Oxidized LDL Upregulates Angiotensin II Type 1 Receptor Expression in Cultured Human Coronary Artery Endothelial Cells
The Potential Role of Transcription Factor NF-κB
Background—We demonstrated earlier that angiotensin II (Ang II), by AT1 receptor activation, upregulates oxidized LDL (ox-LDL) endothelial receptor LOX-1 gene expression and uptake of ox-LDL in human coronary artery endothelial cells (HCAECs). In this study, we investigated the regulation of Ang II receptors (AT1R and AT2R) by ox-LDL and the role of the redox-sensitive transcription factor NF-κB in this process.
Methods and Results—HCAECs were incubated with ox-LDL for 24 hours. Ox-LDL (10 to 40 μg protein/mL) upregulated AT1R but not AT2R, mRNA, or protein. Ox-LDL degraded IκBα in cytoplasm and activated transcription factor NF-κB (P65) in HCAEC nuclear extract. Treatment of cells with the antioxidant α-tocopherol (10 to 50 μmol/L) attenuated ox-LDL–mediated degradation of IκBα and activation of NF-κB (P65) and inhibited the upregulation of AT1R mRNA and protein. The role of NF-κB signal transduction was further examined by use of an NF-κB inhibitor, caffeic acid phenethyl ester (CAPE). Pretreatment of cells with CAPE inhibited ox-LDL–mediated degradation of IκBα and NF-κB activation and inhibited ox-LDL–induced upregulation of AT1R expression. Incubation of cells with both ox-LDL and Ang II increased cell injury, measured as cell viability and LDH release, compared with either ox-LDL or Ang II alone. α-Tocopherol as well as the specific AT1R blocker CV11974 (candesartan) attenuated the cell-injurious effects of ox-LDL.
Conclusions—These observations suggest an important role of ox-LDL–mediated AT1R upregulation in cell injury. In this process, NF-κB activation seems to play a critical role in signal transduction. These findings provide a basis for the use of antioxidants and AT1R blockers in designing therapy of atherosclerosis.
The renin-angiotensin system plays an important role in atherogenesis. Angiotensin II (Ang II) activates at least 2 distinct types of cell-surface receptors, the type 1 (AT1R) and the type 2 (AT2R).1 2 Most studies suggest that AT1R activation mediates most known effects of Ang II in the cardiac tissues.2 3 However, some studies also show that activation of the AT2R receptor mediates myocardial ischemia-reperfusion injury4 and exerts proapoptotic effect on myocytes.5 Both AT1R and AT2R exist in rat coronary artery endothelial cells.6 Recent work from our laboratory7 indicates that both AT1R and AT2R exist in human coronary artery endothelial cells (HCAECs), and AT1R activation induces apoptosis of HCAECs. Another recent study8 showed that activation of AT1R causes myocardial dysfunction during myocardial ischemia-reperfusion in isolated rat hearts.
In atherosclerosis, oxidized LDL (ox-LDL) accumulates in the vessel walls,9 decreases generation of nitric oxide (NO),10 and causes endothelial dysfunction.11 Ox-LDL is cytotoxic12 and acts as a chemotactic factor for monocytes,13 leading to the accumulation of inflammatory cells and the generation of oxygen-derived free radicals that can inactivate endothelium-derived NO.14 Ox-LDL induces apoptosis in vascular smooth muscle cells,15 monocytes/macrophages,16 and endothelial cells.17 On the basis of these considerations, oxidative modification of LDL is considered a key trigger in the initiation and progression of atherosclerosis.18 A recent study by Maziere et al19 demonstrated that ox-LDL induces activation of transcription factor NF-κB in endothelial cells and causes cell injury. Activated NF-κB has indeed been detected in endothelial cells of atherosclerotic plaques.20
There is increasing evidence for an interaction between hyperlipidemia and the renin-angiotensin system in atherogenesis.21 22 23 For example, AT1R expression is upregulated by LDL in vascular smooth muscle cells.21 Ang II facilitates oxidation of LDL22 and its uptake by scavenger receptors on monocytes/macrophages.23 We have recently demonstrated upregulation of specific lectin-like receptors for ox-LDL (LOX-1) in response to Ang II.24 In the present study, we provide evidence that ox-LDL upregulates expression of Ang II type AT1R in cultured HCAECs. We also demonstrate a critical role of transcription factor NF-κB activation in this process.
The methodology for culture of HCAECs has been described previously.17 24 In brief, the initial batch of HCAECs was purchased from Clonetics Corp. The endothelial cells were pure on the basis of morphology and staining for factor VIII and acetylated LDL. HCAECs were cultured in microvascular endothelium growth medium (Clonetics) that consisted of 500 mL endothelial cell basal medium, 5 ng human recombinant epidermal growth factor, 0.5 mg hydrocortisone, 25 mg gentamycin, 50 μg amphotericin B, 6 mg bovine brain extract, and 25 mL FBS. Fourth-generation HCAECs were used in this study.
HCAECs were incubated with ox-LDL (10 and 40 μg protein/mL) for 24 hours, and expression of AT1R and AT2R was determined. Parallel groups of HCAECs were incubated with ox-LDL (40 μg protein/mL) alone or with the antioxidant α-tocopherol (10 and 50 μmol/L) to study degradation of IκBα, NF-κB activation, and AT1R expression. Parallel groups of HCAECs also were pretreated with the antioxidant α-tocopherol, the AT1R blocker CV11974 (candesartan, 10−6 mol/L), or the AT2R blocker PD123319 (10−6 mol/L) for 30 minutes, and HCAECs then were exposed to ox-LDL, Ang II, or both to study degradation of IκB, activation of NF-κB, and induction of cell injury (cell viability and LDH release). To further determine the role of NF-κB activation, cells were incubated with TNF-α (40 ng/mL) and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE), 20 μg/mL, and degradation of IκBα and NF-κB activation and AT1R expression was studied. TNF-α served as a positive control for NF-κB activation by ox-LDL. The concentrations of these reagents were based on previous studies.17 25 26
Preparation of Lipoproteins
Native LDL and ox-LDL were prepared as described earlier.17 LDL was oxidized by exposure to CuSO4 (5 μmol/L free Cu2+) in PBS at 37°C for 24 hours. The thiobarbituric acid–reactive substances content of ox-LDL was 17.5±1.6 versus 0.38±0.11 nmol/100 μg protein in the native-LDL preparation (P<0.01). LDL and ox-LDL were kept in 50 mmol/L Tris-HCl, 0.15 mol/L NaCl, and 2 mmol/L EDTA at pH 7.4 and were used within 10 days of preparation. Endotoxin concentration in the LDL was checked with the E-Toxate kit (Sigma) and found to be consistently <0.005 endotoxin units/mL (lowest detection limit).
RT-PCR for AT1R and AT2R
The detailed methodology for reverse transcription–polymerase chain reaction (RT-PCR) identification of AT1R and AT2R in HCAECs was published recently.24 Essentially, the reverse-transcribed material was amplified with Taq DNA polymerase (Promega) with a primer pair specific to human AT1R receptor (forward primer, 5′-TCATTTACTTTTATATTGTAA-3′; reverse primer, 5′-TGAATTTCATAAGCCTTCTT-3′). PCR product was 532 bp. For PCR, 35 cycles were used at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 2 minutes. The primer pair specific to human AT2R receptor was (forward primer) 5′-AATATGAAG-GGCAACTCCAC-3′ and (reverse primer) 5′-TTAAGAC-ACAAAGGTCTCCAT-3′. The PCR product was 1100 bp. For PCR, 35 cycles were used at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minute.
Immunoprecipitation and Western Blot for AT1R and AT2R
Cell lysates (120 μg) were subjected to immunoprecipitation and then Western analysis. In brief, the cell lysates containing equal amounts of soluble proteins were immunoprecipitated. Precipitates were washed and then resuspended in SDS-PAGE sample buffer and boiled for 5 minutes. Samples were separated by 12% SDS-PAGE, and then transferred to nitrocellulose membranes. After incubation in blocking solution (4% nonfat milk, Sigma), membranes were incubated with primary antibody (polyclonal antibody to AT1R or polyclonal antibody to AT2R, Santa Cruz Laboratory) for 2 hours at room temperature. Membranes were washed and then incubated with 1:3000 dilution secondary antibody (Amersham) for 1 hour, detected with the ECL system, and relative intensities of protein bands were analyzed by MSF-300G Scanner (Microtek Laboratory).27
Preparation of Nuclear Extracts and Western Blot for NF-κB
The detailed methodology for preparation of nuclear extracts and Western blot for NF-κB in HCAECs has been published recently.28 We used a monoclonal antibody to the P65 subunit of NF-κB from mouse to mouse hybrid cells (Boehringer Mannheim). The antibody recognizes an epitope overlapping the nuclear location signal of the P65 subunit and therefore selectively binds the activated form of NF-κB.
Western Blot for IκB
The detailed methodology for Western blot for IκB in HCAECs was published recently.28 The antibody used was a rabbit polyclonal anti-IκBα (Santa Cruz Biotechnology).
Measurement of LDH
HCAEC supernatants were collected for determination of LDH. An enzyme activity method based on oxidation of lactate with measurement of rate of increase in absorbance at 340 nm was used. The activity of LDH was expressed as units per milligram protein.24
A small aliquot of cells was incubated in 0.1% trypan blue for a few minutes, and the cells were viewed under a light microscope. Dead cells are permeable to trypan blue and thus become colored, whereas viable cells do not take up the dye. By counting 100 cells, the percentage of viable cells was calculated.24
All data represent the mean of duplicate samples from 6 independently performed experiments. Data are presented as mean±SD. Statistical significance was determined in multiple comparisons among independent groups of data in which ANOVA and the F test indicated the presence of significant differences. A value of P<0.05 was considered significant.
Ox-LDL and Regulation of AT1R and AT2R in HCAECs
As described earlier,24 AT1R and AT2R mRNA and protein were identified by RT-PCR and Western blot analyses, respectively, in all HCAEC aliquots. Incubation of HCAECs with ox-LDL (10 and 40 μg protein/mL) induced progressive upregulation of AT1R mRNA. Incubation of cells with ox-LDL had no significant effect on AT2R mRNA (Figure 1⇓). Incubation with 80 μg/mL of ox-LDL had a smaller effect on AT1R expression than the 40 μg/mL concentration (data not shown), perhaps a result of the cell-injurious effect of high concentrations of ox-LDL.
Incubation of HCAECs with ox-LDL (10 and 40 μg protein/mL) induced a progressive increase in AT1R protein expression (P<0.01). Incubation of cells with ox-LDL had no significant effect on AT2R protein expression. Results of a representative experiment and summary of data from multiple experiments are shown in Figure 2⇓.
Ox-LDL and Degradation of IκB and Activation of NF-κB and the Effect of α-Tocopherol
Treatment of HCAECs with ox-LDL (40 μg protein/mL) degraded IκBα and activated NF-κB(P65) compared with control (P<0.01). Pretreatment of HCAECs with α-tocopherol (10 and 50 μmol/L) before exposure of cells to ox-LDL consistently inhibited ox-LDL–mediated degradation of IκB and activation of NF-κB compared with ox-LDL alone. The effect of 50 μmol/L concentration was greater than that of 10 μmol/L of α-tocopherol on both IκBα and NF-κB (P65) (Figure 3⇓).
Modulatory Effect of α-Tocopherol on AT1R Expression by Ox-LDL
The presence of α-tocopherol in the culture medium before the cells were exposed to ox-LDL decreased ox-LDL–mediated upregulation of AT1R mRNA (P<0.05 versus ox-LDL alone). The higher concentration of α-tocopherol (50 μmol/L) was more effective than the lower concentration of α-tocopherol (10 μmol/L) in this effect (P<0.01) (Figure 4⇓).
The pretreatment of HCAECs with α-tocopherol before the cells were exposed to ox-LDL also decreased ox-LDL–mediated upregulation of AT1R protein (P<0.01 versus ox-LDL alone). The higher concentration of α-tocopherol (50 μmol/L) was more effective than the lower concentration of α-tocopherol (10 μmol/L) in this effect (P<0.01) (Figure 5⇓). α-Tocopherol alone did not affect AT1R mRNA (Figure 4⇑) and protein expression (Figure 5⇓).
Effect of NF-κB Inhibitor CAPE on ox-LDL– or TNF-α–Mediated NF-κB Activation
To confirm the role of NF-κB in ox-LDL–induced AT1R expression, we used CAPE as a potent inhibitor of NF-κB.26 TNF-α was used as a positive control for NF-κB activation. We found that ox-LDL and TNF-α both significantly degraded IκBα in cytoplasm and activated NF-κB in the nuclear extract. CAPE markedly prevented ox-LDL– or TNF-α–mediated effects. CAPE alone did not affect IκBα degradation and NF-κB activation (Figure 6⇓).
Effect of NF-κB Inhibitor CAPE on AT1R Expression
Ox-LDL upregulated AT1R mRNA and protein as described earlier. TNF-α also significantly induced degradation of IκBα, induced NF-κB activation, and upregulated AT1R mRNA and protein expression. Pretreatment of cells with CAPE markedly attenuated ox-LDL– and TNF-α–induced upregulation of AT1R mRNA and protein expression. CAPE alone did not affect AT1R mRNA and protein expression (Figure 7⇓).
Cell Injury in Response to Ox-LDL and Ang II
Treatment of HCAECs with either ox-LDL or Ang II alone caused a modest increase in LDH release and decrease in cell viability. The presence of both ox-LDL and Ang II further increased cell injury, as measured by cell viability and LDH release, compared with ox-LDL or Ang II alone (P<0.05). Notably, α-tocopherol decreased ox-LDL–induced cell injury. The AT1R blocker CV11974, but not the AT2R blocker PD123319, inhibited ox-LDL–induced cell injury. In control experiments, α-tocopherol (50 μmol/L) decreased cell injury in response to ox-LDL and CV11974 decreased cell injury in response to Ang II. These data are summarized in Figures 8⇓ and 9⇓.
Ox-LDL induces apoptosis and necrosis and upregulates its own receptors, LOX-1, in HCAECs.28 29 The present study indicates that ox-LDL also upregulates the expression of AT1R, but not AT2R, in cultured HCAECs. This study also shows that the degradation of IκBα and activation of NFκB (P65) play an important role in the signal transduction pathway in this action of ox-LDL. The critical role of IκBα and NF-κB became evident in experiments in which α-tocopherol and CAPE inhibited the upregulation of AT1R expression by ox-LDL. The importance of upregulation of AT1R expression in HCAECs became evident from the effects of CV11974, a specific AT1R blocker that markedly decreased the injurious effect of ox-LDL and Ang II.
Ox-LDL and Regulation of Ang II Receptors
Nickenig et al27 showed that hyperlipidemia increases the expression of AT1R receptor in rat cultured vascular smooth muscle cells by 2- to 3-fold. They also showed upregulation of AT1R gene expression when rat vascular smooth muscle cells were exposed to LDL. A recent study from our laboratory demonstrated marked upregulation of AT1R in the atherosclerotic rabbit arteries.30 The importance of AT1R expression in hypercholesterolemic rabbits became evident from increased vasoconstrictor response to Ang II and diminished response to the endothelium-dependent vasodilator acetylcholine.30 Ang II has also been shown to facilitate oxidization of LDL in macrophages.22 These observations suggest an interaction between ox-LDL and Ang II. In the present study, we show that ox-LDL upregulates AT1R mRNA and protein expression, whereas the expression of AT2R remains largely unaffected in cultured HCAECs. We did not further examine the transcriptional and posttranscriptional mechanisms of ox-LDL–induced AT1R expression in this study. We emphasized the effects of ox-LDL on AT1R expression and subsequent functional significance of AT1R expression. We recently provided evidence that Ang II upregulates specific LOX-1 on HCAECs and facilitates the uptake of I125-labeled ox-LDL into these cells; this effect of Ang II can be blocked with specific blockade of AT1R.24 Collectively, these studies provide strong evidence of cross talk between ox-LDL and Ang II in the regulation of cell dysfunction and injury.
Ox-LDL and the Transcription Factor NF-κB
NF-κB is an oncogenic protein that regulates transcription of a variety of cellular genes, including immune and inflammatory response and growth control.31 NF-κB is present in the cytosol as a heterodimer composed of NF-κB1 (P50) and Rel (P65) subunits bound to an inhibitor protein, IκB. Degradation of IκBα protein seems to be necessary for the activation of NF-κB.32 After activation, NF-κB translocates from the cytosol to the nucleus of the cell, binds to specific DNA sequences, and initiates transcription. Maziere et al19 showed that ox-LDL activates NF-κB in fibroblasts, smooth muscle cells, and endothelial cells. Collins33 suggested oxidative activation of endothelial cell transcription factors, especially NF-κB, as a mechanism for changing endothelial cell phenotype and for initiating atherosclerotic lesions. Hernan dez-Presa and colleagues20 provided direct evidence for NF-κB activation in early atherosclerotic lesions. Other studies have shown a critical role of NF-κB activation in apoptosis in myocytes34 and endothelial cells.35 In the present study, we demonstrate that ox-LDL degrades IκBα protein and activates NF-κB in HCAECs. An obvious question relates to the basis of degradation of IκBα and activation of NF-κB by ox-LDL. ox-LDL–induced free radical release may play a critical role in this process. The redox-sensitive nature of NF-κB activation became clear from the observation that a free radical scavenger, α-tocopherol, and the NF-κB inhibitor CAPE not only inhibited ox-LDL–mediated degradation of IκBα and activation of NF-κB but also inhibited the upregulation of AT1R in HCAECs. A recent study26 reported that CAPE inhibits transcription factor NF-κB activation without any effect on other transcription factors, such as AP-1, Oct-1, and TFIID. This study26 also showed that CAPE exerts its effects by inhibiting reactive oxygen intermediates. These observations collectively indicate that activation of NF-κB may be an important signal transduction pathway in the effects of ox-LDL on HCAECs.
Interaction Between ox-LDL and Ang II in Cell Injury
Experimental studies21 24 27 30 suggest that cross talk between hypercholesterolemia and Ang II may have pathophysiological significance. For example, upregulation of AT1R mediated by hypercholesterolemia leads to enhanced Ang II–induced vasoconstriction.27 30 In contrast, Ang II increases oxidation of LDL22 and upregulates ox-LDL endothelial receptor (LOX-1).24
Both Ang II and ox-LDL are important factors in inducing endothelial dysfunction and injury. Work from our laboratory has shown that Ang II7 and ox-LDL17 decrease NO generation and increase lipid peroxidation and LDH release in cultured HCAECs. Both Ang II and ox-LDL enhance anoxia-reoxygenation–mediated HCAEC injury. Work from other laboratories36 37 also suggests that ox-LDL and Ang II cause injury to endothelial cells. In the present study, we demonstrate that ox-LDL and Ang II induce cell injury in a cumulative fashion. The mechanism of ox-LDL–mediated cell injury may be related, at least in part, to the upregulation of AT1R expression. NF-κB activation may play an important role in this process. This became evident in experiments in which α-tocopherol significantly inhibited the actions of ox-LDL in conjunction with inhibition of upregulation of AT1R by ox-LDL. The AT1R blocker CV11974 (candesartan) also attenuated the cell-injurious effects of ox-LDL. These observations suggest that α-tocopherol and AT1R blockers may reduce the adverse effect of cross talk between ox-LDL and Ang II.
In summary, this study shows that ox-LDL upregulates the expression of AT1R, but not AT2R, in cultured human coronary endothelial cells. The cross talk between ox-LDL and Ang II serves to promote injury to coronary endothelial cells. Degradation of IκBα and activation of NF-κB appear to be important signal transduction pathways involved in the action of ox-LDL.
This study was supported by a Merit Review Award from the VA Central Office, a contract with the Department of Defense, and funds from the Swedish Medical Research Council.
- Received March 30, 2000.
- Revision received May 22, 2000.
- Accepted May 22, 2000.
- Copyright © 2000 by American Heart Association
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