Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze–thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task

Journal of Thrombosis and Thrombolysis / Bariatric surgery in morbidly obese individuals affects plasma levels of protein C and thrombomodulin

Obesity is associated with a prothrombotic milieu and an increased risk for thrombotic events. Bariatric surgery is the most effective treatment for obesity resulting in dramatic weight loss and reduced inflammation and extrinsic coagulation pathway activation. Blood samples were drawn from 60 patients undergoing Roux-en-Y gastric bypass surgery before and 1 year after the intervention. Protein C (PC), activated PC (APC), soluble thrombomodulin (TM), soluble E-selectin (E-Sel), prothrombin time (PT) and activated partial thromboplastin time (aPTT) were evaluated. Both PC (187.464.5% before surgery to 118.148% 1 year after surgery, p<0.001) and APC (138.764.4% before surgery to 69.165.7% after surgery, p<0.001) were reduced following surgical intervention. TM showed a similar behavior with a reduction of soluble TM after the procedure from 5.72.6 to 3.21.4 ng/ml (p<0.001). Similarly, soluble E-Sel was reduced after surgery from 26.612.7 to 5.54.1 ng/ml (p<0.001). In contrast, aPTT was not shortened but slightly increased from 29.14.8 s. before surgery to 314.4 s. (p=0.001) after surgery and levels of PT were reduced after surgery to 89.615.5% from an initial 97.513.5% (p<0.001). In conclusion, we demonstrate a reduction of PC and APC 1 year after bariatric surgery accompanied by a reduction in soluble TM and soluble E-Sel. The reduction of PC and APC is not paralleled by a reduction but in contrast by a prolongation of aPTT suggesting a compensatory upregulation of PC during obesity. The reduction of TM and E-Sel might hint towards an improved endothelial function in this cohort of patients.(VLID)365889

Cardioprotective cytokine interleukin33 is upregulated by statins in human cardiac tissue

Interleukin (IL)33 is a member of the IL1 family and is able to act cardioprotective. The aim of this study was to investigate the regulation of IL33 by 3hydroxy3methylglutarylcoenzymeA (HMGCoA) reductase inhibitors (statins) and bisphosphonates (BPs) in human cardiac tissue. The lipophilic fluvastatin, simvastatin, atorvastatin, and lovastatin as well as the nitrogenous BPs alendronate and ibandronate, but not hydrophilic pravastatin increased IL33 mRNA and intracellular IL33 protein levels in both human adult cardiac myocytes (HACM) and fibroblasts (HACF). Additionally, fluvastatin reduced soluble ST2 secretion from HACM. IL33 was also upregulated by the general inhibitor of prenylation perillic acid, a RhoA kinase inhibitor Y27632, and by latrunculin B, but statininduced IL33 expression was inhibited by mevalonate, geranylgeranyl pyrophosphate (GGPP) and RhoA activator U46619. The IL33 promoter was 2.3fold more accessible in statintreated HACM compared to untreated cells (P = 0.037). In explanted hearts of statintreated patients IL33 protein was upregulated as compared with the hearts of nonstatintreated patients (P = 0.048). As IL33 was previously shown to exert cardioprotective effects, one could speculate that such upregulation of IL33 expression in human cardiac cells, which might happen mainly through protein geranylgeranylation, could be a novel mechanism contributing to known cardioprotective effects of statins and BPs.(VLID)481831

Effects of Nicorandil on Inflammation, Apoptosis and Atherosclerotic Plaque Progression

Nicorandil, a balanced vasodilator, is used in the second-line therapy of angina pectoris. In this study, we aimed to illuminate the effects of nicorandil on inflammation, apoptosis, and atherosclerotic plaque progression. Twenty-five LDL-R -/- mice were fed a high-fat diet for 14 weeks. After 6 weeks mice were randomly allocated to treatment with nicorandil (10 mg/kg/day) or tap water. Nicorandil treatment led to a more stable plaque phenotype, displaying an increased thickness of the fibrous cap (p = 0.014), a significant reduction in cholesterol clefts (p = 0.045), and enhanced smooth muscle cell content (p = 0.009). In endothelial cells nicorandil did not reduce the induction of adhesion molecules or proinflammatory cytokines. In H2O2 challenged endothelial cells, pretreatment with nicorandil significantly reduced the percentage of late apoptotic/necrotic cells (p = 0.016) and the ratio of apoptotic to living cells (p = 0.036). Atherosclerotic lesions of animals treated with nicorandil exhibited a significantly decreased content of cleaved caspase-3 (p = 0.034), lower numbers of apoptotic nuclei (p = 0.040), and reduced 8-oxogunanine staining (p = 0.039), demonstrating a stabilizing effect of nicorandil in established atherosclerotic lesions. We suggest that nicorandil has a positive effect on atherosclerotic plaque stabilization by reducing apoptosis

MiRNA Let-7a and Let-7d Are Induced by Globotriaosylceramide via NF-kB Activation in Fabry Disease

Background: Fabry disease is a hereditary genetic defect resulting in reduced activity of the enzyme α-galactosidase-A and the accumulation of globotriaosylceramide (Gb3) in body fluids and cells. Gb3 accumulation was especially reported for the vascular endothelium in several organs. Methods: Three Fabry disease patients were screened using a micro-RNA screen. An in vitro approach in human endothelial cells was used to determine miRNA regulation by Gb3. Results: In a micro-RNA screen of three Fabry patients undergoing enzyme replacement therapy, we found that miRNAs let-7a and let-7d were significantly increased after therapy. We demonstrate in vitro in endothelial cells that Gb3 induced activation of NF-κB and activated downstream targets. In addition, NF-κB activity directly reduced let-7a and let-7d miRNA expression as inhibiting NF-kB nuclear entry abolished the Gb3 effects. Conclusion: We suggest that let-7a and let-7d are potential markers for enzyme activity and inflammation in Fabry disease patients