Novel nuclear hENT2 isoforms regulate cell cycle progression via controlling nucleoside transport and nuclear reservoir.

Nucleosides participate in many cellular processes and are the fundamental building blocks of nucleic acids. Nucleoside transporters translocate nucleosides across plasma membranes although the mechanism by which nucleos(t)ides are translocated into the nucleus during DNA replication is unknown. Here, we identify two novel functional splice variants of equilibrative nucleoside transporter 2 (ENT2), which are present at the nuclear envelope. Under proliferative conditions, these splice variants are up-regulated and recruit wild-type ENT2 to the nuclear envelope to translocate nucleosides into the nucleus for incorporation into DNA during replication. Reduced presence of hENT2 splice variants resulted in a dramatic decrease in cell proliferation and dysregulation of cell cycle due to a lower incorporation of nucleotides into DNA. Our findings support a novel model of nucleoside compartmentalisation at the nuclear envelope and translocation into the nucleus through hENT2 and its variants, which are essential for effective DNA synthesis and cell proliferation.

Microparticles as biomarkers of lung disease: enumeration in biological fluids using lipid bilayer microspheres.

Extracellular vesicles, specifically microparticles (MPs), are rapidly gaining attention for their capacity to act as biomarkers for diagnosis, prognosis, or responsiveness to therapy in lung disease, in keeping with the concept of precision medicine. However, MP analysis by high-sensitivity flow cytometry (FCM) is complicated by a lack of accurate means for MP enumeration. To address this gap, we report here an enhanced FCM MP gating and enumeration technique based on the use of novel engineered lipid bilayer microspheres (LBMs). By comparison of LBM-based MP enumeration with conventional bead- or fluorescent-based FCM enumeration techniques and a gravimetric consumption gold standard, we found LBMs to be superior to commercial bead preparations, showing the smallest fixed bias and limits of agreement in Bland Altman analyses. LBMs had simultaneous capacity to aid FCM enumeration of MPs in plasma, BAL, and cell culture supernatants. LBM enumeration detected differences in MP counts in mice exposed to intraperitoneal lipopolysaccharide or saline. LBMs provided for 1) higher sensitivity for gating MPs populations, 2) reduced background within MP gates, 3) more appropriate size, and 4) an inexpensive alternative amenable to different fluorescent tags. LBM-based MP enumeration was useful for a series of different FCM systems assessed, whereas LBM gating benefited high- but not low-sensitivity FCM systems compared with fluorescence gating. By offering exclusive advantages over current means of gating and enumerating MPs, LBMs are uniquely suited to realizing the potential of MPs as biomarkers in biological lung fluids and facilitating precision medicine in lung disease.

Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrin αIIbß3 in mice.

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.

Cadherin 6 has a functional role in platelet aggregation and thrombus formation.

OBJECTIVE: Thrombosis occurs at sites of vascular injury when platelets adhere to subendothelial matrix proteins and to each other. Platelets express many surface receptor proteins, the function of several of these remains poorly characterized. Cadherin 6 is expressed on the platelet surface and contains an arginine-glycine-aspartic acid motif, suggesting that it might have a supportive role in thrombus formation. The aim of this study was to characterize the role of cadherin 6 in platelet function. METHODS AND RESULTS: Platelet aggregation was inhibited by both antibodies and exogenous soluble cadherin 6. Platelet adhesion to immobilized cadherin 6 was inhibited by arginine-glycine-aspartic acid-serine tetrapeptides. Antibodies to α(IIb)ß(3) inhibited platelet adhesion to cadherin 6. Because platelet aggregation occurs in fibrinogen and von Willebrand factor double-deficient mice, we investigated whether cadherin 6 is an alternative ligand for the integrin α(IIb)ß(3). Platelet aggregation in fibrinogen and von Willebrand factor double-deficient mice was significantly inhibited by an antibody to cadherin 6. In flow-based assays, inhibition of cadherin 6 caused a marked reduction in thrombus formation in both human and mouse blood. CONCLUSIONS: This study demonstrates the role of cadherin 6 as a novel ligand for α(IIb)ß(3) and highlights its function in thrombus formation.

Animal model of fetal and neonatal immune thrombocytopenia: role of neonatal Fc receptor in the pathogenesis and therapy.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder in which maternal antibodies cross the placenta and destroy fetal/neonatal platelets. It has been demonstrated that the neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNIT has not been studied. Here, we developed an animal model of FNIT using combined ß3 integrin-deficient and FcRn-deficient (ß3(-/-)FcRn(-/-)) mice. We found that ß3(-/-)FcRn(-/-) mice are immunoresponsive to ß3(+/+)FcRn(-/-) platelets. The generated antibodies were ß3 integrin specific and were maintained at levels that efficiently induced thrombocytopenia in adult ß3(+/+)FcRn(-/-) mice. FNIT was observed when immunized ß3(-/-)FcRn(+/+) females were bred with ß3(+/+)FcRn(+/+) males, while no FNIT occurred in ß3(-/-)FcRn(-/-) females bred with ß3(+/+)FcRn(-/-) males, suggesting that FcRn is indispensable for the induction of FNIT. We further demonstrated that fetal FcRn was responsible for the transplacental transport of various IgG isotypes. We found that anti-FcRn antibody and intravenous IgG prevented FNIT, and that intravenous IgG ameliorated FNIT through both FcRn-dependent and -independent pathways. Our data suggest that targeting FcRn may be a potential therapy for human FNIT as well as other maternal pathogenic antibody-mediated diseases.

Relative efficacy of steroid therapy in immune thrombocytopenia mediated by anti-platelet GPIIbIIIa versus GPIbα antibodies.

Immune thrombocytopenia (ITP) is characterized by platelet clearance mediated primarily by autoantibodies against the platelet GPIIbIIIa and/or GPIbα. Steroid therapy is a first-line treatment for ITP. However, some patients are refractory to this therapy and currently no method can predict which patients will respond. To evaluate whether steroids are equally efficacious in treating patients with ITP caused by anti-GPIIbIIIa versus anti-GPIbα antibodies, we performed a retrospective study on 176 newly diagnosed patients with acute ITP who had severe bleeding symptoms and were admitted as resident patients to the hospital. The patients were treated first with intravenous administration of high-dose dexamethasone (DXM), followed by oral administration of prednisone. Response to therapy was observed in a majority of patients with antibodies specific for GPIIbIIIa (31/43) or without detectable antibodies against either GPIIbIIIa or GPIbα (36/45). In contrast, the steroid response was significantly lower in patients with anti-GPIbα antibodies (9/34) or with antibodies against both GPIbα and GPIIbIIIa (16/54). The preliminary findings of this study suggest that in future prospective clinical trials including corticosteroids, the anti-GPIbα, and -GPIIbIIIa status should be assessed in order to test its potential relevance in deciding future treatments.

Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von Willebrand factor.

We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWF-independent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre(+), Fn(flox/flox), Fg/VWF(-/-)) mice and littermate control (Cre(-), Fn(flox/flox), Fg/VWF(-/-)) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti-mouse beta(3) integrin antibodies but was not affected by anti-mouse GPIbalpha antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl(3)-injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation.

Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression.

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg(-/-)) mice, and engagement of the C-terminus of the fibrinogen gamma chain with beta3 integrin was required for this process. Furthermore, Fg(-/-) platelets significantly increased P-selectin expression following transfusion into beta3 integrin-deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Non-canonical Wnt signals are modulated by the Kaiso transcriptional repressor and p120-catenin.

Gastrulation movements are critical for establishing the three principal germ layers and the basic architecture of vertebrate embryos. Although the individual molecules and pathways involved are not clearly understood, non-canonical Wnt signals are known to participate in developmental processes, including planar cell polarity and directed cell rearrangements. Here we demonstrate that the dual-specificity transcriptional repressor Kaiso, first identified in association with p120-catenin, is required for Xenopus gastrulation movements. In addition, depletion of xKaiso results in increased expression of the non-canonical xWnt11, which contributes to the xKaiso knockdown phenotype as it is significantly rescued by dominant-negative Wnt11. We further demonstrate that xWnt11 is a direct gene target of xKaiso and that p120-catenin association relieves xKaiso repression in vivo. Our results indicate that p120-catenin and Kaiso are essential components of a new developmental gene regulatory pathway that controls vertebrate morphogenesis.

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter.

Improvements in identification and assessment of extracellular vesicles (EVs) have fuelled a recent surge in EV publications investigating their roles as biomarkers and mediators of disease. Meaningful scientific comparisons are, however, hampered by difficulties in accurate, reproducible enumeration and characterization of EVs in biological fluids. High-sensitivity flow cytometry (FCM) is presently the most commonly applied strategy to assess EVs, yet its utility is limited by variant ability to resolve smaller EVs. Here, we propose the use of 405 nm (violet) wavelength lasers in place of 488 nm (blue) for side scatter (SSC) detection to obtain greater resolution of EVs using high-sensitivity FCM. To test this hypothesis, we modelled EV resolution by violet versus blue SSC in silico and compared resolution of reference beads and biological EVs from plasma and bronchoalveolar lavage (BAL) fluid using either violet or blue wavelength SSC EV detection. Mie scatter modelling predicted that violet as compared to blue SSC increases resolution of small (100-500 nm) spherical particles with refractive indices (1.34-1.46) similar to EVs by approximately twofold in terms of light intensity and by nearly 20% in SSC signal quantum efficiency. Resolution of reference beads was improved by violet instead of blue SSC with two- and fivefold decreases in coefficients of variation for particles of 300-500 nm and 180-240 nm size, respectively. Resolution was similarly improved for detection of EVs from plasma or BAL fluid. Violet SSC detection for high-sensitivity FCM allows for significantly greater resolution of EVs in plasma and BAL compared to conventional blue SSC and particularly improves resolution of smaller EVs. Notably, the proposed strategy is readily implementable and inexpensive for machines already equipped with 405 nm SSC or the ability to accommodate 405/10 nm bandpass filters in their violet detector arrays.

Apolipoprotein A-IV binds αIIbß3 integrin and inhibits thrombosis.

Platelet αIIbß3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet ß3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbß3 integrin, and the direct apoA-IV-αIIbß3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbß3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbß3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.

The p120(ctn)-binding partner Kaiso is a bi-modal DNA-binding protein that recognizes both a sequence-specific consensus and methylated CpG dinucleotides.

The p120(ctn)-binding partner Kaiso is a new member of the POZ-zinc finger family of transcription factors implicated in development and cancer. To understand the role of Kaiso in gene regulation and p120(ctn)-mediated signaling and adhesion, we sought to identify Kaiso-specific DNA binding sequences and potential target genes. Here we demonstrate that Kaiso is a dual specificity DNA-binding protein that recognizes the specific consensus sequence TCCTGCNA as well as methyl-CpG dinucleotides. A minimal core sequence CTGCNA was identified as sufficient for Kaiso binding. Two copies of the Kaiso-binding site are present in the human and murine matrilysin promoters, implicating matrilysin as a candidate target gene for Kaiso. In electrophoretic mobility shift assays, matrilysin promoter-derived oligonucleotide probes formed a complex with GST-Kaiso fusion proteins possessing the zinc finger domain but not with fusion proteins lacking the zinc fingers. We further determined that only Kaiso zinc fingers 2 and 3 were necessary and sufficient for sequence-specific DNA binding. Interestingly, Kaiso also possesses a methyl-CpG-dependent DNA-binding activity distinct from its sequence-specific DNA binding. However, Kaiso has a higher affinity for the TCCTGCNA consensus than for the methyl-CpG sites. Furthermore, the DNA-binding ability of Kaiso with either recognition site was inhibited by p120(ctn). Kaiso thus appears to have two modes of DNA binding and transcriptional repression, both of which may be modulated by its interaction with the adhesion cofactor p120(ctn).

Plasma fibronectin supports hemostasis and regulates thrombosis.

Plasma fibronectin (pFn) has long been suspected to be involved in hemostasis; however, direct evidence has been lacking. Here, we demonstrated that pFn is vital to control bleeding in fibrinogen-deficient mice and in WT mice given anticoagulants. At the site of vessel injury, pFn was rapidly deposited and initiated hemostasis, even before platelet accumulation, which is considered the first wave of hemostasis. This pFn deposition was independent of fibrinogen, von Willebrand factor, ß3 integrin, and platelets. Confocal and scanning electron microscopy revealed pFn integration into fibrin, which increased fibrin fiber diameter and enhanced the mechanical strength of clots, as determined by thromboelastography. Interestingly, pFn promoted platelet aggregation when linked with fibrin but inhibited this process when fibrin was absent. Therefore, pFn may gradually switch from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data indicate that pFn is a supportive factor in hemostasis, which is vital under both genetic and therapeutic conditions of coagulation deficiency. By interacting with fibrin and platelet ß3 integrin, pFn plays a self-limiting regulatory role in thrombosis, suggesting pFn transfusion may be a potential therapy for bleeding disorders, particularly in association with anticoagulant therapy.

A novel murine model of fetal and neonatal alloimmune thrombocytopenia: response to intravenous IgG therapy.

Fetal and neonatal alloimmune thrombo cytopenia (FNAITP) is a life-threatening bleeding disorder caused by maternal antibodies directed against fetal platelet antigens. The immunoreactive epitopes in FNAITP are primarily located in the extracellular regions of the platelet glycoprotein IIIa (beta3 integrin). Here we have established a novel animal model of FNAITP using beta3 integrin-deficient (beta3-/-) mice. We demonstrated first that these mice are immunoresponsive to beta3 integrin; beta3-/- mice transfused with wild-type platelets generated specific anti-beta3 antibodies which were able to induce thrombocytopenia in wild-type mice. Subsequently, beta3-/- female mice (both naive and immunized) were bred with wild-type male mice to recapitulate the features of FNAITP. The titer of generated maternal antibodies correlated with the severity of FNAITP. High titer maternal anti-beta3 anti-bodies caused severe fetal thrombocytopenia, intracranial hemorrhage, and even miscarriage. Furthermore, maternal administration of intravenous immunoglobulin G (IgG) ameliorated FNAITP and down-regulated pathogenic antibodies in both the maternal and fetal circulations.

The catenin p120ctn inhibits Kaiso-mediated transcriptional repression of the beta-catenin/TCF target gene matrilysin.

The POZ-zinc finger transcription factor Kaiso was first identified as a specific binding partner for the Armadillo catenin and cell adhesion cofactor, p120ctn. Kaiso is a unique POZ protein with bi-modal DNA-binding properties; it associates with a sequence-specific DNA consensus Kaiso binding site (KBS) or methylated CpG dinucleotides, and regulates transcription of artificial promoters containing either site. Interestingly, the promoter of the Wnt/beta-catenin/TCF target gene matrilysin possesses two conserved copies of the KBS, which suggested that Kaiso might regulate matrilysin expression. In this study, we demonstrate using chromatin immunoprecipitation analysis that Kaiso associates with the matrilysin promoter in vivo. Minimal promoter assays further confirmed that Kaiso specifically repressed transcription of the matrilysin promoter; mutation of the KBS element or RNAi-mediated depletion of Kaiso abrogated this effect. More importantly, Kaiso blocked beta-catenin-mediated activation of the matrilysin promoter. Consistent with our previous findings, both Kaiso-DNA binding and Kaiso-mediated transcriptional repression of the matrilysin promoter were inhibited by overexpression of wild-type p120ctn, but not by a p120ctn mutant exhibiting impaired nuclear import. Collectively, our data establish Kaiso as a sequence-specific transcriptional repressor of the matrilysin promoter, and suggest that p120ctn and beta-catenin act in a synergistic manner, via distinct mechanisms, to activate matrilysin expression.

NLS-dependent nuclear localization of p120ctn is necessary to relieve Kaiso-mediated transcriptional repression.

The Armadillo catenin p120(ctn) regulates cadherin adhesive strength at the plasma membrane and interacts with the novel BTB/POZ transcriptional repressor Kaiso in the nucleus. The dual localization of p120(ctn) at cell-cell junctions and in the nucleus suggests that its nucleocytoplasmic trafficking is tightly regulated. Here we report on the identification of a specific and highly basic nuclear localization signal (NLS) in p120(ctn). The functionality of the NLS was validated by its ability to direct the nuclear localization of a heterologous beta-galactosidase-GFP fusion protein. Mutating two key positively charged lysines to neutral alanines in the NLS of full-length p120(ctn) inhibited both p120(ctn) nuclear localization as well as the characteristic p120(ctn)-induced branching phenotype that correlates with increased cell migration. However, while these findings and others suggested that nuclear localization of p120(ctn) was crucial for the p120(ctn)-induced branching phenotype, we found that forced nuclear localization of both wild-type and NLS-mutated p120(ctn) did not induce branching. Recently, we also found that one role of p120(ctn) was to regulate Kaiso-mediated transcriptional repression. However, it remained unclear whether p120(ctn) sequestered Kaiso in the cytosol or directly inhibited Kaiso transcriptional activity in the nucleus. Using minimal promoter assays, we show here that the regulatory effect of p120(ctn) on Kaiso transcriptional activity requires the nuclear translocation of p120(ctn). Therefore, an intact NLS in p120(ctn) is requisite for its first identified regulatory role of the transcriptional repressor Kaiso.