Investigating the CYP2B6 rs3745274 and rs3211371 Polymorphisms in Methadone-Responder and Non-Responder Addicts in Iran

Background:Methadone therapy is a major protocol in opioid addiction cases in many health care systems. Population-based studies have shown that in addicted people, the genetic profile affects their response to methadone therapy. Therefore, this study designed to examine the frequency of two SNPs of the CYP2B6 gene (rs3745274 and rs3211371) in addicted cases in two methadone-responders and methadone non-responders groups. Methods:A total of 199 opioid-addicted individuals and 117 unaffected control subjects were genotyped for rs3745274 and rs3211371 polymorphisms of the CYP2B6 gene using the tetra-primer ARMS-PCR. Results:Results of this study revealed the significant association of rs3745274 GG (p < 0.001; OR = 0.027; 95% CI = 0.14-0.49) and GT (p < 0.001; OR = 4.04; 95% CI = 2.26-7.21) genotypes with the risk of addiction in methadone-responders. Also, a significant association between rs3745274 GG (p < 0.001; OR = 0.28; 95% CI = 0.15-0.51) and GT (p < 0.001; OR = 5.1; 95% CI = 2.8-5.28) genotypes and addiction relapse was found in methadone non-responders. Conclusion:Based on our findings, we can conclude that rs3745274 variant of CYP2B6 gene could serve as a potential biomarker, to evaluate the prognosis of addicted people fate under treatment with methadone. Key Words: Addiction, Biomarker, Methadone, Single-nucleotide polymorphism     

Interaction between LINC-ROR and Stemness State in Gastric Cancer Cells with Helicobacter pylori Infection

Background:LINC-ROR, as a cancer-related lncRNA, has vital roles in stem cell survival, pluripotency, differentiation, and self-renewal in hESCs. However, cancer-related molecular mechanisms, its functional roles, and clinical value of LINC-ROR in GC remain unclear. In this study, we aimed to investigate probable interplay between LINC-ROR with SALL4 stemness regulator and their role with the development of the disease. Methods:The mRNA expression profile of LINC-ROR and SALL4 was assessed in tumoral and adjacent non-cancerous tissues of GC patients, using quantitative real-time PCR. Results:Significant LINC-ROR underexpression and SALL4 overexpression were observed in 55.81% and 75.58% (p < 0.0001) of samples, respectively. The expression of LINC-ROR and SALL4 were significantly correlated with each other (p = 0.044). There was an association between the underexpression of LINC-ROR and sex, stage of tumor progression, tumor type, and location of tumor (p < 0.05), and H. pylori infection with SALL4 expression (p = 0.036). There were also significant correlations between concomitant mRNA expression of SALL4 and LINC-ROR in tumors located at distal noncardiac, positive for H. pylori infection, tumors with invasion into the muscle layer of the stomach, and grade II tumor (p < 0.05). Conclusion:The clinical results of the SALL4-LINC-ROR association propose a probable functional interaction between these markers in tumor maintenance and aggressiveness. Our study can help to understand one of the mechanisms involved in the progression of GC through the function of these regulators. Key Words: LINC-ROR, Non-coding RNA, SALL4     

Effect of wheat bran ball-milling on fragmentation and marker extractability of the aleurone layer

Bran (bran_ml) obtained by roller milling of soft (Scipion) and hard (Baroudeur) wheat cultivars was further ball-milled for increasing times and the observed particle size distribution expressed as a dispersion index. Bran (bran_hi) and aleurone layers were also hand-isolated from the same grains and the pattern of size reduction during ball-milling were compared with bran_ml. Bran_ml and bran_hi were found to fracture more rapidly than isolated aleurone layers due to the presence of the highly friable pericarp and the possible mechanical constraints due to tissues surrounding the aleurone layer. Previously identified markers of the aleurone layer cell contents (phytates) and cell walls (p-coumaric acid) were used to determine their water extractabilities from ball-milled samples and the state and degree of dissociation of the aleurone layer, either as an isolated tissue or within bran_ml and bran_hi. The results suggest that ball-milling rapidly induces fractures in walls of cells in the aleurone layer. The partial opening of the cells in the aleurone layer allowed extraction of most (≈70%) of the water-extractable phytates, even though their mean particle size was much larger than the dimensions of the cells. A further increase in extractability of phytates was observed when the particle size was reduced below the aleurone cell dimensions. Although much less soluble, p-coumaric acid followed a similar trend to phytates. The different behaviour of bran_ml and bran_hi was consistent with a weakening effect of the tissues in the former, probably due to the previous milling process. The bran and aleurone layers from both wheat varieties exhibited a similar behaviour.     

Mixed Lineage Kinase Domain-Like Pseudokinase (MLKL) Gene Expression in Human Atherosclerosis with and without Type 2 Diabetes Mellitus

Background:MLKL, one of the main downstream components of the necroptosis or programmed necrosis has recently been demonstrated in advanced atherosclerotic lesions. However, its precise role in the atherosclerosis pathogenesis still requires more elucidation. Our study was set to delineate both the changes in peripheral MLKL gene expression and its influence on disease severity in atherosclerotic patients with and without type 2 DM. Methods:The study involved 50 patients (20 non-diabetics and 30 diabetics) undergoing coronary artery bypass graft and 20 apparently healthy controls. Taqman RT-PCR was used to quantify MLKL mRNA expression levels, while ELISA was employed to estimate serum insulin and hsCRP levels. Results:Compared with the control group, MLKL gene was up regulated significantly in CVD (p ≤ 0.001). Higher MLKL expression was demonstrated in diabetic CVD group than non-diabetic group (p < 0.05). Correlation studies reported positive associations between MLKL and markers of dyslipidemia, inflammation, and insulin resistance. Multiple regression analysis revealed that FBG levels, hsCRP levels, and total WBCs count were significant predictors for MLKL levels. ROC showed a significant diagnostic value of MLKL for CVD. Moreover, regression analysis demonstrated that MLKL and hsCRP were independent predicting factors for the severity of CVD. Conclusion:MLKL is linked to hallmarks of atherosclerosis and could explain increased cardiovascular risk in diabetic patients. Thus, it can be a potential drug target for treatment of atherosclerotic patients. Key Words: Atherosclerosis, Diabetes mellitus, Inflammation, Insulin resistance, Necroptosis     

Investigation of TNF-α and IL-6 Levels in the Sera of Non-Melanoma Skin Cancer Patients

Background:TNF-α and IL-6 are both pleiotropic cytokines playing major roles in cancer-associated cytokine networks. They have previously been investigated for their function in skin malignancies, mostly melanomas, and studies on NMSC patients are relatively rare. In this study, we aimed to assess the associations of serum levels of IL-6 and TNF-α with NMSCs and its clinicopathological features. Methods:This cases-control study was carried out to investigate the serum levels of TNF-α and IL-6 in 70 NMSC patients, in comparison with 30 healthy individuals, by means of flow cytometric bead-based immuneoassay. Results:Serum levels of both TNF-α and IL-6 were significantly higher in NMSC patients (6.470 vs. 4.355 pg/ml; p = 0.0468, respectively), compared to healthy individuals (3.205 vs. 0.000 pg/ml; p = 0.0126, respectively). In the subgroup analysis, SCC patients had higher serum levels of IL-6 compared to healthy individuals (3.445 vs. 0.000 pg/ml; p = 0.0432). No other significant differences were observed in the serum levels of these two cytokines among different clinicopathological subgroups of the patients. Conclusion:The increased levels of TNF-α and IL-6 in NMSC patients can be introduced as an epiphenomenon of a complex cancer-induced cytokine cascade. Key Words: Biomarkers, Cytokines, Interleukin-6, Tumor Necrosis Factor-alpha     

Molecular Investigation of Human Cytomegalovirus and Epstein-Barr virus in Glioblastoma Brain Tumor: A Case-Control Study in Iran

Background:Glioblastoma multiforme is the most invasive and lethal form of brain cancer with unclear etiology. Our study aimed to investigate the molecular prevalence of HCMV and EBV infections in patients with GBM. Methods:This case-control study was conducted on 42 FFPE brain tumor samples from GBM patients and 42 brain autopsies from subjects without neurological disorders. The presence of EBV and HCMV DNA was determined, using PCR and nested-PCR assays, respectively. Results:HCMV DNA was detected in 3 out of 42 (7.1%) of GBM samples and was absent from the control group (p = 0.07). Importantly, EBV DNA was detected in 9 out of 42 (21.4%) brain tissue specimens of GBM subjects, but again in none of the control group (p = 0.001). Conclusion:Our findings indicate that infection with EBV is associated with GBM. Key Words: Brain tumor, Epstein-Barr virus, Glioblastoma, Human cytomegalovirus     

Interaction between Marine-Derived n-3 Long Chain Polyunsaturated Fatty Acids and Uric Acid on Glucose Metabolism and Risk of Type 2 Diabetes Mellitus: A Case-Control Study

The present case-control study explored the interaction between marine-derived n-3 long chain polyunsaturated fatty acids (n-3 LC PUFAs) and uric acid (UA) on glucose metabolism and risk of type 2 diabetes mellitus (T2DM). Two hundred and eleven healthy subjects in control group and 268 T2DM subjects in case group were included. Plasma phospholipid (PL) fatty acids and biochemical parameters were detected by standard methods. Plasma PL C22:6n-3 was significantly lower in case group than in control group, and was negatively correlated with fasting glucose (r = −0.177, p < 0.001). Higher plasma PL C22:6n-3 was associated with lower risk of T2DM, and the OR was 0.32 (95% confidence interval (CI), 0.12 to 0.80; p = 0.016) for per unit increase of C22:6n-3. UA was significantly lower in case group than in control group. UA was positively correlated with fasting glucose in healthy subjects, but this correlation became negative in T2DM subjects. A significant interaction was observed between C22:6n-3 and UA on fasting glucose (p for interaction = 0.005): the lowering effect of C22:6n-3 was only significant in subjects with a lower level of UA. In conclusion, C22:6n-3 interacts with UA to modulate glucose metabolism.     

The Level of Mesenchymal-Epithelial Transition Autophosphorylation is Correlated with Esophageal Squamous Cell Carcinoma Migration

Background:The MET receptor is a critical member of cancer-associated RTKs and plays an important role in different biological activities, including differentiation, migration, and cell proliferation. Methods:In this study, novel MET inhibitors were introduced and applied on esophageal squamous carcinoma cell line KYSE-30, and the level of proliferation and migration, as well as the activated form of MET receptor protein were assessed in the examined cells. The human KYSE-30 cell line was cultured according to ATCC recommendations. The mRNA level of the MET gene was measured in the examined cell line using the quantitative RT-PCR assay. Cytotoxicity evaluation test was performed at different concentrations of heterocyclic anti-MET compounds (i.e. D1, D2, D5, D6, D7, and D8). Finally, the capability of these compounds in MET receptor inhibition was evaluated using the migration assay and Western blot. All experiments were performed in triplicate and repeated three times with similar results. Results:Cell growth and proliferation were significantly inhibited (p ≤ 0.05) by all the above-mentioned compounds. Moreover, the majority of compounds significantly prevented the cell migration (p ≤ 0.05) and inhibited MET autophosphorylation. Interestingly, the level of phosphorylated MET was significantly correlated with KYSE-30 cell migration. Conclusion:The obtained data introduced and confirmed the biological activities of the mentioned novel compounds in KYSE-30 cells and proposed that the therapeutic inhibition of MET with these compounds may be a powerful approach for inhibiting cancer cell migration and proliferation although some structural optimizations are needed to improve their inhibitory functions. Key Words: c-MET, Esophageal squamous cell carcinoma, Receptor tyrosine kinases     

Antioxidant Efficacy of a Spirulina Liquid Extract on Oxidative Stress Status and Metabolic Disturbances in Subjects with Metabolic Syndrome

Lipid peroxidation is associated with the development of some pathologies, such as cardiovascular diseases. Reduction in oxidative stress by antioxidants, such as Arthrospira (formely Spirulina), helps improving this redox imbalance. The aim of the study was to evaluate the effect of the Arthrospira liquid extract “Spirulysat^®” on oxidative markers—in particular, oxidized LDL (oxLDL)/total LDL cholesterol—and isoprostanes and to investigate its impact on lipid and glucose metabolism in the metabolic syndrome subject. A controlled, randomised, double-blind design was conducted in 40 subjects aged 18 to 65 years with metabolic syndrome after a daily intake of Spirulysat^® or placebo for twelve weeks. Blood and urinary samples were collected at three visits (V1, V2, V3) in the two groups for parameters determination. Although the Spirulysat^® group showed a decrease at all visits of the oxLDL/total cholesterol ratio, there was no significant difference compared to the placebo (p = 0.36). The urinary isoprostanes concentration in the Spirulysat^® group was reduced (p = 0.014) at V3. Plasma triglycerides decreased at V3 (p = 0.003) and HDL-cholesterol increased (p = 0.031) at all visits with Spirulysat^®. In conclusion, Spirulysat^® did not change the oxidized LDL (oxLDL)/LDL ratio but decreased the urinary isoprostanes, plasma triglycerides and increased HDL cholesterol, suggesting a beneficial effect on metabolic syndrome.     

Structure of the 4-O-[1-Carboxyethyl]-d-Mannose-Containing O-Specific Polysaccharide of a Halophilic Bacterium Salinivibrio sp. EG9S8QL

The moderately halophilic strain Salinivibrio sp. EG9S8QL was isolated among 11 halophilic strains from saline mud (Emisal Salt Company, Lake Qarun, Fayoum, Egypt). The lipopolysaccharide was extracted from dried cells of Salinivibrio sp. EG9S8QL by the phenol–water procedure. The OPS was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with ¹H and ¹³C NMR spectroscopy, including ¹H,¹H COSY, TOCSY, ROESY, ¹H,¹³C HSQC, and HMBC experiments. The OPS was found to be composed of linear tetrasaccharide repeating units of the following structure: →2)-β-Manp4Lac-(1→3)-α-ManpNAc-(1→3)-β-Rhap-(1→4)-α-GlcpNAc-(1→, where Manp4Lac is 4-O-[1-carboxyethyl]mannose.     

Effect of Triticum monococcum glutenin loci on cookie making quality and on predictive tests for bread making quality

The effects of Triticum monococcum glutenin loci on cookie making quality and predictive tests for bread making quality were evaluated in recombinant substitution lines (RSLs) between chromosome 1A^m from T. monococcum and chromosome 1A from Chinese Spring. All four combinations of high molecular weight (H M_r-GS) and low molecular weight glutenin alleles (L M_r-GS) were studied in a factorial design to evaluate their interactions. Grain protein content was used as a covariable to evaluate the effect of these loci independently of the variation in protein content among lines. No significant interactions were detected indicating an additive effect. RSLs carrying the HM_r -GS from T. monococcum showed a 13·6% increase in SDS sedimentation volume (p=0·004) and a significant reduction in cookie diameter (−5·2%,p =0·02), and cookie quality (−6·8%, p=0·02). RSLs carrying the LM_r -GS from T. monococcum showed a significant decrease in the proportion of polymeric protein (−2·8%, p<0·0001), SDS sedimentation volume (−8·1%,p =0·03) and gluten strength (−16·5%, p=0·01), and a significant increase in cookie quality (5·9%, p=0·05). The T. monococcum LM_r. -GS allele has potential value to be used in soft wheat breeding programs. These results suggest that diploid T. monococcum could be a valuable source for new allelic variation for storage proteins loci and new quality characteristics.     

Source–sink relations and kernel weight differences in maize temperate hybrids

Maize (Zea mays L.) kernel weight (KW) response to changes in assimilate availability per kernel during grain filling suggests that plants establish an early kernel sink potential that place them to grow close to a saturating assimilate availability condition during late grain-filling, meaning source limitations are common only early in kernel development. As maize reproductive efficiency in kernel set is not constant across different plant growth rates (PGR) around flowering, we used PGR per kernel during this period as an indicator of source availability per kernel. We tested whether PGR per kernel during flowering or during the effective grain-filling period were correlated to genotypic and environmental differences in final KW. Plant growth rate during both periods, KW, kernel growth rate during the effective grain-filling period, total duration of grain filling and kernel number per plant were measured in 12 commercial genotypes differing in KW sown at two sites under full irrigation. As expected from the curvilinear response relating kernel number per plant and PGR around flowering, increased PGRs resulted in higher PGR per kernel around this period (r² = 0.86; p < 0.001). Differences in final KW due to genotypes or environments were significantly explained by the PGR per kernel around flowering (r² = 0.40; p < 0.001), and not by the PGR per kernel during the effective grain-filling period. Genotypes differed in kernel growth rate (p < 0.001) and grain-filling duration (p < 0.001). The former was well explained by PGR per kernel around flowering (r² = 0.66; p < 0.001), but showed no relationship with the PGR per kernel during the effective grain-filling period. Grain-filling duration was partially explained (r² = 0.27; p < 0.01) by the ratio between PGR per kernel during the effective grain-filling period and kernel growth rate, but differences in duration were negligible compared to those observed in the ratio (∼41% versus ∼130%, respectively). Together, these results support the importance of source availability per kernel during early grain filling on the determination of maize potential sink capacity and final KW. Early resource availability per kernel was accurately estimated as PGR per kernel around the period of kernel number determination, which helped explain genotypic and environmental differences in maize final KW as well as in kernel growth rate.     

Influence of temperature and length of storage on resistance of potato to tuber rot induced byErwinia chrysanthemi

Tubers of 11 clones ofSolanum tuberosum subsp.andigena (andigena) and 12 cultivars ofS. tuberosum subsp.tuberosum (tuberosum) were inoculated with water suspensions (5.5 × 10⁴ 5.5 × 10⁵, 5.5 × 10⁶ CFU/ml) ofErwinia chrysanthemi. Tubers were inoculated immediately after harvest and after 6 and 16 wk of storage at 4 and 23°C. Tuber rot incidence in andigena and tuberosum increased as inoculum concentration increased. Based on tuber rot severity, clones of andigena were classified as resistant, intermediate, or susceptible and cultivars of tuberosum were classified as intermediate or susceptible. Rot severity increased in all tubers stored at 4°C and in tubers of tuberosum stored at 23°C; rot did not increase in tubers of andigena stored at 23°C. Electrolyte leakage (EL), total sugars (TS), reducing sugars (RS), non-reducing sugars (NRS) and dry matter (DM) were determined in non-inoculated tubers. There was a significant positive correlation between the rate of EL, concentration of RS and tuber rot in andigena and tuberosum. Tuber rot and DM were negatively correlated in tubers of andigena; but they were not correlated in tuberosum. Clones of andigena with low EL, TS, RS, and high DM were resistant to tuber rot, and the incidence of tuber rot in these clones was much less influenced by temperature and length of storage. The influence of temperature and length of storage on susceptibility toE. chrysanthemi may be explained by increased cell membrane permeability; increased leakage of accumulated sugars in potatoes stored at 4°C could favor bacterial proliferation resulting in more disease.     

Comprehensive Analysis of Polyphenols in 55 Extra Virgin Olive Oils by HPLC-ECD and Their Correlation with Antioxidant Activities

In this study, we analyzed eight phenolic compounds (tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acid) in 55 mono- and multivarietal extra virgin olive oil samples by high performance liquid chromatography (HPLC) coupled to a coulometric electrochemical array detector (ECD). The phenolic profile of olive oil samples differed depending on the geographical origin and olive variety. The total reducing capacity (total phenolics) of olive oils ranged from about 40 to 530 mg gallic acid equivalents/kg oil. Tyrosol, hydroxytyrosol and pinoresinol were the most abundant phenolic compounds in olive oils. The antioxidant capacity of the olive oil extracts was determined by ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. Total reducing capacity was significatly correlated with FRAP (R ² ?=?0.91, p?<?0.001) and TEAC (R ² ?=?0.92, p?<?0.001) values. Total reducing capacity, TEAC and FRAP values were significantly correlated with tyrosol, hydroxytyrosol as well as oleuropein concentrations. Hydroxytyrosol, comprising over 40 % of total olive oil phenolics, mainly contributed to the antioxidant activity of olive oils. The present study provides a comprehensive database of polyphenols in olive oils from 9 different countries and four continents.     

Influence of Cultivation Conditions on the Sioxanthin Content and Antioxidative Protection Effect of a Crude Extract from the Vegetative Mycelium of Salinispora tropica

Due to their bioavailability, glycosylated carotenoids may have interesting biological effects. Sioxanthin, as a representative of this type of carotenoid, has been identified in marine actinomycetes of the genus Salinispora. This study evaluates, for the first time, the effect of cultivation temperature (T) and light intensity (LI) on the total cellular carotenoid content (TC), antioxidant activity (AA) and sioxanthin content (SX) of a crude extract (CE) from Salinispora tropica biomass in its vegetative state. Treatment-related differences in TC and SX values were statistically significantly and positively affected by T and LI, while AA was most significantly affected by T. In the S. tropica CE, TC correlated well (R² = 0.823) with SX and somewhat less with AA (R² = 0.777). A correlation between AA and SX was found to be less significant (R² = 0.731). The most significant protective effect against oxidative stress was identified in the CE extracted from S. tropica biomass grown at the highest T and LI (CE-C), as was demonstrated using LNCaP and KYSE-30 human cell lines. The CE showed no cytotoxicity against LNCaP and KYSE-30 cell lines.     

The Highlighted Roles of Metabolic and Cellular Response to Stress Pathways Engaged in Circulating hsa-miR-494-3p and hsa-miR-661 in Alzheimer’s Disease

Background:Among different roles of miRNAs in AD pathogenesis, hsa-miR-494-3p and hsa-miR-661 functions are poorly understood. Methods:To obtain the gene targets, gene networks, gene ontology, and enrichment analysis of the two miRNAs, some web servers were utilized. Furthermore, the expressions of these miRNAs were analyzed by qRT-PCR in 36 blood sera, including 18 Alzheimer’s patients and 18 healthy individuals. Results:The in silico analysis demonstrated the highlighted roles of metabolic and cellular response to stress pathways engaged in circulating hsa-miR-494-3p and hsa-miR-661 in AD. The qRT-PCR analysis showed that the downregulated expression level of hsa-miR-661 was statistically significant (p < 0.05). Also, the ROC curve of hsa-miR-661 displayed the significant AUC (p = 0.01). Conclusion:Based on our findings, the metabolic and cellular responses to stress pathways are closely connected to these two miRNAs functions. Besides, the qRT-PCR and Roc curve determined hsa-miR-661 could be as a biomarker for diagnosis or prognosis of AD patients. Key Words: Alzheimer’s disease, Serum, Circulating microRNAs     

Performance of wheat and triticale varieties in a variable soil water environment IV. Yield components and their association with grain yield

Yield components (Y_c) and their association with grain yield (GY) were studied in seventeen varieties of Triticum aestivum, T. durum and triticale grown for five years in irrigated and unirrigated environments. The varieties were categorized into four contrasting groups based on their drought susceptibility indices (S) and yield potentials (Y_p). The slope (b) and intercept (a) of the linear regression between Y_c and the environmental index, and S values of Y_c were estimated. Their relationships with b, a and S of GY were also studied irrespective of the parameter used. Total dry matter (DM) and the number of grains per m² (GNO) were always positively correlated with GY. GWT (1000-grain weight) was not correlated with GY.There was considerable variation among the groups in their Y_c at different water-availability levels. The group of varieties having low S and moderate Y_p produced more spikes per m² (SPIKES) relative to other groups irrespective of the water availability. Simultaneously, their increased number of grains per spike (GNOSP) in drought environments relative to irrigated environments partially offset the accompanying reduction in SPIKES. The group of varieties characterized by high Y_p and low S had highest yield in irrigated and moderate drought environments because they had both more SPIKES and more GNOSP. By comparison, the varietal group having high Y_p and high S had higher GNOSP but lower SPIKES. Varieties which had low Y_p and were susceptible to drought had the lowest SPIKES, GNOSP and DM.It was concluded that maintenance of SPIKES would provide an advantage in environments varying in soil water availability. Performance in irrigated environments would depend on the maintenance of high GNOSP also. Higher DM production would be advantageous in all environments.     

Coral-Derived Compound WA-25 Inhibits Angiogenesis by Attenuating the VEGF/VEGFR2 Signaling Pathway

Background: WA-25 (dihydroaustrasulfone alcohol, a synthetic derivative of marine compound WE-2) suppresses atherosclerosis in rats by reducing neointima formation. Because angiogenesis plays a critical role in the pathogenesis of atherosclerosis, the present study investigated the angiogenic function and mechanism of WA-25. Methods: The angiogenic effect of WA-25 was evaluated using a rat aortic ring assay and transgenic zebrafish models were established using transgenic Tg(fli-1:EGFP)^y1 and Tg(kdrl:mCherry^ci5-fli1a:negfp^y7) zebrafish embryos. In addition, the effect of WA-25 on distinct angiogenic processes, including matrix metalloproteinase (MMP) expression, endothelial cell proliferation and migration, as well as tube formation, was studied using human umbilical vein endothelial cells (HUVECs). The effect of WA-25 on the endothelial vascular endothelial growth factor (VEGF) signaling pathway was elucidated using qRT-PCR, immunoblot analysis, immunofluorescence and flow cytometric analyses. Results: The application of WA-25 perturbed the development of intersegmental vessels in transgenic zebrafish. Moreover, WA-25 potently suppressed microvessel sprouting in organotypic rat aortic rings. Among cultured endothelial cells, WA-25 significantly and dose-dependently inhibited MMP-2/MMP-9 expression, proliferation, migration and tube formation in HUVECs. Mechanistic studies revealed that WA-25 significantly reduced the VEGF release by reducing VEGF expression at the mRNA and protein levels. In addition, WA-25 reduced surface VEGF receptor 2 (VEGFR2/Flk-1) expression by repressing the VEGFR2 mRNA level. Finally, an exogenous VEGF supply partially rescued the WA-25-induced angiogenesis blockage in vitro and in vivo. Conclusions: WA-25 is a potent angiogenesis inhibitor that acts through the down-regulation of VEGF and VEGFR2 in endothelial cells. General Significance: WA-25 may constitute a novel anti-angiogenic drug that acts by targeting endothelial VEGF/VEGFR2 signaling.