Pro-angiogenic effects of Carthami Flos whole extract in human microvascular endothelial cells in vitro and in zebrafish in vivo.
Aim: Carthami Flos (CF) is a Chinese herb traditionally used fo r cardiovascular disease and bone injury in China with pharmacological effects on improving blood circulation. The aim of this study was to investigate the angiogenic potential of CF whole extract (extracted by boiling with water, followed by ethanol) and the underlying mechanisms in human microvascular endothelial cells (HMEC-1) in vitro and in transgenic TG[(fli1:EGFP).sup.y1]/+ (AB) zebrafish with transgenic endothelial cells expressing EGFP (Enhanced Green Fluorescent Protein) in vivo.
Methods: Effects of CF whole extract on cell proliferation, migration and tube formation in HMEC-1 cells in vitro were detected by MTT assay, wound healing assay and tube formation assay. Its angiogenic effect in zebrafish was investigated by monitoring the sprout number in the sub-intestinal vessel (SIV), and the underlying mechanisms were tested by quantitative real-time PCR.
Results: CF whole extract increased cell proliferation, migration and tube formation in vitro in HMEC 1 cells. Its angiogenic effect was also confirmed in vivo in zebrafish by increasing the sprout number in the SIV. As determined by quantitative real-time PCR, CF whole extract up-regulated the expression of angiogenesis-related genes in zebrafish, including angiogenic and its associated growth factors and receptors (e.g. IGF1, CTGF, NRP2, and VEGFR3), transcription factor (e.g. HIF1A), matrix degradation and endothelial cell migration-related factors (e.g. MMP2, MMP9, TIMP2, PLG and PLAU), cell adhesion molecules (e.g. ITGAV, ITGB3, beta-catenin and PECAM1), tubule formation factors (e.g. ANGPT1, TIE2, PDGFR-B, CDH5, S1PR1, FGF2, Shh, and TGFRB1), and blood vessel maturation/formation factor (e.g. Ephrin B2).
Conclusions: CF whole extract increased angiogenesis in HMEC-1 cells in vitro and in zebrafish in vivo with multiple mechanisms.
Human microvascular endothelial cells
As a critical physiological process, angiogenesis is responsible for forming new blood vessels which can facilitate ischemic sites for tissue repair (Folkman, 1995). Several diseases, such as myocardial ischemia, bone fracture or ischemic chronic wounds (e.g. diabetic limb), are required to promote angiogenesis so that new blood vessels can form and more nutrients and oxygen can be transferred to these ischemia sites (Chim et al., 2013; Jude et al., 2010). Endothelial cells play critical roles in the angiogenic processes such as proliferation, migration and assembly; meanwhile, a number of angiogenic factors have been identified which are supposed to be the targets of potential regulators (Lin et al., 2010). Using small molecules as angiogenic regulators is one of the approaches to promote angiogenesis. Up to now, in vitro human vascular cell models for screening angiogenic compounds can individually mimic most of the above-mentioned mechanisms (e.g. proliferation, migration, and tube formation) of angiogenesis, but not in a single assay. In vivo zebrafish model is a pre-mammalian screening model for testing the angiogenic effects of tested compounds/extracts in the whole interconnected organ systems (Littleton and Hove, 2013). When compared to the chicken embryo chorio-allantoic membrane (CAM), zebrafish model possesses advantages such as convenient and obvious monitoring, high-throughput and low-cost. However, the zebrafish still need other methods to confirm related mechanisms. Thus, vascular cell models and zebrafish model can compensate each other when they are used together.
Carthamus tinctorius L. ("Hong-Hua" in Chinese) belongs to the Asteraceae family, and its florets Carthami Flos (CF) is a traditionally used Chinese herbal medicine in China. According to traditional Chinese medicine (TCM) theory, activating blood circulation and removing blood stasis are the pharmacological principles of CF extract (Lu et al., 2008). In the vascular system, CF can increase blood flow, dilate artery, ameliorate myocardial ischemia, and lower blood pressure (Han et al., 2013; Liu et al., 2012; Siow et al., 2000). CF is commonly used with other medicinal herbs such as Semen Persicae (the dried ripe seed of Primus persica L. and Danshen (the dried root of Salvia miltiorrhiza Bunge) to form herbal pairs for massively increasing blood circulation in the treatment of cardiovascular and cerebrovascular diseases via oral administration (Fu et al., 2012; Liu et al., 2012; Sun et al., 2009). Another traditionally used approach of CF is to cure bruise and bone injury through topical application. CF whole extract (extracted by boiling with water, followed by ethanol) topically used can relieve pain and swelling for bone fracture treatment due to its capability of removing blood stasis (Peng et al., 2010). CF water extract also possesses anti-inflammation activity and activates immune system (Choi et al., 2007; Tien et al., 2010). With regards to the chemical components of the CF water extract, chalcone flavonoids like hydroxysafflor yellow A are considered to be the major compounds responsible for the curative effects. Besides, CF water extract also contains other flavonoids such as kaempferol, quercertin and apigenin (Fan et al., 2009).
Due to the therapeutic effects of CF in the vascular system and bone injury, it was of our interest to explore the pro-angiogenic effects of CF whole extract. Previous study has preliminarily reported the pro-angiogenic effects of CF water extract on CAM without detailed investigations on related mechanisms (Gao et al., 2005). However, there are few reports on the pro-angiogenic effects of CF whole extract in human endothelial cell models or using zebrafish, and the underlying mechanisms are not fully understood. In the current study, the angiogenic effect of CF whole extract was systematically verified in vitro in human microvascular endothelial cell (HMEC-1) culture, including cell proliferation, cell migration, and tube formation. Its angiogenic effect was also confirmed in zebrafish in vivo and angiogenesis-related gene regulation was determined by quantitative real-time PCR. Meanwhile, ginsenoside Rgl (Rgl) severed as positive control in these biological studies (Yue et al., 2005).
Materials and methods
Fetal bovine serum (FBS), trypsin, penicillin and streptomycin were from Gibco (Grand Island, NY, USA). Acetonitrile (HPLC grade) and ethanol were from Fisher Chemicals (Leicester, UK). Matrigel[TM] basement membrane matrix was supplied by BD Biosciences (Franklin Lakes, NJ, USA). The iScript one-step RT-PCR kit and SYBR Green were from Bio-Rad (Hercules, CA, USA). MCDB 131 medium and all other unspecified chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human microvascular endothelial cell line (HMEC-1) was provided by the American Type Culture Collection (ATCC, USA). The chemical standards (purity >97%) of ginsenoside Rgl (Rgl), hydroxysafflor yellow A (HYA) and kaempferol 3-O-[beta]-rutinoside (KOR) were supplied by Tauto Biotech Co. (Shanghai, China).
Plant material and extraction
The raw herb of Carthami Flos (CF) (harvested in Xinjiang Province, China) was supplied by Guangzhou Zhixin Pharmaceutical Co. (Guangzhou, China), and chemically authenticated by thin layer chromatography (TLC) according to Chinese Pharmacopoeia 2010. Its voucher specimen (no. 2013-3415) was kept in the museum of the Institute of Chinese Medicine, The Chinese University of Hong Kong.
The crude herb of CF (60 g) was extracted with water (2000 ml) by refluxing for one hour. After the crude extract was filtered, the supernatant was collected for further procedure; the residue was extracted with 95% ethanol (2000 ml) by refluxing for another hour, and then filtered. The ethanol extract was concentrated under reduced pressure using a vacuum rotary evaporator, and then mixed with the supernatant of water part. After precipitated overnight, the supernatant was collected for freeze-dry. The dried extract was kept in the desiccator before use. Prior to the following biological assays, the whole extract was re-dissolved into the stock solution (800 [micro]g/mL) with respective medium, then filtered by 0.22 [micro]m filter and finally diluted with respective medium to the working concentrations.
CF whole extract (totally dissolved in water at 1 mg/ml and filtered by 0.22 [micro]m filter) was subjected to chemical analysis by Agilent 1290 Infinity HPLC system (Santa Clara, CA, USA) which was equipped with an online degasser, a binary-pump, an autosampler and a diode array detector. HPLC analysis was performed with an Alltima HPLC C18 column (250 mm x 4.6 mm, 5 [micro]m) guarded by a guard column with same stationary phase according to previous report with minor modifications (Fan et al., 2009). The column was maintained at 40[degrees]C, and the flow rate was set at 0.7 ml/min. The mobile phase for qualitative analysis consisted of (A) 0.1% acetate acid and (B) acetonitrile with the following gradient: 2% B from 0 to 10 min; 2-5% B from 10 to 20 min; 5-10% B from 20 min to 70 min; 10-15% B from 70 to 120min; and 15-30% B from 120 to 180 min. The mobile phase for quantitative analysis consisted of (A) 0.1% acetate acid and (B) acetonitrile with the following gradient: 15-20% B from 0 to 4 min; 20-40% B from 4 to 8 min; and 40% B from 8 to 12 min. The UV absorbance was detected at 280 nm. Injection volume was 2 [micro]L. Mass spectrometry (MS) was performed using Agilent 6530 Accurate-Mass QTOF mass spectrometer (Santa Clara, CA, USA), which was equipped with Jet Stream electrospray ionization (ESI) source. Source parameters were set as follows: positive ion mode; gas temperature at 350[degrees]C; drying gas, 11 L/min; nebulizer, 55 psi; capillary, 4200 V. The mass range was set at 100-2000, and data were analyzed using Agilent MassHunter Workstation v.B.05.00 software (Santa Clara, CA, USA).
The concentrations of hydroxysafflor yellow A (HYA) and kaempferol 3-O-[beta]-rutinoside (KOR) in the CF whole extract were simultaneously quantified by a validated method. All calibration curves (ranged from 1 to 40 [micro]g/ml for HYA with [[M+H].sup.+] at m/z 613.1773; ranged from 1 to 20 [micro]g/mL for KOR with [[M+H].sup.+] at m/z 595.1666) with good correlation coefficient ([r.sup.2] > 0.99) were constructed by MS peak areas of the reference standards versus their concentrations. Their intra- and inter-batch precisions were also satisfied with acceptable relative standard deviation (R.S.D. < 3.9%) and accuracy ranged from 93.1% to 104.0%.
Cell viability assay
Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HMEC-1 cells (1 x [10.sup.4] cells per well) were seeded onto the 96 well plates with MCDB medium containing 10% FBS and 0.5% antibiotic overnight, and then the medium was replenished with MCDB medium containing 0.5% FBS and 0.5% antibiotic for another 12 h. After starved, various concentrations (0-800 [micro]g/ml) of CF whole extract were added for 24-h incubation. Subsequently, culture medium was replaced with MTT (0.5 mg/ml in medium) for another 3-h incubation at 37[degrees]C, and then replenished with DMSO (100 [micro]l) to dissolve the formed formazan. The absorbance was measured at 540 nm by a microplate reader. Rg1 (20 [micro]M) served as positive control.
Wound healing assay
Wound healing assay (scratch assay) was performed as described previously (Chan et al., 2011). HMEC-1 cells (1 x [10.sup.5] cells per well) were seeded in MCDB medium supplemented with 10% FBS and 0.5% antibiotic onto 24-well plates overnight. Then, two crosses on cells were scratched with a pipette tip (p200) and then washed with pre-warmed PBS. Different concentrations (0-200 [micro]g/ml) of CF whole extract in MCDB medium supplemented with 0.5% FBS were added for 8-h treatment. Images were taken before and after treatment at a 40x magnification by an inverted microscope (Nikon Eclipse TS100). Data were analyzed with the TScratch software (Geback et al., 2009). Four replicates were conducted for each individual experiment and a total of four experiments were performed. Rg1 (20 [micro]M) served as positive control.
Tube formation assay
HMEC-1 cells (1.5 x [10.sup.4] cells per well) were seeded onto the 96-well plate pre-coated with Matrigel (50 [micro]l) under normal culture condition. Simultaneously, various concentrations (0-200 [micro]g/ml) of CF whole extract in MCDB medium supplemented with 10% FBS were added for 2-h incubation. The formed network of tubes was recorded at a 40 x magnification by the inverted microscope. The total length of tubes in each image was analyzed using Image-Pro Plus 6.0 software (Rockville, MD, USA). Duplicates or triplicates were conducted for each individual experiment and a total of three experiments were performed. Rg1 (20 [micro]M) served as positive control.
Sprout number count in zebrafish
The zebrafish TG[(fli1:EGFP).sup.y1]/+ (AB) line with transgenic endothelial cells expressing EGFP (Enhanced Green Fluorescent Protein) was purchased from the Zebrafish International Resource Centre, University of Oregon, USA. Zebrafish embryos at their 1-4 cell stage were prepared and disinfected with methyl-blue solution (2 [micro]g/ml) according to the previous report (Liu et al., 2013). The handling of zebrafish was approved by Department of Health, Hong Kong SAR according to the guidelines in Care and Use of Animals.
The disinfected embryos were incubated with different concentrations of CF whole extract (diluted by embryo medium), while embryos receiving embryo medium alone acted as negative control. After 72-h post-fertilization (hpf), the morphology of sub-intestinal vessel (SIV) region in zebrafish larvae were assessed by using Olympus IX71S8F-2 inverted florescent microscope (Olympus, Tokyo, Japan). As indication of pro-angiogenesis effect, the mean sprout number in the SIV region was calculated by dividing the sum of the sprout numbers by the total number of embryos in the sample group. Rg1 (20 [micro]M) served as positive control.
mRNA expression in zebrafish by quantitative real-time PCR (qRT-PCR)
After sprout number count, zebrafish was kept in the TriZol. Total RNA from zebrafish larvae were extracted by RNeasy Mini Kit (QIAGEN, MD, USA) according to manufacturer's instructions. Realtime PCR was carried out with iScript one-step RT-PCR kit and SYBR Green using CFX96[TM] Real Time System (Bio-Rad, Hercules, USA). The cycling conditions were as follows: 50[degrees]C for 10 min, 95[degrees]C for 5 min, then 50 cycles of 95[degrees]C for 10 s and 60[degrees]C for 30 sec. Realtime (RT) PCR primers of tested genes were listed in Table 1, and provided by Tech Dragon Limited (Hong Kong) according to our previous study (Liu et al., 2013). [beta]-actin acted as house-keeping gene. All detections were performed in triplicate. Gene expression differences were analyzed using two-cycle threshold calculation.
Data are presented as means [+ or -] standard error of the mean (S.E.M.), and analyzed using GraphPad Prism v.4 software (GraphPad Inc., CA, USA). Statistical significance was assessed by Student t-test or one-way ANOVA followed by Dunnet's post hoc test, where appropriate. The values of p < 0.05 were considered as statistically significant.
Chemical profiles detected by LC-MS/MS
In this study, CF raw herb was extracted by boiling with water, followed by ethanol. As displayed in the extract ion chromatogram (EIC) (Fig. 1), there were two major components hydroxysafflor yellow A (F1YA; [[M+H].sup.+] m/z at 613.1773; error ppm = 0.82) and kaempferol 3-0-[beta]-rutinoside (KOR; [[M+H].sup.+] m/z at 595.1666; error ppm = 0.55) identified in the CF whole extract with same molecular weights and retention times when compared to their chemical standards. According to the validated quantitative method, the contents of HYA and KOR in the extract were 30.3 [+ or -] 2.3 mg/g and 2.7 [+ or -] 0.2 mg/g, respectively. Their contents were higher than those in the water extract (HYA, 2.35-20.74 mg/g; KOR, 0.37-2.05 mg/g) as reported (Fan et al., 2009).
Cell viability assay
After 24-h treatment, CF whole extract significantly increased the cell viability from 50 [micro]g/ml to 200 [micro]g/ml with approximately 10% enhancement, as detected by MTT assay (Fig. 2). This result indicated that CF whole extract could possibly increase the cell proliferation. Flowever, it showed significant toxicity with about 11% decrease of cell viability at high concentration of 800 [micro]g/ml.
Wound healing assay
As shown in Fig. 3, CF whole extract significantly increased cell migration at 25 [micro]g/mL and 50 [micro]g/ml after 8-h treatment. The open wound areas at these two concentrations were smaller than that of control group by about 17%.
Tube formation assay
In the tube formation assay, FIMEC cells formed tubes after 2-h treatment, and CF whole extract at 100 [micro]g/ml significantly enhanced their formation by about 10% (Fig. 4).
Sprout number count in zebrafish SIV region
As shown in Fig. 5, CF whole extract significantly increased the sprout numbers in the SIV region at 100 [micro]g/ml and 200 [micro]g/ml when compared to the control group, but not at lower concentrations.
mRNA expression in zebrafish
As shown in Fig. 6, CF whole extract up-regulated angiogenesis-related genes responsible for proliferation, migration, adhesion and tube formation in endothelial cells. It also stimulated angiogenesis-related cells including pericytes for blood vessel formation.
According to TCM theory, treatment with multiple targets is its basic strategy used in clinic. Likewise, CF whole extract is a mixture of chemicals and it triggered multiple signaling pathways of angiogenesis in zebrafish in vivo as indicated by quantitative real-time PCR (qRT-PCR). The expression of IGF1 (fold change, 4.8), CTGF (fold change, 2.9), NRP2 (fold change, 2.8), and VEGFR3 (fold change, 2.3) was significantly increased. EGFR was decreased (fold change, 0.5) (Fig. 6). IGF1 activates Akt signaling pathway for endothelial cells (EC) proliferation (Piecewicz et al., 2012) ; it also interacted with TGF-beta 1 activation (Rosendahl and Forsberg, 2006). Phosphatidylinositol 3-kinase (PI3K)-Akt-dependent pathways induce CTGF which is responsible for cell adhesion, proliferation and migration (Suzuma et al., 2000). As co-receptor of VEGF, up-regulation of NRP2 and VEGFR3 are supposed to promote EC survival and migration (Favier et al., 2006). Although EGFR gene was down-regulated, its negative effect should be negligible since there is a concomitant 2.3 fold increase in VEGFR3, which shares similar down-stream angiogenic genes.
Transcription factor H1F1A was found to be up-regulated (fold change, 2.4), although its down-stream VEGFA was not affected. HIF1A is associated with overexpression of other angiogenic growth factors such as formation of unstable/leaky vasculature (Hadjipanayi and Schilling, 2013). Meanwhile, it is also regulated by other factors such as PIK3C2[alpha]/Akt/HIF-1[alpha] Pathway (Chai et al., 2013). Therefore its role is not clear at present.
Matrix degradation- and endothelial cell migration-related factors were up-regulated, including: MMP2 (fold change, 4.0), MMP9 (fold change, 4.2), TIMP2 (fold change, 5), PLG (fold change, 2.5) and PLAU (fold change, 6). PLAU is supposed to degrade extracellular matrix, and it mediates direct activation of MMP-9 (a well-known factor increasing cell migration) (Zhao et al., 2008). On the other hand, two factors TIMP2 and PLG, which counteracts angiogenesis, were up-regulated. TIMP2 is able to directly suppress EC proliferation and the activity of MMP2, although we observed increased cell migration due to the up-regulation of MMP9. It is also possible that the up-regulation of TIMP2 can limit uncontrolled angiogenesis (Seo et al., 2003). Likewise, PLG is activated by proteolysis and converted to plasmin and angiostatin which inhibit angiogenesis through diminishing activation of mitogen-activated protein kinases (Redlitz et al., 1999). However, PLG activation can be inhibited by PLAU and MMP9, thus its effect was weakened even though they were all up-regulated.
After extracellular matrix degradation, adhesion molecules assist the development of sprouting blood vessels. Cell adhesion molecules were highly expressed, including ITGAV (fold change, 1.6), ITGB3 (fold change, 2.6), beta-catenin (fold change, 6.2) and PECAM1 (fold change, 3.9). 1TGB3 is a known integrin responsible for cell adhesion and cell-surface-mediated signaling. MMP2 can associate with ITGB3 for promoting vascular invasion (Silletti et al., 2001), and Akt/PKB activates phosphorylation of ITGB3 (Kirk et al., 2000). ITGB3 and ITGAV form heterodimer as the receptor of vitronectin (Liu et al., 2011). Beta-catenin is a subunit of adherens junctions, and it creates and maintains epithelial cell layers by regulating cell growth and adhesion (Lilien and Balsamo, 2005). PECAM1 forms main portion of EC intercellular junctions, and it also forms complex with beta-catenin to modulate EC and fibroblast proliferation (Miragliotta et al., 2008).
Up-regulation of ANGPT1 (fold change, 2.6), TIE-2 (fold change, 2.1), PDGFR-B (fold change, 1.9), CDH5 (fold change, 2.4), S1PR1 (fold change, 7.7), FGF2 (fold change, 3.2), Shh (fold change, 4.1), and TGFRB1 (fold change, 6) indicated enhancement of tubule formation and morphogenesis/smooth muscle cell recruitment and differentiation. Shh can increase angiogenic factor ANGPT1, and its upregulation possibly enhanced ANGPT1-mediating reciprocal interactions with TIE-2/Akt pathway between the endothelium and surrounding matrix and mesenchyme, and subsequent blood vessel maturation and stability (Fiedler et al., 2003; Kim et al., 2000; Suri et al., 1996). SI PR1 induces EC migration through activation of the JAK/STAT3 and FAK/P13K/AKT signaling pathways (Lee et al., 2001). PDGF/PDGFR-B directs cell migration with both sphingosine kinase activation and expression of endothelial differentiation gene-1 (Rosenfeldt et al., 2001). PDGFR-B can also bind to ITGB3 to promote EC proliferation and migration (Borges et al., 2000). FGF2 mediates the activation of TGFB1 to promote cell proliferation and differentiation (Yang et al., 2008). Beta-catenin dissociates from TGFBR1, its tyrosine phosphorylation increases, threonine phosphorylation decreases, and then beta-catenin becomes associated with TGFB1-signaling molecules Smad3 and Smad4 (Tian and Phillips, 2002). Beta-catenin links VE-cadherin(CDH5) junction complex to the cytoskeleton, which is benefit for vascular endothelial cell-cell adhesions and barrier function (Guo et al., 2008).
Up-regulation of Ephrin B2 (fold change, 4.5) indicated that CF whole extract enhance blood vessel maturation/formation of arteries and veins. Endothelial Ephrin B2 associates with PECAM1 (CD31) for endothelial-mesenchymal interactions, thus up-regulation of Ephrin B2 suggested that CF whole extract could enhance loop formation for blood vessel formation (Korff et al., 2006).
In conclusion, the current study indicated that CF whole extract promoted angiogenesis in mammalian cells, via increasing proliferation, migration and tube formation in HMEC-1 cells in vitro. It was also reproduced in the in vivo zebrafish S1V model. These results are consistent with previous findings in chicken embryo CAM model (Gao et al., 2005). The underlying mechanism is likely that CF whole extract up-regulated expression of certain genes for angiogenesis and associated growth factors and receptors, including IGF1, CTGF, NRP2, VEGFR3, HIF1A, MMP2, MMP9, TIMP2, PLG, PLAU, ITGAV, 1TGB3, beta-catenin, PECAM1, ANGPT1, TIE-2, PDGFRB, CDH5, S1PR1, FGF2, Shh, TGFRB1, and Ephrin B2. The probable regulatory mechanism and interaction of these genes and factors are summarized in Fig. 7. Our study is the first to demonstrate that CF works through this network for its angiogenesis effects.
Conflict of interest
The authors have no conflict of interest to disclose.
Received 2 March 2014
Received in revised form 1 May 2014
Accepted 19 June 2014
This study was financially supported by the Innovation and Technology Commission, the Government of Hong Kong SAR, China (Ref. No. GHX/002/11), and Alberta Technology Limited.
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Xuelin Zhou (a,b,c), Wing-Sum Siu (a,b,c), Chak-Hei Fung (a,b,c), Ling Cheng (a,b), Chun-Wai Wong (a,b), Cheng Zhang (a,b), Cheuk-Lun Liu (a,b), Hin-Fai Kwok (a,b), Ching-Po Lau (a,b), Elaine Wat (a,b), Clara Bik-San Lau (a,b,c), Ping-Chung Leung (a,b,c,d), Chun-Hay Ko (a,b,c), *, Leung-Kim Hung (d), **
(a) Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region
(b) State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region
(c) Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong Province, China
(d) Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region
* Corresponding author at: Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region. Tel.: +852 39434143: fax: +852 26035248.
** Corresponding author. Tel.: +852 26322731: fax: +852 26377889.
E-mail addresses: email@example.com (C.-H. Ko), firstname.lastname@example.org (L-K. Hung).
Table 1 Classification of angiogenesis-related genes. Angiogenesis-related Gene names mechanisms Angiogenic and its Epidermal growth factor receptor associated growth (EGFR), Hepatocyte growth factor factors and receptors receptor (Met), Insulin like growth factor 1 (IGF1), Connective tissue growth factor (CTGF), Neuropilin 2 (NRP2), Vascular endothelial growth factor receptor 3 (VEGFR3), VEGFA, Kinase insert domain receptor (KDR) Transcription factors v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1), Flypoxia inducible factor 1 alpha subunit (HIF1A) Matrix Matrix metallopeptidase 2 (MMP-2), degradation/endothelial MMP-9, TIMP metallopeptidase cell migration inhibitor 2 (TIMP2), Plasminogen (PLG), Urokinase-type plasminogen activator (PLAU) Cell adhesion molecules Integrin alpha V (ITGAV), Integrin [beta]-3 (ITGB3), cadherin associated protein [beta] (beta-catenin), Platelet endothelial cell adhesion molecule (PECAM1) Tubule formation and Angiopoietin 1 (ANGPT1), TEK morphogenesis/smooth tyrosine kinase (Tie-2), Platelet muscle cell recruitment derived growth factor and differentiation receptor-[beta] (PDGFR-B), vascular endothelium Type 2 cadherin 5 (CDH5), Sphingosine-l-phosphate receptor 1 (S1PR1), Fibroblast growth factor 2 (FGF2), Fibroblast growth factor receptor 2 (FGFR2), Sonic hedgehog (Shh), Transforming growth factor [beta]1 (TGF[beta]1), Transforming growth factor [beta]1 receptor (TGFRB1) Blood vessel maturation/ Ephrin B2, Ephrin type B receptor 4 formation of arteries (EPHB4) and veins
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|Author:||Zhou, Xuelin; Siu, Wing-Sum; Fung, Chak-Hei; Cheng, Ling; Wong, Chun-Wai; Zhang, Cheng; Liu, Cheuk-L|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Sep 25, 2014|
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