Host-device interactions: exposure of lung epithelial cells and fibroblasts to nickel, titanium, or nitinol affect proliferation, reactive oxygen species production, and cellular signaling.

Endoscopic implantation of medical devices for the treatment of lung diseases, including airway stents, unidirectional valves and coils, is readily used to treat central airway disease and emphysema. However, granulation and fibrotic tissue formation impairs treatment effectiveness. To date little is known about the interaction between implanted devices, often made from metals, such as nickel, titanium or nitinol, and cells in the airways. Here, we study the response of lung epithelial cells and fibroblasts to implant device materials. The adhesion and proliferation of bronchial epithelial cells and lung fibroblasts upon exposure to 10 × 3 × 1 mm pieces of nickel, titanium or nitinol is examined using light and scanning electron microscopy. Pro-inflammatory cytokine mRNA expression and release, signaling kinase activity and intracellular free radical production are assessed. Nitinol, and to a lesser extent nickel and titanium, surfaces support the attachment and growth of lung epithelial cells. Nitinol induces a rapid and significant alteration of kinase activity. Cells directly exposed to nickel or titanium produce free radicals, but those exposed to nitinol do not. The response of lung epithelial cells and fibroblasts depends on the metal type to which they are exposed. Nitinol induces cellular surface growth and the induction of kinase activity, while exposure of lung epithelial cells to nickel and titanium induces free radical production, but nitinol does not.

Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion.

BACKGROUND AND AIMS: The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 ( SMLR1 ), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. APPROACH AND RESULTS: To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 -LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 -LKO mice versus controls. CONCLUSIONS: We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis.

Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.

Activation of a passive, mesoporous silica nanoparticle layer through attachment of bacterially-derived carbon-quantum-dots for protection and functional enhancement of probiotics.

Probiotic bacteria employed for food supplementation or probiotic-assisted antibiotic treatment suffer from passage through the acidic gastro-intestinal tract and unintended killing by antibiotics. Carbon-quantum-dots (CQDs) derived from bacteria can inherit different chemical groups and associated functionalities from their source bacteria. In order to yield simultaneous, passive protection and enhanced, active functionality, we attached CQDs pyrolytically carbonized at 220 â€‹°C from Lactobacillus acidophilus or Escherichia coli to a probiotic strain (Bifidobacterium infantis) using boron hydroxyl-modified, mesoporous silica nanoparticles as an intermediate encapsulating layer. Fourier-transform-infrared-spectroscopy, X-ray-photoelectron-spectroscopy and scanning-electron-microscopy were employed to demonstrate successful encapsulation of B. infantis by silica nanoparticles and subsequent attachment of bacterially-derived CQDs. Thus encapsulated B. infantis possessed a negative surface charge and survived exposure to simulated gastric fluid and antibiotics better than unencapsulated B. infantis. During B. infantis assisted antibiotic treatment of intestinal epithelial layers colonized by E. coli, encapsulated B. infantis adhered and survived in higher numbers on epithelial layers than B. infantis without encapsulation or encapsulated with only silica nanoparticles. Moreover, higher E. coli killing due to increased reactive-oxygen-species generation was observed. In conclusion, the active, protective encapsulation described enhanced the probiotic functionality of B. infantis, which might be considered as a first step towards a fully engineered, probiotic nanoparticle.

Biocompatibility and degradation comparisons of four biodegradable copolymeric osteosynthesis systems used in maxillofacial surgery: A goat model with four years follow-up.

Applying biodegradable osteosyntheses avoids the disadvantages of titanium osteosyntheses. However, foreign-body reactions remain a major concern and evidence of complete resorption is lacking. This study compared the physico-chemical properties, histological response and radiographs of four copolymeric biodegradable osteosynthesis systems in a goat model with 48-months follow-up. The systems were implanted subperiosteally in both tibia and radius of 12 Dutch White goats. The BioSorb FX [poly(70LLA-co-30DLLA)], Inion CPS [poly([70-78.5]LLA-co-[16-24]DLLA-co-4TMC)], SonicWeld Rx [poly(DLLA)], LactoSorb [poly(82LLA-co-18GA)] systems and a negative control were randomly implanted in each extremity. Samples were assessed at 6-, 12-, 18-, 24-, 36-, and 48-month follow-up. Surface topography was performed using scanning electron microscopy (SEM). Differential scanning calorimetry and gel permeation chromatography were performed on initial and explanted samples. Histological sections were systematically assessed by two blinded researchers using (polarized) light microscopy, SEM and energy-dispersive X-ray analysis. The SonicWeld Rx system was amorphous while the others were semi-crystalline. Foreign-body reactions were not observed during the complete follow-up. The SonicWeld Rx and LactoSorb systems reached bone percentages of negative controls after 18 months while the BioSorb Fx and Inion CPS systems reached these levels after 36 months. The SonicWeld Rx system showed the most predictable degradation profile. All the biodegradable systems were safe to use and well-tolerated (i.e., complete implant replacement by bone, no clinical or histological foreign body reactions, no [sterile] abscess formation, no re-interventions needed), but nanoscale residual polymeric fragments were observed at every system's assessment.

Identification of damage associated molecular patterns and extracellular matrix proteins as major constituents of the surface proteome of lung implantable silicone/nitinol devices.

Lung implantable devices have been widely adopted as mechanical interventions for a wide variety of pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation at the implant sites. This study aimed to explore the lung-device interface by identifying the adhered proteome on lung devices explanted from patients with severe emphysema. In this study, scanning electron microscopy is used to visualize the adhesion of cells and proteins to silicone and nitinol surfaces of explanted endobronchial valves. By applying high-resolution mass-spectrometry, the surface proteome of eight explanted valves is characterized, identifying 263 unique protein species to be mutually adsorbed on the valves. This subset is subjected to gene enrichment analysis, matched with known databases and further validated using immunohistochemistry. Enrichment analyses reveal dominant clusters of functionally-related ontology terms associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Matching results show that extracellular matrix proteins and damage-associated molecular patterns are cardinal in the formation of the surface proteome. This is the first study investigating the composition of the adhered proteome on explanted lung devices, setting the groundwork for hypothesis generation and further exploration. STATEMENT OF SIGNIFICANCE: This is the first study investigating the composition of the adhered proteome on explanted lung devices. Lung implantable devices have been widely adopted as mechanical interventions for pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation around the implant sites. We identified the adhered proteome on explanted lung devices using several techniques. We identified 263 unique protein species to be mutually adsorbed on explanted lung devices. Pathway analyses revealed that these proteins are associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Furthermore, we identified that especially extracellular matrix proteins and damage-associated molecular patterns were cardinal in the formation of the surface proteome.

Sample preparation for energy dispersive X-ray imaging of biological tissues.

Traditional electron microscopy (EM) can be complemented with analytical EM to increase objective sample information enabling feature identification. Energy dispersive X-ray (EDX) imaging provides semi-quantitative elemental composition of the sample with high spatial resolution (~10nm) in ultrathin sections. However, EDX imaging of biological samples is still challenging as a routine method because many elements are at the detection limit for this technique. Moreover, samples undergo extensive preparation before analysis, which can introduce disruptive X-ray cross-talk or artifacts. EDX data can, for instance, be skewed by (i) osmium interference with endogenous phosphorus, (ii) chlorine present in EPON-embedded tissues, (iii) lead interference with endogenous sulfur, and (iv) potential spectral overlaps with grid material, contrast agents, and the in-microscope sample holder. Here, we highlight how to circumvent these potential pitfalls and outline how we approach sample preparation and analysis for detection of different elements of interest. Utilization of well-considered a priori sample preparation techniques will best ensure conclusive EDX experiments.

Reversible thrombocytopenia during hibernation originates from storage and release of platelets in liver sinusoids.

Immobility is a risk factor for thrombosis due to low blood flow, which may result in activation of the coagulation system, recruitment of platelets and clot formation. Nevertheless, hibernating animals-who endure lengthy periods of immobility-do not show signs of thrombosis throughout or after hibernation. One of the adaptations of hemostasis in hibernators consists of a rapidly reversible reduction of the number of circulating platelets during torpor, i.e., the hibernation phase with reduction of metabolic rate, low blood flow and immobility. It is unknown whether these platelet dynamics in hibernating hamsters originate from storage and release, as suggested for ground squirrel, or from breakdown and de novo synthesis. A reduction in detaching forces due to low blood flow can induce reversible adhesion of platelets to the vessel wall, which is called margination. Here, we hypothesized that storage-and-release by margination to the vessel wall induces reversible thrombocytopenia in torpor. Therefore, we transfused labeled platelets in hibernating Syrian hamster (Mesocricetus auratus) and platelets were analyzed using flow cytometry and electron microscopy. The half-life of labeled platelets was extended from 20 to 30 h in hibernating animals compared to non-hibernating control hamsters. More than 90% of labeled platelets were cleared from the circulation during torpor, followed by emergence during arousal which supports storage-and-release to govern thrombocytopenia in torpor. Furthermore, the low number of immature platelets, plasma level of interleukin-1α and normal numbers of megakaryocytes in bone marrow make platelet synthesis or megakaryocyte rupture via interleukin-1α unlikely to account for the recovery of platelet counts upon arousal. Finally, using large-scale electron microscopy we revealed platelets to accumulate in liver sinusoids, but not in spleen or lung, during torpor. These results thus demonstrate that platelet dynamics in hibernation are caused by storage and release of platelets, most likely by margination to the vessel wall in liver sinusoids. Translating the molecular mechanisms that govern platelet retention in the liver, may be of major relevance for hemostatic management in (accidental) hypothermia and for the development of novel anti-thrombotic strategies.

Pancreatic beta cell autophagy is impaired in type 1 diabetes.

AIMS/HYPOTHESIS: Pancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. METHODS: Pancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5). RESULTS: We observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3-LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3-LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002). CONCLUSIONS/INTERPRETATION: We provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.

Correction to: Neodymium as an alternative contrast for uranium in electron microscopy.

After publication of our article, it has come to our attention that the legend.

Vps13 is required for timely removal of nurse cell corpses.

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.

Large-scale electron microscopy database for human type 1 diabetes.

Autoimmune ß-cell destruction leads to type 1 diabetes, but the pathophysiological mechanisms remain unclear. To help address this void, we created an open-access online repository, unprecedented in its size, composed of large-scale electron microscopy images ('nanotomy') of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD; www.nanotomy.org). Nanotomy allows analyses of complete donor islets with up to macromolecular resolution. Anomalies we found in type 1 diabetes included (i) an increase of 'intermediate cells' containing granules resembling those of exocrine zymogen and endocrine hormone secreting cells; and (ii) elevated presence of innate immune cells. These are our first results of mining the database and support recent findings that suggest that type 1 diabetes includes abnormalities in the exocrine pancreas that may induce endocrine cellular stress as a trigger for autoimmunity.

Neodymium as an alternative contrast for uranium in electron microscopy.

Uranyl acetate is the standard contrasting agent in electron microscopy (EM), but it is toxic and radioactive. We reasoned neodymium acetate might substitute uranyl acetate as a contrasting agent, and we find that neodymium acetate indeed can replace uranyl acetate in several routine applications. Since neodymium acetate is not toxic, not radioactive and easy to use, we foresee neodymium will replace uranyl in many EM sample preparation applications worldwide.

Loss of MYO5B expression deregulates late endosome size which hinders mitotic spindle orientation.

Recycling endosomes regulate plasma membrane recycling. Recently, recycling endosome-associated proteins have been implicated in the positioning and orientation of the mitotic spindle and cytokinesis. Loss of MYO5B, encoding the recycling endosome-associated myosin Vb, is associated with tumor development and tissue architecture defects in the gastrointestinal tract. Whether loss of MYO5B expression affects mitosis is not known. Here, we demonstrate that loss of MYO5B expression delayed cytokinesis, perturbed mitotic spindle orientation, led to the misorientation of the plane of cell division during the course of mitosis, and resulted in the delamination of epithelial cells. Remarkably, the effects on spindle orientation, but not cytokinesis, were a direct consequence of physical hindrance by giant late endosomes, which were formed in a chloride channel-sensitive manner concomitant with a redistribution of chloride channels from the cell periphery to late endosomes upon loss of MYO5B. Rab7 availability was identified as a limiting factor for the development of giant late endosomes. In accordance, increasing rab7 availability corrected mitotic spindle misorientation and cell delamination in cells lacking MYO5B expression. In conclusion, we identified a novel role for MYO5B in the regulation of late endosome size control and identify the inability to control late endosome size as an unexpected novel mechanism underlying defects in cell division orientation and epithelial architecture.

Exosomes derived from monocytes and from endothelial cells mediate monocyte and endothelial cell activation under high d-glucose conditions.

Diabetes mellitus type 2 (DMT2) is characterized by hyperglycemia and associated with low grade inflammation affecting both endothelial cells and monocytes. Exosomes are nanovesicles, allow communication between endothelial cells and monocytes and have been associated with diabetic complications. In this study we evaluated whether high glucose can activate monocytes and endothelial cells and whether exosomes play a role in this activation. Moreover, we studied whether endothelial cells and monocytes communicate with each other via exosomes under high and basal glncubation. In the first experiment, monomac 6 cells (MM6) were exposed to high glucose (HG; 25 mmol/L) or to exosomes from MM6 exposed to HG (exoMM6-HG) or basal glucose (5.5 mmol/L) (exoMM6-BG). In the second experiment, MM6 were exposed to exosomes from human umbilical vein endothelial cells (HUVECs) and HUVECs to exosomes from MM6. In the third experiment, MM6 and HUVECs were exposed to a mixture of exosomes from MM6 and HUVECs (exoMix). Cell activation was evaluated by measuring the protein surface expression of intracellular adhesion molecule-1 (ICAM-1) by flow cytometry. HG increased ICAM-1 expression in MM6 and monocytic exosomes from HG or BG shown similar effect in HG and BG MM6 cells. Exosomes from HUVECs increased ICAM-1 expression in MM6 cells, incubated under HG or BG, while also exosomes from MM6 increased ICAM-1 expression in HUVECs incubated under HG or BG. The combination of exosomes from both cell types (exoMixHG or exoMixBG) also increased ICAM-1 expression in both type cells in most conditions. However, the exoMixBG reversed the effect of HG in both MM6 and HUVECs. Our results show that HG activated monocytes and endothelial cells and that exosomes play a role in this HG-induced cell ICAM-1 expression. We hypothesize that during DMT2, exosomes may act as a communication mechanism between monocytes and endothelial cells, inducing and maintaining activating of both cell types in the presence of high glucose.

Effect of Dentin Matrix Components on the Mineralization of Human Mesenchymal Stromal Cells.

IMPACT STATEMENT: This research has been conducted with the aim to contribute to the development of treatment modalities for the reconstruction of lost/damaged mineralized tissues. Currently, determining the most appropriate stromal cell population and signaling cues stands at the core of developing effective treatments. We provide new insights into the effect of innate inductive cues found in human dentin matrix components, on the osteogenic differentiation of various human stromal cell types. The effects of dentin extracellular matrix components on umbilical cord mesenchymal stromal cells have not been investigated before. The findings of this study could underpin translational research based on the development of techniques for mineralized tissue engineering and will be of great interest for the readership of Tissue Engineering Part A.

Supramolecular Packing and Macroscopic Alignment Controls Actuation Speed in Macroscopic Strings of Molecular Motor Amphiphiles.

Three-dimensional organized unidirectionally aligned and responsive supramolecular structures have much potential in adaptive materials ranging from biomedical components to soft actuator systems. However, to control the supramolecular structure of these stimuli responsive, for example photoactive, materials and control their actuation remains a major challenge. Toward the design of "artificial muscles", herein, we demonstrate an approach that allows hierarchical control of the supramolecular structure, and as a consequence its photoactuation function, by electrostatic interaction between motor amphiphiles (MA) and counterions. Detailed insight into the effect of various ions on structural parameters for self-assembly from nano- to micrometer scale in water including nanofiber formation and nanofiber aggregation as well as the packing structure, degree of alignment, and actuation speed of the macroscopic MA strings prepared from various metal chlorides solution, as determined by electronic microscopy, X-ray diffraction, and actuation speed measurements, is presented. Macroscopic MA strings prepared from calcium and magnesium ions provide a high degree of alignment and fast response photoactuation. By the selection of metal ions and chain length of MAs, the macroscopic MA string structure and function can be controlled, demonstrating the potential of generating multiple photoresponsive supramolecular systems from an identical molecular structure.

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells.

AIMS/HYPOTHESIS: The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis. METHODS: We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs). RESULTS: ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-κB dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1. CONCLUSIONS/INTERPRETATION: The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy.

Fetoplacental endothelial exosomes modulate high d-glucose-induced endothelial dysfunction.

INTRODUCTION: Gestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, which may be induced by hyperglycemia. We hypothesized that endothelial exosomes, which are extracellular nanovesicles affecting endothelial function, play a role in the high glucose (HG)-induced endothelial dysfunction. METHODS: Exosomes were isolated from HUVECs incubated with basal glucose (5.5 mmol/L; HUVEC- BG; exo-BG) and from HUVECs incubated with HG for 24 h (25 mmol/L; HUVEC-HG; exo-HG) in exosome-free medium. Exosomes were isolated and characterized by ultracentrifugation, sucrose gradient, electron microscopy, nanotracking analysis and Western blotting. HUVEC-BG and HUVEC-HG were exposed to exo-BG and exo-HG in two different concentrations: 5 µg and 1 µg exosome protein/mL. The exosomal effect on endothelial cell function was determined by wound healing assay, expression of endothelial nitric oxide synthase (eNOS), human cationic amino acid transporter type 1 (hCAT-1), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule type 1 (ICAM-1) by Western blotting, qPCR or flow cytometry. RESULTS: HG increased the exosomal release from HUVECs, endothelial wound healing and expression of phosphorylated (P∼Ser1177)-eNOS, hCAT-1, VEGF and ICAM-1. Exo-HG also increased endothelial cell wound healing, P∼Ser1177-eNOS, hCAT-1 and ICAM-1 expression in HUVEC-BG. Exo-BG reverted the effect of HG on endothelial cell wound healing and hCAT-1 mRNA expression to normal values. DISCUSSION: Our results show that HG may induce endothelial dysfunction in HUVECs and that exosomes from HUVEC-HG mimicked some of the effects of HG. This study contributes to the unraveling of the mechanism by which hyperglycemia affects the fetoplacental vasculature in GDM.

There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis is one of the major periodontal agents and it has been recently hailed as a potential cause of the autoimmune disease rheumatoid arthritis. In particular, the peptidylarginine deiminase enzyme of P. gingivalis (PPAD) has been implicated in the citrullination of certain host proteins and the subsequent appearance of antibodies against citrullinated proteins, which might play a role in the etiology of rheumatoid arthritis. The aim of this study was to investigate the extracellular localization of PPAD in a large panel of clinical P. gingivalis isolates. Here we show that all isolates produced PPAD. In most cases PPAD was abundantly present in secreted outer membrane vesicles (OMVs) that are massively produced by P. gingivalis, and to minor extent in a soluble secreted state. Interestingly, a small subset of clinical isolates showed drastically reduced levels of the OMV-bound PPAD and secreted most of this enzyme in the soluble state. The latter phenotype is strictly associated with a lysine residue at position 373 in PPAD, implicating the more common glutamine residue at this position in PPAD association with OMVs. Further, one isolate displayed severely restricted vesiculation. Together, our findings show for the first time that neither the major association of PPAD with vesicles, nor P. gingivalis vesiculation per se, are needed for P. gingivalis interactions with the human host.