Effects of lipoproteins from pre-eclamptic women on umbilical endothelial cell 6-oxo-prostaglandin f1alpha and endothelin 1 synthesis, and nitric oxide synthase 3 mRNA expression.

In order to evaluate whether lipid abnormalities may contribute to endothelial dysfunction in pre-eclampsia, the present study examined the in vitro effects of very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), isolated from women with pre-eclampsia and matched controls, on the endothelial synthesis of 6-oxo-prostaglandin F(1alpha) (6-oxo-PGF(1alpha); a metabolite of prostacyclin) and endothelin 1, and on the expression of nitric oxide synthase 3 (NOS3) mRNA. VLDL, LDL and HDL cholesterol were isolated from 20 pre-eclamptic and 20 age- and gestation-matched normal pregnant women. The lipoproteins (50 microgram/ml) and lipoprotein-free control plasma were incubated for 1, 3 and 6 h at 37 degrees C with a human umbilical endothelial cell line. The synthesis of 6-oxo-PGF(1alpha) and endothelin 1, and NOS3 mRNA expression, were measured at each time point. VLDL from pre-eclamptic women stimulated endothelial cell 6-oxo-PGF(1alpha) synthesis to a lesser extent than that from normal pregnant women (P<0.05). LDL from women with pre-eclampsia also stimulated 6-oxo-PGF(1alpha) synthesis to a lesser extent than LDL from normal pregnant women, but the effect was less sustained. The effect of HDL from women with pre-eclampsia on 6-oxo-PGF(1alpha) synthesis was similar to that of HDL from normal pregnant women. The pre-incubation levels of lipid peroxides in VLDL and LDL were not different between the normal pregnant and pre-eclamptic women, and cannot account for the decrease in 6-oxo-PGF(1alpha) synthesis. VLDL, LDL and HDL from women with pre-eclampsia did not affect endothelial cell synthesis of endothelin 1 or expression of NOS3 mRNA differently from lipoproteins from normal pregnant women. This study suggests that VLDL, and to a lesser extent LDL, from women with pre-eclampsia could potentially contribute to the reduced systemic 6-oxo-PGF(1alpha) synthesis observed in the pre-eclamptic syndrome.

ACE inhibition and fibroblast growth factor in cultured human vascular smooth muscle.

The effects of the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, and basic fibroblast growth factor (bFGF) on DNA synthesis and expression of ACE mRNA were examined in human vascular smooth muscle cells cultured from saphenous vein and internal mammary artery. DNA synthesis was estimated using 3H-thymidine uptake, and ACE mRNA was estimated by rt-PCR. Enalaprilat (0.125 microg/ml, 48 h) decreased 3H-thymidine uptake to 66+/-12% (SE) of the control without enalaprilat (p < 0.05). Basic FGF (10 ng/ml, 24 h) increased uptake by 41 +/- 12% (p < 0.05) while enalaprilat pretreatment (24 h) decreased uptake to 56 +/- 12% of this augmented value (p < 0.025). Basic FGF increased ACE mRNA, a process that was time dependent with an approximately 50% increase after 24 h exposure. Pre-exposure to enalaprilat (24 h) before bFGF reduced ACE mRNA to approximately 50% of that found in the presence of bFGF alone. The results indicate that ACE mRNA is present in human vascular smooth muscle cells and that exposure to an ACE inhibitor reduces DNA synthesis. Basic FGF stimulates DNA synthesis and ACE mRNA expression, and both of these effects are reduced by an ACE inhibitor. The results are consistent with the effects of bFGF being exerted through, or alternatively in concert with, angiotensin II. Further, they suggest that ACE inhibition can reduce the activity of the renin-angiotensin system by inhibiting the production of ACE, or at least the expression of ACE mRNA, in addition to producing enzyme inhibition at the ACE level.

Loss of heterozygosity in asbestos-induced mutations in a human mesothelioma cell line.

The relationship between occupational or environmental exposure to asbestos and the development of mesothelioma, typically after prolonged latency, has been accepted as one of cause and effect. Most studies have concluded that asbestos is not mutagenic to mammalian cells in vitro. We have studied the potential of crocidolite asbestos to induce mutations in a stable mesothelioma cell line, using a mutation assay that measures mutation at the autosomal HLA-A locus and permits clonal growth of mutant cells. The mesothelioma cell line chosen is more akin to the in vivo target cells of asbestos than human peripheral blood lymphocytes used in previous studies. Exposure of mesothelioma cells in culture to both 200 micrograms/ml and 50 micrograms/ml crocidolite for 72 hr did not result in a statistically significant difference in the mutation frequency (MF) in the HLA-A assay when compared to the spontaneous MF in these cells. Mutations in the mesothelioma cells were classified according to their molecular basis. Notwithstanding the lack of statistically significant change in overall MF, molecular analysis of mutants obtained following exposure of mesothelioma cells to crocidolite demonstrated a statistically significant increase in the class of mutations arising from loss of heterozygosity (LOH) events involving the selection locus (HLA-A) and more distal loci. Mutations following exposure to 200 micrograms/ml and 50 micrograms/ml crocidolite showed a greater frequency of LOH than did spontaneous mutants (P < 0.01 and P < 0.001, respectively). These results correlate with those obtained in an earlier study using lymphocytes. The mesothelioma cell-based assay may be useful in detecting the mutagenicity of other asbestiform fibers and man-made fibers.

Asbestos and erionite fibres can induce mutations in human lymphocytes that result in loss of heterozygosity.

Although asbestos and erionite are proven human carcinogens, most studies have concluded that these fibres are not mutagenic to mammalian cells in vitro. We have studied the potential of these fibres and chrysotile fibres to induce mutations in human peripheral lymphocytes, using a mutation assay that measures mutation at the autosomal HLA-A locus. Exposure of lymphocytes in culture to 400 micrograms/ml of crocidolite or erionite for 72 hr did not result in a statistically significant increase in the mutation frequency (MF) in the HLA-A assay, although a trend towards increased MF was observed. Exposure to 400 micrograms/ml chrysotile resulted in no increase in MF; however a significant increase was observed at 50 micrograms/ml. Mutations in somatic cells can be classified according to their molecular basis. Molecular analysis of mutants obtained following exposure of lymphocytes to crocidolite and erionite demonstrated a statistically significant increase in the class of mutations arising from loss-of-heterozygosity (LOH) events involving the selection locus (HLA-A) and more distal loci. Mutations following exposure to crocidolite and erionite showed a greater frequency of LOH than did spontaneous mutants (p < 0.02 and p < 0.005 respectively). Mutants following exposure to chrysotile did not display a significant difference in LOH when compared with spontaneous mutants. Thus, although an increase in overall mutation frequency following fibre exposure did not achieve statistical significance, the modest increase seen following exposure to erionite and crocidolite is translated into a highly significant change in those components of the spectrum of mutations which result in LOH.

Identification of an mRNA species which encodes a voltage-operated Ca2+ channel in rat liver mRNA.

cDNA which encodes part of the alpha 1-subunit of the rabbit skeletal muscle L-type voltage-operated Ca2+ channel (VOCC cDNA) was employed to search for the presence in whole liver and hepatocytes of poly (A+) RNA homologous to mRNA which encodes VOCCs. Such homologous mRNA would be a candidate for mRNA which encodes the putative hepatocyte receptor-activated Ca2+ inflow system (RACIS). Northern blot analysis showed that poly (A+) RNA prepared from intact liver tissue, but not hepatocytes, contained a poly (A+) RNA species comparable in size to that which encodes the alpha 1-subunit of the L-type VOCC. It is concluded (a) that hepatocytes do not possess VOCCs or that the levels of VOCC poly(A+) RNA in hepatocytes are too low to be detected by Northern analysis and (b) that another cell type present in liver tissue does possess a VOCC. In a low stringency screen of a rat liver cDNA library employing VOCC cDNA as a probe, seven positive cDNA clones were obtained. While regions of the 2.3 kb cDNA insert from one of these clones showed sequence similarities with regions of VOCC cDNA, the 2.3 kb sequence did not appear to encode a Ca2+ channel. The present results suggest that the approach of low stringency cDNA library screening is unlikely to allow isolation of RACIS cDNA.