Distribution of TRAP‐positive cells and expression of HIF‐1α, VEGF,and FGF‐2 in the reparative reaction in patients with osteonecrosis of the femoral head

Osteogenesis and angiogenesis are closely associated with the reparative process in bone. In osteonecrosis of the femoral head (ONFH), although the progression of bone resorption by osteoclasts is considered to be followed by femoral head collapse, the reparative reaction remains unknown. In order to investigate the reparative reaction in patients with ONFH, the distribution of TRAP‐ positive cells and expression of HIF‐1α, VEGF, and FGF‐2 were observed in 51 hips in 42 patients. TRAP‐positive cells were detected around the teres insertion and retinaculum in the early radiologic stage, and increased around the new trabecular bone throughout the reparative interface zone in the late collapsed stage. HIF‐1α expression was detected at the fibrosis area and the transitional area, which included the proximal area of the reparative interface zone adjacent to the necrotic zone. VEGF was expressed at the edematous area of the reparative interface zone, while FGF‐2 was detected widely in the reparative interface zone and the normal zone. In the late radiologic stages, HIF‐1α, VEGF, and FGF‐2 were not detected in the necrotic zone, and they acted in angiogenesis in the reparative interface zone, while TRAP‐positive cells increased around the new bone formation in response to remodeling after the collapse. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 694–700, 2009     

Parathyroid hormone induction of cyclooxygenase‐2 in murine osteoblasts: Role of the calcium‐calcineurin‐NFAT pathway

Murine MC3T3‐E1 and MC‐4 cells were stably transfected with ?371/+70 bp of the murine cyclooxygenase‐2 (COX‐2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site‐directed mutations. Mutations were made in (1) the cAMP response element (CRE) at ?57/?52 bp, (2) the activating protein‐1 (AP‐1)–binding site at ?69/?63 bp, (3) the nuclear factor of activated T‐cells (NFAT)–binding site at ?77/?73 bp, and (4) both the AP‐1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP‐1. Single mutation of CRE, AP‐1, or NFAT sites decreased parathyroid hormone (PTH)–stimulated COX‐2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP‐1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c‐Fos, and c‐Jun to an oligonucleotide spanning the NFAT/AP‐1 composite site. Mutation of the NFAT site was less effective than mutation of the AP‐1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP‐1 are more dependent on NFAT than on AP‐1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH‐ and forskolin‐stimulated COX‐2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium‐calcineurin‐NFAT signaling pathway in the PTH induction of COX‐2 and suggest that cross‐talk between the cAMP/PKA pathway and the calcium‐calcineurin‐NFAT pathway may play a role in other functions of PTH in osteoblasts. © 2010 American Society for Bone and Mineral Research     

Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs

Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, Cooper DKC, Bianchi J. Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs. Xenotransplantation 2012; 19: 305–310. © 2012 John Wiley & Sons A/S. Abstract Serum anti‐galactose‐α1,3‐galactose (Gal) IgM and IgG antibody levels were measured by ELISA in α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (78 estimations in 47 pigs). A low level of anti‐Gal IgM was present soon after birth, and rose to a peak at 4–6 m, which was maintained thereafter even in the oldest pigs tested (at >2 yr). Anti‐Gal IgG was also present at birth, peaked at 3 m, and after 6 m steadily decreased until almost undetectable at 20 m. No differences in this pattern were seen between pigs of different gender. Total IgM followed a similar pattern as anti‐Gal IgM, but total IgG did not decrease after 6m. The data provide useful baseline data for future experimental studies in GTKO pigs, e.g., relating to the antibody response to WT pig allografts.     

Evaluation of Hyphecan (1‐4,2‐acetamide‐deoxy‐B‐D‐glucan polymer) on wound healing in a rodent model

Objective: To evaluate the effect of an artificial skin Hyphecan (1‐4,2‐acetamide‐deoxy‐B ‐D ‐glucan polymer) on wound healing in a rodent model. Materials and Method: The prospective study was conducted at a basic science laboratory at a tertiary teaching hospital. Two 4 cm × 4 cm full‐thickness wounds were created on the dorsal surface of 10 Spraque–Dawley rats and covered with Hyphecan and Kaltostat, respectively. Wounds were examined and measured on days 4, 10, 21 and 28, and would continue after day 28 until healed up completely. Punch biopsies (3 mm) were taken on days 4, 10 and 28 for histological examination of the response of healing and repair. Results: Despite the fact that the wound healing rate was similar for both groups on days 4, 10, 21 and 28, the average healing time for the Hyphecan group (29.1 ± 1.7 days) was significantly shorter statistically (P = 0.03) than the Kaltostat group (30.7 ± 2.8 days). Conversely, the marked healing response elicited by Hyphecan on day 4 persisted on days 10 and 28 in contrast to Kaltostat, which had only a mild degree of healing response on days 10 and 28. The study suggests that wounds treated by Hyphecan heal faster than Kaltostat. Conclusion: The findings provide basic scientific evidence supporting the clinical use of Hyphecan in different wounds and might also reduce the cost of wound management as Hyphecan is cheaper than Kaltostat and requires a shorter treatment time.      

MicroRNA‐1 (miR‐1) inhibits chordoma cell migration and invasion by targeting slug

Recent studies have revealed that expression of miRNA‐1 (miR‐1) is frequently down‐regulated in several cancer types including chordoma. Identifying and validating novel targets of miR‐1 is useful for understanding the roles of miR‐1 in chordoma. We aimed to further investigate the functions of miR‐1 in chordoma. Specifically, we assessed whether restoration of miR‐1 affects cell migration and invasion in chordoma, and focused on the miR‐1 potential target Slug gene. Migratory and invasive activities were assessed by wound healing and Matrigel invasion assays, respectively. Cell proliferation was determined by MTT assay. Slug expression was evaluated by Western blot, immunofluorescence, and immunohistochemistry. Restoration of miR‐1 expression suppressed the migratory and invasive activities of chordoma cells. Transfection of miR‐1 inhibited cell proliferation both time‐ and dose‐dependently in chordoma. MiR‐1 transfected cells showed inhibited Slug expression. Slug was over‐expressed in chordoma cell lines and advanced chordoma tissues. In conclusion, we have shown that miR‐1 directly targets the Slug gene in chordoma. Restoration of miR‐1 suppressed not only proliferation, but also migratory and invasive activities, and reduced the Slug expression in chordoma cells. These results collectively indicate that miR‐1/Slug pathway is a potential therapeutic target because of its crucial roles in chordoma cell growth and migration. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1075–1082, 2014.     

NGF promotes mitochondrial function by activating PGC‐1α in TM4 Sertoli cells

Nerve growth factor (NGF), which is required for the survival and differentiation of the nervous system, has been proved to play important roles in male reproductive physiology. Several studies have focused on the roles of NGF in the testes. However, no study has reported on the mechanism of paracrine and autocrine actions of NGF in Sertoli cells. This study showed that NGF stimulated mitochondrial activity and biogenesis in TM4 Sertoli cells. Moreover, our results demonstrated that peroxisome proliferator‐activated receptor‐gamma coactivator‐1α is a possible downstream key target of the NGF signalling pathway. In a 3‐nitropropionic acid cell model, NGF treatment attenuated mitochondrial activity defect and depolarisation. This NGF‐triggered signalling may help in discovering new therapeutic targets for certain male infertility disorders.     

Association of tumour necrosis factor‐α gene (T‐1031C,C‐863A,and C‐857T) polymorphisms with bladder cancer susceptibility and outcome after bacille Calmette‐Guérin immunotherapy

OBJECTIVE

To investigate the association of tumour necrosis factor‐α gene (TNF‐α) polymorphisms T‐1031C, C‐863A, and C‐857T with bladder cancer risk and recurrence after bacille Calmette‐Guérin (BCG) immunotherapy, as TNF‐α regulates inflammatory process influencing bladder cancer susceptibility and outcome of BCG immunotherapy.

PATIENTS AND METHODS

In all, 220 patients with bladder cancer and 206 controls were recruited. Genotyping was done using allele specific‐polymerase chain reaction.

RESULTS

A T‐1031C, CC genotype and haplotype ?1031C/?863C/?857T showed enhanced susceptibility to bladder cancer, with an odds ratio (OR) of 2.23 and 95% confidence interval (CI) of 1.17–4.26; and an OR of 6.05 and 95%CI of 2.46–14.90, respectively. A T‐1031C, CC genotype had a reduced risk of recurrence after BCG treatment (hazard ratio 0.38, 95%CI 0.14–0.98).

CONCLUSION

The present data suggests that T‐1031C (CC) genotype and C/C/T haplotype may confer risk for bladder cancer, moreover T‐1031C (CC) decreased the risk of recurrence after BCG immunotherapy.