Angiogenic and Antiangiogenic mechanisms of high density lipoprotein from healthy subjects and coronary artery diseases patients.

Normal high-density lipoprotein (nHDL) in normal, healthy subjects is able to promote angiogenesis, but the mechanism remains incompletely understood. HDL from patients with coronary artery disease may undergo a variety of oxidative modifications, rendering it dysfunctional; whether the angiogenic effect is mitigated by such dysfunctional HDL (dHDL) is unknown. We hypothesized that dHDL compromises angiogenesis. The angiogenic effects of nHDL and dHDL were assessed using endothelial cell culture, endothelial sprouts from cardiac tissue from C57BL/6 mice, zebrafish model for vascular growth and a model of impaired vascular growth in hypercholesterolemic low-density lipoprotein receptor null(LDLr-/-)mice. MiRNA microarray and proteomic analyses were used to determine the mechanisms. Lipid hydroperoxides were greater in dHDL than in nHDL. While nHDL stimulated angiogenesis, dHDL attenuated these responses. Protein and miRNA profiles in endothelial cells differed between nHDL and dHDL treatments. Moreover, nHDL suppressed miR-24-3p expression to increase vinculin expression resulting in nitric oxide (NO) production, whereas dHDL delivered miR-24-3p to inhibit vinculin expression leading to superoxide anion (O2•-) generation via scavenger receptor class B type 1. Vinculin was required for endothelial nitric oxide synthase (eNOS) expression and activation and modulated the PI3K/AKT/eNOS and ERK1/2 signaling pathways to regulate nHDL- and VEGF-induced angiogenesis. Vinculin overexpression or miR-24-3p inhibition reversed dHDL-impaired angiogenesis. The expressions of vinculin and eNOS and angiogenesis were decreased, but the expression of miR-24-3p and lipid hydroperoxides in HDL were increased in the ischemic lower limbs of hypercholesterolemic LDLr-/- mice. Overexpression of vinculin or miR-24-3p antagomir restored the impaired-angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Collectively, nHDL stimulated vinculin and eNOS expression to increase NO production by suppressing miR-24-3p to induce angiogenesis, whereas dHDL inhibited vinculin and eNOS expression to enhance O2•- generation by delivering miR-24-3p to impair angiogenesis, and that vinculin and miR-24-3p may be therapeutic targets for dHDL-impaired angiogenesis.

Correction: Continuous Positive Airway Pressure Treatment Reduces Mortality in Elderly Patients with Moderate to Severe Obstructive Severe Sleep Apnea: A Cohort Study.

[This corrects the article DOI: 10.1371/journal.pone.0127775.].

Injected Amyloid Beta in the Olfactory Bulb Transfers to Other Brain Regions via Neural Connections in Mice.

Alzheimer's disease (AD) is characterized by progressive neuronal degeneration and pathological accumulation of amyloid plaques in the brain. It has been proposed that the prion-like spreading of amyloid beta (Aß) protein could contribute to the progression of the disease. Olfactory bulb (OB) is one of the earliest brain regions affected in AD and olfaction is easily impaired prior to cognitive symptoms. However, it remains unclear whether Aß accumulation in the OB would spread along olfactory projections to other connected brain regions and trigger further neurodegeneration. In the present study, we experimentally injected recombinant human Aß1-42 (monomers and oligomers, respectively) into the mouse OB and tracked the spreading of Aß to connected brain regions over 3 days. The results showed that both Aß monomers and oligomers were rapidly and readily transferred from the injection site to interconnected brain regions in a neural connection manner and triggered neuronal apoptosis in the affected brain regions. Oligomeric Aß1-42 spread more efficiently and induced more neuronal apoptosis in the affected brain regions compared to monomeric Aß1-42. Therefore, the study provides evidence that Aß peptides can transfer via neural connections and the pattern of Aß peptide spreading provides understanding to manage AD.

Modeling the Pathogenesis of Charcot-Marie-Tooth Disease Type 1A Using Patient-Specific iPSCs.

Charcot-Marie-Tooth disease type 1A (CMT1A), one of the most frequent inherited peripheral neuropathies, is associated with PMP22 gene duplication. Previous studies of CMT1A mainly relied on rodent models, and it is not yet clear how PMP22 overexpression leads to the phenotype in patients. Here, we generated the human induced pluripotent stem cell (hiPSC) lines from two CMT1A patients as an in vitro cell model. We found that, unlike the normal control cells, CMT1A hiPSCs rarely generated Schwann cells through neural crest stem cells (NCSCs). Instead, CMT1A NCSCs produced numerous endoneurial fibroblast-like cells in the Schwann cell differentiation system, and similar results were obtained in a PMP22-overexpressing iPSC model. Therefore, despite the demyelination-remyelination and/or dysmyelination theory for CMT1A pathogenesis, developmental disabilities of Schwann cells may be considered as an underlying cause of CMT1A. Our results may have important implications for the uncovering of the underlying mechanism and the development of a promising therapeutic strategy for CMT1A neuropathy.

A non-invasive risk score for predicting incident diabetes among rural Chinese people: A village-based cohort study.

OBJECTIVE: To develop a new non-invasive risk score for predicting incident diabetes in a rural Chinese population. METHODS: Data from the Handan Eye Study conducted from 2006-2013 were utilized as part of this analysis. The present study utilized data generated from 4132 participants who were ≥30 years of age. A non-invasive risk model was derived using two-thirds of the sample cohort (selected randomly) using stepwise logistic regression. The model was subsequently validated using data from individuals from the final third of the sample cohort. In addition, a simple point system for incident diabetes was generated according to the procedures described in the Framingham Study. Incident diabetes was defined as follows: (1) fasting plasma glucose (FPG) ≥ 7.0 mmol/L; or (2) hemoglobin A1c (HbA1c) ≥ 6.5%; or (3) self-reported diagnosis of diabetes or use of anti-diabetic medications during the follow-up period. RESULTS: The simple non-invasive risk score included age (8 points), Body mass index (BMI) (3 points), waist circumference (WC) (7 points), and family history of diabetes (9 points). The score ranged from 0 to 27 and the area under the receiver operating curve (AUC) of the score was 0.686 in the validation sample. At the optimal cutoff value (which was 9), the sensitivity and specificity were 74.32% and 58.82%, respectively. CONCLUSIONS: Using information based upon age, BMI, WC, and family history of diabetes, we developed a simple new non-invasive risk score for predicting diabetes onset in a rural Chinese population, using information from individuals aged 30 years of age and older. The new risk score proved to be more optimal in the prediction of incident diabetes than most of the existing risk scores developed in Western and Asian countries. This score system will aid in the identification of individuals who are at risk of developing incident diabetes in rural China.

Characterization of the constitutive behavior of municipal solid waste considering particle compressibility.

This paper presents a characterization of the mechanical behavior of municipal solid waste (MSW) under consolidated drained and undrained triaxial conditions. The constitutive model was established based on a deviatoric hardening plasticity model. A power form function and incremental hyperbolic form function were proposed to describe the shear strength and the hardening role of MSW. The stress ratio that corresponds to the zero dilatancy was not fixed but depended on mean stress, making the Rowe's rule be able to describe the stress-dilatancy of MSW. A pore water pressure reduction coefficient, which attributed to the compressibility of a particle and the solid matrix, was introduced to the effective stress formulation to modify the Terzaghi's principle. The effects of particle compressibility and solid matrix compressibility on the undrained behavior of MSW were analyzed by parametric analysis, and the changing characteristic of stress-path, stress-strain, and pore-water pressure were obtained. The applicability of the proposed model on MSW under drained and undrained conditions was verified by model predictions of three triaxial tests. The comparison between model simulations and experiments indicated that the proposed model can capture the observed different characteristics of MSW response from normal soil, such as nonlinear shear strength, pressure dependent stress dilatancy, and the reduced value of pore water pressure.

Alterations in AQP4 expression and polarization in the course of motor neuron degeneration in SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective degeneration of upper and lower motor neurons. The disease progression is associated with the astrocytic environment. Aquaporin-4 (AQP4) water channels are the most abundant AQPs expressed in astrocytes, exerting important influences on central nervous system homeostasis. The present study aimed to characterize the alterations in AQP4 expression and loca-lization in superoxide dismutase 1 (SOD1) G93A transgenic mice. SOD1G93A mice were sacrificed during the presymptomatic, disease onset and end stages and immunostaining was performed on spinal cord sections to investigate neuronal loss, glial activation and AQP4 expression in the spinal cord. It was observed that global AQP4 expression increased in the spinal cord of SOD1G93A mice as the disease progressed. However, AQP4 polarization decreased as the disease progressed, and AQP4 polarized localization at the endfeet of astrocytes was decreased in the spinal ventral horn of SOD1G93A mice at the disease onset and end stages. Meanwhile, motor neuron dege-neration and decreased glutamate transporter 1 expression in astrocytes in SOD1G93A mice were observed as the disease progressed. The results of the present study demonstrated that AQP4 depolarization is a widespread pathological condition and may contribute to motor neuron degeneration in ALS.

High expression levels of the D686N Parkinson's disease mutation in VPS35 induces α-synuclein-dependent toxicity in yeast.

Parkinson's disease (PD) is a common neurodegenerative disorder that affects ~2% of the human population aged >65. α­synuclein serves a role in the pathogenesis of PD as it is a primary component of Lewy bodies, a pathological feature of PD. Endosomal­lysosomal dysfunction may be a key factor involved in the pathophysiology of PD, and may cause PD­associated neurodegeneration via α­synuclein­dependent and ­independent mechanisms. The D620N mutation in the endosomal­lysosomal gene, vacuolar protein sorting­associated protein 35 (VPS35), has been linked to PD. To clarify the underlying cellular mechanism of the VPS35 D620N mutation in PD, cell growth and endosomal­lysosomal functions were investigated in Saccharomyces cerevisiae (sc) yeast cells that exhibited various expression levels of scVPS35, in the presence or absence of non­toxic expression levels of α­synuclein. Overexpression of the scVPS35 D686N mutation (the yeast equivalent of D620N) did not lead to toxicity in yeast. However, the co­expression of high copy numbers of scVPS35 D686N and low copy numbers of α­synuclein caused toxicity, whereas the co­expression of scVPS35 wild­type and α­synuclein did not. In addition, the scVPS35 D686N mutant enhanced α­synuclein aggregation. Fragmentation of vacuoles and subsequent inhibition of lysosome function was evident in yeast cells bearing the scVPS35 mutant. The results of the present study suggested that α­synuclein and scVPS35 were interlinked via the endosomal­lysosome pathway, which is important for the pathogenesis of PD.

Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington's disease.

Huntington's disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington's disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson's and Alzheimer's diseases. To identify potential neuroprotective molecules for Huntington's disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington's disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington's disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results.

Melatonin attenuates hLRRK2-induced sleep disturbances and synaptic dysfunction in a Drosophila model of Parkinson's disease.

Sleep problems are the most common non-motor symptoms in Parkinson's disease (PD), and are more difficult to treat than the motor symptoms. In the current study, the role of human leucine-rich repeat kinase 2 (hLRRK2), the most common genetic cause of PD, was investigated with regards to sleep problems, and the therapeutic potential of melatonin in hLRRK2­associated sleep problems was explored in Drosophila. hLRRK2 was selectively expressed in the mushroom bodies (MBs) in Drosophila and sleep patterns were measured using the Drosophila Activity Monitoring System. MB expression of hLRRK2 resulted in sleep problems, presynaptic dysfunction as evidenced by reduced miniature excitatory postsynaptic current (mEPSC) and excitatory postsynaptic potential (EPSP) frequency, and excessive synaptic plasticity such as increased axon bouton density. Treatment with melatonin at 4 mM significantly attenuated the sleep problems and rescued the reduction in mEPSC and EPSP frequency in the hLRRK2 transgenic flies. The present study demonstrates that MB expression of hLRRK2 in flies recapitulates the clinical features of the sleep disturbances in PD, and that melatonin attenuates hLRRK2-induced sleep disorders and synaptic dysfunction, suggesting the therapeutic potential of melatonin in PD patients carrying LRRK2 mutations.

Continuous Positive Airway Pressure Treatment Reduces Mortality in Elderly Patients with Moderate to Severe Obstructive Severe Sleep Apnea: A Cohort Study.

Obstructive sleep apnea (OSA) is much more prevalent in older people than in middle-aged or young populations, and has been associated with cardiovascular disease. Continuous positive airway pressure (CPAP) is the first-line therapy for OSA, but its long-term clinical benefit in the elderly is unclear. Here, we carried out a prospective cohort study to explore the survival rate and incidence of cardiovascular events in elderly patients with moderate to severe OSA who did or did not receive CPAP treatment. The study included 130 patients (104 male, 26 female; mean age: 77.8 ± 6.2 years) who were followed up for a mean of 5 ± 2.54 years (range, 1-8 years). Thirty-six patients received CPAP and 88 had no CPAP. The results showed that mortality in the untreated group (21.6%) was significantly higher than in the CPAP group (5.6%). Kaplan-Meier survival analysis showed that the survival rate in the CPAP group was 94.4%, which was markedly higher than the rate of 78.4% in the untreated group. The incidence of cardiovascular events was 13.9% in the CPAP group and 55.7% in the untreated group. The present study provides evidence that CPAP can reduce mortality in older patients with moderate to severe OSA, and lead to a good long-term prognosis. The study also indicates that death in older OSA patients is associated with cardiovascular disease and diabetes.

Comparisons of different metabolic syndrome definitions and associations with coronary heart disease, stroke, and peripheral arterial disease in a rural Chinese population.

OBJECTIVES: We estimated the prevalence of metabolic syndrome (MetS) and compared associations of different MetS definitions with coronary heart disease (CHD), stroke, and peripheral arterial disease (PAD) in a rural Chinese population. METHODS: Among 4,748 residents (2,145 men and 2,603 women) aged 30+ years in rural China from 2006 to 2007, the prevalence of MetS was estimated by using five different definitions: modified World Health Organization (WHO), Chinese Diabetes Society (CDS), the updated National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) for Asian-Americans, International Diabetes Federation (IDF), and Joint Interim Statement (JIS). Multivariable logistic regression analyses were implemented to estimate the association between MetS and the prevalence of CHD, stroke and PAD, respectively. RESULTS: Prevalence of MetS in men was 11.5% (WHO), 14.8% (CDS), 32.4% (NCEP-ATP III), 27.5% (IDF) and 39.7% (JIS) and in women was 15.7% (WHO), 20.7% (CDS), 54.2% (NCEP-ATP III), 51.5% (IDF) and 54.2% (JIS), respectively. Respective ORs (95% CI) for associating MetS with CHD in men were 1.79 (1.02-3.17), 1.25 (0.69-2.26), 1.61 (1.01-2.58), 1.84 (1.14-2.96), and 1.53 (0.96-2.43). Corresponding ORs (95% CI) for stroke in men were 2.18 (95% CI 1.20 to 3.97), 2.20 (95% CI 1.25 to 3.89), 1.71 (95% CI 1.02 to 2.84), 1.30 (95% CI 0.77 to 2.23), and 1.61 (95% CI 0.97 to 2.68), respectively. In women, CHD and stroke were significantly associated with MetS using all five definitions of MetS. In addition, PAD was associated with all five MetS definitions in men, but not in women. Only hyperglycemia and BMI were significantly associated with PAD in women. CONCLUSIONS: In this rural Chinese population, the JIS, IDF and CDS criteria may not be more suitable than WHO and updated NCEP-ATPIII definitions for screening high-risk individuals and estimating the risk of CHD and stroke from MetS, especially in men.

Paeonolum protects against MPP(+)-induced neurotoxicity in zebrafish and PC12 cells.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting 2% of the population aged over 65 years old. Mitochondrial defects and oxidative stress actively participate in degeneration of dopaminergic (DA) neurons in PD. Paeonolum, a main component isolated from Moutan cortex, has potent antioxidant ability. Here, we have examined the effects of paeonolum against MPP(+)-induced neurotoxicity in zebrafish and PC12 cells. METHODS: The overall viability and neurodegeneration of DA neurons was assessed in ETvmat2:green fluorescent protein (GFP) transgenic zebrafish, in which most monoaminergic neurons are labeled by GFP. Damage to PC12 cells was measured using a cell viability assay and assessment of nuclear morphology. Intracellular reactive oxygen species (ROS) and the level of total GSH were assessed. The mitochondrial cell death pathway including mitochondrial membrane potential, cytochrome C release and caspase-3 activity were also examined in PC12 cells. RESULTS: Paeonolum protected against MPP(+)-induced DA neurodegeneration and locomotor dysfunction in zebrafish in a concentration-dependent manner. Similar neuroprotection was replicated in the PC12 cellular model of MPP(+) toxicity. Paeonolum attenuated MPP(+)-induced intracellular ROS accumulation and restored the level of total GSH in PC12 cells. Furthermore, paeonolum significantly inhibited the mitochondrial cell death pathway induced by MPP(+). CONCLUSIONS: Collectively, the present study demonstrates that paeonolum protects zebrafish and PC12 cells against MPP(+)-induced neurotoxicity.

Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish.

High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1-36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37-42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.

Protective effects of puerarin against Aß40-induced vascular dysfunction in zebrafish and human endothelial cells.

Aß40-induced vascular dysfunction has been implicated in the pathogenesis of Alzheimer׳s disease (AD). In the present study, we investigated the possible protective effects of puerarin against Aß40-induced vascular damage and impairment to angiogenesis in transgenic TG (fli1:EGFP) zebrafish and human endothelial cells. Aß40 peptides at 5µM caused an obvious reduction of vessel branches in the subintestinal vein basket, induced NADPH oxidase-derived reactive oxygen species and impaired vascular endothelial growth factor (VEGF)-dependent angiogenesis. Pretreatment with puerarin attenuated Aß40-induced vessel reduction and impairment to angiogenesis in a dose-dependent manner. In addition, Aß40 decreased VEGF-dependent phosphorylation of Akt and eNOS, whereas puerarin treatment attenuated these detrimental effects. Furthermore, the restoration of Aß40-induced-angiogenesis impairment by puerarin was abolished by either the PI3 kinase inhibitor LY294002 (10µM) or eNOS inhibitor L-NAME. The present study suggests that puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of the PI3K/Akt/eNOS pathways.

Absence of mutations in exon 6 of the TARDBP gene in 207 Chinese patients with sporadic amyotrohic lateral sclerosis.

Mutations in the TARDBP gene, which encodes the Tar DNA binding protein, have been shown to causes of both familial amyotrophic lateral sclerosis (FALS) and sporadic ALS (SALS). Recently, several novel TARDBP exon 6 mutants have been reported in patients with ALS in Europe and America but not in Asia. To further examine the spectrum and frequency of TARDBP exon 6 mutations, we investigated their frequency in ethnic Chinese patients with sporadic ALS. TARDBP exon 6 was screened by direct sequencing in 207 non-SOD1 SALS patients and 230 unrelated healthy controls but no mutations were identified. Our data indicate that exon 6 mutations in TARDBP are not a common cause of SALS in Han Chinese population from Southern Mainland China.

Motoneuron differentiation of induced pluripotent stem cells from SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder mainly affecting motor neurons. Mutations in superoxide dismutase-1 (SOD-1) account for about 20% of familial ALS patients. A robust supply of motoneurons carrying the mutated gene would help understand the causes of motoneuron death and develop new therapeutics for the disease. Here, we established induced pluripotent stem (iPS) cell lines from SOD1G93A mice and compared their potency in motoneuron generation with normal iPS cells and mouse embryonic stem cells (E14). Our results showed that iPS cells derived from SOD1G93A mice possessed the similar potency in neuronal differentiation to normal iPS cells and E14 cells and can be efficiently driven to motoneuron-like phenotype. These cells exhibited typical neuronal morphology, expressed key motoneuron markers, including ChAT and HB9, and generated repetitive trains of action potentials. Furthermore, these neurons highly expressed human SOD-1 and exhibited shorter neurites compared to controls. The present study provides evidence that ALS-iPS cells can be used as disease models in high-throughput screening and mechanistic studies due to their ability to efficiently differentiate into specific neuronal subtypes.

Xyloketal B exhibits its antioxidant activity through induction of HO-1 in vascular endothelial cells and zebrafish.

We previously reported that a novel marine compound, xyloketal B, has strong antioxidative actions in different models of cardiovascular diseases. Induction of heme oxygenase-1 (HO-1), an important endogenous antioxidant enzyme, has been considered as a potential therapeutic strategy for cardiovascular diseases. We here investigated whether xyloketal B exhibits its antioxidant activity through induction of HO-1. In human umbilical vein endothelial cells (HUVECs), xyloketal B significantly induced HO-1 gene expression and translocation of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) in a concentration- and time-dependent manner. The protection of xyloketal B against angiotensin II-induced apoptosis and reactive oxygen species (ROS) production could be abrogated by the HO-1 specific inhibitor, tin protoporphyrin-IX (SnPP). Consistently, the suppressive effects of xyloketal B on NADPH oxidase activity could be reversed by SnPP in zebrafish embryos. In addition, xyloketal B induced Akt and Erk1/2 phosphorylation in a concentration- and time-dependent manner. Furthermore, PI3K inhibitor LY294002 and Erk1/2 inhibitor U0126 suppressed the induction of HO-1 and translocation of Nrf-2 by xyloketal B, whereas P38 inhibitor SB203580 did not. In conclusion, xyloketal B can induce HO-1 expression via PI3K/Akt/Nrf-2 pathways, and the induction of HO-1 is mainly responsible for the antioxidant and antiapoptotic actions of xyloketal B.

Marine cyclotripeptide X-13 promotes angiogenesis in zebrafish and human endothelial cells via PI3K/Akt/eNOS signaling pathways.

Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or L-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.

dl-3n-Butylphthalide promotes angiogenesis via the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase signaling pathways.

We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.