Hic-5 mediates endothelial sprout initiation by regulating a key surface metalloproteinase

Abstract

During angiogenesis, endothelial cells must coordinate matrix proteolysis with migration. Here, we tested whether the focal adhesion scaffold protein Hic-5 (also known as TGFB1I1) regulated endothelial sprouting in three dimensions. Hic-5 silencing reduced endothelial sprouting and lumen formation, and sprouting defects were rescued by the return of Hic-5 expression. Pro-angiogenic factors enhanced colocalization and complex formation between membrane type-1 matrix metalloproteinase (MT1-MMP, also known as MMP14) and Hic-5, but not between paxillin and MT1-MMP. The LIM2 and LIM3 domains of Hic-5 were necessary and sufficient for Hic-5 to form a complex with MT1-MMP. The degree of interaction between MT1-MMP and Hic-5 and the localization of the complex within detergent-resistant membrane fractions were enhanced during endothelial sprouting, and Hic-5 depletion lowered the surface levels of MT1-MMP. In addition, we observed that loss of Hic-5 partially reduced complex formation between MT1-MMP and focal adhesion kinase (FAK, also known as PTK2), suggesting that Hic-5 bridges MT1-MMP and FAK. Finally, Hic-5 LIM2-LIM3 deletion mutants reduced sprout initiation. Hic-5, MT1-MMP and FAK colocalized in angiogenic vessels during porcine pregnancy, supporting that this complex assembles during angiogenesis in vivo. Collectively, Hic-5 appears to enhance complex formation between MT1-MMP and FAK in activated endothelial cells, which likely coordinates matrix proteolysis and cell motility.

Pericyte ALK5/TIMP3 Axis Contributes to Endothelial Morphogenesis in the Developing Brain

“…Conditioned media from human brain PCs pretreated with ALK5 siRNA or scrambled siRNA was collected at 72 hours post-transfection and applied to human brain microvascular ECs. Three-dimensional invasion experiments were established using collagen matrices (2.5 mg/ml) containing 1 μM sphingosine-1-phosphate (Avanti Polar Lipids), 40 ng/ml VEGF and basic fibroblast growth factor as described previously (Dave et al 2016, Kwak et al 2012). EC invasion cultures were incubated for 20 hours, fixed in 3% gulteraldehyde (Sigma) and stained with toluidine blue (Sigma).…”

Dynamic Expression of Membrane Type 1-Matrix Metalloproteinase (Mt1-mmp/Mmp14) in the Mouse Embryo

“…It is known that MT1-MMP acts as a key pericellular collagenase that controls the ability of VSMCs to degrade and infiltrate 3D barriers of interstitial collagen, including the arterial wall [ 76 ]. Moreover, MT1-MMP has a greater catalytic activity on type III collagen found in visceral and cardiovascular tissues [ 77 , 78 ]. ECM dynamics perform a major influence on umbilical VSMCs fate, although the mechanisms by which the umbilical cord vascular growth happens are poorly understood.…”

Hic-5 regulates Src-induced invadopodia rosette formation and organization

“…, 2012). Moreover, Hic-5 has been found to bridge FAK to MT1-MMP (Dave et al. , 2016) and in a separate study also linked to delivering MT1-MMP at the invadopodia site to facilitate degradation of the matrix (Petropoulos et al.…”

“…, 2012). Moreover, Hic-5 has been shown to regulate delivery of membrane-bound type-1 matrix metalloproteinase MT1-MMP to facilitate degradation of the extracellular matrix in active Src-transformed fibroblasts, as well as in endothelial cells to promote angiogenesis (Dave et al. , 2016; Petropoulos et al.…”