(259d) Optimization of An Elastin Mimetic Peptide as A Template to Engineer Vascular ECM

Elastin is a 70 kDa insoluble protein found in connective tissues such as lungs and blood vessels[1]. It is a key factor in promoting smooth muscle cell (SMC) proliferation and migration throughout the elastin matrix in the medial layer of an artery[2]. The protein has also involved in stimulating further elastin fiber assembly and production in vivo[2]. One of the severe drawbacks of using elastin in a biomaterial is the extreme hydrophobic nature of the protein. As an alternative approach, we are isolating key functions of elastin and engineering them into an elastin mimetic peptide. A 23 amino acid (aa), AAKAAKVGVAPGRGDSAAKAAKK, and a 19 aa, AAKAAKVGVAPGAAKAAKK, peptide were designed to mimic elastin that would be capable of generating functional elastin fibers once incorporated into a tissue engineered vascular graft (TEVG). The hexapeptide sequence, VGVAPG, is a repeat sequence found on elastin that binds to a multi-functional elastin binding protein (EBP) found on surface of SMCs. The binding of this sequence to the EBP has shown to trigger chemotactic cues described above in elastin fiber assembly[2]. Furthermore, RGDS, which has an affinity for the integrin α_vβ₃ on a cell surface, was introduced in the 23 aa sequence to enhance cell adhesion and potentially assist with improving elastin production. The peptides, along with appropriate scrambled sequences, were characterized in vitro in order to assess the deposition of elastin and desmosine, crosslinking domain unique to elastin. Human aortic smooth muscle cells (SMCs) were incubated with either peptides at varying concentrations and the amount of elastin and desmosine deposited was determined after 48 hours. A Fastin assay was used to measure elastin production (Fig 1) whereas a competitive enzyme-linked immunosorbent assay (ELISA) was used to quantify desmosine production (Fig 2). Using immunofluorescent techniques, we were also able to visualize the binding and the crosslinking of either peptides to the cell surface and deposited elastin. The 23 aa sequence is induces recovery of elastin production at highest concentration. With this recovery in elastin, we conclude that there is interplay between α_vβ₃ and the EBP that is influencing elastin production.

1. Patel, A., et al., Elastin biosynthesis: The missing link in tissue-engineered blood vessels. Cardiovascular Research, 2006. 71(1): p. 40-49. 2. Rodgers, U.R. and A.S. Weiss, Cellular interactions with elastin. Pathologie Biologie, 2005. 53(7): p. 390-398.