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R L Nachman

Publications and source records attributed to R L Nachman.

At least 19 recordsLinked to original sources
PubMed1997

Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription-PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (> 1 ng/10(6) cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin-Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis.

Alternative Splicing↗
PubMed1997

Transendothelial migration of CD34+ and mature hematopoietic cells: an in vitro study using a human bone marrow endothelial cell line.

To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 microns microporous membrane inserts and placed in 6-well tissue culture plates. Granulocytecolony stimulating factor (G-CSF)-mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 +/- 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 +/- 0.1 v 7.8% +/- 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% +/- 0.8% v 0.3% +/- 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% +/- 0.4%). While no difference in the expression of the beta 1-integrin very late activation antigen (VLA)-4 and beta 2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.

Antigens, CD34↗
PubMed1996

Effector cell protease receptor-1 is a vascular receptor for coagulation factor Xa.

The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196 --> Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall.

Arthropod Proteins↗
PubMed1996

BMEC-1: a human bone marrow microvascular endothelial cell line with primary cell characteristics.

Bone marrow microvascular endothelial cells (BMEC) are a functional component of the bone marrow stroma and have been shown to release hematopoietic regulatory factors as well as to selectively adhere and support the proliferation and differentiation of CD34+ hematopoietic progenitors. An early passage of these cells was immortalized by transfection with a vector (pSVT) encoding the large T antigen of SV40. The transformed cell line (CDC/CU.BMEC-1) expresses the SV40 transcript, retains the primary cell expression of Ulex europeaus and vWF/ FVIII, and incorporates acetylated low-density lipoprotein. In addition, BMEC-1 mirrors the phenotype of the primary cells with only a few exceptions. Both cell populations express the cellular adhesion molecules ICAM-1 and PECAM and also VCAM-1 and ELAM-1 after upregulation by tumor necrosis factor-alpha. The fibronectin receptor, hyaluronate receptor, collagen receptor, integrins VLA-alpha 3, VLA-alpha 4, and beta 4, endoglin, collagen IV, CD58, and CD61 are also expressed. The only differences are that BMEC-1 expresses higher levels of ICAM-1, CD58, CD34, CD36, and c-kit than the primary cells. The supernatants of primary cell and BMEC-1 contain stem cell factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-11, and G-CSF. The functional significance of these hematopoietic cytokines was demonstrated in transwell cultures. Both cell populations supported the expansion of progeny from CD34+ cell-enriched cord blood mononuclear cells suspended in the upper chamber. These characteristics, plus the fact that BMEC-1 can be maintained independently of exogenous growth factors and exhibit contact inhibition, indicate that this cell line can be used to further define the role of BMEC in hematopoiesis.

Bone Marrow↗
PubMed1996

The role of lipoprotein(a) in atherogenesis and thrombosis.

Lipoprotein(a) [Lp(a)] represents an important independent risk factor for atherosclerotic cardiovascular disease. Lp(a) constitutes a class of low-density lipoprotein-like particles that are structurally heterogeneous due to variability within the distinguishing apoprotein, apolipoprotein(a) [Apo(a)]. Apo(a) bears a high degree of homology to the fibrinolytic zymogen, plasminogen, the parent molecule of the serine protease plasmin. Apo(a) contains a variable number of tandemly repeated triple-loop units called kringles, which appear to mediate Lp(a)'s interactions with fibrin and cell surface receptors. Although the mechanism of its atherogenicity is unknown, Lp(a) has been implicated in the delivery of cholesterol to the injured blood vessel, in blockade of plasmin generation on fibrin and cell surfaces, and as a stimulus for smooth muscle cell proliferation. In addition, new members of the plasminogen/Apo(a) gene family have been defined, creating a potential link between Lp(a) and the control of angiogenesis in both health and disease. Pharmacologic therapy of elevated Lp(a) levels has been only modestly successful; apheresis remains the most effective therapeutic modality.

Animals↗
PubMed1995

Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors.

Endothelial cells are a major component of the bone marrow (BM) microenvironment that regulate the trafficking and homing of hematopoietic progenitor and stem cells. In this paper, we provide evidence that BM endothelial cells (BMECs) also support multilineage hematopoiesis by elaboration of soluble cytokines. Hematopoietic progenitor cells incubated in direct contact with BMEC monolayers, or physically separated by microporous membrane, expanded five-fold to sevenfold at 7 days, in the absence of exogenous cytokines. Flow cytometric analysis of proliferating progenitor cells grown in the presence of BMEC monolayers showed that by day 14 of coculture, 70% to 80% of hematopoietic cells were myeloid, expressing CD15 or CD14, and 14% to 19% were megakaryocytic, expressing GPIIb/IIIa or GPIb. CD34+ cells derived from umbilical cord blood, cultured in the upper chamber of transwell culture plates, as well as the cells grown in direct contact with BMEC monolayers, generated progenitors for up to 70 days. Unstimulated BMEC monolayers constitutively produce interleukin-6, Kit-ligand, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. These data suggest that BMEC regulate proliferation of hematopoietic progenitor cells and long-term culture initiating cells by elaboration of lineage-specific cytokines.

Antigens, CD↗
PubMed1995

Characterization of hematopoietic cells arising on the textured surface of left ventricular assist devices.

BACKGROUND: Textured biomaterial surfaces in implantable left ventricular assist devices induce development of a nonthrombotic neointimal surface and allow elimination of anticoagulation therapy in device recipients. Characterization of the hematopoietic cells formed within the neointimal surfaces of these devices will contribute to our understanding of this unique neointima. METHODS: The blood-contacting surface of seven ThermoCardiosystems left ventricular assist devices was removed, washed with phosphate-buffered saline solution, and digested with 0.1% collagenase for 15 to 20 minutes. The hematopoietic cells released from the explants were isolated and analyzed by flow cytometry and immuno-histochemical staining. RESULTS: More than 80% +/- 6% of hematopoietic cells isolated in this fashion are of myelomonocytic origin and express CD14, CD15, and CD33 surface molecules. Four percent of cells express the CD34 surface marker, which suggests that the neointima is colonized by pluripotent hematopoietic stem cells. Continuous culture of these hematopoietic cells in the presence of the cytokines interleukin-3, c-kit ligand, granulocyte colony-stimulating factor resulted in tenfold expansion by day 7 and 25-fold expansion by day 14. CONCLUSIONS: Pluripotent hematopoietic cells with a high proliferative capacity colonize textured surfaces of left ventricular assist devices and may contribute to the development of a biologically nonthrombogenic neointima.

Antigens, CD↗
PubMed1994

Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion.

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.

Antigens, CD↗
PubMed1993

Hypercoagulable states.

PURPOSE: To describe the major pathophysiologic mechanisms underlying inherited and secondary hypercoagulable states and to evaluate the frequency, natural history, diagnosis, and management of the various clinical disorders. DATA SOURCES AND STUDY SELECTION: Relevant clinical literature obtained from bibliographies in hematology textbooks and from computerized indexes was reviewed. A hypothesis was formed based on this literature review and on recent developments from a number of experimental studies. DATA SYNTHESIS: Hypercoagulable states include various inherited as well as acquired clinical disorders characterized by an increased risk for thromboembolism. Primary hypercoagulable states include relatively rare inherited conditions that lead to disordered endothelial cell thromboregulation. These conditions include decreased thrombomodulin-dependent activation of activated protein C, impaired heparin binding of antithrombin III, or down-regulation of membrane-associated plasmin generation. The major, inherited, inhibitor disease states include antithrombin III deficiency, protein C deficiency, and protein S deficiency and should be considered in patients who have recurrent, familial, or juvenile deep-vein thrombosis or occlusion in an unusual location such as a mesenteric, brachial, or cerebral vessel. Secondary hypercoagulable states may be seen in many heterogeneous disorders. In many of these conditions, endothelial activation by cytokines leads to loss of normal vessel-wall anticoagulant surface functions with conversion to a proinflammatory thrombogenic phenotype. Important clinical syndromes associated with substantial thromboembolic events include the antiphospholipid syndrome, heparin-induced thrombopathy, the myeloproliferative syndromes, and cancer. CONCLUSIONS: Physiologic thromboregulation occurs at the vessel-wall surface. Quantitative and qualitative deficiencies of normal, steady-state endothelial anticoagulant activities are associated with primary hypercoagulable states. Activated endothelial cell surfaces express a thrombogenic phenotype and contribute to secondary or acquired hypercoagulability.

Blood Coagulation Disorders↗
PubMed1992

Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding.

To clarify the role of CD36 as a TSP receptor and to investigate the mechanisms of the TSP-CD36 interaction, transfection studies were performed using CD36-cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membrane surface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminished platelet aggregation. CSVTCG-albumin conjugates support CD36-dependent adhesion of tumor cells. We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding.

Amino Acid Sequence↗
PubMed1991

Lipoprotein (a) regulates plasminogen activator inhibitor-1 expression in endothelial cells. A potential mechanism in thrombogenesis.

Lipoprotein (a) (Lp(a)) is a low density lipoprotein-like particle which contains the plasminogen-like apolipoprotein a. Lp(a) levels are elevated in patients with atherosclerotic coronary artery disease. Recent studies suggest that Lp(a) competitively inhibits plasminogen binding to the endothelial cell and interferes with surface-associated plasmin generation. In this study, we present evidence for the presence of Lp(a) in the microvasculature of inflamed tissue. In addition, we demonstrate that Lp(a) regulates endothelial cell synthesis of a major fibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1). In cultured human endothelial cells, Lp(a) enhanced PAI-1 antigen, activity, and steady-state mRNA levels without altering tissue plasminogen activator activity or mRNA transcript levels. This effect was cell-specific. Although other lipoproteins did not coordinately raise PAI-1 mRNA levels in endothelial cells, low density lipoprotein treatment selectively raised the level of the 3.4-kilobase mRNA species of PAI-1 without a concomitant increase in PAI-1 activity or antigen. Endothelial cell exposure to Lp(a) did not cause generalized endothelial cell activation since the functional activity and mRNA levels for tissue factor, platelet-derived growth factor and interleukin-6 were not elevated following Lp(a) exposure. These data suggest a molecular mechanism whereby Lp(a) may support a specific prothrombotic endothelial cell phenotype, namely by increasing PAI-1 expression.

Cell Line↗
PubMed1991

Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.

Antigens, Differentiation↗
PubMed1991

Endothelial cell fibrinolytic assembly.

Endothelial cells play a critical role in thromboregulation by controlling the assembly of fibrinolytic constituents on the membrane. The assembly system illustrated in FIGURE 6 is characterized by the binding of circulating glu-plasminogen to a membrane receptor (Pathway 1). A membrane-associated protease (possibly plasmin) converts the inactive zymogen into a catalytically more efficient zymogen lys-plasminogen (Pathway 2). T-PA binds to a specific receptor, retains its catalytic activity, and is protected from its natural inhibitor PAI-1. The membrane provides a favorable environment for plasmin generation (Pathway 3) at the vessel surface and contributes to the maintenance of a physiological nonthrombogenic state. The immobilization and surface activation of plasminogen provides an important mechanism for localizing proteolytic activity at the surface of other cells such as macrophages and tumor cells. Lp(a), a plasminogen-like lipoprotein, by competing at the endothelial surface for plasminogen binding down-regulates endothelial cell plasmin generation and may thus promote localized thrombogenesis that over a period of time contributes to progressive atherosclerosis.

Animals↗
PubMed1990

Cytokine-enhanced expression of glycoprotein Ib alpha in human endothelium.

Platelet glycoprotein Ib is a major platelet membrane protein composed of two disulfide-linked chains, termed the alpha and beta chains. The larger alpha chain (GpIb alpha), a platelet receptor for von Willebrand factor, plays a major role in mediating platelet adhesion to the subendothelium. Our laboratories have previously reported synthesis of a protein in human endothelial cells that is immunoprecipitated with polyclonal and monoclonal antibodies to platelet GpIb alpha. Lopez et al. (Lopez, J. A., Chung, D. W., Fujikawa, K., Hagan, F. S., Papayannopoulou, T., and Roth, G. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5615-5619) have reported the cloning of GpIb alpha from a human erythroleukemia (HEL) cell cDNA library. Using this clone as probe, we have isolated two partial GpIb alpha clones from a human umbilical vein endothelial cell lambda gt11 cDNA library. These clones were localized within HEL-derived GpIb alpha cDNA by sequence and restriction enzyme analysis. Additionally, they detected the same message species in HEL and tonsilar RNA that was detected with the HEL GpIb alpha cDNA. Low level GpIb alpha mRNA expression was detected in cultured human umbilical vein endothelial cells, which was increased by treatment of the cells with tumor necrosis factor-alpha. This effect was enhanced by pretreatment with interferon-gamma. Additionally, localization of GpIb alpha in endothelium of fresh tonsilar tissue was demonstrated by immunohistochemistry and in situ hybridization. GpIb alpha may play a role in mediating platelet or other effector cell adhesion to activated endothelium.

Cloning, Molecular↗
PubMed1990

Thrombospondin forms complexes with single-chain and two-chain forms of urokinase.

Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.

Amino Acid Sequence↗