Role of the Wilms’ tumour-1 (WT1) gene in adult angiogenesis
McGregor, Richard James
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In 1899, the German surgeon Max Wilms hypothesised that different cell types in a variety of childhood kidney cancers were all derived from the mesodermal layer during embryonic development. Nearly a century later, the WT1 gene was identified on the short arm of chromosome 11, and was thought to be inactive in ~20% of nephroblastomas (Wilms’ tumours). The expression of WT1 after birth appears to be restricted to a finite number of tissues, namely, the glomerular podocytes, mesothelium and ~1% of bone marrow cells. Emerging evidence suggests WT1 is required not only for development, but also for tissue homeostasis, regeneration, repair and angiogenesis. Interestingly, WT1 has been implicated in the response to myocardial infarction and tumour angiogenesis, yet its precise role remains unclear. This thesis aims to address the hypothesis that activation of the WT1 gene in the vascular endothelium is essential for physiological and pathophysiological angiogenesis in the adult. In order to assess whether Wt1 was expressed in quiescent endothelial cells (ECs) immunofluorescence was used to analyse a variety of tissues in the adult mouse. Whilst Wt1 was detected in renal podocytes, no endothelial Wt1 expression was discovered in the lung, heart, kidney, spleen and gastrocnemius muscle. In contrast, tissues known to undergo physiological angiogenesis (endometrium and breast) did exhibit Wt1 expression in the vascular endothelium. Moreover, tubular EC outgrowths generated by aortic rings embedded in collagen ex vivo were positive for Wt1. The role of Wt1 in ischaemic angiogenesis was assessed using models of hind-limb and coronary ischaemia in the mouse. Wt1 was detected in ECs and non-vascular cells following ischaemic injury by a combination of immunofluorescence and qualitative real-time polymerase chain reaction (qRT-PCR). Using a time course analysis of these experimental models the chronology of this relationship was demonstrated, alongside the association with key angiogenic factors, such as Vegf. Given the findings in ischaemic tissue the C3(1)/Tag transgenic mammary cancer model was used to test the hypothesis that Wt1 would be upregulated in the tumour vasculature. Endothelial Wt1 was up regulated in these tumours compared to healthy control tissue. This finding was mirrored in a sub-set of aggressive breast cancers, confirming that the results obtained in mice can be translated to humans. Quantitative PCR revealed no association between histopathological grade of the tumours, oestrogen receptor status, and WT1 expression. In order to delineate the cell types involved in vessel formation, Wt1+ cells were sorted using fluorescent activated cell sorting (FACS) from transgenic mice with a green fluorescent protein knocked into the Wt1 locus following sponge implantation. Distinct sub-populations of Wt1+ cells were identified, some of which expressed EC and pericyte markers. Moreover, these Wt1+ sub-populations changed in composition and number over time. These findings were confirmed by genetic fate mapping of Wt1+ cells in this model. Finally, a conditional knockout mouse was generated to allow the selective deletion of Wt1 from vascular ECs in the sponge model of angiogenesis. The results demonstrated that deletion of Wt1 from this cellular compartment led to a dramatic reduction in vessel formation supporting a potential role in regulating angiogenesis. These results support the hypothesis that expression of WT1 in the vascular endothelium contributes to the regulation of angiogenesis in tumours and ischaemic tissue, and provides evidence that selective deletion of the gene inhibits new vessel formation. This suggests that targeting WT1 may have a therapeutic benefit in cancer and could aid regeneration of ischaemic tissues following injury in conditions such as myocardial infarction and critical limb ischaemia.