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Hematopoietic Stem Cells Provide Repair Functions after Laser-Induced Bruch's Membrane Rupture Model of Choroidal Neovascularization

2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2006.050697

1525-2191

Tailoi Chan‐Ling, Louise Baxter, Aqeela Afzal, Nilanjana Sengupta, Sergio Caballero, Emilia Rosinova, Maria B. Grant,

Angiogenesis and VEGF in Cancer

Vascular repair by adult hematopoietic stem cells (HSCs) is well-appreciated because these cells are known for their plasticity. We have shown that adult HSCs differentiate into endothelial cells and participate in both retinal and choroidal neovascularization. We asked whether HSCs participated in the wounding response by forming astrocytes, retinal pigment epithelia (RPE), macrophages, and pericytes. Lethally irradiated C57BL6/J mice were reconstituted with HSCs from mice homozygous for green fluorescent protein (GFP) and then subjected to laser-induced rupture of Bruch's membrane. After immunohistochemical examination of ocular tissue, GFP+ astrocytes were observed concentrated along the edge of the laser wound, where they and mural cells closely ensheathed the neovasculature. GFP+ vascular endothelial cells and macrophages/microglia were also evident. Large irregularly shaped GFP+ RPE cells constituted ∼93% of RPE cells adjacent to the edge of the denuded RPE area. In regions farther away from the wound, GFP+ RPE cells were integrated among the GFP− host RPE. Thus, postnatal HSCs can differentiate into cells expressing markers specific to astrocytes, macrophages/microglia, mural cells, or RPE. These studies suggest that HSCs could serve as a therapeutic source for long-term regeneration of injured retina and choroid in diseases such as age-related macular degeneration and retinitis pigmentosa. Vascular repair by adult hematopoietic stem cells (HSCs) is well-appreciated because these cells are known for their plasticity. We have shown that adult HSCs differentiate into endothelial cells and participate in both retinal and choroidal neovascularization. We asked whether HSCs participated in the wounding response by forming astrocytes, retinal pigment epithelia (RPE), macrophages, and pericytes. Lethally irradiated C57BL6/J mice were reconstituted with HSCs from mice homozygous for green fluorescent protein (GFP) and then subjected to laser-induced rupture of Bruch's membrane. After immunohistochemical examination of ocular tissue, GFP+ astrocytes were observed concentrated along the edge of the laser wound, where they and mural cells closely ensheathed the neovasculature. GFP+ vascular endothelial cells and macrophages/microglia were also evident. Large irregularly shaped GFP+ RPE cells constituted ∼93% of RPE cells adjacent to the edge of the denuded RPE area. In regions farther away from the wound, GFP+ RPE cells were integrated among the GFP− host RPE. Thus, postnatal HSCs can differentiate into cells expressing markers specific to astrocytes, macrophages/microglia, mural cells, or RPE. These studies suggest that HSCs could serve as a therapeutic source for long-term regeneration of injured retina and choroid in diseases such as age-related macular degeneration and retinitis pigmentosa. The hematopoietic stem cell (HSC) is the most robust, highly characterized stem cell of the body. From the initial studies showing that irradiated mice could be rescued from death with bone marrow transplants from a healthy donor, it became clear that HSCs could self-renew and produce all of the different cells in the blood.1Till JE McCulloch EA A direct measurement of the radiation sensitivity of normal mouse bone marrow cells.Radiat Res. 1961; 14: 213-222Crossref PubMed Scopus (3263) Google Scholar, 2Spangrude GJ Heimfeld S Weissman IL Purification and characterization of mouse hematopoietic stem cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2249) Google Scholar Approximately 1 of every 10,000 cells in the bone marrow is a stem cell and is defined as being Sca-1+, c-kit+, and lineage-negative (SKL), with ∼1 of 10 SKL cells thought to be a true HSC that can traffic to the bone marrow and perform HSC functions.3Jackson KA Majka SM Wulf GG Goodell MA Stem cells: a minireview.J Cell Biochem Suppl. 2002; 38: 1-6Crossref PubMed Google Scholar One main HSC function involves new blood vessel formation. Angiogenesis represents proliferation and migration of pre-existing fully differentiated endothelial cells that reside within parent vessels.4Folkman J Merler E Abernathy C Williams G Isolation of a tumor factor responsible for angiogenesis.J Exp Med. 1971; 133: 275-288Crossref PubMed Scopus (1368) Google Scholar The finding that bone marrow-derived cells may traffic to sites of neovascularization and differentiate is consistent with vasculogenesis,5Risau W Lemmon V Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis.Dev Biol. 1988; 125: 441-450Crossref PubMed Scopus (333) Google Scholar a critical paradigm for the establishment of vascular networks in the embryo. Asahara and colleagues6Asahara T Murohara T Sullivan A Silver M van der Zee R Li T Isolation of putative progenitor endothelial cells for angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7821) Google Scholar identified the cells derived from the bone marrow capable of differentiating into neovasculature. Injection of HSCs into mice accelerates revascularization of ischemic limbs7Schatteman GC Hanlon HD Jiao C Dodds SG Christy BA Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice.J Clin Invest. 2000; 106: 571-578Crossref PubMed Scopus (434) Google Scholar and wound healing8Sivan-Loukianova E Awad OA Stepanovic V Bickenbach J Schatteman GC CD34+ blood cells accelerate vascularization and healing of diabetic mouse skin wounds.J Vasc Res. 2003; 40: 368-377Crossref PubMed Scopus (153) Google Scholar; thus, these cells are presumed to be key mediators of endothelial repair. Previously, we demonstrated that HSCs differentiate into endothelial cells and participate with resident endothelial cells to form neovascularization in the retina, choroid, and iris.9Grant MB May WS Caballero S Brown GA Guthrie SM Mames RN Byrne BJ Vaught T Spoerri PE Peck AB Scott EW Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization.Nat Med. 2002; 8: 607-612Crossref PubMed Scopus (625) Google Scholar, 10Sengupta N Caballero S Mames RN Butler JM Scott EW Grant MB 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, 11Caballero S, Sengupta N, Mames RN, Scott EW, Grant MB: Hematopoietic stem cell contribution to neovascularization of the iris. Presented at: the Annual Meeting of the Association for Research in Vision and Ophthalmology; April 25–30, 2004; Ft. Lauderdale, FL.Google Scholar Stem cells are self-renewable, pluripotent cells that in adult life proliferate by a characteristic asymmetric division in which one daughter cell is committed to differentiation whereas the other remains a stem cell. These cells are also characterized by their ability to differentiate into various cell types under heterotypic environmental influences. Plasticity refers to the ability of stem cells to acquire a mature phenotype that differs from the tissue of origin.12Grove JE Bruscia E Krause DS Plasticity of bone marrow-derived stem cells.Stem Cells. 2004; 22: 487-500Crossref PubMed Scopus (381) Google Scholar This is true for both embryonic and adult stem cells. Adult bone marrow-derived stem cells, either HSCs or mesenchymal stem cells, are multipotent and have been shown to engraft and express properties of other tissues. HSCs have been shown to transdifferentiate into cell types such as epithelium,13Borue X Lee S Grove J Herzog EL Harris R Diflo T Glusac E Hyman K Theise ND Krause DS Bone marrow-derived cells contribute to epithelial engraftment during wound healing.Am J Pathol. 2004; 165: 1767-1772Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar myocardium,14Balsam LB Wagers AJ Christensen JL Kofidis T Weissman IL Robbins RC Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature. 2004; 428: 668-673Crossref PubMed Scopus (1561) Google Scholar, 15Orlic D Adult bone marrow stem cells regenerate myocardium in ischemic heart disease.Ann NY Acad Sci. 2003; 996: 152-157Crossref PubMed Scopus (110) Google Scholar and liver.16Petersen BE Bowen WC Patrene KD Mars WM Sullivan AK Murase N Boggs SS Greenberger JS Goff JP Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2207) Google Scholar Several studies support the potential of neural stem cells to differentiate into ocular cells such as retinal pigment epithelial (RPE) cells when exposed to RPE-conditioned medium.17Enzmann V Howard RM Yamauchi Y Whittemore SR Kaplan HJ Enhanced induction of RPE lineage markers in pluripotent neural stem cells engrafted into the adult rat subretinal space.Invest Ophthalmol Vis Sci. 2003; 44: 5417-5422Crossref PubMed Scopus (42) Google Scholar These cells were able to attenuate the loss of photoreceptors when transplanted subretinally into Royal College of Surgeons (RCS) rats. This preservation was believed to be mediated either by the cell's ability to phagocytose the host's outer segments or by its ability to secrete soluble factors.18Faktorovich EG Steinberg RH Yasumura D Matthes MT LaVail MM Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor.Nature. 1990; 347: 83-86Crossref PubMed Scopus (647) Google Scholar To date, however, HSCs have not been shown to differentiate into RPE cells in vitro or in vivo. Recent studies have suggested that circulating bone marrow cells can traffic to brain and transdifferentiate into neural cells. Bonilla and co-workers19Bonilla S Silva A Valdes L Geijo E Garcia-Verdugo JM Martinez S Functional neural stem cells derived from adult bone marrow.Neuroscience. 2005; 133: 85-95Crossref PubMed Scopus (64) Google Scholar have explored the potential of adult hematopoietic bone marrow cells to differentiate into cells of oligodendroglial lineage under physiological active-myelinating conditions. They demonstrated that cells from a bone marrow subpopulation enriched in adult hematopoietic progenitor cells (c-kit+) express oligodendroglial-specific markers in vivo after intracerebral transplantation into the neonatal mouse brain. Their results suggest that adult bone marrow cells have the capacity to undergo differentiation from hematopoietic to oligodendroglial cells and support the validity of bone marrow transplantation as an alternative treatment for demyelinating diseases of the central nervous system, including multiple sclerosis. Bone marrow cells traffic to sites of injury because they are attracted by locally produced growth factors and/or cytokines, including stromal-derived growth factor-1 (SDF-1).20Aiuti A Webb IJ Bleul C Springer T Gutierrez-Ramos JC The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood.J Exp Med. 1997; 185: 111-120Crossref PubMed Scopus (1211) Google Scholar, 21Ma Q Jones D Borghesani PR Segal RA Nagasawa T Kishimoto T Bronson RT Springer TA Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice.Proc Natl Acad Sci USA. 1998; 95: 9448-9453Crossref PubMed Scopus (1436) Google Scholar, 22Nagasawa T Hirota S Tachibana K Takakura N Nishikawa S Kitamura Y Yoshida N Kikutani H Kishimoto T Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1.Nature. 1996; 382: 635-638Crossref PubMed Scopus (2029) Google Scholar, 23Tachibana K Hirota S Iizasa H Yoshida H Kawabata K Kataoka Y Kitamura Y Matsushima K Yoshida N Nishikawa S Kishimoto T Nagasawa T The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract.Nature. 1998; 393: 591-594Crossref PubMed Scopus (1326) Google Scholar RPE cells express SDF-1 during healthy conditions and after injury.24Crane IJ Wallace CA McKillop-Smith S Forrester JV CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1α.J Immunol. 2000; 165: 4372-4378PubMed Google Scholar If an SDF-1 gradient is established, cells traffic more efficiently. Conversely, if this gradient is blocked, homing efficiency is reduced to negligible levels.25Butler JM Guthrie SM Koc M Afzal A Caballero S Brooks HL Mames RN Segal MS Grant MB Scott EW SDF-1 is both necessary and sufficient to promote proliferative retinopathy.J Clin Invest. 2005; 115: 86-93Crossref PubMed Scopus (242) Google Scholar Studies illustrate that HSCs and progenitor cells are sensitive to SDF-1, supporting the view that this chemokine is important for stem and progenitor cell migration in both embryonic and adult tissues.26Hattori K Heissig B Rafii S The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1.Leuk Lymphoma. 2003; 44: 575-582Crossref PubMed Scopus (109) Google Scholar Although vascular repair by adult HSCs is well appreciated, these cells are also known for their plasticity and can generate liver, brain, muscle, and fat.12Grove JE Bruscia E Krause DS Plasticity of bone marrow-derived stem cells.Stem Cells. 2004; 22: 487-500Crossref PubMed Scopus (381) Google Scholar, 16Petersen BE Bowen WC Patrene KD Mars WM Sullivan AK Murase N Boggs SS Greenberger JS Goff JP Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2207) Google Scholar, 27Brazelton TR Rossi FM Keshet GI Blau HM From marrow to brain: expression of neuronal phenotypes in adult mice.Science. 2000; 290: 1775-1779Crossref PubMed Scopus (1604) Google Scholar, 28Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Mosca JD Moorman MA Simonetti DW Craig S Marshak DR Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (18305) Google Scholar, 29Kawada H Fujita J Kinjo K Matsuzaki Y Tsuma M Miyatake H Muguruma Y Tsuboi K Itabashi Y Ikeda Y Ogawa S Okano H Hotta T Ando K Fukuda K Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction.Blood. 2004; 104: 3581-3587Crossref PubMed Scopus (537) Google Scholar We therefore asked whether circulating HSCs have the potential not only to form endothelial cells and participate in choroidal neovascularization (CNV) but also to become other cell types that participate in the development of the CNV lesion after rupture of Bruch's membrane. A CNV lesion, whether occurring clinically or after mechanical injury, typically contains numerous cell types that participate in a type of wound healing reaction in the subretinal space. We postulated that the injured subretinal space would provide cues to mobilize HSCs to this region and promote their formation into cell types other than endothelial cells, such as RPE, pericytes, smooth muscle cells, astrocytes, and macrophages. All procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research and were approved by the University of Sydney Animal Ethics Committee. Reconstitution of lethally irradiated C57BL6/J mice with HSCs from GFP+/+ donors was performed as previously described by us.9Grant MB May WS Caballero S Brown GA Guthrie SM Mames RN Byrne BJ Vaught T Spoerri PE Peck AB Scott EW Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization.Nat Med. 2002; 8: 607-612Crossref PubMed Scopus (625) Google Scholar, 10Sengupta N Caballero S Mames RN Butler JM Scott EW Grant MB 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 Induction of CNV lesions by laser rupture of Bruch's membrane was done as previously described.10Sengupta N Caballero S Mames RN Butler JM Scott EW Grant MB 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 Retinas were fixed by immersion in 4% paraformaldehyde in 0.1 mol/L phosphate buffer for 1 hour at 4°C. Retinal whole-mounts were prepared as described previously.30Chan-Ling T Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina.Microsc Res Tech. 1997; 36: 1-16Crossref PubMed Scopus (98) Google Scholar Triple-marker immunohistochemistry was used to co-visualize astrocytes (rabbit polyclonal antibody S-100β from Chemicon, Temecula, CA), mural cells (rabbit polyclonal antibody NG2 from Chemicon), GFP+ cells (anti-GFP from Chemicon), vasculature (GS isolectin B4, Sigma, St. Louis, MO), and RPE (anti-RPE65, gift from J.-X. Ma [Department of Medicine, Oklahoma University, Oklahoma City, OK]; cytokeratin 8/18, clone SD3 from Novocastra, Newcastle-upon-Tyne, UK). A maximum of three primary antibodies were applied to each specimen to determine the progeny of transplanted GFP+ cells in either retinal whole mounts or RPE/choroidal eyecups in treated and untreated tissue. A minimum of three specimens were analyzed per described phenomenon, and conclusions reached were representative of all specimens examined. Retinal whole mounts were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for anti-GFP and 1% Triton X-100 in PBS for S-100Bβ, GFAP, and GS isolectin, and samples were then blocked in 1% bovine serum albumin in PBS. Retinas were incubated overnight at 4°C with primary antibodies, washed with 0.1% Triton X-100 in PBS, incubated for 4 hours at room temperature with the appropriate secondary antibodies, and washed again. For double or triple labeling, this procedure was repeated with the different primary antibodies and appropriate secondary antibodies. Negative controls omitting a primary antibody were performed for each antibody and protocol. Vessels were labeled with biotinylated GS isolectin followed by streptavidin conjugated with Cy5 (Jackson Laboratories, Bar Harbor, ME). All antibodies and streptavidin were diluted with 1% bovine serum albumin in PBS, and washes were performed with 0.1% Triton X-100 in PBS. Retinal whole-mounts were finally mounted with the ganglion cell layer up in glycerol: PBS (2:1, v/v) or Prolong Anti-Fade (Molecular Probes, Eugene, OR). Retinal whole-mounts were examined by both deconvolution and confocal microscopy. For deconvolution fluorescence microscopy and photography, we used a Zeiss microscope (model Axioplan 2 attachment HBO 100) and Axiocam HRm camera. Confocal microscopy was performed with a Leica argon-krypton laser mounted on a Leica DMRBE epifluorescence photomicroscope. Alexa Fluor 488, Cy3, and Cy5 fluorescence was excited sequentially at 488, 550, and 649 nm, respectively. Images were processed with Adobe Photoshop V5.0 software. In regions at or immediately adjacent to the wound, GFP+ HSCs gave rise to S-100β+ astrocytes with predominantly stellate morphology (Figure 1, A, B, D, and E) as typically observed in adult mouse retina. Brighter GFP+/S-100β+ astrocytes with a simple bipolar morphology were observed interspersed among the GFP+ stellate astrocytes (Figure 1, A and B; Figure 2, A–F, M–O). GFP+/S-100β+ astrocytes (Figure 1, D–F) were observed in patches concentrated along the edge of the laser wound (Figure 1D, arrowheads) and were not evident in the control eye. This concentration along the wound edge is suggestive of the ability of HSCs to traffic to sites of neovascularization and exert some rescue effects. The morphology of these GFP+/S-100β+ astrocytes at the edge of a wound was typically simple bipolar (Figure 1, C and F; Figure 2, A–F). Remarkably, astrocyte precursor cells also have a simple bipolar morphology during normal development,31Chu Y Hughes S Chan-Ling T Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma.FASEB J. 2001; 15: 2013-2015PubMed Google Scholar suggestive of a more immature phenotype along the astrocytic differentiation pathway.Figure 2Higher magnification views of HSCs that differentiated into astrocytes in a rodent model of CNV. Although HSC-derived astrocytes ensheathed numerous new blood vessels, astrocytic ensheathment of these new vessels was often incomplete, as shown throughout this panel. A, D, G, J, and M show GFP fluorescence. B, E, H, K, and N show S-100β staining. C, F, I, L, and O show triple-labeling with multiple exposures showing GFP/S-100β and GS isolectin. A–C show a preponderance of bipolar astrocytes aligned along the wound edge. Because bipolar astrocytes are indicative of a more immature astrocyte phenotype, this observation is supportive of HSCs giving rise to immature astrocytes after laser-induced CNV. D–F, G–I, J–L, and M–O show other examples of neovascularization after laser-induced Bruch's membrane rupture. The morphology of the vessels is not typically observed in normal mouse retina. This retinal neovasculature is closely ensheathed by GFP+/S-100β+ astrocytes, supportive of the role played by HSC-derived astrocytes in the wound healing process and induction of blood-retinal barrier properties. Representative GFP+/S-100+ astrocytes are shown by open arrowheads in C, F, I, L, and O. J: Small numbers of GFP+, but not S-100−, cells were evident. Because S-100 is only expressed by mature astrocytes, the GFP+ astrocyte-like cells on the left side of this field of view could represent astrocyte precursor cells before S-100 expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Figure 1F shows an area of retinal neovascularization because retinal blood vessels are not normally found in these layers or with this morphology (Figure 1F, arrows). These GFP+ astrocytes closely ensheathed some of the vasculature in the superficial vascular plexus (Figure 1F; Figure 2, G–L), mimicking the close ensheathment of retinal blood vessels by astrocytes seen normally. On examination at higher magnification, some neovascular segments lacked a continuous astrocytic ensheathment as would be found in a normal adult mouse retina (Figure 2, D–F and J–L). Remarkably, the neovasculature in the deeper layers lacked any S-100β+ astrocytic ensheathment in this field of view (Figure 1F). A quantitative analysis of the number of GFP+/S-100β+ astrocytes within 300 μm of the lesion site showed a mean of 21.0 cells/mm2 (SD = 13.1) determined over eight fields of view using a ×16 objective. The numbers of GFP+/S-100β+ astrocytes showed significant variation with the field of view examined, where the actual counts in the eight fields of view were 18, 18, 10, 25, 51, 18, 10, and 18 cells/mm2. The average number of GFP+/S-100β+ astrocytes farther than 300 μm away from lesions was 1.3 (SD = 2.9, in eight fields of view at ×16). The actual counts in the eight fields of view examined were 8, 0, 0, 0, 3, 0, 0, and 0 cells/mm2. Although the majority of HSC-derived (GFP+/S-100β+) astrocytes appeared to concentrate at the wound margin, a lower number of HSC-derived astrocytes can incorporate into the existing syncytium of resident astrocytes even at distances greater than 300 μm from the edge of the wound. As a negative control, we reacted retinas from control B57/BL6/J mice with an antibody against GFP. No GFP+ cells were evident throughout the entire retina. Examination of the nontreated eyes in the chimeric mouse using an antibody to GFP revealed small numbers of GFP+ vascular endothelial cells (VECs). Activated microglia/macrophages in the retina are characterized by an amoeboid morphology, enhanced expression of Griffonia simplicifolia (GS) isolectin B4, and a noncontact spacing pattern of distribution. Numerous GFP+/GS isolectin B4+ macrophages/microglia were evident in the experimental retina (Figure 3). These cells were regularly distributed and had an amoeboid morphology (Figure 3G). Significant populations of GFP+/GS isolectin B4+ macrophages/microglia were evident over the entire experimental retina, with a slightly higher frequency of distribution in the periphery of the retina (Figure 3). These cells were distributed in a noncontact spacing pattern typical of macrophages/microglia. Such HSC-derived macrophages/microglia were often associated with retinal blood vessels (Figure 3, J–L). A resident population of GFP−/GS isolectin B4+ macrophages/microglia persisted in the experimental retinas (Figure 3I, arrowheads). HSCs differentiated into RPE after laser-induced Bruch's membrane rupture. Closure of the wound was incomplete at time of sacrifice 3 weeks after wounding (Figure 4A). In the area adjacent to the denuded area, RPE cells were present with significantly higher levels of GFP expression than surrounding areas (Figure 4, B–D). A small proportion of these cells had enlarged cell surface area and less regularly distributed pigment granules, resulting in irregularity to the typical hexagonal mosaic pattern. In contrast, in regions that were a substantial distance away from the wound (Figure 4E) or in the periphery (Figure 4F), fewer GFP+ RPE cells were present. These GFP+ RPE cells were smaller and more regular in appearance and formed a heterogeneous hexagonal array. HSC-derived GFP+ RPE cells integrated among the host RPE cells, demonstrating patches of GFP+ and patches of GFP− RPE cells adjacent to each other. These observations suggest that HSCs can differentiate into RPE and participate in wound healing in regions of laser-induced RPE damage. As controls, posterior cups from C57BL6/J mice were subjected to GFP/GS isolectin double-label immunohistochemistry. Figure 4G shows the low level of GFP fluorescence and the regularity of the RPE mosaic in these eyes. Further, RPE cells in control C57BL6/J mice autofluoresce. RPE autofluorescence in control animals has been reported and is due to the presence of secondary inclusion bodies, such as lipofuscin, which have broad-band autofluorescence.32Wassell J Ellis S Burke J Boulton M Fluorescence properties of autofluorescent granules generated by cultured human RPE cells.Invest Ophthalmol Vis Sci. 1998; 39: 1487-1492PubMed Google Scholar In addition, the presence of other fluorophores such as retinoids, flavins, and cytochrome contribute to RPE autofluorescence. Although this level is much lower than that observed in the GFP+ RPE cells (Figure 4, H–J), the level of autofluorescence could be detected and captured photographically. Further, confirming earlier reports, double-nucleated RPE cells were also occasionally observed in control C57BL6/J mice (Figure 4, I and J). Double-nucleated RPE cells could be interpreted as cell fusion or phagocytosis of adjacent cells as opposed to HSC-generating RPE cells. Two RPE-specific markers, RPE-65 and cytokeratin 8/18, were also applied to the laser-induced CNV retinas to provide further support for the conclusion that GFP+ HSCs generate RPE. All RPE cells were positive for RPE-65 and cytokeratin 8/18. Figure 5, A–C, shows GFP+/RPE-65+ HSC-derived RPE cells in the region of the wound whereas Figure 5, D–F, shows GFP+/RPE-65+ HSC-derived RPE cells at the edge of the eyecup. It is unclear why the majority of GFP+ showed a marked increase in the expression of RPE-65. Figure 5, G–I, shows GFP+/cytokeratin 8/18+ HSC-derived RPE cells in the region of the wound. Cytokeratin bundles outline the periphery of all cuboidal RPE cells. The nuclei and larger melanin granules impart a finely vacuolated appearance to the less intensely immunopositive central cytoplasm. We undertook a quantitative analysis of the number of GFP+ RPE cells in two regions: within 400 μm of the lesion site and in a region just internal to the edge of the eyecup. Four fields of view were counted per region using the ×20 objective on the Zeiss deconvolution microscope, resulting in ∼1400 cells being counted per region. A representative field of view adjacent to the lesion is shown in Figure 4A. The average number of GFP+ RPE cells adjacent to the lesions was 1939 ± 488 cells/mm2. The average number of GFP− RPE cells adjacent to the lesions was 173 ± 116 cells/mm2. This equates to a mean of 92.5 ± 3.4% of RPE cells adjacent to the lesion being derived from GFP+ HSCs. The average number of GFP+ RPE cells at the edge of the eyecup was 1010 ± 189 cells/mm2. The average number of GFP− RPE cells at the edge of the eyecup was 1801 ± 372 cells/mm2. This equates to a mean of 36.2 ± 6.8% of RPE cells at the edge of the eyecup being derived from GFP+ HSCs. No GFP+ RPE cells were evident in naïve C57BL6/J mice. Although HSC-derived GFP+ RPE cells concentrated at the wound margin, a significant number (36% of all RPE cells) also incorporated into the existing network of resident RPE cells at the periphery of the eyecup. This finding is consistent with our current understanding that the RPE is the site where the energy from the laser is absorbed and dissipated. Thus, it would be expected that the size of RPE wounds exceeds the actual laser spot size and suggests that laser injury stimulates HSC recruitment well beyond the region of Bruch's membrane rupture. A small number of GFP+/GS isolectin B4+ vascular endothelial strands were evident in the superficial vascular plexus in the experimental retina (Figure 3, A and C), suggesting the possibility of laser-stimulated recruitment into these regions. GFP+ VECs were observed in regions where HSC-derived astrocytes (Figure 3B) and macrophages (Figure 3, A and C) were also evident. Remarkably, GFP+ VECs were more frequently observed in the outer retinal vascular plexus (data not shown), as one