Estrogens and selective estrogen receptor modulators regulate gene and protein expression in the mesenteric arteries

Abstract

Estrogen has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Gene targets of estrogen and SERMs in the vasculature are not well-known. Thus, the present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation.

Figure 1

Differential expression of fatty acid synthase (FAS) in the mesenteric arteries of control (CTL), ethinyl estradiol (EE), estradiol benzoate (EB), equilin (EQ), tamoxifen (TAM), and raloxifene (RAL) treated ovariectomized rats. (A) DNA microarray data (relative expression, n=3/group); (B) real-time reverse transcription-PCR (RT-PCR) relative expression data (n=8/group); (C) Images of immunoblot data for FAS and actin protein for control (lanes 1–3), TAM (lanes 4–6), EE (lanes 7–9), and EQ (lanes 10–12) treated animals. (D) FAS protein expression divided by the actin loading control (relative density, n=6/group). Differential expression for DNA microarray, real time RT-PCR, and immunoblot data in (A), (B) and (D) was determined by ANOVA followed by Dunnett’s Multiple Comparison Test (p<0.05).

Figure 2

Differential expression of stearoyl-CoA desaturase (SCD) in the mesenteric arteries of control (CTL), ethinyl estradiol (EE), estradiol benzoate (EB), equilin (EQ), tamoxifen (TAM), and raloxifene (RAL) treated ovariectomized rats. (A) DNA microarray data (relative expression, n=3/group); (B) real-time reverse transcription-PCR (RT-PCR) relative expression data (n=8/group); (C) Images of immunoblot data for SCD and actin proteinfor control (lanes 1–2), TAM (lanes 3–4), EQ (lanes 5–6), and EE (lanes 7–8) treated animals. (D) SCD protein expression divided by the actin loading control (relative density, n=6/group). Differential expression for DNA microarray, real time RT-PCR, and western blot data in (A), (B) and (D) was determined by ANOVA followed by Dunnett’s Multiple Comparison Test (p<0.05).

Figure 3

Differential expression of soluble epoxide hydrolase (sEH) in the mesenteric arteries of control (CTL), ethinyl estradiol (EE), estradiol benzoate (EB), equilin (EQ), tamoxifen (TAM), and raloxifene (RAL) treated ovariectomized rats. (A) DNA microarray data (relative expression, n=3/group); (B) real-time reverse transcription-PCR (RT-PCR) relative expression data (n=8/group); (C) Images of immunoblot data for sEH and actin for control (lanes 1–2), TAM (lanes 3–4), EQ (lanes 4–5), and EE (lanes 7–8) treated rats. (D) sEH protein expression divided by the actin loading control (relative density, n=6/group). Differential expression for DNA microarray, real time RT-PCR, and western blot data in (A), (B) and (D) was determined by ANOVA followed by Dunnett’s Multiple Comparison Test (p<0.05).

Figure 4

Differential expression of secreted frizzled related protein-4 (SFRP-4) in the mesenteric arteries of control (CTL), ethinyl estradiol (EE), estradiol benzoate (EB), equilin (EQ), tamoxifen (TAM), and raloxifene (RAL) treated ovariectomized rats. (A) DNA microarray data (relative expression, n=3/group); (B) real-time reverse transcription-PCR (RT-PCR) relative expression data (n=8/group); (C) Immunoblot images for control (lanes 1–3), TAM (lanes 4–6), EE (lanes 7–9), and EQ (lanes 10–12) treated animals. (D) SFRP4 protein expression divided by the actin loading control (relative density, n=6/group). Differential expression for DNA microarray, real time RT-PCR, and western blot data in (A), (B) and (C) was determined by ANOVA followed by Dunnett’s Multiple Comparison Test (p<0.05).

Figure 5

Differential expression of insulin-like growth factor-1 (IGF-1) in the mesenteric arteries of control (CTL), ethinyl estradiol (EE), estradiol benzoate (EB), equilin (EQ), tamoxifen (TAM), and raloxifene (RAL) treated ovariectomized rats. (A) DNA microarray data (relative expression, n=3/group); (B) real-time reverse transcription-PCR (RT-PCR) relative expression data (n=8/group); Differential expression for DNA microarray and real time RT-PCR data in (A) and (B) was determined by ANOVA followed by Dunnett’s Multiple Comparison Test (p<0.05).

Figure 6

Differential expression of phospholipase A2 group 1B (PLA2-G1B) in the mesenteric arteries of control (CTL), ethinyl estradiol (EE), estradiol benzoate (EB), equilin (EQ), tamoxifen (TAM), and raloxifene (RAL) treated ovariectomized rats. (A) DNA microarray data (relative expression, n=3/group); (B) real-time reverse transcription-PCR (RT-PCR) relative expression data (n=8/group); Differential expression for DNA microarray and real time RT-PCR data in (A) and (B) was determined by ANOVA followed by Dunnett’s Multiple Comparison Test (p<0.05).