Ya-Ling Yang

Polymorphisms of transforming growth factor-β signaling pathway and Kawasaki disease in the Taiwanese population

Kawasaki disease (KD) is a systemic vasculitis associated with cardiovascular symptom. A previous study in the European descent has indicated that genetic variants of the transforming growth factor-β (TGF-β) pathway are involved in the KD susceptibility and clinical status. This study was conducted to investigate if polymorphisms in TGF-β signaling pathway are associated with KD susceptibility, and the coronary artery lesion formation. A total of 950 subjects (381 KD patients and 569 controls) were investigated to identify 12 single-nucleotide polymorphisms in the TGF-β signaling pathway (rs2796817, rs10482751, rs2027567, rs12029576, rs11466480, rs4776338, rs12901071, rs7162912, rs1438386, rs6494633, rs12910698 and rs4776339) by using TaqMan Allelic Discrimination assay. Our results indicated that rs1438386 in the SMAD3 is significantly associated with the susceptibility of KD. Additionally, both haplotypes of TGFβ2 and SMAD3 were also associated with the risk of KD. This study showed that genetic polymorphisms in TGF-β signaling pathway are associated with KD susceptibility, but not coronary artery lesions formation, or intravenous immunoglobulin treatment response in the Taiwanese population.

Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4

Background Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis. Objectives In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs. Methods We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays. Results After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation. Conclusions Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.

Cholestasis downregulate hepcidin expression through inhibiting IL-6-induced phosphorylation of signal transducer and activator of transcription 3 signaling

Hepcidin is downregulated during progressive cholestasis in biliary atresia, but the mechanism is unknown. To verify whether downregulation of hepcidin is specific to cholestasis irrespective of the patients age, we first analyzed liver hepcidin mRNA and protein expression in adults with primary biliary cirrhosis (PBC) (n=4), non-cholestatic cirrhosis (n=19) and in controls (n=9). We evaluated the tyrosine phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) expressions in the liver sections. A rat model of cholestasis by ligation of the extrahepatic bile duct (BDL) was created, and lipopolysaccharide (LPS)-induced cholangitis in cholestatic rats 2 weeks after BDL was also established to study the modulation of hepcidin by interleukin-6 (IL-6) and STAT3 signaling pathway in these models, using real-time quantitative reverse transcription–PCR, immunohistochemistry, western blotting and enzyme-linked immunosorbent assay (ELISA). An in vitro study of the effect of bile acids on hepcidin expression was carried out to re-confirm the in vivo findings. There was significantly lower hepcidin mRNA and pSTAT3 protein expression in cholestatic cirrhosis compared with non-cholestatic cirrhosis in adults. BDL caused significant decrease in hepcidin and gp130 mRNA expression compared with sham-operated group and normal control. Furthermore, there was significantly lower pSTAT3 protein expression and nuclear translocation in the cholestatic liver from the patients and the BDL rats, which was comparable to lower liver hepcidin mRNA and plasma hepcidin expression. Furthermore, BDL for 2 weeks attenuated the upregulation of hepcidin expression induced by LPS. Hydrophobic bile acid glycochenodeoxycholate inhibited IL-6-induced pSTAT3 expression in primary hepatocytes and resulted in the downregulation of hepcidin mRNA expression. In conclusion, the study shows that cholestasis or its important component—hydrophobic bile acids—can downregulate hepcidin expression through inhibiting IL-6-induced STAT3 phosphorylation and pSTAT3 protein nuclear translocation.

Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice

Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.

Inflammation-Induced Hepcidin is Associated with the Development of Anemia and Coronary Artery Lesions in Kawasaki Disease

PurposeKawasaki disease (KD) is a systemic febrile vasculitis complicated by coronary artery lesions (CAL). Anemia is common in patients with KD and is associated with a prolonged duration of active inflammation. Hepcidin is a central modulator of inflammation-associated anemia, acting via control of iron absorption and a direct inhibitory effect on erythropoiesis. The aims of this study were to investigate the role of inflammation-induced hepcidin in the development of anemia, the occurrence of CAL formation, and IVIG treatment response in patients with KD.MethodsEighty-six KD patients and 30 febrile controls were enrolled. Levels of interleukin (IL)-6 and serum hepcidin were measured in sera by enzyme-linked immunosorbent assay. Hemoglobin and serum iron levels were also measured.ResultsHemoglobin and iron levels were lower in KD patients than in controls (p < 0.001 and p = 0.009, respectively). Serum hepcidin and IL-6 levels were higher in KD patients than in controls (both p < 0.001) before intravenous immunoglobulin (IVIG) treatment. After IVIG treatment, serum hepcidin, IL-6, and hemoglobin levels decreased significantly (all p < 0.001). In addition, the serum hepcidin levels before IVIG treatment were negatively correlated with hemoglobin levels after IVIG treatment (R = −0.188, p = 0.046) and positively correlated with the changes of hemoglobin levels after IVIG treatment (R = 0.269, p = 0.015). Furthermore, serum hepcidin levels were negatively correlated with serum iron levels (R = −0.412, p = 0.002), which were positively correlated with hemoglobin levels (R = 0.210, p = 0.045). Additionally, the change of hepcidin levels was associated with IVIG treatment response and the occurrence of CAL formation.ConclusionsInappropriately raised hepcidin levels impair iron metabolism and are associated with decreased hemoglobin levels in KD patients. Inflammation-induced hepcidin is associated with the development of anemia and disease outcomes in patients with KD.

Microarray Study of Pathway Analysis Expression Profile Associated with MicroRNA-29a with Regard to Murine Cholestatic Liver Injuries

Accumulating evidence demonstrates that microRNA-29 (miR-29) expression is prominently decreased in patients with hepatic fibrosis, which consequently stimulates hepatic stellate cells’ (HSCs) activation. We used a cDNA microarray study to gain a more comprehensive understanding of genome-wide gene expressions by adjusting miR-29a expression in a bile duct-ligation (BDL) animal model. Methods: Using miR-29a transgenic mice and wild-type littermates and applying the BDL mouse model, we characterized the function of miR-29a with regard to cholestatic liver fibrosis. Pathway enrichment analysis and/or specific validation were performed for differentially expressed genes found within the comparisons. Results: Analysis of the microarray data identified a number of differentially expressed genes due to the miR-29a transgene, BDL, or both. Additional pathway enrichment analysis revealed that TGF-β signaling had a significantly differential activated pathway depending on the occurrence of miR-29a overexpression or the lack thereof. Furthermore, overexpression was found to elicit changes in Wnt/β-catenin after BDL. Conclusion: This study verified that an elevated miR-29a level could alleviate liver fibrosis caused by cholestasis. Furthermore, the protective effects of miR-29a correlate with the downregulation of TGF-β and associated with Wnt/β-catenin signal pathway following BDL.

TARC/CCL17 gene polymorphisms and expression associated with susceptibility and coronary artery aneurysm formation in Kawasaki disease

Background:Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. Thymus and activation-regulated chemokine/chemokine ligand 17 (TARC/CCL17) is one of the Th2 chemokines and has been suggested as a candidate gene for conferring susceptibility to Th2 associated with allergy diseases. This study examined the correlation between gene polymorphisms and plasma levels of TARC/CCL17 in patients with KD and the outcomes of KD.Methods:A total of 381 KD patients and 564 controls were subjected to determination of five tagging single-nucleotide polymorphisms of TARC/CCL17. In addition, plasma TARC/CCL17 levels were measured by enzyme-linked immunosorbent assay.Results:Polymorphisms of TARC/CCL17 were significantly different between normal children and patients with KD. A allele of rs4784805 has better intravenous immunoglobulin (IVIG) treatment response to KD. Furthermore, plasma TARC/CCL17 levels were higher in KD patients than that in controls before IVIG treatment. After IVIG treatment, plasma TARC/CCL17 levels decreased significantly.Conclusion:This study provides the first evidence supporting the association between TARC/CCL17 polymorphisms, susceptibility of KD, and IVIG responses in KD patients.

MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

MicroRNA-29 (miR-29) is found to modulate hepatic stellate cells’ (HSCs) activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates were subjected to bile duct-ligation (BDL) to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A) expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

Hepcidin-Induced Iron Deficiency Is Related to Transient Anemia and Hypoferremia in Kawasaki Disease Patients

Kawasaki disease (KD) is a type of systemic vasculitis that primarily affects children under the age of five years old. For sufferers of KD, intravenous immunoglobulin (IVIG) has been found to successfully diminish the occurrence of coronary artery lesions. Anemia is commonly found in KD patients, and we have shown that in appropriately elevated hepcidin levels are related to decreased hemoglobin levels in these patients. In this study, we investigated the time period of anemia and iron metabolism during different stages of KD. A total of 100 patients with KD and 20 control subjects were enrolled in this study for red blood cell and hemoglobin analysis. Furthermore, plasma, urine hepcidin, and plasma IL-6 levels were evaluated using enzyme-linked immunosorbent assay in 20 KD patients and controls. Changes in hemoglobin, plasma iron levels, and total iron binding capacity (TIBC) were also measured in patients with KD. Hemoglobin, iron levels, and TIBC were lower (p < 0.001, p = 0.009, and p < 0.001, respectively) while plasma IL-6 and hepcidin levels (both p < 0.001) were higher in patients with KD than in the controls prior to IVIG administration. Moreover, plasma hepcidin levels were positively and significantly correlated with urine hepcidin levels (p < 0.001) prior to IVIG administration. After IVIG treatment, plasma hepcidin and hemoglobin levels significantly decreased (both p < 0.001). Of particular note was a subsequent gradual increase in hemoglobin levels during the three weeks after IVIG treatment; nevertheless, the hemoglobin levels stayed lower in KD patients than in the controls (p = 0.045). These findings provide a longitudinal study of hemoglobin changes and among the first evidence that hepcidin induces transient anemia and hypoferremia during KD’s acute inflammatory phase.

Comparison of Clinical Characteristics and Neutrophil Values in Omental Infarction and Acute Appendicitis in Children

BACKGROUND Omental infarction is a rare occurrence in children. It is often diagnosed during surgery for suspected appendicitis. This study investigated the use of clinical and laboratory data for distinguishing between omental infarction and acute appendicitis. METHODS Seven patients with surgically and pathologically proven omental infarction and 28 age- and sex-matched patients with acute appendicitis were included in this study. The clinical characteristics, imaging study results and laboratory data were analyzed. RESULTS All 35 patients had right lower quadrant abdominal pain at presentation. The frequency of nausea and fever were significantly lower in the omental infarction group compared with the acute appendicitis group (p< 0.001 and p= 0.018, respectively). In laboratory studies, the white blood cell count, C-reactive protein value and neutrophil percentage were all higher in the acute appendicitis group compared with the omental infarction group (p= 0.001, p< 0.001, and p= 0.008, respectively). It was possible to separate patients with omental infarction from those with acute appendicitis based on a neutrophil percentage of less than 77% (sensitivity 100%, specificity 100%). CONCLUSIONS Results of the current study suggest that omental infarction should be considered as a possible diagnosis in patients presenting with right lower quadrant abdominal pain without nausea or fever, and with a neutrophil percentage below 77%.