Blood-Tissue Gradients of Sphingosine 1-Phosphate (S1P) as a Regulator of Vascular Stability
John Morgan, Jason Spector.
Cornell University Medical College, New York, NY, USA.
BACKGROUND:A major barrier for generalization of tissue engineering beyond thin tissues (thickness < 250 um) is formation of functional, pervasive microvascular systems within tissues to support metabolic demand during laboratory culture and after implantation. Originally informed by studies of vascular growth induced by tumors, clinical and engineering efforts to drive angiogenesis have focused on presentation of a few key growth factors (e.g., VEGF and bFGF). While these factors are potent stimulators of vessel growth, they tend to lead to pathological vascular structures in both network architecture (hyper sprouting) and permeability (leaky). Recently, a lipid signaling molecule carried in the serum, Sphingoside-1 Phosphate (S1P) has emerged as an additional, potent regulator of angiogenesis, but fundamental aspects of its mechanism remain an enigma. Present in micromolar concentrations in serum and nanomolar concentrations in the tissue, a natural gradient of S1P exists across the endothelium. Using both static cultures and perfused microvascular networks with live imaging in vitro we reveal how this gradient of S1P regulates endothelial cell behavior and microvascular stability.
For static cultures, endothelial cell monolayers were cultured on 0.3% 3D Type I collagen matrices in 4mm diameter microwells. Simulated reversal of the physiologic S1P gradient was conducted by loading increasing concentrations of S1P (10 nM, 100 nM, 1000 nM) into the matrix, with none present in the media. Simulated restoration of the gradient was tested by adding increasing concentrations of S1P (10 nM, 100 nM, 1000 nM) into the media while maintaining a fixed concentration in the matrix (10 nM, 100 nM, 1000 nM). For perfusion cultures, microvascular networks were recapitulated in vitro and live imaged as previously described1 , using endothelial cells and pericytes, with similar S1P concentrations in the matrix and media, as described above. Vessels were perfused to achieve physiologic shear stress against the vessel wall. Both cultures were conducted for 1-7 day periods and analyzed via confocal microscopy and immunohistochemistry, to evaluate cell-cell junction integrity, permeability, lumen formation and average invasion depth.
Confocal and fluorescence microscopy, and quantitative image analysis show that incremental reversal of the S1P blood-tissue gradient across in vitro monolayers of static endothelial cells and perfused dynamic microvascular networks, increases endothelium permeability, and induces cell migration and lumen formation. These phenomena occur in increasing magnitude to the S1P concentration difference, and the applied hemodynamic shear stress. Incrementally restoring the gradient attenuates migration and restores monolayer stability, proportionately.
The S1P concentration difference across the endothelium regulates vascular stability and provides a basis for dissection of this differential response and its mediation by the family of S1P G-protein coupled receptors. We hypothesize that this differential response can be used to fine-tune angiogenesis to drive healthy vascular extensions within engineered tissues.
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