The TIMP3/Apelin axis Regulates Atherosclerosis and Heart Metabolism leading to increased mortality in ApoE null Mice

The role of TIMP3 in the context of cardiovascular remodeling is relatively unexplored when considering classical risk factors such as hypercholesterolemia, diabetes and hypertension. To learn more the role of TIMP3 in the progression of cardiovascular disease we combined genetics, metabolomics and in vivo phenotypical analysis using the hypercholesterolemic ApoE null mice to generate ApoE-/-Timp3-/- mice, the latter showing increased atherosclerosis, increased mortality and arrhythmias compared to ApoE-/- mice. We have previously described Timp3-/-mice in ( Fiorentino, L., et al., Regulation of TIMP3 in diabetic nephropathy: a role for microRNAs. Acta Diabetol, 2013) . To generate ApoE-/-Timp3-/- knockout animals we crossbred the 2 strains. Offsprings were then backcrossed into ApoE animals for 6 generations to generate a pure lineage. Collectively, metabolite profiles, gene and protein expression consistently suggested a role for TIMP3 to underlie a decreased activation of PPARalpha/AMPK to dampen fatty acids beta-oxidation eventually leading to atherosclerotic plaque composition vulnerability and perturbation of heart metabolism. mRNA profiling in ApoE-/-Timp3-/- mice revealed a TIMP3 effect to regulate Apelin, which we found decreased in the circulation due to its specific downregulation at the myocardial level but not in other well known sites of expression such as the adipose tissue. mRNA sequencing of the heart of ApoE-/-Timp3-/- mice vs ApoE-/- littermates controls.针对高胆固醇血症、糖尿病与高血压等经典心血管危险因素，金属蛋白酶组织抑制剂3（TIMP3）在心血管重塑中的作用迄今仍鲜有探索。为进一步阐明TIMP3在心血管疾病进展中的作用，我们结合遗传学、代谢组学分析与利用高胆固醇血症ApoE基因敲除（ApoE null）小鼠开展的体内表型分析，构建了ApoE-/-Timp3-/-双基因敲除小鼠。与仅ApoE基因敲除的小鼠相比，该双敲除小鼠表现出动脉粥样硬化加重、死亡率升高以及心律失常表型。我们此前已在文献（Fiorentino, L. 等, Regulation of TIMP3 in diabetic nephropathy: a role for microRNAs. Acta Diabetol, 2013）中报道过Timp3基因敲除小鼠。为构建ApoE-/-Timp3-/-基因敲除动物模型，我们将两种单敲除小鼠品系进行杂交，随后将子代与ApoE基因敲除小鼠回交6代以获得纯合品系。综合代谢谱、基因与蛋白表达分析结果，均可证实TIMP3可通过降低PPARα/AMPK通路的激活水平，抑制脂肪酸β氧化过程，最终导致动脉粥样硬化斑块易损性升高与心脏代谢紊乱。对ApoE-/-Timp3-/-小鼠的mRNA表达谱分析显示，TIMP3可调控Apelin的表达；我们发现，由于Apelin在心肌组织中被特异性下调，其循环水平出现降低，但在脂肪组织等其他已知的Apelin表达位点中未观察到此现象。本研究针对ApoE-/-Timp3-/-小鼠与同窝ApoE-/-对照小鼠的心脏组织开展了mRNA测序。