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Fenretinide prevents the development of osteoporosis in Cftr-KO mice
Journal of Cystic Fibrosis 7 (2008) 222 – 230 www.elsevier.com/locate/jcf
Fenretinide prevents the development of osteoporosis in Cftr-KO mice Zienab Saeed a , Claudine Guilbault a , Juan B. De Sanctis b , Jennifer Henri a , Dominique Marion a , René St-Arnaud c , Danuta Radzioch a,⁎ a
c
McGill University Health Centre, Montreal General Hospital, Montreal, Qc, Canada b Central University of Venezuela, Institute of Immunology, Caracas, Venezuela McGill University, Department of Human Genetics, Shriner's Hospital, Montreal, QC, Canada
Received 21 June 2007; received in revised form 7 September 2007; accepted 10 September 2007 Available online 7 November 2007
Abstract Background: The most recently described phenotype associated with Cystic Fibrosis (CF) is reduced bone mineral density which results in osteopenia and osteoporosis. The etiology of the early onset of osteoporosis in CF patients has remained to be established. It has been suggested that inadequate nutritional absorption of essential fatty acids may play a role in the altered bone metabolism. In this study, we characterized the protective effect of fenretinide [N-(4-hydroxyphenyl) retinamide], a vitamin A derivative, on the early onset of osteoporosis in cystic fibrosis transmembrane conductance regulator knockout (Cftr-KO) mice. Methods: Using micro-computed-tomography we examined the effect of fenretinide on the bone composition and architecture in a Cftr-KO mouse model which was then confirmed with histological analyses. Plasma fatty acids were quantified using thin layer chromatography-ELISA method. Results: Twice-weekly treatments with fenretinide, over four weeks dramatically increased trabecular bone volume compared to controls. This increase in bone volume was also related to an increased concentration of ceramide in the plasma resulting in the down regulation of phospholipidbound AA in Cftr-KO mice. Conclusions: To our knowledge, this is the first time that fenretinide's protective effect against osteoporosis has been demonstrated. The results of this study strongly suggest that fenretinide has potential to be used as a prophylaxis by preventing the early onset of osteoporosis. © 2007 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Keywords: Osteoporosis; Cystic fibrosis; Arachidonic acid; Docosahexaenoic acid; Ceramide; Cftr-KO mice
1. Introduction One of the most recently described phenotypes that has been associated with CF is reduced bone mineral density which results in osteopenia and osteoporosis [1–5]. A number of studies have documented a prevalence (40–70%) of decreased bone density in the CF population [6]. A longitudinal study of 151 adults with CF showed that 34% of patients had a Z-score1 of −2 or less [7]. Bianchi and colleagues reported that 66% (90/ 136) of children and young adults with CF (age 4–24) have low bone mineral density [8]. However, a direct link between the ⁎ Corresponding author. Research Institute of the McGill University Health Center, 1650, Cedar Avenue, Room L11-218, Montreal, Quebec, Canada H3G 1A4. Tel.: +1 514 934 1934x44517; fax: +1 514 934 8260. E-mail address: [email protected] (D. Radzioch). 1 Z-score is the number of standard deviations above or below what's normally expected for someone of the same age, sex, weight, and ethnic or racial origin.
CFTR gene dysfunction and osteoporosis remains to be elucidated [9]. Currently, physicians prescribe a range of vitamins and minerals including: vitamin D, calcium, vitamin K as prophylaxis. Clinical trials are underway with the goal of finding new potential treatments and that might prevent the development of osteoporosis in CF patients including: anti-resorptive agents such as bisphosphates, anabolic agents such as parathyroid hormone (PTH) (extensively reviewed by Aris and colleagues) and human recombinant growth hormones (hrGH) [10,11]. Recently, epidemiological studies and experiments using animals and humans have shown that a high concentration of n-3 essential fatty acids (EFAs) improves bone metabolism [12]. EFA abnormalities in CF have been well-documented since the early 60's [13]. Freedman and colleagues used Cftr-KO mice to show that CF-affected organs have a significantly lower concentration of docosahexaenoic acid (DHA) and a significantly higher concentration of arachidonic acid (AA) [14]. DHA
1569-1993/$ - see front matter © 2007 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. doi:10.1016/j.jcf.2007.09.001
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is an n-3 polyunsaturated fatty acid that regulates membrane functions and displays anti-inflammatory effects in CF. In contrast, AA is an n-6 polyunsaturated fatty acid that has been shown to regulate the production of prostaglandins and leukotrienes, therefore acting as an agonist of the inflammatory process [15]. DHA and AA are the “yin and yang” of fatty acid metabolism, and disruption of the n-3 and n-6 EFA balance results in a number of systemic abnormalities [16]. It has been proposed that increased levels of AA might contribute to the progression of CF lung disease, and that dietary supplementation with high doses of DHA could correct this imbalance [17]. However, the effectiveness of this treatment option is compromised in patients with CF due to the severe malabsorption of lipids displayed by the majority of CF patients [18]. Treatment with fenretinide [N-(4-hydroxyphenyl) retinamide, 4-HPR], a semi-synthetic retinoid leads to the generation of ceramides [19–21], and increases 12-lipoxygenase (12LOX), an enzyme that catalyzes the oxidation of phospholipidbound AA [22,23], suggesting that fenretinide may be able to decrease AA concentrations. Sphingomyelinase, the enzyme that catalyzes the formation of ceramides plays a crucial role as an intracellular messenger in osteoblast differentiation from stroma cells, suggesting that ceramides may also play an important role in modulating bone metabolism [24]. Recently, our laboratory reported that Cftr-KO mice have impaired levels of ceramides and that treatment with fenretinide was able to correct this impairment [25]. We hypothesized that treatment with fenretinide might be able to prevent the development of osteoporosis by decreasing AA concentrations and increasing ceramide concentrations. Therefore the aim of the presented study was to assess the protective role of fenretinide against CF related osteoporosis. 2. Methods
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in 95% ethanol to a 2 μg/μL concentration. Fourty μL of this preparation was incorporated into the Peptamen liquid diet (5 mg/ kg per day per mouse). The prepared food containing fenretinide was then stored in the dark at 4 °C for no more than 3–6 h before its administration to the mice. To ensure that the entire drug dose was consumed, the mice were given 10 mL of fenretinidecontaining Peptamen, which represents 2/3 of the daily mouse food consumption with the completed dose, in the late afternoon. The remaining 5 mL of Peptamen (without fenretinide) was given the following morning. Mice were treated twice a week for 4 weeks. During the treatment period each mouse was kept in a separate cage. The diet for the mock-treated control group was prepared and administered similarly to that described above, by adding the same volume of ethanol to the Peptamen diet but omitting the fenretinide supplementation (vehicle control). 2.3. Micro computed tomography (micro-CT) Mice were euthanized using CO2 inhalation and exsanguinated by cardiac puncture. Femurs, tibiae and lumbar vertebrae were extracted, stripped of soft tissue and fixed overnight in 4% paraformaldehyde. Micro computed tomography (micro-CT) was performed on the left femur after overnight fixation. The distal metaphysis was scanned with a Skycan 1072 instrument (Skyscan, Antwerp, Belgium). Image acquisition was performed at 100 kVand 98 μA, with a 0.9° rotation between frames. The twodimensional images were used to generate three-dimensional reconstructions to obtain quantitative data using the 3D Creator software supplied with the instrument (ANT 3D Creator software, Skyscan, Antwerp, Belgium). The SMI score is an algorithm taking into account the change in surface area for the change in radial expansion of trabecular plate-like and rod-like structures, which is known as the “trabecular bone pattern factorq [26,27]. A SMI score between 1 and 3 is then reported, as the value approaches 3, the quality of the bone worsens.
2.1. Mice 2.4. Histological analysis The inbred mice (11–14 week old) used in our study were bred in the animal facility of the Research Institute of the McGill University Health Centre (from C57BL/6J breeding pairs heterozygous at the Cftr locus). Age- and gender-matched C57BL/6-Cftr+/+(WT) mice (n = 16), and C57BL/6-Cftr−/−(CftrKO) mice (n = 16) were maintained in a murine pathogen- and parasite-free environment. They were kept in cages with sterile maple hardwood bedding (PWI, St-Hyacinthe, QC, Canada) and housed in ventilated racks (Lab Products, Seaford, DE, USA). The liquid diet was freshly prepared every morning. Experimental procedures with the mice were conducted in accordance with the Canadian Council on Animal Care guidelines and with the approval of the Animal Care Committee of the McGill University Health Center, Montreal, Quebec, Canada. Results were pooled from 3 independent experiments. 2.2. Drug administration Fenretinide powder was kindly provided by Dr. Robert Smith from the NIH (Bethesda, MD, USA). Fenretinide was dissolved
After overnight fixation in 4% paraformaldehyde and rinsing in phosphate buffered saline (PBS), right femurs and tibiae were embedded in polymethylmethacrylate (MMA) or a mixture of 50% MMA and 50% glycolmethacrylate (GMA). Serial 4- to 6-μm sections were cut on a modified Leica RM 2155 rotary microtome (Leica Microsystems, Richmond Hill, Ontario, Canada). MMA-embedded tissues were stained with von Kossa and toluidine blue, while 4 μm MMA-GMA sections were stained with tartrate-resistant acidic phosphatase (TRAP). Images were captured using a Carl Zeiss Microscope (Germany) equipped with an AxioCam MRc camera. The left femur and tibia and the lumbar vertebrae were decalcified with 4% EDTA for paraffin embedding after 14 days. Serial 5 μm sections were cut and stained with haematoxylin and eosin (H&E). Osteoblasts lay down new bone lamellae and are active in bone development and in bone remodeling. Osteoblasts were defined as a singlenucleated, rod shaped cells and were identified along the surface of the trabecular lamellae. Osteoclasts have been shown to be involved in bone resorption, by digesting the adjacent bone
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matrix. Osteoclasts were defined as multi-nucleated cells that were much larger then osteoblasts and displayed typical macrophage morphology.
Dunn's multiple comparison post test between groups. Significance was set at a two-tailed P value of ≤ 0.05. 3. Results
2.5. Fatty acid analysis 3.1. Trabecular bone density Plasma was suspended in 1 mM butylated hydroxyanisole (BHA) in chloroform and methanol (2:1 vol) until the analysis was performed to ensure the integrity of the samples. Lipids were extracted from plasma with chloroform/methanol (2:1), as previously described [28]. Phospholipids were identified by thin layer chromatography extraction. Diazomethane was used to esterify the released fatty acids and the esters were identified by GC/MS (Hewlett Packard5880A, WCOT capillary column (Supelco-10, 35 m × 0.5 mm, 1 μm thick)) using commercial standards (Sigma-Aldrich, Oakville, ON, Canada). 2.6. Ceramide/sphingolipid analysis Ceramide concentrations in the plasma of treated and untreated mice were determined by an enzyme-linked immunosorbent assay (ELISA), performed on lipid samples that were separated by thin layer chromatography (TLC), as have previously described and validated in detail elsewhere [29]. Briefly, lipids were extracted from lung homogenates with chloroform/methanol (2:1) containing 1 mM butylated hydroxyanisole (BHA) [30]. The lipid fractions were then dried under nitrogen and resuspended in heptane [31]. Phospholipid separation was performed by TLC, detected by iodine. On these separated lipid samples, the concentration of ceramide was determined by ELISA [32,33]. The phospholipids from the dry silica were re-suspended in ethanol and used to coat Nunc plates specific for lipid binding. Plates were then washed, incubated with blocking buffer for 1 h at 37 °C (PBS, 0.1% Tween 20), and 1% bovine serum albumin (BSA; Sigma, Oakville, ON), and incubated with murine anti-ceramide IgM (Sigma-Aldrich) antibody (Ab) for 1 h at 37 °C. The plates were then washed and incubated with peroxidaseconjugated anti-mouse IgM Ab for 1 h at 37 °C. Finally, the plates were incubated with the peroxidase substrate (TMB; Roche, Laval, QC). The intensity of the colorimetric reaction was determined by spectrophotometry at 405 nm. The levels of ceramide were calculated with reference to a standard curve prepared using purified ceramide (Sigma-Aldrich). Phosphate levels were assessed, as previously described, using the PiBlue™ Phosphate assay kit (Boehringer Ingelheim, Ingelheim, Germany), according to the manufacturer's instructions. 2.7. Statistical analyses Data was analyzed using GraphPad Prism Version 4.03 software (GraphPad Software, San Diego, California, USA). Statistically significant differences between the medians of studied groups were evaluated using the non-parametric Mann– Whitney U test when comparing two groups (Fig. 3). Pair-wise multiple comparisons were used to evaluate the differences between multiple groups using Kruskal–Wallis on ranks and the
Trabecular bone density was measured using micro-CT scans and histology. Fig. 1A illustrated the micro-CT scans of the trabecular bones isolated from Cftr-KO mice femurs and their littermate controls (WT). Results illustrated clearly show evidence of reduced trabecular bone in the Cftr-KO compared to their WT littermate controls. These results also clearly demonstrate that twice-weekly treatment with fenretinide over the course of four weeks (total 8 doses) was able to completely eliminate any signs of osteoporosis in the trabecular bone of Cftr-KO mice. Representative 3D reconstructions and 2D crosssectional scans demonstrate that, before fenretinide treatment, Cftr-KO mice have virtually no trabecular bone (highlighted in black boxes) evident in the cross-sectional image as compared to WT controls. After treatment with fenretinide there is a dramatic increase in trabecular bone in the Cftr-KO mice compared to their WT controls. Micro-CT results were compared with histochemical staining of non-decalcified bone using von Kossa stain, which stains mineralized bone as shown in Fig. 1B. Cftr-KO mice clearly illustrate a decrease in trabecular bone (highlighted in the black box) compared to the WT controls, meanwhile when Cftr-KO mice are treated with fenretinide, the early onset of reduced bone mineral density is avoided. 3.2. Bone composition and architecture To address the osteoporotic changes from the composition and architecture point of view, the trabecular bone was quantified. The following parameters: bone volume fraction (BV/TV%), structural model index (SMI) trabecular separation (TS), and trabecular thickness (TT) were calculated from the left femur using micro-CT scans. Our results illustrate a significant (p = 0.005) lower amount of BV/TV% in the Cftr-KO mice as compared to WT vehiclecontrols as shown in Table 1. Interestingly, this difference is eliminated when Cftr-KO mice are treated with fenretinide (2.2fold increase), which increases their BV/TV% to a level comparable to those observed in the WT mice. This increase in BV/TV% was associated with a statistically significant increase in bone volume (BV) (p = 0.005) and bone number (TN) (p = 0.007). Fenretinide treatment increased BV, 2.8-fold and TN, 2.0-fold compared to Cftr-KO vehicle-control mice. SMI scores (described in detail in the Methods section) shows a significant (p = 0.012) difference between WT and Cftr-KO vehicle-control mice. However, when the Cftr-KO mice are treated with fenretinide, the SMI score is significantly lowered (p = 0.012) to the level of the WT mice resulting in no significant difference between the WT (vehicle controlled) and Cftr-KO (treated). Cftr-KO mice display a significantly (p = 0.009) higher degree of separation when compared to WT vehicle-control mice. However, when mice are treated with fenretinide this difference in
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Fig. 1. A. micro-CT image of femur. Femurs of 5 to 6 mice per group were dissected free of soft tissue, fixed overnight before scanning on a Skyscan 1072 static instrument equipped with 3D Creator analytical software. Representative 3D reconstructions and 2D cross-sectional scans demonstrate a clear difference between vehicle-treated WT and Cftr-KO mice. Cftr-KO mice have much less bone volume then their WT controls. The solid black box illustrates the area of the bone analyzed by micro CT. Fenretinide's positive effect in increasing Cftr-KO bone volume is highlighted by the black square outline on the right. B. von Kossa stains of femur. Bones were embedded in MMA and stained with von Kossa. These were used to confirm the amount of mineralized bone (black stain) to the micro-CT images. Representative slides are shown.
the trabecular bone separation becomes insignificant. Trabecular thickness was also assessed however there was no statistical difference in either the vehicle-controlled groups nor the treated animals. All the micro-CT results observed from the femur were then confirmed by analysis of the L3–L5 vertebrae, which is rich in trabecular bone (data not shown).
3.3. Increased osteoblast formation To establish whether the increase in bone volume observed in the fenretinide-treated Cftr-KO mice results from more efficient bone formation or of less efficient bone resorption we counted the number of osteoblast and osteoclast cells. Table 2
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Table 1 Treatment with fenretinide improves bone composition and architecture in Cftr-KO mice
BV/TV (%)
WT Cftr-KO
BV (mm3)
WT Cftr-KO
TN (1/mm2)
WT Cftr-KO
SMI
WT Cftr-KO
TS (mm)
WT Cftr-KO
TT (mm)
WT Cftr-KO
Vehicle (n = 6)
FEN (n = 5)
p value (^)
Median (25%–75%)
Median (25%–75%)
Vehicle vs FEN
7.78† (5.04, 11.72) 3.82†^ (2.11, 4.85) † p = 0.005 WT-vehicle vs. KO-vehicle 0.26¥ (0.13, 0.36) 0.08¥^ (0.07, 0.12) ¥ p = 0.005 WT-vehicle vs. KO-vehicle 1.61§ (1.07, 2.44) 0.80§^ (0.52, 1.06) § p = 0.005 WT-vehicle vs. KO-vehicle 2.13† (1.80, 2.40) 2.46†^ (2.30, 2.63) † p = 0.012 WT-vehicle vs. KO-vehicle 0.254⁎ (0.194–0.277) 0.284⁎^ (0.272–0.377) ⁎ p = 0.026 WT-vehicle vs. KO-vehicle 0.048 (0.046–0.049) 0.046 (0.042–0.048)
10.20 (8.90–12.59) 8.31^ (7.90–11.95)
ns ^0.007
0.31 (0.24, 0.36) 0.23^ (0.22, 0.34)
ns ^0.005
2.12(1.78, 2.51) 1.62^ (1.54, 2.49)
ns ^0.007
1.90 (1.44, 2.25) 2.16^ (1.85, 2.27)
ns ^0.012
0.201 (0.199–0.227) 0.241^ (0.201–0.259)
ns ^0.009
0.048 (0.047–0.053) 0.049 (0.046–0.054)
ns ns
summarizes the quantification of an average of 3 slides counted per animal in each group from H&E stained slides. Our analysis clearly demonstrates a striking decrease in the number of osteoblasts between the Cftr-KO compared to the WT mice (p = 0.029). Following treatment with fenretinide a significant increase in the number of osteoblasts in the Cftr-KO mice treated as compared to Cftr-KO vehicle-control mice (p = 0.016). Interestingly, no significant difference in number of osteoclasts was found between the fenretinide-treated and untreated Cftr-KO and WT. This finding was corroborated using TRAP staining, as shown as Fig. 2, where the pink stain is positive for tartrate-resistant acidic phosphatase, an enzyme that is specific for osteoclasts cells. No difference between the concentrations of TRAP staining in the Cftr-KO mice compared to the WT controls or fenretinide-treated animals was found. 3.4. Essential fatty acid profiles Fig. 3 panels A–C represent the lipid profiles (AA, DHA, and the AA/DHA ratio) of vehicle-control mice. In all groups a statistical significance is observed between the WT and the CftrKO mice (p = 0.0002, p b 0.0001 and p = 0.0002 respectively). The data in this study illustrates that only 8 treatments with fenretinide effectively reduce the excessive amount of AA consistently found in the plasma of Cftr-KO mice, resulting in no significant difference between WT mice and Cftr-KO mice as shown in Fig. 3D (p = NS). Cftr-KO mice treated with fenretinide also show a positive trend with a 2 fold increase in DHA concentrations then vehicle-control Cftr-KO mice however a statistically significant difference was still observed between the WT and Cftr-KO treated mice as shown in Fig. 3E. When comparing the AA/DHA ratio of vehicle-control Cftr-KO animals to fenretinide-treated Cftr-KO treated mice, it was clearly evident that fenretinide treatment had the ability to decrease the ratio by half, however there was still a statistical difference between the WT and Cftr-KO treated groups (Fig. 3F).
3.5. Ceramide concentrations Ceramide levels in the untreated Cftr-KO mice were significantly lower (p b 0.0001) as compared to their WT littermate controls as shown in Fig. 4. After 4 weeks of semiweekly treatment with fenretinide, the ceramide levels in the treated Cftr-KO mice increased 2.5 fold compared to untreated Cftr-KO mice (p = 0.0002). Although fenretinide did not completely correct the ceramide impairment in the Cftr-KO mice the partial correction was closely associated with the observed protective effect of fenretinide treatment against osteoporosis in these mice. 4. Discussion The data demonstrated in this manuscript illustrates that twice-weekly treatments with fenretinide for 4 weeks, leads to the prevention of the low trabecular bone volume observed in Cftr-KO mice. This increase in trabecular bone density was associated with an increase concentration of ceramide and the down regulation of AA in the plasma. To our knowledge, this is the first time that fenretinide has been documented as an effective prevention of the early onset of osteoporosis in CF. The etiology of early onset osteoporosis in CF patients is not established and most likely involves the complex interactions among several biochemical pathways [34–36]. Aris and Table 2 Treatment with fenretinide number of osteoblasts in Cftr-KO mice Vehicle (n = 4)
FEN (n = 5)
p value (^)
Median (25%–75%)
Median (25%–75%)
vehicle vs FEN
Osteoblast number WT 118⁎ (103–140) 123 (112–143) ns Cftr-KO 71.5⁎^ (56–86) 110^ (105–147) ^0.016 ⁎p = 0.029 WT-vehicle vs KO-vehicle Osteoclast number WT 45.5 (43–50) 50 (44–64) ns Cftr-KO 50.5 (34–66) 54 (48–60) ns
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Fig. 2. Osteoblast and osteoclasts cells quantification in the femur. Bones were decalcified; embedded in paraffin and stained with H&E (inlets). Osteoblasts were identified as single-nucleated, rod shaped cells attached to the trabecular bone as shown by black arrow. Osteoclasts were defined as large multinuclear round (macrophage type) cells attached to the trabecular bone. Multiple slides were used to count the number of osteoblasts and osteoclasts (a representative slide is shown as inlet to TRAP stained slides). Slides were counted at 400× magnification. TRAP staining. Bones were embedded in MMA and stained with TRAP. Multiple slides were analyzed to identify and quantify osteoclasts present in each slide (a representative slide is shown at 200× magnification).
colleagues described the origin of low bone density in patients with CF as the result of several factors including: malnutrition, pancreatic insufficiency, vitamin insufficiency, diabetes, glucocorticoids, inflammatory cytokines, and sex hormone insufficiency [37]. It seems reasonable to assume that the lipid imbalance, one of the hallmarks of CF, would also play an important role in the etiology of osteoporosis. However, all of these factors are intertwined making it difficult to decipher the exact cause, especially when there has been no direct link established between the CFTR gene mutations and reduced bone volume. Research in the CF field was greatly facilitated when the CFTR gene was first cloned, which led to the creation of Cftr-KO mice. Since the first generation of Cftr-KO mice, there have been multiple successful attempts to improve this model, and it is now better suited to the study of CF phenotypes and their treatment [38]. Overall, CF mice still represent the most useful and costefficient animal model available to study such a multifaceted and devastating disease. We have previously reported that the CftrKO mouse model develops spontaneous inflammatory lung disease[39–42]. In addition, we have recently demonstrated that they also exhibit an imbalance between DHA and AA, similar to CF patients [29]. Therefore, it may also represent a very good
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model for studying other CF related phenotypes. Although osteoporotic changes were previously documented in 3 week old CF mice by Dif and colleagues [43], our study shows for the first time using micro-CT technology that adult Cftr-KO mice display a significant defect in BV/TV% compared to their littermate controls. In this manuscript we used our CF mouse model to characterize in a detailed manner the early onset of osteoporosis and to assess the efficiency of fenretinide in treating this condition. Our research demonstrated that Cftr-KO mice develop the osteoporosis phenotype, showing extremely low bone volume and structural abnormalities. Our results suggest that fenretinide not only corrects the quantity of bone but also the quality of the bone structure as well. It is worth noting that using the mouse model to study the etiology of this condition has advantages over human studies because Cftr-KO mice, in contrast to CF patients, are not treated with antibiotics and glucocorticoids. Additionally there is no 1, 25 vitamin D deficiency present in our Cftr-KO mice (data not shown), which allows us to rule out these factors involvement in the development of bone disease. We also measured the change in weight of the Cftr-KO mice before and after treatment of the vehicle-control group and compared it to the treated group and found no statistically significant change in growth caused by the treatment. A skeletal system does not only serve as mechanical support, but rather functions as an active organ that regulates balance and interactions between both local and systemically circulating factors. Bone remodeling is regulated by a number of hormones, cytokines, growth factors, and prostaglandins [44]. Defective fatty acid metabolism has been linked to inflammatory diseases such as asthma and rheumatoid arthritis as well to osteoporosis in elderly people [45–49]. More recently, Gronowitz and colleges reported that the essential fatty acid (n-6/n-3) ratio of children with cystic fibrosis influences bone mineral density, indicating that fatty acid status is important for bone growth [50]. It has previously been suggested that an effective decrease in phospholipid-bound AA concentrations could consequently lead to a decrease in the level of prostaglandin production in bones and an increase in bone formation by improving osteoblast cell function in cell cultures and in periodontal disease in humans [51]. Fenretinide has been shown to catalyze the oxidation of AA so that it can be moved out of the phospholipid membrane and converted to a less harmful metabolite 12-Hydroxyeicosatetraenoic acid (12-HPETE/12HETE) via 12-LOX intracellularly [52]. Ichikawa and colleagues demonstrated that the regulation of the human LOX12 gene is closely associated with bone mineral density [53]. Our results demonstrate that treatment with fenretinide is able to normalize the levels of AA in the plasma of Cftr-KO mice. DHA concentrations were also increased in fenretinide-treated animals, which positively affected a significant decrease in the abnormal AA: DHA ratio typically observed in the Cftr-KO. Taken together, these results demonstrate not only that fenretinide has a protective effect on bone density in Cftr-KO mice, but also provide clues regarding its mechanism of action. Reports had suggested that sphingolipids and ceramide have an important role in modulating bone metabolism [30,50]. Recently our laboratory published Cftr-KO mice have a
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Fig. 3. Lipid profile of Cftr-KO and WT vehicle-control and fenretinide-treated. Mice A–C. Vehicle-control. Concentration's of AA (ng/μg of phosphate), DHA (ng/ μg of phosphate) and the AA/DHA ratio was assessed in WT and Cftr-KO vehicle-control mice. (†) indicates statistical significance between untreated WT and CftrKO vehicle controls. The line represents the median. D–F. Fenretinide Treated. After a total of 8 treatments with fenretinide, AA in the plasma of Cftr-KO mice was decreased significantly to the level observed in the WT. A significant difference between treated Cftr-KO animals and WT animals was no longer detectable (D). After treatment with fenretinide, DHA in the Cftr-KO mice increased 2-fold, illustrating a positive trend (E). Treatments with fenretinide, improved the AA/DHA ratio 2 fold. (†) indicates statistically significant difference between WT and Cftr-KO treated mice. The line represents the median.
ceramide deficiency that was linked to their lung disease phenotype [25]. Our results show, ceramide levels increased 2.5 times after only 8 treatments with fenretinide and subsequently, when analyzing the micro-CT data, we observed that fenretinide treatment also significantly increased the trabecular bone volume and quality of the bone. Matrix vesicles are cell-derived microstructures involved in the initiation of bone mineralization, cartilage and dentin. They are rich in 5-AMP-ase [54] and sphingomyelin, both of which are concentrated in plasma membranes. These membranes are saturated with phospholipids which act as trap for calcium ions [55]. During normal calcification, there is a major influx of calcium and phosphate ions into the cell accompanied by cellular apoptosis and matrix vesicle formation [56]. Matrix vesicles separate from the plasma membrane at sites where there are interactions with the extra cellular matrix, to nucleate mineral formation. These processes coordinate the mineralization during bone development. Kozawa and colleagues hypothesized that sphingomyelinase plays a crucial role as an intercellular messenger in osteoblast differentiation [24]. Our findings strongly suggest that ceramide plays an important role in modulating bone metabolism and that osteoblast proliferation
seems to be increased as well due to fenretinide's effects. We have also proposed a possible mechanism through which fenretinide may be acting to exert this positive effect on bone metabolism as summarized in Fig. 5.
Fig. 4. Ceramide levels in Cftr-KO and WT vehicle-control and treated with fenretinide. Ceramide levels (ng/μg of phosphate) were assessed in plasma isolated from WT and Cftr-KO mice. The ceramide levels in the plasma samples were statistically different p b 0.0001(⁎) between WT and Cftr-KO vehiclecontrol mice. Following treatment with fenretinide, the ceramide levels in the Cftr-KO mice significantly increased p b 0.0002(^).
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Fig. 5. Fenretinide prevents the development of osteoporosis in Cftr-KO mice The chart summarizes the findings from this manuscript. All values are quoted as ranges (minimum and maximum). The diagram depicts biochemical links that might be involved in a fenretinide-induced increase of bone formation in the Cftr-KO mice. Fenretinide stimulates the production of sphingomyelin that subsequently increases ceramide concentrations. Increasing ceramide (a lipid secondary messenger) stimulates lipoxygenase-12 (LOX-12), an enzyme that catalyzes the oxidation of arachidonic acid (AA) to leave the phospholipid bi-layer and become a less harmful metabolite 12-Hydroxyeicosatetraenoic acid (12-HPETE/12-HETE) intracellular.
Overall, treatment with fenretinide represents a unique prophylaxis treatment option by directly increasing ceramide levels, which directly or indirectly decreases excessive AA and consequently increases trabecular bone volume. Based on the results presented in this manuscript, it is clear that fenretinide has potential as a treatment for CF patients, due to its protective role in early onset osteoporosis. It is especially promising since this drug has been reported to have only very mild side effects and has already been approved and currently in clinical trials to treat other disorders, such as neuroblastoma and other cancers. Acknowledgements This work was supported by Canadian Cystic Fibrosis Foundation grants (216714(DR)) and CIHR grant. D.R., C.G., and J.B.S. are inventors on the PCT/CA20061041 patent application entitled “Method for correcting lipid imbalance in a subject", which describes fenretinide-mediated regulation of phopholipids, including ceramides, DHA and AA. None of the authors have a financial relationship with a commercial entity that has a financial interest in the subject of this manuscript. We would like to thank Marie-Christine Guiot for allowing us to use her histology laboratory, Dr. Janet Henderson and technical assistants from the J T N Wong Laboratories for Mineralized Tissue Research for their assistance with micro-CT and histological analyses. We would also like to thank Pierre Camateros for his assistance with statistical analysis and Dr. Emil Skamene for his critical review of this manuscript.
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Fenretinide prevents the development of osteoporosis in Cftr-KO mice Zienab Saeed a , Claudine Guilbault a , Juan B. De Sanctis b , Jennifer Henri a , Dominique Marion a , René St-Arnaud c , Danuta Radzioch a,⁎ a
c
McGill University Health Centre, Montreal General Hospital, Montreal, Qc, Canada b Central University of Venezuela, Institute of Immunology, Caracas, Venezuela McGill University, Department of Human Genetics, Shriner's Hospital, Montreal, QC, Canada
Received 21 June 2007; received in revised form 7 September 2007; accepted 10 September 2007 Available online 7 November 2007
Abstract Background: The most recently described phenotype associated with Cystic Fibrosis (CF) is reduced bone mineral density which results in osteopenia and osteoporosis. The etiology of the early onset of osteoporosis in CF patients has remained to be established. It has been suggested that inadequate nutritional absorption of essential fatty acids may play a role in the altered bone metabolism. In this study, we characterized the protective effect of fenretinide [N-(4-hydroxyphenyl) retinamide], a vitamin A derivative, on the early onset of osteoporosis in cystic fibrosis transmembrane conductance regulator knockout (Cftr-KO) mice. Methods: Using micro-computed-tomography we examined the effect of fenretinide on the bone composition and architecture in a Cftr-KO mouse model which was then confirmed with histological analyses. Plasma fatty acids were quantified using thin layer chromatography-ELISA method. Results: Twice-weekly treatments with fenretinide, over four weeks dramatically increased trabecular bone volume compared to controls. This increase in bone volume was also related to an increased concentration of ceramide in the plasma resulting in the down regulation of phospholipidbound AA in Cftr-KO mice. Conclusions: To our knowledge, this is the first time that fenretinide's protective effect against osteoporosis has been demonstrated. The results of this study strongly suggest that fenretinide has potential to be used as a prophylaxis by preventing the early onset of osteoporosis. © 2007 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Keywords: Osteoporosis; Cystic fibrosis; Arachidonic acid; Docosahexaenoic acid; Ceramide; Cftr-KO mice
1. Introduction One of the most recently described phenotypes that has been associated with CF is reduced bone mineral density which results in osteopenia and osteoporosis [1–5]. A number of studies have documented a prevalence (40–70%) of decreased bone density in the CF population [6]. A longitudinal study of 151 adults with CF showed that 34% of patients had a Z-score1 of −2 or less [7]. Bianchi and colleagues reported that 66% (90/ 136) of children and young adults with CF (age 4–24) have low bone mineral density [8]. However, a direct link between the ⁎ Corresponding author. Research Institute of the McGill University Health Center, 1650, Cedar Avenue, Room L11-218, Montreal, Quebec, Canada H3G 1A4. Tel.: +1 514 934 1934x44517; fax: +1 514 934 8260. E-mail address: [email protected] (D. Radzioch). 1 Z-score is the number of standard deviations above or below what's normally expected for someone of the same age, sex, weight, and ethnic or racial origin.
CFTR gene dysfunction and osteoporosis remains to be elucidated [9]. Currently, physicians prescribe a range of vitamins and minerals including: vitamin D, calcium, vitamin K as prophylaxis. Clinical trials are underway with the goal of finding new potential treatments and that might prevent the development of osteoporosis in CF patients including: anti-resorptive agents such as bisphosphates, anabolic agents such as parathyroid hormone (PTH) (extensively reviewed by Aris and colleagues) and human recombinant growth hormones (hrGH) [10,11]. Recently, epidemiological studies and experiments using animals and humans have shown that a high concentration of n-3 essential fatty acids (EFAs) improves bone metabolism [12]. EFA abnormalities in CF have been well-documented since the early 60's [13]. Freedman and colleagues used Cftr-KO mice to show that CF-affected organs have a significantly lower concentration of docosahexaenoic acid (DHA) and a significantly higher concentration of arachidonic acid (AA) [14]. DHA
1569-1993/$ - see front matter © 2007 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. doi:10.1016/j.jcf.2007.09.001
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is an n-3 polyunsaturated fatty acid that regulates membrane functions and displays anti-inflammatory effects in CF. In contrast, AA is an n-6 polyunsaturated fatty acid that has been shown to regulate the production of prostaglandins and leukotrienes, therefore acting as an agonist of the inflammatory process [15]. DHA and AA are the “yin and yang” of fatty acid metabolism, and disruption of the n-3 and n-6 EFA balance results in a number of systemic abnormalities [16]. It has been proposed that increased levels of AA might contribute to the progression of CF lung disease, and that dietary supplementation with high doses of DHA could correct this imbalance [17]. However, the effectiveness of this treatment option is compromised in patients with CF due to the severe malabsorption of lipids displayed by the majority of CF patients [18]. Treatment with fenretinide [N-(4-hydroxyphenyl) retinamide, 4-HPR], a semi-synthetic retinoid leads to the generation of ceramides [19–21], and increases 12-lipoxygenase (12LOX), an enzyme that catalyzes the oxidation of phospholipidbound AA [22,23], suggesting that fenretinide may be able to decrease AA concentrations. Sphingomyelinase, the enzyme that catalyzes the formation of ceramides plays a crucial role as an intracellular messenger in osteoblast differentiation from stroma cells, suggesting that ceramides may also play an important role in modulating bone metabolism [24]. Recently, our laboratory reported that Cftr-KO mice have impaired levels of ceramides and that treatment with fenretinide was able to correct this impairment [25]. We hypothesized that treatment with fenretinide might be able to prevent the development of osteoporosis by decreasing AA concentrations and increasing ceramide concentrations. Therefore the aim of the presented study was to assess the protective role of fenretinide against CF related osteoporosis. 2. Methods
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in 95% ethanol to a 2 μg/μL concentration. Fourty μL of this preparation was incorporated into the Peptamen liquid diet (5 mg/ kg per day per mouse). The prepared food containing fenretinide was then stored in the dark at 4 °C for no more than 3–6 h before its administration to the mice. To ensure that the entire drug dose was consumed, the mice were given 10 mL of fenretinidecontaining Peptamen, which represents 2/3 of the daily mouse food consumption with the completed dose, in the late afternoon. The remaining 5 mL of Peptamen (without fenretinide) was given the following morning. Mice were treated twice a week for 4 weeks. During the treatment period each mouse was kept in a separate cage. The diet for the mock-treated control group was prepared and administered similarly to that described above, by adding the same volume of ethanol to the Peptamen diet but omitting the fenretinide supplementation (vehicle control). 2.3. Micro computed tomography (micro-CT) Mice were euthanized using CO2 inhalation and exsanguinated by cardiac puncture. Femurs, tibiae and lumbar vertebrae were extracted, stripped of soft tissue and fixed overnight in 4% paraformaldehyde. Micro computed tomography (micro-CT) was performed on the left femur after overnight fixation. The distal metaphysis was scanned with a Skycan 1072 instrument (Skyscan, Antwerp, Belgium). Image acquisition was performed at 100 kVand 98 μA, with a 0.9° rotation between frames. The twodimensional images were used to generate three-dimensional reconstructions to obtain quantitative data using the 3D Creator software supplied with the instrument (ANT 3D Creator software, Skyscan, Antwerp, Belgium). The SMI score is an algorithm taking into account the change in surface area for the change in radial expansion of trabecular plate-like and rod-like structures, which is known as the “trabecular bone pattern factorq [26,27]. A SMI score between 1 and 3 is then reported, as the value approaches 3, the quality of the bone worsens.
2.1. Mice 2.4. Histological analysis The inbred mice (11–14 week old) used in our study were bred in the animal facility of the Research Institute of the McGill University Health Centre (from C57BL/6J breeding pairs heterozygous at the Cftr locus). Age- and gender-matched C57BL/6-Cftr+/+(WT) mice (n = 16), and C57BL/6-Cftr−/−(CftrKO) mice (n = 16) were maintained in a murine pathogen- and parasite-free environment. They were kept in cages with sterile maple hardwood bedding (PWI, St-Hyacinthe, QC, Canada) and housed in ventilated racks (Lab Products, Seaford, DE, USA). The liquid diet was freshly prepared every morning. Experimental procedures with the mice were conducted in accordance with the Canadian Council on Animal Care guidelines and with the approval of the Animal Care Committee of the McGill University Health Center, Montreal, Quebec, Canada. Results were pooled from 3 independent experiments. 2.2. Drug administration Fenretinide powder was kindly provided by Dr. Robert Smith from the NIH (Bethesda, MD, USA). Fenretinide was dissolved
After overnight fixation in 4% paraformaldehyde and rinsing in phosphate buffered saline (PBS), right femurs and tibiae were embedded in polymethylmethacrylate (MMA) or a mixture of 50% MMA and 50% glycolmethacrylate (GMA). Serial 4- to 6-μm sections were cut on a modified Leica RM 2155 rotary microtome (Leica Microsystems, Richmond Hill, Ontario, Canada). MMA-embedded tissues were stained with von Kossa and toluidine blue, while 4 μm MMA-GMA sections were stained with tartrate-resistant acidic phosphatase (TRAP). Images were captured using a Carl Zeiss Microscope (Germany) equipped with an AxioCam MRc camera. The left femur and tibia and the lumbar vertebrae were decalcified with 4% EDTA for paraffin embedding after 14 days. Serial 5 μm sections were cut and stained with haematoxylin and eosin (H&E). Osteoblasts lay down new bone lamellae and are active in bone development and in bone remodeling. Osteoblasts were defined as a singlenucleated, rod shaped cells and were identified along the surface of the trabecular lamellae. Osteoclasts have been shown to be involved in bone resorption, by digesting the adjacent bone
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matrix. Osteoclasts were defined as multi-nucleated cells that were much larger then osteoblasts and displayed typical macrophage morphology.
Dunn's multiple comparison post test between groups. Significance was set at a two-tailed P value of ≤ 0.05. 3. Results
2.5. Fatty acid analysis 3.1. Trabecular bone density Plasma was suspended in 1 mM butylated hydroxyanisole (BHA) in chloroform and methanol (2:1 vol) until the analysis was performed to ensure the integrity of the samples. Lipids were extracted from plasma with chloroform/methanol (2:1), as previously described [28]. Phospholipids were identified by thin layer chromatography extraction. Diazomethane was used to esterify the released fatty acids and the esters were identified by GC/MS (Hewlett Packard5880A, WCOT capillary column (Supelco-10, 35 m × 0.5 mm, 1 μm thick)) using commercial standards (Sigma-Aldrich, Oakville, ON, Canada). 2.6. Ceramide/sphingolipid analysis Ceramide concentrations in the plasma of treated and untreated mice were determined by an enzyme-linked immunosorbent assay (ELISA), performed on lipid samples that were separated by thin layer chromatography (TLC), as have previously described and validated in detail elsewhere [29]. Briefly, lipids were extracted from lung homogenates with chloroform/methanol (2:1) containing 1 mM butylated hydroxyanisole (BHA) [30]. The lipid fractions were then dried under nitrogen and resuspended in heptane [31]. Phospholipid separation was performed by TLC, detected by iodine. On these separated lipid samples, the concentration of ceramide was determined by ELISA [32,33]. The phospholipids from the dry silica were re-suspended in ethanol and used to coat Nunc plates specific for lipid binding. Plates were then washed, incubated with blocking buffer for 1 h at 37 °C (PBS, 0.1% Tween 20), and 1% bovine serum albumin (BSA; Sigma, Oakville, ON), and incubated with murine anti-ceramide IgM (Sigma-Aldrich) antibody (Ab) for 1 h at 37 °C. The plates were then washed and incubated with peroxidaseconjugated anti-mouse IgM Ab for 1 h at 37 °C. Finally, the plates were incubated with the peroxidase substrate (TMB; Roche, Laval, QC). The intensity of the colorimetric reaction was determined by spectrophotometry at 405 nm. The levels of ceramide were calculated with reference to a standard curve prepared using purified ceramide (Sigma-Aldrich). Phosphate levels were assessed, as previously described, using the PiBlue™ Phosphate assay kit (Boehringer Ingelheim, Ingelheim, Germany), according to the manufacturer's instructions. 2.7. Statistical analyses Data was analyzed using GraphPad Prism Version 4.03 software (GraphPad Software, San Diego, California, USA). Statistically significant differences between the medians of studied groups were evaluated using the non-parametric Mann– Whitney U test when comparing two groups (Fig. 3). Pair-wise multiple comparisons were used to evaluate the differences between multiple groups using Kruskal–Wallis on ranks and the
Trabecular bone density was measured using micro-CT scans and histology. Fig. 1A illustrated the micro-CT scans of the trabecular bones isolated from Cftr-KO mice femurs and their littermate controls (WT). Results illustrated clearly show evidence of reduced trabecular bone in the Cftr-KO compared to their WT littermate controls. These results also clearly demonstrate that twice-weekly treatment with fenretinide over the course of four weeks (total 8 doses) was able to completely eliminate any signs of osteoporosis in the trabecular bone of Cftr-KO mice. Representative 3D reconstructions and 2D crosssectional scans demonstrate that, before fenretinide treatment, Cftr-KO mice have virtually no trabecular bone (highlighted in black boxes) evident in the cross-sectional image as compared to WT controls. After treatment with fenretinide there is a dramatic increase in trabecular bone in the Cftr-KO mice compared to their WT controls. Micro-CT results were compared with histochemical staining of non-decalcified bone using von Kossa stain, which stains mineralized bone as shown in Fig. 1B. Cftr-KO mice clearly illustrate a decrease in trabecular bone (highlighted in the black box) compared to the WT controls, meanwhile when Cftr-KO mice are treated with fenretinide, the early onset of reduced bone mineral density is avoided. 3.2. Bone composition and architecture To address the osteoporotic changes from the composition and architecture point of view, the trabecular bone was quantified. The following parameters: bone volume fraction (BV/TV%), structural model index (SMI) trabecular separation (TS), and trabecular thickness (TT) were calculated from the left femur using micro-CT scans. Our results illustrate a significant (p = 0.005) lower amount of BV/TV% in the Cftr-KO mice as compared to WT vehiclecontrols as shown in Table 1. Interestingly, this difference is eliminated when Cftr-KO mice are treated with fenretinide (2.2fold increase), which increases their BV/TV% to a level comparable to those observed in the WT mice. This increase in BV/TV% was associated with a statistically significant increase in bone volume (BV) (p = 0.005) and bone number (TN) (p = 0.007). Fenretinide treatment increased BV, 2.8-fold and TN, 2.0-fold compared to Cftr-KO vehicle-control mice. SMI scores (described in detail in the Methods section) shows a significant (p = 0.012) difference between WT and Cftr-KO vehicle-control mice. However, when the Cftr-KO mice are treated with fenretinide, the SMI score is significantly lowered (p = 0.012) to the level of the WT mice resulting in no significant difference between the WT (vehicle controlled) and Cftr-KO (treated). Cftr-KO mice display a significantly (p = 0.009) higher degree of separation when compared to WT vehicle-control mice. However, when mice are treated with fenretinide this difference in
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Fig. 1. A. micro-CT image of femur. Femurs of 5 to 6 mice per group were dissected free of soft tissue, fixed overnight before scanning on a Skyscan 1072 static instrument equipped with 3D Creator analytical software. Representative 3D reconstructions and 2D cross-sectional scans demonstrate a clear difference between vehicle-treated WT and Cftr-KO mice. Cftr-KO mice have much less bone volume then their WT controls. The solid black box illustrates the area of the bone analyzed by micro CT. Fenretinide's positive effect in increasing Cftr-KO bone volume is highlighted by the black square outline on the right. B. von Kossa stains of femur. Bones were embedded in MMA and stained with von Kossa. These were used to confirm the amount of mineralized bone (black stain) to the micro-CT images. Representative slides are shown.
the trabecular bone separation becomes insignificant. Trabecular thickness was also assessed however there was no statistical difference in either the vehicle-controlled groups nor the treated animals. All the micro-CT results observed from the femur were then confirmed by analysis of the L3–L5 vertebrae, which is rich in trabecular bone (data not shown).
3.3. Increased osteoblast formation To establish whether the increase in bone volume observed in the fenretinide-treated Cftr-KO mice results from more efficient bone formation or of less efficient bone resorption we counted the number of osteoblast and osteoclast cells. Table 2
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Table 1 Treatment with fenretinide improves bone composition and architecture in Cftr-KO mice
BV/TV (%)
WT Cftr-KO
BV (mm3)
WT Cftr-KO
TN (1/mm2)
WT Cftr-KO
SMI
WT Cftr-KO
TS (mm)
WT Cftr-KO
TT (mm)
WT Cftr-KO
Vehicle (n = 6)
FEN (n = 5)
p value (^)
Median (25%–75%)
Median (25%–75%)
Vehicle vs FEN
7.78† (5.04, 11.72) 3.82†^ (2.11, 4.85) † p = 0.005 WT-vehicle vs. KO-vehicle 0.26¥ (0.13, 0.36) 0.08¥^ (0.07, 0.12) ¥ p = 0.005 WT-vehicle vs. KO-vehicle 1.61§ (1.07, 2.44) 0.80§^ (0.52, 1.06) § p = 0.005 WT-vehicle vs. KO-vehicle 2.13† (1.80, 2.40) 2.46†^ (2.30, 2.63) † p = 0.012 WT-vehicle vs. KO-vehicle 0.254⁎ (0.194–0.277) 0.284⁎^ (0.272–0.377) ⁎ p = 0.026 WT-vehicle vs. KO-vehicle 0.048 (0.046–0.049) 0.046 (0.042–0.048)
10.20 (8.90–12.59) 8.31^ (7.90–11.95)
ns ^0.007
0.31 (0.24, 0.36) 0.23^ (0.22, 0.34)
ns ^0.005
2.12(1.78, 2.51) 1.62^ (1.54, 2.49)
ns ^0.007
1.90 (1.44, 2.25) 2.16^ (1.85, 2.27)
ns ^0.012
0.201 (0.199–0.227) 0.241^ (0.201–0.259)
ns ^0.009
0.048 (0.047–0.053) 0.049 (0.046–0.054)
ns ns
summarizes the quantification of an average of 3 slides counted per animal in each group from H&E stained slides. Our analysis clearly demonstrates a striking decrease in the number of osteoblasts between the Cftr-KO compared to the WT mice (p = 0.029). Following treatment with fenretinide a significant increase in the number of osteoblasts in the Cftr-KO mice treated as compared to Cftr-KO vehicle-control mice (p = 0.016). Interestingly, no significant difference in number of osteoclasts was found between the fenretinide-treated and untreated Cftr-KO and WT. This finding was corroborated using TRAP staining, as shown as Fig. 2, where the pink stain is positive for tartrate-resistant acidic phosphatase, an enzyme that is specific for osteoclasts cells. No difference between the concentrations of TRAP staining in the Cftr-KO mice compared to the WT controls or fenretinide-treated animals was found. 3.4. Essential fatty acid profiles Fig. 3 panels A–C represent the lipid profiles (AA, DHA, and the AA/DHA ratio) of vehicle-control mice. In all groups a statistical significance is observed between the WT and the CftrKO mice (p = 0.0002, p b 0.0001 and p = 0.0002 respectively). The data in this study illustrates that only 8 treatments with fenretinide effectively reduce the excessive amount of AA consistently found in the plasma of Cftr-KO mice, resulting in no significant difference between WT mice and Cftr-KO mice as shown in Fig. 3D (p = NS). Cftr-KO mice treated with fenretinide also show a positive trend with a 2 fold increase in DHA concentrations then vehicle-control Cftr-KO mice however a statistically significant difference was still observed between the WT and Cftr-KO treated mice as shown in Fig. 3E. When comparing the AA/DHA ratio of vehicle-control Cftr-KO animals to fenretinide-treated Cftr-KO treated mice, it was clearly evident that fenretinide treatment had the ability to decrease the ratio by half, however there was still a statistical difference between the WT and Cftr-KO treated groups (Fig. 3F).
3.5. Ceramide concentrations Ceramide levels in the untreated Cftr-KO mice were significantly lower (p b 0.0001) as compared to their WT littermate controls as shown in Fig. 4. After 4 weeks of semiweekly treatment with fenretinide, the ceramide levels in the treated Cftr-KO mice increased 2.5 fold compared to untreated Cftr-KO mice (p = 0.0002). Although fenretinide did not completely correct the ceramide impairment in the Cftr-KO mice the partial correction was closely associated with the observed protective effect of fenretinide treatment against osteoporosis in these mice. 4. Discussion The data demonstrated in this manuscript illustrates that twice-weekly treatments with fenretinide for 4 weeks, leads to the prevention of the low trabecular bone volume observed in Cftr-KO mice. This increase in trabecular bone density was associated with an increase concentration of ceramide and the down regulation of AA in the plasma. To our knowledge, this is the first time that fenretinide has been documented as an effective prevention of the early onset of osteoporosis in CF. The etiology of early onset osteoporosis in CF patients is not established and most likely involves the complex interactions among several biochemical pathways [34–36]. Aris and Table 2 Treatment with fenretinide number of osteoblasts in Cftr-KO mice Vehicle (n = 4)
FEN (n = 5)
p value (^)
Median (25%–75%)
Median (25%–75%)
vehicle vs FEN
Osteoblast number WT 118⁎ (103–140) 123 (112–143) ns Cftr-KO 71.5⁎^ (56–86) 110^ (105–147) ^0.016 ⁎p = 0.029 WT-vehicle vs KO-vehicle Osteoclast number WT 45.5 (43–50) 50 (44–64) ns Cftr-KO 50.5 (34–66) 54 (48–60) ns
Z. Saeed et al. / Journal of Cystic Fibrosis 7 (2008) 222–230
Fig. 2. Osteoblast and osteoclasts cells quantification in the femur. Bones were decalcified; embedded in paraffin and stained with H&E (inlets). Osteoblasts were identified as single-nucleated, rod shaped cells attached to the trabecular bone as shown by black arrow. Osteoclasts were defined as large multinuclear round (macrophage type) cells attached to the trabecular bone. Multiple slides were used to count the number of osteoblasts and osteoclasts (a representative slide is shown as inlet to TRAP stained slides). Slides were counted at 400× magnification. TRAP staining. Bones were embedded in MMA and stained with TRAP. Multiple slides were analyzed to identify and quantify osteoclasts present in each slide (a representative slide is shown at 200× magnification).
colleagues described the origin of low bone density in patients with CF as the result of several factors including: malnutrition, pancreatic insufficiency, vitamin insufficiency, diabetes, glucocorticoids, inflammatory cytokines, and sex hormone insufficiency [37]. It seems reasonable to assume that the lipid imbalance, one of the hallmarks of CF, would also play an important role in the etiology of osteoporosis. However, all of these factors are intertwined making it difficult to decipher the exact cause, especially when there has been no direct link established between the CFTR gene mutations and reduced bone volume. Research in the CF field was greatly facilitated when the CFTR gene was first cloned, which led to the creation of Cftr-KO mice. Since the first generation of Cftr-KO mice, there have been multiple successful attempts to improve this model, and it is now better suited to the study of CF phenotypes and their treatment [38]. Overall, CF mice still represent the most useful and costefficient animal model available to study such a multifaceted and devastating disease. We have previously reported that the CftrKO mouse model develops spontaneous inflammatory lung disease[39–42]. In addition, we have recently demonstrated that they also exhibit an imbalance between DHA and AA, similar to CF patients [29]. Therefore, it may also represent a very good
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model for studying other CF related phenotypes. Although osteoporotic changes were previously documented in 3 week old CF mice by Dif and colleagues [43], our study shows for the first time using micro-CT technology that adult Cftr-KO mice display a significant defect in BV/TV% compared to their littermate controls. In this manuscript we used our CF mouse model to characterize in a detailed manner the early onset of osteoporosis and to assess the efficiency of fenretinide in treating this condition. Our research demonstrated that Cftr-KO mice develop the osteoporosis phenotype, showing extremely low bone volume and structural abnormalities. Our results suggest that fenretinide not only corrects the quantity of bone but also the quality of the bone structure as well. It is worth noting that using the mouse model to study the etiology of this condition has advantages over human studies because Cftr-KO mice, in contrast to CF patients, are not treated with antibiotics and glucocorticoids. Additionally there is no 1, 25 vitamin D deficiency present in our Cftr-KO mice (data not shown), which allows us to rule out these factors involvement in the development of bone disease. We also measured the change in weight of the Cftr-KO mice before and after treatment of the vehicle-control group and compared it to the treated group and found no statistically significant change in growth caused by the treatment. A skeletal system does not only serve as mechanical support, but rather functions as an active organ that regulates balance and interactions between both local and systemically circulating factors. Bone remodeling is regulated by a number of hormones, cytokines, growth factors, and prostaglandins [44]. Defective fatty acid metabolism has been linked to inflammatory diseases such as asthma and rheumatoid arthritis as well to osteoporosis in elderly people [45–49]. More recently, Gronowitz and colleges reported that the essential fatty acid (n-6/n-3) ratio of children with cystic fibrosis influences bone mineral density, indicating that fatty acid status is important for bone growth [50]. It has previously been suggested that an effective decrease in phospholipid-bound AA concentrations could consequently lead to a decrease in the level of prostaglandin production in bones and an increase in bone formation by improving osteoblast cell function in cell cultures and in periodontal disease in humans [51]. Fenretinide has been shown to catalyze the oxidation of AA so that it can be moved out of the phospholipid membrane and converted to a less harmful metabolite 12-Hydroxyeicosatetraenoic acid (12-HPETE/12HETE) via 12-LOX intracellularly [52]. Ichikawa and colleagues demonstrated that the regulation of the human LOX12 gene is closely associated with bone mineral density [53]. Our results demonstrate that treatment with fenretinide is able to normalize the levels of AA in the plasma of Cftr-KO mice. DHA concentrations were also increased in fenretinide-treated animals, which positively affected a significant decrease in the abnormal AA: DHA ratio typically observed in the Cftr-KO. Taken together, these results demonstrate not only that fenretinide has a protective effect on bone density in Cftr-KO mice, but also provide clues regarding its mechanism of action. Reports had suggested that sphingolipids and ceramide have an important role in modulating bone metabolism [30,50]. Recently our laboratory published Cftr-KO mice have a
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Fig. 3. Lipid profile of Cftr-KO and WT vehicle-control and fenretinide-treated. Mice A–C. Vehicle-control. Concentration's of AA (ng/μg of phosphate), DHA (ng/ μg of phosphate) and the AA/DHA ratio was assessed in WT and Cftr-KO vehicle-control mice. (†) indicates statistical significance between untreated WT and CftrKO vehicle controls. The line represents the median. D–F. Fenretinide Treated. After a total of 8 treatments with fenretinide, AA in the plasma of Cftr-KO mice was decreased significantly to the level observed in the WT. A significant difference between treated Cftr-KO animals and WT animals was no longer detectable (D). After treatment with fenretinide, DHA in the Cftr-KO mice increased 2-fold, illustrating a positive trend (E). Treatments with fenretinide, improved the AA/DHA ratio 2 fold. (†) indicates statistically significant difference between WT and Cftr-KO treated mice. The line represents the median.
ceramide deficiency that was linked to their lung disease phenotype [25]. Our results show, ceramide levels increased 2.5 times after only 8 treatments with fenretinide and subsequently, when analyzing the micro-CT data, we observed that fenretinide treatment also significantly increased the trabecular bone volume and quality of the bone. Matrix vesicles are cell-derived microstructures involved in the initiation of bone mineralization, cartilage and dentin. They are rich in 5-AMP-ase [54] and sphingomyelin, both of which are concentrated in plasma membranes. These membranes are saturated with phospholipids which act as trap for calcium ions [55]. During normal calcification, there is a major influx of calcium and phosphate ions into the cell accompanied by cellular apoptosis and matrix vesicle formation [56]. Matrix vesicles separate from the plasma membrane at sites where there are interactions with the extra cellular matrix, to nucleate mineral formation. These processes coordinate the mineralization during bone development. Kozawa and colleagues hypothesized that sphingomyelinase plays a crucial role as an intercellular messenger in osteoblast differentiation [24]. Our findings strongly suggest that ceramide plays an important role in modulating bone metabolism and that osteoblast proliferation
seems to be increased as well due to fenretinide's effects. We have also proposed a possible mechanism through which fenretinide may be acting to exert this positive effect on bone metabolism as summarized in Fig. 5.
Fig. 4. Ceramide levels in Cftr-KO and WT vehicle-control and treated with fenretinide. Ceramide levels (ng/μg of phosphate) were assessed in plasma isolated from WT and Cftr-KO mice. The ceramide levels in the plasma samples were statistically different p b 0.0001(⁎) between WT and Cftr-KO vehiclecontrol mice. Following treatment with fenretinide, the ceramide levels in the Cftr-KO mice significantly increased p b 0.0002(^).
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Fig. 5. Fenretinide prevents the development of osteoporosis in Cftr-KO mice The chart summarizes the findings from this manuscript. All values are quoted as ranges (minimum and maximum). The diagram depicts biochemical links that might be involved in a fenretinide-induced increase of bone formation in the Cftr-KO mice. Fenretinide stimulates the production of sphingomyelin that subsequently increases ceramide concentrations. Increasing ceramide (a lipid secondary messenger) stimulates lipoxygenase-12 (LOX-12), an enzyme that catalyzes the oxidation of arachidonic acid (AA) to leave the phospholipid bi-layer and become a less harmful metabolite 12-Hydroxyeicosatetraenoic acid (12-HPETE/12-HETE) intracellular.
Overall, treatment with fenretinide represents a unique prophylaxis treatment option by directly increasing ceramide levels, which directly or indirectly decreases excessive AA and consequently increases trabecular bone volume. Based on the results presented in this manuscript, it is clear that fenretinide has potential as a treatment for CF patients, due to its protective role in early onset osteoporosis. It is especially promising since this drug has been reported to have only very mild side effects and has already been approved and currently in clinical trials to treat other disorders, such as neuroblastoma and other cancers. Acknowledgements This work was supported by Canadian Cystic Fibrosis Foundation grants (216714(DR)) and CIHR grant. D.R., C.G., and J.B.S. are inventors on the PCT/CA20061041 patent application entitled “Method for correcting lipid imbalance in a subject", which describes fenretinide-mediated regulation of phopholipids, including ceramides, DHA and AA. None of the authors have a financial relationship with a commercial entity that has a financial interest in the subject of this manuscript. We would like to thank Marie-Christine Guiot for allowing us to use her histology laboratory, Dr. Janet Henderson and technical assistants from the J T N Wong Laboratories for Mineralized Tissue Research for their assistance with micro-CT and histological analyses. We would also like to thank Pierre Camateros for his assistance with statistical analysis and Dr. Emil Skamene for his critical review of this manuscript.
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