Electron microscopic techniques in the study of cultured vascular cells.
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Blood Platelets↗
Biomedical subjects
E A Jaffe
Publications and source records attributed to E A Jaffe.
Synthesis of prostaglandin I2 (prostacyclin) by cultured human and bovine endothelial cells.
Cultured endothelial cells derived from human umbilical veins or bovine aorta produce a potent inhibitor of platelet aggregation. The inhibitor is synthesized from sodium arachidonate or or prostaglandin endoperoxides by a microsomal enzyme system. Tranylcypromine, a specific antagonist of prostacyclin synthetase, suppresses production of the inhibitor by endothelial cells. The inhibitor, which is ether extractable, has been identified using a two-step thin-layer radiochromatographic procedure and a synthetic prostaglandin I2 standard. With this procedure, we have shown that human and bovine endothelial cells convert sodium [3H]arachidonate to radiolabeled prostaglandin I2 and 6-keto-prostaglandin F1alpha, as wellas prostaglandin E2. Thus, endothelial cells may be non-thrombogenic in vivo because they synthesize and release prostaglandin I2, a potent inhibitor of platelet aggregation.
Animals↗
Myosin in cultured human endothelial cells.
Myosin was isolated from cultured human endothelial cells by extraction with 0.6 M KCl and chromatography on Sepharose 4B. The extracted endothelial cell protein was identified as myosin by the characteristic ATPase profile, that is, the ATPase was activated by Ca2 + and EDTA and inhibited by Mg2 +. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the endothelial cell myosin heavy chain migrated with a molecular weight of 200 000 as did rabbit uterine and human platelet myosin heavy chains. A crude preparation of the endothelial cell myosin reacted immunologically with an antiserum to platelet myosin, a smooth muscle type of myosin. In indirect immunofluorescence studies, antiserum to the purified endothelial cell myosin stained cultured endothelial cells in a fibrillar pattern. The fibrillar pattern was more intense when the endothelial cells were stained with antiserum to platelet myosin. The presence of myosin in the endothelial cell provides a basis for the contractility of these cells. This contractile property may plan an important role in the physiologic function of these cells.
Adenosine Triphosphatases↗
Immunoinhibition of ristocetin-induced platelet aggregation.
Human platelets washed and fixed in paraformaldehyde aggregate in the presence of the antibiotic ristocetin and normal plasma. This aggregation response is abolished after digestion of the fixed platelets with chymotrypsin. Antisera to fixed washed platelets were produced in rabbits and absorbed with chymotrypsin-treated, fixed washed platelets. Monovalent Fab fragments obtained from the isolated gamma-globulin fractions of the antisera blocked ristocetin-induced aggregation of fixed washed platelets in buffer and normal platelets in platelet-rich plasma. By double-antibody immunoprecipitation, it was shown that the antibody which blocked the ristocetin reaction interacted with a platelet membrane surface protein of mol wt 155,000. The results suggest that the glycoprotein I complex on the surface of the human platelet mediates ristocetin-induced von Willebrand factor-dependent platelet aggregation.
Antibodies↗
Synthesis of factor VIII antigen by cultured guinea pig megakaryocytes.
Immunoprecipitates containing guinea pig Factor VIII antigen were prepared from guinea pig plasma with a cross-reacting rabbit anti-human Factor VIII. Monospecific antisera to guinea pig Factor VIII antigen were produced in rabbits by using these washed immunoprecipitates as immunogens. The resulting antisera to guinea pig Factor VIII antigen detected Factor VIII antigen in guinea pig plasma and inhibited the von Willebrand factor activity in guinea pig plasma. This antibody also detected Factor VIII antigen in a solubilized protein mixture prepared from isolated cultured guinea pig megakaryocytes. Cultured guinea pig megakaryocytes were labeled with radio-active leucine. By radioautography, 96.2% of the radio-activity was present in megakaryocytes. The radio-active Factor VIII antigen present in the solubilized cell protein mixture was isolated by immunoprecipitation and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results demonstrate that cultured guinea pig megakaryocytes synthesize Factor VIII antigen which contains the same polypeptide subunit (mol wt 200,000) present in guinea pig plasma Factor VIII antigen.
Animals↗
Synthesis of basement membrane collagen by cultured human endothelial cells.
Studies were performed to determine if cultured human endothelial cells synthesized basement membrane collagen. In culture, endothelial cells were attached to grossly visible membranous structures which on light microscopy were composed of ribbons of dense, amorphous material. On transmission electron microscopy, these membranous structures consisted of amorphous basement membrane, and material morphologically similar to microfibrils and elastic fibers. By immunofluorescence microscopy, these membranous structures stained brightly with antisera to human glomerular basement membrane. Cultured endothelial cells incorporated [3H]proline into protein; 18% of the incorporated [3H]proline was solubilized by purified collagenase. When endothelial cells were cultured with [14C]proline, 7.1% of the incorporated counts were present as [14C]hydroxyproline. Cultured endothelial cells were labeled with [3H]glycine and [3H]proline and digested with pepsin. The resulting fractions on analysis by SDS-polyacrylamide gel electrophoresis contained two radioactive protein peaks of mol wt 94,200 and 120,500. Both these peaks disappeared after digestion with purified collagenase. The peak of mol wt 120,500 corresponds to that of alpha1 (IV) collagen; the peak of the mol wt 94,200 probably corresponds to that of alpha1 (III) collagen. Thus, cultured human endothelial cells synthesize material which is morphologically and immunologically like amorphous basement membrane and biochemically like basement membrane collagen. Cultured endothelial cells probably also synthesize material which is morphologically similar to microfibrils and elastic fibers.
Basement Membrane↗
The platelet-endothelial cell--VIII axis.
Cultured human endothelial cells synthesize and secrete a protein(s) which has factor VIII antigen and von Willebrand factor activity. Subcellular membrane and granule fractions derived from human platelets also contain the factor VIII antigen and von Willebrand factor activity. Circulating platelets constitute a significant reservoir of VIII antigen containing approximately 15% of the amount present in platelet poor plasma. Thus normal platelets contain surface bound as well as intracellularly stored von Willebrand factor, a protein synthesized by endothelial cells which is required for normal platelet function.
Antigens↗
A clinical study of the lupus anticoagulant.
Eighty-three patients with circulating anticoagulants were studied at The New York Hospital. The lupus-type anticoagulant, an inhibitor of the prothrombin activator complex, was demonstrated in 58 patients. The inhibitor was identified using the blood and tissue thromboplastin inhibition tests. Inhibition by the lupus anticoagulant was augmented in 67% of these patients by a cofactor present in normal plasma. The lupus inhibitor was detected primarily because of an unsuspected abnormal coagulation test. One-half of the patients with the lupus-type anticoagulant did not have systemic lupus erythematosus.
Anticoagulants↗
Subcellular platelet factor VIII antigen and von Willebrand factor.
Subcellular membrane and granule fractions derived from human platelets contain factor VIIII antigen and von Willebrand factor activity but not factor VII procoagulant activity. Circulating platelets constitute a significant reservoir of plasma factor VIII antigen, containing approximately 15% of the amount of factor VIII antigen present in platelet-poor plasma. The antibiotic ristocetin, which aggregates human platelets in the presence of von Willebrand factor, nonspecifically precipitates platelet membrane factor VIII antigen. Thus normal platelets contain surface-bound as well as internally stored von Willebrand factor, a protein synthesized by endothelial cells which is necessary for normal platelet function in vivo.
Absorption↗
Synthesis of factor VIII antigen by cultured human endothelial cells.
Endothelial cells have been isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein and grown in tissue culture. These cells have been identified by morphologic and immunologic criteria. It has been demonstrated that these cultured human endothelial cells synthesize and release factor VIII antigen but not factor VIII clot-promoting activity.
Animals↗
Subunit structure of factor VIII antigen synthesized by cultured human endothelial cells.
Cultured human endothelial cells were labeled with (3H)leucine, and the radioactive Factor VIII antigen present in the postculture medium was isolated by double anitbody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol. The Factor VIII antigen synthesized by cultured endothelial cells was found to contain the same single polypeptide subunit (mol wt 225,000) present in plasma Factor VIII antigen. These results suggest that in vivo, the endothelial cell is a major site of synthesis of circulating Factor VIII antigen.
Antigens↗
The identification of HL-A antigens on fresh and cultured human endothelial cells.
Human endothelial cells were obtained from the umbilical cord veins of 16 newborns by methods previously described and tested for HL-A antigens by a microcytotoxicity method. HL-A antigens were present on all endothelial cell lines tested. When the HL-A phenotypes of fresh endothelial cells and autologous fetal lymphocytes were compared, a concordance of 70% was observed. When the HL-A phenotypes of maternal lymphocytes and fresh endothelial cells were compared, a maternal contribution to the endothelial cell phenotype was evident in 72% of the possible commmon antigens. Some HL-A antigens were deleted from 11 of 16 endothelial cell lines that were re-typed after 2 weeks in tissue culture. The majority (90%) of deleted antigens were from the second HL-A locus. When three lines of endothelials cells were again re-typed after 6 weeks in culture, no further changes in antigenicity were noted. These findings: a) demonstrate that HL-A antigens are present on human endothelium and suggest that endothelial cells are actively involved in establishing the immunogenicity of a graft, and b) demonstrate that the HL-A antigens on human endothelial cells may be modulated by in vitro culture.
Adult↗
Synthesis of von Willebrand factor by cultured human endothelial cells.
Cultured human endothelial cells synthesize and secrete a protein(s) which has Factor VIII antigen but which lacks Factor VIII clot-promoting activity (J. Clin. Invest. 52, 2757-2764, 1973). Von Willebrand factor activity has been identified in medium from cultured human endothelial cells. This activity was demonstrated by the ability to correct the defect in platelet adhesiveness of blood obtained from patients with von Willebrand's disease. This activity also supported ristocetin-induced aggregation of washed normal human platelets. The von Willebrand factor activity from cultured endothelial cells has physicochemical and immunologic properties like those of the von Willebrand factor activity and the Factor VIII antigen present in human plasma and the Factor VIII antigen synthesized by human endothelial cells in vitro. Rabbit antibody to chromatographic fractions containing endothelial cell von Willebrand factor inhibits the platelet retention of normal blood in glass bead columns.
Animals↗
Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.
Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.
ABO Blood-Group System↗
Synthesis of antihemophilic factor antigen by cultured human endothelial cells.
Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system.
Animals↗
Thrombotic thrombocytopenic purpura--coagulation parameters in twelve patients.
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Adolescent↗
Uremic levels of oxalic acid suppress replication and migration of human endothelial cells.
Patients with chronic renal failure who undergo hemodialysis experience accelerated atherosclerosis and premature death. Since the end-metabolite, oxalic acid, accumulates in plasma in proportion to the severity of renal failure, we studied whether sodium oxalate (0 to 300 microM) is an endothelial toxin and, therefore, might enhance atherogenesis. Exposure to uremic levels of oxalate (greater than 30 microM) for 9 to 28 days depressed endothelial cell replication by 33% to 84% (mean +/- SD, 54% +/- 15.7%, n = 17 experiments, p = 0.002). In contrast, replication of fibroblasts exposed to 200 microM oxalate for 45 days was not inhibited. The inhibitory effect of oxalate on endothelial cell replication was both dose- and time-dependent (both p less than 0.0001) and was first detected 3 to 7 days after the initial exposure to oxalate. Further, the inhibitory effect was fully reversible upon removal of oxalate, but only if exposure was limited to 5 days or less. Sodium salts of other carboxylic acids (citric, succinic, glyoxylic, and malonic; 200 microM) as well as HCl (200 microM) did not suppress endothelial cell replication. Oxalate also inhibited endothelial cell migration but had no effect on basal, thrombin-induced, or arachidonate-induced prostacyclin production by endothelial cells. Exposure of endothelial cells to sodium oxalate (200 microM) for as little as 24 hours-a time period sufficient to induce delayed, transient inhibition of replication not detectable until approximately 1 week after exposure-inhibited incorporation of 3H-leucine into protein by 40% (p = 0.009). We conclude that sodium oxalate acts as a uremic toxin, inhibiting endothelial cell replication and migration, functions which may be important for constitutive inhibition of atherosclerosis.
6-Ketoprostaglandin F1 alpha↗
Prostacyclin production by endothelial cells. Effects of sera from normal and hyperlipidemic subjects.
The influence of hyperlipidemic sera on prostacyclin (PGI2) production by cultured endothelial cells was assessed by comparing sera from three types of hyperlipidemias with sera from normal subjects. Sera prepared from normal whole blood (WBS), platelet-rich plasma (PRPS), and platelet-poor plasma (PPPS) were also compared. Bovine aortic endothelial cells (BAEC) incubated with 25% WBS increased PGI2 synthesis significantly within 1 hour, with little further increase by 16 hours; human umbilical vein endothelial cells (HUEC) incubated with 25% WBS for 1 hour showed no elevation in PGI2, whereas PGI2 levels increased substantially after 16 hours. PPPS and PRPS stimulated PGI2 synthesis by BAEC equally at 1 hour. However, there was no rise in PGI2 after PPPS in HUEC; PGI2 rose after 16 hours with PRPS and rose further with WBS after 16 hours. Since WBS best enhanced PGI2 production in human endothelial cells, it was chosen for comparison of the effects of hyperlipidemic and normolipidemic sera. PGI2 synthesis by HUEC significantly increased upon incubation with WBS from Types IIb and IV patients in comparison to WBS from Type IIa hypercholesterolemic patients or normal controls. In contrast, WBS from all these hyperlipidemic subjects stimulated PGI2 synthesis by BAEC similarly to WBS from controls. We conclude that incubation of human endothelial cells with WBS containing high levels of atherogenic lipoproteins does not reduce PGI2 formation by the cells. Moreover, the time course and the contribution of lipid, plasma, or cellular factors to PGI2 formation vary according to the cell type tested. Caution should be exercised in extrapolating results achieved with serum and cells from the same species to other settings.
Adult↗