The levels of circulating markers of atherosclerosis and inflammation in subjects with different degrees of body mass index: Soluble CD40 ligand and high-sensitivity C-reactive protein

BACKGROUND: It is well demonstrated that obesity is an independent risk factor for cardiovascular diseases. Recent studies have shown that obesity, insulin resistance and atherosclerosis are closely related phenomena in which low-grade inflammatory state and prothrombotic condition has pivotal roles. It has been shown that CD40-soluble CD40 ligand (sCD40L) interactions might constitute an important mediator for vascular inflammation. The aim of the present study was to assess sCD40L in relation to hs-CRP and cardiovascular risk factors in relation to body mass index (BMI). MATERIALS AND METHODS: Serum sCD40L and hs-CRP concentrations were measured in 52 obese patients and 28 non-obese subjects by ELISA. Insulin resistance was calculated by homeostasis model assessment-insulin resistance (HOMA-IR). We divided the participants into three groups depending in their BMI levels (Group 1: BMI <25 kg/m(2), Group 2: BMI 30-34.9 kg/m(2), Group 3: BMI > or =35 kg/m(2)). RESULTS: We determined that the mean sCD40L of group 3 was significantly higher than group 1 and group 2 (p<0.05, p<0.05, respectively). However, there was no significant correlation between plasma sCD40L levels and BMI. Plasma levels of hs-CRP were higher in obese group than the non-obese group (p<0.001). The levels of sCD40L were not significantly different between the two groups. The mean hs-CRP levels increased gradually in accordance with groups of BMI, there was a strong correlation between hs-CRP levels and BMI (r=0.724, p<0.001). There was no significant correlation between sCD40L and hs-CRP levels in all participants. CONCLUSIONS: It is still a subject for debate whether sCD40L levels are increased or not in obesity. However, the results of this study showed that sCD40L is substantially increased in patients with severe obesity. In terms of causality, the relatively small sample size and cross-sectional design of this study are considered to be the limitation factors.     

Plasma markers of angiogenesis in pregnancy induced hypertension

This study tests the hypothesis that abnormalities in plasma indices of angiogenesis, such as Vascular Endothelial Growth Factor (VEGF) and angiopoietins (Ang-1, Ang-2), as well as their soluble receptors Flt-1 (sFlt-1) and Tie 2 (sTie-2) respectively, are present in women with in pregnancy-induced hypertension (PIH). We also measured platelet levels of VEGF and Ang-1 (pVEGF and pAng-1 respectively). We studied 69 consecutive women with PIH (34 without proteinuria, and 35 with proteinuria, i.e. preeclampsia) who were compared to 64 consecutive women with normotensive pregnancies and 30 normotensive non-pregnant women, in a cross-sectional study. Using ELISA, we measured levels of plasma VEGF, Ang-1 & 2, Tie-2 and sFlt-1, and also the levels of angiogenic markers within the platelet [platelet VEGF (pVEGF) and platelet Ang-1 (pAng1)] by lysing a fixed number of platelets with 0.5% tween. Results show that levels of plasma VEGF, Ang-1, Ang2, sFlt-1 and Tie-2 were significantly different between the study groups. Post hoc analyses revealed plasma Ang-1 was highest in the preeclampsia group (p<0.001), whilst Ang-2 was highest in the normotensive pregnant group (p-=0.018). Plasma Tie-2 was highest in the PIH group. VEGF levels were significantly different between the preeclampsia group and the PIH group (p<0.05). Platelet VEGF levels were higher in the non-pregnant group than in the pregnant group, but there were no significant differences in the platelet levels of Ang-1 between the different groups. Ang-2, sFlt-1 and Tie-2 were undetectable in the platelet lysate in any of the patient groups or controls. Blood pressure was a major determinant of the different angiogenic factors studied. Abnormal indices of angiogenesis are evident in PIH and preeclampsia, with higher levels of sFlt-1 and lower levels of VEGF; in PIH, increased levels of Ang-1 and Tie-2, but reduced Ang-2, are evident compared to normal pregnancy. These abnormalities may have implications for the pathogenesis of PIH and preeclampsia.     

Differences of soluble CD40L in sera and plasma: implications on CD40L assay as a marker of thrombotic risk

INTRODUCTION: Soluble CD40L (sCD40L) ELISA has emerged as a promising predictor of poor outcomes in acute coronary syndrome. Yet many blood processing techniques have been used with little consideration of their effect on the results. METHODS: We measured sCD40L by ELISA in 10 patients with thrombocytopenia and 12 with normal or high platelet counts and 8 healthy controls using three sampling techniques: serum clotted on ice (serum-I) or at room temperature (serum-RT) and platelet poor plasma (PPP). RESULTS: Serum-RT samples, compared to serum-I, gave significantly higher CD40L values (p=0.003), demonstrating that ex vivo sCD40L release by activated platelets is inhibited by cold temperature. Although serum-I and PPP were comparable in patients with normal platelet counts, serum-I gave significantly higher values than PPP in the thrombocytosis group (p=0.01), suggesting that cold inhibition is insufficient in the latter group. To estimate the fraction of sCD40L that was microparticle-bound CD40L (mp-CD40L), 16 samples underwent 0.1-microm filtration. 50.6% of sCD40L was mp-CD40L in serum-RT, whereas 21.3% and 29.9% were observed in serum-I and PPP, respectively. Lastly, plasma sCD40L was assayed in 46 patients with and 35 without thrombosis. Plasma sCD40L did not correlate with platelet count in non-thrombotic, non-inflammatory patients but did (p<0.01) in those with thrombosis. CONCLUSIONS: Sample processing and temperature profoundly affect sCD40L assay. Serum-I and PPP minimize the release of sCD40L ex vivo and better represent sCD40L in vivo. However, PPP may be preferable particularly in patients with thrombocytosis. The existence of mp-CD40L highlights the importance of centrifuge conditions.     

阿尔茨海默病与轻度认知障碍患者血浆sCD40及sCD40L浓度研究

目的 探索阿尔茨海默病(Alzheimer’s disease,AD)及遗忘型轻度认知障碍(amenstic mild cognitive impairment,a MCI)患者血浆可溶性CD40(soluble CD40,s CD40)和可溶性CD40配体(soluble CD40 ligand,s CD40L)浓度变化特点及其临床意义。方法 采用酶联免疫吸附法检测20例AD患者、35例a MCI患者和32名正常对照者血浆s CD40和s CD40L浓度水平,简易精神状态检查表(mini-mental state examination,MMSE)评估患者认知功能。结果 AD组、a MCI组和对照组血浆s CD40浓度中位数(上下四分位数)分别为[123.3(97.4,149.5)pg/m L]、[102.9(63.6,124.0)pg/m L]和[70.66(51.0,90.8)pg/m L],3组间s CD40浓度差异均有统计学意义(P0.05)。AD组、a MCI组和对照组血浆s CD40L浓度中位数(上下四分位数)分别为[537.0(316.0,1134.0)pg/m L]、[316.0(190.0,546.0)pg/m L]和[167.0(107.5,478.0)pg/m L],3组间s CD40L浓度差异均有统计学意义(P0.05)。a MCI组血浆s CD40L浓度与MMSE得分呈负相关(r=-0.74,P0.01)。结论 s CD40和s CD40L浓度水平在AD和a MCI患者血浆中异常增高,因此s CD40和s CD40L可作为AD的早期检测指标,并提示CD40-CD40L信号可能参与AD的发生发展。     

Platelet soluble CD40L and matrix metalloproteinase 9 activity are proinflammatory mediators in Behçet disease patients

Platelets are the major source of plasma-soluble CD40L (sCD40L), an important inflammatory mediator. This study explored the impact of platelet-derived sCD40L on Beh?et disease (BD), an autoinflammatory vasculitis. We also searched for influences by platelet matrix metalloproteinases (MMP) -2 and MMP-9, implicated in several inflammatory diseases, on CD40L shedding from platelet membrane. Platelet activation were studied by flow cytometry and aggregometry, surface expression of CD40L and platelet-leukocyte aggregates by flow cytometry, sCD40L by ELISA, cellular CD40L and CD40 levels by Western blot and MMPs activity by gelatin zymography. The effect of sCD40L on MMP9 expression was studied in cultured MEG-01 cells. Plasma and platelet-released sCD40L levels were higher in BD patients. No differences in platelet activation and in platelet-leukocyte aggregates formation were observed between BD patients and controls. Plasma and platelet MMP-9 levels were increased in BD patients, whereas there was no difference in platelet MMP-2 activity. Since a correlation between plasma sCD40L and platelet MMP-9 activity was observed, we studied the influence of sCD40L on MMP-9 levels in the megakaryoblastic cell line MEG-01. Treatment of MEG-01 cells with recombinant sCD40L increased MMP-9 but did not change MMP-2 levels. In conclusion, sCD40L release from platelets was mediated by MMP-9, and MMP-9 expression was in turn upregulated by sCD40L in the MEG-01 cell line. We conclude that platelets and megakaryocytes might participate in a positive feedback loop occurring between sCD40L and MMP-9 which would contribute to the proinflammatory state observed in BD.     

Increased inflammatory status and higher prevalence of three-vessel coronary artery disease in patients with concomitant coronary and peripheral atherosclerosis

The aim of this study was to determine whether patients with coronary artery disease (CAD) and concomitant peripheral arterial disease (PAD) have a greater inflammatory status than those with CAD alone. To this aim, we evaluated PAD (ankle/brachial pressure index <0.9), and measured plasma levels of C-reactive protein (CRP), interleukin-6 (IL-6) and the soluble forms of intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) in 234 patients who underwent coronary angiography. Median levels of CRP, IL-6 and sICAM-1 were higher in the CAD without PAD (n=134) and CAD+PAD (n=40) groups than in 60 patients without either disease ("controls"). Median CRP values were higher in patients with CAD+PAD than in patients with CAD alone (4.7 mg/L [1.5; 7.6] vs 2.4 mg/L [0.9; 3.8], p < 0.01).Three-vessel CAD was diagnosed in 60% of CAD+PAD patients and in 21% (p< 0.01) of CAD only patients. After adjustment for confounding factors, only PAD was independently associated with three-vessel CAD (p<0.001). This association was maintained after adjustment for IL-6, the only inflammatory parameter significantly associated with three-vessel CAD at univariate analysis (p<0.01). In conclusion, in CAD the coexistence of PAD is associated with a greater inflammatory status and more widespread coronary atherosclerosis. These results could help to explain the high cardiovascular risk of patients with concomitant CAD and PAD and suggest that PAD be included among the variables used to identify CAD patients for further diagnostic evaluation.     

Influence of sample type on soluble CD40 ligand assessment in patients with acute coronary syndromes

BACKGROUND: Several studies have consistently shown that soluble CD40 ligand (sCD40L) concentrations are elevated in patients with acute coronary syndromes (ACS) and that it can be used as a biomarker for risk stratification. However, recently we could demonstrate that sampling techniques have impact on sCD40L measurements. Thus, it was the aim of our prospective study to evaluate the impact of sampling techniques on sCD40L concentrations of patients with acute coronary syndromes compared to controls. METHODS AND RESULTS: We included a total of 60 patients, - 30 with an acute coronary syndrome, 10 with cardiovascular risk factors but no relevant coronary artery disease and 20 healthy individuals. Blood samples were collected in gel filled tubes without additives, EDTA filled tubes, or in citrate filled tubes. In EDTA or citrate plasma samples, sCD40L values were higher in patients with ACS compared to controls. In contrast, no difference in sCD40L values between ACS patients and controls was observed for serum samples. CONCLUSION: Our data show that only plasma samples, but not serum samples are appropriate for sCD40L measurements. In general, preanalytic conditions are crucial for the assessment of sCD40L concentration and, thus, should be carefully considered for future studies.     

Soluble CD40L in peripheral artery disease. Relationship with disease severity, platelet markers and the effects of angioplasty

Although soluble CD40L (sCD40L, possibly derived from platelets and pro-inflammatory in vitro) may be implicated in thrombosis and haemostasis, there are little data in peripheral artery disease (PAD). We hypothesised the following: (a) that sCD40L relates to the clinical severity of PAD; and (b) that peripheral artery angioplasty acutely raises sCD40L levels. sCD40L was compared to established platelet markers soluble P selectin, platelet microparticles and platelet surface expression of CD62 and CD63. We recruited 36 healthy controls, 33 patients with intermittent claudication (IC), and 33 with symptomatically more severe critical limb ischaemia (CLI), measuring plasma markers by ELISA and membrane markers by flow cytometry. Eleven patients with CLI subsequently underwent peripheral artery angioplasty: blood was taken before and 10 minutes after the intervention. Results show that sCD40L was raised in IC at median 68 (IQR 28-333) pg/ml and in CLI at 64 (34-282) pg/mL compared to 35 (IQR 28-55) pg/ml in the healthy controls (p=0.009). Levels were no different between IC and CLI. The same distribution pattern was present for soluble P selectin, %platelets CD62+ve and CD63+ve. sCD40L failed to correlate significantly with ABPI (p=0.264), unlike %platelets CD62+ve (p=0.0032) and CD63+ve (p=0.009). Pre-angioplasty sCD40L level of 72 (35-610) ng/ml rose to 100 ng/ml (IQR=60-237)(p=0.018) post-angioplasty. Plasma sCD40L, in addition to other platelet indices, is raised in peripheral atherosclerosis and is increased by peripheral artery angioplasty, although levels seem unrelated to clinical severity. Failure to correlate with other markers suggest the platelet may not be the sole source of sCD40L, and that other cells may contribute to plasma levels.