Gene array transcript profiling of human endothelial cells identifies pathways regulated by Drotrecogin alfa (activated)

Background

Although the role of Drotrecogin alfa (activated) (recombinant human activated protein C [rhAPC]) in modulating microvascular coagulation through the inhibition of thrombin generation has been well studied in experimental and clinical settings of severe sepsis, little is known about its direct anti-inflammatory effects on vascular endothelial cells. To better understand the molecular mechanisms of action of rhAPC on endothelial cell function during sepsis we used gene array transcript profiling of messenger RNA (mRNA) from primary cultured human umbilical vein endothelial cells (HUVEC) exposed to Drotregocin alfa (activated) in the presence of the central proinflammatory mediator tumor-necrosis factor-alpha (TNF-alpha).

Methods and results

The effect of rhAPC on TNF-alpha-activated HUVEC was assessed using Affymetrix microarrays. Briefly, mRNA from treated cells was isolated and converted to double-stranded copy (c)DNA, which was then used to generate biotinylated cRNA. Biotinylated cRNA was hybridized to Affymetrix oligonucleotide arrays, containing approximately 33,000 human genes. Data analysis was performed using GeneChip 3.1 software. We found that rhAPC reproducibly upregulated TNF-alpha-induced gene expression of the following genes: monocyte chemoattractant protein-1 (MCP-1), platelet-derived growth factor-alpha-chain (PDGF-A), interleukin-6 (IL-6), transforming growth factor-beta receptor II, insulin-like growth factor-binding protein (IGF-BP) and interleukin-8 (IL-8). rhAPC downregulated the following genes induced by TNF-alpha stimulation: the secreted apoptosis related protein-1 (SARP-1), basic fibroblast growth factor (bFGF), lymphotoxin-β, the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Results for IL-6, IL-8 and MCP-1 were confirmed by protein measurements in cell culture supernatants by ELISA as well as by a colorimetric assay for mRNA quantitation (Quantikine assay).

Conclusions

The ability of rhAPC to modulate gene expression of a cluster of proinflammatory genes, genes responsible for cell adhesion and leukocyte trafficking as well as genes involved in endothelial apoptosis, provides insight into the molecular mechanisms contributing to the efficacy of rhAPC in systemic inflammation and sepsis.