Loss of ASAP1 in mice impairs adipogenic and osteogenic differentiation of mesenchymal progenitor cells through dysregulation of FAK/Src and AKT signaling.

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT/GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells.

VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.

VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41+ cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41+ cells in the BM and a significant increase in diploid CD41+ cells, whereas the number of polyploid CD41+ cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.

Delphinidin is a novel inhibitor of lymphangiogenesis but promotes mammary tumor growth and metastasis formation in syngeneic experimental rats.

We have recently demonstrated that the anthocyanidin delphinidin (DEL), one of the most abundant dietary flavonoids, inhibits activation of ErbB and vascular endothelial growth factor receptor family members. These receptors play crucial roles in the context of tumor progression and the outgrowth of blood and lymphatic vessels. Here, we have developed an improved chemical synthesis for DEL in order to study the effects of the aglycon and its degradation product gallic acid (GA) on endothelial and tumor cells in vitro and in vivo. We found that DEL blocked the proliferation in vitro of primary human blood and lymphatic endothelial cells as well as human HT29 colon and rat MT-450 mammary carcinoma cells in a dose-dependent manner. In contrast, its degradation product GA had little effect. At higher concentrations, DEL induced apoptosis of endothelial and tumor cells. Furthermore, DEL potently blocked the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In the MT-450 rat syngeneic breast tumor model, it also significantly reduced angiogenesis and tumor-induced lymphangiogenesis when administered in vivo. These data reveal DEL to be a novel antilymphangiogenesis reagent. Surprisingly, however, the application of DEL unexpectedly promoted tumor growth and metastasis in the MT-450 tumor model, suggesting that the antiproliferative effect of DEL on cultured cells does not necessarily reflect the response of tumors to this anthocyanidin in vivo. Furthermore, while DEL may have utility as a cancer chemopreventative agent, its ability to promote tumor growth once a neoplasm develops also needs to be taken into consideration.

Discovery of a novel tumour metastasis-promoting gene, NVM-1.

We have previously reported that over-expression of a panel of 119 genes correlates with the metastatic potential of pancreatic carcinoma cells. We sought to identify and functionally characterize candidate tumour metastasis promoting genes among this library using a secondary phenotype-assisted screen. Here we report the discovery of the metastasis-promoting function of a hitherto not characterized gene located on chromosome 14 (ORF138), which we have named 'novel metastasis-promoting gene 1' (NVM-1). The NVM-1 transcript is extensively alternatively spliced, is expressed endogenously in a number of different tissues, and is strongly over-expressed at the protein level in a variety of human tumour types. Importantly, NVM-1 expression stimulates the migratory and invasive behaviour of tumour cells and promotes metastasis formation in experimental animals in vivo. Up-regulation of FMNL2 and MT1E and down-regulation of TIMP4 and MHC-I is observed as a consequence of NVM-1 expression. Together these data identify NVM-1 as a gene that is functionally involved in tumour metastasis, and suggest that NVM-1 may constitute a promising therapeutic target for inhibition of tumour metastasis.

Anti-oxidative and inflammatory responses induced by fly ash particles and carbon black in lung epithelial cells.

Combustion-derived nanoparticles as constituents of ambient particulate matter have been shown to induce adverse health effects due to inhalation. However, the components inducing these effects as well as the biological mechanisms are still not fully understood. The fine fraction of fly ash particles collected from the electrostatic precipitator of a municipal solid waste incinerator was taken as an example for real particles with complex composition released into the atmosphere to study the mechanism of early biological responses of BEAS-2B human lung epithelial cells. The studies include the effects of the water-soluble and -insoluble fractions of the fly ash and the well-studied carbon black nanoparticles were used as a reference. Fly ash induced reactive oxygen species (ROS) and increased the total cellular glutathione (tGSH) content. Carbon black also induced ROS generation; however, in contrast to the fly ash, it decreased the intracellular tGSH. The fly ash-induced oxidative stress was correlated with induction of the anti-oxidant enzyme heme oxygenase-1 and increase of the redox-sensitive transcription factor Nrf2. Carbon black was not able to induce HO-1. ROS generation, tGSH increase and HO-1 induction were only induced by the insoluble fraction of the fly ash, not by the water-soluble fraction. ROS generation and HO-1 induction were markedly inhibited by pre-incubation of the cells with the anti-oxidant N-acetyl cysteine which confirmed the involvement of oxidative stress. Both effects were also reduced by the metal chelator deferoxamine indicating a contribution of bioavailable transition metals. In summary, both fly ash and carbon black induce ROS but only fly ash induced an increase of intracellular tGSH and HO-1 production. Bioavailable transition metals in the solid water-insoluble matrix of the fly ash mostly contribute to the effects.

Hyperforin and aristoforin inhibit lymphatic endothelial cell proliferation in vitro and suppress tumor-induced lymphangiogenesis in vivo.

The phloroglucinol derivative hyperforin, a major bioactive constituent of St. John's wort, is increasingly recognized as being able to regulate a variety of pathobiological processes and, thus, to possess potential therapeutic properties. In the context of cancer, hyperforin induces the apoptosis of cancer cells, inhibits angiogenesis and suppresses metastasis formation. Here, we report a new pharmacological function of hyperforin and its stabilized derivative aristoforin, namely the suppression of lymphatic endothelial cell (LEC) growth and lymphangiogenesis. At concentrations less than 10 microM, we found that these compounds induce cell cycle arrest of LECs, and at higher concentrations induce apoptosis. The loss of mitochondrial membrane potential and the activation of caspase-9 during the induction of apoptosis indicate that the intrinsic pathway of apoptosis is stimulated by these compounds, similar to the situation in tumor cells. In thoracic duct ring outgrowth assays, hyperforin and aristoforin both inhibited lymphangiogenesis, as evidenced by the suppression of lymphatic capillary outgrowth. In an in vivo animal model, both compounds were able to inhibit tumor-induced lymphangiogenesis. Together these data substantiate a new role for hyperforin and its derivatives as suppressors of lymphangiogenesis, and support their further investigation as potential anticancer drugs that target tumor growth and metastasis at multiple levels.