Limited Association between Antibodies to Oxidized Low-Density Lipoprotein and Vascular Affection in Patients with Established Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) are at an increased risk of cardiovascular disease. We aimed to evaluate whether antibodies to oxidized low-density lipoprotein (anti-oxLDL) were associated with subclinical atherosclerosis in patients with different SLE phenotypes (lupus nephritis, antiphospholipid syndrome, and skin and joint involvement). Anti-oxLDL was measured by enzyme-linked immunosorbent assay in 60 patients with SLE, 60 healthy controls (HCs) and 30 subjects with anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). Intima-media thickness (IMT) assessment of vessel walls and plaque occurrence were recorded using high-frequency ultrasound. In the SLE cohort, anti-oxLDL was again assessed in 57 of the 60 individuals approximately 3 years later. The levels of anti-oxLDL in the SLE group (median 5829 U/mL) were not significantly different from those in the HCs group (median 4568 U/mL), while patients with AAV showed significantly higher levels (median 7817 U/mL). The levels did not differ between the SLE subgroups. A significant correlation was found with IMT in the common femoral artery in the SLE cohort, but no association with plaque occurrence was observed. The levels of anti-oxLDL antibodies in the SLE group were significantly higher at inclusion compared to 3 years later (median 5707 versus 1503 U/mL, p < 0.0001). Overall, we found no convincing support for strong associations between vascular affection and anti-oxLDL antibodies in SLE.

Plasma osteopontin versus intima media thickness of the common carotid arteries in well-characterised patients with systemic lupus erythematosus.

OBJECTIVE: The progress of accelerated atherosclerosis in systemic lupus erythematosus (SLE) is incompletely understood. Circulating osteopontin (OPN) is increased in autoimmune conditions, e.g. SLE, and its serum concentration was recently reported to associate with subclinical atherosclerosis in SLE, as measured by carotid intima-media thickness. The aim of this study was to investigate whether OPN may be used as a surrogate biomarker of subclinical atherosclerosis in SLE patients with different disease phenotypes. METHODS: We recruited 60 well-characterised SLE cases and 60 age- and sex-matched healthy controls. The SLE cases were divided into three different disease phenotypes: SLE with antiphospholipid syndrome (APS), lupus nephritis, and isolated skin and joint involvement. Plasma OPN was detected by ELISA (Quantikine®, R&D Systems). Common carotid arteries intima media thickness was compared between the studied groups in relation to OPN levels and risk factors for vascular changes. Intima media thickness of common carotid arteries was measured by using a sensitive ultrasound technique (LOGIQ™ E9 ultrasound, GE Healthcare). RESULTS: OPN levels were significantly higher among the entire SLE group (n = 60) compared to the healthy controls (P = 0.03). SLE cases with concomitant APS (n = 20) showed higher OPN levels than the controls (P = 0.004), whereas none of the other two subgroups differed significantly from the healthy controls. OPN and intima media thickness were correlated to several traditional risk factors of atherosclerosis, as well as to SLE-related factors. Yet, no significant correlation was observed between OPN levels and ultrasound findings of the common carotid arteries. CONCLUSIONS: In line with previous studies, we observed increased OPN levels among SLE patients as compared to matched controls. However, the OPN concentrations did not correlate with intima media thickness of the common carotid arteries. Based on our findings, the use of OPN as a surrogate biomarker of subclinical atherosclerosis in SLE subjects, regardless of clinical phenotypes, cannot be recommended.

Computer-Aided Evaluation of Blood Vessel Geometry From Acoustic Images.

A method for computer-aided assessment of blood vessel geometries based on shape-fitting algorithms from metric vision was evaluated. Acoustic images of cross sections of the radial artery and cephalic vein were acquired, and medical practitioners used a computer application to measure the wall thickness and nominal diameter of these blood vessels with a caliper method and the shape-fitting method. The methods performed equally well for wall thickness measurements. The shape-fitting method was preferable for measuring the diameter, since it reduced systematic errors by up to 63% in the case of the cephalic vein because of its eccentricity.

Affected Microcirculation and Vascular Hemodynamics in Takayasu Arteritis.

Introduction: Takayasu arteritis (TAK) is a rare inflammatory disease affecting aorta and its major branches. Ultrasound (US) can detect inflammatory features in the arterial wall, but less is known regarding skin microcirculation and vascular hemodynamics. The aim was to study if assessment of these variables could add valuable information regarding vascular affection in TAK. Methods: 17 patients diagnosed with TAK and 17 age- and sex-matched healthy controls were included. Microcirculatory peak oxygen saturation (OxyP) in the skin after induced ischemia was evaluated with laser Doppler flowmetry/diffuse reflectance spectroscopy. Cerebrovascular reserve capacity (CVR) in the brain was assessed with transcranial Doppler (TCD). Pulse waves were recorded in the radial artery by the aid of applanation tonometry, for calculation of central augmentation index (AIx75). Intima-media thickness (IMT) and stenosis/occlusions were evaluated using US in carotid and central arteries. Results: Reduced OxyP (79 ± 8% vs. 87 ± 4%, p < 0.001) was seen in patients with TAK regardless of significant arterial stenosis/occlusion or not. Increased AIx75 (22.3 ± 13.6 vs. 9.2 ± 16.3, p = 0.01) was seen in TAK patients without significant stenosis/occlusions. No differences were found in CVR, regardless of proximal stenosis. However, signs of a more high-resistance flow profile were seen in arteria cerebri media. Conclusion: Regardless of arterial stenosis or not, impaired microcirculation of the skin and preserved CVR in the brain were found in subjects with TAK. Signs of increased arterial stiffness in the brain and central arteries were observed. The value of these findings for prediction of future cardiovascular events needs to be clarified in further studies.

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells.

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.

The Diagnostic Performance of an Extended Ultrasound Protocol in Patients With Clinically Suspected Giant Cell Arteritis.

OBJECTIVE: To evaluate the diagnostic performance of an extended ultrasound protocol in patients referred under the suspicion of giant cell arteritis (GCA). METHODS: Consecutive patients with suspected GCA were examined with an extended color duplex ultrasound (CDU) protocol during a period of 2 years. The extended CDU protocol included temporal, axillary, subclavian, brachiocephalic, and carotid arteries. The reference was clinically diagnosed GCA, confirmed after ≥6-month follow-up. Hypo- or medium-echogenic, circumferential, homogenous wall thickening, and/or a positive compression sign in temporal arteries, were regarded as typical signs of arteritis. RESULTS: Of the eligible 201 patients, 83 (41%) received a clinical GCA diagnosis at follow-up ≥6 months post CDU examination. Among these cases, 48 (58%) demonstrated inflammation solely in temporal arteries, 8 (10%) showed abnormalities restricted to extra-cranial vessels, and 23 (28%) patients displayed inflammatory changes in both temporal and extra-cranial arteries. Color duplex ultrasound of temporal arteries yielded a diagnostic sensitivity and specificity [95% confidence intervals (CI)] of 86% (76-92%) and 99% (95-99%), respectively. By adding axillary artery examination, the sensitivity increased to 92% (83-97%) while the specificity remained unchanged. Further, inclusion of subclavian artery marginally increased the sensitivity by 1%. Finally, by also including brachiocephalic and common carotid arteries resulted in a sensitivity of 95% (88-99%) and a specificity of 98% (94-99%). CONCLUSIONS: Color duplex ultrasound examination demonstrated a high accuracy in diagnosing patients both with cranial and extra-cranial GCA. Further examination of brachiocephalic and common carotid arteries can increase the sensitivity without affecting the specificity when temporal and axillary findings are indecisive. Finally, the extended CDU protocol allows measurement of the general burden of inflammation, which could be relevant for future monitoring purposes.

Impaired Microcirculation and Vascular Hemodynamics in Relation to Macrocirculation in Patients With Systemic Lupus Erythematosus.

Introduction: Systemic lupus erythematosus (SLE) is associated with premature cardiovascular disease (CVD) and mortality, unexplained by traditional risk factors. Impairment of microcirculation and vascular hemodynamics may represent early signs of vascular affection. We hypothesized that studies of microcirculation and pulse waves may provide additional information, compared to ultrasound (US) alone, for the detection of early vascular disease in SLE. Methods: Sixty well-characterized SLE-patients (52 women, eight men; mean age 43.21 ± 1.3 years) characterized by lupus nephritis (LN; n = 20), antiphospholipid syndrome (APS; n = 20) or skin and joint involvement (n = 20) and 60 healthy controls were included. Microcirculatory peak oxygen saturation (OxyP) was evaluated using a novel combined laser Doppler flowmetry/diffuse reflectance spectroscopy method. Pulse waves were recorded in the radial artery by the aid of applanation tonometry in order to calculate central augmentation index (AIx75). Intima-media thickness (IMT) and plaque occurrence were evaluated using high frequency US, in carotid and central arteries. Results: Lower OxyP (84 ± 8 vs. 87 ± 5 %, p = 0.01) and higher AIx75 (17.3 ± 13.9 vs. 10.0 ± 14.2 %, p = 0.005) were seen in the SLE cohort. OxyP was inversely correlated with IMT in internal carotid artery (ICA), (R = -0.32, p = 0.01). AIx75 correlated with IMT in common carotid artery (CCA), (R = 0.36, p = 0.005), common femoral artery (CFA), (R = 0.43, p = 0.001), and ICA (R = 0.27, p = 0.04). AIx75 correlated negatively with OxyP (R = -0.29, p = 0.02). SLE-patients with plaque had lower OxyP values (80 ± 8 vs. 85 ± 7 %, p < 0.001) and higher AIx75 (23.0 ± 11.6 vs. 15.5 ± 14.2 %, p < 0.001) compared to those without plaque. Conclusion: Impaired microcirculation and vessel hemodynamics were observed in SLE. These methods correlated with IMT and plaque occurrence. The importance of early macro- and micro-circulatory vascular affection for increased risk of CVD in SLE will be followed-up in future studies.

High-Frequency Ultrasound of Multiple Arterial Areas Reveals Increased Intima Media Thickness, Vessel Wall Appearance, and Atherosclerotic Plaques in Systemic Lupus Erythematosus.

Introduction: Despite improved therapies and management, patients with systemic lupus erythematosus (SLE) still have increased risks of cerebrovascular and cardiovascular disease. High-frequency ultrasound (US) provides an opportunity to distinguish atherosclerosis from inflammation in the vessels. We hypothesized that an extended US protocol may add information regarding vascular affection in SLE. Methods: Sixty patients (52 women, 8 men; mean age 43.2 ± 11.3 years) with SLE characterized by either lupus nephritis (LN; n = 20), antiphospholipid syndrome (APS; n = 20), or skin and joint involvement (n = 20) as well as matched healthy controls (n = 60) were included. Intima-media thickness (IMT), assessment of vessel walls, and plaque occurrence were recorded using high-frequency US (GE Logic E9) in common carotid, internal carotid, brachiocephalic, subclavian, axillary, common femoral, and proximal superficial femoral arteries as well as in the aortic arch. Results: For the entire SLE group, IMT was increased in the internal carotid artery (0.52 ± 0.17 vs. 0.45 ± 0.09 mm, p = 0.004), the common femoral artery (0.57 ± 0.23 vs. 0.49 ± 0.11 mm, p < 0.01), the subclavian artery (0.58 ± 0.19 vs. 0.53 ± 0.13 mm, p = 0.02), and the aortic arch (1.21 ± 0.63 vs. 0.98 ± 0.25 mm, p = 0.002) compared to controls. These differences were primarily observed in the APS and LN groups compared to controls. Vessels with increased IMT ≥0.9 mm had a smooth, medium echogenic appearance in areas free of atherosclerotic plaques. Atherosclerotic plaques were detected in 15/60 patients (25%) as compared to 2/60 of the controls (3%). Plaques were predominantly (67%) located in the carotid bifurcation. Multivariate analysis revealed influence of age on IMT in all vessel areas. Furthermore, in the common femoral artery, sagittal abdominal diameter, diastolic blood pressure, and cholesterol all showed association with increased IMT. In the internal carotid artery, male sex and presence of Raynaud phenomenon influenced IMT. Conclusion: Among SLE patients without presence of plaques, an extended US protocol revealed increased wall thickness with predominantly medium echogenic appearance highlighting possibly inflammation or early atherosclerosis. The appearance of vessel walls has not previously been studied in detail. An increased number of plaques were found in SLE compared to age- and sex-matched healthy controls. We found similar risk factors for increased IMT and occurrence of plaques, possibly indicating atherosclerotic mechanisms rather than inflammation.

Vascular ultrasound for monitoring of inflammatory activity in Takayasu arteritis.

BACKGROUND: Takayasu arteritis (TA) is a rare large-vessel arteritis that primarily affects the aorta and its major branches. The aim of this study was to describe the value of high frequency ultrasound for monitoring of inflammatory activity. METHODS: Twenty-five patients, range 11-71 years, diagnosed with TA were investigated with duplex ultrasound (DUS) including follow-up studies. Twenty-five healthy controls were also investigated. Nine patients had newly diagnosed active TA. Sixteen patients had stable/inactive disease at baseline DUS, and TA was diagnosed median 4·5 years previously. Intima-media thickness (IMT), vessel and lumen diameter were measured in the carotid arteries, central neck arteries and the aortic arch. The vessel walls were studied qualitatively. The Takayasu ultrasound index was created for inflammatory activity scoring. RESULTS: Intima-media thickness in common carotid artery (CCA) was (median and 25-75 percentile parenthetic) 2·3 mm (1·7-2·9) in clinically active TA, 1·2 mm (1·1-1·6) in clinically stable TA (P<0·001) and 0·5 mm (0·5-0·6) in healthy controls (P<0·001). Clinically active TA had prominent increase in IMT and/or increased vessel diameter, and/or intramural arteries, and/or hypoechogenic areas interpreted as oedema in the vessel wall. TA in clinical remission was characterized by increased IMT with medium to high echogenicity with or without fibrotic stripes. The Takayasu ultrasound index was higher in patients with active disease versus treated disease, 2·55 (1·60-3·05) versus 1·30 (1·00-1·58), (P = 0·003). CONCLUSION: DUS is an excellent tool to monitor inflammatory changes in the vessel wall in TA. Further DUS studies in larger patient populations are warranted.

Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests.

Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7 days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and ßIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.

LPS-induced iNOS expression in Bv-2 cells is suppressed by an oxidative mechanism acting on the JNK pathway--a potential role for neuroprotection.

Activated microglia cells, observed during chronic inflammation, produce and secrete compounds that at high concentrations or during sustained production might cause neuronal cell death. Inducible nitric oxide synthase (iNOS) is expressed in response to various immunological stimuli and catalyses the formation of the free radical nitric oxide (NO), that at low and regulated levels participate in cell signaling and cytoprotective events, whereas its higher and unregulated production can promote neurotoxicity in cells or in tissues. Regulation of NO production is therefore central for maintaining NO-levels within a safe window. We have analyzed iNOS protein expression and NO production, in murine microglial Bv-2 cells after 16h treatment with the bacterial endotoxin lipopolysaccharide (LPS). We have further analyzed three MAPK pathways, by co-treating the cells with LPS and the inhibitors of ERK1/2, p38 or JNK MAPK activities. To investigate participation of an oxidative regulatory mechanism, cells were also treated with the antioxidant N-acetyl-L-cysteine (NAC). Our results show that LPS-induced iNOS expression in Bv-2 cells is mainly mediated through JNK MAPK. In addition, co-treatment of the Bv-2 cells with LPS and NAC surprisingly further increased the iNOS expression, an effect also found to be mediated through the JNK MAPK pathway. The level of phosphorylated JNK MAPK (p46) was strongly increased by LPS alone and was further increased when combined with NAC. Our data indicate that iNOS and NO production are suppressed by an oxidative mechanism acting on the JNK MAPK pathway and we speculate that it might constitute a potential regulatory mechanism controlling the NO level.