Wnt signaling pathway inhibitors as promising diagnostic serum markers of osteolytic bone metastasis.

Despite the clinical importance of bone metastases, we still know little about their onset and progression and current diagnostic tools lack the sensitivity and specificity required for clear early diagnosis. We therefore need to continue studying the pathogenesis of bone metastatic invasion in order to improve diagnosis. The Wnt pathway has been described as having an important role in bone carcinogenesis and metastatic progression. This study investigated the diagnostic potential of the two main Wnt inhibitors, sclerostin and DKK-1, to improve the detection of osteolytic bone metastases. We measured sclerostin and DKK-1, MMP-2 and MMP-9, the bone resorption marker TRAP5b and the metastatic marker survivin in a control group of healthy patients, in patients with primary tumors and in a group with metastasis. Sclerostin and DKK-1 were clearly high in primary tumor patients and even higher in metastatic patients, compared to controls. The close correlations with metastatic markers and bone resorption markers make sclerostin and DKK-1 promising as new biomarkers in the diagnosis of bone osteolytic metastases.

Circulating sRAGE in the diagnosis of osteolytic bone metastasis.

Despite the clinical importance of metastasis to the skeleton, the diagnostic tools for early detection and monitoring of bone metastasis lack sensitivity and specificity. We evaluated a promising new serum biomarker, the soluble form of the Receptor of Advanced Glycosylated End-products (sRAGE). sRAGE is involved in the Wnt-signaling pathway, and has been reported to reduce the risk of cancer. We investigated the diagnostic potential of sRAGE to improve the detection and monitoring of bone metastasis. We measured sRAGE in the serum of control healthy subjects, patients with primary tumors and patients with bone metastasis. sRAGE was also correlated with the Wnt inhibitors DKK-1 and sclerostin, the bone resorption markers MMP-2, MMP-9 and TRAP5, and the metastatic marker survivin. sRAGE was significantly lower in primary tumor and metastatic patients than in healthy subjects. sRAGE also showed a strong negative correlation with DKK-1, sclerostin, MMP-2, MMP-9, TRAP5b and survivin. These results indicated that sRAGE might play a protective role in bone metastasis progression, and it may diagnostic significance for detecting and monitoring osteolytic metastases.

Why menisci show higher healing rate when repaired during ACL reconstruction? Growth factors release can be the explanation.

PURPOSE: Healing rate of meniscus repair is higher when the suture is associated with anterior cruciate ligament reconstruction. A possible explanation can be a different pattern of release of growth factors between anterior cruciate ligament reconstruction and isolated meniscus surgery. Hypothesis of this study is that the concentrations of bFGF, TGF-ß and platelet-derived growth factor (PDGF) in joint fluid, immediately after single-bundle anterior cruciate ligament reconstruction and arthroscopic partial meniscectomy, can be different. METHODS: Twenty consecutive patients underwent partial medial meniscectomy and twenty consecutive patients underwent single-bundle anterior cruciate ligament reconstruction with hamstring grafts were enrolled in the study. Thirty minutes after the end of the surgical procedure, a sample of joint fluid, as well of venous blood, was collected from all the patients. Concentrations of growth factors were determined by enzyme-linked immunosorbent assay. RESULTS: The peripheral blood concentration of TGF-ß, bFGF and PDGF was comparable between partial meniscectomy and anterior cruciate ligament reconstruction groups. No differences between the two surgical techniques were also found in term of TGF-ß and bFGF joint fluid concentration, whereas joint PDGF concentration of anterior cruciate ligament reconstruction patients was significantly higher than the one found in partial meniscectomy patients. CONCLUSIONS: A significant growth factors release was detected in the knee joint during arthroscopic surgery. PDGF concentration was significantly higher in anterior cruciate ligament reconstructed knee than in the meniscectomy group. PDGF can play an important role enhancing the healing response of meniscus suture and can be one of the biological reasons of the higher meniscal healing rate in anterior cruciate ligament reconstructed knee.

In vitro functional response of human tendon cells to different dosages of low-frequency pulsed electromagnetic field.

PURPOSE: Chronic tendinopathy is a degenerative process causing pain and disability. Current treatments include biophysical therapies, such as pulsed electromagnetic fields (PEMF). The aim of this study was to compare, for the first time, the functional in vitro response of human tendon cells to different dosages of PEMF, varying in field intensity and duration and number of exposures. METHODS: Tendon cells, isolated from human semitendinosus and gracilis tendons (hTCs; n = 6), were exposed to different PEMF treatments (1.5 or 3 mT for 8 or 12 h, single or repeated treatments). Scleraxis (SCX), COL1A1, COL3A1 and vascular endothelial growth factor-A (VEGF-A) expression and cytokine production were assessed. RESULTS: None of the different dosages provoked apoptotic events. Proliferation of hTCs was enhanced by all treatments, whereas only 3 mT-PEMF treatment increased cell viability. However, the single 1.5 mT-PEMF treatment elicited the highest up-regulation of SCX, VEGF-A and COL1A1 expression, and it significantly reduced COL3A1 expression with respect to untreated cells. The treated hTCs showed a significantly higher release of IL-1ß, IL-6, IL-10 and TGF-ß. Interestingly, the repeated 1.5 mT-PEMF significantly further increased IL-10 production. CONCLUSIONS: 1.5 mT-PEMF treatment was able to give the best results in in vitro healthy human tendon cell culture. Although the clinical relevance is not direct, this investigation should be considered an attempt to clarify the effect of different PEMF protocols on tendon cells, in particular focusing on the potential applicability of this cell source for regenerative medicine purpose, both in surgical and in conservative treatment for tendon disorders.

Osteoprotegerin, RANK and RANKL are not modified by acute exercise in elite rugby players.

AIM: The OPG-RANK-RANKL system is a new family of bone metabolism biomarkers belonging to the immune system. In this study, were evaluated these biomarkers in professional rugby players after a single-bout of training session. METHODS: The study has been performed on 30 professional male rugby players during a training camp of the Italian National Team, in July, before the start of the competitive season. Blood drawings were performed before and after training in the same day. Levels of soluble OPG, RANKL RANK in serum specimens were measured by commercially available according to the manufacturers' protocols. RESULTS: All the bone markers examined displayed no significative changes after training session. CONCLUSION: Short exercise is insufficient for modifying serum concentrations of these osteoimmunologic markers, as previously indicated for commonly used bone metabolism markers. Future studies will be conducted over an entire competition season in order to define a common profile of bone markers in rugby players.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor.

BACKGROUND AND PURPOSE: DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH: The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS: A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS: DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.

Iron status evaluation as a marker of postoperative joint infection: a pilot study.

We evaluated the effect of different inflammatory conditions on iron status and, as a consequence, the possible use of iron markers as indicators of infection in the diagnosis of postoperative prosthetic orthopaedic joint infections. The study population was consisted of 26 patients undergoing revision of total hip or total knee joint arthroplasty and subdivided into three groups according to the cause of prosthesis implant failure: 10 as having had previous infection (Group A), 10 patients were categorized as having infection (Group B); and the remaining 6 (Group C) as not having infection. These patients were assayed for mean corpuscular haemoglobin concentration (MCHC) and serum values of iron (Fe), ferritin (Fer), transferrin (Tf), soluble transferrin receptor (sTfR), and transferrin saturation (sat Tf). Septic patients display statistically significant lower serum iron concentration, higher sTfR and ferritin levels, lower, but not statistically significant, MCHC compared to non septic ones. Little differences were observed for Tf, sat Tf, tibc, TfR index, among the three groups of patients. Our study suggests that iron status parameters, in particular serum iron, ferritin, sTfR and TfR index, could be useful tools for the early detection and the diagnosis of orthopaedic prosthetic joint infections.

Platelet rich plasma therapy: inflammatory molecules involved in tissue healing.

Inflammation represents a fundamental aspect of the healing process. Besides their primary role in hemostasis, platelets play an active role in the immunological and inflammatory aspect of tissue healing. Indeed , they can be directly involved in the inflammatory response by the production and release of several inflammatory mediators, including a variety of cytokines, such as TGF-beta, IL-1 beta, CD40L, and chemokines, such as CXCL7, CXCL4, CXCL4L1, CCl5, CXCL1, CXCL8, CXCL5, CXCL12, CCL2, CCL3. Platelet are not only a source of several chemokine involved in the inflammatory response and tissue healing, but they also express chemokine receptors, in particular CCR1 CCR3 CCR4 and CXCR4, thus being able to being able to regulate the inflammatory response associated to the healing process. However, this local inflammation must be taken under control, and platelets can prevent the excess of leukocytes recruitment by anti-inflammatory cytokines, such as TGF-beta. For this biological properties of platelets, platelet rich plasma therapy (PRP) is considered an innovative and promising approach that has been extended to many field of medicine, ranging from non-union defects, bone fractures, spinal fusion, bone implant and osteointegration, joint arthroplasty, to the treatment of several traumatic or degenerative pathologies of tendons, cartilage and ligaments.

High articular levels of the angiogenetic factors VEGF and VEGF-receptor 2 as tissue healing biomarkers after single bundle anterior cruciate ligament reconstruction.

Various factors may account for the positive association between meniscal repair and anterior cruciate ligament reconstruction, one being the modulation of healing response of meniscal fibrochondrocytes by growth factors released with intra-articular bleeding and fibrin clot formation. Analysis of vascular endothelial growth factor (VEGF) and its receptors, VEGFR1 and VEGFR2, may be useful in the clinical assessment of bone and soft-tissue remodeling. We measured systemic and local levels of VEGF (VEGF165), VEGFR1 and VEGFR2 after either arthroscopic partial meniscectomy (APM) or single-bundle anterior cruciate ligament reconstruction (ACLR) in order to determine the local effect of bone tunnelling and notchplasty on the release of these growth factors. The study population included 40 patients: 20 consecutive patients had undergone ACLR with hamstring grafts and 20 had undergone APM. Thirty minutes after the end of the operation, knee joint fluid samples were collected via the drainage tube and at the same time venous blood samples were drawn. In both sets of samples, VEGF, VEGFR1 and VEGFR2 concentrations were determined by enzyme-linked immunosorbent assay (ELISA). No significant differences in VEGF, VEGFR1 or VEGFR2 concentrations in the venous blood were observed between the two treatment groups. In contrast, VEGF and VEGFR2 levels were significantly higher in the knee joint fluid of the ACLR group; furthermore, VEGF and VEGFR1 were significantly higher in the knee joint fluid than in the venous blood, whereas VEGFR2 was lower in the knee joint fluid than in the venous blood. Local release of VEGF and its angiogenetic receptor VEGFR2, but not the negative regulator VEGFR1, was significantly higher after ACLR than after APM, indicating a better vasculogenic potential for enhanced bone-graft and meniscus healing. These results could suggest that VEGF and VEGFRs could be considered as good biomarkers of tissue healing after knee joint surgery.

Matrix metalloproteases MMP-2 and MMP-9: are they early biomarkers of bone remodelling and healing after arthroscopic acromioplasty?

Arthroscopic acromioplasty, one of the most frequent procedures in shoulder surgery, can promote tissue healing process by the release of growth/angiogenic factors from the acromion. Matrix metalloproteinases MMP-2 and MMP-9 are involved in such process. The purpose of this study was to measure MMP-2 and MMP-9 levels in the articular fluid and in the peripheral blood of patients undergoing arthroscopic acromioplasty in order to better understand the local involvement of such factors in the healing process after surgical procedures. Concentrations of MMP-2 and MMP-9 in the subacromial space and peripheral blood collected shortly after surgery were determined by ELISA. MMP-2 and MMP-9 concentrations were measured in the subacromial fluid of 23 patients. In subacromial fluid, the levels between MMP-2 and MMP-9 did not reach statistical significance (127.15±45.56 vs 149.41±53.61 pg/ml, respectively, p>0.05). Peripheral blood levels of MMP-2 (130.75±47.48 pg/ml) were comparable to the subacromial fluid ones (127.15±45.56 pg/ml) whereas MMP-9 level was higher in the subacromial space (149.41±53.61 pg/ml) than in the peripheral blood (67.61±12.62 pg/ml, p<0.001). This work suggests that the measurement of bone specific MMPs (MMP-2 and MMP-9) can be an useful tool to be monitored in parallel with growth factor levels and other bone turnover markers in order to evaluate the bone remodelling and tissue healing processes. This study suggests that the measurement of bone specific MMPs levels, in particular MMP-9, may evaluate the bone remodelling and healing after arthroscopic shoulder acromioplasty.

Molecular aspects of adipokine-bone interactions.

Adipose tissue is an endocrine organ able to produce a wide series of pleiotropic molecules, defined "adipokines". In addition to the regulation of food intake and energy metabolism, adipokines are also implicated in the complex control of bone biology and specifically of bone remodeling. Leptin, the most studied adipokine, promotes satiety and energy expenditure and its circulating levels are proportional to fat mass. Some paradoxical findings originally suggested the involvement of leptin in controlling bone mass. For example, obese postmenopausal women, with elevated circulating leptin and leptin resistance, appear protected against the development of osteoporosis. Moreover, genetically leptin-deficient mice, which are hypogonadal and obese, display a decreased trabecular volume in long bones, but an increased vertebral bone mass, which is reduced by leptin administration. The complex mechanisms of leptin regulation of bone mass appear to involve selected hypothalamic neuronal populations and the sympathetic outflow, with an important role of osteoblastic beta2-adrenergic receptors. Adiponectin is another adipokine, which promotes insulin sensitivity and is reduced in obese and diabetic subjects. Adiponectin appears to exert a negative effect on bone mass and seems to be an independent predictor of lower bone mass. Although the adipokines resistin and visfatin do not seem to significantly affect bone metabolism, the potential impact of them and other adipokines is still to be determined. Moreover, the molecular adipokine-bone interactions should also be considered in the context of the adipokine changes observed in diseases such as obesity and the metabolic syndrome.

Okadaic acid induces apoptosis in Down syndrome fibroblasts.

Down's syndrome (DS) is characterized by several pathological aspects leading to an increased susceptibility to cardiovascular diseases, infections, leukemia, endocrine alterations. DS patients display some of the physiopathological characteristics of aging, observed also in Alzheimer disease (AD), such as abnormalities in lipids metabolism, diabetes, high cholesterol fraction, senile plaques and neurofibrillary tangles. For this reason DS is considered a precocious and accelerated model of senescence, in which increased apoptosis is the main cornerstone. In order to better understand the apoptotic process in pathological cellular aspects of DS, the aim of this study was to investigate the apoptotic response of DS fibroblasts to OA, a toxin that induces malformations and inhibits growth in different cell lines. We focused specifically on the mitochondrial response by investigating changes in mitochondrial membrane potential (evaluate by flow cytometry and fluorescence microscopy using JC-1 probe) and alterations of mitochondrial outer membrane (evaluated by flow cytometry using annexin V/propidium iodide). Results indicates that DS Fibroblasts have a baseline of apoptosis higher than normal fibroblasts and are more susceptible to the pro-apoptotic effect of OA. Understanding the mechanism of apoptosis in DS fibroblasts could provide new insight in the pathogenic mechanism of this pathology and suggest potential therapeutical targets to the clinical treatment at complex diseases associated to this pathology.

Leptin, ciliary neurotrophic factor, leukemia inhibitory factor and interleukin-6: class-I cytokines involved in the neuroendocrine regulation of the reproductive function.

Class-I cytokines represent a large group of molecules involved in different physiological processes including host defence, immune regulation, food intake, energy metabolism and, relevant for this review, reproduction. In this latter respect, here, we focus the attention on four of these molecules, specifically leptin, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). These cytokines present similar three-dimensional fold structure, interact with related class-I receptors, which are expressed in the same regions (i.e., hypothalamus), and activate similar intracellular pathways. Leptin and CNTF share functional similarities, by acting at hypothalamic and pituitary levels, and their receptors are colocalized in the arcuate and paraventricular nuclei of the hypothalamus. For both these molecules, no effect on GnRH migration has been described. LIF has also been shown to affect gonadotropin secretion and here we present the novel observation that it is also able to stimulate GnRH secretion in vitro. Moreover, in the mouse, LIF is prenatally expressed in nasal regions where GnRH neurons originate and start their migration, and in vitro it stimulates intrinsic cell motility and directional migration. The role of the prototypical cytokine, IL-6, on the GnRH-LH axis is not fully clear and additional information seem necessary to better clarify this aspect. In conclusion, the data here discussed suggest that this family of cytokines appears to participate to the complex control of the reproductive function by affecting the development and function of the hypothalamus-pituitary system at different ontogenic times and anatomical sites.

Chemokines and bone remodeling.

Bone remodeling is characterized by spatial and temporal coupling of bone resorption and formation and is necessary for skeletal growth and normal bone structure maintenance. Imbalance of this process is related to metabolic bone disorders such as osteoporosis or rheumatoid arthritis. For this reason, bone remodeling is under the control of several local and systemic factors, including molecules of the immune system. The importance of the interplay of both the skeletal and immune systems is reflected by the emerging interdisciplinary research field, called osteoimmunology, focused on common aspects of osteology and immunology. This review focuses on the role of inflammatory mediators, such as cytokines in bone remodeling and, in particular, a subfamily of chemotactic cytokines or chemokines which are involved not only in several aspects of physiological bone remodeling but also in pathological bone disorders, such as rheumatoid arthritis or osteoporosis. Understanding the role of inflammation and chemokines will provide new insights for the treatment of diseases affecting both skeletal and immune systems, by the development of new therapeutic strategies targeting common inflammatory mediators.

Aplidine, a new anticancer agent of marine origin, inhibits vascular endothelial growth factor (VEGF) secretion and blocks VEGF-VEGFR-1 (flt-1) autocrine loop in human leukemia cells MOLT-4.

The mechanism by which aplidine, a marine natural product in early clinical development as an anticancer agent, induces cell growth inhibition and apoptosis has been investigated in the human leukemia cell line MOLT-4. This cell line is characterized not only by the ability to secrete VEGF, but also for the presence on its surface of the VEGF receptor-1 (VEGFR-1). Previous studies from our laboratory concerned with evaluating early changes in gene expression induced by aplidine in MOLT-4 cells have shown that the drug decreases the expression of VEGFR-1 (Marchini et al. Proc Am Assoc Cancer Res 2000; 41: 833). Here, we report the ability of aplidine to block the VEGF/VEGFR-1 loop. We found that aplidine blocked VEGF secretion that was temporally followed by a decrease in both VEGF and VEGFR-1 production. Aplidine did not directly affect either VEGF transcription or stabilization of its mRNA. Transfection of MOLT-4 cells with an antisense VEGF cDNA construct, resulted in inhibition of colony formations. One clone, transfected with sense VEGF cDNA, secreting 8-10 times more VEGF than parental cells, was less sensitive to aplidine-induced cytotoxicity and apoptosis than control cells. Moreover, addition of VEGF in the medium decreased the activity of aplidine in MOLT-4 cells. These data demonstrate that aplidine inhibits the growth and induces apoptosis in MOLT-4 cells through the inhibition of VEGF secretion which blocks the VEGF/VEGFR-1 autocrine loop necessary for the growth of these cells.

P73a overexpression is associated with resistance to treatment with DNA-damaging agents in a human ovarian cancer cell line.

We examined the consequences of p73alpha overexpression on gene expression and cellular response to anticancer agents in clones from the human ovarian cancer cell line A2780. Using microarray filters, the expression of 588 genes in two clones overexpressing p73alpha (A2780/p73.4 and A2780/ p73.5) in comparison with empty vector-transfected cells was evaluated. There were clear differences in gene expression profiles. Both of the clones showed a marked increase in the expression of genes involved in DNA repair, including genes participating in nucleotide excision repair and mismatch repair. This was confirmed by reverse transcription-PCR and Northern blot analysis and was associated with an increase in the ability of p73alpha-expressing clones to repair two different DDP (cis-dichlorodiammine platinum)-damaged plasmids in a host reactivation assay. p73alpha overexpressing clones were less sensitive than parental cells to alkylating agents treatment or UV radiation but equally sensitive to the topoisomerase I inhibitor topotecan, which indicated that the increase in expression of DNA repair genes has implications for the response to DNA damaging agents.