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Cardiovascular Research 1999 41(2):489-499; doi:10.1016/S0008-6363(98)00312-5
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Evidence for 12-lipoxygenase induction in the vessel wall following balloon injury

Rama Natarajana,*, Hong Peia, Jia-Li Gua, Jonnalegedda M. Sarmab,1,1 and Jerry Nadlera

aDepartment of Diabetes, Endocrinology and Metabolism, Gonda Diabetes Center, City of Hope Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA
bDepartment of Cardiology, City of Hope Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA

* Corresponding author. Tel.: +626-359-8111, ext 2289; fax: +626-301-8136; e-mail: rnatarajan@smtplink.coh.org

Received 5 June 1998; accepted 19 October 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Vascular smooth muscle cell (VSMC) migration and proliferation are key events in the development of atherosclerosis and restenosis following angioplasty. These events are mediated by several growth factors and cytokines whose cellular effects include activation of phospholipases and arachidonic acid metabolism via the lipoxygenase (LO) pathway. Since 12-LO products have potent growth and chemotactic effects, we have examined if 12-LO is upregulated in the neointima of injured rat carotid arteries and also if LO inhibition could attenuate neointimal thickening. Methods: The left common carotid arteries of male Sprague Dawley rats were injured using a 1.8F PTCA balloon catheter. Four–fourteen days after injury, injured and uninjured tissue samples were processed for histology, and immunohistochemistry or polymerase chain reaction (PCR) to examine 12-LO expression. Results: Twelve days after injury, immunohistochemical staining with a 12-LO antibody revealed intense staining in injured left carotid arteries, mainly in neointimal VSMCs and inflammatory cells, but not in the uninjured right arteries. There was also a marked upregulation of 12-LO mRNA (over five-fold by competitive PCR) in the injured arteries. Treatment of the arteries with a LO inhibitor, phenidone, soon after injury resulted in significant inhibition of neointimal thickening. In contrast, a cyclooxygenase inhibitor, ibuprofen, had no effect. Conclusions: These results indicate for the first time that balloon injury results in marked induction of 12-LO mRNA and protein expression in the vessel wall. Furthermore, LO pathway activation may mediate, at least in part, the development of the lesion or plaque instability, suggesting a novel target for therapeutic intervention to block these pathological events.

KEYWORDS Lipid signaling; Lipoxygenase; Balloon angioplasty; Restenosis; Rats; Neointimal thickening; PCR


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Arterial injury leads to the migration of medial vascular smooth muscle cells (VSMCs) to the intima where they proliferate and elaborate extracellular matrix. This plays an important role in the pathogenesis of several cardiovascular diseases, including restenosis and atherosclerosis [1–3]. Neointimal thickening and restenosis occur in nearly 40% of patients who undergo percutaneous transluminal coronary angioplasty [4, 5]. Development of successful modalities to reduce the incidence of restenosis is therefore of great importance. The excessive migration and proliferation of VSMCs after vascular injury are mainly mediated by growth factors and cytokines released by various cells in the vessel wall [3]. However, the role of free lipids released by these factors in the vessel wall are not very clear.

The stimulation of cells by extracellular signals such as growth factors, cytokines or hormones leads to the activation of several phospholipases, which can act on membrane phospholipids to release arachidonic acid [6, 7]. Arachidonic acid is also the precursor for several eicosanoids with potent biological effects, including inflammation and cell growth [8]. The 20-carbon arachidonic acid can be metabolized by pathways such as the cyclooxygenase pathway, which leads to the formation of prostaglandins, and the lipoxygenase (LO) pathway, which forms hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs) and leukotrienes [9]. The lipoxygenases, mainly called 5-, 12- and 15-LOs, are named for their ability to insert molecular oxygen at the 5-, 12-, or 15-carbon atom of arachidonic acid [10]. The 5-LO pathway leads to the formation of 5-HETE and leukotrienes, while the 12- and 15-LOs can form 12- and 15-HETEs. The production of 12- and 15-HETE has been shown in several vascular cells, including cultured VSMCs and endothelial cells, and also in leukocytes. In addition, two major LOs can also interact in transcellular pathways to form compounds called lipoxins, which have been shown to have potent vasoactive and anti-inflammatory effects [11]. They can also be released during atherosclerotic plaque rupture [11].

12-LOs have been isolated and cloned from various sources [12–17]. Two types of 12-LOs can be distinguished immunologically and catalytically [10, 12]. A platelet-type has been cloned from human platelets and the megakaryocytic cell line, HEL [14, 15]. The other 12-LO, namely the leukocyte-type, has been detected in porcine leukocytes [16], rat brain [17], VSMCs [18] and also in human adrenal [19], human monocytes, endothelial and VSMCs [20].

The LO enzymes and their products, such as HETEs, have been implicated in the pathogenesis of atherosclerosis, hypertension and other vascular disorders, since LO products have potent inflammatory, growth and chemotactic properties [8, 21]. Studies indicate that the LO enzyme can mediate the oxidative modification of low density lipoprotein (LDL) to oxidized LDL, which is believed to be the atherogenic form of LDL [22, 23]. Increased levels of HETEs and other LO products have been found in the aortas of atherosclerotic rabbits [24] and in early atherosclerotic lesions [25] but not in later lesions where non-enzymatic oxidation reactions related to lipid peroxidation predominate. However, analysis of the lipid oxidation products in human atherosclerotic lesions revealed that the oxidation of polyunsaturated fatty acids therein was mainly mediated by the LO enzyme [26]. In another recent study, Benz et. al. [27] demonstrated that LDL incubated with fibroblasts overexpressing 15-LO had enhanced levels of lipid hydroperoxides, further implicating the LO enzyme in LDL oxidation. We also have new evidence supporting the presence of a leukocyte-type of 12-LO in early and advanced porcine atherosclerotic lesions [28].

We have recently shown that VSMCs, endothelial cells and monocytes express a leukocyte-type 12-LO [18, 20]. The activity and expression of this LO was markedly upregulated by potent VSMC growth factors and chemotactic agents, such as angiotensin II (AII) and platelet-derived growth factor (PDGF) [18, 29] as well as by inflammatory cytokines [30]. Furthermore, the 12-LO pathway could mediate the hypertrophic effects of AII [31] and the chemotactic effects of PDGF [29]. 12-LO products also had direct hypertrophic effects [31]. In the present study, we have examined the hypothesis that 12-LO may be activated during balloon angioplasty and thus play a role in neointimal thickening.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1995) and is in accordance with the approved guidelines of the Research Animal Care Committee of City of Hope.

Rats (Sprague-Dawley, 400–450 g) were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg). The common carotid and external carotid arteries on the left side were exposed by a 2.5-cm long midline incision on the neck. The external carotid artery was partially cut at about 2–3 mm from the arterial bifurcation with micro scissors. A 1.8F balloon angioplasty catheter with a 2 cm long, 1.5 mm diameter balloon and a 1 cm long 0.014'' diameter leading guide wire (Model ACE 20/1.5-1, SciMed Life Systems, Maple Grove, MN, USA) was introduced through the cut and extended into the common carotid. The balloon was then inflated to a pressure of 1–1.5 atmospheres, passed back and forth throughout the common carotid artery three times for 18 s, to ensure uniformity of the extent of the injury [32]. The balloon was then deflated to about 0.5 atm and withdrawn, while blocking blood flow through the common and internal carotid arteries.

Local delivery of inhibitors was performed using mainly a pluronic gel-F127 (Sigma, St. Louis, MO, USA). Phenidone (LO inhibitor, 100 µmol/l) [33] or ibuprofen (cyclooxygenase inhibitor, 10 µmol/l) were prepared in 25% pluronic gel (Sigma) prepared in cold phosphate-buffered saline (PBS) according to manufacturer's instructions and as described in ref. [34] and kept on ice until used. This gel remains liquid at 4°C and gels at room temperature. Immediately following injury, gel containing the inhibitor was rapidly applied to the distal neck portion of the injured artery, while gel alone was applied to the proximal chest portion of the same artery. The surgical wound was then closed and the animal was allowed to recover in a warm chamber. In some experiments, inhibitors were also delivered by the dwell technique [35, 36]. In this method, the neck portion (distal) of the common carotid artery was temporarily isolated with micro-vascular clips placed on the common and internal carotid arteries. The external carotid artery was cannulated through the cut (see above) with a blunted 23G butterfly needle. The inhibitors, in PBS solution, were introduced into the isolated distal section of the artery under slight pressure and maintained for 10 min. This ensures that the proximal portion of the injured artery does not receive the inhibitor and thus serves as an internal control. At the end of the incubation period the inhibitor solution was suctioned through the canula under slight negative pressure. The canula was removed and the external carotid artery was ligated proximal to the cut and the vascular clips were removed to establish carotid flow. In all cases, each animal served as its own control since the inhibitor-treated and -untreated sections were from the same injured left artery, while the right artery served as the uninjured control.

The animals were maintained for 4–14 days after initial surgery and the injured treated (neck portion of the left common carotid artery), injured untreated (the chest portion of the left common carotid artery) and uninjured (right carotid artery) sections of the carotid arteries were harvested for either histology or biochemical analysis. India ink markings were used to demarcate the treated and untreated sections of the left carotid artery. The arterial bed above the descending aorta was perfused under pressure with cold Dulbecco's PBS solution. The appropriate sections of the carotid arteries were either snap frozen in liquid nitrogen for further analyses or, for histological examination, they were first washed with PBS and then fixed in situ by perfusing the vascular bed with PBS containing 4% paraformaldehyde and 2% glutaraldehyde at a pressure of 100–120 mmHg through the aortic canula. These fixed tissues were paraffin-embedded and sections were stained with hematoxylin–eosin (H and E) or leukocyte-type 12-LO peptide antibody [29]. In all sections, the luminal area, area within the internal elastic lamina (IEL), and the external elastic lamina (EEL) were measured by planimetry and image analysis, and the intimal area (IEL–luminal area) and the medial area (EEL–IEL) were calculated. Results are expressed as ratios of intima-to-media areas.

2.2 Immunohistochemical detection of leukocyte type 12-LO in arterial sections
The immunohistochemical methods were carried out using a modification of the technique of Larsson [37]. Briefly, 4 µm paraffin-embedded tissue sections were mounted on Probe-on slides (Biotek Solutions) and dried for 1 h in a 56°C oven and overnight at 45–50°C, deparaffinized in xylene and rehydrated in graduated alcohol to distilled water. The slides were loaded into a TechmateTM Slide holder and placed into 0.1 mol/l citrate buffer solution for HIER (heat-induced epitope retrieval). A household Black and Decker steamer (model number HS90) was used for the HIER. The slides were steamed in 0.1 M citrate buffer for 20 min and then allowed to cool for 5 min. After treatment with the first and second antibody, slides were stained using a modified ABC technique using 3,3'-diaminobenzidine tetrahydrochloride (DAB) as the chromogen and counterstained using Mayer's hematoxylin. Staining was performed using a Bioteck TechmateTM 1000 Immunostainer (Teckmate, Santa Barbara, CA, USA) with Biotek solutions and an ABC detection system (Teckmate). We used a specific primary antibody (IgG) to the porcine leukocyte 12-LO (1:500) (raised to a peptide derived from the sequence of the porcine leukocyte 12-LO) [16, 29]. Parallel controls were run without primary antibody, and with preimmune normal rabbit IgG. SMCs were detected using a monoclonal antibody to smooth muscle {alpha}-actin (clone 1A4, Sigma; 1:5000), endothelial cells with a specific antibody to human Von Willebrand Factor (1:200, DAKO, Carpinteria, CA, USA) while leukocytes were detected using a specific cross-reacting antibody to human myeloperoxidase (1:200, DAKO). Immunostaining 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.

2.3 12-LO mRNA expression in rat carotid artery tissues by competitive polymerase chain reaction (PCR)
This was performed essentially according to our reported method [38].

2.3.1 RNA preparation and cDNA synthesis
The carotid artery tissue fragments were homogenized with a Polytron in RNA Stat 60 reagent (Tel-Test ‘B’ Inc., Friendwood, TX, USA). Total RNA was isolated using the single-step method according to the manufacturer's instructions. cDNA was prepared as described in ref. [38] using 1 µg of total RNA with 200 U of reverse transcriptase (MMLV; Gibco-BRL) and 50 pmol of random hexamer primers (Perkin Elmer) and dNTPs in a total reaction volume of 20 µl containing reverse transcriptase buffer and 2 U of RNasin. The relative efficiency of cDNA synthesis for each sample was confirmed by comparison of PCR products of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification using GAPDH primers as described [38].

2.3.2 Rat leukocyte 12-LO competitor DNA preparation
The competitor DNA used as internal standard for the competitive PCR was designed to contain the same base pair sequence as the target 12-LO cDNA that would allow efficient priming, but had a portion of the sequence deleted so that the competitor PCR-generated fragment could be easily distinguished by size. The competitor was prepared by using a PCR-based mutagenesis approach to induce a 31-bp deletion of the rat leukocyte 12-LO cDNA fragment as described previously [38]. The rat leukocyte-type 12-LO has been cloned [17] and sequences were derived from this information. The template, rat pineal 12-LO cDNA PUC 19 plasmid, was a gift from Dr. T. Yoshimoto (Tokushima, Japan). The primers used for preparing the deletion 12-LO competitor were as follows: The sense primer was located at nucleotides 324 to 343 (5'-TGGGGCAACTGGAAGGATGG-3'), and the antisense primer was located between nucleotides 658 and 735 (5'-AGAGCGCTTCAGCACCATGGGAACCCATCTTCGTCCAGGAGTTTCG-3'). The PCR reaction was carried out by mixing 12-LO cDNA–PUC 19 plasmid with PCR buffer (Perkin Elmer), 200 µmol/l dNTP, 25 pmol of each primer and 2.5 U of Taq polymerase. PCR was carried out at 94°C for 1 min, 60°C for 1 min and 72°C for 1 min for 24 cycles. The PCR product was purified in Centricon-100 tubes (Amicon, Beverly, MA, USA) and subcloned into the HincII site of pGEM-3Z (+) plasmid (Promega). The sequence of the deletion fragment was confirmed in the positive clones. The competitor plasmid was then purified by cesium chloride/ethidium bromide equilibrium centrifugation.

2.3.3 Competitive PCR
The details of this method have been described previously [38]. The PCR method is specific for leukocyte 12-LO and does not cross-react with platelet 12-LO [19, 20]. The synthesized 12-LO cDNA samples were co-amplified with a constant amount of the competitor (10–7 ng) in the presence of rat 12-LO primers, [{alpha}32P]dCTP and Taq polymerase (Perkin Elmer) in a thermocycler (model 2400, Perkin Elmer). Sense primer was located at 324 to 339 of rat 12-LO (5'-TGGGGCAACTGGAAGG-3') and antisense primer was located at 718 to 735 (5'-AGAGCGCTTCAGCACCAT-3'). The PCR conditions were 94°C for 45 s, 68°C for 1 min and 72°C for 1 min for 35 cycles. The products of PCR were separated by 5% polyacrylamide gel electrophoresis (PAGE). Radioactive bands in the gels were quantitated on a phosphorimager (Molecular Dynamics) using the ImageQuant software.

2.3.4 Data analyses
As described in Section 2, each experimental animal yielded three samples, uninjured right carotid artery, injured untreated, and injured treated left carotid. All values are expressed as mean±SEM. Experimental groups were compared using paired Student's t-tests (for two groups) or ANOVA with Dunnett's post test (for multiple groups) using the INSTAT software (Graph Pad, San Diego, CA, USA). Statistical significance was detected at the 0.05 level.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Evidence of 12-LO expression in the neointima of balloon injured rats
Twelve days after balloon injury to the left common carotid artery, the injured rat arterial segments as well as matched control segments from the uninjured right arteries were fixed in situ and processed for histology and immunohistochemical detection of 12-LO using an antibody specific for porcine leukocyte 12-LO. We have shown that this antibody recognizes 12-LO from many species, including porcine, human and rat [19, 20, 29, 38]. Fig. 1 shows the representative results. H&E-stained sections show that the injured sections (Fig. 1C) have extensive intimal thickening with a narrowed lumen arising from a thick neointima, when compared to the control uninjured artery (Fig. 1A). Furthermore, we performed immunohistochemical staining using a specific antibody to leukocyte 12-LO. Staining was very faint in the control uninjured right carotid arteries (15±2% of total) (Fig. 1B). However, there was intense staining of 12-LO in the injured section, and the staining was particularly strong in the neointimal cells, particularly in VSMCs (68±5%) (Fig. 1D).


Figure 1
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  		Fig. 1 Increased immunostaining with an antibody to leukocyte-type 12-LO in balloon injured rat carotid arteries. (A) and (C) depict H and E staining in uninjured right and injured left common rat carotid artery sections, respectively (400x magnification). (B) and (D) show immunostaining with a specific leukocyte-type 12-LO antibody IgG (1:500).

 
Staining was also observed in the cells closest to the luminal side, which could represent endothelial cells or adherent leukocytes. Since leukocytes are a major source of 12-LO and studies have also shown the presence of 12-LO in endothelial cells [12, 20], we carried out immunohistochemistry using cell-type-specific antibodies: an antibody to {alpha}-actin to identify SMCs, an antibody to human myeloperoxidase (MPX) to identify leukocytes, and an antibody to Von Willebrand Factor to identify endothelial cells. Results are shown in Fig. 2. Fig. 2A–B show staining with endothelial-specific Von Willebrand factor antibody, and indicate a clear unbroken endothelium in the uninjured control right artery (Fig. 2A) but much fainter staining along the lumen in the injured sections, indicating a denuded endothelium. In Fig. 2C–D, strong staining for actin is observed in the media and intima of the uninjured and injured sections (over 95% of the VSMCs). In Fig. 2E–F, staining of inflammatory cells (which include leukocytes) is seen in the injured section in the cells lining the inner lumen as well as scattered cells around the adventitia. These results suggest that 12-LO staining in the media and neointimal cells predominantly occurs in SMCs, while staining closer to the luminal edge may also arise from leukocytes and endothelial cells.


Figure 2
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  		Fig. 2 Cell-type-specific immunocytochemistry. Sections from uninjured right carotid arteries (A, C and E) and from injured left arteries (B, D and F) were stained with antibodies that were specific for endothelial cells (anti-human Von Willebrand Factor; A and B), smooth muscle ({alpha}-actin; C and D) or for leukocytes (myeloperoxidase; E and F).

 
3.2 Time course of induction of 12-LO expression
We next examined the time course of 12-LO expression. 12-LO expression in carotid artery sections was then examined by immunostaining. Fig. 3 shows that 12-LO expression was noted in the arterial neointimal areas as early as four days (35±2% of luminal cells), was clearly evident by seven days (53±3%), and was highly expressed in the neointima by 14 days (74±4% of neointimal cells). A control with normal rabbit IgG is seen in the lowest panel.


Figure 3
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  		Fig. 3 Time course of induction of 12-LO evaluated by immunohistochemistry. Carotid artery tissue specimens were obtained from balloon injured carotid artery specimens, four, seven and fourteen days after injury. Paraffin-embedded sections of these specimens were screened for 12-LO expression by immunohistochemistry. (A) Control uninjured artery; (B) four, (C) seven and (D) 14 days after injury; (E) normal rabbit IgG.

 
In addition, we also examined the levels of leukocyte 12-LO mRNA in injured versus uninjured carotid artery sections from these rats by using a specific competitive PCR [38]. RNA extracted from these samples was reverse transcribed to cDNA and then subjected to competitive PCR [38]. A competitor oligo (10–7 ng) was added to each sample during the PCR reaction to estimate the relative level of 12-LO mRNA expressed [38]. Fig. 4 shows an experiment depicting 12-LO mRNA levels in tissue samples obtained at four, seven and twelve days. The size of the 12-LO PCR product is 312 bp and the competitor is 281 bp long. Only low levels of 12-LO mRNA expression were observed in the control uninjured arterial sections at all time periods. However, at each time period, there was a marked upregulation of 12-LO mRNA in the injured sections. The levels were markedly increased at the four day time period (11-fold over control as obtained by comparing the ratios of 12-LO cDNA/competitor cDNA phosphorimager counts in injured versus uninjured) and remained sustained thereafter (3.4- and 3.6-fold over control at seven and twelve days in the figure shown).


Figure 4
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  		Fig. 4 Time course of 12-LO mRNA expression after balloon injury. Injured and uninjured carotid artery tissue obtained four, seven and twelve days post injury were snap frozen in liquid nitrogen. RNA was extracted from these samples and subjected to competitive PCR to quantitate 12-LO mRNA as described in Section 2. The ratio of amounts of 12-LO cDNA to competitor cDNA was calculated in each case. In the figure shown, this revealed an 11-fold increase in 12-LO mRNA at four days, 3.4-fold at seven days, and 3.6-fold at 12 days. Results shown at four and seven days are representative of two experiments each. At 12 days, results of multiple experiments (n=16) revealed a significant up-regulation of 12-LO mRNA (5.7±1.5-fold over control, p=0.01 by Student's t-test).

 
Data from several experiments at the 12 day time period (16 rats) indicated that there was a significant upregulation of 12-LO mRNA at 12 days following injury (5.7±1.5-fold over control), p=0.01 by Student's t-test).

3.3 Effect of a LO and cyclooxygenase inhibitor on neointimal proliferation
In order to determine the functional significance of increased 12-LO expression, we evaluated whether or not a LO inhibitor would be able to attenuate the neointimal proliferation. We examined the effects of a pharmacological LO inhibitor, phenidone [33], added at a concentration of 100 µmol/l in a pluronic gel [34], which was directly applied to the surface of the vessel immediately following injury. The chest portion of the injured segment in the same rat was also coated with pluronic gel alone to act as a control. Results of a representative experiment are shown in Fig. 5. Tissues were removed 12 days after injury. The injured section (center panel) showed a highly narrowed lumen relative to uninjured (left panel). Furthermore, the lumen size in the phenidone-treated section (right panel) was distinctly larger than in the untreated section (center panel). The ratio of intima-to-media areas of the arterial sections in four separate experiments was measured and the results shown in Fig. 6 indicated that phenidone treatment led to a significant reduction in neointimal proliferation (p<0.01). Fig. 5 also depicts the results of immunohistochemical staining with the 12-LO antibody. As in Fig. 1, staining was weak in the uninjured sections (left panel), but greatly enhanced in the injured sections, particularly in the intima.


Figure 5
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  		Fig. 5 Effect of a LO inhibitor, phenidone, on neointimal thickening. Rats were subjected to balloon injury as described in Section 2. Immediately following injury, they were treated with phenidone (100 µmol/l in a pluronic gel) (distal neck portion of the injured artery). The chest portion of the same artery was treated with gel alone. Twelve days later, the artery sections were fixed, paraffin-embedded and subjected to H and E staining as well as immunostaining with the 12-LO antibody. Sections were obtained as shown. Results shown are representative of experiments done with four rats.

 

Figure 6
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  		Fig. 6 Phenidone treatment in a pluronic gel results in inhibition of neointimal thickening. Intima-to-media ratios from four separate rats using phenidone applied in the pluronic gel. Data shows significant inhibition of neointimal thickening in rats receiving phenidone soon after angioplasty. *, p<0.01 by ANOVA.

 
We also examined the consequences of instilling phenidone (100 µmol/l) in the vessel by the dwell technique, as described in the Section 2. Results are shown in Fig. 7. Here, the arterial sections were analyzed ten days after injury. Again, it is seen that the size of the lumen was distinctly larger in the phenidone-treated section (right panel) compared to the untreated injured section (center panel). Staining was with the 12-LO antibody.


Figure 7
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  		Fig. 7 Effect of phenidone instilled into the vessel wall after injury. The left common carotid artery was balloon injured and a 100 µmol/l freshly prepared solution of phenidone in PBS was instilled into the vessel as described in Section 2. Arteries were harvested ten days after injury. The phenidone-treated section (right panel) shows a marked reduction in neointimal thickening. Results shown are representative of three experiments (three rats).

 
Since phenidone can also have inhibitory effects on the cyclooxygenase pathway [33], we examined the effects of a specific cyclooxygenase inhibitor, ibuprofen, at a concentration of 10 µmol/l instilled into the vessel soon after injury. Sections were examined 12 days after injury. The results shown in Fig. 8 indicate that ibuprofen had no effect on the neointimal thickening. Furthermore, there was also no change in the extent of 12-LO immunostaining in the treated versus untreated arterial sections. These results exclude the cyclooxygenase pathway while implicating the LO pathway in the development of neointimal thickening.


Figure 8
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  		Fig. 8 Effect of a cyclooxygenase inhibitor, ibuprofen, on neointimal thickening. Rats were subjected to balloon angioplasty and soon after injury, they were treated with ibuprofen (10 µmol/l), which was instilled into the vessel by the dwell technique as in Fig. 7. Animals were sacrificed 12 days after injury. Results shown are representative of three experiments.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the current study, we have shown for the first time that there is a marked upregulation of the leukocyte-type 12-LO pathway in balloon injured rat carotid arteries. This was demonstrated both by immunohistochemical methods as well as by competitive PCR. Increased 12-LO expression was observed by four days following injury and remained sustained up to 14 days. Furthermore, our observation of significant inhibition of neointimal thickening by treatment with a LO inhibitor, but not a cyclooxygenase inhibitor, revealed the functional significance of increased 12-LO expression in this animal model of restenosis.

Several lines of evidence suggest that the LO pathway may play a role in the pathogenesis of atherosclerosis. The LO enzymes have been shown to mediate the oxidative modification of LDL [22]. LO enzyme and mRNA have been identified in atherosclerotic lesions [23]. Growth factors and cytokines such as angiotensin II, PDGF, interleukins-1, and -8, which are present in circulation and also copiously released at sites of vascular injury, are potent inducers of 12-LO activity and expression [18, 29, 30]. 12-LO activation was also shown to mediate the hypertrophic effects of angiotensin II, the mitogenic effects of cytokines and the chemotactic effects of PDGF in VSMCs [29–31]. They also have direct and potent growth-promoting and chemotactic effects in VSMCs [21, 31]. However, the functional in vivo role for the 12-LO pathway in mediating cytokine or growth factor action after injury has not been evaluated previously.

A recent study showed that injection of rats with a high dose of aspirin, a cyclooxygenase inhibitor, could reduce neointimal thickening [39]. However, these high doses may be working by blocking pathways other than cyclooxygenase, such as NF-{kappa}B [39, 40], thereby explaining the lack of effect of clinical doses of aspirin on restenosis [41].

The mechanisms by which 12-LO inhibition with agents such as phenidone lead to the reduction in luminal narrowing after carotid artery balloon angioplasty have not been investigated in this study. Furthermore, it is not clear whether the LO pathway leads to the initiation and/or progression of the lesion, as well as plaque instability. Since 12-LO products have chemotactic, mitogenic and hypertrophic properties, they can initiate VSMC migration as well as proliferation. LO products can also directly activate key growth-related kinases, such as the mitogen-activated protein kinase [21]. LO pathway activation can also lead to the induction of oxidative stress [10] and the formation of reactive oxygen species such as superoxide [42]. Studies in animal models have shown evidence of the release of oxidants in response to arterial injury [43]. Furthermore, antioxidants such as probucol have proved effective in reducing restenosis after balloon angioplasty. However, the antioxidant effects of probucol alone could not be attributed to the beneficial effects on restenosis, and the mechanisms involved are still not very clear [44]. It is possible that the actions of phenidone in the current study may be due to an antioxidant effect. However, we have evidence that phenidone is a potent inhibitor of the formation of 12-LO products such as 12-HETE in aortic tissue [45].

Increased 12-LO in VSMCs and macrophages/leukocytes of atherosclerotic blood vessels may also play a role in plaque rupture since LO activation can lead to lipid peroxidation [10] and evidence shows that 12-LO metabolites can mediate the induction of interstitial collagenase (MMP-13) in macrophages [46]. LO products also have inflammatory effects and evidence suggests that certain LO products of arachidonic and linoleic acids can lead to the activation of key inflammatory endothelial cell adhesion molecules, such as VCAM-1, through the activation of NF-{kappa}B [47, 48]. These factors play key roles in the development of atherosclerosis and studies have shown that NF-{kappa}B is activated in VSMCs after balloon injury and mediates adhesion molecule expression [39]. The antiinflammatory lipoxins may also play a role in vessel injury and restenosis. In particular, possible cross-talk between 12-LO activation and lipoxins would be of interest, especially in the context of plaque stability, given the well known effects of lipoxins on monocyte and neutrophil activation [11, 49].

Growth factors and cytokines, which are released by platelets, endothelial cells, monocytes, macrophages and VSMCs during the initiation of the atherogenic lesion, can have autocrine and paracrine effects, leading to the progression of the plaque [3]. Evidence shows that angiotensin II plays a major role in the mediating VSMC proliferation in the injured rat carotid artery [50]. PDGF-mediated VSMC migration is also a key early event in the development of restenosis and atherosclerosis [51]. Since these growth factors are potent inducers of 12-LO, it is possible that their effects in the injured vessel wall are mediated through the 12-LO pathway. Our present results may also be relevant to human restenosis since there is evidence of a leukocyte type 12-LO in human vascular cells [20]. The ability to target appropriate molecules or enzymes such as LO may represent a novel approach to prevent or reduce restenosis and the formation of atherosclerotic lesions as well as plaque rupture. We have recently developed and tested a specific molecular inhibitor, a ribozyme directed to leukocyte 12-LO [52]. This ribozyme very efficiently cleaved 12-LO mRNA and reduced 12-LO protein and product levels in VSMCs [52]. Future studies with this ribozyme and other gene transfer techniques aimed at interrupting 12-LO will be utilized to further evaluate the role of 12-LO activation in restenosis, as well as in atherosclerosis and plaque rupture.

Time for primary review 32 days.


   Acknowledgements
 
These studies were supported by grant PO1 HL55798 (to R.N. and J.N.) jointly funded by the National Institutes of Health and the Juvenile Diabetes Foundation. The authors thank Yanyu Sun for her excellent technical help with the immunohistochemical analyses. We also acknowledge the assistance of City of Hope Anatomic Pathology Core Facility, funded by the Cancer Core Grant (CA-33572-15), and SCIMED Life Systems (Minnesota) for supplying some balloon catheters.


   Notes
 
1 Present address: Department of Cardiology, VA Medical Center, Los Angeles, CA 90073, USA. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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