Predicting failures of suture anchors used for rotator cuff repair: a CT-based 3-dimensional finite element analysis.

BACKGROUND: Failure of inserted anchors has been recognized as one of the major pathomechanisms of re-tearing after rotator cuff repair. OBJECTIVE: To predict the inserted anchor failure using CT-based 3-dimensional finite element method (CT/3D-FEM). METHODS: Among twenty patients who underwent rotator cuff repair, 5 had anchor failure (failed anchor group) and 15 had no anchor failure (stable anchor group). A 3D model of proximal humerus was developed for each patient based on the CT data. A virtual pullout testing of TWINFIX™ anchors inserted into bone at 6 different sites was performed using FEM. Then, mean failure load of 6 anchors for each patient was compared between two groups. Moreover, an optimal cut-off value of the mean failure load was determined for predicting anchor failure. RESULTS: The mean failure load in the failed anchor group (70.3 N) was significantly lower than that in the stable anchor group (119.0 N; p<0.0001). In our method, the optimum cut-off value of the mean failure load was 75.4 N. CONCLUSIONS: Failure of the inserted TWINFIX™ anchor could be predicted using CT/3D-FEM. In this method, there seemed to be a high risk of anchor failure in shoulders with a mean failure load of <75.4 N.

Association of reduced type IX collagen gene expression in human osteoarthritic chondrocytes with epigenetic silencing by DNA hypermethylation.

OBJECTIVE: To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter. METHODS: Expression levels were determined using quantitative reverse transcription-polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation. RESULTS: Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter. CONCLUSION: This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment.

Epigenetic regulation during fetal femur development: DNA methylation matters.

Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1(+) marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.

Regulated transcription of human matrix metalloproteinase 13 (MMP13) and interleukin-1ß (IL1B) genes in chondrocytes depends on methylation of specific proximal promoter CpG sites.

The role of DNA methylation in the regulation of catabolic genes such as MMP13 and IL1B, which have sparse CpG islands, is poorly understood in the context of musculoskeletal diseases. We report that demethylation of specific CpG sites at -110 bp and -299 bp of the proximal MMP13 and IL1B promoters, respectively, detected by in situ methylation analysis of chondrocytes obtained directly from human cartilage, strongly correlated with higher levels of gene expression. The methylation status of these sites had a significant impact on promoter activities in chondrocytes, as revealed in transfection experiments with site-directed CpG mutants in a CpG-free luciferase reporter. Methylation of the -110 and -299 CpG sites, which reside within a hypoxia-inducible factor (HIF) consensus motif in the respective MMP13 and IL1B promoters, produced the most marked suppression of their transcriptional activities. Methylation of the -110 bp CpG site in the MMP13 promoter inhibited its HIF-2α-driven transactivation and decreased HIF-2α binding to the MMP13 proximal promoter in chromatin immunoprecipitation assays. In contrast to HIF-2α, MMP13 transcriptional regulation by other positive (RUNX2, AP-1, ELF3) and negative (Sp1, GATA1, and USF1) factors was not affected by methylation status. However, unlike the MMP13 promoter, IL1B was not susceptible to HIF-2α transactivation, indicating that the -299 CpG site in the IL1B promoter must interact with other transcription factors to modulate IL1B transcriptional activity. Taken together, our data reveal that the methylation of different CpG sites in the proximal promoters of the human MMP13 and IL1B genes modulates their transcription by distinct mechanisms.

Loss of methylation in CpG sites in the NF-κB enhancer elements of inducible nitric oxide synthase is responsible for gene induction in human articular chondrocytes.

OBJECTIVE: To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. METHODS: Expression of iNOS was quantified by quantitative reverse transcriptase-polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. RESULTS: The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at -289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at -5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. CONCLUSION: These findings demonstrate the association between demethylation of specific NF-κB-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.

Suppressors of cytokine signalling (SOCS) are reduced in osteoarthritis.

OBJECTIVES: Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling that function via the JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription). Eight SOCS proteins, SOCS1-SOCS7 and CIS-1 (cytokine-inducible SH2-domain, with similar structure to the other SOCS proteins) have been identified, of which SOCS1, 2, and 3 and CIS-1 are the best characterised. A characteristic feature of osteoarthritis (OA) is increased production by articular chondrocytes of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNFα), which may be induced by mechanotransduction and contribute to cartilage destruction. In this study, we have compared the gene expression of SOCS1, 2, 3 and CIS-1 in healthy and OA human chondrocytes, and also analyzed the effects of IL-1ß and TNFα on the levels of mRNA encoding these SOCS family members. In addition, SOCS2 protein production was assessed and the CpG methylation status of the SOCS2 promoter was analyzed to determine the role of epigenetics in its regulation. METHODS: Femoral heads were obtained after joint replacement surgery for late stage OA and hemiarthroplasty following a fracture of the neck of femur (#NOF). Chondrocytes from the superficial layer of OA cartilage and the deep zone of #NOF cartilage were isolated by sequential treatment with trypsin, hyaluronidase and collagenase B. Total DNA and RNA were extracted from the same chondrocytes, and the levels of SOCS1, 2, 3 and CIS-1 mRNA were determined by qRT-PCR. The percentage of methylation in the CpG sites of the SOCS2 proximal promoter was quantified by pyrosequencing. Alternatively, healthy chondrocytes were isolated from #NOF cartilage and cultured with and without a mixture of IL-1ß and oncostatin M (OSM, both 2.5ng/ml) or TNFα (10ng/ml). The short-term cultures with single cytokine treatment were harvested 24 and 72h after treatment, and the long-term cultures were maintained for 4-5 weeks until confluent with periodical cytokine stimulation. Total RNA was extracted and mRNA levels were determined by qRT-PCR. RESULTS: The SOCS2 and CIS-1 mRNA levels were reduced by approximately 10-fold in OA samples compared to control samples, while SOCS1 and SOCS3 showed similar expression patterns in OA and control chondrocytes. The SOCS2 and CIS-1 mRNA levels declined by 6-fold and 3-fold with long-term treatment with IL-1ß and OSM in combination and TNFα, respectively. There was no significant difference in the CpG methylation status of the SOCS2 promoter between healthy and OA chondrocytes. Similarly, cytokine stimulation did not change the CpG methylation status of the SOCS2 promoter. CONCLUSIONS: This study demonstrates the reduced expression of SOCS2 and CIS-1 in OA, while SOCS1 and SOCS3 were unaffected. The observation that long-term treatment with inflammatory cytokines attenuated the expression of SOCS2 and CIS-1 suggests a potential positive feedback mechanism, and a role of SOCS in the pathology of OA.

The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes--implications for osteoarthritis.

OBJECTIVE: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1ß, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. METHOD: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1ß and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2mM N-acetyl GlcN (Sigma-Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1ß, 2.5 ng/ml OSM and 2mM GlcN, (v) cultured with 1.0 µM BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1ß, 2.5 ng/ml OSM and 1.0 µM BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1ß and MMP13 proximal promoter were quantified by pyrosequencing. RESULT: IL1ß expression was enhanced over 580-fold in articular chondrocytes treated with IL-1ß and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1ß expression by 3-fold. In the presence of BAY, IL-1ß induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the -256 CpG site in the IL1ß promoter was 65% in control cultures and decreased to 36% in the presence of IL-1ß/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1ß/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. CONCLUSION: We demonstrate for the first time that GlcN and BAY can prevent cytokine-induced demethylation of a specific CpG site in the IL1ß promoter and this was associated with decreased expression of IL1ß. These studies provide a potential mechanism of action for OA disease modifying agents via NF-kB and, critically, demonstrate the need for further studies to elucidate the role that NF-kB may play in DNA demethylation in human chondrocytes.

Treatment with recombinant placental growth factor (PlGF) enhances both angiogenesis and arteriogenesis and improves survival after myocardial infarction.

BACKGROUND: Placental growth factor (PlGF), a homolog of vascular endothelial growth factor, is reported to stimulate angiogenesis and arteriogenesis in pathological conditions. It was recently demonstrated that PlGF is rapidly produced in myocardial tissue during acute myocardial infarction (MI). However, the effects of exogenous PlGF administration on the healing process after MI are not fully understood. The purpose of the present study was to examine whether PlGF treatment has therapeutic potential in MI. METHODS AND RESULTS: Recombinant human PlGF (rhPlGF: 10 microg) was administered continuously for 3 days in a mouse model of acute MI. rhPlGF treatment significantly improved survival rate after MI and preserved cardiac function relative to control mice. The numbers of CD31-positive cells and alpha-smooth muscle actin-positive vessels in the infarct area were significantly increased in the rhPlGF group. Endothelial progenitor cells (Flk-1(+)Sca-1(+) cells) were mobilized by rhPlGF into the peripheral circulation. Furthermore, rhPlGF promoted the recruitment of GFP-labeled bone marrow cells to the infarct area, but only a few of those migrating cells differentiated into endothelial cells. CONCLUSIONS: Exogenous PlGF plays an important role in healing processes by improving cardiac function and stimulating angiogenesis following MI. It can be considered as a new therapeutic molecule.

Inflammatory response to acute myocardial infarction augments neointimal hyperplasia after vascular injury in a remote artery.

OBJECTIVE: Percutaneous coronary intervention (PCI) is currently the most widely accepted treatment for acute myocardial infarction (AMI). It remains unclear, however, whether post-AMI conditions might exacerbate neointimal hyperplasia and restenosis following PCI. Given that both a medial smooth muscle cell lineage and a bone marrow (BM)-derived hematopoietic stem cell lineage are now thought to contribute to neointima formation, the primary aims of the present study were to determine whether AMI augments neointimal hyperplasia at sites of arterial injury, and whether BM-derived cells contribute to that process. METHODS AND RESULTS: We simultaneously generated models of AMI and arterial injury in the same mice, some of which had received BM transplantation. We found that AMI augments neointimal hyperplasia at sites of femoral artery injury by approximately 35% (P<0.05), but that while BM-derived cells contributed to neointimal hyperplasia, they did not contribute to the AMI-related augmentation. Expression of interleukin (IL)-6 mRNA was approximately 7-fold higher in the neointimas of mice subjected to both AMI and arterial injury than in those of mice subjected to arterial injury alone. In addition, we observed increased synthesis of tumor necrosis factor (TNF)-alpha within infarcted hearts and TNF-alpha receptor type 1 (TNFR1) within injured arteries. Chronic treatment with pentoxifylline, which mainly inhibits TNF-alpha synthesis, reduced levels of circulating TNF-alpha and attenuated neointimal hyperplasia after AMI. CONCLUSIONS: Conditions after AMI could exacerbate postangioplasty restenosis, not by increasing mobilization of BM-derived cells, but by stimulating signaling via TNF-alpha, TNFR1 and IL-6.

Cardiac expression of placental growth factor predicts the improvement of chronic phase left ventricular function in patients with acute myocardial infarction.

OBJECTIVES: Our aim was to investigate cardiac expression of placental growth factor (PlGF) and its clinical significance in patients with acute myocardial infarction (AMI). BACKGROUND: Placental growth factor is known to stimulate wound healing by activating mononuclear cells and inducing angiogenesis. The clinical significance of PlGF in AMI is not yet known. METHODS: Fifty-five AMI patients and 43 control subjects participated in the study. Peripheral blood sampling was performed on days 1, 3, and 7 after AMI. Blood was also sampled from the coronary artery (CAos) and the coronary sinus (CS), before and after acute coronary recanalization. Cardiac expression of PlGF was analyzed in a mouse AMI model. RESULTS: In AMI patients, peripheral plasma PlGF levels on day 3 were significantly higher than in control subjects. Plasma PlGF levels just after recanalization were significantly higher in the CS than the CAos, which indicates cardiac production and release of PlGF. Peripheral plasma levels of PlGF on day 3 were negatively correlated with the acute phase left ventricular ejection fraction (LVEF), positively correlated with both acute phase peak peripheral monocyte counts and chronic phase changes in LVEF. Placental growth factor messenger ribonucleic acid expression was 26.6-fold greater in a mouse AMI model than in sham-operated mice, and PlGF was expressed mainly in endothelial cells within the infarct region. CONCLUSIONS: Placental growth factor is rapidly produced in infarct myocardium, especially by endothelial cells during the acute phase of myocardial infarction. Placental growth factor might be over-expressed to compensate the acute ischemic damage, and appears to then act to improve LVEF during the chronic phase.