Relative resistance to Mammalian target of rapamycin inhibition in vascular smooth muscle cells of diabetic donors

Abstract

Background: Diabetes mellitus is associated with an increased risk of cardiovascular disease. Intimal thickening, a component of cardiovascular disease, entails the proliferation and migration of vascular smooth muscle cells (VSMCs). Inhibition of the mammalian target of rapamycin (mTOR) blocks VSMC proliferation, in part through an increase in the cyclin-dependent kinase inhibitor, p27(Kip1). The use of mTOR inhibitors, such as rapamycin, is effective clinically in inhibiting intimal thickening. This efficacy is reduced in diabetic subjects, however, suggesting a change in the role of the mTOR pathway in intimal thickening under diabetic conditions.

Methods: To examine whether diabetes induced changes in the role of mTOR in VSMC proliferation, we compared the response to rapamycin of human coronary artery VSMCs from diabetic (DM-huCASMC [human coronary artery smooth muscle cell]) and nondiabetic (ND-huCASMC) subjects.

Results: The DM-huCASMCs exhibited a relative resistance to rapamycin's inhibition of proliferation. Activation of the mTOR effector p70(S6kinase) was inhibited in rapamycin-treated DM-huCASMCs as in ND-huCASMCs. While ND-huCASMCs exhibited the normal increase in p27(Kip1) in response to rapamycin treatment, the DM-huCASMCs did not. Additionally, activation of the extracellular signal response kinase pathway was increased in the DM-huCASMCs, suggesting a potential pathway mediating the mTOR-independent decrease in p27(Kip1).

Conclusion: We conclude that diabetes is accompanied by a relative resistance to the effects of mTOR inhibition on VSMC proliferation through a loss of mTOR's effects on p27(Kip1) levels. These data provide insight into the effects of insulin resistance on the role of mTOR in regulating intimal thickening.

Keywords: Coronary artery disease; TOR serine-threonine kinases; cyclin-dependent kinase inhibitor p27; vascular smooth muscle.

Figure 1.

Rapamycin dose-response curves for proliferation of human coronary artery smooth muscle cells (huCASMCs) from nondiabetic (ND-huCASMC) and diabetic (DM-huCASMC) patients.

Figure 2.

Representative Western blots (WB) of (A) p70^S6kinase and (B) p27^Kip1 in response to incubation with increasing doses of rapamycin. Human coronary artery smooth muscle cells (huCASMCs) in diabetic (DM-huCASMC) and nondiabetic (ND-huCASMC) patients were serum starved overnight (U) and then stimulated with smooth muscle growth medium for 1 hour (p70^S6kinase) or overnight (p27^Kip1). Phosphorylated p70^S6kinase appears as the slower migrating band labeled p-p70, and unphosphorylated p70^S6kinase appears as the faster migrating band labeled p70. β-actin is presented as a loading control for the p27^Kip1 blots. Note: The lower band in the DM-huCASMC p27^Kip1 blot is a nonspecific band.

Figure 3.

Representative Western blots (WB) of (A) phosporylated and total extracellular signal response kinases 1/2 (ERK1/2) and (B) Akt in response to incubation with increasing doses of insulin. Human coronary artery smooth muscle cells (huCASMCs) were serum starved overnight and then stimulated with smooth muscle basal media supplemented with insulin for 10 minutes. Ser473, serine 473; Thr202, threonine 202; Ty204, tyrosine 204.