(1026) Plasminogen Activator Inhibitor 1 promotes endothelial cell inflammatory response

Plasminogen Activator Inhibitor 1 (PAI1) is elevated in preeclampsia. In addition to its prothrombotic activity, emerging evidence suggests that PAI-1 directly contributes to endothelial dysfunction. Our objective was to determine whether PAI1 modulates the endothelial cell response to tumor necrosis factor alpha (TNFα), an inflammatory cytokine that is elevated in preeclampsia.


Study Design:

Human umbilical vein endothelial cells (HUVECs) were transfected with PAI1-specific silencing RNA or non-targeting control, followed by treatment with TNFα (1 ng/mL) for 16 hours. In other experiments, HUVECs were treated with purified recombinant PAI1 (40 ng/µL) prior to TNFα. To rescue the phenotype, HUVECs were infected with a lentivirus that over-expresses PAI1 prior to RNA silencing and TNFα treatment. Gene expression was measured by reverse transcription polymerase chain reaction (RT-PCR). Protein levels were determined by Western blot. Statistical analysis was performed in GraphPad Prism 9. Between-group differences were analyzed by two-way analysis of variance (ANOVA) with post-hoc analysis using Sidak’s test for multiple comparisons. Differences were considered significant if P < 0.05.


Results:

HUVECs treated with TNFα have upregulation of pro-inflammatory genes, but silencing PAI1 significantly blunted the TNFα response for genes including IL6 (Figure a), PTGS2 (b), CXCL8, and EDN1. Reduction of COX2 was confirmed by Western blot (c). Reciprocally, addition of recombinant PAI-1 increased the expression of PTGS2, CXCL8, and IL6. KLF2 is a transcription factor that promotes endothelial cell homeostasis. Silencing PAI-1 increased KLF2 transcription, and addition of recombinant PAI-1 decreased KLF2 transcription (d). Lentiviral over-expression of PAI-1 partially restored the response to TNFα by significantly reducing KLF2 expression in response to TNFα as seen by RT-PCR and Western blot (e, f).


Conclusion:

PAI-1 promotes the endothelial cell response to TNFα, which may be mediated by suppressing KLF2. These findings suggest that PAI-1 may contribute directly to endothelial dysfunction in preeclampsia.