Introduction Inflammation plays an important role in pathogenesis of vascular disease. Estrogen (17β-estradiol, E2) negatively modulates neointima formation in response to vascular injury, in part by attenuating expression of pro-inflammatory mediators and migration of leukocytes (primarily neutrophils and monocyte/macrophages) into injured vessels. Cytokine-induced neutrophil chemoattractant (CINC)-2 is a potent chemokine that causes neutrophil recruitment and provokes neutrophil activation in vitro. This study tested the hypothesis that activated rat aortic smooth muscle cells (RASMC) express CINC-2 and induce neutrophil migration in vitro, and that E2 inhibits these processes by an estrogen receptor (ER) dependent mechanism.
Methods Quiescent cultured RASMCs were pretreated with E2 or vehicle for 24 hours before tumor necrosis factor (TNF)-α was added. After 6 hours of treatment, total RNA was extracted from cells using TRIzol reagent, and SYBR green real-time RT-PCR was used to detect expression of CINC-2 mRNA. Conditioned media was collected and concentrated to measure CINC-2 protein level by ELISA. To assess neutrophil chemotactic activity of conditioned media, in vitro chemotaxis assays were performed using differentiated HL-60 cells in a 96-well modified Boyden chamber appropriate for the evaluation of leukocyte chemotaxis. The nonselective ER antagonist ICI-182780 was given to cells 2 hours prior to E2 incubation to study the mechanism of E2 effect.
Results Steady state of CINC-2 mRNA was undetectable in unstimulated RASMCs; TNF-α (0.1 ng/mL to 2.5 ng/mL) dose dependently stimulated CINC-2 mRNA expression, and E2 pretreatment significantly reduced TNF-α-stimulated CINC-2 expression (by 30%). ICI-182780 completely blocked the E2 effect, indicating ER dependence. ELISA confirmed increased of CINC-2 protein in conditioned media from RASMCs stimulated with TNF-α and E2-induced inhibition of CINC-2 expression. Conditioned media from RASMCs treated with TNF-α produced a 2-fold increase in migration of differentiated HL-60 cells compared to conditioned media from vehicle treated cells. E2 treatment markedly inhibited this effect.
Conclusion This study suggested that the vasoprotective effects of E2 in injured arteries may be mediated, at least in part, by an inhibitory effect on expression of the neutrophil chemoattactant CINC-2 in injured arterial SMCs, with resultant attenuation of neutrophil infiltration and inflammatory damage to the blood vessel.
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