The Effect of Anoxia and Reoxygenation on Synthesis of Nitric Oxide, ICAM-1 and VCAM of Umbilical Vein Endothelium

PURPOSE: The reperfusion flowing ischemia are associated with high systemic complication rates and severe local tissue injuries, which are primarily related to the reperfusion process. Anoxia or hypoxia and reoxygenation are principal components of ischemia and reperfusion (I/R) and in I/R injury model endothelial cell injury is known to be a initial event. The purpose of this study is to examine the changes of the levels of nitric oxide (NO), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM) of the cultured endothelial cells following anoxia and reoxygenation. METHODS: Experimental groups were divided into 4 groups: control group, without any treatment; anoxia group (A-G), treatment with anoxic air (93% N2, 5% CO2, 2% H2) for 20 minutes; reoxygenation group (RO-G), treatment with 100% O2 for 90 minutes; superoxide dismutase (SOD) group, treatment with SOD just before reoxygenation. Endothelial cells were isolated from human umbilical vein and cultured in M-199 medium. Using microelectrode and ELISA we studied the time-course changes of the levels of NO, ICAM-1 and VCAM of 4 groups. RESULTS: The concentration of NO in A-G was lower than that of control group (P<0.05). NO concentration of RO-G at 30 minutes reached the highest level of 4809.01 444.69 nM/1 105 cells/ml (P<0.005) and after then decreased. The concentration of ICAM-1 in A-G was higher than that of control group (P<0.005). ICAM-1 concentration of RO-G at 15 minutes reached the highest level of 7.18 0.62 ng/1 105 cells/ml (P<0.005) and then decreased to the lowest level but after 75 minutes increased again. The concentration of VCAM in A-G was higher than control group (P<0.005). VCAM concentration of RO-G at 15 minutes reached the highest level of 5.50 0.55 ng/1 105 cells/ml (P<0.05) and then decreased to the lowest level at 45 minutes, but after 60 minutes the concentration increased again. SOD group showed a little change of NO, ICAM-1 and VCAM concentration comparing with both A-G and RO-G. CONCLUSION: This study showed that endothelial cell function of reoxygenation group decreased significantly compared with anoxia group. In anoxia and reoxygenation group, the levels of two adhesion molecules of ICAM-1 and VCAM increased faster than those of NO and the change of the level of ICAM-1 was more sensitive than that of VCAM. In reoxygenation group SOD treatment could inhibit the changes of the levels of NO, ICAM-1 and VCAM.

The Effect of Anoxia and Reoxygenation on Synthesis of Nitric Oxide, ICAM-1 and VCAM of Umbilical Vein Endothelium

PURPOSE: The reperfusion flowing ischemia are associated with high systemic complication rates and severe local tissue injuries, which are primarily related to the reperfusion process. Anoxia or hypoxia and reoxygenation are principal components of ischemia and reperfusion (I/R) and in I/R injury model endothelial cell injury is known to be a initial event. The purpose of this study is to examine the changes of the levels of nitric oxide (NO), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM) of the cultured endothelial cells following anoxia and reoxygenation. METHODS: Experimental groups were divided into 4 groups: control group, without any treatment; anoxia group (A-G), treatment with anoxic air (93% N2, 5% CO2, 2% H2) for 20 minutes; reoxygenation group (RO-G), treatment with 100% O2 for 90 minutes; superoxide dismutase (SOD) group, treatment with SOD just before reoxygenation. Endothelial cells were isolated from human umbilical vein and cultured in M-199 medium. Using microelectrode and ELISA we studied the time-course changes of the levels of NO, ICAM-1 and VCAM of 4 groups. RESULTS: The concentration of NO in A-G was lower than that of control group (P<0.05). NO concentration of RO-G at 30 minutes reached the highest level of 4809.01 444.69 nM/1 105 cells/ml (P<0.005) and after then decreased. The concentration of ICAM-1 in A-G was higher than that of control group (P<0.005). ICAM-1 concentration of RO-G at 15 minutes reached the highest level of 7.18 0.62 ng/1 105 cells/ml (P<0.005) and then decreased to the lowest level but after 75 minutes increased again. The concentration of VCAM in A-G was higher than control group (P<0.005). VCAM concentration of RO-G at 15 minutes reached the highest level of 5.50 0.55 ng/1 105 cells/ml (P<0.05) and then decreased to the lowest level at 45 minutes, but after 60 minutes the concentration increased again. SOD group showed a little change of NO, ICAM-1 and VCAM concentration comparing with both A-G and RO-G. CONCLUSION: This study showed that endothelial cell function of reoxygenation group decreased significantly compared with anoxia group. In anoxia and reoxygenation group, the levels of two adhesion molecules of ICAM-1 and VCAM increased faster than those of NO and the change of the level of ICAM-1 was more sensitive than that of VCAM. In reoxygenation group SOD treatment could inhibit the changes of the levels of NO, ICAM-1 and VCAM.

Hormonal and Cytokine Regulation of ICAM-1 gene in FRTL-5 Thyroid Cells: Cloning and Analysis of 5-Regulatory Region of Rat ICAM-1 Gene / 대한내분비학회지

BACKGROUND: We have found abnormal expression of ICAM-1 in thyroid follicular cells from patients with Graves disease and Hashimoto disease. In this report, we present the hormonal regulation of ICAM-1 mRNA expression and the primary structure of 5-regulatory region which is important for transcriptional regulation of ICAM-1 gene. A I.S kb fragment of the 5-regulatory sequences are identified and linked to luciferase as a reporter. METHOD: Those reporter constructs were used to evaluate the expression in response to cytokines and hormones. Deletion analysis of 1.8 kb fragment of ICAM-1 promoter in FRTL-5 cells provide the evidence for the existence of several regulatory elements of enhancer and silencer in ICAM-1 gene transcription in thyroid cells. RESULTS: ICAM-1 mRNA is easily detected by Northern analysis using total RNA from FRTL-5 cells regardless of culture conditions. The transcripts of rat ICAM-1 showed single band of 2.6 kb in length. The FRT cells which was come from early FRTL cell culture did not show ICAM-1 mRNA with usual Northern analysis, We found differential regulation of ICAM-1 RNA level in different culture condition in FRTL-5 cells, The cells maintained at 3H (no hydrocortisone, no insulin, no TSH) condition showed the highest expression level compared to 4H, 5H, or 6H medium. Hydrocortisone markedly decreased the ICAM-1 RNA and insulin partially recovered the hydrocortisone induced repression. TSH which is important in growth and function of FRTL-5 cells could independently downregulate the ICAM-1 RNA levels. Forskolin (10 mM) could mimic the action of TSH on ICAM-1 mRNA. TNF-a and interferon-y increase ICAM-1 expression in FRTL-5 thyroid cells. TSH/forskolin inhibited maximal expression of ICAM-1 by TNF-a and interferon-r. Promoter activity of the ICAM-1 gene was positively regulated by cytokines, TNF-a and IFN-r and negatively regulated by thyroid stimulating hormone. The addition of TSH and FSK caused a 50% decrease in ICAM-1 promoter activity within 24 hour. The TSH and FSK action was mapped at 175 bp and 97 bp of the start of translation. The mutant construct pCAM-175 delGAS which has no GAS sequence showed no TSH mediated suppression of promoter activity. CONCLUSION: These findings suggested that hormones and cytokines differentially regulated the ICAM-1 gene expression and TSH downregulated ICAM-1 gene transcription by inhibiting the activation of IFN-r induced transcription factors which can bind the GAS of ICAM-1 promoter.