The Study Of Smoke-induced Proteolytic Microvesicles In The Progression Of Thinning Fibrous Cap Of Atherosclerosis Plaque

Objectives:Cigarette smoking greatly increases the risk of atherosclerosis plaque rupture and subsequent intravascular thrombosis. However, the underlying mechanisms remain poorly understood. Increasing evidences have shown that the progression of atherosclerotic plaque was associated with accumulation of amounts of microvesicles (MVs) within the plaque lesion. Our group recently found that tobacco smoke extract (TSE) induces human monocytes/macrophages to generate tissue factor-positive MVs which have procoagulant activity. This finding may account for the increases content of tissue factor in atheromata from smokers and their increased risk of thrombotic occlusion after plaque rupture. But it would not necessarily explain the underlying mechanisms of plaque rupture. Therefore, in the present study, we sought to determine whether exposure of human macrophages to TSE induces the release MVs with proteolytic activity, what nature of the proteases on TSE-induced MVs might be, and what the molecular mechanisms might be responsible for their generation.Methods:1. The human THP-1cells were differentiated to macrophages using PMA. Primary human monocyte-derived macrophages (hMDMs) were prepared from fresh buffy coats by selecting monocytes by adherence followed by differentiation into macrophages.2. The proteolytic activities of TSE-induced MVs were assayed by zymogram and flurometric analysis.3. TSE-induced MVs were determined to activate pro-MMP2to gelatinase MMP2by zymogram after co-incubation with pro-MMP12for1hour at37℃.4. Confocal microscopy was used to documents MMP14display and exteriorization of phosphatidylserine (PS) on the surface of TSE-treated macrophage.5. Western Blot was used to detect the expression of MMP14and activation of MAPKs signaling pathways in the TSE-treated macrophages and MVs.6. Flow cytometry were used to detect the generation of TSE-induced total-and MMP14-positive MVs. 7. Pretreatment macrophages with caspase inhibitor intervene the generation of TSE-induced total and proteolytic MVs.8. Pretreatment macrophages with p38or JNK inhibitor intervene the generation of TSE-induced total-and proteolytic MVs.Results1. THP-1cell successfully differentiated to macrophages by PMA and got the hMDMs by adherence.2. TSE-induced MVs significantly cleaved fluorogenic substrate I and degraded the native substrates gelatin and collagen in SDS gels.3. Pre-coincubation of TSE-induced MVs with pro-MMP2can activate the MMP2gelatinase.4. TSE exposure of macrophages significantly increased expression of MMP14in dose-and time-dependent manners. Confocal images showed a huge induction of cell surface MMP14and PS exteriorization on TSE-treated macrophages. The TSE-induced MVs were proved to carry MMP14by Western Blot.5. Flow cytometry results showed that TSE exposure of macrophages significantly increased the generation of total-and MMP14-positive MVs.6. Pretreatment of macrophage with CASPi significantly blocked the generation TSE-induced total-and MMP14-positive MVs and the proteolytic activity without affecting cellular expression of MMP14.7. TSE exposure of macrophages increased the phosphoralytion of3major MAPKs including JNK, p38and ERK. The TSE-induced MMP14expression was associated with the phosphoralytion of JNK and p38, not ERK.8. Pretreatment of macrophage with of JNK or p38inhibitor significantly blocked the generation of TSE-induced total-and MMP14-positive MVs, also blocked the proteolytic acitivity.Conclusions:TSE-induced macrophage-derived MVs carry substantial gelatinolytic and collagenolytic activities which can be attributed to a single, dominant, transmembrane protease of the MMP superfamily, namely MMP14. The production of these MVs relies on the activation of JNK and p38MAPKs, not ERK activation. The new pathogenic factors might play an important role in the formation of atherosclerosis thinning fibrous cap and plaque rupture.