Publications

Abstract Prominin‐1 (Prom1) is recognized as a stem cell marker in several tissues, including blood, neuroepithelium, and gut, and in human and mouse embryos and many cancers. Although Prom1 is routinely used as a marker for isolating stem cells, its biological function remains unclear. Here we use a knockout model to investigate the role of Prom1 in the mammary gland. We demonstrate that complete loss of Prom1 does not affect the regenerative capacity of the mammary epithelium. Surprisingly, we also show that in the absence of Prom1, mammary glands have reduced ductal branching, and an increased ratio of luminal to basal cells. The effects of Prom1 loss in the mammary gland are associated with decreased expression of prolactin receptor and matrix metalloproteinase‐3. These experiments reveal a novel, functional role for Prom1 that is not related to stem cell activity, and demonstrate the importance of tissue‐specific characterization of putative stem cell markers. Developmental Dynamics 240:674–681, 2011. © 2011 Wiley‐Liss, Inc.

GRAZ-01-21 Introduction: Symptoms of generalized inflammatory syndromes are related to polymorphonuclear neutrophil (PMN) adhesion to microvascular endothelium and their consecutive extravazation. The urokinase receptor (u-PAR) has recently been shown to be critically involved in the regulation of the activity of β-integrin function. The role of u-PAR in inflammation has not been fully elucidated. Methods: PMN were percoll-purified from hind limb bone marrow of wild-type (wt) or u-PAR-deficient (−/−) mice and fluorescently labelled. PMN were superfused in a parallel plate flow chamber with a shear rate of 400s−1 over resting or TNFα stimulated murine endothelial cells (ECs) grown on collagen coated coverslips. Firm adhesion in this system is mainly mediated by MAC-1/ICAM-1. PMN adhesion (/20 mm2 of the coverslip) is reported as percentage of wt-PMN adhesion to resting ECs. u-PAR−/− or wt mice were injected i.p. with 30 or 45 μg g−1 bodyweight E. coli endotoxin (LPS) and left ventricular performance (by transthoracic echocardiography), survival time and mortality were recorded. Results: Compared to resting ECs adhesion of WT PMN increased to 172 ± 34% after 6-h stimulation with 100 nM TNFα (n = 5, P < 0.01), while u-PAR−/− PMN completely failed to increase adhesion to activated endothelium (99 ± 26% vs. 73 ± 26%, n = 6/5, P = NS resting vs. TNFα and P < 0.01, u-PAR−/− -TNFα vs. wt-TNFα). 68% of the u-PAR−/− (n = 22) but only 33% of the wt mice (n = 21) survived an injection of 30 μg g−1 LPS i.p. When 45 μg LPS was injected, only a trend for decreased mortality was detected after 72 h (43% vs. 25% survival, u-PAR−/− vs. wt, n = 21/20) but a significantly prolonged time to death was observed (29 ± 5 h vs. 16 ± 6 h, u-PAR−/− vs. wt). Increased survival was not fully explained by differences in LV function as 90 min after LPS injection shortening fraction (69 ± 3% vs. 62 ± 2%, u-PAR vs. wt, n = 5, P < 0.05) and velocity of circumferential fibre shortening (Vcf) were similar or only marginally different between groups. Conclusions: u-PAR expression on PMN is required for adequate PMN adhesion to activated EC. Lack of u-PAR protects mice from endotoxin induced systemic inflammatory responses and death, which is only in part related to cardiac function. Regulation of u-PAR mediated activation of integrins could be a suitable target for reducing inflammatory responses.

Objective— Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset neurovascular disorder caused by stereotyped mutations in the NOTCH3 receptor. Elucidation of its pathobiology is still incomplete and remains a challenge, in part because the available preclinical mouse models to date do not reproduce the full spectrum of CADASIL pathology and clinical disease. Methods and Results— Here, we report a novel knock-in mouse with Arg170Cys substitution in murine Notch3, corresponding to the prevalent Arg169Cys substitution in CADASIL. The Notch3 Arg170Cys mice displayed late-onset, dominant CADASIL arteriopathy with typical granular osmiophilic material deposition and developed brain histopathology including thrombosis, microbleeds, gliosis, and microinfarction. Furthermore, Notch3 Arg170Cys mice experienced neurological symptoms with motor defects such as staggering gait and limb paresis. Conclusion— This model, for the first time, phenocopies the arteriopathy and the histopathologic as well as clinical features of CADASIL and may offer novel opportunities to investigate disease pathogenesis.

The combination of erlotinib and bevacizumab is well tolerated, however, with low clinical activity in patients with chemoresistant UGI cancer. Some patients do benefit from the therapy, and uPAR forms are potential biomarkers in these patients.

Rationale: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/ Slc2a1 ) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood. Objective: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB. Methods and Results: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets. Conclusions: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.

Abstract Placenta growth factor (PlGF; formerly PGF), a vascular endothelial growth factor gene family member, is expressed in human implantation sites by maternal uterine NK (uNK) and fetal trophoblast cells. Lower than normal concentrations of blood and urinary PlGF have been associated with impending onset of pre-eclampsia, a hypertensive disease of late human gestation characterized by limited intravascular trophoblast invasion. In pregnant rodents, delivery of the PlGF antagonist sFlt-1 or S-endoglin induces pre-eclampsia-like lesions. Mice genetically deleted in PlGF reproduce, but neither their implantation sites nor their uNK cell development are described. We combined real-time PCR of endometrium from nonpregnant and gestation day (gd)6–18 C57BL6/J (B6) mice with immunohistology to analyze PlGF expression in normal mouse pregnancy. To estimate the significance of uNK cell-derived PlGF, PlGF message was quantified in mesometrial decidua from pregnant alymphoid Rag2 null/common γ chain null mice and in laser capture-microdissected B6 uNK cells. Histopathologic consequences from PlGF deletion were also characterized in the implantation sites from PlGF null mice. In B6, decidual PlGF expression rose between gd8–16. uNK cells were among several types of cells transcribing PlGF in decidualized endometrium. Immature uNK cells, defined by their low numbers of cytoplasmic granules, were the uNK cells displaying the greatest number of transcripts. PlGF deletion promoted the early differentiation high numbers of binucleate uNK cells (gd8) but had no other significant, morphometrically detectable impact on implantation sites. Thus, in mice, PlGF plays an important role in successful uNK cell proliferation and/or differentiation.