Month: August 2020

The most common approach to genomic analysis starts with the identification of differentially expressed genes and subsequent biological interpretation using Gene Set Enrichment Analysis. According to our definition, Limma analysis of the microarray data identified 2737 differentially expressed genes between 7 GDC-0879 905281-76-7 aortic dissection cases and 5 controls. Of the top 25 differentially expressed genes, 17 genes were up-regulated and 8 were downregulated in cases. However, the common approach may miss important biological pathways, because the inference does not take the pathway or network structure into account and because changes affecting individual features are often of a small magnitude. Thus, a number of statistical approaches have emerged using the pathway/network structure in the inference procedure. Here we used a novel integrative approach to infer network modules, which subsequently facilitated the biological interpretation. By this integrative network algorithm, interactome hotspots associated with aortic dissection were identified. Among them, we found a JAK2 module, which was validated in an independent gene expression microarray data set, enriched for cytokines and receptors, including IL-6, IL-6R, CCL2 and IFNGR2. That many cytokines are implicated in AAD is well supported by previous literature: for instance, IL-6, INF gamma and CCL2, were observed to be significantly increased in AAD patients. The previous microarray study also observed that IL-2, -6 and -8 genes were up-regulated in aortic dissection. It has also been noted that IL-6 is secreted at high levels in human aortic aneurysm disease. These data indicate an inflammatory process characterized by abnormal expression of cytokines in aortic disease. Vascular inflammation is a common pathologic and physiological response in diverse cardiovascular disease processes, including atherosclerosis, myocardial infarction and congestive heart failure. Furthermore, IL-6 is found to be linked with the development of coronary disease and atherosclerosis. Taking all the factors together, it is reasonable to hypothesize that chronic inflammation may probably play a contributory role in the pathogenesis of aortic dissection. However, the precise relationship between inflammation and AAD remains complicated and the specific pattern of the inflammation as well as the key regulators is still pending. Therefore, when JAK2, one of the top 25 genes from Limma analysis, came up also as the hotspot, it was of particular interest to further discuss its potential biological significance. The JAK family plays a critical role in growth, development, survival and differentiation through signal transduction of many cytokine receptors. STAT3, a downstream transducer activated by JAKs, was found to be involved in vascular abnormalities. Frequent cerebral and coronary artery ectasias and aneurysms were observed in patients with STAT3 deficiency.

These results demonstrate that csynuclein can influence cell viability, signal transduction pathways and also stress response. Pro-apoptotic BAX belongs to the Bcl-2 family and plays an important role in the intrinsic apoptotic pathway through binding mitochondrial VDAC, which leads to the release of cytochrome c and finally to the initiating of apoptosis. In an elevated intraocular pressure mouse glaucoma model the expression of BAX was increased in hypertensive eyes in comparisons to control eyes. Also, a BAX deficiency in DBA/2J mouse protects RGC from cell death. The expression of BAX is regulated by transcription factor p53 which in turn is regulated by S100A4, down-regulated in c-synuclein ab treated cells. S100A4 induction in a murine non-metastatic adenocarcinoma cell line leads to an increased expression of BAX and thereby to increased apoptosis. The anti-apoptotic protein BIRC6 belongs to the inhibitor of apoptosis family and is up-regulated in c-synuclein ab treated RGC-5. BIRC6 is up-regulated in tumors and can inhibit active caspase-3. Studies show that overexpression of BIRC6 in mammalian cells inhibits apoptosis. In an ocular hypertensive glaucoma model the over-expression of BIRC4, another member of the IAP family, promotes optic nerve axon survival. VDAC 1/2/3, significantly down-regulated in this study, play an important role in apoptosis-initiation and are located on the outer mitochondrial membrane. They participate in energy balance regulation as well as in the release of pro-apoptotic factors. Studies show that a reduction of VDAC1 levels in endothelial cells attenuates endostatin induced apoptosis. Other proteins, such as active caspase-3, caspase-9 and BAD were down-regulated in this study whereas the active form of ERK called p-ERK1/2 was up-regulated in c-synuclein ab treated RGC-5. The well characterized ERK pathway transfers signals from different membrane receptors into the nucleus. It is composed of different kinases which activate ERK1. Activated ERK1, which is increased in RGC-5 treated with c-synuclein abs, is able to phosphorylate many cytoplasmic as well as nuclear targets, which leads to cell proliferation. An experimental rat glaucoma model shows that the activation of ERK leads to increased survival of rgc after ocular hypertension surgery. A MEK-ERK survival pathway is described, whereby activated MAPK participate in the phosphorylation of BAD and promote cell survival. BAD is a pro apoptotic member of the Bcl-2 family and participates in the initiation of apoptosis. Studies assume an involvement of BAD and active caspase-3 in glaucoma, which leads to the cellular protein cleavage and apoptosis. Studies show that c-synuclein is able to bind transcriptional factors and modulate the transcription of genes and factors such as JunB, MECP2, CREB1 and ATF3. Furthermore csynuclein can GSK1363089 interfere with the mitochondrial apoptosis pathway through transcriptional regulation of kinases and phosphates, which control the phosphorylation status of BAD. Other studies analyzing ab functions, such as hsp27.

Studies suggest an immunological component in the pathology of glaucoma. An increased occurrence of paraproteins and autoantibodies against nuclear antigens like Sjo¨gren’s syndrom A, was demonstrated in glaucoma patients. Furthermore, studies show not only up-regulated, but also down-regulated antibodies in glaucoma patients. In the serum and aqueous humor of glaucoma patients general complex autoantibody patterns against retinal and optic nerve antigens were found but also more specific autoantibody changes such as an up- regulation of abs against e.g. alpha foldrin and Hsp 70, and a downregulation of abs against aB Crystallin and Vimentin leading to the conclusion that there is a role for the autoantibodies in the pathogenesis of glaucoma. Previous studies incubating neuroretinal cells with the serum and the abs of glaucoma patients found changed protein expression patterns in cells incubated with glaucoma serum in comparison to serum from healthy people. Furthermore the cells reacted differently towards the serum after removal of IgG abs. These results underline the hypothesis that changes in the autoantibodies could play a role in the pathogenesis of the disease. One autoantibody down-regulated in glaucoma patients is high throughput screening targeted against c-synuclein. This study aimed to investigate, which effect the down-regulated ab against c-synuclein has on stressed neuroretinal cells. A dose response effect as well as a negative effect of high c-synuclein ab concentrations couldn’t be observed. Studies show that ab-uptake into cells can be saturable. Our immunohistochemical staining results showing small amounts of abs in the cells at one defined time point support the assumption that ab uptake of the used cells is restricted. Furthermore, high ab concentrations not necessarily have a negative effect, as other studies could show that even high concentrations of ab, also internalized by cells, do not have a negative influence on the viability of cells. This could be due to the fact that the binding partners of the abs are saturated and further abs cannot be bound and therefore have no additional effect. When using unspecific abs such as anti-myoglobin abs no protective or negative effect was detected. Studies demonstrate an impact of c-synuclein on apoptotic pathways in RGC. Knocking down c-synuclein in RGC-5 leads to decreased viability through the regulation of kinases and phosphatases. In general, the effect of changes in c-synuclein expression either in vivo or in vitro shows opposing results. In vivo studies show that an up-regulation of c-synuclein can lead to neurodegeneration, which stands in contrast to other reports demonstrating that an overexpression of c-synuclein has no negative effect whereas other studies show that there is no effect on neuronal cells when inactivating csynuclein. Additionally studies show that c-synuclein can participate in signal transduction pathways. In Y79 cells overexpression of synoretin, the bovine orthologous of c-synuclein, induces increased MAPK activity as well as its downstream effector Elk-1.

In this case, the different stages of angiogenesis can be a target for drug administration: cell migration, proliferation and differentiation. Since the differentiation stage of angiogenesis is the final and most important step during which capillary-like tubules are formed, the assays simulating this stage are extensively used to determine the efficacy of anti-angiogenic drugs. Currently used differentiation assays involve the plating of endothelial cells into or onto a gel matrix to investigate the formation of tubule-like structures after 4 to 24 h. The Matrigel assay is a widely used differentiation assay, where a protein mixture derived from mouse Engelbreth-HolmSwarm sarcoma is used. After 12 h of endothelial cell seeding on the Matrigel, the cells start to form cord-like structures, although in many cases, the seeded cells often clump into cell aggregates. Additionally, there is significant debate as to whether these cordlike structures actually contain patent lumina or not. Furthermore, non-endothelial cells like fibroblasts and glioblastoma cells have also been shown to form cord-like structures on Matrigel, and therefore a cautious interpretation of the results in this system is required. A further limitation of these gel assays is their inability to mimic the complete in vivo situation, since the surrounding cells near an angiogenic site are not taken into consideration. In order to test angiostatic drugs efficiently, definitive in vitro assays are necessary to reliably estimate the anti-angiogenic potential of the drugs. 2-D assays should be fast, inexpensive and easy to set up. Furthermore, the assays should optimally imitate the in vivo situation. Most translatable assays would include supporting cells, extracellular matrix and/or basement membrane. Circulating blood would further optimize an in vitro assay. After an estimation of applicability via the 2-D assay, an in vivo assay could then be considered for further analysis. Co-culture models could imitate the in vivo situation in a more reliable manner than Matrigel or other ECM models, since supportive cells adjacent to the angiogenic site in the body also have an influence on vessel development. Several co-culture models have been described so far, using HUVECs or human dermal microvascular endothelial cells as sproutbuilding cells, and different supportive cell types in 2D and 3D. These supportive cells develop an ECM and/or release growth factors that support angiogenesis. As supportive cells in co-culture models, fibroblasts from dermal human breast tissue, HUASMCs, pulmonary RAD001 artery smooth muscle cells, mesenchymal stem cells and fibroblasts from dermal superficial skin layer have been used. All of these supportive cells in co-culture with sprout-forming cells supported the development of capillarylike structures in 2D. These co-cultures provide a reliable estimation regarding the anti-angiogenic potential of a particular drug. Unfortunately, all supportive cells used in the aforementioned assays derive from human tissue cell sources with limited access.

The main cause of death during the early phase of MCS. Moreover, pre-implant levels of IL-6 have been associated with hemodynamic status, as defined by Interagency Registry for Mechanically Assisted Circulatory Support profiles, with higher levels in patients presenting critical INTERMACS profiles. Since the signal pathways, IL-6-dependent, and specific monocyte attracting chemokines, such as IL-8, are proposed as crucial triggers in controlling monocyte activation, an important condition in the development of MOF and of haemostatic complications, it can be assumed that they play a critical role in affecting outcomes during the early phase of LVAD support. The aims of this study were to assess whether preoperative IL-6, IL-8 and neopterin levels affect postoperative inflammatory response and short-term outcomes in LVAD-recipients. The main findings of this study may be summarized as follows: 1) ESHF-patients supported by LVAD with preoperative IL-6 levels higher than 8.3 pg/mL are more susceptible of poor early outcome, longer ICU stay and hospitalisation, when compared to patients with lower IL-6 levels; 2) postoperatively, LVAD-patients with IL-6 levels higher than 8.3 pg/mL showed a more pronounced neopterin and IL-8 release, and MOF severity. Recent advances in MCS, specifically implantable CF-LVAD therapy, are providing alternatives for patients waiting for heart transplantation, for patients who are HT ineligible or anticipated to experience recovery after LV-unloading. Every centre involved in advanced HF treatments has to evaluate patient specific risk profile according to one’s own experience and to data reported by larger studies. With worsening of clinical status, the need for LVAD increases as well as the peri-operative risk, and optimal operative timing becomes difficult. In this setting, clinical indications, absolute or relative contraindications are not universally accepted because of contrasting published data. With regard to risk stratification in ESHF-patients, little is known about baseline inflammatory profiles and their impact on clinical outcome and prognosis, and it’s reasonable to speculate a role of inflammatory system on the outcome of these fragile patients. In the present study, pre-implant levels of IL-6, IL-8 and neopterin were investigated to evaluate the impact of these monocyte-related inflammatory mediators on the inflammatory response and outcome in LVAD patients. IL-8, a known chemokine attracting monocyte on endothelial cells, neopterin, a pteridine produced by PI-103 activated macrophages, and IL-6-dependent signals, mainly associated to progression of HF, are proposed as crucial triggers in controlling monocyte activation and recruitment in vascular inflammation and endothelial dysfunction, important factors for development of MOF. Moreover, neopterin is a key pteridine that links inflammation and redox state in heart failure. Indeed macrophages, stimulated by interferon-gamma, generate neopterin that interferes with reactive species, such as peroxynitrite, inducing myocardial contractile failure. However, in our cohort of LVAD-candidates, only patients with preoperatively elevated IL-6 levels.