Molecular imaging of tissue repair after myocardial infarction : preclinical evaluation of novel 68Ga-labeled PET tracers

Congestive heart failure (HF) develops soon after acute myocardial infarction (AMI) in almost 25% of initial survivors. Modern cardiac imaging methods are useful for HF diagnostics and, possibly, the detection of underlying molecular mechanisms involved in myocardial repair. CD44, a cell-surface glycoprotein, is involved in various cellular functions, including cell proliferation, adhesion, migration and lymphocyte activation. Integrins are transmembrane proteins involved in various signaling pathways related to inflammation, angiogenesis and fibrosis. Expression of proteolytic matrix metalloproteinases 2 and 9 (MMP-2/9) also associates with extracellular matrix remodeling. The purpose of this thesis was to evaluate novel Gallium-68 labeled imaging agents targeting αvβ3 integrin, MMP-2/9, or CD44, for positron emission tomography (PET) imaging of post-MI repair in a surgical rat model. The MMP- 2/9 targeting tracer watarkias also evaluated for imaging of atherosclerotic lesions in a hypercholesterolemic mouse model. In vivo PET imaging, ex vivo biodistribution, ex vivo autoradiography, and immunohistochemistry were utilized to assess tracer stability, uptake in various tissues, as well as uptake correlation with various cellular level processes. Of the studied tracers, αvβ3 integrin targeting tracer showed the most optimal characteristics for imaging of myocardial healing processes. Tracer uptake in the damaged myocardium was clearly visible in vivo, and blood clearance as well as tracer stability were sufficient. The CD44 targeting tracer showed initial potential warranting further development, as the tracer uptake was associated with myocardial inflammation. MMP-2/9 targeted imaging showed significant limitations due to tracer instability and slow clearance. In conclusion, imaging of αvβ3 integrin expression is a potential tool for the purpose of evaluating myocardial repair after MI.Sydänkudoksen infarktinjälkeisen paranemisen molekyylikuvantaminen : uusien 68Ga-leimattujen merkkiaineiden prekliininen arviointi Sydämen vajaatoiminta kehittyy pian akuutin sydäninfarktin jälkeen lähes 25 prosentille eloonjääneistä. Nykyaikaiset sydämen kuvantamismenetelmät ovat hyödyllisiä diagnostiikassa ja mahdollisesti sydänlihaksen muovautumiseen liittyvien molekyylimekanismien havaitsemisessa. Solupinnan glykoproteiini CD44 osallistuu erilaisiin soluvälitteisiin toimintoihin, kuten proliferaatioon, adheesioon, migraatioon ja lymfosyyttien aktivaatioon. Integriinit ovat transmembraaniproteiineja, jotka osallistuvat erilaisiin signalointireitteihin liittyen tulehdukseen, angiogeneesiin ja fibroosiin. Proteolyyttisten matriksin metalloproteinaasi 2:n ja 9:n (MMP-2/9) ilmentyminen liittyy niin ikään solunulkoisen matriksin uudelleenmuovautumiseen. Tämän väitöskirjan tarkoituksena on arvioida uusia Gallium-68-leimattuja koettimia positroniemissiotomografiaa (PET) varten. Tutkitut koettimet kohdistuvat joko αvβ3-integriiniin, MMP-2/9:ään tai CD44:ään. Tutkimus toteutettiin infarktinjälkeisen sydämen vajaatoiminnan kirurgisessa rottamallissa. MMP-2/9-koetinta arvioitiin myös ateroskleroottisten muutosten kuvantamiseen hyperkolesterolemisessa hiirimallissa. αvβ3-integriiniin kohdentuvan merkkiaineen kertymä näkyi selkeästi in vivo, ja veren puhdistuma sekä merkkiaineen stabiilisuus olivat riittävät. CD44 kuvantamiskohteena osoitti alkuvaiheen potentiaalia, joka mahdollistaa jatkokehityksen, sillä merkkiaineen kertymä assosioitui infarktinjälkeiseen tulehdusreaktioon. MMP-2/9- kohdennetulle kuvantamiselle puolestaan ilmeni merkittäviä rajoituksia merkkiaineiden epävakauden ja hitaan veripuhdistuman vuoksi Yhteenvetona voidaan todeta, että αvβ3-integriinifragmentin kuvantaminen on potentiaalinen työkalu sydänlihaksen paranemisprosessien arvioimiseksi akuutin sydäninfarktin jälkeen

Type 2 diabetes enhances arterial uptake of choline in atherosclerotic mice: an imaging study with positron emission tomography tracer 18F-fluoromethylcholine

Additional file 1. Supplemental data

68Ga-DOTA chelate, a novel imaging agent for assessment of myocardial perfusion and infarction detection in a rodent model

BackgroundMagnetic resonance imaging (MRI) with Gadolinium 1,4,7,10-tetraazacyclododecane-N′,N″,N′′′,N″″-tetraacetic acid (Gd-DOTA) enables assessment of myocardial perfusion during first-pass of the contrast agent, while increased retention can signify areas of myocardial infarction (MI). We studied whether Gallium-68-labeled analog, 68Ga-DOTA, can be used to assess myocardial perfusion on positron emission tomography/computed tomography (PET/CT) in rats, comparing it with 11C-acetate.MethodsRats were studied with 11C-acetate and 68Ga-DOTA at 24 hours after permanent ligation of the left coronary artery or sham operation. One-tissue compartmental models were used to estimate myocardial perfusion in normal and infarcted myocardium. After the PET scan, hearts were sectioned for autoradiographic detection of 68Ga-DOTA distribution.Results11C-acetate PET showed perfusion defects and histology showed myocardial necrosis in all animals after coronary ligation. Kinetic modeling of 68Ga-DOTA showed significantly higher k1 values in normal myocardium than in infarcted areas. There was a significant correlation (r = 0.82, P = 0.001) between k1 values obtained with 68Ga-DOTA and 11C-acetate. After 10 minutes of tracer distribution, the 68Ga-DOTA concentration was significantly higher in the infarcted than normal myocardium on PET imaging and autoradiography.ConclusionsOur results indicate that acute MI can be detected as reduced perfusion, as well as increased late retention of 68Ga-DOTA.</p

Glucagon-like peptide-1 receptor expression after myocardial infarction: Imaging study using [68Ga]NODAGA-exendin-4 positron emission tomography

BACKGROUND: Activation of glucagon-like peptide-1 receptor (GLP-1R) signaling protects against cardiac dysfunction and remodeling after myocardial infarction (MI). The aim of the study was to evaluate 68Ga-NODAGA-exendin-4 positron emission tomography (PET) for assessment of GLP-1R expression after MI in rats.METHODS AND RESULTS: Rats were studied at 3 days, 1 and 12 weeks after permanent coronary ligation or a sham-operation. Rats were injected with 68Ga-NODAGA-exendin-4 and scanned with PET and contrast-enhanced computed tomography (CT) followed by digital autoradiography and histology of left ventricle tissue sections. 68Ga-NODAGA-exendin-4 PET/CT showed focally increased tracer uptake in the infarcted regions peaking at 3 days and continuing at 1 week after MI. Pre-treatment with an unlabeled exendin-4 peptide significantly reduced 68Ga-NODAGA-exendin-4 uptake. By autoradiography, 68Ga-NODAGA-exendin-4 uptake was 8.6-fold higher in the infarcted region and slightly increased also in the remote, non-infarcted myocardium at 1 week and 12 weeks post-MI compared with sham. Uptake of 68Ga-NODAGA-exendin-4 correlated with the amount of CD68-positive macrophages in the infarcted area and alpha-smooth muscle actin staining in the remote myocardium.CONCLUSIONS: 68Ga-NODAGA-exendin-4 PET detects up-regulation of cardiac GLP-1R expression during healing of MI in rats and may provide information on the activated repair mechanisms after ischemic myocardial injury.</p

Amyloid-Targeting PET Tracer [18F]Flutemetamol Accumulates in Atherosclerotic Plaques

Atherosclerosis is characterized by the accumulation of oxidized lipids in the artery wall, which triggers an inflammatory response. Oxidized low-density lipoprotein (ox-LDL) presents amyloid-like structural properties, and different amyloid species have recently been recognized in atherosclerotic plaques. Therefore, we studied the uptake of the amyloid imaging agent [ 18 F]Flutemetamol in atherosclerotic plaques. The binding of [ 18 F]Flutemetamol to human carotid artery plaque was studied in vitro. In vivo uptake of the tracer was studied in hypercholesterolemic IGF-II/LDLR − / − ApoB 100/100 mice and C57BL/6N controls. Tracer biodistribution was studied in vivo with PET/CT, and ex vivo by gamma counter and digital ex vivo autoradiography. The presence of amyloid, ox-LDL, and macrophages in the plaques was examined by immunohistochemistry. [ 18 F]Flutemetamol showed specific accumulation in human carotid plaque, especially in areas positive for amyloid beta. The aortas of IGF-II/LDLR − / − ApoB 100/100 mice showed large thioflavin-S-positive atherosclerotic plaques containing ox-LDL and macrophages. Autoradiography revealed 1.7-fold higher uptake in the plaques than in a lesion-free vessel wall, but no difference in aortic tissue uptake between mouse strains were observed in the in vivo PET/CT. In conclusion, [ 18 F]Flutemetamol binds to amyloid-positive areas in human atherosclerotic plaques. Further studies are warranted to clarify the uptake mechanisms, and the potential of the tracer for in vivo imaging of atherosclerosis in patients. </p

Dimeric [Ga-68]DOTA-RGD Peptide Targeting alpha(v)beta(3) Integrin Reveals Extracellular Matrix Alterations after Myocardial Infarction

Siirretty Doriast

Evaluation of [⁶⁸Ga]Ga-DOTA-TCTP-1 for the Detection of Metalloproteinase 2/9 Expression in Mouse Atherosclerotic Plaques

Background: The expression of matrix metalloproteinases 2/9 (MMP-2/9) has been implicated in arterial remodeling and inflammation in atherosclerosis. We evaluated a gallium-68 labeled peptide for the detection of MMP-2/9 in atherosclerotic mouse aorta. Methods: We studied sixteen low-density lipoprotein receptor deficient mice (LDLR-/-ApoB100/100) kept on a Western-type diet. Distribution of intravenously-injected MMP-2/9-targeting peptide, [68Ga]Ga-DOTA-TCTP-1, was studied by combined positron emission tomography (PET) and contrast-enhanced computed tomography (CT). At 60 min post-injection, aortas were cut into cryosections for autoradiography analysis of tracer uptake, histology, and immunohistochemistry. Zymography was used to assess MMP-2/9 activation and pre-treatment with MMP-2/9 inhibitor to assess the specificity of tracer uptake. Results: Tracer uptake was not visible by in vivo PET/CT in the atherosclerotic aorta, but ex vivo autoradiography revealed 1.8 &#177; 0.34 times higher tracer uptake in atherosclerotic plaques than in normal vessel wall (p = 0.0029). Tracer uptake in plaques correlated strongly with the quantity of Mac-3-positive macrophages (R = 0.91, p &lt; 0.001), but weakly with MMP-9 staining (R = 0.40, p = 0.099). Zymography showed MMP-2 activation in the aorta, and pre-treatment with MMP-2/9 inhibitor decreased tracer uptake by 55% (p = 0.0020). Conclusions: The MMP-2/9-targeting [68Ga]Ga-DOTA-TCTP-1 shows specific uptake in inflamed atherosclerotic lesions; however, a low target-to-background ratio precluded in vivo vascular imaging. Our results suggest, that the affinity of gelatinase imaging probes should be steered towards activated MMP-2, to reduce the interference of circulating enzymes on the target visualization in vivo

Amyloid-Targeting PET Tracer [18F]Flutemetamol Accumulates in Atherosclerotic Plaques

Atherosclerosis is characterized by the accumulation of oxidized lipids in the artery wall, which triggers an inflammatory response. Oxidized low-density lipoprotein (ox-LDL) presents amyloid-like structural properties, and different amyloid species have recently been recognized in atherosclerotic plaques. Therefore, we studied the uptake of the amyloid imaging agent [18F]Flutemetamol in atherosclerotic plaques. The binding of [18F]Flutemetamol to human carotid artery plaque was studied in vitro. In vivo uptake of the tracer was studied in hypercholesterolemic IGF-II/LDLR&minus;/&minus;ApoB100/100 mice and C57BL/6N controls. Tracer biodistribution was studied in vivo with PET/CT, and ex vivo by gamma counter and digital ex vivo autoradiography. The presence of amyloid, ox-LDL, and macrophages in the plaques was examined by immunohistochemistry. [18F]Flutemetamol showed specific accumulation in human carotid plaque, especially in areas positive for amyloid beta. The aortas of IGF-II/LDLR&minus;/&minus;ApoB100/100 mice showed large thioflavin-S-positive atherosclerotic plaques containing ox-LDL and macrophages. Autoradiography revealed 1.7-fold higher uptake in the plaques than in a lesion-free vessel wall, but no difference in aortic tissue uptake between mouse strains were observed in the in vivo PET/CT. In conclusion, [18F]Flutemetamol binds to amyloid-positive areas in human atherosclerotic plaques. Further studies are warranted to clarify the uptake mechanisms, and the potential of the tracer for in vivo imaging of atherosclerosis in patients