Original Article[68Ga]Ga-NODAGA-E[(cRGDyK)]2 angiogenesis PET following myocardial infarction in an experimental rat model predicts cardiac functional parameters and development of heart failure
Introduction
Adverse cardiac remodeling after myocardial infarction (MI) is a process of structural and functional changes which could lead to heart failure. Remodeling of the myocardium occurs as a response to inadequate repair after an MI.1, 2, 3 The repair of the myocardium after MI consists of three phases: the acute, the proliferation and scar maturation phase. During the proliferation phase, endothelial cells proliferate and infiltrate the infarcted area, leading to the formation of a dense microvascular network, which supplies oxygen and nutrients to the infarcted area.4,5 This process is known as angiogenesis and is essential to repair after MI.6 The third phase is the maturation phase, in which most of the myofibroblasts transition to a phenotype that promotes scar maturation.3,7 Angiogenesis peaks seven days after MI and slowly decreases over the next 14 to 28 days.8 Since angiogenesis is vital to repair, the targeting and stimulation of angiogenesis has been of clinical interest for many years.
To develop and optimize treatment that promotes angiogenesis, it is crucial to establish a non-invasive method for the real-time monitoring of angiogenesis. Positron emission tomography (PET) is a modality with high sensitivity and acceptable resolution that enables continuous in vivo monitoring of angiogenesis in human subjects. The integrin αvβ3 has been extensively studied and the highest expression of this integrin has been found in activated endothelial cells undergoing angiogenesis.9, 10, 11 The integrin αvβ3 is expressed at low levels in normal healthy tissue like intestinal, vascular and smooth muscle cells.12 Other cell types with expression of integrin αvβ3 is bone resorbing osteoclasts, activated macrophages, angiogenic endothelial cells and migrating smooth muscle cells.13 The tripeptide motif Arg-Gly-Asp (RGD) is a specific ligand to αvβ3 and has been the major peptide used in the molecular imaging of αvβ3, since it is recognized by the α-subunit of the integrin14,15 on the endothelial cell. Several different RGD-based PET tracers have previously been used for imaging integrin, primarily in animal studies. The tracers differ in characteristics like linkers, chelators, radionuclides.16, 17, 18.
The aim of this study was to investigate the emerging PET radiotracer [68Ga]Ga-NODAGA-E[(cRGDyK)]2 ([68Ga]Ga-RGD), as a marker of angiogenesis and its potential use in predicting outcome following MI. This was done by (a) evaluating the binding between αvβ3 and [68Ga]Ga-RGD using surface plasmon resonance (SPR), (b) in vivo animal experiment imaging angiogenesis in a myocardial infarction rodent model using [68Ga]Ga-RGD PET/CT and (c) ex vivo evaluation to confirm infarction (histology), cell distribution (flow cytometry) and verify tracer accumulation using autoradiography and gamma counting.
Section snippets
Binding kinetics
The assessment of real-time biomolecular interaction was performed using a Biacore X100 (Biacore, Uppsala, Sweden), with the determination of binding kinetics of [68Ga]Ga-RGD and integrin αvβ3. To evaluate the assay development and results, vitronectin and fibronectin was used as comparison.
Ethical statement
The Danish Animal Experiments Inspectorate approved experimental protocols (Permit No. 2016-15-0201-00920). All animal procedures performed are in accordance with the guidelines in Directive 2010/63/EU of
Integrin αvβ3, SPR experiments by single cycle kinetics
The binding between integrin αvβ3 and [68Ga]Ga-RGD showed a strong and stable interaction in the presence of Mg2+ and an even slower dissociation rate constant in the presence of Mg2+ and Mn2+ (Figure 2).
Integrin αvβ3 is the natural receptor for vitronectin, and SPR showed likewise a strong interaction with vitronectin in the presence of Mg2+ with no change in the presence of Mg2+ and Mn2+ and thereby validate the SPR analyses of integrin αvβ3 and [68Ga]Ga-RGD.
Fibronectin showed steady state
Discussion
The major finding of this study is that [68Ga]Ga-RGD PET-uptake in the infarcted area correlates with cardiac functional parameters at a later time point. More specifically, we found that the uptake of [68Ga]Ga-RGD in the infarcted area after one week correlated to the EDV and EF measured after four weeks. The [68Ga]Ga-RGD tracer used was recently developed by our group,19, 20, 21, 22 and demonstrated in the present study, a stronger binding to αvβ3 than that of its natural ligand vitronectin.
Study limitations
This study investigated permanent ligation of the LAD as a MI model. To assess the broader applicability of [68Ga]Ga-RGD studies in reperfusion models with transient occlusion of the LAD should be conducted in the future. A reperfusion model would be a more clinically relevant and translate better to investigate acute myocardial infarction in humans.
This study was not designed to investigate heart failure following MI. To establish if [68Ga]Ga-RGD uptake correlates with heart failure, the
Conclusion
This study demonstrates that [68Ga]Ga-RGD has a high affinity for integrin αvβ3 which enables the evaluation of angiogenesis following an MI, using PET/CT. The in vivo RGD uptake after one week correlated to ejection fraction and end diastolic volume after four weeks, indicating that [68Ga]Ga-RGD may be used as an early predictor of cardiac functional parameters and possible development of heart failure after an MI. These encouraging data support a clinical translation.
New knowledge gained:
The PET tracer [68Ga]Ga-NODAGA-E[(cRGDyK)]2 forms a stable and strong binding to the integrin αvβ3 αvβ3, which enable the detection of angiogenesis. The angiogenic response after one week correlated to predictors of early onset of heart failure phenotype.
Author contributions
SB participated in conception of the study as well as collection of the data, analysis, drafting the manuscript and final approval of the manuscript. JKJ, CEG, BF, JSM and AC contributed with data collection, analysis and revision of the manuscript. LRP, EC and CC contributed to data analysis and revision of the manuscript. TB, PH, RSR and AK contributed with conception and design of the study as well critical revision of the manuscript and final approval.
Funding
Open access funding provided by Royal Danish Library. The authors received funding from the European Union’s Horizon 2020 research and innovation program under grant agreements no. 670261 (ERC Advanced Grant) and 668532 (Click-It), the Lundbeck Foundation, the Novo Nordisk Foundation, the Innovation Fund Denmark, the Danish Cancer Society, Arvid Nilsson Foundation, the Neye Foundation, the Research Foundation of Rigshospitalet, the Danish National Research Foundation (grant 126), the Research
Disclosure
A.K. is an inventor on patents covering the PET tracer used (EP3706809A1 and US16/762,873). No other potential conflicts of interest relevant to this article exist.
References (35)
- et al.
Angiogenesis in wound healing
J Invest Dermatol Symposium Proc
(2000) - et al.
Early molecular imaging of interstitial changes in patients after myocardial infarction: Comparison with delayed contrast-enhanced magnetic resonance imaging
J Nucl Cardiol
(2010) - et al.
Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor
J Biol Chem.
(1990) - et al.
Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells
Cell
(1989) - et al.
Comparison of two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2 and 64Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in human xenograft tumors
Nucl Med Biol
(2014) - et al.
Global conformational earrangements in integrin extracellular domains in outside-in and inside-out signaling
Cell
(2002) - et al.
Left ventricular dilation and incident congestive heart failure in asymptomatic adults without cardiovascular disease: Multi-Ethnic Study of Atherosclerosis (MESA)
J Card Fail
(2014) - et al.
Rubidium-82 PET imaging is feasible in a rat myocardial infarction model
J Nucl Cardiol
(2019) - et al.
Lessons from sudden coronary death: A comprehensive morphological classification scheme for atherosclerotic lesions
Arterioscler Thromb Vasc Biol
(2000) - et al.
Intraplaque hemorrhage and progression of coronary atheroma
N Engl J Med
(2003)
Cardiac fibrosis in myocardial infarction—from repair and remodeling to regeneration. Vol. 365, Cell and Tissue Research
The inflammatory response in myocardial injury, repair, and remodelling
Nat Rev Cardiol.
Targeting angiogenesis to restore the microcirculation after reperfused MI
Nat Rev Cardiol
The mechanistic basis of infarct healing
Antioxid Redox Signal
Temporal response and localization of integrins β1 and β3 in the heart after myocardial infarction: Regulation by cytokines
Circulation
The role of αv integrins during angiogenesis: Insights into potential mechanisms of action and clinical development
J Clin Invest.
Noninvasive imaging of myocardial angiogenesis following experimental myocardial infarction
J Clin Invest
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