The platelet-derived growth factor receptor alpha promoter-directed expression of cre recombinase in mouse placenta.

BackgroundNumerous pathologies of pregnancy originate from placental dysfunction. It is essential to understand the functions of key genes in the placenta in order to discern the etiology of placental pathologies. A paucity of animal models that allow conditional and inducible expression of a target gene in the placenta is a major limitation for studying placental development and function.ResultsTo study the platelet-derived growth factor receptor alpha (PDGFRα)-directed and tamoxifen-induced Cre recombinase expression in the placenta, PDGFRα-CreER mice were crossed with mT/mG dual-fluorescent reporter mice. The expression of endogenous membrane-localized enhanced green fluorescent protein (mEGFP) and/or dTomato in the placenta was examined to identify PDGFRα promoter-directed Cre expression. Pregnant PDGFRα-CreER;mT/mG mice were treated with tamoxifen at various gestational ages. Upon tamoxifen treatment, reporter protein mEGFP was observed in the junctional zone (JZ) and chorionic plate (CP). Furthermore, a single dose of tamoxifen was sufficient to induce the recombination.ConclusionsPDGFRα-CreER expression is restricted to the JZ and CP of mouse placentas. PDGFRα-CreER mice provide a useful tool to conditionally knock out or overexpress a target gene in these regions of the mouse placenta

Sox9-Meis1 Inactivation Is Required for Adipogenesis, Advancing Pref-1+ to PDGFRα+ Cells.

Adipocytes arise from the commitment and differentiation of adipose precursors in white adipose tissue (WAT). In studying adipogenesis, precursor markers, including Pref-1 and PDGFRα, are used to isolate precursors from stromal vascular fractions of WAT, but the relation among the markers is not known. Here, we used the Pref-1 promoter-rtTA system in mice for labeling Pref-1+ cells and for inducible inactivation of the Pref-1 target Sox9. We show the requirement of Sox9 for the maintenance of Pref-1+ proliferative, early precursors. Upon Sox9 inactivation, these Pref-1+ cells become PDGFRα+ cells that express early adipogenic markers. Thus, we show that Pref-1+ cells precede PDGFRα+ cells in the adipogenic pathway and that Sox9 inactivation is required for WAT growth and expansion. Furthermore, we show that in maintaining early adipose precursors, Sox9 activates Meis1, which prevents adipogenic differentiation. Our study also demonstrates the Pref-1 promoter-rtTA system for inducible gene inactivation in early adipose precursor populations

Aberrant oligodendroglial-vascular interactions disrupt the blood-brain barrier, triggering CNS inflammation.

Disruption of the blood-brain barrier (BBB) is critical to initiation and perpetuation of disease in multiple sclerosis (MS). We report an interaction between oligodendroglia and vasculature in MS that distinguishes human white matter injury from normal rodent demyelinating injury. We find perivascular clustering of oligodendrocyte precursor cells (OPCs) in certain active MS lesions, representing an inability to properly detach from vessels following perivascular migration. Perivascular OPCs can themselves disrupt the BBB, interfering with astrocyte endfeet and endothelial tight junction integrity, resulting in altered vascular permeability and an associated CNS inflammation. Aberrant Wnt tone in OPCs mediates their dysfunctional vascular detachment and also leads to OPC secretion of Wif1, which interferes with Wnt ligand function on endothelial tight junction integrity. Evidence for this defective oligodendroglial-vascular interaction in MS suggests that aberrant OPC perivascular migration not only impairs their lesion recruitment but can also act as a disease perpetuator via disruption of the BBB

Essential role for PDGF signaling in ophthalmic trigeminal placode induction

Much of the peripheral nervous system of the head is derived from ectodermal thickenings, called placodes, that delaminate or invaginate to form cranial ganglia and sense organs. The trigeminal ganglion, which arises lateral to the midbrain, forms via interactions between the neural tube and adjacent ectoderm. This induction triggers expression of Pax3, ingression of placode cells and their differentiation into neurons. However, the molecular nature of the underlying signals remains unknown. Here, we investigate the role of PDGF signaling in ophthalmic trigeminal placode induction. By in situ hybridization, PDGF receptor β is expressed in the cranial ectoderm at the time of trigeminal placode formation, with the ligand PDGFD expressed in the midbrain neural folds. Blocking PDGF signaling in vitro results in a dose-dependent abrogation of Pax3 expression in recombinants of quail ectoderm with chick neural tube that recapitulate placode induction. In ovo microinjection of PDGF inhibitor causes a similar loss of Pax3 as well as the later placodal marker, CD151, and failure of neuronal differentiation. Conversely, microinjection of exogenous PDGFD increases the number of Pax3+ cells in the trigeminal placode and neurons in the condensing ganglia. Our results provide the first evidence for a signaling pathway involved in ophthalmic (opV) trigeminal placode induction

Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

Adipocyte differentiation of bovine adipose-derived stem cells (ASC) was induced by foetal bovine serum (FBS), biotin, pantothenic acid, insulin, rosiglitazone, dexamethasone and 3-isobutyl-1-methylxanthine, followed by incubation in different media to test the influence of ascorbic acid (AsA), bovine serum lipids (BSL), FBS, glucose and acetic acid on transdifferentiation into functional adipocytes. Moreover, different culture plate coatings (collagen-A, gelatin-A or poly-L-lysine) were tested. The differentiated ASC were subjected to Nile red staining, DAPI staining, immunocytochemistry and quantitative reverse transcription PCR (for NT5E, THY1, ENG, PDGFRα, FABP4, PPARγ, LPL, FAS, GLUT4). Nile red quantification showed a significant increase in the development of lipid droplets in treatments with AsA and BSL without FBS. The presence of BSL induced a prominent increase in FABP4 mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of NT5E, ENG and THY1 mRNA decreased or tended to decrease in the absence of FBS, and ENG was additionally suppressed by AsA. DAPI fluorescence was higher in cells cultured in poly-L-lysine or gelatin-A coated wells. In additional experiments, the multi-lineage differentiation potential to osteoblasts was verified in medium containing ß-glycerophosphate, dexamethasone and 1,25-dihydroxyvitamin D3 using alizarin red staining. In conclusion, bovine ASC are capable of multi-lineage differentiation. Poly-L-lysine or gelatin-A coating, the absence of FBS, and the presence of BSL and AsA favour optimal transdifferentiation into adipocytes. AsA supports transdifferentiation via a unique role in FABP4 induction, but this is not linearly related to the primarily BSL-driven lipid accumulation

Targeting a therapeutic LIF transgene to muscle via the immune system ameliorates muscular dystrophy.

Many potentially therapeutic molecules have been identified for treating Duchenne muscular dystrophy. However, targeting those molecules only to sites of active pathology is an obstacle to their clinical use. Because dystrophic muscles become extensively inflamed, we tested whether expressing a therapeutic transgene in leukocyte progenitors that invade muscle would provide selective, timely delivery to diseased muscle. We designed a transgene in which leukemia inhibitory factor (LIF) is under control of a leukocyte-specific promoter and transplanted transgenic cells into dystrophic mice. Transplantation diminishes pathology, reduces Th2 cytokines in muscle and biases macrophages away from a CD163+/CD206+ phenotype that promotes fibrosis. Transgenic cells also abrogate TGFβ signaling, reduce fibro/adipogenic progenitor cells and reduce fibrogenesis of muscle cells. These findings indicate that leukocytes expressing a LIF transgene reduce fibrosis by suppressing type 2 immunity and highlight a novel application by which immune cells can be genetically modified as potential therapeutics to treat muscle disease

Targeting kinases with anilinopyrimidines: Discovery of N-phenyl-N'-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives as selective inhibitors of class III receptor tyrosine kinase subfamily

Kinase inhibitors are attractive drugs/drug candidates for the treatment of cancer. The most recent literature has highlighted the importance of multi target kinase inhibitors, although a correct balance between specificity and non-specificity is required. In this view, the discovery of multityrosine kinase inhibitors with subfamily selectivity is a challenging goal. Herein we present the synthesis and the preliminary kinase profiling of a set of novel 4-anilinopyrimidines. Among the synthesized compounds, the N-phenyl-N\u2019-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives selectively targeted some members of class III receptor tyrosine kinase family. Starting from the structure of hit compound 19 we synthesized a further compound with an improved affinity toward the class III receptor tyrosine kinase members and endowed with a promising antitumor activity both in vitro and in vivo in a murine solid tumor model. Molecular modeling simulations were used in order to rationalize the behavior of the title compounds

PDGF Receptor-α Does Not Promote HCMV Entry into Epithelial and Endothelial Cells but Increased Quantities Stimulate Entry by an Abnormal Pathway

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128–131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα–transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128–131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128–131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types