Gabriele Jedlitschky

Leeches of the genus Placobdella Blanchard, 1893 (Annelida: Rhynchobdellida: Glossiphoniidae) are mainly distributed across North and Central America, but at least two species are also present in the palearctic region: the European turtle leech Placobdella costata Müller, 1846, and the North African turtle leech Placobdella nabeulensis Ben Ahmed et al., 2023. All species of the genus are hematophagous and feed on vertebrates, but are known to preferentially target aquatic and semi-aquatic reptiles like turtles and snakes. Placobdella ornata Verrill, 1872, is the original source of ornatins, a class of potent platelet aggregation inhibitors. However, the whole repertoire of bioactive peptides, including anticoagulation factors remains largely underexplored for Placobdella , and functional characterizations of putative antithrombotics are scarce. Here we describe the genes and cDNAs that encode putative hirudins and ornatins in both of the palearctic and three American species of the genus Placobdella . A selection of putative hirudins and ornatins was recombinantly expressed and functionally characterized using appropriate coagulation and platelet aggregation assays, and our data confirm the expression of functional hirudins and ornatins in the studied leeches. In addition, our genetic analyses strongly support the hypothesis that blood feeding evolved only once in the evolutionary history of leeches.

The multidrug resistance protein 4 (MRP4/ABCC4) is a versatile efflux pump, known to transport several drugs but also signaling molecules such as cyclic nucleotides and lipid mediators. Based on this substrate spectrum and its broad tissue distribution, MRP4 plays a significant physiological and pathophysiological role in both the cardiovascular and oncological fields. However, the determinants of its gene expression are still incompletely defined. This study aimed to identify key regulatory elements and transcription factors that are essential for basal MRP4 expression. Using luciferase reporter assays with a series of 5′‐deletion constructs, we identified a region upstream of the transcription start site as crucial for basal expression across diverse cell types. This region is evolutionary highly conserved and contains putative binding sites for Sp1 and Ets transcription factors. Site‐directed mutagenesis of both binding elements resulted in a significant decrease in the promoter activity in HeLa and megakaryoblastic M07e cells. The binding of Sp1 to this region was further confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Finally, siRNA knockdown of Sp1 led to a significant decrease in MRP4 protein levels and function. In summary, we show that Sp1 binds to the MRP4 promoter and plays an essential role in the basal expression of MRP4 , with Ets factors also potentially cooperating in this regulation.

Medicinal leeches express a broad variety of anticoagulants and other bioactive factors that are involved in the blood feeding process. For most of the anticoagulants, several genes exist that may encode isoforms of the respective proteins, including hirudins and decorsins. Decorsins negatively affect platelet aggregation, whereas hirudins are potent thrombin inhibitors. Both factors belong to the hirudin superfamily that also includes the group of hirudin-like factors, and all hematophagous leeches analyzed so far contain several gene copies that encode representatives of at least two of the three groups of these factors. Members of the hirudin superfamily may contain only one central globular domain, but others may contain two or more copies. Here we describe the molecular identification and partial functional characterization of a broad variety of putative mono-, bi and multimeric hirudins, decorsins and hirudin-like factors in two Asian medicinal leech species, namely Hirudo nipponia and H. tianjinensis . Some of the monomeric hirudins and decorsins have already been described before, but they represent only a small part of the overall diversity. For the first time, putative monomeric and one oligomeric decorsins of H. tianjinensis were expressed as recombinant proteins, functionally characterized and successfully verified as platelet aggregation inhibitors. In addition we have conducted phylogenetic analyses based on genomic and mitochondrial markers and found convincing evidence that H. nipponia and H. tianjinensis together with members of the genus Whitmania form a monophyletic clade that is clearly distinct from clades that are formed either by European members of the genus Hirudo or by members of the genus Hirudinaria .

Hematophagous leeches rely on a broad diversity of bioactive factors that interfere with the host’s defense systems to secure a successful blood meal. The most prominent examples of such factors are the antithrombotics that address either the primary hemostasis (the platelet aggregation) or the secondary hemostasis (the blood coagulation). Whereas the inhibitors of platelet aggregation mainly cause continuous blood flow, coagulation inhibitors mainly keep the blood fluid within the stomach of the leech. The critical dependency of hematophagous leeches on the accurate action of all antithrombotic factors may explain the presence of multiple genes for each type of these factors that is regularly observed in leech genomes. The genome of Hirudinaria manillensis Lesson, 1842, the Asian buffalo leech, contains five individual genes that encode variants of the coagulation inhibitor hirudin. However, no genes that encode putative decorsins, the archetype of leech-derived inhibitors of platelet aggregation, have been described to date. Here, we report the identification of one monomeric and four multimeric decorsin encoding genes in the genome of H. manillensis. A selection of the putative decorsins was expressed as recombinant proteins, purified and functionally characterized. All but one of these putative decorsins displayed platelet aggregation-inhibitory potencies. Furthermore, we provide a mathematical calculation based on the mechanism of alternative pre-mRNA splicing that illustrates the potential to generate an enormous variety of different factors from one single multimeric ornatin gene.

Glioblastoma (GBM) is still a deadly tumor due to its highly infiltrative growth behavior and its resistance to therapy. Evidence is accumulating that sphingosine-1-phosphate (S1P) acts as an important tumor-promoting molecule that is involved in the activation of the S1P receptor subtype 1 (S1PR1). Therefore, we investigated the effect of ACT-209905 (a putative S1PR1 modulator) on the growth of human (primary cells, LN-18) and murine (GL261) GBM cells. The viability and migration of GBM cells were both reduced by ACT-209905. Furthermore, co-culture with monocytic THP-1 cells or conditioned medium enhanced the viability and migration of GBM cells, suggesting that THP-1 cells secrete factors which stimulate GBM cell growth. ACT-209905 inhibited the THP-1-induced enhancement of GBM cell growth and migration. Immunoblot analyses showed that ACT-209905 reduced the activation of growth-promoting kinases (p38, AKT1 and ERK1/2), whereas THP-1 cells and conditioned medium caused an activation of these kinases. In addition, ACT-209905 diminished the surface expression of pro-migratory molecules and reduced CD62P-positive GBM cells. In contrast, THP-1 cells increased the ICAM-1 and P-Selectin content of GBM cells which was reversed by ACT-209905. In conclusion, our study suggests the role of S1PR1 signaling in the growth of GBM cells and gives a partial explanation for the pro-tumorigenic effects that macrophages might have on GBM cells.

Sphingosine-1-phosphate (S1P) is a versatile signaling lipid involved in the regulation of numerous cellular processes. S1P regulates cellular proliferation, migration, and apoptosis as well as the function of immune cells. S1P is generated from sphingosine (Sph), which derives from the ceramide metabolism. In particular, high concentrations of S1P are present in the blood. This originates mainly from erythrocytes, endothelial cells (ECs), and platelets. While erythrocytes function as a storage pool for circulating S1P, platelets can rapidly generate S1P de novo, store it in large quantities, and release it when the platelet is activated. Platelets can thus provide S1P in a short time when needed or in the case of an injury with subsequent platelet activation and thereby regulate local cellular responses. In addition, platelet-dependently generated and released S1P may also influence long-term immune cell functions in various disease processes, such as inflammation-driven vascular diseases. In this review, the metabolism and release of platelet S1P are presented, and the autocrine versus paracrine functions of platelet-derived S1P and its relevance in various disease processes are discussed. New pharmacological approaches that target the auto- or paracrine effects of S1P may be therapeutically helpful in the future for pathological processes involving S1P.

The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.

Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.