Publications

Placental growth factor (PlGF)-deficient (PlGF−/−) and wild-type mice were kept on a standard-fat or high-fat diet for 15 weeks. With the standard-fat diet, the body weights of PlGF−/− and wild-type mice were comparable, whereas the combined weight of subcutaneous and gonadal adipose tissues was lower in PlGF−/− mice (P = 0.02). With the high-fat diet, PlGF−/− mice had a lower body weight (P < 0.05) and less total subcutaneous plus gonadal adipose tissue (P < 0.0001). Blood vessel size was lower in gonadal adipose tissue of PlGF−/− mice with both the standard-fat and high-fat diet (P < 0.05). Blood vessel density, normalized to adipocyte number, was significantly lower in subcutaneous adipose tissue of PlGF−/− mice fed the high-fat diet (P < 0.01). De novo adipose tissue development in nude mice injected with 3T3-F442A preadipocytes was reduced (P < 0.005) by administration of a PlGF-neutralizing antibody. Bone marrow transplantation from wild-type or PlGF−/− mice to wild-type or PlGF−/− recipient mice revealed significantly lower blood vessel density in PlGF−/− recipient mice without an effect on adipose tissue growth. Thus, in murine models of diet-induced obesity, inactivation of PlGF impairs adipose tissue development, at least in part as a result of reduced angiogenesis.

Expression levels of VEGFR1 and VEGF-B correlate with edema and clinical markers of NP disease and therefore represent potential therapeutic targets.

Variations in the CLL DNA methylome reflect modifications that occur during normal B cell maturation, along with IGHV mutated (M-CLL) and unmutated CLL (U-CLL), retaining an imprint of the DNA methylation signature of memory (m-CLL) and naive B cells (n-CLL), respectively, with a third intermediate epigenetic subgroup (i-CLL) (1-3). To further test the clinical utility of DNA methylation signatures, we performed the first analysis of patients entering clinical trials; we tested treatment-naive CLL patients [n=605] randomized to CLL4 (chemotherapy, CT) (4), ARCTIC and ADMIRE (both chemo-immunotherapy, CIT) (5, 6). We identified n-, i- and m-CLL in 49.3% (n=299), 32.0% (n=195) and 18.5% (n=112) of our patients, respectively. Fewer m-CLL patients were identified in our study compared to published data reflecting the progressive nature of our cohort, with 80% (n=245/305, P<0.001) of U-CLL cases exhibiting the n-CLL signature (i-CLL: 17% and m-CLL: 3%). For M-CLL cases, 9%, 50% and 41% exhibited the n-, i- and m-CLL epigenetic signature, respectively. 68% (80/117, p<0.001) of cases with del(11q), 77% (41/53, p<0.001) with trisomy 12, and 68% (38/56, p=0.03) with TP53 lesions were n-CLL. Cases with NOTCH1 (p=0.01) and SF3B1 (p=0.02) mutations were also enriched in n-CLL. Next, we investigated the impact of methylation signatures on progression-free (PFS) and overall survival (OS). In CT patients, n-, i- and m-CLL patients exhibited a median PFS of 23, 34 and 35 months, and OS of 63, 66 and 106 months, respectively. n-CLL showed significantly shorter PFS than i-CLL (HR 0.64, p<0.001) and m-CLL (HR 0.52, p<0.001), and had the shortest OS, again compared to i-CLL (HR 0.73, p=0.01) and m-CLL (HR 0.33, p<0.001). Ten-year OS differed according to epigenetic signature (P<0.001) and was reached by only 14% of n-CLL patients. Multivariate Cox proportional analysis, controlling for confounding variables (incl. clinical features, IGHV status, TP53, NOTCH1 and SF3B1) in 278 patients, showed that m-CLL was an independent prognostic factor for OS (HR 0.46, p<0.01). In 247 CIT patients, univariate analysis showed that the i- (HR:0.57, p=0.05) and m-CLL (HR:0.3, p=0.002) subgroups displayed longer PFS. In a multivariate model, including TP53 lesions and IGHV status (239 patients), the m-CLL subgroup retained independent prognostic significance (HR:0.25, p<0.001). In conclusion, we provide important evidence that DNA methylation analysis may aid in the identification of patients destined to demonstrate durable remissions when treated with these agents.

Abstract The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

Abstract Background Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo . Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGF δ/δ mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. Results Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGF δ/δ mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGF δ/δ mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGF δ/δ mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGF δ/δ mice, axon outgrowth is significantly reduced compared to wild-type littermates. Conclusions Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

Significance Statement In previous work, the authors demonstrated that short-term loss of integrity of the luminal part of the glycocalyx envelope that covers endothelial cells resulted in impaired glomerular filtration barrier function. In this study, using knockout mice lacking endothelial hyaluronan synthase 2 (the enzyme that produces hyaluronan, the main structural component of the glycocalyx layer), they found that loss of glomerular endothelial hyaluronan leads to mesangiolysis and glomerulosclerosis. Tissue from patients with diabetic nephropathy showed loss of glomerular endothelial hyaluronan in association with lesion formation. The authors also demonstrated that hyaluronan loss results in disturbed signaling of the extracellular matrix stabilizer angiopoietin 1. These findings suggest that the loss of glomerular endothelial hyaluronan in diabetic nephropathy may be a new therapeutic target to restabilize glomerular lesions. Background A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function. Methods To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy. Results Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2. Conclusions Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrix that is required for glomerular structure and function and lost in diabetic nephropathy.

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca 2+ influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca 2+ influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).

Neuroinflammation and dysregulated energy metabolism are linked to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The egl-9 family hypoxia-inducible factor (EGLN) enzymes, also known as prolyl hydroxylase domain (PHD) enzymes, are metabolic sensors regulating cellular inflammation and metabolism. Using an oligonucleotide-based and a genetic approach, we showed that the downregulation of Egln2 protected motor neurons and mitigated the ALS phenotype in two zebrafish models and a mouse model of ALS. Single-nucleus RNA sequencing of the murine spinal cord revealed that the loss of EGLN2 induced an astrocyte-specific downregulation of interferon-stimulated genes, mediated via the stimulator of interferon genes (STING) protein. In addition, we found that the genetic deletion of EGLN2 restored this interferon response in patient induced pluripotent stem cell (iPSC)-derived astrocytes, confirming the link between EGLN2 and astrocytic interferon signaling. In conclusion, we identified EGLN2 as a motor neuron protective target normalizing the astrocytic interferon-dependent inflammatory axis in vivo, as well as in patient-derived cells.