Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Abstract

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.

Development of a three-dimensional model of lung cancer using cultured transformed lung cells

“…32 Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. 40 These findings confirm the notion that a substrate of laminin and collagen type IV stimulates the in vitro expression of differentiated smooth muscle traits at a higher level than does a substrate of fibronectin. 41 We used this marker to help identify endothelial cell differentiation.…”

Cellular differentiation in three-dimensional lung cell cultures

“…29 Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. 36 These findings confirm the notion that a substrate of laminin and collagen type IV stimulates the in vitro expression of differentiated smooth muscle traits at a higher level than does a substrate of fibronectin. 37 We used this marker to help identify endothelial cell differentiation.…”

Disruption of the subendothelial basement membrane during neutrophil diapedesis in an in vitro construct of a blood vessel wall.

“…In vivo, emigrating neutrophils invade the endothelial cell layer and pause atop the basement membrane 3. Although a similar pattern has been reported in extracts ofsubendothelial matrices synthesized by human microvascular endothelial cells, laminin, an additional basement membrane component, cannot be readily detected on SDS gels in these or other systems (40,41,48 until pseudopodia begin to extend through the matrix (1,(14)(15)(16)(17)(18). To determine whether the basement membrane synthesized by cultured endothelial cells retarded neutrophil movement in a similar fashion, the ability of emigrating neutrophils to transmigrate the vessel wall construct was monitored.…”