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(A) MTS assay for cell survival and growth over 72 h expressed as % of day 0 values at the indicated plating densities of first passage WT and CD47 null cells. (B) BrdU assay for DNA synthesis. (C) Percentage of senescence-associated β-galactosidase expression at passage 3. (D) Expression of genes associated with cell immortalization in WT and CD47 null cells. (E) c-Myc mRNA levels in lung endothelial cells of WT and CD47 null mice. (F) CD47 limits c-Myc protein levels. (G) c-Myc expression (red) in WT and CD47−/− endothelial cells. Blue = DAPI nuclear stain. (H) Flow cytometric analysis of c-Myc expression in WT and CD47−/− endothelial cells. (*p < 0.05, **p < 0.01).
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Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form...
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CD47-specific antibodies and fusion proteins that block CD47–SIRPα signaling are employed as antitumor agents for several cancers. Here, we investigated the synergistic antitumor effect of simultaneously targeting CD47 and autophagy in non–small cell lung cancer (NSCLC). SIRPαD1-Fc, a novel CD47-targeting fusion protein, was generated and was found...
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Activation of STING signaling plays an important role in anti-tumor immunity, and we previously reported the anti-tumor effects of STING through accumulation of M1-like macrophages in tumor tissue treated with a STING agonist. However, myeloid cells express SIRPα, an inhibitory receptor for phagocytosis, and its receptor, CD47, is overexpressed in...
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Citations
... [46] Consistent with these observations, our study highlights the importance of THBS1 as not only a chemotactic cytokine for monocytes but also as a polarizing cytokine for a novel subset of pro-fibrotic macrophages with a strong proliferative ability. In contrast to our findings, [27,47,48] other studies in endothelial and epithelial cells have suggested that THBS1 attenuates selfrenewal and proliferation suggesting that there may be other factors at play. This difference could potentially be attributed to variations in cellular signaling pathways between immune cells and epithelial/endothelial cells. ...
With the increasing incidence of kidney diseases, there is an urgent need to develop therapeutic strategies to combat post‐injury fibrosis. Immune cells, including platelets, play a pivotal role in this repair process, primarily through their released cytokines. However, the specific role of platelets in kidney injury and subsequent repair remains underexplored. Here, the detrimental role of platelets in renal recovery following ischemia/reperfusion injury and its contribution to acute kidney injury to chronic kidney disease transition is aimed to investigated. In this study, it is shown that depleting platelets accelerates injury resolution and significantly reduces fibrosis. Employing advanced single‐cell and spatial transcriptomic techniques, macrophages as the primary mediators modulated by platelet signals is identified. A novel subset of macrophages, termed “cycling M2”, which exhibit an M2 phenotype combined with enhanced proliferative activity is uncovered. This subset emerges in the injured kidney during the resolution phase and is modulated by platelet‐derived thrombospondin 1 (THBS1) signaling, acquiring profibrotic characteristics. Conversely, targeted inhibition of THBS1 markedly downregulates the cycling M2 macrophage, thereby mitigating fibrotic progression. Overall, this findings highlight the adverse role of platelet THBS1‐boosted cycling M2 macrophages in renal injury repair and suggest platelet THBS1 as a promising therapeutic target for alleviating inflammation and kidney fibrosis.
... We found that treatment with the CD47 antibody B6H12 altered gene expression in CD47expressing triple-negative MDA-MB-231 breast carcinoma cells, resulting in suppression of breast cancer stem cell (bCSC) characteristics (Kaur et al., 2016). B6H12 treatment decreased expression of Klf4 (Kaur et al., 2016), one of several stem cell transcription factors that are also regulated by TSP1/ CD47 signaling in nonmalignant cells (Kaur et al., 2013). B6H12 inhibited asymmetric division of breast cancer stem cells and induced several differentiation markers. ...
... Frontiers in Cell and Developmental Biology frontiersin.org CD47 antibody B6H12 prevents binding of SIRPα and TSP1 to CD47, and the latter interaction was shown to regulate stem cell differentiation (Kaur et al., 2013;Kaur et al., 2016). Unlike B6H12, our current data showed no inhibitory activity of CC90002 in expression of CD44, CD24 and KLF4. ...
Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.
... To establish the mechanism for enhanced proliferation, we considered transcription factor c-Myc because it is a major regulator of cell growth and proliferation, including in islets (29,30). We, and others, have shown that loss of CD47 permits sustained proliferation of primary murine cells, and CD47 knockdown or blockade acutely increases mRNA and protein expression of c-Myc (and other stem cell transcription factors) in vitro and in vivo (17,31,32). Miap301-treated WT islets had up-regulated c-Myc expression compared with IgG-treated controls (P < 0.001, Fig. 8O). ...
Diabetes is a global public health burden and is characterized clinically by relative or absolute insulin deficiency. Therapeutic agents that stimulate insulin secretion and improve insulin sensitivity are in high demand as treatment options. CD47 is a cell surface glycoprotein implicated in multiple cellular functions including recognition of self, angiogenesis, and nitric oxide signaling; however, its role in the regulation of insulin secretion remains unknown. Here, we demonstrate that CD47 receptor signaling inhibits insulin release from human as well as mouse pancreatic β cells and that it can be pharmacologically exploited to boost insulin secretion in both models. CD47 depletion stimulated insulin granule exocytosis via activation of the Rho GTPase Cdc42 in β cells and improved glucose clearance and insulin sensitivity in vivo. CD47 blockade enhanced syngeneic islet transplantation efficiency and expedited the return to euglycemia in streptozotocin-induced diabetic mice. Further, anti-CD47 antibody treatment delayed the onset of diabetes in nonobese diabetic (NOD) mice and protected them from overt diabetes. Our findings identify CD47 as a regulator of insulin secretion, and its manipulation in β cells offers a therapeutic opportunity for diabetes and islet transplantation by correcting insulin deficiency.
... Consistent with the absence of inhibitory SIRPα signaling that limits clearance of aged RBC, 14,15 cd47 −/− mice derived using CRISPR/Cas9 exhibited hemolytic anemia and splenomegaly. 16 Conversely, CD47-dependent thrombospondin-1 signaling regulates the differentiation of multipotent stem cells in a stage-specific manner, [17][18][19] and both thbs1 −/− and cd47 −/− mouse spleens have more abundant Sox2 + stem cells and higher mRNA expression of the multipotent stem cell transcription factors Myc, Sox2, Oct4, and Klf4. 17 Therefore, both thrombospondin-1-and SIRPα-dependent CD47 signaling could alter erythropoiesis and contribute to spleen enlargement. ...
... 16 Conversely, CD47-dependent thrombospondin-1 signaling regulates the differentiation of multipotent stem cells in a stage-specific manner, [17][18][19] and both thbs1 −/− and cd47 −/− mouse spleens have more abundant Sox2 + stem cells and higher mRNA expression of the multipotent stem cell transcription factors Myc, Sox2, Oct4, and Klf4. 17 Therefore, both thrombospondin-1-and SIRPα-dependent CD47 signaling could alter erythropoiesis and contribute to spleen enlargement. Here, we utilized flow cytometry combined with bulk and single cell transcriptomics to examine extramedullary hematopoiesis in cd47 −/− and thbs1 −/− mice, which revealed cooperative and opposing roles for CD47 and thrombospondin-1 to limit extramedullary erythropoiesis in spleen. ...
... cd47 −/− and sirpa −/− mice treated with CpG 21 and aging cd47 −/− mice, 16 or increased cell numbers could result from the increased stem cell abundance in cd47 −/− spleens. 17 The spleen enlargement was associated with a significantly higher total spleen cell number in a single cell suspension after RBC lysis in cd47 −/− mice compared to WT and thbs1 −/− mice ( Figure 1A). ...
Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in cd47-/- spleens but significantly depleted in thbs1-/- spleens. Single cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc, Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in thbs1-/- spleens relative to basal levels in wild type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.
... It is possible that blockade of TSP1-CD47 signaling in LEC via CD47 deletion/blocking antibodies may contribute to LEC self-renewal via upregulating the expression of c-myc. 47 However, we cannot exclude the engagement of other TSP1's receptors like CD36 or integrins in TSP1-mediated inhibition of LEC proliferation as a significant decrease in cell growth was observed in CD47-silenced cells following TSP1 treatment. TSP1 treatment downregulated VEGFR3 expression in LECs without affecting FLT4 (VEGFR3) mRNA and epsin 1 protein levels. ...
Background:
TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis.
Methods:
Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC Cd47 in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms.
Results:
Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF-C-induced AKT and eNOS activation in LEC and attenuated NO production. Further, Cd47 silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV8-PCSK9-injected LEC-specific Cd47 knockout mice (Cd47ΔLEC) had reduced atherosclerosis in both aorta and aortic root compared with control mice (Cd47ΔWT). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in Cd47ΔLEC in comparison to controls.
Conclusions:
These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC Cd47 attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.
... For instance, TSP1-CD47 signaling promotes aging in human cells and tissues [104] and animals [31,105], and limits the Yamanaka self-renewal transcription factors in human cells [106]. In fact, CD47-null cells grown in serum-free medium de-differentiation [107]. Thus, forced CD47 expression may prematurely drive stem cells out of the cell cycle and into senescence [108]. ...
The use of treatments, such as programmed death protein 1 (PD1) or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies, that loosen the natural checks upon immune cell activity to enhance cancer killing have shifted clinical practice and outcomes for the better. Accordingly, the number of antibodies and engineered proteins that interact with the ligand–receptor components of immune checkpoints continue to increase along with their use. It is tempting to view these molecular pathways simply from an immune inhibitory perspective. But this should be resisted. Checkpoint molecules can have other cardinal functions relevant to the development and use of blocking moieties. Cell receptor CD47 is an example of this. CD47 is found on the surface of all human cells. Within the checkpoint paradigm, non-immune cell CD47 signals through immune cell surface signal regulatory protein alpha (SIRPα) to limit the activity of the latter, the so-called trans signal. Even so, CD47 interacts with other cell surface and soluble molecules to regulate biogas and redox signaling, mitochondria and metabolism, self-renewal factors and multipotency, and blood flow. Further, the pedigree of checkpoint CD47 is more intricate than supposed. High-affinity interaction with soluble thrombospondin-1 (TSP1) and low-affinity interaction with same-cell SIRPα, the so-called cis signal, and non-SIRPα ectodomains on the cell membrane suggests that multiple immune checkpoints converge at and through CD47. Appreciation of this may provide latitude for pathway-specific targeting and intelligent therapeutic effect.
... 35 Furthermore, the interaction of CD47 and TSP-1 enhances the regeneration of stem cells by upregulating transcription factors of stem cells such as KLF4, Sox2, c-Myc and Oct4. 36 SIRPα was identified as an endogenous ligand of CD47. 37 It is also a transmembrane glycoprotein that is mainly expressed on macrophages, monocytes, and DCs. ...
Cancer immunotherapy, mainly including immune checkpoints-targeted therapy and the adoptive transfer of engineered immune cells, has revolutionized the oncology landscape as it utilizes patients’ own immune systems in combating the cancer cells. Cancer cells escape immune surveillance by hijacking the corresponding inhibitory pathways via overexpressing checkpoint genes. Phagocytosis checkpoints, such as CD47, CD24, MHC-I, PD-L1, STC-1 and GD2, have emerged as essential checkpoints for cancer immunotherapy by functioning as “don’t eat me” signals or interacting with “eat me” signals to suppress immune responses. Phagocytosis checkpoints link innate immunity and adaptive immunity in cancer immunotherapy. Genetic ablation of these phagocytosis checkpoints, as well as blockade of their signaling pathways, robustly augments phagocytosis and reduces tumor size. Among all phagocytosis checkpoints, CD47 is the most thoroughly studied and has emerged as a rising star among targets for cancer treatment. CD47-targeting antibodies and inhibitors have been investigated in various preclinical and clinical trials. However, anemia and thrombocytopenia appear to be formidable challenges since CD47 is ubiquitously expressed on erythrocytes. Here, we review the reported phagocytosis checkpoints by discussing their mechanisms and functions in cancer immunotherapy, highlight clinical progress in targeting these checkpoints and discuss challenges and potential solutions to smooth the way for combination immunotherapeutic strategies that involve both innate and adaptive immune responses.
... 35 Furthermore, the interaction of CD47 and TSP-1 enhances the regeneration of stem cells by upregulating transcription factors of stem cells such as KLF4, Sox2, c-Myc and Oct4. 36 SIRPα was identified as an endogenous ligand of CD47. 37 It is also a transmembrane glycoprotein that is mainly expressed on macrophages, monocytes, and DCs. ...
Cancer immunotherapy, mainly including immune checkpoints-targeted therapy and the adoptive transfer of engineered immune cells, has revolutionized the oncology landscape as it utilizes patients’ own immune systems in combating the cancer cells. Cancer cells escape immune surveillance by hijacking the corresponding inhibitory pathways via overexpressing checkpoint genes. Phagocytosis checkpoints, such as CD47, CD24, MHC-I, PD-L1, STC-1 and GD2, have emerged as essential checkpoints for cancer immunotherapy by functioning as “don’t eat me” signals or interacting with “eat me” signals to suppress immune responses. Phagocytosis checkpoints link innate immunity and adaptive immunity in cancer immunotherapy. Genetic ablation of these phagocytosis checkpoints, as well as blockade of their signaling pathways, robustly augments phagocytosis and reduces tumor size. Among all phagocytosis checkpoints, CD47 is the most thoroughly studied and has emerged as a rising star among targets for cancer treatment. CD47-targeting antibodies and inhibitors have been investigated in various preclinical and clinical trials. However, anemia and thrombocytopenia appear to be formidable challenges since CD47 is ubiquitously expressed on erythrocytes. Here, we review the reported phagocytosis checkpoints by discussing their mechanisms and functions in cancer immunotherapy, highlight clinical progress in targeting these checkpoints and discuss challenges and potential solutions to smooth the way for combination immunotherapeutic strategies that involve both innate and adaptive immune responses.
... Consistent with our previous observations of a CD47-dependent enrichment of an MYC target signature in mouse CD8 T cells [23] and CD47-dependent regulation of MYC expression in mouse CD8 T cells and Jurkat T lymphoblasts [28], MYC mRNA in the RNAseq was increased 2.1-fold in CD47 − relative to WT Jurkat cells ( Figure 1E and Supplementary Data S1). However, the other two members of the MYC gene family, MYCN (12-fold) and MYCL (3.5-fold) were also elevated in CD47 − Jurkat cells relative to WT. Increased MYCN expression was confirmed via real-time PCR analysis using four sets of MYCN primers (Supplementary Data S3, Figure 1F). ...
... Consistent with our previous observations of a CD47-dependent enrichment of an MYC target signature in mouse CD8 T cells [23] and CD47-dependent regulation of MYC expression in mouse CD8 T cells and Jurkat T lymphoblasts [28], MYC mRNA in the RNAseq was increased 2.1-fold in CD47 − relative to WT Jurkat cells ( Figure 1E and Data S1). However, the other two members of the MYC gene family, MYCN (12-fold) and MYCL (3.5-fold) were also elevated in CD47 − Jurkat cells relative to WT. Increased MYCN expression was confirmed via real-time PCR analysis using four sets of MYCN primers (Data S3, Figure 1F). ...
... Treatment with 1 μg/mL (2.2 nM) TSP1 did not affect MYC mRNA levels in the absence or presence of activation. Consistent with these results, we previously reported reduced Myc mRNA in positively selected CD8 T cells from the spleen of cd47 −/− mice [28]. Basal Mycn and Mycl mRNA expression was higher in cd47 −/− CD8 + T cells cultured in RPMI medium with 2% FCS for 6 h than in the corresponding WT CD8 + T cells ( Figure 2D,E). ...
Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) noncoding RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival.
... The interaction between TSP-1 and CD47 may inhibit the replication and growth of progenitor cells. Studies have found that TSP-1 or CD47 expression is absent in the lung ECs of 2-to 3-month-old mice, enabling young mouse cells to maintain their replication and passage functions for >6 months [42]. This suggests that these genes play important roles in the aging process. ...
(1) Background: The declined function of peripheral circulating endothelial progenitor cells (EPCs) in aging individuals resulted in decreased endothelial cell regeneration and vascular endothelial function. Improving EPCs function in aging individuals plays an important role in preventing cardiovascular diseases. (2) Methods: Thirty aged (18-month-old) male Sprague-Dawley rats were randomly divided into control and exercise groups. An aerobic exercise intervention was performed 5 days/week for 8 weeks. EPCs functions, miR-21-5p, and TSP-1 expressions were detected after the intervention. The senescence rate, proliferation, and migration of EPCs were examined after overexpression of miR-21-5p and inhibition of TSP-1 expression. (3) Results: The senescence rate, proliferation, and migration of EPCs in exercise groups were significantly improved after exercise intervention. The miR-21-5p expression was increased and the TSP-1 mRNA expression was decreased in the EPCs after the intervention. miR-21-5p overexpression can improve EPCs function and inhibit TSP-1 expression but has no effect on senescence rate. Inhibition of TSP-1 expression could improve the function and reduce the senescence rate. (4) Conclusions: Our results indicate that long-term aerobic exercise can improve the functions of EPCs in aging individuals by downregulating TSP-1 expression via miR-21-5p, which reveals the mechanism of exercise in improving cardiovascular function.
