Eugene F. Howard

Nuclear factor-κB activation during cerebral reperfusion: effect of attenuation with N-acetylcysteine treatment

We examined activation of the transcription factor, nuclear factor-kappaB (NF-kappaB), which participates in the upregulation of endothelial cell adhesion proteins, during reperfusion after temporary middle cerebral artery occlusion (TMCAO). We hypothesized that N-acetylcysteine (NAC), an antioxidant which inhibits NF-kappaB activation, would alter events in brain reperfusion injury. We used a rat model of TMCAO. The left sides of the brains were rendered ischemic for 2 h, and then the area was allowed to reperfuse. The animals were treated with NAC (150 mg/kg) or saline placebo, sacrificed, and activated NF-kappaB was assessed in both the left and right hemispheres, all at varying intervals. Cerebral infarction volume was also measured in each of the hemispheres collected from a separate group of animals. Activated NF-kappaB, consisting of p65 and p50 Rel proteins, was significantly increased 15 min after reperfusion in the affected hemisphere. The activation at 15 min was completely abolished with NAC treatment. NAC treatment 1 h prior to the end of occlusion and at 24 h reduced the percentage infarction volume of the affected hemispheres from 35.5+/-2.8% (S.E.) to 18. 1+/-2.1% (p<0.01). NAC treatment at 1 h after the occlusion (after the NF-kappaB peak) and again at 24 h also significantly reduced the percentage infarction volume from 34.8+/-3.8% to 24.6+/-3.8% (p<0. 05). Thus, while NAC inhibited activation of NF-kappaB at 15 min after reperfusion, the drug acted to reduce cerebral infarction by additional, undefined mechanisms. These results bring into question the various roles of NF-kappaB in cerebral infarction followed by reperfusion.

NF-κB is activated and ICAM-1 gene expression is upregulated during reoxygenation of human brain endothelial cells

Reperfusion injury is mediated, in part, by the upregulated expression of genes in microvascular endothelial cells that encode for inflammatory cytokines and adhesion molecules. The redox-regulated transcription factor, nuclear factor kappa B (NF-kappaB), may play a major role in the induced expression of these genes. In this study we use cultured human brain microvascular endothelial cells (HBMEC) to investigate whether reoxygenation of hypoxic HBMEC results in the activation of NF-kappaB and the upregulation of the adhesion molecule, ICAM-1. When HBMEC were subjected to hypoxia followed by reoxygenation but not hypoxia alone, an NF-kappaB complex composed of p65 and p50 Rel proteins was rapidly activated within 15-30 min. Four hours later, expression of the ICAM-1 gene was significantly upregulated. The antioxidant pyrrolidine dithiocarbamate and the proteasome inhibitor, n-Tosyl-Phe-chloromethyl ketone, blocked both the activation of NF-kappaB and the upregulation of the ICAM-1 gene. These results indicate that NF-kappaB is activated in HBMEC by reoxygenation and may play a significant role in the upregulation of the ICAM-1 gene. Agents which inhibit NF-kappaB activation may be potential therapeutic agents in acute ischemic stroke.

The NF-κB inhibitor diethyldithiocarbamate (DDTC) increases brain cell death in a transient middle cerebral artery occlusion model of ischemia

A transient ischemic middle cerebral artery occlusion model of stroke was used to examine the role of the transcription factor NF-kappaB in cell death as measured by DNA fragmentation and infarction volume. The left middle cerebral artery was occluded for either 30 min or 2 h in rats. One set of animals was pretreated with diethyldithiocarbamate (DDTC), an inhibitor of NF-kappaB, 30 min prior to reperfusion. The animals were reperfused and allowed to survive for 2 or 7 days. DNA fragmentation was assayed by in situ end labeling in the stroke core and penumbral regions. Specific cortical and subcortical regions were measured using quantitative image analysis. DNA fragmentation was seen only on the ischemic side of the brains in all cases. Overall, the DDTC-treated groups showed significantly increased DNA fragmentation within the ischemic side compared to the saline control groups. DDTC treatment also caused an increase in stroke volume based on triphenyl tetrazolium chloride staining. Electrophoretic mobility shift assays showed NF-kappaB activation peaking 15 min following reperfusion and that this activation was blocked by the DDTC treatment. This study suggests that the use of NF-kappaB inhibitors to block cell death following stroke needs to be carefully examined because global inhibitors may not promote neuronal survival.

Hypertonic Mannitol Loading of NF-κB Transcription Factor Decoys in Human Brain Microvascular Endothelial Cells Blocks Upregulation of ICAM-1

BACKGROUND AND PURPOSE An acute inflammatory response exacerbates tissue injury during acute ischemic stroke. The transcription factor nuclear factor (NF)-kappaB plays a key role in endothelial cell activation and the inflammatory response. Targeted genetic disruption of NF-kappaB activation in cerebral endothelial cells may be protective in stroke. We determined whether a NF-kappaB transcription factor decoy (TFD) could block intercellular adhesion molecule (ICAM)-1 upregulation, an indicator of endothelial cell activation. METHODS We modeled ischemia-reperfusion in vitro by exposing cultured human brain microvascular endothelial cells (HBMEC) to tumor necrosis factor (TNF)-alpha and conditions of hypoxia-reoxygenation (H/R). Mannitol was used to load phosphothiorated oligonucleotides containing 3 copies of the kappaB binding sequences (TFDs) into cultured HBMEC. An NF-kappaB TFD, a mutated NF-kappaB TFD, and a scrambled TFD were studied for their effect on ICAM-1 mRNA levels and surface ICAM-1 by ELISA. RESULTS Hyperosmolar loading with mannitol permitted rapid transfection of TFD into endothelial cell nuclei. The NF-kappaB TFD but not the mutated or scrambled TFD competed with a kappaB sequence for binding to nuclear extracts from HBMEC exposed to TNF-alpha. The NF-kappaB TFD blocked the TNF-alpha-induced and H/R-induced increase in ICAM-1 mRNA levels and the upregulation of surface ICAM-1. CONCLUSIONS Mannitol delivers phosphothiorated oligonucleotides into cultured HBMEC. An NF-kappaB decoy blocks both TNF-alpha-induced and H/R-induced ICAM-1 upregulation in HBMEC. Targeted genetic disruption of endothelial NF-kappaB activation may be of benefit in acute ischemic stroke.

Characterization and mapping of DNA sequence homologous to mouse U1a1 snRNA: Localization on chromosome 11 near the Dlb-1 and Re loci

A phage clone which contained a functional U1a1snRNA gene was isolated from a mouse genomic library. A single copy fragment was isolated from the 3′ flanking region of the U1a1gene and used as a hybridization probe for Southern blotted DMAs from recombinant inbred strains of mice, mouse-hamster hybrid cells, and the offspring from backcrosses between BALB/c mice and mice which were heterozygous for the Rex (Re)marker. The results of these experiments prove that the U1a1gene is located on chromosome 11 near the Delb-1and Reloci.

Reduction of T-kininogen messenger RNA levels by dexamethasone in the adjuvant-treated rat

When inflammation is induced in rats following injection of Freunds complete adjuvant, steady state levels of T-I and T-II kininogen mRNAs increase markedly as do plasma levels of T-I and T-II kininogens. When rats are additionally treated with dexamethasone, T-I and T-II steady state mRNA levels and plasma levels of T-kininogens are reduced. The results suggest that dexamethasone may affect the magnitude of T-kininogen gene induction caused by inflammation.

Isolation of low molecular weight nuclear RNA from small numbers of nuclei with cetyltrimethylammonium bromide.

Abstract A method is presented for the isolation of low molecular weight nuclear (LMN) RNAs from small numbers of nuclei. Cetyltrimethylammonium bromide (CTAB) is used to precipitate small quantities of whole nuclear RNA from dilute aqueous solution following phenol-SDS extraction of purified nuclei. No carrier RNA is necessary during the precipitation step. LMN RNAs are separated from whole nuclear RNA by electrophoresis on polyacrylamide gels. No further purification of the RNA is necessary prior to electrophoresis. Both radioactivity and absorbance profiles of the LMN RNAs on the gels can be obtained. Thus, specific activities of labeled LMN RNA species can be estimated.