Figure 1.
Treatment with GRN510 enhances telomerase function in the lung using a bleomycin-induced model of pulmonary fibrosis. A.
Analysis of telomerase activity levels in lung tissue for Tert Het mice dosed with GRN510 at 10 mg/kg/d for 21 days post-Bleomycin treatment (23 days total). Intratracheal injection of Bleomycin was performed on day 0. The mean telomerase activity level for the GRN510 treated and vehicle control mice are shown (n = 4–10 per group). The average activity level for the vehicle control group was arbitrarily assigned a value of 1.0. Asterisk, * denotes significant difference relative to the vehicle control mice (Student's t Test). B. Analysis of the effect of telomerase inhibitor GRN163L on GRN510-mediated telomerase activation in the lung. Mice were dosed with GRN510 (10 mg/kg/d) and/or the telomerase inhibitor GRN163L (13 mg/kg/d) as indicated for 21 days (n = 5–10 for all groups). The average activity level for the vehicle control group was arbitrarily assigned a value of 1.0. Asterisks, (*, **) denotes significance amongst the indicated groups (pvalue≤0.03, 1-way ANOVA; P≤0.01 comparing GRN510+GRN163L treated group to GRN510 treated group Student’s t Test). C. Sample southern blot for analysis of terminal restriction fragment length of lung tissue after GRN510 (TA-telomerase activator) treatment. A control sample for untreated Tert Het mice is represented by 'θ'. Size of molecular weight standards are shown on the left (Kb). D. Quantitative analysis of mean TRF length lung tissue after 3 weeks of GRN510 treatment. For each group, n = 5–10. Asterisk * denotes significance amongst indicated groups; P = 0.03, 1-way ANOVA). E. Mean TRF length analysis of PBS control mice. Analysis was performed after 3 weeks of dosing (all mice dosed as described above). N = 4 per group. Asterisk, * denotes significance amongst groups (P = 0.02, 1-way ANOVA). For all plots, mean ± SEM is shown.
Figure 2.
GRN510 and GRN163L treatments did not affect the structure and the function of the lungs.
Mice were treated with GRN510, GRN163L, and GRN510+ GRN163L for 3 weeks. Expression of α-smooth muscle actin (αSMA) and collagen deposition was evaluated as well as the baseline lung mechanics. A. Representative picorsirius red staining (upper panel) and immunostaining of α−SMA (lower panel) are shown; X200. B. Collagen deposition was quantified using ImagePro software by blinded personnel to the treatment groups. C. Dynamic compliance (ml/cm H2O) was measured. Treatment with the 2 components, alone or in combination indicated no difference in compliance among groups. Statistical analysis was performed by 1-way ANOVA (Prism, GraphPad Software, Inc). N = 4 per group (same animals as assessed in Fig. 1E); For all plots, mean ± SEM is shown.
Figure 3.
Reduced fibrotic response in bleomycin-injured lungs after treatment with GRN510. A.
Tert Het mice were treated with GRN510 for 3 weeks at the indicated dose and subjected to bleomycin-induced fibrosis (day 0). Representative histological sections from the whole left pulmonary lobe were taken from each animal after 3 weeks of dosing and stained with H&E (Left panel), picrosirius red (middle panel), and a-SMA (Right panel). All these stainings indicate that treatment with GRN510 attenuates the bleomycin injury and in a dose dependent manner. B. Levels of picrosirius red staining were quantified with ImagePro software. Asterisk, * denotes significance amongst indicated groups (P = 0.01, 1-way ANOVA) C. Dynamic compliance (ml/cm H2O) was significantly increased in the lungs of bleomycin-treated Tert Het mice. This increase in compliance was attenuated after GRN510 treatment 3 weeks after bleomycin injury (P = 0.01; 1-way ANOVA). N = 4–10 animals per group, values given are mean ± SEM.
Table 1.
Evaluation of collagen content in lung tissues (Ug of collagen/mg of tissue).
Figure 4.
The GRN510-mediated protection from bleomycin-induced fibrosis is dependent on telomerase activation.
Tert Het mice subjected to Bleomycin-induced fibrosis were treated with GRN510 (10 mg/kg/d) and the telomerase inhibitor GRN163L as indicated. A. Representative histological sections were obtained from the whole left pulmonary lobe taken from each animal 11 days after bleomycin-induced injury and stained with H&E (Top panel) or with picrosirius red (Lower panel). To minimize variation, samples were processed under identical conditions. B. Levels of picrosirius red staining were quantified with ImagePro software. Asterisk, * indicates significance amongst indicated groups (P = 0.02, 1-way ANOVA). C. RT-PCR expression levels of procollagen1α mRNA in the lung. Analysis was performed in triplicate for each specimen. Asterisk, * denotes significance amongst indicated groups (P = 0.04, 1-way ANOVA). N = 5–10 animals per group; for all plots, mean ± SEM is shown.
Figure 5.
Reduced cellular senescence in bleomycin-injured lung tissue with GRN510 treatment.
All analyses were performed on the same mice as in Fig. 4. A. Analysis of SA-β galactosidase activity in the lung tissue. Shown are representative paraffin-embedded lung sections after staining for SA-β gal activity (blue staining). Increased SA-β gal activity was detected in the mice challenged with bleomycin and treated with vehicle or GRN510 and GRN163L. In contrast, reduced SA-β gal activity was detected in the mice challenged with bleomycin and treated with GRN510. Positive stained cells are encircled. Magnification 40X. Representative paraffin embedded lung sections immunohistochemically stained for MH2A using enhance blank staining solution (KPL, Gaithersburg, MD); n = 3 different specimens with 3 fields of view analyzed per section; Hematoxylin was used as a negative counter-stain. Magnification of 40X. Circles denote positive staining. B. Analysis of SIRT6 and p21 expression in the lung. Targeted genes and β-actin expression at the mRNA level was determined by qRT-PCR. Analysis was performed in triplicate for each specimen. N = 5–10 animals per group. Asterisk, * indicates significance amongst indicated groups (P = 0.002, 1-way ANOVA). C. Western analysis of p53 expression. A trend in increase expression was associated with bleomycin treatment that was partially reverted by GRN510 treatment. For all plots mean ± SEM is shown.
Figure 6.
The effect of GRN510 in the lung tissue is cell type dependent. A.
Cultured small airway epithelial cells (SAEC) or lung fibroblasts (IMR90) were treated with GRN510 for 48 hours at the indicated doses. Telomerase activity was subsequently assessed in extracts prepared from equal numbers of cells using the TRAP assay. A sample gel analysis of activity is shown. B. Quantitation of telomerase activity for the extracts assessed in A. C. Analysis of expression of the senescence markers P21 and P16 following long term culture of SAEC cells in the presence of GRN510. Cells were grown continuously in the presence of GRN510 at the indicated doses. Expression was assessed using real time RT-PCR. P values represent Student t Test between indicated groups. For all plots mean ± SEM is shown.