Abstract
Background
It is well established that sickle cell disease (SCD) manifests global perturbations of hemostasis. Vaso-occlusion, inflammation and coagulopathy all likely contribute to the protean complications of SCD. Central to both inflammation and coagulation is the monocyte. These cells can be profoundly pro-inflammatory and can express tissue factor on their surface and thus influence both inflammation and coagulation. Monocytosis is common in SCD, as is steady state monocyte activation. Exaggerated monocytic response to stimulus may also contribute to the severity of acute events. Thus, agents that regulate monocyte function are potentially of significant relevance in SCD.
To this end, we have discovered that the heat shock proteins (HSPs) are potential master regulators of these cells. We previously demonstrated that inhibition of one such HSP, HSP90, could completely ablate the profound and hyper-responsive monocytic inflammatory release upon lipopolysacchiride (LPS) challenge. Inhibition of HSP90 also blocked LPS induced NFk-B translocation to the nucleus. Thus, these results suggested a potent role for HSP90 in LPS-induced monocyte based inflammation. However, the role of HSP90 is cytokine-induced monocyte activation was speculative. The role of HSP90 in monocyte tissue factor expression, or reactive oxygen species (ROS) generation remained unknown.
Objectives
We sought to determine the role of HSP90 in regulating the pro-inflammatory, pro-coagulant, and ROS generating potential in monocytes. We then wanted to establish a potential role for HSP90 inhibition.
Methods
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll density separation. Flow cytometry was employed to measure the LPS- or cytokine-induced monocyte tissue factor expression and ROS generation. Monocyte ROS generation was detected using L012 based chemiluminescence or visualized in individual cells using flow cytometry with CELLROX. Inflammatory cytokines and tissue factor gene expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). NFkB translocation to the nucleus was detected via cell fractionation followed by western blotting or indirect immunofluorescence. PBMCs or THP-1 cells were pre-treated with the HSP90 inhibitors 17-DMAG or AUY922 prior to assay. Monocyte- induced endothelial cell permeability was measured using endothelial cell-substrate impedance sensing (ECIS). Unless otherwise stated p<0.05.
Results
At baseline, PBMCs from patients with SCD demonstrated elevated monocyte ROS generation and tissue factor expression than those from healthy controls. Inhibition of significantly reduced these measures of steady state monocyte activation. HSP90 inhibition also inhibited both LPS and cytokine induced tissue mRNA accumulation and subsequent cell surface expression of tissue factor in monocytes. Cytokine- induced ROS generation was significantly interrupted in monocytes upon inhibition of HSP90. A panel of monocyte pro-inflammatory genes could be inhibited with AUY922, whereas the anti-inflammatory cytokine, IL-10, was induced upon HSP90 inhibition with AUY922. Mechanistically, we also noted a profound translocation of NFkB to the monocyte nucleus upon cytokine stimulation. Consistent with the effects on tissue factor expression, pro-inflammatory potential, and ROS generation, this translocation could be completely ablated with HSP90 inhibition. Importantly, HSP90 inhibitors significantly attenuated the LPS-activated monocyte -induced lung microvascular endothelial permeability.
Conclusion
Our data suggest that Hsp90 inhibitors significantly reduced the both pro-inflammatory and pro-coagulatory potential of PBMCs from patients with SCD. These results thus position HSP90 as a potential master regulator of hemostasis, endothelial cell permeability, and thus suggest that HSP90 is an attractive therapeutic target in patients with SCD.
Disclosures:
Kutlar: Celgene Corporation: Research Funding. Meiler:Celgene Corporation: Research Funding.
Activation of endothelial intrinsic NF-κB pathway impairs protein C anticoagulation mechanism and promotes coagulation in endotoxemic mice
