Functional Change of the Endothelial Cells of the Umbilical Vein Following Anoxia and Reoxygenation

Lee, Do Sang; Hur, Jong Il; Sung, Gi Young; Song, Moo Hyung; Kim, Wook; Park, Il Young; Park, Jang Sang; Won, Jong Man

Abstract

BACKGROUND: Successful revascularization and reperfusion of ischemia are associated with high systemic complication rates and severe tissue injuries. Such complications with nonfunction are primarily related to the injuries that occur in the reperfusion process, but to date the exact mechanism is not clear. Anoxia or hypoxia and reoxygenation are principal components of ischemia and reperfusion (I/R) and have distinctive effects on the tissue. In the I/R injury model, endothelial cell injury is known to be an initial event, the microvasculature is highly sensitive, and the hyperadhesiveness of leukocytes to endothelial cells contributes to I/R-induced tissue injury.
METHODS
Experimental groups were divided into 4 groups: a control group without any treatment, an anoxia group (A-G) treated with anoxic air (93% N2, 5% CO2, 2% H2) for 20 minutes, reoxygenation group (RO-G) treated with 100% O2 for 90 minutes, and a superoxide dismutase (SOD) group treated with SOD just before reoxygenation. Endothelial cells were isolated from human umbilical vein and cultured in an M-199 medium. Their purity was determined by immunofluorescent staining of factor VIII related antigen, phase-contrast, and scanning electron microscopy. Using a microelectrode, radio immunoassay, and Emzyme-linked immunosorbent assay (ELISA), we studied the time-course changes of the levels of nitric oxide (NO), prostaglandin I2 (PGI2), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule (VCAM) of the 4 groups.
RESULTS
1) Endothelial cell detachment was found in the A-G and more detachment with lysis was found in the RO-G but no significant detachment and lysis were noted in the SOD group. 2) The concentration of NO in the A-G was lower than that of the control group (P<0.05). The NO concentration of the RO-G reached its highest level of 4809.01+/-444.69 nM/1x10(5) cells/ml at 30 minutes(P<0.005) and decreased after that. 3) The concentration of PGI2 in the A-G was higher than that of the control group (P<0.05). The PGI2 concentration of the RO-G reached its highest level of 64.25+/-2.39 pg/1x10(5) cells/ml at 45 minutes (P<0.005) and decreased after that. 4) The concentration of ICAM-1 in the A-G was higher than that of the control group (P<0.005). The ICAM-1 concentration of the RO-G reached its highest level of 7.18+/-0.62 ng/1x105 cells/ml at 15 minutes (P<0.005), then decreased to its lowest level of 2.53+/-0.31 ng/1x105 cells/ml at 60 minutes, but after 75 minutes increased again. 5) The concentration of VCAM in the A-G was higher than that of the control group (P<0.005). The VCAM concentration of the RO-G reached its highest level of 5.50+/-0.55 ng/1x10(5) cells/ml at 15 minutes (P<0.05), then decreased to its lowest level of 3.15+/-0.40 ng/1x10(5) cells/ml at 45 minutes, but after 60 minutes increased again. 6) The SOD group showed little change of NO, PGI2, ICAM-1, and VCAM concentration compared with both the A-G and the RO-G.
CONCLUSION
This study showed that cell destruction in the reoxygenation group was more severe than that in the anoxia group and that the endothelial cell function of the reoxygenation group decreased signi ficantly compared with that of the anoxia group. In the anoxia and the reoxygenation groups, the levels of the two adhesion molecules ICAM-1 and VCAM increased faster than those of NO and PGI2 and the change in the level of ICAM-1 was more sensitive than that (in the level) of VCAM. In reoxygenation group, SOD treatment could inhibit the changes in the levels of NO, PGI2, ICAM-1, and VCAM.