Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

Purpose: We investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)-derived cells to the neurosensory retina (NSR) and RPE layer. Methods: GFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 µm) with 30 applications were placed 50 to 100 µm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed. Results: Within 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle-dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid. Conclusions: SRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle-dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.

Paracrine modulation of CXCR4 by IGF-1 and VEGF: implications for choroidal neovascularization.

PURPOSE: Modulators of angiogenesis typically work in an orchestrated manner. The authors examined the interaction between insulinlike growth factor (IGF)-1, vascular endothelial growth factor (VEGF), and stromal derived factor (SDF)-1 in vivo and in vitro using angiogenesis models. METHODS: The angiogenic effect of SDF-1, alone or in combination with IGF-1 and VEGF, was assessed in human lung microvascular endothelial cells using capillary tube formation and thymidine incorporation. Immunohistochemical analysis for CD31, SDF-1, and CXCR4 was performed on mouse eyes 2 weeks after the initiation of laser rupture of Bruch's membrane, a choroidal neovascularization (CNV) model. CXCR4 antagonist and CXCR4 blocking antibody were tested on inhibition of CNV lesion size in this model. Real-time PCR was used to determine mRNA levels for SDF-1, VEGF, IGF-1, and their cognate receptors in the retinal pigment epithelium/choroid complex of mice that underwent this CNV model. RESULTS: IGF-1 and VEGF demonstrated an additive effect on SDF-1-induced in vitro angiogenesis. CXCR4 immunoreactivity was present in both normal and laser-injured mice at the laser burn site and at the ganglion cell layer, the anterior portion of the inner nuclear layer, photoreceptors, and choroidal stroma. SDF-1 was observed in identical locations but was not seen in photoreceptors. mRNA levels for SDF-1, VEGF, and IGF-1 and their receptors were increased after laser injury. CXCR4-neutralizing antibody reduced neovascularization when injected subretinally but not intraperitoneally or intravitreally. CONCLUSIONS: The potent proangiogenic factors IGF-1 and VEGF both stimulate SDF-1-induced angiogenesis. Local inhibition of CXCR4 is required for an antiangiogenic effect in CNV lesions.

Regulation of adult hematopoietic stem cells fate for enhanced tissue-specific repair.

The ability to control the differentiation of adult hematopoietic stem cells (HSCs) would promote development of new cell-based therapies to treat multiple degenerative diseases. Systemic injection of NaIO(3) was used to ablate the retinal pigment epithelial (RPE) layer in C57Bl6 mice and initiate neural retinal degeneration. HSCs infected ex vivo with lentiviral vector expressing the RPE-specific gene RPE65 restored a functional RPE layer, with typical RPE phenotype including coexpression of another RPE-specific marker, CRALBP, and photoreceptor outer segment phagocytosis. Retinal degeneration was prevented and visual function, as measured by electroretinography (ERG), was restored to levels similar to that found in normal animals. None of the controls (no HSCs, HSCs alone and HSCs infected with lentiviral vector expressing LacZ) showed these effects. In vitro gene array studies demonstrated that infection of HSC with RPE65 increased adenylate cyclase mRNA. In vitro exposure of HSCs to a pharmacological agonist of adenylate cyclase also led to in vitro differentiation of HSCs to RPE-like cells expressing pigment granules and the RPE-specific marker, CRALBP. Our data confirm that expression of the cell-specific gene RPE65 promoted fate determination of HSCs toward RPE for targeted tissue repair, and did so in part by activation of adenylate cyclase signaling pathways. Expression by HSCs of single genes unique to a differentiated cell may represent a novel experimental paradigm to influence HSC plasticity, force selective differentiation, and ultimately lead to identification of pharmacological alternatives to viral gene delivery.

IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development.

We asked whether the hypoxia-regulated factor, insulin-like growth factor binding protein-3 (IGFBP3), could modulate stem cell factor receptor (c-kit+), stem cell antigen-1 (sca-1+), hematopoietic stem cell (HSC), or CD34+ endothelial precursor cell (EPC) function. Exposure of CD34+ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygen-induced retinopathy (OIR) model. In separate studies, GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration, protection of the developing vasculature from hyperoxia-induced regression, and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion, IGFBP3 mediates EPC migration, differentiation, and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions.

Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells.

Endothelial precursor cells (EPCs) play a key role in vascular repair and maintenance, and their function is impeded in diabetes. We previously demonstrated that EPCs isolated from diabetic patients have a profound inability to migrate in vitro. We asked whether EPCs from normal individuals are better able to repopulate degenerate (acellular) retinal capillaries in chronic (diabetes) and acute (ischemia/reperfusion [I/R] injury and neonatal oxygen-induced retinopathy [OIR]) animal models of ocular vascular damage. Streptozotocin-induced diabetic mice, spontaneously diabetic BBZDR/Wor rats, adult mice with I/R injury, or neonatal mice with OIR were injected within the vitreous or the systemic circulation with fluorescently labeled CD34(+) cells from either diabetic patients or age- and sex-matched healthy control subjects. At specific times after administering the cells, the degree of vascular repair of the acellular capillaries was evaluated immunohistologically and quantitated. In all four models, healthy human (hu)CD34(+) cells attached and assimilated into vasculature, whereas cells from diabetic donors uniformly were unable to integrate into damaged vasculature. These studies demonstrate that healthy huCD34(+) cells can effectively repair injured retina and that there is defective repair of vasculature in patients with diabetes. Defective EPCs may be amenable to pharmacological manipulation and restoration of the cells' natural robust reparative function.

Hematopoietic stem cells provide repair functions after laser-induced Bruch's membrane rupture model of choroidal neovascularization.

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 approximately 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.

Nitric oxide synthases modulate progenitor and resident endothelial cell behavior in galactosemia.

We used knockout animals of either inducible nitric oxide synthase (iNOS(/)) or endothelial NOS (eNOS(/)) to characterize the role of NOS in galactosemia, a model of diabetic retinopathy. NADH oxidase and nitrotyrosine were used as biomarkers of oxidative stress and vascular dysfunction. These animals were engrafted with hematopoietic stem cells (HSC) expressing green fluorescence protein (gfp(+)) to characterize the contribution of HSC and endothelial progenitor cells to neovascularization. Increased NADH oxidase activity and superoxide generation occurred in all galactose-fed mice. eNOS(/) mice demonstrated increased iNOS immunoreactivity in their retinal vasculature. Nitrotyrosine levels were low at baseline in the wild-type (WT) mice, eNOS(/) and iNOS(/) mice, and the galactose-fed iNOS mice and increased following galactose feeding in eNOS(/) and WT. Galactose-fed WT.gfp and iNOS(/).gfp chimeric animals had areas of perfused new vessels composed of gfp(+) cells. In contrast, galactose-fed eNOS(/).gfp mice produced copious, unbranched, nonperfused tubes. Thus, nitric oxide modulates HSC behavior and vascular phenotype in the retina. Although there is increased NADH oxidase and superoxide in galactosemic mice of all isoforms, iNOS is the source of nitric oxide responsible for peroxynitrite and nitrotyrosine formation that leads to the pathology observed in galactosemic mice.

Preventing stem cell incorporation into choroidal neovascularization by targeting homing and attachment factors.

PURPOSE: The primary cause of vision loss in people more than 50 years of age in developed nations is age-related macular degeneration (ARMD). The wet form of ARMD is characterized by choroidal neovascularization (CNV). A prior study has shown that adult hematopoietic stem cells (HSCs) contribute to approximately 50% of newly formed vasculature in CNV. Stromal-derived factor (SDF)-1 is involved with homing of HSCs from bone marrow to target tissue. Vascular endothelial cadherin (VE-cadherin, or CD144) is involved in endothelial cell adhesion. Preventing homing and/or adhesion of progenitor cells to damaged choroid could reduce CNV. METHODS: Adult C57BL/6J mice were lethally irradiated, and then received a transplant of purified c-kit+Sca-1+ HSCs from the bone marrow of green fluorescent protein (gfp) homozygous donor mice. Bruch's membrane rupture by laser photocoagulation was used to induce CNV. Animals were injected subretinally with anti-SDF-1, anti-CD144, or control, before or after laser photocoagulation. The eyes were enucleated, and the neural retinas were separated from the RPE/choroid/sclera complex. All tissues were flatmounted and qualitatively and quantitatively assessed by fluorescence microscopy. RESULTS: CNV lesions from eyes treated with anti-CD144 showed significantly less incorporation of gfp+ cells compared with those treated with anti-SDF-1. Antibody treatment generally reduced the degree of gfp+ stem cell recruitment and incorporation into the CNV lesions, compared with the control. Treatment with either antibody also significantly reduced the size of the CNV lesions. CONCLUSIONS: These results indicate that homing and adhesion of progenitor cells to CNV may be targeted differentially or in combination to prevent CNV.

The role of adult bone marrow-derived stem cells in choroidal neovascularization.

PURPOSE: Age-related macular degeneration (ARMD) is the primary cause of blindness in people aged of 50 years or more. The wet form leads to severe loss of central vision. Recent evidence supports that adult hematopoietic stem cells (HSCs) contribute to preretinal neovascularization. In the current study, it was determined whether HSCs, by producing both blood and blood vessels, provide functional hemangioblast activity during choroidal neovascularization (CNV) in mice. METHODS: Gfp chimeric mice were developed by bone marrow ablation of C57BL/6J mice and reconstitution with donor tissue from gfp(+/+) transgenic mice. Gfp chimeric mice underwent laser rupture of Bruch's membrane and were killed and eyes enucleated at 1, 2, 3, and 4 weeks after laser injury. CNV was examined by confocal microscopy of retinal flatmounts. Because endothelial progenitor cells (EPCs) derive from HSCs, immunocytochemistry was used to quantify relative the EPC contribution to CNV. RESULTS: Laser injury alone was sufficient to induce stem cell recruitment and subsequent CNV. Gfp+ cells formed part of the functional vasculature in the choroid as early as 1 week after injury and were present for the duration of the study. The relative EPC contribution to CNV remained fairly constant throughout the study and constituted almost 50% of the total vasculature. CONCLUSIONS: Adult stem cells are recruited to the choroid in a model of CNV, where they contribute to forming aberrant new vessels. This observation suggests that targeting stem cell recruitment to the eye may offer a novel therapeutic strategy for ARMD.