Figure 1.
Resveratrol Induces Differentiation in HuVSMC.
A, HuVSMC treated with increasing doses of resveratrol for 24 hours prior to western blot assessment with primary antibodies as indicated. Bar graphs represent densitometric quantification. N = 5 experiments. Probability values are indicated above bars: * p<0.05, **p<0.01, ***p<0.001 versus control. B, Cell morphology after resveratrol treatment. HuVSMC treated with 0, 3, 10, 30 and 100 µM resveratrol. 40X magnification. Bar graphs represent mean area change plus standard error of mean. N = 6, Probability values are indicated above bars: **p<0.01, ***p<0.001 versus control. C, HuVSMC pretreated with 3 µM resveratrol for 24 hours contracted the collagen disc, compared to control. Bar graphs represent mean area change plus standard error of mean. N = 2 experiments, in triplicate. Probability values are indicated above bars: * p<0.05 versus control.
Figure 2.
SirT1 Induces Changes in Contractile Proteins Indicative of Differentiation.
A, HuVSMC treated with increasing concentrations of resveratrol for 24 hours before western blot assessment with primary antibodies as indicated. Bar graphs represent densitometric quantification. N = 9 experiments. Probability values are indicated above bars: * p<0.05, **p<0.01, ***p<0.001 versus control. B, HuVSMC were transfected with control small interfering (si) RNA or SirT1 siRNA for 48 hours and then assessed by western blot with primary antibodies as indicated. Bar graphs represent densitometric quantification. N = 2 experiments, in duplicate. Probability values are indicated above bars: * p<0.05, **p<0.01 versus control. C, HuVSMC were transfected with control or SirT1 plasmid for 48 hours and then assessed by western blot with primary antibodies as indicated. At higher exposures, endogenous SirT1 is present in this experiment (data not shown). Bar graphs represent densitometric quantification. N = 3 experiments. Probability values are indicated above bars: **p<0.01 versus control.
Figure 3.
Resveratrol Induced Differentiation in VSMC via SirT1-AKT Signaling.
A, HuVSMC treated with increasing concentrations of resveratrol for 24 hours before western blot assessment with primary antibodies as indicated. Bar graphs represent densitometric quantification. N = 9 experiments. Probability values are indicated above bars: * p<0.05 versus control. B, HuVSMC were treated with 20 µM API-2 one hour prior to resveratrol or control treatment. Twenty-four hours following resveratrol treatment, cells were collected and assessed by western blot with primary antibodies as indicated. Bar graphs represent densitometric quantification. N = 4 experiments. Probability values are indicated above bars: * p<0.05, **p<0.01 versus control. C, HuVSMC were transfected with control siRNA or SirT1 siRNA for 24 hours, prior to treatment with 3 µM resveratrol or control. Twenty-four hours after treatment, cells were collected and assessed by western blot with primary antibodies as indicated. Bar graphs represent densitometric quantification. N = 3 experiments, in duplicate. Probability values are indicated above bars: * p<0.05, **p<0.01, *** p<0.001 versus control or <p<0.01, <<p<0.001 versus control+resveratrol.
Figure 4.
AMPK Inhibits the mTORC1 Pathway to Induce Differentiation.
A, HuVSMC treated with increasing concentrations of resveratrol for 24 hours prior to western blot assessment with primary antibodies as indicated. Bar graphs represent densitometric quantification. Samples have been normalized to GAPDH and then fold change from normalized values have been graphed. N = 5 experiments. Probability values are indicated above bars: * p<0.05 versus control. B, HuVSMC treated with increasing concentrations of resveratrol for 24 hours prior to western blot assessment of primary antibodies as indicated. Bar graphs represent densitometric quantification. Samples have been normalized to GAPDH and then fold change from normalized values have been graphed. N = 3 experiments. Probability values are indicated above bars: * p<0.05, *** p<0.001 versus control. C, HuVSMC were transfected with control siRNA or AMPK α1 siRNA for 24 hours, prior to treatment with 30 µM resveratrol or control. Twenty-four hours after treatment, cells were collected and assessed by western blot with primary antibodies as indicated. Bar graphs represent densitometric quantification. N = 4 experiments. Probability values are indicated above bars: * p<0.05, **p<0.01, *** p<0.001 versus control or <p<0.01, <<p<0.001 versus control+resveratrol.
Figure 5.
Resveratrol-Induced Differentiation in Whole Tissue.
Tibial artery rings cultured for 10 = 3 experiments. Probability values are indicated above bars: *p<0.05, *** p<0.001, versus control.
Figure 6.
Model of Resveratrol Signaling in HuVSMC Differentiation.
Resveratrol stimulates multiple second messengers that lead to AKT activation and the induction of differentiation. Low dose resveratrol stimulates SirT1, which activates AKT to induce differentiation. High dose resveratrol stimulates AMPK, which inhibits the mTORC1 pathway relieving the S6K1 inhibition on IRS-1, allowing activation of AKT and the induction of differentiation. Ultimately, resveratrol induces a multi-signaling cascade that results in robust vascular smooth muscle differentiation.