Inhibition of scleral proteoglycan synthesis blocks deprivation-induced axial elongation in chicks.

A specific inhibitor of proteoglycan synthesis was administered to chicks undergoing the development of form deprivation myopia in order to test the hypothesis that increases in proteoglycan synthesis are responsible for normal and/or deprivation-induced ocular elongation in chicks. Chicks undergoing monocular form deprivation were treated with p-nitrophenyl-beta-D-xylopyranoside (beta-xyloside) via i.p. injection every 8 hr for 5-11 days. Ocular measurements were made at the end of the experiment using high frequency A-scan ultrasound in conjunction with a LabView (v. 5.0) analysis program. Following ultrasound measurements, sclera were isolated and proteoglycans characterized by Sepharose CL-2B and Western blot analyses. Preliminary studies indicated that i.p. administration of beta-xyloside maximally inhibited sulfate incorporation into proteoglycans 8 hr after administration. Beta-xyloside treatment resulted in a significant reduction in the axial length, vitreous chamber depth, and rate of axial elongation of form deprived eyes as compared with form deprived eyes from vehicle treated chicks (P < 0.01, P < 0.05, P < 0.05, respectively). No significant differences were detected in anterior chamber depth, lens thickness, choroid thickness or retina thickness in form deprived eyes of beta-xyloside treated chicks as compared with that of vehicle controls. No significant differences were detected in contralateral non-deprived fellow eyes between beta-xyloside treated and vehicle treated chicks for any ocular measurement. Analysis of proteoglycans indicated that the xyloside treatment resulted in the accumulation of smaller proteoglycans due, in part, to the presence of underglycosylated aggrecan within the scleral matrix. These results indicate that interruption of normal scleral proteoglycan synthesis inhibits form deprivation-induced ocular elongation, supporting the hypothesis that scleral proteoglycan synthesis and accumulation are largely responsible for increases in axial length in form deprived chick eyes.

Identification of choroidal ovotransferrin as a potential ocular growth regulator.

PURPOSE: In an effort to identify choroidal factors potentially involved in the regulation of ocular growth, proteins released into culture medium of organ-cultured choroids were compared between control eyes and eyes recovering from form deprivation myopia. METHODS: The choroids were obtained from the posterior poles of control and recovering chick eyes, and placed into organ culture containing ( 35)S-methionine/(35)S-cysteine. Culture medium was collected after 24 hours and proteins were separated and identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography, immunoprecipitation, western blot analysis and by amino acid sequencing. Choroidal proteins were tested for their effect on scleral proteoglycan synthesis by measuring (35)SO( 4) incorporation into scleral glycosaminoglycans (GAG) in vitro. Choroidal thickness and axial elongation were measured in control and recovering eyes using high frequency A-scan ultrasound. RESULTS: The synthesis of an 80 kD protein was greatly increased in the choroids of recovering eyes compared with those of control eyes. Amino acid sequencing and immunoprecipitation indicated that the newly synthesized 80 kD protein was ovotransferrin (transferrin, conalbumin). Ovotransferrin release into the culture medium by isolated recovering choroids was associated with a decrease in the rate of axial elongation in recovering eyes. When tested in vitro, ovotransferrin (500 ng/micro) inhibited scleral proteoglycan synthesis in the sclera by 62% in a dose-dependent manner. CONCLUSIONS: Chick choroids of recovering eyes synthesize and release ovotransferrin during the recovery from form deprivation myopia. Ovotransferrin significantly inhibited proteoglycan synthesis by the sclera, indicating that ovotransferrin may play a role in slowing the rate of vitreous chamber elongation and facilitating the recovery from induced myopia.

Proteoglycan turnover in the sclera of normal and experimentally myopic chick eyes.

PURPOSE: The turnover of chick scleral proteoglycans from control and form-vision deprived (myopic) eyes was compared in vivo and in explant cultures to determine whether proteoglycan degradation is altered during the development of myopia and to characterize the mechanism of proteoglycan turnover in the sclera. METHODS: Seven-day-old chicks were radiolabeled via an intraperitoneal injection of 35SO4, and monocular form deprivation was induced 48 hours later. After 1, 2, and 3 weeks of form deprivation, birds were killed, and the amount of 35SO4-proteoglycans remaining in different scleral regions was measured in control and deprived eyes. Posterior sclera were also radiolabeled in organ culture containing 35SO4, and radiolabeled scleral proteoglycans were chased into unlabeled medium for 0 to 11 days. 35SO4-labeled proteoglycans within the scleral matrix and those released into the medium were characterized by Sepharose CL-2B chromatography and western blot analysis. RESULTS: The biological half-life of scleral proteoglycans was significantly shorter within the posterior pole of form-deprived eyes (t1/2 = 7.212 days) compared with the same region of control eyes (t1/2 = 9.619 days; P < 0.001), whereas no differences in turnover rates were seen in the anterior sclera or equatorial sclera. When posterior scleral punches were placed in organ culture, 35SO4-labeled proteoglycan turnover rates were similar for control and form-deprived eyes. Chromatographic and western blot analyses indicated that approximately 80% of the total 35SO4 within the posterior sclera is incorporated into the aggrecan. Western blot analyses of aggrecan core protein released into the medium by control and form-deprived scleral punches indicated that the core protein was degraded into a series of smaller fragments of Mr = 102 to 220 kDa. A specific antiserum (anti-FVDIPEN) detected the presence of a 50-kDa C-terminal aggrecan fragment released into the medium, which was generated by the action of the matrix metalloproteinase gelatinase A and/or stromelysin. CONCLUSIONS: The turnover rate of 35SO4-labeled scleral proteoglycans is vision dependent and is accelerated in the posterior sclera of chick eyes during the development of experimental myopia. The loss of proteoglycans from the scleral matrix involves proteolytic cleavage at various sites along the aggrecan core protein through the action, at least in part, of gelatinase A and/or stromelysin.