The muscarinic antagonist gallamine induces proliferation of airway smooth muscle cells regardless of the cell phenotype.

BACKGROUND: Muscarinic receptor antagonists are a usual treatment for chronic airway diseases, with increased bronchoconstriction, like asthma and chronic obstructive pulmonary disease. These diseases are usually accompanied by airway remodeling, involving airway smooth muscle cell (ASMC) proliferation. The purpose of this study was to examine the effect of the muscarinic receptor modulator gallamine on rabbit tracheal ASMC proliferation. METHODS: ASMCs were incubated with gallamine (1 nM-10 mM), atropine (1 fM-10 mM), and/or acetylcholine (1 nM-1 mM), in the presence or absence of FBS (1% or 10%). Cell proliferation was estimated by incorporation of radioactive thymidine, the Cell Titer AQueous One Solution method and cell number counting after Trypan blue exclusion. The mechanisms mediating cell proliferation were studied using the PI3K and MAPK inhibitors LY294002 (20 µM) and PD98059 (100 µM), respectively. Cell phenotype was studied by indirect immunofluorescence for α-actin, Myosin Heavy Chain and desmin. RESULTS: ASMC incubation with the muscarinic receptor allosteric modulator gallamine or the muscarinic receptor antagonist atropine increased methyl-[3H]thymidine incorporation and cell number in a dose-dependent manner. ASMC proliferation was mediated via PI3K and MAPK activation and was transient. Gallamine antagonized the mitogenic effect of 1% FBS. Furthermore, gallamine had a similar effect on contractile ASMCs, without synergizing with or affecting acetylcholine induced proliferation, or altering the percentage of ASMCs expressing contractile phenotype marker proteins. CONCLUSIONS: Gallamine, in the absence of any agonist, has a transient mitogenic effect on ASMCs, regardless of the cell phenotype, mediated by the PI3K and the MAPK signaling pathways.

Diagnostic value of immunohistochemistry for the detection of the BRAF V600E mutation in colorectal carcinoma.

PURPOSE: V600E is the most common activating BRAF mutation in colorectal carcinomas (CRCs). It is a crucial biomarker for patient selection and response to targeted therapy with BRAF V600E inhibitors. Previous studies using immunohistochemistry (IHC) have shown different results. In this study, we evaluated the IHC expression of the mutated BRAF protein in archival material from CRC specimens and correlated it with DNA sequence analysis. METHODS: 51 cases of primary colon adenocarcinoma were stained with BRAF V600E-specific clone VE1 antibody against mutated BRAF protein. DNA sequence analysis was performed and the results were compared. RESULTS: BRAF V600E protein was detected in the cytoplasm of neoplastic cells in 15 of the 51 examined cases (29.4%). The correlation between IHC staining and DNA sequence analysis showed 93.75% sensitivity and 100% specificity. CONCLUSIONS: Our data show that IHC could be used in routine clinical practice as a screening method for BRAF V600E mutant protein detection in CRC patients.

Low Serum Hepcidin in Patients with Autoimmune Liver Diseases.

Hepcidin, a liver hormone, is important for both innate immunity and iron metabolism regulation. As dysfunction of the hepcidin pathway may contribute to liver pathology, we analysed liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases. Hepcidin mRNA levels were determined in liver biopsies obtained from 126 patients with HCV (n = 21), HBV (n = 23), autoimmune cholestatic disease (primary biliary cirrhosis and primary sclerosing cholangitis; PBC/PSC; n = 34), autoimmune hepatitis (AIH; n = 16) and non-alcoholic fatty liver disease (NAFLD; n = 32). Sera sampled on the biopsy day from the same patients were investigated for serum hepcidin levels. Hepatic hepcidin mRNA levels correlated positively with ferritin and negatively with serum γ-GT levels. However, no correlation was found between serum hepcidin and either ferritin or liver hepcidin mRNA. Both serum hepcidin and the serum hepcidin/ferritin ratio were significantly lower in AIH and PBC/PSC patients' sera compared to HBV, HCV or NAFLD (P<0.001 for each comparison) and correlated negatively with serum ALP levels. PBC/PSC and AIH patients maintained low serum hepcidin during the course of their two-year long treatment. In summary, parallel determination of liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases shows that circulating hepcidin and its respective ratio to ferritin are significantly diminished in patients with autoimmune liver diseases. These novel findings, once confirmed by follow-up studies involving bigger size and better-matched disease subgroups, should be taken into consideration during diagnosis and treatment of autoimmune liver diseases.

Long-term exposure to muscarinic agonists decreases expression of contractile proteins and responsiveness of rabbit tracheal smooth muscle cells.

BACKGROUND: Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs). METHODS: ASMCs were serum starved before treatment with muscarinic agonists. Cell phenotype was studied by optical microscopy and indirect immunofluorescence, using smooth muscle α-actin, desmin and SM-Myosin Heavy Chain (SM-MHC) antibodies. [N-methyl-3H]scopolamine binding studies were performed in order to assess M3 muscarinic receptor expression on isolated cell membranes. Contractility studies were performed on isolated ASMCs treated with muscarinic agonists. Proliferation was estimated using methyl-[3H]thymidine incorporation, MTT or cell counting methods. Involvement of PI3K and MAPK signalling pathways was studied by cell incubation with the pathway inhibitors LY294002 and PD98059 respectively. RESULTS: Prolonged culture of ASMCs with acetylcholine, carbachol or FBS, reduced the expression of α-actin, desmin and SM-MHC compared to cells cultured in serum free medium. Treatment of ASMCs with muscarinic agonists for 3-15 days decreased muscarinic receptor expression and their responsiveness to muscarinic stimulation. Acetylcholine and carbachol induced DNA synthesis and increased cell number, of ASMCs that had acquired a contractile phenotype by 7 day serum starvation. This effect was mediated via a PI3K and MAPK dependent mechanism. CONCLUSIONS: Prolonged exposure of rabbit ASMCs to muscarinic agonists decreases the expression of smooth muscle specific marker proteins, down-regulates muscarinic receptors and decreases ASMC contractile responsiveness. Muscarinic agonists are mitogenic, via the PI3K and MAPK signalling pathways.

Endothelial progenitor cells in the pathogenesis of idiopathic pulmonary fibrosis: an evolving concept.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) has been associated with abnormal vascular remodeling. Bone marrow derived endothelial progenitor cells (EPCs) are considered to possess lung tissue repair and vascular remodeling properties. OBJECTIVES: The study aimed to assess early EPCs levels and EPCs endogenous vascular endothelial growth factor (VEGF) expression in IPF. In order to examine alterations in the mobilization of EPCs from the bone marrow we measured plasma VEGF. MAIN RESULTS: Twenty-three patients with IPF and fifteen healthy subjects were included. The number of early EPCs colonies was markedly reduced in IPF patients vs controls (6.00±6.49 vs 49.68±16.73, respectively, p<0.001). EPCs were further decreased in patients presenting systolic pulmonary arterial pressure (sPAP)≥35 mmHg. The number of colonies per well correlated negatively with P((A-a))O(2) (r =  -0.750, p<0.001). Additionally, VEGF mRNA levels were significantly increased in IPF patients. There were no differences observed in VEGF plasma levels in IPF patients when compared to controls. CONCLUSIONS: The current data suggest that inadequate levels of early EPCs may potentially contribute to suppressed repair and recovery of the damaged pulmonary endothelium and thereby may drive the sequence of events in profibrogenic direction. Increased VEGFmRNA levels in the clinical context of IPF may represent a compensatory mechanism to overcome reduced EPCs levels.

Comparison of the electrophysiological properties of the sheep isolated costal and diaphragmatic parietal pleura.

1. Pleural permeability may contribute to pleural fluid turnover. The transmesothelial resistance (R(TM)), is an established surrogate of mesothelial permeability. The aim of the present study was to compare the electrophysiological properties of costal and diaphragmatic parietal pleura. 2. Specimens of the parietal pleura were isolated from 12 adult sheep from the chest wall and the diaphragm. Electrophysiological measurements were conducted with the Ussing system. Specimens of the parietal pleura of both types (diaphragmatic and costal) were compared histologically and total protein content measurements were also made. 3. The R(TM) of the diaphragmatic parietal pleura was significantly higher than that of the costal parietal pleura throughout the experiment. The diaphragmatic parietal pleura contains more cuboidal cells than the costal parietal pleura and its protein content was higher, however this difference was not statistically significant. 4. The costal parietal pleura consists of a more 'leaky' mesothelium than the diaphragmatic pleura. The morphological differences between the two types of parietal pleura may underline the electrophysiological findings.

Identification of MAPK phosphorylation sites and their role in the localization and activity of hypoxia-inducible factor-1alpha.

Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1alpha, involves several post-translational modifications. To study HIF-1alpha phosphorylation, we have used human full-length recombinant HIF-1alpha as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1alpha. Analysis of in vitro phosphorylated HIF-1alpha by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1alpha. When these mutant forms of HIF-1alpha were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1alpha mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1alpha. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1alpha mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1alpha. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1alpha by blocking its CRM1-dependent nuclear export.