(D-159) An In Vitro Model to Study Interaction Between Monocytes and Endothelial Cells in Pediatric Cardiopulmonary Bypass

Introduction:: Cardiopulmonary bypass (CPB) provides surgeons with a bloodless field to operate on the heart, while maintaining the circulation of the blood¹. Pediatric patients with congenital heart diseases (CHDs) often require CPB. In the US, CHDs are the most common type of birth defects and main cause of death in neonates, impacting 40,000 new-born babies each year². 25% of the patients will require immediate surgical intervention with CPB usage³. Despite the demand, current CPB conditions are not well-designed for pediatric patients due to their small size compared to the larger foreign material surface in the circuit⁴. Contact with the tubing material as well as wall shear stress at the material-blood interface can induce activation of inflammatory responses and mechanical trauma, respectively, in the blood cells of pediatric patients⁴. These CPB-induced injuries further lead to activation of leukocytes and endothelium, which can result in postoperative complications, for example, impaired cognitive development in pediatric patients⁵. In a previous study, we have shown that CPB shear specifically upregulated TNF-α and IL-8 in human monocyte-like THP-1 cells through calcium-dependent signaling pathways^6,7. In this study, we build an in vitro CPB model that includes endothelium to further study the interaction between leukocytes and neonatal microvascular endothelial cells. We hypothesize that THP-1 monocyte-like cells activated by CPB shear stress affect the barrier function of the endothelial monolayer and the endothelial cell phenotype.


Materials and Methods:: GFP positive THP-1 cells were maintained at a density of 1x10⁶ cells/ml. Human neonatal dermal microvascular endothelial cells (HNDMVECs) were cultured in a chamber slide or in the upper chamber of a tissue culture insert. An in-vitro CPB circuit was built with a 10-foot-long PVC tubing and a Masterflex pump purchased from Cole-Parmer (Figure 1A). On the day of experiment, THP-1 cells at a concentration of 2x10⁶ cells/ml were sheared in the in-vitro CPB circuit at a flow rate of 10 ml/min, shear stress at 2.1 Pa and a temperature at 36.5 °C. Statically cultured THP-1 cells in PVC flasks or Falcon tubes were used as a control. For ELISA assay, confluent HNDMVECs were treated with 2 hours CPB-treated or static THP-1 cells and incubated at 37°C with 5% CO₂ for 0.5, 1, 3 and 6 hours. Culture media were collected for ELISA of IL-1β, IL-6, IL-8 and TNF-α. Adhesion of CPB-treated and static THP-1 cells to the HNDMVECs was quantified at 1 hour. For transmigration assay, HNDMVECs cultured on the insert were treated with CPB-treated or static THP-1 cells. Cells that migrated to the bottom chamber were quantified after 12 hours. F-actin and VE-cadherin on the HNDMVECs was visualized by immunostaining. For qRT-PCR, 150 ng of RNA was extracted from HNDMVECs treated with human recombinant IL-8 and expression levels of E-selectin, ICAM1, and PECAM1 were measured.


Results, Conclusions, and Discussions:: CPB-treated THP-1 cells showed increased adhesion to the HNDMVECs monolayer than static THP-1 cells (Figure 1B). CPB treatment also promoted the number of THP-1 cells that migrated through endothelial monolayer (Figure 1C). In addition, coculture of CPB-treated THP-1 cells and endothelial cells led to disrupted VE-cadherin formation (Figure 1D1,2), F-actin reorganization (Figure 1D3,4), and upregulated IL-8 release within 1 hour (Figure 1E). Other inflammatory cytokine levels were not affected by the CPB treatment. Preincubating NDMVECs with Reparixin, an inhibitor of CXCR2 IL-8 receptor, inhibited the adhesion of sheared THP-1 cells to the NDMVECs (Figure 1F), while pretreating NDMVECs with IL-8 promoted the adhesion of unsheared cells (Figure 1G). These results suggest that Il-8 signaling pathway played an important role in mediating the adhesion of CPB shear-activated THP-1 cells to the endothelium. We further treated endothelial cells with IL-8 for 1 hour and found increased expression of two adhesion molecules, the intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM1) (Figure 1H).

In conclusion, we demonstrated THP-1 monocyte-like cells activated by shear stress in the in vitro CPB circuit are more likely to adhere to microvascular endothelial cells and infiltrate the endothelial monolayer. Evidently, these inflammation-related events may be specifically driven by IL-8, which is increasingly released in the culture media and leads to the upregulation of adhesion molecules on the endothelial cells. This study may reveal a CPB-specific mechanism of monocyte-endothelium activation and interaction in neonatal patients. Future study will focus on testing currently available drugs that can target the IL-8 signaling as well as formulating treatment plan to counter the inflammatory response after CPB.


Acknowledgements (Optional): : Research reported in this publication was supported by the UW Royalty Research Fund 143524 granted to Dr. Marta Scatena, R35 HL 139602-01 granted to Dr. Cecilia Giachelli and the National Institute of Biomedical Imaging And Bioengineering of the National Institutes of Health under Award Number T32EB032787. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Dr. Nathaniel Peters at UW W. M. Keck Microscopy Center for the necessary training and assistance in acquiring images on the Leica SP8X confocal microscope, which was funded by a S10 OD016240 NIH grant and the UW Student Technology Fee.

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