Calpain 2 is activated downstream of wall shear stress and sphingosine-1-phosphate to induce endothelial cell sprout formation in three dimensional collagen matrices
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abstract
New blood vessel growth is essential for vascularization of wounded tissues. Endothelial cells are integral for this process and are stimulated by growth factors and plateletderived lipids such as sphingosine1phosphate (S1P), while also integrating mechanical signals, such as wall shear stress (WSS). However the molecular signals activated downstream of these cues are not completely defined. Previously, we have established a system to demonstrate that S1P and WSS combine to stimulate primary endothelial cell invasion of 3D collagen matrices. S1P and WSS induced Akt phosphorylation and MMP2 activation that correlated with invasion responses. Here we determined whether calpain regulates S1P and WSSinduced invasion into 3D collagen matrices. WSS and S1P combined to enhance calpain activation. Pharmacological inhibitors of calpain 2, but not calpain 1, significantly reduced S1P and WSSinduced endothelial sprout density and thickness. Also, Akt phosphorylation and MMP2 activation were attenuated by calpain inhibition. To confirm a functional role for calpain 2, short hairpin RNA was delivered using recombinant lentiviruses. Silencing of calpain 2, but not calpain 1, blocked WSS/S1Pinduced EC invasion. These data support an integral role for calpain 2 in mediating S1P and WSSinduced invasion responses in 3D collagen matrices.Grant Funding SourceAmerican Heart Association
