Gene Expression Profiling of Inflamed Human Endothelial Cells and Influence of Activated Protein C

Abstract

Background-During systemic inflammation, activation of vascular endothelium by proinflammatory cytokines leads to hypotension, microvascular thrombosis, and organ damage. Recent data suggest a link between coagulation and inflammation through the activated protein C (APC) pathway. We studied gene expression profiles in human coronary artery endothelial cells (HCAECs) exposed to proinflammatory stimuli and the influence of APC on expression of candidate genes regulated by these stimuli. Methods and Results-HCAECs were stimulated with interleukin-1␤, interferon-␥, and tumor necrosis factor-␣. In gene expression profiling, 400 of 8400 genes were regulated Ͼ2-fold. Verification of selected candidate genes was achieved by measuring expression of mRNA species by real-time polymerase chain reaction, cytokine secretion by ELISA, and metabolites of tetrahydrobiopterin (BH4) biosynthesis by high-performance liquid chromatography. BH4 synthesis, interleukin-6, interleukin-8, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were downregulated by APC at the transcriptional and protein level. Endothelial nitric oxide synthase, endothelial adhesion molecule, and vascular cell adhesion molecule-1 were not affected by APC. Activities of transcription factors c-Fos, FosB, and c-Rel were inhibited by APC in inflamed HCAECs. Conclusions-Our study revealed a novel antiinflammatory mechanism of APC-dependent gene regulation in HCAECs since c-Fos-dependent induction of MCP-1 and ICAM-1 was suppressed. APC downregulates expression and activity of genes related to inflammation, most pronounced under intermediate or mild inflammatory conditions. (Circulation.

Protease-activated Receptor-1 Signaling by Activated Protein C in Cytokine-perturbed Endothelial Cells Is Distinct from Thrombin Signaling

“…Although previous studies have shown that APC administration can attenuate the cytokine perturbation of endothelial cells (38,39), APC appears to be particularly effective in severe sepsis where coagulopathy and thrombin generation is frequent. How can it be explained that thrombin-PAR1 signaling can have proinflammatory effects (8), whereas APC signaling is protective in inflammatory conditions if both thrombin and APC signal through the same receptor?…”

Microparticle-associated endothelial protein C receptor and the induction of cytoprotective and anti-inflammatory effects

“…This suggests that in the absence of inflammatory stimuli, APC may not have a significant influence on NFκB activation and that the main effect of this protein seems to be the preservation of cell viability and endothelial barrier function. Anti-apoptotic effects and permeability barrier protection by APC require EPCR binding and PAR1 activation 17,23,24 and these can be induced through both gene expression and post-translational modification of cellular proteins. The finding that genes related to anti-apoptosis are highly up-regulated in our system could explain the inhibition of staurosporineinduced apoptosis by APC on microparticles observed in the present study.…”

Immune Regulation by Microvascular Endothelial Cells: Directing Innate and Adaptive Immunity, Coagulation, and Inflammation

“…In experimental endotoxininduced inflammation, activated PC inhibits pulmonary vascular injury by inhibiting TNF-␣ release (72) and limits accumulation of activated leukocytes (73). In human EC-based in vitro systems, activated PC modulates expression of genes related to anti-inflammatory and cell survival pathways, including inhibition of NF-B binding to target sites and multiple NF-B-regulated genes, such as cytokines, chemokines, and adhesion molecules (74,75).…”