April 2006
Volume 47, Issue 4
Free
Retinal Cell Biology  |   April 2006
Retinal Pigment Epithelium Damage Enhances Expression of Chemoattractants and Migration of Bone Marrow–Derived Stem Cells
Author Affiliations
  • Yang Li
    From the Department of Ophthalmology and Visual Sciences, the
  • Ryan G. Reca
    Stem Cell Program, Department of Medicine, and the
  • Pelin Atmaca-Sonmez
    From the Department of Ophthalmology and Visual Sciences, the
  • Mariusz Z. Ratajczak
    Stem Cell Program, Department of Medicine, and the
  • Suzanne T. Ildstad
    Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky.
  • Henry J. Kaplan
    From the Department of Ophthalmology and Visual Sciences, the
  • Volker Enzmann
    From the Department of Ophthalmology and Visual Sciences, the
Investigative Ophthalmology & Visual Science April 2006, Vol.47, 1646-1652. doi:10.1167/iovs.05-1092
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      Yang Li, Ryan G. Reca, Pelin Atmaca-Sonmez, Mariusz Z. Ratajczak, Suzanne T. Ildstad, Henry J. Kaplan, Volker Enzmann; Retinal Pigment Epithelium Damage Enhances Expression of Chemoattractants and Migration of Bone Marrow–Derived Stem Cells. Invest. Ophthalmol. Vis. Sci. 2006;47(4):1646-1652. doi: 10.1167/iovs.05-1092.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To characterize chemoattractants expressed by the retinal pigment epithelium (RPE) after sodium iodate (NaIO3)–induced damage and to investigate whether ocular-committed stem cells preexist in the bone marrow (BM) and migrate in response to the chemoattractive signals expressed by the damaged RPE.

methods. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (50 mg/kg) to create RPE damage. At different time points real-time RT-PCR, ELISA, and immunohistochemistry were used to identify chemoattractants secreted in the subretinal space. Conditioned medium from NaIO3-treated mouse RPE was used in an in vitro assay to assess chemotaxis of stem cell antigen-1 positive (Sca-1+) BM mononuclear cells (MNCs). The expression of early ocular markers (MITF, Pax-6, Six-3, Otx) in migrated cells and in MNCs isolated from granulocyte colony–stimulating factor (G-CSF) and Flt3 ligand (FL)–mobilized and nonmobilized peripheral blood (PB) was analyzed by real-time RT-PCR.

results. mRNA for stromal cell–derived factor-1 (SDF-1), C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) was significantly increased, and higher SDF-1 and C3 protein secretion from the RPE was found after NaIO3treatment. A higher number of BMMNCs expressing early ocular markers migrated to conditioned medium from damaged retina. There was also increased expression of early ocular markers in PBMNCs after mobilization.

conclusions. Damaged RPE secretes cytokines that have been shown to serve as chemoattractants for BM-derived stem cells (BMSCs). Retina-committed stem cells appear to reside in the BM and can be mobilized into the PB by G-CSF and FL. These stem cells may have the potential to serve as an endogenous source for tissue regeneration after RPE damage.

In developed countries, age-related macular degeneration (AMD) is the leading cause of permanent vision loss in persons older than 55, and its prevalence increases with age. 1 Approximately 90% of those with macular degeneration have dry AMD, in which atrophy and degeneration of the retinal pigment epithelium (RPE) occurs, presumably through apoptosis associated with the loss of cell attachment. 2 Degeneration of the choriocapillaris and overlying photoreceptors follows. Furthermore, unavoidable removal of the RPE at the time of submacular surgery, one of the potential treatments for wet AMD, occurs at the time of surgery. Attempts to replace the RPE by transplantation of fetal or adult RPE cells have not been successful. 3 4 Therefore, the possibility of targeting bone marrow–derived stem cells (BMSCs) for RPE regeneration is particularly exciting. 
During the past several years, numerous reports have demonstrated that BMSCs have the capacity to regenerate damaged tissues and organs. 5 6 7 8 9 10 However, the mechanisms and factors governing the migration of BMSCs to injured organs are not fully understood. Cytokines and chemokines secreted by the altered tissues are reported to be crucially important in the homing of stem cells to the injured site. 11 Stromal cell–derived factor 1 (SDF-1) is a chemoattractant that plays a central role in the homing process. BMSCs express CXCR4, 12 which is the receptor for SDF-1, on their surfaces. More important, SDF-1 has been shown to be upregulated in damaged tissues and organs, including hypoxic myocardium, liver, and kidney exposed to CCl4. 13 The SDF-1/CXCR4 axis may not be the only pathway involved in stem cell homing. The complement component C3a is found to enhance the response of stem cells to SDF-1 and to the homing process itself. 14  
Two mechanisms may be involved in BMSC-associated tissue regeneration. The concept of stem cell plasticity involves the mobilization and homing of hematopoietic stem cells (HSCs) from the BM to the site of injury and transdifferentiation into cell types of the damaged host organ. This concept has recently been challenged. 15 16 17 An alternative hypothesis has been proposed by Ratajczak et al., 13 18 who have found a small population of CD45 cells in the BM that express the mRNA of various early markers for muscle, neural, and liver cells. This suggests that the BM not only harbors HSCs but that it is also the home for already differentiated tissue-committed stem cells (TCSCs) that can be mobilized into the peripheral blood (PB) and subsequently take part in tissue regeneration. Thus, BMSCs may have the potential to regenerate RPE cells at the site of RPE damage. 
The first aim of our study was to investigate cytokines/chemokines secreted from the damaged RPE that could possibly regulate the homing and migration of stem cells to the altered subretinal space. With the use of a mouse model in which patchy RPE loss was created by intravenous injection of NaIO3, we characterized the changes in the pattern of chemoattractants in the subretinal space after RPE damage. The second aim of our study was to determine whether retina-committed stem cells preexist in the BM and have the potential to migrate in vitro to chemoattractants secreted from damaged RPE. 
Methods
Model of RPE Degeneration
Six- to 8-week-old male C57BL/6 mice, weighing 20 and 25 g each, were purchased from Harlan (Indianapolis, IN). As a negative control for the immunocytochemical study of C3 expression in the eye after NaIO3 injection, C3 knockout mice (C3tm/Crr; Jackson Laboratory, Bar Harbor, ME) were used. The animals were maintained under standard laboratory conditions and were treated according to the regulations in the ARVO Statement for the Use of Animal in Ophthalmic and Vision Research and after approval by the University of Louisville Institutional Animal Care and Use Committee. Mice were briefly restrained (TV-150; Braintree Scientific, Braintree, MA), and a single intravenous injection of sterile 1% solution of sodium iodate in saline (NaIO3; Sigma, St. Louis, MO) was administered into the tail vein of each (50 mg/kg body weight). Saline-injected animals served as controls (0.9% NaCl). 
RPE Preparation
Mice were euthanatized 1, 2, 3, 4, and 7 days after injections of NaIO3 or saline, and eyes were enucleated. Connective tissue was removed, and the eyes were washed twice in PBS and incubated in 2% dispase (Invitrogen, Carlsbad, CA) in DMEM at 37°C for 45 minutes. Then the eyes were washed twice in DMEM plus 10% FBS. After removal of the anterior segment and the neurosensory retina, RPE cells were gently peeled off mechanically with a rounded glass stick. The cells were centrifuged at 1200 rpm for 5 minutes, resuspended, and triturated gently 10 times with DMEM plus 10% FBS with the use of a glass Pasteur pipette. Cells were then centrifuged again at 1200 rpm for 5 minutes, resuspended with lysis buffer, and used in the RT-PCR experiments. 
RPE-Conditioned Medium
For the preparation of RPE-conditioned medium, mice were euthanatized 3, 5, and 7 days after intravenous injection of NaIO3 or saline, and their eyes were enucleated. At these different time points, RPE cells were prepared as described and were plated onto fibronectin-coated (10 μg/mL) six-well cell culture dishes. After culture overnight in DMEM plus 10% FBS to allow the cells to attach, the culture medium was switched to serum-free medium to provide nonproliferating conditions. To ensure a sufficient concentration of cytokine for the in vitro chemotaxis assay, quiescent cells were cultured for an additional 48 hours, and the medium was then collected as RPE-conditioned medium. 
Isolation of Sca-1+ Cells from BM
BM was prepared from 3- to 4-week-old male C57BL/6 mice. Femurs and tibias were dissected and placed in RPMI 1640 culture medium. BM was obtained by slowly flushing medium inside the diaphyseal channel with a syringe through a 25-gauge needle. BM was then homogenized through a 20-gauge needle and filtrated with a nylon filter (70 μM; BD Biosciences, Bedford, MA). Cells were collected, and Sca-1+ cells were isolated with the use of paramagnetic mini-beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol. 
Chemotaxis Assay
The lower chambers of 24-well transwell plates (6.5-mm diameter, 5-μM pore filter; Costar Corning, Cambridge, MA) were filled with 650 μL RPE-conditioned medium. In all, 100 μL aliquots of cell suspension (300,000 cells) were added to the upper chambers. The plates were incubated at 37°C in 5% CO2 for 4 hours. Cells that had transmigrated to the lower chamber were collected and counted by FACS (FACScan; Becton Dickinson). In some of the chemotaxis assays, Sca-1+ cells were preincubated with 1 μM specific CXCR4 antagonist (T-140; Anaspec, San Jose, CA) before they were added to the upper chamber of the transwell. 
Immunocytochemistry
For immunocytochemistry, mice were euthanatized 3 and 7 days after injections of NaIO3 or saline, and the eyes were enucleated and fixed in 4% paraformaldehyde overnight. Whole-eye flat mounts were prepared by removing the anterior segment and the neurosensory retina and making four radial relaxing incisions in the remaining sclera–choroid–RPE complex. Nonspecific binding was blocked with normal serum (Serotec, Raleigh, NC) in PBS/0.1% Triton (Sigma) at room temperature for 60 minutes. Specimens were then washed three times for 5 minutes each in PBS and were incubated overnight at 4°C with Oregon green-conjugated goat anti–mouse C3 primary antibody (1:100; antibody was kindly provided by Daniel J. Allendorf) or rabbit anti–mouse SDF-1 primary antibody (1:100; eBioscience, San Diego, CA) diluted in PBS. After the specimens were washed three more times for 5 minutes each in PBS, immunostaining for SDF-1 was visualized by applying Cy3-conjugated secondary antibody against rabbit IgG (C2181; Sigma) for 1 hour at room temperature. The specimens were washed three final times for 5 minutes each in PBS and were mounted with aqueous mounting medium (Aqua Mount; Lerner, Pittsburgh, PA). RPE tissue without the primary antibody served as negative control. 
ELISA
RPE-conditioned media were harvested at different time points (day 3, day 5, day 7) after NaIO3 or saline injection and were assayed in duplicate for SDF-1 protein with the use of mouse SDF-1 ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. These experiments were repeated five times. Total protein was quantified with the Bradford method, and equal protein amounts were used in the assay. 
Mobilization
Mice were mobilized by subcutaneous injection of 10 μg FL/d from day 1 to day 10 and of 7.5 μg G-CSF/d (generously provided by Amgen, Thousand Oaks, CA) from day 1 to day 6. Growth factors were diluted in saline before injections to a total volume of 100 μL. Control animals were injected with saline only. On day 11, mice were euthanatized, and PB was obtained from the vena cava using a 1-mL heparinized syringe and was enriched for light-density MNCs by Ficoll-Paque centrifugation (Amersham Biosciences, Piscataway, NJ). 
Real-Time RT-PCR
For analysis of SDF-1, C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) mRNA levels in RPE/choroid and for analysis of ocular marker (MITF, Pax-6, Otx, and Six-3) mRNA levels in transmigrated Sca-1+ cells in the chemotaxis assay and in PBMNCs, total mRNA was isolated from the respective sources (RNeasy Mini Kit; Qiagen Inc., Valencia, CA). mRNA was then reverse-transcribed (TaqMan Reverse Transcription Reagents; Applied Biosystems [ABI], Foster City, CA). Detection of SDF-1, C3, HGF, LIF, MITF, Pax-6, Otx, Six-3, and β-microglobulin mRNA levels was performed by real-time RT-PCR (ABI PRISM 7000 Sequence Detection System; ABI). A 25-μL reaction mixture contained 12.5 μL mix (SYBR Green PCR Master Mix), 10 ng cDNA template, 5′-CGT GAG GCC AGG GAA GAG T-3′ forward and 5′-TGA TGA GCA TGG TGG GTT GA-3′ reverse primers for SDF-1, 5′-CCA GCC GGG GAC CTC ACT TGT AG-3′ forward and 5′-GGT CTT CCT GCC GTT TCT CTG TTG-3′ reverse primers for C3, 5′-CCA GAT CCT TGT GGC TCC TAT C-3′ forward and 5′-GGT TAG CGA TTC AGT TCC GTG-3′ reverse primers for HGF, 5′-TTC CCA TCA CCC CTG TAA ATG-3′ forward and 5′-TTG GAG TAC TTG GTC TAG TTC TTA G-3′ reverse primers for LIF, 5′-GGA CTT TCC CTT ATC CCA TTC A-3′ forward and 5′-GTT CTT GCT TGA TGA TCC GAT TC-3′ reverse primers for MITF, 5′-GCA ACC TGG CTA GCG AAA AG-3′ forward and 5′-CCC GTT CAA CAT CCT TAG TTT ATC AT-3′ reverse primers for Pax-6, 5′-CCA ATT TGG GCC GAC TTT G-3′ forward and 5′-GCG TAA GGC GGT TGC TTT AG-3′ reverse primers for Otx, 5′-GAC ACG CCA CAG ACC AAT AGA A-3′ forward and 5′-ATC TCC CCT AAA TCC ACA GTG AGT-3′ reverse primers for Six-3, and 5′-CAT ACG CCT GCA GAG TTA AGC A-3′ forward and 5′-GAT CAC ATG TCT CGA TCC CAG TAG-3′ reverse primers for β-microglobulin. Primers were designed with primer express software. The threshold cycle (Ct), that is, the cycle number at which the amount of amplified gene of interest reached a fixed threshold, was subsequently determined. Relative quantitation of SDF-1, C3, HGF, LIF, MITF, Pax-6, Otx, and Six-3 mRNA expression was calculated with the comparative Ct method. The relative quantitation value of target, normalized to an endogenous control β-microglobulin gene and relative to a calibrator, is expressed as 2−ΔΔCt (fold difference), where ΔCt = Ct of target genes (SDF-1, C3, HGF, LIF, MITF, Pax-6, Otx, Six-3) –Ct of endogenous control gene (β-microglobulin), and ΔΔCt = ΔCt of samples for target gene −ΔCt of calibrator for the target gene. 
Statistical Analysis
Data were presented as mean ± SD or SEM, respectively, and statistical significance was evaluated with an unpaired Student’s t-test. P ≤ 0.05 was considered significant. 
Results
In Vivo Detection of Chemoattractants
RPE damage after NaIO3 injection at 50 mg/kg resulted in patchy RPE loss 19 and enhanced local tissue expression of chemoattractants. The expression of mRNA for all growth factors analyzed (SDF-1, C3, HGF, LIF) was upregulated as early as 24 hours after NaIO3 injection (Fig. 1) . Maximum expression was reached on day 3 with a 3.5-fold increase for SDF-1, a 5-fold increase for C3, a 4.2-fold increase for HGF, and a 4.5-fold increase for LIF (P < 0.05). 
Immunohistochemical analysis for SDF-1 and C3 was performed on flat mounts of the RPE/choroid to assess whether RPE damage after NaIO3 treatment also increased protein levels. Staining for SDF-1 and C3 was evident in areas directly adjacent to sites of RPE damage 3 days after injury, and these cytokines were coexpressed in the same cells (Figs. 2B 2E 2F) . At 7 days, SDF-1 staining was still prominent (Fig. 2C) ; C3 staining was also detectable but at a lower level. Neither SDF-1 nor C3 was seen in normal RPE from control mice (Figs. 2A 2D)or in NaIO3-treated RPE stained without primary antibodies. Negative staining for C3 protein in C3 knockout mice that received NaIO3 demonstrated the specificity of staining for C3. 
We used ELISA to measure SDF-1 protein concentration in conditioned media collected from the RPE of NaIO3-treated and control mice. RPE damage augmented the levels of SDF-1 at all the time points assayed, with a statistically significant increase only on day 3 after NaIO3 injection (Fig. 3)
Detection of Ocular Markers in Mobilized BMSCs
Using real-time RT-PCR to detect the expression of mRNA for early ocular markers on MNCs in the PB (Fig. 4) , we observed a comparable baseline expression of mRNA for these markers in nonmobilized PB in NaCl control and NaIO3-injected mice. The expression of these markers was markedly upregulated after G-CSF– and FL-induced mobilization of MNCs in NaCl control and NaIO3-treated mice. 
In Vitro Chemotactic Analysis
We performed an in vitro chemotaxis assay with conditioned media from damaged RPE to investigate the chemoattractive capability of the upregulated factors on BMSC migration. Sca-1+ MNCs migrated to RPE-conditioned media from NaIO3-treated and control mice. However, Sca-1+ cell migration rates to damaged RPE media from days 3, 5, and 7 after NaIO3 injection were 16%, 43%, and 12% higher, respectively, than to control media. 
Real-time RT-PCR was then used to analyze mRNA expression for early ocular markers—MITF, Otx, Pax-6, and Six-3—in the transmigrated cells (Fig. 5) . RPE-conditioned media from NaIO3-treated mice had statistically significantly higher expression of these markers than control media. 
To demonstrate the role of SDF-1 in the chemoattraction of BMSCs in our experimental in vitro system, cells were preincubated with T140 (a CXCR4 antagonist) before they were used in the chemotactic assay. Figure 6shows that the expression of mRNA for early retinal markers in the transmigrated cells was significantly reduced, suggesting a direct interaction between SDF-1 and the migrating RPE-committed Sca-1+ cells was necessary for chemotaxis. 
Discussion
A number of studies have demonstrated the capacity of BMSCs to engraft and regenerate injured tissues. 5 6 7 8 9 10 RPE degeneration can be created by intravenous injection of NaIO3, which selectively destroys RPE. 20 21 22 This selective effect of NaIO3 on the RPE has been attributed to several mechanisms. First, NaIO3 inhibits various enzymes (eg, triose phosphate dehydrogenase, 23 succinodehydrogenase, lactate dehydrogenase 24 ) in RPE cells; second, it destroys the zonula occludens, the anatomic basis for the blood–retina barrier 25 ; and third, a chemical reaction occurs between sodium iodate and melanin, thereby increasing the ability of melanin to convert glycine to glyoxylate, a potentially cell toxic compound. 26 The effect on the RPE is enhanced by the immediate access of NaIO3 to the pigment epithelial layer through the choriocapillaris, which leads to faster and higher accumulation of the toxin in this tissue. 24 We have reassessed the NaIO3 model of producing RPE damage in the mouse to create a patchy RPE loss similar to that seen in retinal degenerative diseases. 19 Our data show that the extent of RPE degeneration can be varied by using different concentrations of NaIO3. With the use of this model, we provide mechanistic insight into the essential first step of RPE damage-induced migration of BMSCs to the site of injury and reveal a putative role of SDF-1 and other chemokines and cytokines in this process. 
It has been reported that SDF-1 plays an important role in stem cell trafficking between the peripheral circulation and the BM. 27 28 Systemic SDF-1 overexpression induces the mobilization of endothelial cells and HSCs. 29 30 Furthermore, SDF-1 is locally increased within the BM after total body irradiation or chemotherapy, 31 suggesting its role as a signaling molecule to recruit circulating cells to the damaged tissue. The importance of SDF-1 in the homing of stem cells to damaged sites is suggested by the observations that SDF-1 is up-regulated in animal models of liver, limb, and heart damage, and this correlates with adult stem cell recruitment and tissue regeneration. 11 32 33 34 Thus, based on our observations, we hypothesize that the expression of SDF-1 is increased at the site of RPE damage to act as a signaling mediator to guide BMSCs to the injured RPE. 
In agreement with data obtained from other animal models, we found a marked increase in SDF-1 mRNA expression in the altered retina after NaIO3 injection. Moreover, immunohistochemical analysis demonstrated that SDF-1 was abundant in RPE tissues after NaIO3 treatment and was highly expressed around the area of RPE damage. We further found that conditioned media harvested from the RPE of NaIO3-treated mice enhanced Sca-1+ cell migration. Moreover, when these cells were preincubated with T140, an SDF-1 antagonist to CXCR4, less chemoattraction was observed. Together, our data imply that RPE damage by NaIO3 augments the expression and production of SDF-1 and that SDF-1 has the potential to serve as a chemoattractant for BMSCs. 
Because chemotaxis cannot be completely blocked with T140, other stem cell chemoattractants may be involved after RPE damage. In support of this concept, we also found increased expression of mRNA for C3, HGF, and LIF in damaged RPE, and we observed positive immunocytochemical staining for C3 protein with SDF-1 in damaged RPE. Studies have shown that normal HSCs and progenitor cells express a functional complement C3a receptor and that the C3aR–C3a axis plays a role in stem cell recruitment by enhancing the response of stem cells to SDF-1. 14 35 HGF has also been reported to be upregulated in injured liver 11 and ischemic myocardium. 36 In patients with acute liver injury, HGF potentiates the response of immature CD34+ cells to SDF-1 by inducing CXCR4 upregulation, thereby contributing to the recruitment of CD34+ stem cells to the injured liver. 11 Similarly, LIF expression is upregulated in infarcted myocardium 37 and has been reported to induce the regeneration of myocardium after myocardial infarct. 38 Therefore, a local increase in the expression of SDF-1, together with other chemokines and cytokines such as C3, HGF, and LIF, may enable the recruitment of BMSCs to the site of RPE damage. 
Mechanisms underlying BMSC-related tissue repair are not fully understood, and the question remains which type of BMSC has the capability of tissue repair. HSCs are one possibility—they can be mobilized, home to the site of injury, and possibly differentiate—but the concept of transdifferentiation or plasticity of adult stem cells remains controversial. 15 16 17 Our in vitro chemotaxis studies demonstrated that cells expressing early ocular markers were highly enriched within the murine Sca-1+ BMMNC population that migrated in response to conditioned media derived from damaged RPE. Because the study was performed on freshly isolated cells, it excluded the possibility of transdifferentiation of HSCs. Studies by Ratajczak et al. 13 39 provide an alternative explanation based on the observations that the BM contains a small population of CXCR4-positive cells that express mRNA for early muscle, neural, and liver-committed stem/progenitor cells. These TCSCs circulate at a low level in the peripheral circulation and can be mobilized by the hematopoietic growth factor G-CSF. Furthermore, these CXCR4-positive TCSCs migrate in response to SDF-1. 13  
In our studies, we demonstrated early ocular markers (MITF, Pax-6, Otx, Six-3) in BMSCs, implying that retina-committed stem cells may preexist in the BM. G-CSF and FL significantly mobilized this subpopulation of BMSCs into the peripheral circulation. Additionally, we detected a baseline expression of mRNA for early ocular markers in MNCs isolated from murine PB, suggesting that under normal conditions retina-committed stem cells may circulate in PB at low but detectable levels. The expression of mRNA for early ocular markers only slightly increased after NaIO3 treatment, indicating that RPE damage alone is insufficient to release stem cells from the BM into the PB. 
In summary, our in vitro and in vivo data suggest that retina-committed stem cells may reside in the BM and can be mobilized into the PB. Furthermore, these BMSCs have the potential to respond to signals from the damaged RPE. Additional in vivo studies are needed to gain more complete insight into the process of stem cell homing and the potential role of BMSCs to participate in RPE repair. 
 
Figure 1.
 
mRNA expression of chemoattractants in RPE/choroid of mice after intravenous injection of NaIO3 or saline. Data were obtained from three separate experiments and are presented as fold-increase in mRNA expression in NaIO3-treated mice over controls (mean ± SD). RPE/choroid tissue of three mice per group was pooled at each experiment. *P < 0.05.
Figure 1.
 
mRNA expression of chemoattractants in RPE/choroid of mice after intravenous injection of NaIO3 or saline. Data were obtained from three separate experiments and are presented as fold-increase in mRNA expression in NaIO3-treated mice over controls (mean ± SD). RPE/choroid tissue of three mice per group was pooled at each experiment. *P < 0.05.
Figure 2.
 
Immunocytochemical staining for SDF-1 and C3 in whole-eye flat mounts of NaIO3- or saline-treated mice. There was no positive staining for (A) SDF-1 and (D) C3 in normal (control) mice. (B) SDF-1 and (E) C3 were strongly positive in areas around the site of RPE damage 3 days after NaIO3 treatment. (C) SDF-1 was present on day 7 after NaIO3 treatment. (F) Double-labeling for SDF-1 (red fluorescence) and C3 (green fluorescence) on day 3 after NaIO3 treatment was observed as yellow staining of cells.
Figure 2.
 
Immunocytochemical staining for SDF-1 and C3 in whole-eye flat mounts of NaIO3- or saline-treated mice. There was no positive staining for (A) SDF-1 and (D) C3 in normal (control) mice. (B) SDF-1 and (E) C3 were strongly positive in areas around the site of RPE damage 3 days after NaIO3 treatment. (C) SDF-1 was present on day 7 after NaIO3 treatment. (F) Double-labeling for SDF-1 (red fluorescence) and C3 (green fluorescence) on day 3 after NaIO3 treatment was observed as yellow staining of cells.
Figure 3.
 
Concentration of SDF-1 protein detected by ELISA in conditioned media harvested from the RPE of NaIO3 or saline-treated (control) mice. Values are given as mean ± SEM of five independent experiments. The increase in SDF-1 protein concentration was statistically significantly on day 3. *P < 0.05.
Figure 3.
 
Concentration of SDF-1 protein detected by ELISA in conditioned media harvested from the RPE of NaIO3 or saline-treated (control) mice. Values are given as mean ± SEM of five independent experiments. The increase in SDF-1 protein concentration was statistically significantly on day 3. *P < 0.05.
Figure 4.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in MNCs isolated from murine PB. mRNA expression was compared among NaIO3- and NaCl-treated mice with and without mobilization of MNCs by G-CSF and FL. Data represent fold-increase in mRNA expression in mobilized PBMNCs compared with nonmobilized cells. Values are given as mean ± SD of three independent experiments. PBMNCs from five mice per group were pooled at each experiment. *P < 0.05.
Figure 4.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in MNCs isolated from murine PB. mRNA expression was compared among NaIO3- and NaCl-treated mice with and without mobilization of MNCs by G-CSF and FL. Data represent fold-increase in mRNA expression in mobilized PBMNCs compared with nonmobilized cells. Values are given as mean ± SD of three independent experiments. PBMNCs from five mice per group were pooled at each experiment. *P < 0.05.
Figure 5.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in cells migrating to conditioned media harvested from RPE of NaIO3- or NaCl-treated mice. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Values are given as mean of four independent experiments (mean ± SD). *P < 0.05.
Figure 5.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in cells migrating to conditioned media harvested from RPE of NaIO3- or NaCl-treated mice. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Values are given as mean of four independent experiments (mean ± SD). *P < 0.05.
Figure 6.
 
In vitro chemotaxis in the presence of T140 with significant decreases in cell migration (*P < 0.05). The experiment was repeated three times, and the mean ± SD is shown. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Conditioned medium from RPE prepared from six mice per group was used at each time point in the experiments.
Figure 6.
 
In vitro chemotaxis in the presence of T140 with significant decreases in cell migration (*P < 0.05). The experiment was repeated three times, and the mean ± SD is shown. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Conditioned medium from RPE prepared from six mice per group was used at each time point in the experiments.
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Figure 1.
 
mRNA expression of chemoattractants in RPE/choroid of mice after intravenous injection of NaIO3 or saline. Data were obtained from three separate experiments and are presented as fold-increase in mRNA expression in NaIO3-treated mice over controls (mean ± SD). RPE/choroid tissue of three mice per group was pooled at each experiment. *P < 0.05.
Figure 1.
 
mRNA expression of chemoattractants in RPE/choroid of mice after intravenous injection of NaIO3 or saline. Data were obtained from three separate experiments and are presented as fold-increase in mRNA expression in NaIO3-treated mice over controls (mean ± SD). RPE/choroid tissue of three mice per group was pooled at each experiment. *P < 0.05.
Figure 2.
 
Immunocytochemical staining for SDF-1 and C3 in whole-eye flat mounts of NaIO3- or saline-treated mice. There was no positive staining for (A) SDF-1 and (D) C3 in normal (control) mice. (B) SDF-1 and (E) C3 were strongly positive in areas around the site of RPE damage 3 days after NaIO3 treatment. (C) SDF-1 was present on day 7 after NaIO3 treatment. (F) Double-labeling for SDF-1 (red fluorescence) and C3 (green fluorescence) on day 3 after NaIO3 treatment was observed as yellow staining of cells.
Figure 2.
 
Immunocytochemical staining for SDF-1 and C3 in whole-eye flat mounts of NaIO3- or saline-treated mice. There was no positive staining for (A) SDF-1 and (D) C3 in normal (control) mice. (B) SDF-1 and (E) C3 were strongly positive in areas around the site of RPE damage 3 days after NaIO3 treatment. (C) SDF-1 was present on day 7 after NaIO3 treatment. (F) Double-labeling for SDF-1 (red fluorescence) and C3 (green fluorescence) on day 3 after NaIO3 treatment was observed as yellow staining of cells.
Figure 3.
 
Concentration of SDF-1 protein detected by ELISA in conditioned media harvested from the RPE of NaIO3 or saline-treated (control) mice. Values are given as mean ± SEM of five independent experiments. The increase in SDF-1 protein concentration was statistically significantly on day 3. *P < 0.05.
Figure 3.
 
Concentration of SDF-1 protein detected by ELISA in conditioned media harvested from the RPE of NaIO3 or saline-treated (control) mice. Values are given as mean ± SEM of five independent experiments. The increase in SDF-1 protein concentration was statistically significantly on day 3. *P < 0.05.
Figure 4.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in MNCs isolated from murine PB. mRNA expression was compared among NaIO3- and NaCl-treated mice with and without mobilization of MNCs by G-CSF and FL. Data represent fold-increase in mRNA expression in mobilized PBMNCs compared with nonmobilized cells. Values are given as mean ± SD of three independent experiments. PBMNCs from five mice per group were pooled at each experiment. *P < 0.05.
Figure 4.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in MNCs isolated from murine PB. mRNA expression was compared among NaIO3- and NaCl-treated mice with and without mobilization of MNCs by G-CSF and FL. Data represent fold-increase in mRNA expression in mobilized PBMNCs compared with nonmobilized cells. Values are given as mean ± SD of three independent experiments. PBMNCs from five mice per group were pooled at each experiment. *P < 0.05.
Figure 5.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in cells migrating to conditioned media harvested from RPE of NaIO3- or NaCl-treated mice. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Values are given as mean of four independent experiments (mean ± SD). *P < 0.05.
Figure 5.
 
Real-time RT-PCR analysis of mRNA expression for early ocular markers (MITF, Pax-6, Otx, Six-3) in cells migrating to conditioned media harvested from RPE of NaIO3- or NaCl-treated mice. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Values are given as mean of four independent experiments (mean ± SD). *P < 0.05.
Figure 6.
 
In vitro chemotaxis in the presence of T140 with significant decreases in cell migration (*P < 0.05). The experiment was repeated three times, and the mean ± SD is shown. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Conditioned medium from RPE prepared from six mice per group was used at each time point in the experiments.
Figure 6.
 
In vitro chemotaxis in the presence of T140 with significant decreases in cell migration (*P < 0.05). The experiment was repeated three times, and the mean ± SD is shown. Data represent fold-increase in the mRNA expression for early ocular markers in migrated cells in the lower chamber compared with the same number of cells from the input (60,000 Sca-1+ cells). Conditioned medium from RPE prepared from six mice per group was used at each time point in the experiments.
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