Fig. 1. Relative mRNA
AT 101 of t-PA, u-PA and PAI-1 in HUVECs exposed to low, moderate or high shear stress with or without TNF-α for 24 h. Statistical comparisons are made relative to low shear stress without TNF-α. ?P have shown that cytokines and mechanical stress regulate the transcription of a large and diverse set of EC genes [13] and [14]. However, few studies have focused on the interplay between cytokines and shear stress [2], [4], [5], [6] and [7] and to our knowledge none have studied the interplay between cytokines and tensile stress. The uniqueness of the present study is that the interplay between various combinations of biomechanical profiles and the cytokine TNF-α is studied within the same perfusion system. To achieve this goal, the present study used a specially built perfusion system [8]. Our findings indicate that moderate and high shear stress can modify the EC sensitivity to inflammatory stress while low shear stress and tensile stress cannot.Previously, we have studied how EC gene
expression is affected by various combinations of pulsatile and static tensile stress [10]. To our surprise, anti- and prothrombotic genes in ECs seemed to be insensitive to tensile stress. We have previously shown that essential hypertension, which we thought mainly induced an increased tensile stress, was associated with a defective t-PA release capacity in vivo [15] and [16]. Previous studies have shown that genes non-responsive to TNF-α were shifted to a TNF-α sensitive state by biomechanical stress [17]. Therefore, we hypothesised that cytokine stimulation may cause EC to become sensitive to different combinations of tensile stress. However, we could not observe such a mode shift in EC by cytokine stimulation. None, of the studied genes modulated their response to TNF-α by simultaneous tensile stress stimulation.On the contrary, shear stress was a more potent stimulus. Moderate and high shear stress modulated the response to cytokines in all the studied genes. The effect of shear stress and TNF-α on VCAM-1 and eNOS are in accordance with previous reports [4], [5] and [7]. The uniqueness of our study approach was that the effect of different shear stress levels in combination with TNF-α was addressed. The set-up enabled a direct comparison of the effect of TNF-α with unstimulated EC exposed to identical shear stress, which has been a shortage in previous reports. This is important in validating the intergroup significance for different stimulations, e.g. thrombomodulin, eNOS and VCAM-1 in this study. Regarding thrombomodulin, you can easily exaggerate the meaningfulness of the 2–3-fold induction of shear stress on TNF-α stimulated ECs. However, when comparing it to non-inflamed shear stressed ECs the relative quantitative effects are modest. Inflammatory stress was a quantitatively stronger stimulus than shear stress on thrombomodulin, eNOS and VCAM-1.It is interesting that shear stress in combination with TNF-α can have bidirectional effects on different genes, i.e. shear stress attenuated cytokine-induced expression of VCAM-1, opposed the effects on thrombomodulin and eNOS gene expression, additively induced PAI-1 expression, whereas shear stress seemed to totally block the cytokine-suppressive and inducing effects on t-PA and u-PA, respectively. Shear stress showed a dose-dependent suppressing effect on t-PA. TNF-α in combination with low shear stress demonstrated a similar suppressing effect on t-PA, as high shear stress alone. However, our results indicate that moderate and high shear stress neutralize the suppressing effect observed by TNF-α. Shear stress seemed not to have any regulatory effect on u-PA gene expression, while a modestly inducing effect was observed with TNF-α. As for t-PA, high shear stress seemed to block the effect of TNF-α on u-PA. Interestingly, TNF-α and low shear stress showed a similar inducing effect on PAI-1 mRNA as moderate and high shear stress. TNF-α in combination with moderate or high shear stress seemed to have additive effects in inducing PAI-1. Regarding thrombomodulin, shear stress counteracted the suppressive effect exerted by TNF-α on its gene expression.
