BMP7v reduced VEGFR2 and FGFR1 expression in endothelial cells.

<p>(A) Whole cell protein extracts were isolated following 15 or 60 minute PBS or 100 ng/ml BMP7v treatment and subjected to Western blot analysis using antiserum directed against phospho-SMAD1,5,8 (pSMAD1,5,8), phospho-ERK1/2 (pERK1/2), ERK1/2, and β-actin as a loading control. Results shown are representative of three independent experiments. (B) ECFCs were treated with PBS or 2nM BMP7v or BMP4 in ADSC conditioned defined co-culture media for the times indicated. Whole cell protein extracts were isolated following treatment and subjected to Western blot analysis using antiserum directed against phospho-CRAF (pCRAF), phospho-MEK1/2 (pMEK1/2), phospho-ERK1/2 (pERK1/2), and β-actin as a loading control. (C) Whole cell protein extracts were isolated from ECFCs following 72 hours of PBS or 100 ng/ml BMP7v treatment and subjected to Western blot analysis using antiserum directed against VEGFR2, FGFR1, and β-actin as a loading control. Graph represents mean densitometry ± SEM from three independent experiments, and asterisks denote statistically significant (***, <i>p<</i>0.001) differences compared to PBS controls. (D) The ADSC/ECFC co-culture was unstimulated (basal) or stimulated with 10 ng/ml VEGF or bFGF for 96 hours prior to treatment with PBS or 100 ng/ml BMP7v for 72 hours prior to immunohistochemistry for VEGFR2 (VEGF) or FGFR1 (bFGF) (red) and Hoechst 33342 to stain all nuclei (blue). Representative images (5X magnification) are shown. Upon BMP7v treatment, mean staining intensity for VEGFR2 and FGFR1 was statistically decreased (***, <i>p<</i>0.001) compared to PBS controls.