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.

Age-related macular degeneration: beyond anti-angiogenesis.

Recently, anti-vascular endothelial growth factor therapies for neovascular age-related macular degeneration have been developed. These agents, originally developed for their anti-angiogenic mechanism of action, probably also work through an anti-permeability effect in preventing or reducing the amount of leakage from submacular neovascular tissue. Other treatment modalities include laser photocoagulation, photodynamic therapy with verteporfin, and submacular surgery. In reality, these latter treatments can be similarly categorized as anti-angiogenic because their sole aim is destroying or removing choroidal neovascularization (CNV). At the cellular level, CNV resembles stereotypical tissue repair that consists of several matricellular components in addition to neovascularization. In the retina, the clinical term CNV is a misnomer since the term may more appropriately be referred to as aberrant submacular repair. Furthermore, CNV raises a therapeutic conundrum: To complete or correct any reparative process in the body, angiogenesis becomes an essential component. Anti-angiogenic therapy, in all its guises, arrests repair and causes the hypoxic environment to persist, thus fueling pro-angiogenesis and further development of CNV as a component of aberrant repair. However, we realize that anti-vascular endothelial growth factor therapy preserves vision in patients with age-related macular degeneration, albeit temporarily and therefore, repeated treatment is needed. More importantly, however, anti-angiogenic therapy demonstrates that we can at the very least tolerate neovascular tissue beneath the macula and preserve vision in contrast to our historical approach of total vascular destruction. In this clinical scenario, it may be possible to look beyond anti-angiogenesis if our goal is facilitating submacular repair without destroying the neurosensory retina. Thus, in this situation of neovascular tolerance, it may be timely to consider treatments that facilitate vascular maturation, rather than its arrest or destruction. This would neutralize hypoxia, thus removing the stimulus that drives neovascularization and in turn the need for repeated lifelong intravitreal therapy. A pro-angiogenic approach would eliminate neovascular leakage and ultimately complete repair and preserve the neurosensory retina.

Labeling of stem cells with monocrystalline iron oxide for tracking and localization by magnetic resonance imaging.

Precise localization of exogenously delivered stem cells is critical to our understanding of their reparative response. Our current inability to determine the exact location of small numbers of cells may hinder optimal development of these cells for clinical use. We describe a method using magnetic resonance imaging to track and localize small numbers of stem cells following transplantation. Endothelial progenitor cells (EPC) were labeled with monocrystalline iron oxide nanoparticles (MIONs) which neither adversely altered their viability nor their ability to migrate in vitro and allowed successful detection of limited numbers of these cells in muscle. MION-labeled stem cells were also injected into the vitreous cavity of mice undergoing the model of choroidal neovascularization, laser rupture of Bruch's membrane. Migration of the MION-labeled cells from the injection site towards the laser burns was visualized by MRI. In conclusion, MION labeling of EPC provides a non-invasive means to define the location of small numbers of these cells. Localization of these cells following injection is critical to their optimization for therapy.

Polydimethylsiloxane as a substrate for retinal pigment epithelial cell growth.

Retinal pigment epithelial (RPE) cell transplantation represents potential treatment for age-related macular degeneration (AMD). Because delivery of isolated cells can cause serious complications, it is necessary to develop a suitable transplant membrane that could support an intact functioning RPE monolayer. Polydimethylsiloxane (PDMS) possesses the physical properties required for a transplanting device and is widely used clinically. We have investigated the use of PDMS as a potential surface for the growth of healthy RPE monolayers. PDMS discs were surface modified by air and ammonia gas plasma treatments. Dynamic contact angles were measured to determine the changes in wettability. Human ARPE-19 cells were seeded onto untreated and treated samples. Cell number, morphology and monolayer formation, cytotoxicity, and phagocytosis of photoreceptor outer segments (POS) were assessed at set time-points. Air plasma treatment increased the wettability of PDMS. This significantly enhanced cell growth, reaching confluence by day 7. Immunofluorescence revealed well-defined actin staining, monolayer formation, and high cell viability on air plasma treated and untreated surfaces, and to a lesser extent, on ammonia plasma treated. Furthermore, RPE monolayers were able to demonstrate phagocytosis of POS in a time-dependent manner similar to control. PDMS can support an intact functional monolayer of healthy differentiated RPE cells.

The presence of AC133-positive cells suggests a possible role of endothelial progenitor cells in the formation of choroidal neovascularization.

PURPOSE: Recent evidence suggests that vasculogenesis as well as angiogenesis occurs throughout the body during neovascularization. The recruitment of circulating stem cells is a key feature of vasculogenesis. The purpose of the present study was to determine whether markers of endothelial progenitor cells (EPCs) are present in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). METHODS: Surgically excised CNV (n = 9) membranes from patients with AMD were probed with immunohistochemical techniques using the following monoclonal antibodies: AC133 a putative marker of EPCs and hematopoietic stem cells (HSCs); the endothelial cells markers CD31, CD34, and von Willebrand factor (vWF); and cytokeratins and CD68, markers for retinal pigment epithelium (RPE) and macrophages, respectively. After secondary antibody amplification, reactions were visualized with fast red substrate. RESULTS: Six of nine specimens demonstrated cells positive for AC133 that were all found within predominantly cellular regions of the specimens. In the avascular fibrous stromal core of all specimens, the predominant cells were RPE cells and macrophages. The peripheral component of all CNV membranes was highly vascular and showed varying immunoreactivity for all endothelial markers. The greatest immunoreactivity for endothelial markers was observed with CD34 and vWF and least for CD31. CONCLUSIONS: These findings support animal studies that vasculogenesis, in addition to angiogenesis, may contribute to the neovascularization that occurs in AMD.

Angiographic characteristics in patients undergoing macular translocation for subfoveal choroidal neovascularization secondary to age-related macular degeneration.

PURPOSE: To review in a standardized fashion pre- and postoperative fluorescein angiographic characteristics in patients undergoing limited macular translocation (LMT) with scleral imbrication to treat subfoveal choroidal neovascularization (SFCNV) secondary to age-related macular degeneration (AMD). The current study was undertaken to assess any potential effects of the translocation procedure on altering the angiographic characteristics of SFCNV before laser photocoagulation. METHODS: A consecutive series of patients undergoing LMT for AMD was identified retrospectively. The pre- and postoperative fluorescein angiograms were reviewed in a masked fashion. Angiographic characteristics evaluated included pre- and postoperative lesion components, stability of lesion, and the amount of retinal translocation obtained. RESULTS: Eighty-eight patients (90 eyes) had angiograms of adequate quality to permit evaluation. Time between the preoperative and the prelaser angiogram ranged from 2 to 84 days (median 7.5 days). Neovascular complexes remained unchanged or decreased in size in 79% of patients. There was no statistically significant difference in lesion size between the pre- and postoperative periods (P = 0.34). Retinal movement ranged from 160 microm to 3,320 microm (median 960 microm), with 61% of cases undergoing effective translocation (i.e., the fovea was moved away from the neovascular complex). None of the lesion components or demographic factors evaluated affected the amount of translocation obtained. Larger lesions were more likely to remain subfoveal following translocation (P = 0.004). CONCLUSION: The size and lesion characteristics appear relatively stable following translocation. Amount of retinal movement is not associated with angiographic lesion characteristics. Only size was associated with achievement of desired translocation in the final model, with large lesions being less likely to achieve desired translocation. In our study group, the amount of retinal translocation was variable with 61% of cases undergoing effective translocation.