Lymphocytic Microparticles Modulate Angiogenic Properties of Macrophages in Laser-induced Choroidal Neovascularization (original) (raw)
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Molecular medicine reports, 2017
Choroidal neovascularization (CNV) is a serious complication of age‑related macular degeneration. The aim of the present study was to investigate the expression and distribution of M1 and M2 macrophages in a laser‑induced CNV adult mouse model. The mRNA expression levels of M1, M2 and pan macrophage markers, and macrophage‑associated angiogenic cytokines, were determined by reverse transcription‑quantitative polymerase chain reaction. Immunofluorescence studies were performed to determine the location of the macrophages. The expression levels of M1 macrophage markers increased to a greater extent compared with M2 markers in the retinal pigment epithelium (RPE)‑choroid complexes following laser photocoagulation. By contrast, the expression levels of M2 macrophage markers increased primarily in the retinas. Immunofluorescence studies revealed that the increased number of cluster of differentiation (CD)206‑positive cells were located primarily in the retina, whereas the CD80‑positive c...
PLoS ONE, 2014
Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the elderly, and its exsudative subtype critically depends on local production of vascular endothelial growth factor A (VEGF). Mononuclear phagocytes, such as macrophages and microglia cells, can produce VEGF. Their precursors, for example monocytes, can be recruited to sites of inflammation by the chemokine receptor CCR2, and this has been proposed to be important in AMD. To investigate the role of macrophages and CCR2 in AMD, we studied intracellular VEGF content in a laser-induced murine model of choroidal neovascularisation. To this end, we established a technique to quantify the VEGF content in cell subsets from the lasertreated retina and choroid separately. 3 days after laser, macrophage numbers and their VEGF content were substantially elevated in the choroid. Macrophage accumulation was CCR2-dependent, indicating recruitment from the circulation. In the retina, microglia cells were the main VEGF + phagocyte type. A greater proportion of microglia cells contained VEGF after laser, and this was CCR2-independent. On day 6, VEGF-expressing macrophage numbers had already declined, whereas numbers of VEGF + microglia cells remained increased. Other sources of VEGF detectable by flow cytometry included in dendritic cells and endothelial cells in both retina and choroid, and Mü ller cells/astrocytes in the retina. However, their VEGF content was not increased after laser. When we analyzed flatmounts of laser-treated eyes, CCR2-deficient mice showed reduced neovascular areas after 2 weeks, but this difference was not evident 3 weeks after laser. In summary, CCR2dependent influx of macrophages causes a transient VEGF increase in the choroid. However, macrophages augmented choroidal neovascularization only initially, presumably because VEGF production by CCR2-independent eye cells prevailed at later time points. These findings identify macrophages as a relevant source of VEGF in laser-induced choroidal neovascularization but suggest that the therapeutic efficacy of CCR2-inhibition might be limited.
PLoS ONE, 2014
Background: As the murine model of laser-induced choroidal neovascularization (CNV) is becoming the most established and commonly utilized model worldwide for studying the pathogenesis of CNV and its response to treatment, specific operating standards are yet to be clarified. The purpose of this study is to compare the lesion size of CNV in mice with different ages, sex, durations of CNV process, and treated positions of laser spots, to make recommendations that may improve and optimize the quality of the model. Methods and Results: C57/BL6 mice of different ages were treated with diode laser photocoagulation per eye and perfused with PBS containing fluorescein-labeled dextran at different time of observation. Choroid flat mounts, were then examined by fluorescence microscopy for the measurement of CNV area. Messenger-RNA expression levels of several angiogenic cytokines in eye cups of male and female C57BL/6 mice at 5-8 and 16-20 week-old were analyzed by real-time RT-PCR assay. The results showed significantly more CNV area in eyes of female mice compared to male mice with the expression level of several angiogenic cytokines elevated. 16-20-week-old female mice developed the biggest area of CNV. The mean area of CNV increased significantly at the 14 th day after photocoagulation. Laser spots delivered 1PD away from the optic disc induced the biggest area of CNV compared to those 2PD or 3PD away. Interaction of NV was observed in laser spots delivered less than 1PD away from each other. Conclusion: The current results suggest that 16-20-week-old female C57BL/6 mice developed the most distinct CNV lesion size with laser spots delivered 1PD away from the optic disc. The best time to observe and analyze is the 14 th day after photocoagulation.
Investigation of Laser-Induced Choroidal Neovascularization in the Rat
Investigative Ophthalmology & Visual Science, 2003
Choroidal neovascularization plays an important role in pathogenesis of age-related macular degeneration. Induction of neovascularization by laser photocoagulation in the rat fundus is an established animal model in which the effects of new therapeutic approaches are assessed. The purpose of this study was to compare different detection methods of laser-induced neovascularization in the rat. METHODS. Laser spots were applied to the fundus of Long-Evans rats. Ten days after, four different methods were used to detect laser-induced neovascularization: (1) high-resolution angiography with fluorescein isothiocyanate-dextran, (2) immunohistochemical visualization of platelet endothelial cell adhesion molecule (PECAM)-1, (3) visualization of intravascular lumens by peroxidase perfusion in the living rat with subsequent histologic analysis, and (4) histochemical representation of alkaline phosphatase in endothelial cells. RESULTS. At the rim of the laser scars vessel-forming endothelial cells with intravasal dextran and peroxidase were present. Cross-sections demonstrated that these vessels originated from the retina. The center of the scars contained homogenous endothelial cells of choroidal origin, which was confirmed by immunohistochemistry and electron microscopy. In lasertreated eyes without FITC-dextran perfusion, scars showed unspecific fluorescence, making differentiation from specific FITC-dextran-associated fluorescence difficult. CONCLUSIONS. In the rat model of laser-induced neovascularization, newly developed endothelial cells originate from the retina and the choroid. Whereas ring-like surrounding vessels come from the retina, flat endothelial cells in deeper layers are of choroidal origin or may originate from circulating endothelial precursor cells. Dextran angiography has to be regarded critically for visualizing the choriocapillaris and CNV in laser scars. PECAM-1 immunohistochemistry is best for detection and quantification of neovascularization in laser scars. (Invest Ophthalmol Vis Sci. 2003;44:5349 -5354) All flatmounts were prepared as described by McMenamin. 9 The eyes were sectioned at the equator, and the anterior half and the vitreous removed. The retinas were isolated and investigated by light microscopy. The posterior eye segment containing the sclera and choroid was dissected into quarters by four radial cuts and mounted on a slide.
Investigative Ophthalmology & Visual Science, 2003
Macrophage recruitment to the choroid has been proposed to contribute to the pathogenesis of choroidal neovascularization (CNV) in AMD. The study was conducted to determine whether treatment with clodronate liposomes (CL 2 MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV. METHODS. Laser-induced CNV was performed in female 16month-old C57BL/6 mice. Macrophages were depleted by use of CL 2 MDP-lip intraperitoneally and subcutaneously 72 and 24 hours before and every 2 to 3 days after laser injury. Control mice received injections of either PBS alone or PBS liposomes. Blood monocyte and choroidal macrophage depletion were documented by flow cytometry and choroidal flatmount preparation analysis, respectively. Two weeks after laser injury, mice were injected intravenously with fluoresceinated dextran. The right eyes were removed and prepared for flatmount analysis of CNV surface area (in relative disc areas or DA), vascularity (relative fluorescence), and cellularity (propidium iodide stain). The mice were then perfused with 10% formaldehyde, and the left eyes were removed for histopathology. The means of the various parameters for four CNV lesions per eye were calculated. Fluorescein angiography was also performed. RESULTS. Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL 2 MDP-lip-treated mice. Compared with the control, flatmount analysis of macrophage depleted mice demonstrated a significant reduction in size of the CNV area (2.8 Ϯ 0.5 DA vs. 1.4 Ϯ 0.1 DA; P Ͻ 0.043). The treated group also revealed less vascularity (1.6 Ϯ 0.1 units vs. 1.1 Ϯ 0.0 units; P Ͻ 0.0092) and cellularity of CNV lesions (3.3 Ϯ 0.6 DA vs. 1.7 Ϯ 0.1 DA, P Ͻ 0.04). Histopathology revealed that, in the macrophage-depleted group, CNV was smaller in diameter (1270 Ϯ 73 pixels vs. 770 Ϯ 82 pixels, P Ͻ 0.0006) and thickness (120 Ϯ 7 pixels vs. 96 Ϯ 7 pixels, P Ͻ 0.019).
American Journal of Ophthalmology, 2005
Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch's membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3 ؊/؊ mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-2, and -fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser-induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases. The Journal of Immunology, 2005, 174: 491-497.
Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics, 2018
Mononuclear phagocytes (MNPs) are present in neovascular age-related macular degeneration (nv AMD) which is also called choroidal neovascularization (CNV). The number and phenotype of the MNPs depend upon the local environment in the CNV and effect of nv AMD therapy. We investigated ocular cell infiltration and conditions that modulate angiogenesis in a laser-induced mouse CNV model. We developed assays to quantify MNPs in our established mouse CNV model. One MNP assay quantified the number of subretinal cells peripheral to the CNV lesions. A second assay semiquantitatively assesses the number of MNPs localized to the CNV lesion. We used these assays to measure the effect of toll-like receptor-2 (TLR-2) activation, anti-vascular endothelial growth factor (VEGF) therapy, and chemokine (C-C motif) ligand 2 (Ccl2) genetic deletion on MNP infiltration after laser injury. Laser injury induced blood vessel growth and infiltration of MNPs. Systemic administration of a TLR-2 activating pept...
Influence of laser photocoagulation on choroidal capillary cytoarchitecture
British Journal of Ophthalmology, 2001
Aim-To identify if laser photocoagulation induces morphological changes specifically related to the choroidal capillary endothelial processes that protrude into Bruch's membrane. Methods-Two human eyes and one adult macaque monkey eye received retinal laser photocoagulation that was just suprathreshold, before enucleation or exenteration. They were examined by electron microscopy to determine the length of the endothelial processes emanating from the choroidal capillaries in the region around the laser burn. One human and two monkey untreated eyes were used for comparison. Results-In human eyes, there was no increase in the number of processes 15 hours after laser treatment but at 5 days the processes were more numerous and longer within 400-500 µm of the burn than in the untreated half of the same eye. The processes were longer 9 days after photocoagulation in the monkey, when compared with untreated monkeys, and some breached the elastic lamina, a phenomenon not seen in the untreated eyes. Qualitative diVerences were also noted in the endothelial cell processes following photocoagulation. Neovascularisation was not observed. Conclusions-Protrusion of choroidal endothelial cell processes into Bruch's membrane is a normal anatomical feature but the number, length, and morphology of the processes change following mild photocoagulation. It is plausible that these processes may play a part in the clearance of debris from Bruch's membrane, and represent an early stage of angiogenesis. If the latter is true prophylactic laser photocoagulation at just suprathreshold levels may carry a risk of inducing choroidal neovascularisation.