Interaction of heparan sulfate and its fractions with endothelial cells in culture.

In the present work we have investigated the binding capacity, to cell membrane of bovine aortic endothelial cells (BAEC) cultured in vitro, of heparan sulfate (M(r) 11.1 kDa) and its four fractions named A (M(r) 24.5 kDa), B (M(r) 10.7 kDa), C (M(r) 9.1 kDa) and D (M(r) 6.8 kDa). Experiments were performed in comparison with unfractionated heparin (M(r) 12.9 kDa). The binding of the examined compounds was determined by the degree of displacement of [3H]-heparin from the cell membrane. The efficiency of heparan sulfate fractions B, C and D in displacing labelled heparin was found to decrease in function with the decrease of the molecular masses of the compounds. [3H]-heparin could be efficiently displaced mainly by heparin and fraction A, the fraction which has the highest molecular weight and represents only 3.8% of the unfractionated HA 90681. These experimental results may confirm that molecular mass is an important feature for the interaction of glycosaminoglycans with the specific binding sites on the cell surface.

Heparan sulfate fractions: pharmacological properties and effect on smooth muscle cell proliferation in vitro.

In this study we have evaluated the effect of heparan sulfate fractions with different molecular weights on the growth of aortic smooth muscle cells (SMC) of rat in vitro. These fractions were obtained from unfractionated heparan sulfate by a gel filtration technique. The fraction having a molecular weight of 25.5 kDa showed a significant antiproliferative activity only at higher concentrations, while the fractions with molecular weight ranging from 10.7 kDa to 6.8 kDa were effective in reducing cell proliferation at very low concentrations (10(-5) nM and 10(-4) nM). The dose-effect curves are characterized by an initial plateau followed by an increase in effectiveness at the highest concentration. The relationship between molecular weight and antiproliferative activity is discussed.

Effect of heparin derived fractions on the proliferation and protein synthesis of cells in culture.

We investigated the effect of sulfated oligosaccharides derived from depolymerization of heparin on the proliferation and protein synthesis of smooth muscle cells (SMC), hamster kidney (BHK-21) and lung (V-79) fibroblasts, rat hepatoma cells (FAO) and human promyelocytes (HL-60). BHK-21 and FAO showed the highest sensitivity to heparin; V-79 and HL-60 cells were completely resistant. LMWH (Low Molecular Weight Heparin) (MW 4.5 kD) was as effective as unfractionated heparin in reducing cell proliferation. The oligo-derivative 381/1 (MW 2 kD) was effective only on FAO and BHK-21 cells; oligo-derivative 381/2 (MW 1KD) had a negligible effect. The anti-proliferative effect was associated with an increased secretion of some protein classes. This effect was not present in heparin-resistant cells. In conclusion when the molecular size of heparin derivative is reduced below 2 kD (i.e. the size of a hexasaccharide) the anti-proliferative activity decreases dramatically.

Choice and standardization of test protocols in cytotoxicology: A multicentre approach.

A major problem that interferes with the introduction of in vitro tests for toxicological risk assessment is that of defining reliable standardized protocols. This issue was approached in the present study with an interlaboratory comparison of three cytotoxicity assays detecting chemical toxicity as impairment of cell viability in confluent cultures, reduction of colony forming ability, and inhibition of cell proliferation over 3 days of treatment. The study was performed using V79 cells, which are unable to activate indirectly-acting xenobiotics, and six chemicals with different mechanisms of action: two antioxidants (butylated hydroxyanisole and butylated hydroxytoluene), an inhibitor of protein synthesis (cycloheximide), an alkylating agent requiring metabolic activation (cyclophosphamide), an uncoupler of oxidative phosphorylation (dinitrophenol), and a genotoxic metal salt (potassium dichromate). The three tests produced the same rank of relative toxic potency for the tested chemicals, based on LC(50) values. The cell viability test appeared to be the most suited for the screening of unknown chemicals, given its simplicity and better reproducibility.

Effect of heparin-like compounds on the in vitro proliferation and protein synthesis of various cell types.

Previous studies have shown that heparin and heparin-like compounds inhibit the proliferation of arterial smooth muscle cells (SMC) both in vivo and in vitro. This anti-proliferative effect seems to be exerted almost exclusively on arterial SMC and related cell types. In the present study the effect of heparin (HTh) is compared with that of two sulfated glycosaminoglycans with low anticoagulant activity, sulodexide (SDX) and low molecular weight heparin (OP/LMWH) on cell proliferation and protein synthesis of 3 cell types: human arterial smooth muscle cells (SMC), fibroblast-like cells (BHK-21) and epithelial cells (rat hepatoma cells, FAO). HTh, SDX and OP/LMWH (5-100 micrograms/ml) are equally effective in reducing the proliferation of human arterial SMC. This inhibition is dose dependent and reversible. BHK-21 and FAO cells are even more sensitive than SMC to heparin-like compounds. For example 1 microgram/ml of heparin-like compounds is sufficient to produce 40-60% inhibition of FAO cell proliferation. In all types of cells HTh, SDX and OP/LMWH do not reduce the incorporation of 35S-methionine into cellular and medium proteins; they increase the radioactivity incorporated into some proteins secreted into the medium. In the case of SMC this effect is dependent on the concentration and the length of exposure to heparin-like compounds. These findings demonstrate that several cell types are sensitive to the anti-proliferative effect of heparin-like compounds.

Modulation of the synthesis of apolipoproteins in rat hepatoma cells.

The present study was designed to investigate whether plasma lipoproteins and albumin can affect the basal synthetic rate of apolipoproteins in differentiated rat hepatoma cells (Fao) incubated in serum-free medium. The synthesis of apolipoproteins was measured by the incorporation of [35S]methionine into medium lipoproteins isolated by density gradient ultracentrifugation. Under all the experimental conditions used, Fao cells synthesized almost exclusively apolipoprotein E. When cells were incubated in the presence of 5-10% rat plasma the synthesis of apolipoprotein E increased 2-3-fold; lipoprotein-deficient serum had a negligible effect. Fatty acid-poor bovine serum albumin (BSA), which had been found to reduce very-low-density lipoprotein secretion in isolated rat hepatocytes, did not modify the synthesis of apolipoprotein E. When Fao cells were incubated in medium containing rat plasma lipoprotein fractions, the synthesis of apolipoprotein E increased. The d less than 1.090 g/ml plasma lipoprotein fraction had the major stimulatory effect. Increased apolipoprotein E synthesis was observed when cells were incubated in the presence of lipids extracted from rat plasma lipoproteins. These results suggest that the intracellular accumulation of lipoprotein-lipids plays an important role in regulating apolipoprotein E synthesis in Fao cells.

Pseudoxanthoma elasticum (PXE): ultrastructural and biochemical study on proteoglycan and proteoglycan-associated material produced by skin fibroblasts in vitro.

Pseudoxanthoma elasticum is a genetic disease characterized by progressive mineralization of elastic fibers. Previous studies suggested that other components, apart from elastin, might be involved in the alterations of this connective tissue disorder (Martinez-Hernandez and Huffer, 1974; Pasquali Ronchetti et al., 1981; 1986). Evidence is presented that proteoglycan metabolism is altered in PXE-affected patient. Urinary GAGs suggests an increased degradation of glucosamine-containing GAGs in the patient. Pulse and chase experiments on in vitro skin fibroblasts indicated a decreased rate of synthesis of [35SO4] containing GAGs or an increase of their turnover rate in PXE. Moreover, when PGs produced from skin fibroblasts were identified by ultracentrifugation and gel filtration in associative conditions, PXE fibroblasts produced a significantly higher amount of the high molecular weight fraction of sulfated PGs. This high molecular weight material was present both in the medium and in the matrix and disappeared under dissociative conditions or after treatment with hyaluronidase or with pancreas elastase. By electron microscopy, PXE fibroblasts appeared to produce and secrete an enormous amount of toluidine blue 0 positive material organized as filaments and amorphous masses. These data are in agreement with previous observations of the presence of abnormal masses of microfilaments, in the dermis of PXE patients, which were sensitive to hyaluronidase and partially to trypsin and elastase (Pasquali Ronchetti et al., 1986). The results seem to confirm that at least some of the alterations of connective tissues in PXE are due to abnormal PGs metabolism and to their tendency to form abnormal aggregates in the extracellular space.