Matrix density alters zyxin phosphorylation, which limits peripheral process formation and extension in endothelial cells invading 3D collagen matrices

• Pro-angiogenic factors and high matrix density promote robust sprouting responses.
• High matrix density reduces invasion distance and promotes process extension.
• Phorbol ester increases Erk phosphorylation, lumen formation, and multicellularity.
• Zyxin phosphorylation inversely correlates with matrix density.
• Zyxin restricts process extension by invading endothelial cells.

This study was designed to determine the optimal conditions required for known pro-angiogenic stimuli to elicit successful endothelial sprouting responses. We used an established, quantifiable model of endothelial cell (EC) sprout initiation where ECs were tested for invasion in low (1 mg/mL) and high density (5 mg/mL) 3D collagen matrices. Sphingosine 1-phosphate (S1P) alone, or S1P combined with stromal derived factor-1α (SDF) and phorbol ester (TPA), elicited robust sprouting responses. The ability of these factors to stimulate sprouting was more effective in higher density collagen matrices. S1P stimulation resulted in a significant increase in invasion distance, and with the exception of treatment groups containing phorbol ester, invasion distance was longer in 1 mg/mL compared to 5 mg/mL collagen matrices. Closer examination of cell morphology revealed that increasing matrix density and supplementing with SDF and TPA enhanced the formation of multicellular structures more closely resembling capillaries. TPA enhanced the frequency and size of lumen formation and correlated with a robust increase in phosphorylation of p42/p44 Erk kinase, while S1P and SDF did not. Also, a higher number of significantly longer extended processes formed in 5 mg/mL compared to 1 mg/mL collagen matrices. Because collagen matrices at higher density have been reported to be stiffer, we tested for changes in the mechanosensitive protein, zyxin. Interestingly, zyxin phosphorylation levels inversely correlated with matrix density, while levels of total zyxin did not change significantly. Immunofluorescence and localization studies revealed that total zyxin was distributed evenly throughout invading structures, while phosphorylated zyxin was slightly more intense in extended peripheral processes. Silencing zyxin expression increased extended process length and number of processes, while increasing zyxin levels decreased extended process length. Altogether these data indicate that ECs integrate signals from multiple exogenous factors, including changes in matrix density, to accomplish successful sprouting responses. We show here for the first time that zyxin limited the formation and extension of fine peripheral processes used by ECs for matrix interrogation, providing a molecular explanation for altered EC responses to high and low density collagen matrices.