2-Methoxyestradiol protects against pressure overload-induced left ventricular hypertrophy

Abstract

Numerous experimental studies have supported the evidence that 2-methoxyestradiol (2 ME) is a biologically active metabolite that mediates multiple effects on the cardiovascular system, largely independent of the estrogen receptor. 2 ME is a major cytochrome P450 1B1 (CYP1B1) metabolite and has been reported to have vasoprotective and anti-inflammatory actions. However, whether 2 ME would prevent cardiac hypertrophy induced by abdominal aortic constriction (AAC) has not been investigated yet. Therefore, the overall objectives of the present study were to elucidate the potential antihypertrophic effect of 2 ME and explore the mechanism(s) involved. Our results showed that 2 ME significantly inhibited AAC-induced left ventricular hypertrophy using echocardiography. The antihypertrophic effect of 2 ME was associated with a significant inhibition of CYP1B1 and mid-chain hydroxyeicosatetraenoic acids. Based on proteomics data, the protective effect of 2 ME is linked to the induction of antioxidant and anti-inflammatory proteins in addition to the modulation of proteins involved in myocardial energy metabolism. In vitro, 2 ME has shown a direct antihypertrophic effect through mitogen-activated protein kinases- and nuclear factor-κB-dependent mechanisms. The present work shows a strong evidence that 2 ME protects against left ventricular hypertrophy. Our data suggest the potential of repurposing 2 ME as a selective CYP1B1 inhibitor for the treatment of heart failure.

Figure 1

Effect of 2 ME on AAC-induced HW/TL ratio. Sham and AAC rats were treated with 2 ME (5 mg/kg/day) in the mini osmotic pump. Thereafter, HW/TL ratio (in mg per cm) was determined for each animal. The values represent mean ± SEM (n = 6). ⁺P < 0.05 compared to control. *P < 0.05 compared to AAC.

Figure 2

Effect of 2 ME on AAC-mediated induction of fibrotic and apoptotic markers. Sham and AAC rats were treated with 2 ME (5 mg/kg/day) in the mini osmotic pump. Thereafter, (A) the mRNA level of pro III, TGFβ-I, p53 and BAX were quantified using real time-PCR. (B) Fibrotic areas were determined using Trichrome’s stain (intense blue staining), quantified and expressed as % of fibrotic area. The experiment was replicated twice and the values represent mean ± SEM (n = 6). ⁺P < 0.05 compared to control. *P < 0.05 compared to AAC.

Figure 3

Effect of AAC and 2 ME on mid-chain HETE level and the expression of CYP1B1, LOXs and COX protein. Sham and AAC rats were treated with 2 ME (5 mg/kg/day) in the mini osmotic pump and then, (A) mid-chain HETE metabolites were measured using LC–ESI–MS. (B) CYP1B1, 5-LOX, 12-LOX, 15-LOX and COX-2 protein expression levels were determined by Western blot analysis. The experiment was replicated twice and the values represent mean ± SEM (n = 6). ⁺P < 0.05 compared to control. *P < 0.05 compared to AAC.

Figure 4

Effect of 2 ME and AAC on MAPK signaling pathway. Sham and AAC rats were treated with 2 ME (5 mg/kg/day) in the mini osmotic pump. Then, the MAPKs protein phosphorylation, P-JNK, P-p38 and P-ERK1/2 was determined. The values represent mean ± SEM (n = 6). ⁺p < 0.05 compared to control. *p < 0.05 compared to AAC.

Figure 5

Effect of ISO and 2 ME on RL-14 cells viability, hypertrophic genes, and cell surface area. RL-14 cells were exposed to 100 μM ISO in the presence and absence of 0.25 μM 2 ME for 24 h. Thereafter, (A) RL-14 cell viability was determined using MTT and LDH assays. (B) The mRNA level of α-MHC and β-MHC was quantified using real time-PCR. (C) Cell surface area was analyzed by phase contrast imaging. The values represent mean ± SEM (n = 6). ⁺P < 0.05 compared to control. *P < 0.05 compared to ISO.

Figure 6

Effect of 2 ME on ISO-mediated effect on superoxide radical, MAPK and NF-κB signaling pathways. RL-14 cells were treated for 24 h with 100 µM ISO in the presence and absence of 0.25 μM 2 ME. Thereafter, (A) Superoxide anion was determined using DHE assay. (B) MAPKs protein phosphorylation was determined in cytoplasmic protein extracts using PhosphoTracer Elisa Kit (Abcam, Cambridge, UK). (C) NF-κB binding activity was determined using commercially available kit. The values represent mean ± SEM (n = 6). ⁺P < 0.05 compared to control. *P < 0.05 compared to ISO.

Figure 7

Effect of Ang II and 2 ME2 ME on RL-14 cells viability and hypertrophic genes. H9c2 cells were exposed to 10 μM Ang II in the presence and absence of 0.25 μM 2 ME for 24. Thereafter, (A) H9c2 cell viability was determined using MTT and LDH assays. (B) The mRNA level of BNP, β-MHC, TNF-α and IL-6 was quantified using real time-PCR. The values represent mean ± SEM (n = 6). ⁺P < 0.05 compared to control. *P < 0.05 compared to Ang II.

Figure 8

Effect of 2 ME on Ang II-mediated effect on superoxide radical, MAPK and NF-κB signaling pathways. H9c2 cells were treated for 24 h with 10 µM Ang II in the presence and absence of 0.25 μM 2 ME2 ME. Thereafter, (A) Superoxide anion was determined using DHE assay. (B) MAPKs protein phosphorylation was determined in cytoplasmic protein extracts using PhosphoTracer Elisa Kit (Abcam, Cambridge, UK). (C) NF-κB binding activity was determined using commercially available kit. The values represent mean ± SEM (n = 6). ⁺P < 0.05 compared to control. *P < 0.05 compared to Ang II.