J C Swinscoe1, E C Carlson. 1. Department of Anatomy and Cell Biology, University of North Dakota School of Medicine, Grand Forks 58202, USA.
Abstract
OBJECTIVE: To determine which interstitial collagen types may be present in bovine retinal microvessel extracellular matrix (ECM). METHODS: Dissociated bovine microvessels were treated with detergents and the resultant purified ECM monitored by transmission electron microscopy (TEM). Pepsin-extracted soluble ECM collagens were identified by Western blots. Collagens were further purified by neutral salt precipitation and carboxymethyl cellulose (CMC) chromatography before cyanogen bromide (CNBr) peptide mapping and two-dimensional peptide mapping of CMC-generated fractions. Interstitial collagens were localized by immunofluorescence on frozen sections. RESULTS: Transmission electron microscopy of detergent-purified microvessel ECM demonstrated numerous 10-50-nm collagen fibrils associated with basal laminae regardless of vessel diameter. Western blots showed that soluble ECM collagens were strongly positive for type II, moderate for type III, and weak for type I. CNBr peptide maps and two-dimensional maps of neutral salt and CMC-purified fractions confirmed the presence of type II collagen. Immunofluorescence localized type II collagen in large and small vessels of the retina. CONCLUSIONS: Type II collagen is an unexpected major component of bovine microvessel ECM, whereas types I and III are present in minor amounts. Type V collagen is also a substantial ECM component. Accordingly, all four types may contribute to a heterogeneous population of collagenous fibrils identified by TEM in intact isolated retinal microvessel ECM.
OBJECTIVE: To determine which interstitial collagen types may be present in bovine retinal microvessel extracellular matrix (ECM). METHODS: Dissociated bovine microvessels were treated with detergents and the resultant purified ECM monitored by transmission electron microscopy (TEM). Pepsin-extracted soluble ECM collagens were identified by Western blots. Collagens were further purified by neutral salt precipitation and carboxymethyl cellulose (CMC) chromatography before cyanogen bromide (CNBr) peptide mapping and two-dimensional peptide mapping of CMC-generated fractions. Interstitial collagens were localized by immunofluorescence on frozen sections. RESULTS: Transmission electron microscopy of detergent-purified microvessel ECM demonstrated numerous 10-50-nm collagen fibrils associated with basal laminae regardless of vessel diameter. Western blots showed that soluble ECM collagens were strongly positive for type II, moderate for type III, and weak for type I. CNBr peptide maps and two-dimensional maps of neutral salt and CMC-purified fractions confirmed the presence of type II collagen. Immunofluorescence localized type II collagen in large and small vessels of the retina. CONCLUSIONS: Type II collagen is an unexpected major component of bovine microvessel ECM, whereas types I and III are present in minor amounts. Type V collagen is also a substantial ECM component. Accordingly, all four types may contribute to a heterogeneous population of collagenous fibrils identified by TEM in intact isolated retinal microvessel ECM.