TY - THES A1 - Heupel, Wolfgang-Moritz Felix T1 - Role and modulation of cadherins in pathologic processes T1 - Rolle und Modulation von Cadherinen in pathologischen Prozessen N2 - Ca2+ dependent cell adhesion molecules (cadherins) are central for a variety of cell and tissue functions such as morphogenesis, epithelial and endothelial barrier formation, synaptic function and cellular signaling. Of paramount importance for cadherin function is their specific extracellular adhesive trans-interaction. Cadherins are embedded in a cellular environment of intracellular and extracellular regulators that modify cadherin binding in response to various physiological and pathological stimuli. Most experimental approaches used for studying cadherin interaction however lack a physiological proof of principle mostly by not investigating cadherins in their physiological environment. In the present cumulative dissertation, experimental approaches were applied to characterize and modulate vascular endothelial (VE)-cadherin and desmocadherin functions in the (patho-)physiological contexts of endothelial permeability regulation and disturbance of epidermal barrier function, which is typical to the blistering skin disease pemphigus, respectively. Whereas VE-cadherin is a key regulator of the endothelial barrier that separates the blood compartment from the interstitial space of tissues, desmosomal cadherins are crucial for maintenance of epidermal integrity and separation of the external environment from the body’s internal milieu. Cadherin functions were both investigated in cell-free and cell-based conditions: by using biophysical single molecule techniques like atomic force microscopy (AFM), cadherin function could be investigated in conditions, where contributions of intracellular signaling were excluded. These experiments were, however, compared and combined with cell-based experiments in which cadherins of epidermal or endothelial cell cultures were probed by laser force microscopy (laser tweezers), fluorescence recovery after photobleaching (FRAP) and other techniques. The autoimmune blistering skin diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are caused by autoantibodies directed against the extracellular domains of the desmosomal cadherins desmoglein (Dsg) 1 and 3, which are important for epidermal adhesion. The mechanism of autoantibody-induced cell dissociation (acantholysis) in pemphigus, however, is still not fully understood. For the first time, it is shown by AFM force spectroscopy that pemphigus autoantibodies directly inhibit Dsg3 adhesion by steric hindrance but do not inhibit adhesion of Dsg1. However, the full pathogenicity of the autoantibodies depended on cellular signaling processes, since autoantibodies targeting Dsg1 also resulted in loss of cadherin-mediated adhesion in cell-based experiments. However, two other signaling pathways that have been reported to be involved in pemphigus pathogenesis, i.e. epidermal growth factor receptor (EGFR) and c-Src activation, were not found to be important in this context. Furthermore, peptide-based modulators of cadherin functions were generated for Dsg1/3 and VE-cadherin. By comparing Dsg1, Dsg3 and VE-cadherin sequences to published X-ray structures of cadherin trans-interactions, specific amino acid sequences of the binding pockets of these cadherins were identified. Peptide versions of these motifs were synthesized and the antagonistic functions of these “single peptides” were validated by AFM force spectroscopy as well as by cell-based assays. By linking two single peptides in tandem, stabilization of cadherin bonds because of by cross-bridge formation between trans-interacting cadherins was demonstrated. Protective effects of tandem peptides were shown by partly preventing pemphigus autoantibody-induced acantholysis, or in the case of VE-cadherin, by stabilizing endothelial barrier properties against barrier disrupting agents like the Ca2+ ionophore A23187 and an inhibitory VE-cadherin antibody. Most importantly, VE-cadherin tandem peptides abolished microvascular hyperpermeability induced by the physiologic inflammatory agent tumor necrosis factor-α in the rat mesentery in vivo. Both classes of tandem peptides therefore can be considered as a starting point for the generation of potential therapeutic agents that might prevent cell dissociation in pemphigus and breakdown of the endothelial barrier under inflammatory conditions. N2 - Die Familie der Ca2+ - abhängigen Adhäsionsproteine (Cadherine) spielt eine zentrale Rolle bei elementaren zellulären, geweblichen und Entwicklungsprozessen. Eine in der vorliegenden kumulativen Dissertation untersuchte Funktion von Cadherinen ist ihre Rolle beim Aufbau und der Aufrechterhaltung der epidermalen Barriere der Haut und der endothelialen Barriere von Blutgefäßen. Cadherine vermitteln Adhäsion über die extrazelluläre Bindung mit Cadherinen auf der Zelloberfläche angrenzender Zellen. Die durch Cadherine vermittelte Zelladhäsion ist ein dynamischer Prozess, der durch extrazelluläre und intrazelluläre Modulatoren im Zusammenspiel mit vielfältigen physiologischen Prozessen reguliert wird. Vielen Experimentalsystemen fehlt der realistische physiologische und gewebliche Bezug zur funktionellen Bedeutung der untersuchten Eigenschaften der Cadherine. In der vorliegenden kumulativen Dissertation wurden verschiedene Ansätze zur Untersuchung und Modulation von Cadherinen im Hinblick zweier (patho-)physiologischer Prozesse durchgeführt. Zum einen befasst sich die Doktorarbeit mit den Blasen-bildenden Hauterkrankungen der Pemphigus-Gruppe, bei welcher die Funktionsstörung desmosomaler Cadherine im Mittelpunkt steht. Zum anderen wurde das vaskuläre endotheliale (VE)-Cadherin und dessen Rolle bei der Regulation und pathologischen Entgleisung der Gefäßpermeabilität untersucht. Die Funktion dieser Cadherine wurde in der Arbeit sowohl in Zell-freien als auch in Zell-basierten Experimenten analysiert: mittels biophysikalischer Charakterisierung auf Einzelmolekülebene durch Kraftspektroskopie mit dem Atomkraftmikroskop (AFM) konnte die Adhäsion (Transinteraktion) von Cadherinen frei von zellulären Einflüssen isoliert untersucht werden. Diese Einzelmolekülstudien wurden durch Laserkraftmikroskopie (Laserpinzette) und verschiedene zellphysiologische Untersuchungen an epithelialen und endothelialen Zellkulturen und Geweben komplettiert. Bei der autoimmunen Hauterkrankung Pemphigus foliaceus (PF) und Pemphigus vulgaris (PV) bewirken Autoantikörper, die gegen die desmosomale Cadherine Desmoglein (Dsg) 1 und 3 gerichtet sind, eine Zelldissoziation (Akantholyse), die zu einer charakteristischen Blasenbildung auf der Haut der Patienten teils mit Ablösung der Epidermis führt. In der vorliegenden Arbeit wurde mit Hilfe der AFM-Kraftspektroskopie zum ersten Mal gezeigt, dass Pemphigus-Autoantikörper direkt die Dsg3-vermittelte Adhäsion durch sterische Behinderung inhibieren. Zusätzlich wurden auch Unterschiede in der Pathogenität der Autoantikörper in Abhängigkeit von zellulären Signalwegen gefunden. In früheren Studien konnte bereits gezeigt werden, dass neben der vermuteten Hemmung der Cadherinbindung durch die Autoantikörper auch inhibitorische, die Zelladhäsion herabsetzende zytoplasmatische Signalwege für die Pathogenese dieser Krankheit wichtig sind. Daneben belegen Experimente dieser Arbeit, dass die durch Autoantikörper vermittelte Akantholyse in unseren Versuchsbedingungen unabhängig von der in anderen Studien postulierten Beteilung des epidermalen Wachstumsfaktorrezeptors (EGFR) und von c-Src war. In weiteren Experimenten wurden Peptide zur Modulation der Funktion von Dsg1/3 und VE-Cadherin entwickelt. Dazu wurden die Sequenzen von Dsg1, Dsg3 und VE-Cadherin mit bereits beschriebenen Röntgenkristallstrukturen von anderen Cadherinen verglichen und eigene Strukturmodelle auf der Grundlage einer Analogiemodellierung generiert. Auf diese Weise wurden Sequenzabschnitte identifiziert, die für die Cadherin-Transinteraktion wichtig sind. Aus diesen Sequenzen wurden Peptide abgeleitet, die die Cadherinfunktion entweder in einer agonistischen oder antagonistischen Weise beeinflussen sollten. Die inhibitorische Funktion der Einzelpeptide wurde sowohl durch AFM-Kraftspektroskopie als auch in Zell-basierten Laserpinzetten-Studien validiert. Durch das Zusammenfügen von zwei separaten Einzelpeptidsequenzen wurden Tandempeptide erzeugt. Diese sollten die jeweilige Cadherininteraktion durch das Überbrücken benachbarter adhäsiver Cadherindomänen stabilisieren. Das Dsg-spezifische Tandempeptid verhinderte teilweise die durch Autoantikörper hervorgerufene Akantholyse beim Pemphigus und das VE-Cadherin-spezifische Tandempeptid schützte die Endothelbarriere vor Permeabilitätserhöhung durch das Ca2+ - Ionophor A23187 oder durch einen inhibitorischen VE-Cadherin-Antikörper. In in-vivo-Experimenten an perfundierten Mikrogefäßen des Rattenmesenteriums verhinderte das VE-Cadherin-Tandempeptid den Anstieg der Endothelpermeabilität durch den physiologischen Entzündungsmediator Tumornekrosefaktor-α. Die Tandempeptide können als Ausgangspunkt für die Identifikation von spezifischen therapeutischen Agenzien zur Prävention der Akantholyse beim Pemphigus oder Verlust der VE-Cadherin-Bindung bei vaskulärer Hyperpermeabilität angesehen werden. KW - Cadherine KW - Zelladhäsion KW - Pemphigus KW - Desmosom KW - Endothel KW - Zelladhäsion KW - Cadherine KW - Desmogleine KW - VE-Cadherin KW - cell adhesion KW - cadherins KW - desmosomes Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-52716 ER - TY - JOUR A1 - Gerull, Brenda A1 - Brodehl, Andreas T1 - Insights Into Genetics and Pathophysiology of Arrhythmogenic Cardiomyopathy JF - Current Heart Failure Reports N2 - Purpose of Review Arrhythmogenic cardiomyopathy (ACM) is a genetic disease characterized by life-threatening ventricular arrhythmias and sudden cardiac death (SCD) in apparently healthy young adults. Mutations in genes encoding for cellular junctions can be found in about half of the patients. However, disease onset and severity, risk of arrhythmias, and outcome are highly variable and drug-targeted treatment is currently unavailable. Recent Findings This review focuses on advances in clinical risk stratification, genetic etiology, and pathophysiological concepts. The desmosome is the central part of the disease, but other intercalated disc and associated structural proteins not only broaden the genetic spectrum but also provide novel molecular and cellular insights into the pathogenesis of ACM. Signaling pathways and the role of inflammation will be discussed and targets for novel therapeutic approaches outlined. Summary Genetic discoveries and experimental-driven preclinical research contributed significantly to the understanding of ACM towards mutation- and pathway-specific personalized medicine. KW - dilated cardiomyopathy KW - arrhythmogenic cardiomyopathy KW - junctions KW - sudden cardiac death KW - cardiovascular genetics KW - desmosomes Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-269916 SN - 1546-9549 VL - 18 IS - 6 ER - TY - JOUR A1 - Gerull, Brenda A1 - Brodehl, Andreas T1 - Genetic Animal Models for Arrhythmogenic Cardiomyopathy JF - Frontiers in Physiology N2 - Arrhythmogenic cardiomyopathy has been clinically defined since the 1980s and causes right or biventricular cardiomyopathy associated with ventricular arrhythmia. Although it is a rare cardiac disease, it is responsible for a significant proportion of sudden cardiac deaths, especially in athletes. The majority of patients with arrhythmogenic cardiomyopathy carry one or more genetic variants in desmosomal genes. In the 1990s, several knockout mouse models of genes encoding for desmosomal proteins involved in cell–cell adhesion revealed for the first time embryonic lethality due to cardiac defects. Influenced by these initial discoveries in mice, arrhythmogenic cardiomyopathy received an increasing interest in human cardiovascular genetics, leading to the discovery of mutations initially in desmosomal genes and later on in more than 25 different genes. Of note, even in the clinic, routine genetic diagnostics are important for risk prediction of patients and their relatives with arrhythmogenic cardiomyopathy. Based on improvements in genetic animal engineering, different transgenic, knock-in, or cardiac-specific knockout animal models for desmosomal and nondesmosomal proteins have been generated, leading to important discoveries in this field. Here, we present an overview about the existing animal models of arrhythmogenic cardiomyopathy with a focus on the underlying pathomechanism and its importance for understanding of this disease. Prospectively, novel mechanistic insights gained from the whole animal, organ, tissue, cellular, and molecular levels will lead to the development of efficient personalized therapies for treatment of arrhythmogenic cardiomyopathy. KW - arrhythmogenic cardiomyopathy KW - desmosomes KW - animal models of human disease KW - sudden death KW - genetics KW - mouse KW - zebrafish Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-206903 SN - 1664-042X VL - 11 IS - 264 ER - TY - JOUR A1 - Brodehl, Andreas A1 - Meshkov, Alexey A1 - Myasnikov, Roman A1 - Kiseleva, Anna A1 - Kulikova, Olga A1 - Klauke, Bärbel A1 - Sotnikova, Evgeniia A1 - Stanasiuk, Caroline A1 - Divashuk, Mikhail A1 - Pohl, Greta Marie A1 - Kudryavtseva, Maria A1 - Klingel, Karin A1 - Gerull, Brenda A1 - Zharikova, Anastasia A1 - Gummert, Jan A1 - Koretskiy, Sergey A1 - Schubert, Stephan A1 - Mershina, Elena A1 - Gärtner, Anna A1 - Pilus, Polina A1 - Laser, Kai Thorsten A1 - Sinitsyn, Valentin A1 - Boytsov, Sergey A1 - Drapkina, Oxana A1 - Milting, Hendrik T1 - Hemi- and homozygous loss-of-function mutations in DSG2 (desmoglein-2) cause recessive arrhythmogenic cardiomyopathy with an early onset JF - International Journal of Molecular Sciences N2 - About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2–c.378+1G>T) in the first patient and a nonsense mutation (DSG2–p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases. KW - desmoglein-2 KW - desmocollin-2 KW - DSG2 KW - DSC2 KW - ARVC KW - ACM KW - LVNC KW - cardiomyopathy KW - desmosomes KW - desmin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285279 SN - 1422-0067 VL - 22 IS - 7 ER -