Increased Expression of Endothelin-Converting Enzyme-1c Isoform in Response to High Glucose Levels in Endothelial Cells

Abstract

Endothelin-1 (ET-1) is both a potent vasoconstrictor and mitogenic factor that has been implicated as a cause of the micro- and macrovascular complications of diabetes mellitus. The pathway by which the high-glucose environment of diabetes mediates increased levels of endothelins has not been completely elucidated but appears to involve endothelin-converting enzyme (ECE-1), which converts inactive big ET-1 to active ET-1 peptide. To determine the effect of high glucose concentrations on the expression of ECE-1, hybrid endothelial cells (EA.hy926) and human umbilical vein endothelial cells (HUVEC) were both grown in various glucose concentrations. There was a 2-fold increase in ECE-1 immunoreactivity in the EA.hy926 cell line growing in medium containing 22.2 versus 5.5 mmol/l glucose after 24 h, which rose to greater than 20-fold after 5 days. Similar results were seen with HUVEC. Bradykinin or NG-nitro-L-arginine methyl ester did not change the effect of high glucose on ECE-1 protein expression. High glucose induced a 72 and 41% increase in total protein kinase C (PKC) activity in both EA.hy926 cells and HUVEC, respectively, and a 39, 49 and 109% elevation in PKC β1, β2 and δ expression, respectively, in EA.hy926 cells. The increase in ECE-1 expression was inhibited in both cell cultures by GF109203X (5 µmol/l), a general PKC inhibitor, while addition of 10 nmol/l phorbol myristic acid to EA.hy926 cells or HUVEC growing on medium containing 5.5 mmol/l glucose increased ECE-1 expression to a level similar to that of cells conditioned in high glucose. Human ECE-1 protein exists in four different isoforms, termed 1a, 1b, 1c and 1d. Northern blot analysis revealed that only ECE-1c isoform mRNA levels increased. Immunohistochemical staining of EA.hy926 cells grown in high glucose concentrations demonstrated an increase in the ECE-1c isoform, which occurred mainly in the plasma membrane. These results showed that the PKC pathway may play an important role in the glucose-mediated induction of ECE-1 expression. The main isoform to increase in response to high glucose was ECE-1c. This enzyme may be one of the factors contributing to the elevated ET-1 peptide levels observed in diabetes.

2-Amino-phenoxazine-3-one Attenuates Glucose-Induced Augmentation of Embryonic Form of Myosin Heavy Chain, Endothelin-1 and Plasminogen Activator Inhibitor-1 in Human Umbilical Vein Endothelial Cells

“…We confirmed the increased expression of ET-1 at the mRNA level in HUVECs with high ambient glucose (Fig. 3), as has been shown by Keynan et al 33) These results strongly support the views that the changes in the factors such as SMemb, ET-1 and PAI-1 in the endothelial cells may be associated with the angiogenesis and atherosclerosis under hyperglycemic conditions. Furthermore, we found that Phx-3, which is equivalent to questiomycin A, whose actions have not been clarified except for the anti-mycobacterial activity, 22,28) attenuated the increased expression of SMemb, ET-1 and PAI-1 in HUVECs caused by high ambient glucose (Fig.…”

“…ET-1, a potent vasoconstrictor, is implicated as a cause of the micro-and macrovascular complications of diabetes mellitus, 7,8) and is detected in large quantities in HUVECs treated with high glucose. 33) PAI-1, on the other hand accelerates the proliferation of HUVECs through VEGF activation, promoting the angiogenesis 34) and is detectable in HUVECs. 10) 800…”

Role of Endothelium in Vasomotor Responses to Endothelin System and Protein Kinase C Activation in Porcine Retinal Arterioles

“…Whether this is the case in other microvascular beds is unknown. Incubation of vascular endothelial cells with high glucose has been associated with increased ECE expression 32 and ET-1 production. 33 Moreover, since intravitreal treatment with pharmacologic blockade of ECE in vivo has been shown to improve retinal blood flow in early diabetes in rats, 34 increased ECE activity/expression in diabetic retinal arterioles possibly could contribute to the observed flow deficiency.…”

High glucose increases the expression of Gq/11α and PLC-β proteins and associated signaling in vascular smooth muscle cells

“…The levels of ET-1 and ANG II have been shown to be elevated in plasma from both type 1 and type 2 diabetes and also in experimental models (7,15,22), as well as in aortic endothelial and smooth muscle cells in the presence of high glucose (19,21,29), which may contribute to the vascular complications of diabetes.…”