Endothelial Cell Response to Artificial Extracellular Matrix Proteins

• Main Author: Liu, Julie Chih-I
• Format: Others
• Published: 2006
• Online Access: https://thesis.library.caltech.edu/1099/1/00_Title.pdf; https://thesis.library.caltech.edu/1099/2/01_Chapter_1.pdf; https://thesis.library.caltech.edu/1099/3/02_Chapter_2.pdf; https://thesis.library.caltech.edu/1099/4/03_Chapter_3.pdf; https://thesis.library.caltech.edu/1099/5/04_Chapter_4.pdf; https://thesis.library.caltech.edu/1099/6/05_Chapter_5.pdf; https://thesis.library.caltech.edu/1099/7/06_Chapter_6.pdf; https://thesis.library.caltech.edu/1099/8/07_Chapter_7.pdf; https://thesis.library.caltech.edu/1099/9/08_Chapter_8.pdf; https://thesis.library.caltech.edu/1099/10/09_Appendix_A.pdf; https://thesis.library.caltech.edu/1099/11/10_Appendix_B.pdf; https://thesis.library.caltech.edu/1099/12/11_Appendix_C.pdf; Liu, Julie Chih-I (2006) Endothelial Cell Response to Artificial Extracellular Matrix Proteins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ABQJ-TR83. https://resolver.caltech.edu/CaltechETD:etd-03242006-134157 <https://resolver.caltech.edu/CaltechETD:etd-03242006-134157>

<p>Artificial extracellular matrix (aECM) proteins were designed originally for use in small-diameter vascular grafts. Current synthetic grafts fail primarily due to (i) a compliance mismatch between the prostheses and surrounding tissue and (ii) the inability to support the growth of an endothelial cell monolayer. To address these issues, biomimetic proteins were engineered with elastin-like repeats to confer elastomeric behavior and fibronectin cell-binding domains to promote endothelialization. Lysine residues or the non-canonical amino acid, para-azidophenylalanine (pN3phe), serve as crosslinking sites.</p> <p>Human umbilical vein endothelial cell (HUVEC) adhesion to aECM proteins was sequence-specific. Cells bind more strongly and exhibit faster spreading kinetics on aECM proteins containing the RGD versus the CS5 sequence. Furthermore, HUVECs on the former protein exhibited well-formed stress fibers and organized the alpha-v-beta-3 but not the alpha-5-beta-1 integrin into focal adhesions.</p> <p>Although biomaterial design has focused on the sequences of cell-binding domains, elements remote to these bioactive sequences were found to affect cell response to aECM proteins. Proteins containing identical CS5 cell-binding domains differed in their placement of lysine residues that serve as crosslinking sites. Cell adhesion and spreading were more robust on proteins in which lysine residues were located at the termini versus within the elastin cassettes.</p> <p>Crosslinked films of aECM proteins with RGD sequences adhered HUVECs in a sequence-specific manner. Poly(ethylene glycol) was covalently attached to films to reduce nonspecific cell interactions. Increasing the density of RGD in a film resulted in increased cell adhesion and spreading but did not have a significant effect on migration rates.</p> <p>aECM proteins were made photoreactive through the incorporation of pN3phe. Upon exposure to ultraviolet radiation through a patterned mask, proteins were patterned on a non-adhesive background. These two-dimensional patterns then served as templates for cell adhesion.</p> <p>A new technique for studying cells on aECM proteins was developed. Cells were pulsed with homopropargylglycine. Newly synthesized proteins labeled with alkyne-containing amino acids were ligated to 3-azido-7-hydroxycoumarin. Fluorescence microscopy was used to visualize these proteins in a wide variety of mammalian cell types.</p>