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Am J Physiol Endocrinol Metab 290: E92-E102, 2006. First published August 23, 2005; doi:10.1152/ajpendo.00133.2005
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Activation of the 12-lipoxygenase and signal transducer and activator of transcription pathway during neointima formation in a model of the metabolic syndrome

Hong Pei, Jiali Gu, Pushpa-Rekha Thimmalapura, Angeles Mison, and Jerry L. Nadler

Division of Endocrinology, Department of Internal Medicine, University of Virginia, Charlottesville, Virginia

Submitted 23 March 2005 ; accepted in final form 17 August 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin resistance (IR) is associated with an increased risk of cardiovascular diseases. The obese Zucker rat (ZR) is a model of IR that shows markedly increased insulin and triglyceride concentrations without major changes in glucose. In this study, we evaluated the response of obese and lean ZR to carotid balloon injury and determined potential mechanisms and treatments. The neointima-to-media ratio of obese ZR was greater than that of lean ZR, starting at 14 days after injury, and persisted until at least day 30. An enhanced inflammatory response to balloon injury in the obese ZR was reflected by significantly higher ED1-positive macrophage cells in the injured vessel wall compared with that in lean ZR at 3, 7, and 14 days after balloon injury. Inflammatory mediators 12-lipoxygenase (12-LO) and STAT4 were studied in neointimal lesions. Expression of 12-LO RNA was increased beginning at day 7 and showed increases of 4.3-fold on day 14 and 7-fold on day 30 in obese ZR compared with lean animals. Staining of phosphorylated STAT4 (PSTAT4), the activated form of STAT4, in lesions from obese ZR was also increased compared with that in leans. We tested the effects of a novel anti-inflammatory agent, lisofylline (LSF), in the obese ZR. LSF markedly reduced neointimal formation in the obese ZR. LSF also reduced monocyte/macrophage infiltration into the vessel wall and the activation of PSTAT4. These studies suggest both the presence of an exaggerated injury response in the insulin-resistant obese ZR model and that inflammation plays a major role in mediating neointimal growth.

lisofylline; Zucker rat

INSULIN RESISTANCE (IR) is associated with an increased risk of cardiovascular diseases. Studies in both humans (21, 23) and animals (24, 31) show that, after balloon angioplasty, one sees a greater restenosis rate in the insulin-resistant state. The mechanisms of the pathogenesis of exaggerated injury response in diabetes and the metabolic syndrome are not fully understood. However, evidence indicates that the inflammatory response to vascular injury plays a critical role in restenosis in diabetes. Recent studies have shown that activation of inflammatory genes after vascular injury, including selectins such as P-selectin (41) and cell adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (29), results in leukocyte infiltration into the vessel wall. The role of leukocytes in restenosis is likely caused by their capacity to release numerous growth factors, cytokines, and enzymes (15). These factors could further damage the vessel wall, amplify the effects of platelets, cause vasoconstriction, and stimulate further leukocyte adhesion and smooth muscle cell proliferation and migration. Preventing inflammatory cell accumulation by inhibiting inflammatory genes has resulted in reduced neointimal formation after balloon angioplasty (5, 9, 16, 22, 26, 33, 41). We hypothesize that an enhanced inflammatory response to vascular injury might contribute to the excess neointimal formation in insulin-resistant states and that blockade of particular inflammatory pathways could provide a novel therapeutic approach.

It has been shown that 12-lipoxygenase (12-LO) and inflammatory lipids are associated with monocyte binding, oxidation of low-density lipoprotein, smooth muscle cell growth and migration, and inflammatory kinase activation (18). Targeted deletion of 12-LO reduces atherosclerosis in mouse models (10).

Lisofylline (LSF) is a novel anti-inflammatory compound that reduces oxidized lipid signaling and Th-1-mediated inflammatory cytokine production (39). LSF has been shown to inhibit the gene expression and production of TNF, IL-1{beta}, IL-6, macrophage inflammatory protein, and interferon-{gamma} (6, 30). LSF also inhibits IL-12 signaling and IL-12-mediated Th1-type T cell differentiation (3) and prevents the development of autoimmune allergic encephalomyelitis and type 1 diabetes in an animal model (2). One of the main targets of LSF is the blockade of (STAT4) phosphorylation (39). STAT4 is an important transcription factor found in immune cells and the vessel wall, and new data show that STAT4 can contribute to inflammatory gene induction such as interferon-{gamma} and monocyte chemotactic protein-1 (MCP-1) (37).

In this study, we examined the role of the inflammatory response for neointima formation in the injured vessel wall by using the obese ZR model of insulin resistance. We evaluated several candidates for the inflammatory response, including 12-LO and activated STAT4. In addition, we evaluated the effects of LSF on inflammation and neointimal formation in obese ZR after balloon angioplasty.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH Publication No. 85-23, revised 1985) and approved by the Institutional Animal Care and Use Committee at the University of Virginia.

Obese and lean ZRs (female, 10 wk old, from Charles River) were anesthetized with ketamine (80 mg/kg) and xylazine (5 mg/kg). The left common carotid arteries were injured by balloon angioplasty with a 1.8-F PTCA balloon catheter (19) (SciMed Life System, Maple Grove, MN). In brief, the left external carotid artery was tied off distally, and via transverse arteriotomy a 1.8-F PTCA balloon catheter with a 2-cm-long, 1.5-mm diameter balloon was advanced into the common carotid artery. The balloon was then inflated to a pressure of 1.5 atmospheres and passed back and forth throughout the common carotid artery three times for 18 s to ensure uniformity of the extent of the injury. The animals were killed on day 3, 7, 14, or 30 after initial surgery, and the injured left and uninjured right common carotid arteries were harvested for histological, biochemical, or molecular analysis. Body weights of obese and lean ZR were obtained at day 14 after balloon injury. Blood was collected in heparinized tubes 14 days after the injury at the time the animals were killed through heart puncture. The rats were in a fed state. Plasma glucose, cholesterol, and triglycerides were determined with an Olympus analyzer by the Pathology Laboratory in the Medical Center at the University of Virginia. Insulin levels were detected by RIA, with an antibody made specifically against rat insulin (Linco, St. Charles, MO).

To test the effects of LSF [1-(5-R-hydroxyhexyl)-3,7-dimethylxanthine] on neointima formation, one group of obese ZR received LSF (25 mg/kg ip, twice daily) starting at 5 days before balloon angioplasty and continued until the anmimals were killed (14 days after carotid balloon injury). LSF was provided by Dr. Jack Singer (Cell Therapeutic, Seattle, WA).

Morphometric Analysis

The injured arteries (14 and 30 days after balloon angioplasty) were fixed in situ by perfusion with PBS containing 4% paraformaldehyde and 2% glutaraldehyde through the aortic cannula. These fixed tissues were paraffin embedded, and 5-µm cross sections were stained with hematoxylin-eosin. Morphometric analysis was conducted using an Olympus microscope, a digital camera, and image software (Image Pro, Silver Spring, MD). The area of the vessel within the external elastic lamina (EEL), the internal elastic lamina (IEL), and the lumen (luminal area) was measured. The intimal area (IEL-luminal area) and the medial area (EEL-IEL) were calculated. Results are expressed as ratios of intimal-to-medial areas.

Immunohistochemistry

Immunohistochemical (IHC) staining was performed using our standard protocol (7, 19). In brief, 5-µm-thick paraffin-embedded tissue sections were deparaffinized and rehydrated in graduated alcohol to distilled water. Antigens were retrieved using a high-temperature antigen-unmasking technique (Antigen Unmasking Solution, Vector Laboratories, Burlingame, CA). The endogenous peroxidase was quenched using 0.5% H2O2 in methanol (Fisher Scientific) for 30 min at room temperature. The sections were then incubated for 30 min at room temperature with diluted normal blocking serum (Vector Laboratories) and stained at 4°C overnight with different primary antibodies, including 1) Ki67, a monoclonal antibody for rat-proliferating cells (DAKO, Carpinteria, CA); 2) ED1, a monoclonal antibody used to detect rat monocytes/macrophages (Serotec, Raleigh, NC); 3) a primary antibody to leukocyte 12-LO (raised to a peptide derived from the sequence of the porcine leukocyte 12-LO) (17, 40); and 4) PSTAT4, a polyclonal antibody for phosphorylated STAT4 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by a biotinylated second antibody (Vector Laboratories) for 30 min at room temperature and an avidin-biotin-peroxidase complex (ABC; Vector Laboratories) for 30 min at room temperature. Finally, the sections were developed with a diaminobenzidine substrate kit (Vector Laboratories) and counterstained using hematoxylin. IHC results were evaluated quantitatively by viewing sections under a light microscope and determining the percentage of stained cells relative to the total number in four different fields.

Detection of 12-LO mRNA by RT-Competitive PCR

This was performed according to our previously reported method (1, 7, 8, 19).

RNA preparation and cDNA synthesis. The sections of the injured carotid arteries were snap-frozen in liquid nitrogen and then stored at –80°C. Frozen tissue samples were disrupted and homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Total RNA was isolated according to the manufacturer’s instructions, treated with DNase I, and then reverse transcribed to cDNA using SuperScript RNaseH-RT (Invitrogen) (8).

Preparation of competitor DNA for rat leukocyte 12-LO. The competitor DNA used as the internal standard for the competitive PCR was designed so that a portion of the sequence was deleted, resulting in a competitor PCR-generated fragment that could be easily distinguished by size. The 12-LO competitor DNA was prepared as described (1, 7, 8).

Quantitation of 12-LO mRNA by competitive PCR. The quantitative PCR assay was performed as described previously (1, 7, 19), with some modifications. The sequence of PCR primers and the conditions used in the PCR were the same as described previously (1, 7, 19). PCR products of competitor DNA and 12-LO cDNA were separated by 5% polyacrylamide gel and stained with ethidium bromide and photographed. The negatives of ethidium bromide-stained gels were scanned with an AlphaImager 2200 analysis system (Alpha Innotech, San Leandro, CA). To confirm the specificity of the PCR amplication, the 12-LO cDNA fragment was isolated and sequenced. Quantitation of the bands was done by scanning densitometry. The ratio of the added internal standard DNA to the target 12-LO cDNA was then determined.

Data Analyses

All data are expressed as means ± SE. Experimental groups were compared using the unpaired Student’s t-test (for 2 groups) or ANOVA with Dunnett’s posttest (for multiple groups) with the use of Prism software (GraphPad, San Diego, CA). A value of P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Body Weight and Metabolic and Hormonal Features of Obese and Lean ZR

Although obese and lean rats had similar glucose levels, body weight, insulin, and triglyceride levels were significantly higher in obese rats. Short-term use of LSF did not change body weight or metabolic or hormonal features of the obese ZR (Table 1).


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  		Table 1. Body weight and metabolic, and hormonal features of lean and obese Zucker rats

 
Neointimal Formation and Cell Proliferation Responses to Balloon Angioplasty in Obese and Lean ZR

Neointimal formation was found in the injured arteries 7 days after balloon injury in both obese and lean rats. There was a significant difference in the neointimal-to-medial ratios when obese rats are compared with lean rats at day 14 (obese vs. lean, 0.88 ± 0.06 vs. 0.58 ± 0.05, n = 8, P < 0.05) and day 30 (obese vs. lean, 1.37 ± 0.12 vs. 0.89 ± 0.14, n = 8, P < 0.05; Fig. 1A). Representative vessel sections are shown in Fig. 1B.



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  		Fig. 1. A: Zucker obese rats show significantly increased intima-to-media ratios vs. Zucker lean rats at 14 and 30 days after balloon angioplasty. B: representative light microscopy of cross section of injured carotid arteries (hematoxylin-eosin staining). *P < 0.05 vs. obese rats. Bar = 50 µm.

 
The percentage of proliferating cells (Ki67 positive) was significantly higher in obese rats compared with lean rats at 3 days (5.68 ± 0.63 vs. 3.33 ± 0.41, P < 0.05), 7 days (12.34 ± 1.13 vs. 5.08 ± 0.59, P < 0.01), and 14 days (4.97 ± 0.51 vs. 2.91 ± 0.30, P < 0.05) after balloon injury (Fig. 2A). Representative stained sections are shown in Fig. 2B.



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  		Fig. 2. A: percentage of Ki67-positive cells 3, 7, and 14 days after balloon injury in obese and lean rats. B: representative light microscopy of cross section of injured carotid arteries [Ki67 immunohistochemical (IHC) staining]. *P < 0.05; **P < 0.01 vs. obese rats. Bar = 50 µm.

 
Increased 12-LO Expression After Balloon Injury in Obese ZR

Leukocyte 12-LO expression was examined by competitive PCR and immunostaining. Balloon injury induced upregulation of 12-LO mRNA in both obese and lean rats. However, there was significantly greater 12-LO expression in obese rats beginning at 7 days (data not shown). The increase in 12-LO RNA expression was 4.3-fold higher on day 14 and 7-fold higher on day 30 (P < 0.01) compared with lean rats (Fig. 3A). Immunostaining for 12-LO also revealed stronger staining of 12-LO protein in obese rats compared with lean rats (Fig. 3B).



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  		Fig. 3. 12-Lipoxygenase (12-LO) expression in carotid artery tissue. A, top: representative gel staining of competitive RT-PCR products from carotid artery tissue sections after being injured for 7, 14, and 30 days. Top band is 12-LO; bottom band is the competitor. The relative efficiency of cDNA synthesis for each sample was confirmed by comparison of PCR products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification. Bottom: bar graph represents image quantification from 3 experiments and reveals significant increases in 12-LO mRNA. B: representative light microscopy of cross section of injured carotid arteries 30 days after balloon injury (12-LO IHC staining).

 
Enhanced Inflammatory Response to Balloon Injury in Obese ZR

ED1 is a monoclonal antibody that stains rat monocytes-macrophages. We therefore used this reagent to evaluate the presence of these inflammatory cells in the vessel wall. In the uninjured artery, ED1-positive staining cells were absent (data not shown). The percentage of ED1-positive cells was significantly higher in injured vessels from obese rats compared with lean rats starting at day 3 (15.36 ± 1.15 vs. 7.53 ± 0.83, P < 0.01), day 7 (8.21 ± 0.81 vs. 3.55 ± 0.57, P < 0.05), and day 14 (5.23 ± 0.69 vs. 2.91 ± 0.26, P < 0.05; Fig. 4A). In addition, ED1-positive stained cells were still detected 30 days after balloon injury in obese rats but not in lean rats (Fig. 4B).



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  		Fig. 4. A: percentage of ED1-positive cells in injured carotid arteries at 3, 7, and 14 days after balloon injury in obese and lean rats. B: representative light microscopy of cross section of injured carotid arteries (ED1 IHC staining). At 30 days, there are monocytes/macrophages in injured carotid artery in obese rats but not in lean rats. *P < 0.05; **P < 0.01 vs. obese rats. Bar = 50 µm.

 
STAT4 is a transcription factor that is linked to chronic inflammation and autoimmunity. Recent data suggest that STAT4 is present in vascular cells and leads to the induction of proinflammatory genes, including MCP-1. We therefore evaluated whether carotid injury can induce activation of STAT4 in lean or obese rats. We found that levels of PSTAT4, the activated form of STAT4, in the injured vessels were significantly higher in obese rats compared with lean rats at 3 days (41.23 ± 5.23 vs. 7.22 ± 2.23), 7 days (29.85 ± 6.18 vs. 5.32 ± 1.47), and 14 days (8.63 ± 1.17 vs. 1.25 ± 0.15) after balloon injury (P < 0.01; Fig. 5A). Representative stained sections showing PSTAT4 are shown in Fig. 5B.



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  		Fig. 5. A: percentage of phosphorylated (P)STAT4-positive cells in injured carotid arteries at 3, 7, and 14 days after balloon injury in obese and lean rats. B: representative light microscopy of cross section of injured carotid arteries (PSTAT4 IHC staining). **P < 0.01 vs. obese; ***P < 0.001 vs. obese. Bar = 50 µm.

 
Effect of LSF on Neointima Formation, Cell Proliferation, and Inflammatory Response After Balloon Injury

LSF is a novel anti-inflammatory compound that reduces interleukin-12 signaling and STAT4 phosphorylation. We therefore used LSF as a tool to evaluate the role of these inflammatory pathways in the injury response in the obese IR rat. The LSF-treated obese ZR had significantly reduced neointimal formation compared with obese rats treated with vehicle alone (0.88 ± 0.06 vs. 0.35 ± 0.05, P < 0.01, n = 8; Fig. 6A). A representative histological section at 14 days after injury is shown in Fig. 6B. LSF also markedly inhibited cell proliferation after balloon injury in the obese rats. LSF significantly reduced the number of Ki67-positive staining cells 14 days after injury in obese rats (4.97 ± 0.51 vs. 1.27 ± 0.15, P < 0.001; Fig. 7A) and as shown in a representative section (Fig. 7B). Furthermore, LSF decreased ED1-positive staining cells (5.23 ± 0.69 vs. 1.69 ± 0.35, P < 0.01; Fig. 8) and markedly inhibited PSTAT4 expression (8.63 ± 1.17 vs. 1.38 ± 0.22, P < 0.001; Fig. 9) in obese ZR after balloon injury.



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  		Fig. 6. A: Lisofylline (LSF) significantly reduced intima-to-media ratios in obese rats 14 days after balloon angioplasty. B: representative light microscopy of cross section of injured carotid arteries (HE staining). ***P < 0.001 vs. obese. Bar = 50 µm.

 


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  		Fig. 7. A: LSF inhibited cell proliferation in vivo. Percentage of Ki67-positive cells 14 days after balloon injury in obese rats treated with LSF was significantly lower than the untreated group. B: representative light microscopy of cross section of injured carotid arteries (Ki67 IHC staining). ***P < 0.001 vs. obese. Bar = 50 µm.

 


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  		Fig. 8. A: LSF significantly reduced monocyte/macrophage infiltration 14 days after balloon injury. Percentage of ED1-positive cells in the LSF-treated group was significantly lower than in the untreated group 14 days after balloon injury in obese rats. B: representative light microscopy of cross section of injured carotid arteries (ED1 IHC staining). **P < 0.01 vs. obese. Bar = 50 µm.

 


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  		Fig. 9. A: LSF reduced PSTAT4 expression. Percentage of PSTAT4-positive cells 14 days after balloon injury in obese rats treated with LSF was significantly lower than in the untreated group. B: representative light microscopy of cross section of injured carotid arteries (PSTAT4 IHC staining). ***P < 0.001 vs obese. Bar = 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Patients with insulin resistance and type 2 diabetes continue to show relatively high rates of restenosis after angioplasty and stent placement (4, 13, 34, 38). In the present study, we have demonstrated that obese ZR, a model of obesity and the metabolic syndrome (12, 43), show a significantly enhanced neointimal response to balloon angioplasty. These changes were evident at 14 days and remained greater until at least 30 days after angioplasty. Our results are consistent with the findings of other investigators, who also showed an increased neointimal response to endovascular injury in obese ZR (24, 31, 32, 42).

Several mechanisms have been proposed to account, at least in part, for the increased neointimal responses seen in the obese ZR. These include increased vascular smooth muscle proliferation (24, 31), receptor for advanced glycation end product and ligand interactions (42), increased expression of thrombospondin-1 in the vessel wall (35), and P-selectin-mediated leukocyte activation (41). In the present study, we have shown for the first time that the expression of 12-LO and activation of STAT4 are markedly increased together with more ED1-positive cells in the injured vessel in the insulin-resistant obese ZR compared with that seen in lean ZR. Our results provide additional support for the hypothesis that the inflammatory response is greatly enhanced during neointimal formation in the insulin-resistant state after vascular injury.

It is clear that 12-LO and its products play a role in atherosclerosis and vascular injury because of their inflammatory, growth, and chemotactic properties (10, 18, 20, 25, 28). The 12-LO products have chemotatic, mitogenic, and hypertrophic properties, and they can initiate vascular smooth muscle cell migration and proliferation. The 12/15-LO products can also directly activate key growth-related kinases, such as the mitogen-activated protein kinase (20). In a previous study (7, 19), we demonstrated that 12-LO expression is increased in the Sprague-Dawley rat carotid after balloon injury and that molecular blockade of 12-LO reduces neointimal formation. In the present study, we show for the first time that 12-LO expression is markedly increased in the obese ZR carotid compared with the levels seen in the lean ZR after balloon injury.

STATs function as downstream effectors of cytokine and growth factor receptor signaling. STATs are activated by tyrosine and serine phosphorylation. STAT4 is the primary transcription factor utilized by IL-12 and several other cytokines (11, 36). Recent data suggest that STAT4 regulates both local and systemic inflammatory pathways, including chemokines such as MCP-1 (14, 27, 37). In the present study, we have demonstrated that PSTAT4 levels are significantly higher in the injured vessels in obese ZR compared with the levels seen in the lean ZR. These changes are all consistent with the hypothesis that enhanced inflammatory pathways participate in the increases in neointimal formation after vascular injury in the metabolic syndrome. To test this hypothesis, we utilized a novel anti-inflammatory compound, LSF, that blocks STAT4 activation. LSF significantly reduced neointimal formation after vascular injury in the obese ZR. Of interest was the almost complete blockade of macrophage entry into the vascular lesion and marked inhibition of PSTAT4 levels. LSF also significantly reduced cellular proliferation in the neointima, as reflected by the reduction in Ki67 staining in the lesions, and reduction of vascular smooth muscle cell growth in vitro (our unpublished observations). These results suggest that inhibition of inflammation may provide therapeutic benefits to reduce restenosis in subjects with insulin resistance.

In summary, these new results highlight the important role of certain inflammatory pathways in the exaggerated neointimal response to injury in a rat model of the metabolic syndrome. It will be necessary to conduct additional studies using relevant models and cell types to fully understand and characterize the inflammatory molecules and signals leading to vascular disease in the metabolic syndrome. If these results can be translated to humans, agents that reduce chronic inflammation might provide therapeutic benefits to reduce cardiovascular disease in subjects with the metabolic syndrome.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants PO1 HL-55798 and DK-39721.


   ACKNOWLEDGMENTS
 
We thank Starr Palmore and Tina Turner for assistance in preparing the manuscript and the project leaders of National Heart, Lung, and Blood Institute Grant No. PO1 HL-55798 for helpful discussions. We also thank Mellisa Bevard and John Sanders for the immunohistochemistry assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Nadler, Division of Endocrinology, Dept. of Internal Medicine, University of Virginia, PO Box 801405, Charlottesville, VA 22908 (e-mail: jln2n{at}virginia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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