Time Dependent Changes of Nitric Oxide Synthesis Following Anoxia and Reoxygenation of Endothelial Cells of Umbilical Vein

High systemic complication rates and severe tissue injuries are associated with more successful revascularization and reperfusion of ischemia rather than ischemia itself. It is regarded as a "ischemia- reperfusion (I/R) injury". It is well recognized that the microvasculature is highly sensitive to I/R and that the initial damage of endothelial cells contributes to I/R-induced tissue injury. The endothelial cells serve as an important modulator of vascular homeostases by secreting vasoactive materials. One of the important product of endothelial cells, nitric oxide helps to maintain hemostasis through its involvement in coagulation, platelet activation, vascular tone regulation. But The exact role of nitric oxide and time dependent change of its synthesis during the reperfusion injury are not clear. In this study, endothelial cells were isolated from human fetal umbilical vein and cultured in M-199 medium. We observed morphological change of the endothelial cells and time dependent change of nitric oxide synthesis following anoxia and reperfusion injuries. The results were as follows: 1) More significantly, the endothelial cells of anoxia group were flattened and detached than contol group. More severe detachment of endothelial cells was founded in R-O group than anoxia group. There is no significant differences in morphological changes between allopurinol group and anoxia group. 2) The concentration of NO in the anoxia group (2511.62 428.60nM/1 105 cells/ml) was lower than that of the control group (3505.14 192.95nM/1 105 cells/ml) (P<0.005). The NO concentration of the reoxygenation group reached its highest level of 2953.14 90.98 nM/1 105 cells/ml at 180 minutes (P<0.05) and decreased thereafter. There was no significant differences in NO concentration between allopurinol group and anoxia group. In conclusion, morphologic damage of endothelial cells in reoxygenation group was significantly increased compared with anoxia group. Nitric oxide syntheses in reoxygenation group and anoxia group were decreased compared with control group. More advanced study will be needed to elucidate the detailed mechanism of time-dependent change of nitric oxide synthesis during the reperfusion injury.

Morphologic and Time Dependent Changes of Nitric Oxide Synthesis Following Anoxia and Reoxygenation of the Endothelial Cells of the Umbilical Vein

PURPOSE: In order to clarify the exact role of nitric oxide for a ischemia-reperfusion (I/R) injury, we observed morphologic change of endothelial cells and a time dependent change of nitric oxide synthesis following anoxia and reperfusion injuries. METHODS: The experimental groups were divided into 4 sub-groups: a control group without any treatment, an anoxia group treated with anoxic air (93% N2, 5% CO2, 2% H2) for 50 minutes, a reoxygenation group treated with 100% O2 for 480 minutes, and an allopurinol group treated with allopurinol immediately prior to reoxygenation. Endothelial cells were isolated from a human fetal umbilical vein and cultured in M-199 medium. We observed a morphological change of the endothelial cells with inverted light microscopy and we studied the time dependent change of nitric oxide synthesis with microelectrode following anoxia and reperfusion injuries. RESULTS: Most significantly, the endothelial cells of the anoxia group were more flattened and detached than those of the control group. A more severe detachment of endothelial cells was found in the reoxygenation group than in the anoxia group. There was no significant difference in the morphological change between the allopurinol group and the anoxia group. Additionally, the concentration of NO in the anoxia group (2511.62 428.60 nM/1 105 cells/ml) was lower than that of the control group (3505.14 192.95 nM/1 105 cells/ml) (P<0.005). The NO concentration of thereoxygenation group reached its highest level of 2953.14 90.98 nM/1 105 cells/ml at 180 minutes (P<0.05) and decreased thereafter. There was no significant difference in the NO concentration between the allopurinol and anoxia groups. CONCLUSION: The morphologic damage of endothelial cells in the reoxygenation group was significantly increased as compared with the anoxia group. Nitric oxide syntheses in the reoxygenation and anoxia groups was decreased as compared with the control group.

Morphologic and Time Dependent Changes of Nitric Oxide Synthesis Following Anoxia and Reoxygenation of the Endothelial Cells of the Umbilical Vein

PURPOSE: In order to clarify the exact role of nitric oxide for a ischemia-reperfusion (I/R) injury, we observed morphologic change of endothelial cells and a time dependent change of nitric oxide synthesis following anoxia and reperfusion injuries. METHODS: The experimental groups were divided into 4 sub-groups: a control group without any treatment, an anoxia group treated with anoxic air (93% N2, 5% CO2, 2% H2) for 50 minutes, a reoxygenation group treated with 100% O2 for 480 minutes, and an allopurinol group treated with allopurinol immediately prior to reoxygenation. Endothelial cells were isolated from a human fetal umbilical vein and cultured in M-199 medium. We observed a morphological change of the endothelial cells with inverted light microscopy and we studied the time dependent change of nitric oxide synthesis with microelectrode following anoxia and reperfusion injuries. RESULTS: Most significantly, the endothelial cells of the anoxia group were more flattened and detached than those of the control group. A more severe detachment of endothelial cells was found in the reoxygenation group than in the anoxia group. There was no significant difference in the morphological change between the allopurinol group and the anoxia group. Additionally, the concentration of NO in the anoxia group (2511.62 428.60 nM/1 105 cells/ml) was lower than that of the control group (3505.14 192.95 nM/1 105 cells/ml) (P<0.005). The NO concentration of thereoxygenation group reached its highest level of 2953.14 90.98 nM/1 105 cells/ml at 180 minutes (P<0.05) and decreased thereafter. There was no significant difference in the NO concentration between the allopurinol and anoxia groups. CONCLUSION: The morphologic damage of endothelial cells in the reoxygenation group was significantly increased as compared with the anoxia group. Nitric oxide syntheses in the reoxygenation and anoxia groups was decreased as compared with the control group.

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

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.

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.