Advanced glycation end products and lipopolysaccharide synergistically stimulate proinflammatory cytokine ⁄ chemokine production in endothelial cells via activation of both mitogen-activated protein kinases and nuclear factor-jB Jinghua Liu 1 , Shanchao Zhao 1 , Jing Tang 1 , Zhijie Li 1 , Tianyu Zhong 1 , Yawei Liu 1 , Dengyu Chen 1 , Mingzhe Zhao 1 , Yusheng Li 1 , Xiaowei Gong 1 , Peng Deng 1 , Jiang H. Wang 2 and Yong Jiang 1 1 Key Laboratory of Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University, Guangzhou, China 2 Department of Surgery, University College Cork, Cork University Hospital, Cork, Ireland Introduction As a barrier between circulating blood and extra- vascular tissues, endothelial cells are persistently exposed to diverse circulatory mediators, including endogenous proinflammatory mediators and exogenous pathogens Keywords advanced glycation end products; cytokines ⁄ chemokines; lipopolysaccharide; MAP kinases; NF-jB Correspondence J. H. Wang or Y. Jiang, Department of Surgery, University College Cork, Cork University Hospital, Cork, Ireland; Key Laboratory of Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China Fax: +353 21 4901240; +86 20 87277521 Tel: +353 21 4901275; +86 20 61648231 E-mail: jh.wang@ucc.ie; yjiang@fimmu.com (Received 17 April 2009, revised 17 June 2009, accepted 22 June 2009) doi:10.1111/j.1742-4658.2009.07165.x It has been well documented that both endogenous inflammatory mediator advanced glycation end products (AGEs) and exogenous inflammatory inducer lipopolysaccharide play key roles in the initiation and development of inflammatory diseases. However, the combined inflammation-stimulatory effect of AGEs and lipopolysaccharide on endothelial cells, and, further- more, the underlying signal transduction pathways involved, have not been fully elucidated. We found that in vitro co-stimulation with AGE-human serum albumin (HSA) and lipopolysaccharide exhibits a synergistic effect on proinflammatory cytokine ⁄ chemokine interleukin-6, interleukin-8 and monochemoattractant protein-1 production in human umbilical vein endo- thelial cells. Similar to lipopolysaccharide, AGE-HSA stimulation induced mitogen-activated protein kinase phosphorylation and nuclear factor-jB nuclear translocation in human umbilical vein endothelial cells, which was further enhanced by a combination of the two stimulants. Pharmacological inhibitions of each individual signaling pathway, including p38, extracellu- lar signal-regulated kinase 1 ⁄ 2, Jun N-terminal kinase and nuclear factor- jB, revealed that activation of all of these four pathways is necessary for the effective induction of interleukin-6, interleukin-8 and monochemoattr- actant protein-1 by both AGE-HSA and lipopolysaccharide. These results suggest that AGEs and lipopolysaccharide cooperatively induce proinflam- matory cytokine ⁄ chemokine production by activating mitogen-activated protein kinases and nuclear factor-jB in endothelial cells, thus amplifying the inflammatory response and resulting in tissue damage. Abbreviations AGE, advanced glycation end product; ERK, extracellular signal-regulated kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; HSA, human serum albumin; HUVEC, human umbilical vein endothelial cell; IFN, interferon; IL, interleukin; IP, interferon-inducible protein; JNK, Jun N-terminal kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; MCP, monochemoattractant protein; NF, nuclear factor; PDTC, pyrrolidine dithiocarbamate. 4598 FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS and their toxic components [1,2]. Studies have confirmed that endothelial cells are the major target for these inflammatory initiators, which participate in the devel- opment of diseases by promoting proinflammatory cyto- kine ⁄ chemokine release, adhesion molecule expression and reactive oxygen species production [2,3]. Advanced glycation end products (AGEs) are a het- erogeneous group of molecules formed from the non- enzymatic glycation and oxidation reaction between reducing sugars and free amino groups of proteins, lip- ids and nucleic acids [4]. AGEs normally form at a constant but slow rate in the body, and accumulate markedly by aging and diabetes as a result of the increased availability of glucose [4,5]. A large body of evidence suggests that AGEs are important pathogenic mediators for the development of various diseases, such as diabetic complications, Alzheimer’s disease, atherosclerosis and dialysis-related amyloidosis [6–8]. Experimental studies have shown that interaction of AGEs with the receptor for advanced glycation end products activates monocytes and endothelial cells through intracellular signal transduction pathways to induce the expression of cytokines, adhesion molecules and tissue factors [9]. It has been demonstrated that endothelial cells play a critical role in the development of many diseases that involve elevation of AGEs. For example, AGEs are found in the retinal vessels of dia- betic subjects, and their levels correlate with the sever- ity of the subject’s retinopathy [10]. Upon the action of AGEs, endothelial cells upregulate adhesion mole- cule expression and increase proinflammatory cyto- kine ⁄ chemokine release, resulting in firm adhesion and recruitment of leucocytes to the loci of inflammation [7,9]. Lipopolysaccharide (LPS), a major outer membrane component of Gram-negative bacteria, signals through Toll-like receptor 4 and is a potent inducer of systemic inflammatory response by stimulating monocytes ⁄ macrophages to produce proinflammatory cyto- kines ⁄ chemokines [11–13]. Previous studies have revealed that endothelial cells are also a major target for LPS; with the help of soluble CD14 in the circula- tion, LPS directly activates endothelial cells by inter- acting with Toll-like receptor 4, thus ultimately causing endothelial cell dysfunction and damage to the barrier function of blood vessels [11,14]. Clinical studies have shown that patients with high levels of circulating AGEs, such as diabetics and the elderly, are prone to complicating bacterial infections as a result of their depressed immune function [15,16]. In clinical practice, antibiotics and blood dialysis are intensively applied to these patients to control bacterial infection or to remove the metabolic toxic products from the body. However, both therapeutic approaches might elevate LPS in the circulation because bacteria killed by antibiotics release endotoxin ⁄ LPS from the outer membrane and dialysis procedures increase the risk of bacterial infection and ⁄ or blood contamination with LPS [17,18]. It remains unclear whether AGEs and LPS act syner- gistically to amplify the inflammatory response in endo- thelial cells. In the present study, we show that stimulation of human endothelial cells with a combina- tion of AGEs and LPS demonstrates a synergistic effect on proinflammatory cytokine ⁄ chemokine production, which requires both mitogen-activated protein (MAP) kinase and nuclear factor-jB (NF-jB) activation. Results AGE-human serum albumin (HSA) and LPS stimulate a time- and dose-dependent cytokine ⁄ chemokine production To determine the expression profiles of cyto- kines ⁄ chemokines in human umbilical vein endothelial cells (HUVECs) upon stimulation with AGE-HSA and LPS, fourteen cytokines ⁄ chemokines, including tumor necrosis factor-a, interleukin (IL)-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-c, granulocyte- macrophage colony-stimulating factor (GM-CSF), monochemoattractant (MCP)-1, macrophage inflam- matory protein-1, interferon-inducible protein (IP)-10 and RANTES (regulated upon activation, normal T cell expressed and secreted) were analyzed using a LiquiChip work station (Qiagen, Chatsworth, CA, USA). Incubation of HUVECs with AGE-HSA or LPS resulted in a significantly increased production of IL-6, IL-8 and MCP-1 in both a time-dependent (Fig. 1A) and dose-dependent (Fig. 1B,C) fashion. Notably, AGE-HSA and LPS induced a very similar pattern of cytokine ⁄ chemokine expression, although LPS stimula- tion caused a much higher cytokine ⁄ chemokine response than did AGE-HSA (Fig. 1). LPS, but not AGE-HSA, also significantly induced IFN-c, IP-10 and GM-CSF expression in HUVECs (data not shown). However, neither LPS, nor AGE-HSA stimu- lated HUVECs to produce tumor necrosis factor-a, IL-1b, IL-2, IL-4, IL-10 and IL-12 (data not shown). AGE-HSA and LPS have a synergistic effect on the induction of cytokines ⁄ chemokines To determine whether there is any combined effect of AGE-HSA and LPS on the induction of cytokines ⁄ chemokines, we stimulated HUVECs with AGE-HSA J. Liu et al. Glycation end products and lipopolysaccharide in cytokine production FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS 4599 at 25 lg ÆmL )1 , LPS at 10 ngÆmL )1 , or their combina- tion, for up to 24 h. Compared to each single stimulus, co-stimulation with both stimuli led to a much stron- ger response on IL-6, IL-8 and MCP-1 production (Fig. 2A). For example, on stimulation with either AGE-HSA alone or LPS alone, IL-6 increased from 12.1 ± 2.1 pgÆmL )1 of basal level to 48.3 ± 13.2 pgÆmL )1 and 343.3 ± 93.1 pgÆmL )1 , respectively; upon co-stimulation with both stimuli, IL-6 increased to 1783.3 ± 131.8 pgÆmL )1 , which is much higher than the addition of the IL-6 levels induced by each single stimulus (P < 0.01) (Fig. 2A). Consistently, the induc- tion of IL-8 and MCP-1 also showed synergistic effects after stimulation with both stimuli (P < 0.01), but this was not as strong as that of IL-6 (Fig. 2A). However, we failed to demonstrate any synergistic effects on IFN-c, IP-10 and GM-CSF production after AGE- HSA and LPS co-stimulation (data not shown), suggesting that the synergistic induction of cytokines ⁄ chemokines by AGE-HSA and LPS in HUVECs is highly selective. We further performed RT-PCR and found that either AGE-HSA alone or LPS alone increased the mRNA transcripts of IL-6, IL-8 and MCP-1 compared to their low levels in naive cells (Fig. 2B). Convinc- ingly, co-stimulation with AGE-HSA and LPS led to a further increase in gene transcription of these cyto- kines ⁄ chemokines (P < 0.05) (Fig. 2B). Activation of both MAP kinases and NF-jBis required for the AGE-HSA and LPS amplified cytokine ⁄ chemokine response To elucidate the signal mechanisms involved in the AGE-HSA and LPS amplified cytokine ⁄ chemokine response, we first compared the signal transduction pathways in HUVECs induced by LPS with those induced by AGE-HSA. As expected, incubation of HUVECs with LPS led to activation of both MAP kinases (Fig. 3A) and NF-jB (Fig. 3B). Similar to LPS, AGE-HSA stimulation also induced phosphory- lation of p38, extracellular signal-regulated kinase pg·mL –1 LPS (ng·mL –1 ) 0 1 10 100 1000 0 200 400 600 800 1000 * * * * 0 200 400 600 800 IL-6 pg·mL –1 A B C AGE-HSA LPS Incubation time (h) 04 612 24 0 500 1000 1500 2000 2500 3000 * * * * LPS (ng·mL –1 ) 0 1 10 100 1000 0 800 1600 2400 3200 4000 * * * * LPS (ng·mL –1 ) 0 1 10 100 1000 0 400 800 1200 1600 2000 IL-8 Incubation time (h) 04 612 24 AGE-HSA LPS 0 Incubation time (h) 04612 24 600 1200 1800 2400 3000 MCP-1 AGE-HSA LPS pg·mL –1 0 50 12.5 25 50 100 HSA AGE-HSA (µg·mL –1 ) 25 50 75 1 00 125 0 * * * * 0 200 400 600 800 1000 * * * * 0 50 12.5 25 50 100 HSA AGE-HSA 0 500 1000 1500 2000 2500 * * * * 0 50 12.5 25 50 100 HSA AGE-HSA (µg·mL –1 ) (µg·mL –1 ) Fig. 1. (A) Time- and (B, C) dose-dependent cytokine ⁄ chemokine release induced by AGE-HSA or LPS. HUVECs were incubated with various doses of AGE-HSA or LPS for up to 24 h. Cytokines ⁄ chemokines, including IL-6, IL-8 and MCP-1, in the culture supernatants were mea- sured. Data are expressed as the mean ± SD of four independent experiments. *P < 0.05 versus (B) unstimulated cells or (C) HSA. Glycation end products and lipopolysaccharide in cytokine production J. Liu et al. 4600 FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS (ERK) 1 ⁄ 2, Jun N-terminal kinase (JNK) (Fig. 3A) and nuclear translocation of NF-jB (Fig. 3B), although the activated levels of MAP kinases and NF- jB induced by AGE-HSA were less than those induced by LPS. Furthermore, co-stimulation with AGE-HSA and LPS resulted in further increases in phosphory- lated MAP kinases (Fig. 3A) and NF-jB nuclear translocation (Fig. 3B), which is consistent with the observed synergistic induction of cytokines ⁄ chemokines by AGE-HSA and LPS. After demonstrating that both AGE-HSA and LPS activate similar signal transduction pathways in HUVECs, we next aimed to clarify which of these pathways is involved in the AGE-HSA and LPS ampli- fied cytokine ⁄ chemokine response using specific phar- macological inhibitors to block each individual signaling pathway. Blockage of each individual path- way [i.e. p38 with SB203580, ERK1 ⁄ 2 with PD98059, JNK with JNK inhibitor II, and NF-jB with pyrroli- dine dithiocarbamate (PDTC)] significantly attenuated AGE-HSA and LPS co-stimulation-induced IL-6 (Fig. 4A), IL-8 (Fig. 4B) and MCP-1 (Fig. 4C) produc- tion, indicating that the activation of all of these four pathways is necessary for the effective induction of cytokines ⁄ chemokines in HUVECs by AGE-HSA and LPS. Notably, blocking p38 with SB203580 led to a more profound inhibition in IL-6, IL-8 and MCP-1 production compared to blockade of either ERK1 ⁄ 2, JNK or NF-jB, indicating a predominant role of the p38 pathway in the AGE-HSA and LPS amplified inflammatory response. Furthermore, blockade of both MAP kinases and NF-jB with a combination of inhib- itors completely abolished AGE-HSA and LPS co- stimulated IL-6, IL-8 and MCP-1 production (Fig. 4), suggesting that these two pathways act co-operatively. Discussion Previous studies have demonstrated that AGEs are produced from oxidative modification by myeloperoxi- IL-6 (pg·mL –1 ) IL-8 (pg·mL –1 ) 0 200 400 600 800 1000 Control AGE-HSA LPS AGE-HS A + LPS * Control LPS AGE-HSA + LPS 800 0 400 1200 1600 2000 * MCP-1 (pg·mL –1 ) 0 500 1000 1500 2000 2500 Control AGE-HS A LPS AGE-HS A + LPS * AGE-HSA GAPDH IL-6 AGE-HSA + LPS Control AGE-HSA LPS 10 0 2 4 6 8 Induction fold * * * AGE-HSA + LPS Control AGE-HSA LPS IL-8 GAPDH Induction fold 0 2 4 6 8 10 * * * AGE-HSA + LPS Control AGE-HSA LPS MCP-1 GAPDH Induction fold 0 2 4 6 8 10 * * * A B Fig. 2. Synergistic induction of cytokines ⁄ chemokines by AGE-HSA and LPS co-stimulation. HUVECs were incubated with AGE-HSA (25 lgÆmL )1 ), LPS (10 ngÆmL )1 ), or their combination, for 2 and 24 h. (A) IL-6, IL-8 and MCP-1 release from HUVECs 24 h after stimulation was measured and (B) gene expression of IL-6, IL-8 and MCP-1 in HUVECs 2 h after stimulation was assessed by RT-PCR. Data are expressed as the mean ± SD of four (A) or three (B) independent experiments. *P < 0.05 versus AGE-HSA alone or LPS alone. J. Liu et al. Glycation end products and lipopolysaccharide in cytokine production FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS 4601 dase released from the activated leukocytes in the lesion regions of inflammatory diseases [9,19]. When bacterial infection occurs in patients with elevated AGEs, such as diabetics or the elderly, and those receiving long-term dialysis, the endothelial cells are exposed concomitantly to a variety of proinflammatory mediators, especially AGEs and LPS. It has been dem- onstrated that both AGEs and LPS play key roles in the development of inflammatory diseases by stimulat- ing cytokine ⁄ chemokine production from leukocytes and endothelial cells [1,8,9]. However, the combined inflammation-stimulatory effect of AGEs and LPS on endothelial cells, and, furthermore, the underlying signal mechanisms involved, have not been fully under- stood. In the present study, we found that both AGE-HSA and LPS, in a very similar patter, induced a time- and dose-dependent proinflammatory cytokine ⁄ chemokine production in HUVECs, although a much higher cyto- kine ⁄ chemokine response was induced by LPS com- pared to that induced by AGE-HSA. We further demonstrated that AGE-HSA and LPS co-stimulation Control AGE-HSA + LPS AGE-HSA LPS ERK1/2 P-ERK1 0 2 4 6 8 10 * * * Control AGE-HSA + LPS AGE-HSA LPS JNK P-JNK 12 0 2 4 6 8 10 * * * Control AGE-HSA + LPS AGE-HSA LPS p38 P-p38 Induction fold A B 0 2 4 6 8 10 * * * Control AGE-HSA AGE-HSA +LPS LPS Relative nuclear fluorescence intensity Control AGE-HSA AGE-HSA + LPS LPS 2 4 0 6 8 10 12 * * * Fig. 3. AGE-HSA and LPS stimulate MAP kinase phosphorylation and NF-jB nuclear translocation. HUVECs were incubated with AGE-HSA (25 lgÆmL )1 ), LPS (10 ngÆmL )1 ), or their combination, for 30 min. (A). Total (upper panel) and phosphorylated (lower panel) p38, ERK1 ⁄ 2 and JNK were detected by western blot analysis. (B) The nuclear translocation of NF-jB was detected by immunofluorescent staining with a primary antibody against NF-jB p65 and Alexa Fluor 488-conjugated secondary antibody. Data are expressed as the mean ± SD and the results shown represent one experiment from a total of four independent experiments. *P < 0.05 versus unstimulated cells. Glycation end products and lipopolysaccharide in cytokine production J. Liu et al. 4602 FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS exhibited a synergistic effect on the induction of IL-6, IL-8 and MCP-1, but not other cytokines ⁄ chemokines, at both gene and protein levels. This is important for understanding the mechanisms underlying diseases involving AGEs that are complicated with bacterial infection. As proinflammatory mediators, these cyto- kines ⁄ chemokines play an important role in inflamma- tory processes. For example, IL-6 has been shown to promote smooth muscle cell proliferation and increase the permeability of endothelium, thus exaggerating the inflammatory response [20]; the chemoattractants IL-8 and MCP-1 induce leukocyte–endothelial cell adhesion and promote the migration and infiltration of leuko- cytes to the inflammatory loci, which is a key step in inflammation [21,22]. Therefore, the cooperation of AGE-HSA and LPS on the induction of cyto- kines ⁄ chemokines in endothelial cells observed in the present study may amplify the inflammatory reactions by enhancing the production of IL-6, IL-8 and MCP- 1, consequently leading to intensive inflammation and aggravation of the disease. It is well established that proinflammatory media- tors initiate a cytokine⁄ chemokine storm by activating the intracellular signaling pathways, including MAP kinases and NF-jB [12,23,24]. Previous studies have demonstrated that LPS and AGEs activate MAP kinases in various cells, including monocytes ⁄ macro- phages and endothelial cells [12,24,25]. In agreement with previous work, we also observed increased MAP kinase phosphorylation and NF-jB nuclear transloca- tion in HUVECs after either AGE-HSA or LPS stim- ulation, indicating that both AGE-HSA and LPS activate similar intracellular signaling pathways. Moreover, we demonstrated that AGE-HSA and LPS co-stimulation led to a further activation of both MAP kinases and NF-jB, which may explain the observed much greater production of cyto- kines ⁄ chemokines induced by a combination of the two stimulants. We further used specific pharmacolog- ical inhibitors to block each individual intracellular signaling pathway, in an attempt to determine which of these pathways is responsible for the observed up-regulation of cytokine ⁄ cchemokine expression by AGE-HSA and LPS co-stimulation. Blockage of either MAP kinases or NF-jB resulted in a significant reduction in AGE-HSA and LPS co-stimulation- induced IL-6, IL-8 and MCP-1 release from HUVECs. Of note, blocking p38 alone achieved the maximal attenuation in proinflammatory cyto- kine ⁄ chemokine production, indicating that the p38 pathway acts predominately in the AGE-HSA and LPS amplified inflammatory response and, thus, may serve as a main therapeutic target for AGE-related inflammatory diseases. Furthermore, blockade of both MAP kinases and NF-jB completely abolished AGE- HSA and LPS co-stimulated IL-6, IL-8 and MCP-1 production, suggesting that activation of these two pathways is required for the effective induction of these cytokines ⁄ chemokines. A * 0 500 1000 1500 2000 2500 * * * * IL-6 (pg·mL –1 ) AGE-HSA + LPS SB 203580 PD 98059 JNK inhibitor II PDTC − − − − − + − − − − + + − − − + − + − − + − − + − + − − − + + + + + + B 0 AGE-HSA + LPS SB 203580 PD 98059 JNK inhibitor II PDTC − − − − − + − − − − + + − − − + − + − − + − − + − + − − − + + + + + + 200 400 600 800 1000 1200 * * * * * IL-8 (pg·mL –1 ) C * * * 0 500 1000 1500 2000 2500 * * MCP-1 (pg·mL –1 ) AGE-HSA + LPS SB 203580 PD 98059 JNK inhibitor II PDTC − − − − − + − − − − + + − − − + − + − − + − − + − + − − − + + + + + + Fig. 4. Inhibition of either MAP kinases or NF-jB attenuates AGE- HSA and LPS-stimulated cytokine ⁄ chemokine production. HUVECs were pretreated with SB253580 (20 l M), PD98059 (20 lM), JNK inhibitor II (50 n M), PDTC (50 lM), or their combination, for 1 h and then co-stimulated with AGE-HSA (25 lgÆmL )1 ) and LPS (10 ngÆmL )1 ) for 24 h. (A) IL-6, (B) IL-8 and (C) MCP-1 in the culture supernatants were measured. Data are expressed as the mean ± SD from four independent experiments. *P < 0.05 versus AGE-HSA + LPS co-stimulated cells. J. Liu et al. Glycation end products and lipopolysaccharide in cytokine production FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS 4603 In summary, we demonstrate that AGE-HSA and LPS synergistically stimulate IL-6, IL-8 and MCP-1 production in endothelial cells, thus contributing to the development of AGE-related inflammatory dis- eases. Furthermore, activation of both MAP kinases and NF-jB is required for the AGE-HSA and LPS co-stimulation amplified inflammatory response, impli- cating that the blockade of these signal pathways, in particular the p38 pathway, may provide a novel approach for the treatment of AGE-related diseases, such as diabetes, complicated with bacterial infection. Experimental procedures Preparation of AGE-modified proteins AGE-modified proteins were prepared as previously described [6]. Briefly, 1.75 gÆL )1 purified HSA (Sigma- Aldrich, St Louis, MO, USA) was incubated with 0.1 m d-glucose at 37 °C for 8 weeks, and then dialyzed against NaCl ⁄ P i to remove the unbound glucose. HSA incubated without glucose was used as the control. The content of AGEs in the AGE-HSA preparation was 85.24 UÆmg )1 protein, whereas that of the control was less than 0.9 UÆmg )1 protein, indicating that glycation modified protein was successfully obtained. Endotoxin levels in all preparations were measured with an E-toxate kit (Sigma- Aldrich) and found to be below the detection limit (< 0.1 EUÆmL )1 ). Isolation and culture of endothelial cells Primary HUVECs were isolated from normal human umbilical cord veins and cultured as described previously [26]. Briefly, the separated cells were suspended in RPMI 1640 with 20% fetal bovine serum (HyClone, Logan, UT, USA) and plated in tissue culture dishes. The next day, non-attached cells were removed and the medium was replaced with complete RPMI 1640 supplemented with 20% fetal bovine serum, 100 ngÆmL )1 endothelial cell growth factor (Clontech, Mountain View, CA, USA) and antibiotics. Endothelial cells were identified by the charac- teristic monolayer cobblestone appearance and positive staining for von Willebrand factor. AGE-HSA and LPS stimulation and protein kinase inhibition HUVECs were seeded on 96- or six-well culture plates for cytokine ⁄ chemokine detection, RT-PCR or western blot analysis. Cells were incubated with different concentrations of AGE-HSA and LPS (Escherichia coli 0111:B4) (Sigma- Aldrich) for up to 24 h. Recombinant human CD14 (final concentration: 0.1 lgÆmL )1 ) (R&D Systems, Minneapolis, MN, USA) was used in each experiment to enhance the LPS-mediated cell response because of a deficiency of CD14 expression on the endothelial cells [14]. To observe the effect of MAP kinase inhibitor or NF-jB inhibitor on AGE-HSA and LPS-stimulated cell activation, HUVECs were pretreated with PD98059 (20 lm), JNK inhibitor II (50 nm), SB203580 (20 lm) (all from Merck, Darmstadt, Germany), an NF-jB inhibitor PDTC (50 lm) (Sigma-Aldrich), or their combination, 1 h before AGE- HSA and LPS stimulation. The concentration of each inhibitor used was based on the dose–response experiment (data not shown), with the maximal inhibitory effect. For each experiment, cell viability was always more than 90%, as determined by exclusion of trypan blue dye. Cell-free supernatants were collected and stored at )80 °C until analysis. Cytokine ⁄ chemokine measurement Cytokines ⁄ chemokines in the culture supernatants were analyzed simultaneously using a LiquiChip work station, which employs a bead-based xMAP (flexible multi-analyte profiling) technology [27], according to the manufacturer’s instructions. RT-PCR Total RNA was extracted using a single-step method of RNA isolation by acid guanidinium thiocyanate–phenol– chloroform extraction [20]. PCR amplification was per- formed on the resulting cDNAs with specific primers for human IL-6 (forward, 5¢-CAGGAGCCCAGCTATGA ACT-3¢; reverse, 5¢-TAAGTTCTGTGCCCAGTGGA-3¢), IL-8 (forward, 5¢-AGGGTTGCC AGATGCAATAC-3¢; reverse, 5¢-ACACAGCTGGCAATGACAAG-3¢), MCP-1 (forward, 5¢-GTGAGGAGCCACCAACATTT-3¢; reverse, 5¢-GGGGGATCCCAAGTACTGTT-3¢) and GAPDH (for- ward, 5¢-CCCATCACCATCTTCCAGGA-3¢; reverse, 5¢- TGCTTCACCACCTTCTTGAT-3¢)at94°C for 30 s, 56 °C for 15 s and 72 °C for 2 min, for 30 cycles. The expected lengths of the fragments for IL-6, IL-8, MCP-1 and GAPDH were 730, 870, 733 and 520 bp, respectively. Western blot analysis Cells were lysed with the ice-cold lysis buffer (Cell signaling Technology, Danvers, MA, USA) to extract cytoplamic proteins. Equal amounts of protein extracts were subjected to 12% SDS ⁄ PAGE and blotted onto a poly(vinylidene difluoride) membrane. The membrane was blocked and probed overnight at 4 °C with antibodies against total or phosphorylated p38, ERK1 ⁄ 2 or JNK (Cell Signaling Tech- nology), followed by incubation with horseradish peroxi- dase-conjugated secondary antibodies for 1 h at room Glycation end products and lipopolysaccharide in cytokine production J. Liu et al. 4604 FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS temperature. Blots were developed using an ECL detection system (Amersham Biosciences, Little Chalfont, UK). Each image was captured and the intensity of each band was analyzed with a Kodak image workstation (Eastman Kodak, Rochester, NY, USA). Immunofluorescent staining HUVECs were incubated with AGE-HSA (25 lgÆmL )1 ) and ⁄ or LPS (10 ngÆmL )1 ) in the presence or absence of an NF-jB inhibitor PDTC (50 lm). To detect the nuclear translocation of NF-jB, immunofluorescent staining was performed by staining the cells with a primary antibody against NF-jB p65 (Abcam, Cambridge, UK) and Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). The nuclear fluorescent inten- sity was analyzed by Axio Vision (Carl Zeiss, Oberkochen, Germany). Statistical analysis All data are expressed as the mean ± SD and analyzed with the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA). The statistical significance of differ- ences was determined using one-way analysis of variance and t-test. P < 0.05 was considered statistically significant. Acknowledgements This work was supported by the National Key Basic Research Program of China (973 Program) (No. 2002CB513005), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0731), the General Program of the National Natu- ral Science Foundation of China (No. 30670828, No. 30572151 and No. 30670829), the Joint Program of NSFC and GPG (No. U0632004), PSTPGP (No. A1090202) and PSTPGC (No. 2007J1-C0301). References 1 Bannerman DD & Goldblum SE (1999) Direct effects of endotoxin on the endothelium: barrier function and injury. Lab Invest 79, 1181–1199. 2 Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmer- man GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS et al. (1998) Endothelial cells in physi- ology and in the pathophysiology of vascular disorders. Blood 91, 3527–3561. 3 Brownlee M (1995) Advanced protein glycosylation in diabetes and aging. Annu Rev Med 46, 223–234. 4 Bucala R & Cerami A (1992) Advanced glycosylation: chemistry, biology, and implications for diabetes and aging. Adv Pharmacol 23, 1–34. 5 Ulrich P & Cerami A (2001) Protein glycation, diabetes, and aging. Recent Prog Horm Res 56, 1–21. 6 Hou FF, Chertow GM, Kay J, Boyce J, Lazarus JM, Braatz JA & Owen WFJr (1997) Interaction between beta 2-microglobulin and advanced glycation end prod- ucts in the development of dialysis related-amyloidosis. Kidney Int 51, 1514–1519. 7 Yan SF, Ramasamy R, Naka Y & Schmidt AM (2003) Glycation, inflammation, and RAGE: a scaffold for the macrovascular complications of diabetes and beyond. Circ Res 93, 1159–1169. 8 Sun M, Yokoyama M, Ishiwata T & Asano G (1998) Deposition of advanced glycation end products (AGE) and expression of the receptor for AGE in cardiovascu- lar tissue of the diabetic rat. Int J Exp Pathol 79, 207– 222. 9 Basta G, Lazzerini G, Massaro M, Simoncini T, Tanga- nelli P, Fu C, Kislinger T, Stern DM, Schmidt AM & De CR (2002) Advanced glycation end products acti- vate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation 105, 816–822. 10 Miura J, Yamagishi S, Uchigata Y, Takeuchi M, Yamamoto H, Makita Z & Iwamoto Y (2003) Serum levels of non-carboxymethyllysine advanced glycation endproducts are correlated to severity of microvascular complications in patients with Type 1 diabetes. J Diabe- tes Complications 17, 16–21. 11 Leon CG, Tory R, Jia J, Sivak O & Wasan KM (2008) Discovery and development of toll-like receptor 4 (TLR4) antagonists: a new paradigm for treating sepsis and other diseases. Pharm Res 25, 1751–1761. 12 Hippenstiel S, Soeth S, Kellas B, Fuhrmann O, Seybold J, Krull M, Eichel-Streiber C, Goebeler M, Ludwig S & Suttorp N (2000) Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells. Blood 95, 3044–3051. 13 Chow JC, Young DW, Golenbock DT, Christ WJ & Gusovsky F (1999) Toll-like receptor-4 mediates lipo- polysaccharide-induced signal transduction. J Biol Chem 274, 10689–10692. 14 Arditi M, Zhou J, Dorio R, Rong GW, Goyert SM & Kim KS (1993) Endotoxin-mediated endothelial cell injury and activation: role of soluble CD14. Infect Immun 61, 3149–3156. 15 Meyer KC (2001) The role of immunity in susceptibility to respiratory infection in the aging lung. Respir Physiol 128, 23–31. 16 Shah BR & Hux JE (2003) Quantifying the risk of infectious diseases for people with diabetes. Diabetes Care 26, 510–513. 17 Dofferhoff AS, Nijland JH, de Vries-Hospers HG, Mulder PO, Weits J & Bom VJ (1991) Effects of different types and combinations of antimicrobial agents on J. Liu et al. Glycation end products and lipopolysaccharide in cytokine production FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS 4605 endotoxin release from gram-negative bacteria: an in-vitro and in-vivo study. Scand J Infect Dis 23, 745–754. 18 Szeto CC, Kwan BC, Chow KM, Lai KB, Chung KY, Leung CB & Li PK (2008) Endotoxemia is related to systemic inflammation and atherosclerosis in peritoneal dialysis patients. Clin J Am Soc Nephrol 3, 431–436. 19 Anderson MM, Hazen SL, Hsu FF & Heinecke JW (1997) Human neutrophils employ the myeloperoxidase- hydrogen peroxide-chloride system to convert hydroxy- amino acids into glycolaldehyde, 2-hydroxypropanal, and acrolein. A mechanism for the generation of highly reactive alpha-hydroxy and alpha,beta-unsaturated alde- hydes by phagocytes at sites of inflammation. J Clin Invest 99, 424–432. 20 Remick DG, Bolgos G, Copeland S & Siddiqui J (2005) Role of interleukin-6 in mortality from and physiologic response to sepsis. Infect Immun 73, 2751–2757. 21 Tedgui A & Mallat Z (2006) Cytokines in atherosclero- sis: pathogenic and regulatory pathways. Physiol Rev 86, 515–581. 22 Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, Gimbrone MAJr, Luster AD, Luscinskas FW & Rosenzweig A (1999) MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature 398, 8–723. 23 Denk A, Goebeler M, Schmid S, Berberich I, Ritz O, Lindemann D, Ludwig S & Wirth T (2001) Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells. J Biol Chem 276, 28451–28458. 24 Yeh CH, Sturgis L, Haidacher J, Zhang XN, Sherwood SJ, Bjercke RJ, Juhasz O, Crow MT, Tilton RG & Denner L (2001) Requirement for p38 and p44 ⁄ p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion. Diabetes 50, 1495–1504. 25 Berbaum K, Shanmugam K, Stuchbury G, Wiede F, Korner H & Munch G (2008) Induction of novel cyto- kines and chemokines by advanced glycation endprod- ucts determined with a cytometric bead array. Cytokine 41, 198–203. 26 Marin V, Kaplanski G, Gres S, Farnarier C & Bongrand P (2001) Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J Immunol Methods 254, 183–190. 27 Jiang Y, Xu J, Zhou C, Wu Z, Zhong S, Liu J, Luo W, Chen T, Qin Q & Deng P (2005) Characterization of cytokine ⁄ chemokine profiles of severe acute respiratory syndrome. Am J Respir Crit Care Med 171, 850–857. Glycation end products and lipopolysaccharide in cytokine production J. Liu et al. 4606 FEBS Journal 276 (2009) 4598–4606 ª 2009 The Authors Journal compilation ª 2009 FEBS . Advanced glycation end products and lipopolysaccharide synergistically stimulate proinflammatory cytokine ⁄ chemokine production in endothelial cells via activation of both mitogen-activated protein. proinflam- matory cytokine ⁄ chemokine production by activating mitogen-activated protein kinases and nuclear factor-jB in endothelial cells, thus amplifying the in ammatory response and resulting. and monochemoattractant protein- 1 production in human umbilical vein endo- thelial cells. Similar to lipopolysaccharide, AGE-HSA stimulation induced mitogen-activated protein kinase phosphorylation and nuclear factor-jB nuclear