VEGFR2-Specific Ligands Based on Affibody Molecules Demonstrate Agonistic Effects when Tetrameric in the Soluble Form or Immobilized via Spider Silk

Strategies to promote vascularization are being developed in order to improve long-term survival of artificial tissue constructs. Vascular endothelial growth factor A (VEGFA) has an important role in both pathological and physiological angiogenesis, mediated by binding to VEGF receptors (VEGFRs). In nature, signaling can be modulated by presentation of growth factors in either soluble form or bound to the extracellular matrix. Herein, a previously reported VEGFR2-binding antagonistic affibody heterodimer (di-Z_VEGFR2) was formatted into a tetrameric construct (tetra-Z_VEGFR2) with the intention of generating artificial agonistic ligands for VEGFR2 signaling. In vitro cell assays demonstrated that tetra-Z_VEGFR2 induced VEGFR2 phosphorylation and increased cell proliferation, in contrast to di-Z_VEGFR2. In order to simulate matrix-bound factors, both constructs were fused at the genetic level to a partial spider silk protein, 4RepCT. Assembly of the silk fusion proteins onto a solid surface was verified by quartz crystal microbalance with dissipation analysis. Moreover, surface plasmon resonance studies demonstrated retained VEGFR2 binding ability of di-Z_VEGFR2-silk and tetra-Z_VEGFR2-silk after silk-mediated immobilization. Cell culture studies demonstrated that VEGFR2-overexpressing cells adhered to di-Z_VEGFR2-silk and tetra-Z_VEGFR2-silk and had activated VEGFR2 signaling. Altogether, we demonstrate the potential of especially tetra-Z_VEGFR2-silk to promote angiogenesis in tissue-engineering applications. The results from the study also show that molecules can obtain completely new functions when presented on materials, and verifying the biological effects after functionalizing materials is thus always recommended.