title: Engineering fibrin-binding TGF-β1 for sustained signaling and contractile function of MSC based vascular constructs.
abstract: We present a strategy to conjugate TGF-β1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF-β1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF-β1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF-β1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF-β1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF-β1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF-β1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.
authors.affiliation: Bioengineering Laboratory, Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Amherst, NY 14260-4200, USA.
authors.forename: Mao-Shih
authors.initials: MS
authors.lastname: Liang
authors.forename: Stelios T
authors.initials: ST
authors.lastname: Andreadis
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D008907:Mink; D010766:Phosphorylation; D011485:Protein Binding; D015398:Signal Transduction; D023822:Tissue Engineering; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D053773:Transforming Growth Factor beta1; D005337:Fibrin