Fig 1.
FTI-277 inhibits osteogenic differentiation and mineralization of VSMC.
Confluent VSMC were incubated in 10% FCS-DMEM containing βGP (5 mM) and DMSO (1:500) or FTI-277 (20 μM) for 8–10 days. Control cells were incubated in the absence of βGP. RNA was prepared and qPCR for (A) Runx2 (n = 11), (B) Msx2 (n = 11), (C) α-smooth muscle actin (αSMA) (n = 6), and (D) matrix Gla protein (MGP) (n = 12) performed. mRNA expression is shown relative to housekeeping genes, PP1A and RPL12. Data are shown as mean ± SEM and were analyzed using one-way ANOVA and Tukey post-hoc tests. *p<0.05 compared to βGP. (E,F) Confluent VSMC were incubated in 10% FCS-DMEM containing βGP (5 mM) and DMSO (1:500) or FTI-277 (1, 10, 20 μM) and stained with alizarin red after 10 days. (E) Representative phase contrast images; white scale bar = 500 μm. (F) Quantification of mineralization (mean ± SEM; n = 19, βGP; n = 9, βGP + 1 μM FTI-277; n = 15, βGP + 10 μM FTI-277; n = 13, βGP + 20 μM FTI-277). Data were normalized using log10 and analyzed using one-way ANOVA and Tukey post-hoc tests; *p<0.05 compared to βGP. (G,H) Confluent VSMCs were incubated in 10% FCS-DMEM containing βGP (5 mM) and DMSO (1:1000); FTI-277 (10 μM) was added on day 0, 2, 3, 4, 5, 6 or 7. Cells were stained with alizarin red after 8 days. (G) Representative phase contrast images, white scale bar = 500 μm. (H) Quantification of mineralization (mean ± SEM; n = 6). Data were normalized using log10 and analyzed using one-way ANOVA and Tukey post-hoc tests; *p<0.05 compared to βGP.
Fig 2.
FTI-277 induces Akt signaling in VSMC.
(A) VSMCs were incubated in 10% FCS-DMEM ± FTI-277 (10 μM) for 77 hours, in serum-free medium ± FTI-277 for 2 hours, and 10% FCS-DMEM for 5 minutes. Cell lysates were collected and analyzed for pAkt and total Akt by western blotting. (B) VSMCs were incubated in 10% FCS-DMEM containing DMSO (1:1000), or βGP (5 mM) ± FTI-277 (10 μM) for 10 days. Representative phase contrast images of alizarin red stained cells (white scale bar = 500 μm) and western blots of cell lysates for pAkt and total Akt are shown.
Fig 3.
FTI-277 induced Akt signaling is required for inhibition of VSMC mineralization.
VSMCs were incubated in 10% FCS-DMEM containing βGP (5 mM) and DMSO (1:1000), FTI-277 (20 μM), SH6 (10 μM), or FTI-277 plus SH6 for up to 9 days, and (A) analyzed for pAkt and total Akt by western blotting or (B,C) stained with alizarin red. (B) Representative phase contrast images of alizarin red-stained cells; white scale bar = 500 μm. (C) Quantification of mineralization (mean ± SEM; n = 4). Data were normalized using log10 and analyzed using one-way ANOVA and Tukey post-hoc tests; * p<0.05.
Fig 4.
Over-expression of constitutively activated p110 sub-unit of PI3K inhibits mineralization.
Constitutively active p110 sub-unit of PI3K (p110) or empty virus (Empty) were over-expressed in VSMC using adenovirus and cells were incubated in 10% FCS-DMEM containing βGP (5 mM) for 9 days. Controls were incubated without BGP. (A) Representative western blots of cell lysates for pAkt and total Akt. (B) Representative phase contrast images of alizarin red-stained cells (bar = 500 μm). (C) Quantification of mineralization (mean ± SEM; n = 6). Data were normalized using log10 and analyzed using one-way ANOVA and Tukey post-hoc tests; *p<0.05 compared to βGP. (D) qPCR for Runx2 and matrix Gla protein (MGP) was performed. Relative mRNA expression of Runx2 and MGP are shown as fold-change to cells treated with empty virus and βGP (mean ± SEM; n = 6). Data were analyzed using one-way ANOVA and Tukey post-hoc tests; *p<0.05.
Fig 5.
FTI-277 inhibits phosphate-induced apoptosis.
A) Human VSMCs were incubated in serum-free medium containing elevated phosphate (2.6 mM) ± FTI-277 (10 μM) or vehicle control. Cell lysates were collected 2 and 4 hours later and analysed by western blotting. (B) Human VSMCs incubated in serum-free medium containing phosphate (2.6 mM) ± FTI-277 (10 μM) ± SH6 (10 μM) for 12 hours were fixed and stained with DAPI. Apoptotic cells are expressed as a percentage of total cells (mean ± SEM); >750 cells were counted per variable. Data were analyzed by one-way ANOVA and Tukey post-hoc tests; *p<0.05.
Fig 6.
FTI-277 inhibits phosphate-induced mineralization of aortic rings from uraemic rats.
Aortic rings from uraemic rats in end stage renal failure were incubated in control medium (Con; serum-free DMEM), phosphate medium (Pi; serum-free DMEM + 3.3 mM phosphate, 3.75 U/ml alkaline phosphatase), or phosphate medium + FTI-277 (10 μM) for 10 days. (A) Representative alizarin red stained sections of aortic rings from uraemic rats; bar = 500 μm (left panel) and 100 μm (right panel). (B) Quantification of mineralization in the aortic rings using the O-cresolphthalein complexone assay; n = 4 per group. Data are shown as mean ± SEM and were analyzed by one-way ANOVA and Tukey post-hoc tests; *p<0.05.