Dai Iwase

Nerve Growth Factor Regulation by TNF-α and IL-1β in Synovial Macrophages and Fibroblasts in Osteoarthritic Mice

To investigate the role of macrophages as a regulator and producer of nerve growth factor (NGF) in the synovial tissue (ST) of osteoarthritis (OA) joints, the gene expression profiles of several inflammatory cytokines in the ST, including synovial macrophages and fibroblasts, of OA mice (STR/Ort) were characterized. Specifically, real-time polymerase chain reaction analysis was used to evaluate the expression of tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1β, IL-6, and NGF in CD11b+ and CD11b– cells isolated from the ST of a murine OA model. The effects of TNF-α, IL-1β, and IL-6 on the expression of NGF in cultured synovial cells were also examined. The expression of TNF-α, IL-1β, IL-6, and NGF in the ST of STR/Ort was higher than that in C57/BL6J mice. Compared to the CD11b– cell fraction, higher expression levels of TNF-α, IL-1β, and IL-6 were detected in the CD11b+ cell fraction, whereas no differences in the expression of NGF were detected between the two cell fractions. Notably, TNF-α upregulated NGF expression in synovial fibroblasts and macrophages and IL-1β upregulated NGF expression in synovial fibroblasts. IL-1β and TNF-α may regulate NGF signaling in OA joints and be suitable therapeutic targets for treating OA pain.

Activation of calcitonin gene-related peptide signaling through the prostaglandin E2-EP1/EP2/EP4 receptor pathway in synovium of knee osteoarthritis patients

BackgroundCalcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood.MethodsSynovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3–4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1β, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist.ResultsCGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists.ConclusionsPGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

Athletic ability of school-age children after satisfactory treatment of congenital clubfoot.

Background: This is the first study to objectively assess the athletic ability of school-age congenital clubfoot patients. Methods: Forty-six feet of 30 patients (18 boys, 12 girls) were evaluated in this study. Nine patients were treated conservatively, 8 patients underwent percutaneous tenotomy of the Achilles tendon, and 13 patients were treated with extensive soft-tissue release. The mean age at the investigation was 9.2±1.9 years, and the mean follow-up period was 8.3±2.9 years. Athletic ability was evaluated by calculating Z-scores for the patients’ scores in 5 physical fitness tests routinely performed nationwide at elementary schools: 50-meter run; standing long jump; repetition side steps; 20-meter shuttle run; and sit-ups. The Z-scores were calculated based on data published as the nationwide standards. Results: Of the 148 scores recorded for the 5 tests for the 30 clubfoot patients, 143 scores (96.6%) were higher than the −2 SD value. The mean Z-scores were as follows: −0.32 for 50-meter run; −0.16 for standing long jump; −0.24 for 20-meter shuttle run; 0.22 for repetition side steps; and 0.06 for sit-ups. None of the events showed any significant differences among the three treatment groups. Conclusions: Congenital clubfoot with satisfactory treatment did not significantly impair the athletic performance. Level of Evidence: Prognostic level III.

Nerve growth factor regulation and production by macrophages in osteoarthritic synovium

Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)‐associated pain. Although recent studies suggest that tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF‐α and IL‐1β in freshly isolated CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL‐1β and TNF‐α on NGF mRNA expression in cultured CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF‐α and IL‐1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF‐α and IL‐1β mRNA levels in CD14‐positive cells from the SYN of OA patients was significantly higher than that in CD14‐negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14‐positive and ‐negative cells with IL‐1β and TNF‐α enhanced NGF mRNA and protein levels. Expression of NGF, IL‐1β and TNF‐α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL‐1β and TNF‐α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.

Increase and regulation of synovial calcitonin gene-related peptide expression in patients with painful knee osteoarthritis

Background Recent studies suggest that the vasodilatory neuropeptide calcitonin gene-related peptide (CGRP) is localized in the synovial tissue and may be involved in the pathology of hip and knee osteoarthritis (OA). However, the regulation and relationship between pain and CGRP expression levels in the synovial tissue of human OA patients are not fully understood. Methods Synovial tissues were harvested from 74 participants with radiographic knee OA (unilateral Kellgren/Lawrence grades 3–4) during total knee arthroplasty. CGRP-expressing cells in the resected tissue were identified by immunohistochemical analyses. To examine CGRP expression levels, CD14-positive (CD14+) (macrophage-rich cell fraction) and CD14-negative (CD14−; fibroblast-rich cell fraction) cells were isolated from the synovial tissue. To investigate the involvement of prostaglandin E2 (PGE2) in the regulation of CGRP expression, cultured CD14− and CD14+ cells were stimulated with PGE2. In addition, CGRP expression levels in the synovial tissue of OA patients with strong/severe (visual analog scale [VAS]≥6) and mild/moderate pain (VAS<6) were compared. Results CGRP-positive cells were detected in the intimal lining layer and comprised both CD14− and CD14+ cells. CGRP expression in non-cultured CD14− fractions was significantly higher than that in CD14+ fractions. The expression levels of CGRP were significantly increased in cultured CD14− cell fractions treated with exogenous PGE2, compared to untreated CD14− cell fractions. In contrast, treatment with PGE2 did not increase CGRP regardless of whether or not CD14+ cells expressed CGRP. Furthermore, CGRP expression in the VAS≥6 group was also significantly higher than that in the VAS<6 group. Conclusion These findings suggest that CGRP expression in the synovial fibroblasts is regulated by the COX-2/PGE2 pathway and that elevation of synovial CGRP levels may contribute to OA pain.

Expression of calcitonin gene-related peptide in the infrapatellar fat pad in knee osteoarthritis patients

BackgroundThe infrapatellar fat pad (IPFP) has been implicated as a possible source of osteoarthritis (OA) development and knee pain due to the production of inflammatory mediators and the existence of nerve fibers within this structure. Calcitonin gene-related peptide (CGRP) is a vasodilatory neuropeptide that is localized to joint tissues and has recently been implicated in the development of knee OA and OA pain. To date, however, the expression levels of CGRP in the IPFP of human knee OA patients have not been examined.MethodsIFFP and synovial (SYN) tissues were harvested from 100 individuals with radiographic knee OA (unilateral Kellgren/Lawrence [K/L] grades 2–4) during total knee arthroplasty and subjected to immunohistochemical analysis for CGRP localization. In addition, the messenger RNA (mRNA) expression levels of CGRP and cyclooxygenase-2 (COX-2) in the collected tissues were evaluated and compared using real-time PCR analysis of total RNA extracts. CGRP and COX-2 mRNA expression were also compared among individuals with K/L grades 2–4.ResultsCGRP-positive cells were detected in the capillaries within the IPFP and lining layer of SYN tissue. The expression levels of CGRP in the IPFP were positively correlated with COX-2 and were significantly higher than those in SYN tissue. CGRP expression in tissue from the KL4 group was twofold higher than that from the KL2 group.ConclusionsThe IPFP of knee OA patients produces relatively high levels of CGRP, which may be regulated by COX-2 at the transcriptional level. Further studies are needed to determine if CGRP levels are directly linked to OA pathology.

Down-regulation of microsomal prostaglandin E2 synthase-1 in the infrapatellar fat pad of osteoarthritis patients with hypercholesterolemia

BackgroundWhile epidemiological studies have reported a potential role for hypercholesterolemia (HCE) in osteoarthritis (OA), the association between HCE and OA has yet to be clarified. Adipose tissue is a primary locus for cholesterol metabolism and the presence of HCE reportedly causes adipose dysfunction. The knee joint contains adipose tissue in the form of the infrapatellar fat pad (IPFP), which has been shown to contribute to the pathophysiology of OA in the knee via the secretion of inflammatory mediators. However, the effect of HCE on the expression of inflammatory mediators in the IPFP has not been elucidated.MethodsIPFP and synovial tissues (ST) were extracted from 145 subjects with OA, diagnosed by radiography, during total knee arthroplasty. OA patients were divided into three groups according to their total cholesterol levels (Desirable, Borderline high and High) based on the National Cholesterol Education Program Adult Treatment Panel III (NCEPATP III). We examined the expression of cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES1), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 using real-time polymerase chain reaction and compared results among the Desirable, Borderline high and High groups.ResultsThe mRNA expression levels of TNF-α, IL-1β, and IL-6 in ST and the IPFP were not significantly different among the three groups. COX-2 mRNA expression in ST and IPFP was likewise not different among the three groups. While the mRNA expression level of mPGES1 in ST was also not significantly different, that of mPGES1 in the IPFP was significantly lower in the High group than in the Desirable and Borderline high groups.ConclusionmRNA levels of mPGES-1 are reduced in the IPFP of knee OA patients with HCE. Additional studies are need to clarify the effect of mPGES-1 down-regulation in OA pathology.

Regulation of calcitonin gene-related peptide expression through the COX-2/mPGES-1/PGE2 pathway in the infrapatellar fat pad in knee osteoarthritis

BackgroundThe infrapatellar fat pad (IFP) is implicated in knee osteoarthritis (KOA). Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide expressed in joint tissues and synovial tissues (ST), was recently found to be associated with KOA progression and pain. CGRP is expressed in the IFPs of human KOA patients; however, its regulation has not been elucidated.MethodsIFPs and STs were harvested from 138 KOA patients during total knee replacement (TKR) and analyzed for CGRP, cycloxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1) expression using real-time polymerase chain reaction (PCR). To investigate CGRP regulation by prostaglandin E2 (PGE2), adipocytes (Ad) and the stromal vascular fraction (SVF) were harvested from IFPs using collagenase. Synovial cells (SYC) were also harvested from ST and stimulated with vehicle (serum-free culture medium), PGE2, or CGRP.ResultsCGRP, COX-2, and mPGES-1 expression levels were significantly higher in IFPs than STs. PGE2 stimulation increased CGRP expression in Ad, the SVF, and SYC; however, CGRP expression was significantly higher in PGE2-stimulated SVF than PGE2-stimulated SYC. CGRP stimulation had no effect on COX-2 or mPGES-1 expression.ConclusionsCGRP expression in the IFP of KOA patients is regulated by the COX-2/mPGES-1/PGE2 pathway.

Lower limb ischemia due to popliteal artery compression by Baker cyst

A Baker cyst is the most common mass around the knee joint. It is mostly asymptomatic; however, it may cause knee pain or focal swelling because of compression of vein or nerve. Herein, we report a case of Baker cyst obstructing arterial flow and causing intermittent claudication. An attached polycystic mass was found posterior to the popliteal artery. Needle aspiration was ineffective, and the patient experienced recurrent lower leg pain. Surgical resection was performed, and the patient became symptom free. Baker cyst may cause lower limb ischemia through obstruction of arterial flow, requiring surgical intervention.