bluuebunny/crossref_metadata_2025 huggingface dataset:
Cooperative Calcium Signaling Mediated by Glycoprotein (GP) Ib-IX-V, GPVI and Integrin α2β1 during Platelet Adhesion to Collagen under Flow.
Authors: De Marco Luigi , Cozzi Maria Rita , Mazzucato Mario , Battiston Monica , Perrus Martine Jandrot , Ruggeri Zaverio M.
Published: April 2020

We have investigated signaling mediated by GPIb-IX-V, GPVI and α2β1 during platelet adhesion to collagen type I either in the presence or absence of von Willebrand factor (VWF) A1A2A3 domain under flow conditions (wall shear rate 600 or 3000 s−1). We used platelets labeled with FLUO3-AM and real-time videomicroscopy with high-speed image acquisition to analyze concurrently adhesion, translocation and calcium transients in single platelets interacting with the surface. All experiments were performed in the presence of integrillin (100 μg/ml) to block the possible involvement of αIIbβ3. Platelet adhesion and activation at 600 s−1 were similar in the absence or presence of VWF A1A2A3 domain. Calcium signaling consisted of both short lasting peaks (release from intracellular stores) and long sustained waves (transmembrane flux). In our experimental condition blockade of α2β1 or GPVI with monoclonal antibodies reduced platelet adhesion by 50% and the proportion of adherent platelets that became activated by 20–30%. Concomitant inhibition of both receptors decreased platelet adhesion by >90%. Thus, both α2β1 and GPVI participate in the initial stage of platelet adhesion to collagen type I. At the higher shear rate of 3000 s−1, platelet adhesion and activation was 40–50% less than at 600 s−1 in the absence of VWF A1A2A3 domain; addition of the latter enhanced adhesion and activation by 12 to 14-fold, and >80% of the platelets exhibited stable adhesion during the 30 s observation period. Intracellular Ca++ peaks in adherent platelets reached 2–3 μM. Blockade of GP Ib-IX-V inhibited both platelet adhesion and activation by at least 90%. Blockade of α2β1 markedly reduced the number of adhering platelets, which were mostly (>90%) translocating on the surface and exhibited only short lasting Ca++ transients. Blockade of GPVI greatly decreased the number of adherent platelets, most of which, however, were firmly attached to the surface (<15% exhibiting translocation). Calcium transients in the activated platelet consisted of short lasting peaks and rare long lasting waves reaching concentrations <1.5–2 μM. These results suggest that, on a surface of collagen type I, α2β1 is required for normal platelet arrest following tethering through the VWF A1 domain-GPIb-IX-V interaction at higher shear rates, and Ca++ signaling results from the concerted action of the two receptors reinforced by GPVI. The adhesion potential of platelets exposed to collagen, therefore, appears to depend on signaling triggered by different ligand-receptor interactions varying as a function of flow conditions.
Regulation of Platelet Functions by Targeting STAT3 with a g-Quartet Oligonucleotide.
Authors: Zhou Zhou , Guan Yongli , Sun Wensheng , Jin Naijie , Dong Jing-fei
Published: April 2020

Collagen exposed on the subendothelial matrix supports platelet adhesion and aggregation at the site of vessel injury. Its interaction with the platelet membrane receptor GP V/FcRγ chain initiates intracellular signals that recruit the hematopoietic tyrosine kinase Syk to immunoreceptor tyrosine activation motifs (ITAMs) within the FcRγ cytoplasmic tail, leading a cascade of intracellular signal events that eventually activate platelets. In the present study, we demonstrated that collagen and the collagen-related peptide (CRP) stimulation also dose-dependently (2.5–10 mg/ml, 10 min) phosphorylated the signal transducers and activators of transcription 3 (STAT3) at the tyrosine residue 705 (Tyr705) in platelets, indicating that STAT3 participates in collagen-induced platelet activation, likely through a non-transcriptional mechanism. To test this hypothesis, we determined whether a STAT3-blocking G-rich oligonucleotide (T40214, GGGCGGGCGGGCGGGC) blocks platelet activation and aggregation induced by collagen or CRP. T40214 was designed based on G-rich DNA sequences, which were originally identified in the telomeres DNA to have the ability to form the protein-binding rigid G-quartet structures. It was formed through four guanine (G) bases into cyclic Hoogsteen H-bonding. NMR determined the tertiary structure of T40214 as two stacked identical G-quartets in the center and two G-C-G-C loop domains on the top and bottom, respectively. It has previously been shown to inhibit STAT3 phosphorylation in cancer cell lines and to induce tumor apoptosis in vivo. T40214 was delivered into platelets using nano-particle polyethylenimine (PEI) as the carrier. The treatment of washed platelets with T40214 inhibited STAT3 phosphorylation induced by collagen (10 mg/ml, 10 min), while the scramble oligonucleotides did not. Incubation of washed platelets and platelets-rich plasma (PRP) with T40214 inhibited collagen-induced platelets aggregation. Finally, thrombus formation on immobilized collagen was significantly reduced when whole blood treated with T40214 (10ug/ml) under a wall shear stress of 30 dyn/cm2. Results demonstrate a novel role of STAT3 in regulating the collagen-induced intracellular signal and platelet functions. The STAT3-blocking G-quartet offers a new class of potential platelet-regulating reagents derived from screening tertiary structure of the G-rich DNA sequences that form protein-binding G-quartets.
The Calcineurin/NFAT Signaling Pathway Is a Target for the Collagen Type I-Induced Calcium Response in Megakaryocytes and Mediates Expression of Megakaryocytic Genes.
Authors: Kyttaelae Satu , Habermann Ivonne , Bornhaeuser Martin , Ehninger Gerhard , Kiani Alexander
Published: April 2020

Megakaryocytes, as precursor cells of platelets, comprise a cell population crucial for the maintenance of adult hematopoiesis. Since platelets are anuclear cellular fragments, the platelet transcriptome as well as the proteome are largely determined by the megakaryocyte. Calcium signaling in response to agonists such as collagen or thrombin has a central function in platelet activation and therefore likely represents an important signaling pathway in megakaryocytes as well. Indeed, megakaryocytes show considerable elevation of intracellular calcium levels in response to selected platelet agonists, but the downstream effector molecules of calcium signaling in these cells or the transcriptional responses induced are unknown. We here establish calcineurin and the NFAT (Nuclear Factor of Activated T cells) family of transcription factors as components of a calcium-induced signaling cascade in megakaryocytes. In resting megakaryocytes, NFAT is cytoplasmic and inactive, but can be activated by fibrillar collagen type 1, a physiological agonist of platelets and megakaryocytes known to induce a sustained increase in intracellular calcium levels in these cells. In contrast, treatment with SDF-1a, thrombopoietin, or VEGF remained without noticeable effect, presumably because of the short duration of the calcium transients induced by these agents. Collagen-induced NFAT activation in megakaryocytes requires dephosphorylation by calcineurin and is completely sensitive to the calcineurin inhibitor cyclosporin A. Activation of NFAT by collagen was paralleled by the induction of the expression of Down Syndrome Critical Region I (DSCR1) and Fas Ligand (FasL), two genes recently identified as NFAT targets in megakaryocytes. Collagen-induced expression of DSCR1 and FasL occurred in a calcineurin- and NFAT-dependent manner, as it was blocked by both cyclosporin A as well as the specific peptide inhibitor of NFAT, VIVIT. These experiments show that the calcineurin pathway is a target for selected physiological ligands capable of inducing sustained calcium mobilization in megakaryocytes and regulates megakaryocyte gene expression in a cyclosporin A- and NFAT-dependent manner.
The Cytokine Signal Inhibitor Lnk Promotes αIIbβ3 Integrin Outside-In Signaling through β3 Tyrosine Phosphorylation in Platelets.
Authors: Eto Koji , Takizawa Hitoshi , Takaki Satoshi , Nishikii Hidekazu , Oda Atsushi , Nakauchi Hiromitsu
Published: April 2020

Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk−/− mice exhibit a marked increase in numbers of hematopoietic stem cells, megakaryocytes and platelets, presumably due to the lack of negative regulation in thrombopoietin-mediated signals by Lnk. We previously reported that Lnk might play an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk−/− platelets exhibited defects in full spreading on fibrinogen, clot retraction and formation of thrombi on collagen under flow conditions while they showed normal inside-out signaling (Blood, 106 (11):115a, 2005). However the mechanism(s) in which Lnk participates in αIIbβ3 outside-in signaling had not been elucidated. Here we report that in normal platelets Lnk forms a complex with c-Src, Syk, Fyn and adhesion and degranulation promoting adaptor protein (ADAP) but not SLP-76 in a manner dependent on αIIbβ3 ligation and Src kinase activation. c-Src-, but not Syk-, mediated tyrosine phosphorylation of C-terminus in Lnk appeared to be indispensable for the complex formation and Lnk-mediated function. Furthermore we have shown that Lnk is required for the association of Fyn to αIIbβ3 and for β3 subunit tyrosine phosphorylation while activation of non-receptor tyrosine kinases (c-Src and Syk) in proximity to αIIbβ3 is independent of Lnk. Thus, these results provide new insights into Lnk function and the mechanism by which Lnk contributes to integrin signaling in the adhesion responses of platelets.
Distinct Role for Fyn-Kinase in αIIbß3-Mediated Signaling.
Authors: Reddy Kumar B. , Plow Edward F.
Published: April 2020

Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signaling. αIIbβ3 has been shown to interact with multiple SFK members. We analyzed the role of Fyn in αIIbβ3-signaling and hemostasis. Fyn associated with αIIbβ3 in resting as well as thrombin aggregated platelets. In contrast, the interaction between αIIbβ3 and another SFK member, cSrc, was seen predominantly in resting but not in thrombin aggregated platelets. Based on this difference, we explored if the Fyn−/− mice exhibit any abnormalities in hemostasis and platelet function. Fyn−/− mice had significantly prolonged second bleeding times compared to wild-type (WT) controls (3.61 min for Fyn−/− compared to 1.04 min for WT mice, n=11). Platelets from Fyn−/− mice also exhibited delayed spreading on fibrinogen coated surfaces. Fyn, but not cSrc, localized to focal adhesions in CHO cells adherent to fibrinogen via αIIbβ3, and association of Fyn and cSrc depends on different segments of the cytoplasmic tail of the β3 subunit in this model cell system In the CHO cell system, Fyn mediated αIIbβ3-dependent cell spreading in the absence of Rho-GTPase recruitment. Using mutant forms of Fyn, we found that its kinase activity is required for its localization to focal adhesions and to mediate αIIbβ3-dependent cell spreading. These results show previously unidentified roles for Fyn in αIIbβ3-signaling and hemostasis.
Cyclic AMP-Dependent Protein Kinase Mediates Glycoprotein Ibα Shedding from Platelet.
Authors: Dai Kesheng , Yan Rong , Cheng Hong , Bodnar Richard , Ruan Changgeng
Published: April 2020

Extracellular domain of platelet glycoprotein (GP) Ibα contains ligand binding sites for von Willebrand factor (VWF) and α-thrombin. GPIbα binding to VWF exposed at the injured vessel initiates thrombus formation, thus it plays key roles in thrombosis and hemostasis. While a lot of research has been performed to elucidate the critical roles for GPIbα in platelet activation, little is known about the negative regulatory mechanisms of this adhesion receptor. Here we show that inhibition of cAMP-dependent protein kinase (PKA) resulted in the shedding of GPIbα from platelet. GPIbα was shed after platelets were incubated with PKA inhibitors (H89, PKI) in a dose-dependent manner. PKA mediated GPIbα shedding was inhibited by calpain inhibitors (MDL 28170, E64d, calpain inhibitor-I, calpain inhibitor-II) in a dose-dependent manor, suggesting that shedding of GPIbα is a result of calpain cleavage. Time course experiment revealed that PKA mediated GPIbα shedding occurred as a late event, 10 minutes after platelet activation. Flow cytometry and western-blot data suggested that the cleavage site was at N-terminal of residues 484 and 485 on GPIbα. These residues are responsible for disulfide bond linkage to GPIbβ. Though the size of GPIbα shed fragments from platelet treated with H89 was the same as platelet treated with calcium ionophore A23187 or thrombin, yet the intensity of platelet activation, the amount of GPIbα shedding, and redistribution of GPIbα were different, suggesting that the mechanism of PKA inhibition-initiated GPIbα shedding is different from the shedding caused by A23187 or thrombin. Platelets treated with the PKA inhibitor, H89, presented significant decrease in ristocetin induced platelet aggregation and platelet adhesion on VWF under shear stress. In conclusion, these data provide new evidence that inhibition of PKA results in calpain mediated GPIbα shedding which may play a role in limiting thrombus infinite formation after platelet activation.
DYRK3 Encodes an Inhibitor of Erythroid Differentiation and Is Expressed at High Levels in Patients with Anemia.
Authors: Steensma David P. , Porcher Julie C. , Chadderton Antony , Laubscher Kevin , Jaworski Deb , Lager Joanne J. , Bell William , Hoffman Bryan E. , Erickson-Miller Connie L.
Published: April 2020

Background: Many patients (pts) with anemia due to impaired erythropoiesis fail to respond to currently available erythropoiesis-stimulating agents or do not wish to receive these agents due to safety concerns. For these pts, novel approaches are needed. DYRK3, an evolutionarily conserved member of an emerging family of serine-threonine kinases, is expressed at high levels in erythropoietic progenitors; murine models suggest that DYRK3 selectively inhibits red cell production during stress erythropoiesis. We sought to determine DYRK3 expression in pts with anemia, and whether in vitro inhibition of DYRK3 augments erythropoiesis.

Methods: We performed quantitative RT-PCR for DYRK3 and 4 control genes using whole peripheral blood (WPB) from 14 healthy persons, 5 pts with anemia due to multiple myeloma (MM), and 3 pts with anemia of chronic disease (ACD); in addition, peripheral blood mononuclear cells (PBMCs) were assayed in the 3 ACD and 5 MM pts and bone marrow mononuclear cells (BMMCs) in the MM pts. Erythrocyte subpopulations (CD36+, CD71+, and dual CD36+/CD71+) were quantified by flow cytometry. CFU-E growth was measured from PBMCs and BMMCs in the presence of varying concentrations of GSK626616, an orally bioavailable first-generation DYRK3 kinase inhibitor.

Results: Normalized expression of DYRK3 in WPB was 7.2 fold higher in MM pts (p=0.0001) and 3.4 fold higher in pts with ACD (p=0.026) compared to healthy controls. DYRK3 expression was proportional to the degree of anemia, and WPB and PBMC expression of DYRK3 in MM pts correlated well with BMMC expression. The level of DYRK3 expression was proportional to the population of marrow CD36+/CD71+ erythroid progenitors, and inversely proportional to the size of the more mature CD36−/CD71+ population, suggesting that high DYRK3 expression is associated with maturation arrest in humans at a stage of erythroid differentiation roughly corresponding to pre-Ter119pos/CD71high inhibition observed in murine models. Although incubation of pt-derived BMMC or PBMC with GSK626616 at concentrations up to 30 μM, either in the presence or absence of physiological concentrations of erythropoietin, did not augment in vitro CFU-E formation, CFU-E growth overall was poor in the pt samples studied.

Conclusion: DYRK3 is expressed at high levels in pts with anemia due to neoplasia or inflammation, and elevated DYRK3 expression is associated with decreased numbers of CD36−/CD71+ red cells. Further studies of the effects of DYRK3 antagonists on human erythropoiesis in vitro are necessary, and clinical trials in anemic patients will be required to determine if DYRK3 antagonists can reverse DYRK3-associated inhibition.
Detection of Hypoxia-Inducible Gene Manipulation.
Authors: Imagawa Shigehiko , Horie Masaki , Matsumoto Ken , Hirano Ikuo , Suzuki Norio , Yamamoto Masayuki
Published: April 2020

Erythropoietin (Epo) gene expression is controlled by hypoxia-inducible factor-1 (HIF-1); it is positively regulated through the HIF-1 binding site in the Epo enhancer and negatively regulated by GATA, which binds to the GATA site in the Epo promoter. Drugs that inhibit GATA or activate HIF-1 might increase the production of Epo and restore hemoglobin concentration. Epo gene doping and the illicit use of HIF-PHD inhibitor (FG-2216) and GATA inhibitor (K-11706) or HIF-1 activators may be used as new doping practices to increase the number of red blood cells. The broad scope of this research is to develop a system for detecting illegal hypoxia-inducible gene manipulation. We performed hematologic analyses, a treadmill exercise test, microarray and real-time RT-PCR (reverse transcriptase-polymerase chain reaction) to examine the effects of K-11706 and FG-2216 on mice, and compared these effects with those induced by recombinant human Epo (rhEpo) as a positive control. Oral administration of K-11706 for 5 and 8 days, FG-2216 for 5 days, and 14-day intra-peritoneal injection of rhEpo on alternate days significantly increased Epo and hemoglobin concentrations, hematocrit, and endurance performance, respectively, compared with the control. Transgenic lines of mice were generated to evaluate erythropoiesis in living mice. Firefly luciferase reporter line generated with the human β-globin locus control region (Hbb-LCR) is referred as Hbb-LCR-Luc; this mouse model provides a sensitive, noninvasive, and real-time method to monitor erythropoiesis in vivo in response to the administration of K-11706 and FG-2216. The oral administration of K-11706 and FG-2216 for 5 days significantly accumulated Hbb-LCR-Luc activity in the spleen. DNA chip technology was used to investigate the molecular mechanisms responsible for hypoxia-inducible gene manipulation. Total RNA was prepared from the bone marrow cells of mice treated with K-11706 for 28 days, FG-2216 for 5 days, and rhEpo for 14 days, and in untreated mice. The genes that appeared to exhibit a difference in the expression level (increase more or decrease less than five times for K-11706 and rhEpo, two times for FG-2216) were 251 genes for K-11706, 343 genes for FG-2216, and 142 genes for rhEpo. Twenty-eight genes showed a significant difference between the 3 groups, and 4 genes showed it with K-11706 and FG-2216. Among them, real-time RT-PCR revealed that FG-2216 significantly decreased the expression levels of FOS, OSM, IL8rb, Ms4a8a, and Arl5c genes to 0.58, 0.54, 0.56, 0.46, and 0.53-fold compared with the control, respectively. A panel of several candidate genes to detect hypoxia-inducible gene manipulation by real-time RT-PCR will provide a new detection system.
Four Novel Mutations in the Ferrochelatase (FECH) Gene in Patients with Erythropoietic Protoporphyria (EPP).
Authors: Di Pierro Elena , Brancaleoni Valentina , Besana Valeria , Ausenda Sabrina , Care Melanie , Cappellini Maria Domenica
Published: April 2020

Erythropoietic protoporphyria (EPP) is an autosomal dominant disease with incomplete penetrance, due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the insertion of iron in the protoporphyrin molecule in the final step of the heme biosynthetic pathway. The disease is biochemically characterized by increased protoporphyrins in erythrocytes, plasma and faeces and normal levels in urine. The clinical manifestations have a childhood onset, characterized by mild to moderate cutaneous photosensitivity, mild anemia and, in 5–10% of the cases, by progressive hepatic failure. Diagnosis of EPP can be supported by the simple measurement with flow citometry of fluorescent erythrocytes in peripheral blood. The phenotype of EPP results from coinheritance of a mutated allele and a wild-type low expressed allele of the FECH gene that spans about 45 kb on the long arm of chromosome 18. The FECH gene encodes for a precursor of 423 amino acid residues that is targeted to the matrix side of the inner mitochondrial membrane via a 62 amino acid cleavable leader sequence. A single promoter directs both housekeeping and erythroid expression, but two polyadenylation sites produce two mRNAs of different length. The molecular analysis of FECH gene has allowed the identification of more than 100 different mutations responsible for EPP, showing a high genetic heterogeneity. Aim of this study was to search for molecular abnormalities in four patients with clinical and biochemical signs of EPP. Molecular analysis was performed by PCR and direct sequencing of the promoter, the entire coding region and the splicing junctions of the gene. In patients who remained negative for mutations, DNA analysis was completed by long-PCR and chromosome walking. In this study four different molecular defects have been identified: two missense mutations and two large deletions in the first region of gene. One A>G substitution at nucleotide 362 in exon 4 resulting in a Glu to Gly amino acid substitution at position 121; one change C>T in position 782 of exon 7 causing a Ser 261Phe substitution; the c.1-7887_67+2422del10376bp and the c.1-9628_67+2871del12566bp. Even though with different breakpoints, both deletions include a portion of upstream intergenic region, the promoter, the exon 1 and a portion of intron 1 of the FECH gene preventing the expression of the mutated allele. This deletions are probably caused by unequal intragenic recombination between ALU sequences which have been found close to the breakpoints. Recently others deletions involving ALU sequences were described suggesting that large deletions can affect the FECH gene and cause of EPP.
Heart Failure Improvement after Autologous Bone Marrow Mononuclear Cells (ABMMC) Transplantation.
Authors: Carrasco-Yalan Antonio A. , Fernandez-Viña Roberto , Castillo-Aguirre Jorge , Rios Hugo , Alvarez Marco , de Moura R. , Cruz C. , Carrillo A. , Chirinos S. , Ercilla E. , Patel A. , Tumak-Mubarak Jorge
Published: April 2020

Background: Pilot studies suggest that intracoronary transplantation of unselected ABMMC cells may improve Left Ventricular Ejection Fraction (LVEF) in heart failure (HF)

Methods: 18 patients were enrolled and completed 1 year follow up. Patients underwent SPECT evaluation, all had ejection fraction < 35%, 6 patients were randomly allocated to the control group and 12 in the Bone Marrow Cells (BMC) group, median age 65 years old, male/female ratio 17/1; all with ischemic cardiomyopathy. All of cohort had NYHA class of III with maximal medical therapy and median basal LEVF was 28.38% (SD=6.5%). Median number of mononuclear and CD34+ cells infused were 8.1*108 and 1.2*107 respectively in a 50 cc delivered retrograde via coronary sinus approach using balloon occlusion "over wire" for 8 to 10 minutes. No study related adverse events were observed.

Results: After a median time of 21 days, patients in the BMC group had relief of dyspnea symptoms and improvement in functional class. At 1 year, NYHA class improved in 92% of the patients in the BMC group by at least 1 class and no improvement in the control group. Mean improvements of LVEF post BMC transplantation were 6.18% (SD=4.94%) and 6.7% (SD=4.07%) at rest and stress SPECT respectively. Rest LVEF at baseline and after one-year follow up between the BMC and control groups demonstrates significant difference (4.9% vs 1.6%, p=0.006), as well as the comparison of change in stress LVEF in both groups (6.7% vs 0.11%, p<0.001).

Conclusions: Infusion of progenitor cells into the coronary sinus is safe and feasible in the ischemic HF. It is associated with significant improvement in symptoms, functional capacity and LVEF. Larger randomized studies are in progress to build upon this pilot study.

Figure Figure
Autologous Bone-Marrow Stem Cell Support in Acute Anterior Myocardial Infarction.
Authors: Vallisa Daniele , Piepoli Massimo , Cavanna Luigi , Arbasi Cristina , Moroni Carlo , Lazzaro Antonio , Capucci Alessandro , Bernuzzi Patrizia , Arcari AnnaLisa , Bosi Costanza
Published: April 2020

Purpose- Emerging evidence suggests that stem and progenitor cells derived from bone marrow can improve cardiac function and perhaps outcome in patients after acute myocardial infarction (AMI). In this single-centre randomised trial, we evaluated whether intracoronary transfer of autologous bone-marrow stem cells can affect not only global left ventricular (LV) function (nuclear scintigraphy) but also autonomic control (heart rate variability, HRV) and baroreflex sensitivity (BRS) at 12 months’ follow-up.

Methods- After successful percutaneous coronary intervention (PCI) for acute ST-segment elevation myocardial infarction and residual LV dysfunction, 33 patients were randomly assigned to either a control group (n=17) that received optimum postinfarction treatment, or a bone-marrow-stem-cell group (n=16) that received optimum treatment plus intracoronary infusion of autologous bone-marrow stem cells 4±1 days after PCI. Test analyses were done by two investigators blinded for treatment assignment.

Results- Global LVEF at baseline (determined 3±1 days after PCI) was 36.2±9.4 in controls and 35.1±9.8 in the bone-marrow cell group (P=NS). After 12 months, mean global LVEF had increased by 25% in the control group but 41% in the treated group with an anti-remodeling effect in the latter: LVEDV increased by 12% in the control vs 2% in the treated group (P<0.05). This was associated with improved HRV (SD −20ms vs +644 ms in the treated group P<0.01), particularly in the parasympathetic-related oscillation (HF +85.7±216 ms vs. +289.3±366 ms) and higher BRS (+3.4±11.7 vs +8.7±6.3 ms/mmHg) (control vs treated, P<0.05 in all comparisons). Cell transfer did not increase the risk of adverse clinical events, in-stent restenosis, or proarrhythmic effects. In 12 months follow-up 4 exitus (24%) were observed in the control group vs. only 1 (6%) in the treated group

Conclusions- The improvement of left-ventricular systolic function and prognosis in AMI patients by intracoronary transfer of autologous bone-marrow-cells may be mediated by higher vagal tone. (ClinicalTrials.gov number, NCT00437710).
Mesenchymal Stem Cells Improve Wound Healing Via Early Activation of the Matrix Metalloproteinase-9 and Vascular Endothelial Growth Factor: Using a 3-Dimensional Collagen Gel Model.
Authors: Won Jong-Ho , Kim Cheol-Hann , Cho Moon-Kyun , Lee Sang-Cheol , Kim Chan-Kyu , Lee Nam-Su , Hong Dae-Sik , Park Hee-Sook
Published: April 2020

There is great progress on clinical application of stem cells. Stem cells are self-renewing and are potent in differentiating into multiple tissues. It has been reported that mesenchymal stem cells (MSCs) had successfully improved skin-substitute wound healing. We investigate the effects MSCs on wound healing using a 3D collagen gel system in a full thickness large-sized skin defect rat model. Three full thickness skin and tissue defects of 2 x 2 cm in size were excised on the backs of female SD-rats and replaced with 3D collagen gel contains 2 millions of male rat MSCs (rMSCs) on one site. Two different sites were control. One replaced with 3D collagen gel without rMSCs and the other site was empty. Wound size, histology and protein expression were evaluated at 3, 7 and 14 days after injury. Cultured rMSCs were confirmed to be negative for hematopoietic markers by flow cytometry and to be capable of differentiating into osteocytes, chondrocytes, and adipocytes in vitro. The wound size was significantly smaller in the rMSCs-treated sites than control sites (P < 0.01) and the most effect of wound healing was obtained at day 7. A lot of rat Y chromosome positive cells were found in rMSCs treated sites. The expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) were increased on the rMSCs-treated sites at day 3 but on the other hand those levels on the control sites were increased at day 7. This data suggest that MSCs accelerate wound healing and 3D collagen gel model can stably introduce MSCs to the defected skin area. Early activation of MMP-9 as well as previously known VEGF may play crucial roles in dermal remodeling and accelerate the processes of dermal and epidermal wound healing after MSCs treatment.
Megakaryocytes and Functional Platelets Obtained from Human Subcutaneous Adipocytes in an In Vitro Differentiation System.
Authors: Saito Emi , Matsubara Yumiko , Suzuki Hidenori , Ikeda Yasuo , Murata Mitsuru
Published: April 2020

Because of the difficulty in obtaining the sufficient amounts of hematopoietic stem cells from bone marrow, peripheral blood, and cord blood cells, studies of an in vitro experimental system that allows the production of abundant number of megakaryocytes (MKs) and platelets are currently the focus of research. Here, we present a novel system where human subcutaneous adipocytes were successfully differentiated into MK lineages in an in vitro liquid culture system. We also show that subcutaneous preadipocytes could be successfully transfected with vectors, to obtain modified MKs. Primary human subcutaneous preadipocytes (Cambrex Bio Science Walkersvile, Inc. Walkersville, MD, USA) were cultured in conditioned media to differentiate into mature adipocytes. Cells were cultured in serum-free media containing thrombopoietin for differentiation into MK lineages. The MKs or platelets were counted by flow cytometric analysis on day 14 using the relative value of CD41(+)/propidium iodide(+) cells or platelet size CD41(+) cells, respectively, versus 107 subcutaneous preadipocytes on day 0. The MK and platelet cell count was approximately 9600/ 107 and 2200/ 107, respectively. Morphological analysis with electron microscopy demonstrated that MKs, which had typical organelles such as granules, demarcation membrane, and nuclei, and platelets, which had typical contents such as granules, mitochondria, and open canalicular system, were successfully obtained from subcutaneous preadipocytes. We then attempted to transfect subcutaneous preadipocytes with vectors to obtain transformed MKs. Two glycoprotein (GP) Ib alpha polymorphisms, 145Thr/Met and 1–4 repeats of variable number tandem repeat of 13 amino-acid sequences, were used as the marker of gene transfer, which were detected by PCR-restricited fragment length polymorphism (RFLP) and Western blot analysis, respectively. Subcutaneous preadipocytes with the 145Thr/Thr and 1 repeat sequences were transfected with the expression vector carrying GPIb alpha with the 145Met and 4 repeats sequences. PCR-RFLP analysis with gel electrophoresis was performed on each RNA sample from the expression vector-transfected and non-transfected cells. Non-transfected cell sample had bands corresponding to the 145Thr sequence position, while the expression vector-transfected cell sample had bands corresponding to both the 145Thr and Met sequences. Western blot analysis with an anti-GPIb alpha monoclonal antibody, LJ-Ib alpha1 (a generous gift from Dr. ZM Ruggeri, The Scripps Research Institute, La Jolla, CA, USA), showed bands of the expected size corresponding to 1R and 1R/4R. In summary, we established an in vitro culture system to produce MKs and platelets from subcutaneous adipocytes. We were also able to obtain transfected MKs from subcutaneous preadipocytes.
Identification and Characterization of Zebrafish Tissue Factor and Its Role in Embryonic Blood Vessel Development.
Authors: Zhang Jing , Handin Robert I.
Published: April 2020

Tissue factor (TF) is a trans-membrane glycoprotein that is the cellular receptor for coagulation factor VII/VIIa. Activation of factor VII by TF is well established as the primary regulator of blood coagulation. Although TF has been implicated in such diverse processes as angiogenesis and tumor metastasis, these other functions have been difficult to study, as the knockout of murine TF is lethal at day 8.4 of embryonic life. Because of their hardiness, their rapid maturation sequence and their optical clarity, the zebrafish is an increasingly popular model for studying embryonic development. In order to study the role of TF in embryonic development we have cloned zebrafish TF cDNA. The 1.36kb orthologous cDNA predicts an ORF encoding a 293 amino acid (aa) protein with a 26 amino acid (aa) signal peptide. The predicted mature protein contains a 230 aa extra-cellular domain, a 20 aa trans-membrane segment, and a 17 aa cytoplasmic tail. The predicted amino acid sequence of zebrafish TF has 31% and 32% overall identity with human and mouse TF respectively. The zebrafish TF gene, like the human and mouse genes, has 6 exons spanning 14kb of genomic DNA. Whole-mount in situ hybridization with a TF anti-sense Riboprobe revealed that TF is detected as early as 18 hours post-fertilization (hpf) in developing head structures and brain. At 20 hpf, TF is expressed in the vascular system and highly expressed in the hindbrain. By 48 hpf, TF is seen in the brain, heart, liver, lens and gut. To investigate the expression of zebrafish TF in vivo, we generated transgenic zebrafish lines that express enhanced green fluorescent protein (EGFP) under the control of the TF promoter. A ∼ 2.0 kb promoter fragment upstream of the first exon of the TF gene was sufficient to drive expression of EGFP in a manner that recapitulated the expression of native TF. We inhibited TF expression by injecting anti-sense oligonucleotide morpholinos into the Tg(fli1:EGFP) line, in which there is robust expression of EGFP in vascular endothelial cells and their angioblast precursors. At 24 hpf the development of trunk capillary beds was delayed and, at 48 hpf, most of the morphants developed pericardial edema and impaired formation of inter-somitic vessels. These studies demonstrate that TF is expressed in multiple organs during zebrafish embryogenesis and that TF expression is necessary for the proper migration of endothelial cells into developing blood vessels during the process of embryonic vasculogenesis.
An In Vitro and In Vivo Evidence That Interleukin-8 Is a Pivotal Hypoxia-Regulated Pro-Angiopoietic Factor Secreted by Human Rhabdomyosarcomas (RMS) - Therapeutic Implications.
Authors: Wysoczynski Marcin , Wan Wu , Kucia Magda , Ratajczak Janina , Ratajczak Mariusz Z.
Published: April 2020

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood that frequently infiltrates bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1 (Blood2002;100:2597), hepatocyte growth factor (HGF) (Cancer Res. 2003;63:7926) and leukemia inhibitory factor (LIF) (Cancer Res. 2007;67:2131) play an important role in RMS metastasis. Since RMS tumors are very well vascularized we asked if these factors are secreted by RMS cells and if they stimulate angiogenesis. To address this issue, first we evaluated expression of SDF-1, HGF and LIF as well as several other proangiopoietic factors (IL-8, VEGF, FGF-2) in 9 human RMS cell lines. Expression of these factors was evaluated in both normal steady state conditions and in conditions that lead to overexpression of hypoxia inducible factor-1 (HIF-1a) due to hypoxia or exposure to CoCl2. We found by RQ-PCR and confirmed by ELISA assays that from all the factors evaluated, IL-8, which expression was very low in steady state conditions was very highly upregulated in all RMS cells lines during hypoxic conditions (∼30–135 times). This selective upregulation of IL-8 was somehow unique for RMS cells, and we did not observe similar phenomenon in several other solid tumor cell lines, where IL-8 was usually already very highly expressed in steady state conditions. To evaluate a biological significance of IL-8 in RMS angiogenesis, we collected conditioned media (CM) from RMS cells and found that they stimulate in IL-8 dependent manner in HUVEC i) phosphorylation of MAPKp42/44 and AKT, and ii) chemotactic responses. Pre-clearing of RMS-derived CM with antibodies against IL-8 significantly inhibited chemotaxis of HUVEC. Finally, by employing shRNA we inhibited expression of IL-8 in human RH-30 cells and noticed that such cells in vivo, if injected into skeletal muscles of immunodeficient mice, have the reduced ability to grow tumors and chemoattract co-transplanted human endothelial cells. We conclude that IL-8 is a pivotal pro-angiopoietic factor that is released by human RMS cells in hypoxic conditions, and that the targeting of IL-8 may prove to become a novel efficient strategy to inhibit RMS growth.
Reduced Genotoxic Risk Using Foamy Viral Vectors for the Treatment of Canine Leukocyte Adhesion Deficiency
Authors: Bauer Thomas R. , Hai Mehreen , Adler Rima L. , Allen James M. , Tuschong Laura M. , Olson Erik M. , Hickstein Dennis D. , Russell David W.
Published: April 2020

Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.
The Ratio of Serum Transferrin Receptor and Serum Ferritin in the Diagnosis of Iron Deficiency Anaemia in Young Children
Authors: Park EunSil , Kim In-Suk , Seo JiHyun , Lim JaeYoung , Park ChanHoo , Woo HyangOk , Youn HeeShang
Published: April 2020

The incidence of iron deficiency anaemia in 6∼24 month old infants due to increase in iron demand for growth spurt is reported ranged 10 to 40%. However this age group has a common acute illness such as urinary tract infection, pneumonia, and other viral infections. The aim of this study is to evaluate that iron parameter and acute phase reactant are useful parameters in differentiating anaemia by infection from anemia by iron deficiency and the mixed anaemia of these. Among 6–24 months of the infants who visited Gyeongsang Univeristy Hospital for 7 years from 2000 to 2006, 131 infants were enrolled. Hemoglobin(Hb), serum ferritin(SF), serum transferrin receptor(STfR), C reactive protein(CRP), interleukin-6(IL-6), prohepcidine were checked. The subgroup of anaemia of inflammation(AI) was defined as Hb <11 g/dL and SF >50 μg/L, the subgroup of iron deficiency anaemia(IDA) as Hb <11 g/dL and SF <12 μg/L and the normal group as Hb ≥11 and SF ≥12 μg/L. The mean STfR in the subgroup of AI, IDA and normal was 3.89(±2.64), 10.6(±4.95) and 3.96(±1.24), respectively. The mean STfR/Log SF of subgroup was 1.87(±1.55), 36.11(±71.5), 2.31(±0.97), respectively. The mean Log(STfR/SF) was statistically significant between 3 subgroup. All IDA group had Log(STfR/SF) >2.55 whereas in all subjects classified as AI it was <2.55, thus clearly separating two. The mean IL-6 of AI was significantly higher than IDA subgroup and the mean prohepcidine of AI was significantly lower than the normal group. Calculating Log(STfR/SF) is a useful criteria in classification of the iron status. Prohepcine has nothing to do with AI. Iron signal predominant over inflammatory signal in AI.

The Mean(±SD) of STfR, STfR/LogSF, Log (TfR/SF), CRP, IL-6 and Prohepcidine in Subgroups. AI IDA Normal Same letters mean that are not significantly different (P <0.05) AI, anaemia of inflammation; IDA, iron deficiency anaemia Subgroup(%) 33(25) 29(22) 69(53) Hg(g/dL) <11 <11 ≥11 SF(μg/L) >50 <12 ≥12 STfR mean(±SD) 3.89(2.64)a 10.6(4.95) b 3.96(1.24) a STfR/LogSF mean (±SD) 1.87(1.55) a 36.11(71.5) b 2.31(0.97) a Log (TfR/SF) mean(±SD) 1.30(0.56) a 3.29(0.43) b 1.76(0.43) c CRP mean(±SD) 28(39.2) a 7.6(9.6) b 17(28.0) a IL-6 mean(±SD) 6.1(10.5) a 2.0(6.3) b 4.78(11.3) c Prohepcidine mean(±SD) 204(70.5) a 234(144) a 301(120.6) b Fig.1. Log(TfR/SF) in the subgroup. AI, anaemia of inflammation; IDA, Iron deficiency anaemia. Dotted lines indicate the cut-off value at Log(TfR/SF)=2.55 Fig.1. Log(TfR/SF) in the subgroup. AI, anaemia of inflammation; IDA, Iron deficiency anaemia. Dotted lines indicate the cut-off value at Log(TfR/SF)=2.55
The Immunosuppressor Sirolimus Reduces the Number and Maturation of Colony-Forming Units of Erythroid Progenitor Cells.
Authors: Diaz-Ricart Maribel , Arellano Edgar Marcelo , Rovira Jordi , Jou Josep M. , Campistol Josep M. , Vives-Corrons Joan L. , Escolar Gines , Diekmann Fritz
Published: April 2020

Introduction: Kidney transplantation is the treatment of choice in patients with end-stage renal failure. However, the immunosuppression has severe side effects, such as chronic allograft nephropathy, cardiovascular morbidity and neoplasia. An additional effect of inhibiting mTOR is microcytic anemia. We have explored the pathophysiology of this effect.

Methods: Erythroid progenitor cells were isolated from peripheral blood of healthy control persons (HC; n=8), or kidney transplant recipients with chronic sirolimus treatment with (SRL+MC; n=8) or without (SRL-MC; n=8) microcytosis. The isolated progenitor cells were then cultured in a semi-solid medium, containing 3 U/ml erythropoietin, in the absence or presence of SRL (5ng/mL) for 14 days. Burst forming unit erythroid (BFU-E) derived colonies were then counted through an inverted microscope considering that each colony consists of more than 40 cells. Cultures were performed in duplicate and colonies were counted in the entire culture dish.

Results: Hemoglobin was 13.17 (SRL+MC) and 13.23 (SRL-MC) g/dL. RBC count was 5.22 and 4.55x106 /μL in SRL+MC and SRL-MC respectively. MCV was 76 in SRL+MC and 87 fL in SRL-MC. Presence of SRL in the culture medium led to a decrease in the number of colonies in healthy controls and kidney transplant patients (43.2±4.5 vs. 28.5±3.6 BFU-E derived colonies, Mean±SEM, p<0.05). Similar differences were seen if the three groups were analyzed separately (HC, SRL-MC, SRL+MC). This effect was not observed in the presence of tacrolimus, an immunosuppressor with different mechanism of action. Interestingly, culture dishes corresponding to SRL+MC patients contained a higher number of colonies when compared to SRL-MC and HC.

Conclusion: mTOR inhibition leads to a reduced number of erythroid colonies in culture. There is a higher number of circulating progenitor cells in the microcytic patients. Both these observations indicate that microcytosis may be compensated by a higher number of erythrocytes and circulating progenitor cells.
Epoetin Beta (Neorecormon®) Treatment for Anemia of Patients with Non-Myeloid Hematological Malignancy under Chemotherapy: Results of a Large Prospective Cohort Study.
Authors: Haïoun Corinne , Ifrah Norbert , Casassus Philippe , Audhuy Bruno , Aoudjhane Malek , Frayfer Jamile , Rouanet Stephanie , Bleuzen Pascal , Jenabian Arash , Facon Thierry
Published: April 2020

Background: The efficacy of epoetin beta (E) is well documented in clinical trials in anemic cancer patients (pts). This study was conducted to assess E use, efficacy, safety and effect on quality of life in cancer pts, in usual practice.

Methods: This prospective, multicenter, longitudinal, observational French study assessed a 4-month follow-up of informed consent cancer pts (including both solid tumors (ST) and non-myeloid hematological malignancies (H)) treated with E for chemotherapy-related anemia. Data were collected between January 2005 and March 2006. We only present here the results of the H subgroup.

Results: Among 2809 pts, 675 had an H including 325 non-Hodgkin lymphoma (NHL), 181 multiple myeloma (MM) and 80 chronic lymphocytic leukemia (CLL). 52% of pts received their first line of chemotherapy, 24% their second one. At inclusion, Hb levels were distributed as follows: < 9 g/dl: 32%, [9–11[ g/dl, 56%, [11–13[ g/dl, 11%. At inclusion, endogenous erythropoietin rate was monitored for only 8% of pts, ferritin was available for 32%, transferrin saturation for 17% and reticulocytes for 24%. At initiation, pts received a median dose of 30000 U/week of E on a once weekly regimen schedule for 98% of pts. E was associated with iron supplementation in 20% of pts (1% IV). The mean Hb level increased from 10 g/dl [5 – 14] at baseline to 13 g/dl [7 – 17] in the entire cohort of H pts, from 10 g/dl [5 – 14] to 12 g/dl [7 – 16] in NHL pts, from 10 g/dl [6 – 13] to 13 g/dl [8 – 17] in MM pts and from 10 g/dl [6 – 13] to 13 g/dl [7 – 17] in CLL pts. Hb response rate (RR) was 56% (CI95: 51 – 61) in H pts, 53% (CI95: 46 – 60) in NHL pts, 57% (CI95: 48 – 66) in MM pts and 56% (CI95: 41 – 70) in CLL pts. 41% of patients were transfused at inclusion or during the study. Good performance status (PS≤1) (OR=1.6, CI95: 1.1–2.5), non platinum-based regimen (OR=2.4, CI95: 1.2–4.9) and iron supplementation (OR=2.6, CI95: 1.4–5.1) are identified both in univariate and multivariate analysis as predictive factors of Hb response. The mean of FACT score improved from 29 (CI95: 26–31) at initiation to 36 (CI95: 34–38) at the end of study (p<0.0001). Thromboembolic events were reported in 2% of patients.

Conclusion: These results confirm the efficacy and safety of epoetin beta in routine practice for anemic patients with a non-myeloid hematological malignancy receiving chemotherapy. Epoetin beta improves the quality of life measured by FACT score.