© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
Increased arterial expression of a glycosylated haptoglobin isoform after balloon dilation
aExperimental Cardiology Laboratory, University Medical Center, Utrecht, The Netherlands
bInteruniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands
cDepartment of Pathology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands
d.dekleijn{at}hli.azu.nl
* Corresponding author. Experimental Cardiology Laboratory, University Medical Center, Heidelberglaan 100 (room G02.523), 3584 CX Utrecht, The Netherlands. Tel.: +31-30-250-7155; fax: +31-30-252-2693.
Received 25 November 2002; accepted 28 January 2003
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Haptoglobin is a novel cell migration factor that is expressed in arteries after sustained flow changes and involved in arterial restructuring. Arterial restructuring is the major determinant of arterial shrinkage after balloon dilation. Although the function of extrahepatic haptoglobin expression is not yet understood, local haptoglobin expression may provide the tissue with functionally different haptoglobin due to post-translational modifications. We hypothesized that haptoglobin expression is increased during arterial restructuring after balloon dilation and compared glycosylation patterns between arterial and liver haptoglobin. Methods: Arterial haptoglobin expression was studied in rabbits at 0, 2, 7, 14 and 28 days after balloon dilation (n = 36) using real-time polymerase chain reaction, Western blotting and in situ hybridization. Two-dimensional gel electrophoresis and lectin affinity blotting were used to identify liver and arterial haptoglobin glycoforms. Results: Arterial haptoglobin mRNA (5.7-fold, P = 0.01) and protein levels (1.4-fold, P = 0.01) were increased after balloon dilation whereas liver haptoglobin expression remained constant. Haptoglobin was expressed in the adventitia of balloon dilated rabbit arteries, which was confirmed in human atherosclerotic arteries. Comparison between liver and arterial haptoglobin demonstrated the expression of artery-specific haptoglobin glycoforms. Conclusions: This study demonstrates that arterial haptoglobin expression is increased early after balloon dilation whereas liver haptoglobin expression does not change. Furthermore, arterial haptoglobin consists of an unique set of glycoforms compared to haptoglobin produced in the liver.
KEYWORDS Angioplasty; Arteries; Remodeling; Restenosis
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Haptoglobin is an acute phase glycoprotein that is mainly produced in the liver and secreted into the serum [1,2]. There is, however, increasing evidence that haptoglobin is also expressed in various extrahepatic tissues like lung, kidney, skin and heart [3,4]. Furthermore, extrahepatic haptoglobin expression can be induced by lipopolysaccharide (LPS) [5].
Recently, we demonstrated that haptoglobin is locally expressed in the arterial wall after sustained flow changes. Haptoglobin was found to play an important role during cell migration and involved in flow-induced arterial restructuring [6]. Arterial restructuring determines the degree of lumen loss in pathological arterial processes like restenosis [7] and atherosclerosis [8], suggesting a role for haptoglobin in these processes.
Although the function of extrahepatic haptoglobin synthesis is not yet understood, local haptoglobin synthesis may provide tissues with a source of functionally or structurally different haptoglobin. Tissue-specific protein glycosylation has been demonstrated for many proteins and it is known that glycosylation can modify protein function or structure [9]. Serum haptoglobin concentrations increase during inflammation [10] and tissue injury [11] and are accompanied by changes in haptoglobin glycosylation that have been associated with disease development and progression [12]. Thus, increased extrahepatic synthesis and secretion of differentially glycosylated haptoglobin might explain the observed variations in haptoglobin glycosylation during various diseases.
In this study, we hypothesize that arterial haptoglobin synthesis is increased after balloon dilation and characterized by differentially glycosylated arterial haptoglobin compared to liver derived haptoglobin. We show that arterial haptoglobin expression is increased in the adventitial layer early after balloon dilation in rabbits whereas liver haptoglobin expression remains constant. Furthermore, we demonstrate that arteries produce an unique set of haptoglobin glycoforms that is distinctive from liver derived haptoglobin.
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2 Methods |
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2.1 Tissue material
2.1.1 Rabbits
Balloon dilation was performed in the femoral and iliac arteries of 30 New Zealand white rabbits with an average weight of 3 kg. The rabbits were on a normal chow diet. The contra-lateral artery was used as a control artery. Undilated femoral and iliac arteries from six additional rabbits were used to determine haptoglobin baseline levels (=day 0). The rabbits were anesthetized by intramuscular injection of methadone (0.15 ml) and Vetranquil (0.15 ml) followed by intravenous injection of etomidate (1 mg/kg) and ventilated with N2O–O2 and 0.6% Halothane. The arterial tree was accessed through a carotid approach and the catheter was positioned using angiography. A 3.0 mm balloon was inflated 3x30 s with a pressure of 6–8 atm (1 atm=101,325 Pa). Arterial diameter changes at the internal elastic lamina were determined by measuring arterial lumen using angiography after balloon dilation and at termination. Arterial diameter changes were corrected for intimal hyperplasia thickness determined by histology cross-sectional analysis. After 0, 2, 7, 14 and 28 days, the rabbits were sacrificed. A small segment of the artery was fixed for 2 h in 4% paraformaldehyde at room temperature (RT), followed by overnight (o/n) incubation in 15% sucrose in phosphate-buffered saline (PBS). After embedding in Tissue Tec (Sakura), the segment was stored at –80°C. The remaining part of the artery was snap-frozen in liquid nitrogen and stored at –80°C for RNA and protein extraction. Liver samples were snap-frozen in liquid nitrogen and stored at –80°C for RNA and protein extraction. Blood samples were collected at termination; serum was isolated after centrifugation and stored at –80°C.
All investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the ethical committee on animal experiments of the University Medical Center Utrecht.
2.1.2 Human coronary arteries
Coronary arteries (left anterior descending, left circumflex and right coronary arteries), removed directly after heart transplantation, were used to perform in situ hybridization (n = 3). The arteries were fixated for 2 h in 4% paraformaldehyde at RT, followed by o/n incubation in 15% sucrose in PBS. After embedding in Tissue Tec (Sakura), the segment was stored at –80°C.
2.2 RNA and protein extraction
The frozen tissue samples were ground in liquid nitrogen, using a pestle and mortar. RNA and protein were extracted using 1 ml Tri-pureTM Isolation Reagent (Boehringer Mannheim) according to manufacturers protocol. Protein samples for two-dimensional (2D) gel electrophoresis were isolated using 40 mmol/l Tris, pH 7.5. Protein concentrations were determined using the Bio-Rad DC protein assay.
2.3 Real-time polymerase chain reaction (PCR)
Reverse transcription (RT) was carried out with 500 
g total RNA using superscript II (Life) according to the manufacturer's protocol. To confirm the identity of the amplified cDNA products, PCR products were ligated into the pGEM®-T Easy Vector (Promega) and sequenced using the T7 sequenase version 2.0 DNA sequencing kit (Amersham).
PCR amplification was performed using the I-cycler iGTM Real Time PCR (Bio-Rad). Each reaction contained 14 µl cDNA, 200 µmol/l dNTP, 1x reaction buffer (BRL) containing 1:100,000 Cybergreen (Bio-Rad), 2.5 U Taq DNA polymerase (BRL) and 1 µmol/l of each primer. Quantities were determined by comparison with known quantities of the cloned PCR products representing the target mRNAs. Data were corrected for the amount of 18S or β-actin mRNA, which were used as internal standards.
The following oligonucleotides were used as primers: rabbit haptoglobin (forward primer 5'-GAAGCAGTGGGTGAACAAGG-3', reverse primer 5'-TGACAAGATTGTGGCGGGAG-3'), rabbit 18s (forward primer 5'-TCAACACGGGAAACCTCAC-3', reverse primer 5'-ACAAATCGCTCCAGCAAC-3'), human haptoglobin (forward primer 5'-TTCTACACCCTAACTACTCCCAGG-3', reverse primer 5'-TAACCCACACGCCCTACTTC-3'), human β-actin (forward primer 5'-CTGTACGCCAACACAGTGCT-3', reverse primer 5'-TCCACACAGAGTACTTGCGC-3').
2.4 In situ hybridization
Human and rabbit haptoglobin cDNA in pGEM®-T Easy Vector was linearized and used as template to obtain digoxigenin (DIG, Roche) labeled RNA probes according to manufacturers protocol. Tissue segments were cut into 8 µm sections and transferred to Superfrost plus slides (Menzel Glazer) and stored at –80°C until use.
After defrosting, sections were treated with 0.2 mol/l HCl for 20 min at RT, washed three times with PBS for 5 min and treated with proteinase K (Roche 10 µg/ml) for 10–15 min at 37°C in PBS. The sections were washed with PBS and fixed at RT for 5 min in 4% paraformaldehyde and treated twice for 5 min with acetic anhydride in triethylacetate (TEA, 185 µl acetyl anhydride in 0.1 mol/l TEA). Sections were subsequently washed twice in 2xSSC for 5 min at RT, followed by 5 min in 2xSSC/50% formamide at 37°C.
For prehybridization, 100 µl hybridization mix (50% formamide, 1 mg/ml tRNA, 1xDenhardts, 10% dextrane sulfate, 4xSSC) was added to the slide and incubated for 1 h at 46°C. After o/n hybridization at 46°C, sections were washed with 0.1xSSC at 45°C for 15 min, followed by RNAse treatment (40 µg/ml RNAse A, 1 mmol/l EDTA, pH 8.2, 2xSSC) for 15 min at RT and washed again with 0.1xSSC at 45°C for 15 min. Before detection with 1/500 sheep-
-DIG AP (Boehringer), sections were rinsed with 2xSSC and 100 mmol/l Tris, pH 7.4+150 mmol/l NaCl at RT. Detection with NCBI/NBT (Roche) was performed according to manufacturer's protocol.
2.5 One- and two-dimensional gel electrophoresis
For one-dimensional gel electrophoresis, equal amounts of total protein (5 µg/lane) were separated on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel under reducing conditions.
For two-dimensional gel electrophoresis, equal amounts of protein (100 µg for tissue samples, 1 µl for serum) were diluted to 125 µl with rehydration buffer [8 mol/l urea, 2% CHAPS, 50 mmol/l dithiothreitol (DTT), 0.2% Bio-Lyte® 3/10 ampholyte, 0.001% bromophenol blue; Bio-Rad] and used to rehydrate 7 cm IPG dry strips, pH 4–7 (Bio-Rad) at 50 V for 12 h at 20°C. The rehydrated strips were focused by 2 h ramping to 4,000 V, followed by an additional 20,000 V. The strips were placed for 10 min in equilibration solution (6 mol/l urea, 375 mmol/l Tris–HCl, pH 8.8, 30% glycerol, 2% SDS; Bio-Rad) containing 100 mmol/l DTT, followed by 10 min in equilibration buffer containing 135 mmol/l iodoacetamide. The equilibrated strips were run on a 12% SDS–polyacrylamide gel electrophoresis (PAGE) gel.
2.6 Western blotting and lectin affinity blotting
Proteins were electrophoretically transferred onto Hybond ECL (Amersham) and blocked o/n at 4°C in block solution (PBS, 0.1% Tween, 5% non-fat dry milk). The membrane was incubated for 1 h at RT with goat--human haptoglobin (Sigma, 1/1,000), biotinylated GNA (EY Laboratories, 1/1,000) or biotinylated lotus tetragonolobus (Sigma, 1/1,000), followed by 1 h incubation with biotinylated rabbit--goat (DAKO, 1/1,000) and peroxidase labeled streptavidine (SBA, 1/1,000). Detection occurred using ECL (NEN Life Science Products) and exposure to X-Omat Blue XB-1 films (Kodak). As a negative control, the goat--human haptoglobin antibody was substituted by normal goat serum or blocked with 4 µg purified human serum haptoglobin (Sigma).
2.7 Statistics
Data are presented as the ratio of dilated versus control artery or mean±S.D. Statistical analysis for the balloon dilated rabbits was performed using a Wilcoxon matched pairs signed rank sum test. P values <0.05 were considered as statistically significant.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Haptoglobin expression after balloon dilation
The arterial response after balloon dilation is depicted in Fig. 1A. At 2 days after balloon dilation, the balloon dilated arteries were slightly enlarged. Constrictive remodeling started at day 7 after balloon dilation and continued up to 28 days. Neointima formation was first found at 7 days after balloon dilation and progressively continued up to 28 days. Since both arteries reacted similarly to balloon dilation, the data from femoral and iliac arteries were pooled.
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P = 0.04.To measure haptoglobin mRNA and protein expression, real-time PCR and Western blotting was performed. Haptoglobin mRNA expression was significantly increased at 2, 7 and 14 days (P<0.04) after balloon dilation with peak values at 7 days (median=5.7-fold) (Fig. 1B). At 28 days, haptoglobin mRNA levels were returned to baseline levels. Haptoglobin protein levels were significantly increased in the balloon dilated arteries with a maximum at 14 days after balloon dilation (median=1.4-fold, P = 0.01) (Fig. 1C). The signal disappeared after blocking the primary haptoglobin antibody with an excess of purified haptoglobin or substitution with normal goat serum (data not shown).
To identify the cells expressing haptoglobin, we performed in situ hybridization. A strong staining for haptoglobin mRNA in balloon dilated rabbit arteries was observed in cells in the adventitial layer (Fig. 2A); no staining was observed in the media or neointima. The localization of haptoglobin mRNA in rabbit arteries was confirmed by in situ hybridization in human atherosclerotic coronary arteries where haptoglobin mRNA was mainly expressed in adventitial cells although some positive cells were also found in the media and plaque (Fig. 2C). Alternate sections hybridized with a control sense probe showed no staining (Fig. 2B and D).
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3.2 Arterial versus liver haptoglobin expression
To investigate the relative importance of arterial haptoglobin synthesis, haptoglobin mRNA and protein levels were compared before and 2 days after balloon dilation in liver and arterial tissue samples. Haptoglobin mRNA and protein levels increased significantly in the artery after balloon dilation (Fig. 3A and B). However, liver haptoglobin mRNA and protein levels remained constant after balloon dilation (Fig. 3A and B). The maximum levels of arterial haptoglobin mRNA after balloon dilation were 345% compared to control livers whereas arterial haptoglobin protein increased to 209% of liver haptoglobin.
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3.3 Expression of arterial haptoglobin isoforms
To investigate variations in haptoglobin glycosylation, 2D gel electrophoresis and lectin affinity blotting was performed. Haptoglobin is atetrameric glycoprotein consisting of two
- and two β-chains (2β2). After separation of total protein samples on 2D gel, a train of spots was detected representing different glycoforms of haptoglobin β-chain and two smaller spots representing the -chains of haptoglobin. The 2D gels demonstrated that there were no differences in the number or position of arterial haptoglobin β-chains between control (Fig. 4A) and balloon dilated arteries (Fig. 4B) although total haptoglobin protein levels increased after balloon dilation. There were, however, large differences in localization of haptoglobin β-chains in arterial (Fig. 4A and B) and liver samples (Fig. 4C). Using the -chains as a landmark on the gel, arterial haptoglobin β-chains were mainly located towards, pH 4.5 while liver haptoglobin β-chains were located towards pH 5.5. In serum samples, both the liver and arterial haptoglobin β-chains were detected (Fig. 4D).
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As haptoglobin is a glycoprotein, lectin affinity blotting was used to determine differences in haptoglobin glycosylation between liver and arterial haptoglobin. The reactivity to the lectins GNA and lotus tetragonolobus demonstrated that terminal mannose and fucose residues were present on arterial haptoglobin but not on liver haptoglobin (Fig. 5). Serum samples also reacted positive for the two lectins.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Recently, we demonstrated that haptoglobin is a novel cell migration factor and gelatinase-inhibitor that is expressed in arteries after sustained flow changes and involved in arterial restructuring [6]. Furthermore, haptoglobin has been associated with other extracellular matrix related processes like angiogenesis [13] and cancer [14]. Extracellular matrix turnover and arterial restructuring are important processes after balloon dilation. In the present study, we investigated the local expression of haptoglobin after balloon dilation and the presence of arterial haptoglobin glycoforms.
Haptoglobin expression in balloon dilated rabbit arteries was increased within the first 2 weeks after balloon dilation with peak values at 7 days and correlated with the time period for increased MMP expression and cell migration after arterial injury [15,16]. Activation of MMP-2 has been associated with cell migration. There is, however, no linear correlation between active MMP-2 levels and cell migration. High levels of active MMP-2 result in excessive extracellular matrix and impaired cell migration while cells with intermediately active MMP-2 levels demonstrate efficient cell migration [17]. Thus, haptoglobin may provide cells with an additional regulation of MMP-2 activity [6], thereby providing the correct substrate necessary for optimal cell migration.
Haptoglobin mRNA is only expressed by adventitial cells after balloon dilation and not by cells in the media or neointimal layer. These positive cells are probably fibroblasts, which are also the haptoglobin producing cells in the arterial wall after flow changes [6]. The adventitia is the first arterial layer that responds to arterial injury with enhanced extracellular matrix accumulation, cell proliferation [18] and MMP expression [19]. Moreover, adventitial fibroblasts migrate towards the subendothelial space, thereby contributing to neointima formation [20,21]. The adventitial expression of MMPs combined with elevated haptoglobin levels may facilitate the migration of adventitial fibroblasts.
Until recently, the liver was thought to be the major site of haptoglobin synthesis. However, haptoglobin is also expressed at basal levels in extrahepatic tissues and expression can be induced by lipopolysaccharide (LPS) [4,5]. Liver haptoglobin expression can be induced during systemic inflammation and liver injury, which might explain the observed elevations in serum haptoglobin concentrations. However, until now it was unclear whether liver haptoglobin expression is also elevated after local stimulation of haptoglobin in extrahepatic tissues. In this study, we compared the relative levels of liver and arterial haptoglobin synthesis. Arterial haptoglobin expression increased early after balloon dilation at mRNA and protein levels. In contrast, liver haptoglobin mRNA and protein expression remained constant after balloon dilation. We infer that increased local arterial haptoglobin synthesis is responsible for the observed increases in arterial haptoglobin levels and that this is not due to extravasation of haptoglobin from serum.
Previously, an association had been found between altered glycosylation patterns of haptoglobin and the development and progression of various pathological processes [12]. Moreover, the type of glycosylation appeared to have prognostic values in certain pathological diseases [22,23]. However, the liver was thought to be the production site of these abnormally glycosylated haptoglobin forms. In this study, we demonstrate that the rabbit artery produces an unique set of haptoglobin β-chains compared to the liver and that both forms can be detected in serum. This is in accordance with the human situation in liver and serum samples, as found in the SWISS 2D-PAGE database (http://www.expasy.ch/ch2d).
Lectin affinity blotting revealed that only arterial haptoglobin reacted to the lectins GNA and lotus tetragonolobus, confirming that differences in glycosylation are responsible for the specific arterial and liver haptoglobin glycoforms. This is in accordance with another study [24] showing that different haptoglobin β-chains react with different affinity to lectins. Serum samples also reacted positive with the used lectins, demonstrating that arterial haptoglobin is secreted into the serum. Since alterations in glycosylation are known to modify function, structure or targeting of proteins [25–27], it is conceivable that arterial haptoglobin has a different function compared to haptoglobin produced in the liver. A limitation of this study is that it is purely descriptive and the functional role of tissue-specific differences in haptoglobin glycosylation remains to be elucidated.
In summary, we found increased haptoglobin expression in the adventitia of rabbits after balloon dilation. Localization of arterial haptoglobin expression was confirmed in human atherosclerotic coronary arteries, supporting a role for haptoglobin in pathological arterial restructuring. Furthermore, we demonstrated that arteries produce a unique subset of haptoglobin β-chains compared to liver samples. Since human atherosclerotic arteries also express haptoglobin, this might point to a potential role for arterial haptoglobin glycoforms as a serum marker for atherosclerotic disease [28].
Time for primary review 28 days.
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Acknowledgements |
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We thank the heart transplantation team of the University Medical Center Utrecht for providing samples of human coronary arteries. This study was supported by grants from the Netherlands Organization for Scientific Research (NWO 902-16-239 and 902-16-222) and the Netherlands Heart Foundation (99-209).
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