Publications

Essentials of Angiogenesis

Absence of microRNA-155 protects against adverse cardiac inflammation and hypertrophy during pressure overload and prevents heart failure

Primary Graft Dysfunction: Shifting from Static Grading to Temporal Classification

Lipid availability determines fate of skeletal progenitor cells via SOX9

Metabolic and Innate Immune Cues Merge into a Specific Inflammatory Response via the UPR

PAR1 contributes to influenza A virus pathogenicity in mice

Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.

The autophagy-blocker chloroquine promotes tumor vessel normalization by increasing Notch-signaling

S84 Identification of MIR-124a as a major regulator of enhanced endothelial cell glycolysis in pulmonary arterial hypertension

<h3>Introduction</h3> Pulmonary arterial hypertension (PAH) is a rare desease characterised by profound vascular abnormalities in the peripheral arteries of the lung, leading to a progressive increase in pulmonary vascular resistance, right heart failure and death. The disease exists in several forms including a heritable form (HPAH) caused primarily by mutations in bone morphogenetic protein receptor type 2 (BMPR2) and an idiopathic form (IPAH). Endothelial cell (EC) dysfunction is considered a critical initiating factor in the pathobiology of PAH, manifested by increased susceptibility to apoptosis, heightened permeability and enhanced endothelial proliferation. Substantial changes in bioenergetics of ECs, including higher rates of glycolysis, have been reported in PAH patients. However, the mechanisms underlying alterations in energy production have not been identified. <h3>Methods</h3> We measured glycolysis in blood outgrowth endothelial cells (BOECs) from HPAH patients carrying mutations in BMPR2 and IPAH patients to confirm the metabolic abnormalities previously. We also employed an unbiased genome-wide microarray and proteomic screening approach to detect miRNAs and proteins dysregulated in the same groups to determine the mechanisms underlying abnormal endothelial glycolysis. <h3>Results</h3> HPAH and IPAH BOECs recapitulated the metabolic phenotype previously observed in PAECs. These alterations were found to be associated with the downregulation of miR-124 and the upregulation of its known target, splicing factor polypyrimidine-tract-binding protein (PTBP1). We also demonstrated that increased PTBP1 promotes the switching in expression of two forms of pyruvate kinase, PKM1 and PKM2, resulting in an increase of aerobic glycolysis, consequently increasing cell proliferation (mechanism schematized in Figure 1). Overexpression of miR-124, or siRNA silencing of PTPB1, restoring normal expression levels of PKM2, also restored normal proliferation and glycolysis in HPAH BOECs. Finally, we observed reduced miR-124 and increased PTPB1 and PKM2 expression in a well-established rat model of PAH, characterised by endothelial proliferation, supporting the presence of this mechanism <i>in vivo</i>. <h3>Conclusions</h3> Loss of function of BMPR2 results in the downregulation of miR-124 and consequently in the glycolytic abnormalities reported in PAH ECs. Therefore, the manipulation of this miRNA, or its targets, could represent a novel and effective strategy to achieve clinical benefits in the treatment of PAH.

Placental Growth Factor: a Novel, Stromal-Derived Target in Human CML.

Abstract Abstract 42 Although BCR-ABL kinase inhibitors (TKIs) have revolutionized treatment of CML, they do not eradicate disease and treatment resistance can emerge. We recently identified Placental Growth Factor (PlGF) as an additional pathogenic factor in murine BCR-ABL1+ leukemia. PlGF induces proliferation of bone marrow stromal cells and of BCR-ABL1+ cell lines. PlGF is upregulated in blood (PB) and bone marrow (BM) of mice with BCR-ABL1+ leukemia and it's inhibition by a monoclonal antibody (αPlGF) significantly prolongs survival of mice bearing BCR-ABL1-unmutated and -T315I mutant blast crisis (BC) CML (Loges et al., Blood 2008; 112: abstract 1094). Now, we have further characterized the role of PlGF in disease progression, the mechanism of action of αPlGF and validated PlGF as novel target in human CML. We first quantified PB and BM PlGF protein levels at different time points after inoculation of BCR-ABL1 transduced BM cells and found significantly increasing PlGF levels upon disease progression. In line with those data, PB and BM PlGF protein levels correlated with numbers of GFP-marked leukemia cells (r=0.81; p=&lt;0.001 and r=0.85; p=0.001, respectively). We unraveled that PlGF is almost exclusively expressed by the non-hematopoietic (CD45-), non-leukemic (GFP-) stromal cell compartment. In vitro data indicate specific PlGF upregulation by CD45- BM stromal cells upon co-culture with BCR-ABL1+ leukemia cells, but not in hematopoietic cells or endothelial cells. Hence, PlGF is a stromal target induced by interaction with BCR-ABL+ leukemia cells and significantly correlates with disease burden. Neutralization of PlGF by αPlGF and/or its genetic deletion prolong survival of mice bearing BCR-ABL1+ leukemia. Therefore we analyzed the anti-leukemic efficacy of αPlGF treatment by measuring GFP+ leukemia cells in the PB of mice at an early time-point (d14) and at end-stage disease (d28). We found a reduction of leukemia cells by 42 and 51% in mice treated with αPlGF when compared control mice (p&lt;0.05). Furthermore, we analyzed the effect of PlGF inhibition on the leukemic BM microenvironment. Morphometric analysis of CD31 immunostaining revealed reduced microvessel density (MVD) in αPlGF treated mice compared to controls (236+15 vs. 307+17 vessels/mm2; p=0.006). Investigation of reticulin fibers indicated that αPlGF reduces the prominent fibrosis of the BM occurring upon disease progression by 30% (p=0.001), which complements the finding that PlGF expands BM derived stromal cells in vitro. Hence, αPlGF reduces the leukemic burden and normalizes the abnormal leukemic BM by reducing hypervascularization and BM fibrosis. To investigate the relevance of PlGF as novel target molecule in human CML, we determined PlGF plasma levels in healthy controls (n=10), untreated patients in Chronic Phase (CP) upon primary diagnosis (n=32) and patients with BC under treatment with different TKIs (n=9). These analyses revealed 2.1-fold upregulation of PlGF in newly diagnosed patients in CP (p&lt;0.0001) and 3.7-fold increase of PlGF levels in patients with BC (p&lt;0.0001) compared to healthy controls. We then investigated a potential relation between PlGF protein and BCR-ABL1 transcript numbers as determined by QRT-PCR in a single centre (n=43 CP, n=2 Accelerated Phase; treatment with different TKIs, imatinib+interferon, or homoharringtonine). We found a significant correlation between PlGF levels and BCR-ABL1 transcript numbers (r=0.45; p=0.0016), indicating that PlGF represents a disease specific target in human CML. Subsequently, we isolated CD34+ cells from healthy donors and from CML patients in CP and BC and determined PlGF expression by QRT-PCR. These analyses revealed that PlGF expression is equally low in leukemia cells as in healthy CD34+ cells. Thus, elevated circulating PlGF is most likely not secreted by leukemia cells, but by stromal cells. To investigate this hypothesis, we isolated adherent BM stromal cells from patients with newly diagnosed CML and compared PlGF expression levels to those in CD34+ leukemia cells. We found that stromal cells express &gt;7-fold more PlGF than leukemia cells (p=0.003), which corroborates our preclinical data and extends the concept that PlGF is primarily produced by stromal cells in CML patients. In summary inhibition of PlGF may serve as a new candidate to be targeted in combination therapies or in TKI refractory CML. Disclosures: Carmeliet: Thrombogenics: Research Funding.

Supplementary Figure 9 from PlGF/VEGFR-1 Signaling Promotes Macrophage Polarization and Accelerated Tumor Progression in Obesity

&lt;p&gt;Effects of VEGFR-1 signaling ablation on E0771 tumor growth, vascular and immune environment in lean and obese mice.&lt;/p&gt;

Mouse PAI-1 promotes placentation by increasing foetal and maternal angiogenesis

International audience

Use of Axl, a therapeutic target in AML, to mediate stroma-induced chemoresistance.

7027 Background: Axl, the receptor for Growth Arrest-specific protein 6 (Gas6) plays a role in AML pathobiology (Blood Suppl. Nov 2011; 118: 940). Here, we investigated whether Axl represents a therapeutic target in AML. Methods: Gas6 levels were measured by ELISA and immunohistochemistry. Axl expression was detected by flow cytometry. Co-cultures of (murine) BM stroma cells (primary, OP9, S17) with Mv4-11 and OCI-AML5 cell lines were performed. Results: We found (i) higher expression of Axl in AML BM compared to healthy BM donors (66.20 ± 10.87 vs. 0.65 ± 0.10 %; n=8/6; p&lt;0.05); (ii) Axl expression by 68 ± 31% of AML blasts and (iv) higher expression of Axl by CD34 + CD38 - AML stem cells compared to healthy CD34 + CD38 - BM stem cells (58.43 ± 4.63 % vs. 6.00 ± 2.01 %; n=7/6; p&lt;0.05). The Axl inhibitor BGB324 dose-dependently inhibited proliferation of primary AML cells with a mean IC50 of 1.8 µM. Sensitivity to BGB324 (i.e. a lower IC50) correlated with Axl expression on leukemia cells (Pearson’s r = -0.9656, p&lt;0.05). Combination therapy with BGB324 and cytarabine exerted an additive therapeutic effect and BGB324 could chemosensitize cytarabine-resistant AML cells. Analyses of BM sections revealed that Gas6 expression was low in AML cells, similar to healthy hematopoietic cells while it was abundantly expressed in AML BM stromal cells with fibroblastic/mesenchymal morphology (BMDSCs). Gas6 expression was considerably lower in control BMDSCs (86 ± 14 % vs. 20 ± 20 %; n=5/7; p&lt;0.05) thus suggesting a possible paracrine interaction between AML cells and BMDSCs leading to Gas6 upregulation in the stroma compartment. Co-culture experiments indicated specific upregulation of murine (m)Gas6 in BMDSCs via leukemia-cell derived IL-10 and M-CSF. This stroma-derived Gas6 could mediate chemoresistance of AML cells in co-culture, which was abrogated by sAxl or by BGB324. Thus, interaction between stroma-derived Gas6 and Axl + leukemia cells forms a chemoprotective niche for leukemia cells. In line with these findings Axl blockade chemosensitizes Mv4-11 cells for treatment with doxorubicine in vivo. Conclusions: Axl represents a therapeutic target in AML and Axl inhibition by BGB324 holds potential to treat chemosensitive and chemoresistant AML.

Loss or Inhibition of uPA or MMP-9 Attenuates LV Remodeling and Dysfunction after Acute Pressure Overload in Mice

Data from PlGF/VEGFR-1 Signaling Promotes Macrophage Polarization and Accelerated Tumor Progression in Obesity

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Obesity promotes pancreatic and breast cancer progression via mechanisms that are poorly understood. Although obesity is associated with increased systemic levels of placental growth factor (PlGF), the role of PlGF in obesity-induced tumor progression is not known. PlGF and its receptor VEGFR-1 have been shown to modulate tumor angiogenesis and promote tumor-associated macrophage (TAM) recruitment and activity. Here, we hypothesized that increased activity of PlGF/VEGFR-1 signaling mediates obesity-induced tumor progression by augmenting tumor angiogenesis and TAM recruitment/activity.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We established diet-induced obese mouse models of wild-type C57BL/6, VEGFR-1 tyrosine kinase (TK)-null, or PlGF-null mice, and evaluated the role of PlGF/VEGFR-1 signaling in pancreatic and breast cancer mouse models and in human samples.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; We found that obesity increased TAM infiltration, tumor growth, and metastasis in pancreatic cancers, without affecting vessel density. Ablation of VEGFR-1 signaling prevented obesity-induced tumor progression and shifted the tumor immune environment toward an antitumor phenotype. Similar findings were observed in a breast cancer model. Obesity was associated with increased systemic PlGF, but not VEGF-A or VEGF-B, in pancreatic and breast cancer patients and in various mouse models of these cancers. Ablation of PlGF phenocopied the effects of VEGFR-1-TK deletion on tumors in obese mice. PlGF/VEGFR-1-TK deletion prevented weight gain in mice fed a high-fat diet, but exacerbated hyperinsulinemia. Addition of metformin not only normalized insulin levels but also enhanced antitumor immunity.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Targeting PlGF/VEGFR-1 signaling reprograms the tumor immune microenvironment and inhibits obesity-induced acceleration of tumor progression. &lt;i&gt;Clin Cancer Res; 22(12); 2993–3004. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

The role of placental growth factor in the nervous system

Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish

The development of small animal models is of major interest to unravel the pathogenesis and treatment of neurodegenerative diseases, especially because of their potential in large-scale chemical and genetic screening. We have investigated the zebrafish as a model to study amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by the selective loss of motor neurons, caused by mutations in superoxide dismutase 1 (SOD1) in a subset of patients. Overexpression of mutant human SOD1 in zebrafish embryos induced a motor axonopathy that was specific, dose-dependent and found for all mutations studied. Moreover, using this newly established animal model for ALS, we investigated the role of a known modifier in the disease: vascular endothelial growth factor (VEGF). Lowering VEGF induced a more severe phenotype, whereas upregulating VEGF rescued the mutant SOD1 axonopathy. This novel zebrafish model underscores the potential of VEGF for the treatment of ALS and furthermore will permit large-scale genetic and chemical screening to facilitate the identification of new therapeutic targets in motor neuron disease.

Inhibition of Urokinase-Type Plasminogen Activator or Matrix Metalloproteinases Prevents Cardiac Injury and Dysfunction During Viral Myocarditis

Background— Acute viral myocarditis is an important cause of cardiac failure in young adults for which there is no effective treatment apart from general heart failure therapy. The present study tested the hypothesis that increased expression of the proteinases urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) is implicated in cardiac inflammation, injury, and subsequent failure during Coxsackievirus-B3 (CVB3)–induced myocarditis. Methods and Results— First, we showed increased expression and activity of uPA and MMP-9 in wild-type mice at 7 days of CVB3-induced myocarditis. Targeted deletion of uPA, which resulted in reduced MMP activity and cytokine expression or inhibition of MMPs by adenoviral gene overexpression of tissue inhibitor of metalloproteinases-1, decreased cardiac inflammation and reduced myocardial necrosis at 7 days and decreased cardiac fibrosis at 35 days after CVB3 infection. Importantly, loss of uPA or MMP activity prevented CVB3-induced cardiac dilatation and dysfunction, as determined by serial echocardiography. Conclusions— Loss of uPA or MMP activity reduces the cardiac inflammatory response after CVB3 infection, thereby protecting against cardiac injury, dilatation, and failure during CVB3-induced myocarditis.

Angiogenesis and lymphangiogenesis: highlights of the past year

The intensive search for antiangiogenic and proangiogenic mechanisms during the past decade is starting to translate into clinical promise. Further discovery of novel pathways and concepts in angiogenesis may lead to the optimization and refinement of current strategies to improve the clinical benefit and therapeutic safety for a vast number of patients with angiogenesis-related disease.

Glomerulonephritis in mice with genetic deficiencies of the plasminogen system