Rescue of Impaired Fracture Healing in COX-2−/− Mice via Activation of Prostaglandin E2 Receptor Subtype 4
2009; Elsevier BV; Volume: 175; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.081099
ISSN1525-2191
AutoresChao Xie, Bojian Liang, Ming Xue, Angela Lin, Alayna E. Loiselle, Edward M. Schwarz, Robert E. Guldberg, Regis J. O’Keefe, Xinping Zhang,
Tópico(s)Estrogen and related hormone effects
ResumoAlthough the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2−/− mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2−/− mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2−/− callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2−/− mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2−/− fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2−/− mice. Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2−/− mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2−/− mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2−/− callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2−/− mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2−/− fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2−/− mice. Fracture healing is a complex process orchestrated by precise presentation of growth factors and cytokines that control activation, proliferation, and differentiation of the local mesenchymal stem/progenitor cells. Fracture healing begins with hematoma formation and an inflammatory response. The activated stem/progenitor cells proliferate and further differentiate into osteoblasts and chondrocytes. Endochondral bone formation takes place toward the most central avascular region of the callus. Chondrogenesis initiates directly adjacent to the surface of the cortical bone and is surrounded by less-differentiated mesenchymal progenitor cells. The subsequent expansion of the callus involves the conversion of the lingering mesenchymal progenitor cells into chondrocytes and further proliferation and differentiation of chondrocytes into a calcified cartilage template that permits vascular invasion and bone formation. Areas of intramembranous bone formation flank the area of endochondral ossification, particularly along the bone surface furthest from the central fracture site where the blood supply is typically better preserved. 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PGH2 is further metabolized by specific isomerases to produce various derivatives, eg, PGE2, PGD2, PGA2, thromboxanes, and prostacyclins, collectively called prostanoids. Prostanoids are labile molecules that act locally as important microenvironmental hormones mediating autocrine and/or paracrine functions. 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The goal of this study was to further understand the molecular mechanisms and downstream targets of COX-2 during fracture repair. To this end, we used the COX-2−/− mouse model and selective EP agonists that have been shown to enhance bone formation in animal models.14Li M Ke HZ Qi H Healy DR Li Y Crawford DT Paralkar VM Owen TA Cameron KO Lefker BA Brown TA Thompson DD A novel, non-prostanoid EP2 receptor-selective prostaglandin E2 agonist stimulates local bone formation and enhances fracture healing.J Bone Miner Res. 2003; 18: 2033-2042Crossref PubMed Scopus (94) Google Scholar, 16Ke HZ Crawford DT Qi H Simmons HA Owen TA Paralkar VM Li M Lu B Grasser WA Cameron KO Lefker BA DaSilva-Jardine P Scott DO Zhang Q Tian XY Jee WS Brown TA Thompson DD A nonprostanoid EP4 receptor selective prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats.J Bone Miner Res. 2006; 21: 565-575Crossref PubMed Scopus (51) Google Scholar, 26Aguirre JI Leal ME Rivera MF Vanegas SM Jorgensen M Wronski TJ Effects of basic fibroblast growth factor and a prostaglandin E2 receptor subtype 4 agonist on osteoblastogenesis and adipogenesis in aged ovariectomized rats.J Bone Miner Res. 2007; 22: 877-888Crossref PubMed Scopus (25) Google Scholar By performing detailed analyses using micro computed tomography (CT) and histology we show that COX-2 is induced early in the bone-healing milieu. Deficiency in COX-2 impairs initiation and completion of endochondral bone repair. Local injection of an EP2 agonist, CP-463755 marginally improved fracture healing in COX-2−/− mice whereas delivery of an EP4 agonist, CP734432, reversed the defective bone fracture healing in COX-2−/− mice. Further in vitro analyses demonstrated a differential role of EP receptor in chondrogenesis and induction of MMP-9, a matrix metalloproteinase (MMP) essential for angiogenesis during endochondral ossification. Together, the results pinpoint a key role of the COX-2/PGE2/EP4 pathway in fracture repair. COX-2−/− mice were originally obtained from the breeding colony maintained at the University of North Carolina. They were bred to a 129/ola genetic background and intercrossed for about 30 generations. In all experiments, wild-type littermates were used as controls for COX-2−/− mice. All animal surgery procedures were approved by the University of Rochester Institutional Committee of Animal Resources. The nonprostanoid EP4 receptor agonist CP-73443217Yoshida K Oida H Kobayashi T Maruyama T Tanaka M Katayama T Yamaguchi K Segi E Tsuboyama T Matsushita M Ito K Ito Y Sugimoto Y Ushikubi F Ohuchida S Kondo K Nakamura T Narumiya S Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation.Proc Natl Acad Sci USA. 2002; 99: 4580-4585Crossref PubMed Scopus (282) Google Scholar, 27Li M Healy DR Li Y Cameron K Ke HZ Lefker BA Rosati RL Thompson DD CP-463,755. A non-prostanoid EP2 receptor agonist stimulates fracture healing in a rat femoral fracture model.J Bone Miner Res. 2000; 15: S343Crossref Scopus (82) Google Scholar and EP2 receptor agonist CP-463755 were obtained from Pfizer Global Research and Development (Groton, Connecticut). CP-463755 binds selectively to rat EP2 receptor with a binding IC50 of 14 nmol/L for EP2 receptor subtype and >3200 nmol/L for EP1, EP3, and EP4 receptor subtypes.27Li M Healy DR Li Y Cameron K Ke HZ Lefker BA Rosati RL Thompson DD CP-463,755. A non-prostanoid EP2 receptor agonist stimulates fracture healing in a rat femoral fracture model.J Bone Miner Res. 2000; 15: S343Crossref Scopus (82) Google Scholar EP1 agonist (Misoprostol) and PGE2 were purchased from Cayman Chemicals (Ann Arbor, MI). For in vitro studies, all agonists were used at a final concentration of 10−6 M/L. Agonists for EP2 or EP4 receptor were dissolved in 5% ethanol and administered for 14 days at a dose of 20 mg/kg/day via periosteal injection at the fracture site. Drug therapy was initiated on the day of surgery and all animals were sacrificed on day 14 following fracture. To create a closed stabilized mid-diaphyseal femur fracture, mice received anesthesia using a mix of ketamine and xylazine. The skin and the underlying soft tissues over the left knee were incised lateral to the patellar tendon. The tendon was displaced medially, and a small hole was drilled into the distal femur using a 26-gauge needle. A stylus pin from a 25G Quincke Type spinal needle (BD Medical Systems, Franklin Lakes, NJ) was inserted into the intramedullary canal and clipped. The wound was sutured closed. Fractures were created using a three-point bending Einhorn device as previously described.28Bonnarens F Einhorn TA Production of a standard closed fracture in laboratory animal bone.J Orthop Res. 1984; 2: 97-101Crossref PubMed Scopus (609) Google Scholar A Faxitron system (Faxitron X-ray, Wheeling, IL) was used to take X-ray images at the time of surgery and every week following surgery until sacrifice. Fractured femurs were harvested and processed for histological analyses as previously described.18Zhang X Schwarz EM Young DA Puzas JE Rosier RN O'Keefe RJ Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair.J Clin Invest. 2002; 109: 1405-1415Crossref PubMed Scopus (590) Google Scholar Mice were sacrificed at 3, 5, 7, 10, and 14 days after fracture. A normal mid-diaphysis femoral bone segment was used as a nonfractured, day 0 control. Femurs were disarticulated from the hip and trimmed to remove excess muscle and skin. Specimens were stored in 10% neutral buffered formalin for 2 days. The tissues were infiltrated and embedded in paraffin. Alcian blue and OrangeG staining was done as previously described.18Zhang X Schwarz EM Young DA Puzas JE Rosier RN O'Keefe RJ Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair.J Clin Invest. 2002; 109: 1405-1415Crossref PubMed Scopus (590) Google Scholar Histomorphometric analyses were performed using Osteometrics software to determine the area of bone, cartilage, and mesenchyme (a subtraction of total callus from bone and cartilage tissue) formation by manual tracing. Pre-existing cortical bone was excluded from the histomorphometric analysis. The total area for each sample was then used to quantify the percent areas of bone, cartilage, and mesenchyme. At least three nonconsecutive sections were used for histomorphometric analyses. The mean value represented one sample. A minimum of eight samples was included in each group. The mean of the eight samples was used in statistical analyses to determine the composition of the fracture callus. Cortical bone was excluded from the histomorphometric analysis. For RNA analyses, mice were placed in groups of four (n = 4) and sacrificed on days 0, 3, 5, 7, 10, 14, and 21 following surgery. The fracture calluses were carefully dissected free of muscles and soft tissue and immediately snap frozen in a liquid nitrogen bath. Frozen tissue samples were homogenized using a liquid nitrogen-cooled mortar and pestle apparatus, and mRNA was purified via phase separation (TRIzol, Invitrogen, Carlsbad, CA). Exactly 0.5 μg of mRNA per callus was pooled and used in reverse transcription to make single-strand cDNA. Single-strand cDNA was synthesized using a commercial first strand cDNA synthesis kit (Invitrogen). qPCR reaction was performed using SyberGreen (ABgene, Rochester, NY) in a RotorGene real time PCR machine (Corbett Research, Carlsbad, CA). All genes were compared with a standard β-actin control. Data were assessed quantitatively using analysis of variance comparing relative levels of transcript expression as a function of time. The following primers were used for the assessment: Actin: 5′-CCCCACTGAAGCCTACAAAA-3, 5′-GGGAGGCTCCTCCATTC-3′; OC: 5′ TGCTTGTGACGAGCTATCAG-3′, 5′-GAGGACAGGGAGGATCAAGT-3′; col-2: 5′-CCACACCAAATTCCTGTTCA-3′, 5′-ACTGGTAAGTGGGGCAAGAC-3′; col-X: 5′-CTTTGTGTGCCTTTC AATCG-3′, 5′-GTGAGGTACAGCCTACCAGTTTT-3′; MMP9 - 5-TGAATCAGCTGGCTTTTGTG-3, 5-ACCTTCCAGTAGGGGCAACT-3; and COX-2: 5′-CACAGCCTACCAAAACAGCCA-3′, 5′-GCTCAGTTGAACGCCTTTTGA-3′. Femurs were harvested at indicated times and scanned using a Viva microCT system (Scanco Medical, Bassersdorf, Switzerland) at a voxel size of 10.5 μm to image bone or vasculature. New bone formation and vascular volume were measured as previously described.29Duvall CL Robert Taylor W Weiss D Guldberg RE Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury.Am J Physiol Heart Circ Physiol. 2004; 287: H302-H310Crossref PubMed Scopus (192) Google Scholar, 30Duvall CL Taylor WR Weiss D Wojtowicz AM Guldberg RE Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice.J Bone Miner Res. 2007; 22: 286-297Crossref PubMed Scopus (163) Google Scholar Briefly, mice were sacrificed and given serial intracardiac injections of heparinized saline, 10% neutral buffered formalin, and a lead chromate microfil perfusion reagent (Flow Tech, Carver, MA). The whole mice were soaked in 10% neutral buffered formalin overnight at room temperature. The fractured limbs were disarticulated at the hip, and excess soft tissue was removed. Calluses were further fixed in neutral buffered formalin for 2 additional days, and decalcified in 10% EDTA for 21 days. The samples were scanned in a VivaCT Scanner (Scanco Medical AG, Bassersdorf, Switzerland) at high resolution with a 12.5-μm voxel size. An integration time of 300 ms, a current of 145 mA, and an energy setting of 55 kV were used. The threshold was chosen using two-dimensional evaluation of several slices in the transverse anatomical plane so that mineralized callus and vascular contrast reagent were identified while surrounding soft tissue was excluded. An average threshold of 250 was optimal and used uniformly for all samples. Next, each sample was contoured around the external callus and along the edge of the cortical bone. All mineralized tissues above threshold between these two boundaries were included. Thus, external soft tissues and cortical bone including the marrow cavity were excluded. Contouring of images was done every 20 axial slices proximally to distally until the callus was not visible. Histomorphometric analysis based on direct distance transform methods31Hildebrand T Laib A Muller R Dequeker J Ruegsegger P Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus.J Bone Miner Res. 1999; 14: 1167-1174Crossref PubMed Scopus (1098) Google Scholar, 32Hildebrand T Ruegsegger P Quantification of Bone Microarchitecture with the Structure Model Index.Comput Methods Biomech Biomed Engin. 1997; 1: 15-23Crossref PubMed Scopus (896) Google Scholar was subsequently performed on the three-dimensional images to quantify parameters of vascular network morphology, including vasculature volume, vessel thickness, vessel density (defined as an average number of vessels intersected by test lines passing through the three-dimensional image normalized by test line length), vessel spacing, and connectivity.29Duvall CL Robert Taylor W Weiss D Guldberg RE Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury.Am J Physiol Heart Circ Physiol. 2004; 287: H302-H310Crossref PubMed Scopus (192) Google Scholar, 33Guldberg RE Ballock RT Boyan BD Duvall CL Lin AS Nagaraja S Oest M Phillips J Porter BD Robertson G Taylor WR Analyzing bone, blood vessels, and biomaterials with microcomputed tomography.IEEE Eng Med Biol Mag. 2003; 22: 77-83Crossref PubMed Scopus (61) Google Scholar We used four animals for each group. Data sets were examined using statistical assessments including analysis of variance. 35S-UTP-labeled sense and antisense riboprobes against murine COX-2 were synthesized from a plasmid (kindly provided by Dr. Joseph Bonventre) as previously described.34Sapirstein A Saito H Texel SJ Samad TA O'Leary E Bonventre JV Cytosolic phospholipase A2alpha regulates induction of brain cyclooxygenase-2 in a mouse model of inflammation.Am J Physiol Regul Integr Comp Physiol. 2005; 288: R1774-R1782Crossref PubMed Scopus (38) Google Scholar The specific activities of the probes were determined by radioactivity. The sections were incubated in hybridization buffer (50% formamide, 0.3 M/L NaCl, 20 mmol/L Tris-HCl, 5 mmol/L EDTA, 10% dextran sulfate, 0.02% Ficoll, 0.02% bovine serum albumen, 0.02% polyvinyl pyrrolidone, and 0.5 mg/ml yeast RNA) containing riboprobe at 10,000 cpm/μl. Hybridization was performed at 55°C overnight. Emulsion-dipped slides were exposed for about 7 to 14 days depending on the intensity of the signals. Anti-sense probes of COX-2 and Col2a1 were further used as controls for the experiments. Immunohistochemical staining for nuclear antigen proliferating cell nuclear antigen (PCNA) protein was performed using a staining kit purchased from Zymed (S. San Francisco, CA). The staining procedures were followed as instructed by the manufacturer. To determine the number of PCNA+ cells, three sections of each fracture samples were used to count the positive cells at the fracture junctions. The mean of the three sections represented each sample. A group of three fractured samples was included for each time point. The mean from three samples, divided by the average of the area of the periosteal callus was used in statistical analyses. Limb bud mesenchymal cells were isolated from embryos of 11.5 day time-pregnant female CD1 mice (Charles River, Wilmington, MA) as described.35Zhang X Ziran N Goater JJ Schwarz EM Puzas JE Rosier RN Zuscik M Drissi H O'Keefe RJ Primary murine limb bud mesenchymal cells in long-term culture complete chondrocyte differentiation: tGF-beta delays hypertrophy and PGE2 inhibits terminal differentiation.Bone. 2004; 34: 809-817Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar The distal 1/4 subridge (distal tips) of the limbs were pooled and digested with Dispase (1 U/ml) for 1 hour at 37°C. A total of 1 × 105 cells in 10 μl of media were placed in micromass in the center of a 24-well plate and cultured in medium containing 10% fetal bovine serum, 40% Dulbecco's Modified Eagle Medium and 60% F12. PGE2 or EP agonists (Cayman Chemical) were added every other day at a concentration of 1 μmol/L for the time indicated. No ascorbic acid or β-glycerolphosphate was added into the culture. Cartilage nodules on day 20 were stained by 0.1% Alcian blue solution at 37°C for 12 hours. Areas of cartilage were measured by tracing under the microscope using Osteometrics (OsteoMetrics, Inc., Decatur, GA). The experiments were repeated three times and representative results are presented. Bone marrow cells were isolated from 2- to 3-month-old COX-2+/− mice. Femora and tibiae were removed and bone marrow cells were flushed from the marrow cavity. About 5 × 106 bone marrow cells per 10 cm2 were plated on 60-mm culture dishes cultured in α-Minimum Essential Medium containing 15% fetal bovine serum for 5 days, followed by low serum (1% fetal bovine serum) treatment for 24 hours. PGE2 or an agonist of EP1, EP2, or EP4 at 1 μmol/L was added to the culture and proteins were harvested at the indicated time points for Western blot analyses. Three-day-old neonatal wild-type mice were sacrificed by euthanasia. The anterior rib cage and sternum were harvested, and then digested with a 3 mg/ml solution of collagenase D (Roche Applied Science, Basel, Switzerland) for 90 minutes at 37°C. The soft tissue debris was thoroughly removed. The remaining sterna and costosternal junctions were further digested in fresh collagenase D solution in Petri dishes for 5 hours. At the end of the digestion, the cells were resuspended in DMEM with 10% fetal bovine serum. PGE2 (Cayman Chemicals) or EP agonists were added at a concentration of 1 μmol/L for the indicated times following serum starvation of the culture for 24 hours. Fracture calluses derived from wild-type mice were harvested and cultured in serum-free medium overnight. Supernatant was collected and loaded into zymogram gel containing 1 mg/ml gelatin. After electrophoresis, the gel was soaked in 0.25% Triton X-100 for 1 hour and washed in collagenase buffer for 18 hours followed by Coomassie blue staining and de-staining. Culture media bone marrow stromal cells were also collected and used for zymography to determine MMP-9 activity as described.36Leber TM Balkwill FR Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels.Anal Biochem. 1997; 249: 24-28Crossref PubMed Scopus (243) Google Scholar Cells were lysed in Golden lysis buffer supplemented with protease inhibitor (Roche Applied Science). The protein extracts (10 μg) were separated using NuPAGE BisTris gels (Invitrogen). Gels were transferred to a polyvinylidene difluor
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