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Biomedical subjects

G Tobelem

Publications and source records attributed to G Tobelem.

At least 73 records · Page 4Linked to original sources
PubMed1989

Thrombin mitogenic responses and protein phosphorylation are different in cultured human endothelial cells derived from large and microvessels.

It is well established that thrombin induces various biological responses in endothelial cells derived from large vessels. However, little is known about the effects of thrombin on the microvasculature. Protein phosphorylation may be one of the mechanisms by which an extracellular stimulus initiates cellular events like proliferation. Therefore, we have compared the effects of either human alpha-thrombin or phorbol esters (TPA) on the proliferation or protein phosphorylation in endothelial cells derived from large vessels (umbilical vein, HUVEC) or microvessels (omental tissue, HOMEC). In HOMEC, thrombin did not stimulate cell proliferation and protein phosphorylation while TPA slightly reduced the cell proliferation and induced the phosphorylation of a 27-kDa protein. In contrast, in HUVEC, thrombin or TPA markedly enhanced the cell proliferation and stimulated the phosphorylation of a 59-kDa protein. These data indicate that (i) endothelial cells from large and small vessels respond differently to thrombin and (ii) there is a complex and as yet unclear relationship between the proliferation and the protein phosphorylation induced by thrombin.

Biological Transport↗
PubMed1989

Evidence of endoplasmic reticulum-related Ca2+ ATPase in human microvascular endothelial cells.

We have demonstrated by immunological and molecular methods the presence of a reticulum endoplasmic-related Ca2+-ATPase in human omental microvascular endothelial cells (HOME cells). HOME cells reacted positively with a previously characterized sarcoplasmic reticulum Ca2+-ATPase antibody as demonstrated by indirect immunofluorescence. Western blotting revealed that the antibody recognized a 95-100 kDa protein. 35S-Metabolic labeling led to the detection of a similar protein with which the purified sarcoplasmic reticulum Ca2+-ATPase competed. Dot-blotting experiments indicated that a substantial amount of Ca2+-ATPase was present in HOME cell membranes. In addition, Northern blot analysis using a cDNA probe from cardiac sarcoplasmic reticulum showed the presence of mRNA species of 4-kb. As these experiments were conducted in comparison with cell types with well-defined Ca2+-ATPase in HOME cells.

Animals↗
PubMed1989

Essential thrombocythaemia.

Essential thrombocythaemia (ET) is a rare clonal myeloproliferative disorder characterized by a persistent increase in platelet count. The commonly used criteria for the diagnosis, except for the level of the increase in platelet count, are usually those fixed by the Polycythemia Vera Study Group. The average age of onset is around 60 years, both sexes being affected. The symptoms frequently present at diagnosis are related to microcirculatory disturbances (palms, soles, fingers). Neurological symptoms are often observed. Thrombotic complications of the large vessels are less frequent. Haemorrhagic problems are present in about 30% of patients. Bleeding time is normal in most cases, whereas platelet aggregation abnormalities are frequently found. Nil adrenaline aggregation is the most discriminative test. The clinical course is characterized by long intervals without any symptoms; thromboembolic or haemorrhagic episodes can, however, occur, mainly in uncontrolled ET. Development of terminal acute leukaemia has been reported in 34 cases. The expression of the influence of the treatments, 32P or alkylating agents, is very strong. The treatment of ET has to take in consideration the difficult compromise between balancing the necessity of preventing complications and the effects of drug toxicity. The use of recombinant alpha-interferon has recently been proposed and is under investigation. The pathogenesis of thrombocytosis in ET seems to involve an expansion in the megakaryocyte progenitor cell pool. Platelet membrane glycoprotein abnormalities and defective glycosylation of thrombospondin have been shown. Numerous other platelet abnormalities, including decreased alpha-adrenergic receptors, loss of PGD2 receptors and increased Fc receptors, have been reported. Arachidonic metabolism seems to be abnormal and lipoxygenase is defective. Most of the platelet abnormalities seem to be the result of intrinsic defects at the level of an abnormal clone of megakaryocytes. However, causal relationships between the platelet abnormalities and bleeding or thrombosis are not yet clearly demonstrated.

Arachidonic Acid↗
PubMed1989

Platelet-derived growth factor (PDGF) in type 1 diabetes mellitus.

In 10 patients with type 1 diabetes mellitus, platelet-derived growth factor (PDGF) was assessed using an in vitro assay based on the stimulation of DNA synthesis in the 3T3 mouse fibroblast cell line. beta-Thromboglobulin (beta-TG) was used as an index for platelet alpha-granules content. Platelet beta-TG content and PDGF were markedly decreased in diabetic patients while plasma beta-TG was increased as compared with control subjects. In diabetic patients, a significant negative correlation was found between plasma beta-TG level and beta-TG total platelet content, associated with a significant positive correlation between platelet beta-TG content and PDGF. These results suggest that PDGF release might be increased in diabetic subjects. This may account in part for the cell proliferation observed in diabetic angiopathy.

Adult↗
PubMed1989

Prelining of polytetrafluoroethylene grafts with cultured human endothelial cells isolated from varicose veins.

Prelining graft material with autologous functioning endothelial cells might be one of the ultimate requirements to obtain a biocompatible surface. Accordingly, endothelial cells from stripped varicose veins were enzymatically harvested and grown on a fibronectin matrix. Proliferation was investigated in defined medium supplemented with various concentrations of endothelial cell growth supplement (ECGS) (25, up to 150 micrograms/ml) and heparin (10(-8), up to 10(-5)mol/L): optimal growth required both 150 micrograms/ml of ECGS and 10(-5)mol/L heparin. Under these conditions, cell culture achieved cell densities at a confluence of 1.2 +/- 1.1 10(5) cells/cm2 with a doubling time of 1 day. During subcultivation cultured cells consistently exhibited characteristic cobblestone morphology and immunofluorescent staining for factor VIII-related antigen, whereas prostacyclin production determined by enzyme-linked immunosorbent assay for 6-keto-prostaglandin F1 alpha reached 21.1 +/- 1.2 ng/10(6) cells after 15-minute stimulation with 1 U/ml of thrombin. Heparin-containing culture medium-endothelial cell interactions were particularly studied, and with iodine 125-heparin, binding was demonstrated with an apparent dissociation constant (Kd) of 0.36 +/- 0.04 mumol/L. A cold storage technique at -80 degrees C was sought, and freezed cells were used to coat in vitro polytetrafluoroethylene grafts. Protein-treated material allowed cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy. These data validate the feasibility of prelining grafts in vitro with autologous functioning endothelial cells. This approach may be useful in improving the performance of small-caliber vascular grafts according to prostacyclin production and surface-bound heparin of these cells.

Adult↗
PubMed1988

Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells--inhibition of thrombomodulin-dependent protein C activation.

We have investigated the effect of purified immunoglobulin G (IgG) on endothelial cell functions in 16 patients with lupus anticoagulant, 9 of whom had systemic lupus erythematosus (SLE). Spontaneous or thrombin-stimulated secretion of prostacyclin (PGI2) by cultured human endothelial cells from umbilical cord vein (HUVEC) was not inhibited by the patient's IgG. Nor was spontaneous release of tissue plasminogen activator (t-PA) or of its inhibitor (PAI) modified in the presence of patient's IgG. The rate of activation of purified protein C (PC) by HUVEC in the presence of thrombin was significantly lowered by patient's IgG or Fab' fragment (inhibition of 43%). Neutralization of this effect was obtained by incubation of a greater quantity of phospholipids (phosphatidylcholine, phosphatidylserine) with the patient's IgG. Activation of PC was also performed using purified rabbit thrombomodulin (TM) and a similar inhibition of the patient IgG was observed (inhibition of 48%) but the activation of Gla-domainless PC was not modified.

Animals↗
PubMed1988

Transforming growth factor (TGF beta) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding.

Human bone marrow fibroblasts were cultivated and characterized by immunofluorescent staining and electron microscopy. Their interactions with PDGF and TGF beta were studied. While a positive intracellular antifibronectin staining was observed, the cultured cells were not labeled with specific antibodies toward factor VIII von Willebrand factor (F VIII/vWF), desmin, and macrophage antigen. Moreover, electron microscopy excluded the presence of endothelial cells by the absence of Weibel-Palade bodies. The binding of pure human PDGF to the cultured bone marrow fibroblasts was investigated. Addition of an excess of unlabeled PDGF decreased the binding to 75 and 80%, which means that the nonspecific binding represented 20-25% of total binding, whereas epidermal growth factor (EGF) had no effect. Two classes of sites were detected by Scatchard analysis with respectively 21,000 and 37,000 sites per cell, with a KD of 0.3 x 10(-10) M and KD of 0.5 x 10(-9) M. The stimulation of DNA synthesis by PDGF was quantified by [3H]thymidine incorporation. When PDGF was added alone at a concentration of 15 ng/ml, it induced a maximal DNA synthesis of 400%, which increased up to 900%, in the presence of platelet-poor plasma (PPP). On the other hand, PDGF-induced fibroblast proliferation was inhibited in a dose-dependent manner by TGF beta. This inhibition was related to a significantly decreased binding of 125I-labeled PDGF observed in the presence of TGF beta. Our results suggested that PDGF and TGF beta could modulate the growth of bone marrow fibroblasts.

Bone Marrow↗
PubMed1988

Inhibition of human endothelial cell proliferation by heparin and steroids.

Previous works have reported the controversial effects of heparin and steroids on angiogenesis. In this study, we investigated the effect of these compounds on human endothelial cell (EC) growth in vitro. An antiproliferative heparin activity was found in low human serum concentrations (2%). When EC were exposed to heparin (10(-6) M), their proliferation index was reduced in the presence of endothelial cell growth factor added 6 hours or more later. These results suggest that there is an intracellular effect of heparin which reduces 3H-methylthymidine uptake. Hydrocortisone acetate and tetrahydroS induced inhibition of EC growth in a dose-dependent manner. Steroids inhibited proliferation of EC in culture medium in the presence or the absence of growth factor and in different human serum concentrations. These results suggest a possible synergistic antiangiogenic action of heparin plus steroids.

Cell Division↗
PubMed1988

Heparin stimulates fibroblasts growth induced by platelet derived growth factor.

We have demonstrated that 125I unfractionated heparin binds to cultured human skin fibroblasts with a Kd of 1.16 10(-8) M and is internalized partly. A low molecular weight heparin fraction (PK 10169) competed (50%) with 125I unfractionated heparin, but to a lesser extent than cold unfractionated heparin (90%). When the cell proliferation was induced by pure PDGF, heparin markedly potentiated the fibroblast growth. Similar stimulation was observed when the growth was induced by FGF or EGF. Low molecular weight heparin enhanced the fibroblast proliferation induced by PDGF but to a lesser extent than unfractionated heparin. Chondroitin sulfate has no effect. PDGF did not modify the heparin binding on fibroblast cultures either at 4 degrees C or 37 degrees C and did not alter the process of heparin internalization. PDGF binding to the cultured fibroblast (Kd 10.1 +/- 3.4 10(-10) M) was not modified by the presence of heparin when studied at 4 degrees C or 37 degrees C.

Cell Division↗
PubMed1988

Binding of heparin to human microvascular endothelial cells and the effect on proliferation.

We have studied the binding of 125I-heparin to human omental microvascular endothelial cells (HOME cells) and investigated its effect on cell proliferation. At 20 degrees C, the binding reached a steady state from 4 hours onwards. Saturation of 125I-heparin binding occurred at 200 nM. Scatchard analysis indicated one class of binding sites (KD = 0.023 microM, 2 X 10(-6) sites/cell). Using fractionated heparin and other sulfated polysaccharides it was demonstrated that the binding was dependent on the charge and the molecular weight of the compounds. The binding was followed by the partial internalization of the bound ligand (23.8%). Heparin and Stipocus Japonicus mucopolysaccharide (SJAMP) inhibited the proliferation of exponentially growing HOME cells and human umbilical vein endothelial cells (HUVE cells). However, serum-deprived HOME cells were not inhibited by heparin.

Binding, Competitive↗
PubMed1988

Low doses v conventional doses of corticoids in immune thrombocytopenic purpura (ITP): results of a randomized clinical trial in 160 children, 223 adults.

A randomized clinical trial was performed in 160 children and 223 adults with acute immune thrombocytopenic purpura (ITP). The role of corticoids at this phase of the disease is still controversial. Therefore, patients were randomized to receive either conventional doses (1 mg/kg/day) of corticotherapy or low doses (0.25 mg/kg/day) for 3 weeks. The remission, defined by platelet count superior to 100,000/microliter was studied for adults and children after 6 months and 12 months, respectively. The statistical analysis showed no significant difference between the two corticotherapy regimens neither in children nor in adults. Overall, 74% of children and 41% of adults were in remission. For the first time in acute ITP, a randomized prospective trial showed that both in children and adults low dose corticotherapy (0.25 mg/kg/day) proves to have the same efficacy as conventional doses (1 mg/kg/day).

Acute Disease↗