bluuebunny/crossref_metadata_2025 huggingface dataset:
Abcg2 Expression Marks Neural Stem Cells in the Murine Cerebellum.
Authors: Zhou Sheng , Fatima Soghra , Sorrentino Brian P.
Published: October 2019

Neural stem cells have been identified in both the cerebellum and forebrain of fetal and adult mice. These cells can form neurospheres in culture and differentiate into both glia and neurons either in vitro or in vivo. In embryonic day 14 forebrain, neural stem cells are found to exist exclusively in a subpopulation with the Side Population (SP) phenotype, and express Abcg2, a member of the ABC transporter family that is responsible for the SP phenotype in hematopoietic stem cells (HSCs). The expression of Abcg2 in stem cells in the cerebellum has not been characterized. We have generated an Abcg2/GFP knock-in mouse model in which expression of GFP is under control of the endogenous Abcg2 locus and used this model to demonstrate that Abcg2 expression can be used for HSC enrichment. Here we report the use of this mouse model to explore the relationship between Abcg2 expression and neural stem cell function in neonatal cerebellum. Single cells were prepared from cerebellum of 4–9 day old mice by digesting with papain. We then stained the cells with anti-CD45 and anti-Ter119 antibody to exclude the resident hematopoietic cells in subsequent flow cytometry analysis and cell sorting. We found that a small but consistent subpopulation of cells, comprising 0.7±0.12% of total CD45−Ter119- single cell preparations, expressed the Abcg2/GFP allele. To determine whether these GFP+ cells were enriched for neural stem cells, we sorted the CD45−Ter119- cells into GFP+ and GFP− subpopulations and analyzed for their neurosphere forming activity in the presence of epidermal growth factor and basic fibroblast growth factor. We found that the GFP+ subpopulation formed 21 fold more neurospheres compared with the GFP− subpopulation. These neurosphere forming cells can self-renew as evidenced by their capacity to form secondary neurospheres when replated. These results demonstrate that similar to what is seen with HSCs and with embryonic forebrain cells, Abcg2 is expressed in the neural stem cells in neonatal cerebellum, and Abcg2/GFP expression in this mouse model could also be used as a marker to prospectively purify neural stem cells from cerebellum. Ongoing studies are focused on defining the in vivo multilineage differentiation potential of the Abcg2/GFP+ cells and determining whether Abcg2 expression could be used as a marker for purification of medulloblastoma stem cells.
Contribution of HGF to Liver Regeneration by Strong Mobilization of Bone Marrow Stem Cells.
Authors: Tajima Fumihito , Tsuchitya Hiroyuki , Nishikawa Kenichi , Okazaki Toshirou , Shiota Goushi
Published: October 2019

Cell plasticity of bone marrow stem cells to hepatocytes is known, however, the details are still unclear. hepatocyte growth factor (HGF) promotes an increase in liver stem cells in severely injured liver, but intervention to bone marrow stem cell is unclear. We examined a role of HGF in bone marrow stem cell-mediated liver regeneration in order to obtain effective liver regeneration.

First, we found that the phenotype of stem cells, which can differentiate into hepatocytes, is Lin−c-kit+Sca-1+CD34− in bone marrow or Lin−c-kit+Sca−1+CD34+ in peripheral blood mobilized by G-CSF. We transplanted single Lin−c-kit+Sca-1+CD34− bone marrow cell harvested from male EGFP mouse to female wild type mouse, and then, using this GFP-chimera-mouse, we found that bone marrow origin GFP+ and Y chromosome+ hepatic cells were present in liver after acute liver damage. Next, single Lin−c-kit+Sca-1+CD34+ peripheral blood cell, which was mobilized in peripheral blood by administration of G-CSF, was transplanted into the portal vein of the wild type mouse which was given hepatic damage. We found that the GFP-positive cells also expressed albumin.

Second, we investigated whether the HGF can mobilize stem cells from bone marrow to peripheral blood. In peripheral blood of HGF transgenic mice, 1.1% developed CD34+ cells and 20±3 colony forming cells of 1X106 peripheral blood mononuclear cells were shown. Colony forming cells were found in the mouse into which an HGF-expressing adenovirus was administered. After injection of rHGF to mice, a significant time-dependent increase of percentage of CD34+ cells in the PB was noted at the first 3 hours and CD34+ cells were increased in dose-dependent manner of rHGF and reached plateau level at 100 m/kg. The mice having transplantation with PB cells from 100 mg HGF-treated mice for 4days showed engraftment 2 months after transplantation. Upon activation of rHGF in mouse MS-5 stromal cells, phosphorylation c-Met and SCF were up-regulated, while VCAM -1, MMP-9, SDF -1 or CXCR4 were not changed. SCF level in conditioned media was also increased after the HGF stimulation.

Finally, we examined whether the bone marrow stem cells in PB mobilized by HGF transdifferentiate into hepatocytes. Using GFP-chimera-mice given acute liver injury after administration of retorolusine and CCl4, the levels of GFP+ cells in liver of GFP-chimera-mice 2 months after treatment by PBS and HGF were 2.2±1.4% and 12.7±3.6%, respectively (p<0.01). In conclusion, HGF can mobilize stem cells with long-term engraftment capabilities from bone marrow to peripheral blood, resulting in contribution to liver regeneration.
Hematopoietic Stem Cells Neither Reconstitute Satellite Cells nor Myofibers after Direct Injection into Dystrophic Muscle.
Authors: Kuhr Christian S. , Lupu Marilena , Storb Rainer
Published: October 2019

We sought to determine whether wild-type hematopoietic stem cells directly injected into muscle could restore dystrophin expression in a relevant pre-clinical canine model of Duchenne muscular dystrophy (DMD). Recipients that possessed the dystrophin mutation and thus exhibited the DMD phenotype were chosen. They were rendered tolerant to their dog leukocyte antigen (DLA)-matched unaffected littermates through myeloablative conditioning followed by hematopoietic stem cell transplantation (HCT). Donor bone marrow cells were collected (2.19e8 cells/kg) and injected directly under the aponeurosis of the superspinatus muscle of the DMD affected animals. Excisional muscle biopsies 5, 9 and 15 weeks following injection were examined with immunofluorescence and reverse-transcription-PCR for dystrophin protein and mRNA expression (figure), respectively, and failed to demonstrate increases over background. Consistent with these results, clonal satellite cell cultures from these biopsies at the initiation of myotube formation showed no detectable donor contributions. These findings lessen the enthusiasm for strategies relying on hematopoietic stem cell transdifferentiation to repair or regenerate skeletal muscle.

Figure Figure
Identification of Hepatic Progenitors Having No Hematopoietic Potential in Murine Bone Marrow.
Authors: Yamada Yasuhiro , Miyake Hirosada , Nishimoto Eishi , Mitsuya Hiroaki , Yonemura Yuji
Published: October 2019

We recently reported that when Sca-1+ c-Kit− bone marrow cells (BMCs) were co-cultured with fetal liver cells (FLCs) on laminin-coated dishes, alpha-fetoprotein (AFP)-expressing BMCs became completely adherent by day 3 and expressed albumin as assessed with immunochemistry and RNA-PCR (Yamada et al., Exp Hematol. 34: 97, 2006). In the current study, we attempted to further delineate the characteristics of BMCs that differentiate into hepatic-like cells. It was found that AFP-expressing cells were in CD5+ or B220+ lymphoid lineage, mostly Sca-1+CD5+ lineage, expressing AFP. When cKit+Sca-1+ lineage− BMCs (KSLs), which did not express AFP, were cocultured with CD5+ BMCs, both from green fluorescence protein (GFP)-expressing transgenic mice, in the presence of FLCs from ROSA26 mice (X-gal+ FLCs), fractionated cells gave rise to adherent hepatic-like cells, which expressed albumin and cytokeratin 8 (assessed with immunochemistry) and AFP, albumin, transthyretin and dipeptidylpeptidase IV (examined with RNA-PCR). The hepatic-like cells from KSLs and CD5+ BMCs emerged at the frequency of 1 in 50 and 1x103 as assessed with titration assay. However, CD5+ Mac-1− Gr-1− Ter119− BMCs did not differentiate into hepatic-like cells. CD5+ CD45− cells differentiated into hepatic-like cells without fusion at the frequency of 1 in 300 but CD5+ CD45+ cells did not. CD5+ CD45− cells hardly produced hematopoietic colonies as compared with CD5+ CD45+ cells did.

In conclusion, we have shown that KSLs and CD5+ CD45− cells exposed to FLCs are capable of generating hepatic-like cells, which expressed albumin as assessed with immunochemistry and RNA-PCR. We should further explore whether CD5+ CD45− cells emerge from hematopoietic stem cells or mesenchymal stem cells.
A3 Domain Residue Glu1829 Contributes to A2 Subunit Retention in Factor VIIIa.
Authors: Wakabayashi Hironao , Zhou Qian , Varfaj Fatbardha , Fay Philip J.
Published: October 2019

Factor VIII circulates as a heterodimer composed of a heavy chain (HC) and light chain (LC). Thrombin converts factor VIII into the active cofactor, factor VIIIa, by cleaving HC into A1 and A2 subunits. While the A1 subunit maintains a stable interaction with the LC-derived A3C1C2 subunit, the A2 subunit is weakly associated in the trimer and this low affinity interaction accounts for the instability of factor VIIIa activity. Although the majority of binding energy for A2 retention is thought to be derived from its interaction with the A1 subunit, the factor VIII structure model based on homology with ceruloplasmin suggests a binding interface between A2 and A3 domains. While exploring potential calcium binding sites in LC by Ala scanning mutagenesis of acidic amino acids, we encountered one mutant (factor VIII:E1829A) that exhibited an ~10-fold increase in the apparent decay rate of factor VIIIa activity compared with the wild type control, as monitored by a factor Xa generation assay (see figure). This decay rate was significantly reduced by the addition of exogenous A2 subunit. In addition, the activity of factor VIII:E1829A measured by one-stage clotting and two-stage factor Xa generation assays were ~20% and ~10% of wild type factor VIII, respectively, consistent with an assay discrepancy due to increased lability of the mutant factor VIIIa. Affinity values for A1 and A2 subunits for wild type and E1829 A3C1C2 subunits were assessed using subunits purified from the factor VIII proteins. To measure A1 subunit affinity for A3C1C2, wild type or E1829A A3C1C2 subunit (10 nM) was reacted with A1 subunit (0–200 nM), and subsequently factor VIIIa activity from the reconstituted A1/A3C1C2 plus excess A2 subunit (400 nM) was measured by factor Xa generation assay. To measure A2 subunit affinity for A3C1C2, wild type or E1829A A3C1C2 subunit (1 μM) and A1 subunit (100 nM) were reacted and then mixed with A2 (0–1 μM) and regenerated cofactor activity was determined. A1 binding affinity for E1829A-A3C1C2 was comparable to the wild type A3C1C2 (Kd = 20.1 ± 3.4 nM and 15.3 ± 3.7 nM, respectively) while A2 binding affinity was reduced (Kd = 385 ± 54 nM for E1829A, 153 ± 34 nM for wild type). Furthermore, examination of the hemophilia A database shows the mutation of Glu1829 to Gly as a mild phenotype consistent with a functional role for this acidic residue. These results suggest that residue E1829 in the A3 domain contributes to A2 subunit retention following activation of the procofactor, possibly by mediating a binding interaction between the two subunits.

Factor VIIIa Activity Decay Factor VIIIa Activity Decay
Identification and Characterization of a Sodium Ion Binding Site on the Staphylocoagulase-Prothrombin Complex.
Authors: Panizzi Peter R. , Bock Paul E.
Published: October 2019

Sodium ion binding regulates allosterically the pro- and anti-coagulant functions of the central coagulation serine proteinase, thrombin (T). The conversion of the prothrombin (ProT) zymogen into T results in expression of the fibrinogen (Fbg) recognition site (exosite I) and may facilitate binding of a single Na+. Na+ binding stabilizes the “fast” form of T that mediates the efficient conversion of Fbg into fibrin. The “slow” form of T, lacking Na+, shows decreased Fbg substrate activity and slightly enhanced activity towards activation of protein C, which results in a net increase in the anticoagulant activity of T (Di Cera et al., 1995). Conversion of protein C to activated protein C results in cleavage and inactivation of factor Va and VIIIa, which inhibits reactions central to hemostasis. Staphylocoagulase (SC), a conformational activator of ProT, binds ProT with high affinity through proexosite I and imparts T-like activity to the zymogen-activator complex. To determine whether the Na+ binding site is present on the SC-(1-325)·ProT complex, kinetic studies in the presence and absence of Na+ were performed, using the non-interacting cation, choline to maintain constant ionic strength. Results indicate that indeed Na+ binds to the SC-(1-325)·ProT complex with a KD of 17 ± 2 mM and causes an ~7-fold increase in the activity of SC-(1-325)·ProT complex toward a T-specific tripeptide chromogenic substrate. The chromogenic substrate activity of mouse ProT·SC-(1-325), unlike the human zymogen, was independent of Na+, consistent with the recent finding that mouse T lacks a functional Na+ binding site (Bush et al., 2006). Fbg clotting studies indicated that human T has 24-fold increased clotting activity in the presence of Na+, and the SC-(1-325)·T complex has 13-fold enhanced activity. Surprisingly, much unlike the results obtained from the chromogenic substrate studies, the Fbg clotting activity of the SC-(1-325)·ProThuman complex was less dependent on the presence of Na+, as indicated by a 4.1-fold increase in clotting activity for the complex in the presence of Na+. Further studies will be necessary to elucidate the functional significance of the Na+ binding site on the SC·ProT complex and its effect on Fbg recognition and cleavage.
Characterization of an Anti-Factor VIII Monoclonal Antibody with A1 and A3 Epitopes Which Increases Factor VIII Activity.
Authors: Takeyama Masahiro , Nogami Keiji , Soeda Tetsuhiro , Suzuki Tsukasa , Hattori Kunihiro , Tatsumi Kohei , Fujita Yuri , Tanaka Ichiro , Yoshioka Akira , Shima Midori
Published: October 2019

Epitopes of anti-factor VIII (FVIII) neutralizing antibodies distribute all of the FVIII domains. They inhibit FVIII for instance by blocking the FVIII interaction with factor IXa, factor X/factor Xa (FX/FXa), phospholipid, von Willebrand factor, or thrombin. Therefore, localization of such FVIII inhibitory effect gives us useful information for understanding of FVIII structure and function. We expect FVIII enhancing antibody also may be a useful tool. In this study, we found an anti-FVIII monoclonal antibody (named by moAb216) that increased the FVIII cofactor activity. The addition of moAb216 increased FVIII activity by ~1.6-fold dose-dependently in one-stage clotting assay. The increase of FVIII activity in the presence of moAb216 correlated with that of generated thrombin or factor Xa in thrombin or FXa generation based-assay. Blotting analysis revealed that this antibody reacted with only intact FVIII molecule, whilst failed to react with either SDS-treated FVIII, FVIIIa (active-form), or isolated each A1, A2, and A3-C1-C2 subunit. Individual monoclonal antibody, with an epitope of the A1 or A3 acidic region in FVIII, competitively inhibited FVIII binding to moAb216 as well as the increase of FVIII activity by its antibody. However, anti-A2 or anti-C2 monoclonal antibody did not affect. These results supported that this unique antibody, with a discontinuous epitope spanning both acidic regions in the A1 and A3 domains, recognized the native conformation of FVIII. To examine the mechanism(s) of increasing effect by moAb216, we focused on the activation and/or inactivation of FVIII. Rate constants on thrombin- and FXa-catalyzed activation of FVIII in the presence of moAb216 were ~2 and 3-fold greater, respectively, in dose-dependent manners compared with that of FVIII in its absence. On the other hand, the antibody inhibited the activated protein C (APC)-catalyzed FVIII inactivation with ~10-fold lower of inactivation rate constant. SDS-PAGE analysis revealed that moAb216 accelerated the cleavage at Arg372 in the A1-A2 junction by thrombin and FXa, whilst decelerated the cleavage at Arg336 within the A1 domain by APC. In addition, FVIII activity in the presence of moAb216 was more stable following heat denaturation analysis than that in its absence. We demonstrated that the increasing effect of FVIII activity by moAb216 was attributed to the change of cleavage at Arg372 and/or Arg336 in the heavy chain caused by interaction with the A1/A3 domains. Furthermore, moAb216 would be useful as a new replacement therapy for hemophilia A patients, since this increases and stabilizes FVIII activity, and can function even in the presence of anti-FVIII (A2 and C2) inhibitors.
Tyrosine Kinase 2 (Tyk2) Interacts with and Phosphorylates Siva-1, and Auguments the Apoptotic Effect Induced by Siva-1.
Authors: Kakumitsu Haruko , Shimoda Kazuya , Haro Takashi , Shide Kotaro , Kumano Takashi , Oku Seido , Kamezaki Kenjirou , Numata Akihiko , Takenaka Katsuto , Harada Mine
Published: October 2019

Jak-Stat pathway is essential to transduce cytokine signalings. We previously reported that tyrosine kinase 2 (Tyk2), one of the Jak kinases, was essential but Stat1 was dispensable in IFN-alpha-induced B lymphocyte apoptosis. This indicates the existence of other signaling molecules besides Stat1 downstream of activated Tyk2. Therefore, in order to find Tyk2-activated signaling molecules which transduce IFN-alpha signal and induce B lymphocyte apoptosis, we performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Siva-1, which had been originally cloned as a CD27 binding protein. Siva-1 has a death domain (DD) homology region and induces apoptosis in various cells through binding CD27, which belongs to the tumor necrosis factor receptor (TNFR) family. We found that Tyk2 interacts with and phosphorylates Siva-1. Two regions of Siva-1, the N-terminus and the middle portion of the protein containing a death domain homology region, contributed to binding to Tyk2, and two tyrosines of Siva-1, Tyr53 and Tyr162, were phosphorylated by Tyk2. Because Siva-1 is so far thought to be a proapoptotic protein, we assessed whether Tyk2 had any effects on Siva-1-mediated apoptosis through the Tyk2-Siva-1 interaction. First, we established BaF3 cell (IL-3 dependent murine proB cell) lines stably expressing either wild-type Tyk2 (BaF3/WT Tyk2) or constitutively activated Tyk2 which Valine678 was replaced by Phenylalanine (BaF3/V678F Tyk2). In BaF3/V678F Tyk2 cells, Tyk2 and Stats are constitutively activated, and they grow in the absence of IL-3. We transiently transfected either GFP-Siva-1 or control GFP vector into BaF3/parent cells, BaF3/WT Tyk2 cells or BaF3/V678F Tyk2 cells. Thirty-six hours after transfection, we measured annexin V positive cells by flow cytometry on GFP-positive cells. Approximately 24% of both BaF3/parent cells and BaF3/WT Tyk2 cells transfected with GFP-Siva-1 fell into apoptosis, whereas only less than 7% of the cells transfected control vector showed apoptosis. The frequency of this Siva-1-induced apoptosis was increased in BaF3/V678F Tyk2 cells (over 43% vs <10% of expressing control vector), suggesting that activated Tyk2 augmented the apoptotic function of Siva-1. In conclusion, we show that Tyk2 associates with and phosphorylates Siva-1 and that this Tyk2-Siva-1 interaction enhances the apoptotic effect induced by Siva-1. This indicates that Siva-1 might be a probable key molecule downstream of Tyk2 which is activated in response to IFN-alpha.
Human Mesenchymal Cells Regulates Naive and Memory T Regulatory Cells.
Authors: Di Ianni Mauro , Del Papa Beatrice , Moretti Lorenzo , Sportoletti Paolo , Bonifacio Elisabetta , Cecchini Debora , De Ioanni Mariangela , Falzetti Franca , Tabilio Antonio
Published: October 2019

CD4(+)CD25(+)FoxP3(+) T regulatory (T reg) cells constitute 1–2% of peripheral blood cells in adults and function prevalently as immunosuppressors. 70% of T reg are memory/effector cells with a CD45RO+ phenotype. The other 30% have a naive T cell phenotype (CD45RA+). Both sub-populations exert a regulatory function and when sorted and/or purified each inhibits a mixed lymphocyte culture. In the present study, we investigated the effects of human recombinant IL-7 (rIL-7), human mesenchymal cells (hMSC) and hMSC engineered with the IL-7 gene (hMSC/IL-7) on regulatory T cells expressing a memory (CD4/CD25/CD45RO) or naive (CD4/CD25/CD45RA) phenotype. T cells from healthy donors were enriched by immnuselection to provide populations of CD45RA+ cells (95 % ± 2.9) and CD45RO+ cells (97 % ± 0.25). Enriched naive and memory cells were cultured in presence rIL-7 (100 ng/ml), hMSC (ratio 5:1) or hMSC/IL-7 (ratio 5:1). After 7 days’ culture we examined the CD4/CD25+ cells within the naive and memory populations. In the naive T cell population the T reg starting fraction of 0.05 % ± 0.01 of CD4/CD25 positive cells, did not change in the presence of rIL-7 while it rose to 0.2 % ± 0.14 in presence of human MSC and more interestingly reached 1.67 % ± 0.6 in presence of IL-7 engineered human MSC. In the memory T cell population the T reg starting fraction of 0.3 % ± 0.05 of CD4/CD25 positive cells, did not change in presence of rIL-7 while it rose 1.5 % ± 0.9 in the presence of human MSC and more interestingly reached 11.55 % ± 7.5 in the presence of human IL-7 engineered-MSC. We analyzed CD127 expression within different groups. In the naive T reg starting fraction 3 % ± 1.2 expressed CD127 which was down-regulated to 0.96 % ± 0.5 in the presence of rIL-7, to 0.29 % ± 0.2 with human MSC and to 0.37 % ± 0.02 with human IL-7engineered-MSC. Memory T reg cells expressed CD127 in 15% ± 1.2 of the starting fraction which was down-regulated to 1.2 % ± 0.45 in the presence of rIL-7, to 1.32 % ± 0.34 with hMSC and to 4.01% ± 0.74 in presence of hMSC/IL-7. FoxP3 expression was measured by real time quantitative PCR in sort-purified subsets of peripheral blood, identified by staining with a combination of CD4, CD25, CD45RA or CD45RO. In naive T reg FoxP3 expression was increased 1.15 fold in the presence of hMSC and 2.7 fold in presence of hMSC/IL-7. In memory T reg FoxP3 expression was increased 1.14 fold in the presence of hMSC and 2.67 fold in presence of hMSC/IL-7. In conclusion naive T reg cells are IL-7 independent and up-regulated by human MSC. Engineering human MSC with the IL-7 gene enhanced up-regulation. Memory T reg cells are also IL-7 independent and are up-regulated by human MSC. Engineering human MSC with the IL-7 gene markedly increased up-regulation. The different regulatory systems may underlie different functions within the T reg sub-populations.
Inhibition of γδ T Cell Proliferation by CD4+CD25+FOXP3+ Regulatory T Cells (Treg).
Authors: Kunzmann Volker , Kimmel Brigitte , Engert Judith , Wilhelm Martin , Einsele Hermann
Published: October 2019

CD4+CD25+FOXP3+ regulatory T cells (Treg) are a unique population of T cells that maintain immune tolerance by suppressing self-reactive cells. Treg also contribute to the establishment of a dominant tolerance during infections and after allogeneic transplantation and suppress immune response to tumors. Increasing evidence supports the existence of elevated numbers of Treg in both solid tumors and hematological malignancies. In this study we show that Treg may also suppress other arms of an effective immune response. In vitro, purified human CD4+CD25+FOXP3+ Treg (by immunomagnetic selection) directly inhibit phosphoantigen (BrHPP)-mediated proliferation of Vγ9Vδ2 T cells, the major γδ T lymphocyte subset in humans. Importantly, suppression of γδ T cell proliferation by Treg was maintained when Treg where separated from γδ T cells by Transwells, suggesting that the inhibitory function of Treg on γδ T cell proliferation is not cell-contact independent and rather soluble factors produced by Treg contribute to this suppressive effect. However, Treg do neither influence the expression of activation markers (CD69, CD25) nor the production of IFN-γ by Vγ9Vδ2 T cells stimulated with phosphoantigen indicating that not all effector functions of Vγ9Vδ2 T cells are suppressed by Treg. As we have recently reported, phosphoantigen-mediated γδ T cell proliferation is frequently suppressed in cancer patients. This observation prompted us to address the role of Treg in controlling γδ T cell proliferation in cancer patients. An inverse correlation between Treg frequencies (i.e. the ratio between Treg and Vγ9Vδ2 T cells) in peripheral blood and phosphoantigen-mediated γδ T cell proliferation was found (mean Treg/Vγ9Vδ2 T cell ratio in cancer patients with maintained phosphoantigen-mediated γδ T cell proliferation (n=14): 4.06; mean Treg/Vγ9Vδ2 T cell ratio in cancer patients without phosphoantigen-mediated γδ T cell proliferation (n=55): 33.36)). Therefore, the Treg/Vγ9Vδ2 T cell ratio in peripheral blood can predict the capacity of γδ T cells to proliferate in response to phosphoantigens. In conclusion, these findings support a role for Treg in blunting the γδ T cell arm of the innate immune response in cancer patients and highlight the potential of Treg depletion (e.g. by anti-CD25 antibodies, cyclophosphamide or fludarabine) to promote γδ T cell mediated antitumor activity.
A Novel Use of Rehydrated, Lyophilized Platelets Increases Recombinant FVIIa Procoagulant Activity in a Model of Hemophilia.
Authors: Campbell Robert A. , Fischer Thomas H. , Wolberg Alisa S.
Published: October 2019

The anti-bleeding therapy recombinant factor VIIa (rFVIIa) is used to abrogate bleeding in hemophiliacs with inhibitors, bypassing the need for replacement factors. RFVIIa is hypothesized to work by increasing Xa generation on the platelet’s surface. However, high plasma levels of rFVIIa are required, in part due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. One platelet preparative that may be used in this application is rehydrated, lyophilized (RL) platelets. RL platelets are fixed with paraformaldehyde, which allows them to be frozen and lyophilized while retaining their hemostatic capabilities. Previously, we have shown RL platelets are capable of supporting rFVIIa-mediated thrombin generation and that thrombin generation is increased in a rFVIIa dose-dependent manner (Blood, 106:4057, 2005). In this current study, we have characterized the ability of RL platelets to modulate rFVIIa-mediated thrombin generation and fibrin clot formation in a cell-based complete model of hemophilia. The addition of RL platelets with 50 nM rFVIIa increased the thrombin generation rate in hemophilia 2.8-fold more than 50 nM rFVIIa, alone. Further, the addition of RL platelets with 50 nM rFVIIa normalized clot formation and stability in a fibrinolytic environment, which did not occur in the presence of rFVIIa, alone. In contrast, the addition of RL platelets, alone, to hemophilic conditions had minimal to no effect on thrombin generation rate or the onset of clot formation, suggesting that the effects were due to a specific interaction between rFVIIa and RL platelets. When rFVIIa plus RL platelets were added to platelet-rich plasma from patients with hemophilia A in the presence of tissue-type plasminogen activator, clot formation and stability were improved more than the addition of either agent alone. To examine the mechanism of RL platelets’ augmentation of rFVIIa activity, we titrated the phosphatidylserine (PS) binding protein, annexin V, into reactions with RL platelets in the presence of factors Xa, Va, and prothrombin and measured thrombin generation. The addition of annexin V reduced thrombin generation equally in reactions that contained RL platelets stimulated with or without SFLLRN. Further, thrombin generation was similar on RL platelets simulated with or without SFLLRN in the absence of annexin V. These data suggest that RL platelets already have PS exposed on their surface. We conclude that RL platelets can support rFVIIa-mediated thrombin generation in the absence of factor IX and may enhance rFVIIa activity in part due to PS exposure on the RL platelet surface. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa, at lower doses of rFVIIa than are currently required to achieve hemostasis.
Characterization of TFPI and TFPI-beta in Mouse Tissues.
Authors: Maroney Susan A. , Ferrel Josephine P. , Mast Alan E.
Published: October 2019

Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor. The first two Kunitz domains efficiently inhibit factor VIIa and factor Xa and thereby abrogate tissue factor initiated blood coagulation. Two isoforms of TFPI have been identified. Full-length TFPI is thought to indirectly associate with endothelium via binding to a GPI-anchored co-receptor. TFPI-beta is produced by alternative splicing of full-length TFPI. It consists of the first two Kunitz domains followed by a unique C-terminal region with a direct GPI-anchor attachment site. The physiological significance of TFPI-beta is not known. We performed 3′RACE of mouse placental cDNA to determine if additional isoforms of TFPI exist. These studies identified only TFPI and TFPI-beta. Of importance, a full-length form of TFPI containing a direct GPI-anchor attachment site was not identified. This finding supports the hypothesis that full-length TFPI binds to endothelium indirectly through association with a GPI-anchored co-receptor. In order to identify potential physiological roles for TFPI and TFPI-beta, we analyzed mRNA expression and protein production in mouse tissues. Message for both TFPI and TFPI-beta was present in all tissues examined. Real time PCR showed TFPI message 2.5- to 65-fold higher than TFPI-beta message in the different tissues (heart (2.5)>lung (5.1)>testicle (5.8)>spleen (9.3)>thymus (10)>uterus (11)>kidney (26)> liver (35)> placenta (37)>brain (65)). The high relative amount of TFPI-beta in heart, lung and testicle suggest that it may have an important physiological role in these tissues. In situ hybridization studies did not identify vascular bed specific expression of TFPI or TFPI-beta. These studies demonstrated that TFPI and TFPI-beta localize predominantly within endothelial cells with identical staining patterns in all tissues studied. Western blot analysis for TFPI and TFPI-beta in detergent lysates of the different tissues demonstrated the presence of full-length TFPI in all tissues in amounts proportional to the vascularization of the tissue (placenta>>heart>lung>spleen>kidney>liver>>>brain).

TFPI-beta was not observed by western blot analysis in any of the tissues indicating that TFPI-beta message may be transcriptionally repressed. Brain contains much less TFPI than other tissues, yet has high amounts of tissue factor. The low TFPI in the brain could be due to either low numbers of endothelial cells within the brain or to low expression of TFPI by brain endothelial cells. We used real time PCR to compare the level of TFPI and TFPI-beta mRNA expression to that of the endothelial specific proteins VE-cadherin and PECAM. When compared to these markers, the relative amount of TFPI mRNA brain is similar to that present in the heart and lung, indicating that TFPI likely has an important role in prevention of thrombosis within the cerebral vasculature.
The Basic Helix-Loop-Helix TAL1/SCL and Its Partners E47 and LMO2 Act Upstream of the VE-Cadherin Gene in Endothelial Cells.
Authors: Deleuze Virginie , Chalhoub Elias , El-Hajj Rawan , Dohet Christiane , Le Clech Mikael , Huber Philippe , Mathieu Daniele
Published: October 2019

The basic helix-loop-helix protein TAL-1/SCL, essential for the formation of the hematopoietic system, is also required for vascular development and more particularly for embryonic angiogenesis. We previously reported that TAL-1 acts as a positive factor for post-natal angiogenesis by stimulating endothelial morphogenesis. To understand how TAL-1 modulates angiogenesis, we investigated the functional consequences of TAL-1 silencing, mediated by small-interfering RNAs, in human primary endothelial cells (ECs). We found that TAL-1 knockdown impaired in vitro EC tubulomorphogenesis (in 2-D on Matrigel or 3-D in collagen I gel), with the notable absence of cell-cell contacts, a prerequisite for morphogenesis initiation. This cellular deficiency was associated with a dramatic reduction in the vascular-endothelial (VE)-cadherin at intercellular junctions, the major component of endothelial adherens junctions. In contrast, PECAM (or CD31) was present at cell-cell junctions at the same levels as control cells. Importantly, silencing of two known TAL-1-partners in hematopoietic cells, E47 or LMO2, produce the same effects as TAL-1. Accordingly, silencing of TAL-1, as well as E47 and LMO2, provoked down-regulation of VE-cadherin at both the mRNA and protein levels. Transient transfection experiments in HUVECs showed that TAL-1 and E47 regulate the VE-cadherin promoter through a specialized E-box element. Finally, endogenous VE-cadherin transcription could be directly activated in non-endothelial HEK-293 cells that neither express TAL-1 or LMO2, by the sole concomitant ectopic expression of TAL-1, E47 and LMO2. Overall, our data demonstrate that a multiprotein complex containing at least TAL-1, LMO2 and E47 act upstream of the VE-cadherin gene. We are currently performing chromatin immunoprecipitation (ChIP) to investigate whether the TAL-1-containing complex binds in vivo the VE-cadherin promoter. This study identifies VE-cadherin as an upstream TAL-1-target gene in the endothelial lineage, and provides a first clue in TAL-1 function in the control of angiogenesis.
Tumor Vasculogenesis Can Be Derived from the Hematopoietic Stem Cell.
Authors: Otero Jacklyn , Russell Kathryn J. , Fu DongTao , Shi Wenyin , Jorgensen Marda L. , Guthrie Steven M. , Siemann Dietmar , Scott Edward W. , Cogle Christopher R.
Published: October 2019

Blood vessel development is essential to cancer growth and metastasis. Based on our recent findings that the hematopoietic stem cell (HSC) provides functional hemangioblast activity in repairing the ischemic retina, we questioned if the HSC also acts as a pathologic hemangioblast - contributing to tumor vasculogenesis. Using a transplant model of green fluorescent protein (GFP) marrow into wild-type C57BL/6 mice, we injected lung cancer (LLC) and allowed the tumor to grow for 14 days. All tumor specimens demonstrated tumor vessels with marrow-derived endothelial cells. Approximately 25% of tumor vessels contained marrow-derived endothelial cells as demonstrated by GFP, CD31 and vWF expression. Confocal microscopy was used to identify true marrow-derived endothelial cells lining the vascular lumen versus marrow-derived pericytes juxtaposed to endothelial cells. We further questioned whether the tumor neovasculogenesis is from a clonal, self-renewing HSC. Lung cancers grown in recipients of single cell and serially transplanted hematopoietic stem cells (n=9) demonstrate clonal, donor-derived endothelial cells in 5% of tumor vasculature, matching hematopoietic engraftment. Our results indicate that the self-renewing, clonal adult hematopoietic stem cell exhibits pathologic hemangioblast activity, capable of producing both blood and blood vessels within tumors. Given that the HSC and its EPC progeny can contribute to tumor vasculogenesis, we further hypothesized that factors that affect leukocyte trafficking likely affect the pathologic hemangioblast activity of HSC. Thus, we made slight modifications to our transplant and tumor inoculation model by administering GCSF and SCF to mobilize marrow derived EPC. Over the ensuing 14 days, the tumors in the cytokine treated group grew at a much faster rate and to a much larger size than tumors in the control mice. After 14 days of cytokine treatment and tumor growth, microvessel density was not different between cytokine treated mice (n=4) and control mice (n=4); however, marrow-derived tumor vasculogenesis was markedly elevated in the cytokine treated compared to controls (63% vs. 26%). In conclusion, the HSC contributes to blood vessels within lung cancer and strategies targeting HSC/EPC mobilization (such as during the recovery phase of chemotherapy) potentially represent excellent ant-neoplastic opportunities.
Endogenous Cannabinoid-Like Arachidonoyl Serine Protects Against LPS-Induced Endothelial Apoptosis.
Authors: Zhang Xuefeng , Wang Jian Feng , Maor Yehoshua , Kunos George , Groopman Jerome E.
Published: October 2019

The endocannabinoid system is involved in several physiological processes, and has been reported to have neuroprotective and vasoprotective effects. N-Arachidonoyl Serine (ARA-S), a recently identified endocannabinoid, possesses vasodilatory properties and shows protective effects on the endothelium. ARA-S does not bind to the CB1, CB2, or vanilloid TRPV1 receptors, and most likely binds to an endothelial-specific cannabinoid receptor. This receptor has not yet been identified. We previously found that ARA-S can enhance endothelial wound healing in vitro. Now, we demonstrate that ARA-S can protect endothelial cells from lipopolysaccharide (LPS)-mediated apoptosis, a pathological process which commonly occurs during infection and inflammation. Human primary dermal microvascular endothelial cells (HMVEC) expressing both CB1 and CB2 receptors were used in our functional and biochemical assays. Upon pretreatment of HMVEC with ARA-S (1 μM), LPS-induced apoptosis was significantly diminished by 25–30% compared to treatment with the vehicle control, as shown by in situ TUNEL staining and Annexin V/Propidium Iodide (PI) co-staining assays in live cells. This ARA-S-mediated protection was dose-dependent and could be partially blocked by specific CB1 and CB2 antagonists (AM251 and AM630), either individually or in combination. Other receptors besides CB1 and CB2 appeared to be involved in this process, since O-1918, a blocker of the putative third cannabinoid receptor, also inhibited the activity of ARA-S by 80%. To decipher how ARA-S changes the fate of these cells, we compared LPS-induced signaling cascades in HMVEC in the presence or absence of ARA-S. ARA-S activated both Akt and eNOS in the LPS-treated HMVEC, two important molecules for endothelial survival. In contrast, ARA-S inhibited the activities of Caspase 9 and Cytochrome C, which were induced by LPS and cause cell apoptosis. Our findings support a role for novel endocannabinoids like ARA-S that may interact with putative cannabinoid receptors in protecting the cardiovascular system from toxic stimuli like LPS.
Immune Cells Mimic Endothelial Progenitor Colonies.
Authors: Rohde Eva , Malischnik Christina , Schallmoser Katharina , Reinisch Andreas , Lanzer Gerhard , Linkesch Werner , Guelly Christian , Strunk Dirk
Published: October 2019

Colony-forming units of endothelial progenitor cells (“CFU-EC”) have been introduced as a powerful biological marker for vascular function and cumulative cardiovascular risk. The precise mechanisms underlying the colony formation and their cellular composition are unclear. We hypothesized that “CFU-EC” display an immune cell function distinct from circulating endothelial progenitors.

We performed detailed subtractive “CFU-EC” analyses in blood samples from 18 healthy volunteers. The impact of various blood cell types and kinetics of protein and gene expression were studied by cell sorting, flow cytometry, quantitative RT-PCR and full genome microarray analyses. “CFU-EC”-derived soluble factors were determined in multiplex cytokine measurements and tested during endothelial network formation.

“CFU-EC” contained more than 99% CD45+ nucleated hematopoietic cells mainly comprising T cells and monocytes admixed with B and NK cells. Interestingly, purified T-cells plus monocytes formed “CFU-EC” clusters. The complete lack of colony formation after depletion of T cells or monocytes was contrasted by effective “CFU-EC” formation after depletion of CD34+ progenitors. Microarray analyses revealed an activation of immune function-related biological processes without changes in pathways assigned to angiogenesis. Soluble factors derived from “CFU-EC” cultures supported vascular regeneration in vitro. Unravelling “CFU-EC” formation as a result of a functional crosstalk between T cells and monocytes shifts expectations on vascular regenerative medicine. The data support a switch from a sole view on circulating endothelial progenitors towards models that favor the contribution of immune cells to vascular regeneration.
Endothelial Cells Are Not Derived from Hematopoietic Precursor Cells.
Authors: Timmermans Frank , De Smedt Magda , Raedt Robrecht , Plum Jean , Vandekerckhove Bart
Published: October 2019

Endothelial outgrowth cells (EOC) can be generated from mononuclear blood cells. Based on proliferative and functional characteristics, EOC were claimed to derive from an immature endothelial progenitor cell or angioblast. Several investigators have claimed that these cells constitute a subpopulation of CD34+ hematopoietic stem cells(HSC). However, the EOC-precursor is not well defined and its nature remains elusive.

Methods and results: Umbilical cord blood CD34+ cells were sorted into a small (< 1 %) CD34+CD45− non-hematopoietic cell fraction (purity > 99.5%) and CD34+CD45+ HSC (purity > 99.2 %) (n=5). The cell fractions were cultured separately in EBM2/EGM2 medium (Cambrex, Verviers, Belgium) onto gelatine coated 24 wells. EOC were exclusively derived from the CD34+CD45− cell fraction and not from the CD45+ HSC. We further analysed the CD34+CD45− cell fraction for expression of endothelial progenitor genes. Analysis showed the presence of VEGFR2, VE-Cadherine and CD146 on the CD34+CD45− precursor population whereas CD45+ HSC were consistantly negative for these markers. CD133, which was claimed to be a marker for endothelial progenitors was negative on the CD34+CD45− cells. No VEGFR2+ CD133+ cells could be detected either by flowcytometry or at the mRNA level. In adult bone marrow, EOC only derived from CD45− CD31+ cells, and not from the CD45+ HSC or CD45− CD31− mesenchymal cells. CD34+CD45+ HSC or CD14+ CD45+ monocytes generated under the same conditions large flat adherent cells positive for CD31, LDL uptake and the lectin UEA-1. On RT-PCR and real time RT-PCR analysis, cells were positive for VEGFRII, CD146 and VE cadherin. However, membrane staining was consistently negative for VE-cadherin on flowcytometric analysis and positive for monocytic markers such as CD14 and CD45. In functional assays, the majority of the cells were shown to be phagocytic and were unable to form vascular tubes in the matrigel angiogenesis assay. These data demonstrate that monocytes may acquire a phenotype in vitro which is difficult to discriminate from endothelial cells.

Conclusion : Endothelial cell generated in vitro from cord blood or bone marrow derive from a CD45− nonhematopoietic precursor.
Staphylococcus aureus Clumping Factor Mediates Rapid Thrombus Formation under High Shear.
Authors: Kerrigan Steven W. , Loughmann Anthony , Meade Geradine , Foster Timothy J. , Cox Dermot
Published: October 2019

Infective endocarditis (IE) is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Staphylococcus aureus is the most frequent causative organism of IE. Previously we showed that fibrinogen binding to the S. aureus surface protein Clumping Factor A (ClfA) plays an essential role in mediating platelet aggregation. In this study we investigate the role of ClfA in mediating thrombus formation under pathological shear conditions. Bacteria were coated onto flow chamber glass cover slips overnight at room temperature. Platelets in whole blood were stained with a lipophilic dye. Experiments were recorded in real time and visualized using fluorescence and bright-field microscopy. Platelets failed to interact with immobilized S. aureus at a shear rate of <500s−1 but rapidly formed a thrombus at shear rates greater than 800s−1. Deletion of the ClfA gene from the parent S. aureus strain (Newman) resulted in no thrombus formation at any shear rate. Platelets failed to interact with the Gram positive bacterium Lactococcus lactis at all shear rates, suggesting its suitability as a surrogate host for expressing ClfA. L. lactis over-expressing ClfA supported rapid thrombus formation with a similar profile to S. aureus. Fibrinogen binding to ClfA was found to be essential for thrombus formation although fibrinogen-coated surfaces only produced single-platelet adhesion under all shear conditions. Tirofiban and aspirin inhibited thrombus formation. Specific antibody to ClfA was also required as blood depleted of specific anti-ClfA antibody and an antibody against FcγRIIa (IV.3) inhibited thrombus formation. Our results demonstrate for the first time that, platelets interact with immobilized S. aureus under high shear >800s−1 (arterial conditions) but not low shear conditions <500s−1 (venous conditions). We identify S. aureus surface protein ClfA, fibrinogen and specific IgG are essential for thrombus formation. The rapid nature of the thrombus formation may explain why S. aureus is the major cause of IE.
The Tyrosine Kinase, Pyk2/RAFTK, Mediates LPS-Induced IL-8 and MCP-1 Expression in Human Endothelial Cells.
Authors: Anand Appakkudal R. , Ganju Ramesh K.
Published: October 2019

Lipopolysaccharide (LPS), an essential component of the surface of Gram-negative bacteria, has been shown to induce various inflammatory cytokines, including IL-8 and MCP-1, in endothelial cells. However, the mechanism by which LPS mediates the production of these inflammatory cytokines is not well known. In the present study, we investigated the role of proline-rich tyrosine kinase 2 (Pyk2), also known as RAFTK, in LPS-induced IL-8 and MCP-1 expression in endothelial cells. We observed the concentration-dependent and time-dependent tyrosine phosphorylation of Pyk2 following LPS treatment in human umbilical vein endothelial cells (HUVEC). We next analyzed the site of Pyk2 phosphorylation using antibodies specific to the individual phosphorylated tyrosine residues in Pyk2: pY402 (Src binding site), pY881 (Grb2 binding site) and pY580 (autophosphorylation site). LPS stimulation significantly enhanced phosphorylation of the pY402 and pY580 residues, but not of the pY881 site. The role of Pyk2 in LPS-induced IL-8 and MCP-1 expression was further investigated using a recombinant adeno-associated virus (AAV) expressing kinase-defective mutant Pyk2. Endothelial cells infected with the catalytically inactive Pyk2 mutant significantly attenuated LPS-induced IL-8 and MCP-1 production in HUVEC, compared to cells infected with the control AAV vector. Treatment of endothelial cells with a specific inhibitor of Pyk2, Tyrphostin A9, also blocked the expression of IL-8 and MCP-1 in a dose-dependent manner. Both p42/p44 and p38 MAP kinase pathways have been implicated in the production of inflammatory cytokines in endothelial cells. We found that blocking of Pyk2 using Tyrphostin A9 inhibited the phosphorylation of p38 MAP kinase but merely altered the kinetics of p42/p44 MAPK phosphorylation, indicating that p38 MAP kinase is an important downstream molecule of Pyk2 in LPS-induced IL-8 production. The involvement of the p38 MAP kinase pathway was confirmed by the dose-dependent blocking of IL-8 production upon treatment with a specific inhibitor of p38 MAP kinase. In summary, our data illustrate a key role for Pyk2 in LPS-mediated signaling, resulting in IL-8 and MCP-1 secretion in human endothelial cells. These findings may have important implications for the treatment of sepsis and other inflammatory disorders.