Sulfated Hyaluronan Alters Endothelial Cell Activation in Vitro by

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Sulfate hyaluronan alters endothelial cell activation in vitro by controlling the biological activity of the angiogenic factors vascular endothelial growth factor-A and tissue inhibitor of metalloproteinase-3 Sandra Rother, Sergey A. Samsonov, Stephanie Möller, Matthias Schnabelrauch, Jörg Rademann, Joanna Blaszkiewicz, Sebastian Köhling, Johannes Waltenberger, Maria Teresa Pisabarro, Dieter Scharnweber, and Vera Hintze ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01300 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 3, 2017

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ACS Applied Materials & Interfaces

Sulfate hyaluronan alters endothelial cell activation in vitro by controlling the biological activity of the angiogenic factors vascular endothelial growth factor-A and tissue inhibitor of metalloproteinase-3 Sandra Rother1, Sergey A. Samsonov2, Stephanie Moeller3, Matthias Schnabelrauch3, Jörg Rademann4, 5, Joanna Blaszkiewicz4, 5, Sebastian Köhling4, 5, Johannes Waltenberger6, M. Teresa Pisabarro2, Dieter Scharnweber1 and Vera Hintze1 1

Institute of Materials Science, Max Bergmann Center of Biomaterials, Technische Universität

Dresden, 01069 Dresden, Germany 2

Structural Bioinformatics, BIOTEC Technische Universität Dresden, Tatzberg 47-51, 01307 Dresden,

Germany 3

Biomaterials Department, INNOVENT e.V., 07745 Jena, Germany

4

Institute of Pharmacy & Institute of Chemistry and Biochemistry, Freie Universität Berlin, Königin-

Luise-Str. 2, 14195 Berlin, Germany 5

Institute of Medical Physics and Biophysics, Universität Leipzig, Härtelstr. 16/18, 04107 Leipzig,

Germany 6

Department of Cardiovascular Medicine, University of Münster, Albert-Schweitzer-Campus 1, 48149

Münster, Germany

* To whom correspondence should be addressed. E-Mail: [email protected]

Keywords: Hyaluronan/sulfated hyaluronan, Tissue inhibitor of metalloproteinase-3, Vascular endothelial growth factor-A, Glycosaminoglycans, Endothelial cells ACS Paragon Plus Environment

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Abstract Several pathologic conditions such as rheumatoid arthritis, ocular neovascularization, cancer or atherosclerosis are often associated with abnormal angiogenesis, which require innovative biomaterialbased treatment options to control the activity of angiogenic factors. Here, we studied how sulfated hyaluronan (sHA) and over-sulfated chondroitin sulfate derivatives as potential components of functional biomaterials modulate vascular endothelial growth factor-A (VEGF-A) signaling and endothelial cell activity in vitro. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an effective angiogenesis inhibitor, exerts its activity by competing with VEGF-A for binding to VEGF receptor-2 (VEGFR-2). However, even though TIMP-3 and VEGF-A are known to interact with glycosaminoglycans (GAGs), the potential role and mechanism by which GAGs alter the VEGF-A/TIMP-3 regulated VEGFR-2 signaling remains unclear. Combining surface plasmon resonance, immunobiochemical analysis and molecular modeling, we demonstrate the simultaneous binding of VEGF-A and TIMP-3 to sHA-coated surfaces and identified a novel mechanism by which sulfated GAG derivatives control angiogenesis: GAG derivatives block the binding of VEGF-A and TIMP-3 to VEGFR-2 thereby reducing their biological activity in a defined, sulfation-dependent manner. This effect was stronger for sulfated GAG derivatives than for native GAGs. The simultaneous formation of TIMP-3/sHA complexes partially rescues the sHA inhibited VEGF-A/VEGFR-2 signaling and endothelial cell activation. These results provide novel insights into the regulation of angiogenic factors by GAG derivatives and highlight the potential of sHA derivatives for the treatment of diseases associated with increased VEGF-A and VEGFR-2 levels.

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1. Introduction Controlling angiogenic factors is an important goal in regenerative medicine, especially for neovasculature in ischemic tissues and tissue-engineered scaffolds to ensure sufficient supply with oxygen and nutrients1. Vascular endothelial growth factor (VEGF) family members are the major regulators of angiogenesis under both physiological and pathological conditions 2. They are homodimeric glycoproteins consisting of two subunits containing 120 - 200 amino acid residues. VEGF-A165 is the predominant isoform, which contains a heparin (HEP)-binding domain 3,4. Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides able to direct a variety of physiological functions. The variability in the net amount of sulfation, the particular sulfation patterns as well as the type of saccharide monomeric units within the carbohydrate backbone of GAGs leads to heterogenic molecules. These structural differences potentially conveys the complexity of GAG-mediated biological functions regarding their interaction with mediator proteins (e.g., growth factors), cells and extracellular matrix (ECM) components5. After VEGF-A secretion, a significant part is bound to the ECM and cell surface 6. The HEP-binding site of VEGF-A has a high affinity for sulfated GAGs, despite the fact that it only contains four basic residues 7. However, this affinity varies depending on the GAG type. For example, the affinity of VEGF-A for HEP (KD value of 22-42 nM) was reported to be higher compared to heparan sulfate (HS) (KD value of 20 µM) 8. Studies with mice lacking the HEP-binding VEGF-A isoforms demonstrated their requirement to initiate vascular branch formation 9. To induce signaling, VEGF-A can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2 as well as to the neuropilins belonging to a coreceptor family. The KD values for VEGF-A binding to VEGFR-2 are in the range of 75 - 760 pM while the KD value for binding to VEGFR-1 is about 10 pM

10,11

. However, VEGFR-2 is the major

signal transducer in angiogenesis since its tyrosine kinase activity is about 10-fold higher than for


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VEGFR-1 and gene knockout studies revealed the failure of blood-island formation and vasculogenesis in the absence of VEGFR-2 12,13. Tissue inhibitors of metalloproteinases (TIMPs) are a family with 4 members (TIMP-1 to -4) able to inhibit matrix degradation mainly via the inhibition of matrix metalloproteinases (MMPs)

14,15

. In

contrast to the solute TIMPs, TIMP-3 is bound to the ECM due to its interaction with sulfated GAGs (sGAGs)16. Recent studies revealed a dose- and sulfation-dependent binding of TIMP-3 to native sulfated GAGs like chondroitin sulfate (CS) and HEP as well as chemically sulfated hyaluronan (sHA) and CS (sCS) derivatives without changing its inhibitory potential against MMPs

17

. Especially these

GAG derivatives could have a strong clinical potential as component of functional biomaterials to influence biological processes during wound healing like angiogenesis, due to their homogeneous sulfation degree and more defined sulfation pattern compared to native GAGs. Furthermore, in a previous study we presented that the binding of sGAGs to TIMP-3 alters the interaction of TIMP-3 with the endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP-1) cluster II and IV leading to an accumulation of TIMP-3 in vitro 18. Independently of their functions as MMP inhibitors, TIMPs act as signaling molecules with cytokine-like activities. By this they influence various biological processes like cell growth, apoptosis, differentiation, angiogenesis and oncogenesis

19

.

TIMP-3 acts as an inhibitor of angiogenesis by binding to VEGFR-2, thereby blocking VEGF-A binding to its receptor 20. Recent studies revealed that the C-terminal domain of TIMP-3 is required for the specific blockage of VEGF-A binding to VEGFR-2 21. In general, angiogenesis requires a well-orchestrated interplay of different regulatory factors. Thus, studies on the interrelationship between VEGF-A and further angiogenesis regulators are required for the development of new therapeutical strategies e.g. against pathologic conditions associated with components involved in angiogenesis

13

. Since GAGs interact with VEGF-A and TIMP-3 separately,

the analysis of the possible interference of GAGs with VEGF-A and TIMP-3 together on complex signaling pathways like VEGF-mediated angiogenesis is required for a deeper understanding of the ACS Paragon Plus Environment

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molecular mechanisms underlying these processes. Therefore, in our study, the modulatory potential of sGAG derivatives on the TIMP-3-mediated inhibition of VEGF-A binding to VEGFR-2 was investigated using biophysical (i.e. surface plasmon resonance (SPR)), immunobiochemical and with molecular modeling approaches. Furthermore, the biological consequences of sGAG derivatives interference with VEGFR-2 phosphorylation and endothelial cell migration in vitro were analyzed to demonstrate the tuning capacity of sHA derivatives on the activity of VEGF-A as well as TIMP-3. These findings may foster the design of especially sGAG derivative-containing functional biomaterials, e.g. wound dressings, to modulate the activity of VEGF-A by altering the VEGF-A/VEGFR-2 signaling and thereby the endothelial cell activation in a defined manner.

2. Materials and methods 2.1 Materials HA (from Streptococcus, MW = 110 kDa) was purchased from Aqua Biochem (Dessau, Germany). HEP extracted from the porcine intestinal mucosa was available from Sigma-Aldrich (Schnelldorf, Germany). HA-, CS- and HEP-hexasaccharides (dp 6) were obtained from Iduron (Manchester, UK). Recombinant human TIMP-3 (973-TM-010), recombinant human VEGF-A165 (293-VE-010/CF), recombinant human VEGFR-2/Fc chimera (357-KD-050/CF) as well as monoclonal, mouse antihuman TIMP-3 (MAB973) and monoclonal, biotinylated, mouse antihuman TIMP-3 (BAM9731), monoclonal mouse antihuman VEGF (MAB293) and biotinylated, polyclonal, goat antihuman VEGF (BAF293) antibodies were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany). The Series S Senor Chips C1TM and HBS-EP were obtained from GE Healthcare Europe GmbH (Freiburg, Germany). 2.2 Preparation and characterization of oligomeric and polymeric sulfated GAG derivatives


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The polymeric HA and CS derivatives were synthesized and characterized according to previous protocols

22–24

. Analytical characteristics of the used GAG poly- and oligosaccharides are shown in

Table 1 and 2. For the preparation of HA oligosaccharides (Fig. 1), native HA was enzymatically depolymerized by treatment with hyaluronidase from bovine testes (Sigma-Aldrich, Schnelldorf, Germany). The respective dp 4 and dp 6 were extracted and purified by chromatography. Persulfated HA oligosaccharides were synthesized according to the previously published protocol

25

. Disulfated

HA oligosaccharide (sHA1, dp 4) was directly synthesized using sulfur trioxide pyridine complex (6 eq.), purified by anion exchange chromatography (gradient 0 - 0.6 M NaCl) and two times desalted over Sephadex G-10. The medium-sulfated HA oligosaccharide (sHA2∆6s ,dp 4), that is exclusively sulfated at the C2 and C3 position of the glucuronic acid unit, was obtained by temporarily protecting the 4,6-diol moieties with phenyl boronic acid and subsequent persulfation. The mixture was purified by anion exchange chromatography (gradient 0 - 1.2 M NaCl) and two times desalted over Sephadex G-10.


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Figure 1. Sulfated hyaluronan tetrasaccharides. (a) Structure of sHA1 (dp 4), (b) sHA2∆6s (dp 4) and (c) psHA (dp 4). Table 1. Analytical data of synthesized GAG polysaccharides. Respective degree of sulfation (D.S.) per repeating disaccharide unit (D.U.) of GAG, weight-average (Mw) values of the molecular weight as determined by laser light scattering detection. The molecular weight distributions (polydispersity index: PD) based on the values calculated from refraction detection. HAGAG

sHA3-

HA

sHA1

sHA3

Biotin

CS

sCS3

HEP

Biotin

D.S.

-

-

1.0

2.9

3.6

0.8

3.1

2.2

Mw [g mol-1]

48 255

33 000

31 056

20 950

29 000

19 763

19 915

18 000

462´,

462´,

462´,

Sulfate group

62´, 2N, -

-

6

463´,

463´,

4, 6

463´,

distribution

6 62´3´

62´3´

62´3´

Table 2. Analytical data of GAG oligosaccharides. GAG

HA

sHA1

CS

HEP

sHA2∆6s

psHA

D.S.

-

-

1.0

1.0

2.0

2.0

4.0

4.0

Mw [g mol-1]

777

1 204

1 005

1 488

1 800

1 355

1 763

2 573

4

6

4

6

6

4

4

6

-

-

6

4, 6

62´, 2N

2´3´

462´3´

462´3´

Degree of polymerization (dp) Sulfate group distribution


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2.3 Surface plasmon resonance analysis 2.3.1 Immobilization of VEGFR-2 on Sensor Chips A BiacoreTM T100 instrument (GE Healthcare) was used to measure potential binding events. VEGFR-2 was covalently bound to Series S Sensor Chips C1TM via amine coupling at 25 °C according to the manufacturer (GE Healthcare). Injection of 80 µg/ml for 120 sec at a flow rate of 5 µl/min resulted in an immobilization level of approximately 53 RU. A surface treated equally without immobilized VEGFR-2 was used as reference. 2.3.2 SPR analysis of TIMP-3 or VEGF-A binding to immobilized VEGFR-2 in the presence of GAGs HBS-EP containing 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20 was used as running buffer. All interactions were analyzed at 37 °C using a flow rate of 30 µl/min. The running buffer was used for protein and GAG dilutions. After three start-up injections with buffer, 100 nM TIMP-3 alone or after pre-incubation with 100 µM related to the molecular weight of D.U. of polymeric GAGs for 1 h was injected over the sensor chip surface for 120 s at 30 µl/min. To analyze the influence of oligomeric GAGs on the TIMP-3/VEGFR-2 interaction, 20 nM TIMP-3 was preincubated with 40-80 µM D.U. GAG oligosaccharides. The VEGF-A/VEGFR-2 interaction was studied by injecting 20 nM VEGF-A165 after pre-incubation with 20-40 µM D.U. GAG polysaccharide or 40-80 µM D.U. GAG oligosaccharide solutions for 1 h at room temperature using similar conditions. Equal molar concentrations of D.U. were used to compare GAGs since they characterize the potential binding sites for interactions with proteins. Furthermore, GAG solutions without TIMP-3 were injected over the VEGFR-2 surface to study a possible GAG/VEGFR-2 interaction. Binding levels were recorded 10 s before the end of analyte injection relative to a baseline report point to determine a ranking between different GAG derivatives. After 600 s dissociation, the chip surface was regenerated for 200 s with 60 mM HCl (5 µl/min) and for 60 s with 5 M NaCl (30 µl/min), followed by 1000 s stabilization. Sequential injections of 40 nM ACS Paragon Plus Environment

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TIMP-3, 40 nM VEGF-A and 40 µM D.U. GAGs were performed by 120 s injection of each ligand after a short dissociation phase of 30 s at a flow rate of 30 µl/min and a dissociation phase of 600 sec after the third sample injection without regeneration after the first two sample injections. All values represent the mean ± standard deviation of n=3. Evaluation of binding parameters was performed by using the Biacore T100 evaluation software 2.03. Reference and blank subtraction were used to create specific Biacore sensorgrams as described previously 26. 2.3.3 Competitive SPR analysis of immobilized VEGFR-2 and TIMP/VEGF-A/GAG complexes Interaction analysis of a mixture of TIMP-3 and VEGF-A with VEGFR-2 in the presence of GAGs were performed via pre-incubation of equal molar concentrations of TIMP-3 and VEGF-A in the absence or presence of GAG polysaccharides (5 nM - 50 µM D.U.) or GAG oligosaccharides (80 µM D.U.). Injections of TIMP-3 or VEGF-A alone served as further controls to evaluate the binding capacity of VEGFR-2 after regeneration. 2.3.4 Kinetic analysis of the TIMP-3/VEGFR-2 and VEGF-A/VEGFR-2 interaction TIMP-3 or VEGF-A were immobilized onto Series S Sensor Chips C1TM via standard amine-coupling (GE Healthcare) to final immobilization levels of 142 RU TIMP-3 or 33 RU in case of VEGF-A using 10 µg/ml TIMP-3 in 10 mM sodium acetate buffer (pH 4.5) or 0.7 µg/ml VEGF-A in 10 mM sodium acetate buffer (pH 5.5). HBS-EP was used as running buffer and for all sample dilutions. Single cycle kinetics were performed at 37°C with five sequentially injections of 0.44-7.1 nM VEGFR-2 over a TIMP-3 surface or 1.58 - 25.25 nM VEGFR-2 over a VEGF-A surface without regenerations inbetween the sample injections for 240 sec followed by a dissociation phase of 600 sec at a flow rate of 30 µl/min. A cycle with five buffer injects served as blank. After the sequential injects the surfaces were regenerated by injecting 60 mM HCl for 200 sec (5 µl/min) and 5 M NaCl for 60 sec (30 µl/ml). The binding kinetic parameters were determined using the Biacore evaluation software 2.03 and a 1:1 Langmuir binding fitting model. χ2 was used as statistical parameter to assess the fit. It is calculated


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according to the following formula: χ2 =

∑

( )

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with rf being the fitted value at a distinct point, rx

representing the experimental value at this distinct point, n giving the number of data points and p being the number of fitted parameters. This parameter indicates the agreement of the fitted curves with the experimentally obtained data. It should be low in case of a good fit. In addition, the standard error was used to indicate the significance of the determined KD values. 2.4 GAG-coated surfaces and competitive enzyme-linked immunosorbent assay (ELISA) The binding capacity of TIMP-3 to GAG polysaccharides in the presence of VEGF-A was studied using a competitive ELISA with immobilized GAG derivatives and solute ligands. Therefore, biotinylated non- or high-sulfated HA were coated onto 96-well microtiter plates (MaxiSorp®, Nunc) as described previously

27

. In brief, plates were incubated overnight at 4°C with 100 µl 10 µg/ml

NeutrAvidinTM biotin binding protein (Thermo Scientific, Schwerte, Germany) dissolved in PBS per well. After three times washing with 0.05% Tween 2 in PBS, 50 µl 2.5 mM D.U. biotinylated HA derivative was added to each well. After 2 h of incubation at room temperature, the plates were washed as described above and blocked with 1% bovine serum albumin (BSA) in PBS for 2 h. Wells coated with 1% BSA served as controls. Either 50 µl 1.8 nM TIMP-3, 1.8 nM VEGF-A or a solution containing 1.8 nM TIMP-3 and 1.8 nM VEGF-A in 1% BSA in PBS were added per well. After overnight incubation at 4°C, the supernatants were collected and used for indirect measurements of the bound ligands via sandwich ELISA of TIMP-3 or VEGF according to the manufacturer´s protocols (R&D Systems, Wiesenbaden-Nordenstadt, Germany). 2.5 Modeling of TIMP-3 and VEGFR-2 interaction Structures. For modeling, the experimental structures of the VEGF-A/VEGFR-2 complex was used (PDB ID: 2V2A, 3.2 Å). The structure of VEGFR-2 was extracted from this complex and minimized using a standard procedure in MOE

28

. The full TIMP-3 structure was obtained by combining the


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experimentally available structure of its N-terminal domain (PDB ID: 3CKI, 2.30 Å) and the structure of its C-terminal domain obtained by homology modeling as described in our previous work 17. Protein-protein docking. Docking of TIMP-3 to VEGFR-2 was carried out using ZDOCK29 and CLUSPRO 2.0 30 with default parameters. Electrostatic potential calculations. Electrostatic potential isosurfaces were calculated with AMBER14

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using the PBSA program from AmberTools with the grid spacing of 1 Å and visualized

with VMD 32. 2.6 Cell culture of porcine endothelial cells (PAE/KDR) PAE cells stable transfected with VEGFR-2 (KDR) were kindly provided from Prof. Johannes Waltenberger (University Münster, Germany). Cells were cultivated on tissue culture polystyrene in Dulbecco´s Modified Eagle´s Medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum (Lonza, Verviers, Belgium), 1% penicillin/streptomycin (Biochrom AG, Berlin, Germany) and 2 mM L-glutamine (Biochrom AG, Berlin, Germany) at 37°C in a humidified atmosphere with 5% CO2 up to 85-90% confluency before splitting or using in stimulation or migration assays. 2.7 Stimulation assay and quantification of VEGFR-2 phosphorylation 2 x 106 PAE/KDR cells were seeded in 6 cm petri dishes (cellstar, Greiner, Frickenhausen, Germany) and incubated overnight (37°C, 5% CO2) before starving in serum free media containing 0.1% BSA for 2 hours. Cells were incubated with the inhibitor TIMP-3 (50 nM) for 30 min prior stimulation with 1 nM VEGF-A for 10 min. The VEGF-A-induced VEGFR-2 phosphorylation in the absence or presence of TIMP-3 and/or solute 200 µM D.U. sHA3 polysaccharide was quantified by sandwich enzymelinked immunosorbent assays (ELISA) applying the total VEGFR-2 and phospho-VEGFR-2 detection kits (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to the manufacturer´s protocols. The protein concentrations of the respective cell lysates were determined via NanoDrop 1000 (Thermo Scientific, Schwerte, Germany) using the ND-1000 software. ACS Paragon Plus Environment

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2.8 Migration of PAE/KDR cells The effect of VEGF-A in the presence of TIMP-3 and solute GAGs on the cell migration was determined via the OrisTM Cell Migration assay (Platypus Technologies, Hamburg, Germany). 30,000 PAE/KDR cells/well were seeded into 96 well OrisTM plates and allowed to adhere for 4 h. The cells were starving overnight in media without serum containing 0.1% BSA. 2.5 h before starting the experiment, cell proliferation was blocked by the addition of 10µg/ml Mitomycin C. Afterwards, cells were incubated with sample solutions (10 nM VEGF-A, 100 nM TIMP-3, 200 µM D.U. GAG polysaccharides or several mixtures containing two or all three components) for 26 h in a humidified chamber (37°C, 5% CO2). Cell migration was quantified by fluorescent staining of the cells with 0.5 µg/ml Calcein AM in PBS (containing both Ca2+ and Mg2+) for 60 min prior measuring the fluorescence intensity (TECAN reader infinite M1000 PRO, λex = 485 nm, λem = 528 nm) of the exclusion zones after attaching a detection mask to the plate according to the manufacturers protocol. 2.9 Statistics For statistical significance of the obtained data, experiments were performed at least in triplicate. All results are shown as mean ± standard deviation. One-way ANOVA with Tukey test was used to evaluate significant differences between treatment groups. Two-way ANOVA (analysis of variance) with Bonferroni post-test was applied to data with both different treatments and GAGs. The values of the kinetic analysis via SPR represent the mean ± standard error as determined via the Biacore T100 evaluation software 2.03 (GE Healthcare).


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3. Results 3.1 Kinetic analysis of TIMP-3 and VEGF-A binding to VEGFR-2 The kinetic parameters for the TIMP-3/VEGFR-2 and VEGF-A/VEGFR-2 interaction were determined via SPR single cycle kinetic measurements. The obtained curves were fitted to a 1:1 model assuming a 1:1 stoichiometry for both interactions (Fig. 2). For the ligand TIMP-3 a kon of (4.89 ± 0.06) x 106 M-1s1

and a koff of (1.05 ± 0.01) x 10-3 s-1 (χ2 = 2.26) and for VEGF-A a kon of (3.40 ± 0.02) x 105 M-1s-1 and

a koff of (2.50 ± 0.01) x 10-4 s-1 (χ2 = 0.63) were determined. This results in a KD value of 215 pM for the TIMP-3/VEGFR-2 interaction and a KD value of 737 pM for VEGF/VEGFR-2.

Figure 2. Interaction of TIMP-3 and VEGF-A with VEGFR-2. Kinetic analyses of the VEGFR-2 interaction with TIMP-3 (a) and VEGF-A165 (b) via SPR. Increasing concentrations of VEGFR-2 (0.44 - 7.10 nM VEGFR-2 in case of the TIMP-3 surface; 1.58 - 25.25 nM VEGFR-2 in case of the VEGF-A


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surface) were injected over sensor chip surfaces with immobilized TIMP-3 (142 RU) or VEGF-A (33 RU). The obtained sensorgrams are shown in black, while the fitted curves are displayed in blue. 3.2 Sulfated GAGs regulate the TIMP-3/VEGFR-2 interaction The potential influence of sulfated GAGs (Tab. 1, Tab. 2, and Fig. 1) on the interaction of TIMP-3 with VEGFR-2 was analyzed by SPR (Fig. 3). Control experiments revealed no detectable binding of solute GAGs to VEGFR-2 alone. Binding analysis showed that the pre-incubation of TIMP-3 with native and chemically sulfated GAG polysaccharides led to a sulfation-dependent reduction of the TIMP-3 binding to VEGFR-2 in the following order: HA < CS < sHA1 ≈ HEP < sCS3 ≈ sHA3 (Fig. 3 a-c). All analyzed sHA3 concentrations led to a significantly decrease of the TIMP-3/VEGFR-2 complex formation (Figure S1 a). Non-sulfated HA and CS oligosaccharides marginally changed the TIMP-3/VEGFR-2 interaction while the presence of sHA1 tetrasaccharide (dp 4, dp stays for degree of polymerization) reduced the TIMP-3 binding to VEGFR-2 (Fig. 3 d). HEP (dp 6) significantly inhibited the TIMP-3 binding to the receptor. In contrast, sHA2∆6s (dp 4), which is exclusively sulfated at the glucuronic acid unit, had no detectable effect on the interaction even though the degree of sulfation is comparable to HEP. The persulfated HA tetrasaccharide (psHA, dp 4) exhibited the strongest influence even in comparison to their corresponding longer oligosaccharide (psHA, dp 6) (Fig. 3 e).


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Figure 3. Influence of GAGs on the TIMP-3/VEGFR-2 interaction. Representative SPR sensorgrams for the binding of 100 nM TIMP-3 after preincubation with 100 µM related to the molecular weight of disaccharide units (D.U.) of non- or low-sulfated GAGs (a) or high-sulfated GAGs (b) to immobilized VEGFR-2 (124 RU). Normalized binding of TIMP-3 to immobilized VEGFR-2 (124 RU, 69 RU) after pre-incubation with GAG poly- (c) and oligosaccharides (d, e) relative to TIMPACS Paragon Plus Environment

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3 alone. One-way ANOVA: #p < 0.05, ##p < 0.01, ###p < 0.001 vs. TIMP-3 w/o GAGs; *p < 0.05, **p < 0.01 vs. respective treatment; Fig. 3c: a, p < 0.001 vs. TIMP-3 + HA; b, p < 0.001 vs. TIMP-3 + CS; c, p < 0.001 vs. TIMP-3 + HEP; Fig. 3e: a, p < 0.001 vs. TIMP-3 + 40 µM D.U. HEP (dp 6); b, p < 0.001 vs. TIMP-3 + 80 µM D.U. HEP (dp 6); c, p < 0.001 vs. TIMP-3 + 40 µM D.U. sHA2∆6s (dp 4); d, p < 0.001 vs. TIMP-3 + 80 µM D.U. sHA2∆6s (dp 4); e, p < 0.001 vs. TIMP-3 + 40 µM D.U. psHA (dp 4). Electrostatic potential isosurfaces (blue-positive, red-negative) on TIMP-3 (f) and VEGFR-2 (g). Isovalues of 1000 kcal mol-1 e-1 for TIMP-3, 500 kcal mol-1 e-1 and -200 kcal mol-1 e-1 for VEGFR2 were chosen for visualization. Docking results obtained by ZDOCK (h) and CLUSPRO 2.0 (i) for docking TIMP-3 to VEGFR-2: resulting TIMP-3 centers of mass are depicted as red spheres. VEGFR2 is shown in blue cartoon representation. VEGF-A/VEGFR-2 complex (PDB ID: 2V2A) in cartoon representation: VEGF-A is displayed in yellow and VEGFR-2 in blue, respectively (j).

Electrostatic potential calculations for TIMP-3 (Fig. 3 f) and VEGFR-2 (Fig. 3 g), protein-protein docking calculations and analysis of the VEGF-A/VEGFR-2 interface (Fig. 3 h-j) from the experimental complex structure were performed. The obtained data suggest that TIMP-3 can potentially compete with VEGF-A for the VEGF binding site on the surface of VEGFR-2 due to the complementarity of their surficial electrostatic properties. GAGs interacting with a positively charged region on the TIMP-3 surface17 could potentially disrupt the putative interactions between TIMP-3 and VEGFR-2. Sulfated GAGs alter the VEGF-A/VEGFR-2 binding profile Binding of VEGF-A to VEGFR-2 (Fig. 4) decreased in the presence of native and chemically sulfated GAG polysaccharides in a sulfation-dependent manner. While HA and CS had no significant effect on the VEGF-A binding to VEGFR-2, sHA1 significantly blocked VEGF-A binding to its receptor. Native HEP as well as sHA3 and sCS3 almost completely inhibited the VEGF-A/VEGFR-2 interplay (Fig 4 a,


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b). Depending on the sHA3 concentration significant inhibition was detected for 0.2 to 200 µM D.U. but not for 2 nM D.U. (Figure S1 b). The oligosaccharides of HA (dp 4, 6), CS (dp 6) and sHA2∆6s (dp 4) did not affect the binding of VEGF-A to its receptor VEGFR-2 (Fig. 4 c, d). All other examined sHA and HEP (dp 6) oligosaccharides significantly diminished VEGF-A/VEGFR-2 complex formation. Interestingly, HEP (dp 6) had a much stronger impact on this growth factor/receptor interaction than sHA2∆6s, even though their degree of sulfation is comparable. The interference of psHA was stronger than for the other oligosaccharides and further increased with chain length (Fig. 4 d).


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Figure 4. Influence of GAGs on the VEGF-A/VEGFR-2 interaction. Binding of VEGF-A to immobilized VEGFR-2 (53 RU, 69 RU) after pre-incubation with GAG poly- and oligosaccharides. Representative SPR sensorgrams for the binding of 20 nM VEGF-A165 in the presence of 40 µM D.U. chemically sulfated HA derivatives and HEP are displayed in (a). The normalized binding levels relative to VEGF-A alone after pre-incubation with GAG polysaccharides (b), non- and low-sulfated GAG oligosaccharides (c) or high-sulfated oligosaccharides (d) are shown. One-way ANOVA: #p < 0.05, ###p < 0.001 vs. VEGF-A w/o GAGs; *p < 0.05, **p < 0.01 vs. respective treatment. Fig. 4 b: a, p < 0.001 vs. VEGF-A + HA; b, p < 0.001 vs. VEGF-A + CS; c, p < 0.001 vs. VEGF-A + sHA1. Fig. 4 d: a, p < 0.001 vs. VEGF-A + 80 µM D.U. HEP (dp 6); b, p < 0.001 vs. VEGF-A + 40 µM D.U. sHA2∆6s (dp 4); c, p < 0.001 vs. VEGF-A + 80 µM D.U. sHA2∆6s (dp 4); d, p < 0.001 vs. 40 µM D.U. psHA (dp 4). GAGs interfere with the VEGF-A/TIMP-3 competition for VEGFR-2 The interference of sGAGs with the competition of VEGF-A and TIMP-3 for the binding to VEGFR-2 was analyzed after simultaneous pre-incubation of VEGF-A and TIMP-3 with HA and sHA polysaccharides or GAG oligosaccharides (Fig. 5). The binding level of TIMP-3 alone to VEGFR-2 was found to be higher than for VEGF-A alone, while the combination of TIMP-3 and VEGF-A led to a binding response comparable to TIMP-3 alone (Fig. 5 a). Polymeric sHA polysaccharides sulfationdependently reduced the binding response, while non-sulfated HA did not. However, the lowest examined concentration of sHA3 (5 nM D.U.) had no significant impact on the protein-receptor interaction. Higher GAG to protein ratios were required to significantly reduce the binding response of TIMP-3 and VEGF-A for sulfated GAG oligosaccharides compared to their polymeric counterparts (Fig. 5 a, b). sHA oligosaccharides, with the exception of sHA2∆6s, significantly diminish the proteinVEGFR-2 interplay in a sulfation-dependent manner while non-sulfated HA (dp 4, 6), CS (dp 6) and


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HEP (dp 6) did not (Fig. 5 b). Nevertheless, HEP (dp 6) slightly blocked the binding of the VEGF-A and TIMP-3 mixture to VEGFR-2.

Figure 5. Influence of GAGs on the TIMP-3 and VEGF-A interplay with VEGFR-2 and binding of VEGF or TIMP-3 to GAG surfaces. Normalized SPR binding levels of TIMP-3 to immobilized VEGFR-2 (53 RU) after pre-incubation with VEGF-A165 and sHA polysaccharides (a) or GAG oligosaccharides (b) relative to TIMP-3 and VEGF-A alone. One-way ANOVA: #p < 0.05, ##p < 0.01,


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###p < 0.001 vs. TIMP-3 and VEGF-A w/o GAGs; *p < 0.05, **p < 0.01 vs. respective treatment. Fig. 5 a: a, p < 0.001 vs. TIMP-3; b, p < 0.001 vs. VEGF-A; c, p < 0.001 vs. TIMP-3 + VEGF-A + HA; d, p < 0.001 vs. TIMP-3 + VEGF-A + sHA1; e, p < 0.001 vs. TIMP-3 + VEGF-A + 5 nM D.U. sHA3; f, p < 0.001 vs. TIMP-3 + VEGF-A + 500 nM D.U. sHA3. Fig. 5 b: a, p < 0.001 vs. TIMP-3 + VEGF-A + HA (dp 4); b, p < 0.001 vs. TIMP-3 + VEGF-A + HA (dp 6); c, p < 0.001 vs. TIMP-3 + VEGF-A + CS (dp 6); d, p < 0.001 vs. TIMP-3 + VEGF-A + sHA2∆6s (dp 4). Sequential injection of 40 nM TIMP-3, 40 µM D.U. sHA3 and 40 nM VEGF-A (c) or 40 nM VEGF-A165, 40 µM D.U. sHA3 and 40 nM TIMP-3 (d) over sensor chip surfaces with immobilized VEGFR-2 (53 RU, 69 RU, 51 RU). The data represent the normalized binding levels of the respective injected protein relative to VEGF-A alone determined by SPR. Recovery of VEGF-A (e) or TIMP-3 (f) in the absence or presence of equimolar concentrations of TIMP-3 or VEGF-A after binding to GAGs immobilized to polystyrene surfaces as determined by sandwich ELISA. BSA coated wells were used as controls (Ctrl). Two-way ANOVA: ***p < 0.001 vs. respective treatment. The potential influence of the order of binding events was analyzed via SPR as well. Therefore, solutions containing only one analyte (first TIMP-3, second sHA3 and third VEGF in case of Fig. 5 c or first VEGF, second sHA3 and third TIMP-3) were sequentially injected to VEGFR-2 surfaces without regeneration between the injections. Binding analysis revealed no significant differences between the binding levels of the respective proteins or sHA3 depending on the order of their injection (Fig. 5 c compared to Fig. 5 d). sHA3 showed none or only a marginal binding to pre-formed TIMP3/VEGFR-2 or VEGF-A/VEGFR-2 complexes. VEGF-A reduces the binding of TIMP-3 to sulfated GAG surfaces Since VEGF-A and TIMP-3 are both binding partners of native sGAGs, the influence of VEGF-A on the binding of TIMP-3 to HA or sHA3-coated surfaces was studied via competitive ELISA experiments with immobilized GAGs. The remaining VEGF-A or TIMP-3 amounts in the supernatants were


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quantified by sandwich ELISAs (Fig. 5 e/f). TIMP-3 and VEGF-A alone displayed a sulfationdependent binding to GAGs (white bars). The combination of VEGF-A and TIMP-3 (gray bars) did not significantly alter the VEGF-A recovery (Fig. 5 e), while the TIMP-3 recovery markedly increased from about 23% to 62% in the presence of competing VEGF-A in case of immobilized sHA3 (Fig. 5 f). In contrast, the protein recoveries from the BSA- or HA-coated surfaces remained comparable between the different treatments. GAGs modulate VEGF-A/TIMP-3 regulated endothelial cell functions The impact of GAGs on VEGF-A-induced VEGFR-2 phosphorylation was studied with an endothelial cell stimulation assay and afterwards quantified via ELISA (Fig. 6 a). To determine the influence of GAGs on the VEGFR-2 mediated endothelial cell migration, cells were seeded around a physical barrier and after its removal, cells were allowed to migrate into the exclusion zone for 26 h (Fig. 6 b). GAGs alone had no influence on neither VEGFR-2 phosphorylation nor on endothelial cell migration. Also non-sulfated HA did not alter VEGF-A-induced cell migration, while sHA3 strongly reduced receptor phosphorylation and migration. The presence of TIMP-3 in addition to VEGF-A led to a decreased VEGFR-2 phosphorylation and cell migration, which was comparable to the non-treated control cells. A co-treatment with VEGF-A, TIMP-3 and sHA3 led to a significantly increased VEGFR-2 phosphorylation and cell migration compared to the treatment with VEGF-A and TIMP-3. Compared to the TIMP-3 inhibition, sHA3 increased the VEGF-A activity for nearly 35% in the VEGFR-2 phosphorylation assay and for nearly 38% in the cell migration assay. However, the examined GAG concentration could not completely rescue the VEGF-A bioactivity. Additional SPR measurements using the same concentrations as in the in vitro stimulation assay for the VEGFR-2 phosphorylation were performed (Figure S2). Here, 200 µM D.U. sHA3 significantly reduced the binding of 1 nM VEGF-A or 50 nM TIMP-3 to VEGFR-2. By trend, the presence of 1 nM


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VEGF-A, 50 nM TIMP-3 and 200 µM D.U. sHA3 lead to a slightly higher binding response compared to 1 nM VEGF-A and sHA3 or 50 nM TIMP-3 and sHA3.

Figure 6. Influence of GAGs on the VEGF-A/TIMP-3 regulated VEGFR-2 phosphorylation and endothelial cell migration. (a) 1 nM VEGF-A165-induced phosphorylation of VEGFR-2 in the presence or absence of 50 nM TIMP-3 and/or 200 µM D.U. sHA3. VEGFR-2 phosphorylation was normalized to the total amounts of VEGFR-2 relative to the total protein concentrations of the cell ACS Paragon Plus Environment

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lysates. (b) Migration of PAE/KDR endothelial cells after stimulation with 10 nM VEGF-A165 relative to an untreated control in the presence or absence of 100 nM TIMP-3 and 200 µM D.U. GAGs. Oneway ANOVA: #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Ctrl (media w/o VEGF-A, TIMP-3 or sHA3); *p < 0.05, **p < 0.01 vs. respective treatment. Fig. 6 a: a, p < 0.001 vs. VEGF-A; b, p < 0.001 vs. VEGF-A + TIMP-3 + sHA3. Fig. 6 b: a, p < 0.001 vs. VEGF-A; b, p < 0.001 vs. VEGF-A + TIMP-3; c, p < 0.001 vs. VEGF-A + HA.

4. Discussion There is an unmet need for novel functionalized biomaterials to control and re-balance abnormal angiogenesis. VEGF-A signaling through VEGFR-2 represents a key pathway for the regulation of angiogenesis, which can be inhibited by the VEGF competitor TIMP-3

20,33

. Native HEP/HS

proteoglycans are able to bind TIMP-3 and VEGF-A in the pericellular matrix and function as coreceptors for VEGF by modulating the complex affinity of VEGF-A for VEGFR-2 and neuropilin-1. Both pro- and anti-angiogenic effects of GAGs were previously described

34–38

. However, these

contrasting data on the potential role of GAGs are often restricted to HEP, which is known for its heterogeneity regarding the carbohydrate backbone, sulfation degree and pattern

39

. We therefore

examined how TIMP-3, VEGF-A and native GAGs as well as chemically more defined sulfated sGAG derivatives work together in the VEGF-A signaling process to gain a deeper molecular and biological understanding of their interplay. VEGF-A165 was chosen for all experiments since it is the major isoform of VEGF-A containing a highly basic HEP-binding domain 40. Interestingly, our kinetic analysis revealed for the first time that the KD values characterizing the interaction of TIMP-3 and VEGF-A with VEGFR-2 are both in the pM range (~215 pM vs. ~737 pM) (Fig. 2). This is in line with KD values reported in the literature for VEGF-A/VEGFR-2 binding ranging from 75 to 770 pM 8,10,11,41,42.


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Furthermore, we show that the formation of VEGF-A/GAG or TIMP-3/GAG complexes inhibits binding of both proteins to VEGFR-2 in a sulfation-dependent manner (Fig. 3, Fig. 4). Interestingly, even a concentration of 2 nM D.U. sHA3 was sufficient to significantly reduce the binding of TIMP-3 to VEGFR-2, while VEGF-A was less sensitive against this sHA3 concentration (Fig. S1). The binding of sGAGs to TIMP-3 and VEGF-A explains the reduced binding of these proteins since GAGs alone did not interact with VEGFR-2, and TIMP-3 and VEGF-A did not interact with each other in SPR measurements. This is in line with previously reported results 20,35, albeit Di Benedetto et al. detected a direct binding of HEP-albumin to VEGFR-2

38

. Likewise, a sulfation-dependent inhibitory effect of

sHA derivatives on the interaction of bone morphogenic protein (BMP)-2 with its receptor IA was described

43

. The mechanism responsible for the reduced TIMP-3/VEGFR-2 and VEGF-A/VEGFR-2

interplay in the presence of sGAGs in SPR measurements was supported by molecular modeling. The computational results demonstrate that negatively charged GAGs compete with VEGFR-2 for the binding to positively charged regions of TIMP-3. In line with this, it was reported earlier that TIMP-3 has three GAG binding sites located at the N- and C-terminal domains, and that the C-terminal domain is particularly important for the function as a VEGF competitor

17,21

. Furthermore, our SPR sequential

injection analysis indicates that the GAG binding regions of TIMP-3 and VEGF-A are occupied after binding to VEGFR-2 resulting in a strongly decreased sHA3 binding capacity of both proteins (Fig. 5 c/d). Interestingly, GAG polysaccharides interfered stronger than oligosaccharides with VEGF-A or TIMP-3 binding to VEGFR-2 (Fig. 3, Fig. 4), suggesting a stabilizing effect of the GAG/protein interaction and an enhanced sterically hindrance of the receptor binding with increasing GAG chain length. This effect of the chain length was also apparent for psHA (dp 4, dp 6) in case of the VEGF-A/VEGFR-2 interplay, but not for the TIMP-3/VEGFR-2 interaction possibly due to the reported higher binding capacity of TIMP-3 for psHA (dp 4) than for psHA (dp 6)20. In accordance, Cochran et al. reported a higher affinity of HEP (dp 40) for VEGF-A than for low molecular weight HEP (dp 10) (KD of 22 - 42 ACS Paragon Plus Environment

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nM vs. 660 - 840 nM) 8. Teran and Nugent also described that HEP can affect the complex formation of VEGF with its receptors. In contrast to our results, they observed an enhanced binding of solute VEGF-A to immobilized VEGFR-2 after the simultaneous addition of solute HEP without preincubation. The authors suggested the formation of a new synergistic HEP binding region in the VEGF/VEGFR-2 complex

35

. Differences between HEP sources can lead to high structural

variabilities. However, the extent of these differences and the underlying structural requirements of HEP for the inhibiting or promoting effects on the VEGF-A/VEGFR-2 interaction are challenging to assess since the molecular weights were the only chemical characteristics provided by Teran and Nugent and the examined VEGF-A to HEP ratio was not mentioned 35. Regarding the structural characteristics of GAGs required to interfere with the VEGF-A and TIMP-3 interaction with VEGFR-2, we revealed that especially the sulfation of the primary hydroxyl group of the N-acetylglucosamine unit of HA is important for reducing the binding of both proteins to VEGFR2. This impact was especially evident for HA (dp 4) showing that sHA1 (dp 4) blocked the VEGF-A or TIMP-3 interaction with VEGFR-2, while CS (dp 6) with a comparable D.S. or sHA2∆6s (dp 4) with no sulfation of the N-acetylglucosamine residues, did not change the protein-receptor binding profiles in most cases (Fig. 3, Fig. 4). This agrees well with previous results showing that 6-O-desulfated HEP did not interact with VEGF-A, and that it had a lower affinity to TIMP-3 compared to HEP 34,44. Competitive ELISA experiments revealed that VEGF-A is the preferred interaction partner of sHA3, reducing but not completely eliminating the TIMP-3/sHA3 interaction (Fig. 5). In line with this, the affinity of VEGF-A for HEP is 1.4 - 2.7 times higher than the affinity of TIMP-3 for HEP (KD of ~59 nM)

8,44

. Likewise, we observed that poly- and oligomeric HEP interfered stronger with the VEGF-

A/VEGFR-2 interaction than with the TIMP-3/VEGFR-2 binding, e.g. 40 µM D.U. HEP (dp 6) led to a 54% decreased binding of 20 nM VEGF-A to VEGFR-2, while equal concentration reduced the binding of 20 nM TIMP-3 by 24%.


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To better reproduce the natural conditions and to understand the molecular events required for VEGFR2 binding, we performed SPR measurements with solute TIMP-3, VEGF-A and GAGs and immobilized VEGFR-2 together in one experimental set-up. Here, the TIMP-3 binding to VEGFR-2 resulted in a higher response signal compared to equimolar concentrations of VEGF-A alone, which correlates well with the determined kinetic parameters. TIMP-3 and VEGF-A together show no cumulative binding compared to the binding response of both proteins alone due to the competition between both proteins for the binding to VEGFR-2

20

. The combination of all interaction partners

within one SPR approach revealed that sulfated GAG derivatives also concurrently reduce the VEGF-A and TIMP-3 binding to the VEGFR-2 in a sulfation- and concentration-dependent manner (Fig. 5). Interestingly, these effects are more pronounced for psHA (dp 4, dp 6) than for native CS (dp 6) or HEP (dp 6). This corresponds to the results obtained for the impact of GAG polysaccharides on the TIMP-3/VEGFR-2 interaction (Fig. 3) and possibly attributes to the higher negative charge of highsulfated GAG derivatives, which increases the electrostatic component of GAG/mediator binding. Likewise, high-sulfated HA and CS derivatives have a higher binding capacity for TIMP-3 compared to native GAGs like HEP

17

. The same trend was also apparent for VEGF-A/VEGFR-2, even though

the differences between HEP and sCS3 or sHA3 were insignificant (Fig. 4). However, a potential further impact of the carbohydrate backbone cannot be excluded. Taken together, we could reveal beside the sulfation-dependent increase of inhibition that even sHA1 tetrasaccharides were sufficient to interfere with the VEGF-A/VEGFR-2 and TIMP-3/VEGFR-2 complex formation. Furthermore, we could highlight the importance of the GAG carbohydrate backbone since CS had a lower impact on the protein/receptor interplay than sHA1 even though their degree of sulfation is comparable. To further evaluate the biological relevance of these VEGF-A- and TIMP-3-GAG derivative interactions, we performed cell culture experiments with porcine aortic endothelial cells heterotopically expressing VEGFR-2 (PAE/KDR cells). Our results revealed that the binding of sHA3 to VEGF-A ACS Paragon Plus Environment

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strongly reduces its VEGFR-2 mediated bioactivity in vitro (Fig. 6), which is consistent with our SPR data. Ashikari-Hada et al., however, found a 1.7 fold higher VEGFR-2 phosphorylation after stimulation of human endothelial cells with VEGF-A and HEP of unknown origin 37. In our cell culture experiments, sHA3 was not able to completely diminish the VEGF-A induced VEGFR-2 phosphorylation (Fig. 6), while it efficiently blocked the VEGF-A binding to VEGFR-2 in our isolated SPR approach (Fig. S2). This may be due to the fact that there are no other possible sHA3 interaction partners than VEGF-A and TIMP-3 present in SPR measurements. In contrast, during cell culture, sHA3 can interact with several further secreted and or cell membrane-bound proteins leading to sHA3 molecules with partially occupied binding sites. Due to this, the inhibitory potential of 200 µM D.U. sHA3 might be lower in cell culture compared to SPR measurements.

Figure 7. Simplified overview of VEGF-A signaling in the presence or absence of TIMP-3 and GAGs. (a) Successful binding of the VEGF-A dimer to VEGFR-2 initiates the VEGFR-2 dimerization that activates the intracellular tyrosine residue autophosphorylation. This induces the signal transduction cascade stimulating endothelial cell survival, proliferation and migration.

(b) The

extracellular VEGF competitor TIMP-3 inhibits VEGF-A/VEGFR-2 signaling due to binding to


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VEGFR-2 and thereby blocks the interaction of VEGF-A with VEGFR-2 and endothelial cell activation. (c) The interaction of sGAG derivatives with VEGF-A and TIMP-3 reduces the binding of both proteins to VEGFR-2. However, due to the TIMP-3/GAG derivative interaction, the VEGFA/VEGFR-2 interplay is partially rescued enabling a regulated VEGF-A signaling and endothelial cell activation.

Importantly, our data show that even though sHA3 inhibits the VEGF-A/VEGFR-2 interaction, its ability to interact with both VEGF-A and TIMP-3 leads to a partial rescue of the VEGF-A-mediated VEGFR-2 phosphorylation in PAE/KDR cells as determined via ELISA as well as endothelial cell migration assay (Fig. 6). We therefore hypothesize that sGAG derivatives and TIMP-3 have a coregulatory effect on VEGF-A functions and signaling (Fig. 7). However, in cell culture higher TIMP-3 but not VEGF-A concentrations were required to reduce the VEGF-mediated effects than in SPR experiments. This is in accordance to previous studies suggesting that in cell culture only high TIMP-3 doses led to competition for VEGFR-2

20,21

. Nevertheless, the examined concentrations should be

appropriate to reflect possible biological conditions since enhanced TIMP-3 levels should be expected in the presence of sHA derivatives due to their inhibitory effect on the TIMP-3 endocytosis as reported previously18. In summary, our findings show that sHA3-coated surfaces are able to simultaneously bind VEGF-A and TIMP-3 and that GAG derivatives reduce the interaction of both angiogenic factors with VEGFR-2 in a sulfation-dependent manner. Furthermore, we validated our finding in vitro using endothelial cells by showing that sHA3 decreases the TIMP-3-induced inhibition of the VEGF-A activity. Thus, sHA derivatives are promising candidates for the design of synthetic materials for controlling the activity of VEGF-A as well as TIMP-3, which could be beneficial for the treatment of abnormal angiogenesis. However, we recognize potential limitations of our methods. Based on the performed SPR measurements with a mixture of TIMP-3, VEGF-A and GAGs it is not possible to distinguish between ACS Paragon Plus Environment

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the binding response from the TIMP-3/VEGFR-2 interaction and the VEGF-A/VEGFR-2 interplay. Furthermore, we used simplified in vitro set-ups to analyze a highly complex signaling process. Even though our data strongly suggest the potential of sHA3 to alter the interplay of VEGF-A and TIMP-3 with VEGFR-2, extensive in vivo validation is required to proof whether GAG derivative containing biomaterials are able to control the activity of extracellular VEGF-A and TIMP-3 and thereby the response of endothelial cells.

5. Conclusion In this study, we demonstrate the potential of sulfated GAG derivatives to reduce VEGF-A and TIMP-3 activity by inhibiting their binding to VEGFR-2 in a sulfation- and concentration-dependent manner. Interestingly, these GAGs did not affect the inhibitory activity of TIMP-3 against MMPs

18

. The

simultaneous formation of inactive TIMP-3/sHA3 complexes rescues to some extent the sHA3inhibited VEGF-A/VEGFR-2 signaling leading to an activation of endothelial cells. Our in vitro data emphasize the co-modulatory function of sGAG derivatives on VEGFR-2 phosphorylation and endothelial cell migration. These results could potentially have a direct clinical relevance because the regulation of angiogenesis via VEGF signaling is of great clinical importance for tissue regeneration. Increased levels of VEGF and VEGFR-2 and abnormal angiogenesis is present under a variety of pathophysiological conditions e.g. rheumatoid arthritis, ocular neovascularization, cancer or atherosclerosis 45,46. Therefore, especially sGAG derivatives e.g. as parts of functional wound dressings are promising to tune the VEGF-A/VEGFR-2 signaling in a defined manner. Further in vivo validation needs to proof whether these findings translate into improved wound healing under abnormal angiogenesis conditions. ACKNOWLEDGMENTS We gratefully acknowledge financial support by the DFG [TRR 67 A2, A3, A7, A8, Z3].


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COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Supporting Information SPR data showing the concentration-dependent interference of sHA3 with TIMP-3/VEGFR-2 and VEGF-A/VEGFR-2 complex formation; Influence of sHA3 on the binding of VEGF-A and TIMP-3 to VEGFR-2 determined via SPR

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Graphical abstract


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Sulfated Hyaluronan Alters Endothelial Cell Activation in Vitro by

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