Spatial and Temporal Regulation of Angiogenesis

描述（由申请人提供）： 该项目从多个角度关注肿瘤血管的结构和功能：血管生成细胞因子和内源性抑制剂在肿瘤和替代肿瘤血管中的作用和功能，这些因子诱导的基因表达及其在肿瘤血管生成中的作用，以及血管生成细胞因子和血管生成对肿瘤进展的影响。该提案概述了一系列实验，重点是主要的血管生成细胞因子下游的主要信号通路之一，Akt通路，作为许多肿瘤血管异常的介质：它们的大尺寸，差的支持，增加的渗透性和不稳定性。我们的初步数据表明，Akt信号在生理血管生成涉及一个非常精确的开关'上'和'关闭Akt信号。然而，当这种“关闭开关”被抑制时，所产生的血管重现了肿瘤血管、其他血管异常血管和“母血管”的许多方面，“母血管”是Dvorak博士创造的一个术语，用于描述由VEGF-A和其他细胞因子过度表达形成的血管。这项资助申请旨在探索Akt在“母血管”/“肿瘤血管”形成中的作用，以及下游Akt靶点对这些异常血管的个体特征和功能的影响。我们还建议探索TSP-1调节Akt信号转导的新发现背后的机制。这个项目是写了3年，使其能够折叠到现有的资金时间表，这一计划项目。目的：1.验证Akt活性上调足以重现非肿瘤环境中异常血管的结构和功能的假设2。确定介导Akt影响的下游信号通路3.探讨凝血酶敏感蛋白对Akt信号通路的影响机制。 合作机构：马萨诸塞州总医院，马萨诸塞州波士顿 儿童医院，波士顿，MA 目前筹资期间的进展情况：该方案现已完成第二年的工作。在此期间，17篇出版物来自项目相关研究。其中一些已经出现在高影响力的期刊上，11个列出了多个作者谁是这个计划项目的一部分。 总体进展非常出色。 项目1，“新血管的结构和功能”，由哈罗德·德沃夏克博士与劳拉·本杰明博士共同指导。 在特定目标1下的研究中，研究人员描述了VEGF-A与PIGF，VEGF-A/PIGF异源动力因子和FGF的新血管诱导，现已完成，并发表在Nature Medicine上。 具体目标2是在乳腺肿瘤中采用诱导型基因表达，以过表达或不表达具体目标1下研究的血管生成因子。这些研究正在进行中，并已发表了两份出版物。 具体目标2将使用芯片技术来分析在“母”血管中响应VEGF-A或PIGF局部过表达而差异表达的基因。在该项目的前两年中，已经鉴定了几个新基因，并且还鉴定了许多其他潜在的重要基因。两个手稿正在准备描述这项工作。 项目2，决定血管结构和功能的信号通路，是本补充中新提出的项目，并在下文中详细讨论。值得注意的是，Benjamin博士是所列17篇出版物中6篇的合著者，其中几篇与拟议的补充项目2非常相关。 项目3，“胎盘生长因子在正常和肿瘤性皮肤血管生成中的作用”，由Michael Detmar博士领导。具体目标1是使用转基因动物模型研究PIGF在皮肤血管化和实验性皮肤炎症中的作用。已经制备了一种用于在基底表皮角质形成细胞和其他细胞中靶向过表达人PIGF-2的新型转基因小鼠模型。对这些动物的研究导致了一篇发表在《血液》上的论文。具体目标2将使用这些模型来研究化学诱导的皮肤癌。初步结果表明，PIGF在上皮肿瘤发生中起作用。具体目标3将采用转染的PIGF和semaphorin D基因构建体来研究调节血管生成对人鳞状细胞癌异种移植物生长的影响。已经获得了稳定转染的细胞系，以实现这一目标，目前正在收集这些模型的数据。 项目4，“凝血酶敏感蛋白-1抑制血管生成”，由Jack Lawler博士领导，R. Khosravi-far和S.帕兰吉具体目标1旨在表征TSP-1结合的内皮细胞上的CD 36受体复合物。具体目标2将研究TSP-1诱导细胞凋亡的机制。这些目标的工作正在取得进展，但似乎还处于初步阶段。Lawler博士是三篇论文的合著者，这些论文报告了在高影响力期刊上发表的配体TSP-1的工作。 此外，还有四个核心（管理，形态学，细胞生物学和基因组学），它们都是可操作的，并在整个计划项目中发挥作用。如上所述，总体进展情况良好。

DESCRIPTION (provided by applicant): This program project focuses on the structure and function of tumor blood vessels from many angles: roles and functions of angiogenic cytokines and endogenous inhibitors in tumors and in surrogate tumor blood vessels, the gene expression induced by these factors and their role in tumor angiogenesis, and the impact of angiogenic cytokines and angiogenesis on tumor progression. This proposal outlines a series of experiments that focus on one of major signaling pathways downstream of the major angiogenic cytokines, the Akt pathway, as a mediator of many tumor blood vessel abnormalities: their large size, poor support, increased permeability and instability. Our preliminary data demonstrate that Akt signaling in physiological angiogenesis involves a very precise switching 'on' and 'off of the Akt signal. However, when this 'off switch is inhibited, the resulting vessels recapitulate many aspects of tumor blood vessels, vessels of other vascular anomalies and 'mother vessels', a term coined by Dr. Dvorak to describe blood vessels formed by overexpression of VEGF-A and other cytokines. This grant application seeks to explore the role of Akt in 'mother vessel'/'tumor vessel' formation, and the impact of downstream Akt targets on the individual features and functions of these abnormal blood vessels. We also propose to explore the mechanism behind our novel finding that TSP-1 modulates Akt signaling. This project is written for 3 years to enable it to fold into the existing funding schedule of this program project. Aims: 1. Test the hypothesis that upregulation of Akt activity is sufficient to recapitulate the structure and function of abnormal blood vessels reminiscent of tumor vessel in a non-tumor environment 2. Identify the downstream signaling pathways that mediate Akt affects 3. Explore the mechanisms of Thrombospondins on Akt signaling. COLLABORATING INSTITUTIONS (S): Massachusetts General Hospital, Boston, MA Children's Hospital, Boston, MA PROGRESS DURING THE CURRENT FUNDING PERIOD: This program has now completed its second year. During this period 17 publications have resulted from program related studies. A number of these have appeared in high impact journals and 11 list multiple authors who are part of this Program Project. Overall progress has been excellent. Project 1, "Structure and Function of New Blood Vessels," is directed by Dr. Harold Dvorak, in conjunction with Dr. Laura Benjamin. The studies under Specific Aim 1 where the investigators characterized new blood vessel induction by VEGF-A versus PIGF, VEGF-A/PIGF heterodynamers and FGF, are now completed and the work published in Nature Medicine. Specific Aim 2 is to employ inducible gene expression in mammary tumors to overexpress or not express the angiogenic factors studied under Specific Aim 1. These studies are underway and have resulted in two publications. Specific Aim 2 will use chip technology to profile genes that are differentially expressed in 'mother' vessels in response to local overexpression of VEGF-A or PIGF. Several novel genes have been characterized in the first two years of the project and many other potentially important genes also identified. Two manuscripts are in preparation describing this work. Project 2, Signaling Pathways that Determine Vascular Structure and Function, is the newly proposed project in this supplement and is considered in detail below. It is noted that Dr. Benjamin is a co-author of six of the 17 publications listed, several of which are very relevant to proposed supplement Project 2. Project 3, "Role of Placental Growth Factor in Normal and Neoplastic Skin Angiogenesis," is led by Dr. Michael Detmar. Specific Aim 1 is a study of the role of PIGF in skin vasculaturation and in experimental skin inflammation employing transgenic animal models. A novel transgenic mouse model for the targeted overexpression of human PIGF-2 in basal epidermal keratinocytes and other cells has been made. Studies of these animals have resulted in a paper published in Blood. Specific Aim 2 will employ these models to study chemically-induced skin cancer. Initial results suggest that PIGF plays a role in epithelial tumorigenesis. Specific Aim 3 will employ transfected PIGF and semaphorin D gene constructs to study the effects of modulating angiogenesis on the growth of human squamous cell carcinoma xenotransplants. Stably transfected cell lines have been obtained to pursue this aim and data are now being collected on these models. Project 4, "Inhibition of Angiogenesis by Thrombospondin-1," is led by Dr. Jack Lawler with the participation of Dr. R. Khosravi-far and Dr. S. Parangi. Specific Aim 1 seeks to characterize the CD36 receptor complex on endothelial cells to which TSP-1 binds. Specific Aim 2 will study mechanisms involved in TSP-1-induced apoptosis. Work is progressing on these aims, but appears quite preliminary. Dr. Lawler is co-author of three papers that report work on the ligand TSP-1 which have appeared in high impact journals. In addition, there are four cores (Administration, Morphology, Cell Biology, and Genomics) which are all operational and fulfill their role in the overall Program Project. Overall progress, as noted above, has been excellent.