Angiopoietin-2 exocytosis is stimulated by sphingosine-1-phosphate in human blood and lymphatic endothelial cells.

OBJECTIVE: Although diverse functions of angiopoietin-2 (Ang2) have been revealed, little is known about upstream signaling molecules regulating Ang2 exocytosis. We therefore investigated the mechanism of Ang2 exocytosis in human blood and lymphatic endothelial cells (BECs and LECs) by stimulation with sphingosine-1-phosphate (S1P). METHODS AND RESULTS: By immunostaining and ELISA analyses using our newly developed human Ang2-specific antibodies, Ang2 exocytosis from human endothelial cells was examined. Both exogenous and endogenous S1P trigger rapid Ang2 exocytosis in time- and dose-dependent manners. Intriguingly, S1P-induced Ang2 exocytosis is higher in LECs than BECs. These effects of S1P are mainly mediated by the endothelial differentiation gene receptor 1, which subsequently activates its downstream phospholipase C and intracellular calcium mobilization to trigger Ang2 exocytosis. Consistently, S1P also dramatically stimulates Ang2 exocytosis from the ECs of ex vivo-incubated blood vessels. CONCLUSION: These results imply that the rapid secretion of Ang2 by exocytosis from endothelial cells is another possible mechanism underlying S1P-induced angiogenesis and inflammation.

Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts.

Human mesenchymal stromal cells (MSCs) offer great hope for the treatment of tissue degenerative and immune diseases, but their phenotypic similarity to dermal fibroblasts may hinder robust cell identification and isolation from diverse tissue harvests. To identify genetic elements that can reliably discriminate MSCs from fibroblasts, we performed comparative gene and microRNA expression profiling analyses with genome-wide oligonucleotide microarrays. When taken globally, both gene and microRNA expression profiles of MSCs were highly similar to those of fibroblasts, accounting well for their extensive phenotypic and functional overlaps. Scattered expression differences were pooled to yield an MSC-specific molecular signature, consisting of 64 genes and 21 microRNAs whose expressions were at least 10-fold and two-fold higher, respectively, in MSCs compared with fibroblasts. Genes either encoding transmembrane proteins or associated with tumors were relatively abundant in this signature. These data should provide the molecular basis not only for the discovery of novel diagnostic markers discriminating MSCs from fibroblasts, but also for further studies on MSC-specific signaling mechanisms.

Fibroblast activation protein alpha identifies mesenchymal stromal cells from human bone marrow.

Mesenchymal stromal cells (MSCs) have gained widespread popularity in cell therapy, but their development into clinical products has been impeded by the scarcity of cell-specific markers. We previously explored transcriptome and membrane proteome of MSCs, from which fibroblast activation protein alpha (FAP) was recognized as a prime surface marker candidate. The present study showed that FAP was constitutively expressed on MSCs, but not on other cells. FAP immunoselection yielded homogeneous MSCs from cryopreserved bone marrow (BM). These results suggest that FAP serves as a surface protein marker that can singly define MSCs from BM and possibly from other sources.

Proteomic analysis of the hydrophobic fraction of mesenchymal stem cells derived from human umbilical cord blood.

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering, but their application has been impeded by lack of knowledge of their core biological properties. In order to identify MSC-specific proteins, the hydrophobic protein fraction was individually prepared from two different umbilical cord blood (UCB)-derived MSC populations; these were then subjected to two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). Although the 2D gel patterns differed somewhat between the two samples, computer-assisted image analysis identified shared protein spots. 35 spots were reliably identified corresponding to 32 different proteins, many of which were chaperones. Based on their primary sub-cellular locations the proteins could be grouped into 6 categories: extracellular, cell surface, endoplasmic reticular, mitochondrial, cytoplasmic and cytoskeletal proteins. This map of the water-insoluble proteome may provide valuable insights into the biology of the cell surface and other compartments of human MSCs.

Effect of chemical chaperone addition on production and aggregation of recombinant flag-tagged COMP-angiopoietin 1 in Chinese hamster ovary cells.

To investigate the effect of chemical chaperones on the production and aggregation of flag-tagged cartilage oligomeric matrix protein-Angiopoietin1 (FCA1) in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in serum-free media with various chemical chaperones, 1 mM 4-phenylbutyrate (4-PBA), 200 mM proline, 3% glycerol, 2% dimethyl sulfoxide (DMSO), and without chemical chaperone as control. The addition of chemical chaperones enhanced FCA1 production and specific FCA1 productivity, q(FCA)(1). For example, the q(FCA)(1) at 200 mM proline was fourfold higher than that at control. Unlike q(FCA)(1), the aggregation of FCA1 was strongly affected by which chemical chaperone was added. The addition of 2% DMSO and 200 mM proline significantly reduced the proportion of aggregates, but the addition of 1 mM 4-PBA and 3% glycerol was hardly effective. The proportions of aggregates were 29.5 and 55.6% at 2% DMSO and 200 mM proline, respectively, whereas it was 79.6% at control. The exact mechanism how chemical chaperones affected the aggregation of FCA1 was not investigated in this study, and therefore, more extensive works will be needed to clarify why different chemical chaperones behaved differently in reducing the aggregation of FCA1. Among chemical chaperones tested, DMSO was the most effective one in regard to enhancing the production and reducing the aggregation of FCA1 in CHO cells.

Double antiangiogenic protein, DAAP, targeting VEGF-A and angiopoietins in tumor angiogenesis, metastasis, and vascular leakage.

Two vascular growth factor families, VEGF and the angiopoietins, play critical and coordinate roles in tumor progression and metastasis. A single inhibitor targeting both VEGF and angiopoietins is not available. Here, we developed a chimeric decoy receptor, namely double anti-angiogenic protein (DAAP), which can simultaneously bind VEGF-A and angiopoietins, blocking their actions. Compared to VEGF-Trap or Tie2-Fc, which block either VEGF-A or angiopoietins alone, we believe DAAP is a highly effective molecule for regressing tumor angiogenesis and metastasis in implanted and spontaneous solid tumors; it can also effectively reduce ascites formation and vascular leakage in an ovarian carcinoma model. Thus, simultaneous blockade of VEGF-A and angiopoietins with DAAP is an effective therapeutic strategy for blocking tumor angiogenesis, metastasis, and vascular leakage.

Use of NaCl prevents aggregation of recombinant COMP-angiopoietin-1 in Chinese hamster ovary cells.

To investigate the effect of hyperosmotic medium on production and aggregation of the variant of Angiopoietin-1 (Ang1), cartilage oligomeric matrix protein (COMP)-Ang1, in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in shaking flasks. NaCl and/or sorbitol were used to raise medium osmolality in the range of 300-450mOsm/kg. The specific productivity of COMP-Ang1, q(COMP-Ang1), increased as medium osmolality increased. At NaCl-450mOsm/kg, the q(COMP-Ang1) was 7.7-fold higher than that at NaCl-300mOsm/kg, while, at sorbitol-450mOsm/kg, it was 2.9-fold higher than that at sorbitol-300mOsm/kg. This can be attributed to the increased relative mRNA level of COMP-Ang1 at NaCl-450mOsm/kg which was approximately 2.4-fold higher than that at sorbitol-450mOsm/kg. Western blot analysis showed that COMP-Ang1 aggregates started to occur in the late-exponential phase of cell growth. When sorbitol was used to raise the medium osmolality, a severe aggregation of COMP-Ang1 was observed. On the other hand, when NaCl was used, the aggregation of COMP-Ang1 was drastically reduced at NaCl-400mOsm/kg. At NaCl-450mOsm/kg, the aggregation of COMP-Ang1 was hardly observed. This suggests that environmental conditions are critical for the aggregation of COMP-Ang1. Taken together, the use of NaCl-induced hyperosmotic medium to cell culture process turns out to be an efficient strategy for enhancing COMP-Ang1 production and reducing COMP-Ang1 aggregation.

Genome-wide differential gene expression profiling of human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) reside in bone marrow and provide a lifelong source of new cells for various connective tissues. Although human BMSCs are regarded as highly suitable for the development of cell therapeutics and regenerative medicine, the molecular factors and the networks of signaling pathways responsible for their biological properties are as yet unclear. To gain a comprehensive understanding of human BMSCs at the transcriptional level, we have performed DNA microarray-based, genome-wide differential gene expression analysis with the use of peripheral blood-derived mononuclear cells (MNCs) as a baseline. The resulting molecular profile of BMSCs was revealed to share no meaningful overlap with those of other human stem cell types, suggesting that the cells might express a unique set of genes for their stemness. By contrast, the distinct molecular signature, consisting of 92 different genes whose expression strengths are at least 50-fold higher in BMSCs compared with MNCs, was shown to encompass largely a gene subset of umbilical cord blood-derived adherent cells, suggesting that adherent cells derived from bone marrow and umbilical cord blood may be defined by a common set of genes, regardless of their origin. Intriguingly, a large number of these genes, particularly ones for extracellular matrix products, coincide with normal or tumor endothelium-specific markers. Taken together, our results here provide a BMSC-specific genetic catalog that may facilitate future studies on molecular mechanisms governing core properties of these cells.

Membrane proteomic analysis of human mesenchymal stromal cells during adipogenesis.

Mesenchymal stromal cells (MSCs) have proven useful for cell and immune therapy, but the molecular constituents responsible for their functionalities, in particular, those on the plasma membrane, remain largely unknown. Here we employed both gel and nongel based MS to analyze human MSCs' membrane proteome before and after adipogenesis. 2-DE of cells that were pretreated with membrane impermeable fluorescent dyes revealed that both the whole cell proteome and the cell surface subproteome were independent of donors. LC coupled with tandem MS analysis of the plasma membrane-containing fraction allowed us to identify 707 proteins, approximately half of which could be annotated as membrane-related proteins. Of particular interest was a subset of ectodomain-containing membrane-bound proteins that encompass most known surface markers for MSCs, but also contain a multitude of solute carriers and ATPases. Upon adipogenic differentiation, this proteomic profile was amended to include several proteins involved in lipid metabolism and trafficking, at the expense of, most noticeably, ectoenzymes. Our results here provide not only a basis for future studies of MSC-specific molecular mechanisms, but also a molecular inventory for the development of antibody-based cell isolation and identification procedures.