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DOI: 10.1055/s-2006-931602
© Georg Thieme Verlag KG Stuttgart · New York
Chrysin Inhibits Lipopolysaccharide-Induced Angiogenesis via Down-Regulation of VEGF/VEGFR-2(KDR) and IL-6/IL-6R Pathways
Jen-Hwey Chiu, MD, PhD
Institute of Traditional Medicine
School of Medicine
National Yang-Ming University
155, Sec. 2, Li-Nong St.
Peitou
Taipei 112
Taiwan
Republic of China
Phone: +886-2-2826+7178
Fax: 886-2-2822-5044
Email: chiujh@mailsrv.ym.edu.tw
Publication History
Received: December 21, 2005
Accepted: March 23, 2006
Publication Date:
29 May 2006 (online)
Abstract
The relationship between chrysin and inflammation-induced angiogenesis remains unclear. The aim of this study was to evaluate the suppressive effects of chrysin on lipopolysaccharide (LPS)-induced angiogenesis in chicken chorioallantoic membrane (CAM) as well as in human umbilical endothelial cells (HUVEC). The in vivo CAM model was applied to evaluate the percentage of new vessels formation, followed by measuring endothelial migration and tube formation in HUVEC cultures. The mechanisms of the suppressive effect of chrysin on LPS-induced angiogenesis, in terms of VEGF, VEGF receptors (VEGFR), interleukin 6 (IL-6) and IL-6 receptor gene expressions, were analyzed by Western blot, ELISA cytokine assay, and quantitative real time PCR. The results showed that chrysin (10 - 8 - 10 - 5M) inhibited LPS-induced CAM neovascular density. There was a significant down-regulation of VEGF and VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) gene expression by chrysin in LPS-treated HUVEC cultures. Besides, chrysin concentration-dependently inhibited the auto-regulation loop of IL-6/IL-6R in LPS-treated HUVEC cells. We conclude that chrysin suppresses both in vitro and in vivo LPS-induced angiogenesis.
#Introduction
Angiogenesis is an important process in embryonic development, wound healing, and diabetic retinopathy [1]. Previous investigations have shown that inflammation-stimulated angiogenesis plays a crucial role in rheumatoid arthritis, cardiovascular diseases and even cancer formation [2]. There are many molecular events, such as endothelial cell proliferation and differentiation as well as their regulatory mechanisms, involved in the process of angiogenesis [3], [4], [5]. Some growth factors, such as vascular endothelial growth factor (VEGF), related to angiogenesis have been demonstrated to interact with interleukin 6 (IL-6) in a co-culture model [6]. However, the exact mechanisms remain to be elucidated.
VEGF is a key mitogenic factor for vascular endothelial cells and is activated by shear stress, hypoxia, oxygen tension gradient, inflammation and extracellular matrix [7]. VEGF binds to two receptor tyrosine kinases, namely VEGFR-1 (fms-like tyrosine kinase, Flt-1) and VEGFR-2 (kinase-insert-domain-containing receptor, KDR) [8]. Although increasing evidence indicates that angiogenesis is a highly sophisticated and coordinated process, the activation of the VEGF/VEGFR pathway remains the key event of angiogenesis. Recent studies have demonstrated the presence of IL-6/IL-6 receptors and the relationship between IL-6 and VEGF gene expression in endothelial cell cultures [9], [10]. Furthermore, IL-6-soluble IL-6 receptor autocrine regulation has been documented through an increase in monocyte chemotactic protein 1 (MCP-1) in thrombin-activated-HUVEC inflammation reactions [11], [12]. Nevertheless, the impact of an anti-inflammatory compound, chrysin, on angiogenesis or IL-6/IL-6R auto-regulated VEGF gene expression has not been elucidated.
Chrysin (5,7-dihydroxyflavone; Fig. [1]) is a flavonoid extracted from many plants, honey and propolis. It possesses potent anti-inflammatory, anti-cancer and anti-oxidation properties [13], [14], [15]. Recently, chrysin has been demonstrated to suppress the expression of COX-2, tumor necrosis factor-α (TNF-α), IL-1 and IL-6 in lipopolysaccharide (LPS)-induced murine and human macrophage models [15], [16]. Nevertheless, it remains unclear how chrysin modulates LPS-induced angiogenesis. Therefore, the aim of this study was to evaluate the effects of chrysin on LPS-induced angiogenesis in chicken chorioallantoic membrane (CAM), as well as in human umbilical endothelial cells (HUVEC). Furthermore, the inhibitory effect of chrysin on in vivo and in vitro angiogenesis, as well as its effect on IL-6/IL-6R auto-regulated VEGF gene expression, will be presented.
Fig. 1 Structure of chrysin.
Materials and Methods
#Chemical reagents
Chrysin (Fig. [1]) of high purity (> 98 %) was purchased from Wako Pure Chemical Industry (Osaka, Japan). Lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), penicillin, streptomycin, trysolol (Bayer CropScience Co. Ltd) and thalidomide were obtained from Sigma Chemical Company (St. Louis, MO, USA). Basement membrane matrix (Matrigel) was acquired from Becton Dickinson (Bedford, MA, USA). Endothelial cell-based medium (EBM) was purchased from Cambrex Bio Science Walkersville, Inc (Walkersville, MD, USA) and Medium 199 from GIBCO BRL (Gaithersburg, MD, USA).
#Chorioallantoic membrane assay (CAM) in vivo
The in vivo angiogenesis model was performed as previously described [17]. In brief, a small 1 × 1 cm window in the sterilized egg-shell above the new air sac was made to expose the CAM. A sterilized straw (8 mm in internal diameter, 4 mm height) was put into the 1 × 1 cm window per egg under laminar flow, and 150 μL solution for testing (vehicle, LPS 1 μg/mL alone, thalidomide 30 μg/mL, or chrysin plus LPS induction) were loaded into the straw on the CAM surface. The windows were sealed with transparent tape and the eggs were incubated for 48 hours. Blood vessels were photographed with a Nikon digital camera, followed by analysis with image analysis software (Photoimact 8.0; Ulead System, Taiwan). Two independent, blinded investigators counted the blood vessels for each tested group.
#Human umbilical vein endothelial cells (HUVECs) culture
HUVECs were isolated from human umbilical veins and those with any underlying maternal diseases were excluded (such as carriers of hepatitis, diabetes, or hypertension). Human umbilical veins were flushed with cold phosphate-buffered saline (PBS) and human umbilical vein endothelial cells (HUVECs) were isolated as previously described [18]. The cells were maintained in EBM and M199 containing 12 % heat-inactivated fetal bovine serum (FBS), 100 μg/mL penicillin G, and 100 μg/mL streptomycin. In each independent experiment, HUVECs grown before the fifth passage were used for studies. All the cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO2. HUVECs were incubated in EBM and M199 containing 2 % FBS and then applied in each independent experiment.
#MTT assay for cell viability
HUVECs were implanted into a 96-well plate at 1500 cells/well and incubated for 48 hours. After implanting the cells and 2 % EBM overnight, the treated medium was substituted with 2 % EBM and the plates were incubated at 37 °C in 5 % CO2 for another 48 hours. The cell viability was quantitated by UV absorbance at 570 nm.
#Lipopolysaccharide-induced angiogenesis
LPS-induced angiogenesis model was used with some modification as previously described [19], [20]. In short, different concentrations of LPS (1, 10, 100 μg/mL) were added into the HUVEC culture. To test the efficacy of LPS-induced angiogenesis, the differentiation assay was performed to evaluate the percentage of the cellular permeability and new vessel formation, and the optimal doses were adapted according to the results of this assay.
#Migration assay and tube formation assay
Two assays were applied to study the phenotypic alteration of HUVECs, including migration assay and tube formation assay. The HUVEC migration assay was performed with minor modifications as mentioned before [20], [21]. The assay of endothelial matrigel tube formation has been previously described and was performed with minor modifications [22], [23]. Matrigel (250 μL) was coated in each 24-well-multiple-plate and was gelled at 37 °C by incubation for 30 minutes. After incubation, 5 × 104 HUVECs were implanted to each well and the material to be treated was added to a total volume of 1 mL. After 18 hours incubation at 37 °C, 5 % CO2, cell growth was assessed from 3-dimensional photos taken using a reverse-phase-contrast photomicroscope (Nikon, Eclipase, TE2000-U). The quantified analytical method was followed but slightly modified whereby a branch of vascular network was measured and its length scored [24].
#RNA isolation and real-time polymerase chain reaction (PCR)
All RNAs were isolated from 5 × 105/mL HUVECs after trizol reagent (T-9424, Sigma-Aldrich; St Luis, MO, USA) digestion. A Lightcycler (Roche Diagnostics; Mannhein, Germany) was used for the procedures of real-time PCR. The primers used were as follows, forward: 5′-CATCG ACAGA ACAGT CCT-3′, reverse: 5′-CAACT CAAGTC CACAGC-3′ (386 bp) for VEGF; forward: 5′-ACATCC TCGAC GGCAT CT-3′, reverse: 5′-GTGGT TGGGT CAGGG GTG-3′ for IL-6 (337 bp); forward: 5′-GTCCTA GAGCG TGTGG-3′, reverse: 5′-CGCCG TGCCT ACTAGA-3′ for VEGFR-2 (KDR) (336 bp); forward: 5′-AGCAA CAGTC AATGGG-3′, reverse: 5′-GTCGC CTTAC GGAAGC-3′ for VEGFR-1 (Flt-1) (336 bp); forward: 5′-CATAG TGTCC ATGTGCG-3′, reverse: 5′-AACCG TAGTC TGTAG AAAG-3′ (195 bp) for sIL-6R; forward: 5′-CATCAC CATCT TCCAG GAGC-3′, reverse: 5′-GGATG ATGTTC TGGGC TGCC-3′ (405 bp) for GAPDH (internal control) were purchased from Mission Biotech company (Taipei, Taiwan).
#Cytokines assay for VEGF, VEGFR-2, IL-6, and sIL-6Rα
After chrysin treatment of HUVEC for 18 hours, the supernatants were collected and prepared with 60 μL trysolol and 3 mL culture medium. Following centrifugation of the samples, the supernatants were used for the cytokine assay. VEGF, IL-6, and sIL-6Rα were measured by a specific enzyme-linked immunosorbent assay (ELISA) (Quantikine, R&D systems; Minneapolis, MN, USA). The assay procedure was performed according to the guidelines of the data sheets. The microplate reader was set to 450 nm in order to determine the absorbance of optical density of each well within 30 minutes, and the wavelength correction was set to 540 nm.
#Western blot analysis for VEGF, VEGFR-1, VEGFR-2, sIL-6Rα
To evaluate and confirm the expression of all VEGF isoform proteins, the non-soluble (non-secreted) isoforms VEGF189, VEGF206 and total VEGF proteins were measured with a detection ELISA cytokine assay. VEGF, VEGFR-1 (Flt-1) and IL-6R antibodies were purchased from R&D system (Minneapolis, MN, USA). The VEGFR-2 (KDR) antibody came from Cell Signaling Company (Beverly, MA, USA).
#Statistical analysis
All data in different experimental groups were expressed as means ± S.D. All the data shown in the study were obtained in at least 3 independent experiments. Statistical significance was evaluated by analysis of variance (ANOVA) followed by Bonferroni’s test or Wilcoxon signed rank or rank sum test. A P value less than 0.05 was considered as statistically significant.
#Results
Fig. [2] shows the anti-angiogenic effect of chrysin in the chicken CAM model. There was a significantly increased vascular density index upon LPS (1 μg/mL) induction, which could be suppressed by the pretreatment with chrysin in a concentration range of 10 - 8 to 10 - 5 M (Fig. [3]).
To elucidate the anti-angiogenic properties of chrysin observed in the CAM model, mechanistic studies were performed in the HUVEC culture. After HUVECs were induced with LPS (1 μg/mL), the pretreatment with chrysin inhibited cell migration and tube formation in a concentration-dependent manner. (Fig. [4] and Table [1], p < 0.05) In the MTT assay, there was no significant cytotoxicity when cells were treated with different concentrations of chrysin (data not shown).
Since VEGF is a strong inducer of new vessel formation, the VEGF/VEGFR signaling pathway was evaluated. The results showed that VEGF levels in the cultured medium were decreased upon chrysin treatment (data not shown). By Western blot analysis, it was shown that chrysin concentration-dependently inhibited VEGF and VEGFR-2, but not VEGFR-1 expression in LPS-treated HUVEC cultured for18 hours (Fig. [5]). In addition, pretreatment with chrysin decreased the mRNA levels of VEGF and VEGFR-2 in a concentration-dependent manner (Fig. [6]). These data suggest that down-regulation of the de novo synthesis of the VEGF/VEGFR-2 gene expression plays an important role in chrysin-inhibited angiogenesis.
To further elucidate the IL-6/IL-6R auto-regulation loop and anti-angiogenic properties, chrysin was applied to LPS-induced HUVECs. Supernatants and cell lysates were collected for IL-6 ELISA assays and IL-6R Western blot analysis, respectively. The results revealed that there was a concentration-dependent decrease in IL-6 (ELISA) and IL-6R (Western blot) expression in chrysin-treated LPS-induced HUVEC culture (Fig. [7]). Furthermore, quantitative real-time PCR showed that IL-6 mRNA and sIL-6Rα mRNA expression was significantly suppressed by chrysin treatment, in a concentration range of 10 - 6 to 10 - 5 M for the former and of 10 - 7 to 10 - 5 M for the latter, as compared to the vehicle-treated LPS-induced group (data not shown). These data suggested that chrysin inhibited the de novo gene expression of IL-6 and IL-6R.
Fig. 2 Effects of chrysin on chicken chorioallantoic membrane (CAM). New vessel formation in CAM after lipopolysaccharide (LPS; 1 μg/mL) induction with either chrysin (10 - 8 M) or thalidomide (30 μg/mL) was evaluated as described in Materials and Methods. Arrows indicate new vessel formation arising from a second-order of vascular trees.
Fig. 3 Quantification of neovascularization in the CAM model. The effects of chrysin on lipopolysaccharide (LPS)-induced new vessel formation were quantified as described in Materials and Methods. * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).
Fig. 4 Effects of chrysin on tube formation in human umbilical vein endothelial cells (HUVEC). Endothelial tube formation in lipopolysaccharide (LPS; 1 μg/mL) induction with either chrysin (10 - 6 M) or thalidomide (30 μg/mL) was evaluated as described in Materials and Methods. Bars indicate 0.1 mm.
Fig. 5 Effects of chrysin on VEGF and VEGFR-2 proteins expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line. Upper panel shows the qualitative analysis by Western blot as described in Materials and Methods. Middle and lower panels showed quantification results of chrysin on VEGF and VEGFR-2 proteins expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line, respectively. * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).
Fig. 6 Effects of chrysin on VEGF and VEGFR-2 mRNA gene expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line. Upper and lower panels show real-time polymerase chain reaction (PCR) results of chrysin on VEGF and VEGFR-2 mRNA expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line, respectively. The detailed procedures for real-time PCR analysis were described in Materials and Methods and data are presented as fold of vehicle (LPS-treated alone group). * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).
Fig. 7 Effects of chrysin on interleukin 6 (IL-6) and IL-6 receptor (IL-6R) proteins expression on lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line. Upper and lower panels show the results of chrysin on IL-6 and IL-6R protein expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line, respectively. IL-6 in the cultured medium was measured by a specific enzyme-linked immunosorbent assay (ELISA) while IL-6R was determined by Western blot analysis as described in Materials and Methods. The data are presented as pg/mL for IL-6 and fold of vehicle for IL-6R, respectively. * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).
| Groups | Migration assay | Tube formation assay |
| Vehicle | 102 ± 8.7# | 9 ± 2.2# |
| LPS (1 μg/mL) | 174 ± 9.8 | 53 ± 4.7 |
| LPS + Chrysin 10 - 7 M | 96 ± 11.7* | 22 ± 4.6* |
| LPS + Chrysin 10 - 6 M | 80 ± 12.8* | 12 ± 5.9* |
| LPS + Chrysin 10 - 5 M | 59 ± 9.9* | 8 ± 2.2* |
| LPS = lipopolysaccharide. Following seeding of 5 × 104 HUVECs into each well and induction with LPS (1 μg/mL), cells were incubated with different concentrations of chrysin for 18 hours at 37 °C, 5 % CO2 and photographed as described in the Materials and Methods. * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon signed rank test). | ||
Discussion
In this study, we show that chrysin attenuated LPS-induced angiogenesis through inhibition of the VEGF/VEGFR-2 and IL-6/IL-6R pathways. It is generally accepted that inflammation induces angiogenesis and that many flavonoids inhibit the inflammatory process. For example, chrysin has been demonstrated to suppress pro-inflammatory cytokines such as TNF-α, IL-1, IL-6 as well as COX-2 gene expression in LPS-induced murine and human macrophage models [15], [16]. To our knowledge, this is the first report on the inhibitory effect of chrysin on the VEGF/VEGFR-2 and IL-6/IL-6R signaling pathways.
Chicken CAM is a suitable model for in vivo studies of angiogenesis [17], [20], [25]. LPS-induced angiogenesis has been postulated to be mediated through Toll-like receptor (TLRs)-mediated activation of the TNF receptor associated factor 6 (TRAF6)-dependent signaling pathway [20]. Our results showed that LPS increased the vascular density, while thalidomide suppressed the LPS-enhanced new vessels formation. Besides, on using LPS-responsive cells isolated from TLR4-mutant (C3H/HeJ) and wild-type (C3H/HeN) mice for studying the hepatocyte growth in isolated hepatocyte cultures, there was no significant difference between these two lines when treated with Scutellaria baicalensis Georgi, a medicinal herb containing chrysin. This suggested that such a chrysin-containing herb-stimulated hepatocyte growth was not necessarily mediated via the Toll-like receptor 4 (Wang JY, manuscript in revision). However, the exact relationship between TLRs signaling, TRAF6 and chrysin requires further investigation.
A previous report has shown that chrysin has no inhibitory effect on LPS-induced bovine endothelial cytotoxicity [26], while the IC25 of chrysin-induced cytotoxicity (measured with the MTT assay) in the present study was 3 × 10 - 8 M. There is evidence suggesting that the angiogenic effect was promoted through the VEGF/VEGFR-2 (KDR) pathway in the mice model following IL-1α stimulation [27]. Recent investigations concerning the anti-angiogenic properties of many flavonoids, such as genistein, apigenin, and puerarin, have demonstrated that they act through down-regulation of VEGF and KDR gene expressions in various cell lines [28], [29]. The role of VEGFR-1 (Flt-1) has been debated and its precise function is still unclear. Our results showed no significant change in VEGFR-1 protein expression (Western blot analysis) in chrysin-treated HUVEC cells. On the contrary, KDR is a well-known factor and shows a higher kinase activity than Flt-1 [17]. Based on analyses of protein level and mRNA expression, our data revealed that chrysin inhibited the de novo gene expression of VEGF and KDR.
Pro-inflammatory cytokines, such as IL-6, TGF-beta, and the inflammatory response enhance angiogenesis, tumor growth and the correlation with VEGF expression in both in vitro and in vivo models [7]. Previous studies showed that the nuclear factor of IL-6 (NF-IL6) played an important role both in IL-1-induced COX-2 gene expression in the human microvascular endothelial cell line [30] and in chrysin-mediated COX-2 down-regulation in the murine macrophage RAW 264.7 line [15]. Nevertheless, information concerning the relationship between NF-IL6 and angiogenesis from the above-mentioned studies is lacking. It is worth mentioning that the modulated mechanism of IL-6 on the VEGF pathway has been demonstrated through the regulation of MAPK or STAT3 signaling in inflammation-related angiogenesis [9]. In the present study, chrysin is demonstrated to inhibit angiogenesis through down-regulation of the VEGF/VEGFR-2 and IL-6/IL-6R pathways. Taken together, these data provide evidence for the close relationship between IL-6 signaling and neovascularization, which is further supported by our results from the in vivo CAM model showing that the new vessels formation was significantly suppressed by anti-IL-6 treatment.
In summary, chrysin suppresses both in vitro and in vivo LPS-induced angiogenesis through inhibition of the IL-6-mediated VEFG/VEGFR-2 pathway, which may shed light on the clinical application of chrysin in inflammation-related angiogenic diseases.
#References
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Jen-Hwey Chiu, MD, PhD
Institute of Traditional Medicine
School of Medicine
National Yang-Ming University
155, Sec. 2, Li-Nong St.
Peitou
Taipei 112
Taiwan
Republic of China
Phone: +886-2-2826+7178
Fax: 886-2-2822-5044
Email: chiujh@mailsrv.ym.edu.tw
References
- 1 Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995; 1 27-31
- 2 Karin M. Inflammation and cancer: the long reach of Ras. Nat Med. 2005; 11 20-1
- 3 Lubarsky B, Krasnow M A. Tube morphogenesis making and shaping biological tubes. Cell. 2003; 112 19-28
- 4 Kofler S, Nickel T, Weis M. Role of cytokines in cardiovascular disease: a focus on endothelial responses to inflammation. Clin Sci. 2005; 108 205-13
- 5 Risau W. Mechanism of angiogenesis. Nature. 1997; 386 671-4
- 6 Molostvov G, Morris A, Rose P, Basu S, Muller G. The effects of selective cytokines inhibiting drugs (CC-10 004 and CC-1088) on VEGF and IL-6 expression and apoptosis in myeloma and endothelial cell co-cultures. Brit J Haematol. 2004; 124 366-75
- 7 Kozian D H, Augustin H G. Rapid identification of differentially expresses endothelial cell genes by RNA-display. Biochem Biophys Res Commun. 1995; 209 1068-75
- 8 Ferrara N. Vascular endothelial growth factor: Basic science and clinical progress. Endocr Rev. 2004; 25 581-611
- 9 Heinrich P C, Behrmann I, Haan S, Hermanns H M, Muller-Newen G, Schaper F. Principles of interleukin (IL)-6-type cytokine signaling and its regulation. Biochem J. 2003; 374 1-20
- 10 Modur V, Li Y, Zimmerman G A, Prescott S M, Mclintyre T M. Retrograde inflammatory signaling from neutrophils to endothelial cells by soluble interleukin-6 receptor alpha. J Clin Invest. 1997; 100 2752-6
- 11 Cohen T, Nahari D, Cerem L W, Levi B Z. Interleukin 6 induced the expression of vascular endothelial growth factor. J Biol Chem. 1996; 271 736-41
- 12 Morin V, Montero-Julian F A, Gres S, Boulay V, Bongrand P, Farnarier C. et al . The IL-6, soluble IL-6Rα autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation an experimental model involving thrombin. J Immunol. 2001; 167 3435-42
- 13 Monasterio A, Urdaci M C, Pinchuk I V, Lopez-Moratalla N, Martinez-Irujo J J. Flavonoids induce apoptosis in human leukemia U937 cells through caspase- and caspase-calpain-dependent pathways. Nutr Cancer. 2004; 50 90-100
- 14 Weng M S, Ho Y S, Lin J K. Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21(Waf1/Cip1) expression: Involvement of p38 mitogen-activated protein kinase. Biochem Pharmacol. 2005; 69 1815-27
- 15 Woo K J, Jeong Y J, Inoue H, Park J W, Kwon T K. Chrysin suppresses lipopolysaccharide-induced cyclooxygenase-2 expression through the inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activity. FEBS Lett. 2005; 31 705-11
- 16 Hougee S, Sanders A, Faber J, Graus Y M, van den Berg W B, Graessen J. Decreased pro-inflammatory cytokine production by LPS-stimulates PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin due to selective elimination of monocytes/macrophages. Biochem Pharmacol. 2005; 15 241-8
- 17 Yang S H, Lin J K, Huang C J, Chen W S, Li S Y, Chiu J H. Silibinin inhibits angiogenesis via Flt-1, but not KDR, receptor up-regulation. J Surg Res. 2005; 128 140-6
- 18 Jeffe E A, Nachman R L, Becker C G, Minick C R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973; 52 2745-56
- 19 Unger R E, Peters K, Huang Q, Funk A, Paul D, Kirkpatrick C J. Vascularization and gene regulation of human endothelial cells growing on porous polyethersulfone (PES) hollow fiber membranes. Biomaterials. 2005; 26 3461-9
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Jen-Hwey Chiu, MD, PhD
Institute of Traditional Medicine
School of Medicine
National Yang-Ming University
155, Sec. 2, Li-Nong St.
Peitou
Taipei 112
Taiwan
Republic of China
Phone: +886-2-2826+7178
Fax: 886-2-2822-5044
Email: chiujh@mailsrv.ym.edu.tw
Fig. 1 Structure of chrysin.
Fig. 2 Effects of chrysin on chicken chorioallantoic membrane (CAM). New vessel formation in CAM after lipopolysaccharide (LPS; 1 μg/mL) induction with either chrysin (10 - 8 M) or thalidomide (30 μg/mL) was evaluated as described in Materials and Methods. Arrows indicate new vessel formation arising from a second-order of vascular trees.
Fig. 3 Quantification of neovascularization in the CAM model. The effects of chrysin on lipopolysaccharide (LPS)-induced new vessel formation were quantified as described in Materials and Methods. * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).
Fig. 4 Effects of chrysin on tube formation in human umbilical vein endothelial cells (HUVEC). Endothelial tube formation in lipopolysaccharide (LPS; 1 μg/mL) induction with either chrysin (10 - 6 M) or thalidomide (30 μg/mL) was evaluated as described in Materials and Methods. Bars indicate 0.1 mm.
Fig. 5 Effects of chrysin on VEGF and VEGFR-2 proteins expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line. Upper panel shows the qualitative analysis by Western blot as described in Materials and Methods. Middle and lower panels showed quantification results of chrysin on VEGF and VEGFR-2 proteins expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line, respectively. * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).
Fig. 6 Effects of chrysin on VEGF and VEGFR-2 mRNA gene expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line. Upper and lower panels show real-time polymerase chain reaction (PCR) results of chrysin on VEGF and VEGFR-2 mRNA expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line, respectively. The detailed procedures for real-time PCR analysis were described in Materials and Methods and data are presented as fold of vehicle (LPS-treated alone group). * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).
Fig. 7 Effects of chrysin on interleukin 6 (IL-6) and IL-6 receptor (IL-6R) proteins expression on lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line. Upper and lower panels show the results of chrysin on IL-6 and IL-6R protein expression on the lipopolysaccharide (LPS, 1 μg/mL)-induced HUVEC line, respectively. IL-6 in the cultured medium was measured by a specific enzyme-linked immunosorbent assay (ELISA) while IL-6R was determined by Western blot analysis as described in Materials and Methods. The data are presented as pg/mL for IL-6 and fold of vehicle for IL-6R, respectively. * p < 0.05 versus LPS-treated group (one-way ANOVA); # p < 0.05 versus LPS-treated group (Wilcoxon rank sum test).