Publications

Introduction: The majority of the human transcriptome consists of non-coding transcripts which may exert regulatory effects on epigenetic and transcriptional processes. Since laminar flow is an important atheroprotective regulator, we analysed its impact on the expression of long non-coding RNAs (lncRNA) in human endothelial cells (EC) by RNA deep sequencing. Among the significantly up-regulated lncRNAs, we confirmed a 3-fold up-regulation of lncflow2 by laminar flow (p Results: Silencing of lncflow2 by GapmeRs reduced sprout formation of ECs (34.7 % reduction, p

Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P < 0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.

Influx of Ca 2+ ions through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca 2+ permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca 2+ ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo . Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.

Formation and homeostasis of the vascular system requires several coordinated cellular functions, but their precise interplay during development and their relative importance for vascular pathologies remain poorly understood. Here, we investigate the endothelial functions regulated by Cdc42 and their in vivo relevance during angiogenic sprouting and vascular morphogenesis in the postnatal mouse retina. We find that Cdc42 is required for endothelial tip cell selection, directed cell migration and filopodia formation, but dispensable for cell proliferation or apoptosis. While the loss of Cdc42 seem generally compatible with apical-basal polarization and lumen formation in retinal blood vessels, it leads to defective endothelial axial polarization and to the formation of severe vascular malformations in capillaries and veins. Tracking of Cdc42 depleted endothelial cells in mosaic retinas suggests that these capillary-venous malformations arise as a consequence of defective cell migration, when endothelial cells that proliferate at normal rates are unable to re-distribute within the vascular network.

To the editor: Patients recovering from myelotoxic chemotherapy require antibiotic treatment to prevent infectious complications, because of their immunocompromised status. Antibiotics such as doxycylin and tetracyclin are known to inhibit matrix metalloproteinases (MMPs) including MMP-9.[1][1

ABSTRACT Plasminogen activator inhibitor 1 (PAI‐1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI‐1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell‐matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene‐inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microves‐sel outgrowth. Lack of PAI‐1 completely abolished angio‐genesis, demonstrating its importance in the control of plasmin‐mediated proteolysis. Microvessel outgrowth from PAI‐1 ‐/‐ aortic rings could be restored by adding exogenous PAI‐1 (wild‐type serum or purified recombi‐nant PAI‐1). Addition of recombinant PAI‐1 led to a bell‐shaped angiogenic response clearly showing that PAI‐1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI‐1 mutants, we could demonstrate that PAI‐1 promotes an‐giogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin.—Devy, L., Blacher, S., Grignet‐Debrus, C., Bajou, K., Masson, V., Gerard, R. D., Gils, A., Carmeliet, G., Carmeliet, P., Declerck, P. J., Noèl, A., Foidart, J. M. The pro‐ or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (2002)

Abstract Background and Aims Diabetic kidney disease (DKD) has become the leading cause of end-stage kidney disease (ESKD) worldwide. DKD is a microvascular disease characterized by renal endothelial cell (EC) dysfunction. Studies by the Carmeliet lab and others using scRNA-seq highlighted EC heterogeneity in different pathophysiological contexts, including kidney disease. Further, these studies identified similar patterns of gene expression in EC subsets as in immune cells (ICs) raising the question if these immunomodulatory ECs (coined “IMECs”) participate in immune responses, such as during vascular inflammation and associated EC dysfunction. However, while inflammation, oxidative stress and fibrosis are considered to contribute to EC dysfunction in DKD, a comprehensive unbiased understanding of signatures in renal ECs at single cell resolution is lacking. We are characterizing the (patho)-biological role of ECs in dkd by using single nuclei/cell multi-omics. We identified several unexplored genes (lacking functional annotation and publications) with a predicted IMEC function using in-house generated AI-based tools. Their functional role in the context of DKD will be further investigated. Method Droplet-based snRNA-seq (10X Genomics Chromium) was performed on cryopreserved kidney biopsy cores from 6 patients with severe DKD (6 patients (3 females; 3 males), median age 65 yrs; median eGFR: 15 ml/min/1.73 m2; median albuminuria: 1 g/g creatinine) and 6 sex/age-matched healthy controls. Healthy control samples were pre-perfusion biopsies from patients undergoing living kidney donation. EC subclusters were annotated based on the expression of canonical EC marker genes. Differential gene expression analysis for patient samples vs. controls included all ECs and differentially expressed genes (DEGs) were filtered for log2FC &amp;gt; 0.25 or &amp;lt; −0.25 and adjusted p-value of &amp;lt;0.05. Gene set enrichment analysis (GSEA) on the identified DEGs was performed with Enrich, using GO terms. Leveraging generated snRNA-seq data and a curated set of training genes with documented immunomodulatory functions, we used in-house generated AI-based gene discovery and prioritization tools to select the most promising IMEC unexplored candidate drivers of EC dysfunction in DKD. In vitro, the RNA expression levels in HUVECs were analyzed using qRT-PCR after 72 hours of culture under three conditions: control (5.5 mM glucose), hyperglycemic (33 mM glucose), or inflammatory (10 ng/ml TNF-α). Results Cell type annotation showed the presence of ca 6,000 ECs in the snRNA-seq dataset (Fig. 1A). Initial differential gene expression analysis of the EC population in the patient vs control samples identified several potential immunostimulatory genes. Using the in-house AI-based prioritization tools, we identified three potential unexplored IMEC genes, coded KIMEC1, KIMEC2, and KIMEC3 (the identity of the genes cannot be disclosed due to IP reasons). These genes were among the top upregulated, with log2FC values of 0.57, 0.34, and 0.522 (adjusted p-value of &amp;lt;0.05), respectively. Of note, all 3 genes are predicted to be involved in oxidative stress. In addition, they tend to be also upregulated in ECs in vivo in various inflammatory conditions (Fig. 1B.; shown for KIMEC3) and in vitro in hyperglycemic and/or pro-inflammatory culture conditions (shown for KIMEC3 in Fig. 1C–D). Conclusion We identified previously unexplored IMEC genes expressed in renal EC in DKD. Their potential role in mediating vascular inflammation and endothelial cell dysfunction in DKD will be further investigated.