Charakterisierung der Funktion von Smyd1 und PML bei LPS-abhängigen Entzündungsreaktionen in Endothelzellen

Introduction: Sepsis is a global life-threatening condition associated with destructive effects on the organs due to exaggerated immune response. It commonly occurs after the exposure to Lipopolysaccharide (LPS) following infection with Gram-negative bacteria particularly in severely ill patients. LPS leads frequently to endothelial cell dysfunction and plays an important role in the progression of sepsis leading to one or multiple organs failure. Smyd1 is a histone methyltransferase that affects chromatin remodeling and is involved in regulation of cellular development and cancer. PML protein is involved in the oncogenesis of acute promyelocytic leukemia and has a prominent role in many physiological and pathological processes such as tumor suppression, angiogenesis and inflammatory responses. Both Smyd1 and PML were found to be expressed in endothelial cells but little is known about their role in sepsis and inflammation. The aim of this study was to determine the contribution of Smyd1 and PML to the immune response under septic conditions in ECs. Methods: EA.hy926 cells were treated with LPS. The expression of Smyd1 and PML was detected, after reverse transcription of mRNA, via Real time semi-quantitative PCR (RT-qPCR) and on the protein level using Fluorescence-activated cell sorting (FACS). Then, EA.hy926 cells were transfected with Smyd1 and PML carrying plasmids to study their effect on the expression of IL-1, IL-6, IL-8 and NF-κB on the mRNA level with RT-qPCR and/or on the protein level via immunoblotting, Enzyme-linked immunosorbent assay (ELISA) and FACS. Results: Incubation with LPS increased Smyd1 and PML, while incubation with IL-6 increased PML only. Overexpression of Smyd1 increased the expression of IL-1, IL-6, and IL-8, while PMLIV did not induce IL-8. Although Smyd1 and PMLIV appeared to increase the amount and activity of NF-κB, the Smyd1-induced expression of IL-6 was not completely dependent on NF-κB. Conclusions: It was shown in this work that Smyd1 and PML are involved in inflammatory reactions of the endothelial cells. Thereby, we reported an increase of IL-6, IL-8 and IL-1 by Smyd1 which could be in part due to Smyd1 dependent activation of NF-κB signaling pathway. We also confirmed the stimulatory effect PML on IL-6 expression in endothelial cells and revealed an IL-6-dependent increase of PML as well. A heuristic model is being developed in which, after an amplification phase of IL-6 production, the degradation of Smyd1 after its PML-dependent SUMOylation helps to end the acute inflammatory reaction.Einleitung: Die Sepsis als ein Erkrankungszustand mit überschießenden Abwehrreaktionen und daraus resultierendem Multiorganversagen ist weltweit verbreitet und lebensbedrohlich. Hervorgerufen wird sie besonders häufig durch die Freisetzung von Lipopolysacchariden (LPS) aus Gram-negativen Bakterien. Neben ihrer Wirkung auf Abwehrzellen im engeren Sinne wirken LPS auf vaskuläre Endothelzellen. Es kommt zur charakteristischen, inflammatorischen Veränderung des Endothels, die für den Krankheitsverlauf entscheidend ist. Smyd1 ist eine H3K4 Histon-Methyltransferase mit SET Domäne und spielt u.a. eine Rolle bei der Proliferation von Karzinomen. PML-Proteine, ebenfalls im Zusammenhang mit Karzinomen untersucht, bilden Kernkörperchen, in denen die Aktivität von Histonmethyltransferasen wie auch von Histon- Deacetylasen durch SUMOylierungsreaktionen reguliert wird. Sowohl Smyd1 wie PML konnten in Endothelzellen nachgewiesen werden. Ob sie bei inflammatorischen Reaktionen des Endothels eine Rolle spielen können, ist bisher unbekannt. Es war das Ziel dieser Arbeit, Smyd1 und PML in LPS-exponierten Endothelzellen zu untersuchen. Methoden: EA.hy926 Zellen wurden mit LPS oder IL-6 inkubiert oder es wurde Smyd1 oder PMLIV mit Hilfe von Expressionsplasmiden überexprimiert. Für Smyd1, PML, IL-1, IL-6, IL-8 und NF-κB kodierende mRNA wurde durch semi-quantitative real time RT-PCR bestimmt. Zugehörige Proteine wurden durch Immunoblot, FACS oder ELISA bestimmt. Ergebnisse: Inkubation mit LPS erhöhte Smyd1 und PML, während Inkubation mit IL-6 lediglich PML erhöht wurde. Überexpression von Smyd1 erhöhte IL-1, IL-6 und IL-8, während durch PMLIV IL-8 nicht erhöht wurde. Obwohl Smyd1 und PMLIV Menge und Aktivität von NF-κB zu erhöhen schienen, war die Smyd1-induzierte Expression von IL-6 nicht vollständig von NF-κB abhängig. Schlussfolgerungen: Es wurde in dieser Arbeit gezeigt, dass Smyd1 und PML an den inflammatorischen Reaktionen von Endothelzellen beteiligt sind. Dabei berichteten wir über einen Anstieg von IL-6, IL-8 und IL-1 durch Smyd1, der zum Teil auf eine Smyd1-abhängige Aktivierung des NF-κB-Signalwegs zurückzuführen sein könnte. Wir bestätigten auch die stimulierende Wirkung von PML auf die IL-6-Expression in Endothelzellen und zeigten ebenfalls einen IL-6-abhängigen Anstieg von PML. Es wird ein heuristisches Modell entwickelt,in dem nach einer Amplifikationsphase der IL-6 Produktion die Degradation von Smyd1 nach seiner PML- abhängigen SUMOylierung die akute Entzündungsreaktion zu beenden hilft

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-kappa B and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4

Factors associated with abnormal cerebral blood flow in Egyptian children with sickle cell disease

Background: Transcranial Doppler (TCD) is a well-established tool for cerebrovascular assessment. Estimating the flow velocity across the intracranial arteries helps to identify children with sickle cell anaemia who are at risk for stroke. Objective: Our aim is to correlate TCD findings with clinical condition in children with sickle cell disease (SCD) to determine the value of TCD assessment as a predictive tool for stroke in SCD and to identify any association of TCD findings with disease severity, transfusion therapy and treatment administered. Methods: Eighty-five paediatric SCD patients aged from 3 years to 18 years of both genders who were followed up at the Hematology Clinic of New Children’s Hospital at Cairo University were included in this cross-sectional observational study. All our participants underwent routine laboratory investigations and TCD assessments. Results: Oof the 85 patients, two patients (2.3%) died before completing the TCD study and eventually 83 patients were included in the analysis. Seventeen (20.5%) patients had abnormal TCD findings, seven (8.4%) patients showed high-risk findings and 10 (12.1%) patients had conditional flow pattern. Logistic linear regression analysis confirmed that annual frequency of blood transfusion and hydroxyurea (HU) dose were associated with a decreased risk of abnormal TCD findings. Conclusion: The current study demonstrates that our TCD data reproduce the findings of other studies and that it is very likely the results from large trials are applicable for Egyptian children. The annual frequency of blood transfusion and HU dose were associated with a decreased frequency of abnormal TCD findings