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Cardiovascular Research Advance Access originally published online on February 6, 2008
Cardiovascular Research 2008 78(2):395-403; doi:10.1093/cvr/cvn033
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Assessment of {alpha}vβ3 integrin expression after myocardial infarction by positron emission tomography

Takahiro Higuchi1,*, Frank M. Bengel1, Stefan Seidl2, Petra Watzlowik1, Horst Kessler3, Renate Hegenloh4, Sybille Reder1, Stephan G. Nekolla1, Hans J. Wester1 and Markus Schwaiger1

1 Nuklearmedizinische Klinik und Poliklinik der Technischen Universität München, Klinikum rechts der Isar, Ismaninger Straße 22, 81675 Munich, Germany
2 Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, Klinikum rechts der Isar, Munich, Germany
3 Center of Integrated Protein Science in Department Chemie, Lehrstuhl II für Organische Chemie, Technische Universität München, Munich, Germany
4 Abteilung für Gefässchirurgie der Technischen Universität München, Klinikum rechts der Isar, Munich, Germany

* Corresponding author. Tel: +49 89 4140 2968; fax: +49 89 4140 4950. E-mail address: higuchi{at}po2.nsknet.or.jp

Received 22 August 2007; revised 30 January 2008; accepted 4 February 2008

Time for primary review: 21 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Study limitations
 6. Conclusions
 Funding
 References
 
Aims: The purpose of this study was to determine the feasibility of a new positron emission tomography (PET) imaging approach using an 18F-labelled {alpha}vβ3 integrin antagonist (18F-Galacto-RGD) to monitor the integrin expression after myocardial infarction.

Methods and results: Male Wister rats were subjected to 20 min transient left coronary artery occlusion followed by reperfusion. Autoradiographic analysis and in vivo PET imaging were used to determine myocardial 18F-Galacto-RGD uptake at different time points following reperfusion.

Results: PET imaging and autoradiography demonstrated no significant focal myocardial 18F-Galacto-RGD uptake in non-operated control rats and at day 1 after reperfusion. However, focal accumulation in the infarct area started at day 3 (uptake ratio = 1.91 ± 0.22 vs. remote myocardium), peaked between 1 (3.43 ± 0.57) and 3 weeks (3.43 ± 0.95), and decreased to 1.96 ± 0.40 at 6 months after reperfusion. Pretreatment with {alpha}vβ3 integrin antagonist c(-RGDfV-) significantly decreased tracer uptake, indicating the specificity of tracer uptake. The time course of focal tracer uptake paralleled vascular density as measured by CD31 immunohistochemical analysis.

Conclusion: Regional 18F-Galacto-RGD accumulation suggests up-regulation of {alpha}vβ3 integrin expression after myocardial infarction, which peaks between 1 and 3 weeks and remains detectable until 6 months after reperfusion. This new PET tracer is promising for the monitoring of myocardial repair processes.

KEYWORDS Angiogenesis; Infarction; Ischaemia; Reperfusion; Endothelial receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Study limitations
 6. Conclusions
 Funding
 References
 
Angiogenesis is considered an integral part of the repair process after an ischaemic injury and has been an increasing focus of cardiovascular research.1,2 Under the stimulation of hypoxia-induced angiogenic factors such as basic fibroblast growth factor, endothelial cells are activated, proliferate, and express molecules such as {alpha}vβ3 integrins. The {alpha}vβ3 integrins are thought to play an important role in migration, adhesion, and signal transduction.3

Modifications of the angiogenic response to ischaemia by means of cytokines and cell-based therapy are now being explored as potential treatment strategies to limit infarct size and prevent post-ischaemic remodelling.4,5 Attempts have been made to monitor the efficacy of these therapies by the recovery of perfusion or functional parameters,6–8 but results have been variable and sometimes inconclusive, indicating the need for specific imaging tools for monitoring of angiogenesis.

Recently, Meoli et al. first described up-regulated myocardial {alpha}vβ3 integrin expression after myocardial infarction utilizing111In-RP748 single photon emission tomography (SPECT) tracer and an animal model of coronary occlusion.9

18F-labelled glycosylated {alpha}vβ3 integrin antagonist (18F-Galacto-RGD) is a recently introduced positron emission tomography (PET) tracer that has been shown to target specifically {alpha}vβ3 integrin expression.10–12 This compound contains tripeptide sequence arginine–glycine–aspartic acid (RGD) in a constant conformation. It demonstrates high affinity {alpha}vβ3 integrin binding and rapid clearance from blood via the hepato-biliary system, indicating favourable characteristics for non-invasive PET imaging. The feasibility for monitoring of {alpha}vβ3 integrin expression in tumours has been already reported in animal and human clinical studies.10–15

The purpose of this study was to employ this new PET imaging approach to define the time course and intensity of myocardial integrin expression after transient ischaemia and reperfusion in a rat model and to correlate the imaging signal to immunohistochemical staining.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Study limitations
 6. Conclusions
 Funding
 References
 
2.1 Animal model of coronary occlusion and reperfusion
Experimental protocols were approved by the regional governmental commission of animal protection (Regierung von Oberbayern, Germany) and conform with Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Male Wister rats weighing 350–450 g were used in all experiments. Prior to intervention, rats were anaesthetized with intramuscular administration of midazolam (0.1 mg/kg), fentanyl (1 µg/kg), and medetomidin (10 µg/kg) (MMF) and mechanically ventilated. Left thoracotomy was performed to expose the heart. A 7–0 polypropylene suture on a small curved needle was passed through the myocardium beneath the proximal portion of the left coronary artery (LCA). The suture was ligated to occlude the LCA. Twenty minutes after the coronary occlusion, the suture was cut to obtain reperfusion.16,17 The occlusion and reperfusion were confirmed by the regional cyanosis of the myocardial surface and blush over in the risk area. The model of transient 20 min coronary occlusion was selected to induce myocardial infarction in approximately half of the area at risk as previously reported.18,19 Subsequently, the chest was closed, and the animals were allowed to recover. Sham-operated rats were subjected to identical treatment expect that the suture was not ligated.

2.2 In vivo PET imaging
Synthesis of 18F-Galacto-RGD was carried out as described elsewhere.11 A dedicated small-animal PET system (MicroPET Focus 120, SIEMENS Preclinical Solutions, Knoxville, TN) was used for in vivo detection of myocardial 18F-Galacto-RGD accumulation. Rats with ischaemia and reperfusion were imaged at different time points on day 3 (n = 6), 1 week (n = 6), or 3 weeks (n = 4) after reperfusion, as well as control sham-operated rats on 1 week after thoracotomy (n = 5). As a reference for the localization of left ventricular myocardium and infarct myocardium, 13N-ammonia perfusion PET imaging was performed prior to 18F-Galacto-RGD injection. 37 MBq of 13N-ammonia were injected, and 10 min data acquisition was initiated 5 min after tracer injection. Following the 13N-ammonia imaging, 37 MBq of 18F-Galacto-RGD were administrated, and 30 min data acquisition was started 90 min after the tracer injection (Figure 1). All rats were sacrificed immediately after imaging session, and hearts were excised for autoradiographic analysis.


Figure 1
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  		Figure 1 Schematic diagram illustrating the protocol of ischaemia and reperfusion in the experimental rat model following PET imaging. LCA, left coronary artery; IV, intravenous injection.

 
All data were acquired in three-dimensional (3D) list-mode and sorted into a 3D sinogram. The sinogram was reconstructed into a 128 x 128 x 95 voxel image by 2D-filtered back-projection image reconstruction using a ramp filter with the Nyquist limit (0.5 cycles/voxel) as the cut-off frequency. The voxel size equalled 0.43 x 0.43 x 0.80 mm3. Data were normalized and corrected for randoms, dead time, and decay.

2.3 PET image analysis
ASIPro software (Concorde Microsystems Inc., Knoxville, TN, USA) was used for PET image analysis. Regions of interest (ROI) were manually drawn around the focal 18F-Galacto-RGD uptake area in the myocardium on a transverse image. The mean radioactivity concentration within the ROI was expressed as the percentage of injected dose per tissue cubic centimetre (% ID/cc).

2.4 Dual tracer autoradiography
Dual tracer autoradiography was performed in one rat heart to assess area at risk during coronary occlusion and 18F-Galacto-RGD uptake simultaneously. 111 MBq of 18F-Galacto-RGD was administrated 1 week after reperfusion. Two hours after 18F-Galacto-RGD injection, 0.74 MBq of 201Tl was injected via tail vein just after re-occlusion of the LCA for the delineation of the area at risk. LCA re-occlusion was done as described elsewhere.20 One minute after the 201Tl administration, the heart was removed and sliced into 20 µm short-axis sections. Both 18F and 201Tl tracer activities were analysed using µIMAGER (Biospace, Paris).21

2.5 Time course of 18F-Galacto-RGD uptake
Rats after PET imaging (n = 21) and other rats (n = 23) were studied with autoradiography to assess serial changes of myocardial 18F-Galacto-RGD uptake after ischaemic intervention. We analysed rats at different time points after coronary occlusion and reperfusion including day 1 (n = 7), 3 days (n = 6), 1 week (n = 7), 3 weeks (n = 6), 3 months (n = 7), and 6 months (n = 6), and 1 week after a sham-operation (n = 5). All rats received intravenous administration of 37 MBq of 18F-Galacto-RGD and were sacrificed 2 h after the administration. The heart was excised, frozen and embedded in methylcellulose. Serial short axis sections in 1 mm intervals (20 µm thickness each), covering the entire heart were obtained using a cryostat (HM500OM microtome, Micrim, Walldort, Germany). The autoradiographic exposure for visualization of tracer uptake was performed for 12 h at 1–2 h after sacrifice. Distribution of the tracer was determined by analysis of the digitized autoradiographs (Phosphorimager 445 SI, Molecular Dynamics, Sunnyvale, CA, USA). ROI were manually defined for a focal myocardial tracer uptake region and a contra-lateral normal region on a mid-myocardial section. If no focal myocardial tracer accumulation was observed, ROI were placed over the anterolateral wall. The ROI size for sham-operated animals was similar to the same time point for animals with ischaemia. Radioactivity values of each ROI were expressed as background-corrected photostimulated luminesence units per area (in mm2). 18F-Galacto-RGD uptake ratio was calculated by dividing the value of the focal tracer uptake region with that of the contra-lateral normal area.

2.6 Blocking study
To assess the specificity of tracer uptake, 18 mg/kg of non-radiolabelled cyclo(-arginyl-glycinyl-aspartyl-D-phenylalanyl-valinyl-), c(-RGDfV-), a selective antagonist of {alpha}vβ3 integrin,22,23 was injected 10 min before 18F-Galacto-RGD (37 MBq) administration in rats (n = 7) at 1 week after reperfusion. Further PET imaging and autoradiographic analysis were carried out as described above.

2.7 Histology and immunohistochemistry
Hearts with coronary occlusion at day 1 (n = 3), day 3 (n = 4), week 1 (n = 5), week 3 (n = 2), and 6 months (n = 4) after reperfusion were analysed. Haematoxylin and eosin (HE) and immunohistochemistry staining was performed by use of standard techniques on 5 µm thick cryosections of the heart. For the detection endothelial cells and integrin β3 subunit, monoclonal mouse anti-rat platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31, 0.8 µg/mL) antibody (BD PharMingen, San Diego, CA, USA) and monoclonal mouse anti-rat CD61 (integrin β3 subunit, 5 µg/mL) antibody (BD PharMingen) were used. Vascular density was determined by CD31 immunohistochemical staining using a three-point scoring system performed by a pathologist (S.S.) blinded to the results of tracer uptake study (no-increased vasculature = 1, moderately increased = 2, and highly increased = 3).

2.8 Statistical analysis
All results were expressed as mean ± SD. Statistical analysis was done with StatMate III (ATMS Co., Ltd., Tokyo, Japan). Comparison between two groups was made by means of the unpaired Student’s t-test, and multiple group comparisons were made by ANOVA using ranks (Kruskal–Wallis test) followed by Dunn’s multiple contrast hypothesis test to identify differences of each groups. A value of P < 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Study limitations
 6. Conclusions
 Funding
 References
 
3.1 In vivo detection of 18F-Galacto-RGD uptake using small-animal PET imaging
Left ventricular myocardium was visualized by 13N-ammonia images in all animals, and the images were used as a reference for localization of 18F-Galacto-RGD uptake. Homogeneous 13N-ammonia distribution throughout the left ventricular myocardium was observed in sham-operated rats. However, 13N-ammonia images demonstrated focal decrease of tracer accumulation with visual assessment in the anterolateral wall of the reperfused animals (n = 18/22) indicating infarct myocardium.

No focal 18F-Galacto-RGD uptake in the chest region including myocardium was found in control animals and at day 1 after reperfusion. In contrast, focal 18F-Galacto-RGD uptake was observed in the reperfused myocardium of all rats at 1 and 3 weeks after reperfusion. Representative images at 1 week after sham operation and coronary occlusion and reperfusion are shown in Figure 2A. In the regions of 18F-Galacto-RGD uptake, slightly decreased myocardial perfusion was observed by 13N-ammonia perfusion PET.


Figure 2
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  		Figure 2 (A) In vivo PET images of 18F-Galacto-RGD uptake (upper row), 13N-NH3 perfusion (bottom row), and their fusion (middle row) in transverse view of a sham-operated rat heart without coronary occlusion (left column) and a rat with 20 min coronary occlusion 1 week after reperfusion (right column). Tracer accumulation is visible in the chest wall at the surgical incision area in both rats (arrow head), but focal 18F-Galacto-RGD uptake in the myocardium was observed only after coronary occlusion (arrow). (B) An example of time activity curve after tracer administration. (C) Correlation between percentage of injection dose per tissue (% ID/cc) by PET and uptake ratio by autoradiography in the focal 18F-Galacto-RGD uptake area.

 
Examples of time-activity curves of the region of focal tracer uptake, control myocardium, and blood pool are shown in Figure 2B. Tracer accumulation at the focal myocardial uptake area was already seen during the first 20 min, but blood pool activity decrease slowly. Better contrast for imaging was obtained in the later phase after 75 and 115 min.

Is has to be noted that increased 18F-Galacto-RGD uptake was also detected in the chest wall at the site of surgical incision in sham-operated rats and at week 1 and 3, consistent with angiogenesis associated with wound healing (Figure 2A).

Uptake analysis of PET imaging revealed a good linear relationship (r = 0.87) between 18F-Galacto-RGD uptake (%ID/cc) by PET imaging analysis and uptake ratio by autoradiographic analysis (Figure 2C).

3.2 18F-Galacto-RGD uptake changes after ischaemia by autoradiography
There was no focal radioactivity accumulation in the myocardium of non-operated and sham-operated rats. Only homogeneous background activity was seen throughout the myocardium. In study animals at day 1 after reperfusion, no focal tracer accumulation was observed. Focally increased tracer uptake in the infarct area was first observed at 3 days. Then, the focal 18F-Galacto-RGD uptake peaked between 1 and 3 weeks. Subsequently, the uptake showed decreased, but detectable, uptake even 6 months later. Typical autoradiography images and averaged uptake ratios by autoradiography analysis in different time points are shown in Figure 3. The uptake ratios at week 1, 3 weeks, and 3 months were significantly higher in comparison with that of control animals and that of day 1 after reperfusion.


Figure 3
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  		Figure 3 (A) Representative autoradiographs of 18F-Galacto-RGD uptake in the heart after ischaemia and reperfusion with three slices (apex, middle, and base) studied at different time points. (B) Time course of myocardial 18F-Galacto-RGD uptake ratio (vs. remote myocardium) after coronary occlusion and reperfusion by autoradiography.

 
The results of dual tracer autoradiography at week 1 are shown in Figure 4. Increased 18F-Galacto-RGD uptake was localized in the area at risk, and the uptake area matched well with the area of increased vascular density assessed by CD31 immunohistochemical analysis at 1 week after reperfusion. The focal 18F-Galacto-RGD uptake appeared to be most prominent in the border of the infarction area on day 3 (Figure 3A), and then expanded into the centre of infarct area from week 1.


Figure 4
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  		Figure 4 Corresponding images of 201Tl and 18F-Galacto-RGD activities and histology (HE and CD31 immunohistochemical staining) in a rat heart with 20 min coronary occlusion 1 week after reperfusion. 201Tl image demonstrates the area at risk, because tracer was injected during coronary re-occlusion. Intense 18F-Galacto-RGD uptake is seen in the area at risk where increased vascular density is also seen by CD31 immunohistochemical analysis (arrows).

 
3.3 Specificity of tracer uptake
The specific non-radiolabelled {alpha}vβ3 integrin antagonist, c(-RGDfV-),22,23 was able to reduce the tracer uptake caused by coronary occlusion, demonstrating receptor-specific tracer uptake at 1 week after reperfusion. The average tracer uptake ratio assessed in a group of rats treated with the specific antagonist was significantly lower than that of non-treated rats at 1 week after reperfusion (Figure 5). Uptake value assessed by in vivo PET imaging in a group treated with the specific antagonist was significantly lower compared with non-treated rats, consistent with the autoradiographic results.


Figure 5
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  		Figure 5 Results of blocking studies in rats treated with non-radiolabelled c(-RGDfV-) peptide in rats 1 week after reperfusion. Autoradiographs (A), uptake ratio by autoradiography (B), PET images (fusion PET images with 18F-Galacto-RGD in color scale and 13N-ammonia in grey scale) (C), and percentages of tracer injected dose (%ID/cc) as measured by PET (D).

 
3.4 Histology and immunohistochemistory
The increase in vessel formation was not detectable by CD31 immunohistochemical analysis at day 1. At day 3 after reperfusion, there were an increased number of CD31 positive vessels especially at the border zone of the infarcted area. These vessels also displayed a stronger β3 integrin staining than normal myocardium or vascular structures in non-infarcted areas. One and 3 weeks after reperfusion, acute inflammatory cell infiltration decreased; at the same time, the incidence of spindle-shaped fibroblasts increased. The vessels displayed a higher positivity for β3 integrin than normal vessels. After 6 months, the vascular density in the reperfused area remained increased, but considerably lower as compared with 1 or 3 weeks (Figure 6).


Figure 6
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  		Figure 6 Histology with haematoxylin and eosin staining (HE) and immunohistochemical analysis for endothelial cells (CD31) and integrin subunit β3 expression (β3) in normal myocardium and ischaemic myocardium at different time points after coronary occlusion and reperfusion. In normal myocardium, positivity for CD31 and integrin β3 expression in vessels is shown as a reference [arrows in (B) and (C)]. One day after reperfusion, necrotic myocardium with loss of nuclear staining and acute inflammatory reaction dominating neutrophils [arrowheads in (D)] were confirmed by HE at the ischaemic area, but new vessel formation was not detectable by CD31 analysis (E). Additionally, the acute inflammatory cells stained negative for β3 integrin subunit [arrowhead in (F)]. At 3 days, there was still intense inflammatory infiltration with neutrophils and histiocytes in necrotic myocardium (G). An increased amount of very small vessels especially at the border zone of the infarcted area was noted by CD31 analysis [arrows in (H)]. These small vessels had a much stronger positivity for β3 integrin than normal myocardium or vessels in noninfarcted areas [arrows in (I)]. One and 3 weeks after reperfusion, acute inflammatory cell infiltration decreased with increasing amounts of spindle-shaped fibroblasts, and vessels with HE in infarcted area were visible [arrows in (J) and (M)]. There was a high density of vessels in the whole infarcted area [(K), (N)] and the vessels still had a much higher positivity for β3 integrin subunit than normal vessels [arrows in (L) and (O)]. After 6 months, there was a scar with fibroblasts (P). The vascular density was still increased [arrows in (Q)], but if compared with the infarcted area after 1 or 3 weeks, it was reduced. The vessels had a stronger positivity for β3 integrin than in normal myocardium [arrows in (R)].

 
The time course of tracer uptake peaked between 1 and 3 weeks paralleling the vascular density score as determined by CD31 immunohistochemical staining (Figure 7), but did not correlate with intensity of acute inflammatory cell infiltration which peaked at day 3.


Figure 7
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  		Figure 7 Time course of vascular density score after coronary occlusion and reperfusion assessed by CD31 immunohistochemical and 18F-Galacto-RGD uptake ratio by autoradiography.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Study limitations
 6. Conclusions
 Funding
 References
 
This study demonstrated for the first time the feasibility to measure myocardial integrin expression non-invasively with PET and 18F-Galacto-RGD. Focal 18F-Galacto-RGD uptake was observed after a brief episode of coronary occlusion and reperfusion. The increased tracer uptake was blocked by an antagonist of {alpha}vβ3 integrin indicating the specificity of the tracer signal. The time course of tracer uptake was characterized by delayed onset, maximum intensity between 1 and 3 weeks after intervention, followed by a decrease which correlated well with neovascularization as assessed by immunohistochemical CD31 staining. The 18F-Galacto-RGD uptake did not coincide with acute neutrophilic cell infiltration. These data suggest the feasibility of in vivo monitoring of angiogenesis following a myocardial infarction by 18F-Galacto-RGD PET.

The pathophysiological understanding of angiogenesis with regard to molecular, genetic, and cellular mechanisms contributed to the development of therapeutic interventions to stimulate angiogenesis. They include cytokine therapies, gene therapy, and cell transplantation in order to prevent the development of heart failure.24,25 Clinical studies have been initiated and first results of large clinical trials employing recombinant proteins or gene therapy have been published.6,7,26,27 However, the results were variable and raised many methodological questions, including details of the therapeutic strategy (e.g. timing, dose, and route) as well as the use of appropriate end-points.28,29 Angiogenesis is a multi-step, highly regulated process involving numerous growth factors and interactions between different cell types. In vivo longitudinal monitoring of angiogenesis, by molecular imaging techniques, may be advantageous for the challenging task of optimizing therapeutic strategies.

Integrin {alpha}vβ3, which binds with extra-cellular matrix components with an exposed RGD sequence, shows a unique pattern of expression. It is barely detectable in endothelial cells at quiescent state, but shows high expression in vascular endothelial cells30–32 making {alpha}vβ3 integrin a candidate target molecule for monitoring angiogenesis. The role of integrin {alpha}vβ3 has been confirmed by inhibition of blood vessel formation by {alpha}vβ3 integrin antibodies or peptide antagonists in a variety of animal experiments.30,33–35

Non-invasive visualization of {alpha}vβ3 integrin expression after myocardial infarction by imaging was recently reported by Moeli et al.9 using an 111In-labelled SPECT tracer (111In–RP748) in animal experiments. 111In–RP748 uptake in the ischaemic lesions was increased at 2 weeks after coronary occlusion and reperfusion in rats. Additionally, using a canine model of myocardial infarction with 2 h left circumflex coronary artery occlusion followed by reperfusion, tracer uptake were assessed at 8 h, 1 week, and 3 weeks after reperfusion.9 The maximal tracer accumulation was reported at 1 week. Another report assessing angiogenesis in a hindlimb model after ischaemia using a SPECT tracer targeting {alpha}vβ3 integrin was reported by Hua et al.36 They showed selective localization of the tracer 99 mTc-labelled peptide (NC100692) after right femoral artery occlusion. An increased tracer accumulation was first observed study at 3 days and peaked at 7 days after the occlusion. The delayed onset of the {alpha}vβ3 integrin tracer uptake at 3 days after the infarction is well consistent with our results of 18F-Galacto-RGD PET.

18F-Galacto-RGD is a PET tracer, which has some technical advantages: unlike SPECT imaging, PET imaging does not require an extrinsic collimator resulting in higher count sensitivity and spatial resolution. In addition, accurate and well-validated attenuation correction is available with sequentially acquired transmission data. These advantages allow tracer kinetic modelling with dynamic PET acquisition.37

In the present study, the animals were followed for the first time up to 6 months. 18F-Galacto-RGD tracer retention was observed as early as 3 days and peaked between 1 and 3 weeks, but remained still detectable at 6 months, suggesting a delayed but prolonged tissue response to a transient episode of coronary occlusion and reperfusion. These data of a prolonged {alpha}vβ3 integrin up regulation may serve as a baseline for the monitoring of therapeutic interventions in ischaemic heart disease. It also raises an important question in view of increasing use of anti-angiogenic therapies of cancer patients. Such therapy may interfere with the healing process after myocardial infarction if started within weeks after an acute event.

Immunohistochemical analysis indicates a close correlation of the tracer signal with staining of newly formed vessels. Acute inflammatory cell infiltration peaked at day 1 and 3 after reperfusion, which was not associated with increased 18F-Galacto-RGD retention. The negative tracer accumulation at day 1 also indicated minimal effects of unspecific tracer diffusion in damaged leaky vessels, since the vessel damage with ischaemic insult is expected prominent in the acute phase. Furthermore, the blocking studies support the notion that the tracer uptake is specific for the {alpha}vβ3 integrin expression.

The time course of histological changes in this study is consistent with known healing phases after myocardial infarction.38–41 Blankesteijn et al. separates the process of healing after myocardial infarction into a first phase of myocardial cell death, a second phase of acute inflammation, a third phase of formation of granulation tissue, and a fourth phase of scar formation. According to their definition, the third phase of granulation tissue contains many small blood vessels, which were observed as early as 3 days and peaked at 1 week in rats.38,42 Subsequently, the granulation tissue gradually matures into scar tissue which may take up to 1 year after myocardial infarction.38

Leong-Poi et al.43 recently reported that therapy by fibroblast growth factor-2 modified the expression {alpha}v-integrin as assessed by microbubble ultrasound imaging in a rat model of limb infarction. They showed a greater and earlier peak of {alpha}v-integrin expression in the treated group preceding the recovery of blood flow. Imaging studies may therefore be used to assess the effects of pro-angiogenesis protocols on the {alpha}vβ3 integrin expression in the ischaemic heart. Specifically, the microbubbles are pure intravascular tracers targeting molecules only within the intravascular space. On the other hand, 18F-Galacto-RGD, like most nuclear medicine tracers, is diffusible and targeting molecules both in intra- and extravascular space that may increase sensitivity of the imaging. Additionally, PET imaging offers not only the assessment of the protein expression, but also provides the direct comparison with measures of perfusion and ventricular function integrating several factors describing the recovery after myocardial infarction.


   5. Study limitations
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Study limitations
6. Conclusions
 Funding
 References
 
Manual delineation of the focal tracer uptake area in the myocardium was used for the measurement of 18F-Galacto-RGD uptake. Although myocardial perfusion imaging with 13N-ammonia PET was performed in this study, the spatial resolution was not enough to define exactly the area of myocardial infarction and its border zone. Since our rat model of 20 min transient coronary occlusion induced subendocardial infarction ~50% of area at risk, it may require an improved spatial resolution such as delayed enhancement MRI.17

Since there was no available antibody established against rat {alpha}vβ3 integrin for immunohistochemical staining, we used an antibody against β3 integrin subunit in substitution. New vessels displayed a higher positivity for β3 integrin subunit, but further confirmatory studies such as dual tracer fluoroesence immunohistochemical staining against {alpha}v and β3 integrin subunits may be required to clarify the source of {alpha}vβ3 integrin expression in myocardium after an ischaemic event.


   6. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Study limitations
 6. Conclusions
Funding
 References
 
We observed prolonged myocardial 18F-Galacto-RGD uptake after transient coronary occlusion in the rat model. The tracer accumulation started after 3 days, peaked between 1 and 3 weeks, and showed a slow decrease afterwards, with preserved uptake up to 6 months. These findings correlated well with increased density of neovasculature in the infarct myocardium. Non-invasive imaging using PET and the new 18F-Galacto-RGD tracer appears promising for the monitoring of therapies aiming to stimulate angiogenesis in ischaemic heart disease.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Study limitations
 6. Conclusions
 Funding
References
 
This study was funded in part by the EC-FP6-project DiMI (LSHB-CT-2005-512146), the Bavarian Ministry of State for Science, Research and Arts (BayGene), and the Deutsche Forschungsgemeinschaft (DFG BE 2217/4-1 and DFG BE 2217/4-2).


   Acknowledgements
 
The authors are grateful to Dr Marc C. Huisman, Axel Weber, and Dr Thorsten Poethko for their excellent technical assistance and to Axel Martinez-Möller and Dr Antti Saraste for their careful editorial assistance.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Study limitations
 6. Conclusions
 Funding
 References
 

  1. Sasayama S, Fujita M. Recent insights into coronary collateral circulation. Circulation (1992) 85:1197–1204.[Abstract/Free Full Text]
  2. Khurana R, Simons M, Martin JF, Zachary IC. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation (2005) 112:1813–1824.[Abstract/Free Full Text]
  3. Giancotti FG, Ruoslahti E. Integrin signaling. Science (1999) 285:1028–1032.[Abstract/Free Full Text]
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Targeted Imaging Offers Advantages Over Physiological Imaging for Evaluation of Angiogenic Therapy
J. Am. Coll. Cardiol. Img., July 1, 2008; 1(4): 511 - 514.
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