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Intravitreal TSG-6 suppresses laser-induced choroidal neovascularization by inhibiting CCR2+ monocyte recruitment.

Kim SJ, Lee HJ, Yun JH, Ko JH, Choi da Y, Oh JY - Sci Rep (2015)

Bottom Line: An intravitreal injection of TSG-6 suppressed the expression of VEGF and pro-inflammatory cytokines including CCL2, and reduced the size of CNV.Also, the number of Iba(+) and CCR2(+) cells including infiltrating macrophages was markedly lower in the CNV lesion of TSG-6-treated eyes.Further analysis identified CCR2(+) CD11b(+) CD11c(+) cells and CCR2(+) CD11b(-)CD11c(+) cells as the cell populations that were increased by laser injury and reduced by TSG-6 treatment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, Korea [2] Samsung Biomedical Research Institute, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, Korea.

ABSTRACT
Choroidal neovascularization (CNV) is the hallmark of wet age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. Although the pathogenesis of CNV is not clear, a number of studies show that ocular-infiltrating macrophages and inflammation play a critical role in the development of CNV. TNFα-stimulated gene/protein (TSG)-6 is a multifunctional endogenous protein that has anti-inflammatory activities partly by regulating macrophage activation. Therefore, we here investigated the therapeutic potential of TSG-6 in a rat model of CNV induced by laser photocoagulation. Time course analysis showed that the expression of VEGF and pro-inflammatory cytokines in the choroid was up-regulated early after laser injury, and gradually decreased to baseline over 14 days. An intravitreal injection of TSG-6 suppressed the expression of VEGF and pro-inflammatory cytokines including CCL2, and reduced the size of CNV. Also, the number of Iba(+) and CCR2(+) cells including infiltrating macrophages was markedly lower in the CNV lesion of TSG-6-treated eyes. Further analysis identified CCR2(+) CD11b(+) CD11c(+) cells and CCR2(+) CD11b(-)CD11c(+) cells as the cell populations that were increased by laser injury and reduced by TSG-6 treatment. Together, the results demonstrate that TSG-6 inhibits inflammation and CCR2(+) monocyte recruitment into the choroid, and suppresses the development of CNV.

No MeSH data available.


Related in: MedlinePlus

TSG-6 decreases CCR2+ monocyte infiltration and CCL2 production in the choroid.(a, b) Flow cytometric analysis showed that the number of CCR2+ CD11b+ cells and CCR2+ CD11c+ cells was highly increased in the RPE-choroid at day 3 after laser injury, and significantly reduced by an intravitreal injection of TSG-6. (c-e) Further analysis after gating on CCR2 revealed that the number of CCR2+ CD11b+ CD11c+ cells and CCR2+ CD11b−CD11c+ cells in the RPE-choroid was significantly decreased by TSG-6 treatment. However, the number of CCR2+ CD11b+ CD11c− cells was not altered by laser injury or TSG-6 treatment. (f) Real-time RT PCR showed that the expression of CCL2 in the RPE-choroid was significantly suppressed by TSG-6 treatment. n = 4 in each group. Data are presented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.
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f3: TSG-6 decreases CCR2+ monocyte infiltration and CCL2 production in the choroid.(a, b) Flow cytometric analysis showed that the number of CCR2+ CD11b+ cells and CCR2+ CD11c+ cells was highly increased in the RPE-choroid at day 3 after laser injury, and significantly reduced by an intravitreal injection of TSG-6. (c-e) Further analysis after gating on CCR2 revealed that the number of CCR2+ CD11b+ CD11c+ cells and CCR2+ CD11b−CD11c+ cells in the RPE-choroid was significantly decreased by TSG-6 treatment. However, the number of CCR2+ CD11b+ CD11c− cells was not altered by laser injury or TSG-6 treatment. (f) Real-time RT PCR showed that the expression of CCL2 in the RPE-choroid was significantly suppressed by TSG-6 treatment. n = 4 in each group. Data are presented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

Mentions: Monocytes infiltrating the injury site are known as a major source of inflammatory cytokines and angiogenic factors such as VEGF upon laser photocoagulation, and therefore, are critical for CNV development11121314151617. From the previous results (Figs 1,2), we observed that an intraocular injection of TSG-6 suppressed VEGF expression and local inflammation in the laser-injured RPE-choroid. Based on these observations, we hypothesized that TSG-6 might affect the infiltration of inflammatory monocytes in the choroid after laser injury. To test the hypothesis, we analyzed the RPE-choroid and retina for CCR2, CD11b, and CD11c-expressing cells at day 3 after laser photocoagulation. We here evaluated CCR2 expression because CCR2 is highly expressed by migrating inflammatory monocytes, but not by resident microglia343536. We used CD11b and CD11c as markers for myeloid cells or monocytes: macrophages and dendritic cells. Flow cytometric analysis revealed that the number of CCR2+ CD11b+ cells and CCR2+ CD11c+ cells, indicating migrating monocytes, was markedly increased in the RPE-choroid by laser injury, and significantly reduced by an intravitreal injection of TSG-6 (Fig. 3a,b). Further analysis after gating on CCR2 showed that TSG-6 treatment significantly decreased the number of CCR2+ CD11b+ CD11c+ cells and CCR2+ CD11b-CD11c+ cells in the RPE-choroid (Fig. 3c,d); however the number of CCR2+ CD11b+ CD11c- cells was not altered by TSG-6 (Fig. 3d,e). In contrast, the number of CCR2+ cells, either CD11b+ or CD11c+ , was not increased in the retina at day 3 or changed by TSG-6 (Fig. S1), suggesting that monocytes did not infiltrate the retina or that monocyte infiltration did not persist until day 3.


Intravitreal TSG-6 suppresses laser-induced choroidal neovascularization by inhibiting CCR2+ monocyte recruitment.

Kim SJ, Lee HJ, Yun JH, Ko JH, Choi da Y, Oh JY - Sci Rep (2015)

TSG-6 decreases CCR2+ monocyte infiltration and CCL2 production in the choroid.(a, b) Flow cytometric analysis showed that the number of CCR2+ CD11b+ cells and CCR2+ CD11c+ cells was highly increased in the RPE-choroid at day 3 after laser injury, and significantly reduced by an intravitreal injection of TSG-6. (c-e) Further analysis after gating on CCR2 revealed that the number of CCR2+ CD11b+ CD11c+ cells and CCR2+ CD11b−CD11c+ cells in the RPE-choroid was significantly decreased by TSG-6 treatment. However, the number of CCR2+ CD11b+ CD11c− cells was not altered by laser injury or TSG-6 treatment. (f) Real-time RT PCR showed that the expression of CCL2 in the RPE-choroid was significantly suppressed by TSG-6 treatment. n = 4 in each group. Data are presented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.
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f3: TSG-6 decreases CCR2+ monocyte infiltration and CCL2 production in the choroid.(a, b) Flow cytometric analysis showed that the number of CCR2+ CD11b+ cells and CCR2+ CD11c+ cells was highly increased in the RPE-choroid at day 3 after laser injury, and significantly reduced by an intravitreal injection of TSG-6. (c-e) Further analysis after gating on CCR2 revealed that the number of CCR2+ CD11b+ CD11c+ cells and CCR2+ CD11b−CD11c+ cells in the RPE-choroid was significantly decreased by TSG-6 treatment. However, the number of CCR2+ CD11b+ CD11c− cells was not altered by laser injury or TSG-6 treatment. (f) Real-time RT PCR showed that the expression of CCL2 in the RPE-choroid was significantly suppressed by TSG-6 treatment. n = 4 in each group. Data are presented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.
Mentions: Monocytes infiltrating the injury site are known as a major source of inflammatory cytokines and angiogenic factors such as VEGF upon laser photocoagulation, and therefore, are critical for CNV development11121314151617. From the previous results (Figs 1,2), we observed that an intraocular injection of TSG-6 suppressed VEGF expression and local inflammation in the laser-injured RPE-choroid. Based on these observations, we hypothesized that TSG-6 might affect the infiltration of inflammatory monocytes in the choroid after laser injury. To test the hypothesis, we analyzed the RPE-choroid and retina for CCR2, CD11b, and CD11c-expressing cells at day 3 after laser photocoagulation. We here evaluated CCR2 expression because CCR2 is highly expressed by migrating inflammatory monocytes, but not by resident microglia343536. We used CD11b and CD11c as markers for myeloid cells or monocytes: macrophages and dendritic cells. Flow cytometric analysis revealed that the number of CCR2+ CD11b+ cells and CCR2+ CD11c+ cells, indicating migrating monocytes, was markedly increased in the RPE-choroid by laser injury, and significantly reduced by an intravitreal injection of TSG-6 (Fig. 3a,b). Further analysis after gating on CCR2 showed that TSG-6 treatment significantly decreased the number of CCR2+ CD11b+ CD11c+ cells and CCR2+ CD11b-CD11c+ cells in the RPE-choroid (Fig. 3c,d); however the number of CCR2+ CD11b+ CD11c- cells was not altered by TSG-6 (Fig. 3d,e). In contrast, the number of CCR2+ cells, either CD11b+ or CD11c+ , was not increased in the retina at day 3 or changed by TSG-6 (Fig. S1), suggesting that monocytes did not infiltrate the retina or that monocyte infiltration did not persist until day 3.

Bottom Line: An intravitreal injection of TSG-6 suppressed the expression of VEGF and pro-inflammatory cytokines including CCL2, and reduced the size of CNV.Also, the number of Iba(+) and CCR2(+) cells including infiltrating macrophages was markedly lower in the CNV lesion of TSG-6-treated eyes.Further analysis identified CCR2(+) CD11b(+) CD11c(+) cells and CCR2(+) CD11b(-)CD11c(+) cells as the cell populations that were increased by laser injury and reduced by TSG-6 treatment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, Korea [2] Samsung Biomedical Research Institute, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, Korea.

ABSTRACT
Choroidal neovascularization (CNV) is the hallmark of wet age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. Although the pathogenesis of CNV is not clear, a number of studies show that ocular-infiltrating macrophages and inflammation play a critical role in the development of CNV. TNFα-stimulated gene/protein (TSG)-6 is a multifunctional endogenous protein that has anti-inflammatory activities partly by regulating macrophage activation. Therefore, we here investigated the therapeutic potential of TSG-6 in a rat model of CNV induced by laser photocoagulation. Time course analysis showed that the expression of VEGF and pro-inflammatory cytokines in the choroid was up-regulated early after laser injury, and gradually decreased to baseline over 14 days. An intravitreal injection of TSG-6 suppressed the expression of VEGF and pro-inflammatory cytokines including CCL2, and reduced the size of CNV. Also, the number of Iba(+) and CCR2(+) cells including infiltrating macrophages was markedly lower in the CNV lesion of TSG-6-treated eyes. Further analysis identified CCR2(+) CD11b(+) CD11c(+) cells and CCR2(+) CD11b(-)CD11c(+) cells as the cell populations that were increased by laser injury and reduced by TSG-6 treatment. Together, the results demonstrate that TSG-6 inhibits inflammation and CCR2(+) monocyte recruitment into the choroid, and suppresses the development of CNV.

No MeSH data available.


Related in: MedlinePlus