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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.

Background Transforming growth factor (TGF- β 1 ) is postulated to play an important role in maintaining the structure and function of arterial tissue and protection against development of arteriosclerosis. The TGF- β 1 -induced production of a stable extra-cellular matrix-rich plaque phenotype is suggested to be part of the protection against a switch to an unstable rupture-prone arteriosclerotic plaque. Materials and methods This study addresses the question of whether the expression profile and the type of extra-cellular matrix (ECM) generated by TGF- β 1 stimulation have the structural feature of a fibril-rich stable matrix. Seventeen genes codings for ECM components of human coronary smooth muscle cells (SMCs) after a 24-h stimulation by TGF- β 1 have been analyzed. Results Real-time RT-PCR was used to quantify the mRNA of genes under investigation. It was found that after TGF- β 1 stimulation (a) the up-regulation of COL1A1-specific mRNA was associated with increased [ 3 H]proline incorporation into the α -1 and -2 chains of collagen type I, (b) the up-regulation of biglycan- and syndecan-1-specific mRNA corresponded to an increased [ 35 S]sulphate and [4,5- 3 H]leucine incorporation into the biglycan molecule and to an increase of syndecan-1 protein, (c) the up-regulated FGF-2 gene accounted predominantly for the ECM-bound subfraction of FGF-2-protein and (d) fibronectin and thrombospondin exhibited a significantly higher mRNA level. In contrast collagen XIV, a minor collagen type, and the proteoglycan decorin were down-regulated. The down-regulated decorin changed its structure by elongation and reduced GlcA to IdoA epimerization of the dermatan sulphate side-chain as judged by [ 35 S]sulphate metabolic labelling experiments. No significant changes in response to TGF- β 1 were observed for the collagen types III, VI and XVI, for versican, perlecan and the syndecans-2 and -4. Conclusions It was concluded from the data that the TGF- β 1 -induced formation of a highly specific multicomponent extra-cellular matrix on coronary arterial SMCs could provide in vivo mechanical strength to the neointima in arteriosclerotic lesions and to the fibrous cap overlying the lipid core.

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.

Objective: In an in vitro biosensor model (PCT/ET 97/05212), the interplay between different lipoproteins in arteriosclerotic nanoplaque formation, as well as aqueous garlic extract (0.2-5.0 g/l from LI 111 powder) as a possible candidate drug against arterio/atherosclerosis were tested within the frame of a high throughput screening. Methods: The processes described below were studied by ellipsometric techniques quantifying the adsorbed amount (nanoplaque formation) and layer thickness (nanoplaque size). A thorough description of the experimental setup has been given previously. Results: Proteoheparan sulfate (HS-PG) absorption to hydrophobic silica was monoexponential and after approximately 30 min constant. The addition of 2.52 mmol/l Ca2+ led to a further increase in HS-PG adsorption because Ca2+ was bound to the polyanionic glycosaminoglycan (GAG) chains thus screening their negative fixed charges and turning the whole molecule more hydrophobic. Incubation with 0.2 g/l aqueous garlic extract (GE) for 30 min did not change the adsorption of HS-PG. However, the following addition of Ca2+ ions reduced the increase in adsorption by 50.8 % within 40 min. The adsorption of a second Ca2+ step to 10.08 mmol/l was reduced by even 82.1 % within the next 40 min. Having detected this inhibition of receptor calcification, it could be expected that the build-up of the ternary nanoplaque complex is also affected by garlic. The LDL plasma fraction (100 mg/dl) from a healthy probationer showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca2+ concentration, with a strong increase at higher Ca2+ concentration. GE, preferably in a concentration of 1 g/l, applied acutely in the experiment, markedly slowed down this process of ternary aggregational nanoplaque complexation at all Ca2+ concentrations used. In a normal blood Ca2+ concentration of 2.52 mmol/l, the garlic induced reduction of nanoplaque formation and molecular size amounted to 14.8 % and 3.9 %, respectively, as compared to the controls. Furthermore, after ternary complex build-up. GE similar to HLD, was able to reduce nanoplaque formation and size. The incubation time for HDL and garlic was only 30 min each in these experiments. Nevertheless, after this short time the deposition of the ternary complex decreased by 6.2 % resp. 16.5 %, i.e. the complex aggregates were basically resolvable. Conclusions: These experiments clearly proved that garlic extract strongly inhibits Ca2+ binding to HS-PG. In consequence, the formation of the ternary HS-PG/LDL/Ca2+ complex, initially responsible for the nanoplaque´composition and ultimately for the arteriosclerotic plaque generation, is decisively blunted.

Adhesion and recruitment of blood monocytes, processes mediated by cell adhesion molecules including E-selectin, represent an early event in atherogenesis. High density lipoproteins (HDLs) were shown to inhibit cytokine-induced expression of adhesion molecules, but mechanisms underlying this effect are not fully understood. We here investigated the effects of sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), two lysosphingolipids associated with HDL, on TNF-alpha-induced E-selectin expression in human umbilical endothelial cells. We found that HDL, SPC, and LSF inhibited E-selectin expression both on mRNA and protein level. In addition, all three agents reduced the number of E-selectin molecules present on endothelial cell surface. The inhibitory effects of HDL, SPC, and LSF on TNF-alpha-induced E-selectin expression were partially reverted in the presence of suramin, an antagonist of lysosphingolipid receptor EDG-3, or pertussis toxin, an inhibitor of trimeric G proteins. In addition, inhibition of activation of protein kinase Akt with LY294002 but not inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) with U73122 abolished the restrictive effects of HDL-, SPC-, or LSF on E-selectin expression. We conclude that HDL-associated lysosphingolipids may at least partially account for the inhibitory effects of HDL on cytokine-induced expression of adhesion molecules, and that activations of G-protein-coupled receptors and protein kinase Akt are involved in this process.

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells, has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenosis by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate-modified 15-residue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1-2.0 microM block growth and division of cultured human and bovine coronary smooth muscle cells in a dose-dependent manner. The effect is sequence specific at low (0.1-0.5 microM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48-72 h and diminishes thereafter. If the antisense oligodeoxynucleotide medium is replaced by an oligonucleotide-free medium after 24 h, the [3H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitory actions. The antiproliferative potency of the 15-residue antisense oligodeoxynucleotide is markedly enhanced by adding 3-4 nonbase-pairing guanosine residues at the 5'- and 3'-termini of the 15-residue antisense oligonucleotide. The data implicate bFGF in the process of smooth muscle cell proliferation and an effective and specific antiproliferative potency of bFGF-specific antisense oligonucleotides. The results point to possible new therapeutic strategies for the use of antisense methodology in the suppression of post-angioplasty restenosis.

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.

The finding of flow-dependent vasodilatation rests on the basic observation that with an increase in blood flow the vessels become wider, with a decrease the vascular smooth muscle cells contract. Proteoheparan sulphate could be the sensor macromolecule at the endothelial cell membrane-blood interface, that reacts on the shear stress generated by the flowing blood, and that informs and regulates the vascular smooth muscle cells via a signal transduction chain. This anionic biopolyelectrolyte possesses viscoelastic and specific ion binding properties which allow a change of its configuration in dependence on shear stress and electrostatic charge density. The blood flow sensor undergoes a conformational transition from a random coil to an extended filamentous state with increasing flow, whereby Na + ions from the blood are bound. Owing to the intramolecular elastic recoil forces of proteoheparan sulphate the slowing of a flow rate causes an entropic coiling, the expulsion of Na + ions and thus an interruption of the signal chain. Under physiological conditions, the conformation and Na + binding proved to be extremely Ca 2+ -sensitive while K + and Mg 2+ ions play a minor role for the susceptibility of the sensor. Via counterion migration of the bound Na + ions along the sensor glycosaminoglycan side chains and following Na + passage through an unspecific ion channel in the endothelial cell membrane, the signal transduction chain leads to a membrane depolarization with Ca 2+ influx into the cells. This stimulates the EDRF/NO production and release from the endothelial cells. The consequence is vasodilatation.

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.

Objective: Nitric oxide (NO) is a major signalling molecule in the vascular system enhancing vascular smooth muscle cell relaxation and vasodilation. NO donors are the most frequently and repeatedly used drugs for relief from angina pectoris. Methods: We investigated the effects of the synthetic NO donor DETA/NO on cultured human coronary smooth muscle cells. Results: Cells exposed to 100 μM DETA/NO for 48–72 h were channeled into a cell cycle-arrested hypertrophic growth status associated with overexpression of TGF-β1 on both the protein and mRNA levels. Increased TGF-β1 transcription and translation were associated with enhanced synthesis of extracellular matrix components including the collagen types I and III as shown by immunocytochemistry and enhanced incorporation of [3H]proline. Higher incorporation of [35S]sulfate into chondroitin/dermatan sulfate and heparan sulfate containing proteoglycans was observed in DETA/NO treated cells than in controls. The ratio of chondroitin/dermatan sulfate to heparan sulfate did not change significantly. Conclusions: Our results suggest a dual function of the overexpressed TGF-β1. Overexpressed TGF-β1 could stabilize the fibrous cap overlaying atherosclerotic plaques due to the accumulation of extracellular matrix components. However, the findings could also support a proatherogenic role of TGF-β1 resulting from the overexpression of LDL-binding proteoglycans.

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.

The design and use of minimally modified oligonucleotides for specific inhibition of gene expression is discussed. The “minimal” protection strategy is a combination of the end-capping technique and the protection of internal pyrimidine positions which are the major sites of endonuclease degradation. By reducing the number of phosphorothioate modifications needed to make the oligonucleotide resistant to nuclease degradation, non-sequence-specific effects, which are frequently observed with uniformly phosphorothioate-modified oligonucleotides, can be reduced.

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.

Cultured bovine corneal endothelial cells express 5–8 ng basic fibroblast growth factor (bFGF)/mg cell protein and distribute it between the intracellular and pericellular compartment. Confluent cultures retain approximately 80% of the total bFGF intracellularly, whereas 20% is present in the pericellular (trypsin-releasable) compartment. No bFGF can be detected in the culture medium. The presence of 1–2 ng/ml medium of endogenous or exogenous (human recombinant) bFGF is sufficient to support cell growth. Simultaneously, cells incorporate [35S]sulfate and [3H]glucosamine into the sulfated proteoglycans associated with the cell layer at a rate that is three times higher than in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied by a shift in proteoglycan distribution. In control cells, cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans, whereas under the influence of bFGF the amount of heparan sulfate increases to approximately 60%. At the same time, the molecular structure of the heparan sulfate molecule undergoes bFGF-specific changes as indicated by the [35S]oligosaccharide pattern generated by heparitinase I degradation. The proportion of [35S]oligosaccharides with greater than six monosaccharides decreases on account of disaccharides and tetrasaccharides under the influence of bFGF. Pretreatment of bFGF with neutralizing antibodies against bFGF abolishes its biological activity. The results suggest a bFGF-dependent change in the rate of synthesis and structural features of the membrane-associated heparan sulfate in corneal endothelial cells. The modification of the heparan sulfate structure could influence its bFGF-binding and antiproliferative activity.

Die Bildung neointimaler Lasionen istsehrwahrscheinlich der Mechanismus, der bei Restenosierungen nach koronarangiographischen Eingriffen auftritt [1]. Wesentliche Faktoren des damit verbundenen neointimalen Wachstums sind die Proliferation glatter Gefasmuskelzellen (SMC) und die vermehrte Synthese von Bindegewebe. Die Proliferation von SMCs wird durch Angiotensin II, dessen Funktion als blutdruckregulierende Substanz bereits gut bekannt ist, angeregt [5]. In Zellkulturen wurde nachgewiesen, das Angiotensin II zur Hypertrophierung und zu verstarkter Proteinsynthese von SMCs fuhrt [2, 6]. Angiotensin II entsteht aus Angiotensin I mit Hilfe des Angiotensinkonversionsenzyms ACE. Uber Rezeptoren in der Gefaswand reguliert Angiotensin II offensichtlich nicht nur den Blutdruck, sondern direkt oder indirekt, z. B. uber das intrazellulare Kalzium, Zeltvermehrung und Bindegewebesynthese [3, 7, 12]. Aus diesem Grunde sind Hemmer von Angiotensin II moglicherweise von zusatzlichem therapeutischem Nutzen bei der Unterdruckung von Restenosen. Im vorliegenden Fall wurden in ballonisierten Ratten die Wirkungen von zwei Substanzen gepruft und verglichen, die auf verschiedene Weise Angiotensin II-Aktivitaten hemmen. Der ACE-Inhibitor Enalapril hemmt das Angiotensin-Konversionsenzym. Der nichtpeptidische Angiotensin II-Rezeptorantagonist Losartan, der auch unter den Bezeichnungen DuP753, EXP115 und MK954 bekannt ist, bindet an Angiotensin II- Rezeptoren und verhindert die Bindung von Angiotensin II [11].