Tolerating CD47.

Cluster of differentiation 47 (CD47) occupies the outer membrane of human cells, where it binds to soluble and cell surface receptors on the same and other cells, sculpting their topography and resulting in a pleiotropic receptor-multiligand interaction network. It is a focus of drug development to temper and accentuate CD47-driven immune cell liaisons, although consideration of on-target CD47 effects remain neglected. And yet, a late clinical trial of a CD47-blocking antibody was discontinued, existent trials were restrained, and development of CD47-targeting agents halted by some pharmaceutical companies. At this point, if CD47 can be exploited for clinical advantage remains to be determined. Herein an airing is made of the seemingly conflicting actions of CD47 that reflect its position as a junction connecting receptors and signalling pathways that impact numerous human cell types. Prospects of CD47 boosting and blocking are considered along with potential therapeutic implications for autoimmune diseases and cancer.

A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained ß cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

The TSP1-CD47-SIRPα interactome: an immune triangle for the checkpoint era.

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.

Thrombospondin-1, CD47, and SIRPα display cell-specific molecular signatures in human islets and pancreata.

Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα), and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint-blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Fresh islets and pancreatic tissue from nondiabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression were determined. CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, whereas SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by ß cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Human pancreatic exocrine and endocrine tissue expressed CD47, whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.NEW & NOTEWORTHY CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.

Towards a Rational Balanced Pancreatic and Islet Allocation Schema.

Allocation of donated organs for transplantation is a complex process that considers numerous factors such as donor, organ and candidate characteristics and practical issues such as geography. Whole pancreas and isolated islet transplantation are lifesaving for certain individuals with diabetes. Herein, we suggest a revised allocation schema that matches donor characteristics with candidate medical condition while allowing for geographic considerations. It is hoped that adoption of this schema will shorten allocation time, decrease organ waste and optimize the parity between organ donor characteristics and candidate state of health.

A Multiparametric Assessment of Human Islets Predicts Transplant Outcomes in Diabetic Mice.

Prior to transplantation into individuals with type 1 diabetes, in vitro assays are used to evaluate the quality, function and survival of isolated human islets. In addition to the assessments of these parameters in islet, they can be evaluated by multiparametric morphological scoring (0-10 points) and grading (A, B, C, D, and F) based on islet characteristics (shape, border, integrity, single cells, and diameter). However, correlation between the multiparametric assessment and transplantation outcome has not been fully elucidated. In this study, 55 human islet isolations were scored using this multiparametric assessment. The results were correlated with outcomes after transplantation into immunodeficient diabetic mice. In addition, the multiparametric assessment was compared with oxygen consumption rate of isolated islets as a potential prediction factor for successful transplantations. All islet batches were assessed and found to score: 9 points (n = 18, Grade A), 8 points (n = 19, Grade B), and 7 points (n = 18, Grade B). Islets that scored 9 (Grade A), scored 8 (Grade B) and scored 7 (Grade B) were transplanted into NOD/SCID mice and reversed diabetes in 81.2%, 59.4%, and 33.3% of animals, respectively (P < 0.0001). Islet scoring and grading correlated well with glycemic control post-transplantation (P < 0.0001) and reversal rate of diabetes (P < 0.05). Notably, islet scoring and grading showed stronger correlation with transplantation outcome compared to oxygen consumption rate. Taken together, a multiparametric assessment of isolated human islets was highly predictive of transplantation outcome in diabetic mice.

Chronic marijuana usage by human pancreas donors is associated with impaired islet function.

We investigated the effect of chronic marijuana use, defined as 4 times weekly for more than 3 years, on human pancreatic islets. Pancreata from deceased donors who chronically used marijuana were compared to those from age, sex and ethnicity matched non-users. The islets from marijuana-users displayed reduced insulin secretion as compared to islets from non-users upon stimulation with high glucose (AUC, 3.41 ± 0.62 versus 5.14 ±0.47, p<0.05) and high glucose plus KCl (AUC, 4.48 ± 0.41 versus 7.69 ± 0.58, p<0.001). When human islets from chronic marijuana-users were transplanted into diabetic mice, the mean reversal rate of diabetes was 35% versus 77% in animals receiving islets from non-users (p<0.01). Immunofluorescent staining for cannabinoid receptor type 1 (CB1R) was shown to be colocalized with insulin and enhanced significantly in beta cells from marijuana-users vs. non-users (CB1R intensity/islet area, 14.95 ± 2.71 vs. 3.23 ± 0.87, p<0.001). In contrast, CB1R expression was not co-localized with glucagon or somatostatin. Furthermore, isolated islets from chronic marijuana-users appeared hypertrophic. In conclusion, excessive marijuana use affects islet endocrine phenotype and function in vitro and in vivo. Given the increasing use of marijuana, our results underline the importance of including lifestyle when evaluating human islets for transplantation or research.

CD47 (Cluster of Differentiation 47).

CD47, also known as integrin-associated protein, is a constitutively and ubiquitously expressed transmembrane receptor. CD47 is conserved across amniotes including mammals, reptiles, and birds. Expression is increased in many cancers and, in non-malignant cells, by stress and with aging. The up-regulation of CD47 expression is generally epigenetic, whereas gene amplification occurs with low frequency in some cancers. CD47 is a high affinity signaling receptor for the secreted protein thrombospondin-1 (THBS1) and the counter-receptor for signal regulatory protein-α (SIRPA, SIRPα) and SIRPγ (SIRPG). CD47 interaction with SIRPα serves as a marker of self to innate immune cells and thereby protects cancer cells from phagocytic clearance. Consequently, higher CD47 correlates with a poor prognosis in some cancers, and therapeutic blockade can suppress tumor growth by enhancing innate antitumor immunity. CD47 expressed on cytotoxic T cells, dendritic cells, and NK cells mediates inhibitory THBS1 signaling that further limits antitumor immunity. CD47 laterally associates with several integrins and thereby regulates cell adhesion and migration. CD47 has additional lateral binding partners in specific cell types, and ligation of CD47 in some cases modulates their function. THBS1-CD47 signaling in non-malignant cells inhibits nitric oxide/cGMP, calcium, and VEGF signaling, mitochondrial homeostasis, stem cell maintenance, protective autophagy, and DNA damage response, and promotes NADPH oxidase activity. CD47 signaling is a physiological regulator of platelet activation, angiogenesis and blood flow. THBS1/CD47 signaling is frequently dysregulated in chronic diseases.

CD47 and thrombospondin-1 regulation of mitochondria, metabolism, and diabetes.

Thrombospondin-1 (TSP1) is the prototypical member of a family of secreted proteins that modulate cell behavior by engaging with molecules in the extracellular matrix and with receptors on the cell surface. CD47 is widely displayed on many, if not all, cell types and is a high-affinity TSP1 receptor. CD47 is a marker of self that limits innate immune cell activities, a feature recently exploited to enhance cancer immunotherapy. Another major role for CD47 in health and disease is to mediate TSP1 signaling. TSP1 acting through CD47 contributes to mitochondrial, metabolic, and endocrine dysfunction. Studies in animal models found that elevated TSP1 expression, acting in part through CD47, causes mitochondrial and metabolic dysfunction. Clinical studies established that abnormal TSP1 expression positively correlates with obesity, fatty liver disease, and diabetes. The unabated increase in these conditions worldwide and the availability of CD47 targeting drugs justify a closer look into how TSP1 and CD47 disrupt metabolic balance and the potential for therapeutic intervention.

THBS1 (thrombospondin-1).

Thrombospondins are encoded in vertebrates by a family of 5 THBS genes. THBS1 is infrequently mutated in most cancers, but its expression is positively regulated by several tumor suppressor genes and negatively regulated by activated oncogenes and promoter hypermethylation. Consequently, thrombospondin-1 expression is frequently lost during oncogenesis and is correlated with a poor prognosis for some cancers. Thrombospondin-1 is a secreted protein that acts in the tumor microenvironment to inhibit angiogenesis, regulate antitumor immunity, stimulate tumor cell migration, and regulate the activities of extracellular proteases and growth factors. Differential effects of thrombospondin-1 on the sensitivity of normal versus malignant cells to ischemic and genotoxic stress also regulate the responses to tumors to therapeutic radiation and chemotherapy.

CD47 Promotes Age-Associated Deterioration in Angiogenesis, Blood Flow and Glucose Homeostasis.

The aged population is currently at its highest level in human history and is expected to increase further in the coming years. In humans, aging is accompanied by impaired angiogenesis, diminished blood flow and altered metabolism, among others. A cellular mechanism that impinges upon these manifestations of aging can be a suitable target for therapeutic intervention. Here we identify cell surface receptor CD47 as a novel age-sensitive driver of vascular and metabolic dysfunction. With the natural aging process, CD47 and its ligand thrombospondin-1 were increased, concurrent with a reduction of self-renewal transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in arteries from aged wild-type mice and older human subjects compared to younger controls. These perturbations were prevented in arteries from aged CD47-null mice. Arterial endothelial cells isolated from aged wild-type mice displayed cellular exhaustion with decreased proliferation, migration and tube formation compared to cells from aged CD47-null mice. CD47 suppressed ex vivo sprouting, in vivo angiogenesis and skeletal muscle blood flow in aged wild-type mice. Treatment of arteries from older humans with a CD47 blocking antibody mitigated the age-related deterioration in angiogenesis. Finally, aged CD47-null mice were resistant to age- and diet-associated weight gain, glucose intolerance and insulin desensitization. These results indicate that the CD47-mediated signaling maladapts during aging to broadly impair endothelial self-renewal, angiogenesis, perfusion and glucose homeostasis. Our findings provide a strong rationale for therapeutically targeting CD47 to minimize these dysfunctions during aging.

Thrombospondin-1 in maladaptive aging responses: a concept whose time has come.

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth, including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.

An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles.

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Intra-pancreatic tissue-derived mesenchymal stromal cells: a promising therapeutic potential with anti-inflammatory and pro-angiogenic profiles.

BACKGROUND: Human pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS: We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS: IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α. CONCLUSIONS: The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone.

We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a leading cause of death in sickle cell disease (SCD) patients. Hemolysis and oxidative stress contribute to SCD-associated PH. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and, by interacting with its receptor CD47, limits vasodilation of distal pulmonary arteries ex vivo. We hypothesized that the TSP1-CD47 interaction may promote PH in SCD. We found that TSP1 and CD47 are upregulated in the lungs of Berkeley (BERK) sickling (Sickle) mice and patients with SCD-associated PH. We then generated chimeric animals by transplanting BERK bone marrow into C57BL/6J (n = 24) and CD47 knockout (CD47KO, n = 27) mice. Right ventricular (RV) pressure was lower in fully engrafted Sickle-to-CD47KO than Sickle-to-C57BL/6J chimeras, as shown by the reduced maximum RV pressure (P = 0.013) and mean pulmonary artery pressure (P = 0.020). The afterload of the sickle-to-CD47KO chimeras was also lower, as shown by the diminished pulmonary vascular resistance (P = 0.024) and RV effective arterial elastance (P = 0.052). On myography, aortic segments from Sickle-to-CD47KO chimeras showed improved relaxation to acetylcholine. We hypothesized that, in SCD, TSP1-CD47 signaling promotes PH, in part, by increasing reactive oxygen species (ROS) generation. In human pulmonary artery endothelial cells, treatment with TSP1 stimulated ROS generation, which was abrogated by CD47 blockade. Explanted lungs of CD47KO chimeras had less vascular congestion and a smaller oxidative footprint. Our results show that genetic absence of CD47 ameliorates SCD-associated PH, which may be due to decreased ROS levels. Modulation of TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH.

The role of CD47 in pathogenesis and treatment of renal ischemia reperfusion injury.

Ischemia reperfusion (IR) injury is a process defined by the temporary loss of blood flow and tissue perfusion followed later by restoration of the same. Brief periods of IR can be tolerated with little permanent deficit, but sensitivity varies for different target cells and tissues. Ischemia reperfusion injuries have multiple causes including peripheral vascular disease and surgical interventions that disrupt soft tissue and organ perfusion as occurs in general and reconstructive surgery. Ischemia reperfusion injury is especially prominent in organ transplantation where substantial effort has been focused on protecting the transplanted organ from the consequences of IR. A number of factors mediate IR injury including the production of reactive oxygen species and inflammatory cell infiltration and activation. In the kidney, IR injury is a major cause of acute injury and secondary loss of renal function. Transplant-initiated renal IR is also a stimulus for innate and adaptive immune-mediated transplant dysfunction. The cell surface molecule CD47 negatively modulates cell and tissue responses to stress through limitation of specific homeostatic pathways and initiation of cell death pathways. Herein, a summary of the maladaptive activities of renal CD47 will be considered as well as the possible therapeutic benefit of interfering with CD47 to limit renal IR.