Joseph L. E. Bird

Identifying active vascular microcalcification by (18)F-sodium fluoride positron emission tomography.

Vascular calcification is a complex biological process that is a hallmark of atherosclerosis. While macrocalcification confers plaque stability, microcalcification is a key feature of high-risk atheroma and is associated with increased morbidity and mortality. Positron emission tomography and X-ray computed tomography (PET/CT) imaging of atherosclerosis using 18F-sodium fluoride (18F-NaF) has the potential to identify pathologically high-risk nascent microcalcification. However, the precise molecular mechanism of 18F-NaF vascular uptake is still unknown. Here we use electron microscopy, autoradiography, histology and preclinical and clinical PET/CT to analyse 18F-NaF binding. We show that 18F-NaF adsorbs to calcified deposits within plaque with high affinity and is selective and specific. 18F-NaF PET/CT imaging can distinguish between areas of macro- and microcalcification. This is the only currently available clinical imaging platform that can non-invasively detect microcalcification in active unstable atherosclerosis. The use of 18F-NaF may foster new approaches to developing treatments for vascular calcification.

Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be responsible for the maintenance of healthy nucleus pulposus

Study Design. Analysis of proteoglycan synthesis, distribution and assembly of notochordal cells and small nucleus pulposus cells embedded in alginate beads and cultured in presence of [35S]-Na2SO4. Objective. To determine whether the degeneration of the nucleus pulposus of the intervertebral disc is associated with a change in the cell phenotype. Summary of Background Data. The loss of the notochordal cell from the nucleus pulposus is associated with ageing and disc degeneration. The reduction in their numbers after birth in humans and in the chondrodystrophoid dog has been suggested to result from cell death and replacement or differentiation by chondrocytes. The almost total disappearance of the notochordal cells in the nucleus pulposus correlates with early degenerative changes in the disc and a concomitant reduction in proteoglycan content, increased collagen, and loss of water content. The basic mechanism of this accelerated degeneration with ageing is poorly understood. Methods. Nucleus pulposus and anulus fibrosus cells were isolated from the lumbar intervertebral discs of chondrodystrophoid and nonchondrodystrophoid dogs. The cells from the nucleus pulposus were further separated by size into notochordal cells and small nucleus pulposus cells. Cells were embedded in alginate beads and cultured in the presence of [35S]-Na2SO4 to measure proteoglycan size, rate of synthesis, and distribution into the pericellular and intercellular compartments. Results. Large notochordal cells in the nucleus pulposus of chondrodystrophoid dogs formed 13% of the cell population in young dogs and fell to 0.4% in adults, whereas they were the predominant cell type in the nonchondrodystrophoid dogs at all ages. These cells were capable of 1.5-fold greater rate of synthesis of proteoglycans than the small nucleus pulpous cells. Proteoglycans secreted by the large cells were evenly distributed between the pericellular and intercellular compartments,whereas the small cells distributed 3-fold more proteoglycan into the intercellular phase. By size exclusion chromatography, the proteoglycans synthesized by the small cells of the chondrodystrophoid dogs formed large-size aggregates (Kav = 0.1) within the pericellular region, which then moved to the intercellular region over 5 to 10 days. In contrast, proteoglycans secreted by the notochordal cells were capable of rapid migration to the intercellular phase before assembly into large-sized aggregates. The ability to form aggregates was independent of age of the animal. Conclusions. Our model shows that a change in intervertebral disc cell phenotype correlates with the grade of disc degeneration and that the notochordal cells synthesize proteoglycans, which exhibit delayed aggregation than those synthesized by the small nucleus pulposus cells. This implies that the cell type composition of the nucleus pulposus of the chondrodystrophoid and nonchondrodystrophoid dogs produces an extracellular matrix that is assembled in a distinct manner, which may affect tissue integrity.

Evaluation of translocator protein quantification as a tool for characterising macrophage burden in human carotid atherosclerosis

Macrophage presence within atherosclerotic plaque is a feature of instability and a risk factor for plaque rupture and clinical events. Activated macrophages express high levels of the translocator protein/peripheral benzodiazepine receptor (TSPO/PBR). In this study, we investigated the potential for quantifying plaque inflammation by targeting this receptor. TSPO expression and distribution in the plaque were quantified using radioligand binding assays and autoradiography. We show that cultured human macrophages expressed 20 times more TSPO than cultured human vascular smooth muscle cells (VSMCs), the other abundant cell type in plaque. The TSPO ligands [3H](R)-1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H](R)-PK11195) and [3H]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide ([3H]-DAA1106) bound to the same sites in human carotid atherosclerotic plaques in vitro, and demonstrated significant correlation with macrophage-rich regions. In conclusion, our data indicate that radioisotope-labelled DAA1106 has the potential to quantify the macrophage content of atherosclerotic plaque.

In vivo mapping of vascular inflammation using the translocator protein tracer 18F-FEDAA1106.

Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro-2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5-methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18F-FEDAA1106 and 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG, a marker of glucose metabolism) for positron emission tomographic (PET) imaging of vascular inflammation. This was tested using a murine model in which focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18F-FEDAA1106 or 18F-FDG was registered to anatomic data generated by computed tomographic (CT)/CT angiography. Standardized uptake values were significantly increased at cuffed compared to contralateral arteries using either 18F-FEDAA1106 (p < .01) or FDG (p < .05). However, the 18F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the noninflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro- 2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5-methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18F-FEDAA1106 and 2-deoxy-2-[18F]fluoro-D- glucose (18F-FDG, a marker of glucose metabolism) for positron emission tomographic (PET) imaging of vascular inflammation. This was tested using a murine model in which focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18F-FEDAA1106 or 18F-FDG was registered to anatomic data generated by computed tomographic (CT)/CT angiography. Standardized uptake values were significantly increased at cuffed compared to contralateral arteries using either 18F-FEDAA1106 (p < .01) or FDG (p < .05). However, the 18F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the noninflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.

Bridging the gap: ageing, pharmacokinetics and pharmacodynamics

Changes in pharmacokinetics and pharmacodynamics in elderly patients generally result in an increase in the incidence of drug toxicity and adverse drug reactions. Molecular alterations associated with ageing could bring about biological changes, a consequence of which is an altered response to pharmacological agents. Unfortunately, research in this area has yet to progress beyond the cataloguing of the pharmacokinetic and pharmacodynamic changes observed in the elderly. Therefore, real progress in our understanding of pharmacogerontology could be achieved if it were possible to merge pharmacokinetic and pharmacodynamic studies with recent advances in our understanding of the causal processes bringing about ageing changes at the cellular level. Therefore, this review will focus on the mechanisms of ageing in the hope that the information will be of value to those planning independent studies.

Exercise-induced changes in proteoglycan metabolism of equine articular cartilage

The Thoroughbred racehorse undergoes extensive and intensive training regimens which result in repetitive loads being placed on the tissues of the joints. Cartilage is a specialised connective tissue which functions as a low friction surface mediating smooth motion. Since the tissues of articulating joints are subject to impact loading, cartilage must also be capable of resisting and redistributing the forces arising during joint movement. The major macromolecules present in the extracellular matrix of cartilage are the collagens and the large, aggregating proteoglycan (aggrecan). The synthesis and incorporation of newly synthesised molecules into the extracellular matrix and their degradation and removal is a normal homeostatic event. The turnover of matrix components enables the tissue to adapt to changes in joint mechanics which occur during growth, development and ageing of the tissue (Platt et al. 1 9 9 8 ) . Turnover of the extracellular matrix also allows the composition of the tissue to be modified in response to mechanical loading (Sah et al. 1989). The objective of this study was to investigate the effect of short-term, intensive exercise on the metabolism of aggrecan in articular cartilage isolated from the joints of Thoroughbred horses. The results demonstrate that chondrocytes in equine metatarsophalangeal cartilage, in vivo , can alter their metabolic activity in response to changes in their mechanical environment.

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.

Interleukin‐1 Inhibits the Production of Types II, IX, and XI Procollagen mRNA in Cartilage

Articular chondrocytes are remarkable in their capacity to synthesize, organize, and regulate the deposition of their surrounding matrix in a highly ordered and efficient manner. The synthesis and degradation of matrix molecules become balanced in a stable equilibrium. We previously showed that interleukin1 (IL1 ) can alter this balance and induce a rapid net depletion of proteoglycan and eventually collagen.’-3 The present study investigates why collagen production is decreased in the presence of IL-1. Cartilage explants or chondrocyte cultures’ at 2.5 X lo5 cells/cm2 were established as described previously from the condylar ridge of the metacarpophalangeal joints of pigs aged approximately 6 months and grown in Iscove’s medium containing supplements (albumen, transferrin, lipoprotein, ascorbate) but no serum. The amount of newly synthesized collagen was measured4 and found to be 8% of the total radiolabeled protein in unstimulated explants. This decreased to 2.0% after 72-h exposure to IL1 (100 pM). The effect did not persist, and cartilage cultured in control medium following IL1 treatment increased production of collagen and was close to the original rate of synthesis after 4 days’ recovery. It was not clear whether decreased collagen production resulted mainly from decreased synthesis or enhanced degradation. The extent of degradation of extracellular collagen was estimated from the proportion of unlabeled hydroxyproline present in the medium? There was no significant increase in the release of collagen fragments, which represented about 2% of the total and no change in the salt or acid solubility of collagen within the explants during 3 days’ culture with IL-1 (10-350 pM). The distribution and rate of degradation of newly synthesized collagen were determined by measuring the fraction of radiolabeled collagen present as degradation fragments (ethanol-soluble) following a 16-h pulse with [ 14C]-proline.6 Of the total radiolabled hydroxyproline, 18-22% was rapidly degraded in all the cultures, but this was not altered by IL1. Analysis of the collagen synthesized in the presence of IL-1 by SDS-PAGE and CM-cellulose chromatography of [ I4C]-proline labeled pepsinized samples showed that the al(I1) chains produced in the presence of IL-1 had the same mobility as the controls.’ There was no detectable synthesis of type I collagen protein, although small amounts of al(1) trimer cannot be excluded. The steady state levels of collagen mRNA were assessed by Northern blot transfer and hybridization to specific cDNA

Quantitative Phosphor Imaging Autoradiography of Radioligands for Positron Emission Tomography

Imaging using phosphor screens have increasingly been employed for the analysis of radioactive samples in molecular biology, pharmacology, and receptor autoradiography. The major advantages of phosphor screens compared to radiation sensitive film are their greatly increased sensitivity, reducing exposure times with at least one order of magnitude, and their increased linear dynamic range. These features make phosphor screens ideal for imaging short-lived radionuclides, where exposure times are limited, such as (11)C and (18)F widely used to label radioligands for positron emission tomography (PET). Phosphor imaging can also considerably reduce exposure times for weak β-particle emitters such as (3)H. In this chapter, we present methods for the characterization and evaluation of novel PET radioligands using quantitative phosphor imaging autoradiography.

Absence of premature senescence in Werner's syndrome keratinocytes

Werners syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve <20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werners syndrome is a segmental progeroid syndrome.