Platelets induce Pai-1 mRNA expression in monocytes through TGF-BETA

Introduction: Plasminogen activator inhibitor type-1 (PAI-1) is a physiological modulator of fibrinolysis. High plasma PAI-1 is associated with the 4G/5G promoter polymorphism and with increased cardiovascular risk. Here we explored the role of platelets in regulating expression of the PAI-1 gene in monocytes. Methods: Blood from PAI-1 4G/5G genotyped volunteers (n=6) was incubated with the platelet GPVI-specific agonist, cross-linked collagen related peptide (CRP-XL), in the presence or absence of Mab 9E1 that blocks the binding of P-selectin to PSGL1. Monocytes were isolated by +ve selection on CD14 beads and monocyte PAI-1 mRNA expression was measured by real-time PCR. Results: Activation of platelets with CRP-XL resulted in platelets binding to >70% of monocytes and was accompanied by >5000-fold induction of PAI-1 mRNA, peaking at 4hrs. PAI-1 expression was independent of the 4G/5G genotype. Blocking the binding of platelets to monocytes enhanced PAI-1 induction (p<0.05 at 4 hrs). Incubation of isolated monocytes with the releasate from CRP-XL stimulated platelets also led to PAI-1 mRNA expression. The platelet secretome contains >100 different proteins. To identify the soluble factor(s) responsible for induction of PAI-1, neutralizing antibodies to likely candidates were added to monocytes incubated with the platelet releasate. Anti- TGF-beta inhibited platelet releasate-mediated PAI-1 mRNA induction by >80%. Monocyte PAI-1 was also induced by stimulation of PSGL-1 with a P-selectin-Fc chimera, in the absence of platelets, which was also blocked by the TGF-beta antibody. Conclusions: These results suggest that platelets induce PAI-1 mRNA in monocytes predominantly via TGF-beta, released from both platelets, and monocytes via activation by PSGL-1 signalling.This stimulation is independent of 4G/5G genotype

Influence of chylomicron remnants on human monocyte activation in vitro

Background and aims Atherosclerosis is known to be an inflammatory disease and there is increasing evidence that chylomicron remnants (CMR), the lipoproteins which carry dietary fats in the blood, cause macrophage foam cell formation and inflammation. In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is increased, and clearance of CMR from blood may be delayed, however, whether CMR contribute directly to monocyte activation and subsequent egress into the arterial wall has not been established. Here, the contribution of CMR to activation of monocyte pro-inflammatory pathways was assessed using an in vitro model. Methods and results Primary human monocytes and CMR-like particles (CRLP) were used to measure several endpoints of monocyte activation. Treatment with CRLP caused rapid and prolonged generation of reactive oxygen species by monocytes. The pro-inflammatory chemokines MCP-1 and IL-8 were secreted in nanogram quantities by the cells in the absence of CRLP. IL-8 secretion was transiently increased after CRLP treatment, and CRLP maintained secretion in the presence of pharmacological inhibitors of IL-8 production. In contrast, exposure to CRLP significantly reduced MCP-1 secretion. Chemotaxis towards MCP-1 was increased in monocytes pre-exposed to CRLP and was reversed by addition of exogenous MCP-1. Conclusion Our findings indicate that CRLP activate human monocytes and augment their migration in vitro by reducing cellular MCP-1 expression. Our data support the current hypothesis that CMR contribute to the inflammatory milieu of the arterial wall in early atherosclerosis, and suggest that this may reflect direct interaction with circulating blood monocytes.

Dietary fats induce human monocyte activation in vitro

In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is amplified, and migration of monocytes into the walls of the aorta and large arteries is increased, due partly to de novo expression or activation of monocyte adhesion molecules. Although there is increasing evidence that CMRs (chylomicron remnants) are strongly atherogenic, the outcomes of interactions between blood monocytes and circulating CMRs are not known. Here, we have studied the effects of CRLPs (CMR-like particles) on THP-1 human monocyte oxidative burst. The particles induced a significant increase in reactive oxygen species within 1 h, which persisted for 24 h. We suggest that monocyte–CMR interactions may be important in early atherosclerosis when many activated monocytes are found in susceptible areas of the artery wall.