R. Viebahn

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.

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.

Cultivation of porcine hepatocytes in polyurethane nonwovens as part of a biohybrid liver support system.

Many patients suffering from end-stage liver disease cannot be transplanted within reasonable time due to the shortage of donor organs. Bioartificial liver support systems may contribute to the liver regeneration or bridging the time until a liver graft for transplantation becomes available. Nonwovens with integrated oxygenation capacity have been developed and manufactured by melt blow technology using thermoplastic polyurethane. Capillary membranes for oxygenation were integrated into the nonwoven during the processing. The polyurethane nonwoven structures with adapted pore size and high pore volume allow high cell densities in the hepatocyte culture. The three-dimensional cell culture was housed by a flow bioreactor system and was integrated in a closed loop circulation with monitoring possibilities for pressure, pH, temperature, ammonia, and oxygen. Hepatocytes were isolated from rats or pigs by collagenase perfusion and infused into the medium-perfused circulation. Cells showed high viability and hepatocyte specific cytochrome P450-dependent metabolic function in culture (MEGX test).

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.

TNF-α and its receptors mediate graft rejection and loss after liver transplantation

Abstract Tumour necrosis factor-α (TNF-α) plays a pivotal role in the immune response and mediates inflammation by its receptors (TNF-RI and TNF-RII), as observed during rejection episodes and impaired graft function after liver transplantation. TNF-α and its receptors were analysed by an ELISA technique in serum samples from 77 consecutive liver transplantations in 63 patients. Samples were collected preoperatively from donors and recipients and then daily in the first two postoperative weeks. Peak levels of TNF-α and its soluble receptors (sTNF-RI and sTNF-RII) in the first and second postoperative week correlated with the extent of reperfusion injury. Impaired graft functions correlated with high sTNF-RI levels preoperatively (> 5 ng/ml, p = 0.01) and in the postoperative period (> 16 ng/ml, p = 0.02). Significantly increased TNF-α (> 25 pg/ml, p = 0.009) and sTNF-RI levels (> 5 ng/ml, p = 0.05) were found in donors of grafts with a high rejection risk. Elevated levels of TNF-α in the postoperative period correlated with an increased rejection risk (> 90 pg/ml, p = 0.02). The activity of the immune system with high concentrations of TNF-α and its receptors both in the recipient and the transplant donor seems to play an essential role in allograft development.

RANTES in the postoperative course after liver transplantation

Abstract RANTES (regulated upon activation, normal T‐cell expressed and secreted), an inflammatory cytokine, promotes accumulation and activation of leukocytes. In 67 liver transplantations, systemic concentrations of RANTES were correlated to graft survival and incidence of rejection. RANTES levels either increased to highly elevated levels at day 14 (84 ± 64 ng/ml; group 1; n = 43) or remained within the limit of healthy controls (19 ± 11 ng/ml at day 14; group 2; n = 24). The 100‐day graft function rate was 0.91 in group 1 and 0.63 in group 2 (P = 0.002). The risk ratio for rejection during the first 100 days was increased 2.2‐fold in group 2 compared to group 1 (P = 0.02). High postoperative release of RANTES after liver transplantation, a beneficial factor, may reflect a general systemic immunological activation. It can be concluded that high early systemic RANTES levels may play a role in immunological recognition leading to a tolerance of the liver graft.

Endotoxemia in organ donors: graft function following liver transplantation.

Abstract Translocation of endotoxin (LPS) to the portal‐venous system is produced by multiple factors. In the case of normal liver function, LPS is rapidly cleared from the portal blood by Kupffer cells; in impaired liver function, LPS can reach the systemic circulation. The objective of this study was to investigate whether elevated donor endotoxin levels affect graft function in the recipient. LPS levels in donor plasma were measured in 14 consecutive liver transplantations. Grafts with donor LPS levels ≤ 12 pg/ml had a function probability of 100% after 600 days (n = 10). LPS concentrations of > 12 pg/ml in donor plasma led to loss of function in 75% of the liver grafts (n = 4; P = 0.003; Wilcoxon). Elevated LPS values in donor plasma seem to impair the prognosis of the grafts and could predict poor graft function as early as at the time of brain death.

MHC antigen presentation on the surface of hepatocytes: modulation during and after hypoxic stress.

Abstract Presentation and recognition of MHC antigen on the surface of cells is the basic process of initiating rejection and distinguishing “self and not self‘. This is considered to begin when a transplanted organ is reperfused with the blood of the recipient. In this study, the modulation of MHC I antigen presentation during preservation was investigated in a cell culture model using primary hepatocyte cultures of male Wistar rats. By incubation with different preservation solutions used in clinical transplantation, expression of the MHC antigen was observed during cold hypoxia. Primary hepatocyte cultures were isolated from male Wistar rats by a modified Seglen technique and seeded to glass slides, thus obtaining monolayer cultures. After 1 day of resting the cultures were incubated under different conditions using Krebs Henseleit solution (KH), Euro‐Collins solution (EC), HTK solution of Bretschneider (HTK) and University of Wisconsin solution (UW) as incubation media. The conditions of incubation were warm normoxia (37°C, pO2 100 mmHg) and cold hypoxia (4°C, pO2 < 0.1 mmHg), which is the main condition of organ preservation. Incubation time was 6 h. Before starting the incubation reference cultures were fixed and every 60 min several cultures were withdrawn from the experiment and fixed also. MHC expression was studied by staining the cultures with monoclonal antibodies against rat MHC class I and class II. As expected, MHC class II was not present on the surface of hepatocytes, while MHC class I was demonstrated on the controls as well as on the cultures that were incubated using KH and EC independent of temperature and hypoxia or normoxia. At the end of the incubation they were still positive but not as strong as in the beginning. During incubation using HTK and UW, MHC I was not detectable at all phases of the experiment. In conclusion, hypoxic stress did not completely eradicate MHC I expression in rat hepatocytes, while the composition of the preservation medium may result in a hepatocyte surface negative for MHC I. Hydroxyethylstarch may be the substance in UW that covers the MHC antigen, while in HTK, mannitol or the histidine complex may play the same role. MHC negativity of the cells of a preserved transplant is another reason for the benefit of UW or HTK.

Primäre hepatozytenkulturen als modell zur experimentellen untersuchung der leberkonservierung

SummaryPrimary hepatocyte cultures have been used to evaluate data concerning hypoxic liver cell injury. To show the suitability of this method in liver preservation studies hepatocyte cultures were incubated under different conditions: warm normoxia (37°C, pO2 > 70 mm Hg), warm hypoxia (37°C, pO2 < 0.1 mm Hg), cold normoxia (4°C, pO2 > 70 mm Hg) and cold hypoxia (4°C, pO2 < 0.1 mmHg). Incubations were performed in Euro Collins solution (EC), University of Wisconsin solution of Belzer (UW) and histidine ketoglutarat tryptophan solution of Bretschneider (HTK) as well as in Krebs Henseleit buffer (KH) for control incubations. During 12 h of incubation hepatocyte cultures under warm normoxia lost viability continously in EC, UW and HTK while in KH they remained stable. Under warm normoxia all cultures lost 50% of their viability during 12 h of incubation while in cold normoxia loss of viability was mild but significant. Under cold anoxia which is the standard condition of liver preservation the cultured hepatocytes remained unchanged for 12 h in KH, UW and HTK, while in EC most of the cells were dead after 6 h. It is concluded that incubations of primary hepatocyte cultures under different pO2 and temperatures are well suited to contribute to liver preservation studies on a preclinical level and thus may help to save animal experiments.ZusammenfassungPrimäre Leberzellkulturen haben bisher bei biochemischen Untersuchungen zum Pathomechanismus des hypoxischen Leberzellschadens Anwendung gefunden. Um die Eignung der Methode für Untersuchungen zur Leberkonservierung zu prüfen, wurden Leberzellkulturen bei normothermer Normoxie (37°C, pO2 > 70 mm Hg), normothermer Hypoxie (37°C, pO2 < 0,1 mm Hg), hypothermer Normoxie (4°C, pO2 > 70 mm Hg) and hypothermer Hypoxie (4°C, pO2 < 0,1 mm Hg) inkubiert. Bei jeder Inkubationsbedingung wurde Euro-Collins-Lösung (EC), University-of-Wisconsin-Lösung nach Belzer (UW) and Histidin-Tryptophan-Ketoglutarat-Lösung nach Bretschneider (HTK) als Inkubationsmedium verwendet; Kontrollinkubationen erfolgten in Krebs-Henseleit-Puffer (KH). Das Absterbeverhalten der kultivierten Hepatozyten zeigte während 12stündiger Inkubation einen raschen Viabilitätsverlust bei warmer Normoxie in EC, HTK and UW. In KH blieben die Zellen nahezu vollstandig vital. Bei normothermer Hypoxie starben die Leberzellen in allen Methen während der 12stündigen Inkubation zu mindestens 50% ab, während bei hypothermer Normoxie der Vitalitätsverlust deutlich verlangsamt war. Hypotherme Hypoxie schließlich zeigte in KH, HTK and UW eine nahezu vollständig erhaltene Zellviabilität ℏer 12 h, während in EC nach 6 h die meisten Zellen abgestorben waren. Durch diese Ergebnisse zeigt sich die Methode der Inkubation von primären Leberzellkulturen unter verschiedenen Sauerstoffpartialdrucken und Temperaturen als geeignet zur Evaluierung von Konservierungsmedien und ihrer Komponenten in der präklinischen Phase unter Einsparung von Tierversuchen.Primary hepatocyte cultures have been used to evaluate data concerning hypoxic liver cell injury. To show the suitability of this method in liver preservation studies hepatocyte cultures were incubated under different conditions: warm normoxia (37 degrees C, pO2 greater than 70 mm Hg), warm hypoxia (37 degrees C, pO2 less than 0.1 mm Hg), cold normoxia (4 degrees C, pO2 greater than 70 mm Hg) and cold hypoxia (4 degrees C, pO2 less than 0.1 mm Hg). Incubations were performed in Euro Collins solution (EC), University of Wisconsin solution of Belzer (UW) and histidine ketoglutarat tryptophan solution of Bretschneider (HTK) as well as in Krebs Henseleit buffer (KH) for control incubations. During 12 h of incubation hepatocyte cultures under warm normoxia lost viability continuously in EC, UW and HTK while in KH they remained stable. Under warm normoxia all cultures lost 50% of their viability during 12 h of incubation while in cold normoxia loss of viability was mild but significant. Under cold anoxia which is the standard condition of liver preservation the cultured hepatocytes remained unchanged for 12 h in KH, UW and HTK, while in EC most of the cells were dead after 6 h. It is concluded that incubations of primary hepatocyte cultures under different pO2 and temperatures are well suited to contribute to liver preservation studies on a preclinical level and thus may help to save animal experiments.