Apostolia Hatziefthimiou

Non‐genomic effect of testosterone on airway smooth muscle

Recent studies on blood vessels have provided evidence that testosterone may exert direct effects on smooth muscle. However, an acute effect on airway reactivity has not been shown yet. The aim of this study was to assess the direct effect of testosterone on the responsiveness of male adult rabbit airway smooth muscle (ASM), precontracted with 10 μM acetylcholine, 10μM carbachol or 80 mM KCl.

Azithromycin has an antiproliferative and autophagic effect on airway smooth muscle cells.

Azithromycin is used in long-term, low-dose treatment of airway diseases where airway wall remodelling is present. Since it improves total score symptom and respiratory function of such patients, we hypothesise that azithromycins additional clinical benefits are due to an inhibition of airway smooth muscle cell (SMC) proliferation. Rabbit tracheal SMCs were treated with azithromycin (10−5 to 10−6 M) in the presence or absence of 10% fetal bovine serum (FBS). The proliferation was estimated using the Cell Titer 96® AQueous One Solution Assay (Promega, Madison, WI, USA). Cell viability was assessed with Trypan blue staining and flow cytometry after 7-aminoactinomycin D (7-AAD) staining. Induction of autophagy was studied by indirect immmunofluorescence and/or Western blotting with antibodies against human smooth muscle α-actin, beclin 1, light chain 3 and caspase 3. The involvement of the phosphoinositide 3-kinase pathway was investigated with the inhibitors LY294002 and wortmannin. Incubation with azithromycin for 72 h in the presence of FBS reduced SMC proliferation and viability in a dose-dependent manner. Azithromycin treatment was accompanied by the formation of cytoplasmic vacuoles, characteristic of autophagy. All these effects were reversible after azithromycin removal and prevented by the autophagy inhibitor, 3-methyladenine, or LY294002, but not by wortmannin. In conclusion, azithromycin reduces proliferation and causes autophagy of airway SMCs.

The mitogenic effect of testosterone and 17β-estradiol on airway smooth muscle cells.

Airway disease distribution and/or severity exhibit sex differences suggesting that sex hormones are involved in the respiratory system physiology and pathophysiology. The implication of airway smooth muscle cells (ASMCs) in the physiology of the airways and the pathogenetic mechanism of airway remodeling is of great interest. Therefore, we studied the effect of testosterone and 17β-estradiol on ASMC proliferation and the mechanisms involved. Cell proliferation was estimated using the methyl-[³H]thymidine incorporation and Cell Titer 96® AQueous One Solution Assay methods. ASMC isolated from adult male or female rabbit trachea were incubated with testosterone (1 pM-1 μM) or 17β-estradiol (1 pM-1 μM), in the presence or absence of the androgen receptor antagonist flutamide (10 nM) or estrogen receptor antagonist ICI182780 (10 nM), as well as of the PI3K inhibitors LY294002 (20 μM) or wortmannin (1 μM), or the MAPK inhibitors PD98059 (100 μM) or U0126 (1 μM). After 24 h of incubation, testosterone and 17β-estradiol increased methyl-[³H]thymidine incorporation and cell number, in ASMC isolated from male or female animals. The induction of ASMC proliferation by testosterone or 17β-estradiol was inhibited by flutamide or ICI182780 respectively, as well as by LY294002, wortmannin, PD98059 or U0126. In conclusion, testosterone and 17β-estradiol have a mitogenic effect on ASMC, which is receptor-mediated and involves the MAPK and PI3K signaling pathways. Moreover, their effect is the same for ASMC from male and female animals. It is possible that gender-related differences in ASMC remodeling, may be influenced by the different patterns of sex steroid hormone secretion in males and females.

Ranolazine-induced postrepolarization refractoriness suppresses induction of atrial flutter and fibrillation in anesthetized rabbits.

Ranolazine (Ran) is a novel anti-ischemic agent with electrophysiologic properties mainly attributed to the inhibition of late Na+ current and atrial-selective early Na+ current. However, there are only limited data regarding its efficacy and mechanism of action against atrial flutter (Afl) and atrial fibrillation (AF) in intact animals. Therefore, we aimed to investigate the electrophysiologic mechanism of Ran in a rabbit model of inducible atrial tachyarrhythmias elicited by acetylcholine (ACh). Arrhythmias were produced in 19 rabbits by rapid atrial burst pacing during control, after intravenous ACh and after Ran + ACh administration. Recording of right atrial monophasic action potentials (MAPs) and programmed stimulation were utilized to determine the duration of atrial repolarization at various cycle lengths and voltage levels of action potential, including 75% of total MAP duration (MAPD75), effective refractory period (ERP), and postrepolarization refractoriness (PRR = ERP − MAPD75) prior to and after Ran. Control stimulation yielded no arrhythmias or maximal nonsustained runs of Afl/AF. Upon ACh, 17 of 19 rabbits exhibited sustained Afl and AF as well as mixed forms of Afl/AF, while 2 animals revealed none or short runs of nonsustained arrhythmias and were excluded from the study. High-frequency burst pacing during the first 30 minutes after Ran + ACh failed to induce any arrhythmia in 13 of 17 rabbits (76%), while 2 animals displayed sustained Afl/AF and 2 other animals nonsustained Afl/AF. At basic stimulation cycle length of 250 milliseconds, Ran prolonged baseline atrial ERP (80 ± 8 vs 120 ± 9 milliseconds, P < .001) much more than MAPD75 (65 ± 7 vs 85 ± 7 milliseconds, P < .001), leading to atrial PRR which was more pronounced after Ran compared with control measurements (35 ± 11 vs 15 ± 10 milliseconds, P < .001). This in vivo study demonstrates that Ran exerts antiarrhythmic activity by suppressing inducibility of ACh-mediated Afl/AF in intact rabbits. Its action may predominantly be related to a significant increase in atrial PRR, resulting in depressed electrical excitability and impediment of arrhythmia initiation.

Cytokines and Growth Factors Promote Airway Smooth Muscle Cell Proliferation

Chronic airway diseases, such as asthma or chronic obstructive pulmonary disease, are characterized by the presence in the airways of inflammation factors, growth factors and cytokines, which promote airway wall remodelling. The aim of this study was to investigate the effect of cytokines and growth factors on airway smooth muscle cell (ASMC) proliferation, phenotype and responsiveness. Incubation of serum starved human bronchial ASMCs with TNF-α, TGF, bFGF, and PDGF, but not IL-1β, increased methyl-[3H]thymidine incorporation and cell number, mediated by the PI3K and MAPK signalling pathways. Regarding rabbit tracheal ASMC proliferation, TNF-α, IL-1β, TGF, and PDGF increased methyl-[3H]thymidine incorporation in a PI3K- and MAPK-dependent manner. bFGF increased both methyl-[3H]thymidine incorporation and cell number. Moreover, incubation with TGF, bFGF and PDGF appears to drive human ASMCs towards a synthetic phenotype, as shown by the reduction of the percentage of cells expressing SM-α actin. In addition, the responsiveness of epithelium-denuded rabbit tracheal strips to carbachol was not significantly altered after 3-day treatment with bFGF. In conclusion, all the tested cytokines and growth factors increased ASMC proliferation to a different degree, depending on the specific cell type, with bronchial ASMCs being more prone to proliferation than tracheal ASMCs.

Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway

The airway epithelium is responsible for the production of a number of arachidonic acid and non-prostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM-100 microM) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KC1 in the presence or not of 10(-5) Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)-monomethyl L-arginine (L-NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 microM) on KC1 contraction induced a relaxation of 10% of the initial contraction. The additional use of L-NMMA decreased the relaxation to 5% of initial contraction. MB rather than L-NMMA increased the contraction significantly (p<0.01). Epithelium removal increased the contraction induced by KC1 (80 mM) and histamine (1 microM) by about 30% (p<0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma.

TNFα induces expression of HIF-1α mRNA and protein but inhibits hypoxic stimulation of HIF-1 transcriptional activity in airway smooth muscle cells.

Airway smooth muscle cells (ASMCs) participate in tissue remodeling characteristic of airway inflammatory diseases like asthma. Inflammation and hypoxia pathways are often interconnected and the regulatory subunit of the hypoxia inducible factor, HIF‐1α, has been recently shown to be induced by cytokines. Here we investigate the effect of individual or combined treatment of ASMCs with the inflammatory mediator TNFα and/or hypoxia on the expression of HIF‐1α, HIF‐1 targets and inflammation markers. TNFα enhances HIF‐1α protein and mRNA levels, under both normoxia and hypoxia. TNFα‐mediated induction of HIF‐1α gene transcription is repressed by inhibition of the NF‐κB pathway. Despite the up‐regulation of HIF‐1α protein, the transcription of HIF‐1 target genes remains low in the presence of TNFα at normoxia and is even reduced at hypoxia. We show that the reduction in HIF‐1 transcriptional activity by TNFα is due to inhibition of the interaction of HIF‐1α with ARNT and subsequent blocking of its binding to HREs. Comparison between hypoxia and TNFα for their effects on the expression of inflammatory markers shows significant differences: hypoxia up‐regulates the expression of IL‐6, but not RANTES or ICAM, and reduces the induction of VCAM by TNFα. Finally, ex vivo treatment of rabbit trachea strips with TNFα increases HIF‐1α protein levels, but reduces the expression of HIF‐1 targets under hypoxia. Overall, TNFα induces HIF‐1α mRNA synthesis via an NF‐κB dependent pathway but inhibits binding of HIF‐1α to ARNT and DNA, while hypoxia and TNFα have distinct effects on ASMC inflammatory gene expression. J. Cell. Physiol. 228: 1745–1753, 2013.

Resting tension effect on airway smooth muscle: the involvement of epithelium.

We studied the influence of resting tension (RT) on rabbit tracheal smooth muscle (TSM) contractions induced by acetylcholine or KCl as well as the role of epithelium and the endogenously produced nitric oxide, prostanoids and endothelin on these responses. The alteration of RT from 0.5 to 2.5 g increased the responsiveness of TSM to KCl. The presence of atropine decreased KCl-induced contractions obtained only at 2.5 g RT. The removal of epithelium increased acetylcholine-induced contractions, only at 2.5 g RT. At 0.5 g RT, the presence of L-NAME had no effect on acetylcholine-induced contractions while indomethacin decreased contractions induced by 10(-3) M acetylcholine. At 2.5 g RT, the presence of L-NAME increased acetylcholine-induced contractions while indomethacin, BQ-123 and BQ-788 had no effect. These results demonstrate that RT affects the responsiveness of TSM differentially, depending on the agonist or integrity of the epithelium. Airway epithelium modulates acetylcholine-induced contractions, only at 2.5 g RT partly via NO release. At 0.5 g RT, the endogenous production of prostanoids by sources other than epithelium modulates the contractility of TSM to acetylcholine.

Azithromycin reduces the viability of human bronchial smooth muscle cells

The macrolide antibiotic azithromycin has an antiproliferative and autophagic effect on rabbit tracheal smooth muscle cells (SMCs). The purpose of this study is to investigate the effect of azithromycin on human bronchial SMCs. Human bronchial SMCs were treated with azithromycin (10−5 M) in the presence or absence of 10% fetal bovine serum (FBS). Cell number was estimated using the Cell Titer 96 AQueous One Solution Assay. Induction of autophagy was studied by observation of cell morphology in cells treated or not with the autophagy inhibitor, 3-methyladenine (3-MA), as well as by Lysotracker Red staining of lysosomes. Activation of apoptosis was assessed with flow cytometry after annexin staining. Incubation with azithromycin for 24, 48 or 72 h reduced viability in FBS-deprived cells, as well as cells cultured in FBS-containing medium. Azithromycin treatment resulted in the formation of cytoplasmic vacuoles that could not be prevented by 3-MA. Furthermore, 3-MA did not reverse the effect of azithromycin on the viability of SMCs. There was an increase in the number of lysosomes in cells treated with azithromycin. Finally, azithromycin increased the percentage of early apoptotic cells. In conclusion, azithromycin reduces the viability of human bronchial SMCs possibly by leading to apoptotic cell death.

Epithelium-dependent effect of L-glutamate on airways: involvement of prostaglandins

We investigated the effect of the excitatory amino acid (EAA) receptor agonists L-glutamate, N-methyl-D-aspartate (NMDA), (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid on KCl-induced contractions of rabbit tracheal smooth muscle, as well as the role of epithelium and endogenously produced nitric oxide and prostaglandins on these responses. L-Glutamate decreased KCI-induced contractions up to 30%. This effect was attenuated by epithelium removal, tetrodotoxin, methylene blue and indomethacin but not by NG-nitro-L-arginine methyl ester. While NMDA, AMPA and kainic acid had no effect, the combination of NMDA + kainic acid decreased KCI-induced contractions. These results suggest that, in rabbit trachea, L-glutamate has, at least in part, an epithelium-dependent effect mediated via prostaglandin formation and that the EAA receptors involved are non-classical.