Our investigation reveals that the sGC agonists BAY 41-2272 and BAY 58-2667 attenuate protein expression and function of TF in human monocytes stimulated with LPS, as demonstrated by Western blotting, real time RT-PCR, and the TF procoagulant activity assay. This response is associated with a marked reduction of total and surface TF presentation, as assessed by the ICW assay. Furthermore, BAY 41-2272 and BAY 58-2667 reduce TF protein expression in HUVECs, both under resting conditions and after stimulation with TNF-α. This effect is accompanied by a reduction of the TF-dependent procoagulant activity in the lysates of HUVECs under the same experimental conditions. Importantly, the inhibitory effects of both sGC agonists on TF expression are dependent on the presence of functional sGC, because the siRNA-mediated knockdown of the α1 subunit of sGC render stimulated monocytes and HUVECs insensitive to BAY 41-2271 and BAY 58-2667.
In the present study, BAY 41-2272 and BAY 58-2667 increased intracellular cGMP concentrations and decreased the expression of active TF protein, as well as the surface TF presentation in human monocytes and vascular endothelial cells stimulated, respectively, with LPS and TNF-α. These data are in agreement with an earlier investigation demonstrating an inhibitory role of cyclic nucleotides in regulation of TF expression in monocytes.10
Importantly, proinflammatory mediators such as LPS per se have been shown to increase intracellular levels of cGMP,24
as we also found in the present study. Apparently, further elevation of cGMP levels through pharmacological stimulation of sGC produces a self-limiting negative regulation of the inflammatory response. Of note, the observed changes in TF expression and functional activity in monocytes and HUVECs could not be attributed to the differences in the survival of cells after various treatments, as the fraction of apoptotic cells remained unchanged.
The concentrations of the sGC agonists used to achieve an acute pharmacological response in our in vitro investigation were higher than the concentrations previously tested to produce hemodynamic responses in vivo.12–14
However, an important finding was that the effects of BAY 41-2271 and BAY 58-2667 in monocytes were time-dependent, as both compounds elicited a stronger inhibitory activity on TF when the exposure time before the LPS stimulation was extended from 10 to 45 minutes. Thus, it is likely that with chronic administration the effective concentrations of these compounds required to elicit a significant inhibitory effect on TF in vivo would be much lower.
In humans, the promoter of F3
gene contains a binding site for NF-κB, which mediates responses to TNF-α and LPS.25,26
Although TNF-α and LPS can regulate TF expression by virtue of posttranscriptional stabilization of TF mRNA,27,28
upregulation of TF expression in response to inflammatory stimuli is largely dependent on the transcriptional activity of NF-κB. In the present study, we demonstrate that inhibition of TF by the sGC agonists in both monocytes and HUVECs is mediated at the level of the transcriptional regulation of NF-κB and not upstream of IκBα, because the loss of IκBα protein after stimulation with LPS and TNF-α was not affected by BAY 41-2272 and BAY 58-2667. Our findings are in agreement with Pan et al, who have reported that YC-1, a nonspecific nitric oxide–independent stimulator of sGC, reduced the LPS-induced expression of inflammatory cytokines in mononuclear cells via inhibition of NF-κB.29
Similarly, attenuation of the LPS-induced NF-κB activity by YC-1 has been recently shown in microglial cells.30
In circulating blood monocytes, regulation of TF expression and functional activity depends on interactions with other cell types, notably platelets. Platelets play a crucial role in facilitation of the LPS-induced TF expression in monocytes. 31
Furthermore, platelets promote thrombin generation by providing a catalytic surface for the TF/FVIIa/FXa complex and release FVa,32
and can also affect monocyte TF expression by releasing platelet-derived factors and inducing TNF-α.20,33,34
The cross-talk between monocytes and platelets results in synthesis and surface exposure of various adhesion molecules, such as P-selectin, lymphocyte function-associated antigen-1, membrane-activated complex-1,35
and CD40 with its CD40-ligand, which enhance platelet-monocyte interactions and TF expression by monocytes.36,37
Interestingly, BAY 41-2272 has been recently shown to inhibit platelet activity16
and down-regulate the expression of P-selectin,38
supporting our findings that these novel pharmacophores may exert antithrombotic effects by reducing TF expression in circulating blood monocytes and vascular endothelial cells.
The presence of functional sGC is crucial for BAY 41-2272 and BAY 58-2667 to elicit the inhibitory effect on TF expression, because the siRNA-mediated knockdown of the α1 subunit of sGC completely negated this effect. Interestingly, TF protein levels were significantly higher in resting monocytes and HUVECs in which sGCα1 expression was knocked-down as compared to the resting cells nucleofected with control siRNA. Likewise, the TF expression was also greatly enhanced in the sGCα1-deficient cells after stimulation with LPS and TNF-α. These findings indicate that in human monocytes and endothelial cells sGC plays an important regulatory role by controlling the TF system. Furthermore, the downregulation of TF expression and functional activity by BAY 41-2272 and BAY 58-2667 are mediated through the sGC-dependent mechanisms involving the suppression of transcriptional activity of NF-κB. Taken together, our data strongly suggest that inhibition of TF expression and activity by sGC agonists might provide therapeutic benefits in cardiovascular diseases associated with enhanced prothrombotic and inflammatory responses.