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Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth¹

ANDREAS H. ZISCH^2,3, MATTHIAS P. LUTOLF², MARTIN EHRBAR², GEORGE P. RAEBER, SIMONE C. RIZZI, NEIL DAVIES^*, HUGO SCHMÖKEL, DEON BEZUIDENHOUT^*, VALENTIN DJONOV, PETER ZILLA^* and JEFFREY A. HUBBELL

Department of Materials Science and Institute for Biomedical Engineering, Swiss Federal Institute of Technology Zurich and University of Zurich, Switzerland;
^* Department Cardiothoracic Surgery, Cape Heart Centre, University of Cape Town, South Africa; and
Institute of Anatomy, University of Berne, Switzerland

³Correspondence: Institute for Biomedical Engineering, Moussonstrasse 18, 8044, Zurich, Switzerland. E-mail: andreas.zisch{at}mat.ethz.ch

SPECIFIC AIMS

Our research was motivated by the clinical demand for new therapeutic materials to help restore vascular tissue function (therapeutic angiogenesis). We sought to develop a totally synthetic implant matrix with characteristics that would permit exploitation of natural proteolytic programs of tissue repair to liberate a potent angiogenic stimulus (vascular endothelial growth factor, or VEGF) from the implant matrix while simultaneously forming new vascularized tissue in place of the material.

PRINCIPAL FINDINGS

We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploit the activity of VEGF alongside the base matrix functionality for cellular ingrowth, i.e., induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides.

1. Synthetic biointeractive matrices with covalently integrated VEGF
Three-dimensional hydrogel networks were formed by Michael-type conjugate addition reaction using a structural and a biochemical building block. Vinyl sulfone (VS)-functionalized, 4armPEG macromers (PEG-VS) were used. The VS group demonstrates selective reactivity toward thiol moieties. These two structural building blocks were cross-linked via counter-reactive biochemical building blocks (Fig. 1 ). Because the number of functional groups on the PEG is 3, a covalently cross-linked gel forms; the bond formed by the reaction is hydrolytically stable, so the gel networks cannot degrade without the action of proteases on the cross-linking peptide. Sensitivity to network degradation by MMPs was obtained through cross-linking with the peptide sequence GCRDGPQGIWGQDRCG, the domain shown in italics being derived from the (1) chain of collagen I, which represents a fast degrading substrate for several MMP members; the two reactive cysteine residues are underlined. A pendantly grafted peptide sequence promoting cell adhesion, GCGYRGDSPG, was incorporated (the domain shown in italics is derived from the cell adhesion motif of fibronectin; a single reactive cysteine is underlined). A third biological signal was provided through covalent incorporation of VEGF. The covalent conjugation scheme was designed to provide retention of the factor in the matrix until its local release, triggered by active MMPs.

View larger version (23K): [in this window] [in a new window]   Figure 1. Sequestration of VEGF from synthetic, biomimetic hydrogel matrices by cell-demanded, proteolytic activities. A) 3-Dimensional hydrogel networks form by Michael-type addition reaction between thiol-bearing bioactive peptides and conjugated unsaturations presented through multi-armed PEG chains end functionalized with vinyl sulfone (VS). By the design of cross-linking peptides (a substrate for cleavage by MMP-2) and conjugation of cell adhesive peptide motifs (RGD), base functionalities for cellular ingrowth and remodeling were conferred into the otherwise biologically inert network structure. Provision of matrix-conjugated VEGF could stimulate vascularization of newly forming tissue. The covalent conjugation scheme retains the factor until its local liberation by proteases at the surface of cells invading the matrix. B) Unpaired cysteines (*) in the sequence of VEGF121-Cys variants may represent primary sites of reaction with PEG-VS. Both VEGF isoforms contain a plasmin-sensitive cleavage site at amino acid position 110 that serves to release ECM-bound VEGF. By covalent matrix anchorage of VEGF via sites downstream of amino acid 110, these natural sites for VEGF release may retain their functionality. C) Two concurrent proteolytic pathways (MMP-mediated matrix degradation and plasmin-mediated cleavage of the matrix anchor) may release VEGF protein.

Variants of VEGF₁₂₁ and VEGF₁₆₅ were explored for Michael-type conjugation to PEG peptide hydrogel networks. As depicted in Fig. 1B , a mutant VEGF_121-Cys variant was prepared that possesses two unpaired cysteine residues, one presented by the native VEGF₁₂₁ sequence close to the carboxyl terminus of each monomer unit in the dimer and another at the very carboxyl-terminal position. This exogenous cysteine residue was placed adjacent to the strongly positively charged KPRR sequence, which lowers the pK_a of the cysteine residue’s thiol and accelerates Michael-type addition reaction (Fig. 1B ). VEGF₁₂₁ and VEGF₁₆₅ both possess a plasmin-sensitive cleavage site at amino acid position 110 that has been implicated in mediating release of sequestered extracellular matrix (ECM)-bound VEGF as soluble VEGF₁₁₀. By way of carboxyl-terminal matrix anchors, this natural mechanism of sequestration and release may be preserved. Release of VEGF could occur by two distinct proteolytic, cell-dependent pathways: MMP-mediated matrix degradation and plasmin-mediated sequestering by cleavage of the carboxyl-terminal matrix anchor (Fig. 1C ).

We showed that rates of VEGF grafting onto PEG peptide hydrogels could be readily controlled by the duration of the initial Michael-type reactions between PEG-VS and VEGFs ("conditioning"), which preceded hydrogel polymerization. After 60 min initial conditioning, 80% of the VEGF was found covalently retained. Chemotactic (mediated by 20% diffusible VEGF) and haptotactic (mediated by 80% matrix-conjugated VEGF) signals were provided in the matrices that may respectively induce and support endothelial cell invasion. This matrix formulation was used in all ensuing experiments.

2. VEGF-conjugated PEG peptide matrices form new, vascularized tissue
Confirmation that VEGF-conjugated PEG peptide matrices responded to proteolytic programs used by migratory human endothelial cells was obtained in vitro, showing active movement of matrix-embedded human umbilical vein endothelial cells (HUVECs) inside the hydrogel matrix. In vivo blood vessel invasion of VEGF-conjugated PEG peptide hydrogels was studied in grafting experiments in the embryonic chick CAM assay (Fig. 2 A–H) and adult rats (Fig. 2I-L ). In CAM experiments (see Fig. 2B-D ), VEGF_121-Cys- and VEGF₁₆₅-conjugated PEG peptide matrices were found to be proangiogenic. Consistent with local liberation of matrix-conjugated VEGF, microscopic analysis revealed highly localized new blood vessel growth precisely at the site of graft/membrane contact. In stark contrast, diffusive release of soluble VEGF formulated in PEG peptide hydrogels resulted in a strong increase of capillary density in the vast surrounding of the gel graft (Fig. 2B ). Locally confined vessel growth in CAM tissue exposed to matrix-conjugated VEGF was further confirmed by fluorescence microscopic imaging of CAM microvasculature perfused with FITC-dextran (Fig. 2F-H ).

View larger version (144K): [in this window] [in a new window]   Figure 2. VEGF-conjugated PEG peptide hydrogel matrices are able to induce and guide vascularized tissue growth. Grafting studies were performed in the embryonic chick assay and adult rats. A–H) Angiogenic effects of PEG peptide hydrogels formulated with soluble VEGF, conjugated VEGF, or no VEGF. Gels were grafted atop embryonic day 9 chicken CAM. Representative stereomicrographs (A–E) permit views of the responding graft site and surrounding CAM 48 h after grafting. Black arrows: areas of neovessel formation. A) Control gels with no VEGF did not induce new vessels. B) Diffusive release of soluble VEGF121-Cys strongly increases capillary growth in the surrounding of the graft. C) Angiogenic effect of matrix-conjugated VEGF165. Consistent with local liberation of VEGF, a highly localized angiogenic response is obtained precisely at the area of graft/membrane contact. D, E) Higher resolution images of angiogenic effects of gel-conjugated VEGF121-Cys or VEGF165. F–H) Fluorescence microscopic images of CAM vasculature perfused with FITC dextran 48 h after grafting. Images show CAM zones in the vicinity of gel grafts. F) Control gels (no VEGF) did not affect vessel growth. G) Diffusive release of soluble VEGF121-Cys evoked massive, brush-like capillary growth (white arrows) in the surrounding gel. F, H) CAM vasculature around gel grafts formulated with matrix-bound VEGF121-Cys or no VEGF exhibits normal vessel morphology. I–L) Implantation (s.c.) experiments in rats show that these VEGF-conjugated hydrogels are able to induce cell migration and blood vessel growth into the matrix to generate new tissue in place of the material. Porous PU discs were used to contain VEGF-conjugated PEG peptide hydrogel matrices placed s.c. in adult normal rats and retrieved after 14 days. Implant sections were histochemically stained for blood vessels with antibodies specific for rat CD31 (blue) and counterstained with nuclear red to stain nuclei of all cells. Microscopic inspection revealed complete resorption of the filler matrices and replacement by cellularized tissue throughout degradable hydrogel implants. I) VEGF121-Cys-conjugated gel matrix; J) VEGF165-conjugated gel matrix. K) Gel matrix with no VEGF showing poor cell and vessel ingrowth. L) No cell ingrowth or no gel resorption was observed in PEG peptide hydrogels prepared with a scrambled, MMP-insensitive substrate peptide in the polymer backbone.

In rat subcutaneous (s.c.) implantation experiments, highly porous polyurethane (PU) sponges were used to contain PEG peptide hydrogel matrices (Fig. 2I-L ) to allow identification of the implant site and retrieval of hydrogel implants for analysis,. A high degree of cellular ingrowth and significant numbers of blood vessels were detected throughout the implant matrices. No signs of vessel leakiness or edema were detectable either in the implant, fibrous capsule around the implant, or the pocket of s.c. tissue. Histochemical characterization identified fibroblast-like cells and endothelial cells organized as vessels inside the implants, suggesting that MMP-dependent migratory pathways of multiple cell types were activated in vivo. After cell ingrowth, the synthetic matrices appeared to be gradually resorbed by a MMP-dependent, cell-mediated process and coordinately replaced by vascularized tissue. Sensitivity of these hydrogels to MMPs was critical: a complete lack of cell ingrowth was observed in VEGF-conjugated PEG peptide networks prepared using a scrambled, proteolytically insensitive cross-linking sequence, GCRD-GDQGIAGF-DRCG (Fig. 2L ). The presence of VEGF appeared to be an important driving force for cell ingrowth: poor infiltration limited to the implant edges was observed in MMP-sensitive hydrogels prepared with no VEGF (Fig. 2K ).

CONCLUSIONS AND SIGNIFICANCE

In nature, the ECM plays a key role in regulating most morphogenetic processes: it provides immobilized adhesion cues and growth factors within a 3-dimensional structural network. Likewise, cells participating in the morphogenetic process influences the ECM: they proteolytically remodel it to liberate bound growth factors and create a path for forward cell movement, eventually replacing it with new ECM and functional tissue. We sought to mimic the capacity for these bidirectional ECM/cell interactions in synthetic systems, providing an elastic hydrogel matrix that can be proteolytically remodeled by cell-associated MMPs. Adhesion sites bound within the matrix allow exertion of traction at the cell/material interface and cell-associated MMPs act to remodel the matrix to enable cell migration within three dimensions. VEGF₁₆₅ possesses a binding site for heparan sulfate proteoglycans that maintains it in the immobilized state until release by local cellular enzymes. Such matrix associations stabilize the VEGF’s active conformation, protect it from immediate clearance and proteolytic inactivation, and limit its activity to cells that liberate the factor during proteolytic remodeling of the ECM.

Our biochemical, cell biological, and animal studies indicate that VEGF proteins chemically coupled to PEG peptide hydrogel networks could behave similarly to those in the natural ECM. In vivo, such liberation of VEGF by plasmin or MMP-2 is likely to occur in a highly localized manner. We found that angiogenesis evoked by VEGF-modified PEG peptide hydrogel grafts on the CAM was localized precisely to the area of graft/CAM contact. When implanted s.c. in rats, these VEGF-containing matrices were remodeled into native, vascularized tissue.

VEGF dosing is a formidable challenge for clinicians and bioengineers. Complications such as excessive but malformed vessel induction, vascular leakage, and hypotension may arise from exposure to high VEGF concentrations. This untoward behavior drives the development of release systems, avoiding burst and providing sustained exposure to low levels of the growth factor. Our results indicate that this type of synthetic biointeractive implant with integrated VEGF, presented and released upon cellular demand, could be useful in clinical healing of local therapeutic angiogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1041fje; doi: 10.1096/fj.02-1041fje

² These authors contributed equally.

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