GENISTEIN-MEDIATED PROTECTION AGAINST INTERLEUKIN-4-INDUCED INFLAMMATORY PATHWAYS IN HUMAN VASCULAR ENDOTHELIAL CELLS Yong Woo Lee 1, Bernhard Hennig 2, Michal Toborek 1 Departments of Surgery 1 and Animal Sciences 2, University of Kentucky, Lexington, KY ABI PRISM® 7000 Sequence Detection (Applied Biosystems, Foster City, CA) Specific primers and TaqMan probes for PCR amplification: TaqMan® Pre-developed Assay Reagents for IL-1 , MCP-1 and  -actin Assay-on-Demand Products for VCAM-1 and E-selectin Thermal cycler profiles: 50  C for 2 min before the first cycle, 95  C for 15 sec and 60  C for 1 min, 45 times ΔΔC T = [C T of target gene - C T of housekeeping gene] treated group – [C T of target gene - C T of housekeeping gene] untreated control group Real-time reverse transcriptase-polymerase reaction 1.IL-4 induced inflammatory vascular environment through overexpression of a variety of inflammatory mediators, such as pro-inflammatory cytokine (IL-6), chemokine (MCP-1), and adhesion molecules (VCAM-1 and E- selectin). 2. IL-4 stimulated inflammatory reactions, such as leukocyte adhesion and leukocyte transmigration, in human vascular endothelium. 3. Genistein inhibited IL-4-mediated overexpression of IL-6, MCP-1, VCAM-1 and E-selectin in human vascular endothelial cells. 4. Genistein attenuated IL-4-induced inflammatory reactions, such as leukocyte transmigration. SUMMARY The present study indicates that genistein may protect against IL-4-induced inflammatory reactions in human vascular endothelium by inhibiting overexpression of inflammatory mediators. These results may contribute to development of therapeutic strategies for the prevention of initiation and progression of atherosclerosis specifically targeted against inflammatory pathways. CONCLUSION This work was supported by the American Heart Association, Ohio Valley Affiliate. ACKNOWLEDGEMENTS ABSTRACT Inflammatory vascular environment has been implicated in the critical early pathological events in atherogenesis. Recent studies have also demonstrated the potential protective role of genistein in the progression of atherosclerosis. The present study was designed to examine the hypothesis that genistein may attenuate IL-4- induced inflammatory pathways in human vascular endothelium. Real-time RT-PCR showed that exposure of HUVEC to IL-4 markedly and dose-dependently induced mRNA expression of a variety of inflammatory mediators, including interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. IL-4-induced inflammatory gene expression was paralleled by a concomitant production of protein. In addition, pretreatment with genistein dramatically attenuated IL-4-mediated induction of inflammatory mediators at the mRNA and protein levels. Our results suggest that IL-4 can produce inflammatory vascular environment through upregulation of inflammatory mediators and genistein may play a potential protective role in this process. These data may contribute to understanding the cellular and molecular mechanisms involved in IL-4-mediated progression of atherosclerosis and development of therapeutic strategies for the prevention of atherosclerotic lesion development specifically targeted against inflammatory pathways 1.Cells : Primary Human Vascular Endothelial Cells, HUVEC 2.Treatments Interleukin-4: 0.1, 1.0 and 10 ng/ml Genistein: 5, 25 and 50  M 3. Real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) – Gene Specific TaqMan® Assay 4. Enzyme-linked Immunosorbent Assay (ELISA & CELISA) 5. Leukocyte Adhesion Assay 6. Leukocyte Transmigration Assay Experimental Procedures IL-4 upregulates expression of inflammatory mediators in HUVEC (A) (B) (C) (D) IL-4 upregulates the mRNA expression of inflammatory mediators in human vascular endothelial cells. HUVEC were either untreated or treated with the indicated concentrations of IL-4 for 4 h. The mRNA levels of IL-6 (A), MCP-1 (B), VCAM-1 (C), and E-selectin (D) were determined by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Using the 2 –ΔΔCT method, the data are presented as the fold change in gene expression normalized to a housekeeping gene,  -actin, and relative to the untreated control. Data shown are means ± SE of 4 determinations. *Statistically significant compared with the control cultures. (A) (B) (C) (D) IL-4 upregulates the protein expression of inflammatory mediators in human vascular endothelial cells. HUVEC were either untreated or treated with the indicated concentrations of IL-4 for 16 h (IL-6 and MCP-1), 12 h (VCAM-1), or 6 h (E-selectin). The protein levels of IL-6 (A), MCP-1 (B), VCAM-1 (C), and E-selectin (D) were measured by ELISA, respectively. Data are means ± SD of 6 determinations. *Statistically significant compared with the control group (P<0.05). IL-4 induces inflammatory reactions in human vascular endothelium (A) (B) IL-4 upregulates the adhesion of leukocytes to human vascular endothelial cell monolayers (A) and the leukocyte transmigration (B). HUVEC were either untreated or treated with the indicated concentrations of IL-4 for 24 h (Adhesion assay) or 16 h (Transmigration assay). The adherence of calcein AM-labeled THP-1 cells was measured by fluorescent microplate reader using excitation of 490 nm and emission of 517 nm. Transmigration assay was performed by using the Chemicon 96-well Migration Assay kit according to the manufacturer's protocols. Data are means ± SD of 4 determinations. *Statistically significant compared with the control group (P<0.05). Genistein inhibits IL-4-mediated overexpression of inflammatory mediators in HUVEC (A) (B) (C) (D) Genistein inhibits the mRNA expression of inflammatory mediators in human vascular endothelial cells treated with IL-4. HUVEC were pretreated with indicated concentrations of genistein for 1 h and then exposed to 10 ng/ml of IL-4 for 4 h. The mRNA levels of IL-6 (A), MCP-1 (B), VCAM-1 (C), and E- selectin (D) were determined by quantitative real-time RT-PCR. Data are means ± SE of 4 determinations. *Statistically significant compared with the control group (P<0.05). #Values in the groups treated with IL-4 plus genistein are significantly different from the IL-4-treated group (P<0.05). (A) (B) Genistein attenuates the protein expression of inflammatory mediators in human vascular endothelial cells treated with IL-4. HUVEC were pretreated with indicated concentrations of genistein for 1 h and then exposed to 10 ng/ml of IL-4 for 16 h. The protein levels of IL-6 (A) and MCP-1 (B) were measured by ELISA. Data are means ± SD of 4 determinations. *Statistically significant compared with the control group (P<0.05). #Values in the groups treated with IL-4 plus genistein are significantly different from the IL-4-treated group (P<0.05). Genistein treatment attenuates IL-4-induced inflammatory reactions Genistein attenuates the IL-4-induced leukocyte transmigration. HUVEC were pretreated with indicated concentrations of genistein for 1 h and then exposed to 10 ng/ml of IL-4 for 16 h. Transmigration assay was performed by using the Chemicon 96-well Migration Assay kit according to the manufacturer's protocols. Data are means ± SD of 4 determinations. *Statistically significant compared with the control group (P<0.05). #Values in the groups treated with IL-4 plus genistein are significantly different from the IL-4-treated group (P<0.05). Soy isoflavone, Genistein Bioactive polyphenolic phytochemicals from soybean Antioxidant properties Beneficial health effects: Protection against the development of atherosclerosis Cancer chemoprevention Prevention of menopausal symptoms and osteoporosis