Publications

Abstract Context Heavy menstrual bleeding (HMB) is common and debilitating, but the precise endometrial mechanisms causing increased menstrual blood loss (MBL) remain undefined. We have previously identified a role for hypoxia in endometrial repair following progesterone withdrawal. Objective As hypoxia inducible factor 2 alpha (HIF2A) is known to alter vascular function in other tissues, we hypothezised that endometrial HIF2A is involved in premenstrual optimization of endometrial function during the secretory phase to limit MBL. Results Women with objective HMB had higher endometrial HIF2A during the mid-secretory phase when compared to those with normal MBL (P = 0.0269). In a mouse model of simulated menses, genetic or pharmacological manipulation of HIF2A did not significantly affect endometrial breakdown/repair, volume of MBL or endometrial hypoxia. However, 88% of Hif2a heterozygote mice reached early-full repair by 24 hours vs only 65% of wild-type mice. Mean MBL was 0.39 μL (±0.67) in Hif2a heterozygote mice vs 0.98 μL (±0.79) in wild-type mice. Conversely, when we increased HIF2A before menstruation, 11% reached early repair by 8 hours vs 30% of vehicle-treated mice. Mean MBL was 2.61 μL (±1.10) in mice with HIF2A stabilization and 2.24 μL (±1.14) in vehicle-treated mice. These nonsignificant but consistent trends indicate that increased endometrial HIF2A may contribute to delayed endometrial repair and HMB. Conclusions Increased HIF2A in the secretory endometrium is unlikely to be sufficient to account for the phenotype of HMB, but limitation of HIF2 levels may optimize endometrial function at menstruation.

<div>Abstract<p><b>Purpose:</b> BCR-ABL kinase inhibitors are employed successfully for chronic myeloid leukemia (CML) treatment. However, resistant disease and persistence of BCR-ABL1–independent leukemia stem and progenitor cells (LSPC) remain clinical challenges. The receptor tyrosine kinase Axl can mediate survival and therapy resistance of different cancer cells. We investigated the therapeutic potential of Axl inhibition in CML.</p><p><b>Experimental Design:</b> We used primary cells from patients with CML and TKI-sensitive and -resistant BCR-ABL1<sup>+</sup> CML cell lines and a novel ponatinib-resistant cell line KCL-22 PonR. We analyzed the effects of genetic and pharmacologic Axl blockade by the small-molecule Axl inhibitor BGB324 <i>in vitro</i> and <i>in vivo</i>. In BCR-ABL1–unmutated cells, we also investigated BGB324 in combination with imatinib.</p><p><b>Results:</b> We demonstrate overexpression of Axl receptor tyrosine kinase in primary cells of patients with CML compared with healthy individuals and a further increase of Axl expression in BCR-ABL TKI-resistant patients. We show that Axl blockage decreased growth of BCR-ABL TKI-sensitive CML cells including CD34<sup>+</sup> cells and exerts additive effects with imatinib via inhibition of Stat5 activation. BGB324 also inhibits BCR-ABL TKI-resistant cells, including T315I-mutated and ponatinib-resistant primary cells. BGB324 exerted therapeutic effects in BCR-ABL1 T315I-mutated and ponatinib-resistant preclinical mouse models. Notably, BGB324 does not inhibit BCR-ABL1 and consequently inhibits CML independent of BCR-ABL1 mutational status.</p><p><b>Conclusions:</b> Our data show that Axl inhibition has therapeutic potential in BCR-ABL TKI-sensitive as well as -resistant CML and support the need for clinical trials. <i>Clin Cancer Res; 23(9); 2289–300. ©2016 AACR</i>.</p></div>

In the adult organism the cellular distribution of tissue factor (TF) expression corresponds to biological boundary layers forming a hemostatic barrier ready to activate blood coagulation after tissue injury. Whether TF expression might also play a role in development is unknown. To determine the significance of TF in ontogenesis, we examined the pattern of TF expression in mouse development and compared it with the distribution of TF in human post-implantation embryos and fetuses of corresponding gestational age. At early embryonic periods of murine (6.5 and 7.5 pc) and human (stage 5) development, there was strong expression of TF in both ectodermal and entodermal cells. In situ hybridization and immunohistochemistry demonstrated that TF mRNA and protein were expressed widely in epithelial areas with high levels of morphogenic activity during organogenesis. Staining for TF was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express TF in the adult organism. Surprisingly, during renal development and in adults, expression of TF differed between humans and mice. In humans, maturing stage glomeruli were stained for TF whereas in mice, TF was absent from glomeruli but was present in the epithelia of tubular segments. In neuroepithelial cells, there was a substantial expression of TF. Moreover, there was robust TF expression in tissues such as skeletal muscle and pancreas, which do not express it in the adult. In contrast, expression of the physiological ligand for TF, factor VII, was not detectable during early stages of human embryogenesis using immunohistochemistry. The temporal and spatial pattern of TF expression during murine and human development supports the contention that TF serves as an important morphogenic factor during embryogenesis.

Poster P-428<br/>Poster P-428

Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing “outside-in” signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the β3 integrin, thereby amplifying outside-in signaling via αIIbβ3. Blocking the Gas6-R–αIIbβ3 integrin cross-talk might be a novel approach to the reduction of thrombosis.

Abstract Cell recruitment is a multistep process regulated by cytokines, chemokines, and growth factors. Previous work has indicated that the urokinase plasminogen activator receptor (uPAR) may also play a role in this mechanism, presumably by an interaction with the β2 integrin CD11b/CD18. Indeed, an essential role of uPAR in neutrophil recruitment during pulmonary infection has been demonstrated for β2 integrin-dependent respiratory pathogens. We investigated the role of uPAR and urokinase plasminogen activator (uPA) during pneumonia caused by a β2 integrin-independent respiratory pathogen, Streptococcus pneumoniae. uPAR-deficient (uPAR−/−), uPA-deficient (uPA−/−), and wild-type (Wt) mice were intranasally inoculated with 105 CFU S. pneumoniae. uPAR−/− mice showed reduced granulocyte accumulation in alveoli and lungs when compared with Wt mice, which was associated with more S. pneumoniae CFU in lungs, enhanced dissemination of the infection, and a reduced survival. In contrast, uPA−/− mice showed enhanced host defense, with more neutrophil influx and less pneumococci in the lungs compared with Wt mice. These data suggest that uPAR is necessary for adequate recruitment of neutrophils into the alveoli and lungs during pneumonia caused by S. pneumoniae, a pathogen eliciting a β2 integrin-independent inflammatory response. This function is even more pronounced when uPAR is unoccupied by uPA.

Summary Mice with a total deficiency in blood coagulation Factor X (FX) were generated by targeted replacement of an 18-kb fragment of the FX gene, comprising all exons encoding the mature FX protein, with a neor cassette. The genotype distribution among the offspring from heterozygous breeding pairs suggested that FX deficiency resulted in partial embryonic lethality, with approximately one-third of the FX −/− embryos dying around embryonic day (E) 11.5-12.5. Two of 44 non-resorbed FX −/− embryos analyzed at these stages showed signs of massive bleeding, one of which into the brain ventricles, but no histological defects in the vasculature of these embryos or their yolk sac were observed. The remainder of the FX −/− embryos appeared normal and survived to term, but the majority of neonates (90%) died within 5 days, most frequently from intraabdominal bleeding. The remaining FX −/− animals succumbed between postnatal day (P)5 and P20 with intraabdominal, subcutaneous, or intracranial bleeding or a combination thereof. The lethal phenotype of the FX −/− mice illustrates the importance of FX function in embryonic and postnatal survival and demonstrates that these mice serve as effective models of the bleeding disorders observed in severe FX deficiency in humans.