Molecular imaging and immunotherapy in atherosclerotic cardiovascular disease

Atherosclerotic cardiovascular disease (ASCVD) is a disorder that affects both heart and blood vessels. It is caused by atherosclerosis, a chronic, low-grade inflammatory disease, which is characterized by the accumulation of lipids in the arterial wall. Modified lipoproteins contain pro-inflammatory epitopes, such as phosphorylcholine (PC), that interact with inflammatory cells in the vascular wall, thereby accelerating the development of atherosclerotic lesions. The main cause of myocardial infarction (MI) arises from a rupture of an unstable, inflamed atherosclerotic lesion in the coronary artery. The MI induces an intense inflammatory response that is essential for myocardial repair and scar formation, but which is also implicated in the onset of heart failure over time. Molecular imaging techniques can be potentially used for identification of new molecular targets, such as glucagon-like peptide-1 receptor (GLP-1R), that are involved in inflammatory and repair processes. The aim of this thesis was to investigate whether a novel immunotherapy targeting the PC epitope improves vascular function and attenuates atherosclerotic inflammation in mice. A positron emission tomography (PET) tracer 68Ga¬NODAGA-exendin-4 was evaluated for detection and imaging of GLP-1R expression after MI and in atherosclerosis in experimental models. In vivo PET imaging, ultrasound imaging, tissue autoradiography, immunohistological stainings, and cell assays were utilized as the main methods in the studies. This thesis showed that treatment with the PC immunotherapy preserved coronary artery function and attenuated the uptake of an established inflammation tracer, glucose analog 18F-FDG, in atherosclerotic lesions in mice. 68Ga-NODAGA¬exendin-4 PET detected up-regulated cardiac GLP-1R expression during the healing of MI in rats. The uptake of 68Ga-NODAGA-exendin-4 was also increased in inflamed atherosclerotic lesions in mice. In conclusion, PC immunotherapy might represent a potential approach to inhibit the lipid-driven inflammation in atherosclerosis. 68Ga-NODAGA-exendin-4 PET may have implications for studying pharmacological modification of GLP-1R signaling in ASCVD.Molekyylikuvantaminen ja immunoterapia ateroskleroottisissa sydän-ja verisuonitaudeissa Sydän-ja verisuonitautien yleisin syy on ateroskleroosi, mikä on krooninen matala-asteinen tulehduksellinen sairaus, jossa rasvaa kertyy valtimoiden seinämiin. Muokkautuneiden rasvapartikkelien pinnalta on tunnistettu tulehdusreaktiota edistäviä molekyylejä kuten fosforyylikoliini, jotka ovat osallisena valtimoplakkien muodostumiseen. Suurimman osan sydäninfarkteista aiheuttaa epävakaan tulehtuneen ateroskleroottisen plakin repeytyminen sepelvaltimossa. Sydäninfarkti saa aikaan tulehdusreaktion ja arpikudoksen muodostumisen sydänlihaksessa, mikä pitkittyneenä voi johtaa myös sydämen vajaatoimintaan. Molekyylikuvantamisen avulla voisi olla mahdollista tunnistaa ja tutkia uusia kohteita kuten glukagonin kaltainen peptidi-1 (GLP-1) -reseptori, jotka liittyvät sydäninfarktinjälkeiseen tulehdusreaktioon ja arven paranemiseen. Tämän väitöskirjan tarkoituksena oli tutkia koe-eläinmalleissa, miten uusi fosforyylikoliini-molekyyliin kohdentuva immunoterapia vaikuttaa ateroskleroottisten suonten toimintaan ja tulehdukseen. Lisäksi tutkittiin GLP-1-reseptorin ilmentymistä sydäninfarktin jälkeen ja ateroskleroottisissa plakeissa 68Ga¬NODAGA-exendin-4 merkkiaineen ja positroniemissiotomografia (PET)-kuvantamisen avulla. Tutkimusmenetelminä käytettiin in vivo PET-ja ultraäänikuvantamista, autoradiografiaa, kudosvärjäyksiä ja solukokeita. Väitöskirjassa havaittiin, että fosforyylikoliini-immunoterapian jälkeen hiirten sepelvaltimoiden toiminta oli parantunut ja plakkeihin kertyi vähemmän 18F-FDG¬merkkiainetta, mikä kertoo laantuneesta tulehduksesta. 68Ga-NODAGA-exendin-4 PET-kuvantaminen osoitti, että GLP-1-reseptorin ilmentyminen oli lisääntynyt infarktinjälkeisen tulehdusreaktion aikana. 68Ga-NODAGA-exendin-4-merkkiaine kertyi myös selvästi tulehtuneisiin ateroskleroottisiin plakkeihin. Yhteenvetona voidaan todeta, että fosforyylikoliini-immunoterapia saattaisi olla potentiaalinen uusi lääkehoito valtimoplakkien tulehdusreaktion hillitsemiseksi. 68Ga-NODAGA¬exendin-4 PET-kuvantamista voitaisiin hyödyntää GLP-1-reseptoriin kohdentuvien lääkehoitojen tutkimisessa ateroskleroottisissa sydän- ja verisuonitaudeissa

Ateroskleroottisen plakin angiogeneesin kuvantaminen – αvβ3-integriiniin kohdentuvan 99mTc-leimatun peptidin prekliinisiä tutkimuksia

Sepelvaltimotautimuutoksista erityisesti ateroskleroottisten plakkien morfologialla on tärkeä merkitys taudin ennusteen ja kliinisten komplikaatioiden kehittymisen kannalta. Suurin osa sydäninfarkteista aiheutuu hauraan plakin repeytymisestä ja verihyytymän muodostumisesta, joka tukkii sepelvaltimon. Hauraan plakin tunnuspiirteitä ovat muun muassa suuri rasvaydin, ohut sidekudoskatto, tulehdus ja uudisverisuonien muodostuminen eli angiogeneesi. Tällä hetkellä kliinisessä käytössä on menetelmiä, joilla voidaan lähinnä arvioida sepelvaltimotautimuutosten aiheuttamaa suonen ahtauma-astetta. Kehitteillä on uusia molekyylikuvantamismenetelmiä, joiden avulla voitaisiin tunnistaa hauraat plakit mahdollisimman aikaisessa vaiheessa. Mahdollisia menetelmiä ovat positroniemissiontomografia (PET) ja yksifotoniemissiotomografia (gammakuvaus), jotka ovat kajoamattomia ja herkkiä isotooppikuvantamismenetelmiä. Tämän erikoistyön tarkoituksena oli selvittää uuden gammakuvausmerkkiaineen soveltuvuutta ateroskleroottisten plakkien angiogeneesin kuvantamiseen ja plakkien haurauden arvioimiseen ateroskleroosin hiirimallissa.   Työssä käytettävä merkkiaine oli teknetium-99m leimattu synteettinen peptidi (99mTc-Maraciclatidi), joka tunnistaa alfa-v-beeta-3-integriinin (αvβ3). αvβ3-integriini on solun pinnan glykoproteiinireseptori, joka osallistuu uudisverisuonien muodostukseen myös ateroskleroottisissa plakeissa. Ateroskleroottisessa plakissa αvβ3-integriiniä esiintyy makrofageissa ja endoteelisoluissa. Koe-eläiminä käytettiin LDLR-/-ApoB100/100-poistogeenisiä hiiriä, jotka kehittävät sekä spontaanisti että varsinkin rasvadieetillä indusoituna tulehtuneita plakkeja valtimoihin, erityisesti aortankaareen. Kontrollieläiminä käytettiin C57BL/6N-kannan terveitä hiiriä.    Nukutetun hiiren häntälaskimoon injisoitiin 99mTc-Maraciclatidi. Tunti injektion jälkeen hiiret lopetettiin ja radioaktiivisuuskertymä eri kudoksissa (kokokehojaukauma) mitattiin gammalaskijalla. Aortta ja kudoskontrolli (lihas) leikattiin rinnakkaisiksi jääleikkeiksi ja merkkiaineen kertyminen plakkeihin ja terveen suonen seinämään tutkittiin tarkemmin aorttaan keskittyvällä ex vivo -autoradiografialla. Autoradiografian jälkeen jääleikkeet värjättiin histologisilla ja immunohistokemiallisilla menetelmillä, joista voitiin tarkastella plakin morfologiaa ja tulehdusmerkkiaineita (makrofagit, anti-Mac-3-vasta-aine).   Kokokehojakaumamittausten perusteella 99mTc-Maraciclatidi-merkkiaine kertyi enemmän ateroskleroottisten hiirien aorttaan kuin terveiden kontrollihiirien aorttaan. Lisäksi tunnin kuluttua injektiosta radioaktiivisuutta oli huomattavasti vähemmän verenkierrossa kuin aortassa, joten merkkiaine saattaisi soveltua in vivo -kuvantamiseen. Tarkemman ex vivo -autoradiografia-analyysin perusteella merkkiaine kertyi ateroskleroottisiin plakkeihin, mutta myös suonen seinämään sekä ateroskleroottisilla hiirillä että kontrollihiirillä. Lisätutkimuksia 99mTc-Maraciclatidin soveltuvuudesta ateroskleroosin in vivo -kuvantamiseen ja plakkien haurauden arvioimiseen tarvitaan vielä muilla eläinmalleilla

Morbid obesity and type 2 diabetes alter intestinal fatty acid uptake and blood flow

Aims: Bariatric surgery is the most effective treatment to tackle morbid obesity and type 2 diabetes, but the mechanisms of action are still unclear. The objective of this study was to investigate the effects of bariatric surgery on intestinal fatty acid (FA) uptake and blood flow. Materials and Methods: We recruited 27 morbidly obese subjects, of whom 10 had type 2 diabetes and 15 were healthy age-matched controls. Intestinal blood flow and fatty acid uptake from circulation were measured during fasting state using positron emission tomography (PET). Obese subjects were re-studied 6 months after bariatric surgery. The mucosal location of intestinal FA retention was verified in insulin resistant mice with autoradiography. Results: Compared to lean subjects, morbidly obese subjects had higher duodenal and jejunal FA uptake (P </p

Evaluation of [68Ga]Ga-NODAGA-RGD for PET Imaging of Rat Autoimmune Myocarditis

The 68Gallium-labeled 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid conjugated radiolabelled arginine-glycine-aspartic acid peptide ([68Ga]Ga-NODAGA-RGD) is a positron emission tomography (PET) tracer binding to cell surface receptor αvβ3 integrin that is upregulated during angiogenesis and inflammation. We studied whether αvβ3 targeting PET imaging can detect myocardial inflammation in a rat model of autoimmune myocarditis. To induce myocarditis, rats (n = 8) were immunized with porcine cardiac myosin in complete Freund's adjuvant on days 0 and 7. Control rats (n = 8) received Freund's adjuvant alone. On day 21, in vivo PET/CT imaging with [68Ga]Ga-NODAGA-RGD followed by ex vivo autoradiography and immunohistochemistry were carried out. Inflammatory lesions were detected histologically in the myocardium of 7 out of 8 immunized rats. In vivo PET images showed higher [68Ga]Ga-NODAGA-RGD accumulation in the myocardium of rats with inflammation than the non-inflamed myocardium of control rats (SUVmean 0.4 ± 0.1 vs. 0.1 ± 0.02; P = 0.00006). Ex vivo autoradiography and histology confirmed that [68Ga]Ga-NODAGA-RGD uptake co-localized with inflammatory lesions containing αvβ3 integrin-positive capillary-like structures. A non-specific [68Ga]Ga-DOTA-(RGE)2 tracer showed 76% lower uptake than [68Ga]Ga-NODAGA-RGD in the inflamed myocardium. Our results indicate that αvβ3 integrin-targeting [68Ga]Ga-NODAGA-RGD is a potential PET tracer for the specific detection of active inflammatory lesions in autoimmune myocarditis.</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

Effects of dipeptidyl peptidase 4 inhibition on inflammation in atherosclerosis: A 18F-fluorodeoxyglucose study of a mouse model of atherosclerosis and type 2 diabetes

Background and aims: Dipeptidyl peptidase 4 (DPP-4) inhibitors have anti-inflammatory and atheroprotective effects. We evaluated the effects of the DPP-4 inhibitor linagliptin on atherosclerotic plaque and hepatic inflammation using histology and 2-deoxy-2-[18F]-fluoro-d-glucose (18F-FDG), a positron emission tomography tracer of inflammation, in a mouse model of hypercholesterolemia and type 2 diabetes.Methods: Igf2/Ldlr-/-Apob100/100 mice were fed a high-fat diet (HFD) for 8 weeks and then randomly allocated to receive HFD (n = 14), or HFD with added linagliptin (n = 15) for additional 12 weeks. Five mice fed a chow diet were studied as an additional control. At the end of the study, glucose tolerance, aortic and liver uptake of 18F-FDG, and histology were studied.Results: Mice in linagliptin and HFD groups had similar fasting glucose concentrations, but linagliptin improved glucose tolerance. Aortas of linagliptin and HFD groups showed advanced atherosclerotic plaques with no difference in the mean intima-to-media ratio or number of macrophages in the plaques. Autoradiography showed similar 18F-FDG uptake by atherosclerotic plaques in linagliptin and HFD groups (plaque-to-wall ratio: 1.7 ± 0.25 vs. 1.6 ± 0.21; p = 0.24). In the liver, linagliptin reduced the histologic inflammation score but had no effect on 18F-FDG uptake. Compared with chow diet, uptake of 18F-FDG was similar in the aorta, but higher in the liver after HFD.Conclusions: Linagliptin therapy improved glucose tolerance and reduced hepatic inflammation but had no effect on plaque burden or atherosclerotic inflammation, as determined by histology and 18F-FDG uptake, in atherosclerotic mice with type 2 diabetes.   </p

Folate Receptor β Targeted PET Imaging of Macrophages in Autoimmune Myocarditis

Rationale: Currently available imaging techniques have limited specificity for the detection of active myocardial inflammation. Aluminum fluoride-18-labeled 1,4,7-triazacyclononane-N,N′,N″-triacetic acid conjugated folate (18F-FOL) is a positron emission tomography (PET) tracer targeting folate receptor β (FR-β) that is expressed on activated macrophages at sites of inflammation. We evaluated 18F-FOL PET for the detection of myocardial inflammation in rats with autoimmune myocarditis and studied expression of FR-β in human cardiac sarcoidosis specimens. Methods: Myocarditis was induced by immunizing rats (n = 18) with porcine cardiac myosin in complete Freund’s adjuvant. Control rats (n = 6) were injected with Freund’s adjuvant alone. 18F-FOL was intravenously injected followed by imaging with a small animal PET/computed tomography (CT) scanner and autoradiography. Contrast-enhanced high-resolution CT or 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) PET images were used for co-registration. Rat tissue sections and myocardial autopsy samples of 6 patients with cardiac sarcoidosis were studied for macrophages and FR-β. Results: The myocardium of 10 out of 18 immunized rats showed focal macrophage-rich inflammatory lesions with FR-β expression occurring mainly in M1-polarized macrophages. PET images showed focal myocardial 18F-FOL uptake co-localizing with inflammatory lesions (SUVmean, 2.1 ± 1.1), whereas uptake in the remote myocardium of immunized rats and controls was low (SUVmean, 0.4 ± 0.2 and 0.4 ± 0.1, respectively; P </p

Therapeutic Antibody Against Phosphorylcholine Preserves Coronary Function and Attenuates Vascular 18F-FDG Uptake in Atherosclerotic Mice

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of 18F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.</p