BIOMECHANICS OF LEUKOCYTE ADHESION MOLECULES

该申请提出了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在炎症、免疫、癌症转移和其他过程中控制细胞的运输。一些粘附分子，其中包括选择素，专门用于在血流存在下介导粘附。压力驱动的血流与施加在血管壁上的剪切应力相关，这导致白细胞和试图粘附到内皮的其他细胞上的力。据信，剪切应力下的粘附需要具有快速缔合速率（on-rate）的粘附分子，从而导致键的快速形成。 体外实验和建模研究表明，选择素也具有高的键解离速率（解离速率）。 初步数据表明，选择素的解离速率随施加在由选择素结合的细胞上的剪切力（反应顺应性或拉伸强度）而系统地变化。 此外，通过表面结合或膜整合金属蛋白酶的蛋白水解切割，加速至少一种选择素的释放。 目前的建议有四个具体目标。 (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解离速率。 (2)探讨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胞质尾对剪切流下粘附生物力学的影响。 (4)设计和构建珠粒、脂质体和充气气泡（超声造影剂），这些药物在剪切应力下使用白细胞粘附分子与血管壁结合。 每一个目标都是从三个方面着手实现的。 我们建议使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，使用稀疏基底上的单细胞来了解选择素在体外流动室中的生物力学，并使用活体显微镜来研究选择素在活生物体中的生物力学。 我们建议使用分子生物学技术来操纵cDNA、细胞和小鼠以分离每个分子机制。 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，用于在剪切流下在血管系统中递送。 在第一年年底，我们计划测量选择素解离速率，考虑到选择素脱落，并测试了含有选择素的脂质体在剪切下的粘附能力。 时间轴中列出了随后几年的里程碑。

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.