InVivoMAb anti-rat CD47

InVivoMAb anti-rat CD47 (CLONE: OX-101)

Ye F, Hua Y, Keep RF, Xi G, Garton HJL (2021). "CD47 blocking antibody accelerates hematoma clearance and alleviates hydrocephalus after experimental intraventricular hemorrhage" Neurobiol Dis . PubMed

Background CD47, a glycoprotein on red blood cell membranes, inhibits phagocytosis via interaction with signal regulatory protein α on phagocytes. Our previous research has demonstrated that blocking CD47 accelerates hematoma clearance and reduces brain injury after intracerebral hemorrhage. The current study investigated whether phagocytosis or erythrocyte CD47 impacts hematoma resolution and hydrocephalus development after intraventricular hemorrhage (IVH). Methods Adult (3-month-old) male Fischer 344 rats were intraventricularly injected with 200 μl autologous blood, mixed with either CD47 blocking antibody or isotype IgG, or 200 μl saline as control. In subgroups of CD47 blocking antibody treated rats, clodronate liposomes (to deplete microglia/monocyte-derived macrophages) or control liposomes were co-injected. Magnetic resonance imaging (MRI) was used to evaluate ventricular volume and intraventricular T2* lesion volume (estimating hematoma volume). The brains were harvested after 4 or 72 h for histology to evaluate phagocytosis. Results In adult male rats, CD47 blocking antibody alleviated hydrocephalus development by day 3. In addition, the CD47 blocking antibody reduced intraventricular T2* lesion and T2* non-hypointense lesion size after IVH through day 1 to day 3. Erythrophagocytosis was observed as soon as 4 h after IVH and was enhanced on day 3. Furthermore, intra-hematoma infiltration of CD68, heme oxygenase-1 and ferritin positive phagocytes were upregulated by CD47 blockade by day 3. Clodronate liposomes co-injection caused more severe hydrocephalus and weight loss. Conclusion Blocking CD47 in the hematoma accelerated hematoma clearance and alleviated hemolysis and hydrocephalus development after IVH, suggesting CD47 might be valuable in the future treatment for IVH.

Rogers NM, Sharifi-Sanjani M, Yao M, Ghimire K, Bienes-Martinez R, Mutchler SM, Knupp HE, Baust J, Novelli EM, Ross M, St Croix C, Kutten JC, Czajka CA, Sembrat JC, Rojas M, Labrousse-Arias D, Bachman TN, Vanderpool RR, Zuckerbraun BS, Champion HC, Mora AL, Straub AC, Bilonick RA, Calzada MJ, Isenberg JS (2017). "TSP1-CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction" Cardiovasc Res 113(1):15-29. PubMed

Aims: Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1^-/- mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1-CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. Methods and results: We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1-CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47^-/- mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47^-/- pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47^+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1-CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. Conclusions: In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Sharifi-Sanjani M, Shoushtari AH, Quiroz M, Baust J, Sestito SF, Mosher M, Ross M, McTiernan CF, St Croix CM, Bilonick RA, Champion HC, Isenberg JS (2014). "Cardiac CD47 drives left ventricular heart failure through Ca2+-CaMKII-regulated induction of HDAC3" J Am Heart Assoc 3(3):e000670. PubMed

Background: Left ventricular heart failure (LVHF) remains progressive and fatal and is a formidable health problem because ever-larger numbers of people are diagnosed with this disease. Therapeutics, while relieving symptoms and extending life in some cases, cannot resolve this process and transplant remains the option of last resort for many. Our team has described a widely expressed cell surface receptor (CD47) that is activated by its high-affinity secreted ligand, thrombospondin 1 (TSP1), in acute injury and chronic disease; however, a role for activated CD47 in LVHF has not previously been proposed. Methods and results: In experimental LVHF TSP1-CD47 signaling is increased concurrent with up-regulation of cardiac histone deacetylase 3 (HDAC3). Mice mutated to lack CD47 displayed protection from transverse aortic constriction (TAC)-driven LVHF with enhanced cardiac function, decreased cellular hypertrophy and fibrosis, decreased maladaptive autophagy, and decreased expression of HDAC3. In cell culture, treatment of cardiac myocyte CD47 with a TSP1-derived peptide, which binds and activates CD47, increased HDAC3 expression and myocyte hypertrophy in a Ca(2+)/calmodulin protein kinase II (CaMKII)-dependent manner. Conversely, antibody blocking of CD47 activation, or pharmacologic inhibition of CaMKII, suppressed HDAC3 expression, decreased myocyte hypertrophy, and mitigated established LVHF. Downstream gene suppression of HDAC3 mimicked the protective effects of CD47 blockade and decreased hypertrophy in myocytes and mitigated LVHF in animals. Conclusions: These data identify a proximate role for the TSP1-CD47 axis in promoting LVHF by CaKMII-mediated up-regulation of HDAC3 and suggest novel therapeutic opportunities.

Lin Y, Manning PT, Jia J, Gaut JP, Xiao Z, Capoccia BJ, Chen CC, Hiebsch RR, Upadhya G, Mohanakumar T, Frazier WA, Chapman WC (2014). "CD47 blockade reduces ischemia-reperfusion injury and improves outcomes in a rat kidney transplant model" Transplantation 98(4):394-401. PubMed

Background: Ischemia-reperfusion injury (IRI) significantly contributes to delayed graft function and inflammation, leading to graft loss. Ischemia-reperfusion injury is exacerbated by the thrombospondin-1-CD47 system through inhibition of nitric oxide signaling. We postulate that CD47 blockade and prevention of nitric oxide inhibition reduce IRI in organ transplantation. Methods: We used a syngeneic rat renal transplantation model of IRI with bilaterally nephrectomized recipients to evaluate the effect of a CD47 monoclonal antibody (CD47mAb) on IRI. Donor kidneys were flushed with CD47mAb OX101 or an isotype-matched control immunoglobulin and stored at 4°C in University of Wisconsin solution for 6 hr before transplantation. Results: CD47mAb perfusion of donor kidneys resulted in marked improvement in posttransplant survival, lower levels of serum creatinine, blood urea nitrogen, phosphorus and magnesium, and less histological evidence of injury. In contrast, control groups did not survive more than 5 days, had increased biochemical indicators of renal injury, and exhibited severe pathological injury with tubular atrophy and necrosis. Recipients of CD47mAb-treated kidneys showed decreased levels of plasma biomarkers of renal injury including Cystatin C, Osteopontin, Tissue Inhibitor of Metalloproteinases-1 (TIMP1), β2-Microglobulin, Vascular Endothelial Growth Factor A (VEGF-A), and clusterin compared to the control group. Furthermore, laser Doppler assessment showed higher renal blood flow in the CD47mAb-treated kidneys. Conclusion: These results provide strong evidence for the use of CD47 antibody-mediated blockade to reduce IRI and improve organ preservation for renal transplantation.

Bauer PM, Bauer EM, Rogers NM, Yao M, Feijoo-Cuaresma M, Pilewski JM, Champion HC, Zuckerbraun BS, Calzada MJ, Isenberg JS (2012). "Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1" Cardiovasc Res 93(4):682-93. PubMed

Aims: Pulmonary arterial hypertension (PAH) is a progressive lung disease characterized by pulmonary vasoconstriction and vascular remodelling, leading to increased pulmonary vascular resistance and right heart failure. Loss of nitric oxide (NO) signalling and increased endothelial nitric oxide synthase (eNOS)-derived oxidative stress are central to the pathogenesis of PAH, yet the mechanisms involved remain incompletely determined. In this study, we investigated the role activated CD47 plays in promoting PAH. Methods and results: We report high-level expression of thrombospondin-1 (TSP1) and CD47 in the lungs of human subjects with PAH and increased expression of TSP1 and activated CD47 in experimental models of PAH, a finding matched in hypoxic human and murine pulmonary endothelial cells. In pulmonary endothelial cells CD47 constitutively associates with caveolin-1 (Cav-1). Conversely, in hypoxic animals and cell cultures activation of CD47 by TSP1 disrupts this constitutive interaction, promoting eNOS-dependent superoxide production, oxidative stress, and PAH. Hypoxic TSP1 null mice developed less right ventricular pressure and hypertrophy and markedly less arteriole muscularization compared with wild-type animals. Further, therapeutic blockade of CD47 activation in hypoxic pulmonary artery endothelial cells upregulated Cav-1, increased Cav-1CD47 co-association, decreased eNOS-derived superoxide, and protected animals from developing PAH. Conclusion: Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Csányi G, Yao M, Rodríguez AI, Al Ghouleh I, Sharifi-Sanjani M, Frazziano G, Huang X, Kelley EE, Isenberg JS, Pagano PJ (2012). "Thrombospondin-1 regulates blood flow via CD47 receptor-mediated activation of NADPH oxidase 1" Arterioscler Thromb Vasc Biol 32(12):2966-73. PubMed

Objective: Although the matricellular protein thrombospondin-1 (TSP1) is highly expressed in the vessel wall in response to injury, its pathophysiological role in the development of vascular disease is poorly understood. This study was designed to test the hypothesis that TSP1 stimulates reactive oxygen species production in vascular smooth muscle cells and induces vascular dysfunction by promoting oxidative stress. Methods and results: Nanomolar concentrations of TSP1 found in human vascular disease robustly stimulated superoxide (O(2)(•-)) levels in vascular smooth muscle cells at both cellular and tissue level as measured by cytochrome c and electron paramagnetic resonance. A peptide mimicking the C terminus of TSP1 known to specifically bind CD47 recapitulated this response. Transcriptional knockdown of CD47 and a monoclonal inhibitory CD47 antibody abrogated TSP1-triggered O(2)(•-) in vitro and ex vivo. TSP1 treatment of vascular smooth muscle cells activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase organizer subunit p47(phox) and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and NADPH oxidase 1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow after ischemia reperfusion. Conclusions: Our data suggest a highly regulated process of reactive oxygen species stimulation and blood flow regulation promoted through a direct TSP1/CD47-mediated activation of Nox1. This is the first report, to our knowledge, of a matricellular protein acting as a ligand for NADPH oxidase activation and through specific engagement of integrin-associated protein CD47.

Sick E, Niederhoffer N, Takeda K, Landry Y, Gies JP (2009). "Activation of CD47 receptors causes histamine secretion from mast cells" Cell Mol Life Sci 66(7):1271-82. PubMed

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcepsilonRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the betagamma dimer of a G(i) protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G(i) protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G(i) protein complex.

Maxhimer JB, Shih HB, Isenberg JS, Miller TW, Roberts DD (2009). "Thrombospondin-1/CD47 blockade following ischemia-reperfusion injury is tissue protective" Plast Reconstr Surg 124(6):1880-1889. PubMed

Background: Nitric oxide has prosurvival effects that can limit ischemia-reperfusion injuries. However, the matrix glycoprotein thrombospondin-1 is induced following ischemia-reperfusion injury and limits nitric oxide signaling by engaging its cell surface receptor CD47. In this article, the authors examine whether postinjury blocking of this inhibitory signal can protect from ischemia-reperfusion injury in a rat flap model. Methods: A total of 40 tissue flaps were created in rats based solely on the deep inferior epigastric vessels. Microvascular clamps were used to create 45 minutes of ischemia time to the flaps. The flaps were then treated using a monoclonal antibody to CD47 or an isotype-matched control immunoglobulin G1 5 or 30 minutes after clamp removal. Twenty-four or 72 hours postoperatively, the necrotic area of the flap was determined, and serum, deep inferior epigastric vessels, and flaps were harvested for analysis from five rats in each respective group. Results: Treatment with a CD47 antibody 5 minutes after reperfusion significantly reduces flap necrosis compared with immunoglobulin G1 control (9 percent versus 43 percent; p < 0.01). The protective effect is even more dramatic when treatment is delayed until 30 minutes after reperfusion (10 percent versus 88 percent for control; p < 0.01). Markers of neutrophil and endothelial cell activation along with total leukocytes are reduced in CD47 antibody-treated flaps, as are tissue malondialdehyde levels. Levels of cyclic guanosine monophosphate are elevated 72 hours postoperatively in the CD47 antibody-treated deep inferior epigastric vessels versus the control flaps. Conclusions: Therapies targeting the thrombospondin-1 receptor CD47 offer potential for increasing tissue survival in ischemia-reperfusion injuries. The ability to protect when given after ischemia-reperfusion injury enables a broader clinical applicability.

Alblas J, Honing H, de Lavalette CR, Brown MH, Dijkstra CD, van den Berg TK (2005). "Signal regulatory protein alpha ligation induces macrophage nitric oxide production through JAK/STAT- and phosphatidylinositol 3-kinase/Rac1/NAPDH oxidase/H2O2-dependent pathways" Mol Cell Biol 25(16):7181-92. PubMed

Signal regulatory protein alpha (SIRPalpha) is a glycoprotein receptor that recruits and signals via the tyrosine phosphatases SHP-1 and SHP-2. In macrophages SIRPalpha can negatively regulate the phagocytosis of host cells and the production of tumor necrosis factor alpha. Here we provide evidence that SIRPalpha can also stimulate macrophage activities, in particular the production of nitric oxide (NO) and reactive oxygen species. Ligation of SIRPalpha by antibodies or soluble CD47 triggers inducible nitric oxide synthase expression and production of NO. This was not caused by blocking negative-regulatory SIRPalpha-CD47 interactions. SIRPalpha-induced NO production was prevented by inhibition of the tyrosine kinase JAK2. JAK2 was found to associate with SIRPalpha in macrophages, particularly after SIRPalpha ligation, and SIRPalpha stimulation resulted in JAK2 and STAT1 tyrosine phosphorylation. Furthermore, SIRPalpha-induced NO production required the generation of hydrogen peroxide (H(2)O(2)) by a NADPH oxidase (NOX) and the phosphatidylinositol 3-kinase (PI3-K)-dependent activation of Rac1, an intrinsic NOX component. Finally, SIRPalpha ligation promoted SHP-1 and SHP-2 recruitment, which was both JAK2 and PI3-K dependent. These findings demonstrate that SIRPalpha ligation induces macrophage NO production through the cooperative action of JAK/STAT and PI3-K/Rac1/NOX/H(2)O(2) signaling pathways. Therefore, we propose that SIRPalpha is able to function as an activating receptor.

de Vries HE, Hendriks JJ, Honing H, De Lavalette CR, van der Pol SM, Hooijberg E, Dijkstra CD, van den Berg TK (2002). "Signal-regulatory protein alpha-CD47 interactions are required for the transmigration of monocytes across cerebral endothelium" J Immunol 168(11):5832-9. PubMed

Monocyte infiltration into inflamed tissue requires their initial arrest onto the endothelial cells (ECs), followed by firm adhesion and subsequent transmigration. Although several pairs of adhesion molecules have been shown to play a role in the initial adhesion of monocytes to ECs, the mechanism of transendothelial migration is poorly defined. In this study, we have investigated the role of signal-regulatory protein (SIRP)alpha-CD47 interactions in monocyte transmigration across brain ECs. CD47 expression was observed in vivo on cerebral endothelium of both control animals and animals suffering from experimental allergic encephalomyelitis. To investigate whether SIRPalpha-CD47 interactions are instrumental in the trafficking of monocytes across cerebral EC monolayers, in vitro assays were conducted in which the migration of monocytes, but not adhesion, was found to be effectively diminished by blocking SIRPalpha and CD47 on monocytes and ECs, respectively. In this process, SIRPalpha was found to interact solely with its counterligand CD47 on ECs. Overexpression of the CD47 molecule on brain ECs significantly enhanced monocytic transmigration, but did not affect adhesion. SIRPalpha-CD47-mediated transendothelial migration involved Gi protein activity, a known signaling component of CD47. Finally, cross-linking of CD47 on brain ECs induced cytoskeletal reorganization of the endothelium, a process that was Gi protein independent. These data provide the first evidence that the interaction of CD47 with its monocytic counterligand SIRPalpha is of importance in the final step of monocyte trafficking into the brain, a critical event in the development of neuroinflammatory diseases.

Vernon-Wilson EF, Kee WJ, Willis AC, Barclay AN, Simmons DL, Brown MH (2000). "CD47 is a ligand for rat macrophage membrane signal regulatory protein SIRP (OX41) and human SIRPalpha 1" Eur J Immunol 30(8):2130-7. PubMed

The rat OX41 antigen is a cell surface protein containing three immunoglobulin superfamily domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIM). It is a homologue of the human signal-regulatory protein (SIRP) also known as SHPS-1, BIT or MFR. Cell activation-induced phosphorylation of the intracellular ITIM motifs induces association with the tyrosine phosphatases SHP-1 and SHP-2. To identify the physiological OX41 ligand, recombinant OX41-CD4d3+4 fusion protein was coupled to fluorescent beads to produce a multivalent cell binding reagent. The OX41-CD4d3+4 beads bound to thymocytes and concanavalin A-stimulated splenocytes. This interaction was blocked by the monoclonal antibody (mAb) OX101. Affinity chromatography with OX101 mAb and peptide sequencing revealed the rat SIRP ligand to be CD47 (integrin-associated protein). A direct interaction between human SIRP and human CD47 was demonstrated using purified recombinant proteins and surface plasmon resonance ruling out the involvement of other proteins known to be associated with CD47. The affinity of the SIRP/CD47 interaction was K(d) approximately 8 microM at 37 degrees C with a k(off )>/=2.1 s(-1). The membrane-distal SIRP V-like domain was sufficient for binding to CD47.