Identification of tissue of origin in cancer of unknown primary using a targeted bisulfite sequencing panel.

Aim: To construct a targeted bisulfite sequencing panel predicting origin of cancer of unknown primary. Methods: A bisulfite sequencing panel targeting 2793 tissue-specific markers was performed in 100 clinical samples. Results: The authors' prediction model showed 0.85 accuracy for the 'first-ranked' tissue type and 0.93 accuracy for the 'second-ranked' tissue type using 2793 tissue-specific markers and 0.84 accuracy for the 'first-ranked' tissue type and 0.92 accuracy for the 'second-ranked' tissue type when the number of tissue-specific markers was reduced to 514. Conclusion: Targeted bisulfite sequencing is a useful method for predicting the tissue of origin in patients with cancer of unknown primary.

When patients with cancer present with tumors that have migrated from elsewhere in the body, it is difficult for clinicians to identify where the cancer originated. DNA methylation profiling is a promising test to help identify where the cancer originated because it reflects cell of origin and is compatible with formalin-fixed, paraffin-embedded tissues. Because next-generation sequencing has already been implemented in clinical laboratories, the authors developed a targeted bisulfite sequencing panel that could predict the tissue of origin using genomic DNA extracted from formalin-fixed, paraffin-embedded tissues. The authors found that a hybrid capture-based targeted bisulfite sequencing panel is a useful method for predicting the tissue of origin in patients with cancer of unknown primary origin in clinical practice.

MicroRNA-496 inhibits triple negative breast cancer cell proliferation by targeting Del-1.

ABSTRACT: Del-1 has been linked to the pathogenesis of various cancers, including breast cancer. However, the regulation of Del-1 expression remains unclear. We previously reported the interaction between microRNA-137 (miR-137) and the Del-1 gene. In this study, we investigated miR-496 and miR-137 as regulators of Del-1 expression in triple negative breast cancer (TNBC). Del-1 mRNA and miR-496 were measured by quantitative PCR in breast cancer cells (MDA-MB-231, MCF7, SK-BR3, and T-47D) and tissues from 30 patients with TNBC. The effects of miR-496 on cell proliferation, migration, and invasion were determined with MTT, wound healing, and Matrigel transwell assays, respectively. In MDA-MB-231 cells, miR-496 levels were remarkably low and Del-1 mRNA levels were higher than in other breast cancer cell lines. Luciferase reporter assays revealed that miR-496 binds the 3'-UTR of Del-1 and Del-1 expression is downregulated by miR-496 mimics. Furthermore, miR-496 inhibited the proliferation, migration, and invasion of MDA-MB-231 cells. The effects of miR-496 on cell proliferation were additive with those of miR-137, another miRNA that regulates Del-1 expression. Moreover, in the 30 TNBC specimens, miR-496 was downregulated (P < .005) and the levels of Del-1 in the plasma were significantly elevated as compared with in normal controls (P = .0142). The Cancer Genome Atlas (TCGA) data showed the correlation of miR-496 expression with better overall survival in patients with early TNBC. In in silico and in vitro analyses, we showed that Del-1 is a target of miR-496 in TNBC and thereby affects cancer progression. Our findings suggest that miR-496 and miR-137 additively target Del-1 and act as modulating factors in TNBC. They are potentially new biomarkers for patients with TNBC.

Development and Validation of a Next-Generation Sequencing-Based Multigene Assay to Predict the Prognosis of Estrogen Receptor-Positive, HER2-Negative Breast Cancer.

PURPOSE: Multigene assays provide useful prognostic information regarding hormone receptor (HR)-positive breast cancer. Next-generation sequencing (NGS)-based platforms have numerous advantages including reproducibility and adaptability in local laboratories. This study aimed to develop and validate an NGS-based multigene assay to predict the distant recurrence risk. EXPERIMENTAL DESIGN: In total, 179 genes including 30 reference genes highly correlated with the 21-gene recurrence score (RS) algorithm were selected from public databases. Targeted RNA-sequencing was performed using 250 and 93 archived breast cancer samples with a known RS in the training and verification sets, respectively, to develop the algorithm and NGS-Prognostic Score (NGS-PS). The assay was validated in 413 independent samples with long-term follow-up data on distant metastasis. RESULTS: In the verification set, the NGS-PS and 21-gene RS displayed 91.4% concurrence (85/93 samples). In the validation cohort of 413 samples, area under the receiver operating characteristic curve plotted using NGS-PS values classified for distant recurrence was 0.76. The best NGS-PS cut-off value predicting distant metastasis was 20. Furthermore, 269 and 144 patients were classified as low- and high-risk patients in accordance with the cut-off. Five- and 10-year estimates of distant metastasis-free survival (DMFS) for low- versus high-risk groups were 97.0% versus 77.8% and 93.2% versus 64.4%, respectively. The age-related HR for distant recurrence without chemotherapy was 9.73 (95% CI, 3.59-26.40) and 3.19 (95% CI, 1.40-7.29) for patients aged ≤50 and >50 years, respectively. CONCLUSIONS: The newly developed and validated NGS-based multigene assay can predict the distant recurrence risk in ER-positive, HER2-negative breast cancer.

Multiplex Generation, Tracking, and Functional Screening of Substitution Mutants Using a CRISPR/Retron System.

We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by coutilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.

MicroRNA-137 Inhibits Cancer Progression by Targeting Del-1 in Triple-Negative Breast Cancer Cells.

MicroRNAs (miRNAs) can be used to target a variety of human malignancy by targeting their oncogenes or tumor suppressor genes. The developmental endothelial locus-1 (Del-1) might be under miRNA regulation. This study investigated microRNA-137 (miR-137) function and Del-1 expression in triple-negative breast cancer (TNBC) cells and tissues. Del-1 mRNA and miRNA-137 levels were determined via qRT-PCR in breast cancer cells (MDA-MB-231, MCF7, SK-BR3, and T-47D) and tissues from 30 patients with TNBC. The effects of miR-137 on cell proliferation, migration, and invasion were determined using MTT assays, wound healing, and Matrigel transwell assays. The luciferase reporter assay revealed direct binding of miR-137 to the 3'-UTR of Del-1. miR-137 inhibited cell proliferation, migration, and invasion of MDA-MB-231 cells. Among the 30 TNBC specimens, miR-137 was downregulated and Del-1 level in plasma was significantly elevated relative to normal controls. It is concluded that miR-137 regulates Del-1 expression in TNBC by directly binding to the Del-1 gene and cancer progression. The results implicate miR-137 as a new therapeutic biomarker for patients with TNBC.

Overcoming Tamoxifen Resistance by Regulation of Del-1 in Breast Cancer.

BACKGROUND: Hormone receptor-positive breast cancer accounts for nearly two-thirds of breast cancer cases; it ultimately acquires resistance during endocrine treatment and becomes more aggressive. This study evaluated the role of developmental endothelial locus (Del)-1 in tamoxifen-resistant (TAM-R) breast cancer. METHODS: Del-1 expression in recurrent TAM-R breast cancer tissue was evaluated and compared to that in the original tumor tissue from the same patients. Del-1 expression was also evaluated in TAM-R cells by quantitative real-time PCR, western blotting, and enzyme-linked immunosorbent assay. The effects of Del-1 knockdown on the proliferation, migration, and invasion of TAM-R cells was assessed with wound-healing and Matrigel transwell assays. RESULTS: Del-1 was more highly expressed in recurrent breast cancer as compared to the original tumor tissues before initiation of endocrine treatment. Del-1 mRNA was upregulated in TAM-R and small interfering RNA-mediated knockdown of Del-1 suppressed the migration and proliferation of TAM-R cells while partly restoring TAM sensitivity. And the TAM resistance was recovered by knockdown of Del-1. CONCLUSIONS: TAM-R breast cancer is characterized by Del-1 overexpression and tumor progression can be inhibited by Del-1 depletion, which restores TAM sensitivity. Thus, therapeutic strategies that target Del-1 may be effective for the treatment of hormone-resistant breast cancer.

Estimation of health benefits from air quality improvement using the MODIS AOD dataset in Seoul, Korea.

BACKGROUND: Exposure to fine particles in the atmosphere can adversely affect health and even lead to premature death. Recently, South Korea has attracted attention because of its rapid increase in the concentration of Particulate Matter (PM). OBJECTIVES: We estimated the economic benefits of reducing PM10 in Seoul, South Korea, based on MODerate-resolution Imaging Spectroradiometer (MODIS) Aerosol Optical Depth (AOD). Based on the retrieved PM10 data, we estimated its effects on overall health in each district of Seoul, Korea between 2014 and 2015. METHODS: The relationships between MODIS AOD and ground-based PM10 data were identified in different seasons in South Korea between 2012 and 2013 using the linear regression model. The health benefits were estimated by the Benefits Mapping and Analysis Program (Benmap) using the scenarios from the World Health Organization (WHO). RESULTS: The correlation between MODIS AOD and PM10 concentration differed with the season. There was a higher correlation between MODIS AOD and PM10 concentration in winter (R = 0.57) than there was in other seasons. Based on the MODIS AOD, the average annual PM10 concentration in Seoul was higher in 2014 than it was in 2015, at values of 45.7 µg/m3, and 41.6 µg/m3, respectively. The greatest economic benefit of reducing PM10 concentration (WHO annual standard of 20 µg/m3) was in 2014. This benefit was estimated to be 7022 (95% CI: 599, 20496), 2617 (95% CI: 216, 7750), and 1328 (95% CI: -159, 4679) billion KRW for all-cause, cardiovascular, and respiratory mortalities in 2014 and 2015, respectively. CONCLUSIONS: These results demonstrate that, despite considerable improvements in air quality in recent decades, there is still a need for countermeasures to prevent economic loss due to air pollution in Seoul.

Effect of High-dose Vitamin C Combined With Anti-cancer Treatment on Breast Cancer Cells.

BACKGROUND/AIM: The anti-cancer effect of high doses of intravenous vitamin C (high-dose vitamin C) remains controversial despite growing evidence that high-dose vitamin C exerts anti-tumorigenic activity by increasing the amount of reactive oxygen species in cancer cells without meaningful toxicities. Therefore, this study attempted to demonstrate the in vitro anti-cancer activity of high-dose vitamin C in combination with conventional treatment in breast cancer. MATERIALS AND METHODS: The pro-apoptotic effects of high-dose vitamin C (1.25 to 20 mM) with or without anti-cancer agents (eribulin mesylate, tamoxifen, fulvestrant, or trastuzumab) were estimated using an MTT assay to measure the cell viability of a variety of breast cancer cell lines (MCF7, SK-BR3, and MDA-MB-231), as well as normal breast epithelial cells (MCF10A). RESULTS: High-dose vitamin C (≥10 mM) significantly decreased cell viability of all breast cancer cell lines, particularly of MCF-7 cells. The catalase activities of MCF7 and MDA-MD-231 cells were also lower than those of MCF10A cells. Moreover, cell viability of both MCF7 and MDA-MD-231 cells was decreased further when combining high-dose vitamin C and eribulin mesylate, and this was also true for MCF-7 cells when combining high-dose vitamin C with tamoxifen or fulvestrant and for SK-BR3 cells when combining high-dose vitamin C with trastuzumab in comparison with chemotherapy or endocrine therapy alone. CONCLUSION: Combining high-dose vitamin C with conventional anti-cancer drugs can have therapeutic advantages against breast cancer cells.

Effects of Stromal Vascular Fraction on Breast Cancer Growth and Fat Engraftment in NOD/SCID Mice.

BACKGROUND: To overcome unpredictable fat graft resorption, cell-assisted lipotransfer using stromal vascular fraction (SVF) has been introduced. However, its effect on cancer growth stimulation and its oncological safety are debatable. We investigated the effect of SVF on adjacent breast cancer and transplanted fat in a mouse model. METHODS: A breast cancer xenograft model was constructed by injecting 2 × 106 MDA-MB-231-luc breast cancer cells into the right lower back of 40 NOD/SCID mice. Two weeks later, cancer size was sorted according to signal density using an in vivo optical imaging system, and 36 mice were included. Human fat was extracted from the abdomen, and SVFs were isolated using a component isolator. The mice were divided into four groups: A, controls; B, injected with 30 µl SVF; C, injected with 0.5 ml fat and 30 µl saline; group D, injected with 0.5 ml fat and 30 µl SVF. Magnetic resonance imaging and three-dimensional micro-computed tomography volumetric analysis were performed at 4 and 8 weeks. RESULTS: Tumor volume was 43.6, 42.3, 48.7, and 42.4 mm3 at the initial time point and 6780, 5940, 6080, and 5570 mm3 at 8 weeks in groups A, B, C, and D, respectively. Fat graft survival volume after 8 weeks was 49.32% and 62.03% in groups C and D, respectively. At 2-month follow-up after fat grafting in the xenograft model, SVF injection showed an increased fat survival rate and did not increase the adjacent tumor growth significantly. CONCLUSION: Fat grafting with SVF yields satisfactory outcome in patients who undergo breast reconstruction surgery. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Comparison of laser ablation using multidirectional and forward-firing fibers in breast cancer.

BACKGROUND: The aim of this study was to compare the therapeutic effect of laser ablation using the forward-firing fiber and the multidirectional-firing fiber for breast cancer treatment with pathologic results. MATERIAL AND METHODS: An ex vivo study of laser ablation was conducted using normal breast and breast cancer tissue. Each ablated area was demarcated into three zones, and the temperature was measured. Laser ablations using multidirectional and forward-firing types of fiber were compared regarding the shape, diameter and aspect ratio of the ablated lesions. RESULTS: The ablated lesions were classified into three zones: a carbonized zone with complete tissue loss; a coagulated zone with no viable cells; and a non-damaged zone. The shape of the ablated lesion was elliptical using the forward-firing fiber and round using the multidirectional-firing fiber. Compared with normal breast tissue, breast cancer tissue required a more powerful setting for laser ablation to achieve necrosis, and the aspect ratio of the thermal lesion was higher for laser ablation using the multidirectional-firing fiber. CONCLUSIONS: The experimental results on breast tissue have shown that multidirectional-firing fiber is more effective than using forward-firing fibers and that this may prove to be another feasible therapeutic option for management of breast cancer.

Biological function of long noncoding RNA snaR in HER2-positive breast cancer cells.

PURPOSE: Long noncoding RNA, snaR (small NF90-associated RNA), has been reported to be upregulated in various cancer cell lines. We evaluated the additional role of snaR in HER2-positive breast cancer cell lines. METHODS: We explored changes of expression of snaR among the selected long noncoding RNAs which have a potential in cancer proliferation or progression. The proliferation, migration, and invasion of HER2-positive breast cancer cells (SK-BR3) were evaluated by snaR with RNA interruption in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, wound-healing assay, and Transwell assay. RESULTS: The expression of snaR was remarkably upregulated in SK-BR3 cell lines together with ANRIL, while the SFMBT2 was downregulated in SK-BR3 cell lines. Although Nespas, 7SK, PSF inhibiting RNA, mascRNA, Hoxa11as, NRON, AK023948, MER11C, p53 mRNA, CAR Intergenic 10, HUC 1 and 2, ZFAS1, SCA8, and SNHG5 were also upregulated and UCA1 was downregulated, the differences were not dominent. Based on the expression result, we explored the functional role of snaR in HER2-positive breast cancer. Downregulation of snaR with small interfering RNA was identified to significanlty inhibit migration as well as proliferation of SK-BR3 cells. CONCLUSION: In this study, snaR was identified as upregulated and to play a role in cancer progression of HER2-positive breast cancer cells. These results suggest snaR as a potential biomarker for HER2-positive breast cancer.

AQP5 Variants Affect Tumoral Expression of AQP5 and Survival in Patients with Early Breast Cancer.

BACKGROUND: Our previous study showed the association of AQP5 upregulation with cancer proliferation and migration in breast cancer cell lines and with unfavorable prognosis in patients with early breast cancer (EBC). In the current study, we analyzed the association of AQP5 variants or their haplotypes with AQP5 expression and their prognostic impact for survival in patients with EBC. METHODS: Three AQP5 polymorphisms (rs74091166, rs3736309, and rs1964676) were selected based on the SNP database and genotyped using the Sequenom MassARRAY in 374 out of 447 patients with EBC in whom AQP5 expression had been investigated in our previous study. RESULTS: The allele frequencies of the selected variants in the current study were similar to those from Asian data previously reported. In a univariate analysis, both rs74091166 and rs1964676 were statistically associated with survival as a dominant model of minor allele. Moreover, a multivariate survival analysis revealed that the CC genotype of rs1964676 is an independent prognostic marker of survival in EBC patients, regardless of stage, tumor subtype, and adjuvant treatment [hazard ratio = 0.399, 0.384, and 0.205; p = 0.021, 0.027, and 0.016 for disease-free survival (DFS), distant DFS, and disease-specific survival, respectively]. In particular, the CT/TT genotype of rs1964676 showed an association with strong expression of AQP5 (58.6 vs. 26.0%; p = 0.001), without any associations with clinical or pathological characteristics including tumor subtype, stage, or histologic grade. CONCLUSION: The current study suggests AQP5 rs1964676 as a new potential prognostic marker in patients with EBC involved in AQP5 expression.

Long Noncoding RNA snaR Regulates Proliferation, Migration and Invasion of Triple-negative Breast Cancer Cells.

AIM: We evaluated the role of long noncoding ribonucleic acid (lncRNA) in breast cancer cell lines by quantitative reverse transcription-polymerase change reaction. MATERIALS AND METHODS: The effects of small NF90-associated RNA (snaR) with RNA interference on proliferation, migration and invasion of MDA-MB-231 cells were observed by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, wound healing and transwell assay. RESULTS: Among 90 lncRNAs, E2F transcription factor 4, p107/p130-binding (E2F4) antisense, insulin-like growth factor 2 antisense (IGF2AS), snaR, and small nucleolar RNA host gene 5 (SNHG5) were up-regulated in MDA-MB-231 and 7SK, antisense noncoding RNA in the INK4 locus (ANRIL), IGF2AS, Nespas, p53 mRNA, and snaR were up-regulated in MCF-7 cells. Down-regulation of snaR inhibited the proliferation, migration, and invasion of MDA-MD-231 breast cancer cells. CONCLUSION: LncRNA snaR was found to be up-regulated in breast cancer cells, and the cancer progression of MDA-MB-231 cells was significantly suppressed by down-regulation of snaR. Therefore, snaR knockdown has potential as a treatment modality for triple-negative breast cancer.

Aquaporin 3 Expression Predicts Survival in Patients with HER2-positive Early Breast Cancer.

BACKGROUND/AIM: Recent studies have revealed aquaporins (AQPs) as targets for novel anti-tumor therapy since they are likely to play a role in carcinogenesis, tumor progression and invasion. Accordingly, we analyzed the prognostic impact of AQP3 expression and polymorphisms in a number of patients with early breast cancer (EBC). MATERIALS AND METHODS: AQP3 expression was investigated on the basis of the immunohistochemistry of tissue microarray specimens from 447 EBC patients who underwent surgery between 2003 and 2008. We scored the staining intensity (0 through 3) and percentage of positive tumor cells (0 through 4); the staining score was defined as sum of these scores used to categorize the AQP3 expression as negative (0 through 2), weak (3 through 5) or strong (6 or more). For AQP3 polymorphisms, seven single nucleotide polymorphisms (SNPs) (rs10813981, rs34391490, rs2228332, rs2227285, rs591810, rs17553719 and rs3860987) were selected using in silico analysis and genotyped using the Sequenom MassARRAY. RESULTS: A total of 180 (40.3%) patients were identified as AQP3-positive (staining score >2), including 86 (19.2%) cases of strong expression (stating score >5). In a univariate analysis, AQP3 expression was significantly associated with survival for the patients with HER2-over-expressing EBC. Moreover, a multivariate survival analysis revealed that AQP3 expression was an independent prognostic marker of disease-free survival (DFS): hazard ratio (HR)=3.137, 95% confidence interval (CI)=1.079-9.125, p=0.036; distant DFS (DDFS): HR=2.784, 95%CI=0.921-8.414, p=0.070, for the HER2-over-expressing EBC patients. Meanwhile, none of selected AQP3 polymorphisms were related to AQP3 expression in tumor tissue or survival in the current study. CONCLUSION: AQP3 expression in tumor tissue may be considered as a potential prognostic marker in patients with HER2-over-expressing EBC after curative surgery.

Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker.

Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects, and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC. By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13 kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO).

Core binding factor ß of osteoblasts maintains cortical bone mass via stabilization of Runx2 in mice.

Core binding factor beta (Cbfß), the partner protein of Runx family transcription factors, enhances Runx function by increasing the binding of Runx to DNA. Null mutations of Cbfb result in embryonic death, which can be rescued by restoring fetal hematopoiesis but only until birth, where bone formation is still nearly absent. Here, we address a direct role of Cbfß in skeletal homeostasis by generating osteoblast-specific Cbfß-deficient mice (Cbfb(Δob/Δob) ) from Cbfb-floxed mice crossed with mice expressing Cre from the Col1a1 promoter. Cbfb(Δob/Δob) mice showed normal growth and development but exhibited reduced bone mass, particularly of cortical bone. The reduction of bone mass in Cbfb(Δob/Δob) mice is similar to the phenotype of mice with haploinsufficiency of Runx2. Although the number of osteoblasts remained unchanged, the number of active osteoblasts decreased in Cbfb(Δob/Δob) mice and resulted in lower mineral apposition rate. Immunohistochemical and quantitative real-time PCR analyses showed that the expression of osteogenic markers, including Runx2, osterix, osteocalcin, and osteopontin, was significantly repressed in Cbfb(Δob/Δob) mice compared with wild-type mice. Cbfß deficiency also reduced Runx2 protein levels in osteoblasts. The mechanism was revealed by forced expression of Cbfß, which increased Runx2 protein levels in vitro by inhibiting polyubiquitination-mediated proteosomal degradation. Collectively, these findings indicate that Cbfß stabilizes Runx2 in osteoblasts by forming a complex and thus facilitates the proper maintenance of bone mass, particularly cortical bone.

Functional cooperation between vitamin D receptor and Runx2 in vitamin D-induced vascular calcification.

The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D3 using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr(-/-)) and Runx2 carboxy-terminus truncated heterozygous (Runx2(+/ΔC)) mice. Treatment of VSMCs with active vitamin D3 promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D3. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D3 was completely inhibited in Vdr(-/-) or Runx2(+/ΔC) mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D3.

Reconstruction of radial bone defect using gelatin sponge and a BMP-2 combination graft.

Many bioactive molecules like recombinant human bone morphogenetic protein 2 (rhBMP-2) have been developed for mineralized bone grafts, for which proper scaffolds are necessary to successfully apply the bioactive molecules. In this study, we tested the osteogenic efficacy of rhBMP-2 produced in-house in combination with gelatin sponge as the scaffold carrier in a rabbit radial defect model. The efficacy of the rhBMP-2 was determined by alkaline phosphatase activity assay of C2C12 cells. Two groups of ten rabbits each were treated with rhBMP-2/gelatin sponge, or gelatin sponge only. At 4 weeks, rhBMP-2/gelatin sponge grafts showed more bone regeneration than gelatin sponge grafts, as determined by X-ray radiography, micro-computed tomography, and histological analyses. At 8 weeks, rhBMP-2/gelatin sponge grafts exerted much stronger osteogenic effects. The study demonstrates the improved osteogenic efficacy of the rhBMP-2/gelatin sponge grafts in a rabbit radial bone defect model acting as a bone-inductive material.