Effects of 17 β-estradiol on lipopolysacharride-induced intracellular adhesion molecule-1 mRNA expression and Ca²+ homeostasis alteration in human endothelial cells

Abstract

Recent evidence showed that 17 β-estradiol (E₂) decreased cytokine-induced expression of cell adhesion molecules (CAM). Changes in intracellular Ca²+ concentration ([Ca²+](i)) has been shown to be associated with CAM expression in endothelial cells. Here, the effects of E₂ (1 μM, 24 h) on the expression of intracellular adhesion molecule-1 (ICAM-1) and [Ca²+](i) were investigated in a lipopolysaccharide (LPS) (100 ng/mL, 18 h)-stimulated human endothelial cell line, EA.hy926, using real-time PCR and spectrofluorometry, respectively. PCR analysis revealed a significant increase in ICAM-1 expression in calcium ionophore A23187 (1 nM)- or LPS-stimulated cells. Pretreatment of cells with E(2) significantly inhibited LPS-induced ICAM-1 mRNA expression. [Ca²+](i) was monitored in Fura-2AM-loaded cells in the presence and absence of extracellular Ca²+ with thapsigargin (TG, 1 μM), a sarco/endoplasmic reticulum ATPase inhibitor or ATP (100 μM). The extent of TG- or ATP-induced [Ca²+](i) increase was significantly higher in LPS-stimulated cells than in control cells. Pre-treatment of LPS-stimulated cells with E₂ limited the Ca²+ response to the same level as in control cells. Furthermore, ICI 182,780, an estrogen receptor antagonist, attenuated the inhibitory actions of E₂ on ICAM-1 mRNA expression and Ca²+ responses, suggesting that estrogen receptors mediate, at least in part, the effects of estrogen. These data suggest a potential underlying mechanism for the protective effect of E₂ against atherosclerosis.

Fig. 1. Real-time PCR analysis of ICAM-1 mRNA expression in ERA.hy926

The means ± standard deviation of the ICAM-1 mRNA expression in control, LPS-stimulated, E₂/LPS-stimulated, and ICI/E₂/LPS stimulated cells are shown. ICAM-1 mRNA level was normalized to GAPDH. Significant differences among individual treatment groups are indicated (•—•, P<0.05, n=8) as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Fig. 2. Effect of calcium ionophore A23187 on ICAM-1 mRNA expression in EA.hy926

The means ± standard deviation of the ICAM-1 mRNA expression in et al control and calcium ionophore-stimulated cells are shown. ICAM-1 mRNA level was normalized to GAPDH. Significant difference between two groups is indicated (•—•, P<0.05, n=3) as analyzed using the unpaired Student t-test.

Fig. 3. TG-induced Ca²⁺ signals in the presence of extracellular Ca²⁺

A) A representative tracing of the Ca²⁺ signal from EA.hy926 cells in response to TG (1 μM) in the presence of 1.2 mM extracellular Ca²⁺ followed by EGTA (2 mM) treatment. B) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to TG in the presence of extracellular Ca²⁺. Significant differences among individual treatment groups are indicated (•—•, P<0.05, n=5) as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Fig. 4. TG-induced Ca²⁺ signal in the absence of extracellular Ca²⁺

A) A representative tracing of the Ca²⁺ signal from EA.hy926 cells in response to TG (1 μM) in Ca²⁺-free medium, followed by the addition of extracellular Ca²⁺ (1.2 mM) and EGTA (2 mM). B) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to TG in Ca²⁺-free medium. C) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to TG upon addition of extracellular Ca²⁺. Significant differences among individual treatment groups are indicated by (•—•, P<0.05, n=5) as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Fig. 4. TG-induced Ca²⁺ signal in the absence of extracellular Ca²⁺

A) A representative tracing of the Ca²⁺ signal from EA.hy926 cells in response to TG (1 μM) in Ca²⁺-free medium, followed by the addition of extracellular Ca²⁺ (1.2 mM) and EGTA (2 mM). B) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to TG in Ca²⁺-free medium. C) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to TG upon addition of extracellular Ca²⁺. Significant differences among individual treatment groups are indicated by (•—•, P<0.05, n=5) as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Fig. 5. ATP-induced Ca²⁺ signals in the presence of extracellular Ca²⁺

A) A representative tracing of the Ca²⁺ signal from EA.hy926 cells in response to ATP (100 μM) in the presence of 1.2 mM extracellular Ca²⁺, followed by EGTA (2 mM) treatment. B) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to ATP in the presence of extracellular Ca²⁺. Significant dierences among individual treatment groups are indicated (•—•, P<0.05, n=5) as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Fig. 6. ATP-induced Ca²⁺ signal in the absence of extracellular Ca²⁺

A) A representative tracing of the Ca²⁺ signal from EA.hy926 cells in response to ATP (100 μM) in Ca²⁺-free medium, followed by the addition of extracellular Ca²⁺ (1.2 mM) and EGTA (2 mM). B) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to ATP in Ca²⁺-free medium. C) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to ATP upon the addition of extracellular Ca²⁺. Significant differences among individual treatment groups are indicated by (•—•, P<0.05, n=5) as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Fig. 6. ATP-induced Ca²⁺ signal in the absence of extracellular Ca²⁺

A) A representative tracing of the Ca²⁺ signal from EA.hy926 cells in response to ATP (100 μM) in Ca²⁺-free medium, followed by the addition of extracellular Ca²⁺ (1.2 mM) and EGTA (2 mM). B) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to ATP in Ca²⁺-free medium. C) The mean ± standard deviation of the Ca²⁺ AUC traced during the response to ATP upon the addition of extracellular Ca²⁺. Significant differences among individual treatment groups are indicated by (•—•, P<0.05, n=5) as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.