Funktionelle Untersuchungen zur Rolle des RAD50 Proteins in der zellulären Schadensantwort und in der Mitose

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Preeclampsia-Associated Alteration of DNA Methylation in Fetal Endothelial Progenitor Cells

ObjectiveThe pregnancy complication preeclampsia represents an independent risk factor for cardiovascular disease. Our previous research shows a diminished function of fetal endothelial colony-forming cells (ECFC), a proliferative subgroup of endothelial progenitor cells (EPC) in preeclampsia. The aim of this study was to further investigate whether DNA methylation of fetal EPC is affected in preeclampsia.MethodsThe genomic methylation pattern of fetal ECFC from uncomplicated and preeclamptic pregnancies was compared for 865918 CpG sites, and genes were classified into gene networks. Low and advanced cell culture passages were compared to explore whether expansion of fetal ECFC in cell culture leads to changes in global methylation status and if methylation characteristics in preeclampsia are maintained with increasing passage.ResultsA differential methylation pattern of fetal ECFC from preeclampsia compared to uncomplicated pregnancy was detected for a total of 1266 CpG sites in passage 3, and for 2362 sites in passage 5. Key features of primary networks implicated by methylation differences included cell metabolism, cell cycle and transcription and, more specifically, genes involved in cell-cell interaction and Wnt signaling. We identified an overlap between differentially regulated pathways in preeclampsia and cardiovascular system development and function. Cell culture passages 3 and 5 showed similar gene network profiles, and 1260 out of 1266 preeclampsia-associated methylation changes detected in passage 3 were confirmed in passage 5.ConclusionMethylation modification caused by preeclampsia is stable and detectable even in higher cell culture passages. An epigenetically modified endothelial precursor may influence both normal morphogenesis and postnatal vascular repair capacity. Further studies on epigenetic modifications in complicated pregnancies are needed to facilitate development of EPC based therapies for cardiovascular alterations

Vitamin D Effects on the Immune System from Periconception through Pregnancy

Vitamin D is a well-known secosteroid and guardian of bone health and calcium homeostasis. Studies on its role in immunomodulatory functions have expanded its field in recent years. In addition to its impact on human physiology, vitamin D influences the differentiation and proliferation of immune system modulators, interleukin expression and antimicrobial responses. Furthermore, it has been shown that vitamin D is synthesized in female reproductive tissues and, by modulating the immune system, affects the periconception period and reproductive outcomes. B cells, T cells, macrophages and dendritic cells can all synthesize active vitamin D and are involved in processes which occur from fertilization, implantation and maintenance of pregnancy. Components of vitamin D synthesis are expressed in the ovary, decidua, endometrium and placenta. An inadequate vitamin D level has been associated with recurrent implantation failure and pregnancy loss and is associated with pregnancy-related disorders like preeclampsia. This paper reviews the most important data on immunomodulatory vitamin D effects in relation to the immune system from periconception to pregnancy and provides an insight into the possible consequences of vitamin D deficiency before and during pregnancy

Low Ethanol Concentrations Promote Endothelial Progenitor Cell Capacity and Reparative Function

Background. Endothelial progenitor cells (EPCs) are recruited to injured endothelium and contribute to its regeneration. There is evidence that moderate ethanol consumption prevents the development and progression of atherosclerosis in a variety of in vitro and in vivo models and increases the mobilization of progenitor cells. Furthermore, there are studies that identified ethanol at low concentration as a therapeutic tool to mobilize progenitor cells in peripheral blood. At the same time, the cell number of EPCs represents a close link to cardiovascular system constitution and function and contributes to cardiovascular risk. The aim of this study was to evaluate the effect of low dose ethanol on typical features of endothelial colony-forming cells (ECFCs), a proliferative subtype of EPCs. Methods and Results. We tested whether ethanol impacts the functional abilities of ECFC (e.g., migration, tube formation, and proliferation) using in vitro assays, the intercommunication of ECFC by exploring cell surface molecules by flow cytometry, and the expression of (anti-)angiogenic molecules by ELISA. Low concentrations of ethanol concentration promoted migration, proliferation, and tubule formation of ECFC. The expression of the cell surface marker VE-cadherin, a protein which plays an important role in cell-cell interaction, was enhanced by ethanol, while (anti-)angiogenic molecule expression was not impacted. Conclusion. Ethanol at moderate concentrations increases the angiogenic abilities of endothelial progenitor cells thus possibly contributing to vasoprotection

Protective role of RAD50 on chromatin bridges during abnormal cytokinesis

Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ̃4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ̃4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis

Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs’ functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs’ condition in cell therapy in order to ameliorate endothelial dysfunction