H-type blood vessels participate in alveolar bone remodeling during murine tooth extraction healing.

OBJECTIVES: We aimed to investigate whether skeletal-specific H-type blood vessels exist in alveolar bone and how they function in alveolar bone remodeling. MATERIALS AND METHODS: H-type vessels with high expression of CD31 and Endomucin (CD31hi Emcnhi ) were immunostained in alveolar bone. Abundance and age-related changes in CD31hi Emcnhi endothelial cells (H-ECs) were detected by flow cytometry. Osteoprogenitors association with H-type vessels and bone mass were detected in tooth extraction model of alveolar bone remodeling by immunohistofluorescence and micro-CT, respectively. Transcription and expression of H-EC feature genes during in vitro Notch inhibition were measured by RT-qPCR and immunocytofluorescence. RESULTS: We verified that H-type vessels existed in alveolar bone, the abundance of which was highest at infancy age, then decreased but maintained a constant level during aging. In tooth extraction model, H-ECs significantly increased with concomitant perivascular accumulation of Runx2+ osteoprogenitors and gradually augmentation of bone mass. Notch inhibition of in vitro cultured H-ECs resulted in decreased expression levels of Emcn and hes1, but not Pecam1 or Kdr genes, with decreased expression levels of H-EC numbers, accordingly. CONCLUSIONS: The present study suggests that H-type vessels promote osteogenesis during alveolar bone remodeling. Notch signaling pathway regulates expression of Emcn and possibly determines fate and functions of alveolar H-ECs.

Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism.

Osteoporosis and osteoporotic fractures severely compromise quality of life in elderly people and lead to early death. Human umbilical cord mesenchymal stromal cell (MSC)-derived extracellular vesicles (hucMSC-EVs) possess considerable therapeutic effects in tissue repair and regeneration. Thus, in the present study, we investigated the effects of hucMSC-EVs on primary and secondary osteoporosis and explored the underlying mechanisms. Methods: hucMSCs were isolated and cultured. EVs were obtained from the conditioned medium of hucMSCs and determined by using transmission electron microscopy, dynamic light scattering and Western Blot analyses. The effects of hucMSC-EVs on ovariectomy-induced postmenopausal osteoporosis and tail suspension-induced hindlimb disuse osteoporosis in mouse models were assessed by using microcomputed tomography, biomechanical, histochemical and immunohistochemical, as well as histomorphometric analyses. Proteomic analysis was applied between hucMSC-EVs and hucMSCs to screen the candidate proteins that mediate hucMSC-EVs function. The effects of hucMSC-EVs on osteogenic and adipogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), and osteoclastogenesis of the macrophage cell line RAW264.7 in vitro were determined by using cytochemical staining and quantitative real-time PCR analysis. Subsequently, the roles of the key protein in hucMSC-EVs-induced regulation on BMSCs and RAW264.7 cells were evaluated. Results: hucMSCs were able to differentiate into osteoblasts, adipocytes or chondrocytes and positively expressed CD29, CD44, CD73 and CD90, but negatively expressed CD34 and CD45. The morphological assessment revealed the typical cup- or sphere-shaped morphology of hucMSC-EVs with diameters predominantly ranging from 60 nm to 150 nm and expressed CD9, CD63, CD81 and TSG101. The systemic administration of hucMSC-EVs prevented bone loss and maintained bone strength in osteoporotic mice by enhancing bone formation, reducing marrow fat accumulation and decreasing bone resorption. Proteomic analysis showed that the potently pro-osteogenic protein, CLEC11A (C-type lectin domain family 11, member A) was very highly enriched in hucMSC-EVs. In addition, hucMSC-EVs enhanced the shift from adipogenic to osteogenic differentiation of BMSCs via delivering CLEC11A in vitro. Moreover, CLEC11A was required for the inhibitory effects of hucMSC-EVs on osteoclast formation. Conclusion: Our results suggest that hucMSC-EVs serve as a critical regulator of bone metabolism by transferring CLEC11A and may represent a potential agent for prevention and treatment of osteoporosis.

Harmine enhances type H vessel formation and prevents bone loss in ovariectomized mice.

Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a ß-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. METHODS: The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. RESULTS: Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. CONCLUSION: Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.

Omentin-1 prevents inflammation-induced osteoporosis by downregulating the pro-inflammatory cytokines.

Osteoporosis is a frequent complication of chronic inflammatory diseases and increases in the pro-inflammatory cytokines make an important contribution to bone loss by promoting bone resorption and impairing bone formation. Omentin-1 is a newly identified adipocytokine that has anti-inflammatory effects, but little is known about the role of omentin-1 in inflammatory osteoporosis. Here we generated global omentin-1 knockout (omentin-1-/-) mice and demonstrated that depletion of omentin-1 induces inflammatory bone loss-like phenotypes in mice, as defined by abnormally elevated pro-inflammatory cytokines, increased osteoclast formation and bone tissue destruction, as well as impaired osteogenic activities. Using an inflammatory cell model induced by tumor necrosis factor-α (TNF-α), we determined that recombinant omentin-1 reduces the production of pro-inflammatory factors in the TNF-α-activated macrophages, and suppresses their anti-osteoblastic and pro-osteoclastic abilities. In the magnesium silicate-induced inflammatory osteoporosis mouse model, the systemic administration of adenoviral-delivered omentin-1 significantly protects from osteoporotic bone loss and inflammation. Our study suggests that omentin-1 can be used as a promising therapeutic agent for the prevention or treatment of inflammatory bone diseases by downregulating the pro-inflammatory cytokines.

Runx2/miR-3960/miR-2861 Positive Feedback Loop Is Responsible for Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells.

We previously reported that Runx2/miR-3960/miR-2861 regulatory feedback loop stimulates osteoblast differentiation. However, the effect of this feedback loop on the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) remains unclear. Our recent study showed that miR-2861 and miR-3960 expression increases significantly during ß-glycerophosphate-induced osteogenic transdifferentiation of VSMCs. Overexpression of miR-2861 or miR-3960 in VSMCs enhances ß-glycerophosphate-induced osteoblastogenesis, whereas inhibition of miR-2861 or miR-3960 expression attenuates it. MiR-2861 or miR-3960 promotes osteogenic transdifferentiation of VSMCs by targeting histone deacetylase 5 or Homeobox A2, respectively, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Furthermore, overexpression of Runx2 induces miR-2861 and miR-3960 transcription, and knockdown of Runx2 attenuates ß-glycerophosphate-induced miR-2861 and miR-3960 transcription in VSMCs. Thus, our data show that Runx2/miR-3960/miR-2861 positive feedback loop plays an important role in osteogenic transdifferentiation of VSMCs and contributes to vascular calcification.

Anti-hepatoma human single-chain Fv antibody and adriamycin conjugates with potent antitumor activity.

To construct an improved biological missile, an immunoconjugate ADM-Dex-ScFv-SA3 was synthesized, which was composed of a hepatocellular carcinoma-specific, single-chain Fv antibody (ScFv-SA3) and a highly potent cytotoxic drug, adriamycin (ADM), as the warhead. Oxidized Dextran T10 (Dex-T10) was used as a linker to connect these two moieties. The 40 KD soluble anti-hepatoma human Trx-ScFv-SA3 protein was expressed in E. coli BL21 (DE3), using a prokaryotic expression vector, pET21a (+)-Trx-ScFv-SA3-His. It was purified using a His-Tag Ni-Agarose column and identified by western blot. The activity of Trx-ScFv-SA3 was verified by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to confirm that it specifically binds to the hepatocellular carcinoma cell line HepG2. To prepare ADM-Dex-ScFv-SA3, ADM was conjugated to the antibody at a molar ratio of 14.21:1. The antitumor effect of the conjugate was tested by MTT assay, plate colony formation assay and xenografts in a nude mice experimental model. In vitro experiments revealed that ADM-Dex-ScFv-SA3 could bind to tumor cells selectively and inhibit the proliferation and the colony formation ability of HepG2 cells. In vivo experiments showed that ADM-Dex-ScFv-SA3 suppressed the tumor growth and prolonged the median survival time in tumor-bearing mice. Tumor histology slides indicated a significantly slower tumor tissue proliferation in the ADM-Dex-ScFv-SA3 group. These data indicate that the targeted drug, ADM-Dex-ScFv-SA3, may be a highly potent and selective therapy for the treatment of hepatoma.

Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma.

We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.

GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit.

AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC). METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang's liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 µmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed. RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ. CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC.

Preparation of human scFv antibody against nasopharyngeal carcinoma and identification of its specificity.

UNLABELLED: Abstract Conclusion: The selected scFv antibody could specifically recognize and target nasopharyngeal carcinoma (NPC), and could be applied to clinical diagnosis and therapy. OBJECTIVE: The aim was to construct and screen fully human anti-NPC single chain Fv fusion phage libraries, and to identify the specificity of the scFv antibody. METHODS: Peripheral blood mononuclear cells of patients with NPC were immunized in vitro by NPC cells and transformed by Epstein-Barr virus. The total RNAwas used to construct the scFv libraries. By means of ELISA and immunochemistry, the positively bound scFv was selected and identified. The positive scFv was fused to EGFP, and was then expressed in E. coli strain BL21 (DE3) and purified. Furthermore, we observed the binding bioactivity. RESULTS: The fusion protein has the biological activity of binding the NPC cells and emitting green fluorescence. In targeting experiments in vivo, the results showed that the fusion protein can successfully target the NPC.

Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library.

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.

Spaceflight alters the gene expression profile of cervical cancer cells.

Our previous study revealed that spaceflight induced biological changes in human cervical carcinoma Caski cells. Here, we report that 48A9 cells, which were subcloned from Caski cells, experienced significant growth suppression and exhibited low tumorigenic ability after spaceflight. To further understand the potential mechanism at the transcriptional level, we compared gene expression between 48A9 cells and ground control Caski cells with suppression subtractive hybridization (SSH) and reverse Northern blotting methods, and analyzed the relative gene network and molecular functions with the Ingenuity Pathways Analysis (IPA) program. We found 5 genes, SUB1, SGEF, MALAT-1, MYL6, and MT-CO2, to be up-regulated and identified 3 new cDNAs, termed B4, B5, and C4, in 48A9 cells. In addition, we also identified the two most significant gene networks to indicate the function of these genes using the IPA program. To our knowledge, our results show for the first time that spaceflight can reduce the growth of tumor cells, and we also provide a new model for oncogenesis study.

Omentin-1 attenuates arterial calcification and bone loss in osteoprotegerin-deficient mice by inhibition of RANKL expression.

AIMS: Omentin-1 (also known as intelectin-1) is a recently identified visceral adipose tissue-derived cytokine that is inversely related to obesity. Our previous study showed that omentin-1 inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs) in vitro. This study was undertaken to investigate the effects of omentin-1 on arterial calcification and bone metabolism in vivo. METHODS AND RESULTS: In vitro, omentin-1 stimulated production of osteoprotegerin (OPG) and inhibited production of receptor activator for nuclear factor κB ligand (RANKL) in both CVSMCs and osteoblasts. In vivo, adenovirus-mediated over-expression of omentin-1 attenuated arterial calcification and bone loss in OPG(-/-) mice. All these in vitro and in vivo actions were abrogated by blockade of the PI3K-Akt signalling pathway. Furthermore, omentin-1 reduced serum levels of RANKL, tartarate-resistant acid phosphatase-5b and osteocalcin, all of which are increased dramatically in OPG(-/-) mice. CONCLUSION: These data suggest that omentin-1 ameliorates arterial calcification and bone loss in vivo through the regulation of the RANK signalling pathway.

Omentin inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells through the PI3K/Akt pathway.

Arterial calcification is positively associated with visceral adiposity, but the mechanisms remain unclear. Omentin is a novel adipokine that is selectively expressed in visceral adipose tissue. The levels of circulating omentin are decreased in obesity, and they correlate negatively with waist circumference. This study investigated the effects of omentin on the osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Omentin inhibited mRNA expression of alkaline phosphatase (ALP) and osteocalcin; omentin also suppressed ALP activity, osteocalcin protein production, and the matrix mineralization. Furthermore, omentin selectively activated phosphatidylinositol 3-kinase (PI3K) downstream effector Akt. Moreover, inhibition of PI3K or Akt activation reversed the effects of omentin on ALP activity and the matrix mineralization. The present results demonstrate for the first time that omentin can inhibit osteoblastic differentiation of CVSMCs via PI3K/Akt signaling pathway, suggesting that the lower omentin levels in obese (specially visceral obese) subjects contribute to the development of arterial calcification, and omentin plays a protective role against arterial calcification.

Expression and functional characterization of platelet-derived growth factor receptor-like gene.

AIM: To investigate the role of platelet-derived growth factor receptor-like gene (PDGFRL) in the anti-cancer therapy for colorectal cancers (CRC). METHODS: PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in CRC and colorectal normal tissues. PDGFRL prokaryotic expression vector was carried out in Escherichia coli (E. coli), and purified by immobilized metal affinity chromatography. The effect of PDGFRL protein on CRC HCT-116 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), clone counting, cell cycle, and wound healing assay. RESULTS: Both RT-PCR and immunohistochemistry showed that the expression of PDGFRL in colorectal normal tissues was higher than in cancer tissues. Recombinant pET22b-PDGFRL prokaryotic expression vector was successfully expressed in E. coli, and the target protein was expressed in the form of inclusion bodies. After purification and refolding, recombinant human PDGFRL (rhPDGFRL) could efficiently inhibit the proliferation and invasion of CRC HCT-116 cells detected by MTT, clone counting and wound healing assay. Moreover, rhPDGFRL arrested HCT-116 cell cycling at the G0/G1 phase. CONCLUSION: PDGFRL is a potential gene for application in the anti-cancer therapy for CRC.

Construction and selection of human Fab antibody phage display library of liver cancer.

The aim of this study was to construct the fully humanized anti-hepatoma Fab fragment phage libraries and select antibodies against hepatoma specifically. PBMCs of liver cancer patients were immunized in vitro with HpeG(2) cells and were then transformed by Epstein-Barr virus (EBV). After total RNA was extracted, the heavy chain Fd and kappa/lambda light chain were amplified by RT-PCR and cloned into the vector pComb3 to construct the libraries of Fab fragments. The libraries were then panned by HpeG(2) cells. By means of ELISA and immunochemistry, the Fab phage antibodies binding with hepatoma were selected and identified. The Fd and light chain PCR products were subsequently inserted into pComb3, and the volume of Fab libraries reached 1.7 x 10(7). The libraries were enriched about 138-fold by three cycles of panning. 540 phage clones were picked randomly. Using cell ELISA and immunohistochemistry with cultured cells, one clone Fab phage antibody, which had binding activity with hepatoma, was picked out. Fully humanized anti-hepatoma Fab antibody phage display libraries were constructed. One phage clone was selected and confirmed to specifically bind to hepatoma cells. The selected Fab antibody may be further developed and applied to clinical diagnosis and therapy.

Autoantibodies as potential biomarkers for nasopharyngeal carcinoma.

Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma (NPC). We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor-associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage-displayed proteins, 22 have sequence identity with known or putative tumor-associated proteins. The results of immunochemical reactivity of patients' sera with phage-expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors. The autoantibody against phage-expressed protein MAGE, HSP70, Fibronectin, and CD44 measured by ELISA had greater predictive value than that against EBNA-1, respectively. The antibody levels against MAGE in sera positively correlated with the clinical stages of NPC, and the antibody levels against other three proteins partly correlated with the clinical stages of NPC. Our studies suggested that the autoantibodies against tumor-associated antigens in the sera of NPC patients could be used as a screening test for NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy.

BMI-1 autoantibody in serum as a new potential biomarker of nasopharyngeal carcinoma.

BMI-1 is overexpressed in a variety of cancers, which can activate the immune system to produce antibodies in tumor tissues. In this study, we isolated phage expressing BMI-1 protein by screening of a mixture of nasopharyngeal carcinoma (NPC) cDNA T7 phage library and found that the antibody against BMI-1 was elevated in the sera from NPC patients. BMI-1 mRNA was overexpressed at different levels in seven NPC cell lines compared with normal nasopharyngeal epithelial cell line NP69. Histochemistry showed that patient sera were more reactive with BMI-1 than normal sera. Antibody affinity assay using sera from 40 NPC patients and 54 controls showed that BMI-1 antibody was significantly greater in patient sera than in normal controls (patient 0.791 +/- 0.025 and normal 0.488 +/- 0.042; P < 0.001) and the BMI-1 autoantibody be significantly related with the progress of NPC (Benign versus LNPC P=0.001; LNPC versus MNPC P=0.047). Analysis of the results with logistic regression and receiver operating characteristics (ROC) curves showed that BMI-1 antibody was a modest marker for NPC (sensitivity 0.74 and specificity 0.73; AUC = 0.8044). The showed that BMI-1 antibody as a potential marker of NPC may be rational, and could have diagnostic and prognostic value.