BackgroundIn the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34 + cells and mesenchymal stromal cells with regard to different spatial localizations. Design and MethodsHypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay. ResultsIn co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factorindependent manner. ConclusionsWe demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.

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The Ser/Thr-protein kinase Pim-1 has been discovered as a novel transducer of survival- and cell cycle promoting signals in the hematopoietic cell system. Although its significance for proliferation of vascular smooth muscle cells (VSMC) in vitro and neointima formation in vivo has been suggested recently, the mechanism has barely been characterized. This study aimed to foster the understanding of Pim-1 expression and regulation in murine VSMC in response to factors typically present within the atherosclerotic plaque. While oxidative stress, VEGF-A165 and angiotensin II did not have any effect on Pim-1 expression, VSMC strongly increased (3-fold) Pim-1 mRNA upon stimulation with PDGF(bb), followed by its protein upregulation. Half life of Pim-1 RNA and protein were determined to be 25 min and 6 h, respectively. PDGF(bb) induced a strong, 10-fold increase in BrdU-uptake, a marker of proliferation. This was effectively blocked by either Pim-1-specific inhibitor quercetagetin or adenovirally introduced Pim-1 shRNA. We further identified the signaling pathways linking PDGF(bb) to Pim-1 in VSMC: as expected, we determined transcriptional stimulation of Pim-1 via Janus-activated kinase (Jak), but also an additional pathway involving protein kinase C (PKC) and the mitogen-activated protein kinase Mek1/2. Blockade of Akt signaling did, however, not interfere with Pim-1 upregulation, suggesting an independence of either survival system. PDGF(bb)-induced proliferation of VSMC is partly attributed to transcriptionally upregulated Pim-1 and was assigned to distinct cell signaling. Our findings help to understand the fundamental processes of vasculoproliferative diseases thus opening avenues for its prevention and treatment.

The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3´UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3´UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodysplastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche. MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o nproteases. 11,12 Furthermore, CXCL12 expression can be transcriptionally modulated by a variety of cytokines and growth factors, namely transforming growth factor-beta 1 (TGFB1). TGFB1 has a negative growth effect on HSPC which is mediated, in part, by the regulation of CXCL12 arising from stromal cells. 13 Thus, engraftment and response to cytotoxic drugs may vary according to CXCL12 levels provided by niche cells. 14,15 Recently, Pillai et al. suggested that CXCL12 can also be regulated by microRNA (miRNA) in human marrow stromal cells. 16 There are more than 1000 miRNA which form 1-2% of the human genome. Approximately half of human structural genes are predicted to be under miRNA control. In animals, miRNA act by targeting the 3ú ntranslated region (UTR) of genes with the consequence of repressing output of the respective protein. A classical switch interaction is used to avoid protein expression in a particular cell type, whereas tuning interactions are needed to supply the cell with the optimal level of protein. 17 In the hematopoietic system, CXCL12 must be fine-tuned in response to differing physiological requirements.We, therefore, decided to study the interaction of CXCL12 and miRNA by applying a strategy of overexpression of a library to reveal the various miRNA responsible for CXCL12 regulation in primary human bone marrow stromal cells (hBMSC). Moreover, we mapped expression of candidate miRNA in primary hBMSC to understand their physiological function in fine tuning CXCL12 expression in the hematopoietic niche. Methods Cell linesHS5, HS27, HL60, K562, KG1a, and HeLa cell lines were ob...

Arteries and veins show marked differences in their anatomy, physiology and genetic expression pattern. In this study, we analyzed impact of overexpression or downregulation of arterial marker gene Hey2 and venous marker gene COUP-TFII in human venous and arterial endothelial cells on genes involved in arteriovenous differentiation. Lentiviral overexpression of venous marker gene COUP-TFII in arterial endothelial cells led to downregulation of NICD4, arterial marker gene Hey2 and EphrinB2. Downregulation of Hey2 could be mediated by direct binding of COUP-TFII to Hey2 promoter as shown by ChIP, EMSA and promoter analysis. Downregulation of Hey2 by shRNA causes downregulation of EphrinB2 expression. Overexpression of arterial marker Hey2 in venous endothelial cells did not change expression pattern of COUP-TFII. Downregulation of venous marker gene COUP-TFII in venous endothelial cells resulted in upregulation of VEGF-A, Dll4 and EphrinB2 expression. Our data support an important role of Hey2 and COUP-TFII in arteriovenous differentiation of human endothelial cells.