Glycosaminoglycans and Cell Traffic in the Lymphatic Microenvironment

描述（由申请人提供）： 淋巴管细胞从外周流向淋巴结的过程发生在炎症反应和肿瘤形成过程中。在这两种情况下，由淋巴管内皮产生的独特趋化因子（趋化性细胞因子）在驱动免疫细胞或肿瘤细胞的各自转运中发挥核心作用。该提案研究了糖胺聚糖在淋巴微环境中介导趋化因子依赖性细胞运输中的遗传重要性，糖胺聚糖是一类重要的复合碳水化合物，包括硫酸乙酰肝素（HS）和硫酸软骨素（CS）。这些硫酸化聚糖链被拴系到独特的细胞表面结合以及分泌的蛋白聚糖核心蛋白，并且它们与几种趋化因子的富含碱性氨基酸的结构域相互作用。本文的具体重点是由淋巴管内皮产生的糖胺聚糖，因为初步的工作表明淋巴HS（可能还有CS）介导两种关键的趋化因子功能：（i）主要淋巴趋化因子的支架（如CCL 21和CXCL 12）在淋巴周围空间梯度中的表达，这些梯度似乎由聚糖保持，和（2）趋化因子对其运输细胞上的同源受体的独特呈递，其中分泌的淋巴糖胺聚糖可作为趋化因子共受体。这项工作将集中在肿瘤的设置，其中淋巴趋化因子依赖的运输涉及癌细胞以及免疫细胞（重点在树突状细胞迁移）。这一提议解决了淋巴糖胺聚糖生物合成的遗传破坏将改变肿瘤以及树突状细胞以趋化因子依赖性方式向淋巴管系统迁移和向淋巴结运输的能力的假设。目标是：（1）鉴定由淋巴管内皮产生的蛋白聚糖核心蛋白，并表征糖胺聚糖链在建立淋巴趋化因子梯度中的作用。在鉴定束缚淋巴内皮HS和CS链的蛋白聚糖核心蛋白的库后，将纯化聚糖链并测试其结合主要淋巴内皮趋化因子的能力。在淋巴管内皮细胞和基质为基础的试验中，将检查在静态和流动条件下改变糖胺聚糖生物合成对趋化因子梯度形成的影响。(2)评估淋巴糖胺聚糖在肿瘤和树突状细胞趋化因子依赖性迁移中的遗传重要性。在鼠Cre-LoxP模型和siRNA改变的人淋巴内皮中靶向淋巴糖胺聚糖生物合成，并测定肿瘤和树突细胞向靶向内皮迁移的能力。此外，将检查从突变细胞收获的条件培养基寡聚化（聚集）淋巴趋化因子以及支持趋化因子介导的肿瘤和树突细胞迁移信号传导途径的活化的能力。(3)在靶向体内淋巴微环境中糖胺聚糖精细结构的遗传改变背景下表征实时细胞运输。将在携带淋巴糖胺聚糖生物合成基因缺陷的小鼠中检查肿瘤和树突细胞从外周淋巴管向区域淋巴结的运输。在基因靶向小鼠的单独癌模型中，还将检查运输肿瘤和树突细胞的淋巴结定殖。另外的机制研究将检查趋化因子分布和受体结合在体内。这项工作可能揭示了一种新的机制范式，即淋巴微环境中的聚糖如何控制多种趋化因子的分布和作用。它也可能为新的治疗药物的发现建立一个合理的基础。 公共卫生相关性： 趋化因子是驱动特定细胞向趋化因子源迁移的“传感”分子。在通常困扰退伍军人群体的疾病如炎症或癌症的状态下，由血管或淋巴管产生的趋化因子可能会驱动炎症或肿瘤细胞的运输，从而产生有害的后果。在肺癌的情况下，退伍军人痛苦和死亡的主要原因，后果可能包括癌症扩散到淋巴结以及趋化因子驱动的特化（“树突状”）免疫细胞向淋巴结的运动，这反过来又可以促进对肿瘤的免疫“耐受性”。初步研究表明，一类复杂的糖分子（糖胺聚糖）可能是淋巴趋化因子的基础或介导。在此，我们使用最先进的遗传工具阻断淋巴管细胞中这种复合糖的产生，并检查对树突状细胞迁移和淋巴结转移的影响。这项工作可能会导致新的治疗方法，以抑制癌症的传播，发病率和死亡率在我们的退伍军人人口。

DESCRIPTION (provided by applicant): Lymphatic vascular cell traffic from the periphery to lymph nodes occurs during both inflammatory responses as well as neoplasia. In both cases, unique chemokines (chemotactic cytokines) produced by the lymphatic endothelium play central roles in driving the respective transit of either immune cells or tumor cells. This proposal examines the genetic importance of glycosaminoglycans, an important class of complex carbohydrates that includes heparan sulfate (HS) and chondroitin sulfate (CS), in mediating chemokine- dependent cell traffic in the lymphatic microenvironment. These sulfated glycan chains are tethered to unique cell-surface bound as well as secreted proteoglycan core proteins, and they interact with basic amino acid-rich domains of several chemokines. The specific focus herein is on glycosaminoglycans produced by the lymphatic endothelium, as a preliminary body of work suggests that lymphatic HS (and possibly CS) mediate two critical chemokine functions: (i) the scaffolding of major lymphatic chemokines (such as CCL21 and CXCL12) in peri-lymphatic spatial gradients that appear to be held by the glycans, and (2) the unique presentation of chemokines to their cognate receptors on trafficking cells, wherein secreted lymphatic glycosaminoglycans may serve as chemokine co-receptors. This work will focus on the setting of neoplasia, where lymphatic chemokine-dependent trafficking involves both carcinoma cells as well as immune cells (with focus herein on dendritic cell migration). This proposal addresses the hypothesis that genetic disruption of lymphatic glycosaminoglycan biosynthesis will alter the ability of tumor as well as dendritic cells to migrate toward lymphatic vasculature and traffic to lymph nodes in a chemokine-dependent manner. The goals are to: (1) Identify proteoglycan core proteins produced by the lymphatic endothelium, and characterize the roles of glycosaminoglycan chains in establishing lymphatic chemokine gradients. Upon identifying the repertoire of proteoglycan core proteins that tether lymphatic endothelial HS and CS chains, the glycan chains will be purified and tested for their ability to bind to major lymphatic endothelial chemokines. In lymphatic endothelial cell- and matrix-based assays, the effects of genetically altering glycosaminoglycan biosynthesis on chemokine gradient formation in static as well as flow conditions will be examined. (2) Assess the genetic importance of lymphatic glycosaminoglycans in chemokine-dependent migration of tumor and dendritic cells. Lymphatic glycosaminoglycan biosynthesis will be targeted in murine Cre-LoxP models and in siRNA-altered human lymphatic endothelium, and the ability of tumor and dendritic cells to migrate toward the targeted endothelium will be determined. In addition, the ability of conditioned medium harvested from the mutant cells to oligomerize (cluster) lymphatic chemokines as well as support chemokine-mediated activation of tumor and dendritic-cell migration signaling pathways will be examined. (3) Characterize real-time cell trafficking in the setting of genetic alterations that target glycosaminoglycan fine structure in the lymphatic microenvironment in vivo. Trafficking of tumor and dendritic cells from peripheral lymphatic vessels to regional lymph nodes will be examined in mice bearing gene defects in lymphatic glycosaminoglycan biosynthesis. In separate carcinoma models in the gene-targeted mice, lymph node colonization by trafficking tumor and dendritic cells will also be examined. Additional mechanistic studies will examine chemokine distribution and receptor binding in vivo. This work may uncover a novel mechanistic paradigm for how the distribution and actions of multiple chemokines may be controlled by glycans in the lymphatic microenvironment. It may also establish a rational basis for novel therapeutic drug discovery. PUBLIC HEALTH RELEVANCE: Chemokines are "sensing" molecules that drive migration of specific cells toward a chemokine source. In states of disease such as inflammation or cancer that commonly afflict the Veteran population, chemokines produced by blood or lymph vessels may drive the transit of inflammatory or tumor cells with deleterious consequences. In the case of lung cancer, a leading cause of Veteran suffering and death, the consequences may include the spread of cancer to lymph nodes as well as chemokine-driven movement of specialized ("dendritic") immune cells to the nodes, which in turn can promote immune "tolerance" to the tumor. Preliminary work shows that a class of complex sugar molecules (glycosaminoglycans) may underlie or mediate the actions of such lymphatic chemokines. Herein, we block the production of such complex sugars in lymphatic vessel cells using state-of- the-art genetic tools, and examine the effects on dendritic cell migration and lymph node metastasis. This work may lead to novel therapy to inhibit the spread, morbidity, and mortality of cancer in our Veteran population.