NO in Tumor Angiogenesis,Microcirculation & Rad.Therapy

为了优化对实体肿瘤的药物和氧气输送，我们计划研究调节肿瘤血管生成和微循环的机制。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。我们的目标是通过明智地调节肿瘤中的NO水平来改善肿瘤的血流和放射反应。由于肿瘤血管的功能受损以及在直径、长度、弯曲程度和毛细血管间距离方面的异质性，肿瘤血流量的控制尚未实现。然而，通过抑制NO将肿瘤血管转化为更“正常”的功能表型可能会实现这一目标。在血管内皮细胞(诱导)和血管平滑肌细胞(抑制)中，NO对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(目标1)，但抑制血管成熟(目标2)，因此，慢性NO抑制使肿瘤血管“正常化”，使它们对血管扩张剂敏感。NO的生产将同时受到药理学和遗传学方法的影响。异构体选择性一氧化氮合酶(NOS)抑制剂将阐明每个NOS异构体的相对贡献。利用一氧化氮合酶缺陷小鼠，我们将检测宿主基质细胞的贡献。这些肿瘤将包括胶质母细胞瘤、黑色素瘤和原发和转移性肿瘤原位生长的乳腺癌。活体显微镜将使我们能够监测血管密度、直径、曲折程度、血流速度、血管通透性以及对血管活性物质的反应。为了研究体内内皮细胞与壁细胞的相互作用，我们将用多光子激光扫描显微镜观察在壁细胞中表达荧光蛋白的小鼠(αSMA-RFP)和内皮细胞中表达荧光蛋白的小鼠(TIE2-GFP)。最后，将测试慢性NO抑制后血管扩张剂和Carbogen(O2 95%，CO2 5%)呼吸对肿瘤氧合的改善和对辐射的反应(目标3)。在这个项目中获得的洞察力将对改进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)来克服向实体肿瘤输送治疗药物的一些生理障碍，并引入一种新的范式来研究体内细胞与细胞的相互作用。

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.