Type I collagen-integrin interactions in angiogenesis
Angiogenesis, the formation of new blood vessels by branching from existing vessels, is the primary mechanism for vascular extension in adults. Following injury or hypoxia, endothelial cells proteolytically degrade their basement membranes and encounter an interstitial provisional matrix of diverse composition. Type I collagen, a ubiquitous interstitial extracellular matrix (ECM) molecule and its primary receptor, the α2β1 integrin, are heavily implicated in angiogenesis. We have previously shown that type I collagen gels rapidly induce angiogenesis in human umbilical vein endothelial cell (HUVEC) monolayers in an α2β1 integrin-dependent manner. Analysis of protein content showed that these gels predominantly contained polymerized collagen. Collagen polymerization in contact with the cell monolayer was not required for angiogenesis, and agarose gels failed to induce angiogenesis. The α2β1 integrin has two ligand-binding conformations, intermediate and activated. Integrin clustering further influences ligation and signaling. By immunocytochemistry, α2β1 integrins exhibited a widespread distribution concentrated along cell-cell borders in HUVEC monolayers. Soon after angiogenesis was induced by collagen, α2β1 integrins accumulated on the apical cell surface, aligning with collagen fibers. Activated integrins exhibited a widespread distribution that was not altered by the collagen gel. Antibody-coated beads designed to cluster α2β1 integrins induced tube formation in the absence of collagen; antibodies presented in solution were inactive. Antibody-coated beads targeting αvβ3 integrins and PECAM-1 induced tube formation; while anti-α1 antibody- or BSA-coated beads did not. A map of functional domains and human mutations on type I collagen suggests the existence of a Cell Interaction Domain that resides in a critical region of the fibril, encompassing the central α2β1 integrin binding site, the vertebrate collagenase cleavage site, and a nearby glycation site. Non-enzymatic glycation of type I collagen has been shown to affect cell interactions. However, a three-dimensional model of integrin-collagen interactions suggests that the nearby glycation site is unlikely to affect collagen-integrin binding directly. Glycation reduced collagen binding for α1I, α1β1, and αvβ3 integrins in a cell-free system but did not alter the size or the appearance of collagen fibrils. Thus, the Cell Interaction Domain of type I collagen may mediate important aspects of integrin biology including ligation, clustering, and angiogenesis.