👤 Li-Hsin Chien

Hypertensive disorders of pregnancy (HDP) are associated with future cardiovascular disease but mechanisms are not well defined. We examined the association between HDP and atherosclerotic cardiovascular disease (ASCVD) biomarkers 5-10 years after childbirth. Secondary analysis of the NICHD MFMU Network Gestational Diabetes (GDM) Trial Follow-Up study. Participants were recruited and biospecimens obtained 5-10 years after the original trial of treatment for mild GDM. Patients were included if a biospecimen was available and their HDP status was known. We compared patients who experienced HDP to normotensive controls. Outcomes included unadjusted medians and mean concentrations of ASCVD serum biomarkers Apolipoprotein B (ApoB), high-sensitivity C-reactive protein (hs-CRP), and creatinine. Generalized linear models were used to compare concentrations between groups. Of 740 participants in this analysis, 78 had been diagnosed with HDP. Mean duration of follow up after delivery was 7.1 ± 1.3 years for both groups. Unadjusted means of each biomarker were not different between groups. After adjusting for obesity, mean serum creatinine was elevated in women who had been diagnosed with HDP (0.77 mg/dL 95%CI (0.72, 0.82)) compared to controls (0.71 mg/dL 95%CI (0.69, 0.72)), p = 0.02. Adjusted means for ApoB between women with HDP and controls were 64.2 mg/dL (95%CI (59.3, 69.5)) and 60.6 mg/dL (95%CI (58.9, 62.3)), (p = 0.18), and for hs-CRP mg/L were 14.6 (95%CI (11.4, 19.1)) and 14.5 mg/L (95%CI (13.3, 15.9)), (p = 0.91). In patients with HDP compared to normotensive controls, serum creatinine, but not ApoB or hs-CRP, was modestly elevated within 10 years of childbirth. Show less

Pathological progression in sporadic Alzheimer's disease (sAD) initiates with an early rise in soluble amyloid-β (Aβ), preceding plaque formation and neurodegeneration. However, the molecular event triggering this initial accumulation remains unknown. We report that phosphoglycerate dehydrogenase (PHGDH), a consistent biomarker of prodromal sAD, drives Aβ production through a previously unrecognized RNA-binding function. Specifically, PHGDH binds the 3'UTR of Show less

Lipoprotein(a) [Lp(a)] is a recognized risk factor for atherosclerotic cardiovascular disease (ASCVD), but the shape and potential nonlinearity of its association remain uncertain. We assessed the linear and nonlinear associations between Lp(a) levels and ASCVD risk using observational and Mendelian randomization (MR) approaches. We analyzed 351,858 UK Biobank participants (2006-2023), stratified into Lp(a) percentiles: <70th, 70th-<80th, 80th-<90th, and ≥90th. Outcomes included ASCVD events from hospital, primary care, self-report, and death registry data. Cox models estimated the hazard ratios (HRs). MR analyses used a polygenic risk score from 10 Lp(a)-associated single-nucleotide polymorphisms, with nonlinearity tested by doubly ranked MR. Higher Lp(a) levels were associated with increased ASCVD risk. Compared with the <70th percentile, adjusted HRs (95% confidence interval) were 1.11 (1.07-1.16), 1.18 (1.14-1.22), and 1.25 (1.21-1.30) for the 70th-<80th, 80th-<90th, and ≥90th groups. Kaplan-Meier curves diverged early by group. Spline models suggested nonlinearity with an inflection near 130 nmol/L (P=0.007). MR showed a 2% higher ASCVD risk per 10 nmol/L genetically predicted Lp(a) (P<2×10 Elevated Lp(a) concentrations were causally associated with ASCVD risk, showing a predominantly graded relationship with possible nonlinearity at very high levels, supporting routine Lp(a) measurement and the development of Lp(a)-lowering therapies. Show less

There are limited data on the use of whole-exome sequencing (WES) to diagnose severe hypertriglyceridemia. Our aim was to identify candidate genes linked to triglyceride levels via a genome-wide association study (GWAS) and to recruit participants with severe hypertriglyceridemia for WES to assess allelic variants in the candidate genes. A GWAS was conducted involving 120,140 participants to identify lead loci associated with blood triglyceride levels. Following the identification of these lead loci, WES was performed on DNA samples from 29 participants with hypertriglyceridemia whose triglyceride levels exceeded 800 mg/dL to assess variations in the corresponding genes. In the GWAS of 120,140 participants, the apolipoprotein A5 (APOA5) locus on chromosome 11 showed the strongest association with blood triglyceride levels (lead single nucleotide polymorphism [SNP] rs2075291; P=3.07×10 Our study confirms the role of known genetic loci in triglyceride metabolism and hypertriglyceridemia while uncovering novel loci, offering new perspectives on lipid regulation and potential avenues for therapeutic advancements. Show less

BackgroundAlzheimer's disease (AD) is the main cause of dementia in an aging society. Previous studies have demonstrated that non-invasive light flicker and sound with gamma frequency oscillations can modulate AD-related pathology in AD mice, potentially improving patient outcomes. However, the molecular mechanism by which sound with gamma frequency oscillations inhibits the expression of amyloid-β Show less

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect in neonates. While there is compelling evidence of genetic contribution to the etiology of TOF, the contribution of noncoding variants to the development of the defect remains unexplored. Potentially damaging noncoding de novo variants (NC DNVs) were detected from 141 Chinese nonsyndromic TOF trios (CHN-TOF) and compared with those detected in the Pediatric Cardiac Genomics Consortium (PCGC). Bioinformatic analyses on noncoding and previously detected coding DNVs were performed to identify developmental pathways affected in TOF. Chinese but not PCGC-TOF patients showed a notably increased burden of putative damaging NC DNVs (n = 249). In Chinese, NC and coding DNVs were predominantly associated with cardiomyocyte differentiation and with chamber/valve/aorta development, respectively, producing a combined enrichment in NOTCH signaling (p = 1.1 × 10 Show less

Macrophages play a crucial role in coordinating the skeletal muscle repair response, but their phenotypic diversity and the transition of specialized subsets to resolution-phase macrophages remain poorly understood. Here, to address this issue, we induced injury and performed single-cell RNA sequencing on individual cells in skeletal muscle at different time points. Our analysis revealed a distinct macrophage subset that expressed high levels of Gpnmb and that coexpressed critical factors involved in macrophage-mediated muscle regeneration, including Igf1, Mertk and Nr1h3. Gpnmb gene knockout inhibited macrophage-mediated efferocytosis and impaired skeletal muscle regeneration. Functional studies demonstrated that GPNMB acts directly on muscle cells in vitro and improves muscle regeneration in vivo. These findings provide a comprehensive transcriptomic atlas of macrophages during muscle injury, highlighting the key role of the GPNMB macrophage subset in regenerative processes. Our findings suggest that modulating GPNMB signaling in macrophages may represent a promising avenue for future research into therapeutic strategies for enhancing skeletal muscle regeneration. Show less

The intricate involvement of the histaminergic system, encompassing histamine and histamine receptors, in the progression of diverse neoplasias has attracted considerable scrutiny. Histamine receptor H1 (HRH1) was reported to be overexpressed in several cancer types, but its specific functional implications in oral squamous cell carcinoma (OSCC) predominantly remain unexplored. Our findings indicate that dysregulated high levels of HRH1 were correlated with lymph node (LN) metastasis and poor prognoses in OSCC patients. We identified a disintegrin and metalloprotease 9 (ADAM9) as a critical downstream target of HRH1, promoting protumorigenic and prometastatic characteristics both in vitro and in vivo. Molecular investigations revealed that the cyclic increase in the HRH1-ADAM9-Snail/Slug axis promoted progression of the epithelial-to-mesenchymal transition (EMT). Clinical analyses demonstrated significant correlations of HRH1 expression with ADAM9 and with EMT-related markers, with elevated ADAM9 also associated with LN metastasis in OSCC patients. Regarding therapeutic aspects, we discovered that activated STAT3 acts as a compensatory pathway for the long-term HRH1 signaling blockade in OSCC cells. Combining inhibition of HRH1 and STAT3 using their respective inhibitors or short hairpin (sh)RNAs enhanced the tumor-suppressive effects compared to HRH1 inhibition/depletion alone in OSCC cells and a xenograft model. In summary, HRH1 has emerged as a valuable biomarker for predicting OSCC progression, and combined targeting of HRH1 and STAT3 may represent a promising strategy for preventing OSCC progression. Show less

(1) Background: Adipose tissue serves as a central repository for energy storage and is an endocrine organ capable of secreting various adipokines, including leptin and adiponectin. These adipokines exert profound influences on diverse physiological processes such as insulin sensitivity, appetite regulation, lipid metabolism, energy homeostasis, and body weight. Given the integral role of adipose tissue in metabolic regulation, it is imperative to investigate the effects of varying proportions and types of dietary fats on adipocyte function. In addition, our previous study showed that P/S = 5 and MUFA = 60% appeared to be beneficial in preventing white adipose tissue accumulation by decreasing plasma insulin levels and increasing hepatic lipolytic enzyme activities involved in β-oxidation. Therefore, the objective of this study was to explore the effects of a polyunsaturated fatty acid (PUFA) to saturated fatty acid (SFA) ratio of 5 and varying levels of monounsaturated fatty acids (MUFA = 30% or 60%) on lipogenesis. (2) Methods: We cultured 3T3-L1 mouse embryo fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum until confluent. Varying ratios of palmitic acid (PA), oleic acid (OA), and linoleic acid (LA) were first bound with bovine serum albumin (BSA) before being applied to 3T3-L1 adipocytes in low doses and in high doses. (3) Results: Low doses of P/S ratio = 5, MUFA = 60% (M60) fatty acids decreased the accumulation of triglycerides in mature adipocytes by decreasing the mRNA expression of adipogenic factors, such as peroxisome proliferator-activated receptors (PPARs), lipoprotein lipase (LPL), and glucose transporter-4 (GLUT-4), while increasing lipolytic enzyme (hormone-sensitive lipase, HSL) expression when compared to high doses of P/S ratio = 5, MUFA = 60% (M60), low and high doses of P/S ratio = 5, MUFA = 30% (M30). Furthermore, the treatment of M60 in low doses also decreased the secretion of leptin and increased the secretion of adiponectin in adipocytes. (4) Conclusions: The composition of P/S = 5, MUFA = 60% fatty acid in low doses appeared to result in anti-adipogenic effects on 3T3-L1 adipocytes due to the down-regulation of adipogenic effects and the transcription factor. Show less

Lung adenocarcinoma is the most common type of lung cancer. Known risk variants explain only a small fraction of lung adenocarcinoma heritability. Here, we conducted a two-stage genome-wide association study of lung adenocarcinoma of East Asian ancestry (21,658 cases and 150,676 controls; 54.5% never-smokers) and identified 12 novel susceptibility variants, bringing the total number to 28 at 25 independent loci. Transcriptome-wide association analyses together with colocalization studies using a Taiwanese lung expression quantitative trait loci dataset (n = 115) identified novel candidate genes, including FADS1 at 11q12 and ELF5 at 11p13. In a multi-ancestry meta-analysis of East Asian and European studies, four loci were identified at 2p11, 4q32, 16q23, and 18q12. At the same time, most of our findings in East Asian populations showed no evidence of association in European populations. In our studies drawn from East Asian populations, a polygenic risk score based on the 25 loci had a stronger association in never-smokers vs. individuals with a history of smoking (P Show less

Extremely early age at menarche, also called precocious puberty, has been associated with various cardiometabolic traits, but their shared heritability remains unclear. This work aimed to identify new shared genetic variants and their pathways for age at menarche and cardiometabolic traits and to investigate the influence of central precocious puberty on childhood cardiometabolic traits. Using the conjunction false discovery rate method, this study analyzed genome-wide association study data from the menarche-cardiometabolic traits among 59 655 females of Taiwanese ancestry and systemically investigated pleiotropy between age at menarche and cardiometabolic traits. To support the novel hypertension link, we used the Taiwan Puberty Longitudinal Study (TPLS) to investigate the influence of precocious puberty on childhood cardiometabolic traits. We discovered 27 novel loci, with an overlap between age at menarche and cardiometabolic traits, including body fat and blood pressure. Among the novel genes discovered, SEC16B, CSK, CYP1A1, FTO, and USB1 are within a protein interaction network with known cardiometabolic genes, including traits for obesity and hypertension. These loci were confirmed through demonstration of significant changes in the methylation or expression levels of neighboring genes. Moreover, the TPLS provided evidence regarding a 2-fold higher risk of early-onset hypertension that occurred in girls with central precocious puberty. Our study highlights the usefulness of cross-trait analyses for identifying shared etiology between age at menarche and cardiometabolic traits, especially early-onset hypertension. The menarche-related loci may contribute to early-onset hypertension through endocrinological pathways. Show less

Autophagy plays a protective role in the retinal pigment epithelium (RPE) by eliminating damaged organelles in response to reactive oxygen species (ROS). Dual-specificity protein phosphatase 6 (DUSP6), which belongs to the DUSP subfamily, works as a negative-feedback regulator of the extracellular signal-regulated kinase (ERK) pathway. However, the complex interplay between DUSP6 and autophagy induced by ROS in RPE is yet to be investigated. To investigate the relationship between DUSP6 and autophagy, we exposed the ARPE-19 cell line and C57BL/6N mice to sodium iodate (NaIO Show less

Norcantharidin (NCTD) is a demethylated derivative of cantharidin demonstrated to have anti-proliferative, anti-inflammatory, and anti-fibrosis properties. The purpose of the current study is to investigate the underlying mechanisms and signaling pathways affected by NCTD in human ARPE-19 cells. Cell growth and rate of proliferation were assayed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, colony formation assay, and cell cycle distribution/quantification. Cell motility was detected with in vitro migration assay. The level of epithelial-mesenchymal transition (EMT)-related proteins and mRNA (Snail, Slug, E-cadherin) were detected using Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence assay. Overexpression of Snail plasmid was determined by transfection assay. We found that NCTD reduced epidermal growth factor (EGF)-induced ARPE-19 cell viability and proliferation through increasing the p21 and p27 expression and decreasing the cyclin D1 expression. NCTD also inhibited EGF-mediated EMT and cell motility through increased protein and mRNA levels of E-cadherin and decreased Snail in EGF-induced ARPE-19 cells. Overexpression of Snail significantly decreased ARPE-19 cell motility and increased E-cadherin expression in NCTD-treated cells. Additionally, when NCTD was combined with a PI3K inhibitor (LY294002) significantly decreased the p-AKT and Snail expression, and increased the E-cadherin expression of EGF treatment in ARPE-19 cells. The current findings revealed that NCTD suppresses the EGF-induced proliferation, motility, and EMT of ARPE-19 cells through inactivation of the AKT-mediated Snail/E-cadherin pathway. NCTD may be a potential preventive agent for proliferative vitreoretinopathy. Show less

The relationship between apolipoprotein C3 (APOC3) gene polymorphisms and nonalcoholic fatty liver disease (NAFLD) risk has been investigated in many studies, with inconclusive findings. This meta-analysis evaluated the effect of APOC3 promoter region polymorphisms (-455T/C and -482C/T) on NAFLD susceptibility. A comprehensive search of eligible studies up to October 2020 was performed on Medline, Embase, Web of Science, and Google Scholar databases. No restriction was imposed on language, publication date, or publication status. Odds ratios (ORs) with their 95% confidence intervals (CIs) were calculated to assess the combined effect sizes. The levels of heterogeneity, sensitivity, subgroup, and publication bias were analyzed subsequently. This meta-analysis included eight studies, consisting of 1,511 patients with NAFLD and 1,900 controls fulfilling the inclusion criteria and exclusion criteria. The pooled analysis showed significant associations between APOC3 -455T/C polymorphism and NAFLD risk in allelic (OR = 1.33; 95% CI = 1.05-1.67), dominant (OR = 1.34; 95% CI = 1.04-1.72), and recessive (OR = 1.60; 95% CI = 1.06-2.40) models. Ethnicity-based stratification showed that -455T/C polymorphism was significantly associated with NAFLD risk in the non-Asian but not in the Asian population. No association was evident between -482C/T polymorphism and NAFLD risk. Our findings suggest that APOC3 promoter region polymorphism -455T/C may be associated with NAFLD risk in the non-Asian but not in the Asian population. Additional studies with other functional polymorphisms are needed to discover APOC3 gene effects on NAFLD. Show less

Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases. Show less

CHF1/Hey2 is a Notch-responsive basic helix-loop-helix transcription factor involved in cardiac development. Common variants in Hey2 are associated with Brugada syndrome. We hypothesized that absence of CHF1/Hey2 would result in abnormal cellular electrical activity, altered cardiac conduction system (CCS) development, and increased arrhythmogenesis. We isolated neonatal CHF/Hey2-knockout (KO) cardiac myocytes and measured action potentials and ion channel subunit gene expression. We also crossed myocardial-specific CHF1/Hey2-KO mice with cardiac conduction system LacZ reporter mice and stained for conduction system tissue. We also performed ambulatory ECG monitoring for arrhythmias and heart rate variability. Neonatal cardiomyocytes from CHF1/Hey2-KO mice demonstrate a 50% reduction in action potential dV/dT, a 50-75% reduction in SCN5A, KCNJ2, and CACNA1C ion channel subunit gene expression, and an increase in delayed afterdepolarizations from 0/min to 12/min. CHF1/Hey2 cKO CCS-lacZ mice have a ∼3-fold increase in amount of CCS tissue. Ambulatory ECG monitoring showed no difference in cardiac conduction, arrhythmias, or heart rate variability. Wild-type cells or animals were used in all experiments. CHF1/Hey2 may contribute to Brugada syndrome by influencing the expression of SCN5A and formation of the cardiac conduction system, but its absence does not cause baseline conduction defects or arrhythmias in the adult mouse.-Hartman, M. E., Liu, Y., Zhu, W.-Z., Chien, W.-M., Weldy, C. S., Fishman, G. I., Laflamme, M. A., Chin, M. T. Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias. Show less

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation. Show less

This study aimed to investigate whether the body mass index (BMI) in combination with genetic variations in APOE and APOA5_'T' alleles modulates the risk of sHTG. There were 255 moderate HTG (TG ≥2.26 and <5.65 mmol/L) and 176 sHTG (TG ≥5.65 mmol/L) and 304 controls (TG <2.26 mmol/L) were recruited. APOE epsilon alleles were genotyped using sequence-specific primers; the APOA5_'T' allele (c.553G>T, rs2075291) was identified using a restriction site polymorphism. Overweight/obesity was defined as BMI ≥25 kg/m(2) and non-overweight as BMI <25 kg/m(2). Multivariate logistic regression analysis showed, in addition to APOA5_'T' allele, a significant interaction between BMI ≥25 kg/m(2) and APOE4 carriers on the risk of sHTG. Subjects with diagnosis of diabetes, current smoking, hypertension, levels of non-high density lipoprotein, and BMI ≥25 kg/m(2) were significant determinants of sHTG. The odds ratio (95% confidence intervals) of overweight/obese APOE4 carriers for sHTG was 13.56 (4.89-37.59) more than those of non-overweight non-APOE4 carriers, while the odds ratio for sHTG in overweight/obese patients with the APOA5_'T' allele was 15.83 (7.77-32.26) higher than those of non-overweight non-APOA5 carriers. Overweight/obesity may potentiate the genetic variants of the APOE4 and APOA5_'T' alleles on the risk of sHTG. Show less

In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer. In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time. Show less

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation. Show less

The limitations of revolutionary new mutation-specific inhibitors of BRAF(V600E) include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF(V600E) by PLX4720 synergistically induces apoptosis across a spectrum of BRAF(V600E) melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses. Show less

Because the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome small interfering RNA (siRNA) screen in melanoma cells to expand our understanding of the kinases that regulate this pathway. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAF(V600E) mutation, inhibits Wnt/β-catenin signaling in human melanoma cells. Because inhibitors of BRAF(V600E) show promise in ongoing clinical trials, we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAF(V600E) inhibitor PLX4720. We found that endogenous β-catenin was required for PLX4720-induced apoptosis of melanoma cells and that activation of Wnt/β-catenin signaling synergized with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlated with reduced abundance of an endogenous negative regulator of β-catenin, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, siRNA directed against AXIN1 rendered resistant melanoma cell lines susceptible to apoptosis in response to treatment with a BRAF(V600E) inhibitor. Thus, Wnt/β-catenin signaling and AXIN1 may regulate the efficacy of inhibitors of BRAF(V600E), suggesting that manipulation of the Wnt/β-catenin pathway could be combined with BRAF inhibitors to treat melanoma. Show less

The cardiovascular restricted bHLH transcription factor CHF1/Hey2 has been reported to play an important role in regulation of vascular smooth muscle phenotype and gene expression, but the downstream target genes that mediate these effects have not been completely elucidated. We have previously found that loss of CHF1/Hey2 in vascular smooth muscle cells leads to dysregulated expression of the matrix metalloproteinase gene MMP10 after treatment with PDGF. Here we report that loss or knockdown of CHF1/Hey2 in vascular smooth muscle cells leads to increased expression and activity of MMP10 at baseline, suggesting a direct effect of CHF1/Hey2 on MMP10 promoter regulation. To test this hypothesis, we assessed the effects of CHF1/Hey2 on a 2.5 kb MMP10 promoter region upstream of the transcriptional start site. We found that this region contains multiple elements including 12 E-boxes that mediate constitutive activity and repression by CHF1/Hey2 in 293T cells and A7r5 smooth muscle cells. Surprisingly, mutation of these E-boxes not only abolished CHF1/Hey2 repression, but also diminished constitutive expression. In addition, we observed that some of these mutations unmasked an activator function for CHF1/Hey2, which has not been previously described. These findings support the hypothesis that CHF1/Hey2 is an important regulator of MMP10 expression. Show less

Atrial natriuretic factor (ANF) is abundantly expressed in atrial cardiomyocytes throughout ontogeny and in ventricular cardiomyocytes in the developing heart. However, during cardiac failure and hypertrophy, ANF expression can reappear in adult ventricular cardiomyocytes. The transcription factor Nkx2-5 is one of the major transactivators of the ANF gene in the developing heart. We identified Nkx2-5 binding at three 5' regulatory elements (kb -34, -31, and -21) and at the proximal ANF promoter by ChIP assay using neonatal mouse cardiomyocytes. 3C analysis revealed close proximity between the distal elements and the promoter region. A 5.8-kb fragment consisting of these elements transactivated a reporter gene in vivo recapitulating endogenous ANF expression, which was markedly reduced in tamoxifen-inducible Nkx2-5 gene knockout mice. However, expression of a reporter gene was increased and expanded toward the outer compact layer in the absence of the transcription repressor Hey2, similar to endogenous ANF expression. Functional Nkx2-5 and Hey2 binding sites separated by 59 bp were identified in the -34 kb element in neonatal cardiomyocytes. In adult hearts, this fragment did not respond to pressure overload, and ANF was induced in the absence of Nkx2-5. These results demonstrate that Nkx2-5 and its responsive cis-regulatory DNA elements are essential for ANF expression selectively in the developing heart. Show less

Common polymorphism of the apolipoprotein A5 gene (APOA5, c.553G>T) related to metabolic syndrome components, insulin resistance, and carotid atherosclerosis remains unclear. We investigated the associations of the APOA5 c.553G>T gene with various metabolic syndrome components and carotid artery atherosclerosis among family members. A total of 661 participants who provided complete genotyping and carotid artery measures were included in this study. Participants with APOA5 c.553T carrier (GT and TT) were more likely to have higher levels of triglycerides and apolipoprotein B, as well as lower levels of high-density lipoprotein (HDL) cholesterol, than participants with the GG genotype. Individuals who carried T alleles had an increased risk of a high level of triglycerides (multivariate odds ratio [OR], 3.86; 95% confidence interval [CI], 1.98-7.55; P<0.0001) and low levels of HDL cholesterol (OR, 2.32; 95% CI, 1.40-3.86; P=0.0012) compared with those without T alleles. The age was an effect modifier for the association between APOA5 genotype and smoking, alcohol drinking, obesity, and lipid profiles, including total, HDL, and low-density lipoprotein (LDL) cholesterol; triglycerides; and apolipoproteins. In addition, the association between APOA5 genotype and hypertriglyceridemia was significant only in adult groups (OR, 3.53; 95% CI, 1.79-6.94), and the association between APOA5 genotype and low HDL cholesterol was stable in young adolescents (OR, 2.39; 95% CI, 1.19-4.78) and adults (OR, 2.20; 95% CI, 1.17-4.15). Our findings indicated that the APOA5 c.553G>T polymorphism is associated with high triglycerides and low HDL cholesterol but not with other metabolic syndrome components or carotid atherosclerosis in this ethnic Chinese population. Show less

Polymorphism of apolipoprotein A1/C3/A4/A5 gene cluster affected lipid profiles in general population. We reported 6 polymorphisms, APOA1 -75G>A, APOA1 83C>T, APOC3 3175C>G, APOC3 3206G>T, APOA4 127A>G, and APOA5 553G>T in APOA1/C3/A4/A5 gene and the haplotype structures on triglyceride and HDL traits among ethnic Chinese. Overall, there were statistically significant differences in the distribution of APOA1 -75G>A and APOA5 +553G>T genotypes comparing cases with control subjects. For the APOA1 -75 SNP, a lower risk of triglyceride/HDL among subjects with A/A genotype compared with those with the G/G genotype (odds ratio, OR=0.39, 95% CI 0.16-0.92, P=0.04). However, the risk magnitude reduced after multivariate adjustments. For continuous traits, we found that only in APOA5 +553 T allele carriers showed a significant higher triglyceride and a significant lower HDL cholesterol level than subjects with APOA5 +553 G/G genotypes. There were significant differences in overall haplotype frequencies between case and control subjects (P<0.001). There is an important role of APOA1/C3/A4/A5 gene polymorphisms and haplotypes in the development of high triglyceride/HDL ratio in Chinese. Show less

Apolipoprotein A5 gene (APOA5) has been shown to modulate plasma triglyceride concentrations. We investigated 2 distinct APOA1/C3/A5 haplotypes roles for hypertriglyceridemia. We recruited 308 cases of hypertriglyceridemia and 281 normal controls from a hospital. Twelve single nucleotide polymorphisms (SNPs) across the APOA1/C3/A5 gene region were genotyped. One haplotype containing the minor alleles of the APOA5 (-1131T>C, c.553G>T) and APOA1 (-3013C>T,-75G>A) was more prevalent in cases than in controls (11.3% vs. 1.1%, respectively) and was statistically significantly associated with high triglycerides (adjusted odds ratio: 12.83, 95% confidence interval [CI]: 5.1-32.4, P<0.001). Another haplotype that was associated with hypertriglyceridemia (adjusted odds ratio 2.13, 95% CI, 1.37-3.29, P=0.001). Participants carrying both minor alleles of APOA5-1131CC and c.553TT had a 116% higher triglyceride concentration compared with those carrying common allele. The APOA1/C3/A5 haplotype represents an important locus for predicting risk of hypertriglyceridemia among Taiwanese. Show less

Cardiotoxin III (CTX III) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III in human colorectal cancer Colo205 cells. 2. Cardiotoxin III-induced Colo205 cell apoptosis was confirmed by DNA fragmentation (DNA ladder and sub-G1 formation) with an IC(50) of 4 mg/mL at 48 h. 3. Further mechanistic analysis demonstrate that CTX III induced the loss of mitochondrial membrane potential (Dym), cytochrome c release from mitochondria into the cytosol and activation of capase-9, caspase 3, as well as markedly enhancing the expression of Bax, but not Bcl-2, protein in the cells. Moreover, the CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. 4. However, CTX III did not generate the formation of reactive oxygen species and anti-oxidants, including N-acetylcysteine, and catalase could not block CTX III-induced apoptosis in the Colo205 cells. 5. Taken together, these results suggest that CTX III may induce apoptosis through a mitochondrial- and caspase-dependent mechanism and alteration of Bax/Bcl-2 ratio in human colorectal Colo205 cancer cells. Show less

The apolipoprotein A5 gene (APOA5 ) has been shown to play an important role in determining plasma triglyceride concentrations in humans. We describe here a novel variant, c.553G>T, in the apolipoprotein A5 gene that is associated with hypertriglyceridemia. In contrast to some other polymorphisms, which occur in non-coding regions of the gene, this variant occurs within the coding region and causes the change of amino acid sequence (a substitution of a cysteine for a glycine residue). The minor allele frequencies were 0.042 and 0.27 (P<0.001) for control and hypertriglyceridemic patients, respectively. The serum triglyceride level was significantly different among the genotypic groups (G/G 92.5+/-37.8 mg/dl, G/T 106.6+/-34.8 mg/dl, T/T 183.0 mg/dl, P=0.014) in control subjects. Multiple logistic regression revealed individuals carrying the minor allele had age, gender and BMI (body mass index)-adjusted odds ratio of 11.73 (95% confidence interval of 6.617-20.793; P<0.0001) for developing hypertriglyceridemia in comparison to individuals without that allele. These findings suggest the possible use of c.553G>T polymorphisms in APOA5 as prognostic indicators for hypertriglyceridemia susceptibility in Chinese. Show less