Explore the vast range of titles available.

L-selectin (CD62L) is a type-I transmembrane glycoprotein and cell adhesion molecule that is expressed on most circulating leukocytes. Since its identification in 1983, L-selectin has been extensively characterized as a tethering/rolling receptor. There is now mounting evidence in the literature to suggest that L-selectin plays a role in regulating monocyte protrusion during transendothelial migration (TEM). The N-terminal calcium-dependent (C-type) lectin domain of L-selectin interacts with numerous glycans, including sialyl Lewis X (sLe ) for tethering/rolling and proteoglycans for TEM. Although the signals downstream of L-selectin-dependent adhesion are poorly understood, they will invariably involve the short 17 amino acid cytoplasmic tail. In this review we will detail the expression of L-selectin in different immune cell subsets, and its influence on cell behavior. We will list some of the diverse glycans known to support L-selectin-dependent adhesion, within luminal and abluminal regions of the vessel wall. We will describe how each domain within L-selectin contributes to adhesion, migration and signal transduction. A significant focus on the L-selectin cytoplasmic tail and its proposed contribution to signaling via the ezrin-radixin-moesin (ERM) family of proteins will be outlined. Finally, we will discuss how ectodomain shedding of L-selectin during monocyte TEM is essential for the establishment of front-back cell polarity, bestowing emigrated cells the capacity to chemotax toward sites of damage.

Vascular endothelial cell (EC) dysfunction plays a key role in diabetic complications. This study discovers significant upregulation of Quaking-7 (QKI-7) in iPS cell-derived ECs when exposed to hyperglycemia, and in human iPS-ECs from diabetic patients. QKI-7 is also highly expressed in human coronary arterial ECs from diabetic donors, and on blood vessels from diabetic critical limb ischemia patients undergoing a lower-limb amputation. QKI-7 expression is tightly controlled by RNA splicing factors CUG-BP and hnRNPM through direct binding. QKI-7 upregulation is correlated with disrupted cell barrier, compromised angiogenesis and enhanced monocyte adhesion. RNA immunoprecipitation (RIP) and mRNA-decay assays reveal that QKI-7 binds and promotes mRNA degradation of downstream targets CD144, Neuroligin 1 (NLGN1), and TNF-α-stimulated gene/protein 6 (TSG-6). When hindlimb ischemia is induced in diabetic mice and QKI-7 is knocked-down in vivo in ECs, reperfusion and blood flow recovery are markedly promoted. Manipulation of QKI-7 represents a promising strategy for the treatment of diabetic vascular complications. Vascular endothelial cell (EC) dysfunction contributes to the occurrence of diabetic complications. Here the authors report that in diabetic conditions, upregulation of the RNA binding protein QKI-7 in ECs due to the imbalance of RNA splicing factors CUG-BP and hnRNPM contributes to EC dysfunction, and that in vivo QKI-7 silencing improves blood flow recovery in diabetic mice with limb ischemia.

L-selectin is a type I transmembrane cell adhesion molecule expressed on most circulating leukocytes, including neutrophils. Engagement of L-selectin with endothelial-derived ligands initiates neutrophil tethering and rolling behaviour along luminal walls of post-capillary venules, constituting the first step of the multi-step adhesion cascade. There is a large body of evidence to suggest that signalling downstream of L-selectin can influence neutrophil behaviour: adhesion, migration and priming. This review will cover aspects of L-selectin form and function and introduce the “triad of L-selectin regulation”, highlighting the inextricable links between adhesion, signalling and ectodomain shedding and also highlighting the cytosolic proteins that interconnect them. Recent advances in how L-selectin impacts priming, transendothelial migration (TEM) and cell polarity will also be discussed.

The cytokine-driven transcription factor STAT1 (signal transducer and activator of transcription 1) executes anti-microbial and pro-apoptotic functions, and loss-of-function mutations are associated with increased susceptibility to various infections and the development of tumors. A targeted mutation in mice expressing an N-terminally truncated STAT1 protein (STAT1-ΔN) typically develops splenomegaly in animals older than 6 months due to the formation of splenic non-Hodgkin lymphomas. The expression of the STAT1-ΔN variant resulted in the disruption of normal spleen architecture by malignant CD3- and CD20-negative tumor cells, which stained positively for both tyrosine-phosphorylated STAT1 and STAT3. Immunoblotting of lysates from isolated tumor cells revealed the cytokine-independent hyperphosphorylation of both STAT proteins, whereas the expression level of NF-κB was significantly reduced. Gel-shift assays showed that the DNA-binding activity of STAT1-ΔN was increased compared to the wild-type protein. This elevated level of tyrosine-phosphorylated STAT1-ΔN did not further increase upon stimulation of isolated tumor cells with either interferon-γ (IFNγ), lipopolysaccharide (LPS), or the combination of both. Since the truncation mutant was unable to accumulate in the nucleus upon cytokine stimulation, real-time PCR data from tumor tissue as well as from isolated, IFNγ/LPS-treated lymphoma cells demonstrated significantly reduced STAT1-regulated target gene expression despite its observed hyperphosphorylation. The nuclear import defect of tyrosine-phosphorylated STAT1-ΔN was associated with an elevated tyrosine-phosphorylation level of its antagonistic homolog STAT3, which is a known oncogene. These data demonstrate that the lack of STAT1 nuclear accumulation interferes with the functional balance between the two STAT proteins and, thereby, promotes the formation of phospho-STAT3-expressing CD3 -/- CD20 -/- non-Hodgkin lymphomas in the spleens of the diseased animals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses associationwith calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.

A third of patients with critical limb ischemia (CLI) will eventually require limb amputation. Therapeutic neovascularization using unselected mononuclear cells to salvage ischemic limbs has produced modest results. The TIE2‐expressing monocytes/macrophages (TEMs) are a myeloid cell subset known to be highly angiogenic in tumours. This study aimed to examine the kinetics of TEMs in patients with CLI and whether these cells promote neovascularization of the ischemic limb. Here we show that there are 10‐fold more circulating TEMs in CLI patients, and removal of ischemia reduces their numbers to normal levels. TEM numbers in ischemic muscle are two‐fold greater than normoxic muscle from the same patient. TEMs from patients with CLI display greater proangiogenic activity than TIE2‐negative monocytes in vitro . Using a mouse model of hindlimb ischemia, lentiviral‐based Tie2 knockdown in TEMs impaired recovery from ischemia, whereas delivery of mouse macrophages overexpressing TIE2, or human TEMs isolated from CLI patients, rescued limb ischemia. These data suggest that enhancing TEM recruitment to the ischemic muscle may have the potential to improve limb neovascularization in CLI patients. →See accompanying articles http://dx.doi.org/10.1002/emmm.201302695 and http://dx.doi.org/10.1002/emmm.201302794 Graphical Abstract Tie2‐expressing macrophages (TEMs) have the potential to improve revascularization of the ischemic limb and may therefore, represent an attractive novel cell therapy for promoting limb salvage in patients suffering from critical limb ischemia.

Aims Endothelial activation and inflammatory cell infiltration have important roles in the development of cardiac fibrosis induced by renin–angiotensin system activation. NADPH oxidases (Nox proteins) are expressed in endothelial cells (ECs) and alter their function. Previous studies indicated that Nox2 in ECs contributes to angiotensin II (AngII)‐induced cardiac fibrosis. However, the effects of EC Nox4 on cardiac fibrosis are unknown. Methods and results Transgenic (TG) mice overexpressing endothelial‐restricted Nox4 were studied alongside wild‐type (WT) littermates as controls. At baseline, Nox4 TG mice had significantly enlarged hearts compared with WT, with elongated cardiomyocytes (increased by 18.5%, P < 0.01) and eccentric hypertrophy but well‐preserved cardiac function by echocardiography and in vivo pressure–volume analysis. Animals were subjected to a chronic AngII infusion (AngII, 1.1 mg/kg/day) for 14 days. Whereas WT/AngII developed a 2.1‐fold increase in interstitial cardiac fibrosis as compared with WT/saline controls (P < 0.01), TG/AngII mice developed significant less fibrosis (1.4‐fold increase, P > 0.05), but there were no differences in cardiac hypertrophy or contractile function between the two groups. TG hearts displayed significantly decreased inflammatory cell infiltration with reduced levels of vascular cell adhesion molecule 1 in both the vasculature and myocardium compared with WT after AngII treatment. TG microvascular ECs stimulated with AngII in vitro supported significantly less leukocyte adhesion than WT ECs. Conclusions A chronic increase in endothelial Nox4 stimulates physiological cardiac hypertrophy and protects against AngII‐induced cardiac fibrosis by inhibiting EC activation and the recruitment of inflammatory cells. Highlights Mice with endothelium‐specific overexpression of Nox4 (EndoNox4 TG) exhibit eccentric hypertrophy with well‐preserved cardiac function at baseline. EndoNox4 TG mice develop significantly less interstitial cardiac fibrosis in response to chronic pressure AngII stimulation, independent of cardiac hypertrophy. Overexpression of Nox4 in endothelial cells reduces AngII‐induced endothelial activation. An increase in endothelial Nox4 inhibits AngII‐induced recruitment of inflammatory cells in the heart.

The migration of circulating leukocytes toward damaged tissue is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first cellular barrier that is breached in this process. Human CD14 inflammatory monocytes express L-selectin, bestowing a non-canonical role in invasion during TEM. evidence supports a role for L-selectin in regulating TEM and chemotaxis, but the intracellular mechanism is poorly understood. The ezrin-radixin-moesin (ERM) proteins anchor transmembrane proteins to the cortical actin-based cytoskeleton and additionally act as signaling adaptors. During TEM, the L-selectin tail within transmigrating pseudopods interacts first with ezrin to transduce signals for protrusion, followed by moesin to drive ectodomain shedding of L-selectin to limit protrusion. Collectively, interaction of L-selectin with ezrin and moesin fine-tunes monocyte protrusive behavior in TEM. Using FLIM/FRET approaches, we show that ERM binding is absolutely required for outside-in L-selectin clustering. The cytoplasmic tail of human L-selectin contains two serine (S) residues at positions 364 and 367, and here we show that they play divergent roles in regulating ERM binding. Phospho-S364 blocks direct interaction with ERM, whereas molecular modeling suggests phospho-S367 likely drives desorption of the L-selectin tail from the inner leaflet of the plasma membrane to potentiate ERM binding. Serine-to-alanine mutagenesis of S367, but not S364, significantly reduced monocyte protrusive behavior in TEM under flow conditions. Our data propose a model whereby L-selectin tail desorption from the inner leaflet of the plasma membrane and ERM binding are two separable steps that collectively regulate protrusive behavior in TEM.

Nox2 is a ROS-generating enzyme, deficiency of which increases suppression by Tregs in vitro and in an in vivo model of cardiac remodeling. As Tregs have emerged as a candidate therapy in autoimmunity and transplantation, we hypothesized that Nox2 deficiency in Tregs in recipient mice may improve outcomes in a heart transplant model. We generated a potentially novel B6129 mouse model with Treg-targeted Nox2 deletion (Nox2fl/flFoxP3Cre+ mice) and transplanted with hearts from CB6F1 donors. As compared with those of littermate controls, Nox2fl/flFoxP3Cre+ mice had lower plasma levels of alloantibodies and troponin-I, reduced levels of IFN-γ in heart allograft homogenates, and diminished cardiomyocyte necrosis and allograft fibrosis. Single-cell analyses of allografts revealed higher absolute numbers of Tregs and lower CD8+ T cell infiltration in Nox2-deficient recipients compared with Nox2-replete mice. Mechanistically, in addition to a greater suppression of CD8+CD25- T effector cell proliferation and IFN-γ production, Nox2-deficient Tregs expressed higher levels of CCR4 and CCR8, driving cell migration to allografts; this was associated with increased expression of miR-214-3p. These data indicate that Nox2 deletion in Tregs enhances their suppressive ability and migration to heart allografts. Therefore, Nox2 inhibition in Tregs may be a useful approach to improve their therapeutic efficacy.