Specific VEGF sequestering to biomaterials: Influence of serum stability
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)
- et al.
The role of vascular endothelial growth factor in wound healing
J Surg Res
(2009) Fibrinogen and fibrin structure and functions
J Thromb Haemost
(2005)- et al.
Efficient revascularization by VEGF administration via heparin-functionalized nanoparticle-fibrin complex
J Controlled Release
(2010) - et al.
Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors
Peptides
(2007) - et al.
Biopolymeric delivery matrices for angiogenic growth factors
Cardiovasc Pathol
(2003) - et al.
Stimulation of in vivo angiogenesis by in situ crosslinked, dual growth factor-loaded, glycosaminoglycan hydrogels
Biomaterials
(2010) - et al.
Specific VEGF sequestering and release using peptide-functionalized hydrogel microspheres
Biomaterials
(2012) - et al.
Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF
J Biol Chem
(1999) - et al.
Controlling Affinity Binding with Peptide-Functionalized Poly(ethylene glycol) Hydrogels
Adv Funct Mater
(2009) - et al.
A novel preparation method for polymeric microparticles without the use of organic solvents
Int J Pharm
(1998)
Pituitary Follicular Cells Secrete a Novel Heparin-Binding Growth Factor Specific for Vascular Endothelial Cells
Biochem Biophys Res Commun
Roles for VEGF in the adult
Microvasc Res
TGFbeta1 antagonistic peptides inhibit TGFbeta1-dependent angiogenesis
Biochem Pharmacol
Manipulation of hydrogel assembly and growth factor delivery via the use of peptide-polysaccharide interactions
J Control Release
Enhanced angiogenesis through controlled release of basic fibroblast growth factor from peptide amphiphile for tissue regeneration
Biomaterials
Serum and urinary concentrations of heparan sulfate in patients with diabetic nephropathy
Kidney Int
The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules
J Biol Chem
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
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
Peptide Stability in Drug Development: A Comparison of Peptide Reactivity in Different Biological Media
J Pharm Sci
Effect of D -Amino Acid Substitution on the Stability , the Secondary Structure , and the Activity of Membrane-Active Peptide
Biochem Pharmacol
Angiogenesis in ischemic and neoplastic disorders
Annu Rev Med
Mechanisms of angiogenesis and arteriogenesis
Nat Med
Microenvironmental VEGF concentration , not total dose , determines a threshold between normal and aberrant angiogenesis
J Clin Invest
Growth factors and cytokines in wound healing
Wound Repair Regen
Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb
Pharm Res
Preparation and characterization of collagen microspheres for sustained release of VEGF
J Mater Sci
Matrix-Bound VEGF Mimetic Peptides: Design and Endothelial-Cell Activation in Collagen Scaffolds
Adv Funct Mater
Immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphogenesis
Biomacromolecules
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