Role of CD148 Tyrosine Phosphatase in Angiogenesis

描述(申请人提供)：抑制病理性血管生长的抗血管生成疗法是一种很有前途的治疗多种人类疾病的策略。对调节血管新生血管生长的内在分子控制的定义有望为抗血管生成治疗提供一种新的方法。近几十年来，人们已经发现了促进血管生成的内皮细胞表面受体及其激活配体。然而，人们对诱导“抗血管生成(或血管抑制)”信号的内皮受体知之甚少，尽管它们可能是抗血管生成治疗的强大工具。鉴于内皮受体蛋白酪氨酸激酶(RPTKs)在血管生成信号转导中起主要作用，我们推测RPTKs的对偶酶--内皮受体蛋白酪氨酸磷酸酶(RPTPs)可能诱导抗血管生成信号。我们已经从培养的内皮细胞中分离出一种受体类型的PTP-CD148，表明CD148具有抑制内皮生长因子信号、抑制内皮细胞生长和血管生成的强大活性，表明CD148是一个有前景的抗血管生成治疗的分子靶点。然而，CD148的调控机制，包括它的胞外配体(S)，在很大程度上仍不清楚。为了探索CD148的配体(S)，我们将透明质酸标记的CD148引入血管内皮细胞，然后通过生物素表面标记和亲和纯化分离与CD148相互作用的胞外蛋白。对这些蛋白质进行了质谱学鉴定。通过这种方法，我们分离了凝血酶敏感蛋白-1(TSP1)作为CD148的主要相互作用蛋白，并表明可溶性TSP1与CD148的胞外部分高亲和力结合，并作为功能配体发挥作用。因此，在这一应用中，我们通过以下实验来确定TSP1/CD148相互作用的抗血管生成活性。目的#1：确定TSP1的CD148结合区域和多肽序列，并研制CD148特异性TSP1试剂。TSP1含有多种结构元件，可与多种内皮细胞受体结合。为了专门研究TSP1/CD148途径，我们将利用一系列重组蛋白和合成肽以及体外和原位结合实验来确定TSP1的CD148相互作用区域和肽序列。目的#2：探讨TSP1-CD148相互作用对内皮细胞生长和血管生成的影响。在此，我们将评估TSP1/CD148相互作用，通过沉默或阻断，以及在血管内皮细胞培养和血管生成实验中通过激活TSP1/CD148相互作用而激活的内皮细胞信号转导和抗血管生成活性。使用CD148特异性TSP1片段/肽以及全长TSP1作为激动剂。CD148条件基因敲除小鼠、CD148特异性shRNA和可溶性CD148胞外结构域被用来沉默或拮抗这种相互作用。因此，这一应用将探索一条抑制血管生成的新途径，并将为抗血管生成治疗提供新的策略和试剂。

DESCRIPTION (provided by applicant): Anti-angiogenesis therapy to inhibit pathological vessel growth is a promising treatment strategy for a variety of human diseases. Definition of intrinsic molecular controls that regulate angiogenic vessel growth promises a new approach for anti-angiogenesis therapy. In recent decades, extensive efforts have identified the endothelial surface receptors and their activating ligands which promote angiogenesis. However, less is known about the endothelial receptors that induce "anti- angiogenic (or angiostatic)" signals, though they could be powerful tools for anti-angiogenesis treatment. Given the fact that endothelial receptor protein tyrosine kinases (RPTKs) play a major role in transduction of angiogenic signals, we hypothesized that endothelial receptor protein tyrosine phosphatases (RPTPs), counter-enzymes of RPTKs, may induce anti-angiogenic signals. We have isolated a receptor-type PTP, CD148, from cultured endothelial cells and shown that CD148 has a potent activity to suppress endothelial growth factor signals and to inhibit endothelial cell growth and angiogenesis, indicating that CD148 is a promising molecular target of anti-angiogenesis therapy. However, the regulatory mechanisms of CD148, including its extracellular ligand(s), remain largely unknown. To explore the ligand(s) of CD148, we introduced HA-tagged CD148 into endothelial cells, then isolated the CD148-interacting extracellular proteins by biotin-surfac labeling and subsequent affinity purifications. These proteins were identified by mass spectrometry. By this approach, we have isolated thrombospondin-1 (TSP1) as a major CD148-interacting protein, and shown that soluble TSP1 binds at high affinity to the extracellular part o CD148 and acts as a functional ligand. In this application, we therefore determine the anti-angiogenic activity of TSP1/CD148 interaction by the following experiments. Aim #1: Determine the CD148-interacting region and peptide sequence of TSP1 and develop the CD148-specific TSP1 agent. TSP1 contains multiple structural elements and binds to several endothelial receptors. To specifically investigate the TSP1/CD148 pathway, here we will determine the CD148-interacting region and peptide sequence of TSP1 using a series of recombinant proteins and synthetic peptides and in vitro and in situ binding assays. Aim #2: Determine the effects of TSP1-CD148 interaction in endothelial cell growth and angiogenesis. Here, we will evaluate the anti- angiogenic activity and the endothelial signaling activated by TSP1/CD148 interaction, by silencing or blocking as well as by activating its interaction in endothelial culture and in in viv angiogenesis assay. CD148 specific TSP1 fragment/peptide as well as a full length TSP1 is used as the agonist. CD148 conditional knockout mouse, CD148-specific shRNA, and soluble CD148 ectodomain are used to silence or antagonize the interaction. Thus, this application will explore a novel pathway of angiogenesis inhibition and should offer a new strategy and reagent for anti-angiogenesis therapy.