Untersuchungen zur Regulation des intrazellulären Calciums in Physiologie und Pathophysiologie humaner und muriner Erythrozyten

Calcium übernimmt in Erythrozyten, wie auch in vielen anderen Zellen, die wichtige Rolle eines sekundären Botenstoffs. Ziel der vorliegenden Arbeit war es zum besseren Verständnis dieser Rolle in der Physiologie und Pathophysiologie von Erythrozyten beizutragen. Daher wurden im ersten Teilprojekt dieser Arbeit Untersuchungen gemacht zum intrazellulären Ca2+-Gehalt humaner pathologischer Erythrozyten von Patienten mit seltenen hämolytischen Erkrankungen. Es konnte festgestellt werden, dass Erythrozyten von Patienten mit Hereditärer Sphärozytose, Hereditärer Xerozytose und Gardos-Channelopathie signifikant erhöhte Werte an freiem intrazellulären Calcium im Vergleich zu Erythrozyten gesunder Kontrollblutspendern aufweisen. Für die ebenfalls untersuchten Patienten mit Enzymopathien und uncharakterisierter hämolytischer Anämie wurde keine eindeutige Änderung der Ca2+-Konzentration gefunden. Zusammen mit weiteren Daten aus der Literatur deuten diese Resultate darauf hin, dass ein erhöhter intrazellulärer Ca2+-Gehalt in Erythrozyten ursächlich für den verfrühten Abbau der Zellen in hämolytischen Krankheiten ist. Dieser Mechanismus scheint allgemein gültig sein, wobei sich der Signalweg des Ca2+-Einstroms für jede Erkrankung unterscheidet. Zur Entwicklung neuer Medikamente, die auf eine Inhibition dieses Ca2+-Einstroms als bessere Therapiemöglichkeit anämischer Patienten abzielen, bedarf es jedoch weiterer Forschung. Das zweite Teilprojekt dieser Arbeit beschäftigt sich mit dem Lysophosphatidsäure Signalweg, der in murinen und humanen Erythrozyten zu einem Einstrom von extrazellulärem Calcium in die Zellen führt. Diese Erhöhung des intrazellulären Ca2+-Gehalts steht in Verbindung mit der Aggregation der Erythrozyten als aktiver Teil der Blutgerinnung. Der Lysophosphatidsäure Signalweg ist daher auch zur Behandlung von Thrombosen von pharmakologischem Interesse. Im Detail zeigen die an humanen und murinen Erythrozyten durchgeführten Untersuchungen zum Signalweg, dass die Aktivität des am Signalweg beteiligten TRPC6 Kanals, neben der PKCα, durch einen Komplex aus FKB12 und Calcineurin reguliert wird. Zudem konnte gezeigt werden, dass eine zusätzliche mechanosensitive Komponente im Signalweg existiert. Ob es sich dabei um den Kanal Piezo1 handelt, muss in weiteren Untersuchungen bestätigt werden. Auf Basis des Lysophosphatidsäure Signalweges wurde im dritten Teilprojekt die Herkunft des TRPC6 Kanals in der Membran von Erythrozyten untersucht. Für humane Erythrozyten lässt sich der Kanal weder auf Proteom- noch Transkriptomebene detektieren. Auch funktionell kann TRPC6 nur in Erythrozyten, nicht in Retikulozyten nachgewiesen werden. Als Hypothese dieses Projekts wurde untersucht, ob es zu einem Transfer des Kanals von anderen Zellen auf Erythrozyten kommt. Dazu wurden Transfusionsversuche von Erythrozyten aus TRPC6 Knockout Mäusen in Wildtyp Mäuse durchgeführt. Anschließend wurde zur Beobachtung eines möglichen Proteinaustauschs der Lysophosphatidsäureinduzierte Ca2+-Einstrom in den transfundierten Erythrozyten untersucht. Als Grundlage hierfür wurden mehrere Ansätze zur Markierung von Erythrozyten vor der Transfusion getestet. Die Methode der in vivo-Biotinylierung zeigte sich nicht als geeignet zur mikroskopischen Detektion markierter Erythrozyten nach Transfusion. Eine Markierung der Zellen mittels Membranfarbstoffen als auch eine endogene Markierung der Zellen durch Expression eines Fluoreszenzproteins waren hingegen erfolgreich. Durchgeführte Transfusionsversuche auf Basis beider Methoden zeigten einen Anstieg des Lysophosphatidsäure-induzierten Ca2+-Einstroms in TRPC6-Knockout Erythrozyten nach Transfusion. Hierdurch kann auf einen Transfer des TRPC6 Kanals in die Membran der Knockout Erythrozyten von Zellen der Wildtyp Maus geschlossen werden. Der Transfer scheint dabei vermehrt oder auschließlich bei Retikulozyten vorzukommen. Eine Beteiligung der Milz an diesem Transfer wurde ebenfalls untersucht, konnte jedoch nicht belegt werden. Zum Ablauf, Ort und weiterem Verständnis des Proteintransfer müssen zusätzliche Untersuchungen durchgeführt werden. Ebenfalls soll die Frage geklärt werden, ob ein Austausch von Proteinen auch bei humanen roten Blutzellen auftritt.Calcium plays an important role as a second messenger, in red blood cells as well as in many other cell types. The aim of this study was to contribute to a further understanding of this role in the physiology and pathophysiology of red blood cells. In the first part of this thesis, pathologic human red blood cells from patients with rare hereditary anemias were analyzed for their intracellular calcium content. The results of this study showed that red blood cells from patients with hereditary spherocytosis, hereditary xerocytosis and Gardos channelopathy have significantly increased levels of free intracellular calcium in comparison to red blood cells from healthy donors. For patients with enzymopathies and uncharacterized hemolytic anemia there was no clear change in the intracellular calcium. Together with data from the literature, these findings indicate that an increased intracellular calcium content might be responsible for the premature elimination of red blood cells from the blood stream. This mechanism seems to have to a general scope with different pathways of calcium entry in different diseases. More research is needed for the development of drugs aiming on the inhibition of this calcium increase. The second part of this thesis deals with the lysophosphatidic acid signaling pathway, which leads to an uptake of extracellular calcium in human and murine red blood cells. This increase in intracellular calcium is associated with an aggregation of red blood cells as an active part in thrombus formation. The lysophosphatidic acid signaling pathway is therefore also an interesting pharmacological target for the treatment of thrombosis. The results of the experiments on murine and human red blood cells show, that TRPC6, a channel involved in this pathway, is regulated by PKCα and a protein complex consisting of FKB12 and calcineurin. Furthermore, the results hint to the involvement of a mechanosensitive channel in the signaling cascade. To confirm that this channel is Piezo1, further research needs to be done. Based on the lysophosphatidic acid signaling pathway, the origin of the TRPC6 channel in the membrane of red blood cells was explored in the third part of this thesis. For human red blood cells, it was not yet possible to detect the channel on a proteome- or transcriptome level. Functional evidence is also limited to mature red blood cells and missing in reticulocytes. As a hypothesis of this project, a possible transfer of the channel between red blood cells and other cell types was investigated. For this purpose, transfusion experiments of red blood cells from TRPC6 knockout mice to wildtype mice were performed. Subsequently, the lysophosphatidic acid induced calcium entry was analyzed in the transfused cells to visualize the potential transfer of the channel. Beforehand, several methods to label red blood cells for transfusion were tested. The method of in vivo biotinylation was not suitable for the microscopic detection of the cells after transfusion. Labeling the cells with membrane dyes as well as the endogenous expression of a fluorescent protein were successful. The results from the transfusion experiments based on both labeling methods showed an increase in the lysophosphatidic acid induced calcium signal in TRPC6 knockout cells after transfusion. From these results, it can be concluded that there is a transfer of the channel to the membrane of TRPC6 knockout red blood cells from cells of the wildtype mouse. This transfer seems to occur enhanced or exclusively in reticulocytes. The contribution of the spleen in this process was analyzed but could not be proven. More research is needed to resolve further questions, especially whether a transfer of proteins also occurs in human red blood cells

Immunofluorescent Localization Of Basement Membrane In Lesions Of Dermatitis Herpetiformis

Dermatitis herpetiformis (DH) is a blistering disease with a characteristic histology that includes papillary edema, neutrophilic papillary microabscesses, and development of subepidermal blisters. In spite of this pathologic sequence occurring entirely beneath the basement membrane zone, prior studies have indicated that the basement membrane, as defined by periodic acid-Schiff (PAS) or silver stains, lies at the floor of fully formed blisters or is destroyed by the disease process. To more accurately assess its location in primary lesions of DH, the basement membrane was stained using immunofluorescent techniques.Lesional skin from 5 patients with DH was used as substrate for indirect immunofluorescence with sera from patients with bullous pemphigoid (BP) and fluoresceinated antihuman IgG. The BP-stained basement membrane was attached to the roofs of early blisters, where it would be expected from the pathologic sequence of blister formation. PAS stains of the same or serial sections show the basement membrane to be in the roof or at the floor of the blisters. PAS stains of sections from formalin-fixed lesional skin, on the other hand, show the basement membrane to routinely lie at the blister floor, when not destroyed.The BP-stained epidermal basement membrane has greater anatomic and functional significance than either the PAS- or silver-stained basement membrane for two reasons: (1) it corresponds to a specific morphologic structure, the lamina lucida, a part of the epidermis, and remains attached to the rest of the epidermis unless destroyed; and (2) it is antigenic, capable of binding with BP antibodies

Evidence of in vivo exogen protein uptake by red blood cells: a putative therapeutic concept

For some molecular players in red blood cells (RBCs), the functional indications and molecular evidence are discrepant. One such protein is transient receptor potential channel of canonical subfamily, member 6 (TRPC6). Transcriptome analysis of reticulocytes revealed the presence of TRPC6 in mouse RBCs and its absence in human RBCs. We transfused TRPC6 knockout RBCs into wild-type mice and performed functional tests. We observed the “rescue” of TRPC6 within 10 days; however, the “rescue” was slower in splenectomized mice. The latter finding led us to mimic the mechanical challenge with the cantilever of an atomic force microscope and simultaneously carry out imaging by confocal (3D) microscopy. We observed the strong interaction of RBCs with the opposed surface at around 200 pN and the formation of tethers. The results of both the transfusion experiments and the atomic force spectroscopy suggest mechanically stimulated protein transfer to RBCs as a protein source in the absence of the translational machinery. This protein transfer mechanism has the potential to be utilized in therapeutic contexts, especially for hereditary diseases involving RBCs, such as hereditary xerocytosis or Gardos channelopathy

Red Blood Cell Membrane Conductance in Hereditary Haemolytic Anaemias

Electrophysiology; Haemolytic anemia; Hereditary spherocytosisElectrofisiologia; Anemia hemolítica; Esferocitosi hereditariaElectrofisiologia; Anèmia hemolítica; Esferocitosi hereditàriaCongenital haemolytic anaemias are inherited disorders caused by red blood cell membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and metabolic enzyme deficiencies. In many cases, although the causing mutation might be known, the pathophysiology and the connection between the particular mutation and the symptoms of the disease are not completely understood. Thus effective treatment is lagging behind. As in many cases abnormal red blood cell cation content and cation leaks go along with the disease, by direct electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a possible cause. We recorded whole-cell currents from 29 patients with different types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due to mutations in α-spectrin, β-spectrin, ankyrin and band 3 protein; 6 patients with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency and 1 patient with glutathione reductase deficiency), 1 patient with β-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with β-thalassemia and metabolic enzyme deficiencies showed no changes in their membrane conductance, the patients with hereditary spherocytosis and hereditary xerocytosis showed largely variable results depending on the underlying mutation

Lysophosphatidic Acid-Activated Calcium Signaling Is Elevated in Red Cells from Sickle Cell Disease Patients

(1) Background: It is known that sickle cells contain a higher amount of Ca2+ compared to healthy red blood cells (RBCs). The increased Ca2+ is associated with the most severe symptom of sickle cell disease (SCD), the vaso-occlusive crisis (VOC). The Ca2+ entry pathway received the name of Psickle but its molecular identity remains only partly resolved. We aimed to map the involved Ca2+ signaling to provide putative pharmacological targets for treatment. (2) Methods: The main technique applied was Ca2+ imaging of RBCs from healthy donors, SCD patients and a number of transgenic mouse models in comparison to wild-type mice. Life-cell Ca2+ imaging was applied to monitor responses to pharmacological targeting of the elements of signaling cascades. Infection as a trigger of VOC was imitated by stimulation of RBCs with lysophosphatidic acid (LPA). These measurements were complemented with biochemical assays. (3) Results: Ca2+ entry into SCD RBCs in response to LPA stimulation exceeded that of healthy donors. LPA receptor 4 levels were increased in SCD RBCs. Their activation was followed by the activation of Gi protein, which in turn triggered opening of TRPC6 and CaV2.1 channels via a protein kinase Cα and a MAP kinase pathway, respectively. (4) Conclusions: We found a new Ca2+ signaling cascade that is increased in SCD patients and identified new pharmacological targets that might be promising in addressing the most severe symptom of SCD, the VOC

Heavy metal distribution in some French forest soils: evidence for atmospheric contamination

This study is one of very few dealing with the distribution and the origin of heavy metals in French soils from a priori non-polluted forest areas. The abundance of heavy metals measured in these soils decreases as follows: Cr) Zn)Pb)Ni)Cu)Co4Cd. Total concentrations of Pb, Cr and Ni in some soils exceed the European thresholds for non-polluted soils and even the French association of normalization critical values for sludge spreading. The lowest heavy metal contents are observed in acid soils while the highest concentrations are in the calcaric cambisol and in the mollic andosol, which is rather scarce as compared with the other French forest soils. With the exception of the podzol, Cr and Ni concentrations increase with depth in all soil profiles. The distribution pattern of Co, Cu, Zn depends on the soil characteristics. In some acid soils, however, Cu and Zn decrease with depth. Pb and Cd are accumulated in the upper soil horizons. Heavy metals accumulate in deep soil horizons in relation to important clay content in the dystric planosol and stagnic luvisol. The concentration of each heavy metal is always controlled by different parameters (soil pH, iron and aluminum oxide content, clay content, organic matter and cation exchange capacity), which are heavy metal specific. This study highlights the metal-trapping character of andosol and calcaric soil, the weak heavy metal retention in acid soils, the leaching and trapping character in leached clayed soils, and the migration of heavy metals in the podzol. Pb and Cr concentrations indicate a significant enrichment in surface horizons from various soils in areas which receive significant acid atmospheric pollution. Particularly, the highest Pb content is observed in a soil located in the N-NE part of France. Lead isotope ratios measured in the cambic podzol and the calcaric cambisol, exhibit the importance of the anthropogenic sources and particularly the influence of global atmospheric inputs from leaded gasoline compared to regional and local industrial emissions. The anthropogenic Pb contribution is estimated to 83, 30 and 11%, respectively, for surface, intermediate and deep horizons of the cambic podzol located in the northern part of France, and to 68% in surface horizon of the calcaric cambisol located in the Alps