Angela Sheerin

In vivo absorption, metabolism, and urinary excretion of alpha,beta-unsaturated aldehydes in experimental animals. Relevance to the development of cardiovascular diseases by the dietary ingestion of thermally stressed polyunsaturate-rich culinary oils.

Thermal stressing of polyunsaturated fatty acid (PUFA)- rich culinary oils according to routine frying or cooking practices generates high levels of cytotoxic aldehydic products (predominantly trans-2-alkenals, trans,trans-alka-2,4-dienals, cis,trans-alka-2, 4-dienals, and n-alkanals), species arising from the fragmentation of conjugated hydroperoxydiene precursors. In this investigation we demonstrate that typical trans-2-alkenal compounds known to be produced from the thermally induced autoxidation of PUFAs are readily absorbed from the gut into the systemic circulation in vivo, metabolized (primarily via the addition of glutathione across their electrophilic carbon-carbon double bonds), and excreted in the urine as C-3 mercapturate conjugates in rats. Since such aldehydic products are damaging to human health, the results obtained from our investigations indicate that the dietary ingestion of thermally, autoxidatively stressed PUFA-rich culinary oils promotes the induction, development, and progression of cardiovascular diseases.

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification

Little is known about the senescent phenotype of human vascular smooth muscle cells (VSMCs) and the potential involvement of senescent VSMCs in age-related vascular disease, such as atherosclerosis. As such, VSMCs were grown and characterised in vitro to generate senescent VSMCs needed for microarray analysis (Affymetrix). Comparative analysis of the transcriptome profiles of early (14 CPD) and late (39-42 CPD) passage VSMCs found a total of 327 probesets called as differentially expressed: 149 are up-regulated in senescence and 178 repressed (p-value<0.5%, minimum effect size of at least 2-fold differential regulation, explore data at http://www.madras.cf.ac.uk/vsmc). Data mining shows a differential regulation of genes at senescence associated with the development of atherosclerosis and vascular calcification. These included genes with roles in inflammation (IL1beta, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFbeta, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1beta, IL8 and MGP were validated by either, ELISA, Western blot analysis or RT-PCR. These data thus provide the first evidence for a role of VSMC senescence in the development of vascular calcification and provides further support for the involvement of senescent VSMCs in the progression of atherosclerosis.

Multicomponent Spectroscopic Investigations of Salivary Antioxidant Consumption by an Oral Rinse Preparation Containing the Stable Free Radical Species Chlorine Dioxide (CIO2)

A multicomponent evaluation of the oxidative consumption of salivary biomolecules by a commercially-available oral rinse preparation containing an admixture of the stable free radical species chlorine dioxide (ClO2.) with chlorite anion (ClO2-) has been investigated using high resolution 1H NMR spectroscopy. The results obtained demonstrated that ClO2. and/or ClO2- present in this preparation effected the oxidative decarboxylation of salivary pyruvate (to acetate and CO2). Experiments conducted on chemical model systems confirmed the oxidative decarboxylation of pyruvate by this oral rinse, and also demonstrated that urate, thiocyanate anion, and the amino acids cysteine and methionine (precursors to volatile sulphur compounds responsible for oral malodour), were oxidatively consumed. The biochemical, periodontal and therapeutic significance of the results are discussed.

1H-NMR analysis of microbial-derived organic acids in primary root carious lesions and saliva

In addition to lowered pH values, the molecular profile and concentrations of microbial‐derived organic acids in carious dentin are important demineralization parameters involved in the induction, development and progression of dental caries. High‐resolution proton (1H) NMR spectroscopy was employed to examine the organic acid status of primary root carious lesions. 1H‐NMR analysis of post‐neutralized perchloric acid extracts of active carious lesions revealed that at an operating frequency of 600 MHz, the 1H‐NMR‐detectable organic acid composition of carious dentin samples (mean molecular percentage content ± standard error; the mean molecular percentage content is defined here as the mean of the concentration of each 1H‐NMR‐visible organic acid/anion expressed as a percentage of total 1H‐NMR‐detectable organic acid/anion level in each sample) was acetate 51 ± 2%, formate 37 ± 2%, lactate 5 ± 1%, propionate 3 ± 0.8%, pyruvate 2.4 ± 0.3%, n‐butyrate 1.2 ± 0.2%; succinate 0.1 ± 0.1%; iso‐butyrate, n‐ and iso‐valerate, and n‐ and iso‐caproate (total) <0.2%. Further components detectable included alanine, glycine, choline, phosphorylcholine, trimethylamine oxide, methanol, glycolate and assorted saccharides. In view of their high dissociation constants (Ka), our results demonstrate that formic and pyruvic acids (Ka = 1.77 × 10−4 and 3.20 × 10−3 mol/dm3, respectively) contribute substantially to the decreased pH values associated with active caries lesions (cf. lactate Ka = 1.40 × 10−4 mol/dm3), and hence the pathogenesis of primary root caries. Copyright

Cyclin D1 overexpression permits the reproducible detection of senescent human vascular smooth muscle cells.

Abstract:  The senescence of mitotic cells is hypothesized to play a causal role in organismal aging. Cultures of normal human cells become senescent in vitro as a result of a continuous decline in the mitotic fraction from cell turnover. However, one potential barrier to the evaluation of the frequency and distribution of senescent cells in tissues is the absence of a panel of robust markers for the senescent state. In parallel with an analysis of the growth kinetics of human vascular smooth muscle cells, we have undertaken transcriptomic comparisons of early‐ and late‐passage cultures of human vascular smooth muscle cells to identify potential markers that can distinguish between senescent and growth‐competent cells. A wide range of genes are upregulated at senescence in human vascular smooth muscle cells. In particular, we have identified a 12‐fold upregulation of expression in the cyclin D1 message, which is reflected in a concomitant upregulation at the protein level. Quantitative cytochemical analysis of senescent and growing vascular smooth muscle cells indicates that cyclin D1 reactivity is a considerably better marker of replicative senescence than senescence‐associated β‐galactosidase activity. We have applied this new marker (in combination with Ki67, COMET, and TUNEL staining) to the study of human vascular smooth muscle cells treated with resveratrol, a putative anti‐aging molecule known to have significant effects on cell growth.

Altered composition and DNA binding activity of the AP-1 transcription factor during the ageing of human fibroblasts

We have investigated the expression of AP-1 transcription factor proteins during the in-vitro ageing of human fibroblasts. The numbers of these cells that are in the cell cycle gradually decreases up to 45 cumulative population doublings (cPD), thereafter the decline is steeper, until almost all cells enter a post-mitotic state by 60 cPD. We observed that a 34 kd junB species began to replace the 44 kd junB species after 41 cPD. This was followed, after 44 cPD, by a loss of fra1 and both junD species. After 49 cPD there was a gradual decline in the levels of fos and jun proteins, but disproportionately, so that the fos/jun protein ratio also declined. Although fos and jun proteins were still clearly present at 60 cPD, utilisation of the AP-1 DNA consensus sequence could not be demonstrated after 54 cPD. These data indicate that significant changes occur in the composition of the AP-1 transcription factor during ageing, but also that alterations in its DNA binding activity may involve other factors.

Characterization of cellular senescence mechanisms in human corneal endothelial cells

The human cornea is a tri‐laminar structure composed of several cell types with substantial mitotic potential. Age‐related changes in the cornea are associated with declining visual acuity and the onset of overt age‐related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue‐engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over‐expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof‐of‐principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over‐expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC‐specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature‐sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell‐based therapies in the human cornea.

Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence

BackgroundAltered expression of mRNA splicing factors occurs with ageing in vivo and is thought to be an ageing mechanism. The accumulation of senescent cells also occurs in vivo with advancing age and causes much degenerative age-related pathology. However, the relationship between these two processes is opaque. Accordingly we developed a novel panel of small molecules based on resveratrol, previously suggested to alter mRNA splicing, to determine whether altered splicing factor expression had potential to influence features of replicative senescence.ResultsTreatment with resveralogues was associated with altered splicing factor expression and rescue of multiple features of senescence. This rescue was independent of cell cycle traverse and also independent of SIRT1, SASP modulation or senolysis. Under growth permissive conditions, cells demonstrating restored splicing factor expression also demonstrated increased telomere length, re-entered cell cycle and resumed proliferation. These phenomena were also influenced by ERK antagonists and agonists.ConclusionsThis is the first demonstration that moderation of splicing factor levels is associated with reversal of cellular senescence in human primary fibroblasts. Small molecule modulators of such targets may therefore represent promising novel anti-degenerative therapies.

Multicomponent evaluations of the oxidising actions and status of a peroxoborate-containing tooth-whitening system in whole human saliva using high resolution proton NMR spectroscopy

High resolution 1H nuclear magnetic resonance (NMR) spectroscopy was employed to conduct a multicomponent investigation of the oxidation of salivary biomolecules by peroxoborate present in a tooth-whitening dentifrice formulation. The results acquired demonstrated that peroxoborate gave rise to the oxidative decarboxylation of the hydrogen peroxide scavenger pyruvate, a reaction generating acetate and CO2 as products. Experiments performed on chemical model systems confirmed the oxidative consumption of pyruvate by dentifrice-derived peroxoborate, and also revealed that the salivary electron donors cysteine and methionine (precursors to volatile sulphur compounds), were oxidised to cystine and methionine sulphoxide respectively. The biochemical and periodontal significance of these results is discussed.

A facile, stereoselective, one-pot synthesis of resveratrol derivatives

AbstractBackgroundCompounds based on trans-1,2-diphenylethene are the subject of intense interest both for their optical properties and as potential leads for drug discovery, as a consequence of their anticancer, anti-inflammatory and antioxidant properties. Perhaps the best known of these is trans-3,5,4′-trihydroxystilbene (resveratrol), that has been identified as a promising lead in the search for anti-ageing therapeutics.ResultsWe report here a new, convenient, one-pot stereo-selective synthesis of resveratrol and other trans-stilbene derivatives. A wide range of known and novel “Resveralogues” were synthesised by using this simple protocol, including examples with electron donating and electron withdrawing substituents, in uniformly high yield. The structures of all compounds were confirmed by standard methods including 1H and 13C NMR, IR and High Resolution Mass spectroscopy.ConclusionsWe have established a simple and convenient protocol for resveralogue synthesis. It is readily scalable, and sufficiently robust and simple for ready use in automated synthesis or for library development of resveralogues. This supersedes previously reported synthetic methods that required inert conditions, extensive purification and/or costly reagents. Graphical abstractOne-pot preparation of diverse Resveralogues - high yields of product with minimal purification.