Cell Surface-bound Collagenase-1 and Focal Substrate Degradation Stimulate the Rear Release of Motile Vascular Smooth Muscle Cells

Abstract

To migrate in the vessel wall, smooth muscle cells (SMCs) must contend with abundant type I collagen. We investigated the mechanisms used by human SMCs to efficiently migrate on type I collagen, following stimulation with fibroblast growth factor-2 (FGF-2). FGF-2-stimulated migration was inhibited by a hydroxamic acid inhibitor of matrix metalloproteinases and by a neutralizing anti-collagenase-1 antibody. Moreover, migration speed of SMCs plated on mutant collagenase-resistant type I collagen was not increased by FGF-2. Time-lapse video analysis of unstimulated SMCs migrating on collagen revealed discrete phases of leading edge membrane extension and rear retraction, the latter often after rupture of an elongated tail. FGF-2 stimulation yielded a more synchronous, gliding motion with a collagenase-1-mediated decrease in tail ripping. Surface labeling of SMCs with biotin followed by immunoprecipitation revealed that a proportion of active collagenase-1, expressed in response to FGF-2, was bound to the plasma membrane. Pericellular collagen substrate cleavage was verified by immunostaining for neoepitopes generated by collagenase-1 action and was localized to discrete zones beneath the cell tail and the leading edge. These results identify a novel mechanism by which SMC migration on collagen is enhanced, whereby rear release from the substrate is orchestrated by the localized actions of membrane-bound collagenase-1. Migration of vascular smooth muscle cells (SMCs)1 is an important feature of arterial diseases such as atherosclerosis (1). In response to signals within the diseased vessel wall, SMCs translocate from the arterial media to the intima, or from one region in the intima to another, where they accumulate and elaborate extracellular matrix (ECM). These processes can lead to narrowing of the vascular lumen and organ ischemia.In order to migrate in the vessel wall, SMCs must contend with the dense ECM. There is good evidence that SMC migration and vascular lesion formation are accompanied by expression of ECM-degrading enzymes, including matrix metalloproteinases (MMPs, matrixins) (2-6). Furthermore, SMC migration in the artery wall has been inhibited by infusion of a broad spectrum MMP inhibitor (5) and delivery of tissue inhibitor of metalloproteinase-2 (7). These data thus support a role for ECM proteolysis in SMC translocation, although it is not yet established which ECM components need to be degraded.Fibrillar type I collagen is a dominant ECM protein of both the normal and diseased artery wall (8, 9). Unlike many ECM constituents, type I collagen is susceptible to degradation by only a few enzymes. Of these, collagenase-1 (MMP-1), which has been detected in human atherosclerotic lesions (2-4), appears to be the principal SMC-derived collagenolytic enzyme, although collagenase-3 (MMP-13) (10) and cathepsin K (11, 12) may also participate. Collagenase-1 degrades type I collagen by cleaving a single site in the triple helical domain to yield a 3/4 N-terminal fragment and 1/4 C-terminal fragment. At phys...

Type I Collagen Cleavage Is Essential for Effective Fibrotic Repair after Myocardial Infarction

“…These findings suggest a role for collagen cleavage in loosening cell-substrate connections to allow the cell body and tail to efficiently and appropriately follow the leading edge. Previous studies 22,34 have suggested that products of type I collagen cleavage may be promigratory. Collagen cleavage might also be important for ensuring that other promigratory matrix molecules, such as minor collagens or fibronectin, can appropriately interact with the cell.…”

“…[21][22][23] Cells plated onto dishes coated with a polymerized 3D network of mouse tail collagen (0.8 mg/mL) were imaged with an inverted microscope (Zeiss Axiovert S100; Cornwall, ON, Canada or Leica DMI6000 B; Concord, ON, Canada) using modulated contrast optics. Images were digitally acquired every 5 minutes during a 6-to 8-hour recording period, using a high-resolution charge-coupled device camera (Sony XC-75/75CE; EMPIX Imaging, Inc. or Leica DFC420 C video camera; Leica Microsystems Canada, Inc.) and time-lapse software (Northern Eclipse, Empix Imaging, Inc., and Leica Application Suite; Leica Microsystems Canada Inc.).…”

Regulation and characteristics of vascular smooth muscle cell phenotypic diversity

“…47 Moreover, the composition of the collagen fibrils influences the migration properties of the SMCs, which is associated with a different focal adhesion composition and integrin function. 48 Just as fibrillar collagen type I, collagen type IV and laminin have been shown to promote the contractile phenotype. 49 However, laminin-5 was also reported to enhance PDGF-B-stimulated SMC proliferation and migration, 50 providing another example of a protein with ambiguous effects on SMC phenotype.…”

Structural Analysis of the α2 Integrin I Domain/Procollagenase-1 (Matrix Metalloproteinase-1) Interaction

“…Reverse migration to the wound edge is prevented by turning a pro-migratory substrate into a poor migratory substrate (19). Interestingly, a recent report suggested that MMP-1 bound to the surface of smooth muscle cells is important for stimulating migration by enhancing rear release of the integrin from the degraded collagen (42). Although this work did not determine whether the MMP-1 was bound to the ␣ 2 ␤ 1 or ␣ 1 ␤ 1 integrins, the finding is entirely consistent with the model we have presented in this report.…”