Spatial distribution of tumor-resident macrophages as predictive biomarkers in endometrial cancer.

BACKGROUND: To investigate the role of CD47 expression and its relationship with tumor-resident macrophages, specifically at the tumor margin, in patients with type II endometrial cancer. This study aims to elucidate whether CD47 could serve as a prognostic marker and to understand the dynamics between CD47 and macrophages, which could inform new therapeutic strategies. METHODS: A retrospective cohort study was conducted involving 75 patients of type II endometrial. Immunohistochemical analysis was performed to assess CD47 expression and macrophage markers (CD68 and CD163). RESULTS: The study found no direct correlation between CD47 expression levels and overall survival (p = 0.32), challenging its role as an independent prognostic marker in type II endometrial cancer. The higher expression of CD47 had significantly less incidence of endometrioid carcinoma G3 (p = 0.047). The negative correlation between CD47 H-score and the density of CD68-positive macrophages at tumor margin was statistically significant (p = 0.049). A high density of CD68-positive macrophages at the tumor margin but a low density of CD163-positive macrophages at the tumor margin were associated with poorer prognosis (p = 0.036). CONCLUSIONS: The complex interaction between CD47 and macrophages, particularly at the tumor margin, suggests new avenues for targeted therapy in type II endometrial cancer.

Direct-laser writing for subnanometer focusing and single-molecule imaging.

Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing (<0.8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.

Significance of CD47 and Its Association With Tumor Immune Microenvironment Heterogeneity in Ovarian Cancer.

Background: It was reported that tumor heterogeneity and the surrounding tumor microenvironment (TME) in ovarian cancer affects immunotherapy efficacy and patient outcomes. And the TME of ovarian cancer is intrinsically heterogeneous. CD47 plays vital roles in cell functional behavior and immune homeostasis relating to cancer prognosis. But how it affects TME and its contribution to heterogeneity in ovarian cancer has not been fully illustrated. Therefore, we aimed to identify a prognostic biomarker which may help explain tumor immune microenvironment heterogeneity of ovarian cancer. Methods: Cancer single-cell state atlas (CancerSEA) was used to evaluate functional role of CD47. Several bioinformatics database including Oncomine, Gene Expression Profiling Interaction Analysis (GEPIA), Tumor Immune Estimation Resource (TIMER), The Human Protein Atlas (HPA), Ualcan and Kaplan-Meier plotter (KM plotter) were applied to illustrate correlation of CD47 with ovarian cancer prognosis and immune infiltration. Tumor Immune Single-cell Hub (TISCH) single cell database was employed to evaluate correlation of CD47 with tumor microenvironment. GeneMANIA was implemented to identify regulation networks of CD47. Differentially expressed genes (DEGs) between CD47 high and low expression groups were analyzed with R package DESeq2. Kyoto encyclopedia of genes and genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were utilized to explore how CD47 affect the immune related cell signaling pathway. Results: CD47 expression was upregulated and connected to worse OS and PFS in ovarian cancer. Close relation was found between CD47 expression level and immune infiltration in ovarian cancer, especially with Treg cells, Monocytes, Macrophages and T cell exhaustion (P<0.05). The CD47 expression level was relatively low in plasma cells, dendritic cells and Mono/Macro cells of OV_GSE115007, in myofibroblasts, fibroblasts and endothelial cells of OV_GSE118828, compared to malignant cells of OV_GSE118828 dataset. The cell components and distribution in primary and metastatic ovarian cancer are quite distinct, which may lead to TME heterogeneity of ovarian cancer. Conclusion: Our results indicated that CD47 is closely correlated to ovarian cancer immune microenvironment and might induce ovarian cancer heterogeneity. Therefore, CD47 may be used as a candidate prognostic biomarker and provide us with new insights into potential immunotherapy in ovarian cancer patients.

CD47 expression and CD163+ macrophages correlated with prognosis of pancreatic neuroendocrine tumor.

BACKGROUND: Recent studies have suggested the important roles of CD47 and tumor-associated macrophages in the prognosis and immunotherapy of various human malignancies. However, the clinical significance of CD47 expression and CD163+ TAMs in pancreatic neuroendocrine tumor (PanNET) remains unclear. METHODS: In this study, 47 well-differentiated PanNET resection specimens were collected. CD47 expression and CD163+ macrophages were evaluated using immunohistochemistry and correlated with clinicopathologic properties. RESULTS: Positive CD47 staining was seen in all PanNETs as well as adjacent normal islets. Compared to normal islets, CD47 overexpressed in PanNETs (p = 0.0015). In the cohort, lymph node metastasis (LNM), lymphovascular invasion (LVI), and perineural invasion (PNI) were found in 36.2, 59.6, and 48.9% of the cases, respectively. Interestingly, PanNETs with LNM, LVI, or PNI had significantly lower H-score of CD47 than those without LNM (p = 0.035), LVI (p = 0.0005), or PNI (p = 0.0035). PanNETs in patients with disease progression (recurrence/death) also showed a significantly lower expression of CD47 than those without progression (p = 0.022). In contrast, CD163+ macrophage counts were significantly higher in cases with LNM, LVI, and PNI. CONCLUSIONS: Our data suggest relative low CD47 expression and high CD163+ TAMs may act as indicators for poor prognosis of PanNETs.

IFN-γ-Dependent Reduction of Erythrocyte Life Span Leads to Anemia during Mycobacterial Infection.

Anemia is a frequent and challenging complication of mycobacterial infections. We used a model of disseminated Mycobacterium avium infection in mice to investigate the mechanisms of mycobacteria-induced anemia. We found increased formation of RBC in the bone marrow and spleen of infected mice. Infection induced reticulocytosis and the premature egress of immature progenitors to the systemic circulation in an IFN-γ (IFNG)-dependent way. The newly formed RBC had reduced CD47 surface expression and a reduced life span and were phagocytosed in the liver of infected mice, increasing iron recycling in this organ. The increased engulfment and degradation of RBC was independent of IFNG sensing by macrophages. Together, our findings demonstrate that mycobacterial infection alters the formation of erythrocytes, leading to their accelerated removal from circulation and hemolytic anemia. This comprehensive elucidation of the mechanisms underlying mycobacteria-induced anemia has important implications for its efficient clinical management.

Monitoring kinetics reveals critical parameters of IgA-dependent granulocyte-mediated anti-tumor cell cytotoxicity.

Human IgA antibodies effectively engage myeloid cells for the FcαRI-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Established methods to investigate ADCC are the 51chromium and Calcein release assays. Their critical limitations are the end-point measurement, the unspecific release of the probes, the requirement of target cells in suspension and thus do not reflect physiologic conditions of adherently growing cells. Here we report the label-free real-time monitoring of granulocyte-mediated ADCC using an impedance-based method. We investigated the efficacy of an engineered epidermal growth factor receptor (EGFR)-directed IgA2 antibody to engage neutrophils for ADCC against a panel of adherently growing EGFR-expressing cancer cell lines majorly head and neck squamous cell carcinoma (HNSCC). The impedance assay allowed the documentation of the IgA-neutrophil-and FcαRI-signaling dependent ADCC of adherently growing target cells. While at a short-term it provided comparable results to release assays, in the long run real time monitoring also revealed cell-line specific kinetics and long-term efficacy. Although short-term results may depend on EGFR expression, long-term efficacy did not correlate with the surface level of EGFR nor of the myeloid checkpoint CD47 pointing to additional critical parameters to predict the treatment efficacy. Real-time monitoring of neutrophil-mediated ADCC allowed documenting effector cell activity and exhaustion. Along with excess expression of Mac-1 ligands, which may explain the target cell resistance, this eventually leads to tumor cell outgrowth at later time points. In conclusion, the impedance assay provides valuable information on the kinetics, effector cell performance, efficacy and critical parameters of IgA-dependent granulocyte-mediated cytotoxicity and is expected to become an important tool in its evaluation.

Selected CD molecules and the phagocytosis of microvesicles released from erythrocytes ex vivo.

BACKGROUND AND OBJECTIVES: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. MATERIAL AND METHODS: Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. RESULTS: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. CONCLUSION: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype.

Bladder cancer has the highest recurrence rate of all cancers due in part to inadequate transurethral resection. Inadequate resection is caused by the inability of cystoscopes to detect invisible lesions during the resection procedure. To improve detection and resection of nonmuscle invasive bladder cancer, we quantified the ability of a surface-enhanced Raman nanoparticle and endoscope system to classify bladder tissue as normal or cancerous. Both antibody-based (active) and tissue permeability-based (passive) targeting mechanisms were evaluated by topically applying nanoparticles to ex vivo human bladder tissue samples. Multiplexed molecular imaging of CD47 and Carbonic Anhydrase 9 tumor proteins gave a receiver operating characteristic area under the curve (ROC AUC of 0.93 (0.75, 1.00). Furthermore, passively targeted nanoparticles enabled tissue classification with an ROC AUC of 0.93 (0.73, 1.00). Passively targeted nanoparticles penetrated 5-fold deeper and bound to tumor tissue at 3.3-fold higher concentrations in cancer compared to normal bladder urothelium, suggesting the existence of an enhanced surface permeability and retention effect in human bladder cancer.

NLRP3 Inflammasome Activation Regulates Aged RBC Clearance.

The NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome is triggered by various stimuli. Whether the NLRP3 inflammasome is activated during the monocyte clearing of aged or damaged erythrocytes is unknown. This work aimed to determine whether the NLRP3 inflammasome is activated during the THP-1 cell engulfing of aged erythrocytes. In the study, THP-1 cells were treated with PMA and then coincubated with untreated red blood cells (RBCs), 42 °C-treated RBCs, immunoglobulin G (IgG) anti-D-sensitized RBCs, Rhnull/Rhmod RBC sample, hemoglobin, and RBC ghost. The activation of the NLRP3 inflammasome and production of some proinflammatory cytokines were determined using immunoblotting, cytometric bead array, and digital PCR. An NLRP3 inflammasome inhibitor was also used to evaluate the alteration of the NLRP3 activation and RBC clearance rate. The untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin induced the THP-1-cell-mediated activation of the NLRP3 inflammasome and the production of inflammatory cytokines. The RBC clearance rate exhibited a positive correlation with the expression of proinflammatory cytokines. The NLRP3 inflammasome inhibitor reduced the NLRP3 activation and RBC phagocytosis rate. The NLRP3 inflammasome was activated during the clearance of the aged erythrocytes through unopsonized and opsonized pathways. However, the mechanism of such phenomenon needs to be further elucidated. Such mechanism may provide new insight into the assessment of the safety of transfusing long-storage RBC based on cytokine levels.

The CD47 "don't eat me signal" is highly expressed in human ovarian cancer.

OBJECTIVES: The CD47 "don't eat me" signal allows tumor immune evasion. We tested the association of CD47 expression with outcomes in EOC. METHODS: CD47 expression was examined within the TCGA database for ovarian carcinoma. For validation, IHC was performed on a TMA consisting of specimens from 265 patients with EOC. The medical records of the patients were also retrospectively reviewed to correlate demographic and survival data. RESULTS: CD47 was amplified in 15/316 (5%) ovarian serous cancers in TCGA. In the validation cohort, the majority of patients had stage III/IV disease (208/265, 78.4%). CD47 expression was seen in 210/265 (79.2%). Patients were categorized into CD47hi (129/265; 48.7%) versus CD47lo (136/265; 51.3%). Patients with CD47lo tumors were more likely to have a complete response to adjuvant therapy than CD47hi (65% vs 50%, p=0.026). Although there was a trend towards an increase in median OS (37.64 vs 45.26months, p=0.92) in the CD47lo group compared with CD47hi, the difference was not significant. CONCLUSIONS: CD47 is expressed at high frequency in EOC. Patients with CD47lo EOC had a better treatment response to standard therapy, and trended towards improved OS. This demonstrates that while CD47 may be an immunologic shield that may be considered for targeted therapies, it is likely that it operates in concert with other mechanisms of immune evasion. Future studies to evaluate CD47 expression with other known mechanisms of immune escape in the tumor microenvironment may help further define its role.

CD47 expression in cryopreserved equine cutaneous masses and normal skin.

We investigated CD47 expression in cryopreserved sections of equine cutaneous masses and normal skin. CD47 is a cell surface protein expressed on many cell types and overexpressed in some tumors. Interaction of CD47 and signal regulatory protein-alpha (SIRPα) inhibits phagocytosis by macrophages. Formalin-fixed tissues from horses prospectively enrolled in the study were used to establish a histologic diagnosis. Immunohistochemical assays were performed on cryopreserved tissues using anti-CD47 antibodies or IgG control antibodies. CD47 was not expressed on equine normal skin but positivity to CD47 was present in 13 of 24 (54%) masses. Immunotherapy with anti-CD47 antibodies for equine cutaneous tumors that express CD47 warrants further investigation.

Programmed cell removal biomarkers calreticulin and CD47 implicated in oral lichen planus.

OBJECTIVES: To investigate the expression of the programmed cell removal markers, calreticulin (CRT) and CD47, known to be involved in various autoimmune diseases, in patients with oral lichen planus (OLP), and to investigate the association with clinical behavior. MATERIALS AND METHODS: Biopsies of 78 patients with OLP were included. The clinical data were collected from patients' charts. The expression of CRT and CD47 was immunomorphometrically analyzed in the epithelial (CRTep, CD47ep) and inflammatory cells (CRTinf, CD47inf), and the results were correlated with the clinical presentation. RESULTS: The epithelial and inflammatory cells expressed CRT (2.83 ± 6.62 and 5.13 ± 3.72) and CD47 (7.92 ± 4.6 and 10.7 ± 7.16). The expressions of CD47ep and CD47inf were associated (R = 0.64, P < 0.0005) with one another. The expressions of CRTinf and CD47ep were higher in atrophic erosive forms (A/ELP) than in the keratotic form of patients with OLP (6.46 ± 0.76 and 9.38 ± 0.87 vs 4.2 ± 0.61 and 6.84 ± 0.91, respectively, P = 0.002 and P = 0.021). The expression of CRTep was associated with more localized lesions (P < 0.009) and more abundant in males (P = 0.049), and the expression of CRTinf was associated with the presence of skin lesions and symptoms (P < 0.034 and P = 0.047, respectively). Only in A/ELP patients, the expression of CRTep was associated with high expression of CD47ep (R = 0.6, P = 0.004), where both CD47ep and CD47inf were associated with lower age of the patients (R = -0.48, P = 0.03 and R = -0.54, P = 0.01). CONCLUSIONS: The pattern of expression of CRT and CD47 in OLP suggests a general programmed cell removal response in OLP. Symptomatic patients may benefit from CRT/CD47 targeted therapy in the future.

Co-expression of MET and CD47 is a novel prognosticator for survival of luminal breast cancer patients.

Although luminal-type primary breast cancer can be efficiently treated, development of metastatic disease remains a significant clinical problem. We have previously shown that luminal-type circulating tumor cells (CTCs) co-expressing the tyrosine-kinase MET and CD47, a ligand involved in cancer cell evasion from macrophage scavenging, are able to initiate metastasis in xenografts. Here, we investigated the clinical relevance of MET-CD47 co-expression in 255 hormone receptor positive breast tumors by immunohistochemistry and found a 10.3- year mean overall-survival difference between MET-CD47 double-positive and double-negative patients (p<0.001) MET-CD47 co-expression defined a novel independent prognosticator for overall-survival by multivariate analysis (Cox proportional hazards model: HR: 4.1, p<0.002) and CD47 expression alone or in combination with MET was strongly associated with lymph node metastasis. Furthermore, flow cytometric analysis of metastatic patient blood revealed consistent presence of MET+CD47+ CTCs (range 0.8 - 33.3% of CTCs) and their frequency was associated with increased metastatic spread. Finally, primary uncultured CTCs with high MET+CD47+ content showed an enhanced capacity to initiate metastasis in mice. Detection and targeting of MET and CD47 may thus provide a rational basis for risk stratification and treatment of patients with luminal-type breast cancer.

Suppressed expression of homotypic multinucleation, extracellular domains of CD172α (SIRP-α) and CD47 (IAP) receptors in TAMs upregulated by Hsp70-peptide complex in Dalton's lymphoma.

CD172α and CD47 are members of glycoprotein expressed on macrophages and various immune cells, promote immune recognition and T cell stimulation that priming phagocytosis of pathogens and apoptotic bodies and malignant cell. Tumour-releasing immunosuppressive factor promotes tumour growth and transforms the tumour resident M1 phenotype of macrophage to M2 phenotype (TAMs) that promotes tumour progression by downregulating the expression of different surface receptor including CD172α and CD47. Recent studies have reported that CD172α and CD47 are involved in the pathogenesis and promote malignancies such as lymphoma, leukaemia, melanoma, lung cancer and multiple myeloma, and their expression varies during infection and malignancies. Autologous Hsp70 is well recognized for its role in activating macrophages leading to enhance production of inflammatory cytokines. It has been observed that Hsp70 derived from normal tissues do not elicit tumour immunity, while Hsp70 preparation from tumour cell was able to elicit tumour immunity. However, the role of exogenous autologous hsp70 on the formation of giant cells is completely unknown. Therefore, in the present study, we sought to investigate the effect of Hsp70-peptide complex on the expression of CD172α and CD47 receptors in normal peritoneal macrophages (NMO) and TAMs. Finding shows that the expression of CD172α and CD47 enhances in TAMs and it reverts back the suppressed function of TAMs into M1 state of immunoregulatory phenotype that promotes tumour regression by enhanced multinucleation and phagocytosis of malignant cells and significantly enhances the homotypic fusion of macrophages and polykaryon formation in vitro by enhancing the expression of SIRPα and IAP.

'Marker-of-self' functionalization of nanoscale particles through a top-down cellular membrane coating approach.

We investigate the 'marker-of-self' functionalization of nanoparticles through coating of natural RBC membranes. The membrane translocation approach is shown to be highly efficient and bestows nanoparticles with correctly oriented and functional immunomodulatory proteins such as CD47 at equivalent density to natural RBCs.

Biological validation of bio-engineered red blood cell productions.

The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies. For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34(+) cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index. Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test.

Imaging of CD47 expression in xenograft and allograft tumor models.

CD47 functions as a marker of "self" by inhibiting phagocytosis of autologous cells. CD47 has been shown to be overexpressed by various tumor types as a means of escaping the antitumor immune response. The goal of this research was to investigate the utility of CD47 imaging using positron emission tomography (PET) in both human xenograft and murine allograft tumor models. Anti-CD47 antibodies were conjugated with p-isothiocyanatobenzyldesferrioxamine (Df-Bz-NCS) and labeled with 89Zr. We employed xenograft and allograft small-animal models of cancer in biodistribution and PET imaging studies to investigate the specificity and PET imaging robustness of CD47. Ab-Df-Bz-NCS conjugates were labeled with 89Zr with specific activity of 0.9 to 1.6 µCi/µg. Biodistribution studies in the xenograft and allograft model showed similar specific tumor uptake of the antihuman and antimouse CD47 antibodies. However, the tracer retention in the liver, spleen, and kidneys was significantly higher in the allograft-bearing animals, suggesting uptake mediated by the CD47 normally expressed throughout the reticular endothelial system. CD47, a marker of "self," was evaluated as a diagnostic PET biomarker in xenograft and allograft cancer animal models. CD47 imaging is feasible, warranting further studies and immunoPET tracer development.

Immunoenrichment microwave and magnetic proteomics for quantifying CD47 in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating immunoenrichment prior to rapid microwave and magnetic (IM(2) ) sample preparation, might enable correlation of the relative expression of CD47 and other low abundance proteins to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, anti-CD47 antibodies were used to enrich for low abundance CD47 prior to microwave and magnetic proteomics in EAE. Decoding protein expression at each time point, with CD47-immunoenriched samples and targeted proteomic analysis, enabled peptides from the low abundance proteins to be precisely quantified throughout disease progression, including: CD47: 86-99, corresponding to the "marker of self" overexpressed by myelin that prevents phagocytosis, or "cellular devouring," by microglia and macrophages; myelin basic protein: 223-228, corresponding to myelin basic protein; and migration inhibitory factor: 79-87, corresponding to a proinflammatory cytokine that inhibits macrophage migration. While validation in a larger cohort is underway, we conclude that IM(2) proteomics is a rapid method to precisely quantify peptides from CD47 and other low abundance proteins throughout disease progression in EAE. This is likely due to improvements in selectivity and sensitivity, necessary to partially overcome masking of low abundance proteins by high abundance proteins and improve dynamic range.

CD47 regulates the TGF-ß signaling pathway in osteoblasts and is distributed in Meckel's cartilage.

Previously, CD47 gene expression has been shown to increase during mandible development using a micro array technique. To determine the function of CD47 in osteoblasts, CD47 was silenced using siRNA in vitro. The TGF-ß1 and phosphorylated-Smad2 levels and transcription factor genes related to bone metabolism increased dose-dependently with CD47 silencing. Furthermore, we determined the distribution of CD47 in mouse embryonic E13 and E15 in vivo. The CD47-positive cells were localized in Meckel's cartilage and antenatal mandibular bone. These results suggest that TGF-ß1 signaling and mandible development might be regulated by CD47.

CD47-dependent molecular mechanisms of blood outgrowth endothelial cell attachment on cholesterol-modified polyurethane.

We previously showed that blood outgrowth endothelial cells (BOECs) had a high affinity for polyurethane (PU) covalently configured with cholesterol residues (PU-Chol). However, the molecular mechanisms responsible for this enhanced affinity were not determined. CD47, a multifunctional transmembrane glycoprotein involved in cellular attachment, can form a cholesterol-dependent complex with integrin alpha(v)beta(3) and heterotrimeric G proteins. We tested herein the hypothesis that CD47, and the other components of the multi-molecular complex, enhance the attachment of BOECs to PU-Chol. Immunoprecipitation studies, of human and ovine BOECs, demonstrated that CD47 associates with integrin alpha(v) and integrin beta(3) as well as G(alphai-2) protein. The three-fold increase in BOEC attachment to PU-Chol, compared to unmodified PU, was reversed with the addition of blocking antibodies specific for CD47 and integrin alpha(v) and integrin beta(3). Similar results were observed with the addition of methyl-beta-cyclodextrin (MbetaCD), a known disruptor of the CD47 complex as well as of the membrane cholesterol content, to seeded BOEC or PU-Chol films. Reducing CD47 expression, via lentivirus transduced shRNA, decreased BOEC binding to PU-Chol by 50% compared to control groups. These data are the first demonstration of a role for the CD47 cholesterol-dependent signaling complex in BOEC attachment onto synthetic surfaces.