We investigated the effects of high-density lipoprotein (HDL) on the intracellular pH (pH
i), and on the proliferation of human vascular endothelial cells (HUVEC), as well as on their production of ...prostacyclin (PGI
2). The pH
i was slightly acidified when extracellular Ca
2+ was chelated with EGTA. Pretreatment of HUVEC with amiloride, the Na
+/H
+ exchange inhibitor, caused the pH
i to become strongly acidic. The addition of HDL produced a biphasic shift in pH
i, with a brief initial acidification followed by a rapid alkaline shift. The initial decrease in pH
i was abolished in the cells pretreated with EGTA, and subsequent alkalinization was inhibited. The alkalinization of pH
i disappeared in the cells pretreated with amiloride. These results suggest that pH
i depends mainly on Na
+/H
+ exchange and partially on the extracellular Ca
2+ of the HUVEC either in the resting unstimulated state or during HDL stimulation. In contrast, the addition of LDL produced an acidification of pH
i, which was increased by LDL in the Ca
2+-free condition. In the cells pretreated with amiloride, pH
i was not further acidified by LDL. As a result, HDL promoted the proliferation of cells, an action that was inhibited by pretreatment with EGTA. However LDL inhibited cell proliferation, an action unaffected by EGTA pretreatment. The addition of HDL also enhanced the generation of prostacyclin in endothelial cells, the enhancement of PGI
2 generation resulted from an increase in the release of Ca
2+ from storage sites, due not only to an increased production of inositol 1,4,5-trisphosphate (IP
3), but also to the alkalinization of pH
i. These effects may be involved in the mechanism of HDL's anti-atherosclerotic action.
Emergency colonoscopy for lower gastrointestinal bleeding was performed in 145 patients. Total cases were deviled into 70 males and 75 females and mean age was 50.1 years old. One hundred thirty five ...of 145 patients (93.1%) were correctly diagnosed by emergency colonoscopy examination. 78.6 percents of cases were also diagnosed, even if endoscopy was inserted to the sigmoid colon.Ten patients were necessary to manage the bleeding. Eight patients had endoscopic hemostasis, including 6 patients treated with an electrocoagulation method using "Heater Probe" and 2 patients injected with aethoxsclerol. Surgery was performed in 2 patients. Colonoscopy was possible not only the detection of the bleeding point, but also evaluation of the characteristic of the lesions and bleeding condition and, in many cases, it was also possible effective endoscopic hemostasis. Especially, endoscopic hemostasis showed extremely effective role in the treatment for focal colonic hemorrhagic lesions such as arteriovenous malformation and acute hemorr-hagic rectal ulcer. We concluded that colonoscopy was consider to be an important first-choice examina-tion for diagnosis and therapy of the lower gastrointestinal bleeding.
The antithrombotic effects of low molecular weight heparin (LMWH) were evaluated in the experimental microvascular thrombosis model using the hamster cheek pouch and were compared to unfractionated ...heparin (UFH). The platelet-rich thrombus was produced in the venules by the irradiation of the filterd ultraviolet light in combination with the intravascular administration of fluorescein sodium. The antithrombotic activities of these agents were evaluated with respect to the time required for the initiation of thrombus formation (Ti: sec) and the time to stop the blood flow by thrombus (Ts: sec), together with anti-Xa activity (a-Xa: U/ml), anti-IIa activity (a-IIa: U/ml), PT, aPTT and bleeding time (BT: sec) immediately (0), 1 hour (1) and 2 hour (2) after the administration of LMWH or UFH (500 a-XaU/kg). The values of Ti after administration of LMWH or UFH were (0) 122.5±27.8, 114.6±17.5, (1) 97.3±18.9, 87.1±16.8 and (2) 80.1±16.9, 63.2±9.5 (mean±SD) respectively. The values of Ts were (0) 757.2±137.3, 697.7±63.9, (1) 672.1±77.1, 516.1±64.5 and (2) 599.2±47.5, 429.6±54.8, respectively. The levels of a-Xa activity were (0) 0.98±0.05, 0.95±0.03, (1) 0.54±0.08, 0.23±0.12, and (2) 0.24±0.11, 0.08±0.12, respectively. Antithrombotic activity of LMWH was maintained longer than UFH. Significant correlation was found between the antithrombotic activity and a-Xa activity. Moreover, LMWH showed significantly less effect for prolongation of aPTT and BT than UFH. From these results LMWH was suggested to be more beneficial than UFH in clinical use. And this microvascular thrombosis model will contribute to the evaluation of the antithrombotic effects of various agents in vivo.
We developed a new thrombosis model using a microvasculature of hamster cheek pouch for the kinetic evaluation of thrombotic process in relation to endothelial damage. The thrombus was produced in ...the venule with excellent reproducibility by the irradiation of the filtered light with the intravascular administration of fluorescein sodium. The thrombotic process, assessed by two parameters such as Ti (initiation time of thrombus formation) and Ts (stopping time of blood flow by thrombus), was dependent on the light intensity and the dye concentration. In the initial stage of thrombus formation, cytoplasmic organella swelling, vacuole formation, luminal membrane rupture and swelling of the nuclear envelope were observed in endothelium. With progress of thrombus formation, these changes became more obvious and focal endothelial denudation was observed at the advanced stage. The thrombus was developed only on the irradiated vascular endothelium without exposure of basement membrane, which suggested that primary endotherial damage could lead to the thrombus formation. Superoxide dismutase (SOD), scavenger of oxygen radicals, was intravenously applied to evaluate the cytotoxic effects of oxygen radicals on endothelium. Continuous infusion of SOD of 5, 000 and 10, 000U/kg/min dose dependently prolonged Ti from 66.8±9.6 to 121.0±46.1 and 186.2±40.0sec, and Ts from 405.9±32.5 to 792.0±190.5 and 921.8±197.2sec (mean±SD, n=10), respectively. Furthermore, morphological changes of endothelium and platelet were markedly reduced by administration of SOD. From these data, oxygen radicals were suggested to cause the injury of endothelium and lead to thrombus formation.
Anticoagulant profile of low molecular weight heparinoids (KB-101) and its in vivo effects on the experimental DIC (disseminated intravascular coagulation) were investigated in comparison with ...unfractionated heparin (UFH). KB-101 displayed considerably less effects than UFH on the conventional laboratory tests as activated partial thromboplastin, prothrombin and thrombin time. Both of these agents catalyzed the inactivation of factor Xa by antithrombin III (AT III) and the inactivation of thrombin by AT III and heparin cofactor II (HC II). The potencies of KB-101 on the inactivation of these activated coagulation factors were far lesser than those of UFH. On the other hand, the anti-Xa to anti-thrombin activity ratio of KB-101 was about 28 times higher than that of UFH. Either KB-101 and UFH enhanced the antiplasmin activity in plasma, which was less prominent in KB-101 than UFH. The in vivo effects of these heparin on the endotoxin-induced experimental DIC using rats, were assessed by coagulation and fibrinolytic parameters and the degree of fibrin deposition in the renal glomeruli. The inhibitory effects of KB-101 on DIC were proved to be as equivalent to those of UFH. From these results it was concluded that KB-101 showed a higher anti-Xa to anti-thrombin activity ratio than UFH and revealed a potent inhibitory effects on experimental DIC, which suggested its therapeutic efficacy for DIC.
A neutrophil chemotactic factor (human interleukin 8, human granulocyte-macrophage colony-stimulating factor)-producing cell line, named KHM-5M, was established from a patient with an ...undifferentiated thyroid carcinoma, neutrophilia, and malignant pleurisy with many neutrophils and a few malignant cells. The cell line was transplanted into nude rats, and the infiltration of neutrophils was observed in and around the transplanted tumor tissue. Neutrophil chemotactic activity was predicted from the clinical features and pathological findings in this case. The extreme chemotactic activity of the neutrophils was demonstrated in conditioned medium from KHM-5M cells using the modified Boyden chamber technique. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least two neutrophil chemotactic activities in conditioned medium from the cell line were observed. The levels of these activities derived from KHM-5M cells were screened by measuring conditioned medium from the COS cells, which expressed a complementary DNA library from the KHM-5M cells. Chemotactic activities (human interleukin 8, human granulocyte-macrophage colony-stimulating factor) were identified by DNA cloning. These results show that the KHM-5M cells derived from an undifferentiated thyroid carcinoma produce multicytokines and suggest that those cytokines modified some pathological features in this case.
The effects of endothelin (ET) on the function of cultured human umbilical vein endothelial cells (HUVEC) and that of human platelets were investigated with reference to endothelium-derived relaxing ...factor (EDRF) and PGI2. Considering the platelets, ET had no effect on platelet-rich plasma (PRP) aggregation, the generation of thromboxane A2 (TXA2) from platelets, and cytosolic free calcium ion concentration (Ca++i), cAMP content (cAMPi) or cGMP con-tent (cGMPi) in platelets. In contrast, the addition of the solution in which HUVEC had been incubated with ET to PRP produced a decrease in PRP aggregation, TXA2, and Ca++i, and an increase not only in cAMPi but also in cGMPi in platelets. In the HUVEC pretreated with acetylsalicylic acid (aspirin), this increase of cGMPi Was not affected, but the HUVEC-mediated decrease in PRP aggregation, TXA2, and Ca++i induced by ET were not completely abolished. However, the pretreatment of HUVEC with a combination of aspirin and L-NG-monomethyl arginine (LNMMA) as an inhibitor of EDRF completely abolished the HUVEC-mediated decrease in PRP aggregation, TXA2 and Ca++i induced by ET, and also abolished the enhancement of cGMPi and cAMPi in platelets. The PGI2 Of HUVEC was enhanced by ET with no changes in Ca++i, cAMPi and cGMPi. The ET-induced enhancement was remarkably attenuated by pretreating the HUVEC with aspirin, but not with LNMMA. We conclude that ET attenuates the aggregation of platelets through a decrease in TXA2 by an increase in cAMPi Via the increase in PGI2 Of HUVEC, and by an increase in cGMPi Via EDRF.
We purified a protein possessing a potent ability to induce the differentiation of a murine megakaryoblastic cell line, L8057, from the supernatant of a human fibrous histiocytoma cell line, KHM-5M. ...The protein, a homodimer of a molecular weight of 25-kDa, has an N-terminal sequence identical with that of the beta A-chain of inhibin, indicating that it is identical to or highly homologous with activin A. Thus, it is speculated that activin A or its homologue is involved in megakaryocytic differentiation.
Dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells were transfected with expression plasmid constructed from human tissue-type plasminogen activator (t-PA) and DHFR gene. The ...transfected cells were selected with a medium containing methotrexate (MTX, 0-5μM), followed by isolation of several DHFR+ clones. It was definitely obserbed that the expression levels of t-PA were dose dependently elevated on the MTX resistance and the gene copy number. This recombinant t-PA had a similar specific activity and the same N-terminal amino acid sequence to human melanoma cell derived t-PA, and also showed similar characteristics in a neutralization test with a human t-PA, SDS-PAGE, PAS staining, zymography and human plasma clot lysis time assay. The MTX-selected high expression clone could keep constant production of t-PA without MTX for at shortest 5 months. These results strongly suggestted that CHO cells-DHFR gene amplification system was useful for producing a large amount of human glycoprotein with a complicated structure such as t-PA.
Recently the relationship between sulfhydryls and cGMP has been discussed in the several biological processes. While captopril, but not enalapril, is a sulfhydryl-containing angiotensin converting ...enzyme (ACE) inhibitor, we have previously reported that it decreased PGI2 generation in cultured human vascular endothelial cells. So we aimed to investigate the implication of cGMP and sulfhydryls in the regulatory mechanisms of PGI2 generation including with which induced by captopril with reference to Ca++ kinetics, Bradykinin and Ca ionophre A23187 enhanced PGI2 generation, and the cytosolic free Ca++ concentration (Ca++i). And 8-bromo cGMP increased intracellular cGMP concentration (cGMPi), and decreased PGI2 generation without change in Ca++i. Sulfhydryl radical containing compounds such as captopril, N-acetylcysteine and glutathione decreased PGI2 generation via the increased cGMPi. However an ACE inhibitor without sulfhydryl radicals, enalapril had no effect on them. Through these results it was suggested that enalapril might be more beneficial than captopril for the management of hypertensive patients with thromboembolic complications, and is was considered to be an important factor whether the vasoactive substances contain the sulfhydryls or not for the examinination of their effects on PGI2 generation.
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