P2X7 Receptor Regulates Collagen Expression in Human Intestinal Fibroblasts: Relevance in Intestinal Fibrosis.

Intestinal fibrosis is a common complication that affects more than 50% of Crohn´s Disease (CD) patients. There is no pharmacological treatment against this complication, with surgery being the only option. Due to the unknown role of P2X7 in intestinal fibrosis, we aim to analyze the relevance of this receptor in CD complications. Surgical resections from CD and non-Inflammatory Bowel Disease (IBD) patients were obtained. Intestinal fibrosis was induced with two different murine models: heterotopic transplant model and chronic-DSS colitis in wild-type and P2X7-/- mice. Human small intestine fibroblasts (HSIFs) were transfected with an siRNA against P2X7 and treated with TGF-ß. A gene and protein expression of P2X7 receptor was significantly increased in CD compared to non-IBD patients. The lack of P2X7 in mice provoked an enhanced collagen deposition and increased expression of several profibrotic markers in both murine models of intestinal fibrosis. Furthermore, P2X7-/- mice exhibited a higher expression of proinflammatory cytokines and a lower expression of M2 macrophage markers. Moreover, the transient silencing of the P2X7 receptor in HSIFs significantly induced the expression of Col1a1 and potentiated the expression of Col4 and Col5a1 after TGF-ß treatment. P2X7 regulates collagen expression in human intestinal fibroblasts, while the lack of this receptor aggravates intestinal fibrosis.

Autophagy in intestinal fibrosis: relevance in inflammatory bowel disease.

Chronic inflammation is often associated with fibrotic disorders in which an excessive deposition of extracellular matrix is a hallmark. Long-term fibrosis starts with tissue hypofunction and finally ends in organ failure. Intestinal fibrosis is not an exception, and it is a frequent complication of inflammatory bowel disease (IBD). Several studies have confirmed the link between deregulated autophagy and fibrosis and the presence of common prognostic markers; indeed, both up- and downregulation of autophagy are presumed to be implicated in the progression of fibrosis. A better knowledge of the role of autophagy in fibrosis may lead to it becoming a potential target of antifibrotic therapy. In this review we explore novel advances in the field that highlight the relevance of autophagy in fibrosis, and give special focus to fibrosis in IBD patients.

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

Mindfulness Prevents Depression and Psychopathology in Elderly People with Mild to Moderate Alzheimer's Disease: A Randomized Clinical Trial.

BACKGROUND: This longitudinal study addressed whether mindfulness practice prevents psychological and behavioral symptoms, especially mood disorders, in Alzheimer's disease (AD). OBJECTIVE: To assess the incidence of depression in the course of AD and to determine which non-pharmacological treatment (NPT) is most effective in preventing psychopathological symptoms. METHODS: We conducted a longitudinal, non-inferiority and equivalence randomized clinical trial, repeated-measures design, with a control group and three experimental treatments: mindfulness, cognitive stimulation, and relaxation. Each experimental group performed three weekly sessions for two years. The pharmacological treatment of all participants was donepezil (10 mg). Participants were patients with probable AD without diagnosed depression from the public neurology services of the Canary Health Service, Spain. Psychological evaluation was performed using the Geriatric Depression Scale (GDS), Hamilton Depression Rating Scale (HDRS), and Neuropsychiatric Inventory (NPI-Q). The statistical analysis included only patients who attended at least 75% of the sessions. A nonparametric, repeated-measures analysis was performed with Kruskal-Wallis H test and between-group differences with Mann-Whitney U test with Bonferroni correction (p < 0.008). Effect size was calculated with partial eta-squared. RESULTS: The results showed significant differences with large effect sizes (η2p>0.14) between mindfulness and the rest of the experimental groups as well as the control in the GDS, HDRS, and NPI-Q scales. CONCLUSION: Compared to the other experimental groups, only mindfulness prevented the onset of depression and other psychopathologies in early-stage AD. Based on its effectiveness in maintaining cognitive functions and preventing psychopathology, we recommend mindfulness as the first-choice NPT for mild to moderate AD.

Platelet Lipidome Fingerprint: New Assistance to Characterize Platelet Dysfunction in Obesity.

Obesity is associated with a pro-inflammatory and pro-thrombotic state that supports atherosclerosis progression and platelet hyper-reactivity. During the last decade, the platelet lipidome has been considered a treasure trove, as it is a source of biomarkers for preventing and treating different pathologies. The goal of the present study was to determine the lipid profile of platelets from non-diabetic, severely obese patients compared with their age- and sex-matched lean controls. Lipids from washed platelets were isolated and major phospholipids, sphingolipids and neutral lipids were analyzed either by gas chromatography or by liquid chromatography coupled to mass spectrometry. Despite a significant increase in obese patient's plasma triglycerides, there were no significant differences in the levels of triglycerides in platelets among the two groups. In contrast, total platelet cholesterol was significantly decreased in the obese group. The profiling of phospholipids showed that phosphatidylcholine and phosphatidylethanolamine contents were significantly reduced in platelets from obese patients. On the other hand, no significant differences were found in the sphingomyelin and ceramide levels, although there was also a tendency for reduced levels in the obese group. The outline of the glycerophospholipid and sphingolipid molecular species (fatty-acyl profiles) was similar in the two groups. In summary, these lipidomics data indicate that platelets from obese patients have a unique lipid fingerprint that may guide further studies and provide mechanistic-driven perspectives related to the hyperactivate state of platelets in obesity.

IFNγ-Treated Macrophages Induce EMT through the WNT Pathway: Relevance in Crohn's Disease.

BACKGROUND: Fibrosis is a common complication of Crohn's disease (CD) in which macrophages play a central role. Epithelial-mesenchymal transition (EMT) and the WNT pathway have been associated with fibrosis. We aim to analyse the relevance of the tissue microenvironment in macrophage phenotype and the EMT process. METHODS: Intestinal surgical resections are obtained from control and CD patients with stenotic or penetrating behaviour. Cytokine's expression, macrophage phenotype, EMT markers and WNT signalling pathway are determined by WB, RT-PCR, ELISA or Cytometry. U937 cells are treated with IFNγ, TNFα, IL1ß, IL4 or IL10 and co-cultured with HT29 cells and, in some cases, are treated with XAV939 or miFZD4. The expression of macrophage, EMT and WNT pathway markers in U937 or HT29 cells is analysed by WB or RT-PCR. RESULTS: IFNγ, WNT6, CD16 and CD86 are increased in the intestinal tissue of CD patients. IFNγ-treated U937 activated the EMT process and WNT pathway in HT29 cells, and the EMT process is mediated by FZD4. CONCLUSIONS: An IFNγ-rich microenvironment polarises macrophages, which induces EMT through the WNT pathway.

SUCNR1 Mediates the Priming Step of the Inflammasome in Intestinal Epithelial Cells: Relevance in Ulcerative Colitis.

Intestinal epithelial cells (IECs) constitute a defensive physical barrier in mucosal tissues and their disruption is involved in the etiopathogenesis of several inflammatory pathologies, such as Ulcerative Colitis (UC). Recently, the succinate receptor SUCNR1 was associated with the activation of inflammatory pathways in several cell types, but little is known about its role in IECs. We aimed to analyze the role of SUCNR1 in the inflammasome priming and its relevance in UC. Inflammatory and inflammasome markers and SUCNR1 were analyzed in HT29 cells treated with succinate and/or an inflammatory cocktail and transfected with SUCNR1 siRNA in a murine DSS model, and in intestinal resections from 15 UC and non-IBD patients. Results showed that this receptor mediated the inflammasome, priming both in vitro in HT29 cells and in vivo in a murine chronic DSS-colitis model. Moreover, SUNCR1 was also found to be involved in the activation of the inflammatory pathways NFкB and ERK pathways, even in basal conditions, since the transient knock-down of this receptor significantly reduced the constitutive levels of pERK-1/2 and pNFкB and impaired LPS-induced inflammation. Finally, UC patients showed a significant increase in the expression of SUCNR1 and several inflammasome components which correlated positively and significantly. Therefore, our results demonstrated a role for SUCNR1 in basal and stimulated inflammatory pathways in intestinal epithelial cells and suggested a pivotal role for this receptor in inflammasome activation in UC.

Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist.

BACKGROUND: CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. OBJECTIVES: To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2. METHODS: ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin-CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. RESULTS AND CONCLUSION: We developed a CLEC-2-podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)'2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets.

The bone marrow niche from the inside out: how megakaryocytes are shaped by and shape hematopoiesis.

Megakaryocytes (MKs), the largest of the hematopoietic cells, are responsible for producing platelets by extending and depositing long proplatelet extensions into the bloodstream. The traditional view of megakaryopoiesis describes the cellular journey from hematopoietic stem cells (HSCs) along the myeloid branch of hematopoiesis. However, recent studies suggest that MKs can be generated from multiple pathways, some of which do not require transit through multipotent or bipotent MK-erythroid progenitor stages in steady-state and emergency conditions. Growing evidence suggests that these emergency conditions are due to stress-induced molecular changes in the bone marrow (BM) microenvironment, also called the BM niche. These changes can result from insults that affect the BM cellular composition, microenvironment, architecture, or a combination of these factors. In this review, we explore MK development, focusing on recent studies showing that MKs can be generated from multiple divergent pathways. We highlight how the BM niche may encourage and alter these processes using different mechanisms of communication, such as direct cell-to-cell contact, secreted molecules (autocrine and paracrine signaling), and the release of cellular components (eg, extracellular vesicles). We also explore how MKs can actively build and shape the surrounding BM niche.

HIF-Overexpression and Pro-Inflammatory Priming in Human Mesenchymal Stromal Cells Improves the Healing Properties of Extracellular Vesicles in Experimental Crohn's Disease.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have therapeutic potential in the treatment of several immune disorders, including ulcerative colitis, owing to their regenerative and immunosuppressive properties. We recently showed that MSCs engineered to overexpress hypoxia-inducible factor 1-alpha and telomerase (MSC-T-HIF) and conditioned with pro-inflammatory stimuli release EVs (EVMSC-T-HIFC) with potent immunomodulatory activity. We tested the efficacy of EVMSC-T-HIFC to repolarize M1 macrophages (Mφ1) to M2-like macrophages (Mφ2-like) by analyzing surface markers and cytokines and performing functional assays in co-culture, including efferocytosis and T-cell proliferation. We also studied the capacity of EVMSC-T-HIFC to dampen the inflammatory response of activated endothelium and modulate fibrosis. Finally, we tested the therapeutic capacity of EVMSC-T-HIFC in an acute colitis model. EVMSC-T-HIFc induced the repolarization of monocytes from Mφ1 to an Mφ2-like phenotype, which was accompanied by reduced inflammatory cytokine release. EVMSC-T-HIFc-treated Mφ1 had similar effects of immunosuppression on activated peripheral blood mononuclear cells (PBMC) as Mφ2, and reduced the adhesion of PBMCs to activated endothelium. EVMSC-T-HIFc also prevented myofibroblast differentiation of TGF-ß-treated fibroblasts. Finally, administration of EVMSC-T-HIFc promoted healing in a TNBS-induced mouse colitis model in terms of preserving colon length and intestinal mucosa architecture and altering the ratio of Mφ1/ Mφ2 infiltration. In conclusion, EVMSC-T-HIFC have effective anti-inflammatory properties, making them potential therapeutic agents in cell free-based therapies for the treatment of Crohn's disease and likely other immune-mediated inflammatory diseases.

Virtual chromoendoscopy with iSCAN as an alternative method to dye-spray chromoendoscopy for dysplasia detection in long-standing colonic inflammatory bowel disease: a case-control study.

OBJECTIVE: Patients with long-standing colonic inflammatory bowel disease (cIBD) are at increased risk of developing colorectal cancer (CRC). Dye-spray chromoendoscopy (DCE) with targeted biopsies is the preferred technique for surveillance of dysplasia. Virtual chromoendoscopy (VCE) are arising to improve detection rates and adherence to surveillance guidelines, although its role is not yet well defined. The purpose of this study is to assess the effectiveness of VCE with iSCAN as an alternative method for dysplasia detection in cIBD. METHODS: Retrospective case-control study with 191 patients included, 98 in the DCE (Indigo carmine) group and 93 in the VCE (iSCAN, twin-mode 1-3) group. The dysplasia detection and the exploration time were analysed. A logistic regression analysis was performed to ascertain the factors related to colonic dysplasia. RESULTS: A total of 44 dysplastic lesions were detected in 21 (11%) patients. No differences were found in the per lesion and the per patient analysis (dysplastic versus non-dysplastic). Median withdrawal time was shorter in the VCE group than in the DCE group (9 min versus 14 min; p < .001). Location of lesions in the right colon was independently associated with an increased risk for colonic dysplasia (OR = 4.04, 95%CI 1.11-14.65; p = .034) after adjusting for age at inclusion, age at diagnosis, high risk for CRC and Kudo pit pattern. CONCLUSIONS: VCE with iSCAN presents a similar diagnostic performance to conventional DCE in the detection of colonic dysplasia in patients with long-standing cIBD. Furthermore, VCE with iSCAN is a less time-consuming surveillance alternative to DCE.

The PI3Kδ Inhibitor Idelalisib Diminishes Platelet Function and Shows Antithrombotic Potential.

BACKGROUND: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms. METHODS: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time. RESULTS: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug. CONCLUSION: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans.

Clinical Proteomics for the Analysis of Circulating Extracellular Vesicles.

In recent years, technical improvements in proteomics have allowed its rapid application for biomarker discovery, new drug target identification, and the study of disease progression and drug resistance. The clinical potential of circulating extracellular vesicles (EVs) as a source of biomarkers is one of the reasons why several research groups have recently applied proteomics to their study. A large variety of proteomic approaches such as gel-based proteomics and bottom-up and top-down mass spectrometry have been applied to the study of EVs. In this chapter, we will present basic protocols for gel-based and quantitative MS-based approaches applied to the study of EVs.

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults.

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential "developmental hemostatic mismatch risk" associated with transfusions containing plasma exosomes from adults.

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

Firm productivity gains in a period of slow trade liberalization: evidence from Brazil.

Existing literature recognizes the possible role of trade policy and firms' exposure to international trade as determinants of productivity. A strand of the literature sheds light on the effects of trade policy changes on firm-level productivity. Another strand studies the relationship between firms' trade status (exporting production or importing intermediates, but usually not both simultaneously) and firm-level TFP dynamics. However, the analyses that integrate both strands are scarce. This paper aims to disentangle the impact of input and output tariffs on firms' productivity. Further, it analyses whether the impact of changes in tariffs is conditioned by the trade status of the firm (exporting and/or importing). At difference to most previous papers, we carry out our analysis for a large developing country in a period of slow trade liberalization. Thus, in the empirical part, we use data from firms belonging to Brazilian industrial sectors (manufacturing and mining) during 2000-2008. After estimating total factor productivity (TFP) at the firm level using updated methodologies, we estimate both the impact of trade policy and firms' trade status on TFP dynamics. Our results suggest that trade liberalization (through reductions in input or output tariffs) increases TFP, being the effect associated to a reduction in input tariffs greater. Furthermore, the impact of trade policy on TFP spreads among all firms, which could be consistent with the existence of spillovers from trading firms to non-trading firms or with the notion that trade liberalization exerts competitive pressure on all firms, regardless of their initial exposure to international trade. Finally, we also find evidence of a positive effect of both the import and export statuses on TFP.

Metabolite Sensing GPCRs: Promising Therapeutic Targets for Cancer Treatment?

G-protein-coupled receptors constitute the most diverse and largest receptor family in the human genome, with approximately 800 different members identified. Given the well-known metabolic alterations in cancer development, we will focus specifically in the 19 G-protein-coupled receptors (GPCRs), which can be selectively activated by metabolites. These metabolite sensing GPCRs control crucial processes, such as cell proliferation, differentiation, migration, and survival after their activation. In the present review, we will describe the main functions of these metabolite sensing GPCRs and shed light on the benefits of their potential use as possible pharmacological targets for cancer treatment.

Deciphering the secretome of leukocyte-platelet rich fibrin: towards a better understanding of its wound healing properties.

Leukocyte-platelet rich fibrin (L-PRF) is extensively used in the dentistry field and other clinical scenarios due to its regeneration properties. The goal of the present study was to depict the L-PRF secretome and how it changes over time. We obtained L-PRF membranes and cultured them in DMEM. The secretome was collected at days 3, 7 and 21. The secretome at day 3 was analysed by LC-MS/MS and differences over time were analysed by Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH). Overall, 705 proteins were identified in the secretome of L-PRF membranes after 3 days of culture, including growth factors (EGF, PDGFA) and proteins related to platelet and neutrophil degranulation. A total of 202 differentially secreted proteins were quantified by SWATH when comparing secretomes at days 3, 7 and 21. Most of them were enriched at day 3 such as MMP9, TSP1 and CO3. On the contrary, fibrinogen and CATS were found down-regulated at day 3. Growth factor and western blotting analysis corroborated the proteomic results. This is the most detailed proteome analysis of the L-PRF secretome to date. Proteins and growth factors identified, and their kinetics, provide novel information to further understand the wound healing properties of L-PRF.