EUROPEAN TISSUE REPAIR SOCIETY

GROWTH FACTORS II

DESIGN OF A NOVEL PROTEOLYSIS RESISTANT VASCULAR ENDOTHELIAL GROWTH FACTOR 165 VARIANT

Introduction

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific multifunctional cytokine that is a key regulator in physiological and pathological processes of angiogenic remodelling.¹ By differential mRNA splicing, the single human VEGF gene gives rise to at least six protein isoforms VEGF_{121, 145, 165, 183, 189} and ₂₀₆.^2,3 Among them, the 165-amino-acid isoform is the major gene product found in human tissues. The splice variants differ primarily in the presence or the absence of the heparin-binding domains encoded by exons 6 and 7, giving rise to forms that differ in their heparin/heparan-sulfate binding ability, as well as their affinities to VEGF receptors flt-1, flk-1/ KDR and neuropilin-1.^4–9 Whereas VEGF121 does not bind heparan-sulfate and is freely diffusible, VEGF₁₈₉ binds heparin and is primarily associated with the cell surface and extracellular matrix, and VEGF₁₆₅ has intermediate properties.^5,10,11 Furthermore, native VEGF₁₈₉ binds to flt-1 but not to flk-1/KDR.¹² Native VEGF₁₈₉ requires maturation by urokinase (uPA) within the exon 6-encoded sequence to bind to flk-1/KDR and to exert a mitogenic effect on endothelial cells.¹² In contrast, plasmin digestion of VEGF₁₆₅ decreases its mitogenic activity for endothelial cells.¹³ Plasmin digestion of VEGF₁₆₅ yields two fragments: an amino-terminal homodimer (VEGF1-110) containing the flt-1 and the flk-1/KDR receptor binding determinants encoded by exons 3 and 4, respectively, and a carboxyl-terminal polypeptide comprising the neuropilin- 1 binding site encoded by exon 7 (VEGF111–165).¹³ Interestingly, the reduced mitogenic activity of the aminoterminal homodimer VEGF1-110 is similar to that observed for VEGF₁₂₁.^12,13 These findings suggest that differential protease susceptibility, extracellular localization and/or receptor binding may result in distinct functions for different VEGF isoforms.

Recently, we demonstrated that the proteolysis of VEGF₁₆₅ by plasmin is increased in wound fluid collected from chronic non-healing wounds compared with fluid from healing wounds.¹⁴ VEGF plays a critical role during the angiogenic response in tissue repair,^15-17 suggesting that VEGF degradation and loss of its biological activity may contribute to an impaired wound healing response. These results prompted us to introduce amino acid alterations at the plasmin-sensitive cleavage site of VEGF₁₆₅ (Arg110/ Ala111) in order to stabilize the VEGF165 molecule. Substitutions at either site result in VEGF₁₆₅ products that, while maintaining growth-promoting properties, are either fully or partially plasmin-resistant. This type of modification would be expected to increase the period for which topically applied VEGF protein is active in the wound environment, implying a potential clinical application.

Materials and Methods

Site directed Mutagenesis

Mutagenesis was performed using base-mismatched oligonucleotides as indicated: Mut_Ala110 5'-GACCAAAGAAAGATGCCGCAAGACAAG-3', Mut_Gln110 5'-GACCAAAGAAAGATCAGGCAAGACAAG-3', Mut_Pro111 5'-GACCAAAGAAAGATAGACCAAGACAAG-3', Mut_{Lys110-Pro111} 5'-GACCAAAGAAAGATAAGCCAAGAC AAG (nucleotide differences between wild type VEGF₁₆₅ and mutants are indicated in bold) (MWG-Biotech, Ebersberg, Germany). VEGF₁₆₅ mutants were generated using the Gene Editor™ in vitro site-directed mutagenesis system (Promega, Mannheim, Germany). VEGF₁₆₅ mutants were characterized by DNA sequencing using the ABI Prism™ Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit (PE Applied BioSystems, Langen, Germany) and confirmed in both 5' and 3' direction.

Cell culture

COS-1 cells (provided by Dr Ingo Flamme, ZMMK, Cologne) were cultured in Dulbecco‘s modified-Eagle‘s medium (DMEM) containing 10% foetal calf serum (FCS), L-glutamine (2 mM) and penicillin (10 U ml^-1) streptomycin (10 µg ml^-1l). Human umbilical vein endothelial cells (HUVEC) (TEBU, Frankfurt, Germany) were maintained in MCDB 131 (Life Technologies, Eggenstein, Germany) supplemented with 8% FCS, 20 mM glutamine, penicillin (10 U ml^-1), streptomycin (10 µg ml^-1) and ECGS/H growth supplement (PromoCell, Freiburg, Germany).

Purification of VEGF₁₆₅-protein

COS-1 cells were transfected using the Superfect transfection reagent (Qiagen, Hilden, Germany) following the manufacturer’s instructions and cultivated in serum-free medium (DMEM containing 2 mM L-glutamine, penicillin 10 U ml^-1, streptomycin 10 µg ml^-1 and insulin-transferrin sodium selenite (ITS) supplement (Sigma, Deisenhofen, Germany). Conditioned medium (200 ml) was collected at 48 hours and incubated for 4 hours with 5 ml of heparin-sepharose (Pharmacia, Freiburg, Germany) at 4°C. The heparin-sepharose was packed in a column and culture medium was loaded onto the column at a flow rate of 25 ml h^-1. Elution of bound proteins was carried out by 10 mM Tris-HCl, 0.9 M NaCl pH 7.2. Fractions were pooled, desalted (D-Salt™ Excellulose™ Plastic Desalting Columns, Pierce, St. Augustin, Germany) and lyophilised, and the concentration of VEGF₁₆₅ was determined using a commercially-available human VEGF-specific ELISA (R&D Systems, Minneapolis, MN). The assay was performed following the manufacturer’s instructions.

SDS-PAGE and Immunoblotting

SDS-PAGE was performed following the protocol of Laemmli. Purchased recombinant human VEGF₁₆₅ (rVEGF₁₆₅; R&D Systems, Minneapolis, MN) and VEGF₁₆₅ expressed in COS-1 cells were incubated at 37°C (pH 7.6, up to 4 hrs) with wound fluid obtained from non-healing wounds, with plasmin solution (0.01 U ml^-1) (human plasma plasmin, Sigma, Deisenhofen, Germany) or with PBS as a negative control. Reactions were terminated by the addition of Pefabloc® (1 mM final concentration), and frozen at –20°C before analysis by SDS-PAGE or MALDI-TOF-Mass-Spectrometry. For Western blotting, fragments were resolved on 12% non-reducing SDS-PAGE gels and transferred to nitrocellulose (Hybond C-super, Amersham).

VEGF₁₆₅ integrity was determined by detecting immunoreactive products with a polyclonal rabbit antibody that recognizes the amino-terminal VEGF epitopes (raised against a 20-amino-acid amino-terminal peptide) (Santa Cruz Biotech., Santa Cruz, CA). Detection of intact and VEGF-derived degradation products was accomplished using the LumiLight plus chemiluminescence detection system (Roche Diagnostics, Mannheim, Germany). Densitometric analysis of Western blots was performed using a FluroS-Multiimager and Quantity one analysissoftware (Biorad, München, Germany).

Protein sequencing

Protein sequencing was performed by Dr Karlheinz Mann (Max-Planck-Institute of Biochemistry, Martinsried, Germany). For amino-terminal sequencing the PAGE-separated fragments were blotted onto Immobilon-P (Millipore, Bedford, MA) according to Matsudaira.¹⁸ Sequencing was done using the Applied BioSystems Procise 492 sequencer.

MALDI-TOF Mass Spectrometry

The mass-spectrometry analysis of plasmin-induced VEGF degradation was performed by Dr Macht in the Center of Molecular Medicine, Cologne. For the detection of highmolecular weight proteins a voltage of 20kV was used. The analysis of low-molecular weight proteins was performed using a Post Source Decay Mass Spectrometry (PSD-MS) with a voltage of 26.3 kV and a reflector-voltage of 22.5 kV.

Endothelial cell proliferation assay for VEGF

HUVECs were seeded in 96-well plates (4 x 10³ cells/well) in growth medium. After a starvation phase of 16 hours (1% FCS), cells were stimulated by VEGF variants (1 to 15 ng ml^-1) for five days. Cell proliferation was assessed using a BrdU based proliferation assay (Roche Diagnostics, Mannheim, Germany). VEGF mediated mitogenic activity was inhibited by adding a specific VEGF-neutralizing polyclonal goat antiserum (50 µg ml^-1) (R&D Systems). Three separate dose-response experiments were performed. Differences between wild type and mutant proteins were analysed using the unpaired t-test.

Wound fluid

Wound fluid was obtained from three different patients suffering from non-healing wounds due to venous insufficiency (ulcers showed no clinical sign of infection, 25–50 cm² in size, mean age of patients 67 years). Ulcers were covered with a semipermeable polyurethane film (Hyalofilm ®, Hartmann; Heidenheim, Germany). Following collection, fluids were centrifuged (10 min, 13.000g, 4°C) and supernatants were frozen at –80°C until use.

Results and Discussion

In the present study, we elucidated the functional role of the plasmin cleavage site Arg110/Ala111 in the VEGF₁₆₅ molecule for its mitogenic potency. Western blot analysis, amino-terminal sequencing and MALDI-TOF analysis of plasmin digested VEGF₁₆₅ fragments identified Arg110/ Ala111 as the plasmin cleavage site closest to the N-terminus of the molecule. Incubation of VEGF165 in plasmin for four hours reduced its mitogenic potency on HUVEC cells by over 50%. These data suggest a critical role for the plasmin cleavage site Arg110/Ala111 and the carboxylterminal domain of VEGF₁₆₅ in the stimulation of endothelial cell proliferation.

VEGF activity is mediated through two receptors: flt-1 and flk-1/KDR.⁴ Both receptors show different kinase activity, and have separate signal-transduction properties and possibly mediate different functions.^4,19,20 Receptor studies suggest that the major positive signal of VEGF for endothelial proliferation and vascular permeability appears through flk-1/KDR, while that from flt-1 seems to contribute about one tenth of the total signal.^21,22 Recently, Soker et al. have identified a new VEGF receptor, neuropilin-1, that binds VEGF₁₆₅ but not VEGF₁₂₁.²³ When co-expressed in cells with flk-1/KDR, neuropilin-1 enhanced both the binding of VEGF₁₆₅ to flk-1/KDR and VEGF₁₆₅-mediated chemotaxis and proliferation.^7,23 Conversely, inhibition of VEGF₁₆₅ binding to neuropilin-1 inhibits its binding to flk-1/KDR and its mitogenic activity for endothelial cells. These findings suggest that neuropilin- 1 may present VEGF₁₆₅ to the flk-1/KDR receptor in a manner that enhances the effectiveness of flk-1/KDR-mediated signal transduction. VEGF₁₆₅ binds to neuropilin- 1 via the VEGF exon 7 encoded peptide, which comprises most of the heparin-binding site of VEGF₁₆₅. The ability of VEGF₁₆₅ to bind neuropilin-1 may partly explain its greater mitogenic potency relative to VEGF molecules lacking the exon 7 encoded peptides, whether due to proteolysis or alternative splicing.

Recently we demonstrated that proteolysis and inactivation of VEGF₁₆₅ by plasmin is increased in wound fluid collected from non-healing wounds compared with that from healing wounds.¹⁴ These results prompted us to investigate whether VEGF₁₆₅ could be proteolytically stabilized by mutating the plasmin cleavage site Arg110/Ala111. A general consensus sequence of the plasmin cleavage site is not known. Alterations in amino acid residues of VEGF₁₆₅ were based on the notion that plasmin substrate specificity is directed by its negatively charged catalytic domain. Therefore, Arg110 was substituted by a neutral, non-polar, non-cyclic amino acid such as alanine or glutamine. We also replaced Ala111with proline, an amino acid change that might be expected to have effects on the tertiary as well as primary structure of the protein. Westernblot analysis and a cell proliferation assay demonstrated that both the replacement of the Arg110 by a neutral, noncyclic amino acid and the introduction of Pro111 stabilized VEGF165 mitogenic activity in the presence of plasmin. These data indicated that the mutation of the Arg110/ Ala111 site of VEGF₁₆₅ dramatically affected its plasmin sensitivity.

Consistent with these observations, VEGF₁₆₅ mutants were temporarily stabilized when incubated with wound fluid obtained from non-healing wounds. However, inactivation of the plasmin cleavage site by a neutral, noncyclic amino acid was not sufficient to stabilize the VEGF₁₆₅ molecule in the presence of wound fluid. In contrast, altering the plasmin cleavage site by substituting VEGF-Ala111 with the cyclic VEGF-Pro111 increased the VEGF stability in chronic wound fluid. These results suggest that modification of the tertiary structure of the VEGF₁₆₅ protein can temporarily stabilize VEGF165 in the wound fluid of non-healing wounds. However, a direct effect at the level of primary sequence recognition by proteases is also possible.

Little is known about local mechanisms leading to and perpetuating the failure of cutaneous wound healing. A disturbed balance of proteolytic and anti-proteolytic activity characterizes the hostile environment of a chronic wound.^24,25 The failure of certain wounds to resolve successfully may be due to the increased proteolytic degradation of regulatory factors, including growth factors and extracellular matrix molecules.^14,26-28 Increased degradation of topically applied growth factors may also account for the discrepancy between the remarkable efficacy of growth factors in wound healing in animal models and the rather disappointing clinical results in non-healing wounds.^14,27 Hence, controlling the proteolytic activity in the chronic wound environment might be a key strategy for improving wound healing. Topical application of proteolysis resistant growth regulators in chronic wounds might enhance the efficiency of these mediators in the hostile chronic wound environment.

References

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Abbreviations:

VEGF vascular endothelial growth factor;
HUVEC human umbilical venous endothelial cells,
FCS foetal calf serum,
Wt wild type,
Mut mutant

Gereon Lauer, Stephan Sollberg, Melanie Cole,
Thomas Krieg and Sabine A. Eming*
Department of Dermatology,
University of Cologne, Germany

*Corresponding author:
Dr med. Sabine A. Eming
Department of Dermatology,
University of Cologne
Joseph-Stelzmann Str. 9
D–50931 Köln, Germany
Tel: +49 221 478 4500
Fax: +49 221 420 0988
E-mail: Sabine.Eming@uni-koeln.de