Deformation-induced synthesis of endothelin-1 (ET-1) in endothelial cells exposed to high blood pressure may play an important role in vein graft disease and in restenosis following percutaneous transluminal angioplasty. Effective inhibitors of preproendothelin ET-1 (ppET-1) processing to ET-1 are not available, and blockade of ppET-1 expression may therefore emerge as an alternative therapeutic approach. To evaluate this, we investigated deformation-sensitive transcription factors controlling ppET-1 expression in both native (rabbit carotid artery and jugular vein) and cultured endothelial cells (EC; porcine aorta and human umbilical vein). Deformation of both native and cultured endothelial cells for 6 h resulted in a marked increase in ET-1 synthesis which was preceded by a transient (30-60 min) activation of transcription factors activator protein-1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP) beta and/or delta. A decoy oligodeoxynucleotide directed against AP-1 inhibited deformation-induced ppET-1 expression in the rabbit jugular vein as well as in porcine aorta EC and human umbilical vein EC but not in the rabbit carotid artery. Subsequent reporter gene analyses with different rat ppET-1 promoter-luciferase constructs transiently transfected into porcine aorta EC identified a single AP-1 binding site at -110 to -100 bp as the primary response element for deformation-induced ppET-1 expression. Moreover, a C/EBP-specific decoy oligodeoxynucleotide abolished ppET-1 expression in the endothelium of the rabbit carotid artery, but not in the jugular vein where basal ET-1 synthesis was greatly enhanced instead. These findings suggest that the key transcription factors controlling deformation-induced ppET-1 expression in endothelial cells are blood vessel rather than species-specific. In humans, adjunct treatment with an AP-1-specific decoy oligodeoxynucleotide may prove be an interesting gene therapeutic option for the above cardiovascular interventions.
Abstract-To investigate the hypothesis that high blood pressure activates the endothelin system in the vessel wall, isolated segments of the rabbit carotid artery were subjected to different levels of perfusion pressure. Both preproendothelin-1 (ppET-1) mRNA abundance and intravascular ET-1 peptide content were strongly upregulated on raising the intraluminal pressure from 90 to 160 mm Hg for 3 to 12 hours, and this increase in ppET-1 mRNA occurred predominantly in the endothelial cells. Endothelin-converting enzyme-1 and endothelin A receptor (ET A -R) expression were pressure-insensitive, whereas that of the ET B -R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ET B -R expression required RNA synthesis because they were abolished by actinomycin D. The nuclear signaling mechanisms involved therein, however, appeared to be different. Thus, the pressure-induced expression of ppET-1 and activation of CCAAT-enhancer binding proteins  and ␦ were blocked by the tyrosine kinase inhibitor herbimycin A, whereas ET B -R expression and the nuclear translocation of activator protein-1 were abolished by the protein kinase C inhibitor Ro 31-8220. One consequence of these presumably deformation-induced changes in gene expression was an increased rate of apoptosis of the smooth muscle cells in the media that if transferable to the situation in human blood vessels may contribute to hypertension-induced arterial remodeling. (Hypertension. 2000;35:648-654.)
Abstract-Upregulation of endothelin-1 (ET-1) synthesis in venous bypass grafts in response to arterial levels of blood pressure may play a major role in graft failure. To investigate this hypothesis, isolated segments of the rabbit jugular vein were perfused at physiological (0 to 5 mm Hg) and nonphysiological (20 mm Hg) levels of intraluminal pressure.As judged by reverse transcription-polymerase chain reaction analysis (mRNA level), neither endothelin-converting enzyme nor endothelin A receptor expression appeared to be pressure sensitive. In contrast, there was a profound and time-dependent increase in endothelial prepro-ET-1 mRNA and intravascular ET-1 abundance (by ELISA) as well as in smooth muscle endothelin B receptor mRNA and functional protein (by superfusion bioassay) on raising the perfusion pressure from 5 to 20 mm Hg, but not from 0 to 5 mm Hg, for up to 12 hours. Video microscopy analysis revealed that the segments were distended by 75% at 5 mm
Abstract-Deformation-induced endothelin-1 synthesis in endothelial cells may contribute to the intimal hyperplasia of venous bypass grafts. ACE inhibitors and angiotensin II type 1 (AT 1 ) receptor antagonists are capable of reducing vein graft disease. Therefore, the effects of these drugs on endothelial preproendothelin-1 (ppET-1) and smooth muscle endothelin B receptor (ET B -R) expression were investigated in isolated perfused segments of the rabbit jugular vein. Pretreatment with ramiprilat (0.3 mol/L) or irbesartan (0.01 to 1 mol/L) had no effect on basal ppET-1 or ET B -R expression but markedly attenuated the deformation-induced expression of these gene products, and these effects were reversed by the B 2 receptor antagonist icatibant (Hoe 140) and by the NO synthase inhibitor N G -nitro-L-arginine. Candesartan (1 mol/L) mimicked the inhibitory effect of irbesartan. Moreover, reporter gene analysis with a rat ppET-1 promoter-luciferase construct transiently transfected into porcine aortic cultured endothelial cells revealed that the inhibitory effect of both ramiprilat and irbesartan on deformation-induced ppET-1 expression is species independent and mediated at the level of transcription. In addition, RT-PCR analysis detected only AT 1 receptor expression in the endothelium-intact rabbit jugular vein, and neither irbesartan nor ramiprilat affected endothelial NO synthase expression. Thus, ACE inhibitors and AT 1 receptor antagonists are capable of suppressing deformation-induced gene expression in the vessel wall in both an autocrine (ppET-1) and a paracrine (ET B -R) manner via a common mechanism of action that constitutes a B 2 receptor-mediated increase in endothelial NO release. Key Words: blood pressure Ⅲ endothelin-1 Ⅲ gene expression Ⅲ graft failure Ⅲ nitric oxide E xcessive mechanical strain after exposure to arterial levels of blood pressure could trigger the vasculopathy of venous bypass grafts and lead to graft failure. 1 Moreover, pressure overload of the vessel wall seems to be involved in both hypertension-induced arterial remodeling 2 and restenosis after PTCA. 3 Among the various mediators implicated in these putatively deformation-induced changes in smooth muscle cell (SMC) phenotype, endothelin-1 (ET-1), a potent comitogen for vascular SMCs, may play a pivotal role. [3][4][5] Thus, rhythmic deformation has been shown to enhance the synthesis of ET-1 in cultured endothelial cells (ECs), and this effect is presumed to occur at the level of transcription of the preproendothelin-1 (ppET-1) gene. 6,7 To investigate whether a pressure-induced deformation of ECs also affects ppET-1 gene expression in the vessel wall in situ, we developed an experimental model in which isolated rabbit blood vessel segments are perfused at different levels of intraluminal pressure. With the aid of this model, we were recently able to demonstrate that ET-1 synthesis in the endothelium of both the carotid artery and the jugular vein of the rabbit is markedly upregulated on exposure to a supraphysiological increas...
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