(717e) Engineering Clustered VEGF to Promote Angiogenesis in Vitro

The process of angiogenesis, defined by the development of new blood vessels from pre-existing vessels, is essential in tissue remodeling and regeneration. This complex process involves extensive interplay between cells, soluble factors and extracellular matrix (ECM) components. In our laboratory, we are interested in designing biomaterials that can deliver pro-angiogenic agents such as Vascular Endothelial Growth Factor (VEGF) to activate the vascular process and stimulate the formation of a vascular network at ischemic tissue sites. The presence of VEGF at the site of ischemic damage enables specific endothelial cells (ECs) to undergo a phenotypic switch to become tip cells, which lead the way in a branching vessel. Previously our laboratory found that the physical presentation of VEGF (e.g. covalently bound, clustered or soluble) affected the way in which ECs became activated both at the molecular level (receptor phosphorylation) and at the multi-cellular level (tube formation). In particular, we observed that clustered VEGF enhanced the number of branching points and the total network length compared to less clustered VEGF and soluble VEGF, using a bead HUVEC (Human Umbilical Vascular Endothelail Cells) branching assay. Interestingly, the VEGF presentation didn’t affect the total number of sprouts and the tube thickness. To understand the mechanism by which clustered VEGF resulted in enhanced tube branching points, we optimized the formation of VEGF clusters, studied VEGF receptor-2 (VEGFR-2) activation by its phosphorylation at two different tyrosine residues (1175 and 1214), and studied the activation of p38, Erk1/2 and Akt downstream signaling pathways. Briefly, Huvec cells were plated on 2D and were exposed to basic media containing 20 ng/mL of soluble or clustered VEGF and were studied at different time points. We evidenced that VEGF clusters could be generated with a uniform Z-average size was 95.97 ± 0.5 nm with a polydispersity index of 0.246 ± 0.005 using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Interestingly, although the level of receptor activation was at highest for soluble, VEGF clusters achieved sustained phosphorylation. These results indicate that clustered VEGF causes extended activation when compared to soluble VEGF. Moreover, we studied the tip cell phenotype by evaluating their gene expression profile, and evidenced an increased expression of tip cells specific genes Delta-like 4 (Dll4), Jagged-1, CXCL12 and VEGFR3. Dll4 and Jagged-1 are the two ligands in the Notch signaling pathway that regulates the shaping of a branched vascular network. In addition, CXCL12 and VEGFR3 have been associated with the tip cell phenotype. Moreover, the analysis of mRNA levels by qPCR at different time exposure showed an increased expression of these genes at 4 hours in the group exposed to clustered VEGF compared to soluble VEGF. At the cellular level, quantification of DNA content demonstrated that clustered VEGF resulted in enhanced proliferation compared to soluble VEGF at different concentration of VEGF. To continue to study the mechanism by which clustered VEGF induced enhanced branching, we aim to investigate differences in protein expression and distribution as a result of exposure to clustered VEGF and soluble VEGF.