Daichi Morikawa

Cytoplasmic superoxide causes bone fragility owing to low‐turnover osteoporosis and impaired collagen cross‐linking

The aging process correlates with the accumulation of cellular and tissue damage caused by oxidative stress. Although previous studies have suggested that oxidative stress plays a pathologic role in the development of bone fragility, little direct evidence has been found. In order to investigate the pathologic significance of oxidative stress in bones, we analyzed the bone tissue of mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn‐SOD, encoded by the Sod1 gene; Sod1−/−). In this study, we showed for the first time that in vivo cytoplasmic superoxide caused a distinct weakness in bone stiffness and decreased BMD, aging‐like changes in collagen cross‐linking, and transcriptional alterations in the genes associated with osteogenesis. We also showed that the surface areas of osteoblasts and osteoclasts were decreased significantly in the lumbar vertebrae of Sod1−/− mice, indicating the occurrence of low‐turnover osteopenia. In vitro experiments demonstrated that intracellular oxidative stress induced cell death and reduced the proliferation in primary osteoblasts but not in osteoclasts, indicating that impaired osteoblast viability caused the decrease in osteoblast number and suppressed RANKL/M‐CSF osteoclastogenic signaling in bone. Furthermore, treatment with an antioxidant, vitamin C, effectively improved bone fragility and osteoblastic survival. These results imply that intracellular redox imbalance caused by SOD1 deficiency plays a pivotal role in the development and progression of bone fragility both in vivo and in vitro. We herein present a valuable model for investigating the effects of oxidative stress on bone fragility in order to develop suitable therapeutic interventions.

Oxidative stress in skeletal muscle causes severe disturbance of exercise activity without muscle atrophy.

The increase in reactive oxygen species (ROS) levels that occurs during intense exercise has been proposed to be one of the major causes of muscle fatigue. In addition, the accumulation of cellular damage due to ROS is widely regarded to be one of the factors triggering age-related pathological conditions in skeletal muscle. To investigate the pathological significance of oxidative stress in skeletal muscle, we generated skeletal muscle-specific manganese superoxide dismutase-deficient (muscle-Sod2(-/-)) mice. The mutant mice showed severe disturbances in exercise activity, but no atrophic changes in their skeletal muscles. In histological and histochemical analyses, the mutant mice showed centralized nuclei in their muscle fibers and selective loss of enzymatic activity in mitochondrial respiratory chain complexes. In addition, the mutant mice displayed increased oxidative damage and reduced ATP content in their muscle tissue. Furthermore, a single administration of the antioxidant EUK-8 significantly improved exercise activity and increased the cellular ATP level in skeletal muscle. These results imply that the superoxide anions generated in mitochondria play a pivotal role in the progression of exercise intolerance.

Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration

Mechanical stress and aging are major risk factors of cartilage degeneration. Human studies have previously reported that oxidative damage increased, while SOD2 protein was reciprocally downregulated in osteoarthritic degenerated cartilage. However, it remains unclear whether mitochondrial superoxide imbalance in chondrocytes causes cartilage degeneration. We herein demonstrate that mechanical loading promoted mitochondrial superoxide generation and selective Sod2 downregulation in chondrocytes in vivo and that mitochondrial superoxide inducer also downregulated Sod2 expression in chondrocytes in vitro. A genetically manipulated model revealed that Sod2 deficiency in chondrocytes also resulted in mitochondrial superoxide overproduction and dysfunction, thus leading to cartilage degeneration. Intra-articular injection of a permeable antioxidant effectively suppressed the mechanical loading-induced mitochondrial superoxide generation and cartilage degeneration in mice. Our findings demonstrate that mitochondrial superoxide plays a pivotal role in the development and progression of osteoarthritis, and the mitochondrial superoxide balance may therefore be a promising target for the treatment of cartilage degeneration.

Cytoplasmic reactive oxygen species and SOD1 regulate bone mass during mechanical unloading.

Oxidative stress contributes to the pathogenesis of age‐related diseases as well as bone fragility. Our previous study demonstrated that copper/zinc superoxide dismutase (Sod1)‐deficient mice exhibit the induction of intracellular reactive oxygen species (ROS) and bone fragility resulting from low‐turnover bone loss and impaired collagen cross‐linking (Nojiri et al. J Bone Miner Res. 2011;26:2682–94). Mechanical stress also plays an important role in the maintenance of homeostasis in bone tissue. However, the molecular links between oxidative and mechanical stresses in bone tissue have not been fully elucidated. We herein report that mechanical unloading significantly increased intracellular ROS production and the specific upregulation of Sod1 in bone tissue in a tail‐suspension experiment. We also reveal that Sod1 loss exacerbated bone loss via reduced osteoblastic abilities during mechanical unloading. Interestingly, we found that the administration of an antioxidant, vitamin C, significantly attenuated bone loss during unloading. These results indicate that mechanical unloading, in part, regulates bone mass via intracellular ROS generation and the Sod1 expression, suggesting that activating Sod1 may be a preventive strategy for ameliorating mechanical unloading–induced bone loss.

Mitochondrial superoxide in osteocytes perturbs canalicular networks in the setting of age-related osteoporosis

Osteocytes are major bone cells that play a crucial role in maintaining the quality of and healing damage to bone tissue. The number of living osteocytes and canalicular networks declines in an age-dependent manner. However, the pathological effects of mitochondrial redox imbalances on osteocytes and bone metabolism have not been fully elucidated. We generated mice lacking mitochondrial superoxide dismutase 2 (Sod2) in osteocytes. Like an aged bone, Sod2 depletion in the osteocytes positively enhanced the production of cellular superoxide in vivo. A bone morphological analysis demonstrated that the Sod2-deficient femurs showed remarkable bone loss in an age-dependent manner. Interestingly, Sod2 loss induced markedly disorganized osteocytic canalicular networks and decreased the number of live osteocytes. Furthermore, Sod2 deficiency significantly suppressed bone formation and increased bone resorption concomitant with the upregulation of sclerostin and receptor activator of NF-κB ligand (RANKL). In vitro experiments also revealed that treatment with paraquat, a superoxide inducer in mitochondria, promoted the RANKL expression via, in part, ERK phosphorylation. These findings demonstrate that the mitochondrial superoxide induced in osteocytes by Sod2 ablation causes age-related bone loss due to the impairment of canalicular networks and bone metabolism via the deregulation of the sclerostin and RANKL expression.

Contribution of oxidative stress to the degeneration of rotator cuff entheses.

BACKGROUND Rotator cuff degeneration is one of the multiple factors that lead to rotator cuff tears; however, the precise mechanism of such degeneration still remains unclear. In this study, we investigated the supraspinatus tendon enthesis to clarify the link between rotator cuff degeneration and oxidative stress in antioxidant enzyme superoxide dismutase 1 (Sod1)-deficient mice (Sod1(-/-)). METHODS The supraspinatus tendon and humeral head were isolated and fixed to prepare histologic sections from wild-type and Sod1(-/-) male mice at 20 weeks of age. Hematoxylin-eosin staining was performed to assess the histomorphologic structure. To investigate the collagen fibers, we examined spatially aligned collagen fibers using a polarizing microscope and assessed the amount of collagen using immunohistochemical staining. To analyze the tissue elasticity, we measured the tissue acoustic properties using scanning acoustic microscopy. RESULTS The Sod1(-/-) mice showed histologic changes, such as a misaligned 4-layered structure and fragmented tidemark, in the enthesis. Sod1 loss also decreased the amount of brightly diffracted light and type I collagen, indicating collagen downregulation. The scanning acoustic microscopy analysis showed that the speed and attenuation of sound were increased in the nonmineralized fibrocartilage of the Sod1(-/-) mice, suggesting decreased mechanical properties in the supraspinatus enthesis. CONCLUSION Sod1 deficiency-induced degeneration is associated with impaired elasticity in the supraspinatus tendon enthesis, recapitulating human rotator cuff degeneration. These results suggest that intracellular oxidative stress contributes to the degeneration of rotator cuff entheses.

Protective Effects of Vitamin C on Age-Related Bone and Skin Phenotypes Caused by Intracellular Reactive Oxygen Species

Abstract Oxidative stress induced by reactive oxygen species promotes oxidative damage in cells and tissues, resulting in age-related diseases. To resist oxidative stress, cells possess multiple antioxidative systems. An antioxidant enzyme, superoxide dismutase (SOD), is thought to play a central role in these antioxidative systems due to its ability to catalyze cellular superoxide to oxygen and hydrogen peroxide. We have previously reported that Sod1-deficient mice exhibit various age-related diseases such as low-turnover osteopenia and skin thinning associated with collagen malfunction, indicating that intracellular oxidative stress causes collagen downregulation leading to bone and skin atrophy in mice. We found that vitamin C (VC) treatment morphologically reversed the bone and skin phenotypes in the Sod1-/- mice by reducing intracellular oxidative stress. Furthermore, oxidative stress is also correlated with postmenopausal osteoporosis and mechanical unloading-induced bone loss. In this study, VC treatment improved pathologic phenotypes in experimental bone loss models. These results demonstrate that VC is a beneficial antioxidant for improving age-related bone and skin diseases caused by intracellular oxidative stress.

Antioxidant treatment with vitamin C attenuated rotator cuff degeneration caused by oxidative stress in Sod1-deficient mice

Background Rotator cuff degeneration is 1 of several factors that lead to rotator cuff tears; however, the mechanism of this degeneration remains unclear. We previously reported that deficiency of an antioxidant enzyme, superoxide dismutase 1 (Sod1), in mice induced degeneration in supraspinatus tendon entheses, a model that replicates human rotator cuff degeneration. In this study, we analyzed possible effects of vitamin C (VC), a major antioxidant, on the degenerative changes of supraspinatus entheses in Sod1−/− mice. Methods We administered VC or vehicle, distilled water, for 8 weeks to Sod1−/− and wild-type male mice beginning at 12 weeks of age (n = 5-8 per group). When mice were 20 weeks of age, we sectioned rotator cuff tissue samples and performed hematoxylin-eosin and toluidine blue staining for quantitative histologic evaluation. Results VC administration, compared with vehicle administration, attenuated the histologic changes, including a misaligned 4-layered structure, fragmented tidemark, and toluidine blue staining, in the supraspinatus entheses of Sod1−/− mice. In the quantitative histologic evaluation, all parameters were significantly decreased in Sod1−/− mice compared with wild-type mice, except for the number of nonchondrocytes. Conclusion We demonstrated that an antioxidant treatment, VC administration, attenuated the rotator cuff degeneration, similar to that observed in humans, that is caused by oxidative stress in Sod1−/− mice. VC effects included improvements in quantitative histologic parameters and other histologic changes. These results suggest that VC treatment can prevent oxidative stress–induced degeneration of the rotator cuff.