Methotrexate Chemotherapy Causes Growth Impairments, Vitamin D Deficiency, Bone Loss, and Altered Intestinal Metabolism-Effects of Calcitriol Supplementation.

Vitamin D deficiency or insufficiency is prevalent in childhood cancer patients and survivors after chemotherapy; further studies are needed to investigate the underlying aetiology and effectiveness of vitamin D supplementation in preventing chemotherapy-induced bone loss. This study used a rat model of treatment with antimetabolite methotrexate to investigate whether methotrexate chemotherapy causes vitamin D deficiency and if vitamin D supplementation attenuates the resultant bone loss. Methotrexate treatment (five daily injections) decreased serum vitamin D levels (from 52 to <30 ng/mL), reduced body and bone lengthening and tibial trabecular bone volume, and altered intestinal vitamin D metabolism, which was associated with intestinal mucosal damage known to cause malabsorption of nutrients, including dietary vitamin D and calcium. During the early stage after chemotherapy, mRNA expression increased for vitamin D activation enzyme CYP27B1 and for calcium-binding protein TRPV6 in the intestine. During the intestinal healing stage, expression of vitamin D catabolism enzyme CYP24 increased, and that of TRPV6 was normalised. Furthermore, subcutaneous calcitriol supplementation diminished methotrexate-induced bone loss due to its effect suppressing methotrexate-induced increased bone resorption. Thus, in young rats, methotrexate chemotherapy causes vitamin D deficiency, growth impairments, bone loss, and altered intestinal vitamin D metabolism, which are associated with intestinal damage, and vitamin D supplementation inhibits methotrexate-induced bone loss.

Therapeutic Targeting Notch2 Protects Bone Micro-Vasculatures from Methotrexate Chemotherapy-Induced Adverse Effects in Rats.

Intensive cancer chemotherapy is well known to cause bone vasculature disfunction and damage, but the mechanism is poorly understood and there is a lack of treatment. Using a rat model of methotrexate (MTX) chemotherapy (five once-daily dosses at 0.75 mg/kg), this study investigated the roles of the Notch2 signalling pathway in MTX chemotherapy-induced bone micro-vasculature impairment. Gene expression, histological and micro-computed tomography (micro-CT) analyses revealed that MTX-induced micro-vasculature dilation and regression is associated with the induction of Notch2 activity in endothelial cells and increased production of inflammatory cytokine tumour necrosis factor alpha (TNFα) from osteoblasts (bone forming cells) and bone marrow cells. Blockade of Notch2 by a neutralising antibody ameliorated MTX adverse effects on bone micro-vasculature, both directly by supressing Notch2 signalling in endothelial cells and indirectly via reducing TNFα production. Furthermore, in vitro studies using rat bone marrow-derived endothelial cell revealed that MTX treatment induces Notch2/Hey1 pathway and negatively affects their ability in migration and tube formation, and Notch2 blockade can partially protect endothelial cell functions from MTX damage.

Notch2 Blockade Mitigates Methotrexate Chemotherapy-Induced Bone Loss and Marrow Adiposity.

Childhood cancer methotrexate (MTX) chemotherapy often causes bone growth impairments, bone loss, and increased risks of fractures during or after treatment, for which the pathobiology is unclear and there is a lack of specific treatment. Our time course analyses of long bones from rats receiving intensive MTX treatment (mimicking a clinical protocol) found decreased trabecular bone volume, increased osteoclast formation and activity, increased adipogenesis in the expense of osteogenesis from the bone marrow stromal cells at days 6 and 9 following the first of five daily MTX doses. For exploring potential mechanisms, PCR array expression of 91 key factors regulating bone homeostasis was screened with the bone samples, which revealed MTX treatment-induced upregulation of Notch receptor NOTCH2, activation of which is known to be critical in skeletal development and bone homeostasis. Consistently, increased Notch2 activation in bones of MTX-treated rats was confirmed, accompanied by increased expression of Notch2 intracellular domain protein and Notch target genes HEY1, HES1 and HEYL. To confirm the roles of Notch2 signalling, a neutralising anti-Notch2 antibody or a control IgG was administered to rats during MTX treatment. Microcomputed tomography analyses demonstrated that trabecular bone volume was preserved by MTX+anti-Notch2 antibody treatment. Anti-Notch2 antibody treatment ameliorated MTX treatment-induced increases in osteoclast density and NFATc1 and RANKL expression, and attenuated MTX-induced bone marrow adiposity via regulating Wnt/ß-catenin signalling and PPARγ expression. Thus, Notch2 signalling plays an important role in mediating MTX treatment-induced bone loss and bone marrow adiposity, and targeting Notch2 could be a potential therapeutic option.

Methotrexate treatment suppresses osteoblastic differentiation by inducing Notch2 signaling and blockade of Notch2 rescues osteogenesis by preserving Wnt/ß-catenin signaling.

Methotrexate (MTX) is a commonly used antimetabolite in cancer treatment. Its intensive use is linked with skeletal adverse effects such as reduced bone formation and bone loss, and yet little information is available on molecular mechanisms underlying MTX-induced impaired bone formation. This study investigated the effects of MTX treatment at a clinical chemotherapy relevant dose on osteogenic differentiation in MC3T3E1 osteoblastic cells. To investigate the potential mechanisms, the expression of 87 genes regulating osteoblast differentiation and bone homeostasis was screened in MTX-treated versus untreated cells by polymerase chain reaction (PCR) arrays and results illustrated significant upregulation of Notch2 and Notch target genes at both early and late stages of MC3T3E1 differentiation following MTX treatment. To confirm the roles of Notch2 pathway and its potential action mechanisms, MC3T3E1 cells were treated with MTX with an anti-Notch2 neutralizing antibody or control IgG and effects were examined on osteogenesis and activation of the Wnt/ß-catenin pathway. Our results demonstrated that induction of Notch2 activity is associated with MTX adverse effects on osteogenic differentiation and blocking Notch2 rescues osteoblast differentiation by preserving activation of the Wnt/ß-catenin pathway.

miR-6315 Attenuates Methotrexate Treatment-Induced Decreased Osteogenesis and Increased Adipogenesis Potentially through Modulating TGF-ß/Smad2 Signalling.

Methotrexate (MTX) treatment for childhood malignancies has shown decreased osteogenesis and increased adipogenesis in bone marrow stromal cells (BMSCs), leading to bone loss and bone marrow adiposity, for which the molecular mechanisms are not fully understood. Currently, microRNAs (miRNAs) are emerging as vital mediators involved in bone/bone marrow fat homeostasis and our previous studies have demonstrated that miR-6315 was upregulated in bones of MTX-treated rats, which might be associated with bone/fat imbalance by directly targeting Smad2. However, the underlying mechanisms by which miR-6315 regulates osteogenic and adipogenic differentiation require more investigations. Herein, we further explored and elucidated the regulatory roles of miR-6315 in osteogenesis and adipogenesis using in vitro cell models. We found that miR-6315 promotes osteogenic differentiation and it alleviates MTX-induced increased adipogenesis. Furthermore, our results suggest that the involvement of miR-6315 in osteogenesis/adipogenesis regulation might be partially through modulating the TGF-ß/Smad2 signalling pathway. Our findings indicated that miR-6315 may be important in regulating osteogenesis and adipogenesis and might be a therapeutic target for preventing/attenuating MTX treatment-associated bone loss and marrow adiposity.

Gentiopicroside promotes the osteogenesis of bone mesenchymal stem cells by modulation of ß-catenin-BMP2 signalling pathway.

Osteoporosis is characterized by increased bone fragility, and the drugs used at present to treat osteoporosis can cause adverse reactions. Gentiopicroside (GEN), a class of natural compounds with numerous biological activities such as anti-resorptive properties and protective effects against bone loss. Therefore, the aim of this work was to explore the effect of GEN on bone mesenchymal stem cells (BMSCs) osteogenesis for a potential osteoporosis therapy. In vitro, BMSCs were exposed to GEN at different doses for 2 weeks, whereas in vivo, ovariectomized osteoporosis was established in mice and the therapeutic effect of GEN was evaluated for 3 months. Our results in vitro showed that GEN promoted the activity of alkaline phosphatase, increased the calcified nodules in BMSCs and up-regulated the osteogenic factors (Runx2, OSX, OCN, OPN and BMP2). In vivo, GEN promoted the expression of Runx2, OCN and BMP2, increased the level of osteogenic parameters, and accelerated the osteogenesis of BMSCs by activating the BMP pathway and Wnt/ß-catenin pathway, effect that was inhibited using the BMP inhibitor Noggin and Wnt/ß-catenin inhibitor DKK1. Silencing the ß-catenin gene and BMP2 gene blocked the osteogenic differentiation induced by GEN in BMSCs. This block was also observed when only ß-catenin was silenced, although the knockout of BMP2 did not affect ß-catenin expression induced by GEN. Therefore, GEN promotes BMSC osteogenesis by regulating ß-catenin-BMP signalling, providing a novel strategy in the treatment of osteoporosis.

miR-542-3p Attenuates Bone Loss and Marrow Adiposity Following Methotrexate Treatment by Targeting sFRP-1 and Smurf2.

Intensive methotrexate (MTX) treatment for childhood malignancies decreases osteogenesis but increases adipogenesis from the bone marrow stromal cells (BMSCs), resulting in bone loss and bone marrow adiposity. However, the underlying mechanisms are unclear. While microRNAs (miRNAs) have emerged as bone homeostasis regulators and miR-542-3p was recently shown to regulate osteogenesis in a bone loss context, the role of miR-542-3p in regulating osteogenesis and adipogenesis balance is not clear. Herein, in a rat MTX treatment-induced bone loss model, miR-542-3p was found significantly downregulated during the period of bone loss and marrow adiposity. Following target prediction, network construction, and functional annotation/ enrichment analyses, luciferase assays confirmed sFRP-1 and Smurf2 as the direct targets of miR-542-3p. miRNA-542-3p overexpression suppressed sFRP-1 and Smurf2 expression post-transcriptionally. Using in vitro models, miR-542-3p treatment stimulated osteogenesis but attenuated adipogenesis following MTX treatment. Subsequent signalling analyses revealed that miR-542-3p influences Wnt/ß-catenin and TGF-ß signalling pathways in osteoblastic cells. Our findings suggest that MTX treatment-induced bone loss and marrow adiposity could be molecularly linked to miR-542-3p pathways. Our results also indicate that miR-542-3p might be a therapeutic target for preserving bone and attenuating marrow fat formation during/after MTX chemotherapy.

Chemotherapy-Induced Intestinal Microbiota Dysbiosis Impairs Mucosal Homeostasis by Modulating Toll-like Receptor Signaling Pathways.

Chemotherapy-induced intestinal mucositis, a painful debilitating condition affecting up to 40-100% of patients undergoing chemotherapy, can reduce the patients' quality of life, add health care costs and even postpone cancer treatment. In recent years, the relationships between intestinal microbiota dysbiosis and mucositis have drawn much attention in mucositis research. Chemotherapy can shape intestinal microbiota, which, in turn, can aggravate the mucositis through toll-like receptor (TLR) signaling pathways, leading to an increased expression of inflammatory mediators and elevated epithelial cell apoptosis but decreased epithelial cell differentiation and mucosal regeneration. This review summarizes relevant studies related to the relationships of mucositis with chemotherapy regimens, microbiota, TLRs, inflammatory mediators, and intestinal homeostasis, aiming to explore how gut microbiota affects the pathogenesis of mucositis and provides potential new strategies for mucositis alleviation and treatment and development of new therapies.

Roles of MicroRNAs in Osteogenesis or Adipogenesis Differentiation of Bone Marrow Stromal Progenitor Cells.

Bone marrow stromal cells (BMSCs) are multipotent cells which can differentiate into chondrocytes, osteoblasts, and fat cells. Under pathological stress, reduced bone formation in favour of fat formation in the bone marrow has been observed through a switch in the differentiation of BMSCs. The bone/fat switch causes bone growth defects and disordered bone metabolism in bone marrow, for which the mechanisms remain unclear, and treatments are lacking. Studies suggest that small non-coding RNAs (microRNAs) could participate in regulating BMSC differentiation by disrupting the post-transcription of target genes, leading to bone/fat formation changes. This review presents an emerging concept of microRNA regulation in the bone/fat formation switch in bone marrow, the evidence for which is assembled mainly from in vivo and in vitro human or animal models. Characterization of changes to microRNAs reveals novel networks that mediate signalling and factors in regulating bone/fat switch and homeostasis. Recent advances in our understanding of microRNAs in their control in BMSC differentiation have provided valuable insights into underlying mechanisms and may have significant potential in development of new therapeutics.

The frequency window effect of sinusoidal electromagnetic fields in promoting osteogenic differentiation and bone formation involves extension of osteoblastic primary cilia and activation of protein kinase A.

Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here, we optimized the frequency parameter first by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50, and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content, and osteogenesis-related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50, or 100 Hz for 2 months significantly increased whole-body bone mineral density (BMD) and femur microarchitecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency-dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation.

Roles of apoptotic chondrocyte-derived CXCL12 in the enhanced chondroclast recruitment following methotrexate and/or dexamethasone treatment.

Intensive use of methotrexate (MTX) and/or dexamethasone (DEX) for treating childhood malignancies is known to cause chondrocyte apoptosis and growth plate dysfunction leading to bone growth impairments. However, mechanisms remain vague and it is unclear whether MTX and DEX combination treatment could have additive effects in the growth plate defects. In this study, significant cell apoptosis was induced in mature ATDC5 chondrocytes after treatment for 48 h with 10-5 M MTX and/or 10-6 M DEX treatment. PCR array assays with treated cells plus messenger RNA and protein expression confirmation analyses identified chemokine CXCL12 having the most prominent induction in each treatment group. Conditioned medium from treated chondrocytes stimulated migration of RAW264.7 osteoclast precursor cells and formation of osteoclasts, and these stimulating effects were inhibited by the neutralizing antibody for CXCL12. Additionally, while MTX and DEX combination treatment showed some additive effects on apoptosis induction, it did not have additive or counteractive effects on CXCL12 expression and its functions in enhancing osteoclastic recruitment and formation. In young rats treated acutely with MTX, there was increased expression of CXCL12 in the tibial growth plate, and more resorbing chondroclasts were found present at the border between the hypertrophic growth plate and metaphysis bone. Thus, the present study showed an association between induced chondrocyte apoptosis and stimulated osteoclastic migration and formation following MTX and/or DEX treatment, which could be potentially or at least partially linked molecularly by CXCL12 induction. This finding may contribute to an enhanced mechanistic understanding of bone growth impairments following MTX and/or DEX therapy.

The immuno-reactivity of polypseudorotaxane functionalized magnetic CDMNP-PEG-CD nanoparticles.

pH-magnetic dual-responsive nanocomposites have been widely used in drug delivery and gene therapy. Recently, a polypseudorotaxane functionalized magnetic nanoparticle (MNP) was developed by synthesizing the magnetic nanoparticles with cyclodextrin (CD) molecules (CDMNP) via polyethylene glycol (PEG) (CDMNP-PEG-CD). The purpose of this study was to explore the antigenicity and immunogenicity of the nanoparticles in vivo prior to their further application explorations. Here, nanoparticles were assessed in vivo for retention, bio-distribution and immuno-reactivity. The results showed that, once administered intravenously, CDMNP-PEG-CD induced a temporary blood monocyte response and was cleared effectively from the body through the urine system in mice. The introduction of ß-CD and PEG/ß-CD polypseudorotaxane on SiO2 magnetic nanoparticles (SOMNP) limited particle intramuscular dispersion after being injected into mouse gastrocnemius muscle (GN), which led to the prolonged local inflammation and muscle toxicity by CDMNP and CDMNP-PEG-CD. In addition, T cells were found to be more susceptible for ß-CD-modified CDMNP; however, polypseudorotaxane modification partially attenuated ß-CD-induced T cell response in the implanted muscle. Our results suggested that CDMNP-PEG-CD nanoparticles or the decomposition components have potential to prime antigen-presenting cells and to break the muscle autoimmune tolerance.

ß-Catenin signaling is important for osteogenesis and hematopoiesis recovery following methotrexate chemotherapy in rats.

Cancer chemotherapy can significantly impair the bone formation and cause myelosuppression; however, their recovery potentials and mechanisms remain unclear. This study investigated the roles of the ß-catenin signaling pathway in bone and bone marrow recovery potentials in rats treated with antimetabolite methotrexate (MTX) (five once-daily injections, 0.75 mg/kg) with/without ß-catenin inhibitor indocyanine green (ICG)-001 (oral, 200 mg/kg/day). ICG alone reduced trabecular bone volume and bone marrow cellularity. In MTX-treated rats, ICG suppressed bone volume recovery on Day 11 after the first MTX injection. ICG exacerbated MTX-induced decreases on Day 9 osteoblast numbers on bone surfaces, their formation in vitro from bone marrow stromal cells (osteogenic differentiation/mineralization), as well as expression of osteogenesis-related markers Runx2, Osx, and OCN in bone, and it suppressed their subsequent recoveries on Day 11. On the other hand, ICG did not affect MTX-induced increased osteoclast density and the level of the osteoclastogenic signal (RANKL/OPG expression ratio) in bone, suggesting that ICG inhibition of ß-catenin does nothing to abate the increased bone resorption induced by MTX. ICG also attenuated bone marrow cellularity recovery on Day 11, which was associated with the suppressed recovery of CD34+ or c-Kit+  hematopoietic progenitor cell contents. Thus, ß-catenin signaling is important for osteogenesis and hematopoiesis recoveries following MTX chemotherapy.

Regular Supplementation With Resveratrol Improves Bone Mineral Density in Postmenopausal Women: A Randomized, Placebo-Controlled Trial.

Resveratrol, a naturally occurring polyphenol in red grapes and berries, can act as a phytoestrogen. It has been shown to improve both systemic and cerebral circulatory functions, possibly through activation of endothelial estrogen receptors. in vitro and in vivo studies in rodent models also indicate a bone-protective role for resveratrol, particularly in ovariectomized rat models that mimic postmenopausal osteoporosis caused by estrogen deficiency. Hypothesizing a circulatory benefit of resveratrol in bone tissue, we investigated whether resveratrol supplementation could improve bone health in postmenopausal women. The Resveratrol for Healthy Aging in Women (RESHAW) trial was a 24-month randomized, double-blind, placebo-controlled, two-period crossover intervention conducted to evaluate the effects of resveratrol (75 mg twice daily) on cognition, cerebrovascular function, bone health, cardiometabolic markers, and well-being in postmenopausal women. After 12 months of supplementation with resveratrol versus placebo, there were positive effects on bone density in the lumbar spine (+0.016 ± 0.003 g/cm2 ) and neck of femur (+0.005 ± 0.002 g/cm2 ), which were accompanied by a 7.24% reduction in C-terminal telopeptide type-1 collagen levels, a bone resorption marker, compared with placebo. The increase in bone mineral density in the femoral neck resulted in an improvement in T-score (+0.070 ± 0.018) and a reduction in the 10-year probability of major and hip fracture risk. The magnitude of improvement was higher in women with poor bone health biomarker status. Importantly, the improvement in femoral neck T-score with resveratrol correlated with improvement in perfusion. Our subanalysis also revealed that the bone-protective benefit of resveratrol was greater in participants who supplemented with vitamin D plus calcium. Regular supplementation with 75 mg of resveratrol twice daily has the potential to slow bone loss in the lumbar spine and femoral neck, common fracture sites in postmenopausal women without overt osteoporosis. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Bmi deficiency causes oxidative stress and intervertebral disc degeneration which can be alleviated by antioxidant treatment.

The transcriptional repressor Bmi-1 is involved in cell-cycle regulation and cell senescence, the deficiency of which has been shown to cause oxidative stress. This study investigated whether Bmi-1 deficiency plays a role in promoting disc degeneration and the effect of treatment with antioxidant N-acetylcysteine (NAC) on intervertebral disc degeneration. Bmi-1-/- mice were treated with the antioxidant NAC, supplied in drinking water (Bmi-1-/- +NAC). For in vitro experiments, mouse intervertebral discs were cultured under low oxygen tension and serum-limiting conditions in the presence of tumour necrosis factor α and interleukin 1ß in order to mimic degenerative insult. Disc metabolism parameters in these in vitro and in vivo studies were evaluated by histopathological, immunohistochemical and molecular methods. Bmi-1-/- mice showed lower collagen Ⅱ and aggrecan levels and higher collagen Ⅹ levels than wild-type and Bmi-1-/- +NAC mice. Bmi-1-/- mice showed significantly lower superoxide dismutase (SOD)-1, SOD-2, glutathione peroxidase (GPX)-1 and GPX-3 levels than their wild-type littermates and Bmi-1-/- + NAC mice. Relative to Bmi-1-/- mice, the control and Bmi-1-/- +NAC mice showed significantly lower p16, p21, and p53 levels. These results demonstrate that Bmi-1 plays an important role in attenuating intervertebral disc degeneration in mice by inhibiting oxidative stress and cell apoptosis.

p16 deficiency attenuates intervertebral disc degeneration by adjusting oxidative stress and nucleus pulposus cell cycle.

The cell cycle regulator p16 is known as a biomarker and an effector of aging. However, its function in intervertebral disc degeneration (IVDD) is unclear. In this study, p16 expression levels were found to be positively correlated with the severity of human IVDD. In a mouse tail suspension (TS)-induced IVDD model, lumbar intervertebral disc height index and matrix protein expression levels were reduced significantly were largely rescued by p16 deletion. In TS mouse discs, reactive oxygen species levels, proportions of senescent cells, and the senescence-associated secretory phenotype (SASP) were all increased, cell cycling was delayed, and expression was downregulated for Sirt1, superoxide dismutase 1/2, cyclin-dependent kinases 4/6, phosphorylated retinoblastoma protein, and transcription factor E2F1/2. However, these effects were rescued by p16 deletion. Our results demonstrate that p16 plays an important role in IVDD pathogenesis and that its deletion attenuates IVDD by promoting cell cycle and inhibiting SASP, cell senescence, and oxidative stress.

Neck and shoulder pain, lower back pain and leg numbness are conditions that many people will encounter as years go by. This is because intervertebral discs, the padding structures that fit between the bones in the spine, degenerate with age: their cells enter a 'senescent', inactive state, and stop multiplying. A protein known as p16, an important regulator of cell growth and division, is known to accumulate in senescent cells. In fact, in mouse fat tissue, muscles or eyes, removing the cells that contain high levels of p16 delays aging-associated disorders. However, it was still unknown whether deactivating the gene that codes p16 in senescent cells could delay disc degeneration. Here, Che, Li et al. discovered that p16 is highly present in the senescent cells of severely degenerated human intervertebral discs. The cells in the nucleus pulposus, the jelly-like and most critical tissue in the intervertebral discs, were extracted and grown in the lab under conditions that replicate the early stages of damage to the spine. Drugs and genetic manipulations were then used to decrease the amount of p16 in these cells. The experiments showed that reducing the levels of p16 results in the senescent cells multiplying more and showing fewer signs of damage and aging. In addition, the discs of mice in which the gene that codes for p16 had been deleted were less prone to degeneration compared to 'normal' mice in similar conditions. Overall, the work by Che, Li et al. shows that inhibiting p16 in disc cells delays the aging process and reduces the degeneration of intervertebral discs. These findings may one day be applicable to people with intervertebral disc diseases who, for example, could potentially benefit from a gene therapy targeting the cells which produce p16.

Hydrocortisone Suppresses Early Paraneoplastic Inflammation And Angiogenesis To Attenuate Early Hepatocellular Carcinoma Progression In Rats.

BACKGROUND: Inflammation is implicated in both hepatic cirrhosis development and hepatocellular carcinogenesis, and treatment with long-acting glucocorticoid dexamethasone prevented liver carcinogenesis in mice. However, it is unclear whether glucocorticoids have anti-inflammatory effect on hepatocellular carcinoma (HCC) and if short-acting glucocorticoids (with fewer adverse effects) inhibit paraneoplastic inflammation and HCC progression. METHODS: To investigate whether different types of anti-inflammatory agents attenuate HCC progression, the current study compared effects of treatments with hydrocortisone (a short-acting glucocorticoid) or aspirin on HCC progression. HCC was induced in diethylnitrosamine-treated rats which were randomly divided into 4 groups (n=8), respectively receiving orally once daily vehicle, glucuronolactone, glucuronolactone+hydrocortisone, and glucuronolactone+aspirin. Diethylnitrosamine (DEN) was given to rats in drinking water (100mg/L) to induce HCC. At weeks 12 and 16 post-induction, effects were compared on HCC nodule formation, microvessel density, and macrophage infiltration, and levels of paraneoplastic protein expression of tumor necrosis factor (TNF)-α, p38 mitogen-activated protein kinase (p38), phosphorylated p38 (p-p38), nuclear factor (NF)-κB, interleukin (IL)-10, hepatocyte growth factor (HGF), transforming growth factor (TGF)-ß1 and vascular endothelial growth factor (VEGF). RESULTS: Compared to the model and glucuronolactone alone groups, HCC nodule number and microvessel density in the glucuronolactone+hydrocortisone group were significantly lower at week 12. At week 12 but not week 16, significantly lower levels of macrophages, TNF-α, p-p38, NF-κB, IL-10, HGF, TGF-ß1 and VEGF were observed in the paraneoplastic tissue of the glucuronolactone+hydrocortisone group when compared with the control and glucuronolactone groups. CONCLUSION: The results suggest that hydrocortisone treatment reduces macrophage polarization, expression of inflammatory and anti-inflammatory cytokines, and angiogenesis in paraneoplastic tissue, and attenuates early HCC progression. Although hydrocortisone did not have attenuation effect on advanced solid tumor, the current study shows the potential benefits and supports potential clinical use of hydrocortisone in attenuating early progression of HCC, which is through suppressing paraneoplastic inflammation and angiogenesis.

Calmodulin-dependent signalling pathways are activated and mediate the acute inflammatory response of injured skeletal muscle.

KEY POINTS: There is a close relationship between skeletal muscle physiology and Ca2+ /calmodulin (CaM) signalling. Despite the effects of Ca2+ /CaM signalling on immune and inflammatory responses having been extensively explored, few studies have investigated the role of CaM pathway activation on the post-injury muscle inflammatory response. In this study, we investigated the role of CaM-dependent signalling in muscle inflammation in cardiotoxin induced myoinjuries in mice. The Ca2+ /calmodulin-dependent protein kinase II (CaMII), Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV), and nuclear factor of activated T cells (NFAT) pathways are likely to be simultaneously activated in muscle cells and in infiltrating lymphocytes and to regulate the immune behaviours of myofibres in an inflammatory environment, and these pathways ultimately affect the outcome of muscle inflammation. ABSTRACT: Calcium/calmodulin (Ca2+ /CaM) signalling is essential for immune and inflammatory responses in tissues. However, it is unclear if Ca2+ /CaM signalling interferes with muscle inflammation. Here we investigated the roles of CaM-dependent signalling in muscle inflammation in mice that had acute myoinjuries in the tibialis anterior muscle induced by intramuscular cardiotoxin (CTX) injections and received intraperitoneal injections of either the CaM inhibitor calmidazolium chloride (CCL) or CaM agonist calcium-like peptide 1 (CALP1). Multiple inflammatory parameters, including muscle autoantigens and toll-like receptors, mononuclear cell infiltration, cytokines and chemokines associated with peripheral muscle inflammation, were examined after the injury and treatment. CALP1 treatment enhanced intramuscular infiltration of monocytes/macrophages into the damaged tibialis anterior muscle and up-regulated mRNA and protein levels of muscle autoantigens (Mi-2, HARS and Ku70) and Toll-like receptor 3 (TLR3), and mRNA levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), Monocyte chemoattractant protein-1 (MCP1), Monocyte chemoattractant protein-3 (MCP3) and Macrophage inflammatory protein-1(MIP-1α) in damaged muscle. In contrast, CCL treatment decreased the intramuscular cell infiltration and mRNA levels of the inflammatory mediators. After CALP1 treatment, a substantial up-regulation in Ca2+ /calmodulin-dependent protein kinase II (CaMKII), Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV) and nuclear factor of activated T cells (NFAT) activity was detected in CD45+ cells isolated from the damaged muscle. More pro-inflammatory F4/80+ Ly-6C+ cells were detected in CD45-gated cells after CALP1 treatment than in those after CCL treatment or no treatment. Consistently, in interferon-γ-stimulated cultured myoblasts and myotubes, CALP1 treatment up-regulated the activities of CaMKII, CaMKIV and NFAT, and levels of class I/II major histocompatibility complexes (MHC-I/II) and TLR3. Our findings demonstrated that CaM-dependent signalling pathways mediate the injury-induced acute muscle inflammatory response.

Bone marrow sinusoidal endothelium as a facilitator/regulator of cell egress from the bone marrow.

Despite more attention to cell migration from circulation into the bone marrow (BM), particularly homing of haematopoietic stem/progenitor cells, the process and mechanisms of cell mobilisation from the BM into the circulation remain largely underexplored. The process of cell mobilisation or transcellular cell migration from BM into the circulation (cell egress) is a crucial biological process in mammals as it is important to maintain homeostasis of various physiological functions including, but not limited to, the immune system, erythropoiesis, platelet release, and stem cell migration. The BM microvascular system composes of a monolayer of specialized endothelial cells, called sinusoidal endothelial cells (SECs). While it is very well evident that the process of cell egress occurs exclusively through BM SECs, there is a lack of systematic analyses addressing the extent of contribution of BM SECs to the process of cell egress from the BM. Therefore, this review aims to address the potential ability of BM SECs in regulating and/or facilitating the process of cell egress from BM. In this review, we address, firstly, the unique ultra-/structural and molecular features of BM SECs and discuss the possible biological interactions between BM SECs and various egressing cells in physiological conditions. Secondly, we propose the potential role of BM SECs in egress of leukemic cells from BM into the circulation. Finally, we discuss the potential role of BM SECs in homing of haematopoietic stem cells. Collectively, the current review suggests that the BM SECs may not be merely a neutral gatekeeper for cell intravasation and extravasation, but rather is a dynamic trafficking surveillance system, thereby the process of BM cell egress/mobilisation can be regulated.

Retracted: Icariin attenuates methotrexate chemotherapy-induced bone marrow microvascular damage and bone loss in rats.

Methotrexate (MTX), a widely used antimetabolite in paediatric cancer to treatment, has been widely reported to cause bone loss and bone marrow (BM) microvascular (particularly sinusoids) damage. Investigations must now investigate how MTX-induced bone loss and microvasculature damage can be attenuated/prevented. In the present study, we examined the potency of icariin, an herbal flavonoid, in reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with five daily MTX injections (0.75 mg/kg) with and without icariin oral supplementation until Day 9 after the first MTX injection. Histological analyses showed a significant reduction in the bone volume/tissue volume (BV/TV) fraction (%) and trabecular number in the metaphysis trabecular bone of MTX-treated rats, but no significant changes in trabecular thickness and trabecular spacing. However, the BV/TV (%) and trabecular number were found to be significantly higher in MTX + icariin-treated rats than those of MTX alone-treated rats. Gene expression analyses showed that icariin treatment maintained expression of osteogenesis-related genes but suppressed the induction of adipogenesis-related genes in bones of MTX-treated rats. In addition, icariin treatment attenuated MTX-induced dilation of BM sinusoids and upregulated expression of endothelial cell marker CD31 in the metaphysis bone of icariin + MTX-treated rats. Furthermore, in vitro studies suggest that icariin treatment can potentially enhance the survival of cultured rat sinusoidal endothelial cells against cytotoxic effect of MTX and promote their migration and tube formation abilities, which is associated with enhanced production of nitric oxide.