Jeffrey J Olearczyk

Jeffrey E Quigley

Bradford C Mitchell

Tatsuo Yamamoto

In-Hae Kim

John W Newman

Ayala Luria

Bruce D Hammock

John D Imig
email: jdimig@mcw.edu

soluble epoxide hydrolase protects the kidney from damage in hypertensive Goto-Kakizaki rats

Keywords: diabetes, inflammation, eicosanoids, nephropathy, blood pressure

INTRODUCTION

Hypertension is a major risk factor for the development of nephropathy in patients with type II diabetes [START_REF]USRDS: the United States Renal Data System[END_REF]. If left untreated, hypertension exacerbates the progression of nephropathy to end stage renal disease (ESRD), which is the leading cause of mortality in patients with type II diabetes.

It has been estimated that more than three million people in the United States have hypertension and diabetes and that these disease states are co-morbid [START_REF]USRDS: the United States Renal Data System[END_REF]. Although the pathological progression of nephropathy is defined clinically, the initiating and propagating factors that lead to the development of nephropathy and its progression to ESRD have yet to be elucidated. A number of metabolic and hemodynamic factors have been identified that could contribute to the decline in renal function.

Recent findings have also implicated the involvement of a sub-acute inflammatory component that is characterized by an increase in the infiltration of pro-inflammatory cells into renal tissue and an increase in the transcription of nuclear factor-KB mediated pro-inflammatory genes [2,[START_REF] Guijarro | Transcription factor-kappa B (NF-kappa B) and renal disease[END_REF]. This inflammatory response can result in structural alterations within the kidney that perturb glomerular function and lead to an increase in glomerular filtration rate all of which occur early in the progression of nephropathy to ESRD.

Epoxygenase metabolites and their regulation by the soluble epoxide hydrolase (sEH) enzyme have been reported to regulate renal blood flow and blood pressure [START_REF] Imig | Eicosanoid regulation of the renal vasculature[END_REF][START_REF] Capdevila | The CYP P450 arachidonic acid monooxygenases: from cell signaling to blood pressure regulation[END_REF][START_REF] Roman | P-450 metabolites of arachidonic acid in the control of cardiovascular function[END_REF]. In addition to maintaining renal homeostasis, the epoxides, EETs and epoxide hydrolase inhibition are reported to prevent inflammation by inhibiting the expression of cell adhesion molecules and suppressing leukocyte adhesion [START_REF] Node | Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids[END_REF][START_REF] Campbell | New role for epoxyeicosatrienoic acids as anti-inflammatory mediators[END_REF]. Interestingly, increasing epoxygenase metabolite levels or sEH inhibition has been demonstrated to provide cardiovascular and renal protection. Given the renal-protective and anti-inflammatory properties, inhibition of sEH has been identified as a target for the treatment of diseases like hypertension and inflammation [START_REF] Imig | Epoxide hydrolase and epoxygenase metabolites as therapeutic targets for renal diseases[END_REF]. Pharmacological inhibitors of the sEH bind competitively and with high affinity to the catalytic site of the sEH, inhibiting its enzymatic activity [START_REF] Morisseau | Potent urea and carbamate inhibitors of soluble epoxide hydrolases[END_REF]. These urea based sEH inhibitors have been modified to allow for oral administration [START_REF] Kim | Design, synthesis, and biological activity of 1,3-disubstituted ureas as potent inhibitors of the soluble epoxide hydrolase of increased water solubility[END_REF].

Thus, we hypothesized that sEH inhibition could protect the kidney from the development of nephropathy associated with hypertension and type II diabetes. 4 inhibitor on mean arterial pressure and heart rate. In separate experiments, we investigated the effects of AUDA on renal injury and the infiltration of pro-inflammatory cells into the kidney.

MATERIALS AND METHODS

Animals

The Medical College of Georgia Animal Care and Use Committee approved all animal protocols. Twelve-week-old, male Goto-Kakizaki rats (Taconic, Germantown, NY) were divided into three experimental groups; the first group served as a control and received a normal salt diet and tap water (Goto-Kakizaki control), the second group was made hypertensive and received vehicle treatment, and the third group was made hypertensive and administered AUDA dissolved in the drinking water. Age matched, male Wistar rats (Taconic) were included as a comparison group.

Hypertension was induced by continuous infusion of angiotensin II (ANG, 65 ng/min) via an osmotic mini-pump (model 2004, Alzet, Palo Alto, CA). Rats from this group were also fed a high salt diet containing 8% NaCl by weight. AUDA (25 mg/L) was dissolved in a drinking water solution as previously described [START_REF] Moghaddam | Bioactivation of leukotoxins to their toxic diols by epoxide hydrolase[END_REF]. Rats from all of the groups were allowed access to food and water ad libitum during the 14-day treatment period.

Measurement of Blood Pressure

Telemetry transmitters (Data Sciences, Inc., St. Paul, MN) were implanted and data collected as described previously [START_REF] Zhao | Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage[END_REF]. The mean arterial pressure and heart rate were measured once every 5 minutes for the duration of the experimental protocol. Day and night time averages were calculated and plotted. The Biotelemetry Core at the Medical College of Georgia provided assistance with telemetry studies.

Measurement of Urinary Electrolytes, Albumin and MCP-1

Rats were housed in metabolic cages that separate urine from food and feces 24h prior to the completion of the treatment period. The urine was collected in a tube containing 5 mg triphenylphosphine. Urine volumes were measured and the urine aliquoted and stored at -80 °C until analyzed. Concentrations of urinary electrolytes (Na + , Cl -, K + ) were measured using ionselective electrodes (Synchron EL-ISE, Beckman Instruments, Brea, CA). Albumin (Exocell, Inc., Philadelphia, PA) and MCP-1 (BD Biosciences, San Jose, CA) concentrations were measured using enzyme linked immunosorbent assays.

S t a g e 2 ( a ) P O S T -P R I N T

5

Measurement of Plasma Insulin, Cholesterol and Triglycerides

Whole blood was collected into a heparanized syringe and transferred to a centrifuge tube.

The un-coagulated blood was spun for 5 min at 1,000g to sediment red blood cells. The plasma was pipetted off and aliquoted. Aliquots were stored at -80 °C until assayed. Plasma insulin was measured using an enzyme linked immunosorbent assay (Alpco Diagnostics, Windham, NH).

Cholesterol and total triglycerides were measured using a colorimetric assay (WAKO Chemicals, Richmond, VA).

Histology and Immunohistochemistry

At the end of the treatment period, kidneys were isolated and perfused with ice-cold physiological salt solution (composition) followed by a 10% formalin solution to fix the kidney tissue. After perfusion, the kidneys were removed, cut and fixed in 10% formalin solution overnight. The kidney sections were embedded in paraffin and cut into 4 µm slices for use in histology and immunohistochemistry protocols. For histology, formalin-fixed paraffin-embedded kidney slices were deparaffinized, re-hydrated and stained with hematoxylin-eosin. Another series were stained using a Masson Trichrome Kit according to the manufacturer's protocol. For immunohistochemistry, deparaffinized, re-hydrated kidney slices were incubated with a 10% hydrogen peroxide solution in methanol to block endogenase peroxidase activity and then blocked with normal goat serum. Kidney slices were then incubated with a primary antibody that recognizes monocytes/macrophages (mouse anti-rat CD68). Sections were incubated with anti mouse secondary antibody conjugated to HRP and visualized using diaminobenzamine chromogen. Slides were counterstained with hematoxylin.

Evaluations of renal damage from hematoxylin-eosin stained sections were performed without the evaluator having knowledge of the treatment groups. Kidney sections were scored

Measurement of AUBA Levels

To confirm AUDA treated groups, urinary levels of the inactive AUDA metabolite 4-(3adamantan-1-yl-ureido)-butanoic acid (AUBA) was measured in the urine [START_REF] Watanabe | In vitro metabolism of the mammalian soluble epoxide hydrolase inhibitor, 1-cyclohexyl-3-dodecylurea[END_REF]. Briefly, the analytes were separated from other components in the urine by solid phase extraction using a conditioned Oasis-HLB SPE cartridge. The analytes were eluded form the column with ethyl acetate. The ethyl acetate was evaporated under nitrogen and the resulting residue suspended in 50 µl of methanol containing internal standards. The samples were separated using high pressure liquid chromatography. Analyte concentration was determined by comparison to a measured standard curve.

Measurement of Blood Glucose

Non-fasting blood glucose was measured from blood obtained by tail prick. The amount of glucose in the blood was determined using the Accu-chek Advantage blood glucose monitoring system (Roche, Indianapolis, IN).

Real-time Polymerase Chain Reaction (PCR)

Total RNA was prepared from kidney cortex using ultra-pure TRIzol reagent according to the manufacturer's instructions (GIBCO-BRL, Grand Island, NY). Reverse transcription was performed on equal amounts of total RNA (3 µg) using a blend of oligo-dT primers and random hexanucleotide primers to produce a cDNA library for each sample. Real-time PCR reactions were run on an iCycler iQ Real-Time PCR Detection System using iQ Supermix which is optimized for real-time PCR applications (BioRad Laboratories, Inc., Hercules, CA). TaqMan probes (Roche Molecular Systems) and oligonucleotide primers were designed from the published cDNA sequences for MCP-1 and GAPDH using Beacon Designer software (Premier Biosoft International, Palo Alto, CA). Each sample was run in duplicate and the comparative C t method was used to quantify fold increase compared to controls. Probes and primer sequences were used as follows:

MCP

NFκB Transcription Factor Assay

Whole-cell lysates were obtained from kidney cortex using the Nuclear Extract Kit (Active Motif, Carlsbad, CA). Protein concentrations were determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Twenty µg of whole-cell extract was used for the determination of NFκB activity using the TransAM NFκB p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA). Each of the standards and samples were run in duplicate. In addition to the Jurkat cell nuclear extract provided in the kit as a positive control, HeLa cell whole-cell lysate from cells cultured for 5 min in the presence and in the absence of 10µg/ml TNF-α and 10µM Calyculin A were also run. To ensure NFκB specificity, HeLa whole-cell lysate from cells treated with TNF-α and the whole-cell lysate obtained from experimental groups were run in the presence of the wildtype NFκB consensus oligonucleotide and mutated NFκB consensus oligonucleotide. The wild-type consensus oligonucleotide completely blocked NFκB binding with absorbance that was not different from the blank wells (no NFκB p65 standard). Conversely, the mutated consensus oligonucleotide was without effect on NFκB binding (data not shown). The amount of activated NFκB was normalized per µg of cortical protein used in the assay.

Statistics

All data are presented as mean ± SEM. Statistical significance between experimental groups was determined with an ANOVA. In the event that the associated F ratio indicated that changes occurred, a least significant difference test was used to identify individual differences. P values of 0.05 or less were considered statistically significant.

RESULTS

Urinary excretion of AUDA and AUBA were measured to confirm proper sEH inhibition.

AUDA excretion averaged 28 ± 9 ng/d and AUBA excretion averaged 24,049 ± 6,234 mg/d in hypertensive Goto-Kakizaki rats after 14 days of AUDA administration.

It is well documented that a major risk factor in the development and progression of nephropathy in patients with type II diabetes is hypertension [START_REF] Sowers | Diabetes mellitus and associated hypertension, vascular disease, and nephropathy. An update[END_REF][START_REF] Viberti | Predisposition to essential hypertension and the development of diabetic nephropathy[END_REF]. Our laboratory has reported that sEH inhibition lowered blood pressure and provided renal protection in an animal model of In separate studies, body weight, urine volumes and urine electrolytes were measured at the end of the two-week treatment period as well as blood glucose, plasma insulin, total cholesterol and triglycerides. These results are summarized in Table 1. As expected, the Goto-Kakizaki control group has elevated non-fasting blood glucose and is consistent with the Goto-Kakizaki rat as a model of type II diabetes. The Goto-Kakizaki control group also has significantly decreased plasma insulin and these data are congruous with impaired insulin secretion and impaired development of pancreatic islet cells both of which are involved in the pathogenesis of diabetes in the Goto-Kakizaki rat [START_REF] Movassat | Impaired development of pancreatic beta-cell mass is a primary event during the progression to diabetes in the GK rat[END_REF][START_REF] Picarel-Blanchot | Impaired insulin secretion and excessive hepatic glucose production are both early events in the diabetic GK rat[END_REF][START_REF] Kim | Effects of KIOM-79 on hyperglycemia and diabetic nephropathy in type 2 diabetic Goto-Kakizaki rats[END_REF]. Although the Goto-Kakizaki control and hypertensive groups have elevated blood glucose, plasma cholesterol and triglyceride levels, AUDA treatment did not change these levels in hypertensive Goto-Kakizaki rats. Hypertensive Goto-Kakizaki rats demonstrated a significant decrease in body weight and a significant increase in urine volume compared to the Goto-Kakizaki control group. The increased urinary volume was accompanied by an expected increase in sodium and chloride excretion resulting from the angiotensin and high salt diet-induced hypertension. Urinary electrolyte excretion was not altered in the hypertensive Goto-Kakizaki rats administered AUDA. monocytes /mm 2 and Wistar rats averaged 1.6 ± 0.4 monocytes /mm 2 . The number of monocytes/macrophages in the medulla were also determined and found not to be different between any of the treatment groups (data not shown). Inducing hypertension in Goto-Kakizaki rats resulted in a 4-fold increase in monocyte/macrophage infiltration into the kidney cortex (Figure 4).

Interestingly, this increase in monocyte/macrophage infiltration corresponds with the measured increase in urinary albumin excretion (Figure 2). Treatment of hypertensive Goto-Kakizaki rats with AUDA significantly attenuated hypertension-induced increases in monocyte/macrophage infiltration into the kidney cortex.

Activation and infiltration of monocytes/macrophages from the bloodstream into the tissue results in part from increased transcription and production of monocyte chemoattractant protein-1 (MCP-1). To determine if the increased infiltration of monocyte/macrophages into the kidney resulted from an increase in MCP-1 protein, urinary MCP-1 excretion was measured. As depicted in Figure 4, response has been identified that contributes to this progression. This sub-acute inflammatory response is characterized by an increase in the infiltration of pro-inflammatory cells into the kidney and an increase in the expression of pro-inflammatory genes [2,[START_REF] Guijarro | Transcription factor-kappa B (NF-kappa B) and renal disease[END_REF]. The sEH has been identified as a target for the treatment of hypertension and inflammation. Inhibitors of sEH have been reported to decrease blood pressure in hypertension and decrease hypertension-induced renal damage [START_REF] Zhao | Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage[END_REF][START_REF] Yu | Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids[END_REF].

In addition, sEH inhibitors have demonstrated anti-inflammatory properties and were reported to decrease inflammation-induced tissue damage [START_REF] Smith | Attenuation of tobacco smoke-induced lung inflammation by treatment with a soluble epoxide hydrolase inhibitor[END_REF][START_REF] Schmelzer | Soluble epoxide hydrolase is a therapeutic target for acute inflammation[END_REF]. In the present study, we investigated the hypothesis that sEH inhibition protects the kidney from renal damage associated with hypertension and type II diabetes. Hypertension induced in the spontaneously type II diabetic Goto-Kakizaki rat accelerated the development of renal damage and the infiltration of pro-inflammatory cells into the kidney. AUDA treatment decreased renal damage independent of an effect on blood pressure. In addition, AUDA treatment inhibited the infiltration of pro-inflammatory cells into the kidney and decreased the expression of pro-inflammatory genes.

In the present studies, mean arterial blood pressure increased significantly in Goto-Kakizaki rats infused with angiotensin and fed a high fat diet. The oral administration of AUDA to hypertensive Goto-Kakizaki rats had no effect on mean arterial pressure. This result was of particular interest because the administration of AUDA or other sEH inhibitors were reported to decrease blood pressure in animal models of hypertension [START_REF] Zhao | Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage[END_REF]18]. Our laboratory has demonstrated that AUDA treatment decreased blood pressure in hypertensive Sprague-Dawley rats.

In these experiments hypertension was induced with either angiotensin II infusion or angiotensin II infusion with a high salt diet [START_REF] Imig | An Orally Active Epoxide Hydrolase Inhibitor Lowers Blood Pressure and Provides Renal Protection in Salt-Sensitive Hypertension[END_REF]. The urinary excretion of AUDA averaged 121 ± 61 ng/d for the AUDA treated angiotensin II with high salt diet-induced hypertensive group. In the current study, the urinary excretion of AUDA averaged only 28 ± 9 ng/day for the AUDA treated hypertensive Goto-Kakizaki group [START_REF] Imig | An Orally Active Epoxide Hydrolase Inhibitor Lowers Blood Pressure and Provides Renal Protection in Salt-Sensitive Hypertension[END_REF]. Therefore, it is possible that higher AUDA levels than those attained in the current study are required to elicit effects on blood pressure. Although AUDA did not alter mean arterial pressure, AUDA treatment did have beneficial effects on the hypertensive Goto- 12 Kakizaki rats. Therefore the data presented here suggest that the renal protective effects afforded by AUDA occur via a mechanism that is independent of its effects on blood pressure.

Hypertension induced in the Goto-Kakizaki rat resulted in an increase in blood pressure and was accompanied by a measured increase in heart rate during the second week. It was reported that angiotensin II and/or salt sensitive hypertension resulted in a transient decrease in heart rate that returns to control levels by the end of the first week [START_REF] Imig | An Orally Active Epoxide Hydrolase Inhibitor Lowers Blood Pressure and Provides Renal Protection in Salt-Sensitive Hypertension[END_REF]. Therefore, the ability to control heart rate in the spontaneously type II diabetic Goto-Kakizaki rat after the induction of hypertension using angiotensin II and high salt diet is not the same as rats with hypertension alone. The specific contribution of increased heart rate to the development of renal damage in this animal model of hypertension and type II diabetes is not known.

The progression of renal damage in the spontaneously diabetic Goto-Kakizaki rats was accelerated by hypertension. This was evidenced by and increase in urinary albumin excretion, an indicator of renal damage. In addition, hypertension induced morphological changes in the kidney that are characteristic to the development of nephropathy such as tubular dilation and fibroid necrosis of interstitial arterioles. Similar changes were observed in other models of hypertension induced in the Goto-Kakizaki rat. Salt-sensitive hypertension resulted in a 50% increase in albuminuria and administration of deoxycorticosterone acetate salt to Goto-Kakizaki rats, a model of mineralcorticoid-induced experimental hypertension, induced a 4-fold increase in proteinuria [START_REF] Cheng | Endothelial dysfunction and salt-sensitive hypertension in spontaneously diabetic Goto-Kakizaki rats[END_REF][START_REF] Tefescu | Combination of exercise and losartan enhances renoprotective and peripheral effects in spontaneously type 2 diabetes mellitus rats with nephropathy[END_REF]. Likewise, heminephrectomized Goto-Kakizaki rats have increased macrophage infiltration and accelerated renal damage [START_REF] Janssen | Hypertension superimposed on type II diabetes in Goto Kakizaki rats induces progressive nephropathy[END_REF]. Treatment of hypertensive Goto-Kakizaki rats with AUDA inhibited hypertension-induced increases in albumin excretion and prevented the development of many of the morphological changes induced by hypertension. These renal protective effects resulting from epoxide hydrolase inhibition are consistent with data generated in our laboratory using in vivo models of hypertension. Zhao et al. reported that chronic administration of the epoxide hydrolase inhibitor 1-cyclohexyl-3-dodecylurea (CDU) to angiotensin II-induced hypertensive Sprague-Dawley rats decreased urinary albumin excretion and protected the kidney from hypertension-induced damage [START_REF] Zhao | Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage[END_REF]. Similarly, it was reported that AUDA, decreased urinary albumin excretion and renal damage in both angiotensin II-induced and angiotensin II-induced saltsensitive hypertensive Sprague-Dawley rats [START_REF] Imig | An Orally Active Epoxide Hydrolase Inhibitor Lowers Blood Pressure and Provides Renal Protection in Salt-Sensitive Hypertension[END_REF]. Taken together, these data provide support for the hypothesis that AUDA protects the kidney from damage associated with hypertension and type II diabetes. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

13

An inflammatory component has been identified that contributes to the progression of nephropathy to end stage renal disease. This sub-acute inflammatory component is characterized by an increase in the infiltration of inflammatory cells into the kidney and an increase in the expression of pro-inflammatory genes. In the present study, we demonstrated that hypertension exacerbated monocyte/macrophage infiltration in the Goto-Kakizaki kidney. This increased infiltration of inflammatory cells into the kidney cortex was accompanied by an increase in urinary MCP-1 excretion and an increase in MCP-1 gene expression in the kidney cortex. This hypertensioninduced increase in the sub-acute response corresponds with the observed increase in urinary albumin excretion and renal morphological damage. These data are consistent with renal damage that occurs in other experimental models of hypertension and heminephrectomy in the Goto-Kakizaki rat [START_REF] Cheng | Endothelial dysfunction and salt-sensitive hypertension in spontaneously diabetic Goto-Kakizaki rats[END_REF][START_REF] Tefescu | Combination of exercise and losartan enhances renoprotective and peripheral effects in spontaneously type 2 diabetes mellitus rats with nephropathy[END_REF][START_REF] Janssen | Hypertension superimposed on type II diabetes in Goto Kakizaki rats induces progressive nephropathy[END_REF]. Cheng et al. reported an increase in monocyte/macrophage infiltration into the kidney with a corresponding increase in immunostaining for the intracellular adhesion molecule-1 in Goto-Kakizaki rats with salt-sensitive hypertension [START_REF] Cheng | Endothelial dysfunction and salt-sensitive hypertension in spontaneously diabetic Goto-Kakizaki rats[END_REF]. In addition, administration of deoxycorticosterone acetate salt to Goto-Kakizaki rats resulted in an increase in macrophage infiltration into the kidney and was accompanied by an increase in renal immunohistochemical staining for MCP-1 [START_REF] Janssen | Hypertension superimposed on type II diabetes in Goto Kakizaki rats induces progressive nephropathy[END_REF]. Interestingly, treatment of hypertensive Goto-Kakizaki rats with AUDA inhibited the infiltration of monocyte/macrophages into the kidney and decreased gene expression and urinary excretion of MCP-1. Taken together, these data suggest that the development of hypertension in an animal model of type II diabetes exacerbates the progression of renal injury at least in part by inducing a sub-acute inflammatory response and that this response can be attenuated with AUDA treatment.

The sEH enzyme has been identified as a target for the treatment of hypertension and inflammation [START_REF] Imig | Epoxide hydrolase and epoxygenase metabolites as therapeutic targets for renal diseases[END_REF]. In animal models of hypertension, sEH inhibitors are reported to decrease blood pressure and thereby inhibit hypertension-induced renal damage [START_REF] Zhao | Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage[END_REF]18]. The sEH inhibitors 1cyclohexyl-3-dodecylurea and AUDA lowered blood pressure in animal models of hypertension and reduced hypertension-induced renal damage [START_REF] Zhao | Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage[END_REF]. In animal models of systemic inflammation sEH inhibitors are reported to decrease the infiltration of pro-inflammatory cells into tissue and decrease the expression of pro-inflammatory genes. The sEH inhibitor 12-(3-adamantane-1-yl-ureido)dodecanoic acid n-butyl ester (AUDA-nBE), was reported to inhibit tobacco smoke-induced lung inflammation [START_REF] Smith | Attenuation of tobacco smoke-induced lung inflammation by treatment with a soluble epoxide hydrolase inhibitor[END_REF]. In addition, the sEH inhibitors AUDA-nBE and 1-adamantan- Although increased epoxides is the likely mechanism, it has been reported that AUDA in addition to sEH inhibition can activate the peroxisome proliferator-activated receptor-α (PPAR-α); however the concentration of AUDA required for PPAR-α activation is much higher than the concentrations obtained in the current study [START_REF] Fang | Activation of peroxisome proliferator-activated receptor alpha by substituted urea-derived soluble epoxide hydrolase inhibitors[END_REF]. Also, PPAR-α activation would be expected to decrease triglyceride levels, which were unchanged in AUDA treated hypertensive Goto-Kakizaki rats. sEH inhibitors have also been reported to increase the incorporation and retention of the EETs into endothelial phospholipids and enhance the shuttling of the EETs into alternate metabolic pathways [START_REF] Fang | Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells. Implications for the vascular effects of soluble epoxide hydrolase inhibition[END_REF][31][32]. The contribution of these alternate actions of the sEH inhibitors in the current study are not known and therefore might contribute to the renal-protective effects observed.

In the present study, we demonstrated that hypertension, induced in a model of type II diabetes, exacerbates the development of renal damage. In addition, we provide evidence that this progression involves a sub-acute inflammatory response. We demonstrated that sEH inhibition protects the kidney from the development of renal damage independent of any effects on blood

  using the following numeric scale: 0 = no damage, +1 = very mild, +2 = mild, +3 = moderate, +4 = severe. Evaluation of fibrosis and renal injury were further evaluated in Masson Trichrome stained kidney section. Stained sections were visualized by light microscopy and representative digital images of 5 cortex and 3 medulla areas were obtained for each kidney. To quantify the Masson Trichrome staining, the 8 random images from each kidney were assigned random numbers and scored by three blinded observers on a scale of 0 to 10 for collagen deposition, fibrosis and renal injury. Semiquantitative evaluation of the renal inflammatory cell infiltration was also performed without the evaluator having knowledge of the treatment groups. The numbers of CD68 positive S t a g e 2 ( a ) P O S T -P R I N T 6 cells were counted from a given area of kidney and the numbers obtained from each treatment group averaged and plotted.

  -1 probe 5'-FAM-CAC CTG CTG CTA CTC ATT CAC TGG C-BHQ-3'; MCP-1 forward 5'-CAG CCA GAT GCA GTT AAT GC-3'; MCP-1 reverse 5'-GCT TGG TGA CAA ATA CTA CAG C-3'; GAPDH probe 5'-FAM-ACT CCA CGA CAT ACT CAG CAC CAG CA-BHQ-3'; S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 May 2008 as manuscript CS20080039 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039 7 GAPDH forward 5'-CAC GGC AAG TTC AAC GGC-3'; GAPDH reverse 5'-GGT GGT GAA GAC GCC AGT A-3'.

S t a g e 2 (

 2 a ) P O S T -P R I N T hypertension[START_REF] Zhao | Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage[END_REF]18]. To determine if sEH inhibition lowers blood pressure and/or protects the kidney from the renal damage associated with hypertension in type II diabetes, AUDA was administered to hypertensive Goto-Kakizaki rats. Arterial blood pressure and heart rate are represented graphically in Figures1A and 1B, respectively. Treatment of hypertensive Goto-Kakizaki rats with AUDA did not lower arterial blood pressure, 182 ± 20 versus 187 ± 6 mmHg for vehicle and AUDA treated hypertensive Goto-Kakizaki rats, respectively. The arterial blood pressure of Goto-Kakizaki control rats measured 118 ± 2 mmHg. AUDA treatment did not alter heart rate in hypertensive Goto-Kakizaki rats. As predicted, hypertension resulted in an initial decrease in the heart rate of Goto-Kakizaki rats with the heart rate reaching its lowest point after five days. Heart rates measured 280 ± 2 bpm for vehicle treated and 307 ± 3 bpm for AUDA treated hypertensive Goto-Kakizaki groups compared to 368± 12 bpm in Goto-Kakizaki controls on the fifth day. After the fifth day, the heart rate of the hypertensive Goto-Kakizaki rats and the AUDA treated hypertensive Goto-Kakizaki rats increased and averaged significantly higher than the Goto-Kakizaki control rats. The heart rates measured 456 ± 4 and 448 ± 3 bpm for vehicle and AUDA treated hypertensive Goto-Kakizaki groups, respectively and 357 ± 4 bpm for the Goto-Kakizaki control group.

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 May 2008 as manuscript CS20080039 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.9To investigate whether or not soluble epoxide hydrolase inhibition could prevent renal damage associated with hypertension and type II diabetes urinary excretion of albumin was measured, an indicator of renal damage. Urinary albumin excretion averaged 0.9 ± 0.2 mg/d at day 0, 2.5 ± 0.4 mg/d at day 7, and 1.4 ± 0.2 mg/d at day 14 in Goto-Kakizaki controls and at the end of the experimental period this level is comparable to that observed in Wistar rats (1.0 ± 0.4 mg/d).Induction of hypertension in Goto-Kakizaki rats resulted in an increase in urinary albumin excretion at day 7 (8.6 ± 2.6 mg/d) and a 17-fold increase to 22.0 ± 5.0 mg/d at day 14, indicating the development of renal damage Figure2. AUDA treatment inhibited hypertension-induced increases urinary albumin excretion at day 7 (2.6 ± 1.6 mg/d) and day 14 (6.3 ± 2.6 mg/day).To determine the extent of the renal damage semiquantitative analyses of histological sections were made. Representative sections are provided in Figures2 and 3. The Goto-Kakizaki control group showed very mild tubular dilation (+1), tubular atrophy (+1) and glomerular hypertrophy (+1). This is comparable to Wistar rats that did not have any damage, sclerosis or necrosis of glomeruli detected in histological sections (data not shown). Hypertensive Goto-Kakizaki rats demonstrated mild to moderate renal injury with increased fibroid necrosis (+3) and hyaline arteriopathy of the interstitial arterioles (+3), increased tubular dilation (+3), tubular atrophy (+2), cast formation (+2) and increased mononuclear cell infiltration (+2). Hypertension did not alter the glomerular hypertrophy present in the Goto-Kakizaki control group. Treatment with AUDA reduced hypertension induced renal injury scores for fibroid necrosis (+1) and hyaline arteriopathy of the interstitial arterioles (+1). AUDA treatment also decreased tubular dilation (+2), atrophy (+1) and cast formation (+1) as well as decreased the score for mononuclear cell infiltration (+1); however, glomerular hypertrophy scores were unchanged. Masson Trichrome staining was evaluated to assess the extent of renal fibrosis and injury. As depicted in Figure3, there was extensive fibrosis and renal injury in the hypertensive Goto-Kakizaki that was ameliorated by AUDA treatment. Taken together these data provide support for the hypothesis that inhibition of the soluble epoxide hydrolase protects the kidney from renal damage associated with hypertension and type II diabetes independent of any affects on blood pressure.The progression of nephropathy associated with hypertension and type II diabetes involves an inflammatory component that is characterized by increased expression of pro-inflammatory genes and infiltration of pro-inflammatory cells into the kidney interstitium. Recently, sEH inhibition has been identified as a target for the treatment of inflammatory diseases[START_REF] Smith | Attenuation of tobacco smoke-induced lung inflammation by treatment with a soluble epoxide hydrolase inhibitor[END_REF][START_REF] Schmelzer | Soluble epoxide hydrolase is a therapeutic target for acute inflammation[END_REF]. To S t a g e 2 ( a ) P O S T -P R I N T 10 determine if sEH inhibition decreases the infiltration of pro-inflammatory cells into the kidney, the infiltration of monocyte/macrophages was measured. Goto-Kakizaki control averaged 1.3 ± 0.3

the

  Goto-Kakizaki control group had low levels of MCP-1 in the urine. Urinary excretion of MCP-1 was significantly increased in the hypertensive Goto-Kakizaki group. AUDA treatment attenuated hypertension-induced increases in urinary MCP-1 excretion. These data are in agreement with the infiltration of monocytes/macrophages into the kidney. To determine if the increase in urinary MCP-1 excretion resulted from an increase in MCP-1 transcription kidney cortex was obtained and the amount of MCP-1 mRNA measured by PCR. Cortex from the Goto-Kakizaki control group had low levels of MCP-1 gene expression (2 -ΔΔCt = 1.21 ± 0.29). The hypertensive Goto-Kakizaki group demonstrated a 3.4-fold increase in MCP-1 gene expression in the kidney cortex (2 -ΔΔCt = 3.57 ± 0.43), which was inhibited to control values with AUDA treatment (2 -ΔΔCt = 1.14 ± 0.27). Consistent with the other inflammatory data, renal cortical NFκB activity was significantly higher in hypertensive Goto-Kakizaki group and AUDA treatment reduced NFκB activity in hypertensive Goto-Kakizaki rats (Figure5).Taken together, these data suggest that inhibition of the soluble epoxide hydrolase inhibits renal damage by inhibiting the infiltration of pro-inflammatory cells into the kidney associated with hypertension and type II diabetes. S t a g e 2 ( a ) P O S T -P R I N T DISCUSSION Hypertension is a major risk factor for patients with type II diabetes. It has been proposed that hypertension and type II diabetes are co-morbid disease states and that the development of hypertension exacerbates the progression of diabetic nephropathy to ESRD. An inflammatory

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 May 2008 as manuscript CS20080039 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 May 2008 as manuscript CS20080039 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039

  3-(5-(2-(2-ethylethoxy)ethoxy)pentyl)urea decreased liposaccharide-induced increases in plasma concentrations of S t a g e 2 ( a ) P O S T -P R I N T 14pro-inflammatory cytokines like TNF-alpha, IL-6 and MCP-5 in mice[START_REF] Schmelzer | Soluble epoxide hydrolase is a therapeutic target for acute inflammation[END_REF]. In these animal models, the protective effects of the sEH inhibitors are attributed to a decrease in EET metabolism.

  pressure. A possible mechanism by which AUDA treatment attenuates the development of renal damage in this model of hypertension and type II diabetes is via inhibition of the sub-acute inflammatory response. Taken together, the data presented provides support for the hypothesis inhibition of the epoxide hydrolase ameliorates the inflammatory component of nephropathy associated with hypertension and type II diabetes. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 May 2008 as manuscript CS20080039 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039 31. Weintraub, N.L., Fang, X., Kaduce, T.L., VanRollins, M., Chatterjee, P., and Spector. A.A. (1999) Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids. Am. J. Physiol. Heart Circ. Physiol. 277, H2098-H2108 32. Imig, J.D. (2006) Eicosanoids and renal vascular function in diseaes. Cli. Sci. 111, 21-34 S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 May 2008 as manuscript CS20080039 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 Figure 1. The effect of AUDA on mean arterial pressure (MAP, Panel A) and heart rate (HR, Panel B) in hypertensive Goto-Kakizaki (GK) rats (n=4). Data points represent 12 hour day and night averages of MAP and HR in GK control rats (closed squares) and hypertensive GK rats treated with AUDA (closed triangle) or its vehicle (open triangle). Values represent means ± SEM. *, different from GK control rats, P<0.05.

Figure 2 .Figure 3 .

 23 Figure 2. The effects of AUDA treatment on (A) urinary albumin excretion and (B) kidney morphology from hypertensive Goto-Kakizaki (GK) rats (n=6-9). *, different from GK control and Hypertensive GK AUDA groups; , different from GK control group, P<0.05

Figure 4 .Figure 5 .

 45 Figure 4. The effects of AUDA treatment on (A) urinary excretion of MCP-1 and (B) infiltration of pro-inflammatory cells into the kidney cortex of hypertensive Goto-Kakizaki (GK) rats (n=6-7). *, different from GK control and Hypertensive GK AUDA groups; , different from GK control group, P<0.05

S t a g e 2 (

 2 a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 7 May 2008 as manuscript CS20080039 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 1 .

 1

			Goto-Kakizaki
		GK	ANGII/HS	ANGII/HS
		Control	Vehicle	AUDA
	Body Weight (g)	357 ± 10	292 ± 14*	280 ± 9*
	Urine Volume (mL)	16 ± 1	88 ± 7*	79 ± 12*
	Urine Electrolytes 123456Na + (mmol/d)	0.9 ± 0.1	17.0 ± 1.4*	13.7 ± 2.3*
	123456K + (mmol/d) 123456Cl -(mmol/d)	3.5 ± 0.2 1.6 ± 0.1	2.3 ± 0.2* 17.5 ± 1.4*	1.9 ± 0.3* 13.9 ± 2.3*
	Non-fasting blood glucose (mg/dL)	176 ± 24	138 ± 14	144 ± 15
	Plasma insulin (µg/L)	0.8 ± 0.1	0.2 ± 0.1*	0.2 ± 0.1*
	Cholesterol (mg/dL)	67.7 ± 5.3	74.0 ± 9.6	76.1 ± 9.7
	Triglycerides (mg/dL)	30.2 ± 9.7	24.1 ± 7.2	23.7 ± 6.8

*, different from GK control group, P<0.05

THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080039 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Award, AHA Southeast Affiliate Postdoctoral Fellow, National Institutes of Health Grants HL-59699 and HL-74167


Jingti Deng

Christian H James

Lisa Patel #

Alberto Smith

Kevin G Burnand

Hassan Rahmoune

Jonathan R Lamb

Bill Davis
email: bill.g.davis@gsk.com

± From

Human Tribbles Homolog 2 is Expressed in Unstable Regions of Carotid Plaques and Regulates Macrophage IL-10 in Vitro

Keywords: atherosclerosis, unstable plaque, macrophage, tribbles homolog 2, IL-10

Mammalian orthologs of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In this study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated upregulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was upregulated by treatment with oxidised low density lipoprotein (LDL), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression that may contribute to plaque instability.

Introduction

There is increasing evidence that vessel wall inflammation plays a central role in the development of the atherosclerotic plaque, and crucially in the process of plaque destabilisation that results in the local thrombosis underlying the common clinical presentations of atherosclerosis [START_REF] Libby | Inflammation and atherosclerosis[END_REF]. Recruitment of monocytes into the lesion, and their subsequent differentiation into macrophages and transformation into foam cells via ingestion of modified LDL particles plays a central role in determining the inflammatory environment within the plaque. Several endogenous and microbial molecules are able to activate macrophages within the plaque, leading to production of inflammatory cytokines, proteases and oxygen radicals [START_REF] Hansson | Mechanisms of disease: Inflammation, Atherosclerosis and Coronary Artery Disease[END_REF]. The anti-inflammatory cytokine, IL-10, is also produced by macrophages within atherosclerotic plaques [START_REF] Mallat | Expression of Interleukin-10 in advanced human atherosclerotic plaques, relation to inducible nitric oxide synthase expression and cell death[END_REF]; since there are a number of lines of evidence suggesting an atheroprotective role for IL -10 [4-7], control of plaque macrophage IL-10 production may be an important determinant of inflammatory load, and hence of progression towards instability and rupture.

Three mammalian orthologs of the Drosophila Tribbles protein (Trb1, Trb2 and Trb3) have recently been identified as intracellular signalling molecules that resemble serine-threonine kinases but lack canonical ATP-binding sites [START_REF] Hegedus | Tribbles: a family of kinase-like proteins with potent signalling regulatory function[END_REF]. They are thought to act principally as adaptor proteins, with a scaffold-like regulatory function in a number of signalling pathways [START_REF] Kiss-Toth | Human tribbles, a protein family controlling mitogen-activated protein kinase cascades[END_REF][START_REF] Hegedus | Tribbles: novel regulators of cell function; evolutionary aspects[END_REF]. Trb3 has been reported to be a negative regulator of PKB signalling in the liver [START_REF] Du | TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver[END_REF]; it also acts as a scaffold to bind the E3 ligase COP1 to acetyl-coenzyme A carboxylase, promoting ubiquitin-dependent degradation of the latter and thus inhibiting fatty acid synthesis [START_REF] Qi | TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism[END_REF]. Trb1 and Trb3 have also been shown to modulate activity of mitogen activated protein kinase (MAPK) cascades by binding MAPKKs [START_REF] Kiss-Toth | Human tribbles, a protein family controlling mitogen-activated protein kinase cascades[END_REF]. In this context, a recent paper has identified Trb1 as a negative regulator of c-jun N-terminal kinase (JNK) activation by MAPKK4 in vascular smooth muscle cells [START_REF] Sung | Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via MAPK signaling pathways[END_REF]. Less is known about the function of mammalian Trb2; it was originally reported as a labile phosphoprotein upregulated by mitogens in dog thyroid cells [START_REF] Wilkin | Characterization of a phosphoprotein whose mRNA is regulated by the mitogenic pathways in dog thyroid cells[END_REF]. More recently it has been identified as a protein upregulated by inflammatory stimuli in myeloid (THP-1) cells [START_REF] Sung | Regulation of expression and signalling modulator function of mammalian tribbles is cell-type specific[END_REF] and also as an oncogene that inactivates the transcription factor CEBP/α and causes acute myelogenous leukaemia [START_REF] Keeshan | Tribbles homolog 2 inactivates C/EBPalpha and causes acute myelogenous leukemia[END_REF].

Here we show that Trb2 mRNA is expressed at high levels specifically in vulnerable regions of human atherosclerotic plaques, and that expression correlates with that of the macrophage marker CD68. Overexpression of Trb2 in human monocyte-derived macrophages in vitro caused specific suppression of IL-10 biosynthesis. These findings suggest a possible role for Trb2 as a mediator of atherosclerotic plaque destabilisation via negative regulation of macrophage IL-10 synthesis.

Methods

Human carotid endarterectomy samples

Human atherosclerotic plaque samples were collected (with informed consent) from patients undergoing carotid endarterectomy. Classification of plaque segments as stable or unstable, and RNA isolation from these segments, was as previously described [START_REF] Papaspyridonos | Novel candidate genes in unstable areas of human atherosclerotic plaques[END_REF].

Peripheral blood mononuclear cells (PBMC) preparation

Fresh blood (50-100 ml, taken into EDTA) was obtained from healthy volunteers (with informed consent) and stored on ice for a maximum of 60 min. PBMCs were isolated under sterile conditions as described [START_REF] Böyum | Separation of leukocytes from blood and bone marrow[END_REF]. Briefly, whole blood was layered on endotoxin-free Histopaque-1077 medium, and PBMCs present at the Histopaque interface, following centrifugation at 400 g for 30 min at 25 °C, were collected and subjected to brief treatment with cell lysis solution (Promega, Madison, WI 53711-5399). After washing in 50 ml PBS, PBMC pellets were gently resuspended in 5 ml PBS. CD14 + cells were quantified by flow cytometry with fluorescein-conjugated anti-CD14 (Beckman Coulter Inc. Fullerton CA 92835).

Establishment of macrophage cultures and LDL treatment

PBMCs were resuspended in RPMI 1640/5 % human AB serum (Sigma) and plated in 12-well/6-well plates at 3 x 10 5 monocytes/ml. After incubation at 37 o C overnight (in 5 % CO2 in a water-saturated atmosphere), non-adherent cells were removed by repeated PBS wash; adhered monocytes were allowed to differentiate into macrophages by culture in RPMI 1640/5 % human AB serum for 2-4 weeks. For LDL treatment macrophages were exposed to different concentrations of oxidised or native human LDL (Intracel, Frederick MD 21701) in serum-free medium for 4-48 hours.

Quantitative real-time PCR

Single-stranded cDNA was synthesized with random hexamer primers using the Superscript First-Strand Synthesis System, according to the manufacturer's instructions (Invitrogen). mRNA expression was quantified using a TaqMan 7900HT Detection System, with either pre-synthesized primers (Applied Biosystems) for Trb1 (Hs00179769_m1), Trb2 (Hs00222224_m1), Trb3 (Hs00221754_m1) and IL-10 (Hs00174086_m1), or as described for CD3 and CD68 [START_REF] Papaspyridonos | Novel candidate genes in unstable areas of human atherosclerotic plaques[END_REF]. Reactions (performed in a MicroAmp Optical 96-well reaction plate) contained: 1x Master Mix, 200 µM each primer, and 100 µM probe in a volume of 25 µl. PCR conditions were 50 ºC for 2 min, 95 ºC for 10 min, 40 cycles of 95 ºC for 15 min/60 ºC for 1 min. Gene expression data for macrophages were analyzed by normalization against 18S rRNA; those for plaque samples were analyzed by normalization against the geometric mean of the expression of GAPDH, β-actin and β-microglobulin as described [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF].

cDNA cloning and production of recombinant Trb2 and Trb3

Human Trb2 and Trb3 open reading frames (ORF) were amplified by PCR using the primer pairs : Trb2: 5'-GAA GTT ATC AGT CGA CAT GAA CAT ACA CAG GTC TAC-3' and 5'-ATG GTC TAG AAA GCT TGC TCA GTT AAA GAA AGG GTC -3'; Trb3: 5'-GAA GTT ATC AGT CGA CAT GCG AGC CAC CCC TCT GGC T-3′ and 5′-ATG GTC TAG AAA GCT TTC CTA GCC ATA CAG AAC CAC-3' respectively. The forward primer had a unique SalI site; the reverse primer was designed with a unique HindIII site. PCR was carried out for 30 cycles of 15 s at 92°C/30 s at 55°C, followed by 1 min at 72°C, with Pfu Turbo DNA polymerase (Stratagene, La Jolla, Ca.). Resulting Trb cDNAs were ligated into the unique SalI and HindIII sites of linearized pDNR-Dual donor vector with the BD In-Fusion Dry-Down PCR cloning kit (BD Biosciences) to make pDNR-Dual/Trb2/3, and sequenced to verify fidelity of the PCR. Macrophages were transiently transfected with Trb2-and Trb3-expressing plasmid (or vector) with lipofectamine (Invitrogen), according to the manufacturer's instructions, 48 hours prior to cell lysis. Adenovirus constructs were prepared by using BD Adeno-X Expression System 2 (BD Biosciences) according to the manufacturer's protocol. The TRB2/3 genes were transferred from pDNR-Dual/TRB2/3 constructs (donor vector) to pLP-Adeno-X Viral DNA (acceptor vector). After digestion with PacI, the recombinant adenoviral plasmid was used to transfect HEK293 cells. These were maintained in EMEM medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, Paisley, UK) at 37 °C in a tissue culture incubator. Cells were seeded at a density of 1-2 × 10 5 cells/ml, and transfected with adenovirus-Trb2 or -Trb3 constructs or acceptor vector alone (AD0) when they had reached 50 -70 % confluence. After 3-4 days, when approximately 90 % of the cells were detached from the plate, both cells and culture supernatant were collected for virus purification. Adenovirus were isolated by using the freeze-thaw method and purified with an Adeno-X virus purification kit (BD Biosciences); virus titer was determined by using the BD Adeno-X Rapid Titer kit (BD Biosciences).

IgG-OVA complex was prepared by combining IgG fraction of rabbit antiserum to chicken egg albumin (Capple/MP Biomedicals) and OVA (Worthington Biochemical) at final concentrations of 1.5 mg/ml IgG, and 45ug/ml OVA. Where appropriate, macrophages were infected (in serum-free medium, exchanged for fresh serum-containing medium 4 hours after infection) with purified adenovirus-TRB 2/3 (or AD0) 48 hours before stimulation with IgG-OVA complex (40μl/ml) and E. coli lipopolysaccaride (LPS: Sigma, 80ng/ml, added 10 minutes after the IgG-OVA complex), which was for 24 hours (in serum-containing medium). Both cells (for Taqman RT-PCR analysis and Western blot) and culture supernatants (for IL-10 ELISA, measured using an R&D Systems kit according to the manufacturer's instructions) were collected following a further 24 hours incubation.

Anti-TRB2/3 antibody generation

Polyclonal antibodies were developed by immunizing rabbits with synthetic peptides (KLH-coupled) corresponding to amino acids 11 -27 for TRB2 and 314 -333 for TRB3 (Cambridge Research Biochemicals, Billingham , Cleveland, UK). Antibodies were purified using a Protein A immunoaffinity column (Cambridge Research Biochemicals).

Western Blotting 30 µg protein/well of macrophage lysates were separated on NuPAGE 4-12% Bis-Tris gels (Invitrogen). Gels were blotted by using the XCell II blot module (Invitrogen) onto nitrocellulose, and membranes were probed (following blocking for 30 minutes in 5 % non-fat milk) with anti-human-TRB2 or Trb3 antibody (final concentration 0.7 μg/ml) and human-GAPDH antibody (Abcam, Cambridge, UK; 1:2500 dilution) overnight. HRP conjugated secondary antibodies (Abcam) at a dilution of 1:10,000 were used to detect bound primary antibodies; blots were developed with Supersignal West Dura Extended Duration Substrate (Perbio, Cheshire, UK) and Images were captured using a Chemigenius imaging system (Syngene, Cambridge, UK).

Statistics

Data were compared for statistically significant differences by the Wilcoxon Matched-Pairs Signed-Ranks Test. In situations where more than one control was included in the experimental design, the test condition result was compared directly to that for each of the controls in a pairwise manner (since possible differences between controls groups were not of interest). To assess correlations, Rank Correlation coefficients were calculated with associated p-values.

Results and Discussion

Expression of Trb mRNAs in vulnerable regions of human carotid plaques

As part of a wider screen for markers of atherosclerotic plaque vulnerability [START_REF] Papaspyridonos | Novel candidate genes in unstable areas of human atherosclerotic plaques[END_REF] levels of mRNA for Trb1, Trb2 and Trb3 isolated from regions of human carotid plaques classified as unstable by macroscopic examination [START_REF] Papaspyridonos | Novel candidate genes in unstable areas of human atherosclerotic plaques[END_REF] were compared to those from regions classified as stable (using an intra-plaque comparison where each measurement in an unstable region was paired with one in a stable region of the same plaque explant). The results are shown in Figure 1; Table 1 gives correlation coefficients (and associated statistical significance) between mRNA levels of the three Trbs and those for macrophage and T cell markers for the screen. Correlations between mRNA for CD68 and those for some known macrophage proteins that were regulated in vulnerable plaque regions were observed in the wider screen [START_REF] Papaspyridonos | Novel candidate genes in unstable areas of human atherosclerotic plaques[END_REF], suggesting that this may be a useful way of assigning cell type-specific expression to regulated mRNA species. We found a marked increase in Trb2 expression in unstable compared to stable plaque regions (more than 8-fold regulation, p <0.05) and a significant increase in Trb1 expression (1.7 fold regulation, p < 0.0001) but no significant difference in Trb3 mRNA levels. A possible role for Trb1 in atherosclerosis has been suggested by recent observations that Trb1 mRNA is upregulated in coronary arteries from ischaemic heart disease patients, and that enhanced Trb1 expression in vascular smooth muscle cells inhibits proliferation via specific inhibition of the JNK signalling pathway [START_REF] Sung | Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via MAPK signaling pathways[END_REF]. Our data showing that Trb1 mRNA levels are raised in vulnerable regions of human carotid plaques are also suggestive of a specific role in atherosclerosis, although the positive correlations between (mRNA) levels of Trb1 and both CD3 and CD68 in the plaque segments tested (Table 1) suggest that Trb1 may be mainly expressed by T cells and macrophages within the plaque. Whilst the significant regulation of Trb1 mRNA in vulnerable plaque regions supports a proposed role for the protein in atherosclerosis, the dramatic up-regulation of Trb2 mRNA observed raises the possibility of a hitherto unexplored regulatory function for Trb2 in plaque progression and destabilisation. Further, there is a strong, highly statistically significant correlation observed between expression of Trb2 and CD68 in carotid plaque segments (Table 1). Whilst it is possible that the association is indirect (elevated Trb2 levels in another cell type promoting recruitment of macrophages to the vulnerable plaque region), the finding suggests that macrophages are the cell type primarily responsible for the enhanced Trb2 mRNA levels in unstable plaque regions. The level of Trb2 regulation observed is substantially higher than would be predicted on the basis of higher macrophage content in unstable regions alone [START_REF] Papaspyridonos | Novel candidate genes in unstable areas of human atherosclerotic plaques[END_REF], suggesting some specific Trb2 regulation in unstable plaque regions.

Regulation of monocyte-derived macrophage Trb mRNA expression in response to administration of oxidised LDL

In order to test the possible relevance of macrophage Trb expression in atherosclerosis we examined the regulation of Trb mRNA expression in human monocyte-derived macrophages by oxidised LDL. LDL oxidation is considered a key event in atherogenesis; uptake of oxidized LDL (oxLDL) by macrophages results in foam cell formation and ultimately development of the lipid rich, necrotic core of the lesion [START_REF] Bobryshev | Monocyte recruitment and foam cell formation in atherosclerosis[END_REF], whilst lipid products of LDL oxidation act as inflammatory mediators within the plaque that may contribute to instability [START_REF] Hashmi | Role of interleukin-17 and interleukin-17-induced cytokines interleukin-6 and interleukin-8 in unstable coronary artery disease[END_REF]. Regulation of macrophage Trb2 mRNA by oxLDL would therefore provide evidence of specific expression in foam cells, and of a possible proinflammatory role in plaque destabilisation. We found that expression of all 3 Trb mRNAs was altered by 24 hour incubation with oxLDL in a concentration-dependent manner (Figure 2A). Macrophage treatment with 50 μg (total protein)/ml of oxLDL in serum free conditions significantly reduced Trb1 mRNA levels (by over 70 %, p < 0.01), whilst significantly enhancing levels of Trb2 (over 7.5-fold; p < 0.01) and Trb3 (over 4.5-fold, p < 0.01). The time-dependency of Trb mRNA regulation by this concentration of oxLDL is shown in Figure 2 (B, C andD). With all 3 Trb mRNAs the effect of oxLDL treatment was the reverse of that observed with serum withdrawal alone ('control' bars in Figure 2), and in all cases the effect of treatment with non-oxidised LDL (nLDL) was intermediate (although much closer to the control value in the cases of Trb2 and Trb3). As with the carotid plaque analysis, Trb2 mRNA was more variable and more highly regulated than Trb1 or Trb3. These results therefore identify Trb2 as a strongly regulated macrophage protein, and suggest that it may be expressed at high levels by foam cells within atherosclerotic lesions. In contrast, the small negative regulation by oxLDL suggests that macrophage Trb1 expression is less highly regulated; enhanced levels of Trb1 mRNA (Figure 1) may therefore reflect the higher leukocyte content in unstable plaque regions (rather than specific regulation).

Strong up-regulation of Trb2 mRNA in macrophages, specifically in vulnerable regions of carotid plaque tissue in vivo, and in response to oxLDL treatment in vitro, raises the possibility that Trb2 regulation of macrophage signalling pathways may play a role in plaque progression and destabilisation. Whilst comparatively little is known about regulation of mammalian Trb2 expression, a single report shows that mRNA levels are up-regulated in response to IL-1 treatment in a myeloid cell line, THP-1 [START_REF] Sung | Regulation of expression and signalling modulator function of mammalian tribbles is cell-type specific[END_REF] suggesting that Trb2 may be involved in myeloid cell regulation of inflammatory gene expression. Since inflammation within the plaque is a key determinant of vulnerability, and ultimately rupture [START_REF] Hansson | Mechanisms of disease: Inflammation, Atherosclerosis and Coronary Artery Disease[END_REF][START_REF] Hashmi | Role of interleukin-17 and interleukin-17-induced cytokines interleukin-6 and interleukin-8 in unstable coronary artery disease[END_REF][START_REF] Heeneman | Drug-induced immunomodulation to affect the development and progression of atherosclerosis: a new opportunity? Expert[END_REF], we sought to determine directly the effects of Trb2 expression on synthesis and secretion of macrophage inflammatory mediators to provide clues to possible actions of the protein within plaques.

Regulation of macrophage IL-10 mRNA levels by Trb2 overexpression

The mammalian Trb proteins have been shown to play important roles in transcriptional regulation of gene expression in a number of cellular models with possible relevance to human pathologies [START_REF] Kiss-Toth | Human tribbles, a protein family controlling mitogen-activated protein kinase cascades[END_REF][START_REF] Hegedus | Tribbles: novel regulators of cell function; evolutionary aspects[END_REF][START_REF] Qi | TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism[END_REF][START_REF] Keeshan | Tribbles homolog 2 inactivates C/EBPalpha and causes acute myelogenous leukemia[END_REF][START_REF] Kato | TRB3 modulates C2C12 differentiation by interfering with Akt activation[END_REF][START_REF] Selim | Fibrates upregulate TRB3 in lymphocytes independent of PPAR alpha by augmenting CCAAT/enhancer-binding protein beta (C/EBP beta) expression[END_REF]. The specific up-regulation of macrophage Trb2 in both unstable carotid plaque regions and cells treated with oxidised LDL in vitro suggested that expression of Trb2 in atherosclerotic foam cells may contribute to plaque inflammation and destabilisation, in turn suggesting that it may be involved in (transcriptional) regulation of inflammatory mediators. To test this idea we cloned cDNA for human Trb2 (and Trb3 as a control) into a mammalian expression vector and measured the effect of transfection on secretion of cytokines from monocyte-derived macrophages. We were unable to demonstrate any effect (different from transfect ion of vector alone) on secretion of the pro-inflammatory cytokines IL-1β, IL-6 or IL-8 (data not shown). However, TaqMan RT-PCR measurements in transfected macrophages demonstrated that Trb2 expression caused a specific reduction in macrophage IL-10 mRNA levels measured after 24 hours relative to cells expressing Trb3 (p < 0.05) or those transfected with vector alone (p < 0.05) (Figure 3A). Trb2 overexpression caused no enhancement of macrophage apoptosis over this time period (data not shown). It is interesting to note that, concomitant with enhanced levels of Trb2 mRNA in macrophages (Figure 2D), IL-10 mRNA levels are also reduced by oxLDL treatment in vitro (Figure 3B).

We were unable to detect enhancement of macrophage Trb2 and Trb3 expression at the protein level (by Western blot: not shown) using the pDNR-Dual expression system, and we were also unable to demonstrate regulation of IL-10 at the protein level, since the amounts secreted by macrophages in these experiments were below levels of detection for the ELISA kit used (data not shown). In order to overcome these difficulties we used an adenoviral system to express high levels of Trb2 and Trb3 in macrophages, and measured the effects of overexpression on IL-10 secretion stimulated by treating macrophages with aggregated IgG and lipopolysaccharide (IgG/LPS). Titration of virus multiplicity of infection (MOI) used to infect macrophage cultures showed that both anti-Trb2 and anti-Trb3 antibodies detected recombinant protein at MOI = 200 (by Western blot, Figure 4C), and also that the expression of the two mRNAs in macrophages infected at MOI = 200 was similar (Figure 4D). The relative expression data derived from the threshold cycle values shown in Figure 4D (which are approximately proportional to negative log 2 of specific mRNA concentration) indicate that in non-infected cells Trb3 mRNA was approximately 5.7 times more abundant than Trb2 mRNA, and that in infected cells expression of Trb2 and Trb3 mRNA was approximately equivalent (representing 2600-fold enhancement and 460-fold enhancement over uninfected cells for Trb2 and Trb3 respectively). Figure 4 (A andB) show that, both at the protein and mRNA level, overexpression of Trb2 in adenovirus-infected macrophages specifically inhibited IgG/LPS-stimulated IL-10 biosynthesis, whilst overexpression of Trb3 did not have this effect. These data thus confirm and extend the observations presented in Figure 3. Thus, our data indicate that specific suppression of IL-10 is a potentially pro-inflammatory consequence of Trb2 expression in macrophages and, together with the observation of enhanced Trb2 expression associated with macrophages in vulnerable regions of human carotid plaques, suggest a potentially pro-atherogenic role for the protein in vivo. An atheroprotective effect of IL-10 has been demonstrated in animal models [START_REF] Caligiuri | Interleukin-10 deficiency increases atherosclerosis, thrombosis, and low-density lipoproteins in apolipoprotein E knockout mice[END_REF][START_REF] Liu | Inhibition of atherogenesis in LDLR knockout mice by systemic delivery of adeno-associated virus type 2-hIL-10[END_REF], and potentially anti-atherogenic actions have been shown in vitro [START_REF] Halvorsen | Interleukin-10 enhances the oxidized LDL-induced foam cell formation of macrophages by antiapoptotic mechanisms[END_REF][START_REF] Kamimura | Interleukin-10 suppresses tissue factor expression in lipopolysaccharide-stimulated macrophages via inhibition of Egr-1 and a serum response element/MEK-ERK1/2 pathway[END_REF][START_REF] Rubic | Downregulated CD36 and oxLDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10[END_REF]. A recent study has also demonstrated genetic variation in the human IL-10 promoter region to be associated with vascular events [START_REF] Trompet | Genetic variation in the interleukin-10 gene promoter and risk of coronary and cerebrovascular events: the PROSPER study[END_REF]. These findings support the view that suppression of macrophage IL-10 biosynthesis within the plaque could contribute to plaque destabilisation, and that this may be a specific consequence of high macrophage Trb2 expression within the plaque. The precise mechanism whereby Trb2 may act to suppress IL-10 synthesis remains to be determined. One possible pathway is suggested by the recent finding that expression of Trb2 in a myeloid cell line can inactivate the transcription factor C/EBPα [START_REF] Keeshan | Tribbles homolog 2 inactivates C/EBPalpha and causes acute myelogenous leukemia[END_REF]; previous studies have shown that THP-1 cell IL-10 transcription is (at least in part) controlled by C/EBPα binding to CCAAT motifs in the promoter region [START_REF] Brenner | cAMP-induced Interleukin-10 promoter activation depends on CCAAT/enhancer-binding protein expression and monocytic differentiation[END_REF].

In summary, our results demonstrate strongly upregulated Trb2 expression (that correlates with expression of the macrophage marker, CD68) in regions of human atherosclerotic plaque classified as unstable by macroscopic examination (following carotid endarterectomy), and specific suppression of macrophage IL-10 expression as a consequence of Trb2 expression in vitro. These data identify macrophage Trb2 expression as a potentially important determinant in progression of atherosclerosis, adding another possible role in human pathophysiology to the growing list ascribed to members of this recently identified family of signalling molecules. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 Figure 1. Relative expression of Trb mRNAs in stable and unstable regions of human carotid plaques. Results show mRNA levels measured in unstable segment, relative to those in stable segment of the same plaque explant. Mean + SEM n = 16 for Trb1 and Trb3, n = 10 for Trb2; p < 0.05 (*) or p < 0.0001 (**) for unstable vs stable plaque segments.

Figure 2 .

 2 Figure 2. Regulation of macrophage Trb mRNA levels by treatment with oxidised LDL in serumfree medium. A: dose dependency of Trb1, Trb2 and Trb3 mRNA regulation by oxLDL (expressed relative to untreated cells) after 24 hours treatment. Mean + SEM (n = 5 for 5 μg and 10 μg treatment, n = 6 for 25 μg and 50 μg treatment); p < 0.05 (*) or p < 0.01 (**) for Trb1, Trb2 or Trb3 expression in oxLDL-treated vs untreated cells; B, C, D: time course of regulation by serum withdrawal alone ('control'), or by treatment with 50 μg (total LDL protein)/ml nLDL or oxLDL (expressed relative to cells at time = 0 hours) for Trb1, Trb2 and Trb3 respectively. Mean + SEM (n = 7) in all cases; p < 0.05 for oxLDL treatment vs control (*) or native LDL ( + ).

Figure 3 .

 3 Figure 3. A: Regulation of macrophage IL-10 mRNA levels by expression of recombinant Trb2 and Trb3: mRNA levels expressed relative to untransfected cells from same experiment, mean + SEM (n = 6) in all cases; p < 0.05 for Trb2 transfected cells vs vector (*) or Trb3 ( + ) transfected cells. B: regulation of macrophage IL-10 mRNA levels by treatment with oxidised LDL: mRNA levels expressed relative to cells at time = 0 hours. Mean + SEM (n = 6) in all cases; p < 0.05 for oxLDL treatment vs control (*) or native LDL ( + ).

Figure 4 .

 4 Figure 4. Suppression of IL-10 secretion (A) and mRNA levels (B) by adenovirus-mediated Trb2 overexpression in aggregated IgG/LPS-treated macrophages. Mean + SEM (n = 9 for protein measurements, n =.7 for mRNA): p < 0.05 (*), p < 0.005 (***) for Trb2 adenovirus-infected cells ('Trb2') vs non-infected cells ('IgG/LPS'); p < 0.05 ( + ), p < 0.01 ( ++ ) for Trb2 adenovirus-infected cells vs control adenovirus-infected cells ('AD0'); p < 0.05 ( ^), p < 0.01 ( ^^) for Trb2 adenovirusinfected cells vs Trb3 adenovirus-infected cells ('Trb3'). C: Western blots of anti-Trb2 (left) and anti-Trb3 (right) detection of proteins in adenovirus-infected macrophages. 'AD0': control adenovirus-infected macrophages (MOI = 200); '100', '200': MOI used to infect macrophages with Trb2-(left) or Trb3-(right) expressing adenovirus. Approximate positions of molecular weight markers (centre) and Trb2, Trb3 and GAPDH indicated. D: Relative expression of Trb2 and Trb3 mRNA in non-infected ('control cells') and adenovirus-infected ('Trb2/3-expressing') macrophages (MOI = 200) as detected by TaqMan RT-PCR measurements (threshold cycle is proportional to negative log 2 mRNA concentration; see text for details). Mean + SEM (n = 7).

Figure 1 12

 1 Figure 1

13 Clinical

 13 Figure 2

14 Clinical

 14 Figure 3

Figure 4 IL 15 Clinical

 415 Figure 4

Table 1 .

 1 Rank Correlation Coefficients (and Associated Statistical Significance) Between Levels of Trb mRNAs and those of Leukocyte Markers in Human Carotid Plaque Tissue

		Trb1	Trb2	Trb3
	CD3	0.5213 (P < 0.005)	0.3808 (NS)	0.0323 (NS)
	CD68	0.5244 (P < 0.005)	0.8146 (P < 0.0001)	0.3195 (NS)
	NS: not significant

Clinical Science Immediate Publication. Published on 21 Jul 2008 as manuscript CS20080058 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080058 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Clinical Science Immediate Publication. Published on 21 Jul 2008 as manuscript CS20080058 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080058 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Clinical Science Immediate Publication. Published on 21 Jul 2008 as manuscript CS20080058 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080058 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We thank Joanna Ward and Layla Whitworth for excellent technical assistance, and Sam Miller for very helpful advice on statistics.