Martina Rudelius

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

Deep vein thrombosis initiation is mediated by cross talk between monocytes, neutrophils, and platelets.

Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo

Platelet—collagen interactions play a fundamental role in the process of arterial thrombosis. The major platelet collagen receptor is the glycoprotein VI (GPVI). Here, we determined the effects of a soluble dimeric form of GPVI on platelet adhesion in vitro and in vivo. We fused the extracellular domain of GPVI with the human immunoglobulin Fc domain. The soluble dimeric form of GPVI (GPVI‐Fc) specifically bound to immobilized collagen. Binding of GPVI‐Fc to collagen was inhibited competitively by soluble GPVI‐Fc, but not control Fc lacking the external GPVI domain. GPVI‐Fc inhibited the adhesion of CHO cells that stably express human GPVI and of platelets on collagen and attenuated thrombus formation under shear conditions in vitro. To test the effects of GPVI‐Fc in vivo, arterial thrombosis was induced in the mouse carotid artery, and platelet—vessel wall interactions were visualized by intravital fluorescence microscopy. Infusion of GPVI‐Fc but not of control Fc virtually abolished stable arrest and aggregation of platelets following vascular injury. Importantly, GPVI‐Fc but not control Fc, was detected at areas of vascular injury. These findings further substantiate the critical role of the collagen receptor GPVI in the initiation of thrombus formation at sites of vascular injury and identify soluble GPVI as a promising antithrombotic strategy.

Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

Platelet Adhesion Via Glycoprotein IIb Integrin Is Critical for Atheroprogression and Focal Cerebral Ischemia An In Vivo Study in Mice Lacking Glycoprotein IIb

Background—The platelet glycoprotein (GP) IIb/IIIa integrin binds to fibrinogen and thereby mediates platelet aggregation. Here, we addressed the role of GP IIb for platelet adhesion and determined the relevance of platelet GP IIb for the processes of atherosclerosis and cerebral ischemia-reperfusion (I/R) injury. Methods and Results—GP IIb−/− mice were generated and bred with ApoE−/− animals to create GP IIb−/−ApoE−/− mice. Platelet adhesion to the mechanically injured or atherosclerotic vessel wall was monitored by in vivo video fluorescence microscopy. In the presence of GP IIb, vascular injury and early atherosclerosis induced platelet adhesion in the carotid artery (CA). In contrast, platelet adhesion was significantly reduced in the absence of GP IIb integrin (P<0.05). To address the contribution of platelet GP IIb to atheroprogression, we determined atherosclerotic lesion formation in the CA and aortic arch (AA) of GP IIb+/+ApoE−/− or GP IIb−/−ApoE−/− mice. Interestingly, the absence of GP IIb attenuated lesion formation in CA and AA, indicating that platelets, via GP IIb, contribute substantially to atherosclerosis. Next, we assessed the implication of GP IIb for cerebral I/R injury. We observed that after occlusion of the middle cerebral artery, the cerebral infarct size was drastically reduced in mice lacking GP IIb compared with wild-types. Conclusions—These findings show for the first time in vivo that GP IIb not only mediates platelet aggregation but also triggers platelet adhesion to exposed extracellular matrices and dysfunctional endothelial cells. In a process strictly involving GP IIb, platelets, which are among the first blood cells to arrive at the scene of endothelial dysfunction, contribute essentially to atherosclerosis and cerebral I/R injury.

Platelets contribute to postnatal occlusion of the ductus arteriosus

The ductus arteriosus (DA) is a fetal shunt vessel between the pulmonary artery and the aorta that closes promptly after birth. Failure of postnatal DA closure is a major cause of morbidity and mortality particularly in preterm neonates. The events leading to DA closure are incompletely understood. Here we show that platelets have an essential role in DA closure. Using intravital microscopy of neonatal mice, we observed that platelets are recruited to the luminal aspect of the DA during closure. DA closure is impaired in neonates with malfunctioning platelet adhesion or aggregation or with defective platelet biogenesis. Defective DA closure resulted in a left-to-right shunt with increased pulmonary perfusion, pulmonary vascular remodeling and right ventricular hypertrophy. Our findings indicate that platelets are crucial for DA closure by promoting thrombotic sealing of the constricted DA and by supporting luminal remodeling. A retrospective clinical study revealed that thrombocytopenia is an independent predictor for failure of DA closure in preterm human newborns, indicating that platelets are likely to contribute to DA closure in humans.

Endothelial-like cells expanded from CD34+ blood cells improve left ventricular function after experimental myocardial infarction

Mobilization and recruitment of endothelial progenitor cells (EPC) contributes to vasculogenesis in vivo. So far, applications for cell therapy are limited by the number of available cells. Expansion of EPC or their progeny may, therefore, facilitate its therapeutic use in ischemic disease. The aim of this study was to expand CD34+ EPC‐derived progeny from different sources, characterize them, and investigate their potential for use in therapeutic vasculogenesis. CD34+ cells from G‐CSF‐mobilized peripheral blood (PB) and cord blood (CB) were isolated using immunomagnetic beads and cultured in endothelial cell medium. Cells were expanded up to 16 (PB) and up to 46 (CB) population doublings, respectively. Immunophenotypic and mRNA expression analyses showed a high degree of similarity between the cultured cells and human umbilical vein endothelial cells (HUVEC). By day 14 after transplantation, transplanted human CD31‐positive EPC‐derived cells were detected. These cells expressed the proliferation marker Ki67 and formed vessel‐like structures in ischemic myocardium. Most strikingly, transplantation of EPC‐derived cells improved left ventricular function after experimental ischemia, as shown by echocardiography. In conclusion, cells cultured from CD34+ EPC can be expanded in vitro to clinically relevant numbers. In vivo, these cells proliferate, form vascular structures, and improve left ventricular function after experimental myocardial infarction. Therefore, in vitro expanded EPC‐derived endothelial cells may be beneficial in the treatment of ischemic disease.

Therapeutic Implications for the Induced Levels of Chk1 in Myc-Expressing Cancer Cells

Purpose: The transcription factor c-Myc (or “Myc”) is a master regulator of pathways driving cell growth and proliferation. MYC is deregulated in many human cancers, making its downstream target genes attractive candidates for drug development. We report the unexpected finding that B-cell lymphomas from mice and patients exhibit a striking correlation between high levels of Myc and checkpoint kinase 1 (Chk1). Experimental Design: By in vitro cell biology studies as well as preclinical studies using a genetically engineered mouse model, we evaluated the role of Chk1 in Myc-overexpressing cells. Results: We show that Myc indirectly induces Chek1 transcript and protein expression, independently of DNA damage response proteins such as ATM and p53. Importantly, we show that inhibition of Chk1, by either RNA interference or a novel highly selective small molecule inhibitor, results in caspase-dependent apoptosis that affects Myc-overexpressing cells in both in vitro and in vivo mouse models of B-cell lymphoma. Conclusion: Our data suggest that Chk1 inhibitors should be further evaluated as potential drugs against Myc-driven malignancies such as certain B-cell lymphoma/leukemia, neuroblastoma, and some breast and lung cancers. Clin Cancer Res; 17(22); 7067–79. ©2011 AACR.

Breast cancers: MR imaging of folate-receptor expression with the folate-specific nanoparticle P1133.

PURPOSE To assess the capability of the folate receptor (FR)-targeted ultrasmall superparamagnetic iron oxide (USPIO) P1133 to provide FR-specific enhancement of breast cancers on magnetic resonance (MR) images. MATERIALS AND METHODS This study was approved by the institutional Animal Care and Use Committee. The FR-targeted contrast agent P1133 was incubated with various FR-positive human breast cancer cell lines, with and without free folic acid (FFA) as a competitor. Labeling efficiencies were evaluated with MR imaging and inductively coupled plasma mass spectrometry. Subsequently, six athymic rats with implanted FR-positive MDA-MB-231 breast cancers underwent MR imaging at 3 T before and up to 1 hour and 24 hours after injection of P1133. Six athymic rats with implanted FR-positive MDA-MB-231 cancers injected with the non-FR-targeted USPIO P904 and nine athymic rats with implanted FR-negative A549 lung cancers injected with P1133 (n = 6) or P904 (n = 3) served as controls. Data of the in vitro studies were compared for significant differences with the Wilcoxon test for two independent samples. Tumor signal-to-noise-ratios (SNRs) were compared between different experimental groups by using the Kruskal-Wallis test and were correlated with histopathologic findings. Differences with P < .05 were considered significant. RESULTS FR-positive breast cancer cells showed a significant P1133 uptake which was inhibited by FFA. MDA-MB-231 cells showed the highest level of P1133 uptake and the strongest T2 effect on MR images. In vivo, all tumors showed an initial perfusion effect. At 24 hours after injection, only MDA-MB-231 tumors injected with P1133 showed significantly decreased SNR data compared with baseline data (P < .05). MR findings were confirmed by using histopathologic findings. CONCLUSION The FR-targeted USPIO P1133 demonstrates a specific retention in FR-positive breast cancers. Because FR expression correlates with tumor aggressiveness and prognosis, persistent P1133 tumor enhancement may be used as a noninvasive indicator for tumors with poor outcome.

Protein Kinase C-β-Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B Cells In Vivo

Summary Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-βII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-β knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-βII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.

Highly efficient paramagnetic labelling of embryonic and neuronal stem cells

Recent developments in stem cell and gene therapy will require methods to monitor stem cell survival and integration repeatedly and non-invasively with a high temporal and spatial resolution in vivo. The aim of this study was to visualise embryonic and neuronal stem cells with standard contrast agents using a conventional clinical 1.5-Tesla scanner. We therefore modified standard transfection protocols including lipofection (Lipofectin and Lipofectamine) and calcium phosphate transfection for the efficient uptake of paramagnetic particles [gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA)] in stem cells. Using this approach we obtained intracellular labelling efficiencies of up to 83%. Neither the proliferation capacity nor the differentiation efficiency was affected. Identical differentiation of labelled and unlabelled embryonic and neuronal cells was observed. The established labelling techniques used in this study displayed high labelling efficiencies in embryonic and neuronal stem cells without any alterations of cellular biology; therefore this approach might be a suitable method for targeting stem cells.