Fig 1.
Aging increases senescence marker gene expression in human Airway Smooth Muscle (ASM) cells.
mRNA was isolated from human ASM and p21, p16, and p53 gene expression was assessed by quantitative PCR. Compared to the young, p21 gene expression was increased in middle aged ASM cells (A). Senescent marker p16 was increased in middle aged ASM (B). Protein analysis for p21 (D), p53 (E) or p-p53 (F) normalized to GAPDH showed consistent changes for p53. Data are shown as mean ± SEM from N = 5–6 samples. Fold change was quantified against the average value of the samples from young individuals. * indicates significance P ≤ 0.05.
Fig 2.
Aging increases senescence markers in ASM.
Immunofluorescence staining for p21 a marker of cell cycle arrest (A) showed increased levels in elderly ASM. γH2AX, a marker for DNA damage, increased in elderly ASM (B). Staining for senescence-associated beta galactosidase (β-Gal) increased in both middle age and elderly ASM cells (C). Data shown as mean ± SEM from n = 5–7 patients. * indicates significantly different p≤0.05.
Fig 3.
SASP release from ASM changes with age.
Media were collected from ASM grown to confluence and serum starved for 4 days. Cytokine concentrations were measured via ELISA. CCL2 secretion was decreased in elderly ASM (A). IL-8 secretion increased in elderly ASM (B). IL-6 release decreased in middle aged ASM cells (C). Data shown as mean ± SEM from n = 7–9 patients. *indicates significant difference P≤0.05.
Fig 4.
Aging alters ASM proliferation and extracellular matrix (ECM) deposition.
To measure proliferation cells were plated in 1% serum and allowed to grow for 3 days. Cells were then stained for Ki67 and positive cells relative to total counts (DAPI) were measured. Proliferation decreased in elderly ASM cells (A). A modified Li-Cor In-Cell Western technique (semiquantitative immunofluorescence) was used to quantify deposition of ECM proteins by ASM grown to confluence and serum starved for 4 days. Aging increased deposition of collagen III (C), and fibronectin (D). Immunoblots showed aging-associated decrease in matrix modifying proteins MMP9 (E) and MMP2 (F). Data shown as mean ± SEM from n = 4–7 patients. * indicates significant difference p≤0.05.
Fig 5.
Acetylcholine-induced [Ca2+]i in aging human ASM cells.
Representative tracings in young <45, middle age 45–65, and elderly>65 human ASM (A&B). ACh (10μM, black arrow) was applied to Fura 2 AM loaded cells in 2 mM Ca2+ HBSS. Baseline intracellular calcium was not changed (C). Peak (E) and amplitude (G) of [Ca2+]i was increased in elderly ASM. To measure changes in [Ca2+]i release following contractile agonist stimulation independent of Ca2+ influx ACh (10μM) was applied to Fura 2 AM loaded cells in 0 mM Ca2+ HBSS (B). Baseline [Ca2+]i was not changed (D). Peak (F) and amplitude (H) of [Ca2+]i increased in elderly ASM. Data represents mean ± SEM from n of 6–7 patients. * indicates significant effect (p≤0.05).
Fig 6.
Histamine induced [Ca2+]i increased in elderly human ASM.
Representative tracings in young <45, middle age 45–65, and elderly>65 human ASM (A). Histamine (10μM, black arrow) was applied to Fura 2 AM loaded cells in 2 mM Ca2+ HBSS. Baseline [Ca2+]i was not changed with age(C). After histamine peak (E) and amplitude (G) of [Ca2+]i was increased in elderly ASM. To measure changes in [Ca2+]i release following contractile agonist stimulation independent of influx in ASM histamine (10μM) was applied to Fura 2 AM loaded cells in 0 mM Ca2+ HBSS. Baseline in[Ca2+]i was not changed (D). After histamine peak F) and amplitude (H) of [Ca2+]i in elderly ASM. Data represents mean ± SEM from N of 6–7 patients. * indicates significant effect (p≤0.05).
Fig 7.
Store Operated Calcium Entry (SOCE) is not changed with age.
Representative traces for SOCE in ASM (A). In the representative trace individual lines show the variations in cellular responses for a single well measured. The rate of Ca2+ influx was not changed with age (B). Similarly, the amplitude (C) of Ca2+ influx was not changed with age. Data represents mean ± SEM from N of 4–5 patients. * indicates significant effect (p≤0.05).
Fig 8.
ASM Ca2+ signaling receptors expression decreased with age.
Total protein was isolated from young <45, middle age 45–65, and elderly >65 ASM. Wes analysis was used to measure changes in protein expression. Graphs represent relative expression vs GAPDH. ASM isolated from elderly patients (>65) had significantly decreased M3 muscarinic receptor (A), histamine receptor (B), SERCA2 (G), and Orai1 (H) expression. Fold change was quantified using an average of young. Data shown as mean ± SEM from n = 4–7 patients. * indicates significant effect (p≤0.05).