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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 reduces CNV development and VEGF production in the choroid after laser injury.After laser injury to Bruch’s membrane, either recombinant TSG-6 or PBS was injected into the vitreous cavity of rat eyes. At 1, 3, 7, and 14 days after injury, the whole RPE-choroids and retinas were separated and subjected to assays. (a) Representative photographs of the lectin staining of the whole RPE-choroid-scleral flat mounts at day 7 showed that CNV was induced by laser injury, and TSG-6 treatment reduced CNV development. Original magnification x 200. Scale bar: 100 μm. (b) Bar graphs showed quantification of the CNV size in TSG-6 and PBS-treated eyes. The size of CNV was significantly smaller in the TSG-6-treated rats than in the PBS-treated controls. Data are presented as mean + SEM from three independent experiments (each with six rats per group). The SEM represents the standard error of the mean values per animal (n = 18). (c) Real-time RT PCR showed that the mRNA level of VEGF highly increased in the RPE-choroid at day 1 after injury, and decreased to normal over 14 days. The level of VEGF transcript in the RPE-choroid was significantly lower in TSG-6-treated eyes than in PBS-treated controls at days 1, 3, and 7. However, the expression of VEGF was not either increased in the retina by laser or affected by TSG-6 treatment. Data represent mean ±/+ SD from three independent experiments, each with six rats per group. RQ means a ratio of mRNA levels relative to those in normal eyes. (d) Western blotting of the RPE-choroidal tissues at day 3 and subsequent densitometry demonstrated that the amount of VEGF protein induced by injury was significantly decreased by TSG-6 treatment. Data are shown as mean band density normalized relative to β-actin + SD, and represent three independent experiments, each with six rats per group. *p < 0.05, **p < 0.01.
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f1: TSG-6 reduces CNV development and VEGF production in the choroid after laser injury.After laser injury to Bruch’s membrane, either recombinant TSG-6 or PBS was injected into the vitreous cavity of rat eyes. At 1, 3, 7, and 14 days after injury, the whole RPE-choroids and retinas were separated and subjected to assays. (a) Representative photographs of the lectin staining of the whole RPE-choroid-scleral flat mounts at day 7 showed that CNV was induced by laser injury, and TSG-6 treatment reduced CNV development. Original magnification x 200. Scale bar: 100 μm. (b) Bar graphs showed quantification of the CNV size in TSG-6 and PBS-treated eyes. The size of CNV was significantly smaller in the TSG-6-treated rats than in the PBS-treated controls. Data are presented as mean + SEM from three independent experiments (each with six rats per group). The SEM represents the standard error of the mean values per animal (n = 18). (c) Real-time RT PCR showed that the mRNA level of VEGF highly increased in the RPE-choroid at day 1 after injury, and decreased to normal over 14 days. The level of VEGF transcript in the RPE-choroid was significantly lower in TSG-6-treated eyes than in PBS-treated controls at days 1, 3, and 7. However, the expression of VEGF was not either increased in the retina by laser or affected by TSG-6 treatment. Data represent mean ±/+ SD from three independent experiments, each with six rats per group. RQ means a ratio of mRNA levels relative to those in normal eyes. (d) Western blotting of the RPE-choroidal tissues at day 3 and subsequent densitometry demonstrated that the amount of VEGF protein induced by injury was significantly decreased by TSG-6 treatment. Data are shown as mean band density normalized relative to β-actin + SD, and represent three independent experiments, each with six rats per group. *p < 0.05, **p < 0.01.

Mentions: To evaluate clinical effects of TSG-6 on CNV development, we intravitreally injected either recombinant TSG-6 (400 ng/2 μl phosphate buffered solution; PBS) or the same volume of PBS in rats right after laser photocoagulation to Bruch’s membrane. At 1, 3, 7, and 14 days after laser injury, the whole RPE-choroidal and retinal tissues were separated and subjected to assays. We found that the area of CNV as analyzed by isolectin B4-staining in the RPE-choroid-sclera flat mounts was significantly smaller at day 7 in the TSG-6-treated rats, compared to the PBS-treated controls (Fig. 1a,b). Since the development of CNV depends on local production of VEGF1733, we further evaluated the mRNA and protein levels of VEGF in the retina and RPE-choroid. Real-time RT PCR showed that the expression of VEGF was highly up-regulated in the RPE-choroid at day 1 after injury, and decreased thereafter (Fig. 1c). TSG-6 treatment significantly reduced the level of VEGF transcript in the RPE-choroid at days 1, 3, and 7 (Fig. 1c). Similarly, western blotting of the RPE-choroidal tissue at day 3 showed that the amount of VEGF protein was markedly less in TSG-6-treated eyes than in PBS-treated eyes (Fig. 1d). By contrast, the expression of VEGF was not induced in the retina by laser photocoagulation (Fig. 1c).


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 reduces CNV development and VEGF production in the choroid after laser injury.After laser injury to Bruch’s membrane, either recombinant TSG-6 or PBS was injected into the vitreous cavity of rat eyes. At 1, 3, 7, and 14 days after injury, the whole RPE-choroids and retinas were separated and subjected to assays. (a) Representative photographs of the lectin staining of the whole RPE-choroid-scleral flat mounts at day 7 showed that CNV was induced by laser injury, and TSG-6 treatment reduced CNV development. Original magnification x 200. Scale bar: 100 μm. (b) Bar graphs showed quantification of the CNV size in TSG-6 and PBS-treated eyes. The size of CNV was significantly smaller in the TSG-6-treated rats than in the PBS-treated controls. Data are presented as mean + SEM from three independent experiments (each with six rats per group). The SEM represents the standard error of the mean values per animal (n = 18). (c) Real-time RT PCR showed that the mRNA level of VEGF highly increased in the RPE-choroid at day 1 after injury, and decreased to normal over 14 days. The level of VEGF transcript in the RPE-choroid was significantly lower in TSG-6-treated eyes than in PBS-treated controls at days 1, 3, and 7. However, the expression of VEGF was not either increased in the retina by laser or affected by TSG-6 treatment. Data represent mean ±/+ SD from three independent experiments, each with six rats per group. RQ means a ratio of mRNA levels relative to those in normal eyes. (d) Western blotting of the RPE-choroidal tissues at day 3 and subsequent densitometry demonstrated that the amount of VEGF protein induced by injury was significantly decreased by TSG-6 treatment. Data are shown as mean band density normalized relative to β-actin + SD, and represent three independent experiments, each with six rats per group. *p < 0.05, **p < 0.01.
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Related In: Results  -  Collection

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f1: TSG-6 reduces CNV development and VEGF production in the choroid after laser injury.After laser injury to Bruch’s membrane, either recombinant TSG-6 or PBS was injected into the vitreous cavity of rat eyes. At 1, 3, 7, and 14 days after injury, the whole RPE-choroids and retinas were separated and subjected to assays. (a) Representative photographs of the lectin staining of the whole RPE-choroid-scleral flat mounts at day 7 showed that CNV was induced by laser injury, and TSG-6 treatment reduced CNV development. Original magnification x 200. Scale bar: 100 μm. (b) Bar graphs showed quantification of the CNV size in TSG-6 and PBS-treated eyes. The size of CNV was significantly smaller in the TSG-6-treated rats than in the PBS-treated controls. Data are presented as mean + SEM from three independent experiments (each with six rats per group). The SEM represents the standard error of the mean values per animal (n = 18). (c) Real-time RT PCR showed that the mRNA level of VEGF highly increased in the RPE-choroid at day 1 after injury, and decreased to normal over 14 days. The level of VEGF transcript in the RPE-choroid was significantly lower in TSG-6-treated eyes than in PBS-treated controls at days 1, 3, and 7. However, the expression of VEGF was not either increased in the retina by laser or affected by TSG-6 treatment. Data represent mean ±/+ SD from three independent experiments, each with six rats per group. RQ means a ratio of mRNA levels relative to those in normal eyes. (d) Western blotting of the RPE-choroidal tissues at day 3 and subsequent densitometry demonstrated that the amount of VEGF protein induced by injury was significantly decreased by TSG-6 treatment. Data are shown as mean band density normalized relative to β-actin + SD, and represent three independent experiments, each with six rats per group. *p < 0.05, **p < 0.01.
Mentions: To evaluate clinical effects of TSG-6 on CNV development, we intravitreally injected either recombinant TSG-6 (400 ng/2 μl phosphate buffered solution; PBS) or the same volume of PBS in rats right after laser photocoagulation to Bruch’s membrane. At 1, 3, 7, and 14 days after laser injury, the whole RPE-choroidal and retinal tissues were separated and subjected to assays. We found that the area of CNV as analyzed by isolectin B4-staining in the RPE-choroid-sclera flat mounts was significantly smaller at day 7 in the TSG-6-treated rats, compared to the PBS-treated controls (Fig. 1a,b). Since the development of CNV depends on local production of VEGF1733, we further evaluated the mRNA and protein levels of VEGF in the retina and RPE-choroid. Real-time RT PCR showed that the expression of VEGF was highly up-regulated in the RPE-choroid at day 1 after injury, and decreased thereafter (Fig. 1c). TSG-6 treatment significantly reduced the level of VEGF transcript in the RPE-choroid at days 1, 3, and 7 (Fig. 1c). Similarly, western blotting of the RPE-choroidal tissue at day 3 showed that the amount of VEGF protein was markedly less in TSG-6-treated eyes than in PBS-treated eyes (Fig. 1d). By contrast, the expression of VEGF was not induced in the retina by laser photocoagulation (Fig. 1c).

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