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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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Macrophages are not involved in HSV-1–induced lymphangiogenesis. (A) Mice expressing GFP under the proximal human VEGF-A promoter were infected with HSV-1 and examined for expression of GFP and the pan-leukocyte marker CD45. No colocalization between GFP reporter and CD45 was observed, despite the observation of numerous CD45 cells as well as GFP+ cells, indicating that leukocytes, including macrophages, are not an appreciable source of VEGF-A during HSV-1 infection. Shown is an image of a cornea at day 5 PI representative of two experiments. n = 6. The images were acquired with a 400× objective. Bars, 50 µm. (B) Macrophages were depleted by subconjunctival injection with either clodronate or control PBS containing liposomes. At day 5 PI, corneal F4/80+ macrophages were quantitated by flow cytometry in control and clodronate-treated groups. Clodronate treatment significantly reduced the number of corneal F480+ cells (*, P < 0.05). (C) Corneas were examined for expression of the lymphatic vessel antigen LYVE-1 and quantified as a measure of lymphatic vessel area. No significant differences in lymphatic vessel area or structure (not depicted) were observed between control and clodronate-treated groups. B and C are summaries of two experiments. Error bars represent the SEM based on the results of each cornea sample summarized for both experiments. n = 6. (D) Chimeric mice expressing GFP in bone marrow–derived cells were infected with HSV-1. Corneas were examined for expression of GFP (green) and LYVE-1 (red) to detect possible structural contributions from bone marrow–derived cells. However, no appreciable structural contribution was observed. Data are representative of two experiments. n = 8 animals. Bars, 100 µm.
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fig5: Macrophages are not involved in HSV-1–induced lymphangiogenesis. (A) Mice expressing GFP under the proximal human VEGF-A promoter were infected with HSV-1 and examined for expression of GFP and the pan-leukocyte marker CD45. No colocalization between GFP reporter and CD45 was observed, despite the observation of numerous CD45 cells as well as GFP+ cells, indicating that leukocytes, including macrophages, are not an appreciable source of VEGF-A during HSV-1 infection. Shown is an image of a cornea at day 5 PI representative of two experiments. n = 6. The images were acquired with a 400× objective. Bars, 50 µm. (B) Macrophages were depleted by subconjunctival injection with either clodronate or control PBS containing liposomes. At day 5 PI, corneal F4/80+ macrophages were quantitated by flow cytometry in control and clodronate-treated groups. Clodronate treatment significantly reduced the number of corneal F480+ cells (*, P < 0.05). (C) Corneas were examined for expression of the lymphatic vessel antigen LYVE-1 and quantified as a measure of lymphatic vessel area. No significant differences in lymphatic vessel area or structure (not depicted) were observed between control and clodronate-treated groups. B and C are summaries of two experiments. Error bars represent the SEM based on the results of each cornea sample summarized for both experiments. n = 6. (D) Chimeric mice expressing GFP in bone marrow–derived cells were infected with HSV-1. Corneas were examined for expression of GFP (green) and LYVE-1 (red) to detect possible structural contributions from bone marrow–derived cells. However, no appreciable structural contribution was observed. Data are representative of two experiments. n = 8 animals. Bars, 100 µm.

Mentions: As HSV-1–induced lymphangiogenesis is dependent on VEGF-A but not VEGF-C/D, we sought to determine if macrophages expressed VEGF-A during infection. Transgenic mice expressing GFP under the proximal VEGF-A promoter (pVEGF-A–GFP) were infected with HSV-1, harvested at days 1, 3, and 5 PI, and examined for colocalization of GFP with the pan-leukocyte marker CD45. Although both CD45 and GFP+ cells were present, expression of GFP by CD45+ cells was not observed (Fig. 5 A), indicating that leukocytes were not an appreciable source of VEGF-A during HSV-1 infection.


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

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

Macrophages are not involved in HSV-1–induced lymphangiogenesis. (A) Mice expressing GFP under the proximal human VEGF-A promoter were infected with HSV-1 and examined for expression of GFP and the pan-leukocyte marker CD45. No colocalization between GFP reporter and CD45 was observed, despite the observation of numerous CD45 cells as well as GFP+ cells, indicating that leukocytes, including macrophages, are not an appreciable source of VEGF-A during HSV-1 infection. Shown is an image of a cornea at day 5 PI representative of two experiments. n = 6. The images were acquired with a 400× objective. Bars, 50 µm. (B) Macrophages were depleted by subconjunctival injection with either clodronate or control PBS containing liposomes. At day 5 PI, corneal F4/80+ macrophages were quantitated by flow cytometry in control and clodronate-treated groups. Clodronate treatment significantly reduced the number of corneal F480+ cells (*, P < 0.05). (C) Corneas were examined for expression of the lymphatic vessel antigen LYVE-1 and quantified as a measure of lymphatic vessel area. No significant differences in lymphatic vessel area or structure (not depicted) were observed between control and clodronate-treated groups. B and C are summaries of two experiments. Error bars represent the SEM based on the results of each cornea sample summarized for both experiments. n = 6. (D) Chimeric mice expressing GFP in bone marrow–derived cells were infected with HSV-1. Corneas were examined for expression of GFP (green) and LYVE-1 (red) to detect possible structural contributions from bone marrow–derived cells. However, no appreciable structural contribution was observed. Data are representative of two experiments. n = 8 animals. Bars, 100 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Macrophages are not involved in HSV-1–induced lymphangiogenesis. (A) Mice expressing GFP under the proximal human VEGF-A promoter were infected with HSV-1 and examined for expression of GFP and the pan-leukocyte marker CD45. No colocalization between GFP reporter and CD45 was observed, despite the observation of numerous CD45 cells as well as GFP+ cells, indicating that leukocytes, including macrophages, are not an appreciable source of VEGF-A during HSV-1 infection. Shown is an image of a cornea at day 5 PI representative of two experiments. n = 6. The images were acquired with a 400× objective. Bars, 50 µm. (B) Macrophages were depleted by subconjunctival injection with either clodronate or control PBS containing liposomes. At day 5 PI, corneal F4/80+ macrophages were quantitated by flow cytometry in control and clodronate-treated groups. Clodronate treatment significantly reduced the number of corneal F480+ cells (*, P < 0.05). (C) Corneas were examined for expression of the lymphatic vessel antigen LYVE-1 and quantified as a measure of lymphatic vessel area. No significant differences in lymphatic vessel area or structure (not depicted) were observed between control and clodronate-treated groups. B and C are summaries of two experiments. Error bars represent the SEM based on the results of each cornea sample summarized for both experiments. n = 6. (D) Chimeric mice expressing GFP in bone marrow–derived cells were infected with HSV-1. Corneas were examined for expression of GFP (green) and LYVE-1 (red) to detect possible structural contributions from bone marrow–derived cells. However, no appreciable structural contribution was observed. Data are representative of two experiments. n = 8 animals. Bars, 100 µm.
Mentions: As HSV-1–induced lymphangiogenesis is dependent on VEGF-A but not VEGF-C/D, we sought to determine if macrophages expressed VEGF-A during infection. Transgenic mice expressing GFP under the proximal VEGF-A promoter (pVEGF-A–GFP) were infected with HSV-1, harvested at days 1, 3, and 5 PI, and examined for colocalization of GFP with the pan-leukocyte marker CD45. Although both CD45 and GFP+ cells were present, expression of GFP by CD45+ cells was not observed (Fig. 5 A), indicating that leukocytes were not an appreciable source of VEGF-A during HSV-1 infection.

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