Inhibition of murine placental and embryonic growth by the angiogenesis inhibitor TNP‐470
Authors: Rutland C. S. , Cooper S. , Mitchell C. A. 
Published: March 2003

Angiogenesis is critical for both placental and embryonic growth and development. TNP‐470 inhibits endothelial cell growth and migration in vitro and causes spontaneous abortion when a single dose of 30 mg/kg is administered at E1 or E7 (Klauber et al. Nature Medical3, 1997), however, its affect during the later stages of pregnancy is unknown.

To determine the bioactivity and endothelial specificity of TNP‐470 we performed proliferation and apoptosis assays on human umbilical vein endothelial cells (HUVEC) and a placental trophoblast cell line (SGHPL4) in vitro. TNP‐470 decreased proliferation in both the HUVEC (10 ng/mL − 100 µg/mL) and PL4 cells (100 µg/mL) in a dose dependent manner. No significant effect was seen on apoptosis.

To assess the affect of TNP‐470 in vivo, pregnant mice were injected with TNP‐470 at doses of 0, 3 and 30 mg/kg body weight every other day between E10.5 and E18.5. An hour before culling at E18.5, 1 µCi/g body weight tritiated thymidine was administered (IP), in order to assess the proportion of cells in the S‐phase of the cell cycle. The viability and numbers of embryos was not compromised, however, at a dose of 30 mg/kg TNP‐470 maternal weight was significantly decreased at both E17.5 (P < 0.0495) and E18.5 (P < 0.0335).

Placental and embryonic weights and dimensions were significantly reduced when TNP‐470 was administered (see Table), although morphometric studies showed that fractional proportions of each cell type comprising the placenta were unaffected. Preliminary studies of placentae from mice injected with 30 mg/kg TNP‐470 show a decrease in the numbers of labelled endothelial and trophoblast cells. Electron microscopic studies examining the effects of TNP‐470 on trophoblast invasion of the maternal blood space are in progress. All animal work was carried out with Home Office and Institutional approval and where necessary Schedule 1 methods were used for culling.

Control (n = 130)
3 mg/kg TNP‐470 (n = 78)
30 mg/kg TNP‐470 (n = 113)

Placenta weight (g)
 0.083 (0.022)
 0.083 (0.024) ns
 0.075 (0.014)*

Placenta depth (mm)
 2.05 (0.44)
 1.91 (0.52)*
 2.01 (0.38) ns

Placenta length (mm)
 8.19 (0.66)
 7.46 (0.68)**
 7.35 (1.46)**

Embryo weight (g)
 1.21 (0.10)
 0.99 (0.17)**
 0.97 (0.13)**

Embryo crown‐rump length (mm)
22.0 (1.4)
19.6 (1.7)**
19.8 (1.3)**

Embryo abdominal anteroposterior diameter (mm)
 7.8 (0.6)
 6.4 (0.5)**
 6.8 (0.7)**

Embryo abdominal transverse diameter (mm)
 6.6 (0.6)
 5.5 (0.7)**
 5.8 (0.7)**

All values expressed as mean (SD). ** = statistically different (P < 0.0001). * = statistically different (P < 0.05). ns = no statistically significant difference.

This study demonstrates that the effects of TNP‐470 are not endothelial cell specific and that placental endothelial and trophoblast proliferation is affected both in vitro and in vivo. Furthermore when TNP‐470 is administered at doses of 3 or 30 mg/kg in the latter half of murine pregnancy, the result is a highly reproducible model of intrauterine growth restriction.