Yvonne Alexander

Evaluation of carotid plaque inflammation in patients with active rheumatoid arthritis using 18F-fluorodeoxyglucose PET-CT and MRI: a pilot study

BACKGROUND Rheumatoid arthritis is associated with a 50% increased risk in cardiovascular mortality. Inflammation is thought to accelerate atherosclerosis and might also lead to an inflammatory rupture-prone plaque phenotype. We tested the hypothesis that patients with active rheumatoid arthritis also have carotid plaque inflammation and that plaque inflammation correlates with clinical and serological markers of inflammation. METHODS Patients with active rheumatoid arthritis, defined as the Disease Activity Score in 28 joints (DAS28) score of more than 3·2, were recruited to a single centre study in the UK. Patients with carotid plaque on ultrasound underwent carotid MRI followed by (18)F-fluorodeoxyglucose ((18)F-FDG) PET-CT. Scans were co-registered and analysed by a physicist, masked to clinical information. The maximum standardised uptake values (SUV(max)) were measured in the plaque area. The association of SUV with DAS28, C-reactive protein, and CD4+CD28- T-cell frequency was tested with non-parametric statistics. Ethics approval and informed consent were obtained. FINDINGS Scans were done in 13 patients, nine of whom were women. Median age was 60 years (IQR 57-65), disease duration was 11 years (6-25), and DAS28 score was 4·52 (4·32-5·13). None had a history or symptoms of clinical cardiovascular disease or took statins. All plaques caused less than 70% stenosis, and tracer uptake in plaque was seen on PET in all 13 patients. Median SUV(max) was 2·18 (IQR 2·00-2·65), and all cases had an SUV(max) greater than 1·6 (the threshold for defining carotid plaque inflammation). There was a significant association with SUV(max) and C-reactive protein (r=0·58, p=0·04) and quartiles of CD4+CD28- T-cell frequency (p=0·045), but not with low-density lipoprotein concentrations (r=-0·49, p=0·09) or DAS28 score (r=0·38, p=0·20). No association was found with age (r=0·13, p=0·69) or sex (p=0·64). INTERPRETATION In this small pilot study, plaque inflammation was seen in all patients and correlated with C-reactive protein. Whether this finding represents simultaneous joint and plaque inflammation, which might improve on treatment of joint disease, remains to be determined. CD4+CD28- T-cells are known to predict cardiovascular events in patients with angina. Their association with plaque inflammation in this study suggests a possible role in cardiovascular risk prediction in rheumatoid arthritis. Larger studies are warranted to investigate these findings further. FUNDING North West England MRC Clinical Pharmacology and Therapeutics Clinical Research Fellowship, National Institute for Health Research, AstraZeneca-University of Manchester Strategic Alliance Fund.

Novel Methods for Accurate Identification, Isolation, and Genomic Analysis of Symptomatic Microenvironments in Atherosclerotic Arteries

A challenge facing surgeons is identification and selection of patients for carotid endarterectomy or coronary artery bypass/surgical intervention. While some patients with atherosclerosis develop unstable plaques liable to undergo thrombosis, others form more stable plaques and are asymptomatic. Identification of the cellular signaling mechanisms associated with production of the inflammatory, hemorrhagic lesions of mature heterogenic plaques will help significantly in our understanding of the differences in microenvironment associated with development of regions susceptible to rupture and thrombosis and may help to predict the risk of plaque rupture and guide surgical intervention to patients who will most benefit. Here, we demonstrate detailed and novel methodologies for successful and, more importantly, accurate and reproducible extraction, sampling, and analysis of micro-regions in stable and unstable coronary/carotid arteries. This information can be applied to samples from other origins and so should be useful for scientists working with micro-isolation techniques in all fields of biomedical science.

179 The Role Of Microparticles in Carotid Disease

Introduction Endothelial microparticles (EMPs) are released from dysfunctional endothelial cells. We hypothesised that patients with unstable carotid plaque have higher levels of circulating microparticles compared to patients with stable plaques which could be related to a specific cytokine profile. Methods Circulating EMPs and inflammatory cytokine levels were measured in seventy patients with significant carotid disease undergoing carotid endarterectomy and 20 healthy controls. Fifty one (73%) patients had symptomatic disease whilst 19 (27%) were asymptomatic. EMPs (CD31+/ Annexin V+ CD42b-) were quantified using flow cytometry. Immunohistological analysis of carotid plaques for CD68+, CD206+ macrophages, TNF-α smooth muscle actin and osteopontin was performed, together with Alizarin red staining for detection of calcific deposits. Bioplex assays were used for cytokine analysis. Plaques were graded according to the American Heart Association plaque scoring system. Results Significantly higher EMP levels were observed in symptomatic patients compared to controls, p = 0.01, while no differences were noted in EMP levels in asymptomatic vs controls p = 0.11. The higher EMP levels appeared to associate with the unstable plaques which also had a significantly higher level of CD68+ macrophages compared to stable plaques (AHA I-IV) and higher circulating levels of Chemokine ligand-9 (CXCL-9) (p < 0.004). Of note, macrophage inhibitory factor (MIF) was among the chemokines that were elevated in the circulation of patients with stable plaques, whether a lack of this factor in unstable plaques has direct effects on phenotype remains to be elucidated. Other factors elevated in patients with stable plaques were IL-16, cutaneous T-cell attracting chemokine (CTACK), and stem-cell growth factor-b (SCGF-b) (p value of 0.05, 0.035 and 0.002 respectively). Conclusion Circulatory EMP and specific inflammatory cytokine levels are raised in patients with unstable plaques, while MIF, a potentially protective factor was elevated in serum of patients with stable plaques. These data could have major implications for the development of a diagnostic tool whereby EMPs together with markers of macrophage activity could act in a combined manner as biomarkers of plaque vulnerability and stroke susceptibility.

147 QRISK3 improves identification of endothelial dysfunction and cardiovascular disease risk in patients with systemic lupus erythematosus

Introduction Systemic Lupus Erythematosus (SLE) is an inflammatory autoimmune condition associated with endothelial dysfunction, elevating the risk of cardiovascular disease 50-fold in young women. The QRISK2 algorithm is used currently to predict the 10 year cardiovascular risk in the UK population; however, an updated QRISK3 model was recently released, which considers inflammatory variables such as SLE and steroid prescription. This study aims to elucidate whether QRISK3 is more representative of endothelial dysfunction and cardiovascular risk in SLE patients and if novel biomarkers of SLE disease activity, including endothelial microvesicles (EMVs), correlate with increased risk. Methods QRISK scores of SLE patients (n=109) and controls (n=29) were determined using clinical data. Potential markers of SLE-specific endothelial dysfunction were quantified in a smaller patient cohort (n=60) using flow cytometry and ELISA techniques and included; CD144 +EMVs, high sensitivity C-reactive protein (hsCRP) and triglycerides. Results QRISK3 scores were significantly elevated in SLE patients (average 5.1%) compared to controls (0.3%; p<0.001), newly identifying 19% of patients as _x0018_high risk’ (QRISK3 vs QRISK2: 30 vs 9; p<0.001). _x0018_Missed’ high risk patients had increased likelihood of lupus nephritis, corticosteroid prescription and positive anti-cardiolipin antibody test compared to low risk patients. Levels of EMVS, hsCRP and triglycerides were significantly elevated in the missed group (p<0.05) whereas rates of antiplatelet (38.1%) and statin (23.8%) prescription were low. Abstract 143 Figure 1 Abstract 143 Figure 2 Conclusion QRISK3 identifies significantly more SLE patients at high cardiovascular risk than QRISK2, and is associated with standard and novel markers of SLE-specific inflammation and endothelial dysfunction. The use of QRISK3 as a predictor of cardiovascular risk will support improved management of vascular health and assist in prevention of premature mortality in SLE patients.

183 Novel glycomimetics inhibit gly-ldl induced smooth muscle cell calcification via creb

Advanced glycated end-products (AGEs) are known drivers of cardiovascular complications such as vascular calcification, which is currently untreatable, and are increased in diabetic subjects in part due to oxidative stress and poor glycaemic control. Our previous studies using smooth muscle cells (SMCs) isolated from patients with peripheral arterial disease (PAD) have implicated a number of signalling pathways involved in their osteogenic differentiation in vitro, including OPG/RANK. We have also shown that alterations in the cell surface glycocalyx regulates cell function, suggesting that non-sugar glycosaminoglycan mimics can potentially modulate cell phenotypes. We aim to investigate how modified LDL and PAD serum affects the progression of calcification of SMCs in vitro and whether this pathology can be prevented by novel glycosaminoglycan mimics, using qPCR, ELISA, Alkaline phosphatase (ALP) activity, and Western blotting. Gly-LDL (10 µg/ml) increased an early marker of calcification (ALP activity) at 4 days and enhanced calcification at 21 days, compared to controls (p<0.05) as shown by alizarin red staining. PAD serum accelerated calcification, which was apparent after 10 days (p<0.05). The glycomimetics significantly inhibited both gly-LDL and PAD serum-induced mineralisation, and reduced gly-LDL induced ALP activity at day 4 (p<0.05). In the gly-LDL-treated SMCs, secreted levels of osteocalcin (OCN), a promoter of osteogenic differentiation, were reduced when treated with the glycomimetics, whereas osteopontin (OPN) and osteoprotegerin (OPG), inhibitors of calcification, were increased compared to gly-LDL alone. A similar trend was observed in PAD serum-treated SMCs, with glycomimetics reducing OCN and increasing OPN secretion. A phospho-kinase array analysis of gly-LDL-treated SMCs was performed to identify underlying mechanism of action. Gly-LDL increased phosphorylation of cyclic AMP response element-binding protein (CREB), TOR, and the SRC proteins LYN, YES and CHK-2 compared with untreated control, which was attenuated with the addition of the glycomimetics. A number of upstream activators of CREB were targeted using known pharmacological inhibitors in SMCs treated with gly-LDL. The MEK inhibitor U0126 accelerated calcification, increasing both ALP activity and expression of receptor for AGEs (RAGE), a key receptor implicated in vascular calcification, compared to gly-LDL alone, suggesting that MEK may be a mediator of mineralisation process via phosphorylation of CREB. Glycomimetics have potential as an anti-calcification strategy, inhibiting mineralisation in SMCs induced by both gly-LDL and patient serum in vitro. The protective effect of glycomimetics against calcification may occur via regulation of CREB phosphorylation and subsequent modulation of downstream osteogenic markers, including upregulation of OPN and OPG and reduction of OCN, leading to the development of therapeutics to treat vascular calcification.

173 Infused silica nanoparticles compromise vascular function in small mesenteric arteries

Introduction The emergence of nanomedicine, involving the intravenous injection of tracking agents (such as dye-encapsulated silica nanoparticles [SiNPs]) for imaging diagnostic and therapeutic purposes may hold a biosafety concern specifically to the vascular system. Our previous findings suggest that SiNPs of 100 and 200 nm size have no detrimental effect on conduit arterial function. However, their effect on small size vessels, which play an important role in controlling blood perfusion into tissues, has not been investigated. Methods and results Vasoconstrictor and vasodilator responses of mesenteric arteries (MAs) from male Wistar rats were assessed using pressure myography before and 30 min after intravascular infusion of SiNPs. Our data show that SiNPs (100 nm size at 5.32 × 1011 NPs/mL dose) compromised the contractile responses to phenylephrine (Phe) (n = 5; p < 0.01). SiNPs also attenuated the endothelium-dependent (acetylcholine) dilator responses in high potassium (KPSS) and Phe-pre-contracted vessels following their rapid uptake into the cytoplasm of endothelial cells ex vivo as well as in vivo (At 100 nM Ach, the mean percentage dilation was 78.77 ± 8.58% vs. 36.30 ± 5.07% after incubation in PSS and SiNPs respectively, p < 0.001). The acute exposure to SiNPs did not alter the endothelium-independent relaxation to sodium nitroprusside (n = 4). SiNPs were biodistributed in trace amounts into different tissues; including MAs, aortic vessel, liver and spleen in vivo. The physiological changes were accompanied by a decrease in the phosphorylated levels of both extracellular-signal-regulated kinase (ERK) and protein kinase Akt demonstrated by proteomic analysis. Conclusions Findings suggest that SiNPs may affect microvessel function via modulation of the ERK/AKT pathway. Our data highlight the need to identify strategies that can help minimise possible detrimental effects of nanoparticles on arterial function.

214 Glycated LDL (glyc-LDL) Promotes Osteogenic Differentiation of Vascular Smooth Muscle Cells

Introduction It is well established that glyc-LDL is elevated in the serum of diabetic patients compared to healthy subjects, and that vascular calcification is common in diabetes. Furthermore, recent studies suggest that advanced glycation end products may play a pathogenic role in vascular calcification. We hypothesise that glyc-LDL promotes the osteogenic differentiation of vascular smooth muscle cells in vitro. Methods LDL was isolated from human serum by sequential density gradient ultracentrifugation and incubated at 37°C with a range of glucose concentrations at different time points to determine the optimum glycation conditions. The glyc-LDL was measured using an in-house ELISA method. Bovine Aortic Smooth Muscle Cells (BAoSMCs) were incubated with native or glycated LDL in the presence of osteogenic media and mineral deposition was determined using alizarin red staining and alkaline phosphatase (ALP) activity, which is an early marker of osteogenesis. Results The optimum conditions for LDL glycation was determined as 80mM glucose for 7 days, so these conditions were used for the production of glycated LDL throughout the study. BAoSMCs incubated in osteogenic media exhibited mineralisation after 7 days as determined by alizarin red staining. This calcification was significantly enhanced by treatment with glyc-LDL, but not by native LDL. Furthermore, we found that ALP activity was significantly elevated as early as day 4 in glyc-LDL treated cells, compared to those incubated in native LDL. The glyc-LDL-induced mineralisation was not attenuated in the presence of osteoprotegrin (OPG) nor in the presence of two novel small RAGE antagonist peptides, which were designed to prevent RAGE binding with several of its most important ligands, including HMGB-1, S100P and S1004A. Conclusion We have shown that human-derived glyc-LDL accelerates vascular calcification in vitro. This process does not appear to be attenuated by the addition of OPG, suggesting AGE-induced accelerated calcification occurs through a pathway independent of the RANKL/OPG signalling activity. Further studies with a range of RAGE inhibitors should allow the identification of the molecular pathways implicated in glyc-LDL induced calcification to enable the development of therapeutic peptides effective in blocking of RAGE-ligand interaction, which is prevalent in many pathologies.