The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing

Abstract

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

Graphical abstract

Figure 1.

VWF-deficient mouse shows impaired wound healing and reduced angiogenesis. Full-thickness back-skin wounds were made in WT and VWF-deficient mice. After 5 days, (A) wound closure and (B) granulation tissue area were evaluated by histomorphometry. (WT, n = 6; VWF-deficient, n = 7). (C) Wound histology (hematoxylin and eosin staining). Black arrows indicate tips of the epithelium tongue. The granulation tissue (pink-violet) is characterized by infiltration of granulocytes with nuclei stained in dark-violet or black. Muscle under the wounds is stained in red; fat tissue appears as transparent bubbles. Scale bar = 800 µm. Proliferation of (D) CD31⁺CD45^– ECs and (E) SMA⁺CD45^– SMCs assessed by Ki67⁺ marker was determined using flow cytometry. The amounts of (F) VEGF-A and (G) FGF-2 in the wounds were quantified by enzyme-linked immunosorbent assay (ELISA). (H-I) Representative high-magnification image by immunofluorescence and (J) quantification of VEGF-A in mouse skin wound healing sections from WT and VWF-deficient mice; sections are costained for VWF (green), SMA (red), and CD31 (yellow) to visualize blood vessels; nuclei are identified by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 10 μm. (J) Quantification represents the ratio between the sum of pixel intensity for VEGF-A signal and DAPI volume (μm³) (n = 2 fields per mouse; n = 5 mice per genotype). Graphical data are mean ± standard error of the mean (SEM), Statistical comparisons were carried out using the Mann-Whitney U test. AU, arbitrary unit. *P < .05; **P < .01.

Figure 2.

Human plasma-derived VWF binds promiscuously to GFs with high affinity. VWF binding to (A) GFs and (B) chemokines were measured by ELISA. A450 nm represents absorbance at 450 nm. Signals from VEGF-A121 served as a baseline, and bovine serum albumin (BSA) served as a negative control (n = 4; data are mean ± standard deviation [SD]). Affinity (K_D values are shown) of VWF against (C) VEGF-A165, (D) PDGF-BB, (E) NT-3, and (F) PDGF-DD was measured by surface plasmon resonance (SPR). SPR chips were functionalized with VWF (∼2000 resonance units [RU]), and the individual GF was flowed over the chips at indicated concentrations. Curves represent the specific responses (in RU) to VWF obtained. Experimental curves were fitted with (C,F) 1:1 Langmuir fit model and (D-E) heterogeneous ligand-parallel reactions binding. Binding kinetics values (dissociation constants [K_D] and rate constants [k_on and k_off]) determined from the fitted curves are shown.

Figure 3.

VWF and GF colocalize in ECs and interact in human plasma. (A) Representative high-magnification immunofluorescence images of VWF (green) and VEGF-A (red) expression in the wound, shown as single grayscale channels, in HUVECs; nuclei are identified by DAPI (blue). The white box identifies zoom area shown in (B). Scale bar = 20 μm. (C) VWF was quantified by ELISA in total HUVEC lysates (left) or in cell culture supernatants (right) in the presence or absence of phorbol myristate acetate (PMA) stimulation. Data expressed as ng/mg are normalized to total protein levels (mg) (n = 4; data are mean ± SEM). (D) VEGF-A was quantified by ELISA in total HUVEC lysates (left) or in cell culture supernatants (right) in the presence or absence of PMA stimulation. Data expressed as pg/mg are normalized to total protein levels (mg) (n = 4; data are mean ± SEM). (E) Human plasma or positive control (recombinant VWF + recombinant VEGF-A or FGF-2) was subjected to immunoprecipitation with anti-human VEGF-A antibody or anti-human FGF-2 antibody. Western blotting was performed with collected proteins using anti-human VWF antibody. Mann-Whitney U test was used for analysis. *P < .05.

Figure 4.

The HBD within the A1 domain of VWF mediates GF binding. (A) The location of the A1 domain and HBD within VWF. (B-C) Affinity of VEGF-A, PlGF, PDGF-BB, FGF-2, or CXCL-12 against (B) recombinant VWF A1 domain protein or (C) VWF A1 HBD peptide. ELISA plates were coated with 10 µg/mL recombinant VWF A1 domain protein or 10 µg/mL VWF A1 HBD peptide and further incubated with a 1-μg/mL VEGF-A, PlGF, PDGF-BB, FGF-2, or CXCL-12 solution. Bound GF or chemokine was detected by using a specific antibody for each GF or chemokine (n = 4; data are mean ± SD). Analysis of variance (ANOVA) with Tukey’s test or Mann-Whitney U test was performed. *P < .05; **P < .01.

Figure 5.

R1341 mutations observed in VWD type 2B patients impaires VWF-GF binding. (A) Binding of VEGF-A165, PDGF-BB, and FGF-2 to VWF A1 HBDs with R1341 substitutions. (n = 4; data are mean ± SD). (B) Binding of VEGF-A165, PDGF-BB, and FGF-2 to recombinant human VWF (rhVWF) with R1341Q substitution (n = 4; data are mean ± SD). ANOVA with (A) Tukey’s test and (B) Mann-Whitney U test. **P < .01; *P < .05.

Figure 6.

VWF HBD-functionalized fibrin matrices enhance GFs retention in vitro and in VWF-deficient mice. (A-B) GF retention in fibrin matrix. Graph showing the cumulative release of (A) VEGF-A165 or (B) PDGF-BB over 5 days (n = 4; data are mean ± SEM). (C-E) Full-thickness back-skin wounds in VWF-deficient mice were treated with fibrin only or fibrin functionalized with α₂PI_1–8-VWF HBD. (C) Five days after the wound treatment, the number of CD31⁺/CD45^– ECs was determined by using flow cytometry (data are mean ± SEM). (D-E) The amounts of (D) VEGF-A and (E) PDGF-BB in the wounds were quantified by ELISA and normalized by the weight of the wound tissue (data are mean ± SEM). (A-B) Mann-Whitney U test for each time point, (C) ANOVA with Tukey’s test, and (D-E) Mann-Whitney U test. **P < .01; *P < .05.

Figure 7.

Delivering GFs within VWF HBD–functionalized fibrin matrices enhances skin wound healing in diabetic mice. Full-thickness back-skin wounds were treated with combined 100 ng of VEGF-A165 and 50 ng of PDGF-BB. Four groups were tested: fibrin only, fibrin functionalized with α₂PI_1–8-VWF HBD only, fibrin containing GFs only, and fibrin functionalized with α₂PI_1–8-VWF HBD containing GFs. After 7 days, (A) wound closure and (B) granulation tissue area were evaluated by histomorphometry (n = 11-13 per treatment group; data are mean ± SEM). Five days after the wound treatment, (C) the frequency of CD31⁺CD45^– ECs within total alive cells and (D) proliferation of SMA⁺CD45^– SMCs assessed by Ki67⁺ marker was determined by using flow cytometry (data are mean ± SEM). *P < .05; **P < .01, ANOVA with Tukey’s test.