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.
Inhibition of pathological blood vessel growth (angiogenesis) is thought to be a promising therapeutic strategy for various human diseases including diabetes, cancer, and atherosclerosis. The proposed study will explore a novel anti-angiogenesis pathway, which is mediated by CD148 phosphatase and its newly identified ligand (activating protein), thrombospondin-1. The results obtained through this study offer a new strategy and agent for anti-angiogenesis therapy.
|Takahashi, Keiko; Matafonov, Anton; Sumarriva, Katherine et al. (2014) CD148 tyrosine phosphatase promotes cadherin cell adhesion. PLoS One 9:e112753|