Skip Navigation

Cardiovascular Research 2004 64(1):115-124; doi:10.1016/j.cardiores.2004.05.013
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sandhu, R.
Right arrow Articles by Stewart, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sandhu, R.
Right arrow Articles by Stewart, D. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Reciprocal regulation of angiopoietin-1 and angiopoietin-2 following myocardial infarction in the rat*

Reena Sandhua,1, Krystyna Teichert-Kuliszewskaa,1, Sukriti Nagb,c, Gerald Proteaua, Malcolm J. Robba, Andrew I.M. Campbella, Michael A. Kuliszewskia, Michael J.B. Kutryka,d and Duncan J. Stewart*,a,c,d

aTerrence Donnelly Heart Center, St. Michael's Hospital, Toronto, Ontario, Canada
bThe Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada
cThe Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
dThe Department of Medicine, University of Toronto, Toronto, Ontario, Canada

* Corresponding author. Division of Cardiology, Terrence Donnelly Heart Center, St. Michael's Hospital, 30 Bond Street, Room 7081, Queen Wing, Toronto, Ontario, Canada M5B 1W8. Tel.: +1-416-864-5724; fax: +1-416-864-5914. E-mail address: stewartd{at}smh.toronto.on.ca

Received 4 February 2004; revised 5 May 2004; accepted 19 May 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: This study sought to characterize changes in the angiopoietin system in a rat model of myocardial infarction (MI). Background: Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) bind to the endothelial-specific receptor tyrosine kinase, TIE-2. Ang-2 has been suggested to be an antagonist of TIE-2, possibly acting to release endothelial cells from the tonic stabilizing influence of Ang-1. However, on prolonged exposure, Ang-2 has been shown to acquire agonistic activity at TIE-2, raising the possibility that this isoform may play a direct role in neovascularization. Methods: Sprague–Dawley rats were subjected to left coronary ligation and myocardial tissues were harvested from the infarct and peri-infarct regions, or from non-infarcted myocardium. Changes in gene expression were determined by RT-PCR and confirmed by Northern analysis. Changes in protein expression were confirmed by Western analysis and immunocytochemistry, and TIE-2 activity was determined by immunoprecipitation with anti-TIE-2 and antiphosphotyrosine immunoblotting. Results: At 24 h, Ang-1 mRNA and protein expression within the infarct and peri-infarct regions were decreased compared to non-infarcted myocardium, whereas Ang-2 mRNA levels were markedly increased and TIE-2 expression was unchanged. Immunohistochemical staining revealed Ang-1 and TIE-2 immunoreactivity localized to vascular endothelium. In the infarct territory, Ang-2 immunostaining was localized primarily to invading leukocytes at 24 h. At 1 week, Ang-1 expression was partially restored, whereas Ang-2 expression remained elevated. At the time of peak elevation in Ang-2, Tie2 phosphorylation was found to be markedly increased, consistent with receptor activation. Conclusions: Thus, myocardial ischemia induced by left coronary artery ligation resulted in a sustained increase in Ang-2 expression and a reciprocal decrease in Ang-1, consistent with a predominant role for Ang-2 in the angiogenic response to MI.

KEYWORDS Angiogenesis; Infarction; Ischemia; Growth factors; Endothelial receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Angiopoietins represent a new family of angiogenic factors which bind the endothelial-selective receptor tyrosine kinase, TIE-2 [1]. Two principal angiopoietin isoforms have been identified, angiopoietin-1 (Ang-1), which activates TIE-2 and angiopoietin-2 (Ang-2), a putative endogenous TIE-2 receptor antagonist, thought to be a competitive inhibitor of the actions of Ang-1 [2]. Transgenic mice lacking either TIE-2 or Ang-1 exhibit embryonic lethality. Morphological evaluation of TIE-2- or Ang-1-deficient mice have demonstrated diminished vessel branching, poorly organized subendothelial matrix, loosening of endothelial cell contacts and generalized lack of recruitment of pericytes [3,4]. Transgenic overexpression of Ang-2 during embryogenesis also leads to a lethal phenotype similar to that seen in embryos lacking either Ang-1 or TIE-2 [2], consistent with an antagonistic action of Ang-2 in vivo. These data support a role for Ang-1/TIE-2 interactions in the expansion of the primitive vascular plexus into a hierarchical network of mature vessels composed of endothelial and medial smooth muscle cells and/or pericytes. Indeed, the angiopoietins appear to act in a complementary and coordinated fashion with VEGF, increasing the complexity and maturity of the neovasculature [5].

The angiopoietins also appear to play an important role in postnatal neovascularization. In the adult, Ang-1 and TIE-2 appear to be widely expressed in the quiescent vasculature, whereas Ang-2 is expressed only at sites of vascular remodeling [2]. In vitro studies have shown that hypoxia upregulates Ang-2 expression [6,7] and downregulates Ang-1 [8]. In a corneal micro-pocket model of angiogenesis, Asahara et al [9] showed that exogenous co-administration of Ang-1 with VEGF produced more numerous blood vessels than VEGF alone, whereas Ang-2 increased the length but not density of neovessels. In a rabbit hindlimb model of ischemia, direct intramuscular injection of Ang-1 plasmid DNA augmented collateral formation [10].

However, to date the effects of ischemia on Ang-1, Ang-2 or the TIE-2 receptor expression in vivo have not been defined. Detailed characterization of the spatial and temporal changes in endogenous Ang-1, Ang-2 and TIE-2 expression induced by myocardial ischemia is essential for the elucidation of the role of this new family of angiogenic mediators in coronary collateral vessel formation. In this study, we have looked at the changes in the angiopoietin system in the rat heart induced by permanent coronary artery occlusion. We now report that myocardial infarction (MI) was associated with a substantial increase in Ang-2 expression which was sustained for at least 1 week, whereas Ang-1 expression was decreased. These results suggest that the endogenous angiopoietin system, in particular Ang-2, may play an important role in neovascularization following MI and ischemia.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This 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 1996) and with the Canadian Council of Animal Care guidelines under a protocol approved by the Institutional Animal Care and Use Committee Guidelines at St. Michael's Hospital.

2.1. Experimental preparation
Male Sprague–Dawley rats (250–450 g) were anesthetized with a combination of ketamine (120 mg/kg) and xylazine (17 mg/kg) administered intraperitoneally, and ventilated with room air (Harvard Apparatus). The left main coronary artery was ligated near its origin using a 6.0 proline suture. Animals were subjected to permanent coronary artery occlusion for 24 h, 1 week or 6 weeks. Rats subjected to transient ischemia received 5 min of temporary coronary artery occlusion followed by 24 h of reperfusion by slipping a section of blunt ended PE190 tubing over the two loose ends of the suture to form a snare and clamping it tightly for 5 min against the myocardium. The presence of occlusion was confirmed by observing epicardial cyanosis in the area subserved by the occluded artery [11]. After the appropriate time-points the rats were re-anesthetized using ketamine/xylazine, and the hearts were rapidly excised and rinsed in room-temperature saline. The atria and right ventricular free wall were dissected free of the left ventricle (LV). The LV ventricle was divided into three regions: (1) infarct zone, (2) peri-infarct zone consisting of normal appearing myocardium surrounding the infarct zone, and (3) normal zone distant from the infarct and peri-infarct zones. The infarct zone could be readily distinguished from the normal myocardium surrounding it by its distinctive pale coloration. In the sham-operated and normal control animals, the entire left ventricle was quick-frozen. For immunohistochemistry, rats were sacrificed with 1.2 ml of T-61. The hearts were pressure-fixed in situ with 3% paraformaldehyde in phosphate buffered saline at 100 mm Hg.

2.2. Cells culture
Human THP-1 monocyte and human umbilical cord endothelial cell (HUVEC) were obtained from American Type Culture Collection (ATCC). HUVEC were grown in Kaighn's F12K medium containing endothelial cell growth factor (ECGF, Roche) and 10% fetal bovine serum. THP-1 monocytes were cultured in RPMI medium according to ATCC recommendations, and were differentiated to macrophages in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 162 nM), as described previously [12]. Confluent cultures of monocytes, macrophages and ECs were incubated in a humidified atmosphere of 5% CO2 in air at 37 °C (normoxia). For comparison, HUVEC were exposed to hypoxia (95%N2/5%CO2) for 24 h. Ang-1 and Ang-2 mRNA expression was determined by real-time quantitative RT-PCR.

2.3. Semi-quantitative (RT-PCR)
Total RNA was isolated using Trizol Reagents (Invitrogen) and 6 µg of total RNA was reverse transcribed in 60 µl volumes using Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen) with 1 µg random primers (Invitrogen). For each RT product, aliquots (2–10 µl) of the final reaction volume were amplified in four parallel PCR reactions using Ang-1,- Ang-2-, TIE-2-, or GAPDH-specific primers and Taq polymerase (Pharmacia Biotech Amersham). Partially degenerate oligonucleotides were used for Ang-2, as described previously [6]. The following primers were used: Ang-1 forward: 5'-CACGACAGACCAGTACAACACAAACG-3', reverse: 5'-GACGACTGTTGTTGGTGGTAGCTCT-3'; Ang-2 forward: 5'-GT(T/G)GA(T/C)TT(T/C)CAGAG(A/G/T/C)AC(A/G/T/C)CC(T/C)TTCCA-3', reverse: 5'-CGA(A/G)TAGCC(T/G)GA(A/G/T/C)CC(T/C)TTCCA-3'; TIE-2 forward: 5'-CAGGACCTTCACAACAGCTTCTATCGGACT-3', reverse: 5'-CTGTCGAAGAATGTCACTAAGGGTCCAAGC-3'; GAPDH forward: 5'-CTCTAAGGCTGTGGGCAAGGTCAT-3', reverse: 5'-GAGATCCACCACCCTGTTGCTGTA-3'.

The expected size of PCR products for Ang-1, Ang-2, TIE-2, and GAPDH were 570, 450, 322, and 343 bp, respectively. Quantification of RT-PCR products was done by ethidium bromide staining and densitometry. Results were evaluated as the ratio between the target genes and GAPDH. For the permanent occlusion experiments, five to seven animals were used for each time-point.

2.4. Real-time quantitative RT-PCR
Reverse transcription (RT) was carried out with 0.5 µg of total RNA using Omniscript reverse transcriptase according to the manufacturer's instruction (Qiagen). One microliter of each RTs was added to the real-time PCR amplification mixture (final volume, 30 µl) containing 15 µl of 2 x SYBR Green I Master Mix buffer (Applied Biosystems) and 0.5 µM forward and reverse primer. The following primers were used: VEGF165 forward: 5'-CATAGGAGAGATGAGCTTCCTGC-3', reverse: 5'-AACAAGGCTCACAGTGATTTTCTG-3'; Ang-1 forward: 5'-GCCACTTGAGAATTACATTGTGG-3', reverse: 5'-CGCGGATTTTATGCTCTAATCAACTG-3'; Ang-2 forward: 5'-GTCTCCCAGCTGACCAGTGGG-3', reverse: 5'-TACCACTTGATACCGTTGAAC-3'. GAPDH was amplified in parallel with the genes of interest using primers from Applied Biosystems. Reactions were run on an ABI PRISM 7900HT Sequence Detector (Applied Biosystems, Foster City, CA). The cycling conditions were as follows: 95 °C, 10 min (1 x) [95 °C, 15 s, 60 °C, 60 s] (40 x). Following the amplification, a dissociation curve was generated by increasing the temperature from 50 to 95 °C at a maximum ramp setting of 0.25 °C/min to ensure only a single product was amplified. Data were analyzed using SDS 2.1 (Applied Biosystems) to obtain the threshold cycle (Ct) values. Results were calculated as the expression difference between samples comparing the peri-infarct or infarct myocardium to the normal LV, normalized to the gene of reference (GAPDH). We used the 2T–{Delta}{Delta}C method to determine the relative gene expression between groups, as described by Livak and Schmittgen [13].

2.5. Northern blotting
Ten micrograms of total RNA was electrophoresed on 1.1% formaldehyde-agarose gel and then transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech), covalently linked, using a UV cross-linker system (Stratagene, La Jolla, CA) and then hybridized with Ang-1, Ang-2, TIE-2, and GAPDH probes. A 800-bp HindIII/XbaI restriction enzyme fragment, excised from a pFLAG-CMV-1 expression vector containing the full length of human Ang-1 (kindly provided by Dr. G.Y. Koh, Woosuk University, Chonju, South Korea), was used as an Ang-1 probe. The Ang-2 and TIE-2 probes were generated by RT-PCR, with primers described above. GAPDH cDNA was obtained from Ambion (Austin, TX USA). cDNA probes were labeled to a specific activity of at least 1 x 109 cpm/µg with {alpha}-[32P]dCTP (Amersham) by the random decamer (10-mer) primer extension-labeling standard technique (DECAprime II DNA labeling kit, Ambion). Northern blotting was performed as previously described [14].

2.6. Immunoblotting
Frozen tissue samples were homogenized in RIPA lysis buffer according to standard procedures (Santa Cruz Biotechnology, Santa Cruz, CA). Total protein (150 µg) was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 4–20% Tris–glycine gels (Invitrogen), subsequently transferred to nitrocellulose membranes and blocked in 5% non-fat milk in TBST buffer (10 mM/l Tris 150 mM/l NaCl, pH 7.5) and 0.1% Tween 20. The membranes were then incubated overnight with goat anti-rat polyclonal antibodies to Ang-1 or Ang-2 (Santa Cruz Biotechnology) at a dilution of 1:200, in TBS with 2% skim milk, followed by an anti-goat IgG secondary antibody conjugated to horseradish peroxidase (1:2000, 1 h; Santa Cruz Biotechnology). Angiopoietin-specific bands (70 kDa) were visualized using an enhanced chemiluminescence substrate system ECL (Amersham Pharmacia Biotech). Densitometry was performed and the intensity of each band was analyzed using the Molecular Analyst software (Imaging Densitometer, Bio-Rad). Six animals were studied in each group.

2.7. TIE-2 receptor phosphorylation
Tissue samples were homogenized in RIPA buffer and 500 µg of total protein from each tissue lysate were immunoprecipitated with a TIE-2 polyclonal antibody (Santa Cruz Biotechnology) as previously described [15]. The membranes were probed with a TIE-2 monoclonal antibody (clone Ab33, Upstate Biotechnology; 0.5 µg/ml) for total TIE-2 signal and then with an anti-phosphotyrosine antibody (4G10, Upstate Biotechnology; 1:3000) for receptor phosphorylation state as described [15]. Bands were visualized using the ECL system and densitometric analysis was performed as described above. This experiment was performed in triplicate.

2.8. Immunohistochemistry
Transverse slabs of the heart containing the infarcted area were processed for paraffin sectioning using standard techniques. Sections (6 µm) were stained with hematoxylin and eosin for histological analysis and adjacent sections were used for immunohistochemistry. The indirect streptavidin–biotin peroxidase method was used and paraffin sections were pretreated with 0.5% pepsin for 30 min at 37 °C prior to overnight incubation in primary antibody at 4 °C. Dilutions of antibodies (Santa Cruz Biotechnology) were as follows: goat anti-Ang-1 and Ang-2, 1:100; and rabbit anti-TIE-2, 1:550. The controls for immunohistochemistry included: (1) omission of the primary antibody, and (2) use of non-immune serum in place of the primary antibody. Immunohistochemistry was not performed for the 6-week time-point as there were no changes in either RNA or protein expression for any of the genes studied.

Double labeling for Ang-1 and Ang-2 was also done using link antibodies conjugated with fluorochrome dyes. Sections were first reacted with antibody to Ang-2 overnight at 4 °C and then with a biotinylated link antibody followed by streptavidin-CyTM3 (Jackson Immunoresearch Laboratories, Westgrove, PA). Hence the presence of Ang-2 was detected by red fluorescence. Sections were then blocked with normal rabbit serum and incubated for 2 h with Ang-1 antibody at room temperature followed by rabbit anti-goat-Alexa FluorTM488 antibody (Molecular Probes, Eugene, OR). Hence Ang-1 was detected by green fluorescence. These sections were analyzed using a Nikon Optiphot microscope and a MRC 600 confocal laser scanning microscope (Bio-Rad). Images were merged using Adobe Photoshop 6.0 software. Co-localization of Ang-1 and Ang-2 was indicated by yellow fluorescence.

2.9. Statistical analysis
Comparison of levels of expression and phosphorylation in the infarct and peri-infarct regions to that in the normal myocardium of the same heart was performed by paired t-tests. For all other comparisons, a one-way ANOVA with post hoc Tukey's HSD test (also known as Tukey's A) was used. p<0.05 was accepted as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Myocardial ischemia model
Fifty-one animals surviving the initial perioperative period following permanent left coronary artery occlusion were entered into the study. Of these, 44 animals survived to the predetermined end-study (85%), 39 of which exhibited clear evidence of myocardial infarction and were subsequently used for further analysis. Of the five rats subjected to transient ischemia, all exhibited clear cyanosis during the period of occlusion. In addition, nine animals were sacrificed without prior surgery and served as normal controls. Six additional animals were subjected to thoracotomy with placement of a suture in the myocardium without coronary artery ligation and served as sham-operated controls. In rats subjected to permanent coronary artery occlusion, infarct size was measured in histological sections in a subset of hearts at 1 day, 3 days and 1 week, and showed variation in size ranging from 15% to 60% of the left ventricle with an average size of approximately 40% of the left ventricle.

3.2. Alterations in angiopoietin–TIE-2 system after permanent coronary artery occlusion
3.2.1. RT-PCR
Twenty-four hours after MI, Ang-1 mRNA levels were consistently reduced in the infarct and peri-infarct zones (Fig. 1) to 46±11% and 87±6% of non-infarcted LV, respectively (p<0.005). At the same time, Ang-2 levels were increased in both the infarct and peri-infarct zones (241±71% and 222±63% of non-infarcted LV, respectively, p<0.05). There were no significant changes in TIE-2 expression in either the infarct or peri-infarct zone at this time-point. No differences in expression of Ang-1 and Ang-2 were found in different regions of the LV from sham-operated animals (data not shown).


Figure 1
View larger version (56K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Ang-1, Ang-2, and TIE-2 mRNA expression in the rat myocardium 24 h and 1 week after permanent coronary artery occlusion. Panel A shows representative RT-PCR results from LV samples taken from the non-infarcted (normal) myocardium (NI), or within the peri-infarct (PI) or infarct (I) zones of three MI rats and two sham-treated animals after 24 h. Summary data of semi-quantitative RT-PCR results at 24 h and 1 week after permanent coronary artery occlusion are shown in panel B. Expression of all genes was normalized to the GAPDH signal of the same sample. Each bar represents mean data from five to seven rats per time-point±S.E.M. * indicates p<0.05 as compared to the normal myocardium in the same heart.

 
The decrease in Ang-1 and the increase in Ang-2 persisted for at least 1 week after coronary artery ligation in the infarct zone, whereas, in the peri-infarct zone, levels of both Ang-1 and Ang-2 had returned to baseline values by 1 week. TIE-2 mRNA levels in the infarct zone remained unchanged at 1 week (137±22%, p=0.16).

At 6 weeks post-infarction, mean values for Ang-1, Ang-2, and TIE-2 levels were not significantly different from those in the non-infarcted myocardium (data not shown). As well, there were no significant differences in Ang-1, Ang-2, or TIE-2 levels at any time-point measured either in non-infarcted myocardium from MI animals or normal myocardium from sham-operated or normal rats. Representative Northern blots and summary data for the densitometric analysis at 24 h post MI are presented in Fig. 2. The findings from Northern analysis were similar to the RT-PCR findings.


Figure 2
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Representative composite Northern blots (panel A) showing Ang-1, Ang-2, and TIE-2 mRNA expression in normal myocardium from sham-operated rats, compared to samples taken from non-infarcted myocardium (NI) and from within the peri-infarct (PI) and infarct (I) zones at 24 h after permanent coronary artery occlusion. Summary data for Ang-1, Ang-2, and TIE-2 expression at 24 h after permanent coronary artery occlusion is shown in panel B (n=3–5 for each group). Expression of each gene is normalized to the GAPDH signal in the same sample. Data are shown as mean±S.E.M. *p<0.05 compared to normal myocardium.

 
3.3. Immunohistochemistry
Control rats showed selective endothelial immunostaining for Ang-1 in all cardiac vessels by both immunocytochemistry (Fig. 3A) and immunofluorescence (Fig. 4A), whereas there was no immunoreactivity with the Ang-2 antibody (Fig. 3B). As well, Ang-1 immunostaining was not observed when non-immune serum was substituted for primary antibody. Rats subjected to 24 h of coronary artery occlusion showed marked reduction in endothelial Ang-1 immunoreactivity in vessels in the infarct zone (Fig. 3C). However, polymorphonuclear leukocytes present in the infarcted region stained strongly for both Ang-1 and Ang-2, the latter showing more consistent and homogeneous pattern of immunostaining (Fig. 3C,D). Merged confocal images showed co-localization of Ang-1 and Ang-2 in the polymorphonuclear leukocytes (Fig. 4B). As well, a few vessels in the peri-infarct zone showed endothelial Ang-2 immunoreactivity (Table 1). In contrast, vessels of all sizes distant from the infarct zone showed strong endothelial Ang-1 immunoreactivity, similar to those of controls and were non-reactive for Ang-2 (Table 1). Ang-1 and Ang-2 immunoreactivity persisted in fewer inflammatory cells present within the lesion at 3 days. As well, Ang-1 immunoreactivity was seen in occasional necrotic myofibres, and neovessels within the infarct and the surrounding regions (Table 1). At this time, an increasing number of vessels within the infarct showed endothelial Ang-2 immunoreactivity as compared with the 24-h time period. At 1 week, most vessels within the infarct, including the neovessels, showed definite endothelial Ang-1 immunoreactivity (Fig. 3E). Neovessels were identified as clusters of vessels at the margin of the infarct having varying lumen diameters and wall thickness, as shown well in Figs. 3E,F and 4C,DGo. Merged confocal images showed endothelial co-localization of Ang-1 and Ang-2 both in vessels within the infarct as well as in neovessels (Fig. 4C,D). The early decreases in Ang-1 and increases in Ang-2 protein expression in the infarct and peri-infarct zones were confirmed by Western blot analysis (Fig. 5).


Figure 3
View larger version (130K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Immunolocalization of Ang-1 (A, C, E) and Ang-2 (B, D, F) are shown in control hearts (A, B) and post-infarction (C–F). All myocardial vessels of control rats show endothelial Ang-1 immunostaining (A) and lack of Ang-2 (B). At 24 h post-infarction, there is loss of endothelium in vessels within the infarct and loss of EC Ang-1 (arrowheads, C) while occasional peri-infarct vessels show endothelial Ang-2 (arrowheads, D). The polymorphonuclear leukocytes are immunoreactive for both Ang-1 and Ang-2 (C, D). At 7 days post-infarction, most neovessels show endothelial Ang-1 immunostaining (E) and many peri-infarct vessels still show endothelial Ang-2 (arrowheads, F). A, B, x 250; C–F, x 500. These data are representative of at least three separate experiments.

 

Figure 4
View larger version (185K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Merged confocal images dual stained for Ang-1 and Ang-2. (A) All myocardial vessels of control rats show endothelial Ang-1 (green) but not Ang-2 (red). (B) At 24 h post-infarct, microvessels in the peri-infarct zone show endothelial Ang-1 (green). The merged picture shows co-localization (yellow) of Ang-1 and Ang-2 in polymorphonuclear leukocytes while the inset shows only Ang-2 immunoreactivity in the same cells. (C) At day 7 post-infarction, peri-infarct vessels show co-localization of Ang-1 and Ang-2 (yellow, arrowheads). Neovessels, which are clustered and have varying diameters, also show co-localization of Ang-1 and Ang-2 (yellow, arrowhead) while in other areas they show endothelial Ang-1 only (D). Scale bar=50 µm. The images are representative of three replicates.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Semi-quantitative grading of the number of myocardial vessels per 7.36 mm2 showing endothelial immunoreactivity (IR) for angiopoietin-1 and -2 for sham and MI rat hearts

 

Figure 5
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Representative Western blots showing Ang-1 and Ang-2 protein expression at 24 h post permanent coronary artery occlusion (panel A). In the first lane the top panel shows purified protein (Ang-2, 20 ng/lane) and the bottom panel shows a normal LV sample from a sham-operated animal (Ang-1) as additional controls. Ang-1 and Ang-2 appear as a single band at 70 kDa. Summary data for angiopoietin protein expression at 24 h and 1 week after coronary occlusion is shown in panel B. Each bar represents mean±S.E.M. for four separate experiments, with the exception of Ang-2 at 24 h, which included six animals in each group. *p<0.05; **p<0.01, compared to normal myocardium of the same heart.

 
3.4. Angiopoietin expression in inflammatory cells
To further explore the role of inflammatory cells in the de novo production of angiopoietins post myocardial infarction, we exposed THP-1 monocytes to TPA for 48 h. In the monocyte cell line, basal levels of both angiopoietins were very low; however, when THP-1 cells were activated by TPA to undergo differentiation into adherent macrophage-like cells, there was a 2.7-fold increase in Ang-1 (p=0.07, n=4) and a 6.95-fold increase in Ang-2 mRNA expression (p=0.03, n=4). Furthermore, in HUVEC exposed to hypoxia, Ang-2 levels increased while Ang-1 levels decreased (data not shown) in agreement with previously reported findings [6,8].

3.5. TIE-2 receptor autophosphorylation post MI
The phosphorylation state of TIE-2 was determined at 24 h, a time period at which the greatest changes in Ang-1 and Ang-2 expression were observed. As shown in Fig. 6A, there was only a modest phosphotyrosine signal in the non-infarcted myocardium. However, in the infarct and peri-infarct regions there was a profound increase in TIE-2 phophorylation reaching levels >10-fold baseline. As shown in Fig. 6B, there was a strong relationship between increases in Ang-2 protein expression and increased TIE-2 phosphorylation, with an R2 value approaching unity.


Figure 6
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Relationship between increased Ang-2 protein expression and TIE-2 phosphorylation. Panel A shows representative immunoblots of Ang-2, phosphorylated TIE-2 (pTIE-2), and total TIE-2. In panel B, the fold-increase in pTIE-2 is plotted against the increase in Ang-2 protein expression in both the peri-infarct and infarct regions for three separate animals.

 
3.6. Relationship between the expression profiles of VEGF and Ang-2
To obtain a quantitative measure of VEGF expression relative to Ang-1 and Ang-2 in our model, we used quantitative real-time RT-PCR. The time course of changes in the expression of VEGF and its receptors has previously been described in detail [16]. In agreement with the previous report, VEGF mRNA levels were significantly increased in both the infarct and peri-infarct zones 24 h after MI (Fig. 7), correlating with elevated Ang-2 mRNA. However, if anything the magnitude of increase in Ang-2 was greater than that for VEGF. As before, Ang-1 mRNA levels were consistently reduced in the infarct and peri-infarct zones. Results from real-time RT-PCR also confirmed data previously obtained by semi-quantitative RT-PCR for Ang-1 and Ang-2 expression patterns (Figs. 1 and 2)Go.


Figure 7
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Changes in VEGF, Ang-1, and Ang-2 mRNA expression as detected by real-time PCR in the rat myocardium 24 h after permanent coronary artery occlusion. Results were calculated as the fold expression difference between infarct or peri-infarct comparing to non-infarcted (normal) LV when normalized to GAPDH signal of the same sample using mathematical 2T–{Delta}{Delta}C model [13]. Each bar represents mean data from three rats±S.D. * indicates p<0.05 as compared to the normal myocardium in the same heart.

 
3.7. Alterations the angiopoietin–TIE-2 system after transient coronary artery occlusion
After 5 min of transient coronary artery occlusion followed by 24 h reperfusion, Ang-1 mRNA expression was modestly, but significantly, decreased in the ischemic zone (84±3% of normal LV, p=0.05). Ang-2 and TIE-2 levels were not significantly altered in either the ischemic or peri-ischemic zone (Fig. 8).


Figure 8
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Summary data of semi-quantitative RT-PCR results for Ang-1, Ang-2, and TIE-2 24 h after transient coronary artery occlusion. For analysis, the expression level of all genes was normalized to the GAPDH signal of the same sample, and each bar represents five rats per time point. Data are shown as mean±S.E.M. * indicates p<0.05 compared to the normal myocardium of the same heart.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present report describes changes in the spatial and temporal patterns of Ang-1, Ang-2, and TIE-2 expression in response to myocardial ischemia and infarction. In the rats subjected to permanent coronary artery occlusion, Ang-2 expression was increased in the infarct and peri-infarct regions of myocardium relative to the non-infarcted myocardium. The greatest increases in Ang-2 were observed 24 h after MI, prior to the onset of neovascularization in this model, and persisted for up to 1 week, a time period during which neovascularization was prominent [17–19].

The profound increase in myocardial Ang-2 expression was localized mainly to the periphery of the infarct and associated with invading leukocytes, which also exhibited heterogeneous expression of Ang-1. To our knowledge, this is the first report of angiopoietin expression by inflammatory cells in the heart; however, Ang-2 immunostaining has been demonstrated in alveolar macrophages in a mouse acute lung injury model [20]. The mechanism of induction of angiopoietin expression by leukocytes post MI is not yet known. Hypoxia and cytokines, including VEGF, basic fibroblast growth factor (bFGF) and tumour necrosis factor-{alpha} (TNF-{alpha}) [6,7], have been shown to upregulate endothelial Ang-2 expression in various cell types. Indeed, activation of THP-1 monocytes with TPA in vitro markedly increased the expression of Ang-2, and less so, Ang-1, consisting with the pattern of expression observed in inflammatory cells within the infarct zone (Fig. 3). Previous studies have demonstrated that the activated leukocytes produce a number of different angiogenic peptides such as IL-8 [21], Gro-{alpha} [22], TNF-{alpha} [23], transforming growth factor-β (TGF-β) [24], as well as VEGF [21,25]. These cytokines appear to be involved in the classic acute phase response to injury, which may be critical for tissue repair and healing.

Ang-2 expression was also evident in endothelial cells at the forefront of vessels invading the infarct zone, a pattern similar to that seen in highly angiogenic glioblastomas [26]. In the ovary, high levels of both Ang-2 and VEGF were observed during growth and neovascularization of the follicle, whereas Ang-2 expression alone was associated with involution of the follicle and vascular regression [2]. This pattern of expression has led to the suggestion that Ang-2 may act in concert with VEGF as a permissive factor in the earliest stages of angiogenesis by releasing endothelial cells from the inhibitory actions of Ang-1 [2,27]. However, increased Ang-2 (and decreased Ang-1) expression in the rat MI model persisted for more than 1 week after the onset of ischemia, too long to be involved solely in the initiation of the angiogenic response. As well, since Ang-1 has been shown to potentiate angiogenesis in response to VEGF in vivo [9], the continued expression of a TIE-2 antagonist would seem to be counter-productive to efficient neovascularization.

In the present study, the activation state of TIE-2 was determined by immunoprecipitation and phosphotyrosine immunoblotting at a time-point corresponding to the maximal decreases in Ang-1 and increases in Ang-2 expression. Rather than a decreased level of Tie2 activity, there was marked activation of TIE-2 which was highly correlated with the increase in Ang-2 expression. Interestingly, at least two groups have recently reported that Ang-2 may promote angiogenesis in vitro and increase endothelial cells survival [15,28] through the direct activation of endothelial TIE-2 in certain conditions, including during prolonged exposure [15] or at high concentrations [28]. Thus, Ang-2 may have dual activities in myocardial ischemia [29], initially blocking the effect of Ang-1 by acting as a TIE-2 antagonist, enhancing EC activation in response to VEGF, and then later contributing to the stabilization and maturation of newly formed neovessels as a context-dependent agonist of the TIE-2 receptor. To our knowledge, this is the first demonstration of activation of TIE-2 associated with a selective increase in Ang-2 expression in vivo.

In contrast to Ang-2, Ang-1 was downregulated throughout the period of neovascularization of the healing infarct. This is in agreement with a previous report describing prolonged reduction in Ang-1 expression following MI in the rat using a cDNA micro-array approach [30]. Hypoxia has also been reported to downregulate Ang-1 in rat glioma cells in vitro [8]; however, systemic hypoxemic hypoxia in the rat has been shown either to increase [31] or decrease [32] Ang-1 expression, whereas Ang-2 was transiently increased [31]. Ang-1 production was also downregulated by a number of angiogenic growth factors, including epidermal growth factor (EGF), transforming growth factor (TGF-β), and platelet-derived growth factor (PDGF) [8]. In the current study, we also observed a significant decrease in Ang-1 expression after transient ischemia and reperfusion. Coronary artery occlusion of 5 min or less has been shown previously to increase expression of bFGF and VEGF [33]. Capillary density can also be increased by such short episodes of transient ischemia, persisting for up to 1 month after the acute ischemic event [33]. The period of transient ischemia used in the current study was not associated with infarction in this model [33], and therefore, this permits the examination of the effects of ischemia on the angiopoietin–TIE-2 system, independent from any changes that occur secondary to myocardial infarction.

This investigation represents the first comprehensive analysis of the alterations in expression and tissue distribution of the angiopoietin system in a relevant model of myocardial ischemia and infarction. Our results indicate that there are profound and sustained changes in Ang-1 and Ang-2 gene expression, and provide a basis for a more complete understanding of the potential role of this novel angiogenic system in neovascularization and tissue repair following MI. In particular, they suggest a critical role for Ang-2 in the angiogenic response to myocardial infarction, and support further research into the complex interactions between Ang-1 and Ang-2 at the TIE-2 receptor, and into their functional relevance in vivo.


   Acknowledgements
 
DJS is the Dexter H.C. Man Chair of Cardiology at the University of Toronto.


   Notes
 
1 RS and KT-K contributed equally to this study. Back

* This work was supported by grants from the Canadian Institutes of Health Research (Award #62695) and the Heart and Stroke Foundation of Ontario (Award #NA-4789). Back

Time for primary review 27 days


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

  1. Davis S., Aldrich T.H., Jones P.F., et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell (1996) 87:1161–1169.[CrossRef][ISI][Medline]
  2. Maisonpierre P.C., Suri C., Jones P.F., et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science (1997) 277:55–60.[Abstract/Free Full Text]
  3. Dumont D.J., Gradwohl G., Fong G.H., et al. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev (1994) 8:1897–1909.[Abstract/Free Full Text]
  4. Suri C., Jones P.F., Patan S., et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell (1996) 87:1171–1180.[CrossRef][ISI][Medline]
  5. Thurston G., Suri C., Smith K., et al. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science (1999) 286:2511–2514.[Abstract/Free Full Text]
  6. Mandriota S.J., Pepper M.S. Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia. Circ. Res (1998) 83:852–859.[Abstract/Free Full Text]
  7. Oh H., Takagi H., Suzuma K., Otani A., Matsumura M., Honda Y. Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells. J. Biol. Chem (1999) 274:15732–15739.[Abstract/Free Full Text]
  8. Enholm B., Paavonen K., Ristimaki A., et al. Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia. Oncogene (1997) 14:2475–2483.[CrossRef][ISI][Medline]
  9. Asahara T., Chen D., Takahashi T., et al. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ. Res (1998) 83:233–240.[Abstract/Free Full Text]
  10. Shyu K.G., Manor O., Magner M., Yancopoulos G.D., Isner J.M. Direct intramuscular injection of plasmid DNA encoding angiopoietin-1 but not angiopoietin-2 augments revascularization in the rabbit ischemic hindlimb. Circulation (1998) 98:2081–2087.[Abstract/Free Full Text]
  11. Liu Y., Downey J.M. Ischemic preconditioning protects against infarction in rat heart. Am. J. Physiol (1992) 263:H1107–H1112.[ISI][Medline]
  12. Tsuchiya S., Kobayashi Y., Goto Y., et al. Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res (1982) 42:1530–1536.[Abstract/Free Full Text]
  13. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) Method. Methods (2001) 25:402–408.[CrossRef][ISI][Medline]
  14. Babaei B., Teichert-Kuliszewska K., Monge J.-C., Mohamed F., Bendeck M.P., Stewart D.J. Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor. Circ. Res (1998) 82:1007–1015.[Abstract/Free Full Text]
  15. Teichert-Kuliszewska K., Maisonpierre P.C., Jones N., et al. Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2. Cardiovasc. Res (2001) 49:659–670.[Abstract/Free Full Text]
  16. Li J., Brown L.F., Hibberd M.G., Grossman J.D., Morgan J.P., Simons M. VEGF, flk-1, and flt-1 expression in a rat myocardial infarction model of angiogenesis. Am. J. Physiol (1996) 270:H1803–H1811.[Medline]
  17. Fishbein M.C., Maclean D., Maroko P.R. Experimental myocardial infarction in the rat: qualitative and quantitative changes during pathologic evolution. Am. J. Pathol (1978) 90:57–70.[Abstract]
  18. Sun Y., Weber K.T. Infarct scar: a dynamic tissue. Cardiovasc. Res (2000) 46:250–256.[Abstract/Free Full Text]
  19. Sun M., Opavsky A., Stewart D.J., et al. Temporal response and localization of integrins β1 and β3 in the heart following myocardial infraction. Circulation (2003) 107:1046–1052.[Abstract/Free Full Text]
  20. Karmpaliotis D., Kosmidou I., Ingenito E.P., et al. Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol (2002) 283:L585–L595.[Abstract/Free Full Text]
  21. Cassatella M.A. The production of cytokines by polymorphonuclear neutrophils. Immunol. Today (1995) 16:21–26.[CrossRef][ISI][Medline]
  22. Gasperini S., Calzetti F., Russo M.P., De Gironcoli M., Cassatella M.A. Regulation of GRO alpha production in human granulocytes. J. Inflam (1995) 45:143–151.[ISI][Medline]
  23. Dubravec D.B., Spriggs D.R., Mannick J.A., Rodrick M.L. Circulating human peripheral blood granulocytes synthesize and secrete tumor necrosis factor alpha. Proc. Natl. Acad. Sci. USA (1990) 87:6758–6761.[Abstract/Free Full Text]
  24. Grotendorst G.R., Smale G., Pencev D. Production of transforming growth factor beta by human peripheral blood monocytes and neutrophils. J. Cell. Physiol (1989) 140:396–402.[CrossRef][ISI][Medline]
  25. Gaudry M., Bregerie O., Andrieu V., El Benna J., Pocidalo M.A., Hakim J. Intracellular pool of vascular endothelial growth factor in human neutrophils. Blood (1997) 90:4153–4161.[Abstract/Free Full Text]
  26. Stratmann A., Risau W., Plate K.H. Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis. Am. J. Pathol (1998) 153:1459–1466.[Abstract/Free Full Text]
  27. Hanahan D. Signaling vascular morphogenesis and maintenance. Science (1997) 277:48–50.[Free Full Text]
  28. Kim I., Kim J.H., Moon S.O., Kwak H.J., Kim N.G., Koh G.Y. Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Oncogene (2000) 19:4549–4552.[CrossRef][ISI][Medline]
  29. Davis S., Papadopoulos N., Aldrich T.H., et al. Angiopoietins have distinct modular domains essential for receptor binding, dimerization and superclustering. Nat. Struct. Biol (2002) 10:38.[CrossRef][ISI]
  30. Stanton L.W., Garrard L.J., Damm D., et al. Altered patterns of gene expression in response to myocardial infarction. Circ. Res (2000) 86:939–945.[Abstract/Free Full Text]
  31. Ray P.S., Estrada-Hernandez T., Sasaki H., Zhu L., Maulik N. Early effects of hypoxia/reoxygenation on VEGF, Ang-1, Ang-2 and their receptors in the rat myocardium: implications for myocardial angiogenesis. Mol. Cell. Biochem (2000) 213:145–153.[CrossRef][ISI][Medline]
  32. Abdulmalek K., Ashur F., Ezer N., Ye F., Magder S., Hussain S.N.A. Differential expression of Tie-2 receptors and angiopoietins in response to in vivo hypoxia in rats. Am. J. Physiol. Lung Cell. Mol. Physiol (2001) 281:L582–L590.[Abstract/Free Full Text]
  33. Xie Z., Gao M., Koyama T. Effects of transient coronary occlusion on the capillary network in the left ventricle of rat. Jpn. J. Physiol (1997) 47:537–543.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 FASEB J.Home page
S. M. Dallabrida, N. S. Ismail, E. A. Pravda, E. M. Parodi, R. Dickie, E. M. Durand, J. Lai, F. Cassiola, R. A. Rogers, and M. A. Rupnick
Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy
FASEB J, August 1, 2008; 22(8): 3010 - 3023.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
H.-J. Cho, N. Lee, J. Y. Lee, Y. J. Choi, M. Ii, A. Wecker, J.-O. Jeong, C. Curry, G. Qin, and Y.-s. Yoon
Role of host tissues for sustained humoral effects after endothelial progenitor cell transplantation into the ischemic heart
J. Exp. Med., December 24, 2007; 204(13): 3257 - 3269.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
T. Gustafsson, H. Rundqvist, J. Norrbom, E. Rullman, E. Jansson, and C. J. Sundberg
The influence of physical training on the angiopoietin and VEGF-A systems in human skeletal muscle
J Appl Physiol, September 1, 2007; 103(3): 1012 - 1020.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
Y. Reiss, J. Droste, M. Heil, S. Tribulova, M. H.H. Schmidt, W. Schaper, D. J. Dumont, and K. H. Plate
Angiopoietin-2 Impairs Revascularization After Limb Ischemia
Circ. Res., July 6, 2007; 101(1): 88 - 96.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K.-G. Shyu
Enhancement of new vessel formation by Angiopoietin-2/Tie2 signaling in endothelial progenitor cells: A new hope for future therapy?
Cardiovasc Res, December 1, 2006; 72(3): 359 - 360.
[Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Daly, E. Pasnikowski, E. Burova, V. Wong, T. H. Aldrich, J. Griffiths, E. Ioffe, T. J. Daly, J. P. Fandl, N. Papadopoulos, et al.
Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells
PNAS, October 17, 2006; 103(42): 15491 - 15496.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. I. Dedkov, W. Zheng, and R. J. Tomanek
Compensatory growth of coronary arterioles in postinfarcted heart: regional differences in DNA synthesis and growth factor/receptor expression patterns
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1686 - H1693.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
K. Kobayashi, T. Kondo, N. Inoue, M. Aoki, M. Mizuno, K. Komori, J. Yoshida, and T. Murohara
Combination of In Vivo Angiopoietin-1 Gene Transfer and Autologous Bone Marrow Cell Implantation for Functional Therapeutic Angiogenesis
Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1465 - 1472.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. L. Lindsey, G. P. Escobar, L. W. Dobrucki, D. K. Goshorn, S. Bouges, J. T. Mingoia, D. M. McClister Jr., H. Su, J. Gannon, C. MacGillivray, et al.
Matrix metalloproteinase-9 gene deletion facilitates angiogenesis after myocardial infarction
Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H232 - H239.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sandhu, R.
Right arrow Articles by Stewart, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sandhu, R.
Right arrow Articles by Stewart, D. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us