Abstract
Endothelial cells are often present at inflammation sites. This is the case of endothelial cells of the blood-brain barrier (BBB) of patients afflicted with neurodegenerative disorders such as Alzheimer's, Parkinson's, or multiple sclerosis, as well as in cases of bacterial meningitis, trauma, or tumor-associated ischemia. Inflammation is a known modulator of gene expression through the activation of transcription factors, mostly NF-κB. RLIP76 (a.k.a. RALBP1), an ATP-dependent transporter of electrophile-glutathione conjugates, modulates BBB permeability through the regulation of tight junction function, cell adhesion, and exocytosis. Genes and pathways regulated by RLIP76 are transcriptional targets of tumor necrosis factor alpha (TNF-α) pro-inflammatory molecule, suggesting that RLIP76 may also be an inflammation target. To assess the effects of TNF-α on RLIP76, we faced the problem of choosing reference genes impervious to TNF-α. Since such genes were not known in human BBB endothelial cells, we subjected these to TNF-α, and measured by quantitative RT-PCR the expression of housekeeping genes commonly used as reference genes. We find most to be modulated, and analysis of several inflammation datasets as well as a metaanalysis of more than 5000 human tissue samples encompassing more than 300 cell types and diseases show that no single housekeeping gene may be used as a reference gene. Using three different algorithms, however, we uncovered a reference geneset impervious to TNF-α, and show for the first time that RLIP76 expression is induced by TNF-α and follows the induction kinetics of inflammation markers, suggesting that inflammation can influence RLIP76 expression at the BBB. We also show that MRP1 (a.k.a. ABCC1), another electrophile-glutathione transporter, is not modulated in the same cells and conditions, indicating that RLIP76 regulation by TNF-α is not a general property of glutathione transporters. The reference geneset uncovered herein should aid in future gene expression studies in inflammatory conditions of the BBB.
Highlights
Endothelial cells line the lumen of the entire circulatory system including all blood and lymphatic vessels, myocardium, renal glomeruli, and the blood-brain barrier (BBB)
Since such genes were not known in human BBB endothelial cells, we subjected these to tumor necrosis factor α (TNF-α), and measured by quantitative reverse transcription (RT)-PCR the expression of housekeeping genes commonly used as reference genes
Once we identified a reference geneset, we used it as a Normalization Factor to assess expression modulation by TNF-α of additional test genes
Summary
Endothelial cells line the lumen of the entire circulatory system including all blood and lymphatic vessels, myocardium, renal glomeruli, and the blood-brain barrier (BBB). Endothelial cells have been reported to be exposed to the pro-inflammatory tumor necrosis factor α (TNF-α) cytokine in acute systemic conditions such as inflammation in response to bacteremia [1, 2], acute localized inflammation following myocardial infarction [1, 3], ischemic stroke [4], angioplasty [5], or post-ischemic reperfusion injury [6, 7]. At the blood-brain barrier (BBB), endothelial cells can be exposed to inflammation associated with brain-specific injuries and disorders such as Alzheimer's disease [25], bacterial meningitis [26], brain edema due to head trauma [27], ischemia and hypoxia [28, 29], tumors [30], epilepsy [31, 32], multiple sclerosis [28, 29], or Parkinson's disease [33, 34]. Inflammation modulates gene expression in many tissues, including endothelial cells [24, 35,36,37,38], via mechanisms that involve activation of transcription factors, most importantly NF-κB [39,40,41,42,43]
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