BMP-2 Levels Research Articles

Regulatory expression of bone morphogenetic protein 6 by 2,2′-dipyridyl

BackgroundExpression of hepcidin, a hormone produced by hepatocytes which negatively regulates the circulating iron levels, is known to be positively regulated by BMP6, a member of transforming growth factor (TGF)-β family. Previous studies have shown that iron status is sensed by sinusoidal endothelial cells of hepatic lamina, leading to the modulation of BMP6 expression. MethodsISOS-1, HUVEC, F-2, and SK-HEP1 endothelial cells were treated with either iron or 2,2′-dipyridyl (2DP), a cell-permeable iron-chelator, and expression level of Bmp6 was examined. To identify factors affecting Bmp6 transcription, stimulus screening for regulator of transcription (SSRT) was developed. ResultsTreatment with iron slightly increased the expression levels of Bmp6, while 2DP unexpectedly increased Bmp6 expression in a dose-dependent manner. 2DP-induced Bmp6 expression was resistant to co-treatment with iron. 2DP-induced Bmp6 expression was also detected in HUVEC, F-2 cells, and SK-HEP1 cells. Luciferase-based reporter assays indicated that forced expression of JunB increased the transcription of Bmp6. 2DP induced phosphorylation of JunB; co-treatment with SP600125 blocked the 2DP-induced Bmp6 expression partially. JunB-induced Bmp6 transcription was not affected by mutations of putative JunB-responsive elements. Some endoplasmic reticulum stress inducers increased the expression of Bmp6. SSRT revealed pathways regulating Bmp6 transcription positively and negatively. Hepa1–6 liver cells and C2C12 myogenic cells were prone to 2DP induced Bmp6 expression. ConclusionsThe present study reveals non‑iron-regulated Bmp6 expression in endothelial cells. General significanceRegulatory expression of Bmp6 may be important as a key step for fine tuning of BMP activity.

Transcription of ribosomal rnas and translational capacity are regulated by BMP-7, via BAPX1/NKX3.2 in chondrocytic cells

Purpose: During osteoarthritis (OA) progression the articular chondrocyte undergoes a phenotypic switch in which the chondrocyte acquires a catabolic and hypertrophy-like state. Bone morphogenetic protein (BMP)-7 (or OP-1) is known for its anti-catabolic and pro-anabolic properties in OA chondrocytes, and induces a hypertrophy-suppressive cellular change in OA chondrocytes. It is expected that the protein translational capacity of the chondrocyte is increased after exposure to BMP-7 which might explain the pro-anabolic shift. The cellular availability of maturated ribosomal RNAs (rRNA) is rate-limiting in the assembly of ribosomes and thus the cells protein translational capacity. In addition, we have previously shown that the phenotypic changes by BMP-7 in chondrocytes are, at least partly, induced via bagpipe homeobox homolog 1 (BAPX1/NKX3.2). We therefore hypothesize that BMP-7 enhances the translational capacity of articular chondrocytes via BAPX1/NKX3.2-dependent synthesis of rRNAs. Methods: OA human articular chondrocytes (HACs) were isolated from OA cartilage following total knee arthroplasty (with ethical permission). SW1353 cells and OA HACs were exposed to BMP-7 (1 nM) and expression levels of rRNAs (18S, 5.8S, 28S), crucial co-factors in rRNA transcription (upstream binding factor 1 (UBF-1) and treacle ribosome biogenesis factor 1 (TCOF1)), as well as BAPX1/NKX3.2) were determined by RT-qPCR (and immunoblotting for BAPX1/NKX3.2). BAPX1/NKX3.2 overexpression and knockdown were achieved via transfection of a FLAG-BAPX1/NKX3.2 plasmid or a BAPX1/NKX3.2 siRNA duplex. Overall translational capacity following BMP-7 exposure, BAPX1/NKX3.2 overexpression or knockdown was directly assessed by SUNsET assays. Alterations in rRNA gene transcription were determined by transfecting SW1353 cells and OA HACs with a 47S promoter reporter plasmid (pNL1.2[NlucP]) to determine transcriptional activity of the 47S pre-rRNA gene. Results: Exposure to BMP-7 or BAPX1/NKX3.2 overexpression lead to increased overall protein translational capacity. BAPX1/NKX3.2 knockdown resulted in reduced translational capacity, which could not be restored by BMP-7 treatment. BMP-7 stimulation lead to increased 18S and 5.8S rRNA, as well as UBF-1 expression levels, which correlated with increased BAPX1/NKX3.2 mRNA and protein expression. Overexpression of BAPX1/NKX3.2 resulted in increased rRNA and UBF-1 expression levels, and reciprocally knockdown of BAPX1/NKX3.2 resulted in decreased rRNA and UBF-1 expression levels. Finally, BMP-7 exposure or overexpression of BAPX1/NKX3.2 resulted in increased 47S pre-rRNA promoter activity and BAPX1/NKX3.2 knockdown resulted in an antagonistic reduction of 47S pre-rRNA gene transcription. Conclusions: BMP-7 induces the overall protein translational capacity. This enhanced anabolic state is accompanied with increased cellular levels of maturated rRNAs and concomitant induction of factors involved in the transcription of rRNAs. In line with these results, 47S pre-rRNA transcription is increased after exposure to BMP-7 or BAPX1/NKX3.2 overexpression. Here we show that the anabolic shift seen by BMP-7 works via a BAPX1/NKX3.2 dependent manner. The functionality of increased transcriptional activity of the 47S pre-rRNA gene will be determined by quantification of ribosome levels after BMP-7 exposure. Our data provide important novel insight into the mechanism behind the anabolic properties of BMP-7 and may provide a new molecular cue to target the chondrocyte phenotype in OA.

CircPSM3 inhibits the proliferation and differentiation of OA chondrocytes by targeting miRNA-296-5p.

Osteoarthritis (OA) is a common chronic bone and joint disease. Circular RNA is a type of non-coding RNA that forms a circular structure with covalent bonds. There is growing evidence that circRNA can function as a functional RNA and play an important role in the occurrence and development of osteoarthritis chondrocytes. However, the exact role of circRNA on OA remains to be studied. Quantificational real-time polymerase chain reaction (qRT-PCR) was used to determine the expression levels of CircPSM3 and miRNA-296-5p in OA chondrocytes. Cell proliferation was detected by the Cell Counting Kit (CCK8), and BMP2, BMP4, BMP6 and RUN2 molecular levels in OA chondrocytes were detected by qRT-PCR and Western Blot (WB). Direct targets of CircPSM3 and miRNA-296-5p in OA chondrocytes were measured by Luciferase reporter assay. CircPSM3 expression was upregulated in OA cartilage tissue and cells. Low expression of CircPSM3 promoted the proliferation and cell differentiation of OA chondrocytes. Meanwhile, miRNA-296-5p was down-regulated in OA cartilage tissue and cells. The Luciferase reporter gene showed that CircPSM3 could target miRNA-296-5p. The expression level of CircPSM3 and miRNA-296-5p showed a negative correlation. Further research found that a high expression of miRNA-296-5p could effectively promote the proliferation and cell differentiation of OA chondrocytes. Furthermore, miRNA-296-5p inhibitors reversed the effect of si-CircPSM3 on the proliferation and differentiation of OA chondrocytes, while miRNA-296-5p inhibitors enhanced the effect of si-CircPSM3 on the proliferation and differentiation of OA chondrocytes. CircPSM3 was upregulated in OA chondrocytes. CircPSM3 participated in the proliferation and differentiation of OA chondrocytes through targeted binding to miRNA-296-5p. CircPSM3 may become a potential therapeutic target for osteoarthritis treatment.

Postnatal Conditional Deletion of Bmal1 in Osteoblasts Enhances Trabecular Bone Formation Via Increased BMP2 Signals.

A large number of studies in recent years indicated the involvement of peripheral circadian clock in varied pathologies. However, evidence regarding how peripheral clocks regulate bone metabolism is still very limited. The present study aimed to investigate the direct role of Bmal1 (the key activator of peripheral circadian clock system) in vivo during bone developmental and remodeling stages using inducible osteoblast-specific Bmal1 knockout mice. Unexpectedly, the removal of Bmal1 in osteoblasts caused multiple abnormalities of bone metabolism, including a progressive increase in trabecular bone mass in as early as 8 weeks, manifested by an 82.3% increase in bone mineral density and 2.8-fold increase in bone volume per tissue volume. As mice age, an increase in trabecular bone mass persists while cortical bone mass decreases by about 33.7%, concomitant with kyphoscoliosis and malformed intervertebral disk. The increased trabecular bone mass is attributed to increased osteoblast number and osteoblast activity coupled with decreased osteoclastogenesis. Remarkably, the ablation of Bmal1 in osteoblasts promoted the expression level of Bmp2 and phosphorylation of SMAD1, whereas the attenuation of BMP2/SMAD1 signaling partially alleviated the effects of Bmal1 deficiency on osteoblast differentiation and activity. The results revealed that Bmal1 was a transcriptional silencer of Bmp2 by targeting the Bmp2 promoter. The peripheral clock gene Bmal1 in osteoblasts was crucial to coordinate differential effects on trabecular and cortical bones through regulating BMP2/SMAD1 during bone development, thus providing novel insights into a key role of osteoblast Bmal1 in homeostasis and integrity of adult bones. © 2020 American Society for Bone and Mineral Research.

Can zaprinast and avanafil induce the levels of angiogenesis, bone morphogenic protein 2, 4 and 7 in kidney of ovariectomised rats?

Objective: This study investigated effects of zaprinast and avanafil on angiogenesis, vascular endothelial growth factor (VEGF), bone morphogenic protein (BMP) 2, 4 and 7.Methods: Female rats were randomly divided into four groups (n = 6). Sham; abdomen was approximately 2 cm opened and closed. Ovariectomised (OVX); abdomen was opened 2 cm and the ovaries were cut. OVX + zaprinast and OVX + avanafil groups; after the same procedure with OVX, 10 mg/kg zaprinast and avanafil were orally administered for 2 month, respectively. Angiogenesis and the levels of VEGF, BMP2, 4 and 7 were determined.Results: VEGF, BMP2, 4 and 7 levels in OVX + zaprinast and especially OVX + avanafil groups were higher than the sham and OVX (p < .05). However, only VEGF and BMP2 levels in OVX + zaprinast group were significant according to sham (p < .05). Also, angiogenesis in OVX + zaprinast and OVX + avanafil groups was dominant according to sham and OVX (p < .05).Conclusions: Zaprinast and avanafil induced BMP2, 4 and 7 levels synergistically with increased VEGF and angiogenesis in renal tissue.

The effect of trace elements on BMP-2, BMP-7 and STRO-1+ cells in hip replacement

To explore the correlation between the trace elements in the proximal femur and BMP-2, BMP-7 and STRO-1+ cells in hip replacement, and analyze the therapeutic effect of prosthesis loosening in clinic. Fifty-one patients undergone the first hip replacement in xxx hospital from August 2016 to August 2019 were selected as the study subjects, including 26 females and 25 males, aged 52–89 years. The bone marrow mesenchymal stem cells (BMSCs) were cultured in vitro for flow cytometry, and the string-1+ in BMSCs was detected and analyzed. After that, the expression of bone morphogenetic protein 2 (BMP-2) and bone morphogenetic protein 7 (BMP-7) in the cells were detected by enzyme-linked immunosorbent assay, the content of trace elements in the supernatant was detected by radioimmunoassay, and the collected data were analyzed statistically. In the analysis of the content of trace elements, it was found that the correlation between trace elements was dependent on the separation area, and all trace elements had no correlation with BMP2. Ca2+, Mg2+ were correlated with the level of BMP7 and Ca2+, VD3 was correlated with the percentage of STOR-1+ cells. Further analysis showed that the correlation between trace elements was dependent on bone mineral density (BMD) area, and there was a positive correlation between vitamin D3 (VD3), parathyroid hormone (PTH), zinc, and BMD in zone 7. To sum up, it is found that trace elements may be related to prosthesis loosening, which provides experimental basis for the treatment of prosthesis loosening later.

Toxic effects of dechlorane plus on the common carp (Cyprinus carpio) embryonic development

Dechlorane Plus (DP) is a widely used chlorinated flame retardant, which has been extensively detected in the environment. Although DP content in the surface water is low, it can pose a continuous exposure risk to aquatic organisms due to its strong bioaccumulation. Considering that the related studies on the toxicity mechanism of DP exposure are limited, the effect of DP on carp embryo development was evaluated. In the present work, carp embryos were exposed to different concentrations (0, 30, 60, and 120 μg/L) of DP at 3 h post-fertilization (hpf). The expression levels of neural and skeletal development-associated genes, such as sox2, sox19a, Mef2c and BMP4, were detected with quantitative PCR, and the changes in different developmental toxicity endpoints were observed. Our results demonstrated that the expression levels of sox2, sox19a, Mef2c and BMP4 were significantly altered and several developmental abnormalities were found in DP-exposed carp embryos, such as DNA damage, increased mortality rate, delayed hatching time, reduced hatching rate, decreased body length, and increased morphological deformities. In addition, the activities of reactive oxygen species and malondialdehyde were remarkably higher in 60 and 120 μg/L DP exposure groups than in control group. These results suggest that DP can exhibit a unique modes of action, which lead to aberration occurrence in the early development stage of common carps, which may be related to some gene damage and oxidative stress. Besides, the parameters evaluated here can be used as tools to access the environmental risk for biota and humans exposed to DP.

GREM1 inhibits osteogenic differentiation, senescence and BMP transcription of adipose-derived stem cells

ABSTRACT Purpose: Adipose-derived stem cells (ADSCs) are ideal for cell-based therapies to support bone regeneration. It is vital to understand the critical genes and molecular mechanisms involved in the functional regulation of ADSCs for enhancing bone regeneration. In the present study, we investigated the Gremlin 1 (GREM1) effect on ADSCs osteogenic differentiation and senescence. Materials and methods: The in vitro ADSCs osteogenic differentiation potential was evaluated by determining alkaline phosphatase (ALP) activity, mineralization ability, and the expression of osteogenic markers. Cell senescence is determined by SA-β-gal staining, telomerase assay, and the expression of aging markers. Results: GREM1 overexpression in ADSCs reduced ALP activity and mineralization, inhibited the expression of osteogenic related genes OCN, OPN, DSPP, DMP1, and BSP, and key transcription factors, RUNX2 and OSX. GREM1 knockdown in ADSCs enhanced ALP activity and mineralization, promoted the expression of OCN, OPN, DSPP, DMP1, BSP, RUNX2, and OSX. GREM1 overexpression in ADSCs reduced the percent SA-β-Gal positive cells, P16 and P53 expressions, and increased telomerase activity. GREM1 knockdown in ADSCs increased the percentage of SA-β-Gal positive cells, P16 and P53 expressions, and reduced telomerase activity. Furthermore, GREM1 reduced the mRNA expression levels of BMP2, BMP6, and BMP7. Conclusions: In summary, our findings suggested that GREM1 inhibited ADSCs senescence and osteogenic differentiation and antagonized BMP transcription.

TGF\u03b2-mediated expression of TGF\u03b2-activating integrins in SSc monocytes: disturbed activation of latent TGF\u03b2?

IntroductionThe pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFβ signaling. TGFβ is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFβ in serum of SSc patients and investigated if this correlates with integrin expression on monocytes.MethodsA TGFβ/SMAD3- or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFβ and BMP levels respectively. A pan-specific TGFβ1/2/3 neutralizing antibody was used to confirm TGFβ signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFβ1 or a small molecule inhibitor of TGFBR1: SB-505124.ResultsSSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFβ levels were not different. Addition of a pan-specific TGFβ1/2/3 neutralizing antibody fully inhibited SMAD3-reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3, ITGB5, and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFβ1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFβ receptor inhibitor SB-505124 had the opposite effect.ConclusionTotal TGFβ serum levels are not different between SSc patients and controls, but TGFβ activity is. This coincides with a reduced expression of TGFβ-activating integrins in monocytes of SSc patients. Because TGFβ regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.

Changes of Wnt/β-catenin signalling, BMP2, and BMP4 in the jejunum during ageing in rats

Background and study aimsThe renewal of intestinal epithelium is maintained by intestinal stem cells (ISCs). Studies have found an age-dependent increase of Esg+/Dl+ progenitor cells in the midgut of Drosophila. However, changes of ISCs and the molecular regulation in mammalian animals with age are yet unknown. The aim of this study was to find out the changes of ISCs and molecular regulation in mammalian animals during the process of ageing. Material and methodsThirty Sprague–Dawley rats were divided into three groups: young (3 months old), adult (6 months old), and ageing (24 months old). Levels of PCNA, Bmi1, β-catenin and BMP4 were examined by Immunohistochemistry staining. Levels of Bmi1, GSK-3β, Dkk1 and BMP2 were determined by Western Blot. ResultsOur results showed that the proliferation of ISCs was decreased and the number of intestinal stem cells declined in ageing rats. The niches of ISCs, including Wnt signalling pathway and some proteins of Bone morphogenetic protein (BMP) signalling pathway, were downregulated in the jejunum of ageing rats. ConclusionOur study indicated that age-related decreased proliferation of intestinal stem cells in the jejunum could be associated with the alleviation of niches, including Wnt signalling pathway and some proteins of BMP signalling pathway.

3D Printed Hydroxyapatite/Silk Fibroin/Polycaprolactone Artificial Bone Scaffold and Bone Tissue Engineering Materials Constructed with Double-Transfected Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor Mesenchymal Stem Cells to Repair Rabbit Radial Bone Defects

To examine the effect of bone tissue engineering material constructed via 3D printed nanohydroxyapatite/silk fibroin/polycaprolactone (nHA/SF/PCL) artificial bone scaffolds and doubletransfected BMP-2/VEGF mesenchymal stem cells in repairing rabbit radial bone defects, 60 New Zealand rabbits were randomly selected, and with the establishment of a 15 mm bone defect model upon the middle of the anterior right radius bone. The rabbits were randomly divided into 4 groups, with each group composed of 15 rabbits: Group A (nHA/SF/PCL BMP-2/VEGF), Group B (simple nHA/SF/PCL), Group C (simple BMP-2/VEGF), Group D (blank control). Our team recorded the eating, movement, redness, exudation, and so forth across each sample group following the operation. The bone protein expression levels were measured via western blotting following 12 weeks post-operation. Histological observation was carried performed via tissue sections. Following the operation procedure, the wound healed healthily across the 4 experimental groups, no serious infections occurred, only a few rabbits exhibited redness, exudation, pus, and other phenomena, and there was no significant difference present in each group (P &gt; 0.05). The A group took the shortest time to recover its autonomous activity ability, followed by the B group, and the D group took the longest time to recover its autonomous activity ability. From western blotting tests, the A group possessed the highest BMP-2 and OCN expression levels, and have significant difference existed with the B, C, and D group (P &lt; 0.05). The order of expression level of BMP-2 and OCN across the other three groups were group C &gt; group B &gt; group D, respectively. The results of a histological section performed at 12 weeks post-operation revealed that there was no immune response present and no adverse inflammatory reaction occurring within each experimental group. Among the rabbits, the A group exhibited an apparent plate layer new bone formation, bone trabeculae were arranged regularly, and there was no obvious space within the new bone tissue; furthermore, the repair of the bone defect was completed. The B group still exhibited some bone scaffold remaining within the implantation area, and there was a small gap between the new bone tissue, and several bone defects had not been repaired. The C group of bone defects presented only a small number of new bone formation, and bone defect repair effect was not obvious. The D group still possessed large bone defects and exhibited poor repair of bone defects. The nHA/SF/PCL composite of artificial bone scaffold and double-transfected BMP-2/VEGF mesenchymal stem cells was effective in repairing radial bone defects within rabbits, and was superior to using nHA/SF/PCL bone scaffolds alone.

IL-23 induces the expression of pro-osteogenic factors in osteoclasts

AbstractBackground The mechanism for the new bone formation in ankylosing spondylitis (AS) is still unclear. Although it has been demonstrated that IL-23 plays a pivotal role in the pathophysiology of AS, IL-23 has no direct effects on osteoblasts but modulates the function of osteoclasts.Aims To explore whether IL-23 indirectly facilitates new bone formation through osteoclasts in AS, here we analyzed whether IL-23 enhances the expression levels of pro-osteogenic factors by osteoclasts.Methods Mononuclear cells were harvested from mouse bone marrow and cultured in the presence of M-CSF (50 ng/ml) and RANKL (30 ng/ml) to trigger the production of osteoclasts. Protein and mRNA expression levels of Semaphorin 4D, Ephrin B2, BMP2, BMP6, SPHK1, HtrA1 and Wnt10b were measured using Western blot and qRT-PCR.Results Primary mononuclear cells were transformed into osteoclasts with RANKL and M-CSF. The increased expression of NFATc1 and TRAP together with TRAP staining of&gt;3 nuclei were used to identify mature osteoclasts. The mRNA expression levels of BMP2, Ephrin B2 and SPHK1 were enhanced by 1.46, 2.1 and 2.46 folds after exposure to IL-23. Confirmation of increased levels of Ephrin B2 and SPHK1 in IL-23-stimulated osteoclasts was provided by Western blot analysis. IL-23 had no effects on the expression of BMP6 or Wnt10b, or on the anti-osteogenic factors Semaphorin 4D or HtrA1.Conclusions IL-23 induces osteoclasts to express pro-osteogenic factors rather than anti-osteogenic factors, suggesting IL-23 might indirectly promote the differentiation of osteoblasts through activated osteoclasts in ankylosing spondylitis.

The dose-time effects of fluoride on the expression and DNA methylation level of the promoter region of BMP-2 and BMP-7 in rats

Skeletal fluorosis is a chronic metabolic bone disease caused by excessive exposed to fluoride. Recent studies have shown that fluoride causes abnormal bone metabolism through disrupting the expression of Bone Morphogenetic Proteins (BMPs). However, the relationship between fluoride and BMPs is not fully understood, and the mechanism of fluoride on BMPs expression is still unclear. This study investigated the dose-time effects of fluoride on BMP-2 and BMP-7 levels and DNA methylation status of the promoter regions of these two genes in peripheral blood of rats. Eighty Wistar male rats were randomly divided into four groups and treated for 1 month and 3 months with distilled water (control), 25 mg/L, 50 mg/L or 100 mg/L of sodium fluoride (NaF). Rats exposed to fluoride had higher protein expression of BMP-2 and BMP-7 in plasma at 1 month and 3 months. An increase in BMP-2 expression was also observed with an increase of fluoride exposure time. Significant hypomethylation was observed in 2 CpG sites (CpGs) of BMP-2 and 1 CpG site of BMP-7 promoter regions in the fluoride treatment groups. It concludes that fluoride has a dose-response effect on BMP-2 in fluorosis rats, and fluoride-induced hypomethylation of specific CpGs may play an essential role in the regulation of BMP-2 and BMP-7 expression in rats.

Thymic Reconstitution after Damage Is Influenced By Zinc Status

T cell reconstitution, which is dependent on thymus function, is significantly delayed after hematopoietic stem cell transplant; leading to morbidity and mortality due to infection and possibly even malignant relapse. Zinc, the second most abundant trace metal in the body, is crucial for thymic function: in fact, zinc deficiency of all causes lead to thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). However, the role of zinc in thymic reconstitution after chemotherapy and hematopoietic stem cell transplantation (HSCT) has not been investigated. Using a murine model of thymic function after acute damage, we found that mice showed reduced thymic cellularity after three weeks of a zinc deficient diet, and lower regenerative capacity after a sublethal dose of total body irradiation (SL-TBI) (Fig. a). Conversely, zinc supplementation in normal dietary condition caused increased thymic regeneration at time-points after a mouse model of allogeneic-HSCT (Fig. b). As a result, mice that received zinc supplementation demonstrated more naive T cells and more recent thymic emigrants in peripheral blood (Fig. c) after allo-HSCT. Mechanistically, we found that zinc supplementation could stimulates the proliferation of thymic epithelial cells (TECs), which are the main cell type responsible for supporting for T-cell development, likely via induction in the production of BMP4, a pro-regenerative cytokine produced by endothelial cells (EC) (Fig. d). In fact, treatment with the BMP-receptor antagonist dorsomorphin abrogated the effect of zinc supplementation (Fig. e), and ex vivo propagated ECs (exECs) were directly induced to produce BMP4 when stimulated in vitro for 24 hours with zinc sulfate (Fig. f). Following damage there is a switch in the availability of zinc from inside the cellular fraction to the extracellular fraction (Fig g), suggesting that zinc is normally used and stored in developing T-cells but after damage they release zinc in the extracellular space after cell death due to TBI. This release is enough to trigger the production of regenerating factors from radio resistant cells, such as EC. Consistent with this, we found higher levels of intracellular zinc in thymocytes from zinc-supplemented mice before TBI and higher levels of zinc in the extracellular fraction after damage (Fig h). Moreover, while there was no difference in BMP4 expression in cocultures of exEC in presence of SN from control and zinc-treated mice at day 0, BMP4 levels increased in the presence of SN harvested from mice that had previously received TBI (Fig i). In conclusion, we herein demonstrate that zinc is important for thymic regeneration, and zinc supplementation offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT.

BMP2 Modified by the m6A Demethylation Enzyme ALKBH5 in the Ossification of the Ligamentum Flavum Through the AKT Signaling Pathway.

Ossification of the ligamentum flavum (OLF) is characterized by a process of ectopic bone formation in the ligamentum flavum. The definitive pathophysiology of OLF still remains unclear, but the epigenetic m6A modification plays an important role in OLF. In addition, no studies have reported the function of ALKBH5 in OLF development. In this study, we investigated the function of the m6A demethylation enzyme ALKBH5 in OLF. To evaluate the function of ALKBH5, OLF tissues and normal ligamentum flavum tissues were collected. In vitro methods, including HE, IHC and western blotting assays, were used to evaluate the association of ALKBH5 with OLF. In addition, we verified the effects of ALKBH5 on osteogenesis using alizarin red and ALP staining. MeRIP q-PCR was performed to investigate the methylation level of BMP2. Moreover, the mechanism of ALKBH5-mediated regulation of the ossification of the ligamentum flavum cells through the AKT signaling pathway was also verified. The present study showed that the expression of ALKBH5 increased in OLF tissues. The overexpression of ALKBH5 increased the expression of osteogenic genes and promoted the ossification of ligamentum flavum cells. Furthermore, BMP2 was significantly enriched in the ligamentum flavum cells of the anti-m6A group compared with those of the IgG group. The overexpression of ALKBH5 led to the activation of p-AKT, and BMP2 was regulated by ALKBH5 through the AKT signaling pathway. ALKBH5 promoted the osteogenesis of the ligamentum flavum cells through BMP2 demethylation and AKT activation. ALKBH5 was shown to be an important demethylation enzyme in OLF development.

The diabetic dental pulp repair: involvement of vascular endothelial growth factor and bone morphogenetic protein 2

Introduction/Objective. We aimed to investigate the effects of diabetes mellitus (DM) on rat dental pulp repair by measuring time-dependent changes in expressions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP 2) following direct pulp capping. Methods. Two groups, each of 20 Wistar rats, received either streptozotocin (for DM induction) or the same volume of sterile saline. A week later, the pulp of maxillary and mandibular right incisors in diabetic and non-diabetic groups were exposed and capped with calcium hydroxide in order to provoke reparative response. The levels of VEGF and BMP 2 were determined in the pulp tissue lysates one and seven days after the pulp capping, using enzyme-linked immunosorbent assays. Results. Diabetic state per se increased VEGF level, with a peak at first day after the pulp capping (19.3 ? 0.9 pg/mg, p &lt; 0.001), but did not affect BMP 2 levels. Significant increase of BMP 2 expression was noticed on the seventh day in capped pulp, but only in diabetic rats (16.7 ? 1 pg/mg, p = 0.001). Positive correlation between VEGF and BMP 2 was found on the seventh day following capping, only in diabetic pulp (r = 0.905, p = 0.003). Conclusion. Diabetes-induced increase in VEGF expression reflects changes in the inflammatory phase of pulp repair in DM. Increase in BMP 2 expression suggest that stimulating effect of calcium hydroxide appears seven days after diabetic pulp capping.

Black Tea (Camellia sinensis) Extract Induced Changes on Placenta can Alter Fetal and Neonatal Bone Health in Experimental Animal Model

Tea (Camellia sinensis) being the most consumed beverage worldwide. The safety evaluation of tea needs to be monitored during pregnancy, prenatal and postnatal developmental period beside its beneficial roles toward health and disease. Retardation of growth of fetus and neonates are common in preeclampsia. Present study was to evaluate the role of Black Tea extract (BTE) on placental and apoptotic markers in pregnant Wister albino rats and to correlate it the growth of fetus and pups. Among three experimental groups, Group 1 was pregnant female rats treated with saline, were the control group. Group 2 and Group 3 were pregnant female rats treated with 50 mg and 100 mg BTE/kg/day, p.o. respectively throughout prenatal and postnatal periods. Expressions of BMP-7, MMP-2 and VEGFR2 in placenta were examined by flow cytometry; Bax, Bcl-2 and caspase-3 expressions in uteri and placenta were observed by IHC. Bone health of fetus and pup were checked by histology, bone-cartilage double staining and estimation of bone mineral density by ICP-MS. Experimental data were subjected to the ANOVA; expressed as mean ± standard deviation with significance (P &lt; 0.05) between the controls and the treated groups (n = 6). BTE increased the level of MMP-2, Bax and caspase-3; decreased the level of BMP-7 in placenta. In fetus and pups, BTE significantly decreased the concentration of Ca2+, P, Mg2+ and Zn2+ in bone and decreased the rate of ossification were observed. This study confirmed BTE induced preeclampsia retarded the fetal and neonatal bone health in experimental animal model

Cyclosporine A impairs bone repair in critical defects filled with different osteoconductive bone substitutes

The aim of this study was to assess the influence of cyclosporine administration on the repair of critical-sized calvaria defects (CSDs) in rat calvaria filled with diverse biomaterials. Sixty animals were divided into two groups: the control (CTR) group (saline solution) and the cyclosporine (CCP) group (cyclosporine, 10 mg/kg/day). These medications were administered daily by gavage, beginning 15 days before the surgical procedure and lasting until the day the animals were euthanized. A CSD (5 mm Ø) was made in the calvaria of each animal, which was allocated to one of 3 subgroups, according to the biomaterial used to fill the defect: coagulum (COA), deproteinized bovine bone (DBB), or biphasic calcium phosphate ceramics of hydroxyapatite and β-phosphate tricalcium (HA/TCP). Euthanasia of the animals was performed 15 and 60 days after the surgical procedure (n = 5 animals/period/subgroup). Bone repair (formation) assessment was performed through microtomography and histometry, while the analyses of the expression of the BMP2, Osteocalcin, and TGFβ1 proteins were performed using immunohistochemistry. The CSDs not filled with biomaterials demonstrated lower bone formation in the CCP group. At 15 days, less bone formation was observed in the CSDs filled with DBB, a smaller volume of mineralized tissue was observed in the CSDs filled with HA/TCP, and the expression levels of BMP2 and osteocalcin were lower in the CCP group compared to the CTR group. The use of cyclosporine impaired bone repair in CSD, and this effect can be partially explained by the suppression of BMP2 and osteocalcin expression.

Effect of anatomical location (mandible vs maxilla) of dental implants on the BMP-2, BMP-7, sRANKL and OPG levels in periimplant crevicular fluid during osseointegration. A pilot study

Background:The aim of this study was to investigate levels of bone morphogenetic protein-2 (BMP-2), BMP-7, soluble receptor activator of nuclear factor-kB ligand (sRANKL) and osteoprotegerin (OPG) in the peri-implant crevicular fluid (PICF) of implants placed in both maxilla and mandible during the osseointegration period.Methods:Thirty-three patients (17 females and 16 males; mean age 47.03±11.23 years) were included in this study. A total of 33 implants were placed in both of maxilla (Group 1/n=18) and mandible (group 2/n=15). Peri-implant crevicular fluid (PICF) samples, modified plaque index (MPI), gingival index (GI) and probing depth (PD) measurements were obtained at 1 and 3 months after surgery. PICF levels of BMP-2/-7, sRANKL and OPG were analyzed by ELISA.Results:No complications were observed during the healing period. No significant differences were observed in the PICF levels of sRANKL, OPG, BMP-2 and BMP-7 and evaluated clinical parameters between groups at any time point (p&amp;gt;0.05). While PICF volume of group 2 was greater than group 1 at first month, PICF volume of group 1 was greater than group 2 at 3 months (p&amp;lt;0.05). There was a positive correlation between sRANKL levels and PICF volume (p&amp;lt;0.05) and a strong correlation between BMP-2 and BMP-7 (p&amp;lt;0.01).Conclusions:The results of this pilot study didn’t show any significant difference in PICF levels of BMP-2, BMP-7, sRANKL, and OPG in terms of anatomic location of dental implants. Further well-designed studies should be carried out to evaluate the relationship between bone related biomarkers and anatomic location of dental implants.

Multiple cytokine analyses of aqueous humor from the patients with retinitis pigmentosa

PurposeCataracts are the most common eye complications of retinitis pigmentosa (RP). This study aimed to investigate the cytokine profiles of the aqueous humor of RP with cataracts. MethodsThe aqueous humor was collected from RP eyes with cataract (RP group, n = 20) and age-related cataract eyes (ARC group, n = 20) during cataract surgery. The levels of 37 mediators were measured with multiplex fluorescent bead-based immunoassay and compared across groups. The correlation among chemokines, growth factors, and cytokines was analyzed with Spearman’s rank correlation coefficient. ResultsTwelve cytokines (IL-1α, IL-1β, IL-4, IL-10, TNF-α, IFN-γ, EGF, GM-CSF, PDGF-AB/BB, TGF-α, BMP-9, and E-selection) were below the limit of detection, and the detection rate of IL-6 was significantly higher in RP group than in the ARC group (P < 0.01). Compared with those in the control group, the aqueous humor levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-(IL-)8, interferon gamma-induced protein (IP)-10, hepatocyte growth factor (HGF), platelet-derived growth factor AA (PDGF-AA), matrix metalloproteinase-2 (MMP-2), MMP3, MMP-7, MMP-8, plasminogen activator inhibitor-1 (PAI-1), and thrombospondin-2 (TSP-2) in the RP group increased significantly (P < 0.01). A lower level of BMP-4 in the aqueous humor was observed in the RP patients than in the controls (P < 0.05). ConclusionsSignificantly increased levels of PDGF-AA, MMP2, MMP3, MMP-7, MMP-8, PAI-1, and TSP-2 and lower levels of BMP-4 were found in the aqueous humor of RP patients. This result indicates a disturbance of the extracellular matrix (ECM) and cytokines in RP patients and suggests a possible role of these cytokines in the pathogenesis of capsular contraction syndrome (CCS) in RP patients.