Modulation of gap junctional intercellular communication between human smooth muscle cells by leukocyte-derived growth factors and cytokines in relation to atherogenesis

In this thesis, the effect of leukocyte-derived growth factors and cytokines on GJIC between SMC was investigated. GJIC is regarded as an important mechanism in the control of cell growth, cell differentiation and tissue homeostasis. Disturbance of SMC growth control is regarded to be a key event in the pathogenesis of atherosclerosis in which growth factors and cytokines are thought to play a central role. In the present study, cultured human SMC were incubated with (human) recombinant growth factors and cytokines. TNFα, IFN-γ, PDGF, bFGF and IL-6 were chosen as representatives of several classes of growth modulating factors. These growth factors and cytokines are known to be products of macrophages and/or T lymphocytes and have been detected in human atherosclerotic lesions. After an incubation period, GJIC between SMC was measured. In addition, human SMC were co-cultured with J774A.1 murine macrophages or human monocyte-macrophages in the Transwell-COL cell culture system, to account for the complexity of macrophage secretion patterns. After removal of the macrophages, GJIC between the co-cultured SMC was determined.The experiments described in chapter 2 and 3 clearly demonstrate that all factors tested reduced GJIC between SMC with ~20 - 50%, except for bFGF which strongly increased GJIC. Furthermore, these experiments revealed that effects of growth factors and cytokines on GJIC are not univocal and thus cannot be generalized. PDGF, IL-6 and bFGF caused transient effects on GJIC, whereas in experiments with TNFαor IFN-γ, a persistent inhibition of GJlC was obtained.The most remarkable result of the study described in chapter 4 was that upon combining TNFα and IFN-γ, GJIC between SMC strongly reduced (up to 86%) in an additive or synergistic manner. Upon long term incubation with the combination of TNFα and IFN-γ, some SMC did not communicate with neighbouring cells at all. This may result in an escape from growth control mechanisms, which, in turn, may lead to disturbance of SMC proliferation, a key event in atherosclerosis. In incubations with other combinations of growth factors and cytokines, (antagonistic) interactive effects on GJIC were observed.The present investigation provided evidence that reactive oxygen species may play a role in cytokine-induced inhibition of GJIC between SMC. Experiments described in chapter 2 revealed that pretreatment of SMC with antioxidants like ascorbic acid, α-tocopherol or GSH prevented the inhibition of GJIC upon exposure of SMC to TNFα. Studies with SOD (chapter 4)demonstrated that the superoxide radical may be involved in GJIC reduction by TNFα, since incubation with SOD, even hours after the addition of TNFα, restored GJIC to control values. Furthermore, SOD partly restored IFN-γeffects on GJIC in the short - but not in the long term. When SMC were incubated with TNFα and IFN-γsimultaneously for 24 h, high levels of SOD could not even partly counteract the strong inhibition of GJIC caused by these cytokines. Thus, other, superoxide-unrelated mechanisms may affect GJIC more predominantly in long term incubations with the combination of TNFα, and IFN-γ, One such mechanism may be represented by the reduced Cx43 staining which was observed in immunofluorescence studies on SMC cultures incubated with these cytokines (chapter 4) ,which may be an indication for the reduced presence of functional gap junction channels.PDGF-AA, PDGF-BB, IL-6, IFN-γ, TNFαand bFGF all stimulated SMC proliferation in our cell culture system, as individual factors as well as in combinations (chapter 3 and 4). Upon comparing these cell proliferation results with GJIC data, a complex relationship between modulation of GJIC, cell proliferation and the process of atherosclerosis is suggested.Experiments described in chapter 5 demonstrated that macrophages cultured on pore membrane inserts modulate GJIC between SMC co-cultured in Transwell-COL cell culture chambers. Since these results were obtained in an indirect co-culture system which prevents direct cell-cell contact, it was hypothesized that soluble factors, released by macrophages, may be involved in the modulation of GJIC between SMC. At this moment, one can only speculate about the nature of the factors involved in this macrophage-dependent modulation of GJIC. The results clearly indicate that the source and activation state of macrophages were of importance in these co-culture experiments. Therefore, further research should be aimed at studying the effect of different types of macrophages on GJIC between co-cultured SMC. Heterogeneity in atheroma macrophages exists; the most noticeable difference being the presence of 'normal' macrophages and the presence of macrophage-derived foam cells, which are likely to differ in endocytic and secretory repertoire. Furthermore, macrophages should be exposed to different (patho-)physiological agents with relevance to the process of atherosclerosis, in order to study their effects on GJIC between SMC in even more detail.The present study provides a good starting point for further research aimed at the understanding of mechanisms by which enviromnental contaminants or drugs might interfere with atherogenesis. It is already known that widespread food chain and cigarette smoke contaminants like for example benzo[a]pyrene, polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo- p -dioxin may affect the pathogenesis of atherosclerosis in several ways, for instance by damaging SMC DNA, disrupting endothelial barrier function, or by modulation of plasma cholesterol and lipoprotein levels). Furthermore, chemicals like components in cigarette smoke condensate may modulate GJIC between SMC. Considering that growth factors and cytokines like TNFα, and IFN-γ, may have marked effects on GJIC, one may assume that enviromnental contaminants and drugs capable of affecting the expression of growth factors and cytokines or their receptors may interfere with GJIC in an indirect manner. In the case of atherogenesis for instance, chemicals may stimulate growth factor- and cytokine production by SMC and/or macrophages which, in turn, may influence homologous GJIC between SMC. In addition, exogenous chemicals may influence heterologous GJIC between macrophages and SMC as well; either directly, or indirectly via the induction of growth factor and cytokine expression by these cells. As a consequence, macrophage-derived reactive substances will have more - or just less- impact on SMC functioning.Relatively short exposures to enviromnental contaminants or drugs in individuals in which plaques have already passed some critical phases in the atherosclerotic process might enhance the severity of the lesions. Further research along this line may also lead to the identification of nutritional or chemical factors that may have beneficial (protectiveregressive) effects on the development of atherosclerotic lesions.Modulation of GJIC by growth factors and cytokines may affect a response-to-injury. On the other hand, modulation of GJIC may also play a role in the monoclonal expansion of cells. Therefore, the response-to-injury hypothesis and the monoclonal theory may be compatible is some respects, as was previously suggested by Zwijsen.The results of the present study may also be applicable to other pathophysiological phenomena, in which growth factors and cytokines may play a prominent role in the onset or progression of the disease. Proliferative diseases like pulmonary fibrosis, glomerulosclerosis and liver cirrhosis share some pathobiologic mechanisms with atherosclerosis, including leukocyte infiltration, mesenchymal cell proliferation and enhanced matrix synthesis. Leukocyte-derived growth factors and cytokines may modulate GJIC between the mesenchymal cells concerned, which in turn may result in abnormal cell proliferation. It is known that certain chemicals may contribute to the development of these diseases. The mechanisms by which these chemicals act may be linked to the processes studied and discussed in this thesis.Overall, the information presented in this thesis concerning the possible role of growth factors and cytokines in the pathophysiology of atherosclerosis provides a useful instrument to study possible modulatory effects of chemicals on the process of atherosclerosis via the mechanisms mentioned above

Dietary fatty acids: is it time to change the recommendations

Limiting the saturated fatty acid (SAFA) consumption forms the basis of dietary fat recommendations for heart health, despite several meta-analyses demonstrating no link be- tween dietary SAFA and the risk of cardiovascular disease (CVD). Three experts on dietary fat and health discussed the evidence of reducing SAFA intake at a symposium of the Federation of European Nutrition Societies in Berlin, Germany, October 23, 2015. Ronald P. Mensink, Maastricht University, the Netherlands, discussed the evidence linking dietary fatty acids and CVD risk. He emphasized the impor- tance of the replacement nutrient(s) when SAFA intake is re- duced. Julie Lovegrove, University of Reading, UK, addressed the question of whether higher intakes of unsaturated fatty acids are beneficial. She discussed the replacement of SAFA by polyunsaturated fatty acids (PUFA) and monounsaturat- ed fatty acids (MUFA), noting the reduction in CVD risk with PUFA replacement and in CVD risk markers with MUFA re- placement of SAFA. Ursula Schwab, University of Eastern Finland, Kuopio, Finland, discussed the importance of di- etary patterns in achieving reduced risk of CVD, observing that several dietary patterns following the principles of a health-promoting diet and adapted to local customs, food preferences and seasonality are effective in reducing the risk of CVD, type 2 diabetes and other chronic diseases. This pa- per summarizes the symposium presentations

In-line near infrared spectroscopy during freeze-drying as a tool to measure efficiency of hydrogen bond formation between protein and sugar, predictive of protein storage stability

Sugars are often used as stabilizers of protein formulations during freeze-drying. However, not all sugars are equally suitable for this purpose. Using in-line near-infrared spectroscopy during freeze-drying, it is shown here that hydrogen bond formation during freeze-drying, under secondary drying conditions in particular, can be related to the preservation of the functionality and structure of proteins during storage. The disaccharide trehalose was best capable of forming hydrogen bonds with the model protein, lactate dehydrogenase, thereby stabilizing it, followed by the molecularly flexible oligosaccharide inulin 4kDa. The molecularly rigid oligo- and polysaccharides dextran 5kDa and 70kDa, respectively, formed the least amount of hydrogen bonds and provided least stabilization of the protein. It is concluded that smaller and molecularly more flexible sugars are less affected by steric hindrance, allowing them to form more hydrogen bonds with the protein, thereby stabilizing it better

The development and psychometric evaluation of the Questionnaire Epistemic Trust (QET):A self-report assessment of epistemic trust

Epistemic trust (ET) refers to the predisposition to trust information as authentic, trustworthy and relevant to the self. Epistemic distrust - resulting from early adversity - may interfere with openness to social learning within the therapeutic encounter, reducing the ability to benefit from treatment. The self-report Questionnaire Epistemic Trust (QET) is a newly developed instrument that aims to assess ET. This study presents the first results on the psychometric properties of the QET in both a community and a clinical sample. Our findings indicate that the QET is composed of four meaningful subscales with good to excellent internal consistency. The QET shows relevant associations with related constructs like personality functioning, symptom distress and quality of life. QET scores clearly distinguish between a clinical and community sample and are associated with the quality of the therapeutic alliance. The QET provides a promising, brief and user-friendly instrument that could be used for a range of clinical and research purposes. Future studies with larger samples are needed to strengthen construct validity and to investigate the value of the QET to predict differential treatment responses or to study mechanisms of change

Adding New Tasks to a Single Network with Weight Transformations using Binary Masks

Visual recognition algorithms are required today to exhibit adaptive abilities. Given a deep model trained on a specific, given task, it would be highly desirable to be able to adapt incrementally to new tasks, preserving scalability as the number of new tasks increases, while at the same time avoiding catastrophic forgetting issues. Recent work has shown that masking the internal weights of a given original conv-net through learned binary variables is a promising strategy. We build upon this intuition and take into account more elaborated affine transformations of the convolutional weights that include learned binary masks. We show that with our generalization it is possible to achieve significantly higher levels of adaptation to new tasks, enabling the approach to compete with fine tuning strategies by requiring slightly more than 1 bit per network parameter per additional task. Experiments on two popular benchmarks showcase the power of our approach, that achieves the new state of the art on the Visual Decathlon Challenge

Plasma oxyphytosterols most likely originate from hepatic oxidation and subsequent spill-over in the circulation

We evaluated oxyphytosterol (OPS) concentrations in plasma and various tissues of two genetically modified mouse models with either increased cholesterol (apoE KO mice) or increased cholesterol and plant sterol (PS) concentrations (apoExABCG8 dKO mice). Sixteen female apoE KO and 16 dKO mice followed the same standard, low OPS-chow diet. Animals were euthanized at 36 weeks to measure PS and OPS concentrations in plasma, brain, liver and aortic tissue. Cholesterol and oxysteml (OS) concentrations were analyzed as reference for sterol oxidation in general. Plasma campesterol (24.1 +/- 4.3 vs. 11.8 +/- 3.0 mg/dL) and sitosterol (67.4 +/- 12.7 vs. 4.9 +/- 1.1 mg/dL) concentrations were severely elevated in the dKO compared to the apoE KO mice (p < 0.001). Also, in aortic and brain tissue, PS levels were significantly elevated in dKO. However, plasma, aortic and brain OPS concentrations were comparable or even lower in the dKO mice. In contrast, in liver tissue, both PS and OPS concentrations were severely elevated in the dKO compared to apoE KO mice (sum OPS: 7.4 +/- 1.6 vs. 4.1 +/- 0.8 ng/mg, p < 0.001). OS concentrations followed cholesterol concentrations in plasma and all tissues suggesting ubiquitous oxidation. Despite severely elevated PS concentrations, OPS concentrations were only elevated in liver tissue, suggesting that OPS are primarily formed in the liver and plasma concentrations originate from hepatic spill-over into the circulation