Bernard Jacobson

Isolation and identification of stimulatory and inhibitory cell growth factors in bovine vitreous.

Abstract Using Bio-Gel P-10 chromatography and an endothelial cell assay system, we have identified and partially characterized stimulatory and inhibitory cell growth factors in adult, newborn and fetal bovine vitreous. The sensitivity of these two factors to boiling and trypsin treatment suggest that they are proteins. The stimulatory factor appears near the void volume (mol. wt cut-off 13000) and increases endothelial and smooth muscle cell, as well as fibroblast proliferation. It appears related to ischemic and/or necrotic retinal tissue. The inhibitory factor, estimated to be of 6200 mol. wt, appears in the retarded volume and inhibits endothelial and smooth muscle cells to a greater degree than fibroblasts. It appears native to the vitreous and may be a product of hyalocytes which, in culture, produce an endothelial cell inhibitory factor.

The synthesis of hyaluronic acid in vitreous: I. Soluble and particulate transferases in hyalocytes

The cortical tissue layer of the vitreous, which is contiguous to the retina and ciliary body, contains a uniform population of cells called hyalocytes. Homogenates of hyalocytes isolated from the posterior vitreous were separated into soluble and particulate fractions by ultra-centrifugation, and both fractions were found to contain nucleotide sugar transferase activity. The soluble hyalocyte extract contains endogenous hyaluronic acid, which acts as acceptor of radioactivity from UDP-[ 14 ]C]GlcUA and UDP-[ 14 C]GlcNAc. When endogenous hyaluronic acid is removed and no exogenous acceptor added, the soluble enzyme is able to synthesize oligosaccharide from the added nucleotide sugars, both of which are required for optimal incorporation of radioactivity. The particulate transferase is also able to catalyze the incorporation of radioactivity from UDP-[ 14 C]GlcUA into chromatographically immobile material. Although the soluble transferase showed the same activity whether synthesizing new oligosaccharide or transferring sugar residues to polymeric hyaluronic acid, the particulate transferase evidenced a greater capacity for oligosaccharide synthesis than for catalyzing transfer to polymer.

Inhibition of vascular endothelial cell growth and trypsin activity by vitreous

Vitreous from bovine, human and chick embryo has been found to contain a trypsin inhibitory activity. Chymotrypsin-inhibitory activity was also identified in bovine and chick embryo vitreous. Following either ultrafiltration or Bio Gel P-10 chromatography, these activities appear in fractions having a molecular weight greater than 10000 MW (ultrafiltration) or greater than 13000 MW (P-10 void volume), and are separable from low molecular weight aortic endothelial cell growth inhibitory activity present either in the ultrafiltrate or P-10 retarded volume. Treatment of the trypsin inhibitory fraction with hyaluronidase had no effect on trypsin inhibition, nor did addition of hyaluronic acid inhibit trypsin. Chick embryo vitreous and hyalocyte-conditioned medium were found to contain aortic endothelial cell growth inhibitory activity in both the void volume and retarded volume fractions following Bio Gel P-10 chromatography. Both the 6200 MW bovine vitreous endothelial cell growth inhibitor and the high molecular weight chick embryo vitreous endothelial cell growth inhibitor (greater than 13000 MW) were similar, in that most of the activity did not bind to heparin linked to Sepharose CL-6B.

Lysosomal enzymes associated with vitreous hyalocyte granules. 1. Intracellular distribution patterns of enzymes

Utilizing a combination of centrifugal and enzymatic digestion techniques, a method was developed for the isolation of the subcellular granules of calf vitreous hyalocytes. The granules of varying size were separated according to their sedimentation properties into “nuclear”, “granular” and “mitochondrial-microsomal” fractions. The association of lysosomal enzyme activity with the granules was first indicated by the histochemical demonstration of acid phosphatase activity, followed by in vitro colorimetric determinations for the hydrolytic enzymes acid phosphatase and β-glucuronidase, and for alkaline phosphatase. Enzymatic activities were present only after the cells were mechanically disrupted, and the various fractions had distinctive differences in their enzyme distribution. A structure-linked latency of enzyme activity was demonstrated in the individual fractions, maximum activity being dependent on fragmentation of the granular membrane. Confirmation of the composition and morphology of the separated constituents at each step of the procedures was obtained by light and electron microscopic studies.

The synthesis of hyaluronic acid in vitreous *: II. The presence of soluble transferase and nucleotide sugar in the acellular vitreous gel

The acellular portion of bovine vitreous has been found to contain a transferase that catalyzes the incorporation of radioactivity from UDP-[ 14 C]GlcUA into endogenous hyaluronic acid and oligosaccharide. The transferase was prepared by ammonium sulfate precipitation of protein from the vitreous gel. The gel preparations were isolated by two methods that would preclude contamination of the gel by enzymes resulting from broken cells. A nucleotide pyrophosphatase activity also was identified. Injection of [1- 14 C]glucosamine into the anterior chamber of calf eyes led to the isolation of labeled UDP- N -acetylhexosamine from the acellular central vitreous. Analysis indicated the radioactive hexosamine to be glucosamine rather than galactosamine. The large excess of unlabeled glucosamine found, over and above that added as chromatographic standard, indicates that UDP- N -acetylglucosamine is endogenous to the vitreous gel. In contrast, no galactosamine over and above the amount added as standard was found. Oligosaccharides labeled in the glucosamine and glucuronoic moieties were isolated from the central vitreous 1 hr and 25 min after injection of [ 14 C]glucose and [ 14 C]glucosamine into the anterior chamber of the eye. Radioactive polysaccharide was not found in this preparation. The experimental results may be interpreted to indicate the initiation of hyaluronic acid synthesis by the intracellular production of low molecular weight material, followed by the continuation of this process in the extracellular matrix.

Degradation of glycosaminoglycans by extracts of calf vitreous hyalocytes

The ability of extracts of calf vitreous hyalocytes to catalyze the degradation of glycosaminoglycans was studied by incubation with radioactively labeled substrates. The degradation of the polymeric substrates to lower molecular weight products was assayed by three methods: (1) paper chromatographic separation of low molecular weight, mobile digestion products from undigested, high molecular weight material which remains at or near the origin; (2) loss of the ability of the glycosaminoglycan to be precipitated by cetylpyridinium chloride; (3) gel chromatography to separate low molecular weight digestion products, which appear in the retarded volume, from undegraded, high molecular weight material, which is eluted in the void volume. The acidic pH optimum of the reaction suggests a lysosomal origin of the enzyme activity.

Synthesis and degradation of UDP-glucuronic acid in the hyalocytes of calf vitreous *

Uridine 5′-diphosphoglucose dehydrogenase, which catalyzes the oxidation of uridine 5′-diphosphoglucose to uridine 5′-diphosphoglucuronic acid, has been identified in extracts of calf vitreous hyalocytes. In addition, an enzyme system that degrades uridine 5′-diphosphoglucuronic acid to uridine and free glucuronic acid also has been identified. The role of these enzymes in vitreous metabolism is discussed.