Insulin Determines Transforming Growth Factor ß Effects on Hepatocyte Nuclear Factor 4α Transcription in Hepatocytes.

Loss of hepatocyte nuclear factor 4α (HNF4α) expression is frequently observed in end-stage liver disease and associated with loss of vital liver functions, thus increasing mortality. Loss of HNF4α expression is mediated by inflammatory cytokines, such as transforming growth factor (TGF)-ß. However, details of how HNF4α is suppressed are largely unknown to date. Herein, TGF-ß did not directly inhibit HNF4α but contributed to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CREB-binding protein/p300 to the HNF4α promoter. The recruitment of CREB-binding protein/p300 is indispensable for CCAAT/enhancer-binding protein α (C/EBPα) binding, another essential requirement for constitutive HNF4α expression in hepatocytes. Consistent with the in vitro observation, 67 of 98 patients with hepatic HNF4α expressed both phospho-SMAD2 and C/EBPα, whereas 22 patients without HNF4α expression lacked either phospho-SMAD2 or C/EBPα. In contrast to the observed induction of HNF4α, SMAD2/3 inhibited C/EBPα transcription. Long-term TGF-ß incubation resulted in C/EBPα depletion, which abrogated HNF4α expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBPα promoter was abolished by insulin. Two-thirds of patients without C/EBPα lacked membrane glucose transporter type 2 expression in hepatocytes, indicating insulin resistance. Taken together, these data indicate that hepatic insulin sensitivity is essential for hepatic HNF4α expression in the condition of inflammation.

Comparison of Tumour-Specific Phenotypes in Human Primary and Expandable Pancreatic Cancer Cell Lines.

There is an ongoing need for patient-specific chemotherapy for pancreatic cancer. Tumour cells isolated from human tissues can be used to predict patients' response to chemotherapy. However, the isolation and maintenance of pancreatic cancer cells is challenging because these cells become highly vulnerable after losing the tumour microenvironment. Therefore, we investigated whether the cells retained their original characteristics after lentiviral transfection and expansion. Three human primary pancreatic cancer cell lines were lentivirally transduced to create expandable (Ex) cells which were then compared with primary (Pri) cells. No obvious differences in the morphology or epithelial-mesenchymal transition (EMT) were observed between the primary and expandable cell lines. The two expandable cell lines showed higher proliferation rates in the 2D and 3D models. All three expandable cell lines showed attenuated migratory ability. Differences in gene expression between primary and expandable cell lines were then compared using RNA-Seq data. Potential target drugs were predicted by differentially expressed genes (DEGs), and differentially expressed pathways (DEPs) related to tumour-specific characteristics such as proliferation, migration, EMT, drug resistance, and reactive oxygen species (ROS) were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We found that the two expandable cell lines expressed similar chemosensitivity and redox-regulatory capability to gemcitabine and oxaliplatin in the 2D model as compared to their counterparts. In conclusion, we successfully generated expandable primary pancreatic cancer cell lines using lentiviral transduction. These expandable cells not only retain some tumour-specific biological traits of primary cells but also show an ongoing proliferative capacity, thereby yielding sufficient material for drug response assays, which may provide a patient-specific platform for chemotherapy drug screening.

Association between Cystatin C and Cardiac Function in Acute Myocardial Infarction Patients: A Real-World Analysis.

Background: Acute myocardial infarction (AMI), as well as its long-term and short-term complications, is known to present with high morbidity and mortality. Cardiac function deterioration and ventricular remodelling after AMI are known to be correlated to worse long-term outcomes. However, the underlying mechanism remains elusive and there is a shortage of serum prediction markers. This study investigates the relationship between in-hospital Cystatin C (CysC) and cardiac function and subsequent prognosis among AMI patients. Research Design and Methods. We measured admission CysC and cardiac function parameters, including ejection fraction (EF) and pro-BNP value in 5956 patients diagnosed with AMI. Simple and multiregression analyses were performed to investigate the correlation between CysC and cardiac function in AMI patients. Major adverse cardiovascular events (MACE), cardiovascular, and all-cause mortality were documented, and 351 participants with high cystatin (≥1.09 mg/L) and 714 low cystatin (<1.09 mg/L) were investigated for survival analysis during a 48-month follow-up. Results: 5956 patients with AMI were enrolled in the initial observational analysis, and 1065 patients of the whole cohort were included in the follow-up survival analysis. The admission CysC level was found to be significantly positively correlated to the pro-BNP level (R square = 0.2142, 95% CI 4758 to 5265, p < 0.0001) and negatively correlated to the EF value (R square = 0.0095, 95% CI -3.503 to -1.605, p < 0.0001). Kaplan-Meier survival analysis revealed significantly increased MACE incidence (HR = 2.293, 95% CI 1.400 to 3.755, p < 0.0001), cardiovascular mortality (HR = 3.016, 95% CI 1.694 to 5.371, p = 0.0002), and all-cause mortality (HR = 3.424, 95% CI 2.010 to 5.835, p < 0.0001) in high-admission CysC cohort with AMI at the end of 4-year follow-up. Conclusions: Admission CysC is negatively correlated with cardiac function in AMI patients and acts as a novel predictor for MACE incidence in the whole population. Further studies are needed to investigate the specific mechanism of CysC in the cardiac function deterioration among AMI patients.

Identification of the Key Genes and Potential Therapeutic Compounds for Abdominal Aortic Aneurysm Based on a Weighted Correlation Network Analysis.

BACKGROUND: There is still an unmet need for therapeutic drugs for patients with an abdominal aortic aneurysm (AAA), especially for candidates unsuitable for surgical or interventional repair. Therefore, the purpose of this in silico study is to identify significant genes and regulatory mechanisms in AAA patients to predicate the potential therapeutic compounds for significant genes. METHODS: The GSE57691 dataset was obtained from Gene Expression Omnibus (GEO) and used to identify the differentially expressed genes (DEGs) and weighted correlation network analysis (WGCNA). The biological function of DEGs was determined using gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). AAA-related genes were obtained from the Comparative Toxicogenomics Database (CTD) using the keywords: aortic aneurysm and abdominal. The hub genes in AAA were obtained by overlapping DEGs, WGCNA-based hub genes, and CTD-based genes. The diagnostic values of hub genes were determined using ROC curve analysis. Hereby, a TF-miRNA-hub gene network was constructed based on the miRnet database. Using these data, potential therapeutic compounds for the therapy of AAA were predicted based on the Drug Gene Interaction Database (DGIdb). RESULTS: A total of 218 DEGs (17 upregulated and 201 downregulated) and their biological function were explored; 4093 AAA-related genes were derived by text mining. Three hub modules and 144 hub genes were identified by WGCNA. asparagine synthetase (ASNS), axin-related protein 2 (AXIN2), melanoma cell adhesion molecule (MCAM), and the testis-specific Y-encoded-like protein 1 (TSPYL1) were obtained as intersecting hub genes and the diagnostic values were confirmed with ROC curves. As potential compounds targeting the hub genes, asparaginase was identified as the target compound for ASNS. Prednisolone and abiraterone were identified as compounds targeting TSPYL1. For MCAM and TSPYL1, no potential therapeutic compound could be predicted. CONCLUSION: Using WGCNA analysis and text mining, pre-existing gene expression data were used to provide novel insight into potential AAA-related protein targets. For two of these targets, compounds could be predicted.

MicroRNAs Influence the Migratory Ability of Human Umbilical Vein Endothelial Cells.

To identify miRNAs that are involved in cell migration in human umbilical vein endothelial cells (HUVECs), we employed RNA sequencing under high glucose incubation and text mining within the databases miRWalk and TargetScanHuman using 83 genes that regulate HUVECs migration. From both databases, 307 predicted miRNAs were retrieved. Differentially expressed miRNAs were determined by exposing HUVECs to high glucose stimulation, which significantly inhibited the migratory ability of HUVECs as compared to cells cultured in normal glucose. A total of 35 miRNAs were found as differently expressed miRNAs in miRNA sequencing, and 4 miRNAs, namely miR-21-3p, miR-107, miR-143-3p, and miR-106b-5p, were identified as overlapping hits. These were subjected to hub gene analysis and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG), identifing 71 pathways which were influenced by all four miRNAs. The influence of all four miRNAs on HUVEC migration was phenomorphologically confirmed. miR21 and miR107 promoted migration in HUVECs while miR106b and miR143 inhibited migration. Pathway analysis also revealed eight shared pathways between the four miRNAs. Protein-protein interaction (PPI) network analysis was then performed to predict the functionality of interacting genes or proteins. This revealed six hub genes which could firstly be predicted to be related to HUVEC migration.

High-content analysis of microRNAs involved in the phenotype regulation of vascular smooth muscle cells.

In response to vascular injury vascular smooth muscle cells (VSMCs) alternate between a differentiated (contractile) and a dedifferentiated (synthetic) state or phenotype. Although parts of the signaling cascade regulating the phenotypic switch have been described, the role of miRNAs is still incompletely understood. To systematically address this issue, we have established a microscopy-based quantitative assay and identified 23 miRNAs that induced contractile phenotypes when over-expressed. These were then correlated to miRNAs identified from RNA-sequencing when comparing cells in the contractile and synthetic states. Using both approaches, six miRNAs (miR-132-3p, miR-138-5p, miR-141-3p, miR-145-5p, miR-150-5p, and miR-22-3p) were filtered as candidates that induce the phenotypic switch from synthetic to contractile. To identify potentially common regulatory mechanisms of these six miRNAs, their predicted targets were compared with five miRNAs sharing ZBTB20, ZNF704, and EIF4EBP2 as common potential targets and four miRNAs sharing 16 common potential targets. The interaction network consisting of these 19 targets and additional 18 hub targets were created to facilitate validation of miRNA-mRNA interactions by suggesting the most plausible pairs. Furthermore, the information on drug candidates was integrated into the network to predict novel combinatorial therapies that encompass the complexity of miRNAs-mediated regulation. This is the first study that combines a phenotypic screening approach with RNA sequencing and bioinformatics to systematically identify miRNA-mediated pathways and to detect potential drug candidates to positively influence the phenotypic switch of VSMCs.

A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.

BACKGROUND AND AIMS: It remains unknown how patients with liver failure maintain essential albumin levels. Here, we delineate a hierarchical transcription regulatory network that ensures albumin expression under different disease conditions. APPROACH AND RESULTS: We examined albumin levels in liver tissues and serum in 157 patients, including 84 with HCC, 38 decompensated cirrhosis, and 35 acute liver failure. Even in patients with liver failure, the average serum albumin concentrations were 30.55 g/L. In healthy subjects and patients with chronic liver diseases, albumin was expressed in hepatocytes. In patients with massive hepatocyte loss, albumin was expressed in liver progenitor cells (LPCs). The albumin gene (ALB) core promoter possesses a TATA box and nucleosome-free area, which allows constitutive RNA polymerase II binding and transcription initiation. Chromatin immunoprecipitation assays revealed that hepatocyte nuclear factor 4 alpha (HNF4α), CCAAT/enhancer-binding protein alpha (C/EBPα), and forkhead box A2 (FOXA2) bound to the ALB enhancer. Knockdown of either of these factors reduced albumin expression in hepatocytes. FOXA2 acts as a pioneer factor to support HNF4α and C/EBPα. In hepatocytes lacking HNF4α and C/EBPα expression, FOXA2 synergized with retinoic acid receptor (RAR) to maintain albumin transcription. RAR nuclear translocation was induced by retinoic acids released by activated HSCs. In patients with massive hepatocyte loss, LPCs expressed HNF4α and FOXA2. RNA sequencing and quantitative PCR analyses revealed that lack of HNF4α and C/EBPα in hepatocytes increased hedgehog ligand biosynthesis. Hedgehog up-regulates FOXA2 expression through glioblastoma family zinc finger 2 binding to the FOXA2 promoter in both hepatocytes and LPCs. CONCLUSIONS: A hierarchical regulatory network formed by master and pioneer transcription factors ensures essential albumin expression in various pathophysiological conditions.

Acrolein-inducing ferroptosis contributes to impaired peripheral neurogenesis in zebrafish.

Introduction: Diabetes mellitus (DM) is associated with physiological disorders such as delayed wound healing, diabetic retinopathy, diabetic nephropathy, and diabetic peripheral neuropathy (DPN). Over 50% of diabetic patients will develop DPN, characterized by motor dysfunction and impaired sensory nerve function. In a previous study, we have uncovered acrolein (ACR) as an upstream initiator which induced impaired glucose homeostasis and microvascular alterations in zebrafish. Whether ACR has specific effects on peripheral neurogenesis and mediates DPN, is still waiting for clarification. Methods: To evaluate the function of ACR in peripheral nerve development, in vivo experiments were performed in Tg(hb9:GFP) zebrafish. In addition, a series of rescue experiments, metabolomics assessment, and bioinformatics analysis was performed aimed at identifying the molecular mechanisms behind ACR's function and impaired neurogenesis. Results: Impaired motor neuron development was confirmed in wild-type embryos treated with external ACR. ACR treated embryos displayed ferroptosis and reduction of several amino acids and increased glutathione (GSH). Furthermore, ferroptosis inducer caused similarly suppressed neurogenesis in zebrafish embryos, while anti-ACR treatment or ferroptosis inhibitor could successfully reverse the detrimental phenotypes of ACR on neurogenesis in zebrafish. Discussion: Our data indicate that ACR could directly activate ferroptosis and impairs peripheral neurogenesis. The data strongly suggest ACR and activated ferroptosis as inducers and promising therapeutic targets for future DPN studies.

Biphasic Effects of Blue Light Irradiation on Human Umbilical Vein Endothelial Cells.

Blue light regulates biological function in various cells, such as proliferation, oxidative stress, and cell death. We employed blue light illumination on human umbilical vein endothelial cells utilizing a LED device at 453 nm wavelength and revealed a novel biphasic response on human umbilical vein endothelial cells (HUVECs). The results showed that low fluence blue light irradiation promoted the fundamental cell activities, including cell viability, migration and angiogenesis by activating the angiogenic pathways such as the VEGF signaling pathway. In contrast, high fluence illumination caused the opposite effect on those activities by upregulating pro-apoptotic signaling cascades like ferroptosis, necroptosis and the p53 signaling pathways. Our results provide an underlying insight into photobiomodulation by blue light and may help to implement potential treatment strategies for treating angiogenesis-dependent diseases.

Weighted Gene Co-Expression Network Analysis Reveals Key Genes and Potential Drugs in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a prevalent aortic disease that causes high mortality due to asymptomatic gradual expansion and sudden rupture. The underlying molecular mechanisms and effective pharmaceutical therapy for preventing AAA progression have not been fully identified. In this study, we identified the key modules and hub genes involved in AAA growth from the GSE17901 dataset in the Gene Expression Omnibus (GEO) database through the weighted gene co-expression network analysis (WGCNA). Key genes were further selected and validated in the mouse dataset (GSE12591) and human datasets (GSE7084, GSE47472, and GSE57691). Finally, we predicted drug candidates targeting key genes using the Drug-Gene Interaction database. Overall, we identified key modules enriched in the mitotic cell cycle, GTPase activity, and several metabolic processes. Seven key genes (CCR5, ADCY5, ADCY3, ACACB, LPIN1, ACSL1, UCP3) related to AAA progression were identified. A total of 35 drugs/compounds targeting the key genes were predicted, which may have the potential to prevent AAA progression.

Loss of GRB2 associated binding protein 1 in arteriosclerosis obliterans promotes host autophagy.

BACKGROUND: Arteriosclerosis obliterans (ASO) is a major cause of adult limb loss worldwide. Autophagy of vascular endothelial cell (VEC) contributes to the ASO progression. However, the molecular mechanism that controls VEC autophagy remains unclear. In this study, we aimed to explore the role of the GRB2 associated binding protein 1 (GAB1) in regulating VEC autophagy. METHODS: In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression. Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima. Gain- and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1. RESULTS: The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor (0.80 vs. 0.20, t = 6.43, P < 0.05). The expression level of GAB1 mRNA (1.00 vs. 0.24, t = 7.41, P < 0.05) and protein (0.72 vs. 0.21, t = 5.97, P < 0.05) was significantly decreased in ASO group as compared with the control group. Loss of GAB1 led to a remarkable decrease in LC3II (1.19 vs. 0.68, t = 5.99, P < 0.05), whereas overexpression of GAB1 significantly led to a decrease in LC3II level (0.41 vs. 0.93, t = 7.12, P < 0.05). Phosphorylation levels of JNK and p38 were significantly associated with gain- and loss-of-function of GAB1 protein. CONCLUSION: Loss of GAB1 promotes VEC autophagy which is associated with ASO. GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.

GAS2L1 Is a Potential Biomarker of Circulating Tumor Cells in Pancreatic Cancer.

Pancreatic cancer is a malignant disease with high mortality and a dismal prognosis. Circulating tumor cell (CTC) detection and characterization have emerged as essential techniques for early detection, prognostication, and liquid biopsy in many solid malignancies. Unfortunately, due to the low EPCAM expression in pancreatic cancer CTCs, no specific marker is available to identify and isolate this rare cell population. This study analyzed single-cell RNA sequencing profiles of pancreatic CTCs from a genetically engineered mouse model (GEMM) and pancreatic cancer patients. Through dimensionality reduction analysis, murine pancreatic CTCs were grouped into three clusters with different biological functions. CLIC4 and GAS2L1 were shown to be overexpressed in pancreatic CTCs in comparison with peripheral blood mononuclear cells (PBMCs). Further analyses of PBMCs and RNA-sequencing datasets of enriched pancreatic CTCs were used to validate the overexpression of GAS2L1 in pancreatic CTCs. A combinatorial approach using both GAS2L1 and EPCAM expression leads to an increased detection rate of CTCs in PDAC in both GEMM and patient samples. GAS2L1 is thus proposed as a novel biomarker of pancreatic cancer CTCs.

Left innominate vein stenosis in an asymptomatic population: a retrospective analysis of 212 cases.

BACKGROUND: Although left innominate vein (LIV) stenosis has been demonstrated to be attributed to compression by adjacent anatomical structures, most of the studies are focusing on hemodialysis patients with clinical symptoms compatible with LIV stenosis. The goal of this study was to retrospectively investigate the incidence of LIV stenosis and its influencing factors in an asymptomatic, non-hemodialysis population, which has rarely been performed. METHODS: From Jan 2013 to Dec 2014, 212 consecutive cases undergoing a chest multi-detector computed tomography (MDCT) angiography were enrolled. LIV stenosis was defined as loss of the area of the LIV (that is, 1 - compression degree) >25%. Multivariate logistic regression analysis was performed to explore the independent risk factors associated with LIV stenosis. RESULTS: LIV stenosis occurred in 35.4% of cases (75/212), with the median loss of the area of the LIV of 36.2% (interquartile range 30.2-49.8%). There were significant differences in age (62.5 ± 11.7 vs. 58.6 ± 14.3 years; P = 0.041), BMI (23.9 ± 2.9 vs. 23.0 ± 3.3, P = 0.036), the frequency of crossing site of LIV over the origin of the aortic arch (54.7 vs. 24.8%, P < 0.001), and the space between aortic arch and sternum [mean ± SD, 11.6 ± 4.2 mm vs. median, 14.1 (interquartile range 11.9-16.3) mm, P < 0.001] between patients with and without LIV stenosis, but only the latter two were confirmed as independent factors by the multivariate logistic regression analysis [crossing site of LIV over the aortic arch, OR (95% CI) = 2.632 (1.401, 4.944), P = 0.003; space between the aortic arch and sternum, OR (95% CI) = 0.841 (0.770, 0.919), P < 0.001]. CONCLUSION: The patients with an older age, high BMI, LIV crossing over the origin of the aortic arch, or smaller space between aortic arch and sternum may have high risks for LIV stenosis. They should be paid more attention to exclude LIV stenosis preoperatively using MDCT angiography to prevent venous access dysfunction and symptomatic development by fistula creation when hemodialysis is required.

Neutrophil Extracellular Traps Promote Hypercoagulability in Patients With Sepsis.

BACKGROUND: Patients with sepsis commonly exhibit a hypercoagulability with high risk of venous thromboembolism (VTE). Neutrophil extracellular traps (NETs) are found to trigger inflammation and coagulation. We aim to determine whether NETs promoted the hypercoagulability and early anticoagulation reduced NETs releasing during sepsis. METHODS: In this prospective study, septic patients between September 2013 and June 2015 were included. Patients of age <18 years, acute organ failure, pregnancy, coagulation disorders, receiving anticoagulation before admission were excluded. Blood was sampled in 52 sepsis and 10 non-sepsis patients and 40 healthy controls, clinical, and hematological parameters were collected. The ability of plasma and platelets to prime neutrophils to release NETs and contribution of NETs to coagulation were assessed. NETs releasing was compared in patients with or without early coagulation, and its correlation with the risk of VTE was also evaluated. RESULTS: NETs formation in septic patients was significantly higher than controls and non-sepsis patients. Neutrophils from septic patients had significantly enhanced NETs releasing compared with those from controls or non-sepsis patients. Plasma or platelets obtained from patients induced control neutrophils to release NETs. Notably, NETs released by neutrophils from septic patients significantly increased the potency of control plasma to generate thrombin and fibrin, and this effect was attenuated by administration of DNase I. Post-treatment NETs releasing in septic patients receiving early anticoagulation within 6 h was significantly lower than patients without early anticoagulation. The NETs formation correlated positively with the VTE risk, rather than the parameters of inflammation or disease severity. CONCLUSIONS: The systemic inflammation during sepsis primes neutrophils to release NETs with increased risk of VTE. Early anticoagulation (6 h) reduces NETs releasing and may improve the coagulopathy of septic patients.