The effects of sulfated secondary bile acids on intestinal barrier function and immune response in an inflammatory in vitro human intestinal model.

Dysbiosis-related perturbations in bile acid (BA) metabolism were observed in inflammatory bowel disease (IBD) patients, which was characterized by increased levels of sulfated BAs at the expense of secondary BAs. However, the exact effects of sulfated BAs on the etiology of IBD are not investigated yet. Therefore, we aimed to investigate the effects of sulfated deoxycholic acid (DCA), sulfated lithocholic acid (LCA) and their unsulfated forms on intestinal barrier function and immune response. To this end, we first established a novel in vitro human intestinal model to mimic chronic intestinal inflammation as seen during IBD. This model consisted of a co-culture of Caco-2 and HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation to represent the mucus layer. A pro-inflammatory environment was created by combining the co-culture with LPS-activated dendritic cells (DCs) in the basolateral compartment. The presence of activated DCs caused a decrease in transepithelial electrical resistance (TEER), which was slightly restored by LCA and sulfated DCA. The expression of genes related to intestinal epithelial integrity and the mucus layer were slightly, but not significantly increased. These results imply that sulfated BAs have a minor effect on intestinal barrier function in Caco-2 and HT29-MTX-E12 cells. When exposed directly to DCs, our results point towards anti-inflammatory effects of secondary BAs, but to a minor extent for sulfated secondary BAs. Future research should focus on the importance of proper transformation of BAs by bacterial enzymes and the potential involvement of BA dysmetabolism in IBD progression.

Bradykinin augments EGF-induced airway smooth muscle proliferation by activation of conventional protein kinase C isoenzymes.

This study aims to investigate the effects of bradykinin, alone and in combination with growth factors on proliferation of cultured bovine tracheal smooth muscle cells. Bradykinin did not induce mitogenic responses by itself, but concentration-dependently augmented growth factor-induced [3H]thymidine incorporation and cell proliferation. The bradykinin effect was mediated by bradykinin B2 receptors, and not dependent on cyclo-oxygenase. Bradykinin-induced synergism with epidermal growth factor (EGF) could be suppressed by the protein kinase C (PKC) inhibitors GF 109203X (Bisindolylmaleimide I; specific for conventional and novel PKCs) and Gö 6976 (12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; specific for conventional PKCs). In addition, sole activation of PKC using Phorbol 12-myristate 13-acetate (PMA) was sufficient for a synergistic interaction with EGF. In contrast to bradykinin however, PMA was mitogenic by itself which was not at all affected by Gö 6976, but abolished by GF 109203X. Bradykinin transiently activated the p42/p44 MAP kinase pathway, whereas PMA-induced activation of p42/p44 mitogen activated protein (MAP) kinase was sustained. Neither the combination of bradykinin and EGF nor that of PMA and EGF induced synergistic activation of p42/p44 MAP kinase, however. These results show that bradykinin B2 receptor-stimulation augments growth factor-induced mitogenic responses of airway smooth muscle cells through activation of conventional PKC isozymes. In addition, the results show that PKC isozyme-specificity underlies stimulus-specific differences in mitogenic capacity for bradykinin and PMA.

Acetylcholine: a novel regulator of airway smooth muscle remodelling?

Increased airway smooth muscle mass is a pathological feature that asthma and chronic obstructive pulmonary disease (COPD) have in common. This increase has gained renewed interest in view of recent developments showing that airway smooth muscle, instead of solely being a contractile partner, is capable of interacting dynamically with its environment, especially under inflammatory conditions. Airway smooth muscle cells are able to proliferate, to migrate, and to secrete chemokines, cytokines, extracellular matrix proteins and growth factors, and most importantly, to adapt to these functions by changing its phenotype from contractile to proliferative/synthetic. Conversely, switching to a (hyper)contractile phenotype may also occur. A vast number of inflammatory stimuli regulate these functions and exert their effects via excitatory G(q) or G(i)-coupled receptors. Since acetylcholine activates muscarinic M(2) and M(3) receptors in the airway smooth muscle cell membrane, which are coupled to G(i) and G(q) proteins, respectively, and since acetylcholine release may be enhanced in airway inflammation, a pathophysiological role of acetylcholine related to the above processes and exceeding contraction could be envisaged. In this review, evidence in favour of this hypothesis, based on recent data that show a role for muscarinic receptors in modulating airway smooth muscle proliferation, contractility and contractile protein expression is discussed. Based on these findings, we postulate that endogenous acetylcholine contributes to airway remodeling in asthma and COPD.

Insulin induces a hypercontractile airway smooth muscle phenotype.

This study aims to investigate the effects of insulin on bovine tracheal smooth muscle phenotype in vitro. Contractility of muscle strips and DNA-synthesis ([3H]thymidine incorporation) of isolated cells were used as parameters for smooth muscle phenotyping. Insulin (1 microM) was mitogenic for bovine tracheal smooth muscle and potentiated DNA-synthesis induced by other growth factors. In contrast, after pretreatment of unpassaged bovine tracheal smooth muscle cells in culture, the mitogenic response induced by growth factors was strongly diminished, with no difference in the basal incorporation. Pretreatment of bovine tracheal smooth muscle strips in organ culture with insulin increased maximal contraction to methacholine and KCl. These results show that insulin acutely augments DNA-synthesis in the presence of other growth factors. In contrast, insulin pretreatment induces a hypercontractile phenotype with a decreased mitogenic capacity. This mechanism may be involved in the putative negative association between asthma and type I diabetes. In addition, these findings may have implications for the use of aerosolized insulin in diabetes mellitus.

Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent.

Growth factors have been implicated in the pathophysiology of asthma. However, the putative effects of these growth factors on human airway smooth muscle tone are still largely unknown. We performed contraction experiments using human bronchial smooth muscle ring preparations. The growth factor insulin-like growth factor-1 (IGF-1) induced a slowly developing sustained contraction, which was dependent on Rho-kinase, since contraction was almost completely inhibited by (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632; 1 microM). Angiotensin II, a G(q)-coupled receptor agonist which can act as a growth factor as well, induced a biphasic contraction, the sustained phase of which was also almost completely inhibited by Y-27632. We conclude that angiotensin II and IGF-1 induce a Rho-kinase-dependent sustained contraction of human bronchial smooth muscle. Since growth factors are associated with pathophysiological conditions such as asthma, inhibition of Rho-kinase could be effective under these conditions.

Postprandial dietary lipid-specific effects on human peripheral blood mononuclear cell gene expression profiles.

BACKGROUND: Dietary polyunsaturated fatty acids (PUFAs) have a variety of beneficial effects, and immune cells play an important role in these effects. The mechanisms of action of PUFAs are still not completely understood, but it is known that PUFAs can influence the expression of a broad set of genes. OBJECTIVE: The objective was to determine the postprandial effects of intake of different fatty acids on the gene expression profiles of peripheral blood mononuclear cells (PBMCs). DESIGN: In a single-blind crossover study, 21 healthy male volunteers consumed shakes enriched in PUFAs, monounsaturated fatty acids (MUFAs), or saturated fatty acids (SFAs) in random order. Blood samples were collected before and at several time points after intake. Whole-genome gene expression profiles of PBMCs were examined before and 6 h after intake of the PUFA and SFA shakes. In addition, ex vivo incubation of human PBMCs with different fatty acids was performed. RESULTS: Whole-genome expression analysis showed distinct differences between PUFA and SFA consumption. PUFA intake decreased the expression of genes in liver X receptor signaling, whereas SFA intake increased the expression of these genes. PUFA intake also increased the expression of genes related to cellular stress responses. MUFA intake had an intermediate effect on several genes. Ex vivo experiments showed a direct effect of free fatty acids on PBMC gene expression. CONCLUSION: This study showed that PBMCs can reveal fatty acid-specific gene expression profiles in young healthy men after the consumption of different fatty acids, as evidenced by the opposite effects of PUFA and SFA intakes on the expression of genes involved in liver X receptor signaling. This trial was registered at www.clinicaltrials.gov as NCT01000194.

Muscarinic M3-receptors mediate cholinergic synergism of mitogenesis in airway smooth muscle.

Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [(3)H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M(3)-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 microM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1-10 microM), but only in cells having functional muscarinic M(3)-receptors. This synergism could be suppressed significantly by the selective M(3)-receptor antagonists DAU 5884 (0.1 microM) and 4-DAMP (10 nM), but not at all by the M(2)-subtype selective antagonist gallamine (10 microM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M(3)-receptors in bovine tracheal smooth muscle cells.