Qiao

A Metabolic Shift Favoring Sphingosine 1-Phosphate at the Expense of Ceramide Controls Glioblastoma Angiogenesis

Background: The sphingolipid metabolite sphingosine 1-phosphate (S1P) is a potent angiogenic factor. Results: S1P content is 9-fold higher in glioblastomas compared with normal brain, and S1P production is necessary for glioblastoma cells to trigger endothelial cell angiogenesis. Conclusion: Excessive S1P synthesis is a major contributor to glioblastoma angiogenesis. Significance: Inhibiting S1P synthesis may be a valuable antiangiogenic approach in glioblastoma. Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.

A fluorescent assay for ceramide synthase activity

The sphingolipids are a diverse family of lipids with important roles in membrane compartmentalization, intracellular signaling, and cell-cell recognition. The central sphingolipid metabolite is ceramide, formed by the transfer of a variable length fatty acid from coenzyme A to a sphingoid base, generally sphingosine or dihydrosphingosine (sphinganine) in mammals. This reaction is catalyzed by a family of six ceramide synthases (CerS1-6). CerS activity is usually assayed using either radioactive substrates or LC-MS/MS. We describe a CerS assay with fluorescent, NBD-labeled sphinganine as substrate. The assay is readily able to detect endogenous CerS activity when using amounts of cell or tissue homogenate protein that are lower than those reported for the radioactive assay, and the Michaelis-Menten constant was essentially the same for NBD-sphinganine and unlabeled sphinganine, indicating that NBD-sphinganine is a good substrate for these enzymes. Using our assay, we confirm that the new clinical immunosuppressant FTY720 is a competitive inhibitor of CerS activity, and show that inhibition requires the compounds lipid tail and amine headgroup. In summary, we describe a fluorescent assay for CerS activity that circumvents the need to use radioactive substrates, while being more accessible and cheaper than LC-MS based assays.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Significance In this report we describe the generation of tissue-regenerative multipotent stem cells (iMS cells) by treating mature bone and fat cells transiently with a growth factor [platelet-derived growth factor–AB (PDGF-AB)] and 5-Azacytidine, a demethylating compound that is widely used in clinical practice. Unlike primary mesenchymal stem cells, which are used with little objective evidence in clinical practice to promote tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming tumors. This method can be applied to both mouse and human somatic cells to generate multipotent stem cells and has the potential to transform current approaches in regenerative medicine. Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Disparate in vivo efficacy of FTY720 in xenograft models of Philadelphia positive and negative B-lineage acute lymphoblastic leukemia.

Most patients with acute lymphoblastic leukemia (ALL) respond well to standard chemotherapy-based treatments. However a significant proportion of patients, particularly adult patients, relapse with the majority dying of leukemia. FTY720 is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of hematological malignancies. Using human ALL xenografts in NOD/SCIDγc−/− mice, we show for the first time that three Ph+ human ALL xenografts responded to FTY720 with an 80±12% (p = 0.048) reduction in overall disease when treatment was commenced early. In contrast, treatment of mice with FTY720 did not result in reduced leukemia compared to controls using four separate human Ph− ALL xenografts. Although FTY720 reactivated PP2A in vitro, this reactivation was not required for death of Ph− ALL cells. The plasma levels of FTY720 achieved in the mice were in the high nanomolar range. However, the response seen in the Ph+ ALL xenografts when treatment was initiated early implies that in vivo efficacy may be obtained with substantially lower drug concentrations than those required in vitro. Our data suggest that while FTY720 may have potential as a treatment for Ph+ ALL it will not be a useful agent for the treatment of Ph− B-ALL.

SMAD1 and SMAD5 Expression Is Coordinately Regulated by FLI1 and GATA2 during Endothelial Development.

ABSTRACT The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1+63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortas in vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cells in vitro. In contrast, CD31+ cKit− endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reduced Smad1 but not Smad5 transcript levels. This is suggestive of a degree of in vivo selection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage.

Arrested Hematopoiesis and Vascular Relaxation Defects in Mice with a Mutation in Dhfr

ABSTRACT Dihydrofolate reductase (DHFR) is a critical enzyme in the folate metabolism pathway and also plays a role in regulating nitric oxide (NO) signaling in endothelial cells. Although both coding and noncoding mutations with phenotypic effects have been identified in the human DHFR gene, no mouse model is currently available to study the consequences of perturbing DHFR in vivo. In order to identify genes involved in definitive hematopoiesis, we performed a forward genetic screen and produced a mouse line, here referred to as Orana, with a point mutation in the Dhfr locus leading to a Thr136Ala substitution in the DHFR protein. Homozygote Orana mice initiate definitive hematopoiesis, but expansion of progenitors in the fetal liver is compromised, and the animals die between embryonic day 13.5 (E13.5) and E14.5. Heterozygote Orana mice survive to adulthood but have tissue-specific alterations in folate abundance and distribution, perturbed stress erythropoiesis, and impaired endothelium-dependent relaxation of the aorta consistent with the role of DHFR in regulating NO signaling. Orana mice provide insight into the dual roles of DHFR and are a useful model for investigating the role of environmental and dietary factors in the context of vascular defects caused by altered NO signaling.

Identification of Novel Embryonic Vascular Stem-like cells and their role in Coronary Vasculature development and repair

Several studies have attempted to repair the damaged spinal cord (SC) by stimulating neurogenesis or neuroplasticity, with limited success. Sonic hedgehog (Shh) is involved in neural induction and stem cell functioning, but recent findings also suggest its role in regeneration and functional recovery. TDP-43 is a nuclear protein involved in mRNA processing. Recently, TDP-43 has been found in the cytoplasmic inclusions observed in amyotrophic lateral sclerosis. Substantial attention has been devoted to the toxic effects of TDP-43, whereas the functional role of this protein remains poorly investigated. We used a mouse model of neurotoxic motoneuron depletion to study the role of the above-described factors in the compensatory changes occurring after the lesion. The injection of cholera toxin-B saporin into the gastrocnemius muscle caused a partial motoneuron death accompanied by a glial reaction and an impairment of locomotion. Interestingly, motor activity was significantly restored as soon as one month later. Moreover, we observed an activitydependent modification of Shh and synaptic proteins (synapsin-I and AMPA receptors). Notably, the motor performance of lesioned animals correlated with the expression of synapsin-I and Shh. Conversely, the expression of Shh significantly correlated with the levels of synapsin-I, GluR2 and TDP-43. The results suggest that Shh and TDP-43 are crucial part of a complex mechanism of neuroplasticity in a mouse model of SC motoneuron disease.

Genetic Fate Mapping of Mesenchymal Stem-Like Cells in the Aorta-Gonad Mesonephros (AGM) and Their Contribution to Definitive Hematopoiesis

Preimplantation development in rodent and primate establishes the founding cell population of the foetus in the epiblast and segregates two extraembryonic lineages, trophoblast and hypoblast. Most of our current knowledge about these cell-fate decisions is derived from studies in mouse. However, transcriptional profiling of human embryos has suggested substantial differences to the mouse paradigm. Here, we set out to delineate the primate-specific aspects of preimplantation development. We present a high-quality single-cell RNA-seq dataset from zygote to late blastocyst in marmoset (Callithrix jacchus). In addition, we generated stage-matched samples in mouse (Mus musculus) and re-analysed three human single-cell datasets. Weighted gene network analysis independently identified the establishment of epiblast and hypoblast transcriptional modules. NANOG, SOX2, TDGF1 and TFCP2L1 were highly expressed in the epiblast of all three species. In contrast, KLF17, ARGFX, KHDC3L, LEFTY2 and CTSF represented primate-specific factors of the pluripotency network in vivo. Global features of epiblast and hypoblast segregation included the ERK cascade, apoptosis and extracellular matrix, while we identified elevated levels of BMP and WNT signalling components in primates. Strikingly, the mouse epiblast marker Otx2 is specifically expressed in human and marmoset hypoblast. Our cross-species analysis approach demarcates conserved and primate-specific features of mammalian preimplantation development and provides a rich resource for comparative embryology.