PARACRINE REGULATION OF ANGIOGENESIS BY MURAL CELLS

壁细胞对血管生成的旁分泌调节

• 批准号: 6873025
• 负责人: ROBERTO NICOSIA
• 金额: $ 25.2万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873025
• 关键词: RNase protection assay; adeno associated virus group; angiogenesis; angiopoietins; aorta; blood vessels; cell differentiation; chemotaxis; confocal scanning microscopy; hormone regulation /control mechanism; immunocytochemistry; immunomagnetic separation; laboratory rat; mesenchyme; mitogen activated protein kinase; northern blottings; paracrine; polymerase chain reaction; stem cells; tissue /cell culture; transfection /expression vector; vascular endothelial growth factors; vascular endothelium; vascular smooth muscle; western blottings

Angiogenesis plays a critical role in the revascularization of ischemic organs and in the progression of cancer, atherosclerosis, rheumatoid arthritis, and diabetic retinopathy. The outcome of angiogenesis depends on neovessel survival. Developing vessels uses the angiopoietin/Tie2 system to acquire a stabilizing layer of mural cells (smooth muscle cells/pericytes). The mechanisms regulating this process are, however, poorly understood because the Tie2 receptor is reportedly expressed in endothelial cells but not in mural cells. Using the rat aorta model of angiogenesis we found that Tie2 is transiently expressed in intimal- derived mesenchymal cells that have the capacity to differently into mural cells. Tie2+ mural precursor cells migrate and secrete matrix metalloproteinases in response to Ang-1 which they produce, and Ang-2, which is produced by endothelial cells. Based on the additional observation that the intimal/subintimal layers of the rat aorta contain Flk- 1+ and Tie2+ non-endothelial mesenchymal cells and have angioinformative properties we postulate that Tie2+ mural-precursor cells arise from vascular progenitor cells capable of both endothelial and mural cell differentiation. We linked mural cell recruitment to the p38 MAPK signaling pathway by demonstrating that pharmacologic inhibition of p28, which is activated upon Tie2 stimulation, abrogates mural cell development resulting in naked neovessels. Based on these observations the specific aims of this grant focus on the following hypotheses. 1) The aortic wall. contains vascular progenitor cells capable of both endothelial and mural cell differentiation. 2. The muscular wall of blood vessels originates from Tie2+ mural precursor cells. 3. Mural cell recruitment during angiogenesis is selectively mediated by the p38 MAPK pathway. Vascular progenitor cells of neonatal, young and old rat aortas will be identified by immunohistochemistry and confocal microscopy and isolated by surface marker-based magnetic beads technology. Their angioformative and mural cell differentiation properties will be studied in models of vascular organ culture, microvessel assembly and chemotaxis. The role of Tie2 and p38 MAPK pathway in mural cell recruitment will be studied by gene transfer technology using adeno-associated viral vectors carrying wild type or dominant-negative genes Mural cell recruitment will be analyzed by Immunohistochemistry, confocal microscopy and image analysis. Gene/protein expression and function will be evaluated by Northern and Western analysis, RT-PCR, kinase assays, gel zymography, in situ hybridization, and immunohistochemistry,. These studies will define key cellular and molecular mechanisms of vessel wall development. This knowledge may lead to novel approaches for the stabilization of neovessels in ischemic conditions, the induction of vascular regression in angiogenesis-dependent disorders, and the bioengineering of blood vessels for therapeutic applications.

血管生成在缺血器官的血运重建以及癌症、动脉粥样硬化、类风湿性关节炎和糖尿病视网膜病变的进展中发挥着关键作用。血管生成的结果取决于新血管的存活。发育中的血管使用血管生成素/Tie2 系统来获得稳定的壁细胞层（平滑肌细胞/周细胞）。然而，人们对调节这一过程的机制知之甚少，因为据报道 Tie2 受体在内皮细胞中表达，但在壁细胞中不表达。使用血管生成的大鼠主动脉模型，我们发现 Tie2 在内膜来源的间充质细胞中瞬时表达，这些细胞具有不同程度地分化为壁细胞的能力。 Tie2+ 壁前体细胞响应其产生的 Ang-1 和内皮细胞产生的 Ang-2，迁移并分泌基质金属蛋白酶。基于大鼠主动脉内膜/内膜下层含有 Flk-1+ 和 Tie2+ 非内皮间充质细胞并具有血管信息特性的额外观察，我们假设 Tie2+ 壁前体细胞源自能够同时具有内皮细胞和壁细胞的血管祖细胞。细胞分化。我们通过证明 p28 的药理学抑制（在 Tie2 刺激后被激活）消除壁细胞发育，从而产生裸露的新血管，将壁细胞募集与 p38 MAPK 信号通路联系起来。根据这些观察，这笔赠款的具体目标集中在以下假设上。 1）主动脉壁。含有能够分化内皮细胞和壁细胞的血管祖细胞。 2. 血管的肌肉壁起源于Tie2+壁前体细胞。 3.血管生成过程中壁细胞的募集是由p38 MAPK途径选择性介导的。新生、年轻和年老大鼠主动脉的血管祖细胞将通过免疫组织化学和共聚焦显微镜进行鉴定，并通过基于表面标记的磁珠技术进行分离。它们的血管形成和壁细胞分化特性将在血管器官培养、微血管组装和趋化性模型中进行研究。将使用携带野生型或显性失活基因的腺相关病毒载体通过基因转移技术研究Tie2和p38 MAPK通路在壁细胞募集中的作用。将通过免疫组织化学、共聚焦显微镜和图像分析来分析壁细胞募集。基因/蛋白质表达和功能将通过 Northern 和 Western 分析、RT-PCR、激酶测定、凝胶酶谱、原位杂交和免疫组织化学进行评估。这些研究将定义血管壁发育的关键细胞和分子机制。这些知识可能会带来新的方法来稳定缺血条件下的新血管、诱导血管生成依赖性疾病的血管消退以及用于治疗应用的血管生物工程。