Abstract
The Wilms’ tumor suppressor gene (WT1) is widely expressed during neovascularization, but it is almost entirely absent in quiescent adult vasculature. However, in vessels undergoing angiogenesis, WT1 is dramatically upregulated. Studies have shown Wt1 has a role in both tumor and ischemic angiogenesis, but the mechanism of Wt1 action in angiogenic tissue remains to be elucidated. Here, we describe two methods for induction of in vivo angiogenesis (subcutaneous sponge implantation, femoral artery ligation) that can be used to assess the influence of Wt1 on new blood vessel formation. Subcutaneously implanted sponges stimulate an inflammatory and fibrotic response including cell infiltration and angiogenesis. Femoral artery ligation creates ischemia in the distal hindlimb and produces an angiogenic response to reperfuse the limb which can be quantified in vivo by laser Doppler flowmetry. In both of these models, the role of Wt1 in the angiogenic process can be assessed using histological/immunohistochemical staining, molecular analysis (qPCR) and flow cytometry. Furthermore, combined with suitable genetic modifications, these models can be used to explore the causal relationship between Wt1 expression and angiogenesis and to trace the lineage of cells expressing Wt1. This approach will help to clarify the importance of Wt1 in regulating neovascularization in the adult, and its potential as a therapeutic target.
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References
Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature 473(7347):298–307. doi:10.1038/nature10144
Khurana R, Simons M, Martin JF, Zachary IC (2005) Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 112(12):1813–1824. doi:10.1161/CIRCULATIONAHA.105.535294
Vasuri F, Fittipaldi S, Buzzi M et al (2012) Nestin and WT1 expression in small-sized vasa vasorum from human normal arteries. Histol Histopathol 27(9):1195–1202
Katuri V, Gerber S, Qiu X et al (2014) WT1 regulates angiogenesis in Ewing Sarcoma Oncotarget 15:5(9) 2436–49
Dohi S, Ohno S, Ohno Y et al (2010) WT1 expression correlates with angiogenesis in endometrial cancer tissue. Anticancer Res 3192:3187–3192
Wagner K, Wagner N, Bondke A, Nafz B (2002) The Wilms’ tumor suppressor Wt1 is expressed in the coronary vasculature after myocardial infarction 1. FASEB J:1117–1119. doi:10.1096/fj.01
Small GR, Hadoke PWF, Sharif I et al (2005) Preventing local regeneration of glucocorticoids by 11beta-hydroxysteroid dehydrogenase type 1 enhances angiogenesis. Proc Natl Acad Sci U S A 102(34):12165–12170. doi:10.1073/pnas.0500641102
Andrade SP, Fan TP, Lewis GP (1987) Quantitative in-vivo studies on angiogenesis in a rat sponge model. Br J Exp Pathol 68(6):755–766
Barclay GR, Tura O, Samuel K et al (2012) Systematic assessment in an animal model of the angiogenic potential of different human cell sources for therapeutic revascularization. Stem Cell Res Ther 3(4):23. doi:10.1186/scrt114
Niiyama H, Huang NF, Rollins MD, Cooke JP (2009) Murine model of hindlimb ischemia. J Vis Exp 23:2–4. doi:10.3791/1035
Limbourg A, Korff T, Napp LC, Schaper W, Drexler H, Limbourg FP (2009) Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat Protoc 4(12):1737–1748, Available at: http://dx.doi.org/10.1038/nprot.2009.185
Emanueli C, Minasi A, Zacheo A et al (2001) Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia. Circulation 103(1):125–132. doi:10.1161/01.CIR.103.1.125
Dar A, Domev H, Ben-Yosef O et al (2012) Multipotent vasculogenic pericytes from human pluripotent stem cells promote recovery of murine ischemic limb. Circulation 125(1):87–99. doi:10.1161/CIRCULATIONAHA.111.048264
Biscetti F, Pitocco D, Straface G et al (2011) Glycaemic variability affects ischaemia-induced angiogenesis in diabetic mice. Clin Sci (Lond) 121(12):555–564. doi:10.1042/CS20110043
Chau Y-Y, Bandiera R, Serrels A et al (2014) Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source. Nat Cell Biol 16(4):367–375. doi:10.1038/ncb2922
You D, Cochain C, Loinard C et al (2008) Hypertension impairs postnatal vasculogenesis role of antihypertensive agents. Hypertension 51(6):1537–1544. doi:10.1161/HYPERTENSIONAHA.107.109066
Hosen N, Shirakata T, Nishida S et al (2007) The Wilms’ tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis. Leukemia 21(8):1783–1791. doi:10.1038/sj.leu.2404752
Wagner K-D, Cherfils-Vicini J, Hosen N et al (2014) The Wilms’ tumour suppressor Wt1 is a major regulator of tumour angiogenesis and progression. Nat Commun 5:5852. doi:10.1038/ncomms6852
Kirkby NS, Duthie KM, Miller E et al (2012) Non-endothelial cell endothelin-B receptors limit neointima formation following vascular injury. Cardiovasc Res 95(1):19–28. doi:10.1093/cvr/cvs137
Acknowledgment
R.O. is funded by a British Heart Foundation studentship and RMcG by a Wellcome-Trust-funded ECAT research fellowship. The authors are grateful for support from the Edinburgh British Heart Foundation Centre for Research Excellence (CoRE).
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McGregor, R.J., Ogley, R., Hadoke, P., Hastie, N. (2016). In Vivo Assays for Assessing the Role of the Wilms’ Tumor Suppressor 1 (Wt1) in Angiogenesis. In: Hastie, N. (eds) The Wilms' Tumor (WT1) Gene. Methods in Molecular Biology, vol 1467. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4023-3_8
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DOI: https://doi.org/10.1007/978-1-4939-4023-3_8
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