Role of CD148 Tyrosine Phosphatase in Angiogenesis

描述（由申请人提供）：抑制病理性血管生长的抗血管生成治疗是一种有前途的治疗多种人类疾病的策略。定义的内在分子控制，调节血管生成的血管生长的承诺，一个新的方法，抗血管生成治疗。 近几十年来，广泛的努力已经确定了内皮细胞表面受体及其活化配体，促进血管生成。然而，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有力工具。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的事实，我们假设内皮受体蛋白酪氨酸磷酸酶（RPTPs），RPTKs的反酶，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型PTP，CD 148，并且显示CD 148具有抑制内皮生长因子信号的有效活性，并且抑制内皮细胞生长和血管生成，表明CD 148是抗血管生成治疗的有希望的分子靶点。然而，CD 148的调节机制，包括其细胞外配体，仍然是未知的。 为了探索CD 148的配体，我们将HA标记的CD 148引入内皮细胞，然后通过生物素表面标记和随后的亲和纯化分离与CD 148相互作用的胞外蛋白。这些蛋白质通过质谱鉴定。通过这种方法，我们已经分离出血小板反应蛋白-1（TSP 1）作为主要的CD 148相互作用蛋白，并显示可溶性TSP 1以高亲和力结合到CD 148的细胞外部分，并作为功能性配体。因此，在本申请中，我们通过以下实验确定了TSP 1/CD 148相互作用的抗血管生成活性。目的#1：确定TSP 1的CD 148相互作用区域和肽序列，并开发CD 148特异性TSP 1试剂。TSP 1含有多种结构元件，并与几种内皮受体结合。为了具体研究TSP 1/CD 148通路，在这里，我们将使用一系列重组蛋白和合成肽以及体外和原位结合测定来确定TSP 1的CD 148相互作用区域和肽序列。目的#2：确定TSP 1-CD 148相互作用在内皮细胞生长和血管生成中的作用。在此，我们将评估抗血管生成活性和由TSP 1/CD 148相互作用激活的内皮信号传导，通过沉默或阻断以及通过激活内皮培养物和体内血管生成测定中的其相互作用。CD 148特异性TSP 1片段/肽以及全长TSP 1用作激动剂。CD 148条件性敲除小鼠、CD 148特异性shRNA和可溶性CD 148胞外域用于沉默或拮抗相互作用。因此，本研究将探索一条新的血管生成抑制途径，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.