0.5 up to 13.7 9 1.3 ng PAI-1mg of total proteins n = 6. ATOR or FLU at 1.0 mmoll did not signifi-
cantly alter this IL-1a-induced increase in PAI-1 syn- thesis, as the decrease did not exceed 8 data not
shown.
We also investigated if ATOR or FLU altered the synthesis of t-PA. TNFa is known to decrease the basal
synthesis of t-PA in HUVEC [33], which was also observed herein by a significant P B 0.01 decrease in
t-PA accumulation − 52 when compared with un- stimulated cells Table 2. Pretreatment of HUVEC
cultivated in 20 FCS with either FLU or ATOR reduced the inhibitory effect of TNFa on t-PA release.
However, because of some wide dispersion of t-PA levels among the different HUVEC preparations, the
only significant reversal effect of statins i.e. no statisti- cal significant decrease versus control; P \ 0.05 was
observed with ATOR at 2.0 mmoll − 22 and with FLU at 1.0 mmoll − 15.
4. Discussion
The beneficial effect of statins on cardiovascular dis- eases appears over and above the well established and
proven decrease in circulating LDL-cholesterol. By in- terfering with prenylation of transducing proteins,
statins may alter the expression of proteins implicated in vascular function [9,10]. The dysregulation of the
fibrinolytic potential of endothelial cells is critically involved in the development and progression of
atherothrombosis [38]. In this study, we analyzed whether the two statins ATOR and FLU altered the
levels of PAI-1 in HUVEC placed in basal and proinflammatory conditions. Clearly, both statins sig-
nificantly decreased the synthesis of PAI-1 in HUVEC cultivated in the presence of 20 serum. The effects of
ATOR and FLU were not statistically different one from the other, although FLU tended to act at lower
concentrations than ATOR, which may reflect their different hydrophobic properties. These results are in
line with those of Essig et al. [32] who showed, in a rat transformed endothelial cell line, that lovastatin down-
regulated PAI-1 expression, as regards both activity and mRNA levels. The inhibitory effect of FLU and
ATOR on PAI-1 antigen release is reflected by a de- crease in PAI-1 mRNA level and is prevented by addi-
tion of mevalonate. This strongly suggests that statins exerted their effect on PAI-1 gene expression through
inhibition of the mevalonate pathway. Intermediate metabolites of this pathway, such as farnesylpyrophos-
phate or GGPP, are involved in the prenylation of transducing proteins, allowing proper interaction with
the membrane and consequently an optimal transmis- sion of the information through the cytoskeleton. In
this way, it was demonstrated that prenylation of Rho GTPases by GGPP was inhibited by lovastatin [39].
Interestingly, in rat endothelial cells, GGPP was able to prevent the lovastatin-induced down-regulation of PAI-
1 synthesis [32]. It was proposed that lovastatin induced a defect in the prenylation of Rho protein that in turn
altered its interaction with the cytoskeleton [32]. Inter- estingly, we also observed with HUVEC that GGPP
almost completely reversed the down-modulating effect of statins. However, in HUVEC, cytoskeletal reorgani-
zation itself does not seem to be responsible for the decrease in PAI-1 synthesis, as we observed that the
disrupter of actin filaments, cytochalasin D, did not significantly alter PAI-1 synthesis. This result can be
analyzed in the light of the results of Zohar et al. [40], who showed in NIH3T3 cells that Rho-effector
molecules regulating actin structure are distinct from those signaling to the nucleus. The effect of statins on
the activation of PAI-1 synthesis by proinflammatory cytokines TNFa and IL-1a was modest as the optimal
decrease we observed − 17 to − 20 was with TNFa. This suggests that FLU or ATOR does not
interfere with the proinflammatory cytokines-triggered pathway leading to PAI-1 gene activation. One may
thus hypothesize that, in vivo, statins would signifi- cantly regulate PAI-1 production in normal or mildly
activated endothelial cells rather than those lining an advanced inflamed atherosclerotic lesion. This, how-
ever, does not rule out the possibility that the signalling pathway triggered by other types of inducers of PAI-1
synthesis present in the atherosclerosic lesion can be down-regulated by statins. A different behavior was
observed with t-PA synthesis in HUVEC. In basal conditions i.e. 20 FCS, ATOR and FLU did not
significantly alter t-PA release. However, in HUVEC and in rat endothelial cells incubated in low serum
concentration and treated with lovastatin, Essig et al. [32] observed an increase in t-PA synthesis. In this
study, we also observed that HUVEC incubated in 1 FCS increased the t-PA release in the presence of statin
with a more marked effect of FLU. However, at this stage of investigation, the pathophysiological relevance
of the relationships between statins, serum depletion and t-PA synthesis remains to be clarified. Interestingly,
ATOR and FLU reduce the TNFa-induced decrease in t-PA antigen accumulation, suggesting that these statins
interfere with some factors of the TNFa-triggered transduction pathway that down-regulate t-PA gene
transcription. The differential regulation of PAI-1 and t-PA syntheses by ATOR and FLU in basal and
TNFa-stimulated conditions underlines the complex regulation that statins exert on cell function and
trafficking.
These results, taken together with those of the litera- ture, emphasize the original cholesterol-independent ef-
fects of statins that tend to correct endothelial dysfunction.
5. Note added in proof
