Expression and Activity of Tissue Transglutaminase in the Response to Gelatin-based Hydrogels in vitro and in vivo


Expression and Activity of Tissue Transglutaminase in the Response to Gelatin-based Hydrogels in vitro and in vivo

Ullm, S.; Wodtke, R.; Tondera, C.; Löser, R.; Pietzsch, J.

Tissue transglutaminase (TGase 2) is proposed to be important for biomaterial-cell or -tissue interactions due to its presence and versatile functions in the extracellular environment. For example, TGase 2 catalyzes the crosslinking of proteins of the extracellular matrix through its Ca2+-dependent transamidase activity or enhances the interactions between fibronectin, integrins, and syndecan-4 via its role as scaffold protein. Through these functions, TGase 2 mediates the adhesion, migration and motility of cells. Additionally, TGase 2 is a key player during the development of fibrosis. Consequently, TGase 2 might be a potential target for the improvement and visualization of tissue regeneration following biomaterial implantation. Here, we studied the expression pattern and activity of TGase 2 in the response to gelatin-based hydrogels with tailorable elastic properties and degradation behavior due to different degrees of crosslinking.
Solutions of 10 wt.-% gelatin were crosslinked with 3- (G10_LNCO3) or 8-fold (G10_LNCO8) excess of isocyanate groups of lysine diisocyanate ethyl ester compared to amino groups of gelatin. Primary human aortic endothelial cells (HAEC) and human leukemia THP-1 and HL-60 cells, differentiated to macrophages (Mɸ) or granulocytes (Gɸ), were seeded either directly on the hydrogel films or cultivated with material eluates in order to simulate hydrolytic or enzymatic hydrogel degradation in vivo. Expression levels of intracellular and secreted TGase 2 were analyzed via Western blotting [1]. Additionally, hydrogels were implanted subcutaneously in immunocompetent, hairless SKH-1 mice. TGase 2 expression at the implantation site was detected ex vivo by immunohistochemistry at several time points up to 112 days after implantation. The presence of activatable TGase 2 in tissue sections was confirmed in vitro by incorporation of fluorescently labelled cadaverine derivatives [2] at sites of TGase 2 expression. To prove that the incorporation is caused by TGase 2, the staining was performed in the presence and absence of a recently described irreversible inhibitor for TGase 2 [3].
HAEC showed high expression and secretion of TGase 2, independent from hydrogel contact. THP-1 Mɸ highly expressed TGase 2, and increased its secretion after direct hydrogel contact. In contrast, HL-60 Mɸ and Gɸ exhibited enhanced expression of TGase 2 after direct contact to the hydrogels, without secreting TGase 2. These results demonstrate that TGase 2 expression and secretion by Mɸ and Gɸ are influenced by contact to the hydrogel surface structure and not by hydrogel degradation products. Additionally, the hydrogels with higher content of diurealysine junction units (G10_LNCO8) induced higher effects than G10_LNCO3. Ex vivo analysis of tissue sections by immunohistochemistry revealed enhanced expression levels of activatable TGase 2 around the hydrogels 14 and 21 days after implantation, indicating a role of TGase 2 in hydrogel integration and tissue remodelling.
Our results revealed that contact to the gelatin-based hydrogels influenced TGase 2 expression and secretion by Mɸ and Gɸ in vitro, as well as expression of activatable TGase 2 in vivo. This underlines TGase 2 to be a promising theranostic target during processes of biomaterial integration.
References:
[1] Ullm et al. Biomaterials 2014, 35, 9755-9766
[2] Hauser et al. Amino Acids 2016, DOI 10.1007/s00726-00016-02192-00725
[3] Wityak et al. ACS Med. Chem. Lett. 2012, 3, 1024-1028

  • Poster
    Transglutaminases in Human Disease Processes, 10.-15.07.2016, Girona, Spanien

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Publ.-Id: 25076