Bovine Vitreous Research Articles

Molecular mechanisms of posterior vitreous detachment

To determine a molecular mechanism for posterior vitreous detachment and contraction of vitreous gel, the effects of serum components on bovine vitreous were systematically studied. Incubation of vitreous gel with calf serum caused a contraction of the gel accompanied by the release of a waterlike liquid. The low molecular weight fraction (M(r) < 10,000) of serum was ineffective in this process, whereas incubation of the vitreous with the high molecular weight fraction resulted in changes in the gel structure similar to those caused by serum alone. Individually, transglutaminase (factor XIIIa) was found to be most effective in inducing gel contraction. The effect was more pronounced when transglutaminase was used with fibronectin. Since transglutaminase is known to promote collagen-collagen or collagen-fibronectin-collagen cross-links, the contraction of the vitreous in this case can be attributed to the formation of similar cross-links.

Isolation and characterization of the chains of type V/type XI collagen present in bovine vitreous

Previous studies show that the collagen fibrils of the mammalian vitreous humor are assembled largely from type II collagen with smaller amounts of type IX collagen and either type V or type XI collagen. In this paper, we report the separation of two chains of type V/type XI collagen from type II collagen by heparin-Sepharose chromatography. These chains were characterized by sequencing of selected cyanogen bromide or tryptic peptides with subsequent comparison of these sequences with cDNA-derived amino acid sequences of the alpha 1(V), alpha 1(XI), alpha 2(V), and alpha 2(XI) chains. The results show that vitreous fibrils are assembled from molecules containing the alpha 1(XI) and alpha 2(V) chains. These results, together with recent results from other laboratories, indicate that type V and type XI collagens are not separate collagen types but are part of a larger collagen family in which chains of both type V and type XI collagens participate in the formation of a variety of native molecules.

Age-related changes in the molecular properties of vitreous collagen.

To understand the molecular events underlying rheological vitreous changes, age-related changes in the molecular properties of bovine and human vitreous collagen were monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The adult bovine vitreous showed an increase in the high molecular-weight components of the collagen compared with the young samples. In addition, the apparent molecular size of the alpha-chain of the adult type II collagen was slightly larger than that of the young samples. The same tendency also was observed in human vitreous. The increase of high-molecular-weight components of the vitreous collagen could be attributed to intermolecular cross-links of the peptide chains.

An analysis of two fibril systems in the mammalian vitreous.

The vitreous of most mammals contains low concentrations of three polymeric macromolecules, collagen, hyaluronan and beaded fibrils. To determine whether the beaded fibrils may have a localized function, or whether all three of these polymers could contribute to the structure and/or strength of the vitreous gel, the distribution of collagen and beaded fibrils in the central and cortical regions of the bovine vitreous was studied. The polymers were separated by isopycnic centrifugation and analyzed to determine the protein ratios. Both collagen and beaded fibrils are present in each region, but the concentration of the beaded fibril is greatest in the posterior cortical volume.

Extraction and characterisation of the intact form of bovine vitreous type IX collagen

We provide the first biochemical characterisation of intact type IX collagen extracted from bovine vitreous. It possesses a shortened alpha 1(IX) chain (M(r) 64K) compared to its cartilage counterpart (M(r) 84K). All the vitreous type IX collagen is in a proteoglycan form, its glycosaminoglycan constituent being a chondroitin/dermatan sulphate component of M(r) 15-60K attached to the alpha 2(IX) chain. This contrasts with previous findings in chick vitreous where a very long glycosaminoglycan chain of M(r) approximately 350K was demonstrated.

Mammalian vitreous humor contains networks of hyaluronan molecules: Electron microscopic analysis using the hyaluronan-binding region (G1) of aggrecan and link protein

Vitreous humor from human, bovine, and chicken eyes was analyzed by rotary shadowing to characterize further the supramolecular organization of the gel-like matrix which forms this tissue. Extensive filamentous networks, distinct from collagen fibrils, were found in both human and bovine vitreous but not in chicken vitreous. The networks consisted of branching structures of various diameters, due to variable numbers of hyaluronan molecules being laterally associated with each other and apparently giving rise to a three-dimensional lattice. These networks could be decorated in a specific and regular manner by the hyaluronan-binding region called G1 purified from bovine nasal septum cartilage. The extent of decoration of hyaluronan was dependent on the relative concentration of G1. In the presence of an excess of G1 the networks were destabilized giving rise to individual unbranched hyaluronan chains of varying length that were saturated with G1. One or more globular proteins, as yet uncharacterized, were seen interacting with the hyaluronan networks, often at branch points. These proteins may serve to stabilize the three-dimensional structure of the matrix although highly ordered networks were also observed without globular proteins. Link protein, which also binds to hyaluronan, bound to the networks in a fashion clearly distinct from G1. Neither G1 nor link protein bound directly to human or bovine vitreous collagen fibrils. However, link protein did bind extensively to the glycos-aminoglycan coat of chicken vitreous collagen fibrils described previously (D. W. Wright, and R. Mayne J. Ultrastruct. Mol. Struct. Res. 100, 224–234, 1988), while G1 did not. Digestion of the chicken vitreous collagen fibrils with Streptomyces hyaluronidase did not result in the removal of the glycosaminoglycan coat of the collagen fibrils nor did it affect the binding of G1 or link protein to the fibrils, indicating that hyaluronan is not a component of this structure. These studies demonstrate that proteins with specific binding properties can be used as probes to investigate the structure of the native vitreous humor gel from several species and suggest that this method potentially can be used for structural studies of other connective tissue matrices.

Extraction of the intact form of type IX collagen from mammalian vitreous

The vitreous body is known to contain collagen types 11, IX and V/XI [ 1,21. Type IX collagen is also found in cartilage and in this tissue is aligned in a D-periodic distribution along the surface of 131, and covalently linked to [4], the major type I1 collagen fibrils. The cartilage form consists of three collagenous domains (Col 1 to 3), four non-collagenous domains (NC 1 to 4) and frequently has a chondroitin/dermatan sulphate chain(s) linked to the a2(IX) chain within the NC3 domain [5]. Type IX collagen is likely to be of importance in the interaction of the major collagen fibrils with other extracellular matrix components by virtue of both its glycosaminoglycan chain and its highly cationically charged (PI 9.7), aminoterminal NC4 domain (which together with the Col 3 domain protrudes from the fibril surface) [6]. Recently a new form of type IX collagen has been characterised from adult chick vitreous [7]. This form is also aligned along the surface of the major type 11 collagen fibrils in a D-periodic distribution, but has a much shortened NC4 domain and an extraordinarily long chondroitin sulphate chain (Mr -350,000), as compared to the cartilage form. The chick vitreous gel is morphologically 181 and biochemically different from mammalian vitreous 2,7 I , the major GAG component of the former being chondroitin sulphate, whereas in mammalian vitreous it is hyaluronan. Therefore the aim of this study was to extract the intact form of type IX collagen from bovine vitreous and compare it with that from bovine cartilage, to determine whether the mammalian vitreous collagen also possesses these unique properties. Intact collagens were extracted from pooled bovine vitreous samples using techniques previously established for cartilage 191. Briefly, following ultracentrifugation, intact collagens were extracted from the insoluble residue by sequential extraction with 0.05M Tris/HCl, pH 7.4, containing 1M-NaC1, 0.5M-and 4Mguanidinium chloride (GuHC1) respectively, each in the presence of proteinase inhibitors. The collagens in the 1M-NaC1 extracts were precipitated by increasing the salt concentration to 4.5M NaCl and the precipitate resuspended in 1M-NaC1. Aliquots of the purified 1M-NaC1, 0.5Mand 4M-GuHC1 extracts were dialysed against chondroitin ABC lyase buffer (O.05M-Tris/HCll 0.06M sodium acetate, pH8, with proteinase inhibitors) and half of each sample treated with chondroitin ABC lyase (0.015 units/ml) for 24 hrs at 18'C, to remove the GAG chain from the a2(IX) chain. Treated and untreated samples were subjected to SDS-PAGE under reducing and non-reducing conditions, and imunoblotted with polyclonal antibodies directed against type IX collagen. Results were compared with those of similarlyextracted cartilage type IX collagen. The NaC1, 0.5Mand 4M-GuHC1 extracts largely comprised of collagen types I1 and IX in their native intact triple-helical forms, that had not been crosslinked within the insoluble matrix of the vitreous. When analysed under non-reducing conditions, the extracted vitreous type IX collagen did not penetrate the resolving gel prior to chondroitin ABC lyase treatment but after digestion migrated as_ a high-molecularweight species of Mr 200K. This is in contrast to the unreduced cartilage type IX collagen that migrates with an Mr of 270K before, and 220K after, chondroitin ABC lyase digestion. The GAG chain of the vitreous collagen is therefore much larger than that of the cartilage form. When analysed under reducing conditions the vitreous collagen comprised a shorter a1 (IX) chain (Mr 64K), an apparently larger a3(IX) chain (Mr 78K), but following chondroitin ABC lyase treatment, a similar a2(IX) chain (Mr 67K) when compared to the corresponding chains of the cartilage collagen (Mr 84K, 72K and 66K respectively). Since the shorter al(1X) chain and larger GAG chain of the vitreous collagen are similarly found in both chick and bovine eyes, the structural diversity of type IX collagen is dependent to a greater extent on the tissue rather than animal species.

Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment epithelium

Insulin-like growth factors (IGFs) I and II are mitogenic polypeptides structurally homologous to insulin, which are thought to mediate important neurobiologic actions in the CNS. The purpose of this study was to determine if cultured bovine retinal pigment epithelial cells (RPE) express IGF receptors and secrete soluble IGF binding proteins, and to characterize these receptors and binding proteins. We also characterized the soluble IGF binding proteins present in juvenile and adult rat vitreous and serum, as well as those in fetal bovine vitreous and serum, in order to facilitate identification of the RPE IGF binding protein, and to determine potential destinations for this protein once produced. Affinity labeling was used to characterize insulin, IGF-I and IGF-II receptors. Western radioligand blotting and immunoprecipitation were used to characterize IGF binding proteins. We found that RPE cells in culture express virtually no insulin receptors, and only modest amounts of IGF-I receptors. IGF-II receptors were abundantly expressed. Additionally, RPE cells secrete a soluble IGF binding protein which is immunologically related to IGFBP-2, the primary IGF binding protein produced in the central nervous system. Bovine vitreous was found to contain a mixture of IGF binding proteins (IGFBP). The most prominent IGFBP in this mixture is immunologically related to IGFBP-2. Likewise, juvenile and adult rat vitreous contained only one IGF binding protein that was shown to be immunologically related to IGFBP-2. Juvenile rat vitreous contained more binding activity corresponding to IGFBP-2 than did adult vitreous, suggesting developmental regulation. These data suggest that IGFs and their binding proteins may have important, and as yet undefined, roles in retinal neurophysiology.

The origin of the intrinsic glycoproteins of the rabbit vitreous body: An immunohistochemical and autoradiographic study

A cartilage matrix glycoprotein (CMGP), previously identified in human and bovine vitreous, now has been found in the vitreous body of rabbits aged 1–22 months by immunohistochemical techniques. Epithelial cells of the inner layer of the ciliary epithelium contain material that has immunologic crossreactivity with a specific antibody to CMGP. These cells also secrete glycoproteins, as determined by autoradiography after intravitreal injection of [ 3H]fucose. Approximately 14 bands, representing intrinsic glycoproteins containing fucose residues, can be identified in fluorograms of SDS-polyacrylamide gels of vitreous bodies from 6- and 22-month-old rabbits. Fluorograms of gels of samples of vitreous and ciliary bodies from several time points after intravitreal injection of [ 3H]fucose reveal at least seven comigrating protein bands and also demonstrate turnover of the labeled ciliary body glycoproteins. These results suggest that the inner layer of the ciliary epithelium is the source of the glycoproteins of the vitreous body and that these glycoproteins undergo turnover, probably throughout the entire life of the animals.

Isolation and partial characterization of neutrophil elastase inhibitors from bovine vitreous and aorta.

Bovine vitreous body and aorta contain extractable leukocyte elastase inhibitors, which were purified by gel filtration and affinity chromatography on agarose-pancreatic elastase. The purified inhibitor preparation from aorta was resolved by polyacrylamide gel electrophoresis into a main band migrating slightly faster than commercial Trasylol and a more weakly stained band migrating close to chymotrypsinogen. The purified inhibitor preparation from both sources inhibited, in a competitive fashion, purified human leukocyte elastase and was ineffective against bovine trypsin and leukocyte cathepsin G or collagenase. These inhibitors from vitreous body and aorta were distinguishable by several criteria from serum inhibitors.

Partial purification of a 5.7K glycoprotein from bovine vitreous which inhibits both angiogenesis and collagenase activity

An inhibitor of angiogenic activity similar to that described by Raymond and Jacobson (1) has been partially purified and shown to be a glycoprotein of molecular mass 5,700. This inhibitor gave rise to an avascular zone on the chick vitelline plexus and also negated the action of a low M r angiogenic factor. When the angiogenic inhibitor was incubated with mammalian collagenase it inhibited the enzyme activity by nearly 70%. The relevance of these findings to the role of collagenase in angiogenesis is discussed.

Regulation of cell growth by vitreous humour.

Extracts of normal vitreous have been found to inhibit angiogenesis in two animal models: tumour-induced neovascularization in the rabbit corneal micropocket and retinal extract-induced angiogenesis in the chick chorioallantoic membrane assay. Using in vitro assays, we have found recently that an extract of bovine vitreous, free of hyaluronic acid, inhibits proliferation of cells in the aortic wall, i.e. endothelium and smooth muscle cells, as well as capillary and corneal endothelium. The inhibition is dose-dependent, as determined by either cell count or [3H]thymidine incorporation, and not due to cytotoxicity, as demonstrated with a double-label thymidine assay. The inhibitor is trypsin-sensitive and heat-stable (95 degrees C for 10 min). Conversely, proliferation of pericytes, lens epithelium and fibroblasts (dermal and corneal) was stimulated by the vitreous extract. This mitogenic activity was heat-labile. Growth of pigment epithelium and several tumour cell lines was unaffected. The data demonstrate that normal vitreous contains a heat-stable growth inhibitor specific for endothelium and smooth muscle cells, and a non-specific heat-labile mitogen. The paradoxical effect of this antiangiogenic factor on arterial and capillary contractile cells, smooth muscle and pericytes, suggests a basic difference in the regulation of the two vasculatures. The results suggest that a substance in normal vitreous may be important in controlling neovascularization that results from diabetic and other retinopathies, and could be useful for inhibiting tumour-induced angiogenesis.

Glycosaminoglycan metabolism of cultured fibroblasts from bovine vitreous.

Glycosaminoglycan metabolism of cultured vitreal cells of bovine origin was investigated with the following results: Cultured vitreal cells distributed the synthesized macromolecules after [35S] sulfate incorporation into the culture medium (extracellular pool), the membrane-bound proteoglycans (pericellular pool), and within the cells (intraocular pool). No extracellular plateau was reached after 3 days; the pericellular and intracellular pools showed a steady state of radioactivity after 48 and 24 h, respectively. Pulse-chase experiments exhibited a rapid turnover of intracellular polysaccharides and a slower metabolism of pericellular polymers. Pinocytosis, adsorption and degradation of vitreal and corneal cell secretion by vitreal cells were observed to a comparable extent. Chondroitin sulfate isomers and heparan sulfate could be detected in minor proportions. The relevance of glycosaminoglycan metabolism in the replacement of cultured vitreal cells into the vitreous cavity is discussed.

In vitro effect of hyaluronate on lysosomal enzyme activities in the bovine ciliary body and iris

The effect of hyaluronate on lysosomal enzyme activities in bovine ciliary body and iris was studied in vitro. Hyaluronates from both the human umbilical cord and bovine vitreous inhibited the activities of cathepsin b and acid phosphatase. Cathepsin d, β-N-acetyl-glucosaminidase, β- d-glucuronidase and α- l-fucosidase activities were not affected by the hyaluronate.

The molecular weight of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle eyes

The molecular weight distribution of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle has been determined by gel chromatography. The eluate from the column was monitored by a radioassay, whereby the molecular weight distribution of 15–20 μg samples could be analysed. Control experiments were carried out with radioactively-labelled hyaluronate added to bovine material to estimate the degradation of the polymer during handling of the ocular fluids. It was shown that in vitro degradation does not appreciably affect the results. The analyses show a considerable polydispersity of the hyaluronate preparations. There is also a marked variation in the degree of polymerization between the two species. Hyaluronate from rabbit vitreous has a weight-average molecular weight ( M w ) of 2–3 × 10 6 while adult bovine vitreous displays a value of about 5–8 × 10 5. The hyaluronate of bovine vitreous varies with age. In newborn calf, a value of 3 × 10 6 was registered. This value dropped to about 5 × 10 5 in old cattle. The hyaluronate in the aqueous humour of rabbit showed a considerably higher molecular weight than that of the vitreous indicating that part of the hyaluronate in the anterior segment originates elsewhere than the vitreous. The differences between hyaluronate from the aqueous humour of adult cattle and that from the vitreous were more complex. As with the rabbit, a relatively large proportion of hyaluronate in the aqueous humour was of high-molecular weight, but, in contrast, the aqueous humour also contained material which had a lower degree of polymerization than the hyaluronate in the vitreous. The proportion of high-molecular weight material in bovine aqueous humour seemed to be lower in the summer than in the winter.

A new chromatographic method for the fractionation of hyaluronic acid

Sodium hyaluronate from rooster comb, umbilical cord, bovine vitreous and a special commercial hyaluronic acid preparation, “Healon R” have been fractionated at 4°C on DEAE-Sephacel columns using salt gradients from 0→0.4M NaCl. All samples examined displayed a high degree of polydispersity. The number of well separated fractions observed in these chromatograms ranges from 10–30 depending on the source of the material. A molecular weight distribution effect does not seem to be the main factor controlling this fractionation as no differences could be observed when the limiting viscosity numbers (η) for numerous fractions isolated from the same sample were compared with each other. As no galactosamine, sulfate or phosphate is found directly linked to these fractions, it is suggested that disaggregation of an hyaluronate molecular aggregate could be the basis for this fractionation.

Vitreous structure. VI: Age-related changes in the thermal stability and crosslinks of vitreous, articular cartilage and tendon collagens

Thermal stability studies were performed on samples of fetal, young and adult bovine vitreous, articular cartilage and tendon collagens. The major age-related change observed was a decrease in the quantity of tendon collagen solubilized by heat alone, whereas very small quantities of vitreous and articular cartilage collagens were rendered soluble by heat alone at all ages. Vitreous collagen and tendon collagens had similar heat-denaturation profiles, although at all ages vitreous was slightly more stable than tendon. The denaturation of articular cartilage collagen occurred at significantly higher temperatures that that of vitreous and tendon collagens. The relative proportions of reducible crosslinks in fetal and adult vitreous, articular cartilage and tendon collagens were also determined. In all fetal collagens dihydrolysinonorleucine was the major reduced crosslink detected. Fetal articular cartilage and tendon collagen had about twice the amount of dihydroxylysinonorleucine as vitreous collagen but when the adult samples were compared vitreous collagen contained a 3.4-times higher content of dihydroxylysinonorleucine than articular cartilage and only trace amounts of this crosslink were detected in tendon collagen. The 3-hydroxypyridinium content of adult vitreous collagen was also determined and was found to be about half that of articular cartilage collagen. These results indicate that there is maturation of hydroxylysino-5-ketonorleucine to 3-hydroxypyridinium crosslinks in a similar manner to that proposed for cartilage collagen. The presence of dihydroxylysinonorleucine crosslinks in the adult vitreous collagen, however, indicates that either the maturation process is much slower or incomplete or that there is a much more active synthesis of collagen containing immature hydroxylysino-5-ketonorleucine crosslinks in the vitreous than in articular cartilage.

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

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.

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 fine structure of the hyaloid arteriole in bovine vitreous

The purpose of this study is the descriptive analysis of the morphology of the bovine fetal and neonatal hyaloid arteriole as seen with the transmission electron microscope. Gross and minute details of intra- and extracellular formations in the wall of this arteriole are noted during its developing and atrophying stages. During the last period of the first trimester and through mid-gestation the endothelial cells and the primitive myocytes have the ultrastructural characteristics of actively seretory cells. It is presumed that they are involved in the synthesis of the elaborate lamellar systems of the extracellular matrix. Observations are made on the appearance and the staining characteristics of the elaborate multi-layered basal lamina-like membranes located between the intima and muscularis and between the several muscle cell layers. The composition and eventual fate of the non-degenerated portion of the hyaloid arteriole is discussed and its relation to the wall of Cloquet's canal is postulated.