Cartilage tissues regulate systemic aging via ectonucleotide pyrophosphatase/phosphodiesterase 1 in mice.

Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.

Amyloid deposits derived from transthyretin in the ligamentum flavum as related to lumbar spinal canal stenosis.

Amyloidosis is a protein conformational disorder with the distinctive feature of extracellular accumulation of amyloid fibrils that come from different proteins. In the ligamentum flavum of the lumbar spine, amyloid deposits were frequently found in elderly patients with lumbar spinal canal stenosis and were at least partially formed by wild-type transthyretin. However, how amyloid deposits in the ligamentum flavum affect lumbar spinal canal stenosis has remained unclear. In this study, we analyzed clinical, pathologic, and radiologic findings of patients with lumbar spinal canal stenosis who had amyloid deposits in the ligamentum flavum. We studied 95 ligamentum flavum specimens obtained from 56 patients with lumbar spinal canal stenosis and 21 ligamentum flavum specimens obtained from 19 patients with lumbar disk herniation. We evaluated histopathologic findings and clinicoradiologic manifestations, such as thickness of the ligamentum flavum and lumbar spinal segmental instability. We found that all 95 ligamentum flavum specimens resected from patients with lumbar spinal canal stenosis had amyloid deposits, which we classified into two types, transthyretin-positive and transthyretin-negative, and that transthyretin amyloid formation in the ligamentum flavum of patients with lumbar spinal canal stenosis was an age-associated phenomenon. The amount of amyloid in the ligamentum flavum was related to clinical manifestations of lumbar spinal canal stenosis, such as thickness of the ligamentum flavum and lumbar spinal segmental instability, in the patients with lumbar spinal canal stenosis with transthyretin-positive amyloid deposits. To our knowledge, this report is the first to show clinicopathologic correlations in transthyretin amyloid deposits of the ligamentum flavum. In conclusion, transthyretin amyloid deposits in the ligamentum flavum may be related to the pathogenesis of lumbar spinal canal stenosis in elderly patients.

Stromal expression of neutrophil gelatinase-associated lipocalin correlates with poor differentiation and adverse prognosis in oral squamous cell carcinoma.

AIMS: Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily. Although its overexpression in various cancers has been reported, little is known about its expression and clinical significance in oral squamous cell carcinoma (OSCC). This study aimed to elucidate the clinical significance of NGAL in OSCC. METHODS AND RESULTS: We investigated NGAL expression immunohistochemically in tumour cells and stromal cells in 96 OSCC tissues. NGAL expression in tumour cells correlated significantly with histological tumour cell differentiation, as shown by its specific distribution in the horn pearl-forming keratinized tumour cells, but not with other major clinicopathological parameters. We found NGAL(+) cells in the stroma that were predominantly myeloperoxidase-positive neutrophils. The number of such NGAL-expressing stromal cells was associated significantly with poor differentiation and reduced overall survival in OSCC. The prognostic value of stromal NGAL expression was significant in a univariate analysis, while only a trend was found in multivariate analyses. CONCLUSIONS: This study is the first to show the clinical significance of stromal NGAL expression, which may be an indicator of poor prognosis and more aggressive histological grade in OSCC. Our data suggest that NGAL expression in tumour cells and expression in stroma are associated in different ways with OSCC differentiation.

The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice.

Osteosarcoma is rare but is the most common bone tumor. Diagnostic tools such as magnetic resonance imaging development of chemotherapeutic agents have increased the survival rate in osteosarcoma patients, although 5-year survival has plateaued at 70%. Thus, development of new treatment approaches is needed. Here, we report that IL-17, a proinflammatory cytokine, increases osteosarcoma mortality in a mouse model with AX osteosarcoma cells. AX cell transplantation into wild-type mice resulted in 100% mortality due to ectopic ossification and multi-organ metastasis. However, AX cell transplantation into IL-17-deficient mice significantly prolonged survival relative to controls. CD4-positive cells adjacent to osteosarcoma cells express IL-17, while osteosarcoma cells express the IL-17 receptor IL-17RA. Although AX cells can undergo osteoblast differentiation, as can patient osteosarcoma cells, IL-17 significantly inhibited that differentiation, indicating that IL-17 maintains AX cells in the undifferentiated state seen in malignant tumors. By contrast, IL-17RA-deficient mice transplanted with AX cells showed survival comparable to wild-type mice transplanted with AX cells. Biopsy specimens collected from osteosarcoma patients showed higher expression of IL-17RA compared to IL-17. These findings suggest that IL-17 is essential to maintain osteosarcoma cells in an undifferentiated state and could be a therapeutic target for suppressing tumorigenesis.

Remnant tissue enhances early postoperative biomechanical strength and infiltration of Scleraxis-positive cells within the grafted tendon in a rat anterior cruciate ligament reconstruction model.

When ruptured, ligaments and tendons have limited self-repair capacity and rarely heal spontaneously. In the knee, the Anterior Cruciate Ligament (ACL) often ruptures during sports activities, causing functional impairment and requiring surgery using tendon grafts. Patients with insufficient time to recover before resuming sports risk re-injury. To develop more effective treatment, it is necessary to define mechanisms underlying ligament repair. For this, animal models can be useful, but mice are too small to create an ACL reconstruction model. Thus, we developed a transgenic rat model using control elements of Scleraxis (Scx), a transcription factor essential for ligament and tendon development, to drive GFP expression in order to localize Scx-expressing cells. As anticipated, Tg rats exhibited Scx-GFP in ACL during developmental but not adult stages. Interestingly, when we transplanted the flexor digitorum longus (FDP) tendon derived from adult Scx-GFP+ rats into WT adults, Scx-GFP was not expressed in transplanted tendons. However, tendons transplanted from adult WT rats into Scx-GFP rats showed upregulated Scx expression in tendon, suggesting that Scx-GFP+ cells are mobilized from tissues outside the tendon. Importantly, at 4 weeks post-surgery, Scx-GFP-expressing cells were more frequent within the grafted tendon when an ACL remnant was preserved (P group) relative to when it was not (R group) (P vs R groups (both n = 5), p<0.05), and by 6 weeks, biomechanical strength of the transplanted tendon was significantly increased if the remnant was preserved (P vsR groups (both n = 14), p<0.05). Scx-GFP+ cells increased in remnant tissue after surgery, suggesting remnant tissue is a source of Scx+ cells in grafted tendons. We conclude that the novel Scx-GFP Tg rat is useful to monitor emergence of Scx-positive cells, which likely contribute to increased graft strength after ACL reconstruction.

A machine learning-based scoring system and ten factors associated with hip fracture occurrence in the elderly.

Hip fractures are fragility fractures frequently seen in persons over 80-years-old. Although various factors, including decreased bone mineral density and a history of falls, are reported as hip fracture risks, few large-scale studies have confirmed their relevance to individuals older than 80, and tools to assess contributions of various risks to fracture development and the degree of risk are lacking. We recruited 1395 fresh hip fracture patients and 1075 controls without hip fractures and comprehensively evaluated various reported risk factors and their association with hip fracture development. We initially constructed a predictive model using Extreme Gradient Boosting (XGBoost), a machine learning algorithm, incorporating all 40 variables and evaluated the model's performance using the area under the receiver operating characteristic curve (AUC), yielding a value of 0.87. We also employed SHapley Additive exPlanation (SHAP) values to evaluate each feature importance and ranked the top 20. We then used a stepwise selection method to determine key factors sequentially until the AUC reached a plateau nearly equal to that of all variables and identified the top 10 sufficient to evaluate hip fracture risk. For each, we determined the cutoff value for hip fracture occurrence and calculated scores of each variable based on the respective feature importance. Individual scores were: serum 25(OH)D levels (<10 ng/ml, score 7), femoral neck T-score (<-3, score 5), Barthel index score (<100, score 3), maximal handgrip strength (<18 kg, score 3), GLFS-25 score (≥24, score 2), number of falls in previous 12 months (≥3, score 2), serum IGF-1 levels (<50 ng/ml, score 2), cups of tea/day (≥5, score -2), use of anti-osteoporosis drugs (yes, score -2), and BMI (<18.5 kg/m2, score 1). Using these scores, we performed receiver operating characteristic (ROC) analysis and the resultant optimal cutoff value was 7, with a specificity of 0.78, sensitivity of 0.75, and AUC of 0.85. These ten factors and the scoring system may represent tools useful to predict hip fracture.

Transthyretin amyloid deposition in ligamentum flavum (LF) is significantly correlated with LF and epidural fat hypertrophy in patients with lumbar spinal stenosis.

Lumbar spinal stenosis (LSS) is a degenerative disease characterized by intermittent claudication and numbness in the lower extremities. These symptoms are caused by the compression of nerve tissue in the lumbar spinal canal. Ligamentum flavum (LF) hypertrophy and spinal epidural lipomatosis in the spinal canal are known to contribute to stenosis of the spinal canal: however, detailed mechanisms underlying LSS are still not fully understood. Here, we show that surgically harvested LFs from LSS patients exhibited significantly increased thickness when transthyretin (TTR), the protein responsible for amyloidosis, was deposited in LFs, compared to those without TTR deposition. Multiple regression analysis, which considered age and BMI, revealed a significant association between LF hypertrophy and TTR deposition in LFs. Moreover, TTR deposition in LF was also significantly correlated with epidural fat (EF) thickness based on multiple regression analyses. Mesenchymal cell differentiation into adipocytes was significantly stimulated by TTR in vitro. These results suggest that TTR deposition in LFs is significantly associated with increased LF hypertrophy and EF thickness, and that TTR promotes adipogenesis of mesenchymal cells. Therapeutic agents to prevent TTR deposition in tissues are currently available or under development, and targeting TTR could be a potential therapeutic approach to inhibit LSS development and progression.

Interleukin-6 signalling regulates vascular endothelial growth factor-C synthesis and lymphangiogenesis in human oral squamous cell carcinoma.

Lymph node metastasis is associated with resistance to conventional therapy and poor survival of patients with oral squamous cell carcinoma (OSCC). Although lymphangiogenesis is well known to be associated with the occurrence of lymph node metastasis in various cancers, the precise mechanisms of lymphangiogenesis in OSCC are largely unknown. IL-6, a potent pro-inflammatory cytokine, has been shown to play active roles in various cancers, including OSCC. This study aimed to investigate the involvement of IL-6 signalling in lymphatic metastasis and to evaluate the efficacy of tocilizumab, a humanized anti-human IL-6 receptor antibody, as an anti-lymphangiogenic agent for OSCC. This investigation confirmed that levels of expression of IL-6 protein and VEGF-C mRNA in OSCC tissues were significantly correlated with lymph node metastasis in patients with OSCC, as assessed by immunohistochemical analysis and real-time quantitative RT-PCR. In vitro studies showed that IL-6 regulated VEGF-C mRNA expression in a human OSCC cell line, SAS cells, through the phosphoinositide 3-kinase-Akt pathway. In addition, treatment with tocilizumab led to markedly reduced VEGF-C mRNA expression and OSCC-related lymphangiogenesis in SAS xenografts. Together, these data suggest that tocilizumab acted as expected: it inhibited lymph node metastasis in OSCC by reducing tumour lymphangiogenesis.

Early and Mid-Term Outcomes after Vascular Reconstruction for Patients with Lower-Extremity Soft-Tissue Malignant Tumors.

Objective: To evaluate limb-salvage surgery including vascular resection for lower-extremity soft-tissue sarcomas and carcinomas for adult patients. Materials and Methods: Eight consecutive patients (median age, 59 years) who underwent vascular replacement during surgery for malignant tumors in the lower limbs between November 2006 and March 2018 were evaluated. Patient data were retrospectively obtained in a computerized database. Arterial and venous reconstructions were performed for seven patients, with one additional patient receiving venous reconstruction only. Autologous-vein (n=6) and synthetic bypasses were used for arterial repairs, whereas only autologous veins were implanted for venous repairs. Results: Morbidity was 62.5%, and in-hospital mortality was 12.5%. At a median follow-up of 24 months, the primary patency rates of arterial and venous reconstructions were 85.7% and 62.5%, respectively. Limb salvage was achieved in all cases. Conclusion: Early and mid-term bypass patency rates, the high percentage of limb salvage, and the oncologic outcome underline the efficacy of en bloc resection of soft-tissue tumors involving major vessels of the lower limbs. The anticipated need for vascular resection and reconstruction should not be a contraindication to sarcoma and carcinoma resections. However, efforts to achieve better control over systemic spread are required for long-term survival.

Knee osteoarthritis associated with different kinds of amyloid deposits and the impact of aging on type of amyloid.

Amyloidosis is a protein conformational disorder in which amyloid fibrils accumulate in the extracellular space and induce organ dysfunction. Recently, two different amyloidogenic proteins, transthyretin (TTR) and apolipoprotein A-I (Apo A-I), were identified in amyloid deposits in knee joints in patients with knee osteoarthritis (OA). However, clinicopathological differences related to those two kinds of amyloid deposits in the knee joint remain to be clarified. Here, we investigated the clinicopathological features related to these knee amyloid deposits associated with knee OA and the biochemical characteristics of the amyloid deposits. We found that all of our patients with knee OA had amyloid deposits in the knee joints, especially in the meniscus, and those deposits were primarily derived from TTR and/or Apo A-I. Some patients with knee OA, however, had unclassified amyloid deposits. One of our interesting observations concerned the different effects of aging on each type of amyloid formed. The frequency of formation of ATTR deposits clearly increased with age, but that of AApo A-I deposits decreased. Furthermore, we found that ∼16% of patients with knee OA developed ATTR/AApo A-I double deposits in the meniscus. Amyloid deposition may therefore be a common histopathological feature associated with knee OA. Also, aging may induce ATTR formation in the knee joint in elderly patients with knee OA, whereas AApo A-I formation may be inversely correlated with age.

Downregulation of midkine induces cisplatin resistance in human oral squamous cell carcinoma.

The presence of drug-resistant cancer cells has been associated with poor clinical outcomes. Cisplatin is one of the most effective chemotherapeutic agents commonly used for several malignancies including oral squamous cell carcinoma (OSCC). Although cisplatin resistance is a major obstacle in cancer treatment, mechanisms by which it develops are not well understood. Midkine (MK), a heparin-binding growth factor, has various cancer-related functions. In this study, we investigated whether MK is involved in cisplatin resistance in OSCC. We demonstrated that the Sa-3R cell line, which is OSCC cisplatin-resistant, exhibited lower MK expression with slow growth compared with its parent, Sa-3 cells. In Sa-3 cells, downregulation of MK expression significantly reduced cisplatin sensitivity, cell growth, and the expression of cyclin D1 and cyclin E1. MK knockdown suppressed cellular cisplatin accumulation via induction of ATP-binding cassette efflux transporters. These data suggest that MK may play important roles in cisplatin resistance in OSCC by modulating both cell growth and intracellular cisplatin accumulation.

Therapeutic approaches targeting midkine suppress tumor growth and lung metastasis in osteosarcoma.

Midkine (MK) plays important roles in tumorigenesis, however, the biological function of MK and whether MK can be a therapeutic target in osteosarcoma are unclear. Here, we found that osteosarcoma tissues showed high MK expression. MK knockdown by small interfering RNA significantly induced apoptosis in osteosarcoma cells, whereas recombinant MK increased cell proliferation. Inhibition of MK signaling by anti-MK monoclonal antibody (anti-MK mAb) suppressed growth of osteosarcoma cells both in vitro and in vivo. Moreover, inhibition of MK function significantly suppressed lung metastasis in xenograft transplantation model. Targeting MK by anti-MK mAb may have value in the treatment of osteosarcoma.

Wild-type transthyretin-derived amyloidosis in various ligaments and tendons.

Transthyretin-derived amyloid deposition is commonly found in intercarpal ligaments of patients with senile systemic amyloidosis. However, the frequency of transthyretin-derived amyloid deposits in ligaments of other tissues remains to be elucidated. This study aimed to determine the frequency of amyloid deposition and the precursor proteins of amyloid found in orthopedic disorders. We studied 111 specimens from patients with carpal tunnel syndrome (flexor tenosynovium specimens), rotator cuff tears (rotator cuff tendon specimens), and lumbar canal stenosis (yellow ligament specimens). To identify amyloid precursor proteins, we used immunohistochemical staining with antibodies that react with transthyretin, immunoglobulin light chain, amyloid A protein, and ß(2)-microglobulin. By means of Congo red staining, we identified 47 (42.3%) amyloid-positive samples, 39 of which contained transthyretin-derived amyloid (18 flexor tenosynovium specimens, 5 rotator cuff tendon specimens, and 16 yellow ligament specimens). Genetic testing and/or clinical findings suggested that all patients with transthyretin amyloid deposits did not have familial amyloidotic polyneuropathy. The occurrence of amyloid deposition in those tissues depended on age. These results suggest that transthyretin-derived amyloid deposits may occur more frequently in various ligaments and tendons than originally expected. In the future, such amyloid deposits may aid determination of the pathogenesis of ligament and tendon disorders in older patients.

Spinal multifocal amyloidosis derived from wild-type transthyretin.

Abstract Spinal amyloidosis can occur as a part of systemic amyloidosis or as localized amyloidomas. However, the exact pathogenesis of the spinal amyloidosis remains to be fully understood. Transthyretin (TTR) is an amyloidogenic protein causing two kinds of amyloid diseases. One is senile systemic amyloidosis (SSA), which is caused by wild-type (WT) TTR and primarily affects cardiac functions. The other type is familial amyloidosis, which is mainly induced by mutated TTR. We report here the first case of multifocal spinal TTR amyloidosis derived from WT TTR with radiculomyelopathy and destructive spondylosis. The data and clinical manifestations suggest that the patient may develop SSA. Clinical manifestations of TTR-related amyloidosis may vary more than we previously thought. In spinal amyloidosis, WT TTR is one of the candidate precursor proteins for the disease.

Midkine expression in malignant salivary gland tumors and its role in tumor angiogenesis.

The aims of this study were to investigate midkine (MK) expression patterns in salivary gland tumors (SGTs) and to evaluate the correlation between MK expression and the degree of malignancy. We performed immunohistochemistry to examine MK expression in specimens of adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), and pleomorphic adenoma (PA). In addition, we performed immunohistochemistry for CD31 and measured microvessel density (MVD), which is an indicator of angiogenesis. Immunohistochemistry showed that MK protein expression was significantly higher in specimens of malignant SGTs (ACC [P<0.01] and MEC [P<0.001]) than in benign SGT (PA) samples. Furthermore, MVD values tended to be higher in cases that exhibited high expression of MK, which indicated a significant correlation between the degree of MK expression and MVD (P<0.001). These results suggest that MK may play important roles in malignant transformation and tumor angiogenesis in SGTs.

Midkine expression is correlated with an adverse prognosis and is down-regulated by p53 in oral squamous cell carcinoma.

Midkine (MK) expression has been documented to be inversely correlated with the prognosis of patients with various tumors, but the mechanism of this relationship has not been well characterized. Recent studies have also correlated p53 expression with prognosis of patients with oral squamous cell carcinoma (OSCC). We evaluated the relationship between MK expression and clinicopathological features of patients with OSCC to clarify the influence of p53 status on MK expression in OSCC cells. Our results showed that patients with MK over-expression in OSCC cells had a significantly lower 5-year survival rate compared with patients with low MK expression. Immunohistochemical analyses demonstrated overexpression of MK protein in OSCC samples with mutant p53. Cell culture experiments with human lingual squamous cell carcinoma revealed that the MK gene was regulated by the wild-type p53 gene. Thus, MK expression may affect prognosis via the p53 status and mutation of the p53 gene, and MK may be an attractive target for therapeutic intervention in patients with cancer cells with mutant p53.