扰流诱导内皮细胞异常增殖和炎症反应的表观遗传机制- - DNA甲基转移酶的作用

Shear stress, the tangential component of the hemodynamic forces acting on the vascular wall, has been shown to play crucial roles in the regulation of vascular endothelial function, as well as the formation and progression of atherosclerosis. Previous studies by us, our collaborators, and others have elucidated that atheroprone shear stress modulates dysfunction of vascular endothelial cells (ECs), including aberrant proliferation, inflammation, migration, increased permeability, and releasing of some pro-atherogenic molecules, by the mediation of several molecules and factors, e.g. microRNAs and histone deacetylases (HDACs), which are epigenetic mediators regulating gene expression without altering the underlying DNA sequences. One of the most important molecules that have been found in our recent study is another epigenetic mediator, DNA methyltransferase 1 (DNMT1), whose expression and activation could be induced by atheroprone oscillatory shear stress (OS) in ECs and its pharmacological inhibition attenuated OS-induced DNA hypermethylaion, proliferation, and inflammatory responses of ECs. With the new finding, we hypothesize that DNMT1 governs the effects of OS on endothelial functions and atheroma formation. Through the activation of upstream mechanotransduction, signaling and regulatory pathways, e.g. integrins, mTOR, and TLRs, OS-induced DNMT1 methylates DNA both globally and at specific loci, resulting in the changes of gene expressions and functional consequences. Three specific aims are proposed to test the guiding hypothesis. Aim 1 will profile the DNMT1-mediated genome-wide DNA methylation patterns in ECs in response to OS versus atheroprotective pulsatile shear stress (PS). Aim 2 will elucidate the regulatory pathways and functional consequences that involved in or resulted from the activation of DNMT1 by OS. Aim 3 will validate the roles of DNMT1 in endothelial functions and atherogenesis in vivo. The results from the present study will help to identify the hitherto poorly understood connection between DNA methylation and atheroprone hemodynamic forces applied to the vascular endothelium and to discover new target biomarkers highly associated with the translational and clinical medicine research with great potentials for biotechnological development, thereby facilitating the development of new therapeutic strategies for the disease.

血液动力学的重要因素- - 剪应力，在血管内皮细胞功能以及动脉粥样硬化发生过程中具关键作用。申请人及其合作者在近年的研究中发现，微小RNA和组蛋白去乙酰化酶等表观遗传调控因子，介导了促动脉硬化发生血液扰流产生的振荡剪力诱导的内皮细胞功能紊乱。在最近的研究中，我们又发现另外一个重要的表观遗传调控因子，DNA甲基转移酶1（DNMT1），在内皮细胞中被振荡剪力上调；DNMT1抑制后能削弱振荡剪力引起的DNA异常甲基化、增殖、和炎症反应。我们推测：振荡剪力激活DNMT1，导致全基因组DNA异常甲基化，从而改变内皮细胞基因表达和血管生理稳态。本研究将探讨1）不同类型剪力以DNMT1介导的基因组DNA甲基化谱；2）促动脉硬化发生的振荡剪力激活DNMT1的分子机制及其功能； 3）利用动物血流扰流模型和动脉硬化模型验证DNMT1的血管病生理学意义。研究结果将增进关于机械生物信号转导机制及动脉硬化发生机制的了解

临床医学证据显示动脉粥样硬化斑块好发于血管弯曲或分支处，其中一个重要原因是由于该处血管内扰流引起的振荡型剪力（OS）诱导内皮表达促动脉粥样硬化基因，导致内皮功能障碍。我们的前期研究已经证明相对于脉冲型高剪切力（PS），振荡型剪力（OS）能够诱导血管内皮细胞DNA甲基转移酶1（DNMT1）的高表达。同时在大鼠体内扰流模型中我们也验证了这一实验结果。在此基础上，我们的研究证明OS可以持续性地上调内皮细胞中DNMT1的表达，而PS只是瞬时上调内皮细胞中DNMT1的表达，随着时间的推移其表达恢复到基础水平。同时在小鼠颈总动脉部分结扎模型扰流作用区域内皮以及ApoE敲除小鼠动脉粥样硬化斑块内皮中，我们发现和对照组相比，DNMT1以及其作用产物5-meC表达显著增加。特异性敲低血管内皮细胞中的DNMT1可以显著抑制扰流诱导的血管内皮细胞增殖，迁移以及炎性反应的异常增强，甚至缓解了动脉粥样硬化的发生。而这一过程至少部分通过其下游靶基因细胞周期调控因子（cyclin A）和结缔组织生长因子（CTGF）实现。通过功能抑制实验，我们确定了integrin-β3/Shc/FAK、ERK1/2以及PI3K/mTOR/p70S6K参与了转导OS诱导DNMT1表达的信号通路中。同时在体内和体外使用mTOR抑制剂雷帕霉素可以抑制扰流对于内皮细胞DNMT1的诱导。我们的实验结果证明了扰流影响内皮细胞功能和促进动脉粥样硬化是通过mTOR/DNMT1途径实现的。本研究增进了对于机械生物信号转导机制及动脉硬化发生机制的了解。课题的研究结果将有助于阐明表观遗传对于内皮细胞功能的调节在动脉粥样硬化病理发生发展过程中的作用，并为预防和治疗动脉粥样硬化提供新的靶点。