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

Abstract

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 1mg/mL compared to 5mg/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 5mg/mL compared to 1mg/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.

Coculturing Human Islets with Proangiogenic Support Cells to Improve Islet Revascularization at the Subcutaneous Transplantation Site

“…Established in vitro models of angiogenesis involve the monitoring of capillary-like structure formation in threedimensional gels mimicking the extracellular matrix, such as Matrigel, 39 fibrin gel, 9,40,41 or collagen gel. 42 Using two of these model systems, Matrigel and fibrin gel, we demonstrate the induction of sprout formation of CIs over time. The observation that control islets sprout in fibrin gel is in line with previous reports.…”

“…9,36,41,43 To our knowledge, no studies have monitored sprouting of control islets in Matrigel. The improved performance in the sprouting capacity of uncoated islets in fibrin compared to Matrigel could be explained by either the difference in matrix composition or stiffness, shown to influence sprout formation of mature endothelial cells both in vitro 42,[44][45][46][47] and in vivo, 33 or the expression of a specific set of fibrinolytic enzymes in intraislet endothelial cells, specifically enabling successful remodeling of the fibrin matrix. [47][48][49] Our results further indicate that hMSCs, either naive or preconditioned, significantly enhance sprout formation in vitro compared to HUVEC-hMSC-CIs or control islets.…”

CCL19 suppresses angiogenesis through promoting miR-206 and inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway in colorectal cancer

“…HUVECs were allowed to invade for 24 h at 37 °C with 5% CO 2 before fixing in 3% glutaraldehyde (Sigma-Aldrich) in PBS and staining with 0.1% toluidine blue (Sigma-Aldrich) in 30% methanol. Finally, collagen matrices were photographed by microscopy and evaluated as described by Colette A. Abbey 49 .…”

Rebooting the collagen gel: Artificial hydrogels for the study of epithelial mesenchymal transformation

“…Recent studies revealed in endothelial cells that the phosphorylation levels of zyxin inversely correlated with collagen matrix stiffness, identifying this focal adhesion protein to be mechanosensitive (Abbey and Bayless, 2014). Integrin-mediated signaling was further found to be transduced by the adhesion-localized Rac1 and cdc42 specific GTPase activating protein cdGAP that senses and responds to mechanical cues in the ECM to coordinate directed cell motility (Wormer et al, 2014).…”