Indirect-immunofluorescence studies were performed on cultured dermal fibroblasts from patients with Pseudoxanthoma Elasticum (PXE), an inherited connective-tissue disorder the pathogenesis of which is still unknown. Apparent abnormalities of cytoskeletal structures were revealed by using phalloidin and specific antibodies to alfa-smooth muscle actin and to vimentin. Altered expression of integrin receptors for different extracellular matrix components seems to be present in the pathological cells as preliminary data suggest by using antibodies against alfa subunits of integrins. This study was designed to test the presence of abnormal cell-matrix interactions responsible for the clinical features and involved in the pathogenesis of the PXE disease.

Some glycosaminoglycans (GAGs), such as heparin and heparin-like compounds inhibit the proliferation of several cell types, including smooth muscle cells, cervical epithelial cells and fibroblasts (1-3). In order to establish which domain of the hepar-in molecule is specifically responsible for the anti-proliferative activity, several strategies have been adopted such as: chemical modification or fractionation of the heparin molecule into low molecular weight fragments or synthesis of oligosaccharides with a defined chemical structure (4-6).In the present study we attempted to determine the role of N- and 0- linked sulfate groups on the anti-proliferative and anticoagulant effect of heparin. To that purpose we modified the molecule to produce N-desulfated, 0-desulfated compounds. The anti-proliferative activity of these modified heparins was compared to that of low molecular weight heparins obtained by depolymerization, heparan sulfate as N-acetylated compound with glucuronic acid and heparin. Since the anti-proliferative effect of heparin depends also on the cell type, we used two different cell types: BHK-21 (hamster fibroblasts) and human arterial SMC (smooth muscle cells), that were found to exhibit a high or intermediate sensitivity to heparin (3,7).

A group of cytotoxicity tests that detect alterations in cell metabolism were applied to obtain a preliminary classification of test chemicals, based on their main mechanism of toxicity. V79 cells were exposed to toxic compounds in 'acute' treatments (up to 2 hr) and the specificity of different endpoints of cytotoxicity was compared. We tested five directly acting chemicals: butylated hydroxyanisole, butylated hydroxytoluene, cycloheximide, potassium dichromate [Cr(VI)] and dinitrophenol using four assays measuring; [(3)H]thymidine uptake and incorporation into DNA, [(3)H]leucine incorporation into proteins, ATP pool size and cellular energy charge, and oxygen consumption. In the thymidine-uptake test the radioactivity of the nucleotide pool was hardly affected by low concentrations of the toxic chemicals and was reduced by about 20-30% at the 10(-4)m concentration, except by butylated hydroxytoluene; the latter which caused a marked inhibition of [(3)H]thymidine uptake. Thymidine incorporation into DNA was more specifically altered, showing a net inhibition by potassium dichromate, in a concentration-dependent fashion, and by butylated hydroxytoluene. Protein synthesis was more inhibited by potassium dichromate and butylated hydroxytoluene than by the specific inhibitor, cycloheximide. All chemicals reduced ATP concentration, but the energy charge was scarcely affected, reflecting a parallel decrease of the other adenine nucleotides. The assay measuring early changes in oxygen consumption produced the expected results with dinitrophenol, the antioxidants and potassium dichromate, and showed that cycloheximide also inhibits mitochondrial respiration, in agreement with the observed fall of intracellular ATP. All the chemicals tested induced a range of effects on the metabolic parameters analysed but specific pathways of toxicity may be inferred by comparing the results of the individual tests.