Persistent Osteopenia in Adult Cystic Fibrosis Transmembrane

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					Persistent Osteopenia in Adult Cystic Fibrosis
Transmembrane Conductance Regulator–deficient Mice
Christina K. Haston1, Wei Li2, Ailian Li2, Melanie Lafleur1, and Janet E. Henderson2
1
Meakins-Christie Laboratories and 2J.T.N. Wong Laboratories for Mineralised Tissue Research, Department of Medicine, McGill University,
Montreal, Quebec, Canada


Rationale: A loss of function mutation in the cystic fibrosis trans-
membrane conductance regulator gene is believed to be an in-                             AT A GLANCE COMMENTARY
dependent risk factor for bone disease in patients with cystic fibrosis.
Objectives: The objective of this work was to use congenic mice as                       Scientific Knowledge on the Subject
a preclinical model to examine the bone phenotype of Cftr2/2 mice                        Bone disease in patients with cystic fibrosis is multifacto-
and control littermates at 8, 12, and 28 weeks of age.                                   rial, but the contribution of the cystic fibrosis transmem-
Methods: The bone phenotype of control and Cftr2/2 mice was                              brane conductance regulator (CFTR) gene to bone disease
evaluated by quantitative imaging, histologic and histomorphomet-                        remains undefined.
ric analyses, and serum levels of bone biomarkers.
Measurements and Main Results: At 12 weeks of age, Cftr2/2 mice were
smaller, had lower bone mineral density, cortical bone thinning, and                     What This Study Adds to the Field
altered trabecular architecture compared with Cftr1/1 or Cftr1/2
control mice. In skeletally mature 28-week-old mice, there were                          We show that adult Cftr-deficient mice have low femoral
persistent deficits in cortical and trabecular bone structure in Cftr2/2                  bone mineral density and compromised bone architecture
mice despite significant, quantifiable improvements. Cftr2/2 mice                          in the absence of other overt disease symptoms. The results
also had lower serum insulin-like growth factor-I levels at 12 weeks                     suggest that deficiency of Cftr contributes to the osteopenic
of age than did control mice, whereas parathyroid hormone and                            phenotype of Cftr2/2 mice.
25-hydroxyvitamin D levels were not significantly different.
Conclusions: Persistent osteopenia and structural abnormalities in
adult Cftr2/2 mice, in the absence of overt respiratory and gastro-
intestinal disease, suggest that loss of Cftr function has a direct
impact on bone metabolism in Cftr2/2 mice that is not sex specific or                   physical inactivity, and steroid therapy. Studies of serum bio-
subject to haplotype insufficiency.                                                     markers for bone turnover in patients with CF suggest that bone
                                                                                       resorption exceeds formation (2). It has also been suggested
Keywords: bone disease; lung disease; preclinical model; genetically                   that low BMD in adults with CF may arise from a combination
modified mouse                                                                          of insufficient bone accrual during puberty (7) and subsequent
                                                                                       bone loss in young adulthood (2). King and coworkers (8) have
Cystic fibrosis (CF) is a disease in which mutations in the cystic
                                                                                       shown that the common DF508 mutation in the chloride ion
fibrosis transmembrane conductance regulator gene (CFTR) in
                                                                                       channel encoded by CFTR is an independent risk factor for
humans lead to abnormalities affecting principally the lung,
                                                                                       bone disease in the CF population, in addition to malnutrition
intestine, and pancreas (1). Patients with CF may also suffer
                                                                                       and lung disease and Sermet-Gaudelus and coworkers (9) report
from bone disease, characterized by low bone mineral density
                                                                                       low BMD in children with CF who are younger than 6 years of
(BMD) and increased fracture rates (2). In general, suscepti-
                                                                                       age. The mechanisms through which the primary CF defect influ-
bility to fracture is determined by a combination of factors
                                                                                       ences bone metabolism, however, remain undefined.
including BMD, geometry, microarchitecture, and bone cell
                                                                                          Mouse models that exhibit the altered electrophysiology
activity (3, 4). The diagnosis and response to treatment of
                                                                                       expected to arise from mutations in Cftr have been extensively
osteopenia and osteoporosis, therefore, are currently monitored
                                                                                       characterized (10), but there have been relatively few studies
with tests that determine BMD and bone architecture (using
                                                                                       investigating their bone phenotype (11, 12). Dif and coworkers
peripheral quantitative micro–computed tomography) and bone
                                                                                       (11) evaluated 3-week-old female UNC Cftr-deficient mice,
cell activity (using serum biomarker assays).
                                                                                       which carry a mutation that results in the absence of the Cftr
    In cystic fibrosis, according to the consensus statement on
                                                                                       protein in a mixed genetic background (13). They showed the
bone health (2), bone disease arises from a variety of factors
                                                                                       weanling mice to have lower BMD, reduced cortical bone width,
including decreased vitamin absorption, pancreatic insuffi-
                                                                                       and thinner trabeculae compared with their wild-type littermates.
ciency, altered sex hormone production, chronic lung infection
                                                                                       This study suggested that bone disease in weanling Cftr-deficient
resulting in increased levels of bone-active cytokines (5, 6),
                                                                                       mice was due primarily to the absence of Cftr, as the mice had
                                                                                       not yet developed pancreatic insufficiency or lung disease and
                                                                                       had not been subjected to therapeutic doses of steroids.
                                                                                          Under physiological conditions, skeletal maturity in mice has
(Received in original form May 3, 2007; accepted in final form November 9, 2007)        been determined to occur at about 16 weeks of age. Comparable
Supported by the CCFF and Canadian Institutes of Health Research, the Basic            to the situation seen in humans, skeletal maturity in the mouse
                                                                 ´
Research and Therapy (BREATHE) Program, Valorisation Recherche Quebec, and             marks the cessation of longitudinal and appositional bone growth.
                             ´   ´
Fonds de la Recherche en Sante Quebec.
                                                                                       At this stage, net bone acquisition is replaced by remodeling to
Correspondence and requests for reprints should be addressed to Christina              maintain bone mass. Given the changes in bone physiology with
Haston, Ph.D., Meakins-Christie Laboratories, 3626 rue St. Urbain, Montreal, PQ,
H2X 2P2 Canada. E-mail: christina.haston@mcgill.ca
                                                                                       age, the current study was undertaken to determine how loss of
                                                                                       function of the gene encoding the cystic fibrosis transmembrane
Am J Respir Crit Care Med Vol 177. pp 309–315, 2008
Originally Published in Press as DOI: 10.1164/rccm.200705-659OC on November 15, 2007   conductance regulator, Cftr, influences the bone phenotype in
Internet address: www.atsjournals.org                                                  skeletally mature, juvenile, and adult CF mice.
310                                                       AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 177                 2008


METHODS                                                                       Cftr genotype, using a single section from three different mice per
                                                                              group. Osteoclast numbers were determined by counting the number of
Mice                                                                          TRAP-positive cells present in five individual fields of bone at a final
Animal procedures were performed in accordance with McGill Uni-               magnification of 3400, and normalizing this count to the bone volume.
versity (Montreal, PQ, Canada) guidelines set by the Canadian Council         Osteoblast cell numbers were similarly determined by examination of
on Animal Care (Ottawa, ON, Canada). Congenic BALB Cftr UNC1/2                von Kossa–stained sections.
mice were originally obtained from J. Hu of the Hospital for Sick
Children (Toronto, ON, Canada) and maintained in a breeding colony            Blood Analysis
at the Meakins-Christie Laboratories of McGill University. The Cftr1/2        Blood was collected by cardiac puncture of anesthetized animals and
mice were intercrossed to produce Cftr1/1, Cftr1/2, and Cftr2/2 mice,         centrifuged, and the supernatant serum was stored at –858C until
which were identified by genotyping as described previously (14). We           biochemical analyses for bone biomarkers were undertaken with the
used mice that are congenic in the BALB background to control for pos-        following commercial assays: radioimmunoassay for 25-hydroxyvitamin
sible effects of background strain variation on the CF phenotype (14–16).     D (ImmunoDiagnostic Systems, Tyne and Wear, UK), ELISA for
To circumvent premature death of Cftr2/2 mice as a result of intestinal       insulin-like growth factor-I (Quantikine mouse IGF-I; R&D Systems,
disease, control and Cftr2/2 mice were fed standard chow and received         Minneapolis, MN), and ELISAs for parathyroid hormone and osteo-
PegLyte (polyethylene glycol [17.8 mmol/L] and electrolytes; Phar-            calcin (Immunotopics, Inc., San Clemente, CA).
mascience, Montreal, PQ, Canada) in their drinking water as described
previously (17). PegLyte is used clinically to cleanse the intestinal tract   Statistical Analysis
while preserving electrolyte balance. To determine the effect of PegLyte      Results are expressed as means 6 SD and differences between groups of
on bone growth in normal mice, one group of female Cftr1/1 mice was           mice were determined by the Student t test and the Mann-Whitney test.
fed regular drinking water. Mixed sex groups were used for experiments
conducted at 12 weeks (Cftr2/2: 12 wk, five males and three females
                                                                              RESULTS
[5 M/3 F]; control: 12 wk, 8 M/6 F) and 28 weeks (Cftr2/2: 28 wk, 1 M/7 F;
control: 28 wk, 1 M/5 F).                                                     Effect of PegLyte, Sex, and Haplotype on 12-Week-Old
                                                                              Control Mice
Radiologic Imaging
Phenotyping procedures were performed essentially as described pre-           Intervention with PegLyte to minimize lethality due to the gas-
viously (18, 19). At the indicated ages mice were weighed and killed with     trointestinal phenotype enabled the survival of all the Cftr2/2
a lethal dose of anesthetic before capturing high-resolution X-rays of the    mice to adulthood. To determine whether this treatment altered
femora and vertebrae with a Faxitron general science radiography              body weight or skeletal development of control mice we assessed
system (model MX-20/DC2; Faxitron X-ray Corporation, Wheeling,                the morphology, mineral density, and structure of femoral bone
IL), equipped with an FPX-2 imaging system (MedOptics/DALSA Life              in 12-week-old mice receiving PegLyte in their drinking water,
Sciences, Waterloo, ON, Canada). X-rays were used to qualitatively            and compared the results with those of age- and sex-matched
assess bone morphology and mineral density and to measure the lengths         control mice receiving plain water. As shown in Table 1, a small
of femora and vertebrae. The right femur was measured from the plateau
                                                                              but significant decrease in bone volume and altered trabecular
of the knee to the tip of the femoral head and the vertebral measurement
represents the length from the proximal end of lumbar vertebra 3 (L3) to      architecture was seen in the control mice receiving PegLyte
the distal end of L5 (L3–L5). A Lunar PIXImus 1.46 instrument (GE             compared with untreated control mice.
Healthcare Lunar, Madison, WI) was then used to measure BMD of the               To eliminate the possibility that haplotype insufficiency
intact animal before dissecting bones free of soft tissue. Bones were fixed    might contribute to a skeletal phenotype we analyzed the bones
overnight in 4% paraformaldehyde, rinsed three times with phosphate-          of each of four 3-month-old female Cftr1/1 and Cftr1/2 mice.
buffered saline, and scanned with a SkyScan 1072 micro–computed               No significant differences in morphology, BMD, or architecture
tomography (micro-CT) instrument (SkyScan, Antwerp, Belgium) to               were observed between Cftr1/1 and Cftr1/2 mice (data not
assess bone morphometry. Image acquisition was performed at 45 kV,            shown). These data are consistent with a previous report for
222 mA for a 2.24-second exposure at 350 for femur and 330 for lumbar
                                                                              weanling 3-week-old littermates (11). Cftr1/1 and Cftr1/2 mice
vertebrae with a 0.98 rotation between frames. These two-dimensional
images were used to reconstruct three-dimensional images for quantita-
                                                                              were subsequently used interchangeably as control animals.
tive analysis, using 3D Creator software supplied with the instrument.
                                                                              Age-related Changes in Bone Morphology and BMD in
The area of interest selected for quantification of trabecular bone was
located immediately below the growth plate and extended for 1 mm              Control and Cftr2/2 Mice
toward the diaphysis of the femur or the midpoint of the vertebra.            In a previous study it was reported that 3-week-old weanling
Reported parameters include the following: bone volume as a percentage        Cftr-deficient mice had reduced BMD and osteopenia charac-
of tissue volume (BV/TV); the structure model index (SMI), which is the       terized by increased bone resorption relative to formation (11).
ratio of rodlike structures to platelike structures, with the ideal ratio
                                                                              Table 2 confirms and expands on the previous data by showing
being 1.0. A high ratio is indicative of a reduction in trabecular
connectivity; the thickness of individual trabeculae (TrTh); trabecular
                                                                              that the highly significant reduction in body weight, bone length,
separation (TrSp), which is an indirect measure of trabecular thinning;       and BMD in Cftr2/2 mice compared with control mice persists
and the number of trabeculae in a given area (TrNo).                          at 8 and 12 weeks of age. These changes were apparent in both
                                                                              male (n 5 5) and female (n 5 3) Cftr2/2 mice compared with
Histology and Histochemistry                                                  their sex-matched control mice at the age of 12 weeks and were
After micro-CT analysis the right femur of each of four mice from each        not significantly different between 12-week-old male and female
group was embedded in polymethylmethacrylate at low temperature               Cftr2/2 mice (data not shown). Skeletally mature 28-week-old
and 4-mm sections were cut. Sections were stained with von Kossa              Cftr2/2 mice had largely normalized with respect to skeletal
to identify mineralized bone and counterstained with toluidine blue to        morphology and BMD in the vertebra, although the femoral
identify unmineralized tissue. Midsagittal sections, corresponding to         BMD remained significantly lower.
the micro-CT images, were cut and images were captured at a magni-
fication of 32.5, using a Zeiss Axioskop microscope (Carl Zeiss                Age-related Changes in Trabecular Bone Architecture in
Canada Ltd, Toronto, ON, Canada) equipped with a Zeiss AxioCam                Control and Cftr2/2 Mice
MRc camera. Adjacent sections were stained for alkaline phosphatase
(ALP) activity in osteoblasts or tartrate-resistant acid phosphatase          To further explore the differences in the skeletal phenotypes of
(TRAP) in osteoclasts as described previously (18). Cell numbers were         control and Cftr2/2 mice we assessed architectural parameters
scored by three independent investigators, blinded to mouse age and           by micro-CT and bone composition by classic histology. At 12
Haston, Li, Li, et al.: Bone Phenotype of Adult Cftr2/2 Mice                                                                                               311

                  TABLE 1. EFFECT OF SEX AND TREATMENT WITH PEGLYTE ON BONE CHARACTERISTICS OF
                  12-WEEK-OLD Cftr1/1 AND Cftr1/2 CONTROL MICE
                                                                                     1 PegLyte                                 P Value

                                                  – PegLyte                                                                              PegLyte:
                  Parameter                     Female (n 5 5)        Female (n 5 6)         Male (n 5 8)       PegLyte: – vs. 1      Female vs. Male

                  Body weight, g                22.44   6   2.91      21.48   6   1.43      28.91   6   1.18         NS                   ,0.001
                  Femur length, cm              1.498   6   0.026     1.457   6   0.027     1.495   6   0.050       ,0.029                 NS
                  Vertebral length, cm          0.918   6   0.056     0.955   6   0.024     1.003   6   0.025        NS                   ,0.004
                  Femur BMD, mg/cm2             70.06   6   1.90      69.67   6   3.88      72.48   6   6.58         NS                    NS
                  Vertebral BMD, mg/cm2         53.20   6   1.51      55.83   6   2.40      59.30   6   3.35         NS                    NS
                  BV/TV, %                      16.20   6   1.67      12.36   6   2.58      15.82   6   1.34        ,0.019                ,0.007
                  SMI                            1.37   6   0.66       1.76   6   0.20       1.56   6   0.14         NS                   ,0.050
                  TrTh, mm                       59.4   6   1.14      56.67   6   2.16      51.38   6   1.51        ,0.032                ,0.001
                  TrSp, mm                      213.4   6   7.40      240.7   6   21.30     204.9   6   33.88       ,0.024                ,0.043
                  TrNo, n                        2.82   6   0.19       2.17   6   0.42       3.09   6   0.26        ,0.012                ,0.001

                     Definition of abbreviations: BMD 5 bone mineral density; BV/TV 5 bone volume as a percentage of tissue volume; NS 5 not
                  significant; SMI 5 structure model index (i.e., the ratio of rodlike structures to platelike structures); TrNo 5 number of trabeculae
                  in a given area; TrSp 5 trabecular separation, an indirect measure of trabecular thinning; TrTh 5 thickness of individual trabeculae.
                     BALB Cftr1/1 and Cftr1/2 mice were maintained with or without PegLyte in their drinking water until 12 weeks of age, the time
                  of death. Bone lengths were measured on the basis of high-resolution X-rays (Faxitron general science radiography system model
                  MX-20/DC2) and bone mineral density (BMD) was determined with a Lunar PIXImus densitometer, as described in METHODS. Bone
                  volume; the ratio of platelike to rodlike structures; and trabecular thickness, separation, and number were quantified by micro–
                  computed tomography. Values represent means 6 SD.


weeks of age (Figure 1), reconstruction of micro-CT images into                           little difference between control and Cftr2/2 mice, except for
a three-dimensional model (Figure 1a) shows little difference                             a marginal increase in TRAP staining in the Cftr2/2 mice. By
between the control (Figure 1, top) and Cftr2/2 (Figure 1,                                28 weeks of age (Figure 2) there appears to be an increase in
bottom) mice, except perhaps for a thinner diaphyseal shaft in                            trabecular bone (Figure 2b) and cortical thickness (Figure 2c) in
the Cftr2/2 mice. Two-dimensional coronal sections adjacent to                            the Cftr2/2 mice (Figure 2, bottom) as evidenced by micro-CT
the growth plate (Figure 1b) show the reduced trabecular bone                             and von Kossa staining (Figure 2e). ALP activity (Figure 2f) is
and cortical thinning in the diaphysis (Figure 1c) of Cftr2/2 mice                        greatly increased in Cftr2/2 mice compared with control mice
compared with control mice. These data are corroborated in the                            and TRAP activity (Figure 2g) also appears up-regulated.
matched, midsagittal micro-CT (Figure 1d) and von Kossa– and                                  Quantitative micro-CT analysis of bone at 8 weeks (skele-
toluidine blue–stained histologic sections (Figure 1e). Adjacent                          tally immature), 12 weeks (juvenile), and 28 weeks (skeletally
sections stained for ALP (Figure 1f) and TRAP (Figure 1g) to                              mature adult) of age is shown in Table 3. All parameters except
localize osteoblasts and osteoclasts, respectively, show there is                         for trabecular thickness changed significantly between 8 and
                                                                                          12 weeks in the control mice and there was minimal significant
TABLE 2. AGE-RELATED CHANGES IN MORPHOLOGY AND                                            change after that time. In contrast, bone architecture in the
MINERAL DENSITY OF BONE TISSUE OF CONTROL AND                                             Cftr2/2 mice did not change significantly between 8 and 28 weeks
Cftr2/2 MICE                                                                              of age, despite the significant increase in ALP- and TRAP-positive
                                                                                          cells. Thus, at 8 weeks of age, trabecular bone volume in Cftr2/2
Age (wk)          Parameter              Control            Cftr2/2        P Value
                                                                                          femora is similar to that in control littermates, despite the signif-
8*         Number                           6                   6                         icant difference in BMD, but it was significantly reduced at 12 and
           Body weight, g            18.30 6 2.79       14.55   6 1.65     ,0.018         28 weeks of age, primarily because of reduced numbers of tra-
           Femur length, cm                —                    —            —            beculae. As shown in Figure 3A, there was a significant increase
           Vertebral length, cm            —                    —            —
           Femur BMD, mg/cm2         55.60 6 5.86       43.00   6 1.00     ,0.003
                                                                                          in TRAP-positive cells in Cftr2/2 mice at both 12 and 28 weeks,
           Vertebral BMD, mg/cm2     47.00 6 5.05       42.60   6 3.13      NS            whereas the increase in ALP-positive cells did not reach signifi-
12         Number                          14                   8                         cance until 28 weeks. The alteration in bone cell activity in Cftr2/2
           Body weight, g            25.73 6 4.01†      16.89   6 2.21     ,0.001         mice was reflected in a decrease in cortical width of the bones of
           Femur length, cm          1.479 6 0.045      1.381   6 0.100    ,0.001         Cftr2/2 mice compared with that seen in control mice. The dis-
           Vertebral length, cm      0.982 6 0.034      0.884   6 0.053    ,0.001         crepancy in cortical bone width between control and Cftr2/2 mice
           Femur BMD, mg/cm2         71.27 6 5.58†      47.24   6 7.23     ,0.001
           Vertebral BMD, mg/cm2     57.81 6 3.38†      43.04   6 4.86     ,0.001
                                                                                          appeared to diminish over time, as shown in Figure 3B.
28         Number                           8                   6                             To determine whether alterations in the bone phenotype of
           Body weight, g            27.42 6 2.69       21.85   6 2.05‡    ,0.004         Cftr2/2 mice were due in part to changes in the circulating levels
           Femur length, cm          1.529 6 0.074      1.458   6 0.100     NS            of bone-active factors, we examined serum biomarkers in
           Vertebral length, cm      1.006 6 0.039      0.978   6 0.062‡    NS            representative mice at 12 and 28 weeks of age. As shown in
           mg/cm2                    73.20 6 6.48       59.00   6 5.40‡    ,0.001         Table 4, Cftr2/2 mice had lower levels of IGF-I at 12 weeks and
           mg/cm2                    64.76 6 9.69‡      59.17   6 7.20‡     NS
                                                                                          osteocalcin at 28 weeks compared with age-matched control
  Definition of abbreviations: BMD 5 bone mineral density; NS 5 not significant.            mice. No significant differences were seen in the calciotropic
  BALB control and Cftr2/2 mice were maintained on PegLyte-treated drinking               hormones parathyroid hormone and 25-hydroxyvitamin D.
water to 12 or 28 weeks of age, the time of death. Bone lengths were measured on
the basis of high-resolution X-rays (Faxitron MX-20/DC2) and BMD was de-
termined with a Lunar PIXImus densitometer, as described in METHODS. Values               DISCUSSION
represent means 6 SD.
  * Eight-week-old mice were all female and received normal drinking water.               This study was undertaken to determine how loss of function of
  †
    P , 0.05; significantly different from 8-week-old mice.                                the Cftr gene influences bone development and metabolism. We
  ‡
    P , 0.05; significantly different from 12-week-old mice.                               have shown that at 12 weeks of age, Cftr2/2 male and female
312                                              AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 177                 2008




                                                                                              Figure 1. Architecture and composition
                                                                                              of distal femur of 12-week-old control
                                                                                              and Cftr2/2 mice. Bones from control
                                                                                              (top) and Cftr2/2 (bottom) mice were
                                                                                              dissected free of soft tissue, fixed, and
                                                                                              scanned with a SkyScan 1072 micro–
                                                                                              computed tomography (micro-CT) sys-
                                                                                              tem equipped with 3D-Creator analytical
                                                                                              software. Three-dimensional reconstruc-
                                                                                              tion (a) and two-dimensional cross-
                                                                                              sectional scans (b–d) demonstrated fewer
                                                                                              trabeculae and cortical thinning of Cftr2/2
                                                                                              bones. Plastic-embedded sections of the
                                                                                              same bones, stained with von Kossa and
                                                                                              counterstained with toluidine blue (e) and
                                                                                              photographed at original magnifications
                                                                                              of 32.5 and 320 (inset), confirmed the
                                                                                              micro-CT data. Adjacent sections stained
                                                                                              for alkaline phosphatase (f, brown) and
                                                                                              tartrate-resistant acid phosphatase (g,
                                                                                              red) showed little difference between con-
                                                                                              trol and Cftr2/2 mice in osteoblast (brown)
                                                                                              or osteoclast (red) activity along the edges
                                                                                              of bony trabeculae. CF 5 cystic fibrosis
                                                                                              (Cftr2/2 mice).




mice are smaller, have low BMD, cortical bone thinning, and        absence of overt, life-threatening lung and pancreatic disease
compromised trabecular architecture compared with wild-type        or steroid hormone treatment and in the presence of significant
or heterozygous control mice. At 28 weeks of age, when the         alterations in bone cell numbers and activity. The data thus
mice are skeletally mature, there are persistent deficits in        support the conjecture that loss of Cftr function has a direct
cortical and trabecular bone structure despite significant, quan-   impact on skeletal metabolism that is not sex specific or subject
tifiable improvements. These differences were seen in the           to haplotype insufficiency in a mouse model.




                                                                                          Figure 2. Architecture and composition of
                                                                                          distal femur of 28-week-old control and
                                                                                          Cftr2/2 mice. Bones from control (top) and
                                                                                          Cftr2/2 (bottom) mice were processed as
                                                                                          described in Figure 1. Trabecular bone (b–
                                                                                          d) and cortical width (c and d) were increased
                                                                                          in Cftr2/2 mice as compared with 12-week-
                                                                                          old Cftr2/2 mice, although the values still had
                                                                                          not normalized relative to control mice. A
                                                                                          significant increase in alkaline phosphatase
                                                                                          activity (f, brown) and tartrate-resistant acid
                                                                                          phosphatase activity (g, red) was seen along
                                                                                          the edges of bony trabeculae in Cftr2/2 mice
                                                                                          compared with control littermates. CF 5
                                                                                          cystic fibrosis (Cftr2/2 mice).
Haston, Li, Li, et al.: Bone Phenotype of Adult Cftr2/2 Mice                                                                                                      313

TABLE 3. AGE-RELATED CHANGES IN FEMUR BONE                                            TABLE 4. SERUM LEVELS OF BONE BIOMARKERS IN 12- AND
ARCHITECTURE OF CONTROL AND Cftr2/2 MICE                                              28-WEEK-OLD CONTROL AND Cftr2/2 MICE
Age (wk)       Parameter            Control              Cftr2/2           P Value    Age (wk)           Parameter             Control          Cftr2/2        P Value

8*             Number                  6                    6                         12             Number                       4                 5
               BV/TV, %          9.02 6 2.56           7.75 6   2.28        NS                       25 VitD, ng/ml            47 6   5        49   6   10       NS
               SMI               2.18 6 0.14           2.13 6   0.21        NS                       IGF-I, ng/ml             411 6   36      208   6   97      ,0.05
               TrTh, mm         57.20 6 3.42          49.20 6   2.78       ,0.003                    PTH, pg/ml                23 6   4        27   6   15       NS
               TrSp, mm         297.2 6 77.10         334.2 6   35.3        NS                       Osteocalcin, ng/ml        50 6   5        67   6   24       NS
               TrNo              1.74 6 0.43           1.57 6   0.41        NS        28             Number                       5                 4
12             Number                 14                    8                                        25 VitD, ng/ml            49 6   5        41   6   20       NS
               BV/TV, %         14.34 6 2.58†          5.83 6   2.71       ,0.001                    IGF-I, ng/ml             459 6   18      389   6   102      NS
               SMI               1.65 6 0.19†          2.16 6   0.24       ,0.001                    PTH, pg/ml                24 6   6        20   6   6        NS
               TrTh, mm         53.64 6 3.23          46.75 6   3.45       ,0.001                    Osteocalcin, ng/ml        67 6   15       49   6   14      ,0.05
               Tr Sp, mm        220.2 6 33.6†         307.2 6   65.3       ,0.001
               TrNo              2.70 6 0.57†          1.23 6   0.51       ,0.001       Definition of abbreviations: 25 VitD 5 25-hydroxyvitamin D; IGF-I 5 insulin-like
28             Number                  8                    6                         growth factor-I; NS 5 not significant; PTH 5 parathyroid hormone.
               BV/TV, %         13.17 6 3.22           7.11 6   1.84       ,0.001       BALB control and Cftr2/2 mice were maintained on PegLyte-treated drinking
               SMI               1.52 6 0.31           1.97 6   0.12       ,0.005     water to 12 or 28 weeks of age, the time of death. Blood was drawn by cardiac
               TrTh, mm         59.38 6 2.77‡         47.67 6   2.42       ,0.001     puncture from anesthetized animals and serum biomarkers were assessed with
               TrSp, mm         280.0 6 54.4‡         266.0 6   20.1        NS        a commercial radioimmunoassay assay for 25-hydroxyvitamin D and commercial
               TrNo              2.22 6 0.55           1.48 6   0.33       ,0.013     ELISAs for IGF-I, parathyroid hormone, and osteocalcin. Values represent medians
                                                                                      6 95% confidence interval.
  Definition of abbreviations: BV/TV 5 bone volume as a percentage of tissue
volume; NS 5 not significant; SMI 5 structure model index (i.e., the ratio of
rodlike structures to platelike structures); TrNo 5 number of trabeculae in a given   complications with associated altered levels of inflammatory
area; TrSp 5 trabecular separation, an indirect measure of trabecular thinning;       cytokines (6) and did not receive corticosteroids, which could
TrTh 5 thickness of individual trabeculae.
  BALB control and Cftr2/2 mice were maintained on PegLyte-treated drinking
                                                                                      lead to severe osteopenia when combined with structural abnor-
water to 12 or 28 weeks of age, the time of death. Bone architectural parameters      malities conferred by mutations in the CF gene. In contrast to the
were quantified by micro–computed tomography, as described in METHODS.                 BMD data, quantitative micro-CT shows that body size is not the
Values represent means 6 SD.                                                          major determinant of the observed differences in the skeletal
  * Eight-week-old mice were all female and received normal drinking water.           phenotypes of control and Cftr2/2 mice. At 8, 12, and 28 weeks
  †
    P , 0.05; significantly different from 8-week-old mice.                            the Cftr2/2 mice weighed 20, 44, and 19% less than control mice,
  ‡
    P , 0.05; significantly different from 12-week-old mice.
                                                                                      respectively. However, most micro-CT parameters, which reveal
                                                                                      changes in trabecular bone quantity and architecture that are
Impact of PegLyte on Survival and Bone Phenotype of
                                                                                      indicative of metabolic bone disease, were significantly different
Cftr2/2 Mice
                                                                                      at 28 weeks, but not at 8 weeks, when the differences in body
In this study, we used PegLyte in the drinking water to extend the                    weight were approximately equivalent.
life span of Cftr2/2 mice as described by Gawenis and coworkers                           Using quantitative techniques such as neutron activation and
(12). The intervention had little direct effect on bone develop-                      bone ash analysis, Gawenis and coworkers did not detect differ-
ment, as evidenced by attainment of nearly normal bone lengths                        ences in the chemical composition of bones from Cftr2/2 and
by 28 weeks of age in Cftr2/2 mice, but did allow for examination                     control mice and concluded that Cftr does not play a direct role
of adult mice. It was reported by Gawenis and coworkers that,                         in bone mineralization (12). Our own studies, as well as those of
after correcting for body weight, BMD did not differ between                          Dif and coworkers (11), support this conjecture, as there was no
UNC Cftr2/2 mice and control littermate mice. Our own                                 evidence of increased osteoid in either study. We also reported
observations support their data by showing no significant de-                          normal circulating levels of parathyroid hormone and 25-hydrox-
crease in femoral or vertebral BMD after correcting for differ-                       yvitamin D, suggesting that mineral ion homeostasis and absorp-
ences in body weight in the Cftr2/2 mice. These observations are                      tion of vitamin D via the gut were normal in Cftr2/2 mice during
somewhat surprising given the severe vertebral osteopenia                             pre- and postnatal bone development. Malabsorption would have
observed in young adult patients with CF. This discrepancy                            resulted in low vitamin D and an excess of osteoid, as seen in
might be explained by the fact that the Cftr2/2 mice receiving                        vitamin D–deficient rickets (20). PegLyte treatment therefore
PegLyte did not display overt gastrointestinal or respiratory                         had an additional indirect beneficial effect on bone by allowing


                                                                                                                     Figure 3. Trabecular bone cell numbers and
                                                                                                                     cortical bone width of femur of 12- and 28-
                                                                                                                     week-old control and Cftr2/2 mice. Osteo-
                                                                                                                     blasts were counted on representative von
                                                                                                                     Kossa– and toluidine blue–stained sections,
                                                                                                                     and osteoclasts on representative tartrate-re-
                                                                                                                     sistant acid phosphatase–stained sections, of
                                                                                                                     plastic-embedded femoral bone from three or
                                                                                                                     four female mice per group (A). Cortical width
                                                                                                                     was measured at four points at the midpoint
                                                                                                                     of the femoral diaphysis of three female mice
                                                                                                                     per group (B). Values are expressed as means
                                                                                                                     6 SD. *P , 0.05, significantly different from
                                                                                                                     control data. CF 5 cystic fibrosis (Cftr2/2
                                                                                                                     mice); OB 5 osteoblasts; OC 5 osteoclasts.
314                                                 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 177                          2008


for improved absorption of calcium and vitamin D, which may be        data, the 28-week-old Cftr2/2 mice demonstrated nearly normal
compromised in patients with CF (21).                                 vertebral BMD. This discrepancy is most likely due to the
                                                                      absence of steroidal treatment in the mice and its presence as
Improvement of Osteopenia over Time in BALB Cftr2/2 Mice              a confounding variable in the interpretation of the clinical
In the current study we show that the bone phenotype of 12-           findings. The increase in osteoclast numbers and the absence
week-old (juvenile) BALB Cftr2/2 mice resembles that of the           of excessive osteoid (osteomalacia) are common features of CF
3-week-old (weanling) UNC Cftr2/2 mice studied by Dif and             and adult Cftr2/2 mouse phenotypes.
coworkers. The defects in Cftr2/2 mice included low BMD;                 At 12 weeks of age, there was a significant reduction in
reduced cortical bone width; and fewer, thinner trabeculae            circulating IGF-I in Cftr2/2 mice compared with control mice.
compared with control littermates. The osteopenic phenotype           This deficiency was coincident with the largest discrepancy in
of 3-week-old Cftr2/2 mice was attributed to a decrease in bone-      body weight and bone mass between control and Cftr2/2 mice.
forming surfaces and in osteoclast numbers in trabecular bone.        IGF-I is a major regulator of somatic cell and bone growth and
However, the mixed genetic background (14–16) and the                 has been identified as an independent predictor of low BMD in
immature bone age may have been confounding factors in the            patients with CF (23) and compromised growth in Cftr2/2 mice
interpretation of data in the Dif study. In 28-week-old Cftr2/2       (24). Work has used genome-wide scanning of F2 mice derived
mice, osteopenia was seen in the presence of increased numbers        from C57BL and C3H strains, with low and high BMD,
of trabecular osteoblasts and osteoclasts, suggesting a high          respectively, to identify quantitative trait loci linked to both
turnover defect. Alternatively, the relative increase in osteo-       serum IGF-I and bone acquisition (25). Adult female congenic
blasts and osteoclasts in the 28-week-old mice could reflect           mice carrying a chromosome 10 locus from C3H on a C57BL
a ‘‘catch-up’’ phenomenon, resulting from normalization of            background had significantly higher circulating levels of IGF-I
IGF-I levels, as discussed below. Studies examining dynamic           in association with higher femoral BMD and trabecular num-
labeling of mineralization fronts and including in-depth histo-       ber. In BALB Cftr2/2 mice, there was a significant increase in
morphometric analyses will be required to resolve this issue.         serum IGF-I between 12 and 28 weeks and a concomitant
    Cortical thinning in 3-week-old Cftr2/2 mice (11) was             increase in BMD and trabecular number, suggesting that the
attributed to a relative increase in cortical osteoclasts, although   late-onset ‘‘growth spurt’’ in Cftr2/2 mice could have been
the cortical osteoblast surfaces were not documented. In 12-          mediated by circulating IGF-I. Of additional interest in this
week-old Cftr2/2 mice, despite the significant reduction in            respect are the reports of reduced peroxisome proliferator–
cortical width, we could not detect any significant differences        activated receptor-g in Cftr2/2 mice (26) and the negative
in the number of osteoclasts or osteoblasts in cortical bone          regulation of IGF-I by activation of this receptor (27). It is
compared with the control mice. The cortical width of Cftr2/2         conceivable that the rise in IGF-I at 28 weeks was related to
bones in the study by Dif and coworkers was approximately             an age-dependent downregulation of peroxisome proliferator–
50% that of control mice at 3 weeks of age, compared with 75%         activated receptor-g in Cftr2/2 mice.
at 12 weeks and 85% at 28 weeks of age in our own work. These            The precise molecular mechanisms by which mutations in
observations, together with those documenting a gradual in-           CFTR can alter bone cell function remain to be elucidated,
crease in trabecular and cortical BMD and improvement in              although insight may be gained from studies of other chloride
bone architectural parameters between 12 and 28 weeks of age,         channels implicated in defective bone metabolism. For exam-
suggest that attainment of peak bone mass may have been               ple, loss of function of the chloride channel-7 (ClC-7) results in
delayed in Cftr-deficient mice. However, it is also important to       a defect in chloride conductance required for efficient proton
note that trabecular bone volume remained almost 50% lower            pumping by the H1-ATPase of the osteoclasts (28). The
in Cftr2/2 mice at 28 weeks, with reductions in the number and        catabolic activity of osteoclasts depends on the active transport
thickness of individual trabeculae and an increase in the ratio of    of hydrogen ions into the extracellular space adjacent to bone to
platelike to rodlike structures measured. These features, along       solubilize bone mineral. Loss of chloride channel-7 activity
with the continued elevation in osteoblasts and osteoclasts,          therefore leads to accumulation of bone that would otherwise
support the conjecture that bone turnover is increased in Cftr2/2     be resorbed, or to osteopetrosis (28). Similarly, Kajiya and
mice compared with age-matched control mice.                          coworkers (29) reported that the K1/Cl2 cotransporter-1 ex-
    The trajectory of weight gain and bone accrual in control         pressed in mouse osteoclasts also participates in H1 extrusion
mice was greatest between 8 and 12 weeks of age, which would          during bone resorption, thus providing a second example of
roughly correspond with childhood and adolescence in humans,          a chloride channel involved in the catabolic activity of osteo-
and then slowed thereafter. In contrast, Cftr2/2 mice gained          clasts. In our current work, bone acquisition continued in mice
little weight or bone mass between 8 and 12 weeks whereas             up to 28 weeks of age in the presence of a significant increase in
significant increases in both were observed between 12 and 28          osteoclast and osteoblast activity. It is therefore unlikely that
weeks. This failure to keep pace with age-matched control mice        there was a significant defect in either cell type, although CFTR
recapitulates the clinical situation, in which low bone mass in       protein has been shown to be present in both (30), but rather
adult patients with CF has been attributed to a failure to thrive     that a change in the bone microenvironment influenced their
and accrue bone during childhood and adolescence (7). Sup-            activity. Additional in vitro and molecular studies are required
porting this, through histomorphometric analyses, Elkin and           to resolve this issue.
coworkers (5) revealed bone harvested from adult patients with
CF with low BMD to have reduced trabecular bone volume                Conflict of Interest Statement: None of the authors has a financial relationship
compared with age- and sex-matched healthy control subjects,          with a commercial entity that has an interest in the subject of this manuscript.
which was ascribed to low bone formation. Furthermore,
a second study, in which histomorphometric analysis of bone
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