Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS-MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS-MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (␤A4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila melanogaster is used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in f ly tissue culture cells as well as in transgenic f ly lines. After expression of full-length APP forms, secretion of APP but not of ␤A4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the ␤A4 region, transmembrane domain, and cytoplasmic tail, we observed ␤A4 release in f lies and f ly-tissue culture cells. Consequently, we showed a ␥-secretase activity in f lies. Interestingly, transgenic f lies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion͞signaling pathways in Drosophila, independently of ␤A4 generation.The amyloid precursor protein (APP) is a ubiquitously expressed, integral membrane protein (1, 2). APP belongs to a protein family with two other members in mammals: the amyloid precursor-like protein 1 and 2 (APLP1 and APLP2, refs. 3-5). The central role of APP in the pathogenesis of Alzheimer's disease (AD, ref. 6) and its evolutionary conservation suggest important biological functions for the protein. Two homologues have been found in invertebrates: the amyloid protein-like protein 1 (APL-1) in Caenorhabditis elegans (7) and the amyloid precursor protein-like protein (APPL) in Drosophila melanogaster (8). Flies deficient for expression of APPL show phototaxis impairment and can be rescued by the expression of human APP (9). None of the homologous members of the APP protein family exhibit sequence similarities within the ␤-amyloid region of APP that encodes the characteristic 4-kDa proteinaceous component in vascular deposits and amyloid plaques of AD, ␤A4 (6). The ␤A4 peptide is cleaved from APP by unknown proteases termed ␤-secretase, which generates the N terminus and ␥-secretase, which releases the C terminus by cleaving within the transmembrane domain of APP. Differential cleavage by ␥-secretase produces ␤A4 of 40 or 42 amino acid residues in length with the 42-aa peptide being increased by distinct mutations in the APP and presenilin genes causing early onset familial AD (reviewed in ref. 6). A third cleavage of APP by ␣-secretase occurs within the ␤A4 domain and precludes ␤A4 formation.The 110-to 130-kDa ectodomain of APP generated by ␣-or ␤-secretase is secreted into the extracellular space.Despite the availability of APPL-deficient flies (9), APPnull mutants, or transgenic mice expressing human APP (10-12), the physiologi...

The chromatin protein Polycomb (PC) is necessary for keeping homeotic genes repressed in a permanent and heritable manner. PC is part of a large multimeric complex (PcG proteins) involved in generating silenced chromatin domains at target genes, thus preventing their inappropriate expression. In order to assess the intranuclear distribution of PC during mitosis in different developmental stages as well as in the germ line we generated transgenic fly lines expressing a PC-GFP (Green Fluorescent Protein) fusion protein. Rapidly dividing nuclei were found to display a rather homogeneous PC-GFP distribution. However, with increasing differentiation a pronounced subnuclear pattern was observed. In all investigated diploid somatic tissues the bulk of PC-GFP fusion protein is depleted from the chromosomes during mitosis: however, a detectable fraction remains associated. In the male germ line in early spermatogenesis, PC-GFP was closely associated with the chromosomal bivalents and gradually lost at later stages. Interestingly, we found that PC is associated with the nucleolus in spermatocytes, unlike somatic nuclei. In contrast to mature sperm showing no PC-GFP signal the female germ line retains PC in the germinal vesicle.

During embryonic development, the lymphatic system emerges by transdifferentiation from the cardinal vein. Although lymphatic and blood vasculature share a close molecular and developmental relationship, they display distinct features and functions. However, even after terminal differentiation, transitions between blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) have been reported. Since phenotypic plasticity and cellular differentiation processes frequently involve epigenetic mechanisms, we hypothesized that DNA methylation might play a role in regulating cell type-specific expression in endothelial cells. By analyzing global gene expression and methylation patterns of primary human dermal LEC and BEC, we identified a highly significant set of genes, which were differentially methylated and expressed. Pathway analyses of the differentially methylated and upregulated genes in LEC revealed involvement in developmental and transdifferentiation processes. We further identified a set of novel genes, which might be implicated in regulating BEC-LEC plasticity and could serve as therapeutic targets and/or biomarkers in vascular diseases associated with alterations in the endothelial phenotype.

Wound healing is a multistage process involving collaborative efforts of different cell types and distinct cellular functions. Among others, the high metabolic activity at the wound site requires the formation and sprouting of new blood vessels (angiogenesis) to ensure an adequate supply of oxygen and nutrients for a successful healing process. Thus, a cutaneous wound healing model was established to identify new factors that are involved in vascular formation and remodeling in human skin after embryonic development. By analyzing global gene expression of skin biopsies obtained from wounded and unwounded skin, we identified a small set of genes that were highly significant differentially regulated in the course of wound healing. To initially investigate whether these genes might be involved in angiogenesis, we performed siRNA experiments and analyzed the knockdown phenotypes using a scratch wound assay which mimics cell migration and proliferation in vitro. The results revealed that a subset of these genes influence cell migration and proliferation in primary human endothelial cells (EC). Furthermore, histological analyses of skin biopsies showed that two of these genes, ALBIM2 and TMEM121, are colocalized with CD31, a well known EC marker. Taken together, we identified new genes involved in endothelial cell biology, which might be relevant to develop therapeutics not only for impaired wound healing but also for chronic inflammatory disorders and/or cardiovascular diseases.