Effect of RANTES on Vascular Contractile and Relaxant Responses in Rat Carotid Arteries

Abstract

Although regulated-upon-activation normal T-cell expressed and secreted (RANTES), a chemokine originally identified as a T-cell-specific gene product, has been shown to influence various cellular processes, its effects on vascular reactivity remain unclear. Therefore, we investigated the direct effects of prolonged RANTES exposure on responses to various vasoactive agents in isolated rat carotid arteries. Contractile responses to serotonin, isotonic high K⁺, noradrenaline, the thromboxane A₂ analog U46619, and endothelin-1 were similar between the control and RANTES-treated groups (100 ng/mL for approximately 24 h). However, RANTES treatment impaired acetylcholine-induced relaxation, whereas relaxations induced by the nitric oxide donor sodium nitroprusside, the ATP-sensitive potassium channel activator cromakalim, and the large-conductance calcium–activated potassium channel activator NS19504 were unaffected. Acute incubation with the nitric oxide synthase inhibitor N^G-nitro-l-arginine abolished acetylcholine-induced relaxation and eliminated the differences between the control and RANTES-treated groups. Furthermore, the cyclooxygenase inhibitor indomethacin also abolished the differences in acetylcholine-induced relaxation between the 2 groups. Co-treatment with the antioxidant N-acetyl-l-cysteine enhanced acetylcholine-induced relaxation in the presence of RANTES, while co-treatment with the C–C motif chemokine receptor 5 antagonist maraviroc slightly improved the relaxation response. These findings suggest that RANTES impairs acetylcholine-induced relaxation, likely due to the reduction of nitric oxide bioavailability and the unmasking of vasoconstrictor prostanoids through increased reactive oxygen species.

INTRODUCTION

Vascular tone is precisely regulated by physical, neural, hormonal, and endothelial stimuli that elicit contractile and relaxant responses in vascular smooth muscle cells.^1–7) In the vascular system, both vascular smooth muscle cells and endothelial cells play critical roles in the remodeling processes involved in the initiation and progression of vascular diseases.^{1–4,8–13)} Therefore, identifying the causative factors and underlying molecular signaling pathways responsible for the dysregulation of vascular responses to various vasoactive substances is essential for a comprehensive understanding of the pathogenesis and management of vascular dysfunction.

The regulated-upon-activation normal T-cell expressed and secreted (RANTES; also known as C–C chemokine ligand 5) is a soluble factor secreted by various cell types, including macrophages, activated T cells, smooth muscle cells, endothelial cells, trophoblast cells, and platelets.^14–16) RANTES plays a key role in inflammation by recruiting T cells, eosinophils, dendritic cells, mast cells, natural killer cells, macrophages, and basophils to sites of inflammation and infection.^9,17,18) It is also upregulated in vascular wall cells and cooperates in leukocyte recruitment to injured arteries during vascular remodeling. RANTES has been implicated in the pathogenesis of atherosclerosis and organ damage.^19–24) It interacts with chemokine receptors, such as C–C motif chemokine receptor 1 (CCR1), CCR3, CCR4, and CCR5, as well as G-protein-coupled receptor 75, thereby modulating a range of cellular functions and being involved in various inflammatory disorders.^18,25) Although recent studies suggest a role for RANTES in vascular function,^26,27) limited information is available regarding its direct effects on vascular tone regulation, including contraction and relaxation. In this study, we hypothesized that exposure to RANTES would alter vascular function in the carotid arteries of rats. To test this hypothesis, we employed an organ culture model of the intact vascular wall,^28–32) as RANTES may exert indirect effects on vascular function through interactions with nonvascular cells and tissues.

MATERIALS AND METHODS

Animals and Organ Culture Method

Male Wistar rats were obtained from Japan SLC Inc. (Hamamatsu, Shizuoka, Japan) and housed under standard laboratory conditions with free access to food and water. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and the International Guiding Principles for Biomedical Research Involving Animals. All procedures complied with animal welfare regulations, adhered to the 3R (Replacement, Reduction, and Refinement), and were approved by the Laboratory Animal Use and Management Committee of the Institute of Kyushu University of Medical Science (Approval Numbers: 6-1-33 and 7-1-09).

Organ culture was performed as previously described.^28–30) In all experiments, non-fasted rats (9–12 weeks old) were anesthetized with isoflurane via a nose cone for surgical procedures and euthanized via thoracotomy followed by exsanguination. After euthanasia, the common carotid arteries were carefully and aseptically isolated, cleaned, and cut into arterial rings (2 mm in length, approximately 1 mm in diameter) in ice-cold, oxygenated, filtered, modified Krebs–Henseleit solution. The arterial rings were then placed in low-glucose serum–free Dulbecco’s modified Eagle’s medium supplemented with 1.0% penicillin–streptomycin, in the absence or presence of RANTES (100 ng/mL),²⁶⁾ and cultured for approximately 24 h in a humidified incubator at 37°C with 95% air and 5% CO₂. To investigate the effect of the antioxidant N-acetyl-l-cysteine, arterial rings were co-treated with either vehicle (0.1% (v/v) ultrapure water) or N-acetyl-l-cysteine (10⁻³ mol/L) in the presence of RANTES (100 ng/mL) and cultured for approximately 24 h. In another set of experiments, arterial rings were co-treated with either vehicle (0.1% (v/v) dimethyl sulfoxide) or the CCR5 antagonist maraviroc (4 × 10⁻⁵ mol/L)²⁶⁾ along with RANTES (100 ng/mL), and similarly cultured for approximately 24 h.

Measurement of Isometric Force in the Carotid Artery

Cultured carotid arterial rings, pretreated as described, were mounted on a pair of stainless-steel pins in a well-oxygenated (95% O₂, 5% CO₂) organ bath containing modified Krebs–Henseleit solution maintained at 37°C. The endothelium-intact rings were stretched to an optimal resting tension of 9.8 mN, as previously reported,^30,33) and allowed to equilibrate for at least 20 min. Isometric force generation was measured using an isometric transducer (model TB-611T; Nihon Kohden, Tokyo, Japan), consistent with prior studies involving rat carotid arteries. Arterial viability was assessed by contraction in response to high K⁺ (80 mmol/L) and relaxation in response to acetylcholine following precontraction with phenylephrine. To evaluate contractile responses, concentration–response curves were generated for serotonin (10⁻⁹–10^−4.5 mol/L), high K⁺ (10–80 mmol/L), noradrenaline (10⁻⁹–10⁻⁵ mol/L), the thromboxane–prostanoid receptor agonist U46619 (10⁻¹⁰–10⁻⁶ mol/L), and endothelin-1 (10⁻¹⁰–10^−7.5 mol/L). For relaxation studies, carotid arterial rings were precontracted with U46619 (10⁻⁷–10^−6.5 mol/L), and concentration–response curves were obtained for acetylcholine (10⁻⁹–10⁻⁵ mol/L), the nitric oxide donor sodium nitroprusside (10⁻¹⁰–10⁻⁵ mol/L), the ATP-sensitive K⁺ channel activator cromakalim (10⁻⁹–10⁻⁵ mol/L), and the large-conductance Ca²⁺-activated K⁺ channel activator NS19504 (10⁻⁹–10⁻⁴ mol/L). To assess the role of nitric oxide synthase and cyclooxygenase in acetylcholine-induced relaxation, rings were incubated with the nitric oxide synthase inhibitor N^G-nitro-l-arginine (10⁻⁴ mol/L) or the cyclooxygenase inhibitor indomethacin (10⁻⁵ mol/L) 30 min before precontraction, and the inhibitors remained present throughout the experiment.

Statistical Analysis

Data are presented as the mean ± standard error of the mean, with n representing the number of rats. Vasocontractile responses are expressed as absolute force (mN) per milligram of ring tissue, whereas vasorelaxant responses are expressed as the percentage reduction from the precontraction level induced by U46619. Data were analyzed using GraphPad Prism software (San Diego, CA, U.S.A.). For the measurement of pD₂, the individual concentration–response curves were fitted using a nonlinear regression-fitting program with a standard slope with GraphPad Prism software. An unpaired Student’s t-test or an unpaired Student’s t-test with Welchi’s correction was used for 2-group comparisons, as necessary (GraphPad Prism software). Statistical significance of the concentration–response curves was determined using repeated-measures two-way ANOVA, followed by Sidak’s multiple comparisons test. A p-value of <0.05 was considered statistically significant.

RESULTS

Effects of Prolonged Treatment with RANTES on Carotid Arterial Contractions

To determine whether RANTES affects contractile responses, we examined concentration–response curves for serotonin (Fig. 1A), high K⁺ (Fig. 1B), noradrenaline (Fig. 1C), U46619 (Fig. 1D), and endothelin-1 (Fig. 1E) in carotid arteries cultured with (RANTES group) or without (control group) RANTES. Prolonged treatment with RANTES had no effect on contractions induced by any of these vasoconstrictors.

Fig. 1. Concentration–Response Curves for Contractions of Carotid Arteries Induced by Serotonin (A), Isotonic K⁺ (B), Noradrenaline (C), U46619, a Thromboxane–Prostanoid Receptor Agonist (D), and Endothelin-1 (E) in Cultured Arteries Treated with RANTES (100 ng/mL for Approximately 24 h) (RANTES) or Left Untreated (Control)

Data are presented as the mean ± standard error of the mean, with the number of determinations indicated in parentheses.

Effects of Prolonged Treatment with RANTES on Carotid Arterial Relaxations

To investigate whether RANTES affects relaxant responses, we assessed concentration–response curves for acetylcholine (Fig. 2A), sodium nitroprusside (Fig. 2B), cromakalim (Fig. 2C), and NS19504 (Fig. 2D) in arteries cultured with (RANTES group) or without (control group) RANTES. RANTES treatment impaired acetylcholine-induced relaxation [E_max (% relaxation to U46619): control (n = 9) 50.3 ± 2.3 vs. RANTES (n = 9) 39.3 ± 4.1, p < 0.05; pD₂: control (n = 9) 6.92 ± 0.04 vs. RANTES (n = 9) 6.53 ± 0.11, p < 0.05], whereas relaxations induced by sodium nitroprusside, cromakalim, and NS19504 were unaffected.

Fig. 2. Concentration–Response Curves for Relaxations of Carotid Arteries Induced by Acetylcholine (A), Sodium Nitroprusside, a Nitric Oxide Donor (B), Cromakalim, an Activator of ATP-Sensitive Potassium Channels (C), and NS19504, an Activator of Large-Conductance Calcium-Activated Potassium Channels (D) in Cultured Arteries Treated with RANTES (100 ng/mL for Approximately 24 h) (RANTES) or Left Untreated (Control)

Data are presented as the mean ± standard error of the mean, with the number of determinations indicated in parentheses. * p < 0.05, control vs. RANTES.

Effects of Nitric Oxide Synthase and Cyclooxygenase Inhibitors on Acetylcholine-Induced Relaxation in Carotid Arteries Treated with and without RANTES

To investigate the roles of nitric oxide and prostanoids in acetylcholine-induced relaxation in carotid arteries treated with or without RANTES, we assessed concentration–response curves for acetylcholine in the presence of the nitric oxide synthase inhibitor N^G-nitro-l-arginine and the cyclooxygenase inhibitor indomethacin. As shown in Fig. 3A, acute incubation of N^G-nitro-l-arginine abolished the acetylcholine-induced relaxation and eliminated the differences in relaxation responses between the control and RANTES-treated groups. Similarly, indomethacin abolished the difference in acetylcholine-induced relaxation between the 2 groups [E_max (% relaxation to U46619): control (n = 7) 49.0 ± 4.3 vs. RANTES (n = 7) 51.9 ± 5.3, p > 0.05; pD₂: control (n = 7) 6.99 ± 0.17 vs. RANTES (n = 7) 6.92 ± 0.10, p > 0.05] (Fig. 3B).

Fig. 3. Effect of RANTES on Acetylcholine-Induced Relaxation in Rat Cultured Arteries under Nitric Oxide Synthase or Cyclooxygenase Inhibition

Concentration–response curves for acetylcholine-induced relaxation of carotid arteries treated with RANTES (100 ng/mL for approximately 24 h) (RANTES) or left untreated (control), in the presence of N^G-nitro-l-arginine (10⁻⁴ mol/L) (A) or indomethacin (10⁻⁵ mol/L) (B). Data are presented as the mean ± standard error of the mean, with the number of determinations indicated in parentheses.

Effects of Co-treatment with RANTES and Antioxidant on Acetylcholine-Induced Relaxation

To investigate whether an antioxidant could enhance acetylcholine-induced relaxation in carotid arteries treated with RANTES, we assessed concentration–response curves for acetylcholine in arteries co-treated with RANTES and either vehicle or the antioxidant N-acetyl-l-cysteine. As shown in Fig. 4, co-treatment with N-acetyl-l-cysteine increased acetylcholine-induced relaxation compared with vehicle treatment in RANTES-treated carotid arteries [E_max (% relaxation to U46619): vehicle (n = 9) 44.5 ± 6.0 vs. N-acetyl-l-cysteine (n = 9) 62.3 ± 5.7, p < 0.05; pD₂: vehicle (n = 9) 6.6 ± 0.12 vs. N-acetyl-l-cysteine (n = 9) 6.79 ± 0.09, p > 0.05].

Fig. 4. Effect of Co-treatment with an Antioxidant and RANTES on Acetylcholine-Induced Relaxation in Rat Cultured Arteries

Concentration–response curves for acetylcholine in carotid arteries co-treated with RANTES (100 ng/mL for approximately 24 h) and either vehicle (0.1% (v/v) ultrapure water) or the antioxidant N-acetyl-l-cysteine (10⁻³ mol/L). Data are presented as the mean ± standard error of the mean, with the number of determinations indicated in parentheses. * p < 0.05, vehicle vs. N-acetyl-l-cysteine.

Effects of Co-treatment with RANTES and CCR5 Antagonist on Acetylcholine-Induced Relaxation

To examine the relationship between RANTES and CCR5, one of its primary receptors, on acetylcholine-induced relaxation in rat carotid arteries, we assessed concentration–response curves for acetylcholine in arteries co-treated with RANTES and either vehicle or the CCR5 antagonist maraviroc. As shown in Fig. 5, co-treatment with maraviroc slightly, but not significantly, enhanced acetylcholine-induced relaxation compared with vehicle treatment in RANTES-treated carotid arteries [E_max (% relaxation to U46619): vehicle (n = 9) 45.0 ± 2.7 vs. maraviroc (n = 9) 51.1 ± 6.8, p > 0.05; pD₂: vehicle (n = 9) 6.4 ± 0.13 vs. maraviroc (n = 9) 6.71 ± 0.08; p > 0.05].

Fig. 5. Effect of Co-treatment with a C–C Motif Chemokine Receptor 5 Antagonist and RANTES on Acetylcholine-Induced Relaxation in Rat Cultured Arteries

Concentration–response curves for acetylcholine in carotid arteries co-treated with RANTES (100 ng/mL for approximately 24 h) and either vehicle (0.1% (v/v) dimethyl sulfoxide) or the CCR5 antagonist maraviroc (4 × 10⁻⁵ mol/L). Data are presented as the mean ± standard error of the mean, with the number of determinations indicated in parentheses.

DISCUSSION

Herein, we investigated the effects of prolonged treatment with RANTES on vascular contractile and relaxant responses in rat carotid arteries. The major findings are as follows: 1) prolonged treatment with RANTES did not affect contractile responses induced by either receptor-dependent or receptor-independent stimuli; 2) RANTES impaired acetylcholine-induced relaxation but had no effect on relaxations induced by a nitric oxide donor, an ATP-sensitive K⁺ channel activator, or a large-conductance Ca²⁺-activated K⁺ channel activator; 3) the difference in acetylcholine-induced relaxation between control and RANTES-treated arteries was abolished by an inhibitor of either nitric oxide synthase or cyclooxygenase; and 4) co-treatment with an antioxidant enhanced acetylcholine-nduced relaxation in the presence of RANTES, while co-treatment with a CCR5 antagonist slightly increased the relaxation response.

Because RANTES is a chemokine that promotes the recruitment and activation of various inflammatory cells, it can be challenging to evaluate its direct effects on vascular components in vivo. The organ culture technique offers a suitable and unique approach to investigate the direct effects of specific substances on vascular function without interference from nonvascular tissues.^28–32) In this study, to assess whether RANTES directly and specifically affects vascular function, carotid arteries were exposed to RANTES using an organ culture system, consistent with our previous studies.^28–30) Prolonged RANTES treatment did not alter either receptor-dependent (i.e., serotonin, noradrenaline, U46619, and endothelin-1) or receptor-independent (i.e., high K⁺) contractions. This suggests that RANTES does not influence Gq-coupled receptor–mediated signaling, voltage-gated Ca²⁺ channel-mediated calcium influx, or downstream Ca²⁺-dependent contraction pathways in vascular smooth muscle cells. Additionally, ATP-sensitive K⁺ channels and large-conductance Ca²⁺-activated K⁺ channels play key roles in vascular tone regulation.^34–36) In this study, RANTES treatment did not affect relaxations induced by activators of these channels, indicating that RANTES does not impair their functional activity in rat carotid arteries.

Additionally, RANTES impaired acetylcholine-induced relaxation but did not affect relaxation mediated by a nitric oxide donor in rat carotid arteries precontracted with U46619. Among the endothelium-derived relaxing factors, namely nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factors, nitric oxide plays a particularly important role in regulating vascular tone in large arteries.³⁾ In our carotid artery model, acetylcholine-induced relaxation was completely abolished by nitric oxide synthase inhibition in both the control and RANTES-treated groups. Additionally, acetylcholine-induced relaxation persisted under cyclooxygenase inhibition, and the differences between the control and RANTES-treated groups were eliminated in this condition. These findings indicate that RANTES-induced impairment of acetylcholine-mediated relaxation may be attributable to reduced nitric oxide bioavailability rather than reduced prostacyclin production.

Reactive oxygen species are known to reduce nitric oxide bioavailability in blood vessels³⁷⁾ and impair acetylcholine-induced relaxation in various arteries.^13,38) In this study, the antioxidant N-acetyl-l-cysteine enhanced acetylcholine-induced relaxation in RANTES-treated carotid arteries, suggesting that increased oxidative stress contributes to the RANTES-induced impairment of endothelium-dependent relaxation. This is supported by several studies demonstrating that RANTES promotes reactive oxygen species generation.^26,39) In the present study, indomethacin treatment promoted an increase in acetylcholine-mediated relaxation in RANTES-treated carotid arteries. Indomethacin treatment abolished the difference in relaxation between the control and RANTES-treated groups. These data suggest that the contribution of the contractile component by prostanoids in acetylcholine-mediated relaxation may be unmasked in carotid arteries subjected to prolonged RANTES treatment. Indeed, constrictor prostanoids could interrupt endothelium-dependent relaxation.^40,41) Moreover, cyclooxygenase enzymes not only produce prostanoids but also reactive oxygen species,⁴²⁾ and in turn, reactive oxygen species can enhance cyclooxygenase activation, ultimately leading to endothelial dysfunction, including disruption of nitric oxide.⁴²⁾ Taken together, the aforementioned data and relevant evidence suggest that the impairment of acetylcholine-induced relaxation by RANTES was due to the reduction of nitric oxide bioavailability and the unmasking of vasoconstrictor prostanoids through increased reactive oxygen species. Future studies are warranted to further elucidate the interactions among RANTES, cyclooxygenase, nitric oxide, and reactive oxygen species in the carotid artery.

RANTES can bind to multiple receptors, including CCR1, CCR3, CCR4, CCR5, and G-protein–coupled receptor 75. In this study, the CCR5 antagonist maraviroc slightly, though not significantly, improved acetylcholine-induced relaxation in RANTES-treated arteries. Costa et al.²⁶⁾ reported that RANTES impaired endothelial function and increased inflammation, nuclear factor-κB activation, and reduced nicotinamide adenine dinucleotide phosphate oxidase 1-derived reactive oxygen species generation via CCR5 in mouse aortas and endothelial cells. However, it is important to note that CCR5 blockade did not completely prevent RANTES-induced endothelial dysfunction.²⁶⁾ Taken together with our results, this indicates that RANTES-mediated impairment of acetylcholine-induced relaxation may involve activation of other receptor(s) in addition to CCR5.

This study has certain limitations. First, we focused exclusively on vascular reactivity in a single arterial site, the carotid artery, and assessed responses at only one time point and a single concentration of RANTES. Time-course studies involving multiple arterial regions are required to better understand the role of RANTES in vascular function across different vascular beds. Second, we were unable to determine the precise cellular targets of RANTES, such as whether its effects are mediated via endothelial cells, vascular smooth muscle cells, or both. Further studies are needed to clarify the underlying mechanisms of RANTES-induced impairment of acetylcholine-mediated relaxation, to assess the contractile reactivities in vessels without endothelium to investigate the direct effects of RANTES on smooth muscle function, and to achieve a more comprehensive understanding of its vascular actions.

Several reports have suggested a relationship between circulating RANTES levels and vascular dysfunction in humans.^27,43–45) For example, Mikolajczyk et al.²⁷⁾ found that in a cohort of individuals with metabolic syndrome and other risk factors for coronary artery disease, serum RANTES levels were significantly inversely correlated with flow-mediated dilation. Additionally, a positive correlation was observed between serum RANTES levels and von Willebrand factor, an established marker of endothelial injury. Because endothelial dysfunction plays a key role in the initiation and progression of atherosclerosis, targeting RANTES may offer a potential preventive and therapeutic strategy against the disease. However, as the source of circulating RANTES in patients is not limited to platelets, further research is needed to explore the associations among RANTES, platelet activation, and vascular function.

In conclusion, this study demonstrated that RANTES impairs acetylcholine-induced relaxation in the rat carotid artery. These findings may encourage further research into RANTES as a potential therapeutic target for vasculopathy.

Acknowledgments

This work was supported in part by a Grant from the Daiwa Securities Foundation. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Conceptualization: TM; data curation: TM, TN, NT-T; formal analysis: TM; investigation: TM; methodology: TM; project administration: TM; resources: TM, NT-T; visualization: TM, TN, NT-T; writing—original draft: TM; writing—review and editing: TM, TN, NT-T.

Conflict of Interest

The authors declare no conflict of interest.

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