NO in Tumor Angiogenesis,Microcirculation & Rad.Therapy

为了优化实体肿瘤的药物和氧气输送，我们计划研究肿瘤血管生成和微循环的调节机制。本项目将重点研究一氧化氮（NO）在肿瘤血管生成和血管成熟中的作用。我们的目标是通过合理调节肿瘤中的NO水平来改善肿瘤血流和辐射反应。由于肿瘤血管在直径、长度、弯曲度和毛细血管间距离方面的功能受损和异质性，肿瘤血流的控制尚未实现。然而，通过抑制NO向肿瘤血管中更“正常”的功能表型转化可能实现这一目标。NO对血管内皮细胞（诱导）和平滑肌细胞（抑制）细胞增殖和迁移的影响不同。我们假设NO诱导肿瘤血管生成（Aim 1），但抑制血管成熟（Aim 2），因此，慢性NO抑制使肿瘤血管“正常化”，使其对血管扩张剂敏感。一氧化氮的产生将被药理学和遗传方法操纵。同种异构体选择性NO合成酶（NOS）抑制剂将阐明每种NOS异构体的相对贡献。使用NOS缺陷小鼠，我们将检查宿主基质细胞的贡献。肿瘤包括胶质母细胞瘤、黑色素瘤和原位生长的原发性和转移性肿瘤乳腺癌。活体显微镜将允许我们监测血管密度、直径、弯曲度、血流速率、血管通透性和对血管活性药物的反应。为了研究内皮-壁细胞在体内的相互作用，将通过多光子激光扫描显微镜观察在壁细胞（alphaSMA-RFP）和内皮细胞（TIE2-GFP）中表达荧光蛋白的小鼠。最后，将测试慢性NO抑制后血管扩张剂和碳（O2 95%, CO2 5%）呼吸对肿瘤氧合和辐射反应的改善（Aim 3）。在这个项目中获得的见解将对改善肿瘤治疗方案具有重要意义。它将提出一种新的策略（即肿瘤血管正常化），以克服治疗药物递送到实体肿瘤的一些生理障碍，并引入一种新的范式来研究体内细胞-细胞相互作用。

In order to optimize drug and oxygen delivery to solid tumors, we plan to investigate the mechanisms, which regulate tumor angiogenesis and microcirculation. This project will focus on the role of nitric oxide (NO) in tumor angiogenesis and vessel maturation. Our goal is to improve tumor blood flow and radiation response by judicious modulation of NO levels in tumors. Control of tumor blood flow has not been achieved due to the fact that tumor vessels are functionally impaired and heterogeneous with respect to diameter, length, tortuosity, and inter-capillary distance. However, transformation to a more "normal" functional phenotype in tumor blood vessels by suppressing NO may realize this goal. Effects of NO on cell proliferation and migration are different in vascular endothelial cells (induction) and smooth muscle cells (inhibition). We hypothesize that NO induces angiogenesis in tumors (Aim 1), but inhibits vessel maturation (Aim 2), and thus, chronic NO inhibition "normalizes" tumor vessels, making them sensitive to vasodilators. NO production will be manipulated by both pharmacological and genetic approaches. Isoform-selective NO synthase (NOS) inhibitors will clarify the relative contribution of each NOS isoform. With the use of NOS deficient mice, we will examine the contribution of host stromal cells. The tumors will include glioblastoma, melanoma, and breast carcinoma grown in orthotopic sites for primary and metastatic tumors. Intravital microscopy will allow us to monitor vessel density, diameter, tortuosity, blood flow rate, vascular permeability, and response to vasoactive agents. To study endothelial-mural cell interaction in vivo, mice expressing fluorescent protein in mural cells (alphaSMA-RFP to be developed in this project) and endothelial cells (TIE2-GFP) will be visualized by multiphoton laser-scanning microscopy. Finally, improvement of tumor oxygenation and response to radiation by a vasodilator and carbogen (O2 95 percent, CO2 5 percent) breathing after chronic NO suppression will be tested (Aim 3). The insight gained in this project will have significant implications for improving tumor treatment protocols. It will suggest a novel strategy (i.e. normalization of tumor vessels) to overcome some of the physiological barriers to the delivery of therapeutic agents to solid tumors and introduce a new paradigm to study cell-cell interaction in vivo.