Dorothea I. Axel

Local Paclitaxel Delivery for the Prevention of Restenosis: Biological Effects and Efficacy In Vivo

OBJECTIVE The aim of this study was to evaluate the potential of paclitaxel to prevent restenosis in vivo. BACKGROUND Paclitaxel (Taxol) is a microtubule-stabilizing compound with potent antitumor activity. It influences the cytoskeleton equilibrium by increasing the assembly of altered microtubules, thereby inducing cellular modifications that result in reduced proliferation, migration and signal transduction. METHODS Before the in vivo study, delivery efficiency was determined with radiolabeled paclitaxel in porcine hearts. After induction of a defined plaque in the right carotid arteries of 76 New Zealand rabbits by electrical stimulation, 27 animals underwent balloon dilation and subsequent local paclitaxel delivery (10 ml, 10 micromol/liter) with a double-balloon catheter. Twenty-nine animals served as control with angioplasty only, 10 animals underwent local delivery of vehicle only (0.9% NaCl solution) and 10 animals were solely electrostimulated. Vessels were excised one, four, and eight weeks after intervention. RESULTS The extent of stenosis in paclitaxel-treated animals was significantly reduced compared with balloon-dilated control animals (p = 0.0012, one, four and eight weeks after intervention: 14.6%, 24.6% and 20.5%, vs. 24.9%, 33.8% and 43.1%, respectively). Marked vessel enlargement compared with balloon-dilated control animals could be observed (p = 0.0001, total vessel area after one, four and eight weeks: paclitaxel group: 1.983, 1.700 and 1.602 mm2, control: 1.071, 1.338 and 1.206 mm2, respectively). Tubulin staining and electron microscopy revealed changes in microtubule assembly, which were limited to the intimal area. Vasocontractile function after paclitaxel treatment showed major impairment. CONCLUSIONS Local delivery of paclitaxel resulted in reduced neointimal stenosis and enlargement in vessel size. Both these effects contribute to a preservation of vessel shape and are likely to be caused by a structural alteration of the cytoskeleton.

Cartilage Oligomeric Matrix Protein (Thrombospondin-5) Is Expressed by Human Vascular Smooth Muscle Cells

Abstract—Cartilage oligomeric matrix protein (COMP/thrombospondin [TSP]-5) belongs to the thrombospondin gene family and is an extracellular glycoprotein found predominantly in cartilage and tendon. To date, there is limited evidence of COMP/TSP-5 expression outside of the skeletal system. The aim of the present study was to investigate the expression of COMP/TSP-5 in cultured human vascular smooth muscle cells and human arteries. COMP/TSP-5 mRNA and protein expression was detected in cultured human vascular smooth muscle cells with both Northern blotting and immunoprecipitation. Serum, as well as transforming growth factor (TGF)-&bgr;1 and TGF-&bgr;3, stimulated COMP/TSP-5 mRNA expression. COMP/TSP-5 was detected in normal as well as atherosclerotic and restenotic human arteries with immunohistochemistry. The majority of COMP/TSP-5 was expressed in close proximity to vascular smooth muscle cells. In vitro attachment assays demonstrated strong adhesion of smooth muscle cells to COMP/TSP-5–coated surfaces, with the majority of cells spreading and forming stress fibers. In addition, COMP/TSP-5 supported the migration of smooth muscle cells in vitro. The present study shows that COMP/TSP-5 is present in human arteries and may play a role in the adhesion and migration of vascular smooth muscle cells during vasculogenesis and in vascular disease settings such as atherosclerosis.

All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells

OBJECTIVE The vitamin-A derivative all-trans retinoic acid (atRA) is a potent regulator of cell growth, differentiation, and matrix formation of various cell types and plays an important role in embryogenesis. However, sparse data are available about its effects on human vessel diseases. Thus, we studied the effects of atRA on human arterial smooth muscle cell (haSMC) and endothelial cell (haEC) proliferation, migration, differentiation and extracellular matrix (ECM) turnover in mono- and transfilter cocultures. METHODS Effects of atRA on human arterial cells in monocultures were determined using cell counting assays, BrdU-ELISA and MTT-tests. In transfilter cocultures haSMC-growth was studied under the stimulatory effect of proliferating haEC. Using Northern blot analysis, effects of atRA on mRNA expression of ECM-proteins were examined while protein expression and activity of matrix metalloproteinases were determined by Western blotting and zymography. RESULTS atRA caused a dose dependent inhibition of haSMC-growth in monocultures (IC(50) at 0.022 microM) whereas haEC-growth was inhibited less potently (IC(50) at 97 microM). In addition, proliferation and migration of haSMC through a porous membrane were inhibited dose dependently by micromolar atRA-doses after non-stop and single dose application of atRA on the endothelial side of the complex transfilter coculture system. Immunostainings and Northern blotting demonstrated an enhanced alpha-smooth muscle actin and heavy chain myosin expression in haSMC after atRA-treatment. Whereas mRNA-expression of the glycoproteins thrombospondin-1 and fibronectin were decreased, collagen-1 mRNA expression was even slightly stimulated. Transcription of biglycan and TGF-beta1 were not influenced in a specific manner. Finally, protein expression and activity of the matrix metalloproteinases MMP-2 and MMP-9 were inhibited significantly by atRA. CONCLUSIONS atRA was found to be a potent inhibitor of both haSMC-proliferation and -migration, even in coculture with haEC releasing growth factors. In addition, redifferentiation, ECM synthesis and ECM degradation were regulated by atRA which also influence haSMC migration and intima formation. Thus, atRA-treatment seems to be a promising strategy for the inhibition of processes involved both in atherosclerosis and restenosis.

Effects of cerivastatin on human arterial smooth muscle cell proliferation and migration in transfilter cocultures

Statins competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity reducing mevalonate synthesis. In this study, antiproliferative and antimigratory effects of the new compound cerivastatin were analyzed and compared with classic statins of the first and second generation using mono- and cocultures of human arterial smooth muscle (haSMC) and endothelial (haEC) cells. Effects on the mitotic index and mitochondrial activity of haEC and haSMC monocultures were tested using BrdU enzyme-linked immunosorbent assay (ELISA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) tests, respectively. In lactate dehydrogenase (LDH) assays, cytotoxicity of statins was studied. Transfilter cocultures were performed for 14 days to evaluate haSMC growth under the stimulatory effect of proliferating haEC, which release growth factors [e.g., platelet-derived growth factor (PDGF)]. The hydrophobic statins simvastatin, lovastatin, and atorvastatin significantly inhibited haSMC and haEC growth in monocultures at 0.5-50 microM. However, most potent effects were exerted by cerivastatin in 10- to 30-fold lower doses without any significant cytotoxicity. More important, cerivastatin showed also significant effects on haSMC proliferation and migration in transfilter cocultures at extremely low doses (IC50, 0.04-0.06 microM), even when applied exclusively to the endothelial side and in the presence of low-density lipoprotein (LDL). Addition of mevalonate abolished the effects of cerivastatin completely. Even in the presence of growth-stimulating haEC and LDL, cerivastatin was found to be the most potent inhibitor of haSMC proliferation and migration in doses that also can be reached in human serum after oral drug administration. The results support the concept that statins seems to influence additional cellular mechanisms beyond cholesterol reduction, which might also have a relevance for the prevention of restenosis.

Effect of HMG-CoA reductase inhibitors on extracellular matrix expression in human vascular smooth muscle cells

Abstract Clinical studies have shown that treatment with 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors can stabilize atherosclerotic plaques and slow their progression. One determinant of plaque stability and size is the composition of the vascular extracellular matrix. The aim of this study was to evaluate the effects of different HMG-CoA reductase inhibitors on the expression of major components of the vascular extracellular matrix in smooth muscle cells.Cultured human vascular smooth muscle cells were incubated for 24–72 h with the HMG-CoA reductase inhibitors lovastatin (1–50 μmol/L), simvastatin (0.05–20 μmol/L), and pravastatin ( 1–100 μmol/L). RNA expression of the extracellular matrix proteins thrombospondin-1, fibronectin, collagen type I, and biglycan as well as expression of the cytokine TGF-β1 was determined by Northern blotting. Extracellular matrix protein secretion was visualized by immunofluorescence. In addition, cell proliferation and viability were measured using BrDU-ELISAs, MTT-tests, and direct cell counting.Expression of thrombospondin-1 was significantly decreased after 24 h incubations with lovastatin in concentrations as low as 1 μmol/L. Coincubation with the cholesterol precursor mevalonate completely reversed this effect. The downregulation of thrombospondin-1 expression occured in the same concentration range that also inhibited cell proliferation. In contrast, lovatatin did not affect expression of fibronectin, whereas collagen type I and biglycan expression decreased only after long incubations with high, toxic lovastatin concentrations. Simvastatin, but not the very hydrophilic compound pravastatin, had a similar effect on extracellular matrix expression as lovastatin.In summary, lovastatin and simvastatin predominantly decrease the expression of the glycoprotein thrombospondin-1, which is functionally associated with smooth muscle cell migration and proliferation. In contrast, expression of plaque-stabilizing extracellular proteins such as collagen type I and biglycan are much less affected.

Induction of Cell-Rich and Lipid-Rich Plaques in a Transfilter Coculture System with Human Vascular Cells

Cell-to-cell interactions are mainly involved in the control of the proliferation, migration, differentiation and function of different cell types in a wide range of tissues. In the arterial vessel wall, human arterial endothelial cells (haEC) and smooth muscle cells (haSMC) coexist in close contact with each other. In atherogenesis, haSMC can migrate from the media to the subintimal space to form fibromuscular and atheromatous plaques. In the present study, a transfilter coculture system is described, in which the interface between haSMC and confluent or proliferative haEC can be studied in detail. Cells were cocultured on the opposite sides of a porous filter which separates both cell types like the internal elastic lamina in vivo. In cocultures containing proliferative haEC, haSMC growth was significantly stimulated (33.4 +/- 5.7 cells/section, p < 0.05) compared to haSMC monocultures (22.9 +/- 2.5 cells/section) and cocultures containing confluent haEC (15.6 +/- 2.9 cells/section). If confluent haEC were injured mechanically, haSMC growth increased highly significantly (71.3 +/- 16.8 cells/section, p < 0.001). Thus, cell-rich proliferates containing 5-7 layers of haSMC embedded in extracellular matrix were formed after 14 days. On the other hand, after haSMC migration to the endothelial side had occurred, the addition of LDL and monocytes to cocultures with arterial media explants and haEC resulted in the formation of lipid-rich, low-cellular structures. After 28 days, characteristic in vitro plaque growth was induced; the plaque contained a lipid core with predominantly necrotic cells, extracellular lipid accumulations, atypically shaped lipid-loaded haSMC and macrophages, similar to in vivo foam cells, as well as an increased amount of extracellular matrix (collagen I, III and IV). These areas were surrounded by typical fibromuscular caps consisting of smooth muscle alpha-actin-positive haSMC. Finally, the formation of capillaries by haEC could also be observed within these structures.

Toxicity, uptake kinetics and efficacy of new transfection reagents: increase of oligonucleotide uptake.

Human arterial smooth muscle cell (haSMC) proliferation is stimulated by platelet-derived growth factor (PDGF) release of human arterial endothelial cells (haEC) whereas transforming growth factor-β1 (TGF-β1) secretion by haSMC promotes extracellular matrix formation. Inhibitory concepts with antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxicity and efficacy of new transfection reagents were examined. MTT tests showed that high doses >1.6 μg/ml of the liposome Cytofectin GSV® (CF) and the dendrimer SuperFect® (SF) reduced mitochondrial activity of haEC after ≥4 h transfection whereas viability of haSMC was not influenced. DAC-30® showed significant toxic effects on haEC and haSMC at each dose after ≥4 h and Lipofectin® (LF) caused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that ‘naked’ ASO were not incorporated intracellularly whereas transfection within CF or SF resulted in a strong cytoplasmic and nuclear labeling after 2–5 h. With DAC-30®, only a slight cytoplasmic fluorescence was found. SF caused an unexpected stimulation of endothelial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endothelial PDGF-B and smooth muscle TGF-β1 mRNA expression and synthesis after transfection for 3–5 h using 0.1–1.0 μM ASO versus control oligonucleotides. We conclude that Cytofectin GSV® is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin®. Cytofectin GSV® might offer a promising tool for antisense strategies in the treatment of vascular disorders.

Loss of cyclin A and G1-cell cycle arrest are a prerequisite of ceramide-induced toxicity in human arterial endothelial cells

BACKGROUND Ceramide is an important messenger of TNF- and lipid-induced apoptosis. We previously demonstrated the adverse effect of TNF in the process of reendothelialization as well as the dependence of its effect on cell-cycle regulation. The current study was designed to investigate the linkage between ceramide induced toxicity and growth arrest in human endothelial cells. METHODS AND RESULTS Cultured human arterial endothelial cells (HAEC) served as an in-vitro model to test the cellular effects of C2-ceramide (C2). C2-induced cell death in HAECs occurred time- and dose-dependently. The LD(50) in subconfluent cells was three times lower than in confluent cell layers (25 vs. 75 microM). C2 caused up to 70% inhibition of BrdU and [3H]thymidine incorporation at non-toxic concentrations as a result of G1 cell-cycle arrest. Downregulation of cyclin A and p21(Cip1/Waf1) protein expression was observed independently of C2-toxicity, while expression of other cell-cycle regulatory genes was not affected. Inhibition of cyclin A protein expression by sequence-specific antisense-oligonucleotides was paralleled by significant growth-inhibition. The protein phosphatase inhibitor okadaic acid induced endothelial cell proliferation, which was completely abrogated by C2. In contrast, aphidicolin-synchronized endothelial cells demonstrated elevated cyclin A levels along with 30% higher BrdU-incorporation and 70% less C2-toxicity. G1-arrested cells, however, showed significantly enhanced C2-toxicity, lack of cyclin A expression and induction of uncleaved caspase-3 (CPP32). CONCLUSIONS Ceramide abrogates endothelial cell proliferation independently of apoptosis or necrosis at low concentrations (<or=10 microM) through loss of cyclin A expression with subsequent G1 cell-cycle arrest. Synchronization of HAECs in S-phase with aphidicolin overcomes C2-induced G1-arrest and partially blocks ceramide toxicity. These findings demonstrate the dependence of ceramide toxicity on cell cycle regulation, suggesting a strong bidirectional relationship between cell-cycle control and cell death in vessel biology.

Downregulation of β-adrenergic receptors by low density lipoproteins and its prevention by β-adrenergic receptor antagonists

Objective: Vasodilation by β-adrenergic receptors of smooth muscle cells appears to be impaired early after the onset of hypercholesteremia. The aim of this study was to analyze the modulation of β-adrenergic receptor density and adenylyl cyclase activity in the presence of moderately elevated concentrations of LDL. The effects of β1- and β2-adrenergic receptor antagonists on LDL-induced receptor changes were studied. Methods and results: Media explants of porcine coronary arteries were incubated with moderately elevated LDL concentrations (0.7–3.9 mmol/l). The density of β-adrenergic receptors was determined in plasma membranes using the radioligand [125I]iodocyanopindolol. LDL (3.9 mmol/l) resulted in a decrease of β-adrenergic receptor density (control 137±5 vs. 89±7 fmol/mg protein, P <0.01). After removal of LDL and cultivation for an additional 3 days β-adrenergic receptors increased to 129±5 fmol/mg. In the presence of the β1- or β2-adrenergic receptor antagonists the LDL-mediated decrease was inhibited. Addition of metoprolol after 3 days of LDL incubation caused a restoration of receptor density. The basal, isoproterenol- and forskolin-stimulated adenylyl cyclase activities were increased after LDL incubation by 180, 110 or 80%, respectively. Conclusion: Moderately elevated LDL levels decreased β-adrenergic receptor density while adenylyl cyclase activity was simultaneously increased. β1- or β2-adrenergic receptor antagonists prevented this receptor decrease and might preserve the β-adrenergic receptor density in the presence of moderately elevated LDL levels.