Hans L.A. Nienhuis

Skin autofluorescence is increased in systemic lupus erythematosus but is not reflected by elevated plasma levels of advanced glycation endproducts

OBJECTIVES To examine whether skin advanced glycation endproducts (AGEs) accumulation, plasma levels of AGEs-N(epsilon)-carboxymethyllysine (CML) and N(epsilon)-carboxyethyllysine (CEL)-and serum levels of soluble receptor for AGEs (sRAGE) are elevated in SLE patients compared with controls, and whether these parameters are related to disease activity and endothelial cell (EC) activation. METHODS Ten SLE patients (9 women, age 34 +/- 13 yrs, mean +/- s.d.) and 10 age- and sex-matched controls were included. Patients were analysed during inactive as well as active disease. Skin AGE accumulation was estimated using ultraviolet-A (UV-A) light for measurement of autofluorescence obtained by Excitation-Emission matrix Scanner (AF-EEMS). Levels of CML and CEL were determined by tandem mass spectrometry. Levels of sRAGE and of soluble vascular cell adhesion molecule-1 (sVCAM-1) were determined by ELISAs. RESULTS Skin AF-EEMS was increased in SLE patients compared with controls (P < 0.05). Levels of CML and CEL were comparable between patients and controls and were not influenced by disease activity. sRAGE and sVCAM-1 levels were higher in quiescent SLE patients compared with controls (P < 0.05) and increased further during active disease (P < 0.05). In patients with quiescent disease and controls, sRAGE levels correlated to sVCAM-1 levels (r = 0.579, P = 0.007). CONCLUSIONS Skin AGEs and levels of sRAGE and sVCAM-1 were elevated in SLE patients, whereas levels of CML and CEL were comparable with controls. As sRAGE even further increased during endothelial activation, it might be hypothesized that sRAGE acts as a decoy receptor. Why this proposed mechanism is insufficient to prevent increased AGE accumulation in the skin of SLE patients has to be established.

AGE and their receptor RAGE in systemic autoimmune diseases: an inflammation propagating factor contributing to accelerated atherosclerosis.

Systemic autoimmune diseases are associated with inflammation, and oxidative stress favouring the formation of advanced glycation endproducts (AGE), able to modulate cellular functions by activation of receptor for advanced glycation endproducts (RAGE). As RAGE expression is increased in an inflammatory milieu, present in patients with systemic autoimmune diseases, these patients are especially prone for the deleterious effects of AGE. Interaction of AGE with RAGE leads to intracellular signalling, and subsequent expression of adhesion molecules, chemokines, pro-inflammatory cytokines and up-regulation of RAGE itself. The AGE–RAGE interaction might act as a pro-inflammatory loop in these patients, contributing to chronic low grade inflammation rendering these individuals susceptible for development of accelerated atherosclerosis.

The Prevalence and Management of Systemic Amyloidosis in Western Countries.

Background: Amyloidosis has been a mystery for centuries, but research of the last decennia has clarified many of the secrets of this group of diseases. A protein-based classification of amyloidosis helps to understand problems that were part of the obsolete clinical classification in primary, secondary, and familial amyloidosis. All types of amyloid are secondary to some underlying precursor-producing process: each type is caused by a misfolded soluble precursor protein that becomes deposited as insoluble amyloid fibrils. Summary: The incidence of amyloidosis is not well documented, but probably falls between 5 and 13 per million per year. Prevalence data are scarce, but one UK study indicates about 20 per million inhabitants. Amyloidosis can be localized (amyloid deposited in the organ or tissue of precursor production) or systemic (amyloid at one or more sites distant from the site of precursor production). The major systemic types of amyloidosis are AL (associated with a light chain-producing plasma cell dyscrasia), AA (associated with longstanding inflammation), wild-type ATTR (associated with normal transthyretin and old age), and hereditary ATTR (associated with a transthyretin mutation) amyloidosis. Imaging techniques, such as cardiac ultrasound, magnetic resonance imaging, bone scintigraphy, and serum amyloid P component scintigraphy, are useful both for diagnosing amyloidosis and for assessing disease severity. Serologic markers are useful for detecting organ disease and disease monitoring during follow-up. Current treatment modalities are directed against the ongoing supply of precursor proteins and thereby aim to stop further accumulation of amyloid. Novel treatment modalities, such as interference with amyloid formation and even removal of amyloid, are being studied. A well-thought and planned monitoring during follow-up helps to assess the effect of treatment and to early detect possible progression of amyloidosis. Key Messages: Clinical management comprises histologic proof of amyloid, evidence of systemic deposition, reliable typing, precursor assessment, severity of organ disease, risk assessment and prognosis, choice of treatment, and planned monitoring during follow-up. Facts from East and West: (1) AL amyloidosis is the most prevalent type of amyloidosis accounting for 65% of the amyloidosis-diagnosed patients in the UK and for 93% of the amyloidosis-diagnosed patients in China. The predisposition of men over women to develop AL amyloidosis might be higher in China than in Western countries (2:1 vs. 1.3:1). Both in the East and West, incidence increases with age. At the time of diagnosis, edema is twice as frequent and the proportion of renal involvement is higher in Chinese compared to Western patients. (2) Melphalan followed by autologous stem cell transplantation (ASCT) is the current standard therapy but is restricted to eligible patients. The efficacy and safety of bortezomib combined with dexamethasone were proven in Western patients and recently confirmed in a Chinese cohort. Recent studies in China and the US indicate that bortezomib induction prior to ASCT increases the response rate. Thalidomide and lenalidomide have shown benefit, but toxicity and lack of clinical evidence exclude these agents from first-line therapy. The green tea extract epigallocatechin-3-gallate is under investigation as an inhibitor of AL amyloid formation and a compound that might dissolve amyloid.

Small artery elasticity is decreased in patients with systemic lupus erythematosus without increased intima media thickness

IntroductionThe objectives of this study were to determine small arterial elasticity (SAE) in systemic lupus erythematosus (SLE) and to investigate its relationship with intima media thickness (IMT), accumulation of advanced glycation end products (AGEs), endothelial activation and inflammation.MethodsThirty SLE patients with inactive disease and 30 age- and sex-matched healthy controls were included. Twenty patients with essential hypertension (EH) served as positive control. SAE was assessed by pulse-wave analysis using tonometric recordings of the radial artery. IMT of the carotid arteries was measured by ultrasound. AGE accumulation was assessed with an AGE-reader. Endothelial activation markers and C-reactive protein (CRP) were determined by enzyme-linked immunosorbent assay (ELISA).ResultsSAE was decreased in SLE (P = 0.01) and further decreased in EH (P < 0.01) compared to healthy controls. IMT was increased in EH (P < 0.05), but not in SLE. AGE accumulation was increased in SLE (P < 0.05) and further increased in EH (P < 0.01) compared to healthy controls. Endothelial activation markers and CRP were increased in SLE but not in EH. SAE related to AGE accumulation (r = -0.370, P < 0.05), CRP (r = -0.429, P < 0.05) and creatinine clearance (r = 0.440, P < 0.05), but not to IMT and endothelial activation markers. In multivariate analysis SLE was an independent predictor of SAE.ConclusionsSAE is decreased in SLE patients without increased IMT, independently of traditional cardiovascular risk factors. Longitudinal studies are needed to investigate whether SAE, endothelial activation and AGE accumulation are early markers for cardiovascular disease in SLE.

Is Small Artery Elasticity Decreased Prior to Intima-Media Thickening in Patients with Longstanding Rheumatoid Arthritis?

Objective. To determine small artery elasticity (SAE) in patients with longstanding rheumatoid arthritis (RA) in comparison to healthy controls, and to investigate its relation to markers of endothelial cell activation, disease activity, joint damage, and the presence of atherosclerosis. Methods. Forty-nine patients with RA and 50 age- and sex-matched healthy controls were studied. Traditional cardiovascular risk factors and disease-related factors were recorded. SAE was measured noninvasively by pulse-wave analysis (PWA). Endothelial activation was assessed by measuring levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and von Willebrand factor (vWF). Carotid intima-media thickness (IMT), as an indicator of subclinical atherosclerosis, was assessed using ultrasonography. Results. Patients with RA had higher body mass index, blood pressure, and triglyceride levels and were more often cigarette smokers compared to controls. SAE was decreased in RA patients compared to controls and was inversely related with age, smoking, blood pressure, vWF, sVCAM-1, high sensitivity C-reactive protein, and IMT. Presence of RA was independently related to SAE in multivariate linear regression analysis. SAE was inversely related with the Health Assessment Questionnaire score. No correlation was found between SAE and other disease activity markers and damage. IMT in patients and controls was not different. Conclusion. Small artery elasticity was decreased in patients with longstanding RA. The presence of RA was independently associated with SAE. Whereas IMT in patients with RA was not increased, we hypothesize that endothelial dysfunction, reflected by decreased SAE, is present prior to IMT thickening in these patients.

Increased accumulation of advanced glycation endproducts in patients with Wegener’s granulomatosis

Wegeners granulomatosis (WG) is a systemic autoimmune disease characterised by an increased risk for cardiovascular disease owing to accelerated atherosclerosis which cannot be fully explained by traditional risk factors.1 Non-traditional risk factors are therefore probably involved. Among these, accumulation of advanced glycation endproducts (AGEs) might be important. AGEs are implicated in vascular pathology2 and are independent predictors of cardiovascular mortality in patients with diabetes and those receiving haemodialysis.3 4 In this study, AGE accumulation, atherosclerosis and levels of the receptor for AGEs (sRAGE) were measured in 24 consecutive patients with WG who fulfilled the American College of Rheumatology criteria5 with inactive disease and 21 controls (table 1). AGE accumulation was assessed as previously described6 and intima media thickness (IMT) in both carotid …

Frequency of and Prognostic Significance of Cardiac Involvement at Presentation in Hereditary Transthyretin-Derived Amyloidosis and the Value of N-Terminal Pro-B-Type Natriuretic Peptide

The aim of this study is to assess the prevalence of cardiac involvement in hereditary transthyretin-derived (ATTRm) amyloidosis at the time of diagnosis and to determine the diagnostic and clinical value of N-terminal pro-B-type natriuretic peptide (NT-proBNP). The University Medical Center Groningen is the national center of expertise for amyloidosis. All consecutive patients between 1994 and 2016 with ATTRm amyloidosis were followed prospectively. Baseline was set at the time of the first positive biopsy. All patients underwent a standard cardiac and neurologic work-up. Cardiac involvement was defined by otherwise unexplained left and/or right ventricular wall hypertrophy on cardiac ultrasound and/or advanced conduction disturbances. Seventy-seven patients had ATTRm amyloidosis and were included in the study. The TTR V30M mutation was present in 30 patients (39%). In both the V30M and the non-V30M groups, the neurologic presentation dominated (77% vs 51%), whereas cardiac presentation was infrequent (7% vs 15%). Clinical work-up showed that cardiac involvement was present at baseline in 51% of all patients irrespective of genotype and was associated with increased overall mortality (hazard ratio 5.95, 95% confidence interval 2.12 to 16.7), independent from clinical confounders. At a cutoff level of 125 ng/L, NT-proBNP had a sensitivity of 92% for establishing cardiac involvement. In conclusion, irrespective of the frequent noncardiac presentation of ATTRm amyloidosis, cardiac involvement is already present at diagnosis in half of the patients and is associated with increased mortality. NT-proBNP is a useful marker to determine cardiac involvement in this disease.

Late onset cardiomyopathy as presenting sign of ATTR A45G amyloidosis caused by a novel TTR mutation (p.A65G)

OBJECTIVE The clinical description of a novel TTR gene mutation characterized by a late onset amyloid cardiomyopathy. METHODS AND RESULTS A 78-year-old man of Dutch origin with recent surgery for bilateral carpal tunnel syndrome (CTS) was admitted to our hospital because of heart failure with preserved ejection fraction (55%). Cardiac ultrasound showed thickened biventricular walls, and cardiac magnetic resonance imaging also showed late gadolinium enhancement. Early signs of a polyneuropathy were found by neurophysiological testing. A few months later, his 72-year-old sister was admitted to an affiliated hospital because of heart failure caused by a restrictive cardiomyopathy. In both patients, a subcutaneous abdominal fat aspirate was stained with Congo red and DNA was analyzed by direct sequencing of exons 1 to 4 of the transthyretin (TTR) gene. Both fat aspirates revealed transthyretin-derived (ATTR) amyloid. 99mTc-diphosphonate scintigraphy further confirmed cardiac ATTR amyloidosis in the male patient. DNA analysis of both patients showed a novel TTR mutation c.194C>G that encodes for the gene product TTR (p.A65G) ending up as the mature protein TTR A45G. The 56-year-old daughter of the male patient had the same TTR mutation. A full diagnostic workup did not reveal any signs of amyloidosis yet. CONCLUSIONS A novel amyloidogenic TTR mutation was found in a Dutch family. The clinical presentation of ATTR A45G amyloidosis in the affected family members was heart failure due to a late-onset cardiomyopathy. The systemic nature of this disease was reflected by bilateral CTS and by early signs of a polyneuropathy in the index patient.

High mobility group box 1 levels are not associated with subclinical carotid atherosclerosis in patients with granulomatosis with polyangiitis but are reduced by glucocorticoids and statins

Background/Purpose: MicroRNAs (miRs) are a novel class of posttranscriptional regulators. A single miR can have profound effects on cell activation due to its ability to modulate multiple pathways at once. We have previously shown that miR-155 is upregulated in rheumatoid arthritis (RA) synovial macrophages and promotes the development of autoimmunity and joint inflammation. Pre-clinical arthritis may be associated with lung changes e.g. bronchial wall thickening, thus the aim of this study was to investigate the contribution of miR-155 regulated pathways to lung homeostasis. Methods: Normal human lung tissue was tested by in situ hybridisation with miR-155 and control probes. To model the fibrotic response, WT and miR-155 / mice were given bleomycin (0.06 unit/mouse) intranasally. Intervention included intraperitoneal injections of the Liver X Receptor (LXR) agonist (GW3965 daily; 40 mg/kg). End-points included bronchial lavage (BAL) cytology, lung tissue histology, evaluation of the expression of inflammatory and fibrotic genes by qPCR and concentrations of soluble mediators in serum and BAL fluid by multiplex assays. The validation of miR-155 binding to LXR, and the LXR response element in collagen gene promoters were performed with reporter assays. Results: In situ hybridisation showed an abundant expression of miR-155 in the normal human lung suggesting that this miR may contribute to normal lung homeostasis. miR-155 / mice developed more severe bleomycininduced lung fibrosis compared to WT mice, as seen by increased collagen 1a/3a mRNA expression and protein deposition in the lungs, as well as accumulation of macrophages and lymphocytes in BAL. Gene expression analysis of lung extracts revealed an increase in the M2 pro-fibrotic macrophage markers Arginase 2, IL-13R and Ym1. In addition, the levels of pro-fibrotic cytokines such as VEGF and bFGF were significantly higher in BAL and serum of miR-155 / mice. Primary lung fibroblast lines derived from miR-155 / mice showed higher proliferation rates and motility compared to WT cells in wound healing assays. Computational analysis followed by functional luciferase assays revealed that the transcription activator LXR alpha is a direct target of miR-155 in the lungs. Expression of LXR alpha was significantly upregulated in the lungs of naive miR-155 / mice and was further increased in mice given bleomycin compared to similarly treated WT controls. Injection of the LXR agonist to WT mice increased LXR expression and mirrored the same phenotypic response to bleomycin as the miR-155 deficient mice; shown by increased collagen deposition and M2 macrophage and fibroblast activation. Promoter analysis revealed that LXRs could directly induce collagen production by binding to col1a and col3a promoters. / Conclusion: miR-155 appears important for lung homeostasis, likely by fine tuning levels of LXR thereby protecting from excessive remodelling. Given this and the emerging contribution of miR-155 to development of autoimmunity, this miR may act as a master-switch determining the duration of inflammation and the initiation of remodelling, as well as the balance between the immune and auto-immune responses.Background/Purpose: High mobility group box 1 (HMGB1) is a non-histone DNA binding protein that is passively released by dying cells or actively secreted by immunocompetent cells and the receptor for advanced glycation end-products (RAGE) is one of its receptors. Higher levels of HMGB1 have been found in patients with granulomatosis with polyangiitis (GPA) with active disease whereas higher HMGB1 and lower soluble (sRAGE) levels have been found in patients with acute atherosclerotic events suggesting sRAGE acts as a decoy receptor. This study aims to evaluate HMGB1 levels in relation to subclinical carotid atherosclerosis in GPA, and the impact of therapy on HMGB1 levels. Methods: A cross-sectional study was performed on 23 GPA patients during a quiescent phase of the disease in comparison to 20 matched controls. All study participants underwent carotid ultrasound to assess atherosclerotic plaques and intima-media thickness (IMT) and were tested for traditional risk factors for atherosclerosis, serum HMGB1 levels (ELISA-Shino Test, Kanagawa, Japan), and sRAGE levels (ELISA RD P = 0.978), HDLcholesterol (1.41 ± 0.37 vs. 1.51±0.33 mmol/L; P = 0.359), LDLcholesterol (3.01±0.79 vs. 3.29±0.82 mmol/L; P = 0.267), and a similar frequency of smoking (8.7% vs. 5.0%; P = 0.635), family history of premature coronary artery disease (CAD) (39.1% vs. 40.0%; P = 0.954), and obesity (4.3% vs. 10.0%; P = 0.446). Hypertension was only found in GPA patients (39.1% vs. 0.0%; P = 0.002) while no study participants had diabetes. Overt cardiovascular disease was found only in 13.0% of GPA patients. Statins were prescribed for 21.7% of GPA patients and 5.0% of controls (P = 0.127). Among GPA patients, prednisolone was being used by 34.8% with a median daily dose of 5.0mg (2.5-15.0) and azathioprine by 34.8%. Only two GPA patients used statins and prednisolone concomitantly. Carotid plaques were found in 30.4% of GPA patients and in 15.0% of controls (P = 0.203) and the overall IMT was similar in GPA patients and in controls (0.833±0.256 vs. 0.765±0.133mm; P = 0.861). Median serum HMGB1 levels were similar between GPA patients and controls [2.13ng/mL (1.11-7.22) vs. 2.42ng/mL (0.38-6.75); P = 0.827] as well as mean sRAGE levels (1256.1±559.6 vs. 1483.3±399.8pg/mL; P = 0.155). No correlations were found between HMGB1 and sRAGE ( = 0.068; P = 0.681) and between HMGB1 and maximum IMT in carotid arteries ( = -0.067; P = 0.720). GPA patients on prednisolone (1.77±0.76 vs. 3.53±2.06ng/ mL; P = 0.017) and statins (1.39±0.28 vs. 3.34±1.94ng/mL; P = 0.001) presented significantly lower serum HMGB1 levels whereas no difference in mean HMGB1 levels was found regarding azathioprine use (2.89±2.28 vs. 2.93±1.75; P = 0.970). Conclusion: No association was found between subclinical atherosclerosis in carotid arteries and HMGB1 levels in GPA patients. Furthermore, the use of either prednisone or statins was associated with lower HMGB1 levels in GPA patients. These findings suggest that the anti-inflammatory properties of statins include effects on serum HMGB1 levels in GPA.

Serum levels of soluble receptor for advanced glycation end products in AA amyloidosis are increased and related to loss of renal function

Protein misfolding and aggregation diseases, such as e.g. Alzheimer’s disease, are associated by the accumulation of a disease-related protein. The pathogenic mechanisms involved in these confor- mational diseases are only poorly understood. Luminescent-conjugated polymers (LCPs) have been shown as a sensitive tool for detection of amyloid deposits. In contrast to commonly used amyloidotropic dyes such as thioflavins or Congo Red, LCPs are composed of flexible polythiophene chains which allow rotation of the molecule. Upon binding to amyloids, the LCPs alter their spectral properties in a conformation dependent manner. However, there is still limited information available on the binding mechanism and binding properties of the LCPs to amyloid fibrils and oligomers.We have produced recombinant human Aβ1-42 (recAβ1-42) protein in Escherichia coli and purified it by conventional chromatographic techniques in large quantities. The recAβ-protein was incubated in the presence of SDS to induce formation of homogenous, globular Aβ-oligomers with a size of approximately 60 kDa, known as Aβ-globulomers. We present first biophysical and spectroscopic data used to study the binding and structural properties of the complex formed by the globulomers and LCPs with various charged side chains. These data will provide a more detailed knowledge of the binding mode of amyloidogenic probes which is essential for understanding the structural char- acteristics of amyloid fibrils detected by thesemolecules.