| Summary: | 碩士 === 國立臺灣大學 === 化學工程學研究所 === 94 === During atherogenesis, inflammation plays an important role. Thrombomodulin (TM), in vascular endothelial cells, has anti-coagulation and anti-inflammation properties. Transcription factor, Kruppel-like factor 2 (KLF2) has been shown to participate in the regulation of the expression of TM and endothelial nitric oxide synthase and thus is important for regulating thrombotic function. Endothelial cells are constantly under the influence of flow-induced hear stress and the atherosclerotic lesions are closely related to this hemodynamic effect. In the present study, we focused on the study of gene expression of TM and
KLF2 in endothelial cells exposed to shear stress. Human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) were used to probe into the signaling mechanisms involved in this shear-induced TM and KLF2 expression. ECs exposed to shear stress of 25 dyn/cm2 transiently induced the TM mRNA expression with peak induction about 1.6-folds after exposure to shear stress for 4 to 6 hrs before it returned to basal level. However, KLF2 mRNA expression in those ECs was found to be a sustained one with peak induction more than 85-folds. While mRNA expression of TM and KLF2 were significantly induced in shear-induced ECs, protein expression of TM and KLF2 had no obvious increase. Furthermore, TM mRNA and KLF2 mRNA were
significantly increased in ECs exposed to higher shear stress (25 dyn/cm2) in contrast to those ECs exposed to low shear stress (4.5 dyn/cm2) indicating that TM mRNA induction is sensitive to shear force. However, the protein expression of TM and KLF2 from shear-treated ECs show no significant difference as compared to those control ECs. This indicates that protein stability may be involved in those shear-treated ECs. ECs under shear flow condition constantly release nitric oxide (NO). The role of NO in this shear-induced TM and KLF2 mRNA expression was examined. ECs exposed to a NO donor (NOC18) significantly suppressed the TM and KLF2 mRNA level with a dose-dependent manner. Consistently, shear stress to ECs increased the TM promoter activity. Interestingly, ECs pretreated with an eNOS inhibitor (L-NAME) did not enhance the shear-induced TM promoter activity.
Furthermore, the protection effect of shear flow on cytokine-treated ECs was examined. ECs treated with tumor necrosis factor (TNF) greatly reduced the TM and KLF2 expression. ECs pretreated with TNF followed with shear flow, however, significantly attenuated the TNF-induced
suppression of TM mRNA levels. In contrast, the basal KLF2 and the shear-induced KLF2 mRNA levels were not affected by TNF pretreatment.
In summary, shear flow to ECs increases the TM and KLF2 mRNA expression in a dose-dependent manner. However, this induction can not be reflected by the TM protein levels indicating that the stability of TM mRNA and protein may be involved. Furthermore, shear flow exerts its protective effect by attenuating the TNF-induced suppression of TM mRNA expression. ECs treated with NO suppress the TM and KLF2 mRNA expression. Although the detailed up-regulation mechanism of TM remains unclear, this study suggests that shear flow plays an important role in regulating TM expression and consequently affects endothelial integrity.
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