Calpain-mediated vimentin cleavage occurs upstream of MT1-MMP membrane translocation to facilitate endothelial sprout initiation.

Endothelial cells normally line the vasculature and remain quiescent. However, these cells can be rapidly stimulated to undergo morphogenesis and initiate new blood vessel formation given the proper cues. This study reports a new mechanism for initiating angiogenic sprout formation that involves vimentin, the major intermediate filament protein in endothelial cells. Initial studies confirmed vimentin was required for sphingosine 1-phosphate (S1P)- and growth factor (GF)-induced endothelial cell invasion, and vimentin was cleaved by calpains during invasion. Calpains were predominantly activated by GF and were required for sprout initiation. Because others have reported membrane type 1-matrix metalloproteinase (MT1-MMP) is required for endothelial sprouting responses, we tested whether vimentin and calpain acted upstream of MT1-MMP. Both calpain and vimentin were required for successful MT1-MMP membrane translocation, which was stimulated by S1P. In addition, vimentin complexed with MT1-MMP in a manner that required both the cytoplasmic domain of MT1-MMP and calpain activation, which increased the soluble pool of vimentin in endothelial cells. Altogether, these data indicate that pro-angiogenic signals converge to activate calpain-dependent vimentin cleavage and increase vimentin solubility, which act upstream to facilitate MT1-MMP membrane translocation, resulting in successful endothelial sprout formation in three-dimensional collagen matrices. These findings help explain why S1P and GF synergize to stimulate robust sprouting in 3D collagen matrices.

ADAM17 co-purifies with TIMP-3 and modulates endothelial invasion responses in three-dimensional collagen matrices.

In this study, we investigated potential mechanisms through which the known anti-angiogenic factor, tissue inhibitor of metalloproteinase-3 (TIMP-3) blocks angiogenesis. As a strategy to identify TIMP-3 binding proteins, we used tandem affinity purification, employing recombinant adenoviruses constructed to deliver TIMP-3 fused to C-terminal S and His tags (TIMP-3-S-His) or TIMP-1-S-His control to endothelial cells prior to extraction. Western blotting of final eluates revealed robust binding of A Disintegrin and Metalloproteinase (ADAM) 17 and a slight association of ADAM15 to TIMP-3, but not TIMP-1 control. To confirm a functional requirement for ADAM15 and 17 in mediating angiogenic events, a model of endothelial cell invasion was utilized. Silencing of ADAM17, but not ADAM15, expression using small interfering RNA (siRNA) interfered with invasion, resulting in decreased density of invading cells and decreased invasion distance. Stable EC lines expressing short hairpin RNA directed to ADAM17 were similarly inhibited. To confirm these results, dominant negative mutants (DeltaMPs) of ADAM10, ADAM15 or ADAM17 were delivered using recombinant lentiviruses. Expression of ADAM17 DeltaMP, but not ADAM10 or ADAM15 DeltaMP, decreased invasion density and distance. Further, time-lapse analyses revealed ADAM17 DeltaMP cells exhibited far greater numbers of protruding sprouts compared to control, suggesting an inability of extended processes to retract properly. Immunofluorescence analyses revealed ADAM17 localized to bifurcations in invading sprouts. These data jointly indicate a role for ADAM17 in modulating endothelial sprouting events during angiogenesis.

Molecular profile of endothelial invasion of three-dimensional collagen matrices: insights into angiogenic sprout induction in wound healing.

Sprouting angiogenesis is a multistep process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and sprout stabilization. Such complexity is consistent with a requirement for orchestration of individual gene expression alongside multiple signaling pathways. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to develop multicellular invading sprouts, we analyzed differential gene expression with time using a defined in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate, basic FGF, and VEGF. Gene expression changes were confirmed by real-time PCR and Western blot analyses. A cohort of cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated, whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, a disintegrin and metalloproteinase with thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt and Notch pathways was observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. This study has enhanced the molecular road map of gene expression changes that occur during endothelial invasion and highlighted the utility of three-dimensional models to study EC morphogenesis.