Choroidal neovascularization in transgenic mice expressing prokineticin 1: an animal model for age-related macular degeneration  Nobushige Tanaka, Masahito Ikawa, Nathan L. Mata, Inder M. Verma  Molecular Therapy  Volume 13, Issue 3, Pages 609-616 (March 2006) DOI: 10.1016/j.ymthe.2005.08.024 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 (A) Diagram of lentiviral vector (LV-Rho-hPK1) used for generation of transgenic mice expressing hPK1 in retina. Rho, bovine rhodopsin gene promoter sequence. The lentiviral vector carries a posttranscriptional regulatory element of the woodchuck hepatitis virus (wPRE) [30], a central polypurine tract (cPPT) [31] of human immunodeficiency virus-1, and self-inactivating deletion mutations (open triangle) [32]. Arrow indicates BamHI site, a unique site in the vector. RSV, promoter sequence from respiratory syncytial virus; ψ, packing signal. (B) Lysates of eyes derived from each line of transgenic mice were subjected to Western blot analysis. hPK1 expression was confirmed in lines 1 and 5. Molecular Therapy 2006 13, 609-616DOI: (10.1016/j.ymthe.2005.08.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Fluorescein angiography. Age-matched wild-type control mice (8 months of age) and transgenic mice (lines 1 and 5) were perfused with FITC-conjugated dextran through the left ventricle. Retinal tissues were stripped from the eyecup and then both (A, B) RPE–choroid–sclera complex and (C, D) retina were flat-mounted. (A) Pictures of the RPE–choroid–sclera complex taken under bright field show intact RPE layer in each eye. (B) Choroidal angiography. Accumulation of fluorescein was observed in the choroidal vessels of transgenic mice (B′, Bʺ), but not in that of control (B). Retinal angiography. (C) Low- and (D) high-magnification pictures are shown. (C) No obvious fluorescein accumulation was observed in the retinal capillary. (D) In the deep plexus layer of the retinal capillary, no evidence of retinal microvascular changes, such as aneurysm and tuft of neovascular vessels, were detected in either control or transgenics. Molecular Therapy 2006 13, 609-616DOI: (10.1016/j.ymthe.2005.08.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Electron microscopy. Retinal blood vessels of a transgenic mouse (8 months of age, line 1) were examined. The retinal vessel in the deep plexus layer had a lumen (*) lined with thick continuous retinal endothelium (En) similar to controls (data not shown). Although the retinal endothelium was thinner at the sites indicated by arrows, no evidence of induced fenestration was observed. Photoreceptor cells had dark nuclei at the outer nuclear layer (ONL) and bipolar cells had relatively light nuclei at the inner nuclear layer (INL). Molecular Therapy 2006 13, 609-616DOI: (10.1016/j.ymthe.2005.08.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Transverse sections of eyes derived from age-matched wild-type control mice (8 months of age) and transgenic mice (lines 1 and 5). The number and the diameter of choroidal vessels (arrows) were increased in transgenic mice compared to controls. Note that the choroidal vessels are uneven in diameter and in vascular wall thickness. OS, outer segment of photoreceptor cell; RPE, retinal pigment epithelium. Molecular Therapy 2006 13, 609-616DOI: (10.1016/j.ymthe.2005.08.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 (A) Cryosections of eyes derived from age-matched wild-type control mice (8 months of age) and transgenic mice (lines 1 and 5). Arrows indicate RPE cell layer. Immunohistochemical analysis for (B) endothelial cell and (C) hPK1. (B) The sections shown in A were stained with FITC-conjugated GSA-I-B4 lectin, which binds to the endothelial cells of nonprimates. Each vessel was FITC-positive (arrowhead), thus ruling out artificial vessels. (C) Each section was reacted with anti-hPK1 antibody and then visualized by RR-conjugated anti-rabbit IgG antibody. hPK1 expression was observed, especially at the interphotoreceptor matrix, in both lines of transgenic mice (C′, Cʺ). Molecular Therapy 2006 13, 609-616DOI: (10.1016/j.ymthe.2005.08.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Analysis of lipofuscin fluorophores (A2E and its precursor A2PE) in the eyes of age-matched wild-type control mice (8 months of age) and transgenic mice (line 1). Representative chromatographic tracings obtained from posterior eyecup, including retina/RPE, phospholipid extracts of (A) control mice and (B) transgenic mice are shown. The arrows indicate the peaks derived from A2E, A2PE, and internal standard. Identification of A2E and A2PE was confirmed by spectral analysis of the eluted peaks (B, inset). Quantitative analyses revealed (C) a 2.4-fold increase in A2E and (D) a 3.4-fold increase in A2PE in transgenic mice relative to control mice. Molecular Therapy 2006 13, 609-616DOI: (10.1016/j.ymthe.2005.08.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions