Manigrasso MB, O'Connor JPAccelerated fracture healing in mice lacking the 5-lipoxygenase gene. Acta Orthop 81:748-755

Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School and Graduate School of Biological Sciences, Newark, USA.
Acta Orthopaedica (Impact Factor: 2.77). 11/2010; 81(6):748-55. DOI: 10.3109/17453674.2010.533931
Source: PubMed


Cyclooxygenase-2 (COX-2) promotes inflammation by synthesizing pro-inflammatory prostaglandins from arachidonic acid. Inflammation is an early response to bone fracture, and ablation of COX-2 activity impairs fracture healing. Arachidonic acid is also converted into leukotrienes by 5-lipoxygenase (5-LO). We hypothesized that 5-LO is a negative regulator of fracture healing and that in the absence of COX-2, excess leukotrienes synthesized by 5-LO will impair fracture healing.
Fracture healing was assessed in mice with a targeted 5-LO mutation (5-LO(KO) mice) and control mice by radiographic and histological observations, and measured by histomorphometry and torsional mechanical testing. To assess effects on arachidonic acid metabolism, prostaglandin E2, F2α, and leukotriene B4 levels were measured in the fracture calluses of control, 5-LO(KO) COX-1(KO), and COX-2(KO) mice by enzyme linked immunoassays.
Femur fractures in 5-LO(KO) mice rapidly developed a cartilaginous callus that was replaced with bone to heal fractures faster than in control mice. Femurs from 5-LO(KO) mice had substantially better mechanical properties after 1 month of healing than did control mice. Callus leukotriene levels were 4-fold higher in mice homozygous for a targeted mutation in the COX-2 gene (COX-2(KO)), which indicated that arachidonic acid was shunted into the 5-LO pathway in the absence of COX-2.
These experiments show that 5-LO negatively regulates fracture healing and that shunting of arachidonic acid into the 5-LO pathway may account, at least in part, for the impaired fracture healing response observed in COX-2(KO) mice.

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Article: Manigrasso MB, O'Connor JPAccelerated fracture healing in mice lacking the 5-lipoxygenase gene. Acta Orthop 81:748-755

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    • "Genetic ablation or pharmacological inhibition of cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) significantly affects bone regeneration during fracture healing. Loss of COX-2 activity impairs fracture healing, while loss of 5-LO activity accelerates healing in rodent models [1]–[5]. COX-2 and 5-LO catalyze critical steps in the conversion of arachidonic acid into prostaglandins and leukotrienes, respectively [6], [7]. "
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    ABSTRACT: This study investigated the localization of critical enzymes involved in arachidonic acid metabolism during the initial and regenerative phases of mouse femur fracture healing. Previous studies found that loss of cyclooxygenase-2 activity impairs fracture healing while loss of 5-lipoxygenase activity accelerates healing. These diametric results show that arachidonic acid metabolism has an essential function during fracture healing. To better understand the function of arachidonic acid metabolism during fracture healing, expression of cyclooxygenase-1 (COX-1), cyclooxygenase -2 (COX-2), 5-lipoxygenase (5-LO), and leukotriene A4 hydrolase (LTA4H) was localized by immunohistochemistry in time-staged fracture callus specimens. All four enzymes were detected in leukocytes present in the bone marrow and attending inflammatory response that accompanied the fracture. In the tissues surrounding the fracture site, the proportion of leukocytes expressing COX-1, COX-2, or LTA4H decreased while those expressing 5-LO remained high at 4 and 7 days after fracture. This may indicate an inflammation resolution function for 5-LO during fracture healing. Only COX-1 was consistently detected in fracture callus osteoblasts during the later stages of healing (day 14 after fracture). In contrast, callus chondrocytes expressed all four enzymes, though 5-LO appeared to be preferentially expressed in newly differentiated chondrocytes. Most interestingly, osteoclasts consistently and strongly expressed COX-2. In addition to bone surfaces and the growth plate, COX-2 expressing osteoclasts were localized at the chondro-osseous junction of the fracture callus. These observations suggest that arachidonic acid mediated signaling from callus chondrocytes or from callus osteoclasts at the chondro-osseous junction regulate fracture healing.
    PLoS ONE 02/2014; 9(2):e88423. DOI:10.1371/journal.pone.0088423 · 3.23 Impact Factor
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    • "However, the role of LOX in healing is controversial. While healing is improved in a few experimental models after LOX inhibition, e.g. after bone fracture [20], we found impaired healing after MI. There are a few in vitro reports linking 5-LOX−/− to fibroblast function. "
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    ABSTRACT: Early healing after myocardial infarction (MI) is characterized by a strong inflammatory reaction. Most leukotrienes are pro-inflammatory and are therefore potential mediators of healing and remodeling after myocardial ischemia. The enzyme 5-lipoxygenase (5-LOX) has a key role in the transformation of arachidonic acid in leukotrienes. Thus, we tested the effect of 5-LOX on healing after MI. After chronic coronary artery ligation, early mortality was significantly increased in 5-LOX(-/-) when compared to matching wildtype (WT) mice due to left ventricular rupture. This effect could be reproduced in mice treated with the 5-LOX inhibitor Zileuton. A perfusion mismatch due to the vasoactive potential of leukotrienes is not responsible for left ventricular rupture since local blood flow assessed by magnetic resonance perfusion measurements was not different. However, after MI, there was an accentuation of the inflammatory reaction with an increase of pro-inflammatory macrophages. Yet, mortality was not changed in chimeric mice (WT vs. 5-LOX(-/-) bone marrow in 5-LOX(-/-) animals), indicating that an altered function of 5-LOX(-/-) inflammatory cells is not responsible for the phenotype. Collagen production and accumulation of fibroblasts were significantly reduced in 5-LOX(-/-) mice in vivo after MI. This might be due to an impaired migration of 5-LOX(-/-) fibroblasts, as shown in vitro to serum. In conclusion, a lack or inhibition of 5-LOX increases mortality after MI because of healing defects. This is not mediated by a change in local blood flow, but through an altered inflammation and/or fibroblast function.
    Archiv für Kreislaufforschung 07/2013; 108(4):367. DOI:10.1007/s00395-013-0367-8 · 5.41 Impact Factor
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    • "The accelerated fracture repair was associated with enhanced chondrogenesis, suggesting that inhibition of 5-LO accelerates endochondral bone formation. However, these previous studies also showed early increases in peripheral fracture site bone formation, which is initially mediated by direct bone formation from the periosteum and would be consistent with the current findings.1,2 "
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    ABSTRACT: Recent studies have shown that modulating inflammation-related lipid signalling after a bone fracture can accelerate healing in animal models. Specifically, decreasing 5-lipoxygenase (5-LO) activity during fracture healing increases cyclooxygenase-2 (COX-2) expression in the fracture callus, accelerates chondrogenesis and decreases healing time. In this study, we test the hypothesis that 5-LO inhibition will increase direct osteogenesis. Bilateral, unicortical femoral defects were used in rats to measure the effects of local 5-LO inhibition on direct osteogenesis. The defect sites were filled with a polycaprolactone (PCL) scaffold containing 5-LO inhibitor (A-79175) at three dose levels, scaffold with drug carrier, or scaffold only. Drug release was assessed in vitro. Osteogenesis was assessed by micro-CT and histology at two endpoints of ten and 30 days. Using micro-CT, we found that A-79175, a 5-LO inhibitor, increased bone formation in an apparent dose-related manner. These results indicate that 5-LO inhibition could be used therapeutically to enhance treatments that require the direct formation of bone.
    02/2013; 2(2):41-50. DOI:10.1302/2046-3758.22.2000066
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