Modulation of PPAR Expression and Inflammatory Interleukin-6 Production by Chronic Glucose Increases Monocyte/Endothelial Adhesion
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Division of Endocrinology and Metabolism, University of Virginia, Charlottesville.
ABSTRACT
Objective— We have previously reported increased monocyte adhesion to human aortic endothelial cells (HAECs) cultured in 25 mmol/L glucose (HG) compared with normal glucose (NG) (5.5 mmol/L). In this study, we explored mechanisms that contribute to increased monocyte adhesion by elevated glucose.
Methods and Results— We found that HAECs cultured in HG have increased production of the chemokine interleukin-6 (IL-6). We examined whether IL-6 directly modulated monocyte adhesion to EC. Inhibition of IL-6 using a neutralizing antibody significantly reduced glucose-mediated monocyte adhesion by 50%, and addition of IL-6 directly to human EC stimulated monocyte adhesion. PPAR has been reported to negatively regulate expression of IL-6 in vascular cells, so we examined PPAR-associated signaling in EC. A known PPAR agonist, Wy14,643, prevented glucose-mediated IL-6 production by EC and reduced glucose-mediated monocyte adhesion by 40%. HG-cultured HAEC had a 50% reduction in expression of PPAR compared with control EC. Primary aortic EC isolated from PPAR knockout (KO) mice showed increased monocyte adhesion compared with EC isolated from control mice. PPAR KO EC also had increased production of IL-6. Finally, we measured IL-6 levels in diabetic db/db mice and found significant 6-fold elevations in IL-6 levels in db/db EC.
Conclusions— These data indicate that IL-6 production is increased in diabetes and contributes to early vascular inflammatory changes. PPAR protects EC from glucose-mediated monocyte adhesion, in part through regulation of IL-6 production.
Key Words: endothelium ? monocytes ? PPAR ? atherosclerosis ? interleukin-6
Introduction
Endothelial cell (EC) activation by oxidative and inflammatory stresses and monocyte recruitment and adhesion are key early events in atherosclerosis. During inflammation, monocytes are recruited to sites of EC injury and roll along the vascular endothelium, where they become activated by soluble or surface-bound chemokines.1–3 The monocytes adhere firmly to the endothelium and transmigrate through the EC monolayer.4,5 Vascular cell adhesion molecule-1 (VCAM-1) and an alternatively spliced form of fibronectin, CS-1, have been shown to mediate monocyte adhesion to human EC.6,7 Monocytes are the primary inflammatory cell type that has been localized to human atherosclerotic plaques.8,9
Diabetes is an independent risk factor in the development of atherosclerosis. Atherosclerosis remains a primary complication of patients with type 2 diabetes.10–12 We have previously shown that glucose increases monocyte adhesion to EC in vitro.7 However, there has been limited investigation as to the mechanism of glucose action on monocyte/EC interactions. Chemokines, including IL-6 and IL-8, play an important role in mediating local inflammatory effects in EC. We have recently shown a 2-fold induction of IL-8 secretion by EC cultured in chronic elevated glucose.13 We, and others, have also shown that IL-8 is an important mediator of monocyte rolling and firm adhesion in the vessel wall.1413,15 In addition, IL-6 may play a pro-inflammatory role in the vessel wall.16,17 Kaplanski et al have suggested that during chronic inflammation, such as occurs in diabetes, IL-6 functions to mediate monocyte recruitment to the vessel wall, thus participating in disease pathogenesis.16 Recent studies have indicated that IL-6 production is negatively regulated by PPAR.18–20 However, no study has definitively shown a role for IL-6 in mediating monocyte/endothelial interactions, particular in the context of diabetes.
PPAR is a nuclear hormone receptor that controls gene expression by interacting with specific DNA response elements (PPRE) in specific genes. PPAR partners with the retinoic acid receptor RXR to form a heterodimeric complex that binds PPRE elements in DNA. PPAR is activated by several fatty acids, as well as by fibrates.21,22 PPAR is believed to be anti-inflammatory on activation because PPAR knockout (KO) mice are more prone to inflammation.22,23 PPAR regulates expression of COX-2, IL-1?, IFN, TNF, IL-6, IL-8, and MCP-1, mostly to downregulate expression of these pro-inflammatory molecules,18,24,25 except in a few cases.26 PPAR inhibits expression of IL-6 via negative regulation of nuclear factor-kappa B and AP-1.19,27 The PPAR agonist, Wy14,643, reduces IL-6 expression in vivo in C57BL/6J mice.28 Furthermore, PPAR KO mice have increased production of IL-6 mRNA in aorta in response to a bacterial lipopolysaccharide (LPS) challenge.19 PPAR activation has been shown to reduce atherosclerosis in mice, although the role of PPAR in mediating atherosclerosis remains unclear.29,30 Recent studies by Semenkovich et al reported that deficiency of PPAR in low-density lipoprotein receptor-deficient mice inhibited development of atherosclerosis.26
In this study, we examined additional mechanisms for glucose regulation of monocyte adhesion to EC. We examined glucose-mediated regulation of PPAR expression and its impact on events related to monocyte adhesion. We also examined the role of IL-6 in mediating monocyte adhesion to EC in the setting of diabetes.
Methods
Reagents
Tissue culture media and reagents were purchased from Invitrogen. Fetal bovine serum was obtained from Hyclone. Calcein-AM was purchased from Molecular Probes. Human and mouse IL-6 ELISA kits were obtained from R&D Systems. Wy 14 643 was purchased from Biomol. Human IL-6 neutralizing antibody, human IL-8 neutralizing antibody, recombinant human IL-6, and recombinant human IL-8 were all purchased from R&D Systems. WEHI78/24 cells were a kind gift from Dr Judith A. Berliner (UCLA). Pioglitazone (Takeda Pharmaceuticals) was a kind gift from Dr Milagros Huerta (University of Virginia). Antibody to human PPAR was purchased from Affinity BioReagents, and antibody to histone H1 was purchased from Santa-Cruz Biotechnology (sc-8030).
Human EC Culture
Human aortic endothelial cells (HAEC) were obtained from aortic rings of explanted donor hearts. Use of HAEC was approved by the University of Virginia Institutional Review Board, and all procedures were performed in accordance with University Institutional Review Board guidelines. Briefly, HAEC were cultured for 7 days in medium 199 containing 20% heat-inactivated FBS, 20 μg/mL endothelial cell growth supplement (ECGS), and 90 μg/mL heparin in the absence (NG) and presence of 25 mmol/L D-glucose (HG) or 25 mmol/L L-glucose (as an osmotic control). The 7-day, 25-mmol/L HG incubation condition was chosen because monocyte adhesion to EC was maximal at this concentration of glucose and time-point of incubation.31
Mouse Aortic EC Isolation
Aortic EC from PPAR KO mice23 (Jackson Laboratories stock 003580) on a 129S4/SvJae background, PPAR control 129S1/SvImJ mice (Jackson Laboratories stock 002448), diabetic B6.Cg-m+/+Leprdb (db/db) mice on a C57BL/6J background (Jackson Laboratories stock 000697), and control C57BL/6J mice (Jackson Laboratories stock 000664) were harvested from mouse aorta after modifications of the method described previously.32 These methods were approved and performed under the guidelines established by the University of Virginia Animal Care and Use Committee. Briefly, the aorta is excised, all periadventitial fat is removed under a magnifying scope, and the aortic pieces are placed onto Matrigel in DMEM plus 15% HI-FBS. The EC grow out from these aortic explants. After 3 days, the explants are removed, and the EC are grown to confluence as described.33 EC are routinely used from passages 2 to 4. For the studies using diabetic db/db EC, cells were isolated as described and cultured for 1 passage in DMEM containing 15% heat-inactivated FBS, 60 μg/mL ECGS, and 100 μg/mL heparin, and 5.5 mmol/L glucose before performing assays. We have previously reported that diabetic db/db EC retain memory of their diabetic milieu during short-term passage in culture.33
Monocyte Adhesion Assays
Monocyte adhesion assays using human cells were performed as described previously.7 Briefly, NG- and HG-cultured HAEC were cultured to confluence as described into 48-well plates. HAEC were rinsed with 1% M199. Human primary monocytes were isolated from healthy normal volunteers using a modification of the Recalde method34 and labeled with Calcein AM (Molecular Probes) for 10 minutes at 37°C. Labeled human primary monocytes (50 000/well) were added to HAEC monolayers and incubated for 30 minutes at 37°C. Unbound monocytes were rinsed away, cells were fixed in 1% glutaraldehyde, and bound labeled monocytes were counted within a 10x10 grid using epifluorescence microscopy. Cells were incubated at 37°C with 10 U/mL recombinant human TNF for 4 hours as a control to show maximal monocyte adhesion in our assays. For subsets of studies, NG- and HG-cultured HAEC were incubated at 37°C with 100 μmol/L Wy14,643 or 5 μmol/L pioglitazone for 4 hours before adhesion assays. For IL-6 studies, HAEC were incubated at 37°C with recombinant human IL-6 (5 ng/mL) for 4 hours, IL-6 neutralizing antibody (0.1 μg/mL) for 4 hours, or an irrelevant antibody (-mouse IgG; 0.1 μg/mL) for 4 hours before performing a monocyte adhesion assay. For IL-8 studies, HAEC were incubated at 37°C with either recombinant human IL-8 (5 ng/mL) for 4 hours or IL-8 neutralizing antibody (20 μg/mL for 2 hours) before performing a monocyte adhesion assay.
Mouse Assays
Our laboratory has recently developed a monocyte adhesion assay that uses primary mouse aortic EC and WEHI 78/24 mouse monocytes.13 WEHI 78/24 is a mouse monocyte cell line that has been well characterized.35 WEHI are cultured in 10% heat-inactivated FBS in DMEM containing 4.5 g/L glucose. WEHI are labeled with Calcein AM (Molecular Probes) immediately before experiments according to manufacturer’s instructions. Mouse aortic EC are incubated with 35 000 fluorescent WEHI cells/well for 30 minutes at 37°C. Nonadherent cells are rinsed and the cells fixed with 1% glutaraldehyde. The number of attached monocytes present within a 10x10 grid is counted using epifluorescence microscopy.
Enzyme-Linked Immunosorbent Assay for Human and Mouse IL-6
Supernatants from cultured EC were collected, aliquoted to prevent repetitive thawing, and stored at –20°C. Enzyme-linked immunosorbent assay (ELISA) for human and mouse IL-6 in supernatants was performed using ELISA kits according to the manufacturer’s instructions. Supernatants were used undiluted in quadruplicate wells/sample. IL-6 levels in supernatant were determined using a standard curve. For normalization purposes, EC lysates were harvested using sodium dodecyl sulfate (SDS) lysis buffer containing phenylmethanesulfonyl fluoride and protease inhibitor cocktail (Sigma) and total cell protein was measured using a BioRad protein assay. IL-6 secretion into media was represented as picograms released into media/mg total cell protein to normalize for possible cell number differences under each experimental condition.
Nuclear Protein Extraction and Immunoblotting of PPAR
HAEC were cultured in NG and HG as described. After rinsing the monolayer of cells twice with PBS, cells were harvested using a cell scraper and lysed using cell lysis buffer (10 mmol/L HEPES pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.1 mmol/L DTT, and 10 μL of Sigma protease inhibitor cocktail). Cells were incubated on ice for 15 minutes. Then 10% Igepal CA-630 was added to a final concentration of 0.6%, and cells were vortexed vigorously and centrifuged at 10 000g for 3 minutes. Cell pellets were resuspended in extraction buffer (20 mmol/L HEPES pH 7.9, 1.5 mmol/L MgCl2, 0.42 mol/L NaCl, 0.2 mmol/L EDTA, 25% glycerol with Sigma protease inhibitor cocktail, and 0.1 mmol/L DTT) and agitated on a vortex mixer for 30 minutes. The nuclear extracts were separated by centrifugation at 20 000g for 5 minutes and aliquots were stored in –70°C. Protein was quantitated using a BioRad protein assay. SDS-PAGE of 25 μg nuclear protein was performed using 4% to 12% NuPAGE gels (Invitrogen). An antibody to human PPAR was incubated with the blots overnight at 4°C at a 1:1000 dilution. Blots were also incubated with antibody to histone H1 at a 1:500 dilution. Blots were normalized to histone H1 expression using densitometry (ZeroD Scan software; Stratagene).
Statistical Analyses
Data for all experiments were analyzed by ANOVA and Fisher protected least significant difference test using the Statview 6.0 software program. Data are represented as the mean±SE of 5 different experiments performed in quadruplicate unless otherwise stated in the Figure legends. Significance was reported at P0.05.
Results
Glucose Increases IL-6 Production by HAEC
We have previously reported increased production of IL-8 in HAEC mediated by chronic elevated glucose.13 In the current study, we found that HAEC cultured chronically for 7 days have increased production of IL-6 protein (Figure 1). HAEC cultured in 25 mmol/L L-glucose for 7 days as an osmotic control showed no change in IL-6 production. These data suggest that chronic elevated glucose, as occurs in patients with diabetes, causes endothelial activation through increased production of IL-6 and IL-8. Both IL-6 and IL-8 are involved in mediating monocyte recruitment to endothelium during vascular inflammation.16,17
Figure 1. Glucose increases endothelial production of IL-6. HAEC were cultured in 5.5.mM (NG), 25 mmol/L L-glucose (L-glucose), or 25 mmol/L D-glucose (HG) for 7 days. Media was collected and secreted IL-6 was measured using an ELISA for human IL-6. L-glucose had no effect on IL-6 levels in EC. *P<0.0001 versus NG by ANOVA. Data represent the mean±SE of 5 experiments.
IL-6 Directly Stimulates Monocyte Adhesion to EC
We have previously reported that chronic elevated glucose increases monocyte adhesion to HAEC.7,13 These data are illustrated in Figure 2, in which HG-cultured HAEC (HG) have a 2-fold increase in monocyte adhesion compared with EC cultured in 5.5 mmol/L glucose (NG). There was no effect of L-glucose on monocyte adhesion to EC. TNF is used as a positive control to indicate maximal monocyte adhesion in the assay.
Figure 2. Neutralizing antibody to human IL-6 reduces glucose-mediated monocyte adhesion. HAEC were cultured in NG, HG, or L-glucose as described in Methods. Before an adhesion assay, NG- and HG-cultured HAEC were incubated for 4 hours at 37°C with anti-human IL-6 antibody (+IL-6Ab) or anti-human IL-8 antibody (+IL-8Ab). NG-cultured cells were also incubated in the presence of recombinant human IL-6 (+IL6) or recombinant human IL-8 (+IL8) as described in Methods before performing a monocyte adhesion assay. L-glucose had no effect on monocyte adhesion. NG-HAEC were incubated with 10 U/mL recombinant human TNF for 4 hours as a positive control (+TNF) to show maximal monocyte adhesion. HG cells were also incubated with an irrelevant antibody (+CTRAb) as a negative control. *Significantly higher than NG, P<0.001; **significantly lower than HG, P<0.009. Data represent the mean±SE of 4 experiments performed in quadruplicate.
Several studies have suggested that IL-6 may mediate monocyte recruitment to activated endothelium.16 However, whether IL-6 directly can stimulate monocyte/EC adhesion is unclear. We tested this hypothesis and found that addition of recombinant human IL-6 to HAEC directly stimulated monocyte adhesion by 50% (NG+IL6; Figure 2). Furthermore, inhibition of endothelial IL-6 action through use of an IL-6 neutralizing antibody significantly reduced glucose-mediated monocyte adhesion to HAEC by 50% (HG versus HG+IL6Ab; Figure 2), suggesting that IL-6 action contributes significantly to glucose-mediated monocyte adhesion to EC.
We have previously reported that IL-8 plays a major role in glucose-stimulated monocyte adhesion.13 In this study, we directly compared the contributions of IL-6 and IL-8 in glucose-mediated monocyte adhesion. As shown in Figure 2, addition of recombinant human IL-8 to HAEC directly stimulated monocyte/endothelial interactions (NG versus NG+IL8). In addition, inhibition of endothelial IL-8 action through use of an IL-8 neutralizing antibody significantly reduced glucose-mediated monocyte adhesion by 50% (HG versus HG+IL8Ab; Figure 2). Thus, IL-6 is an important contributor, yet not the sole contributor, to glucose-stimulated monocyte adhesion. IL-6 and IL-8 contribute almost equally to regulate glucose-mediated monocyte adhesion.
Glucose Decreases PPAR Expression in EC
PPAR expression may negatively regulate IL-6 production in an anti-inflammatory manner.19 In Figure 1, we found an increase in IL-6 production in HG-cultured HAEC, so one explanation for an increase in IL-6 production could be a decrease in PPAR expression or function. We tested whether PPAR expression in HAEC was reduced by chronic elevated glucose. We found that PPAR protein levels were reduced by 50% in HAEC cultured in chronic glucose (HG) (P<0.001; Figure 3).
Figure 3. Glucose decreases PPAR expression in EC. HAEC were cultured in NG or HG as described in Methods. In subsets of cells, NG- or HG-cultured HAEC were incubated with 100 μmol/L Wy14,643 for 4 hours at 37°C (+Wy). Nuclear extracts were isolated as described in Methods. Western immunoblotting using 25 μg nuclear protein was performed using an antibody specific for PPAR, and blots were normalized to histone H1 levels. #Significantly higher than NG, P<0.01; **significantly lower than NG, P<0.001; *significantly higher than HG, P<0.01 by ANOVA. Data represent the mean±SE of 3 experiments.
We also found that the known PPAR agonist, Wy14,643 prevented complete downregulation of PPAR protein in response to glucose in EC (HG+Wy; Figure 3). Wy14,643 also significantly increased PPAR protein levels in normal HAEC (NG+Wy; Figure 3). We have recently found that Wy14,643 increases PPAR mRNA expression in EC (data not shown). Regulation of PPAR mRNA transcription by Wy14,643 has been reported by Sterchele and Mukherjee.36,37 These data suggest that Wy14,643 acts as an agonist and also regulates PPAR expression, probably through transcriptional processes.
The PPAR agonist Wy14,643 completely blocked glucose-mediated IL-6 production in HAEC (Figure 4A). Wy14,643 also significantly reduced glucose-mediated monocyte adhesion to HAEC by 40% (Figure 4B). TNF was used as a positive control to show maximal adhesion in this assay. We also examined the effects of the PPAR agonist pioglitazone on monocyte adhesion. We found no reduction in glucose-mediated monocyte adhesion using 5 μmol/L pioglitazone for 4 hours (Figure 4B). These data indicate that glucose increases monocyte adhesion in part through downregulation of PPAR expression with a resulting increase in production of IL-6. These data also suggest that IL-6 is responsible for 50% of glucose-mediated monocyte adhesion.
Figure 4. PPAR agonist Wy14,643 reduces glucose-mediated monocyte adhesion to HAEC. A, Endothelial IL-6 secretion. HAEC were cultured for 7 days in 5.5 mmol/L (NG) or 25 mmol/L glucose (HG). HG-HAEC were treated with 100 μmol/L Wy14, 643 for 4 hours (HG+Wy). Media was collected and secreted IL-6 measured using an ELISA for human IL-6. Values were normalized to total cell protein. *Significantly higher than NG, P<0.005; **significantly lower than HG, P<0.008 by ANOVA. B, Monocyte adhesion. HG-cultured HAEC were pre-treated for 4 hours at 37°C with 100 μmol/L Wy14,643 (HG+Wy), 5 μmol/L pioglitazone (HG+Pio), or DMSO as a vehicle control (HG+Vehicle). NG-HAEC were treated for 4 hours with 10 U/mL recombinant human TNF (+TNF) as a positive control. Normal human primary monocytes were added for a monocyte adhesion assay as described in Methods. *Significantly higher than NG, P<0.0001; **significantly lower than HG, P<0.01 by ANOVA. Data represent the mean±SE of 7 experiments.
PPAR-Deficient Mice Have Increased Monocyte/Endothelial Interactions and Increased IL-6 Expression
To further examine the role of endothelial PPAR in glucose-mediated monocyte adhesion, we used primary aortic EC isolated from PPAR KO mice on a B6,129 S4 background. EC usually do not bind monocytes unless they are activated. As shown in Figure 5, monocyte adhesion was significantly increased 2-fold in basal, unstimulated EC isolated from PPAR KO mice compared with EC from control mice (control strain 129S1/svImJ; P<0.009). Furthermore, we measured IL-6 production in control and PPAR KO aortic EC. We found a dramatic 4-fold increase in IL-6 production in aortic EC of PPAR KO mice compared with control mice (Figure 5) and a 2-fold increase in IL-6 mRNA levels in PPAR KO mice (data not shown). The Wy compound had no effect on monocyte adhesion or IL-6 production in PPAR KO mice (data not shown). This is consistent with earlier data from Gonzalez et al who found no effects of Wy14,643 on cellular parameters in the PPAR KO mice.38–41 We also found that exposing PPAR KO EC to elevated glucose (25 mmol/L for 7 days) increased monocyte adhesion but had no effect on IL-6 production (data not shown). This is important in that we have shown regulation of monocyte adhesion by additional chemokines that are not regulated by PPAR, including IL-8.13 However, IL-6 production appears to be primarily regulated by PPAR. Taken together, our data using PPAR KO mice indicate that PPAR expression is important for regulation of monocyte/endothelial adhesion, and that this regulation occurs, at least in part, through modulation of endothelial IL-6 production.
Figure 5. EC from PPAR KO mice have increased expression of IL-6 and bind more monocytes than control mouse EC. Mouse aortic endothelial cells from aorta of PPAR KO (PPARKO) and control mice (CTR) were isolated as described in Methods. All mouse aortic endothelial cells were cultured in DMEM containing 5.5 mmol/L glucose for 7 days. A, Endothelial IL-6 secretion. Media was collected and secreted murine IL-6 was measured using an ELISA for mouse IL-6. Values were normalized to total cell protein. *P<0.0001 versus CTR by ANOVA. B, Monocyte adhesion. Assays were performed using WEHI78/24 cells. *Significantly higher than CTR by ANOVA, P<0.005. Data represent the mean±SE of 4 experiments.
Diabetic Mice Also Show Increased Production of IL-6
We wanted to determine that IL-6 levels were also increased in diabetic db/db mice to illustrate the importance of this chemokine pathway in diabetes in vivo. EC were freshly isolated from mouse aortas of diabetic db/db and control mice and cultured for 1 passage in 5.5 mmol/L glucose. IL-6 levels were measured in control and diabetic EC. We found a significant 6-fold increase in IL-6 production in diabetic db/db mouse EC (Figure 6). We also found that IL-6 mRNA was elevated in db/db EC (data not shown), suggesting that IL-6 is regulated at the level of mRNA abundance in diabetic db/db mouse EC. EC from db/db mice also display increased monocyte adhesion (Figure 6). We have previously reported increased monocyte adhesion to EC in diabetic db/db mice.33 Thus, these data indicate a significant role for IL-6 in mediating monocyte adhesion to EC in the setting of diabetes.
Figure 6. IL-6 production and monocyte adhesion is increased in diabetic db/db mouse EC. EC from C57BL/6J control mice (B6 CTR) and diabetic db/db (db/db) mice were isolated and cultured for 1 passage as described in Methods. A, Endothelial IL-6 secretion. Media was collected and secreted IL-6 was measured using an ELISA for mouse IL-6. Values were normalized to total cell protein. *P<0.0001 versus B6 CTR by ANOVA. Data represent the mean±SE of 4 experiments. B, Monocyte adhesion. Assay was performed as described in Methods. *Significantly higher than B6 CTR, P<0.0001. Date represent the mean±SE of 10 experiments performed in quadruplicate.
Discussion
The signaling pathways by which glucose modulates monocyte/endothelial adhesion remain unclear. This is the first study that links glucose-mediated changes in endothelial activation to downregulation of PPAR. EC cultured in chronic glucose showed decreased expression of PPAR and increased monocyte adhesion. The studies shown in Figure 5 illustrate that absence of PPAR in murine EC results in increased monocyte adhesion. These data collectively indicate the importance of PPAR as an anti-inflammatory molecule in EC for early events of atherogenesis. In the current study, we found increased production of the pro-inflammatory chemokine IL-6 by EC in response to chronic elevated glucose. IL-6 production by EC was blocked by a PPAR agonist Wy14,643. These novel results indicate that glucose-stimulated production of IL-6 in endothelium in diabetes is mediated through inhibition of PPAR expression and/or action. In the present study we focused on PPAR, because we have found that expression of PPAR is low in human aortic EC.42 Furthermore, levels of PPAR in human EC were not increased in response to glucose (data not shown). We found no effect of pioglitazone, a PPAR agonist, on reducing glucose-mediated monocyte adhesion in HAEC treated for 4 hours at 37°C with 5 μmol/L pioglitazone (Figure 4). However, longer pretreatment of HAEC for 24 hours with 5 μmol/L pioglitazone slightly reduced glucose-mediated monocyte adhesion to HAEC, although the trend was not significant (data not shown). This would be consistent with another study that found that pioglitazone (20 μmol/L for 48 hours) reduced monocyte/EC interactions in response to elevated shear stress.43 However, other studies have not examined the role of pioglitazone in reducing glucose-stimulated monocyte adhesion. Because PPAR levels in HAEC appear not to be regulated by glucose, and because there is a minimal effect of pioglitazone on monocyte adhesion in our case, taken together, our results indicate that PPAR regulation is important for glucose-mediated endothelial activation. However, detailed studies of the interaction between glucose and PPAR in EC are needed and are underway in the laboratory.
PPAR may play a pro-inflammatory or anti-inflammatory role in the vessel wall depending on the state of oxidative stress in vascular cells.44,45 On the pro-inflammatory side, we have recently found chronic exposure of EC to glucose dramatically increases levels of reactive oxygen species in the cell.13 Previous work by our colleagues illustrated that PPAR stimulated MCP-1 and IL-8 synthesis in response to oxidized phospholipids and minimally modified low-density lipoprotein.46 Interestingly, PPAR KO mice on the apoE KO background were shown to have less atherosclerosis than control apoE KO mice.26 This study suggests a pro-inflammatory role for PPAR in mediating atherosclerosis. The authors suggested that the reduction of atherosclerosis in the absence of PPAR was caused by increased insulin sensitivity and enhanced glucose control. However, these investigators did not examine early events contributing to atherosclerosis that are relevant to the current study, such as monocyte/EC interactions and IL-6 production. Nevertheless, collectively, the data from these studies suggest a pro-inflammatory role for PPAR. However, many more groups have shown that PPAR is anti-inflammatory and that activation of PPAR by fibrate agonists reduces atherosclerosis development in mice.29 Cunard et al found that feeding C57BL/6J mice the PPAR agonist Wy14,643 reduced T cell and monocyte activation, which are key early events in formation of atherosclerotic plaques.28 Duez et al found that fenofibrate reduced aortic cholesterol content in apoE-deficient mice but did not reduce lesion area in the aorta.47 However, when these apoE KO mice were crossed with human apoAI transgenic mouse, fenofibrate dramatically reduced atherosclerotic lesion development.47 Mechanisms for the reduced atherosclerosis in response to PPAR activation include inhibition of T cell and monocyte activation,29 reduction of endothelial MCP-1 expression, and activation of apoAI.47 Although in most cases it is believed that PPAR exerts an anti-inflammatory effect, the type of inflammation (acute versus chronic) and the cause of inflammation (hyperlipidemia, hyperglycemia, oxidation) could be of importance in determining the anti- versus pro-inflammatory nature of PPAR.
To examine the direct role of PPAR in modulating monocyte adhesion, we used EC isolated from PPAR KO mice. Gonzalez et al have shown that PPAR KO mice have a significantly greater response to inflammatory stimuli compared with control mice.23 In adhesion assays, we found that EC isolated from PPAR KO mice bound a greater number of monocytes than did EC from control mice (Figure 5). These data indicate that in the absence of PPAR, EC are already activated to bind monocytes; thus, the PPAR KO EC are in a pro-inflammatory state. Additional studies will be needed to identify the molecular mechanisms leading to the enhanced monocyte adhesion observed in the PPAR KO EC.
We also show for the first time to our knowledge that EC from diabetic db/db mice have a significant upregulation in IL-6 production (Figure 6). We have previously reported that monocyte adhesion to EC is increased in db/db mice.33 These new data on IL-6 suggest that IL-6 plays a role in mediating monocyte adhesion in diabetic db/db mice. Further studies in these mice are needed to address this question. Nevertheless, these data suggest that IL-6 may be an important contributor to early vascular inflammatory events in diabetes in vivo.
In addition to the PPAR–IL-6 signaling pathway, there are several additional pathways in vascular EC that contribute to glucose-mediated monocyte adhesion. We have previously shown regulation of IL-8 synthesis in EC by chronic elevated glucose.13 We have found that IL-8 is a primary regulator of monocyte adhesion to EC in response to glucose, and it accounts for 50% of glucose-mediated monocyte adhesion (Figure 2). The regulation of IL-8 production in EC by glucose does not appear to be through PPAR. However, there is significant cross-talk between PPAR and other signaling pathways, including MAP kinases; therefore, PPAR may be indirectly involved in production of other chemokines.48 We have recently shown upregulation of the 12/15 lipoxygenase pathway in diabetic db/db mice33 as well as in human EC cultured chronically in elevated glucose.7 This inflammatory pathway most certainly contributes to monocyte/EC interactions in diabetes, although the exact mechanisms remain unclear. The role that these lipoxygenase eicosanoid products play in mediating monocyte/EC interactions in diabetes is currently being studied in the laboratory.
In summary, our data indicate that glucose-mediated induction of IL-6 and subsequent acceleration of monocyte adhesion occurs through modulation of levels of endothelial PPAR. These results support further development of modulators of PPAR expression or action to reduce accelerated cardiovascular disease caused by diabetes.
Acknowledgments
The authors thank Dr Judith Berliner (UCLA) for the gift of WEHI78/24 cells, David T. Bolick for assistance with endothelial cell culture, Dr Milagros Huerta (University of Virginia) for the gift of the pioglitazone, and Dr Jerry L. Nadler (University of Virginia) for helpful discussions. This work is supported by NIH PO1 HL55798-08 (CCH), American Heart Association Mid-Atlantic Affiliate (CCH), and Jeffress Foundation Memorial Trust fund (CCH).
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ABSTRACT
Objective— We have previously reported increased monocyte adhesion to human aortic endothelial cells (HAECs) cultured in 25 mmol/L glucose (HG) compared with normal glucose (NG) (5.5 mmol/L). In this study, we explored mechanisms that contribute to increased monocyte adhesion by elevated glucose.
Methods and Results— We found that HAECs cultured in HG have increased production of the chemokine interleukin-6 (IL-6). We examined whether IL-6 directly modulated monocyte adhesion to EC. Inhibition of IL-6 using a neutralizing antibody significantly reduced glucose-mediated monocyte adhesion by 50%, and addition of IL-6 directly to human EC stimulated monocyte adhesion. PPAR has been reported to negatively regulate expression of IL-6 in vascular cells, so we examined PPAR-associated signaling in EC. A known PPAR agonist, Wy14,643, prevented glucose-mediated IL-6 production by EC and reduced glucose-mediated monocyte adhesion by 40%. HG-cultured HAEC had a 50% reduction in expression of PPAR compared with control EC. Primary aortic EC isolated from PPAR knockout (KO) mice showed increased monocyte adhesion compared with EC isolated from control mice. PPAR KO EC also had increased production of IL-6. Finally, we measured IL-6 levels in diabetic db/db mice and found significant 6-fold elevations in IL-6 levels in db/db EC.
Conclusions— These data indicate that IL-6 production is increased in diabetes and contributes to early vascular inflammatory changes. PPAR protects EC from glucose-mediated monocyte adhesion, in part through regulation of IL-6 production.
Key Words: endothelium ? monocytes ? PPAR ? atherosclerosis ? interleukin-6
Introduction
Endothelial cell (EC) activation by oxidative and inflammatory stresses and monocyte recruitment and adhesion are key early events in atherosclerosis. During inflammation, monocytes are recruited to sites of EC injury and roll along the vascular endothelium, where they become activated by soluble or surface-bound chemokines.1–3 The monocytes adhere firmly to the endothelium and transmigrate through the EC monolayer.4,5 Vascular cell adhesion molecule-1 (VCAM-1) and an alternatively spliced form of fibronectin, CS-1, have been shown to mediate monocyte adhesion to human EC.6,7 Monocytes are the primary inflammatory cell type that has been localized to human atherosclerotic plaques.8,9
Diabetes is an independent risk factor in the development of atherosclerosis. Atherosclerosis remains a primary complication of patients with type 2 diabetes.10–12 We have previously shown that glucose increases monocyte adhesion to EC in vitro.7 However, there has been limited investigation as to the mechanism of glucose action on monocyte/EC interactions. Chemokines, including IL-6 and IL-8, play an important role in mediating local inflammatory effects in EC. We have recently shown a 2-fold induction of IL-8 secretion by EC cultured in chronic elevated glucose.13 We, and others, have also shown that IL-8 is an important mediator of monocyte rolling and firm adhesion in the vessel wall.1413,15 In addition, IL-6 may play a pro-inflammatory role in the vessel wall.16,17 Kaplanski et al have suggested that during chronic inflammation, such as occurs in diabetes, IL-6 functions to mediate monocyte recruitment to the vessel wall, thus participating in disease pathogenesis.16 Recent studies have indicated that IL-6 production is negatively regulated by PPAR.18–20 However, no study has definitively shown a role for IL-6 in mediating monocyte/endothelial interactions, particular in the context of diabetes.
PPAR is a nuclear hormone receptor that controls gene expression by interacting with specific DNA response elements (PPRE) in specific genes. PPAR partners with the retinoic acid receptor RXR to form a heterodimeric complex that binds PPRE elements in DNA. PPAR is activated by several fatty acids, as well as by fibrates.21,22 PPAR is believed to be anti-inflammatory on activation because PPAR knockout (KO) mice are more prone to inflammation.22,23 PPAR regulates expression of COX-2, IL-1?, IFN, TNF, IL-6, IL-8, and MCP-1, mostly to downregulate expression of these pro-inflammatory molecules,18,24,25 except in a few cases.26 PPAR inhibits expression of IL-6 via negative regulation of nuclear factor-kappa B and AP-1.19,27 The PPAR agonist, Wy14,643, reduces IL-6 expression in vivo in C57BL/6J mice.28 Furthermore, PPAR KO mice have increased production of IL-6 mRNA in aorta in response to a bacterial lipopolysaccharide (LPS) challenge.19 PPAR activation has been shown to reduce atherosclerosis in mice, although the role of PPAR in mediating atherosclerosis remains unclear.29,30 Recent studies by Semenkovich et al reported that deficiency of PPAR in low-density lipoprotein receptor-deficient mice inhibited development of atherosclerosis.26
In this study, we examined additional mechanisms for glucose regulation of monocyte adhesion to EC. We examined glucose-mediated regulation of PPAR expression and its impact on events related to monocyte adhesion. We also examined the role of IL-6 in mediating monocyte adhesion to EC in the setting of diabetes.
Methods
Reagents
Tissue culture media and reagents were purchased from Invitrogen. Fetal bovine serum was obtained from Hyclone. Calcein-AM was purchased from Molecular Probes. Human and mouse IL-6 ELISA kits were obtained from R&D Systems. Wy 14 643 was purchased from Biomol. Human IL-6 neutralizing antibody, human IL-8 neutralizing antibody, recombinant human IL-6, and recombinant human IL-8 were all purchased from R&D Systems. WEHI78/24 cells were a kind gift from Dr Judith A. Berliner (UCLA). Pioglitazone (Takeda Pharmaceuticals) was a kind gift from Dr Milagros Huerta (University of Virginia). Antibody to human PPAR was purchased from Affinity BioReagents, and antibody to histone H1 was purchased from Santa-Cruz Biotechnology (sc-8030).
Human EC Culture
Human aortic endothelial cells (HAEC) were obtained from aortic rings of explanted donor hearts. Use of HAEC was approved by the University of Virginia Institutional Review Board, and all procedures were performed in accordance with University Institutional Review Board guidelines. Briefly, HAEC were cultured for 7 days in medium 199 containing 20% heat-inactivated FBS, 20 μg/mL endothelial cell growth supplement (ECGS), and 90 μg/mL heparin in the absence (NG) and presence of 25 mmol/L D-glucose (HG) or 25 mmol/L L-glucose (as an osmotic control). The 7-day, 25-mmol/L HG incubation condition was chosen because monocyte adhesion to EC was maximal at this concentration of glucose and time-point of incubation.31
Mouse Aortic EC Isolation
Aortic EC from PPAR KO mice23 (Jackson Laboratories stock 003580) on a 129S4/SvJae background, PPAR control 129S1/SvImJ mice (Jackson Laboratories stock 002448), diabetic B6.Cg-m+/+Leprdb (db/db) mice on a C57BL/6J background (Jackson Laboratories stock 000697), and control C57BL/6J mice (Jackson Laboratories stock 000664) were harvested from mouse aorta after modifications of the method described previously.32 These methods were approved and performed under the guidelines established by the University of Virginia Animal Care and Use Committee. Briefly, the aorta is excised, all periadventitial fat is removed under a magnifying scope, and the aortic pieces are placed onto Matrigel in DMEM plus 15% HI-FBS. The EC grow out from these aortic explants. After 3 days, the explants are removed, and the EC are grown to confluence as described.33 EC are routinely used from passages 2 to 4. For the studies using diabetic db/db EC, cells were isolated as described and cultured for 1 passage in DMEM containing 15% heat-inactivated FBS, 60 μg/mL ECGS, and 100 μg/mL heparin, and 5.5 mmol/L glucose before performing assays. We have previously reported that diabetic db/db EC retain memory of their diabetic milieu during short-term passage in culture.33
Monocyte Adhesion Assays
Monocyte adhesion assays using human cells were performed as described previously.7 Briefly, NG- and HG-cultured HAEC were cultured to confluence as described into 48-well plates. HAEC were rinsed with 1% M199. Human primary monocytes were isolated from healthy normal volunteers using a modification of the Recalde method34 and labeled with Calcein AM (Molecular Probes) for 10 minutes at 37°C. Labeled human primary monocytes (50 000/well) were added to HAEC monolayers and incubated for 30 minutes at 37°C. Unbound monocytes were rinsed away, cells were fixed in 1% glutaraldehyde, and bound labeled monocytes were counted within a 10x10 grid using epifluorescence microscopy. Cells were incubated at 37°C with 10 U/mL recombinant human TNF for 4 hours as a control to show maximal monocyte adhesion in our assays. For subsets of studies, NG- and HG-cultured HAEC were incubated at 37°C with 100 μmol/L Wy14,643 or 5 μmol/L pioglitazone for 4 hours before adhesion assays. For IL-6 studies, HAEC were incubated at 37°C with recombinant human IL-6 (5 ng/mL) for 4 hours, IL-6 neutralizing antibody (0.1 μg/mL) for 4 hours, or an irrelevant antibody (-mouse IgG; 0.1 μg/mL) for 4 hours before performing a monocyte adhesion assay. For IL-8 studies, HAEC were incubated at 37°C with either recombinant human IL-8 (5 ng/mL) for 4 hours or IL-8 neutralizing antibody (20 μg/mL for 2 hours) before performing a monocyte adhesion assay.
Mouse Assays
Our laboratory has recently developed a monocyte adhesion assay that uses primary mouse aortic EC and WEHI 78/24 mouse monocytes.13 WEHI 78/24 is a mouse monocyte cell line that has been well characterized.35 WEHI are cultured in 10% heat-inactivated FBS in DMEM containing 4.5 g/L glucose. WEHI are labeled with Calcein AM (Molecular Probes) immediately before experiments according to manufacturer’s instructions. Mouse aortic EC are incubated with 35 000 fluorescent WEHI cells/well for 30 minutes at 37°C. Nonadherent cells are rinsed and the cells fixed with 1% glutaraldehyde. The number of attached monocytes present within a 10x10 grid is counted using epifluorescence microscopy.
Enzyme-Linked Immunosorbent Assay for Human and Mouse IL-6
Supernatants from cultured EC were collected, aliquoted to prevent repetitive thawing, and stored at –20°C. Enzyme-linked immunosorbent assay (ELISA) for human and mouse IL-6 in supernatants was performed using ELISA kits according to the manufacturer’s instructions. Supernatants were used undiluted in quadruplicate wells/sample. IL-6 levels in supernatant were determined using a standard curve. For normalization purposes, EC lysates were harvested using sodium dodecyl sulfate (SDS) lysis buffer containing phenylmethanesulfonyl fluoride and protease inhibitor cocktail (Sigma) and total cell protein was measured using a BioRad protein assay. IL-6 secretion into media was represented as picograms released into media/mg total cell protein to normalize for possible cell number differences under each experimental condition.
Nuclear Protein Extraction and Immunoblotting of PPAR
HAEC were cultured in NG and HG as described. After rinsing the monolayer of cells twice with PBS, cells were harvested using a cell scraper and lysed using cell lysis buffer (10 mmol/L HEPES pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.1 mmol/L DTT, and 10 μL of Sigma protease inhibitor cocktail). Cells were incubated on ice for 15 minutes. Then 10% Igepal CA-630 was added to a final concentration of 0.6%, and cells were vortexed vigorously and centrifuged at 10 000g for 3 minutes. Cell pellets were resuspended in extraction buffer (20 mmol/L HEPES pH 7.9, 1.5 mmol/L MgCl2, 0.42 mol/L NaCl, 0.2 mmol/L EDTA, 25% glycerol with Sigma protease inhibitor cocktail, and 0.1 mmol/L DTT) and agitated on a vortex mixer for 30 minutes. The nuclear extracts were separated by centrifugation at 20 000g for 5 minutes and aliquots were stored in –70°C. Protein was quantitated using a BioRad protein assay. SDS-PAGE of 25 μg nuclear protein was performed using 4% to 12% NuPAGE gels (Invitrogen). An antibody to human PPAR was incubated with the blots overnight at 4°C at a 1:1000 dilution. Blots were also incubated with antibody to histone H1 at a 1:500 dilution. Blots were normalized to histone H1 expression using densitometry (ZeroD Scan software; Stratagene).
Statistical Analyses
Data for all experiments were analyzed by ANOVA and Fisher protected least significant difference test using the Statview 6.0 software program. Data are represented as the mean±SE of 5 different experiments performed in quadruplicate unless otherwise stated in the Figure legends. Significance was reported at P0.05.
Results
Glucose Increases IL-6 Production by HAEC
We have previously reported increased production of IL-8 in HAEC mediated by chronic elevated glucose.13 In the current study, we found that HAEC cultured chronically for 7 days have increased production of IL-6 protein (Figure 1). HAEC cultured in 25 mmol/L L-glucose for 7 days as an osmotic control showed no change in IL-6 production. These data suggest that chronic elevated glucose, as occurs in patients with diabetes, causes endothelial activation through increased production of IL-6 and IL-8. Both IL-6 and IL-8 are involved in mediating monocyte recruitment to endothelium during vascular inflammation.16,17
Figure 1. Glucose increases endothelial production of IL-6. HAEC were cultured in 5.5.mM (NG), 25 mmol/L L-glucose (L-glucose), or 25 mmol/L D-glucose (HG) for 7 days. Media was collected and secreted IL-6 was measured using an ELISA for human IL-6. L-glucose had no effect on IL-6 levels in EC. *P<0.0001 versus NG by ANOVA. Data represent the mean±SE of 5 experiments.
IL-6 Directly Stimulates Monocyte Adhesion to EC
We have previously reported that chronic elevated glucose increases monocyte adhesion to HAEC.7,13 These data are illustrated in Figure 2, in which HG-cultured HAEC (HG) have a 2-fold increase in monocyte adhesion compared with EC cultured in 5.5 mmol/L glucose (NG). There was no effect of L-glucose on monocyte adhesion to EC. TNF is used as a positive control to indicate maximal monocyte adhesion in the assay.
Figure 2. Neutralizing antibody to human IL-6 reduces glucose-mediated monocyte adhesion. HAEC were cultured in NG, HG, or L-glucose as described in Methods. Before an adhesion assay, NG- and HG-cultured HAEC were incubated for 4 hours at 37°C with anti-human IL-6 antibody (+IL-6Ab) or anti-human IL-8 antibody (+IL-8Ab). NG-cultured cells were also incubated in the presence of recombinant human IL-6 (+IL6) or recombinant human IL-8 (+IL8) as described in Methods before performing a monocyte adhesion assay. L-glucose had no effect on monocyte adhesion. NG-HAEC were incubated with 10 U/mL recombinant human TNF for 4 hours as a positive control (+TNF) to show maximal monocyte adhesion. HG cells were also incubated with an irrelevant antibody (+CTRAb) as a negative control. *Significantly higher than NG, P<0.001; **significantly lower than HG, P<0.009. Data represent the mean±SE of 4 experiments performed in quadruplicate.
Several studies have suggested that IL-6 may mediate monocyte recruitment to activated endothelium.16 However, whether IL-6 directly can stimulate monocyte/EC adhesion is unclear. We tested this hypothesis and found that addition of recombinant human IL-6 to HAEC directly stimulated monocyte adhesion by 50% (NG+IL6; Figure 2). Furthermore, inhibition of endothelial IL-6 action through use of an IL-6 neutralizing antibody significantly reduced glucose-mediated monocyte adhesion to HAEC by 50% (HG versus HG+IL6Ab; Figure 2), suggesting that IL-6 action contributes significantly to glucose-mediated monocyte adhesion to EC.
We have previously reported that IL-8 plays a major role in glucose-stimulated monocyte adhesion.13 In this study, we directly compared the contributions of IL-6 and IL-8 in glucose-mediated monocyte adhesion. As shown in Figure 2, addition of recombinant human IL-8 to HAEC directly stimulated monocyte/endothelial interactions (NG versus NG+IL8). In addition, inhibition of endothelial IL-8 action through use of an IL-8 neutralizing antibody significantly reduced glucose-mediated monocyte adhesion by 50% (HG versus HG+IL8Ab; Figure 2). Thus, IL-6 is an important contributor, yet not the sole contributor, to glucose-stimulated monocyte adhesion. IL-6 and IL-8 contribute almost equally to regulate glucose-mediated monocyte adhesion.
Glucose Decreases PPAR Expression in EC
PPAR expression may negatively regulate IL-6 production in an anti-inflammatory manner.19 In Figure 1, we found an increase in IL-6 production in HG-cultured HAEC, so one explanation for an increase in IL-6 production could be a decrease in PPAR expression or function. We tested whether PPAR expression in HAEC was reduced by chronic elevated glucose. We found that PPAR protein levels were reduced by 50% in HAEC cultured in chronic glucose (HG) (P<0.001; Figure 3).
Figure 3. Glucose decreases PPAR expression in EC. HAEC were cultured in NG or HG as described in Methods. In subsets of cells, NG- or HG-cultured HAEC were incubated with 100 μmol/L Wy14,643 for 4 hours at 37°C (+Wy). Nuclear extracts were isolated as described in Methods. Western immunoblotting using 25 μg nuclear protein was performed using an antibody specific for PPAR, and blots were normalized to histone H1 levels. #Significantly higher than NG, P<0.01; **significantly lower than NG, P<0.001; *significantly higher than HG, P<0.01 by ANOVA. Data represent the mean±SE of 3 experiments.
We also found that the known PPAR agonist, Wy14,643 prevented complete downregulation of PPAR protein in response to glucose in EC (HG+Wy; Figure 3). Wy14,643 also significantly increased PPAR protein levels in normal HAEC (NG+Wy; Figure 3). We have recently found that Wy14,643 increases PPAR mRNA expression in EC (data not shown). Regulation of PPAR mRNA transcription by Wy14,643 has been reported by Sterchele and Mukherjee.36,37 These data suggest that Wy14,643 acts as an agonist and also regulates PPAR expression, probably through transcriptional processes.
The PPAR agonist Wy14,643 completely blocked glucose-mediated IL-6 production in HAEC (Figure 4A). Wy14,643 also significantly reduced glucose-mediated monocyte adhesion to HAEC by 40% (Figure 4B). TNF was used as a positive control to show maximal adhesion in this assay. We also examined the effects of the PPAR agonist pioglitazone on monocyte adhesion. We found no reduction in glucose-mediated monocyte adhesion using 5 μmol/L pioglitazone for 4 hours (Figure 4B). These data indicate that glucose increases monocyte adhesion in part through downregulation of PPAR expression with a resulting increase in production of IL-6. These data also suggest that IL-6 is responsible for 50% of glucose-mediated monocyte adhesion.
Figure 4. PPAR agonist Wy14,643 reduces glucose-mediated monocyte adhesion to HAEC. A, Endothelial IL-6 secretion. HAEC were cultured for 7 days in 5.5 mmol/L (NG) or 25 mmol/L glucose (HG). HG-HAEC were treated with 100 μmol/L Wy14, 643 for 4 hours (HG+Wy). Media was collected and secreted IL-6 measured using an ELISA for human IL-6. Values were normalized to total cell protein. *Significantly higher than NG, P<0.005; **significantly lower than HG, P<0.008 by ANOVA. B, Monocyte adhesion. HG-cultured HAEC were pre-treated for 4 hours at 37°C with 100 μmol/L Wy14,643 (HG+Wy), 5 μmol/L pioglitazone (HG+Pio), or DMSO as a vehicle control (HG+Vehicle). NG-HAEC were treated for 4 hours with 10 U/mL recombinant human TNF (+TNF) as a positive control. Normal human primary monocytes were added for a monocyte adhesion assay as described in Methods. *Significantly higher than NG, P<0.0001; **significantly lower than HG, P<0.01 by ANOVA. Data represent the mean±SE of 7 experiments.
PPAR-Deficient Mice Have Increased Monocyte/Endothelial Interactions and Increased IL-6 Expression
To further examine the role of endothelial PPAR in glucose-mediated monocyte adhesion, we used primary aortic EC isolated from PPAR KO mice on a B6,129 S4 background. EC usually do not bind monocytes unless they are activated. As shown in Figure 5, monocyte adhesion was significantly increased 2-fold in basal, unstimulated EC isolated from PPAR KO mice compared with EC from control mice (control strain 129S1/svImJ; P<0.009). Furthermore, we measured IL-6 production in control and PPAR KO aortic EC. We found a dramatic 4-fold increase in IL-6 production in aortic EC of PPAR KO mice compared with control mice (Figure 5) and a 2-fold increase in IL-6 mRNA levels in PPAR KO mice (data not shown). The Wy compound had no effect on monocyte adhesion or IL-6 production in PPAR KO mice (data not shown). This is consistent with earlier data from Gonzalez et al who found no effects of Wy14,643 on cellular parameters in the PPAR KO mice.38–41 We also found that exposing PPAR KO EC to elevated glucose (25 mmol/L for 7 days) increased monocyte adhesion but had no effect on IL-6 production (data not shown). This is important in that we have shown regulation of monocyte adhesion by additional chemokines that are not regulated by PPAR, including IL-8.13 However, IL-6 production appears to be primarily regulated by PPAR. Taken together, our data using PPAR KO mice indicate that PPAR expression is important for regulation of monocyte/endothelial adhesion, and that this regulation occurs, at least in part, through modulation of endothelial IL-6 production.
Figure 5. EC from PPAR KO mice have increased expression of IL-6 and bind more monocytes than control mouse EC. Mouse aortic endothelial cells from aorta of PPAR KO (PPARKO) and control mice (CTR) were isolated as described in Methods. All mouse aortic endothelial cells were cultured in DMEM containing 5.5 mmol/L glucose for 7 days. A, Endothelial IL-6 secretion. Media was collected and secreted murine IL-6 was measured using an ELISA for mouse IL-6. Values were normalized to total cell protein. *P<0.0001 versus CTR by ANOVA. B, Monocyte adhesion. Assays were performed using WEHI78/24 cells. *Significantly higher than CTR by ANOVA, P<0.005. Data represent the mean±SE of 4 experiments.
Diabetic Mice Also Show Increased Production of IL-6
We wanted to determine that IL-6 levels were also increased in diabetic db/db mice to illustrate the importance of this chemokine pathway in diabetes in vivo. EC were freshly isolated from mouse aortas of diabetic db/db and control mice and cultured for 1 passage in 5.5 mmol/L glucose. IL-6 levels were measured in control and diabetic EC. We found a significant 6-fold increase in IL-6 production in diabetic db/db mouse EC (Figure 6). We also found that IL-6 mRNA was elevated in db/db EC (data not shown), suggesting that IL-6 is regulated at the level of mRNA abundance in diabetic db/db mouse EC. EC from db/db mice also display increased monocyte adhesion (Figure 6). We have previously reported increased monocyte adhesion to EC in diabetic db/db mice.33 Thus, these data indicate a significant role for IL-6 in mediating monocyte adhesion to EC in the setting of diabetes.
Figure 6. IL-6 production and monocyte adhesion is increased in diabetic db/db mouse EC. EC from C57BL/6J control mice (B6 CTR) and diabetic db/db (db/db) mice were isolated and cultured for 1 passage as described in Methods. A, Endothelial IL-6 secretion. Media was collected and secreted IL-6 was measured using an ELISA for mouse IL-6. Values were normalized to total cell protein. *P<0.0001 versus B6 CTR by ANOVA. Data represent the mean±SE of 4 experiments. B, Monocyte adhesion. Assay was performed as described in Methods. *Significantly higher than B6 CTR, P<0.0001. Date represent the mean±SE of 10 experiments performed in quadruplicate.
Discussion
The signaling pathways by which glucose modulates monocyte/endothelial adhesion remain unclear. This is the first study that links glucose-mediated changes in endothelial activation to downregulation of PPAR. EC cultured in chronic glucose showed decreased expression of PPAR and increased monocyte adhesion. The studies shown in Figure 5 illustrate that absence of PPAR in murine EC results in increased monocyte adhesion. These data collectively indicate the importance of PPAR as an anti-inflammatory molecule in EC for early events of atherogenesis. In the current study, we found increased production of the pro-inflammatory chemokine IL-6 by EC in response to chronic elevated glucose. IL-6 production by EC was blocked by a PPAR agonist Wy14,643. These novel results indicate that glucose-stimulated production of IL-6 in endothelium in diabetes is mediated through inhibition of PPAR expression and/or action. In the present study we focused on PPAR, because we have found that expression of PPAR is low in human aortic EC.42 Furthermore, levels of PPAR in human EC were not increased in response to glucose (data not shown). We found no effect of pioglitazone, a PPAR agonist, on reducing glucose-mediated monocyte adhesion in HAEC treated for 4 hours at 37°C with 5 μmol/L pioglitazone (Figure 4). However, longer pretreatment of HAEC for 24 hours with 5 μmol/L pioglitazone slightly reduced glucose-mediated monocyte adhesion to HAEC, although the trend was not significant (data not shown). This would be consistent with another study that found that pioglitazone (20 μmol/L for 48 hours) reduced monocyte/EC interactions in response to elevated shear stress.43 However, other studies have not examined the role of pioglitazone in reducing glucose-stimulated monocyte adhesion. Because PPAR levels in HAEC appear not to be regulated by glucose, and because there is a minimal effect of pioglitazone on monocyte adhesion in our case, taken together, our results indicate that PPAR regulation is important for glucose-mediated endothelial activation. However, detailed studies of the interaction between glucose and PPAR in EC are needed and are underway in the laboratory.
PPAR may play a pro-inflammatory or anti-inflammatory role in the vessel wall depending on the state of oxidative stress in vascular cells.44,45 On the pro-inflammatory side, we have recently found chronic exposure of EC to glucose dramatically increases levels of reactive oxygen species in the cell.13 Previous work by our colleagues illustrated that PPAR stimulated MCP-1 and IL-8 synthesis in response to oxidized phospholipids and minimally modified low-density lipoprotein.46 Interestingly, PPAR KO mice on the apoE KO background were shown to have less atherosclerosis than control apoE KO mice.26 This study suggests a pro-inflammatory role for PPAR in mediating atherosclerosis. The authors suggested that the reduction of atherosclerosis in the absence of PPAR was caused by increased insulin sensitivity and enhanced glucose control. However, these investigators did not examine early events contributing to atherosclerosis that are relevant to the current study, such as monocyte/EC interactions and IL-6 production. Nevertheless, collectively, the data from these studies suggest a pro-inflammatory role for PPAR. However, many more groups have shown that PPAR is anti-inflammatory and that activation of PPAR by fibrate agonists reduces atherosclerosis development in mice.29 Cunard et al found that feeding C57BL/6J mice the PPAR agonist Wy14,643 reduced T cell and monocyte activation, which are key early events in formation of atherosclerotic plaques.28 Duez et al found that fenofibrate reduced aortic cholesterol content in apoE-deficient mice but did not reduce lesion area in the aorta.47 However, when these apoE KO mice were crossed with human apoAI transgenic mouse, fenofibrate dramatically reduced atherosclerotic lesion development.47 Mechanisms for the reduced atherosclerosis in response to PPAR activation include inhibition of T cell and monocyte activation,29 reduction of endothelial MCP-1 expression, and activation of apoAI.47 Although in most cases it is believed that PPAR exerts an anti-inflammatory effect, the type of inflammation (acute versus chronic) and the cause of inflammation (hyperlipidemia, hyperglycemia, oxidation) could be of importance in determining the anti- versus pro-inflammatory nature of PPAR.
To examine the direct role of PPAR in modulating monocyte adhesion, we used EC isolated from PPAR KO mice. Gonzalez et al have shown that PPAR KO mice have a significantly greater response to inflammatory stimuli compared with control mice.23 In adhesion assays, we found that EC isolated from PPAR KO mice bound a greater number of monocytes than did EC from control mice (Figure 5). These data indicate that in the absence of PPAR, EC are already activated to bind monocytes; thus, the PPAR KO EC are in a pro-inflammatory state. Additional studies will be needed to identify the molecular mechanisms leading to the enhanced monocyte adhesion observed in the PPAR KO EC.
We also show for the first time to our knowledge that EC from diabetic db/db mice have a significant upregulation in IL-6 production (Figure 6). We have previously reported that monocyte adhesion to EC is increased in db/db mice.33 These new data on IL-6 suggest that IL-6 plays a role in mediating monocyte adhesion in diabetic db/db mice. Further studies in these mice are needed to address this question. Nevertheless, these data suggest that IL-6 may be an important contributor to early vascular inflammatory events in diabetes in vivo.
In addition to the PPAR–IL-6 signaling pathway, there are several additional pathways in vascular EC that contribute to glucose-mediated monocyte adhesion. We have previously shown regulation of IL-8 synthesis in EC by chronic elevated glucose.13 We have found that IL-8 is a primary regulator of monocyte adhesion to EC in response to glucose, and it accounts for 50% of glucose-mediated monocyte adhesion (Figure 2). The regulation of IL-8 production in EC by glucose does not appear to be through PPAR. However, there is significant cross-talk between PPAR and other signaling pathways, including MAP kinases; therefore, PPAR may be indirectly involved in production of other chemokines.48 We have recently shown upregulation of the 12/15 lipoxygenase pathway in diabetic db/db mice33 as well as in human EC cultured chronically in elevated glucose.7 This inflammatory pathway most certainly contributes to monocyte/EC interactions in diabetes, although the exact mechanisms remain unclear. The role that these lipoxygenase eicosanoid products play in mediating monocyte/EC interactions in diabetes is currently being studied in the laboratory.
In summary, our data indicate that glucose-mediated induction of IL-6 and subsequent acceleration of monocyte adhesion occurs through modulation of levels of endothelial PPAR. These results support further development of modulators of PPAR expression or action to reduce accelerated cardiovascular disease caused by diabetes.
Acknowledgments
The authors thank Dr Judith Berliner (UCLA) for the gift of WEHI78/24 cells, David T. Bolick for assistance with endothelial cell culture, Dr Milagros Huerta (University of Virginia) for the gift of the pioglitazone, and Dr Jerry L. Nadler (University of Virginia) for helpful discussions. This work is supported by NIH PO1 HL55798-08 (CCH), American Heart Association Mid-Atlantic Affiliate (CCH), and Jeffress Foundation Memorial Trust fund (CCH).
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