Renin-Angiotensin System Modulates Oxidative Stress–Induced Endothelial Cell Apoptosis in Rats
The role of the renin-angiotensin system in oxidative stress–induced apoptosis of endothelial cells (ECs) was investigated using a rat model and cultured ECs. EC apoptosis was induced by 5-minute intra-arterial treatment of a rat carotid artery with 0.01 mmol/L H2O2 and was evaluated at 24 hours by chromatin staining of en face specimens with Hoechst 33342. Although activity of angiotensin-converting enzyme in arterial homogenates was not increased, administration of an angiotensin-converting enzyme inhibitor temocapril for 3 days before H2O2 treatment inhibited EC apoptosis, followed by reduced neointimal formation 2 weeks later. Also, an angiotensin II type 1 (AT1) receptor blocker (olmesartan) inhibited EC apoptosis, whereas angiotensin II administration accelerated apoptosis independently of blood pressure. Next, cultured ECs derived from a bovine carotid artery were treated with H2O2 to induce apoptosis, as evaluated by DNA fragmentation. Combination of angiotensin II and H2O2 dose-dependently increased EC apoptosis and 8-isoprostane formation, a marker of oxidative stress. Conversely, temocapril and olmesartan reduced apoptosis and 8-isoprostane formation induced by H2O2, suggesting that endogenous angiotensin II interacts with H2O2 to elevate oxidative stress levels and EC apoptosis. Neither an AT2 receptor blocker, PD123319, affected H2O2-induced apoptosis, nor a NO synthase inhibitor, NG-nitro-l-arginine methyl ester, influenced the effect of temocapril on apoptosis in cell culture experiments. These results suggest that AT1 receptor signaling augments EC apoptosis in the process of oxidative stress–induced vascular injury.
Stress-induced injury of vascular endothelial cells (ECs) is considered to be an initial event in the development of atherosclerosis.1 In particular, oxidative stress has been implicated in endothelial injury caused by oxidized LDL and smoking, as well as hypertension, diabetes, and ischemia reperfusion.1–3⇓⇓ This notion is supported by the findings that the production of reactive oxygen species is upregulated in vascular lesions4,5⇓ and that lesion formation such as endothelial dysfunction is accelerated by superoxide anion6 and, in contrast, is attenuated by free radical scavengers, including vitamin E7 and superoxide dismutase.8
The renin-angiotensin system (RAS) is known to play a pivotal role in the process of vascular lesion formation such as atherosclerosis and restenosis after angioplasty. The expression of RAS components renin,9 angiotensinogen,10 angiotensin-converting enzyme (ACE),11,12⇓ and angiotensin II (Ang II) receptors13 is upregulated in vascular lesions. Also, RAS inhibitors attenuate neointimal formation after vascular injury in animals12,14⇓ and endothelial dysfunction in humans.15,16⇓ The interaction between oxidative stress and the RAS, factors essential for the development of vascular disease, needs to be addressed. It has been demonstrated that RAS activation induces oxidative stress17–20⇓⇓⇓ and can enhance EC apoptosis in vitro.20,21⇓ However, it has not been elucidated whether the RAS plays a role in oxidative stress–induced vascular injury in vivo, particularly in EC apoptosis, an initial and important process in atherosclerosis.1,22,23⇓⇓
In this study, we first tested whether the RAS would augment EC apoptosis induced by brief exposure to H2O2 and the subsequent neointimal formation using a rat model.24 Next, we used an in vitro model of H2O2-induced EC apoptosis to clarify the underlying cellular mechanism.
H2O2 Treatment of Carotid Artery
Ten- to 12-week-old male Wistar rats (Japan Clea; Tokyo, Japan) were used in this study. Maintenance of rats and surgical procedures for H2O2 treatment were performed as described previously.24 Methods are detailed in the online data supplement (available online at http://www.hypertensionaha.org). All of the experimental protocols were approved by the animal research committee of the Kyorin University School of Medicine.
Animal Groups and Blood Pressure Measurement
An ACE inhibitor, temocapril (10 mg/kg per day; donated by Sankyo Co, Ltd; Tokyo, Japan), or vehicle (40% ethanol) was administered orally using a feeding tube daily for 3 days. Separately, an Ang II type 1 (AT1) receptor blocker, olmesartan (1 mg/kg per day; donated by Sankyo Co, Ltd), or vehicle (40% ethanol) was administered orally for 3 days. Ang II was administered for 3 days using an osmotic minipump (Model 103D; Alza Corporation) prefilled with Ang II (0.7 mg/kg per day; Sigma), and implanted subcutaneously in the back. Hydralazine (25 mg/kg per day; Sigma) was orally administered alone for 5 days and subsequently with or without Ang II for 3 days before H2O2 treatment to abolish the effect of Ang II on blood pressure. On the last day of drug administration, blood pressure was measured with the animals in a conscious state by the tail-cuff method (BP-98A; Softron), and then H2O2 treatment was performed.
Measurement of ACE Activity and Ang II Concentration
At various time points after H2O2 treatment, the carotid arteries were dissected, weighed, and stored at −80°C. Pooled samples (n=6 to 10 for a pool) were homogenized with a polytron homogenizer in distilled water and centrifuged at 25 000g for 30 minutes at 4°C. ACE activity and Ang II concentration in the supernatants were measured using a colorimetric assay12 and a sensitive radioimmunoassay, respectively. The values were calibrated by the tissue wet weight. ACE activity in the cell lysates of cultured ECs was measured using a colorimetric assay and calibrated by the protein concentration.
Evaluation of EC Apoptosis and Neointimal Formation in Carotid Artery
EC apoptosis was evaluated at 24 hours after H2O2 treatment as described previously.24 Neointimal formation in the common carotid artery was evaluated 2 weeks after H2O2 treatment as described previously.24 Methods are detailed in the online data supplement.
Induction of EC Apoptosis in Culture
ECs isolated from bovine carotid artery25 were used at the fifth to seventh passage. When the cells had grown to 80% confluence, ECs were pretreated for 24 hours with culture medium containing the reagents that were tested in the experiments. Subsequently, after washing twice with Hank’s balanced salt solution, the cells were exposed to H2O2 (0.01 to 0.2 mmol/L) diluted in Hank’s balanced salt solution for 1.5 hours at 37°C to induce apoptosis. The cells were washed twice with Hank’s balanced salt solution and then cultured in culture medium containing the reagents until assay.
The effects of temocapril, olmesartan, a NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME; Sigma), an Ang II type 2 (AT2) receptor blocker, PD123319 (Research Biochemical International), and Ang II (Sigma) were examined by adding them into the medium throughout the experiments.
Measurement of EC Apoptosis and Oxidative Stress Markers in Culture
For quantitative determination of apoptosis, we measured DNA fragmentation and caspase-3 activity at 24 hours after H2O2 treatment. DNA fragmentation was evaluated by histone-associated DNA fragments using a photometric enzyme immunoassay (EIA; Cell Death Detection ELISA; Roche) according to manufacturer instructions. Caspase-3 activity was measured using a colorimetric kit (Caspase-3 Colorimetric Activity Assay Kit; Chemicon) based on its activity to digest the substrate DVED according to manufacturer instructions.
Formation of 8-isoprostane (8-iso prostaglandin F2α) was measured using a commercially available EIA kit (Cayman Chemical). Culture supernatants were diluted with EIA buffer when necessary and were applied to EIA according to manufacturer instructions. Intracellular oxidative stress levels were measured using 2′,7′-dichlorofluorescein (DCF) as described previously,26 and the intensity values were calculated using the Metamorph software.
Real-Time Polymerase Chain Reaction
Real-time polymerase chain reaction (PCR) to quantify AT1 receptor mRNA in cultured ECs was performed using SYBR Green I (Sigma) and the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Methods are detailed in the online data supplement.
The values are expressed as mean±SEM in the text and figure data were analyzed using 1-factor ANOVA. If a statistically significant effect was found, Newman–Keuls test was performed to isolate the difference between the groups. Differences with a value of P<0.05 were considered statistically significant.
ACE Activity in Carotid Artery After H2O2 Treatment
We examined whether H2O2 treatment would activate ACE and stimulate Ang II synthesis in the carotid artery. As shown in Figure 1A, ACE activity in tissue homogenates was not increased at 1 to 3 hours and, rather, was decreased at 24 hours, probably because of EC denudation.24 Low ACE activity in the de-endothelialized artery is consistent with the previous finding11,12⇓ and was confirmed by measurement of ACE activity in the rat carotid artery, in which ECs were denuded ex vivo using a cotton swab (data not shown). In contrast, ACE activity was significantly increased at 1 week after H2O2 treatment, reflecting neointimal formation.11,12,24⇓⇓ Ang II concentration in arterial homogenates showed similar changes to ACE activity after H2O2 treatment (Figure 1B).
Effect of RAS Inhibitors and Ang II on EC Apoptosis After H2O2 Treatment in Rats
The effects of an ACE inhibitor, temocapril, and an AT1 receptor blocker, olmesartan, on EC apoptosis were examined at 24 hours after H2O2 treatment because the peak of apoptosis was observed at 6 to 24 hours.24 Administration of 10 mg/kg per day temocapril or 1 mg/kg per day olmesartan for 3 days before H2O2 treatment did not significantly change body weight, heart rate, or blood pressure, but this dose of temocapril effectively inhibited plasma ACE activity (data not shown). The number and percentage of apoptotic cells, as determined using en face specimens with Hoechst 33342 staining, were significantly decreased by temocapril compared with vehicle (Figure 2A; supplemental Figure I, available online at http://www.hypertensionaha.org). Olmesartan showed a comparable inhibitory effect on EC apoptosis (Figure 2B).
Ang II was administered for 3 days in combination with hydralazine to eliminate the effect of Ang II on blood pressure. Consequently, systolic blood pressure was higher in rats administered Ang II alone (161±5 mm Hg; P<0.01) than in the other groups of rats: 123±3 mm Hg in the vehicle group, 129±7 mm Hg in the Ang II plus hydralazine group, and 114±4 mm Hg in the hydralazine group. In contrast to RAS inhibitors, Ang II administration augmented EC apoptosis independent of the pressor effect because coadministration of hydralazine did not influence EC apoptosis (Figure 2C).
Inhibitory Effect of Temocapril on Neointimal Formation
We examined whether inhibition of EC apoptosis by temocapril would result in a reduction of neointimal formation. To do so, histological analysis of the carotid artery was performed 2 weeks after H2O2 treatment. Temocapril significantly decreased the neointimal area and the intima/media area ratio: intima/media area ratio was 0.18±0.02 in the vehicle group versus 0.12±0.02 in the temocapril group (n=9; P<0.05; supplemental Figure II). Because temocapril was administered for only 3 days before H2O2 treatment, it is suggested that inhibition of EC apoptosis may play a mechanistic role in attenuation of neointimal formation, although ACE inhibitors have various effects such as anti-inflammation and antimigration as well.
Effect of RAS Inhibitors on H2O2-Induced EC Apoptosis in Culture
To reproduce oxidative stress–induced EC apoptosis in culture, we applied 0.2 mmol/L H2O2 to cultured ECs derived from a bovine carotid artery for 1.5 hours based on dose- and time-response experiments. EC apoptosis, as determined by DNA fragmentation and caspase-3 activity, was induced at 24 hours after H2O2 treatment. Comparable to in vivo experiments, temocapril inhibited EC apoptosis in a dose-dependent manner (Figure 3A and 3B). The inhibitory effect on EC apoptosis was mimicked by 10 μmol/L olmesartan (Figure 3C), but an AT2 receptor blocker, PD123319, did not influence EC apoptosis (supplemental Figure IIIA). The involvement of NO in the effect of temocapril was examined using an NO synthase inhibitor, l-NAME, because ACE inhibitors stimulate NO production via the inhibition of bradykinin degradation.12 However, l-NAME did not influence the effect of temocapril (supplemental Figure IIIB).
To make the interaction between H2O2 and Ang II clear, dose response and combined effects of both agents on EC apoptosis and 8-isoprostane formation, a marker of oxidative stress, were examined. As shown in Figures 3D and 4⇓A, combination of Ang II and H2O2 dose-dependently stimulated EC apoptosis and 8-isoprostane formation. Conversely, temocapril and olmesartan restrained 8-isoprostane formation (Figure 4B) and intracellular DCF formation (Figure 4C; supplemental Figure IV) induced by H2O2, suggesting that endogenous Ang II also interacts with H2O2 to elevate oxidative stress levels.
ACE activity and the expression of AT1 receptor mRNA in cultured ECs were determined. ACE activity calibrated by the protein concentration was not changed after H2O2 treatment: 106±9% at 3 hours and 103±8% at 24 hours after H2O2 treatment compared with the values at baseline and 3 hours after vehicle treatment (100±3% and 96±13%, respectively; n=3). The relative amount of the AT1 receptor to the housekeeping gene G3PDH, as measured by real-time PCR analysis, was not significantly changed after H2O2 treatment: 91±2% at 1.5 hours during the treatment, 99±5% at 3 hours, and 102±4% at 6 hours after H2O2 treatment compared with vehicle treatment (100±6%; n=3). Considering negative regulation in vascular smooth muscle cells27,28⇓ together, upregulation of the AT1 receptor is not likely to occur in response to H2O2 treatment.
This study was conducted to elucidate the role of the RAS in oxidative stress–induced EC apoptosis using a rat model and cultured ECs. Treatment with H2O2 did not increase ACE activity or Ang II in the rat carotid artery during the acute phase. However, administration of an ACE inhibitor, temocapril, and an AT1 receptor blocker, olmesartan, inhibited EC apoptosis in vivo. Furthermore, we demonstrated using cultured ECs that combination of Ang II and H2O2 dose-dependently increased EC apoptosis and 8-isoprostane formation. In addition, temocapril and olmesartan reduced but not canceled EC apoptosis and 8-isoprostane formation induced by H2O2, suggesting that endogenous Ang II interacts with H2O2 to elevate oxidative stress levels and EC apoptosis.
In vascular lesions such as atherosclerosis and intimal hyperplasia, the production of reactive oxygen species4,5⇓ as well as the components of the RAS9–12⇓⇓⇓ are upregulated, suggesting a possible interaction between them. A number of investigations have clarified that Ang II induces oxidative stress in vascular cells. Ang II stimulates the production of reactive oxygen species in ECs by upregulating the subunits of NAD(P)H oxidase: gp91 phox17 and p47 phox.18 It has been reported that the RAS enhances EC apoptosis in vitro20,21⇓ and contributes to endothelial dysfunction in patients with renovascular hypertension through the oxidant-dependent mechanism.19 Conversely, it remains unknown whether oxidative stress could regulate the RAS; only 1 report has shown the modulation of ACE by oxidative stress.29 Usui et al29 reported that the inhibition of NO synthesis by chronic administration of l-NAME in rats augmented superoxide production and ACE activity in aortic ECs, and these effects were eliminated by treatment with antioxidants. In the present study, ACE activity in the carotid artery was not increased until 24 hours after H2O2 treatment. We also found that ACE activity was not changed after H2O2 treatment in cell culture experiments. Furthermore, the expression of AT1 receptor mRNA in cultured ECs, as measured using real-time PCR, was not increased after H2O2 treatment. Together, it is not likely that Ang II production or its receptor expression was upregulated in response to H2O2.
However, an ACE inhibitor, temocapril, and an AT1 receptor blocker, olmesartan, inhibited H2O2-induced EC apoptosis in rats as well as in cell culture experiments. No influence of l-NAME on the antiapoptotic effect of temocapril in cell culture studies indicates that the effect of temocapril was attributable to the inhibition of Ang II synthesis. An AT2 receptor blocker, PD123319, did not influence H2O2-induced EC apoptosis either. This result appears to be inconsistent with the previous finding30 but suggests a minimal contribution of the AT2 receptor in H2O2-induced EC apoptosis or minimal expression of the AT2 receptor in the cultured ECs used in the present study. Reduction in 8-isoprostane formation by temocapril and olmesartan suggests that endogenous Ang II adds to the oxidative stress levels on top of exogenous H2O2; otherwise temocapril and olmesartan would have antioxidant effects independent of Ang II through currently unknown mechanisms, although the in vivo role of bradykinin/NO in the effect of ACE inhibitors and that of the AT2 receptor remain to be addressed.
Administration of Ang II provided evidence that Ang II can interact with H2O2 to elevate oxidative stress levels and induce EC apoptosis. In rat experiments, a high and pressor dose of Ang II was used in combination with hydralazine31 because 3-day administration of lower doses of Ang II (0.1 to 0.2 mg/kg per day) did not show significant effects on EC apoptosis (data not shown). The cell culture experiments to examine the effect of submaximal doses of Ang II and H2O2 on apoptosis and 8-isoprostane formation gave us clear information that AT1 receptor signaling augments EC apoptosis by an interaction with oxidative stress. Although the doses of H2O2 and the time duration of exposure were optimized on the basis of the time- and dose-response experiments, the conditions in cell culture studies were different from those in animal studies. However, it has been reported that cigarette smoke, oxidized lipoproteins, and polymorphonuclear leukocytes, which play important roles in atherogenesis, can generate H2O2 concentrations of 0.05 to 0.2 mmol/L in vitro.32 These reports suggest that the dosages of H2O2 used in the present study do not far exceed the physiological range, although direct comparison of physiological or pathophysiological conditions with those in our experiments may be inappropriate.
Considering the stimulatory effect of Ang II on free radical production,17–19⇓⇓ our finding that endogenous Ang II exacerbates EC apoptosis induced by exogenous H2O2 is not surprising. In fact, a number of reports have shown experimentally that RAS inhibitors can reduce the production of reactive oxygen species in pathological conditions such as peripheral arteries in rats with chronic heart failure,33 rat diabetic nephropathy,34 and kidney mitochondria in aged rats.35 In the clinical setting, it is reported that administration of an AT1 receptor blocker (losartan) to patients with chronic renal disease reduced urinary excretion of oxidized albumin and malondialdehyde.36 Also, 4-week treatment with losartan or an ACE inhibitor (ramipril) in patients with coronary artery disease diminished the response of endothelium-dependent vasodilation to intracoronary administration of antioxidant vitamin C in parallel with improvement of basal endothelium-dependent vasodilation,37 indicating that RAS inhibitors can improve endothelial function in association with a reduction of oxidative stress. In the present study, we investigated EC apoptosis, an important process that leads to endothelial dysfunction and atherosclerosis22,23⇓ using an in vivo model. Moreover, our finding that RAS inhibitors attenuated EC apoptosis suggests broad end-organ protective effects of RAS inhibitors, which have been used for the treatment of hypertension and heart failure.
We found using an in vivo model and cultured ECs that Ang II elevated oxidative stress levels and increased EC apoptosis, whereas RAS inhibitors restrained them. These findings will add new information for cardiovascular research and the clinical application of RAS inhibitors.
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Culture and Sports of Japan (13670741), and by Health and Labor Sciences Research Grants (H15-Choju-013 and H15-Choju-015) from the Ministry of Health, Labor and Welfare of Japan. We thank Mariko Sawano for her excellent technical assistance.
- Received October 26, 2004.
- Revision received December 13, 2004.
- Accepted March 24, 2005.
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