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18.05.2018 | Original Paper | Ausgabe 4/2018 Open Access

Angiogenesis 4/2018

Improved recovery from limb ischaemia by delivery of an affinity-isolated heparan sulphate

Angiogenesis > Ausgabe 4/2018
Selina Poon, Xiaohua Lu, Raymond A. A. Smith, Pei Ho, Kishore Bhakoo, Victor Nurcombe, Simon M. Cool
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1007/​s10456-018-9622-9) contains supplementary material, which is available to authorized users.


Peripheral arterial disease is a major cause of limb loss and its prevalence is increasing worldwide. As most standard-of-care therapies yield only unsatisfactory outcomes, more options are needed. Recent cell- and molecular-based therapies that have aimed to modulate vascular endothelial growth factor-165 (VEGF165) levels have not yet been approved for clinical use due to their uncertain side effects. We have previously reported a heparan sulphate (termed HS7) tuned to avidly bind VEGF165. Here, we investigated the ability of HS7 to promote vascular recovery in a murine hindlimb vascular ischaemia model. HS7 stabilised VEGF165 against thermal and enzyme degradation in vitro, and isolated VEGF165 from serum via affinity-chromatography. C57BL6 mice subjected to unilateral hindlimb ischaemia injury received daily intramuscular injections of respective treatments (n = 8) and were assessed over 3 weeks by laser Doppler perfusion, magnetic resonance angiography, histology and the regain of function. Mice receiving HS7 showed improved blood reperfusion in the footpad by day 7. In addition, they recovered hindlimb blood volume two- to fourfold faster compared to the saline group; the greatest rate of recovery was observed in the first week. Notably, 17% of HS7-treated animals recovered full hindlimb function by day 7, a number that grew to 58% and 100% by days 14 and 21, respectively. This was in contrast to only 38% in the control animals. These results highlight the potential of purified glycosaminoglycan fractions for clinical use following vascular insult, and confirm the importance of harnessing the activity of endogenous pro-healing factors generated at injury sites.

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Fig. S1—Elution of VEGF165 from HS-tagged columns The affinity-chromatography of growth factors from FBS using HS-tagged columns was performed twice. Densitometry analysis of each replicate was performed separately and represented here as “Replicate 1” (shown in Fig. 2B) and “Replicate 2”. In both replicates, HS7-tagged columns captured higher amounts of VEGF165 compared to the HSft-tagged columns (EPS 1498 KB)
Fig. S2—Comparison of number of regenerating myofibres The total number of regenerating myofibres from H&E stained sections in a cross section of gastrocnemius tissue were counted and presented as mean number ± standard deviation per square millimetre (mm2). Values were from two independent observers. The bottom panel shows representative cross section images from saline vehicle and HS7 (30 µg) groups. Regenerating myofibres were identified by their centrally located nuclei. White arrows indicate some of the regenerating myofibres. Scale bar = 100 µm. Treatment group size: n = 3 (EPS 74233 KB)
Fig. S3—Comparison of number of αSMA+ vessels The number of αSMA+ vessels from quadriceps was counted from random field-of-views and presented as mean number ± standard deviation per square millimetre (mm2). The observer was blinded to the treatment the animals received. Treatment group size: n = 3 (EPS 433 KB)
Fig. S4—Effect of heparin and HS on TLR4 (a) SPR curves showed no interaction of TLR4 with heparin immobilised on the sensor chip; in comparison, VEGF165 showed dose-dependent interaction on the same SPR chip. (b) HS (10 µg/ml) was incubated with the murine macrophage cell line RAW264.7 and phosphorylated proteins in the TLR4-signalling cascade were probed. Two replicates of the experiment were performed and represented here as “Replicate 1” and “Replicate 2”. Both replicates demonstrated similar findings (EPS 6442 KB)
Table S1 (DOCX 104 KB)
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