Altered ratios of pro- and anti-angiogenic VEGF-A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma.

Xi Ye,Neil J Sebire,David O Bates,Patrick Watts,Joyce Bischoff,Yassir Abou-Rayyah,Alison Ritchie,Amanda J Churchill

doi:10.1002/path.4715

Abstract

Infantile haemangioma (IH), the most common neoplasm in infants, is a slowly resolving vascular tumour. Vascular endothelial growth factor A (VEGF‐A), which consists of both the pro‐ and anti‐angiogenic variants, contributes to the pathogenesis of IH. However, the roles of different VEGF‐A variants in IH progression and its spontaneous involution is unknown. Using patient‐derived cells and surgical specimens, we showed that the relative level of VEGF‐A165b was increased in the involuting phase of IH and the relative change in VEGF‐A isoforms may be dependent on endothelial differentiation of IH stem cells. VEGFR signalling regulated IH cell functions and VEGF‐A165b inhibited cell proliferation and the angiogenic potential of IH endothelial cells in vitro and in vivo. The inhibition of angiogenesis by VEGF‐A165b was associated with the extent of VEGF receptor 2 (VEGFR2) activation and degradation and Delta‐like ligand 4 (DLL4) expression. These results indicate that VEGF‐A variants can be regulated by cell differentiation and are involved in IH progression. We also demonstrated that DLL4 expression was not exclusive to the endothelium in IH but was also present in pericytes, where the expression of VEGFR2 is absent, suggesting that pericyte‐derived DLL4 may prevent sprouting during involution, independently of VEGFR2. Angiogenesis in IH therefore appears to be controlled by DLL4 within the endothelium in a VEGF‐A isoform‐dependent manner, and in perivascular cells in a VEGF‐independent manner. The contribution of VEGF‐A isoforms to disease progression also indicates that IH may be associated with altered splicing. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Highlights

Infantile haemangioma (IH) is the most common neoplasm of infancy [1,2]
We used a commonly used vascular endothelial growth factor receptor (VEGFR) inhibitor, PTK787, and identified that proliferation of both haemangioma stem cell (HemSC) and haemangioma endothelial cell (HemEC) was significantly inhibited by PTK787 at concentrations specific to VEGFRs (Figure 1D), supporting the notion that active vascular endothelial growth factor (VEGF)-A signalling is important for IH
Using primers spanning exons 7 and 8b, which distinguish the pro- and anti-angiogenic Vascular endothelial growth factor A (VEGF-A) variants (Figure 2B), we found that the relative VEGF-A165b:VEGF-A165a ratio was higher in HemECs when compared with HemSCs, at both the cDNA and protein levels (Figure 2C, D)

Summary

Introduction

Infantile haemangioma (IH) is the most common neoplasm of infancy [1,2]. The natural history of IH is a rapid proliferating phase within the first 12 months of life, followed by spontaneous involution over 7–10 years [2]. HemSCs proliferation and vasculogenesis are VEGF-A-dependent [5] and HemECs from some patients demonstrate a high level of VEGF receptor 2 (VEGFR2) activation compared with normal endothelial cells [6]. Pre-mRNA alternative splicing controls whether the mRNA contains both exon 8a and 8b or exon 8b alone. The final translated protein product is dependent on this alternative splicing [7,8]. Proximal splicing of exon 8 leads to the translation of the first six amino acids in exon 8a, forming the pro-angiogenic VEGF-Axxxa family. Distal splicing removes exon 8a from the transcript and results in the translation of the first six amino acids in exon 8b, forming the anti-angiogenic VEGF-Axxxb. VEGF-Ax appears to have similar properties to VEGF-A165b, in that it antagonizes VEGF-A165a-mediated VEGFR2 activation and does not bind to Nrp-1 [9]

Methods

Results

Conclusion