Publications

Aims: Thrombospondin-2 (TSP-2) modulates matrix integrity and myocyte survival in the hypertensive or ageing heart. Whether TSP-2 may affect cardiac inflammation and injury, in particular during acute viral myocarditis, is completely unknown. Methods and results: Therefore, mortality, cardiac inflammation, and function were assessed in TSP-2-null (KO) and wild-type (WT) mice in human Coxsackie virus B3 (CVB3)-induced myocarditis. TSP-2 KO had an increased mortality when compared with WT mice during viral myocarditis. The absence of TSP-2 resulted in increased cardiac inflammation and injury at 14 days, which resulted in depressed systolic function [fractional shortening (FS); 34 ± 2.6 in WT vs. 24 ± 1.8 in KO mice, P< 0.05] and increased cardiac dilatation (end-diastolic dimensions, mm; 3.7 ± 0.09 in WT vs. 4.8 ± 0.06 in KO mice, P< 0.05) 35 days post-infection. Lack of TSP-2 resulted in a decreased activation of the anti-inflammatory T-regulatory cells, as indicated by a lower number of CD25-positive T-cells, and significantly decreased gene expression of regulatory T-cell markers, Foxp3 and CTLA-4. Finally, overexpression of TSP-2 in WT hearts using cardiotropic vectors derived from adeno-associated virus serotype 9 (AAV9) inhibited cardiac inflammation and injury at 14 days and improved cardiac function at 35 days post-CVB3 infection when compared with control AAV9. Conclusion: TSP-2 has a protective role against cardiac inflammation, injury, and dysfunction in acute viral myocarditis. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2012.

Summary The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.

Abstract GVHD is the single most important complication of allogeneic bone marrow transplantation (BMT), mediated by donor-derived alloreactive T cells. Gas6, a vitamin K-dependent protein, interacts with phospholipid membranes via its γ-carboxyglutamic acid-containing tail and binds to the receptor tyrosine kinases Tyro3, Axl and Mer via its carboxy-terminal globular domain. Gas6 has been implicated in reversible growth arrest, survival, proliferation, cell adhesion, hemostasis and erythropoiesis. Preliminary experiments have shown that Gas6 deficient (Gas6-/-) mice have a leukocyte trafficking defect into inflamed tissues. We hypothesized that donor Gas6-/- T cells might induce less GVHD than wild-type (WT) T cells in MHC-mismatched BMT. We therefore compared the ability of Gas6-/- FVB (H-2q) bone marrow cells to produce GVHD in lethally irradiated MHC haplo-mismatched Gas6-/- and WT B6 mice (H-2b). Gas6-/- and WT B6 received 9 Gy total body irradiation and 107 cells from bone marrow of FVB mice (H-2q) of the same genotype. (Gas6-/- B6 mice received Gas6-/- FVB cells and WT B6 mice were injected with WT FVB cells.) On Day 28, recipients were sacrificed and chimerism was confirmed by expression of H-2q haplotype in recipient bone marrow. Neither group had clinical evidence of GVHD. However, histologic studies revealed lesions in the livers of both genotypes, which were notably less severe in Gas6-/- mice. The size and number of portal space leukocyte infiltrates were reduced in Gas6-/- mice (40 ± 6.6 % of portal spaces infiltrated), compared to WT mice (76.8 ± 6.5 % of portal spaces infiltrated, P < 0.01). Apoptosis of liver cells was also reduced in Gas6-/- mice. We hypothesized that diminished leukocyte trafficking in the Gas6-/- mice may be due to defects in cytokine-induced expression of adhesion molecules. We therefore isolated primary capillary endothelial cells from Gas6-/- and WT mice, and assessed their response to TNF-α. In contrast to WT endothelial cells, Gas6-/- endothelial cells responded without increases in VCAM-1, ECAM-1, E-selectin, IL-1 or IL-6. This resistance to TNF-α was reversed when the Gas6-/- endothelial cells were co-treated with recombinant Gas6, suggesting that the effect of Gas6 on vascular endothelial function is critical for normal leukocyte trafficking in GVHD. Overall, these results provide strong rationale for considering Gas6 and/or its receptors as a potential therapeutic target to minimize GVHD.

Gene targeting studies in mice have elucidated a functional role of placental growth factor PlGF, a homologue of VEGF, in pathological angiogenesis. Loss of PlGF, while not causing any vascular defects during embryonic development, reproduction or normal adult life, impaired angiogenesis in pathological conditions, such as cancer. We generated a neutralizing anti- mPlGF monoclonal antibody which inhibits PlGF binding to its receptor Flt1. Here we report the effect of this anti-mPlGF mAb on tumor angiogenesis and growth in syngeneic, subcutaneous B16 melanoma, CT26 colon and orthotopic Panc02 pancreatic tumor models. Treatment was initiated when tumors reached a size of 60mm³ or at 3 days after orthotopic tumor cell inoculation. Compared to a control antibody, intraperitoneal administration of anti-mPlGF mAb every other day significantly suppressed the growth of primary B16 melanoma tumors (end-stage tumor volume: 1,470±81mm³ after anti-PlGF vs. 2,660±81mm³ after control; p=0.001), colon CT26 tumors (729±73mm³ vs. 1,533±113mm³; p=0.001) and pancreatic Panc02 tumors (1,230±220mm³ vs. 2,313±290mm³; p=0.007). Histological examination on CD31-stained tumor sections revealed that the vessel density in anti-mPlGF mAb treated tumors was reduced. Moreover, unlike VEGF inhibitors, the anti- mPlGF mAb failed to affect pregnancy or skin wound healing, did not induce hypertension or pruning of quiescent vessels in the trachea and thyroid gland, and failed to elevate plasma levels of PAI–1, a marker of thrombotic risk. These findings may offer novel therapeutic opportunities for the treatment of cancer.

Einleitung: Der angiogene Wachstumsfaktor Placental growth factor (PlGF), ein Homolog von VEGF, stimuliert über VEGF Rezeptor-1 die Proliferation von Endothelzellen und die Aktivität angiogener Makrophagen und trägt somit zur Tumorangiogenese bei. Wir demonstrierten jüngst das therapeutische Potential eines anti-PlGF Antikörpers (αPlGF), Tumorwachstum und Metastasierung effizient zu inhibieren.

Abstract Airway hyper-responsiveness (AHR) affects 55-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the underlying mechanisms of this highly prevalent AHR in a hemoglobinopathy remain unknown. We hypothesized that Placenta Growth Factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates this AHR. We studied AHR in allergen-exposed PlGF-/- and SCD mice. Sickle mice genetically deficient in PlGF were not viable. PlGF augmented AHR, lung inflammation and blood/lung eosinophilia via IL-13 (a Th2-cytokine) and increased 5-lipoxygenase (a leukotriene synthetic enzyme) expression. AHR, or pulmonary inflammation and leukotriene levels were blunted in PlGF-/- mice or in PlGFWT mice treated with anti-PlGF-Ab, and was rescued by intratracheal administration of leukotrienes to PlGF-/- mice. Notably, Th2-mediated Stat-6 activation further increased PlGF expression from lung epithelium, eosinophils and macrophages, linking the PlGF-leukotriene pathway in a positive feedback loop. Th2 response is classically seen in asthma, and indeed, expression of PlGF and its downstream genes was increased in respiratory epithelial cells of asthma patients. Like patients with SCD, sickle hematopoietic chimeras developed increased AHR and had higher leukotriene levels; and both were abrogated by anti-PlGF-Ab or zileuton (5-lipoxygenase inhibitor) phenocopying the blunted AHR in PlGF-/- mice, and indicating that sickle erythroid-cells/RBC potentiate AHR. Overall, PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. Both zileuton, licensed for asthma and anti-PlGF-Ab, in clinical trials for cancer, could be promising therapies to reduce the pulmonary morbidity in SCD. Disclosures Jonckx: ThromboGenics: Employment.