The Role Of Tryptase In ApoE-/-Mice Atherosclerosis Plague Hemorrhage

In recent years, the incidents of acute cardiovascular events caused by atherosclerosis have highly increased, while in the process plague hemorrhage took an important role which has a close relation with plague angiogenesis.Tryptase is an inartificial protease with multiplicate biological functions and nearly concerned with the pathogenesis of atherosclerosis. Trypase can cause neutrophil recruitment to promote inflammation, stimulate endothelial cells to synthesize and release of IL-1? and IL-8that induce leukocytes infiltration and stimulate peripheral blood monocytes to produce and secrete IL-6, TNF-a and IL-1? by activating PAR-2on cell membrane, so as to affect the occurrence and development of vascular inflammation of atherosclerosis. It's been reported recently that inhibition of tryptase activity decreased mast cell's ability to stimulate capillary-like lumen structure formation of dermal endothelial cells. Tryptase has been considered as a novel angiogenic factor. However, the role of tryptase on atherosclerosis plague hemorrhage is still unclear. In this study we use lenti-virus carrier to observe the effect of tryptase on plague hemorrhage in vivo and in vitro.This study includes three parts:Part I:Construct lent-virus carriers to overexpress or knock down tryptase expression in vivo and in vitro.Firstly, tryptase gene sequence was synthesized and cloned into plasmids for lenti-virus carrier construction. Then, siRNA for tryptase were designed and cloned into lenti-virus carrier. These carriers were constructed successfully and prepared for the following experiments.Part II:The effect of tryptase on atherosclerosis plague hemorrhage in ApoE-/-mice.The animal model of plague hemorrhage was established using ApoE-/-mice. After cuffing-cervical artery operation, the mice were randomly divided into6groups to study the effect of tryptase or siRNA lenti-virus carrier on plagues hemorrhage. HE staining showed that plague area was much bigger in over-expressed tryptase group than others and artery stenosis was more serious. All groups' artery stenosis from the cuff-side is over90%except for the siRNA group. Tryptase promotes plague hemorrhage distinctively,50%mice in over-expressed tryptase group have plague hemorrhage, while only1mouse in siRNA group. The immunohistochamitry of the cervical artery plague showed that expressions of CD31(represent microvascular angiogenesis) in tryptase overexpression group are highest among all groups, while lowest in siRNA group. PAI expression in tryptase overexpression group was lowest while tPA was highest, which was completely contrary to siRNA group. These results suggested tryptase can promote plague angiogensis and hemorrhage by regulating PAI and tPA.Part III:The effect of tryptase on bEend.3cells growth, migration and capillary-like tubes formation.Mast cell line P815cells were trasnsfected by Lenti-tryptase. The P815-conditioned medium was collected and used for culturing endothelial cell line bEnd.3cells. bEnd.3cell growth, migraration,capillary-like tubes formation, and PAI and tPA expressions were observed. The results showed that over-expressed tryptase P815-conditioned medium promote bEnd.3cell growth, migration and capillary-like tubes formation ability, which suggested tryptase can promote microvessl angiogensis. Realtime RT-PCR and Western Blot assays showed that mRNA and protein expression level of PAI is lower in over-expressed tryptase-conditioned medium than control cultured bEnd.3cells while tPA is higher, which is completely contrary to the control groups. Our in vitro data indicated typtase may influence plague hemorrhage by regulating PAI and tPA.In conclusion, the results in vivo and in vitro study suggest that trypase can promote atherosclerosis plague angiogensis and hemorrhage by regulating PAI and tPA expression. Thus, inhibiting tryptase expression in mast cells may be a new therapeutic strategy for atherosclerosis.