Role of WT1 in Ischaemic Angiogenesis

Abstract

Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis fails to consistently induce sufficient reperfusion to facilitate repair, targeted manipulation of angiogenesis is clinically desirable. The Wilms’ tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that WT1 in the endothelium influences the angiogenic function of endothelial cells. WT1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from WT1-GFP reporter mice. Inducible deletion of WT1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p<0.05 relative to controls). In vivo, WT1 expression was evident in vascular endothelial and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia, often in cells expressing epithelial and mesenchymal markers simultaneously. However, VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density or expression of angiogenic factors (qRT-PCR) in sponges inserted subcutaneously in mice (20 days). To further understand the role of WT1 in angiogenesis, transcriptomic RNA expression analysis was performed in WT1+ and WT1- cells isolated (FACs) from sponges after implantation in WT1-GFP mice. WT1+ cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including angiogenesis (p=3.11x10-8), wound healing (p=3.45x10-7) and epithelial-to-mesenchymal transition (EMT) (p=5.86x10-4). These results shed new light on the role of WT1 in ischaemic angiogenesis. In concurrence with previously published work, we show that deletion of endothelial WT1 can delay angiogenesis however, WT1 is not just instrumental in endothelial cells in this context. WT1 has a broader role in tissue repair in ischaemia, in part through regulation of cell transition (EMT). This work has improved our understanding of the regulatory role of WT1 in angiogenesis and repair, while revealing a number of novel insights into the function of WT1. This highlights WT1 as a potentially beneficial therapeutic target to facilitate regeneration in cardiovascular disease.