Vasculoprotective effects of rosiglitazone through modulating renin-angiotensin system in vivo and vitro

Abstract

Background: The peroxisome proliferator-activated receptor-γ (PPARγ) agonist rosiglitazone has been suggested to exert cardiovascular protection through the improvement of lipid metabolism, anti-inflammation, anti-proliferation etc. However, whether renin-angiotensin system (RAS) is involved in the vascular protective effects of PPARγ agonists is not fully understood. The present study aimed to investigate the effects of the renin-angiotensin system in vascular protection mediated by PPARγ agonists.

Objective: To investigate the actions of the renin-angiotensin system in vascular protection mediated by activation of PPARγ in vivo and in vitro.

Methods: Rats were fed a regular diet (n = 8), a cholesterol-rich diet plus methylthiouracil (80 mg/Kg/day, n = 10), a cholesterol-rich diet plus methylthiouracil and rosiglitazone (4 mg/kg/day, n = 10). The rosiglitazone treatment was started from one month after the start of cholesterol-rich diet plus methylthiouracil, and lasted five months. Cultured vascular smooth muscle cells (VSMCs) were pretreated with 1 μmol/L angiotensin II (ANG II) for 6 h and randomly divided into the control group; the ANG II group (1 μmol/L ANG II); the groups respectively treated with different concentration rosiglitazone (20, 30, 50) μmol/L for 12 h; the groups treated with 30 μmol/L rosiglitazone for (6, 12, 24) h. Morphology changes of the aortic tissues were observed by hematoxylin and eosin stain. The VSMC growth was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Angiotensin II and expression of angiotensin receptors were determined by radioimmunoassay, reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry.

Results: After 6 months, lipid deposition, VSMC proliferation and migration toward intima were observed in aortic tissues in the rats on a cholesterol-rich diet plus methylthiouracil, while these pathological changes induced by the cholesterol-rich diet were significantly suppressed by rosiglitazone. In addition, VSMC proliferation induced by ANG II was markedly inhibited by rosiglitazone. Rosiglitazone markedly down-regulated expression of angiotensin type 1 receptor (AT1R) and up-regulated expression of angiotensin type 2 receptor (AT2R) in the aortic tissues and ANG II-treated VSMCs.

Conclusions: The present study demonstrated that PPARγ agonist rosiglitazone suppressed ANG II-induced VSMC proliferation in vitro and early atherosclerotic formation evoked by cholesterol-rich diet in vivo. These vasculoprotective effects of rosiglitazone were mediated at least partially by reduction in local tissue ANG II concentration, down-regulation of AT1R expression and up-regulation of AT2R expression both at the mRNA and protein levels.

Figure 1

Representative slides of hematoxylin-eosin staining of proximal aortic tissues. Con: regular diet group (200 ×), showing integrated endothelial cell layer and regular arrangement of smooth muscle cells; Cho-a: cholesterol-rich diet plus methylthiouracil (80 mg/Kg/day) group (200 ×); Cho-b: cholesterol-rich diet plus methylthiouracil group (400 ×), showing smooth muscle cell proliferation, migration toward intima (broken arrow), and lipid deposition (solid arrow); Ros: cholesterol-rich diet plus methylthiouracil and rosiglitazone treatment (200 ×). Smooth muscle cell proliferation and lipid deposition were barely observed in the Ros group.

Figure 2

In vitro cultured VSMC features. Cultured VSMCs seen under in phase contrast microscope, were spindle or fusiform shape, showing valley-like feature (2A, 10×20); Unique cordlike filaments and macula densa in cytoplasm were observed under transmission electron microscope (2B and 2C, 20×10³); Immunocytochemical staining of VSMC α-actin clearly showed brown myofilaments of distribution along cell longitudinal axis. Cell nucleus were not stained (2D, 10×40).

Figure 3

Immunohistochemical staining of protein expression of angiotensin II type 1 receptor (AT₁R) and angiotensin II type 2 receptor (AT₂R) in aortic tissues. A and F: the negative controls of AT₁R and AT₂R for the immunohistochemistry, respectively, showing no positive staining. B and G: the regular diet group; C and H: the cholesterol-rich diet plus methylthiouracil (80 mg/Kg/day) group; D and I: group with cholesterol-rich diet plus methylthiouracil and rosiglitazone treatment. Rosiglitazone treatment markedly attenuated AT₁R immunoreactivity induced by the cholesterol-rich diet plus methylthiouracil (D), while increased AT₂R immunoreactivity (I). (Magnification: 400×).

Figure 4

Protein expression of angiotensin II type 1 receptor (C: AT₁R, 43 kDa) and type 2 receptor (D: AT₂R, 44 kDa) in aortic tissues. Representative blots from aortic samples in each group are shown at the top. The bar graph shows protein expression of AT₁R or AT₂R relative to internal control ß-actin (45 kDa) from 4 separate experiments in each group. Con: the regular diet group; Cho: the cholesterol-rich diet plus methylthiouracil group; Ros: the group on cholesterol-rich diet plus methylthiouracil and rosiglitazone treatment. Note that rosiglitazone treatment led to the marked reduction in protein expression of AT₁R, while further increase in protein expression of AT₂R compared with the Cho group. *P < 0.01 versus Con group; ^‡P < 0.01 versus Cho group.

Figure 5

mRNA expression of angiotensin II type 1 receptor (C: AT₁R, 352-bp fragment) and type 2 receptor (D: AT₂R, 418-bp fragment) in aortic tissues. Representative samples of AT₁R and AT₂R mRNA from aortic samples in each group are shown at the top. The bar graph shows mRNA expression of AT₁R or AT₂R relative to internal control ß-actin (232-bp fragment) from 4 replicate experiments in each group. Con: the regular diet group; Cho: the cholesterol-rich diet plus methylthiouracil group; Ros: the group on cholesterol-rich diet plus methylthiouracil and rosiglitazone treatment. Note that the increase in AT₁R mRNA caused by the cholesterol-rich diet plus methylthiouracil was markedly suppressed by rosiglitazone treatment (P < 0.01). However, mRNA expression of AT₂R was further increased (P < 0.01). *P < 0.01 versus Con group; ^‡P < 0.01 versus Cho group.

Figure 6

Effects of rosiglitazone treatment on protein expression of ANG II-induced angiotensin II type 1 receptor (A: AT₁R, 43 kDa) and type 2 receptor (B: AT₂R, 44 kDa) in vitro cultured VSMCs. (C): VSMCs were pretreated with 1 μmol/L ANG II for 6 h and subsequently treated for 12 h as follows: Con, ANG II, ANG II plus different concentration rosiglitazones (20, 30, 50) μmol/L; (D): VSMCs were pretreated with 1 μmol/L ANG II for 6 h and subsequently treated with 30 μmol/L rosiglitazone for (6, 12, 24) h, respectively. Representative blots from each experimental group are shown at the top. The bar graph shows protein expression of AT₁R or AT₂R relative to internal control ß-actin (45 kDa) from 3 separate experiments in each group. *P < 0.01 versus Con group, ** P < 0.05 versus Con group; ^‡P < 0.01 versus ANG II.

Figure 7

Effects of rosiglitazone treatment on mRNA expression of ANG II-induced angiotensin II type 1 receptor (A: AT₁R) and type 2 receptor (B: AT₂R) in cultured VSMCs. (C): VSMCs were pretreated with 1 μmol/L ANG II for 6 h and subsequently treated for 12 h as follows: Con, ANG II, ANG II plus different concentration rosiglitazones (20, 30, 50) μmol/L; (D):VSMCs were pretreated with 1 μmol/L ANG II for 6 h and subsequently treated with 30 μmol/L rosiglitazone for (6, 12, 24) h, respectively. Representative samples of AT₁R and AT₂R mRNA from each experimental group are shown at the top. The bar graph shows mRNA expression of AT₁R or AT₂R relative to internal control ß-actin (232-bp fragment) from 3 separate experiments in each group. ** P < 0.05 versus Con group, *P < 0.01 versus Con group; ^†P < 0.05 versus ANG II, ^‡P < 0.01 versus ANG II.