PPARγ Inhibits VSMC Proliferation and Migration via Attenuating Oxidative Stress through Upregulating UCP2

Abstract

Increasing evidence showed that abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are common event in the pathophysiology of many vascular diseases, including atherosclerosis and restenosis after angioplasty. Among the underlying mechanisms, oxidative stress is one of the principal contributors to the proliferation and migration of VSMCs. Oxidative stress occurs as a result of persistent production of reactive oxygen species (ROS). Recently, the protective effects of peroxisome proliferator-activated receptor γ (PPARγ) against oxidative stress/ROS in other cell types provide new insights to inhibit the suggests that PPARγ may regulate VSMCs function. However, it remains unclear whether activation of PPARγ can attenuate oxidative stress and further inhibit VSMC proliferation and migration. In this study, we therefore investigated the effect of PPARγ on inhibiting VSMC oxidative stress and the capability of proliferation and migration, and the potential role of mitochondrial uncoupling protein 2 (UCP2) in oxidative stress. It was found that platelet derived growth factor-BB (PDGF-BB) induced VSMC proliferation and migration as well as ROS production; PPARγ inhibited PDGF-BB-induced VSMC proliferation, migration and oxidative stress; PPARγ activation upregulated UCP2 expression in VSMCs; PPARγ inhibited PDGF-BB-induced ROS in VSMCs by upregulating UCP2 expression; PPARγ ameliorated injury-induced oxidative stress and intimal hyperplasia (IH) in UCP2-dependent manner. In conclusion, our study provides evidence that activation of PPARγ can attenuate ROS and VSMC proliferation and migration by upregulating UCP2 expression, and thus inhibit IH following carotid injury. These findings suggest PPARγ may represent a prospective target for the prevention and treatment of IH-associated vascular diseases.

Fig 1. PDGF-BB-stimulated-VSMC showed increased proliferation and migration as well as ROS.

VSMC from WT mice in basal conditions displayed low concentration of ROS production, PDGF-BB (20 μg/l) treatment significantly elevated ROS level (a). Proliferation and migration of VSMC showed significant increase in response to PDGF-BB compared with con group (b and c). NAC (10mmol/l) significantly reduced the intracellular ROS production and VSMC proliferation and migration induced by PDGF-BB. (*P<0.05 vs. con; #P<0.05 vs. PDGF). Con, wild-type VSMCs in basal conditions; PDGF, PDGF-BB, platelet derived growth factor-BB; NAC, N-acetylcysteine; ROS, reactive oxygen species.

Fig 2. PPARγ inhibited PDGF-BB-induced VSMC proliferation, migration and oxidative stress.

Cultured VSMCs were incubated with RSG (10μmol/L) for different times (0, 1, 6, 12 or 24 h). PPARγ expression increased in time-dependent manner, with an obvious effect at 6 h and the peak at 12 h that sustained after 24 h (a). (*P<0.05 vs. 0 h). RSG treatment for 12 h significantly counteracted ROS generation induced by PDGF-BB in VSMCs, while PPARγ inhibitor GW9662 diminished the effect of RSG (b). VSMCs treated with RSG showed reduced proliferation and PCNA expression, VSMC migration and MMP9 expression in response to PDGF-BB, which was reversed by GW9662 (c and d). (*P<0.05 vs. con; #P<0.05 vs. PDGF; ΔP<0.05 vs. PDGF+RSG). Con, wild-type VSMCs in basal conditions; PDGF, PDGF-BB, platelet derived growth factor-BB; ROS, reactive oxygen species; RSG, rosiglitazone; GW9662, PPARγ inhibitor; PCNA, proliferating cell nuclear antigen; MMP9, matrix metalloproteinase 9.

Fig 3. PPARγ activation upregulated UCP2 expression in VSMCs.

Cultured VSMCs were incubated with RSG (10 μmol/l) for different times (0, 1, 6, 12 or 24 h). The level of UCP2 expression increased in time-dependent manner with an obvious effect at 6 h and the peak at 12 h that sustained after 24 h (a). (*P<0.05 vs. 0 h). RSG (10 μmol/l) treatment significantly upregulated the UCP2 expression which was reversed by PPARγ inhibitor GW9662 (5 μmol/l) detected respectively by immunofluorescence (b, green-UCP2, blue-DAPI) and western blot (c). (*P<0.05 vs. con; #P<0.05 vs. RSG). Con, wild-type VSMCs in basal conditions; PDGF, PDGF-BB, platelet derived growth factor-BB; ROS, reactive oxygen species; UCP2, uncoupling protein 2; RSG, rosiglitazone.

Fig 4. PPARγ inhibited PDGF-BB-induced ROS in VSMCs by upregulating UCP2 expression.

UCP2 expression in cultured WT- and UCP2^-/- − VSMCs in response to PDGF-BB (20 μg/l) and RSG (10 μmol/l) was detected by western blot (a). Compared to WT-VSMCs, UCP2^-/- -VSMCs displayed much higher ROS level (b), enhanced proliferation (c) and migration (d) in response to PDGF-BB. The RSG treatment failed to impeded the PDGF-induced ROS production and VSMC proliferation and migration in UCP2^-/- -VSMCs (b-d). (*P<0.05 vs. con; #P<0.05 vs. PDGF; ΔP<0.05 vs. WT-VSMCs treated with PDGF and RSG). Con, wild-type VSMCs in basal conditions; ROS, reactive oxygen species; UCP2, uncoupling protein 2; RSG, rosiglitazone; UCP2^-/- VSMC, VSMCs from UCP2^-/- mice; WT-VSMC, VSMCs from wild-type mice.

Fig 5. PPARγ ameliorated injury-induced oxidative stress and intimal hyperplasia in UCP2-dependent manner.

RSG significantly increased UCP2 expression in injured arteries (In) from WT mice but not in UCP2^-/- mice (a). Carotid arteries ROS was detected by DHE staining (b). The increased ROS by wire injury was also reduced by RSG in WT mice but not in UCP2^-/- mice. Hematoxylin and eosin staining on cross-sections from representative injured carotid arteries are presented (c). Carotid wire injury induced IH with increased intima/media ratio in WT mice. RSG inhibited the wire-injury-induced IH in WT mice but not in UCP2^-/- mice. (*P<0.05 vs. sham; #P<0.05 vs. WT+In; ΔP<0.05 vs. WT+In+RSG). Sham, sham operation; WT, wild-type mice; In, carotid wire injury; ROS, reactive oxygen species; RSG, rosiglitazone; UCP2^-/-, UCP2-dificient mice.