Angiogenesis is the process of forming blood vessels from the pre-existing vasculature1. It is necessary in the process of development, but is also crucial for the recovery of damaged or pathological tissue1–3. However, abnormal angiogenesis is associated with several diseases, including cancer, cardiovascular disease, diabetes, and age-related macular degeneration1. Thus, modulators of angiogenesis, such as vascular endothelial growth factor and its related molecules (inhibitors such as pegaptanib sodium, ranibizumab, bevacizumab, and antibodies) have been approved for treating diseases described above1. Other growth factors such as platelet-derived growth factor, fibroblast growth factor, angiopoietins, and thymosin-β4 have also been shown to be modulators of angiogenesis1–4.
Beta-thymosins (thymosin-β) comprise a structurally related family that is highly conserved in the animal kingdom4,5. There are 16 types of beta-thymosins, but only 3 types are found in humans (thymosin-β4, thymosin-β10, and thymosin-β15)6,7. Although highly homologous in genetic structure, these three proteins are derived from different genes and thus, are distinct products. NMR studies have revealed that thymosin-β4 is mostly unstructured in aqueous solutions with some preferential α-helix structures8,9. Thymosin β-4 controls the assembly and disassembly of actin filaments through its initial interaction with G-actin8,9. Thus, the mechanism by which thymosin-β4 influences angiogenesis, cell differentiation, morphogenesis, cell migration, and organogenesis is assumed to be linked to the maintenance of a dynamic equilibrium between G-actin and F-actin8–10.
As such, thymosin β-4 is a promising drug candidate for many diseases. However, unlike other angiogenic modulators, thymosin-β4 does not act through receptors on the cell surface but acts directly on cells11. This is a major obstacle to the use of thymosin-β4 as a drug target. For this reason, we combined the well-known cell-penetrating peptide TAT and thymosin-β4 for intracellular translocation from the extracellular area12,13.
The recombinant protein produced in E. coli by molecular cloning techniques revealed the angiogenic effects of thymosin-β4 in vitro and in vivo. Here, we provide first experimental evidence that recombinant TAT- thymosin-β4 is an angiogenic modulator of the VEGFR2 signaling pathway.