Toll-Like Receptor 4 Inhibitor TAK-242 Augments Acetylcholine-Induced Relaxation in Superior Mesenteric Arteries of the Streptozotocin-Induced Diabetic Rat

Abstract

Although vascular dysfunction is a key event in the development of diabetic complications, and abnormal toll-like receptor 4 (TLR4) may contribute to the pathophysiology of vascular diseases, the direct relationships between TLR4 and vascular function in diabetic arteries are still poorly understood. Thus, to investigate whether pharmacological blockade of TLR4 affects vascular function in the superior mesenteric artery (SMA) of streptozotocin (STZ)-induced diabetic rats, the SMA was isolated from male Wistar rat injected once with STZ (65 mg/kg, 27–34 weeks) which was treated with TAK-242 (10⁻⁶ M), a TLR4 inhibitor, for approximately 1 d using organ culture techniques. After incubation, functional and biochemical studies were performed. In the functional study, treatment with TAK-242 increased acetylcholine (ACh)-induced relaxation of the diabetic SMA in the intact condition. Sodium nitroprusside (SNP)-induced relaxation was also increased in the TAK-242-treated group compared with the vehicle-treated group. Under cyclooxygenase (COX) blockade by indomethacin (10⁻⁵ M), ACh-induced relaxation was similar in the vehicle- and TAK-242-treated groups. In addition, ACh-induced relaxation in the combined presence of the nitric oxide (NO) synthase inhibitor, N^G-nitro-L-arginine (L-NNA) (10⁻⁴ M), and indomethacin (10⁻⁵ M) was similar in the vehicle- and TAK-242-treated groups. The productions of thromboxane (TX) B₂ in cultured medium in the presence of ACh (10⁻⁵ M) were lower in the TAK-242-treated group than in the vehicle-treated group. These data suggested that TAK-242 could augment endothelium-dependent relaxation by partly suppressing vasoconstrictor TXA₂ or increasing NO signaling. TLR4 inhibition may be a novel therapeutic strategy to assist in the management of diabetes-associated vascular complications.

INTRODUCTION

Long-term diabetes leads to vascular dysfunction, including endothelial and smooth muscle dysfunction.¹⁾ The mechanisms underlying diabetes-associated vascular complications are multifactorial and complex.^1,2) Therefore, understandings on the molecular basis of the initiation and development of vascular dysfunction in diabetes is an urgent issue for the management of diabetes-associated systematic complications.

Activation of vascular toll-like receptors (TLRs) is related to the development of vascular complications observed in hypertension and diabetes.³⁾ Among TLRs, for example, TLR4 activation causes vascular dysfunction in hypertension,³⁾ and blockade of TLR4 can decrease inflammation and oxidative stress in the cardiovascular systems, thereby blunting vascular abnormalities and reducing organ damage.⁴⁾ In addition, high glucose leads to inflammation, production of reactive oxygen species (ROS), and nuclear factor-kappa B (NF-κB) activation via TLR4 signaling in endothelial⁵⁾ and smooth muscle⁶⁾ cells. Inhibition of TLR4 signaling suppressed mesenteric contraction in the diabetic rat.⁶⁾ Although the regulation of TLR4 signaling may play a key role in controlling diabetes-associated vascular abnormalities, the relationship between TLR4 and the relaxation response remains unclear.

Several animal models have been used to investigate the pathophysiology of diabetes.⁷⁾ The streptozotocin (STZ)-induced diabetic model is one of the classical major models.⁷⁾ Although there are many reports suggesting alterations of vascular functions in STZ-diabetic models, responsiveness to endogenous substances [e.g., acetylcholine (ACh)] in vessels varies depending on the species, vessel types, duration of disease, and sex. For instance, ACh-mediated relaxation was impaired in the STZ-diabetic rat aorta,⁸⁾ rat mesenteric artery,⁹⁾ and mouse aorta.¹⁰⁾ ACh-induced endothelium-derived hyperpolarizing factor (EDHF)-type relaxation was reduced in the superior mesenteric artery (SMA) of STZ-diabetic rats¹¹⁾ and mice.¹²⁾ However, no study has assessed the direct effects of inhibition of TLR4 on vasorelaxation in STZ-induced diabetic rats with long-term disease.

Here, we hypothesized that inhibition of TLR4 affects endothelium-dependent vasorelaxation in STZ-diabetic rats. We aimed to investigate the effects of long-term treatment with TAK-242, a cell-permeable small-molecule inhibitor of TLR4 by binding to the intracellular Cys747 residue of TLR4 and interfering with interactions between TLR4 and its adaptor molecules including Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) or Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-β-related adaptor molecule (TRAM),^13,14) on ACh-induced relaxation and related components in the SMA of STZ-diabetic rats by using the organ culture method, which is a useful tool for directly evaluating the vascular effects of target substances on vascular functions without complicated interactions among other humoral and neuronal factors and other cells, including immune cells.¹⁵⁾

MATERIALS AND METHODS

Animals, Organ Culture, and Vascular Function

Male Wistar rats 7 weeks of age received a single injection through the tail vein of 65 mg/kg STZ (Sigma-Aldrich, St. Louis, MO, U.S.A.) dissolved in a citrate buffer, as in our previous studies.¹¹⁾ Water and food were given ad libitum until the rats were sacrificed at 34–40 weeks of age (i.e., 27–34 weeks after STZ injection). Experiments were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals from the Committee for the Care and Use of Laboratory Animals of Hoshi University (Tokyo, Japan).

All experiments, including organ culture,¹⁵⁾ vascular functional study,^11,15) and release of thromboxane (TX) B₂,¹⁰⁾ were based on our previous studies. Body weight and blood glucose were measured in nonfasted STZ-induced rats at sacrifice (body weight, 350.2 ± 10.5 g; glucose, 470.8 ± 14.7 mg/dL; n = 24). The rats were anesthetized with isoflurane and euthanized by thoracotomy and exsanguination. After euthanasia, the SMA was rapidly isolated, cleaned, cut into arterial rings in an oxygenated modified Krebs–Henseleit solution (KHS; composition in mmol/L: NaCl 118.0, KCl 4.7, NaHCO₃ 25.0, CaCl₂ 1.8, NaH₂PO₄ 1.2, MgSO₄ 1.2, and glucose 11.0). Subsequently, the arterial rings were placed in Dulbecco’s modified Eagle’s medium (low-glucose, Gibco BRL, Grand Island, NY, U.S.A.) containing antibiotics and 1.0% fetal bovine serum (Biological Industries, Kibbutz Beit Kaemek, Israel) in the presence of vehicle (1.0% dimethyl sulfoxide) or TAK-242 (10⁻⁶ M,^13,16,17) Calbiochem, San Diego, CA, U.S.A.) and incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37°C for approximately 1 d. After incubation, the rings were, suspended in organ bath as reported previously.^11,15) The concentration–response curves for ACh (Daiichi-Sankyo Pharmaceuticals, Tokyo, Japan) in intact (without inhibitor), in the presence of 10⁻⁵ M indomethacin (Sigma-Aldrich), a cyclooxygenase (COX) inhibitor, or in the combined presence of 10⁻⁴ M N^G-nitro-L-arginine (L-NNA, Sigma-Aldrich), a nitric oxide synthase (NOS) inhibitor, plus 10⁻⁵ M indomethacin and for sodium nitroprusside (SNP, Wako Pure Chemical Industries, Ltd., Osaka, Japan) were obtained in the SMAs. Inhibitors were treated for 30 min before applying U46619 (Cayman Chemical, Ann Arbor, MI, U.S.A.) and were presented thereafter. Relaxations are presented as the percentage in the contraction induced by U46619. There were no differences in U46619-induced contraction between vehicle and TAK-242 groups in each condition (data not shown).

Measurement of TXB₂ Release

After organ-cultured SMA was treated with TAK-242 (10⁻⁶ M) or vehicle (1.0% dimethyl sulfoxide) for approximately 1 d, the SMA was stimulated with ACh (10⁻⁶ M) for 20 min. Subsequently, the SMA was weighed and the cultured medium was rapidly frozen in liquid N₂ and stored at −80°C for later analysis. The levels of TXB₂, a metabolite of TXA₂, in the medium were measured by an enzyme immunoassay kit (Item No. 501020, Cayman Chemical).

Statistical Analysis

The data were expressed as means ± standard error of the mean (S.E.M.), with n representing the number of animals used in the experiments. The concentration–response curves were fitted using a nonlinear regression-fitting program with a standard slope for each curve (GraphPad Prism ver. 8.0; GraphPad Software Inc., San Diego, CA, U.S.A.). The significance of differences between the two groups was determined by Student’s t-test. Statistical comparisons between concentration–response curves were made using a two-way repeated ANOVA with Bonferroni’s multiple comparisons test. The results were considered significant when the p value was less than 0.05.

RESULTS

After treatment of SMAs of STZ-diabetic rats with vehicle or TAK-242 for 1 d, exposure of SMA rings to ACh (10⁻⁹–10⁻⁵ M) led to a concentration-dependent relaxation in both the vehicle- and the TAK-242-treated groups (Fig. 1A). The ACh-induced relaxation was greater in the TAK-242 group than in the vehicle group, at intermediate concentrations of ACh (i.e., 10⁻⁷ M), the ACh-induced relaxation was significantly stronger in rings from the TAK-242 group than in those from the vehicle group (Fig. 1A). The maximal ACh-induced relaxation was significantly greater in the TAK-242 group than in the vehicle group (Fig. 1B). The pD₂ values for the ACh-induced relaxations were 7.41 ± 0.11 (n = 11) and 7.56 ± 0.09 (n = 12) in vehicle and TAK-242 group, respectively (p > 0.05). SNP-induced relaxation was slightly greater in SMA rings from the TAK-242 group than in SMA rings from the vehicle group (Fig. 1C). The maximal SNP-induced relaxation was significantly greater in the TAK-242 group than in the vehicle group (Fig. 1D). The pD₂ values for the SNP-induced relaxations were 7.94 ± 0.15 (n = 11) and 7.99 ± 0.10 (n = 12) in vehicle and TAK-242 group, respectively (p > 0.05).

Fig. 1. Effect of TAK-242 on Relaxations Induced by ACh and SNP in the SMA of STZ-Diabetic Rats

Concentration–response curves (A, C) and maximal relaxation (B, D) for ACh (A, B) and SNP (C, D) in STZ-diabetic SMAs treated with vehicle (1.0% dimethyl sulfoxide) or TAK-242 (10⁻⁶ M) for 1 d. n = 11–12. * p < 0.05 vs. Vehicle. ACh, acetylcholine; SMA, superior mesenteric artery; SNP, sodium nitroprusside; STZ, streptozotocin.

COX-derived prostanoids play a key role in the regulation of vascular tone.¹⁾ It is known that prostacyclin is one of the endothelium-derived relaxing factors (EDRFs), and that vasoconstrictor prostanoids exert an endothelium-derived contracting factor (EDCF) in various conditions, including aging, diabetes, and hypertension.¹⁾ To investigate the component of COX-derived prostanoids that is mediated by ACh-induced relaxation, concentration–response curves for ACh were assessed in SMA rings precontracted by U46619 in the presence of indomethacin (Figs. 2A, B). The concentration–response curve (Fig. 2A) and the maximal response (Fig. 2B) for ACh were similar in the vehicle and TAK-242 groups.

Fig. 2. Effect of TAK-242 on Relaxations Induced by ACh in the SMA of STZ-Diabetic Rats under Cyclooxygenase Inhibition (A, B) or Cyclooxygenase/Nitric Oxide Synthase Inhibitions (C, D)

Concentration–response curves (A, C) and maximal relaxation (B, D) for ACh in the presence of indomethacin (10⁻⁵ M) (A, B) and indomethacin (10⁻⁵ M) plus L-NNA (10⁻⁴ M) (C, D) in STZ-diabetic SMAs treated with vehicle (1.0% dimethyl sulfoxide) or TAK-242 (10⁻⁶ M) for 1 d. n = 8 or 12. ACh, acetylcholine; L-NNA, N^G-nitro-L-arginine; ns, not significant; SMA, superior mesenteric artery; SNP, sodium nitroprusside; STZ, streptozotocin.

An another EDRF, endothelium-derived hyperpolarizing factor (EDHF), is proposed to be a substance and/or electrical signal [i.e., endothelium-derived hyperpolarization (EDH)], depending on the vessel types.^1,18) The action of EDHF or EDH is to hyperpolarize vascular smooth muscle cells, resulting in relaxation.^1,18) Classically, vascular relaxation induced by ACh in the presence of a combination of NOS inhibitor and COX inhibitor is used to determine EDHF/EDH-induced, endothelium-dependent relaxation.^1,18) To investigate the effects of TAK-242 treatment on the components of EDHF/EDH-mediated relaxation in the SMA of diabetic rats, concentration–response curves for ACh were assessed in SMA rings precontracted by U46619 in the presence of indomethacin plus L-NNA (Figs. 2C, D). The concentration–response curve (Fig. 2C) and the maximal response (Fig. 2D) to ACh were similar in the vehicle- and TAK-242-treated groups.

Finally, we examined whether generation of TXB₂, a metabolite of TXA₂, in the SMA differed between the vehicle- and TAK-242-treated groups (Fig. 3). Production of TXB₂ was significantly lower in the TAK-242 group than in the vehicle group.

Fig. 3. Release of TXB₂ in ACh-Stimulated STZ-Diabetic SMAs Treated with TAK-242 (10⁻⁶ M) or Vehicle (1.0% Dimethyl Sulfoxide)

n = 8. * p < 0.05 vs. vehicle. ACh, acetylcholine; SMA, superior mesenteric artery; STZ, streptozotocin.

DISCUSSION

This study investigated the direct effects of the TLR4 inhibitor TAK-242 on vascular relaxant function in the isolated SMA of STZ-diabetic rats. We evaluated whether treatment with TAK-242 for approximately 1 d augmented ACh-induced relaxation and whether this beneficial effect was due to a normalization of NO signaling and/or a reduction of TXA₂ production.

A growing body of evidence suggests that the relationship between cellular events related to innate immune responses including the activation of TLRs and vascular functions and pathogenesis of diabetes.³⁾ TLR4 is present not only in endothelial cells,⁵⁾ but also in vascular smooth muscle cells.^6,19) Based on studies in TLR4-deficient mice, a growing body of evidence suggests that TLR4 plays an important role in the development of cardiovascular and metabolic diseases. TLR4-deficient mice are protected from several cardiovascular diseases, such as heart failure, ischemia/reperfusion injury, and cardiac hypertrophy.²⁰⁾ Benhamou et al.²¹⁾ observed that hemorrhagic shock followed by resuscitation largely impaired ACh-induced, NO-mediated mesenteric relaxations, whereas in TLR4-deficient mice, it did not impair relaxation. Deletion of TLR4 could prevent N^G-nitro-L-arginine methyl ester (L-NAME)-induced hypertension, release of damage-associated molecular patterns, ROS generation by smooth muscle cells, and impairment of the NO-soluble guanylate cyclase (sGC)-cyclic GMP signaling.²²⁾ In addition, deletion of TLR4 has been shown to protect endothelial dysfunction in arteries of type 2 diabetic mice,²³⁾ suppress circulating pro-inflammatory cytokines in STZ-diabetic mice.²⁴⁾ Although, the exact role of TLR4 in the vascular functions of chronic diabetic arteries remains unclear, signaling by TLR4 may partly contributes to the development of diabetic vascular dysfunction, because blockade of TLR4 treated with CLI-095, also known as TAK-242, suppressed the augmented noradrenaline-mediated contraction of mesenteric arteries in STZ-diabetic rats.⁶⁾ To assess the direct effect of TLR4 inhibitors on vascular function in chronic diabetes, we used organ culture techniques, which provide a useful method to maintain constant conditions and purely and directly evaluate vascular functions that are affected by substances or drugs without complicated interactions by other neuro-humoral factors and other cells, such as immune cells.¹⁵⁾

A novel and potentially important findings of the present study was that TAK-242 enhances ACh-induced relaxation in the SMA of STZ-diabetic rats. In general, stimulation of the endothelium (e.g., by ACh) can release EDRFs including NO, prostacyclin, and EDHF/EDH,¹⁾ and their contributions to relaxation depend on the vessel type. For example, NO contributes greatly in large arteries, and the contribution of EDHF/EDH gradually increases as vessel diameter decreases.¹⁾ Although prostacyclin is another EDRF, it may be unrelated to relaxation induced by ACh in the SMA of STZ-diabetic rats. This possibility is supported by previous reports that vasodilator prostanoids do not make a prominent contribution to endothelium-dependent relaxation in the SMA.^12,25) In chronic diseases including hypertension and diabetes, and aging, vasoconstrictor prostanoids rather than vasodilator prostanoids play a role in alteration of the responses induced by ACh.^1,25) In the present study, indomethacin abolished the difference in ACh-induced relaxations between the vehicle and TAK-242 groups. Moreover, TAK-242 reduced the generation of TXA₂ in SMAs stimulated with ACh. Furthermore, TAK-242 slightly increased SNP-induced relaxation in the SMA of STZ-diabetic rats. Based on our data and published evidence, we suggest that the enhancement of ACh-induced relaxation by treatment with TAK-242 is due to increased NO signaling and/or suppression of TXA₂ generation, but not affecting EDHF/EDH signaling.

Although the molecular mechanisms underlying the enhancement of ACh-induced relaxation by TAK-242 treatment in the SMA of STZ-diabetic rats remain unclear, oxidative stress might be involved. Activation of TLR4 induced generation of ROS in endothelial cells²⁶⁾ and vascular smooth muscle cells.⁶⁾ Decreased bioavailability of NO⁸⁾ or increased generation of TXA₂ was observed in arteries from STZ-diabetic models, and these changes were partly due to increased oxidative stress.^8,10) Previous reports also suggest that impaired ACh-induced relaxation was increased by reducers of oxidative stress in STZ-diabetic vessels.^8,27) Although ROS induced by TLR4 activation during chronic diabetes may be a causal factor of decreased NO signaling and increased TXA₂ generation, further investigation will be required to determine the underlying mechanisms.

In summary, we have shown that TAK-242 increases ACh-induced relaxation in the SMA of STZ-diabetic rats by increased NO signaling and suppressing TXA₂ production. Because TAK-242 enhances endothelium-dependent relaxation in STZ-diabetic rats, it is expected to be employed as a therapeutic agent for diabetes-associated vasculopathies.

Acknowledgments

We would like to thank Yurika Ezaki, Yurina Mae, Hiyori Yokoyama, Marina Ito, Tamayo Hashimoto, Amane Kurakata, Yuzuki Sato, and Tomoki Katome for their excellent technical assistance. This work was supported in part by Grants JSPS KAKENHI Grant numbers JP18K06861 (to TM), JP17K08318 (to KT), and JP18K06974 (to TK), and The Promotion and Mutual Aid Corporation for Private Schools of Japan.

Author Contributions

TM designed the study. TM, KT, MK, TK, and KT conducted the experiments and analyzed the data. TM and TK wrote the manuscript. All authors have read and approved the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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