Control of endothelial cell differentiation and proliferation for vascular tissue engineering

Therapeutic vascularization for tissue engineering and ischemic disease requires identification of an appropriate cell source and an ability to control its growth after implantation. Basic fibroblast growth factor (bFGF) is a potent angiogenic molecule, but its mitogenic effects on multiple cell types limit its therapeutic use. Human umbilical vein and microvascular endothelial cells (HUVECs and HMVECs) were retrovirally transduced with a chimeric FGF receptor-1 (FGFR1), and the effects of synthetic receptor-dimerizing ligands were studied. Chemical dimerization of FGFR1 stimulated proliferation in both HUVECs and HMVECs. Dimerizer-induced proliferation was MEK-dependent and was accompanied by MAP kinase phosphorylation, indicating the chimeric receptor uses signaling pathways similar to endogenous FGFR1. Although bFGF stimulated migration in HUVECs (which natively express FGFR1 and FGFR4), chemical dimerization of FGFR1 did not; this suggests FGFR4 may control migration in these cells. The ability to selectively activate receptor subtypes should facilitate the study of signaling pathways in vitro and in vivo beyond what can be accomplished with nonselective natural ligands.; Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering. PECAM1-positive cells were purified from differentiating hESCs by fluorescence and magnetic sorting methods. These cells expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous PHEMA biomaterial scaffolds and implanted in vivo. To enhance endothelial cell differentiation above a baseline of ∼2% in embryoid body (EB) spontaneous differentiation, EBs were treated with vascular endothelial growth factor (VEGF), which markedly increased expression of endothelial cell proteins PECAM1, vWF, and VE-Cadherin. PECAM1 expression peaked around days 10--14 for both control and VEGF-treated EBs. VEGF administration for shorter times yielded fewer endothelial cells, although preliminary results absolve VEGF-induced proliferation of causing this effect. Monolayer serum-free differentiation of endothelial cells was also achieved with the sequential addition of activin A, BMP4, and VEGF to the cultures. These enrichment methods increase endothelial cell yield and efficiency, facilitating applications for revascularization as well as basic studies of human endothelial biology.