The
isoform-specific role of human
apolipoprotein E (
apoE) has been assessed in a mouse model of ocular herpes. Female, age-matched transgenic mice knocked-in for the human allele
apoE3 or
apoE4 and their parent C57Bl/6 mice were inoculated corneally with HSV-1 strain KOS. Ocular HSV-1 pathogenesis was monitored through viral replication and
clinical progression of stromal opacity and neovascularization by
slit-lamp examination. Establishment of latency was determined by analysis of HSV-1
DNA (copy number) by specific real-time PCR in the cornea, trigeminal ganglia (TG), and brain. Representative groups of transgenic mice were sacrificed for the analysis of gene expression of
vascular endothelial growth factor (
VEGF) by reverse-transcription PCR, and
apoE expression by Western blot analysis. At 6days post-
infection (P.I.), the ocular infectious HSV-1 titer was significantly higher (p<0.05) in
apoE4 mice compared with
apoE3 and C57Bl/6 mice.
Corneal neovascularization in
apoE4 mice was significantly higher (p<0.05) than
apoE3 and C57Bl/6 mice. The onset of
corneal opacity in
apoE4 mice was accelerated during days 9-11 P.I.; however, no significant difference in severity was seen on P.I. days 15 and beyond. At 28 days P.I., infected mice of all genotypes had no significant differences in copy numbers (range 0-15) of HSV-1
DNA in their corneas, indicating that HSV-1
DNA copy numbers in cornea are independent of
apoE isoform regulation. At 28 days P.I., both
apoE4 and C57Bl/6 mice had a significantly higher (p=0.001) number of copies of HSV-1
DNA in TG compared with
apoE3.
ApoE4 mice also had significantly higher (p=0.001) copies of HSV-1
DNA in their TGs compared with C57Bl/6 mice. In brain, both
apoE4 and C57Bl/6 mice had significantly higher numbers (p<or=0.03) of copies of HSV-1
DNA compared with
apoE3 mice. However, the number of HSV-1
DNA copies in the brain of C57Bl/6 mice was not significantly different than that of
apoE4 (p=0.1). Comparative molecular analysis between
apoE3 and
apoE4 mice on selected days between 7 and 28 P.I., inclusive, revealed that the corneas of
apoE4 mice expressed
VEGF. None of the corneas in the
apoE3 mice expressed
VEGF during this time. Western blot analysis showed proteolytic cleavage of the
apoE protein in the corneas of the
apoE4 mice. Through days 14-28 P.I., a approximately 29 kDa C-terminal truncated
apoE fragment was present in the corneas of
apoE4 mice, but not in
apoE3 mice.
ApoE4 is a risk factor for ocular herpes, in part, through increased replication of virus in the eye, an earlier onset in clinical opacity, significantly higher neovascularization, and increased HSV-1
DNA load in TG and brain than that of
apoE3. Increased pathogenesis of ocular herpes in
apoE4 mice was also mediated, in part through up-regulated expression of
VEGF and
apoE proteolysis in the cornea. This is the first report linking a human gene,
apoE4, as a risk factor for ocular herpes pathogenesis in a transgenic mouse model.