Vascular Endothelin-B Receptor System In Vivo Plays a Favorable Inhibitory Role in Vascular Remodeling After Injury Revealed by Endothelin-B Receptor–Knockout Mice

Endothelin (ET)-1, a vasoconstrictor peptide produced by endothelial cells,¹ acts by binding to its 2 subtypes of receptors, endothelin type A (ET_A) and type B (ET_B) receptors.² In the vessels, ET_A receptors are located primarily on vascular smooth muscle cells (VSMCs) to induce vasoconstriction and cell proliferation.² ET_B receptors are expressed not only on vascular endothelial cells but also on VSMCs.³ Endothelial ET_B receptors activate the release of endothelium-derived relaxation factors, such as NO,⁴ a potent vasodilator with an antiproliferative action on VSMCs, whereas the ET_B receptors on VSMCs induce vasoconstriction and proliferation of VSMCs.³ In animal models with vascular disease, such as restenosis after balloon injury, selective ET_A receptor antagonists and ET_A/B dual receptor antagonists are reported to effectively inhibit VSMC proliferation and neointimal hyperplasia.^5–8 Therefore, ET_A receptor antagonism is expected to be an important therapeutic target for vascular diseases. However, the long-term effect of selective blockade of ET_B receptors on vascular diseases has not been reported. Analysis of the effects of ET_B receptor blockade on vascular diseases is important in further understanding the pathophysiological role of the ET system in vascular diseases and in determining whether a selective ET_A receptor antagonist or an ET_A/B dual receptor antagonist is more favorable for the treatment of vascular remodeling.

The purpose of this study was to reveal how the ET-1–ET_B receptor system is involved in vascular remodeling after injury in vivo. To obtain direct evidence concerning the pathophysiological role of the ET_B receptor, we used endogenous ET_B receptor–knockout (KO) mice. Because targeted disruption or naturally occurring mutations of the mouse ET_B receptor gene result in a white-spotted coat and aganglionic megacolon, the latter leading to juvenile death, ^9,10 the mice received a tissue-specific ET_B receptor transgene driven by the human dopamine β-hydroxylase (DβH) gene promoter.^11–13 Thus, the “rescued” KO mice lack functional ET_B receptors in their vessels but exhibit normal enteric development because of adrenergic tissue-specific ET_B receptor expression by the transgene and survive into adulthood.^11–13

Recently, a murine model of vascular remodeling produced by complete ligation of the carotid artery, ie, a blood flow cessation model, was described.^14,15 This has been widely accepted as a useful model characterized by vascular remodeling and proliferative VSMC-dominant intimal hyperplasia induced by cessation of blood flow in the carotid artery. We performed complete ligation of the right common carotid artery in both KO and wild-type (WT) mice and investigated the pathophysiological role of ET_B receptors in vascular remodeling in comparison between KO and WT mice.

Methods

Mice

The generation of heterozygous ET_B receptor–knockout mice (ET_B^+/−) on a C57BL/6J genetic background was described previously.^9,10 We generated the human DβH gene promoter–regulated ET_B receptor transgene (DβH-ET_B) and ET_B^+/− with DβH-ET_B as described previously.^11–13 As a result of crossing between ET_B^+/− with DβH-ET_B, we obtained 3 genotypes: ET_B^+/+ with DβH-ET_B (WT), ET_B^+/− with DβH-ET_B (heterozygous KO mice), and ET_B^−/− with DβH-ET_B (KO mice). KO mice exhibit a white coat that distinguished them from heterozygous KO and WT mice. We distinguished WT from heterozygous KO mice by genomic polymerase chain reaction (PCR) for a targeted allele. PCR was performed on DNA isolated from tail biopsy specimens as described previously.^11,16

Blood Pressure Measurements and Biochemical Assays

We measured blood pressure with a programmable sphygmomanometer using the tail-cuff method as described in our previous article.¹⁷ To investigate the effect of a low-salt diet on blood pressure, WT and KO mice were each divided into 2 groups. One group was given a standard diet (0.24% sodium), and the other was given a low-salt diet (0.0046% sodium). The average blood pressure from 5 independent readings was obtained.

Blood samples were collected, and blood glucose was immediately measured. Plasma concentrations of total cholesterol, angiotensin II, arginine vasopressin, and norepinephrine were measured as described in our previous articles.^18,19

Carotid Artery Ligation

We performed complete ligation of the right common carotid artery of mice, as described in previous articles.^14,15 In brief, 18- to 20-week-old male mice were anesthetized with sodium pentobarbital (50 mg/kg IP), and a cervical incision was performed. The right common carotid artery and its bifurcation into the external and internal carotid arteries were exposed, and the common carotid artery was ligated near the bifurcation. The left common carotid artery was only exposed as a control.

Treatment With Selective ET_A or ET_B Receptor Antagonist

We administered the ET_B receptor antagonist A-192621 orally (30 mg · kg^{− 1} · d⁻¹) to WT mice from 2 days before operation. Furthermore, the ET_A receptor antagonist TA-0201 (0.5 mg · kg⁻¹ · d⁻¹) was infused continuously by an osmotic minipump (model 2004, Alza Co) implanted subcutaneously in each WT and KO mouse from 2 days before operation.

Morphometric Analysis

The bilateral carotid arteries excised 14 days after operation were fixed in 4% paraformaldehyde and embedded in paraffin. A total of 20 transverse sections per animal were cut at 200-μm intervals from the bifurcation to the proximal end and stained with hematoxylin-eosin or Masson’s trichrome. We assessed the right carotid artery morphometrically at the position displaying the smallest lumen and the left carotid artery at the contralateral position as the control side. The borders of the internal lumen, internal elastic lamina (IEL), and external elastic lamina (EEL) were traced on a digitizing board with Apple Macintosh commercial software, Adobe Photoshop 5.0 (Adobe Inc). The luminal area, IEL area, and EEL area were measured with Macintosh image analyzing software, NIH Image version 1.61. The intimal area was calculated by subtracting the luminal area from the IEL area, and the medial area was calculated by subtracting the IEL area from the EEL area. The vessel size was defined by EEL area. The ratio of intimal to medial area (I/M ratio) was calculated by dividing intimal area by medial area. The stenotic ratio was calculated by dividing intimal area by IEL area. Average values were obtained from morphometric analysis of each section of 5 to 11 animals per group.

Quantification of mRNA Levels by Reverse Transcription–PCR

RNA isolation and RT-PCR were performed as in our previous studies.¹⁶ The gene specific primers were as follows: ET-1 (sense): 5′ -GCTGTTCGTGACTTTCCA-3′; ET-1 (antisense): 5′ -GTGGCAGAAGTAGACACA-3′; β-actin (sense): 5′ -GAAGA-TCCTGACCGAGCGTA-3′; β-actin (antisense): 5′ -CGTACTC-CTGCTTGCTGATCC-3′.

The PCR protocol for ET-1 was as follows: initial denaturation at 94°C for 5 minutes, followed by 38 cycles of 15 seconds of denaturation at 94°C, annealing at 58°C for 20 seconds, and extension at 72°C for 45 seconds. The PCR protocol for β-actin was as follows: 22 cycles of 15 seconds of denaturation at 94°C, annealing at 64°C for 30 seconds, and extension at 72°C for 45 seconds.

Measurement of Tissue NO_x Level

NO_x (NO₂⁻ and NO₃⁻) level in plasma or artery was measured as described in our previous article, with minor modification.²⁰ The method was based on the Griess reaction.²¹

Data Analysis

Data are given as mean±SEM. All statistical comparisons were performed by use of a commercially available statistical package for Apple Macintosh computer (StatView, version 4.5, Abacus Concepts, Inc). For multiple comparisons, results were analyzed by 1-way ANOVA followed by Fisher’s post hoc test. For comparisons between 2 values, unpaired Student’s t test or nonparametric Kruskal-Wallis analysis was used when appropriate. Differences were considered significant at a value of P< 0.05.

Results

Characteristics of KO Mice

First, we confirmed the genotypes of WT and homozygous KO mice with an introduced tissue-specific ET_B receptor transgene. As shown in Figure 1, the DβH-gene promoter–flanking ET_B receptor transgene was documented in both WT and KO mice, whereas endogenous ET_B receptor gene was detected only in WT mice. The targeted allele was specifically detected in KO mice.

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We measured blood pressure in both WT and KO mice from 14 to 16 weeks of age. We found that systolic blood pressure was significantly elevated in KO mice compared with WT mice at 14 weeks old (122.9±1.6 versus 113.6±3.3 mm Hg, P<0.05; KO, n=16; WT, n=22). The elevated blood pressure of KO mice was significantly decreased by 2-week treatment with a low-salt diet compared with that of KO mice with a standard diet (118.3±1.3 versus 125.6±1.9 mm Hg, P< 0.05, both n=8). Blood pressure of WT mice did not respond to the low-salt diet (WT with low-salt diet, 115.9±2.1 mm Hg versus WT with standard diet, 116.6±1.9 mm Hg, both n= 11). Therefore, it is suggested that KO mice develop salt-sensitive hypertension. We performed the following experiments using WT and KO mice fed a low-salt diet under normal blood pressure, because high blood pressure is a major factor affecting vascular remodeling.

Vascular Remodeling in Blood Flow Cessation Model

At 14 days after ligated operation, each parameter of body weight, systolic blood pressure, blood glucose, and plasma total cholesterol level was not significantly different between WT and KO mice treated with a low-salt diet (Table 1). Circulating levels of angiotensin II, arginine vasopressin, and norepinephrine were also not significantly different between the 2 groups (Table 1). It was suggested that the physiological and biochemical parameters affecting vascular remodeling were almost comparable in the 2 groups.

In the nonligated (control) artery, intimal thickening was not observed in either WT or KO mice, and the vessel size, luminal area, and medial thickness were not significantly different between WT and KO mice (Figure 2, a and b; Figure 3, a and b). In contrast, the ligated artery exhibited luminal narrowing, which was primarily a result of neointimal hyperplasia and medial thickening, in both WT and KO mice (Figure 2, d and e; Figure 3, c and d). The maximal stenotic lesion was observed 1.23±0.27 mm (WT, n=7; KO, n=7) proximal to the bifurcation in the ligated artery. The extent of each neointimal hyperplasia or vascular stenosis was more greatly enhanced in KO than in WT mice. Furthermore, long-term treatment with the ET_B receptor antagonist A-192621 (30 mg · kg⁻¹ · d⁻¹ for 16 days) increased neointimal formation and decreased luminal area in the ligated artery of WT mice to almost same extent as those in KO mice (Figure 2, e and f). This indicates that the findings obtained from genetic inhibition of ET_B receptors in KO mice are reproduced in the ligated artery in mice treated pharmacologically with an ET_B receptor antagonist.

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Morphometric analysis of the ligated artery revealed that the intimal area (Figure 4a) and the I/M ratio (Figure 4c) were significantly increased in KO mice compared with those in WT mice. These were also increased in WT mice treated with A-192621 to almost same extent as in KO mice, but the differences were not statistically significant. The medial area was comparable in WT, KO, and A-192621–treated WT mice (Figure 4b). The stenotic ratio (Figure 4d) was significantly increased in KO and WT mice treated with A-192621 compared with that in WT mice. Various calculated values are summarized in Table 2. These findings indicate that neointimal hyperplasia and vascular stenosis were accelerated by the inhibition of ET_B receptors independent of blood pressure.

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Expression Level of ET-1 mRNA and NO_x Level in Carotid Artery

Seven days after ligation, the gene expression level of ET-1 mRNA was greatly increased in the ligated artery compared with that in the nonligated artery (Figure 5a). However, the expression level of ET-1 mRNA in the ligated artery of KO mice was comparable to that of WT mice.

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Then, we measured the NO_x level in plasma and the artery. Plasma NO_x level was slightly, but not significantly, lower in KO than in WT mice (19.9±3.4 versus 25.1±3.9 μmol/L; WT, n=7; KO, n=6) (Figure 5b). However, tissue NO_x level in the ligated artery of KO mice was significantly lower than that of WT mice (168.8±21.2 versus 268.4±40.6 nmol/g tissue, P<0.05, both n=8) (Figure 5c). These results indicate that the gene expression of ET-1 mRNA was increased similarly in the ligated arteries of both mice, whereas the tissue NO_x level was significantly decreased in the ligated artery of KO mice.

Effect of Long-Term Treatment With Selective ET_A Receptor Antagonist on Vascular Remodeling

To study the influence of the ET_A receptor system, we administered TA-0201 (0.5 mg · kg⁻¹ · d^{− 1}) to KO and WT mice from 2 days before ligation. Body weight, blood pressure, and other biochemical data were not significantly different between WT and KO mice (data not shown). As shown in Figure 6, treatment with TA-0201 for 16 days markedly inhibited luminal narrowing and neointimal formation in each group. Medial area and vessel size of the ligated arteries in both mice were not affected by treatment. Morphometric analysis revealed that the stenotic ratio of WT mice treated with TA-0201 was significantly lower than that of KO mice treated with TA-0201 (25.1±6.7% versus 50.1±7.4%, P<0.05, both n=6) (Figure 6e). Intimal area and I/M ratio were lower in WT mice treated with TA-0201 than in KO mice treated with TA-0201, but the differences were not statistically significant (intimal area, 9954.9±3234.0 versus 17,585.4±2892.0 μm², both n=6) (I/M ratio, 0.27±0.08 versus 0.49±0.07, both n=6). No significant difference was observed in the medial area between TA-0201–treated groups (medial area, 34,564.9±2159.1 μm² in WT+TA versus 36,170.5±2902.6 μm² in KO+ TA, both n=6).

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Discussion

Previous studies reported that the ET-1 expression is increased in restenotic lesions after balloon injury and in atherosclerosis.^22–24 Moreover, previous observations using pharmacological reagents suggested that the ET_A receptor system contributes to vasoconstriction² and the development of vascular diseases.^5–8 However, there has been little information concerning the pathophysiological role of ET_B receptors in vivo. The present study showed that vascular remodeling with neointimal formation was accelerated in the proximal carotid artery after ligation in rescued KO mice. We also demonstrated that long-term treatment with a selective ET_B receptor antagonist, A-192621, in WT mice aggravated vascular stenosis to almost same extent as in KO mice, suggesting that the enhancement of vascular remodeling by inhibition of the ET_B receptor system is a response seen not only in gene-manipulated mice but also in pharmacologically treated mice. Furthermore, we demonstrated that the gene expression of ET-1 mRNA was similarly increased, whereas the tissue NO_x level was significantly decreased, in the ligated artery of KO mice. Our observations revealed that inactivation of the ET_B receptor system leads to accelerated neointimal formation and vascular stenosis with a significant decrease in NO production in the injured artery.

In this study, we have demonstrated that inactivation of vascular ET_B receptors accelerates pathological vascular remodeling in which gene expression of ET-1 mRNA was comparably increased, whereas tissue NO_x level was significantly decreased, compared with that of WT mice. Previous studies reported that the gene expression of ET-1 and endothelial NO synthase was reciprocally regulated by flow or shear stress in cultured endothelial cells.^25,26 Therefore, our observations suggest that decreased blood flow initially induces an increase in ET-1 expression and a decrease in NO production, resulting in an imbalance between ET-1 and NO levels in lesions. Furthermore, it is also suggested that failure of ET_B receptor–mediated NO release in KO mice causes a significant decrease in tissue NO_x level and consequently leads to pathological aggravation in KO mice. Our suggestion is supported by a previous study showing that endothelial NO synthase–deficient mice display abnormal vascular remodeling after vascular ligation.²⁷ Therefore, it is suggested that inactivation of ET_B receptor–mediated NO release is an important mechanism responsible for the pathological phenotype in KO mice.

In addition to the reduction of the vascular tissue NO_x level in KO mice, other possible mechanisms underlying the enhancement of vascular remodeling in KO mice are considered. Neointimal formation is associated with balances between smooth muscle cell growth and cell death. It has been reported that ET_B receptors promote stretch-induced apoptosis in cultured VSMCs.²⁸ Therefore, a loss of ET_B receptor– mediated apoptosis of VSMCs may contribute to the aggravated neointimal hyperplasia in KO mice. Alternatively, the following consideration is also possible. The ET_B receptor system has also been reported to activate the release of other endothelium-derived relaxation factors, such as prostacyclin. Thus, a decrease in release of other endothelium-derived relaxation factors as well as NO may be involved in the enhancement of vascular remodeling in KO mice.

Clinical Implications and Study Limitations

In the clinical field, ET receptor antagonists are potential drugs for the treatment of vascular disease. However, it remains unclear whether a selective ET_A receptor antagonist or an ET_A/B dual receptor antagonist is more favorable for the treatment of vascular remodeling. Our results suggest that in the case of long-term administration of a selective ET_B receptor antagonist, we should consider the unfavorable effects on vascular remodeling. Moreover, from our results, it seems likely that selective ET_A receptor antagonists are superior to ET_A/B dual receptor antagonists in their therapeutic effect on vascular remodeling. A previous study reported that long-term treatment with a selective ET_A receptor antagonist restored NO–mediated endothelial function and inhibited atherosclerosis in apolipoprotein E–deficient mice.²⁹ Moreover, there are also recent reports that blockade of both ET_A and ET_B receptors is insufficient to inhibit intimal hyperplasia after balloon injury in porcine coronary artery, in contrast to the results of selective ET_A receptor antagonism.^30,31 These studies suggest that selective inhibition of ET_A receptors is better for the treatment of vascular diseases, because selective ET_A receptor antagonism alternatively activated the ET_B receptor system, which plays a vasculoprotective role in injured artery through NO activation. Our findings revealed by KO mice were in accordance with these previous reports.

In the blood flow cessation model used in this study, ^14,15 a major cause of the vascular lesion formation is no-flow stimulus, whereas restenosis after coronary angioplasty in humans is triggered primarily by mechanical endothelial injury. Moreover, although it seems certain that a decrease in blood flow is associated with the cause of human vascular diseases,^32,33 the no-flow phenomenon that occurred in the ligated carotid artery is not generally seen in the human diseased artery, such as in restenosis or atherosclerosis. Thus, we address some differences in the pathogenesis of vascular remodeling between humans and this experimental model as a study limitation.

Conclusions

Neointimal formation and vascular stenosis caused by the cessation of blood flow were markedly accelerated in the carotid artery of KO mice. The phenomenon was attributed in part to a decrease in NO release originating from ET_B receptor deficiency in KO mice. We also observed that long-term treatment with an ET_B receptor antagonist worsened vascular remodeling in WT mice. Moreover, long-term ET_A receptor blockade significantly attenuated vascular stenosis in both WT and KO mice. These results suggest that the ET_A receptor system plays an aggravating role, whereas the ET_B receptor system plays a favorable inhibitory role, at least partly through NO release, in vascular remodeling after injury. These findings provide a novel insight into the pathophysiological roles of the subtypes of ET receptors in vascular diseases and a potential clinical indication for using ET receptor antagonists.

Footnotes