动脉粥样硬化是以炎症细胞浸润动脉血管壁为主要特征的慢性血管炎症。中性粒细胞是帮助器官启动和维持免疫反应的关键参与者，也是第一个穿过血管内皮细胞进入组织的细胞类型。在动脉粥样硬化过程中，中性粒细胞的募集是一个炎症级联反应，首先血液里流动的中性粒细胞被激活的内皮细胞捕获，在内皮细胞表面滚动，然后稳定粘附，最后跨内皮迁移到炎症部位。上述过程受到趋化因子、粘附分子和生理力学因素（血管刚度、流体剪切）的共同调控，是一个典型的力学-生物学耦合过程。此外，中性粒细胞和内皮细胞上的粘附分子的作用可介导微丝骨架动态重组，进而促进中性粒细胞的跨血管内皮迁移。Ca²⁺被作为细胞内的第二信使，参与调控中性粒细胞跨内皮迁移过程。动脉粥样硬化病理过程中，血管刚度增加，同时中性粒细胞募集也增加。但是，中性粒细胞募集如何受到血管刚度的调控，仍不清楚。本文采用生物力学、生物材料和活细胞成像相结合的手段，分别从炎症反应的两个对象（中性粒细胞和血管内皮细胞）入手，研究表达在内皮细胞表面的粘附分子E-选择素（E-selectin）通过内皮细胞骨架动态重组进而调控中性粒细胞迁移，以及基底刚度对中性粒细胞胞内钙响应的调控，深化了对动脉粥样硬化环境下炎症反应的认识。本文工作主要包括以下两方面：

  1. 研究了E-selectin调控的内皮细胞微丝骨架重组对中性粒细胞跨内皮迁移的影响：本文建立了体外中性粒细胞跨内皮迁移模型，采用脂多糖处理脐静脉内皮细胞模拟在体的炎症内皮细胞，通过原子力显微镜等手段量化了E-selectin调控下内皮细胞胞间连接的力学性质变化，及其对中性粒细胞跨内皮迁移的影响。研究发现E-selectin负向调控中性粒细胞跨内皮迁移，中性粒细胞在E-selectin敲除的内皮细胞上表现出更快更高的跨内皮迁移动力学，这种负向调控主要是通过E-selectin调控内皮细胞骨架动态重组来实现。敲除E-selectin可减少内皮细胞胞间连接处的分支状肌动蛋白，增加应力纤维，进而增加胞间连接处的细胞间隙，导致胞间连接不稳定，促进中性粒细胞从此处穿过内皮而迁移。这个过程是E-selectin通过调控微丝骨架结合蛋白Arp 2/3和Cortactin介导的内皮细胞骨架重组和片状伪足生成来完成的。另此外，E-selectin敲除后，内皮细胞上表达的P-选择素（P-selectin）对E-selectin介导的中性粒细胞粘附功能起补偿作用。该部分研究表明E-selectin可通过调控内皮细胞骨架动态重组改变胞间连接整合性，从而负调控中性粒细胞的跨内皮迁移，深化了对内皮细胞骨架重组影响中性粒细胞募集的生物力学机制的认识。

  2. 研究了基底刚度对中性粒细胞胞内钙响应的影响。本文将HUVEC接种于5 kPa、34.88 kPa这两种不同刚度的聚丙烯酰胺水凝胶来模拟生理病理条件下的血管刚度，并以玻璃基底作为对照，观察不同基底刚度对人中性粒细胞胞内钙响应的影响。研究发现，中性粒细胞胞内钙响应表现出随基底刚度升高而增加的现象。采用原子力显微镜检测基底刚度对内皮细胞刚度的影响，发现内皮细胞刚度随基底刚度增加而增加，而在三种基底刚度上内皮细胞表面主要粘附分子E/P-selectin，ICAM-1的表达足量，说明中性粒细胞胞内钙响应具有刚度依赖性、且不受粘附分子表达的影响。进一步采用抑制剂阻断研究中性粒细胞刚度依赖的钙响应调控机制，结果显示内皮细胞上的E-/P-selectin可与中性粒细胞上的配体分子CD44和PSGL-1相互作用，通过Syk/Src信号通路激活β₂整合素，从而增强中性粒细胞胞内钙响应。此外，细胞骨架和机械敏感的钙离子通道也参与该过程。本研究揭示了动脉粥样硬化等疾病过程中力学微环境调控钙响应的分子机制，对于深入了解心血管活动和疾病发生有重要意义。

  综上，本文工作定量诠释了E-selectin和基底刚度对内皮细胞单层上中性粒细胞募集的生物力学调控和力学-生物学耦合机制，对深化认识动脉粥样硬化的免疫调控机制具有重要的基础意义。

  Atherosclerosis is a chronic vascular inflammation characterized by inflammatory cells infiltrating the arterial wall. Polymorphonuclear neutrophils (PMNs) are the key participants in the initiation and maintenance of immune responses and are also the first cell to enter the tissues through vascular endothelial cells (ECs). PMN extravasation is a complex multistep process involving tethering, rolling, adhesion, crawling and finally transmigration through ECs, which is a typical mechanical-biological coupling process regulated by chemokines, adhesion molecules and mechanical factors such as vascular stiffness and shear flow. Besides, the remodeling of actin network mediated by adhesion molecules in both PMNs and ECs is required for the PMN extravasation. The calcium flux also functions in the signaling of PMN recruitment as the second messenger. The blood vessel wall becomes stiff, and PMN extravasation increases in the process of atherosclerosis. However, the mechanism how the vascular stiffness alteration affects the behavior of the PMN extravasation is still unknown. In this dissertation, the roles of E-selectin in PMN transmigration via endothelial actin cytoskeleton remodeling and how substrate stiffness affects neutrophil calcium response were studied using the approaches that coordinate mechanobiology, biological materials and live-cell imaging, which attempted to further the understanding of inflammatory response in the process of atherosclerosis. The major works done were summarized as follows:

  1. To elucidate how E-selectin-induced actin remodeling in ECs contributes to PMN transmigration, an in vitro model was proposed to test the transendothelial migration (TEM) dynamics of PMNs on the surface of LPS-treated HUVEC monolayer with or without shRNA E-selectin transfection. E-selectin-induced inter-endothelial junction integrity was characterized by immunostaining and atomic force microscopy (AFM) test. Data showed that E-selectin-knockdown promotes faster and higher migration of PMN, implying that E-selectin on HUVECs negatively regulates TEM dynamics of PMNs. E-selectin-knockdown also destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions, thus expediting PMN transmigration. This process is attributed to actin binding protein Arp 2/3- and cortactin-mediated actin remodeling and lamellipodia formation. Besides, P-selectins possess the overlapped functions in E-selectin-mediated neutrophil adhesion and transmigration. These results presented the evidences that E-selectin negatively regulates neutrophil transmigration due to actin remodeling and endothelial plasticity, which furthers the understanding of actin remodeling in the PMN transmigration from the biomechanical viewpoint and provides a new perspective for understanding the mechanisms of PMN transmigration in inflammatory conditions.

  2. To understand how substrate stiffness affects neutrophil calcium response, an in vitro cellular inflammation model by pre-culturing HUVECs on a polyacrylamide (PA) substrate (5 or 34.88 kPa) or glass surface was conducted. AFM test indicated that HUVEC elasticity is increased with the increased substrate stiffness. PMNs were then loaded on the surface of HUVEC monolayer and calcium flux of PMNs was recorded. Data showed that neutrophil calcium spike is presented in a stiffness-dependent manner since the expression of adhesion molecules such as E-/P-selectin and ICAM-1 were sufficient to verify the stiffness-dependent manner of calcium response, indicating that stiffness dependence of calcium spike is not affected by expressions of cellular adhesive molecules. Furthermore, the interaction of E-/P-selectin on activated HUVECs with their ligands CD44 and PSGL-1 can activate β₂ integrin to mediate the stiffness-dependent calcium response in PMNs through Syk/Src signaling pathway. Besides, F-actin/myosin II and tension-activated calcium ion channels are also involved in initiating such calcium response. These results shed light on the molecular mechanism by which mechanical microenvironment governs calcium response, and are biologically significant in understanding the features of cardiovascular diseases regulated by mechanical factors.

  Collectively, this work elucidated quantitatively the biomechanical regulation and mechano-biological coupling of neutrophil recruitment onto endothelial monolayer and provided an insight in understanding the immunoregulation on atherosclerosis