Senescent endothelial cells promote pathogenic neutrophil trafficking in inflamed tissues.

Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

PTPN23 binds the dynein adaptor BICD1 and is required for endocytic sorting of neurotrophin receptors.

Signalling by target-derived neurotrophins is essential for the correct development of the nervous system and its maintenance throughout life. Several aspects concerning the lifecycle of neurotrophins and their receptors have been characterised over the years, including the formation, endocytosis and trafficking of signalling-competent ligand-receptor complexes. However, the molecular mechanisms directing the sorting of activated neurotrophin receptors are still elusive. Previously, our laboratory identified Bicaudal-D1 (BICD1), a dynein motor adaptor, as a key factor for lysosomal degradation of brain-derived neurotrophic factor (BDNF)-activated TrkB (also known as NTRK2) and p75NTR (also known as NGFR) in motor neurons. Here, using a proteomics approach, we identified protein tyrosine phosphatase, non-receptor type 23 (PTPN23), a member of the endosomal sorting complexes required for transport (ESCRT) machinery, in the BICD1 interactome. Molecular mapping revealed that PTPN23 is not a canonical BICD1 cargo; instead, PTPN23 binds the N-terminus of BICD1, which is also essential for the recruitment of cytoplasmic dynein. In line with the BICD1-knockdown phenotype, loss of PTPN23 leads to increased accumulation of BDNF-activated p75NTR and TrkB in swollen vacuole-like compartments, suggesting that neuronal PTPN23 is a novel regulator of the endocytic sorting of neurotrophin receptors.

Tetanus toxin entry. Nidogens are therapeutic targets for the prevention of tetanus.

Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in nonvaccinated areas. TeNT targets the neuromuscular junction (NMJ) with high affinity, yet the nature of the TeNT receptor complex remains unknown. Here, we show that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Inhibition of the TeNT-nidogen interaction by using small nidogen-derived peptides or genetic ablation of nidogens prevented the binding of TeNT to neurons and protected mice from TeNT-induced spastic paralysis. Our findings demonstrate the direct involvement of an extracellular matrix protein as a receptor for TeNT at the NMJ, paving the way for the development of therapeutics for the prevention of tetanus by targeting this protein-protein interaction.

Mapping nucleolar and spliceosome localization sequences of neuregulin1-beta3.

Mitogenic growth factors are generally cell surface associated or secreted proteins, which produce effects by binding to cell surface receptor tyrosine kinases. More recently, it has become clear that some of these proteins can accumulate in the nucleus, where they are proposed to have transcriptional activity. We show here that neuregulin1 (NRG1-beta), an EGF-like growth factor, localizes to the cell nuclei of a human breast cancer. We also show that a nonsecreted isoform of this family of ligands, neuregulin1-beta3, localizes to two distinct compartments within the nucleus, nucleoli, and SC35-positive speckles. Importantly, localization of NRG-beta3 to either structure is receptor-independent, as it occurs in cells lacking its cognate receptors, erbB-3 and erbB-4, and is unaffected by removal of the receptor-binding domain. A panel of deletion mutants was used to demonstrate that the first 21 amino acids of the N-terminus are essential for nucleolar localization, while targeting to nuclear speckles requires residues 49-79 of the 241 amino acid protein. These observations support the idea that secretion and subsequent cell surface receptor binding of mitogenic growth factors are not a prerequisite for nuclear localization and that nonsecreted ligands may have highly specific functions in defined nuclear compartments.

VEGF164(165) as the pathological isoform: differential leukocyte and endothelial responses through VEGFR1 and VEGFR2.

PURPOSE: Vascular endothelial growth factor (VEGF) induces angiogenesis and vascular permeability and is thought to be operative in several ocular vascular diseases. The VEGF isoforms are highly conserved among species; however, little is known about their differential biological functions in adult tissue. In the current study, the inflammatory potential of two prevalent VEGF isoform splice variants, VEGF(120(121)) and VEGF(164(165)), was studied in the transparent and avascular adult mouse cornea. METHODS: Controlled-release pellets containing equimolar amounts of VEGF(120) and VEGF(164) were implanted in corneas. The mechanisms underlying this differential response of VEGF isoforms were explored. The response of VEGF in cultured endothelial cells was determined by Western blot analysis. The response of VEGF isoforms in leukocytes was also investigated. RESULTS: VEGF(164) was found to be significantly more potent at inducing inflammation. In vivo blockade of VEGF receptor (VEGFR)-1 significantly suppressed VEGF(164)-induced corneal inflammation. In vitro, VEGF(165) more potently stimulated intracellular adhesion molecule (ICAM)-1 expression on endothelial cells, an effect that was mediated by VEGFR2. VEGF(164) was also more potent at inducing the chemotaxis of monocytes, an effect that was mediated by VEGFR1. In an immortalized human leukocyte cell line, VEGF(165) was found to induce tyrosine phosphorylation of VEGFR1 more efficiently. CONCLUSIONS: Taken together, these data identify VEGF(164(165)) as a proinflammatory isoform and identify multiple mechanisms underlying its proinflammatory biology.

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia.

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.

Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis.

Branching morphogenesis in the mammalian lung and Drosophila trachea relies on the precise localization of secreted modulators of epithelial growth to select branch sites and direct branch elongation, but the intercellular signals that control blood vessel branching have not been previously identified. We found that VEGF(120/120) mouse embryos, engineered to express solely an isoform of VEGF-A that lacks heparin-binding, and therefore extracellular matrix interaction domains, exhibited a specific decrease in capillary branch formation. This defect was not caused by isoform-specific differences in stimulating endothelial cell proliferation or by impaired isoform-specific signaling through the Nrp1 receptor. Rather, changes in the extracellular localization of VEGF-A in heparin-binding mutant embryos resulted in an altered distribution of endothelial cells within the growing vasculature. Instead of being recruited into additional branches, nascent endothelial cells were preferentially integrated within existing vessels to increase lumen caliber. The disruption of the normal VEGF-A concentration gradient also impaired the directed extension of endothelial cell filopodia, suggesting that heparin-binding VEGF-A isoforms normally provide spatially restricted stimulatory cues that polarize and thereby guide sprouting endothelial cells to initiate vascular branch formation. Consistent with this idea, we found opposing defects in embryos harboring only a heparin-binding isoform of VEGF-A, including excess endothelial filopodia and abnormally thin vessel branches in ectopic sites. We conclude that differential VEGF-A isoform localization in the extracellular space provides a control point for regulating vascular branching pattern.

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts.

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.