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. 2016 Feb;71(2):153-60.
doi: 10.1093/gerona/glu241. Epub 2015 Jan 7.

Senescent Cells Contribute to the Physiological Remodeling of Aged Lungs

Cheresa Calhoun  1 , Pooja Shivshankar  1 , Mirna Saker  2 , Lauren B Sloane  3 , Carolina B Livi  4 , Zelton D Sharp, Carlos J Orihuela  5 , Serge Adnot  2 , Eric S White  6 , Arlan Richardson  3 , Claude Jourdan Le Saux  7
Affiliations

Senescent Cells Contribute to the Physiological Remodeling of Aged Lungs

Cheresa Calhoun et al. J Gerontol A Biol Sci Med Sci. 2016 Feb.

Abstract

Age-associated decline in organ function governs life span. We determined the effect of aging on lung function and cellular/molecular changes of 8- to 32-month old mice. Proteomic analysis of lung matrix indicated significant compositional changes with advanced age consistent with a profibrotic environment that leads to a significant increase in dynamic compliance and airway resistance. The excess of matrix proteins deposition was associated modestly with the activation of myofibroblasts and transforming growth factor-beta signaling pathway. More importantly, detection of senescent cells in the lungs increased with age and these cells contributed toward the excess extracellular matrix deposition observed in our aged mouse model and in elderly human samples. Mechanistic target of rapamycin (mTOR)/AKT activity was enhanced in aged mouse lungs compared with those from younger mice associated with the increased expression of the histone variant protein, MH2A, a marker for aging and potentially for senescence. Introduction in the mouse diet of rapamycin, significantly blocked the mTOR activity and limited the activation of myofibroblasts but did not result in a reduction in lung collagen deposition unless it was associated with prevention of cellular senescence. Together these data indicate that cellular senescence significantly contributes to the extracellular matrix changes associated with aging in a mTOR 1-dependent mechanism.

Keywords: Aging; Rapamycin; Remodeling; Senescence; mTOR pathway.

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Figures

Figure 1.
Figure 1.
Aged lungs are associated with a profibrotic environment and changes in lung mechanics in wild-type C57BL/6J (WT). (A) PSR staining and quantitation of collagen deposition in 8-, 24-, and 32-mo old lung tissues. Arrows indicate the collagen deposition. Magnification ×200. The bar graph represents the quantification of the collagen deposition using ImagePro software. (B) Representative photomicrographs of IHC staining for OPN and TNC-c indicative of an increase in extracellular matrix proteins expression in the parenchyma and vessel walls in mice older than 12-mo compared with 3-mo old mice. Magnification ×400. (C) Endotracheal intubation was performed on deeply anesthetized mice. Ventilation was set at 150 breaths/min (normal breathing rate) with a positive end-expiratory pressure of 2 cmH2O. Airway resistance and dynamic compliance were significantly increased overtime in the lungs of the animals from 8 to 32 mo of age; dynamic elastance did not vary over time (n = 8–10 animals per age group). Data presented are mean ± SEM. One-way analysis of variance Tukey post-test, *p < .05. IHC = immunohistochemistry; OPN = osteopontin; PSR = picrosirius red; TNC-c = tenascin-c.
Figure 2.
Figure 2.
Cellular senescence increased in mouse lungs with age. (A) DNA damage response signaling pathway. Densitometric quantifications of p53, p21, and γH2x WB analysis carried out with whole lung homogenates of 8-, 24-, and 32-mo old male C57B/6J mice. Relative levels were calculated relative to actin level as the loading control. (B) Oxidative stress signaling pathway. Densitometric quantification of pRb, p16, and MH2A WB analysis. Relative levels were calculated relative to actin level as the loading control analysis carried out with whole lung homogenates of 8-, 24-, and 32-mo old male C57B/6J mice. (C) Representative IHC staining of MH2A in 8-, 24-, and 32-month old male mouse lungs. n = 5; statistical significance: p value *<.05; **<.01; ***<.005. IHC = immunohistochemistry; WB = Western blot.
Figure 3.
Figure 3.
Extracellular matrix proteins are secreted by senescent cells. Representative microphotographs of the detection of p16-positive cells in young mice (2–3 mo old) and mature (older than 12 mo) by IHC. Arrows are indicative of positive-stained cells. Bar graph representation of the percentage of positive cells counting using ImageJ software in the parenchyma and vessel walls. Original magnification ×400. n = 5 animals and at least five sections per animal. OPN and TNC-c protein expression was assessed by immunofluorescence staining (red) in relation to the senescence status of the cells evaluated by immunofluorescence staining of p16 (green). Arrows are indicative of positive-stained cells. Statistical significance: p value **<.01; ***<.005 between young and mature animals; $$<.01 between parenchyma and vessel walls. n = 3. IHC= immunohistochemistry; OPN = osteopontin; TNC-c = tenascin-c.
Figure 4.
Figure 4.
AKT/mTOR activation in aged mouse lungs. Western blot analyses of AKT/mTOR pathway activation (A) Akt, (B) mTOR, (C) mTOR downstream effector p70S6 kinase and densitometric quantitation carried out in whole lung homogenates of lungs isolated from 8-, 24-, and 32-mo old male mice. One-way ANOVA Tukey post-test: *p value <.05 and **p value<.01. n = 3. ANOVA = analysis of variance; mTOR = mechanistic target of rapamycin.
Figure 5.
Figure 5.
Oral rapamycin treatment reduces mTOR activation and cellular senescence in mouse lungs. (A) WB analyses and quantitation of phospho-p70S6 kinase/total p70S6 kinase in whole lung homogenates of mice from control (Eudragit), rapamycin fed for short-term (ST rapa) and long-term (LT rapa) for a duration of 3wk and 4 mo, respectively, whereas 14 and 42 ppm denotes the concentration of rapamycin fed to mice for the same amount to time (4 mo). (B) Positive p16 cells in the parenchyma and vessel walls as indicated by the arrows. (C) The quantification of the positive p16 cells in the parenchyma and the vessel walls was determined using ImageJ software. N = 5 animals per treatment group/2–3 slides per animal. Statistical significance: p value *<.05; **<.01; ***<.001 demonstrate statistical significance of the rapamycin treatment compared with Eudragit diet within a tissue compartment, and p value $<.05 demonstrates statistical significance between two tissue compartment within the same treatment group. mTOR = mechanistic target of rapamycin; WB = Western blot.
Figure 6.
Figure 6.
Rapamycin inhibits profibrotic environment in the lungs in a dose-dependent manner. (A and B) PSR staining and quantitation of collagen deposition in the lung tissue sections from control mice (Eudragit), rapamycin fed for short-term (St rapa) and long-term (Lt rapa) for a duration of 3wk and 4 mo, respectively, whereas 14 and 42 ppm denotes the concentration of rapamycin fed to mice for the same amount to time (4 mo). (C) Representative WB of α-SMA and their densitometric quantitations carried out using the whole lung homogenates of mice from control, rapamycin fed for short-term (14 ppm ST) and long-term (14 ppm LT). p value *<.05; **<.01 between control and treatment group and p value ###<.001 between treatment group. α-SMA = α-smooth muscle actin; PSR = picrosirius red; WB = Western blot.
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