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Effects of alpha-thrombin on superoxide dismutase levels in human cerebral microvascular endothelial cells
C M Van Gelder1, J M Doherty, M A Shatos
1University of Vermont College of Medicine, Department of Biochemistry, Burlington, USA.
Background:
Sequelae of traumatic brain injury include generation of oxygen-free radicals and fibrin deposition, which worsen the initial injury. Superoxide dismutases (SODs) scavenge and bind to the free-radical superoxide anion (O2-), potentially defending against oxidative stress. In the present study, we investigated the production of SOD within human cerebral microvascular endothelial (HCME) cells after exposure to alpha-thrombin, hypothesizing that manganese SOD (MnSOD) expression is increased. Our aims were to determine whether alterations in SOD are observed at the mRNA level, to examine whether a particular species is preferentially expressed, and to determine the requirement of the active site of alpha-thrombin.
Methods:
HCME cells were characterized and grown to confluence. Control cells and cells exposed to 10 nmol/L alpha-thrombin were harvested for mRNA isolation using reverse transcriptase-polymerase chain reaction. Quantitation of mRNA production determined the levels of copper-zinc SOD and MnSOD. Active site blocked alpha-thrombin was used as a negative control and determined the specificity of the response.
Results:
The cells in culture were identified as endothelial after fulfilling criteria, such as positive immunocytochemical staining for factor VIII/von Willebrand factor antigen and binding of Ulex europaeus agglutinin-1 lectin. Levels of MnSOD mRNA were elevated at all time points in response to alpha-thrombin, whereas the cytosolic form was undetectable. HCME cells that were exposed to active site-blocked alpha-thrombin produced mRNA levels of MnSOD that were increased above those of controls, but this increase was half that of mRNA levels of MnSOD produced by HCME cells that were exposed to alpha-thrombin.
Conclusion:
Our study showed for the first time that alpha-thrombin partially modulates SOD in HCME cells, causing a preferential increase in MnSOD. Further investigation into secondary brain injury will provide insights into the role of alpha-thrombin in the mechanism of free radical-induced alterations, potentially improving the outcome of patients with head injury.