Detection of Inflammatory Circulating Endothelial Cells Using Human Umbilical Vein Endothelial Cells Detached from Culture Dishes by Tumor Necrosis Factor-alpha as Control Cells

Junpei Tochikubo, Naoyuki Matsuda, Yoshimi Ota, Tomoko Higashi, Yudai Takatani, Masato Inaba, Yushi Adachi and Norihiko Shiiya

Background: Systematic inflammatory response syndrome (SIRS) with disseminated intravascular coagulation (DIC) is a severe disorder in critically ill patients and closely related to progression of vascular endothelial injury. Early diagnosis is required to prevent the progression of organ dysfunction. Circulating endothelial cells (CECs) might increase in vascular endothelial injury and play an important role in DIC diagnosis. However, the CEC detection method has not been standardized. This study aimed to establish a method for CEC detection in a critical care setting. We used human umbilical vein endothelial cells (HUVECs) detached from culture dishes by tumor necrosis factor-alpha (TNF-α) as control cells for CECs.
Methods: Cultured HUVECs were incubated in medium with TNF-α (100 ng/mL), and cells detached from culture dishes after 24 h were used as TNF-HUVECs. Cell surface molecules of normal HUVECs, TNF-HUVECs, and blood cells were analyzed using flow cytometry (FC) to search for appropriate markers for detecting CECs. Normal HUVECs and TNF-HUVECs were added to the blood and detected using FC and the immunobead method (IB) for comparing two methods. CECs were measured in healthy volunteers and intensive care unit (ICU) patients using FC.
Results: CD146 and CD105 were highly expressed in HUVECs and superior for separation of UVECs from whole blood cells. Mean detection rates of normal HUVECs were 75% in FC and 82% in IB. However, mean detection rates of TNF-HUVECs were 64% in FC and 27% in IB (p < 0.05). Mean CEC counts from 20 healthy volunteers and 16 ICU patients were 2.8 cells/mL and 4.3 cells/mL, respectively. In one ICU patient with SIRS-induced DIC, CECs were elevated by 49 cells/mL.
Conclusion: CD146 and CD105 are suitable for detecting endothelial cells from blood. FC is superior to IB for detecting endothelial cells in severe inflammatory states.