BIOMECHANICS OF LEUKOCYTE ADHESION MOLECULES

本申请提出分子生物力学领域的跨学科生物工程研究。白细胞和内皮粘附分子控制着炎症、免疫、癌症转移和其他过程中细胞的运输。一些粘附分子，其中包括选择素，专门用于在血流存在时介导粘附。压力驱动的血流与施加在血管壁上的剪切应力有关，这导致白细胞和其他试图粘附在内皮上的细胞受到压力。人们认为，剪切应力下的粘附需要具有快速缔合速率（on-rates）的粘附分子，从而导致键的快速形成。体外实验和模型研究表明，选择也具有高的键解离率（off-rates）。初步数据表明，选择素的脱失率随选择素结合的细胞所受的剪切力（反应顺应性或拉伸强度）而系统性地变化。此外，至少一种选择素的释放可通过表面结合或膜一体化金属蛋白酶的蛋白水解裂解而加速。目前的提议有四个具体目标。(1)测定L-、P-和e -选择素与其天然配体结合的键寿命和表观脱配率。(2)确定l -选择素脱落在调节白细胞粘附和选择素动力学中的作用。(3)确定选择素长度及其细胞质尾部对剪切流动下黏附生物力学的影响。(4)设计和制造利用白细胞粘附分子在剪切应力下与血管壁结合的微珠、脂质体和充气气泡（超声造影剂）。每一个目标都以三管齐下的方式实现。我们建议利用激光捕获技术直接测量选择素键的生物力学和动力学参数，利用稀疏底物上的单细胞在体外流动室中了解选择素的生物力学，并利用活体显微镜在生物体背景下研究选择素的生物力学。我们建议使用分子生物学技术来操纵cDNA，细胞和小鼠来分离每个分子机制。我们将利用所获得的见解来设计基于脂质体的靶向药物递送系统和超声造影剂微泡，以便在剪切流下在血管系统中递送。在第一年结束时，我们计划测量选择素的脱落率，考虑到选择素的脱落，并测试含选择素脂质体在剪切下的粘附能力。时间表中列出了接下来几年的里程碑。

This application proposes interdisciplinary bioengineering research in the area of molecular biomechanics. Leukocyte and endothelial adhesion molecules govern the trafficking of cells in inflammation, immunity, cancer mestastasis and other processes. Some adhesion molecules, among them the selectins, are specialized to mediate adhesion in the presence of blood flow. Pressure-driven blood flow is associated with a shear stress exerted on the vessel wall, which results in a force on leukocytes and other cells trying to adhere to the endothelium. It is believed that adhesion under shear stress requires adhesion molecules with rapid association rates (on-rates), resulting in rapid formation of bonds. In vitro experiments and modeling studies indicate that the selectins also have high rates of bond dissociation (off-rates). Preliminary data suggest that the off-rates of selectins vary systematically with the shearing force exerted on the cell bound by the selectin (reactive compliance or tensile strength). In addition, the release of at least one of the selectins is accelerated by proteolytic cleavage by a surface-bound or membrane integral metalloproteinase. The current proposal has four specific aims. (1) To measure the bond lifetimes and apparent off-rates of L-, P- and E-selectin bound to their natural ligands. (2) To determine the role of L-selectin shedding in regulating leukocyte adhesion and selectin kinetics. (3) To determine the impact of the selectin length and their cytoplasmic tail for the biomechanics of adhesion under shear flow. (4) To design and build beads, liposomes and gas-filled bubbles (ultrasound contrast agents) that use leukocyte adhesion molecules to bind to vessel walls under shear stress. Each of these aims is approached in a three-pronged fashion. We propose to use laser trapping technology to directly measure biomechanical and kinetic parameters of selectin bonds, use single cells on sparse substrates to understand the biomechanics of selectins in an in vitro flow chamber, and use intravital microscopy to study selectin biomechanics in the context of the living organism. We propose to use molecular biology techniques to manipulate cDNA, cells, and mice to isolate each molecular mechanism. We will use the insights gained to design liposome-based targeted drug delivery systems and ultrasound contrast microbubbles for delivery in the vascular system under shear flow. At the end of the first year, we plan to have measurements of selectin off-rates, taking into account selectin shedding, and have tested selectin-containing liposomes for their ability to adhere under shear. Milestones for the following years are listed in the timeline.