The peptidase inhibitor CGS-26303 increases endothelin converting enzyme-1 expression in endothelial cells through accumulation of big endothelin-1.

BACKGROUND AND PURPOSE: CGS-26303 inhibits endothelin converting enzyme (ECE)-1 more specifically than phosphoramidon. We have studied the effect of CGS-26303 on ECE-1 expression in bovine aortic endothelial cells. METHODS: ECE-1 activity and big endothelin (ET)-1 levels were measured by ELISA, ECE-1 expression using western and northern blot and promoter activity using transfection assays. KEY RESULTS: ECE-1 activity was completely inhibited by CGS-26303 25 microM and phosphoramidon 100 microM. CGS-26303 and phosphoramidon, though not thiorphan, a neutral endopeptidase (NEP) inhibitor, stimulated ECE-1 expression in cells (maximal effect at 16 h, 25 microM). Cycloheximide abolished that effect. CGS-26303 induced ECE-1 mRNA expression and ECE-1 promoter activity. CGS-35066, a selective ECE-1 inhibitor, mimicked the effects of CGS-26303, suggesting that the effect was specific to ECE-1 inhibition. Big ET-1 accumulated in the cells and in the supernatants after CGS-26303 treatment. Neither exogenously added ET-1 nor the blockade of their receptors with bosentan modified ECE-1 protein. When big ET-1 was added to cells, significant increases in ECE-1 protein content and ECE-1 promoter activity were found. Bosentan did not block those effects. CGS-26303 did not modify prepro-ET-1 expression. CGS-26303 and big ET-1 induced the same effects in human endothelial cells, at lower doses. CONCLUSIONS: These results suggest that the accumulation of big ET-1 is responsible for the effects of CGS-26303 on ECE-1 and they did not depend on NEP blockade. Changes in ECE-1 protein after the administration of CGS-26303 could lead to a decreased response in long-term treatments.

Decreased nitric oxide synthesis in human endothelial cells cultured on type I collagen.

Endothelial dysfunction, considered as a defective vascular dilatation after certain stimuli, is characteristic of different pathological conditions, such as hypertension, atherosclerosis, or diabetes. A decreased synthesis or an increased degradation of nitric oxide (NO) has been postulated as the mechanism responsible for this alteration. The present experiments were designed to test the hypothesis that the presence of an abnormal extracellular matrix in vessel walls could be responsible for the decreased NO synthesis observed in these pathological conditions. Experiments were performed in cultured human umbilical vein endothelial cells (HUVECs) grown on type IV (Col. IV) or type I (Col. I) collagen. Cells seeded on Col. I showed decreased nitrite synthesis, nitric oxide synthase activity, eNOS protein content, and eNOS mRNA expression when compared with cells grown on Col. IV. Moreover, cells grown on Col. I failed to respond to glucose oxidase activation of the eNOS system. In both cases, the changes in the eNOS mRNA expression seemed to depend on the modulation of eNOS promoter activity. The downregulation of eNOS induced by Col. I was blocked by D6Y, a peptide that interferes with the Col. I-dependent signals through integrins, as well as by specific anti-integrin antibodies. Moreover, a decreased activation of integrin-linked kinase (ILK) may explain the effects observed in Col. I-cultured cells because the activity of this kinase was decreased in these cells and ILK modulation prevented the Col. I-induced changes in HUVECs. Taken together, these findings may contribute to explaining the basis of endothelial dysfunction in some vascular diseases.

Hydrogen peroxide regulation of bovine endothelin-converting enzyme-1.

Vascular injury leads to the production of reactive oxygen species (ROS), but the mechanisms by which ROS contribute to vascular pathology are not completely understood. We hypothesized that ROS increase endothelin converting enzyme (ECE-1) expression. We found that glucose oxidase (GO) increases ECE-1 mRNA, protein, and activity in bovine aortic endothelial cells. Catalase abolishes this effect. Glucose oxidase treatment of endothelial cells transactivates the ECE-1 promoter. The ECE-1 promoter element that mediates this response to GO is located between -444 and -216 bp. This region contains a STAT response element, and GO activates STAT-3 binding to this STAT response element. Our data suggest that STAT3 mediates hydrogen peroxide induction of ECE-1 expression.

The role of hydrogen peroxide in the contractile response to angiotensin II.

In the last years, reactive oxygen species (ROS) have been proposed as mediators of proliferative/hypertrophic responses to angiotensin II (Ang II), both in vivo and in vitro. However, the hypothesis that the Ang II-dependent cell contraction could be mediated by ROS, particularly H2O2, has not been tested. Present experiments were devoted to test this hypothesis and to analyze the possible mechanisms involved. Catalase (CAT) prevented the increased myosin light chain phosphorylation and the decreased planar cell surface area (PCSA) induced by 1 microM Ang II in cultured rat vascular smooth muscle cells (VSMC). This preventive effect of CAT was also detected when 1 microM platelet-activating factor (PAF) was used as a contractile agonist instead of Ang II. Similar results were found when using horseradish peroxidase as an H2O2 scavenger or cultured rat mesangial cells. In vascular smooth muscle cells, CAT modified neither the binding of labeled Ang II nor the Ang II-induced inositol 1,4,5-trisphosphate (IP3) synthesis. However, it completely abolished the Ang II-dependent calcium peak, in a dose-dependent fashion. CAT-loaded cells (increased intracellular CAT concentration over 3-fold) did not show either a decreased PCSA or an increased intracellular calcium concentration after Ang II treatment. Ang II stimulated the H2O2 synthesis by cultured cells, and the presence of CAT in the extracellular compartment significantly diminished the Ang II-dependent increased intracellular H2O2 concentration. The physiological importance of these findings was tested in rat thoracic aortic rings: CAT prevented the contraction elicited by Ang II. In summary, present experiments point to H2O2 as a critical intracellular metabolite in the regulation of cell contraction.

Mechanisms involved in the relaxation of bovine aortic endothelial cells.

The importance of endothelial cell contraction in the regulation of vascular biology is being increasingly recognized. Our group has demonstrated that reactive oxygen species, particularly hydrogen peroxide, which are released in pathological conditions such as ischemia-reperfusion, are able to induce contraction in bovine aortic endothelial cells (BAEC). The cGMP-dependent relaxation of contractile cells depends on the ability of the cyclic nucleotide to interfere with intracellular calcium; however, this is not the only mechanism involved. The present experiments were designed to analyse the mechanism by which cGMP induces relaxation in BAEC. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, as well as atrial natriuretic (ANP) and C-type natriuretic (CNP) peptides, activators of particulate guanylate cyclase, blunted the hydrogen peroxide-induced contraction of BAEC and myosin light chain phosphorylation. The inhibitory effect was more marked with SNP and CNP than with ANP, and the action of SNP and CNP were partially reversed by blocking soluble and particulate guanylate cyclases, respectively. Dibutyryl cGMP (db-cGMP), a cGMP analogue, mimicked the effect of SNP and CNP. Cyclic GMP-dependent protein kinase (cGK) protein levels and activity were measured. Hydrogen peroxide induced a significant reduction in cGK activity without any change in protein level. This effect was completely reversed by preincubation with db-cGMP. Calyculin A, a myosin light chain phosphatase inhibitor, prevented the cGMP-induced relaxation of BAEC. SNP, CNP and db-cGMP also partially prevented the hydrogen peroxide-induced increase in intracellular calcium levels. Catalase completely blocked this effect. In summary, the present results support a role for those metabolites which activate guanylate cyclases in the relaxation of BAEC, and suggest that the cGMP-induced BAEC relaxation could be due, at least partially, to the stimulation of cGK and/or myosin light chain phosphatase activity, and to calcium blockade.

Imbalance in endothelial vasoactive factors as a possible cause of cyclosporin toxicity: a role for endothelin-converting enzyme.

Cyclosporin A (CsA) is a powerful, widely used immunosuppressant, but it is not devoid of serious clinical side effects such as hypertension and nephrotoxicity. To clarify the mechanisms involved in the genesis of these side effects, we studied the effects of chronic CsA administration on the expression of some endothelial vasoactive factors in the aorta and kidney. For this purpose rats were treated for 30 days with 50 mg/kg/day CsA, and hypertension and renal insufficiency developed. In rats receiving CsA, the mRNA expression of pre-pro-endothelin-1 increased, whereas that of endothelial nitric oxide (NO) synthase decreased, both in the aorta and in the renal cortex (increases in pre-pro-endothelin-1 mRNA in aorta and renal cortex, respectively: 275%+/-18%, 300%+/-27%; decreases in endothelial NO synthase mRNA in aorta and renal cortex respectively: 40%+/-8%, 42%+/-6%). Moreover, long-term CsA treatment also induced an up-regulation of the endothelin-converting enzyme 1 mRNA expression (156% vs. control rats) in the renal cortex, with a significantly increased protein content and enzyme activity. In contrast, no changes were detected in endothelin-converting enzyme 1 mRNA expression in aortas from rats receiving the drug. This imbalance between endothelin-1 and NO systems could explain the hypertension and the deranged kidney function observed after long-term CsA treatment in rats.

Superoxide regulation of endothelin-converting enzyme.

Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.

Reactive oxygen species induce proliferation of bovine aortic endothelial cells.

The effects of reactive oxygen species (ROS) on different cellular types are variable. In some conditions they can be harmful metabolites, but they can also act as intracellular messengers that are able to activate different transcription factors. Based on previous reports in which ROS were shown to stimulate the proliferation of mesenchymal cells, this study was carried out to assess this effect on bovine aortic endothelial cells (BAECs). When cells were incubated with glucose oxidase (GO), an enzyme that generates H2O2 continuously, a significant increase in BAEC proliferation was detected. BAEC proliferation was measured by the incorporation of [3H]-thymidine in the DNA of BAECs, and also by an increase in the number of cells. The effect observed with GO was maximal at 8-24 h. Catalase abolishes proliferation. We also tested the ability of GO to phosphorylate tyrosine residues in endothelial cell proteins. A significant increase in tyrosine phosphorylation was found, which might constitute the molecular basis for proliferative effect of GO. In conclusion, these results demonstrate the ability of H2O2 to stimulate BAEC proliferation at least under certain experimental conditions. We suggest a general activation of the cascade of tyrosine phosphorylation as one of the possible cellular mechanisms responsible for GO-induced BAEC proliferation.

Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide.

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.

CsA and FK506 up-regulate eNOS expression: role of reactive oxygen species and AP-1.

Cyclosporine A (CsA) and FK506 increase endothelial nitric oxide synthase (eNOS) mRNA expression in cultured bovine aortic endothelial cells (BAEC). CsA appears to increase eNOS mRNA levels mainly by increasing the rate of transcription, although a small contribution of mRNA stabilization could not be ruled out. CsA and FK506 induced an increase of ROS synthesis with the fluorescent probe used, DHR123. The ROS generating system glucose oxidase (GO) increased the expression of eNOS mRNA in BAEC. This upregulation of eNOS mRNA by CsA or GO was abrogated by catalase. As AP-1 is a redox-sensitive transcription factor and the bovine eNOS promoter has an AP-1 consensus sequence, a role of this factor in the up-regulation of eNOS mRNA was studied. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. The potential participation of ROS and the transcription factor AP-1 in the regulation of eNOS gene expression is suggested.

Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells.

1. Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultured aortic endothelial cells (BAEC). As some effects of CsA may be mediated by reactive oxygen species (ROS), present experiments were devoted to test the hypothesis that the CsA-induced eNOS up-regulation could be dependent on an increased synthesis of ROS. 2. CsA induced a dose-dependent increase of ROS synthesis, with the two fluorescent probes used, DHR123 (CsA 1 microM: 305+/-7% over control) and H2DCFDA (CsA 1 microM: 178+/-6% over control). 3. Two ROS generating systems, xanthine plus xanthine oxidase (XXO) and glucose oxidase (GO), increased the expression of eNOS mRNA in BAEC, an effect which was maximal after 8 h of incubation (XXO: 168+/-21% of control values. GO: 208+/-18% of control values). The ROS-dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. 4. The effect of CsA on eNOS mRNA expression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. 5. The potential participation of the transcription factor AP-1 was explored. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. 6. The present results support a role for ROS, particularly superoxide anion and hydrogen peroxide, as mediators of the CsA-induced eNOS mRNA up-regulation. Furthermore, they situate ROS as potential regulators of gene expression in endothelial cells, both in physiological and pathophysiological situations.

Regulation of endothelial NO synthase expression by cyclosporin A in bovine aortic endothelial cells.

The introduction of cyclosporin A (CsA) is considered a cornerstone advance in immunosuppression. However, serious side effects such as hypertension have fostered an important body of research regarding their pathophysiology. Although the participation of several vasoactive factors in the hypertensive response has been described, recent attention has focused on endothelium-derived vasoactive factors, and several reports describe an overproduction of endothelin-1 and a deficient endothelium-dependent vasodilation. In the case of the latter, no definitive clues for the precise molecular mechanisms have been provided. We demonstrate that endothelial cells in culture synthesize more NO in the presence of CsA for 24 h in a concentration-response manner. This augmentation is correlated with a threefold increase in the endothelial constitutive NO synthase (ecNOS) transcript, which is time dependent and maximal at 24 h. The CsA-induced increase in ecNOS mRNA expression was blocked by actinomycin D but unaltered by cycloheximide. Levels of ecNOS protein were also enhanced by CsA after 24 h. These data establish that NO synthesis is moderately enhanced in endothelial cells exposed to CsA for long periods of time and describe a new mode of regulating ecNOS gene expression.

Recombinant human erythropoietin does not regulate the expression of endothelin-1 and constitutive nitric oxide synthase in vascular endothelial cells.

Erythropoietin (rHuEPO)-induced hypertension represents a clinical problem in hemodialysis units. However, the underlying molecular mechanisms of this side effect are still unclear. In order to assess the potential participation of the endothelial-derived vasoactive factors, nitric oxide and endothelin-1, bovine aortic endothelial cells were incubated in the presence of rHuEPO. The latter (0.1-10 U/ml, 1-24 h) did not modify the synthesis of nitric oxide and endothelin-1 or the expression of nitric oxide synthase and pre-pro-endothelin-1 mRNAs. These results suggest that endothelial-derived vasoactive factors are not a key component in the hypertensive response observed in certain patients after the administration of rHuEPO.