Thrombomodulin regulation in human brain microvascular endothelial cells in vitro: role of cytokines and shear stress.

Thrombomodulin (TM), an important determinant of blood vessel homeostasis, is expressed on the luminal vascular endothelial cell surface and is released into serum in response to circulatory signals. This includes the cerebrovascular endothelium, where the anti-coagulant and anti-inflammatory properties of TM are thought to be critical to the brain microcirculation and blood-brain barrier (BBB) integrity. Much is still unknown however about how circulatory stimuli may regulate TM activity within the brain microvasculature. To address this, the current short paper investigated the effects of opposing regulatory signals, namely cytokines (TNF-α, IL-6) and laminar shear stress, on the cellular levels and release of TM in cultured human brain microvascular endothelial cells (HBMvECs). Treatment of confluent HBMvECs with either TNF-α or IL-6 (100ng/ml, 18h) reduced TM protein levels by up to 70%, whilst inducing TM release into media by up to 4.4 and 5.5 fold, respectively. The effects of either cytokine (0-100ng/ml) on TM protein levels (6 or 18h) and release (0-18h) were also found to be concentration- and time-dependent. Either cytokine (100ng/ml, 24-72h) also reduced TM mRNA levels by >50%. When exposed to laminar shear stress for 24h at 8dyn/cm(2) (SI unit equivalent=0.8Pa), TM protein levels were upregulated by 65% in parallel with a 2-fold increase in TM mRNA levels. Shear stress also proved to be a much more potent stimulus for TM release from HBMvECs, yielding media TM levels of 1000pg/10(5) cells, when compared to 175 and 210pg/10(5) cells for TNF-α and IL-6, respectively, after parallel 18h treatments. Finally, shear-conditioned media was found to completely block thrombin-induced permeabilization of HBMvECs, confirming the functional efficacy of released TM. In summary, our data indicate that TM is differentially regulated within cultured HBMvECs by humoral and biomechanical signals.