OC 75.2 - Endothelial cell origin determines the fibrinolytic response to proinflammatory stimuli and downstream thrombus stability

Background: Inflammatory cytokines, observed in pathogenic infections and chronic inflammatory states, may differently alter the microenvironment and shape thrombus architecture and stability in the vasculature.

Aims: Examine the impact of endothelial cells (ECs) from different vascular beds in dictating the fibrinolytic response to proinflammatory stimuli.

Methods: Human umbilical vein ECs (HUVECs) and coronary artery ECs (HCAECs) cultured at 100 or 500 s-1 respectively, were treated ±10 ng/mL TNFα for 24 h. Expression and secretion of plasminogen activator inhibitor-1 (PAI 1) and tissue-type plasminogen activator (tPA) were quantified by qPCR or ELISA. Plasma clot lysis (CL) on ECs ±300 pM tPA was monitored at 405 nm. Real-time whole blood thrombus dynamics ±30 nM tPA on endothelialised biochips was monitored by inclusion of PE-anti-CD42b and AlexaFluor647-fibrin(ogen) and mean fluorescent intensity determined.

Results: HCAECs secreted significantly more PAI-1 than HUVECs, however, basal tPA secretion was similar (Figure 1). TNFα-stimulation of HCAECs significantly increased PAI-1 and tPA expression and secretion. In contrast, there was no effect on HUVECs. CL was significantly delayed on HUVECs (10±3 min) and HCAECs (14±3 min) compared to the no cell control (Figure 2) and was normalised by inclusion of an inhibitory antibody to PAI 1. TNFα-stimulation further delayed CL on HCAECs in a PAI-1-dependent manner. Whole blood thrombus composition on endothelialised biochips varied according to EC type and shear rate; dense fibrin was evident on HUVECs, whereas on HCAECs thrombi were platelet-rich. TNFα-stimulation increased fibrin accumulation on both EC types. The proinflammatory environment promotes a hypofibrinolytic state in thrombi formed on HCAECs, consistent with increased PAI 1 secretion.

Conclusion(s): HCAECs respond more potently to a proinflammatory environment than HUVECs, as evidenced by the high PAI-1 expression and secretion. Endothelial-derived PAI-1 exacerbates hypofibrinolysis which is amplified by proinflammatory stimuli. Determining the response of vascular beds to inflammatory stimuli could permit more targeted antithrombotic strategies.