An In vitro Model to Study the Monocyte-Endothelial Interaction in Pediatric Cardiopulmonary Bypass

Abstract

Cardiopulmonary bypass (CPB) is a technique used in open-heart surgeries to create a bloodless field in the heart and maintain circulation in pediatric patients with congenital heart diseases (CHDs). Due to unoptimized settings in pediatric CPB, perioperative and postoperative complications often occur. However, there is no effective treatment to counteract them. Therefore, a CPB-specific mechanism needs to be determined for the development of targeted therapy. Shear stress in the bypass tubing was identified as a major factor that induces mechanical trauma and inflammatory response of the blood components. Previous study recognized monocytes as the blood cell population that responded the most to the CPB shear. However, the inflammatory response of CPB also involves endothelium that actively mediates the monocyte insult. In this dissertation, I further investigated the endothelial response to CPB-activated monocytes and cytokines derived from CPB-activated monocytes. Monocyte adhesion and transmigration assays were developed as a part of the in vitro CPB mode. Monocytes activated by CPB shear are more likely to adhere to and transmigrate through the monolayer of human neonatal dermal microvascular endothelial cells (HNDMVECs). Treatment of CPB-activated monocytes disrupted the VE-cadherin and cytoskeleton arrangement of confluent endothelium. The monocyte-endothelial cell interaction is accompanied by increasing IL-8 level in the co-culture media. The correlation between IL-8 and monocyte adhesion is studied by loss and gain of function experiments. Inhibiting IL-8 signaling on endothelial cells with reparixin resulted in significant decrease of monocyte adhesion. Treating HNDMVECs with IL-8 induced the adhesion of naïve monocytes. Results of qPCR showed IL-8 treatment selectively upregulated intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression in the HNDMVECs, which mediated the monocyte adhesion. Inhibiting the IL-8 expression in HNDMVECs resulted in reduced monocyte adhesion, suggesting that IL-8 produced by endothelial cells played an important role during cellular interactions. To investigate the cytokines derived from CPB-activated monocytes and the effect of anti-inflammatory drugs including dexamethasone and FK506, the conditioned media from CPB-activated monocytes with or without drug treatment were collected and assayed. ELISA showed CPB shear caused increasing production of IL-8 and tumor necrosis factor α (TNF-α) from the monocytes. Both cytokines expression and production can be reduced by dexamethasone and FK506 with the FK506 being more effective. Luminex multiplex assay showed that dexamethasone and FK506 alter the cytokine release profile of sheared monocytes in different ways. I further studied the effect of conditioned media on endothelial cells. Conditioned media derived from sheared monocytes disrupted the VE-cadherin and cytoskeleton arrangement in confluent HNDMVECs. Live and dead assays showed that treating HNDMVECs with shear conditioned media led to lower viability. Finally, the shear conditioned media treatment resulted in increasing naïve monocyte adhesion mediated by upregulated adhesion molecules including E-selectin and ICAM-1. The study in this dissertation provided insight into the mechanisms that led to CPB-induced inflammatory response of monocytes and endothelial cells. Therapies that target the IL-8 and TNF-α pathways may be developed to treat CPB-related complications.