Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: ( a ). Cytoscape network visualization of the genes which are significantly correlated with CD47 expression in both human and murine atherosclerotic plaque reveals a high number of TNF-α-related factors (indicated in blue), including ligands, receptors, and downstream signaling factors. ( b ). PANTHER pathway analysis of those genes which were (a) significantly associated with CD47 expression in mouse and human vascular tissue and (b) have been previously associated with atherosclerosis through the STAGE study , identifies “ inflammation mediated by chemokine and cytokine signaling pathway ” as the most over-abundant pathway associated with CD47 expression in vascular tissue. ( c ). Using the Hybrid Mouse Diversity Panel (HMDP), which correlates aortic gene expression with Luminex cytokine array data of plasma samples from over 100 inbred strains of mice, we found that vascular CD47 expression is positively correlated with three inflammatory cytokines in vivo, including TNF-α, IL-2 and CXCL1. Correlation data shown for CD47 and TNF-α. ( d ). Co-expression studies confirm that TNF-α and CD47 expression are positively correlated in human carotid endarterectomy samples from the BiKE validation study. The Pearson correlation coefficient was determined assuming a Gaussian distribution and P values were determined using a two-tailed test. ( e ). Experiments with primarily cultured mouse aortic SMCs indicate that TNF-α reproducibly induces CD47 mRNA upregulation, while a number of other classical pro-atherosclerotic stimuli have no significant effect. Notably, CXCL1, IL4, TGFβ and IL-2 fail to induce CD47 expression in vitro, as assessed by ANOVA. ( f ). Additional studies suggest that the effect of TNF-α on CD47 expression persists in the presence of oxidized LDL, as occurs in the atherosclerotic plaque. ( g ). Western blotting confirms that TNF-α induces CD47 expression in vascular cells at the protein level. For gel source data, see . ( h ). Immunocytochemistry studies of HCASMCs confirm that CD47 expression is induced on the cell surface of TNF-α treated cells. TNF-α effect is assessed by co-staining for HMGB1, and antibody specificity is confirmed with isotype control and recombinant CD47 peptide quenching assays. ( i ). Multiple assays (including FACS, Taqman and immunocytochemistry studies) reveal that CD47 expression is downregulated on vascular SMCs during programmed cell death, as has previously been observed with inflammatory cells. ( j ). Confirmatory assays in cultured human coronary artery SMC reveal that TNF-α induces changes similar to those observed in murine cells , including an induction of CD47 under physiological conditions and a blunting of its expected downregulation during apoptosis. ( k ). TNF-α’s capacity to impair CD47 downregulation during programmed cell death is also observed in mouse SMCs simultaneously exposed to pro-apoptotic stimuli and oxidized LDL. ( l ). No correlation between CD47 and other candidate cytokines was observed in the BiKE biobank, further supporting a specific relationship between CD47 and TNF-α. ( m ). Representative FACS-based apoptosis panels from cells exposed to the conditions used in confirm that TNF-α suppresses efferocytosis despite increasing programmed cell death. Comparisons made by two-tailed t tests, unless otherwise specified. *** = P < 0.001, * = P < 0.05. Error bars represent the SEM.

Article Snippet: Antibody specificity was confirmed using isotype control and by preincubating the anti-CD47 Ab with recombinant CD47 antigen (R&D Systems) in a 1:5 ratio for 16h at 4°C before application.

Techniques: Expressing, Gene Expression, Luminex, Clinical Proteomics, In Vivo, Biomarker Discovery, Two Tailed Test, Cell Culture, In Vitro, Western Blot, Immunocytochemistry, Staining, Control, Recombinant

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: In vivo serological data and additional in silico and bioinformatic data ( a ). Complete serological studies (including blood count, liver function studies, basic metabolic panel, and fasting glucose) from the 4 week apoE −/− -AngII atherosclerosis model indicate that anti-CD47 Ab induces a significant reduction in hemoglobin and compensatory reticulocytosis, consistent with prior reports 4 , 7 . The erythrophagocytosis of senescent RBCs appears to be self-limited, and no anemia was observed in the chronic atherosclerosis model or the reduced dose model (P = 0.54 and 0.57, respectively). No significant difference in any other serum marker is observed except for an increase in serum creatinine, which does not deviate outside of the reference range. Metabolic parameters and leukocyte differential data from the 12 week chronic atherosclerosis model are displayed at the bottom of the table. ( b ). Additional Upstream Regulator Analysis (URA) bioinformatic analyses of the Cytoscape data displayed in Extended Data Figure 7a performed within the Ingenuity Pathway Analysis (IPA) software identifies a number of TNF-α related factors (indicated in red) which are predicted to mediate transcriptional regulatory roles in the gene network shown in that panel. P-values were determined from Fisher’s Exact Test by comparing overlap of co-expressed genes with known upstream regulators from the Ingenuity Knowledge Base. ( c ). Several additional DAVID -based bioinformatics analyses including ( KEGG , SMART , PANTHER and GO analyses) confirm the association between CD47 and inflammatory signaling related to the TNF-α pathway (indicated in red). Blue panels indicate the –logp10 value for each identified factor. ( d ). Transcription factor binding site prediction algorithms identify several putative NFKB family binding sites within the CD47 promoter, as displayed in Extended Data Figure 8a . ( d ). List of primers used in this study.

Article Snippet: Antibody specificity was confirmed using isotype control and by preincubating the anti-CD47 Ab with recombinant CD47 antigen (R&D Systems) in a 1:5 ratio for 16h at 4°C before application.

Techniques: In Vivo, In Silico, Marker, Software, Binding Assay