Molecular Regulation of Vascular Regression

血管退化的分子调控

基本信息

批准号：
8212754
负责人：
ROBERTO NICOSIA
金额：
--
依托单位：
VA PUGET SOUND HEALTHCARE SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8212754
关键词：
Ablation Age related macular degeneration Angiogenic Switch Animals Antibodies Aorta Applications Grants Area Atherosclerosis Biological Assay Blocking Antibodies Blood Vessel Tissue Blood Vessels Blood capillaries Cancer Patient Caring Cells Cessation of life Chronic Kidney Failure Clinical Collagen Confocal Microscopy Coronary Arteriosclerosis Cytolysis Development Diabetic Retinopathy Disease Disease Progression Dissection Endothelial Cells Enzyme-Linked Immunosorbent Assay Enzymes Event Extracellular Matrix Gel Gelatinase A Gene Expression Profile Genes Goals Grant Granulation Tissue Growth Hemangioma Hypertension Immune system Implant In Situ Nick-End Labeling In Vitro Integrin beta3 Integrins Interferon Type I Interferon-alpha Interferon-beta Interferons Ischemia Knowledge Laboratories Lead Life Malignant Neoplasms Matrix Metalloproteinase Inhibitor Matrix Metalloproteinases Mediating Methods Microarray Analysis Mission Modeling Molecular Molecular Analysis Morbidity - disease rate Mus Outcome Pathologic Pathologic Processes Patients Phase Phenotype Physiological Play Procedures Process Proteolysis Psoriasis Rattus Receptor Gene Recombinants Regulation Research Resistance Resolution Reverse Transcriptase Polymerase Chain Reaction Rheumatoid Arthritis Role Scleroderma Source Staging Stromelysin 1 System Time Tissues Tube Veterans Western Blotting Work Wound Healing angiogenesis base cancer therapy capillary design human MMP14 protein immunocytochemistry in vivo innovation insight macrophage matrix metalloproteinase 12 neovascularization neovasculature novel strategies novel therapeutic intervention overexpression patient population prevent public health relevance research study response response to injury subcutaneous tissue repair tumor tumor progression type I interferon receptor vessel regression

项目摘要

DESCRIPTION (provided by applicant): Angiogenesis, the formation of new blood vessels, plays a critical role in the progression of cancer and other pathologic processes, but it also ameliorates coronary artery disease and other ischemic conditions caused by inadequate neovascularization. The outcome of angiogenesis depends on the capacity of neovessels to regress or survive. There is however a gap in our understanding of the molecular mechanisms regulating the fate of postangiogenic neovessels. We have reproduced the process of vascular regression by culturing rat or mouse aortic rings in collagen gels under chemically defined culture conditions. In this ex vivo culture system aortic rings generate microvessels which regress over time due to matrix metalloproteinase (MMP)-mediated proteolysis of the extracellular matrix (ECM). We have identified MMP-14 as one of the enzymes responsible for vascular regression, and shown that involution of neovessels can be accelerated with anti-21 and anti-23 integrin antibodies. We have successfully duplicated the vascular regression process in vivo by implanting aortic rings in the subcutaneous space of syngeneic rats or mice. We recently found that vascular regression and lysis of the ECM in the ex vivo aortic ring model are associated with perivascular accumulation of macrophages and overexpression of MMP-3, MMP- 12 and MMP-14. Macrophages isolated from regressing cultures inhibited the sprouting of freshly cut aortic rings. Molecular analysis of regressing aortic cultures demonstrated upregulated expression of type I interferons (INF-1 and IFN-2) and IFN response genes. Recombinant IFNs blocked the angiogenic response of the aortic explants and caused involution of neovessels. Since macrophages are required for angiogenic sprouting, we hypothesize that these cells change over time from an angiogenic to an angiostatic phenotype. We postulate that angiostatic macrophages are the main source of angiolytic MMPs. Finally we propose that type I IFNs promote the vascular regression process including the transformation of macrophages into an angiostatic and angiolytic phenotype. Specific aims of this proposal are designed to investigate the following hypotheses: (1) Macrophages switch from an angiogenic to an angiostatic phenotype during vascular regression. (2) MMP-mediated vascular regression (angiolysis) following angiogenesis requires macrophages. (3) Interferons promote vascular regression following angiogenesis. Proposed experiments will be performed with in vitro, ex vivo and in vivo assays of angiogenesis. For in vitro studies we will use a capillary tube formation assay with isolated endothelial cells. The rat and mouse aortic models of angiogenesis will be used for ex vivo studies of angiogenesis and vascular regression. For studies in the live animal, we will use our newly developed in vivo assay in which subcutaneously implanted aortic rings stimulate formation of a richly vascularized granulation tissue which involutes over time. Our research plan includes genetically modified mice with disrupted MMP-3, MMP-12, MMP-14, or type I IFN receptor genes. Proposed experiments employ quantitative real-time RT-PCR, immunocytochemistry, confocal microscopy, Western blotting, TUNEL, ELISA, and pharmacological methods of ex vivo and in vivo macrophage ablation. Functional studies will be performed with anti-INF-1 and -IFN-2 blocking antibodies. Experiments proposed in this grant will further our understanding of the cellular and molecular mechanisms responsible for vascular regression following angiogenesis. This knowledge may contribute to the development of novel therapeutic approaches for the treatment of cancer and other angiogenesis-related disorders. PUBLIC HEALTH RELEVANCE: This grant is designed to identify key mechanisms of vascular regression following blood vessel growth (angiogenesis). Understanding how the fate of angiogenic neovessels is regulated at a molecular level has important clinical implications. Cancer patients will benefit from targeted disruption of the tumor vasculature with therapies aimed at inducing vascular regression. The same consideration applies to patients with wet age related macular degeneration or diabetic retinopathy. On the other hand, inducing the formation of regression-resistant blood vessels will benefit patients with coronary artery disease or other ischemic conditions. Studies proposed in this grant will contribute to the VA care mission since many diseases afflicting the veteran patient population are associated with abnormal persistence or loss of angiogenic neovessels.

描述（由申请人提供）：血管生成是新血管的形成，在癌症和其他病理过程的进展中起着至关重要的作用，但它也可以改善因新生血管形成而引起的冠状动脉疾病和其他缺血性疾病。血管生成的结果取决于新固定剂回归或生存的能力。但是，我们对调节后传管疾病新固定命运的分子机制的理解存在差距。我们通过在化学定义的培养条件下培养大鼠或小鼠主动脉环的培养大鼠或小鼠主动脉环的过程。在此离体培养系统中，主动脉环会产生微血管，由于基质金属蛋白酶（MMP）介导的细胞外基质（ECM）介导的蛋白水解会随着时间的流逝而回归。我们已经将MMP-14确定为负责血管回归的酶之一，并表明可以用抗21和抗23整联蛋白抗体加速新固定固定的酶。我们通过将主动脉环植入合成大鼠或小鼠的皮下空间中，成功地在体内复制了血管回归过程。我们最近发现，离体主动脉环模型中ECM的血管回归和裂解与巨噬细胞的血管周期积累以及MMP-3，MMP-12和MMP-14的过表达有关。从回归培养物中分离出来的巨噬细胞抑制了新鲜切割的主动脉环的发芽。回归主动脉培养物的分子分析表明，I型干扰素（INF-1和IFN-2）和IFN反应基因的上调表达上调。重组IFNS阻断了主动脉植体的血管生成反应，并引起了Neovessels的参与。由于血管生成需要巨噬细胞，因此我们假设这些细胞会随着时间的流逝从血管生成变为血管静脉表型。我们假设血管遗传性巨噬细胞是血管生成MMP的主要来源。最后，我们提出I型IFN促进了血管回归过程，包括将巨噬细胞转化为血管静脉和血管静脉表型。该提案的具体目的旨在研究以下假设：（1）巨噬细胞在血管回归期间从血管生成到血管静脉表型。（2）血管生成后MMP介导的血管回归（血管溶）需要巨噬细胞。（3）干扰素在血管生成后促进血管回归。提出的实验将通过体外，离体和血管生成的体内测定法进行。对于体外研究，我们将使用带有分离的内皮细胞的毛细管形成测定法。血管生成的大鼠和小鼠主动脉模型将用于血管生成和血管再生的体内研究。对于活动物的研究，我们将使用我们新开发的体内测定法，其中皮下植入的主动脉环刺激了随着时间的推移涉及的丰富血管化颗粒组织的形成。我们的研究计划包括具有干扰的MMP-3，MMP-12，MMP-14或I型IFN受体基因的转基因小鼠。拟议的实验采用定量实时RT-PCR，免疫细胞化学，共焦显微镜，蛋白质印迹，TUNEL，ELISA和药理学方法的外体和体内巨噬细胞消融。功能研究将使用抗INF-1和-IFN-2阻断抗体进行。该赠款中提出的实验将进一步了解我们对负责血管生成后血管回归的细胞和分子机制的理解。这些知识可能有助于发展新型治疗方法，以治疗癌症和其他与血管生成有关的疾病。公共卫生相关性：该赠款旨在确定血管生长后血管回归的关键机制（血管生成）。了解在分子水平上如何调节血管生成的命运具有重要的临床意义。癌症患者将受益于肿瘤脉管系统的靶向破坏，旨在诱导血管回归的疗法。同样的考虑也适用于与湿年龄相关的黄斑变性或糖尿病性视网膜病的患者。另一方面，诱导耐回归血管的形成将使冠状动脉疾病或其他缺血性疾病的患者受益。该赠款中提出的研究将有助于VA护理任务，因为许多困扰退伍军人群体的疾病与血管生成的新固定剂的持久性异常或丧失有关。