All trans retinoic acid alleviates coronary stenosis by regulating smooth muscle cell function in a mouse model of Kawasaki disease

Coronary artery (CA) stenosis is a detrimental and often life-threatening sequela in Kawasaki disease (KD) patients with coronary artery aneurysm (CAA). Therapeutic strategies for these patients have not yet been established. All-trans-retinoic acid (atRA) is a modulator of smooth muscle cell functions. The purpose of this study was to investigate the effect of atRA on CA stenosis in a mouse model of KD. Lactobacillus casei cell wall extract (LCWE) was intraperitoneally injected into 5-week-old male C57BL/6 J mice to induce CA stenosis. Two weeks later, the mice were orally administered atRA (30 mg/kg) 5 days per week for 14 weeks (LCWE + atRA group, n = 7). Mice in the untreated group (LCWE group, n = 6) received corn oil alone. Control mice were injected with phosphate-buffered saline (PBS, n = 5). Treatment with atRA significantly suppressed CA inflammation (19.3 ± 2.8 vs 4.4 ± 2.8, p < 0.0001) and reduced the incidence of CA stenosis (100% vs 18.5%, p < 0.05). In addition, atRA suppressed the migration of human coronary artery smooth muscle cells (HCASMCs) induced by platelet-derived growth factor subunit B homodimer (PDGF-BB). In conclusion, atRA dramatically alleviated CA stenosis by suppressing SMC migration. Therefore, it is expected to have clinical applications preventing CA stenosis in KD patients with CAA.

www.nature.com/scientificreports/ leading to CA stenosis 14 . Since the LCWE-induced vasculitis model closely resembled the pathological features found in human KD autopsy cases, we therefore decided to use this mouse as a model for chronic CA stenosis. All-trans-retinoic acid (atRA) is a natural derivative of vitamin A that inhibits cell proliferation and migration and has anti-inflammatory properties [15][16][17] . Currently, atRA is a conventional therapy for the management of acute promyelocytic leukemia (APL) 18 . Recent experimental studies have reported that atRA treatment significantly reduces the formation of atherosclerosis in a high-fat diet-induced rabbit model 19 . Moreover, it has also been reported that atRA reduces neointimal formation in the carotid artery after balloon withdrawal injury in a rat model 20 . In addition, the synthetic retinoid Am80 significantly ameliorates Candida albicans water-soluble fraction (CAWS)-induced vasculitis through the inhibition of neutrophil migration and activation 21 . Based on previous animal studies, we hypothesized that atRA could suppress CA stenosis by modulating the properties of vascular smooth muscle cells (VSMCs) in a KD mouse model. In this study, we investigated the effect of atRA on LCWE-induced CA stenosis in a mouse model of KD.

Results
Preliminary evaluation of LCWE-induced CA stenosis. The mice were sacrificed 2 (n = 5), 4 (n = 6), 8 (n = 6) and 16 weeks (n = 7) after LCWE injection to investigate the natural history of LCWE-induced CA stenosis. Elastica van Gieson (EVG) staining revealed that CA intimal formation was first observed at 2 weeks and that the intimal thickness gradually increased over time. In addition to the increased intimal thickness, maximum vessel luminal narrowing was observed at 16 weeks after LCWE administration (Fig. 1b). Therefore, we chose to begin atRA treatment 2 weeks after LCWE injection and continued treatment for the next 14 weeks, which coincided with the onset of intimal formation and the time of maximum CA stenosis in this mouse model (Fig. 1c,d). None of the control mice that were injected with phosphate-buffered saline (PBS) exhibited pathological changes ( Fig. 1a) (n = 3 in each group).
Effect of atRA on CA inflammation and stenosis. Next, we evaluated the effects of atRA on CA inflammation. atRA (20 mg/kg) was orally administered 5 days per week from 2 to 16 weeks after LCWE injection. Inflammatory cells predominantly infiltrated the aortic root, and bilateral CAs were observed in LCWEinduced mice compared to PBS-treated mice. atRA significantly suppressed CA inflammation (19.3 ± 2.8 vs 4.4 ± 2.8, p < 0.0001) (Fig. 2). Mice injected with LCWE exhibited CA stenosis in addition to vasculitis. We next assessed the effects of atRA on CA stenosis using three parameters. Representative microphotographs showing LCWE-induced CA intimal formation are shown in Fig. 3a. atRA significantly reduced intimal incidence (100% vs 18.5%, p < 0.05), intimal thickness (100.5 ± 18 vs 11.5 ± 9.3 μm, p < 0.01), and the % of CA stenosis (67.5 vs 7.6%, p < 0.01) (Fig. 3b-d). α-Smooth muscle actin (αSMA)-positive cells in the thickened intima of the CA were predominantly observed in mice injected with LCWE. Proliferating cell nuclear antigen (PCNA)-positive and matrix metalloproteinase (MMP)-9-positive cells were localized on the surface of the neointima but were not observed in mice treated with atRA. The isotype control for each staining revealed no evidence of positively stained cells or regions in cardiac sections from LCWE-injected mice (Fig. 4a). Figure 4 shows the quantification of the number of positively stained cells in the intima for αSMA and PCNA (Fig. 4b,c), and the positively stained area in the intima for MMP-9 (Fig. 4d). Compared with mice injected with PBS, LCWE-injected mice had significantly increased numbers of αSMA-positive cells (393 ± 53 vs 1.6 ± 0.4, p < 0.0001), PCNA (209 ± 63 vs 1.0 ± 0.8, p = 0.006) and MMP-9-positive area (21,775 ± 285 μm 2 vs 465 ± 285 μm 2 , p = 0.002). However, the enhancement of αSMA, PCNA, and MMP-9 expression by LCWE was completely abolished by atRA administration.
Effect of atRA on LCWE-induced elastin degradation through the suppression of MMP-9. Changes in the proliferative phenotype of SMCs precede elastolysis and are thought to play an important role in the development of intimal hyperplasia 22 . Therefore, assessing elastin degradation is extremely important for regulating intimal formation in the vessel wall. Next, we investigated the effect of atRA on the frequency of elastic breaks in the tunica media. LCWE-injected mice had more frequent interruptions and weakening of elastic fibers than mice that were administered PBS (Fig. 5a). atRA significantly reduced the elastin break scores of the external elastic lumina (EEL) (28 ± 1 vs 6.9 ± 3.4, p < 0.0001) and internal elastic lumina (IEL) (21.2 ± 1.7 vs 3.6 ± 2.1, p < 0.0001) (Fig. 5b,c). The potent electrolytic protein MMP-9, was increased in the serum of LCWE-induced mice (1.226 ± 0.18 ng/ml) compared with PBS-injected mice (0.697 ± 0.12 ng/ml, p = 0.66). This LCWE-induced increase in MMP-9 was significantly suppressed in atRA-treated mice (0.674 ± 0.12 ng/ml, p = 0.035 vs LCWE group) (Fig. 5d).
Inhibitory effects of atRA on SMC migration and MMP-9 and TNF-α production in vitro. Next, human coronary artery smooth muscle cells (HCASMCs) were used to investigate the effect of atRA on HCASMC migration (Fig. 6a). The cell migration assay revealed that platelet-derived growth factor subunit B homodimer (PDGF-BB) stimulation increased the area covered by migrated cells (n = 16, 515,703 μm 2 ) compared to that of medium alone (n = 8, 443,594 μm 2 , p = 0.04). Cells were then treated with 0.1, 1.0, and 10 nM atRA for 72 h. The areas covered by migrated cells after treatment with 0.1 and 1 nM atRA were 407,610 (n = 16) and 424,162 μm 2 (n = 16), respectively. While these concentrations of atRA induced significant reductions of 21% (p < 0.0001) and 18% (p = 0.002), respectively, compared to those in the PDGF-BB-treated group, a greater reduction of 49% was observed in the 10 nM atRA treatment group (n = 16, p < 0.0001 vs. PDGF-BB, 0.1, 1 nM atRA treatment) (Fig. 6b). To examine the direct effects of atRA on MMP-9 and TNF-α production in HCASMCs, ELISA analysis was conducted. Although total MMP-9 activity was not changed by the PDGF-BB group (0.51 ± 0.03 ng/ml) compared to the PBS group (0.45 ± 0.03 ng/ml, p = 0.145), atRA in addition to PDGF-BB significantly decreased www.nature.com/scientificreports/ . The values are expressed as the mean ± SD. *p < 0.05 vs PBS (16w), **p < 0.0001 vs PBS (8w). All the data were generated from 1 experiment at the same timeframe. www.nature.com/scientificreports/ total MMP-9 activity (0.39 ± 0.01 ng/ml, p = 0.003 vs PDGF-BB). There were no differences in TNF-α concentration among the groups (Fig. 6c).

Discussion
In the present study, we found that atRA dramatically reduced intimal hyperplasia and alleviated CA stenosis in an LCWE-induced model of KD vasculitis. CA stenosis is clinically caused by thrombus formation or intimal hyperplasia and induces cardiac events such as cardiac ischemia, MI, and even sudden death 2,4 . The clinical . The values are expressed as the mean ± SD. *p < 0.05, **p < 0.0001. All the data were generated from 1 experiment at the same timeframe. The data are expressed as the mean ± SD. *p < 0.01, **p < 0.05. All the data were generated from 1 experiment at the same timeframe. www.nature.com/scientificreports/ definition of myocardial infarction (MI) denotes the presence of acute myocardial injury detected by abnormal cardiac biomarkers in the setting of evidence of acute myocardial ischemia. In addition, MI is defined pathologically as myocardial cell death due to prolonged ischemia 23 . Vascular smooth muscle proliferation plays a pivotal role in the development of intimal hypertrophy, causing CA stenosis in KD patients with CAA, as evidenced by autopsy studies 11 . Therefore, this is the first report focused on the prevention of CA stenosis by regulating the properties of SMCs in a mouse CA arteritis model. atRA is the most active metabolite of vitamin A. Numerous studies have reported that atRA has biological effects on various types of tumors, including breast and lung cancer and APL 24 . In recent years, atRA has been used as the standard therapeutic drug for the treatment of adult APL and pediatric neuroblastoma 25 . On the other hand, several basic experimental studies of cardiovascular disorders have shown that atRA has antiproliferative and antimigratory effects in animal models of intimal hyperplasia. Miano et al. showed that atRA reduced neointimal formation and promoted favorable geometric remodeling of the rat carotid artery after balloon withdrawal injury 20 . In addition, Zhang et al. showed that atRA suppressed neointimal hyperplasia and inhibited VSMC proliferation and migration through direct activation of AMP-activated protein kinase (AMPK) and inhibition of mTOR signaling 26 . Therefore, we hypothesized that atRA might exert beneficial effects on the CA stenosis mouse model we developed in recent years. These previous data indicated that atRA improved intimal proliferation mainly associated with αSMA-positive cells. However, the effectiveness of atRA on cardiovascular disorders in clinical practice has not yet been verified. In addition, several clinical studies have investigated the relationship between retinol binding protein 4 (RBP4) and KD. Kimura et al. showed that RBP4, which is a candidate diagnostic marker, was decreased in patients with acute KD 27 . Recently, Yang et al. reported that KD patients had significantly lower RBP4 levels than healthy controls, suggesting that RBP4, which is a main retinol transport protein, is closely associated with markers of inflammation and thrombogenesis in children with KD 28,29 .
Notably, compared to untreated mice, mice treated with atRA had significantly reduced CA inflammatory scores. This anti-inflammatory effect was consistent with the data reported by Miyabe et al. Am80, a retinoic acid receptor (RAR) agonist, has been shown to ameliorate mouse vasculitis induced by CAWS by suppressing neutrophil migration and activation 21 . In our study, the underlying pathophysiological mechanism of the www.nature.com/scientificreports/ anti-inflammatory effect of atRA remains unclear, but it is hypothesized that the SMC phenotype predisposes patients to increased proliferation and migration and contributes to persistent inflammation of the vessel wall. VSMC phenotypic switching is characterized by loss of a contractile phenotype that lacks VSMC markers including αSMA, calponin and SM22/tagln and acquires increased capacity for cell proliferation, migration and secretion of various extracellular matrix proteins and cytokines 30 . In contrast, it was unexpected that an increase in αSMA-positive cells in the CA intima was observed in mice injected with LCWE. However, this phenotypic switch is considered the hallmark of vascular repair, a reversible process wherein VSMCs return to their contractile state after completing vessel repair 31 . This may be a possible reason why abundant αSMA-positive cells were observed at the site of CA stenosis in LCWE-injected mice. We found that atRA significantly decreased elastin breaks and suppressed serum MMP-9 activity. A previous study by Axel et al. revealed that atRA inhibited human SMC proliferation and significantly inhibited the protein expression and activity of MMP-2 and MMP-9 in vitro 32 . More recently, Xiao et al. reported that atRA attenuated the progression of angiotensin II-induced abdominal aortic aneurysms by downregulating MMP-2 and MMP-9 expression in abdominal aortic tissue in apolipoprotein E-knockout mice 33 . In addition, Bunton et al. reported that phenotypic alterations in VSMCs preceded elastolysis in a mouse model of Marfan syndrome 22 . Therefore, it is reasonable to hypothesized that atRA protects against elastin degradation through the downregulation of MMP-9 activity, which in turn results in the suppression of proliferative phenotypic switching and the inhibition of intimal hyperplasia. . The data are expressed as the mean ± SD. *p < 0.0001, **p < 0.05. All the data were generated from 1 experiment at the same timeframe. www.nature.com/scientificreports/ Moreover, there was no significant increase in serum MMP-9 levels in LCWE-injected mice compared to PBS-infected mice. However, cardiac sections of LCWE-infected mice showed that local expression of MMP-9 was clearly enhanced. This is a noteworthy finding, and we believe that higher expression of MMP-9 predisposes patients to facilitate elastin breakdown and cause CA stenosis. Therefore, it may be hypothesized that serum MMP-9 had already peaked at the time of sacrifice, but local MMP-9 expression remained.
In vitro, we showed direct inhibitory effects of atRA on migration using HCASMCs stimulated with PDGF-BB. Several in vivo and in vitro studies have investigated the pathways that regulate the migration or proliferation of VSMCs. Day et al. first reported that atRA inhibited airway SMC migration by modulating the phosphatidylinositol 3 kinase (PI3K)/Akt pathway 34 . In addition, Zhang et al. demonstrated that atRA might inhibit neointimal hyperplasia and suppress VSMC proliferation and migration by direct activation of AMPactivated protein kinase (AMPK). They concluded that AMPK might be the pharmacological target of ATRA and that activation of AMPK by atRA may be a novel treatment strategy for atherosclerosis 26 . More recently, Yu et al. reported that atRA prevented vein graft stenosis by inhibiting Rb-E2F-mediated cell cycle progression in human vein SMCs 35 . It was also reported that the Rb-E2F pathway was required for PDGF-BB-induced VSMC proliferation 36 . Our results confirmed that atRA inhibited PDGF-BB-induced HCASMC migration, suggesting an association between the Rb-E2F pathway and the antiproliferative effect of atRA. Our research has some limitations. First, it was not possible to clearly determine whether atRA had a significant effect on inflammatory suppression or the induction of vascular repair. This is because pathological evaluations at different time points and cytokine/chemokine measurements during the experimental periods were not performed. One possibility remains that atRA provides complete protection from the development of CA inflammation and stenosis during the experimental period. Treatment with atRA was started relatively early in this mouse model. Therefore, it was considered necessary to select a schedule for later administration of atRA treatment when CA inflammation had already developed. Second, the therapeutic goal of KD patients with CAA is to not only promote the regression of CA aneurysms but also prevent further cardiac events such as acute MI and sudden death. Thus, the beneficial effect of atRA on the suppression of intimal hyperplasia may lead to protection against CA stenosis and these harmful events. On the other hand, excessive inhibition of intimal hyperplasia may delay aneurysmal regression, resulting in a residual aneurysm. Therefore, it is important to induce favorable SMC proliferation rather than completely suppressing intimal hyperplasia leading to CA stenosis. Additional research is needed to elucidate the mechanism of the beneficial effects of atRA. Third, no experiments were conducted in this study to investigate the distribution or type of retinoic acid receptors that could be expressed at the site of coronary arteritis. Moreover, we did not examine any specific signaling for retinoic acid receptors that could be measured in CA. Further research will be needed to clarify the mechanism by which atRA directly or indirectly influences vascular inflammation and SMC phenotypic switching via the retinoic acid signaling pathway. Fourth, it was not clear whether retinoic acid inhibits vascular stenosis by suppressing immune cell infiltration or directly regulating the SMC phenotype. We assumed that both of these potential mechanisms were involved in this beneficial effect of atRA. An In vitro study reported by Xu et at. reported that atRA inhibited lipopolysaccharide-induced proinflammatory cytokines (TNF-α, IL-1β and IL-6) 37 . Most of these cytokines are closely associated with the pathophysiology of Kawasaki vasculitis. Furthermore, it should be emphasized that both IL-1α and IL-1β are directly associated with the LCWE-induced KD mouse model of vasculitis 38,39 . In addition, Fujiu et al. showed that the synthetic retinoid Am80 suppresses smooth muscle phenotypic modulation and in-stent neointima www.nature.com/scientificreports/ formation by inhibiting KLF5 40 . Therefore, additional in vivo and in vitro experiments will be needed in the future to more clearly distinguish the main benefits of atRA for anti-inflammatory effects or regulating the SMC phenotype. Finally, for clinical application, Miyabe et al. reported that a synthetic retinoid, Am80 significantly suppressed CAWS-induced vasculitis via regulation of neutrophil migration and activation, suggesting that Am80 was expected to be useful in the acute phase of KD vasculitis in this study 21 . On the other hand, our research focuses on the ability of atRA to regulate SMC function, including the regulation of SMC migration and MMP-9 activity associated with CA stenosis. Compared to Miyabe's study, our study provided a different mechanism of atRA that is beneficial to patients with CA aneurysms in terms of preventing cardiac events such as angina pectoris and MI. This is a new discovery in our current experiment.
In conclusion, atRA dramatically reduced CA inflammation and stenosis by suppressing the production of MMP-9 and the migratory properties of SMCs by regulating cellular functions. Therefore, atRA, which has both anti-inflammatory effects and the ability to repair the vascular wall, is expected to have clinical applications to prevent CA stenosis in KD patients with CAA.

Methods
Experimental protocol. Five-week-old male C57BL/6 J mice were purchased from CLEA Japan (Tokyo, Japan) and maintained in an environment with a 12 h light/12 h dark cycle under specific pathogen-free conditions. First, we examined the natural course of coronary stenosis in mice after LCWE administration. The mice were sacrificed on weeks 2, 4, 8, and 16 after intraperitoneal injection of 1000 µg of LCWE (n = 5 to 7 in each group). Control mice received PBS (n = 3 in each group). Cardiac tissue was harvested, and histological assessments were performed in detail as follows. Based on the pathological features of CA stenosis observed in the preliminary natural course experiment, the effects of atRA on CA stenosis in this mouse model of KD were examined. Five-week-old male C57BL/6 J mice (n = 13) were intraperitoneally injected with 1000 μg of LCWE. Two weeks later, these mice were divided into two groups. Mice were orally administered atRA (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 20 mg/kg (n = 7) or an equivalent volume (0.2 ml) of corn oil (n = 6) using a disposable flexible-type gastric tube (Fuchigami, Kyoto, Japan) 5 days per week for 16 weeks. The same dose of atRA has been shown previously to inhibit neointimal hyperplasia and suppress the proliferation and migration of VSMCs in mice 26 . Control mice (n = 5) were injected with PBS instead of LCWE. All mice were killed at 18 weeks after LCWE or PBS administration. All mouse experimental procedures were performed in accordance with institutional guidelines and regulations of Saitama Children's Medical Center, Japan. This animal experiment was performed with the approval of the Animal Experimental Ethics Committee of Saitama Children's Medical Center (No: 2020-003). All experiments were performed in accordance with relevant ARRIVE guidelines. LCWE preparation. LCWE was prepared as previously described 14,41 . Briefly, Lactobacillus Paracasei subsp. paracasei (ATCC 11578; American Type Culture Collection, Manassas, VA, USA) was cultured in MRS broth (BD Difco, Franklin Lakes, NJ, USA) for 48 h at 37 °C. The cells were harvested and washed with PBS, after which the cells were disrupted in 2 packed volumes of 4% sodium dodecyl sulfate (SDS) overnight at room temperature. Cell wall fragments were extensively washed 10 times with PBS to remove any residual SDS. The SDS-treated cell wall fragments were sonicated (5 g of packed wet weight in 15 ml of PBS) for 2 h using a Q500 sonicator with a ¾" diameter probe at an amplitude setting of 70% (QSonica LLC, CT, USA). During sonication, the cell wall fragments were maintained by cooling in a dry ice/ethanol bath. The supernatant was centrifuged for 1 h at 20,000g at 4 °C, and the supernatant containing the cell wall extract was used for the experiments. The concentration of LCWE was determined based on the rhamnose content as measured by a phenol-sulfuric acid colorimetric assay and adjusted to 5000 µg/ml. To induce coronary arteritis/stenosis in mice, 1000 µg (0.2 ml) of the LCWE preparation was injected intraperitoneally.
Histological evaluation. As a preliminary natural course experiment, we measured the luminal area and intimal thickness of the CA artery at 2, 4, 8, and 16 weeks after administration of PBS or LCWE. The luminal area was measured in a total of 6 coronary arteries per animal and expressed as the average luminal area (μm 2 ) for each CA. Intimal formation was defined as the thickened intima afferently overhanging the inside of the internal elastic lamina (IEL). We chose to use intimal thickness as an indicator of intimal formation. The length of the intima at four sites was measured from CA and averaged, followed by counting 6 coronary arteries for each animal and representing the average values of the thickened intima of each CA.
An inflammatory assessment of the CAs was performed as described previously 14 . Briefly, 2.5-μm sections of cardiac tissue were stained with hematoxylin and eosin (H&E) and EVG. Then, we selected 5 consecutive sections slightly distal to the bifurcation of the bilateral CAs. The intensity of coronary arteritis was scored with the following four criteria: 0, no inflammation; 1, inflammatory cells in only the adventitia; 2, inflammatory cells in both the intima and adventitia; and 3, panvasculitis as previously described 42 . Ultimately, the inflammatory score is expressed as the total score of 10 CAs per individual animal. For the quantitative parameters of CA stenosis, we evaluated the incidence of neointima, intimal thickness and % of CA stenosis. The incidence of neointima implied the frequencies of individuals with intimal thickening of the CA and expressed as percentages in each group. Intimal thickness was previously described as above. % of CA stenosis was calculated from the formula [all regions within IEL − Remaining lumen region/All regions within IEL × 100 (%)].
Elastin breaks were scored on the following scale: score 0, no interruption of elastic fibers; score 1, elastic breaks ≤ 10; 2, elastic breaks ≻10; and score 3, obscuration or disappearance of elastic fibers. Elastin breaks were defined as interruptions in the elastin fiber and the reappearance of the fiber and are expressed as the total number of breaks in five consecutive sections. The IEL and EEL of the bilateral CAs were evaluated. In addition, coronary stenosis was assessed according to three different parameters, including the incidence of neointimal www.nature.com/scientificreports/