Molecular Regulation of Vascular Regression

血管退化的分子调控

• 批准号: 8397562
• 负责人: ROBERTO NICOSIA
• 金额: --
• 依托单位: VA PUGET SOUND HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 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/8397562
• 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

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.

描述（由申请人提供）： 血管生成，即新血管的形成，在癌症和其他病理过程的进展中起着至关重要的作用，但它也可以改善冠状动脉疾病和由于新血管形成不足而引起的其他缺血性疾病。血管生成的结果取决于新血管消退或存活的能力。然而，我们对调节血管生成后新生血管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在胶原凝胶中培养大鼠或小鼠主动脉环来重现血管退化的过程。在这种离体培养系统中，主动脉环产生微血管，由于基质金属蛋白酶（MMP）介导的细胞外基质（ECM）蛋白水解作用，微血管随着时间的推移而退化。我们已经确定 MMP-14 是负责血管消退的酶之一，并表明抗 21 和抗 23 整合素抗体可以加速新血管的退化。我们通过将主动脉环植入同种大鼠或小鼠的皮下空间，成功地在体内复制了血管退化过程。 我们最近发现离体主动脉环模型中ECM的血管退化和溶解与血管周围巨噬细胞的积累以及MMP-3、MMP-12和MMP-14的过度表达有关。从回归培养物中分离出的巨噬细胞抑制了新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 反应基因的表达上调。重组干扰素阻断主动脉外植体的血管生成反应并导致新血管退化。 由于巨噬细胞是血管生成萌芽所必需的，因此我们假设这些细胞随着时间的推移从血管生成表型转变为血管抑制表型。我们假设血管抑制巨噬细胞是溶血管 MMP 的主要来源。最后，我们提出I型干扰素促进血管消退过程，包括巨噬细胞转化为血管抑制和血管溶解表型。该提案的具体目的旨在研究以下假设：（1）巨噬细胞在血管消退过程中从血管生成表型转变为血管抑制表型。 (2)血管生成后MMP介导的血管消退(血管溶解)需要巨噬细胞。 (3)干扰素促进血管生成后的血管消退。 拟议的实验将通过血管生成的体外、离体和体内测定进行。对于体外研究，我们将使用分离的内皮细胞的毛细管形成测定。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消退的离体研究。对于活体动物的研究，我们将使用我们新开发的体内测定，其中皮下植入的主动脉环刺激形成富含血管的肉芽组织，随着时间的推移，肉芽组织会逐渐退化。我们的研究计划包括 MMP-3、MMP-12、MMP-14 或 I 型 IFN 受体基因被破坏的转基因小鼠。拟议的实验采用定量实时 RT-PCR、免疫细胞化学、共聚焦显微镜、蛋白质印迹、TUNEL、ELISA 以及离体和体内巨噬细胞消融的药理学方法。将使用抗-INF-1 和-IFN-2 阻断抗体进行功能研究。 本次资助中提出的实验将进一步加深我们对血管生成后血管消退的细胞和分子机制的理解。这些知识可能有助于开发治疗癌症和其他血管生成相关疾病的新治疗方法。