Preliminary evaluation of LCWE-induced CA stenosis
The mice were sacrificed 2, 4, 8 and 16 weeks 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 1). 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. None of the control mice that were injected with phosphate-buffered saline (PBS) exhibited pathological changes (data not shown).
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 LCWE-induced 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 stimulated 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 CA stenosis rate (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 stimulated 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 (Fig. 4).
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 hyperplasia20. 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-stimulated 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 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 μm2) compared to that of medium alone (n=8, 443,594 μm2, 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 μm2 (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).
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 death2,4. 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 studies9. 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 APL21. In recent years, atRA has been used as the standard therapeutic drug for the treatment of adult APL and pediatric neuroblastoma22. 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 injury18. 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 signaling23. 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 KD24. 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 KD25,26.
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 activation19. In our study, the underlying pathophysiological mechanism of the 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.
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 vitro27. 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 mice28. In addition, Bunton et al. reported that phenotypic alterations in VSMCs preceded elastolysis in a mouse model of Marfan syndrome20. 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.
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 pathway29. In addition, Zhang et al. demonstrated that atRA might inhibit neointimal hyperplasia and suppress VSMC proliferation and migration by direct activation of AMP-activated 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 atherosclerosis23. More recently, Yu et al. reported that atRA prevented vein graft stenosis by inhibiting Rb-E2F-mediated cell cycle progression in human vein SMCs30. It was also reported that the Rb-E2F pathway was required for PDGF-BB-induced VSMC proliferation31. 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 we did not observe pathological features or changes in proinflammatory cytokines and chemokines over time. 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.
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.