Elsevier

Acta Biomaterialia

Volume 9, Issue 11, November 2013, Pages 8823-8831
Acta Biomaterialia

Specific VEGF sequestering to biomaterials: Influence of serum stability

https://doi.org/10.1016/j.actbio.2013.06.033Get rights and content

Abstract

Vascular endothelial growth factor (VEGF) was originally discovered as a tumor-derived factor that is able to induce endothelial cell behavior associated with angiogenesis. It has been implicated during wound healing for the induction of endothelial cell proliferation, tube formation and blood vessel remodeling. However, previous investigations into the biological effect of VEGF concluded that a particular range of growth factor concentrations are required for healthy vasculature to form, motivating recent studies to regulate VEGF activity via molecular sequestering to biomaterials. Numerous VEGF sequestering strategies have been developed, and they have typically relied on extracellular matrix mimicking moieties that are not specific for VEGF and can affect many growth factors simultaneously. We describe here a strategy for efficient, specific VEGF sequestering with poly(ethylene glycol) (PEG) microspheres, using peptides designed to mimic VEGF receptor type 2 (VEGFR2). By immobilizing two distinct peptides with different serum stabilities, we examined the effect of serum on the specific interaction between peptide-containing PEG microspheres and VEGF. We addressed the hypothesis that VEGF sequestering in serum-containing solutions would be influenced by the serum stability of the VEGF-binding peptide. We further hypothesized that soluble VEGF could be sequestered in serum-containing cell culture media, resulting in decreased VEGF-dependent proliferation of human umbilical vein endothelial cells. We show that soluble VEGF concentration can be effectively regulated in serum-containing environments via specific molecular sequestering, which suggests potential clinical applications.

Introduction

During wound healing, vascular endothelial growth factor (VEGF) is released by activated platelets, neutrophils and macrophages [1], and stimulates an angiogenesis cascade that ultimately leads to new blood vessel formation [2]. Events in the angiogenesis cascade including induction of endothelial cell proliferation are highly dependent on local VEGF activity, motivating numerous studies to deliver VEGF and induce blood vessel formation in a healing wound. Clinical trials focused on VEGF protein and gene delivery show that leaky, immature vasculature often forms below and above an optimal VEGF concentration range [3], [4], demonstrating a need to carefully regulate the VEGF dosage. Recent investigations have concluded that maintaining VEGF levels in an optimal concentration range results in mature blood vessel formation in vivo [5], [6], which motivates the need for developing biomaterials that regulate VEGF activity.

One mechanism by which nature regulates VEGF-dependent signaling involves sequestering and release from the extracellular matrix (ECM). Natural ECMs are composed of numerous VEGF-binding molecules, including collagens [7], [8], [9], glycoproteins (e.g. fibronectin [10]) and proteoglycans [11]. Sequestered VEGF165, the most commonly studied VEGF isoform due to its role in wound healing, can be released from the ECM by proteolytic remodeling of ECM components [12]. Investigators have recently mimicked the function of the natural ECM using synthetic biomaterials that bind to VEGF via affinity interactions. For example, Tan et al. [13] demonstrated that heparan sulfate proteoglycansincorporated into collagen microspheres can bind to VEGF and regulate VEGF-dependent endothelial cell behavior in vitro, and Chung et al. [14] showed that microspheres with immobilized heparin and heparin–fibrin conjugates [15] can influence VEGF release kinetics in vitro and induce neovascularization in vivo. These studies and others [9], [16], [17], [18], [19], [20] clearly show that ECM mimicking molecules (e.g. heparin, proteoglycans) can bind to numerous growth factors, providing an adaptable mechanism for growth factor regulation [21]. In addition, our group recently showed that peptide mimics of VEGF receptor type 2 (VEGFR2) immobilized within a synthetic hydrogel can specifically sequester VEGF, and thereby selectively regulate VEGF-dependent endothelial cell behavior in vitro [22], [23]. This receptor mimicking approach is particularly attractive, as it allows one to regulate activity of a specific growth factor, even in heterogeneous environments like biological fluids. However, previous studies have not explored in detail the context-dependence of growth factor sequestering, and little is known about the role of biological fluids in growth factor regulation in vitro or in vivo. Biological fluids such as blood serum contains significant quantities of growth factors and other proteins [24], [25], which may decrease the specificity and affinity of growth factor sequestering. Therefore, there is a need to more clearly understand the serum dependence of growth factor sequestering biomaterials.

Here we examined the effect of serum on VEGF binding to polymeric microspheres containing specific, VEGF-binding peptides. These peptides were derived from VEGFR2 [26], [27] and were chosen based on their differing serum stability, which allowed us to characterize the influence of serum on VEGF binding and associated VEGF regulation. Specifically, we explored a wild-type VEGF-binding peptide as well as a derivative of this peptide that included four D-substituted amino acids, which provide enhanced peptide stability against protease-mediated degradation [27]. We hypothesized that peptide stability in serum would influence VEGF sequestering. Specifically, we reasoned that the increased serum stability of the D-substituted VEGF-binding peptide (VBP) would increase VEGF sequestering relative to the wild-type VEGF-binding peptide (VBPWT). In addition, we hypothesized that VEGF-binding microspheres would reduce VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation in culture, resulting in a novel, biology-inspired mechanism for “knocking down” VEGF signaling in vitro.

Section snippets

Peptide synthesis and characterization

Two peptides identified from a previous study, VBP sequence CEFdAdYdLdIDFNWEYPASK and the wild-type VBPWT sequence CELNVGIDFNWEYPASK [26], [27], and a peptide with the same amino acids but in a scrambled sequence (Scramble), CDAdPYNFdEFAWEYdVISLdK, were synthesized using standard Fmoc solid-phase peptide synthesis on MBHA Rink amide resin, as previously described [23]. All amino acids (EMD Novabiochem), were protected at the N-terminus with an Fmoc protecting group. Initial deprotection was

Results

Binding of VEGF to microspheres was specific and dependent on the peptide content. Microspheres containing either of the peptides VBP or VBPWT, which are designed to mimic VEGFR2, sequestered significantly higher amounts of soluble VEGF than microspheres containing a scrambled version of VBP (Scramble) or no peptide (Blank). In addition, VBP microspheres sequestered significantly more VEGF than VBPWT microspheres at each peptide density, except for the highest density tested (3.1%).

Discussion

Regulation of growth factor activity is an important function of the extracellular matrix, which is composed of numerous growth-factor-binding proteins, such as collagens [7], [8], [9], glycoproteins (e.g. fibronectin [10]) and proteoglycans (e.g. perlecan [11]). This regulation is particularly important during angiogenesis, during which growth factors stimulate endothelial cells to migrate, proliferate and eventually undergo tube formation [2], [36]. Many previous approaches have used soluble

Conclusion

In the current study, we have investigated the serum-dependence of VEGF sequestering to biomaterials containing VEGFR2-mimicking peptides. We observed high-affinity binding in the presence of serum, which significantly reduced VEGF-dependent HUVEC proliferation in culture. Consequently, this strategy of incorporating receptor-mimicking peptides into a biomaterial is effective for demonstrating specific high-affinity binding of a growth factor. Although many biomaterial formulations currently

Acknowledgements

The authors acknowledge support from the National Institutes of Health (T32 HL007936-12, RO1HL093282, and R21 EB016381).

References (58)

  • N. Ferrara et al.

    Pituitary Follicular Cells Secrete a Novel Heparin-Binding Growth Factor Specific for Vascular Endothelial Cells

    Biochem Biophys Res Commun

    (1989)
  • A.S.R. Maharaj et al.

    Roles for VEGF in the adult

    Microvasc Res

    (2007)
  • S. Serratì et al.

    TGFbeta1 antagonistic peptides inhibit TGFbeta1-dependent angiogenesis

    Biochem Pharmacol

    (2009)
  • L. Zhang et al.

    Manipulation of hydrogel assembly and growth factor delivery via the use of peptide-polysaccharide interactions

    J Control Release

    (2006)
  • H. Hosseinkhani et al.

    Enhanced angiogenesis through controlled release of basic fibroblast growth factor from peptide amphiphile for tissue regeneration

    Biomaterials

    (2006)
  • H. Yokoyama et al.

    Serum and urinary concentrations of heparan sulfate in patients with diabetic nephropathy

    Kidney Int

    (1999)
  • H. Gitay-Goren et al.

    The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules

    J Biol Chem

    (1992)
  • S. Soker et al.

    Variations in the size and sulfation of heparin modulate the effect of heparin on the binding of VEGF165 to its receptors

    Biochem Biophys Res Commun

    (1994)
  • L. Hetian et al.

    A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelial growth factor to its kinase domain receptor

    J Biol Chem

    (2002)
  • M.F. Powell et al.

    Peptide Stability in Drug Development: A Comparison of Peptide Reactivity in Different Biological Media

    J Pharm Sci

    (1992)
  • S.Y. Hong et al.

    Effect of D -Amino Acid Substitution on the Stability , the Secondary Structure , and the Activity of Membrane-Active Peptide

    Biochem Pharmacol

    (1999)
  • G.L. Semenza

    Angiogenesis in ischemic and neoplastic disorders

    Annu Rev Med

    (2003)
  • P. Carmeliet

    Mechanisms of angiogenesis and arteriogenesis

    Nat Med

    (2000)
  • C.R. Ozawa et al.

    Microenvironmental VEGF concentration , not total dose , determines a threshold between normal and aberrant angiogenesis

    J Clin Invest

    (2004)
  • S. Barrientos et al.

    Growth factors and cytokines in wound healing

    Wound Repair Regen

    (2008)
  • Q. Sun et al.

    Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb

    Pharm Res

    (2005)
  • N. Nagai et al.

    Preparation and characterization of collagen microspheres for sustained release of VEGF

    J Mater Sci

    (2010)
  • T.R. Chan et al.

    Matrix-Bound VEGF Mimetic Peptides: Design and Endothelial-Cell Activation in Collagen Scaffolds

    Adv Funct Mater

    (2011)
  • A.Y. Wang et al.

    Immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphogenesis

    Biomacromolecules

    (2008)
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