bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.
Homocysteine Inhibits Cyclin A Promoter Methylation Via DNMT3 Inactivation in Human Endothelial Cells.
Authors: Jamaluddin S. , Chen Irene , Yang Fan , Liu Xiaoming , Schafer Andrew I. , Durante William , Yang Xiao-feng , Wang Hong 
Published: October 2019

Abstract
Hyperhomocysteinemia is a significant and independent risk factor for cardiovascular disease. We reported previously that homocysteine (Hcy) inhibits endothelial cell (EC) growth by transcriptional inhibition of the cyclin A gene. This is associated with an increase in S-adenosylhomocysteine, a potent inhibitor of methyltransferase. We hypothesized that Hcy inhibits EC growth and cyclin A transcription via hypomethylation and studied the effect of Hcy on epigenetic regulation of the cyclin A gene in EC. We found that the levels of cyclin A mRNA were significantly suppressed by azacytidne (AZC), a potent DNA methyl transferase (DNMT) inhibitor, in human umbilical vein EC (HUVEC). The cyclin A promoter (−518/256) is rich in GC content (59.1%), and has a CpG island spanning a 477 bp region (−277/200). Bisulphite sequencing followed by PCR amplification of the cyclin A core (−267/37) promoter, which contains 23 CpG sites serving as potential methylation sites, showed that Hcy (50 mM) eliminated methylation at two CpG sites, position 1 and 35, in the cyclin A promoter. Hcy selectively inhibited the activity of DNA methyl transferase 1 (DNMT1) by 40%, and had no effect on DNA methyl transferase 3 (DNMT3) activity in HUVEC. Furthermore, chromatin immunoprecipitation (ChIP) assays demonstrated that Hcy reduced the binding of methyl CpG binding protein 2 (MeCP2) and increased the binding of acetylated histone H3 and H4 in the cyclin A promoter in HUVEC but not in human aortic smooth muscle cells. The binding of MeCP2 to the cyclin A promoter was completely suppressed by AZC and trichostatin A, a histone deacetylase (HDAC) inhibitor, indicating that HDAC and DNA methylation mediate MeCP2 binding to cyclin A promoter. Finally, adenovirus tranduced DNMT1 overexpression, but not DNMT3, reversed Hcy-induced growth inhibition. In conclusion, we found that Hcy inhibits EC growth by suppressing cyclin A transcription, and that Hcy exerts this action by inhibiting DNA methylation through the suppression of DNMT1 and HDAC activity.
Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Crisis of Chronic Myeloid Leukemia (CML) and Their Emergence Following Therapy with Bcr-Abl Kinase Inhibitors.
Authors: Jones Dan , Luthra Rayjalakshmi , Kantarjian Hagop M. , Breeden Megan , O’Brien Susan , Faderl Stefan , Cortes Jorge , Thomas Deborah 
Published: October 2019

Abstract
Bcr-Abl kinase domain (KD) point mutations are detected in the dominant clone(s) in approximately 45% of CML at the time of disease resistance, developing after an average of 20–35 months of imatinib therapy. However, low numbers of Philadelphia chromosome (Ph)+ tumor cells with KD mutations could be present at earlier timepoints providing a pool of potential resistant subclones. Since current therapy of Ph+ ALL relies on imatinib maintenance therapy, the pattern of Bcr-Abl KD mutations in this tumor is an important and understudied phenomenon. We assessed the frequency and levels of Bcr-Abl KD mutations at different points in ALL, including at diagnosis, upon relapse and following salvage therapy with kinase inhibitors. We performed Bcr-Abl KD mutational analysis by direct sequencing in 25 cases of Ph+ ALL at the time of diagnosis and 25 cases upon disease persistence/relapse. For comparison, we analyzed 22 cases of lymphoid blast crisis of CML (LyBC), most of which transformed following long-term imatinib monotherapy. To track the emergence of mutated clones, we also performed more sensitive analysis for the T315I mutation by pyrosequencing (5% sensitivity) and allele-specific oligonucleotide probe (ASO) PCR (1:500 sensitivity). KD mutations were not seen by direct sequencing in ALL cases at diagnosis. The T315I mutation was also not detected by pyrosequencing (n =25) or ASO-PCR (n = 10) in newly diagnosed ALL. In contrast, Bcr-Abl KD mutations (Y253H in 3, Q252H, T315I, F317L, E355Q, H396R in 1 each) were seen in 8 of 25 (32%) relapsed/persistent ALL, occurring in patients who had been receiving imatinib for a median of 14 months (range 2–26). An additional 3 patients treated with dasatinib or nilotinib for relapse subsequently developed KD mutations (T315I and Y253H, and F317L) after 1, 4 and 9 months of second therapy. KD mutations were seen in 16 of 22 (73%) patients with lymphoid blast crisis, including T315I in 7, E255K and M244V in 2 each, and Y253H, V299L, F311I, E355G, F359V in 1 each. All KD mutations in LyBC developed following imatinib or nilotinib therapy. As with CML, kinase inhibitor therapy particularly in the relapse/salvage setting is the primary risk factor for emergence of Bcr-Abl KD mutations in Ph+ ALL. There is a high frequency of Bcr-Abl KD mutations associated the lymphoid transformation of CML. However, Bcr-Abl KD mutations develop more rapidly in persistent or relapsed Ph+ ALL than in CML and there is a higher frequency of Y253H mutations noted. These findings will likely have consequences for the timing and dosages of imatinib and other kinase inhibitors in maintenance and relapsed ALL regimens.