Table 1.
Primer Sequence Used for qRT-PCR.
Table 2.
SBP, BW and vascular morphology changes in Ang II-infused rats.
Fig 1.
In vivo induction of KLF5 in response to Ang II and altered expression in response to rosiglitazone.
(A) KLF5 and cyclin D1 expression as measured by real-time RT-PCR in the thoracic aorta of Ang II-infused rats (150 ng/kg/min) with or without rosiglitazone. Values are expressed as fold induction compared with control group. (B) Immunohistochemical analysis of KLF5 and cyclin D1 expression in each group. Images are representative of 6 animals studied in each group. Scale bar = 50 μm. Quantification results are presented as gray scale levels and mean ± S.E.M. data of 24 measurements in 6 slides. (C) PPAR-γ mRNA expression was analyzed by real-time RT-PCR. (D) PPAR-γ activation was analyzed by DNA-binding assay. *P<0.05 vs. control; #P<0.05 vs. Ang II alone. Ros: rosiglitazone.
Fig 2.
Effects of PPAR-γ activation on cell proliferation and cell cycle in Ang II-stimulated vascular smooth muscle cells (VSMCs).
Growth-arrested VSMCs were switched to fresh medium containing 0.5% of serum when pretreated with or without PPAR-γ antagonist GW9662 (3 μM), BADGE (1 μM) for 30 min or PPAR-γ specific siRNA for 48 hrs prior to the addition of rosiglitazone (1, 5 and 10 μM) or PPAR-γ nature ligand 15-d-PGJ2 (1, 5 and 10 μM). Ang II (0.1 μM) was then added for 24 hrs. (A). CCK8 assays (values represent the mean ± S.E.M. (n = 12) were carried out to assess cell proliferation. Ros = rosiglitazone; 15d = 15-d-PGJ; Ang II = Angiotensin. *P<0.05 vs. control; #P<0.05 vs. Ang II; †P<0.05 vs. Ang II + Ros.
Fig 3.
PPAR-γ agonists inhibit KLF5 and cyclin D1 expression in Ang II-stimulated VSMCs.
Cells were pretreated with increasing concentrations of rosiglitazone (1, 5 and 10 μM) or 15-d-PGJ2 (1, 5 and 10 μM) for 1 hr and then stimulated with Ang II (0.1 μM) for 24 hr. (A) Real-time RT-PCR analysis of KLF5 mRNA expression in VSMCs. Results are expressed as fold increase over control group, and data are mean ± S.E.M. of 3 independent experiments. GAPDH served as an internal control. (B) Immunocytochemical staining of KLF5 staining in VSMCs. Scale bar: 100 mm. (C) Western blot analysis of KLF5 protein expression in VSMCs. Results are representative of 3 independent experiments. β-actin served as an internal control. Data are mean ± S.E.M. of 3 experiments. (D) Real-time RT-PCR analysis of cyclin D1 mRNA expression in VSMCs. Results are expressed as fold increase over control group, and data are mean ± S.E.M. of 3 independent experiments. GAPDH served as an internal control. (*P <0.05 vs. control; #P <0.05 vs. Ang II). Ros = rosiglitazone; 15d = 15-d-PGJ; Ang II = Angiotensin.
Fig 4.
Relationship between effect of rosiglitazone on Ang II-induced cell proliferation and KLF5 expression in VSMCs.
VSMCs were pretreated with KLF5 siRNA for 1 hr prior to the addition of rosiglitazone (5 μM) for 1 hr, and subsequently stimulated with Ang II (0.1 μM) for 24 hrs. CCK8 assays (values represent the mean ± S.E.M. n = 12) were carried out to assess cell proliferation. *P<0.05 vs. control; #P<0.05 vs. Ang II; †P<0.05 vs. Ang II + Ros.
Fig 5.
Suppression of KLF5 expression is mediated by PPAR-γ activation.
Cells were pretreated with or without PPAR-γ antagonist GW9662 (3 mM) or BADGE (1 μM) for 30 min or PPAR-γ specific siRNA for 48 hrs prior before the addition of PPAR-γ activator rosiglitazone (Ros) (5μM), 15-d-PGJ2 (15D) (5μM) or pioglitazone (Pio) (50 μM) for 1 hr, and subsequently stimulated with Ang II (0.1 μM) for 24 hrs. (A) Representative immunoblots for PPAR-γ and β-actin from 3 separate experiments. PPAR-γ protein expression is shown as fold increase over control group. Results are mean ± S.E.M. β-actin was used as an internal control. (B) PPAR-γ activation was analyzed by DNA-binding assay. NSB: non-specific binding, C1: competitor binding, PC: positive control binding. Results are mean ± S.E.M. of 3 independent experiments, expressed as OD 450. (C) Western blot analysis of KLF5 protein expression in VSMCs. Representative western blot (upper panel), and data are mean ± S.E.M. (bottom panel) of 3 independent experiments. Results are expressed as fold increase over control group. β-actin served as an internal control. (D) Real-time RT-PCR analysis of KLF5 mRNA expression in response to different treatment in VSMCs. Results are fold increase over control, and data are mean ± S.E.M. of 3 independent experiments. GAPDH served as an internal control. (*P<0.05 vs. control; # P<0.05 vs. Ang II; †P<0.05 vs. Ang II + Ros).
Fig 6.
Blockade of AT1 with losartan was not involved in the inhibitory effect of rosiglitzone on Ang II-induced KLF5 expression in VSMCs.
Western blot analysis (upper panel), and data are mean ± S.E.M. (bottom panel) of 3 independent experiments. Results are expressed as fold increase over control group. β-actin served as an internal control (*P<0.05 vs. control; #P<0.05 vs. Ang II).
Fig 7.
PKC signaling pathway in the effect of PPAR-γ agonist on Ang II-induced KLF5 expression.
(A) and (B). VSMCs were pretreated with rosiglitazone (5 μM) for 1hr, and incubated with Ang II (0.1 μM) for 30 min; phosphorylated PKCε and PKCζ were then detected by western blot. Representative western blot of p-PKCε and p-PKCζ, and data of mean ± S.E.M. of 3 experiments (bottom panel) were shown. (C). VSMCs were pretreated with PKCζ siRNA for 48 hrs prior to the addition of rosiglitazone (5 μM) for 1 h, and subsequently stimulated with Ang II (0.1 μM) for 24 h. A representative western blot of KLF5, and data of mean ± S.E.M. of 3 experiments (bottom panel) were shown. All results are expressed as fold increase over control group. β-actin served as an internal control. (*P<0.05 vs. control; #P<0.05 vs. Ang II).
Fig 8.
ERK signaling pathway in the effects of PPAR-γ agonist on Ang II-mediated KLF5 expression.
(A) VSMCs were subjected to ERK1/2 inhibitor PD098059 (1 μM) for 30min, followed by treatment of rosiglitazone (5 μM) for further 1 h, and finally stimulated with Ang II (0.1 mM) for 24 h. A representative western blot of KLF5 (upper panel), and data of mean ± S.E.M. of 3 experiments (bottom panel) were shown. (B) VSMCs were pretreated with rosiglitazone (5 μM) for 1h, and incubated with Ang II (0.1 μM) for 30 min, phosphorylated ERK1/2 were then detected by western blot. A representative western blot of ERK1/2 and p-ERK1/2, and data of mean ± S.E.M. of 3 experiments (bottom panel) were shown. Results are expressed as fold increase over control group. β-actin served as an internal control. (*P<0.05 vs. control; #P<0.05 vs. Ang II.)
Fig 9.
Effect of PPAR-γ agonist on Egr activity.
Egr activation was analyzed by DNA-binding assay. Results are mean ± S.E.M. of 3 independent experiments, expressed as relative light units (RLUs). (*P<0.05 vs. control; #P<0.05 vs. Ang II).
Fig 10.
Agonist-induced activation of PPAR-γ suppresses Ang II-induced KLF5 expression, likely by interfering with the Ang II/PKCζ/ERK1/2/Egr pathway.