Maternal administration of anti-angiogenic agents, TNP-470 and Angiostatin4.5, induces fetal microphthalmia

Abstract

Purpose: Agents specifically targeting the vasculature as a mode of therapy are finding increasing use in the clinic, primarily in the treatment of colon cancer (Avastin) and age-related macular degeneration (Lucentis). We have previously shown that maternal administration of angiogenic inhibitors (TNP-470 [O-[chloroacetyl-carbamoyl]fumagillol, initially called AGM-1470], the first angiogenic inhibitor to undergo clinical trials, and Angiostatin(4.5), currently in phase I-III clinical trials) cause fetal growth restriction and/or placental abnormalities. During a rapid growth phase of ocular development in the mouse (embryonic days 12 to 19 [E12-E19]), the placenta mediates the metabolic requirements of the fetus and consequently may impact upon the growth of the highly oxygen sensitive fetal eye.

Methods: We injected pregnant dams (between E10.5 - E18.5) with anti-angiogenic agents, which caused either a placental insufficiency type of IUGR (intrauterine growth restriction; i.e., TNP-470) or frank placental pathology (Angiostatin(4.5) [AS(4.5)]), and assessed changes in absolute ocular dimensions, tissue types, and vascular profiles using stereological techniques.

Results: The experiments showed that ocular volumes were significantly reduced in fetal mice where dams were treated with either TNP-470 or AS(4.5). Furthermore, TNP-470 specifically caused a reduction in hyaloid blood vessel length and volume, the only intraocular vascular circulation in fetal mice.

Conclusions: These experiments support the hypothesis that the angiogenic inhibitors (specifically TNP-470 and AS(4.5)) induce microphthalmia either indirectly by their known effects on placental morphology (and/or function) or directly via altering microvascular growth in the fetus. These results also warrant further investigation of a new experimental paradigm linking placental pathology-related fetal growth restriction and microphthalmia.

Figure 1

Hyaloid blood vessel dimensions from E18.5 mice treated with vehicle-only, TNP-470, or AS_4.5 solution. The graphs show ocular volume (A) where the asterisk indicates p<0.04, vascular cross sectional area (B) where the asterisk indicates p<0.01, vascular length (C) where the asterisk indicates p<0.001, and vascular volume (D) where the asterisk indicates p<0.001 in vehicle-only (control), TNP-470-treated, and AS_4.5-treated animals. Statistical comparisons between groups were performed using t-test for equality of means.

Figure 2

Photomicrographs of the eye and associated structures from E18.5 mice treated with vehicle-only, TNP-470, or AS_4.5. Photomicrographs of representative E18.5 murine eyes stained with toluidine blue are as follows: vehicle-only treated (A), TNP-470-treated eyes (B), and AS_4.5-treated eyes (C). The ocular cross-sectional area is clearly smaller in the TNP-470- and AS_4.5-treated groups when compared with controls (compare with Figure 1). In the images, the scale bar=500 μm.

Figure 3

Photomicrographs of the lens and hyaloid vasculature in eyes from E18.5 mice treated with either vehicle-only, TNP-470, or AS_4.5 solution. Low power micrographs (A-C) and matching high power detail (D-F) of the lens and hyaloid vasculature in eyes from E18.5 mice stained with toluidine blue are displayed. Dams were treated with either vehicle-only (control: A,D), TNP-470 (B,E), or AS_4.5 (C,F)_. In high power light micrographs (D-F), the hyaloid vessels are clearly visible on the lens surface (arrows) in addition to hyalocytes (arrowheads). Scale bars on A-C=100 µm and on D-F=50 µm.