Untersuchungen zum Einfluss von Scherspannung, Inflammation und BET-Inhibition auf humane epitheliale Zellen
Studies on the influence of shear stress, inflammation and BET inhibition on human epithelial cells
by Johannes Jarausch
Date of Examination:2025-01-14
Date of issue:2025-01-09
Advisor:Prof. Dr. Abdul Rahman Asif
Referee:Prof. Dr. Abdul Rahman Asif
Referee:Prof. Dr. Karl Toischer
Persistent Address:
http://dx.doi.org/10.53846/goediss-10979
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Abstract
English
Atherosclerosis is an important cause of some of the most common causes of death, cardiovascular disease. In addition to dyslipidemia, inflammation is one of the key causes of remodeling processes in the endothelium, which can ultimately lead to the final stage of the disease with plaque formation and atherothrombotic occlusion. One possible therapeutic approach, which is being considered for a number of diseases with an inflammatory component, is the inhibition of bromodomain and extraterminal domain (BET) proteins with corresponding inhibitors (BETi). BET proteins are important transcriptional regulator proteins. They bind to histones and transcription factors and serve as transcriptional cofactors. In addition, lipid-lowering properties have already been described for a BETi and it has already been clinically tested. Although BETis are in clinical trials for various diseases, their exact effect has not been fully characterized due to the broad influence that BET proteins have on transcriptional control and the inflammatory cascade. It is therefore useful to identify proteins and signaling pathways relevant to the pathogenesis of atherosclerosis and the effects of BETis. This promotes understanding of the disease and enables new therapeutic approaches.In this way, potential targets for precision therapies can be identified in order to replicate the positive effects of BETi administration, but also potential weaknesses and side effects of the therapy can be identified. The aim of this work was therefore to create an experimental basis to test and better understand the therapy with BETis in an in vitro model of atherosclerosis. To achieve this, a model was established in which endothelial shear stress (ESS) could be applied using a viscometer in order to approximate the intravascular conditions in the arterial vascular system. Human umbilical vein cells (HUVECs) were chosen for this purpose. In this setting, an inflammation model was established to simulate an environment typical in the initial phase of atherosclerotic diseases. The cells were then treated with the non-selective BETi JQ1 under these conditions. The model was validated in different steps using microscopic morphology, immunofluorescence microscopy, quantitative real-time PCR (qRT-PCR) and electrochemiluminescence immunoassay (ECLIA). Finally, the different established treatment methods were combined and tested against each other in eight different treatment groups. Two control groups with and without ESS were created, as well as two groups in which the cells were put into an inflammatory state, with and without ESS. Furthermore, two groups were treated with JQ1 only, with and without ESS, and finally cells in two groups were treated with JQ1 and an inflammatory state was induced, with and without ESS. To gain a comprehensive insight into the changes in the treated cells, the use of data-independent acquisition-mass spectrometry (DIA-MS) was chosen. After creating a library, this makes it possible to make a reliable quantitative statement about changes in the overall proteome of the cells. 4037 proteins were identified, of which 3316 were available for further statistical analysis after rigorous statistical filtering. For the first time, global, unbiased proteomic data of HUVEC cells under the influence of ESS, BET inhibition with JQ1 and inflammation were generated. Significantly differentially expressed proteins were found when comparing the above treatment groups. Using ANOVA-like tests, 1815 differentially expressed proteins were identified across all treatment groups. When comparing the two untreated control groups with and without ESS, 35 differentially expressed proteins were found, among which the overexpressed sodium-hydrogen exchanger 3 regulator 2 (SLC9A3R2), a scaffold protein thought to have a protective function against hypertension, stood out. In addition, the significantly overrepresented signaling pathways fluid shear stress and atherosclerosis and complement and coagulation cascades were identified. A comparison of the non-sheared control group with the group treated with JQ1 alone revealed 962 differentially expressed proteins. In particular, the inhibition of Ras-related protein Rab-5B (Rab5B), a protein that plays a role in angiogenesis, was described. In addition, the treatment showed a particular influence on the signaling pathways regulated exocytosis, response to wounding and cell activation.When comparing the group in which inflammation was induced under ESS with the group in which an attempt was made to suppress this effect under ESS with JQ1, 955 differentially expressed proteins were found. In particular, an additive effect of ESS and JQ1 on the expression of SLC9A3R2 was found, which underlines the atheroprotective potential of BET inhibition in vessels. Furthermore, 186 significantly enriched signaling pathways affecting cell cycle, transcription and metabolic processes were identified. In particular, prolyl 4-hydroxylase subunit alpha-1 (P4HA1), whose increased suppression under TNFα and JQ1 and ESS could possibly lead to dysregulation of the extracellular matrix and inhibition of angiogenesis. Furthermore, the overexpression of high mobility group protein B1 (HMGB1) under TNFα and JQ1 and ESS should be noted, which may support an inflammatory state and induce increased cell death. These are potentially negative findings. The suppression of Sorbin And SH3 Domain Containing 2 (SORBS2) should also be noted. Together with other studies, this may illustrate the therapeutic potential of BETis in diabetics. In summary, this work was the first to investigate the impact of shear stress, inflammation and BET inhibition on human endothelial cells in a very broad, non-targeted proteomic approach. A library of 4037 proteins was generated. Significantly altered proteins were also identified for different treatment groups and possible induced or suppressed signaling pathways can be identified. These data may form the basis for identifying potential candidates for diagnostic or prognostic biomarkers or for possible therapeutic approaches in the treatment of atherosclerosis and/or for understanding the pathophysiology of shear stress and JQ1 treatment on endothelial cells. Finally, potentially deleterious effects of JQ1 could be demonstrated, which in turn calls for further, more detailed research on the impact of BETi therapy on cardiovascular disease.
Keywords: HUVEC; shear stress; endothelial; proteomic; BRD4; JQ1; DIA-MS; BET-Inhibitor; atherosclerosis
