Distinct influence of the anthracycline derivative doxorubicin on the differentiation efficacy of mESC-derived endothelial progenitor cells.

Cardiotoxicity is a highly relevant, because often life-threatening, adverse effect of doxorubicin (Doxo)-based anticancer therapy. Here, we investigated the Doxo-response of cardiovascular stem/progenitor cells employing a mouse embryonic stem cell (mESC)-based in vitro differentiation model. Endothelial progenitor cells revealed a pronounced Doxo sensitivity as compared to mESC, differentiated endothelial-like (EC) and cardiomyocyte-like cells (CM) and CM progenitors, which rests on the activation of senescence. Doxo treatment of EC progenitors altered protein expression of individual endothelial markers, actin cytoskeleton morphology, mRNA expression of genes related to mitochondrial functions, autophagy, apoptosis, and DNA repair as well as mitochondrial DNA content, respiration and ATP production in the surviving differentiated EC progeny. By contrast, LDL uptake, ATP-stimulated Ca2+ release, and cytokine-stimulated ICAM-1 expression remained unaffected by the anthracycline treatment. Thus, exposure of EC progenitors to Doxo elicits isolated and persistent dysfunctions in the surviving EC progeny. In conclusion, we suggest that Doxo-induced injury of EC progenitors adds to anthracycline-induced cardiotoxicity, making this cell-type a preferential target for pharmacoprotective and regenerative strategies.

Profiling of a suramin-derived compound library at recombinant human P2Y receptors identifies NF272 as a competitive but non-selective P2Y2 receptor antagonist.

Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y2 receptor antagonist with a Ki of 19 µM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 receptors. Up to 250 µM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.

mESC-based in vitro differentiation models to study vascular response and functionality following genotoxic insults.

Because of high exposure to systemic noxae, vascular endothelial cells (EC) have to ensure distinct damage defense and regenerative mechanisms to guarantee vascular health. For meaningful toxicological drug assessments employing embryonic stem cell (ESC)-based in vitro models, functional competence of differentiated progeny and detailed knowledge regarding damage defense mechanisms are essential. Here, mouse ESCs (mESC) were differentiated into functionally competent vascular cells (EC and smooth muscle cells [SMC]). mESC, EC, and SMC were comparatively analyzed regarding DNA repair and DNA damage response (DDR). Differentiation was accompanied by both congruent and unique alterations in repair and DDR characteristics. EC and SMC shared the downregulation of genes involved cell cycle regulation and repair of DNA double-strand breaks (DSBs) and mismatches, whereas genes associated with nucleotide excision repair (NER), apoptosis, and autophagy were upregulated when compared with mESC. Expression of genes involved in base excision repair (BER) was particularly low in SMC. IR-induced formation of DSBs, as detected by nuclear γH2AX foci formation, was most efficient in SMC, the repair of DSBs was fastest in EC. Together with substantial differences in IR-induced phosphorylation of p53, Chk1, and Kap1, the data demonstrate complex alterations in DDR capacity going along with the loss of pluripotency and gain of EC- and SMC-specific functions. Notably, IR exposure of early vascular progenitors did not impair differentiation into functionally competent EC and SMC. Summarizing, mESC-based vascular differentiation models are informative to study the impact of environmental stressors on differentiation and function of vascular cells.

Comparing the gene expression profile of stromal cells from human cord blood and bone marrow: lack of the typical "bone" signature in cord blood cells.

With regard to the bone-regenerative capacity, bone marrow stromal cells (BMSC) can still be termed the "gold standard." Nevertheless, neonatal stromal cells from cord blood (CB) feature advantages concerning availability, immaturity, and proliferation potential. The detailed gene expression analysis and overexpression of genes expressed differentially provide insight into the inherent capacity of stromal cells. Microarray and qRT-PCR analyses revealed closely related gene expression patterns of two stromal cell populations derived from CB. In contrast to the CB-derived cell types, BMSC displayed high expression levels of BSP, OSX, BMP4, OC, and PITX2. Lentiviral overexpression of BSP but not of OSX in CB-cells increased the capacity to form a mineralized matrix. BMP4 induced the secretion of proteoglycans during chondrogenic pellet culture and extended the osteogenic but reduced the adipogenic differentiation potential. BMSC revealed the typical osteogenic gene expression signature. In contrast, the CB-derived cell types exhibited a more immature gene expression profile and no predisposition towards skeletal development. The absence of BSP and BMP4-which were defined as potential key players affecting the differentiation potential-in neonatal stromal cells should be taken into consideration when choosing a cell source for tissue regeneration approaches.