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Functional und structural insights into TIMP-3- glycosaminoglycan interactions - implications for the design of functional biomaterials improving chronic wound treatment

Sandra Rother1, Sergey A. Samsonov2, Tommy Hofmann3, Ute Hempel4, Joanna Blaszkiewicz5, Jörg Rademann5, Stephanie Moeller6, Matthias Schnabelrauch6, Stefan Kalkof3, Martin Von Bergen3, 7, Mayte T. Pisabarro2, Dieter Scharnweber1 and Vera Hintze1
  • 1 TU Dresden, Max Bergmann Center of Biomaterials, Germany
  • 2 BIOTEC, TU Dresden, Structural Bioinformatics, Germany
  • 3 Helmholtz-Center for Environmental Research-UFZ, Department of Proteomics, Germany
  • 4 TU Dresden, Institute of Physiological Chemistry, Carl Gustav Carus Faculty of Medicine, Germany
  • 5 Freie Universität Berlin, Institute of Pharmacy & Institute of Chemistry and Biochemistry, Germany
  • 6 INNOVENT e.V., Biomaterials Department, Germany
  • 7 Helmholtz-Center for Environmental Research-UFZ, Department of Metabolomics, Germany

Introduction: In developed countries the life expectancy increases and therefore the number of multi-morbidities, which lead to impaired wound healing. One important aspect of remodeling during wound healing is related to extracellular matrix (ECM) degradation. This process is regulated via the interplay of matrix metalloproteinases (MMPs) and their counterparts, tissue inhibitors of metalloproteinases (TIMPs). Hence, chronic wounds are often associated with increased MMP and decreased TIMP levels[1]. Among TIMPs, TIMP‑3 is unique as it is bound to sulfated glycosaminoglycans (sGAGs) in the ECM and thought to be the main regulator of matrix turnover. Its cellular up-take is mediated by the endocytic receptor LRP-1[2]. GAGs, as functional components of the ECM, also influence ECM turnover, since sGAGs like heparin modulate the activities of both, TIMP-3[2] and MMPs[3]. Sulfated hyaluronans (sHAs) are promising in this respect as they also support e.g. fibroblast proliferation when used in collagen-based artificial ECMs (aECMs)[4]. Hence, the purpose of this study was to elucidate the influence of chemically sulfated GAGs on ECM homeostasis and in particular the molecular mechanisms by which they affect TIMP activity and up-take.  

Experimental Methods: Enzyme kinetic analyses were used to determine the residual activity of MMP-1/-2 after pre-incubation with native and chemically sGAGs, TIMP‑3 or combinations of both. The molecular binding mechanism was analyzed via surface plasmon resonance (SPR). TIMP-3 or LRP-1 were immobilized on sensor chips and GAG derivatives were injected. Furthermore, GAGs pre-incubated with TIMP-3 were injected over the LRP-1 surface. Computational approaches and H/D exchange experiments were performed to determine GAG possible binding sites on TIMP-3. Finally in vitro studies with human mesenchymal stromal cells (hMSCs) were supposed to reveal the biological consequence of the interaction between TIMP-3 and GAG derivatives.

Results and Discussion: SPR analysis reveals a dose- and sulfation-dependent binding of GAGs to immobilized TIMP-3. This interaction does however not affect the inhibitory potential of TIMP-3 against MMP‑1/-2, even after pre-incubation with GAGs, indicating that GAGs do not affect the activity of TIMP-3.

This is in line with the respective GAG binding sites on TIMP-3 determined by molecular modelling and H/D exchange mass spectroscopy. The former indicates that the GAG-binding regions of TIMP-3 do not overlap with the MMP-binding region. At the same time sGAGs interfere with the TIMP-3/LRP‑1 complex formation leading to a reduced binding of TIMP-3 to this endocytic receptor. This is supported by in vitro experiments with hMSCs revealing the accumulation of TIMP-3 in the media after treatments with sHA and a co-localization of TIMP-3 and sHA.

Conclusion: Sulfated GAGs are promising candidates for the treatment of chronic wounds since they sequester TIMP-3 and inhibit its internalization without altering the inhibitory potential of TIMP-3 towards MMPs. By slowing down matrix degradation processes sGAGs as part of biomaterials could prolong their beneficial presence in the wound and thereby improve chronic wound healing.

We acknowledge financial support by the DFG [TRR 67 A2, A3, A7, A8, B1, Z4].

References:
[1] Lobmann R et al., Diabetologia 45:1011-6, 2002
[2] Troeberg L et al., Chem Biol. 21:1300-9, 2014
[3] Yu and Woessner, Anal. Biochem. 293:38-42, 2001
[4] van der Smissen A et al., Biomaterials. 32:8938-8946, 2011

Keywords: Extracellular Matrix, Bioactivity, biofunctionalization, matrix-cell interaction

Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.

Presentation Type: Poster

Topic: Protein interactions with biomaterials

Citation: Rother S, Samsonov SA, Hofmann T, Hempel U, Blaszkiewicz J, Rademann J, Moeller S, Schnabelrauch M, Kalkof S, Von Bergen M, Pisabarro MT, Scharnweber D and Hintze V (2016). Functional und structural insights into TIMP-3- glycosaminoglycan interactions - implications for the design of functional biomaterials improving chronic wound treatment. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01675

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Received: 27 Mar 2016; Published Online: 30 Mar 2016.