Jens Fischer

Differential regulation of thrombospondin-1 and fibronectin by angiotensin II receptor subtypes in cultured endothelial cells

OBJECTIVES Angiotensin II (ANG II) can modulate cellular proliferation in various cell types via AT(1) and AT(2) receptors. In the present study, we investigated the effect of the angiotensin AT(1) and AT(2) receptors on DNA-synthesis as well as on the expression of the extracellular matrix (ECM) components, thrombospondin-1 (TSP-1) and fibronectin (FN) in endothelial cells (EC). METHODS The experiments were performed in microvascular EC derived from rat heart (CEC) and macrovascular EC derived from bovine aorta (BAEC). The experiments were performed in cells of the second and third passage and the expression of AT(1) and AT(2) receptors was verified by binding studies, Northern analysis or RT-PCR. Quiescent rat CEC and BAEC were stimulated to proliferate by the addition of 25 ng/ml bFGF, while ANG II (10(-7) M) and the selective ANG II receptor antagonists, Losartan (10(-5) M) and PD123177 (10(-6) M) or the AT(2) agonist, CGP42112A (10(-7) M) were added 16 h later. RESULTS ANG II induced a dose-dependent decrease of DNA-synthesis in BAEC measured by [3H]-thymidine incorporation. This inhibitory effect of ANG II was prevented by the addition of the AT(2) receptor antagonist PD123177 (10(-6) M), demonstrating, that the inhibition of DNA synthesis is mediated by the AT(2) receptor. In the presence of Losartan, stimulation of both, CEC and BAEC, with ANG II resulted in a marked increase of TSP-1 mRNA levels, which was maximal between 3 and 6 h in rat CEC and after 9 h in BAEC. In addition, TSP-1 was clearly induced by the AT(2) agonist CGP42112A. In contrast, blockade of the AT(2) receptor by the selective AT(2) antagonist, PD123177 (10(-6) M), resulted in a pronounced down regulation of FN mRNA 9 h after the stimulation. CONCLUSIONS The present results suggest that the ANG II receptor subtype AT(2) mediates growth inhibition in macrovascular EC similar to what has been shown before in microvascular rat EC and that AT(2) receptors mediates remodeling of the endothelial ECM by upregulation of TSP-1 expression in both macro- and micro-vascular endothelial cells.

Vascular smooth muscle cell phenotype influences glycosaminoglycan composition and growth effects of extracellular matrix

Rat neonatal and neointimal vascular smooth muscle cells differ dramatically from adult medical vascular smooth muscle cells in their growth properties, with the neonatal and neointimal cells exhibiting growth in the absence of exogenously added growth factors. Since it has been hypothesized that extra-cellular matrix proteoglycans may influence the growth and differentiation of vascular smooth muscle cells, we examined the ability of matrix derived from these cells to influence vascular smooth muscle cell proliferation. To produce test matrices, cells were grown to confluence and removed by brief alkali treatment. Test cells were seeded onto these matrices and the rates of growth in a growth-factor-deficient medium determined. Compared to plastic wells, matrix from neonatal or neointimal cells stimulated the growth of vascular smooth muscle cells. Interestingly, matrix from adult cells was less efficient at promoting growth. Enzymatic digestion of extracellular matrix heparan sulfate, but not of other glycosaminoglycans, further increased the growth-stimulatory effect of extracellular matrix, suggesting that matrix heparan sulfate acts as a growth inhibitor. Consistent with this, biochemical analysis showed that the adult matrix contained a higher percentage of heparan sulfate compared with neonatal or neointimal matrix. These results suggest that autocrine production of heparan sulfate proteoglycans may play an important role in growth regulation of vascular smooth muscle cells during normal vascular development and differentiation as well as in pathological response to injury.

Drug-induced lysosomal storage of sulphated glycosaminoglycans.

1. Certain compounds (e.g., the immunomodulator tilorone and congeners) are able to induce lysosomal storage of sulphated glycosaminoglycans (GAG), thus, producing cytological and biochemical alterations reminiscent of the inherited mucopolysaccharidoses. The drug-induced GAG storage has been studied in cultured fibroblasts of several species and in rats, and it is likely to occur also in humans. 2. The cytological hallmarks of GAG storage are enlarged lysosomes congested with material that is intensely stained by cationic dyes. With respect to fixation techniques, one has to keep in mind that the GAGs are highly water-soluble and are leached during conventional fixation and tissue processing. Biochemically, the elevation of GAG contents in tissues and cultured fibroblasts is due to storage of dermatan sulphate, predominantly. 3. The molecular structure of the potent inducers of GAG storage is characterized by a planar tricyclic aromatic ring system that is symmetrically substituted with two side chains of 4-5 sigma bond length, each carrying a protonizable nitrogen atom. The lysosomal storage of GAG is accompanied by lysosomal accumulation of the inducing drug, with the molar ratio of drug to GAG-disaccharide unit amounting to > 1:1. The reversibility of GAG storage is rather slow. 4. The pathogenic mechanisms underlying the drug side effects are discussed and the following hypothesis is put forward: The compounds in question are lysosomotropic weak bases. They get trapped in the acidic lysosomes and accumulate highly there. Physicochemical data suggest that the drugs form complexes with the sulphated GAGs, particularly with dermatan sulphate: The positively charged nitrogen atoms of the drug side chains interact with the negative charges of sulphate and carboxy groups of the GAGs, thereby crosslinking at least two GAG helices. Moreover, the interlinking drug molecules form parallel stacks resulting from interaction of the aromatic pi-electrons of the planar ring systems. This further stabilizes the complexes. The GAGs within the complexes are thought to be resistant to the degrading lysosomal enzymes. 5. Drug-induced GAG storage has not been directly demonstrated in man. Yet, clinical reports on keratopathy and basophilic cytoplasmic inclusions in blood lymphocytes of tilorone-treated patients suggest that this drug side effect may also occur in man.

Drug-induced glycosaminoglycan storage: Dose-dependent changes in the pattern of accumulated glycosaminoglycans in cultured bovine and human fibroblasts

The present study determines the amounts and patterns of glycosaminoglycans stored in cultured corneal fibroblasts after treatment with tilorone and three related compounds. The compounds have immunomodulatory properties and have been shown to impair the lysosomal degradation of glycosaminoglycans as a side effect. This side effect has been described as drug-induced mucopolysaccharidosis because the induced lysosomal storage of glycosaminoglycans leads to cellular lesions resembling those in patients with inherited mucopolysaccharidosis. In the present study, the dose-dependency of glycosaminoglycan storage was analyzed after treatment (96 hr) of bovine corneal fibroblasts. The investigated drug concentrations ranged from low concentrations inducing cytological lesions typical of drug-induced mucopolysaccharidosis to high concentrations at the borderline of cytotoxicity. The intracellular amounts of dermatan sulfate, heparan suflate, and chondroitin sulfate were quantified by densitometric scanning of Alcian Blue-stained bands after electrophoresis. All investigated compounds induced a predominant dermatan sulfate storage (3-4-fold accumulation) at low drug concentrations. With rising drug concentrations, a shift of the pattern of stored glycosaminoglycans was observed, characterized by the additional accumulation of heparan sulfate (up to 5-fold of control levels). In cultured human fibroblasts, tilorone also caused a marked dermatan sulfate storage, reaching maximum values at 5 microM and marked heparan sulfate storage at 20 microM. The present data provide evidence: (a) that selective dermatan sulfate accumulation is a characteristic feature of drug-induced glycosaminoglycan storage in cultured bovine and human fibroblasts, if these cells are treated with low concentrations (< or = 5 microM), that are likely to reflect the situation in vivo; and (b) that additional heparan sulfate storage is induced in vitro only by treatment with high concentrations that induce nonspecific cellular lesions.

Lysosomal glycosaminoglycan storage as induced by dicationic amphiphilic drugs: Investigation into the mechanisms underlying the slow reversibility

Several dicationic amphiphilic compounds, such as the immunomodulator tilorone and analogues, impair the lysosomal catabolism of sulphated glycosaminoglycans (GAGs). Thereby they cause lysosomal GAG storage in rats and in cultured fibroblasts of several species including man. The GAG storage is rather slowly reversible in vivo; it persists for months after discontinuance of drug treatment. In the present study, we investigated the mechanisms underlying the slow reversibility. Cultured bovine corneal fibroblasts were pretreated for 4 days with tilorone (5 and 20 microM) or with compound CL-90.100 (3 and 10 microM) and further cultured in drug-free medium for periods up to 11 days. The intracellular GAG storage was analysed biochemically and demonstrated histochemically. The subcellular drug distribution (CL-90.100) was demonstrated by fluorescence microscopy. Dermatan sulphate (DS) provided the predominant contribution towards the GAG storage. After pretreatments with the low, as well as the high concentrations of either drug, the storage of DS was irreversible during the period of observation, whereas the minor storage of heparan sulphate was resolved. The enhanced secretion of the lysosomal enzyme beta-hexosaminidase (E.C. 3.2.1.52) caused by pretreatment with the high concentration of tilorone was also readily reversible. Thus, enzyme deprivation could not be the explanation for the sustained DS storage. The localization of the drug-related fluorescence within perinuclear cell organelles, presumably lysosomes, resembled that of the stored GAGs as visualized by histochemical staining. Both, the fluorescence and the positive GAG staining persisted with unchanged intracellular distribution throughout the recovery period. The present results suggest that the persistence of the DS storage is due to the formation of long-lived, non-degradable DS-drug complexes within the lysosomes.

Lysosomal storage of sulfated glycosaminoglycans induced by two bis-aminomethyl anthrachinones.

Abstract Several immunomodulatory drugs, all of them symmetrically substituted dicationic amphiphilic compounds, are known to cause lysosomal storage of sulfated glycosaminoglycans (GAGs) in intact animals and cultured fibroblasts. The storage is due to impaired GAG degradation. The standard compound is tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one). In the present study two bis-aminomethyl anthrachinones were examined for their ability to induce lysosomal GAG storage in cultured bovine corneal fibroblasts. For reference, a bis-aminoethoxy-anthrachinone compound (RMI-10.024) was included, which is known to be a potent inducer of lysosomal GAG storage. The present morphological, radiochemical, and biochemical results show that the bis-aminomethyl anthrachinone compounds investigated cause lysosomal storage of GAGs, although with significantly lower potencies than the bis-aminoethoxy anthrachinone. Dermatan sulfate contributed approximately 90% to the drug-induced increment of intracellular GAGs. The present results suggest that the length of the side chains, i. e., the distance between the aromatic ring system and the protonizable nitrogen of the side chains, and the position of the side chains relative to the aromatic ring system are important molecular features influencing the potency of inducing lysosomal GAG storage.

Time course of the tilorone-induced lysosomal accumulation of sulphated glycosaminoglycans in cultured fibroblasts

Dicationic amphiphilic drugs such as the immunomodulator tilorone [2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one] are accumulated in lysosomes and disturb the degradation of sulphated glycosaminoglycans (GAGs) thus leading to generalized lysosomal GAG storage (mainly dermatan sulphate) in vivo and in cultured cells. In the present study, the time course of the tilorone-induced GAG storage was determined in cultured bovine corneal fibroblasts by a radiochemical approach and by morphological examination. In contrast to the rapid lysosomal accumulation of the drug as reported previously, it took approximately 42 h to reach 50% of the GAG storage obtained after 96 h. This is thought to be due to the fact that the temporal development of storage of undigested GAGs depends on the natural delivery of GAGs towards the lysosomal apparatus.