**1. Introduction**

726 Biomedical Science, Engineering and Technology

Yang, P., Wang, J., Gong, G., Sun, X., Zhang, R., Du, Z., Liu, Y., Li, R., Ding, F., Tang, B., Dai,

Yang, D., Wang, C.E., Zhao, B., Li, W., Ouyang, Z., Liu, Z., Yang, H., Fan, P., O'Neill, A., Gu,

Yu, G., Chen, J., Yu, H., Liu, S., Chen, J., Xu, X., Sha, H., Zhang, X., Wu, G., Xu, S., Cheng, G.

Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J.,

Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I.I., Thomson, J.A. 2009. Human

Zaidi, A., Schmoeckel, M., Bhatti, F., Waterworth, P., Tolan, M., Cozzi, E., Chavez, G.,

Zakhartchenko, V., Mueller, S., Alberio, R., Schernthaner, W., Stojkovic, M., Wenigerkind,

Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G.M., Arlotta, P. 2011. Efficient

Zhao, J., Ross, J.W., Hao, Y., Spate, L.D., Walters, E.M., Samuel, M.S., Rieke, A., Murphy,

Zhao, J., Hao, Y., Ross, J.W., Spate, L.D., Walters, E.M., Samuel, M.S., Rieke, A., Murphy,

Zhao, X. Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Guo, C.L., Ma, Q.W., Wang, L.,

Zhu, C., Li, B., Yu, G., Chen, J., Yu, H., Chen, J., Xu, X., Wu, Y., Zhang, A., Cheng, G. 2009.

postnatal fibroblasts. Mol Reprod Dev 60: 362-369.

transcription. Nat Biotechnol 29(2): 149-153.

complementation. Nature 461(7260): 86-90.

transfer. Biol Reprod 81(3): 525-530.

Reprogram 12(1): 75-83.

18(2):163-171.

One 3(10): e3453.

87: 1019-1027.

1917-1920.

324(5928): 797-801.

Transplantation 65: 1584.

Genet 19(20): 3983-3994.

Y., Li, N. 2008. Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin. PloS

W., Yi, H., Li, S., Lai, L., Li, X.J. 2010. Expression of Huntington's disease protein results in apoptotic neurons in the brains of cloned transgenic pigs. Hum Mol

2006. Functional disruption of the prion protein gene in cloned goats. J Gen Virol

Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):

induced pluripotent stem cells free of vector and transgene sequences. Science

Langford, G., Thiru, S., Wallwork, J., White, D., Friend, P. 1998. Life-supporting pig-to primate renal xenotransplantation using genetically modified donors.

H., Wanke, R., Lassnig, C., Mueller, M., Wolf, E., Brem, G. 2001. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and

construction of sequence-specific TAL effectors for modulating mammalian

C.N., Prather, R.S. 2009a. Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear

C.N., Prather, R.S. 2010. Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell

Zeng, F., Zhou, Q. 2009b. iPS cells produce viable mice through tetraploid

Production of Prnp -/- goats by gene targeting in adult fibroblasts. Transgenic Res

Blood and lymphatic vessels are present in all tissues, and play important roles for their function, homeostasis and maintenance. Angiogenesis, the growth of new blood vessels, is therefore highly important during development, but is largely not observed in the adult, except for during the female reproduction cycle and during wound healing. In pathological situations, however, angiogenesis may be turned on, and in this case contribute to the onset and progression of most severe human pathologies characterized by high mortality, including cancer, diabetes, obesity and retinopathies (Carmeliet, 2003) or is insufficiently activated such as in the case of myocardial infarction and stroke (Y Cao et al, 2005). Thus, angiogenesis is one of the largest and fastest evolving areas of research today. Angiogenesis is a highly complicated process, involving many different cell types, and it is therefore highly recommended that researchers use *in vivo* animal models for their studies. Accordingly, today there are many *in vivo* models available.

The aim of this chapter is to give insights into the most commonly used *in vivo* angiogenesis models in both mice and zebrafish. We will provide detailed descriptions and discussions of the adipose tissue-, tumor-, ischemic hind limb- wound healing- and corneal micropocket angiogenesis models in mice and developmental-, tumor-, hypoxia-induced retinal- and regenerating tail fin angiogenesis models in zebrafish. We will provide a base for comparison between the different assays to quickly identify which model is best suited for a particular research focus.

### **1.1 Basic mechanisms of angiogenesis**

Angiogenesis is a multistep process which is tightly regulated by an intimate balance between pro- and anti-angiogenic factors. Angiogenesis is stimulated by angiogenic factors the most commonly studied being members of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor (TGF) or platelet derived growth factor (PDGF) families in the tissue. These factors either act locally, or are

<sup>\*</sup> J. Honek1, K. Hosaka1, P. Rouhi1, S. Lim1, H. Ji1, Z. Cao2, E. M. Hedlund1, J. Zhang1 and Y. Cao1,2

*<sup>1</sup> Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden,* 

*<sup>2</sup> Institution of Medicine and Health, Linköping University, Linköping, Sweden.* 

released into the blood stream and activate angiogenesis through binding to their corresponding receptors on pre-existing endothelial cells and/or perivascular cells (Y Cao, 2008). Upon ligand binding to the receptors, a cascade of intracellular signal transduction is triggered resulting in activation of the endothelial cell and initiation of the cellular mechanisms required for angiogenesis including basal membrane degradation, endothelial cell (EC) proliferation and migration, formation of tube like structures, and finally vascular maturation by coverage with smooth muscle cells or pericytes to ensure stability (Davis & Senger, 2005; Jensen et al, 2007).

The dynamics of angiogenesis as well as the morphology of the resulting vasculature differs depending on which factor induces the process (R Cao, 2004). In the case of the most studied angiogenic factor, VEGF, a mature vessel may be exposed to a gradient of VEGF induced by hypoxia in a nearby tissue such as a tumor. VEGF activates VEGF receptor 2 (VEGFR2) on endothelial cells leading to production of matrix-metalloproteases (MMPs) which disrupts EC-pericyte contacts, degrade the basal membrane and induce a tip-cell behavior in one cell, which is allowed to form cell processes important for cell migration such as filopodia and lammelopodia. The tip cell will via lateral inhibition signal to surrounding cells that they should not adopt this phenotype thus leading to an ordered sprouting of just one or a few neovessels from the original mother-vessel. The tip cell will start migrating up the angiogenic factor gradient, but remain in contact with the underlying and proliferating endothelial cells (stalk cells) and thus retain the connection to the original vessel. At a certain point, the tip cell will meet other tip cells, anastomose – i.e. fuse – and thus form a circulation loop. The endothelial chords will lumenize, allowing blood to flow through the vessel and the vessels will mature by recruiting perivascular mural cells which are tightly associated with the endothelial cells thus providing stability and trophic factors for the endothelial cells, leading to their maturation and re-entry into quiescence (Risau 1997).

### **1.2 Angiogenesis in physiology and pathology**

During development the first vessels – the aorta and the cardinal vein – are formed by vasculogenesis - i.e. the *de novo* formation of blood vessels by aggregation and vascular morphogenesis of single endothelial progenitor cells. Following this process, the majority of the blood vessels in the body are formed by angiogenic expansion of this initial, primitive circulation loop (Risau 1997). Thus angiogenesis is very important during development. In adults however, most tissues and therefore also the vasculature have stopped growing and instead adopted a quiescent state. In some cases such as during the female reproductive cycle and during wound healing, angiogenesis is needed to regenerate the tissue, but otherwise the erroneous or insufficient induction of angiogenesis in adults is usually associated with pathology. For example, as a tumor is a growing tissue and therefore need blood vessels for supplying energy, angiogenesis is induced after the tumor has reached a certain size (approximately 1 cubic centimetre). Similarly in growing fat tissue new blood vessels are needed when the distance from the existing blood vessels are too large for efficient oxygenation. By blocking such tumor- or adipose-angiogenesis, it is possible to inhibit the growth of these tissues and therefore anti-angiogenic drugs have potential as treatment for cancer and obesity. In patients with retinopathy retinal hypoxia is a consequence of blocked or disrupted blood vessels and

released into the blood stream and activate angiogenesis through binding to their corresponding receptors on pre-existing endothelial cells and/or perivascular cells (Y Cao, 2008). Upon ligand binding to the receptors, a cascade of intracellular signal transduction is triggered resulting in activation of the endothelial cell and initiation of the cellular mechanisms required for angiogenesis including basal membrane degradation, endothelial cell (EC) proliferation and migration, formation of tube like structures, and finally vascular maturation by coverage with smooth muscle cells or pericytes to ensure stability (Davis &

The dynamics of angiogenesis as well as the morphology of the resulting vasculature differs depending on which factor induces the process (R Cao, 2004). In the case of the most studied angiogenic factor, VEGF, a mature vessel may be exposed to a gradient of VEGF induced by hypoxia in a nearby tissue such as a tumor. VEGF activates VEGF receptor 2 (VEGFR2) on endothelial cells leading to production of matrix-metalloproteases (MMPs) which disrupts EC-pericyte contacts, degrade the basal membrane and induce a tip-cell behavior in one cell, which is allowed to form cell processes important for cell migration such as filopodia and lammelopodia. The tip cell will via lateral inhibition signal to surrounding cells that they should not adopt this phenotype thus leading to an ordered sprouting of just one or a few neovessels from the original mother-vessel. The tip cell will start migrating up the angiogenic factor gradient, but remain in contact with the underlying and proliferating endothelial cells (stalk cells) and thus retain the connection to the original vessel. At a certain point, the tip cell will meet other tip cells, anastomose – i.e. fuse – and thus form a circulation loop. The endothelial chords will lumenize, allowing blood to flow through the vessel and the vessels will mature by recruiting perivascular mural cells which are tightly associated with the endothelial cells thus providing stability and trophic factors for the endothelial cells, leading to their maturation

During development the first vessels – the aorta and the cardinal vein – are formed by vasculogenesis - i.e. the *de novo* formation of blood vessels by aggregation and vascular morphogenesis of single endothelial progenitor cells. Following this process, the majority of the blood vessels in the body are formed by angiogenic expansion of this initial, primitive circulation loop (Risau 1997). Thus angiogenesis is very important during development. In adults however, most tissues and therefore also the vasculature have stopped growing and instead adopted a quiescent state. In some cases such as during the female reproductive cycle and during wound healing, angiogenesis is needed to regenerate the tissue, but otherwise the erroneous or insufficient induction of angiogenesis in adults is usually associated with pathology. For example, as a tumor is a growing tissue and therefore need blood vessels for supplying energy, angiogenesis is induced after the tumor has reached a certain size (approximately 1 cubic centimetre). Similarly in growing fat tissue new blood vessels are needed when the distance from the existing blood vessels are too large for efficient oxygenation. By blocking such tumor- or adipose-angiogenesis, it is possible to inhibit the growth of these tissues and therefore anti-angiogenic drugs have potential as treatment for cancer and obesity. In patients with retinopathy retinal hypoxia is a consequence of blocked or disrupted blood vessels and

Senger, 2005; Jensen et al, 2007).

and re-entry into quiescence (Risau 1997).

**1.2 Angiogenesis in physiology and pathology** 

also here lead to induction of retinal angiogenesis. The newly formed blood vessels are however often immature and prone to rupture which leads to micro-hemorrhages and thereby drives the pathological progression to advanced states of the disease (Carmeliet, 2003; Folkman, 1995). On the other hand, in other hypoxic tissues such as the hypoxic heart tissue downstream of a blocked coronary artery, it is important to speed up the angiogenic revascularization of the tissue in order to secure sufficient oxygen for the highly metabolically active myocardium such that it may sustain its function (Y Cao, 2010).

These aspects of physiological and pathological angiogenesis are accurately modeled by the angiogenesis models we will discuss in this chapter.

### **1.3 Mouse models of angiogenesis**

Mouse models are the primary *in vivo* tools used in biomedical research. Most research institutions have their own mouse or rodent facilities, and today several strains have been genetically purified through serial in-breeding, which allow sophisticated investigations such as transplantation and grafting experiments to be done in a wildtype animal with a fully functional immune system. Furthermore, genetically manipulated mice have been generated for most of the genes important for angiogenesis and vascular biology. Such mice include conditional or global knock-outs or mice over-expressing angiogenic factors in defined tissues. As the murine and human vasculatures are highly similar, these tools have been very valuable in defining the role of angiogenesis and distinct angiogenic factors in the onset and progression of various human diseases.

Here we present five mouse assays of pathological angiogenesis that have shown tremendous power in the study of mechanisms behind human diseases. These are the adipose-angiogenesis models used to study obesity and diabetes, the xenograft models used to study cancer biology, the ischemia models used to study myocardial infarction and stroke, the wound healing models and the cornea micropocket models used to study basic mechanisms of angiogenesis and lymphangiogenesis.
