FIG. 3.

Lens-CM induces apoptosis of HUVE cells. (A) HUVE cells were cultured in M199 containing 5% FBS with various organ-CM at different stages (days after birth) or BMP4 (50 ng/ml). After 24 h of culture, TUNEL-positive cells were counted. Data are shown as the mean percent apoptosis ± standard error from several independent experiments (upper panel). Representative fields of HUVE cells cultured in M199 with (b and d) or without (a and c) Lens-CM from day 8 rats (8L-CM), stained with TUNEL reagents (a and b) or Hoechst 33258 (c and d) are shown (lower panels). (B) Effect of Noggin and the BMP4-specific neutralizing antibody on Lens-CM-induced apoptosis of HUVE cells. Cells were cultured with or without Noggin (250 ng/ml), anti-BMP4 antibody (250 ng/ml) or BMP4 (50 ng/ml) under the conditions indicated for 24 h at 37°C. Excess amounts of BMP4 (500 ng/ml) were added when cultured with 8L-CM as indicated. Data are expressed as in panel A. (C) Effect of VEGF on Lens-CM-induced apoptosis of HUVE cells. Cells were cultured with or without VEGF (50 ng/ml) or BMP4 (50 ng/ml) for 24 h at 37°C under the conditions indicated. Data are expressed as in panel A. Abbreviations: Ab, anti-BMP4 antibody; B, BMP4; Ig, normal mouse IgG; N, Noggin; V, VEGF.