Abluminal stimulation of sphingosine 1-phosphate receptors 1 and 3 promotes and stabilizes endothelial sprout formation.

Local delivery of lipid mediators has become a promising new approach for therapeutic angiogenesis and regenerative medicine. In this study, we investigated how gradient stimulation (either abluminal/distal or luminal/proximal) of engineered microvessels with sphingosine 1-phosphate (S1P) receptor-subtype-targeted molecules affects endothelial sprout growth using a microfluidic device. Our studies show that distal stimulation of microvessels with FTY720, an S1P1/3 selective agonist, promotes both arterial and venular sprout growth, whereas proximal stimulation does not. Using novel pharmacological antagonists of S1P receptor subtypes, we further show that S1P3 functionality is necessary for VEGF-induced sprouting, and confirmed these findings ex vivo using a murine aortic ring assay from S1P3-deficient mice. S1P3 agonist stimulation enhanced vascular stability in both cell types via upregulation of the interendothelial junction protein VE-cadherin. Lastly, S1P3 activation under flow promoted endothelial sprouting and branching while decreasing migratory cell fate in the microfluidic device. We used an in vivo murine dorsal skinfold window chamber model to confirm S1P3's role in neovascular branching. Together, these data suggest that a distal transendothelial gradient of S1P1/3-targeted drugs is an effective technique for both enhancing and stabilizing capillary morphogenesis in angiogenic applications.

Laminin- and basement membrane-polycaprolactone blend nanofibers as a scaffold for regenerative medicine.

Mimicking one or more components of the basement membrane (BM) holds great promise for overcoming insufficiencies in tissue engineering therapies. We have electrospun laminin nanofibers (NFs) isolated from the murine Engelbreth-Holm Swarm (EHS) tumor and evaluated them as a scaffold for embryonic stem cell culture. Seeded human embryonic stem cells were found to better maintain their undifferentiated, colony environment when cultured on laminin NFs compared to laminin mats, with 75% remaining undifferentiated on NFs. Mouse embryonic stem cells cultured on 10% laminin-polycaprolactone (PCL) NFs maintained their colony formation for twice as long without passage compared to those on PCL or gelatin substrates. In addition, we have established a protocol for electrospinning reconstituted basement membrane aligned (RBM)-PCL NFs within 10° of angular deviation. Neuron-like PC12 cells show significantly greater attachment (p < 0.001) and percentage of neurite-extending cells in vitro on 10% RBM-PCL NFs when compared to 1% and 0% RBM-PCL NFs (p < 0.015 and p < 0.001, respectively). Together, these results implicate laminin- and RBM-PCL scaffolds as a promising biomimetic substrate for regenerative medicine applications.