Fibroblasts protect 621–101 cells against apoptosis.

<p>(a) Culture of either 621–101 cells or fibroblasts on glass in serum free conditions results in cell death. Co-culture of the two cell types under the same conditions results in formation of viable cell colonies. (b) Under serum-free conditions, 621–101 cells have a basal rate of apoptosis of 8% assessed by TUNEL positive nuclear staining (arrowed). TUNEL positivity is reduced by half in the presence of fibroblast conditioned medium (*p = 0.032).</p

Characteristics of wild-type fibroblast-like cells obtained from LAM lungs.

<p>(a) Explant culture of lung tissue from patients with LAM yields a fibroblast-like population with spindle shaped morphology under phase contrast microscopy (phase) and immunoreactivity to anti-alpha Smooth Muscle Actin, and fibroblast markers FSP, S100A4 and TE-7 (these cells are also DAPI stained for clarity). (b) Western blot of lysates of fibroblast like-cells from two donors with LAM compared with TSC-2 mutation bearing 621–101 cells. Unlike 621–101 cells fibroblast-like cells have full length tuberin, and suppression of phospho-S6 in the absence of serum.</p

LAM nodules express fibroblast markers.

<p>LAM nodules react with antibodies against alpha-Smooth Muscle Actin, Fibroblast Surface Protein (FSP), Fibroblast Activation Protein (FAP) and S100A4. Left panels are x20 magnification and right are inset area at x40 taken from serial sections of a representative donor. Fibroblast markers display different expression patterns within LAM tissue, with anti-FSP reacting with 30–70% of cells within a nodule, anti-FAP detecting the majority of cells and S100A4 detecting only small nests of cells within nodules.</p

FSP is expressed by variable numbers of cells within LAM nodules.

<p>There is heterogeneity between different LAM lung donors in the number of anti-FSP immunoreactive cells in LAM nodules. Panels (a) and (b) at x10 magnification, (c) at x20, (d) at x40. (e) and (f) show anti-tuberin reactive and non-reactive cells (arrowed) within nodules from two donors, x20.</p

Expression of tenascin-C and MMP-1 in the airway.

<p>Endobronchial biopsies from seven patients with asthma and five controls stained for MMP-1 and tenascin-C. Representative images of smooth muscle bundles and epithelium are shown with positive staining visible as violet colouration. Original magnification ×100.</p

Asthma ASM cells secrete greater quantities of MMP-1 protein than control cells and control by ECM.

<p>(a) Six asthma derived and six control ASM cultures were incubated for 24 hours with and without the addition of soluble tenascin-C (10 µg/ml). The supernatant was harvested and levels of total MMP-1 assessed by ELISA. Expression levels were analysed using an unpaired one-tailed t-test (*P<0.05. **P<0.01). (b) ASM cells from four asthma and three control donors were incubated with a β3 integrin subunit function blocking antibody prior to stimulation with tenascin-C (10 µg/ml). Supernatant was harvested after 24 hours and assayed for MMP-1 expression. Expression levels for control and asthma ASM were separately normalised to an equivalent concentration of an isotype control, stimulated with tenascin-C (10 µg/ml). (c) Three control ASM cell cultures were seeded on ECM deposited by ASM cells from six asthma and six control donors. After 24 hours total MMP-1 was assessed by ELISA. Expression was analysed using a 1 way ANOVA with <i>post-hoc</i> Dunnett’s Multiple comparison test (**P<0.01. ***P≤0.001). (c) Serum starved ASM cells from four asthma and three control donors were seeded onto tissue culture plastic for six hours and TNC or COL1A1 mRNA assessed by qRT-PCR. Levels are expressed as mean fold change in cycle threshold number relative to GAPDH and compared by paired t-test (*P<0.05).</p

Tenascin-C induced MMP-1 secretion is mediated by the β1 and β3 integrin subunits.

<p>(a) ASM cells processed for flow cytometry were incubated with antibodies against the integrin subunits β1, β3 and the αvβ5 receptor prior to incubation with a fluorescently conjugated secondary antibody. Histogram depicts fluorescence intensity detected by flow cytometry (one representative experiment of three shown) for antigens (shaded) and their respective isotype control (broken line). (b) ASM cells were incubated with the same antibodies as listed above prior to stimulation with tenascin-C (10 µg/ml). Supernatant was harvested after 24 hours and assayed for MMP-1 protein. Expression levels analysed using a 2-way ANOVA with <i>post-hoc</i> Dunnett’s Multiple comparison test (*P≤0.05. ****P≤0.0001). Graph depicts results normalised to the isotype control (stimulated with tenascin-C (10 µg/ml)) at equivalent concentration, representative experiment of three separate donors is shown.</p

Induction of MMP-1 protein by tenascin-C is ERK1/2, JNK and p38 dependent.

<p>(a) ASM cells were stimulated with soluble tenascin-C (10 µg/ml). Lysates were harvested at 20 minutes and assayed by immunoblot for a panel of phosphorylated signalling proteins using proteome profiler. (b) ASM cells from three normal donors were stimulated with soluble tenascin-C (10 µg/ml). Lysates were harvested at 20 minutes and 24 hours, with levels of total and phosphorylated signalling proteins assayed by Western Blotting. (c) ASM cells from three normal donors were incubated for 30 minutes with inhibitors of JNK (SP600125, 10 µmol), MEK1/2 (U0126, 10 µmol), Rac1 (NS23766, 100 µmol), p38 (SB208350, 10 µmol), mammalian target of rapamycin complex 1 (rapamycin 10 nmol), PI3K (wortmanin 100 nmol) or glycogen synthase kinase αβ (GSK3αβ, SB216763 10 µmol) prior to stimulation with tenascin-C (10 µg/ml). Supernatant was harvested after 24 hours and assayed for MMP-1 protein. Expression levels were normalised to vehicle control (0.02% DMSO, tenascin-C (10 µg/ml)) and analysed using a 1-way ANOVA with <i>post-hoc</i> Dunnett’s multiple comparison test (*P≤0.05. ***P<0.001).</p

MMP expression and activation enhances ASM contraction.

<p>(a) Bradykinin-stimulated contraction of 3D collagen gels containing ASM cells is concentration dependent. (b) Ilomastat pretreatment (10 µmol) reduces bradykinin induced contraction when compared with vehicle control. Changes are analysed using a two way Repeated-Measures ANOVA with post-hoc bonferroni multiple comparison (c) siRNA against MMP-1 reduces MMP-1 expression in 3D collagen gel supernatants and analysed using a one way ANOVA with post-hoc Dunnet’s multiple comparison test (d) siRNA against MMP-1 reduces ASM contraction compared with transfection of a control siRNA and analysed by two way Repeated-Measures ANOVA with post-hoc bonferroni multiple comparison test. Graphs represent mean +/−SE of four replicate gels from three donors repeated on three occasions. (**P<0.01. ***P≤0.001).</p

Literature-based and estimated parameter values used in the ASM growth model.

<p>(^* indicates the values estimated from <i>in vitro</i> experiments.)</p