Non-receptor type, proline-rich protein tyrosine kinase 2 (Pyk2) is a possible therapeutic target for Kawasaki disease.

Kawasaki disease (KD) is a paediatric vasculitis whose pathogenesis remains unclear. Based on experimental studies using a mouse model for KD, we report here that proline-rich protein tyrosine kinase 2 (Pyk2) plays a critical role in the onset of KD-like murine vasculitis. The mouse model for KD was prepared by administrating a Candida albicans water-soluble fraction (CAWS). Unlike CAWS-treated WT mice, CAWS-treated Pyk2-Knockout (Pyk2-KO) mice did not develop apparent vasculitis. A sustained increase in MIG/CXCL9 and IP-10/CXCL10, both of which have potent angiostatic activity, was observed in CAWS-treated Pyk2-KO mice. CAWS-induced activation of STAT3, which negatively regulates the expression of these chemokines, was also attenuated in macrophages derived from Pyk2-KO mice. The present study suggests that defects in Pyk2 suppress KD-like experimental vasculitis, presumably through CXCL9- and CXCL10-dependent interference with neo-angiogenesis. Since Pyk2-KO mice show no life-threatening phenotype, Pyk2 may be a promising therapeutic molecular target for KD.

The Application of a Modified d-ROMs Test for Measurement of Oxidative Stress and Oxidized High-Density Lipoprotein.

Reactive oxygen species (ROS) are involved in the initiation and progression of atherosclerosis. ROS-derived hydroperoxides, as an indicator of ROS production, have been measured by using the diacron reactive oxygen metabolites (d-ROMs) test, which requires iron-containing transferrin in the reaction mixture. In this study we developed a modified d-ROMs test, termed the Fe-ROMs test, where iron ions were exogenously added to the reaction mixture. This modification is expected to exclude the assay variation that comes from different blood iron levels in individuals. In addition, this Fe-ROMs test was helpful for determining the class of plasma lipoproteins that are hydroperoxidized. Low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and high-density lipoprotein (HDL) were purified by use of an LDL/VLDL purification kit and the dextran sulfate-Mg2+ precipitation method, respectively; their hydroperoxide contents were assessed by performing the Fe-ROMs test. The majority of the hydroperoxides were detected only in the HDL fraction, not in the LDL/VLDL. Further detailed analysis of HDLs by size-exclusion high-performance liquid chromatography revealed that the hydroperoxide-containing molecules were small-sized HDLs. Because HDL was shown to be the principal vehicle for the plasma hydroperoxides, this Fe-ROMs test is a beneficial method for the assessment of oxidized-HDL levels. Indeed, Fe-ROMs levels were strongly associated with the levels of oxidized HDL, which were determined by performing the malondialdehyde-modified HDL enzyme immunoassay. In conclusion, the Fe-ROMs test using plasma itself or the HDL fraction after dextran sulfate-Mg2+ precipitation is useful to assess the functionality of HDL, because the oxidation of HDL impairs its antiatherogenic capacity.

Transcriptional Analysis of Intravenous Immunoglobulin Resistance in Kawasaki Disease Using an Induced Pluripotent Stem Cell Disease Model.

BACKGROUND: Approximately 10-20% of Kawasaki disease (KD) patients are resistant to intravenous immunoglobulin (IVIG) treatment. Further, these patients are at a particularly high risk of having coronary artery abnormalities. The mechanisms of IVIG resistance in KD have been analyzed using patient leukocytes, but not patient vascular endothelial cells (ECs). The present study clarifies the mechanisms of IVIG resistance in KD using an induced pluripotent stem cell (iPSC) disease model.Methods and Results:Dermal fibroblasts or peripheral blood mononuclear cells from 2 IVIG-resistant and 2 IVIG-responsive KD patients were reprogrammed by the episomal vector-mediated transduction of 6 reprogramming factors. KD patient-derived iPSCs were differentiated into ECs (iPSC-ECs). The gene expression profiles of iPSC-ECs generated from IVIG-resistant and IVIG-responsive KD patients were compared by RNA-sequencing analyses. We found that the expression ofCXCL12was significantly upregulated in iPSC-ECs from IVIG-resistant KD patients. Additionally, Gene Set Enrichment Analysis (GSEA) revealed that gene sets involved in interleukin (IL)-6 signaling were also upregulated. CONCLUSIONS: The first iPSC-based model for KD is reported here. Our mechanistic analyses suggest thatCXCL12, which plays a role in leukocyte transmigration, is a key molecule candidate for IVIG resistance and KD severity. They also indicate that an upregulation of IL-6-related genes may be involved in this pathogenesis.

Involvement of mannose-binding lectin in the pathogenesis of Kawasaki disease-like murine vasculitis.

Kawasaki disease (KD) is a paediatric idiopathic vasculitis. In this study, on the basis of studies using an established animal model for KD, we report that mannose-binding lectin (MBL) is involved in the pathogenesis of the disease. KD-like experimental murine vasculitis was induced by intraperitoneally administering a Candida albicans water-soluble extract (CAWS). MBL-A gradually increased in the serum of the model mice treated with CAWS. Deposition of MBL-A and MBL-C was observed in the aortic root, including the coronary arteries, which is a predilection site in experimental vasculitis. Corresponding to the distribution patterns of MBLs, marked deposition of C3/C3-derived peptides was also observed. Regarding the self-reactivity of MBLs, we observed that MBLs interacted with core histones to activate the lectin pathway. These results suggest that some types of pathogens provoke the MBL-dependent complement pathway (lectin pathway) to cause and/or exacerbate KD-like vasculitis.

Platelet activation dynamics evaluated using platelet-derived microparticles in Kawasaki disease.

BACKGROUND: Little is known about the platelet dynamics and the effect of antiplatelet therapy in Kawasaki disease (KD). The aim of this study was to clarify platelet activation dynamics in acute-phase KD patients by assaying platelet-derived microparticles (PDMPs). METHODS AND RESULTS: The PDMP level in 18 patients with acute KD was measured on ELISA. Of the 18 patients, 14 were receiving oral aspirin and i.v. immunoglobulin (IVIG) and 4, oral aspirin alone. Blood samples were drawn before, immediately after, and 10-14 days after IVIG infusion; thereafter, at 1, 2, and 3 months after the onset of disease. PDMP level before aspirin treatment was significantly higher in acute-phase KD patients than in the control subjects with common febrile diseases (P<0.01). In the acute-phase KD patients, IVIG significantly decreased PDMP level; the PDMP level was not lower on the similar day of KD in the patients who did not receive IVIG. Eight patients' PDMP level rebounded after aspirin was discontinued. CONCLUSIONS: Platelets are activated during acute-phase KD, which confirms the importance of antiplatelet therapy. In addition, platelet activation continues as long as 2 or 3 months after the acute phase, the time at which aspirin is commonly discontinued, and the timing of aspirin discontinuation should therefore be evaluated in each individual patient.

Utility of whole-blood aggregometry for evaluating anti-platelet therapy for Kawasaki disease.

BACKGROUND: Anti-platelet therapy for Kawasaki disease (KD) is often done without monitoring drug efficacy. The aim of this study was to investigate the utility of whole-blood aggregometry to evaluate the efficacy of anti-platelet therapy for KD. METHODS: Of 37 late-phase KD patients included in the present study, 20 were prescribed anti-platelet drugs. Platelet-rich plasma (PRP) aggregation with collagen as the stimulus was measured using an optical aggregometer. The area under the curve of small and large size aggregations was calculated, and categorized into five classes: -2, -1, 0, 1, and 2. Whole-blood aggregation with collagen or adenosine 5'-diphosphate (ADP) as stimulus was evaluated using the platelet aggregation threshold index (PATI), which is the concentration of stimulus that induces a whole-blood aggregation rate of 50%. RESULTS: In both collagen- and ADP-induced aggregation, there was a negative correlation between PATI and class determination using the PRP technique (collagen, rs = -0.870, P < 0.0001; ADP, rs = -0.620, P < 0.0001). Moreover, the PATI in collagen- and ADP-induced aggregation was significantly higher in the anti-platelet drug therapy group than in the untreated group (collagen, P < 0.0001; ADP, P = 0.0002). The serum thromboxane B2 level in the anti-platelet drug therapy group was also significantly lower than that in the untreated group (P < 0.0001). PATI was significantly higher in those treated with thienopyridine drug combinations than those without drug therapy (P = 0.0036). CONCLUSIONS: Whole-blood aggregometry is useful for monitoring the efficacy of anti-platelet therapy for KD.

Dynamics of reactive oxygen metabolites and biological antioxidant potential in the acute stage of Kawasaki disease.

BACKGROUND: The dynamics of oxidation/reduction control system activities using reactive oxygen metabolites (ROM) and biological antioxidant potential (BAP) in acute stage patients was evaulated to understand the mechanism of vascular injury and remodeling in Kawasaki disease (KD). METHODS AND RESULTS: ROM, BAP, high-sensitivity C-reactive protein (hs-CRP), interleukin-1,2,6, and tumour necrosis factor-α in 19 KD patients were measured. ROM decreased in good correlation only with hs-CRP (P<0.05) at 2 weeks after intravenous immunoglobulin (IVIG). Patients were further classified as responding well (Group A) or responding poorly (Group B) to IVIG. Both treatment groups had significantly higher ROM values than the control group (P<0.01). ROM decreased in Group A both immediately and 2 weeks after the IVIG treatment (P<0.05), but it did not decrease in Group B until 2 weeks post-treatment (P<0.01). BAP levels were unremarkable in Group A, but were significantly lower in Group B than in both other groups (P<0.05). BAP increased in Group A 2 weeks after IVIG treatment (P<0.01), but remained low in Group B (P<0.01). CONCLUSIONS: Acute stage KD patients suffer from obvious hyperoxidant stress, and improved in response to IVIG treatment in most patients. Blood BAP level might be a useful index for predicting responsiveness to IVIG the treatment.

Histopathological Characteristics of Post-inflamed Coronary Arteries in Kawasaki Disease-like Vasculitis of Rabbits.

Kawasaki disease (KD) is a systemic vasculitis in infants that develops predominantly in the coronary arteries. Despite the clinically transient nature of active inflammation in childhood albeit rare complications (e.g., coronary artery aneurysm), KD has recently been suggested to increase the incidence of ischemic heart diseases in young adulthood. However, little is known about the histopathology of the coronary artery long after development of the acute KD vasculitis. To address this, we conducted histological studies of rabbit coronary arteries in adolescent phase after induction of the KD-like vasculitis induced by horse serum administration. After a transmural infiltration of inflammatory cells in acute phase at day 7, the artery exhibited a gradual decrease in the number of inflammatory cells and thickening of the intima during the chronic phase up to day 90, where proteoglycans were distinctly accumulated in the intima with abundant involvement of α-smooth muscle actin (α-SMA)-positive cells, most of which accompanied expression of VCAM-1 and NF-κB. Distinct from classical atherosclerosis, inflammatory cells, e.g., macrophages, were barely detected during the chronic phase. These observations indicate that the KD-like coronary arteritis is followed by intimal thickening via accumulation of proteoglycans and proliferation of α-SMA-positive cells, reflecting aberrant coronary artery remodeling.

The involvement of the vasa vasorum in the development of vasculitis in animal model of Kawasaki disease.

BACKGROUND: Kawasaki Disease (KD) involves a diffuse and systemic vasculitis of unknown etiology that mainly affects infants and children. Although a considerable number of analyses of the clinical, histopathological and molecular biological details underlying the mechanism responsible for the development of coronary arterial lesions, it is still poorly understood.The purpose of this study was to analyze the state of angiogenesis, vasculogenesis and the distribution of blood vessels using an animal model of KD like vasculitis. We investigated the involvement of the vasa vasorum from the adventitia in the vascular involvement and the development of the disease state by performing sequential histopathology, scanning electron microscopy (SEM) and micro computed tomography (CT) studies using a murine model of vasculitis induced by the Candida albicans water-soluble fraction (CAWS). METHODS: To prepare the animal model of KD like vasculitis, CAWS was intraperitoneally injected into C57BL/6N mice for five consecutive days as reported by Ohno et al. We observed the changes of the vasa vasorum at the aorta and the orifices of the coronary arteries by SEM and micro CT, and also compared the neovascularization at the media and adventitia of the aorta by an immunohistochemical analysis. RESULTS: As previously reported, obvious inflammation was detected two weeks after the injection of CAWS, and also intimal thickening was observed three weeks after the injection. We found that the vasa vasorum in the adventitia of the aorta was increased in the model mice. The vasa vasorum started increasing one week after the injection of CAWS, before any obvious vasculitis was microscopically detected. CONCLUSION: The present results indicate that the vasculitis in Kawasaki disease starts as a disorder of the vasa vasorum.

Preventive effects of green tea catechins on spontaneous stroke in rats.

BACKGROUND: Green tea catechins possess potent antioxidative properties and protect against various oxidative diseases. Malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) develop severe hypertension and spontaneous stroke at early ages. We previously reported that ingestion of green tea catechins prevents cerebral ischemic damage in a middle cerebral artery occlusion and reperfusion rat stroke model, in association with increased plasma epigallocatechin-gallate (EGCG) concentrations. In this study, we examined whether tea catechin intake decreases the incidence of spontaneous stroke in M-SHRSP. MATERIAL/METHODS: Male M-SHRSP ingested 0.5% green tea catechin extract (Polyphenon E) in their drinking water beginning at 5 weeks of age, and blood pressure, heart rate, and locomotor activity were continuously monitored from 8 weeks using a telemetry system. Stroke onset was assessed by the appearance of neurologic symptoms, body weight loss, and circadian rhythm disturbances in heart rate, blood pressure, and locomotor activity. RESULTS: Tea catechin ingestion significantly delayed stroke onset by 10 days compared to the control group. Although there was no difference in blood pressure at 10 weeks, the rate of in blood pressure increase in the tea catechin group was significantly smaller than that in the control group. Plasma NO2- and NO3- concentrations increased after stroke in both groups without significant difference between the two groups. Plasma EGCG concentration significantly decreased at post-stroke compared with that of pre-stroke. CONCLUSIONS: Continuous ingestion of green tea catechins from an early age prevented the development of spontaneous stroke in M-SHRSP, probably by inhibiting the further development of high blood pressure at later ages.

Repeated in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure affects male gonads in offspring, leading to sex ratio changes in F2 progeny.

The effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the reproductive system of male rat offspring (F1) and the sex ratio of the subsequent generation (F2) were examined. Female Holtzman rats were gavaged with an initial loading dose of 400 ng/kg TCDD prior to mating, followed by weekly maintenance doses of 80 ng/kg during mating, pregnancy, and the lactation period. Maternal exposure to TCDD had no significant effects on fetus/pup (F1) mortality, litter size, or sex ratio on gestation day (GD) 20 or postnatal day (PND) 2. The TCDD concentration in maternal livers and adipose tissue on GD20 was 1.21 and 1.81 ng/kg, respectively, and decreased at weaning to 0.72 in the liver and 0.84 in the adipose tissue. In contrast, the TCDD concentration in pup livers was 1.32 ng/kg on PND2 and increased to 1.80 ng/kg at weaning. Ventral prostate weight of male offspring was significantly decreased by TCDD exposure on PND28 and 120 compared with that of controls. Weight of the testes, cauda epididymides, and seminal vesicle, and sperm number in the cauda epididymis were not changed by TCDD exposure at PND120. TCDD- or vehicle-exposed male offspring were mated with unexposed females. The sex ratio (percentage of male pups) of F2 offspring was significantly reduced in the TCDD-exposed group compared with controls. These results suggest that in utero and lactational TCDD exposures affect the development of male gonads in offspring (F1), leading to changes in the sex ratio of the subsequent generation (F2).