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YAKUGAKU ZASSHI 128(9) 1293―1299 (2008) 2008 The Pharmaceutical Society of Japan 1293
―Regular Articles― Scutellarin Isolated from Erigeron multiradiatus Inhibits High Glucose-mediated Vascular In‰ammation
Pei LUO,a Zheng-Huai TAN,b Zhi-Feng ZHANG,a Hao ZHANG,a Xian-Fu LIU,c and Zheng-Ji MO,a
aWest China School of Pharmacy, Sichuan University, No. 17, Section 3, Ren-Min-Nan-Lu Road, Chengdu, Sichuan 610041, China, bSichuan Institute of Chinese Materia Medica, Chengdu, Sichuan, China, and cGanzi Institute of Drugs Control, Sichuan 626000, China
(Received January 29, 2008; Accepted May 26, 2008)
Erigeron multiradiatus (Lindl.) Benth is a traditional Tibetan medicine herb long used to treat various diseases related to in‰ammation. Our previous phytochemical studies on E. multiradiatus resulted in the isolation of scutellarin, which is a known ‰avone glucuronide with comprehensive pharmacological actions. In present study, we investigated the inhibition action of scutellarin on high glucose-induced vascular in‰ammation in human endothelial cells (ECV304 cells). Consistent with previous reports, exposure of ECV304 cells to high glucose for 24 h caused an increase of intercel- lular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1), and promoted cell adhesion between monocyte and ECV304 cells. However, pretreatment with scutellarin (0.1 and 1 mM) reversed these eŠects in a concentration-dependent manner. Scutellarin was able to inhibit the activation of NF-kB induced by high glucose in ECV304 cells. Furthermore, although oral administration of scutellarin (10 and 50 mg/kg) did not produce signiˆcant antihyperglycemic action, it lowered the serum MCP-1 levels signiˆcantly in alloxan-induced diabetic mice. Therefore, our results suggest that scutellarin has anti-in‰ammation eŠect that may aŠord some protection against hyperglycemia- induced vascular in‰ammatory both in vitro and in vivo.
Key words―scutellarin; adhesion molecule; nuclear factor-kappa B; endothelial cell
up-regulation of endothelial cell adhesion molecules INTRODUCTION including ICAM-1 and MCP-1 is mediated by interac- Diabetes vascular complication, due to its high tions between monocytes and endothelial cells via ac- prevalence, is becoming probably the most important tivation of nuclear factor-kappa B (NF-kB).9,10) metabolic disease and is widely recognized as one of Therefore suppression of vascular in‰ammation the leading causes of morbidity and mortality might be beneˆcial for the treatment of diabetic vas- worldwide.1,2) Diabetic chronic hyperglycemia, as a cular complications. common fate for patients with diabetes mellitus, in- Erigeron multiradiatus (Lindl.) Benth is naturally duces endothelial cell dysfunction, which can be de- distributed in the northern and southwestern moun- ˆned as a partial or complete loss of vascular tain regions of China as well as Sikkim, Nepal, and homeostasis including hemostasis, antigen presenta- Afghanistan.11) This folk medicine has been widely tion, permeability, in‰ammatory function, and an- used under ``meiduoluomi'' for the treatment of giogenesis.38) Over the past decade, accumulating hypopepsia, enteronitis, diarrhea, hepatitis, adeno- evidence has shown that endothelial dysfunction lymphitis, rheumatism, and hemiparalysis.12) Our closely related with vascular in‰ammatory process previous phytochemistry studies reported that E. mul- might represent an early stage of vasculopathy that tiradiatus comprised a mixture of diŠerent com- can lead to diabetic vascular disorders. Recently, pounds such as ‰avonoids, phenolic acids, monoter- reports have shown that the diabetic condition eleva- penes, sterols, etc.13,14) We successfully isolated tion of intracellular glucose causes overproduction of scutellarin (Fig. 1) from the extract of E. multiradia- adhesion molecules leading to leukocyte-endothelium tus.14,15) Scutellarin is a known ‰avone glucuronide adhesion, which is considered one of the earliest with comprehensive pharmacological actions, pri- events in vascular in‰ammation process. Therefore, marily derived from Erigeron breviscapus (Vant.) Hand-mazz. Since the 1970s, several studies showed e-mail: mzj-121＠scu.edu.cn the eŠectiveness of scutellarin on dilating blood ves- hon p.2 [100%]
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from Sigma Chemical Co. (St. Louis, MO, USA). The other chemicals used were of the highest analyti- cal grade commercially available. Cell Culture and Treatment ECV304 Cells, a spontaneously transformed line derived from the hu- man umbilical vein endothelial cell,19) were purchased from CCTCC (China). The cells were maintained at
37°C in a humidiˆed atmosphere of 5％ CO2 on cul- Fig. 1. Chemical Structure of Scutellarin ture plates with 10％ fetal bovine serum-supplement- ed DMEM medium. All the subsequent procedures sels, improving microcirculation, increasing cerebral were carried out under these conditions. ECV304 cells blood ‰ow, and inhibiting platelet aggregation.1618) were pretreated with a series of concentrations of Scutellarin preparations have been suggested for clini- scutellarin (0.1 and 1 mM) for 24 h then stimulated cal use to treat diabetes with vascular complications. with 30 mM glucose for 24 h. Cell treatment with However, as the main constituent in those prepara- scutellarin was conducted as follows: control (5.5 tions, scutellarin has not been investigated for its mM glucose),HG(30 mM glucose), low-concentra- eŠect on hyperglycemia-induced endothelial dysfunc- tion scutellarin (30 mM glucose plus 0.1 mM scutella- tion. Therefore the purpose of the present study was rin), high-concentration scutellarin (30 mM glucose to verify scutellarin's inhibitory eŠect against vascu- plus 1 mM scutellarin). All the glucose used in the lar in‰ammation both in high glucose-cultured hu- present study was D-glucose. man endothelial cells (ECV304 cells) and alloxan-in- Cell Viability and Morphology The MTT assay duced diabetic mice. for cell survival assessment was performed as described by Mosmann.20) ECV304 cells were seeded MATERIALS AND METHODS at 2×104 cell/well onto 96-well plates and preincu- Plant Material E. multiradiatus samples were bated for 24 h. Following incubation with various collected in natural growth sites in Luhuo, Sichuan, treatments, a volume of 200 mlof0.5mg/ml MTT in China. These samples were authenticated by Prof. DMEM medium was added to each well. After incu- Hao Zhang (West China School of Pharmacy, bation at 37°C for 4 h, the MTT solution was removed Sichuan University, Chengdu, P. R. China.) using from the medium. The resultant formazan crystals geographical origin identiˆcation, macroscopic iden- were dissolved in 150 ml of dimethylulfoxide tiˆcation, and microscopic identiˆcation. A voucher (DMSO) and the absorbance in each well was then specimen was deposited at Herbarium Center of West read at 570 nm using a microplate reader (BioRad China, School of Pharmacy, Sichuan University 3550, Bio-Rad Laboratories). Survival of control (NO. E12025). groups not exposed to high glucose was deˆned as 100 Extraction and Isolation E. multiradiatus (the ％. The numbers of surviving cells in the treated content of scutellarin is 1.1％ w/w) was extracted groups were expressed as a percent of control groups. with methanol. The yield (％,w/w dry plant materi- Values are given as averages for triplicate determina- al) of methanol extract is 20.3％. The extracts were tions. For the morphology study, cells were grown on subjected to column chromatographic separation on 6-well plates, treated with glucose and scutellarin, and macroporous resins and Sephadex LH-20; pure com- observed under light microscopy (Nikon, Japan) pound was obtained and yielded (％,w/w) 32.3％. then photographed. The chemical structure of the isolated compound Monocyte Adhesion Assay The monocyte-en- was identiˆed by comparison of spectral properties dothelial cell adhesion assay was modiˆed from that (MS,1H-,13C-NMR) with those reported in the of Cybulsky and Gimbrone.21) 5×106 ECV304 cells/ literature.14) well were seeded onto 24-well plate and incubated Reagents Cell culture materials were purchased with diŠerent treatments. After endothelial monolay- from Gibco-BRL (MD, USA).3-(4,5-dimethylthia- ers were washed three times with PBS, 1×105 mono- zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cytes/well were added and microplates incubated at and dimethylsulfoxide (DMSO) were purchased 37°C for 60 min. The monocytes used for monocyte- hon p.3 [100%]
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endothelial cell adhesion assay were isolated from supernatant was used as nucleus extract. The nucleus peripheral human blood as described previously.22) extracts were then analyzed for protein content using Theseafter the plates were sealed, inverted, and cen- Bradford assay. trifuged for 5 min to separate nonadherent mono- The above extract (70 mg) was separated on a 12％ cytes. Adherent cells were quantiˆed by protein-quan- sodium dodecyl sulfate (SDS) polyacrylamide gel titation methods. Monocyte adherence was calculated and transferred to PVDF membrane. Blots were then
as the ratio of the protein quantitation of adhered washed with H2O, blocked with 5％ skimmed milk monocytes to that of the total monocytes (1×105 powder in TBST [10 mM Tris-HCl (pH 7.6),150 cells) added. mM NaCl, 0.05％ Tween-20] for 1 h, and immunob- Cell ELISA Enzyme immunoassay was used to lotted using against NF-kBp65andb-actin proteins determine the level of adhesion molecule expression (1：1000 at 4°Covernight) antibody (Santa Cruz on ECV304 cells as previously described.23) Con‰uent Biotechnology, U.S.A). Then, the membrane was cells were grown on 96-well plate and stimulated with washed and primary antibodies were detected with 30 mM glucose in the presence or absence of diŠerent goat anti-rabbit-IgG conjugated to horseradish concentrations of scutellarin (0.1 and 1 mM).Ac- peroxidase detected by an enhanced chemilumines- cording to the manufacturer's protocol, after in- cence procedure (Amersham, Buckinghamshire, Eng- cubating endothelial cells under various conditions, land). the cells were washed 3 times with PBS and then ˆxed Experimental Animals Kunming mice weighing by 1％ paraformaldehyde and exposed to the antiad- 2025 g of either sex obtained from Animal House, hesion molecule antibody directed against ICAM-1, Pharmacy Discipline, Sichuan University, Chengdu, MCP-1 (puriˆed mouse anti-human ICAM-1, were used. The animals were fed with a rodent stan- MCP-1 antibody) at room temperature for 60 min, dard diet with free access water ad libitum and were washed three times with PBS, and incubated for 1 h housed in rooms maintained at 25±1°Cwitha12h with a horseradish-peroxidase-conjugated goat an- light/dark cycle following international recommenda- timouse IgG. The content of ICAM-1, MCP-1 was tions. The Animal Ethics Committees of the Faculty quantiˆed by adding a peroxidase substrate solution of Medicine, Sichuan University, approved all ex- and incubated at 37°C for 30 min. The reaction was perimental protocols, in accordance with ``Principles
stopped by addition of 5 NH2SO4, and the optical of Laboratory Animal Care and Use in Research'' density of each well was read using a microplate read- (Ministry of Health, Beijing, China). er. The expressions were calculated as the ratio of the Injecting alloxan at a dose of 60 mg/kg in- absorbance associated with various medium-treated travenously induced hyperglycemia in mice. Then, the versus control. mice were kept under observation and after 48 h were Preparation of Nuclear Extract and Western Blot tested for serum glucose. Animals with a blood glu- Analysis Cells were pretreated with scutellarin for cose level ＞16.7 mmol/l were considered diabetic 24 h and stimulated with high glucose for 24 h. After and equally divided into 4 groups of 8 animals each. treatment, the cells were harvested and washed twice Another 8 normal mice served as control. The 5 with ice-cold PBS, and then resuspended in 1 ml of groups were used in the antidiabetic study. Normal the same buŠer. Nuclear extracts were prepared on ice control group and model control group animals by the method previously described by Kim et al.24) received orally distilled water. Group 3 was ad- After centrifugation at 13000 rpm, the cell pellet was ministered pioglitazone (5mg/kg); group 4 scutella- suspended in ice-cold buŠer A (10 mmol/lHEPES rin (10 mg/kg), and group 5 scutellarin (50 mg/kg).
pH, 1.5 mmol/lMgCl2,0.2mmol/l KCl 0.2 mmol/l The animals were treated for 3 weeks. On the day 22, phenylmethylsulphonyl‰uoride, 0.5 mmol/l ditho- the animals were killed by decapitation and blood was threitol), vortexed for 10 s, then centrifuged at 13000 collected from the arterial jugular and serum separat- rpm for 5 min. The nuclear pellet was then washed in ed for estimation of blood glucose and MCP-1 levels 1mlbuŠer (20 mM HEPES, pH 7.9, 25％ glycerol, (mouse MCP-1 ELISA kit from ShangHai Lengton
0.42 M NaCl, 1.5 mM MgCl2,0.2mMEDTA) at Bioscience Co. LTD). 4200 g for 3 min, resuspended in 30 ml buŠer, rotated Statistical Analysis Statistical analysis of each at 4°C for 30 min, then centrifuged for 20 min. The set of data was carried out using oneway ANOVA hon p.4 [100%]
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and Duncan's multiple range test with SPSS11.5, and vascular complications.25) It has been shown previ- p values ＜0.05 were considered statistically sig- ously that hyperglycemia induces increased adhesion niˆcant. of leukocytes (especially monocytes) to endothelial cells as an early event in atherosclerosis and certain RESULTS AND DISCUSSION in‰ammatory disorders in diabetes-induced en- EŠect of Scutellarin on ECV304 Cells Viability and dothelial dysfunction.26) Inhibition of adhesion of Morphological Changes In this study, we carried monocytes is a pharmacological approach to attenu- out experiments to evaluate the cytotoxicity of scutel- ate hyperglycemia-induced vascular in‰ammatory larin on ECV304 cells. The results suggest that scutel- disorders. To determine the eŠect of scutellarin on en- larin does not signiˆcantly alter viability of ECV304 dothelial cell-monocyte interaction, we examined the cells until reaching a concentration of 100 mM (data adhesion of monocytes to ECV304 cells induced by not shown). As Fig. 2 shows, treatment with 30 mM high glucose. ECV304 cell monolayers were treated glucose signiˆcantly caused cytotoxicity against with 5.5 and 30 mM of glucose for 24 h and adhesion ECV304 cells as detected by MTT assay. However, assays performed with monocytes. As shown in Fig. pre-treating the cells with diŠerent concentrations of 3, the control group (5.5 mM glucose) showed scutellarin (0.1 and 1 mM) for 24 h markedly sup- minimal binding to monocytes, but when were treated pressed the damage of high glucose in a dose-depen- with high glucose (30 mM) for 24 h, the adhesion of dent manner and maximum eŠects (91.7％) were ob- monocytes to ECV304 cells was found signiˆcantly to served at the highest concentration (1 mM). Morpho- increase (69％ vs. 25％, n＝3, p＜0.01). On the other logical observation by phase microscopy showed that hand, the pretreatment with scutellarin (0.1 and 1 the cells were reduced by the treatment with 30 mM mM) signiˆcantly attenuated the adherence of mono- glucose. However, the morphology of scutellarin (0.1 cytes in a concentration-dependent manner (55％, and 1 mM) treated-cells was not diŠerent from that of 38％ vs. 69％, n＝3, p＜0.05 or p＜0.01). 5.5 mM glucose-treated cells. EŠects of Scutellarin on Level of High Glucose- EŠects of Scutellarin on Monocyte-Endothelial induced ICAM-1 and MCP-1 in ECV304 Cells Cell Adhesion Mediated by High Glucose There ICAM-1 and MCP-1 have been implicated as hav- is strong evidence for an involvement of monocyte ac- ing an important role in the mechanism of in‰amma- tivation in the pathogenesis of diabetes-associated tion in vascular disease.27) According to Takami et
Fig. 2. Cell Viability Cells were pre-cultured in serum-free medium in the presence or absence Fig. 3. EŠects of Scutellarin on High Glucose-induced Mono- of scutellarin (0.1 and 1 mM) for 24 h, and then stimulated with 30 mM glu- cyte Adhesion to ECV304 cells cose for 24 h. p＜0.05 compared with 5.5 mM glucose-treated values; #p＜ p＜0.05 compared with 5.5 mM glucose-treated values; #p＜0.05 com- 0.05 compared with 30 mM glucose-treated values. pared with 30 mM glucose-treated values. hon p.5 [100%]
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al.,28) expression of ICAM-1 was increased on the ba- blocked high glucose-induced increase of adhesion sis of diabetic vascular diseases. Baumgartner-Parzer molecules expression as regards the same glucose con- et al.29) also reported that incubation of human um- ditions where no scutellarin was added. bilical vein endothelial cells in 30 mM glucose for 24 h EŠect of Scutellarin on High Glucose-induced NF- increased ICAM-1 expression. Many epidemiological kB Activation We evaluated the eŠect of scutella- studies have shown that the concentration of soluble rinonhighglucose-inducedNF-kB activation by ICAM-1 in patients with type 2 diabetes is signiˆcant- western blotting analysis for NF-kB p65 protein level. ly higher than that in healthy subjects.30) In addition, NF-kB exists in the cytoplasm as a latent inactive MCP-1, a major chemotactic molecule of monocyte transcription factor form bound to an inhibitory pro- tra‹cking, generated within the vessel wall, has been tein, I-kB. Upon stimulation of cells, NF-kBis observed in a variety of cell types including mono- released rapidly from I-kB, translocates the active cytes, vascular smooth muscle cells, and vascular en- dimer p50 and p65 to the nucleus, and activates target dothelial cells in response to several diŠerent stimuli gene expression of several cytokines and chemotactic including hyperglycemia.31) Recent ˆndings seem to and matrix proteins involved in in‰ammation suggest that hyperglycemia could accelerate MCP-1 responses.32,33) In‰ammation at the cellular level can production in vascular cells. Thus glucose induction be described as an increase in the proin‰ammatory of MCP-1 might underlie the higher risk of cardiovas- transcription factor nuclear factor NF-kBinthe cular diseases in hyperglycemic states. To investigate nucleus, with concomitant decreases in its inhibitors, whether scutellarin inhibits vascular in‰ammation in- I-kB. Previous studies have shown that high glucose- duced by hyperglycemia, we evaluated its eŠect on induced cell adhesion molecules expression may de- high glucose-caused expression of ICAM-1 and pend on activation of NF-kB. Morigi et al.34) also MCP-1 in ECV304 cells using ELISA. As shown in reported that leukocyte adhesion to endothelial cells Fig. 4, cell ELISA results showed that high glucose increases in conditions of high glucose concentration (30 mM) increased ICAM-1 and MCP-1 contents in in a NF-kB-dependent fashion.34) Accumulating evi- comparison with the 5.5 mM condition (p＜0.01). dence suggests that NF-kB, known to be the primary Pretreatment with both concentrations of scutellarin transcription factor responsible for in‰ammation during hyperglycemia, may be a key step in disease progression in microvascular complications in diabetes.35) As shown in Fig. 5, translocation of NF- kB in the nuclear fractions of ECV304 cells was sig- niˆcantly increased by treatment with high glucose. However, scutallarin at a concentration ＞0.1 mM decreased high glucose-induced p65 NF-kB expres- sion levels. EŠect of Scutellarin on Blood Glucose and MCP-1 Level in Alloxan-induced Diabetic Mice At the third week, in the model control there was severe hyperglycemia as compared with the normal control
Fig. 4. EŠect of Scutellarin on High Glucose-induced In- crease of ICAM-1 and MCP-1 Expression Induced in ECV304 Cells p＜0.05 compared with 5.5 mM glucose-treated values; #p＜0.05 com- Fig. 5. EŠect of Scutellarin on High Glucose-induced NF-kB pared with 30 mM glucose-treated values. Activation in ECV304 Cells hon p.6 [100%]
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Table 1. EŠect of Scutellarin on Blood Glucose and MCP-1 Level in Alloxan-induced Diabetic Mice
Pioglitazone Scutellarin Scutellarin Samples Normal Model (5mg/kg) (10 mg/kg) (50 mg/kg) Blood glucose (mmol/l) 6.8±1.4 25.3±4.5 14.6±4.2# 24.6±3.6 23.0±5.1 MCP-1 (pg/ml) 3.24±0.57 5.11±0.46 4.36±0.48# 5.20±0.84 4.25±0.26#
Values are mean±S.E.M., p＜0.01 when compared with Normal, # p＜0.05 when compared to Model.
(p＜0.001). Comparing with the model control, 2) Feener E. P., King G. L., Heart Fail Monit., pioglitazone signiˆcantly lowered the elevated blood 1,7482 (2001). glucose levels, but there was no signiˆcant decline in 3) Rojas A., Morales M. A., Life Sci., 76,715 the two scutellarin-treated groups. It was also ob- 730 (2004). served that due to diabetes there was an increase in se- 4) Gokce G., Haznedaroglu M. Z., J. Ethno- rum MCP-1 levels in model groups (p＜0.01).The pharmacol., 115,122130 (2008). pioglitazone treated animals showed a signiˆcant 5) Kannel W. B., McGee D. L., JAMA, 241, reversal in the levels as compared with the model 20352038 (1979). animals. Scutellarin (50 mg/kg) treatment showed a 6) Nomura S., Suzuki M., Katsura K., Xie G. L., similar reducing capacity. On the contrary, scutellarin Miyazaki Y., Miyake T., Kido H., Kagawa (10 mg/kg) did not show any eŠect in lowering the H., Fukuhara S., Atherosclerosis, 116,23540 MCP-1 level, as shown in Table 1. (1995). 7) Tk áac I., Kimball B. P., Lewis G., UŠelman CONCLUSION K., Steiner G., Arterioscler. Thromb. Vasc. In conclusion, the present data suggest that scutel- Bio., 17,36333638 (1997). larin isolated from E. multiradiatus might inhibit 8) Lum H., Malik A. B., Am.J.Physiol., 267, high glucose-induced monocyte-endothelial cells ad- 223241 (1994) hesion by decreasing ICAM-1 and MCP-1 levels in 9) Lopes-Virella M. F., Virella G., Diabetes, 41, ECV304 cells as well as lower the elevated MCP-1 lev- Suppl 2, 8691 (1992). el in alloxan-induced diabetic mice. Here, we could 10) Quagliaro L., Piconi L., Assaloni R., Mar- conclude that the antidiabetic activity of scutellarin tinelli L., Motz E., Ceriello A., Diabetes, 52, may not be attributed to its antihyperglycemic action, 27952804 (2003). butmightbeassociatedwithdecreasedexpressionof 11) Ling R., Chen Y. L., Flora of China, 74,299 NF-kB, which is believed an important factor in the 311 (1985). progression of vascular disease in diabetes. Therefore 12) ``Dimaer･Danzengpengcuo,'' Jingzhu Herbal, our study suggests that the inhibitory eŠect of scutel- Science and Technology Press, Shanghai, larin on high glucose-induced vascular in‰ammation 1986, pp. 115116. is mediated via decreasing in‰ammatory factor levels 13) Zhang Y. J., Li L. Q., Yang P. Q., Zhang H., and reducing NF-kB nuclear translocation, and this Acta Pharm. Sin., 33,836838 (1988). may provide therapeutical beneˆts in diabetic vascu- 14) Zhang Y. J., Li L. Q., Yang P. Q., Zhang H., lar in‰ammation-related endothelial dysfunction. Chin. Trad. Herbal Drugs, 29,798800 (1988). Acknowledgements This work was co-ˆnanced 15) Zhang Z. F., Zhang H., Hu Y. H., Liu X. F., by the Sichuan Youth Science & Technology Founda- Chin. J. Ethnomed. Ethnopharm., 61,111 tion (05ZQ026034) and Applied fundamental Study 112 (2003). of Science and Technology Bureau of Sichuan 16) Xu M., Huang Z. H., Chin.J.NewDrugs Province (No. 2006Z08081). Clin. Rem., 10,260263 (1991). 17) LiuY.M.,LinA.H.,ChenH.,ZengF.D., REFERENCES Acta.Pharm.Sin., 38,775778 (2003). 1) Calles-Escandon J., Cipolla M., Endocr. 18) Chen X. X., He B., J. Chi. Pharm. Sci., 7,91 Rev., 22,3652 (2001). 93 (1998). hon p.7 [100%]
No. 9 1299
19) Moreno-Manzano V., Ishikawa Y., Lucio- J. Immunol., 173,36473652 (2004). Cazana J., Kitamura M., J. Biol. Chem., 274, 28) Takami S., Yamashita S., Kihara S., Kameda- 2025120258 (1999). Takemura K., Matsuzawa Y. Atherosclerosis, 20) Mosmann T., J. Immunol. Methods, 65,55 138,3541 (1998). 63 (1983). 29) Cominacini L., Fratta Pasini A., Garbin U., 21) Cybulsky M. I., Gimbrone M. A., Science, Davoli A., De Santis A., Campagnola M., 251,788791 (1991). Rigoni A., Zenti M. G., Moghetti P., Lo Cas- 22) GoldrosenM.H.,BiddleW.C.,PancookJ., cio V., Diabetologia, 38,11221124 (1995). Bakshi S., Vanderheyden J. L., Fritzberg A. 30) Davies M. J., Gordon J. L., Gearing A. J. H., R., Morgan A. C., Foon K. A., Cancer Res., J. Pathol., 171,223229 (1993). 50,79737978 (1990). 31) Shanmugam N., Reddy M. A., Guha M., 23) Rothlein R., Czajkowski M., O'Neill M. M., Natarajan R., Diabetes, 52,12561264 Marlin S. D., Mainolˆ E., Merluzzi V. J., J. (2003). Immunol., 141,16651669 (1988). 32) Gosh S., Baltimore D., Nature, 344,678682 24) Kim S. O., Kundu J. K., Shin Y. K., Park J. (1990). H., Cho M. H., Kim T. Y., Surh Y. J., On- 33) Iwasaki Y., Asai M., Yoshida M., Nigawara cogene, 24,25582567 (2005). T., Kambayashi M., Nakashima N., J. Clin. 25) Zardi E. M., Zardi D. M., Cacciapaglia F., Endocrinol. Metab., 89,34493454 (2004). Dobrina A., Amoroso A., Picardi A., Afeltra 34) MorigiM.,AngiolettiS.,ImbertiB.,Donadel- A., Int. Immunopharmacol., 5,437459 li R., Micheletti G., Figliuzzi M., Remuzzi A., (2005). Zoja C., Remuzzi G., J. Clin. Invest., 101, 26) Kim J. A., Berliner J. A., Natarajan R. D., 19051915 (1998). Nadler J. L., Diabetes, 43,11031107 (1994). 35) Auwardt R. B., Mudge S. J., Chen C., Power 27) Boisvert J., Edmondson S., Krummel M. F., D. A., Exp. Nephrol., 8,144151 (2008).