Johan F. Jongkind

De novo synthesis of glutathione in human fibroblasts during in vitro ageing and in some metabolic diseases as measured by a flow cytometric method

A flow cytometric method to determine cellular GSH contents has been developed. This method is fast and simple and enables the determination of GSH contents in intact cells. Results obtained with the new method correlate well with the results obtained by a specific biochemical assay for GSH (r = 0.9984; n = 7). The method has been used to determine GSH recovery rates in cultured fibroblasts from healthy subjects and from patients with Werners syndrome, Spielmeyer-Vogt syndrome and Fanconis anemia. No obvious differences in GSH recovery rates were observed. GSH recovery rates were also not affected after in vitro ageing. Experiments with cells deficient in GSH synthetase revealed that the observed GSH recovery is exclusively due to de novo synthesis.

Reversible inhibition of DNA and protein synthesis by cumene hydroperoxide and 4-hydroxy-nonenal

To test the possible role of lipid peroxidation in the process of in vitro ageing, human diploid skin fibroblasts were cultured with the lipophilic hydroperoxide cumene hydroperoxide (Chp) or the breakdown product of lipid peroxidation 4-hydroxy-2,3-trans-nonenal (HNE). Both compounds inhibited cellular DNA and protein synthesis in a dose-dependent way. Cells exposed to Chp or to HNE during growth inhibition recovered DNA and protein synthesis within 24 h upon removal of Chp or HNE from the culture medium. Continuously proliferating cells showed only a partial recovery of DNA and protein synthesis. Pre-culturing cells with the lipophilic free radical scavenger vitamin E did not abolish the effect of Chp upon DNA synthesis. Cellular levels of reduced glutathione (GSH) rose slightly during 1 week of culture with HNE, but remained unaltered with Chp. Neither ATP levels nor cellular energy charges were affected during culture with Chp or HNE. So, DNA synthesis is not impaired due to a shortage of nucleotides nor does GSH protect DNA synthesis against the effects of Chp or HNE. These results suggest that oxygen free-radical induced lipid peroxidation is not the cause of the irreversible loss of proliferation occurring during in vitro ageing.

Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting: Morphology, enzyme activity and proliferative capacity

Abstract In cultures of human fibroblasts the percentage of bright autofluorescent (AF) cells increases with increasing passage number. These autofluorescent cells were isolated using a FACS II cell sorter and compared with sorted non-fluorescent (NF) cells. The AF cells showed an increase in population doubling time (2.3-fold), cell protein (1.9-fold), and in specific activities of the lysosomal enzymes: β-hexosaminidase (4.2-fold), β-galactosidase (3.8-fold) and acid phosphatase (2.5-fold). The specific activities of two non-lysosomal enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase had increased only slightly (1.1-fold) respectively (1.5-fold). The autofluorescence in the AF cells was restricted to small round organelles. The distribution and size of these autofluorescence granules were similar to the acid phosphatase-containing granules in the cytochemically stained cells. Electronmicroscopical examination showed that these AF cells contained a large amount of small electron-dense granules containing amorphosmophilic material. These granules which were positive for the acid phosphatase reaction, were classified as secondary lysosomes. The low percentage of the sorted AF cells which incorporate [ 3 H]thymidine during a 24 h test period (19%) as compared with the labelling percentage of sorted NF cells (73%) from the same culture, indicate that the autofluorescent cells in a ‘young’ culture have a very limited remaining proliferative capacity. The results imply, that by flow sorting it is possible to isolate ‘aged’ cells with characteristics of ‘phase III’ cells out of non-aged fibroblast cultures.

Glutathione metabolism of human vascular endothelial cells under peroxidative stress.

Glutathione (GSH) plays an important role in the cellular defense against (per-)oxidative stress. The capacity of this cellular defense system may be related to the oxygen tension, cells are normally subjected to in vivo; therefore, we studied the de novo synthesis of glutathione, and the redox turnover under peroxidative stress, in human umbilical vein and artery endothelial cells (HUVEC, HUAEC) and human skin fibroblasts. De novo synthesis in these cell types was studied in vitro by measuring the time course of intracellular GSH recovery after depletion with diamide. For fibroblasts, the initial rate of de novo synthesis after GSH depletion was twice that of the endothelial cell strains. In the endothelial cells (HUVEC, HUAEC) the original intracellular GSH level is reached within 40 min. while in the same time span, the GSH level in fibroblasts returned to 75% of control level. The activity of the hexose monophosphate shunt (HMS) was determined under oxidative stress as a measure for the coupled redox turnover of intracellular GSH. Under control conditions the HMS in endothelial cells was twice as high as in fibroblasts. Cumene hydroperoxide (40 microM) induced a three-fold increase in HMS in both HUVEC and HUAEC, while fibroblasts exhibited an increase of 83%. During the same peroxidative stress, the intracellular GSH concentration of HUVEC, HUAEC and fibroblasts stayed at control level. So with respect to GSH metabolism there were no differences between the two endothelial cell strains. In comparison with the endothelial cells, the fibroblasts were less susceptible toward oxidative stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme assays at the single cell level using a new type of microfluorimeter

SummaryA direct method for the microfluorimetric determination of NADPH2 and 4-methylumbelliferon in the sub-picomole range is described. By means of an inverted fluorescence microscope with epi-illumination it has been possible to measure low concentrations of these fluorophores in volumes down to 17 nl. The aqueous microvolumes to be measured are suspended in oil to prevent evaporation. Since the droplets are sunk on thin teflon foil, to attain a spherical form, they can be measured in a reproducible way. The excitation of the microdroplet takes place from below through the teflon foil, while the fluorescence emission follows the same route back to a photomultiplier. The possibilities of this method are illustrated with the biochemical measurements of glucose-6-phosphate dehydrogenase (G6PD) activity in single fibroblasts. It was possible to distinguish between normal and G6PD deficient cells on a single cell level. The microfluorimetric measurement of lysosomal enzymes with 4-methylumbelliferyl substrates is discussed.

Patients with combined hypercholesterolemia-hypertriglyceridemia show an increased monocyte-endothelial cell adhesion in vitro: triglyceride level as a major determinant.

Hypercholesterolemia (HC) is one of the primary risk factors for atherosclerosis. Patients with familial hypercholesterolemia (FH) or combined hypercholesterolemia-hypertriglycerinemia (CHH) are at risk to develop premature atherosclerosis. Animal models have revealed that diet-induced HC in vivo leads to an increased adhesion of monocytes to the endothelium of the vessel wall. Changes in the monocytes, endothelial cells, or serum components may lead to the increased monocyte adhesion that results in atherosclerotic plaque formation. In the present study, we investigated the binding of the monocyte in an in vitro system. Incubation of freshly isolated monocytes from CHH patients with cultured human umbilical vein endothelial cells (HUVEC) gave a significant 60% increase in monocyte adhesion when compared with monocytes from healthy subjects. No such increase was observed using monocytes from nontreated FH patients. These data suggest that CHH results in in vivo alterations of the monocytes that lead to an increased in vitro adhesion to HUVEC, and that an increased level of plasma triglycerides is the major determinant, since HC alone does not induce this alteration.

Characterization of oxygen-tolerant Chinese hamster ovary cells. II: Energy metabolism and antioxidant status

Further characteristics of an oxygen-tolerant variant of Chinese hamster ovary cells (CHO-99) capable of stable proliferation at 99% O2/1% CO2, and O2 level that is lethal to the parental line (CHO-20), are described. Previous work has revealed that CHO-99 cells have 2- to 4-fold increased activities of superoxide dismutases, catalase and glutathione peroxidase, and substantially increased relative volumes of mitochondria and peroxisomes. To document possible additional mechanisms of O2 tolerance we compared CHO-20 cells growing at 20% O2 (normoxia) and CHO-99 cells at 99% O2 (normobaric hyperoxia). We show the following: (1) the estimated total (oxidative and glycolytic) ATP production in CHO-99 cells was 36% decreased. ATP production through oxidative phosphorylation was 52% lower in CHO-99 cells, while the relative contribution from glycolysis was increased from 6% to 30%. The ATP content was 29% lower in CHO-99 cells, the adenylate energy charge being also significantly decreased, indicating that energy production through oxidative phosphorylation is compromised in CHO-99 cells. Cyanide-resistant respiration was 4-fold higher in CHO-99 cells, probably reflecting, at least partly, the increased peroxisomal activity in these cells. (2) The level of reduced glutathione was several fold increased in CHO-99 cells, oxidized glutathione being unaltered; (NADPH + NADP+) levels were elevated 2.7-fold, while the ratio of NADPH to NADP+ was increased almost two-fold. These changes were associated with a 50% increased metabolism of glucose through the hexose monophosphate pathway. (3) No evidence was obtained for an increased steady-state level of endogenous lipid peroxidation in CHO-99 cells, in spite of a 50% increased content of polyunsaturated fatty acids in the phospholipid fraction.

Hypertriglyceridemia Enhances Monocyte Binding to Endothelial Cells in NIDDM

OBJECTIVE The mechanisms by which diabetes leads to rapidly progressive atherosclerosis are not fully understood. Adherence of monocytes to the arterial wall is an early event in the development of atherosclerotic lesions. RESEARCH DESIGN AND METHODS The binding of freshly isolated monocytes from patients with NIDDM, IDDM, and healthy control subjects to a monolayer of endothelial cells obtained from human umbilical vein was investigated. RESULTS Endothelial adherence of monocytes from normolipidemic patients with IDDM (15.8 ± 4.5%) or NIDDM (16.9 ± 4.6%) was comparable to that of monocytes from a control population (15.3 ± 3.5%). In patients with NIDDM with a serum triglyceride concentration > 2.5 mmol/l, the percentage of cells that adhere to endothelial cells in vitro was significantly increased (23.3 ± 3.1%). Glycemic control did not correlate with monocyte adherence. The presence of symptomatic atherosclerotic disease, age, or sex was not associated with a change in monocyte binding in vitro. CONCLUSIONS The results suggest that in NIDDM hypertriglyceridemia should be treated to reduce the high risk for atherosclerosis.

Monocytes from patients with combined hypercholesterolemia-hypertriglyceridemia and isolated hypercholesterolemia show an increased adhesion to endothelial cells in vitro: II. Influence of intrinsic and extrinsic factors on monocyte binding

One of the primary risk factors for atherosclerosis is hypercholesterolemia. Patients with isolated hypercholesterolemia or combined hypercholesterolemia-hypertriglyceridemia are at risk to develop premature atherosclerosis. Diet-induced hypercholesterolemia in animals leads to an increased adhesion of monocytes to and transmigration through the intact endothelium of the vessel wall. In the present study, we investigated in vitro binding of freshly isolated monocytes from patients and healthy controls to a monolayer of endothelial cells obtained from human umbilical vein. All four diagnosed patient groups with isolated or combined hypercholesterolemia showed a significant increase in monocyte binding as compared with the control group (familial hypercholesterolemia [FH], +41%; polygenic hypercholesterolemia [PH] +35%; familial combined hypercholesterolemia [FCH], +47%; nonfamilial combined hypercholesterolemia-hypertriglyceridemia [CHH], +67%). In a longitudinal study it was observed that diet or medication induced a decrease in cholesterol and triglycerides; however, these therapeutic conditions did not diminish in vitro monocyte binding in the patient groups. There was no correlation between monocyte binding and plasma cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, or lipoprotein(a) within hyperlipidemic patient groups. The presence of heart and vessel disease in hyperlipidemic patients was not associated with a change in monocyte binding. The adhesion to endothelial cells of monocytes from smoking patients with combined hypercholesterolemia (27%) was significantly higher (+23%) than that of monocytes from nonsmoking patients. Cytofluorimetric analysis of monocytes from FCH and CHH patients for specific monocyte differentiation markers and integrins did not show differences as compared with monocytes from healthy controls.

Vascular Cells Under Peroxide Induced Oxidative Stress: A Balance Study on in Vitro Peroxide Handling by Vascular Endothelial and Smooth Muscle Cells

Enzymes such as glutathione peroxidase and catalase play an important role in the cellular defence against (per)oxidative stress. Balance- and inhibitor-studies were undertaken with in vitro cultured human vascular endothelial cells (EC) and smooth muscle cells (SMC) to assay the relative importance of these enzymes in the handling of cumene hydroperoxide (Chp) and hydrogen peroxide (H2O2). Low concentrations of Chp (up to 80 microM) could be removed to near completion within the first hour of incubation by stimulation of the hexose monophosphate shunt (HMS) of both cell types. The HMS activity reached a plateau upon incubation with higher concentrations of Chp (> 80 microM). The non-converted Chp in the higher concentrations could be detected quantitatively in the incubation solution. After inhibition of the glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), incubation with Chp (40 microM) did not result in a stimulation of the HMS activity. Moreover the added Chp could be recovered from the medium. So Chp is exclusively handled by the GSH-redox cycle. When low concentrations of H2O2 (up to 80 microM) were added to EC or SMC approximately 50% of the peroxide loss could not be accounted for. Inhibitor studies with aminotriazole proved that catalase was responsible for the handling of this unaccounted H2O2. In both ECs and SMCs at lower concentrations of H2O2 the GSH-redox cycle was as effective as catalase and at higher H2O2 concentrations the catalase pathway plays the major role.