Angiotensin II Induces Apoptosis of Human Endothelial Cells
Protective Effect of Nitric Oxide
Abstract Angiotensin II (Ang II) importantly contributes to the pathobiology of atherosclerosis. Since endothelial injury is a key event early in the pathogenesis of atherosclerosis, we tested the hypothesis that Ang II may injure endothelial cells by activation of cellular suicide pathways leading to apoptosis. Human umbilical venous endothelial cells (HUVECs) were incubated with increasing doses of Ang II for 18 hours. Apoptosis of HUVECs was measured by ELISA specific for histone-associated DNA fragments and confirmed by DNA laddering and nuclear staining. Ang II dose-dependently induced apoptosis of HUVECs. Simultaneous blockade of both the AT1 and AT2 receptor prevented Ang II–induced apoptosis, whereas each individual receptor blocker alone was not effective. Selective agonistic stimulation of the AT2 receptor also dose-dependently induced apoptosis. Ang II–mediated as well as selective AT2 receptor stimulation–mediated apoptosis was associated with the activation of caspase-3, a central downstream effector of the caspase cascade executing the cell death program. Specific inhibition of caspase-3 activity abrogated Ang II–induced apoptosis. In addition, the NO donors sodium nitroprusside and S-nitrosopenicillamine completely inhibited Ang II–induced apoptosis and eliminated caspase-3 activity. Thus, Ang II induces apoptosis of HUVECs via activation of the caspase cascade, the central downstream effector arm executing the cell death program. NO completely abrogated Ang II–induced apoptosis by interfering with the activation of the caspase cascade.
Angiotensin II importantly contributes to the pathobiology of atherosclerosis and vascular disease not only via its role in hypertension but also via its direct effects on vascular cell growth and migration.1,2 Ang II has previously been shown to promote the growth of VSMCs via activation of several growth factors like fibroblast growth factor and platelet-derived growth factor.2 These growth-promoting effects of Ang II for VSMCs appear to be mediated by the AT1 receptor.3 However, recent data indicate that Ang II represents a bifunctional growth factor by simultaneously stimulating proliferative and antiproliferative pathways.4 Indeed, in endothelial cells, Ang II mediates an antigrowth effect via stimulation of the AT2 receptor.5 The mechanisms underlying the antimitogenic action of Ang II in endothelial cells are unknown.
Cellular proliferation is in large part determined by the balance between cell division and cell death by apoptosis. Apoptosis refers to the morphological alterations exhibited by “actively” dying cells that include cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation.6 The effector arm of the signal transduction pathway executing the cell death program is composed of cysteine proteases belonging to the ICE/CPP32 family, which have been recently termed caspases.7 Caspase-3, also referred to as CPP32/Yama,8,9 has been shown to play an important role as a downstream member of the protease cascade, where various cell death pathways converge into the same effector pathway.10 On activation of the protease cascade, the caspase-3 proenzyme is proteolytically cleaved into the p17 and p12 subunits, which then heterodimerize to form the active enzyme.8
We have previously demonstrated that caspase-3 is activated in both the TNF-α–mediated and growth factor withdrawal–induced apoptotic signal transduction pathways in human endothelial cells.11,12 Therefore, the present study was designed to investigate whether the growth inhibitory effects of Ang II on HUVECs involve activation of the caspase cascade suggestive of the execution of the cell death program. In addition, since previous studies have consistently shown a countervailing balance between Ang II and NO with respect to the effects of these factors in the regulation of vessel tone and VSMC growth,13,14 we examined any potentially antagonistic effects of NO on Ang II–induced apoptosis of HUVECs.
Materials and Methods
HUVECs, endothelial basal medium, and supplements were purchased from Cell Systems/Clonetics, and FCS was from GIBCO. [32P]dCTP was obtained from Amersham Klenow polymerase and cell death detection assay were from Boehringer Mannheim. PD123177, caspase-3-inhibitor, and substrate were obtained from Biomol. Ang II was from Sigma, and CGP42112 was from Neosystem Laboratoire. The antibody against CPP32(p20) was from Santa Cruz (clone D027). The guanylate cyclase inhibitor NS202815 was kindly donated by Dr R. Busse (Frankfurt, Germany).
HUVECs were cultured in endothelial basal medium supplemented with hydrocortisone (1 μg/mL), bovine brain extract (12 μg/mL), gentamicin (50 μg/mL), amphotericin B (50 ng/mL), epidermal growth factor (10 ng/mL), and 10% fetal calf serum until the third passage. After detachment with trypsin, cells were grown for at least 18 hours. All experiments were performed in the presence of complete medium including 10% fetal calf serum. Shear exposure was performed as previously outlined.11,16 The rat smooth muscle cell line A-10 (DSMZ, Braunschweig, Germany) or human vascular smooth muscle cells were cultivated in DMEM with 20% fetal calf serum and 1% penicillamine and streptomycin. Before the experiment, the smooth muscle cells were starved for 48 hours in the absence of fetal calf serum.
DNA fragmentation analysis was carried out as recently described.11,12 Cells were scraped off the plates and centrifuged at 700g for 10 minutes, washed with PBS, and resuspended in incubation buffer. The histone-associated DNA fragments were linked to the anti-histone antibody from mouse, and the DNA part of the nucleosome was linked to the anti–DNA-peroxidase. The amount of peroxidase retained in the immunocomplex was determined photometrically.
DNA Isolation and Klenow Labeling
Cells (1×106), including detached cells, were removed from the culture flask and collected by centrifugation (10 minutes at 700g), washed with PBS, and incubated in lysis buffer (5 mmol/L Tris-HCl, pH 8, 20 mmol/L EDTA, and 0.5% Triton X-100) for 15 minutes at 4°C. After centrifugation for 20 minutes at 20 000g at 4°C, the supernatants were treated with RNase A for 1 hour at 37°C. A final concentration of 0.5 mg/mL proteinase K and 1% SDS was added, and the samples were incubated overnight at 65°C. After isolation of DNA by phenol-chloroform extraction, the DNA was precipitated with 70% isopropanol and 0.1 mol/L NaCl. The resulting pellet was resolved in TE buffer (10 mmol/L Tris-HCl, pH 8, and 1 mmol/L EDTA), and the DNA samples were incubated with 5 U Klenow polymerase and 0.5 μCi [32P]dCTP according to Rösl.17 The reaction was terminated by the addition of 10 mmol/L EDTA, and the unincorporated nucleotides were removed by Sephadex G-50 columns. Labeled DNA fragments were separated on a 1.8% agarose gel, transferred to nitrocellulose membranes, and exposed to x-ray film.
Determination of Cell Viability
Cell viability was detected as described previously.18 HUVECs (1×105 cells/mL) were grown in 96-well plates for 24 hours. After incubation with apoptotic stimuli for 18 hours, cells were treated with MTT (0.5 mg/mL) for 4 hours at 37°C. The cell culture medium was removed, cells were lysed in 2-isopropanol containing 0.04 mol/L HCl, and the amount of MTT was photometrically determined.
Cells were washed with PBS and fixed in 4% formaldehyde. Cells were stained with DAPI (0.2 μg/mL in 10 mmol/L Tris-HCl, pH 7, 10 mmol/L EDTA, and 100 mmol/L NaCl) for 30 minutes. Then cells were washed with PBS, and nuclei were analyzed by fluorescence microscopy. Two independent investigators counted apoptotic nuclei in a total cell number of 600 nuclei and expressed the result as apoptotic nuclei/600×100%.
Measurement of Caspase-3–Like Protease Activity
For detection of caspase-3–like activity, HUVECs (5×105 cells) were lysed in buffer (1% Triton X-100, 0.32 mol/L sucrose, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 2 mmol/L dithiothreitol, and 10 mmol/L Tris-HCl, pH 8) for 15 minutes at 4°C, followed by centrifugation (20 000g for 10 minutes). Caspase-3–like activity was detected in resulting supernatants by measuring the proteolytic cleavage of the fluorogenic substrate AMC-DEVD and AMC as standard in assay buffer containing 100 mmol/L HEPES, 10% sucrose, 0.1% CHAPS, pH 7.5, and 10 mmol/L dithiothreitol (excitation wavelength, 380 nm; emission wavelength, 460 nm).19 Enzyme activity was calculated as mol AMC×mg protein−1×s−1. Specificity for caspase-3–like enzymatic activity was demonstrated by inhibition with 10 nmol/L Ac-DEVD-CHO.8,19 Protein content was analyzed using the Bio-Rad assay.
HUVECs were incubated, and protein was prepared as described for detection of caspase-3–like activity. Proteins (80 μg protein/slot) were resolved on 15% SDS-polyacrylamide gels and were blotted on nitrocellulose membranes by means of a semidry blotting system (3 mA/cm2 for 30 minutes; buffer consisted of 48 mmol/L Tris, 39 mmol/L glycine, 0.037% SDS, and 20% methanol). The membranes were washed twice with TBS (50 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, and 2.5 mmol/L KCl), and unspecific binding was blocked overnight at 4°C with 3% BSA in TBS/0.1% Tween 20. The antibody against the p17 subunit of human caspase-3 was added in a final dilution of 1:200 in TBS/3% BSA/0.1% Tween 20 for 1 hour at room temperature. After it was washed three times with TBS/Tween 20, the horseradish peroxidase–conjugated anti-goat IgG antibody (1:2000 in TBS/3% BSA/0.1% Tween 20) was incubated for 1 hour, and enhanced chemiluminescence was performed according to the instructions of the manufacturer.
Statistical analysis was performed with ANOVA followed by a modified least significant difference (Bonferroni) test (SPSS-Software).
Ang II Induces Apoptosis of HUVECs
Incubation of HUVECs with Ang II dose-dependently induced apoptosis as determined by an ELISA specific for histone-associated DNA fragments (Fig 1a⇓). Maximal DNA fragmentation was obtained after incubation for 18 hours, whereas prolonged incubation for 24 hours did not lead to a further increase of DNA fragmentation (data not shown). Ang II–induced apoptosis was confirmed by demonstration of the typical DNA-laddering pattern (Fig 1b⇓) as well as by visual analysis of DAPI-stained cells (Fig 1c⇓). Quantification after fluorescence staining revealed apoptosis in 4.5±1.7% of the cells after treatment with Ang II versus apoptosis in 1.7±2% of the control cells (n=4, P<.05). Furthermore, the appearance of DNA fragments correlated with a decrease of cell viability to 83±6% at 1 μmol/L Ang II compared with control cells. Necrosis was excluded by determination of the LDH release, which was not affected by Ang II treatment (105±5% apoptosis after 18-hour incubation with 1 μmol/L Ang II compared with control cells). In contrast to the induction of apoptosis in HUVECs, Ang II did not render rat smooth muscle cells apoptotic under similar conditions (Fig 1a⇓). In addition, human smooth muscle cells were also not affected by 1 μmol/L Ang II (102±9% apoptosis compared with control cells).
To characterize the angiotensin receptor subtype involved in the stimulation of apoptosis in HUVECs, we examined the influence of AT1 and AT2 receptor antagonists on Ang II–induced apoptosis. Ang II (1 μmol/L)–triggered apoptosis was not significantly affected by the specific AT1 and AT2 receptor antagonists, EXP3174 (1 and 10 μmol/L) and PD123177 (1 and 10 μmol/L), respectively (Fig 1d⇑). However, simultaneous blockade of both AT1 and AT2 receptors by the combination of EXP3174 and PD123177 completely prevented Ang II–mediated apoptosis (Fig 1d⇑). Similar effects were observed by stimulating apoptosis with 0.1 μmol/L Ang II (data not shown). Neither of the two antagonists alone nor their combination affected DNA fragmentation in the absence of Ang II (data not shown). Control experiments further ensured that the solvent ethanol did not influence apoptosis and viability of endothelial cells (DNA fragmentation, 212±68% of control in the presence of Ang II).
In order to further investigate a potential involvement of AT2 receptor activation in the stimulation of apoptosis signal transduction, we additionally investigated the effects of the specific AT2 receptor agonist CGP42112.5 As illustrated in Fig 1e⇑, CGP42112 dose-dependently induced apoptosis.
To rule out the possibility that Ang II processing into shorter peptides20 accounts for the proapoptotic effect of Ang II, we tested the Ang II peptides Ang(1–7) and Ang(3–8). Neither of the peptides induced apoptosis when incubated for 18 hours at a concentration of 1 μmol/L [Ang(1–7), 92±27% of control; Ang(3–8), 108±19% of control; n=3].
To finally ensure that AT1 and AT2 receptors are expressed in the HUVECs under study, AT1 and AT2 mRNA was detected by means of reverse-transcriptase PCR. Specific mRNA transcripts for both receptor subtypes were demonstrated in HUVECs by PCR analysis (oligonucleotides: AT1, 5′-gccctgtccacaatatcttgc/5′-tgtaagattgcttcagccagc5; AT2, 5′-cttcatttaatagctgtatgat/5′-ttgtggtttaaatacaaagca), which exhibited the predicted sizes (AT1, 507 bp; AT2, 590 bp).
Involvement of Caspase-3–Like Protease
The caspase proteases play a key role in apoptotic processes in HUVECs.12,21 Therefore, the effect of Ang II on caspase-3–like activity was investigated. Ang II as well as the specific AT2 receptor agonist CGP42112 activated caspase-3–like activity to 134±9% and 146±25%, respectively, compared with unstimulated cells. In addition, CGP42112 stimulated the proteolytical cleavage of caspase-3 into its active subunits, p12 and p17, as assessed by Western blot using an antibody that prevalently reacts with the active p17 subunit (Fig 2⇓). Most important, the specific peptide caspase-3 inhibitor Ac-DEVD-CHO completely inhibited Ang II–stimulated apoptosis (Fig 3a⇓), documenting the involvement of caspase-3–like enzymes in Ang II–induced DNA fragmentation. In addition, the Ang II–induced decrease of cell viability (83±6% of control) was completely prevented by 100 μmol/L Ac-DEVD-CHO (102±7% of control).
NO Inhibits Ang II–Induced Apoptosis
Since we have previously shown that NO inhibits caspase-3–like activity in HUVECs,12 we investigated the effects of the NO donors SNP and SNAP. SNP (10 μmol/L) as well as SNAP (10 μmol/L) significantly reduced Ang II–induced apoptosis (Fig 3a⇑). Inhibition of Ang II–induced apoptosis by NO was observed in the presence of AT1 or AT2 receptor blockade (Fig 3b⇑), suggesting that the inhibitory effect of NO was due to interference with AT1 and AT2 receptor–mediated signal transduction. In addition, the specific stimulation of AT2 receptor–induced DNA fragmentation with CGP42112 was completely prevented by the NO donors SNP and SNAP (Fig 3c⇑). To determine the effect of endogenous NO, endothelial cells were exposed to laminar shear stress for 6 hours, which led to an increase of endothelial NO synthase protein levels (data not shown and References 12 and 1612 16 ). Preexposure of shear stress for 6 hours completely prevented further induction of apoptosis by Ang II (94±18% after preexposure to shear stress compared with 189±46%).
Inhibition of Ang II–induced apoptosis by NO appeared to be cGMP independent, since the cGMP analogue 8-bromo-cGMP did not affect Ang II–induced or CGP42112-induced DNA fragmentation (Fig 3a⇑ and 3c⇑, respectively). In addition, the guanylate cyclase inhibitor NS202815 did not affect the inhibitory effect of NO on Ang II–induced apoptosis (113±24% of control in the presence of 1 μmol/L Ang II, 10 μmol/L SNP, and 1 μmol/L NS2028; P<.05 versus Ang II). In order to elucidate the mechanism underlying the protective effect of NO, we investigated the influence on caspase-3–like activity and cleavage. SNP and SNAP abrogated the AT2 receptor–triggered caspase-3–like activity and additionally prevented CGP42112-induced cleavage into the active subunits (Fig 2⇑ and 3d⇑).
The results of the present study demonstrate that Ang II induces apoptosis of human endothelial cells via activation of the caspase cascade, as evidenced by an increase of caspase-3–like enzyme activity and proteolytical cleavage of caspase-3 into its active subunits, p12 and p17. Furthermore, inhibition of caspase-3–like activity completely abrogated Ang II–induced apoptosis of HUVECs, indicating that activation of the caspase cascade plays a central role in executing the Ang II–stimulated cell death program. Importantly, NO antagonized the effects of Ang II to induce apoptosis of HUVECs, further supporting the concept of the countervailing influences of NO and Ang II in vascular biology. These findings considerable extend the role of Ang II as a potentially important contributor to the pathobiology of atherosclerosis.
The growth-promoting effects of Ang II have generally been attributed to stimulation of the AT1 receptor.3 However, recent studies in nonendothelial cells have demonstrated that Ang II is also capable of inducing apoptosis via stimulation of the AT2 receptor,22 which is primarily expressed during ontogenesis.23 Moreover, it has been suggested that the antigrowth effect of Ang II on endothelial cells is mediated by the AT2 receptor,5 although the underlying mechanisms have not been elucidated thus far. The results of the present study for the first time demonstrate that Ang II induces the execution of the cell death program in HUVECs. Endothelial cells have been shown to express both AT1 and AT2 receptor subtypes.5 Indeed, stimulation of the AT2 receptor by high concentrations of the AT2-selective analogue CGP42112 in the present study not only induced DNA fragmentation (indicative of the induction of apoptosis) but also significantly increased caspase-3–like activity and led to the proteolytical cleavage of caspase-3. Thus, activation of the cell death program by Ang II clearly involves stimulation of the AT2 receptor in HUVECs. However, blocking the AT2 receptor did not abolish the proapoptotic effects of Ang II, suggesting that Ang II–induced apoptosis of HUVECs is not exclusively mediated by the AT2 receptor but depends on the complex interplay between both the AT1 and AT2 receptor, because simultaneously blocking both receptors eliminated Ang II–induced apoptosis. Given the pleiotropic AT1 receptor–mediated effects of Ang II on a variety of second-messenger systems, such as protein kinase C, calcium, mitogen-activated kinases, and other tyrosine kinases, which all have been implicated in the regulation of cellular survival signals,24,25 it is not surprising that blocking of individual receptors did not result in a complete reversal of the effects of Ang II. In addition, it is still an open debate whether more than two Ang II receptors are present in endothelial cells with as-yet-unknown functions and, probably more important, unknown affinities for the receptor blockers currently used. In addition, shorter fragments of Ang II were identified in human plasma and in tissues with distinct effects mediated via non-AT1 and non-AT2 receptors.20 However, the processed Ang II fragments Ang(1–7) and Ang(3–8) did not affect endothelial cell apoptosis, suggesting that the proapoptotic effect of Ang II is not mediated via its metabolites. Regardless of the specific type of Ang II receptor responsible for the proapoptotic effects of Ang II, the results of the present study unambiguously demonstrate that the divergent signaling pathways coupled to the AT1 and AT2 receptor types converge into the activation of the caspase cascade, executing the cell death program in HUVECs on stimulation with Ang II.
The intracellular signal transduction mechanisms following Ang II receptor stimulation leading to activation of the caspase cascade in HUVECs remain to be determined. In nonendothelial cells, Ang II has been shown to dephosphorylate MAP kinase via AT2-mediated activation of MAP kinase phosphatase,22 thereby counteracting the effects of growth-stimulatory signals. Whether such a mechanism is operative in endothelial cells is unknown at present. Moreover, there are no data providing support for a link between dephosphorylation of MAP kinases and activation of the caspase cascade. Ang II has been shown to induce the production of oxygen radicals, namely, superoxide anion, in a variety of cells, including endothelial cells.26 The generation of reactive oxygen species has been demonstrated to be involved in mediating apoptosis via activation of the cell death program and stimulation of caspase activity.27,28 However, Ang II–induced oxidative stress is mediated via activation of the AT1 receptor in human endothelial cells and, therefore, most likely cannot account for the activation of the caspase cascade by the selective AT2 receptor agonist observed in the present study.
NO donors completely abrogated Ang II–induced apoptosis in HUVECs and prevented cleavage of caspase-3. NO was equipotent in inhibiting Ang II–induced apoptosis compared with the effects of the specific tetrapeptide aldehyde inhibitor of caspase-3. These findings not only underscore the pivotal role of the caspase cascade as the downstream effector arm executing the Ang II–induced cell death program in HUVECs but also point toward the potent effects of NO to inhibit this effector pathway of apoptosis. Indeed, we have previously shown that NO inhibited TNF-α–triggered apoptosis in HUVECs via S-nitrosylation of the reactive cysteine group within the active center of caspase-3 and caspase-1, thereby abrogating enzyme activity.12 The results of the present study considerably extend these findings by demonstrating that NO-mediated inhibition of the caspase cascade exerts an important physiological function, namely, antagonizing the proapoptotic effects of Ang II in endothelial cells. Thus, the present study provides another example for the countervailing effects of Ang II and NO in vascular biology.
The results of the present study are apparently contradictory to recently published data by Pollman et al,14 who demonstrated that Ang II exerts antiapoptotic effects and directly antagonized NO-induced apoptosis in cultured VSMCs. However, these apparent discrepancies are easily reconciled. First, cultured VSMCs exclusively express AT1 and no AT2 receptors29; second, the doses of NO used to induce apoptosis of VSMCs in the study by Pollman et al exceeded 50 μmol/L. It is well known that high doses of NO induce apoptosis via a direct DNA-damaging mechanism,30 whereas low concentrations of NO have been shown to be protective against apoptosis.12,31–33 Thus, the findings of the present study illustrate that it is extremely important not only to interpret the effects of Ang II in a cell type–specific manner but also to recognize the role of NO as a bifunctional modulator of cell fate capable of either inhibiting or stimulating cell death, depending on the concentration of NO applied. Nevertheless, since the endothelial NO synthase is known to produce small amounts of NO,34 the countervailing autocrine effects between NO and Ang II in endothelial cells in vivo will most likely be inhibition of Ang II–induced apoptosis by NO.
The demonstration that Ang II induces apoptosis of endothelial cells may have important clinical implications by extending the potential mechanisms involved in the well-established role of Ang II to be a major contributor to the pathobiology of atherosclerosis.35 Whereas previous studies have mainly focused on the effect of Ang II to promote growth and migration of VSMCs as a key feature of atherosclerotic lesion development,36 the present study demonstrates that Ang II causes an injurious insult leading to apoptotic death of endothelial cells. Lesion-prone regions are characterized by an increased endothelial cell turnover rate,37 which most likely is due to an increase of apoptotic cell death, suggesting a mechanistic link between endothelial cell turnover and the susceptibility to atherosclerotic plaque development.38 Indeed, we have recently shown that oxidized low density lipoprotein, which is a well-established triggering molecule in the atherosclerotic process, also induces apoptosis of HUVECs.21 Taken together, the demonstration that Ang II induces apoptosis of endothelial cells may provide a mechanistic clue linking activity of the renin-angiotensin system with the “response-to-injury” hypothesis of atherogenesis. Activation of the renin-angiotensin system has been repeatedly shown to be associated with the presence of coronary atherosclerosis, especially in patients without additional risk factors.39,40
In summary, the results of the present study demonstrate that Ang II induces apoptosis of human endothelial cells via activation of the caspase cascade, the central downstream effector arm executing the cell death program in response to a variety of stimuli. In accordance with the countervailing effects of Ang II and NO in the regulation of vessel tone and cell growth, NO completely abrogated Ang II–induced apoptosis of HUVECs by interfering with the activation of the caspase cascade. Thus, NO protects endothelial cells from being driven into cell death by Ang II. These findings have important implications not only with respect to the countervailing balance of NO and Ang II to determine vascular lesion formation but may also provide a mechanistic clue linking Ang II with the “response-to-injury” hypothesis of atherogenesis.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|CHO||=||Chinese hamster ovary|
|MAP kinase||=||mitogen-activated protein kinase|
|PCR||=||polymerase chain reaction|
|VSMC||=||vascular smooth muscle cell|
This study was supported by grants from the Deutsche Forschungsgemeinschaft Di 600/2–2. Dr Dimmeler has a fellowship from the Deutsche Forschungsgemeinschaft. We would like to thank Christine Goebel for expert technical assistance.
- Received June 26, 1997.
- Accepted September 25, 1997.
- © 1997 American Heart Association, Inc.
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