Deciphering tumour tissue organization by 3D electron microscopy and machine learning.

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumour tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also found anatomical connections between the blood capillary and the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for future investigations in the emerging onconanotomy field.

CD154 Induces Interleukin-6 Secretion by Kidney Tubular Epithelial Cells under Hypoxic Conditions: Inhibition by Chloroquine.

Inflammation is a major contributor to tubular epithelium injury in kidney disorders, and the involvement of blood platelets in driving inflammation is increasingly stressed. CD154, the ligand of CD40, is one of the mediators supporting platelet proinflammatory properties. Although hypoxia is an essential constituent of the inflammatory reaction, if and how platelets and CD154 regulate inflammation in hypoxic conditions remain unclear. Here, we studied the control by CD154 of the proinflammatory cytokine interleukin- (IL-) 6 secretion in short-term oxygen (O2) deprivation conditions, using the HK-2 cell line as a kidney tubular epithelial cell (TEC) model. IL-6 secretion was markedly stimulated by CD154 after 1 to 3 hours of hypoxic stress. Both intracellular IL-6 expression and secretion were stimulated by CD154 and associated with a strong upregulation of IL-6 mRNA and increased transcription. Searching for inhibitors of CD154-mediated IL-6 production by HK-2 cells in hypoxic conditions, we observed that chloroquine, a drug that has been repurposed as an anti-inflammatory agent, alleviated this induction. Therefore, CD154 is a potent early stimulus for IL-6 secretion by TECs in O2 deprivation conditions, a mechanism likely to take part in the deleterious inflammatory consequences of platelet activation in kidney tubular injury. The inhibition of CD154-induced IL-6 production by chloroquine suggests the potential usefulness of this drug as a therapeutic adjunct in conditions associated with acute kidney injury.

A Role for CD154, the CD40 Ligand, in Granulomatous Inflammation.

Granulomatous inflammation is a distinctive form of chronic inflammation in which predominant cells include macrophages, epithelioid cells, and multinucleated giant cells. Mechanisms regulating granulomatous inflammation remain ill-understood. CD154, the ligand of CD40, is a key mediator of inflammation. CD154 confers a proinflammatory phenotype to macrophages and controls several macrophagic functions. Here, we studied the contribution of CD154 in a mouse model of toxic liver injury with carbon tetrachloride and a model of absorbable suture graft. In both models, granulomas are triggered in response to endogenous persistent liver calcified necrotic lesions or by grafted sutures. CD154-deficient mice showed delayed clearance of carbon tetrachloride-induced liver calcified necrotic lesions and impaired progression of suture-induced granuloma. In vitro, CD154 stimulated phagocytosis of opsonized erythrocytes by macrophages, suggesting a potential mechanism for the altered granulomatous inflammation in CD154KO mice. These results suggest that CD154 may contribute to the natural history of granulomatous inflammation.

CD40 signaling and hepatic steatosis: Unanticipated links.

Obesity predisposes to an increased risk of nonalcoholic fatty liver disease (NAFLD). Hepatic steatosis is the key pathological feature of NAFLD and has emerged as a metabolic disorder in which innate and adaptive arms of the immune response play a central role in disease pathogenesis. Recent studies have revealed unexpected relationships between CD40 signaling and hepatic steatosis in high fat diet rodent models. CD154, the ligand of CD40, is a mediator of inflammation and controls several critical events of innate and adaptive immune responses. In the light of these reports, we discuss potential links between CD40 signaling and hepatic steatosis in NAFLD.

CD154 Induces Matrix Metalloproteinase-9 Secretion in Human Podocytes.

Matrix remodeling is a key feature of glomerulosclerosis secondary to diabetes or hypertension. Podocytes contribute to glomerular basement membrane (GBM) turnover by producing matrix components and matrix remodelling enzymes, including matrix metalloproteinases (MMPs). The CD40/CD154 signaling pathway modulates matrix remodeling through the synthesis of MMPs and tissue inhibitors of MMPs. Platelets are a primary blood reservoir of CD154. Here we studied, the impact of the CD154/CD40 pathway on MMP-9 expression by cultured human podocytes. The role of CD40/CD154 was evaluated upon exposure of podocytes to recombinant human CD154 (rhCD154) or activated platelet supernatants from healthy human subjects. We first showed by protein and mRNA expression that CD40 was synthesized by podocytes and detectable on kidney tissue sections. CD40 expression was acquired during podocyte differentiation and enhanced upon exposure to rhCD154. In podocytes, rhCD154 induced an increase of MMP-9 production as shown by RT-PCR, Western blot and and gelatin zymography. Activated platelet supernatants induced MMP-9 mRNA synthesis in podocytes, an effect reduced by anti-CD40 antibody. Our results underscore a potential role for platelets through the CD40/CD154 signaling pathway in the control of GBM synthesis and degradation, via its regulatory role on MMP-9 production. CD154 secretion by activated platelets may contribute to GBM alterations in proteinuric nephropathies. J. Cell. Biochem. 117: 2737-2747, 2016. © 2016 Wiley Periodicals, Inc.

Beneficial Effect of Covalently Grafted α-MSH on Endothelial Release of Inflammatory Mediators for Applications in Implantable Devices.

Intravascular devices for continuous glucose monitoring are promising tools for the follow up and treatment of diabetic patients. Limiting the inflammatory response to the implanted devices in order to achieve better biocompatibility is a critical challenge. Herein we report on the production and the characterization of gold surfaces covalently derivatized with the peptide α-alpha-melanocyte stimulating hormone (α-MSH), with a quantifiable surface density. In vitro study demonstrated that the tethered α-MSH is able to decrease the expression of an inflammatory cytokine produced by endothelial cells.

New frontiers for platelet CD154.

The role of platelets extends beyond hemostasis. The pivotal role of platelets in inflammation has shed new light on the natural history of conditions associated with acute or chronic inflammation. Beyond the preservation of vascular integrity, platelets are essential to tissue homeostasis and platelet-derived products are already used in the clinics. Unanticipated was the role of platelets in the adaptative immune response, allowing a renewed conceptual approach of auto-immune diseases. Platelets are also important players in cancer growth and dissemination. Platelets fulfill most of their functions through the expression of still incompletely characterized membrane-bound or soluble mediators. Among them, CD154 holds a peculiar position, as platelets represent a major source of CD154 and as CD154 contributes to most of these new platelet attributes. Here, we provide an overview of some of the new frontiers that the study of platelet CD154 is opening, in inflammation, tissue homeostasis, immune response, hematopoiesis and cancer.

IQGAP1 interacts with components of the slit diaphragm complex in podocytes and is involved in podocyte migration and permeability in vitro.

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.

A protective role for CD154 in hepatic steatosis in mice.

UNLABELLED: Inflammation and lipid metabolism pathways are linked, and deregulation of this interface may be critical in hepatic steatosis. The importance of the dialog between inflammatory signaling pathways and the unfolded protein response (UPR) in metabolism has been underlined. Herein, we studied the role of CD154, a key mediator of inflammation, in hepatic steatosis. To this end, Balb/c mice, wild-type or deficient in CD154 (CD154KO), were fed a diet rich in olive oil. In vitro, the effect of CD154 was studied on primary hepatocyte cultures and hepatocyte-derived cell lines. Results showed that CD154KO mice fed a diet rich in olive oil developed hepatic steatosis associated with reduced apolipoprotein B100 (apoB100) expression and decreased secretion of very low-density lipoproteins. This phenotype correlated with an altered UPR as assessed by reduced X-Box binding protein-1 (XBP1) messenger RNA (mRNA) splicing and reduced phosphorylation of eukaryotic initiation factor 2α. Altered UPR signaling in livers of CD154KO mice was confirmed in tunicamycin (TM) challenge experiments. Treatment of primary hepatocyte cultures and hepatocyte-derived cell lines with soluble CD154 increased XBP1 mRNA splicing in cells subjected to either oleic acid (OA) or TM treatment. Moreover, CD154 reduced the inhibition of apoB100 secretion by HepG2 cells grown in the presence of high concentrations of OA, an effect suppressed by XBP1 mRNA silencing and in HepG2 cells expressing a dominant negative form of inositol requiring ER-to-nucleus signaling protein-1. The control of the UPR by CD154 may represent one of the mechanisms involved in the pathophysiology of hepatic steatosis. CONCLUSION: Our study identifies CD154 as a new mediator of hepatic steatosis.

The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis.

OBJECTIVES: Altered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease. METHODS: A cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied. RESULTS: Platelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation. CONCLUSIONS: Platelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.

Magnetic targeting of magnetoliposomes to solid tumors with MR imaging monitoring in mice: feasibility.

PURPOSE: To establish the feasibility of magnetoliposome tumor targeting with an extracorporeal magnet. MATERIALS AND METHODS: Animal experiments were performed in compliance with Institut National de la Santé Et de la Recherche Médicale animal protection guidelines and were approved by local government authorities. Magnetophoresis was used to measure the velocity of magnetoliposomes constituted of polyethylene glycol-lipids and anionic maghemite nanocrystals in a calibrated magnetic field in vitro. For in vivo studies, 38 male Swiss nude mice bearing a PC3 human prostate carcinoma tumor in each flank received an intravenous injection of magnetoliposomes (n = 27), saline (n = 9), or nonencapsulated superparamagnetic particles (n = 2) after a small magnet with a magnetic field of 0.3 T and a field gradient of 11 T/m was fixed to the skin above one tumor. The animals were examined at magnetic resonance (MR) imaging with eight different sequences, iron doses (13 mice), and magnet-application durations (12 mice). Their excised tumors were then stained with Perls Prussian blue and hematoxylin-eosin and were examined histologically. With use of the paired Student t test, signal intensity, tumor surface enhancement, and number of stained cells were compared between the control and magnet-exposed tumors to determine significant differences (P

Activation of mesangial cells by platelets in systemic lupus erythematosus via a CD154-dependent induction of CD40.

BACKGROUND: Platelets are potential contributors to glomerular injury via the release of chemotactic and/or mitogenic mediators upon activation or through direct CD154/CD40-dependent interaction with cell components of the glomerulus. We examined whether platelets could activate mesangial cells and the potential role of the platelet-associated CD154. METHODS: Thrombin-activated platelets from systemic lupus erythematosus (SLE) patients or from disease or healthy controls were grown with human mesangial cells in the presence or not of a neutralizing anti-CD154 antibody either in contact or in a noncontact setting, the platelets and mesangial cells being separated by a pore size semipermeable membrane. The induction of mesangial cell surface antigens was assayed by flow cytometry. The quantification of mesangial cell proliferation was performed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the production of transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor (PDGF) and soluble CD40 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Activated platelets from patients with SLE could induce an up-regulation of the expression of CD40 on mesangial cells with a concomitant release of soluble CD40. This induction required a direct contact between platelets and mesangial cells and was dependent upon the platelet-associated CD154. Pathologic consequences of the up-regulation of CD40 were a CD40-dependent stimulation of the proliferation of mesangial cells and a CD40-dependent increased production of TGF-beta1 by these cells. CONCLUSION: Platelets from patients with SLE can activate mesangial cells through CD40/CD154 interactions, leading to an induction of proliferation of the mesangial cells and an enhanced production of TGF-beta1, a profibrotic cytokine.

[Platelet-associated CD154: a new interface in haemostasis and in the inflammatory reaction]. / Le CD154 plaquettaire: une nouvelle interface dans l'hémostase et la réaction inflammatoire.

Blood platelets play a crucial part in the blood clotting process by forming the platelet plug. Recent evidence indicates that they are likely to play a key role in the inflammatory reaction via CD154/CD40 interactions. CD40 was known to be widely expressed, for instance on cells of the vasculature including endothelial cells, smooth muscle cells and macrophages. It was also known that the triggering of CD40 on these cells led to the acquisition of an activated pro-inflammatory and pro-coagulant phenotype. It was subsequently shown that platelets express CD154 which is cryptic in unstimulated platelets but is expressed at the platelet surface upon platelet activation. When expressed at the platelet surface and exposed to CD40-expressing vascular cells, the platelet-associated CD154 triggers a variety of pro-inflammatory and pro-coagulant responses including induction of adhesion receptors, release of cytokines and chemokines, induction of tissue factor and of metalloproteinases. Platelet-associated CD154 is also involved in platelet/platelet interactions during platelet aggregation. Furthermore, in vivo models have emphasized the critical role of the platelet-associated CD154 in the progression of atherosclerotic disease and in the stabilization of arterial thrombi. Recent data show that CD40-bearing cells involved in fibrosis such as hepatic stellate cells and glomerular mesangial cells also respond to platelet-associated CD154, thus suggesting a new mechanism by which platelets may be instrumental in the inflammatory diseases of the liver or the kidney. Finally, platelet-associated CD154 has been shown to have immune competence both in vitro and in vivo, observations that open new fields of research on the potential implications of platelets in the immune response and auto-immune diseases.

Platelet-associated CD154 in immune thrombocytopenic purpura.

CD40-ligand (CD154) is expressed on activated CD4+ T lymphocytes and is essential for the T cell-dependent activation of B lymphocytes. CD154 is also expressed at the activated platelet surface. In this study, we show that platelet-associated CD154 is increased in immune thrombocytopenic purpura (ITP), a disease characterized by an autoimmune response against proteins of the platelet membrane. CD154 and its messenger RNA were also present in increased amounts in the megakaryocytes of patients with ITP. We found that platelet-associated CD154 is competent to induce the CD40-dependent proliferation of B lymphocytes, and we observed an in vitro CD154-dependent production of antibodies to the GPIIb/IIIa complex (integrin alphaIIbbeta3) when platelets and peripheral blood B lymphocytes from ITP patients with circulating anti-GPIIb/IIIa antibody were cultured together. Therefore, platelet-associated CD154 expression is increased in ITP and is able to drive the activation of autoreactive B lymphocytes in this disease.

In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver.

PURPOSE: To evaluate in vivo magnetic resonance (MR) imaging with a conventional 1.5-T system for depiction and tracking of intravascularly injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs). MATERIALS AND METHODS: This study was conducted in accordance with French law governing animal research and met guidelines for animal care and use. Rat MSCs were labeled with SPIO and transfection agent. Relaxation rates at 1.5 T, cell viability, proliferation, differentiation capacity, and labeling stability were assessed in vitro as a function of SPIO concentration. MSCs were injected into renal arteries of healthy rats (labeled cells in four, unlabeled cells in two) and portal veins of rats treated with carbon tetrachloride to induce centrolobular liver necrosis (labeled cells and unlabeled cells in two each). Follow-up serial T2*-weighted gradient-echo MR imaging and R2* mapping were performed. MR imaging findings were compared histologically. RESULTS: SPIO labeling caused a strong R2* effect that increased linearly with iron dose; R2* increase for cells labeled for 48 hours with 50 microg of iron per milliliter was 50 sec(-1) per million cells per milliliter. R2* was proportional to iron load of cells. SPIO labeling did not affect cell viability (P > .27). Labeled cells were able to differentiate into adipocytes and osteocytes. Proliferation was substantially limited for MSCs labeled with 100 microg Fe/mL or greater. Label half-life was longer than 11 days. In normal kidneys, labeled MSCs caused signal intensity loss in renal cortex. After labeled MSC injection, diseased liver had diffuse granular appearance. Cells were detected for up to 7 days in kidney and 12 days in liver. Signal intensity loss and fading over time were confirmed with serial R2* mapping. At histologic analysis, signal intensity loss correlated with iron-loaded cells, primarily in renal glomeruli and hepatic sinusoids; immunohistochemical analysis results confirmed these cells were MSCs. CONCLUSION: MR imaging can aid in monitoring of intravascularly administered SPIO-labeled MSCs in vivo in kidney and liver.

In vivo studies of translational repression mediated by the granulocyte-macrophage colony-stimulating factor AU-rich element.

The AU-rich element (ARE) controls the turnover of many unstable mRNAs and their translation. The granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE is known to be a destabilizing element, but its role in translation remains unclear. Here we studied in vivo the role of the GM-CSF ARE on the mRNA and protein expressions of an enhanced green fluorescent protein reporter gene. The GM-CSF ARE had a repressor effect on translation independently of its effect on mRNA levels. In the context of an internal ribosome entry site, the GM-CSF ARE still repressed translation but was no longer functional as a destabilizing element. Gel retardation assays showed that poly(A)-binding protein is displaced from the poly(A) tail when the ARE is present in the 3'-untranslated region. These data suggest that the GM-CSF ARE controls translation and mRNA decay by interfering with poly(A)-binding protein-mediated mRNA circularization.

Flt3-ligand induces adhesion of haematopoietic progenitor cells via a very late antigen (VLA)-4- and VLA-5-dependent mechanism.

The adhesion of haematopoietic progenitor cells (HPC) to the bone marrow microenvironment is a process regulated by cytokines. In this study, we have shown that flt3-ligand (FL), a growth factor that controls early haematopoiesis, regulated the function and expression of the beta-1 integrins, very late antigen (VLA)-4 and VLA-5 on HPC. The modulation of the adhesiveness of HPC by FL was studied by adhesion assays on umbilical vein endothelial cells (HUVEC). Stimulation by FL induced two peaks of increased adhesiveness of HPC. The first peak was at around 30 min and was mechanistically related to an activation of the beta-1 integrins, mainly VLA-4 and VLA-5. The second peak was at around 12 h and was related to increased expression of VLA-4 and VLA-5. The control of HPC adhesiveness by FL is a previously unreported property of FL that may be important for the homing and the retention of flt3-expressing HPC within the bone marrow microenvironment.

Increased soluble and platelet-associated CD40 ligand in essential thrombocythemia and reactive thrombocytosis.

CD40 ligand (CD40L) is expressed on activated CD4(+) T lymphocytes and at the activated platelet surface. A circulating soluble form of CD40L (sCD40L) is generated by the way of a proteolytic cleavage. We measured sCD40L in the plasma of either healthy subjects; patients with inflammatory disorders and low, normal, or high platelet count (reactive thrombocytosis); or patients with essential thrombocythemia (ET). A tight correlation was found between the platelet count and plasma sCD40L. ET patients had the highest levels of sCD40L. Platelet-associated CD40L was increased in ET and reactive thrombocytosis, conditions associated with increased platelet regeneration. Platelet-associated CD40L was released upon platelet activation. In conclusion, platelets appear as a reservoir of CD40L that may be a major contributor to circulating sCD40L. Platelet-associated CD40L may be a potential marker of platelet regeneration.

Iron particle labeling of haematopoietic progenitor cells: an in vitro study.

We present a method for labeling bone marrow haematopoietic progenitor cells with iron particles. Labeling was assessed by magnetic resonance imaging and electron microscopy. Labeling with iron particles could allow the following by imaging techniques of haematopoietic cells in physiologic and pathologic conditions such as the engraftment of haematopoietic progenitor cells or the migration of myelomonocytic cells in inflammatory diseases.