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VEGF-A expression by HSV-1-infected cells drives corneal lymphangiogenesis.

Wuest TR, Carr DJ - J. Exp. Med. (2009)

Bottom Line: HSV-1-elicited lymphangiogenesis was strictly dependent on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands.Macrophages played no role in the induction of lymphangiogenesis and were not a detectable source of VEGF-A.Our results indicate that HSV-1 directly induces vascularization of the cornea through up-regulation of VEGF-A expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
Inflammatory lymphangiogenesis plays a crucial role in the development of inflammation and transplant rejection. The mechanisms of inflammatory lymphangiogenesis during bacterial infection, toll-like receptor ligand administration, and wound healing are well characterized and depend on ligands for the vascular endothelial grow factor receptor (VEGFR) 3 that are produced by infiltrating macrophages. But inflammatory lymphangiogenesis in nonlymphoid tissues during chronic viral infection is unstudied. Herpes simplex virus 1 (HSV-1) infection of the cornea is a leading cause of blindness and depends on aberrant host immune responses to antigen within the normally immunologically privileged cornea. We report that corneal HSV-1 infection drives lymphangiogenesis and that corneal lymphatics persist past the resolution of infection. The mechanism of HSV-1-induced lymphangiogenesis was distinct from the described mechanisms of inflammatory lymphangiogenesis. HSV-1-elicited lymphangiogenesis was strictly dependent on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands. Macrophages played no role in the induction of lymphangiogenesis and were not a detectable source of VEGF-A. Rather, using VEGF-A reporter transgenic mice, we have identified infected epithelial cells as the primary source of VEGF-A during HSV-1 infection. Our results indicate that HSV-1 directly induces vascularization of the cornea through up-regulation of VEGF-A expression.

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The dominant source of VEGF-A during HSV-1 infection is virus-infected cells. To determine the source of VEGF-A, pVEGF-GFPs were infected with HSV-1. For VEGF-A transcription, we used a reporter mouse line expressing GFP under the proximal VEGF-A promoter. Corneas were harvested at days 1, 3, and 5 PI with HSV-1. (A) GFP (green) was visualized using secondary detection along with HSV-1 antigen (red), shown here from a cornea at 24 h PI. Reporter expression was observed exclusively in HSV-1 antigen-positive cells. Although cells expressing GFP reporter were positive for HSV-1 antigen, in cells expressing the highest levels of GFP, HSV-1 antigen staining was predominantly perinuclear, possibly reflecting a particular stage in viral replication. GFP HI cells are highlighted by arrows. Images were acquired with a 400× objective. Data are representative of two experiments. n = 6. Bars, 50 µm. (B) High-objective (600×) image of GFP HI and LO expressing HSV-1 antigen-positive cells. Bars, 20 µm. (C) Although the vast majority of cells infected with HSV-1 through day 5 PI are corneal epithelial cells, stromal cells expressing HSV-1 antigen also expressed GFP reporter for VEGF-A. Bars, 20 µm.
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fig7: The dominant source of VEGF-A during HSV-1 infection is virus-infected cells. To determine the source of VEGF-A, pVEGF-GFPs were infected with HSV-1. For VEGF-A transcription, we used a reporter mouse line expressing GFP under the proximal VEGF-A promoter. Corneas were harvested at days 1, 3, and 5 PI with HSV-1. (A) GFP (green) was visualized using secondary detection along with HSV-1 antigen (red), shown here from a cornea at 24 h PI. Reporter expression was observed exclusively in HSV-1 antigen-positive cells. Although cells expressing GFP reporter were positive for HSV-1 antigen, in cells expressing the highest levels of GFP, HSV-1 antigen staining was predominantly perinuclear, possibly reflecting a particular stage in viral replication. GFP HI cells are highlighted by arrows. Images were acquired with a 400× objective. Data are representative of two experiments. n = 6. Bars, 50 µm. (B) High-objective (600×) image of GFP HI and LO expressing HSV-1 antigen-positive cells. Bars, 20 µm. (C) Although the vast majority of cells infected with HSV-1 through day 5 PI are corneal epithelial cells, stromal cells expressing HSV-1 antigen also expressed GFP reporter for VEGF-A. Bars, 20 µm.

Mentions: To clarify the cell source, pVEGF-A–GFP mice were infected with 105 HSV-1 and expression of GFP and HSV-1 antigen was examined. GFP reporter expression was localized exclusively to cells coexpressing HSV-1 antigen at day 1, 3, and 5 PI (Fig. 7 A, day 1 PI) and was primarily observed in corneal epithelial cells. However, an occasional HSV-1–infected stromal cell also expressed GFP VEGF-A reporter (Fig. 7 C). VEGF-A reporter expression could be categorized between either GFP HI or LO with differential HSV-1 antigen expression between the two groups (Fig. 7 B). In GFP HI cells, HSV-1 antigen expression was slightly lower than that observed in GFP LO cells, and HSV-1 antigen staining was primarily perinuclear in these cells. In contrast, GFP LO cells typically expressed high levels of HSV-1 antigen, which was diffuse throughout the cytoplasm. At this point, the delineation between high and low GFP-expressing cells is unclear and may be the result of differential stages within the cycle of viral replication. However, both GFP HI and LO cells coexpressed HSV-1 antigen and, thus, the sole source of observable reporter expression was HSV-1–infected cells. Therefore, the contribution from leukocytes or other uninfected cell populations to VEGF-A expression appear negligible.


VEGF-A expression by HSV-1-infected cells drives corneal lymphangiogenesis.

Wuest TR, Carr DJ - J. Exp. Med. (2009)

The dominant source of VEGF-A during HSV-1 infection is virus-infected cells. To determine the source of VEGF-A, pVEGF-GFPs were infected with HSV-1. For VEGF-A transcription, we used a reporter mouse line expressing GFP under the proximal VEGF-A promoter. Corneas were harvested at days 1, 3, and 5 PI with HSV-1. (A) GFP (green) was visualized using secondary detection along with HSV-1 antigen (red), shown here from a cornea at 24 h PI. Reporter expression was observed exclusively in HSV-1 antigen-positive cells. Although cells expressing GFP reporter were positive for HSV-1 antigen, in cells expressing the highest levels of GFP, HSV-1 antigen staining was predominantly perinuclear, possibly reflecting a particular stage in viral replication. GFP HI cells are highlighted by arrows. Images were acquired with a 400× objective. Data are representative of two experiments. n = 6. Bars, 50 µm. (B) High-objective (600×) image of GFP HI and LO expressing HSV-1 antigen-positive cells. Bars, 20 µm. (C) Although the vast majority of cells infected with HSV-1 through day 5 PI are corneal epithelial cells, stromal cells expressing HSV-1 antigen also expressed GFP reporter for VEGF-A. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC2812544&req=5

fig7: The dominant source of VEGF-A during HSV-1 infection is virus-infected cells. To determine the source of VEGF-A, pVEGF-GFPs were infected with HSV-1. For VEGF-A transcription, we used a reporter mouse line expressing GFP under the proximal VEGF-A promoter. Corneas were harvested at days 1, 3, and 5 PI with HSV-1. (A) GFP (green) was visualized using secondary detection along with HSV-1 antigen (red), shown here from a cornea at 24 h PI. Reporter expression was observed exclusively in HSV-1 antigen-positive cells. Although cells expressing GFP reporter were positive for HSV-1 antigen, in cells expressing the highest levels of GFP, HSV-1 antigen staining was predominantly perinuclear, possibly reflecting a particular stage in viral replication. GFP HI cells are highlighted by arrows. Images were acquired with a 400× objective. Data are representative of two experiments. n = 6. Bars, 50 µm. (B) High-objective (600×) image of GFP HI and LO expressing HSV-1 antigen-positive cells. Bars, 20 µm. (C) Although the vast majority of cells infected with HSV-1 through day 5 PI are corneal epithelial cells, stromal cells expressing HSV-1 antigen also expressed GFP reporter for VEGF-A. Bars, 20 µm.
Mentions: To clarify the cell source, pVEGF-A–GFP mice were infected with 105 HSV-1 and expression of GFP and HSV-1 antigen was examined. GFP reporter expression was localized exclusively to cells coexpressing HSV-1 antigen at day 1, 3, and 5 PI (Fig. 7 A, day 1 PI) and was primarily observed in corneal epithelial cells. However, an occasional HSV-1–infected stromal cell also expressed GFP VEGF-A reporter (Fig. 7 C). VEGF-A reporter expression could be categorized between either GFP HI or LO with differential HSV-1 antigen expression between the two groups (Fig. 7 B). In GFP HI cells, HSV-1 antigen expression was slightly lower than that observed in GFP LO cells, and HSV-1 antigen staining was primarily perinuclear in these cells. In contrast, GFP LO cells typically expressed high levels of HSV-1 antigen, which was diffuse throughout the cytoplasm. At this point, the delineation between high and low GFP-expressing cells is unclear and may be the result of differential stages within the cycle of viral replication. However, both GFP HI and LO cells coexpressed HSV-1 antigen and, thus, the sole source of observable reporter expression was HSV-1–infected cells. Therefore, the contribution from leukocytes or other uninfected cell populations to VEGF-A expression appear negligible.

Bottom Line: HSV-1-elicited lymphangiogenesis was strictly dependent on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands.Macrophages played no role in the induction of lymphangiogenesis and were not a detectable source of VEGF-A.Our results indicate that HSV-1 directly induces vascularization of the cornea through up-regulation of VEGF-A expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
Inflammatory lymphangiogenesis plays a crucial role in the development of inflammation and transplant rejection. The mechanisms of inflammatory lymphangiogenesis during bacterial infection, toll-like receptor ligand administration, and wound healing are well characterized and depend on ligands for the vascular endothelial grow factor receptor (VEGFR) 3 that are produced by infiltrating macrophages. But inflammatory lymphangiogenesis in nonlymphoid tissues during chronic viral infection is unstudied. Herpes simplex virus 1 (HSV-1) infection of the cornea is a leading cause of blindness and depends on aberrant host immune responses to antigen within the normally immunologically privileged cornea. We report that corneal HSV-1 infection drives lymphangiogenesis and that corneal lymphatics persist past the resolution of infection. The mechanism of HSV-1-induced lymphangiogenesis was distinct from the described mechanisms of inflammatory lymphangiogenesis. HSV-1-elicited lymphangiogenesis was strictly dependent on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands. Macrophages played no role in the induction of lymphangiogenesis and were not a detectable source of VEGF-A. Rather, using VEGF-A reporter transgenic mice, we have identified infected epithelial cells as the primary source of VEGF-A during HSV-1 infection. Our results indicate that HSV-1 directly induces vascularization of the cornea through up-regulation of VEGF-A expression.

Show MeSH
Related in: MedlinePlus