利用微流控芯片技术研究AcSDKP对血管损伤后的重塑作用与机制

Drug-eluting stents (DES) have significantly improved the safety and efficacy of percutaneous coronary intervention (PCI). However, a major limitation of stents lies in the in-stent restenosis (ISR) caused by neointimal hyperplasia. To protect against restenosis and stent-thrombosis, novel strategies are needed for enhancing re-endothelialization while inhibiting thrombus formation, inflammation and proliferation of smooth muscle cells. AcSDKP (N-acetyl-seryl-aspartyl-lysyl-proline), a tetrapeptide, has been shown to act as a potent antifibrotic agent via suppressing myofibroblast differentiation. AcSDKP is also able to induce post-ischemic neovascularization and vascular remodeling through promoting the proliferation and migration of endothelial cells. Thus, AcSDKP may be a promising agent for use in DES. In this proposal, we aim to employ the mouse femoral artery wire injury model to examine the effect of AcSDKP on re-endothelialization. Given that hemodynamic forces generated by the blood flow are of central importance in influencing the function of endothelial cells, we intend to develop a microfluidic device with integrated electrical impedance sensing apparatus, thereby mimicking the physiological shear-flow conditions in the blood vessel. Through real-time monitoring endothelial cell growth and migration, this microfluidic technology will provide an easy-to-use and reproducible in vitro platform for evaluating the mechanistic effects of AcSDKP upon endothelial functions during vascular remodeling. In addition, we will further examine the signaling pathways by which AcSDKP influences the production of NO and regulates endothelial cell proliferation and migration. This study will help us to understand the molecular basis for AcSDKP’s action in improving vascular healing and its potential in protecting against in-stent restenosis.

血管内支架再狭窄源于血管损伤后的修复反应，而如何促进血管的再内皮化，抑制内膜增生，从而降低血栓发生率，是目前改进药物洗脱支架（DES）的关键环节。我们前期研究发现N-乙酰丝氨酸-天冬氨酸-赖氨酸-脯氨酸（AcSDKP）能够抑制成纤维细胞的增殖，减轻梗死区域的纤维化，同时诱导血管内皮细胞的生长和迁移，因此我们推测它具有加速再内皮化、促进血管损伤修复的潜在作用。本项目拟利用导丝诱导股动脉损伤的小鼠模型，验证AcSDKP对血管损伤后的稳态维持和重塑作用；同时基于血流动力学因素对血管内皮细胞形态和功能的重要影响，构建并利用模拟血管血流微环境的微流控生物芯片，结合电阻抗传感技术，实时检测在剪切力作用下AcSDKP对血管内皮细胞生长和迁移的影响；进一步探讨四肽调节血管内皮细胞生长迁移以及NO合成与释放的信号转导机制。本研究有望发现AcSDKP促血管再内皮化的新机制，使它成为临床上改进DES的有效途径。

血管内支架再狭窄源于血管损伤后的修复反应，如何促进血管的再内皮化，抑制内膜增生，从而降低血栓发生率，是目前改进药物洗脱支架（DES）的关键环节。本项目基于血流动力学因素对血管内皮细胞形态和功能的重要影响，构建了能够模拟血管血流微环境的微流控生物芯片，检测不同的剪切力对血管内皮细胞生物功能的影响。遗憾的是AcSDKP在研究过程中，因为其容易被降解，在血液中半衰期短，在我们的模型中没有得到很理想的效果。但是，我们在血管再内皮化的调节机制领域还是做了以下几个工作：1）合作研究发现NEDD8-活化酶（NAE）选择性抑制剂MLN4924能够显著抑制新生内膜增生和血管平滑肌细胞的积累，增加血管壁的细胞凋亡；2）利用巨噬细胞中特异敲除内质网应激中的感应蛋白IRE1的MφKO小鼠合作研究揭示了IRE1在代谢调节以及炎症免疫反应的双重调控作用及其潜在机制；3）利用肝脏特异性敲除IRE1的小鼠，发现肝细胞中特异性敲除IRE1能明显抑制STAT3磷酸化水平及其介导的肝细胞增殖；4）为了研究IRE1对血管损伤后再内皮化的可能作用机制，我们通过IRE1flox/flox小鼠与VE-Cadherin-Cre小鼠杂交获得了内皮细胞特异敲除的IRE1endo-/-小鼠。研究表明血管内皮细胞特异性敲除IRE1导致小鼠血管内皮生理功能不良，利用导丝诱导股动脉损伤的小鼠模型，验证IRE1对血管损伤后的稳态维持和重塑作用，发现血管内皮细胞中特异性敲除IRE1血管再内皮明显减缓，加重了损伤血管新生内膜增生。本研究有望发现IRE1通路对血管再内皮化的调节的新机制，使它成为临床上改进DES的有效靶点。