(722f) Covalent Immobilization of Transforming Growth Factor-β1 (TGF-β1) for Enhanced Vascular Functionality In Vitro Perhaps Through Prolonged Activation of TGF-β1 Pathway

In general, growth factor delivery to 3D tissue constructs is limited by diffusion which causes delivery in vivo limited by low efficiency, lack of targeting and side effects. Here we report a novel method to covalently conjugate transforming growth factor-β1 (TGF-β1), which involved in many physiological processes, in fibrin hydrogels. Specifically, TGF-β1 was modified to generate a fusion protein containing the factor XIII recognition sequence (NQEQVSP) and a plasmin cutting site (GYISR). During fibrin polymerization factor XIII links the glutamine (Q) in this sequence to leucine (L303) on α-chain of fibrinogen, thereby conjugating TGF-b1 covalently to the hydrogel. Two peptides NQEQVSP with and without plasmin cleavage sites GYIRSR amino acids sequences were fused to TGF-β1 between the latency associated peptide and mature-TGF-β1, and they were termed LNG-TGF-β1 and LN-TGF-β1 respectively. These fusion proteins were expressed in Chinese Hamster Ovary (CHO) cells and purified by chromatography and they had similar bioactivity to commercially derived TGF-β1 as shown by TGF-b1 dependent promoter (PAI) activity and by inhibition of cell proliferation. In addition, fusion TGF-b1 proteins bound to fibrinogen in a factor XIII dose-dependent manner and this binding prevented TGF-b1 release from fibrin hydrogels. Next we investigated the effects of TGF-b1 immobilization on the function of the vascular wall. To this end, we fabricated vascular constructs with immobilized TGF-b1 using fibrin hydrogels containing smooth muscles cells (SMC) derived from mesenchymal stem cells (MSCs). Interestingly, vascular reactivity in response to receptor and non-receptor mediated pathways was significantly higher in the constructs containing immobilized TGF-b1 as compared to those treated with the soluble growth factor. Also, the elastic modulus of the same constructs significantly increased over time during the 8-week culture period. To further investigate whether these functional effects of immobilized TGF-b1 were due to differences in the signaling response, we measured Smad2 phosphorylation as a function of time in 3D vascular constructs. Interestingly, cells in fibrin gels with conjugated TGF-b1 showed prolonged Smad2 phosphorylation at least up to 96hr, while treatment with soluble TGF-b1 induced only transient phosphorylation for one day. This result suggests that the enhanced contractility and improved mechanical properties of the vascular constructs might be attributed to prolonged activation of the TGF-b1 pathway.