Risk Factors of Coronary Artery Abnormalities and Resistance to Intravenous Immunoglobulin Plus Corticosteroid Therapy in Severe Kawasaki Disease: An Analysis of Post RAISE.

BACKGROUND: Coronary artery abnormalities (CAAs) still occur in patients with Kawasaki disease receiving intensified treatment with corticosteroids. We aimed to determine the risk factors of CAA development and resistance to intensified treatment in Post RAISE (Prospective Observational Study on Stratified Treatment With Immunoglobulin Plus Steroid Efficacy for Kawasaki Disease)-the largest prospective cohort of Kawasaki disease patients to date. METHODS: In Post RAISE, 2648 consecutive patients with Kawasaki disease were enrolled. The present study analyzed 724 patients predicted to be intravenous immunoglobulin (IVIG) nonresponders (Kobayashi score ≥5) who received intensified treatment consisting of IVIG plus prednisolone. The association between the baseline characteristics and CAA at 1 month after disease onset was examined. The association between the baseline characteristics and treatment resistance was also investigated. RESULTS: Maximum Z score at baseline ≥2.5 (odds ratio, 3.4 [95% CI, 1.5-7.8]), age at fever onset <1 year (odds ratio, 3.4 [95% CI, 1.6-7.4]), and nonresponsiveness to IVIG plus prednisolone treatment (odds ratio, 6.8 [95% CI, 3.3-14.0]) were independent predictors of CAA development. Nonresponsiveness to IVIG plus prednisolone was significantly associated with 8 baseline variables. Baseline total bilirubin (odds ratio, 1.4 [95% CI, 1.2-1.7]) was the only significant independent predictor other than the variables included in the Kobayashi score, enabling treatment resistance to be identified at diagnosis. The area under the ROC curve was 0.74 (95% CI, 0.69-0.79). At a cutoff point of 1.0, the sensitivity and specificity for predicting treatment resistance were 71% and 65%, respectively. CONCLUSIONS: In Post RAISE, younger age at fever onset, a larger maximum Z score at baseline, and nonresponsiveness to IVIG plus prednisolone were risk factors significantly associated with CAA development. Nonresponders were able to be identified at diagnosis based on the total bilirubin value. To prevent CAA, more intensified or adjunctive therapies using other agents, such as pulsed methylprednisolone, ciclosporin, infliximab, and Anakinra, should be considered for patients with these risk factors. Registration: URL: https://www.umin.ac.jp/ctr/; Unique identifier: UMIN000007133.

Compound heterozygous variants in the ABCG8 gene in a Japanese girl with sitosterolemia.

Sitosterolemia is an autosomal recessive disorder that affects lipid metabolism and is characterized by elevated serum plant sterol levels, xanthomas, and accelerated atherosclerosis. In this study, we report a novel nonsense single-nucleotide variant, c.225G > A (p.Trp75*), and an East Asian population-specific missense multiple-nucleotide variant, c.1256_1257delTCinsAA (p.Ile419Lys), in the ABCG8 gene in a compound heterozygous state observed in a Japanese girl with sitosterolemia.

Inositol 1,4,5-trisphosphate receptor 2 as a novel marker of vasculature to delineate processes of cardiopulmonary development.

Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) are intracellular Ca2+ channels that are essential for many tissues and organs. We discovered that IP3R2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IP3R2 locus. Whole-mount and section LacZ staining showed that IP3R2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IP3R2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IP3R2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IP3R-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IP3R2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.

Efficacy and safety of intravenous immunoglobulin plus prednisolone therapy in patients with Kawasaki disease (Post RAISE): a multicentre, prospective cohort study.

BACKGROUND: The RAISE study showed that additional prednisolone improved coronary artery outcomes in patients with Kawasaki disease at high risk of intravenous immunoglobulin (IVIG) resistance. However, no studies have been done to test the steroid regimen used in the RAISE study. We therefore aimed to verify the efficacy and safety of primary IVIG plus prednisolone. METHODS: We did a multicentre, prospective cohort study at 34 hospitals in Japan. We included patients diagnosed with Kawasaki disease according to the Japanese diagnostic criteria, and excluded those who were treated at other hospitals before being transferred to a participating hospital. Patients who were febrile at diagnosis received primary IVIG (2 g/kg per 24 h) and oral aspirin (30 mg/kg per day) until the fever resolved, followed by oral aspirin (5 mg/kg per day) for 2 months after Kawasaki disease onset. We stratified patients using the Kobayashi score into predicted IVIG non-responders (Kobayashi score ≥5) or predicted IVIG responders (Kobayashi score <5). For predicted non-responders, each hospital independently decided whether to add prednisolone (intravenous injection of 2 mg/kg per day for 5 days) to the primary IVIG treatment, according to their respective treatment policy, and we further divided these patients based on the primary treatment received. The primary endpoint was the incidence of coronary artery abnormalities determined by two-dimensional echocardiography at 1 month after the primary treatment in predicted non-responders treated with primary IVIG plus prednisolone. Coronary artery abnormalities were defined according to the criteria of the Japanese Ministry of Health and Welfare and of the American Heart Association (AHA). This study is registered with the University Hospital Medical Information Network Clinical Trials Registry, number UMIN000007133. FINDINGS: From July 1, 2012, to June 30, 2015, we enrolled 2628 patients with Kawasaki disease, of whom 724 (27·6%) were predicted IVIG non-responders who received IVIG plus prednisolone as primary treatment. 132 (18·2%) of 724 patients did not respond to primary treatment. Among patients with complete data, coronary artery abnormalities were present in 40 (incidence rate 5·9%, 95% CI 4·3-8·0) of 676 patients according to the AHA criteria or in 26 (3·8%, 2·5-5·6) of 677 patients according to the Japanese criteria. Serious adverse events were reported in 12 (1·7%) of 724 patients treated with primary IVIG plus prednisolone; two of these patients had hypertension and bacteraemia that was probably related to prednisolone. One patient died possibly due to severe inflammation from the Kawasaki disease itself. INTERPRETATION: Primary IVIG plus prednisolone therapy in this study had an effect similar to that seen in the RAISE study in reducing the non-response rate and decreasing the incidence of coronary artery abnormalities. A primary IVIG and prednisolone combination therapy might prevent coronary artery abnormalities and contribute to lowering medical costs. FUNDING: Tokyo Metropolitan Government Hospitals and the Japan Kawasaki Disease Research Center.

Pharyngeal arch artery defects and lethal malformations of the aortic arch and its branches in mice deficient for the Hrt1/Hey1 transcription factor.

The aortic arch and major branch arteries are formed from the three pairs of pharyngeal arch arteries (PAAs) during embryonic development. Their morphological defects are clinically observed as isolated diseases, as a part of complicated cardiovascular anomalies or as a manifestation of multi-organ syndromes such as 22q11.2 deletion syndrome. Although numerous genes have been implicated in PAA formation and remodeling, detailed mechanisms remain poorly understood. Here we report that the mice null for Hrt1/Hey1, a gene encoding a downstream transcription factor of Notch and ALK1 signaling pathways, show perinatal lethality on the C57BL/6N, C57BL/6N × C57BL/6J or C57BL/6N × 129X1/SvJ background. Hrt1/Hey1 null embryos display abnormal development of the fourth PAA (PAA4), which results in congenital vascular defects including right-sided aortic arch, interruption of the aortic arch and aberrant origin of the right subclavian artery. Impaired vessel formation occurs randomly in PAA4 of Hrt1/Hey1 null embryos, which likely causes the variability of congenital malformations. Endothelial cells in PAA4 of null embryos differentiate normally but are structurally disorganized at embryonic day 10.5 and 11.5. Vascular smooth muscle cells are nearly absent in the structurally-defective PAA4, despite the appropriate migration of cardiac neural crest cells into the fourth pharyngeal arches. Endothelial expression of Jag1 is down-regulated in the structurally-defective PAA4 of null embryos, which may be one of the mechanisms underlying the suppression of vascular smooth muscle cell differentiation. While the direct downstream phenomena of the Hrt1/Hey1 deficiency remain to be clarified, we suggest that Hrt1/Hey1-dependent transcriptional regulation has an important role in PAA formation during embryonic development.

The chemokine receptor CXCR7 functions to regulate cardiac valve remodeling.

CXCR7 (RDC1), a G-protein-coupled receptor with conserved motifs characteristic of chemokine receptors, is enriched in endocardial and cushion mesenchymal cells in developing hearts, but its function is unclear. Cxcr7 germline deletion resulted in perinatal lethality with complete penetrance. Mutant embryos exhibited aortic and pulmonary valve stenosis due to semilunar valve thickening, with occasional ventricular septal defects. Semilunar valve mesenchymal cell proliferation increased in mutants from embryonic day 14 onward, but the cell death rate remained unchanged. Cxcr7 mutant valves had increased levels of phosphorylated Smad1/5/8, indicating increased BMP signaling, which may partly explain the thickened valve leaflets. The hyperproliferative phenotype appeared to involve Cxcr7 function in endocardial cells and their mesenchymal derivatives, as Tie2-Cre Cxcr7(flox/-) mice had semilunar valve stenosis. Thus, CXCR7 is involved in semilunar valve development, possibly by regulating BMP signaling, and may contribute to aortic and pulmonary valve stenosis.

Hand2 function in second heart field progenitors is essential for cardiogenesis.

Cardiogenesis involves the contributions of multiple progenitor pools, including mesoderm-derived cardiac progenitors known as the first and second heart fields. Disruption of genetic pathways regulating individual subsets of cardiac progenitors likely underlies many forms of human cardiac malformations. Hand2 is a member of the basic helix loop helix (bHLH) family of transcription factors and is expressed in numerous cell lineages that contribute to the developing heart. However, the early embryonic lethality of Hand2-null mice has precluded lineage-specific study of its function in myocardial progenitors. Here, we generated and used a floxed allele of Hand2 to ablate its expression in specific cardiac cell populations at defined developmental points. We found that Hand2 expression within the mesoderm-derived second heart field progenitors was required for their survival and deletion in this domain recapitulated the complete Hand2-null phenotype. Loss of Hand2 at later stages of development and in restricted domains of the second heart field revealed a spectrum of cardiac anomalies resembling forms of human congenital heart disease. Molecular analyses of Hand2 mutant cells revealed several genes by which Hand2 may influence expansion of the cardiac progenitors. These findings demonstrate that Hand2 is essential for survival of second heart field progenitors and that the graded loss of Hand2 function in this cardiac progenitor pool can cause a spectrum of congenital heart malformation.

Mechanisms underlying early development of pulmonary vascular obstructive disease in Down syndrome: An imbalance in biosynthesis of thromboxane A2 and prostacyclin.

Patients with Down syndrome (DS) and a left-to-right shunt often develop early severe pulmonary hypertension (PH) and pulmonary vascular obstructive disease (PVOD); the pathophysiological mechanisms underlying the development of these complications are yet to be determined. To investigate the mechanisms, we evaluated the biosynthesis of thromboxane (TX) A(2) and prostacyclin (PGI(2)) in four groups of infants, cross-classified as shown below, by measuring the urinary excretion levels of 11-dehydro-TXB(2) and 2,3-dinor-6-keto-PGF(1alpha): DS infants with a left-to-right shunt and PH (D-PH, n = 18), DS infants without congenital heart defect (D-C, n = 8), non-DS infants with a left-to-right shunt and PH (ND-PH, n = 12), and non-DS infants without congenital heart defect (ND-C, n = 22). The urinary excretion ratios of 11-dehydro-TXB(2) to 2,3-dinor-6-keto-PGF(1alpha) in the D-PH, D-C, ND-PH, and ND-C groups were 7.69, 4.71, 2.10, and 2.27, respectively. The ratio of 11-dehydro-TXB(2) to 2,3-dinor-6-keto-PGF(1alpha) was higher in the presence of DS (P < 0.001), independently of the presence of PH (P = 0.297). The predominant biosynthesis of TXA(2) over PGI(2), leading to vasoconstriction, was observed in DS infants, irrespective of the presence/absence of PH. This imbalance in the biosynthesis of vasoactive eicosanoids may account for the rapid progression of PVOD in DS infants with a left-to-right shunt.

Molecular embryology for an understanding of congenital heart diseases.

Congenital heart diseases (CHD) result from abnormal morphogenesis of the embryonic cardiovascular system and usually involve defects in specific structural components of the developing heart and vessels. Therefore, an understanding of "Molecular Embryology", with specific focus on the individual modular steps involved in cardiovascular morphogenesis, is particularly relevant to those wishing to have a better insight into the origin of CHD. Recent advances in molecular embryology suggest that the cardiovascular system arises from multiple distinct embryonic origins, and a population of myocardial precursor cells in the pharyngeal mesoderm anterior to the early heart tube, denoted the "second heart field", has been identified. Discovery of the second heart field has important implications for the interpretation of cardiac outflow tract development and provides new insights into the morphogenesis of CHD.

Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling.

Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a coculture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen, and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. Myocardial beta1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of beta1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.

Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2.

MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.

Sonic hedgehog is essential for first pharyngeal arch development.

The secreted protein sonic hedgehog (Shh) is essential for normal development of many organs. Targeted disruption of Shh in mouse leads to near complete absence of craniofacial skeletal elements at birth, and mutation of SHH in human causes holoprosencephaly (HPE), frequently associated with defects of derivatives of pharyngeal arches. To investigate the role of Shh signaling in early pharyngeal arch development, we analyzed Shh mutant embryos using molecular markers and found that the first pharyngeal arch (PA1) was specifically hypoplastic and fused in the midline, and remaining arches were well formed at embryonic day (E) 9.5. Molecular analyses using specific markers suggested that the growth of the maxillary arch and proximal mandibular arch was severely defective in Shh-null PA1, whereas the distal mandibular arch was less affected. TUNEL assay revealed an increase in the number of apoptotic signals in PA1 of Shh mutant embryos. Ectodermal expression of fibroblast growth factor (Fgf)-8, a cell survival factor for pharyngeal arch mesenchyme, was down-regulated in the PA1 of Shh mutants. Consistent with this observation, downstream transcriptional targets of Fgf8 signaling in neural crest-derived mesenchyme, including Barx1, goosecoid, and Dlx2, were also down-regulated in Shh-null PA1. These results demonstrate that epithelial-mesenchymal signaling and transcriptional events coordinated by Shh, partly via Fgf8, is essential for cell survival and tissue outgrowth of the developing PA1.