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

Axel, Dorothea I.; Brehm, Bernhard R.; Wolburg-Buchholz, Karin; Betz, E.; Köveker, G.; Karsch, Karl R.

doi:10.1159/000159160

Cited by 42 publications

(23 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previously we could demonstrate that in transfilter cocultures activated haEC (e.g. after mechanical injury) significantly induce proliferation and migration of cocultured haSMC by the secretion of PDGF-AB and that TGF-β 1 release from haSMC stimulates matrix synthesis leading to the formation of fibromuscular proliferates [24, 25]. Controversy exists regarding the capability of ASO to enter cells and the exact ASO uptake mechanisms.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Toxicity, Uptake Kinetics and Efficacy of New Transfection Reagents: Increase of Oligonucleotide Uptake

Axel¹,

Spyridopoulos²,

Riessen³

et al. 2000

J Vasc Res

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

“…Human vascular cells were isolated from specimens of the iliac artery obtained from donors for liver transplantation, as described previously [25]. haEC were subcultured using the EGM kit without heparin (Promocell, Heidelberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Toxicity, Uptake Kinetics and Efficacy of New Transfection Reagents: Increase of Oligonucleotide Uptake

Axel¹,

Spyridopoulos²,

Riessen³

et al. 2000

J Vasc Res

Self Cite

View full text Add to dashboard Cite

show abstract

“…To obtain high-titer virus preparations, EC-propagated virus stocks were inoculated with fibroblasts for a single round of infection, harvested at 100% cytopathic effect (CPE), and made cell free by centrifugation. HAEC were isolated from adult human iliac arteries of bypass recipients by mechanically removing the endothelial layer as previously described (1,2) and were cultured in EGM-2 medium (Clonetics, Walkersville, Md.) containing 2% fetal calf serum on culture flasks (Greiner, Frickenhausen, Germany) coated with 2% gelatin.…”

Section: Efficient Lytic Infection Of Haec By Ec-propagated Hcmv Stramentioning

confidence: 99%

Efficient Lytic Infection of Human Arterial Endothelial Cells by Human Cytomegalovirus Strains

Kahl

Siegel-Axel

Stenglein³

et al. 2000

J Virol

View full text Add to dashboard Cite

Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) inEndothelial cells (EC) are major targets of human cytomegalovirus (HCMV) during acute infection of an immunocompromised host (18). In addition to contributing to hematogenous viral dissemination (7,8,13,19,23), infected EC may trigger direct vascular injury if viral induced cytopathogenicity occurs. In support of these in vivo data, susceptibility of cultured human venous EC (HUVEC) to productive lytic HCMV infection has been demonstrated (25). However, studies of interactions of HCMV with human arterial EC (HAEC) have generated conflicting results. Whereas reports about noncytopathic persistence of HCMV in cultured aortic EC (6) raised the possibility of different susceptibilities of EC depending on their vascular origin, Knight et al. (9) have described similar cytopathologies of HCMV in HUVEC, HAEC, and microvascular EC with regard to adhesion molecule induction. The respective usage of fibroblast-propagated and EC-propagated HCMV strains might provide an explanation for the discrepancies. While such interstrain differences in the cytopathic potential of HCMV in HUVEC are well established (11,20,22,24), the lytic potentials of various HCMV strains in HAEC have not yet been determined.In the present investigation we have tested the hypothesis that interstrain differences in viral host cell tropism, rather than properties inherent to EC of different vascular origins, determine the outcome of HCMV infection. To this end we have performed a detailed analysis of viral replication kinetics, long-term evolution of cytopathology, and lytic end points, comparing EC derived from human adult iliac artery, placental microvasculature, and umbilical vein following inoculation with various HCMV strains. Efficient lytic infection of HAEC by EC-propagated HCMV strains.In a first set of experiments, HCMV strains VHL/E (kindly provided by W. J. Waldman) (24) and TB40/E (22) were tested for cytopathic potential in HAEC. Both strains were propagated in HUVEC to preserve the natural endothelial cytopathogenicity of the original isolates. To obtain high-titer virus preparations, EC-propagated virus stocks were inoculated with fibroblasts for a single round of infection, harvested at 100% cytopathic effect (CPE), and made cell free by centrifugation. HAEC were isolated from adult human iliac arteries of bypass recipients by mechanically removing the endothelial layer as previously described (1, 2) and were cultured in EGM-2 medium (Clonetics, Walkersville, Md.) containing 2% fetal calf serum on culture flasks (Greiner, Frickenhausen, Germany) coated with 2% gelatin. For infection, cells were preincubated for 2 h in heparin-free medium, inoculated with cell-free virus at multiplicities of infection (MOI) of 0.1, 1, and 10, and incubated for 90 min before removal of the inoculum. The medium was replaced at 48-h intervals. The kinetics of viral antigen expression in HAEC cultures was analyzed by indirect immunoperoxidase detection of imm...

show abstract

“…Previously, we described some mono-and coculture systems having the advantage that primary cultures of human arterial endothelial (haEC) and smooth muscle cells (haSMCs) are isolated from human arteries and used in very early passages to preserve most of the in vivo properties of normal arterial cells in men [135 -137]. Furthermore, we developed a transfilter coculture model to imitate the morphology of the arterial vessel wall enabling cell-to-cell interactions by direct cell-cell contacts through lamellipodia and by the secretion of PDGF-AB, TGF-b1, and other mediators [136,138]. When cerivastatin was applied continously to the endothelial side of transfilter cocultures for 2 weeks, a dose-dependent inhibition of haSMC proliferation on the opposite filter side could be observed [139].…”

Section: Cerivastatin and Smooth Muscle Cell Proliferation/migrationmentioning

confidence: 99%

Cerivastatin: a cellular and molecular drug for the future?

Di¹

2003

Cellular and Molecular Life Sciences (CMLS)

View full text Add to dashboard Cite

The 'statin story' began in 1987 when the first-generation, fungal HMG-CoA reductase inhibitor lovastatin received FDA approval in the USA. Ten years later, the sixth compound of this class came onto the world market--the fully synthetic statin cerivastatin. A number of clinical studies had confirmed its high pharmacological efficacy, its excellent pharmacokinetic properties with fast and nearly complete absorption after oral uptake, a linear kinetic over a broad concentration range, and its favorable safety profile. The greatest advantages, of cerivastatin, however, are its lipophilicity, its high bioavailability of about 60% after oral application and its potency at 100-fold lower doses compared to other lipophilic statins. Nevertheless, the most exciting findings are certainly its non-lipid-related, pleiotropic effects at the cellular and molecular level. Statin therapy was also found to reduce mortality in cases where cholesterol levels or atherosclerotic plaque formation remained unaltered. However, cerivastatin improves endothelial dysfunction, possesses anti-inflammatory, antioxidant, anticoagulant, antithrombotic, antiproliferative, plaque-stabilizing, immunmodulatory, and angiogenic effects, and may even prevent tumor growth, Alzheimer's disease, and osteoporosis. Most of these effects seem to be based on the inhibition of isoprenoid synthesis. Although cerivastatin is no longer on the market because of some problematic side effects, it could be one of the most potent cellular and molecular drugs for the future.

show abstract

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

Cited by 42 publications

References 28 publications

Toxicity, Uptake Kinetics and Efficacy of New Transfection Reagents: Increase of Oligonucleotide Uptake

Toxicity, Uptake Kinetics and Efficacy of New Transfection Reagents: Increase of Oligonucleotide Uptake

Efficient Lytic Infection of Human Arterial Endothelial Cells by Human Cytomegalovirus Strains

Cerivastatin: a cellular and molecular drug for the future?

Contact Info

Product

Resources

About