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Background: To investigate the risk factors for enterococcal intra-abdominal infections (EIAIs) and the association between EIAIs and outcomes in intensive care unit (ICU) patients. Methods: We rev...

Endothelial hyperpermeability is the initial event in the development of diabetic microvascular complications, and advanced glycation end products (AGEs) are suggested to cause much of the endothelial hyperpermeability associated with diabetes mellitus, but the molecular mechanism remains to be characterized. β‐catenin reportedly plays dual functions in maintaining normal endothelial permeability by serving both as an adhesive component and a signal transduction component. Here, we found that AGEs induced the phosphorylation of β‐catenin at residues Y654 and Y142 and the endothelial hyperpermeability was reversed when the two residues were blocked. In mechanism, phosphorylation of Y654 was blocked by Src inactivation, whereas phosphorylation of Y142 was reduced by a focal adhesion kinase inhibitor. β‐catenin Y654 phosphorylation induced by AGEs facilitated the dissociation of vascular endothelial (VE)‐cadherin/β‐catenin and the impairment of adherens junctions (AJs), whereas β‐catenin Y142 phosphorylation favoured the dissociation of β‐catenin and α‐catenin. Further investigation revealed that β‐catenin Y142 phosphorylation was required for AGEs‐mediated β‐catenin nuclear translocation, and this nuclear‐located β‐catenin subsequently activated the TCF/LEF pathway. This pathway promotes the transcription of the Wnt target, ADAM10 (a disintegrin and metalloprotease 10), which mediates VE‐cadherin shedding and leads to further impairment of AJs. In summary, our study showed the role of β‐catenin Y654 and Y142 phosphorylation in AGEs‐mediated endothelial hyperpermeability through VE‐cadherin/β‐catenin/α‐catenin dissociation and up‐regulation of ADAM10, thereby advancing our understanding of the underlying mechanisms of AGEs‐induced microvascular hyperpermeability.

Background Sepsis-associated acute kidney injury (SA-AKI) is a common problem in critically ill patients and is associated with high morbidity and mortality. Early prediction of the survival of hospitalized patients with SA-AKI is necessary, but a reliable and valid prediction model is still lacking. Methods We conducted a retrospective cohort analysis based on a training cohort of 2066 patients enrolled from the Multiparameter Intelligent Monitoring in Intensive Care Database III (MIMIC III) and a validation cohort of 102 patients treated at Nanfang Hospital of Southern Medical University. Least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analysis were used to identify predictors for survival. Areas under the ROC curves (AUC), the concordance index (C-index) and calibration curves were used to evaluate the efficiency of the prediction model (SAKI) in both cohorts. Results The overall mortality of SA-AKI was approximately 18%. Age, admission type, liver disease, metastatic cancer, lactate, BUN/SCr, admission creatinine, positive culture and AKI stage were independently associated with survival and combined in the SAKI model. The C-index in the training and validation cohorts was 0.73 and 0.72. The AUC in the training cohort was 0.77, 0.72, and 0.70 for the 7-day, 14-day and 28-day probability of in-hospital survival, respectively, while in the external validation cohort, it was 0.83, 0.73 and 0.67. SAPSII and SOFA scores showed poorer performance. Calibration curves demonstrated a good consistency. Conclusions Our SAKI model has predictive value for in-hospital mortality of SA-AKI in critically ill patients and outperforms generic scores.

The effect of long intergenic non-protein coding RNAs (lncRNAs) was verified in prostate cancer (PCa), but the mechanism of LINC01146 in PCa is unclear. Bioinformatics was applied to analyze LINC01146 expression in PCa and predict target genes of LINC01146, followed by the verification of qRT-PCR, RNA pull-down and co-immunoprecipitation (Co-IP). The correlation between LINC01146 expression and clinicopathological characteristics was investigated. The location of LINC01146 in PCa cells was detected by fluorescence in situ hybridization (FISH). After interference with LINC01146 or/and F11 receptor (F11R) or treated with transforming growth factor beta 1 (TGF-β1), the function of LINC01146 in PCa in vitro or in vivo was determined by CCK-8, colony formation, flow cytometry, scratch test, transwell assay, xenograft experiment and western blot. LINC01146 and F11R were over-expressed in PCa and positively correlated with poor prognosis. LINC01146 located in the cytoplasm and combined with F11R. LINC01146 overexpression impeded apoptosis, facilitated viability, proliferation, migration and invasion in PCa cells in vitro, promoted tumor growth in vivo, downregulated E-cadherin, Bax and Cleaved caspase-3, and upregulated N-cadherin, Vimentin and PCNA, but LINC01146 silencing did the opposite. F11R was positively regulated by LINC01146 and F11R depletion negated the effect of LINC01146 overexpression on malignant phenotypes of PCa cells. The expression of LINC01146 and F11R was regulated by TGF-β1. The promoting role of TGF-β1 in migration, invasion and F11R in PCa cells was reversed by LINC01146 silencing. LINC01146 upregulated F11R to facilitate malignant phenotypes of PCa cells, which was regulated by TGF-β.

This study aimed to assess the gastric emptying capacity in nondiabetic patients with end-stage kidney disease (ESKD) by ultrasound.Consecutive hemodialysis patients with ESKD (Compared with the controls, patients with ESKD, on both dialysis and non-dialysis days, had significantly larger antral areas when examined in the supine position (Nondiabetic patients with ESKD had significantly delayed gastric emptying. Hemodialysis might improve gastric emptying and reduce gastric emptying delay.

Transthyretin (TTR) is associated with several human amyloid diseases. Various kinetic stabilizers have been developed to inhibit the dissociation of TTR tetramer and the formation of amyloid fibrils. Most of them are bisaryl derivatives, natural flavonoids, crown ethers and carborans. In this review article, we focus on TTR tetramer stabilizers, genetic therapeutic approaches and fibril remodelers. The binding modes of typical bisaryl derivatives, natural flavonoids, crown ethers and carborans are discussed. Based on knowledge of the binding of thyroxine to TTR tetramer, many stabilizers have been screened to dock into the thyroxine binding sites, leading to TTR tetramer stabilization. Particularly, those stabilizers with unique binding profiles have shown great potential in developing the therapeutic management of TTR amyloidogenesis.

The digestive tract is replete with complex and diverse microbial communities that are important for the regulation of multiple pathophysiological processes in humans and animals, particularly those involved in the maintenance of intestinal homeostasis, immunity, inflammation, and tumorigenesis. The diversity of bile acids is a result of the joint efforts of host and intestinal microflora. There is a bidirectional relationship between the microbial community of the intestinal tract and bile acids in that, while the microbial flora tightly modulates the metabolism and synthesis of bile acids, the bile acid pool and composition affect the diversity and the homeostasis of the intestinal flora. Homeostatic imbalances of bile acid and intestinal flora systems may lead to the development of a variety of diseases, such as inflammatory bowel disease (IBD), colorectal cancer (CRC), hepatocellular carcinoma (HCC), type 2 diabetes (T2DM), and polycystic ovary syndrome (PCOS). The interactions between bile acids and intestinal flora may be (in)directly involved in the pathogenesis of these diseases.

The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.

Ropivacaine, one of the most commonly used local anesthetics in clinical practice, has shown potent antitumor activity in multiple types of cancer cells. However, its effect on cervical cancer cell growth remains unknown. In the present study, it was found that ropivacaine inhibited cervical cancer cell growth by suppressing cell cycle progression and promoting cell apoptosis, as determined by CCK‑8 assay, cell cycle and apoptosis analyses. Western blot analysis and luciferase assay demonstrated that ropivacaine abrogated the phosphorylation and transcriptional activation of signal transducer and activator of transcription 3 (STAT3), and that STAT‑3C overexpression reversed the inhibition of cervical cancer cell viability mediated by ropivacaine. Furthermore, our results revealed that the increased expression of maternally expressed gene 2 (MEG2) caused by ropivacaine led to STAT3 dephosphorylation. Finally, we found that ropivacaine upregulated MEG2 by decreasing the expression of microRNA‑96 (miR‑96). Taken together, our results describe a novel mechanism for the anticancer activity of ropivacaine and suggest ropivacaine as a potential therapeutic agent for cervical cancer patients.

Endoplasmic reticulum (ER) stress-induced endothelial cell (EC) apoptosis has been implicated in a variety of human diseases. In addition to being regarded as an NADPH oxidase (NOX) inhibitor, apocynin (APO) exhibits an anti-apoptotic effect in various cells. The present study aimed to identify the protective role of apocynin in ER stress-mediated EC apoptosis and the underlying mechanisms. We found that ER stress resulted in a significant increase in c-Jun N-terminal kinase phosphorylation, and elicited caspase 3 cleavage and apoptosis. However, apocynin obviously attenuated EC apoptosis and this effect was partly dependent on ER stress sensor inositol-requiring enzyme 1α (IRE1α). Importantly, apocynin upregulated IRE1α expression in both protein and mRNA levels and promoted the pro-survival XBP1 splicing. Our results suggest that apocynin protects ECs against ER stress-induced apoptosis via IRE1α involvement. These findings may provide a novel mechanistic explanation for the anti-apoptotic effect of apocynin in ER stress.

A natural ursolic compound, 2α,3β-dihydroxy-urs-12-en-28-oic acid (corosolic acid, CRA) was isolated from the root of Actinidia valvata Dunn. (A. valvata Radix). Since a large number of triterpenoid compound has marked anticancer effects toward various types of cancer cell lines in vitro, this study was carried out to investigate the anticancer effect of CRA in human gastric cancer cell line BGC823 cells and the underlying apoptotic mechanism of CRA was examined in BGC823 cell lines. The results showed that CRA significantly suppressed the viability of BGC823 cells in a concentration- and time-dependent manner. CRA also significantly increased the sub G1 population by cell cycle analysis in a concentration dependent manner. Exposure to CRA decreased p65, bcl-2, Fas, smac mRNA and protein expression, and increased IκBα, bax, survivin mRNA and protein expression. Results of immunofluorescence staining and EMSA further indicated CRA induced apoptosis by inhibiting nuclear translocation of nuclear factor NF-κB subunit p65. Consistently overall, our findings suggest that CRA induces apoptosis via inhibition of NF-κB (p65) expression level and activation of IκBα in BGC cells as a potent anticancer candidate for gastric cancer treatment.

ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.Methods and resultsUsing intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition.ConclusionThe FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

This study aimed to investigate the efficacy, safety, and prognostic factors of drug-eluting beads transarterial chemoembolization (DEB-TACE) in treating Chinese patients with liver cancer. A total of 367 liver cancer patients from 24 medical centers were consecutively enrolled in this multiple-center, prospective cohort study, including 275 hepatocellular carcinoma (HCC) cases, 37 intrahepatic cholangiocarcinoma (ICC) cases, and 55 secondary liver cancer cases. All the patients received CalliSpheres® DEB-TACE treatment. Treatment response, overall survival (OS), change of liver function, and adverse events (AEs) were assessed. DEB-TACE treatment achieved 19.9% complete response (CR) and 79.6% objective response rate (ORR), with mean OS of 384 days [95% confidence interval (CI): 375–393 days]. CR and ORR were both higher in HCC patients compared with primary ICC patients and secondary liver cancer patients, while no difference was discovered in OS. Portal vein invasion was an independent risk factor for CR, while portal vein invasion, previous conventional TACE (cTACE) treatment, and abnormal blood creatinine (BCr) were independent risk factors for ORR. In addition, largest nodule size ≥5.0 cm, abnormal albumin (ALB), and abnormal total bilirubin (TBIL) independently correlated with unfavorable OS. Most liver function indexes were recovered to baseline levels at 1–3 months after DEB-TACE. Common AEs were pain, fever, vomiting, and nausea; most of them were at mild grade. CalliSpheres® DEB-TACE is efficient and well tolerated in Chinese liver cancer patients. Portal vein invasion, previous cTACE treatment, largest nodule size, abnormal BCr, ALB, and TBIL correlate with worse prognosis independently.

Idiopathic renal hypouricemia is a rare hereditary condition. Type 2 renal hyperuricemia (RHUC2) is caused by a mutation in the SLC2A9 gene, which encodes a high-capacity glucose and urate transporter, glucose transporter (GLUT)9. RHUC2 predisposes to exercise-induced acute renal failure (EIARF) and nephrolithiasis, which is caused by a defect in renal tubular urate transport and is characterized by increased clearance of renal uric acid. In the present study a case of a 35-year-old Chinese man with EIARF is reported. The patient had isolated renal hypouricemia, with a serum uric acid level of 21 µmol/l and a fractional excretion of uric acid of 200%. The mutational analysis revealed a homozygous mutation (c.857G>A in exon 8) in the SLC2A9 gene. The patient's family members carried the same mutation, but were heterozygous and clinically asymptomatic. In conclusion, to the best of our knowledge, this is the first report of a RHUC2 patient with a GLUT9 mutation, p.W286X, which may be a pathogenic mutation of RHUC2. Further investigation into the functional role of GLUT9 in this novel SLC2A9 mutation is required.

The purpose of this study was to investigate the efficacy and safety of drug-eluting beads transarterial chemoembolization (DEB-TACE) treatment in Chinese hepatocellular carcinoma (HCC) patients and the prognostic factors for treatment response as well as survival. A total of 275 HCC patients were included in this prospective study. Treatment response was assessed by modified Response Evaluation Criteria in Solid Tumors (mRECIST), and progression-free survival (PFS) as well as overall survival (OS) were determined. Liver function and adverse events (AEs) were assessed before and after DEB-TACE operation. Complete response (CR), partial response (PR), and objective response rate (ORR) were 22.9%, 60.7%, and 83.6%, respectively. The mean PFS was 362 (95% CI: 34.9–375) days, the 6-month PFS rate was 89.4 ± 2.1%, while the mean OS was 380 (95% CI: 370–389) days, and the 6-month OS rate was 94.4 ± 1.7%. Multivariate logistic regression revealed that portal vein invasion (p = 0.011) was an independent predictor of worse clinical response. Portal vein invasion (p = 0.040), previous cTACE treatment (p = 0.030), as well as abnormal serum creatinine level (BCr) (p = 0.017) were independent factors that predicted worse ORR. In terms of survival, higher Barcelona Clinic Liver Cancer (BCLC) stage (p = 0.029) predicted for worse PFS, and abnormal albumin (ALB) (p = 0.011) and total serum bilirubin (TBIL) (p = 0.009) predicted for worse OS. The number of patients with abnormal albumin, total protein (TP), TBIL, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were augmented at 1 week posttreatment and were similar at 1–3 months compared with baseline. The most common AEs were pain, fever, nausea, and vomiting, and no severe AEs were observed in this study. DEB-TACE was effective and tolerable in treating Chinese HCC patients, and portal vein invasion, previous cTACE treatment, abnormal BCr, ALB, and TBIL appear to be important factors that predict worse clinical outcome.

In a previous study, we demonstrated the role of polydatin (PD) in protecting against multiple organ dysfunction in sepsis. The aim of this study is to investigate whether PD protects against lipopolysaccharide (LPS)-induced endothelial barrier disruption through SIRT3 activation and to disclose the underlying mechanisms. Wild-type mice were injected with LPS and Evans Blue assay was performed to evaluate vascular permeability. Primary human umbilical vein endothelial cells (HUVECs) were stimulated with LPS. Endothelial permeability was evaluated by transendothelial electrical resistance (TER) and FITC-dextran leakage. SIRT3 activity was determined by a Deacetylase Fluorometric kit, and protein expression level of SIRT3 was detected by western blotting. Mitochondrial function was evaluated by determination of ROS level, mitochondrial membrane potential and mPTP opening. In endotoxemic mice, PD pretreatment attenuated vascular leakage in multiple organs while SIRT3 inhibition with 3-TYP reversed the effects of PD. PD treatment in late sepsis also exhibited barrier protective effects. In HUVECs, PD alleviated LPS-induced F-actin rearrangement, cadherin-catenin complex dissociation and endothelial hyperpermeability, whereas 3-TYP or SIRT3 siRNA attenuated the protective effects of PD. PD enhanced SIRT3 deacetylase activity, and attenuated LPS-induced decrease in SIRT3 expression as well. Furthermore, gain-of-function and loss-of-function strategies also confirmed the role of SIRT3 in enhancing endothelial barrier integrity. It was further ascertained that PD enhanced SIRT3-mediated deacetylation of SOD2 and cyclophilin D (CypD), thus suppressing mitochondrial dysfunction and subsequent endothelial barrier dysfunction. In addition, it was revealed that RAGE was involved in LPS-regulated SIRT3 signaling. Our results suggest that polydatin protects against LPS-induced endothelial barrier disruption dependent on SIRT3 and can be applied as a potential therapy for sepsis.

Recent studies have shown that activation of liver X receptors (LXRs) attenuates the development of atherosclerosis, not only by regulating lipid metabolism but also by suppressing inflammatory signaling. Sphingosine 1-phosphate receptor 2 (S1PR2), an important inflammatory gene product, plays a role in the development of various inflammatory diseases. It was proposed that S1PR2 might be regulated by LXR-α. In the present study, the effect of LXR-α on tumor necrosis factor-α (TNF-α)-induced S1PR2 expression in human umbilical vein endothelial cells (HUVECs) was investigated and the underlying mechanism was explored. The results demonstrated that TNF-α led to an increase in S1PR2 expression and triggered a downregulation of LXR-α expression in HUVECs as well. Downregulation of LXR-α with specific small interfering RNA (siRNA) remarkably enhanced the primary as well as TNF-α-induced expression of S1PR2 in HUVECs. Activation of LXR-α by agonist GW3965 inhibited both primary and TNF-α-induced S1PR2 expression. GW3965 also attenuated S1PR2-induced endothelial barrier dysfunction. The data further showed that TNF-α induced a significant decrease in miR-130a-3p expression. Overexpression of miR-130a-3p with mimic product reduced S1PR2 protein expression, and inhibition of miR-130a-3p by specific inhibitor resulted in an increase in S1PR2 protein expression. Furthermore, activation of LXRs with agonist enhanced the expression of miR-130a-3p, and knockdown of LXR-α by siRNA suppressed miR-130a-3p expression. These results suggest that LXR-α might downregulate S1PR2 expression via miR-130a-3p in quiescent HUVECs. Stimulation of TNF-α attenuates the activity of LXR-α and results in enhanced S1PR2 expression.

Background The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury. Actin cytoskeleton has been implicated to be critical in regulation of epithelial barrier. Here, we performed in vivo and in vitro study to investigate role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced ALI. Methods For in vivo studies, 6–8-week-old C57 mice were used, Bronchoalveolar lavage Fluid /Blood fluorescent ratio, wet-to-dry lung weight ratio, as well as protein concentrations and neutrophil cell counts in BALF were detected as either directly or indirectly indicators of pulmonary alveolar barrier dysfunction. And hematoxylin and eosin staining was performed to estimate pulmonary injury. The in vitro explorations of transepithelial permeability were achieved through transepithelial electrical resistance measurement and testing of FITC-Dextran transepithelial flux in A549. In addition, cytoskeletal rearrangement was tested through F-actin immunostaining. And SB203580 was used to inhibit p38 MAPK activation, while siRNA was administered to genetically knockdown specific protein. Results We showed that LPS triggered activation of p38 MAPK, rearrangement of cytoskeleton which resulted in severe epithelial hyperpermeability and lung edema. A549 pretreated with TLR4 siRNA、p38 MAPK siRNA and its inhibitor SB203580 displayed a lower permeability and fewer stress fibers formation after LPS stimulation, accompanied with lower phosphorylation level of p38 MAPK and Hsp27, which verified the involvement of TLR4-p38 MAPK-Hsp27 in LPS-evoked alveolar epithelial injury. Inhibition of p38 MAPK activity with SB203580 in vivo attenuated pulmonary edema formation and hyperpermeability in response to LPS. Conclusions Our study demonstrated that LPS increased alveolar epithelial permeability both in vitro and in vivo and that TLR4- p38 MAPK- Hsp27 signal pathway dependent actin remolding was involved in this process.

Objective Endothelial hyperpermeability is a hallmark of acute lung injury in response to sepsis. The imbalance between adherence junction (AJ) mediated cell-cell adherence forces and stress fiber driven contractile forces contributes to increased endothelial permeability. Here, we spotlight the effects of β-catenin Y654 andY142 phosphorylation on HMGB1-mediated endothelial barrier leakage. Approach and results Our results showed that phospho-deficiencies at both β-catenin Y654and Y142ameliorated pulmonary vascular dysfunction in male C57 mice receiving a cecal ligation and puncture operation. In vitro analysis indicated that high mobility group box-1 protein (HMGB1) triggered β-catenin Y654 and Y142 phosphorylation, causing β-catenin translocation and adherence junction (AJ) disruptions as well as cytoskeleton rearrangement. In addition,β-catenin Y654 dephosphorylation attenuated HMGB1-mediated dissociation of VE-cadherin/β-catenin and, hence, partially prevented endothelial hyperpermeability. β-catenin Y142 dephosphorylation abolished HMGB1-induced uncoupling of β-catenin and α-catenin, suppressed cytoskeletal reassembly and, hence, alleviated endothelial hyperpermeability. Further investigation demonstrated that RAGE and Src were required forβ-catenin Y654 phosphorylation in response to HMGB1, while FAK was responsible for HMGB1-triggered β-catenin Y142 phosphorylation. Conclusions In sum, this study revealed the role of β-catenin Y654 and Y142 phosphorylation in HMGB1-mediated endothelial hyperpermeability through dysregulation between adherence and contractile forces. This result advances understanding of the mechanisms underlying pulmonary vascular hyperpermeability in sepsis.

Background The association between body mass index (BMI) and clinical outcomes of gastric cancer were still under debate. The aim of the present study was to investigate the impact of BMI on intraoperative conditions, postoperative complications and prognosis of gastric cancer. Methods From October 2008 to March 2015, 1210 gastric cancer patients treated with D2 gastrectomy were enrolled in the present study. Patients were divided into three groups: low BMI group (BMI 0.05). High BMI was associated with increased blood loss and operation time, and deceased number of retrieved lymph nodes (all P 0.05). Conclusions BMI was inversely associated with tumor size, tumor depth, LNM and tumor stage. High BMI was associated with increased blood loss and operation time, and deceased number of retrieved lymph nodes. Low BMI was associated with decreased rate of postoperative fever and decreased survival.

We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen–activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product–induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase–targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product–induced myosin light chain phosphorylation. Advanced glycation end product–induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product–induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product–induced endothelial hyperpermeability.

It was previously reported that low‑dose sphingosine‑1‑phosphate (S1P) enhanced endothelial barrier integrity, whereas high‑dose S1P induced endothelial monolayer hyperpermeability responses. A number of studies have revealed the underlying molecular mechanisms of the physiological‑dose of S1P on barrier‑protective effect. However, little work has been performed to determine the effect of S1P‑induced endothelial barrier responses. In the present study, the role of moesin phosphorylation in excessive S1P‑induced endothelial hyperpermeability was investigated by western blotting, fluorescence staining and transendothelial electrical resistance detection. It was revealed that S1P induced moesin phosphorylation in a time‑ and concentration‑dependent manner. In addition, it was confirmed that high‑dose S1P‑induced moesin phosphorylation occurred via S1P receptor 2 (S1PR2). Moesin phosphorylation was required for S1P‑induced F‑actin rearrangement and endothelial barrier disruption. The results suggested that the S1PR2‑moesin axis is involved in high‑dose S1P‑induced endothelial barrier responses. The results of the present study may provide novel therapeutic targets for endothelial injury‑associated vascular disorders.

Background/Aims: Disruption of endothelial barrier integrity in response to advanced glycation end products (AEGs) stimulation contributes to vasculopathy associated with diabetes mellitus. Mammalian diaphanous-related formin (mDia1) has been reported to bind to the cytoplasmic domain of the receptor for advanced glycation end products (RAGE), which induces a series of cellular processes. This study directly evaluated the participation of mDia1 in AGE-induced hyperpermeability and revealed the precise intracellular signal transductions of this pathological process. Methods: Human umbilical vein endothelial cells (HUVECs) were used in the in vitro studies. Trans-endothelial electric resistance and permeability coefficient for dextran (Pd) were measured to analyze cell permeability. Western blotting, immunofluorescence staining and flow cytometry assay were performed to investigate the underlying mechanism. Dextran flux across the mesentery in mice was monitored to investigate in vivo microvascular permeability. Results: we found that AGEs evoked Nox4 membrane translocation, reactive oxygen species production, phosphorylation of Src and VE-cadherin, dissociation of adherens junctions and eventual endothelial hyperpermeability through RAGE-mDia1 binding. Cells overexpressing mDia1 by recombinant adenovirus infection showed stronger cellular responses induced by AGEs. Down-regulation of mDia1 by infection with an adenovirus encoding siRNA or blockade of RAGE-mDia1 binding by transfection with RAGE mutant plasmids into HUVECs abolished these AGE-induced effects. Furthermore, knockdown of mDia1 using an adenovirus or genetical knockout of RAGE in C57 mice rescued AGE-evoked microvascular hyperpermeability. Conclusion: Our study revealed that mDia1 plays a critical role in AGE-induced microvascular hyperpermeability through binding to RAGE.

Several mitogen-activated protein kinases (MAPKs) are activated during thermal injury, and the p38 MAPK is specifically involved in endothelial cell (EC) actin and myosin rearrangement (stress-fiber formation) with ensuing cellular contraction and enhanced vessel permeability. Inhibition of p38 MAPK and extracellular signal-related kinase MAPK by their inhibitors SB203580 and PD98059, respectively, significantly reduces burn serum-induced EC stress-fiber formation, whereas SB203580 also inhibits burn serum-induced EC tight-junction damage and thereby general blood vessel hyperpermeability. The JNK MAPK inhibitor, SP600125, on the contrary, influences neither stress-fiber formation nor EC tight-junction damage. Extracellular signal-related kinase MAPK inhibition significantly decreases burn serum-induced Monocyte chemotactic protein-1 (MCP-1) release, whereas SB203580 and SP600125 have only limited such effects. Western blotting, real-time reverse transcriptase-polymerase chain reaction, and confocal laser scanning microscopy proved that SP600125 significantly inhibits burn serum-induced intercellular adhesion molecule 1 expression, whereas SB203580 depresses the expression of P selectin. In vivo studies, using the dominant negative adenoviral approach of MAPK kinase 3b and MAPK kinase 6b to block p38 MAPKs, and MKK4 and MKK7 to block JNK MAPKs, show that the latter MAPKs are involved in the regulation of P selectin and intercellular adhesion molecule 1 expression, respectively, following thermal injury. Taken together, the results suggest that several MAPKs play important, although different, roles in general EC alterations following burn injuries.

Advanced glycation end products (AGEs) accumulated in different pathological conditions have the potent capacity to alter cellular properties that include endothelial structural and functional regulations. The disruption of endothelial barrier integrity may contribute to AGE-induced microangiopathy and macrovasculopathy. Previous studies have shown that AGEs induced the rearrangement of actin and subsequent hyperpermeability in endothelial cells (ECs). However, the mechanisms involved in this AGE-evoked EC malfunction are not well understood. This study directly evaluated the involvement of moesin phosphorylation in AGE-induced alterations and the effects of the RhoA and p38 MAPK pathways on this process. Using immortalized human dermal microvascular ECs (HMVECs), we first confirmed that the ezrin/radixin/moesin (ERM) protein moesin is required in AGE-induced F-actin rearrangement and hyperpermeability responses in ECs by knockdown of moesin protein expression with small interfering RNA. We then detected AGE-induced moesin phosphorylation by Western blot analysis. The mechanisms involved in moesin phosphorylation were analyzed by blocking AGE receptor binding and inhibiting Rho and MAPK pathways. AGE-treated HMVECs exhibited time- and dose-dependent increases in the Thr558phosphorylation of moesin. The increased moesin phosphorylation was attenuated by preadministrations of AGE receptor antibody, Rho kinase (ROCK), or p38 inhibitor. Suppression of p38 activation via the expression of dominant negative mutants with Ad.MKK6b or Ad.p38α also decreased moesin phosphorylation. The activation of the p38 pathway by transfection of HMVECs with an adenoviral construct of dominant active MKK6b resulted in moesin phosphorylation. These results suggest a critical role of moesin phosphorylation in AGE-induced EC functional and morphological regulations. Activation of the ROCK and p38 pathways is required in moesin phosphorylation.

Disorders of angiogenesis are related to microangiopathies during the development of diabetic vascular complications, but the effect of advanced glycation end products (AGEs) on angiogenesis and the mechanism has not been completely unveiled. We previous demonstrated that moesin belonging to the ezrin-radixin-moesin (ERM) protein family protein played a critical role in AGE-induced hyper-permeability in human umbilical vein endothelial cells (HUVECs). Here, we investigated the impact of moesin on AGE-induced HUVEC proliferation, migration, and tubulogenesis. Silencing of moesin decreased cell motility and tube formation but not cell proliferation. It also attenuated cellular F-actin reassembly. Further, phosphorylation of threonine at the 558 amino acid residue (Thr 558) in moesin suppressed AGE-induced HUVEC proliferation, migration, and tube formation, while the activating mutation of moesin at Thr 558 enhanced HUVEC angiogenesis. Further, the inhibition of either RhoA activity by adenovirus or ROCK activation with inhibitor Y27632 decreased AGE-induced moesin phosphorylation and subsequently suppressed HUVEC angiogenesis. These results indicate that the Thr 558 phosphorylation in moesin mediates endothelial angiogenesis. AGEs promoted HUVEC angiogenesis by inducing moesin phosphorylation via RhoA/ROCK pathway.

The disruption of microvascular barrier in response to advanced glycation end products (AGEs) stimulation contributes to vasculopathy associated with diabetes mellitus. Here, to study the role of Src and its association with moesin, VE-cadherin and focal adhesion kinase (FAK) in AGE-induced vascular hyperpermeability, we verified that AGE induced phosphorylation of Src, causing increased permeability in HUVECs. Cells over-expressed Src displayed a higher permeability after AGE treatment, accompanied with more obvious F-actin rearrangement. Activation of Src with pcDNA3/flag-SrcY530F alone duplicated these effects. Inhibition of Src with siRNA, PP2 or pcDNA3/flag-SrcK298M abolished these effects. The pulmonary microvascular endothelial cells (PMVECs) isolated from receptor for AGEs (RAGE)-knockout mice decreased the phosphorylation of Src and attenuated the barrier dysfunction after AGE-treatment. In vivo study showed that the exudation of dextran from mesenteric venules was increased in AGE-treated mouse. This was attenuated in RAGE knockout or PP2-pretreated mice. Up-regulation of Src activity induced the phosphorylation of moesin, as well as activation and dissociation of VE-cadherin, while down-regulation of Src abolished these effects. FAK was also proved to interact with Src in HUVECs stimulated with AGEs. Our studies demonstrated that Src plays a critical role in AGE-induced microvascular hyperpermeability by phosphorylating moesin, VE-cadherin and FAK respectively.

Cell apoptosis induced by heat stress is regulated by a complex signaling network. We previously reported that a p53-dependent pathway is involved. Here, we present evidence that NF-κB signaling plays a crucial role in preventing heat stress-induced early apoptosis. Human umbilical vein endothelial cells (HUVECs) were examined and increased phosphorylation of p65 and IκBα were detected, without IκBα degradation. When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, an association between HSP27 and p65 was identified which may enhance NF-κB activation. When HSP27 was overexpressed, pretreatment of HUVECs with the antioxidant, apocynin, or N-acetyl cysteine, suppressed apoptosis. Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise. In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

To investigate the molecular mechanisms of continuous venovenous hemofiltration (CVVH) combined with ulinastatin (ULI) (CVVH-ULI) for the treatment of septic shock.Human umbilical endothelial cells (HUVECs) were incubated with serums isolated from normal healthy people (control), septic shock patients treated with conventional therapy (CT) or treated with CVVH combined with ULI (CVVH-ULI). Endothelial permeability was evaluated by the leakage of FITC-labeled albumin. The morphological changes of F-actin was evaluated by Rhodamine-phalloidin. The phosphorylated levels of p38 were determined by Western blot. Cells were then treated with p38inhibitor (SB203580), or DMSO, followed by incubation with serum from septic shock patients treated with conventional therapy. Endothelial permeability and F-actin rearrangements were also evaluated as noted above.Serum from CT group increased endothelial permeability, F-actin rearrangements, and phosphorylated levels of p38, which were inhibited by CVVH-ULI treatment. Moreover, in CT group, the serum-induced endothelial hyperpermeability and F-actin rearrangements were inhibited by SB203580, the inhibitor of p38.CVVH combined with ulinastatin decreases endothelial hyperpermeability induced by septic shock through inhibiting p38 MAPK pathways.

Increased vascular permeability leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is central to the pathogenesis of heatstroke. Protease-activated receptor 1 (PAR1), the receptor for thrombin, plays a key role in disruption of endothelial barrier function in response to extracellular stimuli. However, the role of PAR1 in heat stress-induced endothelial hyper-permeability is unknown. In this study, we measured PAR1 protein expression in heat-stressed human umbilical venous endothelial cells (HUVECs), investigated the influences of PAR1 on endothelial permeability, F-actin rearrangement, and moesin phosphorylation by inhibiting PAR1 with its siRNA, neutralizing antibody (anti-PAR1), specific inhibitor(RWJ56110), and Xuebijing injection (XBJ), a traditional Chinese medicine used for sepsis treatment, and evaluated the role of PAR1 in heatstroke-related ALI/ARDS in mice by suppressing PAR1 with RWJ56110, anti-PAR1and XBJ. We found that heat stress induced PAR1 protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA. PAR1 mediated moesin phosphorylation, which caused F-actin rearrangement and disruption of endothelial barrier function. To corroborate findings from in vitro experiments, we found that RWJ56110 and the anti-PAR1 significantly decreased lung edema, pulmonary microvascular permeability, protein exudation, and leukocytes infiltrations in heatstroke mice. Additionally, XBJ was found to suppress PAR1-moesin signal pathway and confer protective effects on maintaining endothelial barrier function both in vitro and in vivo heat-stressed model, similar to those observed above with the inhibition of PAR1. These results suggest that PAR1 is a potential therapeutic target in heatstroke.

The disruption of endothelial integrity and the occurrence of angiogenesis in response to AGEs contribute greatly to micro- and macrovascular complications associated with DM. Among human dermal, brain, and retinal vascular ECs, activation of ERM, moesin, by phosphorylation of Thr-558 is involved in AGE-induced hyperpermeability and angiogenesis via the Rho and ROCK (Rho/ROCK) and p38 pathways. Src also plays an important role in AGE-induced endothelial barrier dysfunction by phosphorylating moesin, VE-cadherin, and FAK. Furthermore, recent studies have demonstrated that ROS serve as a key mediator of the AGE-induced endothelial response. ROS inhibition would greatly benefit ECs. This review focuses on the role of moesin in microvascular permeability and angiogenesis, and on the involvement of Src and ROS in endothelial barrier disruption.

Endothelial functional dysregulation and barrier disruption contribute to the initiation and development of sepsis. The receptor for advanced glycation end products (RAGE) has been demonstrated to be involved in the pathogenesis of sepsis. The present study aimed to investigate the role of RAGE in lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation in endothelial cells and the consequent endothelial hyperpermeability. LPS-induced upregulation of RAGE protein expression in human umbilical vein endothelial cells (HUVECs) was detected by western blotting. Activation of NF-κB was revealed using western blotting and immunofluorescent staining. LPS-elicited endothelial hyperpermeability was explored by transendothelial electrical resistance (TER) assay and endothelial monolayer permeability assay. The blocking antibody specific to RAGE was used to confirm the role of RAGE in LPS-mediated NF-κB activation and endothelial barrier disruption. We found that LPS upregulated the protein expression of RAGE in a dose- and time-dependent manner in HUVECs. Moreover, LPS triggered a significant phosphorylation and degradation of IκBα, as well as NF-κB p65 nuclear translocation. Moreover, we observed a significant increase in endothelial permeability after LPS treatment. However, the RAGE blocking antibody attenuated LPS-evoked NF-κB activation and endothelial hyperpermeability. Our results suggest that RAGE plays an important role in LPS-induced NF-κB activation and endothelial barrier dysfunction.

To investigate the clinical features and current therapy of atrial fibrillation (AF) of inpatients in Urumqi, China.The clinical data of inpatients diagnosed with AF from January, 2008 to December, 2012, in 12 hospitals in Urumqi were retrospectively analyzed.Totally 1 310 AF inpatients were enrolled in this study with the age of (64.8 ± 3.3) years old and a men to women ratio of 1.39. Most patients were in age groups of 61-70 years (26.5%) and 71-80 years (27.6%). More patients with paroxysmal AF were at cardiac function class I-II (75.2%), while more patients with persistent AF were at cardiac function class III-IV (31.0%) (both P values0.05). The most common co-morbidities of AF were hypertension (49.2%), coronary heart disease (38.5%), diabetes mellitus (20.1%). Compared with patients of chronic AF, the patients of paroxysmal AF had higher success rates in amiodarone conversation and sinus rhythm maintenance after ablation (44.8% vs 29.9%, 87.5% vs 68.9%, P values0.05). Among the 1 310 inpatients, 992 patients (75.7%) received antithrombotic therapy. There were statistically significant differences in CHA2DS2 score and incidence rate of cerebral infarction among patients receiving aspirin, warfarin or rivaroxaban/other anticoagulation drugs [2(1, 3) vs 3(2, 4) vs 3(2, 5) and 6.3% vs 23.8% vs 30.2%, both P values0.05].Our results of AF inpatients' age, gender, related disease distribution, AF types, incidence of stoke, therapeutic and epidemiological features are in accordance with the domestic and abroad reports.

What is the central question of this study? Why do different doses of sphingosine-1-phosphate (S1P) induce distinct biological effects in endothelial cells? What is the main finding and its importance? S1P at physiological concentrations preserved endothelial barrier function by binding to S1P receptor 1, then triggering Ca(2+) release from endoplasmic reticulum through phosphoinositide phospholipase C and inositol triphosphate, and consequently strengthening tight junction and F-actin assembly through Rac1 activation. Excessive S1P induced endothelial malfunction by activating S1P receptor 2 and RhoA/ROCK pathway, causing F-actin and tight junction disorganisation. Extracellular Ca(2+) influx was involved in this process. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid in plasma, and its plasma concentration can be adjusted through a complex metabolic process. The alterations in S1P levels and the activation of receptors collaboratively regulate distinct biological effects. This study was performed to investigate comparatively the effect of different concentrations of S1P on endothelial barrier function and to explore the roles of S1P receptors (S1PRs), Rho GTPases and calcium in S1P-induced endothelial responses. Endothelial barrier function was studied using transendothelial electric resistance and a resistance meter in human umbilical vein endothelial cells. Specific agonists or antagonists were applied to control the activation of S1P receptors and the release of calcium from different cellular compartments. The results indicated that at physiological concentrations, S1P preserved endothelial barrier function by binding with S1PR1. The activation of S1PR1 triggered the release of intracellular Ca(2+) from the endoplasmic reticulum through the PI-phospholipase C and inositol trisphosphate pathways. Consequently, the Rho GTPase Rac1 was activated, strengthening the assembly of tight junction proteins and F-actin. However, excessive S1P induced endothelial barrier dysfunction by activating S1PR2 followed by the RhoA/RhoA kinase pathway, causing the disorganization of F-actin and the disassembly of the tight junction protein ZO-1. An influx of extracellular Ca(2+) was involved in this process. These data suggest that physiological and excessive amounts of S1P induce different responses in human umbilical vein endothelial cells; the activation of the 1PR1-PLC-IP3 R-Ca(2+) -Rac1 pathway governs the low-dose S1P-enhanced endothelial barrier integrity, and the activation of S1PR2-calcium influx-RhoA/ROCK dominates the high-dose S1P-induced endothelial monolayer hyperpermeability response.

OBJECTIVESThe purpose of this study was to design a more efficient protocol for the electrical cardioversion of atrial arrhythmias.BACKGROUNDGuidelines for electrical cardioversion of atrial arrhythmias recommend starting with low energy shocks, which are often ineffective.METHODSWe recorded the sequence of shocks in 1,838 attempts at cardioversion for atrial fibrillation (AF) and 678 attempts at cardioversion for atrial flutter. These data were used to calculate the probability of success for each shock of a standard series and the probability of success with a single shock at each intensity. In 150 cases, a rhythm strip with the time of each shock allowed us to calculate the time expended on unsuccessful shocks.RESULTSWe analyzed the effects of 5,152 shocks delivered to patients for AF and 1,238 shocks delivered to patients for atrial flutter. The probability of success on the first shock in AF of >30 days duration was 5.5% at 30 days duration, shocks of 180 days. In those with AF for >180 days, the initial use of a 360 J shock was associated with the eventual use of less electrical energy than with an initial shock of ≤100 J (581 ± 316 J vs. 758 ± 433 J, p < 0.01, Mann-Whitney Utest).CONCLUSIONSAn initial energy setting of ≥360 J can achieve cardioversion of AF more efficiently in patients than traditional protocols, particularly with AF of longer duration.

To characterize the nature and timing of atrial ectopics preceding clinical episodes of paroxysmal atrial fibrillation.Holter recordings (n= 177, 60 patients, 58% male, mean age 61.7 +/- 11.5 years) were performed on patients with paroxysmal atrial fibrillation. These were subjected to standard analysis and recordings containing atrial fibrillation episodes suitable for analysis were identified (n = 74). Beat interval files differentiating sinus rhythm from atrial fibrillation were generated and atrial ectopics were identified. Atrial ectopics preceding atrial fibrillation were found to be more frequent (5.07 +/- 7.39 min(-1)) and more premature (ratio of coupling interval to that of surrounding sinus cycles = 0.56 +/- 0.08) compared to ectopics occurring remote from atrial fibrillation episodes (frequency = 3.60 +/- 7.32 min(-1) P = 5 x 10(-24), prematurity ratio = 0.60 +/- 0.10, P = 2 x 10(-73)). Atrial ectopic coupling interval frequency histograms were generated and analysed visually and by an automated statistically based test. Many ectopics were seen to occur at one coupling interval in 27 recordings (in eight this occurred only preceding atrial fibrillation onset, while in a further 19 cases this was also seen remote from atrial fibrillation onset). Overall 45% of ectopics preceding atrial fibrillation episodes occurred in isolation, 13% as part of a bigeminal rhythm, 22% as couplets and 20% as runs. This pattern did not differ from that seen remote from atrial fibrillation episodes.Paroxysmal atrial fibrillation is preceded by ectopics of a fixed coupling interval in a significant proportion of patients. If, as seems likely, this is a marker of 'focally mediated' atrial fibrillation, then Holter techniques may provide a useful screening tool with which to identify patients suitable for fuller electrophysiological assessment.

Heart rate variability (HRV) analysis from 24-hour ambulatory ECG has been widely used in risk stratification of patients after myocardial infarction (MI). The accuracy of HRV assessment is known to potentially vary when different commercial systems are used. However, the consistency of HRV measurements has never been fully investigated. Twenty-six post-MI patients (mean age 59 +/- 8 years, 22 men) were studied, of whom 13 succumbed to sudden cardiac death (SCD) within 1 year and 13 remained alive for at least 3 years (MI survivors). Each patient had a 24-hour Holter ECG recorded before hospital discharge. HRV analysis was performed four times from the same recordings using three different Holter tape analysis systems (Marquette, Reynolds, and CardioData) by four independent operators (CardioData system was used twice, once in the United Kingdom and once in the United States). Mean normal-to-normal RR intervals (mNN) and 3 HRV parameters (SDNN, RMSSD, and HRV triangular index [HRVi]) were derived from each recording. The consistency of mNN and HRV measurements was evaluated by coefficient of variance (CV) and by the Bland-Altman method. The results demonstrated that (1) all indices measured by different systems were statistically similar (P = NS) except the measurement of RMSSD (P = 0.01), (2) the measurements of mNN were highly reproducible with a maximum mean difference of 1.8 +/- 13.8 ms and maximum limits of agreement from -14.6 to +15.6 ms. The maximum mean differences were--1.8 +/- 1.4 unit and 4.4 +/- 9.6 ms for HRVi and SDNN, respectively, and RMSSD was less reproducible with a maximum mean difference of--11.1 +/- 11.5 ms, and limits of agreement from -16.2 to +9.6 ms; and (3) the consistency of mNN (CV 0.9% +/- 0.9%) was significantly higher than that of HRVi, SDNN, and RMSSD (P < 0.0001). The consistency of HRVi was similar to that of SDNN (4.8% +/- 2.1% vs 5.7% +/- 4.8%, P = 0.4), and the consistency of RMSSD (26.6% +/- 13.3%) was significantly lower than that of the other measurements (P < 0.00001). In conclusion, the measurements of mNN by different analytical systems are the most consistent among the parameters studied. The global 24-hour measurements of HRV (SDNN and HRVi) are highly reproducible, whereas the measurement of short-term HRV components (RMSSD) is significantly less reproducible.

Changes in the RR interval within episodes of paroxysmal atrial fibrillation (PAF) have not been fully characterized. A database of 177 24-hour Holter recordings were created from patients with PAF in the CRAFT studies. PAF episodes ofor = 1 minute duration containingor = 20% noise and preceded byor = 1 minute of sinus rhythm withor = 20% noise were selected. Sections of each AF episode containing 10 and 25 RR intervals were identified at the onset, middle, and termination of each episode. Descriptive characteristics (mean, SD, and RMSSD of RR intervals) were calculated within each section, and compared using a nonparametric, paired Wilcoxon test. In 25 patients (17 men, 60.6 +/- 12.2 years old), 231 episodes from 44 recordings met the selection criteria. The mean RR interval increased slightly between the onset and mid-portion of AF episodes (565.9 +/- 128.3 vs 580.3 +/- 144.7 ms, P0.001). The RR interval at the termination of AF was significantly greater than that at the start (627.1 +/- 156.1 vs 565.9 ms, P10-11) or mid-portion (627.1 +/- 156.1 vs 580.3 +/- 144.7 ms, P10-13). SD of the RR interval increased significantly between onset and mid-portion (111.1 +/- 60.2 vs 118.2 +/- 66.7 ms, P0.001) and more substantially between mid-portion and termination (118.2 +/- 66.7 vs 201.8 +/- 93.7 ms, P10-21). During paroxysms of AF, the mean RR interval and the variability of RR intervals increases. Termination of a paroxysm is preceded by a marked increase in RR interval variability.

This study seeks to elucidate whether there was a common mode of initiation of paroxysmal atrial fibrillation (PAF) episodes that might suggest new therapies.A library of 177 digitized and analyzed 24-hour Holter recordings from PAF pharmacotherapy trials was studied. All noise-free PAF episodesor =0.5 minutes were identified. PAF episodes and the preceding 2 minutes of sinus rhythm were printed as tachograms and visually inspected. Heart rate and ectopic beat behavior were used to characterize modes of PAF onset by comparing half-minute segments of the final 2 minutes of sinus rhythm.Thirty-four recordings (from 19 patients, aged 61.7 +/- 11.5 years) provided 231 PAF episodes suitable for analysis. No patients had a consistent mode of PAF onset. This was confirmed by systematic analysis of the five patients with the most episodes. Overall, a highly significant increase in ectopic beats, from 1.34 to 6.52 min(-1) (p0.001) was found, but heart rate did not significantly change (mean heart rate at onset = 64 beats/min). PAF was initiated by a solitary ectopic beat in more than half of the cases. No consistent evidence for short-long-short sequences, seen in ventricular arrhythmias, was found.The mode of onset of atrial fibrillation is inconsistent, both across a population with PAF and within individuals. This has implications for understanding the mechanisms of atrial fibrillation onset in human beings and for the treatment of the disorder.

To determine whether the automatic measurement of the QT interval is consistent with the manual measurement, this study evaluated the reproducibility and agreement of both methods in 70 normal subjects and 54 patients with hypertrophic cardiomyopathy. The mean, minimum, and maximum QT interval and QT dispersion were computed in a set of 6 consecutive electrocardiograms (3 in the supine and 3 in the standing position) obtained from each subject. The automatic method determined the T-wave end as the intersect of the least-squares-fit line around the tangent to the T-wave downslope with the isoelectric baseline. Manual measurements were obtained using a high-resolution digitizing board. QT dispersion was defined as the difference between the maximum and minimum QT interval and as standard deviations of the QT interval duration in all and precordial leads. In patients with hypertrophic cardiomyopathy, the absolute values of the QT interval and QT dispersion were significantly higher than those in normal subjects (p

Background: Prolongation of the QT interval and increased QT dispersion have been proposed to be associated with arrhythmic risk after myocardial infarction. However, controversy remains regarding the prognostic value of ventricular repolarization abnormalities in the risk stratification of patients surviving acute myocardial infarction. Hypothesis and objective: The QT interval is sensitive to myocardial ischaemia, and exercise-induced ischaemia may change the QT interval regionally, resulting in increased QT dispersion. This study examined whether there are abnormalities of ventricular repolarization during exercise and whether assessment of the exercise-induced changes in QT interval duration and dispersion would be able to differentiate patients at high risk from those at low risk of sudden cardiac death after myocardial infarction. Methods: Twenty-six post-myocardial infarction patients (mean age 54.5±8.9 years, 22 men) were retrospectively studied. Thirteen patients who died suddenly (SCD patients) during a follow-up of 39±6 months were compared to 13 patients who remained event-free, i.e. no ventricular tachyarrhythmias, no reinfarction, no by-pass (MI survivors). The two groups were pair-matched for age, gender, site of infarction, left ventricular ejection fraction and use of beta blocker. A further 13 patients with chest pain, normal coronary arteriograms and negative exercise test results were studied as controls. They were age and gender matched with the post-infarction patients. A 12-lead exercise ECG was recorded from each patient before, during and after exercise. QT and RR interval were measured on the exercise ECGs at each stage and QT dispersion was defined as the difference between the maximum and minimum QT intervals across the 12-lead ECG. Results: There were no significant differences in RR, QT and QTc (Bazett's and Fridericia's correction) intervals, or QT dispersion between any groups before exercise. A significant difference in QT and QT dispersion was found at peak exercise between post-infarction patients and controls ( P =0.03 and P =0.0001, respectively), but no difference was observed between SCD patients and MI survivors. The maximum QTc at peak exercise was longer in SCD patients compared with MI survivors ( P =0.02) and a maximum QTc>440 ms (Bazett's correction) was common in SCD patients but not in MI survivors or controls (62%, 15%, 15%, P =0.01). The differences in QT, QTc or QT dispersion observed at peak exercise were no longer significant after exercise. Conclusions: Exercise-induced prolongation of the QTc interval differentiates patients at high risk of sudden cardiac death from those at low risk, whereas exercise-induced changes in QT dispersion failed to identify patients at high risk of sudden cardiac death after myocardial infarction.

Aims Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs). Main methods AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry. Key findings AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation. Significance This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.

A construction of a purpose designed graphical display is demonstrated in a study investigating the circadian distribution of patterns of RR interval sequences preceding episodes of paroxysmal atrial fibrillation (PAF). Based on a comparison with a (80%, 120%) range around the median of preceding 10 RR intervals, each RR interval is classified as normal, short, or long. Classifications of RR intervals in n-tuplets (n = 1, ...,5) preceding PAF episodes are used to compute probabilities of individual types of sequences occurring within 4-hour periods of the day (between 1 am, 5 am, 9 am, 1 pm, 5 pm, and 9 pm). Graphical representation of the data is proposed using a hierarchy of bar graphs. The graphical system has been filled with data of 327 atrial fibrillation episodes recorded in 46 24-hour ECGs in PAF patients. The graphical analysis supports a link between PAF initiation and cardiac autonomic status.

Background The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs. Methods Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D were applied in endothelial cells. Results The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses. Conclusion These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.

Microvascular barrier damage, induced by thermal injury, imposes life-threatening problems owing to the pathophysiological consequences of plasma loss and impaired perfusion that finally may lead to multiple organ failure. The aim of the present study was to define the signaling role of selected mitogen-activated protein kinases (MAPKs) in general vessel hyperpermeability caused by burns and to look for a potential gene therapy. Rearrangement of cytoskeletons and cell tight junctions were evaluated by phalloidin labeling of actin and immunocytochemical demonstration of the ZO-1 protein, whereas blood vessel permeability was evaluated by a fluorescence ratio technique. The p38 MAPK inhibitor SB203580 largely blocked burn serum-induced stress-fiber formation and tight-junction damage. Using the adenoviral approach to transfect dominant negative forms of p38 MAPKs, we found that p38alpha and p38delta had similar effects. The in vivo part of the study showed that transfection of these two constructs significantly lowered general venular hyperpermeability and enhanced the survival of burned animals. Because the p38 MAPK pathway seems to play a crucial role in burn-induced vascular hyperpermeability, general transfection with p38 MAP dominant negative constructs might become a new therapeutic method to block burn-induced plasma leakage.

It has been well appreciated that aldosterone (Aldo) plays a direct profibrotic role in the kidney but the underlying mechanism is unclear. We examined the role of Aldo in epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Exposure of human renal proximal tubular cells to Aldo for 48 h dose dependently induced EMT as evidenced by conversion to the spindle-like morphology, loss of E-cadherin, and de novo expression of α-smooth muscle actin (SMA); the effect was noticeable at 50 nM and maximal at 100 nM. The EMT was completely blocked by the selective mineralocorticoid receptor (MR) antagonist eplerenone. Aldo time dependently increased intracellular reactive oxygen species (ROS) production that was detectable at 15 min and peaked (2.3-fold) at 60 min, as assessed by 2′,7′-dichlorofluorescin diacetate fluorescence. Aldo-induced oxidative stress and EMT were both abolished by the mitochondrial respiratory chain complex I inhibitor rotenone, but not the NADPH oxidase inhibitor apocynin. Aldo induced phosphorylation of ERK1/2 that was completely blocked by rotenone. Male 129-C57/BL6 mice were treated with deoxycorticosterone acetate (DOCA) salt (subcutaneous implantation of 50 mg of DOCA pellet plus 1% NaCl as drinking fluid) for 3 wk and animals were treated with vehicle or rotenone (600 ppm in diet) for the last week. DOCA salt induced a 2.5-fold increase in α-SMA and a 30% reduction of E-cadherin, as assessed by real-time RT-PCR, that were both restricted to renal epithelial cells, as determined by immunohistochemistry. In contrast, DOCA salt-induced changes in α-SMA and E-cadherin were completely blocked by treatment with rotenone. These observations suggest that Aldo induces EMT via MR-mediated, mitochondrial-originated, ROS-dependent ERK1/2 activation in renal tubular epithelial cells.

Abnormal repolarization is associated with arrhythmogenesis. Because of controversies in existing methodology, new computerized methods may provide more reliable tools for the noninvasive assessment of myocardial repolarization from the surface electrocardiogram (ECG). Measurement of the interval between the peak and the end of the T wave (TpTe interval) has been suggested for the detection of repolarization abnormalities, but its clinical value has not been fully studied. The intrasubject reproducibility and reliability of automatic measurements of QT, QT peak, and TpTe interval and dispersion were assessed in 70 normal subjects, 49 patients with acute myocardial infarction (5th day; MI), and 37 patients with hypertrophic cardiomyopathy (HC). Measurements were performed automatically in a set of 10 ECGs obtained from each subject using a commercial software package (Marquette Medical Systems, Milwaukee, WI, U.S.A.). Compared to normal subjects, all intervals were significantly longer in HC patients (P < 0.001 for QT and QTp; p < 0.05 for TpTe); in MI patients, this difference was only significant for the maximum QT and QTp intervals (P < 0.05). In both patient groups, the QT and QTp dispersion was significantly greater compared to normal subjects (P < 0.05) but no consistent difference was observed in the TpTe dispersion among all three groups. In all subjects, the reproducibility of automatic measurement of QT and QTp intervals was high (coefficient of variation, CV, 1%-2%) and slightly lower for that of TpTe interval (2%-5%; p < 0.05). The reproducibility of QT, QTp, and TpTe dispersion was lower (12%-24%, 18%-28%, 16%-23% in normal subjects, MI and HC patients, respectively). The reliability of automatic measurement of QT, QTp, and TpTe intervals is high but the reproducibility of the repeated measurements of QT, QTp and TpTe dispersion is comparatively low.

The complexity of the T wave assessed by principal component analysis (PCA) has been proposed to reflect abnormal repolarization, which may be arrhythmogenic. To determine whether PCA can differentiate patients with hypertrophic cardiomyopathy (HCM) from normal subjects and whether PCA is of prognostic importance in HCM, 112 patients with HCM (41 +/- 14 years, 64 males) and 72 healthy subjects (39 +/- 9 years, 41 males) were studied. Patients with sinus node dysfunction, AV conduction block, flat T waves, QRS > 140 ms, and those < 15 years were excluded from this study. Standard 12-lead ECGs were recorded digitally using the MAC-VU system (Marquette Medical Systems). PCA parameters were computed using the QT Guard software package by Marquette. PCA ratio was significantly greater in HCM patients than in normal controls (23.9% +/- 12.4% vs 16.1% +/- 7.6%, P < 0.0001) and was correlated with QT-end dispersion (r = 0.24, P = 0.01) and QT peak (Q point to T peak) dispersion (r = 0.35, P < 0.0001). HCM patients with syncope (n = 23) had increased PCA ratios compared with those without syncope (29.1% +/- 11.5% vs 22.5% +/- 12.3%, P = 0.01). PCA ratio was similar in patients with and without nonsustained ventricular tachycardia on Holter (25.9% +/- 11.4% vs 22.7% +/- 12.1%, P = 0.2), as well as in patients treated with amiodarone or sotalol versus those not on therapy. In conclusion, assessment of the complexity of the T wave by PCA differentiates HCM patients from normal subjects. PCA ratio correlated with QT dispersion and an increased PCA ratio was associated with a history of syncope in HCM.