A label-free activatable biosensor for in situ detection of exosomal microRNAs based on DNA-AgNCs and hairpin type nucleic acid probes.

Exosomal microRNA (miRNA) is a potential biomarker for cancer diagnosis, metastasis, and treatment. In situ detection of exosomal miRNA is an attractive option due to its simplicity and high accuracy. However, in situ exosomal miRNA detection has encountered challenges because of the low target abundance of targets and limited probe permeability. Herein, a label-free and activatable biosensor was developed for in situ exosomal miRNA assays by utilizing hairpin-shaped nucleic acid probes and DNA-hosted silver nanoclusters (DNA-AgNCs). The probe is directly internalized into the exosomes, and then hybridized with the target miRNA-21. Subsequently, the DNA-AgNCs are pulled closer to the G-rich sequence, ultimately leading to in situ red fluorescence activation. The biosensor not only can detect exosomal miRNA-21 but also distinguish cancer cells from normal cells. Under optimal reaction conditions, the detection limit (LOD) of exosomal miRNA-21 is 1.53 × 107 particles per mL. Furthermore, DNA-AgNCs are used as label-free signal elements for in situ detection of exosomal miRNAs for the first time, expanding the application of nanomaterials in this field. This strategy does not require tedious RNA extraction steps and expensive instruments, and may develop into a non-invasive diagnostic tool for ovarian cancer.

Relationships among the Dosage of Erythropoiesis-Stimulating Agents, Erythropoietin Resistance Index, and Mortality in Maintenance Hemodialysis Patients.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) constitute an important treatment option for anemia in hemodialysis (HD) patients. We investigated the relationships among the dosage of ESA, erythropoietin resistance index (ERI) scores, and mortality in Chinese MHD patients. METHODS: This multicenter observational retrospective study included MHD patients from 16 blood purification centers (n = 824) who underwent HD in 2011-2015 and were followed up until December 31, 2016. We collected demographic variables, HD parameters, laboratory values, and ESA dosages. Patients were grouped into quartiles according to ESA dosage to study the effect of ESA dosage on all-cause mortality. The ERI was calculated as follows: ESA (IU/week)/weight (kg)/hemoglobin levels (g/dL). We also compared outcomes among the patients stratified into quartiles according to ERI scores. We used the Cox proportional hazards model to measure the relationships between the ESA dosage, ERI scores, and all-cause mortality. Using propensity score matching, we compared mortality between groups according to ERI scores, classified as either > or ≤12.80. RESULTS: In total, 824 patients were enrolled in the study; 200 (24.3%) all-cause deaths occurred within the observation period. Kaplan-Meier analyses showed that patients administered high dosages of ESAs had significantly worse survival than those administered low dosages of ESAs. A multivariate Cox regression identified that high dosages of ESAs could significantly predict mortality (ESA dosage >10,000.0 IU/week, HR = 1.59, 95% confidence intervals (CIs) (1.04, 2.42), and p = 0.031). Our analysis also indicated a significant increase in the risk of mortality in patients with high ERI scores. Propensity score matching-analyses confirmed that ERI > 12.80 could significantly predict mortality (HR = 1.56, 95% CI [1.11, 2.18], and p = 0.010). CONCLUSIONS: Our data suggested that ESA dosages >10,000.0 IU/week in the first 3 months constitute an independent predictor of all-cause mortality among Chinese MHD patients. A higher degree of resistance to ESA was related to a higher risk of all-cause mortality.

Identification of Prognosis-Related RNA-Binding Proteins to Reveal the Role of RNA-Binding Proteins in the Progression and Prognosis of Colon Cancer.

BACKGROUND: RNA binding proteins (RBPs) are now under discussion as novel promising bio-markers for patients with colon cancer. The purpose of our study is to identify several RBPs related to the progression and prognosis of colon cancer and to further investigate the mechanism of their influence on tumor progression. METHODS: The transcriptome data of colon cancer and clinical characteristics were downloaded from The Cancer Genome Atlas (TCGA) database. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) were performed to elucidate the gene functions and relative pathways. Cox and Lasso regression analyses were used to analyze the effect of immune genes on the prognosis of colon cancer. An immune risk scoring model was constructed based on the statistical correlation between hub immune genes and survival. Meanwhile, multivariate Cox regression analysis was utilized to investigate whether the immune gene risk score model was an independent factor for predicting the prognosis of colon cancer. A nomogram was constructed to comprehensively predict the survival rate of colon cancer. P < 0.05 was considered statistically significant. RESULTS: The results showed that 473 RBPs exhibited differential expression between normal and colon cancer tissues (P < 0.05). Univariate Cox regression analysis revealed 25 RBPs statistically correlated with colon cancer-related survival risk (P < 0.05). In addition, a 10-RBPs based risk scoring model was constructed through multivariate Cox regression analysis. A K-M curve indicated that high-risk patients were associated with poor outcomes (P < 0.001). A ROC curve indicated that the immune risk score model was reliable in predicting survival risk (5-year overall survival (OS), area under curve (AUC) = 0.782). Our model showed satisfying AUC and survival correlation in the validation dataset (5-year OS, AUC = 0.744). Furthermore, multivariate Cox regression analysis confirmed that the immune risk score model was an independent factor for predicting the prognosis of colon cancer. Finally, we found that 10-RBPs and risk scores were significantly associated with clinical factors and prognosis and were involved in multiple oncogenic pathways. CONCLUSION: Collectively, RBPs play an essential role in the progression and prognosis of colon cancer by regulating multiple biological pathways. Furthermore, the RBP risk score was an independent predictive factor of colon cancer, indicating poor survival.

High expression of keratin 6C is associated with poor prognosis and accelerates cancer proliferation and migration by modulating epithelial-mesenchymal transition in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is a more frequent subtype of lung cancer and most cases are discovered in the late stages. The proliferation and metastasis of LUAD are pivotal for disease progression. Despite unremitting deeper understanding of LUAD biology, the mechanisms involved in the proliferation and metastasis of LUAD remain unclear. The objective of our article was to inquiry the expression and the function of keratin 6C (KRT6C) in LUAD cells. METHODS: First, the expression level and prognostic value of KRT6C in LUAD tissues were analyzed on the basis of the data acquired from TCGA database. Through qRT-PCR, the expression level of KRT6C on LUAD cell lines (A549, H1299, PC-9) and human normal lung cell line MRC-5 was tested. After that, CCK8 and colony formation assays was utilized to detect cell proliferation. In addition, to explore the influence of KRT6C on LUAD migration and invasion ability, scratch wound healing and transwell assays were utilized. Through western blotting, the protein expression levels of KRT6C, PCNA, E-cadherin, N-cadherin, Snail and Vimentin were detected. RESULTS: The outcomes revealed that KRT6C was highly expressed in LUAD tissues and cell lines. Besides, elevated level of KRT6C was related to worse prognosis in LUAD patients. Ablation of KRT6C restrained proliferation, migration and invasion of A549 cells. KRT6C deficiency augmented the expression of E-cadherin as well as reduced the expression of N-cadherin, Snail and Vimentin. CONCLUSION: Above all, these consequences indicated that depletion of KRT6C suppressed A549 cell proliferation, migration and invasion, which might be achieved by regulating EMT. In general, KRT6C is identified as a potential therapeutic target for LUAD.

[Effect of down-regulation of fatty acid synthase expression on proliferation, migration and invasion of bladder carcinoma UMUC3 cell lines].

OBJECTIVE: To investigate the effects of fatty acid synthase (FASN) on proliferation, migration and invasion of bladder cancer UMUC3 cell lines and possible mechanism. METHODS: The expression levels of FASN protein in 30 cases of bladder cancer and 15 cases of normal bladder tissues were detected by Immunohistochemistry. FASN siRNA and nonsense siRNA were transfected into UMUC3 cell lines by lipofectamine 2000 respectively, and the stable siFASN and siControl cell lines were successfully obtained after screening and identification for several times. The siFASN cell lines were set as the experimental group, while the siControl cell lines were set as the control group. The expressions of FASN protein and mRNA in the experimental group and the control group were detected by Western blot and real-time quantitative PCR (RT-PCR) respectively. Cell proliferation activities in two groups were detected by MTT assay and cell invasion and migration in two groups were detected by cell scratch test and Transwell invasive assays respectively. RESULTS: FASN protein was overexpressed in bladder cancer tissues, and it was closely correlated with pathological stage and grade (P＜0.05). Compared with the siControl group, the expressions of FASN mRNA and protein in the siFASN group cell lines were decreased significantly (P＜0.05). The cell proliferation ability, the migration ability and the number of transmembrane cells of siFASN group cell lines were reduced significantly (P＜0.05). CONCLUSION: The FASN overexpression may play an essential role in the development and progression of bladder cancer. Down-regulation of FASN expression can inhibit the proliferation, migration and invasion of bladder cancer cells, and inhibition of FASN expression is expected to be a new treatment for bladder cancer.

Overexpression of prostaglandin E2 EP4 receptor improves cardiac function after myocardial infarction.

BACKGROUND: Prostaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We recently reported that PGE2 via its EP3 receptor could reduce cardiac contractility of isolated myocytes and the working heart preparation. We thus hypothesized that there is an imbalance in the EP3/EP4 ratio towards EP3 in the failing heart and that overexpression of EP4 in a mouse model of heart failure would improve cardiac function. METHODS AND RESULTS: Our hypothesis was tested in a mouse model of myocardial infarction (MI) with the use of AAV9-EP4 driven by the myosin heavy chain promoter to overexpress EP4 in the cardiac myocytes. Echocardiography was performed to assess cardiac function. We found that overexpression of EP4 improved shortening fraction (p = 0.0025), ejection fraction (p = 0.0003), and reduced left ventricular dimension at systole (p = 0.0013). Overexpression of EP4 also significantly reduced indices of cardiac hypertrophy and interstitial collagen fraction. Animals treated with AAV9-EP4 also had a significant decrease in TNFα mRNA expression and in the number of macrophages and T cells migrated post MI coupled with a reduction in the expression of iNOS. CONCLUSION: Overexpression of EP4 improves cardiac function post MI. This may be mediated through reductions in adverse cardiac remodeling or via inhibition of cytokine/chemokine production.

Ac-SDKP decreases mortality and cardiac rupture after acute myocardial infarction.

The natural peptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) decreases inflammation in chronic diseases such as hypertension and heart failure. However, Ac-SDKP effects on acute inflammatory responses during myocardial infarction (MI) are unknown. During the first 72 hours post-MI, neutrophils, M1 macrophages (pro-inflammatory), and M2 macrophages (pro-resolution) and release of myeloperoxidase (MPO) and matrix metalloproteinases (MMP) are involved in cardiac rupture. We hypothesized that in the acute stage of MI, Ac-SDKP decreases the incidence of cardiac rupture and mortality by preventing immune cell infiltration as well as by decreasing MPO and MMP expression. MI was induced by ligating the left descending coronary artery in C57BL/6 mice. Vehicle or Ac-SDKP (1.6 mg/kg/d) was infused via osmotic minipump. Cardiac immune cell infiltration was assessed by flow cytometry, cardiac MPO and MMP levels were measured at 24-48 hrs post-MI. Cardiac rupture and mortality incidence were determined at 7 days post-MI. In infarcted mice, Ac-SDKP significantly decreased cardiac rupture incidence from 51.0% (26 of 51 animals) to 27.3% (12 of 44) and mortality from 56.9% (29 of 51) to 31.8% (14 of 44). Ac-SDKP reduced M1 macrophages in cardiac tissue after MI, without affecting M2 macrophages and neutrophils. Ac-SDKP decreased MMP-9 activation in infarcted hearts with no changes on MPO expression. Ac-SDKP prevents cardiac rupture and decreases mortality post-acute MI. These protective effects of Ac-SDKP are associated with decreased pro-inflammatory M1 macrophage infiltration and MMP-9 activation.

Photoluminescent polyphosphazene nanoparticles for in situ simvastatin delivery for improving the osteocompatibility of BMSCs.

Nanomedicines have found promising applications in regulating the biological behaviors of cells because of the cell endocytosis effect. To enhance the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), which is one of the key issues in relation to bone regeneration, a biodegradable simvastatin-bearing polyphosphazene prodrug was synthesized and made into nanoparticles (NPs). At the same time, photoluminescent tryptophan ethyl ester and hydrolyzable glycine ethyl ester were introduced as co-substituted side groups onto the polyphosphazene backbone. The resultant polymer, poly(simvastatin-co-ethyl tryptophanato-co-ethyl glycinato)phosphazene (PTGP-SIM), displayed the expected features of photoluminescence, degradability and sustained SIM release. Endocytosis of PTGP-SIM NPs by BMSCs and the location of internalized NPs, were visualized via the inherent photoluminescence features of PTGP-SIM. Thus, simvastatin was released inside the cells directly along with polymer degradation and could play a role in promoting osteogenic differentiation efficiently at quite a low local concentration. From the results, the present study suggested a very promising biomaterial for use as a flexible and functional carrier for bioactive components, which could find wide applications in relation to tissue regeneration.

Cell studies of hybridized carbon nanofibers containing bioactive glass nanoparticles using bone mesenchymal stromal cells.

Bone regeneration required suitable scaffolding materials to support the proliferation and osteogenic differentiation of bone-related cells. In this study, a kind of hybridized nanofibrous scaffold material (CNF/BG) was prepared by incorporating bioactive glass (BG) nanoparticles into carbon nanofibers (CNF) via the combination of BG sol-gel and polyacrylonitrile (PAN) electrospinning, followed by carbonization. Three types (49 s, 68 s and 86 s) of BG nanoparticles were incorporated. To understand the mechanism of CNF/BG hybrids exerting osteogenic effects, bone marrow mesenchymal stromal cells (BMSCs) were cultured directly on these hybrids (contact culture) or cultured in transwell chambers in the presence of these materials (non-contact culture). The contributions of ion release and contact effect on cell proliferation and osteogenic differentiation were able to be correlated. It was found that the ionic dissolution products had limited effect on cell proliferation, while they were able to enhance osteogenic differentiation of BMSCs in comparison with pure CNF. Differently, the proliferation and osteogenic differentiation were both significantly promoted in the contact culture. In both cases, CNF/BG(68 s) showed the strongest ability in influencing cell behaviors due to its fastest release rate of soluble silicium-relating ions. The synergistic effect of CNF and BG would make CNF/BG hybrids promising substrates for bone repairing.

Diagnostic and prognostic roles of serum vitronectin in hepatitis B-related hepatocellular carcinoma.

BACKGROUND: Vitronectin (VN) might be involved in the progression of hepatocellular carcinoma (HCC). OBJECTIVE: This study was designed to evaluate the diagnostic and prognostic value of serum vitronectin among HCC patients. METHODS: A total of 105 patients with HCC, 91 with liver cirrhosis, 102 with chronic hepatitis, and 100 healthy subjects were recruited. Serum VN and alpha-fetoprotein (AFP) levels were measured. RESULTS: Serum VN levels were significantly higher in HCC patients than in the other groups. Based on area under receiver operating characteristic curve, serum VN had similar diagnostic value, compared with serum AFP, in distinguishing HCC from the groups, and also improved the diagnostic value of AFP alone. Serum VN levels were associated with the degree of histological differentiation, multiple foci, vascular tumor thrombosis and tumor node metastasis stage. Serum VN was an independent predictor for early recurrence and disease-free survival. Moreover, serum VN possessed similar prognostic predictive performance as compared to serum AFP and also significantly enhanced the prognostic value of AFP alone. CONCLUSIONS: Elevated serum VN levels represented high diagnostic value and had close relation to clinicopathological factors and early recurrence, suggesting that serum VN might be a useful diagnostic and prognostic marker for HCC.

Cardiac-deleterious role of galectin-3 in chronic angiotensin II-induced hypertension.

Galectin-3 (Gal-3), a member of the ß-galactoside lectin family, has an important role in immune regulation. In hypertensive rats and heart failure patients, Gal-3 is considered a marker for an unfavorable prognosis. Nevertheless, the role and mechanism of Gal-3 action in hypertension-induced target organ damage are unknown. We hypothesized that, in angiotensin II (ANG II)-induced hypertension, genetic deletion of Gal-3 prevents left ventricular (LV) adverse remodeling and LV dysfunction by reducing the innate immune responses and myocardial fibrosis. To induce hypertension, male C57BL/6J and Gal-3 knockout (KO) mice were infused with ANG II (3 µg·min-1·kg-1 sc) for 8 wk. We assessed: 1) systolic blood pressure by plethysmography, 2) LV function and remodeling by echocardiography, 3) myocardial fibrosis by histology, 4) cardiac CD68+ macrophage infiltration by histology, 5) ICAM-1 and VCAM-1 expression by Western blotting, 6) plasma cytokines, including interleukin-6 (IL-6), by enzyme-linked immunosorbent assay, and 7) regulatory T (Treg) cells by flow cytometry as detected by their combined expression of CD4, CD25, and FOXP3. Systolic blood pressure and cardiac hypertrophy increased similarly in both mouse strains when infused with ANG II. However, hypertensive C57BL/6J mice suffered impaired ejection and shortening fractions. In these mice, the extent of myocardial fibrosis and macrophage infiltration was greater in histological sections, and cardiac ICAM-1, as well as plasma IL-6, expression was higher as assessed by Western blotting. However, all these parameters were blunted in Gal-3 KO mice. Hypertensive Gal-3 KO mice also had a higher number of splenic Treg lymphocytes. In conclusion, in ANG II-induced hypertension, genetic deletion of Gal-3 prevented LV dysfunction without affecting blood pressure or LV hypertrophy. This study indicates that the ANG II effects are, in part, mediated or triggered by Gal-3 together with the related intercellular signaling (ICAM-1 and IL-6), leading to cardiac inflammation and fibrosis.

Ac-SDKP suppresses TNF-α-induced ICAM-1 expression in endothelial cells via inhibition of IκB kinase and NF-κB activation.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases. We previously showed that, in angiotensin II-induced hypertension, Ac-SDKP decreased the activation of nuclear transcription factor NF-κB, whereas, in experimental autoimmune myocarditis and hypertension animal models, it also reduced the expression of endothelial leukocyte adhesion molecule ICAM-1. However, the mechanisms by which Ac-SDKP downregulated ICAM-1 expression are still unclear. TNF-α is a proinflammatory cytokine that induces ICAM-1 expression in various cell types via TNF receptor 1 and activation of the classical NF-κB pathway. We hypothesized that in endothelial cells Ac-SDKP suppresses TNF-α-induced ICAM-1 expression by decreasing IKK phosphorylation that as a consequence leads to a decrease of IκB phosphorylation and NF-κB activation. To test this hypothesis, human coronary artery endothelial cells were treated with Ac-SDKP and then stimulated with TNF-α. We found that TNF-α-induced ICAM-1 expression was significantly decreased by Ac-SDKP in a dose-dependent manner. Ac-SDKP also decreased TNF-α-induced NF-κB translocation from cytosol to nucleus, as assessed by electrophoretic mobility shift assay, which correlated with a decrease in IκB phosphorylation. In addition, we found that Ac-SDKP decreased TNF-α-induced IKK phosphorylation and IKK-ß expression. However, Ac-SDKP had no effect on TNF-α-induced phosphorylation of p38 MAP kinase or ERK. Thus we conclude that Ac-SDKP inhibition of TNF-α activation of canonical, i.e., IKK-ß-dependent, NF-κB pathway and subsequent decrease in ICAM-1 expression is achieved via inhibition of IKK-ß.

Melatonin inhibits the Migration of Colon Cancer RKO cells by Down-regulating Myosin Light Chain Kinase Expression through Cross-talk with p38 MAPK.

BACKGROUND: Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. MATERIALS AND METHODS: The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). RESULTS: The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. CONCLUSIONS: Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.

Farnesol activates the intrinsic pathway of apoptosis and the ATF4-ATF3-CHOP cascade of ER stress in human T lymphoblastic leukemia Molt4 cells.

In this study, we demonstrate that treatment of T lymphoblastic leukemic Molt4 cells with farnesol activates the apoptosome via the intrinsic pathway of apoptosis. This induction was associated with changes in the level of intracellular potassium and calcium, the dissipation of the mitochondrial and plasma membrane potential, release of cytochrome c, activation of several caspases, and PARP cleavage. The induction of apoptosis by farnesol was inhibited by the addition of the pan-caspase inhibitor Z-VAD-fmk and by the exogenous expression of the anti-apoptotic protein Bcl2. Analysis of the gene expression profiles by microarray analysis revealed that farnesol increased the expression of several genes related to the unfolded protein response (UPR), including CHOP and CHAC1. This induction was associated with the activation of the PERK-eIF2α-ATF3/4 cascade, but not the XBP-1 branch of the UPR. Although farnesol induced activation of the ERK1/2, p38, and JNK pathways, inhibition of these MAPKs had little effect on farnesol-induced apoptosis or the induction of UPR-related genes. Our data indicate that the induction of apoptosis in leukemic cells by farnesol is mediated through a pathway that involves activation of the apoptosome via the intrinsic pathway and induction of the PERK-eIF2α-ATF3/4 cascade in a manner that is independent of the farnesol-induced activation of MAPKs.

Profibrotic Role for Interleukin-4 in Cardiac Remodeling and Dysfunction.

Elevated interleukin-4 (IL-4) levels are associated with cardiac fibrosis in hypertension and heart failure in both patients and experimental animals. We hypothesized that chronically elevated IL-4 induces cardiac fibrosis, resulting in a predisposition of the heart to angiotensin II-induced damage. Wild-type Balb/c (WT, high circulating IL-4) and IL-4-deficient Balb/c mice (IL-4(-/-)) were used. WT mice exhibited cardiac fibrosis (evidenced by an increase in expression of procollagen genes/interstitial collagen fraction), enlarged left ventricle chamber, and declined cardiac function associated with a greater number of mast cells and macrophages in the heart compared with IL-4(-/-). In contrast, IL-4(-/-) mice had normal cardiac architecture/function while showing a 57.9% reduction in heart interstitial collagen compared with WT, despite elevated proinflammatory cytokines in heart tissue. In response to angiotensin II administration, IL-4(-/-) had reduced interstitial myocardial fibrosis and were protected from developing dilated cardiomyopathy, which was seen in WT mice. This was associated with increased macrophage infiltration into the hearts of WT mice, despite a similar degree of hypertension and increased cardiac transforming growth factor-ß1 in both groups. In vitro data demonstrated that IL-4 upregulates procollagen genes and stimulates collagen production in mouse cardiac fibroblasts. This process is mediated by signal transducer and activator of transcription 6 signaling pathway via IL-4 receptor alpha. This study not only establishes a causal relationship between IL-4 and cardiac fibrosis/dysfunction, but also reveals a critical role for IL-4 in angiotensin II-induced cardiac damage. IL-4 could serve as an additional target for the treatment of cardiac fibrosis.

Activation of angiotensin II type 2 receptor suppresses TNF-α-induced ICAM-1 via NF-кB: possible role of ACE2.

ANG II type 2 receptor (AT2) and ANG I-converting enzyme 2 (ACE2) are important components of the renin-ANG system. Activation of AT2 and ACE2 reportedly counteracts proinflammatory effects of ANG II. However, the possible interaction between AT2 and ACE2 has never been established. We hypothesized that activation of AT2 increases ACE2 activity, thereby preventing TNF-α-stimulated ICAM-1 expression via inhibition of NF-κB signaling. Human coronary artery endothelial cells were pretreated with AT2 antagonist PD123319 (PD) or ACE2 inhibitor DX600 and then stimulated with TNF-α in the presence or absence of AT2 agonist CGP42112 (CGP). We found that AT2 agonist CGP increased both ACE2 protein expression and activity. This effect was blunted by AT2 antagonist PD. ICAM-1 expression was very low in untreated cells but greatly increased by TNF-α. Activation of AT2 with agonist CGP or with ANG II under concomitant AT1 antagonist reduced TNF-α-induced ICAM-1 expression, which was reversed by AT2 antagonist PD or ACE2 inhibitor DX600 or knockdown of ACE2 with small interfering RNA. AT2 activation also suppressed TNF-α-stimulated phosphorylation of inhibitory κB (p-IκB) and NF-κB activity. Inhibition of ACE2 reversed the inhibitory effect of AT2 on TNF-α-stimulated p-IκB and NF-κB activity. Our findings suggest that stimulation of AT2 reduces TNF-α-stimulated ICAM-1 expression, which is partly through ACE2-mediated inhibition of NF-κB signaling.

Human Sulfatase-1 Improves the Effectiveness of Cytosine Deaminase Suicide Gene Therapy with 5-Fluorocytosine Treatment on Hepatocellular Carcinoma Cell Line HepG2 In Vitro and In Vivo.

BACKGROUND: Human sulfatase-1 (Hsulf-1) is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans (HSPGs), altering the binding of several growth factors and cytokines to HSPG to regulate cell proliferation, cell motility, and apoptosis. We investigated the role of combined cancer gene therapy with Hsulf-1 and cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene on a hepatocellular carcinoma (HCC) cell line, HepG2, in vitro and in vivo. METHODS: Reverse transcription polymerase chain reaction and immunohistochemistry were used to determine the expression of Hsulf-1 in HCC. Cell apoptosis was observed through flow cytometry instrument and mechanism of Hsulf-1 to enhance the cytotoxicity of 5-FC against HCC was analyzed in HCC by confocal microscopy. We also establish a nude mice model of HCC to address the effect of Hsulf-1 expression on the CD/5-FC suicide gene therapy in vivo. RESULTS: A significant decrease in HepG2 cell proliferation and an increase in HepG2 cell apoptosis were observed when Hsulf-1 expression was combined with the CD/5-FC gene suicide system. A noticeable bystander effect was observed when the Hsulf-1 and CD genes were co-expressed. Intracellular calcium was also increased after HepG2 cells were infected with the Hsulf-1 gene. In vivo studies showed that the suppression of tumor growth was more pronounced in animals treated with the Hsulf-1 plus CD than those treated with either gene therapy alone, and the combined treatment resulted in a significant increase in survival. CONCLUSIONS: Hsulf-1 expression combined with the CD/5-FC gene suicide system could be an effective treatment approach for HCC.

Fractalkine neutralization improves cardiac function after myocardial infarction.

NEW FINDINGS: What is the central question of this study? What is the cardioprotective role of fractalkine neutralization in heart failure and what are the mechanisms responsible? What is the main finding and its importance? The concentration of fractalkine is increased in the left ventricle of mice with myocardial infarction, similar to the increases in plasma from heart failure patients. The present study shows a clear beneficial effect of neutralizing fractalkine in a model of myocardial infarction, which results in increased survival. Such an approach may be worthwhile in human patients. Concentrations of the chemokine fractalkine (FKN) are increased in patients with chronic heart failure, and our previous studies show that aged mice lacking the prostaglandin E2 EP4 receptor subtype (EP4-KO) have increased cardiac FKN, with a phenotype of dilated cardiomyopathy. However, how FKN participates in the pathogenesis of heart failure has rarely been studied. We hypothesized that FKN contributes to the pathogenesis of heart failure and that anti-FKN treatment prevents heart failure induced by myocardial infarction (MI) more effectively in EP4-KO mice. Male EP4-KO mice and wild-type littermates underwent sham or MI surgery and were treated with an anti-FKN antibody or control IgG. At 2 weeks post-MI, echocardiography was performed and hearts were excised for determination of infarct size, immunohistochemistry and Western blot of signalling molecules. Given that FKN protein levels in the left ventricle were increased to a similar extent in both strains after MI and that anti-FKN treatment improved survival and cardiac function in both strains, we subsequently used only wild-type mice to examine the mechanisms whereby anti-FKN is cardioprotective. Myocyte cross-sectional area and interstitial collagen fraction were reduced after anti-FKN treatment, as were macrophage migration and gelatinase activity. Activation of ERK1/2 and p38 MAPK were reduced after neutralization of FKN. In vitro, FKN increased fibroblast proliferation. In conclusion, increased FKN contributes to heart failure after MI. This effect is not exacerbated in EP4-KO mice, suggesting that there is no link between FKN and lack of EP4. Overall, inhibition of FKN may be important to preserve cardiac function post-MI.

Deletion of interleukin-6 prevents cardiac inflammation, fibrosis and dysfunction without affecting blood pressure in angiotensin II-high salt-induced hypertension.

OBJECTIVE: Inflammation has been proposed as a key component in the development of hypertension and cardiac remodeling associated with different cardiovascular diseases. However, the role of the proinflammatory cytokine interleukin-6 in the chronic stage of hypertension is not well defined. Here, we tested the hypothesis that deletion of interleukin-6 protects against the development of hypertension, cardiac inflammation, fibrosis, remodeling and dysfunction induced by high salt diet and angiotensin II (Ang II). METHODS: Male C57BL/6J and interleukin-6-knock out (KO) mice were implanted with telemetry devices for blood pressure (BP) measurements, fed a 4% NaCl diet, and infused with either vehicle or Ang II (90 ng/min per mouse subcutaneously) for 8 weeks. We studied BP and cardiac function by echocardiography at baseline, 4 and 8 weeks. RESULTS: Myocyte cross-sectional area (MCSA), macrophage infiltration, and myocardial fibrosis were also assessed. BP increased similarly in both strains when treated with Ang II and high salt (Ang II-high salt); however, C57BL/6J mice developed a more severe decrease in left ventricle ejection fraction, fibrosis, and macrophage infiltration compared with interleukin-6-KO mice. No differences between strains were observed in MCSA, capillary density and MCSA to capillary density ratio. CONCLUSION: In conclusion, absence of interleukin -6 did not alter the development of Ang II-high salt-induced hypertension and cardiac hypertrophy, but it prevented the development of cardiac dysfunction, myocardial inflammation, and fibrosis. This indicates that interleukin-6 plays an important role in hypertensive heart damage but not in the development of hypertension.