Therapy-resistant tumor microvascular endothelial cells contribute to treatment failure in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a devastating disease with high mortality and poor prognosis. Cancer stem cells (CSCs) have recently been defined as a fraction of tumor cells highly resistant to therapy and subsequently considered to be responsible for tumor recurrence. These cells have been characterized in GBM and suggested to reside in and be supported by the tumor microvascular niche. Here we evaluated the response of tumor microvascular endothelial cells (tMVECs) to radio- and chemotherapy, and analyzed how this affects their interaction with CSCs. Our data demonstrate that tMVECs exhibit extreme resistance to both therapies, with the main response to irradiation being senescence. Importantly, senescent tMVECs can be detected in human GBM samples as well as in mice upon irradiation. Even though permanently arrested, they are still viable and able to support CSC growth with the same efficacy as non-senescent tMVECs. Intriguingly, GBM CSCs themselves are capable of differentiating into cells with similar features as tMVECs that subsequently undergo senescence when exposed to radiation. This indicates that endothelial-like cells are therapy resistant and, more importantly, support expansion of GBM cells.

Vagus nerve stimulation inhibits activation of coagulation and fibrinolysis during endotoxemia in rats.

BACKGROUND: Sepsis and endotoxemia are associated with concurrent activation of inflammation and the hemostatic mechanism, which both contribute to organ dysfunction and death. Electrical vagus nerve stimulation (VNS) has been found to inhibit tumor necrosis factor (TNF)-alpha release during endotoxemia in rodents. OBJECTIVE: To determine the effect of VNS on activation of coagulation and fibrinolysis. METHODS: Rats received a sublethal i.v. dose of lipopolysaccharide (LPS) after electrical VNS or sham stimulation. Activation of coagulation and fibrinolysis, as well as cytokine release, was measured before LPS injection and 2, 4 and 6 h thereafter. RESULTS: LPS induced activation of the coagulation system (increases in the plasma concentrations of thrombin-antithrombin complexes and D-dimer, and a decrease in antithrombin) and biphasic changes in the fibrinolytic system [early rises of plasminogen activator activity and tissue-type plasminogen activator, followed by a delayed increase in plasminogen activator inhibitor type 1 (PAI-1)]. VNS strongly inhibited all LPS-induced procoagulant responses and more modestly attenuated the fibrinolytic response. In addition, VNS attenuated the LPS-induced increases in plasma and splenic concentrations of the proinflammatory cytokines TNF-alpha and interleukin-6 (IL-6), while not influencing the release of the anti-inflammatory cytokine IL-10. CONCLUSION: These data illustrate a thus far unrecognized effect of VNS and suggest that the cholinergic anti-inflammatory pathway not only impacts on inflammation but also on the coagulant-anticoagulant balance.

Evaluation of a novel bioartificial liver in rats with complete liver ischemia: treatment efficacy and species-specific alpha-GST detection to monitor hepatocyte viability.

BACKGROUND/AIMS: There is an urgent need for an effective bioartificial liver system to bridge patients with fulminant hepatic failure to liver transplantation or to regeneration of their own liver. Recently, we proposed a bioreactor with a novel design for use as a bioartificial liver (BAL). The reactor comprises a spirally wound nonwoven polyester fabric in which hepatocytes are cultured (40 x 10(6) cells/ml) as small aggregates and homogeneously distributed oxygenation tubing for decentralized oxygen supply and CO2 removal. The aims of this study were to evaluate the treatment efficacy of our original porcine hepatocyte-based BAL in rats with fulminant hepatic failure due to liver ischemia (LIS) and to monitor the viability of the porcine hepatocytes in the bioreactor during treatment. The latter aim is novel and was accomplished by applying a new species-specific enzyme immunoassay (EIA) for the determination of porcine alpha-glutathione S-transferase (alpha-GST), a marker for hepatocellular damage. METHODS: Three experimental groups were studied: the first control group (LIS Control, n = 13) received a glucose infusion only; a second control group (LIS No-Cell-BAL, n = 8) received BAL treatment without cells; and the treated group (LIS Cell-BAL, n = 8) was connected to our BAL which had been seeded with 4.4 x 10(8) viable primary porcine hepatocytes. RESULTS/CONCLUSIONS: In contrast to previous comparable studies, BAL treatment significantly improved survival time in recipients with LIS. In addition, the onset of hepatic encephalopathy was significantly delayed and the mean arterial blood pressure significantly improved. Significantly lower levels of ammonia and lactate in the LIS Cell-BAL group indicated that the porcine hepatocytes in the bioreactor were metabolically activity. Low pig alpha-GST levels suggested that our bioreactor was capable of maintaining hepatocyte viability during treatment. These results provide a rationale for a comparable study in LIS-pigs as a next step towards potential clinical application.

Does the extend of the culture time of primary hepatocytes in a bioreactor affect the treatment efficacy of a bioartificial liver?

The purpose of this study was to investigate whether the efficacy of our novel extracorporeal bioartificial liver (BAL) to support rats with complete liver ischemia (LIS) could be improved by extending the culture time of freshly isolated porcine hepatocytes from 14 hours to 38 hours. The results showed that survival as well as porcine hepatocyte integrity improved, the onset of coma delayed, and the ammonia levels decreased in LIS rats of the 38 hour group compared to the 14 hour group, but no statistically significant differences were observed. In the 38 hour group, but not the 14 hour group, the onset of hepatic encephalopathy was significantly delayed and ammonia metabolism significantly improved compared to the LIS rats in control groups that only received a glucose infusion or were connected to a BAL without cells. In conclusion, prolonged hepatocyte recovery favoured all investigated parameters, although not all observed effects were statistically significant. More research is required to find out how long primary hepatocytes should be cultured in a bioreactor for optimal BAL support.

Commercially available media for flushing extracorporeal bioartificial liver systems prior to connection to the patient's circulation: an in vitro comparative study in two and three dimensional porcine hepatocyte cultures.

Extracorporeal bioartificial liver (BAL) systems based on hepatocytes need to be flushed before clinical application, as hepatocyte culture media are not approved for medical use. Commercially available 0.9% NaCl solution and hemofiltration solution (both supplemented with 10% human albumin) were investigated in vitro to test their potential to wash BAL systems with minimal stress for the cultured hepatocytes. After a 2 hour incubation, the lidocaine metabolising capacity and release of liver enzymes were assessed. As hepatocytes have been cultured in bioreactors in either two or three dimensional cell configurations, we tested the media in respectively hepatocyte monolayers cultures and in our newly developed bioreactor in which hepatocytes reorganise as small hepatocyte aggregates. The three dimensional hepatocyte cultures tolerated both media well, and no significant differences were seen compared with hepatocytes cultured in Williams' E (reference hepatocyte culture medium). The two dimensional hepatocyte cultures tolerated the supplemented hemofiltration solution and the reference medium equally well, but the condition of the porcine hepatocytes monolayer cultures was significantly impaired when incubated with the supplemented physiological saline solution. In conclusion, as a supplemented physiological saline solution may have detrimental effects on the condition of the hepatocytes, the more complex hemofiltration solution (bicarbonate buffered, glucose, essential minerals) was considered the better alternative for flushing bioartificial liver systems.

Memantine, a noncompetitive NMDA receptor antagonist improves hyperammonemia-induced encephalopathy and acute hepatic encephalopathy in rats.

The aim of this study was to investigate the possible role of N-methyl-D-aspartate (NMDA)-receptor overactivity in two different experimental rat models of encephalopathy: subacute encephalopathy caused by severe hyperammonemia in portacaval-shunted rats (AI-PCS rats) and acute hepatic encephalopathy caused by complete liver ischemia (LIS rats). The effect of the noncompetitive NMDA-receptor antagonist memantine (intraperitoneal [i.p.] 10-20 mg/kg bw or intravenous [i.v.] 5 mg/kg bw) was studied on the severity of encephalopathy by assessment of clinical grading and electroencephalogram (EEG) spectral analysis, on plasma ammonia concentrations, amino acid concentrations in cerebrospinal fluid (CSF), intracranial pressure (ICP), and brain water content. Both rat models developed encephalopathy within 3 to 6 hours, associated with increased CSF glutamate and aspartate concentrations and increased ICP and brain water content. Memantine administration in AI-PCS and LIS rats resulted in a significant improvement in clinical grading and less slowing of EEG activity (P < .05), and smaller increases in CSF glutamate (P < .05) concentrations. Moreover, ICP and brain water content were significantly lower in memantine-treated AI-PCS rats than in untreated AI-PCS rats (P < .05). Memantine had no significant effect on ICP and brain water content in LIS rats, and on ammonia concentrations in both models. These results indicate that NMDA-receptor activation might be involved in the pathogenesis of hyperammonemia-induced encephalopathy and of acute hepatic encephalopathy caused by LIS.