17β-Estradiol Promotes Angiogenesis of Rat Cardiac Microvascular Endothelial Cells In Vitro

Abstract

BACKGROUND The formation of new blood vessels, known as angiogenesis, is critical for recovery from ischemic heart disease, and estrogen is considered an important factor in this process. Here, we investigated the effects of 17β-estradiol (17β-E2) on proliferation and migration of cardiac microvascular endothelial cells (CMECs) in vitro. MATERIAL AND METHODS Rat CMECs were isolated and cultured with 17β-E2 (0.001-1 µmol/l) in the absence or presence of the estrogen antagonist tamoxifen. Then, the expression level of estrogen receptor alpha was evaluated by using immunofluorescence assay, RT-PCR, and Western blot. Cell proliferation was detected by methyl thiazolyl tetrazolium analysis and the cell migration was verified by a scraping assay and quantified by a Transwell chamber assay. CMEC differentiation was examined using a tube formation assay. Vascular endothelial growth factor (VEGF) secretion was detected by enzyme-linked immunosorbent assay. RESULTS CMECs exhibited homogenous, polygonal, exhibited contact inhibition, and had characteristically ovoid nuclei with 1 or 2 nucleoli, and the cytoplasm exhibited red fluorescence after staining for von Willebrand factor. 17β-E2 treatment upregulated estrogen receptor alpha expression in CMECs. 17β-E2 treatment significantly promoted the proliferation, migration, tubular structure formation, and VEGF secretion in CMECs. The maximal proliferation occurred in the presence of 0.01 µmol/l 17β-E2. Furthermore, estrogen and VEGF were found to synergistically stimulate angiogenesis. CONCLUSIONS Our data show that 17β-E2 promotes angiogenesis in vitro and suggests that estrogen treatment as a novel therapeutic modality in the management of arterial insufficiency.

Figure 1

Culture of CMECs isolated from adult male Sprague-Dawley rat heart tissue (left ventricle). (A) Primary isolated rat CMECs (100×). (B) Rat CMECs at passage 2 (100×). (C) Immunostaining for CD34 in rat CMECs (100×). (D) Immunostaining for von Willebrand factor in rat CMECs (200×).

Figure 2

ER expression is detected in rat CMECs. Show fluorescence immunostaining of estrogen receptor alpha in rat CMECs (100×). Immunofluorescent staining in control samples lacking anti-ER alpha. Immunofluorescent staining (green) in samples treated with anti-ER alpha.

Figure 3

Effect of 17β-estradiol (17β-E₂) on rat CMEC proliferation. (A) Cell cultures were treated with the indicated concentration of 17β-E₂, and cell proliferation was quantified using the MTT assay. * P<0.05 vs. control, *** P<0.001 vs. control; n=6. (B) Tamoxifen inhibition of 17β-E₂-induced cell proliferation. Cell cultures were untreated or treated with 17β-E₂ (0.01 μmol/l) in the absence or presence of tamoxifen (Tamo; 0.1 μmol/l) *** P<0.001 vs. control; n=6.

Figure 4

Effect of 17β-estradiol (17β-E₂) on rat CMEC migration. (A) Cell cultures were untreated (Normal) or treated with 17β-E₂ (0.01 μmol/l) in the absence or presence of tamoxifen (Tamo; 0.1 μmol/l). Representative images of CMEC migration across wound edges 24 h after scraping of the culture surface are shown. (B) Cell migration was determined using a Transwell chamber assay; migration was quantified by counting cells after 24 h. Data represent the mean ±S.E.M. from 4 independent experiments. Data were compared using ANOVA. ^### P<0.001 vs. control.

Figure 5

17β-estradiol (17β-E₂) induces the formation of tube-like structures in CMEC cultures. Cell cultures were untreated (Control) or treated with 17β-E₂ (0.01 μmol/l) in the absence or presence of tamoxifen (Tamo; 0.1 μmol/l) for 48 h. Representative images are shown.

Figure 6

17 β-estradiol (17β-E₂) induces VEGF expression in CMECs. Cell cultures were untreated or treated with 17β-E₂ (0.01 μmol/l) in the absence or presence of tamoxifen (Tamo; 0.1 μmol/l) for 48 h, and VEGF expression in culture supernatants was analyzed by ELISA. Data represent the means ±S.E.M. from 4 independent experiments. Data were compared by ANOVA. *** P<0.001 vs. control.