Refine
Year of publication
Document Type
- Doctoral Thesis (31)
Has Fulltext
- yes (31)
Is part of the Bibliography
- no (31)
Keywords
- vascular endothelial cells (2)
- AF4 (1)
- AMPK, nuclear receptors, PPAR, LXR, fatty acid oxidation, ABCA1, human macrophages (1)
- Acute leukemia, chromosomal translocation, MLL, KMT2A, AF4-MLL, reciprocal fusion protein, MLL-r leukemia, (1)
- DDX6 (1)
- HIV Humanized Mice (1)
- Leukemia (1)
- Metabolismus (1)
- Nukleäre Rezeptoren (1)
- P-TEFb (1)
Institute
- Biochemie, Chemie und Pharmazie (19)
- Pharmazie (10)
- Biochemie und Chemie (3)
The EMT-transcription factor ZEB1 has been intensively studied in solid cancers, where it is expressed at the invasive front and in cancer-associated fibroblasts (CAFs). In tumour cells, ZEB1 has been involved in multiple steps of cancer progression including stemness, metastasis and therapy resistance, yet its role in the tumour-microenvironment is largely unknown. Here, the role of Zeb1 in CAFs was investigated using mouse models reflecting different tumour stages in immunocompetent fibroblast specific Zeb1 KO mice. Fibroblast-specific depletion of Zeb1 accelerated tumour growth in the inflammation driven AOM/DSS tumour initiation model, reduced tumour growth and invasion in the sporadic AOM/P53 model and reduced liver metastasis in a progressed orthotopic transplantation model. Immunohistochemical and single cell RNA-sequencing analysis showed that Zeb1 ablation resulted in attenuated expression of the myofibroblast marker aSMA and reduced ECM deposition, indicating a shift among fibroblast subpopulations. Modulation of CAFs was furthermore associated with increased inflammatory signaling in fibroblasts resulting in immune infiltration into primary tumours and exaggerated inflammatory signaling in T cells, B cells and macrophages. These changes in the tumour microenvironment were associated with increased efficacy of immune checkpoint inhibition therapy. In summary, Zeb1 expression in CAFs was identified as a potential target to block immunosuppression and metastatic dissemination in colon cancer.
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all "cancer genes" were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: proximity of genes able to produce prematurely terminated transcripts, which leads to the production of transspliced fusion RNAs, and finally, the induction of DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced.
Cytochrome P450 enzymes are a large superfamily of membrane-bound heme-containing monooxygenases. They are essential for the oxidative metabolism of endogenous substrates such as steroids and fatty acids, and biotransformation of xenobiotic substrates such as pollutants and drugs. Although the highest expression of CYPs is found in the liver, their cardiovascular expression is not negligible with CYP450 subfamilies being responsible for the production of vasoactive lipids. Of importance, the enzymatic activity of all microsomal CYP450 isoenzymes is dependent on the cytochrome P450 reductase (POR), an electron donor.
In the first part of this work, the role of cytochrome P450 monooxygenases on the biotransformation of organic nitrates was investigated. Recombinant SupersomesTM were selected and incubated with NTG and PETN, where nitrite release was measured as a nitric oxide (NO) footprint. The capacity of the recombinant POR/CYP450 system to release nitrite from NO prodrugs was shown to be CYP-specific and dose-dependent. To study the involvement of CYP450 enzymes in the vascular biotransformation of organic nitrates in vivo, a smooth muscle-cell specific, inducible knockout model of POR (smcPOR-/-) was generated. Organ chamber experiments revealed that the vascular POR/CYP450 system had no impact on the dilator response of NTG and PETN. In line with previous publications, inhibition of ALDH2, known as the main enzyme responsible for the activation of NTG and PETN, and/or abolishment of the endogenous NO production did not reveal a contribution of the POR/CYP450 system to the dilator response of NTG and PETN. To better understand these results, we looked at the expression of the hepatic and vascular expression of the POR/CYP450 system where the hepatic was increased by 10- to 40-fold as shown by Western blot analysis. We concluded that due to insufficient vascular expression of CYP450 enzymes their contribution to the bioactivation of NTG and PETN is only minor.
The second part of this work focused on the cardiac relevance of endothelial isoenzymes. For that purpose, an endothelial cell-specific, tamoxifen-inducible knockout model of POR was generated and characterized in the present study. RNA-sequencing of the heart of healthy mice revealed that the CYP450 expression is cell-specific with cardiac endothelial cells (ECs) exhibiting an enrichment in the expression of the Cyp4 family (ω-oxidation of fatty acids) and of the Cyp2 family (production of EETs). Under non-stredded conditions (i.e. 30 days after inducing the knockout by tamoxifen feeding), endothelial deletion of POR was associated with cardiac remodelling as observed by an increase in the ratio of heart weight to body weight and an increase in the cardiomyocyte area. RNA-sequencing of cardiac ECs suggested that loss of POR might alter ribosomal biogenesis and protein synthesis, which could potentially affect the cardiac contractility in ecPOR-/- mice. Metabolomics from cardiac tissue of CTL and ecPOR-/- mice were not indicative for an important metabolic function of the endothelial POR/CYP450 system in the heart. The combination of transverse aortic constriction (TAC) with endothelial deletion of POR accelerates the development of heart failure in mice as detected by a reduction in cardiac output and stroke volume. These effects were mediated most likely by a reduction in vascular EETs production, which increases vascular stiffness, resulting in cardiac remodeling.
Identifizierung potenzieller Taspase1 Inhibitoren für die Behandlung von t(4,11) akuter Leukämie
(2022)
Leukämie ist die häufigste bösartige Krebserkrankung im Kindes- und Jugendalter. Bei einem Kind von 1120 Kindern wird Leukämie diagnostiziert, dabei trifft diese Diagnose Jungen 30 % häufiger als Mädchen. Die Krankheitssymptome treten bei den Kindern noch vor dem Schulalter auf und am häufigsten haben die Kinder mit der akuten Form zu kämpfen. Bei einer Diagnose mit einer akuten lymphatischen Leukämie (ALL) haben die Kinder meist eine gute Prognose, während bei der akuten myeloischen Leukämie (AML) eine deutlich schlechtere.1
Die t(4;11)(q21;q23) Translokation ist aufgrund ihres häufigen Auftretens und der damit schlechten verbundenen Prognose eines der bekanntesten strukturellen Chromosomen-anomalien bei akuten Leukämien. Diese Translokation wurde das erste Mal 1977 von Oshimura et al. beschrieben.2 Bei einer t(4;11)-Translokation ist das Chromosom 4 und das Chromosom 11 involviert. Auf Chromosom 4 ist das AF4-Gen lokalisiert (AFF1) und auf dem Chromosom 11 liegt das MLL-Gen (ALL-1, HRX, hTRX, KMT2A).
Taspase1 wurde als ein proteolytisch prozessierendes Enzym identifiziert, das sich in Wirbellosen und Vertebraten zusammen mit Mitgliedern der Trithorax/MLL/KMT2A-Protein¬familie koevolviert hat. Taspase1 prozessiert nicht nur das MLL und MLL2, deren Fusions¬proteine AF4-MLL, sondern auch den Transkriptionsfaktor IIA (TFIIA) sowohl in vitro als auch in vivo.3
Die Dimerisierung von Taspase1 löst eine intrinsische Serinproteasefunktion aus, die zum katalytischen Rest Thr234 führt, der die Konsensussequenz Q-3X-2D-1•G1X2D3D4 katalysiert, die in Mitgliedern der MLL-Familie sowie im Transkriptionsfaktor TFIIA vorhanden ist. Taspase1 ist kein klassisches Enzym, da es seine Zielproteine stöchiometrisch hydrolysiert. Diese Eigenschaft macht es nahezu unmöglich, in einem klassischen Screening-Setup nach potenziellen Inhibitoren zu screenen.
In dieser Arbeit wurde ein Homogeneous time-resolved fluorescence HTRF-Reporter-Assays etabliert. Das etablierte Testsystem ermöglicht erstmalig die Untersuchung von Substanzen zusammen mit Taspase1 Monomere, die in einem zellfreien System (cfs) hergestellt wurden. Durch die Expression non monomeren Taspase1 Proteinen sollten Inhibitoren durch das etablierte Screening-Verfahren gefunden werden, die sowohl (1) Dimerisierung, (2) Autoaktivierung oder (3) Substratbindung selektiv blockieren können. Die durchgeführten Experimente führten zur Identifikation eines ersten Taspase1-Inhibitors, Closantel sodium. Closantel sodium ist ein U.S. Food and Drug Administration (FDA) zugelassenes Medikament, das Taspase1 auf nicht-kovalente Weise bindet. Die erzielten Daten zeigen, dass Closantel sodium den Dimerisierungsschritt und/oder die intrinsische Serinproteasefunktion blockiert. Closantel sodium hemmte die Spaltung des eingesetzten CS2-Substratproteins mit einem IC50 zwischen 1,6 und 3,9 µM, je nachdem, welches Taspase1-Präparat in dem HTRF Screening Assay ver¬wendeten (cfs- oder E.coli-produziert). Die Daten weisen darauf hin, dass Closantel sodium als allosterischer Inhibitor gegen die Taspase1 fungiert. Taspase1 wird zur Aktivierung der AF4-MLL-Onkofusionsproteine benötigt und wird auch in mehreren soliden Tumoren überexprimiert. Daher könnte dieser neue Inhibitor für die weitere Validierung von Taspase1 als Ziel für die Krebstherapie und für das Design potenterer Liganden für zukünftige klinische Anwendungen nützlich sein.
Standard cancer therapy research targets tumor cells while not considering the damage on the tumor microenvironment (TME) and its associated implications in impairing therapy response. Employing patients-derived organoids (PDOs) and matched stroma cells or a novel murine preclinical rectal cancer model of local radiotherapy, it was demonstrated that tumor cells-derived IL-1α polarizes cancer-associated fibroblasts towards an inflammatory (iCAFs) phenotype. While numerous studies in different tumor entities highlighted the molecular heterogeneity of CAFs, so far there are no clear findings on their functional heterogeneity and relevance in therapy resistance and response. The present study molecularly characterized iCAFs subpopulation among RCA patients as well as the preclinical mouse model and importantly unraveled the detailed molecular mechanism underlying their contribution to impair therapy response. Mechanistically, iCAFs were demonstrated to be characterized by an upregulation of nitric oxide synthase (iNOS) which triggered accumulation of reactive nitrogen species (RNS) and subsequently an oxidative DNA damage response (DDR). Such a baseline IL-1α-driven DNA damage further sensitized iCAFs to a p53-mediated therapy induced senescence (TIS) causing extensive extracellular matrix (ECM) changes and induction of senescence associated secretory phenotype (SASP) that favored tumor progression and hindered tumor cell death. Moreover, iCAFs reversibility and repolarization into more quiescent like phenotype was demonstrated upon IL-1 signaling inhibition by anakinra, a recombinant IL-1 receptor antagonist (IL1RA). Accordingly, treating mice with anakinra or specific deletion of Il1r1 in CAFs sensitized stroma-rich resistant tumors to chemoradiotherapy (CRT). Similarly, targeting CAFs senescence by senotherapy (venetoclax chemical) or employing Trp53 deficient mice reverted therapy resistance among non-responsive tumors in vivo by reducing ECM deposition and consequently favoring CD8+ T cells intratumoral infiltration posttherapy. Importantly, rectal cancer patients that do not completely respond to neoadjuvant therapy displayed an iCAFs senescence program post-CRT. Moreover, these patients presented a baseline increased CAFs content, a dominant iCAFs signature that correlated with poorer disease-free survival (DFS) and a significantly reduced circulating IL1RA serum levels. While reduced pretherapeutic IL1RN gene expression predicted poor prognosis among RCA patients, IL1RA serum levels were associated with rs4251961 (T/C) single nucleotide polymorphism (SNP) in the IL1RN gene. Finally, functional validation assays revealed that conditioned media of PDOs drove inflammatory polarization of fibroblasts and consequently rendered them sensitive to RNS-mediated DNA damage and TIS. Collectively, the study highlighted a crucial and novel role of a CAFs subset, iCAFs, in therapy resistance among RCA patients, shedding light on their functional relevance by identifying IL-1 signaling as an appealing target for their repolarization and successful targeting. Therefore, it makes sense to combine the newly demonstrated and thoroughly proven therapeutic approach of targeting IL-1 signaling in combination with conventional CRT and possibly immunotherapy. This might have a major impact on RCA therapy and be of immense relevance for other stroma-rich tumors.
The majority of B-cell precursor acute leukemias in infants are associated with the chromosomal translocation t(4;11)(q21;q23), resulting in the fusion of the mixed-lineage leukemia (MLL) and ALL1-fused gene of chromosome 4 (AF4) genes. While the fusion protein MLL-AF4 is expressed in all t(4;11) patients and essential for leukemia progression, the distinct role of the reciprocal fusion protein AF4-MLL, that is expressed in only 50-80% of t(4;11) leukemia patients (Meyer et al., 2018), remains unclear. In addition, t(4;11) leukemia could so far exclusively be generated in vivo in the presence of AF4-MLL and independent of the co-expression of MLL-AF4 (Bursen et al., 2010).
In a multifactorial approach inhibiting histone deacetylases (HDACs) and expressing the dominant negative mutation of Taspase1 (dnTASP1), both MLL fusion proteins were targeted simultaneously to evaluate a possible cooperative effect between MLL-AF4 and AF4-MLL during the progression of leukemia. Of note, neither HDACi nor dnTASP1 expression negatively affect endogenous MLL, but rather endorse its function hampered by the MLL fusion proteins (Ahmad et al., 2014; Bursen et al., 2004; Zhao et al., 2019). The mere expression of dnTASP1 failed to induce apoptosis, whereas dnTASP1 could elevate apoptosis levels significantly in HDACi-treated t(4;11) cells underlining the therapeutic potential of co-inhibiting both MLL fusion proteins.
Next, the impact of inhibiting either MLL-AF4 or AF4-MLL in vivo was resolved using whole transcriptome analysis. In PDX cells obtained by the Jeremias Laboratory (Völse, 2020) that co-expressed both t(4;11) fusion proteins, the knock-down of MLL-AF4 revealed the down-regulation of pivotal hemato-malignant factors. The expression of dnTASP1 led to massive deregulation of cell-cycle genes in vivo. Considering that the inhibition of particularly MLL-AF4 but not AF4-MLL impaired leukemic cell growth in vivo (Völse, 2020), the results of this work suggest a cooperative effect between both fusion proteins, while the loss of AF4-MLL during leukemia progression appears not essential.
Thereafter, a possible short-term role of AF4-MLL during the establishment of t(4;11) leukemia was analyzed. For this purpose, an in vitro t(4;11) model was constructed to investigate the transforming potential of transiently expressed AF4-MLL in cells constitutively expressing MLL-AF4, putatively reflecting the situation in vivo. Due to the lack of a leukemic background of the applied cell line, the aim was to investigate the long-term potential of AF4-MLL to significantly alter the epigenome rather than mimicking the development of leukemia. Strikingly, short-term-expressed AF4-MLL in cooperation with MLL-AF4 exerted durable epigenetic effects on gene transcription and chromatin accessibility. The here obtained in vitro data suggest a clonal evolutionary process initiated by AF4-MLL in a cooperative manner with MLL-AF4. Importantly, no long-term changes in chromatin accessibility could be observed by the transient expression of either MLL-AF4 or AF4-MLL alone.
All in all, considering endogenous MLL, MLL-AF4 and AF4-MLL in a targeted treatment is a promising approach for a more tailored therapy against t(4;11) leukemia, and AF4-MLL is suggested to act in a cooperative manner with MLL-AF4 especially during the development of a t(4;11) leukemia.
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in early childhood. Despite recent advances in the treatment regimes of rhabdomyosarcoma, the 5-year survival is still alarmingly low for the more aggressive metastasizing alveolar rhabdomyosarcoma subtype. Novel treatment strategies are needed in order to increase the overall survival rate. Hallmarks of cancer include evade cell death induction and evade immune system surveillance. This is mediated in part by up-regulation of inhibitor of apoptosis (IAP) proteins. With the development of Smac mimetic compounds mimicking the endogenous IAP antagonist Smac, this tumor evasion mechanism became exploitable.
In this PhD thesis, a combinatory approach for a putative treatment option of RMS will be presented. Here, the Smac mimetic compound BV6 will be used as a pre-treatment of RMS cells. This leads to a sensitizing effect within the tumor cells, increasing the killing efficacy of natural killer (NK) cells.
Subtoxic concentrations of BV6 were chosen to sensitize RMS cells. To remodel the solid tumor characteristics of RMS, a multicellular RMS tumor spheroid culture model was used.
In both tumor spheroids and conventional monolayer cell culture BV6 induced the degradation of IAP proteins (cIAP1, cIAP2, in spheroids XIAP). Further, BV6 led to the activation of both, the canonical and non-canonical NF-κB signaling pathways.
This was demonstrated by an increased IκBα and p65 phosphorylation, and nuclear translocation of p-p65, indicative for an active canonical NF-κB signaling. On the other side, cIAP degradation led to the stabilization and accumulation of NIK and downstream partial degradation of p100 to p52 and its nuclear translocation, indicating non-canonical NF-κB signaling pathway activity. A bulk RNA sequencing approach of BV6 treated RH30 cells validated the NF-κB signaling involvement and identified 182 differentially expressed genes. Among the interesting target genes are NFKBIA (IκBα),BIRC3 (cIAP2), NFKB2 (p100), CCL5 and SSTR2. SSTR2 was thoroughly validated as being up-regulated on a transcriptional and on protein level. Here, SSTR2A, one of the two alternative splicing variants, is up-regulated and opens a hypothetical targeted treatment strategy, as SSTR2 expression is not associated with RMS, but rather described with neuroendocrine tumor entities. In addition, CCL5 was thoroughly validated as a BV6 induced target. Again, the up-regulated mRNA transcription was validated by an increased translation and by increased secretion of CCL5. As CCL5 being associated as pro-migratory and activating of NK cells, CRISPR/Cas9 mediated CCL5 knock-out studies were performed to evaluate the influence of CCL5 within a BV6 pre-treatment and NK cell co-cultivation setting. It was shown that CCL5 knock-out does not rescue BV6 pre-treated RMS spheroids from NK cell attack and killing.
The previous mentioned transcriptional activity by BV6 stimulation was NIK mediated as knock-down of NIK reduced the mRNA transcription of several interesting genes.
However, NIK mediated down-stream signaling had no influence on the BV6 induced sensitizing effect towards NK cell mediated attack. A NIK knock-down had no rescue effect upon BV6 pre-treatment and NK cell co-treatment.
As cIAP proteins are present in receptor bound complexes, e.g. complex I at the TNF receptor 1 (TNFR1), a putative involvement of death receptors in general was evaluated.
Indeed, BV6 treatment of RMS cells could increase the surface presentation of DR5, a death receptor ligating TRAIL. Functionally, co-treatment of BV6 with TRAIL led to an additive cell death inducting effect. However, within the NK cell co-cultivation setting, addition of a neutralizing TRAIL anitbody could not rescue BV6 pre-treated RMS spheroids from NK cell killing. A similar effect was observed when neutralizing TNFα by adding Enbrel during the NK cell co-cultivation. BV6 sensitization of RMS spheroids seems to be independent of death receptors.
In addition to activating NF-κB, BV6 as a Smac mimetic is supposed to be able to release caspases bound by IAP proteins. Indeed, BV6 pre-treatment of RMS spheroids and co-cultivation with NK cells could cleave and thereby activate the executioner caspase-3. Further, treatment with a pan-caspase inhibitor, zVAD.fmk, could reduce the BV6 mediated sensitizing effect towards NK cell attack in RD spheroids.
Taken together, BV6 does induce a thoroughly validated NF-κB signaling pathway, leading to a NIK mediated transcriptional signature change. However, the NF-κB activation might not be responsible for the observed sensitization. Further, BV6 in combination with NK cells led to a seemingly death receptor independent, caspase dependent cell death induction of RMS spheroids. Although the mechanism remains partially con-cealed, a therapeutic benefit by combining a cell death sensitizing compound, i.e. BV6, with cytotoxic lymphocytes is evident.
KMT2A-rearrangements are causative for 70-80% all infant acute lymphoblastic leukemias (Pieters et al., 2019, 2007). Among these, the translocation t(4;11)(q21;23) generating the oncogenic fusion genes KMT2A::AFF1 and AFF1::KMT2A is the most frequent one, accounting for almost every second case of KMT2A-r infant ALL (Meyer et al., 2018). Despite passing a multimodal chemotherapy, 64% of patients achieve an event including relapse or death within four years from diagnosis, and overall survival three years from relapse remains poor with only 17% (Driessen et al., 2016; Pieters et al., 2019, 2007). Vari-ous studies have shown that relapse and therapy resistance were not mediated by chemotherapy-induced mutagenesis as there was no accumulation of secondary mutations in the dominant leukemic clone between diagnosis and relapse (Agraz-Doblas et al., 2019; Andersson et al., 2015; Bardini et al., 2011; Dobbins et al., 2013; Driessen et al., 2013; Mullighan et al., 2007).
Intriguingly, exclusively infant t(4;11) ALL patients were reported to subdivide in two groups depending on the level of HOXA gene cluster expression (Trentin et al., 2009). The HOXAlo group displayed a high expression of IRX1 and the HOXAhi group a low expression of IRX1 (Symeonidou and Ottersbach, 2021; Trentin et al., 2009). Importantly, the HOXAlo/IRX1hi group was characterized to possess a strongly ele-vated relapse incidence compared to the HOXAhi/IRX1lo group (Kang et al., 2012; Stam et al., 2010). IRX1 was identified to upregulate the Early growth response genes EGR1, EGR2 and EGR3 (Kühn et al., 2016).
The doctoral project “EGR-mediated relapse mechanisms in infant t(4;11) acute lymphoblastic leuke-mia” aimed to investigate a potential correlation between the HOXAlo-IRX1-EGR axis and relapse development in infant t(4;11) ALL. The primary objective was to clarify through which molecular mechanism(s) relapse development despite continuous chemotherapy could be achieved. In this context, the role of the EGR genes has been investigated. In addition, this project aimed to disclose molecular targets which could offer novel therapeutic interventions to interfere with therapy resistance and relapse formation.
The vascular endothelium is a monolayer of endothelial cells that builds the inner lining of the blood vessels and constitutes a regulatory organ within the physiological system to sustain homeostasis. Endothelial cells participate in physiological processes including inflammation and angiogenesis. Dysregulation of these processes, however, can evoke or maintain pathological disorders, including cardiovascular and chronic inflammatory diseases or cancer. Although pathological inflammation and angiogenesis represent treatable conditions, current pharmacotherapeutic approaches are frequently not satisfying since their long-term application can evoke therapy resistance and thus reduced clinical efficacy. Consequently, there is an ongoing demand for the discovery of new therapeutic targets and drug leads. Considering that endothelial cells play a critical role in both angiogenesis and inflammation, the vascular endothelium represents a promising target for the treatment of diseases.
Vioprolide A is a secondary metabolite isolated from the myxobacterium Cystobacter violaceus Cb. vi35. Recently, vioprolide A was identified to interact with NOP14, a nucleolar protein involved in ribosome biogenesis. Ribosome biogenesis is an indispensable cellular event that ensures adequate homeostasis. Abnormal alterations in the ribosome biogenesis, referred to as ribosomopathies, however, can lead to an overall increase in the risk of developing cancer. Accordingly, several studies have outlined the involvement of NOP14 in cancer progression and metastasis, and vioprolide A has been demonstrated to exert anti-cancer effects in vitro. However, the impact of vioprolide A and NOP14 on the endothelium has been neglected so far, although endothelial cells are crucially involved in inflammation and angiogenesis under both physiological and pathological conditions.
In the present study, the effect of vioprolide A on inflammatory and angiogenic actions was analysed. In vivo, the laser-induced choroidal neovascularization (CNV) assay outlined a strong inhibitory effect of vioprolide A on both inflammation and angiogenesis. Furthermore, intravital microscopy of the cremaster muscle in mice revealed that vioprolide A strongly impaired the TNF-induced leukocyte-endothelial cell interaction in vivo.
In further experiments, the specific effect of vioprolide A on activation processes of primary human umbilical vein endothelial cells (HUVECs) was examined. According to the in vivo results, vioprolide A decreased the leukocyte-endothelial cell interaction in vitro through downregulating the cell surface expression and total protein expression of ICAM-1, VCAM-1 and E-selectin. Vioprolide A evoked its anti-inflammatory actions via a dual mechanism: On the one hand, the expression of pro-inflammatory proteins, including TNFR1 and cell adhesion molecules, was lowered through a general downregulation of de novo protein synthesis. The inhibition of de novo protein synthesis is most likely linked to the interaction with and inhibition of NOP14 by vioprolide A in HUVECs. On the other hand, the natural product prevented the nuclear translocation and promotor activity of the pro-inflammatory transcription factor NF-ĸB. Interestingly, most anti-inflammatory compounds that interfere with the NF-ĸB signaling pathway prevent NF-ĸB nuclear translocation through recovering or stabilizing the inhibitory IĸB proteins. Vioprolide A, however, decreased rather than stabilized the IĸB proteins and prevented NF-ĸB nuclear translocation through interfering with its importin-dependent nuclear import. By performing siRNA-mediated knockdown experiments, we evaluated the role of NOP14 in inflammatory processes in HUVECs and could establish a causal link between the anti-inflammatory actions of vioprolide A and the deletion of NOP14.
Besides exerting anti-inflammatory actions, we found that vioprolide A potently decreased the angiogenic key features proliferation, migration and sprouting of endothelial cells. Mechanistically, the natural product interfered with pro-angiogenic signaling pathways. Vioprolide A reduced the protein level of growth factor receptors, including VEGFR2, which is the most prominent receptor responsible for angiogenic signaling in endothelial cells. This effect was based on the general inhibition of de novo protein synthesis by the natural product. Downregulation of growth factor receptors impaired the activation of downstream signaling intermediates, including the MAPKs ERK, JNK and p38. To our surprise, however, activation of Akt, another downstream effector of VEGFR2, was increased rather than decreased. Furthermore, vioprolide A lowered the nuclear translocation of the transcriptional coactivator TAZ, which is regulated by the evolutionary conserved Hippo signaling pathway. Interestingly, however, and in contrast to NF-ĸB, TAZ nuclear translocation in mammalian cells seems to be independent of importins. In this context, we found that vioprolide A reduced both the protein level and nuclear localization of MAML1, which is needed to retain TAZ in the nucleus after its successful translocation.
...
Während hohe Spiegel von reaktiven Sauerstoffspezies (reactive oxygen species, ROS) in Form von oxidativem Stress schädliche Auswirkungen auf den Körper haben können, zeigen aktuelle Forschungsarbeiten, dass Redox-Modifikationen an Thiolresten von Proteinen reversible Signalprozesse steuern können. Dieses Prinzip der posttranslationalen Proteinmodifikation durch Redox-Signale scheint auch bei der Verarbeitung und Chronifizierung von Schmerzen von Bedeutung zu sein. Über die potenziellen Redox-modulierten Zielstrukturen im nozizeptiven System ist jedoch bisher nur wenig bekannt.
Ein potentielles Redoxtarget im nozizeptiven System ist das kleine EF-Hand Ca2+-bindende Protein S100A4. Wie die anderen Familienmitglieder der S100-Proteinfamilie enthält S100A4 Cysteinreste, die in der Lage sind, redoxabhängig modifiziert zu werden. Studien an menschlichen Biopsien nach Gehirnverletzungen und an Mäusen in Verletzungsmodellen konnten zeigen, dass S100A4 neuroprotektiv wirkt. Darüber hinaus kann S100A4 sezerniert werden und vermittelt extrazellulär insbesondere regulatorische Funktionen innerhalb der Angiogenese, bei der Zellmigration sowie bei zellulären Differenzierungsprozessen. Die Funktionen von S100A4 im nozizeptiven System sind jedoch weitgehend unbekannt. In Vorarbeiten zu diesem Projekt wurde in einem Proteom-Screen beobachtet, dass S100A4 nach einer peripheren Nervenverletzung redoxabhängig im verletzten Nervengewebe hochreguliert wird. Darauf basierend wurde im Rahmen dieser Arbeit die Lokalisation von S100A4 innerhalb des nozizeptiven Systems sowie die funktionelle Bedeutung nach peripherer Nervenverletzung genauer untersucht.
Anhand von Immunfluoreszenzaufnahmen konnte gezeigt werden, dass S100A4 basal in Subpopulationen Peripherin- und NF200-positiver sensorischer Neurone lokalisiert ist. Interessanterweise führt eine Nervenverletzung nicht nur zu einer deutlichen Steigerung der S100A4-Expression im Bereich der Verletzungsstelle, sondern auch zu einer Änderung des neuronalen Verteilungsmusters. Die funktionelle Bedeutung von S100A4 für die Verarbeitung von Schmerzen wurde anhand von Verhaltenstests an Mäusen näher charakterisiert. Dafür wurden gewebsspezifische S100A4 Knockout Mäuse (Adv-S100A4-/-) und globale S100A4 Knockout Mäuse (S100A4-/-) generiert. In Modellen der akuten Nozizeption zeigten sowohl Adv-S100A4-/- als auch S100A4-/- Mäuse eine normale Reaktion auf thermische und mechanische Stimuli. Im „Spared Nerve Injury“ (SNI) Modell für periphere Neuropathien zeigten die S100A4-/- Mäuse eine im Vergleich zu wildtypischen (WT) Mäusen signifikant reduzierte mechanische Hyperalgesie, während bei den gewebsspezifischen Adv-S100A4-/- Mäusen kein verändertes Schmerzverhalten beobachtet werden konnte. Im „Crush Injury“ Modell für periphere Neuropathien war die mechanische Hyperalgesie der S100A4-/- Mäuse im Vergleich zu WT Tieren jedoch nicht verändert. Zusätzlich zur mechanischen Hyperalgesie wurden auch weitere Methoden der Quantifizierung des Schmerzverhaltens (Sciatic Functional Index, Brush Test und Wühlverhalten) etabliert. Allerdings war auch hier das Verhalten der S100A4-/- Mäuse mit dem der WT Mäuse vergleichbar. Darüber hinaus war das durch Applikation eines ROS-Donors induzierte nozizeptive Verhalten von S100A4-/- und WT Mäusen ähnlich. Man kann daher schlussfolgern, dass nach einer peripheren Nervenverletzung die S100A4-Expression insbesondere im Bereich der Verletzungsstelle hochreguliert wird. Dem gegenüber scheint S100A4 jedoch für die Schmerzverarbeitung funktionell nur von untergeordneter Bedeutung zu sein.
Ein weiteres potentielles Redoxtarget im nozizeptiven System ist die lösliche Epoxidhydrolase (soluble epoxide hydrolase, sEH). Die funktionelle Bedeutung von sEH für die Schmerzverarbeitung wurde bereits in früheren Studien belegt, da eine Behandlung mit sEH-Inhibitoren bei Ratten zu einer reduzierten Hypersensitivität in inflammatorischen und neuropathischen Schmerzmodellen führte. Während die analgetische Wirkung von sEH-Inhibitoren bereits gut bekannt ist, wurde eine redoxabhängige Modulation der sEH-Aktivität im nozizeptiven System in bisherigen Forschungsarbeiten kaum untersucht. Bestimmte Elektrophile können die sEH inhibieren, indem sie an das redoxaktive Cystein an Position 521 der sEH binden. Forschungsarbeiten konnten in diesem Zusammenhang bereits zeigen, dass die Cys521-vermittelte Inhibition von sEH durch das Prostaglandin 15d-PGJ2 oder 9-/10-Nitrooleonsäure (NO2-OA) im kardiovaskulären System zu einer Dilatation der Koronargefäße und einer Reduktion des Blutdrucks führt. Im Rahmen dieser Arbeit wurde untersucht, ob es durch eine redoxabhängige Hemmung der sEH-Funktion auch innerhalb des nozizeptiven Systems zu einer veränderten Schmerzreaktion bei Mäusen kommt. Um diese Fragestellung beantworten zu können, wurden sEH-Knockin (sEH-KI) Mäuse verwendet, deren redox-sensitives Cystein 521 durch ein Serin ersetzt wurde. Bei diesen Knockin-Mäusen können Elektrophile wie 15d-PGJ2 oder 9-/10-NO2-OA keine Enzyminhibition erzeugen. Die Charakterisierung der sEH-KI Mäuse zeigte sowohl in akuten als auch inflammatorischen Schmerzmodellen (Formalin Test und Zymosan-Pfotenentzündungsmodell) keinen Zusammenhang der Redoxmodifikation mit dem Schmerzverhalten der Mäuse. Auch in neuropathischen und viszeralen Schmerzmodellen (SNI-Modell und Modell der Zymosan-induzierten Peritonitis) konnte kein verändertes Schmerzverhalten der sEH-KI-Mäuse im Vergleich zu Kontrolltieren beobachtet werden. Darüber hinaus war das nozizpetive Verhalten nach Applikation von 15d-PGJ2 bei sEH-KI und WT Mäusen vergleichbar. Die redoxabhängige Modulation der sEH an Cystein 521 scheint demnach, im Gegensatz zum kardiovaskulären System, im nozizeptiven System keine Rolle zu spielen.
Investigating the inhibition of anti-apoptotic BCL-2 family proteins in pediatric cancer cells
(2020)
Cancer is amongst the leading causes of death in childhood. Rhabdomyosarcoma (RMS) is the most frequently occurring soft tissue sarcoma in children and adolescents. It presumably arises from mesenchymal progenitors of skeletal muscle cells and presents with different subtypes that differ both histologically and genetically. Osteosarcoma (OS) and Ewing sarcoma (ES) are the most frequently diagnosed pediatric bone tumors. Even though the prognosis of these cancer entities improved significantly during recent decades, the survival rates are currently stagnating. Especially, dismal prognosis of relapsed and metastasizing cases of these malignancies urgently call for novel treatment options. BCL-2 proteins are vital guardians that control intrinsic apoptosis. Furthermore, it was shown that BCL-2 proteins critically regulate apoptosis in pediatric solid tumors. BH3 mimetics are small molecules that bind and inhibit anti-apoptotic BCL-2 proteins. They have already been investigated as cancer therapeutics for several years and show first encouraging clinical results. Therefore, we hypothesized that targeting BCL-2, MCL-1 and BCL-XL might be a promising approach to treat RMS, OS and ES.
In this study, we aimed to comprehensively evaluate the potential of anti-apoptotic BCL-2 family proteins as therapeutic targets for pediatric solid tumors such as RMS, OS and ES.
Notably, RMS, OS and ES cells largely expressed the most relevant BCL-2 family protein members. However, cells were widely insensitive to single pharmacological inhibition of either BCL-XL, BCL-2 or MCL-1 by A-1331852, ABT-199 and S63845, respectively. This finding was independent of their BCL-2 family protein expression levels. Significantly, co-administration of A-1331852 and S63845 induced cell death in RMS, OS and ES cell lines in a highly synergistic manner. Transient silencing of MCL-1 and/or BCL-XL verified the co-dependency of RMS cells on these proteins for survival. Importantly, A-1331852/S63845 co-treatment was more efficient in causing cell death in RMS, OS and ES cells than either inhibitor combined with ABT-199. Efficacy of A-1331852/S63845 co-treatment could be additionally demonstrated in a primary sample of pediatric malignant epithelioid mesothelioma.
Mechanistically, concomitant A-1331852/S63845 treatment mediated rapid intrinsic apoptosis involving swift loss of the mitochondrial outer membrane potential as well as activation of caspases-3, -8 and -9. An observed caspase dependent loss of MCL-1 might further amplify the A-1331852/S63845 triggered pro-death signaling. Furthermore, we identified BAX and BAK as key mediators of apoptosis caused by dual inhibition of MCL-1 and BCL-XL. A-1331852/S63845 induced cell death was relying on BAX and/or BAK in a cell line dependent manner. Interestingly, treatment with A-1331852 and S63845 liberated BAK from its interaction with MCL-1 and BCL-XL. Moreover, BAX and BAK were activated and interacted with each other to form a pore in the outer mitochondrial membrane. Further, in RD cells BIM and NOXA partially contributed to A-1331852/S63845 mediated cell death. Consistently, in this cell line BIM and NOXA were disrupted from their binding to BCL-XL and MCL-1 by A-1331852 and S63845, respectively. However, BH3 only proteins were not involved in A-1331852/S63845 induced cell death in Kym-1 cells. Therefore, we concluded that BH3 only proteins played only a marginal and cell line dependent role in mediating cell death caused by MCL-1 and BCL-XL co-repression.
Notably, A-1331852/S63845 co-treatment spared non-malignant fibroblasts, myoblasts and peripheral blood mononuclear cells, which suggests a therapeutic window for its application in vivo. Besides, we could demonstrate that sequential BH3 mimetic treatment still significantly induced cell death, albeit to minor extents compared to its dual administration. Importantly, we successfully evaluated concomitant treatment with A-1331852 and S63845 in multicellular RMS spheroids and in an in vivo embryonic chicken model of RMS. These findings stress the high transcriptional relevance of A-1331852/S63845 as an emerging novel cancer regimen.
Collectively, the thesis at hand explored the great potential of co-treatment with A-1331852 and S63845 in pediatric solid tumors and unveiled the underlying molecular mechanisms of cell death in RMS. Together, the current investigations support further preclinical and clinical studies to evaluate the effect of dual MCL-1 and BCL-XL targeting in pediatric solid tumors.
Chronic inflammation is considered to be a cause of the autoimmune diseases such as rheumatoid arthritis, Alzheimer’s disease, multiple sclerosis, etc. The search for effective compounds with anti-inflammatory properties to combat these diseases is still ongoing. Natural compound narciclasine, derived from plants of Narcissus species, demonstrated its anti-inflammatory activity in in vivo arthritis models. Further investigation of narciclasine’s anti-inflammatory activity together with its impact on the interaction between leukocytes and endothelial cells was the main focus of this PhD thesis.
Narciclasine reduced the infiltration of monocytes and neutrophils to the abdomen and the concentration of the pro-inflammatory cytokines TNF, IL-6 and IL-1β. Together with this, it reduced acute visceral pain caused by zymosan injection. Narciclasine interfered with leukocyte-endothelial cell interaction in both in vivo and in vitro models. In vivo microscopy revealed that the compound reduced rolling, adhesion and transmigration of leukocytes in the vessels of an injured murine cremaster muscle. This observation was confirmed in the in vitro models for adhesion and transmigration where narciclasine reduced the level of leukocyte’s interaction with HUVECs. Narciclasine demonstrated profound anti-inflammatory properties based on its interference with leukocyte-endothelium interaction by downregulation of endothelial cell adhesion molecules expression (ICAM-1, VCAM-1, E-selectin, CX3CL1) and shutdown of NF-κB pathway. All these effects were a result of the TNF receptor 1 protein translation blocking by narciclasine.
In this work the ability of the compound to reduce visceral pain, downregulate the expression of the endothelial cell adhesion molecules and to interfere with the interaction between leukocytes and endothelial cells was demonstrated for narciclasine for the first time. Obtained results open a promising insight into the understanding of narciclasine’s anti-inflammatory properties and justify further investigation of its potential for treatment of inflammatory diseases.
In the recent years, myxobacteria have emerged as a novel source of natural compounds with structural diversity and biological activity for drug discovery. In this work, the two myxobacterial compounds archazolid and vioprolide were characterized for their potential pharmacological effects in vascular endothelial cells. Archazolid is a wellestablished v-ATPase inhibitor found in Archangium gephyra and Cystobacter spec. As the v-ATPase represents a promising target in cancer treatment, the effects of archazolid have been intensively studied in cancer cells, but rarely in endothelial cells. Vioprolide is an antifungal and cytotoxic metabolite obtained from Cystobacter violaceus. There are only few studies on vioprolide, most of them focusing on its biosynthesis. Preliminary studies revealed that it inhibited TNF-induced expression of ICAM-1, indicating possible anti-inflammatory properties. As the endothelium plays an important role in cancer and inflammation, it represents an attractive drug target. Therefore, the archazolid and vioprolide were investigated regarding their effects on endothelial cells.
V-ATPase inhibition by archazolid resulted in anti-tumor and anti-metastatic effects in vitro and in vivo. Archazolid was used to study the consequences of v-ATPase inhibition in endothelial cells that might contribute to the anti-metastatic activities observed in vivo. To analyze the impact of archazolid on the interaction endothelial and cancer cells, in vitro cell adhesion and transmigration assays were performed using primary HUVEC or immortalized HMEC-1 and different cancer cell types (MDA-MB-231, PC-3 and Jurkat cells). For these experiments, only the endothelial cells were treated with archazolid. VATPase inhibition by archazolid led to an increased adhesion of the metastatic breast cancer cell line MDA-MB-231 and prostate cancer cell line PC-3 onto endothelial cells whereas the adhesion of Jurkat cells was unaffected. Interestingly, archazolid treatment of HUVECs decreased the transendothelial migration of MDA-MB-231 cells. Endothelial ICAM-1, VCAM-1, E-selectin and N-cadherin are potential ligands of interacting cancer cells. Therefore, the mRNA and surface protein levels of these cell adhesion molecules were measured via qRT-PCR and flow cytometry, respectively. These adhesion molecules were not responsible for the archazolid-induced cancer cell adhesion, as archazolid treatment of HUVECs did not upregulate their mRNA or surface expression. Instead, cell adhesion assays using a monoclonal antibody against integrin subunit β1 showed that β1-integrins expressed on MDA-MB-231 and PC-3 cells mediated the archazolid-induced cancer cell adhesion. Cell adhesion assays onto plastic coated with ECM components which are the major ligands of β1-integrins, revealed that MDA-MB231 and PC-3 cells preferably interact with collagen. So next, we investigated the influence of archazolid on surface collagen levels in HUVECs by immunostaining, which demonstrated an increase of nearly 50 % upon archazolid treatment. We confirmed the hypothesis that the expression and activity of cathepsin B, a lysosomal enzyme that degrades extracellular matrix components including collagen, was inhibited by archazolid in endothelial cells. Finally, overexpression of cathepsin B reduced the cancer cell adhesion on archazolid-treated HUVECs, but also in control cells, indicating a negative correlation between cathepsin B expression and cancer cell adhesion.
The influence of vioprolide on the interaction of endothelial cells with leukocytes was analyzed by in vitro cell adhesion assays using HUVECs and primary monocytes, THP-1 or Jurkat cells. Vioprolide inhibited the adhesion of these cells onto TNF-activated HUVECs. In addition, the endothelial-leukocyte interaction was observed in vivo by intravital microscopy in the mouse cremaster muscle. Vioprolide prevented the TNFinduced firm adhesion and transmigration of leukocytes, while leukocyte rolling was not affected. ICAM-1, VCAM-1 and E-selectin are cell adhesion molecules, which are upregulated by TNF and mediate leukocyte adhesion onto endothelial cells. Therefore, flow cytometric analysis was performed to measure their surface expression. Vioprolide significantly decreased TNF-induced expression of surface ICAM-1, VCAM-1 and E-selectin, which was in line with the in vitro results. In vivo, vioprolide may act in a different way on E-selectin expression, so that leukocyte rolling, which is governed by E-selectin, remained unaffected. qRT-PCR experiments revealed that the mRNA expression of ICAM-1 and VCAM-1 were also reduced by vioprolide, indicating a regulation on transcriptional level. In contrast, the mRNA expression of E-selectin was not decreased at the timepoint when surface protein expression was diminished. The induction of these cell adhesion molecules is mainly mediated by the transcription factor NFκB. A Dual-Luciferase® reporter assay was used to study the impact of vioprolide on the TNF-induced NFκB promotor activity. Vioprolide blocked the TNF-induced NFκB promotor activity while the TNF-induced IκBα degradation and nuclear translocation of the NFκB subunit p65 was not altered by vioprolide. Western blot analysis revealed that vioprolide had no effect on the activation of MAPK (p38, JNK) and AKT by TNF, which could interfere with the NFκB-dependent gene expression.
Taken together, archazolid and vioprolide are interesting myxobacterial compounds with different modes of actions. The study suggests that the v-ATPase inhibitor archazolid impairs the expression and activity of cathepsin B in endothelial cells, which leads to a higher amount of collagen on the endothelial surface. As a result, the adhesion of β1-integrin expressing metastatic cancer cells onto archazolid-treated endothelial cells increased while transendothelial migration was reduced. Further, archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Vioprolide was able to prevent TNF-induced endothelial-leukocyte interaction in vitro and in vivo by interfering with NFκB-dependent gene expression. Further research is required to enlighten the underlying mechanism and the direct target of vioprolide.
Lange ging man davon aus, dass die Physiologie der Thyroidhormone weitestgehend erforscht ist und nahm an, dass sämtliche Thyroidhormon-Wirkungen auf einer Bildung von L-Thyroxin (T4) und einer anschließenden Deiodierung zu Triiodthyronin (T3) beruhen, welches an die nukleären Thyroidhormon Rezeptoren (THRs) bindet. Über die THRs werden genomische Signalwege vermittelt, die während der Wachstums- und Entwicklungsphase essentiell sind. Beim Erwachsenen werden zudem vorwiegend katabole Stoffwechsel-Prozesse induziert. Jedoch zeigte sich in den letzten 20 Jahren, dass die Signalwege der Thyroidhormone komplexer sind als bisher angenommen. Vor allem die Metabolite des in der Schilddrüse gebildeten T4s, zeigen ein breites Interaktions-Profil mit anderen molekularen Zielstrukturen. Thyronamine, die decarboxylierten Thyroidhormon-Metabolite, binden beispielsweise den G-Protein-gekoppelten Trace Amine Associated Receptor 1 (TAAR1). Wird dieser Rezeptor aktiviert, kommt es innerhalb kürzester Zeit zu einem rapiden Abfall der Köpertemperatur, sowie zu einer akuten Bradykardie. Die durch oxidative Deaminierung gebildeten Iodthyroacetate Tetraiodthyroacetat (TETRAC) und Triiodthyroacetat (TRIAC) sind Antagonisten des Membran-Rezeptors Integrin αVβ3 und besitzen antiproliferative und pro-apoptotische Eigenschaften.
In dieser Arbeit sollte die Hypothese untersucht werden, ob Thyroidhormone neben diesen neuen zumeist nicht-genomischen Signalwegen, auch THR-unabhängige genomische Wirkmechanismen besitzen.
Mit Hilfe eines Gal4-Luciferase-Reportergen-Assays wurde in einem Screening die Aktivität einiger Thyroidhormone und Thyroidhormon-Metabolite an elf THR-ähnlichen Rezeptoren und den drei Retinoid X Rezeptor (RXR)-Subtypen untersucht. Es konnte detektiert werden, dass Thyroidhormone, vor allem TETRAC, potente Peroxisom-Proliferator-aktivierter Rezeptor (PPAR)γ-Agonisten sind, die zum Teil zusätzlich dessen Heterodimer-Partner RXR aktivieren können. Diese PPARγ- und RXR-Aktivität wurde zunächst mit Hilfe eines Coaktivator-Rekrutierungs-Assays, einer Isothermen Titrationskalorimetrie (ITC) und einer Kristallstrukturanalyse genauer charakterisiert. Zum einen konnte nachgewiesen werden, dass sowohl PPARγ, als auch RXR in artifizielleren Testsystemen durch Thyroidhormone aktiviert werden. Zum anderen konnte die für permissive Heterodimere, wie das PPARγ/RXR-Heterodimer, typische additive Transaktivierungs-Effizienz nach Bindung beider Heterodimer-Partner bestätigt werden. Außerdem zeigte die Untersuchung der Kristallstruktur von TETRAC und PPARγ, dass Thyroidhormone einen abweichenden Bindungsmodus im Vergleich zu anderen PPARγ Agonisten, wie den Glitazonen und entsprechende Fettsäuren oder Fettsäuremimetika, besitzen.
Die Evaluation der biologischen Relevanz der PPARγ/RXR-Heterodimer-Aktivierung ergab zudem, dass TETRAC, als potentester PPARγ-Agonist, in der Lage ist die Differenzierung von Präadipocyten zu Adipocyten zu induzieren. Außerdem wurde die mRNA-Expression wichtiger PPARγ-regulierter Gene in Hepatozyten trotz knockdown beider THR-Isoformen signifikant durch Thyroidhormone induziert.
Für eine erste Abschätzung einer möglichen physiologischen Relevanz der PPARγ/RXR-Aktivierung durch Thyroidhormone, wurde die Bildung von TETRAC nach Inkubation von Hepatozyten mit T4 quantifiziert. Es konnte festgestellt werden, dass ausreichend TETRAC in den Hepatozyten gebildet werden kann, um PPARγ zu aktivieren. Auch in einem in vivo-Experiment, bei dem Mäusen ein mit Brom substituiertes T4-Analog (Br-T4) appliziert wurde, um Interferenzen mit der endogenen Thyroidhormon-Produktion zu verhindern, konnte gezeigt werden, dass die PPARγ-regulierte Genexpression in den Lebern der Tiere induziert wurde. Dies deutete auf eine physiologisch relevante Bildung von Br-TETRAC hin, da Br-TETRAC analog zu TETRAC eine hohe Bindungs-Aktivität an PPARγ besaß, während Br-T4 keine Aktivität an diesem Rezeptor aufwies.
Die Ergebnisse dieser Arbeit deuten darauf hin, dass Thyroidhormone neben den THR-vermittelten Effekten auch andere genomische Wirkmechanismen besitzen, indem sie das PPARγ/RXR-Heterodimer aktivieren. Diese biologische Aktivität könnte sowohl eine physiologische als auch eine pharmakologische Relevanz besitzen. Die beiden T4-Metabolite T3 und TETRAC sind in der Lage komplementäre Signalwege zu induzieren. Wird T4 deiodiert kommt es zur Bildung von T3, welches den THR aktiviert. Durch oxidative Deaminierung des T4s bildet sich TETRAC, das wiederum PPARγ bindet und aktiviert. Durch die vermehrte Bildung von TETRAC und anschließende Aktivierung von PPARγ könnte die katabole Wirkung der THR-Signalwege abgeschwächt werden und so eine Art negative Rückkopplung gewährleistet werden. Die physiologische Bedeutung der Interaktion von Thyroidhormonen mit PPARγ/RXR muss jedoch noch genauer untersucht werden.
Aber auch pharmakologisch könnte die Iodthyroacetat-Aktivität an PPARγ eine Rolle spielen. TETRAC könnte durch seinen individuellen Bindungsmodus als Leitstruktur für neue PPARγ-Partialagonisten mit verbessertem Nebenwirkungs-Profil dienen. Außerdem wird das Thyroidhormon-Derivat TRIAC schon jetzt als Leitstruktur für die Entwicklung von Thyroidhormon-Analoga mit THRβ-Selektivität verwendet. Durch die zusätzliche PPARγ-Aktivität könnte zukünftig ein dualer THRβ/PPARγ-Agonist bei Erkrankungen, die mit einer Insulinresistenz einhergehen, Verwendung finden.
Zusammenfassend stellt die Entdeckung der Aktivität von Thyroidhormonen an PPARγ und RXR einen weiteren Baustein im komplexen System der Thyroidhormone dar.
Stickstoff (NO), Kohlenmonoxid (CO) und Schwefelwasserstoff (H2S) gehören zur Gruppe der Gasotransmitter. Dabei handelt es sich um kleine gasförmige Signalmoleküle, welche innerhalb des Körpers gebildet werden und dort wichtige physiologische Funktionen bei der Regulation der Apoptose, der Proliferation, der Entzündungsreaktion und der Genexpression übernehmen. Aufgrund ihrer Membranpermeabilität ist die Wirkung der Gasotransmitter nicht an die Interaktion mit spezifischen membranständigen Rezeptoren gebundenen. Je nach Organ, Gewebe und Konzentration können diese Mediatoren unterschiedliche Prozesse beeinflussen und teils sogar gegenteilige Wirkungen hervorrufen.H2S beispielsweise kann im Verlauf der Leukozytenadhäsion im Epithelium anti-inflammatorisch, bei Brandwunden oder rheumatischen Erkrankungen jedoch pro-inflammatorisch wirken. Im Kreislaufsystem hingegen bewirkt H2S durch die Aktivierung von ATP-abhängigen K+-Kanälen und die damit zusammenhängende Vasorelaxion der glatten Muskelzellen einen eindeutig protektiven Effekt.
H2S kann je nach Substrat und Zelltyp durch eines von 3 Enzymen gebildet werden. Die Cystathionin-γ-Lyase (CSE) und die Cystathionin-β-Synthase (CBS) nutzen L-Cystein als Substrat für die Synthese von H2S. Das dritte H2S-bildende Enzym, die 3-Mercaptopyruvate Sulfurtransferase (3-MST) verwendet α-Ketoglutarat als Substrat, welches zuvor von der Cystein-Aminotransferse (CAT) aus L-Cystein synthetisiert wurde. Während die beiden Enzyme CSE und CBS im Zytosol der Zelle zu finden sind, ist die 3-MST hauptsächlich in den Mitochondrien der Zelle zu finden. Im Gegensatz zur CBS, welche eher ein konstitutiv exprimiertes Protein ist, wird die Expression der CSE auf der Transkriptionsebene durch u.a. Entzündungsmediatoren wie TNF-α oder Wachstumsfaktoren wie PDGF-BB induziert.
Ein Ziel der Arbeit war es, die Wirkung von H2S bei der Wundheilung, bei entzündlichen glomerulären Erkrankungen der Niere und beim Schlaganfall zu untersuchen. Für diesephänotypische Analysen stand ein Knockoutmodell für die CSE zur Verfügung.
Zudem wurden in dieser Arbeit Untersuchungen mit einem Knockoutmodell für das zytoskeletäre Protein durchgeführt. Bei Clp36 (PDLIM1) handelt es sich um ein PDLIM-Protein (PDZ and LIM domain protein),welches durch die Gasotransmitter NO und H2S auf transkriptioneller und translationaler Ebene reguliert wird ist und aufgrund seiner Assoziation mit dem Zytoskelett dynamische Vorgänge der Zelle moduliert. Es ist bereits bekannt, dass Clp36 ein negativer Regulator des Glykoprotein VI (GPVI), welches eine wichtige Rolle bei der Aktivierung von Thrombozyten spielt, ist.
Beide Knockoutmodelle wurden in murinen Mesangiumzellen der Niere und in Krankheitsmodellen der Haut (kutane Wundheilung)und des Gehirns (Schlaganfall mit dem MCAO-Modell) analysiert.
Neben nicht signifikanten Effekten im MCAO-Modell, konnten sowohl Effekte des CSE-, als auch des CLP36-KOs auf die Migration und Proliferation und im Falle der CSE auch auf die Adhäsion der murinen Mesangiumzellen beobachtet werden. Die Depletion von Clp36 führte zu einer Verringerung der Migrations- und einer Erhöhung der Proliferationsrate, wohingegen die Depletion der CSE zu einer Erhöhung der Migrations-, Proliferations- und Adhäsionsrate führte. Die vielversprechendsten Ergebnisse konnten im Tiermodell der kutanen Wundheilung generiert werden. Untersucht wurde die Expression der H2S-produzierenden Enzyme CSE, CBS und 3-MST. Alle drei Enzyme zeigten im Tiermodell keine transkriptionelle Regulation und blieben auch während der akuten Entzündungsphase und der proliferativen Phase der Wundheilung unverändert. Es konnte jedoch gezeigt werden, dass die Expression der CSE in der späten Phase der Wundheilung signifikant anstieg, wenn die Proliferation innerhalb des Granulationsgewebes und der Neoepidermis geringer wurde. Die Vermutung, dass H2S in dieser Phase eine wichtige Rolle spielt, konnte durch die Analyse der CSE-KO Mäuse bekräftigt werden, da dort der Verlust der CSE offenbar durch die CBS kompensiert wurde.
In immunhistochemischen Untersuchungen konnten insbesondere follikuläre Keratinozyten der Neo-Epidemis als Quelle der CSE-Expression identifiziert werden. Durch in-vitro Studien auf mRNA und Proteinebene in HaCaT Zellen wurde gezeigt, dass H2S die Keratinozyten-Differenzierung beeinflusst. Der langsam freisetzendeH2S-Donor GYY4137 konnte in humanen Keratinozyten zu einer signifikanten Erhöhung der Ca2+- induzierten Expression der frühen Keratinozyten-Differenzierungsmarker Cytokeratin 10 (CK10) und Involucrin (IVN) beitragen.
Im Laufe dieser Arbeit konnte der molekulare Mechanismus hinter diesen Beobachtungen noch nicht geklärt werden.
Durch weitere Versuche meiner Arbeitsgruppe konnte jedoch gezeigt werden, dass die GYY4137-abhängige Induktion der CK10-Expression durch eine verstärkte Bindung der RNA-Polymerase II an den CK10 Promotor zustande kommt.
The three major autoimmune diseases (ADs) of the liver are primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). All of those diseases show an aggressive immune reaction resulting in the destruction of liver tissue and finally to the development of hepatic fibrosis.
PSC is an autoimmune mediated disease of unknown etiology. It is characterized by inflammation of intra- and extrahepatic bile ducts. The progressive destruction of the bile ducts can lead to liver cirrhosis and finally to liver failure. Clinical signs for PSC are increased alkaline phosphatase (AP) and gamma glutamyltransferase (GGT) levels, presence of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) and bile ducts with characteristic strictures and dilations of the biliary tree as well as onion skin fibrosis surrounding the damaged bile ducts. Currently, there is no established treatment for PSC patients. The administration of ursodeoxycholic acid (UDCA) is being use as a therapy. However, it merely serves a symptomatic treatment to reduce serum AP and GGT as well as the formation of gallstones. In the advanced stage of PSC, liver transplantation is the last therapeutic option. Mdr2-/- mice are an excepted mouse model for human PSC. Such mice show lymphocytes infiltration into the liver, bile duct lesions, as well as the presence of the typical onion skin-like pericholangitis and periductal fibrosis.
AIH is a rare chronic autoimmune disease of the liver that results from the loss of self-tolerance to hepatocytes and leads to destruction of the hepatic parenchyma with the onset of cirrhosis. Clinical signs for AIH are elevated alanine aminotransferase (ALT) and aspartate transaminase (AST) levels, hypergammaglobulinemia and different types of autoantibodies. In addition, interphase hepatitis with lymphocytic and plasmacellular infiltrates in the periportal field are characteristic for AIH. Two different subtypes of AIH exist and depending on their autoantibody profile they can be distinguished into AIH type 1 which is characterized by the presence of anti-nuclear (ANA) and/or anti-smooth muscular (SMA) autoantibodies, and AIH type 2 showing liver/kidney microsomal autoantibodies (LKM-1). LKM-1 recognizes the major autoantigen, the 2D6 isoform of the cytochrome P450 enzyme family (CYP2D6). One mouse model for AIH is the CYP2D6 model in which the injection of Ad-2D6 leads to a breakdown of the immune tolerance by the destruction of hepatocytes.
There are some patients with autoimmune diseases of the liver who have both cholestatic and hepatic liver enzymes and histological features suggestive of two different liver diseases. These patients are diagnosed with an overlap syndrome (OS).
In my thesis I generated an animal model with characteristics of both diseases, which would mimic features of human PSC-AIH OS. Mdr2-/- mice which spontaneously develop PSC were infected with Ad-2D6 to trigger the autoimmune-driven hepatic injury. Pathogenesis of PSC-AIH OS mice was compared to mice with solitary PSC or AIH. Naïve FVB wild type mice have been used as healthy controls. The characterization of the PSC-AIH OS model was done by analyzing serological parameters like ALT, AP, different antibodies like pANCA, LKM-1 like CYP2D6 and total IgG. Additionally, fibrosis and cholangitis were analyzed by immunohistochemistry and Western blotting. Moreover, cellular infiltrations of CD4+ and CD8+ T cells, dendritic cells (DCs), monocytes/macrophages and neutrophils were determined with immunohistochemistry. Finally, the overall immune balance in the liver and the frequency of CYP specific T cells were analyzed via flow cytometry. Our new mouse model indeed represents the characteristics of both PSC and AIH and mimics features of the human PSC-AIH OS. It allows studying the development of a PSC-AIH OS and how the two overlapping diseases are influencing one another. In a second approach I wanted to induce CYP2D6-specific tolerance in AIH mice. Therefore, I tried four different approaches, namely intranasal peptide administration, injection of tolerogenic DCs, antigen-coupled splenocytes, and Ag-coupled nanoparticles (NP) and evaluated their potential to induce CYP2D6 specific Treg with the capacity to prevent AIH in mice. Unfortunately, the intranasal peptide administration and also the injection of tolerogenic DCs did not increase the amount of CYP2D6 specific Treg which would lead to a reduction of the frequency of inflammatory T cells. Surprisingly, the injection of antigen-coupled splenocytes showed the opposite effect characterized by a very strong cytokine secretion in the tolerized mice. The use of NPs led to an increase in CYP2D6 specific Treg as well as in decrease in the frequency of inflammatory T cells and finally has the potential for a therapeutic approach.
In summary, the generated PSC-AIH OS model represents many clinical signs which can also be observed in PSC-AIH OS patients. This model can be used to study the etiology of this overlap syndrome and further to test potential therapeutic approaches. The different immune tolerance induction pathways which I tried in the AIH model show that NPs have to potential to induce immune tolerance but this approach has to be refined and the outcome has to be characterized in more detail.
Pretubulysin (PT), a biosynthetic precursor of the myxobacterial compound tubulysin D, was recently identified as a novel microtubule-targeting agent (MTA) causing microtubule destabilization. MTAs are the most frequently used chemotherapeutic drugs. They are well studied regarding their direct cytotoxic effects against various tumors as well as for their anti-angiogenic and vascular-disrupting action addressing endothelial cells of the tumor vasculature. However, the impact of MTAs on endothelial cells of the non-tumor vasculature has been largely neglected, although tumor cell interactions with the healthy endothelium play a crucial role in the process of cancer metastasis. Besides their use as potent anti-cancer drugs, some MTAs such as colchicine are traditionally used or recommended for the therapy of inflammatory diseases. Here, too, the role of endothelial cells has been largely neglected, although the endothelium is crucially involved in regulating the process of inflammation.
In the present study, the impact of PT on tumor-endothelial cell interactions was therefore analyzed in vitro to gain insights into the mechanism underlying its anti-metastatic effect that was recently confirmed in vivo. In the second part of this work, the influence of PT and other MTAs, namely the microtubule-destabilizing compounds vincristine (VIN) and colchicine (COL) and the microtubule-stabilizing drug paclitaxel (PAC), on leukocyte-endothelial cell interactions was investigated in vitro and in vivo (only PT). It is important to mention that in all in vitro experiments solely endothelial cells and not tumor cells or leukocytes were treated with the MTAs to strictly focus on the role of the endothelium in the action of these compounds.
The impact of PT on tumor-endothelial cell interactions was analyzed in vitro by cell adhesion and transendothelial migration assays as well as immunocytochemistry using the breast cancer cell line MDA-MB-231 and primary human umbilical vein endothelial cells (HUVECs). The treatment of HUVECs with PT increased the adhesion of MDA cells onto the endothelial monolayer, whereas their transendothelial migration was reduced by the compound. Thereafter, the influence of PT on the endothelial cell adhesion molecules (CAMs) E-selectin, N-cadherin, ICAM-1, VCAM-1 and galectin-3 and on the CXCL12/CXCR4 chemokine system was examined, since they might be involved in the PT-triggered tumor cell adhesion. Interestingly, although PT induced the upregulation of ICAM-1, VCAM-1, N-cadherin and CXCL12, cell adhesion assays using neutralizing antibodies or the CXCL12 inhibitor AMD3100 revealed that all these molecules were dispensable for the PT-evoked tumor cell adhesion. As PT induces the formation of interendothelial gaps and MDA cells might adhere onto components of the underlying extracellular matrix (ECM), the precise location of MDA cells attached to the PT-treated endothelial monolayer was investigated. Instead of a direct interaction between tumor and endothelial cells, this work showed that MDA cells preferred to adhere to the ECM component collagen that was exposed within PT-triggered endothelial gaps. Both the PT-evoked increase in tumor cell adhesion onto and the decrease in trans-endothelial migration were completely abolished when β1-integrins were blocked on MDA cells. Similar results were obtained when endothelial cells were treated with VIN and COL but not PAC, indicating that the observed effects of PT depend on its microtubule-destabilizing activity.
The impact of PT, VIN, COL and PAC on leukocyte-endothelial cell interactions was analyzed in vivo (only PT) by intravital microscopy of the mouse cremaster muscle and in vitro by cell adhesion assays using the monocyte-like cell line THP-1 and TNFα-activated human dermal microvascular endothelial cells (HMEC-1). While PT did not affect the rolling of leukocytes on the endothelium, their firm adhesion onto and transmigration through the activated endothelium was reduced by PT in vivo. In accordance, the treatment of HMEC-1 with PT, VIN and COL decreased the TNFα-induced adhesion of THP-1 cells onto the endothelial monolayer, whereas PAC had no influence on this process. Thereafter, the influence of PT, VIN, COL and PAC on endothelial ICAM-1 and VCAM-1 was examined, since these molecules are substantially involved in the firm adhesion of leukocytes onto the endothelium. The cell surface protein expression of ICAM-1 and VCAM-1 was reduced by PT, VIN and COL in activated endothelial cells, whereas PAC did only slightly affect the TNFα-induced upregulation of VCAM-1. As the pro-inflammatory transcription factor NFκB plays a crucial role in the TNFα-induced expression of these CAMs, the impact of the MTAs on the NFκB promotor activity was investigated. While PT, VIN and COL decreased the activation of NFκB in activated endothelial cells, PAC did not affect this process. However, in contrast to the strong effects regarding the cell surface protein expression of ICAM-1 and VCAM-1, the effects of PT, VIN and COL on the NFκB activity was rather low. Thus, the used MTAs might also affect other relevant signaling pathways and/or the intracellular transport of CAMs might be influenced by the impact of the MTAs on the microtubule network.
Taken together, the current study provides – at least in part – an explanation for the anti-metastatic potential of PT and gives first insights into the use of PT and VIN as anti-inflammatory drugs. Moreover, this work highlights the endothelium as an attractive target for the development of new anti-cancer and anti-inflammatory drugs.
The enzyme acetyl-CoA carboxylase (ACC) plays a fundamental role in the fatty acid metabolism. It regulates the first and rate limiting step in the biosynthesis of fatty acids by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA and exists as two different isoforms, ACC1 and ACC2. In the last few years, ACC has been reported as an attractive drug target for treating different diseases, such as insulin resistance, hepatic steatosis, dyslipidemia, obesity, metabolic syndrome and nonalcoholic fatty liver disease. An altered fatty acid metabolism is also associated with cancer cell proliferation. In general, the inhibition of ACC provides two possibilities to regulate the fatty acid metabolism: It blocks the de novo lipogenesis in lipogenic tissues and stimulates the mitochondrial fatty acid β-oxidation. Surprisingly, the role of ACC in human vascular endothelial cells has been neglected so far. This work aimed to investigate the role of the ACC/fatty acid metabolism in regulating important endothelial cell functions like proliferation, migration and tube formation.
To investigate the function of ACC, the ACC-inhibitor soraphen A as well as an siRNA-based approach were used. This study revealed that ACC1 is the predominant isoform both in human umbilical vein endothelial cells (HUVECs) and in human dermal microvascular endothelial cells (HMECs). Inhibition of ACC via soraphen A resulted in decreased levels of malonyl-CoA and shifted the lipid composition of endothelial cell membranes. Consequently, membrane fluidity, filopodia formation and the migratory capacity were attenuated. Increasing amounts of longer acyl chains within the phospholipid subgroup phosphatidylcholine (PC) were suggested to overcompensate the shift towards shorter acyl chains within phosphatidylglycerol (PG), which resulted in a dominating effect on regulating the membrane fluidity. Most importantly, this work provided a link between changes in the phospholipid composition and altered endothelial cell migration. The antimigratory effect of soraphen A was linked to a reduced amount of PG and to an increased amount of polyunsaturated fatty acids (PUFAs) within the phospholipid cell membrane. This link was unknown in the literature so far. Interestingly, a reduced filopodia formation was observed upon ACC inhibition via soraphen A, which presumably caused the impaired migratory capacity.
This work revealed a relationship between ACC/fatty acid metabolism, membrane lipid composition and endothelial cell migration. The natural compound soraphen A emerged as a valuable chemical tool to analyze the role of ACC/fatty acid metabolism in regulating important endothelial cell functions. Furthermore, regulating endothelial cell migration via ACC inhibition promises beneficial therapeutic perspectives for the treatment of cell migration-related disorders, such as ischemia reperfusion injury, diabetic angiopathy, macular degeneration, rheumatoid arthritis, wound healing defects and cancer.
Aim: Long noncoding RNAs (lncRNAs) belong to the interface of epigenetics and exhibit diverse functions. Their features depend on their sequence, genomic location and tertiary structure. The aim was to identify novel lncRNAs and characterise their physiological functions and mechanisms in endothelial cells. Three different approaches were performed:
The hypothesis that pseudogene-annotated lncRNA NONHSAT073641 regulates the expression of their parental gene platelet activating factor acetylhydrolase 1b regulatory subunit 1 (PAFAH1B1) was examined.
The physiological functions and in vivo relevance of most lncRNAs are still unknown, therefore a part of this work aimed to identify lncRNAs in response to a pathophysiological stimulus (high amplitude stretch) in endothelial cells.
The long intergenic noncoding RNA antisense to S1PR1 (LISPR1) gene, is located within the promotor of sphingosine-1-phosphate receptor 1 (S1PR1) and shares a part of the promotor region. This study examined additionally the hypothesis that LISPR1 controls the S1PR1 expression in endothelial cells.
Methods: The angiogenic functions of NONHSAT073641 and LISPR1 were examined with spheroid-outgrowth and scratch wound assays. Furthermore, stretch experiments were performed in order to identify differently expressed lncRNAs in human umbilical vein endothelial cells (HUVECs). In addition, the in vivo relevance of both lncRNAs was examined in samples from pulmonary arterial hypertension patients. Knockdown (e.g. LNA GapmeRs), knockout (CRISPR/ Cas9) and overexpression experiments (e.g. CRISPR activation) were performed to analyse target genes. The molecular mechanism of LISPR1 was investigated with RNA and Chromatin immunoprecipitation.
Results: NONHSAT073641 and PAFAH1B1 exhibited angiogenic function in endothelial cells. It could be observed that NONHSAT073641 is not regulating the expression of PAFAH1B1. The pro-angiogenic feature of PAFAH1B1 might be attributed to the target gene matrix Gla protein (MGP). NONHSAT073641 and PAFAH1B1 were significantly induced in CTEPH samples and might be important in the development of this disease. It could be speculated that NONHSAT073641 is regulating the expression of the cell-cycle regulator BCL2L11 as has been investigated in mice.
LISPR1 is a cis-acting lncRNA which maintains S1PR1 gene transcription by intercepting the transcriptional repressor ZNF354C and enabling Polymerase II (PolII) to bind. ZNF354C regulates S1PR1 expression in HUVECs. However, the role of ZNF354C in pulmonary arterial hypertension (PAH) is unknown. LISPR1 and S1P1 receptor were both significantly depleted in COPD samples. It can be assumed that due to higher S1P production, the signalling is attenuated through reduction of the lncRNA LIPSR1 and thus the receptor S1P1.
The stretch experiments present a possible in vitro model in order to mimic the condition of endothelial cells during high blood pressure, such as in PAH. Referring to published data, it could be confirmed that stretching of endothelial cells alters the gene expression, which is on the other hand linked to cardiovascular disease. In cardiovascular disease mechanical stretch altered genes, which are participating in the vascular remodelling process. The role of differently expressed lncRNAs (TGFβ2-AS1, CTD-2033D15.2, INHBA-AS1, RP11-393I2.4, TAPT1-AS1, TPM1-AS1, CFLAR-AS1 and HIF1α-AS2) upon mechanical stretch is yet not clarified.
Conclusion: NONHSAT073641 and LISPR1 are important for the endothelial angiogenic function. Both lncRNAs were deregulated in PAH samples. The pathophysiological stimulus had an impact on the expression of different lncRNAs (e.g. TGFβ2-AS1) and pathways (e.g. TGF-β) in endothelial cells.