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Background Nitric oxide (NO) – a major signalling molecule of the vascular system – is constitutively produced in endothelial cells (EC) by the endothelial NO synthase (eNOS). Since a reduced NO synthesis is an early sign of endothelial dysfunction and NO delivering drugs are used to substitute the impaired endothelial NO production, we addressed the effect of exogenous NO on eNOS in human umbilical venous endothelial cell cultures. Materials and methods The synthetic NO donor DETA/NO (trade name, but in the following we refer to detNO), that releases NO in a strictly first order reaction with a half life of 20 h, was used in our experiments. Results Short-term (20–30 min) detNO treatment of EC increases the Ser 1177 phosphorylation of the constitutively expressed endothelial NOS and the production of endogenous NO generated by eNOS from [ 3 H]arginine. The phosphorylation of eNOS is Akt-dependent and completely reverted by the phosphatidylinositol3 kinase (PI-3K) inhibitor LY294002. A prolonged continuous exposure of EC to detNO 150 μmol L –1 over a period of 24–48 h causes a reversible cell cycle arrest at G1-phase associated with a larger cell volume and increased cell protein content (hypertrophic phenotype of EC). The eNOS protein and mRNA of the hypertrophic cells and the generation of endogenous NO are reduced but eNOS phosphorylation could still be elevated by stimulation with vascular endothelial growth factor. Conclusions Our data explain clinical studies describing a short-term but not a long-term benefit of NO treatment for patients with cardiovascular risk factors. The results could be a rational approach to develop a generation of NO donors accomplishing a retarded release from NO donors that mimic the low continuous pulsatile stressinduced release of endogenous NO.

Ca2+-induced deposition of low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL) at proteoheparan sulfate-modified surfaces was investigated as a function of beta-amyloid (Abeta) presence and apolipoprotein E isoform. Presence of beta-amyloid resulted in an increased deposition, as did the E4/E4 isoform compared to the corresponding E3/E3 isoform. The results are compatible with findings reported in literature on plaque formation in Alzheimer's disease, and suggest that, although simplistic, the present model system may have some potential in biosensor studies of Alzheimer plaque formation.

Glycosaminoglycans are regular constituents of the arterial wall and essential for its structure and function. The arteriosclerosis-dependent changes of glycosaminoglycans were investigated, the degree of arteriosclerosis was monitored by the cholesterol content of the tissue. Histological characterization was achieved by electron microscopy. Total glycosaminoglycans were isolated from 33 delipidated segments of human aorta thoracica after exhaustive proteolytic digestion, and fractionated into the individual glycosaminoglycans by a multistep purification procedure. Chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and hyaluronate (HA) were identified and quantified by chemical and enzymatic analysis. The concentration of total and individual glycosaminoglycans, expressed as mg/g delipidated dry weight of tissue, decreased significantly with increasing cholesterol content of tissue (p = 0.0005-0.005). The extent of decrease differed between the individual glycosaminoglycans as indicated by a shift in the CS/DS:HA:HS ratio from 47:32:21 in low cholesterol aortic segments to 59:29:12 in cholesterol-rich specimens. Determination of the relative molecular masses (Mr) revealed 58 kDa for CS/DS and 92 kDa for HS with a (statistically not significant) increase of the molecular mass of CS/DS and a decrease of HS with increasing cholesterol content. The copolymeric CS/DS glycosaminoglycans were disintegrated enzymatically into CS and DS containing fragments. A significantly higher relative DS content (p = 0.01) was found in cholesterol-rich arterial tissue (32.5%) as compared with low cholesterol tissue samples (28.8%). Cell culture experiments revealed that human arterial HS is able to inhibit the proliferation of cultured human arterial smooth muscle cells. The HS concentration required for a 30% inhibition of smooth muscle cell proliferation was in the same order as the tissue concentration of HS. This confirms the function of HS as an endogenous inhibitor of cell division and its impact for the development of atherosclerosis.

In the vessel walls, the connective tissue components surrounding the smooth muscle cells display a high binding capacity for small cations which has been ascribed to the presence of polyanionic proteoglycans. 23Na+ and 39K+ NMR measurements indicate that, while monovalent cations exert merely competitive interaction, divalent cations induce a conformational transition changing specifically the affinities for other ion species. Mg2+ or Ca2+ ions cause such a configurational change in the physiologic concentration range which promotes allosteric, cooperative binding of K+ or Na+ ions, respectively, to vascular connective tissue. The geometry of narrow tissue clefts in the interstitial compartment of the vessel wall entails that an increase of extracellular Mg2+ (Ca2+) concentration effects a decrease of external K+ (Na+) concentration in the vicinity of vascular smooth muscle cell membranes. Membrane hyperpolarization and vasorelaxation is the result. Proteoglycans are viscoelastic, anionic biopolyelectrolytes which consist of many highly sulphated and carboxylated glycosaminoglycan chains that are covalently attached to the protein core. The physicochemical and functional properties of the proteoglycans are due to the fact that in solution they are strongly hydrated anionic macromolecules. With an external strain, such a compound can go from a randomly coiled to an oriented state. The intensity of the blood flow can cause such a conformational transition of proteoheparan sulphate, integrated in the membrane of smooth muscle and endothelial cells. This conformational change is combined with an increase in binding sites for Na+ ions. When flow is reduced, inner molecular, elastic recoil forces guarantee entropic coiling. Sodium ions are released into the solution. Heparan sulphate shows Ca2+-dependent conformational transitions. These transitions are correlated with an increased or decreased local mobility of the molecular chains as the correlation times prove. Between 1 and 2.5 mmol/l [Ca2+]o, not only are Ca2+ ions bound but Na+ ions are also bound in an allosteric, cooperative manner. Above 2.5 mmol/l [Ca2+]o, there is a competitive expulsion of Na+ ions from their binding sites on the heparan sulphate by Ca2+ ions. These findings support the hypothesis that proteoglycans, integrated in the cell membrane, fulfill the mechano-chemical requirements of a flow sensor.

Cultured arterial smooth muscle cells synthesize two proteoheparan sulfate species. One is found associated with the cells, whereas the other is excreted into the medium. The two proteoheparan sulfates have similar hydrodynamic sizes but differ in the M r of their core proteins. The cell-associated proteoheparan sulfate has a M r of 92,000 while that of soluble proteoheparan sulfate is 38,000. The cell-associated and the soluble proteoheparan sulfate species differ in their ability to suppress the proliferation of smooth muscle cells. When added to the culture medium 2–5 μg/ml of the cell-associated and 20–25 μg/ml of the soluble proteoheparan sulfate species inhibit the growth of smooth muscle cells half maximally. The antiproliferative potency of both species resides in the heparan sulfate chains. Commercially available heparin has no antiproliferative effect and is not able to prevent the antiproliferative action of cellular heparan sulfate. In contrast to heparin, none of the heparan sulfate preparations has anticoagulant activity. Smooth muscle cells endocytose the soluble heparan sulfate at a rate three to four times higher than that of the cell-associated heparan sulfate. The data suggest that the cell-associated and the soluble proteoheparan sulfate species are separate and possibly genetically distinct molecules. Furthermore, the structural determinants for antiproliferative activity and the recognition sites for endocytotic uptake appear to be different.

Syndecans are transmembranous heparan sulfate proteoglycans abundant in the surface of all adherent mammalian cells and involved in vital cellular functions. In this study, we found syndecan-1, -2, -3, and -4 to be constitutively expressed by human umbilical vein endothelial cells. The exposure of the ectodomains of syndecan-1 and -4 to the cell surface and their constitutive shedding into the extracellular compartment was measured by immunoassays. In the presence of plasmin and thrombin, shedding was accelerated and monitored by detection and identification of (35)S-labeled proteoglycans. To elucidate the cleavage site of the syndecan ectodomains, we used a cell-free in vitro system with enzyme and substrate as the only reactants. For this purpose, we constructed recombinant fusion proteins of the syndecan-1 and -4 ectodomain together with maltose-binding protein and enhanced yellow fluorescent protein as reporter proteins attached to the N and C termini via oligopeptide linkers. After protease treatment of the fusion proteins, the electrophoretically resolved split products were sequenced and cleavage sites of the ectodomain were identified. Plasmin generated cleavage sites at Lys(114) downward arrowArg(115) and Lys(129) downward arrowVal(130) in the ectodomain of syndecan-4. In thrombin proteolysates of the syndecan-4 ectodomain, the cleavage site Lys(114) downward arrowArg(115) was also identified. The cleavage sites for plasmin and thrombin within the syndecan-4 ectodomain were not present in the syndecan-1 ectodomain. Cleavage of the syndecan-1 fusion protein by thrombin occurred only at a control cleavage site (Arg downward arrowGly) introduced into the linker region connecting the ectodomain with the enhanced yellow fluorescent protein. Because both plasmin and thrombin are involved in thrombogenic and thrombolytic processes in the course of the pathogenesis of arteriosclerosis, the detachment of heparan sulfate-bearing ectodomains could be relevant for the development of arteriosclerotic plaques and recruitment of mononuclear blood cells to the plaque.

Inhibitors of HMG-CoA reductase (statins) reveal important pharmacological effects in addition to reducing the plasma LDL cholesterol level. In the pathogenesis of arteriosclerosis, transendothelial migration of various leukocytes including monocytes is a crucial step. We, therefore, investigated the expression of E -selectin, intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in vascular endothelial cells as influenced by lovastatin. Human umbilical vein endothelial cells (HUVECs) express significant amounts of selectins and cell adhesion molecules (CAMs) within a few hours after stimulation with TNF-α. This effect is potentiated by 100–200% when the cells are pretreated with 0.1–2.5 μM lovastatin. The lovastatin-mediated increase in the cytoplasm and at the cell surface is dose-dependent and significant at lovastatin concentrations comparable to plasma levels in patients under lovastatin treatment. The lovastatin-potentiated increase of E -selectin and CAMs is correlated with a corresponding increase of selectin- and CAM-specific mRNA. We conclude that, in vivo, statin treatment could trigger an enhanced recruitment of macrophages that might support the cholesteryl ester efflux from the arteriosclerotic plaque.

Hypertrophy of vascular smooth muscle cells occurs during hypertension-induced remodelling of arteries and during development of arteriosclerosis and restenosis following angioplasty but the pathogenesis of the hypertrophic status is not yet fully understood. In a previous study we demonstrated that the synthetic non-sulfated, non-toxic heparin-mimicking compound RG-13577 is capable of inducing a cell cycle-arrested hypertrophic phenotype of coronary smooth muscle cells. In this study we clarify the mode of action of RG-13577 and demonstrate that the RG-13577-induced hypertrophy is associated with an increased expression of TGF-beta1 as indicated by an increase in TGF-beta1-specific protein and mRNA level. Furthermore we show that RG-13577-treated hypertrophic smooth muscle cells maintain full metabolic activity as indicated by a continuous de novo synthesis of protein and proteoglycans and that the RG-13577-induced growth arrest is caused not only by a higher expression of TGF-beta, but also by a reduced response of RG-treated cells to the mitogenic activity of bFGF, PDGF and EGF. The growth inhibitory activity of RG-13577 is reduced in the presence of neutralizing antibodies against TGF-beta. TGF-beta itself has anti-proliferative activity in serum-depleted medium. The RG-13577 effect is reversible since incubation of hypertrophic cells in RG-13577-free medium restores cell volume and [3H]thymidine incorporation to the values of untreated control cells within 4 days. We conclude, that the active metabolic status of RG-13577-treated cells in association with the overexpression of TGF-beta could promote repair processes of injured arteries after angioplasty without stimulating cell proliferation.

Lovastatin has been proven to effectively lower circulating LDL cholesterol and to exert antiproliferative effects on various cell lines, the latter effect being only incompletely understood. We found that lovastatin modulates the signal transducing phosphorylation cascade in vascular smooth muscle cells in a mevalonate-independent manner. Lovastatin was found to distinctively increase total phosphotyrosine levels in smooth muscle cells, an effect which could not be restored by mevalonate. At a concentration of 5 micromol/L lovastatin had a highly specific effect on the mitogen-activated protein kinase pathway. The expression of p42/44 mitogen-activated protein kinase (MAPK) was clearly reduced, but could be restored by addition of mevalonate, while the phosphorylation of p44 was mildly suppressed and the phosphorylation of p42 MAPK was reduced to non-detectable levels. While the phosphorylation of p44 MAPK could partially be restored by addition of mevalonate, the reduced phosphorylation of p42 MAPK could not be restored by addition of excessive doses of mevalonate or stimulation of the cells with basic fibroblast growth factor. Concurrently the expression of the GTP-binding Ras protein was significantly elevated at 5 and 20 micromol/L lovastatin, this effect being attenuated by addition of mevalonate to cell cultures. The data indicate that lovastatin is capable of modulating cellular signaling independently of the cholesterol synthesis pathway.

Studies on the mode of action of basic fibroblast growth factor (bFGF) identified an essential role of heparan sulfate and heparin-like molecules in the formation of distinct bFGF-heparan sulfate-bFGF-receptor complexes that are required for bFGF-induced signal transduction. In coronary smooth muscle cells that express 6-8 ng bFGF mg(-1) cell protein, the heparan sulfate chains of membrane-associated proteoheparan sulfate are implicated in bFGF signaling and thus are involved in the regulation of proliferation and differentiation of vascular smooth muscle cells. We studied the mode of action of a synthetic non-sulfated heparin-mimicking compound termed RG-13,577 (poly-4-hydroxyphenoxy acetic acid, Mr approximately 5 kD) and found a dose-dependent antiproliferative effect that was characterized by a block of G(1)/S-phase transition indicated by a marked (80%) reduction of [3H]thymidine incorporation at a concentration of 5 microg ml(-1) RG-13,577. Cell cycle analysis showed a block of cell division in the G(1)-phase. In response to RG-13,577 the cells were converted into a hypertrophic growth status within 72 h as judged from a doubling of the cellular protein content and measurement of cell and nucleus size. The increased cell protein content resulted from a de novo synthesis and was also associated with an increase in the incorporation of [35S]sulfate into cell-associated proteoglycans, including the proteoheparan sulfate coreceptor of bFGF. In contrast, the compound-induced G(1)-phase arrest was associated with an extensive downregulation of the cellular and pericellular bFGF level. The reduced bFGF content was accompanied by downregulation of the bFGF signaling-involved protein kinase C-alpha and MAP kinase, abrogation of MAP kinase phosphorylation and overexpression of protein kinase C-gamma. RG-13,577 failed to elicit apoptotic reactions at a concentration range of 0.5-10 microg ml(-1) and its effect was reversible upon removal of the compound. It appears that RG-13,577 induces a phenotype transformation of coronary SMC into a metabolically active hypertrophic status that could promote repair processes after balloon angioplasty (PTCA) without stimulating cell proliferation. Development of non-toxic polyanionic compounds may provide an effective strategy to inhibit cell proliferation associated with restenosis following balloon angioplasty and coronary artery bypass surgery.

The pathogenesis of arteriosclerosis and of restenosis after angioplasty is linked with an inflammatory and fibroproliferative response of the arterial tissue. We have induced a non-infectious inflammation by implanting a silicon–copper cuff around rat carotid arteries. The copper ions released from the oxidized copper initiate and mimic all morphological features of post-angioplasty restenotic and arteriosclerotic lesions. The copper-induced lesions were analyzed by electron and light microscopy, immunohistochemical methods and quantified by morphometry. During the first phase of copper-induced tissue reaction (3 days), macrophages and polymorphonuclear leucocytes invaded through the endothelium, accumulated in the subendothelial space and triggered the proliferation of smooth muscle cells which then migrated from the tunica media through the lamina elastica interna into the intima. Within 3 weeks, the accumulated smooth muscle cells, macrophages, leucocytes and newly synthesized extracellular matrix formed a circular mostly eccentric fibrotic thickening that narrows the vessel lumen by 30–40%. The accompanying structural disorganization of the medial layer led to focal rupture and aneurysm-like dilatation of the vessel wall in 3 of 11 animals between day 20 and 43. The neointima progressively increased in thickness over time leading to corresponding reduction of the vessel lumen. The carotid arteries of control animals and animals treated with copper-free silicon cuffs showed no abnormal pathological appearence. Our results show that inflammation-inducing agents can contribute to and simulate restenosis- and arteriosclerosis-like lesions and that the copper-cuff model may be useful in the exploration of new approaches to intervention.

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells (SMCs), plays a pivotal role in the pathogenesis of arteriosclerosis and restenosis. Heparin in nanogram quantities may promote or even be required for binding of bFGF to its cognate receptor. Conversely, heparin in microgram doses is a strong inhibitor of arterial SMC replication in vitro and in vivo. Bovine coronary SMCs (cSMCs) express bFGF, bFGF receptor (FGF-R1), and cell membrane–integrated proteoheparan sulfate (HSPG). These three molecules are known to form a trimolecular complex that promotes signal transduction and mitogenesis. The bFGF synthesized by cSMCs is distributed to an intracellular and a pericellular compartment. Resting cultured cells retain about 80% of their bFGF intracellularly; 20% is found in the pericellular region. During proliferation, 70% to 80% of total bFGF is expressed in the pericellular compartment. Trypsinization generates soluble forms of the complex of bFGF with the ectodomains of the bFGF receptor and cell membrane–integrated HSPG in the pericellular compartment, thus allowing quantification of pericellular bFGF by a highly specific enzyme immunoassay. Standard heparin inhibits the proliferation of cSMCs by up to 80% in a concentration range between 10 and 100 μg/mL medium in a dose-dependent manner but increases the protein content of cSMCs compared with proliferating control cells. The heparin-induced increase in cellular protein content includes a 60% to 100% increase in the expression of pericellular bFGF, FGF-R1, and cell membrane–integrated HSPG. Thus, under heparin treatment, the heparan sulfate side chains of cell membrane–integrated HSPG incorporate more [ 35 S]sulfate, and the proportion of [ 35 S]heparan sulfate among total glycosaminoglycans increases from 36% to 52%. Fluorescence-activated cell sorting analysis and [ 3 H]thymidine incorporation experiments provide evidence for multiple effects of heparin, including blocks at early and late checkpoints of the cell cycle in heparin-treated cells. These results indicate that heparin, despite its antiproliferative potency, stimulates the expression of all components of the bFGF system even in coronary SMCs in which growth is inhibited.

Insulin-like growth factor I and II (IGF-I and -II) — two 7.65- and 7.47-kDa polypeptides belonging to the somatomedine family — are regular constituents of human blood plasma. Both factors exert mitogenic activity on a variety of cell types including arterial smooth muscle cells. In the present study, the effect of IGF-I and -II on cultured bovine coronary smooth muscle cells (cSMC) was assessed. Human recombinant IGF-I and IGF-II added to cSMC cultured in a medium containing 10% fetal bovine serum (FBS) decreased the cell number and [ 3 H]thymidine incorporation in a dose dependent fashion up to 40% and 43% compared to control cells (100%). At the same time, the expression of basic fibroblast growth factor (bFGF) increased from 60 pg/5 × 10 4 cells (control) to 75 (IGF-I) and 113 pg5 x 104 cells (IGF-II). In parallel with enhanced bFGF expression, the bFGF receptor content per cell and the [ 35 S]sulfate incorporation into extracellular and cell-associated proteoglycans also increased under the influence of IGF-I and -II. In contrast, with low serum concentration (0.1% FBS) the addition of IGF-I and -II to bovine cSMC cultures resulted in a slight increase in cell number, protein content and [ 3 H]thymidine incorporation as described in previous studies. These results suggest that the mitogenic activity of IGF-I and -II towards coronary smooth muscle cells depends on culture conditions. In the presence of 10% fetal bovine serum that mimics in vivo conditions. IGF-I and -II did not necessarily act as mitogenic factors but inhibited the proliferation of cSMC in vitro possibly by modulating and antagonizing the action of other growth factors. Irrespective of the inhibition of cell division, the cellular bFGF, the bFGF receptor and the bFGF activity-related proteoheparan sulfate were overexpressed under the influence of IGF.

Human corneas from normal (healthy) donors and patients with keratoconus were either metabolically labelled under organ culture conditions or investigated without preincubation. The sulfated proteoglycans were isolated from a 4 M guanidinium chloride/2% Triton X 100 extract. Two predominant proteoglycans were obtained from normal cornea after digestion of total sulfated proteoglycans with chondroitin ABC-lyase or endo-beta-galactosidase. One had an overall mass of 150 kDa, two dermatan sulfate chains (Mr approximately 50 kDa) with an iduronic acid content of 24%-28% and, after chondroitin ABC-lyase digestion, a core protein of 48 kDa. The other proteoglycan had an overall mass of 110 kDa, one keratan sulfate chain of approximately 60 kDa and, following endo-beta-galactosidase (keratanase) digestion, a core protein of 46 kDa. Each proteoglycan population was further fractionated into two subpopulations by chromatography on concanavalin A-Sepharose. The dermatan sulfate- and keratan sulfate-containing proteoglycans isolated from keratan and healthy cornea had comparable Mr values and core proteins with identical molecular weights, but the ratio of dermatan sulfate/keratan sulfate proteoglycan was increased in keratoconic cornea and the keratan sulfate chains of two keratan sulfate proteoglycans from keratoconic cornea were considerably shorter (Mr 44 and 33 kDa) than those from normal corneas.

Cultured bovine arterial smooth muscle cells express 6 to 7 ng basic fibroblast growth factor (bFGF)/mg cell protein and distribute it to two compartments. About 80% of total bFGF remain intracellularly, 20% are present in the pericellular (trypsin-releasable) compartment. No bFGF can be detected in the culture medium. All bFGF fractions have full biological activity. They are quantified by a highly specific immunoassay system and identified after sodium dodecyl sulfate polyacrylamide electrophoresis as a 18 kDa double band by immunoblotting. During exponential growth the intracellular concentration of bFGF decreases from about 130 pg to 20 to 40 pg/10(5) cells. Simultaneously the pericellular bFGF increases to 60-70% of total bFGF, but declines continuously with increasing cell density, whereas the intracellular bFGF reincreases under these conditions. The pericellular bFGF is known to form complexes with (membrane integrated) proteoheparan sulfate which undergoes structural changes during transition from subconfluent to confluent growth status. After metabolic labeling of the cells with [35S]sulfate and [3H]glucosamine, the 35S/3H ratio of heparan sulfate oligosaccharides increases from 1.58 during proliferation to 2.47 in growth-inhibited cells. The results indicate that the bFGF-induced proliferation of arterial smooth muscle cells depends on the pericellular localization of bFGF and on a specific molecular organization of the cell surface heparan sulfate. Depending on its specific structural characteristics heparan sulfate may promote or inhibit bFGF receptor binding and signal transduction.

The proteoglycans and glycosaminoglycans of four human chondrosarcomas with different degrees of malignancy (I–III) have been studied. The hydrodynamic size of proteoglycan subunits and the tissue concentration of total glycosaminoglycans decreased with increasing grade of malignancy. The glycosaminoglycan distribution pattern of all chondrosarcomas showed a similar ratio of chondroitin-4-sulfate: chondroitin-6-sulfate but an increasing portion of keratan sulfate from grade I (6.5%) to grade III (19.2%). Determinations of the molecular weight ( M r values) of glycosaminoglycans were made after 3 H labeling by alkaline reduction of proteoglycans in the presence of NaB 3 H 4 . The M r of [ 3 H]chondroitin sulfate isomeres decreased markedly from grade I (35,500) to grade III (15,100) while the chain length of [ 3 H]keratan sulfate showed minor variations ( M r 5600–6200). The previously reported decrease in the molecular weight of keratan sulfate with increasing degree of malignancy ( FS. Pal, W. Strider, R. Margolis, G. Gallo, S. Lee-Huang, and L. Rosenberg, 1978 , J. Biol. Chem. 253 , 1279–1289) was not observed.

Preincubation of hepatoma cells and human skin fibroblasts in the presence of the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide resulted in a dose-dependent suppression of [14C]mevalonolactone incorporation into cholesterol. At a calmodulin antagonist concentration of 25 mumol, the incorporation of [14C]mevalonolactone into cellular cholesterol was suppressed to about 30% (hepatoma cells) and 10% (human skin fibroblasts) of control values. When the total nonsaponifiable [14C]lipids were separated and analyzed by two-dimensional thin layer chromatography, an accumulation of [14C]desmosterol was observed along with reduced formation of [14C]cholesterol. However, when cells were preincubated in the presence of [14C]dihydrolanosterol, [14C]cholesterol formation was not inhibited by the calmodulin antagonists. About 25% of the cell-associated dihydrolanosterol radioactivity was converted to cholesterol in both control and calmodulin antagonist-pretreated cells. The data suggest that calmodulin antagonists prevent the conversion of desmosterol into cholesterol by inhibiting sterol delta 24 reductase and that the enzymes catalyzing sterol ring modifications are not affected by the inhibitors.

on in vitro incubation of articular and epiphysial cartilage of the ulna of the domestic pig 70 to 80 per cent of [U-14C] glucose was metabolised to 14C-lactate, but cartilage of the epiphysial plate produced up to five times as much 14C-CO2 as articular cartilage, and the specific radioactivity of 14C (or 35S)-chondroitin 4 (6)-sulphate isolated from epiphysial cartilage (following 35S-sulphate incorporation) was about twice as high as that of articular cartilage. Six weeks after an osteotomy on both sides of the proximal epiphysial plate of the left ulna, the glucose uptake, lactate production, and the specific radioactivity of the glycosaminoglycans displayed no significant differences when compared with those of the corresponding epiphysial plate of the control right ulna, whereas a 50 per cent increase in the oxidation of 14C-glucose to 14c-co2 was observed.

The LDL receptor synthesis of human skin fibroblasts in the presence of the specific calmodulin antagonists trifluoperazine, condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) (W-7) was studied. Labelling of cells with [35S]methionine followed by immunoprecipitation of radioactive LDL receptor protein with monospecific antibodies revealed that calmodulin antagonists caused a 3-fold increase in the radioactivity of the LDL receptor protein as compared with values found in control cells. A corresponding increase of high-affinity binding and internalization of 125I-labelled LDL was observed. The drugs did not influence the overall protein synthesis or the half-life of the LDL receptor. A concomitant suppression of cholesterol synthesis from [14C]mevalonolactone was found to be an independent effect. The calmodulin antagonist-produced stimulation of LDL receptor synthesis could not be simulated by preincubation of cells with cyclic nucleotide analogues, cholera toxin or 3-isobutyl-1-methylxanthine, known as specific effectors of adenylate cyclase and cyclic nucleotide phosphodiesterase, respectively. Modulation of calcium concentration in the incubation medium had no reproducible effect on the rate of LDL receptor synthesis. The results implicate calmodulin as an intracellular suppressor of LDL receptor synthesis in human skin fibroblasts.

The (high-affinity receptor)-mediated uptake of homologous low-density (low-rho) lipoproteins by cultured human arterial smooth muscle cells or human skin fibroblasts is controlled by the sialic acid content of low-rho lipoprotein particles. This conclusion is derived from the following results. 1. Gangliosides incubated with native low-rho lipoproteins associate with low-rho lipoprotein particles. Low-rho lipoproteins modified by associated GLac1, GGtet1, and GGtet2b + GGtet3 gangliosides are internalized by arterial smooth muscle cells at a rate up to 80% lower than native low-rho lipoproteins or those preincubated with desialized gangliosides. 2. The inhibitory effect of gangliosides is specific for high affinity uptake and not detectable on skin fibroblasts deficient in low-rho-lipoprotein receptor. 3. Desialyzed low-rho lipoproteins are internalized by smooth muscle cells up to 100% faster than native low-rho lipoproteins, the enhancement of uptake corresponding to the degree of desialization.

Sulfated glycosaminoglycans were extracted from arteriosclerotic and adjacent nonarteriosclerotic areas of human aortas from persons ages 28 to 83 years; the glycosaminoglycans were compared with the cholesterol and triglyceride content of the tissues. Sulfated glycosaminoglycans were isolated after proteolytic digestion of defatted arterial tissue and were quantified after reductive labeling with NaB3H4. The amount of glycosaminoglycans in the aorta increased with the age of the person and the cholesterol content (degree of arteriosclerosis) of the aorta. The proportion of chondroitin sulfate/dermatan sulfate increased significantly with age and cholesterol content, whereas the corresponding amounts of heparan sulfate decreased.

The cuprolinic blue (CB) staining method has been used to visualize and characterize proteoglycans (PG) in the extracellular matrix (ECM) of normal and atherosclerotic human arteries. Arterial tissues of 13 individuals (1-83 years of age) were obtained by autopsy. For electron microscopic visualization of PGs staining with CB was performed in the presence of a critical electrolyte concentration of 0.3 M MgCl2. Under these conditions CB selectively interacts with the polysulfated glycosaminoglycan (GAG) side chains of the molecules. Removal of PG side chains by GAG-degrading enzymes prior to CB staining selectively prevented the formation of chondroitin sulfate (CS)-rich and dermatan sulfate (DS)-rich PG-CB precipitates. The DS-rich type of PG is mainly associated with collagen fibrils, the CS-rich type of PG is preferentially localized in nonfibrous areas of the ECM (soluble matrix). When normal arterial tissues are compared with those affected by atherosclerosis quantitative and qualitative changes of PG-CB precipitates are detected. In fibrous plaques a strong accumulation of a large CS-rich type of precipitate close by smooth muscle cells (SMC) and foam cells is observed. In addition, these precipitates are significantly longer in fibrous plaques than in adjacent normal media (116 nm vs. 100 nm; P less than 0.001). This alteration is independent of the age of the donor. Small DS-rich PG-CB precipitates associated with collagen fibrils show strong variations in their length, but not a significant tendency towards elongated precipitates in atherosclerosis. The present results demonstrate that ultracytochemical and morphometric analysis are useful in providing information on the diverse types, locations, interactions, and possibly of molecular changes of PGs in normal and atherosclerotic human arteries.

Tyrosine O-sulfate residues were detected in the protein core of sulfated proteoglycans. When cultured skin fibroblasts and arterial smooth muscle cells were incubated in the presence of [35S]sulfate, dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan isolated from the culture medium contained tyrosine [35S]sulfate ester which accounted for 0.03%-0.82% of total 35S radioactivity incorporated into the sulfated proteoglycans. This corresponds to a tyrosine sulfation of every second (fibroblasts) and every 10th (smooth muscle cells) dermatan sulfate proteoglycan molecule. [3H]Tyrosine labeling of fibroblast dermatan sulfate proteoglycan gave a similar stoichiometry. However, the relative proportion of tyrosine [35S]sulfate in proteoglycans from arterial tissue was about 10 times higher than in that from cultured arterial cells. Pulse chase experiments with [35S]sulfate revealed that tyrosine sulfation is a late event in the biosynthesis of dermatan sulfate proteoglycan from fibroblasts and occurs immediately prior to secretion. Cultured skin fibroblasts from a patient with a progeroid variant (Kresse et al. 1987, Am. J. Hum. Gen. 41, 436-453) which exhibit a partial deficiency to synthesize dermatan sulfate proteoglycan were shown to form and to secrete a tyrosine-sulfated but glycosaminoglycan-free protein core, thus confirming a selective and independent [35S]sulfate labeling of the protein core.

The effects of calmodulin antagonists on the amount of LDL receptor (LDL-R) mRNA in cultured human fibroblasts was examined by hybridization with a fragment of LDL-R cDNA. In a ‘Northern’ blot the fragment hybridized to a 5.3-kilobase RNA, as expected for LDL-R mRNA. The concentration of this RNA was increased in preparations from cells that were treated with trifluoperazine or W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide]. The selectivity of the increase was established by using a probe for beta-actin mRNA. In dot-blot hybridization it was observed that the calmodulin antagonists cause 2-4-fold relative increase in the amount of LDL-R mRNA.

Skin fibroblasts cultured from patients with Sanfilippo's disease type B and Hurler's disease, preincubated with [35S]sulfate, were subjected to high resolution autoradiography. A 4-day pulse followed by a 24 h chase resulted in a labelling of 61% of the cell nuclei by silver grains, thus indicating the existence of ethanol-insoluble [35S]sulfate-containing material within the nuclei, especially within their chromatin-rich peripheral zone. A computer-calculated statistical evaluation of the autoradiographic results showed that the silver grains overlying the cell nuclei originated from [35S]radioactivity within the nuclei and not from an overall background or cross fire effects of cytoplasmic radiation sources. Chemical analyses of chloroform/methanol extracts and pronase digests of the [35S]labelled cells provided evidence that neither [35S]sulfatides nor [35S]glycopeptides contribute substantially to the cellular [35S]radioactivity. The results strongly suggest the association of sulfated glycosaminoglycans with cell nuclei.

Muzolimine, 3-amino-1-(3,4-dichloro-α-methylbenzyl)-2-pyrazolin-5-one, an antihypertensive and diuretic drug, accumulates in the arterial tissue of rats and dogs after oral administration. Two weeks after the administration of 3 mg [14C]muzolimine, the aorta of rats contained 60–300 times more 14C-radioactivity/weight unit than the skin or tail tendon. The 14C-radioactivity was exclusively bound to the isolated aortic elastin and corresponded to 0.04% of the applied muzolimine dose. Up to ca 250 ng bound muzolimine/mg elastin was found in the aorta of dogs treated with non-labelled muzolime for 52 weeks. The elastin-bound [14C]muzolimine was not extractable by organic solvents or by weak acids or bases but was released in a soluble form by pancreatic elastase and extracted from the elastase digest by dichloromethane. In the dichloromethane extract muzolimine was detected by HPLC and HPTLC, and was identified by mass spectrometry. Muzolimine pretreatment of rats for 2 months did not influence the elastin content of arterial tissue or [3H]glycine incorporation into aortic elastic under organ culture conditions, but after labelling the elastin with [4,5-3H]lysine, the [3H]desmosine and [3H]-isodesmosine isolated from the elastin of muzolimine-pretreated rats and incorporated under organ culture conditions was lower than that of control animals. In addition, aortic elastin of rats pretreated for 2 months with 800 ppm muzolimine in the diet was more resistant to elastase degradation. This effect might give some implications for muzolimine in the therapy of cardiovascular disorders with impaired arterial elastin metabolism.

1. Human skin fibroblasts internalize homologous sulphated proteoglycans by adsorptive endocytosis. Endocytosis rate is half maximal when the concentration of the proteoglycans is 0.1 nM. At saturation, a single fibroblast may endocytose up to 8 × 10(6) proteoglycan molecules/h. 2. The kinetics of prote;glycan binding to the cell surface suggest the presence of 6 × 10(5) high-affinity binding sites per cell. The bulk of sulphated proteoglycans associates to low-affinity binding sites on the cell surface. 3. Glycosaminoglycans and other anionic macromolecules inhibit endocytosis of sulphated proteoglycans non-competitively. The lack of interaction of glycosaminoglycans with the cell-surface receptors for sulphated proteoglycans suggests that the protein core of proteoglycans is essential for binding to the cell surface. 4. The effects of trypsin, cell density, serum concentration and medium pH on endocytosis and degradation of endocytosed sulphated proteoglycans is described. 5. A comparison of the number of the high-affinity binding sites and the number of molecules endocytosed with respect to time suggests a recycling of the proteoglycan receptors between the cell surface and the endocytotic vesicles and/or the lysosomes.

Bovine corneal tissue incorporated 14C into lactate, glycogen and acid glycosaminoglycans when incubated in vitro in the presence of [U-14C]glucose either under exposure to light or in the dark, but the uptake of [14C]glucose and its incorporation into glycogen was higher by about 30% when exposed to light. In the dark, on the other hand, a higher rate of [14C]lactate production and 14C-incorporation into acid glycosaminoglycans was observed. The statistically significant differences suggest a light dependent activation and suppression of corneal glucose metabolism.

Comparative studies of the chemistry and metabolism of human and bovine arterial and venous tissue had the following results (values for bovine tissue in parenthesis): 1. In arterial tissue the contents of collagen were 21% (37%) and of elastin 20% (31%) referred to the dry weight. In veins collagen amounted to 47% (30%) and elastin to 7% (18%). The mucopolysaccharide concentration was higher in arterial (1.2 and 1.5 resp.) than in venous tissue (0.4 and 0.2% resp.). Hyaluronate, chondroitin 6-sulphate (chondroitin 4-sulphate), dermatan sulphate and heparan sulphate were identified. In arterial tissue chondroitin sulphate was predominant with 59 % and 49 % resp. In human venous tissue the main component was dermatan sulphate (65%), in bovine veins it was hyaluronate (75%). No dermatan sulphate was found in bovine venous tissue. The number of cells was higher in arteries (4.5 × 107 nuclei/g w. wt.) than in veins (2.8 × 107). 2. On in vitro incubation bovine arterial tissue metabolized 73 %, venous tissue 32% of the added [U-I4C] glucose within 12 h. In this period 2.8 and 2.4 mg lactate resp./g wet tissue were formed by arteries and veins. The specific radioactivity of lactate synthesized in arterial tissue reached that of the added [U-I4C] glucose whereas lactate formed by veins had only 36 % of the specific activity of the [U-I4C] glucose. The specific radioactivity of the acid mucopolysaccharides was 5–6 times higher in venous tissue than in arterial tissue. After labelling with 35S-sulphate a 3-fold higher specific radioactivity of venous sulphated mucopolysaccharides was observed. 3. After incubation of bovine venous tissue in the presence of I4C-acetate the specific radioactivity of phospholipids was 45 % higher than that of arterial tissue in contrast to a 50 % lower labelling of cholesterol in venous tissue.

Aortic endothelial cells from control and streptozotocin diabetic minipigs were cultured. Both groups of cells exhibited the typical cobblestone-like appearance and gap junction formation. Endothelial cells derived from diabetic minipigs differed, however, from those from control animals by a higher rate of proliferation and a higher percentage of large and often multinucleated cells. In these cells the specific binding of low-density lipoproteins (LDL) to coated pits on the cell surface, the LDL uptake, and the intracellular transport of LDL to lysosomes were visualized by gold-labeled LDL complexes. The binding, internalization, and degradation of LDL by subconfluent, non-contact-inhibited endothelial cells was quantified using 125I-labeled LDL. The LDL metabolism of endothelial cells derived from diabetic animals was increased by about 40% compared to endothelial cells derived from nondiabetic animals.

The ability of acidic alpha-mannosidases from pig kidney, bovine liver, human urine and placenta, and jack bean to correct the deranged metabolism of cultured skin fibroblasts from manosidosis patients is studied. One cause for the different corrective abilities are, as shown for other systems, the different rates of endocytosis of the enzyme forms into the cells. Besides this, it is demonstrated for the first time, that there are differences in the intracellular corrective activity of the internalized enzymes, which can be explained by different specificities of the enzymes against the storage material.

Arterial smooth muscle cells cultured from normotensive and hypertensive rats incorporated [35S]sulfate into the extracellular and pericellular sulfated proteoglycans and endocytose extracellular [35S]proteoglycans at a significantly higher rate in the phase of logarithmic growth than did nondividing cells. 35S incorporation into proteoglycans was positively correlated with [3H]thymidine incorporation into the cellular TCA-precipitable material. The rates of [35S]proteoglycan synthesis and endocytosis per cell pr day were higher in smooth muscle cells from hypertensive than from normotensive animals, the observed differences being related to a higher average protein content of smooth muscle cells cultured from hypertensive rats as compared with cells of normotensive animals. Gel filtration under dissociative conditions separated the [35S]proteoglycans into high and low molecular weight fractions (A, B) differing in glycosaminoglycan composition and their ability to be endocytosed by smooth muscle cells. The relative proportion of the high molecular weight proteoglycan fraction A decreased continuously from sparse to confluent cell cultures.

From human hyaline cartilage (processus xyphoid) preincubated in the presence of [35S]sulfate, proteoglycans were extracted by 4M guanidinium chloride and divided into 6 age groups. Fractionation of proteoglycans by gel filtration under dissociative conditions resulted in two proteoglycan fractions (a and b) with different hydrodynamic volumes. The higher molecular weight fraction a contained chondroitin sulfate, the fraction b keratan sulfate as predominant glycosaminoglycan, the chondroitin sulfate/keratan sulfate ratio decreasing with increasing age in either fraction. The relative portion of proteoglycan fraction b and its 35S-labelling increased with increasing age. From the specific 35S radioactivities of the chondroitin sulfate and keratan sulfate preparations, the occurrence of two independent proteoglycan populations is suggested. A precursor-product relationship between proteoglycan fraction a and b could be excluded.

Human skin fibroblasts incubated in lipoprotein-deficient medium in the presence of 50–100 μM of the calcium channel blockers verapamil or diltiazem incorporated up to 2.5 times more [ 35 S]methionine into immunoprecipitable LDL receptor protein than did control cells. Verapamil was found to be more potent in this regard than diltiazem. The calcium channel blockers did not influence the overall synthesis of cellular proteins or the half-life of the LDL receptor, and they were not able to prevent the suppression of LDL receptor synthesis caused by exogenous LDL or 25-hydroxycholesterol. The calcium channel blocker-induced stimulation of LDL receptor synthesis was accompanied by a corresponding increase in binding and internalization of [ 125 I]LDL, but the degradation of internalized lipoprotein was slightly decreased. The results suggest that intracellular Ca 2+ levels modulate LDL receptor metabolism in human skin fibroblasts.

Binding of homologous low density lipoproteins (LDL) to specific cell-membrane receptors initiates regulation of cholesterol metabolism in normal human fibroblasts, but minor regulatory effects are observed in receptor-deficient cells. After enzymic removal of sialic acid residues, however, the desialized LDL aquires the ability to regulate cholesterol biosynthesis and cholesterol esterification in receptor-deficient cells as effective as native LDL in normal cells. This is indicated by a suppression of hydroxymethylglutaryl-CoA reductase activity, by a reduced incorporation of [14C]acetate into cholesterol and by a stimulation of the [3H]oleate incorporation into cholesterol esters in both normal and receptor deficient cells. Competitive binding and internalisation experiments revealed that the action of desialized LDL in normal and receptor-deficient cells is receptor-specific. The results suggest that desialisation of LDL facilitates its binding to cell membrane receptors, its internalisation and subsequent regulation of cellular cholesterol metabolism, this effect becoming significant in fibroblasts with reduced number of LDL receptors.