The Role And Mechanism Of TFPI-2 On Stability Of Atherosclerotic Plaque

Unstable angina, myocardial infarction or cardiac sudden death is often triggered by the rupture of a vulnerable coronary atherosclerotic plaque, which during its development can expose the thrombogenic core to luminal blood flow, frequently resulting in thrombosis and ischemic cardiac events. During this process, the integrity of the fibrous cap was shown to play a crucial role in plaque development and disruption. The structural integrity of plaques seems to depend on a balance between synthesis and degradation of the extracellular matrix (ECM) which is mainly regulated by proteinases such as matrix metalloproteinases (MMPs) including interstitial collagenase or MMP-1, gelatinase A or MMP-2, gelatinase B or MMP-9, membrane-type MMP (MT-MMP) or MMP-14. MMPs are secreted as inactive zymogens that are activated by other proteinases, such as plasmin. The activation and accumulation of monocyte-macrophage and the fomation and infiltration of foam cells were shown to play another important role in plaque development and disruption. Atherosclerosis can be considered to represent an inflammatory response of macrophages to 'invading' pathogenic lipoproteins in the arterial wall. The formation of foam cells from lipid-accumulated macrophages is a critical step in atherogenesis, which is closely related to the pathogenesis of atherosclerosis. Many studies indicated that more macrophages and foam cells infiltrated and accumulated in ruptured atherosclerotic plaque, compared to stable plaque. Macrophages weakened the ECM of fabrious caps via secretion proteinase including matrix metalloproteinases families like MMPs, collagenase, gelatinase and stromelysin, thus the atherosclerotic plaques presented ulcer, crack and then ruptured, which switched on blood clotting, vessel blockage.Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor, mainly synthesized by endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and syncytiotrophoblasts. TFPI-2 associates through arginine-madiated ionic interactions with ECM, and recombinant TFPI-2 exhibits strong inhibitory activity toward trypsin, plasmin, factorⅪa, and plasma kallikrein, and weaker inhibitory activity for factorⅦa-tissue factor complex, factorⅨa-polylysine, and cathepsin G. In addition, TFPI-2, as well as its first Kunitz-type domain, has been reported to inhibit MMPs, and TFPI-2 seems to be plasmin inhibition and thus of proMMP-1, proMMP-3, and proMMP-13 activation. Thus TFPI-2 is a inhibitor of MMPs and potent inhibitor of both matrix-bound and cell-associated plasmin, suggesting that TFPI-2 could regulate extracellular proteolysis, ECM remodeling and consequently might stabilize atherosclerotic plaque.Although the importance of TFPI-2 as an inhibitor of thrombin generation remains unclear, a recent report has implicated that the interaction of TFPI-2 with TF:FVIIa in promoting cell migration, although the physiological relevance of this process is yet to be elucidated. A further action of TFPI-2 as a vascular smooth muscle cell mitogen has also been suggested. Furthermore, TFPI-2 may represent a mechanism for negative feedback regulation and modulation of its pro-angiogenic action on endothelial cells and might be an important regulator of aberrant angiogenesis, suggesting that TFPI-2 might be a potential therapeutic for angiogenic disease processes. Apoptosis, the programmed cell death, is critical for the development and maintenance of healthy tissues. A recent report has implicated TFPI-2 activated both intrinsic and extrinsic caspase-mediated, proapoptotic signaling pathways and induced apoptosis in U-251 cells. In our recent study, we found that during U937-derived foam cells formation the mRNA and protein levels of TFPI-2 peaked at 6 hours post treatment oxLDL and decreased gradually in a time dependent manner. A recent report described the apparent downregulation of TFPI-2 in macrophages of atherosclerotic tissues, which also suggested the loss of TFPI-2 might promote plaque formation and/or complication. We consequently suppose that TFPI-2 might promote macrophage apoptosis and inhibit foam cell formation.Therefore we performed a detailed study of upregulating the expression of TFPI-2 in apoE-/-mice to examine the possible significance of its in vivo functions. First of all, we made an animal model of sustained TFPI-2 upregulation by means of intramscular injection plus electroporation in apoE-/-mice. Then our in vivo study were carried out as follow:A. In animal models using ApoE knock-out mice upregulated TFPI-2 with transfection, we surveyed the diversity of plaque's stablity (including the continuity of fabrious caps, buried fabrious caps, the thickness of fabrious caps, the size of lipid core, etc). B. Meanwhile we observed what roles TFPI-2's upregulation played on MMPs'expresion and activities and on PI3K, NF-κB, PPAR-y activating levels of cell signal pathway.Futhermore, we treated U937 derived macrophages and foam cells with different concentration of recombinant TFPI-2 protein (0,1.25,2.5,5.0,10.0 p.g/mL) in vitro. We observed the proliferation and apoptosis of U937 derived macrophages with BrdU marking method, Hoechst stain, Annexin V/PI stain and flow cytometry, and assayed Fas/FasL cell signal pathway. We also surveyed U937 derived foam cell formation by oil red O stain and high performance liquid chromatography (HPLC), and determined foam cell formation pathway like PPARγ, ABCG1, ApoA I, etc.Initially, we successfully constructed an animal model of sustained TFPI-2 upregulation by means of intramscular injection plus electroporation in apoE-/-mice. After electroporation, the TFPI-2 level in mice serum was enhanced for 2～3 folds, so did the TFPI-2 level in atherosclerotic plaque. Furemore, the results in vivo showed that upregulation of TFPI-2 in apoE-/-mice could reduce lipid core size, buried fabrious caps and rupture numbers of atherosclerotic plaque, and increase the thickness of fabrious caps via downregulating expression of MMPs and inhibiting phosphorylation of PI3Ky/PKB signal pathway.Meanwhile, our results in vitro demonstrated that TFPI-2 had minor effect on macrophage proliferation but could promote macrophages apoptosis in a dose-dependent manner through Caspase-8 activation mediated by Fas/FasL, which might be related to MMPs downregulation in macrophages by TFPI-2. Furmore, TFPI-2 could reduce foam cells formation in a dose-dependent manner via PPAR-y activation mediated cholesterol efflux to ApoA, I which might be related to the inhibition of MMPs'expression and activities in macrophages by TFPI-2.Our research preliminarily definted TFPI-2's effects on stabilizing arterosclerotic plaque and its possible mechanism for the first time, and our results will provide a new target on prevention and treatment of artherosclerosis.