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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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Provision of sR4 reduces corneal inflammation and angiogenesis.WT mice were infected with HSV-1 RE and treated with sR4 of vehicle (PBS) from day 2 pi. to 14 pi. 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 decrease in sR4 treated mice compared to WT 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. (D) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between sR4 treated and control mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from three different independent experiments (n = 2 and each sample is representative of 6 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.g005: Provision of sR4 reduces corneal inflammation and angiogenesis.WT mice were infected with HSV-1 RE and treated with sR4 of vehicle (PBS) from day 2 pi. to 14 pi. 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 decrease in sR4 treated mice compared to WT 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. (D) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between sR4 treated and control mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from three different independent experiments (n = 2 and each sample is representative of 6 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test.

Mentions: In order to further evaluate the effect of sR4 in the progression of HSV-1-induced angiogenesis, WT animals were treated subconjunctivally with sR4 or vehicle starting on day 2 pi. and repeated daily until day 14 pi. (Fig 4A). As shown in Fig 4B, sR4 treatment caused reduced levels of angiogenesis and SK development with the maximum reduction observed at the higher dose used for treatment (SK score control:4 vs SK score treated: 2–3, angiogenesis score control:14 vs angiogenesis score control: to 8–10). That the sR4 treatment was effective at reducing lesions was also evident in the pictures and histological sections (Fig 4C, 4D and 4E). The extent of vascularization and inflammatory ocular reaction were compared by sacrificing treated and control animals at day 15 pi., following collagenase digestion and recovering corneal cells for FACS analysis. As is evident, the number of CD31+ endothelial cells were reduced around 3 fold in animals treated with sR4 compared to the controls (p = 0.02) (Fig 5A). In addition CD4+, and neutrophils were reduced around 3 and 2.5 fold respectively, (p = 0.02 and p = 0.01) in treated compared with control mice (Fig 5B and 5C). Finally, pools of 6 corneas HSV-1 infected sR4 treated and non treated were collected at day 15 pi. and processed to prepare RNA. As is evident in Fig 6D, animals treated with sR4 showed reduced of IL-1β, IL-6 and CXCL-1 transcripts compared with infected controls (p = 0.02, p = 0.01 and p = 0.01, respectively). Thus, taken together our results demonstrate that sR4 is a useful protein to modulate the extent of angiogenesis as well as the consequent immunopathology 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)

Provision of sR4 reduces corneal inflammation and angiogenesis.WT mice were infected with HSV-1 RE and treated with sR4 of vehicle (PBS) from day 2 pi. to 14 pi. 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 decrease in sR4 treated mice compared to WT 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. (D) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between sR4 treated and control mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from three different independent experiments (n = 2 and each sample is representative of 6 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test.
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Related In: Results  -  Collection

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pone.0141925.g005: Provision of sR4 reduces corneal inflammation and angiogenesis.WT mice were infected with HSV-1 RE and treated with sR4 of vehicle (PBS) from day 2 pi. to 14 pi. 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 decrease in sR4 treated mice compared to WT 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. (D) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between sR4 treated and control mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from three different independent experiments (n = 2 and each sample is representative of 6 corneas). ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistical levels of significance were analyzed by t test.
Mentions: In order to further evaluate the effect of sR4 in the progression of HSV-1-induced angiogenesis, WT animals were treated subconjunctivally with sR4 or vehicle starting on day 2 pi. and repeated daily until day 14 pi. (Fig 4A). As shown in Fig 4B, sR4 treatment caused reduced levels of angiogenesis and SK development with the maximum reduction observed at the higher dose used for treatment (SK score control:4 vs SK score treated: 2–3, angiogenesis score control:14 vs angiogenesis score control: to 8–10). That the sR4 treatment was effective at reducing lesions was also evident in the pictures and histological sections (Fig 4C, 4D and 4E). The extent of vascularization and inflammatory ocular reaction were compared by sacrificing treated and control animals at day 15 pi., following collagenase digestion and recovering corneal cells for FACS analysis. As is evident, the number of CD31+ endothelial cells were reduced around 3 fold in animals treated with sR4 compared to the controls (p = 0.02) (Fig 5A). In addition CD4+, and neutrophils were reduced around 3 and 2.5 fold respectively, (p = 0.02 and p = 0.01) in treated compared with control mice (Fig 5B and 5C). Finally, pools of 6 corneas HSV-1 infected sR4 treated and non treated were collected at day 15 pi. and processed to prepare RNA. As is evident in Fig 6D, animals treated with sR4 showed reduced of IL-1β, IL-6 and CXCL-1 transcripts compared with infected controls (p = 0.02, p = 0.01 and p = 0.01, respectively). Thus, taken together our results demonstrate that sR4 is a useful protein to modulate the extent of angiogenesis as well as the consequent immunopathology 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