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Novel Protective Properties of IGFBP-3 Result in Enhanced Pericyte Ensheathment, Reduced Microglial Activation, Increased Microglial Apoptosis, and Neuronal Protection after Ischemic Retinal Injury

2011; Elsevier BV; Volume: 178; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2010.12.031

1525-2191

Jennifer L. Kielczewski, Ping Hu, Lynn C. Shaw, Sergio Li Calzi, Robert N. Mames, Tom A. Gardiner, Eleanore A. McFarland, Tailoi Chan‐Ling, Maria B. Grant,

Glaucoma and retinal disorders

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3–mediated response was examined using green fluorescent protein–positive (GFP+) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3–expressing plasmid resulted in increased differentiation of GFP+ hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP+ HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity. This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3–mediated response was examined using green fluorescent protein–positive (GFP+) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3–expressing plasmid resulted in increased differentiation of GFP+ hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP+ HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity. Diabetic retinopathy is the leading cause of adult blindness, and retinopathy of prematurity is a leading cause of pediatric blindness.1Chen J. Smith L.E. Retinopathy of prematurity.Angiogenesis. 2007; 10: 133-140Crossref PubMed Scopus (480) Google Scholar, 2Mohamed Q. Gillies M.C. Wong T.Y. Management of diabetic retinopathy: a systematic review.JAMA. 2007; 298: 902-916Crossref PubMed Scopus (669) Google Scholar, 3Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders.N Engl J Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3391) Google Scholar, 4Robinson G.S. Pierce E.A. Rook S.L. Foley E. Webb R. Smith L.E. Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.Proc Natl Acad Sci U S A. 1996; 93: 4851-4856Crossref PubMed Scopus (350) Google Scholar Insulin-like growth factor-1 (IGF-1) contributes to the pathogenesis of both conditions because unregulated levels of IGF-1 can lead to pathologic ocular neovascularization.5Rosenthal R. Wohlleben H. Malek G. Schlichting L. Thieme H. Bowes Rickman C. Strauss O. Insulin-like growth factor-1 contributes to neovascularization in age-related macular degeneration.Biochem Biophys Res Commun. 2004; 323: 1203-1208Crossref PubMed Scopus (54) Google Scholar, 6Grant M.B. Mames R.N. Fitzgerald C. Ellis E.A. Aboufriekha M. Guy J. Insulin-like growth factor I acts as an angiogenic agent in rabbit cornea and retina: comparative studies with basic fibroblast growth factor.Diabetologia. 1993; 36: 282-291Crossref PubMed Scopus (180) Google Scholar, 7Grant M.B. Mames R.N. Fitzgerald C. Ellis E.A. Caballero S. Chegini N. Guy J. Insulin-like growth factor I as an angiogenic agent: in vivo and in vitro studies.Ann N Y Acad Sci. 1993; 692: 230-242Crossref PubMed Scopus (65) Google Scholar, 8Grant M.B. Afzal A. Spoerri P. Pan H. Shaw L.C. Mames R.N. The role of growth factors in the pathogenesis of diabetic retinopathy.Expert Opin Investig Drugs. 2004; 13: 1275-1293Crossref PubMed Scopus (157) Google Scholar The effects of IGF-1 are mediated by the IGF-1 receptor and are modulated by complex interactions with six different IGF binding proteins (IGFBPs), which function as transporter proteins and as storage pools for IGF-1 in tissues. Of the six different IGFBPs, IGFBP-3 is the most abundant, carrying more than 75% of serum IGF-1 and IGF-2 in heterotrimeric complexes.9Yamada P.M. Lee K.W. Perspectives in mammalian IGFBP-3 biology: local vs. systemic action.Am J Physiol Cell Physiol. 2009; 296: C954-C976Crossref PubMed Scopus (132) Google Scholar, 10Firth S.M. Baxter R.C. Cellular actions of the insulin-like growth factor binding proteins.Endocr Rev. 2002; 23: 824-854Crossref PubMed Scopus (1441) Google Scholar The IGFBP-3 has autocrine and paracrine actions that affect cell mobility and survival, and it is regulated by hypoxia.10Firth S.M. Baxter R.C. Cellular actions of the insulin-like growth factor binding proteins.Endocr Rev. 2002; 23: 824-854Crossref PubMed Scopus (1441) Google Scholar Depending on the cellular milieu and local levels, IGFBP-3 can be either proangiogenic or antiangiogenic.11Granata R. Trovato L. Lupia E. Sala G. Settanni F. Camussi G. Ghidoni R. Ghigo E. Insulin-like growth factor binding protein-3 induces angiogenesis through IGF-I- and SphK1-dependent mechanisms.J Thromb Haemost. 2007; 5: 835-845Crossref PubMed Scopus (86) Google Scholar Abnormally low levels of IGFBP-3 occur in the serum of low-birth-weight infants, and normalization of these levels is being considered as a strategy to prevent the development of retinopathy of prematurity.12Lofqvist C. Chen J. Connor K.M. Smith A.C. Aderman C.M. Liu N. Pintar J.E. Ludwig T. Hellstrom A. Smith L.E. IGFBP3 suppresses retinopathy through suppression of oxygen-induced vessel loss and promotion of vascular regrowth.Proc Natl Acad Sci U S A. 2007; 104: 10589-10594Crossref PubMed Scopus (148) Google Scholar Previously, we showed that IGFBP-3 facilitates remodeling of the retinal vasculature after ischemic injury in the oxygen-induced retinopathy (OIR) model.13Chang K.H. Chan-Ling T. McFarland E.L. Afzal A. Pan H. Baxter L.C. Shaw L.C. Caballero S. Sengupta N. Li Calzi S. Sullivan S.M. Grant M.B. IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development.Proc Natl Acad Sci U S A. 2007; 104: 10595-10600Crossref PubMed Scopus (112) Google Scholar, 14Kielczewski J.L. Jarajapu Y.P. McFarland E.L. Cai J. Afzal A. Li Calzi S. Chang K.H. Lydic T. Shaw L.C. Busik J. Hughes J. Cardounel A.J. Wilson K. Lyons T.J. Boulton M.E. Mames R.N. Chan-Ling T. Grant M.B. Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.Circ Res. 2009; 105: 897-905Crossref PubMed Scopus (75) Google Scholar Under conditions of hypoxia, IGFBP-3 expression by ischemic tissue can stimulate hematopoietic stem cell (HSC) migration, thus promoting the recruitment of these cells from the circulation into areas of ischemia, where they contribute to endothelial repair.13Chang K.H. Chan-Ling T. McFarland E.L. Afzal A. Pan H. Baxter L.C. Shaw L.C. Caballero S. Sengupta N. Li Calzi S. Sullivan S.M. Grant M.B. IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development.Proc Natl Acad Sci U S A. 2007; 104: 10595-10600Crossref PubMed Scopus (112) Google Scholar, 15Annabi B. Lee Y.T. Turcotte S. Naud E. Desrosiers R.R. Champagne M. Eliopoulos N. Galipeau J. Beliveau R. Hypoxia promotes murine bone-marrow–derived stromal cell migration and tube formation.Stem Cells. 2003; 21: 337-347Crossref PubMed Scopus (250) Google Scholar Lofqvist et al12Lofqvist C. Chen J. Connor K.M. Smith A.C. Aderman C.M. Liu N. Pintar J.E. Ludwig T. Hellstrom A. Smith L.E. IGFBP3 suppresses retinopathy through suppression of oxygen-induced vessel loss and promotion of vascular regrowth.Proc Natl Acad Sci U S A. 2007; 104: 10589-10594Crossref PubMed Scopus (148) Google Scholar showed that IGFBP-3–deficient mice have increased retinal vessel loss when subjected to the OIR model compared with wild-type mice. We demonstrated that IGFBP-3 increases recruitment of green fluorescent protein–positive (GFP+) bone marrow–derived HSCs into the injured retina, and we found that the recruitment and migratory effects of IGFBP-3 in the endothelium are nitric oxide mediated.14Kielczewski J.L. Jarajapu Y.P. McFarland E.L. Cai J. Afzal A. Li Calzi S. Chang K.H. Lydic T. Shaw L.C. Busik J. Hughes J. Cardounel A.J. Wilson K. Lyons T.J. Boulton M.E. Mames R.N. Chan-Ling T. Grant M.B. Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.Circ Res. 2009; 105: 897-905Crossref PubMed Scopus (75) Google Scholar In addition, we found that IGFBP-3 decreases apoptosis of endothelial cells in ischemic retinal vessels. Although IGFBP-3 demonstrates beneficial effects in restoring healthy endothelium after injury,12Lofqvist C. Chen J. Connor K.M. Smith A.C. Aderman C.M. Liu N. Pintar J.E. Ludwig T. Hellstrom A. Smith L.E. IGFBP3 suppresses retinopathy through suppression of oxygen-induced vessel loss and promotion of vascular regrowth.Proc Natl Acad Sci U S A. 2007; 104: 10589-10594Crossref PubMed Scopus (148) Google Scholar, 13Chang K.H. Chan-Ling T. McFarland E.L. Afzal A. Pan H. Baxter L.C. Shaw L.C. Caballero S. Sengupta N. Li Calzi S. Sullivan S.M. Grant M.B. IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development.Proc Natl Acad Sci U S A. 2007; 104: 10595-10600Crossref PubMed Scopus (112) Google Scholar, 14Kielczewski J.L. Jarajapu Y.P. McFarland E.L. Cai J. Afzal A. Li Calzi S. Chang K.H. Lydic T. Shaw L.C. Busik J. Hughes J. Cardounel A.J. Wilson K. Lyons T.J. Boulton M.E. Mames R.N. Chan-Ling T. Grant M.B. Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.Circ Res. 2009; 105: 897-905Crossref PubMed Scopus (75) Google Scholar a paucity of information exists regarding the effect of IGFBP-3 on supporting cells of the vasculature (specifically, pericytes and microglia) and neurons. To address this issue, we undertook the present study to assess the cytoprotective effects of IGFBP-3 and found that IGFBP-3 demonstrates neuroprotective, antiapoptotic, and anti-inflammatory effects after retinal injury. All the animals were treated in accordance with the Guiding Principles in the Care and Use of Animals (NIH) and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6J.gfp transgenic mice homozygous for GFP+ were obtained from The Jackson Laboratory (Bar Harbor, ME) and were housed and bred in the institutional animal care facilities at the University of Florida. Eighteen adult chimeric mice were used for immunohistochemical studies. Pregnant C57BL/6 mice were purchased from The Jackson Laboratory. We used 32 mouse pups for transmission electron microscopy experiments, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analysis. The GFP+ chimeric mice were generated as previously reported.16Grant M.B. May W.S. Caballero S. Brown G.A. Guthrie S.M. Mames R.N. Byrne B.J. Vaught T. Spoerri P.E. Peck A.B. Scott E.W. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization.Nat Med. 2002; 8: 607-612Crossref PubMed Scopus (615) Google Scholar Briefly, to generate the C57BL/6J.gfp chimeric mice, femoral and tibial bone marrow were harvested from GFP+ transgenic (homozygous) male mice. Antibodies to c-kit (CD117) and Sca-1 (both from BD Pharmingen, San Diego, CA) were used to isolate a select population of undifferentiated bone marrow cells. These cells were then separated using flow cytometry (FACSCalibur flow cytometer; BD Biosciences, San Jose, CA) to obtain a population of cells that was GFP+/c-kit+/Sca-1+. The purity of the GFP+/c-kit+/Sca-1+ cell population obtained from fluorescence-activated cell sorting typically resulted in a 95% hemangioblast population. These cells were then injected into the retro-orbital sinuses of lethally irradiated (9.5 Gy from a Cs137 source) 6- to 8-week-old adult female mice. After 3 months, GFP+ cell engraftment was confirmed by flow cytometric analysis of mouse peripheral blood as previously described.17Sengupta N. Caballero S. Mames R.N. Butler J.M. Scott E.W. Grant M.B. The role of adult bone marrow–derived stem cells in choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 4908-4913Crossref PubMed Scopus (141) Google Scholar, 18Sengupta N. Caballero S. Mames R.N. Timmers A.M. Saban D. Grant M.B. Preventing stem cell incorporation into choroidal neovascularization by targeting homing and attachment factors.Invest Ophthalmol Vis Sci. 2005; 46: 343-348Crossref PubMed Scopus (64) Google Scholar Mice with a GFP+ engraftment greater than 80% were used for experimentation. Three groups of animals were used in the experiments, all consisting of C57BL/6J.gfp chimeric mice: laser only (n = 6), laser with IGFBP-3 injection (n = 6), and IGFBP-3 injection only (n = 6). Mice were anesthetized with ketamine and xylazine before laser treatment. Animals were photocoagulated by delivering 35 spots at 150 W for 1.0 second with an argon green laser to the retinal vessels of the right eye at a distance of 1 disk diameter from the optic nerve head. This laser injury model has been used previously.14Kielczewski J.L. Jarajapu Y.P. McFarland E.L. Cai J. Afzal A. Li Calzi S. Chang K.H. Lydic T. Shaw L.C. Busik J. Hughes J. Cardounel A.J. Wilson K. Lyons T.J. Boulton M.E. Mames R.N. Chan-Ling T. Grant M.B. Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.Circ Res. 2009; 105: 897-905Crossref PubMed Scopus (75) Google Scholar, 16Grant M.B. May W.S. Caballero S. Brown G.A. Guthrie S.M. Mames R.N. Byrne B.J. Vaught T. Spoerri P.E. Peck A.B. Scott E.W. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization.Nat Med. 2002; 8: 607-612Crossref PubMed Scopus (615) Google Scholar Retinal vessels were occluded in an attempt to cause ischemia: laser applications were applied repeatedly to the same point on the vessel, as needed, to whiten the area of treatment and damage the vasculature. A total of 2 μL of IGFBP-3 plasmid (2 μg/μL), under the control of a proliferating endothelial cell–specific promoter (ETe) composed of a 7 × 46-mer multimerized endothelin enhancer upstream of a human Cdc6 promoter,19Shaw L.C. Pan H. Afzal A. Calzi S.L. Spoerri P.E. Sullivan S.M. Grant M.B. Proliferating endothelial cell-specific expression of IGF-I receptor ribozyme inhibits retinal neovascularization.Gene Ther. 2006; 13: 752-760Crossref PubMed Scopus (27) Google Scholar was placed into liposomes and was injected intravitreally into the right eye of the 12 adult female mice, as noted previously herein. All three groups of mice were sacrificed 3 weeks after laser and/or IGFBP-3 treatment. We used the mouse model of OIR developed by Smith et al.20Smith L.E. Wesolowski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar Mice were injected intravitreally with a plasmid expressing mouse IGFBP-3 (2 μg/μL) under the control of the proliferating endothelial cell–specific promoter or an “empty” cloning vector at postnatal day (P) 1 in a volume of 0.5 μL per eye. By using a proliferating endothelial cell–specific promoter, IGFBP-3 expression was targeted to areas of neovascularization as previously described.19Shaw L.C. Pan H. Afzal A. Calzi S.L. Spoerri P.E. Sullivan S.M. Grant M.B. Proliferating endothelial cell-specific expression of IGF-I receptor ribozyme inhibits retinal neovascularization.Gene Ther. 2006; 13: 752-760Crossref PubMed Scopus (27) Google Scholar At P7, the pups were placed into high oxygen (75%) for 5 days and then were subsequently returned to room air at P12. The animals were euthanized immediately on removal from hyperoxia at P12 or 5 days after returning to room air at P17. Eyes from mice injected with plasmid expressing IGFBP-3 (n = 16) were compared with those from mice injected with empty cloning vector (n = 16) and/or the uninjected eye of the same animal. The IGFBP-3–injected eyes (n = 6) and the contralateral uninjected control eyes (n = 6) of mouse pups at P12 and P17 were enucleated and immersion fixed in 2.5% glutaraldehyde in 0.1 M cacodylate at pH 7.2 with 10 mmol/L MgCl2 (Electron Microscopy Sciences, Hatfield, PA) for 1 hour at room temperature. Whole eye globes were then washed with PBS. Tissue blocks were treated with 1% osmium tetroxide, dehydrated in ethanol, and embedded in Spurr's resin as previously reported.21Gardiner T.A. Stitt A.W. Anderson H.R. Archer D.B. Selective loss of vascular smooth muscle cells in the retinal microcirculation of diabetic dogs.Br J Ophthalmol. 1994; 78: 54-60Crossref PubMed Scopus (58) Google Scholar Mice were euthanized by anesthetizing them with isoflurane followed by cervical dislocation. Eyes were enucleated and fixed with 4% paraformaldehyde/PBS for 1 hour. The neural retina was dissected from the posterior cup and was washed with PBS. Neural retinas were stained as previously reported for detection of NG2 (Chemicon International, Temecula, CA) for pericytes and S-100 (Sigma-Aldrich, St. Louis, MO) for detection of astrocytes and their somas to covisualize GFP+ cells in the vasculature of flat-mounted retinas.22Hughes S. Chan-Ling T. Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo.Invest Ophthalmol Vis Sci. 2004; 45: 2795-2806Crossref PubMed Scopus (157) Google Scholar, 23Chan-Ling T. Chu Y. Baxter L. Weible Ii M. Hughes S. In vivo characterization of astrocyte precursor cells (APCs) and astrocytes in developing rat retinae: differentiation, proliferation, and apoptosis.Glia. 2009; 57: 39-53Crossref PubMed Scopus (27) Google Scholar Retinas were imaged via confocal microscopy using a Leica argon krypton laser mounted on a Leica DMRBE epifluorescence photomicroscope using Leica imaging software (Leica Microsystems GmbH, Wetzlar, Germany). Fluorescence microscopy was also performed using OpenLab 4.0 imaging software (Weltham, MA). Images were processed using Adobe Photoshop 5.0 software (Adobe Systems Inc., San Jose, CA). Astrocytes [S-100 or glial fibrillary acidic protein (GFAP)],22Hughes S. Chan-Ling T. Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo.Invest Ophthalmol Vis Sci. 2004; 45: 2795-2806Crossref PubMed Scopus (157) Google Scholar pericytes (NG2),24Ozerdem U. Grako K.A. Dahlin-Huppe K. Monosov E. Stallcup W.B. NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis.Dev Dyn. 2001; 222: 218-227Crossref PubMed Scopus (473) Google Scholar and endothelial cells and microglia (both stained for GS isolectin B4)25Medana I.M. Hunt N.H. Chan-Ling T. Early activation of microglia in the pathogenesis of fatal murine cerebral malaria.Glia. 1997; 19: 91-103Crossref PubMed Scopus (92) Google Scholar, 26Chan-Ling T. Halasz P. Stone J. Development of retinal vasculature in the cat: processes and mechanisms.Current Eye Res. 1990; 9: 459-478Crossref PubMed Scopus (105) Google Scholar, 27Htain W.W. Leong S.K. Ling E.A. A comparative Mac-1 immunocytochemical and lectin histochemical study of microglial cells in the normal and athymic mice.Glia. 1994; 12: 44-51Crossref PubMed Scopus (30) Google Scholar, 28Maddox D.E. Shibata S. Goldstein I.J. Stimulated macrophages express a new glycoprotein receptor reactive with Griffonia simplicifolia I-B4 isolectin.Proc Natl Acad Sci U S A. 1982; 79: 166-170Crossref PubMed Scopus (117) Google Scholar were visualized using triple-marker immunohistochemical analysis on retinal whole mounts and cryosections in the midperipheral and peripheral regions of the retina. Retinas of OIR animals were examined using a confocal microscope (Zeiss LSM 510 META; Carl Zeiss MicroImaging GmbH, Jena, Germany). Images were processed using Adobe Photoshop CS software (Adobe Systems Inc.). Retinas were examined after exposure to a high-oxygen environment (P12) and after 5 days' return to room air (P17). Pericyte, astrocyte, microglial, and neuronal cell death were assessed using TUNEL (F. Hoffman–La Roche, Basel, Switzerland) on retinal whole mounts and transverse sections. Oxygen-induced retinopathy animals were stained with an in situ cell death detection system based on the TUNEL method29Hughes S. Chan-Ling T. Roles of endothelial cell migration and apoptosis in vascular remodelling during development of the central nervous system.Microcirculation. 2000; 7: 317-333Crossref PubMed Scopus (67) Google Scholar to label nuclei undergoing DNA fragmentation, followed by GS isolectin B4 for vascular endothelial cells and activated microglia (Sigma-Aldrich), NG2 for pericytes (Chemicon International), OX42 for macrophages (Serotec, Oxford, United Kingdom), and NeuN or βIII tubulin for neurons (Chemicon International). Representative fields of view (area = 0.0625 mm2) from the midperipheral and peripheral retinas were counted using a ×40 objective as the field of view for analysis. In each field of view, TUNEL+/GS lectin+ vascular endothelial cells, TUNEL+/NG2+ pericytes, TUNEL+/S-100+ astrocytes, and TUNEL+/NeuN+ neurons were counted, as previously described in Chan-Ling T et al.30Chan-Ling T. Baxter L. Weible II, M.W. Hughes S. In vivo characterization of astrocyte precursor cells (APCs) and astrocytes in developing rat retinae; differentiation, proliferation and apoptosis.Glia. 2009; 57: 39-53Crossref PubMed Scopus (55) Google Scholar Data are reported as mean ± SE, with n = 4 per experimental group. The frequency of NG2 pericyte ensheathment of retinal vessels was determined using a modified method previously described for determining the desmin ensheathment ratio.31Chan-Ling T. Page M.P. Gardiner T. Baxter L. Rosinova E. Hughes S. Desmin ensheathment ratio as an indicator of vessel stability: evidence in normal development and in retinopathy of prematurity.Am J Pathol. 2004; 165: 1301-1313Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Four representative fields of view from the midperipheral retina were counted using a ×40 objective as the field of view for analysis. Each confocal image was overlaid with a 23 × 23 equally spaced grid using Adobe Photoshop CS. The grid was superimposed onto each image. The occurrence of NG2 labeling relative to lectin labeling at the 529 intersection points yielded the percentage of pericyte ensheathment. After visualization of the vessels and activated microglia with GS isolectin B4, four fields of views were captured using a ×40 objective, and the number of microglia was determined in each field of view. The average number of cells counted per field of view (area = 0.0625 mm2) was multiplied by 16 to obtain the number of cells per square millimeter. T-tests were performed. Differences were considered to be statistically significant at P < 0.05. The contribution of bone marrow–derived cells to vascular repair has been well appreciated regarding their ability to repair endothelium. We previously showed that IGFBP-3 can promote differentiation of HSCs into endothelial cells after laser-induced mouse retinal injury.14Kielczewski J.L. Jarajapu Y.P. McFarland E.L. Cai J. Afzal A. Li Calzi S. Chang K.H. Lydic T. Shaw L.C. Busik J. Hughes J. Cardounel A.J. Wilson K. Lyons T.J. Boulton M.E. Mames R.N. Chan-Ling T. Grant M.B. Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.Circ Res. 2009; 105: 897-905Crossref PubMed Scopus (75) Google Scholar Less well understood is the ability of these cells to become perivascular cells, such as pericytes and astrocytes; however, their plasticity is generally believed to remain context and injury determined.32Ritter M.R. Banin E. Moreno S.K. Aguilar E. Dorrell M.I. Friedlander M. Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy.J Clin Invest. 2006; 116: 3266-3276Crossref PubMed Scopus (212) Google Scholar, 33Otani A. Kinder K. Ewalt K. Otero F.J. Schimmel P. Friedlander M. Bone marrow–derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis.Nat Med. 2002; 8: 1004-1010Crossref PubMed Scopus (290) Google Scholar, 34Friedlander M. Dorrell M.I. Ritter M.R. Marchetti V. Moreno S.K. El-Kalay M. Bird A.C. Banin E. Aguilar E. Progenitor cells and retinal angiogenesis.Angiogenesis. 2007; 10: 89-101Crossref PubMed Scopus (52) Google Scholar, 35Barber C.L. Iruela-Arispe M.L. The ever-elusive endothelial progenitor cell: identities, functions and clinical implications.Pediatr Res. 2006; 59: 26R-32RCrossref PubMed Scopus (66) Google Scholar, 36Chan-Ling T. Baxter L. Afzal A. Sengupta N. Caballero S. Rosinova E. Grant M.B. Hematopoietic stem cells provide repair functions after laser-induced Bruch’s membrane rupture model of choroidal neovascularisation.Am J Pathol. 2006; 168: 1031-1044Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar To address this issue, we generated GFP+ chimeric mice and laser injured their retinal vessels. Immediately after laser injury, IGFBP-3–expressing plasmid was injected into the vitreous. Three weeks after injury, retinas from mice injected with the IGFBP-3 plasmid demonstrated increased incorporation of GFP+ cells into the retinal vasculature in the absence or presence of laser injury (Figure 1, Figure 2, D–I). The GFP+ cells differentiated into pericytes (Figure 1) and astrocytes (Figure 2) as determined by immunohistochemical analysis. Although not quantified, IGFBP-3–injected, laser-treated retinas showed a striking increase in GFP+ cell differentiation into pericytes (NG2) and astrocytes (S-100) compared with the laser-only–treated retinas (Figure 1, Figure 2, D–F versus A–C). Previously, we showed that IGFBP-3 increases astrocytic ensheathment in the OIR model,14Kielczewski J.L. Jarajapu Y.P. McFarland E.L. Cai J. Afzal A. Li Calzi S. Chang K.H. Lydic T. Shaw L.C. Busik J. Hughes J. Cardounel A.J. Wilson K. Lyons T.J. Boulton M.E. Mames R.N. Chan-Ling T. Grant M.B. Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.Circ Res. 2009; 105: 897-905Crossref PubMed Scopus (75) Google Scholar and this is consistent with the present study, which shows IGFBP-3–mediated HSC differentiation into astrocytes, which, in turn, would promote astrocytic ensheathment.Figure 2Overexpression of IGFBP-3 by vascular endothelial cells results in GFP+ HSC differentiation into astrocytes in an adult ocular laser model. A–I: The green stain represents GFP+ cells, and the red stain represents S-100 for the detection of astrocytes. A–C: Laser-only retinas showing S-100 immunoreactivity. GFP+ HSC differentiated into S-100+ astrocytes (white arrows) in very low numbers in the laser-only eyes. D–F: Overexpression of IGFBP-3 + laser retinal injury showing markedly increased S-100 immunoreactivity. The GFP+ HSCs gave rise to S-100+ astrocytes (white arrows) that were more evident in laser + IGFBP-3–overexpressing eyes compared with laser-only eyes. G–I: The IGFBP-3–overexpressing eyes showing GFP+ cell differentiation into S-100+ astrocytes (white arrows). The scale bar in C is applied throughout A–I.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because we observed that IGFBP-3 injection can increase bone marrow–derived progenitor cell differentiation into pericytes in the OIR model, we asked whether IGFBP-3 could affect pericyte morphology and apoptosis in newly forming vessels. The developing mouse vasculature is in a state of constant cell turnover. Hence, it is an ideal model for studying apoptosis and cellular remodeling. Compared with contralateral uninjected eyes, IGFBP-3–injected eyes showed pericytes with significantly greater NG2 immunoreactivity and NG2 ensheathment in the superficial and outer plexus (Figure 3, A, B, E, and F versus C, D, G, and H). Quantification of the frequ