Abstract
The expression profile of a number of genes in the rat mesocecum linked to sickle (SS) red blood cell (RBC) adhesion was examined by microarray analysis. The abnormal adherence of SS RBC to the vascular endothelium has been proposed to play an important role in vaso-occlusion in a double capacity; first, by actual physical blockage of the post capillary venules and, second, by inducing the expression of pleiotropic genes that could lead to further modification of the SS phenotype. The goals of these experiments were to identify these pleiotropic genes expressed upon adhesion of SS RBC, to understand how they contribute to the SS phenotype and, to identify potential targets for therapy. The rat mesocecum preparation was prepared as described (Kaul et al, Blood95:368, 2000) and platelet activating factor (PAF) was used to induce endothelial adhesion receptors to accentuate human SS RBC binding. Rat cecum/ mesocecum RNA was prepared by Qiagen RNeasy mini-kit and fluorescent labeled cDNA hybridized to 32K oligonucleotide microarrays followed by analysis using Gene Pix Pro 4.1. The effect of SS RBC perfusion on PAF-treated tissues vs untreated tissues was compared to PAF-only tissues vs untreated tissues using hierarchical clustering. SS RBC adhesion was analyzed as a function of venular diameter and was significantly different (p<0.00001) in the PAF treated tissues as revealed by a significantly higher Y-intercept (work quoted above). Of the 533 transcripts that were up-regulated and 353 transcripts that were down-regulated more than 2 fold, 68 were up-regulated and 32 were down-regulated more than 4-fold; of this subset, 91 transcripts were compared by Cluster analysis. Many of the up-regulated genes detected in the PAF treated SS RBC adherent tissues were associated with inflammation such as phospholipase A2, group IIA; glutathione peroxidase 2; sialyltransferase 4C; and integrin beta 4. Interestingly, a number of proteasome subunits and ubiquitin D were also highly up-regulated; these genes were only present in the SS RBC perfused PAF treated mesocecum and could then be considered potential targets for specific therapies e.g. proteasome inhibitors. Recent studies also indicate that the ubiquitin system controls NF-kappaB pathways that, in turn, control integrin expression that can increase cell adhesion. The genes that were down-regulated included apolipoprotein B; cytochrome P450 Cyp4b1; cyclin G associated kinase; and metallothionein 2 (MT2). The down-regulation of MT2 is interesting because it is known to be highly regulated in response to the plasma zinc concentration; due to the chronic inflammatory/oxidative condition present in SS disease, zinc may be depleted and feedback negatively on MT2 synthesis. SS RBC adhesion however, not just PAF treatment, is necessary for MT2 downregulation and may, paradoxically, increase zinc availability since MT expression and zinc depletion are inversely correlated. Overall, the results suggest that SS RBC adhesion regulates a number of mesocecum genes involved in the inflammatory response, regulation of oxidative damage, increased intracellular protein degradation, decreased vesicular transport and regulation of zinc ion concentration. These pleiotropic genes are candidates for epistatic (modifier) genes if found to be polymorphic in different individuals and ethnicities. Enhancing or interfering with these specific genes or metabolic pathways may open up new therapeutic strategies for SS disease.
Proteasome Inhibitors and Their Pharmacokinetics, Pharmacodynamics, and Metabolism
