Bone Marrow Transplantation Transfers Age-Related Susceptibility to Neovascular Remodeling in Murine Laser- Induced Choroidal Neovascularization

Citation: Espinosa-Heidmann DG, Malek G, Mettu PS, et al. Bone marrow transplantation transfers age-related susceptibility to neovascular remodeling in murine laser-induced choroidal neovascularization. Invest Ophthalmol Vis Sci. 2013;54:7439-7449. DOI:10.1167/iovs.13-12546 PURPOSE. Neovascular remodeling (NVR), the progression of small capillaries into large-caliber arterioles with perivascular fibrosis, represents a major therapeutic challenge in neovascular age-related macular degeneration (AMD). Neovascular remodeling occurs after laser-induced choroidal neovascularization (CNV) in aged but not young mice. Additionally, bone marrowderived cells, including macrophages, endothelial precursor cells, and mesenchymal precursor cells, contribute to CNV severity. In this study, we investigated the impact of aged bone marrow transplantation (BMT) on the degree of fibrosis, size, and vascular morphology of CNV lesions in a mouse model of laser-induced CNV. METHODS. Young (2 months) and old (16 months) mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow isolated from either young or old donors. Laser CNV was induced 1 month following transplant, and eyes were analyzed via choroidal flat mounts and immunohistochemistry 1 month postlaser. The identity of cells infiltrating CNV lesions was determined using specific markers for the labeled transplanted cells (GFPþ), macrophages (F4/80þ), perivascular mesenchymal-derived cells (smooth muscle actin, SMAþ), and endothelial cells (CD31þ). RESULTS. Bone marrow transplantation from aged mice transferred susceptibility to NVR into young recipients. Inversely, transplantation of young marrow into old mice prevented NVR, preserving small size and minimal fibrosis. Mice with NVR demonstrated a greater relative contribution of marrow-derived SMAþ perivascular mesenchymal cells as compared to other cells. CONCLUSIONS. Our findings indicate that the status of bone marrow is an important determining factor of neovascular severity. Furthermore, we find that perivascular mesenchymal cells, rather than endothelial cells, derived from aged bone marrow may contribute to increased CNV severity in this murine model of experimental neovascularization

Photoreceptor synapses degenerate early in experimental choroidal neovascularization

Severe visual loss in patients with age-related macular degeneration is associated with the development of choroidal neovascularization (CNV). The pathogenic mechanisms for CNV formation have been extensively investigated, but remarkably little research has addressed the mechanisms for dysfunction of the retina in CNV. Using laser-induced CNV in mice, we evaluated the mechanisms of retinal dysfunction. At 3 days, 1 week, 2 weeks, and 4 weeks after laser application, retinas under experimental CNV were characterized physiologically (ERG recordings, synaptic uptake of the exocytotic marker FM1-43, and light-induced translocation of transducin), histologically, and immunohistochemically. ERG amplitudes were reduced by 20% at 1 week after CNV. Depolarization-induced FM1-43 uptake in photoreceptor synapses was selectively reduced by 45% at 1 week after CNV. Although photoreceptor outer segments were shortened by 36%, light adaptation as measured by transducin translocation was mostly preserved. Early in CNV (3 days to 1 week), Muller cells demonstrated induction of c-fos and pERK expression. Also, the density of macrophage-like, F4/80 immunoreactive cells increased approximately 3-fold. Minimal photoreceptor death occurred during the first week, and was variable thereafter. At later times in CNV formation (> or =2 weeks), expression of photoreceptor synaptic markers was reduced in the outer plexiform layer, indicating loss of photoreceptor synaptic terminals. ERG amplitudes, synaptic uptake of FM1-43, and the induction of c-fos and pERK in Muller cells were altered within 1 week of experimental CNV, suggesting that during CNV formation, deficits in retinal function, in particular photoreceptor synaptic function, precede degeneration of photoreceptor terminals and photoreceptor cell death

Blood-derived macrophages infiltrate the retina and activate Muller glial cells under experimental choroidal neovascularization

Inflammation is a major mechanism in the pathogenesis of age-related macular degeneration, the most important cause of blindness in the elderly. Previous studies have focused on the role of macrophages in regulating the growth of pathological new vessels over the retina, called choroidal neovascularization (CNV). However, no research has been done to evaluate the role of inflammation as a mechanism of vision loss and retinal degeneration in the retina underlying CNV. In other neuropathological conditions, hematogenous macrophages and/or resident microglia contribute to neurodegeneration. We have combined laser-induced CNV in mice and bone marrow transplantation with GFP-labeled bone marrow to determine the relative role of recruited blood-derived macrophages versus resident microglia in the retina associated with CNV. Using these chimeric mice, we have found that many GFP-labeled cells infiltrated the retina underlying CNV but not the retina unaffected by CNV. Immunostaining for the cell adhesion molecules VCAM 1, ICAM 1, and PECAM was strongly upregulated in retinal blood vessels under CNV. All GFP-labeled cells were immunoreactive for the macrophage marker F4/80. Most (70%) of the F4/80 immunoreactive cells were GFP-labeled under CNV. The density of resident microglia did not increase. Most GFP-labeled cells were found in close proximity to activated Muller cells. Depleting circulating macrophages with clodronic acid diminished the density of F4/80 immunoreactive cells as well as the density of pERK immunoreactive Muller cells in the retina under CNV. Thus, recruitment of blood-derived macrophages more than resident microglia seems to be associated with CNV

The Role of Aging, High Fat Diet and Blue Light Exposure in an Experimental Mouse Model for Basal Laminar Deposit Formation

We sought to investigate the role of aging as a susceptibility factor for the capacity of dietary fat intake to increase the development of subretinal deposits. Mice of various ages (2, 9 and 16 months) were fed a normal diet or a diet high in saturated and unsaturated fats for a total four and a half months. Some eyes were also exposed to non-phototoxic levels of blue-green light. The outer retina and choroid were examined by light and transmission electron microscopy, and the characteristics, frequency and severity of subRPE deposits was determined. Aged mice fed normal diets developed only very mild subretinal deposits. However, many eyes of mice aged 9 months or older at the time of initiation of diet developed frequent basal laminar deposits of moderate severity, and only 16 month old mice developed more severe deposits after exposure to blue-green light. Some eyes in this older group also developed endothelial invasion into Bruch's membrane. None of the eyes developed classic drusen or linear deposits. These observations demonstrate that age increases the capacity of dietary fat, especially in the presence of environmental light, to induce subRPE deposits

Quantitative enumeration of vascular smooth muscle cells and endothelial cells derived from bone marrow precursors in experimental choroidal neovascularization

Choroidal neovascularization (CNV) is characterized by the subretinal invasion of a pathologic new vessel complex from the choriocapillaris. Although CNV is traditionally considered to consist of endothelial cells, the cellular population of CNV is likely more complex in nature, comprising several different cell types. In addition, recent studies suggest that the CNV cell population has a dual origin (circulating versus resident populations). In this study we sought to determine the contribution and origin of different cell types in experimental CNV. Laser-induced CNV was performed on chimeric mice generated by reconstituting C57BL/6 mice with bone marrow from green fluorescent protein (GFP)-transgenic mice. In these mice, bone marrow-derived cells are GFP-labeled. Immunofluorescence staining was used to examine both flatmount preparations of the choroid and cross sections of the posterior pole for macrophages, endothelial cells, vascular smooth muscle cells, retinal pigment epithelial (RPE) cells, lymphocytes, or neutrophils at day 3, 7, 14 and 28 post-laser ( n=5 per group). Cell types present in CNV included macrophages (20% of the cells in CNV), endothelial cells (25%), vascular smooth muscle cells (11%), RPE cells (12%) and non-labeled cells (32%). The macrophage population was mostly derived from circulating monocytes at all timepoints studied (70% were GFP labeled), while endothelial and vascular smooth muscle cells were partly bone marrow derived (50–60% were GFP labeled), and RPE cells appeared to be entirely derived from preexisting tissue resident cells. These results demonstrate that bone marrow-derived progenitor cells contribute significantly to the vascular and inflammatory components of CNV. Knowledge of the cellular composition and origin might help understand the pathogenic mechanisms controlling CNV severity as well as indicate potential targets for therapeutic intervention

Gender and estrogen supplementation increases severity of experimental choroidal neovascularization

Observational clinical studies suggest that post-menopausal women may be at risk for more severe age-related macular degeneration, and that estrogen loss due to menopause may contribute. We sought to determine the effect of gender and estrogen status on the severity of choroidal neovascularization (CNV) in a mouse model for experimental choroidal neovascularization. Laser-induced CNV was performed in mice with or without estrogen supplementation. At various times, eyes were removed for analysis of severity of CNV lesions or for extraction of choroidal mRNA to evaluate iNOS, TNF-α, MMP-9, and ER-α expression, which are molecules relevant to angiogenic processes. Also, splenic macrophages were analysed for iNOS to determine the effect of estrogen treatment in vitro. Finally, laser-induced CNV was performed in iNOS −/− mice. Our result showed that aged female mice had significantly larger CNV than age-matched males. Ovariectomy in adult mice did not increase severity, but paradoxically estrogen supplementation after ovariectomy did increase CNV severity. More severe CNV were associated with a significant decrease in choroidal iNOS mRNA. Splenic macrophages from estrogen supplemented mice showed a significant increased in TNF-α mRNA expression (eight fold difference compared to the control) but only a mild change in iNOS mRNA levels (2–3 fold difference). In vitro data further showed that nitric oxide production in splenic macrophages at different estrogen levels was not different from controls. Finally, CNV severity was significantly more severe in iNOS −/− mice, compared to iNOS +/+ mice after laser treatment. In conclusion, aged female mice developed more severe CNV than do males. Estrogen replacement seems to increase severity, possibly by suppressing the upregulation of choroidal iNOS and activating macrophages. The putative beneficial or detrimental role of estrogen biology in age-related macular degeneration must be more carefully evaluated and may vary with the stage of age-related macular degeneration (atrophic or neovascular) as well as with the specific target cell type (monocytes vs. endothelial cell or vascular smooth muscle cell)