Citations of BMS158
Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs
Andonegui,Graciela; Bonder,Claudine S.; Green,Francis; Mullaly,Sarah C.; Zbytnuik,Lori; Raharjo,Eko; Kubes,Paul
Journal of Clinical Investigation 2003;111:1011-1020.
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Abstract
The rapid and selective accumulation of neutrophils into the lungs is thought to underlie the pulmonary failure that leads to sepsis-related death. In this study we investigated whether neutrophil TLR4 is important in LPS-induced pulmonary neutrophil recruitment by creating chimeric mice (transferring bone marrow between TLR4+/+ and TLR4-/- mice). In TLR4+/+ mice receiving TLR4-/- bone marrow, 6 weeks after transplant TLR4 was absent in all circulating leukocytes as well as in resident macrophages (these mice were termed LeukocyteTLR4-/-), and these cells were completely nonresponsive to LPS. In TLR4-/- mice receiving TLR4+/+ bone marrow, endothelial cells but not leukocytes were deficient in TLR4 (EndotheliumTLR4-/-). Surprisingly, systemic LPS (0.5 mg/kg) induced a dramatic increase in neutrophil sequestration into the lungs of LeukocyteTLR4-/- mice over the first 4 hours. Concomitantly, numbers of circulating leukocytes decreased by 90%. By contrast, EndotheliumTLR4-/- mice showed very little increase in neutrophil sequestration in the lungs, suggesting that endothelium rather than leukocyte TLR4 was important. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte-endothelial cell interactions in LPS-treated EndotheliumTLR4-/- mice than in LPS-treated LeukocyteTLR4-/- mice. This is consistent with less sequestration of leukocytes into the lungs of EndotheliumTLR4-/- mice. In conclusion, our data challenge the view that LPS directly activates neutrophils to trap in lungs and suggest a far more important role than previously appreciated for the endothelial cells
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Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells
Artwohl,Michaela; Lindenmair,Andrea; Sexl,Veronika; Maier,Christina; Rainer,Georg; Freudenthaler,Angelika; Huttary,Nicole; Wolzt,Michael; Nowotny,Peter; Luger,Anton; Baumgartner-Parzer,Sabina M.
Journal of Lipid Research 2008;49:2627-2640.
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Abstract
Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 {micro}mol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms
2358
Scavenger Receptor-A-Targeted Leukocyte Depletion Inhibits Peritoneal Ovarian Tumor Progression
Bak,S.Peter; Walters,Julie Jo; Takeya,Motohiro; Conejo-Garcia,Jose R.; Berwin,Brent L.
Cancer Research 2007;67:4783-4789.
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Abstract
Immunosuppressive leukocytes are emerging as a critical factor in facilitating tumor progression. These leukocytes are converted by the tumor microenvironment to become tolerogenic, facilitate metastasis, and to aid in neovascularization. The predominant variety of suppressive leukocytes found in human and murine ovarian cancer are called vascular leukocytes (VLC), due to sharing functions and cell surface markers of both dendritic cells and endothelial cells. Using the ID8 murine model of ovarian cancer, the aim of this study was to test the efficacy of VLC elimination as an ovarian tumor therapy. We show that carrageenan-mediated depletion of peritoneal tumor-associated leukocytes inhibits ovarian tumor progression. We then identified scavenger receptor-A (SR-A) as a cell surface receptor that is robustly and specifically expressed within human and murine ovarian tumor ascites upon VLCs. Administration of anti-SR-A immunotoxin to mice challenged with peritoneal ID8 tumors eliminated tumor-associated VLCs and, importantly, substantially inhibited peritoneal tumor burden and ascites accumulation. Moreover, the toxin required targeting to SR-A because mice that received untargeted toxin did not exhibit inhibition of tumor progression. We conclude that SR-A constitutes a novel and specific target for efficacious immunotherapeutic treatment of peritoneal ovarian cancer. [Cancer Res 2007;67(10):4783-9]
774
Human endothelial cells derived from circulating progenitors display specific functional properties as compared to mature vessel wall endothelial cells
Bompais,Heidi; Chagraoui,Jalila; Canron,Xavier; Crisan,Mihaela; Liu,Xu Hui; Anjo,Aurora; Tolla-Le Port,Carine; Leboeuf,Marylene; Charbord,Pierre; Bikfalvi,Andreas; Uzan,Georges
Blood 2003;103:2003-2008.
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Abstract
Endothelial progenitor cells (EPCs) were shown to be present in the systemic circulation and cord blood. We investigated if EPCs display specific properties as compared to mature endothelial cells. Human cord blood CD34+ cells were isolated and adherent cells were amplified under endothelial conditions. Expression of specific markers identified them as endothelial cells, also called EPDCs (endothelial progenitor-derived cells). When compared to mature endothelial cells, HUVECs and HBMECs, endothelial markers were expressed to the same extent except for KDR which is expressed more in EPDCs. They display a higher proliferation potential. Functional studies demonstrated that EPDCs were more sensitive to angiogenic factors, which afford these cells greater protection against cell death as compared to HUVECs. Moreover, EPDCs exhibit more hematopoietic supportive activity than HUVECs. Finally, studies in NOD/SCID mice demonstrated that human circulating EPCs are able to colonize a Matrigel plug. EPDCs display the morphology and phenotype of endothelial cells. Their functional features indicate, however, that although these cells have undergone some differentiation steps, they still have the properties of immature cells, suggesting greater tissue repair capabilities. Future use of in vitro amplified peripheral blood EPDCs may constitute a challenging strategy for cell therapy
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Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
Boyer,Laurent; Doye,Anne; Rolando,Monica; Flatau,Gilles; Munro,Patrick; Gounon,Pierre; Clement,Rene; Pulcini,Celine; Popoff,Michel R.; Mettouchi,Amel; Landraud,Luce; Dussurget,Olivier; Lemichez,Emmanuel
Journal of Cell Biology 2006;173:809-819.
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Abstract
The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus
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Peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial cells in association with downregulation of vascular endothelial cadherin
Dewi,Beti Ernawati; Takasaki,Tomohiko; Kurane,Ichiro
Journal of General Virology 2008;89:642-652.
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Abstract
Plasma leakage is one of the characteristic features of dengue haemorrhagic fever. The interaction among peripheral blood mononuclear cells (PBMCs), dengue virus and endothelial cells was analysed in vitro. Human umbilical vein endothelial cells (HUVECs) were infected with dengue-2 virus (DV-2) at an m.o.i. of 0.5 p.f.u. per cell. PBMCs were added to DV-2-infected HUVECs, and transendothelial electrical resistance (TEER) and transalbumin permeability were assessed. Dengue virus infection at an m.o.i. of 0.5 p.f.u. per cell alone did not decrease the TEER, but addition of PBMCs decreased the TEER, increased the albumin permeability and induced morphological changes of HUVECs. The extent of the decrease was more profound with adherent PBMCs than with non-adherent PBMCs. The expression of vascular endothelial cadherin (VE-cadherin) was examined using real-time RT-PCR and immunofluorescence. Addition of PBMCs to DV-2-infected HUVECs decreased the levels of mRNA transcripts and cell-surface expression of VE-cadherin. The results indicate that PBMCs increased the permeability of DV-2-infected HUVECs and that the increased permeability was concomitant with morphological change and the decrease in VE-cadherin expression. The results suggest that functional impairment of the DV-2-infected HUVEC monolayer was caused by interaction with PBMCs
2449
Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical..
Eggermann,J.; Kliche,S.; Jarmy,G.; Hoffmann,K.; Mayr-Beyrle,U.; Debatin,K.M.; Waltenberger,J.; Beltinger,C.
Cardiovascular Research 2003;58:478-486.
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Abstract
Objective: Endothelial progenitor cells (EPC) can contribute to vascular repair and targeted tumour therapy. Little is known about generating EPC from human umbilical cord blood. We therefore compared methods for purification of EPC from human umbilical cord blood. Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation and used either unselected or after CD34 preselection. Unselected mononuclear cells were cultured for 9 days. Culture-dish-adherent (CDAC) and non-adherent (CDNAC) CD34+ cells were cultured separately for 4 weeks. Surface markers were assessed by immunofluorescence staining and FACS analysis. Results: In unselected mononuclear cells, VEGF-R2 and VE-cadherin expression increased up to day 6. They stained positive with UEA-1 and took up acetylated LDL. Expression of CD45 and CD14 decreased over time, but remained strong. CD133 and CD34 were not expressed. CD34+-CDNAC acquired an endothelial phenotype over time with an increase of VEGFR-2 and von Willebrand factor (vWF). CD45 and CD14 decreased, while CD34 and the progenitor-cell marker CD133 remained strongly expressed. CD34+-CDAC showed a strong increase in VEGFR-2, CD133, CD34 and vWF, while CD14 decreased, and CD45 did not change. Conclusion: Putative EPC can be obtained from human umbilical cord blood. When selected for CD34, cells can be differentiated in culture to express markers of mature endothelial cells, while keeping progenitor markers. In contrast, short-term culture of unselected mononuclear cells leads to an endothelioid-monocytoid phenotype devoid of progenitor markers. Thus, the outgrowth from CD34-selected cells appears to be superior to short-term culture of unselected mononuclear cells with regard to endothelial cell-lineage specific differentiation of cells with a progenitor marker profile.
940
Transplantation of Blood-Derived Progenitor Cells After Recanalization of Chronic Coronary Artery Occlusion: First Randomized and Placebo-Controlled Study
Erbs,Sandra; Linke,Axel; Adams,Volker; Lenk,Karsten; Thiele,Holger; Diederich,Klaus Werner; Emmrich,Frank; Kluge,Regine; Kendziorra,Kai; Sabri,Osama; Schuler,Gerhard; Hambrecht,Rainer
Circulation Research 2005;97:756-762.
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Abstract
Transplantation of blood-derived circulating progenitor cells (CPC) has been shown to improve myocardial regeneration after myocardial infarction. It remains unclear whether CPC transplantation exerts beneficial effects also in patients with chronic myocardial ischemia. We initiated a randomized, double-blind, placebo-controlled study evaluating the impact of intracoronary infusion of CPCs on coronary vasomotion and left ventricular (LV) function in patients after recanalization of chronic coronary total occlusion (CTO). After recanalization of CTO, 26 patients (age, 63{+/-}2 years; LV ejection fraction, 53{+/-}2%) were randomly assigned to the treatment (intracoronary transplantation of CPCs) or control group. Coronary flow reserve in response to adenosine (2.4 mg/min) was measured in the target vessel at the beginning of the study and after 3 months. LV function and infarct size were assessed by MRI and metabolism by 18F deoxyglucose positron emission tomography. CPC application resulted in an increase in coronary flow reserve by 43% from 2.3{+/-}0.3 to 3.3{+/-}0.5 (P<0.05 versus beginning and control). At 3 months, the number of hibernating segments in the target region (from 2.9{+/-}0.6 to 2.0{+/-}0.6 segments, P<0.05 versus beginning and control) had declined in the treatment group, whereas no significant changes were observed in the control group. MRI revealed a reduction in infarct size by 16% and an increase in LV ejection fraction by 14% in the treatment group (from 51.7{+/-}3.7 to 58.9{+/-}3.2%; P<0.05 versus beginning and control) because of an augmented wall motion in the target region. Hence, intracoronary transplantation of CPCs after recanalization of CTO results in an improvement of macro- and microvascular function and contributes to the recruitment of hibernating myocardium
486
Platelet Endothelial Cell Adhesion Molecule-1 and Vascular Endothelial Cadherin Cooperatively Regulate Fibroblast Growth Factor-induced Modulations of Adherens Junction Functions
Halama,Thomas; Groger,Marion; Pillinger,Manuela; Staffler,Gunther; Prager,Elisabeth; Stockinger,Hannes; Holnthoner,Wolfgang; Lechleitner,Sonja; Wolff,Klaus; Petzelbauer,Peter
J Investig Dermatol 2001;116:110-117.
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Abstract
Cellular adherens junctions are formed by cadherins linked to proteins of the catenin family. In endothelial cells, not only vascular endothelial cadherin but also platelet endothelial cell adhesion molecule-1 localizes into junctions and associates with beta-catenin. To explore a putative cooperation of platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin, we analyzed transfectants expressing either platelet endothelial cell adhesion (CD31 cells) or vascular endothelial cadherin (CD144 cells) or both molecules (CD31/CD144 cells), and, for comparison, human umbilical vein endothelial cells. Basic fibroblast growth factor completely dissociated vascular endothelial cadherin/beta-catenin complexes and robustly moved beta-catenin into the nucleus in CD144 cells, whereas in CD31/CD144 cells as well as in human umbilical vein endothelial cells, fibroblast growth factor only partially dissociated the junctional complex followed by a significantly reduced nuclear translocation of beta-catenin. In contrast, in CD31 cells, the subcellular distribution of beta-catenin remained unaffected by fibroblast growth factor. As a functional consequence, fibroblast growth factor induced a complete collapse of the F-actin network in CD144 cells, a limited rearrangement of F-actin fibers in CD31/CD144 cells and no F-actin rearrangement in CD31 cells. We also analyzed the effect of fibroblast growth factor-induced rearrangement of junctions on junction permeability for leukocytes: in line with our observation that vascular endothelial cadherin was required for cells to respond to fibroblast growth factor, only in CD31/CD144 cells, but not in CD31 cells, leukocyte transmigration was significantly enhanced by fibroblast growth factor. In conclusion platelet endothelial cell adhesion molecule-1 cooperates with vascular endothelial cadherin in a mutual fashion; platelet endothelial cell adhesion molecule-1 reduces and temporarily limits fibroblast growth factor-induced dissociation of vascular endothelial cadherin/beta-catenin complexes, but requires vascular endothelial cadherin to control leukocyte transmigration in dependence of fibroblast growth factor.
1596
Kidney Transplantation Substantially Improves Endothelial Progenitor Cell Dysfunction in Patients with End-Stage Renal Disease
Herbrig,K.; Gebler,K.; Oelschlaegel,U.; Pistrosch,F.; Foerster,S.; Wagner,A.; Gross,P.; Passauer,J.
American Journal of Transplantation 2006;6:2922-2928.
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Abstract
Endothelial progenitor cells (EPC) are involved in endothelial repair and maintenance. Dysfunction of EPC may contribute to accelerated arteriosclerosis in chronic kidney disease. Kidney transplantation (KTx) improves both survival and endothelial function of dialysis patients. In a prospective study, we tested to which extent KTx changes EPC biology. We studied number and function (migratory activity, adhesion to extracellular matrix proteins and to mature endothelial cells [EC]) of EPC in 20 patients during dialysis and 3, 6, 9 and 12 months after KTx. Twenty-two healthy volunteers served as matched controls. Circulating precursor populations were measured by flow cytometric analysis. Cytokines relevant for EPC mobilization were monitored. Compared to the dialysis state, KTx increased the migration of EPC to approximately 2-fold. Adhesion to fibronectin and to collagen type IV was significantly increased after KTx. An improved adhesion rate of EPC to mature EC was observed. The number of EPC decreased. The amount of precursor populations showed no difference compared to the pretransplant state. Our study shows an improved function of EPC after KTx. This finding indicates an improved potential for endothelial repair which in turn may contribute to enhanced endothelial function and reduced cardiovascular morbidity after KTx
1361
Endothelial dysfunction in patients with rheumatoid arthritis is associated with a reduced number and impaired function of endothelial progenitor cells
Herbrig,K.; Haensel,S.; Oelschlaegel,U.; Pistrosch,F.; Foerster,S.; Passauer,J.
Annals of the Rheumatic Diseases 2006;65:157-163.
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Abstract
Background: Rheumatoid arthritis (RA) is associated with increased morbidity and mortality attributable to accelerated atherosclerosis and cardiovascular events. Objective: To determine the role played by endothelial progenitor cells (EPC) in the defence system against arteriosclerosis. Methods: The number and function of EPC in 13 young patients with RA with low disease activity (DAS28 3.5 (0.3)) and 13 healthy control subjects was studied. Endothelial function was investigated by agonist-induced, endothelium dependent vasodilatation measured by the forearm blood flow technique. Migratory activity and adhesion of EPC to tumour necrosis factor {alpha} (TNF{alpha}) activated mature endothelial cells and components of the extracellular matrix were tested in vitro. Putative precursor populations (CD34+, CD34+/CD133+, and CD34+/KDR+ haematopoietic stem cells) were measured by flow cytometric analysis. Results: Acetylcholine-induced, endothelium dependent vasodilatation was reduced by about 50% in patients with RA, indicating endothelial dysfunction, whereas endothelium-independent vasodilatation in response to glyceryl trinitrate was at control level. Significantly reduced numbers of EPC were found in the patients compared with controls. Migratory activity of EPC was decreased in patients with RA. Adhesion to mature endothelial cells after activation with TNF{alpha} was enhanced only in controls. The adhesion to matrix proteins and the number of putative precursor cell lineages was comparable in both groups. Conclusion: Endothelial dysfunction in patients with RA with low grade inflammation is associated with a reduced number and partial dysfunction of EPC. Further studies are needed to explore whether interventions that potentially ameliorate the number and function of EPC also improve endothelial function in these patients
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Increased total number but impaired migratory activity and adhesion of endothelial progenitor cells in patients on long-term hemodialysis
Herbrig,Kay; Pistrosch,Frank; Oelschlaegel,Uta; Wichmann,Gunnar; Wagner,Andrea; Foerster,Sarah; Richter,Susanne; Gross,Peter; Passauer,Jens
American Journal of Kidney Diseases 2004;44:› Link
Abstract
Background: Endothelial progenitor cells (EPCs), derived from bone marrow, contribute to vessel repair and neovascularization. Because uremia is a state of endothelial dysfunction associated with high cardiovascular mortality, as well as a state of reduced hematopoiesis, we studied the number and function of EPCs in patients on long-term hemodialysis (HD) therapy. Methods: We counted the number of EPCs in 20 HD patients and 16 healthy volunteers. To assess EPC function, we measured migratory activity, adhesion to matrix proteins, and adhesion to endothelial cells. Furthermore, we measured blood levels of vascular endothelial growth factor (VEGF) and granulocyte-macrophage colony-stimulating factor, factors known to influence EPC kinetics. Circulating precursor cells (CD34+, CD34+/CD133+, CD34+/KDR+ cells) were counted by means of flow cytometric analysis. Results: The number of EPCs in HD patients was significantly elevated compared with controls (459.7 ± 92 versus 364.8 ± 77.4 EPC/high-power field). However, migratory activity was markedly decreased in HD patients (47.5 ± 27.7 versus 84.7 ± 3.2 EPC/high-power field). EPCs of HD patients showed impaired adhesion to extracellular matrix and endothelial cells. VEGF blood levels in HD patients were 2-fold greater compared with controls. The number of circulating CD34+ and CD34+/133+ cells was reduced in HD patients. There were no differences in total numbers of CD34+/KDR+ cells. Conclusion: This study shows an elevated number, but pronounced functional impairment, of EPCs in patients on long-term HD therapy. The latter may result in limited endothelial repair, which, in turn, may contribute to endothelial dysfunction in this particular group of patients.
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Therapeutical potential of blood-derived progenitor cells in patients with peripheral arterial occlusive disease and critical limb ischaemia
Lenk,Karsten; Adams,Volker; Lurz,Philipp; Erbs,Sandra; Linke,A.; Gielen,Stephan; Schmidt,Andrej; Scheinert,Dierck; Biamino,Giancarlo; Emmrich,Frank; Schuler,Gerhard; Hambrecht,Rainer
European Heart Journal 2005;26:ehi285› Link
Abstract
Aims Despite considerable advances in the therapy of patients with peripheral arterial occlusive disease (PAOD) and critical limb ischaemia (CLI), a substantial number remain, in whom amputation has to be considered the only and final option. Recent evidence from animal models of hind limb ischaemia suggests that neovascularization induced by circulating blood-derived progenitor cells (CPCs) may permit limb salvage. It remains unclear, however, whether an intra-arterial application of autologous CPCs in patients with infrapopliteal PAOD and CLI is safe, feasible, and of potentially beneficial effects.Methods and results Seven patients with critical PAOD were treated with an intra-arterial infusion of autologous CPCs (39 {+/-} 24x106) isolated from peripheral blood. Pre-interventional stimulation with G-CSF and CPC application was well tolerated. Twelve weeks after CPC administration, the pain-free walking distance increased from 6 {+/-} 13 to 195 {+/-} 196 m. A significant increase in the ankle-brachial index, transcutaneous O2, flow-dependent vasodilation, flow reserve in response to adenosine, and endothelium-dependent vasodilation was observed.Conclusion These preliminary data in a small series of patients with CLI without surgical or interventional options indicate that CPC application is safe, feasible, and may improve both functional and clinical indices
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LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration
Liu,Lixin; Cara,Denise C.; Kaur,Jaswinder; Raharjo,Eko; Mullaly,Sarah C.; Jongstra-Bilen,Jenny; Jongstra,Jan; Kubes,Paul
Journal of Experimental Medicine 2005;201:409-418.
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Abstract
Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1-/-) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-{alpha} and interleukin-1{beta}) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1-/- chimeric mice using bone marrow transplantation revealed that in mice with Lsp1-/- endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1-/- neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1-/- animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1-/- mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration
547
Human adipose tissue as a source of Flk-1^+ cells: new method of differentiation and expansion
Martinez-Estrada,O.M.; Munoz-Santos,Y.; Julve,J.; Reina,M.; Vilaro,S.
Cardiovascular Research 2005;65:328-333.
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Abstract
Objective The low number of postnatal endothelial progenitor cells (EPC) in the circulation limits their therapeutic application in cardiovascular medicine. Processed lipoaspirate (PLA) cells differentiate into osteoid, adipose, muscle, and cartilaginous cells. This study examines the potential of PLA cells as a source of EPCs. Methods PLA cells obtained from human lipoaspirates were cultured for 1 week in serum-depleted medium to form three-dimensional cell clusters (3DCC). The phenotype of 3DCC-derived cells was assessed by immunofluorescense staining and FACS analysis. Results Flow cytometry showed that 45±5% of cells derived from the 3DCC expressed Flk-1, a marker of early EPC, whilst only 4±0.5% of freshly isolated PLA were Flk-1+. The proportion of Flk-1+ cells increased to 98±2% during culture in hematopoietic stem cell medium. When cultured in an endothelial cell (EC)-specific medium, Flk-1+ cells also expressed Ve-cadherin, von Willebrand's factor (vW), and a lectin receptor, and took up low-density lipoprotein. Incorporation into an endothelial cell tubular network confirmed their functional activity. Conclusion This report describes the first isolation and culture of Flk-1+ cells from human adipose tissue. The feasibility of the extraction and culture of these cells in increased numbers suggests that such autologous cells will be useful for applications ranging from basic research to cell-based therapies.
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Blood Monocytes Mimic Endothelial Progenitor Cells
Rohde,Eva; Malischnik,Christina; Thaler,Daniela; Maierhofer,Theresa; Linkesch,Werner; Lanzer,Gerhard; Guelly,Christian; Strunk,Dirk
Stem Cells 2006;24:357-367.
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Abstract
The generation of endothelial progenitor cells (EPCs) from blood monocytes has been propagated as a novel approach in the diagnosis and treatment of cardiovascular diseases. Low-density lipoprotein (LDL) uptake and lectin binding together with endothelial marker expression are commonly used to define these EPCs. Considerable controversy exists regarding their nature, in particular, because myelomonocytic cells share several properties with endothelial cells (ECs). This study was performed to elucidate whether the commonly used endothelial marker determination is sufficient to distinguish supposed EPCs from monocytes. We measured endothelial, hematopoietic, and progenitor cell marker expression of monocytes before and after angiogenic culture by fluorescence microscopy, flow cytometry, and real-time reverse transcription-polymerase chain reaction. The function of primary monocytes and monocyte-derived supposed EPCs was investigated during vascular network formation and EC colony-forming unit (CFU-EC) development. Monocytes cultured for 4 to 6 days under angiogenic conditions lost CD14/CD45 and displayed a commonly accepted EPC phenotype, including LDL uptake and lectin binding, CD31/CD105/CD144 reactivity, and formation of cord-like structures. Strikingly, primary monocytes already expressed most tested endothelial genes and proteins at even higher levels than their supposed EPC progeny. Neither fresh nor cultured monocytes formed vascular networks, but CFU-EC formation was strictly dependent on monocyte presence. LDL uptake, lectin binding, and CD31/CD105/CD144 expression are inherent features of monocytes, making them phenotypically indistinguishable from putative EPCs. Consequently, monocytes and their progeny can phenotypically mimic EPCs in various experimental models
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Impaired VE-Cadherin/{beta}-Catenin Expression Mediates Endothelial Cell Degeneration in Dilated Cardiomyopathy
Schafer,Romana; Abraham,Dietmar; Paulus,Patrick; Blumer,Roland; Grimm,Michael; Wojta,Johann; Aharinejad,Seyedhossein
Circulation 2003;108:1585-1591.
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Abstract
Background-- The cross-talk between vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang), and VE-cadherin coregulates endothelial cell (EC) survival. Cardiac expression of VEGF-A but not its receptor KDR is blunted in dilated cardiomyopathy (DCM). Whether VE-cadherin/Ang function is affected in DCM is unknown. Methods and Results-- The myocardial expression of VE-cadherin/{beta}-catenin, Ang-1, Ang-2, and their receptor Tie-2 was examined in DCM, ischemic cardiomyopathy (ICM), and in control subjects through the use of real-time RT-PCR, Western blotting, and immunocytochemistry. EC degeneration was quantified by TEM. RNA interference against VE-cadherin and VEGF deprivation and stimulation were applied to cultured DCM myocardium and human microvascular ECs to examine the interplay between VEGF, VE-cadherin/{beta}-catenin, and Ang-2. Analysis of tissue sections with similar rates of EC degeneration in both patient groups showed that VE-cadherin/{beta}-catenin expression was downregulated in DCM only (P<0.05). Although Ang-1 was not changed, Ang-2 expression was downregulated and Tie-2 protein expression was upregulated both in DCM and ICM (P<0.05). The ratio of degenerated to normal ECs was significantly higher in DCM versus ICM (P<0.05). Targeted VE-cadherin gene silencing in cultured human ECs resulted in similar degenerative effects observed in myocardial ECs of DCM patients. In vitro experiments indicated that VE-cadherin/{beta}-catenin expression is independent of VEGF. Conclusions-- These results indicate for the first time that the EC survival is impaired in myocardium of patients with DCM involving VE-cadherin/{beta}-catenin, probably independent of VEGF. Targeting VE-cadherin might be of benefit to counteract the selective EC pathology in DCM
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The Origin and in Vivo Significance of Murine and Human Culture-Expanded Endothelial Progenitor Cells
Sharpe,Emerson E.,III; Teleron,Amylynn A.; Li,Bin; Price,James; Sands,Mark S.; Alford,Kathy; Young,Pampee P.
American Journal of Pathology 2006;168:1710-1721.
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Abstract
In adults highly purified populations of early hematopoietic progenitors or cells derived from ex vivo expanded unmobilized human peripheral blood mononuclear cells contribute to new blood vessel formation. However, the source of these culture-expanded endothelial progenitor cells (CE-EPCs) remains controversial. We demonstrate that ex vivo expansion of unmobilized human peripheral blood generated CE-EPCs with similar numbers, kinetics, and antigen expression profile as compared to plating unfractionated CD34+/lin--enriched bone marrow mononuclear cells. Both CE-EPC populations uniformly co-expressed myeloid and endothelial markers, suggesting that peripheral blood progenitor enumeration does not correlate with the numbers of early outgrowth CE-EPCs. Using purified myeloid subpopulations obtained from mice harboring the lacZ transgene driven by an endothelial-specific promoter, we showed that the immature myeloid lineage marker CD31+ cells generated CE-EPCs with fourfold greater frequency than mature myeloid populations. Biphenotypic cells co-expressing myeloid/endothelial antigens were not detected in circulating human or murine peripheral blood or bone marrow but were associated with murine tumors. Unlike CE-EPCs, CD14+ leukocytes admixed within tumors did not generate vWF-positive blood vessels during a similarly defined period of tumor growth, but some leukocytes up-regulated the endothelial marker VE-cadherin. Taken together, the data suggest that the local neovascular microenvironment may facilitate vasculogenesis by promoting endothelial differentiation and that CE-EPCs may accelerate such vasculo-genesis
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Two consecutive high-fat meals affect endothelial-dependent vasodilation, oxidative stress and cellular microparticles in healthy men
TUSHUIZEN,M.E.; NIEUWLAND,R.; SCHEFFER,P.G.; STURK,A.; Heine,R.J.; DIAMANT,M.
Journal of Thrombosis and Haemostasis 2006;4:1003-1010.
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Abstract
Summary. Background: A large body of evidence has accumulated indicating a relation between postprandial hyperglycemia and hypertriglyceridemia, and the risk of cardiovascular disease. Objective: We studied possible mechanisms underlying the postprandial proatherogenic state by exposing healthy males to two consecutive high-fat mixed meals. Patients/methods: Seventeen healthy males [age 25.4 +- 3 years, body mass index 23.6 +- 2 kg m-2] were studied during two randomized visits. During the meal visit, subjects consumed standardized meals (50 g of fat, 55 g of carbohydrates and 30 g of proteins) as breakfast and 4 h later as lunch. During the control visit, subjects remained fasted. Prior to each blood collection (before and every 2 h after the first meal), flow-mediated dilation (FMD) of the brachial artery was measured. Results: Although within the normal range, postprandial plasma glucose and triacylglycerol concentrations increased significantly, especially after the second meal, as compared with baseline (4.8 +- 0.3 to 5.4 +- 0.4, 0.8 +- 0.2 to 1.7 +- 0.7 mmol L-1, respectively; both P < 0.05) and the fasting visit. After the second meal, FMD was significantly impaired (6.9% vs. 3.7%, P < 0.05) whereas oxidized low-density lipoprotein (oxLDL)/LDL cholesterol ratio and malondialdehyde concentrations were markedly elevated (both P < 0.01). Finally, an increase in total microparticle (MP) numbers was observed during the meal visit (P < 0.05). Conclusions: In healthy males, after two consecutive fat-rich meals, mild elevations in plasma glucose and triacylglycerol were paralleled by impaired FMD, increased markers of oxidative stress and circulating MPs, in particular, after the second meal. These findings may have consequences for subjects with postprandial dysmetabolism, including those with Type 2 diabetes
1432
Changes in circulating mesenchymal stem cells, stem cell homing factor, and vascular growth factors in patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention
Wang,Y.; Johnsen,H.E.; Mortensen,S.; Bindslev,L.; Sejersten Ripa,R.; Haack-Sorensen,M.; Jorgensen,E.; Fang,W.; Kastrup,J.
Heart 2006;92:768-774.
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Abstract
Objective: To investigate the spontaneous occurrence of circulating mesenchymal stem cells (MSC) and angiogenic factors in patients with ST elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI). Design: In 20 patients with STEMI, blood samples were obtained on days 1, 3, 7, 14, 21, and 28 after the acute PCI. Fifteen patients with a normal coronary angiography formed a control group. MSC (CD45-/CD34-), plasma stromal derived factor 1 (SDF-1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2) were measured by multiparametric flow cytometry and enzyme linked immunosorbent assay (ELISA). Results: Circulating CD45-/CD34- cells were significantly decreased on day 7 compared with day 3. Cell counts normalised one month after the acute onset of STEMI. The changes were mainly seen in patients with a large infarction. Plasma SDF-1 increased significantly from day 3 to day 28, and VEGF-A and FGF-2 increased significantly from day 7 to day 28. Conclusions: Spontaneous sequential fluctuations in MSC and the increase in vascular growth factor concentrations after STEMI suggest that the optimal time for additional stem cell therapy is three weeks after a myocardial infarction to obtain the maximum effects by stimulating endogenous growth factors on the delivered stem cells
529
Subcellular distribution of Wnt-1 at adherens junctions and actin-rich densities in endothelial cells
Wechezak,A.R.; Coan,D.E.
Experimental Cell Research 2003;288:335-343.
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Abstract
The Wnt family of signaling proteins functions in embryonic development and mammalian oncogenesis. It is unknown whether these molecules have a role in normal, postdevelopmental, homeostatic processes. Possessing a putative signal sequence and potential glycosylation sites, Wnt-1 is believed to be secreted and remain associated with the cell surface and extracellular matrix. While it has been suggested that Wnt proteins may target cytoskeletal structures more directly, no definitive studies have identified an intracellular association and function for these molecules. Here, we report that Western blots of lysates from retinoic-acid-differentiated P19 cells and bovine endothelial cells indicate the presence of a 45-kDa Wnt-1 protein. In endothelium, Wnt-1 was present in both the Triton X soluble and the insoluble cell fractions. Immunocytochemical labeling localized Wnt-1 to adherens junctions, codistributing with ß-catenin. Wnt-1 also was detected at actin-rich densities (ARDs) within basal cell regions. In wounded monolayers, ARDs delineated the distal margins of cells undergoing directed migration. Transfection with antisense oligonucleotides to Wnt-1 resulted in reduced cohesion of wound edge cells, abnormal protrusive activity, and random movement. Our data indicate that Wnt-1 protein is present in postdevelopmental endothelial cells where it associates with cytoskeletal elements and may retain function as a tissue polarity gene.
1116
Targeted release of oncolytic measles virus by blood outgrowth endothelial cells in situ inhibits orthotopic gliomas
Wei,J.; Wahl,J.; Nakamura,T.; Stiller,D.; Mertens,T.; Debatin,K.M.; Beltinger,C.
Gene Ther 2007;14:1573-1586.
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Abstract
Malignant gliomas remain largely incurable despite intensive efforts to develop novel therapies. Replicating oncolytic viruses have shown great promise, among them attenuated measles viruses of the Edmonston B strain (MV-Edm). However, host immune response and the infiltrative nature of gliomas limit their efficacy. We show that human blood outgrowth endothelial cells (BOECs), readily expandable from peripheral blood, are easily infected by MV-Edm and allow replication of MV-Edm while surviving long enough after infection to serve as vehicles for MV-Edm (BOEC/MV-Edm). After intravenous and peritumoral injection, BOEC/MV-Edm deliver the viruses selectively to irradiated orthotopic U87 gliomas in mice. At the tumor, MV-Edm produced by the BOECs infect glioma cells. Subsequent spread from tumor cell to tumor cell leads to focal infection and cytopathic effects that decrease tumor size and, in the case of peritumoral injection, prolong survival of mice. Since MV-Edm within BOECs are not readily neutralized and because BOEC/MV-Edm search and destroy glioma cells, BOEC/MV-Edm constitute a promising novel approach for glioma therapy.
2261
Both Cell Fusion and Transdifferentiation Account for the Transformation of Human Peripheral Blood CD34-Positive Cells Into Cardiomyocytes In Vivo
Zhang,Sui; Wang,Dachun; Estrov,Zeev; Raj,Sean; Willerson,James T.; Yeh,Edward T.H.
Circulation 2004;110:3803-3807.
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Abstract
Background-- Adult human peripheral blood CD34-positive (CD34+) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients. Methods and Results-- We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34+ cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34+ cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA+ and troponin T+ or Nkx2.5+ cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA-, troponin T+ cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34+ cells contained human X chromosome only. Conclusions-- Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo
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