Guided vasculogenic sprouting induced by the immobilized fusion construct CaM-VEGF120
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This project is intended to utilize an immobilized bio-active first generation fusion constructed cytokine inducing in receptive cell lines guided vasculogenic development. This research through the assembly, expression and purification of a bio-active molecule the CaM-VEGF120 fusion construct permitted the creation of a first generation smart-gel platform. Cell culture bringing together HUVECs or cBOECs with soluble or immobilized CaM-VEGF120 coupled with a type-I collagen platform are the main components intended to induce guided vascular sprouting. Purification of the CaM-VEGF120 was achieved utilizing HIC coupled with size exclusion chromotography. Mass Spectrometry and cellular augmentation noted by survivability and proliferation suggests the correct CaM-VEGF120 properties were achieved. Cell culture interactive changes were recorded utilizing fluorescent and phase microscopy. The 66 KDa dimeric CaM-VEGF120 was able to phosphorylate the cytoplasmic Tyr1175 localized to the C-terminal portion of the transmembrane VEGFR2. GNP immobilized CaM-VEGF120 induced VEGFR2 expressing cell lines as were imaged over a week’s period recording vascular pseudo-tube formation. These events resulting from contact with the immobilized CaM-VEGF120 and VEGFR2 induced activity thus presenting in vitro guided vascular pseudo-tube development. This research is being pursued utilizing HUVEC and cBOECs as guided vascular pseudo-tube structural formation is possible. This successful model implies a first generation model for physiological vascular development having therapeutic applications.