CD47-binding Peptide Research Articles

CD47 Binding on Vascular Endothelial Cells Inhibits IL-17-Mediated Leukocyte Adhesion.

To address the conflicting role of thrombospondin (TSP)-1 reported in acute and chronic pathologies, this study investigated the role of TSP-1 in regulating leukocyte recruitment and regulation of VCAM-1 expression using mouse models of uveitis. The spontaneously increased VCAM-1 expression and leukocyte adhesion in retinas of TSP-1-deficient mice suggested a TSP-1-mediated regulation of VCAM-1 expression. In a chronic uveitis model, induced by immunizing wild-type mice with specific interphotoreceptor retinoid-binding protein (IRBP) peptide, topically applied TSP-1-derived CD47-binding peptide significantly reduced the clinical disease course and retinal leukocyte adhesion as compared to the control peptide-treated group. In contrast, in LPS-mediated acute uveitis, TSP-1 deficiency significantly reduced the retinal leukocyte adhesion. The results of our in vitro study, using vascular endothelial cell (EC) cultures, demonstrate that unlike TNF-α, VCAM-1 expression induced by IL-17 is associated with a reduced expression of endogenous TSP-1. Such reduced endogenous TSP-1 expression in IL-17-stimulated ECs helps limit the CD36-mediated increased VCAM-1 expression, while favoring CD47-mediated inhibition of VCAM-1 expression and leukocyte adhesion. Thus, our study identifies TSP-1:CD47 interaction as a molecular pathway that modulates IL-17-mediated VCAM-1 expression, contributing to its anti-inflammatory effect in chronic inflammatory conditions.

AAV-mediated expression of 3TSR inhibits tumor and metastatic lesion development and extends survival in a murine model of epithelial ovarian carcinoma.

An integral step in the development of solid tumors is the recruitment of blood vessels to fuel tumor growth. Antiangiogenic therapies can inhibit this process and control solid tumor growth. Thrombospondin-1 is an antiangiogenic protein possessing three type I repeats (3TSR) near the center of the protein and a CD47-binding peptide (CD47) in its C-terminus. Previously, we showed that treatment with recombinant 3TSR induces tumor regression, normalizes tumor vasculature, and improves uptake of chemotherapy drugs in an orthotopic, syngeneic mouse model of advanced stage epithelial ovarian cancer (EOC). While effective, this intervention required daily intraperitoneal injections. To circumvent this, here we employ adeno-associated virus (AAV) gene therapy vectors to express 3TSR alone or in combination with the CD47-binding peptide of TSP-1 and evaluate the impact on tumor development and survival in a mouse model of EOC. A single intraperitoneal injection of 1 × 1011 vg of AAV expressing 3TSR, CD47-binding peptide, or 3TSR + CD47 effectively suppressed primary tumor growth; however, only AAV-3TSR was able to inhibit development of secondary lesions at 90-days post-tumor implantation and significantly improve survival. Taken together, AAV-mediated expression of 3TSR appears safe and effective at inhibiting tumor development and represents a novel, less invasive approach for treating ovarian carcinoma.

Corrigendum

Liver InternationalVolume 34, Issue 4 p. 647-647 CorrigendumFree Access Corrigendum This article corrects the following: The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells Lakshmi Venkatraman, Lisa Tucker-Kellogg, Volume 33Issue 9Liver International pages: 1386-1397 First Published online: June 26, 2013 First published: 29 December 2013 https://doi.org/10.1111/liv.12448Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Reference number 19 in 1 cited a 2008 paper by Cheluvappa et al., but should have cited the 2008 thesis by Cheluvappa instead. Therefore the correct reference number 19 should be: Cheluvappa R. Pathophysiology of Liver Sinusoidal Endothelial Cells. 2008. PhD Thesis, University of Sydney. Reference 1Venkatraman L, Lisa Tucker-Kellogg L. The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells. Liver Int 2013; 33: 1386– 97. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume34, Issue4April 2014Pages 647-647 ReferencesRelatedInformation

Thrombospondin-1 is a CD47-dependent endogenous inhibitor of hydrogen sulfide signaling in T cell activation

Thrombospondin-1 is a potent suppressor of T cell activation via its receptor CD47. However, the precise mechanism for this inhibition remains unclear. Because H2S is an endogenous potentiator of T cell activation and is necessary for full T cell activation, we hypothesized that thrombospondin-1 signaling through CD47 inhibits T cell activation by antagonizing H2S signaling. Primary T cells from thrombospondin-1 null mice were more sensitive to H2S-dependent activation assessed by proliferation and induction of interleukin-2 and CD69 mRNAs. Exogenous thrombospondin-1 inhibited H2S responses in wild type and thrombospondin-1 null T cells but enhanced the same responses in CD47 null T cells. Fibronectin, which shares integrin and glycosaminoglycan binding properties with thrombospondin-1 but not CD47 binding, did not inhibit H2S signaling. A CD47-binding peptide derived from thrombospondin-1 inhibited H2S-induced activation, whereas two other functional sequences from thrombospondin-1 enhanced H2S signaling. Therefore, engaging CD47 is necessary and sufficient for thrombospondin-1 to inhibit H2S-dependent T cell activation. H2S stimulated T cell activation by potentiating MEK-dependent ERK phosphorylation, and thrombospondin-1 inhibited this signaling in a CD47-dependent manner. Thrombospondin-1 also limited activation-dependent T cell expression of the H2S biosynthetic enzymes cystathionine β-synthase and cystathionine γ-lyase, thereby limiting the autocrine role of H2S in T cell activation. Thus, thrombospondin-1 signaling through CD47 is the first identified endogenous inhibitor of H2S signaling and constitutes a novel mechanism that negatively regulates T cell activation.

Thrombospndin 1 accelerates VEGFR2 trafficking and directs towards lysosomes for degradation

In a recent study we found that CD47 constitutively associates with VEGFR2 based on FRET and receptor co-localization. CD47 ligation with either TSP1 or a peptide derived from the CD47 binding site of TSP1 (7N3) abolishes FRET and co-localization with VEGFR2, and inhibits phosphorylation of VEGFR2 at Y1175 and its downstream target Akt at S473. We found that association of VEGFR2 with the early endosomal markers EEA1 and Rab5 is decreased with TSP1 treatment, whereas association with LAMP1 and Rab7 in late endosomes is enhanced. Confocal live imaging showed that TSP1 directs VEGFR2 to the lysosomal compartment and can be inhibited with NH4Cl. Surface labeling with biotin and cycloheximide treatment showed that VEGFR2 degrades faster in TSP1-treated cells in the presence of VEGF. This indicates that TSP1 binding to CD47 inhibits VEGFR2 signaling by accelerating trafficking of the VEGF-VEGFR2 complex towards lysosomes. Current antiangiogenic therapies are directed towards inhibition of VEGF signaling. The data presented here indicates that VEGF signaling can also be blocked be enhancing VEGFR2 degradation by TSP1 treatment or using a CD47 binding peptide. This provides a new mechanism for the anti-angiogenic activity of TSP1. Analogs of the 7N3 peptide could be useful small molecule adjuvants to current anti-angiogenic therapies.

Thrombospondin-1 Inhibits VEGF Receptor-2 Signaling by Disrupting Its Association with CD47

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.

The CD47 Pathway Is Deregulated In Human Immune Thrombocytopenia (ITP).

AbstractAbstract 3776The CD47 antigen is a transmembrane glycoprotein ubiquitously expressed on hematopoietic and non-hematopoietic cells. It serves as a receptor for Thrombospondin (TSP) and a ligand for signal regulatory protein-alpha (SIRP-alpha) receptor, acting, respectively, as a regulator of apoptosis and as antagonistic to phagocyte activity. Ligation of CD47 with antibodies, its natural physiological ligand TSP or the specific CD47-binding peptide 41NK induces apoptosis in nucleated blood cells. This apoptosis is characterized by mitochondrial damage and the exposure of phosphatydilserine on the outerleaflet of the plasma membrane. Interaction of SIRP-alpha with CD47 is important also for the regulation of phagocytosis. SIRP-alpha is an immunoglobulin superfamily member and is predominantly expressed in neurons, dendritic cells (DCs) and monocytes/macrophages. Phagocytes engulf foreign cells but not “self” in part because “self” cells express CD47 as a ligand for SIRP-alpha, which inhibits phagocytosis. Thus CD47 functions as a “don't eat me” signal. Based on studies in mice, a novel mechanism of platelet destruction involving the CD47/SIRP-alpha system has been recently suggested in Immune Thrombocytopenia (ITP). Specifically, it has been demonstrated that: 1) platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in immune thrombocytopenia in a mouse model; 2) interaction between platelet CD47 and macrophage SIRP-alpha is important in regulating normal platelet turnover and FcgammaR-mediated clearance of IgG-sensitized platelets; 3) CD47-deficient platelets have a shortened half-life in the circulation of CD47 wild-type mice and are also more sensitive to Fcgamma receptor-mediated clearance, both in vivo and in vitro. However, the role of CD47 pathway in the pathogenesis of human ITP has not yet been studied. Therefore, the main purpose of the present study was to evaluate whether alterations of this system (platelets/phagocytes) might play a pathogenetic role in human ITP. In particular, we investigated whether in ITP: i) platelets are more susceptible to CD47-induced cell death; ii) expression of CD47 on fresh and in vitro aged platelets is reduced; iii) the platelet phagocytic capacity of CD14-derived DCs and macrophages is differentially modulated in the presence or absence of antibodies against CD47 and SIRP-alpha. Phenotypical and functional analysis of the expression of CD47 on platelets and SIRP-αlpha on CD14-derived/circulating DCs and on CD14-derived macrophages was performed in 32 ITP patients. Patients were newly diagnosed (14 cases) or with persistent (15 cases) or chronic (3 cases) ITP. At the time of the study, patients with persistent or chronic ITP were off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 49×109/L (range 14–98). We found that in healthy subjects CD47 expression increased in in vitro aged platelets and ligation of CD47 with anti-CD47 antibody induced a dose-dependent increase of platelet apoptosis. Immature and mature CD14-derived DCs and circulating myeloid DCs were strongly positive for SIRP-α. Conversely, we demonstrated that in ITP: 1) CD47 expression was unchanged in freshly isolated and in vitro aged platelets; 2) increased platelet apoptosis was not due to the activation of the CD47-induced cell death pathway, which instead was shown to be blocked; 3) the blockage of SIRP-αlpha on immature CD14-derived DCs or CD47 on platelets by specific antibodies failed to modify platelet uptake/phagocytosis of DCs; in contrast, targeting platelet CD47 with specific antibody significantly increases platelet phagocytosis of CD14-derived macrophages. In conclusion, our data demonstrate that in ITP the increased platelet clearance is not due to reduced CD47 expression on platelets. However, platelets from ITP patients are not healthy because 1) apoptosis is increased; 2) platelet apoptosis is independent from CD47 death signal; 3) CD47 expression is not modified by in vitro ageing/apoptosis. In addition, we show that the CD47 pathway plays a role in platelet phagocytosis of macrophages, but not in DCs. We conclude that in ITP patients platelet homeostasis is differentially modulated by the CD47 pathway. Disclosures:No relevant conflicts of interest to declare.

Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPalpha (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.

Ligation of CD47 mediates phosphatidylserine expression on erythrocytes and a concomitant loss of viability in vitro

CD47 ligation has been shown to induce phosphatidylserine (PS) expression as part of a death pathway in nucleated blood cells. Using Annexin V binding assays we showed that ligation of CD47 with the specific CD47-binding peptide 41NK, anti-CD47 monoclonal antibody, and its natural ligand thrombospondin-1 also induced PS expression on enucleated erythrocytes. PS expression was associated with a concomitant loss of erythrocyte viability in vitro. Further characterisation of the CD47-PS signalling pathway on erythrocytes may help develop clinical strategies to further preserve the life of blood donations and improve our understanding of certain types of haemolytic anaemias.

Novel CD47-dependent intercellular adhesion modulates cell migration

CD47 is a ubiquitously expressed plasma membrane protein, also known as Integrin Associated Protein, that modulates cell adhesion both through alteration of the avidity of integrin binding and through interaction with its own ligands, the extracellular matrix protein thrombospondin (TSP) and the plasma membrane response regulator SIRPalpha1. We now show that CD47 expression on fibroblasts can induce intercellular adhesion resulting in cell aggregation in the absence of active integrins, SIRPalpha1 binding, and detectable TSP. CD47-expressing cells preferentially bind to other CD47-expressing cells, and intercellular adhesion requires stimulation by serum or a CD47-binding peptide from TSP. Cell-cell adhesion is inhibited by pertussis toxin and C. difficile toxin B, and both adherent and aggregating CD47-expressing fibroblasts have more rac in the GTP bound state than CD47-deficient cells. Spontaneous migration of Jurkat lymphocytes through a fibroblast monolayer is decreased by fibroblast expression of CD47, consistent with an increased barrier function of the CD47 expressing cells. The lymphocyte chemoattractant SDF-1alpha stimulates migration of Jurkat cells through this monolayer only if both the lymphocytes and fibroblasts express CD47, and the inhibition of migration by a CD47-interacting peptide from TSP similarly requires CD47 expression on both cell types. Thus, signaling dependent on both heterotrimeric and rho family GTPases can induce CD47 to participate in cell-cell interactions independent of known ligands that enhance intercellular adhesion and modulate cell migration.

CD47 Ligation Induces a Rapid Caspase‐Independent Apoptosis‐Like Cell Death in Human Monocytes and Dendritic Cells

CD47 is a versatile cell-surface molecule expressed on nearly all haematopoietic cells. In its capacity as a thrombospondin-1 (TSP-1) receptor, CD47 has recently been shown to mediate cell death in certain cells, for example, activated but not resting T cells. Here, we have investigated the possibility that human monocytes and dendritic cells (DCs) undergo cell death, following CD47 ligation. Using the TSP-1-derived CD47-binding peptide 4N1K, we found that both freshly isolated monocytes and monocyte-derived DCs underwent a rapid, caspase-independent cell death. This was characterized by the simultaneous presence of phosphatidylserine exposure, plasma membrane permeability, reduced mitochondrial membrane potential and highly fragmented DNA. Not all cells were sensitive to 4N1K-induced apoptosis; a plateau of cell death reached at an average of 38% of the monocyte and DCs populations. The results presented here, thus, show that CD47 can mediate a rapid apoptosis-like cell death of human monocytes and DCs.

β1 Integrin- and Proteoglycan-Mediated Stimulation of T Lymphoma Cell Adhesion and Mitogen-Activated Protein Kinase Signaling by Thrombospondin-1 and Thrombospondin-1 Peptides

Abstract Cell-cell and cell-matrix interactions play important regulatory roles in lymphocyte homeostasis. Thrombospondin-1 (TSP1) is a matricellular protein that differentially promotes the adhesion of resting and activated T cells. In this work, we show that adhesion of Jurkat T cells on substrates coated with TSP1 or TSP1-derived peptides is mediated by β1 integrins, CD47, and heparan sulfate proteoglycans. Interactions with TSP1 or TSP1 peptides stimulated CD3-induced Ras activation and tyrosine phosphorylation of several T cell proteins. The signals from TSP1 and its derived peptides differentially synergized with activation of the TCR to induce phosphorylation of linker for activation of T cells (LAT) and extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 kinases. The phosphorylation of ERK in the presence of full-length TSP1 was transient and dependent on a β1 integrin receptor. Interestingly, peptides derived from the type 1 repeats of TSP1 and a CD47-binding peptide from the carboxyl-terminal domain of TSP1 also stimulated mitogen-activated protein (MAP) kinase phosphorylation. Moreover, the TSP1 heparin-binding peptide synergized with Ab-ligated TCR to transduce signals to the nucleus, detected by activation of AP-1- and Elk-dependent transcription. This TSP1 peptide-dependent activation of AP-1 was inhibited by both heparin and the MAP/ERK kinase inhibitor PD98059, providing a functional link between adhesion molecule interaction and nuclear transactivation events via the MAP kinase pathways. These findings have implications for the role of extracellular TSP1 and TSP1 fragments in the regulation of T cell function during hemostasis, wound repair, and other inflammatory responses.