidase for one hour at 37°C. After rinsing to remove the enzyme, 1 flg/ml of tritiated HA was added and the cornea was incubated for 90 minutes at room temperature. One cornea from each pair of eyes was incubated in the presence of 100 flg/ml of unlabeled HA (Healon®) to give an estimate of unspecific binding. The corneas were dissolved in 1 M KOH. Radioactivity was determined by liquid scintillation counting.
Table 1. Amount of HA on explanted corneas. HA (~g/g)
Number
Human
1.20 ± 0.85*
12
Human endothelium removed
0.28 ± 0.09
3
Rabbit
0.69 ± 0.07
14
