Expression of capillary basement membrane components during sequential phases of wound angiogenesis

Before capillaries sprout to form new vessels in a wound, the endothelial cells are sequestered from the surrounding stromal or provisional matrix by a well organized protein envelope called the basement membrane (BM). After breaching the BM, endothelial cells are exposed to the wound provisional matrix and begin to migrate and proliferate. Endothelial derived basement membrane proteins and molecules of the provisional matrix are mutually accessible to the endothelial cell surface during migration. Eventually, new capillaries again segregate in a formed envelope of basement membrane and resume a tubular morphology. Endothelial cell recognition of the architecture and concentration of basement membrane ligands appears to be an important determinant of capillary morphology during angiogenesis. In this study, we characterized the molecular composition, expression and steady-state transcript levels of BM proteins during sequential stages of wound angiogenesis. Specific analysis of rat capillary BM transcripts was achieved by employing a space-filling wound model which did not have non-capillary BM. Invading capillaries appeared between days 3 and 5 and matured by day 12. Occasionally, vessels larger than capillaries were observed to form by a process resembling vasculogenesis. Steady-state transcript levels for subunits of all major BM proteins studied were readily measured by day 3, and laminin and type IV collagen immunostaining were evident at the periphery of all vessels studied. From vessel initiation to regression, the transcript levels most changed were the alpha 1 and alpha 2 transcripts of type IV collagen; after an early peak, they exhibited a sharp three-fold decline as the response progressed. Conversely, entactin and laminin subunits did not decline as the response progressed, suggesting an increasing ratio of expression relative to type IV collagen. Perlecan expression was inconsistent, but it appeared to decline during the late phase of the response. Laminin beta 1 and gamma 1, but not alpha 1 or beta 2, transcripts were expressed by forming capillaries, providing evidence that the laminin 1, 3, 4 and 7 isotypes are not expressed by growing capillaries. These results also demonstrate that the steady-state ratios of type IV collagen transcripts to laminin and entactin transcripts are greatest during the early proliferative-migratory phase of angiogenesis but decrease significantly in later phases, when vessel maturation and tube formation predominate.