Promethazine Downregulates Wnt/ß-Catenin Signaling and Increases the Biomechanical Forces of the Injured Achilles Tendon in the Early Stage of Healing.

BACKGROUND: Wnt/ß-catenin signaling suppresses the differentiation of cultured tenocytes, but its roles in tendon repair remain mostly elusive. No chemical compounds are currently available to treat tendon injury. HYPOTHESIS: We hypothesized that the inhibition of Wnt/ß-catenin signaling would accelerate tendon healing. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon-derived cells (TDCs) were isolated from rat Achilles tendons. The right Achilles tendon was injured via a dermal punch, while the left tendon was sham operated. A Wnt/ß-catenin inhibitor, IWR-1, and an antihistamine agent, promethazine (PH), were locally and intramuscularly injected, respectively, for 2 weeks after surgery. The healing tendons were histologically and biomechanically evaluated. RESULTS: The amount of ß-catenin protein was increased in the injured tendons from postoperative weeks 0.5 to 2. Inhibition of Wnt/ß-catenin signaling by IWR-1 in healing tendons improved the histological abnormalities and decreased ß-catenin, but it compromised the biomechanical properties. As we previously reported that antihistamine agents suppressed Wnt/ß-catenin signaling in human chondrosarcoma cells, we examined the effects of antihistamines on TDCs. We found that a first-generation antihistamine agent, PH, increased the expression of the tendon marker genes Mkx and Tnmd in TDCs. Intramuscular injection of PH did not improve histological abnormalities, but it decreased ß-catenin in healing tendons and increased the peak force and stiffness of the healing tendons on postoperative week 2. On postoperative week 8, however, the biomechanical properties of vehicle-treated tendons became similar to those of PH-treated tendons. CONCLUSION: IWR-1 and PH suppressed Wnt/ß-catenin signaling and improved the histological abnormalities of healing tendons. IWR-1, however, compromised the biomechanical properties of healing tendons, whereas PH improved them. CLINICAL RELEVANCE: PH is a candidate repositioned drug that potentially accelerates tendon repair.

Meclozine ameliorates skeletal muscle pathology and increases muscle forces in mdx mice.

We screened pre-approved drugs for the survival of the Hu5/KD3 human myogenic progenitors. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, promoted the proliferation and survival of Hu5/KD3 cells. Meclozine increased expression of MyoD, but reduced expression of myosin heavy chain and suppressed myotube formation. Withdrawal of meclozine, however, resumed the ability of Hu5/KD3 cells to differentiate into myotubes. We examined the effects of meclozine on mdx mouse carrying a nonsense mutation in the dystrophin gene and modeling for Duchenne muscular dystrophy. Intragastric administration of meclozine in mdx mouse increased the body weight, the muscle mass in the lower limbs, the cross-sectional area of the paravertebral muscle, and improved exercise performances. Previous reports show that inhibition of phosphorylation of ERK1/2 improves muscle functions in mouse models for Emery-Dreifuss muscular dystrophy and cancer cachexia, as well as in mdx mice. We and others previously showed that meclozine blocks the phosphorylation of ERK1/2 in cultured cells. We currently showed that meclozine decreased phosphorylation of ERK1/2 in muscles in mdx mice but not in wild-type mice. This was likely to be one of the underlying mechanisms of the effects of meclozine on mdx mice.

Outcomes of pediatric anterior cruciate ligament reconstructions in patients with and without bucket handle meniscus repairs.

The purpose of this study was to report the outcomes of concomitant bucket handle meniscal tear (BHMT) repair and anterior cruciate ligament (ACL) reconstruction and to compare the outcomes with those after isolated ACL reconstruction in patients aged ≤16 years. Patients in our database from 2013 to 2017 were retrospectively analyzed. Patients were assigned to one of two treatment groups based on the presence of BHMTs: no meniscal tear group (group A) and BHMT group (group B). All BHMTs were repaired using the combined inside-out with all-inside technique. This study included 64 knees divided into two groups: 47 knees in group A and 17 knees in group B. There was a significant difference in the interval between ACL injury and surgery between groups A and B (69 vs. 150 days, respectively; P < 0.001). Mean postoperative International Knee Documentation Committee and Lysholm scores in group A were slightly, although significantly, improved compared to those in group B (96.5 vs. 92.6, respectively; P < 0.05, and 98 vs. 95, respectively; P < 0.05). There were no significant differences in postoperative anteroposterior laxity and graft failure rate between the groups. In group B, four patients (23.5%) required surgery for incomplete meniscal healing. Postoperative International Knee Documentation Committee and Lysholm scores of patients with BHMTs were significantly lower than those of patients without any meniscal tear, although with significant improvement in the amount of instability. Level of evidence was Level III.

Biceps tenotomy versus soft-tissue tenodesis in females aged 60 years and older with rotator cuff tears.

BACKGROUND: Recently, to treat the long head of the biceps tendon lesions in addition to rotator cuff repair has been recommended. However, the differences in clinical outcomes between biceps tenotomy and tenodesis for middle-aged and elderly females remains unclear. The purpose of this study was to compare the outcomes of biceps tenotomy and soft-tissue tenodesis that were performed concurrently with arthroscopic rotator cuff repair in ≥60-year-old females. METHODS: Female shoulders that underwent arthroscopic rotator cuff repair in our institute in 2016 were retrospectively reviewed. This study included 66 shoulders with concurrent biceps tenotomy or soft-tissue tenodesis: tenotomy group, 41 shoulders; soft-tissue tenodesis group, 25 shoulders. Clinical scores, biceps pain (visual analogue scale, VAS), Popeye deformity, and biceps strength (%contralateral side) were compared between the two groups. RESULTS: The mean age was significantly higher in the tenotomy group than the soft-tissue tenodesis group (72 ± 4 and 68 ± 6 years, respectively; P = 0.002). There were no significant differences in post-operative JOA and UCLA scores between the groups. VAS for biceps pain was significantly higher at postoperative 6 months in the tenotomy group than the soft-tissue tenodesis group (2.9 ± 2.5 and 1.7 ± 1.6, respectively, P = 0.03), though there were no significant differences at postoperative 3, 12, and ≥24 months. Subjective evaluation of Popeye deformity was not significantly different between the groups. Postoperative biceps strength was significantly lower in the tenotomy group than the soft-tissue tenodesis group (89.9% and 102.8%, respectively, P = 0.02). CONCLUSIONS: Both biceps tenotomy and soft-tissue tenodesis concurrent with rotator cuff repair in ≥60-year-old female patients resulted in good outcomes. Shoulders with soft-tissue tenodesis demonstrated earlier improvement in postoperative biceps pain and better postoperative biceps strength than those with tenotomy. There were no differences in objective and subjective Popeye deformity between tenotomy and soft-tissue tenodesis. The LHB procedures, tenotomy or tenodesis, can be selected depending on surgeons' preference.

Long-Term Survivorship of Closed-Wedge High Tibial Osteotomy for Severe Knee Osteoarthritis: Outcomes After 10 to 37 Years.

BACKGROUND: High tibial osteotomy (HTO) was developed as a joint-preserving procedure to treat relatively young patients with isolated medial compartmental knee osteoarthritis (OA). Long-term survivorship after HTO is important to determine whether patients will need additional surgery. PURPOSE: To determine the long-term (>35-year) survivorship and prognostic factors for closed-wedge HTO (CWHTO) for severe medial OA. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: We retrospectively evaluated patients who underwent CWHTO for severe medial knee OA between 1983 and 2009 at our institution, Nagoya University Graduate School of Medicine (Nagoya, Japan). Patient demographics, follow-up period, and pre- and postoperative femoral-tibial angle (FTA) were reviewed. The patients or the relatives of the patients were interviewed by telephone to record postoperative status, including conversion to total knee arthroplasty (TKA). RESULTS: Of the 74 CWHTO procedures performed, we evaluated 56 procedures in 45 patients (mean age at time of surgery, 56.8 years). The mean follow-up period was 17.1 years. Nine knees (16.1%) underwent conversion to TKA. The mean time to TKA conversion was 15.6 years. Kaplan-Meier analysis revealed a 10-year survival rate of 90.1%, a 15-year rate of 83.8%, a 20-year rate of 75.9%, and a 35-year rate of 75.9%. Log-rank test showed that age ≥55 years (P = .044), body mass index (BMI) ≥25 kg/m2 (P = .0016), and preoperative FTA <185° (P = .0034) were risk factors associated with TKA conversion. Multivariate analyses adjusted for age and sex identified BMI ≥25 kg/m2 (hazard ratio [HR], 13.4; 95% CI, 1.7-106.9; P = .014) and preoperative FTA <185° (HR, 4.2; 95% CI, 1.1-16.6; P = .04) as risk factors associated with TKA conversion. CONCLUSION: The survival rate of CWHTO for severe medial knee OA was 90.1% at 10 years, 83.8% at 15 years, and 75.9% at 20 years and 35 years. Furthermore, a BMI ≥25 kg/m2 and FTA <185° were the independent risk factors associated with TKA conversion after CWHTO.

Assessment of the Preserved Biceps Tendon After Arthroscopic Rotator Cuff Repair in Patients ≤ 55 Years.

PURPOSE: We assessed hypertrophy of preserved long head of the biceps tendon (LHBT) and vascularity in the bicipital groove after arthroscopic rotator cuff repair in ≤55-year-old patients and compared postoperative pain between shoulders with or without vascularity in the bicipital groove. METHODS: Patients who underwent arthroscopic rotator cuff repair between 2015 and 2017 were reviewed. Inclusion criteria were arthroscopic rotator cuff repair and ≤55 years old. Exclusion criteria were a history of contralateral rotator cuff repair, revision surgery, partial repair or superior capsular reconstruction, shoulder dislocation or fracture, torn LHBT at surgery, LHBT tenodesis, retears, <1-year follow-up, and incomplete follow-up data. Cross-sectional area (CSA) of the LHBT and vascularity in the bicipital groove were examined preoperatively and 1 year after surgery using ultrasonography. Shoulder pain at postoperative 1 year was assessed using the pain subscore of the University of California at Los Angeles scale. The data were compared between shoulders with negative and positive vascularity. RESULTS: Fifty-seven shoulders were included in this study. There was no side-to-side difference in preoperative CSA. No difference was found between preoperative and postoperative CSA in the affected shoulders. Postoperative vascularity was identified in 28 (49%) shoulders. Mean pain score was significantly higher in the negative vascularity group than the positive vascularity group (9 and 8, respectively; P = .002). CONCLUSIONS: The preserved LHBT did not show hypertrophy 1 year after arthroscopic repair of medium-sized or smaller posterosuperior rotator cuff tear in ≤55-year-old patients. However, 49% of the shoulders postoperatively demonstrated lower-grade vascularity in the bicipital groove. Healthy LHBT can be preserved in ≤55-year-old patients with posterosuperior medium-sized or smaller rotator cuff tears. LEVEL OF EVIDENCE: III, retrospective comparative prognostic trial.

Deep flexion helps to avoid popliteal artery injury during all-inside lateral meniscal repair: A cadaveric study.

BACKGROUND: Arthroscopic meniscus repair rarely leads to major complications such as popliteal artery injury. The distance between the suturing device and the popliteal artery, and the risk of popliteal artery injury at different knee flexion angles during all-inside lateral meniscal repair remain unclear. METHODS: All-inside devices were inserted into 10 human cadaveric knees at the posterior horn of the lateral meniscus through the anterolateral portal at 60°, 90°, and 120° knee flexion; posterior segment of the lateral meniscus through the anterolateral portal at 60°, 90°, and 120°; and anteromedial portal at 90°. Distance and positional relationship between the device and popliteal artery were measured radiographically. RESULTS: In posterior horn repair through the anterolateral portal, the median distance increased from 5.7 mm at 60° to 9.1 mm at 90° (P = 0.63) and 18.0 mm at 120° (P = 0.02). The device pushed the wire at 60° in three cases, 90° in one case, and 120° in 0 cases. In posterior segment repair through the anterolateral portal, the median distance was 12.6 mm at 60°, 10.4 mm at 90°, and 18.3 mm at 120° (P = 0.08). The median distance at 90° was 18.1 mm through the anteromedial portal, the same as that at 120° through the anterolateral portal (P = 0.43), but greater than that at 90° through the anterolateral portal (P = 0.04). The wire was not pushed in any case. CONCLUSION: Although all-inside repair of the posterior part of the lateral meniscus through the anterolateral portal is risky, deeper knee flexion reduces the risk of popliteal artery injury.

4-Methylumbelliferone suppresses catabolic activation in anterior cruciate ligament-derived cells via a mechanism independent of hyaluronan inhibition.

BACKGROUND: The anterior cruciate ligament (ACL) has a key role as a dynamic stabilizer of the knee joints, and ACL dysfunction caused by traumatic or degenerative rupture accelerates osteoarthritis progression. Thus, it is important to prevent the degenerative rupture of the ACL. 4-Methylumbelliferone (4-MU), a pre-approved drug, exerts anti-inflammatory effects in osteoarthritis chondrocytes. It was originally used as an inhibitor of hyaluronan synthesis in chondrocytes. METHODS: In this study, we investigated whether 4-MU affects the expression of catabolic factors, such as matrix metalloproteinase (MMP)-1, MMP-3, and interleukin (IL)-6, in ACL-derived cells and ACL explant cultures using immunohistochemistry, real-time RT-qPCR, and capillary western immunoassay. Furthermore, the hyaluronan concentration was evaluated using a colorimetric assay. Statistical analyses were conducted using analysis of variance for multi-group comparisons, followed by Tukey or Tukey-Kramer post hoc test. RESULTS: Our results revealed, for the first time, that 4-MU suppressed the IL-ß-induced upregulation of pro-catabolic factors, such as MMP-1, MMP-3, and IL-6, in ACL-derived cells. This suppressive effect was also observed in the cultured ligament tissues in ex vivo experiments. 4-MU also reversed an enhanced dependence on glycolysis in IL-1ß-activated ACL-derived cells. Furthermore, we found that the suppressive effects of 4-MU were exerted directly and not through the inhibition of hyaluronan synthesis. CONCLUSIONS: We conclude that 4-MU could be an effective and useful treatment for knee osteoarthritis, owing to its anti-inflammatory effect on, not only chondrocytes but also on ligament cells.

Association Between Knee Alignment and Meniscal Tear in Pediatric Patients with Anterior Cruciate Ligament Injury.

BACKGROUND: The present study aimed to evaluate the association between elapsed time from anterior cruciate ligament (ACL) injury to surgical treatment and the incidence of meniscal tears in a cohort of patients ≤16 years old with varus-aligned and non-varus-aligned knees. METHODS: The study cohort included 123 patients ≤16 years old who underwent primary ACL reconstruction between January 2016 and March 2020. Knee alignment was expressed as the hip-knee-ankle angle (HKAA), as measured preoperatively on an anteroposterior view of 3-dimensional computed tomography of the full length of the lower limb. Varus alignment was defined as an HKAA ≥181.0°, and non-varus alignment was defined as an HKAA <181.0°. Patients were divided into groups according to knee alignment and the elapsed time from injury to surgical treatment: early-treatment group (<60 days) and delayed-treatment group (≥60 days). RESULTS: A total of 64 varus-aligned and 59 non-varus-aligned knees were identified. Among patients with varus-aligned knees, those in the delayed-treatment group showed a significantly lower rate of lateral meniscal tears (6 of 30, 20%) compared with those in the early-treatment group (17 of 34, 50%; p = 0.015). Among patients with non-varus-aligned knees, there was no significant difference in meniscal tears of any type between the early and delayed-treatment groups. Among patients without medial meniscal injury identified on initial magnetic resonance imaging, those with varus-aligned knees in the delayed-treatment group showed a significantly higher rate of medial meniscal tears at the time of the surgical procedure (8 of 20, 40%) compared with those with non-varus-aligned knees (1 of 18, 6%; p = 0.015). CONCLUSIONS: Delayed ACL reconstruction in patients ≤16 years old with varus-aligned knees might be associated with an increased incidence of secondary medial meniscal tears. Accordingly, earlier ACL reconstruction in patients with varus-aligned knees should be considered. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Delayed anterior cruciate ligament reconstruction increases the incidence of medial meniscal bucket handle tears and medial compartment chondral injuries in patients aged 40 years and older.

INTRODUCTION: No widely accepted evidence-based indications exist for the initial surgical management of patients with anterior cruciate ligament (ACL) injuries ≥ 40 years old, and treatment for these patients remains controversial. This study aimed to evaluate the association between elapsed time from ACL injury to surgery and the incidence of meniscal tears and chondral injury in patients aged ≥ 40 years. MATERIALS AND METHODS: The patients who underwent primary ACL reconstruction were divided into two groups based on elapsed time from injury to surgery: early group, < 12 months; and delayed group, ≥ 12 months. Patient records were reviewed for incidence and types of meniscal tears and chondral injuries in each group. Chondral injury grades were evaluated with International Cartilage Regeneration and Joint Preservation Society (ICRS) Criteria. RESULTS: This study evaluated 67 knees in the early group and 33 knees in the delayed group. Mean ages in each group were 46.9 ± 6.5 and 46.9 ± 6.0. The delayed group showed significantly higher rates of medial meniscal tear [31 of 33, 93.9% vs 29 of 67, 43.3%; P < 0.0001; odds ratio (OR), 20.31; 95% confidence interval (CI), 4.49-91.9], medial femoral condyle chondral injuries ≥ ICRS grade II (15 of 33, 45.5% vs 8 of 67, 11.9%; P < 0.001; OR, 6.15; 95% CI 2.24-16.83), and medial tibial chondral injuries ≥ ICRS grade II (7 of 33, 21.2% vs 3 of 67, 4.5%; P < 0.05; OR, 5.74; 95% CI 1.38-23.9) compared with the early group. With respect to types of medial meniscal tear, the delayed group showed a significantly higher frequency of bucket handle tears (11 of 33, 33.3%) compared with the early group (2 of 67, 3.0%; P < 0.0001; OR, 16.25; 95% CI 3.34-79.1). CONCLUSIONS: Delayed ACL reconstruction was associated with increased incidence of chondral injuries and medial meniscal tears, particularly bucket handle tears in this cohort. LEVEL OF EVIDENCE: Level III.

Incidence of Medial and Lateral Meniscal Tears After Delayed Anterior Cruciate Ligament Reconstruction in Pediatric Patients.

BACKGROUND: The treatment of pediatric anterior cruciate ligament (ACL) injuries is controversial, and no clear management guidelines have been established. PURPOSE: To evaluate the association between elapsed time from ACL injury to surgery and the incidence of meniscal tears and chondral injuries in patients aged ≤16 years. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Between December 2012 and April 2019, a total of 207 consecutive knees in 207 patients aged ≤16 years underwent primary ACL reconstruction and were included in this study. Patients were divided into 1 of 2 groups (early group [≤150 days] and delayed group [>150 days]) based on the time between injury and surgery. Patient records, including arthroscopic findings identified by 2 experienced knee surgeons at the time of surgery, were reviewed for demographic information, incidence and types of medial and lateral meniscal tears, and chondral injuries and their locations in each group. RESULTS: There were 180 knees in the early group and 27 knees in the delayed group. The delayed group showed a significantly higher rate of medial meniscal tears than the early group: 16 of 27 (59.2%) and 46 of 180 (25.6%), respectively (odds ratio [OR], 4.24 [95% CI, 1.83-9.33]; P = .0011). The delayed group had a significantly lower rate of lateral meniscal tears than the early group: 6 of 27 (22.2%) and 90 of 180 (50.0%), respectively (OR, 0.29 [95% CI, 0.11-0.70]; P = .007). The delayed group had significantly higher rates of chondral injuries in the medial femoral condyle and the medial tibial plateau than the early group: 8 of 27 (29.6%) and 25 of 180 (13.9%), respectively (OR, 2.61 [95% CI, 1.03-6.62]; P = .049), and 2 of 27 (7.4%) and 1 of 180 (0.6%), respectively (OR, 14.32 [95% CI, 1.58-208.10]; P = .045). CONCLUSION: Delayed ACL reconstruction was associated with an increased incidence of medial chondral injuries and medial meniscal tears but with a decreased incidence of lateral meniscal tears.

Fluoxetine ameliorates cartilage degradation in osteoarthritis by inhibiting Wnt/ß-catenin signaling.

Abnormal activation of the Wnt/ß-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/ß-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/ß-catenin activity in the presence of 10 µM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/ß-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/ß-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total ß-catenin and a LiCl-induced decrease of phosphorylated ß-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of ß-catenin with Axin1, which is a scaffold protein forming the degradation complex for ß-catenin. Fluoxetine suppressed LiCl-induced ß-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed ß-catenin accumulation.

Expression of tenocyte lineage-related factors in regenerated tissue at sites of tendon defect.

BACKGROUND: The healing mechanism of ruptured or injured tendons is poorly understood. To date, some lineage-specific factors, such as scleraxis and tenomodulin, have been reported as markers of tenocyte differentiation. Because few studies have focused on tenocyte lineage-related factors with respect to the repaired tissue of healing tendons, the aim of this study was to investigate their expression during the tendon healing process. METHODS: Defects were created in the patellar tendons of rats, and the patellae and patellar tendons were harvested at 3 days and at 1, 2, 3, 6, 12, and 20 weeks after surgery. They were studied using micro-computed tomography, and paraffin-embedded sections were then prepared for histological evaluation. Reverse transcription-polymerase chain reactions were performed to analyze the expression of genes related to the tenocyte lineage, chondrogenesis, and ossification. RESULTS: Repaired tissue became increasingly fibrous over time and contained a greater number of vessels than normal tendons, even in the later period. Safranin O staining revealed the existence of proteoglycan at 1 week and its persistence through 20 weeks. Ossification was detected in all tendons at 12 weeks. The expression of tenocyte lineage-related genes was high at 1 and 2 weeks. Chondrogenic genes were up-regulated until 6 weeks. Runt-related transcription factor 2, an osteogenic gene, was up-regulated at 20 weeks. CONCLUSIONS: In our tendon defect model, cells participating in the tendon healing process appeared to differentiate toward tenocyte lineage only in the early phase, and chondrogenesis seemed to occur from the early phase onward. To improve tendon repair, it will be necessary to promote and maintain tenogenesis and to inhibit chondrogenesis, especially in the early phase, in order to avoid erroneous differentiation of stem cells.

Role of S100A12 in the pathogenesis of osteoarthritis.

S100A12 is a member of the S100 protein family, which are intracellular calcium-binding proteins. Although there are many reports on the involvement of S100A12 in inflammatory diseases, its presence in osteoarthritic cartilage has not been reported. The purpose of this study was to investigate the expression of S100A12 in human articular cartilage in osteoarthritis (OA) and to evaluate the role of S100A12 in human OA chondrocytes. We analyzed S100A12 expression by immunohistochemical staining of cartilage samples obtained from OA and non-OA patients. In addition, chondrocytes were isolated from knee cartilage of OA patients and treated with recombinant human S100A12. Real-time RT-PCR was performed to analyze mRNA expression. Protein production of matrix metalloproteinase 13 (MMP-13) and vascular endothelial growth factor (VEGF) in the culture medium were measured by ELISA. Immunohistochemical analyses revealed that S100A12 expression was markedly increased in OA cartilages. Protein production and mRNA expression of MMP-13 and VEGF in cultured OA chondrocytes were significantly increased by treatment with exogenous S100A12. These increases in mRNA expression and protein production were suppressed by administration of soluble receptor for advanced glycation end products (RAGE). Both p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors also suppressed the increases in mRNA expression and protein production of MMP-13 and VEGF. We demonstrated marked up-regulation of S100A12 expression in human OA cartilages. Exogenous S100A12 increased the production of MMP-13 and VEGF in human OA chondrocytes. Our data indicate the possible involvement of S100A12 in the development of OA by up-regulating MMP-13 and VEGF via p38 MAPK and NF-κB pathways.

Regulation of prostaglandin E(2) synthesis in cells derived from chondrocytes of patients with osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a disorder that causes pain and degeneration of the joint over a chronic time course. Chondrocytes in OA play important roles in maintaining the homeostasis of the joint while they produce many cytokines and pathological mediators, including interleukin-1beta (IL-1beta), cyclooxygenases (COX), and prostaglandin E(2) (PGE(2)). To elucidate the mechanisms of pain due to OA, the pathway of PGE(2) synthesis was analyzed using cells derived from chondrocytes obtained from patients with OA. METHODS: Chondrocytes were isolated from cartilage samples obtained at the time of joint replacement surgery from patients with OA. The chondrocytes at the second passage were cultured with or without IL-1beta, dexamethasone (DEX), or COX inhibitors such as NS-398, meloxicam, and indomethacin. Reverse transcription-polymerase chain reaction and Western blotting analysis were performed to study the levels of mRNA and protein, respectively. An enzyme-linked immunosorbent assay was performed to investigate the translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus, and Western blotting analysis was performed to study the phosphorylation of mitogen-activated protein kinases. RESULTS: IL-1beta markedly enhanced the expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) at both the mRNA and protein levels. The up-regulation was suppressed by DEX or COX inhibitors. IL-1beta strongly increased the translocation of NF-kappaB to the nucleus and the phosphorylation of extracellular-signal-regulated kinase, p38, and c-Jun amino-terminal kinase; but the up-regulation was not inhibited by DEX or COX inhibitors. Interestingly, in a dose-dependent manner, PGE(2) recovered mPGES-1 expression from suppression by DEX, whereas it did not restore the expression of COX-2 in the presence of DEX and IL-1beta. CONCLUSIONS: These results suggested that in cells derived from OA chondrocytes different mechanisms of regulation exist between mPGES-1 and COX-2, and the expression of mPGES-1 was, at least partially, regulated through the autocrine positive feedback by PGE(2).

A 12,000-rad porcine radiation hybrid (IMNpRH2) panel refines the conserved synteny between SSC12 and HSA17.

Reverse or bidirectional Zoo-FISH suggests that synteny between porcine chromosome 12 (SSC12) and human chromosome 17 (HSA17) is completely conserved. The construction of a high-resolution radiation hybrid (RH) map for SSC12 provides a unique opportunity to determine whether chromosomal synteny is reflected at the molecular level by comparative gene mapping of SSC12 and HSA17. We report an initial, high-resolution RH map of SSC12 on the 12,000-rad IMNpRH2 panel using CarthaGene software. This map contains a total of 320 markers, including 20 microsatellites and 300 ESTs/genes, covering approximately 4836.9 cR12,000. The markers were ordered in 16 linkage groups at LOD 6.0 using framework markers previously mapped on the IMpRH7000-rad SSC12 and porcine genetic maps. Ten linkage groups ordered more than 10 markers, with the largest containing 101 STSs. The resolution of the current RH map is approximately 15.3 kb/cR on SSC12, a significant improvement over the second-generation EST SSC12 RH7000-rad map of 103 ESTs and 15 framework markers covering approximately 2287.2 cR7000. Compared to HSA17, six distinct segments were identified, revealing macro-rearrangements within the apparently complete synteny between SSC12 and HSA17. Further analysis of the order of 245 genes (ESTs) on HSA17 and SSC12 also revealed several micro-rearrangements within a synteny segment. A high-resolution SSC12 RH12,000-rad map will be useful in fine-mapping QTL and as a scaffold for sequencing this chromosome.

Cloning and characterization of porcine common gamma chain gene.

The common gamma chain, which was originally identified as a component of interleukin-2 receptors (IL-2R), plays a key role in differentiation of T lymphocytes and natural killer (NK) cells. In the present study, cDNA of the porcine common gamma chain gene and its genomic DNA were molecularly cloned and characterized. The porcine common gamma chain gene was found to consist of 8 exons, spanning approximately 3.7 kb, and to encode a 368-amino acid polypeptide. The amino acid sequence showed 82.4%, 71.1%, 86.1%, and 84.8% similarities with that of human, murine, bovine, and canine chains, respectively. The common gamma chain gene was assigned to swine chromosome Xq13 by FISH analysis and was consistent with the result of radiation hybrid (RH) mapping. When various porcine tissues were examined for the expression of this gene, the expression was observed in lymphocytes and lymphocyte-related tissues. Since GATA, T cell factor-1 (TCF-1), Ets-1, activated protein2 (AP-2), and Ikaros2 binding motifs were demonstrated in the 5' upstream region of this gene, promoter activity was investigated using luciferase gene as a reporter. The results indicate that the Ets-1 binding motif in the segment from -95 to -59 (major transcription initiation site: +1) was an essential cis-acting regulatory element for the common gamma chain gene in lymphoid cells.