M. Cristina Kenney

The presence of EC collagen and type IV collagen in bovine Descemet's membranes

When bovine Descemets membranes (DMs) were characterized after limited pepsinization the major component in DM was found to be endothelial cell (EC) collagen. Phenol extractions of the undigested pepsin residue recovered only type IV collagen. This study provides evidence that EC collagen may be produced by corneal endothelial cells in vivo.

Modulation of rabbit keratocyte production of collagen, sulfated glycosaminoglycans and fibronectin by retinol and retinoic acid

This study elucidates the biochemical response of rabbit corneal keratocytes (fibroblasts) to retinol and retinoic acid in their production of collagen, fibronectin, sulfated glycosaminoglycans, collagenase, and [3H]thymidine incorporation. The morphologic appearance of cultured keratocytes was not altered by retinoid treatment. Collagen production and [3H]thymidine incorporation demonstrated a parallel decline in response to retinoids. Collagen type was unaffected as was collagenase activity. Retinoids had minimal effect on cell layer-associated 35S-labeled glycosaminoglycans, however medium-soluble 35S-glycosaminoglycans were increased. The most dramatic effect was in fibronectin synthesis which was increased 2-3-fold. These data demonstrate that rabbit keratocytes alter their biosynthesis of extracellular matrices in response to retinoids. This may be significant in corneal pathology, since the delicate balance of these extracellular macromolecules is responsible for corneal integrity and stability.

Identification of collagens isolated from bovine Descemet's membrane

In this study collagens were isolated and identified from a morphologically pure preparation of bovine Descemets membrane (DM) which was obtained by gentle scraping of the cornea, sieving and subsequent treatment with detergents. An alternative procedure of DM isolation with forceps resulted in stromal contamination of the preparation as verified by light and transmission electron microscopy, and gel electrophoresis. The amino acid profile of collagens isolated by pepsinization and salt precipitation from the pure sample was similar to the analysis of the intact bovine DM. Polyacrylamide gel electrophoresis of this collagen under non-reducing conditions resulted in five major bands: 300 000 daltons (300 K), 200 000 daltons (200 K), 100 000 daltons (100 K) and lower molecular weight fractions (50 K1 and 50 K2). Individual collagen chains were isolated from preparative polyacrylamide gels and characterized by 125I two dimensional peptide mapping patterns. This data suggests that (1) the majority of collagen fragments seen in bovine DM pepsin supernatant are derived from a single genetically distinct collagen molecule, and (2) that type I and V collagens are not major components of bovine DM.

Vitamin A augments collagen production by corneal endothelial cells

When isolated confluent corneal endothelial cells were cultured in delipidized serum, a marked reduction in collagen production was observed. Supplementation of such cultures with vitamin A as either retinol or retinoic acid at concentrations of 10(-6)-10(-7) M was capable of significantly increasing collagen production. In addition, when cultured in normal (non-delipidized) serum, both retinol and retinoic acid were capable of further increasing collagen production by corneal endothelial cells. Such augmentation of collagen production was relatively specific as total protein synthesis was not altered to the same extent, nor was it merely a reflection of changes in total cell number, as such cell numbers were similar in all treatment groups.

Stability of the collagen phenotype and decreased collagen production in serial subcultures of rabbit corneal endothelial cells

The effect of serial subculture upon collagen biosynthesis by rabbit corneal endothelial cells has been studied to determine if the collagen phenotype is stable. Nearly confluent first through fourth endothelial cell cultures were labeled for 48 hr with [3H]proline and newly synthesized radioactive collagens were treated with pepsin and purified by neutral and acid salt precipitation. Cellular production of collagen (c/min/μg DNA) was greatest in primary cultures and decreased with subsequent passage. No differences were observed in the distribution of radioactive peptides separated by sodium dodecyl sulfate gel electrophoresis. This suggests that the collagen phenotype is qualitatively stable during the first four serial cultures. The [3H]proline content and the electrophoretic patterns of these collagens clearly distinguish them from those produced by fetal rabbit fibroblasts (i.e. type I and III). In summary, the progeny of rabbit corneal endothelial cells produced decreased amounts of qualitatively similar type(s) of collagen synthesized by their parent primary cultures.

Production of collagen by cells isolated from a retrocorneal fibrous membrane rabbit model

The morphological and biosynthetic characteristics of cells from an experimentally induced rabbit retrocorneal fibrous membrane (RCFM) model were investigated. By transmission electron microscopy the cells within the RCFM demonstrated overlapping cytoplasmic processes and intercellular junctions, neither of which are fibroblast-like characteristics. The extracellular matrix within the RCFM had a fibrillar and amorphous component. Collagenous biosynthetic products of primary cultures of RCFM cells were compared to normal corneal endothelial cells, which produce mainly type IV collagen and a small amount of type V collagen, and fibroblasts, which produce types I, III and V collagens. The collagenous components produced by the RCFM cells were a combination of types I, V and low molecular weight fragments of type IV collagen. Therefore, these morphological and biochemical data suggest that RCFM cells are a type of modified corneal endothelial cell that produce collagens distinctly different from normal corneal endothelial cells.

Altered rabbit endothelial cell phenotypic expression in response to human fibronectin.

Abstract When rabbit corneal endothelial cells were cultured in excess concentrations of human fibronectin an altered phenotypic expression was observed. Cell appearance was changed radically and collagen synthesis was specifically inhibited in a dose-response fashion. This study provides further evidence that fibronectin may be one of the developmental signals which act a molecular level and is capable of interspecies activity.

Isolation and identification of basement membrane collagen purified from dissociated rabbit renal tubules

Abstract In the present study collagens were isolated and identified from morphologically pure basement membrane material. Preparations of rabbit renal tubules devoid of contaminating glomeruli were obtained by homogenization and sieving of kidney cortices. Cellular material was removed by sequential detergent solubilization and the purity of the resultant tubular basement membrane was verified by transmission electron microscopy. The collagenous component of this ultrastructurally pure starting material was isolated by limited pepsinization and salt precipitation. Polyacrylamide gel electrophoresis of this collagen under nonreducing conditions resulted in four major bands: 300,000 (γ component), 100,000 (100K), 80,000 (80K), and 50,000 (50K). Individual collagen fractions of each of these molecular weights were then isolated from preparative polyacrylamide gels. Identification by their electrophoretic properties and cyanogen bromide peptide patterns leads us to believe that: (i) the 100K is composed of the C chain of type IV collagen; (ii) the 80K and 50K are derived from the genetically distinct D chain of type IV collagen; (iii) the γ component is structurally related to the 100K, 80K, and 50K; and (iv) A and B chains (type V collagen) are not major components of rabbit renal tubular basement membranes.

Structural Properties of Type I Collagen Isolated from Chickens with Scoliosis

This study examines biochemically the Type I collagen isolated from skin of chickens that develop idiopathic scoliosis. Previous studies indicate a defect in collagen exists in these chickens. Alpha 1 (I) and alpha 2 chains were separated by gel filtration and carboxymethyl cellulose column chromatography and were then subjected to the analytical techniques of sodium dodecyl sulfate gel electrophoresis, Staphylococcus aureus V8 protease digestion, cyanogen bromide peptide mapping and amino acid analyses. In all categories, the scoliotic alpha 1 (I) and alpha 2 chains were identical to alpha chains isolated from normal chickens. These data suggest that the altered properties of collagen solubility and connective tissue stress relaxation seen in these scoliotic chickens are not a manifestation of an altered primary structure of the alpha chains or post-translational modification affecting chromatographic elution profiles or electrophoretic migration patterns.

Fibronectin initiates fibrin production by rabbit corneal endothelial cells in vitro

Confluent rabbit corneal endothelial cells incubated in the absence of serum do not produce fibrinogen. When exogenous fibronectin is added to these cultures, fibrinogen production is observed. Fibronectin concentrations stimulate fibrinogen synthesis by endothelial cells in a dose-response fashion. This direct interaction of fibronectin and fibrinogen may be important in both wound healing processes and pathological states.