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Cardioprotective effects of carvedilol on acute autoimmune myocarditis: anti-inflammatory effects associated with antioxidant property

Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

Submitted 15 June 2003 ; accepted in final form 25 August 2003

	ABSTRACT

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Carvedilol, a new -blocker with antioxidant properties, has been shown to be cardioprotective in experimental models of myocardial damage. We investigated whether carvedilol protects against experimental autoimmune myocarditis (EAM) because of its suppression of inflammatory cytokines and its antioxidant properties. We orally administered a vehicle, various doses of carvedilol, racemic carvedilol [R(+)-carvedilol, an enantiomer of carvedilol without -blocking activity], metoprolol, or propranolol to rats with EAM induced by porcine myosin for 3 wk. Echocardiographic study showed that the three -blockers, except R(+)-carvedilol, suppressed left ventricular fractional shortening and decreased heart rates to the same extent. Carvedilol and R(+)-carvedilol, but not metoprolol or propranolol, markedly reduced the severity of myocarditis at the two different doses and suppressed thickening of the left ventricular posterior wall in rats with EAM. Only carvedilol suppressed myocardial mRNA expression of inflammatory cytokines and IL-1 protein expression in myocarditis. In addition, carvedilol and R(+)-carvedilol decreased myocardial protein carbonyl contents and myocardial thiobarbituric acid-reactive substance products in rats with EAM. The in vitro study showed that carvedilol and R(+)-carvedilol suppressed IL-1 production in LPS-stimulated U937 cells and that carvedilol and R(+)-carvedilol, but not metoprolol or propranolol, suppressed thiobarbituric acid-reactive substance products in myocardial membrane challenged by oxidative stress. It was also confirmed that probucol, an antioxidant, ameliorated EAM in vivo. Carvedilol protects against acute EAM in rats, and the superior cardioprotective effect of carvedilol compared with metoprolol and propranolol may be due to suppression of inflammatory cytokines associated with the antioxidant properties in addition to the hemodynamic modifications.

myocarditis; -blocker

Carvedilol, a novel multifunctional neurohormonal antagonist, has been shown to provide greater benefit than traditional -blockers in chronic heart failure because of its antioxidant actions, which synergize with its nonspecific - and ₁-blocking effects (19). When carvedilol and metoprolol were recently compared in clinical trials for heart failure, each showed beneficial -blocker effects and decreased levels of serum thiobarbituric acid-reactive substance (TBARS) (14). However, it was demonstrated that the superior cardioprotection of carvedilol compared with metoprolol and propranolol is induced by its anti-inflammatory action in ischemia-reperfusion models (3, 27). Nevertheless, it remains to be determined whether carvedilol protects against autoimmune myocarditis.

The present study was undertaken to assess whether carvedilol attenuates myocardial inflammation and decreases the severity of myocarditis in the acute stage of EAM in rats. In addition, to determine whether the effects of carvedilol are attributable solely to the hemodynamic effect or whether its antioxidant property with immunomodulation may also be involved, the effects of propranolol, metoprolol, a racemic carvedilol [R(+)-carvedilol, an enantiomer of carvedilol without -blocking activity], and an antioxidant, probucol, were also assessed in the same animal model.

	MATERIALS AND METHODS

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In Vivo Study

Immunization. Acute EAM was induced by porcine cardiac myosin in 7-wk-old Lewis rats, as previously described (13, 24). Control rats were immunized with Freund's complete adjuvant alone.

Medication. EXPERIMENT I. To analyze the hemodynamics of three -blockers on normal rats in which heart rate (HR) alone was affected, 7-wk-old rats were divided into seven groups for the oral administration of 1) carvedilol at 10 mg·kg^–1·day^–1 (Car-10, n = 3), 2) carvedilol at 20 mg·kg^–1·day^–1 (Car-20, n = 3), 3) metoprolol at 75 mg·kg^–1·day^–1 (Met-75, n = 3), 4) metoprolol at 150 mg·kg^–1·day^–1 (Met-150, n = 3), 5) propranolol at 30 mg·kg^–1·day^–1 (Pro-30, n = 3), 6) propranolol at 60 mg·kg^–1·day^–1 (Pro-60, n = 3), or 7) vehicle (0.5% methylcellulose, n = 3) for 3 wk. Blood pressure and HR were determined by the tail-cuff method using a photoelectric tail-cuff detection system (model BP-98A, Softron, Tokyo, Japan) on days 1, 7, 14, and 21. To analyze the cardioprotective effect of three -blockers on acute EAM, rats with myocarditis were divided into seven groups: 1) Car-10 (n = 8), 2) Car-20 (n = 7), 3) Met-75 (n = 7), 4) Met-150 (n = 7), 5) Pro-30 (n = 7), 6) Pro-60 (n = 7), and 7) vehicle (n = 8). The drugs were administrated for 3 wk. All of the rats were killed on day 22.

EXPERIMENT II. To analyze the effects of three -blockers on cardiac function in rats with acute EAM, where HR and blood pressure were affected, we performed an echocardiographic study. Rats with EAM were divided into nine groups for the oral administration of 1) carvedilol at 10 mg·kg^–1·day^–1 (Car-10, n = 6), 2) carvedilol at 20 mg·kg^–1·day^–1 (Car-20, n = 7), 3) R(+)-carvedilol at 10 mg·kg^–1·day^–1 (R-Car-10, n = 7), 4) R(+)-carvedilol at 20 mg·kg^–1·day^–1 (R-Car-20, n = 7), 5) metoprolol at 180 mg·kg^–1·day^–1 (Met-180, n = 7), 6) metoprolol at 360 mg·kg^–1·day^–1 (Met-360, n = 6), 7) propranolol at 90 mg·kg^–1·day^–1 (Pro-90, n = 6), 8) probucol at 200 mg·kg^–1·day^–1 (n = 7), or 9) vehicle (n = 7) for 3 wk. Normal rats treated with the drugs and vehicle (Table 1) were also prepared. Transthoracic echocardiography was performed on the day before the rats were killed on day 21, as previously described (9). Carvedilol and racemic carvedilol [catalog no. BM14.190R(+)] were provided by Daiichi Pharmaceutical (Tokyo, Japan).

The protocol was approved by the Institutional Animal Research Committee of Kyoto University.

Histopathology. At death, macroscopic findings and pericardial effusion were graded. Microscopic findings for myocardial damage and cellular infiltration were graded on a scale of 0–4, as described previously (24).

Detection of myocardial oxidized proteins. Oxidative inactivation of enzymes and oxidative modification of proteins by metal-catalyzed oxidation reactions are accompanied by the generation of protein carbonyl derivatives. Oxidized protein was detected using an oxidized protein detection kit (OxyBlot, Oncor), as described previously (23). The OxyBlot kit provides reagents for sensitive immunodetection of carbonyl groups, which represent a hallmark of the oxidation status of all proteins.

TBARS assay. Lipid peroxide formation in the myocardium was determined using a modified thiobarbituric acid method for estimating malondialdehyde, as described previously (25).

Ribonuclease protection assay. The total mRNA was extracted from the myocardium, and the cytokine mRNA levels were measured by ribonuclease protection assay, as described previously (24).

Western blotting. Myocardial IL-1 protein expression was detected by Western blotting, as described previously (23).

Immunohistochemistry. An immunoperoxidase technique was used to perform immunohistochemistry for IL-1, as described previously (24).

In Vitro Study

Membrane preparations and TBARS measurement. Rat ventricular membrane preparations were prepared as reported previously (28). Membrane preparations were preincubated at 15°C with carvedilol, R(+)-carvedilol, metoprolol, or propranolol for 30 min. Lipid peroxidation was initiated by the addition of 0.1 ml of 25 mM FeCl₂ and 1 mM ascorbic acid. TBARS were then measured, and the data are presented as percentages of Fe²⁺-ascorbate-stimulated TBARS content.

Cytokine assays. U937 cells, human monoblast cells, were stimulated with 10 µg/ml lipopolysaccharide (LPS). Carvedilol, R(+)-carvedilol, metoprolol, or propranolol was added to the medium 30 min before LPS stimulation. After 48 h, IL-1, tumor necrosis factor-, and IL-10 in the medium were assayed by enzyme-linked immunosorbent assay using commercially available kits (R & D Systems).

Statistics

Values are means ± SD. Statistical significance was determined by one-way analysis of variance, followed by Fisher's protected least-significant difference test. P < 0.05 was considered statistically significant.

	RESULTS

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HR, Blood Pressure, and Heart Weight-to-Body Weight Ratio in Normal Rats

Experiment I. We treated normal rats with various doses of carvedilol, metoprolol, or propranolol for 3 wk to determine which drugs affected HR alone. The HRs in the treated groups were significantly decreased by 8.5–18.1% compared with the vehicle group (Table 1), but there was no significant difference among the six treated groups. There were no significant differences in blood pressure among the seven groups. It was suggested that the two doses of the three drugs have the same -blocking property.

Experiment II. We conducted this experiment to determine which drugs affected HR and blood pressure. High doses of R(+)-carvedilol (R-Car-20), metoprolol (Met-360), and propranolol (Pro-90) reduced blood pressure. Probucol did not affect blood pressure (Table 1).

Histopathology, Heart Weight-to-Body Weight Ratio, and Hemodynamics in Rats With Acute EAM

Experiment I. No rats died during the study period. At the time of death, on day 22, the hearts showed severe and diffuse discolored myocarditis with massive pericardial effusion. Extensive injuries to myocytes with inflammatory changes and multinucleated giant cells (Fig. 1B) were observed. Treatment with carvedilol markedly reduced the severity of the disease, as assessed by measuring heart weight-to-body weight ratio and microscopic score (Table 2, Fig. 1), but metoprolol and propranolol only slightly reduced, or tended to reduce, the severity of the disease, although all three -blockers at the high dose decreased HR to the same extent.

View larger version (145K): [in this window] [in a new window]   Fig. 1. Histopathology and immunohistochemical staining for IL-1 in the heart. A and C: histopathology in control animal (grade 0). B and D: representative histopathology in a vehicle-treated rat with myocarditis. Marked diffuse myocardial necrosis and cellular infiltration with multinuclear giant cells (arrows) are shown in inflammatory regions (grade 4). E and F: representative histopathology in a rat with myocarditis treated with carvedilol at 10 and 20 mg·kg–1·day–1 (Car-10 and Car-20). Small foci of cellular infiltrations in inflammatory regions (arrows) are shown (grade 1). G and H: representative histopathology in a rat with myocarditis treated with metoprolol at 75 and 150 mg·kg–1·day–1 (Met-75 and Met-150). Myocardial inflammation is transmural (grade 3). I and J: representative histopathology in a rat with myocarditis treated with propranolol at 30 and 60 mg·kg–1·day–1 (Pro-30 and Pro-60). Myocardial inflammation is transmural (grade 3). Hematoxylin and eosin; original magnification x100 (A and B; inset x200) and x10 (C–J). K–P: immunohistochemical staining for IL-1. IL-1 was strongly stained in infiltrating inflammatory cells (arrowheads in inset in L), and only carvedilol administration reduced IL-1 expression in inflammatory lesions. Original magnification x100; inset x200.

View this table: [in this window] [in a new window]   Table 2. Hemodynamics and histopathology by -blockers treatment in rats with EAM

Experiment II. High doses of metoprolol (Met-360) and propranolol (Pro-90), which reduced blood pressure in normal rats, did not reduce the severity of myocarditis (Table 2). The two doses of R(+)-carvedilol ameliorated myocarditis significantly. Probucol also reduced the severity of myocarditis slightly but significantly.

Echocardiographic Study

In this animal model, fractional shortening (FS) of the left ventricle in rats with EAM did not decrease on day 21 compared with normal control (Table 3). Left ventricular posterior wall thickness (PWT) increased and, thus, left ventricular end-diastolic diameter decreased in rats with EAM on day 21.

The three -blockers suppressed FS to the same extent (Table 3). Probucol and the two doses of R(+)-carvedilol did not affect FS significantly. R(+)-carvedilol suppressed PWT compared with rats with EAM. In summary, carvedilol, R(+)-carvedilol, and probucol, all of which showed antioxidant activity, suppressed the increase of left ventricular mass-to-body weight ratio compared with EAM rats.

Changes in Oxidized Proteins in Myocardium

In rats with acute EAM, myocardial protein carbonyl contents were markedly increased (Fig. 2). Thus it was suggested that cellular protein oxidative damage was increased in acute EAM. The increased protein carbonyl contents were reduced by carvedilol, but not by metoprolol or propranolol, treatment compared with the untreated rats, suggesting that carvedilol could protect cellular proteins from oxidative damage.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Changes in myocardial protein carbonyl contents. A: Oxyblot analysis; 10 µg of each protein sample were loaded. Controls, rat immunized with Freund's complete adjuvant alone; Myocarditis, vehicle-treated rat with myocarditis; Car-20, rat with myocarditis treated with carvedilol at 20 mg·kg–1·day–1; Met-150, rat with myocarditis treated with metoprolol at 150 mg·kg–1·day–1; Pro-60, rat with myocarditis treated with propranolol at 60 mg·kg–1·day–1. MW, molecular weight markers. B: densitometric analysis of relative protein carbonyl contents. In rats with myocarditis, protein carbonyl contents were increased markedly and were decreased by carvedilol, but not by metoprolol and propranolol, treatment. Values are means ± SE from 4 animals and represented as percentage of controls. *P < 0.01 vs. control; P < 0.01 vs. myocarditis.

TBARS Products in Myocardium

Myocardial TBARS contents were significantly higher (P < 0.01) in EAM than in control rats: 112.4 ± 9.6 vs. 51.5 ± 5.6 nmol/g heart (n = 5). TBARS contents were significantly lower in groups treated with low and high doses of carvedilol: 66.8 ± 8.9 and 64.6 ± 7.9 nmol/g heart for Car-10 and Car-20, respectively (n = 5, P < 0.01 vs. vehicle-treated rats with myocarditis). In contrast, there was no difference in TBARS content among groups treated with low and high dose of metoprolol or propranolol: 98.8 ± 11.9, 106.9 ± 12.4, 101.1 ± 9.7, and 96.5 ± 15.4 nmol/g heart for Met-75, Met-150, Pro-30, and Pro-60, respectively (n = 5, P = not significant vs. vehicle-treated rats with myocarditis).

Ribonuclease Protection Assay

In controls, myocardial mRNA expression of cytokines was detected only for macrophage inhibitory factor and interferon-. In vehicle-treated rats with acute EAM, mRNAs of Th₁ cytokines (e.g., IL-18), Th₂ cytokines (e.g., IL-6), and proinflammatory cytokines (e.g., macrophage inhibitory factor, interferon-, IL-1, IL-1, and IL-1 receptor type a) were markedly upregulated, and mRNA expressions of IL-12 p35, IL-12 p40, and IL-10 were slightly upregulated. In summary, treatment with carvedilol, but not metoprolol or propranolol, markedly reduced expression of cytokine mRNAs (Fig. 3).

View larger version (87K): [in this window] [in a new window]   Fig. 3. Ribonuclease protection assay for mRNAs of Th1 (e.g., IL-18), Th2 (e.g., IL-6), and proinflammatory [e.g., macrophage inhibitory factor (MIF), IFN-, IL-1, IL-1, and IL-1 receptor type a (IL-1Ra)] cytokines. In controls, myocardial mRNA expression of cytokines was detected only for MIF and IFN-. In vehicle-treated rats with myocarditis, mRNAs of Th1, Th2, and proinflammatory cytokines were markedly upregulated and mRNA expressions of IL-12 p35, IL-12 p40, and IL-10 were slightly upregulated compared with intact hearts. Treatment with carvedilol, but not metoprolol or propranolol, reduced expressions of cytokine mRNAs. L32 and GAPDH are housekeeping genes. A representative finding of 3 distinct experiments is shown.

IL-1 Expression in Rats With EAM

Western blotting showed 3.2-fold upregulation of myocardial IL-1 in vehicle-treated EAM rats compared with controls (Fig. 4). Treatment with carvedilol, but not metoprolol or propranolol, decreased the upregulated IL-1 expression. Immunohistochemistry showed that IL-1 was strongly stained in infiltrating inflammatory cells (Fig. 1, arrowheads) and that only carvedilol administration reduced IL-1 expression in the inflammatory lesions (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 4. Myocardial IL-1 protein expression. A: Western blot analysis; 10 µg of each protein sample were loaded. Vehicle, vehicle-treated rats with myocarditis; see Fig. 2 legend for explanation of other groups. B: densitometric analysis of relative protein levels. In rats with myocarditis, IL-1 protein expression was markedly increased and was decreased by carvedilol, but not by metoprolol and propranolol, treatment. Values are means ± SD from 4 animals and represented as percentage of controls. *P < 0.01 vs. controls; P < 0.01 vs. vehicle-treated rats with myocarditis.

Protection From Lipid Peroxidation in Ventricular Membrane

We also examined TBARS contents of the heart membrane in vitro in the absence or presence of carvedilol, R(+)-carvedilol, metoprolol, or propranolol. Addition of FeCl₂-ascorbate to membrane preparations caused a 7.5-fold increase in TBARS. Only carvedilol and R(+)-carvedilol protected from this lipid peroxidation in a dose-dependent manner. However, metoprolol and propranolol were ineffective at concentrations as high as 100 µM (Fig. 5A).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Protection from lipid peroxidation and suppression of LPS-induced IL-1 production by carvedilol and R(+)-carvedilol in vitro. A: incubation of ventricular membrane preparations for 30 min with the system FeCl2-ascorbate caused a 7.5-fold increase in thiobarbituric acid-reactive substances (TBARS). Three -blockers and R(+)-carvedilol were added in increasing concentrations before oxidative stress. TBARS formation was quantitated as described in MATERIALS AND METHODS. Values are presented as percentage of Fe2+-ascorbate-stimulated TBARS content. Carvedilol and R(+)-carvedilol protected from lipid peroxidation in a dose-dependent manner. Metoprolol and propranolol were ineffective at concentrations as high as 100 µM. Values are means ± SD of 3 independent experiments. *P < 0.01 vs. absence of drugs. B: IL-1 production was increased by LPS stimulation in U937 cells. Carvedilol and R(+)-carvedilol, but not metoprolol or propranolol, suppressed IL-1 production in a dose-dependent manner. Values are means ± SD of 4 independent experiments. *P < 0.01 vs. absence of drugs.

Effect of Carvedilol on Proinflammatory Cytokines in U937 Cells

IL-1 production was increased by LPS stimulation in U937 cells. Tumor necrosis factor- and IL-10 production were undetected. Carvedilol and R(+)-carvedilol, but not metoprolol or propranolol, suppressed LPS-induced IL-1 production in a dose-dependent manner (Fig. 5B).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present findings clearly demonstrate that although the three -blockers showed the same extent of negative inotropic and negative chronotropic effects on acute EAM, carvedilol markedly reduced the severity of acute EAM in rats at the two different doses. In contrast, metoprolol and propranolol only slightly reduced or did not reduce the severity of the disease. The present results also showed that the superior cardioprotection by carvedilol compared with metoprolol and propranolol might be due to suppression of inflammatory cytokines associated with the antioxidant property.

The most interesting finding in the present study was that treatment with carvedilol, but not metoprolol and propranolol, suppressed the mRNA expression of inflammatory cytokines and IL-1 protein expression in myocarditis. Also, only carvedilol and R(+)-carvedilol suppressed IL-1 production in vitro. Accordingly, the superior beneficial effects of carvedilol compared with metoprolol and propranolol in EAM may be partly due to the suppression of inflammatory cytokines.

Another interesting finding was the concomitant antioxidant effect of carvedilol. Administration of carvedilol, but not metoprolol or propranolol, decreased but did not fully eliminate myocardial protein carbonyl contents, a marker of cellular protein injury, and also decreased myocardial TBARS production in rats with EAM. The same effect also occurred in myocardial membrane challenged by oxidative stress ex vivo. This suggested that only carvedilol protected cellular proteins from oxidative damage and cellular membrane lipid from peroxidation. The antioxidant property might be unique to carvedilol and nadolol (3), another -blocker (15). The anti-oxidant activity of carvedilol has been attributed to the carbazole moiety of the drug (4). Indeed, R(+)-carvedilol, an enantiomer of carvedilol without -blocking activity, also ameliorated acute EAM in vivo and antioxidant activity in vitro. Probucol, an antioxidant, also ameliorated EAM to a certain degree. It is possible, therefore, that the greater reduction in myocardial inflammation and necrosis produced by carvedilol than by metoprolol and propranolol may be due to the synergistic effects of the antioxidant and anti-inflammatory properties of the drug on acute EAM.

Carvedilol blocks three (₁, ₂, and ₁) adrenergic receptors and, therefore, possesses a more comprehensive sympatholytic action than the other -blockers (6, 26). It is unlikely that the cardioprotection of carvedilol demonstrated in this study is achieved by -receptor blocking action per se because of the absence of such protection by metoprolol and propranolol in the present study. In addition, R(+)-carvedilol, an enantiomer of carvedilol without -blocking activity (16), was also cardioprotective. Although the present study did not precisely elucidate the role of ₁-receptor blocking action per se in EAM, it is also unlikely that ₁-blockade is predominantly involved in the cardioprotection of carvedilol, because the vasodilatory effect of carvedilol is no longer prominent during chronic treatment (22).

Concerning the relative doses of the three -blockers used in the present study, we compared the drug effects at similar blood pressure levels in the resting state in experiment I. To make a further comparison of -blocking efficacy, we performed experiment II to ramp up the doses of metoprolol and propranolol in which HRs and blood pressure were affected and compared the negative inotropic action by echocardiography. The three -blockers presented the same negative inotropic and negative chronotropic actions. In addition, we have investigated the effects of the three -blockers on cardiac function and ventricular remodeling using echocardiography. The FS in rats with EAM did not decrease significantly on day 21 compared with normal control rats in this animal model. The three -blockers suppressed FS to the same extent. Probucol did not affect FS significantly. PWT increased in rats with EAM, and only carvedilol and R(+)-carvedilol suppressed the increase in PWT. According to the characteristic of this model (13), i.e., that the inflammatory process continues until 3 wk and thereafter decreases and then ventricular remodeling begins, the PWT differences among the three -blockers might be attributed to the anti-inflammatory effect of carvedilol during the acute stage of EAM. As a result, carvedilol, R(+)-carvedilol, and probucol, all of which have antioxidant activity, exhibited an antihypertrophic effect in this animal model.

Although the molecular mechanisms of antioxidant effects of carvedilol for myocardial contractile proteins are not understood, recent studies suggest that the drug protects against oxidation of sarcoplasmic reticulum Ca²⁺-ATPase, inhibits oxidative damage to amino acids, and also reduces oxidative stress in the myocardium in patients with DCM (2, 5, 17). It was reported that cytokines or reactive oxygen species induce the activation of NF-B (5, 21). Accordingly, it is possible that carvedilol may inhibit the activator signal transduction pathways of NF-B.

Although supportive therapy is the first line of treatment for patients with myocarditis (7, 11, 12, 24), only limited success has been reported for treatment of inflammatory myocardial diseases with corticosteroid and intravenous immunoglobulin (15, 24). Although several studies (1, 8, 19) have demonstrated highly significant positive effects on total mortality as well as all-cause hospitalization in patients with stable chronic heart failure, the effect of those -blockers in acute inflammatory myocardial diseases, such as myocarditis and recent-onset cardiomyopathy, have not been fully explored. Alternatively, carvedilol may be promising for the treatment of myocarditis patients, especially with autoimmune and giant cell origins, because of its potent anti-inflammatory action, as demonstrated in the present study. In conclusion, carvedilol protects against acute EAM in rats, and the superior cardioprotective effects of carvedilol compared with metoprolol and propranolol may be attributed to the suppression of inflammatory cytokines associated with the antioxidant properties.

	ACKNOWLEDGMENTS

 
The authors thank M. Nimata for technical help.

GRANTS

This study was supported in part by research grants from Japanese Education of Science and Welfare, Shimizu Immunology Foundation, and Cardiovascular Research Foundation.

	FOOTNOTES

 

Address for reprint requests and other correspondence: C. Kishimoto, Dept. of Cardiovascular Medicine, Graduate School of Medicine, Kyoto Univ., 54 Kawaracho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: kkishi{at}kuhp.kyoto-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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