Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering
Anneke Hecke1, Hilary Brooks, Matthieu Meryet-Figuière
1Laboratoire des Défenses Antivirales et Antitumorales, UMR 5124 CNRS, Montpellier, France.
Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.
Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.
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