Astaxanthin induces angiogenesis through Wnt/[beta]-catenin signaling pathway.
Objective: In the present study, we sought to elucidate whether astaxanthin contributes to induce angiogenesis and its mechanisms.
Materials and methods: To this end, we examined the role of astaxanthin on human brain microvascular endothelial cell line (HBMEC) and rat aortic smooth muscle cell (RASMC) proliferation, invasion and tube formation in vitro. For study of mechanism, the Wnt/[beta]-catenin signaling pathway inhibitor IWR-1-endo was used. HMBECs and RASMCs proliferation were tested by cell counting. Scratch adhesion test was used to assess the ability of invasion. A matrigel tube formation assay was performed to test capillary tube formation ability. The Wnt/[beta]-catenin pathway activation in HMBECs and RASMCs were tested by Western blot.
Results: Our data suggested that astaxanthin induces angiogenesis by increasing proliferation, invasion and tube formation in vitro. Wnt and f)-catenin expression were increased by astaxanthin and counteracted by IWR-1-endo in HMBECs and RASMCs. Tube formation was increased by astaxanthin and counteracted by IWR-1-endo.
Conclusions: It may be suggested that astaxanthin induces angiogenesis in vitro via a programmed Wnt/[beta]-catenin signaling pathway.
Angiogenesis, the growth of new blood vessels, is a crucial force for shaping the nervous system and protecting it from disease. Angiogenesis and neurogenesis are prominent features of neurological disease, either as pathophysiological factors or as responses to injury. Neurovascular responses have a critical role as the damaged central nervous system transitions from initial injury into repair (Zhang and Chopp, 2009). Reestablishment of functional microvasculature enhances neurogenesis and functional recovery after stroke (Dalkara et al., 2011; Pries and Secomb, 2014). Enhanced angiogenesis should provide new opportunities for stroke recovery (Zeng et al., 2014).
The Wnt/[beta]-catenin signaling plays a prominent role in cell differentiation, adhesion, survival and apoptosis, and is involved in organ development, neurogenesis, and tissue fibrosis, among other functions. [beta]-catenin, a key component of the Wnt signaling pathway, interacts with the TCF/LEF family of transcription factors and activates transcription of Wnt target genes (Akiyama, 2000; Satoh and Kuroda, 2000). The lack of a single [beta]-catenin allele shows already abnormal blood vessel development and leads to embryonic lethality.
Astaxanthin is a xanthophyll carotenoid and belongs to a larger class of phytochemicals known as terpenes. Natural astaxanthin is produced and extracted from Haematococcus microalgae (Lorenz and Cysewski, 2000). It has shown that astaxanthin has antioxidant and anti-inflammatory effects (Pashkow et al., 2008). It has also shown protective potential against cerebral ischemia injury (Ergul et al., 2012). In the present study, we therefore investigated the hypothesis that astaxanthin induced angiogenesis in cerebral endothelial cells and smooth muscle cells and regulated Wnt/[beta]-catenin signaling pathway through activating [beta]-catenin.
Materials and methods
Astaxanthin (purity > 98.0%, molecular formula [C.sub.40][H.sub.52][O.sub.4]: 596.86). A stock solution of astaxanthin was made in DMSO at a concentration of 30 mM. The following pharmacologic agents were used: Wnt/[beta]-catenin signaling pathway inhibitor IWR-1-endo (Calbiochem).
Human brain microvascular endothelial cell (HEMEC) culture
A human brain microvascular endothelial cell line (HBMEC) were seeded at 60%-70% confluence and kept at 37[degrees]C in 5% C[O.sub.2]. Culture media comprised RPMI 1640 containing 10% fetal bovine serum, 10% Nu-Serum, 2 mM L-glutamine, 1 mM pyruvate, essential amino acids and vitamins.
Rat aortic smooth muscle cell (RASMC) culture
Rat aortic smooth muscle cells (RASMCs) were seeded at 60%-70% confluence and kept at 37[degrees]C in 5% C02. Culture media comprised DMEM 10% fetal bovine serum, 10% Nu-Serum, 2 mM L-glutamine, 1 mM pyruvate, essential amino acids and vitamins. RASMC cultures were obtained by enzymatic dissociation of the aortas obtained from SD rats. The isolation and culture procedure was based on a previous described method (Bochaton-Piallat et al., 1992). Cell characterization was performed based on both cell morphology and indirect immunohistochemistry staining of [alpha]-SMA. Tightly confluent monolayers of RASMCS from 2nd to 10th passage were used in all the experiments.
For in vitro proliferation assays, HBMECs were seeded into 96-well (1 x [10.sup.4] cells/well) flat bottom plates with medium alone (control) or medium containing different concentrations of astaxanthin (3,10 and 30 [micro]M). RASMCs were seeded into 96-well (8 x [10.sup.3] cells/well) flat bottom plates with medium alone (control) or medium containing different concentrations of astaxanthin (1,3 and 10 [micro]M). Cell proliferation was tested by cell counting. Briefly, for HBMECs, serum-starved cells were treated with astaxanthin for 24 h. Similarly, for RASMCs, serum-starved cells were treated with astaxanthin for 48 h. The number of cells were counted by cell counting instrument, and calculated as a ratio against untreated cells. In addition, serum-starved HBMECs were treated with astaxanthin for 24 h, and serum-starved RASMCs were treated with astaxanthin for 48 h, then collected HBMECs and RASMCs, flow cytometry analyzed cell-cycle distribution and counted the proportion of cells in S phase and G0/G1 phase.
Scratch adhesion test
HBMECs and RASMCs were seeded in 6-well plates (5 x [10.sup.4] cells/well) until the cells were fused to more than 90%, and discarded the culture liquid, then washed twice with PBS, added RPMI 1640 medium diluted with 3, 10 and 30 [micro]M astaxanthin in HBMECs, and DMEM medium diluted with 1, 3 and 10 [micro]M of astaxanthin in RASMCs, respectively, then used 200 [micro]l pipette tip to scratch and pictured after 24 h, and 48 h, measured the distance and counted in five random fields (x 100). The results were expressed as fold decrease over the control.
Matrigel tube formation assay for angiogenesis
The standard matrigel assay was used to assess the spontaneous formation of capillary-like structures in vitro. HBMECs (3 x [10.sup.4] cells/well) containing different concentrations of astaxanthin (3, 10 and 30 [micro]M), and RASMCs (2 x [10.sup.4] cells/well) containing different concentrations of astaxanthin (1, 3 and 10 [micro]M) were seeded in 24-well plates in serum-free media previously coated with growth factor-reduced matrigel matrix (BD Bioscience, San Jose, CA, USA). Wnt/[beta]-catenin signaling pathway inhibitor IWR-1-endo (CAS 1127442-82-3, 5 [micro]M) were incubated at 37[degrees]C for 12 h. Tube formations was determined in four random fields (x 200) from each well. Data were analyzed as tube formation versus untreated control wells.
In vitro oxygen and glucose deprivation model
To mimic the oxygen and glucose deprivation in vitro, HBMECs were incubated in a hypoxia solution for 6 h. The hypoxia solution contained 0.9 mM Na[H.sub.2]P[O.sub.4], 6.0 mM NaHC[O.sub.3], 1.0 mM Ca[Cl.sub.2], 1.2 mM MgS[O.sub.4], 40 mM Natrium lacticum, 20 mM HEPES, 98.5 mM NaCl, 10.0 mM KCl (pH adjusted to 6.8) and was bubbled with N2 for 30 min before application. The p[O.sub.2] of the hypoxia solution was adjusted to reach a level of 64.0 kPa. Hypoxic condition was produced by placing the plates of cultured HBMECs in a hypoxic incubator (Kendro, Germany) and oxygen was adjusted to 1.0% and C[O.sub.2] to 5.0%. Prior to hypoxia, HBMECs were pretreated with various concentrations (3,10 and 30 [micro]M) of astaxanthin for 24 h. Normal culture (RPMI 1640 containing 2% fetal bovine serum (FBS) under 20% oxygen and 5% C[O.sub.2]) served as a negative control, the hypoxia solution culture served as the control.
Determination of cell viability and LDH leakage
HBMECs were incubated with or without astaxanthin in a hypoxia solution for 6 h and HBMECs viability was assessed using an MTT assay. LDH, an indicator of cell injury, was detected according to the description of the LDH assay kit (Zhongsheng Bioreagent, PR China). LDH leakage rate (%) = Ae/At x 100%. Ae indicated extracellular LDH (cells culture fluid), at indicated intracellular and extracellular LDH (cells lysate).
Western blotting analysis
HBMECs were cultured for 24 h, and RASMCs were cultured for 48 h, then washed twice with ice cold PBS on ice and lysed in NP40 lysis buffer (Biosource, Camarillo, CA, USA) (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM [Na.sub.3]V[O.sub.4], 1% NP-40 and 0.02% Na[N.sub.3]) supplemented with 1 mM PMSF and 1 x protease inhibitor cocktail (Sigma, Saint Louis, MO, USA). Equal amounts of cell protein (40 [micro]g) were separated by SDS-PAGE and analyzed by Western blot using specific antibodies to Wnt7a, Wnt5a, GSK3[beta], [beta]-catenin, cyclin D1, Caspase-3 and [beta]-actin (as a loading control). Optical densities of the bands were scanned and quantified with a Gel Doc2000 (Bio-Rad Laboratories Ltd.). Data were normalized against those of the corresponding [beta]-actin bands. Results were expressed as fold increase over control.
All of the experiments were performed in triplicate. Quantitative data from experiments were expressed as mean [+ or -] SD. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test. P < 0.05 was considered statistically significant.
Astaxanthin augments proliferation, migration and tube formation HBMECs were incubated with different concentrations of astaxanthin (3-30 [micro]M) for 24 h and RASMCs were incubated with different concentrations of astaxanthin (1-10 [micro]M) for 48 h, respectively. The assay of proliferation, migration and tube formation as the markers of angiogenesis in vitro were examined. HBMEC and RASMC displayed a basal migration in absence of astaxanthin, respectively, while HBMECs and RASMCs treated with astaxanthin, and displayed a faster migration and induced HBMECs and RASMCs proliferation in a concentration-dependent manner, as shown in Fig. 1A and Fig. 1B. Along with increased proliferation, astaxanthin also enhanced cells migration as quantified with a scratch adhesion test, as shown in Fig. 1 A, IB, 1C and Fig. ID. Flow cytometry analysis confirmed that astaxanthin induced a significant increase in the proliferative S phase while decreasing the resting C0/G1 phase of the cell cycle, as shown in Fig. 1E and Fig. 1F. Matrigel assays showed astaxanthin induced tube formation in a concentration-dependent manner, as shown in Fig. 2A, 2B, 2C and Fig. 2D. In all of proliferation, migration and tube formation assays, the most effective concentrations of astaxanthin appeared to peak around 10 [micro]M for HBMECs, and appeared to peak around 3 [micro]M for RASMCs.
Effects of astaxanthin on cultured HBMECs against hypoxia-induced cytotoxicity
As estimated by the MTT assay, cell viability was markedly decreased after hypoxia for 6 h, as shown in Table 1. However, HBMECs were incubated with astaxanthin; cell viability was significantly increased in a concentration-dependent manner. To further investigate the protective effect of astaxanthin, LDH leakage rate was estimated; a significant increase of LDH leakage rate in HBMECs was observed after hypoxia 6 h. Incubation with various concentrations of astaxanthin significantly inhibited hypoxia-induced LDH release in a concentration-dependent manner.
Caspase 3, a key protein is presented in the process of apoptosis. The results of Western blotting showed that the Caspase 3 expression was increased significantly after oxygen and glucose deprivation (OGD) in vitro. However, Caspase 3 expression was reduced in a concentration-dependent manner after incubation with astaxanthin for 24 h, as shown in Fig. 3A and Fig. 3B.
Effects of astaxanthin on Wnt/f)-catenin signaling pathway in HBMECs with normal oxygen and OGD
In order to prove whether astaxanthin plays a protective role in normal oxygen and OGD through the signaling in HBMECs, the protein expressions were assessed which were related with Wnt/[beta]-catenin signaling pathway. HBMECs were incubated with astaxanthin (3-30 [micro]M) for 24 h robustly leading to a rapid reduce in p-GSK3[beta], and increase wnt7a, [beta]-catenin, cyclin D1 expression, as shown in Fig. 4A and Fig. 4C. HBMECs were pre-incubated with astaxanthin 10 [micro]M for 24 h, and then OGD for 6 h, similarly leading to a reduction in p-GSK3[beta], and increase wnt7a, [beta]-catenin, cyclin D1 expression relatively, compared with the OGD condition, as shown in Fig. 4B and Fig. 4D.
Astaxanthin up-regulates wnt5a, [beta]-catenin, cyclin D1. reduce p-GSK3[beta] expression in RASMC with normal oxygen
To directly link astaxanthin-induced signaling with angiogenesis in RASMCs, the protein expression related with Wnt/[beta]-catenin signaling pathway were assessed in RASMCs. RASMCs were incubated with astaxanthin (1-10 [micro]M) for 48 h robustly leading to a rapid decrease in p-GSK3[beta] and increase wnt5a, [beta]-catenin and cyclin D1 expression, as shown in Fig. 5A and Fig. 5B.
Astaxanthin induced angiogenesis via Wnt/[beta]-catenin signaling pathway
In order to investigate whether astaxanthin induced angiogenesis via Wnt/[beta]-catenin signaling pathway, the Wnt/[beta]-catenin signaling pathway inhibitor IWR-1-endo was used in Matrigel tube formation assay and western blotting.
HBMECs were incubated with astaxanthin 10 [micro]M for 24 h in 24-well plates in serum-free media previously coated with growth factor-reduced matrigel matrix, and then IWR-1-endo with 5 [micro]M continue to develop 8 h. IWR-1-endo inhibit angiogenesis in matrigel tube formation assay ,as shown in Fig. 6A and Fig. 6C. RASMCs (2 x [10.sup.4]) were incubated with astaxanthin 3 [micro]M for 48 h in 24-well plates in serum-free media previously coated with growth factor-reduced matrigel matrix then IWR-1-endo with 5 [micro]M continue to incubate for 8 h. IWR-1-endo inhibits angiogenesis in matrigel tube formation assay, as shown in Fig. 6B and Fig. 6D.
HBMECs were incubated with astaxanthin 10 [micro]M for 24 h, and then IWR-1-endo with 5 [micro]M continue to cultivate 8 h, robustly activated Wnt/[beta]-catenin signaling pathway, leading to a rapid increase in P-GSK3[beta], reduce the wnt7a, [beta]-catenin and cyclin D1 expression, as shown in Fig. 7A and Fig. 7C. Similarly, the use of IWR-1-endo increased the expression of p-GSK3[beta], reduced the expression of wnt7a, [beta]-catenin, cyclin D1 in RASMCs, as shown in Fig. 7B and Fig. 7D.
It has shown that astaxanthin is a potent neuroprotectant which reduces neuronal death in many experimental models of stroke and brain injury (Shen et al., 2009). The major finding of the present study is that astaxanthin induce proliferation, migration and tube formation in HBMECs and RASMCs in vitro. The mechanisms of this phenomenon appear to involve the Wnt/[beta]-catenin signaling. These data provide a mechanistic basis for the potential application of astaxanthin as a candidate therapy for neurovascular repair.
Endothelial progenitor cells (EPCs) possess robust therapeutic angiogenic potential, yet is limited in the capacity to develop into fully mature vasculature. Mature vasculature requires the presence of supporting elements, such as smooth muscle cells (SMCs) which are essentially vascular pericytes to enhance the angiogenic performance of EPCs (Shudo et al., 2013; Yang and Proweller, 2011). It is essential to investigate MBEC and VSMC how to play synergistic role in angiogenesis. Previous studies have confirmed that Wnt family involved in multiple signaling pathways to regulate cell proliferation, differentiation, survival and migration, and closely related to the occurrence and development of angiogenesis (Masckauchan et al., 2005; Reis and Liebner, 2013). It demonstrated that GSK-3[beta] plays a key role in regulating embryonic development, cell proliferation, adhesion and survival, and inflammation by converting multiple signaling pathways (Cheng et al., 2011; Takahashi-Yanaga, 2013). GSK-3[beta] is constitutively active in quiescent cells and its kinase activity is negatively regulated by the phosphorylation of an N-terminal serine residue (Ser9) which can be modulated by Wnt/[beta]-catenin signaling (Glass and Karsenty, 2006). Inhibition of GSK-3[beta] is beneficial in preventing inflammation and protecting vascular structures during hypoxia (Hummler et al., 2013). Wnt suppresses GSK-3[beta] phosphorylation and causes activation of [beta]-catenin. When different concentrations of astaxanthin cultivate HBMECs and RASMCs, the expression of p-GSK3[beta] decreased obviously, while [beta]-catenin expression increased significantly. Similarly, the use of IWR-1-endo inhibits the activity of p-GSK-[beta]. However, compared to astaxanthin, astaxanthin plus IWR-1-endo didn't decrease p-GSK3[beta] expression and increase [beta]-catenin expression, as is shown in Fig. 4, Fig. 5 and Fig. 7.
As demonstrated Wnt/[beta]-catenin signaling pathway utilizes [beta]-catenin as an effector to transmit a receptor mediated signal from the cytosol to the nucleus where it interacts with and activates the Tcf/Lef transcription factors (Masckauchan et al., 2005; Nagaoka et al., 2013; Reis and Liebner, 2013). In our study, astaxanthin up-regulated [beta]-catenin expression in HBMECs and RASMCs, the major effects of astaxanthin appeared to take place via anti-phosphorylation rather than absolute alterations of protein levels. It has recently shown that the nuclear presence of [beta]-catenin resulted in the significant enhancement of TCF-dependent promoter activity and activation of the [beta]-catenin downstream targets, Cyclin D1 (Ripple et al., 2014) is a key regulatory protein in the cell cycle, playing a critical role in the transition from G1 to S phase of the cell cycle (Fu et al., 2004; Pestell, 2013). As is shown in Fig. 1, Fig. 2 and Fig. 7, astaxanthin significantly promote the expression of Cyclin D1 in HBMECs and RASMCs, launching cells division, proliferation and migration, inducing the occurrence of angiogenesis. It suggested that astaxanthin promoted proliferation and migration of MBEC and VSMC, and might play synergistic role in angiogenesis.
As demonstrated that apoptosis and survival coexist in the process of vascular endothelial cell adhesion, proliferation and migration (Cardone et al., 1998; Omura et al., 2013). How to make those cells survive? Reducing the apoptosis is very important. The results showed that oxygen glucose deprivation increased Caspase-3 activity in cultured HBMECs. Moreover, treatment with astaxanthin inhibited oxygen glucose deprivation induced cell apoptosis.
Taken together, our findings suggest that astaxanthin may be a novel way to induce angiogenesis and play synergistic effect in HBMECs and RASMCs. But there are several important caveats to keep in mind. First, although our data provides cellular and pharmacologic proof of principle for astaxanthin in cerebral angiogenesis, in vivo validation of these mechanisms remain to be obtained. The proangiogenic utility of astaxanthin as a potential stroke recovery therapy should be explored in future experiments.
In summary, the present study provides mechanistic evidence that astaxanthin induces angiogenesis in cerebral endothelial cells and RASMCs via Wnt/[beta]-catenin signaling. Further in vivo and clinical exploration of these pathways is warranted to validate these experimental findings and develop astaxanthin as a potential neurovascular repair therapy for stroke and brain injury.
Conflict of interest
The authors have no conflicts of interest to declare.
Abbreviations HBMEC human brain microvascular endothelial cell line RASMC rat aortic smooth muscle cell SMCs smooth muscle cells OGD oxygen and glucose deprivation EPCs endothelial progenitor cells FBS fetal bovine serum
Received 23 March 2015
Revised 21 May 2015
Accepted 24 May 2015
The study was supported by National Natural Science Foundation of China (Grant NO.: 31170321) and in part financially supported by National Natural Science Foundation of China (Grant NO.: 31170391) and Taishan Scholar Project to Xuri Li.
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Yangyang Xu, Jie Zhang Wanglin Jiang *, Shuping Zhang
School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, 264003, PR. China
* Corresponding authors. Tel.: +86 535 6912036; fax: +86 535 6912036.
E-mail addresses: firstname.lastname@example.org (W. Jiang), email@example.com (S. Zhang).
Table 1 Effects of astaxanthin on the viability and LDH leakage in HBMECs exposed to oxygen-glucose deprivation. Groups OGD Content ([micro]M) Cell viability (%) Normal - -- 100 [+ or -] 6.4 Control + -- 54.5 [+ or -] 4.2 (a) + 3 69.3 [+ or -] 2.6 (b) Astaxanthin + 10 73.3 [+ or -] 2.2 (b) + 30 70.8 [+ or -] 1.8 (b) Groups OGD LDH leakage (%) Normal - 2.1 [+ or -] 0.7 Control + 21.2 [+ or -] 3.6 (a) + 15.4 [+ or -] 1.7 (b) Astaxanthin + 12.6 [+ or -] 1.1 (b) + 14.4 [+ or -] 1.1 (b) After 6 h oxygen-glucose deprivation (OGD) followed by 12 h incubation with astaxanthin, cell viability and LDH leakage were assessed. Normal: no oxygen-glucose deprivation (OGD); Control: OGD. Values are mean [+ or -] S.D. (n = 6). Significance was determined by one-way ANOVA followed by Dunnett's test. (a) P < 0.01 versus normal group; (b) P < 0.01 versus control group.
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|Author:||Xu, Yangyang; Jiang, Jie Zhang Wanglin; Zhang, Shuping|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jul 15, 2015|
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