Contribution of vascular endothelial growth factor in the neovascularization process during the pathogenesis of herpetic stromal keratitis

Abstract

This report analyzes the role of vascular endothelial growth factor (VEGF)-induced angiogenesis in the immunoinflammatory lesion stromal keratitis induced by ocular infection with herpes simplex virus (HSV). Our results show that infection with replication-competent, but not mutant, viruses results in the expression of VEGF mRNA and protein in the cornea. This a rapid event, with VEGF mRNA detectable by 12 h postinfection (p.i.) and proteins detectable by 24 h p.i. VEGF production occurred both in the virus-infected corneal epithelium and in the underlying stroma, in which viral antigens were undetectable. In the stroma, VEGF was produced by inflammatory cells; these initially were predominantly polymorphonuclear leukocytes (PMN), but at later time points both PMN and macrophage-like cells were VEGF producers. In the epithelium, the major site of VEGF-expressing cells in early infection, the infected cells themselves were usually negative for VEGF. Similarly, in vitro infection studies indicated that the cells which produced VEGF were not those which expressed virus. Attesting to the possible role of VEGF-induced angiogenesis in the pathogenesis of herpetic stromal keratitis were experiments showing that VEGF inhibition with mFlt(1-3)-immunoglobulin G diminished angiogenesis and the severity of lesions after HSV infection. These observations are the first to evaluate VEGF-induced angiogenesis in the pathogenesis of stromal keratitis. Our results indicate that the control of angiogenesis represents a useful adjunct to therapy of herpetic ocular disease, an important cause of human blindness.

FIG. 1

Angiogenesis and lesion scores at different times after HSV infection. BALB/c mice were infected on the slightly scarified corneal surface with HSV KOS or GFP-HSV (KOS background). Angiogenesis and HSK lesion scores were documented at different time points p.i. as described in Materials and Methods. (a) Correlation of angiogenesis and HSK lesion scores in the process of HSK. (b) GFP-HSV replicates on the infected corneal surface at day 1 p.i. The image was taken under a GFP filter by stereomicroscopy and an image system. Magnification, ×20. (c) Angiogenic sprouting was evident at day 1 p.i. as shown by the India ink perfusion technique. Magnification, ×50. (d) At day 8 p.i., neovascularization was around halfway toward the center of the cornea. (e) Intense neovascularization was seen in HSV-infected cornea at day 16 p.i. Magnification, ×20.

FIG. 2

Necessity for replicating virus to induce angiogenesis and HSK. BALB/c mice were infected (with wt KOS, UV-inactivated KOS [UV-KOS], ICP4^−/−, or ICP8^−/−) or only scratched on the corneal epithelium as a trauma control. Angiogenesis scores (a) and HSK lesion scores (b) were recorded as described in the text. Mild angiogenesis was observed at day 1 postinjury and then faded away in trauma control eyes (data not shown).

FIG. 3

Presence of VEGF in corneal lysates at different times after HSV infection. At different time points p.i., the infected (wt KOS [low, 5 × 10⁵ PFU; high, 5 × 10⁷ PFU], ICP4^−/−, or ICP8^−/−) and control (trauma or alkali) corneas (n = 4) were isolated and stored at −80°C until further use. The corneal lysates were assayed by ELISA for detection of mVEGF164. VEGF levels in the wt virus-infected corneas were correlated with the titers of the infected virus in the preclinical phase (P ≤ 0.05). VEGF in trauma control corneas could not be detected beyond 48 h. VEGF levels in UV-inactivated-HSV-infected corneas at all time points were similar to those in trauma control and ICP4^−/−- and ICP8^−/−-infected corneas (data not shown).

FIG. 4

Expression of corneal VEGF mRNA at various times after HSV infection. Mice were infected as described in the text. At different time points p.i., the eyes were enucleated and the corneas (n = 4) were isolated and stored at −80°C in Tri-reagent. Total RNA was extracted from the samples and reverse transcribed into cDNA. PCR was performed to detect the isoforms (VEGF120, -164, and -188) of VEGF in HSV-infected corneas. β-Actin served as the positive control and standard for semiquantitative RT-PCR.

FIG. 5

Demonstration of virus-infected and VEGF-expressing cells in vivo and in vitro. (a and b) Frozen sections of corneas 24 h p.i. with GFP-HSV. (a) Corneal epithelial cells infected with virus. (b) A different area of the same cornea stained for VEGF. The white arrow indicates a virus-infected cell lacking VEGF expression. The blue arrows indicate VEGF-expressing cells with no virus infection. (c and d) Thioglycolate-elicited PEC infected with GFP-HSV at an MOI of 2 for 18 h and stained with VEGF. Virus-infected cells (white arrows) and VEGF-expressing cells (blue arrows) are distinct. (c) Neutrophils; (d) macrophages.

FIG. 6

VEGF expression in corneal sections at different times after HSV infection. Eyes were collected at different times p.i., and frozen sections were processed for VEGF detection as described in Materials and Methods. (a) VEGF-positive corneal epithelial cells and a few infiltrating cells in the corneal stroma at day 1 p.i.; (b) absence of VEGF-positive cells in the epithelium and both negative and positive (arrow) cells in the stroma (day 8 p.i.); (c) VEGF-positive cells tend to gather at sites of the neovascularization (day 15 p.i.).

FIG. 7

Effect of VEGF inhibition on severity of HSK. Mice (n = 5) were treated every other day with mFlt(1–3)-IgG at 12.5 mg/kg starting 1 h prior to HSV-1 infection. Mouse IgG served as a control. (a and b) The HSK lesion scores (a) and angiogenesis scores (b) were determined as described in Materials and Methods. (c) Corneal images of control (left) and mFlt(1–3)-IgG-treated (right) mice at day 15 p.i. The experiments were done twice with similar results, and the results of one such experiment are shown.