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Robo 4 Counteracts Angiogenesis in Herpetic Stromal Keratitis.

Gimenez F, Mulik S, Veiga-Parga T, Bhela S, Rouse BT - PLoS ONE (2015)

Bottom Line: However, in some circumstances, damage to the eye can result in neovascularization that impairs vision.This outcome can occur when herpes simplex virus type 1 (HSV-1) causes the immunoinflammatory lesion stromal keratitis (SK).Our results indicate that sR4 could represent a useful therapeutic tool to counteract corneal angiogenesis and help control the severity of SK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Avenue, Knoxville, TN, 37996, United States of America.

ABSTRACT
The cornea is a complex tissue that must preserve its transparency to maintain optimal vision. However, in some circumstances, damage to the eye can result in neovascularization that impairs vision. This outcome can occur when herpes simplex virus type 1 (HSV-1) causes the immunoinflammatory lesion stromal keratitis (SK). Potentially useful measures to control the severity of SK are to target angiogenesis which with herpetic SK invariably involves VEGF. One such way to control angiogenesis involves the endothelial receptor Robo4 (R4), which upon interaction with another protein activates an antiangiogenic pathway that counteracts VEGF downstream signaling. In this study we show that mice unable to produce R4 because of gene knockout developed significantly higher angiogenesis after HSV-1 ocular infection than did infected wild type (WT) controls. Moreover, providing additional soluble R4 (sR4) protein by subconjunctival administration to R4 KO HSV-1 infected mice substantially rescued the WT phenotype. Finally, administration of sR4 to WT HSV-1 infected mice diminished the extent of corneal angiogenesis compared to WT control animals. Our results indicate that sR4 could represent a useful therapeutic tool to counteract corneal angiogenesis and help control the severity of SK.

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Robo4 deficient mice present more inflammation and vascularization.Robo4 knockout (R4 KO) and WT mice were infected with HSV-1 RE and at day 15 pi. corneas were collected and pooled for analysis by flow cytometry or Q-RT-PCR. The frequency and total cell number per cornea for (A) endothelial cells (CD31+) gated on total CD45- cells infiltrate, (B) CD4+ T cells (CD4+) (gated on total CD45+ cells infiltrate) and (C) neutrophils (Ly6G+ CD11b+ gated on total CD45+ cells infiltrate) show significant increase in R4 KO mice. Data are a combination of 3 independent experiments and show mean values ± SEM (n = 7 and each sample is representative of 2 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test. (E) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between WT and Robo4 KO mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from two different independent experiments (n = 3 and each sample is representative of 5 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test.
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pone.0141925.g002: Robo4 deficient mice present more inflammation and vascularization.Robo4 knockout (R4 KO) and WT mice were infected with HSV-1 RE and at day 15 pi. corneas were collected and pooled for analysis by flow cytometry or Q-RT-PCR. The frequency and total cell number per cornea for (A) endothelial cells (CD31+) gated on total CD45- cells infiltrate, (B) CD4+ T cells (CD4+) (gated on total CD45+ cells infiltrate) and (C) neutrophils (Ly6G+ CD11b+ gated on total CD45+ cells infiltrate) show significant increase in R4 KO mice. Data are a combination of 3 independent experiments and show mean values ± SEM (n = 7 and each sample is representative of 2 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test. (E) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between WT and Robo4 KO mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from two different independent experiments (n = 3 and each sample is representative of 5 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test.

Mentions: To evaluate the role of R4 in HSV-1 induced SK, the outcome of infection was compared over a 15 day time period in ocularly infected R4 KO and WT mice. While in R4 KO mice SK lesions started to be evident from day 9 pi. onward as in WT mice, angiogenesis and SK scores were significantly higher in R4 KO mice and peaked at day 15 pi. (p = 0.001, for both scores) (Fig 1A and 1B). Examination of histological sections at day 15 pi. also showed increased lesion severity in the R4 KO animals compared to WT sections (Fig 1C). In independent experiments of the same design, corneal tissues were collected from both groups on day 15 pi. and collagen digested to recover cells for FACS analysis. The numbers of CD31+ corneal endothelial cells (blood vessels) and SK lesion inducing CD4+ T cells increased around 2 fold for both cell types (p = 0.04 and p = 0.02, respectively) (Fig 2A and 2B). Additionally neutrophils were increased by approximately 3 fold (p = 0.02) (Fig 2C). Finally, at the same time point, pools of corneas from WT and R4 KO mice were collected for the measurement by Q-RT-PCR of proinflammatory cytokines. These included IL-1β, IL-6, and CXCL-1 all of which were significantly increased in R4 KO compared to WT mice (p = 0.01, p = 0.002 and p = 0.04, respectively) (Fig 2D). In conclusion, these results show that R4 plays a role to limit the extent of angiogenesis that follows HSV-1 infection.


Robo 4 Counteracts Angiogenesis in Herpetic Stromal Keratitis.

Gimenez F, Mulik S, Veiga-Parga T, Bhela S, Rouse BT - PLoS ONE (2015)

Robo4 deficient mice present more inflammation and vascularization.Robo4 knockout (R4 KO) and WT mice were infected with HSV-1 RE and at day 15 pi. corneas were collected and pooled for analysis by flow cytometry or Q-RT-PCR. The frequency and total cell number per cornea for (A) endothelial cells (CD31+) gated on total CD45- cells infiltrate, (B) CD4+ T cells (CD4+) (gated on total CD45+ cells infiltrate) and (C) neutrophils (Ly6G+ CD11b+ gated on total CD45+ cells infiltrate) show significant increase in R4 KO mice. Data are a combination of 3 independent experiments and show mean values ± SEM (n = 7 and each sample is representative of 2 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test. (E) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between WT and Robo4 KO mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from two different independent experiments (n = 3 and each sample is representative of 5 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test.
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pone.0141925.g002: Robo4 deficient mice present more inflammation and vascularization.Robo4 knockout (R4 KO) and WT mice were infected with HSV-1 RE and at day 15 pi. corneas were collected and pooled for analysis by flow cytometry or Q-RT-PCR. The frequency and total cell number per cornea for (A) endothelial cells (CD31+) gated on total CD45- cells infiltrate, (B) CD4+ T cells (CD4+) (gated on total CD45+ cells infiltrate) and (C) neutrophils (Ly6G+ CD11b+ gated on total CD45+ cells infiltrate) show significant increase in R4 KO mice. Data are a combination of 3 independent experiments and show mean values ± SEM (n = 7 and each sample is representative of 2 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test. (E) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between WT and Robo4 KO mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from two different independent experiments (n = 3 and each sample is representative of 5 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test.
Mentions: To evaluate the role of R4 in HSV-1 induced SK, the outcome of infection was compared over a 15 day time period in ocularly infected R4 KO and WT mice. While in R4 KO mice SK lesions started to be evident from day 9 pi. onward as in WT mice, angiogenesis and SK scores were significantly higher in R4 KO mice and peaked at day 15 pi. (p = 0.001, for both scores) (Fig 1A and 1B). Examination of histological sections at day 15 pi. also showed increased lesion severity in the R4 KO animals compared to WT sections (Fig 1C). In independent experiments of the same design, corneal tissues were collected from both groups on day 15 pi. and collagen digested to recover cells for FACS analysis. The numbers of CD31+ corneal endothelial cells (blood vessels) and SK lesion inducing CD4+ T cells increased around 2 fold for both cell types (p = 0.04 and p = 0.02, respectively) (Fig 2A and 2B). Additionally neutrophils were increased by approximately 3 fold (p = 0.02) (Fig 2C). Finally, at the same time point, pools of corneas from WT and R4 KO mice were collected for the measurement by Q-RT-PCR of proinflammatory cytokines. These included IL-1β, IL-6, and CXCL-1 all of which were significantly increased in R4 KO compared to WT mice (p = 0.01, p = 0.002 and p = 0.04, respectively) (Fig 2D). In conclusion, these results show that R4 plays a role to limit the extent of angiogenesis that follows HSV-1 infection.

Bottom Line: However, in some circumstances, damage to the eye can result in neovascularization that impairs vision.This outcome can occur when herpes simplex virus type 1 (HSV-1) causes the immunoinflammatory lesion stromal keratitis (SK).Our results indicate that sR4 could represent a useful therapeutic tool to counteract corneal angiogenesis and help control the severity of SK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Avenue, Knoxville, TN, 37996, United States of America.

ABSTRACT
The cornea is a complex tissue that must preserve its transparency to maintain optimal vision. However, in some circumstances, damage to the eye can result in neovascularization that impairs vision. This outcome can occur when herpes simplex virus type 1 (HSV-1) causes the immunoinflammatory lesion stromal keratitis (SK). Potentially useful measures to control the severity of SK are to target angiogenesis which with herpetic SK invariably involves VEGF. One such way to control angiogenesis involves the endothelial receptor Robo4 (R4), which upon interaction with another protein activates an antiangiogenic pathway that counteracts VEGF downstream signaling. In this study we show that mice unable to produce R4 because of gene knockout developed significantly higher angiogenesis after HSV-1 ocular infection than did infected wild type (WT) controls. Moreover, providing additional soluble R4 (sR4) protein by subconjunctival administration to R4 KO HSV-1 infected mice substantially rescued the WT phenotype. Finally, administration of sR4 to WT HSV-1 infected mice diminished the extent of corneal angiogenesis compared to WT control animals. Our results indicate that sR4 could represent a useful therapeutic tool to counteract corneal angiogenesis and help control the severity of SK.

Show MeSH
Related in: MedlinePlus