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J Appl Physiol 102: 1632-1639, 2007. First published January 11, 2007; doi:10.1152/japplphysiol.00833.2006
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Variation in senescent-dependent lung changes in inbred mouse strains

Kewu Huang, Wayne Mitzner, Richard Rabold, Brian Schofield, Hannah Lee, Shyam Biswal, and Clarke G. Tankersley

Johns Hopkins Bloomberg School of Public Health, Department of Environmental Health Sciences, Baltimore, Maryland

Submitted 28 July 2006 ; accepted in final form 10 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies from our laboratories showed lung development differences between inbred strains of mice. In the present study, the C57BL/6J (B6) and DBA/2J (D2) strains were examined for senescent-dependent differences with respect to the lung structure and function. Specifically, we hypothesize that senescent changes in lung vary between strains due to identifiable gene expression differences. Quasi-static pressure-volume curves and respiratory impedance measurements were performed on 2- and 20-mo-old B6 and D2 mice. Lung volume at 30 cmH2O (V30) pressure was significantly (P < 0.01) increased with age in both strains, but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. In addition, decreased elastic recoil pressure at 50% of V30 and a reduction in airway resistance as a function of positive end-expiratory pressure were observed in 20-mo-old D2 mice but not in B6 mice. Morphometric analysis of lung parenchyma showed significant decreases in elastic fiber content with age in both strains, but the collagen content was significantly (P < 0.01) increased with age in D2 but not B6 mice at 20 mo. Furthermore, using quantitative RT-PCR methods, gene expression differences between strains suggested that D2 mice significantly (P < 0.05) downregulated the expressions of elastin (Eln) and procollagen I, III, and VI (Col1a1, Col3a1, and Col6a3) in lung tissue at 20 mo of age. These age-dependent changes were accompanied by an increased gene expression in matrix metalloproteinase 9 (Mmp9) in D2 and an increase in tissue inhibitor of matrix metalloproteinase (Timp1 and Timp4) in B6 mice. In conclusion, the results from the present study demonstrate that lung mechanics of both strains show significant age-dependent changes. However, changes in D2 mice are accelerated relative to B6 mice. Moreover, gene expression differences appear to be involved in the strain-specific changes of lung mechanic properties.

C57BL/6J; DBA/2J; aging; lung mechanics; extracellular matrix

AGING EFFECTS on lung structure and mechanics in humans have been investigated and reviewed in several previous reports (6, 27, 40). In general, senescent-dependent changes in lung mechanics are summarized by a reduction in lung elastic recoil and an increase in residual volume in the absence of a change in total lung capacity (TLC) (6). Other studies have demonstrated that progressive decline in lung function with age is variable among individuals, implying that there are genetic determinants involved in the senescent process (5, 34). However, in humans it is difficult to address the interaction between genetic determinants and aging per se since recruiting senescent volunteers without significant exposure to smoking or air pollution is unfeasible. Alternatively, senescent-dependent changes in lung structure and mechanics have been investigated in rodent models. In Sprague-Dawley rats, for example, a leftward shift in the pressure-volume (P-V) curve was observed, and lung elastance was reduced at high lung volume in the aged group (23). Hirai and colleagues (13) showed senescent-dependent increases in static compliance, dynamic compliance, and TLC in a model of accelerated aging. In short, little is known about the effects of aging per se on alveolar architectural structure, the maintenance of extracellular matrix (ECM), and the consequences of senescence on lung mechanics in humans or in animal models. In particular, the interactive effects between genetic predisposition and aging on lung structure have not been studied.

Numerous studies have shown substantial variation in lung structure and mechanics among inbred mouse strains (16, 30, 38). A comparison of lung structure in male C57BL/6J (B6) and DBA/2J (D2) mice, for example, showed that D2 mice had a larger alveolar air proportion, smaller mean chord length (MCL) of ducts, and smaller fixed lung volume per fresh lung weight (16). These observations lead to a difference in the recoil properties of the lung between the two mouse strains. In addition, B6 and D2 inbred mice have been routinely used to study lung pathophysiological effects of exposure to cigarette smoke (4), bleomycin (7), ozone (32), mycobacterium (2), and allergen (43). These two strains have been shown to be widely different in their genetic characteristics. However, little information is available on the effects of aging per se in altering lung structure and mechanics in these two strains. Therefore, the purpose of this study is to investigate the interactive effects of aging and genetic predisposition, which may lead to individual variation in onset and severity of senescent changes in lung mechanics. We hypothesize that the genetic determinants, which regulate strain differences in lung structure and mechanics in younger B6 and D2 mice, are exacerbated with senescence. Furthermore, the interaction between genetic variability and the aging process might accelerate adverse changes in lung structure and mechanics. To address this question, quasi-static P-V curves, positive end-expiratory pressure (PEEP)-dependence of airway and tissue impedance, elastic fiber and collagen content in lung tissue, and gene expression levels related to lung ECM were studied in adult (2 mo) and aged (20 mo) B6 and D2 mice.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.   Male B6 and D2 strains of mice were purchased from Jackson Laboratories and the aged mouse colonies at the National Institute on Aging. The two strains of mice were studied at 2 and 20 mo of age. The animals were housed in a facility at the Johns Hopkins University School of Public Health. The temperature was maintained at ~21.5°C, and the light-dark cycle was 12:12 h, beginning at 0700. The animals were housed in cages and fed ad libitum with a pelleted stock diet. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee and complied with the American Physiological Society Guidelines.

Respiratory impedance measurements.   Animals were anesthetized with intraperitoneal injections of pentobarbital sodium at a dose of 80 mg/kg body wt. After the trachea was cannulated, the mouse was connected via the tracheal cannula to a computer-controlled small animal ventilator (FlexiVent, Montreal, Canada) while in a supine position. All mice were mechanically ventilated with 100% O2 at 150 breaths/min and a tidal volume of 10 ml/kg, and each animal was paralyzed with an intraperitoneal injection of 0.05 ml succinylcholine (9 mg/ml). Stable breathing in each animal was attained after 3 min, at which time a deep inspiration was applied at an airway pressure of 30 cmH2O. The impedance of the respiratory system was measured 2 min after the deep inspiration and then fitted by Flexivent software to a constant-phase model (11, 12) to provide measures of airways resistance (R), tissue damping (G), tissue elastance (H), and tissue hysteresivity (eta). For the measurement of impedance, a computer-generated volume signal composed of 19 mutually primed sinusoids ranging from 0.25 to 19.625 Hz was applied to the airway opening. Measurements were made at four different PEEP levels of 2, 5, 10, and 15 cmH2O with 1-min intervals between each PEEP level.

P-V curve.   After the impedance measurements, mice were mechanically ventilated with 100% O2 at a PEEP of 2 cmH2O for 1 min, and the cannula was sealed with a stopcock for 3 min to degas the lung. Quasi-static P-V curves were then performed ex vivo with the respiratory system intact. The rate of inflation and deflation was standardized by a dual-infusion withdraw pump (model 900-610, Harvard Apparatus, Dover, MA), and the airway pressure was measured by using a differential pressure transducer (model 8510B-2, Endevco). The initial inflation rate was controlled at ~0.75 ml/min to ensure that all lung regions opened before being switched to a rate of 2 ml/min for the remaining inflation-deflation maneuvers. The pressure limits of the inflation and deflation airway pressures were 30 and –10 cmH2O, respectively. The volumes on deflation at 0 cmH2O (V0) and 30 cmH2O (V30) were considered to represent functional residual capacity (FRC) and TLC, respectively. Compliance of the intact respiratory system was computed from the slopes of the P-V relationships between 5 and 0 cmH2O (Crs5–0) and between 15 and 10 cmH2O (Crs15–10) on deflation.

MCL of alveoli and lung tissue density.   After the P-V curve was completed, the cannulated trachea was connected to a 10-cmH2O column of Zenker's fixative (mercuric chloride/acetic acid fixative, EM Science, Gibbstown, NJ) for 4 h. To more consistently control the selection of sample sections, only the left lungs were processed and analyzed. We selected lung sections by a standardized procedure as described by Soutiere et al. (36). From the left lung, the top 3-mm section was removed and discarded, and then three serial, 2-mm sections were removed and processed for histology. Blocks were washed in 70% alcohol twice followed by 80% alcohol preservation, then embedded in paraffin in methacrylate. For the measurement of MCL, 5-µm-thick sections were cut and stained with 0.05% toluidine blue. From each section, seven nonoverlapping 676 µm x 505 µm fields were sampled, deliberately avoiding the large airways and blood vessels. Each of these regions was photographed for digital analysis, and conventional morphometric methods were used to determine MCL of alveoli using NIH Image software (version 1.62). For consistency with previous studies (36), chord lengths <8 µm or >250 µm were excluded. The lung tissue density (TD) was computed as the percentage of the total area of the lung tissue divided by the total area of the histological field. Like the MCL measurements, 21 histological samples were obtained from each animal and averaged for a given animal.

Quantification of elastic fiber and collagen content.   From these same tissue blocks, additional 5-µm sections were analyzed for elastic fiber and collagen content in lung parenchymal tissue. Sections were stained with acid orcein alcoholic solution for elastic fiber content, and additional sections were stained with 0.05% fast green FCF and picrosirius red for collagen content. Seven nonoverlapping 676 µm x 505 µm fields were sampled, deliberately avoiding the large airways and blood vessels. To quantify elastic fiber and collagen content in the lung parenchyma, color thresholding was performed using Image-pro plus 5.1 software as described elsewhere (10, 26, 31). In the present study, color thresholding was used to separate the histological samples into pixels associated with different tissue staining procedures. Each image, stained for either elastic fibers or collagen, was composed of contrasting pixels representing the color of interest (i.e., elastic fibers or collagen) against a background color (i.e., other tissue or air space). The areas of elastic fiber [elastic fiber density relative to lung parenchyma (ELP)] and collagen [collagen density relative to lung parenchyma (CLP)] in the tissue samples were determined by computing the percentage as normalized to the total area of the tissue (10). Twenty-one histological samples were obtained from each animal (n = 6 mice per strain for each age group) and averaged for a given animal.

Quantitative RT-PCR.   Total RNA isolation was prepared from the lungs of 2- and 20-mo-old B6 and D2 male mice (n = 3 per strain per age group) using the TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA), and 3 µg of total RNA was reverse transcribed to complementary DNA (cDNA) using random primers and MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA). Using 100 ng of cDNA as a template, quantification was performed by an ABI Prism 7000 Sequence Detector (Applied Biosystems) using the TaqMan 5' nuclease activity from the TaqMan Universal PCR Master Mix, fluorogenic probes (Applied Biosystems) and oligonucleotide primers (Invitrogen). TaqMan assays were repeated twice for each of 10 selected genes related to lung ECM in each lung sample. These genes were as follows: elastin (Eln); procollagen I (Col1a1), III (Col3a1), and VI (Col6a3); matrix metalloproteinase 9 (Mmp9) and 12 (Mmp12); and tissue inhibitor of metalloproteinase 1 (Timp1), 2 (Timp2), 3 (Timp3), and 4 (Timp4). The mRNA expression levels of all samples were normalized to the levels for the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the same sample, and relative fold changes were calculated using the 2–{Delta}{Delta}Ct method, where Ct is cycle threshold (33).

Statistical analysis.   Differences between B6 and D2 mice were analyzed by two-way ANOVA (Graphpad Prism, Version 4.03). Mean comparisons between strains and age groups were further tested by a Bonferroni post test. Statistical significance was accepted at P value of 0.05. Results were expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The body weight and lung volume parameters are shown in Table 1. There were no detectable differences observed between the strains in body weight within each age group. However, body weight increased significantly with age in both strains of mice (P < 0.01). Whereas FRC remained unchanged with age in B6 mice, FRC at 20 mo of age in D2 mice was significantly (P < 0.01) increased relative to younger mice, and it was also significantly greater compared with 20-mo-old B6 mice (0.58 ± 0.08 vs. 0.35 ± 0.02 ml, P < 0.05). Likewise, the V30 increased significantly with age in both strains (P < 0.01), but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. Crs5–0 was also increased with age in both strains (P < 0.01); however, there were no detectable differences (P > 0.05) observed between strains within the same age group. The Crs15–10 was increased with age in only B6 mice (P < 0.01) and was significantly (P < 0.01) lower in D2 compared with B6 mice at both 2 and 20 mo of age.


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  		Table 1. Body weight and lung volume parameters of mice

 
Figure 1, A and B, shows quasi-static P-V curves normalized to V30 in B6 and D2 mice, respectively. Leftward shifts in the deflation limb of the P-V curves were observed from 2 to 20 mo of age in B6 (F statisticdf=1 = 5.0, P < 0.05) and D2 (F statisticdf=1 = 13.1, P < 0.01) mice. This shift was quantified by measuring the elastic recoil pressures at 50% of V30 (PEL). The PEL was significantly (P < 0.05) lower in D2 compared with B6 mice within the same age group; however, a significant (P < 0.05) decrease with age was seen only in D2 mice (3.19 ± 0.61 vs. 1.60 ± 0.48 cmH2O; Fig. 1C).


Figure 1
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  		Fig. 1. Static pressure-volume (P-V) curves normalized by the volume achieved at 30 cmH2O (i.e., percent of V30) in C57BL/6J (B6) (A) and DBA/2J (D2) mouse strains (B). C: changes of elastic recoil pressure at 50% of V30 in both strains of mice. Data are expressed as means ± SE. *P < 0.05, **P < 0.01, D2 vs. B6 at same age; {dagger}P < 0.05, 20 mo vs. 2 mo in same strain.

 
The respiratory impedance data at a PEEP level of 2 cmH2O are shown in Table 2. At 2 mo of age, R was significantly (P < 0.01) higher in D2 than in B6 mice, but the strain differences were eliminated in 20-mo-old mice due to a significant (P < 0.01) age-dependent decrease in D2 mice. Thus there was a significant interactive effect of age on R between the strains (F statisticdf=1 = 32.23, P < 0.001). Both G and H were significantly (P < 0.01) diminished with age in both stains. An age-dependent increase in eta was significant (P < 0.01) only in D2 mice.


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  		Table 2. Respiratory impedance parameters of mice with PEEP of 2 cmH2O

 
In Fig. 2A, the strain variation in R is shown for different age groups at increasing levels of PEEP. With increasing PEEP, R progressively decreased in each group. In contrast to B6 mice, D2 mice showed a significant age-dependent decrease in R at all PEEP levels. For example, at a PEEP level of 2 cmH2O, R was significantly (P < 0.01) decreased from 2.29 ± 0.19 to 0.84 ± 0.05 cmH2O·s/ml from 2 to 20 mo of age in D2 mice. However, R was only slightly (P > 0.05) decreased from 0.82 ± 0.05 to 0.74 ± 0.06 cmH2O·s/ml in B6 mice of similar age. Figure 2B shows the pressure sensitivity of R as a function of increasing PEEP levels. In D2 mice, the pressure sensitivity of R (from 2-cmH2O to 5-cmH2O PEEP levels) was significantly (P < 0.01) decreased at 20 mo age. There was no age-dependent change in pressure sensitivity of R between 2- and 20-mo-old B6 mice (P > 0.05). There was also a significant interactive effect of age on the pressure sensitivity of R between the strains (F statisticdf=1 = 20.28, P < 0.001). As shown in Fig. 2C, this unique age-dependent change in pressure sensitivity for D2 mice was not exclusively attributable to the elevated R observed in this strain at a younger age.


Figure 2
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  		Fig. 2. Airways resistance (R) as a function of increasing levels of positive end-expiratory pressure (PEEP) in B6 and D2 mouse strains. A: airway resistance. B: slope of the relationship between airways resistance as a function of increasing levels of PEEP from 2 to 5 cmH2O. C: percent change in airways resistance between PEEP levels of 2 cmH2O and 5 cmH2O normalized for the resistance at 2 cmH2O. Data are expressed as means ± SE. **P < 0.01 vs. same-age B6 mice. {dagger}{dagger}P < 0.01, 20 mo vs. 2 mo in same strain.

 
Figure 3A shows age-dependent changes in alveolar MCL in B6 and D2 mice. The MCL was consistently higher (9–11%) in both strains at 20 mo of age compared with the younger age group (F statisticdf=1 = 5.74, P < 0.05). Figure 3B shows the lung TD in both mouse strains. Although there were no lung TD differences observed with age in B6 mice, TD decreased significantly (P < 0.05) from 2 mo (33.3 ± 2.1 %) to 20 mo (27.0 ± 1.7 %) of age in D2 mice.


Figure 3
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  		Fig. 3. A: mean chord length (MCL) of alveoli. B: lung parenchymal tissue density (TD) in B6 and D2 mouse strains. There was consistently higher (~9–11%) MCL in both strains at 20 mo compared with 2 mo of age (F statisticdf=1 = 5.74, P < 0.05). Data are expressed as means ± SE. {dagger}P < 0.05, 20 mo vs. 2 mo in same strain.

 
Figure 4 shows age-dependent changes in ELP and CLP for B6 and D2 mice. The ELP decreased significantly (P < 0.01) from 2 to 20 mo in both strains (Fig. 4A). However, there were no detectable strain differences observed in ELP within each age group. In Fig. 4B, age-dependent changes in CLP are illustrated. The CLP increased significantly (P < 0.01) in D2 mice with age, but the CLP was not significantly (P > 0.05) changed with age in B6 mice. In addition, CLP was significantly (P < 0.05) higher in D2 mice relative to B6 mice in both age groups.


Figure 4
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  		Fig. 4. Elastic fiber density (ELP; A) and collagen density (CLP; B) relative to lung parenchyma in B6 and D2 mouse strains. Data are expressed as means ± SE. *P < 0.05, **P < 0.01, D2 vs. B6 at same age; {dagger}{dagger}P < 0.01, 20 mo vs. 2 mo in same strain.

 
Table 3 reports age-dependent changes in the gene expression profiles related to lung ECM for B6 and D2 mice. At 20 mo of age, the expression levels of Col1a1, Col3a1, Col6a3, and Eln genes were significantly (P < 0.05) decreased in D2 mice but not in B6 mice. In 2-mo-old mice, the expression level of only the Col1a1, Col3a1, and Col6a3 genes were significantly (P < 0.05) higher in D2 relative to B6 mice. There was also a significant interactive effect of age on the expression levels of the Col3a1 gene between the strains (F statisticdf=1 = 6.13, P < 0.05).


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  		Table 3. Fold changes in gene expression levels related to strain differences in lung extracellular matrix as a function of age

 
The expression levels of the Mmp9 gene were modestly increased with age in both strains, but the increase was significantly (P < 0.01) greater in 20-mo-old D2 mice. The expression level of the Timp1 and Timp4 genes were significantly (P < 0.05) increased with age in B6 mice but not in D2 mice. In addition, there were significant interactive effects of age on the expression levels of the Mmp12 (F statisticdf=1 = 7.56, P < 0.05) and Timp1 (F statisticdf=1 = 14.78, P < 0.01) genes between the strains.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, several age-dependent changes occurred in lung mechanics that were common to both B6 and D2 mice, including age-dependent increases in V30 and Crs5–0. In addition, respiratory impedance parameters G and H were also decreased with age in both strains. These common age-dependent changes in both B6 and D2 mice were associated with a decrease in elastin fiber content in the lung parenchyma. On the other hand, there were several notable age-dependent changes unique to the D2 strain. These unique characteristics in D2 mice included decreases in TD and PEL, which are emphysematous-like changes that are observed with age in humans (41) and other animal models (19). While there were no significant changes in MCL in either strain, the collagen content in D2 mice was significantly elevated at 20 mo of age. These exceptional age-dependent changes in the lungs of D2 mice were accompanied by a substantial reduction in R as a function of PEEP (Fig. 2). Taken together, the results support the hypothesis that D2 mice are characterized by exaggerated age-dependent changes in lung function and structure compared with B6 mice.

A similar strain comparison of lung structure was performed in younger male B6 and D2 mice by Kida and coworkers (16). The results observed by these investigators with respect to MCL are relatively similar to those shown in 2-mo-old B6 and D2 from the present study. Our present study quantified many additional structural and mechanical characteristics of the lung between B6 and D2. The most notable strain difference was the measurement of R at relatively low levels of PEEP (Fig. 2). There was a threefold greater R in D2 mice compared to B6 mice. A survey of the literature indicates a broad range of baseline resistances among a variety of inbred strains from 0.3 cmH2O·s/ml (1) to 1.5 cmH2O·s/ml (35). The variability in baseline R among different studies may be attributable to strain variation or methodological differences, such as variable anesthetic effects and the use of paralyzing agents. The present study also shows that the PEEP dependence of R in 2-mo-old D2 mice was substantially greater than B6 mice. These results indicate that there are robust genetic determinants that regulate the strain variation in R between B6 and D2 mice. Whereas others (18) have shown that the D2 strain is more hyperresponsive to bronchoprovocative agents compared with the B6 mice, these investigators did not measure baseline resistance. In fact, the higher baseline R in the D2 strain by itself could possibly account for an increased responsiveness to methacholine. Although the genetic determinants are of great interest for future studies, the focus of the present study is to explore the interactions between genetic influences and differential aging processes in the lung. Since age-dependent changes in R are dramatic in D2 mice but not in B6 mice (Fig. 2), the results suggest that there is a potent interaction of aging, which plays a substantial role on the genetic regulation of R between the strains. The age-dependent decline in R as a function of PEEP for D2 mice further suggests that a loss of interdependence between the lung parenchyma and the airway tree is one important manifestation of accelerated aging in this strain. This unique aging effect in the lungs of D2 mice is not solely explicated by the greater R observed in this strain at 2 mo of age (Fig. 2C).

In addition to apparent strain and aging effects in the airways, significant strain variation between B6 and D2 mice was evident in the lung parenchyma at 2 mo of age. The strain differences were especially apparent in the PEL (Fig. 1C) and the compliance of the lung at high pressure (Table 1). The reduced PEL in 2-mo-old D2 mice indicated a significantly lower distensibility of the lung parenchyma relative to B6 mice. There was a further reduction in PEL with age in D2 mice, and the strain difference was exaggerated at 20 mo of age. The D2 strain showed a greater age-dependent leftward shift in the deflation curve than the B6 mice (Fig. 1), and there was a further reduction in PEL with age in this strain. Since the age-dependent change in lung volume did not differ between strains, the 60% lower PEL in D2 mice compared with B6 mice at 20 mo represents a volume-independent effect of aging. This strain-specific loss of elasticity with age observed in D2 mice did not occur in B6 mice. As a result, there was also a significant age-dependent increase in FRC in D2 mice. For example, the rise in FRC in D2 mice with age is threefold greater than in B6 mice (93% in D2 vs. 30% in B6). Therefore, not only do genetic determinants regulate volume-independent differences between B6 and D2 strains, but also the lung parenchyma of D2 mice is more vulnerable to the effects of accelerated aging compared with B6 mice.

Elastin and collagen are important structural proteins composing the alveolar structure and the lung parenchyma and are most important in determining the mechanical properties of lung (35, 37). Thus it is reasonable to postulate that strain differences in elastin and/or collagen with senescence between B6 and D2 might also contribute to the accelerating emphysematous-like changes in D2 mice. Traditionally, elastic fiber degradation was considered a key factor contributing to loss of elastic recoil in the pathogenesis of aging and emphysema (20, 24, 28). However, the strain variation that we observed in elastic recoil pressure as a function of age (Fig. 1C) was not accompanied by parallel changes in ELP (Fig. 4A). Collectively, these results demonstrated that ELP was not the only factor or even a primary factor in the greater loss in elastic recoil found in the D2 strains. Other studies (3, 14, 21) have shown similar inconsistencies. Taken together, these studies make it clear that the changes in lung mechanics involve a complex interaction between several structural proteins in the extracellular matrix.

Consistent with this conjecture, we found age-dependent lung changes unique to D2 mice, particularly in the loss of lung tissue (TD) and the increase in collagen content in the lung parenchyma at 20 mo of age. This loss of lung tissue with increased collagen content in parenchyma must have some effect on the aging differences in lung mechanics at 20 mo in D2 compared with B6 mice. Although age-related changes in collagen content of human and animal lungs have been reported (25, 39), the results have varied widely. In human lungs, various environmental factors including pollutant and cigarette smoke exposures may have affected the aging process. Several studies have shown that D2 inbred mice are more susceptible to lung pathophysiological effects following exposure to cigarette smoke (4) and bleomycin (7) than B6 mice. In the present study, we found that collagen content increased significantly with age between 2 and 20 mo in D2 mice but not in B6 mice (Fig. 4B). Chung and colleagues (7) showed that D2 mice were uniquely susceptible to bleomycin-induced increases in collagen content, leading to prolonged fibrosis compared with B6 mice. These results strongly suggested that genetic determinants lead to greater susceptibility to collagen deposition and persistent fibrosis. The present study suggests that similar changes may occur in D2 mice with senescence.

The disparity between the D2 and B6 strains of mice in senescent-dependent lung changes is likely attributable to a complex interaction between genetic predisposition and ongoing aging processes. To begin to address this, we tested whether there were strain-specific age-dependent differences in ECM-related gene expression levels. We observed age-dependent downregulation in gene expression levels for Eln, Col1a1, Col3a1, and Col6a3 genes in both strains, but the extent of downregulation was more dramatic in D2. These findings are consistent with the hypothesis that organ-specific genes modulate the rate of aging, and decreased gene expression levels of structural proteins in the ECM can parallel accelerated aging specific to that organ. That is, the slowing of ECM protein synthesis is one of the most commonly observed biochemical changes during aging (29). From the present study, the results indicate that the lung of D2 mice is more susceptible to accelerated aging than B6 mice. Although we cannot show a causal link, it is clear that this accelerated aging of the lung is correlated with the downregulation of Eln, Col1a1, Col3a1, and Col6a3 gene expression levels in D2 mice. One additional factor that can also affect the shape of the P-V curve is surface tension. We did not measure surfactant levels in the strains and are unaware of published data that show changes in surfactant in the aging lung or among different mouse strains. Thus the potential importance of age- and strain-dependent alterations in surfactant remains to be determined. However, to the extent that such changes exist, it is uncertain how this factor might influence the observed age-dependent changes.

In general, protein turnover is hindered by an age-dependent decrease in proteolytic activity (29). For the lung, matrix metalloproteinases (Mmps) are an important family of proteolytic enzymes and can be broadly classified on the basis of substrate specificity (9, 42). The key characteristic of Mmps is their ability to degrade structural proteins of the ECM, and Mmp9 and Mmp12 play a significant role in the development of lung destructive diseases, such as emphysema (17, 22). Clinical data have also shown elevated Mmp9 and Mmp12 levels in bronchoalveolar lavage fluid as well as in bronchial biopsies of chronic obstructive pulmonary disease patients when compared with controls (8, 15). In the present study, we found that the gene expression levels of Mmp9 were significantly (P < 0.05) increased with age only in 20-mo-old D2 mice and not in B6 mice. That is, the lungs from 20-mo-old D2 mice expressed 6.6 times more Mmp9 compared with the expression levels in 2-mo-old D2 mice and 3.4 times more than 20-mo-old B6 mice (Table 3). These findings suggest that the proteolytic activity of Mmp9 may be a significant contributing factor in the accelerated aging of the lung in D2 mice. In contrast, the expression levels of Timp1 and Timp4 genes were significantly increased in 20-mo-old B6 mice, suggesting that these may provide important inhibitory effects on the age-dependent degradation of the lung in this strain. The results also show that the gene expression levels of Mmp12 were not significantly different with age in either strain, although there was a significant (P < 0.05) interaction between age and strain; that is, the gene expression of Mmp12 was increased with age in B6, whereas it was decreased with age in D2 (Table 3). Our findings are consistent with the hypothesis that an imbalance between Mmp9 and its tissue inhibitors accelerates lung aging and may induce a greater lung tissue remodeling in D2 compared with B6 mice.

In conclusion, our results suggest that there are strain-specific differences in lung senescence between B6 and D2 mice. These data further suggest that robust genetic determinants regulate the rate of age-dependent changes in the lung, with D2 mice demonstrating accelerated aging compared with B6 mice. The physiological changes in R as a function of lung inflation in the D2 mice are most notable and indicate a dramatic age-dependent change in the interdependence between the lung parenchyma and the airways. The decline in the recoil pressure in D2 mice further suggested that the accelerated aging of the lung in D2 mice involves the loss of parenchymal elasticity. While both strains showed similar declines in elastic fiber content, the D2 strain demonstrated a greater age-dependent increase in collagen content. This parenchymal remodeling is a consequence of a complex interaction between genetic predisposition and aging. Since these changes observed in mice mimic those seen in human subjects, these genetic models may be useful in studying specific genetic processes that are generally applicable to the aging mammalian lung.


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 METHODS
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This study was supported by National Institute on Aging Grant AG-21057 (C. G. Tankersley) and National Heart, Lung, and Blood Institute Grants HL-010342 (C. G. Tankersley) and HL-081205 (S. Biswal).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. G. Tankersley, Div. of Physiology, Bloomberg School of Public Health, The Johns Hopkins Univ., 615 N. Wolfe St., Baltimore, MD 21205 (e-mail: ctankers{at}jhsph.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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