Ileal mucus viscoelastic properties differ in Crohn's disease.

Crohn's disease (CD) is an inflammatory bowel disease (IBD) that can affect any part of the gastrointestinal tract, frequently involving the terminal ileum. While colonic mucus alterations in CD patients have been described, terminal ileal mucus and its mechanobiological properties have been neglected. Our study is the first of its kind to decipher the viscoelastic and network properties of ileal mucus. With that aim, oscillatory rheological shear measurements based on an airway mucus protocol that was thoroughly validated for ileal mucus were performed. Our pilot study analyzed terminal ileum mucus from controls (n=14) and CD patients (n=14). Mucus network structure was visualized by scanning electron microscopy (SEM). Interestingly, a statistically significant increase in viscoelasticity as well as a decrease in mesh size was observed in ileal mucus from CD patients compared to controls. Furthermore, rheological data were analyzed in relation to study participants' clinical characteristics, revealing a noteworthy trend between non-smokers and smokers. In conclusion, this study provides the first data on the viscoelastic properties and structure of human ileal mucus in the healthy state and in Crohn's disease, demonstrating significant alterations between groups and highlighting the need for further research on mucus and its effect on the underlying epithelial barrier.

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on sputum viscoelastic properties, airway infection and inflammation in patients with cystic fibrosis.

BACKGROUND: Recent studies demonstrated that the triple combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and reduces pulmonary exacerbations in cystic fibrosis (CF) patients with at least one F508del allele. However, effects of ETI on downstream consequences of CFTR dysfunction, i.e. abnormal viscoelastic properties of airway mucus, chronic airway infection and inflammation have not been studied. The aim of this study was to determine the longitudinal effects of ETI on airway mucus rheology, microbiome and inflammation in CF patients with one or two F508del alleles aged ≥12 years throughout the first 12 months of therapy. METHODS: In this prospective observational study, we assessed sputum rheology, the microbiome, inflammation markers and proteome before and 1, 3 and 12 months after initiation of ETI. RESULTS: In total, 79 patients with CF and at least one F508del allele and 10 healthy controls were enrolled in this study. ETI improved the elastic modulus and viscous modulus of CF sputum at 3 and 12 months after initiation (all p<0.01). Furthermore, ETI decreased the relative abundance of Pseudomonas aeruginosa in CF sputum at 3 months and increased the microbiome α-diversity at all time points. In addition, ETI reduced interleukin-8 at 3 months (p<0.05) and free neutrophil elastase activity at all time points (all p<0.001), and shifted the CF sputum proteome towards healthy. CONCLUSIONS: Our data demonstrate that restoration of CFTR function by ETI improves sputum viscoelastic properties, chronic airway infection and inflammation in CF patients with at least one F508del allele over the first 12 months of therapy; however, levels close to healthy were not reached.

A novel thiol-saccharide mucolytic for the treatment of muco-obstructive lung diseases.

BACKGROUND: Mucin disulfide cross-links mediate pathologic mucus formation in muco-obstructive lung diseases. MUC-031, a novel thiol-modified carbohydrate compound, cleaves disulfides to cause mucolysis. The aim of this study was to determine the mucolytic and therapeutic effects of MUC-031 in sputum from patients with cystic fibrosis (CF) and mice with muco-obstructive lung disease (ßENaC-Tg mice). METHODS: We compared the mucolytic efficacy of MUC-031 and existing mucolytics (N-acetylcysteine (NAC) and recombinant human deoxyribonuclease I (rhDNase)) using rheology to measure the elastic modulus (G') of CF sputum, and we tested effects of MUC-031 on airway mucus plugging, inflammation and survival in ßENaC-Tg mice to determine its mucolytic efficacy in vivo. RESULTS: In CF sputum, compared to the effects of rhDNase and NAC, MUC-031 caused a larger decrease in sputum G', was faster in decreasing sputum G' by 50% and caused mucolysis of a larger proportion of sputum samples within 15 min of drug addition. Compared to vehicle control, three treatments with MUC-031 in 1 day in adult ßENaC-Tg mice decreased airway mucus content (16.8±3.2 versus 7.5±1.2 nL·mm-2, p<0.01) and bronchoalveolar lavage cells (73 833±6930 versus 47 679±7736 cells·mL-1, p<0.05). Twice-daily treatment with MUC-031 for 2 weeks also caused decreases in these outcomes in adult and neonatal ßENaC-Tg mice and reduced mortality from 37% in vehicle-treated ßENaC-Tg neonates to 21% in those treated with MUC-031 (p<0.05). CONCLUSION: MUC-031 is a potent and fast-acting mucolytic that decreases airway mucus plugging, lessens airway inflammation and improves survival in ßENaC-Tg mice. These data provide rationale for human trials of MUC-031 in muco-obstructive lung diseases.

An optimized protocol for assessment of sputum macrorheology in health and muco-obstructive lung disease.

Background: Airway mucus provides important protective functions in health and abnormal viscoelasticity is a hallmark of muco-obstructive lung diseases such as cystic fibrosis (CF). However, previous studies of sputum macrorheology from healthy individuals and patients with CF using different experimental protocols yielded in part discrepant results and data on a systematic assessment across measurement settings and conditions remain limited. Objectives: The aim of this study was to develop an optimized and reliable protocol for standardized macrorheological measurements of airway mucus model systems and native human sputum from healthy individuals and patients with muco-obstructive lung disease. Methods: Oscillatory rheological shear measurements were performed using bovine submaxillary mucin (BSM) at different concentrations (2% and 10% solids) and sputum samples from healthy controls (n = 10) and patients with CF (n = 10). Viscoelastic properties were determined by amplitude and frequency sweeps at 25°C and 37°C with or without solvent trap using a cone-plate geometry. Results: Under saturated atmosphere, we did not observe any temperature-dependent differences in 2% and 10% BSM macrorheology, whereas in the absence of evaporation control 10% BSM demonstrated a significantly higher viscoelasticity at 37°C. Similarly, during the measurements without evaporation control at 37°C we observed a substantial increase in the storage modulus G' and the loss modulus G″ of the highly viscoelastic CF sputum but not in the healthy sputum. Conclusion: Our data show systematically higher viscoelasticity of CF compared to healthy sputum at 25°C and 37°C. For measurements at the higher temperature using a solvent trap to prevent evaporation is essential for macrorheological analysis of mucus model systems and native human sputum. Another interesting finding is that the viscoelastic properties are not much sensitive to the applied experimental deformation and yield robust results despite their delicate consistency. The optimized protocol resulting from this work will facilitate standardized quantitative assessment of abnormalities in viscoelastic properties of airway mucus and response to muco-active therapies in patients with CF and other muco-obstructive lung diseases.

Age-Related Differences in Structure and Function of Nasal Epithelial Cultures From Healthy Children and Elderly People.

The nasal epithelium represents the first line of defense against inhaled pathogens, allergens, and irritants and plays a key role in the pathogenesis of a spectrum of acute and chronic airways diseases. Despite age-dependent clinical phenotypes triggered by these noxious stimuli, little is known about how aging affects the structure and function of the airway epithelium that is crucial for lung homeostasis and host defense. The aim of this study was therefore to determine age-related differences in structural and functional properties of primary nasal epithelial cultures from healthy children and non-smoking elderly people. To achieve this goal, highly differentiated nasal epithelial cultures were established from nasal brushes at air-liquid interface and used to study epithelial cell type composition, mucin (MUC5AC and MUC5B) expression, and ion transport properties. Furthermore, we determined age-dependent molecular signatures using global proteomic analysis. We found lower numeric densities of ciliated cells and higher levels of MUC5AC expression in cultures from children vs. elderly people. Bioelectric studies showed no differences in basal ion transport properties, ENaC-mediated sodium absorption, or CFTR-mediated chloride transport, but detected decreased calcium-activated TMEM16A-mediated chloride secretory responses in cultures from children vs. elderly people. Proteome analysis identified distinct age-dependent molecular signatures associated with ciliation and mucin biosynthesis, as well as other pathways implicated in aging. Our data identified intrinsic, age-related differences in structure and function of the nasal epithelium and provide a basis for further studies on the role of these findings in age-dependent airways disease phenotypes observed with a spectrum of respiratory infections and other noxious stimuli.

BMP9 Promotes an Epithelial Phenotype and a Hepatocyte-like Gene Expression Profile in Adult Hepatic Progenitor Cells.

Bone morphogenetic protein 9 (BMP9), a member of the TGF-ß superfamily, has emerged as a new player in chronic liver diseases (CLDs). Its levels increase in the fibrotic liver where it promotes fibrogenesis. It also regulates hepatic progenitor cells (oval cells in rodents), a cell population that contributes to repopulate the liver and recover functionality upon severe damage, but it can also be pro-fibrogenic, depending upon the hepatic microenvironment. Here we analyze the effect of chronic exposure to BMP9 in oval cells. We show that cells chronically treated with BMP9 (B9T-OC) display a more epithelial and hepatocyte-like phenotype while acquiring proliferative and survival advantages. Since our previous studies had revealed a functional crosstalk between BMP9 and the HGF/c-Met signaling pathways in oval cells, we analyzed a possible role for HGF/c-Met in BMP9-induced long-term effects. Data evidence that active c-Met signaling is necessary to obtain maximum effects in terms of BMP9-triggered hepatocytic differentiation potential, further supporting functionally relevant cooperation between these pathways. In conclusion, our work reveals a novel action of BMP9 in liver cells and helps elucidate the mechanisms that serve to increase oval cell regenerative potential, which could be therapeutically modulated in CLD.

Polyglycerol-Based Mucus-Inspired Hydrogels.

The mucus layer is a hydrogel network that covers mucosal surfaces of the human body. Mucus has important protective properties that are related to its unique rheological properties, which are based on mucins being the main glycoprotein constituents. Mucin macromolecules entangle with one another and form a physical network that is instrumental for many important defense functions. Mucus derived from various human or animal sources is poorly defined and thus not suitable for many application purposes. Herein, a synthetic route is fabricated to afford a library of compositionally defined mucus-inspired hydrogels (MIHs). MIHs are synthesized by thiol oxidation to render disulfide bonds between the crosslinker ethoxylated trimethylolpropane tri(3-mercaptopropionate) (THIOCURE ETTMP 1300) and the linear precursors, dithiolated linear polyglycerol (LPG(SH)2 ) or polyethylene glycol (PEG(SH)2 ) of different molecular weights. The mixing ratio of linear polymers versus crosslinker and the length of the linear polymer are varied, thus delivering a library of compositionally defined mucin-inspired constructs. Their viscoelastic properties are determined by frequency sweeps at 25 and 37 °C and compared to the corresponding behavior of native human mucus. Here, MIHs composed of a 10:1 ratio of LPG(SH)2 and ETTMP 1300 are proved to be the best comparable to human airway mucus rheology.

A Signaling Crosstalk between BMP9 and HGF/c-Met Regulates Mouse Adult Liver Progenitor Cell Survival.

During chronic liver disease, hepatic progenitor cells (HPC, oval cells in rodents) become activated, proliferate, and differentiate into cholangiocytes and/or hepatocytes contributing to the final outcome of the regenerative process in a context-dependent fashion. Here, we analyze the crosstalk between the hepatocyte growth factor (HGF)/c-Met signaling axis, key for liver regeneration, and bone morphogenetic protein (BMP)9, a BMP family ligand that has emerged as a critical regulator of liver pathology. Our results show that HGF/c-Met signaling blocks BMP9-mediated apoptotic cell death, while it potentiates small mothers against decapentaplegic (SMAD)1 signaling triggered by BMP9 in oval cells. Interestingly, HGF-induced overactivation of SMAD1, -5, -8 requires the upregulation of TGF-ß type receptor activin receptor-like kinase (ALK)1, and both ALK1 and SMAD1 are required for the counteracting effect of HGF on BMP9 apoptotic activity. On the other hand, we also prove that BMP9 triggers the activation of p38MAPK in oval cells, which drives BMP9-apoptotic cell death. Therefore, our data support a model in which BMP9 and HGF/c-Met signaling axes establish a signaling crosstalk via ALK1 that modulates the balance between the two pathways with opposing activities, SMAD1 (pro-survival) and p38 mitogen-activated protein kinases (p38MAPK; pro-apoptotic), which determines oval cell fate. These data help delineate the complex signaling network established during chronic liver injury and its impact on the oval cell regenerative response.

Clathrin switches transforming growth factor-ß role to pro-tumorigenic in liver cancer.

BACKGROUND & AIMS: Upon ligand binding, tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), are recruited into clathrin-coated pits for internalization by endocytosis, which is relevant for signalling and/or receptor degradation. In liver cells, transforming growth factor-ß (TGF-ß) induces both pro- and anti-apoptotic signals; the latter are mediated by the EGFR pathway. Since EGFR mainly traffics via clathrin-coated vesicles, we aimed to analyse the potential role of clathrin in TGF-ß-induced signalling in liver cells and its relevance in liver cancer. METHODS: Real-Time PCR and immunohistochemistry were used to analyse clathrin heavy-chain expression in human (CLTC) and mice (Cltc) liver tumours. Transient knockdown (siRNA) or overexpression of CLTC were used to analyse its role on TGF-ß and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGFB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). RESULTS: Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-ß phosphorylating SMADs (canonical signalling) but showed impairment in the anti-apoptotic signals (EGFR transactivation). Experiments of loss or gain of function in HCC cells reveal an essential role for clathrin in inhibiting TGF-ß-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-ß signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CONCLUSIONS: This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-ß pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-ß-targeted therapy. LAY SUMMARY: Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Cltc), altering the cellular response to TGF-ß in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CLTC expression in HCC human samples could help select patients that would benefit from therapies targeting TGF-ß.

c-Met Signaling Is Essential for Mouse Adult Liver Progenitor Cells Expansion After Transforming Growth Factor-ß-Induced Epithelial-Mesenchymal Transition and Regulates Cell Phenotypic Switch.

Adult hepatic progenitor cells (HPCs)/oval cells are bipotential progenitors that participate in liver repair responses upon chronic injury. Recent findings highlight HPCs plasticity and importance of the HPCs niche signals to determine their fate during the regenerative process, favoring either fibrogenesis or damage resolution. Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) are among the key signals involved in liver regeneration and as component of HPCs niche regulates HPCs biology. Here, we characterize the TGF-ß-triggered epithelial-mesenchymal transition (EMT) response in oval cells, its effects on cell fate in vivo, and the regulatory effect of the HGF/c-Met signaling. Our data show that chronic treatment with TGF-ß triggers a partial EMT in oval cells based on coexpression of epithelial and mesenchymal markers. The phenotypic and functional profiling indicates that TGF-ß-induced EMT is not associated with stemness but rather represents a step forward along hepatic lineage. This phenotypic transition confers advantageous traits to HPCs including survival, migratory/invasive and metabolic benefit, overall enhancing the regenerative potential of oval cells upon transplantation into a carbon tetrachloride-damaged liver. We further uncover a key contribution of the HGF/c-Met pathway to modulate the TGF-ß-mediated EMT response. It allows oval cells expansion after EMT by controlling oxidative stress and apoptosis, likely via Twist regulation, and it counterbalances EMT by maintaining epithelial properties. Our work provides evidence that a coordinated and balanced action of TGF-ß and HGF are critical for achievement of the optimal regenerative potential of HPCs, opening new therapeutic perspectives. Stem Cells 2019;37:1108-1118.

Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

BACKGROUND & AIMS: Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. METHODS: WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. RESULTS: Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. CONCLUSIONS: We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases.

Dual role of protein tyrosine phosphatase 1B in the progression and reversion of non-alcoholic steatohepatitis.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. Protein tyrosine phosphatase 1B (PTP1B), a negative modulator of insulin and cytokine signaling, is a therapeutic target for type 2 diabetes and obesity. We investigated the impact of PTP1B deficiency during NAFLD, particularly in non-alcoholic steatohepatitis (NASH). METHODS: NASH features were evaluated in livers from wild-type (PTP1BWT) and PTP1B-deficient (PTP1BKO) mice fed methionine/choline-deficient diet (MCD) for 8 weeks. A recovery model was established by replacing MCD to chow diet (CHD) for 2-7 days. Non-parenchymal liver cells (NPCs) were analyzed by flow cytometry. Oval cells markers were measured in human and mouse livers with NASH, and in oval cells from PTP1BWT and PTP1BKO mice. RESULTS: PTP1BWT mice fed MCD for 8 weeks exhibited NASH, NPCs infiltration, and elevated Fgf21, Il6 and Il1b mRNAs. These parameters decreased after switching to CHD. PTP1B deficiency accelerated MCD-induced NASH. Conversely, after switching to CHD, PTP1BKO mice rapidly reverted NASH compared to PTP1BWT mice in parallel to the normalization of serum triglycerides (TG) levels. Among NPCs, a drop in cytotoxic natural killer T (NKT) subpopulation was detected in PTP1BKO livers during recovery, and in these conditions M2 macrophage markers were up-regulated. Oval cells markers (EpCAM and cytokeratin 19) significantly increased during NASH only in PTP1B-deficient livers. HGF-mediated signaling and proliferative capacity were enhanced in PTP1BKO oval cells. In NASH patients, oval cells markers were also elevated. CONCLUSIONS: PTP1B elicits a dual role in NASH progression and reversion. Additionally, our results support a new role for PTP1B in oval cell proliferation during NAFLD.

BMP-9 interferes with liver regeneration and promotes liver fibrosis.

OBJECTIVE: Bone morphogenetic protein (BMP)-9, a member of the transforming growth factor-ß family of cytokines, is constitutively produced in the liver. Systemic levels act on many organs and tissues including bone and endothelium, but little is known about its hepatic functions in health and disease. DESIGN: Levels of BMP-9 and its receptors were analysed in primary liver cells. Direct effects of BMP-9 on hepatic stellate cells (HSCs) and hepatocytes were studied in vitro, and the role of BMP-9 was examined in acute and chronic liver injury models in mice. RESULTS: Quiescent and activated HSCs were identified as major BMP-9 producing liver cell type. BMP-9 stimulation of cultured hepatocytes inhibited proliferation, epithelial to mesenchymal transition and preserved expression of important metabolic enzymes such as cytochrome P450. Acute liver injury caused by partial hepatectomy or single injections of carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) into mice resulted in transient downregulation of hepatic BMP-9 mRNA expression. Correspondingly, LPS stimulation led to downregulation of BMP-9 expression in cultured HSCs. Application of BMP-9 after partial hepatectomy significantly enhanced liver damage and disturbed the proliferative response. Chronic liver damage in BMP-9-deficient mice or in mice adenovirally overexpressing the selective BMP-9 antagonist activin-like kinase 1-Fc resulted in reduced deposition of collagen and subsequent fibrosis. CONCLUSIONS: Constitutive expression of low levels of BMP-9 stabilises hepatocyte function in the healthy liver. Upon HSC activation, endogenous BMP-9 levels increase in vitro and in vivo and high levels of BMP-9 cause enhanced damage upon acute or chronic injury.

BMP Signalling at the Crossroad of Liver Fibrosis and Regeneration.

Bone Morphogenetic Proteins (BMPs) belong to the Transforming Growth Factor-ß (TGF-ß) family. Initially identified due to their ability to induce bone formation, they are now known to have multiple functions in a variety of tissues, being critical not only during development for tissue morphogenesis and organogenesis but also during adult tissue homeostasis. This review focus on the liver as a target tissue for BMPs actions, devoting most efforts to summarize our knowledge on their recently recognized and/or emerging roles on regulation of the liver regenerative response to various insults, either acute or chronic and their effects on development and progression of liver fibrosis in different pathological conditions. In an attempt to provide the basis for guiding research efforts in this field both the more solid and more controversial areas of research were highlighted.

Dissecting the role of epidermal growth factor receptor catalytic activity during liver regeneration and hepatocarcinogenesis.

UNLABELLED: Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, ΔEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-ß pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-ß through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in ΔEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, ΔEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because ΔEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. CONCLUSION: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (Hepatology 2016;63:604-619).

BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation.

The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells.

Unusual death of a transvestite: identification of crime weapon and survival time*.

The authors report a case of a transvestite found murdered near his automobile with several lacerated contused wounds to the face and cranial fractures. Autopsy revealed that the cause of death was a serious head trauma with subdural and subarachnoidal hemorrhages. In order to identify the crime weapon, a scanning electron microscopy (SEM) was used which revealed metallic residue on the skin fragments with the same molecular composition of the car paint. As for survival time, antibody anti-beta-amyloid precursor protein (APP) was applied to brain fragments and brainstem tissue, allowing for axonal varicosities (which form 2 to 3 h following death) to be observed under the optic microscope. So, by using SEM we understood that the fatal cranial-encephalic lesions were the result of the victim's head being repeatedly struck against the car door while anti-betaAPP led to the understanding that the time elapsed between injury and death was less than 2 to 3 h.

Sudden cardiac death and myocardial ischemia indicators: a comparative study of four immunohistochemical markers.

The postmortem diagnosis of acute myocardial infarction represents a current challenge for forensic pathologists, particularly when death occurs within minutes to a few hours after the ischemic insult. Among the adult population the single most important cause of sudden cardiac death (SCD) is the well-known atherosclerotic coronary artery disease, commonly asymptomatic or unrecognized. The recognition of early myocardial damage using routine hematoxylin and eosin (H&E) staining is possible only if death has occurred at least 6 hours after the onset of the ischemic injury. The usefulness of immunohistochemical markers to the diagnosis of early myocardial damage has been recently suggested because most of them can be visible even serologically as early as few minutes after the beginning of the symptoms. To evaluate the usefulness of plasma and cellular antigens, their distribution patterns have been studied among a group of 18 SCD cases in which a myocardial ischemia was strongly suspected. For the present study, 4 markers have been selected on the basis of their different diagnostic potential as follows: among the plasma markers the C5b-9 and fibronectin, among the cellular markers the myoglobin and cardiac troponin. The results show that only the study of multiple markers such as those selected can provide enough evidence of myocardial ischemia and/or necrosis, supporting the final diagnosis of SCD. No single immunohistochemical staining is ideal for diagnosing early myocardial ischemia but a set of markers can improve the ability of forensic pathologists to detect ischemic areas when no macroscopic or microscopic evidence of necrosis is available. However, the interpretation of data obtained in each individual cannot be isolated from the overall assessment of the factors (cardiopulmonary resuscitation and/or agonal artifacts) that can affect the expression of each marker.