in cells near the retinal blood vessels. In oxygen induced retinopathy mice model, the expression of Robo4 was up-regulated while the other Robo or Slit genes were not (Jones et al. 2008).

Three mice models were setup to investigate the roles of Slit-Robo signaling in ocular angiogenesis. Laser-induced choroidal neovascularization, which mimics age-related macular degeneration, is commonly used to study choroidal angiogenesis in the mouse. Intravitreal administration of Slit2 reduced choroidal neovascularization in wild type mice; however, the effect was lost in Robo4 null mice. Oxygen induced retinopathy is the most common used animal model for retinal neovascularization that mimics the ischemia-induced angiogenesis observed in proliferative diabetic retinopathy and retinopathy of prematurity. Similarly, intravitreal administration of recombinant Slit2 markedly reduced area of isolectin labeled retinal neovascularization and of FITC-dextran leakage from retinal vasculature in wild type but not in Robo4 null mice. The third model used is VEGF induced retinal permeability which mimics macular edema. Similarly, intravitreal administration of recombinant Slit2 markedly reduced leakage of Evans blue from retinal vasculature in wild type mice. This effect was not observed in Robo4 null mice. These finding suggested that Slit-Robo Signaling can inhibit ocular angiogenesis in Robo4 dependent pattern. Considering the expression of robo4 on stalk cells but not tip cells, Slit-Robo signaling may provides a tonic pathway that stabilized ocular blood vessels (Jones et al. 2008).

52.5 Signaling Pathway of Slit-Robo System in Angiogenesis

There is controversy on the Slit-Robo binding in vascular system. Overexpressed human Slit2 coimmunoprecipitated with Robo4, indicating direct binding of Slit2 to Robo4 (Park et al. 2003). Overexpression of Robo4 blocked migration of endothelial cells towards VEGF and FGF and that this effect was dependent on Slit2 binding to the extracellular domain of Robo4 (Seth et al. 2005). The inhibitory effects of Slit2 on endothelial cells and ocular angiogenesis are dependent on Robo4 and loss in Robo4 null mice (Jones et al. 2008). These results suggested that Robo4 is a receptor for Slit2 on endothelial cells. However, it was also reported that Robo1 forms a heterodimeric complex with Robo4. Robo1 is essential for Robo4-mediated filopodia induction (Sheldon et al. 2009). Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP (Kaur et al. 2008).

Since Slit-Robo signaling play important role in cell migration in both neuron and endothelial cells, the signaling pathway of Slit-Robo1 in neuron may also provides cue for downstream signaling of Slit-Robo4 in endothelial cell. The intracellular signaling that regulates the actin cytoskeleton is a good candidate of the downstream signaling of Slit-Robo. Robo4 can bind the Mena, an actin regulatory protein, suggesting that Robo4, like Robo1, can control cell movement by locally remodel the actin cytoskeleton (Park et al. 2003). Addition of Slit2 to endothelial cells led to the inhibition of FAK phosphorylation (Seth et al. 2005). Robo4

knockdown endothelial cells show up regulation of Rho small guanosine triphosphatase (GTPase). Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in Robo4 knockdown cells. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which are involved with Robo4 signaling in endothelial cells (Kaur et al. 2008).

VEGF and PDGF signaling are key pathways in angiogenesis; therefore it is necessary to test the interaction of Slit-Robo signaling with VEGF and PDGF signaling. Treatment of endothelial cells with Slit2 reduced VEGF-165 stimulated phosphorylation of the Src family of nonreceptor tyrosine kinases and Src-dependent activation of the Rho family small GTPase Rac1 (Jones et al. 2008). Recombinant N terminal cleavage of Slit2 prevented PDGF-mediated activation of GTPase Rac1 and formation of lamellipodia in smooth muscle cells, both of which are involved in cell motility (Liu et al. 2006).

52.6 Perspective

Slit-Robo signaling has been shown to play an important role in angiogenesis including ocular angiogenesis. The application of Slit can inhibit ocular angiogenesis in disease models and counteract the ability of VEGF in mice. However, controversy exists still whether it is pro-angiogenic or anti-angiogenic signaling The details of Slit-Robo signaling are not fully understood, as well as how these signaling events affect the role of Slit-Robo signaling in pathogenesis of ocular angiogenesis. Therefore, more investigation is required to better understand the role of Slit-Robo in angiogenesis and develop novel therapeutic approaches for ocular angiogenesis diseases like age related macular degeneration, diabetic retinopathy and macular edema.

Acknowledgments We thank the following support to HC: Kaisi Funds at Sun Yat-sen University, JSIEC Starting grants; to ST: National Key Science and Technology Project from ‘Tenth Five-Year Plan’ of China, National Natural Science Foundation of China; to KZ: National Institutes of Health Grants R01EY14428, R01EY14448, P30EY014800, and GCRCM01-RR00064, Foundation Fighting Blindness, the Macular Vision Research Foundation, Veterans Affairs Merit Award, and Research to Prevent Blindness to NRL: the Ruth L. Kirschstein National Research Service Award.

References

Adams RH (2006) Nerve cell signposts in the blood vessel roadmap. Circ Res 98:440–442 Bedell VM, Yeo SY, Park KW et al (2005) roundabout4 is essential for angiogenesis in vivo. Proc

Natl Acad Sci U S A 102:6373–6378

Campochiaro PA, Hackett SF (2003) Ocular neovascularization: a valuable model system. Oncogene 22:6537–6548

Dallol A, Forgacs E, Martinez A et al (2002) Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers. Oncogene 21:3020–3028

Friedman DS, O’Colmain BJ, Munoz B et al (2004) Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 122:564–572

Gariano RF, Gardner TW (2005) Retinal angiogenesis in development and disease. Nature 438:960–966

Hohenester E, Hussain S, Howitt JA (2006) Interaction of the guidance molecule slit with cellular receptors. Biochem Soc Trans 34:418–421

Huminiecki L, Gorn M, Suchting S et al (2002) Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis. Genomics 79:547–552

Jones CA, London NR, Chen H et al (2008) Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. Nat Med 14:448–453

Kaur S, Samant GV, Pramanik K et al (2008) Silencing of directional migration in roundabout4 knockdown endothelial cells. BMC Cell Biol 9:61

Kidd T, Brose K, Mitchell KJ et al (1998) Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92:205–215 Li HS, Chen JH, Wu W et al (1999) Vertebrate slit, a secreted ligand for the transmembrane protein

roundabout, is a repellent for olfactory bulb axons. Cell 96:807–818

Liu D, Hou J, Hu X et al (2006) Neuronal chemorepellent Slit2 inhibits vascular smooth muscle cell migration by suppressing small GTPase Rac1 activation. Circ Res 98:480–489

Narayan G, Goparaju C, Arias-Pulido H et al (2006) Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression. Mol Cancer 5:16

Park KW, Morrison CM, Sorensen LK et al (2003) Robo4 is a vascular-specific receptor that inhibits endothelial migration. Dev Biol 261:251–267

Rosenfeld PJ, Brown DM, Heier JS et al (2006) Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 355:1419–1431

Rothberg JM, Jacobs JR, Goodman CS et al (1990) Slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev 4:2169–2187

Seth P, Lin Y, Hanai J et al (2005) Magic roundabout, a tumor endothelial marker: expression and signaling. Biochem Biophys Res Commun 332:533–541

Sheldon H, Andre M, Legg JA et al (2009) Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleationpromoting factors. FASEB J 23:513–522

Suchting S, Heal P, Tahtis K et al (2005) Soluble Robo4 receptor inhibits in vivo angiogenesis and endothelial cell migration. FASEB J 19:121–123

Varma R, Macias GL, Torres M et al (2007) Biologic risk factors associated with diabetic retinopathy: the Los Angeles Latino Eye Study. Ophthalmology 114:1332–1340

Wang B, Xiao Y, Ding BB et al (2003) Induction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity. Cancer Cell 4:19–29

Zou HD, Zhang X, Xu X et al (2005) Prevalence study of age-related macular degeneration in Caojiadu blocks, Shanghai. Zhonghua Yan Ke Za Zhi 41:15–19

Part V

Animal Models of Retinal Degeneration

Chapter 53

Evaluation of Retinal Degeneration in P27KIP1

Null Mouse

Yumi Tokita-Ishikawa, Ryosuke Wakusawa, and Toshiaki Abe

Abstract

Purpose: p27kip1 is well-known as a cell cycle inhibitor and also plays an important role for cell differentiation. We hypothesized that if we caused retinal degeneration in a p27(–/–) mouse, then the appropriate method of restoration may be different from that of wild mice and therefore suggest a therapeutic methodology for retinal regeneration.

Methods: Histological and electrophysiological (ERG) examination was performed on p27(–/–) mice retina. We injected N-methy-N-nitrosourea (MNU) to induce retinal degeneration. BrdU was used to identify the dividing cells in the retina.

Results: Thicker retina were observed in the p27(–/–) mice when compared to those of the p27(–/+) mice or wild type mice. Almost all retinal layers were thick and optic nerves were also enlarged. A statistically significant decrease of a and b waves amplitudes of ERG was observed in p27(–/–) mice when compared to those of the other mice. BrdU and nestin positive cells were present at the outer nuclear layer with no difference between p27(–/–) and wild type mice after MNU injection.

Conclusion: p27(–/–) mice showed thicker retina and less retinal function than those of other mice. The MNU-induced retinal degeneration in p27(–/–) mice closely resembled the reaction of the other mice with no retinal regeneration observed in our experimental condition.

T. Abe (B)

Division of Clinical Cell Therapy, School of Medicine, Tohoku University, 1-1 Seiryomachi Aobaku, Miyagi, Sendai 980-8574 Japan

e-mail: to-shi@oph.med.tohoku.ac.jp

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_53,C Springer Science+Business Media, LLC 2010