Fucoidan extract derived from Undaria pinnatifida inhibits angiogenesis by human umbilical vein endothelial cells

Phytomedicine 19 (2012) 797–803

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Fucoidan extract derived from Undaria pinnatifida inhibits angiogenesis by human umbilical vein endothelial cells Fang Liu a , Jia Wang a , Alan K. Chang b , Bing Liu a , Lili Yang a , Qiaomei Li a , Peisheng Wang a , Xiangyang Zou a,∗ a b

Department of Biotechnology, Dalian Medical University, Dalian, 116044 Liaoning Province, China School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China

a r t i c l e

i n f o

Keywords: Angiogenesis Fucoidan Undaria pinnatifida Human umbilical vein endothelial cells VEGF-A

a b s t r a c t In recent years, anti-angiogenic therapy has become an effective strategy for inhibiting tumor growth. Fucoidan is a class of fucose-enriched sulfated polysaccharides found in brown algae, and it is known to have strong anti-tumor property. Using a human umbilical vein endothelial cells (HUVEC)-based cell culture model, the present study investigated the anti-angiogenic activity of fucoidan extracted from the brown seaweed Undaria pinnatifida. Treatment of HUVECs with various concentrations of fucoidan resulted in significant inhibition of cell proliferation, cell migration, tube formation and vascular network formation. However, significant inhibition of cell proliferation only occurred with longer treatment time (48 h instead of 24 h or less). About 40% of cell proliferation and cell migration and 61% of tube formation by HUVECs were inhibited by 400 ␮g/ml fucoidan, the maximum concentration tested. These results appeared to suggest that modulation of angiogenesis by fucoidan might not occur through growth inhibition and apoptosis. Ex vivo angiogenesis assay demonstrated that at 100 ␮g/ml, fucoidan caused significant reduction in microvessel outgrowth. Western blot and RT-PCR analyses indicated that at 400 ␮g/ml, fucoidan significantly reduced the expression of the angiogenesis factor VEGF-A in the suppression of angiogenesis activity. Our results showed that fucoidan isolated from U. pinnatifida may have a new therapeutic potential in the prevention angiogenesis-related diseases. © 2012 Elsevier GmbH. All rights reserved.

Introduction Angiogenesis is the formation of new blood capillaries from existing vessels by action of vascular endothelial cells. It is essential for a diverse range of physiological processes, including embryonic development, tissue organ regeneration, menstrual cycle, and wound healing. Under these conditions, angiogenesis is highly regulated and transient. However, several pathological states such as diabetic retinopathy, rheumatoid arthritis, cancer growth, tumor metastases, and transplantation are driven by persistent unregulated angiogenesis (Klagsbrun and D’Amore 1991). Angiogenesis is not only necessary for the growth of cancer but also for tumor transplantation and metastasis. There are two stages in tumor growth; the first stage involves the absence of blood vessel formation, whereas the second stage requires the formation of new capillaries. Under the second stage, tumor growth requires angiogenesis to supply nutrients and oxygen, and to remove waste products when the solid tumors grow to 1–2 mm (Folkman 1974). In addition, the

∗ Corresponding author. Tel.: +86 411 86110296. E-mail address: [email protected] (X. Zou). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2012.03.015

tumor also utilizes the newly formed blood vessels as conduits to disseminate invasive tumor cells (Nelson 1998). Angiogenesis is the result of a complex effect on cell–cell and cell–matrix interactions. It mainly involves the degradation of extracellular matrix (ECM), endothelial cells proliferation, migration and tube formation, as well as sprouting of new capillary branches (Folkman and Shing 1992). The complex process is regulated by stimulatory and inhibitory factors (Hanahan and Folkman 1996; Hanahan et al. 1996). Angiogenesis is an essential component for the growth and progression of neoplasms, and no doubt blocking it is an effective strategy for inhibiting tumor growth. Hence, anti-angiogenic therapy has become a promising approach in the development of novel anticancer therapy as well as therapies for other pro-angiogenic diseases. Vascular endothelial growth factor (VEGF) is a highly conserved dimeric heparin-binding glycoprotein and it appears to have a particularly important role in angiogenesis (Ferrara 1993). VEGF-A, a pro-survival factor for endothelial cells (ECs), is believed to be a well known angiogenic growth factor in angiogenesis, and it can act both by inducing EC proliferation and by stimulating EC migration (Ferrara et al. 2003; Levy et al. 1995; Alon et al. 1995; Rousseau et al. 2000a,b; Chavakis and Dimmeler 2002).

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Undaria (Undaria pinnatifida, Wakame), a large brown algae, is widely distributed in China. Fucoidans, which represent a class of fucose-enriched sulfated polysaccharides found in the ECM of brown seaweed, have recently been reported to have various biological activities, such as antitumor, antiviral, antioxidative, anti-inflammatory, anti-angiogenic and anti-adhesion properties. Recently, fucoidan has attracted a lot of clinical attention due to its strong anti-tumor potential, which has been intensively investigated. We therefore, wished to determine if fucoidan also has anti-angiogenic activity, since anti-angiogenesis has been widely considered as an approach toward preventing tumor growth through starving the tumor cells of nutrients. In this study, we demonstrated the anti-angiogenic effect of fucoidan on human umbilical vein endothelial cells (HUVECs) using both in vitro and ex vivo experiments, and attempted to examine the mechanism by which fucoidan may affect angiogenesis. Materials and methods Materials Dimethylsulfoxide (DMSO), RPMI 1640 medium, antibiotics (penicillin and streptomycin), trypsin, fetal bovine serum (FBS) and EDTA were obtained from Hyclone. Matrigel and MTT reagent were purchased from Sigma. Transwell plate was obtained from Corning. Rabbit anti-VEGF-A antibody was supplied by Beijing Biosynthesis Biotechnology Co., Ltd. Mouse anti-␤-actin antibody, anti-rabbit IgG antibody and anti-mouse IgG antibody were purchased from ZhongShan GoldenBridge Biotechnology Co., Ltd. HUVECs, obtained from the Cell Bank of the Chinese Academy of Sciences, were maintained in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 100 ng/ml each of penicillin and streptomycin. The cells were cultured at 37 ◦ C in a humidified atmosphere with 5% CO2 . Cells from three to six passages were used for experiments. Six-week old female SD-rats (Specific pathogen Free, SPF) were obtained from the Animal Facility of Dalian Medical University. Preparation and analyses of the fucoidan The marine brown algae Undaria pinnatifida that was collected from the coast of Dalian (Dalian, China) on April in 2009, was used in this study. The fucoidan extract from U. pinnatifida sporophyll was prepared by enzymatic hydrolysis and alcohol precipitation. The main fucoidan fraction was separated by using DEAE Cellulose52 (Whatman, England) and Sephadex G-100 (Pharmacia, Sweden) column chromatography. For Fourier-transform infrared spectroscopy analysis, pellets prepared from a mixture of polysaccharide powder and potassium bromide (KBr) were analyzed by a Nicolet 510P FT-IR spectrometer in the range 4000–600 cm−1 . The contents of total carbohydrate, sulfate radical and uronic acid were measured by the phenol–sulfuric acid reaction, BaCl2 –gelation and sulfuric acid–carbazole colorimetric method, respectively. The protein content was scanned by a UV-spectrophotometer (UV-2100) in the range of 260–350 nm. The molecular weight of the sample was evaluated by size exclusion chromatography using a TSK-gel G3000PWXL (TOSOH, Tokyo, Japan). The optical rotation was measured at 589 nm by the WZZ-1 polarimeter at 20 ◦ C.

with 10% FBS for another 24 or 48 h at 37 ◦ C in the presence of 5% CO2 atmosphere. This was followed by addition of 20 ␮l of 5 mg/ml MTT. After 4 h, 150 ␮l DMSO was added to each well to solublize the formazan product. The absorbance (A) of the plate was measured at 490 nm, and the inhibition ratio (%) was calculated by the equation, I % = (1 − (Atreated − Ablank )/(Acontrol − OD blank )) × 100%, and expressed as the average of three parallel experiments. In vitro migration assay The chemotactic motility of HUVECs was assayed by using Transwell (diameter: 6.5 mm; pore size: 8 ␮m). Briefly, HUVECs were first starved for 24 h in RPMI 1640 medium and then trypsinized and suspended in RPMI 1640 medium containing 0.l% FBS to a final density of 4 × 105 cells/ml. Cell suspension (100 ␮l) was treated with different concentrations (100, 200, and 400 ␮g/ml) of fucoidan. The fucoidan was loaded into the upper wells while fresh RPMI 1640 medium containing l0% FBS (500 ␮l) were placed in the lower wells. The chamber was incubated at 37 ◦ C for 12 h. Nonmigrating cells on the upper surface of the filter were removed by wiping with a cotton swab. Cells were fixed and stained with 70% ethanol for 5 min and then with 10% Giemsa for 15 min. Each culture was photographed at a magnification of 100× with a microscope (Olympus, Japan). Migration was quantitatively measured by counting the cells that migrated to the lower side of the filter. Five fields were counted for each assay to obtain the average. The percentage of cell inhibition was calculated by the equation, I% = [1 − (the number of migrating cellstreated /the number of migrating cellscontrol )] × 100%. Tube formation assay Formation of capillary tube-like structures by HUVECs was assessed in a matrigel-based assay as previously described (Ashton et al. 1999). Briefly, the thawed matrigel was dispensed into a refrigerated 96-well plate at 20 ␮l per well, and the plate was then incubated at 37 ◦ C for 1 h to allow the gel to set. HUVECs were trypsinized, resuspended in RPMI 1640 only or RPMI 1640 containing different concentrations of fucoidan (100, 200, and 400 ␮g/ml) and then seeded onto the matrigel at a density of 1 × 104 cells/well. The plate was incubated at 37 ◦ C for 24 h in a humidified 5% CO2 atmosphere. Tube formation was examined and photographed by phase-contrast microscopy from five different fields (×100). Inhibition of tube formation was calculated by the equation, I% = [1 − (tube quantitytreated /tube quantitycontrol )] × 100%. New vascular network damaging assay New vascular network damaging assay was based on the formation of capillary tube. Briefly, solid gels were prepared in a 96-well plate according to the manufacturer guidelines. 1 × 105 cells/ml were seeded (100 ␮l/well) onto the surface of the matrigel, and maintained at 37 ◦ C in a humidified 5% CO2 . After 24 h, the cells from each well were treated with medium or 400 ␮g/ml fucoidan. The cultures were photographed by phase-contrast microscopy from five randomly chosen fields (×100) after 24 and 48 h of incubation. Rat aortic rings angiogenesis assay

HUVECs proliferation inhibition assay The effect of fucoidan on growth inhibition was measured by MTT assay. Briefly, HUVECs were seeded in a 96-well plate at a density of 1 × 104 cells/well in triplicate. After 24 h, the cells were treated with different concentrations of fucoidan supplemented

Six-week old female SD-rats were kept in a metal cage (two animals per cage) in the presence of controlled temperature (24 ± 1 ◦ C) with 12 h light:dark cycle (lights on, 08:00–20:00). They were given free access to diets and deionized water. The rats were maintained according to the Guide for the Care and use of Laboratory Animals

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Fig. 1. Elution curves of the purified fucoidan by Sephadex G-100 column chromatography.

established by Dalian Medical University. Rat aortic ring activity was tested using a previously described method (Nicosia and Ottinetti 1990a,b). Thoracic aorta was separated and washed with RPMI 1640 medium, and then cut into 1-mm slices and seeded on the matrigel in a refrigerated 96-well plate at 50 ␮l per well. The chamber was allowed to solidify at 37 ◦ C for 1 h. Fucoidan was then added to the well at different concentrations (100, 200, and 400 ␮g/ml). Matrigel containing aortic ring not treated with fucoidan was used as negative control. The plate was incubated at 37 ◦ C for 8 days and the medium was changed on the fourth day. At the end of the incubation the plate was photographed at four randomly chosen fields (×100). The capillary length was analyzed using Image-Pro Plus (Media Cybernetics, USA). Inhibition of the length of capillary was calculated as: I% = [1 − (capillary lengthtreated /capillary lengthcontrol )] × 100%.

Tris-buffered saline and 0.1% Tween-20) containing 5% nonfat dry milk for 2 h at room temperature. It was then incubated with polyclonal anti-VEGF-A (1:300) for 16 h at 4 ◦ C, followed by washing in TBS for three times. The membrane was incubated in TBST buffer containing 5% nonfat dry milk and HRP-conjugated secondary antibody at a 1:10,000 dilution. After that, the membrane was washed three times in TBST followed by detection with chemiluminescence agent. Statistical analysis Statistical analysis of data was performed with SPSS11.5 software. Each experiment was repeated at least three times. Data are expressed as mean ± SD, and Student’s t-test was used to determine the significance of differences in multiple comparisons. P < 0.01 was considered to be statistically significant.

Semi-quantitative RT-PCR HUVECs were treated with fucoidan (100, 200 or 400 ␮g/ml) for 48 h and total RNA was isolated from the cells using a Total RNA isolation Kit as described by the manufacturer (Takara, Japan). To generate cDNA for amplification, 100 ng of RNA was used in a two-step reverse-transcriptase polymerase chain reaction (RT-PCR) kit as described by the manufacturer (Takara, Japan). Estimations of VEGF-A were normalized using the housekeeping gene GAPDH. GAPDH was amplified using the GAPDH forward primer (5 -ACCACAGTCCATGCCATCAC-3 ) and GAPDH-reverse primer (5 TCCACCACCCTG TTGCTGTA-3 ), whereas VEGF-A was amplified using the VEGF-A forward primer, (5 -GGGCCTCCGAAACCATGAAC3 ), and VEGF-A reverse primer (5 -CTGGTTCCCGAAACCCTGAG-3 ). For GAPDH amplification, the PCR condition consisted of 30 cycles of 94 ◦ C for 30 s, 58 ◦ C for 30 s, and 72 ◦ C for 1 min. For VEGF-A amplification, the PCR condition consisted of 35 cycles of 94 ◦ C for 45 s, 57 ◦ C for 45 s, and 72 ◦ C for 1 min. PCR-amplified products were resolved by 1% agarose gel and visualized by staining with ethidium bromide. Western blot The level of VEGF-A expression in HUVECs was also evaluated by Western blot. HUVECs seeded in the plates were treated without or with 400 ␮g/ml fucoidan for 48 h. The medium was removed and the cells were washed with PBS and then resuspended in 100 ␮l lysis buffer. The cell suspension was kept at 4 ◦ C for 20 min, and then centrifuged at 13,000 × g for 10 min. The supernatant was then subjected to SDS-PAGE using 12% gel. Equal amount of proteins were loaded for each sample (without and with fucoidan treatment) protein bands in the gel were transferred onto nitrocellulose membrane. The membrane was blocked with TBST buffer (20 mM

Results Preparation and properties of fucoidan extract from U. pinnatifida The fucoidan purified from U. pinnatifida was a beige fibrous powder (purity > 90%). Infrared spectrum analysis of the sample revealed strong absorption peaks at around 1630 cm−1 , about 845 cm−1 and 820 cm−1 , which are characteristic of sulfated residues, fucose and galactose, respectively. This fucoidan sample consisted mainly of carbohydrates (59.2%), sulfates (21%) and uronic acid (9.13%) with fucose and galactose constituting the monosaccharide composition, and the percentage of protein content was detected to be 0.13% (Fig. 1). The molecular weight of the purified fucoidan was about 9.52 × 104 Da. The optical rotation of the fucoidan (0.6 mg/ml) showed a value of 0.99◦ at 20 ◦ C. HUVECs proliferation inhibition To assess the effect of fucoidan on the proliferation of HUVECs, these cells were treated with different concentrations of fucoidan (0, 25, 50, 100, 200 and 400 ␮g/ml) for 24 h or 48 h and their proliferation was measured. As shown in Fig. 2, cells treated with fucoidan at concentrations up to 400 ␮g/ml for 24 h showed some growth inhibition (<20%). However, the inhibition was not statistically significant when compared to untreated cells. By increasing the treatment time to 48 h, growth inhibition exerted by the same concentrations of fucoidan became stronger and statistically significant (p < 0.05). Fucoidan therefore inhibited the growth of HUVECs in a dose-dependent manner, when the cells were treated with the polysaccharide for 48 h. As much as 40% of growth was inhibited by the maximum concentration (400 ␮g/ml) of fucoidan tested.

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Fig. 2. Effect of fucoidan on HUVECs proliferation. HUVECs were treated with different concentrations of fucoidan for 24 h and 48 h, and cell proliferation was then measured by using MTT assay. Data represent mean ± SD from five independent experiments. *p < 0.05 and **p < 0.01 as compared to the control (by one-way ANOVA).

Fucoidan inhibit HUVECs migration Migration of HUVECs plays an important role in angiogenesis, which is a chemical chemotaxis process. As shown in Fig. 3, fucoidan induced the in vitro migration of HUVECs in a dose-dependent manner. There was more HUVECs migration in the absence of fucoidan. In contrast, treatment of HUVECs with fucoidan at 100, 200 or 400 ␮g/ml led to significant reduction (p < 0.05, p < 0.01) in the number of migrating cells compared to control. Fucoidan inhibit HUVECs tube formation The effect of fucoidan on the inhibition of HUVECs tube formation was investigated by seeding the cells for 24 h and measuring Fig. 4. Inhibitory effect of fucoidan on HUVECs tube formation. (A) Microscopic photograph of tube formation on reconstituted basement membrane gel after 24 h of incubation. (a) control; (b) fucoidan, 100 ␮g/ml; (c) fucoidan, 200 ␮g/ml; (d) fucoidan, 400 ␮g/ml; (B) quantitative analysis of the dose-dependent inhibition exerted by fucoidan on tube formation by HUVECs. Data represent the mean ± SD from three independent experiments. *p < 0.05 and **p < 0.01, as compared to the control (by one-way ANOVA).

the number of the tubes formed in the matrigel after treatment with different concentrations of fucoidan. HUVECs grown on reconstituted basement membrane migrated, attached to each other, and formed tube structures as shown in Fig. 4A, and it was clear that HUVECs that were treated with fucoidan formed incomplete and fluffy tubular structures. The number of tubes formed was also much less compared to those formed by cells treated with RPMI 1640 medium only. Approximately 61% of tube formation by HUVECs was inhibited by the maximum concentration (400 ␮g/ml) of fucoidan tested (Fig. 4B). Taken together, the result showed that fucoidan clearly inhibited tube formation by HUVECs, and the inhibition was dependent on the dosage of fucoidan. Effect of fucoidan on the formation of new vascular network HUVECs formed reticulate capillary tube in two-dimensional matrigel after 24 h incubation. Tubes developed from cells grown on fucoidan treated (400 ␮g/ml) membrane for 24 h and 48 h showed no significant change whereas tubes from cells grown on untreated membrane showed strong promoting effect on HUVEC new vascular structures at 48 h (Fig. 5). These results suggested that fucoidan could prevent the growth of existing capillary tube. Fig. 3. Effect of fucoidan on HUVECs migration. (A) Migrated cells were counted (after 12 h incubation) from five microscopic fields at 100×. (a) Control; (b) fucoidan, 100 ␮g/ml; (c) fucoidan, 200 ␮g/ml; (d) fucoidan, 400 ␮g/ml; (B) quantitative analysis of the dose-dependent inhibition of migration of HUVECs by fucoidan. Data represent the mean ± SD from three independent experiments. *p < 0.05 and **p < 0.01, as compared to the control (by one-way ANOVA).

The effect of fucoidan on rat aortic rings angiogenesis To investigate whether fucoidan could suppress angiogenesis of rat aortic ring, rat aortic tissue was placed on the matrigel to allow

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Fig. 5. Inhibition of exerted by fucoidan on new vascular network formation by HUVECs. HUVECs were treated with 400 ␮g/ml fucoidan for 24 h and 48 h. a, b and c represent control groups; a1, b1 and c1 represent 400 ␮g/ml fucoidan-treated groups. 0, 24 and 48 h beneath the panels indicated the treatment times.

it to form capillary tubes. This model is similar to an in vivo assay. Microvessels appeared after 3 days and became elongated after 5–6 days (Fig. 6A). Inhibition of microvessel formation by fucoidan was observed even at low concentration (100 ␮g/ml). At 400 ␮g/ml, fucoidan inhibited about 61% of microvessel formation compared to control (Fig. 6B). These results suggested that fucodian suppressed ex vivo microvessel formation in a dose-dependent manner.

Fig. 6. Inhibition of rat aortic ring ex vivo angiogenesis by fucoidan. Rat aortic rings (1 mm) were seeded on matrigel in 96-well plates, and then treated with medium only or medium containing various concentrations of fucoidan for 8 days. (A) Microscopic photograph of capillary formation. (a) control; (b) fucoidan, 100 ␮g/ml; (c) fucoidan, 200 ␮g/ml; (d) fucoidan, 400 ␮g/ml; (B) microvessel length was measured on day 8 of the culture. Quantification of the inhibition exerted by fucoidan on vessel outgrowth arising from rat aortic ring. Data represent mean ± SD from three independent experiments. *p < 0.05 and **p < 0.01, as compared to the control (by one-way ANOVA).

Fucoidan down-regulates VEGF-A The expressions of two different isoforms of VEGF-A (VEGF165 and VEGF121) were analyzed by RT-PCR. HUVECs treated with fucoidan showed reduced level of VEGF-A mRNA compared to untreated cells, as measured by RT-PCR (Fig. 7A). Quantitation of the mRNA levels of both VEGF-A isoforms showed a reduction of about three folds in the case of VEGF165 and two folds in the case of VEGF121 (Fig. 7B), indicating that the effect of fucoidan on VEGFA was somewhat isoform specific. The effect of fucoidan on the expression of VEGF-A by HUVECs was also evaluated by western blot (Fig. 8A). VEGF-A was detected by western blot as a 45-kDa band. At 400 ␮g/ml, fucoidan decreased the level of VEGF-A protein by at least three folds compared to control (Fig. 8B). This result suggested that inhibition of VEGF-A by fucoidan may contribute to a reduced level of angiogenesis by HUVECs.

Fig. 7. Inhibition of VEGF-A expression in HUVECs by fucoidan. Total RNA was isolated from HUVECs treated with fucoidan (100, 200 or 400 ␮g/ml) for 48 h and then subjected to VEGF-A RT-PCR. (A) VEGF-A mRNA expression as detected by RT-PCR in HUVECs. (B) Quantitative RT-PCR for VEGF-A mRNA. Values are expressed as a ratio of VEGF-A to GAPDH expression. Numerical RT-PCR data analyzed by quantity one. Data represent mean ± SD from three independent experiments. Significance are considered either at p < 0.05 (*) or p < 0.01 (**) using the one-way ANOVA.

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Fig. 8. Inhibition of VEGF-A expression in HUVECs by fucoidan. (A) HUVECs were treated with fucoidan (400 ␮g/ml) for 48 h and cell extract was prepared and subjected to western blot using anti-VEGF-A antibody. ␤-Actin is shown as loading control. (B) Quantitative Western for VEGF-A expression in HUVECs. Values are expressed as a ratio of VEGF-A to ␤-actin expression. *p < 0.05 and **p < 0.01, as compared to the control (independent sample t test).

Discussion It is shown that angiogenesis is not only needed for the growth of tumor but also required for tumor transplantation and metastasis (Folkman 1971). Tumor growth requires angiogenesis to supply nutrients and oxygen and by removing waste products (O’Reilly 1997). Thus inhibition of tumor angiogenesis is one of the promising strategies in the development of anticancer therapy. Recently, it has been well documented that some anti-angiogenic compounds are present in seaweeds such as Grateloupia longifolid (Zhang et al. 2006), Sargassum stenophyllum (Dias et al. 2005), Fucus vesiculosus (Koyanagi et al. 2003), Codium cylindricum (Matsubara et al. 2003), Laminaria japonica (Xu et al. 1999). Enzyme-digested fucoidan extracts from Mozuku of Cladosiphon novae-caledoniae kylin can inhibit the invasion and angiogenesis of tumor cells in vitro, as well as suppressing the growth of various anchorage-dependent and independent cancer cells (Ye et al. 2005; Matsuda et al. 2010). Low molecular weight fucoidan (LMWF) can increase HUVEC migration and tube formation, and decrease VSMC migration in vitro through the modulation of matrix metalloproteinase-2 expression (Hlawaty et al. 2011; Lake et al. 2006). Previous study has shown that fucoidan can induce apoptosis in HT-29 and HCT116 human colon cancer cells, and that this phenomenon is mediated by both the death receptor-mediated and mitochondria-mediated apoptotic pathways (Kim et al. 2010). Siphonaxanthin from the green algae Codium fragile can significantly suppress HUVEC proliferation, tube formation and microvessel outgrowth, but its effect on chemotaxis is not significant (Ganesan et al. 2010). Moreover, anticancer activity has been observed among the oversulfated F5–30 K , F>30 K and F<5 K fractions, and the results suggest that the anticancer activity of fucoidans can be significantly improved by lowering their molecular weights and by improving the binding properties of the sulphate groups, possibly through changing the molecular conformation (Cho et al. 2011; You et al. 2010). However, until now, there has been no study investigating the anti-angiogenesis activity of fucoidan isolated from U. pinnatifida. Angiogenesis is a complicated process involving the degradation of extracellular matrix (ECM), endothelial cells proliferation, migration, differentiation, tube formation and sprouting of new capillary branches, which are regulated by a wide array of proangiogenic factors and anti-angiogenic factors (Bussolino et al.

1997). In our experiments, the effect of fucoidan on growth and apoptosis of HUVECs was not substantial when the cells were treated with the polysaccharide for 24 h (p > 0.05). However, at 100–400 ␮g/ml, fucoidan markedly suppressed cell migration and tube formation, and damaged new vascular network as assessed by a series of in vitro models, suggesting that fucoidan had antiangiogenesis activity. The anti-angiogenic activity of fucoidan was further demonstrated using an ex vivo angiogenic model employing rat aortic ring, in which fucoidan was shown to significantly inhibit the formation of microvessels in a dose-dependent manner consistent with the fact that fucoidan possesses anti-angiogenic property. A variety of anti-angiogenic agents (several of which are not known) with diverse mechanism of action is currently undergoing clinical trials (Ferrara and Kerbel 2005). The signaling pathway of angiogensis is a complex balance between stimulatory and inhibitory factors, involving multiple cell factors. VEGF is a highly conserved dimeric heparin-binding glycoprotein and it appears to have a particularly important role in angiogenesis (Ferrara 1993). The VEGF family of proteins consists of many members, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, PLGF (placenta growth factor) (Neufeld et al. 1999; Takahashi and Shibuya 2005). VEGF-A is a secreted glycoprotein with multiple splice variants (Houck et al. 1991; Relf et al. 1997). It is well known that VEGF165 and VEGF121 are the most abundant splice variants. VEGF165, but not VEGF121, is known to bind heparin and heparin sulfates, since VEGF121 lacks a heparin binding motif. They are the soluble secreted proteins, which can integrate easily with the target cells, consequently promoting EC proliferation and stimulating EC migration. VEGF121 has weaker mitogenic and migration inducing effect than VEGF165 (Ferrara et al. 2003). The anti-angiogenic effect of fucoidan appeared to be related to its inhibition of VEGFA expression in HUVECs, as shown by RT-PCR and western blot (Figs. 7 and 8), and such inhibition in turn resulted in the suppression of vascular tubes formation. There are many signaling molecules that participate in angiogenesis (Wamer et al. 2000; Lgarashi et al. 1998; Rousseau et al. 2000a,b), and although some of these have been identified and characterized, it still remains to be determined what other factors or molecules are involved in the regulation of angiogenesis. Signaling molecules, such as platelet-derived growth factor (PDGF) (Lindhal et al. 1997), fibroblast growth factor (FGF) (Mori et al. 2008), matrix metalloproteinases (MMPs) (Romanic et al. 2001), basic fibroblast factor (bFGF) (Smolej et al. 2005) and the angiopoietins (Ang) (Yancopoulos et al. 2000), may also contribute to the anti-angiogenic property of fucoidan, since these molecules are involved in tumor angiogenesis through autocrine or paracrine pathways. The data present in this study are likely to stimulate further interest in elucidating the molecular mechanism for angiogenesis. Therefore, further investigation is needed to unravel the mechanism associated with the anti-angiogenesis property of fucoidan. In conclusion, our study showed that fucoidan significantly inhibited angiogenesis in vitro and ex vivo, suggesting that fucoidan may be a potent anti-angiogenetic factor, but further investigation is warranted to obtain more information on the mechanism by which fucoidan exerts its anti-angiogenesis activity. Our study also opens up a new line of research on the anti-cancer potential of fucoidan, especially, in relation to its anti-angiogenic property.

Acknowledgements This work was supported by Research Fund from Education Department of Liaoning Province of the People’s Republic of China (no. 2009A199), the Science and Technology Department

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