Vaggelis Harokopos

The RNA-Binding Protein Elavl1/HuR Is Essential for Placental Branching Morphogenesis and Embryonic Development

ABSTRACT HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuRs requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuRs mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuRs absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny.

Soluble TNF Mediates the Transition from Pulmonary Inflammation to Fibrosis

Background Fibrosis, the replacement of functional tissue with excessive fibrous tissue, can occur in all the main tissues and organ systems, resulting in various pathological disorders. Idiopathic Pulmonary Fibrosis is a prototype fibrotic disease involving abnormal wound healing in response to multiple sites of ongoing alveolar epithelial injury. Methodology/Principal Findings To decipher the role of TNF and TNF-mediated inflammation in the development of fibrosis, we have utilized the bleomycin-induced animal model of Pulmonary Fibrosis and a series of genetically modified mice lacking components of TNF signaling. Transmembrane TNF expression is shown to be sufficient to elicit an inflammatory response, but inadequate for the transition to the fibrotic phase of the disease. Soluble TNF expression is shown to be crucial for lymphocyte recruitment, a prerequisite for TGF-b1 expression and the development of fibrotic lesions. Moreover, through a series of bone marrow transfers, the necessary TNF expression is shown to originate from the non-hematopoietic compartment further localized in apoptosing epithelial cells. Conclusions These results suggest a primary detrimental role of soluble TNF in the pathologic cascade, separating it from the beneficial role of transmembrane TNF, and indicate the importance of assessing the efficacy of soluble TNF antagonists in the treatment of Idiopathic Pulmonary Fibrosis.

The pattern of inflammatory/anti-inflammatory cytokines and chemokines in type 1 diabetic patients over time

Abstract Aims. To evaluate the profile of pro- and anti-inflammatory cytokines in type 1 diabetes mellitus (T1DM) and the way they are connected in co-regulated networks and determine whether disease duration influences their pattern. Methods. Plasma levels of 20 cytokines and soluble CD40 (sCD40) from 44 uncomplicated patients and 22 healthy controls (HCs) were measured using enzyme-linked immunosorbent assay (ELISA) and protein array technology. Results. Patients showed significantly higher levels of sCD40, IL-1a, IL-2, IL-4, IL-5, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1a, MIP-1b, regulated on activation normal T cell expressed and secreted (RANTES), matrix metalloproteinase (MMP)-9, and a trend to higher IL-6 than did HCs. RANTES and sCD40 discriminated significantly between diabetics and HCs. In patients with disease duration >6 months, cytokines were organized in two clusters mainly regulated by Th17 and Th1/Th2 cells respectively, while in those with disease duration ≤6 months a set of Th1-cytokines was separated apart from the second cluster. Monocyte chemotactic protein (MCP)-1 was revealed as the most discriminant factor between patients with disease duration of more than and less than 6 months. Conclusions. A parallel elevation of both inflammatory and anti-inflammatory cytokines was observed in patients compared with HCs. In T1DM patients with disease duration ≤6 months, Th1-cytokines were organized on a separate cluster, suggesting a possible role of Th1 cells in the progress of beta-cell destruction during the first period of the disease.

Cytoskeletal Rearrangements in Synovial Fibroblasts as a Novel Pathophysiological Determinant of Modeled Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology–assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease.

Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis

Background: Despite intense research efforts, the aetiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. Gelsolin, an actin-binding protein that modulates cytoskeletal dynamics, was recently highlighted as a likely disease modifier through comparative expression profiling and target prioritisation. Methods: To decipher the possible role of gelsolin in pulmonary inflammation and fibrosis, immunocytochemistry on tissue microarrays of human patient samples was performed followed by computerised image analysis. The results were validated in the bleomycin-induced animal model of pulmonary inflammation and fibrosis using genetically-modified mice lacking gelsolin expression. Moreover, to gain mechanistic insights into the mode of gelsolin activity, a series of biochemical analyses was performed ex vivo in mouse embryonic fibroblasts. Results: Increased gelsolin expression was detected in lung samples of patients with idiopathic interstitial pneumonia as well as in modelled pulmonary inflammation and fibrosis. Genetic ablation of gelsolin protected mice from the development of modelled pulmonary inflammation and fibrosis attributed to attenuated epithelial apoptosis. Conclusions: Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis, while the caspase-3-mediated gelsolin fragmentation was shown to be an apoptotic effector mechanism in disease pathogenesis and a marker of lung injury.

Hepatocyte autotaxin expression promotes liver fibrosis and cancer

Autotaxin (ATX) is a secreted lysophospholipase D that catalyzes the production of lysophosphatidic acid (LPA), a pleiotropic growth‐factor–like lysophospholipid. Increased ATX expression has been detected in various chronic inflammatory disorders and different types of cancer; however, little is known about its role and mode of action in liver fibrosis and cancer. Here, increased ATX expression was detected in chronic liver disease (CLD) patients of different etiologies, associated with shorter overall survival. In mice, different hepatotoxic stimuli linked with the development of different forms of CLDs were shown to stimulate hepatocyte ATX expression, leading to increased LPA levels, activation of hepatic stellate cells (HSCs), and amplification of profibrotic signals. Hepatocyte‐specific, conditional genetic deletion and/or transgenic overexpression of ATX established a liver profibrotic role for ATX/LPA, whereas pharmacological ATX inhibition studies suggested ATX as a possible therapeutic target in CLDs. In addition, hepatocyte ATX ablation and the consequent deregulation of lipid homeostasis was also shown to attenuate hepatocellular carcinoma (HCC) development, thus implicating ATX/LPA in the causative link of cirrhosis and HCC. Conclusion: ATX is a novel player in the pathogenesis of liver fibrosis and cancer and a promising therapeutic target. (Hepatology 2017;65:1369‐1383).

Mast cells mediate malignant pleural effusion formation

Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

A critical role for gelsolin in ventilator-induced lung injury.

Mechanical ventilation, an essential life-support modality of patients with acute lung injury (ALI) or the acute respiratory distress syndrome (ARDS), exerts its detrimental effects through largely unknown mechanisms. Gelsolin (GSN), an actin-binding protein and a substrate of caspase-3, was recently shown to play a major role in bleomycin- or lipopolysaccharide-induced lung injury. To dissect a possible role of GSN in the pathogenesis of ventilator-induced lung injury (VILI), genetically modified mice lacking GSN expression and wild-type controls underwent mechanical ventilation with high tidal volumes. GSN was found up-regulated in the airways upon VILI, and its genetic ablation led to almost complete disease protection as manifested by reduced edema formation, reduced lung injury, attenuated epithelial apoptosis, diminished cytokine expression, and impaired neutrophil infiltration. GSN fragmentation was shown to be an effector mechanism in VILI-induced apoptosis, while GSN expression was shown to be necessary for efficient neutrophil infiltration, which was found to be a prerequisite for VILI induction in this model. Therefore, intracellular GSN and GSN-mediated responses were shown to be an important player in the pathogenesis of VILI.

Mutant KRAS promotes malignant pleural effusion formation

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

Kresoxim-methyl primes Medicago truncatula plants against abiotic stress factors via altered reactive oxygen and nitrogen species signalling leading to downstream transcriptional and metabolic readjustment

Highlight The fungicide kresoxim-methyl displays novel priming properties against key abiotic stress factors (drought and salinity) by modifying reactive oxygen and nitrogen species signalling, inducing osmoprotection through increased proline biosynthesis and suppressing proteolysis.