Detection of calcification in atherosclerotic plaques using optical imaging

PET imaging, using the bone tracer Na18F, allows the non-invasive location of atherosclerotic plaques that are at risk of rupture. However, the spatial resolution of PET is only 4-5 mm, limiting the mechanistic information this technique can provide. In this thesis, the use of fluorescence and Raman imaging to elucidate the mechanism of micro-calcification within atherosclerotic plaques has been investigated. A number of fluorescent probes to detect fluoride and calcium have been synthesised. One of the fluoride probes has been shown to be selective for fluoride however, the concentration of fluoride required to activate the probe is order of magnitudes higher than the amount of Na18F used for PET imaging making it problematic to use for future studies. On the other hand, a calcium probe has been shown to: selectively bind to hydroxyapatite (HAP); permit visualisation and quantification of HAP in both vascular and bone cell models; and effectively stain cultured aortic sections and whole mouse aorta for OPT imaging. Building on these preliminary data, fluorescence imaging and immunohistochemistry (IHC) imaging of both healthy and atherosclerotic tissue that were previously subjected to PET imaging, were successfully carried out showing the ability of the probe to detect HAP in human vascular tissue. IHC staining for Osteoprotegerin (OPG) and Osteopontin (OPN), two bone proteins recently detected in vascular tissue, showed the co-localization of OPG with the probe. Conversely, the OPN was shown to localize in areas surrounding high OPG and probe signal. To determine the exact composition of vascular calcification, Raman spectroscopy was also used. It is believed that the biosynthetic pathway to HAP passes through a series of transitional states; each of these has different structural characteristics which can be studied using Raman spectroscopy. In particular, HAP has a strong characteristic Raman peak at 960 cm-1. An increase in HAP concentration has been detected by Raman in both calcified cell models and aortic sections. When human vascular tissue was analysed, an additional peak at 973 cm-1 was present suggesting the presence of whitlockite (WTK) in this tissue as well as HAP

Ex vivo 18F-fluoride uptake and hydroxyapatite deposition in human coronary atherosclerosis

Early microcalcification is a feature of coronary plaques with an increased propensity to rupture and to cause acute coronary syndromes. In this ex vivo imaging study of coronary artery specimens, the non-invasive imaging radiotracer, 18F-fluoride, was highly selective for hydroxyapatite deposition in atherosclerotic coronary plaque. Specifically, coronary 18F-fluoride uptake had a high signal to noise ratio compared with surrounding myocardium that makes it feasible to identify coronary mineralisation activity. Areas of 18F-fluoride uptake are associated with osteopontin, an inflammation-associated glycophosphoprotein that mediates tissue mineralisation, and Runt-related transcription factor 2, a nuclear protein involved in osteoblastic differentiation. These results suggest that 18F-fluoride is a non-invasive imaging biomarker of active coronary atherosclerotic mineralisation

<i>Aza</i>-Conjugate Addition Methodology for the Synthesis of <i>N</i>‑Hydroxy-isoindolin-1-ones

Aryl-aldehydes containing <i>ortho</i>-substituted propiolate fragments react with hydroxylamine to afford carbinolamine intermediates that undergo intramolecular <i>aza</i>-conjugate addition reactions to afford <i>N</i>-hydroxy-2.3-dihydro-isoindolin-1-ones that can be reduced to their corresponding isoindolin-1-ones and isoindoles