Reduction of oxidative stress and AT1 receptor expression by the selective oestrogen receptor modulator idoxifene

Abstract

1. The beneficial vasoprotective effects of oestrogens are hampered by their side effects on secondary sexual organs. Selective oestrogen receptor modulators (SERM) such as idoxifene may exert beneficial vascular effects without influencing cancerogenesis in breast or uterus. 2. In order to investigate vascular effects of selective oestrogen receptor modulators, we examined the impact of idoxifene on production of reactive oxygen species as well as AT1 receptor expression in vascular smooth muscle cells (VSMC). 3. Idoxifene caused a concentration- and time-dependent down-regulation of AT1 receptor mRNA expression, as assessed by Northern analysis. The maximal effect was reached with 10 micromol l(-1) idoxifene after a 4 h incubation period (33+/-7% of control levels). Western blots showed a similar down-regulation of AT1 receptor protein to 36+/-11% of control levels. 4. Confocal laserscanning microscopy using the redox sensitive marker 2',7'-dichlorofluorescein (DCF) and measurement of NAD(P)H oxidase activity in cell homogenates revealed that idoxifene effectively blunted the angiotensin II-induced production of reactive oxygen species. 5. In order to investigate the signal transduction involved in SERM-induced modulation of AT1 receptor expression, VSMC were preincubation with PD98059, genistein, wortmannin, or N(omega)-Nitro-L-arginine. The results suggested that idoxifene caused AT1 receptor down-regulation through nitric oxide-dependent pathways. 6. In conclusion, idoxifene reduces angiotensin II-evoked oxidative stress in VSMC. This could in part be explained by idoxifene-induced down-regulation of AT1 receptor expression. These results demonstrate that the selective oestrogen receptor modulator idoxifene may exert beneficial vascular effects which could be useful for therapeutic regimen in postmenopausal women at risk for cardiovascular diseases.

Figure 1

Effect of idoxifene on AT1 receptor mRNA expression. (A) Representative autoradiography of a Northern hybridization of a AT1 receptor cDNA probe to RNA isolated from VSMC stimulated for 4 h with increasing concentrations of idoxifene (I) and 1 μmol l⁻¹ 17β-Estradiol (E). Also displayed are the GAPDH signals detected from the same membrane. (B) Densitometric analysis showing the time dependency of the effect of 10 μmol l⁻¹ idoxifene on the expression of the AT1 receptor mRNA in VSMC. Mean±s.e. n=8, *P<0.05. (C) Effect of idoxifene at various concentrations on the expression of the AT1 receptor mRNA in VSMC. Densitometric analysis. Mean±s.e. n=3, *P<0.05. (D) Effect of increasing concentrations of 17β-Estradiol and raloxifene on AT1 receptor mRNA expression. Densitometric analysis. Mean±s.e. n=3, *P<0.05.

Figure 2

Effect of idoxifene on AT1 receptor protein expression. Western blot of proteins isolated from VSMC stimulated for 12 h with different concentrations of idoxifene. Representative autoradiogram and densitometric analysis. mean±s.e. n=3, *P<0.05.

Figure 3

Signal transduction pathways involved in AT1 receptor regulation induced by idoxifene. VSMC were co-incubated with idoxifene (4 h) with the following compounds. PD=PD98059 (1 μmol l⁻¹), Geni=Genistein (1 μmol l⁻¹), Wort=wortmannin (1 μmol l⁻¹), or NNLA=N^ω-Nitro-L-arginine (10 μmol l⁻¹). AT1 receptor mRNA was assessed after isolation of total RNA and Northern blot. The densitometric analysis of the Northern blots quantifying AT1 receptor mRNA is depicted. mean±s.e. n=3, *P<0.05 Idoxifene, PD, Wort or Geni versus control, **P<0.05 NNLA versus Idoxifene, PD, Wort or Geni.

Figure 4

Effect of idoxifene on angiotensin II-induced intracellular production of reactive oxygen species in VSMC. (A) VSMC were preincubated for 12 h with 10 μmol l⁻¹ idoxifene (Idox) or vehicle (control), followed by a 3 h incubation with 1 μmol l⁻¹ angiotensin II (AngII). Free radical production is visualized through DCF fluorescence. (B) Quantitative analysis of signal intensities are shown as mean±s.e., four different experiments with five groups of 25 randomly selected cells each, *P<0.05 AngII versus control, **P<0.05 AngII versus AngII+idoxifene. (C) VSMC were preincubated for 12 h with 10 μmol l⁻¹ raloxifene (ralox) or vehicle (control), followed by a 3 h incubation with 1 μmol l⁻¹ angiotensin II (AngII). Quantitative analysis of signal intensities are shown as mean±s.e. of four different experiments with five groups of 25 randomly selected cells each, *P<0.05 AngII versus control, **P<0.05 AngII versus AngII+raloxifene.

Figure 5

Effect of idoxifene on NAD(P)H oxidase activity in VSMC. VSMC were preincubated for 12 h with 10 μmol l⁻¹ idoxifene (Idox) or vehicle (Control), followed by a 4 h incubation with 1 μmol l⁻¹ angiotensin II (AngII). Superoxide release of cell homogenates was measured via lucigenin enhanced chemiluminescence. mean±s.e. n=3, *P<0.05 AngII versus control, **P<0.05 AngII versus AngII+idoxifene.