Association between weight cycling and risk of colorectal cancer: a prospective cohort study.

BACKGROUND: Weight cycling is the repeated episodes manifesting intentional weight loss and subsequent unintentional weight gain. Whether the frequency and magnitude of weight cycling is associated with colorectal cancer risk independent of body mass index (BMI) remains unknown. METHODS: Two prospective cohort studies, Nurses' Health Study I and Health Professionals Follow-up Study, followed 85,562 participants from 1992 to 2014. Participants completed a questionnaire regarding the frequency and magnitude of intentional weight loss in the past 4 years at the baseline. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox proportional hazard model. RESULTS: We identified 1626 colorectal cancer cases during up to 22 years of follow-up. In the pooled analysis of HPFS and NHS, compared to non-weight cycling, moderate weight cycling (≥3 times of intentional weight loss of ≥2.3-4.4 kg) was associated with a reduced risk of colorectal cancer after adjustment for confounders, including attained BMI after weight cycling (HR = 0.82, 95% CI 0.69, 0.97). However, no significant association was observed in mild weight cyclers and in severe weight cyclers. CONCLUSIONS: Moderate weight cycling was associated with a lower risk of colorectal cancer independent of BMI. This finding needs further studies for replication and putative biological mechanisms.

O-glycans on death receptors in cells modulate their sensitivity to TRAIL-induced apoptosis through affecting on their stability and oligomerization.

The TNF-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in cells by signaling through the O-glycosylated death receptors (DR4 and DR5), but the sensitivity to TRAIL-induced apoptosis of cells varies, and the attributes of this phenomenon are complex. Human carcinoma cells often express truncated O-glycans, Tn (GalNAcα1-Ser/Thr), and Sialyl-Tn (Siaα2-6GalNAcα1-Ser/Thr, STn) on their surface glycoproteins, yet molecular mechanisms in terms of advantages for tumor cells to have these truncated O-glycans remain elusive. Normal extended O-glycan biosynthesis is regulated by a specific molecular chaperone Cosmc through assisting of the correct folding of Core 1 ß3 Galactosyltransferase (T-synthase). Here, we use tumor cell lines harboring mutations in Cosmc, and therefore expressing Tn and STn antigens to study the role of O-glycans in TRAIL-induced apoptosis. Expression of Tn and STn in tumor cells attenuates their sensitivity to TRAIL treatment; when transfected with wild-type Cosmc, these tumor cells thus express normal extended O-glycans and become more sensitive to TRAIL treatment. Mechanistically, Tn/STn antigens impair homo-oligomerization and stability of DR4 and DR5. These results represent the first mechanistic insight into how O-glycan structures on cell surface modulate their sensitivity to apoptotic stimuli, suggesting expression of Tn/STn may offer tumor cell survival advantages through altering DR4 and/or DR5 activity.

Comparing Verum and Sham Acupuncture in Fibromyalgia Syndrome: A Systematic Review and Meta-Analysis.

OBJECTIVES: Acupuncture is often used for relieving symptoms of fibromyalgia syndrome (FMS). Our aim is to ascertain whether verum acupuncture is more effective than sham acupuncture in FMS. METHODS: We collected RCTs to investigate the effects of verum acupuncture and sham acupuncture on pain, sleep quality, fatigue, and general status in FMS patients. The databases used for data retrieval were PubMed, Central Cochrane, EMBASE, PsycINFO, CNKI, VIP, OASIS, KoreaMed, and RISS. Selection/exclusion from the retrieved records was performed according to prespecified criteria, and the final selected records were assessed according to the Cochrane risk of bias tool. The results of the included trials were synthesized on the basis of outcomes, and subgroup analysis depended on the type of add-on sham acupuncture that was performed. RESULTS: Ten RCTs (690 participants) were eligible, and eight RCTs were eventually included in the meta-analysis. The synthesis showed a sizable effect of verum acupuncture compared with sham acupuncture on pain relief (standardized mean difference (SMD) -0.49, Z = 3.26, P=0.001; I 2 = 59%), improving sleep quality (SMD -0.46, Z = 3.24, P=0.001; I 2 = 0%), and reforming general status (SMD -0.69, Z = 6.27, P < 0.00001; I 2 = 4%). However, efficacy on fatigue was insignificant (SMD -0.10, Z = 0.51, P=0.61; I 2 = 46%). When compared with a combination of simulation and improper location of needling, the effect of verum acupuncture for pain relief was the most obvious. CONCLUSIONS: Verum acupuncture is more effective than sham acupuncture for pain relief, improving sleep quality, and reforming general status in FMS posttreatment. However, evidence that it reduces fatigue was not found.

Reliability, Accuracy, and Use Frequency of Evaluation Methods for Amount of Tongue Coating.

OBJECTIVE: To classify the evaluation methods for amount of tongue coating (TC) and investigate their reliability, accuracy, and frequency of use. METHODS: Articles published from 1985 to 2015 were searched for evaluation methods for the amount of TC in PubMed and the Cochrane Library. Only clinical researches were included except protocol articles. The methods were classified according to their characteristics. RESULTS: Finally, 113 articles were selected. The evaluation method for the amount of TC from the articles was classified into 4 types: intuitive, specificative, computerized, and weighing TC. The reliability in the intuitive and specificative methods (κ =0.33-0.92) showed varying levels among the studies. In general, the amount of TC calculated by the specificative method (Spearman's r=0.68-0.80) was more strongly related to the directly measured value than to the value estimated by the computerized method (Pearson's r=0.442). The number of articles published on this topic has increased consistently, and the specificative method was the most frequently used. Despite the higher reliability of the computerized method, it has not been widely used. CONCLUSIONS: The high prevalence of the specificative method would continue in clinical practice because of its convenience and accuracy. However, to establish higher reliability, the limitation of the subjectivity of the assessors should be overcome through calibration training. In the computerized method, novel algorithms are needed to obtain a higher accuracy so that it can help the practitioners confidently estimate the amount of TC.

Comprehensive analysis of N-glycans in IgG purified from ferrets with or without influenza A virus infection.

Influenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, N-glycosylation of IgGs plays a critical role in antigen binding and effector functions. The ferret is the most commonly used animal model for studying influenza pathogenesis, virus transmission, and vaccine development, but its IgG structure and functions remain largely undefined. Here we show that ferret IgGs are N-glycosylated and that their N-glycan structures are diverse. Using a comprehensive strategy based on MS and ultra-HPLC analyses in combination with exoglycosidase digestions, we assigned 42 N-glycan structures in ferret IgGs. We observed that N-glycans of ferret IgGs consist mainly of complex-type glycans, including some high-mannose and hybrid glycans, similar to those observed in human IgG. The complex-type glycans of ferret IgGs were primarily core-fucosylated. Furthermore, a fraction of N-glycans carried bisecting GlcNAc. Ferret IgGs also had a minor fraction of glycans carrying α2-6Neu5Ac(s). We noted that, unlike human IgG, ferret IgGs have αGal epitopes on some N-glycans. Interestingly, influenza A infection caused prominent changes in the N-glycans of ferret IgG, mainly because of an increase in bisecting GlcNAc and F1A2G0 and a corresponding decrease in F1A2G1. This suggests that the glycosylation of virus-specific IgG may play a role in its functionality. Our study highlights the need to further elucidate the structure-function relationships of IgGs in universal influenza vaccine development.

Comparative Analysis of Tongue Indices between Patients with and without a Self-Reported Yin Deficiency: A Cross-Sectional Study.

We investigated the hypothesis that Yin-deficient patients have a reddened tongue with less coating. We screened 189 participants aged 20 to 49 years, complaining of headache. To classify patients in terms of Yin deficiency, we used two self-reporting Yin-deficiency questionnaires (Yin-Deficiency Questionnaire and Yin-Deficiency Scale) and diagnosis by a doctor. Based on the tests, a total of 33 subjects were assigned to a Yin-deficient group and 33 subjects were assigned to a nondeficient control group. Tongue images were acquired using a computerized tongue diagnostic system, for evaluating tongue indices. The tongue coating percentage and tongue redness were calculated as the mean a⁎ value of both the whole tongue area (WT a⁎) and the tongue body area (TB a⁎). The tongue coating percentage of the Yin-deficient group (34.79 ± 10.76) was lower than that of the nondeficient group (44.13 ± 14.08). The WT a⁎ value of the Yin-deficient group (19.39 ± 1.52) was significantly higher than that of the nondeficient group (18.21 ± 2.06). However, the difference in the TB a⁎ value between the two groups was not significant. In conclusion, we verified that Yin-deficient patients had less tongue coating and tended to have a more reddish tongue than nondeficient patients.

Involvement of Heme Oxygenase-1 in Orexin-A-induced Angiogenesis in Vascular Endothelial Cells.

The cytoprotective enzyme heme oxygenase-1 (HO-1) influences endothelial cell survival, proliferation, inflammatory response, and angiogenesis in response to various angiogenic stimuli. In this study, we investigate the involvement of HO-1 in the angiogenic activity of orexin-A. We showed that orexin-A stimulates expression and activity of HO-1 in human umbilical vein endothelial cells (HUVECs). Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin-A-induced angiogenesis in vivo and ex vivo. Orexin-A-stimulated endothelial tube formation and chemotactic activity were also blocked in SnPP-treated vascular endothelial cells. Orexin-A treatment increased the expression of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and antioxidant response element (ARE) luciferase activity, leading to induction of HO-1. Collectively, these findings indicate that HO-1 plays a role as an important mediator of orexin-A-induced angiogenesis, and provide new possibilities for therapeutic approaches in pathophysiological conditions associated with angiogenesis.

Spatial dynamics of TRAIL death receptors in cancer cells.

TNF-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells without harming most normal cells. Currently, multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL (rhTRAIL) and agonistic antibodies that target death receptors (DRs) 4 or 5. It is encouraging that these products have shown a tolerated safety profile in early phase studies. However, their therapeutic potential is likely limited by the emergence of tumor drug resistance phenomena. Increasing evidence indicates that TRAIL DRs are deficient on the plasma membrane of some cancer cells despite their total protein expression. Notably, the lack of surface DR4/DR5 is sufficient to render cancers resistant to TRAIL-induced apoptosis, regardless of the status of other apoptosis signaling components. The current review highlights recent findings on the dynamic expression of TRAIL death receptors, including the regulatory roles of endocytosis, autophagy, and Ras GTPase-mediated signaling events. This information could aid in the identification of novel predictive biomarkers of tumor response as well as the development of combinational drugs to overcome or bypass tumor drug resistance to TRAIL receptor-targeted therapies.

Visfatin promotes cell and tumor growth by upregulating Notch1 in breast cancer.

Overexpression of Notch1 has been associated with breast cancer. We recently showed that visfatin stimulates breast cancer cell proliferation and invasion. The present study was undertaken to determine whether Notch1 signaling is affected by visfatin and to characterize the functional role of the visfatin-Notch1 axis in breast cancer. Visfatin and Notch1 were expressed at higher levels in breast tumors than in matched control tissues. Visfatin induced Notch1 expression in MDA-MB-231 breast cancer cell line and in nontransformed MCF10A mammary epithelial cells, whereas visfatin depletion reduced Notch1 mRNA and protein levels. Depletion of Notch1 in MDA-MB-231 cells attenuated cell growth in vitro and in vivo; visfatin depletion produced similar effects, but was less potent. Additionally, Notch1 depletion inhibited cell proliferation induced by visfatin. Analysis of the signaling pathways underlying visfatin-mediated Notch1 upregulation revealed that visfatin activated NF-κB p65. Blockade of NF-κB signaling suppressed the effects of visfatin on Notch1 upregulation and breast cancer cell proliferation. Breast tumors expressing high levels of NF-κB p65 exhibited increased expression of Notch1. Our results demonstrate that the visfatin-Notch1 axis contributes to breast tumor growth through the activation of the NF-κB pathway. Study of the visfatin-Notch1 axis may offer new therapeutic directions for breast cancer.

Hypoxia regulates the expression of the neuromedin B receptor through a mechanism dependent on hypoxia-inducible factor-1α.

The neuromedin B receptor (NMB-R), a member of the mammalian bombesin receptor family, is frequently overexpressed in various tumors. In the present study, we found that exposure to hypoxic conditions increases the levels of NMBR mRNA and protein in breast cancer cells, which are tightly regulated by hypoxia-inducible factor-1α (HIF-1α). We confirmed the effect of HIF-1α on NMBR transcription by performing an NMBR promoter-driven reporter assay and then identified a functional hypoxia-responsive element (HRE) in the human NMBR promoter region. Further, the binding of HIF-1α to the NMBR promoter was corroborated by electrophoretic mobility shift and chromatin immunoprecipitation assays, which showed that HIF-1α specifically and directly bound to the NMBR promoter in response to hypoxia. Immunohistochemical analysis of a xenograft and a human breast cancer tissue array revealed a significant correlation between NMB-R and HIF-1α expression. Taken together, our findings indicate that hypoxia induces NMB-R expression through a novel mechanism to regulate HIF-1α expression in breast cancer cells.

Glycyrrhetinic acid inhibits Porphyromonas gingivalis lipopolysaccharide-induced vascular permeability via the suppression of interleukin-8.

OBJECTIVE: Porphyromonas gingivalis is a major periodontopathogen that plays a role in the pathogenesis of periodontal disease. In this study, we investigated the effect of 18alpha-glycyrrhetinic acid (18α-GA), a natural triterpenoid compound derived from licorice root extract, on P. gingivalis lipopolysaccharide (LPS)-induced vascular permeability, which is a hallmark of inflammatory diseases such as periodontitis. METHODS: The inhibitory effects of 18α-GA on endothelial permeability were determined by measuring in vivo and in vitro endothelial permeability. Endothelial cells were pretreated with 18α-GA before exposure to P. gingivalis LPS, and total RNA or proteins were extracted and analyzed by reverse transcription polymerase chain reaction or western blotting. RESULTS: Porphyromonas gingivalis LPS-induced endothelial permeability was significantly inhibited by 18α-GA both in vivo and in vitro. 18α-GA reduces P. gingivalis LPS-induced gap formation of endothelial cells. Importantly, 18α-GA modulated the expression and secretion of interleukin-8 (IL-8), a key inducer of vascular permeability, by downregulating nuclear factor-κB (NF-κB). 18α-GA suppressed P. gingivalis LPS-stimulated inhibitor of kappa B (IκB) kinase activation, IκBα phosphorylation, and nuclear translocation of NF-κB. CONCLUSIONS: Overall, these findings suggest that 18α-GA significantly reduces P. gingivalis LPS-induced vascular permeability by repressing NF-κB-dependent endothelial IL-8 production, suggesting its therapeutic potential in P. gingivalis-related vascular diseases.

Upregulation of thromboxane synthase mediates visfatin-induced interleukin-8 expression and angiogenic activity in endothelial cells.

Thromboxane synthase (TXAS) is an enzyme that catalyzes the synthesis of thromboxane A(2) (TXA(2)). Overexpression of TXAS is associated with a variety of vascular diseases. Recently, we reported that visfatin, a novel adipokine, exhibits angiogenic actions. In this study, we showed that visfatin increased mRNA and protein levels of TXAS and stimulated TXA(2) biosynthesis in vascular endothelial cells. In addition, visfatin induced the expression and secretion of interleukin-8 (IL-8), which is blocked by a TXAS inhibitor and by the transfection of siRNA specific for TXAS. Furthermore, the inhibition of TXAS activity and blockade of the IL-8 receptor attenuated visfatin-induced endothelial angiogenesis. Together, these results showed that visfatin promoted IL-8 production by upregulation of TXAS, leading to angiogenic activation in endothelial cells.

Curcumin down-regulates visfatin expression and inhibits breast cancer cell invasion.

Obesity is frequently associated with breast cancer. Such associations are possibly mediated by adipokines. Visfatin, an adipokine, has recently been shown to be related to the development and progression of breast cancer. Therefore, the down-regulation of visfatin may be a novel strategy for breast cancer therapy. Curcumin has anticancer activities by modulating multiple signaling pathways and genes. The purpose of this study was to investigate whether visfatin gene expression is affected by curcumin in human breast cancer cells and to characterize the functional role of visfatin in breast cancer. We found that the mRNA and protein levels of visfatin were down-regulated by curcumin in MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cells, along with decreased activity of constitutive nuclear factor (NF)-κB. We confirmed the repressive effect of curcumin on visfatin transcription by performing a visfatin promoter-driven reporter assay and identified two putative NF-κB-binding sites on visfatin promoter that are important for this effect. EMSA and chromatin immunoprecipitation analysis indicated the binding of p65 to the visfatin promoter, which was effectively blocked by curcumin. Enforced expression of p65 protein increased visfatin promoter activity, whereas blocking NF-κB signaling suppressed visfatin gene expression. Visfatin could enhance the invasion of MDA-MB-231 cells and also attenuate curcumin-induced inhibition of cell invasion; on the other hand, visfatin knockdown by small interfering RNA led to the reduction of cell invasion. Our data demonstrate, for the first time, that curcumin down-regulates visfatin gene expression in human breast cancer cells by a mechanism that is, at least in part, NF-κB dependent and suggest that visfatin may contribute to breast cancer cell invasion and link obesity to breast cancer development and progression.

Neuromedin B receptor antagonist suppresses tumor angiogenesis and tumor growth in vitro and in vivo.

Neuromedin B (NMB), a member of the mammalian bombesin-like peptide family, and its receptor were aberrantly expressed in vascularized solid tumors. Here, the NMB receptor (NMB-R) antagonist PD168368 specifically inhibited both NMB-induced in vivo and in vitro angiogenesis. In addition, PD168368 showed growth inhibitory effects on MDA-MB-231 breast cancer cells by inducing cell cycle arrest and apoptosis. Furthermore, PD168368 effectively suppressed tumor growth in a xenograft model of breast tumor in vivo. Overall, NMB-R antagonist exhibited a significant antitumor activity by simultaneously inhibiting neovascularization and cancer cell growth, thereby suggesting that NMB-R could be a potential therapeutic target for cancer treatment.

Notch1 mediates visfatin-induced FGF-2 up-regulation and endothelial angiogenesis.

AIMS: Our aims were to determine the role of Notch1 in mediating visfatin-induced angiogenesis and to explore potential target genes involved. METHODS AND RESULTS: Inhibition of Notch signalling attenuated visfatin-induced angiogenesis in vitro, ex vivo, and in vivo. Visfatin increased γ-secretase activity, Notch1 cleavage and activation, and Hes1 gene induction. Visfatin also stimulated fibroblast growth factor-2 (FGF-2) gene expression in a Notch1-dependent manner. Enforced expression of active Notch1 intracellular domain increased FGF-2 protein levels and stimulated endothelial tube formation, whereas blocking Notch1 signalling or knockdown of Notch1 by small interfering RNA suppressed visfatin-induced FGF-2 up-regulation and angiogenesis. Reporter analysis of FGF-2 promoter revealed the presence of CSL (CBF-1, suppressor of hairless, LAG-1)-binding site, and chromatin immunoprecipitation analysis demonstrated the binding of Notch1-CSL complex to this site in response to visfatin. CONCLUSION: Our data provide the first example of Notch1-dependent endothelial FGF-2 induction by visfatin and of Notch1 activation in visfatin-stimulated endothelial angiogenesis, suggesting that the signalling axis of visfatin/Notch1/angiogenic factors like FGF-2 might be a valuable target for pathological angiogenesis.

Angiogenic role of orexin-A via the activation of extracellular signal-regulated kinase in endothelial cells.

Orexin-A, a neuropeptide originally discovered in the hypothalamus, is found in peripheral organs, as well as in the central nervous system, and is involved in the regulation of food intake, energy homeostasis, and cardiovascular functions. In this study, we report that orexin-A induces invivo neovascularization in a mouse Matrigel plug and ex vivo sprouting of endothelial cells in rat aortic rings. We also show that orexin-A increases migration and tube formation in human umbilical vein endothelial cells (HUVECs), and this effect is mediated by orexin receptors on endothelial cells. Moreover, orexin-A activates the extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs, which is closely linked to angiogenic responses. The inhibition of ERK activation significantly suppresses orexin-A-stimulated endothelial angiogenesis. Taken together, our results indicate that orexin-A functions as a new proangiogenic peptide and requires MEK/ERK-dependent pathway for its angiogenic actions. These results suggest orexin-A and its receptor may act as important modulators of angiogenesis under pathophysiological conditions.

Thymosin ß4 induces the expression of vascular endothelial growth factor (VEGF) in a hypoxia-inducible factor (HIF)-1α-dependent manner.

Thymosin ß4 has multi-functional roles in cell physiology, but little is known about its mechanism(s) of action. We previously reported that thymosin ß4 stimulated angiogenesis through the induction of vascular endothelial growth factor (VEGF). To identify the mechanism of VEGF induction by thymosin ß4, we have used a luciferase assay system with VEGF in the 5' promoter region. We also analyzed the effect of thymosin ß4 on VEGF mRNA stability and on the expression and stability of hypoxia-inducible factor (HIF)-1α. We found that thymosin ß4 induces VEGF expression by an increase in the stability of HIF-1α protein. Analysis of the expression patterns of thymosin ß4 and HIF-1α in colon cancer tissue microarray showed that thymosin ß4 and HIF-1α co-localized in these biopsies. These data show that thymosin ß4 induces the expression of VEGF indirectly by increasing the protein stability of HIF-1α.

Thromboxane A(2) increases endothelial permeability through upregulation of interleukin-8.

Thromboxane A(2) (TXA(2)), a major prostanoid formed from prostaglandin H(2) by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA(2) mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-kappaB (NF-kappaB). U46619 induced the activation of NF-kappaB through IkappaB kinase (IKK) activation, IkappaB phosphorylation and NF-kappaB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-kappaB activation in endothelial cells.

Visfatin through STAT3 activation enhances IL-6 expression that promotes endothelial angiogenesis.

Signal transducer and activator of transcription 3 (STAT3) acts as a mediator and biomarker in endothelial activation. We have recently shown that a novel adipokine visfatin promotes endothelial angiogenesis. The present study was to determine whether visfatin affects STAT3 activity and to explore the potential target gene(s). Here, we found that visfatin induced the activation of STAT3, as characterized by increased tyrosine phosphorylation, nuclear translocation, and DNA-binding activity in human endothelial cells. In addition, visfatin significantly upregulated mRNA and protein levels of endothelial interleukin-6 (IL-6), which was blocked by a specific inhibitor of STAT3 signaling and by the transfection of siRNA specific for STAT3. Furthermore, visfatin-induced angiogenesis was reduced by the inhibition of STAT3 signaling or neutralization of IL-6 function, as measured by tube formation, rat aortic ring assay, and mouse Matrigel plug assay. Taken together, our results provide the first example of STAT3-dependent endothelial IL-6 induction by visfatin and of the role of IL-6 in mediating visfatin-induced angiogenesis.

Neuromedin B induces angiogenesis via activation of ERK and Akt in endothelial cells.

Neuromedin B (NMB) is one of the bombesin-like peptides in mammals. Recently, bombesin-like peptides have been characterized as growth factors in highly vascularized tumors. In this study, we report that NMB potently stimulates in vivo neovascularization in a mouse Matrigel plug and the sprouting of endothelial cells ex vivo in rat aortic rings. In addition, NMB increases the migration and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with NMB activates the extracellular signal-regulated kinase 1/2 (ERK(1/2)), Akt, and endothelial nitric oxide synthase (eNOS) and increases the level of NO production in a dose- and time-dependent manner. Furthermore, ERK activation and angiogenic sprouting in response to NMB are significantly blocked by the MEK inhibitor. Inhibition of phosphatidylinositol 3-kinase (PI3K) suppresses the NMB-stimulated tubular formation of HUVECs, along with reduction in the phosphorylation of Akt and eNOS. Taken together, these results indicate that NMB is a novel angiogenic peptide, and its angiogenic activity is mediated by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent pathways. This study suggests that NMB may play important roles in mediating a variety of pathophysiological angiogenesis.