Angiotensin II increases the release of endothelin-1 from human cultured endothelial cells but does not regulate its circulating levels.
We investigated the effect of angiotensin II on endothelin-1 secretion in vitro and in vivo. In vivo, angiotensin II was given intravenously to 23 essential hypertensive and 8 control subjects according to different protocols: Study A, 1.0 ng x min-1 x kg-1 and 3.0 ng x min-1 x kg-1 angiotensin II for 30 min each; Study B, 1.0 ng x min-1 x kg-1 and 3.0 ng x min-1 x kg-1 angiotensin II for 120 min each; Study C, 3.0 ng x min-1 x kg-1 angiotensin II for 30 min followed by a dose increment of 3.0 ng x min-1 x kg-1 every 30 min until mean blood pressure levels increased by 25 mmHg; Study D, 1.0 ng x min-1 x kg-1 followed by 3.0 ng x min-1 x kg-1 angiotensin II for 60 min each on two different NaCl diets (either 20 mmol NaCl/day or 220 mmol NaCl/day, both for 1 week). In all in vivo studies neither plasma nor urine endothelin-1 levels changed with angiotensin II infusion. In contrast, angiotensin II (10(-9), 10(-8), 10(-7) mol/l) stimulated endothelin-1 secretion from cultured human vascular endothelial cells derived from umbilical cord veins in a time- and dose-dependent manner. The in vitro angiotensin II effects were abolished by candesartan cilexetil, an inhibitor of the membrane-bound AT1 receptor, and also by actinomycin D, an RNA synthesis inhibitor, and cycloheximide, a protein synthesis inhibitor, indicating that endothelin-1 release depended on AT1 receptor subtype and de novo protein synthesis. Our findings indicate that angiotensin II regulates endothelin-1 release by cultured endothelial cells through an AT1 receptor-dependent pathway, but does not influence circulating endothelin-1 levels in vivo. (+info)
A technique for dual determination of cytotoxic and helper lymphocyte precursor frequency by a miniaturized dye release method.
Helper (HTLPf) and cytotoxic (CTLPf) lymphocyte precursor frequency assays are increasingly used in bone marrow stem cell and organ transplant compatibility testing. Current techniques require large cell numbers and radioisotopes. To improve the technique, we developed a miniaturized fluorescent read-out combined HTLPf/CTLPf limiting dilution assay. The assay requires only 5 x 10(6) stimulators, 2 x 10(6) responders and 0.24 x 10(6) target cells in Terasaki plates (40 microl/well). For the HTLPf, culture supernatants from each well were assayed for IL-2 production. The IL-2-dependent proliferation of the mouse 9.12 cell line was detected by a semi-automated fluorescent dye technique. After addition of rhIL-2 (recombinant human IL-2) on days 3 and 7, CTLPs were detected on day 10 by measuring the lysis of dye-labeled targets. Results were comparable to standard radioisotope-based techniques. The assay had a coefficient of variation of approximately 30%. The assay detected helper CD4 cells, pure cytotoxic CD8, helper CD8 cells and helper/cytotoxic CD8 cells. Discrimination was demonstrated between HLA-matched related and non-related pairs. The ease of testing and small cell numbers required should facilitate further evaluation of HTLPf and CTLPf for compatibility testing in unrelated donor transplantation and monitoring immune responses following adoptive transfer of lymphocytes. (+info)
Generation and characterization of aggrecanase. A soluble, cartilage-derived aggrecan-degrading activity.
A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix. (+info)
The cellular ecology of progressive neoplastic transformation: a clonal analysis.
A comparison was made of the competence for neoplastic transformation in three different sublines of NIH 3T3 cells and multiple clonal derivatives of each. Over 90% of the neoplastic foci produced by an uncloned transformed (t-SA') subline on a confluent background of nontransformed cells were of the dense, multilayered type, but about half of the t-SA' clones produced only light foci in assays without background. This asymmetry apparently arose from the failure of the light focus formers to register on a background of nontransformed cells. Comparison was made of the capacity for confluence-mediated transformation between uncloned parental cultures and their clonal derivatives by using two nontransformed sublines, one of which was highly sensitive and the other relatively refractory to confluence-mediated transformation. Transformation was more frequent in the clones than in the uncloned parental cultures for both sublines. This was dramatically so in the refractory subline, where the uncloned culture showed no overt sign of transformation in serially repeated assays but increasing numbers of its clones exhibited progressive transformation. The reason for the greater susceptibility of the pure clones is apparently the suppression of transformation among the diverse membership that makes up the uncloned parental culture. Progressive selection toward increasing degrees of transformation in confluent cultures plays a major role in the development of dense focus formers, but direct induction by the constraint of confluence may contribute by heritably damaging cells. In view of our finding of increased susceptibility to transformation in clonal versus uncloned populations, expansion of some clones at the expense of others during the aging process would contribute to the marked increase of cancer with age. (+info)
Micronucleus test using cultured new born rat astrocytes.
Micronuclei is induced in cytoplasm as a consequence of the formation of chromosomal fragments or remaining chromosomes during cell division by the cause of clastogens or spindle poisons, and is used as an indicator of genotoxicity screening tests. There are few short-term genotoxicity screening tests using brain cells. We attempted to establish a new in vitro micronucleus test (MN test) system by use of central nervous system cells. Primary cultured astrocytes were prepared from newborn male Sprague-Dawley (SD) rats. In growth curve of astrocytes, doubling time was determined to be 31 h. In time study, the highest frequency of micronuclei was observed at 48 h, 72 h and 6 h-exposure-66 h-recovery by vincristine (VCR), mitomycin C (MMC) without metabolic activation system and cyclophosphamide (CPM) with metabolic activation system, respectively. Dose-response relationships between micronucleus frequency and concentrations of MMC, VCR and CPM were observed, respectively. It is suggested that the in vitro MN test using new born rat-astrocytes could be used as a screening test of environmental and occupational genotoxic chemicals in the central nervous system cells. (+info)
In-vitro fertilization and culture of mouse embryos in vitro significantly retards the onset of insulin-like growth factor-II expression from the zygotic genome.
In this study, the effect of in-vitro fertilization (IVF) and culture of mouse embryos in vitro on the normal expression of insulin-like growth factor-II (IFG-II) ligand and receptor was examined. The expression of IGF-II increased in a linear fashion at least up to the 8-cell stage of development. IGF-II expression in embryos collected fresh from the reproductive tract was significantly (P < 0.001) greater than in embryos fertilized in the reproductive tract and cultured in vitro (in-situ fertilized: ISF), and its expression was further reduced (P < 0.001) in IVF embryos at all development stages tested. The expression of IGF-II was significantly (P < 0.001) lower when embryos were cultured individually in 100 microl drops compared with culture in groups of 10 in 10 microl drops of medium. The addition of platelet activating factor to culture medium partially overcame this density-dependent decline of expression. Culture of ISF and IVF zygotes also caused the onset of new IGF-II mRNA transcription from the zygotic genome to be significantly (P < 0.001) retarded, until at least the 8-cell stage of development. This effect was greater (P < 0.05) for IVF than for ISF embryos. Neither IVF nor culture had any obvious effect on IFG-II/mannose-6-phosphate receptor (IGF-IIr) mRNA expression. (+info)
Isolation and characterization of a new human breast cancer cell line, KPL-4, expressing the Erb B family receptors and interleukin-6.
A new human breast cancer cell line, KPL-4, was recently isolated from the malignant pleural effusion of a breast cancer patient with an inflammatory skin metastasis. This cell line can be cultured under serum-free conditions and is tumorigenic in female athymic nude mice. Flow cytometric analysis revealed the expression of Erb B-1, -2 and -3. Dot blot hybridization showed a 15-fold amplification of the erb B-2. Reverse transcription-polymerase chain reaction analysis showed a detectable level of mRNA expression of all the Erb B family receptors. In addition, all the receptors were autophosphorylated under a serum-supplemented condition. Unexpectedly, transplanted KPL-4 tumours induced cachexia of recipient mice. A high concentration of interleukin-6 (IL-6) was detected in both the culture medium and the serum of mice. The weight of tumours significantly correlated with the serum IL-6 level. The antiproliferative effect of a humanized anti-Erb B-2 monoclonal antibody, rhuMAbHER2, was investigated. This antibody significantly inhibited the growth of KPL-4 cells in vitro but modestly in vivo. Loss of mouse body weight was partly reversed by rhuMAbHER2. These findings suggest that KPL-4 cells may be useful in the development of new strategies against breast cancer overexpressing the Erb B family receptors and against IL-6-induced cachexia. (+info)
Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.
The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2 novel plaque cell lines exhibiting various features of plaque SMC biology; pdSMC2 may represent an earlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion. (+info)