Aart C. Strang

Pro-atherogenic lipid changes and decreased hepatic LDL receptor expression by tocilizumab in rheumatoid arthritis

OBJECTIVES Blocking the interleukin-6 pathway by tocilizumab (TCZ) has been associated with changes in the lipoprotein profile, which could adversely impact cardiovascular (CV) risk in patients with rheumatoid arthritis (RA). In the present study, we addressed the effect of TCZ on lipoproteins in both fasting and non-fasting state in RA patients and tested the effect of TCZ on LDL receptor (LDLr) expression in vitro. METHODS Twenty patients with active RA and an inadequate response to TNF blockers received monthly TCZ intravenously. On week 0, 1 and 6 blood was drawn before and after an oral fat load, the lipid profiles and HDL antioxidative capacity were measured. Effects of TCZ on LDLr expression in transfected HepG2 cells were subjected. RESULTS After 6 weeks of TCZ, total cholesterol increased by 22% (4.8 ± 0.9 to 5.9 ± 1.3 mmol/L; p < 0.001), LDLc by 22% (3.0 ± 0.6 to 3.6 ± 0.8 mmol/L; p < 0.001) and HDLc by 17% (1.4 ± 0.4 to 1.7 ± 0.7 mmol/L; p < 0.016). Fasting triglycerides (TG) increased by 48% (1.0 ± 0.4 to 1.4 ± 0.8 mmol/L; p = 0.011), whereas postprandial incremental area under the curve TG increased by 62% (p = 0.002). Lipid changes were unrelated to the change in disease activity or inflammatory markers. No difference in HDL antioxidative capacity was found. In vitro, LDLr expression in cultured liver cells was significantly decreased following TCZ incubation (P < 0.001). CONCLUSIONS TCZ adversely impacts on both LDLc as well as fasting and postprandial TG in patients with RA. The changes in hepatic LDLr expression following TCZ imply that adverse lipid changes may be a direct hepatic effect of TCZ. The net effect of TCZ on CV-morbidity has to be confirmed in future clinical trials.

Thresholds for Arterial Wall Inflammation Quantified by 18F-FDG PET Imaging: Implications for Vascular Interventional Studies

Objectives This study assessed 5 frequently applied arterial 18fluorodeoxyglucose (18F-FDG) uptake metrics in healthy control subjects, those with risk factors and patients with cardiovascular disease (CVD), to derive uptake thresholds in each subject group. Additionally, we tested the reproducibility of these measures and produced recommended sample sizes for interventional drug studies. Background 18F-FDG positron emission tomography (PET) can identify plaque inflammation as a surrogate endpoint for vascular interventional drug trials. However, an overview of 18F-FDG uptake metrics, threshold values, and reproducibility in healthy compared with diseased subjects is not available. Methods 18F-FDG PET/CT of the carotid arteries and ascending aorta was performed in 83 subjects (61 ± 8 years) comprising 3 groups: 25 healthy controls, 23 patients at increased CVD risk, and 35 patients with known CVD. We quantified 18F-FDG uptake across the whole artery, the most-diseased segment, and within all active segments over several pre-defined cutoffs. We report these data with and without background corrections. Finally, we determined measurement reproducibility and recommended sample sizes for future drug studies based on these results. Results All 18F-FDG uptake metrics were significantly different between healthy and diseased subjects for both the carotids and aorta. Thresholds of physiological 18F-FDG uptake were derived from healthy controls using the 90th percentile of their target to background ratio (TBR) value (TBRmax); whole artery TBRmax is 1.84 for the carotids and 2.68 in the aorta. These were exceeded by >52% of risk factor patients and >67% of CVD patients. Reproducibility was excellent in all study groups (intraclass correlation coefficient >0.95). Using carotid TBRmax as a primary endpoint resulted in sample size estimates approximately 20% lower than aorta. Conclusions We report thresholds for physiological 18F-FDG uptake in the arterial wall in healthy subjects, which are exceeded by the majority of CVD patients. This remains true, independent of readout vessel, signal quantification method, or the use of background correction. We also confirm the high reproducibility of 18F-FDG PET measures of inflammation. Nevertheless, because of overlap between subject categories and the relatively small population studied, these data have limited generalizability until substantiated in larger, prospective event-driven studies. (Vascular Inflammation in Patients at Risk for Atherosclerotic Disease; NTR5006)

Unexpected arterial wall and cellular inflammation in patients with rheumatoid arthritis in remission using biological therapy: a cross-sectional study

BackgroundIncreasing numbers of patients (up to 40 %) with rheumatoid arthritis (RA) achieve remission, yet it remains to be elucidated whether this also normalizes their cardiovascular risk. Short-term treatment with TNF inhibitors lowers arterial wall inflammation, but not to levels of healthy controls. We investigated whether RA patients in long-term remission are characterized by normalized inflammatory activity of the arterial wall and if this is dependent on type of medication used (TNF-inhibitor versus nonbiological disease-modifying antirheumatic drugs (DMARDs)).MethodsArterial wall inflammation, bone marrow and splenic activity (index of progenitor cell activity) was assessed with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) in RA patients in remission (disease activity score (DAS28) <2.6 for >6 months) and healthy controls. We performed ex vivo characterization of monocytes using flow cytometry and a transendothelial migration assay.ResultsOverall, arterial wall inflammation was comparable in RA patients (n = 23) in long-term remission and controls (n = 17). However, RA subjects using current anti-TNF therapy (n = 13, disease activity score 1.98[1.8–2.2]) have an almost 1.2-fold higher 18F-FDG uptake in the arterial wall compared to those using DMARDs (but with previous anti-TNF therapy) (n = 10, disease activity score 2.24[1.3–2.5]), which seemed to be predominantly explained by longer duration of their rheumatic disease in a multivariate linear regression analysis. This coincided with increased expression of pro-adhesive (CCR2) and migratory (CD11c, CD18) surface markers on monocytes and a concomitant increased migratory capacity. Finally, we found increased activity in bone marrow and spleen in RA patients using anti-TNF therapy compared to those with DMARDs and controls.ConclusionsA subset of patients with RA in clinical remission have activated monocytes and increased inflammation in the arterial wall, despite the use of potent TNF blocking therapies. In these subjects, RA disease duration was the most important contributor to the level of arterial wall inflammation. This increased inflammatory state implies higher cardiovascular risk in these patients, who thus may require more stringent CV risk management.

The Genetics of High-Density Lipoprotein Metabolism: Clinical Relevance for Therapeutic Approaches

The risk for coronary artery disease (CAD) is inversely correlated with high-density lipoprotein (HDL) cholesterol plasma levels. These plasma HDL cholesterol levels are influenced by the activity of a number of enzymes and receptors, and therefore, variations in the genes encoding for these proteins may consequently result in an altered CAD risk. Identification of such pivotal players in HDL cholesterol metabolism that are also strongly associated with CAD risk is crucial for the materialization of novel therapeutic modalities. A large amount of knowledge has been obtained by studies involving families with extreme HDL phenotypes specific to molecular defects. In fact, thus far, monogenetic defects have been described in the genes coding for apolipoprotein A-I, adenosine triphosphate-binding cassette transporter A1, cholesterol ester transfer protein, the lack of endothelial lipase (LIPG), phospholipid transfer protein, and lecithin-cholesterol acyltransferase. Despite the fact that the total number of carriers of such mutations is rather small, much can be gained by extensively studying the metabolic and vascular consequences of these mutations. Surrogate markers for atherosclerosis have proved to be useful to overcome this sample size limitation and have been widely exploited to study families with decreased or increased HDL cholesterol levels in order to correlate HDL cholesterol phenotypes to atherosclerotic burden in cases and controls. Apart from such extreme phenotype approaches, novel population-based genome-wide association studies have been used to decipher the link between genetic loci and HDL cholesterol levels, and the identification of novel HDL cholesterol-related genes is eagerly awaited. These might be instrumental in the ongoing fight against atherosclerosis.

Increasing the Spatial Resolution of 3T Carotid MRI Has No Beneficial Effect for Plaque Component Measurement Reproducibility.

Purpose Different in-plane resolutions have been used for carotid 3T MRI. We compared the reproducibility, as well as the within- and between reader variability of high and routinely used spatial resolution in scans of patients with atherosclerotic carotid artery disease. Since no consensus exists about the optimal segmentation method, we analysed all imaging data using two different segmentation methods. Materials and Methods In 31 patient with carotid atherosclerosis a high (0.25 × 0.25 mm2; HR) and routinely used (0.50 × 0.50 mm2; LR) spatial resolution carotid MRI scan were performed within one month. A fully blinded closed and a simultaneously open segmentation were used to quantify the lipid rich necrotic core (LRNC), calcified and loose matrix (LM) plaque area and the fibrous cap (FC) thickness. Results No significant differences were observed between scan-rescan reproducibility for HR versus LR measurements, nor did we find any significant difference between the within-reader and between-reader reproducibility. The same applies for differences between the open and closed reads. All intraclass correlation coefficients between scans and rescans for the LRNC, calcified and LM plaque area, as well as the FC thickness measurements with the open segmentation method were excellent (all above 0.75). Conclusions Increasing the spatial resolution at the expense of the contrast-to-noise ratio does not improve carotid plaque component scan-rescan reproducibility in patients with atherosclerotic carotid disease, nor does using a different segmentation method.

Stents eluting 6-mercaptopurine reduce neointima formation and inflammation while enhancing strut coverage in rabbits

Background The introduction of drug-eluting stents (DES) has dramatically reduced restenosis rates compared with bare metal stents, but in-stent thrombosis remains a safety concern, necessitating prolonged dual anti-platelet therapy. The drug 6-Mercaptopurine (6-MP) has been shown to have beneficial effects in a cell-specific fashion on smooth muscle cells (SMC), endothelial cells and macrophages. We generated and analyzed a novel bioresorbable polymer coated DES, releasing 6-MP into the vessel wall, to reduce restenosis by inhibiting SMC proliferation and decreasing inflammation, without negatively affecting endothelialization of the stent surface. Methods Stents spray-coated with a bioresorbable polymer containing 0, 30 or 300 μg 6-MP were implanted in the iliac arteries of 17 male New Zealand White rabbits. Animals were euthanized for stent harvest 1 week after implantation for evaluation of cellular stent coverage and after 4 weeks for morphometric analyses of the lesions. Results Four weeks after implantation, the high dose of 6-MP attenuated restenosis with 16% compared to controls. Reduced neointima formation could at least partly be explained by an almost 2-fold induction of the cell cycle inhibiting kinase p27Kip1. Additionally, inflammation score, the quantification of RAM11-positive cells in the vessel wall, was significantly reduced in the high dose group with 23% compared to the control group. Evaluation with scanning electron microscopy showed 6-MP did not inhibit strut coverage 1 week after implantation. Conclusion We demonstrate that novel stents coated with a bioresorbable polymer coating eluting 6-MP inhibit restenosis and attenuate inflammation, while stimulating endothelial coverage. The 6-MP-eluting stents demonstrate that inhibition of restenosis without leaving uncovered metal is feasible, bringing stents without risk of late thrombosis one step closer to the patient.

Effect of Anti-ApoA-I Antibody-Coating of Stents on Neointima Formation in a Rabbit Balloon-Injury Model

Background and Aims Since high-density lipoprotein (HDL) has pro-endothelial and anti-thrombotic effects, a HDL recruiting stent may prevent restenosis. In the present study we address the functional characteristics of an apolipoprotein A-I (ApoA-I) antibody coating in vitro. Subsequently, we tested its biological performance applied on stents in vivo in rabbits. Materials and Methods The impact of anti ApoA-I- versus apoB-antibody coated stainless steel discs were evaluated in vitro for endothelial cell adhesion, thrombin generation and platelet adhesion. In vivo, response to injury in the iliac artery of New Zealand white rabbits was used as read out comparing apoA-I-coated versus bare metal stents. Results ApoA-I antibody coated metal discs showed increased endothelial cell adhesion and proliferation and decreased thrombin generation and platelet adhesion, compared to control discs. In vivo, no difference was observed between ApoA-I and BMS stents in lumen stenosis (23.3±13.8% versus 23.3±11.3%, p=0.77) or intima surface area (0.81±0.62 mm2 vs 0.84±0.55 mm2, p=0.85). Immunohistochemistry also revealed no differences in cell proliferation, fibrin deposition, inflammation and endothelialization. Conclusion ApoA-I antibody coating has potent pro-endothelial and anti-thrombotic effects in vitro, but failed to enhance stent performance in a balloon injury rabbit model in vivo.

Superior in vivo compatibility of hydrophilic polymer coated prosthetic vascular grafts

Purpose Protein adsorption, cell adhesion and graft patency was compared in hydrophilic versus hydrophobic polymer-coated prosthetic vascular grafts. We hypothesize that in vivocompatibility of hydrophilic polymer-coated prosthetic vascular grafts is superior to in vivo compatibility of hydrophobic grafts. Methods A pairwise side-to-side common carotid artery interposition graft was placed eight female landrace goats (mean weight 55 kg). Protein adsorption was assessed using Western Blot in two hydrophilic and two hydrophobic grafts harvested after three days. Graft patency was monitored for 28 days in six goats with continuous wave Doppler ultrasonography. Adherence of endothelial cells, leukocytes and platelets was determined with ELISA and compared between the two graft types after 28 days. Results After three days, more ApoA-l, albumin and VEGF and less fibrin adsorbed to hydrophilic grafts. After 28 days, compared to hydrophobic grafts, higher numbers of endothelial cells were present on hydrophilic grafts (P=0.016), and less thrombocytes and leukocytes (P=0.012 and 0.024, respectively). Two out of eight hydrophobic grafts lost patency, while none of the hydrophilic grafts failed (P=0.157). Conclusions Hydrophilic polymer-coated vascular grafts have superior in vivo compatibility when compared to hydrophobic grafts as characterized by reduced platelet and leukocyte adherence as well as higher endothelialization.

Guideline treatment results in regression of atherosclerosis in type 2 diabetes mellitus

Background: Efficacy of guideline cardiovascular disease prevention regimens may differ between patients with or without type II diabetes mellitus. We therefore compared change in carotid artery wall dimensions in type II diabetes mellitus and non-type II diabetes mellitus patients with a history of a major cardiovascular disease event, using magnetic resonance imaging. Methods: Thirty type II diabetes mellitus patients and 29 age- and sex-matched non-diabetes mellitus patients with a history of stroke or myocardial infarction and a carotid artery stenosis (15%–70%) were included. In all patients, treatment was according to cardiovascular risk management guidelines. At baseline and follow-up, carotid artery vessel wall dimensions were measured using 1.5 T magnetic resonance imaging. Results: After 2 years of follow-up, total wall volume of the carotid artery in type II diabetes mellitus patients decreased by 9.6% (p = 0.016). In contrast, stabilization rather than regression of carotid artery wall dimensions was observed in non-diabetes mellitus patients over a 2-year period. Body mass index was identified as a predictor of total wall volume decrease. Conclusions: Guideline treatment arrests atherogenesis in non-diabetes mellitus patients and even decreases vessel wall dimensions in type II diabetes mellitus patients. Baseline body mass index predicts cardiovascular disease prevention efficacy expressed as decrease in total wall volume. These data emphasize the importance of optimal cardiovascular-prevention, particularly in diabetes patients with a high body mass index.