Improving the diagnosis and treatment of atherosclerosis: targeting calcification as a basis for patient management

The presence of atherosclerotic calcification serves as a surrogate marker for plaque burden and a prognostic marker of cardiovascular risk. Additionally, calcification plays a critical role in plaque stability and heavily calcified lesions are associated with increased transcatheter therapy failures. The purpose of this thesis is to advance calcification-based cardiovascular patient management. Specifically, an improved understanding of the capabilities of clinical image- and blood-based biomarkers to distinguish between high risk patients and high-risk plaques based on calcified content is required. Additionally, more accurate stiffness properties for calcified and non-calcified tissue constituents will optimise computational predictions of plaque rupture and device-tissue interactions. Calcifications within ex vivo atherosclerotic lesions were quantified using micro computed tomography (micro-CT), clinical CT, magnetic resonance (MR), intravascular ultrasound (IVUS) and optical coherence tomography (OCT). Coronary artery calcium (CAC) scores were obtained from non-contrast chest CT scans. Circulating blood biomarkers of vascular calcification were measured using commercial immunoassays. Nanomechanical techniques were employed to characterise the stiffness of calcified and non-calcified tissue portions. These biomechanical techniques were coupled micro-CT, scanning electron microscopy, energy dispersive x-ray spectroscopy and histological analyses to confirm the biological content of the regions of interest being examined. Agatston calcium scores correlate well with calcification volumes and are therefore good markers of atherosclerotic burden. However, calcified particle distributions are not estimated and larger calcified particles have higher maximum x-ray attenuation densities. An assessment into the effect of decreasing CT resolution on measures of calcification revealed the inaccuracies acquired for calcification volume, density and particle measurements. Additionally, a heterogeneous distribution of calcium density was identified. The efficacy of coronary-derived calcium scores or the fraction of low- or high-density calcium to differentiate between symptomatic or asymptomatic carotid plaques was investigated. Neither the Agatston, Volume or Density-Volume coronary calcium scores could differentiate between carotid plaques based on patient preoperative cerebrovascular symptoms. However, asymptomatic plaques contained significantly lower levels of low-density calcification and higher levels of high-density calcification. Clinical CT also exhibited the closest correlation to micro-CT for measures of calcified content. No differences were observed between circulating blood-biomarkers of vascular calcification or bone formation with patient endarterectomy or number of diagnosed atherosclerotic locations groups. Moderate and weak negative associations were observed between dephospho-uncarboxylated Matrix Gla Protein and coronarty artery calcium (CAC) density scores, and between percent undercarboxylated osteocalcin and CAC scores or total volume of calcification for certain participant subgroups only. There is a clear distinction between the elastic modulus of calcification with respect to radiographic density. Furthermore, there is no difference in the behaviours of carotid versus lower extremity calcification. This study confirms the hypothesis that the mechanical properties of calcification are similar to that of human bone tissues (17–25 GPa). Moreover, greater than 6 orders of magnitude compliance mismatch exists between the calcified and non-calcified portions of carotid atherosclerotic plaques. Collectively, this research demonstrates an improved understanding of the use of atherosclerotic calcification measurements in clinical diagnostics and the biomechanics of advanced calcified atherosclerotic plaques

The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells

Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm2 but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4–6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm2, which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutic

Identification of nanoparticle properties for optimal drug delivery across a physiological cell barrier

Nanoparticles (NPs) represent an attractive strategy to overcome difficulties associated with the delivery of therapeutics. Knowing the optimal properties of NPs to address these issues could allow for improved in vivo responses. This work investigated NPs prepared from 5 materials of 3 sizes and 3 concentrations applied to a cell barrier model. The NPs permeability across a cell barrier and their effects on cell barrier integrity and cell viability were evaluated. The properties of these NPs, as determined in water (traditional) vs. media (realistic), were compared to cell responses. It was found that for all cellular activities, NP properties determined in media was the best predictor of the cell response. Notably, ZnO NPs caused significant alterations to cell viability across all 3 cell lines tested. Importantly, we report that the zeta potential of NPs correlates significantly with NP permeability and NP-induced changes in cell viability. NPs with physiological-based zeta potential of −12 mV result in good cell barrier penetration without considerable changes in cell viability.</p

A blood biomarker and clinical correlation cohort study protocol to diagnose sports-related concussion and monitor recovery in elite rugby

Introduction In professional rugby, sports-related concussion (SRC) remains the most frequent time loss injury. Therefore, accurately diagnosing SRC and monitoring player recovery, through a multi-modal assessment process, is critical to SRC management. In this protocol study, we aim to assess SRC over multiple time points post-injury to determine the value of multi-modal assessments to monitor player recovery. This is of significance to minimise premature return-to-play and, ultimately, to reduce the long-term effects associated with SRC. The study will also establish the logistics of implementing such a study in a professional setting to monitor a player’s SRC recovery. Methods and analysis All players from the participating professional rugby club within the Irish Rugby Football Union are invited to participate in the current study. Player assessment includes head injury assessment (HIA), neuropsychometric assessment (ImPACT), targeted biomarker analysis and untargeted biomarker analysis. Baseline HIA, ImPACT, and blood draws are performed prior to the start of playing season. During the baseline tests, player’s complete consent forms and an SRC history questionnaire. Subsequently, any participant that enters the HIA process over the playing season due to a suspected SRC will be clinically assessed (HIA and ImPACT) and their blood will be drawn within 3 days of injury, 6 days post-injury, and 13 days post-injury. Ethics and dissemination Ethical approval was attained from the Science and Engineering Research Ethics Committee, University of Limerick (Approval Code: 2018_06_11_S&E). On completion of the study, further manuscripts will be published to present the results of the tests and their ability to measure player recovery from SRC. Trial registration number NCT04485494