Regulation of the endothelin system by shear stress in human endothelial cells

The endothelial cells in situ are permanently exposed to shear stress, the dragging frictional force created by flowing blood. Physiological degrees of shear stress are involved in the regulation of vascular tone, and are considered as possible pathophysiological mechanisms for the localization of arteriosclerotic plaques (Glagov et al. 1988; Traub & Berk, 1998). Shear stress acting on endothelial cells is higher in arterial vessels (∼15-30 dyn cm−2 (∼1.5-3 N m−2)), compared with shear stress in venous vessels (∼1 dyn cm−2). These differences in shear stress may contribute to altered endothelial gene expression. A variety of genes have been described to be regulated by shear stress. These include genes encoding transcription factors, factors affecting coagulation, migration of leucocytes, smooth muscle proliferation, lipoprotein uptake and metabolism, cytoskeletal structure, apoptosis and release of vasoactive substances (Davies et al. 1997). Arterial laminar shear stress was shown to induce the endothelial generation of vasodilators nitric oxide (NO) and prostacyclin (Frangos et al. 1985; Uematsu et al. 1995). Interestingly, the vasoconstrictor angiotensin II generating angiotensin-converting enzyme is downregulated by arterial laminar shear stress (Rieder et al. 1997). However, the effect of shear stress on the expression of genes of the endothelin-1 system in human endothelial cells is less clearly understood. The endothelin family includes peptides which are the most potent vasoconstrictors known to date (Yanagisawa et al. 1988). Three endothelin isoforms have been identified: endothelin-1 (ET-1), ET-2 and ET-3 (Inoue et al. 1989a). Of these three isoforms, ET-1 was the first to be cloned, is the most abundant in circulation, and is the one studied in most detail. The biosynthesis of human ET-1 includes: (1) the expression of the 212 amino acid preproendothelin-1 (ppET-1) (Itoh et al. 1988; Inoue et al. 1989b); (2) the proteolytic cleavage to form big-ET-1 (39 amino acids) by a furin convertase (Denault et al. 1995); and (3) in the final key step, the cleavage of big-ET-1 into the active ET-1 peptide of 21 amino acids by the recently cloned metalloprotease endothelin-converting enzyme-1 (ECE-1) (Xu et al. 1994; Turner & Murphy, 1996). ET-1 binds to endothelin type A (ETA) and type B (ETB) receptors (Huggins et al. 1993). ETA receptors are present on vascular smooth muscle cells and induce ET-1-mediated vasoconstriction (Hosoda et al. 1991). On the other hand, most probably two ETB receptor subtypes are present on endothelial and vascular smooth muscle cells. The endothelial subtype mediates vasodilatation and is sensitive to the ETA and/or ETB non-selective antagonist PD142893 (Ogawa et al. 1991), while the smooth muscle cell-specific subtype causes vasoconstriction and is resistant to this antagonist (Warner et al. 1993). The data regarding the regulation of endothelin synthesis and release by shear forces in endothelial cells are controversial. Initial reports described a shear stress-dependent induction of endothelin production (Yoshizumi et al. 1989; Morita et al. 1993). Other groups found no significant changes in ET-1 release (Noris et al. 1995), or a downregulation of ppET-1 mRNA and endothelin release by shear stress in human and bovine endothelial cells (Sharefkin et al. 1991; Malek & Izumo, 1992; Kuchan & Frangos, 1993). Therefore, the time- and dose-dependent regulation of ppET-1 mRNA as well as endothelin peptide release by shear stress in human umbilical vein endothelial cells (HUVEC) was studied. In addition, the effect of shear stress on ECE-1 isoforms and the endothelial ETB receptor was analysed. Finally, we investigated the involvement of NO and protein kinases in shear stress-dependent regulation of the endothelin system in human endothelial cells.