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Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC Aug 1, 2013.
Published in final edited form as:
PMCID: PMC3480341
NIHMSID: NIHMS386596
Marked Acceleration of Atherosclerosis following Lactobacillus casei induced Coronary Arteritis in a Mouse Model of Kawasaki Disease
Shuang Chen, M.D., Ph.D.,1 Young Ho Lee, PhD.,1 Timothy R. Crother, Ph.D.,1 Michael Fishbein, M.D.,2 Wenxuan Zhang, MS.,1 Atilla Yilmaz, M.D.,3 Kenichi Shimada, Ph.D.,1 Danica J Schulte, M.D.,1 Thomas J.A. Lehman, M.D.,4 Prediman K. Shah, M.D.,5 and Moshe Arditi, M.D.1,5
1Division of Pediatric Infectious Diseases and Immunology, Burns and Allen Research Institute, Cedars-Sinai Medical Center and David Geffen School of Medicine at UCLA, Los Angeles, California
2Department of Pathology, David Geffen School of Medicine at UCLA, Los Angeles, CA
3Clinic of Internal Medicine I, Department of Cardiology, University Hospital Jena Germany
4Pediatric Rheumatology, Hospital for Special Surgery, Cornell University NY, NY
5Division of Cardiology, Oppenheimer Atherosclerosis Research Center, Cedars-Sinai Heart Institute and Medical Center, Los Angeles, CA
Correspondence: Moshe Arditi, MD; Professor of Pediatrics, David Geffen School of Medicine at the University of California, Los Angeles; Division of Pediatric Infectious Diseases and Immunology, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Room 4221, Los Angeles, CA 90048. Tel.: (310) 423-4471, Fax: (310) 423-8284, moshe.arditi/at/cshs.org
Objective
To investigate if Lactobacillus casei cell wall extract (LCWE)-induced Kawasaki Disease (KD) accelerates atherosclerosis in hypercholesterolemic mice.
Method and Resuslts
Apoe−/− or Ldlr−/− mice were injected with LCWE (KD mice) or PBS, fed high fat diet for 8 weeks, and atherosclerotic lesions in aortic sinuses (AS), arch (AC) and whole aorta were assessed. KD mice had larger, more complex aortic lesions with abundant collagen, and both extracellular and intracellular lipid and foam cells, compared to lesions in control mice despite similar cholesterol levels. Both Apoe−/− KD and Ldlr−/− KD mice showed dramatic acceleration in atherosclerosis vs. controls, with increases in en face aortic atherosclerosis and plaque size in both the AS and AC plaques. Accelerated atherosclerosis was associated with increased circulating IL-12p40, IFN-γ, TNF-α, and increased macrophage, DC, and T cell recruitment in lesions. Furthermore, daily injections of the IL-1Ra, which inhibits LCWE induced KD vasculitis, prevented the acceleration of atherosclerosis.
Conclusions
Our results suggest an important pathophysiologic link between coronary arteritis/vasculitis in the KD mouse model and subsequent atherosclerotic acceleration, supporting the concept that a similar relation may also be present in KD patients. These results also suggest that KD in childhood may predispose to accelerated and early atherosclerosis as adults.
Keywords: atherosclerosis, coronary disease, Kawasaki Disease, Interleukin 1 beta, IL-1 Receptor antagonist, mouse model of Kawasaki, vasculitis
Kawasaki Disease (KD) is a multisystem inflammatory disease with unknown etiology that results in an acute febrile syndrome, most common amongst children under the age of five 1. KD represents the leading cause of acquired heart disease among children2. The disease brings about its most detrimental effects via acute coronary arteritis, often accompanied by the development of coronary artery aneurysms in approximately 25% of untreated patients. The vasculitis and coronary arteritis are characterized histologically by inflammatory cell infiltration and destruction of extracellular matrix, especially elastic tissue in vascular media, with resultant coronary artery aneurysm formation 3. Long term cardiovascular complications among survivors of childhood KD are reported with increasing frequency4,5, 6. There are data suggesting that premature atherosclerosis and cardiovascular disease occur with increased frequency among survivors of childhood KD 5, 79.
Atherosclerosis is a lipid-driven, chronic inflammatory disease of the vessel wall in which both innate and adaptive immune responses play a role10. Immune cells and their mediators directly cause the chronic arterial inflammation that is a hallmark of atherosclerosis. It is clinically and experimentally reported that post-inflammatory vascular remodeling induces the development of arteriosclerosis or early onset of atherosclerosis11. There is evidence that clinical or subclinical vasculitis that occurs in KD may be the precipitating factor in lasting sequelae of the disease, namely atherosclerosis of the coronary and systemic arteries12. As the first cohort of patients diagnosed with KD are reaching middle age, epidemiological evidence is mounting that show greater incidence of cardiac events amongst adults with a history of KD4 . In a scientific statement from the American Heart Association’s expert panels, KD was listed among the eight pediatric diseases that is associated with high risk for accelerated atherosclerosis in children 13. Children with coronary aneurysms, and even those in whom coronary dilatation was never detected following KD, appear to be at increased risk for future atherosclerotic coronary artery disease 13. Recent reports further suggest that KD patients may be at increased risk for accelerated atherosclerosis 7, 8, 12, 14. However, there are conflicting clinical studies on this association and whether KD is a risk factor for accelerated atherosclerosis still remains controversial 12,15, 16. McCrindle et al concluded that vessels in post-KD teenage patients were not significantly altered and thus posed no increased cardiovascular risk15. In contrast, Della Pozza et al found that there were indeed significant changes in vascular profile, specifically an increase in carotid artery intima-media- thickness 16. These and other recent studies addressing the association between KD and atherosclerosis have come to opposing conclusions.
To address these conflicting results and explore the possibility that vasculitis observed during KD predisposes to accelerated development of atherosclerosis, we took advantage of a well-established mouse model of Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis and KD, which mimics histopathologically the coronary lesions observed in KD patients. We evaluated the effects of KD vasculitis on progression of atherosclerotic changes in mice genetically predisposed to develop atherosclerosis on high fat diet, including apolipoprotein E knockout (Apoe−/−) or low density lipoprotein receptor knockout (Ldlr−/−) models of atherosclerosis. Here we show that mouse with LCWE-induced coronary arteritis (KD group) in hypercholesterolemic atherosclerosis models develop a dramatic acceleration in atherosclerosis compared to non-KD control group, despite similar serum cholesterol levels. We also observed that prevention of coronary arteritis and vasculitis with IL-1Ra treatment in the KD mouse mode significantly inhibited the acceleration of atherosclerosis in hypercholesterolemic mouse models of atherosclerosis.
Mice
Apolipoprotein E (Apoe−/−), low-density lipoprotein receptor (Ldlr−/−) mice (all on C57BL/6 background) were purchased from Jackson Laboratory (Bar Harbor, ME). All animals were housed under specific pathogen-free conditions at the animal center of the Cedars-Sinai Medical Center. Experiments were conducted under approved IACUC protocols. Each of experimental group had n= 12 mice unless noted otherwise.
Reagents
Recombinant human IL-1 receptor antagonist (IL-1Ra) (Anakinra-Kineret, Amgen), recombinant mouse IL-1β (Sigma, St. Louis, MO), IL-1Ra was used at 25 mg/kg or 500 μg/mouse given i.p. The dose was based on our published study showing almost complete protection from coronary lesions17.
Atherosclerosis Development In LCCWE-Induced Coronary Arteritis Model
Group B L. casei (ATCC 11578) cell wall extract was prepared as previously described18 Five-week-old Apoe−/− or Ldlr−/− mice were injected i.p. with 250 μg of LCWE in PBS to induce KD or PBS alone (controls) as previously described17,18. Five mice from each group were sacrificed 14 days later to confirm the coronary arteritis, hearts were removed, coronary arteries were identified in serial sections (6 μm), and stained with H&E as described in our early publication18. Other mice from each group were fed a high fat diet containing 0.15% cholesterol starting at 14 days after LCWE or PBS injection. Following 8 weeks of high fat diet mice were sacrificed, heart and aorta were harvested, and the aortic root and aorta enface preparations were examined. To prevent any gender effect we used only male Apoe−/− or Ldlr−/−mice in both groups.
Assessment of Atherosclerotic Lesions in the Aorta and Aortic Sinus
Mice were anesthetized and aortas were excised from the aortic arch to the iliac bifurcation. Whole aortas en face and aortic sinus were prepared and stained with Oil red O as previously described19,20. Lesions areas were quantified with Image-Pro Plus (Media Cybernetics, Silver Spring, MD). Image analysis was performed by a trained observer who was blinded to the genotypes of mice as previously described 19, 20,21. The lesion area and lipid-stained areas in the aortic sinus were measured. Lipid content in aortic root plaques was expressed as aortic sinus lesion area or as percent of plaque area. The lesion area in the aorta en face preparations was expressed as a percent of the aortic surface area as previously reported19.
Assessment of DCs, Macrophages and T Cells in the Coronary artery and Aortic Sinus
Heart sections were immunohistochemically analyzed for the presence of mDCs, pDCs, macrophages, and T cells expression. For this purpose we used the following rat anti-mouse antibodies (Abs): anti-MIDC-8 Ab (Serotec) specific for mature mDCs, anti-PDCA-1 Ab specific for pDCs, anti-F4/80 Ab (Serotec), a specific marker for macrophages (4), and anti-CD3 Ab for T cells. For negative control, a mixture of different isotype antibodies (IgG2a and IgG2b) were used (Serotec). Immunostainings of serial cross-sections were performed using the catalyzed signal amplification kit according to manufacturer’s instructions (CSA System, DakoCytomation, Hamburg, Germany) as described earlier 22. Brown staining was obtained by incubation with 3, 3′-diaminobenzidine tetrahydrochloride (DAB).
Computer-Assisted Image Analysis
Digital images were taken at a magnification of 200x with a charge-coupled device camera (Nikon DXM 1200) of representative areas of coronary lesions, aortic root, and myocardium. mDCs, pDCs, macrophages, and T cells were quantified in different areas (0.2 mm2) by computer-assisted histomorphometry (Image-J) as described before 22 .
Serum Levels of Cytokines
IL-12p40, TNFα and IFNγ concentrations in the sera of mice were measured by ELISAs according to the manufacturer’s instructions (BD Biosciences).
Statistical Analysis
Results are reported as mean ± SEM. All data were analyzed using Prism 4.03 Statistical Program. A probability value of p<0.05 was considered statistically significant. We used the two-tailed Student’s t-test (at 95% confidence interval) to compare unpaired samples between experimental groups or one-way ANOVA with Tukey’s post-hoc test for multiple comparison. All data analyzed was normally distributed. *: p<0.05, **: p<0.01, ***: p<0.001.
LCCWE Injection Accelerates Atherosclerotic Plaque Development In Apoe−/− or Ldlr−/− Mice fed high-fat diet
In order to directly investigate whether induction of vasculitis and coronary arteritis in the KD mouse model accelerates the development of atherosclerosis in the presence of high-fat diet, we injected five-week-old Apoe−/− mice with either 250 μg LCWE or PBS intraperitoneally. Two weeks later, five mice from each group were sacrificed to confirm the development of vasculitis and coronary arteritis. 100% of the mice that received LCWE injection demonstrated coronary arteritis as expected (Fig. 1). Another 15 mice from each group were fed a high cholesterol diet, starting two weeks following the LCCWE injection, and continued for 8 weeks before sacrifice. (Fig. 1A). At that time, the heart, aortic arch, great vessels, and aorta were harvested revealing to naked eye considerable atherosclerotic plaques as whitish patches along the arteries, particularly in the proximal regions in KD group, but not in the control group (Fig. 1B). Aortic sinus, aortic arch and en face aorta were stained for lipids with Oil Red O. KD mouse developed significantly increased atherosclerotic lesions in the en face aorta compared to control mouse group (p<0.001, Fig. 1C). Following morphometric studies, KD mouse had significantly increased total atherosclerotic lesion area and lipid accumulation in the aortic sinus (p < 0.001; p<0.01; Fig. 1D). Additionally, the 3 branches of the aortic arch also showed significantly increased lesions as measured by total plaque area and lipid accumulation (p< 0.01; Fig. 1E). In 6/15 mice, the innominate artery (the first branch coming off the aortic arch) was nearly completely occluded (Fig. 1E). Importantly, these differences were independent of serum cholesterol levels as the two groups had similar blood cholesterol levels and same lipoprotein profiles. These results strongly indicate that an initial vascular insults, such as KD arteritis and vasculitis, leads to significantly accelerated atherosclerosis in this mouse model during subsequent high-fat feeding.
Figure 1
Figure 1
LCWE injection induces acceleration of atherosclerosis in Apoe−/− mice with 8 weeks of high fat diet. (A) Schematic for LCWE-induced KD mouse model, and Apoe−/− KD mouse model to study acceleration of atherosclerosis. (B) (more ...)
We next investigated the serum concentration levels of several pro-atherogenic cytokines to better understand the mechanisms by which LCWE induced KD might lead to accelerated atherosclerosis. LCWE injected Apoe−/− KD mice had significantly increased circulating concentrations of IFN-γ, IL-12p40, and TNF-α (Fig. 1F) compared to PBS injected mice placed on high fat diet. These results appear most consistent with the interpretation that at least part of the acceleration of atherosclerosis observed in Apoe−/− KD mice may be mediated by a general increase in circulating levels of pro-atherogenic inflammatory cytokines.
Since Apoe−/− mice was reported to display certain immune defects23,24, we repeated the above experiment using Ldlr−/− mice, another widely studied murine model of atherosclerosis. As in the Apoe−/− group, Ldlr−/− mice that first developed KD vasculitis prior to high fat diet also developed significantly accelerated atherosclerosis (Fig. 2B). Quantification of the lesion area of aortic sinus and aortic arch plaques revealed a significant increase in lesion size in KD Ldlr−/− mice compared to PBS littermate controls (p< 0.01; Fig. 2B and C). KD Ldlr−/− mice also developed significantly increased lipid accumulation in both the aortic sinus plaque, aortic arch lesions (p < 0.01; Fig. 2B and C) and total lesion area in the en face aorta (p < 0.01; Fig. 2D) compared to non-KD, control Ldlr−/− mice. The serum cholesterol levels (Supplemental Table 1) and serum lipoprotein profiles were equal between the KD and non-KD Ldlr−/−groups.
Figure 2
Figure 2
LCWE injection induces acceleration of atherosclerosis in Ldlr−/− mice with 8 weeks of high fat diet. (A) Schematic for Ldlr−/− KD mouse model to investigate acceleration of atherosclerosis. (B) Light photographs showing (more ...)
Examination of H&E and trichrome/elastin stained histologic sections of the aortic root showed marked differences between the Apoe−/− KD and Apoe−/− non-KD groups. Apoe−/− KD mice developed larger, more complex aortic lesions with abundant collagen, and extracellular as well as intracellular lipid (Fig. 3) compared with Apoe−/− non-KD control group. The aortic lesions in the control group were smaller and composed primarily of intracellular lipids in foam cells (Fig. 3). In the Apoe−/− KD group there were coronary lesions that resembled those of the aorta with variable degrees of luminal narrowing. For the most part, the coronary arteries in the control PBS group were normal or had minimal lesions.
Figure 3
Figure 3
Trichrome/Elastin Stain of Aortic Sinus and Coronary Arteries of Apoe−/− KD and Apoe−/− non-KD Mice Fed High-Fat Diet. With trichome/elastin stain the muscle tissue is red, collagen is blue, and foam cells are pink. Top (more ...)
LCWE-Induced Acceleration of Atherosclerosis Is Associated with Increased Numbers of Activated DCs, T-Cells, and Macrophages in Aortic Sinus Plaques
Infiltration of immune cells into atherosclerotic lesions plays an important role in plaque development. DCs directly control the innate and adaptive immune responses that occur during inflammatory diseases such as atherosclerosis25, 26 and their functions in innate and adaptive immunity. 27 DCs are present in normal arteries, but the numbers of activated DCs increase as atherosclerosis develops2830. Indeed, recent data indicate that both myeloid and plasmacytoid DCs (mDCs and pDCs) are present in increased amounts in human plaques31, 32. We reasoned that in the Apoe−/− KD mice feed mice high-fat diet for 8 weeks, the number of infiltrating activated DC numbers in the aortic sinus plaques would increase further when compared to Apoe−/− non-KD mice. To test this hypothesis, we performed immunohistochemical staining using MIDC-8 Ab to quantitatively measure numbers of mature, activated myeloid DCs (mDCs), and PDCA-1 Ab for pDCs. As anticipated, Apoe−/− KD mice developed significantly increased numbers of activated mDCs and pDC in the aortic sinus plaques compared to Apoe−/− non-KD mice. (Fig. 4A and B). Additionally, we examined the coronary artery, as coronary arteritis is a key component of KD. Indeed, we also saw increased DCs and pDCs at the coronary artery (Fig. 4A and B). These data suggest that acceleration of atherosclerosis induced by LCWE-induced KD vasculitis is accompanied by increased numbers of activated mDCs recruited into the plaques.
Fig. 4
Fig. 4
Apoe−/− KD Mice Show Increased infiltration of mDC, pDC, macrophage, and CD3 T cells in the Aortic Sinus and coronary arteries. (A–D) representative and quantitative analysis of mDC, pDC, T cell and macrophage immunoreactivity (more ...)
In addition to DCs, both macrophages (Mϕ) and T cells participate in the development of atherosclerotic plaques33 atherosclerosis, and coronary artery disease 34. Therefore, we also examined the extent of macrophage infiltration with F4/80 immunostaining and T cell infiltration with CD3 immunostaining in the coronary lesions and aortic sinus plaques. Apoe−/− KD mice had significantly increased T cell numbers in coronary lesions and aortic sinus plaques (p<0.05; Fig. 4C) as well as macrophage in coronary lesions when compared to Apoe−/− non-KD control mice (p<0.05, Fig. 4D).
LCWE Injection Accelerates Atherosclerotic Plaque Development In Apoe−/− Mice even when fed Regular Chow
As discussed above, LCWE injection induced acceleration of atherosclerosis in hypercholesterolemic Apoe−/− mice (Apoe−/− KD mice) following high fat diet. To investigate if KD vasculitis provide a strong stimulus for accelerated atherosclerosis even in the absence of high-fat diet, we repeated the above experiment in LCWE-injected Apoe−/− mice, but fed them regular chow at day 14, after extract injection and kept for 8 weeks before sacrifice. Quantification of the lesion area of aortic sinus plaques revealed a significant increase in atherosclerotic lesion size in Apoe−/− KD mice compared to Apoe−/− non-KD mice (p< 0.01; Fig. 5A). Apoe−/− KD mice had a significantly increased lipid accumulation in both the aortic sinus plaque lesions (p<0.01; Fig. 5A and C) and total lesion area in the en face aorta (p <0.01; Fig. 5D), as well as in the aortic arch compared to Apoe−/− non-KD mice (p<0.05; Fig. 5B). Serum cholesterol concentrations (Supplemental Table 1), and lipoprotein profiles (data not shown) were again similar in LCWE-injected and PBS control mice.
Fig. 5
Fig. 5
LCWE injection Induces Acceleration of Atherosclerosis in Apoe−/− mice fed 8 weeks with Regular Chow. (A) Schematic for and Apoe−/− KD on regular chow. (B) Quantification of aortic sinus lesion size, lipid content in lesions (more ...)
Treatment with IL-1 Receptor Antagonist (IL-1Ra), significantly inhibits KD vasculitis-induced acceleration of atherosclerosis in Apoe−/− Mice
We have recently shown that Caspase-1 and IL-1β signaling pathway is critical for the LCWE-induced KD mouse model, and that IL-1Ra treatment effectively blocks LCWE-induced vasculitis, coronary arteritis and myocarditis17. Therefore, we next investigated whether IL-1Ra given for prevention or treatment of the acute KD vasculitis can also inhibit or ameliorate the ensuing accelerated atherosclerosis that we observe in the Apoe−/− KD mice. We injected IL-1Ra (Kineret, Amgen) (500μg) daily (i.p) into Apoe−/− mice from 1 day prior to LCWE or PBS injection to day 5, as we recently described17. Five mice were sacrificed on day 7 after extract injection and their hearts were harvested for analysis to study the effect of IL-1Ra on LCWE-induced coronary lesions. As expected and reported 17, the incidence of KD vasculitis was significantly decreased in IL-1Ra-treated mice, compared to PBS treated controls (Supplemental Fig. 1). Additional LCWE-injected 10 Apoe−/− mice from each group were either treated with IL1Ra or given PBS injections and fed a high cholesterol diet for 8 weeks before sacrifice. We observed that IL-1Ra treated Apoe−/− KD group had significantly reduced acceleration of atherosclerosis compared to PBS treated Apoe−/− KD group: IL-1Ra treated Apoe−/− KD mice demonstrated a reduction in the atherosclerotic lesion development in both the aortic sinus and aortic arch, had less lipid accumulation in aortic sinus and aortic arch plaques, and a had reduced size of atherosclerotic lesions in the aorta compared with the PBS treated Apoe−/− KD mice (Fig. 6A–C). Additionally, IL-1Ra treatment resulted in a reduction in the serum levels of TNFα compared to PBS treated group (Fig. 6D). Taken together, these data demonstrate that LCWE-induced KD vasculitis significantly accelerates atherosclerotic lesion development in Apoe−/− mice fed high-fat diet, and that initial treatment of the KD vasculitis by IL-1Ra, can prevent the accelerated atherogenesis seen in Apoe−/− KD mice.
Figure 6
Figure 6
IL-1R Antagonist (IL-Ra) Protects Against KD Vasculitis-induced Acceleration of Atherosclerosis. (A) Following LCWE injection, Apoe−/− mice were administrated daily with 500 μg IL-1Ra or same volume of PBS (i.p) from day -1 to (more ...)
Kawasaki Disease is the leading cause of pediatric acquired heart disease in the United States, and hospital admissions attributed to KD are increasing across the country35. Although KD is considered to be an acute and self-limiting disease in the majority of cases, the coronary artery damages caused by KD and the diffuse vascular inflammation that is pathognomonic for this disease may have long-term sequelae12. Multiple studies have shown that patients with KD and persistent coronary artery aneurysm after the acute phase of disease have various vascular abnormalities, generalized vascular disease, and enduring inflammation7. The most prominent histological feature of coronary lesions after the acute phase of illness is intimal thickening, consisting of smooth muscle cells and extracellular matrix that is the result of cell migration through disrupted internal elastic intima6. Even when coronary artery lesions regress to normal form on echocardiogram or angiogram, they are virtually always associated with intimal thickening in all forms of lesion5. Excessive intimal thickening has the potential to develop into stenosis or promote thrombus formation5. Another important sequelae of KD, that is frequently discussed but is still controversial is the potential for accelerated development of atherosclerosis. Compounding the risk factor for accelerated atherosclerosis is the observation that KD is associated with altered lipid metabolism (in particular, lower HDL cholesterol) that persists beyond clinical resolution of disease 36. The observation of low plasma HDL concentrations after KD is particularly important because the vasculitis in KD has a predilection for the coronary arteries at sites identical to those most often affected in atherosclerosis 37, 38. For these children, intensive cardiovascular risk reduction is of critical importance. Frequently, awareness of the risk for premature atherosclerosis is often limited when the main focus is on timely diagnosis and acute medical care. Endothelial dysfunction is considered an initial event in the development of atherosclerotic plaques, as it promotes the migration of leukocytes and monocytes into the vessel wall, where macrophage interactions with T-cells play an important pathogenic role39. Other autoimmune vasculitic disorders such as systemic lupus erythematosus and Rheumatoid arthritis have also been associated with increased atherosclerosis leading to increased morbidity and mortality due to cardiovascular disease 4044. The potential mechanisms whereby KD patients would be at increased risk for accelerated atherosclerosis include: (1) arterial damage secondary to the acute disease process that alters the vascular structure itself, predisposing these vessels to development of atherosclerosis, and (2) enduring inflammation and vasculitis that promotes atherosclerotic processes15. To study whether patients with a history of KD are at increased risk for atherosclerosis or other vascular abnormalities, researchers have turned to non-invasive techniques in assessing post-KD patients. One such technique is flow-mediated dilation (FMD), which measures nitric oxide mediated vasodilation of the brachial artery on ultrasonography. Decreased FMD occur in children with a history of KD, as well as other conditions that predispose to the development of atherosclerosis such as diabetes mellitus and family history of premature coronary artery disease8. Another technique to study vascular integrity is by measuring carotid intima-media thickness (cIMT), where increased IMT, or thickening of vascular walls, correlates with development of atherosclerosis8. Studies that use these tests and others to assess KD patients have been largely conflicting in regards to whether or not patients with a history of KD show evidence of long-term vascular damage. McCrindle et al found that children with a history of KD did not have significantly decreased FMD, while Della Pozza et al found that they had significantly increased IMT15, 16 as did Notos et. al14. Many of these studies were hampered by small sample sizes, short duration of follow-up, and complicated by countless risk factors other than KD that influence vascular health, including dyslipidemia and diabetes mellitus. These discrepancies may also be related to differing KD characteristics during the acute phase of disease and subsequent treatment, and racial disparities45. As KD was only described approximately 40 years ago, there have yet to be any large-scale epidemiological studies that address these issues.
In view of conflicting clinical data, we wished to directly investigate if KD vasculitis accelerates the development of atherosclerosis using the combination of the LCWE-induced KD mouse model and the hypercholesterolemic mouse models such as Apoe−/− and Ldlr−/− mice. The LCWE model of KD has been shown to be a valuable tool in the immunopathological studies of this disease, as it mimics the histopathology of the KD coronary arteritis, vasculitis and myocarditis and even reliably predict human intravenous immunoglobulin (IVIG) treatment responses4648. Therefore, the Apoe−/− KD mouse model used in the present study has the potential to predict the acceleration of atherosclerosis in KD patients. In our study, acceleration of atherosclerosis was observed at the same sites where we saw the initial vasculitis, i.e. coronary artery and aorta. It remains to be determined if other vessels that have been reported to develop vasculitis also develop accelerated atherosclerosis. We found that in Apoe−/− KD and Ldlr−/− KD mice fed high fat diet, developed significantly accelerated atherosclerosis as measured in their aortic sinus and aortic arch plaques compared to Apoe−/− non-KD control mice. Apoe−/− KD mice also had significantly increased lipid accumulation in the aortic sinus plaques, aortic arch lesions and increased total atherosclerotic lesion area in aorta compared to Apoe−/− non-KD mice despite similar level of serum cholesterol levels between the groups. While human KD has been associated with additional risk factors for atherosclerosis, such as increased lipid profiles, the KD mouse model provides compelling evidence that initial vascular injury predisposes to accelerated atherosclerosis, particularly in the presence of hypercholesterolemia.
Furthermore, the accelerated atherosclerosis seen in the present study was associated with an increase in levels of cytokines IFN-γ, IL-12, p40, and TNF-α. As these cytokines have been associated with pathogenesis of atherosclerosis, we can conclude that this increase in cytokine levels caused by LCCWE injection may in part contribute to the accelerated atherosclerosis observed. Immune cells and their mediators are critical players in atherogenesis and contribute to the chronic arterial inflammation that is a hallmark of the disease. The inflammatory response is mediated by components of the innate immune system, including macrophages and dendritic cells (DCs)49,50 and by components of the adaptive immune system, including T lymphocytes33, 51. We observed an increase in DCs, macrophages, and T cells within the lesion areas, which is consistent with human data characterizing atherosclerotic lesions5. Together these findings are consistent with the fact that atherosclerotic processes are accelerated in Apoe−/− KD mice fed high-fat diet.
Secretion of IL-1β, a potent pyrogen that elicits a strong pro-inflammatory response52 is tightly controlled by a diverse class of cytosolic complexes known as53 inflammasomes. It is well established that IL-1β plays a critical role in chronic inflammatory diseases such as atherosclerosis54,55, 56. IL-1β signaling is mediated through the type I IL-1 receptor (IL-1RI). Additionally, the IL-1β receptor antagonist (IL-1Ra), an endogenous molecule, can bind the IL-1β receptor and prevent normal IL-1 signaling57. Recombinant IL-1Ra (Anakinra) has been approved for the treatment of various inflammatory diseases, such as rheumatoid arthritis58, and anti IL-1β mAb is currently in Phase III clinical trials for atherosclerosis. IL-1β has been associated with the pathogenesis of KD in our previous studies17 as well as by others, and in recent years its key role in vascular wall inflammation has been appreciated even further59. Indeed, we have recently shown that blocking IL-1β in the LCWE-induced KD mouse by IL-1Ra can effectively block coronary arteritis, vasculitis and myocarditis17. In an attempt to modulate the KD vasculitis-mediated acceleration of atherosclerosis, we treated LCWE-injected Apoe−/− mice with IL-1Ra, and observed that mice treated with IL-1Ra developed significantly less atherosclerosis. This protection is most likely due to the IL-1Ra-mediated blocking of the initial KD vasculitis. It should be noted, however, that IL-1Ra was not completely protective for accelerated atherosclerosis in the Apoe−/− KD mice. This may be due to residual EC dysfunction despite treatment for prevention of KD vasculitis. Recent studies suggest that statin treatment may also be beneficial in children with a history of KD45. In a pilot study of 11 children with a history of KD complicated with persistent coronary arterial abnormality, the investigators found that when these children were treated with oral simvastatin for three months they exhibited a significant reduction in hs-CRP and a significant increase in FMD of the brachial arteries45. These findings suggest that novel anti-inflammatory therapies are needed not only for IVIG-resistant KD patients, but perhaps also to prevent potential acceleration of atherosclerosis and the resulting long-term cardiac complications in KD patients.
The present study supports the possibility that KD patients maybe at increased risk for developing accelerated atherosclerosis and larger clinical studies with longer follow up in these patients will be needed to prove this association clinically. Until than, our findings support the current AHA recommendations that children with a history of KD should be carefully monitored for known risk factors of atherosclerosis, and potentially treated for accelerated development of atherosclerosis60. The observations from the current study, together with a recently published manuscript17, suggest that IL-1β signaling may play an important role in the development of LCWE-induced KD vasculitis as well as in the accelerated atherosclerosis that we observed in the Apoe−/− KD mice. These findings provide a justification for undertaking clinical studies to investigate whether FDA approved anti-IL-1β agents may provide benefit in KD-induced coronary arteritis and in KD -induced acceleration of atherosclerosis as well.
Acknowledgments
We would like to thank Ganghua Huang and Polly Sun for their technical assistance.
Funding Sources
Supported by grants from the National Institute of Health (HL66436 and AI1072726 to MA; AI070162 to DJS; and AHA 2060145 to SC.
Footnotes
The authors have declared that no conflict of interest exists.
Disclosures
None.
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