Doxycycline's effect on ocular angiogenesis: an in vivo analysis

Abstract

Purpose: To determine the in vivo effect of doxycycline on choroidal angiogenesis and pterygium growth by using a choroidal neovascular (CNV) murine model, a directed in vivo angiogenesis assay (DIVAA) and a pterygium murine model.

Design: Experimental study.

Participants: Three murine models were investigated with 4 mice minimum per group and 22 maximum per group.

Methods: Mice received water with or without doxycycline. For the CNV, the neovascular lesion volume was determined in choroid-retinal pigment epithelial flat mounts using confocal microscopy 7 days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells were implanted in the flanks of mice subcutaneously. After 11 days, neovascularization (NV) was quantified using spectrofluorometry after murine tail-vein injection of fluorescein isothiocyanate-labeled dextran. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells in the nasal subconjunctival space in athymic nude mice. Doxycycline was started on day 6 at 50 mg/kg per day; corneal lesions that resulted from the injections were compared at days 6 and 15.

Main outcome measures: The Student t-test was used to evaluate the data for the CNV and DIVAA models and histologic preparations were used to evaluate pterygia lesions.

Results: There was significantly less NV and lesion volume with doxycycline taken in drinking water versus plain water. With doxycycline treatment, the laser-induced CNV showed a maximal 66% decrease in choroidal blood vessel volume (P< or =0.008) and the DIVAA showed a 30% reduction of blood vessel growth and migration (P<0.004). Histologic preparations demonstrated that pterygium cell lesions regressed when mice were administered doxycycline for 9 days.

Conclusions: Doxycycline significantly inhibited angiogenesis in 3 murine models. The most dramatic effect was found in the CNV model followed by the pterygia epithelial cell DIVAA model. The anterior segment pterygium model also showed regression histologically. This suggests that doxycycline may be successful as an adjunctive treatment for CNV and pterygia in humans; clinical trials would be necessary to determine if there is a benefit.

Figure 1

Doxycycline (doxy) reduced pterygium epithelial cell and human dermal microvascular endothelial cell (HDMEC) migration. Boyden migration assays of pterygium cells (A) and HDMEC (B). Cells were incubated in serum-free medium with or without doxy at concentrations ranging from 20–500 micrograms/ml.

Figure 2

Doxycycline (doxy) inhibits choroidalneovascularization (CNV). Laser model of CNV in mice given plain water (left panel) or 50 mg/kg/day doxy added daily (rightpanel) for 7 days. Representative confocal microscope Z-series from neovessel projections (red) of choroid-retinal pigment epithelial (RPE) flat mounts at 7 days post-laser are shown. In green phalloidin outlines the RPE cytoskeleton. The arrows at the bottom left represent the X (green), Y (red) and Z (blue) axes.

Figure 3

Doxycycline (doxy) reduces neovascular lesion volume by 66%. Laser model of choroidalneovascularization (CNV) in mice given doxy at doses of 0.5 (n=15), 5 (n=12), and 50 mg/kg (n=22) added daily to drinking water for 7 days. Tap water (n=22) was used as control. Plot shows volume of CNV lesions after daily administrations of effectors as indicated in the X-axis. Each point corresponds to one CNV lesion. The horizontal bars correspond to median values. The hatched horizontal line is a projection of the median value of the control mice.

Figure 4

Pterygium epithelial cells are angiogenic in Directed In Vivo Angiogenesis Assay (DIVAA). Mice were implanted with silicone tubes containing human pterygium epithelial cells, Matrigel with phosphate buffered saline (negative control) or Matrigel with basic fibroblastic growth factor (positive control) for 11 days. n=6 replicates/group. Neovascularization in the angioreactors was quantified using a spectrofluorimeter.

Figure 5

Doxycycline (doxy) inhibits angiogenesis in the Directed In Vivo Angiogenesis Assay (DIVAA) model. DIVAA of mice implanted with silicone tubes containing human pterygium epithelial cells and given plain water or doxy for 11 days. n=7 for plain water, n=7 for 30 mg/kg/day doxy, and n=6 for 5 mg/kg/day doxy. Neovascularization in the angioreactors was quantified using a spectrofluorimeter.

Figure 6

An experimental nude mouse model of human pterygium epithelial cells. Photograph of mice six days after nasal para-limbalsubconjunctival injection of human pterygium epithelial cells. Eye (left panel) has noticeable lesion, while eye (right panel) has little if any pterygium cell involvement.

Figure 7

Histology of pterygium mouse model shows features of human pterygium pathogenesis, with migrating cells and neovascularization (neo-vessels). Top panel: anterior segment of mouse six days after nasal para-limbalsubconjunctival injection of human pterygium epithelial cells. Bottom left panel: cornea of mouse six days after nasal para-limbalsubconjunctival injection of Matrigel only. Bottom right panel: cornea of mouse six days after nasal para-limbalsubconjunctival injection of human pterygium epithelial cells. Magnification= 10× top panel; 40× bottom panels.

Figure 8

Histology of anterior segment of human pterygium (upper panels) and normal human conjunctiva and cornea (lower panels).

Figure 9

Doxycycline (doxy) causes regression of experimental pterygium epithelial cell lesions in a nude mouse model. Upper panels show mice 6 days post injection of pterygium cells; doxy (50 mg/kg/day) was started on this day in drinking water. Bottom panels show mice 15 days post injection of pterygium cells and after receiving doxycycline for 9 days. Mice receiving plain water did not show regression of lesions (data not shown).

Figure 10

Doxycycline (doxy) inhibits pterygium cell-generated gelatinolytic activity in vitro. Zymogram of media from cultured human pterygium cells grown in the presence or absence of doxy. Three concentrations of doxy were used. No doxy: medium from cells not treated with doxy. MMP-9 was used as a standard.