Inhibition of glucose use improves structural recovery of injured Achilles tendon in mice.

Injured tendons do not regain their native structure except at fetal or very young ages. Healing tendons often show mucoid degeneration involving accumulation of sulfated glycosaminoglycans (GAGs), but its etiology and molecular base have not been studied substantially. We hypothesized that quality and quantity of gene expression involving the synthesis of proteoglycans having sulfated GAGs are altered in injured tendons and that a reduction in synthesis of sulfated GAGs improves structural and functional recovery of injured tendons. C57BL6/j mice were subjected to Achilles tendon tenotomy surgery. The injured tendons accumulated sulfate proteoglycans as early as 1-week postsurgery and continued so by 4-week postsurgery. Transcriptome analysis revealed upregulation of a wide range of proteoglycan genes that have sulfated GAGs in the injured tendons 1 and 3 weeks postsurgery. Genes critical for enzymatic reaction of initiation and elongation of chondroitin sulfate GAG chains were also upregulated. After the surgery, mice were treated with the 2-deoxy-d-glucose (2DG) that inhibits conversion of glucose to glucose-6-phosphate, an initial step of glucose metabolism as an energy source and precursors of monosaccharides of GAGs. The 2DG treatment reduced accumulation of sulfated proteoglycans, improved collagen fiber alignment, and reduced the cross-sectional area of the injured tendons. The modulus of the 2DG-treated groups was higher than that in the vehicle group, but not of statistical significance. Our findings suggest that mucoid degeneration in injured tendons may result from the upregulated expression of genes involved the synthesis of sulfate proteoglycans and can be inhibited by reduction of glucose utilization.

Control of glucose metabolism is important in tenogenic differentiation of progenitors derived from human injured tendons.

Glucose metabolism is altered in injured and healing tendons. However, the mechanism by which the glucose metabolism is involved in the pathogenesis of tendon healing process remains unclear. Injured tendons do not completely heal, and often induce fibrous scar and chondroid lesion. Because previous studies have shown that tendon progenitors play roles in tendon repair, we asked whether connective tissue progenitors appearing in injured tendons alter glucose metabolism during tendon healing process. We isolated connective tissue progenitors from the human injured tendons, obtained at the time of primary surgical repair of rupture or laceration. We first characterized the change in glucose metabolism by metabolomics analysis using [1,2-13C]-glucose using the cells isolated from the lacerated flexor tendon. The flux of glucose to the glycolysis pathway was increased in the connective tissue progenitors when they proceeded toward tenogenic and chondrogenic differentiation. The influx of glucose to the tricarboxylic acid (TCA) cycle and biosynthesis of amino acids from the intermediates of the TCA cycle were strongly stimulated toward chondrogenic differentiation. When we treated the cultures with 2-deoxy-D-glucose (2DG), an inhibitor of glycolysis, 2DG inhibited chondrogenesis as characterized by accumulation of mucopolysaccharides and expression of AGGRECAN. Interestingly, 2DG strongly stimulated expression of tenogenic transcription factor genes, SCLERAXIS and MOHAWK under both chondrogenic and tenogenic differentiation conditions. The findings suggest that control of glucose metabolism is beneficial for tenogenic differentiation of connective tissue progenitors.

The effect of cup medialization and lateralization on hip range of motion in total hip arthroplasty.

BACKGROUND: There is little description of the effect of cup position on the hip range of motion in total hip arthroplasty. The purpose is to evaluate the effect of cup medialization/lateralization with a compensatory increase/decrease in femoral offset on the hip range of motion, and whether the bone morphology of the anterior inferior iliac spine affects hip range of motion in total hip arthroplasty. METHODS: Using the CT data of 100 patients (male; 30, female; 70), 3D-dynamic motion analysis was performed in four scenarios with cup medialization/lateralization with the same/decreased global offset. We calculated the range of motion before component impingement and bony impingement in flexion, internal rotation and external rotation using the software. Furthermore, we measured bony morphological features of anterior inferior iliac spine, and we analyzed the correlations among them. FINDINGS: We found that the cup medialization with the same stem offset had negative effects on hip range of motion in flexion and internal rotation due to bony impingement, whereas cup medialization caused external rotation to significantly decrease with the same global offset. On the other hand, cup lateralization with the same global offset had negative effects on flexion and internal rotation, whereas external rotation increased. Furthermore, there were negative correlations among flexion and laterally large and steep anterior inferior iliac spine. INTERPRETATION: Our results demonstrated that the advantage of cup medialization can depend on the individual anatomy such as bony morphology of anterior inferior iliac spine in flexion.

Modulating Glucose Metabolism and Lactate Synthesis in Injured Mouse Tendons: Treatment With Dichloroacetate, a Lactate Synthesis Inhibitor, Improves Tendon Healing.

BACKGROUND: Tendon injuries are common problems among athletes. Complete recovery of the mechanical structure and function of ruptured tendons is challenging. It has been demonstrated that upregulation of glycolysis and lactate production occurs in wounds, inflammation sites, and cancerous tumors, and these metabolic changes also control growth and differentiation of stem and progenitor cells. Similar metabolic changes have been reported in human healing tendons. In addition, lactate production has increased in progenitors isolated from injured tendons after treatment with IL-1ß. It is thought that the metabolic changes play a role in tendon healing after injury. HYPOTHESIS: Glucose metabolism is altered during tendon injury and healing, and modulation of this altered metabolism improves tendon repair. STUDY DESIGN: Controlled laboratory study. METHODS: The authors used the tendon injury model involving a complete incision of the Achilles tendon in C57BL/6J female mice and studied alterations of glucose metabolism in injured tendons with [U-13C]glucose and metabolomics analysis 1 and 4 weeks after surgery. They also examined the effects of dichloroacetate (DCA; an indirect lactate synthesis inhibitor) treatment on the recovery of structure and mechanical properties of injured tendons 4 weeks after surgery in the same mouse model. RESULTS: Significant changes in glucose metabolism in tendons after injury surgery were detected. 13C enrichment of metabolites and intermediates, flux through glycolysis, and lactate synthesis, as well as tricarboxylic acid cycle activity, were acutely increased 1 week after injury. Increased glycolysis and lactate generation were also found 4 weeks after injury. DCA-treated injured tendons showed decreased cross-sectional area and higher values of modulus, maximum stress, and maximum force when compared with vehicle-treated injured tendons. Improved alignment of the collagen fibers was also observed in the DCA group. Furthermore, DCA treatment reduced mucoid accumulation and ectopic calcification in injured tendons. CONCLUSION: The findings indicate that injured tendons acutely increase glycolysis and lactate synthesis after injury and that the inhibition of lactate synthesis by DCA is beneficial for tendon healing. CLINICAL RELEVANCE: Changing metabolism in injured tendons may be a therapeutic target for tendon repair.

Anterior Inferior Iliac Spine Bone Morphology in Hip Dysplasia and Its Effect on Hip Range of Motion in Total Hip Arthroplasty.

BACKGOUND: Despite the fact that femoral impingement against the anterior inferior iliac spine (AIIS) is increasingly recognized, there is no description of morphologic features of the AIIS in hip dysplasia and their effect on hip range of motion (ROM) in total hip arthroplasty (THA). The purpose is to evaluate the bone morphology of the AIIS in hip dysplasia and whether its morphology affects hip ROM in THA. METHODS: Computed tomography-based simulation software was used to create 3-dimensional bone models and perform virtual simulations. Using the computed tomographic data of 85 patients (male: n = 25, female: n = 60, mean age: 60.9) with hip osteoarthritis due to dysplasia, we measured the straight, vertical, and horizontal distances between the anteroinferior edge of the AIIS and the center of rotation in sagittal and axial views. The anterior and lateral versions of the AIIS were also measured. We calculated the ROM of flexion (Flex), and internal rotation (Int-R) in THA in the software, and analyzed the correlations among them. RESULTS: The AIIS prominence is bigger and extends more anteriorly and laterally in males than in females. Furthermore, the taller the patient, the more the AIIS extends anteriorly and laterally. We found that Flex and Int-R decreased inversely proportional to the size and lateral version of the AIIS. CONCLUSION: Our results demonstrated that the AIIS bone morphology substantially affects the ROM of Flex and Int-R especially in patients with laterally large AIIS bony anatomy in THA. Furthermore, our result indicates that the morphologic features of AIIS in hip dysplasia may be different between males and females.

The influence of stem offset and neck shaft angles on the range of motion in total hip arthroplasty.

PURPOSE: To evaluate the influence of stem offset and neck shaft angles on the range of motion before component impingement (ROMCI) and bony impingement (ROMBI), and the types of impingement in total hip arthroplasty (THA). METHODS: Using the computed tomography data of 101 patients who underwent THA, three-dimensional dynamic motion analysis was performed using a modular implant (Kinectiv(®) stem) that enabled adjustment of offset and leg length independently. We defined offset as horizontal offset (HO) and leg length as vertical offset (VO), and measured the ROMCI and ROMBI in flexion (Flex), internal rotation (Int-R) and external rotation (Ext-R) with the configuration of each horizontal/vertical offset. RESULTS: We found that HO lengthening increased the ROMCI and ROMBI in Flex and Int-R by delaying bony impingement, although excessive lengthening had minimal effect. On the contrary, VO lengthening decreased the ROMCI and ROMBI in Flex and ROMCI in Int-R. As for Ext-R, VO lengthening had positive effects on the ROMCI and ROMBI, whereas lengthening of HO had negative effects on the ROMCI and ROMBI. CONCLUSIONS: We demonstrated that the appropriate long offset with a low shaft angle increased the ROM in Flex and Int-R, and a high neck shaft angle increased the ROM in Ext-R. We should use implants properly in accordance with the types of impingement for avoiding dislocations in THA.

Low femoral antetorsion as a risk factor for bony impingement after bipolar hemiarthroplasty.

INTRODUCTION: Reports of dislocation after bipolar hemiarthroplasty (BHA) abound in literature, and several studies have mentioned the factors that are associated with an increased risk of dislocation. However, there is no report detailing the pattern of impingement in BHA and how femoral antetorsion can affect the range of motion (ROM) after BHA. PURPOSE: The purpose of this study was to evaluate the pattern of impingement in BHA and whether femoral antetorsion affects the ROM after BHA using three-dimensional (3D) dynamic motion analysis. METHODS: Using the computed tomography (CT) data of 60 patients (60 hips), including 31 men and 29 women who underwent BHA for the treatment of idiopathic osteonecrosis (ION) of the femoral head, we calculated the antetorsion of the femoral neck, ROM of flexion (Flex), internal rotation (Int-R), and external rotation (Ext-R) using a CT-based 3D simulation software. We evaluated the pattern of impingement and the relationship between femoral antetorsion and ROM in BHA. As for the implant position in the 3D simulation software, the anteversion of the femoral implant was set to be the same as the natural antetorsion of the femoral neck and neck length was set to be the standard neck in all cases. RESULTS: This study revealed the mechanism of impingement in BHA: (1) bone to bone impingement and (2) implant to bone impingement. We found a significant decrease in the ROM of Flex and Int-R inversely proportional to the femoral antetorsion. In patients with lower femoral antetorsion, the ROM of Flex and Int-R decreased due to bony impingement (the anterior great trochanteric region of the femur impinges on the anteroinferior edge of the anteroinferior iliac spine). Whereas, high anteversion of the femoral implant may decrease the ROM of Ext-R; however, our results also showed that even the lowest ROM of Ext-R with 10° hip extension was over 40°. CONCLUSIONS: We demonstrated that lower femoral antetorsion substantially affects the ROM of Flex and Int-R due to bony impingement. For these patients, there should be consideration given to retaining femoral "anterior offset" in BHA.

Low femoral antetorsion and total hip arthroplasty: a risk factor.

PURPOSE: The purpose of this study was to evaluate whether femoral antetorsion affects the range of motion (ROM) following total hip arthroplasty (THA) using 3D dynamic analysis. METHODS: Using 3D computed tomography (CT) data of 71 patients (71 hips) who underwent THA, we calculated antetorsion of the femoral neck, flexion range of motion (Flex ROM), internal rotation (Int-R) and external rotation (Ext-R). Evaluation of the relationship between antetorsion, ROM and the impingement site was performed. As for implant position, anteversion of the femoral implant was set to be the same as natural antetorsion of the femoral neck, and the acetabular component was set 45° of total anteversion in all cases. RESULTS: We found a significant decrease in Flex ROM and Int-R inversely proportional to femoral antetorsion. In patients with lower antetorsion, Flex ROM and Int-R decreased due to bony impingement (the anterior great trochanteric region of the femur impinges on the anteroinferior edge of the anteroinferior iliac spine). However, in Ext-R, there was no relationship between ROM and femoral antetorsion. CONCLUSIONS: We demonstrated that lower femoral antetorsion substantially affects Flex ROM and Int-R due to bony impingement. For these patients, consideration must be given to retaining femoral anterior offset in THA.

Transcriptional regulation of VEGFA by the endoplasmic reticulum stress transducer OASIS in ARPE-19 cells.

BACKGROUND: Vascular endothelial growth factor-A (VEGFA) is the main mediator of angiogenesis. Angiogenesis plays important roles not only in many physiological processes, but also in the pathophysiology of many diseases. VEGFA is one of the therapeutic targets of treatment for ocular diseases with neovascularization. Therefore, elucidation of the regulatory mechanisms for VEGFA expression is important for the development of pharmaceutical drugs. Recent studies have demonstrated that the unfolded protein response is involved in the transcriptional regulation of VEGFA. However, the precise regulation of VEGFA in the human retina is not fully understood. PRINCIPAL FINDINGS: When human retinal pigment epithelial cells, ARPE-19, were exposed to endoplasmic reticulum stressors, VEGFA mRNA was significantly upregulated. The unfolded protein response-related transcription factors XBP1, ATF4, ATF6, and OASIS were expressed in ARPE-19 cells. To determine which transcription factors preferentially contribute to the induction of VEGFA expression after endoplasmic reticulum stress, we carried out reporter assays using an approximately 6-kbp 5'-upstream region of the human VEGFA gene. Among these transcription factors, OASIS acted most effectively on the VEGFA promoter in ARPE-19 cells. Based on data obtained for certain deleted and mutated reporter constructs, we determined that OASIS promoted VEGFA expression by acting on a cyclic AMP-responsive element-like site located at around -500 bp relative to the VEGFA transcription start site. Furthermore, we confirmed that OASIS directly bound to the promoter region containing this site by chromatin immunoprecipitation assays. CONCLUSIONS AND SIGNIFICANCE: We have demonstrated a novel regulatory mechanism for VEGFA transcription by OASIS in human retinal pigment epithelial cells. Chemical compounds that regulate the binding of OASIS to the promoter region of the VEGFA gene may have potential as therapeutic agents for ocular diseases with neovascularization.

The endoplasmic reticulum stress transducer BBF2H7 suppresses apoptosis by activating the ATF5-MCL1 pathway in growth plate cartilage.

BBF2H7 (box B-binding factor 2 human homolog on chromosome 7) is a basic leucine zipper transmembrane transcription factor that belongs to the cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) family. This novel endoplasmic reticulum (ER) stress transducer is localized in the ER and is cleaved in its transmembrane region in response to ER stress. BBF2H7 has been shown to be expressed in proliferating chondrocytes in cartilage during the development of long bones. The target of BBF2H7 is Sec23a, one of the coat protein complex II components. Bbf2h7-deficient (Bbf2h7(-/-)) mice exhibit severe chondrodysplasia, with expansion of the rough ER in proliferating chondrocytes caused by impaired secretion of extracellular matrix (ECM) proteins. We observed a decrease in the number of proliferating chondrocytes in the cartilage of Bbf2h7(-/-) mice. TUNEL staining of the cartilage showed that apoptosis was promoted in Bbf2h7(-/-) chondrocytes. Atf5 (activating transcription factor 5), another member of the CREB/ATF family and an antiapoptotic factor, was also found to be a target of BBF2H7 in chondrocytes. ATF5 activated the transcription of Mcl1 (myeloid cell leukemia sequence 1), which belongs to the antiapoptotic B-cell leukemia/lymphoma 2 family, to suppress apoptosis. Finally, we found that the BBF2H7-ATF5-MCL1 pathway specifically suppressed ER stress-induced apoptosis in chondrocytes. Taken together, our findings indicate that BBF2H7 is activated in response to ER stress caused by synthesis of abundant ECM proteins and plays crucial roles as a bifunctional regulator to accelerate ECM protein secretion and suppress ER stress-induced apoptosis by activating the ATF5-MCL1 pathway during chondrogenesis.

Unfolded protein response, activated by OASIS family transcription factors, promotes astrocyte differentiation.

OASIS is a member of the CREB/ATF family of transcription factors and modulates cell- or tissue-specific unfolded protein response signalling. Here we show that this modulation has a critical role in the differentiation of neural precursor cells into astrocytes. Cerebral cortices of mice specifically deficient in OASIS (Oasis(-/-)) contain fewer astrocytes and more neural precursor cells than those of wild-type mice during embryonic development. Furthermore, astrocyte differentiation is delayed in primary cultured Oasis(-/-) neural precursor cells. The transcription factor Gcm1, which is necessary for astrocyte differentiation in Drosophila, is revealed to be a target of OASIS. Introduction of Gcm1 into Oasis(-/-) neural precursor cells improves the delayed differentiation of neural precursor cells into astrocytes by accelerating demethylation of the Gfap promoter. Gcm1 expression is temporally controlled by the unfolded protein response through interactions between OASIS family members during astrocyte differentiation. Taken together, our findings demonstrate a novel mechanism by which OASIS and its associated family members are modulated by the unfolded protein response to finely control astrocyte differentiation.

The endoplasmic reticulum stress transducer OASIS is involved in the terminal differentiation of goblet cells in the large intestine.

OASIS is a basic leucine zipper transmembrane transcription factor localized in the endoplasmic reticulum (ER) that is cleaved in its transmembrane region in response to ER stress. This novel ER stress transducer has been demonstrated to express in osteoblasts and astrocytes and promote terminal maturation of these cells. Additionally, OASIS is highly expressed in goblet cells of the large intestine. In this study, we investigated the roles of OASIS in goblet cell differentiation in the large intestine. To analyze the functions of OASIS in goblet cells, we examined morphological changes and the expression of goblet cell differentiation markers in the large intestine of Oasis(-/-) mice. By disrupting the Oasis gene, the number of goblet cells and production of mucus were decreased in the large intestine. Oasis(-/-) goblet cells showed abnormal morphology of mucous vesicles and rough ER. The expression levels of mature goblet cell markers were lower, and conversely those of early goblet cell markers were higher in Oasis(-/-) mice, indicating that differentiation from early to mature goblet cells is impaired in Oasis(-/-) mice. To determine the association of OASIS with other factors involved in goblet cell differentiation, in vitro experiments using a cell culture model were performed. We found that OASIS was activated in response to mild ER stress that is induced in differentiating goblet cells. Knockdown of the Oasis transcript perturbed goblet cell terminal differentiation. Together, our data indicate that OASIS plays crucial roles in promoting the differentiation of early goblet cells to mature goblet cells in the large intestine.