Abstract
The vascular endothelium continually senses and responds to biochemical and mechanical stimuli to appropriately initiate angiogenesis. We have shown previously that fluid wall shear stress (WSS) and sphingosine 1-phosphate (S1P) cooperatively initiate the invasion of human umbilical vein endothelial cells into collagen matrices (Kang, H., Bayless, K. J., and Kaunas, R. (2008) Am. J. Physiol. Heart Circ. Physiol. 295, H2087-2097). Here, we investigated the role of calpains in the regulation of endothelial cell invasion in response to WSS and S1P. Calpain inhibition significantly decreased S1P- and WSS-induced invasion. Short hairpin RNA-mediated gene silencing demonstrated that calpain 1 and 2 were required for WSS and S1P-induced invasion. Also, S1P synergized with WSS to induce invasion and to activate calpains and promote calpain membrane localization. Calpain inhibition results in a cell morphology consistent with reduced matrix proteolysis. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown by others to regulate endothelial cell invasion, prompting us to test whether calpain acted upstream of MT1-MMP. S1P and WSS synergistically activated MT1-MMP and induced cell membrane localization of MT1-MMP in a calpain-dependent manner. Calpain activation, MT1-MMP activation and MT1-MMP membrane localization were all maximal with 5.3 dynes/cm(2) WSS and S1P treatment, which correlated with maximal invasion responses. Our data show for the first time that 5.3 dynes/cm(2) WSS in the presence of S1P combine to activate calpains, which direct MT1-MMP membrane localization to initiate endothelial sprouting into three-dimensional collagen matrices.
Highlights
Wall shear stress (WSS) and sphingosine 1-phosphate (S1P) combine to promote endothelial sprouting and angiogenesis
Because calpain activity is modulated in Endothelial cells (ECs) by growth factors and mechanical stimuli, we investigated the functional requirement for calpains in initiating EC sprouting events induced by WSS and S1P in three-dimensional collagen matrices
ECs were stimulated with S1P and 5.3 dynes/cm2 WSS in the presence of vehicle control, ALLN, or calpain inhibitor III, which inhibit calpains 1 and 2
Summary
Wall shear stress (WSS) and sphingosine 1-phosphate (S1P) combine to promote endothelial sprouting and angiogenesis. Endothelial cells (ECs) in the vascular system must continually sense and respond to both biochemical and mechanical stimuli within their microenvironment to appropriately initiate angiogenesis. Proangiogenic factors such as sphingosine 1-phosphate (S1P), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor are potent stimulators of new blood vessel growth (2, 6 – 8). Because calpain activity is modulated in ECs by growth factors and mechanical stimuli, we investigated the functional requirement for calpains in initiating EC sprouting events induced by WSS and S1P in three-dimensional collagen matrices. We demonstrate for the first time an ability of calpain to regulate MT1-MMP membrane localization in ECs following treatment with S1P and WSS
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