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

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)

Macrophage activation associated with chronic murine cytomegalovirus infection results in more severe experimental choroidal neovascularization.

The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication

Eyes of mice with chronic MCMV infection show more severe CNV as determined by histopathologic analysis.

<p>Groups of adult C57BL/6 mice were inoculated intraperitoneally with either MCMV or UV-inactivated virus. At 6 days, 6 weeks, or 12 weeks after inoculation, all mice were subjected to laser treatment to induce CNV, and, four weeks later, retinal sections were subjected to histopathologic analysis. (<b>A</b>) Hematoxylin and eosin-stained histopathologic sections of representative individual mouse retinas four weeks after laser treatment showing areas of CNV (white outlines and arrows). (Magnification = 100×) Mouse inoculated with UV-inactivated MCMV (control); mouse infected with MCMV for 6 days prior to laser treatment; mouse infected with MCMV for 6 weeks prior to laser treatment; mouse infected with MCMV for 12 weeks prior to laser treatment. (<b>B</b>) Comparative quantitative analysis of all animals of each group for size of CNV areas as determined by histopathologic analysis. Asterisk indicates statistical significance when compared with control (<i>p</i> = <0.0004).</p

Ganciclvir treatment reduces levels of VEGF mRNA production by MCMV-infected IC-21 macrophages.

<p>Monolayers of IC-21 mouse macrophages were either mock infected or infected with MCMV (2.5 PFU/cell), treated at 1 hour postinfection with 0, 15, 30, or 60 uM of ganciclovir, and harvested at 24 hour postinfection. (<b>A</b>) Quantitative real-time PCR assay for comparison of levels of VEGF mRNA production. Three independent experiments were performed. (<b>B</b>) Immunostaining for detection of cytoplasmic VEGF production. VEGF = Red; DAPI = Blue (Magnification = 200×).</p

Ganciclvir treatment reduces levels of VEGF mRNA and TNF-α mRNA production by splenic macrophages collected from chronically infected mice.

<p>Groups of adult C57BL/6 mice were inoculated intraperitoneally with either MCMV or mock-infected with maintenance medium. At 12 weeks after inoculation, groups of MCMV-infected or mock-infected mice were treated intraperitoneally with ganciclovir for 7 days (40 mg/kg/day). Parallel groups of untreated control MCMV-infected or mock-infected mice received daily intraperitoneal injections of phosphate-buffered saline for 7 days. Following the 7-day regimen of ganciclovir or phosphate-buffered saline treatment, splenic macrophages were collected from treated and untreated MCMV-infected animal groups and compared with splenic macrophages collected from treated and untreated mock-infected animal groups by quantitative real-time RT-PCR assay for comparison of levels of TNF-α mRNA and VEGF mRNA production. Asterisks indicate statistical significance when compared with parallel treated or untreated mock-infected animals. (p = ≤0.009). Gray bars = TNF-α mRNA; Black bars = VEGF mRNA.</p

MCMV infection stimulates production of VEGF mRNA and VEGF protein in monolayers of IC-21 mouse macrophages.

<p>Monolayers of IC-21 mouse macrophages were either mock infected or infected with MCMV (2.5 PFU/cell) and analyzed for amounts of VEGF mRNA and VEGF protein at 24 hour and/or 48 hour postinfection. Asterisks indicate statistical significance when compared with mock-infected monolayers. (<b>A</b>) Quantitative RT-PCR assay for VEGF mRNA (black bars) and TNF-α mRNA (gray bars) in monolayers of IC-21 macrophages either mock-infected, treated with LPS, inoculated with UV-inactivated virus, or infected with MCMV at 24 hr or 48 hr after infection and compared with mock-infected monolayers. (<i>p</i> = ≤0.04) (<b>B</b>) ELISA for VEGF protein (black bars) in monolayers of IC-21 macrophages infected with MCMV at 48 hr postinfection and compared with mock-infected monolayers (<i>p</i> = 0.01).</p

MCMV infection of monolayers of IC-21 mouse macrophages results in VEGF production by virus-infected and bystander uninfected cells.

<p>Monolayers of IC-21 mouse macrophages were either mock infected or infected with MCMV (2.5 PFU/cell). At 24 hour postinfection (MCMV 24 hr pi) or 48 hour postinfection (MCMV 48 hr pi), all mock-infected and MCMV-infected cells were harvested, fixed, and reacted with either control rabbit IgG or anti-VEGF rabbit IgG as primary antibody for visualization of VEGF production. All cells were counterstained with DAPI to visualize cell nuclei. VEGF = Red; DAPI = Blue (Magnification = 200×) White circle emphasizes focus of cells at 48 hr postinfection undergoing cytopathology and plaque formation. Quantification of cells exhibiting positive staining for VEGF was accomplished by using mean values of the number of cells showing obvious red cytoplasmic staining within five random microscopic fields of view.</p