18F-GP1, a Novel PET Tracer Designed for High-Sensitivity, Low-Background Detection of Thrombi.

Thromboembolic diseases such as myocardial infarction, stroke, transient ischemic attacks, and pulmonary embolism are major causes of morbidity and mortality worldwide. Glycoprotein IIb/IIIa (GPIIb/IIIa) is the key receptor involved in platelet aggregation and is a validated target for therapeutic approaches and diagnostic imaging. The aim of this study was to develop and characterize a specific small-molecule tracer for PET imaging that binds with high affinity to GPIIb/IIIa receptors and has suitable pharmacokinetic properties to overcome limitations of previous approaches. Methods: Binding of 18F-GP1 to GPIIb/IIIa receptors was investigated in competition binding assays and autoradiography using a fresh cardiac thrombus from an explanted human heart. The clot-to-blood ratio for 18F-GP1 was investigated by an in vitro blood flow model. Biodistribution and thrombus detection was investigated in cynomolgus monkeys after insertion of a roughened catheter into either the vena cava or the aorta. Results:18F-GP1 is an 18F-labeled small molecule for PET imaging of thrombi. The half maximal inhibitory concentration of 18F-GP1 to GPIIb/IIIa was 20 nM. 18F-GP1 bound to thrombi with a mean clot-to-blood ratio of 95. Binding was specific and can be displaced by excess nonradioactive derivative. Binding was not affected by anticoagulants such as aspirin or heparin. 18F-GP1 showed rapid blood clearance and a low background after intravenous injection in cynomolgus monkeys. Small arterial, venous thrombi, thrombotic depositions on damaged endothelial surface, and small cerebral emboli were detected in vivo by PET imaging. Conclusions:18F-GP1 binds specifically with high affinity to the GPIIb/IIIa receptor involved in platelet aggregation. Because of its favorable preclinical characteristics, 18F-GP1 is currently being investigated in a human clinical study.

Comparing the global mRNA expression profile of human atrial and ventricular myocardium with high-density oligonucleotide arrays.

OBJECTIVES: The knowledge of chamber-specific gene expression in human atrial and ventricular myocardium is essential for the understanding of myocardial function and the basis for the identification of putative therapeutic targets in the treatment of cardiac arrhythmia and heart failure. In this study the gene expression pattern of human left atrial and ventricular myocardium was analyzed. METHODS: Global mRNA expression patterns with high-density oligonucleotide arrays between left atrial and left ventricular myocardium of 6 patients with heart failure undergoing heart transplantation were compared. Clustering of microarray data confirmed chamber-specific gene expression profiles. Genes similarly expressed in all patients were further analyzed, and data were confirmed by means of real-time polymerase chain reaction and Western blot analysis. RESULTS: Of 22,215 genes examined, 7115 transcripts were found to be expressed in all 12 human myocardial samples. One hundred twenty-five genes were differentially expressed between left atrial and left ventricular specimens in all patients examined. Novel genes preferentially expressed in human atria were identified. Interestingly, several potassium channels of subfamily K are more highly expressed in atria than in ventricles. Members of the potassium inwardly rectifying channel of subfamily J were found to be more highly expressed in human ventricular myocardium. Finally, chronic atrial fibrillation was associated with reduced atrial expression of the potassium channel TWIK-1, suggesting potential contribution of the corresponding current to electrical remodeling. CONCLUSIONS: Human atria and ventricles show specific gene expression profiles. Our data provide the basis of a comprehensive understanding of chamber-specific gene expression in diseased human hearts and will support the identification of therapeutic targets in the treatment of arrhythmia and heart failure.

A nonradioactive 96-well plate assay for the detection of hypoxia-inducible factor prolyl hydroxylase activity.

The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel-Lindau protein-elongin B-elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.