Effects of 17β-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine
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Objectives: When administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17β-estradiol (17β-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs). Methods: VSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction. Results: Following pre-treatment for 24 hours, 17β-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17β-E2 (10−7 M) inhibited PAI-1 gene expression, and ICI 182,780—a specific estrogen receptor antagonist—blocked the effects of 17β-E2 on the PAI-1 protein. 17β-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17β-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription. Conclusions: In VSMCs stimulated with lysoPC, 17β-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17β-E2 suppressed PAI- 1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.
Abstract
Objectives: When administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17β-estradiol (17β-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs). Methods: VSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction. Results: Following pre-treatment for 24 hours, 17β-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17β-E2 (10−7 M) inhibited PAI-1 gene expression, and ICI 182,780—a specific estrogen receptor antagonist—blocked the effects of 17β-E2 on the PAI-1 protein. 17β-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17β-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription. Conclusions: In VSMCs stimulated with lysoPC, 17β-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17β-E2 suppressed PAI- 1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.
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