Introduction
Vascular insufficiency affects one in ten people over 70 years of age and one in six over 80 [
1]. With age as a major contributing factor, the number of people diagnosed with peripheral arterial disease (PAD) increased by over 23% in the decade between 2000 and 2010 [
1]. Current treatment options include either changes in lifestyle for patients experiencing intermittent claudication or surgical intervention such as open surgical bypass or catheter-based revascularization for patients at risk of limb loss. With concerns growing around the long-term patency of surgical procedures [
2], and the ongoing failure of procedural revascularization in a significant proportion of patients [
3], clinicians are also focusing efforts on alternative strategies. Novel attempts to induce new vessel formation at sites of tissue ischaemia have concentrated on the use of O
2 therapy [
4], stem cell [
5] or growth factor therapy [
6]. They all aim to increase the local concentration of growth factors to stimulate angiogenesis. More recently, restoring balance in the glycosaminoglycan (GAG) fraction of vessel extracellular matrix (ECM) has proven effective for the induction of new vessels in embryonic models [
7]. Such GAG-based treatments are novel because they act by sequestering, and then bio-activating powerful endogenous mitogens naturally produced at sites of tissue ischaemia to promote the formation of new vessels, all without the risks inherent with exogenous growth factor therapy.
Angiogenesis is a complex process that involves temporal regulation of growth factor signalling to initiate neovascularization and remodelling at the compromised site. The most actively studied growth factor for angiogenic therapy has been vascular endothelial growth factor-A (VEGF-A) [
8]. Neovascularization in the ischaemic limb can be attenuated with the use of neutralising antibodies against VEGF [
9], highlighting its critical role during vessel recovery in the early stages of revascularization. Most investigations have focused on the 165-isoform of VEGF-A (VEGF
165) due to its intrinsic potency. While the use of VEGF-A has proven successful for treating ischaemia in animal models [
10], only modest benefits have been reported in human clinical trials [
11], mainly owing to the short half-life of the growth factor [
12,
13] when confronted with fluctuations in inflammatory state, pH, temperature, oxidation states and proteolytic factors within sites of damage [
14]. In attempts to overcome the poor pharmacokinetics, multiple large doses of growth factors are frequently administered, which often result in unwanted toxicity and adverse off-target events [
15].
The binding of VEGF to receptors on endothelial cells to initiate the angiogenesis cascade [
16] is dependent on particular forms of its carbohydrate cofactor, heparan sulphate (HS) [
17]. Such a crucial role suggests the possibility of developing these sugars for angiogenic therapy. However, targeting the binding of HS to particular proteins is necessary for driving desirable outcomes, because the inherent heterogeneity of pericellular HS leads to the binding of a plethora of factors that can otherwise disrupt angiogenic progression. We previously reported a VEGF
165-binding HS (HS7) that was isolated via affinity-chromatography using the heparin-binding domain of VEGF
165 as an affinity substrate [
18]. HS7 demonstrated increased affinity for VEGF
165 and potentiated endothelial cell proliferation and vessel tube formation in an embryonic model. The most striking observation was the ability of HS7 alone to promote blood vessel formation in the chick embryo chorioallantoic membrane assay without the addition of exogenous VEGF
165 [
18]. Here, we sought to determine whether VEGF
165 could be protected from thermal and enzymatic degradation by HS7. In a hypoxic environment, where VEGF expression is elevated to promote angiogenesis and reperfusion of blood supply to the injured site, one of the factors determining the extent of the angiogenic response is its stability. VEGF is very susceptible to both thermal and enzymatic degradation in vivo. Brandner et al. have previously demonstrated that VEGF can be protected against thermal degradation by heparin [
19].
To further exemplify the proangiogenic potential of HS7, we induced hindlimb ischaemia in mice and investigated the ability of HS7 to restore blood flow. Our results demonstrate that HS7 by itself can restore blood volume at ischaemic sites by increasing blood vessel density. The results support the continued development of glycosaminoglycans as a means of therapeutic angiogenesis.
Methods
Materials
HS7 used in this paper was isolated from crude porcine mucosal HS (HS
pm, Lot# HO-10697; Celsus Laboratories, Cincinnati, OH) using the methods previously described [
18]. Recombinant human VEGF
165 (VEGF
165) and Toll-like receptor-4 (TLR4), and biotinylated antibody against VEGF
165 were from R&D Systems (Minneapolis, MN). Horseradish peroxidase (HRP)-conjugated streptavidin was from Invitrogen. Plasmin, general chemicals and reagents were purchased from Sigma-Aldrich. Human umbilical vein endothelial cells (HUVECs) (Merck Millipore) were provided at passage 1 and maintained with the EndoGRO™-Low Serum culture media kit (Merck Millipore). HUVECs from passages 2–8 were used for all experiments.
Thermal degradation and plasmin proteolysis
To investigate thermal stability, 5.2 mmol/L VEGF165 was pre-incubated with or without respective HS (1 µg/µl) on ice for 10 min. The reactions were then transferred to 37 °C for further incubation, or − 80 °C for subsequent freeze–thaw treatment. For reactions incubated at 37 °C, aliquots of each reaction were collected at stipulated time points and stored at − 80 °C.
In addition to thermal degradation, VEGF165 is also susceptible to enzymatic digest by plasmin in vivo. To determine the stability of VEGF165 against plasmin digest, 5.2 mmol/L VEGF165 was pre-incubated on ice for 10 min with or without respective HS (1 µg/µL). Plasmin was added to the reactions (final activity 0.5 mU/µL) and then further incubated for 4 h at 37 °C.
At the end of the respective assays, 4× native gel loading buffer (40 mmol/L Tris–HCl pH 8, 400 mmol/L KCl, 40% v/v glycerol, 0.4% v/v NP-40) was added to the thermal and proteolytic degradation reactions, resolved on 4–12% Bis-Tris gelsNuPAGE, Novex, Life Technologies) and blotted onto nitrocellulose membranes. Membranes were blocked and incubated with a biotinylated anti-VEGF antibody, followed by HRP-conjugated streptavidin. Immunoreactive bands were visualised using the LumiGLO® Chemiluminescent Substrate Kit.
VEGF-VEGFR2 signal transduction
HUVECs were seeded in 12-well plates at a density of 190,000 cells per well and cultured in EndoGRO™-Low Serum complete culture media kit lacking HS, rEGF and LS-growth supplement for 24 h. VEGF165 (treated as described in Thermal degradation and plasmin proteolysis) was added to cells and incubated for 10 min at 37 °C. Cells were washed with ice-cold 1× PBS and lysed in ice-cold RIPA lysis buffer containing protease inhibitor cocktail and 2 mmol/L sodium orthovanadate. The lysate was collected and incubated on ice for 20 min and clarified. Total protein content was determined using the BCA quantification method with the Pierce™ BCA Protein Assay Kit (Thermo Scientific). For each reaction, 10 µg of protein was resolved on 4–12% Bis-Tris gels under reducing conditions and blotted onto nitrocellulose membranes. Membranes were subsequently blocked and probed with specific primary antibodies (Cell Signaling Technology Inc.) followed by appropriate HRP-conjugated secondary antibodies (Jackson Immunoresearch). Immunoreactive bands were visualised using the LumiGLO® Chemiluminescent Substrate Kit (KPL, USA).
Cell proliferation
The BrdU Cell Proliferation Kit (Roche) was used to assay cell proliferation. HUVECs were seeded in 96-well plates at a density of 50 cells/mm2 (square millimetre) and cultured in EndoGRO™-Low Serum complete culture media kit lacking HS, rhEGF and LS-growth supplement for 24 h. VEGF165 (treated as described in Thermal degradation and plasmin proteolysis) was added to cells and incubated at 37 °C. After 24 h, BrdU was added to cells and incorporated for 24 h. BrdU detection was performed according to the manufacturer’s protocol.
Affinity fractionation using HS-tagged chromatography columns
The interaction of HS with growth factors in serum was investigated using a modified affinity fractionation protocol. To prepare the column, lyophilised cyanogen bromide-activated Sepharose 4B (CNBr-Sepharose) (GE Healthcare) was rehydrated in 1 mmol/L HCl. The CNBr-Sepharose slurry was incubated with HS in binding buffer (100 mmol/L NaHCO3, 500 mmol/L NaCl, pH 8.3) at a ratio of 2:1 HS to CNBr-Sepharose to obtain HS-tagged Sepharose beads that were loaded onto chromatography columns and washed with wash buffer (1× PBS, 1 mol/L NaCl), followed by equilibration buffer (50 mmol/L Tris, 200 mmol/L NaCl, 2 mmol/L CaCl2, 2 mmol/L MgCl2, pH 7.4). The fractionation sample was prepared by adding 500 ng VEGF165 and 500 ng BMP-2 (bone morphogenetic protein-2) to fetal bovine serum (FBS), then diluted in equilibration buffer. The samples were added to the columns (HS7-tagged or HSft-tagged columns) and washed with equilibration buffer via gravity-flow. A first elution was done using Elution Buffer 1 (50 mmol/L Tris, 500 mmol/L NaCl, 2 mmol/L CaCl2, 2 mmol/L MgCl2, pH 7.4) to remove weak binding proteins to the HS-tagged column. A second elution was then performed using Elution Buffer 2 (50 mmol/L Tris, 1 mol/L NaCl, 2 mmol/L CaCl2, 2 mmol/L MgCl2, pH 7.4) to elute proteins that firmly bound to the column. Samples eluted with Elution Buffer 2 were prepared for immunoblotting as previously described using native gel loading buffer.
Surface-plasmon resonance (SPR)-based measurement of protein binding to heparin-coated SA sensor chip
Protein binding to a streptavidin (SA) sensor chip was performed as previously described [
18]. Briefly, biotinylated heparin was immobilised onto a streptavidin (SA) sensor chip. Protein binding to the heparin support was detected using a BIACORE T100 surface-plasmon resonance instrument as per the manufacturer’s protocols (GE Healthcare, Sweden). Toll-like receptor 4 (TLR4) was prepared in HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 0.1%
v/
v Tween-20) and applied to the sensor chip at a flow rate of 30 µL/min for 120 s, followed by washing with running buffer for 600 s. The sensor chip was regenerated between consecutive applications of TLR4 at different concentrations. VEGF
165 was applied separately as a comparison.
Signal transduction in RAW264.7
RAW264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, high glucose, pyruvate preparation, Gibco™, Thermo Fisher Scientific) supplemented with 10% v/v FBS and 100 µg/mL streptomycin and 100 IU/mL penicillin. For signal transduction, RAW264.7 were seeded in 6-well plates at a density of \(2 \times {10^6}\) cells per well. Lipopolysaccharide (LPS) at 50 ng/mL, or HS at 10 µg/mL were added to cells and incubated for 20 min at 37 °C. Cells were then washed and lysate was collected and quantified as described in the section on “VEGF-VEGFR2 signal transduction”. For each treatment condition, 20 µg of lysate was resolved on 4–12% Bis-Tris gels under reducing conditions, blotted onto nitrocellulose membranes, and probed with specific antibodies followed by appropriate HRP-conjugated secondary antibodies. Immunoreactive bands were visualised using the LumiGLO® Chemiluminescent Substrate Kit.
Efficacy study of HS7 in murine model of hindlimb ischaemia
All animal-related procedures were in accordance with Institutional Animal Care and Use Committee guidelines at the Biological Resource Centre, A*STAR, Singapore (IACUC #130842). Unilateral hindlimb ischaemia was induced in the right hindlimb of C57BL/6N mice (male, 10 weeks old, each weighing 20–25 g) as described previously [
20]. Male mice were chosen to exclude the effect of hormonal influences in the angiogenic healing response. Also, the C57BL/6 strain was shown to exhibit elevated expression of the murine VEGF-A-164 isoform following ischaemia, which shares 89% homology with corresponding regions of human VEGF
165 and could stimulate HUVEC proliferation [
8,
9]. Briefly, the mice were anaesthetised with an intraperitoneal injection of Ketamine (150 mg/kg) and Xylazine (10 mg/kg). A 1-cm incision was made in the right hindlimb from the medial thigh to the knee. The external iliac artery was isolated, ligated twice using 7/0 polypropylene suture (Premilene, Braun, Melsungen AG) and then transected between the two ligations. The mice received subcutaneous injections of Enrofloxacin (10 mg/kg) once daily for 5 days and of Buprenorphine (0.1 mg/kg) twice daily for 3 days after the surgery. The animals were divided into four treatment groups. One day after surgery and for a further 7 days, animals were injected daily with 3 µg HS7 (
n = 8), 30 µg HS7 (
n = 8) or 30 µg flowthrough HS (HS
ft,
n = 8) intramuscularly at three different sites on the operated limb (vastus lateralis, vastus medialis and gastrocnemius). All HSs were delivered in 60 µL of PBS. Controls (
n = 8) received PBS (60 µL) only. Postoperative loss and gain of function, during the treatment and recovery phases were recorded and categorised numerically from 1 to 4, with 1 showing loss of function and 4 showing recovery of function (Table
1). A list of animals is shown in the Supplemental Table (Table S1).
Table 1
Hindlimb functional score
4 | Full recovery |
3 | Able to flex toes, but lacks fine motor control |
2 | Able to support weight on the hindlimb |
1 | Dragging hindlimb |
Laser Doppler flow imaging (LDI)
Blood perfusion in the plantar foot was assessed using a PeriScan PIM 3 laser Doppler system (Perimed AB, Sweden) preoperatively, postoperatively and on days 3, 7, 14 and 21. Animals were anaesthetised with 2% isoflurane and placed in a prone position connected to a continuous flow of isoflurane (1–3%) for the duration of the scan. Images were analysed using PIMSoft software (Perimed AB, Sweden). The footpad was used as the region of interest; the perfusion rate in the footpad of the right (ligated) limb was normalised to the contralateral (non-ligated) limb.
Magnetic resonance angiography (MRA)
The hindlimb vasculature of the animals was acquired using time-of-flight (TOF) magnetic resonance angiography (MRA) with a Bruker Biospec 9.4T scanner (Bruker, Germany). A flow compensated gradient-echo TOF protocol, with a spatial resolution of 0.109 millimetres per pixel (mm/pixel) × 0.109 mm/pixel, 0.35 mm slice thickness and 150 slices, was applied at ultra-high field to characterise the vascular signal above the signals originating from the surrounding stationary tissues. Signals originating from the stationary tissues were saturated with very short repetition time such that the longitudinal magnetization of these tissues did not have time to recover, thereby weakening their signal. This favours the inflow effect.
The MRA data were analysed using ImageJ software. Blood vessels were segmented by thresholding. The total volume of blood was calculated by multiplying the total vessel area by the slice thickness. 80 slices covering the start of the thigh to the lower calf were used for blood volume quantification. Image slices were stacked using Onis 2.5 (DigitalCore Co. Ltd., Japan) to create three-dimensional views of the vasculature.
Immunohistochemistry and histomorphometric analysis
Quadriceps, hamstring and gastrocnemius tissues were harvested on day 8 post-surgery. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Serial transverse sections from all tissues were obtained and stained for visualisation of von Willebrand factor (vWF) with rabbit anti-human vWF antibody (Dako, USA) using the LEICA Bond Auto Stain according to the manufacturer’s instructions, and also for haemotoxylin and eosin (H&E) staining for visualisation of muscle morphology. Transverse sections from quadriceps tissue were obtained and stained for visualisation of α-smooth muscle actin (αSMA) using rabbit anti-αSMA antibody (Abcam, Catalogue ab5694) following standard immunohistochemistry protocols. Images for quantification of vWF-stained and αSMA-stained vessels were captured under brightfield using the Metafer 4 automated captured system (MetaSystems, Germany). Vessels positive for vWF and αSMA were counted from six random fields of view per section and represented as the number of vWF-positive (vWF
+) or αSMA
+ cells ± standard deviation per square millimetre of area quantified. For display purposes only, representative images were captured on the ZEISS AxioImage Z1 (ZEISS, Germany) and uniform adjustments were made on ImageJ to enhance contrast. The number of regenerating myofibres, defined as having a centrally located nuclei [
21], was counted from H&E sections. Data were presented as the mean number of regenerating myofibres ± standard deviation per square millimetre of area quantified. All measurements were made on unprocessed images.
Statistical analysis
Data on proliferation were reported as the mean ± standard deviation of three independent experiments. Data on LDI and MRA were reported as the mean fold-change ± standard deviation. Statistical analyses were performed with GraphPad Prism 7.0a (GraphPad Software Inc., San Diego, CA). Unpaired t test, or one-way and two-way ANOVA with Tukey’s multiple comparisons were performed where appropriate.
Discussion
In this study, we demonstrate that treatment of ischaemic limbs with a blood vessel ECM-mimicking HS variant (HS7), one that avidly binds VEGF165, can accelerate subsequent revascularization and functional recovery. This particular HS7 variant was purified from HSpm via affinity-chromatography targeting the heparin-binding domain of VEGF165.
VEGF
165 is the most abundant isoform in its family and exists as a homodimer in vivo. When the homodimer binds two copies of its receptor, VEGF receptor-2, angiogenesis is initiated. Thus, maintaining the stability of the VEGF
165 homodimer is central to unlocking the therapeutic effects of this potent angiogenic growth factor. Moreover, achieving this by targeting endogenously produced ligands represents a novel means of regulating the body’s angiogenic cascade. Data from the in vitro study demonstrate that HS7 binds and sustains homodimeric VEGF
165 for up to 6 h, whereas complexation with a low-affinity binding HS variant (HS
ft) did not improve the stability of the homodimer. That is, an HS variant with increased affinity for a particular growth factor can enhance the growth factor’s bioactivity. Similar increased stability in FGF-2 (fibroblast growth factor-2) [
26] and VEGF [
19] has been observed in the presence of heparin, a highly sulphated form of HS [
27]; however, due to its powerful anticoagulant activity [
28], the administration of heparin raises strong concerns about associated bleeding [
29], let alone its myriad of ancillary, adverse clinical reactions. Heparin-bonded bypasses, or stent grafts for below-knee bypasses, have also brought about heparin-induced thrombocytopenia in patients [
30]. Furthermore, while the high sulphation of heparin affords the ability to bind and stabilise a broad range of proteins, its propensity for indiscriminate binding also carries the substantial risk of activating various off-target biological processes, so leading to unfavourable healing outcomes.
In addition to thermal stability, studies in vitro show that HS7 also reduced VEGF
165 susceptibility to proteolysis by plasmin, an enzyme present in injured tissue that plays several roles during angiogenesis, including the release of ECM proteins, activation of matrix metalloproteinases and modulating the availability of major angiogenic growth factors. VEGF-induced angiogenesis in plasminogen-deficient mice is known to be compromised, for example [
31]. However, VEGF
165 is also susceptible to plasmin, with a resultant reduction in its mitogenic activity by the removal of its heparin-binding domain [
32]. Our results demonstrate that HS7 reduced VEGF
165 susceptibility to plasmin, reemphasizing the importance of the heparin-binding domain of VEGF
165 for its angiogenic activity, as others have previously indicated [
23,
32,
33].
Our previous report showed that HS7 alone was not able to stimulate endothelial cell proliferation [
18], thus making the selection of C57BL/6 mice for this proof-of-concept study crucial [
9]. Also, the period of treatment coincided with the time-frame when VEGF is stably detected in the ligated tissue [
9]. Based on our MRA data, even 3 µg of HS7 was sufficient to boost blood flow in the ischaemic limb and increase vWF
+ vessel number by day 8. It was, however, insufficient to recover perfusion in the plantar foot as well as 30 µg of HS7. This appears to represent a dose–response criterion that has to be considered for future trials. The observation of more regenerating myofibres and αSMA
+-vessels after only one week of treatment with HS7 was encouraging as it indicated that HS7 delivery could promote the recovery that was subsequently observed in our functional assessments of limb usage (Fig.
5). The presence of more αSMA
+-vessels indicated the presence of mature vessels that could contribute to the higher blood flow quantified in HS7-treated animals. The comparison of HS
ft treatment outcomes was of particular interest. Based on our in vitro findings, HS
ft had much reduced stabilising effects on VEGF
165. This might explain the minimal effect HS
ft had on accelerating blood reperfusion in the ischaemic limb, even though its interaction with other angiogenic factors upregulated during ischaemia seems highly likely. Recovery from ligation was similar to the saline group and could represent the normal rate of blood flow recovery that occurs in C57BL/6 mice [
34,
35]. A concern with the use of HS7 as a treatment for vascular ischaemia was the potential for HS7 to bind to a host of other proteins in vivo via electrostatic interaction, or for proteins in serum to disrupt the HS7-VEGF
165 interaction. Therefore, we deemed it encouraging when affinity-chromatography of FBS pre-loaded with both BMP-2 and VEGF
165 demonstrated that HS7-tagged columns had a higher affinity for VEGF
165 (Fig.
2b). This confirmed that the HS7-VEGF
165 interaction was maintained despite the presence of proteins in serum that could potentially hinder this interaction. Such data lend support to our hypothesis that HS7 was able to bind and potentiate the endogenous ligands like VEGF
165 to promote reperfusion recovery. However, while the HS7-column bound more VEGF
165 by virtue of its increased affinity for the growth factor, it does not answer the question of whether they co-localise in vivo, which necessitates further investigations.
Recent reports have highlighted the possibility of HS-activating Toll-like receptor 4, so promoting inflammatory events that could initiate angiogenesis at ischaemic sites [
36,
37]. Indeed, inflammation is observed in patients suffering from critical limb ischaemia with underlying atherosclerotic pathology [
38]. However, our SPR data revealed no interaction between TLR4 and heparin (Fig. S4a) immobilised on a SA chip support, in contrast with the experiments that showed VEGF
165 binding to immobilised heparin in a dose-dependent manner. Incubation of RAW264.7 with HS also showed that the TLR4 signal transduction pathway was not activated, whereas phosphorylation was observed when cells were exposed to LPS as a positive control (Fig. S4b).
The use of HS on its own for the treatment of limb ischaemia is in contrast to studies investigating gene-, protein- or cell-based treatments for therapeutic angiogenesis in similar animal models [
34,
39‐
41]. The assumptions of such studies still rely on increasing the local concentration of angiogenic growth factors to stimulate neovascularization. In contrast, similar studies in cutaneous wounds [
42] and pressure ulcers [
43] in rats using the HS glycosaminoglycan mimetic OTR4120 support the finding that external applications of HS potentiate the endogenous healing capacity of tissues experiencing damage. The administration of another synthetic glycosaminoglycan, OTR4131, accelerated neovascularization in rat limb ischaemia as well as muscle regeneration [
44]. These and other studies postulated that the binding of synthetic glycosaminoglycans to VEGF might potentiate the growth factor’s angiogenic activity [
45] through a combination of mechanisms shown by our in vitro assays in the current report, as well as from past investigations [
18]. We show here that HS7 prolongs the active, dimeric VEGF
165 for a longer period than the low-affinity HS
ft or in its absence. Also, the use of an affinity-tuned HS to enhance the activity of VEGF
165 should decrease the risk of off-target effects of HS, as seen in the case of heparin, which binds non-discriminately to a large number of proteins. Synthetic glycosaminoglycans may replace endogenous HS species that are degraded in wound sites, but they presumably bind a large number of factors, including agonists and antagonists, unless they are carefully filtered. Naturally occurring HS species carry targeted binding motifs that synthetic GAGs cannot fully mimic.
Using a murine hindlimb ischaemia model, we showed that HS7 improved blood flow recovery. Despite the positive outcomes, we recognise the inherent limitations of the study. One of them is the use of 10-week old C57BL/6N mice, which is akin to replicating the presence of active repair cascades that occur in young, healthy adolescents or adults [
46]. PAD and critical limb ischaemia are more prevalent in aged and unhealthy populations, such as patients with diabetes [
47,
48]. It is therefore crucial that the efficacy of HS7 is further validated in animals models with accompanying comorbidities [
46]. For HS7 to be a suitable therapeutic agent, amounts of endogenous VEGF expression in the patient become crucial. Healing capacity is observed to decrease with age in a mouse wound model, due to reduced gene expression of growth factors and their receptors [
49]. The combined delivery of VEGF
165 and HS7 may still be required in some patients. HS7 stabilisation of ligands also offers the additional advantage of reducing the high doses of exogenous growth factor required, providing a possible solution to the side effects that accompany the use of growth factors in revascularization therapy.