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Captopril and enalaprilat decrease antioxidant defences in human endothelial cells and are unable to protect against apoptosis
Cell Biology International 27 (2003) 825–830
Cell Biology International www.elsevier.com/locate/cellbi
Captopril and enalaprilat decrease antioxidant defences in human endothelial cells and are unable to protect against apoptosis Agne`s Mailloux 1, Benjamin Deslandes 1, Michel Vaubourdolle 1, Bruno Baudin 1,2* 2
1 Service de Biochimie A, Hoˆpital Saint-Antoine AP-HP, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France GRECAN (EA-1772) UFR des Sciences Pharmaceutiques, Universite´ de Caen-Basse Normandie, boulevard Becquerel, 14032 Caen Cedex, France
Received 27 January 2003; revised 12 May 2003; accepted 8 July 2003
Abstract Angiotensin-converting enzyme (ACE) inhibitors were shown to improve endothelial dysfunction in various human diseases and some of these inhibitors have been proposed as enhancers of antioxidant defences. We measured glutathione peroxidase (GPX), superoxide dismutase (SOD) and malondialdehyde (MDA) in human endothelial cells treated with captopril or enalaprilat, two ACE inhibitors, and we showed that both inhibitors decreased GPX and SOD activities but not MDA, the end-product of lipoperoxidation. Captopril and enalaprilat were also unable to protect against etoposide-induced apoptosis in endothelial cells, indicating that they cannot be considered as protective drugs for the endothelium, in particular in clinical situations involving oxidative stress or apoptosis. Moreover, when used at high concentration captopril, but not enalaprilat, was toxic for endothelial cells with both necrotic and apoptotic effects. 2003 Elsevier Ltd. All rights reserved.
1. Introduction Endothelial dysfunctions are common denominators in vascular diseases, as particularly induced by changes in anti-haemostatic properties of the endothelium, changes in the control of the vascular tone and of the permeability to plasma lipoproteins, as well as the acquisition of a hyper-adhesiveness to blood leucocytes and increased cytokine and growth factor production. These maladaptive changes in endothelial functions in response to abnormal stimuli are involved in the pathogenesis of atherosclerosis, hypertension, heart failure, diabetes and glomerulonephritis (De Caterina, 2000; Drexler, 1994, 1997; Stehouwer et al., 1997), also in the toxicity of drugs such as anticancer drugs in the course of cancer chemotherapy (Baudin, 1995; Lazo, 1986). Endothelial dysfunction can be modified by angiotensinconverting enzyme (ACE) inhibitors, ACE being an ecto-enzyme anchored in plasma membrane of endothelial cells controlling vascular tone and permeability by converting angiotensin I into angiotensin II and by * Corresponding author E-mail address: [email protected] (B. Baudin). 1065-6995/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00162-8
degrading bradykinin (Baudin, 2002). In particular ACE inhibitors can improve endothelial dysfunction in patients with coronary disease, dyslipidemia, hypertension and IgA nephropathy whereas more disparate results were found in patients with heart failure and diabetes (Brown and Vaughan, 1998; Hernandez et al., 1998; Mancini, 2000). The mechanisms of this specific protection of the vascular endothelium is not yet understood although some ACE inhibitors have been described as enhancers of endogenous antioxidant defences (De Cavanagh et al., 1995, 2000, 2001). In this report, we analysed the role of two ACE inhibitors, captopril and enalaprilat (the de-esterified active form of the pro-drug enalapril), to protect human umbilical vein endothelial cells (HUVECs) in culture by enhancing antioxidant defences. We have shown that both ACE inhibitors not only did not enhance glutathione peroxidase (GPX) or superoxide dismutase (SOD) activities in endothelial cells but on the contrary decreased GPX activity; they also did not modify malondialdehyde (MDA) concentration measured as thiobarbituric acid-reactive substances. Moreover captopril used at high concentration was cytotoxic and led to endothelial cell apoptosis. As a corollary, both
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ACE inhibitors were unable to protect the cells against apoptosis triggered by etoposide, an anticancer drug and an inductor of apoptosis in both tumour cells and endothelial cells (Lock and Stribinskiene, 1996; Mailloux et al., 2001).
2. Materials and methods 2.1. Endothelial cell culture Primary cultures of HUVECs were obtained in 35 mm Petri dishes according to the method of Jaffe et al. (1973) using collagenase (Boehringer-Mannheim, Germany) digestion (at 0.2%) and growth in Medium 199 supplemented with 20% fetal calf serum (Invitrogen, Cergy-Pontoise, France) in a humidified 95% air/5% CO2 atmosphere at 37 (C (Mailloux et al., 2001). Cells were characterized by a typical cobblestone appearance by optical microscopy and by the presence of von Willebrand factor and angiotensin-converting enzyme (ACE) (Baudin et al., 1997). Only primary cultures were used; two days after cell confluency, the medium was changed and supplemented for 24 h, although not in controls, with captopril (Sigma, St Louis, MO, USA) or enalaprilat (Parke-Davis France, Orle´ans), at concentrations used in in vitro studies (Bartosz et al., 1997; Odaka and Mizuochi, 2000), without calf serum but 1% Ultroser G medium (Sigma). In the experiment to search for a protection of apoptosis by captopril, the cells were pre-treated with ACE inhibitor for 24 h; then the cells were rinsed in medium and treated for another 24 h in 0.1 mg/ml etoposide containing medium. At the end of the experiments, culture supernatants were collected for determination of lactate dehydrogenase (LDH) activity. For the assessment of oxidative stress by both determinations of GPX and SOD activities, and by MDA concentration measurement, 2–3106 cells were scraped in pure water after two washes in PBS and studied by phase-contrast microscopy. Protein concentration was also determined on these samples using protein assay kit DCII from Bio-Rad (Hercules, CA, USA) and bovine serum-albumin as the standard. The samples were kept frozen at 80 (C until assay. 2.2. Determination of oxidative stress and relative enzyme activities Malondialdehyde (MDA) was fluorimetrically measured after reaction with thiobarbituric acid using the MDA kit from Sobioda (Grenoble, France) and fluorescence reading on the model F-2000 spectrofluorimeter from Hitachi (Tokyo, Japan); malondialdehydetetra-ethyl-acetal was used as a standard. MDA was expressed in nmol/mg of proteins (Mailloux et al., 2001). Glutathione peroxidase (GPX) activity was determined according to the method of Paglia and Valentine (1967)
using reduced glutathione and cumene-hydroperoxide as substrates. The decrease of the absorbance at 340 nm is proportional to the reduction of the oxidized glutathione by NADPH,H+ as catalysed by glutathionereductase (kit Ransel RS 505 from Randox, Antrim, UK); for this determination the model DU-70 spectrophotometer from Beckman-Coulter (Fullerton, CA, USA) was used with a kinetic mode program and regulation at 37 (C; the control (Ransel SC 692) was within the reference values. This assay specifically determines selenium-GPX activity. Superoxide dismutase (SOD or CuZn-SOD) was determined by a method using a competition between SOD and an iodo-nitrotetrazolium (INT) chloride for dismutation of superoxide anion as produced by the action of xanthine oxidase on xanthine; SOD decreases the transformation of INT into a red formazan dye which was measured at 505 nm on DU-70 spectrophotometer at 37 (C (kit Ransod SD 125 from Randox); a standard curve was established from purified SOD provided by the manufacturer; the control (Ransod SD 126) was within the reference values. Both GPX and SOD activities were expressed in units per milligram of proteins (U/mg). 2.3. Other determinations Lactate dehydrogenase (LDH) activity was determined in cell supernatants on a Beckman-Coulter automated Synchron CX-4 CE using pyruvate as the substrate and at 37 (C; activity was expressed in mU/24 h/106 cells. For the assessment of apoptosis, we used the Cell Death ELISA kit from Boehringer based on the detection of oligonucleosomes (18020 bp) characteristic of internucleosomal DNA fragmentation during apoptosis; the specificity for oligonucleosomes was assessed by double reactivity with anti-histones and anti-DNA monoclonal antibodies. For this determination, cellular monolayers were rinsed in serum-free medium and then lysed in a specific solution (Mailloux et al., 2001). Results were expressed as the differential absorbance between 405 and 490 nm; a control provided by the manufacturer was positive in the series. 2.4. Statistical analysis The results were expressed as the meanSD of at least four determinations. Non-parametric Mann– Whitney statistics available in Graphpad Prism were used to establish the significance of between-group differences; P values <0.05 were considered significant. 3. Results 3.1. Effects of ACE inhibitors on oxidative stress and antioxidant defences in HUVECs Neither enalaprilat nor captopril modified the intracellular content in MDA of HUVECs. In contrast, when
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3.2. Effects of ACE inhibitors on necrosis and apoptosis of HUVECs Used at 1 or 10 mmoles/l enalaprilat was not toxic towards HUVECs as well as morphologically (not shown) as judged by LDH activity whereas captopril was toxic at 10 mmoles/l with cell retraction, decrease in protein concentration (not shown) and LDH activity increment in cell supernatants (Fig. 2). To better understand the toxic effect of captopril on HUVECs, 10 mmoles/l captopril was applied on to confluent cells for 24 h; thereafter the oligonucleosomes characteristic of apoptosis were measured showing that, used at this concentration, captopril induced apoptosis in HUVECs (Fig. 3); used at a lower concentration, it was ineffective as for necrosis. Moreover, captopril was unable to protect HUVECs towards apoptosis subsequently triggered by 0.1 mg/ml etoposide, a concentration of this anticancer drug known to induce apoptosis in this model (Mailloux et al., 2001); the effects of etoposide and captopril were cumulative by enhancing both necrosis and apoptosis (Fig. 3). As for captopril, enalaprilat was unable to protect HUVECs against etoposide-induced apoptosis but, conversely to captopril, it did not trigger necrosis nor apoptosis.
4. Discussion
Fig. 1. Effects of 10 mmol/l captopril or enalaprilat on malondialdehyde (MDA), glutathione peroxidase (GPX) activity, and superoxide dismutase (SOD) activity in HUVECs after 24 h of incubation with the drugs.
used at 10 mmoles/l both ACE inhibitors decreased GPX activity; lower concentrations were ineffective (not shown). Captopril but not enalaprilat decreased SOD activity; a lower concentration of captopril was not effective (Fig. 1). Thus, both inhibitors tend to decrease rather to enhance oxidant defences in HUVECs and are unable to diminish the spontaneous lipid peroxidation as determined by the measure of thiobarbituric acidderivative substances. ACE inhibitors do not seem to be useful for protection towards an oxidative stress in endothelial cells.
Angiotensin-converting enzyme (ACE) inhibitors are widely prescribed for the treatment of hypertension and congestive heart failure; they also delay the progression of chronic renal failure and of diabetic nephropathy (Brown and Vaughan, 1998; Mancini, 2000). In addition, they have been shown to retard the development of atherosclerosis in experimental models (Ambrosioni et al., 1992; Jacobsson et al., 1994; Kowala et al., 1998) and to improve endothelial dysfunction in patients with coronary artery disease (Pfeffer et al., 1992; The SOLVD Investigators, 1992). The mechanism underlying these pharmacological effects of ACE inhibitors are not fully understood; the potentiation of bradykinin and of free radical scavenger action by the ACE inhibitors have been postulated. In particular, De Cavanagh et al. (2000) showed that enalapril and captopril treatments increased antioxidant enzymes and non-enzymatic antioxidants in several mouse tissues; furthermore, in erythrocytes the augmentation of antioxidants by ACE inhibitors was associated with protection against oxidant damage. On the other hand, ACE inhibitors decrease bradykinin degradation; bradykinin is a potent vasodilator known to stimulate the release of nitric oxide (Cannon, 1998); however the participation of nitric oxide as a modulator of oxidant defences is controversial since, in different systems, it was able to either increase (Kuo et al., 1996; Moellering et al., 1999) or decrease (Mak et al., 1996) antioxidant defences.
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Fig. 2. Concentration-dependent cytotoxicity of captopril, but not of enalaprilat, towards HUVECs as judged on lactate dehydrogenase (LDH) activity increment in cell supernatants after 24 h of incubation with the drugs. LDH is expressed in mU/24 h/106 cells.
Fig. 3. Effects of 10 mmoles/l captopril on necrosis and apoptosis in HUVECs pre-treated or not with etoposide; LDH activity in cell supernatants is expressed in mU/24 h/106 cells and oligonucleosomes in cell lysates (Oligo-C) in A as stated in Materials and methods. *P<0.05 versus 0/0; P<0.05 versus 0.1/0 and versus 0/10 (etoposide/captopril).
In this study we report that enalapril and captopril are unable to enhance antioxidant enzymes, namely glutathione peroxidase (GPX) and superoxide dismutase (SOD), in human vascular endothelial cells, these particular cells being the major site of synthesis of ACE and an important target of ACE inhibitors (Baudin et al., 1997; Baudin and Drouet, 1989). Both ACE inhibitors were also unable to decrease the spontaneous intracellular production of malondialdehyde (MDA), the main product of lipid peroxidation, that has been shown to be increased, for example, in endothelial cells treated by doxorubicin, an anticancer drug (Mailloux et al., 2001). Doxorubicin, a quinone-containing anthracycline antineoplastic drug, is used in the treatment of a wide spectrum of human cancers, but the development of severe cardiac toxicity compromises its clinical
effectiveness (Lefrak et al., 1973). It is metabolically activated to a free radical state and interacts with molecular oxygen to generate superoxide radicals (Sinha et al., 1987; Yin et al., 1998); it is particularly the case in vascular and cardiac endothelial cells (Kang et al., 2000; Keizer et al., 2000; Mailloux et al., 2001). Therefore the protective effects of antioxidants and metal chelators against doxorubicin-induced cardio-toxicity have been investigated (Doroshow et al., 1981; Unverferth et al., 1985; Van Vleet et al., 1980). In particular, temocapril and zofenopril, new ACE inhibitors, were shown to prevent doxorubicin-induced cardiomyopathy in a rat model (Sacco et al., 2001; Tokudome et al., 2000), but our results are not consistent with a putative role of ACE inhibitors in endothelial cell protection related to the enhancement of antioxidant defences. Temocapril
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could better prevent endothelial dysfunction via nitric oxide production as shown in diabetes-induced endothelial abnormalities (Pieper and Siebeneich, 2000). Captopril also appeared to be beneficial as a protective agent against cardio-toxicity related to doxorubicin (Al-Shabanad et al., 1998). This ACE inhibitor, the first described, is a thiol compound which can react with superoxide anion radical acting as a scavenger, or with hydroxyl radical (Al-Shabanad et al., 1997; Aruoma et al., 1991; Bartosz et al., 1997; Benzie and Tomlison, 1998); but captopril as well as enalaprilat, which does not contain a thiol group, failed to decrease MDA concentration in endothelial cells. Moreover, captopril appeared to be toxic for endothelial cells when used at high concentration and this toxicity could be rather related to its thiol group reacting as a mercaptan for cellular proteins and reduced glutathione, the main intracellular non-enzymatic antioxidant defence. Captopril was also described as preventing apoptosis by interfering with Fas signalling, or with other pathways for activation of cell death, but in other cell types than endothelial cells (Odaka and Mizuochi, 2000; Uhal et al., 1998); the blockade of angiotensin II function may play a role in the regulation of cell death by apoptosis (Filippatos and Uhal, 2003). Thereby we tried to protect endothelial cell by ACE inhibitors against etoposide induced-apoptosis, etoposide being an anti-cancer drug that we previously described for inducing caspase-3 activity (a pro-apoptotic enzyme) and for decreasing bcl-2 concentration (an anti-apoptotic protein) in human endothelial cell (Mailloux et al., 2001). Captopril as well as enalaprilat was unable to protect the cells, and conversely captopril induced apoptosis when it was used at the concentration that also led to cell necrosis with LDH release in supernatant. We also showed (personal communication) that other thiol-containing substances such as N-acetylcysteine were unable to protect HUVECs towards apoptosis, that was also the case for nitroso-captopril which brings the dual potential for NO production and ACE inhibition; we also showed that etoposide did not induce apoptosis by activating Fas pathway although HUVECs expressed Fas. Nevertheless, the concentrations of both ACE inhibitors used in this study are above the maximal plasma concentrations that can be obtained after per os administration (Richer et al., 1984). A number of drugs largely used in therapy are toxic towards endothelial cells, and particularly in vitro from concentrations that can be obtained in vivo to concentrations usually not reached (Baudin, 1995; Baudin et al., 1996; Mailloux et al., 2001). Taking all data together, we can conclude that both ACE inhibitors, captopril and enalapril, cannot be considered as protective drugs for the endothelium, either against an oxidative stress as in the course of cancer chemotherapy, for example using doxorubicin, or against apoptosis as induced by many anti-cancer drugs,
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also in clinical situations where both oxidative stress and apoptosis have been involved, e.g. atherosclerosis, ischaemic diseases, auto-immunity and inflammation. When ACE inhibitors can protect against endothelial dysfunction, other pathways than oxidative stress and apoptosis might be engaged, for example nitric oxide production leading to vasorelaxation or cytoprotective prostaglandins production since, in clinical situations, they are able to improve endothelial dysfunction, for example in the treatment of allogenic bone marrow transplantation and for the prevention of liver venoocclusive disease (Scrobohaci et al., 1991). Otherwise, ACE inhibitors could protect against doxorubicininduced cardiac impairment at the level of cardiac myocytes but not the cardiac endothelial cells (Maeda et al., 1997). Our results also point to potential toxicity towards vascular endothelium of captopril when used at high dose; even when such concentrations are obtained with difficulty in human therapy, captopril has been used at higher concentrations than those reached in vivo in some protocols such as for transplant preservation (Fisher et al., 2000). The toxicity of captopril can be related to its thiol group since enalaprilat was not toxic towards endothelial cells nor induced apoptosis, even at high concentration.
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Cell Biology International www.elsevier.com/locate/cellbi
Captopril and enalaprilat decrease antioxidant defences in human endothelial cells and are unable to protect against apoptosis Agne`s Mailloux 1, Benjamin Deslandes 1, Michel Vaubourdolle 1, Bruno Baudin 1,2* 2
1 Service de Biochimie A, Hoˆpital Saint-Antoine AP-HP, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France GRECAN (EA-1772) UFR des Sciences Pharmaceutiques, Universite´ de Caen-Basse Normandie, boulevard Becquerel, 14032 Caen Cedex, France
Received 27 January 2003; revised 12 May 2003; accepted 8 July 2003
Abstract Angiotensin-converting enzyme (ACE) inhibitors were shown to improve endothelial dysfunction in various human diseases and some of these inhibitors have been proposed as enhancers of antioxidant defences. We measured glutathione peroxidase (GPX), superoxide dismutase (SOD) and malondialdehyde (MDA) in human endothelial cells treated with captopril or enalaprilat, two ACE inhibitors, and we showed that both inhibitors decreased GPX and SOD activities but not MDA, the end-product of lipoperoxidation. Captopril and enalaprilat were also unable to protect against etoposide-induced apoptosis in endothelial cells, indicating that they cannot be considered as protective drugs for the endothelium, in particular in clinical situations involving oxidative stress or apoptosis. Moreover, when used at high concentration captopril, but not enalaprilat, was toxic for endothelial cells with both necrotic and apoptotic effects. 2003 Elsevier Ltd. All rights reserved.
1. Introduction Endothelial dysfunctions are common denominators in vascular diseases, as particularly induced by changes in anti-haemostatic properties of the endothelium, changes in the control of the vascular tone and of the permeability to plasma lipoproteins, as well as the acquisition of a hyper-adhesiveness to blood leucocytes and increased cytokine and growth factor production. These maladaptive changes in endothelial functions in response to abnormal stimuli are involved in the pathogenesis of atherosclerosis, hypertension, heart failure, diabetes and glomerulonephritis (De Caterina, 2000; Drexler, 1994, 1997; Stehouwer et al., 1997), also in the toxicity of drugs such as anticancer drugs in the course of cancer chemotherapy (Baudin, 1995; Lazo, 1986). Endothelial dysfunction can be modified by angiotensinconverting enzyme (ACE) inhibitors, ACE being an ecto-enzyme anchored in plasma membrane of endothelial cells controlling vascular tone and permeability by converting angiotensin I into angiotensin II and by * Corresponding author E-mail address: [email protected] (B. Baudin). 1065-6995/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00162-8
degrading bradykinin (Baudin, 2002). In particular ACE inhibitors can improve endothelial dysfunction in patients with coronary disease, dyslipidemia, hypertension and IgA nephropathy whereas more disparate results were found in patients with heart failure and diabetes (Brown and Vaughan, 1998; Hernandez et al., 1998; Mancini, 2000). The mechanisms of this specific protection of the vascular endothelium is not yet understood although some ACE inhibitors have been described as enhancers of endogenous antioxidant defences (De Cavanagh et al., 1995, 2000, 2001). In this report, we analysed the role of two ACE inhibitors, captopril and enalaprilat (the de-esterified active form of the pro-drug enalapril), to protect human umbilical vein endothelial cells (HUVECs) in culture by enhancing antioxidant defences. We have shown that both ACE inhibitors not only did not enhance glutathione peroxidase (GPX) or superoxide dismutase (SOD) activities in endothelial cells but on the contrary decreased GPX activity; they also did not modify malondialdehyde (MDA) concentration measured as thiobarbituric acid-reactive substances. Moreover captopril used at high concentration was cytotoxic and led to endothelial cell apoptosis. As a corollary, both
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ACE inhibitors were unable to protect the cells against apoptosis triggered by etoposide, an anticancer drug and an inductor of apoptosis in both tumour cells and endothelial cells (Lock and Stribinskiene, 1996; Mailloux et al., 2001).
2. Materials and methods 2.1. Endothelial cell culture Primary cultures of HUVECs were obtained in 35 mm Petri dishes according to the method of Jaffe et al. (1973) using collagenase (Boehringer-Mannheim, Germany) digestion (at 0.2%) and growth in Medium 199 supplemented with 20% fetal calf serum (Invitrogen, Cergy-Pontoise, France) in a humidified 95% air/5% CO2 atmosphere at 37 (C (Mailloux et al., 2001). Cells were characterized by a typical cobblestone appearance by optical microscopy and by the presence of von Willebrand factor and angiotensin-converting enzyme (ACE) (Baudin et al., 1997). Only primary cultures were used; two days after cell confluency, the medium was changed and supplemented for 24 h, although not in controls, with captopril (Sigma, St Louis, MO, USA) or enalaprilat (Parke-Davis France, Orle´ans), at concentrations used in in vitro studies (Bartosz et al., 1997; Odaka and Mizuochi, 2000), without calf serum but 1% Ultroser G medium (Sigma). In the experiment to search for a protection of apoptosis by captopril, the cells were pre-treated with ACE inhibitor for 24 h; then the cells were rinsed in medium and treated for another 24 h in 0.1 mg/ml etoposide containing medium. At the end of the experiments, culture supernatants were collected for determination of lactate dehydrogenase (LDH) activity. For the assessment of oxidative stress by both determinations of GPX and SOD activities, and by MDA concentration measurement, 2–3106 cells were scraped in pure water after two washes in PBS and studied by phase-contrast microscopy. Protein concentration was also determined on these samples using protein assay kit DCII from Bio-Rad (Hercules, CA, USA) and bovine serum-albumin as the standard. The samples were kept frozen at 80 (C until assay. 2.2. Determination of oxidative stress and relative enzyme activities Malondialdehyde (MDA) was fluorimetrically measured after reaction with thiobarbituric acid using the MDA kit from Sobioda (Grenoble, France) and fluorescence reading on the model F-2000 spectrofluorimeter from Hitachi (Tokyo, Japan); malondialdehydetetra-ethyl-acetal was used as a standard. MDA was expressed in nmol/mg of proteins (Mailloux et al., 2001). Glutathione peroxidase (GPX) activity was determined according to the method of Paglia and Valentine (1967)
using reduced glutathione and cumene-hydroperoxide as substrates. The decrease of the absorbance at 340 nm is proportional to the reduction of the oxidized glutathione by NADPH,H+ as catalysed by glutathionereductase (kit Ransel RS 505 from Randox, Antrim, UK); for this determination the model DU-70 spectrophotometer from Beckman-Coulter (Fullerton, CA, USA) was used with a kinetic mode program and regulation at 37 (C; the control (Ransel SC 692) was within the reference values. This assay specifically determines selenium-GPX activity. Superoxide dismutase (SOD or CuZn-SOD) was determined by a method using a competition between SOD and an iodo-nitrotetrazolium (INT) chloride for dismutation of superoxide anion as produced by the action of xanthine oxidase on xanthine; SOD decreases the transformation of INT into a red formazan dye which was measured at 505 nm on DU-70 spectrophotometer at 37 (C (kit Ransod SD 125 from Randox); a standard curve was established from purified SOD provided by the manufacturer; the control (Ransod SD 126) was within the reference values. Both GPX and SOD activities were expressed in units per milligram of proteins (U/mg). 2.3. Other determinations Lactate dehydrogenase (LDH) activity was determined in cell supernatants on a Beckman-Coulter automated Synchron CX-4 CE using pyruvate as the substrate and at 37 (C; activity was expressed in mU/24 h/106 cells. For the assessment of apoptosis, we used the Cell Death ELISA kit from Boehringer based on the detection of oligonucleosomes (18020 bp) characteristic of internucleosomal DNA fragmentation during apoptosis; the specificity for oligonucleosomes was assessed by double reactivity with anti-histones and anti-DNA monoclonal antibodies. For this determination, cellular monolayers were rinsed in serum-free medium and then lysed in a specific solution (Mailloux et al., 2001). Results were expressed as the differential absorbance between 405 and 490 nm; a control provided by the manufacturer was positive in the series. 2.4. Statistical analysis The results were expressed as the meanSD of at least four determinations. Non-parametric Mann– Whitney statistics available in Graphpad Prism were used to establish the significance of between-group differences; P values <0.05 were considered significant. 3. Results 3.1. Effects of ACE inhibitors on oxidative stress and antioxidant defences in HUVECs Neither enalaprilat nor captopril modified the intracellular content in MDA of HUVECs. In contrast, when
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3.2. Effects of ACE inhibitors on necrosis and apoptosis of HUVECs Used at 1 or 10 mmoles/l enalaprilat was not toxic towards HUVECs as well as morphologically (not shown) as judged by LDH activity whereas captopril was toxic at 10 mmoles/l with cell retraction, decrease in protein concentration (not shown) and LDH activity increment in cell supernatants (Fig. 2). To better understand the toxic effect of captopril on HUVECs, 10 mmoles/l captopril was applied on to confluent cells for 24 h; thereafter the oligonucleosomes characteristic of apoptosis were measured showing that, used at this concentration, captopril induced apoptosis in HUVECs (Fig. 3); used at a lower concentration, it was ineffective as for necrosis. Moreover, captopril was unable to protect HUVECs towards apoptosis subsequently triggered by 0.1 mg/ml etoposide, a concentration of this anticancer drug known to induce apoptosis in this model (Mailloux et al., 2001); the effects of etoposide and captopril were cumulative by enhancing both necrosis and apoptosis (Fig. 3). As for captopril, enalaprilat was unable to protect HUVECs against etoposide-induced apoptosis but, conversely to captopril, it did not trigger necrosis nor apoptosis.
4. Discussion
Fig. 1. Effects of 10 mmol/l captopril or enalaprilat on malondialdehyde (MDA), glutathione peroxidase (GPX) activity, and superoxide dismutase (SOD) activity in HUVECs after 24 h of incubation with the drugs.
used at 10 mmoles/l both ACE inhibitors decreased GPX activity; lower concentrations were ineffective (not shown). Captopril but not enalaprilat decreased SOD activity; a lower concentration of captopril was not effective (Fig. 1). Thus, both inhibitors tend to decrease rather to enhance oxidant defences in HUVECs and are unable to diminish the spontaneous lipid peroxidation as determined by the measure of thiobarbituric acidderivative substances. ACE inhibitors do not seem to be useful for protection towards an oxidative stress in endothelial cells.
Angiotensin-converting enzyme (ACE) inhibitors are widely prescribed for the treatment of hypertension and congestive heart failure; they also delay the progression of chronic renal failure and of diabetic nephropathy (Brown and Vaughan, 1998; Mancini, 2000). In addition, they have been shown to retard the development of atherosclerosis in experimental models (Ambrosioni et al., 1992; Jacobsson et al., 1994; Kowala et al., 1998) and to improve endothelial dysfunction in patients with coronary artery disease (Pfeffer et al., 1992; The SOLVD Investigators, 1992). The mechanism underlying these pharmacological effects of ACE inhibitors are not fully understood; the potentiation of bradykinin and of free radical scavenger action by the ACE inhibitors have been postulated. In particular, De Cavanagh et al. (2000) showed that enalapril and captopril treatments increased antioxidant enzymes and non-enzymatic antioxidants in several mouse tissues; furthermore, in erythrocytes the augmentation of antioxidants by ACE inhibitors was associated with protection against oxidant damage. On the other hand, ACE inhibitors decrease bradykinin degradation; bradykinin is a potent vasodilator known to stimulate the release of nitric oxide (Cannon, 1998); however the participation of nitric oxide as a modulator of oxidant defences is controversial since, in different systems, it was able to either increase (Kuo et al., 1996; Moellering et al., 1999) or decrease (Mak et al., 1996) antioxidant defences.
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Fig. 2. Concentration-dependent cytotoxicity of captopril, but not of enalaprilat, towards HUVECs as judged on lactate dehydrogenase (LDH) activity increment in cell supernatants after 24 h of incubation with the drugs. LDH is expressed in mU/24 h/106 cells.
Fig. 3. Effects of 10 mmoles/l captopril on necrosis and apoptosis in HUVECs pre-treated or not with etoposide; LDH activity in cell supernatants is expressed in mU/24 h/106 cells and oligonucleosomes in cell lysates (Oligo-C) in A as stated in Materials and methods. *P<0.05 versus 0/0; P<0.05 versus 0.1/0 and versus 0/10 (etoposide/captopril).
In this study we report that enalapril and captopril are unable to enhance antioxidant enzymes, namely glutathione peroxidase (GPX) and superoxide dismutase (SOD), in human vascular endothelial cells, these particular cells being the major site of synthesis of ACE and an important target of ACE inhibitors (Baudin et al., 1997; Baudin and Drouet, 1989). Both ACE inhibitors were also unable to decrease the spontaneous intracellular production of malondialdehyde (MDA), the main product of lipid peroxidation, that has been shown to be increased, for example, in endothelial cells treated by doxorubicin, an anticancer drug (Mailloux et al., 2001). Doxorubicin, a quinone-containing anthracycline antineoplastic drug, is used in the treatment of a wide spectrum of human cancers, but the development of severe cardiac toxicity compromises its clinical
effectiveness (Lefrak et al., 1973). It is metabolically activated to a free radical state and interacts with molecular oxygen to generate superoxide radicals (Sinha et al., 1987; Yin et al., 1998); it is particularly the case in vascular and cardiac endothelial cells (Kang et al., 2000; Keizer et al., 2000; Mailloux et al., 2001). Therefore the protective effects of antioxidants and metal chelators against doxorubicin-induced cardio-toxicity have been investigated (Doroshow et al., 1981; Unverferth et al., 1985; Van Vleet et al., 1980). In particular, temocapril and zofenopril, new ACE inhibitors, were shown to prevent doxorubicin-induced cardiomyopathy in a rat model (Sacco et al., 2001; Tokudome et al., 2000), but our results are not consistent with a putative role of ACE inhibitors in endothelial cell protection related to the enhancement of antioxidant defences. Temocapril
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could better prevent endothelial dysfunction via nitric oxide production as shown in diabetes-induced endothelial abnormalities (Pieper and Siebeneich, 2000). Captopril also appeared to be beneficial as a protective agent against cardio-toxicity related to doxorubicin (Al-Shabanad et al., 1998). This ACE inhibitor, the first described, is a thiol compound which can react with superoxide anion radical acting as a scavenger, or with hydroxyl radical (Al-Shabanad et al., 1997; Aruoma et al., 1991; Bartosz et al., 1997; Benzie and Tomlison, 1998); but captopril as well as enalaprilat, which does not contain a thiol group, failed to decrease MDA concentration in endothelial cells. Moreover, captopril appeared to be toxic for endothelial cells when used at high concentration and this toxicity could be rather related to its thiol group reacting as a mercaptan for cellular proteins and reduced glutathione, the main intracellular non-enzymatic antioxidant defence. Captopril was also described as preventing apoptosis by interfering with Fas signalling, or with other pathways for activation of cell death, but in other cell types than endothelial cells (Odaka and Mizuochi, 2000; Uhal et al., 1998); the blockade of angiotensin II function may play a role in the regulation of cell death by apoptosis (Filippatos and Uhal, 2003). Thereby we tried to protect endothelial cell by ACE inhibitors against etoposide induced-apoptosis, etoposide being an anti-cancer drug that we previously described for inducing caspase-3 activity (a pro-apoptotic enzyme) and for decreasing bcl-2 concentration (an anti-apoptotic protein) in human endothelial cell (Mailloux et al., 2001). Captopril as well as enalaprilat was unable to protect the cells, and conversely captopril induced apoptosis when it was used at the concentration that also led to cell necrosis with LDH release in supernatant. We also showed (personal communication) that other thiol-containing substances such as N-acetylcysteine were unable to protect HUVECs towards apoptosis, that was also the case for nitroso-captopril which brings the dual potential for NO production and ACE inhibition; we also showed that etoposide did not induce apoptosis by activating Fas pathway although HUVECs expressed Fas. Nevertheless, the concentrations of both ACE inhibitors used in this study are above the maximal plasma concentrations that can be obtained after per os administration (Richer et al., 1984). A number of drugs largely used in therapy are toxic towards endothelial cells, and particularly in vitro from concentrations that can be obtained in vivo to concentrations usually not reached (Baudin, 1995; Baudin et al., 1996; Mailloux et al., 2001). Taking all data together, we can conclude that both ACE inhibitors, captopril and enalapril, cannot be considered as protective drugs for the endothelium, either against an oxidative stress as in the course of cancer chemotherapy, for example using doxorubicin, or against apoptosis as induced by many anti-cancer drugs,
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also in clinical situations where both oxidative stress and apoptosis have been involved, e.g. atherosclerosis, ischaemic diseases, auto-immunity and inflammation. When ACE inhibitors can protect against endothelial dysfunction, other pathways than oxidative stress and apoptosis might be engaged, for example nitric oxide production leading to vasorelaxation or cytoprotective prostaglandins production since, in clinical situations, they are able to improve endothelial dysfunction, for example in the treatment of allogenic bone marrow transplantation and for the prevention of liver venoocclusive disease (Scrobohaci et al., 1991). Otherwise, ACE inhibitors could protect against doxorubicininduced cardiac impairment at the level of cardiac myocytes but not the cardiac endothelial cells (Maeda et al., 1997). Our results also point to potential toxicity towards vascular endothelium of captopril when used at high dose; even when such concentrations are obtained with difficulty in human therapy, captopril has been used at higher concentrations than those reached in vivo in some protocols such as for transplant preservation (Fisher et al., 2000). The toxicity of captopril can be related to its thiol group since enalaprilat was not toxic towards endothelial cells nor induced apoptosis, even at high concentration.
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